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Sample records for ccr5-tropic chimeric virus

  1. CCR5 Disruption in Induced Pluripotent Stem Cells Using CRISPR/Cas9 Provides Selective Resistance of Immune Cells to CCR5-tropic HIV-1 Virus.

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    Kang, HyunJun; Minder, Petra; Park, Mi Ae; Mesquitta, Walatta-Tseyon; Torbett, Bruce E; Slukvin, Igor I

    2015-12-15

    The chemokine (C-C motif) receptor 5 (CCR5) serves as an HIV-1 co-receptor and is essential for cell infection with CCR5-tropic viruses. Loss of functional receptor protects against HIV infection. Here, we report the successful targeting of CCR5 in GFP-marked human induced pluripotent stem cells (iPSCs) using CRISPR/Cas9 with single and dual guide RNAs (gRNAs). Following CRISPER/Cas9-mediated gene editing using a single gRNA, 12.5% of cell colonies demonstrated CCR5 editing, of which 22.2% showed biallelic editing as determined by a Surveyor nuclease assay and direct sequencing. The use of dual gRNAs significantly increased the efficacy of CCR5 editing to 27% with a biallelic gene alteration frequency of 41%. To ensure the homogeneity of gene editing within cells, we used single cell sorting to establish clonal iPSC lines. Single cell-derived iPSC lines with homozygous CCR5 mutations displayed the typical characteristics of pluripotent stem cells and differentiated efficiently into hematopoietic cells, including macrophages. Although macrophages from both wild-type and CCR5-edited iPSCs supported CXCR4-tropic virus replication, macrophages from CCR5-edited iPSCs were uniquely resistant to CCR5-tropic virus challenge. This study demonstrates the feasibility of applying iPSC technology for the study of the role of CCR5 in HIV infection in vitro, and generation of HIV-resistant cells for potential therapeutic applications.

  2. Pathogenic infection of Macaca nemestrina with a CCR5-tropic subtype-C simian-human immunodeficiency virus

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    Song Ruijiang

    2009-07-01

    Full Text Available Abstract Background Although pig-tailed macaques (Macaca nemestrina have been used in AIDS research for years, less is known about the early immunopathogenic events in this species, as compared to rhesus macaques (Macaca mulatta. Similarly, the events in early infection are well-characterized for simian immunodeficiency viruses (SIV, but less so for chimeric simian-human immunodeficiency viruses (SHIV, although the latter have been widely used in HIV vaccine studies. Here, we report the consequences of intrarectal infection with a CCR5-tropic clade C SHIV-1157ipd3N4 in pig-tailed macaques. Results Plasma and cell-associated virus was detectable in peripheral blood and intestinal tissues of all four pig-tailed macaques following intrarectal inoculation with SHIV-1157ipd3N4. We also observed a rapid and irreversible loss of CD4+ T cells at multiple mucosal sites, resulting in a marked decrease of CD4:CD8 T cell ratios 0.5–4 weeks after inoculation. This depletion targeted subsets of CD4+ T cells expressing the CCR5 coreceptor and having a CD28-CD95+ effector memory phenotype, consistent with the R5-tropism of SHIV-1157ipd3N4. All three animals that were studied beyond the acute phase seroconverted as early as week 4, with two developing cross-clade neutralizing antibody responses by week 24. These two animals also demonstrated persistent plasma viremia for >48 weeks. One of these animals developed AIDS, as shown by peripheral blood CD4+ T-cell depletion starting at 20 weeks post inoculation. Conclusion These findings indicate that SHIV-1157ipd3N4-induced pathogenesis in pig-tailed macaques followed a similar course as SIV-infected rhesus macaques. Thus, R5 SHIV-C-infection of pig-tailed macaques could provide a useful and relevant model for AIDS vaccine and pathogenesis research.

  3. HIV-1 with multiple CCR5/CXCR4 chimeric receptor use is predictive of immunological failure in infected children.

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    Cavarelli, Mariangela; Karlsson, Ingrid; Zanchetta, Marisa; Antonsson, Liselotte; Plebani, Anna; Giaquinto, Carlo; Fenyö, Eva Maria; De Rossi, Anita; Scarlatti, Gabriella

    2008-09-29

    HIV-1 R5 viruses are characterized by a large phenotypic variation, that is reflected by the mode of coreceptor use. The ability of R5 HIV-1 to infect target cells expressing chimeric receptors between CCR5 and CXCR4 (R5(broad) viruses), was shown to correlate with disease stage in HIV-1 infected adults. Here, we ask the question whether phenotypic variation of R5 viruses could play a role also in mother-to-child transmission (MTCT) of HIV-1 and pediatric disease progression. Viral isolates obtained from a total of 59 HIV-1 seropositive women (24 transmitting and 35 non transmitting) and 28 infected newborn children, were used to infect U87.CD4 cells expressing wild type or six different CCR5/CXCR4 chimeric receptors. HIV-1 isolates obtained from newborn infants had predominantly R5(narrow) phenotype (n = 20), but R5(broad) and R5X4 viruses were also found in seven and one case, respectively. The presence of R5(broad) and R5X4 phenotypes correlated significantly with a severe decline of the CD4+ T cells (CDC stage 3) or death within 2 years of age. Forty-three percent of the maternal R5 isolates displayed an R5(broad) phenotype, however, the presence of the R5(broad) virus was not predictive for MTCT of HIV-1. Of interest, while only 1 of 5 mothers with an R5X4 virus transmitted the dualtropic virus, 5 of 6 mothers carrying R5(broad) viruses transmitted viruses with a similar broad chimeric coreceptor usage. Thus, the maternal R5(broad) phenotype was largely preserved during transmission and could be predictive of the phenotype of the newborn's viral variant. Our results show that R5(broad) viruses are not hampered in transmission. When transmitted, immunological failure occurs earlier than in children infected with HIV-1 of R5(narrow) phenotype. We believe that this finding is of utmost relevance for therapeutic interventions in pediatric HIV-1 infection.

  4. HIV-1 with multiple CCR5/CXCR4 chimeric receptor use is predictive of immunological failure in infected children.

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    Mariangela Cavarelli

    Full Text Available BACKGROUND: HIV-1 R5 viruses are characterized by a large phenotypic variation, that is reflected by the mode of coreceptor use. The ability of R5 HIV-1 to infect target cells expressing chimeric receptors between CCR5 and CXCR4 (R5(broad viruses, was shown to correlate with disease stage in HIV-1 infected adults. Here, we ask the question whether phenotypic variation of R5 viruses could play a role also in mother-to-child transmission (MTCT of HIV-1 and pediatric disease progression. METHODOLOGY/PRINCIPAL FINDINGS: Viral isolates obtained from a total of 59 HIV-1 seropositive women (24 transmitting and 35 non transmitting and 28 infected newborn children, were used to infect U87.CD4 cells expressing wild type or six different CCR5/CXCR4 chimeric receptors. HIV-1 isolates obtained from newborn infants had predominantly R5(narrow phenotype (n = 20, but R5(broad and R5X4 viruses were also found in seven and one case, respectively. The presence of R5(broad and R5X4 phenotypes correlated significantly with a severe decline of the CD4+ T cells (CDC stage 3 or death within 2 years of age. Forty-three percent of the maternal R5 isolates displayed an R5(broad phenotype, however, the presence of the R5(broad virus was not predictive for MTCT of HIV-1. Of interest, while only 1 of 5 mothers with an R5X4 virus transmitted the dualtropic virus, 5 of 6 mothers carrying R5(broad viruses transmitted viruses with a similar broad chimeric coreceptor usage. Thus, the maternal R5(broad phenotype was largely preserved during transmission and could be predictive of the phenotype of the newborn's viral variant. CONCLUSIONS/SIGNIFICANCE: Our results show that R5(broad viruses are not hampered in transmission. When transmitted, immunological failure occurs earlier than in children infected with HIV-1 of R5(narrow phenotype. We believe that this finding is of utmost relevance for therapeutic interventions in pediatric HIV-1 infection.

  5. A chimeric measles virus with a lentiviral envelope replicates exclusively in CD4+/CCR5+ cells

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    Mourez, Thomas; Mesel-Lemoine, Mariana; Combredet, Chantal; Najburg, Valerie; Cayet, Nadege; Tangy, Frederic

    2011-01-01

    We generated a replicating chimeric measles virus in which the hemagglutinin and fusion surface glycoproteins were replaced with the gp160 envelope glycoprotein of simian immunodeficiency virus (SIVmac239). Based on a previously cloned live-attenuated Schwarz vaccine strain of measles virus (MV), this chimera was rescued at high titers using reverse genetics in CD4+ target cells. Cytopathic effect consisted in the presence of large cell aggregates evolving to form syncytia, as observed during SIV infection. The morphology of the chimeric virus was identical to that of the parent MV particles. The presence of SIV gp160 as the only envelope protein on chimeric particles surface altered the cell tropism of the new virus from CD46+ to CD4+ cells. Used as an HIV candidate vaccine, this MV/SIVenv chimeric virus would mimic transient HIV-like infection, benefiting both from HIV-like tropism and the capacity of MV to replicate in dendritic cells, macrophages and lymphocytes.

  6. Involvement of both the V2 and V3 Regions of the CCR5-Tropic Human Immunodeficiency Virus Type 1 Envelope in Reduced Sensitivity to Macrophage Inflammatory Protein 1α

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    Maeda, Yosuke; Foda, Mohamed; Matsushita, Shuzo; Harada, Shinji

    2000-01-01

    To determine whether C-C chemokines play an important role in the phenotype switch of human immunodeficiency virus (HIV) from CCR5 to CXCR4 usage during the course of an infection in vivo, macrophage inflammatory protein (MIP)-1α-resistant variants were isolated from CCR5-tropic (R5) HIV-1 in vitro. The selected variants displayed reduced sensitivities to MIP-1α (fourfold) through CCR5-expressing CD4-HeLa/long terminal repeat–β-galactosidase (MAGI/CCR5) cells. The variants were also resistant to other natural ligands for CCR5, namely, MIP-1β (>4-fold) and RANTES (regulated upon activation, normal T-cell expressed and secreted) (6-fold). The env sequence analyses revealed that the variants had amino acid substitutions in V2 (valine 166 to methionine) and V3 (serine 303 to glycine), although the same V3 substitution appeared in virus passaged without MIP-1α. A single-round replication assay using a luciferase reporter HIV-1 strain pseudotyped with mutant envelopes confirmed that mutations in both V2 and V3 were necessary to confer the reduced sensitivity to MIP-1α, MIP-1β, and RANTES. However, the double mutant did not switch its chemokine receptor usage from CCR5 to CXCR4, indicating the altered recognition of CCR5 by this mutant. These results indicated that V2 combined with the V3 region of the CCR5-tropic HIV-1 envelope modulates the sensitivity of HIV-1 to C-C chemokines without altering the ability to use chemokine receptors. PMID:10644351

  7. Chemokine (C-C motif) receptor 5-using envelopes predominate in dual/mixed-tropic HIV from the plasma of drug-naive individuals.

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    Irlbeck, David M; Amrine-Madsen, Heather; Kitrinos, Kathryn M; Labranche, Celia C; Demarest, James F

    2008-07-31

    HIV-1 utilizes CD4 and either chemokine (C-C motif) receptor 5 (CCR5) or chemokine (C-X-C motif) receptor 4 (CXCR4) to gain entry into host cells. Small molecule CCR5 antagonists are currently being developed for the treatment of HIV-1 infection. Because HIV-1 may also use CXCR4 for entry, the use of CCR5 entry inhibitors is controversial for patients harboring CCR5-using and CXCR4-using (dual/mixed-tropic) viruses. The goal of the present study was to determine the proportion of CCR5-tropic and CXCR4-tropic viruses in dual/mixed-tropic virus isolates from drug-naïve patients and the phenotypic and genotypic relationships of viruses that use CCR5 or CXCR4 or both. Fourteen antiretroviral-naive HIV-1-infected patients were identified as having population coreceptor tropism readout of dual/mixed-tropic viruses. Intrapatient comparisons of coreceptor tropism and genotype of env clones were conducted on plasma virus from each patient. Population HIV-1 envelope tropism and susceptibility to the CCR5 entry inhibitor, aplaviroc, were performed using the Monogram Biosciences Trofile Assay. Twelve env clones from each patient were analyzed for coreceptor tropism, aplaviroc sensitivity, genotype, and intrapatient phylogenetic relationships. Viral populations from antiretroviral-naive patients with dual/mixed-tropic virus are composed primarily of CCR5-tropic env clones mixed with those that use both coreceptors (R5X4-tropic) and, occasionally, CXCR4-tropic env clones. Interestingly, the efficiency of CXCR4 use by R5X4-tropic env clones varied with their genetic relationships to CCR5-tropic env clones from the same patient. These data show that the majority of viruses in these dual/mixed-tropic populations use CCR5 and suggest that antiretroviral-naive patients may benefit from combination therapy that includes CCR5 entry inhibitors.

  8. Maraviroc (UK-427,857), a Potent, Orally Bioavailable, and Selective Small-Molecule Inhibitor of Chemokine Receptor CCR5 with Broad-Spectrum Anti-Human Immunodeficiency Virus Type 1 Activity

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    Dorr, Patrick; Westby, Mike; Dobbs, Susan; Griffin, Paul; Irvine, Becky; Macartney, Malcolm; Mori, Julie; Rickett, Graham; Smith-Burchnell, Caroline; Napier, Carolyn; Webster, Rob; Armour, Duncan; Price, David; Stammen, Blanda; Wood, Anthony

    2005-01-01

    Maraviroc (UK-427,857) is a selective CCR5 antagonist with potent anti-human immunodeficiency virus type 1 (HIV-1) activity and favorable pharmacological properties. Maraviroc is the product of a medicinal chemistry effort initiated following identification of an imidazopyridine CCR5 ligand from a high-throughput screen of the Pfizer compound file. Maraviroc demonstrated potent antiviral activity against all CCR5-tropic HIV-1 viruses tested, including 43 primary isolates from various clades a...

  9. CD4 is expressed on a heterogeneous subset of hematopoietic progenitors, which persistently harbor CXCR4 and CCR5-tropic HIV proviral genomes in vivo.

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    Nadia T Sebastian

    2017-07-01

    Full Text Available Latent HIV infection of long-lived cells is a barrier to viral clearance. Hematopoietic stem and progenitor cells are a heterogeneous population of cells, some of which are long-lived. CXCR4-tropic HIVs infect a broad range of HSPC subtypes, including hematopoietic stem cells, which are multi-potent and long-lived. However, CCR5-tropic HIV infection is limited to more differentiated progenitor cells with life spans that are less well understood. Consistent with emerging data that restricted progenitor cells can be long-lived, we detected persistent HIV in restricted HSPC populations from optimally treated people. Further, genotypic and phenotypic analysis of amplified env alleles from donor samples indicated that both CXCR4- and CCR5-tropic viruses persisted in HSPCs. RNA profiling confirmed expression of HIV receptor RNA in a pattern that was consistent with in vitro and in vivo results. In addition, we characterized a CD4high HSPC sub-population that was preferentially targeted by a variety of CXCR4- and CCR5-tropic HIVs in vitro. Finally, we present strong evidence that HIV proviral genomes of both tropisms can be transmitted to CD4-negative daughter cells of multiple lineages in vivo. In some cases, the transmitted proviral genomes contained signature deletions that inactivated the virus, eliminating the possibility that coincidental infection explains the results. These data support a model in which both stem and non-stem cell progenitors serve as persistent reservoirs for CXCR4- and CCR5-tropic HIV proviral genomes that can be passed to daughter cells.

  10. CD4 is expressed on a heterogeneous subset of hematopoietic progenitors, which persistently harbor CXCR4 and CCR5-tropic HIV proviral genomes in vivo.

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    Sebastian, Nadia T; Zaikos, Thomas D; Terry, Valeri; Taschuk, Frances; McNamara, Lucy A; Onafuwa-Nuga, Adewunmi; Yucha, Ryan; Signer, Robert A J; Riddell, James; Bixby, Dale; Markowitz, Norman; Morrison, Sean J; Collins, Kathleen L

    2017-07-01

    Latent HIV infection of long-lived cells is a barrier to viral clearance. Hematopoietic stem and progenitor cells are a heterogeneous population of cells, some of which are long-lived. CXCR4-tropic HIVs infect a broad range of HSPC subtypes, including hematopoietic stem cells, which are multi-potent and long-lived. However, CCR5-tropic HIV infection is limited to more differentiated progenitor cells with life spans that are less well understood. Consistent with emerging data that restricted progenitor cells can be long-lived, we detected persistent HIV in restricted HSPC populations from optimally treated people. Further, genotypic and phenotypic analysis of amplified env alleles from donor samples indicated that both CXCR4- and CCR5-tropic viruses persisted in HSPCs. RNA profiling confirmed expression of HIV receptor RNA in a pattern that was consistent with in vitro and in vivo results. In addition, we characterized a CD4high HSPC sub-population that was preferentially targeted by a variety of CXCR4- and CCR5-tropic HIVs in vitro. Finally, we present strong evidence that HIV proviral genomes of both tropisms can be transmitted to CD4-negative daughter cells of multiple lineages in vivo. In some cases, the transmitted proviral genomes contained signature deletions that inactivated the virus, eliminating the possibility that coincidental infection explains the results. These data support a model in which both stem and non-stem cell progenitors serve as persistent reservoirs for CXCR4- and CCR5-tropic HIV proviral genomes that can be passed to daughter cells.

  11. Limited protective effect of the CCR5Delta32/CCR5Delta32 genotype on human immunodeficiency virus infection incidence in a cohort of patients with hemophilia and selection for genotypic X4 virus

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    Iversen, Astrid K N; Christiansen, Claus Bohn; Attermann, Jørn

    2003-01-01

    The relationship among CCR5 genotype, cytomegalovirus infection, and disease progression and death was studied among 159 human immunodeficiency virus (HIV)-infected patients with hemophilia. One patient (0.6%) had the CCR5Delta32/CCR5Delta32 genotype (which occurs in approximately 2% of the Scand......The relationship among CCR5 genotype, cytomegalovirus infection, and disease progression and death was studied among 159 human immunodeficiency virus (HIV)-infected patients with hemophilia. One patient (0.6%) had the CCR5Delta32/CCR5Delta32 genotype (which occurs in approximately 2...

  12. Resistance of a human immunodeficiency virus type 1 isolate to a small molecule CCR5 inhibitor can involve sequence changes in both gp120 and gp41

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    Anastassopoulou, Cleo G.; Ketas, Thomas J.; Depetris, Rafael S.; Thomas, Antonia M.; Klasse, Per Johan; Moore, John P.

    2011-01-01

    Here, we describe the genetic pathways taken by a human immunodeficiency virus type 1 (HIV-1) isolate, D101.12, to become resistant to the small molecule CCR5 inhibitor, vicriviroc (VCV), in vitro. Resistant D101.12 variants contained at least one substitution in the gp120 V3 region (H308P), plus one of two patterns of gp41 sequence changes involving the fusion peptide (FP) and a downstream residue: G514V+V535M or M518V+F519L+V535M. Studies of Env-chimeric and point-substituted viruses in peripheral blood mononuclear cells (PBMC) and TZM-bl cells showed that resistance can arise from the cooperative action of gp120 and gp41 changes, while retaining CCR5 usage. Modeling the VCV inhibition data from the two cell types suggests that D101.12 discriminates between high- and low-VCV affinity forms of CCR5 less than D1/85.16, a resistant virus with three FP substitutions.

  13. Inhibition of human immunodeficiency virus replication by a dual CCR5/CXCR4 antagonist

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    Princen, Katrien; Hatse, Sigrid; Vermeire, Kurt

    2004-01-01

    Here we report that the N-pyridinylmethyl cyclam analog AMD3451 has antiviral activity against a wide variety of R5, R5/X4, and X4 strains of human immunodeficiency virus type 1 (HIV-1) and HIV-2 (50% inhibitory concentration [IC(50)] ranging from 1.2 to 26.5 microM) in various T-cell lines, CCR5...... at the virus entry stage. AMD3451 dose-dependently inhibited the intracellular Ca(2+) signaling induced by the CXCR4 ligand CXCL12 in T-lymphocytic cells and in CXCR4-transfected cells, as well as the Ca(2+) flux induced by the CCR5 ligands CCL5, CCL3, and CCL4 in CCR5-transfected cells. The compound did...... not interfere with chemokine-induced Ca(2+) signaling through CCR1, CCR2, CCR3, CCR4, CCR6, CCR9, or CXCR3 and did not induce intracellular Ca(2+) signaling by itself at concentrations up to 400 microM. In freshly isolated monocytes, AMD3451 inhibited the Ca(2+) flux induced by CXCL12 and CCL4...

  14. Influence of the CCR2-V64I Polymorphism on Human Immunodeficiency Virus Type 1 Coreceptor Activity and on Chemokine Receptor Function of CCR2b, CCR3, CCR5, and CXCR4

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    Lee, Benhur; Doranz, Benjamin J.; Rana, Shalini; Yi, Yanji; Mellado, Mario; Frade, Jose M. R.; Martinez-A., Carlos; O’Brien, Stephen J.; Dean, Michael; Collman, Ronald G.; Doms, Robert W.

    1998-01-01

    The chemokine receptors CCR5 and CXCR4 are used by human immunodeficiency virus type 1 (HIV-1) in conjunction with CD4 to infect cells. In addition, some virus strains can use alternative chemokine receptors, including CCR2b and CCR3, for infection. A polymorphism in CCR2 (CCR2-V64I) is associated with a 2- to 4-year delay in the progression to AIDS. To investigate the mechanism of this protective effect, we studied the expression of CCR2b and CCR2b-V64I, their chemokine and HIV-1 coreceptor ...

  15. CD4-independent use of the CCR5 receptor by sequential primary SIVsm isolates

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    Thorstensson Rigmor

    2007-07-01

    Full Text Available Abstract Background CD4-independence has been taken as a sign of a more open envelope structure that is more accessible to neutralizing antibodies and may confer altered cell tropism. In the present study, we analyzed SIVsm isolates for CD4-independent use of CCR5, mode of CCR5-use and macrophage tropism. The isolates have been collected sequentially from 13 experimentally infected cynomolgus macaques and have previously been shown to use CCR5 together with CD4. Furthermore, viruses obtained early after infection were neutralization sensitive, while neutralization resistance appeared already three months after infection in monkeys with progressive immunodeficiency. Results Depending whether isolated early or late in infection, two phenotypes of CD4-independent use of CCR5 could be observed. The inoculum virus (SIVsm isolate SMM-3 and reisolates obtained early in infection often showed a pronounced CD4-independence since virus production and/or syncytia induction could be detected directly in NP-2 cells expressing CCR5 but not CD4 (CD4-independent-HIGH. Conversely, late isolates were often more CD4-dependent in that productive infection in NP-2/CCR5 cells was in most cases weak and was revealed only after cocultivation of infected NP-2/CCR5 cells with peripheral blood mononuclear cells (CD4-independent-LOW. Considering neutralization sensitivity of these isolates, newly infected macaques often harbored virus populations with a CD4-independent-HIGH and neutralization sensitive phenotype that changed to a CD4-independent-LOW and neutralization resistant virus population in the course of infection. Phenotype changes occurred faster in progressor than long-term non-progressor macaques. The phenotypes were not reflected by macrophage tropism, since all isolates replicated efficiently in macrophages. Infection of cells expressing CCR5/CXCR4 chimeric receptors revealed that SIVsm used the CCR5 receptor in a different mode than HIV-1. Conclusion Our

  16. Limited protective effect of the CCR5Δ32/CCR5Δ32 genotype on human immunodeficiency virus infection incidence in a cohort of patients with hemophilia and selection for genotypic X4 virus

    DEFF Research Database (Denmark)

    Iversen, Astrid K. N.; Christiansen, Claus Bohn; Attermann, Jørn

    2003-01-01

    The relationship among CCR5 genotype, cytomegalovirus infection, and disease progression and death was studied among 159 human immunodeficiency virus (HIV)–infected patients with hemophilia. One patient (0.6%) had the CCR5Δ32/CCR5Δ32 genotype (which occurs in ∼2% of the Scandinavian population...

  17. [The expression of periphery blood leucocyte CCR3 and CCR5 in the children with Epstein-Barr virus associated infectious mononucleosis].

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    Qi, Tie-xiong; Gao, Guo-hua; Liu, Shi-hua

    2010-10-01

    To explore the expression of periphery blood leucocyte CCR3 and CCR5 and to comprehend T helper cell in the Children with Epstein-Barr virus associated infectious mononucleosis. We defined the children according to the diagnosis criterion through Paul-Bunnell test inspecting the children's periphery blood unusual lymphocyte and detecting their anti-EBV-CA-IgM, anti-EBV-CA-IgG and anti-EBV-NA-IgG by ELISA and counted the ratio of CCR3 + and CCR5 + cells in lymphocytes with flow cytometry. The ratio of unusual lymphocyte in IM was higher than that of the healthy control group (P < 0.05). The ratio of CCR3 + cells in IM group was higher than that of the healthy control group (P < 0.05). The ratio of CCR5 + cells in IM group was significantly lower than that of the healthy control group. CCR3 + had direct interrelation with fever continued time and the ratio of unusual lymphocyte. There was a negative interrelation between CCR5 and fever continued time (P < 0.05). Children infectious of IM expressed higher level of CCR3 + and lower level of CCR5 + and there was a tendency of Th2 polarization with over production of T helper cell divide imbalance. CCR3 + and CCR5 + may be important targets to judge the degree of seriousness of IM.

  18. Maraviroc (UK-427,857), a potent, orally bioavailable, and selective small-molecule inhibitor of chemokine receptor CCR5 with broad-spectrum anti-human immunodeficiency virus type 1 activity.

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    Dorr, Patrick; Westby, Mike; Dobbs, Susan; Griffin, Paul; Irvine, Becky; Macartney, Malcolm; Mori, Julie; Rickett, Graham; Smith-Burchnell, Caroline; Napier, Carolyn; Webster, Rob; Armour, Duncan; Price, David; Stammen, Blanda; Wood, Anthony; Perros, Manos

    2005-11-01

    Maraviroc (UK-427,857) is a selective CCR5 antagonist with potent anti-human immunodeficiency virus type 1 (HIV-1) activity and favorable pharmacological properties. Maraviroc is the product of a medicinal chemistry effort initiated following identification of an imidazopyridine CCR5 ligand from a high-throughput screen of the Pfizer compound file. Maraviroc demonstrated potent antiviral activity against all CCR5-tropic HIV-1 viruses tested, including 43 primary isolates from various clades and diverse geographic origin (geometric mean 90% inhibitory concentration of 2.0 nM). Maraviroc was active against 200 clinically derived HIV-1 envelope-recombinant pseudoviruses, 100 of which were derived from viruses resistant to existing drug classes. There was little difference in the sensitivity of the 200 viruses to maraviroc, as illustrated by the biological cutoff in this assay (= geometric mean plus two standard deviations [SD] of 1.7-fold). The mechanism of action of maraviroc was established using cell-based assays, where it blocked binding of viral envelope, gp120, to CCR5 to prevent the membrane fusion events necessary for viral entry. Maraviroc did not affect CCR5 cell surface levels or associated intracellular signaling, confirming it as a functional antagonist of CCR5. Maraviroc has no detectable in vitro cytotoxicity and is highly selective for CCR5, as confirmed against a wide range of receptors and enzymes, including the hERG ion channel (50% inhibitory concentration, >10 microM), indicating potential for an excellent clinical safety profile. Studies in preclinical in vitro and in vivo models predicted maraviroc to have human pharmacokinetics consistent with once- or twice-daily dosing following oral administration. Clinical trials are ongoing to further investigate the potential of using maraviroc for the treatment of HIV-1 infection and AIDS.

  19. Dengue virus requires the CC-chemokine receptor CCR5 for replication and infection development.

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    Marques, Rafael E; Guabiraba, Rodrigo; Del Sarto, Juliana L; Rocha, Rebeca F; Queiroz, Ana Luiza; Cisalpino, Daniel; Marques, Pedro E; Pacca, Carolina C; Fagundes, Caio T; Menezes, Gustavo B; Nogueira, Maurício L; Souza, Danielle G; Teixeira, Mauro M

    2015-08-01

    Dengue is a mosquito-borne disease that affects millions of people worldwide yearly. Currently, there is no vaccine or specific treatment available. Further investigation on dengue pathogenesis is required to better understand the disease and to identify potential therapeutic targets. The chemokine system has been implicated in dengue pathogenesis, although the specific role of chemokines and their receptors remains elusive. Here we describe the role of the CC-chemokine receptor CCR5 in Dengue virus (DENV-2) infection. In vitro experiments showed that CCR5 is a host factor required for DENV-2 replication in human and mouse macrophages. DENV-2 infection induces the expression of CCR5 ligands. Incubation with an antagonist prevents CCR5 activation and reduces DENV-2 positive-stranded (+) RNA inside macrophages. Using an immunocompetent mouse model of DENV-2 infection we found that CCR5(-/-) mice were resistant to lethal infection, presenting at least 100-fold reduction of viral load in target organs and significant reduction in disease severity. This phenotype was reproduced in wild-type mice treated with CCR5-blocking compounds. Therefore, CCR5 is a host factor required for DENV-2 replication and disease development. Targeting CCR5 might represent a therapeutic strategy for dengue fever. These data bring new insights on the association between viral infections and the chemokine receptor CCR5. © 2015 John Wiley & Sons Ltd.

  20. Allosteric and orthosteric sites in CC chemokine receptor (CCR5), a chimeric receptor approach

    DEFF Research Database (Denmark)

    Thiele, Stefanie; Steen, Anne; Jensen, Pia C

    2011-01-01

    -allosteric molecules. A chimera was successfully constructed between CCR5 and the closely related CCR2 by transferring all extracellular regions of CCR2 to CCR5, i.e. a Trojan horse that resembles CCR2 extracellularly but signals through a CCR5 transmembrane unit. The chimera bound CCR2 (CCL2 and CCL7), but not CCR5...... preserved, the allosteric enhancement of chemokine binding was disrupted. In summary, the Trojan horse chimera revealed that orthosteric and allosteric sites could be structurally separated and still act together with transmission of agonism and antagonism across the different receptor units....

  1. Vectors expressing chimeric Japanese encephalitis dengue 2 viruses.

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    Wei, Y; Wang, S; Wang, X

    2014-01-01

    Vectors based on self-replicating RNAs (replicons) of flaviviruses are becoming powerful tool for expression of heterologous genes in mammalian cells and development of novel antiviral and anticancer vaccines. We constructed two vectors expressing chimeric viruses consisting of attenuated SA14-14-2 strain of Japanese encephalitis virus (JEV) in which the PrM/M-E genes were replaced fully or partially with those of dengue 2 virus (DENV-2). These vectors, named pJED2 and pJED2-1770 were transfected to BHK-21 cells and produced chimeric viruses JED2V and JED2-1770V, respectively. The chimeric viruses could be passaged in C6/36 but not BHK-21 cells. The chimeric viruses produced in C6/36 cells CPE 4-5 days after infection and RT-PCR, sequencing, immunofluorescence assay (IFA) and Western blot analysis confirmed the chimeric nature of produced viruses. The immunogenicity of chimeric viruses in mice was proved by detecting DENV-2 E protein-specific serum IgG antibodies with neutralization titer of 10. Successful preparation of infectious clones of chimeric JEV-DENV-2 viruses showed that JEV-based expression vectors are fully functional.

  2. Chemokine Receptor CCR5 Antagonist Maraviroc: Medicinal Chemistry and Clinical Applications

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    Xu, Guoyan G.; Guo, Jia; Wu, Yuntao

    2015-01-01

    The human immunodeficiency virus (HIV) causes acquired immumodeficiency syndrome (AIDS), one of the worst global pandemic. The virus infects human CD4 T cells and macrophages, and causes CD4 depletion. HIV enters target cells through the binding of the viral envelope glycoprotein to CD4 and the chemokine coreceptor, CXCR4 or CCR5. In particular, the CCR5-utilizing viruses predominate in the blood during the disease course. CCR5 is expressed on the surface of various immune cells including macrophages, monocytes, microglia, dendric cells, and active memory CD4 T cells. In the human population, the CCR5 genomic mutation, CCR5Δ32, is associated with relative resistance to HIV. These findings paved the way for the discovery and development of CCR5 inhibitors to block HIV transmission and replication. Maraviroc, discovered as a CCR5 antagonist, is the only CCR5 inhibitor that has been approved by both US FDA and the European Medicines Agency (EMA) for treating HIV/AIDS patients. In this review, we summarize the medicinal chemistry and clinical studies of Maraviroc. PMID:25159165

  3. CCR5 Signal Transduction in Macrophages by Human Immunodeficiency Virus and Simian Immunodeficiency Virus Envelopes

    OpenAIRE

    Arthos, James; Rubbert, Andrea; Rabin, Ronald L.; Cicala, Claudia; Machado, Elizabeth; Wildt, Kathryne; Hanbach, Meredith; Steenbeke, Tavis D.; Swofford, Ruth; Farber, Joshua M.; Fauci, Anthony S.

    2000-01-01

    The capacity of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) envelopes to transduce signals through chemokine coreceptors on macrophages was examined by measuring the ability of recombinant envelope proteins to mobilize intracellular calcium stores. Both HIV and SIV envelopes mobilized calcium via interactions with CCR5. The kinetics of these responses were similar to those observed when macrophages were treated with MIP-1β. Distinct differences in the capacity o...

  4. Flexible use of CCR5 in the absence of CXCR4 use explains the immune deficiency in HIV-1 infected children.

    Science.gov (United States)

    Cavarelli, Mariangela; Karlsson, Ingrid; Ripamonti, Chiara; Plebani, Anna; Fenyo, Eva Maria; Scarlatti, Gabriella

    2010-10-23

    CCR5-using HIV-1 (R5 viruses) are usually isolated during acute infection from both adults and children. We have recently demonstrated that R5 viruses with a flexible use of CCR5 (called R5broad) can be detected in children close to birth and are predictive of a fast immunological failure. The aim of the present work was to investigate viral phenotype variation during disease progression in HIV-1 infected children, six slow and eight fast progressors. A total of 74 viral isolates obtained sequentially from 14 HIV-1 infected children were tested for their ability to infect U87.CD4 cells expressing a set of six different CCR5/CXCR4 chimeric receptors or wild-type coreceptors. The sensitivity of 35 R5 viruses to inhibition with the CC-chemokine RANTES (regulated upon activation, normal T-cell expressed and secreted) was evaluated in a peripheral blood mononuclear cells based assay. Viral evolution to R5broad or to R5X4 phenotype occurred with one exception, in all children, although at a different time point according to rate of disease progression. Immune deficiency in the children was significantly associated with the appearance of R5broad phenotype or R5X4 viruses. Analysis of the sensitivity to inhibition by RANTES revealed a significant correlation between the R5broad phenotype and an augmented resistance to this CC-chemokine. We demonstrate that the viral evolution to a more flexible CCR5-use is sufficient to explain the immunological failure in the absence of CXCR4 usage. These results warrant detailed analysis of the R5 phenotype in forthcoming clinical studies introducing CCR5 inhibitors for the treatment of pediatric HIV-1 infection.

  5. Functional analysis of the CC chemokine receptor 5 (CCR5) on virus-specific CD8+ T cells following coronavirus infection of the central nervous system

    International Nuclear Information System (INIS)

    Glass, William G.; Lane, Thomas E.

    2003-01-01

    Intracranial infection of C57BL/6 mice with mouse hepatitis virus (MHV) results in an acute encephalomyelitis followed by a demyelinating disease similar in pathology to the human disease multiple sclerosis (MS). T cells participate in both defense and disease progression following MHV infection. Expression of chemokine receptors on activated T cells is important in allowing these cells to traffic into and accumulate within the central nervous system (CNS) of MHV-infected mice. The present study evaluated the contributions of CCR5 to the activation and trafficking of virus-specific CD8 + T cells into the MHV-infected CNS mice. Comparable numbers of virus-specific CD8 + T cells derived from immunized CCR5 +/+ or CCR5 -/- mice were present within the CNS of MHV-infected RAG1 -/- mice following adoptive transfer, indicating that CCR5 is not required for trafficking of these cells into the CNS. RAG1 -/- recipients of CCR5 -/- -derived CD8 + T cells exhibited a modest, yet significant (P ≤ 0.05), reduction in viral burden within the brain which correlated with increased CTL activity and IFN-γ expression. Histological analysis of RAG1 -/- recipients of either CCR5 +/+ or CCR5 -/- -derived CD8 + T cells revealed only focal areas of demyelination with no significant differences in white matter destruction. These data indicate that CCR5 signaling on CD8 + T cells modulates antiviral activities but is not essential for entry into the CNS

  6. Prognostic value of a CCR5 defective allele in pediatric HIV-1 infection.

    Science.gov (United States)

    Romiti, M L; Colognesi, C; Cancrini, C; Mas, A; Berrino, M; Salvatori, F; Orlandi, P; Jansson, M; Palomba, E; Plebani, A; Bertran, J M; Hernandez, M; de Martino, M; Amoroso, A; Tovo, P A; Rossi, P; Espanol, T; Scarlatti, G

    2000-01-01

    A deletion of 32 base pairs in the CCR5 gene (delta32 CCR5) has been linked to resistance to HIV-1 infection in exposed adults and to the delay of disease progression in infected adults. To determine the role of delta32 CCR5 in disease progression of HIV-1 infected children born to seropositive mothers, we studied a polymerase chain reaction in 301 HIV-1 infected, 262 HIV-1 exposed-uninfected and 47 HIV-1 unexposed-uninfected children of Spanish and Italian origin. Infected children were further divided into two groups according to their rate of HIV-1 disease progression: rapid progressors who developed severe clinical and/or immunological conditions within the second year of life, and delayed progressors with any other evolution of disease. Among the latter were the long-term, non-progressors (LTNP) who presented with mild or no symptoms of HIV-1 infection above 8 years of age. Viral phenotype was studied for 45 delayed progressors. No correlation was found between delta32 CCR5 and mother-to-child transmission of HIV-1. However, the frequency of the deletion was substantially higher in LTNP, compared with delayed (p = 0.019) and rapid progressors (p = 0.0003). In children carrying the delta32 CCRS mutation, the presence of MT-2 tropic virus isolate was associated with a severe immune suppression (p = 0.028); whereas, the presence of MT-2 negative viruses correlated with LTNP (p = 0.010). Given the rapidity and simplicity of the assay, the delta32 CCR5 mutation may be a useful predictive marker to identify children with delayed disease progression who, consequently, may not require immediate antiretroviral treatment.

  7. Influence of the CCR-5/MIP-1 α axis in the pathogenesis of Rocio virus encephalitis in a mouse model.

    Science.gov (United States)

    Chávez, Juliana H; França, Rafael F O; Oliveira, Carlo J F; de Aquino, Maria T P; Farias, Kleber J S; Machado, Paula R L; de Oliveira, Thelma F M; Yokosawa, Jonny; Soares, Edson G; da Silva, João S; da Fonseca, Benedito A L; Figueiredo, Luiz T M

    2013-11-01

    Rocio virus (ROCV) caused an outbreak of human encephalitis during the 1970s in Brazil and its immunopathogenesis remains poorly understood. CC-chemokine receptor 5 (CCR5) is a chemokine receptor that binds to macrophage inflammatory protein (MIP-1 α). Both molecules are associated with inflammatory cells migration during infections. In this study, we demonstrated the importance of the CCR5 and MIP-1 α, in the outcome of viral encephalitis of ROCV-infected mice. CCR5 and MIP-1 α knockout mice survived longer than wild-type (WT) ROCV-infected animals. In addition, knockout mice had reduced inflammation in the brain. Assessment of brain viral load showed mice virus detection five days post-infection in wild-type and CCR5-/- mice, while MIP-1 α-/- mice had lower viral loads seven days post-infection. Knockout mice required a higher lethal dose than wild-type mice as well. The CCR5/MIP-1 α axis may contribute to migration of infected cells to the brain and consequently affect the pathogenesis during ROCV infection.

  8. The Effects of the Recombinant CCR5 T4 Lysozyme Fusion Protein on HIV-1 Infection.

    Directory of Open Access Journals (Sweden)

    Qingwen Jin

    Full Text Available Insertion of T4 lysozyme (T4L into the GPCR successfully enhanced GPCR protein stability and solubilization. However, the biological functions of the recombinant GPCR protein have not been analyzed.We engineered the CCR5-T4L mutant and expressed and purified the soluble recombinant protein using an E.coli expression system. The antiviral effects of this recombinant protein in THP-1 cell lines, primary human macrophages, and PBMCs from different donors were investigated. We also explored the possible mechanisms underlying the observed antiviral effects.Our data showed the biphasic inhibitory and promotion effects of different concentrations of soluble recombinant CCR5-T4L protein on R5 tropic human immunodeficiency virus-1 (HIV-1 infection in THP-1 cell lines, human macrophages, and PBMCs from clinical isolates. We demonstrated that soluble recombinant CCR5-T4L acts as a HIV-1 co-receptor, interacts with wild type CCR5, down-regulates the surface CCR5 expression in human macrophages, and interacts with CCL5 to inhibit macrophage migration. Using binding assays, we further determined that recombinant CCR5-T4L and [125I]-CCL5 compete for the same binding site on wild type CCR5.Our results suggest that recombinant CCR5-T4L protein marginally promotes HIV-1 infection at low concentrations and markedly inhibits infection at higher concentrations. This recombinant protein may be helpful in the future development of anti-HIV-1 therapeutic agents.

  9. CCR2-64I polymorphism is not associated with altered CCR5 expression or coreceptor function.

    Science.gov (United States)

    Mariani, R; Wong, S; Mulder, L C; Wilkinson, D A; Reinhart, A L; LaRosa, G; Nibbs, R; O'Brien, T R; Michael, N L; Connor, R I; Macdonald, M; Busch, M; Koup, R A; Landau, N R

    1999-03-01

    A polymorphism in the gene encoding CCR2 is associated with a delay in progression to AIDS in human immunodeficiency virus (HIV)-infected individuals. The polymorphism, CCR2-64I, changes valine 64 of CCR2 to isoleucine. However, it is not clear whether the effect on AIDS progression results from the amino acid change or whether the polymorphism marks a genetically linked, yet unidentified mutation that mediates the effect. Because the gene encoding CCR5, the major coreceptor for HIV type 1 primary isolates, lies 15 kb 3' to CCR2, linked mutations in the CCR5 promoter or other regulatory sequences could explain the association of CCR2-64I with slowed AIDS pathogenesis. Here, we show that CCR2-64I is efficiently expressed on the cell surface but does not have dominant negative activity on CCR5 coreceptor function. A panel of peripheral blood mononuclear cells (PBMC) from uninfected donors representing the various CCR5/CCR2 genotypes was assembled. Activated primary CD4(+) T cells of CCR2 64I/64I donors expressed cell surface CCR5 at levels comparable to those of CCR2 +/+ donors. A slight reduction in CCR5 expression was noted, although this was not statistically significant. CCR5 and CCR2 mRNA levels were nearly identical for each of the donor PBMC, regardless of genotype. Cell surface CCR5 and CCR2 levels were more variable than mRNA transcript levels, suggesting that an alternative mechanism may influence CCR5 cell surface levels. CCR2-64I is linked to the CCR5 promoter polymorphisms 208G, 303A, 627C, and 676A; however, in transfected promoter reporter constructs, these did not affect transcriptional activity. Taken together, these findings suggest that CCR2-64I does not act by influencing CCR5 transcription or mRNA levels.

  10. Production and immunogenicity of chimeric virus-like particles containing the spike glycoprotein of infectious bronchitis virus.

    Science.gov (United States)

    Lv, Lishan; Li, Xiaoming; Liu, Genmei; Li, Ran; Liu, Qiliang; Shen, Huifang; Wang, Wei; Xue, Chunyi; Cao, Yongchang

    2014-01-01

    Infectious bronchitis virus (IBV) poses a severe threat to the poultry industry and causes heavy economic losses worldwide. Vaccination is the most effective method of preventing infection and controlling the spread of IBV, but currently available inactivated and attenuated virus vaccines have some disadvantages. We developed a chimeric virus-like particle (VLP)-based candidate vaccine for IBV protection. The chimeric VLP was composed of matrix 1 protein from avian influenza H5N1 virus and a fusion protein neuraminidase (NA)/spike 1 (S1) that was generated by fusing IBV S1 protein to the cytoplasmic and transmembrane domains of NA protein of avian influenza H5N1 virus. The chimeric VLPs elicited significantly higher S1-specific antibody responses in intramuscularly immunized mice and chickens than inactivated IBV viruses. Furthermore, the chimeric VLPs induced significantly higher neutralization antibody levels than inactivated H120 virus in SPF chickens. Finally, the chimeric VLPs induced significantly higher IL-4 production in mice. These results demonstrate that chimeric VLPs have the potential for use in vaccines against IBV infection.

  11. Live Zika virus chimeric vaccine candidate based on a yellow fever 17-D attenuated backbone

    OpenAIRE

    Nougairede, Antoine; Klitting, Raphaelle; Aubry, Fabien; Gilles, Magali; Touret, Franck; De Lamballerie, Xavier

    2018-01-01

    Zika virus (ZIKV) recently dispersed throughout the tropics and sub-tropics causing epidemics associated with congenital disease and neurological complications. There is currently no commercial vaccine for ZIKV. Here we describe the initial development of a chimeric virus containing the prM/E proteins of a ZIKV epidemic strain incorporated into a yellow fever 17-D attenuated backbone. Using the versatile and rapid ISA (Infectious Subgenomic Amplicons) reverse genetics method, we compared diff...

  12. An anti-CCR5 monoclonal antibody and small molecule CCR5 antagonists synergize by inhibiting different stages of human immunodeficiency virus type 1 entry

    International Nuclear Information System (INIS)

    Safarian, Diana; Carnec, Xavier; Tsamis, Fotini; Kajumo, Francis; Dragic, Tatjana

    2006-01-01

    HIV-1 coreceptors are attractive targets for novel antivirals. Here, inhibition of entry by two classes of CCR5 antagonists was investigated. We confirmed previous findings that HIV-1 isolates vary greatly in their sensitivity to small molecule inhibitors of CCR5-mediated entry, SCH-C and TAK-779. In contrast, an anti-CCR5 monoclonal antibody (PA14) similarly inhibited entry of diverse viral isolates. Sensitivity to small molecules was V3 loop-dependent and inversely proportional to the level of gp120 binding to CCR5. Moreover, combinations of the MAb and small molecules were highly synergistic in blocking HIV-1 entry, suggesting different mechanisms of action. This was confirmed by time course of inhibition experiments wherein the PA14 MAb and small molecules were shown to inhibit temporally distinct stages of CCR5 usage. We propose that small molecules inhibit V3 binding to the second extracellular loop of CCR5, whereas PA14 preferentially inhibits subsequent events such as CCR5 recruitment into the fusion complex or conformational changes in the gp120-CCR5 complex that trigger fusion. Importantly, our findings suggest that combinations of CCR5 inhibitors with different mechanisms of action will be central to controlling HIV-1 infection and slowing the emergence of resistant strains

  13. CCR5 and CXCR3 are dispensable for liver infiltration, but CCR5 protects against virus-induced T-cell-mediated hepatic steatosis

    DEFF Research Database (Denmark)

    Holst, P J; Orskov, C; Qvortrup, K

    2007-01-01

    CCR5 and CXCR3 are important molecules in regulating the migration of activated lymphocytes. Thus, the majority of tissue-infiltrating T cells found in the context of autoimmune conditions and viral infections express CCR5 and CXCR3, and the principal chemokine ligands are expressed within inflam...... of CCR5 is associated with the induction of CD8(+) T-cell-mediated immunopathology consisting of marked hepatic microvesicular steatosis....

  14. CCL5, CCR1 and CCR5 in murine glioblastoma: immune cell infiltration and survival rates are not dependent on individual expression of either CCR1 or CCR5

    OpenAIRE

    Pham, Kien; Luo, Defang; Liu, Che; Harrison, Jeffrey K.

    2012-01-01

    Glioblastoma multiforme (GBM) is the most malignant brain tumor. Microglia/macrophages are found within human GBM where they likely promote tumor progression. We report that CCL5, CCR1, and CCR5 are expressed in glioblastoma. Individual deletion of CCR1 or CCR5 had little to no effect on survival of tumor bearing mice, or numbers of glioblastoma-infiltrated microglia/macrophages or lymphocytes. CCL5 promoted in vitro migration of wild type, CCR1- or CCR5-deficient microglia/macrophages that w...

  15. Interaction of small molecule inhibitors of HIV-1 entry with CCR5

    International Nuclear Information System (INIS)

    Seibert, Christoph; Ying Weiwen; Gavrilov, Svetlana; Tsamis, Fotini; Kuhmann, Shawn E.; Palani, Anandan; Tagat, Jayaram R.; Clader, John W.; McCombie, Stuart W.; Baroudy, Bahige M.; Smith, Steven O.; Dragic, Tatjana; Moore, John P.; Sakmar, Thomas P.

    2006-01-01

    The CC-chemokine receptor 5 (CCR5) is the major coreceptor for macrophage-tropic (R5) HIV-1 strains. Several small molecule inhibitors of CCR5 that block chemokine binding and HIV-1 entry are being evaluated as drug candidates. Here we define how CCR5 antagonists TAK-779, AD101 (SCH-350581) and SCH-C (SCH-351125), which inhibit HIV-1 entry, interact with CCR5. Using a mutagenesis approach in combination with a viral entry assay to provide a direct functional read out, we tested predictions based on a homology model of CCR5 and analyzed the functions of more than 30 amino acid residues. We find that a key set of aromatic and aliphatic residues serves as a hydrophobic core for the ligand binding pocket, while E283 is critical for high affinity interaction, most likely by acting as the counterion for a positively charged nitrogen atom common to all three inhibitors. These results provide a structural basis for understanding how specific antagonists interact with CCR5, and may be useful for the rational design of new, improved CCR5 ligands

  16. CCR5 antibodies HGS004 and HGS101 preferentially inhibit drug-bound CCR5 infection and restore drug sensitivity of Maraviroc-resistant HIV-1 in primary cells

    International Nuclear Information System (INIS)

    Latinovic, Olga; Reitz, Marvin; Le, Nhut M.; Foulke, James S.; Faetkenheuer, Gerd; Lehmann, Clara; Redfield, Robert R.; Heredia, Alonso

    2011-01-01

    R5 HIV-1 strains resistant to the CCR5 antagonist Maraviroc (MVC) can use drug-bound CCR5. We demonstrate that MVC-resistant HIV-1 exhibits delayed kinetics of coreceptor engagement and fusion during drug-bound versus free CCR5 infection of cell lines. Antibodies directed against the second extracellular loop (ECL2) of CCR5 had greater antiviral activity against MVC-bound compared to MVC-free CCR5 infection. However, in PBMCs, only ECL2 CCR5 antibodies HGS004 and HGS101, but not 2D7, inhibited infection by MVC resistant HIV-1 more potently with MVC-bound than with free CCR5. In addition, HGS004 and HGS101, but not 2D7, restored the antiviral activity of MVC against resistant virus in PBMCs. In flow cytometric studies, CCR5 binding by the HGS mAbs, but not by 2D7, was increased when PBMCs were treated with MVC, suggesting MVC increases exposure of the relevant epitope. Thus, HGS004 and HGS101 have antiviral mechanisms distinct from 2D7 and could help overcome MVC resistance.

  17. High frequency of the CCR5delta32 variant among individuals from an admixed Brazilian population with sickle cell anemia

    Directory of Open Access Journals (Sweden)

    J.A.B. Chies

    2003-01-01

    Full Text Available Homozygous sickle cell disease (SCD has a wide spectrum of clinical manifestations. In Brazil, the main cause of death of individuals with SCD is recurrent infection. The CCR5delta32 allele, which confers relative resistance to macrophage-tropic HIV virus infection, probably has reached its frequency and world distribution due to other pathogens that target macrophage in European populations. In the present investigation a relatively higher prevalence (5.1% of the CCR5delta32 allele was identified, by PCR amplification using specific primers, in 79 SCD patients when compared to healthy controls (1.3% with the same ethnic background (Afro-Brazilians. Based on a hypothesis that considers SCD as a chronic inflammatory condition, and since the CCR5 chemokine receptor is involved in directing a Th1-type immune response, we suggest that a Th1/Th2 balance can influence the morbidity of SCD. If the presence of the null CCR5delta32 allele results in a reduction of the chronic inflammation state present in SCD patients, this could lead to differential survival of SCD individuals who are carriers of the CCR5delta32 allele. This differential survival could be due to the development of less severe infections and consequently reduced or less severe vaso-occlusive crises.

  18. Production of infectious chimeric hepatitis C virus genotype 2b harboring minimal regions of JFH-1.

    Science.gov (United States)

    Murayama, Asako; Kato, Takanobu; Akazawa, Daisuke; Sugiyama, Nao; Date, Tomoko; Masaki, Takahiro; Nakamoto, Shingo; Tanaka, Yasuhito; Mizokami, Masashi; Yokosuka, Osamu; Nomoto, Akio; Wakita, Takaji

    2012-02-01

    To establish a cell culture system for chimeric hepatitis C virus (HCV) genotype 2b, we prepared a chimeric construct harboring the 5' untranslated region (UTR) to the E2 region of the MA strain (genotype 2b) and the region of p7 to the 3' UTR of the JFH-1 strain (genotype 2a). This chimeric RNA (MA/JFH-1.1) replicated and produced infectious virus in Huh7.5.1 cells. Replacement of the 5' UTR of this chimera with that from JFH-1 (MA/JFH-1.2) enhanced virus production, but infectivity remained low. In a long-term follow-up study, we identified a cell culture-adaptive mutation in the core region (R167G) and found that it enhanced virus assembly. We previously reported that the NS3 helicase (N3H) and the region of NS5B to 3' X (N5BX) of JFH-1 enabled replication of the J6CF strain (genotype 2a), which could not replicate in cells. To reduce JFH-1 content in MA/JFH-1.2, we produced a chimeric viral genome for MA harboring the N3H and N5BX regions of JFH-1, combined with a JFH-1 5' UTR replacement and the R167G mutation (MA/N3H+N5BX-JFH1/R167G). This chimeric RNA replicated efficiently, but virus production was low. After the introduction of four additional cell culture-adaptive mutations, MA/N3H+N5BX-JFH1/5am produced infectious virus efficiently. Using this chimeric virus harboring minimal regions of JFH-1, we analyzed interferon sensitivity and found that this chimeric virus was more sensitive to interferon than JFH-1 and another chimeric virus containing more regions from JFH-1 (MA/JFH-1.2/R167G). In conclusion, we established an HCV genotype 2b cell culture system using a chimeric genome harboring minimal regions of JFH-1. This cell culture system may be useful for characterizing genotype 2b viruses and developing antiviral strategies.

  19. Targeting Spare CC Chemokine Receptor 5 (CCR5) as a Principle to Inhibit HIV-1 Entry*

    OpenAIRE

    Jin, Jun; Colin, Philippe; Staropoli, Isabelle; Lima-Fernandes, Evelyne; Ferret, Cécile; Demir, Arzu; Rogée, Sophie; Hartley, Oliver; Randriamampita, Clotilde; Scott, Mark G. H.; Marullo, Stefano; Sauvonnet, Nathalie; Arenzana-Seisdedos, Fernando; Lagane, Bernard; Brelot, Anne

    2014-01-01

    International audience; : CCR5 binds the chemokines CCL3, CCL4, and CCL5 and is the major coreceptor for HIV-1 entry into target cells. Chemokines are supposed to form a natural barrier against human immunodeficiency virus, type 1 (HIV-1) infection. However, we showed that their antiviral activity is limited by CCR5 adopting low-chemokine affinity conformations at the cell surface. Here, we investigated whether a pool of CCR5 that is not stabilized by chemokines could represent a target for i...

  20. In vivo emergence of vicriviroc resistance in a human immunodeficiency virus type 1 subtype C-infected subject.

    Science.gov (United States)

    Tsibris, Athe M N; Sagar, Manish; Gulick, Roy M; Su, Zhaohui; Hughes, Michael; Greaves, Wayne; Subramanian, Mani; Flexner, Charles; Giguel, Françoise; Leopold, Kay E; Coakley, Eoin; Kuritzkes, Daniel R

    2008-08-01

    Little is known about the in vivo development of resistance to human immunodeficiency virus type 1 (HIV-1) CCR5 antagonists. We studied 29 subjects with virologic failure from a phase IIb study of the CCR5 antagonist vicriviroc (VCV) and identified one individual with HIV-1 subtype C who developed VCV resistance. Studies with chimeric envelopes demonstrated that changes within the V3 loop were sufficient to confer VCV resistance. Resistant virus showed VCV-enhanced replication, cross-resistance to another CCR5 antagonist, TAK779, and increased sensitivity to aminooxypentane-RANTES and the CCR5 monoclonal antibody HGS004. Pretreatment V3 loop sequences reemerged following VCV discontinuation, implying that VCV resistance has associated fitness costs.

  1. The frequency of CCR5 promoter polymorphisms and CCR5 32 mutation in Iranian populations

    Directory of Open Access Journals (Sweden)

    Mohammad Zare-Bidaki

    2015-04-01

    Full Text Available Evidence showed that chemokines serve as pro-migratory factors for immune cells. CCL3, CCL4 and CCL5, as the main CC  chemokines subfamily members, activate immune cells through binding to CC chemokine receptor 5 or CCR5. Macrophages, NK cells and T lymphocytes express CCR5 and thus, affected CCR5 expression or functions could be associated with altered immune responses. Deletion of 32 base pairs (D 32 in the exon 1 of the CCR5 gene, which is known as CCR5 D 32 mutation causes down regulation and malfunction of the molecule. Furthermore, it has been evidenced that three polymorphisms in the promoter region of CCR5 modulate its expression. Altered CCR5 expression in microbial infection and immune related diseases have been reported by several researchers but the role of CCR5 promoter polymorphisms and CCR5 D 32 mutation in Iranian patients suffering from these diseases are controversial. Due to the fact that Iranian people have different genetic backgrounds compared to other ethnics, hence, CCR5 promoter polymorphisms and CCR5 D 32 mutation association with the diseases may be different in Iranian patients. Therefore, this review addresses the most recent information regarding the prevalence as well as association of the mutation and polymorphisms in Iranian patients with microbial infection and immune related diseases as along with normal population.

  2. The case for selection at CCR5-Delta32.

    Directory of Open Access Journals (Sweden)

    2005-11-01

    Full Text Available The C-C chemokine receptor 5, 32 base-pair deletion (CCR5-Delta32 allele confers strong resistance to infection by the AIDS virus HIV. Previous studies have suggested that CCR5-Delta32 arose within the past 1,000 y and rose to its present high frequency (5%-14% in Europe as a result of strong positive selection, perhaps by such selective agents as the bubonic plague or smallpox during the Middle Ages. This hypothesis was based on several lines of evidence, including the absence of the allele outside of Europe and long-range linkage disequilibrium at the locus. We reevaluated this evidence with the benefit of much denser genetic maps and extensive control data. We find that the pattern of genetic variation at CCR5-Delta32 does not stand out as exceptional relative to other loci across the genome. Moreover using newer genetic maps, we estimated that the CCR5-Delta32 allele is likely to have arisen more than 5,000 y ago. While such results can not rule out the possibility that some selection may have occurred at C-C chemokine receptor 5 (CCR5, they imply that the pattern of genetic variation seen atCCR5-Delta32 is consistent with neutral evolution. More broadly, the results have general implications for the design of future studies to detect the signs of positive selection in the human genome.

  3. The case for selection at CCR5-Delta32.

    Directory of Open Access Journals (Sweden)

    Pardis C Sabeti

    2005-11-01

    Full Text Available The C-C chemokine receptor 5, 32 base-pair deletion (CCR5-Delta32 allele confers strong resistance to infection by the AIDS virus HIV. Previous studies have suggested that CCR5-Delta32 arose within the past 1,000 y and rose to its present high frequency (5%-14% in Europe as a result of strong positive selection, perhaps by such selective agents as the bubonic plague or smallpox during the Middle Ages. This hypothesis was based on several lines of evidence, including the absence of the allele outside of Europe and long-range linkage disequilibrium at the locus. We reevaluated this evidence with the benefit of much denser genetic maps and extensive control data. We find that the pattern of genetic variation at CCR5-Delta32 does not stand out as exceptional relative to other loci across the genome. Moreover using newer genetic maps, we estimated that the CCR5-Delta32 allele is likely to have arisen more than 5,000 y ago. While such results can not rule out the possibility that some selection may have occurred at C-C chemokine receptor 5 (CCR5, they imply that the pattern of genetic variation seen at CCR5-Delta32 is consistent with neutral evolution. More broadly, the results have general implications for the design of future studies to detect the signs of positive selection in the human genome.

  4. Role of the chemokine receptors CCR1, CCR2 and CCR4 in the pathogenesis of experimental dengue infection in mice.

    Directory of Open Access Journals (Sweden)

    Rodrigo Guabiraba

    Full Text Available Dengue virus (DENV, a mosquito-borne flavivirus, is a public health problem in many tropical countries. Recent clinical data have shown an association between levels of different chemokines in plasma and severity of dengue. We evaluated the role of CC chemokine receptors CCR1, CCR2 and CCR4 in an experimental model of DENV-2 infection in mice. Infection of mice induced evident clinical disease and tissue damage, including thrombocytopenia, hemoconcentration, lymphopenia, increased levels of transaminases and pro-inflammatory cytokines, and lethality in WT mice. Importantly, infected WT mice presented increased levels of chemokines CCL2/JE, CCL3/MIP-1α and CCL5/RANTES in spleen and liver. CCR1⁻/⁻ mice had a mild phenotype with disease presentation and lethality similar to those of WT mice. In CCR2⁻/⁻ mice, lethality, liver damage, levels of IL-6 and IFN-γ, and leukocyte activation were attenuated. However, thrombocytopenia, hemoconcentration and systemic TNF-α levels were similar to infected WT mice. Infection enhanced levels of CCL17/TARC, a CCR4 ligand. In CCR4⁻/⁻ mice, lethality, tissue injury and systemic inflammation were markedly decreased. Despite differences in disease presentation in CCR-deficient mice, there was no significant difference in viral load. In conclusion, activation of chemokine receptors has discrete roles in the pathogenesis of dengue infection. These studies suggest that the chemokine storm that follows severe primary dengue infection associates mostly to development of disease rather than protection.

  5. Polymorphisms of CCL3L1/CCR5 genes and recurrence of hepatitis B in liver transplant recipients.

    Science.gov (United States)

    Li, Hong; Xie, Hai-Yang; Zhou, Lin; Wang, Wei-Lin; Liang, Ting-Bo; Zhang, Min; Zheng, Shu-Sen

    2011-12-01

    The genetic diversity of chemokines and chemokine receptors has been associated with the outcome of hepatitis B virus infection. The aim of this study was to evaluate whether the copy number variation in the CCL3L1 gene and the polymorphisms of CCR5Δ32 and CCR5-2459A→G (rs1799987) are associated with recurrent hepatitis B in liver transplantation for hepatitis B virus infection-related end-stage liver disease. A total of 185 transplant recipients were enrolled in this study. The genomic DNA was extracted from whole blood, the copy number of the CCL3L1 gene was determined by a quantitative real-time PCR based assay, CCR5Δ32 was detected by a sizing PCR method, and a single-nucleotide polymorphism in CCR5-2459 was detected by restriction fragment length polymorphism PCR. No CCR5Δ32 mutation was detected in any of the individuals from China. Neither copy number variation nor polymorphism in CCR5-2459 was associated with post-transplant re-infection with hepatitis B virus. However, patients with fewer copies (CCR5 genes might be more likely to have recurrence of hepatitis B after transplantation.

  6. Both Cerebral and Hematopoietic Deficiencies in CCR2 Result in Uncontrolled Herpes Simplex Virus Infection of the Central Nervous System in Mice.

    Science.gov (United States)

    Menasria, Rafik; Canivet, Coraline; Piret, Jocelyne; Gosselin, Jean; Boivin, Guy

    2016-01-01

    CCR2 is a chemokine receptor expressed on the surface of blood leukocytes, particularly «Ly6Chi» inflammatory monocytes and microglia. Signaling through this receptor is thought to influence the immune activity of microglia as well as monocytes egress from the bone marrow (BM) and their trafficking into the central nervous system (CNS) in several neurological diseases. During experimental herpes simplex virus 1 (HSV-1) encephalitis (HSE), CCR2 deficiency has been reported to exacerbate the outcome of the disease. However, the precise contribution of CCR2 expressed in cells of the CNS or peripheral monocytes in the protection against HSE remains unclear. To dissect the differential role of CCR2 during HSE, chimeric mice with receptor deficiency in the brain or blood cells were generated by transplanting wild-type (WT) C57BL/6 or CCR2-/- BM-derived cells in CCR2-/- (WT→CCR2-/-) and WT (CCR2-/-→WT) mice, respectively. Our results indicate that following intranasal infection with 1.2x106 plaque forming units of HSV-1, CCR2 deficiency in hematopoietic cells and, to a lesser extent, in CNS exacerbates the outcome of HSE. Mortality rates of CCR2-/- (71.4%) and CCR2-/-→WT (57.1%) mice were significantly higher than that of WT (15.3%; Pdeficiencies in CCR2 resulted in increased infectious viral titers and wider dissemination of HSV antigens in the brain as well as an overproduction of inflammatory cytokines and chemokines including IL-1β, IL-6, CCL2, CCL3 and CCL5. Furthermore, CCR2 deficiency in the hematopoietic system altered monocytes egress from the BM and their recruitment to the CNS, which may contribute to the failure in HSV-1 containment. Collectively, these data suggest that CCR2 expressed on cells of CNS and especially on peripheral monocytes is important for the control of HSV-1 replication and inflammatory environment during experimental HSE.

  7. A cooperative interaction between nontranslated RNA sequences and NS5A protein promotes in vivo fitness of a chimeric hepatitis C/GB virus B.

    Directory of Open Access Journals (Sweden)

    Lucile Warter

    Full Text Available GB virus B (GBV-B is closely related to hepatitis C virus (HCV, infects small non-human primates, and is thus a valuable surrogate for studying HCV. Despite significant differences, the 5' nontranslated RNAs (NTRs of these viruses fold into four similar structured domains (I-IV, with domains II-III-IV comprising the viral internal ribosomal entry site (IRES. We previously reported the in vivo rescue of a chimeric GBV-B (vGB/III(HC containing HCV sequence in domain III, an essential segment of the IRES. We show here that three mutations identified within the vGB/III(HC genome (within the 3'NTR, upstream of the poly(U tract, and NS5A coding sequence are necessary and sufficient for production of this chimeric virus following intrahepatic inoculation of synthetic RNA in tamarins, and thus apparently compensate for the presence of HCV sequence in domain III. To assess the mechanism(s underlying these compensatory mutations, and to determine whether 5'NTR subdomains participating in genome replication do so in a virus-specific fashion, we constructed and evaluated a series of chimeric subgenomic GBV-B replicons in which various 5'NTR subdomains were substituted with their HCV homologs. Domains I and II of the GBV-B 5'NTR could not be replaced with HCV sequence, indicating that they contain essential, virus-specific RNA replication elements. In contrast, domain III could be swapped with minimal loss of genome replication capacity in cell culture. The 3'NTR and NS5A mutations required for rescue of the related chimeric virus in vivo had no effect on replication of the subgenomic GBneoD/III(HC RNA in vitro. The data suggest that in vivo fitness of the domain III chimeric virus is dependent on a cooperative interaction between the 5'NTR, 3'NTR and NS5A at a step in the viral life cycle subsequent to genome replication, most likely during particle assembly. Such a mechanism may be common to all hepaciviruses.

  8. Distribution of the CCR5delta32 allele (gene variant CCR5) in Rondônia, Western Amazonian region, Brazil

    Science.gov (United States)

    de Farias, Josileide Duarte; Santos, Marlene Guimarães; de França, Andonai Krauze; Delani, Daniel; Tada, Mauro Shugiro; Casseb, Almeida Andrade; Simões, Aguinaldo Luiz; Engracia, Vera

    2012-01-01

    Since around 1723, on the occasion of its initial colonization by Europeans, Rondonia has received successive waves of immigrants. This has been further swelled by individuals from northeastern Brazil, who began entering at the beginning of the twentieth century. The ethnic composition varies across the state according to the various sites of settlement of each wave of immigrants. We analyzed the frequency of the CCR5Δ32 allele of the CCR5 chemokine receptor, which is considered a Caucasian marker, in five sample sets from the population. Four were collected in Porto Velho, the state capital and the site of several waves of migration. Of these, two, from the Hospital de Base were comprised of HB Mothers and HB Newborns presenting allele frequencies of 3.5% and 3.1%, respectively, a third from the peri-urban neighborhoods of Candelária/Bate-Estaca (1.8%), whereas a fourth, from the Research Center on Tropical Medicine/CEPEM (0.6%), was composed of malaria patients under treament. The fifth sample (3.4%) came from the inland Quilombola village of Pedras Negras. Two homozygous individuals (CCR5Δ32/CCR5Δ32) were detected among the HB Mother samples. The frequency of this allele was heterogeneous and higher where the European inflow was more pronounced. The presence of the allele in Pedras Negras revealed European miscegenation in a community largely comprising Quilombolas. PMID:22481870

  9. CCR5 Gene Disruption via Lentiviral Vectors Expressing Cas9 and Single Guided RNA Renders Cells Resistant to HIV-1 Infection

    Science.gov (United States)

    Liu, Jingjing; Zhang, Di; Kimata, Jason T.; Zhou, Paul

    2014-01-01

    CCR5, a coreceptor for HIV-1 entry, is a major target for drug and genetic intervention against HIV-1. Genetic intervention strategies have knocked down CCR5 expression levels by shRNA or disrupted the CCR5 gene using zinc finger nucleases (ZFN) or Transcription activator-like effector nuclease (TALEN). In the present study, we silenced CCR5 via CRISPR associated protein 9 (Cas9) and single guided RNAs (sgRNAs). We constructed lentiviral vectors expressing Cas9 and CCR5 sgRNAs. We show that a single round transduction of lentiviral vectors expressing Cas9 and CCR5 sgRNAs into HIV-1 susceptible human CD4+ cells yields high frequencies of CCR5 gene disruption. CCR5 gene-disrupted cells are not only resistant to R5-tropic HIV-1, including transmitted/founder (T/F) HIV-1 isolates, but also have selective advantage over CCR5 gene-undisrupted cells during R5-tropic HIV-1 infection. Importantly, using T7 endonuclease I assay we did not detect genome mutations at potential off-target sites that are highly homologous to these CCR5 sgRNAs in stably transduced cells even at 84 days post transduction. Thus we conclude that silencing of CCR5 via Cas9 and CCR5-specific sgRNAs could be a viable alternative strategy for engineering resistance against HIV-1. PMID:25541967

  10. Solution Structure of LC4 Transmembrane Segment of CCR5

    OpenAIRE

    Miyamoto, Kazuhide; Togiya, Kayo

    2011-01-01

    CC-chemokine receptor 5 (CCR5) is a specific co-receptor allowing the entry of human immunodeficiency virus type 1 (HIV-1). The LC4 region in CCR5 is required for HIV-1 entry into the cells. In this study, the solution structure of LC4 in SDS micelles was elucidated by using standard 1H two-dimensional NMR spectroscopy, circular dichroism, and fluorescdence quenching. The LC4 structure adopts two helical structures, whereas the C-terminal part remains unstructured. The positions in which LC4 ...

  11. Characterization of the virus-cell interactions by HIV-1 subtype C variants from an antiretroviral therapy-naïve subject with baseline resistance to the CCR5 inhibitor maraviroc

    DEFF Research Database (Denmark)

    Jakobsen, Martin Roelsgaard

    The CCR5 inhibitor maraviroc (MVC) exerts its antiviral activity by binding to- and altering the conformation of the CCR5 extracellular loops such that HIV-1 gp120 no longer recognizes CCR5. Viruses that have become resistant to MVC through long-term in vitro culture, or from treatment failure...... in vivo, can use the MVCbound form of CCR5 for HIV-1 entry via adaptive alterations in gp120. Partial baseline resistance to another CCR5 inhibitor through this mechanism, AD101, has been noted recently in one subject (1). Here, we identified and characterized envelope (Env) clones with baseline...

  12. CCR5 internalisation and signalling have different dependence on membrane lipid raft integrity.

    Science.gov (United States)

    Cardaba, Clara Moyano; Kerr, Jason S; Mueller, Anja

    2008-09-01

    The chemokine receptor, CCR5, acts as a co-receptor for human immunodeficiency virus entry into cells. CCR5 has been shown to be targeted to cholesterol- and sphingolipid-rich membrane microdomains termed lipid rafts or caveolae. Cholesterol is essential for CCL4 binding to CCR5 and for keeping the conformational integrity of the receptor. Filipin treatment leads to loss of caveolin-1 from the membrane and therefore to a collapse of the caveolae. We have found here that sequestration of membrane cholesterol with filipin did not affect receptor signalling, however a loss of ligand-induced internalisation of CCR5 was observed. Cholesterol extraction with methyl-beta-cyclodextrin (MCD) reduced signalling through CCR5 as measured by release of intracellular Ca(2+) and completely abolished the inhibition of forskolin-stimulated cAMP accumulation with no effect on internalisation. Pertussis toxin (PTX) treatment inhibited the intracellular release of calcium that is transduced via Galphai G-proteins. Depletion of cholesterol destroyed microdomains in the membrane and switched CCR5/G-protein coupling to a PTX-independent G-protein. We conclude that cholesterol in the membrane is essential for CCR5 signalling via the Galphai G-protein subunit, and that integrity of lipid rafts is not essential for effective CCR5 internalisation however it is crucial for proper CCR5 signal transduction via Galphai G-proteins.

  13. Replacement of the V3 domain in the surface subunit of the feline immunodeficiency virus envelope glycoprotein with the equivalent region of a T cell-tropic human immunodeficiency virus type 1 results in a chimeric surface protein that efficiently binds to CXCR4.

    Science.gov (United States)

    González, Silvia A; Falcón, Juan I; Affranchino, José L

    2014-03-01

    Feline immunodeficiency virus (FIV) and the T cell-tropic strains of human immunodeficiency virus type 1 (HIV-1) share the use of the chemokine receptor CXCR4 for cell entry. To study this process further we developed a cell surface binding assay based on the expression of a soluble version of the FIV SU C-terminally tagged with the influenza virus hemagglutinin epitope (HA). The specificity of the assay was demonstrated by the following evidence: (1) the SU-HA protein bound to HeLa cells that express CXCR4 but not to MDCK cells that lack this chemokine receptor; and (2) binding of the SU-HA to HeLa cells was blocked by incubation with the CXCR4 antagonist AMD3100 as well as with the anti-CXCR4 monoclonal antibody (MAb) 12G5. Deletion of the V3 region from the FIV SU glycoprotein abolished its ability to bind CXCR4-expressing cells. Remarkably, substitution of the V3 domain of the FIV SU by the equivalent region of the HIV-1 NL4-3 isolate resulted in efficient cell surface binding of the chimeric SU protein to CXCR4. Moreover, transfection of MDCK cells with a plasmid encoding human CXCR4 allowed the association of the chimeric SU-HA glycoprotein to the transfected cells. Interestingly, while cell binding of the chimeric FIV-HIV SU was inhibited by an anti-HIV-1 V3 MAb, its association with CXCR4 was found to be resistant to AMD3100. Of note, the chimeric FIV-HIV Env glycoprotein was capable of promoting CXCR4-dependent cell-to-cell fusion.

  14. CCR2+ and CCR5+ CD8+ T cells increase during viral infection and migrate to sites of infection

    DEFF Research Database (Denmark)

    Nansen, A; Marker, O; Bartholdy, C

    2000-01-01

    Chemokines and their receptors play a critical role in the selective recruitment of various leukocyte subsets. In this study, we correlated the expression of multiple chemokine and CC chemokine receptor (CCR) genes during the course of intracerebral (i.c.) infection with lymphocytic choriomeningi......Chemokines and their receptors play a critical role in the selective recruitment of various leukocyte subsets. In this study, we correlated the expression of multiple chemokine and CC chemokine receptor (CCR) genes during the course of intracerebral (i.c.) infection with lymphocytic...... a rapidly lethal, T cell-independent encephalitis, and infection resulted in a dramatic early up-regulation of chemokine gene expression. Similar marked up-regulation of chemokine expression was not seen until late after LCMV infection and required the presence of activated T cells. Cerebral CCR gene...... expression was dominated by CCR1, CCR2 and CCR5. However, despite a stronger initial chemokine signal in VSV-infected mice, only LCMV-induced T cell-dependent inflammation was found to be associated with substantially increased expression of CCR genes. Virus-activated CD8+ T cells were found to express CCR2...

  15. Differential evolution of a CXCR4-using HIV-1 strain in CCR5wt/wt and CCR5∆32/∆32 hosts revealed by longitudinal deep sequencing and phylogenetic reconstruction.

    Science.gov (United States)

    Le, Anh Q; Taylor, Jeremy; Dong, Winnie; McCloskey, Rosemary; Woods, Conan; Danroth, Ryan; Hayashi, Kanna; Milloy, M-J; Poon, Art F Y; Brumme, Zabrina L

    2015-12-03

    Rare individuals homozygous for a naturally-occurring 32 base pair deletion in the CCR5 gene (CCR5∆32/∆32) are resistant to infection by CCR5-using ("R5") HIV-1 strains but remain susceptible to less common CXCR4-using ("X4") strains. The evolutionary dynamics of X4 infections however, remain incompletely understood. We identified two individuals, one CCR5wt/wt and one CCR5∆32/∆32, within the Vancouver Injection Drug Users Study who were infected with a genetically similar X4 HIV-1 strain. While early-stage plasma viral loads were comparable in the two individuals (~4.5-5 log10 HIV-1 RNA copies/ml), CD4 counts in the CCR5wt/wt individual reached a nadir of 250 cells/mm(3) in the CCR5∆32/∆32 individual. Ancestral phylogenetic reconstructions using longitudinal envelope-V3 deep sequences suggested that both individuals were infected by a single transmitted/founder (T/F) X4 virus that differed at only one V3 site (codon 24). While substantial within-host HIV-1 V3 diversification was observed in plasma and PBMC in both individuals, the CCR5wt/wt individual's HIV-1 population gradually reverted from 100% X4 to ~60% R5 over ~4 years whereas the CCR5∆32/∆32 individual's remained consistently X4. Our observations illuminate early dynamics of X4 HIV-1 infections and underscore the influence of CCR5 genotype on HIV-1 V3 evolution.

  16. [Biological characteristics of a chimeric rabies virus expressing canine parvovirus VP2 protein].

    Science.gov (United States)

    Niu, Xue-Feng; Liu, Xiao-Hui; Sun, Zhao-Jin; Shi, He-He; Chen, Jing; Jiang, Bido; Sun, Jing-Chen; Guo, Xiao-Feng

    2009-09-01

    To obtain a bivalence vaccine against canine rabies virus and canine parvovirus, a chimeric rabies virus expressing canine parvovirus VP2 protein was generated by the technique of reverse genetics. It was shown that the chimeric virus designated as HEP-Flury (VP2) grew well on BHK-21 cells and the VP2 gene could still be stably expressed after ten passages on BHK-21 cells. Experiments on the mice immunized with the chimeric virus HEP-Flury (VP2) demonstrated that specific antibodies against rabies virus and canine parvovirus were induced in immunized mice after vaccination with the live chimeric virus.

  17. CCR5 Targeted Cell Therapy for HIV and Prevention of Viral Escape

    Directory of Open Access Journals (Sweden)

    Gero Hütter

    2015-07-01

    Full Text Available Allogeneic transplantation with CCR5-delta 32 (CCR5-d32 homozygous stem cells in an HIV infected individual in 2008, led to a sustained virus control and probably eradication of HIV. Since then there has been a high degree of interest to translate this approach to a wider population. There are two cellular ways to do this. The first one is to use a CCR5 negative cell source e.g., hematopoietic stem cells (HSC to copy the initial finding. However, a recent case of a second allogeneic transplantation with CCR5-d32 homozygous stem cells suffered from viral escape of CXCR4 quasi-species. The second way is to knock down CCR5 expression by gene therapy. Currently, there are five promising techniques, three of which are presently being tested clinically. These techniques include zinc finger nucleases (ZFN, clustered regularly interspaced palindromic repeats/CRISPR-associated protein 9 nuclease (CRISPR/Cas9, transcription activator-like effectors nuclease (TALEN, short hairpin RNA (shRNA, and a ribozyme. While there are multiple gene therapy strategies being tested, in this review we reflect on our current knowledge of inhibition of CCR5 specifically and whether this approach allows for consequent viral escape.

  18. Neutralizing antibody and anti-retroviral drug sensitivities of HIV-1 isolates resistant to small molecule CCR5 inhibitors

    International Nuclear Information System (INIS)

    Pugach, Pavel; Ketas, Thomas J.; Michael, Elizabeth; Moore, John P.

    2008-01-01

    The small molecule CCR5 inhibitors are a new class of drugs for treating infection by human immunodeficiency virus type 1 (HIV-1). They act by binding to the CCR5 co-receptor and preventing its use during HIV-1-cell fusion. Escape mutants can be raised against CCR5 inhibitors in vitro and will arise when these drugs are used clinically. Here, we have assessed the responses of CCR5 inhibitor-resistant viruses to other anti-retroviral drugs that act by different mechanisms, and their sensitivities to neutralizing antibodies (NAbs). The rationale for the latter study is that the resistance pathway for CCR5 inhibitors involves changes in the HIV-1 envelope glycoproteins (Env), which are also targets for NAbs. The escape mutants CC101.19 and D1/85.16 were selected for resistance to AD101 and vicriviroc (VVC), respectively, from the primary R5 HIV-1 isolate CC1/85. Each escape mutant was cross-resistant to other small molecule CCR5 inhibitors (aplaviroc, maraviroc, VVC, AD101 and CMPD 167), but sensitive to protein ligands of CCR5: the modified chemokine PSC-RANTES and the humanized MAb PRO-140. The resistant viruses also retained wild-type sensitivity to the nucleoside reverse transcriptase inhibitor (RTI) zidovudine, the non-nucleoside RTI nevirapine, the protease inhibitor atazanavir and other attachment and fusion inhibitors that act independently of CCR5 (BMS-806, PRO-542 and enfuvirtide). Of note is that the escape mutants were more sensitive than the parental CC1/85 isolate to a subset of neutralizing monoclonal antibodies and to some sera from HIV-1-infected people, implying that sequence changes in Env that confer resistance to CCR5 inhibitors can increase the accessibility of some NAb epitopes. The need to preserve NAb resistance may therefore be a constraint upon how escape from CCR5 inhibitors occurs in vivo

  19. Priming B cell-mediated anti-HIV envelope responses by vaccination allows for the long-term control of infection in macaques exposed to a R5-tropic SHIV

    International Nuclear Information System (INIS)

    Buckner, Clarisa; Gines, Leoned G.; Saunders, Cheryl J.; Vojtech, Lucia; Srivastava, Indresh; Gettie, Agegnehu; Bohm, Rudolph; Blanchard, James; Barnett, Susan W.; Safrit, Jeffrey T.; Stamatatos, Leonidas

    2004-01-01

    The potential of vaccine-elicited anti-HIV envelope antibodies to control HIV-infection was evaluated by immunizing macaques with the HIV envelope protein and transiently depleting them of their CD8+ cells before intravenous challenge with the pathogenic CCR5-tropic SIV/HIV chimeric virus, SHIV SF162P4 . Although sterilizing immunity was not achieved, all vaccinated animals effectively controlled infection and remained free of disease for the duration of observation (over 3 years). In contrast, during the same period, the control animals progressed to disease. Both the vaccinees and the controls developed robust cell-mediated antiviral and neutralizing antibody responses following infection. A comparative analysis of these responses suggests that the more effective long-term control of infection by the vaccinated animals is due to the more rapid development of anti-HIV envelope antibodies. These studies suggest that priming by vaccination of B cell anti-HIV envelope responses maybe crucial for the long-term control of HIV infection

  20. Elucidating a Key Anti-HIV-1 and Cancer-Associated Axis: The Structure of CCL5 (Rantes) in Complex with CCR5

    Science.gov (United States)

    Tamamis, Phanourios; Floudas, Christodoulos A.

    2014-06-01

    CCL5 (RANTES) is an inflammatory chemokine which binds to chemokine receptor CCR5 and induces signaling. The CCL5:CCR5 associated chemotactic signaling is of critical biological importance and is a potential HIV-1 therapeutic axis. Several studies provided growing evidence for the expression of CCL5 and CCR5 in non-hematological malignancies. Therefore, the delineation of the CCL5:CCR5 complex structure can pave the way for novel CCR5-targeted drugs. We employed a computational protocol which is primarily based on free energy calculations and molecular dynamics simulations, and report, what is to our knowledge, the first computationally derived CCL5:CCR5 complex structure which is in excellent agreement with experimental findings and clarifies the functional role of CCL5 and CCR5 residues which are associated with binding and signaling. A wealth of polar and non-polar interactions contributes to the tight CCL5:CCR5 binding. The structure of an HIV-1 gp120 V3 loop in complex with CCR5 has recently been derived through a similar computational protocol. A comparison between the CCL5 : CCR5 and the HIV-1 gp120 V3 loop : CCR5 complex structures depicts that both the chemokine and the virus primarily interact with the same CCR5 residues. The present work provides insights into the blocking mechanism of HIV-1 by CCL5.

  1. Discovery and mapping of an intracellular antagonist binding site at the chemokine receptor CCR2

    DEFF Research Database (Denmark)

    Zweemer, Annelien J M; Bunnik, Julia; Veenhuizen, Margo

    2014-01-01

    be divided into two groups with most likely two topographically distinct binding sites. The aim of the current study was to identify the binding site of one such group of ligands, exemplified by three allosteric antagonists, CCR2-RA-[R], JNJ-27141491, and SD-24. We first used a chimeric CCR2/CCR5 receptor...

  2. Characterization of the neutralization determinants of equine arteritis virus using recombinant chimeric viruses and site-specific mutagenesis of an infectious cDNA clone

    International Nuclear Information System (INIS)

    Balasuriya, Udeni B.R.; Dobbe, Jessika C.; Heidner, Hans W.; Smalley, Victoria L.; Navarrette, Andrea; Snijder, Eric J.; MacLachlan, N. James

    2004-01-01

    We have used an infectious cDNA clone of equine arteritis virus (EAV) and reverse genetics technology to further characterize the neutralization determinants in the GP5 envelope glycoprotein of the virus. We generated a panel of 20 recombinant viruses, including 10 chimeric viruses that each contained the ORF5 (which encodes GP5) of different laboratory, field, and vaccine strains of EAV, a chimeric virus containing the N-terminal ectodomain of GP5 of a European strain of porcine reproductive and respiratory syndrome virus, and 9 mutant viruses with site-specific substitutions in their GP5 proteins. The neutralization phenotype of each recombinant chimeric/mutant strain of EAV was determined with EAV-specific monoclonal antibodies and EAV strain-specific polyclonal equine antisera and compared to that of their parental viruses from which the substituted ORF5 was derived. The data unequivocally confirm that the GP5 ectodomain contains critical determinants of EAV neutralization. Furthermore, individual neutralization sites are conformationally interactive, and the interaction of GP5 with the unglycosylated membrane protein M is likely critical to expression of individual epitopes in neutralizing conformation. Substitution of individual amino acids within the GP5 ectodomain usually resulted in differences in neutralization phenotype of the recombinant viruses, analogous to differences in the neutralization phenotype of field strains of EAV and variants generated during persistent infection of EAV carrier stallions

  3. Opposing effects of CXCR3 and CCR5 deficiency on CD8+ T cell-mediated inflammation in the central nervous system of virus-infected mice

    DEFF Research Database (Denmark)

    de Lemos, Carina; Christensen, Jeanette Erbo; Nansen, Anneline

    2005-01-01

    and therefore protect mice against the otherwise fatal CD8+ T cell-mediated immune attack. Contrary to expectations, the accumulation of mononuclear cells in cerebrospinal fluid was only slightly delayed compared with mice with normal expression of both receptors. Even more surprising, CXCR3/CCR5 double-deficient......T cells play a key role in the control of viral infection in the CNS but may also contribute to immune-mediated cell damage. To study the redundancy of the chemokine receptors CXCR3 and CCR5 in regulating virus-induced CD8+ T cell-mediated inflammation in the brain, CXCR3/CCR5 double-deficient mice...... mice were more susceptible to intracerebral infection than CXCR3-deficient mice. Analysis of effector T cell generation revealed an accelerated antiviral CD8+ T cell response in CXCR3/CCR5 double-deficient mice. Furthermore, while the accumulation of CD8+ T cells in the neural parenchyma...

  4. 78 FR 16505 - Prospective Grant of Exclusive License: Chimeric West Nile/Dengue Viruses

    Science.gov (United States)

    2013-03-15

    ... Grant of Exclusive License: Chimeric West Nile/Dengue Viruses AGENCY: Centers for Disease Control and.... Provisional Application 61/049,342, filed 4/30/2008, entitled ``Engineered, Chimeric West Nile/Dengue Viruses;'' PCT Application PCT/US2009/041824, filed 4/27/2009, entitled ``Engineered, Chimeric WN/Flavivirus as...

  5. Chimeric SV40 virus-like particles induce specific cytotoxicity and protective immunity against influenza A virus without the need of adjuvants

    International Nuclear Information System (INIS)

    Kawano, Masaaki; Morikawa, Katsuma; Suda, Tatsuya; Ohno, Naohito; Matsushita, Sho; Akatsuka, Toshitaka; Handa, Hiroshi; Matsui, Masanori

    2014-01-01

    Virus-like particles (VLPs) are a promising vaccine platform due to the safety and efficiency. However, it is still unclear whether polyomavirus-based VLPs are useful for this purpose. Here, we attempted to evaluate the potential of polyomavirus VLPs for the antiviral vaccine using simian virus 40 (SV40). We constructed chimeric SV40-VLPs carrying an HLA-A ⁎ 02:01-restricted, cytotoxic T lymphocyte (CTL) epitope derived from influenza A virus. HLA-A ⁎ 02:01-transgenic mice were then immunized with the chimeric SV40-VLPs. The chimeric SV40-VLPs effectively induced influenza-specific CTLs and heterosubtypic protection against influenza A viruses without the need of adjuvants. Because DNase I treatment of the chimeric SV40-VLPs did not disrupt CTL induction, the intrinsic adjuvant property may not result from DNA contaminants in the VLP preparation. In addition, immunization with the chimeric SV40-VLPs generated long-lasting memory CTLs. We here propose that the chimeric SV40-VLPs harboring an epitope may be a promising CTL-based vaccine platform with self-adjuvant properties. - Highlights: • We constructed chimeric SV40-VLPs carrying an influenza virus-derived CTL epitope. • Chimeric SV40-VLPs induce influenza-specific CTLs in mice without adjuvants. • Chimeric SV40-VLPs induce heterosubtypic protection against influenza A viruses. • Chimeric SV40-VLPs induce long-lasting memory CTLs. • Chimeric SV40-VLPs is a promising vaccine platform with self-adjuvant properties

  6. Chimeric SV40 virus-like particles induce specific cytotoxicity and protective immunity against influenza A virus without the need of adjuvants

    Energy Technology Data Exchange (ETDEWEB)

    Kawano, Masaaki [Department of Allergy and Immunology, Faculty of Medicine, Saitama Medical University, Moroyama-cho, Iruma-gun, Saitama 350-0495 (Japan); Morikawa, Katsuma [Department of Biological Information, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8501 (Japan); Suda, Tatsuya [Department of Microbiology, Faculty of Medicine, Saitama Medical University, Moroyama-cho, Iruma-gun, Saitama 350-0495 (Japan); Laboratory for Immunopharmacology of Microbial Products, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392 (Japan); Ohno, Naohito [Laboratory for Immunopharmacology of Microbial Products, Tokyo University of Pharmacy and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392 (Japan); Matsushita, Sho [Department of Allergy and Immunology, Faculty of Medicine, Saitama Medical University, Moroyama-cho, Iruma-gun, Saitama 350-0495 (Japan); Allergy Center, Saitama Medical University, Moroyama-cho, Iruma-gun, Saitama 350-0495 (Japan); Akatsuka, Toshitaka [Department of Microbiology, Faculty of Medicine, Saitama Medical University, Moroyama-cho, Iruma-gun, Saitama 350-0495 (Japan); Handa, Hiroshi, E-mail: handa.h.aa@m.titech.ac.jp [Solutions Research Laboratory, Tokyo Institute of Technology, Midori-ku, Yokohama 226-8503 (Japan); Matsui, Masanori, E-mail: mmatsui@saitama-med.ac.jp [Department of Microbiology, Faculty of Medicine, Saitama Medical University, Moroyama-cho, Iruma-gun, Saitama 350-0495 (Japan)

    2014-01-05

    Virus-like particles (VLPs) are a promising vaccine platform due to the safety and efficiency. However, it is still unclear whether polyomavirus-based VLPs are useful for this purpose. Here, we attempted to evaluate the potential of polyomavirus VLPs for the antiviral vaccine using simian virus 40 (SV40). We constructed chimeric SV40-VLPs carrying an HLA-A{sup ⁎}02:01-restricted, cytotoxic T lymphocyte (CTL) epitope derived from influenza A virus. HLA-A{sup ⁎}02:01-transgenic mice were then immunized with the chimeric SV40-VLPs. The chimeric SV40-VLPs effectively induced influenza-specific CTLs and heterosubtypic protection against influenza A viruses without the need of adjuvants. Because DNase I treatment of the chimeric SV40-VLPs did not disrupt CTL induction, the intrinsic adjuvant property may not result from DNA contaminants in the VLP preparation. In addition, immunization with the chimeric SV40-VLPs generated long-lasting memory CTLs. We here propose that the chimeric SV40-VLPs harboring an epitope may be a promising CTL-based vaccine platform with self-adjuvant properties. - Highlights: • We constructed chimeric SV40-VLPs carrying an influenza virus-derived CTL epitope. • Chimeric SV40-VLPs induce influenza-specific CTLs in mice without adjuvants. • Chimeric SV40-VLPs induce heterosubtypic protection against influenza A viruses. • Chimeric SV40-VLPs induce long-lasting memory CTLs. • Chimeric SV40-VLPs is a promising vaccine platform with self-adjuvant properties.

  7. CCR2, CCR5, and CXCL12 variation and HIV/AIDS in Papua New Guinea.

    Science.gov (United States)

    Mehlotra, Rajeev K; Hall, Noemi B; Bruse, Shannon E; John, Bangan; Zikursh, Melinda J Blood; Stein, Catherine M; Siba, Peter M; Zimmerman, Peter A

    2015-12-01

    Polymorphisms in chemokine receptors, serving as HIV co-receptors, and their ligands are among the well-known host genetic factors associated with susceptibility to HIV infection and/or disease progression. Papua New Guinea (PNG) has one of the highest adult HIV prevalences in the Asia-Pacific region. However, information regarding the distribution of polymorphisms in chemokine receptor (CCR5, CCR2) and chemokine (CXCL12) genes in PNG is very limited. In this study, we genotyped a total of nine CCR2-CCR5 polymorphisms, including CCR2 190G >A, CCR5 -2459G >A and Δ32, and CXCL12 801G >A in PNG (n=258), North America (n=184), and five countries in West Africa (n=178). Using this data, we determined previously characterized CCR5 haplotypes. In addition, based on the previously reported associations of CCR2 190, CCR5 -2459, CCR5 open reading frame, and CXCL12 801 genotypes with HIV acquisition and/or disease progression, we calculated composite full risk scores, considering both protective as well as susceptibility effects of the CXCL12 801 AA genotype. We observed a very high frequency of the CCR5 -2459A allele (0.98) in the PNG population, which together with the absence of Δ32 resulted in a very high frequency of the HHE haplotype (0.92). These frequencies were significantly higher than in any other population (all P-valuesnew insights regarding CCR5 variation in the PNG population, and suggest that the collective variation in CCR2, CCR5, and CXCL12 may increase the risk of HIV/AIDS in a large majority of Papua New Guineans. Copyright © 2015 Elsevier B.V. All rights reserved.

  8. Molekulare Klonierung, stabile Transfektion und funktionelle Expression der murinen Chemokinrezeptoren Ccr2 und Ccr5

    OpenAIRE

    Simonis, Christopher

    2009-01-01

    The two chemokine receptors CCR2 and CCR5 play important roles in the recruitment and activation of monocytes/macrophages and T-lymphocytes at sites of infection and inflammation. To further examine their function, I cloned the two murine chemokine receptors Ccr2 and Ccr5 from genomic mouse DNA by a PCR-based cloning strategy and functionally expressed them in stably transfected CHO-cells. These cells were used to generate the first monoclonal antibodies against Ccr2 and Ccr5.

  9. Frequencies of CCR5-D32, CCR2-64I and SDF1-3’A mutations in Human Immunodeficiency Virus (HIV seropositive subjects and seronegative individuals from the state of Pará in Brazilian Amazonia

    Directory of Open Access Journals (Sweden)

    Fernanda Andreza de Pinho Lott Carvalhaes

    2005-12-01

    Full Text Available The distribution of genetic polymorphisms of chemokine receptors CCR5-delta32, CCR2-64I and chemokine (SDF1-3’A mutations were studied in 110 Human Immunodeficiency Virus type 1 (HIV-1 seropositive individuals (seropositive group and 139 seronegative individuals (seronegative group from the population of the northern Brazilian city of Belém which is the capital of the state of Pará in the Brazilian Amazon. The CCR5-delta32 mutation was found in the two groups at similar frequencies, i.e. 2.2% for the seronegative group and 2.7% for the seropositive group. The frequencies of the SDF1-3’A mutation were 21.0% for the seronegative group and 15.4% for the seropositive group, and the CCR2-64I allele was found at frequencies of 12.5% for the seronegative group and 5.4% for the seropositive group. Genotype distributions were consistent with Hardy-Weinberg expectations in both groups, suggesting that none of the three mutations has a detectable selective effect. Difference in the allelic and genotypic frequencies was statistically significant for the CCR2 locus, the frequency in the seronegative group being twice that found in the seropositive group. This finding may indicate a protective effect of the CCR2-64I mutation in relation to HIV transmission. However, considering that the CCR2-64I mutation has been more strongly associated with a decreased risk for progression for AIDS than to the resistance to the HIV infection, this could reflect an aspect of population structure or a Type I error.

  10. A recombinant chimeric La Crosse virus expressing the surface glycoproteins of Jamestown Canyon virus is immunogenic and protective against challenge with either parental virus in mice or monkeys.

    Science.gov (United States)

    Bennett, R S; Gresko, A K; Nelson, J T; Murphy, B R; Whitehead, S S

    2012-01-01

    La Crosse virus (LACV) and Jamestown Canyon virus (JCV), family Bunyaviridae, are mosquito-borne viruses that are endemic in North America and recognized as etiologic agents of encephalitis in humans. Both viruses belong to the California encephalitis virus serogroup, which causes 70 to 100 cases of encephalitis a year. As a first step in creating live attenuated viral vaccine candidates for this serogroup, we have generated a recombinant LACV expressing the attachment/fusion glycoproteins of JCV. The JCV/LACV chimeric virus contains full-length S and L segments derived from LACV. For the M segment, the open reading frame (ORF) of LACV is replaced with that derived from JCV and is flanked by the untranslated regions of LACV. The resulting chimeric virus retained the same robust growth kinetics in tissue culture as observed for either parent virus, and the virus remains highly infectious and immunogenic in mice. Although both LACV and JCV are highly neurovirulent in 21 day-old mice, with 50% lethal dose (LD₅₀) values of 0.1 and 0.5 log₁₀ PFU, respectively, chimeric JCV/LACV is highly attenuated and does not cause disease even after intracerebral inoculation of 10³ PFU. Parenteral vaccination of mice with 10¹ or 10³ PFU of JCV/LACV protected against lethal challenge with LACV, JCV, and Tahyna virus (TAHV). The chimeric virus was infectious and immunogenic in rhesus monkeys and induced neutralizing antibodies to JCV, LACV, and TAHV. When vaccinated monkeys were challenged with JCV, they were protected against the development of viremia. Generation of highly attenuated yet immunogenic chimeric bunyaviruses could be an efficient general method for development of vaccines effective against these pathogenic viruses.

  11. Solution structure of LC4 transmembrane segment of CCR5.

    Directory of Open Access Journals (Sweden)

    Kazuhide Miyamoto

    Full Text Available CC-chemokine receptor 5 (CCR5 is a specific co-receptor allowing the entry of human immunodeficiency virus type 1 (HIV-1. The LC4 region in CCR5 is required for HIV-1 entry into the cells. In this study, the solution structure of LC4 in SDS micelles was elucidated by using standard 1H two-dimensional NMR spectroscopy, circular dichroism, and fluorescence quenching. The LC4 structure adopts two helical structures, whereas the C-terminal part remains unstructured. The positions in which LC4 binds to the HIV-1 inhibitory peptide LC5 were determined by docking calculations in addition to NMR data. The poses showed the importance of the hydrophobic interface of the assembled structures. The solution structure of LC4 elucidated in the present work provides a structural basis for further studies on the HIV-1 inhibitory function of the LC4 region.

  12. Solution structure of LC4 transmembrane segment of CCR5.

    Science.gov (United States)

    Miyamoto, Kazuhide; Togiya, Kayo

    2011-01-01

    CC-chemokine receptor 5 (CCR5) is a specific co-receptor allowing the entry of human immunodeficiency virus type 1 (HIV-1). The LC4 region in CCR5 is required for HIV-1 entry into the cells. In this study, the solution structure of LC4 in SDS micelles was elucidated by using standard 1H two-dimensional NMR spectroscopy, circular dichroism, and fluorescence quenching. The LC4 structure adopts two helical structures, whereas the C-terminal part remains unstructured. The positions in which LC4 binds to the HIV-1 inhibitory peptide LC5 were determined by docking calculations in addition to NMR data. The poses showed the importance of the hydrophobic interface of the assembled structures. The solution structure of LC4 elucidated in the present work provides a structural basis for further studies on the HIV-1 inhibitory function of the LC4 region.

  13. HIV-1 resistance conferred by siRNA cosuppression of CXCR4 and CCR5 coreceptors by a bispecific lentiviral vector

    Directory of Open Access Journals (Sweden)

    Akkina Ramesh

    2005-01-01

    Full Text Available Abstract Background RNA interference (RNAi mediated by small interfering RNAs (siRNAs has proved to be a highly effective gene silencing mechanism with great potential for HIV/AIDS gene therapy. Previous work with siRNAs against cellular coreceptors CXCR4 and CCR5 had shown that down regulation of these surface molecules could prevent HIV-1 entry and confer viral resistance. Since monospecific siRNAs targeting individual coreceptors are inadequate in protecting against both T cell tropic (X4 and monocyte tropic (R5 viral strains simultaneously, bispecific constructs with dual specificity are required. For effective long range therapy, the bispecific constructs need to be stably transduced into HIV-1 target cells via integrating viral vectors. Results To achieve this goal, lentiviral vectors incorporating both CXCR4 and CCR5 siRNAs of short hairpin design were constructed. The CXCR4 siRNA was driven by a U6 promoter whereas the CCR5 siRNA was driven by an H1 promoter. A CMV promoter driven EGFP reporter gene is also incorporated in the bispecific construct. High efficiency transduction into coreceptor expressing Magi and Ghost cell lines with a concomitant down regulation of respective coreceptors was achieved with lentiviral vectors. When the siRNA expressing transduced cells were challenged with X4 and R5 tropic HIV-1, they demonstrated marked viral resistance. HIV-1 resistance was also observed in bispecific lentiviral vector transduced primary PBMCs. Conclusions Both CXCR4 and CCR5 coreceptors could be simultaneously targeted for down regulation by a single combinatorial lentiviral vector incorporating respective anti-coreceptor siRNAs. Stable down regulation of both the coreceptors protects cells against infection by both X4 and R5 tropic HIV-1. Stable down regulation of cellular molecules that aid in HIV-1 infection will be an effective strategy for long range HIV gene therapy.

  14. Toll-Like Receptor 2 Ligation Enhances HIV-1 Replication in Activated CCR6+ CD4+ T Cells by Increasing Virus Entry and Establishing a More Permissive Environment to Infection.

    Science.gov (United States)

    Bolduc, Jean-François; Ouellet, Michel; Hany, Laurent; Tremblay, Michel J

    2017-02-15

    In this study, we investigated the effect of Toll-like receptor 2 (TLR2) ligation on the permissiveness of activated CD4 + T cells to HIV-1 infection by focusing our experiments on the relative susceptibility of cell subsets based on their expression of CCR6. Purified primary human CD4 + T cells were first subjected to a CD3/CD28 costimulation before treatment with the TLR2 agonist Pam3CSK4. Finally, cells were inoculated with R5-tropic HIV-1 particles that permit us to study the effect of TLR2 triggering on virus production at both population and single-cell levels. We report here that HIV-1 replication is augmented in CD3/CD28-costimulated CCR6 + CD4 + T cells upon engagement of the cell surface TLR2. Additional studies indicate that a higher virus entry and polymerization of the cortical actin are seen in this cell subset following TLR2 stimulation. A TLR2-mediated increase in the level of phosphorylated NF-κB p65 subunit was also detected in CD3/CD28-costimulated CCR6 + CD4 + T cells. We propose that, upon antigenic presentation, an engagement of TLR2 acts specifically on CCR6 + CD4 + T cells by promoting virus entry in an intracellular milieu more favorable for productive HIV-1 infection. Following primary infection, HIV-1 induces an immunological and structural disruption of the gut mucosa, leading to bacterial translocation and release of microbial components in the bloodstream. These pathogen-derived constituents include several agonists of Toll-like receptors that may affect gut-homing CD4 + T cells, such as those expressing the chemokine receptor CCR6, which are highly permissive to HIV-1 infection. We demonstrate that TLR2 ligation in CD3/CD28-costimulated CCR6 + CD4 + T cells leads to enhanced virus production. Our results highlight the potential impact of bacterial translocation on the overall permissiveness of CCR6 + CD4 + T cells to productive HIV-1 infection. Copyright © 2017 American Society for Microbiology.

  15. Chemokine receptor CCR5 in interferon-treated multiple sclerosis

    DEFF Research Database (Denmark)

    Sellebjerg, F; Kristiansen, Thomas Birk; Wittenhagen, P

    2007-01-01

    OBJECTIVE: To study the relationship between CC chemokine receptor CCR5 expression and disease activity in multiple sclerosis (MS) patients treated with beta-interferon (IFN-beta). METHODS: The CCR5 Delta32 allele and a CCR5 promoter polymorphism associated with cell surface expression of CCR5 were...

  16. HIV-1 entry inhibition by small-molecule CCR5 antagonists: A combined molecular modeling and mutant study using a high-throughput assay

    International Nuclear Information System (INIS)

    Labrecque, Jean; Metz, Markus; Lau, Gloria; Darkes, Marilyn C.; Wong, Rebecca S.Y.; Bogucki, David; Carpenter, Bryon; Chen Gang; Li Tongshuang; Nan, Susan; Schols, Dominique; Bridger, Gary J.; Fricker, Simon P.; Skerlj, Renato T.

    2011-01-01

    Based on the attrition rate of CCR5 small molecule antagonists in the clinic the discovery and development of next generation antagonists with an improved pharmacology and safety profile is necessary. Herein, we describe a combined molecular modeling, CCR5-mediated cell fusion, and receptor site-directed mutagenesis approach to study the molecular interactions of six structurally diverse compounds (aplaviroc, maraviroc, vicriviroc, TAK-779, SCH-C and a benzyloxycarbonyl-aminopiperidin-1-yl-butane derivative) with CCR5, a coreceptor for CCR5-tropic HIV-1 strains. This is the first study using an antifusogenic assay, a model of the interaction of the gp120 envelope protein with CCR5. This assay avoids the use of radioactivity and HIV infection assays, and can be used in a high throughput mode. The assay was validated by comparison with other established CCR5 assays. Given the hydrophobic nature of the binding pocket several binding models are suggested which could prove useful in the rational drug design of new lead compounds.

  17. Chimeric SV40 virus-like particles induce specific cytotoxicity and protective immunity against influenza A virus without the need of adjuvants.

    Science.gov (United States)

    Kawano, Masaaki; Morikawa, Katsuma; Suda, Tatsuya; Ohno, Naohito; Matsushita, Sho; Akatsuka, Toshitaka; Handa, Hiroshi; Matsui, Masanori

    2014-01-05

    Virus-like particles (VLPs) are a promising vaccine platform due to the safety and efficiency. However, it is still unclear whether polyomavirus-based VLPs are useful for this purpose. Here, we attempted to evaluate the potential of polyomavirus VLPs for the antiviral vaccine using simian virus 40 (SV40). We constructed chimeric SV40-VLPs carrying an HLA-A*02:01-restricted, cytotoxic T lymphocyte (CTL) epitope derived from influenza A virus. HLA-A*02:01-transgenic mice were then immunized with the chimeric SV40-VLPs. The chimeric SV40-VLPs effectively induced influenza-specific CTLs and heterosubtypic protection against influenza A viruses without the need of adjuvants. Because DNase I treatment of the chimeric SV40-VLPs did not disrupt CTL induction, the intrinsic adjuvant property may not result from DNA contaminants in the VLP preparation. In addition, immunization with the chimeric SV40-VLPs generated long-lasting memory CTLs. We here propose that the chimeric SV40-VLPs harboring an epitope may be a promising CTL-based vaccine platform with self-adjuvant properties. © 2013 Elsevier Inc. All rights reserved.

  18. Why CCR2 and CCR5 blockade failed and why CCR1 blockade might still be effective in the treatment of rheumatoid arthritis

    OpenAIRE

    Lebre, M.C.; Vergunst, C.E.; Choi, I.Y.K.; Aarrass, S.; Oliveira, A.S.F.; Wyant, T.; Horuk, R.; Reedquist, K.A.; Tak, P.P.

    2011-01-01

    BACKGROUND: The aim of this study was to provide more insight into the question as to why blockade of CCR1, CCR2, and CCR5 may have failed in clinical trials in rheumatoid arthritis (RA) patients, using an in vitro monocyte migration system model. METHODOLOGY/PRINCIPAL FINDINGS: Monocytes from healthy donors (HD; n = 8) or from RA patients (for CCR2 and CCR5 antibody n = 8; for CCR1 blockade n = 13) were isolated from peripheral blood and pre-incubated with different concentrations of either ...

  19. An infectious bat-derived chimeric influenza virus harbouring the entry machinery of an influenza A virus.

    Science.gov (United States)

    Juozapaitis, Mindaugas; Aguiar Moreira, Étori; Mena, Ignacio; Giese, Sebastian; Riegger, David; Pohlmann, Anne; Höper, Dirk; Zimmer, Gert; Beer, Martin; García-Sastre, Adolfo; Schwemmle, Martin

    2014-07-23

    In 2012, the complete genomic sequence of a new and potentially harmful influenza A-like virus from bats (H17N10) was identified. However, infectious influenza virus was neither isolated from infected bats nor reconstituted, impeding further characterization of this virus. Here we show the generation of an infectious chimeric virus containing six out of the eight bat virus genes, with the remaining two genes encoding the haemagglutinin and neuraminidase proteins of a prototypic influenza A virus. This engineered virus replicates well in a broad range of mammalian cell cultures, human primary airway epithelial cells and mice, but poorly in avian cells and chicken embryos without further adaptation. Importantly, the bat chimeric virus is unable to reassort with other influenza A viruses. Although our data do not exclude the possibility of zoonotic transmission of bat influenza viruses into the human population, they indicate that multiple barriers exist that makes this an unlikely event.

  20. Vaccinia virus recombinants expressing chimeric proteins of human immunodeficiency virus and gamma interferon are attenuated for nude mice.

    OpenAIRE

    Giavedoni, L D; Jones, L; Gardner, M B; Gibson, H L; Ng, C T; Barr, P J; Yilma, T

    1992-01-01

    We have developed a method for attenuating vaccinia virus recombinants by expressing a fusion protein of a lymphokine and an immunogen. Chimeric genes were constructed that coded for gamma interferon (IFN-gamma) and structural proteins of the human immunodeficiency virus type 1 (HIV-1). In this study, we describe the biological and immunological properties of vaccinia virus recombinants expressing chimeric genes of murine or human IFN-gamma with glycoprotein gp120, gag, and a fragment of gp41...

  1. Antigen-driven C–C Chemokine-mediated HIV-1 Suppression by CD4+ T Cells from Exposed Uninfected Individuals Expressing the Wild-type CCR-5 Allele

    Science.gov (United States)

    Furci, Lucinda; Scarlatti, Gabriella; Burastero, Samuele; Tambussi, Giuseppe; Colognesi, Claudia; Quillent, Caroline; Longhi, Renato; Loverro, Patrizia; Borgonovo, Barbara; Gaffi, Davide; Carrow, Emily; Malnati, Mauro; Lusso, Paolo; Siccardi, Antonio G.; Lazzarin, Adriano; Beretta, Alberto

    1997-01-01

    Despite repeated exposure to HIV-1, certain individuals remain persistently uninfected. Such exposed uninfected (EU) people show evidence of HIV-1–specific T cell immunity and, in rare cases, selective resistance to infection by macrophage-tropic strains of HIV-1. The latter has been associated with a 32–base pair deletion in the C–C chemokine receptor gene CCR-5, the major coreceptor of macrophage-tropic strains of HIV-1. We have undertaken an analysis of the HIV-specific T cell responses in 12 EU individuals who were either homozygous for the wild-type CCR-5 allele or heterozygous for the deletion allele (CCR-5Δ32). We have found evidence of an oligoclonal T cell response mediated by helper T cells specific for a conserved region of the HIV-1 envelope. These cells produce very high levels of C–C chemokines when stimulated by the specific antigen and suppress selectively the replication of macrophage-tropic, but not T cell–tropic, strains of HIV-1. These chemokine-producing helper cells may be part of a protective immune response that could be potentially exploited for vaccine development. PMID:9236198

  2. Appraising the performance of genotyping tools in the prediction of coreceptor tropism in HIV-1 subtype C viruses

    Directory of Open Access Journals (Sweden)

    Crous Saleema

    2012-09-01

    Full Text Available Abstract Background In human immunodeficiency virus type 1 (HIV-1 infection, transmitted viruses generally use the CCR5 chemokine receptor as a coreceptor for host cell entry. In more than 50% of subtype B infections, a switch in coreceptor tropism from CCR5- to CXCR4-use occurs during disease progression. Phenotypic or genotypic approaches can be used to test for the presence of CXCR4-using viral variants in an individual’s viral population that would result in resistance to treatment with CCR5-antagonists. While genotyping approaches for coreceptor-tropism prediction in subtype B are well established and verified, they are less so for subtype C. Methods Here, using a dataset comprising V3 loop sequences from 349 CCR5-using and 56 CXCR4-using HIV-1 subtype C viruses we perform a comparative analysis of the predictive ability of 11 genotypic algorithms in their prediction of coreceptor tropism in subtype C. We calculate the sensitivity and specificity of each of the approaches as well as determining their overall accuracy. By separating the CXCR4-using viruses into CXCR4-exclusive (25 sequences and dual-tropic (31 sequences we evaluate the effect of the possible conflicting signal from dual-tropic viruses on the ability of a of the approaches to correctly predict coreceptor phenotype. Results We determined that geno2pheno with a false positive rate of 5% is the best approach for predicting CXCR4-usage in subtype C sequences with an accuracy of 94% (89% sensitivity and 99% specificity. Contrary to what has been reported for subtype B, the optimal approaches for prediction of CXCR4-usage in sequence from viruses that use CXCR4 exclusively, also perform best at predicting CXCR4-use in dual-tropic viral variants. Conclusions The accuracy of genotyping approaches at correctly predicting the coreceptor usage of V3 sequences from subtype C viruses is very high. We suggest that genotyping approaches can be used to test for coreceptor tropism in HIV-1

  3. Preclinical safety and efficacy of an anti–HIV-1 lentiviral vector containing a short hairpin RNA to CCR5 and the C46 fusion inhibitor

    Directory of Open Access Journals (Sweden)

    Orit Wolstein

    2014-01-01

    Full Text Available Gene transfer has therapeutic potential for treating HIV-1 infection by generating cells that are resistant to the virus. We have engineered a novel self-inactivating lentiviral vector, LVsh5/C46, using two viral-entry inhibitors to block early steps of HIV-1 cycle. The LVsh5/C46 vector encodes a short hairpin RNA (shRNA for downregulation of CCR5, in combination with the HIV-1 fusion inhibitor, C46. We demonstrate here the effective delivery of LVsh5/C46 to human T cell lines, peripheral blood mononuclear cells, primary CD4+ T lymphocytes, and CD34+ hematopoietic stem/progenitor cells (HSPC. CCR5-targeted shRNA (sh5 and C46 peptide were stably expressed in the target cells and were able to effectively protect gene-modified cells against infection with CCR5- and CXCR4-tropic strains of HIV-1. LVsh5/C46 treatment was nontoxic as assessed by cell growth and viability, was noninflammatory, and had no adverse effect on HSPC differentiation. LVsh5/C46 could be produced at a scale sufficient for clinical development and resulted in active viral particles with very low mutagenic potential and the absence of replication-competent lentivirus. Based on these in vitro results, plus additional in vivo safety and efficacy data, LVsh5/C46 is now being tested in a phase 1/2 clinical trial for the treatment of HIV-1 disease.

  4. Oral CCR5 inhibitors: will they make it through?

    Science.gov (United States)

    Biswas, Priscilla; Nozza, Silvia; Scarlatti, Gabriella; Lazzarin, Adriano; Tambussi, Giuseppe

    2006-05-01

    The therapeutic armamentarium against HIV has recently gained a drug belonging to a novel class of antiretrovirals, the entry inhibitors. The last decade has driven an in-depth knowledge of the HIV entry process, unravelling the multiple engagements of the HIV envelope proteins with the cellular receptorial complex that is composed of a primary receptor (CD4) and a co-receptor (CCR5 or CXCR4). The vast majority of HIV-infected subjects exhibit biological viral variants that use CCR5 as a co-receptor. Individuals with a mutated CCR5 gene, both homo- and heterozygotes, appear to be healthy. For these and other reasons, CCR5 represents an appealing target for treatment intervention, although certain challenges can not be ignored. Promising small-molecule, orally bioavailable CCR5 antagonists are under development for the treatment of HIV-1 infection.

  5. CXCR3 Directs Antigen-Specific Effector CD4+ T Cell Migration to the Lung During Parainfluenza Virus Infection

    DEFF Research Database (Denmark)

    Kohlmeier, Jacob E; Cookenham, Tres; Miller, Shannon C

    2009-01-01

    effector CD4(+) T cell migration to the lungs. To assess the role of CCR5 and CXCR3 in vivo, we directly compared the migration of Ag-specific wild-type and chemokine receptor-deficient effector T cells in mixed bone marrow chimeric mice during a parainfluenza virus infection. CXCR3-deficient effector CD4......(+) T cells were 5- to 10-fold less efficient at migrating to the lung compared with wild-type cells, whereas CCR5-deficient effector T cells were not impaired in their migration to the lung. In contrast to its role in trafficking, CXCR3 had no impact on effector CD4(+) T cell proliferation, phenotype......, or function in any of the tissues examined. These findings demonstrate that CXCR3 controls virus-specific effector CD4(+) T cell migration in vivo, and suggest that blocking CXCR3-mediated recruitment may limit T cell-induced immunopathology during respiratory virus infections....

  6. CCR1

    African Journals Online (AJOL)

    inflammatory protein II; Bioinformatics; Protease digestion; HEK293 cells; Radioligand binding. Tropical ... development of many diseases. ... diseases, antagonism of CCR1 has become an ..... importance of molecular conformation in terms of.

  7. CCL5 and CCR5 interaction promotes cell motility in human osteosarcoma.

    Directory of Open Access Journals (Sweden)

    Shih-Wei Wang

    Full Text Available BACKGROUND: Osteosarcoma is characterized by a high malignant and metastatic potential. CCL5 (previously called RANTES was originally recognized as a product of activated T cells, and plays a crucial role in the migration and metastasis of human cancer cells. It has been reported that the effect of CCL5 is mediated via CCR receptors. However, the effect of CCL5 on migration activity and integrin expression in human osteosarcoma cells is mostly unknown. METHODOLOGY/PRINCIPAL FINDINGS: Here we found that CCL5 increased the migration and expression of αvβ3 integrin in human osteosarcoma cells. Stimulation of cells with CCL5 increased CCR5 but not CCR1 and CCR3 expression. CCR5 mAb, inhibitor, and siRNA reduced the CCL5-enhanced the migration and integrin up-regulation of osteosarcoma cells. Activations of MEK, ERK, and NF-κB pathways after CCL5 treatment were demonstrated, and CCL5-induced expression of integrin and migration activity was inhibited by the specific inhibitor and mutant of MEK, ERK, and NF-κB cascades. In addition, over-expression of CCL5 shRNA inhibited the migratory ability and integrin expression in osteosarcoma cells. CONCLUSIONS/SIGNIFICANCE: CCL5 and CCR5 interaction acts through MEK, ERK, which in turn activates NF-κB, resulting in the activations of αvβ3 integrin and contributing the migration of human osteosarcoma cells.

  8. Stable gene transfer of CCR5 and CXCR4 siRNAs by sleeping beauty transposon system to confer HIV-1 resistance

    Directory of Open Access Journals (Sweden)

    Akkina Ramesh

    2008-07-01

    Full Text Available Abstract Background Thus far gene therapy strategies for HIV/AIDS have used either conventional retroviral vectors or lentiviral vectors for gene transfer. Although highly efficient, their use poses a certain degree of risk in terms of viral mediated oncogenesis. Sleeping Beauty (SB transposon system offers a non-viral method of gene transfer to avoid this possible risk. With respect to conferring HIV resistance, stable knock down of HIV-1 coreceptors CCR5 and CXCR4 by the use of lentiviral vector delivered siRNAs has proved to be a promising strategy to protect cells from HIV-1 infection. In the current studies our aim is to evaluate the utility of SB system for stable gene transfer of CCR5 and CXCR4 siRNA genes to derive HIV resistant cells as a first step towards using this system for gene therapy. Results Two well characterized siRNAs against the HIV-1 coreceptors CCR5 and CXCR4 were chosen based on their previous efficacy for the SB transposon gene delivery. The siRNA transgenes were incorporated individually into a modified SB transfer plasmid containing a FACS sortable red fluorescence protein (RFP reporter and a drug selectable neomycin resistance gene. Gene transfer was achieved by co-delivery with a construct expressing a hyperactive transposase (HSB5 into the GHOST-R3/X4/R5 cell line, which expresses the major HIV receptor CD4 and and the co-receptors CCR5 and CXCR4. SB constructs expressing CCR5 or CXCR4 siRNAs were also transfected into MAGI-CCR5 or MAGI-CXCR4 cell lines, respectively. Near complete downregulation of CCR5 and CXCR4 surface expression was observed in transfected cells. During viral challenge with X4-tropic (NL4.3 or R5-tropic (BaL HIV-1 strains, the respective transposed cells showed marked viral resistance. Conclusion SB transposon system can be used to deliver siRNA genes for stable gene transfer. The siRNA genes against HIV-1 coreceptors CCR5 and CXCR4 are able to downregulate the respective cell surface proteins

  9. Chimeric viruses containing the N-terminal ectodomains of GP5 and M proteins of porcine reproductive and respiratory syndrome do not change the cellular tropism of equine arteritis virus

    Science.gov (United States)

    Equine arteritis virus (EAV) and porcine reproductive and respiratory syndrome virus (PRRSV) are members of family Arteriviridae; they share many biological properties but differ significantly in cellular tropism. Using an infectious cDNA clone of EAV, we engineered a panel of six chimeric viruses b...

  10. Genetic Polymorphism at CCL5 Is Associated With Protection in Chagas' Heart Disease: Antagonistic Participation of CCR1+ and CCR5+ Cells in Chronic Chagasic Cardiomyopathy.

    Science.gov (United States)

    Batista, Angelica Martins; Alvarado-Arnez, Lucia Elena; Alves, Silvia Marinho; Melo, Gloria; Pereira, Isabela Resende; Ruivo, Leonardo Alexandre de Souza; da Silva, Andrea Alice; Gibaldi, Daniel; da Silva, Thayse do E S Protásio; de Lorena, Virginia Maria Barros; de Melo, Adriene Siqueira; de Araújo Soares, Ana Karine; Barros, Michelle da Silva; Costa, Vláudia Maria Assis; Cardoso, Cynthia C; Pacheco, Antonio G; Carrazzone, Cristina; Oliveira, Wilson; Moraes, Milton Ozório; Lannes-Vieira, Joseli

    2018-01-01

    Chronic cardiomyopathy is the main clinical manifestation of Chagas disease (CD), a disease caused by Trypanosoma cruzi infection. A hallmark of chronic chagasic cardiomyopathy (CCC) is a fibrogenic inflammation mainly composed of CD8 + and CD4 + T cells and macrophages. CC-chemokine ligands and receptors have been proposed to drive cell migration toward the heart tissue of CD patients. Single nucleotide polymorphisms (SNPs) in CC-chemokine ligand and receptor genes may determine protein expression. Herein, we evaluated the association of SNPs in the CC-chemokines CCL2 (rs1024611) and CCL5 (rs2107538, rs2280788) and the CCL5/RANTES receptors CCR1 (rs3181077, rs1491961, rs3136672) and CCR5 (rs1799987) with risk and progression toward CCC. We performed a cross-sectional association study of 406 seropositive patients from endemic areas for CD in the State of Pernambuco, Northeast Brazil. The patients were classified as non-cardiopathic (A, n  = 110) or cardiopathic (mild, B1, n  = 163; severe, C, n  = 133). Serum levels of CCL5 and CCL2/MCP-1 were elevated in CD patients but were neither associated with risk/severity of CCC nor with SNP genotypes. After logistic regression analysis with adjustment for the covariates gender and ethnicity, CCL5 -403 (rs2107538) CT heterozygotes (OR = 0.5, P -value = 0.04) and T carriers (OR = 0.5, P -value = 0.01) were associated with protection against CCC. To gain insight into the participation of the CCL5-CCR5/CCR1 axis in CCC, mice were infected with the Colombian T. cruzi strain. Increased CCL5 concentrations were detected in cardiac tissue. In spleen, frequencies of CCR1 + CD8 + T cells and CD14 + macrophages were decreased, while frequencies of CCR5 + cells were increased. Importantly, CCR1 + CD14 + macrophages were mainly IL-10 + , while CCR5 + cells were mostly TNF + . CCR5-deficient infected mice presented reduced TNF concentrations and injury in heart tissue. Selective blockade of CCR1 (Met

  11. Genetic Polymorphism at CCL5 Is Associated With Protection in Chagas’ Heart Disease: Antagonistic Participation of CCR1+ and CCR5+ Cells in Chronic Chagasic Cardiomyopathy

    Science.gov (United States)

    Batista, Angelica Martins; Alvarado-Arnez, Lucia Elena; Alves, Silvia Marinho; Melo, Gloria; Pereira, Isabela Resende; Ruivo, Leonardo Alexandre de Souza; da Silva, Andrea Alice; Gibaldi, Daniel; da Silva, Thayse do E. S. Protásio; de Lorena, Virginia Maria Barros; de Melo, Adriene Siqueira; de Araújo Soares, Ana Karine; Barros, Michelle da Silva; Costa, Vláudia Maria Assis; Cardoso, Cynthia C.; Pacheco, Antonio G.; Carrazzone, Cristina; Oliveira, Wilson; Moraes, Milton Ozório; Lannes-Vieira, Joseli

    2018-01-01

    Chronic cardiomyopathy is the main clinical manifestation of Chagas disease (CD), a disease caused by Trypanosoma cruzi infection. A hallmark of chronic chagasic cardiomyopathy (CCC) is a fibrogenic inflammation mainly composed of CD8+ and CD4+ T cells and macrophages. CC-chemokine ligands and receptors have been proposed to drive cell migration toward the heart tissue of CD patients. Single nucleotide polymorphisms (SNPs) in CC-chemokine ligand and receptor genes may determine protein expression. Herein, we evaluated the association of SNPs in the CC-chemokines CCL2 (rs1024611) and CCL5 (rs2107538, rs2280788) and the CCL5/RANTES receptors CCR1 (rs3181077, rs1491961, rs3136672) and CCR5 (rs1799987) with risk and progression toward CCC. We performed a cross-sectional association study of 406 seropositive patients from endemic areas for CD in the State of Pernambuco, Northeast Brazil. The patients were classified as non-cardiopathic (A, n = 110) or cardiopathic (mild, B1, n = 163; severe, C, n = 133). Serum levels of CCL5 and CCL2/MCP-1 were elevated in CD patients but were neither associated with risk/severity of CCC nor with SNP genotypes. After logistic regression analysis with adjustment for the covariates gender and ethnicity, CCL5 −403 (rs2107538) CT heterozygotes (OR = 0.5, P-value = 0.04) and T carriers (OR = 0.5, P-value = 0.01) were associated with protection against CCC. To gain insight into the participation of the CCL5CCR5/CCR1 axis in CCC, mice were infected with the Colombian T. cruzi strain. Increased CCL5 concentrations were detected in cardiac tissue. In spleen, frequencies of CCR1+ CD8+ T cells and CD14+ macrophages were decreased, while frequencies of CCR5+ cells were increased. Importantly, CCR1+CD14+ macrophages were mainly IL-10+, while CCR5+ cells were mostly TNF+. CCR5-deficient infected mice presented reduced TNF concentrations and injury in heart tissue. Selective blockade of CCR1 (Met-RANTES therapy) in

  12. Genetic Polymorphism at CCL5 Is Associated With Protection in Chagas’ Heart Disease: Antagonistic Participation of CCR1+ and CCR5+ Cells in Chronic Chagasic Cardiomyopathy

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    Angelica Martins Batista

    2018-04-01

    Full Text Available Chronic cardiomyopathy is the main clinical manifestation of Chagas disease (CD, a disease caused by Trypanosoma cruzi infection. A hallmark of chronic chagasic cardiomyopathy (CCC is a fibrogenic inflammation mainly composed of CD8+ and CD4+ T cells and macrophages. CC-chemokine ligands and receptors have been proposed to drive cell migration toward the heart tissue of CD patients. Single nucleotide polymorphisms (SNPs in CC-chemokine ligand and receptor genes may determine protein expression. Herein, we evaluated the association of SNPs in the CC-chemokines CCL2 (rs1024611 and CCL5 (rs2107538, rs2280788 and the CCL5/RANTES receptors CCR1 (rs3181077, rs1491961, rs3136672 and CCR5 (rs1799987 with risk and progression toward CCC. We performed a cross-sectional association study of 406 seropositive patients from endemic areas for CD in the State of Pernambuco, Northeast Brazil. The patients were classified as non-cardiopathic (A, n = 110 or cardiopathic (mild, B1, n = 163; severe, C, n = 133. Serum levels of CCL5 and CCL2/MCP-1 were elevated in CD patients but were neither associated with risk/severity of CCC nor with SNP genotypes. After logistic regression analysis with adjustment for the covariates gender and ethnicity, CCL5 −403 (rs2107538 CT heterozygotes (OR = 0.5, P-value = 0.04 and T carriers (OR = 0.5, P-value = 0.01 were associated with protection against CCC. To gain insight into the participation of the CCL5CCR5/CCR1 axis in CCC, mice were infected with the Colombian T. cruzi strain. Increased CCL5 concentrations were detected in cardiac tissue. In spleen, frequencies of CCR1+ CD8+ T cells and CD14+ macrophages were decreased, while frequencies of CCR5+ cells were increased. Importantly, CCR1+CD14+ macrophages were mainly IL-10+, while CCR5+ cells were mostly TNF+. CCR5-deficient infected mice presented reduced TNF concentrations and injury in heart tissue. Selective blockade of CCR1 (Met-RANTES therapy

  13. Vpx complementation of 'non-macrophage tropic' R5 viruses reveals robust entry of infectious HIV-1 cores into macrophages.

    Science.gov (United States)

    Mlcochova, Petra; Watters, Sarah A; Towers, Greg J; Noursadeghi, Mahdad; Gupta, Ravindra K

    2014-03-21

    It is now known that clinically derived viruses are most commonly R5 tropic with very low infectivity in macrophages. As these viruses utilize CD4 inefficiently, defective entry has been assumed to be the dominant restriction. The implication is that macrophages are not an important reservoir for the majority of circulating viruses. Macrophage infection by clinical transmitted/founder isolates was 10-100 and 30-450 fold less efficient as compared to YU-2 and BaL respectively. Vpx complementation augmented macrophage infection by non-macrophage tropic viruses to the level of infectivity observed for YU-2 in the absence of Vpx. Augmentation was evident even when Vpx was provided 24 hours post-infection. The entry defect was measured as 2.5-5 fold, with a further 3.5-10 fold block at strong stop and subsequent stages of reverse transcription as compared to YU-2. The overall block to infection was critically dependent on the mechanism of entry as demonstrated by rescue of infection after pseudotyping with VSV-G envelope. Reverse transcription in macrophages could not be enhanced using a panel of cytokines or lipopolysaccharide (LPS). Although the predominant block to clinical transmitted/founder viruses is post-entry, infectivity is determined by Env-CD4 interactions and can be rescued with VSV-G pseudotyping. This suggests a functional link between the optimal entry pathway taken by macrophage tropic viruses and downstream events required for reverse transcription. Consistent with a predominantly post-entry block, replication of R5 using viruses can be greatly enhanced by Vpx. We conclude therefore that entry is not the limiting step and that macrophages represent clinically relevant reservoirs for 'non-macrophage tropic' viruses.

  14. Extracellular Disulfide Bridges Serve Different Purposes in Two Homologous Chemokine Receptors, CCR1 and CCR5

    DEFF Research Database (Denmark)

    Rummel, Pia Cwarzko; Thiele, Stefanie; Hansen, Laerke Smidt

    2013-01-01

    interact with residues in the main binding crevice, we show that the 7TM-conserved bridge is essential for all types of ligand-mediated activation, whereas the chemokine-conserved bridge is dispensable for small-molecule activation in CCR1. However, in striking contrast to previous studies in other...... chemokine receptors, high affinity CCL3 chemokine binding was maintained in the absence of either bridge. In CCR5, the closest homolog to CCR1, a completely different dependency was observed as neither chemokine activation nor binding was retained in the absence of either bridge. In contrast, both bridges...... where dispensable for small-molecule activation. This indicates that CCR5 activity is independent of extracellular regions, whereas in CCR1, preserved folding of ECL2 is necessary for activation. These results indicate that conserved structural features in a receptor subgroup, does not necessarily...

  15. HIV-1 and SIV Predominantly Use CCR5 Expressed on a Precursor Population to Establish Infection in T Follicular Helper Cells

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    John Zaunders

    2017-04-01

    Full Text Available BackgroundT follicular helper (Tfh cells are increasingly recognized as a major reservoir of HIV infection that will likely need to be addressed in approaches to curing HIV. However, Tfh express minimal CCR5, the major coreceptor for HIV-1, and the mechanism by which they are infected is unclear. We have previously shown that macaque Tfh lack CCR5, but are infected in vivo with CCR5-using SIV at levels comparable to other memory CD4+ T cells. Similarly, human splenic Tfh cells are highly infected with HIV-1 DNA. Therefore, we set out to examine the mechanism of infection of Tfh cells.MethodologyTfh and other CD4+ T cell subsets from macaque lymph nodes and spleens, splenic Tfh from HIV+ subjects, and tonsillar Tfh from HIV-uninfected subjects were isolated by cell sorting prior to cell surface and molecular characterization. HIV proviral gp120 sequences were submitted to genotypic and phenotypic tropism assays. Entry of CCR5- and CXCR4-using viruses into Tfh from uninfected tonsillar tissue was measured using a fusion assay.ResultsPhylogenetic analysis, genotypic, and phenotypic analysis showed that splenic Tfh cells from chronic HIV+ subjects were predominantly infected with CCR5-using viruses. In macaques, purified CCR5+PD-1intermediate(int+ memory CD4+ T cells were shown to include pre-Tfh cells capable of differentiating in vitro to Tfh by upregulation of PD-1 and Bcl6, confirmed by qRT-PCR and single-cell multiplex PCR. Infected PD-1int cells survive, carry SIV provirus, and differentiate into PD-1hi Tfh after T cell receptor stimulation, suggesting a pathway for SIV infection of Tfh. In addition, a small subset of macaque and human PD-1hi Tfh can express low levels of CCR5, which makes them susceptible to infection. Fusion assays demonstrated CCR5-using HIV-1 entry into CCR5+ Tfh and pre-Tfh cells from human tonsils.ConclusionThe major route of infection of Tfh in macaques and humans appears to be via a CCR5-expressing pre-Tfh population

  16. CCR3, CCR5, CCR8 and CXCR3 expression in memory T helper cells from allergic rhinitis patients, asymptomatically sensitized and healthy individuals

    DEFF Research Database (Denmark)

    Holse, Mille; Assing, Kristian; Poulsen, Lars K.

    2006-01-01

    Chemokine receptors have been suggested to be preferentially expressed on CD4+ T cells with CCR3 and CCR8 linked to the T helper (Th) 2 subset and CCR5 and CXCR3 to the Th1 subset, however this remains controversial....

  17. Use of homologous recombination in yeast to create chimeric bovine viral diarrhea virus cDNA clones

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    Sandra Arenhart

    Full Text Available Abstract The open reading frame of a Brazilian bovine viral diarrhea virus (BVDV strain, IBSP4ncp, was recombined with the untranslated regions of the reference NADL strain by homologous recombination in Saccharomyces cerevisiae, resulting in chimeric full-length cDNA clones of BVDV (chi-NADL/IBSP4ncp#2 and chi-NADL/IBSP4ncp#3. The recombinant clones were successfully recovered, resulting in viable viruses, having the kinetics of replication, focus size, and morphology similar to those of the parental virus, IBSP4ncp. In addition, the chimeric viruses remained stable for at least 10 passages in cell culture, maintaining their replication efficiency unaltered. Nucleotide sequencing revealed a few point mutations; nevertheless, the phenotype of the rescued viruses was nearly identical to that of the parental virus in all experiments. Thus, genetic stability of the chimeric clones and their phenotypic similarity to the parental virus confirm the ability of the yeast-based homologous recombination to maintain characteristics of the parental virus from which the recombinant viruses were derived. The data also support possible use of the yeast system for the manipulation of the BVDV genome.

  18. Impact of CCR5 Delta32/+ deletion on herpes zoster among HIV-1-infected homosexual men

    NARCIS (Netherlands)

    Krol, Anneke; Lensen, Ruud; Veenstra, Jan; Prins, Maria; Schuitemaker, Hanneke; Coutinho, Roel A.

    2006-01-01

    The association between the presence of CCR5 Delta32 heterozygosity and incidence of clinical herpes zoster was studied among 296 homosexual men from the Amsterdam cohort study (ACS) infected with human immunodeficiency virus type I (HIV-1) with an estimated date of seroconversion. Of them 63 were

  19. CCR5 is a suppressor for cortical plasticity and hippocampal learning and memory.

    Science.gov (United States)

    Zhou, Miou; Greenhill, Stuart; Huang, Shan; Silva, Tawnie K; Sano, Yoshitake; Wu, Shumin; Cai, Ying; Nagaoka, Yoshiko; Sehgal, Megha; Cai, Denise J; Lee, Yong-Seok; Fox, Kevin; Silva, Alcino J

    2016-12-20

    Although the role of CCR5 in immunity and in HIV infection has been studied widely, its role in neuronal plasticity, learning and memory is not understood. Here, we report that decreasing the function of CCR5 increases MAPK/CREB signaling, long-term potentiation (LTP), and hippocampus-dependent memory in mice, while neuronal CCR5 overexpression caused memory deficits. Decreasing CCR5 function in mouse barrel cortex also resulted in enhanced spike timing dependent plasticity and consequently, dramatically accelerated experience-dependent plasticity. These results suggest that CCR5 is a powerful suppressor for plasticity and memory, and CCR5 over-activation by viral proteins may contribute to HIV-associated cognitive deficits. Consistent with this hypothesis, the HIV V3 peptide caused LTP, signaling and memory deficits that were prevented by Ccr5 knockout or knockdown. Overall, our results demonstrate that CCR5 plays an important role in neuroplasticity, learning and memory, and indicate that CCR5 has a role in the cognitive deficits caused by HIV.

  20. Mechanism of attenuation of a chimeric influenza A/B transfectant virus.

    Science.gov (United States)

    Luo, G; Bergmann, M; Garcia-Sastre, A; Palese, P

    1992-08-01

    The ribonucleoprotein transfection system for influenza virus allowed us to construct an influenza A virus containing a chimeric neuraminidase (NA) gene in which the noncoding sequence is derived from the NS gene of influenza B virus (T. Muster, E. K. Subbarao, M. Enami, B. P. Murphy, and P. Palese, Proc. Natl. Acad. Sci. USA 88:5177-5181, 1991). This transfectant virus is attenuated in mice and grows to lower titers in tissue culture than wild-type virus. Since such a virus has characteristics desirable for a live attenuated vaccine strain, attempts were made to characterize this virus at the molecular level. Our analysis suggests that the attenuation of the virus is due to changes in the cis signal sequences, which resulted in a reduction of transcription and replication of the chimeric NA gene. The major finding concerns a sixfold reduction in NA-specific viral RNA in the virion, causing a reduction in the ratio of infectious particles to physical particles compared with the ratio in wild-type virus. Although the NA-specific mRNA level is also reduced in transfectant virus-infected cells, it does not appear to contribute to the attenuation characteristics of the virus. The levels of the other RNAs and their expression appear to be unchanged for the transfectant virus. It is suggested that downregulation of the synthesis of one viral RNA segment leads to the generation of defective viruses during each replication cycle. We believe that this represents a general principle for attenuation which may be applied to other segmented viruses containing either single-stranded or double-stranded RNA.

  1. Chimeric rabies viruses for trans-species comparison of lyssavirus glycoprotein ectodomain functions in virus replication and pathogenesis.

    Science.gov (United States)

    Genz, Berit; Nolden, Tobias; Negatsch, Alexandra; Teifke, Jens-Peter; Conzelmann, Karl-Klaus; Finke, Stefan

    2012-01-01

    The glycoprotein G of lyssaviruses is the major determinant of virus pathogenicity and serves as a target for immunological responses to virus infections. However, assessment of the exact contribution of lyssavirus G proteins to observed differences in the pathogenicity of lyssavirus species is challenging, since the direct comparison of natural lyssaviruses does not allow specific ascription to individual virus proteins or domains. Here we describe the generation and characterization of recombinant rabies viruses (RABV) that express chimeric G proteins comprising of a RABV cytoplasma domain fused to transmembrane and ectodomain G sequences of a virulent RABV (challenge virus standard; CVS-11) or two European bat lyssaviruses (EBLV- and EBLV-2). These "envelope-switched" recombinant viruses were recovered from cDNAs. Similar growth kinetics and protein expression in neuroblastoma cell cultures and successful targeting of primary neurons showed that the chimeric G proteins were able to replace the authentic G protein in a RABV based virus vector. Inoculation of six week old CD-1 mice by the intracranial (i. c.) route of infection further demonstrated that all recombinant viruses were able to spread in the brain and to induce disease. The "envelope-switched" RABV therefore represent an important tool to further investigate the influence of lyssavirus ectodomains on virus tropism, and pathogenicity.

  2. Relevance of CCL3/CCR5 axis in oral carcinogenesis.

    Science.gov (United States)

    da Silva, Janine Mayra; Moreira Dos Santos, Tálita Pollyanna; Sobral, Lays Martin; Queiroz-Junior, Celso Martins; Rachid, Milene Alvarenga; Proudfoot, Amanda E I; Garlet, Gustavo Pompermaier; Batista, Aline Carvalho; Teixeira, Mauro Martins; Leopoldino, Andréia Machado; Russo, Remo Castro; Silva, Tarcília Aparecida

    2017-08-01

    The chemokine CCL3 is a chemotactic cytokine crucial for inflammatory cell recruitment in homeostatic and pathological conditions. CCL3 might stimulate cancer progression by promoting leukocyte accumulation, angiogenesis and tumour growth. The expression of CCL3 and its receptors CCR1 and CCR5 was demonstrated in oral squamous cell carcinoma (OSCC), but their role was not defined. Here, the functions of CCL3 were assessed using a model of chemically induced tongue carcinogenesis with 4-nitroquinoline-1-oxide (4NQO). Lineages of OSCC were used to analyse the effects of CCL3 in vitro . The 4NQO-induced lesions exhibited increased expression of CCL3, CCR1 and CCR5. CCL3 -/- and CCR5 -/- mice presented reduced incidence of tongue tumours compared to wild-type (WT) and CCR1 -/- mice. Consistently, attenuated cytomorphological atypia and reduced cell proliferation were observed in lesions of CCL3 -/- and CCR5 -/- mice. OSCC from CCL3 -/- mice exhibited lower infiltration of eosinophils and reduced expression of Egf, Fgf1, Tgf-β1, Vegfa, Vegfb, Itga-4, Vtn, Mmp-1a, Mmp-2 and Mmp-9 than WT mice. In vitro , CCL3 induced invasion and production of CCL5, IL-6, MMP -2, -8, -9. Blockage of CCL3 in vitro using α-CCL3 or Evasin-1 (a CCL3-binding protein) impaired tumour cell invasion. In conclusion, CCL3/CCR5 axis has pro-tumourigenic effects in oral carcinogenesis. The induction of inflammatory and angiogenic pathways and eosinophils recruitment appear to be the underlying mechanism explaining these effects. These data reveal potential protective effects of CCL3 blockade in oral cancer.

  3. Frequency of CCR5 Delta-32 Mutation in Human Immunodeficiency Virus (HIV-seropositive and HIV-exposed Seronegative Individuals and in General Population of Medellin, Colombia

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    Francisco J Díaz

    2000-04-01

    Full Text Available Repeated exposure to human immunodeficiency virus (HIV does not always result in seroconversion. Modifications in coreceptors for HIV entrance to target cells are one of the factors that block the infection. We studied the frequency of Delta-32 mutation in ccr5 gene in Medellin, Colombia. Two hundred and eighteen individuals distributed in three different groups were analyzed for Delta-32 mutation in ccr5 gene by polymerase chain reaction (PCR: 29 HIV seropositive (SP, 39 exposed seronegative (ESN and 150 individuals as a general population sample (GPS. The frequency of the Delta-32 mutant allele was 3.8% for ESN, 2.7% for GPS and 1.7% for SP. Only one homozygous mutant genotype (Delta-32/Delta-32 was found among the ESN (2.6%. The heterozygous genotype (ccr5/Delta-32 was found in eight GPS (5.3%, in one SP (3.4% and in one ESN (2.6%. The differences in the allelic and genotypic frequencies among the three groups were not statistically significant. A comparison between the expected and the observed genotypic frequencies showed that these frequencies were significantly different for the ESN group, which indirectly suggests a protective effect of the mutant genotype (Delta-32/Delta-32. Since this mutant genotype explained the resistance of infection in only one of our ESN persons, different mechanisms of protection must be playing a more important role in this population.

  4. The chemokine receptor CCR5 in the central nervous system.

    Science.gov (United States)

    Sorce, Silvia; Myburgh, Renier; Krause, Karl-Heinz

    2011-02-01

    The expression and the role of the chemokine receptor CCR5 have been mainly studied in the context of HIV infection. However, this protein is also expressed in the brain, where it can be crucial in determining the outcome in response to different insults. CCR5 expression can be deleterious or protective in controlling the progression of certain infections in the CNS, but it is also emerging that it could play a role in non-infectious diseases. In particular, it appears that, in addition to modulating immune responses, CCR5 can influence neuronal survival. Here, we summarize the present knowledge about the expression of CCR5 in the brain and highlight recent findings suggesting its possible involvement in neuroprotective mechanisms. Copyright © 2011. Published by Elsevier Ltd.

  5. Critical amino acids within the human immunodeficiency virus type 1 envelope glycoprotein V4 N- and C-terminals contribute to virus entry.

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    Yan Li

    Full Text Available The importance of the fourth variable (V4 region of the human immunodeficiency virus 1 (HIV-1 envelope glycoprotein (Env in virus infection has not been well clarified, though the polymorphism of this region has been found to be associated with disease progression to acquired immunodeficiency syndrome (AIDS. In the present work, we focused on the correlation between HIV-1 gp120 V4 region polymorphism and the function of the region on virus entry, and the possible mechanisms for how the V4 region contributes to virus infectivity. Therefore, we analyzed the differences in V4 sequences along with coreceptor usage preference from CCR5 to CXCR4 and examined the importance of the amino acids within the V4 region for CCR5- and CXCR4-tropic virus entry. In addition, we determined the influence of the V4 amino acids on Env expression and gp160 processing intracellularly, as well as the amount of Env on the pseudovirus surface. The results indicated that V4 tended to have a shorter length, fewer potential N-linked glycosylation sites (PNGS, greater evolutionary distance, and a lower negative net charge when HIV-1 isolates switched from a coreceptor usage preference for CCR5 to CXCR4. The N- and C-terminals of the HIV-1 V4 region are highly conserved and critical to maintain virus entry ability, but only the mutation at position 417 in the context of ADA (a R5-tropic HIV-1 strain resulted in the ability to utilize CXCR4. In addition, 390L, 391F, 414I, and 416L are critical to maintain gp160 processing and maturation. It is likely that the hydrophobic properties and the electrostatic surface potential of gp120, rather than the conformational structure, greatly contribute to this V4 functionality. The findings provide information to aid in the understanding of the functions of V4 in HIV-1 entry and offer a potential target to aid in the development of entry inhibitors.

  6. Partial protective effect of CCR5-Delta 32 heterozygosity in a cohort of heterosexual Italian HIV-1 exposed uninfected individuals

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    Cauda Roberto

    2006-09-01

    Full Text Available Abstract Despite multiple sexual exposure to HIV-1 virus, some individuals remain HIV-1 seronegative (exposed seronegative, ESN. The mechanisms underlying this resistance remain still unclear, although a multifactorial pathogenesis can be hypothesised. Although several genetic factors have been related to HIV-1 resistance, the homozigosity for a mutation in CCR5 gene (the 32 bp deletion, i.e. CCR5-Delta32 allele is presently considered the most relevant one. In the present study we analysed the genotype at CCR5 locus of 30 Italian ESN individuals (case group who referred multiple unprotected heterosexual intercourse with HIV-1 seropositive partner(s, for at least two years. One hundred and twenty HIV-1 infected patients and 120 individuals representative of the general population were included as control groups. Twenty percent of ESN individuals had heterozygous CCR5-Delta 32 genotype, compared to 7.5% of HIV-1 seropositive and 10% of individuals from the general population, respectively. None of the analysed individuals had CCR5-Delta 32 homozygous genotype. Sequence analysis of the entire open reading frame of CCR5 was performed in all ESN subjects and no polymorphisms or mutations were identified. Moreover, we determined the distribution of C77G variant in CD45 gene, which has been previously related to HIV-1 infection susceptibility. The frequency of the C77G variant showed no significant difference between ESN subjects and the two control groups. In conclusion, our data show a significantly higher frequency of CCR5-Delta 32 heterozygous genotype (p = 0.04 among the Italian heterosexual ESN individuals compared to HIV-1 seropositive patients, suggesting a partial protective role of CCR5-Delta 32 heterozygosity in this cohort.

  7. Frequency of CCR5Δ32 allele in healthy Bosniak population.

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    Grażyna Adler

    2014-08-01

    Full Text Available Recent evidence has demonstrated the role of CCR5Δ32 in a variety of human diseases: from infectious and inflammatory diseases to cancer. Several studies have confirmed that genetic variants in chemokine receptor CCR5 gene are correlated with susceptibility and resistance to HIV infection. A 32-nucleotide deletion within the CCR5 reading frame is associated with decreased susceptibility to HIV acquisition and a slower progression to AIDS. Mean frequency of CCR5Δ32 allele in Europe is approximately 10%. The highest allele frequency is observed among Nordic populations (about 12% and lower in the regions of Southeast Mediterranean (about 5%. Although the frequency of CCR5Δ32 was determined in numerous European populations, there is a lack of studies on this variant in the Bosnia and Hercegovina population. Therefore, the aim of our study was to assess the frequency of CCR5Δ32 allele in the cohort of Bosniaks and compare the results with European reports. CCR5Δ32 was detected by sequence-specific PCR in a sample of 100 healthy subjects from Bosnia and Herzegovina (DNA collected 2011-2013.  Mean age of the cohort being 58.8 (±10.7 years, with 82% of women. We identified 17 heterozygotes and one mutant homozygote in study group, with mean ∆32 allele frequency of 9.5%. CCR5∆32 allele frequency among Bosniaks is comparable to that found in Caucasian populations and follows the pattern of the north-southern gradient observed for Europe. Further studies on larger cohorts with adequate female-to-male ratio are necessary. 

  8. Abnormally high levels of virus-infected IFN-gamma+ CCR4+ CD4+ CD25+ T cells in a retrovirus-associated neuroinflammatory disorder.

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    Yoshihisa Yamano

    Full Text Available BACKGROUND: Human T-lymphotropic virus type 1 (HTLV-1 is a human retrovirus associated with both HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP, which is a chronic neuroinflammatory disease, and adult T-cell leukemia (ATL. The pathogenesis of HAM/TSP is known to be as follows: HTLV-1-infected T cells trigger a hyperimmune response leading to neuroinflammation. However, the HTLV-1-infected T cell subset that plays a major role in the accelerated immune response has not yet been identified. PRINCIPAL FINDINGS: Here, we demonstrate that CD4(+CD25(+CCR4(+ T cells are the predominant viral reservoir, and their levels are increased in HAM/TSP patients. While CCR4 is known to be selectively expressed on T helper type 2 (Th2, Th17, and regulatory T (Treg cells in healthy individuals, we demonstrate that IFN-gamma production is extraordinarily increased and IL-4, IL-10, IL-17, and Foxp3 expression is decreased in the CD4(+CD25(+CCR4(+ T cells of HAM/TSP patients as compared to those in healthy individuals, and the alteration in function is specific to this cell subtype. Notably, the frequency of IFN-gamma-producing CD4(+CD25(+CCR4(+Foxp3(- T cells is dramatically increased in HAM/TSP patients, and this was found to be correlated with disease activity and severity. CONCLUSIONS: We have defined a unique T cell subset--IFN-gamma(+CCR4(+CD4(+CD25(+ T cells--that is abnormally increased and functionally altered in this retrovirus-associated inflammatory disorder of the central nervous system.

  9. Chemokine receptor CCR5 in interferon-treated multiple sclerosis

    DEFF Research Database (Denmark)

    Sellebjerg, F; Kristiansen, T B; Wittenhagen, P

    2007-01-01

    To study the relationship between CC chemokine receptor CCR5 expression and disease activity in multiple sclerosis (MS) patients treated with beta-interferon (IFN-beta).......To study the relationship between CC chemokine receptor CCR5 expression and disease activity in multiple sclerosis (MS) patients treated with beta-interferon (IFN-beta)....

  10. Phenotypic expressions of CCR5-Delta 32/Delta 32 homozygosity

    NARCIS (Netherlands)

    Nguyen, GT; Carrington, M; Beeler, JA; Dean, M; Aledort, LM; Blatt, PM; Cohen, AR; DiMichele, D; Eyster, ME; Kessler, CM; Konkle, B; Leissinger, C; Luban, N; O'Brien, SJ; Goedert, JJ; O'Brien, TR

    1999-01-01

    Objective: As blockade of CC-chemokine receptor 5 (CCR5) has been proposed as therapy for HIV-1, we examined whether the CCR5-Delta 32/Delta 32 homozygous genotype has phenotypic expressions other than those related to HIV-1. Design: Study subjects were white homosexual men or men with hemophilia

  11. Haplotypes in CCR5-CCR2, CCL3 and CCL5 are associated with natural resistance to HIV-1 infection in a Colombian cohort.

    Science.gov (United States)

    Vega, Jorge A; Villegas-Ospina, Simón; Aguilar-Jiménez, Wbeimar; Rugeles, María T; Bedoya, Gabriel; Zapata, Wildeman

    2017-06-01

    Variants in genes encoding for HIV-1 co-receptors and their natural ligands have been individually associated to natural resistance to HIV-1 infection. However, the simultaneous presence of these variants has been poorly studied. To evaluate the association of single and multilocus haplotypes in genes coding for the viral co-receptors CCR5 and CCR2, and their ligands CCL3 and CCL5, with resistance or susceptibility to HIV-1 infection. Nine variants in CCR5-CCR2, two SNPs in CCL3 and two in CCL5 were genotyped by PCR-RFLP in 35 seropositive (cases) and 49 HIV-1-exposed seronegative Colombian individuals (controls). Haplotypes were inferred using the Arlequin software, and their frequency in individual or combined loci was compared between cases and controls by the chi-square test. A p' value ;0.05 after Bonferroni correction was considered significant. Homozygosis of the human haplogroup (HH) E was absent in controls and frequent in cases, showing a tendency to susceptibility. The haplotypes C-C and T-T in CCL3 were associated with susceptibility (p'=0.016) and resistance (p';0.0001) to HIV-1 infection, respectively. Finally, in multilocus analysis, the haplotype combinations formed by HHC in CCR5-CCR2, T-T in CCL3 and G-C in CCL5 were associated with resistance (p'=0.006). Our results suggest that specific combinations of variants in genes from the same signaling pathway can define an HIV-1 resistant phenotype. Despite our small sample size, our statistically significant associations suggest strong effects; however, these results should be further validated in larger cohorts.

  12. CCR5 controls immune and metabolic functions during Toxoplasma gondii infection.

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    Giuliano Bonfá

    Full Text Available CCR5, an important receptor related to cell recruitment and inflammation, is expressed during experimental Toxoplasma gondii infection. However, its role in the immunopathology of toxoplasmosis is not clearly defined yet. Thus, we inoculated WT and CCR5(-/- mice with a sub lethal dose of the parasite by oral route. CCR5(-/- mice were extremely susceptible to infection, presenting higher parasite load and lower tissue expression of IL-12p40, IFN-γ, TNF, IL-6, iNOS, Foxp3, T-bet, GATA-3 and PPARα. Although both groups presented inflammation in the liver with prominent neutrophil infiltration, CCR5(-/- mice had extensive tissue damage with hepatocyte vacuolization, steatosis, elevated serum triglycerides and transaminases. PPARα agonist Gemfibrozil improved the vacuolization but did not rescue CCR5(-/- infected mice from high serum triglycerides levels and enhanced mortality. We also found intense inflammation in the ileum of CCR5(-/- infected mice, with epithelial ulceration, augmented CD4 and decreased frequency of NK cells in the gut lamina propria. Most interestingly, these findings were accompanied by an outstanding accumulation of neutrophils in the ileum, which seemed to be involved in the gut immunopathology, once the depletion of these cells was accompanied by reduced local damage. Altogether, these data demonstrated that CCR5 is essential to the control of T. gondii infection and to maintain the metabolic, hepatic and intestinal integrity. These findings add novel information on the disease pathogenesis and may be relevant for directing future approaches to the treatment of multi-deregulated diseases.

  13. Identification of specific regions in hepatitis C virus core, NS2 and NS5A that genetically interact with p7 and co-ordinate infectious virus production.

    Science.gov (United States)

    Gouklani, H; Beyer, C; Drummer, H; Gowans, E J; Netter, H J; Haqshenas, G

    2013-04-01

    The p7 protein of hepatitis C virus (HCV) is a small, integral membrane protein that plays a critical role in virus replication. Recently, we reported two intergenotypic JFH1 chimeric viruses encoding the partial or full-length p7 protein of the HCV-A strain of genotype 1b (GT1b; Virology; 2007; 360:134). In this study, we determined the consensus sequences of the entire polyprotein coding regions of the wild-type JFH1 and the revertant chimeric viruses and identified predominant amino acid substitutions in core (K74M), NS2 (T23N, H99P) and NS5A (D251G). Forward genetic analysis demonstrated that all single mutations restored the infectivity of the defective chimeric genomes suggesting that the infectious virus production involves the association of p7 with specific regions in core, NS2 and NS5A. In addition, it was demonstrated that the NS2 T23N facilitated the generation of infectious intergenotypic chimeric virus encoding p7 from GT6 of HCV. © 2012 Blackwell Publishing Ltd.

  14. Chimeric vaccine composed of viral peptide and mammalian heat-shock protein 60 peptide protects against West Nile virus challenge.

    Science.gov (United States)

    Gershoni-Yahalom, Orly; Landes, Shimon; Kleiman-Shoval, Smadar; Ben-Nathan, David; Kam, Michal; Lachmi, Bat-El; Khinich, Yevgeny; Simanov, Michael; Samina, Itzhak; Eitan, Anat; Cohen, Irun R; Rager-Zisman, Bracha; Porgador, Angel

    2010-08-01

    The protective efficacy and immunogenicity of a chimeric peptide against West Nile virus (WNV) was evaluated. This virus is the aetiological agent of West Nile fever, which has recently emerged in the western hemisphere. The rapid spread of WNV throughout North America, as well as the constantly changing epidemiology and transmission of the virus by blood transfusion and transplantation, have raised major public-health concerns. Currently, there are no effective treatments for WNV or vaccine for human use. We previously identified a novel, continuous B-cell epitope from domain III of the WNV envelope protein, termed Ep15. To test whether this epitope can protect against WNV infection, we synthesized a linear chimeric peptide composed of Ep15 and the heat-shock protein 60 peptide, p458. The p458 peptide is an effective carrier peptide for subunit vaccines against other infectious agents. We now report that mice immunized with the chimeric peptide, p458-Ep15, were resistant to lethal challenges with three different WNV strains. Moreover, their brains were free of viral genome and infectious virus. Mice immunized with Ep15 alone or with p431-Ep15, a control conjugate, were not protected. The chimeric p458-Ep15 peptide induced WNV-specific immunoglobulin G antibodies that neutralized the virus and induced the secretion of interferon-gammain vitro. Challenge of chimeric peptide-immunized mice considerably enhanced WNV-specific neutralizing antibodies. We conclude that this chimeric peptide can be used for formulation of a human vaccine against WNV.

  15. Biological characterization of HIV type 1 envelope V3 regions from mothers and infants associated with perinatal transmission.

    Science.gov (United States)

    Matala, E; Hahn, T; Yedavalli, V R; Ahmad, N

    2001-12-10

    Our previous study has shown that the human immunodeficiency virus type 1 (HIV-1) envelope V3 region minor genotypes of infected mothers were transmitted to their infants and predominated initially as a homogeneous virus population in the infants (Ahmad N, Baroudy BM, Baker RC, et al.: J Virol 1995;69:1001-1012). Here we have characterized the biological properties, including cellular tropism, replication efficiency, cytopathic effects, and coreceptor utilization, of these V3 region isolates from mothers and infants. Nineteen V3 region sequences from three mother-infant pairs, including the minor variants of mothers and the major variants of infants as characterized in our previous study, were reciprocally inserted into an HIV-1 infectious molecular clone, pNL4-3, and chimeric viruses were generated by DNA transfections into HeLa cells. Equal amounts of chimeric viruses were then used to infect T lymphocyte cell lines (A3.01 and MT-2), primary blood lymphocytes (PBLs), primary monocyte-derived macrophages (MDMs), and coreceptor cell lines. We found that the V3 region chimeras failed to replicate in T lymphocyte cell lines but replicated in MDMs and PBLs, albeit at reduced levels compared with R5 laboratory HIV-1 strains. In addition, the V3 region chimeras were able to infect the HOS-CD4(+)CCR5(+) cell line, suggesting CCR5 coreceptor utilization. Moreover, the V3 region chimeras were unable to induce syncytia in MT-2 cells, indicative of non-syncytium-inducing (NSI) phenotypes. In conclusion, the HIV-1 minor genotypes of infected mothers with macrophage-tropic and NSI or R5 phenotypes are transmitted to their infants and are initially maintained with the same properties.

  16. A transgenic plant cell-suspension system for expression of epitopes on chimeric Bamboo mosaic virus particles.

    Science.gov (United States)

    Muthamilselvan, Thangarasu; Lee, Chin-Wei; Cho, Yu-Hsin; Wu, Feng-Chao; Hu, Chung-Chi; Liang, Yu-Chuan; Lin, Na-Sheng; Hsu, Yau-Heiu

    2016-01-01

    We describe a novel strategy to produce vaccine antigens using a plant cell-suspension culture system in lieu of the conventional bacterial or animal cell-culture systems. We generated transgenic cell-suspension cultures from Nicotiana benthamiana leaves carrying wild-type or chimeric Bamboo mosaic virus (BaMV) expression constructs encoding the viral protein 1 (VP1) epitope of foot-and-mouth disease virus (FMDV). Antigens accumulated to high levels in BdT38 and BdT19 transgenic cell lines co-expressing silencing suppressor protein P38 or P19. BaMV chimeric virus particles (CVPs) were subsequently purified from the respective cell lines (1.5 and 2.1 mg CVPs/20 g fresh weight of suspended biomass, respectively), and the resulting CVPs displayed VP1 epitope on the surfaces. Guinea pigs vaccinated with purified CVPs produced humoral antibodies. This study represents an important advance in the large-scale production of immunopeptide vaccines in a cost-effective manner using a plant cell-suspension culture system. © 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

  17. Accelerated in vivo proliferation of memory phenotype CD4+ T-cells in human HIV-1 infection irrespective of viral chemokine co-receptor tropism.

    Directory of Open Access Journals (Sweden)

    Yan Zhang

    Full Text Available CD4(+ T-cell loss is the hallmark of HIV-1 infection. CD4 counts fall more rapidly in advanced disease when CCR5-tropic viral strains tend to be replaced by X4-tropic viruses. We hypothesized: (i that the early dominance of CCR5-tropic viruses results from faster turnover rates of CCR5(+ cells, and (ii that X4-tropic strains exert greater pathogenicity by preferentially increasing turnover rates within the CXCR4(+ compartment. To test these hypotheses we measured in vivo turnover rates of CD4(+ T-cell subpopulations sorted by chemokine receptor expression, using in vivo deuterium-glucose labeling. Deuterium enrichment was modeled to derive in vivo proliferation (p and disappearance (d* rates which were related to viral tropism data. 13 healthy controls and 13 treatment-naive HIV-1-infected subjects (CD4 143-569 cells/ul participated. CCR5-expression defined a CD4(+ subpopulation of predominantly CD45R0(+ memory cells with accelerated in vivo proliferation (p = 2.50 vs 1.60%/d, CCR5(+ vs CCR5(-; healthy controls; P<0.01. Conversely, CXCR4 expression defined CD4(+ T-cells (predominantly CD45RA(+ naive cells with low turnover rates. The dominant effect of HIV infection was accelerated turnover of CCR5(+CD45R0(+CD4(+ memory T-cells (p = 5.16 vs 2.50%/d, HIV vs controls; P<0.05, naïve cells being relatively unaffected. Similar patterns were observed whether the dominant circulating HIV-1 strain was R5-tropic (n = 9 or X4-tropic (n = 4. Although numbers were small, X4-tropic viruses did not appear to specifically drive turnover of CXCR4-expressing cells (p = 0.54 vs 0.72 vs 0.44%/d in control, R5-tropic, and X4-tropic groups respectively. Our data are most consistent with models in which CD4(+ T-cell loss is primarily driven by non-specific immune activation.

  18. A stable CC-chemokine receptor (CCR)-5 tropic virus is correlated with the persistence of HIV RNA at less than 2.5 copies in successfully treated naïve subjects.

    Science.gov (United States)

    Parisi, Saverio Giuseppe; Andreis, Samantha; Mengoli, Carlo; Scaggiante, Renzo; Cruciani, Mario; Ferretto, Roberto; Manfrin, Vinicio; Panese, Sandro; Basso, Monica; Boldrin, Caterina; Bressan, Stefania; Sarmati, Loredana; Andreoni, Massimo; Palù, Giorgio

    2013-07-11

    To determine if tropism for CXCR4 or CCR5 correlates with cellular HIV DNA load, residual viraemia and CD4 count in 219 successfully treated naive subjects with HIV infection enrolled in five infectious diseases units in Northeastern Italy. A subset of subjects, achieving plasma HIV RNA level <50 copies/ml after initiation of first-line therapy and maintaining it until follow-up time points, was retrospectively selected from a prospective cohort. Blood samples were collected before the beginning of therapy (T0), at the first follow-up time (T1) and, when available, at a second (T2) follow-up time. HIV DNA, CD4 count and plasma viraemia were available from all 219 patients at T0 and T1, and in 86 subjects at T2, while tropism determinations were available from 109 subjects at T0, 219 at T1, and from 86 subjects at T2. Achieving residual viraemia <2.5 copies/ml at T1 correlated with having the same condition at T2 (p = 0.0007). X4 tropism at T1 was negatively correlated with the possibility of achieving viraemia<2.5 copies/ml at T2 (p = 0.0076). T1-T2 tropism stability was significant (p <0.0001). T0 tropism correlated with T1 and T2 tropism (p < 0.001); therefore the stability of the tropism over the two follow-up periods was significant (p = 0.0003). An effective viremic suppression (viraemia<2.5 copies/ml) correlated with R5 coreceptor affinity (p= 0.047). The tropism of archived virus was stable during an effective treatment, with 15-18% of subjects switching over time, despite a viraemia<50 copies/ml. R5 tropism and its stability were related to achieving and maintaining viraemia<2.5 copies/ml.

  19. Increased brain damage after ischaemic stroke in mice lacking the chemokine receptor CCR5

    Science.gov (United States)

    Sorce, S; Bonnefont, J; Julien, S; Marq-Lin, N; Rodriguez, I; Dubois-Dauphin, M; Krause, KH

    2010-01-01

    Background and purpose: The chemokine receptor CCR5 is well known for its function in immune cells; however, it is also expressed in the brain, where its specific role remains to be elucidated. Because genetic factors may influence the risk of developing cerebral ischaemia or affect its clinical outcome, we have analysed the role of CCR5 in experimental stroke. Experimental approach: Permanent cerebral ischaemia was performed by occlusion of the middle cerebral artery in wild-type and CCR5-deficient mice. Locomotor behaviour, infarct size and histochemical alterations were analysed at different time points after occlusion. Key results: The cerebral vasculature was comparable in wild-type and CCR5-deficient mice. However, the size of the infarct and the motor deficits after occlusion were markedly increased in CCR5-deficient mice as compared with wild type. No differences between wild-type and CCR5-deficient mice were elicited by occlusion with respect to the morphology and abundance of astrocytes and microglia. Seven days after occlusion the majority of CCR5-deficient mice displayed neutrophil invasion in the infarct region, which was not observed in wild type. As compared with wild type, the infarct regions of CCR5-deficient mice were characterized by increased neuronal death. Conclusions and implications: Lack of CCR5 increased the severity of brain injury following occlusion of the middle cerebral artery. This is of particular interest with respect to the relatively frequent occurrence of CCR5 deficiency in the human population (1–2% of the Caucasian population) and the advent of CCR5 inhibitors as novel drugs. PMID:20423342

  20. Identification of dual-tropic HIV-1 using evolved neural networks.

    Science.gov (United States)

    Fogel, Gary B; Lamers, Susanna L; Liu, Enoch S; Salemi, Marco; McGrath, Michael S

    2015-11-01

    Blocking the binding of the envelope HIV-1 protein to immune cells is a popular concept for development of anti-HIV therapeutics. R5 HIV-1 binds CCR5, X4 HIV-1 binds CXCR4, and dual-tropic HIV-1 can bind either coreceptor for cellular entry. R5 viruses are associated with early infection and over time can evolve to X4 viruses that are associated with immune failure. Dual-tropic HIV-1 is less studied; however, it represents functional antigenic intermediates during the transition of R5 to X4 viruses. Viral tropism is linked partly to the HIV-1 envelope V3 domain, where the amino acid sequence helps dictate the receptor a particular virus will target; however, using V3 sequence information to identify dual-tropic HIV-1 isolates has remained difficult. Our goal in this study was to elucidate features of dual-tropic HIV-1 isolates that assist in the biological understanding of dual-tropism and develop an approach for their detection. Over 1559 HIV-1 subtype B sequences with known tropisms were analyzed. Each sequence was represented by 73 structural, biochemical and regional features. These features were provided to an evolved neural network classifier and evaluated using balanced and unbalanced data sets. The study resolved R5X4 viruses from R5 with an accuracy of 81.8% and from X4 with an accuracy of 78.8%. The approach also identified a set of V3 features (hydrophobicity, structural and polarity) that are associated with tropism transitions. The ability to distinguish R5X4 isolates will improve computational tropism decisions for R5 vs. X4 and assist in HIV-1 research and drug development efforts. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  1. [CCR5, CCR2, apoe, p53, ITGB3 and HFE gene polymorphism in Western Siberia long-livers].

    Science.gov (United States)

    Ivanoshchuk, D E; Mikhaĭlova, S V; Kulikov, I V; Maksimov, V N; Voevoda, M I; Romashchenko, A G

    2012-01-01

    In order to estimate the distribution of some polymorphisms for the CCR5, CCR2, apoE, p53, ITGB3, and HFE genes in Russian long-livers from Western Siberia, a sample of 271 individuals (range 90-105 years) was examined. It was demonstrated that carriage of the delta32 polymorphism for the CCR5 gene, V64/polymorphism for the CCR2 gene, e2/e3/e4 for the apoE gene, L33P for the ITGB3 gene, as well as H63D and S65C polymorphisms for the HFE gene does not influence on predisposition to the longevity; carriage of the 282 Y allele for the HFE gene negatively influences on the longevity; carriage of the heterozygous genotype for the R72P polymorphism for the p53 gene correlates with the longevity of elderly people.

  2. CCR6-dependent positioning of memory B cells is essential for their ability to mount a recall response to antigen

    Science.gov (United States)

    Elgueta, Raul; Marks, Ellen; Nowak, Elizabeth; Menezes, Shinelle; Benson, Micah; Raman, Vanitha S.; Ortiz, Carla; O’Connell, Samuel; Hess, Henry; Lord, Graham M.; Noelle, Randolph

    2014-01-01

    Chemokine-dependent localization of specific B cell subsets within the immune microarchitecture is essential to insure successful cognate interactions. While cognate interactions between T cells and memory B cells (Bmem)5 are essential for the secondary humoral immune responses, the chemokine response patterns of Bmem cells are largely unknown. In contrast to naïve B cells, this study shows that antigen-specific Bmem cells have heightened expression of CCR6 and a selective chemotactic response to the CCR6 ligand, CCL20. While CCR6 appears be non-essential for the initial clonal expansion and maintenance of Bmem, CCR6 is essential for the ability of Bmem to respond to a recall response to their cognate antigen. This dependency was deemed intrinsic by studies in CCR6-deficient mice and in bone-marrow chimeric mice where CCR6 deficiency was limited to the B cell lineage. Finally, the mis-positioning of CCR6-deficient Bmem was revealed by immunohistological analysis with an altered distribution of CCR6-deficient Bmem from the marginal and perifollicular to the follicular/germinal center area. PMID:25505290

  3. Effects of CCR5-delta32 and CCR2-64I alleles on disease progression of perinatally HIV-1-infected children: an international meta-analysis.

    Science.gov (United States)

    Ioannidis, John P A; Contopoulos-Ioannidis, Despina G; Rosenberg, Philip S; Goedert, James J; De Rossi, Anita; Espanol, Teresa; Frenkel, Lisa; Mayaux, Marie-Jeanne; Newell, Marie-Louise; Pahwa, Savita G; Rousseau, Christine; Scarlatti, Gabriella; Sei, Shizuko; Sen, Luisa; O'Brien, Thomas R

    2003-07-25

    Among perinatally infected children, the effects of certain alleles of the CCR5 and CCR2 genes on the rate of disease progression remain unclear. We addressed the effects of CCR5-delta32 and CCR2-64I in an international meta-analysis. Genotype data were contributed from 10 studies with 1317 HIV-1-infected children (7263 person-years of follow-up). Time-to-event analyses were performed stratified by study and racial group. Endpoints included progression to clinical AIDS, death, and death after the diagnosis of clinical AIDS. The time-dependence of the genetic effects was specifically investigated. There was large heterogeneity in the observed rates of disease progression between different cohorts. For progression to clinical AIDS, both CCR5-delta32 and CCR2-64I showed overall non-significant trends for protection [hazard ratios 0.84, 95% confidence interval (CI) 0.58-1.23; and 0.87, 95% CI 0.67-1.14, respectively]. However, analyses of survival showed statistically significant time-dependence. No deaths occurred among CCR5-delta32 carriers in the first 3 years of life, whereas there was no protective effect (hazard ratio 0.95; 95% CI 0.43-2.10) in later years (P=0.01 for the time-dependent model). For CCR2-64I, the hazard ratio for death was 0.69 (95% CI 0.39-1.21) in the first 6 years of life and 2.56 (95% CI 1.26-5.20) in subsequent years (P<0.01 for the time-dependent model). CCR5-delta32 and CCR2-64I offered no clear protection after clinical AIDS had developed. The CCR5-delta32 and CCR2-64I alleles are associated with a decreased risk of death among perinatally infected children, but only for the first years of life.

  4. Molecularly engineered live-attenuated chimeric West Nile/dengue virus vaccines protect rhesus monkeys from West Nile virus

    International Nuclear Information System (INIS)

    Pletnev, Alexander G.; St Claire, Marisa; Elkins, Randy; Speicher, Jim; Murphy, Brian R.; Chanock, Robert M.

    2003-01-01

    Two molecularly engineered, live-attenuated West Nile virus (WN) vaccine candidates were highly attenuated and protective in rhesus monkeys. The vaccine candidates are chimeric viruses (designated WN/DEN4) bearing the membrane precursor and envelope protein genes of WN on a backbone of dengue 4 virus (DEN4) with or without a deletion of 30 nucleotides (Δ30) in the 3' noncoding region of DEN4. Viremia in WN/DEN4- infected monkeys was reduced 100-fold compared to that in WN- or DEN4-infected monkeys. WN/DEN4-3'Δ30 did not cause detectable viremia, indicating that it is even more attenuated for monkeys. These findings indicate that chimerization itself and the presence of the Δ30 mutation independently contribute to the attenuation phenotype for nonhuman primates. Despite their high level of attenuation in monkeys, the chimeras induced a moderate-to-high titer of neutralizing antibodies and prevented viremia in monkeys challenged with WN. The more attenuated vaccine candidate, WN/DEN4-3'Δ30, will be evaluated first in our initial clinical studies

  5. Lack of association between the chemokine receptor 5 polymorphism CCR5delta32 in rheumatoid arthritis and juvenile idiopathic arthritis

    Directory of Open Access Journals (Sweden)

    Kvien Tore K

    2007-06-01

    Full Text Available Abstract Background The chemokine receptor CCR5 has been detected at elevated levels on synovial T cells, and a 32 bp deletion in the CCR5 gene leads to a non-functional receptor. A negative association between the CCR5Δ32 and rheumatoid arthritis (RA has been reported, although with conflicting results. In juvenile idiopathic arthritis (JIA, an association with CCR5 was recently reported. The purpose of this study was to investigate if the CCR5Δ32 polymorphism is associated with RA or JIA in Norwegian cohorts. Methods 853 RA patients, 524 JIA patients and 658 controls were genotyped for the CCR5Δ32 polymorphism. Results The CCR5Δ32 allele frequency was 11.5% in the controls vs. 10.4% in RA patients (OR = 0.90; P = 0.36 and 9.7% in JIA patients (OR = 0.85; P = 0.20. No decreased homozygosity was observed for CCR5Δ32, as previously suggested. Conclusion Our data do not support an association between the CCR5Δ32 allele and Norwegian RA or JIA patients. Combining our results with those from a recently published meta-analysis still provide evidence for a role for CCR5Δ32 in RA, albeit substantially weaker than the effect first reported.

  6. Chimeric plant virus particles administered nasally or orally induce systemic and mucosal immune responses in mice

    DEFF Research Database (Denmark)

    Brennan, F.R.; Bellaby, T.; Helliwell, S.M.

    1999-01-01

    The humoral immune responses to the D2 peptide of fibronectin-binding protein B (FnBP) of Staphylococcus aureus, expressed on the plant virus cowpea mosaic virus (CPMV), were evaluated after mucosal delivery to mice. Intranasal immunization of these chimeric virus particles (CVPs), either alone...

  7. An avirulent chimeric Pestivirus with altered cell tropism protects pigs against lethal infection with classical swine fever virus

    International Nuclear Information System (INIS)

    Reimann, Ilona; Depner, Klaus; Trapp, Sascha; Beer, Martin

    2004-01-01

    A chimeric Pestivirus was constructed using an infectious cDNA clone of bovine viral diarrhea virus (BVDV) [J. Virol. 70 (1996) 8606]. After deletion of the envelope protein E2-encoding region, the respective sequence of classical swine fever virus (CSFV) strain Alfort 187 was inserted in-frame resulting in plasmid pA/CP7 E 2alf. After transfection of in vitro-transcribed CP7 E 2alf RNA, autonomous replication of chimeric RNA in bovine and porcine cell cultures was observed. Efficient growth of chimeric CP7 E 2alf virus, however, could only be demonstrated on porcine cells, and in contrast to the parental BVDV strain CP7, CP7 E 2alf only inefficiently infected and propagated in bovine cells. The virulence, immunogenicity, and 'marker vaccine' properties of the generated chimeric CP7 E 2alf virus were determined in an animal experiment using 27 pigs. After intramuscular inoculation of 1 x 10 7 TCID 50 , CP7 E 2alf proved to be completely avirulent, and neither viremia nor virus transmission to contact animals was observed; however, CSFV-specific neutralizing antibodies were detected from day 11 after inoculation. In addition, sera from all animals reacted positive in an E2-specific CSFV-antibody ELISA, but were negative for CSFV-E RNS -specific antibodies as determined with a CSFV marker ELISA. After challenge infection with highly virulent CSFV strain Eystrup, pigs immunized with CP7 E 2alf were fully protected against clinical signs of CSFV infection, viremia, and shedding of challenge virus, and almost all animals scored positive in a CSFV marker ELISA. From our results, we conclude that chimeric CP7 E 2alf may not only serve as a tool for a better understanding of Pestivirus attachment, entry, and assembly, but also represents an innocuous and efficacious modified live CSFV 'marker vaccine'

  8. The CCL3L1-CCR5 genotype influences the development of AIDS, but not HIV susceptibility or the response to HAART

    Energy Technology Data Exchange (ETDEWEB)

    Bhattacharya, Tanmoy [Los Alamos National Laboratory; Stanton, Jennifer [NORTHWESTERN UNIV; Kim, Eun - Young [NORTHWESTERN UNIV; Kunstman, Kevin [NORTHWESTERN UNIV; Phair, John [NORTHWESTERN UNIV; Jacobson, Lisa P [JOHNS HOPKINS UNIV; Wolinsky, Steven M [NORTHWESTERN UNIV

    2008-01-01

    A selective advantage against infectious diseases such as HIV/AIDS is associated with differences in the genes relevant to immunity and virus replication. The CC chemokine receptor 5 (CCR5), the principal coreceptor for HIV, and its chemokine ligands, including CCL3L1, influences the CD4+ target cells susceptibility to infection. The CCL3L1 gene is in a region of segmental duplication on the q-arm of human chromosome 17. Increased numbers of CCL3L1 gene copies that affect the gene expression phenotype might have substantial protective effects. Here we show that the population-specific CCL3L1 gene copy number and the CCR5 {Delta}32 protein-inactivating deletion that categorizes the CCL3L1-CCR5 genotype do not influence HIV/AIDS susceptibility or the robustness of immune recovery after the initiation of highly active antiretroviral therapy (HAART).

  9. A Simplified Technique for Evaluating Human "CCR5" Genetic Polymorphism

    Science.gov (United States)

    Falteisek, Lukáš; Cerný, Jan; Janštová, Vanda

    2013-01-01

    To involve students in thinking about the problem of AIDS (which is important in the view of nondecreasing infection rates), we established a practical lab using a simplified adaptation of Thomas's (2004) method to determine the polymorphism of HIV co-receptor CCR5 from students' own epithelial cells. CCR5 is a receptor involved in inflammatory…

  10. Targeted induction of interferon-λ in humanized chimeric mouse liver abrogates hepatotropic virus infection.

    Science.gov (United States)

    Nakagawa, Shin-ichiro; Hirata, Yuichi; Kameyama, Takeshi; Tokunaga, Yuko; Nishito, Yasumasa; Hirabayashi, Kazuko; Yano, Junichi; Ochiya, Takahiro; Tateno, Chise; Tanaka, Yasuhito; Mizokami, Masashi; Tsukiyama-Kohara, Kyoko; Inoue, Kazuaki; Yoshiba, Makoto; Takaoka, Akinori; Kohara, Michinori

    2013-01-01

    The interferon (IFN) system plays a critical role in innate antiviral response. We presume that targeted induction of IFN in human liver shows robust antiviral effects on hepatitis C virus (HCV) and hepatitis B virus (HBV). This study used chimeric mice harboring humanized livers and infected with HCV or HBV. This mouse model permitted simultaneous analysis of immune responses by human and mouse hepatocytes in the same liver and exploration of the mechanism of antiviral effect against these viruses. Targeted expression of IFN was induced by treating the animals with a complex comprising a hepatotropic cationic liposome and a synthetic double-stranded RNA analog, pIC (LIC-pIC). Viral replication, IFN gene expression, IFN protein production, and IFN antiviral activity were analyzed (for type I, II and III IFNs) in the livers and sera of these humanized chimeric mice. Following treatment with LIC-pIC, the humanized livers of chimeric mice exhibited increased expression (at the mRNA and protein level) of human IFN-λs, resulting in strong antiviral effect on HBV and HCV. Similar increases were not seen for human IFN-α or IFN-β in these animals. Strong induction of IFN-λs by LIC-pIC occurred only in human hepatocytes, and not in mouse hepatocytes nor in human cell lines derived from other (non-hepatic) tissues. LIC-pIC-induced IFN-λ production was mediated by the immune sensor adaptor molecules mitochondrial antiviral signaling protein (MAVS) and Toll/IL-1R domain-containing adaptor molecule-1 (TICAM-1), suggesting dual recognition of LIC-pIC by both sensor adaptor pathways. These findings demonstrate that the expression and function of various IFNs differ depending on the animal species and tissues under investigation. Chimeric mice harboring humanized livers demonstrate that IFN-λs play an important role in the defense against human hepatic virus infection.

  11. Virulence, immunogenicity and vaccine properties of a novel chimeric pestivirus

    DEFF Research Database (Denmark)

    Rasmussen, Thomas Bruun; Uttenthal, Åse; Reimann, Ilona

    2007-01-01

    A chimeric pestivirus of border disease virus Gifhorn and bovine viral diarrhea virus CP7 (Meyers et al., 1996) was constructed. Virulence, immunogenicity and vaccine properties of the chimeric virus were studied in a vaccination–challenge experiment in pigs. The chimeric virus proved...... to be avirulent and neither chimeric virus nor viral RNA was detected in serum after vaccination. The safety of the vaccine was tested by horizontal transmission to sentinel pigs, which remained uninfected. The vaccine efficacy was examined by challenge infection with classical swine fever virus (CSFV) Eystrup...

  12. Lentiviral Gag assembly analyzed through the functional characterization of chimeric simian immunodeficiency viruses expressing different domains of the feline immunodeficiency virus capsid protein.

    Directory of Open Access Journals (Sweden)

    María J Esteva

    Full Text Available To gain insight into the functional relationship between the capsid (CA domains of the Gag polyproteins of simian and feline immunodeficiency viruses (SIV and FIV, respectively, we constructed chimeric SIVs in which the CA-coding region was partially or totally replaced by the equivalent region of the FIV CA. The phenotypic characterization of the chimeras allowed us to group them into three categories: the chimeric viruses that, while being assembly-competent, exhibit a virion-associated unstable FIV CA; a second group represented only by the chimeric SIV carrying the N-terminal domain (NTD of the FIV CA which proved to be assembly-defective; and a third group constituted by the chimeric viruses that produce virions exhibiting a mature and stable FIV CA protein, and which incorporate the envelope glycoprotein and contain wild-type levels of viral genome RNA and reverse transcriptase. Further analysis of the latter group of chimeric SIVs demonstrated that they are non-infectious due to a post-entry impairment, such as uncoating of the viral core, reverse transcription or nuclear import of the preintegration complex. Furthermore, we show here that the carboxyl-terminus domain (CTD of the FIV CA has an intrinsic ability to dimerize in vitro and form high-molecular-weight oligomers, which, together with our finding that the FIV CA-CTD is sufficient to confer assembly competence to the resulting chimeric SIV Gag polyprotein, provides evidence that the CA-CTD exhibits more functional plasticity than the CA-NTD. Taken together, our results provide relevant information on the biological relationship between the CA proteins of primate and nonprimate lentiviruses.

  13. Engineering HIV-resistant human CD4+ T cells with CXCR4-specific zinc-finger nucleases.

    Directory of Open Access Journals (Sweden)

    Craig B Wilen

    2011-04-01

    Full Text Available HIV-1 entry requires the cell surface expression of CD4 and either the CCR5 or CXCR4 coreceptors on host cells. Individuals homozygous for the ccr5Δ32 polymorphism do not express CCR5 and are protected from infection by CCR5-tropic (R5 virus strains. As an approach to inactivating CCR5, we introduced CCR5-specific zinc-finger nucleases into human CD4+ T cells prior to adoptive transfer, but the need to protect cells from virus strains that use CXCR4 (X4 in place of or in addition to CCR5 (R5X4 remains. Here we describe engineering a pair of zinc finger nucleases that, when introduced into human T cells, efficiently disrupt cxcr4 by cleavage and error-prone non-homologous DNA end-joining. The resulting cells proliferated normally and were resistant to infection by X4-tropic HIV-1 strains. CXCR4 could also be inactivated in ccr5Δ32 CD4+ T cells, and we show that such cells were resistant to all strains of HIV-1 tested. Loss of CXCR4 also provided protection from X4 HIV-1 in a humanized mouse model, though this protection was lost over time due to the emergence of R5-tropic viral mutants. These data suggest that CXCR4-specific ZFNs may prove useful in establishing resistance to CXCR4-tropic HIV for autologous transplant in HIV-infected individuals.

  14. Elucidation of the CCR1- and CCR5-binding modes of MIP-1α by application of an NMR spectra reconstruction method to the transferred cross-saturation experiments

    Energy Technology Data Exchange (ETDEWEB)

    Yoshiura, Chie; Ueda, Takumi; Kofuku, Yutaka; Matsumoto, Masahiko; Okude, Junya; Kondo, Keita; Shiraishi, Yutaro; Shimada, Ichio, E-mail: shimada@iw-nmr.f.u-tokyo.ac.jp [The University of Tokyo, Graduate School of Pharmaceutical Sciences (Japan)

    2015-12-15

    C–C chemokine receptor 1 (CCR1) and CCR5 are involved in various inflammation and immune responses, and regulate the progression of the autoimmune diseases differently. However, the number of residues identified at the binding interface was not sufficient to clarify the differences in the CCR1- and CCR5-binding modes to MIP-1α, because the NMR measurement time for CCR1 and CCR5 samples was limited to 24 h, due to their low stability. Here we applied a recently developed NMR spectra reconstruction method, Conservation of experimental data in ANAlysis of FOuRier, to the amide-directed transferred cross-saturation experiments of chemokine receptors, CCR1 and CCR5, embedded in lipid bilayers of the reconstituted high density lipoprotein, and MIP-1α. Our experiments revealed that the residues on the N-loop and β-sheets of MIP-1α are close to both CCR1 and CCR5, and those in the C-terminal helix region are close to CCR5. These results suggest that the genetic influence of the single nucleotide polymorphisms of MIP-1α that accompany substitution of residues in the C-terminal helix region, E57 and V63, would provide clues toward elucidating how the CCR5–MIP-1α interaction affects the progress of autoimmune diseases.

  15. Chimeric exchange of coronavirus nsp5 proteases (3CLpro) identifies common and divergent regulatory determinants of protease activity.

    Science.gov (United States)

    Stobart, Christopher C; Sexton, Nicole R; Munjal, Havisha; Lu, Xiaotao; Molland, Katrina L; Tomar, Sakshi; Mesecar, Andrew D; Denison, Mark R

    2013-12-01

    Human coronaviruses (CoVs) such as severe acute respiratory syndrome CoV (SARS-CoV) and Middle East respiratory syndrome CoV (MERS-CoV) cause epidemics of severe human respiratory disease. A conserved step of CoV replication is the translation and processing of replicase polyproteins containing 16 nonstructural protein domains (nsp's 1 to 16). The CoV nsp5 protease (3CLpro; Mpro) processes nsp's at 11 cleavage sites and is essential for virus replication. CoV nsp5 has a conserved 3-domain structure and catalytic residues. However, the intra- and intermolecular determinants of nsp5 activity and their conservation across divergent CoVs are unknown, in part due to challenges in cultivating many human and zoonotic CoVs. To test for conservation of nsp5 structure-function determinants, we engineered chimeric betacoronavirus murine hepatitis virus (MHV) genomes encoding nsp5 proteases of human and bat alphacoronaviruses and betacoronaviruses. Exchange of nsp5 proteases from HCoV-HKU1 and HCoV-OC43, which share the same genogroup, genogroup 2a, with MHV, allowed for immediate viral recovery with efficient replication albeit with impaired fitness in direct competition with wild-type MHV. Introduction of MHV nsp5 temperature-sensitive mutations into chimeric HKU1 and OC43 nsp5 proteases resulted in clear differences in viability and temperature-sensitive phenotypes compared with MHV nsp5. These data indicate tight genetic linkage and coevolution between nsp5 protease and the genomic background and identify differences in intramolecular networks regulating nsp5 function. Our results also provide evidence that chimeric viruses within coronavirus genogroups can be used to test nsp5 determinants of function and inhibition in common isogenic backgrounds and cell types.

  16. High-throughput screening of effective siRNAs using luciferase-linked chimeric mRNA.

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    Shen Pang

    Full Text Available The use of siRNAs to knock down gene expression can potentially be an approach to treat various diseases. To avoid siRNA toxicity the less transcriptionally active H1 pol III promoter, rather than the U6 promoter, was proposed for siRNA expression. To identify highly efficacious siRNA sequences, extensive screening is required, since current computer programs may not render ideal results. Here, we used CCR5 gene silencing as a model to investigate a rapid and efficient screening approach. We constructed a chimeric luciferase-CCR5 gene for high-throughput screening of siRNA libraries. After screening approximately 900 shRNA clones, 12 siRNA sequences were identified. Sequence analysis demonstrated that most (11 of the 12 sequences of these siRNAs did not match those identified by available siRNA prediction algorithms. Significant inhibition of CCR5 in a T-lymphocyte cell line and primary T cells by these identified siRNAs was confirmed using the siRNA lentiviral vectors to infect these cells. The inhibition of CCR5 expression significantly protected cells from R5 HIV-1JRCSF infection. These results indicated that the high-throughput screening method allows efficient identification of siRNA sequences to inhibit the target genes at low levels of expression.

  17. Association of TGFβ1, TNFα, CCR2 and CCR5 gene polymorphisms in type-2 diabetes and renal insufficiency among Asian Indians

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    Gupta Arvind

    2007-04-01

    Full Text Available Abstract Background Cytokines play an important role in the development of diabetic chronic renal insufficiency (CRI. Transforming growth factor β1 (TGF β1 induces renal hypertrophy and fibrosis, and cytokines like tumor necrosis factor-alpha (TNFα, chemoattractant protein-1 (MCP-1, and regulated upon activation and normal T cell expressed and secreted (RANTES mediate macrophage infiltration into kidney. Over expression of these chemokines leads to glomerulosclerosis and interstitial fibrosis. The effect of MCP-1 and RANTES on kidney is conferred by their receptors i.e., chemokine receptor (CCR-2 and CCR-5 respectively. We tested association of nine single nucleotide polymorphisms (SNPs from TGFβ1, TNFα, CCR2 and CCR5 genes among individuals with type-2 diabetes with and without renal insufficiency. Methods Type-2 diabetes subjects with chronic renal insufficiency (serum creatinine ≥ 3.0 mg/dl constituted the cases, and matched individuals with diabetes of duration ≥ 10 years and normoalbuminuria were evaluated as controls from four centres in India. Allelic and genotypic contributions of nine SNPs from TGFβ1, TNFα, CCR2 and CCR5 genes to diabetic CRI were tested by computing odds ratio (OR and 95% confidence intervals (CI. Sub-analysis of CRI cases diabetic retinopathy status as dependent variable and SNP genotypes as independent variable in a univariate logistic regression was also performed. Results SNPs Tyr81His and Thr263Ile in TGF β1 gene were monomorphic, and Arg25Pro in TGF β1 gene and Δ32 polymorphism in CCR5 gene were minor variants (minor allele frequency A SNP of CCR5 gene has been observed and the allele 59029A seems to confer predisposition to development of diabetic CRI (OR 1.39; CI 1.04–1.84. In CRI subjects a compound group of genotypes "GA and AA" of SNP G>A -800 was found to confer predisposition for proliferative retinopathy (OR 3.03; CI 1.08–8.50, p = 0.035. Conclusion Of the various cytokine gene

  18. CCR5 delta32, matrix metalloproteinase-9 and disease activity in multiple sclerosis

    DEFF Research Database (Denmark)

    Sellebjerg, Finn; Madsen, Hans O; Jensen, Claus V

    2000-01-01

    Chemokines and matrix metalloproteinases (MMPs) appear to be crucial in leukocyte recruitment to the central nervous system in multiple sclerosis (MS). CCR5 delta32, a truncated allele of the CC chemokine receptor CCR5 gene encoding a non-functional receptor, did not confer protection from MS. CCR5...... delta32 was, however, associated with a lower risk of recurrent clinical disease activity. High CSF levels of MMP-9 activity were also associated with recurrent disease activity. These results directly link intrathecal inflammation to disease activity in patients with MS, suggesting that treatments...... targeting CCR5 or treatment with MMP inhibitors may attenuate disease activity in MS...

  19. Deficient Fas expression by CD4+ CCR5+ T cells in multiple sclerosis

    DEFF Research Database (Denmark)

    Julià, Eva; Montalban, Xavier; Al-Zayat, Hammad

    2006-01-01

    OBJECTIVE: To evaluate whether T cells expressing CCR5 and CXCR3 from multiple sclerosis (MS) patients are more resistant to apoptosis. METHODS: Expression of CD69, TNF-R1, Fas, FasL, bcl-2, and bax was investigated in 41 MS patients and 12 healthy controls by flow cytometry in CD4+ and CD8+ T...... cells expressing CCR5 and CXCR3. RESULTS: In MS patients, the percentage of CD69 was increased and Fas expression decreased in CD4+ CCR5+ T cells. INTERPRETATION: The lower Fas expression in activated CD4+ CCR5+ T cells might contribute to disease pathogenesis by prolonging cell survival and favoring...

  20. Development and characterization of novel chimeric monoclonal antibodies for broad spectrum neutralization of rabies virus.

    Directory of Open Access Journals (Sweden)

    Pan Kyeom Kim

    Full Text Available Current post-exposure prophylaxis for rabies virus infection has several limitations in terms of supply, cost, safety, and efficacy. Attempts to replace human or equine rabies immune globulins (HRIG or ERIG have been made by several companies and institutes. We developed potent monoclonal antibodies to neutralize a broad spectrum of rabies viruses by screening hybridomas received from the U.S. Centers for Disease Control and Prevention (CDC. Two kinds of chimeric human antibodies (chimeric #7 and #17 were constructed by cloning the variable regions from selected hybridomas and the constant region of a human antibody. Two antibodies were bound to antigenic site III and I/IV, respectively, and were able to neutralize 51 field isolates of rabies virus that were isolated at different times and places such as Asia, Africa, North America, South America, and Australia. These two antibodies neutralize rabies viruses with high efficacy in an in vivo test using Syrian hamster and mouse models and show low risk for adverse immunogenicity.

  1. Development and characterization of novel chimeric monoclonal antibodies for broad spectrum neutralization of rabies virus.

    Science.gov (United States)

    Kim, Pan Kyeom; Keum, Sun Ju; Osinubi, Modupe O V; Franka, Richard; Shin, Ji Young; Park, Sang Tae; Kim, Man Su; Park, Mi Jung; Lee, Soo Young; Carson, William; Greenberg, Lauren; Yu, Pengcheng; Tao, Xiaoyan; Lihua, Wang; Tang, Qing; Liang, Guodong; Shampur, Madhusdana; Rupprecht, Charles E; Chang, Shin Jae

    2017-01-01

    Current post-exposure prophylaxis for rabies virus infection has several limitations in terms of supply, cost, safety, and efficacy. Attempts to replace human or equine rabies immune globulins (HRIG or ERIG) have been made by several companies and institutes. We developed potent monoclonal antibodies to neutralize a broad spectrum of rabies viruses by screening hybridomas received from the U.S. Centers for Disease Control and Prevention (CDC). Two kinds of chimeric human antibodies (chimeric #7 and #17) were constructed by cloning the variable regions from selected hybridomas and the constant region of a human antibody. Two antibodies were bound to antigenic site III and I/IV, respectively, and were able to neutralize 51 field isolates of rabies virus that were isolated at different times and places such as Asia, Africa, North America, South America, and Australia. These two antibodies neutralize rabies viruses with high efficacy in an in vivo test using Syrian hamster and mouse models and show low risk for adverse immunogenicity.

  2. Chimeric infectious DNA clones, chimeric porcine circoviruses and uses thereof

    OpenAIRE

    2011-01-01

    The present invention relates to infectious DNA clones, infectious chimeric DNA clones of porcine circovirus (PCV), vaccines and means of protecting pigs against viral infection or postweaning multisystemic wasting syndrome (PMWS) caused by PCV2. The new chimeric infectious DNA clone and its derived, avirulent chimeric virus are constructed from the nonpathogenic PCV1 in which the immunogenic ORF gene of the pathogenic PCV2 replaces a gene of the nonpathogenic PCV1, preferably in the same pos...

  3. The discovery of tropane-derived CCR5 receptor antagonists.

    Science.gov (United States)

    Armour, Duncan R; de Groot, Marcel J; Price, David A; Stammen, Blanda L C; Wood, Anthony; Perros, Manos; Burt, Catherine

    2006-04-01

    The development of compound 1, a piperidine-based CCR5 receptor antagonist with Type I CYP2D6 inhibition, into the tropane-derived analogue 5, is described. This compound, which is devoid of CYP2D6 liabilities, is a highly potent ligand for the CCR5 receptor and has broad-spectrum activity against a range of clinically relevant HIV isolates. The identification of human ether a-go-go-related gene channel inhibition within this series is described and the potential for QTc interval prolongation discussed. Furthermore, structure activity relationship (SAR) around the piperidine moiety is also described.

  4. Inhibition of HIV-1 infection by synthetic peptides derived CCR5 fragments

    International Nuclear Information System (INIS)

    Imai, Masaki; Baranyi, Lajos; Okada, Noriko; Okada, Hidechika

    2007-01-01

    HIV-1 infection requires interaction of viral envelope protein gp160 with CD4 and a chemokine receptor, CCR5 or CXCR4 as entry coreceptor. We designed HIV-inhibitory peptides targeted to CCR5 using a novel computer program (ANTIS), which searched all possible sense-antisense amino acid pairs between proteins. Seven AHBs were found in CCR5 receptor. All AHB peptides were synthesized and tested for their ability to prevent HIV-1 infection to human T cells. A peptide fragment (LC5) which is a part of the CCR5 receptor corresponding to the loop between the fifth and sixth transmembrane regions (amino acids 222-240) proved to inhibit HIV-1 IIIB infection of MT-4 cells. Interaction of these antisense peptides could be involved in sustaining HIV-1 infectivity. LC5 effectively indicated dose-dependent manner, and the suppression was enhanced additively by T20 peptide, which inhibits infection in vitro by disrupting the gp41 conformational changes necessary for membrane fusion. Thus, these results indicate that CCR5-derived AHB peptides could provide a useful tool to define the mechanism(s) of HIV infection, and may provide insight which will contribute to the development of an anti-HIV-1 reagent

  5. Adverse effect of the CCR5 promoter -2459A allele on HIV-1 disease progression

    DEFF Research Database (Denmark)

    Knudsen, T B; Kristiansen, T B; Katzenstein, T L

    2001-01-01

    /G transition that has been discovered recently, have also been shown to influence HIV progression. Since genetic linkages make these polymorphisms interdependent variables, the aim of the present study was to isolate and evaluate the effect on HIV disease progression for each of these mutations independently......HIV positive individuals heterozygous for a 32 basepair deletion in the CCR5 encoding gene (CCR5 Delta32) have a reduced number of CCR5 receptors on the cell surface and a slower progression towards AIDS and death. Other human polymorphisms, such as the CCR2 64I and the CCR5 promoter -2459 A...

  6. Chimeric Hemagglutinin Constructs Induce Broad Protection against Influenza B Virus Challenge in the Mouse Model.

    Science.gov (United States)

    Ermler, Megan E; Kirkpatrick, Ericka; Sun, Weina; Hai, Rong; Amanat, Fatima; Chromikova, Veronika; Palese, Peter; Krammer, Florian

    2017-06-15

    Seasonal influenza virus epidemics represent a significant public health burden. Approximately 25% of all influenza virus infections are caused by type B viruses, and these infections can be severe, especially in children. Current influenza virus vaccines are an effective prophylaxis against infection but are impacted by rapid antigenic drift, which can lead to mismatches between vaccine strains and circulating strains. Here, we describe a broadly protective vaccine candidate based on chimeric hemagglutinins, consisting of globular head domains from exotic influenza A viruses and stalk domains from influenza B viruses. Sequential vaccination with these constructs in mice leads to the induction of broadly reactive antibodies that bind to the conserved stalk domain of influenza B virus hemagglutinin. Vaccinated mice are protected from lethal challenge with diverse influenza B viruses. Results from serum transfer experiments and antibody-dependent cell-mediated cytotoxicity (ADCC) assays indicate that this protection is antibody mediated and based on Fc effector functions. The present data suggest that chimeric hemagglutinin-based vaccination is a viable strategy to broadly protect against influenza B virus infection. IMPORTANCE While current influenza virus vaccines are effective, they are affected by mismatches between vaccine strains and circulating strains. Furthermore, the antiviral drug oseltamivir is less effective for treating influenza B virus infections than for treating influenza A virus infections. A vaccine that induces broad and long-lasting protection against influenza B viruses is therefore urgently needed. Copyright © 2017 American Society for Microbiology.

  7. Could FIV zoonosis responsible of the breakdown of the pathocenosis which has reduced the European CCR5-Delta32 allele frequencies?

    Science.gov (United States)

    Faure, Eric

    2008-01-01

    Background In Europe, the north-south downhill cline frequency of the chemokine receptor CCR5 allele with a 32-bp deletion (CCR5-Δ32) raises interesting questions for evolutionary biologists. We had suggested first that, in the past, the European colonizers, principally Romans, might have been instrumental of a progressively decrease of the frequencies southwards. Indeed, statistical analyses suggested strong negative correlations between the allele frequency and historical parameters including the colonization dates by Mediterranean civilisations. The gene flows from colonizers to native populations were extremely low but colonizers are responsible of the spread of several diseases suggesting that the dissemination of parasites in naive populations could have induced a breakdown rupture of the fragile pathocenosis changing the balance among diseases. The new equilibrium state has been reached through a negative selection of the null allele. Results Most of the human diseases are zoonoses and cat might have been instrumental in the decrease of the allele frequency, because its diffusion through Europe was a gradual process, due principally to Romans; and that several cat zoonoses could be transmitted to man. The possible implication of a feline lentivirus (FIV) which does not use CCR5 as co-receptor is discussed. This virus can infect primate cells in vitro and induces clinical signs in macaque. Moreover, most of the historical regions with null or low frequency of CCR5-Δ32 allele coincide with historical range of the wild felid species which harbor species-specific FIVs. Conclusion We proposed the hypothesis that the actual European CCR5 allelic frequencies are the result of a negative selection due to a disease spreading. A cat zoonosis, could be the most plausible hypothesis. Future studies could provide if CCR5 can play an antimicrobial role in FIV pathogenesis. Moreover, studies of ancient DNA could provide more evidences regarding the implications of

  8. A novel self-replicating chimeric lentivirus-like particle.

    Science.gov (United States)

    Jurgens, Christy K; Young, Kelly R; Madden, Victoria J; Johnson, Philip R; Johnston, Robert E

    2012-01-01

    Successful live attenuated vaccines mimic natural exposure to pathogens without causing disease and have been successful against several viruses. However, safety concerns prevent the development of attenuated human immunodeficiency virus (HIV) as a vaccine candidate. If a safe, replicating virus vaccine could be developed, it might have the potential to offer significant protection against HIV infection and disease. Described here is the development of a novel self-replicating chimeric virus vaccine candidate that is designed to provide natural exposure to a lentivirus-like particle and to incorporate the properties of a live attenuated virus vaccine without the inherent safety issues associated with attenuated lentiviruses. The genome from the alphavirus Venezuelan equine encephalitis virus (VEE) was modified to express SHIV89.6P genes encoding the structural proteins Gag and Env. Expression of Gag and Env from VEE RNA in primate cells led to the assembly of particles that morphologically and functionally resembled lentivirus virions and that incorporated alphavirus RNA. Infection of CD4⁺ cells with chimeric lentivirus-like particles was specific and productive, resulting in RNA replication, expression of Gag and Env, and generation of progeny chimeric particles. Further genome modifications designed to enhance encapsidation of the chimeric virus genome and to express an attenuated simian immunodeficiency virus (SIV) protease for particle maturation improved the ability of chimeric lentivirus-like particles to propagate in cell culture. This study provides proof of concept for the feasibility of creating chimeric virus genomes that express lentivirus structural proteins and assemble into infectious particles for presentation of lentivirus immunogens in their native and functional conformation.

  9. Targeted induction of interferon-λ in humanized chimeric mouse liver abrogates hepatotropic virus infection.

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    Shin-ichiro Nakagawa

    Full Text Available BACKGROUND & AIMS: The interferon (IFN system plays a critical role in innate antiviral response. We presume that targeted induction of IFN in human liver shows robust antiviral effects on hepatitis C virus (HCV and hepatitis B virus (HBV. METHODS: This study used chimeric mice harboring humanized livers and infected with HCV or HBV. This mouse model permitted simultaneous analysis of immune responses by human and mouse hepatocytes in the same liver and exploration of the mechanism of antiviral effect against these viruses. Targeted expression of IFN was induced by treating the animals with a complex comprising a hepatotropic cationic liposome and a synthetic double-stranded RNA analog, pIC (LIC-pIC. Viral replication, IFN gene expression, IFN protein production, and IFN antiviral activity were analyzed (for type I, II and III IFNs in the livers and sera of these humanized chimeric mice. RESULTS: Following treatment with LIC-pIC, the humanized livers of chimeric mice exhibited increased expression (at the mRNA and protein level of human IFN-λs, resulting in strong antiviral effect on HBV and HCV. Similar increases were not seen for human IFN-α or IFN-β in these animals. Strong induction of IFN-λs by LIC-pIC occurred only in human hepatocytes, and not in mouse hepatocytes nor in human cell lines derived from other (non-hepatic tissues. LIC-pIC-induced IFN-λ production was mediated by the immune sensor adaptor molecules mitochondrial antiviral signaling protein (MAVS and Toll/IL-1R domain-containing adaptor molecule-1 (TICAM-1, suggesting dual recognition of LIC-pIC by both sensor adaptor pathways. CONCLUSIONS: These findings demonstrate that the expression and function of various IFNs differ depending on the animal species and tissues under investigation. Chimeric mice harboring humanized livers demonstrate that IFN-λs play an important role in the defense against human hepatic virus infection.

  10. CCR5 receptor antagonists: discovery and SAR study of guanylhydrazone derivatives.

    Science.gov (United States)

    Wei, Robert G; Arnaiz, Damian O; Chou, Yuo-Ling; Davey, Dave; Dunning, Laura; Lee, Wheeseong; Lu, Shou-Fu; Onuffer, James; Ye, Bin; Phillips, Gary

    2007-01-01

    High throughput screening (HTS) led to the identification of the guanylhydrazone of 2-(4-chlorobenzyloxy)-5-bromobenzaldehyde as a CCR5 receptor antagonist. Initial modifications of the guanylhydrazone series indicated that substitution of the benzyl group at the para-position was well tolerated. Substitution at the 5-position of the central phenyl ring was critical for potency. Replacement of the guanylhydrazone group led to the discovery of a novel series of CCR5 antagonists.

  11. Modification of a loop sequence between α-helices 6 and 7 of virus capsid (CA protein in a human immunodeficiency virus type 1 (HIV-1 derivative that has simian immunodeficiency virus (SIVmac239 vif and CA α-helices 4 and 5 loop improves replication in cynomolgus monkey cells

    Directory of Open Access Journals (Sweden)

    Adachi Akio

    2009-08-01

    Full Text Available Abstract Background Human immunodeficiency virus type 1 (HIV-1 productively infects only humans and chimpanzees but not cynomolgus or rhesus monkeys while simian immunodeficiency virus isolated from macaque (SIVmac readily establishes infection in those monkeys. Several HIV-1 and SIVmac chimeric viruses have been constructed in order to develop an animal model for HIV-1 infection. Construction of an HIV-1 derivative which contains sequences of a SIVmac239 loop between α-helices 4 and 5 (L4/5 of capsid protein (CA and the entire SIVmac239 vif gene was previously reported. Although this chimeric virus could grow in cynomolgus monkey cells, it did so much more slowly than did SIVmac. It was also reported that intrinsic TRIM5α restricts the post-entry step of HIV-1 replication in rhesus and cynomolgus monkey cells, and we previously demonstrated that a single amino acid in a loop between α-helices 6 and 7 (L6/7 of HIV type 2 (HIV-2 CA determines the susceptibility of HIV-2 to cynomolgus monkey TRIM5α. Results In the study presented here, we replaced L6/7 of HIV-1 CA in addition to L4/5 and vif with the corresponding segments of SIVmac. The resultant HIV-1 derivatives showed enhanced replication capability in established T cell lines as well as in CD8+ cell-depleted primary peripheral blood mononuclear cells from cynomolgus monkey. Compared with the wild type HIV-1 particles, the viral particles produced from a chimeric HIV-1 genome with those two SIVmac loops were less able to saturate the intrinsic restriction in rhesus monkey cells. Conclusion We have succeeded in making the replication of simian-tropic HIV-1 in cynomolgus monkey cells more efficient by introducing into HIV-1 the L6/7 CA loop from SIVmac. It would be of interest to determine whether HIV-1 derivatives with SIVmac CA L4/5 and L6/7 can establish infection of cynomolgus monkeys in vivo.

  12. CCR5 signalling, but not DARC or D6 regulatory, chemokine receptors are targeted by herpesvirus U83A chemokine which delays receptor internalisation via diversion to a caveolin-linked pathway.

    Science.gov (United States)

    Catusse, Julie; Clark, David J; Gompels, Ursula A

    2009-07-30

    Herpesviruses have evolved chemokines and chemokine receptors, which modulate the recruitment of human leukocytes during the inflammatory response to infection. Early post-infection, human herpesvirus 6A (HHV-6A) infected cells express the chemokine receptor U51A and chemokine U83A which have complementary effects in subverting the CC-chemokine family thereby controlling anti-viral leukocyte recruitment. Here we show that, to potentiate this activity, the viral chemokine can also avoid clearance by scavenger chemokine receptors, DARC and D6, which normally regulate an inflammatory response. Conversely, U83A delays internalisation of its signalling target receptor CCR5 with diversion to caveolin rich membrane domains. This mechanism can redirect displaced human chemokines to DARC and D6 for clearance of the anti-viral inflammatory response, leaving the viral chemokine unchecked. Cell models for competitive binding assays were established using radiolabeled human chemokines and cold U83A on CCR5, DARC or D6 expressing cells. Flow cytometry was used to assess specific chemotaxis of CCR5 bearing cells to U83A, and internalisation of CCR5 specific chemokine CCL4 after stimulation with U83A. Internalisation analyses were supported by confocal microscopy of internalisation and co-localisation of CCR5 with caveosome marker caveolin-1, after virus or human chemokine stimulation. U83A displaced efficiently human chemokines from CCR5, with a high affinity of 0.01nM, but not from DARC or D6. Signalling via CCR5 resulted in specific chemoattraction of primary human leukocytes bearing CCR5. However, U83A effective binding and signalling to CCR5 resulted in delayed internalisation and recycling up to 2 hours in the absence of continual re-stimulation. This resulted in diversion to a delayed caveolin-linked pathway rather than the rapid clathrin mediated endocytosis previously shown with human chemokines CCL3 or CCL4. U83A diverts human chemokines from signalling, but not

  13. CCR5 signalling, but not DARC or D6 regulatory, chemokine receptors are targeted by herpesvirus U83A chemokine which delays receptor internalisation via diversion to a caveolin-linked pathway

    Directory of Open Access Journals (Sweden)

    Gompels Ursula A

    2009-07-01

    Full Text Available Abstract Background Herpesviruses have evolved chemokines and chemokine receptors, which modulate the recruitment of human leukocytes during the inflammatory response to infection. Early post-infection, human herpesvirus 6A (HHV-6A infected cells express the chemokine receptor U51A and chemokine U83A which have complementary effects in subverting the CC-chemokine family thereby controlling anti-viral leukocyte recruitment. Here we show that, to potentiate this activity, the viral chemokine can also avoid clearance by scavenger chemokine receptors, DARC and D6, which normally regulate an inflammatory response. Conversely, U83A delays internalisation of its signalling target receptor CCR5 with diversion to caveolin rich membrane domains. This mechanism can redirect displaced human chemokines to DARC and D6 for clearance of the anti-viral inflammatory response, leaving the viral chemokine unchecked. Methods Cell models for competitive binding assays were established using radiolabeled human chemokines and cold U83A on CCR5, DARC or D6 expressing cells. Flow cytometry was used to assess specific chemotaxis of CCR5 bearing cells to U83A, and internalisation of CCR5 specific chemokine CCL4 after stimulation with U83A. Internalisation analyses were supported by confocal microscopy of internalisation and co-localisation of CCR5 with caveosome marker caveolin-1, after virus or human chemokine stimulation. Results U83A displaced efficiently human chemokines from CCR5, with a high affinity of 0.01nM, but not from DARC or D6. Signalling via CCR5 resulted in specific chemoattraction of primary human leukocytes bearing CCR5. However, U83A effective binding and signalling to CCR5 resulted in delayed internalisation and recycling up to 2 hours in the absence of continual re-stimulation. This resulted in diversion to a delayed caveolin-linked pathway rather than the rapid clathrin mediated endocytosis previously shown with human chemokines CCL3 or CCL4

  14. The expression and genetic immunization of chimeric fragment of Hantaan virus M and S segments

    International Nuclear Information System (INIS)

    Zhang Fanglin; Wu Xingan; Luo Wen; Bai Wentao; Liu Yong; Yan Yan; Wang Haitao; Xu Zhikai

    2007-01-01

    Hemorrhagic fever with renal syndrome (HFRS), which is characterized by severe symptoms and high mortality, is caused by hantavirus. There are still no effective prophylactic vaccines directed to HFRS until now. In this research, we fused expressed G2 fragment of M segment and 0.7 kb fragment of S segment. We expect it could be a candidate vaccine. Chimeric gene G2S0.7 was first expressed in prokaryotic expression system pGEX-4T. After inducing expressed fusion proteins, GST-G2S0.7 was induced and its molecular weight was about 100 kDa. Meanwhile, the fusion protein kept the activity of its parental proteins. Further, BALB/c mice were vaccinated by the chimeric gene. ELISA, cell microculture neutralization test in vitro were used to detect the humoral immune response in immunized BALB/c mice. Lymphocyte proliferation assay was used to detect the cellular immune response. The results showed that the chimeric gene could simultaneously evoke specific antibody against nucleocapsid protein (NP) and glycoprotein (GP). And the immunized mice of every group elicited neutralizing antibodies with different titers. But the titers were low. Lymphocyte proliferation assay results showed that the stimulation indexes of splenocytes of chimeric gene to NP and GP were significantly higher than that of control. It suggested that the chimeric gene of Hantaan virus containing G2 fragment of M segment and 0.7 kb fragment of S segment could directly elicit specific anti-Hantaan virus humoral and cellular immune response in BALB/c mice

  15. CCL3L1 copy number, CCR5 genotype and susceptibility to tuberculosis

    OpenAIRE

    Carpenter, Danielle; Taype, Carmen; Goulding, Jon; Levin, Mike; Eley, Brian; Anderson, Suzanne; Shaw, Marie-Anne; Armour, John AL

    2014-01-01

    Background: Tuberculosis is a major infectious disease and functional studies have provided evidence that both the chemokine MIP-1α and its receptor CCR5 play a role in susceptibility to TB. Thus by measuring copy number variation of CCL3L1, one of the genes that encode MIP-1α, and genotyping a functional promoter polymorphism -2459A > G in CCR5 (rs1799987) we investigate the influence of MIP-1α and CCR5, independently and combined, in susceptibility to clinically active TB in three populatio...

  16. Peripheral Blood CCR4+CCR6+ and CXCR3+CCR6+ CD4+ T Cells Are Highly Permissive to HIV-1 Infection

    OpenAIRE

    Gosselin, Annie; Monteiro, Patricia; Chomont, Nicolas; Diaz-Griffero, Felipe; Said, Elias A.; Fonseca, Simone; Wacleche, Vanessa; El-Far, Mohamed; Boulassel, Mohamed-Rachid; Routy, Jean-Pierre; Sekaly, Rafick-Pierre; Ancuta, Petronela

    2009-01-01

    There is limited knowledge on the identity of primary CD4+ T cell subsets selectively targeted by HIV-1 in vivo. In this study, we established a link between HIV permissiveness, phenotype/homing potential, and lineage commitment in primary CD4+ T cells. CCR4+CCR6+, CCR4+CCR6−, CXCR3+CCR6+, and CXCR3+CCR6− T cells expressed cytokines and transcription factors specific for Th17, Th2, Th1Th17, and Th1 lineages, respectively. CCR4+CCR6+ and CXCR3+CCR6+ T cells expressed the HIV coreceptors CCR5 a...

  17. The chemokine receptor CCR5 Δ32 allele in natalizumab-treated multiple sclerosis

    DEFF Research Database (Denmark)

    Møller, M; Søndergaard, Helle B; Koch-Henriksen, N

    2014-01-01

    OBJECTIVE: The chemokine receptor CCR5 may be important for the recruitment of pathogenic T cells to the CNS in multiple sclerosis (MS). We hypothesized that this chemokine receptor might still be important for T-cell migration during treatment with anti-very late antigen (VLA)-4 antibody. We...... impact on the frequency of relapses 1 year prior to natalizumab treatment or during the first 48 weeks of treatment. The multiple sclerosis severity score (MSSS) was significantly lower at baseline in patients carrying CCR5 Δ32 (P = 0.031). CONCLUSIONS: CCR5 Δ32 is not associated with lower disease...

  18. Correlating HIV tropism with immunological response under combination antiretroviral therapy.

    Science.gov (United States)

    Bader, J; Schöni-Affolter, F; Böni, J; Gorgievski-Hrisoho, M; Martinetti, G; Battegay, M; Klimkait, T

    2016-09-01

    A significant percentage of patients infected with HIV-1 experience only suboptimal CD4 cell recovery while treated with combination therapy (cART). It is still unclear whether viral properties such as cell tropism play a major role in this incomplete immune response. This study therefore intended to follow the tropism evolution of the HIV-1 envelope during periods of suppressive cART. Viruses from two distinct patient groups, one with good and another one with poor CD4 recovery after 5 years of suppressive cART, were genotypically analysed for viral tropism at baseline and at the end of the study period. Patients with CCR5-tropic CC-motif chemokine receptor 5 viruses at baseline tended to maintain this tropism to the study end. Patients who had a CXCR4-tropic CXC-motif chemokine receptor 4 virus at baseline were overrepresented in the poor CD4 recovery group. Overall, however, the majority of patients presented with CCR5-tropic viruses at follow-up. Our data lend support to the hypothesis that tropism determination can be used as a parameter for disease progression even if analysed long before the establishment of a poorer immune response. Moreover, the lasting predominating CCR5-tropism during periods of full viral control suggests the involvement of cellular mechanisms that preferentially reduce CXCR4-tropic viruses during cART. © 2016 British HIV Association.

  19. Gating function of isoleucine-116 in TM-3 (position III:16/3.40) for the activity state of the CC-chemokine receptor 5 (CCR5)

    DEFF Research Database (Denmark)

    Steen, A; Sparre-Ulrich, A H; Thiele, Stefanie

    2014-01-01

    TM receptors - it is a leucine indicating an altered function. Here, we describe the significance of this position and its possible interaction with TM-3 for CCR5 activity. EXPERIMENTAL APPROACH: The effects of [L203F]-CCR5 in TM-5 (position V:13/5.47), [I116A]-CCR5 in TM-3 (III:16/3.40) and [L203F...... ) with a threefold increase in agonist potency. In silico, [I116A]-CCR5 switched χ1-angle in [L203F]-CCR5. Furthermore, [I116A]-CCR5 was constitutively active to a similar degree as [L203F]-CCR5. Tyr(244) in TM-6 (VI:09/6.44) moved towards TM-5 in silico, consistent with its previously shown function for CCR5...... in the active state, a mechanism proposed previously for the β2 -adrenoceptor. The results provide an understanding of chemokine receptor function and thereby information for the development of biased and non-biased antagonists and inverse agonists....

  20. Novel chimeric foot-and-mouth disease virus-like particles harboring serotype O VP1 protect guinea pigs against challenge.

    Science.gov (United States)

    Li, Haitao; Li, Zhiyong; Xie, Yinli; Qin, Xiaodong; Qi, Xingcai; Sun, Peng; Bai, Xingwen; Ma, Youji; Zhang, Zhidong

    2016-02-01

    Foot-and-mouth disease is a highly contagious, acute viral disease of cloven-hoofed animal species causing severe economic losses worldwide. Among the seven serotypes of foot-and-mouth disease virus (FMDV), serotype O is predominant, but its viral capsid is more acid sensitive than other serotypes, making it more difficult to produce empty serotype O VLPs in the low pH insect hemolymph. Therefore, a novel chimeric virus-like particle (VLP)-based candidate vaccine for serotype O FMDV was developed and characterized in the present study. The chimeric VLPs were composed of antigenic VP1 from serotype O and segments of viral capsid proteins from serotype Asia1. These VLPs elicited significantly higher FMDV-specific antibody levels in immunized mice than did the inactivated vaccine. Furthermore, the chimeric VLPs protected guinea pigs from FMDV challenge with an efficacy similar to that of the inactivated vaccine. These results suggest that chimeric VLPs have the potential for use in vaccines against serotype O FMDV infection. Copyright © 2015 Elsevier B.V. All rights reserved.

  1. Chimeric Lyssavirus Glycoproteins with Increased Immunological Potential

    Science.gov (United States)

    Jallet, Corinne; Jacob, Yves; Bahloul, Chokri; Drings, Astrid; Desmezieres, Emmanuel; Tordo, Noël; Perrin, Pierre

    1999-01-01

    The rabies virus glycoprotein molecule (G) can be divided into two parts separated by a flexible hinge: the NH2 half (site II part) containing antigenic site II up to the linear region (amino acids [aa] 253 to 275 encompassing epitope VI [aa 264]) and the COOH half (site III part) containing antigenic site III and the transmembrane and cytoplasmic domains. The structural and immunological roles of each part were investigated by cell transfection and mouse DNA-based immunization with homogeneous and chimeric G genes formed by fusion of the site II part of one genotype (GT) with the site III part of the same or another GT. Various site II-site III combinations between G genes of PV (Pasteur virus strain) rabies (GT1), Mokola (GT3), and EBL1 (European bat lyssavirus 1 [GT5]) viruses were tested. Plasmids pGPV-PV, pGMok-Mok, pGMok-PV, and pGEBL1-PV induced transient expression of correctly transported and folded antigens in neuroblastoma cells and virus-neutralizing antibodies against parental viruses in mice, whereas, pG-PVIII (site III part only) and pGPV-Mok did not. The site III part of PV (GT1) was a strong inducer of T helper cells and was very effective at presenting the site II part of various GTs. Both parts are required for correct folding and transport of chimeric G proteins which have a strong potential value for immunological studies and development of multivalent vaccines. Chimeric plasmid pGEBL1-PV broadens the spectrum of protection against European lyssavirus genotypes (GT1, GT5, and GT6). PMID:9847325

  2. Molecular anatomy of CCR5 engagement by physiologic and viral chemokines and HIV-1 envelope glycoproteins: differences in primary structural requirements for RANTES, MIP-1 alpha, and vMIP-II Binding.

    Science.gov (United States)

    Navenot, J M; Wang, Z X; Trent, J O; Murray, J L; Hu, Q X; DeLeeuw, L; Moore, P S; Chang, Y; Peiper, S C

    2001-11-09

    Molecular analysis of CCR5, the cardinal coreceptor for HIV-1 infection, has implicated the N-terminal extracellular domain (N-ter) and regions vicinal to the second extracellular loop (ECL2) in this activity. It was shown that residues in the N-ter are necessary for binding of the physiologic ligands, RANTES (CCL5) and MIP-1 alpha (CCL3). vMIP-II, encoded by the Kaposi's sarcoma-associated herpesvirus, is a high affinity CCR5 antagonist, but lacks efficacy as a coreceptor inhibitor. Therefore, we compared the mechanism for engagement by vMIP-II of CCR5 to its interaction with physiologic ligands. RANTES, MIP-1 alpha, and vMIP-II bound CCR5 at high affinity, but demonstrated partial cross-competition. Characterization of 15 CCR5 alanine scanning mutants of charged extracellular amino acids revealed that alteration of acidic residues in the distal N-ter abrogated binding of RANTES, MIP-1 alpha, and vMIP-II. Whereas mutation of residues in ECL2 of CCR5 dramatically reduced the binding of RANTES and MIP-1 alpha and their ability to induce signaling, interaction with vMIP-II was not altered by any mutation in the exoloops of the receptor. Paradoxically, monoclonal antibodies to N-ter epitopes did not block chemokine binding, but those mapped to ECL2 were effective inhibitors. A CCR5 chimera with the distal N-ter residues of CXCR2 bound MIP-1 alpha and vMIP-II with an affinity similar to that of the wild-type receptor. Engagement of CCR5 by vMIP-II, but not RANTES or MIP-1 alpha blocked the binding of monoclonal antibodies to the receptor, providing additional evidence for a distinct mechanism for viral chemokine binding. Analysis of the coreceptor activity of randomly generated mouse-human CCR5 chimeras implicated residues in ECL2 between H173 and V197 in this function. RANTES, but not vMIP-II blocked CCR5 M-tropic coreceptor activity in the fusion assay. The insensitivity of vMIP-II binding to mutations in ECL2 provides a potential rationale to its inefficiency as an

  3. Enhancement of mucosal immune responses by chimeric influenza HA/SHIV virus-like particles

    International Nuclear Information System (INIS)

    Guo Lizheng; Lu Xiaoyan; Kang, S.-M.; Chen Changyi; Compans, Richard W.; Yao Qizhi

    2003-01-01

    To enhance mucosal immune responses using simian/human immunodeficiency virus-like particles (SHIV VLPs), we have produced novel phenotypically mixed chimeric influenza HA/SHIV VLPs and used them to immunize C57BL/6J mice intranasally. Antibody and cytotoxic T-cell (CTL) responses as well as cytokine production in both systemic and mucosal sites were compared after immunization with SHIV VLPs or chimeric HA/SHIV VLPs. By using enzyme-linked immunosorbent assay (ELISA), the levels of serum IgG and mucosal IgA to the HIV envelope protein (Env) were found to be highest in the group immunized with chimeric HA/SHIV VLPs. Furthermore, the highest titer of serum neutralizing antibody against HIV Env was found with the group immunized with chimeric HA/SHIV VLPs. Analysis of the IgG1/IgG2a ratio indicated that a T H 1-oriented immune response resulted from these VLP immunizations. HA/SHIV VLP-immunized mice also showed significantly higher CTL responses than those observed in SHIV VLP-immunized mice. Moreover, a MHC class I restricted T-cell activation ELISPOT assay showed a mixed type of T H 1/T H 2 cytokines in the HA/SHIV VLP-immunized mice, indicating that the chimeric VLPs can enhance both humoral and cellular immune responses to the HIV Env protein at multiple mucosal and systemic sites. The results indicate that incorporation of influenza HA into heterotypic VLPs may be highly effective for targeting vaccines to mucosal surfaces

  4. Association between the CCR5 32-bp deletion allele and late onset of schizophrenia

    DEFF Research Database (Denmark)

    Rasmussen, Henrik Berg; Timm, Sally; Wang, August G

    2006-01-01

    OBJECTIVE: The 32-bp deletion allele in chemokine receptor CCR5 has been associated with several immune-mediated diseases and might be implicated in schizophrenia as well. METHOD: The authors genotyped DNA samples from 268 schizophrenia patients and 323 healthy subjects. Age at first admission...... of the deletion allele in the latter subgroup of patients. CONCLUSIONS: These findings suggest that the CCR5 32-bp deletion allele is a susceptibility factor for schizophrenia with late onset. Alternatively, the CCR5 32-bp deletion allele may act as a modifier by delaying the onset of schizophrenia without...

  5. Preferential susceptibility of Th9 and Th2 CD4+ T cells to X4-tropic HIV-1 infection.

    Science.gov (United States)

    Orlova-Fink, Nina; Chowdhury, Fatema Z; Sun, Xiaoming; Harrington, Sean; Rosenberg, Eric S; Yu, Xu G; Lichterfeld, Mathias

    2017-10-23

    The functional polarization of CD4 T cells determines their antimicrobial effector profile, but may also impact the susceptibility to infection with HIV-1. Here, we analyzed the susceptibility of CD4 T cells with different functional polarization to infection with X4 and R5-tropic HIV-1. CD4 T cells with a Th1, Th2, Th17, and Th9 polarization were subjected to in-vitro infection assays with X4, R5, or vesicular stomatitis virus-G protein-pseudotyped HIV-1. In addition, we sorted differentially polarized CD4 T-cell subsets from individuals treated with antiretroviral therapy and analyzed the tropism of viral env sequences. Th9-polarized CD4 T cells and, to a lesser extent, Th2-polarized CD4 T cells expressed higher surface levels of CXCR4, and are more permissive to X4-tropic infection in vitro. In contrast, Th1 and Th17 CD4 T cells exhibited stronger surface expression of CCR5, and were more susceptible to infection with R5-tropic viruses. Correspondingly, the distribution of X4-tropic viral sequences in antiretroviral therapy-treated HIV-1-infected patients was biased toward Th9/Th2 cells, whereas R5-tropic sequences were more frequently observed in Th17 cells. CD4 T-cell polarization is associated with a distinct susceptibility to X4 and R5-tropic HIV-1 infection.

  6. CCL3L1 copy number, CCR5 genotype and susceptibility to tuberculosis.

    Science.gov (United States)

    Carpenter, Danielle; Taype, Carmen; Goulding, Jon; Levin, Mike; Eley, Brian; Anderson, Suzanne; Shaw, Marie-Anne; Armour, John A L

    2014-01-09

    Tuberculosis is a major infectious disease and functional studies have provided evidence that both the chemokine MIP-1α and its receptor CCR5 play a role in susceptibility to TB. Thus by measuring copy number variation of CCL3L1, one of the genes that encode MIP-1α, and genotyping a functional promoter polymorphism -2459A > G in CCR5 (rs1799987) we investigate the influence of MIP-1α and CCR5, independently and combined, in susceptibility to clinically active TB in three populations, a Peruvian population (n = 1132), a !Xhosa population (n = 605) and a South African Coloured population (n = 221). The three populations include patients with clinically diagnosed pulmonary TB, as well as other, less prevalent forms of extrapulmonary TB. Copy number of CCL3L1 was measured using the paralogue ratio test and exhibited ranges between 0-6 copies per diploid genome (pdg) in Peru, between 0-12 pdg in !Xhosa samples and between 0-10 pdg in South African Coloured samples. The CCR5 promoter polymorphism was observed to differ significantly in allele frequency between populations (*A; Peru f = 0.67, !Xhosa f = 0.38, Coloured f = 0.48). The case-control association studies performed however find, surprisingly, no evidence for an influence of variation in genes coding for MIP-1α or CCR5 individually or together in susceptibility to clinically active TB in these populations.

  7. CCR5 gene polymorphism is a genetic risk factor for radiographic severity of rheumatoid arthritis.

    Science.gov (United States)

    Han, S W; Sa, K H; Kim, S I; Lee, S I; Park, Y W; Lee, S S; Yoo, W H; Soe, J S; Nam, E J; Lee, J; Park, J Y; Kang, Y M

    2012-11-01

    The chemokine receptor [C-C chemokine receptor 5 (CCR5)] is expressed on diverse immune effecter cells and has been implicated in the pathogenesis of rheumatoid arthritis (RA). This study sought to determine whether single-nucleotide polymorphisms (SNPs) in the CCR5 gene and their haplotypes were associated with susceptibility to and severity of RA. Three hundred fifty-seven patients with RA and 383 healthy unrelated controls were recruited. Using a pyrosequencing assay, we examined four polymorphisms -1118 CTAT(ins) (/del) (rs10577983), 303 A>G (rs1799987), 927 C>T (rs1800024), and 4838 G>T (rs1800874) of the CCR5 gene, which were distributed over the promoter region as well as the 5' and 3' untranslated regions. No significant difference in the genotype, allele, and haplotype frequencies of the four selected SNPs was observed between RA patients and controls. CCR5 polymorphisms of -1118 CTAT(del) (P = 0.012; corrected P = 0.048) and 303 A>G (P = 0.012; corrected P = 0.048) showed a significant association with radiographic severity in a recessive model, and, as a result of multivariate logistic regression analysis, were found to be an independent predictor of radiographic severity. When we separated the erosion score from the total Sharp score, the statistical significance of CCR5 polymorphisms showed an increase; -1118 CTAT(ins) (/del) (P = 0.007; corrected P = 0.028) and 303 A>G (P = 0.007; corrected P = 0.028). Neither SNPs nor haplotypes of the CCR5 gene showed a significant association with joint space narrowing score. These results indicate that genetic polymorphisms of CCR5 are an independent risk factor for radiographic severity denoted by modified Sharp score, particularly joint erosion in RA. © 2012 John Wiley & Sons A/S.

  8. Lack of CCR5 modifies glial phenotypes and population of the nigral dopaminergic neurons, but not MPTP-induced dopaminergic neurodegeneration.

    Science.gov (United States)

    Choi, Dong-Young; Lee, Myung Koo; Hong, Jin Tae

    2013-01-01

    Constitutive expression of C-C chemokine receptor (CCR) 5 has been detected in astrocytes, microglia and neurons, but its physiological roles in the central nervous system are obscure. The bidirectional interactions between neuron and glial cells through CCR5 and its ligands were thought to be crucial for maintaining normal neuronal activities. No study has described function of CCR5 in the dopaminergic neurodegeneration in Parkinson's disease. In order to examine effects of CCR5 on 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced dopaminergic neurodegeneration, we employed CCR5 wild type (WT) and knockout (KO) mice. Immunostainings for tyrosine hydroxylase (TH) exhibited that CCR5 KO mice had lower number of TH-positive neurons even in the absence of MPTP. Difference in MPTP (15mg/kg×4 times, 2hr interval)-mediated loss of TH-positive neurons was subtle between CCR5 WT and KO mice, but there was larger dopamine depletion, behavioral impairments and microglial activation in CCR5 deficient mice. Intriguingly, CCR5 KO brains contained higher immunoreactivity for monoamine oxidase (MAO) B which was mainly localized within astrocytes. In agreement with upregulation of MAO B, concentration of MPP+ was higher in the substantia nigra and striatum of CCR5 KO mice after MPTP injection. We found remarkable activation of p38 MAPK in CCR5 deficient mice, which positively regulates MAO B expression. These results indicate that CCR5 deficiency modifies the nigrostriatal dopaminergic neuronal system and bidirectional interaction between neurons and glial cells via CCR5 might be important for dopaminergic neuronal survival. Copyright © 2012 Elsevier Inc. All rights reserved.

  9. Autologous Hematopoietic Stem Cells transplantation and genetic modification of CCR5 m303/m303 mutant patient for HIV/AIDS.

    Science.gov (United States)

    Esmaeilzadeh, Abdolreza; Farshbaf, Alieh; Erfanmanesh, Maryam

    2015-03-01

    HIV and AIDS is one of the biggest challenges all over the world. There are an approximately 34 million people living with the virus, and a large number of them become infected each year. Although there are some antiviral drugs for HIV viral load reduction, they are not sufficient. There is no cure for AIDS. Nowadays natural resistance or immunity has absorbed attentions. Because in some HIV positive patients progression trend is slow or even they indicate resistance to AIDS. One of the most interesting approaches in this category is CCR5 gene. CCR5 is a main cc-chemokine co-receptor that facilitates HIV-1 entry to macrophage and CD4(+) T cells. To now, many polymorphisms have been known by CCR5 gene that produces a truncated protein with no function. So, HIV-1 could not entry to immune-cells and the body resistant to HIV/AIDS. Δ32/Δ32 and m303/m303 homozygotes are example of mutations that could create this resistance mechanism. There is a new treatment, such as Hematopoietic Stem Cell transplantation (HSCT) in Berlin and Boston patients for Δ32/Δ32 mutation. It could eliminate co-receptor antagonist and highly-active-anti retroviral therapy (HAART) drugs problems such as toxicity, low safety and side-effects. Now there, the aim of this hypothesis will be evaluation of a new mutation CCR5 m303/m303 as autologous HSCT. This novel hypothesis indicates that autologous HSCT for m303/m303 could be effective treatment for anyone HIV/AIDS affected patient worldwide. Copyright © 2015 Elsevier Ltd. All rights reserved.

  10. Lymphotropic Virions Affect Chemokine Receptor-Mediated Neural Signaling and Apoptosis: Implications for Human Immunodeficiency Virus Type 1-Associated Dementia

    Science.gov (United States)

    Zheng, Jialin; Ghorpade, Anuja; Niemann, Douglas; Cotter, Robin L.; Thylin, Michael R.; Epstein, Leon; Swartz, Jennifer M.; Shepard, Robin B.; Liu, Xiaojuan; Nukuna, Adeline; Gendelman, Howard E.

    1999-01-01

    Chemokine receptors pivotal for human immunodeficiency virus type 1 (HIV-1) infection in lymphocytes and macrophages (CCR3, CCR5, and CXCR4) are expressed on neural cells (microglia, astrocytes, and/or neurons). It is these cells which are damaged during progressive HIV-1 infection of the central nervous system. We theorize that viral coreceptors could effect neural cell damage during HIV-1-associated dementia (HAD) without simultaneously affecting viral replication. To these ends, we studied the ability of diverse viral strains to affect intracellular signaling and apoptosis of neurons, astrocytes, and monocyte-derived macrophages. Inhibition of cyclic AMP, activation of inositol 1,4,5-trisphosphate, and apoptosis were induced by diverse HIV-1 strains, principally in neurons. Virions from T-cell-tropic (T-tropic) strains (MN, IIIB, and Lai) produced the most significant alterations in signaling of neurons and astrocytes. The HIV-1 envelope glycoprotein, gp120, induced markedly less neural damage than purified virions. Macrophage-tropic (M-tropic) strains (ADA, JR-FL, Bal, MS-CSF, and DJV) produced the least neural damage, while 89.6, a dual-tropic HIV-1 strain, elicited intermediate neural cell damage. All T-tropic strain-mediated neuronal impairments were blocked by the CXCR4 antibody, 12G5. In contrast, the M-tropic strains were only partially blocked by 12G5. CXCR4-mediated neuronal apoptosis was confirmed in pure populations of rat cerebellar granule neurons and was blocked by HA1004, an inhibitor of calcium/calmodulin-dependent protein kinase II, protein kinase A, and protein kinase C. Taken together, these results suggest that progeny HIV-1 virions can influence neuronal signal transduction and apoptosis. This process occurs, in part, through CXCR4 and is independent of CD4 binding. T-tropic viruses that traffic in and out of the brain during progressive HIV-1 disease may play an important role in HAD neuropathogenesis. PMID:10482576

  11. Medicinal chemistry of small molecule CCR5 antagonists for blocking HIV-1 entry: a review of structural evolution.

    Science.gov (United States)

    Tian, Ye; Zhang, Dujuan; Zhan, Peng; Liu, Xinyong

    2014-01-01

    CCR5, a member of G protein-coupled receptors superfamily, plays an important role in the HIV-1 entry process. Antagonism of this receptor finally leads to the inhibition of R5 strains of HIV entry into the human cells. The identification of CCR5 antagonists as antiviral agents will provide more option for HAART. Now, more than a decade after the first small molecule CCR5 inhibitor was discovered, great achievements have been made. In this article, we will give a brief introduction of several series of small molecule CCR5 antagonists, focused on their appealing structure evolution, essential SAR information and thereof the enlightenment of strategies on CCR5 inhibitors design.

  12. Upregulation of the Chemokine Receptor CCR2B in Epstein‒Barr Virus-Positive Burkitt Lymphoma Cell Lines with the Latency III Program

    Directory of Open Access Journals (Sweden)

    Svetlana Kozireva

    2018-05-01

    Full Text Available CCR2 is the cognate receptor to the chemokine CCL2. CCR2–CCL2 signaling mediates cancer progression and metastasis dissemination. However, the role of CCR2–CCL2 signaling in pathogenesis of B-cell malignancies is not clear. Previously, we showed that CCR2B was upregulated in ex vivo peripheral blood B cells upon Epstein‒Barr virus (EBV infection and in established lymphoblastoid cell lines with the EBV latency III program. EBV latency III is associated with B-cell lymphomas in immunosuppressed patients. The majority of EBV-positive Burkitt lymphoma (BL tumors are characterized by latency I, but the BL cell lines drift towards latency III during in vitro culture. In this study, the CCR2A and CCR2B expression was assessed in the isogenic EBV-positive BL cell lines with latency I and III using RT-PCR, immunoblotting, and immunostaining analyses. We found that CCR2B is upregulated in the EBV-positive BL cells with latency III. Consequently, we detected the migration of latency III cells toward CCL2. Notably, the G190A mutation, corresponding to SNP CCR2-V64I, was found in one latency III cell line with a reduced migratory response to CCL2. The upregulation of CCR2B may contribute to the enhanced migration of malignant B cells into CCL2-rich compartments.

  13. The impact of CCR5-Δ32 deletion on C-reactive protein levels and cardiovascular disease

    DEFF Research Database (Denmark)

    Dinh, Khoa Manh; Pedersen, Ole B; Petersen, Mikkel S

    2015-01-01

    BACKGROUND AND PURPOSE: The C-C chemokine receptor 5-Δ32 deletion (CCR5-Δ32) has been associated with lower levels of C-reactive protein (CRP), but the effect on cardiovascular diseases is uncertain. This study addresses the impact of CCR5-Δ32 on the risk of low-grade inflammation...... and hospitalization with cardiovascular diseases in a large cohort of blood donors. METHODS: Genotyping of 15,206 healthy participants from The Danish Blood Donor Study for CCR5-Δ32 was performed and combined with CRP measurements and questionnaire data. Cardiovascular disease diagnoses were identified by ICD-10......: In this cohort, carriers of the CCR5-Δ32 deletion had normal CRP levels but a borderline significant increased risk of cardiovascular diseases....

  14. Response to Vicriviroc in Treatment-Experienced Subjects Using an Enhanced Sensitivity Co-receptor Tropism Assay: Reanalysis of AIDS Clinical Trials Group A5211

    Science.gov (United States)

    Su, Zhaohui; Gulick, Roy M.; Krambrink, Amy; Coakley, Eoin; Hughes, Michael D.; Han, Dong; Flexner, Charles; Wilkin, Timothy J.; Skolnik, Paul R.; Greaves, Wayne L.; Kuritzkes, Daniel R.; Reeves, Jacqueline D.

    2009-01-01

    The enhanced sensitivity Trofile assay was used to re-test co-receptor usage at study screening and entry for the 118 ACTG A5211 treatment-experienced subjects who had CCR5-tropic (R5) virus by the original Trofile assay at study screening. Among 90 vicriviroc recipients, a significantly (P<0.001) greater mean reduction in HIV-1 RNA was observed in 72 subjects with R5 virus versus 15 subjects reclassified with dual/mixed-tropic viruses at screening: −1.11 vs. −0.09 (day 14), −1.91 vs. −0.57 (week 24) log10 copies/mL, respectively. Results suggest that the enhanced sensitivity assay is a better screening tool for determining patient eligibility for CCR5 antagonist therapy. PMID:19874179

  15. Transmitted/founder and chronic subtype C HIV-1 use CD4 and CCR5 receptors with equal efficiency and are not inhibited by blocking the integrin α4β7.

    Directory of Open Access Journals (Sweden)

    Nicholas F Parrish

    Full Text Available Sexual transmission of human immunodeficiency virus type 1 (HIV-1 most often results from productive infection by a single transmitted/founder (T/F virus, indicating a stringent mucosal bottleneck. Understanding the viral traits that overcome this bottleneck could have important implications for HIV-1 vaccine design and other prevention strategies. Most T/F viruses use CCR5 to infect target cells and some encode envelope glycoproteins (Envs that contain fewer potential N-linked glycosylation sites and shorter V1/V2 variable loops than Envs from chronic viruses. Moreover, it has been reported that the gp120 subunits of certain transmitted Envs bind to the gut-homing integrin α4β7, possibly enhancing virus entry and cell-to-cell spread. Here we sought to determine whether subtype C T/F viruses, which are responsible for the majority of new HIV-1 infections worldwide, share biological properties that increase their transmission fitness, including preferential α4β7 engagement. Using single genome amplification, we generated panels of both T/F (n = 20 and chronic (n = 20 Env constructs as well as full-length T/F (n = 6 and chronic (n = 4 infectious molecular clones (IMCs. We found that T/F and chronic control Envs were indistinguishable in the efficiency with which they used CD4 and CCR5. Both groups of Envs also exhibited the same CD4+ T cell subset tropism and showed similar sensitivity to neutralization by CD4 binding site (CD4bs antibodies. Finally, saturating concentrations of anti-α4β7 antibodies failed to inhibit infection and replication of T/F as well as chronic control viruses, although the growth of the tissue culture-adapted strain SF162 was modestly impaired. These results indicate that the population bottleneck associated with mucosal HIV-1 acquisition is not due to the selection of T/F viruses that use α4β7, CD4 or CCR5 more efficiently.

  16. Antifibrotic Effects of the Dual CCR2/CCR5 Antagonist Cenicriviroc in Animal Models of Liver and Kidney Fibrosis.

    Directory of Open Access Journals (Sweden)

    Eric Lefebvre

    Full Text Available Interactions between C-C chemokine receptor types 2 (CCR2 and 5 (CCR5 and their ligands, including CCL2 and CCL5, mediate fibrogenesis by promoting monocyte/macrophage recruitment and tissue infiltration, as well as hepatic stellate cell activation. Cenicriviroc (CVC is an oral, dual CCR2/CCR5 antagonist with nanomolar potency against both receptors. CVC's anti-inflammatory and antifibrotic effects were evaluated in a range of preclinical models of inflammation and fibrosis.Monocyte/macrophage recruitment was assessed in vivo in a mouse model of thioglycollate-induced peritonitis. CCL2-induced chemotaxis was evaluated ex vivo on mouse monocytes. CVC's antifibrotic effects were evaluated in a thioacetamide-induced rat model of liver fibrosis and mouse models of diet-induced non-alcoholic steatohepatitis (NASH and renal fibrosis. Study assessments included body and liver/kidney weight, liver function test, liver/kidney morphology and collagen deposition, fibrogenic gene and protein expression, and pharmacokinetic analyses.CVC significantly reduced monocyte/macrophage recruitment in vivo at doses ≥20 mg/kg/day (p < 0.05. At these doses, CVC showed antifibrotic effects, with significant reductions in collagen deposition (p < 0.05, and collagen type 1 protein and mRNA expression across the three animal models of fibrosis. In the NASH model, CVC significantly reduced the non-alcoholic fatty liver disease activity score (p < 0.05 vs. controls. CVC treatment had no notable effect on body or liver/kidney weight.CVC displayed potent anti-inflammatory and antifibrotic activity in a range of animal fibrosis models, supporting human testing for fibrotic diseases. Further experimental studies are needed to clarify the underlying mechanisms of CVC's antifibrotic effects. A Phase 2b study in adults with NASH and liver fibrosis is fully enrolled (CENTAUR Study 652-2-203; NCT02217475.

  17. Analysis of the CCR5 gene coding region diversity in five South American populations reveals two new non-synonymous alleles in Amerindians and high CCR5*D32 frequency in Euro-Brazilians

    Directory of Open Access Journals (Sweden)

    Angelica B.W. Boldt

    2009-01-01

    Full Text Available The CC chemokine receptor 5 (CCR5 molecule is an important co-receptor for HIV. The effect of the CCR5*D32 allele in susceptibility to HIV infection and AIDS disease is well known. Other alleles than CCR5*D32 have not been analysed before, neither in Amerindians nor in the majority of the populations all over the world. We investigated the distribution of the CCR5 coding region alleles in South Brazil and noticed a high CCR5*D32 frequency in the Euro-Brazilian population of the Paraná State (9.3%, which is the highest thus far reported for Latin America. The D32 frequency is even higher among the Euro-Brazilian Mennonites (14.2%. This allele is uncommon in Afro-Brazilians (2.0%, rare in the Guarani Amerindians (0.4% and absent in the Kaingang Amerindians and the Oriental-Brazilians. R223Q is common in the Oriental-Brazilians (7.7% and R60S in the Afro-Brazilians (5.0%. A29S and L55Q present an impaired response to b-chemokines and occurred in Afro- and Euro-Brazilians with cumulative frequencies of 4.4% and 2.7%, respectively. Two new non-synonymous alleles were found in Amerindians: C323F (g.3729G > T in Guarani (1.4% and Y68C (g.2964A > G in Kaingang (10.3%. The functional characteristics of these alleles should be defined and considered in epidemiological investigations about HIV-1 infection and AIDS incidence in Amerindian populations.

  18. The effect of the CCR5-delta32 deletion on global gene expression considering immune response and inflammation

    Directory of Open Access Journals (Sweden)

    Hütter Gero

    2011-10-01

    Full Text Available Abstract Background The natural function of the C-C chemokine receptor type 5 (CCR5 is poorly understood. A 32 base pair deletion in the CCR5 gene (CCR5-delta32 located on chromosome 3 results in a non-functional protein. It is supposed that this deletion causes an alteration in T-cell response to inflammation. For example, the presence of the CCR5-delta32 allele in recipients of allografts constitutes as an independent and protective factor associated with a decreased risk of graft-versus-host disease (GVHD and graft rejection. However, the mechanism of this beneficial effect of the deletion regarding GVHD is unknown. In this survey we searched for a CCR5-delta32 associated regulation of critical genes involved in the immune response and the development of GVHD. Methods We examined CD34+ hematopoietic progenitor cells derived from bone marrow samples from 19 healthy volunteers for the CCR5-delta32 deletion with a genomic PCR using primers flanking the site of the deletion. Results 12 individuals were found to be homozygous for CCR5 WT and 7 carried the CCR5-delta32 deletion heterozygously. Global gene expression analysis led to the identification of 11 differentially regulated genes. Six of them are connected with mechanisms of immune response and control: LRG1, CXCR2, CCRL2, CD6, CD7, WD repeat domain, and CD30L. Conclusions Our data indicate that the CCR5-delta32 mutation may be associated with differential gene expression. Some of these genes are critical for immune response, in the case of CD30L probably protective in terms of GVHD.

  19. A chimeric measles virus with canine distemper envelope protects ferrets from lethal distemper challenge.

    Science.gov (United States)

    Rouxel, Ronan Nicolas; Svitek, Nicholas; von Messling, Veronika

    2009-08-06

    CDV infects a broad range of carnivores, and over the past decades it has caused outbreaks in a variety of wild carnivore populations. Since the currently available live-attenuated vaccine is not sufficiently safe in these highly susceptible species, we produced a chimeric virus combining the replication complex of the measles Moraten vaccine strain with the envelope of a recent CDV wild type isolate. The resulting virus did not cause disease or immunosuppression in ferrets and conferred protection from challenge with a lethal wild type strain, demonstrating its potential value for wildlife conservation efforts.

  20. Persistent replication of a hepatitis C virus genotype 1b-based chimeric clone carrying E1, E2 and p6 regions from GB virus B in a New World monkey.

    Science.gov (United States)

    Suzuki, Saori; Mori, Ken-Ichi; Higashino, Atsunori; Iwasaki, Yuki; Yasutomi, Yasuhiro; Maki, Noboru; Akari, Hirofumi

    2016-01-01

    The development of effective hepatitis C virus (HCV) vaccines is essential for the prevention of further HCV dissemination, especially in developing countries. Therefore the aim of this study is to establish a feasible and immunocompetent surrogate animal model of HCV infection that will help in evaluation of the protective efficacy of newly developing HCV vaccine candidates. To circumvent the narrow host range of HCV, an HCV genotype 1b-based chimeric clone carrying E1, E2 and p6 regions from GB virus B (GBV-B), which is closely related to HCV, was generated. The chimera between HCV and GBV-B, named HCV/G, replicated more efficiently as compared with the HCV clone in primary marmoset hepatocytes. Furthermore, it was found that the chimera persistently replicated in a tamarin for more than 2 years after intrahepatic inoculation of the chimeric RNA. Although relatively low (chimeric RNA was found in the pellet fraction obtained by ultracentrifugation of the plasma at 73 weeks, indicating production of the chimeric virus. Our results will help establish a novel non-human primate model for HCV infection on the basis of the HCV/G chimera in the major framework of the HCV genome. © 2015 The Societies and John Wiley & Sons Australia, Ltd.

  1. Biased small-molecule ligands for selective inhibition of HIV-1 cell entry via CCR5

    DEFF Research Database (Denmark)

    Berg, Christian; Spiess, Katja; von Lüttichau, Hans Rudolf

    2016-01-01

    Since the discovery of HIV's use of CCR5 as the primary coreceptor in fusion, the focus on developing small-molecule receptor antagonists for inhibition hereof has only resulted in one single drug, Maraviroc. We therefore investigated the possibility of using small-molecule CCR5 agonists as HIV-1...

  2. Molecular Mechanism of Action for Allosteric Modulators and Agonists in CC-chemokine Receptor 5 (CCR5)

    DEFF Research Database (Denmark)

    Karlshøj, Stefanie; Amarandi, Roxana Maria; Larsen, Olav

    2016-01-01

    The small molecule metal ion chelators bipyridine and terpyridine complexed with Zn(2+) (ZnBip and ZnTerp) act as CCR5 agonists and strong positive allosteric modulators of CCL3 binding to CCR5, weak modulators of CCL4 binding, and competitors for CCL5 binding. Here we describe their binding site......Terp binds deeply in the major binding pocket and, in contrast to ZnBip, interacts directly with the Trp-248(VI:13/6.48) microswitch, contributing to its 8-fold higher potency. The impact of Trp-248 was further confirmed by ZnClTerp, a chloro-substituted version of ZnTerp that showed no inherent agonism...

  3. Response to vicriviroc in treatment-experienced subjects, as determined by an enhanced-sensitivity coreceptor tropism assay: reanalysis of AIDS clinical trials group A5211.

    Science.gov (United States)

    Su, Zhaohui; Gulick, Roy M; Krambrink, Amy; Coakley, Eoin; Hughes, Michael D; Han, Dong; Flexner, Charles; Wilkin, Timothy J; Skolnik, Paul R; Greaves, Wayne L; Kuritzkes, Daniel R; Reeves, Jacqueline D

    2009-12-01

    The enhanced-sensitivity Trofile assay (Monogram Biosciences) was used to retest coreceptor use at both study screening and study entry for 118 treatment-experienced subjects in AIDS Clinical Trials Group A5211 who had CCR5-tropic (R5) virus detected by the original Trofile assay at study screening. Among 90 recipients of vicriviroc, a significantly (P< .001) greater mean reduction in HIV-1 RNA was observed in 72 subjects with R5 virus versus 15 subjects reclassified as having dual/mixed-tropic viruses at screening: -1.11 versus -0.09 log(10) copies/mL at day 14 and -1.91 versus -0.57 log(10) copies/mL at week 24, respectively. Results suggest that the enhanced-sensitivity assay is a better screening tool for determining patient eligibility for CCR5 antagonist therapy.

  4. Monocytes infiltrate the pancreas via the MCP-1/CCR2 pathway and differentiate into stellate cells.

    Directory of Open Access Journals (Sweden)

    Kazuko Ino

    Full Text Available Recent studies have shown that monocytes possess pluripotent plasticity. We previously reported that monocytes could differentiate into hepatic stellate cells. Although stellate cells are also present in the pancreas, their origin remains unclear. An accumulation of enhanced green fluorescent protein (EGFP(+CD45(- cells was observed in the pancreases and livers of chimeric mice, which were transplanted with a single hematopoietic stem cell isolated from EGFP-transgenic mice and treated with carbon tetrachloride (CCl4. Because the vast majority of EGFP(+CD45(- cells in the pancreas expressed stellate cell-associated antigens such as vimentin, desmin, glial fibrillary acidic protein, procollagen-I, and α-smooth muscle actin, they were characterized as pancreatic stellate cells (PaSCs. EGFP(+ PaSCs were also observed in CCl4-treated mice adoptively transferred with monocytes but not with other cell lineages isolated from EGFP-transgenic mice. The expression of monocyte chemoattractant protein-1 (MCP-1 and angiotensin II (Ang II increased in the pancreas of CCl4-treated mice and their respective receptors, C-C chemokine receptor 2 (CCR2 and Ang II type 1 receptor (AT1R, were expressed on Ly6C(high monocytes isolated from EGFP-transgenic mice. We examined the effect of an AT1R antagonist, irbesartan, which is also a CCR2 antagonist, on the migration of monocytes into the pancreas. Monocytes migrated toward MCP-1 but not Ang II in vitro. Irbesartan inhibited not only their in vitro chemotaxis but also in vivo migration of adoptively transferred monocytes from peripheral blood into the pancreas. Irbesartan treatment significantly reduced the numbers of EGFP(+F4/80(+CCR2(+ monocytic cells and EGFP(+ PaSCs in the pancreas of CCl4-treated chimeric mice receiving EGFP(+ bone marrow cells. A specific CCR2 antagonist RS504393 inhibited the occurrence of EGFP(+ PaSCs in injured mice. We propose that CCR2(+ monocytes migrate into the pancreas possibly via the

  5. Phenotype Variation in Human Immunodeficiency virus Type 1 Transmission and Disease Progression

    Directory of Open Access Journals (Sweden)

    Mariangela Cavarelli

    2009-01-01

    Full Text Available Human immunodeficiency virus type I (HIV-1 infects target cells through interaction with the CD4 molecule and chemokine receptors, mainly CCR5 and CXCR4. Viral isolates can be phenotypically classified based on the co-receptor they utilize to infect target cells. Thus, R5 and X4 virus use respectively CCR5 and CXCR4, whereas R5X4 virus can use either CCR5 or CXCR4. This review describes the central role played by co-receptor expression and usage for HIV-1 cell tropism, transmission and pathogenesis. We discuss various hypotheses proposed to explain the preferential transmission of R5 viruses and the mechanisms driving the change of HIV-1 co-receptor usage in the course of infection. Recent insights in the intrinsic variability of R5 viruses and their role in influencing disease progression in both adults and children are also discussed.

  6. Phenotype variation in human immunodeficiency virus type 1 transmission and disease progression.

    Science.gov (United States)

    Cavarelli, Mariangela; Scarlatti, Gabriella

    2009-01-01

    Human immunodeficiency virus type I (HIV-1) infects target cells through interaction with the CD4 molecule and chemokine receptors, mainly CCR5 and CXCR4. Viral isolates can be phenotypically classified based on the co-receptor they utilize to infect target cells. Thus, R5 and X4 virus use respectively CCR5 and CXCR4, whereas R5X4 virus can use either CCR5 or CXCR4. This review describes the central role played by co-receptor expression and usage for HIV-1 cell tropism, transmission and pathogenesis. We discuss various hypotheses proposed to explain the preferential transmission of R5 viruses and the mechanisms driving the change of HIV-1 co-receptor usage in the course of infection. Recent insights in the intrinsic variability of R5 viruses and their role in influencing disease progression in both adults and children are also discussed.

  7. The Isolation of Novel Phage Display-Derived Human Recombinant Antibodies Against CCR5, the Major Co-Receptor of HIV

    Science.gov (United States)

    Shimoni, Moria; Herschhorn, Alon; Britan-Rosich, Yelena; Kotler, Moshe; Benhar, Itai

    2013-01-01

    Abstract Selecting for antibodies against specific cell-surface proteins is a difficult task due to many unrelated proteins that are expressed on the cell surface. Here, we describe a method to screen antibody-presenting phage libraries against native cell-surface proteins. We applied this method to isolate antibodies that selectively recognize CCR5, which is the major co-receptor for HIV entry (consequently, playing a pivotal role in HIV transmission and pathogenesis). We employed a phage screening strategy by using cells that co-express GFP and CCR5, along with an excess of control cells that do not express these proteins (and are otherwise identical to the CCR5-expressing cells). These control cells are intended to remove most of the phages that bind the cells nonspecifically; thus leading to an enrichment of the phages presenting anti-CCR5-specific antibodies. Subsequently, the CCR5-presenting cells were quantitatively sorted by flow cytometry, and the bound phages were eluted, amplified, and used for further successive selection rounds. Several different clones of human single-chain Fv antibodies that interact with CCR5-expressing cells were identified. The most specific monoclonal antibody was converted to a full-length IgG and bound the second extracellular loop of CCR5. The experimental approach presented herein for screening for CCR5-specific antibodies can be applicable to screen antibody-presenting phage libraries against any cell-surface expressed protein of interest. PMID:23941674

  8. Increased frequency of CCR4+ and CCR6+ memory T-cells including CCR7+CD45RAmed very early memory cells in granulomatosis with polyangiitis (Wegener's)

    OpenAIRE

    Fagin, Ursula; Pitann, Silke; Gross, Wolfgang L; Lamprecht, Peter

    2012-01-01

    Introduction Chemokine receptors play an important role in mediating the recruitment of T cells to inflammatory sites. Previously, small proportions of circulating Th1-type CCR5+ and Th2-type CCR3+ cells have been shown in granulomatosis with polyangiitis (GPA). Wondering to what extent CCR4 and CCR6 expression could also be implicated in T cell recruitment to inflamed sites in GPA, we investigated the expression of CCR4 and CCR6 on T cells and its association with T cell diversity and polari...

  9. Prolonged control of replication-competent dual- tropic human immunodeficiency virus-1 following cessation of highly active antiretroviral therapy

    Directory of Open Access Journals (Sweden)

    Salgado Maria

    2011-12-01

    Full Text Available Abstract Background While initiation of highly active antiretroviral therapy (HAART during primary HIV-1 infection occasionally results in transient control of viral replication after treatment interruption, the vast majority of patients eventually experience a rebound in plasma viremia. Results Here we report a case of a patient who was started on HAART during symptomatic primary infection and who has subsequently maintained viral loads of + T cells. In addition, he does not have any known protective HLA alleles. Thus it is unlikely that he was destined to become a natural elite controller or suppressor. The mechanism of control of viral replication is unclear; he is infected with a CCR5/CXCR4 dual-tropic virus that is fully replication-competent in vitro. In addition, his spouse, who transmitted the virus to him, developed AIDS. The patient's CD4+ T cells are fully susceptible to HIV-1 infection, and he has low titers of neutralizing antibodies to heterologous and autologous HIV-1 isolates. Furthermore, his CD8+ T cells do not have potent HIV suppressive activity. Conclusion This report suggests that some patients may be capable of controlling pathogenic HIV-1 isolates for extended periods of time after the cessation of HAART through a mechanism that is distinct from the potent cytotoxic T lymphocyte (CTL mediated suppression that has been reported in many elite suppressors.

  10. CCR6-dependent positioning of memory B cells is essential for their ability to mount a recall response to antigen.

    Science.gov (United States)

    Elgueta, Raul; Marks, Ellen; Nowak, Elizabeth; Menezes, Shinelle; Benson, Micah; Raman, Vanitha S; Ortiz, Carla; O'Connell, Samuel; Hess, Henry; Lord, Graham M; Noelle, Randolph

    2015-01-15

    Chemokine-dependent localization of specific B cell subsets within the immune microarchitecture is essential to ensure successful cognate interactions. Although cognate interactions between T cells and memory B cells (B(mem)) are essential for the secondary humoral immune responses, the chemokine response patterns of B(mem) cells are largely unknown. In contrast to naive B cells, this study shows that Ag-specific B(mem) cells have heightened expression of CCR6 and a selective chemotactic response to the CCR6 ligand, CCL20. Although CCR6 appears be nonessential for the initial clonal expansion and maintenance of B(mem), CCR6 is essential for the ability of B(mem) to respond to a recall response to their cognate Ag. This dependency was deemed intrinsic by studies in CCR6-deficient mice and in bone marrow chimeric mice where CCR6 deficiency was limited to the B cell lineage. Finally, the mis-positioning of CCR6-deficient B(mem) was revealed by immunohistological analysis with an altered distribution of CCR6-deficient B(mem) from the marginal and perifollicular to the follicular/germinal center area. Copyright © 2015 by The American Association of Immunologists, Inc.

  11. Efficient, trans-complementing packaging systems for chimeric, pseudoinfectious dengue 2/yellow fever viruses

    International Nuclear Information System (INIS)

    Shustov, Alexandr V.; Frolov, Ilya

    2010-01-01

    In our previous studies, we have stated to build a new strategy for developing defective, pseudoinfectious flaviviruses (PIVs) and applying them as a new type of vaccine candidates. PIVs combined the efficiency of live vaccines with the safety of inactivated or subunit vaccines. The results of the present work demonstrate further development of chimeric PIVs encoding dengue virus 2 (DEN2V) glycoproteins and yellow fever virus (YFV)-derived replicative machinery as potential vaccine candidates. The newly designed PIVs have synergistically functioning mutations in the prM and NS2A proteins, which abolish processing of the latter proteins and make the defective viruses capable of producing either only noninfectious, immature and/or subviral DEN2V particles. The PIV genomes can be packaged to high titers into infectious virions in vitro using the NS1-deficient YFV helper RNAs, and both PIVs and helpers can then be passaged as two-component genome viruses at an escalating scale.

  12. DMPD: Macrophage activation through CCR5- and CXCR4-mediated gp120-elicited signalingpathways. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 12960231 Macrophage activation through CCR5- and CXCR4-mediated gp120-elicited sign...82. Epub 2003 Jul 22. (.png) (.svg) (.html) (.csml) Show Macrophage activation through CCR5- and CXCR4-media...on through CCR5- and CXCR4-mediated gp120-elicited signalingpathways. Authors Lee C, Liu QH, Tomkowicz B, Yi

  13. Investigating the association of chemokine receptor 5 (CCR5 polymorphism with cervical cancer in human papillomavirus (HPV positive patients - DOI: 10.4025/actascihealthsci.v30i2.944 Investigating association of chemokine receptor 5 (CCR5 polymorphism with cervical cancer in human papillomavirus (HPV suggestive patients - DOI: 10.4025/actascihealthsci.v30i2.944

    Directory of Open Access Journals (Sweden)

    Sueli Donizete Borelli

    2008-12-01

    Full Text Available HPV is one of the most frequent causes for the development of cervical cancer. It is known that chemokines are important determinants of early inflammatory responses. The CC chemokine receptor 5 (CCR5 gene is involved in the chemotaxis of leukocytes toward inflammation sites. In the present study, polymerase chain reactions (PCR in genomic DNA samples, using specific CCR5 oligonucleotide primers surrounding the breakpoint deletion, detected a 225 bp product from the normal CCR5 allele and a 193 bp product from the 32 bp deletion allele. The wild type genotype was prevalent in both group, but it was not statistically significant, with χ2 = 1.519 (2 degrees of freedom; p > 0.05. As there are a small number of 32 allele carriers, further studies are needed to clarify the role of CCR5 in the cervical cancer.HPV is the most responsible of cervical cancer. It is known that chemokines are important determinants of the early inflammatory response. The CC chemokine receptor 5 (CCR5 gene is involved in the chemotaxis of leukocytes toward inflammation sites. In the present study, polymerase chain reactions (PCR in genomic DNA samples, using specific CCR5 oligonucleotide primers surrounding the breakpoint deletion, detected a 225bp product from the normal CCR5 allele and a 193bp product from the 32bp deletion allele. The wild type genotype was prevalent in both group, but it wasn’t statistically significant with χ² =1,519 (2 degrees of freedom; p>0.05. Once there is a small number of 32 allele carriers, further studies are needed to clarify the role of CCR5 in the cervical cancer.

  14. The neurovirulence and neuroinvasiveness of chimeric tick-borne encephalitis/dengue virus can be attenuated by introducing defined mutations into the envelope and NS5 protein genes and the 3' non-coding region of the genome

    International Nuclear Information System (INIS)

    Engel, Amber R.; Rumyantsev, Alexander A.; Maximova, Olga A.; Speicher, James M.; Heiss, Brian; Murphy, Brian R.; Pletnev, Alexander G.

    2010-01-01

    Tick-borne encephalitis (TBE) is a severe disease affecting thousands of people throughout Eurasia. Despite the use of formalin-inactivated vaccines in endemic areas, an increasing incidence of TBE emphasizes the need for an alternative vaccine that will induce a more durable immunity against TBE virus (TBEV). The chimeric attenuated virus vaccine candidate containing the structural protein genes of TBEV on a dengue virus genetic background (TBEV/DEN4) retains a high level of neurovirulence in both mice and monkeys. Therefore, attenuating mutations were introduced into the envelope (E 315 ) and NS5 (NS5 654,655 ) proteins, and into the 3' non-coding region (Δ30) of TBEV/DEN4. The variant that contained all three mutations (vΔ30/E 315 /NS5 654,655 ) was significantly attenuated for neuroinvasiveness and neurovirulence and displayed a reduced level of replication and virus-induced histopathology in the brains of mice. The high level of safety in the central nervous system indicates that vΔ30/E 315 /NS5 654,655 should be further evaluated as a TBEV vaccine.

  15. Homology-Directed Recombination for Enhanced Engineering of Chimeric Antigen Receptor T Cells

    Directory of Open Access Journals (Sweden)

    Malika Hale

    2017-03-01

    Full Text Available Gene editing by homology-directed recombination (HDR can be used to couple delivery of a therapeutic gene cassette with targeted genomic modifications to generate engineered human T cells with clinically useful profiles. Here, we explore the functionality of therapeutic cassettes delivered by these means and test the flexibility of this approach to clinically relevant alleles. Because CCR5-negative T cells are resistant to HIV-1 infection, CCR5-negative anti-CD19 chimeric antigen receptor (CAR T cells could be used to treat patients with HIV-associated B cell malignancies. We show that targeted delivery of an anti-CD19 CAR cassette to the CCR5 locus using a recombinant AAV homology template and an engineered megaTAL nuclease results in T cells that are functionally equivalent, in both in vitro and in vivo tumor models, to CAR T cells generated by random integration using lentiviral delivery. With the goal of developing off-the-shelf CAR T cell therapies, we next targeted CARs to the T cell receptor alpha constant (TRAC locus by HDR, producing TCR-negative anti-CD19 CAR and anti-B cell maturation antigen (BCMA CAR T cells. These novel cell products exhibited in vitro cytolytic activity against both tumor cell lines and primary cell targets. Our combined results indicate that high-efficiency HDR delivery of therapeutic genes may provide a flexible and robust method that can extend the clinical utility of cell therapeutics.

  16. Chimeric human parainfluenza virus bearing the Ebola virus glycoprotein as the sole surface protein is immunogenic and highly protective against Ebola virus challenge

    International Nuclear Information System (INIS)

    Bukreyev, Alexander; Marzi, Andrea; Feldmann, Friederike; Zhang Liqun; Yang Lijuan; Ward, Jerrold M.; Dorward, David W.; Pickles, Raymond J.; Murphy, Brian R.; Feldmann, Heinz; Collins, Peter L.

    2009-01-01

    We generated a new live-attenuated vaccine against Ebola virus (EBOV) based on a chimeric virus HPIV3/ΔF-HN/EboGP that contains the EBOV glycoprotein (GP) as the sole transmembrane envelope protein combined with the internal proteins of human parainfluenza virus type 3 (HPIV3). Electron microscopy analysis of the virus particles showed that they have an envelope and surface spikes resembling those of EBOV and a particle size and shape resembling those of HPIV3. When HPIV3/ΔF-HN/EboGP was inoculated via apical surface of an in vitro model of human ciliated airway epithelium, the virus was released from the apical surface; when applied to basolateral surface, the virus infected basolateral cells but did not spread through the tissue. Following intranasal (IN) inoculation of guinea pigs, scattered infected cells were detected in the lungs by immunohistochemistry, but infectious HPIV3/ΔF-HN/EboGP could not be recovered from the lungs, blood, or other tissues. Despite the attenuation, the virus was highly immunogenic, and a single IN dose completely protected the animals against a highly lethal intraperitoneal challenge of guinea pig-adapted EBOV

  17. Chimeric avian paramyxovirus-based vector immunization against highly pathogenic avian influenza followed by conventional Newcastle disease vaccination eliminates lack of protection from virulent ND virus

    Directory of Open Access Journals (Sweden)

    C. Steglich

    2014-01-01

    Full Text Available Recently, we described a chimeric, hemagglutinin of highly pathogenic avian influenza virus (HPAIV H5 expressing Newcastle disease virus (NDV-based vector vaccine (chNDVFHNPMV8H5 in which NDV envelope glycoproteins were replaced by those of avian paramyxovirus-8 (APMV-8. This chimeric vaccine induced solid protection against lethal HPAIV H5N1 even in chickens with maternal antibodies against NDV (MDA+. However, due to the absence of the major NDV immunogens it failed to induce protection against Newcastle disease (ND. Here, we report on protection of MDA+ chickens against HPAI H5N1 and ND, by vaccination with chNDVFHNPMV8H5 either on day 1 or day seven after hatch, and subsequent immunization with live attenuated NDV seven days later. Vaccination was well tolerated and three weeks after immunization, challenge infections with highly pathogenic NDV as well as HPAIV H5N1 were carried out. All animals remained healthy without exhibiting any clinical signs, whereas non-vaccinated animals showed morbidity and mortality. Therefore, vaccination with chNDVFHNPMV8H5 can be followed by NDV vaccination to protect chickens from HPAIV as well as NDV, indicating that the antibody response against chNDVFHNPMV8H5 does not interfere with live ND vaccination.

  18. Virulence of a chimeric recombinant infectious haematopoietic necrosis virus expressing the spring viraemia of carp virus glycoprotein in salmonid and cyprinid fish

    Science.gov (United States)

    Emmenegger, Eveline; Biacchesi, Stéphane; Mérour, Emilie; Glenn, Jolene. A; Palmer, Alexander D.; Brémont, Michel; Kurath, Gael

    2018-01-01

    Infectious haematopoietic necrosis virus (IHNV) and spring viraemia of carp virus (SVCV) are both rhabdoviruses of fish, listed as notifiable disease agents by the World Organization for Animal Health. Recombinant rhabdoviruses with heterologous gene substitutions have been engineered to study genetic determinants and assess the potential of these recombinant viruses for vaccine development. A recombinant IHNV (rIHNV), containing the full-length genome of a European IHNV strain, was modified by deleting the glycoprotein (G) gene and replacing it with a European SVCV G-gene to make the rIHNV-Gsvcv. The chimeric rIHNV-Gsvcv level of virulence in rainbow trout, common carp and koi was assessed, and its ability to induce a protective immune response in surviving koi against wild-type SVCV infection was tested. The rIHNV-Gsvcv infection of trout led to high mortality, ranging from 78% to 92.5%, after immersion. In contrast, no deaths occurred in juvenile common carp after infection with rIHNV-Gsvcv by either immersion or intraperitoneal (IP) injection. Similarly, koi infected with rIHNV-Gsvcv via IP injection had little to no mortality (≤9%). Koi that survived initial infection with a high dose of recombinant virus rIHNV-Gsvcv were protected against a virulent SVCV challenge resulting in a high relative per cent survival of 82.5%.

  19. Frequency of CCR5delta32 in Brazilian populations

    Directory of Open Access Journals (Sweden)

    A.E. Vargas

    2006-03-01

    Full Text Available A sample of 103 randomly chosen healthy individuals from Alegrete, RS, Brazil, was tested for the CCR5delta32 allele, which is known to influence susceptibility to HIV-1 infection. The CCR5delta32 allele was identified by PCR amplification using specific primers flanking the region of deletion, followed by electrophoresis on a 3% agarose gel. The data obtained were compared to those reported for other populations and interpreted in terms of Brazilian history. The individuals studied came from a highly admixed population. Most of them were identified as white (N = 59, while blacks and browns (mulattoes were N = 13 and N = 31, respectively. The observed frequencies, considering the white, black and brown samples (6.8, 3.8, and 6.4%, respectively, suggest an important European parental contribution, even in populations identified as black and brown. However, in Brazil as a whole, this allele shows gradients indicating a relatively good correlation with the classification based on skin color and other physical traits, used here to define major Brazilian population groups.

  20. Structure-Activity Relationships and Identification of Optmized CC-Chemokine Receptor CCR1, 5, and 8 Metal-Ion Chelators

    DEFF Research Database (Denmark)

    Chalikiopoulos, Alexander; Thiele, Stefanie; Malmgaard-Clausen, Mikkel

    2013-01-01

    Chemokine receptors are involved in trafficking of leukocytes and represent targets for autoimmune conditions, inflammatory diseases, viral infections, and cancer. We recently published CCR1, CCR8, and CCR5 agonists and positive modulators based on a three metal-ion chelator series: 2,2'-bipyridi...

  1. Virus-Like Particles of Chimeric Recombinant Porcine Circovirus Type 2 as Antigen Vehicle Carrying Foreign Epitopes

    Directory of Open Access Journals (Sweden)

    Huawei Zhang

    2014-12-01

    Full Text Available Virus-like particles (VLPs of chimeric porcine circovirus type 2 (PCV2 were generated by replacing the nuclear localization signal (NLS; at 1–39 aa of PCV2 capsid protein (Cap with classical swine fever virus (CSFV T-cell epitope (1446–1460 aa, CSFV B-cell epitope (693–716 aa and CSFV T-cell epitope conjugated with B-cell epitope. The recombinant proteins were expressed using the baculovirus expression system and detected by immunoblotting and indirect immunofluorescence assay. The abilities to form PCV2 VLPs were confirmed by transmission electron microscopy. Immunogenicities of the three recombinant proteins were evaluated in mice. Our Results indicated that Cap protein NLS deletion or substitution with CSFV epitopes did not affect the VLPs assembly. Three chimeric Cap proteins could form VLPs and induce efficient humoral and cellular immunity against PCV2 and CSFV in mice. Results show that PCV2 VLPs can be used as an efficient antigen carrier for delivery of foreign epitopes, and a potential novel vaccine.

  2. The CC-chemokine receptor 5 (CCR5) is a marker of, but not essential for the development of human Th1 cells

    DEFF Research Database (Denmark)

    Odum, Niels; Bregenholt, S; Eriksen, K W

    1999-01-01

    The CC-chemokine receptor 5 (CCR5) has recently been described as a surface marker of human T cells producing type 1 (Th1) cytokines. Here we confirm that CCR5 is expressed on human Th1 but not on Th2 T-cell clones. Using intracellular cytokine staining, we show that alloantigen specific CD4+ T...

  3. Identification and characterization of HIV-2 strains obtained from asymptomatic patients that do not use CCR5 or CXCR4 coreceptors

    International Nuclear Information System (INIS)

    Azevedo-Pereira, J.M.; Santos-Costa, Q.; Mansinho, K.; Moniz-Pereira, J.

    2003-01-01

    In vivo, human immunodeficiency virus type 2 (HIV-2) infection reveals several unique characteristics when compared to HIV-1 infection, the most remarkable of which is the extraordinarily long asymptomatic period. Here we describe two HIV-2 primary isolates, obtained from asymptomatic individuals, which do not infect any coreceptor-expressing cell lines tested. In those cells, we show that the absence of replication is directly related to cell entry events. Furthermore, productive infection observed in peripheral blood mononuclear cells (PBMC) was not inhibited by natural ligands and monoclonal antibodies directed to CCR5 and CXCR4. Finally, viral entry efficiency and viral progeny production of these viruses are markedly impaired in PBMC, indicating a reduced replicative fitness of both viruses. In conclusion, our data suggest that in some HIV-2 asymptomatic individuals, the circulating viruses are unable to use the major coreceptors to infect PBMC. This fact should have important implications in HIV-2 pathogenesis and transmission

  4. Characterization of Chemokine Receptor Utilization of Viruses in the Latent Reservoir for Human Immunodeficiency Virus Type 1

    Science.gov (United States)

    Pierson, Theodore; Hoffman, Trevor L.; Blankson, Joel; Finzi, Diana; Chadwick, Karen; Margolick, Joseph B.; Buck, Christopher; Siliciano, Janet D.; Doms, Robert W.; Siliciano, Robert F.

    2000-01-01

    Latently infected resting CD4+ T cells provide a long-term reservoir for human immunodeficiency virus type 1 (HIV-1) and are likely to represent the major barrier to virus eradication in patients on combination antiretroviral therapy. The mechanisms by which viruses enter the latent reservoir and the nature of the chemokine receptors involved have not been determined. To evaluate the phenotype of the virus in this compartment with respect to chemokine receptor utilization, full-length HIV-1 env genes were cloned from latently infected cells and assayed functionally. We demonstrate that the majority of the viruses in the latent reservoir utilize CCR5 during entry, although utilization of several other receptors, including CXCR4, was observed. No alternative coreceptors were shown to be involved in a systematic fashion. Although R5 viruses are present in the latent reservoir, CCR5 was not expressed at high levels on resting CD4+ T cells. To understand the mechanism by which R5 viruses enter latent reservoir, the ability of an R5 virus, HIV-1 Ba-L, to infect highly purified resting CD4+ T lymphocytes from uninfected donors was evaluated. Entry of Ba-L could be observed when virus was applied at a multiplicity approaching 1. However, infection was limited to a subset of cells expressing low levels of CCR5 and markers of immunologic memory. Naive cells could not be infected by an R5 virus even when challenged with a large inoculum. Direct cell fractionation studies showed that latent virus is present predominantly in resting memory cells but also at lower levels in resting naive cells. Taken together, these findings provide support for the hypothesis that the direct infection of naive T cells is not the major mechanism by which the latent infection of resting T cells is established. PMID:10933689

  5. Molecular Mechanism of Action for Allosteric Modulators and Agonists in CC-chemokine Receptor 5 (CCR5).

    Science.gov (United States)

    Karlshøj, Stefanie; Amarandi, Roxana Maria; Larsen, Olav; Daugvilaite, Viktorija; Steen, Anne; Brvar, Matjaž; Pui, Aurel; Frimurer, Thomas Michael; Ulven, Trond; Rosenkilde, Mette Marie

    2016-12-23

    The small molecule metal ion chelators bipyridine and terpyridine complexed with Zn 2+ (ZnBip and ZnTerp) act as CCR5 agonists and strong positive allosteric modulators of CCL3 binding to CCR5, weak modulators of CCL4 binding, and competitors for CCL5 binding. Here we describe their binding site using computational modeling, binding, and functional studies on WT and mutated CCR5. The metal ion Zn 2+ is anchored to the chemokine receptor-conserved Glu-283 VII:06/7.39 Both chelators interact with aromatic residues in the transmembrane receptor domain. The additional pyridine ring of ZnTerp binds deeply in the major binding pocket and, in contrast to ZnBip, interacts directly with the Trp-248 VI:13/6.48 microswitch, contributing to its 8-fold higher potency. The impact of Trp-248 was further confirmed by ZnClTerp, a chloro-substituted version of ZnTerp that showed no inherent agonism but maintained positive allosteric modulation of CCL3 binding. Despite a similar overall binding mode of all three metal ion chelator complexes, the pyridine ring of ZnClTerp blocks the conformational switch of Trp-248 required for receptor activation, thereby explaining its lack of activity. Importantly, ZnClTerp becomes agonist to the same extent as ZnTerp upon Ala mutation of Ile-116 III:16/3.40 , a residue that constrains the Trp-248 microswitch in its inactive conformation. Binding studies with 125 I-CCL3 revealed an allosteric interface between the chemokine and the small molecule binding site, including residues Tyr-37 I:07/1.39 , Trp-86 II:20/2.60 , and Phe-109 III:09/3.33 The small molecules and CCL3 approach this interface from opposite directions, with some residues being mutually exploited. This study provides new insight into the molecular mechanism of CCR5 activation and paves the way for future allosteric drugs for chemokine receptors. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. Infection of Common Marmosets with GB Virus B Chimeric Virus Encoding the Major Nonstructural Proteins NS2 to NS4A of Hepatitis C Virus.

    Science.gov (United States)

    Zhu, Shaomei; Li, Tingting; Liu, Bochao; Xu, Yuxia; Sun, Yachun; Wang, Yilin; Wang, Yuanzhan; Shuai, Lifang; Chen, Zixuan; Allain, Jean-Pierre; Li, Chengyao

    2016-09-15

    A lack of immunocompetent-small-primate models has been an obstacle for developing hepatitis C virus (HCV) vaccines and affordable antiviral drugs. In this study, HCV/GB virus B (GBV-B) chimeric virus carrying the major nonstructural proteins NS2 to NS4A (HCV NS2 to -4A chimera) was produced and used to infect common marmosets, since HCV NS2 to NS4A proteins are critical proteases and major antigens. Seven marmosets were inoculated intrahepatically with HCV NS2 to -4A chimera RNA for primary infection or intravenously injected with chimera-containing serum for passage infection. Three animals used as controls were injected with phosphate-buffered saline (PBS) or GBV-B, respectively. Six of seven HCV NS2 to -4A chimera-infected marmosets exhibited consistent viremia and one showed transient viremia during the course of follow-up detection. All six infected animals with persistent circulating viremia presented characteristics typical of viral hepatitis, including viral RNA and proteins in hepatocytes and histopathological changes in liver tissue. Viremia was consistently detected for 5 to 54 weeks of follow-up. FK506 immunosuppression facilitated the establishment of persistent chimera infection in marmosets. An animal with chimera infection spontaneously cleared the virus in blood 7 weeks following the first inoculation, but viral-RNA persistence, low-level viral protein, and mild necroinflammation remained in liver tissue. The specific antibody and T-cell response to HCV NS3 in this viremia-resolved marmoset was boosted by rechallenging, but no viremia was detected during 57 weeks of follow-up. The chimera-infected marmosets described can be used as a suitable small-primate animal model for studying novel antiviral drugs and T-cell-based vaccines against HCV infection. HCV infection causes approximately 70% of chronic hepatitis and is frequently associated with primary liver cancer globally. Chimpanzees have been used as a reliable primate model for HCV infection

  7. ChimericSeq: An open-source, user-friendly interface for analyzing NGS data to identify and characterize viral-host chimeric sequences

    Science.gov (United States)

    Shieh, Fwu-Shan; Jongeneel, Patrick; Steffen, Jamin D.; Lin, Selena; Jain, Surbhi; Song, Wei

    2017-01-01

    Identification of viral integration sites has been important in understanding the pathogenesis and progression of diseases associated with particular viral infections. The advent of next-generation sequencing (NGS) has enabled researchers to understand the impact that viral integration has on the host, such as tumorigenesis. Current computational methods to analyze NGS data of virus-host junction sites have been limited in terms of their accessibility to a broad user base. In this study, we developed a software application (named ChimericSeq), that is the first program of its kind to offer a graphical user interface, compatibility with both Windows and Mac operating systems, and optimized for effectively identifying and annotating virus-host chimeric reads within NGS data. In addition, ChimericSeq’s pipeline implements custom filtering to remove artifacts and detect reads with quantitative analytical reporting to provide functional significance to discovered integration sites. The improved accessibility of ChimericSeq through a GUI interface in both Windows and Mac has potential to expand NGS analytical support to a broader spectrum of the scientific community. PMID:28829778

  8. ChimericSeq: An open-source, user-friendly interface for analyzing NGS data to identify and characterize viral-host chimeric sequences.

    Directory of Open Access Journals (Sweden)

    Fwu-Shan Shieh

    Full Text Available Identification of viral integration sites has been important in understanding the pathogenesis and progression of diseases associated with particular viral infections. The advent of next-generation sequencing (NGS has enabled researchers to understand the impact that viral integration has on the host, such as tumorigenesis. Current computational methods to analyze NGS data of virus-host junction sites have been limited in terms of their accessibility to a broad user base. In this study, we developed a software application (named ChimericSeq, that is the first program of its kind to offer a graphical user interface, compatibility with both Windows and Mac operating systems, and optimized for effectively identifying and annotating virus-host chimeric reads within NGS data. In addition, ChimericSeq's pipeline implements custom filtering to remove artifacts and detect reads with quantitative analytical reporting to provide functional significance to discovered integration sites. The improved accessibility of ChimericSeq through a GUI interface in both Windows and Mac has potential to expand NGS analytical support to a broader spectrum of the scientific community.

  9. A Linear Epitope in the N-Terminal Domain of CCR5 and Its Interaction with Antibody.

    Directory of Open Access Journals (Sweden)

    Benny Chain

    Full Text Available The CCR5 receptor plays a role in several key physiological and pathological processes and is an important therapeutic target. Inhibition of the CCR5 axis by passive or active immunisation offers one very selective strategy for intervention. In this study we define a new linear epitope within the extracellular domain of CCR5 recognised by two independently produced monoclonal antibodies. A short peptide encoding the linear epitope can induce antibodies which recognise the intact receptor when administered colinear with a tetanus toxoid helper T cell epitope. The monoclonal antibody RoAb 13 is shown to bind to both cells and peptide with moderate to high affinity (6x10^8 and 1.2x107 M-1 respectively, and binding to the peptide is enhanced by sulfation of tyrosines at positions 10 and 14. RoAb13, which has previously been shown to block HIV infection, also blocks migration of monocytes in response to CCR5 binding chemokines and to inflammatory macrophage conditioned medium. A Fab fragment of RoAb13 has been crystallised and a structure of the antibody is reported to 2.1 angstrom resolution.

  10. Sensitive luminescent reporter viruses reveal appreciable release of hepatitis C virus NS5A protein into the extracellular environment.

    Science.gov (United States)

    Eyre, Nicholas S; Aloia, Amanda L; Joyce, Michael A; Chulanetra, Monrat; Tyrrell, D Lorne; Beard, Michael R

    2017-07-01

    The HCV NS5A protein is essential for viral RNA replication and virus particle assembly. To study the viral replication cycle and NS5A biology we generated an infectious HCV construct with a NanoLuciferase (NLuc) insertion within NS5A. Surprisingly, beyond its utility as a sensitive reporter of cytoplasmic viral RNA replication, we also observed strong luminescence in cell culture fluids. Further analysis using assembly-defective viruses and subgenomic replicons revealed that infectious virus production was not required for extracellular NS5A-NLuc activity but was associated with enrichment of extracellular NS5A-NLuc in intermediate-density fractions similar to those of exosomes and virus particles. Additionally, BRET analysis indicated that intracellular and extracellular forms of NS5A may adopt differing conformations. Importantly, infection studies using a human liver chimeric mouse model confirmed robust infection in vivo and ready detection of NLuc activity in serum. We hypothesise that the presence of NS5A in extracellular fluids contributes to HCV pathogenesis. Copyright © 2017 Elsevier Inc. All rights reserved.

  11. The role of CC chemokine receptor 5 in antiviral immunity

    DEFF Research Database (Denmark)

    Nansen, Anneline; Christensen, Jan Pravsgaard; Andreasen, Susanne Ørding

    2002-01-01

    The CC chemokine receptor CCR5 is an important coreceptor for human immunodeficiency virus (HIV), and there is a major thrust to develop anti-CCR5-based therapies for HIV-1. However, it is not known whether CCR5 is critical for a normal antiviral T-cell response. This study investigated the immune...

  12. Evidence favoring the involvement of CC chemokine receptor (CCR) 5 in T-lymphocyte accumulation in optic neuritis

    DEFF Research Database (Denmark)

    Sørensen, Torben Lykke; Ransohoff, R M; Jensen, J

    2003-01-01

    To define the relationships between levels of chemokine receptor (CCR)5+ T-cells in blood and cerebrospinal fluid (CSF) of optic neuritis (ON) and control patients (CON).......To define the relationships between levels of chemokine receptor (CCR)5+ T-cells in blood and cerebrospinal fluid (CSF) of optic neuritis (ON) and control patients (CON)....

  13. Effects of sequence changes in the HIV-1 gp41 fusion peptide on CCR5 inhibitor resistance

    International Nuclear Information System (INIS)

    Anastassopoulou, Cleo G.; Ketas, Thomas J.; Sanders, Rogier W.; Johan Klasse, Per; Moore, John P.

    2012-01-01

    A rare pathway of HIV-1 resistance to small molecule CCR5 inhibitors such as Vicriviroc (VCV) involves changes solely in the gp41 fusion peptide (FP). Here, we show that the G516V change is critical to VCV resistance in PBMC and TZM-bl cells, although it must be accompanied by either M518V or F519I to have a substantial impact. Modeling VCV inhibition data from the two cell types indicated that G516V allows both double mutants to use VCV-CCR5 complexes for entry. The model further identified F519I as an independent determinant of preference for the unoccupied, high-VCV affinity form of CCR5. From inhibitor-free reversion cultures, we also identified a substitution in the inner domain of gp120, T244A, which appears to counter the resistance phenotype created by the FP substitutions. Examining the interplay of these changes will enhance our understanding of Env complex interactions that influence both HIV-1 entry and resistance to CCR5 inhibitors.

  14. Plant-derived chimeric virus particles for the diagnosis of primary Sjögren syndrome

    Directory of Open Access Journals (Sweden)

    Elisa eTinazzi

    2015-12-01

    Full Text Available Plants are ideal for the production of protein-based nanomaterials because they synthesize and assemble complex multimeric proteins that cannot be expressed efficiently using other platforms. Plant viruses can be thought of as self-replicating proteinaceous nanomaterials generally stable and easily produced in high titers. We used Potato virus X (PVX chimeric virus particles (CVPs and Cowpea mosaic virus (CPMV empty virus-like particles (eVLPs to display a linear peptide (lipo derived from human lipocalin , which is immunodominant in Sjögren’s syndrome (SjS and is thus recognized by autoantibodies in SjS patient serum. These virus-derived nanoparticles (VNPs were thus used to develop a diagnostic assay for SjS based on a direct enzyme linked immunosorbent assay (ELISA format. We found that PVX-lipo formulations were more sensitive than the chemically synthesized immunodominant peptide and equally specific when used to distinguish between healthy individuals and SjS patients. Our novel assay therefore allows the diagnosis of SjS using a simple, low-invasive serum test, contrasting with the invasive labial biopsy required for current tests. Our results demonstrate that nanomaterials based on plant viruses can be used as diagnostic reagents for SjS, and could also be developed for the diagnosis of other diseases.

  15. Functional impact of HIV coreceptor-binding site mutations

    International Nuclear Information System (INIS)

    Biscone, Mark J.; Miamidian, John L.; Muchiri, John M.; Baik, Sarah S.W.; Lee, Fang-Hua; Doms, Robert W.; Reeves, Jacqueline D.

    2006-01-01

    The bridging sheet region of the gp120 subunit of the HIV-1 Env protein interacts with the major virus coreceptors, CCR5 and CXCR4. We examined the impact of mutations in and adjacent to the bridging sheet region of an X4 tropic HIV-1 on membrane fusion and entry inhibitor susceptibility. When the V3-loop of this Env was changed so that CCR5 was used, the effects of these same mutations on CCR5 use were assayed as well. We found that coreceptor-binding site mutations had greater effects on CXCR4-mediated fusion and infection than when CCR5 was used as a coreceptor, perhaps related to differences in coreceptor affinity. The mutations also reduced use of the alternative coreceptors CCR3 and CCR8 to varying degrees, indicating that the bridging sheet region is important for the efficient utilization of both major and minor HIV coreceptors. As seen before with a primary R5 virus strain, bridging sheet mutations increased susceptibility to the CCR5 inhibitor TAK-779, which correlated with CCR5 binding efficiency. Bridging sheet mutations also conferred increased susceptibility to the CXCR4 ligand AMD-3100 in the context of the X4 tropic Env. However, these mutations had little effect on the rate of membrane fusion and little effect on susceptibility to enfuvirtide, a membrane fusion inhibitor whose activity is dependent in part on the rate of Env-mediated membrane fusion. Thus, mutations that reduce coreceptor binding and enhance susceptibility to coreceptor inhibitors can affect fusion and enfuvirtide susceptibility in an Env context-dependent manner

  16. Preclinical and clinical investigation of a CCR5 antagonist, AZD5672, in patients with rheumatoid arthritis receiving methotrexate

    NARCIS (Netherlands)

    Gerlag, Daniëlle M.; Hollis, Sally; Layton, Mark; Vencovský, Jiří; Szekanecz, Zoltán; Braddock, Martin; Tak, Paul P.; Oparanov, Boycho; Stoilov, Rumen; Yaneva, Tanya; Batalov, Anastas; Arteaga, Edgardo Tobias; Escalante, William Otero; Velez, Patricia; Restrepo, Jose Molina; Augustinova, Sevda; Blahova, Anna; Dvorak, Zdenek; Novosad, Libor; Rosa, Jan; Stehlikova, Helena; Vitek, Petr; Balazs, Tibor; Seregely, Katalin; Szombati, Istvan; Tarjan, Katalin; Csengei, Gabor; Galeazzi, Mauro; Saleniece, Sarmite; Saulite-Kandevica, Daina; Coleiro, Bernard; Badurski, Janusz; Brzosko, Marek; Chudzik, Dariusz; Gruszecka-Marczynska, Katarzyna; Hensel, Joanna; Pokrzywnicka-Gajek, Ines; Korpanty-Danda, Joanna; Sochocka-Bykowska, Malgorzata; Tlustochowicz, Witold; Stopinska-Polaszewska, Maria; Gluszko, Piotr; Nedelcovici, Corina; Radulescu, Florin; Gavrila, Mirea; Tanasescu, Coman; Korshunov, Nikolay; Matsievskaia, Galina; Damjanov, Nemanja; Dimic, Aleksandar

    2010-01-01

    To investigate both the preclinical effects of blocking the chemokine receptor CCR5 and the clinical effects of this approach on the signs and symptoms of rheumatoid arthritis (RA) in patients with active disease. Preclinical evaluations of AZD5672, a small-molecule antagonist of CCR5, were

  17. DIVA vaccine properties of the live chimeric pestivirus strain CP7_E2gif

    DEFF Research Database (Denmark)

    von Rosen, Tanya; Rangelova, Desislava Yordanova; Nielsen, Jens

    2014-01-01

    Live modified vaccines to protect against classical swine fever virus (CSFV), based on chimeric pestiviruses, have been developed to enable serological Differentiation of Infected from Vaccinated Animals (DIVA). In this context, the chimeric virus CP7_E2gif vaccine candidate is unique as it does...

  18. Co-Introduced Functional CCR2 Potentiates In Vivo Anti-Lung Cancer Functionality Mediated by T Cells Double Gene-Modified to Express WT1-Specific T-Cell Receptor

    Science.gov (United States)

    Asai, Hiroaki; Fujiwara, Hiroshi; An, Jun; Ochi, Toshiki; Miyazaki, Yukihiro; Nagai, Kozo; Okamoto, Sachiko; Mineno, Junichi; Kuzushima, Kiyotaka; Shiku, Hiroshi; Inoue, Hirofumi; Yasukawa, Masaki

    2013-01-01

    Background and Purpose Although gene-modification of T cells to express tumor-related antigen-specific T-cell receptor (TCR) or chimeric antigen receptor (CAR) has clinically proved promise, there still remains room to improve the clinical efficacy of re-directed T-cell based antitumor adoptive therapy. In order to achieve more objective clinical responses using ex vivo-expanded tumor-responsive T cells, the infused T cells need to show adequate localized infiltration into the tumor. Methodology/Principal Findings Human lung cancer cells variously express a tumor antigen, Wilms' Tumor gene product 1 (WT1), and an inflammatory chemokine, CCL2. However, CCR2, the relevant receptor for CCL2, is rarely expressed on activated T-lymphocytes. A HLA-A2402+ human lung cancer cell line, LK79, which expresses high amounts of both CCL2 and WT1 mRNA, was employed as a target. Normal CD8+ T cells were retrovirally gene-modified to express both CCR2 and HLA-A*2402-restricted and WT1235–243 nonapeptide-specific TCR as an effector. Anti-tumor functionality mediated by these effector cells against LK79 cells was assessed both in vitro and in vivo. Finally the impact of CCL2 on WT1 epitope-responsive TCR signaling mediated by the effector cells was studied. Introduced CCR2 was functionally validated using gene-modified Jurkat cells and human CD3+ T cells both in vitro and in vivo. Double gene-modified CD3+ T cells successfully demonstrated both CCL2-tropic tumor trafficking and cytocidal reactivity against LK79 cells in vitro and in vivo. CCL2 augmented the WT1 epitope-responsive TCR signaling shown by relevant luciferase production in double gene-modified Jurkat/MA cells to express luciferase and WT1-specific TCR, and CCL2 also dose-dependently augmented WT1 epitope-responsive IFN-γ production and CD107a expression mediated by these double gene-modifiedCD3+ T cells. Conclusion/Significance Introduction of the CCL2/CCR2 axis successfully potentiated in vivo anti-lung cancer

  19. Co-introduced functional CCR2 potentiates in vivo anti-lung cancer functionality mediated by T cells double gene-modified to express WT1-specific T-cell receptor.

    Directory of Open Access Journals (Sweden)

    Hiroaki Asai

    Full Text Available BACKGROUND AND PURPOSE: Although gene-modification of T cells to express tumor-related antigen-specific T-cell receptor (TCR or chimeric antigen receptor (CAR has clinically proved promise, there still remains room to improve the clinical efficacy of re-directed T-cell based antitumor adoptive therapy. In order to achieve more objective clinical responses using ex vivo-expanded tumor-responsive T cells, the infused T cells need to show adequate localized infiltration into the tumor. METHODOLOGY/PRINCIPAL FINDINGS: Human lung cancer cells variously express a tumor antigen, Wilms' Tumor gene product 1 (WT1, and an inflammatory chemokine, CCL2. However, CCR2, the relevant receptor for CCL2, is rarely expressed on activated T-lymphocytes. A HLA-A2402(+ human lung cancer cell line, LK79, which expresses high amounts of both CCL2 and WT1 mRNA, was employed as a target. Normal CD8(+ T cells were retrovirally gene-modified to express both CCR2 and HLA-A*2402-restricted and WT1(235-243 nonapeptide-specific TCR as an effector. Anti-tumor functionality mediated by these effector cells against LK79 cells was assessed both in vitro and in vivo. Finally the impact of CCL2 on WT1 epitope-responsive TCR signaling mediated by the effector cells was studied. Introduced CCR2 was functionally validated using gene-modified Jurkat cells and human CD3(+ T cells both in vitro and in vivo. Double gene-modified CD3(+ T cells successfully demonstrated both CCL2-tropic tumor trafficking and cytocidal reactivity against LK79 cells in vitro and in vivo. CCL2 augmented the WT1 epitope-responsive TCR signaling shown by relevant luciferase production in double gene-modified Jurkat/MA cells to express luciferase and WT1-specific TCR, and CCL2 also dose-dependently augmented WT1 epitope-responsive IFN-γ production and CD107a expression mediated by these double gene-modified CD3(+ T cells. CONCLUSION/SIGNIFICANCE: Introduction of the CCL2/CCR2 axis successfully potentiated in

  20. Human Eosinophils Express Functional CCR7

    Science.gov (United States)

    Ueki, Shigeharu; Estanislau, Jessica; Weller, Peter F.

    2013-01-01

    Human eosinophils display directed chemotactic activity toward an array of soluble chemokines. Eosinophils have been observed to migrate to draining lymph nodes in experimental models of allergic inflammation, yet it is unknown whether eosinophils express CCR7, a key chemokine receptor in coordinating leukocyte trafficking to lymph nodes. The purpose of this study is to demonstrate expression of CCR7 by human eosinophils and functional responses to CCL19 and CCL21, the known ligands of CCR7. Human eosinophils were purified by negative selection from healthy donors. CCR7 expression of freshly purified, unstimulated eosinophils and of IL-5–primed eosinophils was determined by flow cytometry and Western blot. Chemotaxis to CCL19 and CCL21 was measured in transwell assays. Shape changes to CCL19 and CCL21 were analyzed by flow cytometry and microscopy. Calcium fluxes of fluo-4 AM–loaded eosinophils were recorded by flow cytometry after chemokine stimulation. ERK phosphorylation of CCL19- and CCL21-stimulated eosinophils was measured by Western blot and Luminex assay. Human eosinophils expressed CCR7 as demonstrated by flow cytometry and Western blots. Eosinophils exhibited detectable cell surface expression of CCR7. IL-5–primed eosinophils exhibited chemotaxis toward CCL19 and CCL21 in a dose-dependent fashion. Upon stimulation with CCL19 or CCL21, IL-5–primed eosinophils demonstrated dose-dependent shape changes with polarization of F-actin and exhibited calcium influxes. Finally, primed eosinophils stimulated with CCL19 or CCL21 exhibited increased phosphorylation of ERK in response to both CCR7 ligands. We demonstrate that human eosinophils express CCR7 and have multipotent responses to the known ligands of CCR7. PMID:23449735

  1. Genetic Susceptibility to Cardiac and Digestive Clinical Forms of Chronic Chagas Disease: Involvement of the CCR5 59029 A/G Polymorphism.

    Science.gov (United States)

    de Oliveira, Amanda Priscila; Bernardo, Cássia Rubia; Camargo, Ana Vitória da Silveira; Ronchi, Luiz Sérgio; Borim, Aldenis Albaneze; de Mattos, Cinara Cássia Brandão; de Campos Júnior, Eumildo; Castiglioni, Lílian; Netinho, João Gomes; Cavasini, Carlos Eugênio; Bestetti, Reinaldo Bulgarelli; de Mattos, Luiz Carlos

    2015-01-01

    The clinical manifestations of chronic Chagas disease include the cardiac form of the disease and the digestive form. Not all the factors that act in the variable clinical course of this disease are known. This study investigated whether the CCR5Δ32 (rs333) and CCR5 59029 A/G (promoter region--rs1799987) polymorphisms of the CCR5 gene are associated with different clinical forms of chronic Chagas disease and with the severity of left ventricular systolic dysfunction in patients with chronic Chagas heart disease (CCHD). The antibodies anti-T. cruzi were identified by ELISA. PCR and PCR-RFLP were used to identify the CCR5Δ32 and CCR5 59029 A/G polymorphisms. The chi-square test was used to compare variables between groups. There was a higher frequency of the AA genotype in patients with CCHD compared with patients with the digestive form of the disease and the control group. The results also showed a high frequency of the AG genotype in patients with the digestive form of the disease compared to the other groups. The results of this study show that the CCR5Δ32 polymorphism does not seem to influence the different clinical manifestations of Chagas disease but there is involvement of the CCR5 59029 A/G polymorphism in susceptibility to the different forms of chronic Chagas disease. Besides, these polymorphisms do not influence left ventricular systolic dysfunction in patients with CCHD.

  2. Genetic Susceptibility to Cardiac and Digestive Clinical Forms of Chronic Chagas Disease: Involvement of the CCR5 59029 A/G Polymorphism.

    Directory of Open Access Journals (Sweden)

    Amanda Priscila de Oliveira

    Full Text Available The clinical manifestations of chronic Chagas disease include the cardiac form of the disease and the digestive form. Not all the factors that act in the variable clinical course of this disease are known. This study investigated whether the CCR5Δ32 (rs333 and CCR5 59029 A/G (promoter region--rs1799987 polymorphisms of the CCR5 gene are associated with different clinical forms of chronic Chagas disease and with the severity of left ventricular systolic dysfunction in patients with chronic Chagas heart disease (CCHD. The antibodies anti-T. cruzi were identified by ELISA. PCR and PCR-RFLP were used to identify the CCR5Δ32 and CCR5 59029 A/G polymorphisms. The chi-square test was used to compare variables between groups. There was a higher frequency of the AA genotype in patients with CCHD compared with patients with the digestive form of the disease and the control group. The results also showed a high frequency of the AG genotype in patients with the digestive form of the disease compared to the other groups. The results of this study show that the CCR5Δ32 polymorphism does not seem to influence the different clinical manifestations of Chagas disease but there is involvement of the CCR5 59029 A/G polymorphism in susceptibility to the different forms of chronic Chagas disease. Besides, these polymorphisms do not influence left ventricular systolic dysfunction in patients with CCHD.

  3. Antiviral propierties of 5,5'-dithiobis-2-nitrobenzoic acid and bacitracin against T-tropic human immunodeficiency virus type 1

    Directory of Open Access Journals (Sweden)

    Rodriguez-Padilla Cristina

    2011-03-01

    Full Text Available Abstract Bacitracin and the membrane-impermeant thiol reagent 5,5'-dithiobis-2-nitrobenzoic acid (DTNB are agents known to inhibit protein disulfide isomerase (PDI, a cell-surface protein critical in HIV-1 entry therefore they are fusion inhibitors (FI. Here we investigated the possibility that Bacitracin and or DTNB might have other antiviral activities besides FI. By means of residual activity assays, we found that both compounds showed antiviral activity only to viruses T-tropic HIV-1 strain. Cell-based fusion assays showed inhibition on HeLa-CD4-LTR-β-gal (CD4 and HL2/3 cells treated with Bacitracin, and DTNB with the latest compound we observed fusion inhibition on both cells but strikingly in HL2/3 cells (expressing Env indicating a possible activity on both, the cell membrane and the viral envelope. A time-of-addition experiment showed that both compounds act on HIV entry inhibition but DTNB also acts at late stages of the viral cycle. Lastly, we also found evidence of long-lasting host cell protection in vitro by DTNB, an important pharmacodynamic parameter for a topical microbicide against virus infection, hours after the extracellular drug was removed; this protection was not rendered by Bacitracin. These drugs proved to be leading compounds for further studies against HIV showing antiviral characteristics of interest.

  4. Travel to tropical areas: Zika virus disease

    CERN Multimedia

    CERN Medical Service

    2016-01-01

    Transmitted by the bite of a certain species of mosquitoes (Aedes), the Zika virus is spreading quickly in tropical areas of Central America, the Caribbean and South America.   Although no specific treatment nor vaccine is currently available, the most effective preventive measures are those focused on avoiding mosquito bites. There are no travel restrictions in place at present. However it is recommended that pregnant women defer travel plans to countries affected by the Zika virus. For further information on symptoms and prevention measures, please click on the Zika virus link or contact the Medical Service.

  5. Bioinformatic analysis of neurotropic HIV envelope sequences identifies polymorphisms in the gp120 bridging sheet that increase macrophage-tropism through enhanced interactions with CCR5

    International Nuclear Information System (INIS)

    Mefford, Megan E.; Kunstman, Kevin; Wolinsky, Steven M.; Gabuzda, Dana

    2015-01-01

    Macrophages express low levels of the CD4 receptor compared to T-cells. Macrophage-tropic HIV strains replicating in brain of untreated patients with HIV-associated dementia (HAD) express Envs that are adapted to overcome this restriction through mechanisms that are poorly understood. Here, bioinformatic analysis of env sequence datasets together with functional studies identified polymorphisms in the β3 strand of the HIV gp120 bridging sheet that increase M-tropism. D197, which results in loss of an N-glycan located near the HIV Env trimer apex, was detected in brain in some HAD patients, while position 200 was estimated to be under positive selection. D197 and T/V200 increased fusion and infection of cells expressing low CD4 by enhancing gp120 binding to CCR5. These results identify polymorphisms in the HIV gp120 bridging sheet that overcome the restriction to macrophage infection imposed by low CD4 through enhanced gp120–CCR5 interactions, thereby promoting infection of brain and other macrophage-rich tissues. - Highlights: • We analyze HIV Env sequences and identify amino acids in beta 3 of the gp120 bridging sheet that enhance macrophage tropism. • These amino acids at positions 197 and 200 are present in brain of some patients with HIV-associated dementia. • D197 results in loss of a glycan near the HIV Env trimer apex, which may increase exposure of V3. • These variants may promote infection of macrophages in the brain by enhancing gp120–CCR5 interactions

  6. Bioinformatic analysis of neurotropic HIV envelope sequences identifies polymorphisms in the gp120 bridging sheet that increase macrophage-tropism through enhanced interactions with CCR5

    Energy Technology Data Exchange (ETDEWEB)

    Mefford, Megan E., E-mail: megan_mefford@hms.harvard.edu [Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Boston, MA (United States); Kunstman, Kevin, E-mail: kunstman@northwestern.edu [Northwestern University Medical School, Chicago, IL (United States); Wolinsky, Steven M., E-mail: s-wolinsky@northwestern.edu [Northwestern University Medical School, Chicago, IL (United States); Gabuzda, Dana, E-mail: dana_gabuzda@dfci.harvard.edu [Department of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, Boston, MA (United States); Department of Neurology (Microbiology and Immunobiology), Harvard Medical School, Boston, MA (United States)

    2015-07-15

    Macrophages express low levels of the CD4 receptor compared to T-cells. Macrophage-tropic HIV strains replicating in brain of untreated patients with HIV-associated dementia (HAD) express Envs that are adapted to overcome this restriction through mechanisms that are poorly understood. Here, bioinformatic analysis of env sequence datasets together with functional studies identified polymorphisms in the β3 strand of the HIV gp120 bridging sheet that increase M-tropism. D197, which results in loss of an N-glycan located near the HIV Env trimer apex, was detected in brain in some HAD patients, while position 200 was estimated to be under positive selection. D197 and T/V200 increased fusion and infection of cells expressing low CD4 by enhancing gp120 binding to CCR5. These results identify polymorphisms in the HIV gp120 bridging sheet that overcome the restriction to macrophage infection imposed by low CD4 through enhanced gp120–CCR5 interactions, thereby promoting infection of brain and other macrophage-rich tissues. - Highlights: • We analyze HIV Env sequences and identify amino acids in beta 3 of the gp120 bridging sheet that enhance macrophage tropism. • These amino acids at positions 197 and 200 are present in brain of some patients with HIV-associated dementia. • D197 results in loss of a glycan near the HIV Env trimer apex, which may increase exposure of V3. • These variants may promote infection of macrophages in the brain by enhancing gp120–CCR5 interactions.

  7. Selective elimination of HIV-1-infected cells by Env-directed, HIV-1-based virus-like particles

    International Nuclear Information System (INIS)

    Peretti, Silvia; Schiavoni, Ilaria; Pugliese, Katherina; Federico, Maurizio

    2006-01-01

    We recently showed that both replicating and resting cells cultivated with ganciclovir (GCV) were killed when challenged with vesicular stomatitis virus G glycoprotein pseudotyped HIV-1-based virus-like particles (VLPs) carrying the Nef7 (i.e., an HIV-1 Nef mutant incorporating in virions at high levels)/herpes simplex virus-1 thymidine kinase (HSV-TK) fusion product. On this basis, a novel anti-HIV therapeutic approach based on Nef7/TK VLPs expressing X4 or R5 HIV cell receptor complexes has been attempted. We here report that (CD4-CXCR4) and (CD4-CCR5) Nef7-based VLPs efficiently enter cells infected by X4- or R5-tropic HIV-1 strains, respectively. Importantly, the delivery of the VLP-associated Nef7/TK led to cell death upon GCV treatment. Of interest, VLPs were effective also against non-replicating, HIV-1-infected primary human monocyte-derived macrophages. HIV-targeted VLPs represent a promising candidate for the treatment of persistently HIV-1-infected cells that are part of virus reservoirs resistant to HAART therapies

  8. HIV-infected individuals with the CCR delta32/CCR5 genotype have lower HIV RNA levels and higher CD4 cell counts in the early years of the infection than do patients with the wild type. Copenhagen AIDS Cohort Study Group

    DEFF Research Database (Denmark)

    Katzenstein, T L; Eugen-Olsen, J; Hofmann, B

    1997-01-01

    The relations among serum HIV RNA levels, CD4 cell counts, presence of the mutant CCR5-allele in heterozygous form, and clinical outcome was analyzed in 96 patients from the Copenhagen AIDS Cohort. In the early years of the infection, patients with the CCR5 delta32/CCR5 genotype had significantly...

  9. Enhanced stability of a chimeric hepatitis B core antigen virus-like-particle (HBcAg-VLP) by a C-terminal linker-hexahistidine-peptide.

    Science.gov (United States)

    Schumacher, Jens; Bacic, Tijana; Staritzbichler, René; Daneschdar, Matin; Klamp, Thorsten; Arnold, Philipp; Jägle, Sabrina; Türeci, Özlem; Markl, Jürgen; Sahin, Ugur

    2018-04-13

    Virus-like-particles (VLPs) are attractive nanoparticulate scaffolds for broad applications in material/biological sciences and medicine. Prior their functionalization, specific adaptations have to be carried out. These adjustments frequently lead to disordered particles, but the particle integrity is an essential factor for the VLP suitability. Therefore, major requirements for particle stabilization exist. The objective of this study was to evaluate novel stabilizing elements for functionalized chimeric hepatitis B virus core antigen virus-like particles (HBcAg-VLP), with beneficial characteristics for vaccine development, imaging or delivery. The effects of a carboxy-terminal polyhistidine-peptide and an intradimer disulfide-bridge on the stability of preclinically approved chimeric HBcAg-VLPs were assessed. We purified recombinant chimeric HBcAg-VLPs bearing different modified C-termini and compared their physical and chemical particle stability by quantitative protein-biochemical and biophysical techniques. We observed lower chemical resistance of T = 3- compared to T = 4-VLP (triangulation number) capsids and profound impairment of accessibility of hexahistidine-peptides in assembled VLPs. Histidines attached to the C-terminus were associated with superior mechanical and/or chemical particle stability depending on the number of histidine moieties. A molecular modeling approach based on cryo-electron microscopy and biolayer interferometry revealed the underlying structural mechanism for the strengthening of the integrity of VLPs. Interactions triggering capsid stabilization occur on a highly conserved residue on the basis of HBcAg-monomers as well as on hexahistidine-peptides of adjacent monomers. This new stabilization mechanism appears to mimic an evolutionary conserved stabilization concept for hepadnavirus core proteins. These findings establish the genetically simply transferable C-terminal polyhistidine-peptide as a general stabilizing element

  10. An E2-Substituted Chimeric Pestivirus With DIVA Vaccine Properties

    DEFF Research Database (Denmark)

    Rasmussen, Thomas Bruun; Uttenthal, Åse; Nielsen, Jens

    An advantage of the use of chimeric pestiviruses as modified live vaccines against classical swine fever (CSF) resides in their capacity to be manipulated to achieve the characteristics desired for safe and efficacious DIVA vaccines. We have recently generated a new chimeric virus, Riems26_E2gif...... vaccinated pigs were protected. This new chimeric pestivirus represents a C-strain based DIVA vaccine candidate that can be differentiated based on CSFV E2 specific antibodies....

  11. Simulated digestion for testing the stability of edible vaccine based on Cucumber mosaic virus (CMV) chimeric particle display Hepatitis C virus (HCV) peptide.

    Science.gov (United States)

    Vitti, Antonella; Nuzzaci, Maria; Condelli, Valentina; Piazzolla, Pasquale

    2014-01-01

    Edible vaccines must survive digestive process and preserve the specific structure of the antigenic peptide to elicit effective immune response. The stability of a protein to digestive process can be predicted by subjecting it to the in vitro assay with simulated gastric fluid (SGF) and simulated intestinal fluid (SIF). Here, we describe the protocol of producing and using chimeric Cucumber mosaic virus (CMV) displaying Hepatitis C virus (HCV) derived peptide (R9) in double copy as an oral vaccine. Its stability after treatment with SGF and SIF and the preservation of the antigenic properties were verified by SDS-PAGE and immuno western blot techniques.

  12. [Maraviroc: clinical trials results].

    Science.gov (United States)

    Chidiac, C; Katlama, C; Yeni, P

    2008-03-01

    Just over a decade after identification of chemokine receptors CCR5 and CXCR4 as coreceptors for HIV, maraviroc (Celsentri), the first CCR5 antagonist, has recently obtained its Marketing Authorization in the United States and Europe, for treatment of treatment-experienced adult patients infected with only CCR5-tropic HIV-1 detectable. CCR5 antagonists, after fusion inhibitor enfuvirtide available since 2003, also belong to entry inhibitors. These molecules, unlike previous antiretrovirals, do not target the virus but its target cell by blocking viral penetration. Maraviroc has shown its clinical efficacy in patients failing other antiretroviral classes. Its safety profile was similar to placebo in two large phase III trials. However, careful assessment of both hepatic and immunologic safety of this new therapeutic class is needed. Viral tropism testing has to be investigated before using maraviroc in the clinic, because CCR5 antagonists are not active against CXCR4 viruses. For the moment indicated for the treatment-experienced patient population, maraviroc could in the future benefit to other types of patients, depending on ongoing trials results.

  13. 27-Hydroxycholesterol and 7alpha-hydroxycholesterol trigger a sequence of events leading to migration of CCR5-expressing Th1 lymphocytes

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Sun-Mi, E-mail: lala1647@hanmail.net [Department of Pharmacology, Pusan National University, School of Medicine, Yangsan, Gyeongnam 626-870 (Korea, Republic of); Kim, Bo-Young, E-mail: kimboyoung@pusan.ac.kr [Department of Pharmacology, Pusan National University, School of Medicine, Yangsan, Gyeongnam 626-870 (Korea, Republic of); Lee, Sae-A, E-mail: saeah486@nate.com [Department of Pharmacology, Pusan National University, School of Medicine, Yangsan, Gyeongnam 626-870 (Korea, Republic of); Eo, Seong-Kug, E-mail: vetvirus@chonbuk.ac.kr [Laboratory of Microbiology, College of Veterinary Medicine and Bio-Safety Research Institute, Chonbuk National University, Jeonju, Jeonbuk 561-756 (Korea, Republic of); Yun, Yungdae, E-mail: yunyung@ewha.ac.kr [Department of Life Science, Ewha Womans University, Seoul 120-750 (Korea, Republic of); Kim, Chi-Dae, E-mail: chidkim@pusan.ac.kr [Department of Pharmacology, Pusan National University, School of Medicine, Yangsan, Gyeongnam 626-870 (Korea, Republic of); Kim, Koanhoi, E-mail: koanhoi@pusan.ac.kr [Department of Pharmacology, Pusan National University, School of Medicine, Yangsan, Gyeongnam 626-870 (Korea, Republic of)

    2014-02-01

    Th1 lymphocytes are predominant in atherosclerotic lesions. However, mechanisms involved in the Th1 predominance are unknown. We have investigated the possibility of Th1 lymphocyte recruitment in a cholesterol-rich milieu. A high cholesterol diet resulted in enhanced expression of CCR5 ligands, including CCL3 and CCL4, but not of proatherogenic CXCR3 ligands, in atherosclerotic arteries of ApoE{sup −/−} mice. 27-Hydroxycholesterol and 7α-hydroxycholesterol, cholesterol oxides (oxysterols) detected in abundance in atherosclerotic lesions, greatly induced the transcription of CCL3 and CCL4 genes in addition to enhancing secretion of corresponding proteins by THP-1 monocytic cells. However, an identical or even higher concentration of cholesterol, 7β-hydroxycholesterol, and 7-ketocholsterol did not influence expression of these chemokines. Conditioned media containing the CCR5 ligands secreted from THP-1 cells induced migration of Jurkat T cells expressing CCR5, a characteristic chemokine receptor of Th1 cells, but not of Jurkat T cells that do not express CCR5. The migration of CCR5-expressing Jurkat T cells was abrogated in the presence of a CCR5-neutralizing antibody. 27-Hydroxycholesterol and 7α-hydroxycholesterol enhanced phosphorylation of Akt. Pharmacological inhibitors of phosphoinositide-3-kinase/Akt pathways blocked transcription as well as secretion of CCL3 and CCL4 in conjunction with attenuated migration of CCR5-expressing Jurkat T cells. This is the first report on the involvement of cholesterol oxides in migration of distinct subtype of T cells. We propose that 27-hydroxycholesterol and 7α-hydroxycholesterol can trigger a sequence of events that leads to recruitment of Th1 lymphocytes and phosphoinositide-3-kinase/Akt pathways play a major role in the process. - Graphical abstract: Th1 lymphocytes are predominant in atherosclerotic lesions. However, mechanisms involved in the Th1 predominance are unknown. We have investigated the possibility of

  14. 27-Hydroxycholesterol and 7alpha-hydroxycholesterol trigger a sequence of events leading to migration of CCR5-expressing Th1 lymphocytes

    International Nuclear Information System (INIS)

    Kim, Sun-Mi; Kim, Bo-Young; Lee, Sae-A; Eo, Seong-Kug; Yun, Yungdae; Kim, Chi-Dae; Kim, Koanhoi

    2014-01-01

    Th1 lymphocytes are predominant in atherosclerotic lesions. However, mechanisms involved in the Th1 predominance are unknown. We have investigated the possibility of Th1 lymphocyte recruitment in a cholesterol-rich milieu. A high cholesterol diet resulted in enhanced expression of CCR5 ligands, including CCL3 and CCL4, but not of proatherogenic CXCR3 ligands, in atherosclerotic arteries of ApoE −/− mice. 27-Hydroxycholesterol and 7α-hydroxycholesterol, cholesterol oxides (oxysterols) detected in abundance in atherosclerotic lesions, greatly induced the transcription of CCL3 and CCL4 genes in addition to enhancing secretion of corresponding proteins by THP-1 monocytic cells. However, an identical or even higher concentration of cholesterol, 7β-hydroxycholesterol, and 7-ketocholsterol did not influence expression of these chemokines. Conditioned media containing the CCR5 ligands secreted from THP-1 cells induced migration of Jurkat T cells expressing CCR5, a characteristic chemokine receptor of Th1 cells, but not of Jurkat T cells that do not express CCR5. The migration of CCR5-expressing Jurkat T cells was abrogated in the presence of a CCR5-neutralizing antibody. 27-Hydroxycholesterol and 7α-hydroxycholesterol enhanced phosphorylation of Akt. Pharmacological inhibitors of phosphoinositide-3-kinase/Akt pathways blocked transcription as well as secretion of CCL3 and CCL4 in conjunction with attenuated migration of CCR5-expressing Jurkat T cells. This is the first report on the involvement of cholesterol oxides in migration of distinct subtype of T cells. We propose that 27-hydroxycholesterol and 7α-hydroxycholesterol can trigger a sequence of events that leads to recruitment of Th1 lymphocytes and phosphoinositide-3-kinase/Akt pathways play a major role in the process. - Graphical abstract: Th1 lymphocytes are predominant in atherosclerotic lesions. However, mechanisms involved in the Th1 predominance are unknown. We have investigated the possibility of Th1

  15. Challenge Pools of Hepatitis C Virus Genotypes 1–6 Prototype Strains: Replication Fitness and Pathogenicity in Chimpanzees and Human Liver–Chimeric Mouse Models

    Science.gov (United States)

    Bukh, Jens; Meuleman, Philip; Tellier, Raymond; Engle, Ronald E.; Feinstone, Stephen M.; Eder, Gerald; Satterfield, William C.; Govindarajan, Sugantha; Krawczynski, Krzysztof; Miller, Roger H.; Leroux-Roels, Geert; Purcell, Robert H.

    2010-01-01

    Chimpanzees represent the only animal model for studies of the natural history of hepatitis C virus (HCV). To generate virus stocks of important HCV variants, we infected chimpanzees with HCV strains of genotypes 1–6 and determined the infectivity titer of acute-phase plasma pools in additional animals. The courses of first- and second-passage infections were similar, with early appearance of viremia, HCV RNA titers of >104.7 IU/mL, and development of acute hepatitis; the chronicity rate was 56%. The challenge pools had titers of 103–105 chimpanzee infectious doses/mL. Human liver–chimeric mice developed high-titer infections after inoculation with the challenge viruses of genotypes 1–6. Inoculation studies with different doses of the genotype 1b pool suggested that a relatively high virus dose is required to consistently infect chimeric mice. The challenge pools represent a unique resource for studies of HCV molecular virology and for studies of pathogenesis, protective immunity, and vaccine efficacy in vivo. PMID:20353362

  16. Differential between Protein and mRNA Expression of CCR7 and SSTR5 Receptors in Crohn's Disease Patients

    Directory of Open Access Journals (Sweden)

    Nathalie Taquet

    2009-01-01

    Full Text Available Crohn's disease (CD is a multifactorial chronic inflammatory bowel disease of unknown cause. The aim of the present study was to explore if mRNA over-expression of SSTR5 and CCR7 found in CD patients could be correlated to respective protein expression. When compared to healthy donors, SSTR5 was over-expressed 417 ± 71 times in CD peripheral blood mononuclear cells (PBMCs. Flow cytometry experiments showed no correlation between mRNA and protein expression for SSTR5 in PBMCs. In an attempt to find a reason of such a high mRNA expression, SSTR5 present on CD PBMCs were tested and found as biologically active as on healthy cells. In biopsies of CD intestinal tissue, SSTR5 was not over-expressed but CCR7, unchanged in PBMCs, was over-expressed by 10 ± 3 times in the lamina propria. Confocal microscopy showed a good correlation of CCR7 mRNA and protein expression in CD intestinal biopsies. Our data emphasize flow and image cytometry as impossible to circumvent in complement to molecular biology so to avoid false interpretation on receptor expressions. Once confirmed by further large-scale studies, our preliminary results suggest a role for SSTR5 and CCR7 in CD pathogenesis.

  17. Attenuation of pathogenic Rift Valley fever virus strain through the chimeric S-segment encoding sandfly fever phlebovirus NSs or a dominant-negative PKR.

    Science.gov (United States)

    Nishiyama, Shoko; Slack, Olga A L; Lokugamage, Nandadeva; Hill, Terence E; Juelich, Terry L; Zhang, Lihong; Smith, Jennifer K; Perez, David; Gong, Bin; Freiberg, Alexander N; Ikegami, Tetsuro

    2016-11-16

    Rift Valley fever is a mosquito-borne zoonotic disease affecting ruminants and humans. Rift Valley fever virus (RVFV: family Bunyaviridae, genus Phlebovirus) causes abortions and fetal malformations in ruminants, and hemorrhagic fever, encephalitis, or retinitis in humans. The live-attenuated MP-12 vaccine is conditionally licensed for veterinary use in the US. However, this vaccine lacks a marker for the differentiation of vaccinated from infected animals (DIVA). NSs gene is dispensable for RVFV replication, and thus, rMP-12 strains lacking NSs gene is applicable to monitor vaccinated animals. However, the immunogenicity of MP-12 lacking NSs was not as high as parental MP-12. Thus, chimeric MP-12 strains encoding NSs from either Toscana virus (TOSV), sandfly fever Sicilian virus (SFSV) or Punta Toro virus Adames strain (PTA) were characterized previously. Although chimeric MP-12 strains are highly immunogenic, the attenuation through the S-segment remains unknown. Using pathogenic ZH501 strain, we aimed to demonstrate the attenuation of ZH501 strain through chimeric S-segment encoding either the NSs of TOSV, SFSV, PTA, or Punta Toro virus Balliet strain (PTB). In addition, we characterized rZH501 encoding a human dominant-negative PKR (PKRΔE7), which also enhances the immunogenicity of MP-12. Study done on mice revealed that attenuation of rZH501 occurred through the S-segment encoding either PKRΔE7 or SFSV NSs. However, rZH501 encoding either TOSV, PTA, or PTB NSs in the S-segment uniformly caused lethal encephalitis. Our results indicated that the S-segments encoding PKRΔE7 or SFSV NSs are attenuated and thus applicable toward next generation MP-12 vaccine candidates that encode a DIVA marker.

  18. Complexity and dynamics of HIV-1 chemokine receptor usage in a multidrug-resistant adolescent.

    Science.gov (United States)

    Cavarelli, Mariangela; Mainetti, Lara; Pignataro, Angela Rosa; Bigoloni, Alba; Tolazzi, Monica; Galli, Andrea; Nozza, Silvia; Castagna, Antonella; Sampaolo, Michela; Boeri, Enzo; Scarlatti, Gabriella

    2014-12-01

    Maraviroc (MVC) is licensed in clinical practice for patients with R5 virus and virological failure; however, in anecdotal reports, dual/mixed viruses were also inhibited. We retrospectively evaluated the evolution of HIV-1 coreceptor tropism in plasma and peripheral blood mononuclear cells (PBMCs) of an infected adolescent with a CCR5/CXCR4 Trofile profile who experienced an important but temporary immunological and virological response during a 16-month period of MVC-based therapy. Coreceptor usage of biological viral clones isolated from PBMCs was investigated in U87.CD4 cells expressing wild-type or chimeric CCR5 and CXCR4. Plasma and PBMC-derived viral clones were sequenced to predict coreceptor tropism using the geno2pheno algorithm from the V3 envelope sequence and pol gene-resistant mutations. From start to 8.5 months of MVC treatment only R5X4 viral clones were observed, whereas at 16 months the phenotype enlarged to also include R5 and X4 clones. Chimeric receptor usage suggested the preferential usage of the CXCR4 coreceptor by the R5X4 biological clones. According to phenotypic data, R5 viruses were susceptible, whereas R5X4 and X4 viruses were resistant to RANTES and MVC in vitro. Clones at 16 months, but not at baseline, showed an amino acidic resistance pattern in protease and reverse transcription genes, which, however, did not drive their tropisms. The geno2pheno algorithm predicted at baseline R5 viruses in plasma, and from 5.5 months throughout follow-up only CXCR4-using viruses. An extended methodological approach is needed to unravel the complexity of the phenotype and variation of viruses resident in the different compartments of an infected individual. The accurate evaluation of the proportion of residual R5 viruses may guide therapeutic intervention in highly experienced patients with limited therapeutic options.

  19. Simvastatin Inhibits IL-5-Induced Chemotaxis and CCR3 Expression of HL-60-Derived and Human Primary Eosinophils.

    Science.gov (United States)

    Fu, Chia-Hsiang; Tsai, Wan-Chun; Lee, Ta-Jen; Huang, Chi-Che; Chang, Po-Hung; Su Pang, Jong-Hwei

    2016-01-01

    IL-5-induced chemotaxis of eosinophils is an important feature of allergic airway inflammatory diseases. Simvastatin, a lipid lowering agent, has been shown to exhibit anti-inflammatory and anti-allergic effects. Our aim was to investigate the effect of simvastatin on IL-5-induced eosinophil chemotaxis and its regulatory mechanisms. Eosinophils were derived by treating HL-60 clone 15 (HC15) cells with butyric acid (BA) in an alkaline condition or through direct isolation from human peripheral blood. The expressions of CC chemokine receptor 3 (CCR3) and interleukin (IL)-5 receptors (IL5Rα and β) were analyzed using RT/real-time PCR. The granular proteins were stained using fast green. Eotaxin-induced chemotaxis was measured using a transwell migration assay. CCR3 protein expression was revealed by immunocytochemistry. An animal model of allergic rhinitis was established by challenging Sprague-Dawley® rats repeatedly with ovalbumin. Butyric acid significantly increased the expression of IL5Rα and IL5Rβ, CCR3 and granular proteins in HC15 cells, indicating the maturation of eosinophils (BA-E cells). IL-5 further enhanced the CCR3 expression at both the mRNA and protein levels and the eotaxin-induced chemotaxis of BA-E cells. Simvastatin inhibited the effects of IL-5 on BA-E cells, but not in the presence of mevalonate. Similar results were also exhibited in human primary eosinophils. In vivo animal studies further confirmed that oral simvastatin could significantly suppress the infiltration of eosinophils into turbinate tissues of allergic rats. Therefore, simvastatin was demonstrated to inhibit IL-5-induced CCR3 expression and chemotaxis of eosinophils mediated via the mevalonate pathway. We confirmed that simvastatin also reduced eosinophilic infiltration in allergic rhinitis.

  20. Genetic Susceptibility to Cardiac and Digestive Clinical Forms of Chronic Chagas Disease: Involvement of the CCR5 59029 A/G Polymorphism

    OpenAIRE

    de Oliveira, Amanda Priscila; Bernardo, C?ssia Rubia; Camargo, Ana Vit?ria da Silveira; Ronchi, Luiz S?rgio; Borim, Aldenis Albaneze; Brand?o de Mattos, Cinara C?ssia; de Campos J?nior, Eumildo; Castiglioni, L?lian; Netinho, Jo?o Gomes; Cavasini, Carlos Eug?nio; Bestetti, Reinaldo Bulgarelli; de Mattos, Luiz Carlos

    2015-01-01

    The clinical manifestations of chronic Chagas disease include the cardiac form of the disease and the digestive form. Not all the factors that act in the variable clinical course of this disease are known. This study investigated whether the CCR5Δ32 (rs333) and CCR5 59029 A/G (promoter region--rs1799987) polymorphisms of the CCR5 gene are associated with different clinical forms of chronic Chagas disease and with the severity of left ventricular systolic dysfunction in patients with chronic C...

  1. Engineering HIV-1-resistant T-cells from short-hairpin RNA-expressing hematopoietic stem/progenitor cells in humanized BLT mice.

    Directory of Open Access Journals (Sweden)

    Gene-Errol E Ringpis

    Full Text Available Down-regulation of the HIV-1 coreceptor CCR5 holds significant potential for long-term protection against HIV-1 in patients. Using the humanized bone marrow/liver/thymus (hu-BLT mouse model which allows investigation of human hematopoietic stem/progenitor cell (HSPC transplant and immune system reconstitution as well as HIV-1 infection, we previously demonstrated stable inhibition of CCR5 expression in systemic lymphoid tissues via transplantation of HSPCs genetically modified by lentiviral vector transduction to express short hairpin RNA (shRNA. However, CCR5 down-regulation will not be effective against existing CXCR4-tropic HIV-1 and emergence of resistant viral strains. As such, combination approaches targeting additional steps in the virus lifecycle are required. We screened a panel of previously published shRNAs targeting highly conserved regions and identified a potent shRNA targeting the R-region of the HIV-1 long terminal repeat (LTR. Here, we report that human CD4(+ T-cells derived from transplanted HSPC engineered to co-express shRNAs targeting CCR5 and HIV-1 LTR are resistant to CCR5- and CXCR4- tropic HIV-1-mediated depletion in vivo. Transduction with the combination vector suppressed CXCR4- and CCR5- tropic viral replication in cell lines and peripheral blood mononuclear cells in vitro. No obvious cytotoxicity or interferon response was observed. Transplantation of combination vector-transduced HSPC into hu-BLT mice resulted in efficient engraftment and subsequent stable gene marking and CCR5 down-regulation in human CD4(+ T-cells within peripheral blood and systemic lymphoid tissues, including gut-associated lymphoid tissue, a major site of robust viral replication, for over twelve weeks. CXCR4- and CCR5- tropic HIV-1 infection was effectively inhibited in hu-BLT mouse spleen-derived human CD4(+ T-cells ex vivo. Furthermore, levels of gene-marked CD4(+ T-cells in peripheral blood increased despite systemic infection with either

  2. Neutralization of X4- and R5-tropic HIV-1 NL4-3 variants by HOCl-modified serum albumins

    Directory of Open Access Journals (Sweden)

    Schwalbe Birco

    2010-06-01

    Full Text Available Abstract Background Myeloperoxidase (MPO, an important element of the microbicidal activity of neutrophils, generates hypochlorous acid (HOCl from H2O2 and chloride, which is released into body fluids. Besides its direct microbicidal activity, HOCl can react with amino acid residues and HOCl-modified proteins can be detected in vivo. Findings This report is based on binding studies of HOCl-modified serum albumins to HIV-1 gp120 and three different neutralization assays using infectious virus. The binding studies were carried out by surface plasmon resonance spectroscopy and by standard ELISA techniques. Virus neutralization assays were carried out using HIV-1 NL4-3 virus and recombinant strains with CXCR4 and CCR5 coreceptor usage. Viral infection was monitored by a standard p24 or X-gal staining assay. Our data demonstrate that HOCl-modified mouse-, bovine- and human serum albumins all bind to the HIV-1 NL4-3 gp120 (LAV glycoprotein in contrast to non-modified albumin. Binding of HOCl-modified albumin to gp120 correlated to the blockade of CD4 as well as that of V3 loop specific monoclonal antibody binding. In neutralization experiments, HOCl-modified serum albumins inhibited replication and syncytium formation of the X4- and R5-tropic NL4-3 isolates in a dose dependent manner. Conclusions Our data indicate that HOCl-modified serum albumin veils the binding site for CD4 and the V3 loop on gp120. Such masking of the viral gp120/gp41 envelope complex might be a simple but promising strategy to inactivate HIV-1 and therefore prevent infection when HOCl-modified serum albumin is applied, for example, as a topical microbicide.

  3. CCL3L1-CCR5 genotype improves the assessment of AIDS Risk in HIV-1-infected individuals.

    Science.gov (United States)

    Kulkarni, Hemant; Agan, Brian K; Marconi, Vincent C; O'Connell, Robert J; Camargo, Jose F; He, Weijing; Delmar, Judith; Phelps, Kenneth R; Crawford, George; Clark, Robert A; Dolan, Matthew J; Ahuja, Sunil K

    2008-09-08

    Whether vexing clinical decision-making dilemmas can be partly addressed by recent advances in genomics is unclear. For example, when to initiate highly active antiretroviral therapy (HAART) during HIV-1 infection remains a clinical dilemma. This decision relies heavily on assessing AIDS risk based on the CD4+ T cell count and plasma viral load. However, the trajectories of these two laboratory markers are influenced, in part, by polymorphisms in CCR5, the major HIV coreceptor, and the gene copy number of CCL3L1, a potent CCR5 ligand and HIV-suppressive chemokine. Therefore, we determined whether accounting for both genetic and laboratory markers provided an improved means of assessing AIDS risk. In a prospective, single-site, ethnically-mixed cohort of 1,132 HIV-positive subjects, we determined the AIDS risk conveyed by the laboratory and genetic markers separately and in combination. Subjects were assigned to a low, moderate or high genetic risk group (GRG) based on variations in CCL3L1 and CCR5. The predictive value of the CCL3L1-CCR5 GRGs, as estimated by likelihood ratios, was equivalent to that of the laboratory markers. GRG status also predicted AIDS development when the laboratory markers conveyed a contrary risk. Additionally, in two separate and large groups of HIV+ subjects from a natural history cohort, the results from additive risk-scoring systems and classification and regression tree (CART) analysis revealed that the laboratory and CCL3L1-CCR5 genetic markers together provided more prognostic information than either marker alone. Furthermore, GRGs independently predicted the time interval from seroconversion to CD4+ cell count thresholds used to guide HAART initiation. The combination of the laboratory and genetic markers captures a broader spectrum of AIDS risk than either marker alone. By tracking a unique aspect of AIDS risk distinct from that captured by the laboratory parameters, CCL3L1-CCR5 genotypes may have utility in HIV clinical management

  4. Ancient DNA Investigation of a Medieval German Cemetery Confirms Long-Term Stability of CCR5-Δ32 Allele Frequencies in Central Europe.

    Science.gov (United States)

    Bouwman, Abigail; Shved, Natallia; Akgül, Gülfirde; Rühli, Frank; Warinner, Christina

    2017-04-01

    The CCR5-Δ32 mutation present in European populations is among the most prominently debated cases of recent positive selection in humans. This allele, a 32-bp deletion that renders the T-cell CCR5 receptor nonfunctional, has important epidemiological and public health significance, as homozygous carriers are resistant to several HIV strains. However, although the function of this allele in preventing HIV infection is now well described, its human evolutionary origin is poorly understood. Initial attempts to determine the emergence of the CCR5-Δ32 allele pointed to selection during the 14th-century Black Death pandemic; however, subsequent analyses suggest that the allele rose in frequency more than 5,000 years ago, possibly through drift. Recently, three studies have identified populations predating the 14th century CE that are positive for the CCR5-Δ32 allele, supporting the claim for a more ancient origin. However, these studies also suggest poorly understood regional differences in the recent evolutionary history of the CCR5-Δ32 allele. Here a new hydrolysis-probe-based real-time PCR assay was designed to ascertain CCR5 allele frequency in 53 individuals from a 10th- to 12th-century CE church and convent complex in central Germany that predates outbreaks of the Black Death pandemic. High-confidence genotypes were obtained for 32 individuals, and results show that CCR5-Δ32 allele frequency has remained unchanged in this region of Central Europe over the last millennium, suggesting that there has been no strong positive selective pressure over this time period and confirming a more ancient origin for the allele.

  5. Characterization of oligosaccharide structures on a chimeric respiratory syncytial virus protein expressed in insect cell line Sf9

    International Nuclear Information System (INIS)

    Wathen, M.W.; Aeed, P.A.; Elhammer, A.P.

    1991-01-01

    The oligosaccharide structures added to a chimeric protein (FG) composed of the extracellular domains of respiratory syncytial virus F and G proteins, expressed in the insect cell line Sf9, were investigated. Cells were labeled in vivo with [ 3 H]glucosamine and infected wit a recombinant baculovirus containing the FG gene. The secreted chimeric protein was isolated by immunoprecipitation and subjected to oligosaccharide analysis. The FG protein contains two types of O-linked oligosaccharides: GalNAc and Galβ1-3GalNAc constituting 17 and 66% of the total number of structures respectively. Only one type of N-linked oligosaccharide, constituting the remaining 17% of the structures on FG, was detected: a trimannosyl core structure with a fucose residue linked α1-6 to the asparagine-linked N-acetylglucosamine

  6. Chemokine Ligand 5 (CCL5 and chemokine receptor (CCR5 genetic variants and prostate cancer risk among men of African Descent: a case-control study

    Directory of Open Access Journals (Sweden)

    Kidd LaCreis R

    2012-11-01

    Full Text Available Abstract Background Chemokine and chemokine receptors play an essential role in tumorigenesis. Although chemokine-associated single nucleotide polymorphisms (SNPs are associated with various cancers, their impact on prostate cancer (PCA among men of African descent is unknown. Consequently, this study evaluated 43 chemokine-associated SNPs in relation to PCA risk. We hypothesized inheritance of variant chemokine-associated alleles may lead to alterations in PCA susceptibility, presumably due to variations in antitumor immune responses. Methods Sequence variants were evaluated in germ-line DNA samples from 814 African-American and Jamaican men (279 PCA cases and 535 controls using Illumina’s Goldengate genotyping system. Results Inheritance of CCL5 rs2107538 (AA, GA+AA and rs3817655 (AA, AG, AG+AA genotypes were linked with a 34-48% reduction in PCA risk. Additionally, the recessive and dominant models for CCR5 rs1799988 and CCR7 rs3136685 were associated with a 1.52-1.73 fold increase in PCA risk. Upon stratification, only CCL5 rs3817655 and CCR7 rs3136685 remained significant for the Jamaican and U.S. subgroups, respectively. Conclusions In summary, CCL5 (rs2107538, rs3817655 and CCR5 (rs1799988 sequence variants significantly modified PCA susceptibility among men of African descent, even after adjusting for age and multiple comparisons. Our findings are only suggestive and require further evaluation and validation in relation to prostate cancer risk and ultimately disease progression, biochemical/disease recurrence and mortality in larger high-risk subgroups. Such efforts will help to identify genetic markers capable of explaining disproportionately high prostate cancer incidence, mortality, and morbidity rates among men of African descent.

  7. Regulation of CCR7-dependent cell migration through?CCR7 homodimer formation

    OpenAIRE

    Kobayashi, Daichi; Endo, Masataka; Ochi, Hirotaka; Hojo, Hironobu; Miyasaka, Masayuki; Hayasaka, Haruko

    2017-01-01

    The chemokine receptor CCR7 contributes to various physiological and pathological processes including T cell maturation, T cell migration from the blood into secondary lymphoid tissues, and tumor cell metastasis to lymph nodes. Although a previous study suggested that the efficacy of CCR7 ligand-dependent T cell migration correlates with CCR7 homo- and heterodimer formation, the exact extent of contribution of the CCR7 dimerization remains unclear. Here, by inducing or disrupting CCR7 dimers,...

  8. Anti-Bovine Programmed Death-1 Rat–Bovine Chimeric Antibody for Immunotherapy of Bovine Leukemia Virus Infection in Cattle

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    Tomohiro Okagawa

    2017-06-01

    Full Text Available Blockade of immunoinhibitory molecules, such as programmed death-1 (PD-1/PD-ligand 1 (PD-L1, is a promising strategy for reinvigorating exhausted T cells and preventing disease progression in a variety of chronic infections. Application of this therapeutic strategy to cattle requires bovinized chimeric antibody targeting immunoinhibitory molecules. In this study, anti-bovine PD-1 rat–bovine chimeric monoclonal antibody 5D2 (Boch5D2 was constructed with mammalian expression systems, and its biochemical function and antiviral effect were characterized in vitro and in vivo using cattle infected with bovine leukemia virus (BLV. Purified Boch5D2 was capable of detecting bovine PD-1 molecules expressed on cell membranes in flow cytometric analysis. In particular, Biacore analysis determined that the binding affinity of Boch5D2 to bovine PD-1 protein was similar to that of the original anti-bovine PD-1 rat monoclonal antibody 5D2. Boch5D2 was also capable of blocking PD-1/PD-L1 binding at the same level as 5D2. The immunomodulatory and therapeutic effects of Boch5D2 were evaluated by in vivo administration of the antibody to a BLV-infected calf. Inoculated Boch5D2 was sustained in the serum for a longer period. Boch5D2 inoculation resulted in activation of the proliferation of BLV-specific CD4+ T cells and decrease in the proviral load of BLV in the peripheral blood. This study demonstrates that Boch5D2 retains an equivalent biochemical function to that of the original antibody 5D2 and is a candidate therapeutic agent for regulating antiviral immune response in vivo. Clinical efficacy of PD-1/PD-L1 blockade awaits further experimentation with a large number of animals.

  9. POTENTIAL EXPANSION OF TROPICAL VIRUSES IN SERBIA AND EUROPE

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    Zorana Deljanin

    2015-12-01

    Full Text Available The current, hot topic is the risk of introducing new vector-borne diseases and harmful ectoparasites into Europe, or of the geographic extension of the existing ones. There are many doubts that global warming affects the transfer of a number of vectorborne diseases. Special emphasis was placed on spreading the Lyme disease, tick-borne encephalitis, West Nile fever and leishmaniasis, the recurrence of malaria and dengue fever. Climate models predict a 2-5ºC temperature increase and a significant increase in rainfall in Europe in the following years. However, non-environmental variables such as socio-economic situation and agriculture should be considered. The main problem can be expected when new viruses emerge. As they change, their mutations can enter into the population and thus have “the greater potential” for the spread of the epidemic. The control network of the health system in our country and in Europe is very dense, and the outbreak of the virus can be always registered, giving the authorities enough time to take measures. Although modeling studies indicate that climate change could increase the risk of transmission of vector-transmitted diseases in Serbia and Europe, historical analyses indicate that, at least for malaria, socio-economic conditions in combination with effective surveillance and early treatment are likely to prevent further spread, which is the main task of the Public Health Institutes. Tropical medicine experts said that the so-called supervirus causes mutations of the virus, and represents the greatest danger for human population. The circumstances that allow such a development already exist, an additional climate change is not necessary. The challenge for future research is the mechanism of tropical viruses and their persistence in endemic foci in temperate climate area in Europe.

  10. Effectiveness and cost of treatment with maraviroc in HIV infection

    Directory of Open Access Journals (Sweden)

    Viola Sacchi

    2009-12-01

    Full Text Available Since 1995, life expectancy and quality of life of HIV patients improved significantly due to the use of Highly Active Anti-Retroviral Therapy (HAART, consisting of different combinations of three classes of antiretroviral agents, nucleoside and non-nucleoside reverse transcriptase inhibitors, and protease inhibitors. Recently, new treatment options for individuals developing resistance to these drugs have become available, with the appearance of new drug classes like integrase inhibitors, fusion inhibitors and CCR5 antagonists. Maraviroc is the first antiretroviral agent belonging to the latter drug class approved for clinical use. CCR5 receptor antagonists act by blocking the interaction of the HIV virus with the CCR5 chemokine receptor, a co-receptor essential to the entry process of R5-tropic viruses. The drug is indicated, in combination with other antiretroviral products, for treatment-experienced adult patients infected with only CCR5-tropic HIV-1 detectable virus strains. Results of main phase III clinical trials indicate that maraviroc, in combination with optimized background therapy (OBT, causes significantly greater reductions in viral load and increases in CD4+ cell count, as compared to OBT alone in this kind of patients. In Italy, the monthly cost of maraviroc therapy is about € 780. A number of economic evaluations, performed for different settings, demonstrate that the therapy including maraviroc is cost-effective if compared to OBT alone, determining an ICER generally below the threshold of three times the GDP per capita. In the Italian context, the ICER determined by OBT + maraviroc vs OBT alone is approximately 45,000 €/LYG.

  11. Nanoparticles containing siRNA to silence CD4 and CCR5 reduce expression of these receptors and inhibit HIV-1 infection in human female reproductive tract tissue explants

    Directory of Open Access Journals (Sweden)

    Susan K. Eszterhas

    2011-09-01

    Full Text Available Human Immunodeficiency Virus-type 1 (HIV- 1 binds to CD4 and CCR5 receptors on target cells in the human female reproductive tract. We sought to determine whether reducing levels of messenger RNA (mRNA transcripts that encode these receptors in female reproductive tract cells could protect mucosal tissue explants from HIV- 1 infection. Explants prepared from the endometrium, endocervix, and ectocervix of hysterectomy tissues from HIV-1 sero-negative women were exposed to nanoparticles containing CD4- and CCR5-specific short-interfering RNA (siRNA sequences. Explants were then exposed two days later to HIV-1, and HIV-1 reverse transcripts were measured five days post-infection. Explants treated with nanoparticles containing CD4- and CCR5-specific siRNA showed reduced levels of CD4 and CCR5 transcripts, and significantly lower levels of HIV-1 reverse transcripts compared to those treated with an irrelevant siRNA. In female reproductive tract explants and in peripheral blood cell cultures, siRNA transfection induced the secretion of IFN-alpha (IFN-α, a potent antiviral cytokine. In female mice, murine-specific Cd4-siRNA nanoparticles instilled within the uterus significantly reduced murine Cd4 transcripts by day 3. Our findings demonstrate that siRNA nanoparticles reduce expression of HIV-1 infectivity receptors in human female reproductive tract tissues and also inhibit HIV-1 infection. Murine studies demonstrate that nanoparticles can penetrate the reproductive tract tissues in vivo and silence gene expression. The induction of IFN-α after siRNA transfection can potentially contribute to the antiviral effect. These findings support the therapeutic development of nanoparticles to deliver siRNA molecules to silence host cell receptors in the female reproductive tract as a novel microbicide to inhibit mucosal HIV-1 transmission.

  12. CCL3L1-CCR5 genotype influences durability of immune recovery during antiretroviral therapy of HIV-1–infected individuals

    Science.gov (United States)

    Ahuja, Sunil K; Kulkarni, Hemant; Catano, Gabriel; Agan, Brian K; Camargo, Jose F; He, Weijing; O'Connell, Robert J; Marconi, Vincent C; Delmar, Judith; Eron, Joseph; Clark, Robert A; Frost, Simon; Martin, Jeffrey; Ahuja, Seema S; Deeks, Steven G; Little, Susan; Richman, Douglas; Hecht, Frederick M; Dolan, Matthew J

    2008-01-01

    The basis for the extensive variability seen in the reconstitution of CD4+ T cell counts in HIV-infected individuals receiving highly active antiretroviral therapy (HAART) is not fully known. Here, we show that variations in CCL3L1 gene dose and CCR5 genotype, but not major histocompatibility complex HLA alleles, influence immune reconstitution, especially when HAART is initiated at <350 CD4+ T cells/mm3. The CCL3L1-CCR5 genotypes favoring CD4+ T cell recovery are similar to those that blunted CD4+ T cell depletion during the time before HAART became available (pre-HAART era), suggesting that a common CCL3L1-CCR5 genetic pathway regulates the balance between pathogenic and reparative processes from early in the disease course. Hence, CCL3L1-CCR5 variations influence HIV pathogenesis even in the presence of HAART and, therefore, may prospectively identify subjects in whom earlier initiation of therapy is more likely to mitigate immunologic failure despite viral suppression by HAART. Furthermore, as reconstitution of CD4+ cells during HAART is more sensitive to CCL3L1 dose than to CCR5 genotypes, CCL3L1 analogs might be efficacious in supporting immunological reconstitution. PMID:18376407

  13. Determination of Coreceptor Usage of Human Immunodeficiency Virus Type 1 from Patient Plasma Samples by Using a Recombinant Phenotypic Assay

    Science.gov (United States)

    Trouplin, Virginie; Salvatori, Francesca; Cappello, Fanny; Obry, Veronique; Brelot, Anne; Heveker, Nikolaus; Alizon, Marc; Scarlatti, Gabriella; Clavel, François; Mammano, Fabrizio

    2001-01-01

    We developed a recombinant virus technique to determine the coreceptor usage of human immunodeficiency virus type 1 (HIV-1) from plasma samples, the source expected to represent the most actively replicating virus population in infected subjects. This method is not subject to selective bias associated with virus isolation in culture, a step required for conventional tropism determination procedures. The addition of a simple subcloning step allowed semiquantitative evaluation of virus populations with a different coreceptor (CCR5 or CXCR4) usage specificity present in each plasma sample. This procedure detected mixtures of CCR5- and CXCR4-exclusive virus populations as well as dualtropic viral variants, in variable proportions. Sequence analysis of dualtropic clones indicated that changes in the V3 loop are necessary for the use of CXCR4 as a coreceptor, but the overall context of the V1-V3 region is important to preserve the capacity to use CCR5. This convenient technique can greatly assist the study of virus evolution and compartmentalization in infected individuals. PMID:11119595

  14. Reanalysis of Coreceptor Tropism in HIV-1–Infected Adults Using a Phenotypic Assay with Enhanced Sensitivity

    Science.gov (United States)

    Goetz, Mathew Bidwell; Leduc, Robert; Skowron, Gail; Su, Zhaohui; Chan, Ellen S.; Heera, Jayyant; Chapman, Doug; Spritzler, John; Reeves, Jacqueline D.; Gulick, Roy M.; Coakley, Eoin

    2011-01-01

    The enhanced-sensitivity Trofile assay (TF-ES; Monogram Biosciences) was used to retest coreceptor tropism samples from 4 different cohorts of HIV-1–infected patients. Nine percent to 26% of patients with CCR5-tropic virus by the original Trofile assay had CXCR4-using virus by TF-ES. Lower CD4 cell counts were associated with CXCR4-using virus in all cohorts. PMID:21427401

  15. Development of transgenic watermelon resistant to Cucumber mosaic virus and Watermelon mosaic virus by using a single chimeric transgene construct.

    Science.gov (United States)

    Lin, Ching-Yi; Ku, Hsin-Mei; Chiang, Yi-Hua; Ho, Hsiu-Yin; Yu, Tsong-Ann; Jan, Fuh-Jyh

    2012-10-01

    Watermelon, an important fruit crop worldwide, is prone to attack by several viruses that often results in destructive yield loss. To develop a transgenic watermelon resistant to multiple virus infection, a single chimeric transgene comprising a silencer DNA from the partial N gene of Watermelon silver mottle virus (WSMoV) fused to the partial coat protein (CP) gene sequences of Cucumber mosaic virus (CMV), Cucumber green mottle mosaic virus (CGMMV) and Watermelon mosaic virus (WMV) was constructed and transformed into watermelon (cv. Feeling) via Agrobacterium-mediated transformation. Single or multiple transgene copies randomly inserted into various locations in the genome were confirmed by Southern blot analysis. Transgenic watermelon R(0) plants were individually challenged with CMV, CGMMV or WMV, or with a mixture of these three viruses for resistance evaluation. Two lines were identified to exhibit resistance to CMV, CGMMV, WMV individually, and a mixed inoculation of the three viruses. The R(1) progeny of the two resistant R(0) lines showed resistance to CMV and WMV, but not to CGMMV. Low level accumulation of transgene transcripts in resistant plants and small interfering (si) RNAs specific to CMV and WMV were readily detected in the resistant R(1) plants by northern blot analysis, indicating that the resistance was established via RNA-mediated post-transcriptional gene silencing (PTGS). Loss of the CGMMV CP-transgene fragment in R1 progeny might be the reason for the failure to resistant CGMMV infection, as shown by the absence of a hybridization signal and no detectable siRNA specific to CGMMV in Southern and northern blot analyses. In summary, this study demonstrated that fusion of different viral CP gene fragments in transgenic watermelon contributed to multiple virus resistance via PTGS. The construct and resistant watermelon lines developed in this study could be used in a watermelon breeding program for resistance to multiple viruses.

  16. Influence of CCR7 ligand DNA preexposure on the magnitude and duration of immunity

    International Nuclear Information System (INIS)

    Lee, Yunsang; Seong, Kug Eo; Rouse, Richard J.D.; Rouse, Barry T.

    2003-01-01

    The CC chemokine receptor (CCR) 7 ligands CCL21 and CCL19 were recently described as essential elements for establishing the microenvironment needed to initiate optimal immune responses in secondary lymphoid tissues. In the present study we have kinetically investigated the primary responses of naive DO11.10 TCR-transgenic CD4+ T cells (OVA323-339 peptide specific) adoptively transferred into normal BALB/c mice given plasmid DNA encoding CCR7 ligands. The primary responses of CD4+ Tg-T cells in CCR7 ligand DNA recipients occurred more promptly, reaching levels higher than those observed in vector controls. In line with enhanced specific immunity, the T-cell population in CCR7 ligand recipients underwent more in vivo cell division following Ag stimulation, and a higher percentage of Ag-specific T cells expressed an activation phenotype. Moreover, the enhanced primary responses of naive CD4+ T cells appeared to act via affects on migration and maturation of CD11c+ dendritic cells in the draining lymph nodes. In addition following mucosal challenge of herpes simplex virus-immune mice with virus, those that had received CCL21 or CCL19 during priming contained a higher frequency of responding CD4 T cells in lymph nodes and the site of infection. Moreover, CCL21- and CCL19-treated mice showed less severe disease and better survival following challenge. Our results are discussed in terms of the relevance of CCR7 ligand preimmunization to improve vaccine

  17. Capsid proteins from field strains of foot-and-mouth disease virus confer a pathogenic phenotype in cattle on an attenuated, cell-culture-adapted virus

    DEFF Research Database (Denmark)

    Bøtner, Anette; Kakker, Naresh K.; Barbezange, Cyril

    2011-01-01

    Chimeric foot-and-mouth disease viruses (FMDVs) have been generated from plasmids containing full-length FMDV cDNAs and characterized. The parental virus cDNA was derived from the cell-culture-adapted O1Kaufbeuren B64 (O1K B64) strain. Chimeric viruses, containing capsid coding sequences derived...... cells than the rescued parental O1K B64 virus. The two chimeric viruses displayed the expected antigenicity in serotype-specific antigen ELISAs. Following inoculation of each virus into cattle, the rescued O1K B64 strain proved to be attenuated whereas, with each chimeric virus, typical clinical signs...... region within the O1K B64 strain that inhibits replication in cattle. These chimeric infectious cDNA plasmids provide a basis for the analysis of FMDV pathogenicity and characterization of receptor utilization in vivo....

  18. Molecular characterization and expression analysis of four fish-specific CC chemokine receptors CCR4La, CCR4Lc1, CCR4Lc2 and CCR11 in rainbow trout (Oncorhynchus mykiss).

    Science.gov (United States)

    Qi, Zhitao; Holland, Jason W; Jiang, Yousheng; Secombes, Christopher J; Nie, Pin; Wang, Tiehui

    2017-09-01

    The chemokine and chemokine receptor networks regulate leukocyte trafficking, inflammation, immune cell differentiation, cancer and other biological processes. Comparative immunological studies have revealed that both chemokines and their receptors have expanded greatly in a species/lineage specific way. Of the 10 human CC chemokine receptors (CCR1-10) that bind CC chemokines, orthologues only to CCR6, 7, 9 and 10 are present in teleost fish. In this study, four fish-specific CCRs, termed as CCR4La, CCR4Lc1, CCR4Lc2 and CCR11, with a close link to human CCR1-5 and 8, in terms of amino acid homology and syntenic conservation, have been identified and characterized in rainbow trout (Oncorhynchus mykiss). These CCRs were found to possess the conserved features of the G protein-linked receptor family, including an extracellular N-terminal, seven TM domains, three extracellular loops and three intracellular loops, and a cytoplasmic carboxyl tail with multiple potential serine/threonine phosphorylation sites. Four cysteine residues known to be involved in forming two disulfide bonds are present in the extracellular domains and a DRY motif is present in the second intracellular loop. Signaling mediated by these receptors might be regulated by N-glycosylation, tyrosine sulfation, S-palmitoylation, a PDZ ligand motif and di-leucine motifs. Studies of intron/exon structure revealed distinct fish-specific CCR gene organization in different fish species/lineages that might contribute to the diversification of the chemokine ligand-receptor networks in different fish lineages. Fish-specific trout CCRs are highly expressed in immune tissues/organs, such as thymus, spleen, head kidney and gills. Their expression can be induced by the pro-inflammatory cytokines, IL-1β, IL-6 and IFNγ, by the pathogen associated molecular patterns, PolyIC and peptidoglycan, and by bacterial infection. These data suggest that fish-specific CCRs are likely to have an important role in immune

  19. Enhanced Expression of Anti-CD19 Chimeric Antigen Receptor in piggyBac Transposon-Engineered T Cells

    Directory of Open Access Journals (Sweden)

    Daisuke Morita

    2018-03-01

    Full Text Available Adoptive T cell therapy using chimeric antigen receptor (CAR-modified T cells is a promising cancer immunotherapy. We previously developed a non-viral method of gene transfer into T cells using a piggyBac transposon system to improve the cost-effectiveness of CAR-T cell therapy. Here, we have further improved our technology by a novel culture strategy to increase the transfection efficiency and to reduce the time of T cell manufacturing. Using a CH2CH3-free CD19-specific CAR transposon vector and combining irradiated activated T cells (ATCs as feeder cells and virus-specific T cell receptor (TCR stimulation, we achieved 51.4% ± 14% CAR+ T cells and 2.8-fold expansion after 14 culture days. Expanded CD19.CAR-T cells maintained a significant fraction of CD45RA+CCR7+ T cells and demonstrated potent antitumor activity against CD19+ leukemic cells both in vitro and in vivo. Therefore, piggyBac-based gene transfer may provide an alternative to viral gene transfer for CAR-T cell therapy.

  20. Chimeric L2-Based Virus-Like Particle (VLP Vaccines Targeting Cutaneous Human Papillomaviruses (HPV.

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    Bettina Huber

    Full Text Available Common cutaneous human papillomavirus (HPV types induce skin warts, whereas species beta HPV are implicated, together with UV-radiation, in the development of non-melanoma skin cancer (NMSC in immunosuppressed patients. Licensed HPV vaccines contain virus-like particles (VLP self-assembled from L1 major capsid proteins that provide type-restricted protection against mucosal HPV infections causing cervical and other ano-genital and oro-pharyngeal carcinomas and warts (condylomas, but do not target heterologous HPV. Experimental papillomavirus vaccines have been designed based on L2 minor capsid proteins that contain type-common neutralization epitopes, to broaden protection to heterologous mucosal and cutaneous HPV types. Repetitive display of the HPV16 L2 cross-neutralization epitope RG1 (amino acids (aa 17-36 on the surface of HPV16 L1 VLP has greatly enhanced immunogenicity of the L2 peptide. To more directly target cutaneous HPV, L1 fusion proteins were designed that incorporate the RG1 homolog of beta HPV17, the beta HPV5 L2 peptide aa53-72, or the common cutaneous HPV4 RG1 homolog, inserted into DE surface loops of HPV1, 5, 16 or 18 L1 VLP scaffolds. Baculovirus expressed chimeric proteins self-assembled into VLP and VLP-raised NZW rabbit immune sera were evaluated by ELISA and L1- and L2-based pseudovirion (PsV neutralizing assays, including 12 novel beta PsV types. Chimeric VLP displaying the HPV17 RG1 epitope, but not the HPV5L2 aa53-72 epitope, induced cross-neutralizing humoral immune responses to beta HPV. In vivo cross-protection was evaluated by passive serum transfer in a murine PsV challenge model. Immune sera to HPV16L1-17RG1 VLP (cross- protected against beta HPV5/20/24/38/96/16 (but not type 76, while antisera to HPV5L1-17RG1 VLP cross-protected against HPV20/24/96 only, and sera to HPV1L1-4RG1 VLP cross-protected against HPV4 challenge. In conclusion, RG1-based VLP are promising next generation vaccine candidates to target

  1. A closed-tube assay for genotyping of the 32-bp deletion polymorphism in the chemokine receptor 5 (CCR5) gene

    DEFF Research Database (Denmark)

    Rasmussen, Henrik Berg; Werge, Thomas

    2007-01-01

    We have developed a closed-tube assay for determination of the chemokine receptor type 5 (CCR5) 32-bp deletion allele, which protects against infections with HIV and modulates susceptibility to a variety of inflammatory diseases. This assay utilizes dissociation analysis of amplified products...

  2. African Ancestry Influences CCR5 –2459G>A Genotype-Associated Virologic Success of Highly Active Antiretroviral Therapy

    Science.gov (United States)

    Cheruvu, Vinay K.; Igo, Robert P.; Jurevic, Richard J.; Serre, David; Zimmerman, Peter A.; Rodriguez, Benigno; Mehlotra, Rajeev K.

    2014-01-01

    Introduction In a North American, HIV-positive, highly active antiretroviral therapy (HAART)-treated, adherent cohort of self-identified white and black patients, we previously observed that chemokine (C-C motif) receptor 5 (CCR5) –2459G>A genotype had a strong association with time to achieve virologic success (TVLS) in black but not in white patients. Methods Using 128 genome-wide ancestry informative markers, we performed a quantitative assessment of ancestry in these patients (n = 310) to determine (1) whether CCR5 –2459G>A genotype is still associated with TVLS of HAART when ancestry, not self-identified race, is considered and (2) whether this association is influenced by varying African ancestry. Results We found that the interaction between CCR5 –2459G>A genotype and African ancestry (≤0.125 vs. ≥0.425 and A genotype and TVLS was stronger in patients with African ancestry ≥0.71 than in patients with African ancestry ≥0.452, in both Kaplan-Meier (log-rank P = 0.039 and 0.057, respectively, for AA, GA, and GG) and Cox proportional hazards regression (relative hazard for GG compared with AA 2.59 [95% CI, 1.27–5.22; P = 0.01] and 2.26 [95% CI, 1.18–4.32; P = 0.01], respectively) analyses. Conclusions We observed that the association between CCR5 –2459G>A genotype and TVLS of HAART increased with stronger African ancestry. Understanding the genomic mechanisms by which African ancestry influences this association is critical, and requires further studies. PMID:24714069

  3. Development of an inflammation imaging tracer, 111In-DOTA-DAPTA, targeting chemokine receptor CCR5 and preliminary evaluation in an ApoE-/- atherosclerosis mouse model.

    Science.gov (United States)

    Wei, Lihui; Petryk, Julia; Gaudet, Chantal; Kamkar, Maryam; Gan, Wei; Duan, Yin; Ruddy, Terrence D

    2018-02-07

    Chemokine receptor 5 (CCR5) plays an important role in atherosclerosis. Our objective was to develop a SPECT tracer targeting CCR5 for imaging plaque inflammation by radiolabeling D-Ala-peptide T-amide (DAPTA), a CCR5 antagonist, with 111 In. 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) conjugated DAPTA (DOTA-DAPTA) was labeled with 111 In. Cell uptake studies were conducted in U87-CD4-CCR5 and U87-MG cells. Biodistribution was determined in C57BL/6 mice. Autoradiography, en face and Oil Red O (ORO) imaging studies were performed in ApoE -/- mice. DOTA-DAPTA was radiolabeled with 111 In with high radiochemical purity (> 98%) and specific activity (70 MBq·nmol). 111 In-DOTA-DAPTA exhibited fast blood and renal clearance and high spleen uptake. The U87-CD4-CCR5 cells had significantly higher uptake in comparison to the U87-MG cells. The cell uptake was reduced by three times with DAPTA, indicating the receptor specificity of the uptake. Autoradiographic images showed significantly higher lesion uptake of 111 In-DOTA-DAPTA in ApoE -/- mice than that in C57BL/6 mice. The tracer uptake in 4 month old ApoE -/- high fat diet (HFD) mice with blocking agent was twofold lower than the same mice without the blocking agent, demonstrating the specificity of the tracer for the CCR5 receptor. 111 In-DOTA-DAPTA, specifically targeting chemokine receptor CCR5, is a potential SPECT agent for imaging inflammation in atherosclerosis.

  4. CCR 20th Anniversary Commentary: Chimeric Antigen Receptors-From Model T to the Tesla.

    Science.gov (United States)

    Hwu, Patrick

    2015-07-15

    The research article by Kershaw and colleagues, published in the October 15, 2006, issue of Clinical Cancer Research, presents one of the first clinical trials to utilize chimeric antigen receptors. Subsequent studies have shown promise for the treatment of patients with lymphoid malignancies, but further progress will require optimization, including the identification of more specific antigens for solid tumors. ©2015 American Association for Cancer Research.

  5. Generation and Characterization of HIV-1 Transmitted and Founder Virus Consensus Sequence from Intravenous Drug Users in Xinjiang, China.

    Science.gov (United States)

    Li, Fan; Ma, Liying; Feng, Yi; Hu, Jing; Ni, Na; Ruan, Yuhua; Shao, Yiming

    2017-06-01

    HIV-1 transmission in intravenous drug users (IDUs) has been characterized by high genetic multiplicity and suggests a greater challenge for HIV-1 infection blocking. We investigated a total of 749 sequences of full-length gp160 gene obtained by single genome sequencing (SGS) from 22 HIV-1 early infected IDUs in Xinjiang province, northwest China, and generated a transmitted and founder virus (T/F virus) consensus sequence (IDU.CON). The T/F virus was classified as subtype CRF07_BC and predicted to be CCR5-tropic virus. The variable region (V1, V2, and V4 loop) of IDU.CON showed length variation compared with the heterosexual T/F virus consensus sequence (HSX.CON) and homosexual T/F virus consensus sequence (MSM.CON). A total of 26 N-linked glycosylation sites were discovered in the IDU.CON sequence, which is less than that of MSM.CON and HSX.CON. Characterization of T/F virus from IDUs highlights the genetic make-up and complexity of virus near the moment of transmission or in early infection preceding systemic dissemination and is important toward the development of an effective HIV-1 preventive methods, including vaccines.

  6. Emerging tropical diseases in Australia. Part 5. Hendra virus

    DEFF Research Database (Denmark)

    Tulsiani, Suhella; Graham, G C; Moore, P R

    2011-01-01

    gene of the virus and the discovery that the virus had an exceptionally large genome subsequently led to HeV being assigned to a new genus, Henipavirus, along with Nipah virus (a newly emergent virus in pigs). The regular outbreaks of HeV-related disease that have occurred in Australia since 1994 have...

  7. Induction of human immunodeficiency virus neutralizing antibodies using fusion complexes.

    Science.gov (United States)

    Zipeto, Donato; Matucci, Andrea; Ripamonti, Chiara; Scarlatti, Gabriella; Rossolillo, Paola; Turci, Marco; Sartoris, Silvia; Tridente, Giuseppe; Bertazzoni, Umberto

    2006-05-01

    Human immunodeficiency virus-1 (HIV-1) infects cells by membrane fusion that is mediated by the envelope proteins gp120/gp41 and the cellular receptors CD4 and CCR5. During this process, some conserved viral epitopes are temporarily exposed and may induce a neutralizing antibody response when fixed in the fusogenic conformation. These transient structures are conserved and may be effective antigens for use in an anti-HIV-1 vaccine. In this study we tested different conditions of preparation of fusion complexes inducing neutralizing antibodies against both R5 and X4 tropic HIV-1 strains. Cell lines expressing HIV-1 gp120/gp41 and CD4-CCR5 were prepared and conditions for producing fusion complexes were tested. Complexes produced at different temperature and fixative combinations were used to immunize mice. Results indicated that (a) fusion complexes prepared at either 21 degrees C, 30 degrees C or 37 degrees C were immunogenic and induced neutralizing antibodies against both R5 and X4 HIV-1 heterologous isolates; (b) after extensive purification of antibodies there was no cytotoxic effect; (c) complexes prepared at 37 degrees C were more immunogenic and induced higher titers of neutralizing antibodies than complexes prepared at either 21 degrees C or 30 degrees C; (d) the fixative used did not affect the titer of neutralizing antibodies except for glutaraldehyde which was ineffective; (e) the neutralizing activity was retained after CD4-CCR5 antibody removal. The production of higher titers of neutralizing antibody with fusion complexes prepared at 37 degrees C, as compared to lower temperatures, may be related to the induction of antibodies against many different conformation intermediates that subsequently act synergistically at different steps in the fusion process.

  8. CCR+: Metadata Based Extended Personal Health Record Data Model Interoperable with the ASTM CCR Standard.

    Science.gov (United States)

    Park, Yu Rang; Yoon, Young Jo; Jang, Tae Hun; Seo, Hwa Jeong; Kim, Ju Han

    2014-01-01

    Extension of the standard model while retaining compliance with it is a challenging issue because there is currently no method for semantically or syntactically verifying an extended data model. A metadata-based extended model, named CCR+, was designed and implemented to achieve interoperability between standard and extended models. Furthermore, a multilayered validation method was devised to validate the standard and extended models. The American Society for Testing and Materials (ASTM) Community Care Record (CCR) standard was selected to evaluate the CCR+ model; two CCR and one CCR+ XML files were evaluated. In total, 188 metadata were extracted from the ASTM CCR standard; these metadata are semantically interconnected and registered in the metadata registry. An extended-data-model-specific validation file was generated from these metadata. This file can be used in a smartphone application (Health Avatar CCR+) as a part of a multilayered validation. The new CCR+ model was successfully evaluated via a patient-centric exchange scenario involving multiple hospitals, with the results supporting both syntactic and semantic interoperability between the standard CCR and extended, CCR+, model. A feasible method for delivering an extended model that complies with the standard model is presented herein. There is a great need to extend static standard models such as the ASTM CCR in various domains: the methods presented here represent an important reference for achieving interoperability between standard and extended models.

  9. Evolution of R5 and X4 human immunodeficiency virus type 1 gag sequences in vivo: evidence for recombination

    International Nuclear Information System (INIS)

    Rij, Ronald P. van; Worobey, Michael; Visser, Janny A.; Schuitemaker, Hanneke

    2003-01-01

    Human immunodeficiency virus type 1 (HIV-1) infection is in general established by CCR5-utilizing (R5) virus variants, which persist throughout the course of infection. R5 HIV-1 variants evolve into CXCR4-utilizing (X4) HIV-1 variants in approximately half of the infected individuals. We have previously observed an ongoing genetic evolution with a continuous divergence of envelope gp120 sequences of coexisting R5 and X4 virus variants over time. Here, we studied evolution of gag p17 sequences in two patients who developed X4 variants in the course of infection. In contrast to the envelope gp120 sequences, gag p17 sequences of R5 and X4 virus populations intermingled in phylogenetic trees and did not diverge from each other over time. Statistical evaluation using the Shimodaira-Hasegawa test indicated that the different genomic regions evolved along different topologies, supporting the hypothesis of recombination. Therefore, our data imply that recombination between R5 and X4 HIV-1 variants occurs in vivo

  10. Analysis of Staphylococcal cassette chromosome mec in Staphylococcus haemolyticus and Staphylococcus sciuri: identification of a novel ccr gene complex with a newly identified ccrA allotype (ccrA7).

    Science.gov (United States)

    Urushibara, Noriko; Paul, Shyamal Kumar; Hossain, Mohammad Akram; Kawaguchiya, Mitsuyo; Kobayashi, Nobumichi

    2011-06-01

    Methicillin resistance in staphylococci is conferred by the acquisition in its chromosome of the mecA gene, which is located on a mobile genetic element called staphylococcal cassette chromosome mec (SCCmec). Genetic type of SCCmec is defined by combination of mec gene complex class and cassette chromosome recombinase gene (ccr) allotype. In this study, we analyzed genetic diversity of the SCCmec in 11 Staphylococcus haemolyticus strains and a Staphylococcus sciuri strain, which were recently isolated from clinical specimens in Bangladesh. Among these strains, only two S. haemolyticus strains were proved to have the known types of SCCmec, that is, SCCmec V (class C2 mec-ccrC) and VII (class C1 mec-ccrC). Five S. haemolyticus strains were assigned two unique mec-ccr gene complexes combination; that is, class C1 mec-ccrA4B4 (four isolates) and class A mec-ccrC (one isolate). In the remaining four S. haemolyticus strains with class C1 mec, no known ccr allotypes could be detected. A single S. sciuri strain with class A mec complex carried a ccrA gene belonging to a novel allotype designated ccrA7, together with ccrB3. The ccrA7 gene in the S. sciuri strain showed 61.7%-82.7% sequence identity to the ccrA gene sequences published so far, and 75.3% identity to ccrA3, which is a component of the type 3 ccr complex (ccrA3-ccrB3) in methicillin-resistant Staphylococcus aureus. The results of the present study indicated that mec gene complex and ccr genes in coagulase-negative staphylococci are highly divergent, and distinct from those of common methicillin-resistant S. aureus. Identification of the novel ccrA7 allotype combined with ccrB3 suggested an occurrence of recombination between different ccr complexes in nature.

  11. Relation of circulating concentrations of chemokine receptor CCR5 ligands to C-peptide, proinsulin and HbA1c and disease progression in type 1 diabetes

    DEFF Research Database (Denmark)

    Pfleger, C.; Kaas, A.; Hansen, L.

    2008-01-01

    Th1 related chemokines CCL3 and CCL5 and Th2 related CCL4 as ligands of the receptor CCR5 contribute to disease development in animal models of type 1 diabetes. In humans, no data are available addressing the role of these chemokines regarding disease progression and remission. We investigated...... longitudinally circulating concentrations of CCR5 ligands of 256 newly diagnosed patients with type 1 diabetes. CCR5 ligands were differentially associated with beta-cell function and clinical remission. CCL5 was decreased in remitters and positively associated with HbA1c suggestive of a Th1 associated...... of CCR5 by therapeutic agents such as maraviroc may provide a new therapeutic target to ameliorate disease progression in type 1 diabetes. (C) 2008 Elsevier Inc. All rights reserved Udgivelsesdato: 2008/7...

  12. CCR presentations at AACR | Center for Cancer Research

    Science.gov (United States)

    CCR presentations at AACR Several CCR scientists will present their research at the AACR Annual Meeting in Washington, D.C., between April 1-5, 2017. Selected oral presentations are listed below. A full list of abstracts can be found on the AACR website.

  13. The synergistic effect of combined immunization with a DNA vaccine and chimeric yellow fever/dengue virus leads to strong protection against dengue.

    Directory of Open Access Journals (Sweden)

    Adriana S Azevedo

    Full Text Available The dengue envelope glycoprotein (E is the major component of virion surface and its ectodomain is composed of domains I, II and III. This protein is the main target for the development of a dengue vaccine with induction of neutralizing antibodies. In the present work, we tested two different vaccination strategies, with combined immunizations in a prime/booster regimen or simultaneous inoculation with a DNA vaccine (pE1D2 and a chimeric yellow fever/dengue 2 virus (YF17D-D2. The pE1D2 DNA vaccine encodes the ectodomain of the envelope DENV2 protein fused to t-PA signal peptide, while the YF17D-D2 was constructed by replacing the prM and E genes from the 17D yellow fever vaccine virus by those from DENV2. Balb/c mice were inoculated with these two vaccines by different prime/booster or simultaneous immunization protocols and most of them induced a synergistic effect on the elicited immune response, mainly in neutralizing antibody production. Furthermore, combined immunization remarkably increased protection against a lethal dose of DENV2, when compared to each vaccine administered alone. Results also revealed that immunization with the DNA vaccine, regardless of the combination with the chimeric virus, induced a robust cell immune response, with production of IFN-γ by CD8+ T lymphocytes.

  14. The puzzling role of CXCR4 in human immunodeficiency virus infection.

    Science.gov (United States)

    Vicenzi, Elisa; Liò, Pietro; Poli, Guido

    2013-01-01

    The human immunodeficiency virus type-1 (HIV-1) is the etiological agent of the acquired immunodeficiency syndrome (AIDS), a disease highly lethal in the absence of combination antiretroviral therapy. HIV infects CD4(+) cells of the immune system (T cells, monocyte-macrophages and dendritic cells) via interaction with a universal primary receptor, the CD4 molecule, followed by a mandatory interaction with a second receptor (co-receptor) belonging to the chemokine receptor family. Apart from some rare cases, two chemokine receptors have been evolutionarily selected to accomplish this need for HIV-1: CCR5 and CXCR4. Yet, usage of these two receptors appears to be neither casual nor simply explained by their levels of cell surface expression. While CCR5 use is the universal rule at the start of every infection regardless of the transmission route (blood-related, sexual or mother to child), CXCR4 utilization emerges later in disease coinciding with the immunological deficient phase of infection. Moreover, in most instances CXCR4 use as viral entry co-receptor is associated with maintenance of CCR5 use. Since antiviral agents preventing CCR5 utilization by the virus are already in use, while others targeting either CCR5 or CXCR4 (or both) are under investigation, understanding the biological correlates of this "asymmetrical" utilization of HIV entry co-receptors bears relevance for the clinical choice of which therapeutics should be administered to infected individuals. We will here summarize the basic knowledge and the hypotheses underlying the puzzling and yet unequivocal role of CXCR4 in HIV-1 infection.

  15. Dual R3R5 tropism characterizes cerebrospinal fluid HIV-1 isolates from individuals with high cerebrospinal fluid viral load.

    Science.gov (United States)

    Karlsson, Ulf; Antonsson, Liselotte; Ljungberg, Bengt; Medstrand, Patrik; Esbjörnsson, Joakim; Jansson, Marianne; Gisslen, Magnus

    2012-09-10

    To study the use of major and alternative coreceptors by HIV-1 isolates obtained from paired plasma and cerebrospinal fluid (CSF) samples. Paired plasma and CSF isolates from HIV-1-infected individuals with varying clinical, virologic, and immunologic parameters were assessed for the ability to infect indicator cells expressing a panel of coreceptors with documented expression in the central nervous system (CNS). HIV-1 isolates obtained from plasma and CSF in 28 individuals with varying viral load, CD4 T-cell counts, and with or without AIDS-defining disease were analyzed for the ability to infect NP2.CD4 cells stably expressing a panel of HIV coreceptors (CCR5, CXCR4, CCR3, CXCR6, GPR1, APJ, ChemR23, RDC-1 or BLT1). All isolates from both plasma and CSF utilized CCR5 and/or CXCR4. However, the ability to use both CCR3 and CCR5 (R3R5) was more pronounced in CSF isolates and correlated with high CSF viral load and low CD4 T-cell count. Notably, four out of five CSF isolates of subtype C origin exhibited CXCR6 use, which coincided with high CSF viral load despite preserved CD4 T-cell counts. The use of other alternative coreceptors was less pronounced. Dual-tropic R3R5 HIV-1 isolates in CSF coincide with high CSF viral load and low CD4 T-cell counts. Frequent CXCR6 use by CSF-derived subtype C isolates indicates that subtype-specific differences in coreceptor use may exist that will not be acknowledged when assessing plasma virus isolates. The findings may also bare relevance for HIV-1 replication within the CNS, and consequently, for the neuropathogenesis of AIDS.

  16. Tears from children with chronic hepatitis B virus (HBV) infection are infectious vehicles of HBV transmission: experimental transmission of HBV by tears, using mice with chimeric human livers.

    Science.gov (United States)

    Komatsu, Haruki; Inui, Ayano; Sogo, Tsuyoshi; Tateno, Akihiko; Shimokawa, Reiko; Fujisawa, Tomoo

    2012-08-15

    Body fluids such as saliva, urine, sweat, and tears from hepatitis B virus (HBV) carriers are potential sources of HBV transmission. Thirty-nine children and 8 adults who were chronically infected with HBV were enrolled. Real-time polymerase chain reaction was used for the quantification of HBV DNA. HBV DNA was detected in 73.7% of urine samples (14 of 19), 86.8% of saliva samples (33 of 38), 100% of tear samples (11 of 11), and 100% of sweat samples (9 of 9). Mean HBV DNA levels (±SD) in urine, saliva, tears, and sweat were 4.3 ± 1.1 log copies/mL, 5.9 ± 1.2 log copies/mL, 6.2 ± 0.7 log copies/mL, and 5.2 ± 0.6 log copies/mL, respectively. A statistically significant correlation was observed between the HBV DNA level in serum specimens and HBV DNA levels in saliva and tear specimens (r = 0.88; P Tear specimens from a child were injected intravenously into 2 human hepatocyte-transplanted chimeric mice. One week after inoculation, both chimeric mice had serum positive for HBV DNA. The levels of HBV DNA in tear specimens from young children were high. Tears were confirmed to be infectious, using chimeric mice. Strict precautions should be taken against direct contact with body fluids from HBV carriers with high-level viremia.

  17. Early transduction produces highly functional chimeric antigen receptor-modified virus-specific T-cells with central memory markers: a Production Assistant for Cell Therapy (PACT) translational application

    OpenAIRE

    Sun, Jiali; Huye, Leslie E; Lapteva, Natalia; Mamonkin, Maksim; Hiregange, Manasa; Ballard, Brandon; Dakhova, Olga; Raghavan, Darshana; Durett, April G; Perna, Serena K; Omer, Bilal; Rollins, Lisa A; Leen, Ann M; Vera, Juan F; Dotti, Gianpietro

    2015-01-01

    Background Virus-specific T-cells (VSTs) proliferate exponentially after adoptive transfer into hematopoietic stem cell transplant (HSCT) recipients, eliminate virus infections, then persist and provide long-term protection from viral disease. If VSTs behaved similarly when modified with tumor-specific chimeric antigen receptors (CARs), they should have potent anti-tumor activity. This theory was evaluated by Cruz et al. in a previous clinical trial with CD19.CAR-modified VSTs, but there was ...

  18. Expression of chimeric HCV peptide in transgenic tobacco plants ...

    African Journals Online (AJOL)

    Expression of chimeric HCV peptide in transgenic tobacco plants infected with recombinant alfalfa mosaic virus for development of a plant-derived vaccine against HCV. AK El Attar, AM Shamloul, AA Shalaby, BY Riad, A Saad, HM Mazyad, JM Keith ...

  19. Variation in HIV-1 R5 macrophage-tropism correlates with sensitivity to reagents that block envelope: CD4 interactions but not with sensitivity to other entry inhibitors

    Directory of Open Access Journals (Sweden)

    Simmonds Peter

    2008-01-01

    Full Text Available Abstract Background HIV-1 R5 viruses cause most of the AIDS cases worldwide and are preferentially transmitted compared to CXCR4-using viruses. Furthermore, R5 viruses vary extensively in capacity to infect macrophages and highly macrophage-tropic variants are frequently identified in the brains of patients with dementia. Here, we investigated the sensitivity of R5 envelopes to a range of inhibitors and antibodies that block HIV entry. We studied a large panel of R5 envelopes, derived by PCR amplification without culture from brain, lymph node, blood and semen. These R5 envelopes conferred a wide range of macrophage tropism and included highly macrophage-tropic variants from brain and non-macrophage-tropic variants from lymph node. Results R5 macrophage-tropism correlated with sensitivity to inhibition by reagents that inhibited gp120:CD4 interactions. Thus, increasing macrophage-tropism was associated with increased sensitivity to soluble CD4 and to IgG-CD4 (PRO 542, but with increased resistance to the anti-CD4 monoclonal antibody (mab, Q4120. These observations were highly significant and are consistent with an increased affinity of envelope for CD4 for macrophage-tropic envelopes. No overall correlations were noted between R5 macrophage-tropism and sensitivity to CCR5 antagonists or to gp41 specific reagents. Intriguingly, there was a relationship between increasing macrophage-tropism and increased sensitivity to the CD4 binding site mab, b12, but decreased sensitivity to 2G12, a mab that binds a glycan complex on gp120. Conclusion Variation in R5 macrophage-tropism is caused by envelope variation that predominantly influences sensitivity to reagents that block gp120:CD4 interactions. Such variation has important implications for therapy using viral entry inhibitors and for the design of envelope antigens for vaccines.

  20. Colorectal carcinoma metastases: Detection with In-111-labeled monoclonal antibody CCR 086

    International Nuclear Information System (INIS)

    Abdel-Nabi, H.H.; Levine, G.; Lamki, L.M.; Murray, J.L.; Tauxe, W.N.; Shah, A.N.; Patt, Y.Z.; Doerr, R.J.; Klein, H.A.; Gona, J.

    1990-01-01

    A phase I/II clinical trial with indium-111-labeled antimucin murine monoclonal antibody (MoAb) CCR 086 was conducted. Seventeen patients with histologically proved colorectal carcinoma and known metastatic disease underwent external scintigraphy after administration of 5.5 mCi (203.5 MBq) of In-111 CCR 086 at doses of 5 and 20 mg. Of 25 known lesions, 17 were detected (sensitivity, 68%). The smallest detected lesion in the lung was 1 cm and in the liver was 1.5 cm. The serum half-life of In-111-labeled CCR 086 MoAb was approximately 64 hours. The formation of human antimouse antibody (HAMA) was detected in the serum of four of five patients who received 20 mg of MoAb. No HAMAs were detected in four patients receiving 5 mg of MoAb. No side effects were encountered. Because of effective detection of liver and lung metastases with lower doses (5-20 mg) of CCR 086 conjugated with In-111, further investigations are warranted to assess clinical and therapeutic potentials of CCR 086 in the management of colorectal cancer

  1. Circulating precursor CCR7(lo)PD-1(hi) CXCR5⁺ CD4⁺ T cells indicate Tfh cell activity and promote antibody responses upon antigen reexposure.

    Science.gov (United States)

    He, Jing; Tsai, Louis M; Leong, Yew Ann; Hu, Xin; Ma, Cindy S; Chevalier, Nina; Sun, Xiaolin; Vandenberg, Kirsten; Rockman, Steve; Ding, Yan; Zhu, Lei; Wei, Wei; Wang, Changqi; Karnowski, Alexander; Belz, Gabrielle T; Ghali, Joanna R; Cook, Matthew C; Riminton, D Sean; Veillette, André; Schwartzberg, Pamela L; Mackay, Fabienne; Brink, Robert; Tangye, Stuart G; Vinuesa, Carola G; Mackay, Charles R; Li, Zhanguo; Yu, Di

    2013-10-17

    Follicular B helper T (Tfh) cells support high affinity and long-term antibody responses. Here we found that within circulating CXCR5⁺ CD4⁺ T cells in humans and mice, the CCR7(lo)PD-1(hi) subset has a partial Tfh effector phenotype, whereas CCR7(hi)PD-1(lo) cells have a resting phenotype. The circulating CCR7(lo)PD-1(hi) subset was indicative of active Tfh differentiation in lymphoid organs and correlated with clinical indices in autoimmune diseases. Thus the CCR7(lo)PD-1(hi) subset provides a biomarker to monitor protective antibody responses during infection or vaccination and pathogenic antibody responses in autoimmune diseases. Differentiation of both CCR7(hi)PD-1(lo) and CCR7(lo)PD-1(hi) subsets required ICOS and BCL6, but not SAP, suggesting that circulating CXCR5⁺ helper T cells are primarily generated before germinal centers. Upon antigen reencounter, CCR7(lo)PD-1(hi) CXCR5⁺ precursors rapidly differentiate into mature Tfh cells to promote antibody responses. Therefore, circulating CCR7(lo)PD-1(hi) CXCR5⁺ CD4⁺ T cells are generated during active Tfh differentiation and represent a new mechanism of immunological early memory. Copyright © 2013 Elsevier Inc. All rights reserved.

  2. The Central Conserved Region (CCR) of Respiratory Syncytial Virus (RSV) G Protein Modulates Host miRNA Expression and Alters the Cellular Response to Infection.

    Science.gov (United States)

    Bakre, Abhijeet A; Harcourt, Jennifer L; Haynes, Lia M; Anderson, Larry J; Tripp, Ralph A

    2017-07-03

    Respiratory Syncytial Virus (RSV) infects respiratory epithelial cells and deregulates host gene expression by many mechanisms including expression of RSV G protein (RSV G). RSV G protein encodes a central conserved region (CCR) containing a CX3C motif that functions as a fractalkine mimic. Disruption of the CX3C motif (a.a. 182-186) located in the CCR of the G protein has been shown to affect G protein function in vitro and the severity of RSV disease pathogenesis in vivo. We show that infection of polarized Calu3 respiratory cells with recombinant RSV having point mutations in Cys173 and 176 (C173/176S) (rA2-GC12), or Cys186 (C186S) (rA2-GC4) is associated with a decline in the integrity of polarized Calu-3 cultures and decreased virus production. This is accompanied with downregulation of miRNAs let-7f and miR-24 and upregulation of interferon lambda (IFNλ), a primary antiviral cytokine for RSV in rA2-GC12/rA2-GC4 infected cells. These results suggest that residues in the cysteine noose region of RSV G protein can modulate IFN λ expression accompanied by downregulation of miRNAs, and are important for RSV G protein function and targeting.

  3. Identification of full-length transmitted/founder viruses and their progeny in primary HIV-1 infection

    Energy Technology Data Exchange (ETDEWEB)

    Korber, Bette [Los Alamos National Laboratory; Hraber, Peter [Los Alamos National Laboratory; Giorgi, Elena [Los Alamos National Laboratory; Bhattacharya, T [Los Alamos National Laboratory

    2009-01-01

    Identification of transmitted/founder virus genomes and their progeny by is a novel strategy for probing the molecular basis of HIV-1 transmission and for evaluating the genetic imprint of viral and host factors that act to constrain or facilitate virus replication. Here, we show in a cohort of twelve acutely infected subjects (9 clade B; 3 clade C), that complete genomic sequences of transmitted/founder viruses could be inferred using single genome amplification of plasma viral RNA, direct amplicon sequencing, and a model of random virus evolution. This allowed for the precise identification, chemical synthesis, molecular cloning, and biological analysis of those viruses actually responsible for productive clinical infection and for a comprehensive mapping of sequential viral genomes and proteomes for mutations that are necessary or incidental to the establishment of HIV-1 persistence. Transmitted/founder viruses were CD4 and CCR5 tropic, replicated preferentially in activated primary T-Iymphocytes but not monocyte-derived macrophages, and were effectively shielded from most heterologous or broadly neutralizing antibodies. By 3 months of infection, the evolving viral quasispecies in three subjects showed mutational fixation at only 2-5 discreet genomic loci. By 6-12 months, mutational fixation was evident at 18-27 genomic loci. Some, but not all, of these mutations were attributable to virus escape from cytotoxic Tlymphocytes or neutralizing antibodies, suggesting that other viral or host factors may influence early HIV -1 fitness.

  4. Pushing it back. Dating the CCR5–32 bp deletion to the Mesolithic in Sweden and its implications for the Meso\\Neo transition

    Directory of Open Access Journals (Sweden)

    Kerstin Lidén

    2006-12-01

    Full Text Available Genetic variation in the chemokine receptor gene CCR5 has received considerable scientific interest during the last few years. Protection against HIV-infection and AIDS, together with specific geographic distribution are the major reasons for the great interest in CCR5 32bp deletion. The event for the occurrence of this mutation has been postulated by coalescence dating to the 14th century, or 5000 BP. In our prehistoric Swedish samples we show that the frequency of 32pb deletion in CCR5 in the Neolithic population does not deviate from the frequency in a modern Swedish population, and that the deletion existed in Sweden already during the Mesolithic period.

  5. CCR 20th anniversary commentary: a chimeric antibody, C225, inhibits EGFR activation and tumor growth.

    Science.gov (United States)

    Mendelsohn, John; Prewett, Marie; Rockwell, Patricia; Goldstein, Neil I

    2015-01-15

    Murine mAb 225 was effective against the EGFR tyrosine kinase and inhibited tumor growth in preclinical studies. A phase I trial showed safety, tumor localization, and satisfactory pharmacokinetics. Human:murine chimeric C225 retained biologic activity, which was essential for the conduct of subsequent combination therapy trials and eventual regulatory approval. ©2015 American Association for Cancer Research.

  6. Moderate restriction of macrophage-tropic human immunodeficiency virus type 1 by SAMHD1 in monocyte-derived macrophages.

    Science.gov (United States)

    Taya, Kahoru; Nakayama, Emi E; Shioda, Tatsuo

    2014-01-01

    Macrophage-tropic human immunodeficiency virus type 1 (HIV-1) strains are able to grow to high titers in human monocyte-derived macrophages. However, it was recently reported that cellular protein SAMHD1 restricts HIV-1 replication in human cells of the myeloid lineage, including monocyte-derived macrophages. Here we show that degradation of SAMHD1 in monocyte-derived macrophages was associated with moderately enhanced growth of the macrophage-tropic HIV-1 strain. SAMHD1 degradation was induced by treating target macrophages with vesicular stomatitis virus glycoprotein-pseudotyped human immunodeficiency virus type 2 (HIV-2) particles containing viral protein X. For undifferentiated monocytes, HIV-2 particle treatment allowed undifferentiated monocytes to be fully permissive for productive infection by the macrophage-tropic HIV-1 strain. In contrast, untreated monocytes were totally resistant to HIV-1 replication. These results indicated that SAMHD1 moderately restricts even a macrophage-tropic HIV-1 strain in monocyte-derived macrophages, whereas the protein potently restricts HIV-1 replication in undifferentiated monocytes.

  7. Display of neutralizing epitopes of Canine parvovirus and a T-cell epitope of the fusion protein of Canine distemper virus on chimeric tymovirus-like particles and its use as a vaccine candidate both against Canine parvo and Canine distemper.

    Science.gov (United States)

    Chandran, Dev; Shahana, Pallichera Vijayan; Rani, Gudavelli Sudha; Sugumar, Parthasarthy; Shankar, Chinchkar Ramchandra; Srinivasan, Villuppanoor Alwar

    2009-12-10

    Expression of Physalis mottle tymovirus coat protein in Escherichia coli was earlier shown to self-assemble into empty capsids that were nearly identical to the capsids formed in vivo. Amino acid substitutions were made at the N-terminus of wild-type Physalis mottle virus coat protein with neutralizing epitopes of Canine parvovirus containing the antigenic sites 1-2, 4 and 6-7 and T-cell epitope of the fusion protein of Canine distemper virus in various combinations to yield PhMV1, PhMV2, PhMV3, PhMV4 and PhMV5. These constructs were cloned and expressed in E. coli. The chimeric proteins self-assembled into chimeric tymovirus-like particles (TVLPs) as determined by electron microscopy. The TVLPs were purified by ultracentrifugation and injected into guinea pigs and dogs to determine their immunogenicity. Initial immunogenicity studies in guinea pigs indicated that PhMV3 gave a higher response in comparison to the other TVLPs for both CPV and CDV and hence all further experiments in dogs were done with PhMV3. HI was done against different isolates obtained from various parts of the country. Protective titres indicated the broad spectrum of the vaccine. In conclusion the study indicated that the above chimeric VLP based vaccine could be used in dogs to generate a protective immune response against diseases caused by both Canine parvo and Canine distemper virus.

  8. Asn 362 in gp120 contributes to enhanced fusogenicity by CCR5-restricted HIV-1 envelope glycoprotein variants from patients with AIDS

    Directory of Open Access Journals (Sweden)

    Wang Bin

    2007-12-01

    Full Text Available Abstract Background CCR5-restricted (R5 human immunodeficiency virus type 1 (HIV-1 variants cause CD4+ T-cell loss in the majority of individuals who progress to AIDS, but mechanisms underlying the pathogenicity of R5 strains are poorly understood. To better understand envelope glycoprotein (Env determinants contributing to pathogenicity of R5 viruses, we characterized 37 full-length R5 Envs from cross-sectional and longitudinal R5 viruses isolated from blood of patients with asymptomatic infection or AIDS, referred to as pre-AIDS (PA and AIDS (A R5 Envs, respectively. Results Compared to PA-R5 Envs, A-R5 Envs had enhanced fusogenicity in quantitative cell-cell fusion assays, and reduced sensitivity to inhibition by the fusion inhibitor T-20. Sequence analysis identified the presence of Asn 362 (N362, a potential N-linked glycosylation site immediately N-terminal to CD4-binding site (CD4bs residues in the C3 region of gp120, more frequently in A-R5 Envs than PA-R5 Envs. N362 was associated with enhanced fusogenicity, faster entry kinetics, and increased sensitivity of Env-pseudotyped reporter viruses to neutralization by the CD4bs-directed Env mAb IgG1b12. Mutagenesis studies showed N362 contributes to enhanced fusogenicity of most A-R5 Envs. Molecular models indicate N362 is located adjacent to the CD4 binding loop of gp120, and suggest N362 may enhance fusogenicity by promoting greater exposure of the CD4bs and/or stabilizing the CD4-bound Env structure. Conclusion Enhanced fusogenicity is a phenotype of the A-R5 Envs studied, which was associated with the presence of N362, enhanced HIV-1 entry kinetics and increased CD4bs exposure in gp120. N362 contributes to fusogenicity of R5 Envs in a strain dependent manner. Our studies suggest enhanced fusogenicity of A-R5 Envs may contribute to CD4+ T-cell loss in subjects who progress to AIDS whilst harbouring R5 HIV-1 variants. N362 may contribute to this effect in some individuals.

  9. Human glial chimeric mice reveal astrocytic dependence of JC virus infection

    DEFF Research Database (Denmark)

    Kondo, Yoichi; Windrem, Martha S; Zou, Lisa

    2014-01-01

    with humanized white matter by engrafting human glial progenitor cells (GPCs) into neonatal immunodeficient and myelin-deficient mice. Intracerebral delivery of JCV resulted in infection and subsequent demyelination of these chimeric mice. Human GPCs and astrocytes were infected more readily than...... that was chimeric for human astrocytes and GPCs. JCV effectively propagated in these mice, which indicates that astroglial infection is sufficient for JCV spread. Sequencing revealed progressive mutation of the JCV capsid protein VP1 after infection, suggesting that PML may evolve with active infection...

  10. Correlations between polymorphisms in the uridine diphosphate-glucuronosyltransferase 1A and C-C motif chemokine receptor 5 genes and infection with the hepatitis B virus in three ethnic groups in China.

    Science.gov (United States)

    Zhang, Chan; He, Yan; Shan, Ke-Ren; Tan, Kui; Zhang, Ting; Wang, Chan-Juan; Guan, Zhi-Zhong

    2018-02-01

    Objective To determine whether genetic polymorphisms in the uridine diphosphate-glucuronosyltransferase 1A ( UGT1A) and the C-C motif chemokine receptor 5 ( CCR5) genes are associated with hepatitis B virus (HBV) infection in Yi, Yao and Han ethnic groups in the Guizhou Province of China. Methods The study enrolled subjects with and without HBV infection. Whole blood was used for DNA genotyping using standard techniques. The study determined the frequencies of several polymorphic alleles ( UGT1A6 [rs2070959], UGT1A1 [rs8175347], CCR5-59029 [rs1799987] and CCR5Δ32 [rs333]) and then characterized their relationship with HBV infection. Results A total of 404 subjects were enrolled in the study: 138 from the Yao group, 101 from the Yi group and 165 from the Han group. There was a significant difference in the frequency of UGT1A1 rs8175347 polymorphisms among the three groups. The rates of 7TA carriers of UGT1A1 rs8175347 in all three groups were significantly higher than the other genotypes. Individuals with genotype AA of UGT1A6 rs2070959 in the Yi group had a higher risk for HBV infection than in the Yao and Han groups. The frequency of genotype GG in CCR5-59029 in the Yao group was significantly higher than in the Yi group. The genotypes of CCR5Δ32 were not associated with HBV infection. Conclusion These findings provide genetic and epidemiological evidence for an association of UGT1A and CCR5-59029 polymorphisms with HBV infection in Chinese Yi and Yao populations.

  11. Clinical Case Registries (CCR)

    Data.gov (United States)

    Department of Veterans Affairs — The Clinical Case Registries (CCR) replaced the former Immunology Case Registry and the Hepatitis C Case Registry with local and national databases. The CCR:HIV and...

  12. Induction of experimental autoimmune encephalomyelitis in C57BL/6 mice deficient in either the chemokine macrophage inflammatory protein-1alpha or its CCR5 receptor

    DEFF Research Database (Denmark)

    Tran, E H; Kuziel, W A; Owens, T

    2000-01-01

    -type mice in Th1 cytokine gene expression, the kinetics and severity of disease, and infiltration of the central nervous system by lymphocytes, macrophages and granulocytes. RNase protection assays showed comparable accumulation of mRNA for the chemokines interferon-inducible protein-10, RANTES, macrophage...... and its CCR5 receptor in the induction of EAE by immunizing C57BL / 6 mice deficient in either MIP-1alpha or CCR5 with myelin oligodendrocyte glycoprotein (MOG). We found that MIP-1alpha-deficient mice were fully susceptible to MOG-induced EAE. These knockout animals were indistinguishable from wild...... chemoattractant protein-1, MIP-1beta, MIP-2, lymphotactin and T cell activation gene-3 during the course of the disease. CCR5-deficient mice were also susceptible to disease induction by MOG. The dispensability of MIP-1alpha and CCR5 for MOG-induced EAE in C57BL / 6 mice supports the idea that differential...

  13. Vaccination with recombinant RNA replicon particles protects chickens from H5N1 highly pathogenic avian influenza virus.

    Directory of Open Access Journals (Sweden)

    Stefan J Halbherr

    Full Text Available Highly pathogenic avian influenza viruses (HPAIV of subtype H5N1 not only cause a devastating disease in domestic chickens and turkeys but also pose a continuous threat to public health. In some countries, H5N1 viruses continue to circulate and evolve into new clades and subclades. The rapid evolution of these viruses represents a problem for virus diagnosis and control. In this work, recombinant vesicular stomatitis virus (VSV vectors expressing HA of subtype H5 were generated. To comply with biosafety issues the G gene was deleted from the VSV genome. The resulting vaccine vector VSV*ΔG(HA was propagated on helper cells providing the VSV G protein in trans. Vaccination of chickens with a single intramuscular dose of 2×10⁸ infectious replicon particles without adjuvant conferred complete protection from lethal H5N1 infection. Subsequent application of the same vaccine strongly boosted the humoral immune response and completely prevented shedding of challenge virus and transmission to sentinel birds. The vaccine allowed serological differentiation of infected from vaccinated animals (DIVA by employing a commercially available ELISA. Immunized chickens produced antibodies with neutralizing activity against multiple H5 viruses representing clades 1, 2.2, 2.5, and low-pathogenic avian influenza viruses (classical clade. Studies using chimeric H1/H5 hemagglutinins showed that the neutralizing activity was predominantly directed against the globular head domain. In summary, these results suggest that VSV replicon particles are safe and potent DIVA vaccines that may help to control avian influenza viruses in domestic poultry.

  14. Significance of CCL2, CCL5 and CCR2 polymorphisms for adverse prognosis of Japanese encephalitis from an endemic population of India.

    Science.gov (United States)

    Chowdhury, Purvita; Khan, Siraj Ahmed

    2017-10-20

    Japanese encephalitis (JE) is a major contributor for viral encephalitis in Asia. Vaccination programme has limited success for largely populated JE endemic countries like India and disease exposure is unavoidable. Involvement of chemokines and its co-receptors for adverse prognosis of JE have been documented both in vitro and in vivo. Identification of the genetic predisposing factor for JE infection in humans is crucial but not yet established. Therefore, we investigated the association of single nucleotide polymorphisms (SNPs) in chemokines (CCL2 and CCL5) and its co-receptors (CCR2 and CCR5) with their protein level for JE. The study enrolled 87 symptomatic JE cases (mild: severe = 24:63) and 94 asymptomatic controls. Our study demonstrated that CCL2 (rs1024611G), CCL5 (rs2280788G) and CCR2 (rs1799864A) significantly associated with JE (Odds ratio = 1.63, 2.95 and 2.62, respectively and P = 0.045, P = 0.05 and P = 0.0006, respectively). The study revealed that rs1024611G allele was associated with elevated level of CCL2. CCL5 elevation associated with JE mortality having a Cox proportional hazard of 1.004 (P = 0.033). In conclusion, SNPs of chemokine viz. CCL2 (rs1024611G) and its receptor CCR2 (rs1799864A) significantly associated with JE which may serve as possible genetic predisposing factor and CCL5 protein level may act as marker for disease survival.

  15. Reanalysis of coreceptor tropism in HIV-1-infected adults using a phenotypic assay with enhanced sensitivity.

    Science.gov (United States)

    Wilkin, Timothy J; Goetz, Mathew Bidwell; Leduc, Robert; Skowron, Gail; Su, Zhaohui; Chan, Ellen S; Heera, Jayyant; Chapman, Doug; Spritzler, John; Reeves, Jacqueline D; Gulick, Roy M; Coakley, Eoin

    2011-04-01

    The enhanced-sensitivity Trofile assay (TF-ES; Monogram Biosciences) was used to retest coreceptor tropism samples from 4 different cohorts of HIV-1-infected patients. Nine percent to 26% of patients with CCR5-tropic virus by the original Trofile assay had CXCR4-using virus by TF-ES. Lower CD4 cell counts were associated with CXCR4-using virus in all cohorts. © The Author 2011. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved.

  16. Seamless modification of wild-type induced pluripotent stem cells to the natural CCR5Δ32 mutation confers resistance to HIV infection.

    Science.gov (United States)

    Ye, Lin; Wang, Jiaming; Beyer, Ashley I; Teque, Fernando; Cradick, Thomas J; Qi, Zhongxia; Chang, Judy C; Bao, Gang; Muench, Marcus O; Yu, Jingwei; Levy, Jay A; Kan, Yuet Wai

    2014-07-01

    Individuals homozygous for the C-C chemokine receptor type 5 gene with 32-bp deletions (CCR5Δ32) are resistant to HIV-1 infection. In this study, we generated induced pluripotent stem cells (iPSCs) homozygous for the naturally occurring CCR5Δ32 mutation through genome editing of wild-type iPSCs using a combination of transcription activator-like effector nucleases (TALENs) or RNA-guided clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 together with the piggyBac technology. Remarkably, TALENs or CRISPR-Cas9-mediated double-strand DNA breaks resulted in up to 100% targeting of the colonies on one allele of which biallelic targeting occurred at an average of 14% with TALENs and 33% with CRISPR. Excision of the piggyBac using transposase seamlessly reproduced exactly the naturally occurring CCR5Δ32 mutation without detectable exogenous sequences. We differentiated these modified iPSCs into monocytes/macrophages and demonstrated their resistance to HIV-1 challenge. We propose that this strategy may provide an approach toward a functional cure of HIV-1 infection.

  17. Inhibitory function of adapter-related protein complex 2 alpha 1 subunit in the process of nuclear translocation of human immunodeficiency virus type 1 genome

    International Nuclear Information System (INIS)

    Kitagawa, Yukiko; Kameoka, Masanori; Shoji-Kawata, Sanae; Iwabu, Yukie; Mizuta, Hiroyuki; Tokunaga, Kenzo; Fujino, Masato; Natori, Yukikazu; Yura, Yoshiaki; Ikuta, Kazuyoshi

    2008-01-01

    The transfection of human cells with siRNA against adapter-related protein complex 2 alpha 1 subunit (AP2α) was revealed to significantly up-regulate the replication of human immunodeficiency virus type 1 (HIV-1). This effect was confirmed by cell infection with vesicular stomatitis virus G protein-pseudotyped HIV-1 as well as CXCR4-tropic and CCR5-tropic HIV-1. Viral adsorption, viral entry and reverse transcription processes were not affected by cell transfection with siRNA against AP2α. In contrast, viral nuclear translocation as well as the integration process was significantly up-regulated in cells transfected with siRNA against AP2α. Confocal fluorescence microscopy revealed that a subpopulation of AP2α was not only localized in the cytoplasm but was also partly co-localized with lamin B, importin β and Nup153, implying that AP2α negatively regulates HIV-1 replication in the process of nuclear translocation of viral DNA in the cytoplasm or the perinuclear region. We propose that AP2α may be a novel target for disrupting HIV-1 replication in the early stage of the viral life cycle

  18. A novel mechanism of RNase L inhibition: Theiler's virus L* protein prevents 2-5A from binding to RNase L

    Science.gov (United States)

    Drappier, Melissa; Elliott, Ruth; Zhang, Rong; Weiss, Susan R.; Silverman, Robert H.

    2018-01-01

    The OAS/RNase L pathway is one of the best-characterized effector pathways of the IFN antiviral response. It inhibits the replication of many viruses and ultimately promotes apoptosis of infected cells, contributing to the control of virus spread. However, viruses have evolved a range of escape strategies that act against different steps in the pathway. Here we unraveled a novel escape strategy involving Theiler’s murine encephalomyelitis virus (TMEV) L* protein. Previously we found that L* was the first viral protein binding directly RNase L. Our current data show that L* binds the ankyrin repeats R1 and R2 of RNase L and inhibits 2’-5’ oligoadenylates (2-5A) binding to RNase L. Thereby, L* prevents dimerization and oligomerization of RNase L in response to 2-5A. Using chimeric mouse hepatitis virus (MHV) expressing TMEV L*, we showed that L* efficiently inhibits RNase L in vivo. Interestingly, those data show that L* can functionally substitute for the MHV-encoded phosphodiesterase ns2, which acts upstream of L* in the OAS/RNase L pathway, by degrading 2-5A. PMID:29652922

  19. Novel recombinant chimeric virus-like particle is immunogenic and protective against both enterovirus 71 and coxsackievirus A16 in mice.

    Science.gov (United States)

    Zhao, Hui; Li, Hao-Yang; Han, Jian-Feng; Deng, Yong-Qiang; Zhu, Shun-Ya; Li, Xiao-Feng; Yang, Hui-Qin; Li, Yue-Xiang; Zhang, Yu; Qin, E-De; Chen, Rong; Qin, Cheng-Feng

    2015-01-19

    Hand-foot-and-mouth disease (HFMD) has been recognized as an important global public health issue, which is predominantly caused by enterovirus 71 (EV-A71) and coxsackievirus A16 (CVA16). There is no available vaccine against HFMD. An ideal HFMD vaccine should be bivalent against both EV-A71 and CVA16. Here, a novel strategy to produce bivalent HFMD vaccine based on chimeric EV-A71 virus-like particles (ChiEV-A71 VLPs) was proposed and illustrated. The neutralizing epitope SP70 within the capsid protein VP1 of EV-A71 was replaced with that of CVA16 in ChiEV-A71 VLPs. Structural modeling revealed that the replaced CVA16-SP70 epitope is well exposed on the surface of ChiEV-A71 VLPs. These VLPs produced in Saccharomyces cerevisiae exhibited similarity in both protein composition and morphology as naive EV-A71 VLPs. Immunization with ChiEV-A71 VLPs in mice elicited robust Th1/Th2 dependent immune responses against EV-A71 and CVA16. Furthermore, passive immunization with anti-ChiEV-A71 VLPs sera conferred full protection against lethal challenge of both EV-A71 and CVA16 infection in neonatal mice. These results suggested that this chimeric vaccine, ChiEV-A71 might have the potential to be further developed as a bivalent HFMD vaccine in the near future. Such chimeric enterovirus VLPs provide an alternative platform for bivalent HFMD vaccine development.

  20. Preclinical and Clinical Development of a YFV 17 D-Based Chimeric Vaccine against West Nile Virus

    Directory of Open Access Journals (Sweden)

    Gustavo H. Dayan

    2013-12-01

    Full Text Available Substantial success has been achieved in the development and implementation of West Nile (WN vaccines for horses; however, no human WN vaccines are approved. This review focuses on the construction, pre-clinical and clinical characterization of ChimeriVax-WN02 for humans, a live chimeric vaccine composed of a yellow fever (YF 17D virus in which the prM-E envelope protein genes are replaced with the corresponding genes of the WN NY99 virus. Pre-clinical studies demonstrated that ChimeriVax-WN02 was significantly less neurovirulent than YF 17D in mice and rhesus and cynomolgus monkeys. The vaccine elicited neutralizing antibody titers after inoculation in hamsters and monkeys and protected immunized animals from lethal challenge including intracerebral inoculation of high dose of WN NY99 virus. Safety, viremia and immunogenicity of ChimeriVax-WN02 were assessed in one phase I study and in two phase II clinical trials. No safety signals were detected in the three clinical trials with no remarkable differences in incidence of adverse events (AEs between vaccine and placebo recipients. Viremia was transient and the mean viremia levels were low. The vaccine elicited strong and durable neutralizing antibody and cytotoxic T cell responses. WN epidemiology impedes a classical licensure pathway; therefore, innovative licensure strategies should be explored.

  1. The Role of Natural Antibodies to CC Chemokine Receptor 5 in HIV Infection

    Directory of Open Access Journals (Sweden)

    Assunta Venuti

    2017-10-01

    Full Text Available The CC chemokine receptor 5 (CCR5 is responsible for immune and inflammatory responses by mediation of chemotactic activity in leukocytes, although it is expressed on different cell types. It has been shown to act as co-receptor for the human and simian immunodeficiency viruses (HIV-1, HIV-2, and SIV. Natural reactive antibodies (Abs recognizing first loop (ECL1 of CCR5 have been detected in several pools of immunoglobulins from healthy donors and from several cohorts of either HIV-exposed but uninfected subjects (ESN or HIV-infected individuals who control disease progression (LTNP as well. The reason of development of anti-CCR5 Abs in the absence of autoimmune disease is still unknown; however, the presence of these Abs specific for CCR5 or for other immune receptors and mediators probably is related to homeostasis maintenance. The majority of anti-CCR5 Abs is directed to HIV binding site (N-terminus and ECL2 of the receptor. Conversely, it is well known that ECL1 of CCR5 does not bind HIV; thus, the anti-CCR5 Abs directed to ECL1 elicit a long-lasting internalization of CCR5 but not interfere with HIV binding directly; these Abs block HIV infection in either epithelial cells or CD4+ T lymphocytes and the mechanism differs from those ones described for all other CCR5-specific ligands. The Ab-mediated CCR5 internalization allows the formation of a stable signalosome by interaction of CCR5, β-arrestin2 and ERK1 proteins. The signalosome degradation and the subsequent de novo proteins synthesis determine the CCR5 reappearance on the cell membrane with a very long-lasting kinetics (8 days. The use of monoclonal Abs to CCR5 with particular characteristics and mode of action may represent a novel mode to fight viral infection in either vaccinal or therapeutic strategies.

  2. Virus tropical: Presencia y relevancia del personaje autobiográfico femenino en la novela gráfica colombiana

    Directory of Open Access Journals (Sweden)

    Felipe Gómez Gutiérrez

    2015-04-01

    Abstract Here I analyze Virus Tropical (Colombia, 2009, a graphic novel by PowerPaola. Despite having achieved great popularity for a Latin American graphic novel –with national and international publications in both print and electronic formats–, it still has not been properly considered or analyzed in traditional academic media; particularly in Latin America where the study of comics is considered a matter of minor importance, and therefore discussions about Virus Tropical were relegated to alternative, communitarian spaces, such as public libraries or bookstores. Because Virus Tropical poses a challenge to the notion of the male comic book creator, because of its use of visual language to defy stereotypical female characters, and because of the way it engages issues of contemporary comic production, in this article I argue for a sound academic engagement with comics like this one.

  3. HIV restriction by APOBEC3 in humanized mice.

    Directory of Open Access Journals (Sweden)

    John F Krisko

    2013-03-01

    Full Text Available Innate immune restriction factors represent important specialized barriers to zoonotic transmission of viruses. Significant consideration has been given to their possible use for therapeutic benefit. The apolipoprotein B mRNA editing enzyme catalytic polypeptide 3 (APOBEC3 family of cytidine deaminases are potent immune defense molecules capable of efficiently restricting endogenous retroelements as well as a broad range of viruses including Human Immunodeficiency virus (HIV, Hepatitis B virus (HBV, Human Papilloma virus (HPV, and Human T Cell Leukemia virus (HTLV. The best characterized members of this family are APOBEC3G (A3G and APOBEC3F (A3F and their restriction of HIV. HIV has evolved to counteract these powerful restriction factors by encoding an accessory gene designated viral infectivity factor (vif. Here we demonstrate that APOBEC3 efficiently restricts CCR5-tropic HIV in the absence of Vif. However, our results also show that CXCR4-tropic HIV can escape from APOBEC3 restriction and replicate in vivo independent of Vif. Molecular analysis identified thymocytes as cells with reduced A3G and A3F expression. Direct injection of vif-defective HIV into the thymus resulted in viral replication and dissemination detected by plasma viral load analysis; however, vif-defective viruses remained sensitive to APOBEC3 restriction as extensive G to A mutation was observed in proviral DNA recovered from other organs. Remarkably, HIV replication persisted despite the inability of HIV to develop resistance to APOBEC3 in the absence of Vif. Our results provide novel insight into a highly specific subset of cells that potentially circumvent the action of APOBEC3; however our results also demonstrate the massive inactivation of CCR5-tropic HIV in the absence of Vif.

  4. Cryptic nature of a conserved, CD4-inducible V3 loop neutralization epitope in the native envelope glycoprotein oligomer of CCR5-restricted, but not CXCR4-using, primary human immunodeficiency virus type 1 strains.

    Science.gov (United States)

    Lusso, Paolo; Earl, Patricia L; Sironi, Francesca; Santoro, Fabio; Ripamonti, Chiara; Scarlatti, Gabriella; Longhi, Renato; Berger, Edward A; Burastero, Samuele E

    2005-06-01

    The external subunit of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env), gp120, contains conserved regions that mediate sequential interactions with two cellular receptor molecules, CD4 and a chemokine receptor, most commonly CCR5 or CXCR4. However, antibody accessibility to such regions is hindered by diverse protective mechanisms, including shielding by variable loops, conformational flexibility and extensive glycosylation. For the conserved neutralization epitopes hitherto described, antibody accessibility is reportedly unrelated to the viral coreceptor usage phenotype. Here, we characterize a novel, conserved gp120 neutralization epitope, recognized by a murine monoclonal antibody (MAb), D19, which is differentially accessible in the native HIV-1 Env according to its coreceptor specificity. The D19 epitope is contained within the third variable (V3) domain of gp120 and is distinct from those recognized by other V3-specific MAbs. To study the reactivity of MAb D19 with the native oligomeric Env, we generated a panel of PM1 cells persistently infected with diverse primary HIV-1 strains. The D19 epitope was conserved in the majority (23/29; 79.3%) of the subtype-B strains tested, as well as in selected strains from other genetic subtypes. Strikingly, in CCR5-restricted (R5) isolates, the D19 epitope was invariably cryptic, although it could be exposed by addition of soluble CD4 (sCD4); epitope masking was dependent on the native oligomeric structure of Env, since it was not observed with the corresponding monomeric gp120 molecules. By contrast, in CXCR4-using strains (X4 and R5X4), the epitope was constitutively accessible. In accordance with these results, R5 isolates were resistant to neutralization by MAb D19, becoming sensitive only upon addition of sCD4, whereas CXCR4-using isolates were neutralized regardless of the presence of sCD4. Other V3 epitopes examined did not display a similar divergence in accessibility based on

  5. The biological characteristics of anti-CD71 mouse/human chimeric antibody

    International Nuclear Information System (INIS)

    Wang Shuo; Jiang Lin; Lei Ping; Zhu Huifen; Shen Guanxin; Cui Wuren; Wang Yanggong

    2002-01-01

    Objective: To study the biological characteristics of an anti-CD71 mouse/human chimeric antibody (D2C). Methods: Analysis of the chimeric Ab production in culture supernatant was made by the standard concentration curve method with ELISA. The antibody was purified by DEAE-Sephredax-A50 ion-exchange chromatography and was confirmed by SDS-PAGE. The competition inhibition studies for binding to the same epitope on CD71 were performed between the chimeric Ab(D2C) in the culture supernatant was about 0.5-5 μg/ml in 5-day cultures when seeded at 1 x 10 5 cells/5ml compared with 12.5-25 μg/ml in the supernatant from their parental monoclonal Ab(7579). The purified chimeric Ab(D2C) from mouse ascetics was 1-2 mg/ml. The SDS-PAGE analysis of purified chimeric Ab(D2C) with discontinuous system confirmed two protein bands of 55 kDa and 25 kDa. It was clear that both chimeric Ab(D2C) and murine monoclonal Ab (7579) compete effectively to join the same epitope of CD71 each other. The chimeric antibody's affinity constant (Ka), quantitated by Scatchard analysis, is about 9.34-9.62 x 10 9 L/mol. Conclusion: The chimeric Ab(D2C) produced from the transfectomas is stable. The binding capacity of the chimeric Ab(D2C) to the antigen (CD71) was retained

  6. Imaging of alpha(v)beta(3) expression by a bifunctional chimeric RGD peptide not cross-reacting with alpha(v)beta(5).

    Science.gov (United States)

    Zannetti, Antonella; Del Vecchio, Silvana; Iommelli, Francesca; Del Gatto, Annarita; De Luca, Stefania; Zaccaro, Laura; Papaccioli, Angela; Sommella, Jvana; Panico, Mariarosaria; Speranza, Antonio; Grieco, Paolo; Novellino, Ettore; Saviano, Michele; Pedone, Carlo; Salvatore, Marco

    2009-08-15

    To test whether a novel bifunctional chimeric peptide comprising a cyclic Arg-Gly-Asp pentapeptide covalently bound to an echistatin domain can discriminate alpha(v)beta(3) from alpha(v)beta(5) integrin, thus allowing the in vivo selective visualization of alpha(v)beta(3) expression by single-photon and positron emission tomography (PET) imaging. The chimeric peptide was preliminarily tested for inhibition of alpha(v)beta(3)-dependent cell adhesion and competition of 125I-echistatin binding to membrane of stably transfected K562 cells expressing alpha(v)beta(3) (Kalpha(v)beta(3)) or alpha(v)beta(5) (Kalpha(v)beta(5)) integrin. The chimeric peptide was then conjugated with diethylenetriaminepentaacetic acid and labeled with 111In for single-photon imaging, whereas a one-step procedure was used for labeling the full-length peptide and a truncated derivative, lacking the last five C-terminal amino acids, with 18F for PET imaging. Nude mice bearing tumors from Kalpha(v)beta(3), Kalpha(v)beta(5), U87MG human glioblastoma, and A431 human epidermoid cells were subjected to single-photon and PET imaging. Adhesion and competitive binding assays showed that the novel chimeric peptide selectively binds to alpha(v)beta(3) integrin and does not cross-react with alpha(v)beta(5). In agreement with in vitro findings, single-photon and PET imaging studies showed that the radiolabeled chimeric peptide selectively localizes in tumor xenografts expressing alphavbeta3 and fails to accumulate in those expressing alpha(v)beta(5) integrin. When 18F-labeled truncated derivative was used for PET imaging, alphavbeta3- and alpha(v)beta(5)-expressing tumors were visualized, indicating that the five C-terminal amino acids are required to differentially bind the two integrins. Our findings indicate that the novel chimeric Arg-Gly-Asp peptide, having no cross-reaction with alphavbeta5 integrin, allows highly selective alphavbeta3 expression imaging and monitoring.

  7. Crystal Structures of Yeast-Produced Enterovirus 71 and Enterovirus 71/Coxsackievirus A16 Chimeric Virus-Like Particles Provide the Structural Basis for Novel Vaccine Design against Hand-Foot-and-Mouth Disease.

    Science.gov (United States)

    Lyu, Ke; He, Ya-Ling; Li, Hao-Yang; Chen, Rong

    2015-06-01

    Human enterovirus 71 (EV71) and coxsackievirus A16 (CVA16) are the two major causative agents for hand-foot-and-mouth disease (HFMD). Previously, we demonstrated that a virus-like particle (VLP) for EV71 produced from Saccharomyces cerevisiae is a potential vaccine candidate against EV71 infection, and an EV71/CVA16 chimeric VLP can elicit protective immune responses against both virus infections. Here, we presented the crystal structures of both VLPs, showing that both the linear and conformational neutralization epitopes identified in EV71 are mostly preserved on both VLPs. The replacement of only 4 residues in the VP1 GH loop converted strongly negatively charged surface patches formed by portions of the SP70 epitope in EV71 VLP into a relatively neutral surface in the chimeric VLP, which likely accounted for the additional neutralization capability of the chimeric VLP against CVA16 infection. Such local variations in the amino acid sequences and the surface charge potential are also present in different types of polioviruses. In comparison to EV71 VLP, the chimeric VLP exhibits structural changes at the local site of amino acid replacement and the surface loops of all capsid proteins. This is consistent with the observation that the VP1 GH loop located near the pseudo-3-fold junction is involved in extensive interactions with other capsid regions. Furthermore, portions of VP0 and VP1 in EV71 VLP are at least transiently exposed, revealing the structural flexibility of the VLP. Together, our structural analysis provided insights into the structural basis of enterovirus neutralization and novel vaccine design against HFMD and other enterovirus-associated diseases. Our previous studies demonstrated that the enterovirus 71 (EV71) virus-like particle (VLP) produced from yeast is a vaccine candidate against EV71 infection and that a chimeric EV71/coxsackievirus A16 (CVA16) VLP with the replacement of 4 amino acids in the VP1 GH loop can confer protection against both

  8. Roles of CcrA and CcrB in Excision and Integration of Staphylococcal Cassette Chromosome mec, a Staphylococcus aureus Genomic Island▿

    OpenAIRE

    Wang, Lei; Archer, Gordon L.

    2010-01-01

    The gene encoding resistance to methicillin and other β-lactam antibiotics in staphylococci, mecA, is carried on a genomic island, SCCmec (for staphylococcal cassette chromosome mec). The chromosomal excision and integration of types I to IV SCCmec are catalyzed by the site-specific recombinases CcrA and CcrB, the genes for which are encoded on each element. We sought to identify the relative contributions of CcrA and CcrB in the excision and integration of SCCmec. Purified CcrB but not CcrA ...

  9. Expression and purification of chimeric peptide comprising EGFR B-cell epitope and measles virus fusion protein T-cell epitope in Escherichia coli.

    Science.gov (United States)

    Wu, Meizhi; Zhao, Lin; Zhu, Lei; Chen, Zhange; Li, Huangjin

    2013-03-01

    Chimeric peptide MVF-EGFR(237-267), comprising a B-cell epitope from the dimerization interface of human epidermal growth factor receptor (EGFR) and a promiscuous T-cell epitope from measles virus fusion protein (MVF), is a promising candidate antigen peptide for therapeutic vaccine. To establish a high-efficiency preparation process of this small peptide, the coding sequence was cloned into pET-21b and pET-32a respectively, to be expressed alone or in the form of fusion protein with thioredoxin (Trx) and His(6)-tag in Escherichia coli BL21 (DE3). The chimeric peptide failed to be expressed alone, but over-expressed in the fusion form, which presented as soluble protein and took up more than 30% of total proteins of host cells. The fusion protein was seriously degraded during the cell disruption, in which endogenous metalloproteinase played a key role. Degradation of target peptide was inhibited by combined application of EDTA in the cell disruption buffer and a step of Source 30Q anion exchange chromatography (AEC) before metal-chelating chromatography (MCAC) for purifying His(6)-tagged fusion protein. The chimeric peptide was recovered from the purified fusion protein by enterokinase digestion at a yield of 3.0 mg/L bacteria culture with a purity of more than 95%. Immunogenicity analysis showed that the recombinant chimeric peptide was able to arouse more than 1×10(4) titers of specific antibody in BALB/c mice. Present work laid a solid foundation for the development of therapeutic peptide vaccine targeting EGFR dimerization and provided a convenient and low-cost preparation method for small peptides. Copyright © 2012 Elsevier Inc. All rights reserved.

  10. Role of CCL-2, CCR-2 and CCR-4 in cerulein-induced acute pancreatitis and pancreatitis-associated lung injury.

    Science.gov (United States)

    Frossard, Jean Louis; Lenglet, Sébastien; Montecucco, Fabrizio; Steffens, Sabine; Galan, Katia; Pelli, Graziano; Spahr, Laurent; Mach, Francois; Hadengue, Antoine

    2011-05-01

    Acute pancreatitis is an inflammatory process of variable severity. Leucocytes are thought to play a key role in the development of pancreatitis and pancreatitis-associated lung injury. The interactions between inflammatory cells and their mediators are crucial for determining tissue damage. Monocyte chemoattractant protein-1 (or CCL-2), CCR-2 and CCR-4 are chemokines and chemokine receptors involved in leucocyte trafficking. The aim of the study was to evaluate the role of the CCL-2, CCR-2 and CCR-4 chemokine receptors in the pathogenesis of cerulein-induced pancreatitis and pancreatitis-associated lung injury. To address the role of CCL-2, CCR-2 and CCR-4 that attracts leucocytes cells in inflamed tissues, pancreatitis was induced by administering supramaximal doses of cerulein in mice that do not express CCL-2, CCR-2 or CCR-4. The severity of pancreatitis was measured by serum amylase, pancreatic oedema and acinar cell necrosis. Lung injury was quantitated by evaluating lung microvascular permeability and lung myeloperoxidase activity. Chemokine and chemokine-receptor expression were quantitated by real-time PCR. The nature of inflammatory cells invading the pancreas and lungs was studied by immunostaining. The authors have found that pancreas CCL-2 and CCR-2 levels rise during pancreatitis. Both pancreatitis and the associated lung injury are blunted, but not completely prevented, in mice deficient in CCL-2, whereas the deficiency in either CCR-2 or CCR-4 does not reduce the severity of both the pancreatitis and the lung injury. The amounts of neutrophils and monocyte/macrophages (MOMA)-2 cells were significantly lower in mice deficient in CCL-2 compared with their sufficient littermates. These results suggest that CCL-2 plays a key role in pancreatitis by modulating the infiltration by neutrophils and MOMA-2 cells, and that its deficiency may improve the outcome of the disease.

  11. PA from an H5N1 highly pathogenic avian influenza virus activates viral transcription and replication and induces apoptosis and interferon expression at an early stage of infection

    Directory of Open Access Journals (Sweden)

    Wang Qiang

    2012-06-01

    Full Text Available Abstract Background Although gene exchange is not likely to occur freely, reassortment between the H5N1 highly pathogenic avian influenza virus (HPAIV and currently circulating human viruses is a serious concern. The PA polymerase subunit of H5N1 HPAIV was recently reported to activate the influenza replicon activity. Methods The replicon activities of PR8 and WSN strains (H1N1 of influenza containing PA from HPAIV A/Cambodia/P0322095/2005 (H5N1 and the activity of the chimeric RNA polymerase were analyzed. A reassortant WSN virus containing the H5N1 Cambodia PA (C-PA was then reconstituted and its growth in cells and pathogenicity in mice examined. The interferon promoter, TUNEL, and caspase 3, 8, and 9 activities of C-PA-infected cells were compared with those of WSN-infected cells. Results The activity of the chimeric RNA polymerase was slightly higher than that of WSN, and C-PA replicated better than WSN in cells. However, the multi-step growth of C-PA and its pathogenicity in mice were lower than those of WSN. The interferon promoter, TUNEL, and caspase 3, 8, and 9 activities were strongly induced in early infection in C-PA-infected cells but not in WSN-infected cells. Conclusions Apoptosis and interferon were strongly induced early in C-PA infection, which protected the uninfected cells from expansion of viral infection. In this case, these classical host-virus interactions contributed to the attenuation of this strongly replicating virus.

  12. Contemporary Avian Influenza A Virus Subtype H1, H6, H7, H10, and H15 Hemagglutinin Genes Encode a Mammalian Virulence Factor Similar to the 1918 Pandemic Virus H1 Hemagglutinin

    OpenAIRE

    Qi, Li; Pujanauski, Lindsey M.; Davis, A. Sally; Schwartzman, Louis M.; Chertow, Daniel S.; Baxter, David; Scherler, Kelsey; Hartshorn, Kevan L.; Slemons, Richard D.; Walters, Kathie-Anne; Kash, John C.; Taubenberger, Jeffery K.

    2014-01-01

    ABSTRACT Zoonotic avian influenza virus infections may lead to epidemics or pandemics. The 1918 pandemic influenza virus has an avian influenza virus-like genome, and its H1 hemagglutinin was identified as a key mammalian virulence factor. A chimeric 1918 virus expressing a contemporary avian H1 hemagglutinin, however, displayed murine pathogenicity indistinguishable from that of the 1918 virus. Here, isogenic chimeric avian influenza viruses were constructed on an avian influenza virus backb...

  13. In vivo evolution of HIV-1 co-receptor usage and sensitivity to chemokine-mediated suppression.

    Science.gov (United States)

    Scarlatti, G; Tresoldi, E; Björndal, A; Fredriksson, R; Colognesi, C; Deng, H K; Malnati, M S; Plebani, A; Siccardi, A G; Littman, D R; Fenyö, E M; Lusso, P

    1997-11-01

    Following the identification of the C-C chemokines RANTES, MIP-1alpha and MIP-1beta as major human immunodeficiency virus (HIV)-suppressive factors produced by CD8+ T cells, several chemokine receptors were found to serve as membrane co-receptors for primate immunodeficiency lentiretroviruses. The two most widely used co-receptors thus far recognized, CCR5 and CXCR4, are expressed by both activated T lymphocytes and mononuclear phagocytes. CCR5, a specific RANTES, MIP-1alpha and MIP-1 receptor, is used preferentially by non-MT2-tropic HIV-1 and HIV-2 strains and by simian immunodeficiency virus (SIV), whereas CXCR4, a receptor for the C-X-C chemokine SDF-1, is used by MT2-tropic HIV-1 and HIV-2, but not by SIV. Other receptors with a more restricted cellular distribution, such as CCR2b, CCR3 and STRL33, can also function as co-receptors for selected viral isolates. The third variable region (V3) of the gp120 envelope glycoprotein of HIV-1 has been fingered as a critical determinant of the co-receptor choice. Here, we document a consistent pattern of evolution of viral co-receptor usage and sensitivity to chemokine-mediated suppression in a longitudinal follow-up of children with progressive HIV-1 infection. Viral isolates obtained during the asymptomatic stages generally used only CCR5 as a co-receptor and were inhibited by RANTES, MIP-1alpha and MIP-1beta, but not by SDF-1. By contrast, the majority of the isolates derived after the progression of the disease were resistant to C-C chemokines, having acquired the ability to use CXCR4 and, in some cases, CCR3, while gradually losing CCR5 usage. Surprisingly, most of these isolates were also insensitive to SDF-1, even when used in combination with RANTES. An early acquisition of CXCR4 usage predicted a poor prognosis. In children who progressed to AIDS without a shift to CXCR4 usage, all the sequential isolates were CCR5-dependent but showed a reduced sensitivity to C-C chemokines. Discrete changes in the V3 domain

  14. Simian Immunodeficiency Virus (SIV-Specific Chimeric Antigen Receptor-T Cells Engineered to Target B Cell Follicles and Suppress SIV Replication

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    Kumudhini Preethi Haran

    2018-03-01

    Full Text Available There is a need to develop improved methods to treat and potentially cure HIV infection. During chronic HIV infection, replication is concentrated within T follicular helper cells (Tfh located within B cell follicles, where low levels of virus-specific CTL permit ongoing viral replication. We previously showed that elevated levels of simian immunodeficiency virus (SIV-specific CTL in B cell follicles are linked to both decreased levels of viral replication in follicles and decreased plasma viral loads. These findings provide the rationale to develop a strategy for targeting follicular viral-producing (Tfh cells using antiviral chimeric antigen receptor (CAR T cells co-expressing the follicular homing chemokine receptor CXCR5. We hypothesize that antiviral CAR/CXCR5-expressing T cells, when infused into an SIV-infected animal or an HIV-infected individual, will home to B cell follicles, suppress viral replication, and lead to long-term durable remission of SIV and HIV. To begin to test this hypothesis, we engineered gammaretroviral transduction vectors for co-expression of a bispecific anti-SIV CAR and rhesus macaque CXCR5. Viral suppression by CAR/CXCR5-transduced T cells was measured in vitro, and CXCR5-mediated migration was evaluated using both an in vitro transwell migration assay, as well as a novel ex vivo tissue migration assay. The functionality of the CAR/CXCR5 T cells was demonstrated through their potent suppression of SIVmac239 and SIVE660 replication in in vitro and migration to the ligand CXCL13 in vitro, and concentration in B cell follicles in tissues ex vivo. These novel antiviral immunotherapy products have the potential to provide long-term durable remission (functional cure of HIV and SIV infections.

  15. Increased levels of CCR7(lo)PD-1(hi) CXCR5+ CD4+ T cells, and associated factors Bcl-6, CXCR5, IL-21 and IL-6 contribute to repeated implantation failure.

    Science.gov (United States)

    Gong, Qiaoqiao; Zhu, Yuejie; Pang, Nannan; Ai, Haiquan; Gong, Xiaoyun; La, Xiaolin; Ding, Jianbing

    2017-12-01

    In vitro fertilization-embryo transfer (IVF-ET) can be used by infertile couples to assist with reproduction; however, failure of the embryo to implant into the endometrial lining results in failure of the IVF treatment. The present study investigated the expression of chemokine receptor 7 (CCR7)(lo) programmed death-1(PD-1)(hi) chemokine receptor type 5 (CXCR5) + cluster of differentiation 4 (CD4) + T cells and associated factors in patients with repeated implantation failure (RIF). A total of 30 females with RIF and 30 healthy females were enrolled in the current study. Flow cytometry was used to detect the proportion of CCR7(lo)PD-1(hi) CXCR5 + CD4 + T cells in the peripheral blood. Cytokine bead arrays were performed to detect the levels of interleukin (IL)-6, -4 and -2 in the serum. ELISAs were used to detect the level of IL-21 in the serum. Quantitative real time polymerase chain reaction analysis and immunohistochemistry were used to investigate the expression of B-cell lymphoma 6 (Bcl-6), chemokine receptor type 5 (CXCR5) and IL-21 in the endometrium. The results revealed that the percentage of CCR7(lo)PD-1(hi) CXCR5 + CD4 + T cells was increased in the RIF group compared with the control group during the mid luteal phase. The mRNA and protein levels of Bcl-6, IL-21 and CXCR5 in the endometrium and the concentrations of IL-21 and IL-6 in the serum were significantly increased in the RIF group; however, no significant difference was observed between the two groups in regards to the expression of IL-4 and IL-2. Furthermore, a significant positive correlation was identified between the percentage of CCR7(lo)PD-1(hi) CXCR5 + CD4 + T cells and IL-21 and IL-6 levels. The expression of IL-21 also had a positive correlation with Bcl-6 and CXCR5 expression in the RIF group. These results suggest that increased levels of CCR7(lo)PD-1(hi) CXCR5 + CD4 + T cells and associated factors contribute to RIF and could therefore be a potential therapeutic target.

  16. Overcoming hERG affinity in the discovery of maraviroc; a CCR5 antagonist for the treatment of HIV.

    Science.gov (United States)

    Price, David A; Armour, Duncan; de Groot, Marcel; Leishman, Derek; Napier, Carolyn; Perros, Manos; Stammen, Blanda L; Wood, Anthony

    2008-01-01

    Avoiding cardiac liability associated with blockade of hERG (human ether a go-go) is key for successful drug discovery and development. This paper describes the work undertaken in the discovery of a potent CCR5 antagonist, maraviroc 34, for the treatment of HIV. In particular the use of a pharmacophore model of the hERG channel and a high throughput binding assay for the hERG channel are described that were critical to elucidate SAR to overcome hERG liabilities. The key SAR involves the introduction of polar substituents into regions of the molecule where it is postulated to undergo hydrophobic interactions with the ion channel. Within the CCR5 project there appeared to be no strong correlation between hERG affinity and physiochemical parameters such as pKa or lipophilicity. It is believed that chemists could apply these same strategies early in drug discovery to remove hERG interactions associated with lead compounds while retaining potency at the primary target.

  17. Characterization of hepatitis C virus recombinants with chimeric E1/E2 envelope proteins and identification of single amino acids in the E2 stem region important for entry

    DEFF Research Database (Denmark)

    Carlsen, Thomas H R; Scheel, Troels K H; Ramirez, Santseharay

    2013-01-01

    The hepatitis C virus (HCV) envelope proteins E1 and E2 play a key role in host cell entry and represent important targets for vaccine and drug development. Here, we characterized HCV recombinants with chimeric E1/E2 complexes in vitro. Using genotype 1a/2a JFH1-based recombinants expressing 1a....../release. Studies of E1/E2 heterodimerization showed no differences in intracellular E1/E2 interaction for chimeric constructs with or without E2 stem region mutations. Interestingly, the E2 stem region mutations allowed efficient entry, which was verified in 1a-E1/1b-E2 HCV pseudoparticle assays. A CD81 inhibition...

  18. More about the Viking hypothesis of origin of the delta32 mutation in the CCR5 gene conferring resistance to HIV-1 infection.

    Science.gov (United States)

    Lucotte, Gérard; Dieterlen, Florent

    2003-11-01

    The chemokine receptor CCR5 constitutes the major coreceptor for the HIV-1, because a mutant allele of the CCR5 gene named delta32 was shown to provide to homozygotes a strong resistance against infection. In the present study the frequency of the delta32 allele was collected in 36 European populations and in Cyprus, and the highest allele frequencies were found in Nordic countries. We constructed an allele map of delta32 frequencies in Europe; the map is in accordance to the Vikings hypothesis of the origin of the mutation and his dissemination during the eighth to the tenth centuries.

  19. Generation of a Lineage II Powassan Virus (Deer Tick Virus) cDNA Clone: Assessment of Flaviviral Genetic Determinants of Tick and Mosquito Vector Competence.

    Science.gov (United States)

    Kenney, Joan L; Anishchenko, Michael; Hermance, Meghan; Romo, Hannah; Chen, Ching-I; Thangamani, Saravanan; Brault, Aaron C

    2018-05-21

    The Flavivirus genus comprises a diverse group of viruses that utilize a wide range of vertebrate hosts and arthropod vectors. The genus includes viruses that are transmitted solely by mosquitoes or vertebrate hosts as well as viruses that alternate transmission between mosquitoes or ticks and vertebrates. Nevertheless, the viral genetic determinants that dictate these unique flaviviral host and vector specificities have been poorly characterized. In this report, a cDNA clone of a flavivirus that is transmitted between ticks and vertebrates (Powassan lineage II, deer tick virus [DTV]) was generated and chimeric viruses between the mosquito/vertebrate flavivirus, West Nile virus (WNV), were constructed. These chimeric viruses expressed the prM and E genes of either WNV or DTV in the heterologous nonstructural (NS) backbone. Recombinant chimeric viruses rescued from cDNAs were characterized for their capacity to grow in vertebrate and arthropod (mosquito and tick) cells as well as for in vivo vector competence in mosquitoes and ticks. Results demonstrated that the NS elements were insufficient to impart the complete mosquito or tick growth phenotypes of parental viruses; however, these NS genetic elements did contribute to a 100- and 100,000-fold increase in viral growth in vitro in tick and mosquito cells, respectively. Mosquito competence was observed only with parental WNV, while infection and transmission potential by ticks were observed with both DTV and WNV-prME/DTV chimeric viruses. These data indicate that NS genetic elements play a significant, but not exclusive, role for vector usage of mosquito- and tick-borne flaviviruses.

  20. High throughput generation and characterization of replication-competent clade C transmitter-founder simian human immunodeficiency viruses.

    Directory of Open Access Journals (Sweden)

    Debashis Dutta

    Full Text Available Traditional restriction endonuclease-based cloning has been routinely used to generate replication-competent simian-human immunodeficiency viruses (SHIV and simian tropic HIV (stHIV. This approach requires the existence of suitable restriction sites or the introduction of nucleotide changes to create them. Here, using an In-Fusion cloning technique that involves homologous recombination, we generated SHIVs and stHIVs based on epidemiologically linked clade C transmitted/founder HIV molecular clones from Zambia. Replacing vif from these HIV molecular clones with vif of SIVmac239 resulted in chimeric genomes used to generate infectious stHIV viruses. Likewise, exchanging HIV env genes and introducing N375 mutations to enhance macaque CD4 binding site and cloned into a SHIVAD8-EO backbone. The generated SHIVs and stHIV were infectious in TZMbl and ZB5 cells, as well as macaque PBMCs. Therefore, this method can replace traditional methods and be a valuable tool for the rapid generation and testing of molecular clones of stHIV and SHIV based on primary clinical isolates will be valuable to generate rapid novel challenge viruses for HIV vaccine/cure studies.

  1. The CCR4-NOT complex physically and functionally interacts with TRAMP and the nuclear exosome.

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    Nowel Azzouz

    Full Text Available BACKGROUND: Ccr4-Not is a highly conserved multi-protein complex consisting in yeast of 9 subunits, including Not5 and the major yeast deadenylase Ccr4. It has been connected functionally in the nucleus to transcription by RNA polymerase II and in the cytoplasm to mRNA degradation. However, there has been no evidence so far that this complex is important for RNA degradation in the nucleus. METHODOLOGY/PRINCIPAL FINDINGS: In this work we point to a new role for the Ccr4-Not complex in nuclear RNA metabolism. We determine the importance of the Ccr4-Not complex for the levels of non-coding nuclear RNAs, such as mis-processed and polyadenylated snoRNAs, whose turnover depends upon the nuclear exosome and TRAMP. Consistently, mutation of both the Ccr4-Not complex and the nuclear exosome results in synthetic slow growth phenotypes. We demonstrate physical interactions between the Ccr4-Not complex and the exosome. First, Not5 co-purifies with the exosome. Second, several exosome subunits co-purify with the Ccr4-Not complex. Third, the Ccr4-Not complex is important for the integrity of large exosome-containing complexes. Finally, we reveal a connection between the Ccr4-Not complex and TRAMP through the association of the Mtr4 helicase with the Ccr4-Not complex and the importance of specific subunits of Ccr4-Not for the association of Mtr4 with the nuclear exosome subunit Rrp6. CONCLUSIONS/SIGNIFICANCE: We propose a model in which the Ccr4-Not complex may provide a platform contributing to dynamic interactions between the nuclear exosome and its co-factor TRAMP. Our findings connect for the first time the different players involved in nuclear and cytoplasmic RNA degradation.

  2. The geographic spread of the CCR5 Delta32 HIV-resistance allele.

    Directory of Open Access Journals (Sweden)

    John Novembre

    2005-11-01

    Full Text Available The Delta32 mutation at the CCR5 locus is a well-studied example of natural selection acting in humans. The mutation is found principally in Europe and western Asia, with higher frequencies generally in the north. Homozygous carriers of the Delta32 mutation are resistant to HIV-1 infection because the mutation prevents functional expression of the CCR5 chemokine receptor normally used by HIV-1 to enter CD4+ T cells. HIV has emerged only recently, but population genetic data strongly suggest Delta32 has been under intense selection for much of its evolutionary history. To understand how selection and dispersal have interacted during the history of the Delta32 allele, we implemented a spatially explicit model of the spread of Delta32. The model includes the effects of sampling, which we show can give rise to local peaks in observed allele frequencies. In addition, we show that with modest gradients in selection intensity, the origin of the Delta32 allele may be relatively far from the current areas of highest allele frequency. The geographic distribution of the Delta32 allele is consistent with previous reports of a strong selective advantage (>10% for Delta32 carriers and of dispersal over relatively long distances (>100 km/generation. When selection is assumed to be uniform across Europe and western Asia, we find support for a northern European origin and long-range dispersal consistent with the Viking-mediated dispersal of Delta32 proposed by G. Lucotte and G. Mercier. However, when we allow for gradients in selection intensity, we estimate the origin to be outside of northern Europe and selection intensities to be strongest in the northwest. Our results describe the evolutionary history of the Delta32 allele and establish a general methodology for studying the geographic distribution of selected alleles.

  3. SCH-C (SCH 351125), an orally bioavailable, small molecule antagonist of the chemokine receptor CCR5, is a potent inhibitor of HIV-1 infection in vitro and in vivo.

    Science.gov (United States)

    Strizki, J M; Xu, S; Wagner, N E; Wojcik, L; Liu, J; Hou, Y; Endres, M; Palani, A; Shapiro, S; Clader, J W; Greenlee, W J; Tagat, J R; McCombie, S; Cox, K; Fawzi, A B; Chou, C C; Pugliese-Sivo, C; Davies, L; Moreno, M E; Ho, D D; Trkola, A; Stoddart, C A; Moore, J P; Reyes, G R; Baroudy, B M

    2001-10-23

    We describe here the identification and properties of SCH-C (SCH 351125), a small molecule inhibitor of HIV-1 entry via the CCR5 coreceptor. SCH-C, an oxime-piperidine compound, is a specific CCR5 antagonist as determined in multiple receptor binding and signal transduction assays. This compound specifically inhibits HIV-1 infection mediated by CCR5 in U-87 astroglioma cells but has no effect on infection of CXCR4-expressing cells. SCH-C has broad and potent antiviral activity in vitro against primary HIV-1 isolates that use CCR5 as their entry coreceptor, with mean 50% inhibitory concentrations ranging between 0.4 and 9 nM. Moreover, SCH-C strongly inhibits the replication of an R5-using HIV-1 isolate in SCID-hu Thy/Liv mice. SCH-C has a favorable pharmacokinetic profile in rodents and primates with an oral bioavailability of 50-60% and a serum half-life of 5-6 h. On the basis of its novel mechanism of action, potent antiviral activity, and in vivo pharmacokinetic profile, SCH-C is a promising new candidate for therapeutic intervention of HIV infection.

  4. Differential impact of lytic viruses on prokaryotic morphopopulations in a tropical estuarine system (Cochin estuary, India).

    Science.gov (United States)

    Jasna, Vijayan; Pradeep Ram, Angia Sriram; Parvathi, Ammini; Sime-Ngando, Telesphore

    2018-01-01

    Our understanding on the importance of viral lysis in the functioning of tropical estuarine ecosystem is limited. This study examines viral infection of prokaryotes and subsequent lysis of cells belonging to different morphotypes across a salinity gradient in monsoon driven estuarine ecosystem (Cochin estuary, India). High standing stock of viruses and prokaryotes accompanied by lytic infection rates in the euryhaline/mesohaline region of the estuary suggests salinity to have an influential role in driving interactions between prokaryotes and viruses. High prokaryotic mortality rates, up to 42% of prokaryote population in the pre-monsoon season is further substantiated by a high virus to prokaryote ratio (VPR), suggesting that maintenance of a high number of viruses is dependent on the most active fraction of bacterioplankton. Although myoviruses were the dominant viral morphotype (mean = 43%) throughout the study period, there was significant variation among prokaryotic morphotypes susceptible to viral infection. Among them, the viral infected short rod prokaryote morphotype with lower burst estimates (mean = 18 viruses prokaryote-1) was dominant (35%) in the dry seasons whereas a substantial increase in cocci forms (30%) infected by viruses with high burst size (mean = 31 viruses prokaryote-1) was evident during the monsoon season. Such preferential infections of prokaryotic morphopopulations with respect to seasons can have a strong and variable impact on the carbon and energy flow in this tropical ecosystem.

  5. Perforator chimerism for the reconstruction of complex defects: A new chimeric free flap classification system.

    Science.gov (United States)

    Kim, Jeong Tae; Kim, Youn Hwan; Ghanem, Ali M

    2015-11-01

    Complex defects present structural and functional challenges to reconstructive surgeons. When compared to multiple free flaps or staged reconstruction, the use of chimeric flaps to reconstruct such defects have many advantages such as reduced number of operative procedures and donor site morbidity as well as preservation of recipient vessels. With increased popularity of perforator flaps, chimeric flaps' harvest and design has benefited from 'perforator concept' towards more versatile and better reconstruction solutions. This article discusses perforator based chimeric flaps and presents a practice based classification system that incorporates the perforator flap concept into "Perforator Chimerism". The authors analyzed a variety of chimeric patterns used in 31 consecutive cases to present illustrative case series and their new classification system. Accordingly, chimeric flaps are classified into four types. Type I: Classical Chimerism, Type II: Anastomotic Chimerism, Type III: Perforator Chimerism and Type IV Mixed Chimerism. Types I on specific source vessel anatomy whilst Type II requires microvascular anastomosis to create the chimeric reconstructive solution. Type III chimeric flaps utilizes the perforator concept to raise two components of tissues without microvascular anastomosis between them. Type IV chimeric flaps are mixed type flaps comprising any combination of Types I to III. Incorporation of the perforator concept in planning and designing chimeric flaps has allowed safe, effective and aesthetically superior reconstruction of complex defects. The new classification system aids reconstructive surgeons and trainees to understand chimeric flaps design, facilitating effective incorporation of this important reconstructive technique into the armamentarium of the reconstruction toolbox. Copyright © 2015 British Association of Plastic, Reconstructive and Aesthetic Surgeons. Published by Elsevier Ltd. All rights reserved.

  6. The efficacy of chimeric vaccines constructed with PEP-1 and Ii-Key linking to a hybrid epitope from heterologous viruses.

    Science.gov (United States)

    Liu, Xue-lan; Shan, Wen-jie; Xu, Shan-shan; Zhang, Jin-jing; Xu, Fa-zhi; Xia, Sheng-lin; Dai, Yin

    2015-09-01

    The heterologous epitope-peptide from different viruses may represent an attractive candidate vaccine. In order to evaluate the role of cell-permeable peptide (PEP-1) and Ii-Key moiety from the invariant chain (Ii) of MHC on the heterologous peptide chimeras, we linked the two vehicles to hybrid epitopes on the VP2 protein (aa197-209) of the infectious bursal disease virus and HN protein (aa345-353) of the Newcastle disease virus. The chimeric vaccines were prepared and injected into mice. The immune effects were measured by indirect ELISA. The results showed that the vehicle(s) could significantly boost immune effects against the heterologous epitope peptide. The Ii-Key-only carrier induced more effective immunological responses, compared with the PEP-1 and Ii-Key hybrid vehicle. The carrier-peptide hybrids all showed strong colocalization with major histocompatibility complex (MHC) class II molecules compared with the epitope-peptide (weakly-binding) after co-transfection into 293T cells. Together, our results lay the groundwork for designing new hybrid vaccines based on Ii-Key and/or PEP-1 peptides. Copyright © 2015 The International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.

  7. Biological and genetic evolution of HIV type 1 in two siblings with different patterns of disease progression.

    Science.gov (United States)

    Ripamonti, Chiara; Leitner, Thomas; Laurén, Anna; Karlsson, Ingrid; Pastore, Angela; Cavarelli, Mariangela; Antonsson, Liselotte; Plebani, Anna; Fenyö, Eva Maria; Scarlatti, Gabriella

    2007-12-01

    To investigate the immunological and virological factors that may lead to different patterns of disease progression characteristic of HIV-1-infected children, two HIV-1-infected siblings, a slow and a fast progressor, were followed prospectively before the onset of highly active antiretroviral therapy. Viral coreceptor usage, including the use of CCR5/CXCR4 chimeric receptors, macrophage tropism, and sensitivity to the CC-chemokine RANTES, has been studied. An autologous and heterologous neutralizing antibody response has been documented using peripheral blood mononuclear cells- and GHOST(3) cell line-based assays. Viral evolution was investigated by env C2-V3 region sequence analysis. Although both siblings were infected with HIV-1 of the R5 phenotype, their viruses showed important biological differences. In the fast progressor there was a higher RANTES sensitivity of the early virus, an increased trend to change the mode of CCR5 receptor use, and a larger genetic evolution. Both children developed an autologous neutralizing antibody response starting from the second year with evidence of the continuous emergence of resistant variants. A marked viral genetic and phenotypic evolution was documented in the fast progressor sibling, which is accompanied by a high viral RANTES sensitivity and persistent neutralizing antibodies.

  8. Heterologous prime-boost immunization of Newcastle disease virus vectored vaccines protected broiler chickens against highly pathogenic avian influenza and Newcastle disease viruses.

    Science.gov (United States)

    Kim, Shin-Hee; Samal, Siba K

    2017-07-24

    Avian Influenza virus (AIV) is an important pathogen for both human and animal health. There is a great need to develop a safe and effective vaccine for AI infections in the field. Live-attenuated Newcastle disease virus (NDV) vectored AI vaccines have shown to be effective, but preexisting antibodies to the vaccine vector can affect the protective efficacy of the vaccine in the field. To improve the efficacy of AI vaccine, we generated a novel vectored vaccine by using a chimeric NDV vector that is serologically distant from NDV. In this study, the protective efficacy of our vaccines was evaluated by using H5N1 highly pathogenic avian influenza virus (HPAIV) strain A/Vietnam/1203/2004, a prototype strain for vaccine development. The vaccine viruses were three chimeric NDVs expressing the hemagglutinin (HA) protein in combination with the neuraminidase (NA) protein, matrix 1 protein, or nonstructural 1 protein. Comparison of their protective efficacy between a single and prime-boost immunizations indicated that prime immunization of 1-day-old SPF chicks with our vaccine viruses followed by boosting with the conventional NDV vector strain LaSota expressing the HA protein provided complete protection of chickens against mortality, clinical signs and virus shedding. Further verification of our heterologous prime-boost immunization using commercial broiler chickens suggested that a sequential immunization of chickens with chimeric NDV vector expressing the HA and NA proteins following the boost with NDV vector expressing the HA protein can be a promising strategy for the field vaccination against HPAIVs and against highly virulent NDVs. Copyright © 2017 Elsevier Ltd. All rights reserved.

  9. CCR5Δ32 Polymorphism Associated with a Slower Rate Disease Progression in a Cohort of RR-MS Sicilian Patients

    Directory of Open Access Journals (Sweden)

    Rosalia D'Angelo

    2011-01-01

    Full Text Available Multiple sclerosis (MS disease is carried through inflammatory and degenerative stages. Based on clinical feaures, it can be subdivided into three groups: relapsing-remitting MS, secondary progressive MS, and primary progressive MS. Multiple sclerosis has a multifactorial etiology with an interplay of genetic predisposition, environmental factors, and autoimmune inflammatory mechanism in which play a key role CC-chemokines and its receptors. In this paper, we studied the frequency of CCR5 gene Δ32 allele in a cohort of Sicilian RR-MS patients comparing with general Sicilian population. Also, we evaluate the association between this commonly polymorphism and disability development and age of disease onset in the same cohort. Our results show that presence of CCR5Δ32 is significantly associated with expanded disability status scale score (EDSS but not with age of disease onset.

  10. Influence of Acyclic Nucleoside Phosphonate Antivirals on Gene Expression of Chemokine Receptors CCR5 and CXCR4

    Czech Academy of Sciences Publication Activity Database

    Potměšil, P.; Holý, Antonín; Zídek, Zdeněk

    2015-01-01

    Roč. 61, č. 1 (2015), s. 1-7 ISSN 0015-5500 R&D Projects: GA ČR GA305/03/1470; GA MŠk 1M0508 Institutional support: RVO:61388963 ; RVO:68378041 Keywords : acyclic nucleoside phosphonate * HIV * CCR5 * CXCR4 * cytokine * RT-PCR Subject RIV: CC - Organic Chemistry; FR - Pharmacology ; Medidal Chemistry (UEM-P) Impact factor: 0.833, year: 2015

  11. Functional replacement of Wheat streak mosaic virus HC-Pro with the corresponding cistron from a diverse array of viruses in the family Potyviridae

    International Nuclear Information System (INIS)

    Stenger, Drake C.; French, Roy

    2004-01-01

    Helper component-proteinase (HC-Pro) of Wheat streak mosaic virus strain Sidney 81 (WSMV-Sidney 81) was systematically replaced with the corresponding cistron derived from four strains of WSMV (Type, TK1, CZ, and El Batan 3), the tritimovirus Oat necrotic mottle virus (ONMV), the rymoviruses Agropyron mosaic virus (AgMV) and Hordeum mosaic virus (HoMV), or the potyviruses Tobacco etch virus (TEV) and Turnip mosaic virus (TuMV). These HC-Pro proteins varied in amino acid sequence identity shared with HC-Pro of WSMV-Sidney 81 from high (strains of WSMV at ∼86-99%) to moderate (ONMV at 70%) to low (rymoviruses and potyviruses at ∼15-17%). Surprisingly, all chimeric viral genomes examined were capable of systemic infection of wheat upon inoculation with RNA transcripts produced in vitro. HC-Pro replacements derived from tritimoviruses did not alter host range relative to WSMV-Sidney 81, as each of these chimeric viruses was able to systemically infect wheat, oat, and corn line SDp2. These results indicate that differences in host range among tritimoviruses, including the inability of ONMV to infect wheat or the inability of WSMV strains Type and El Batan 3 to infect SDp2 corn, are not determined by HC-Pro. In contrast, all chimeric viruses bearing HC-Pro replacements derived from rymoviruses or potyviruses were unable to infect SDp2 corn and oat. Collectively, these results indicate that HC-Pro from distantly related virus species of the family Potyviridae are competent to provide WSMV-Sidney 81 with all functions necessary for infection of a permissive host (wheat) and that virus-host interactions required for systemic infection of oat and SDp2 corn are more stringent. Changes in symptom severity or mechanical transmission efficiency observed for some chimeric viruses further suggest that HC-Pro affects virulence in WSMV

  12. Induction of protective immunity in swine by recombinant bamboo mosaic virus expressing foot-and-mouth disease virus epitopes

    Directory of Open Access Journals (Sweden)

    Lin Na-Sheng

    2007-09-01

    Full Text Available Abstract Background Plant viruses can be employed as versatile vectors for the production of vaccines by expressing immunogenic epitopes on the surface of chimeric viral particles. Although several viruses, including tobacco mosaic virus, potato virus X and cowpea mosaic virus, have been developed as vectors, we aimed to develop a new viral vaccine delivery system, a bamboo mosaic virus (BaMV, that would carry larger transgene loads, and generate better immunity in the target animals with fewer adverse environmental effects. Methods We engineered the BaMV as a vaccine vector expressing the antigenic epitope(s of the capsid protein VP1 of foot-and-mouth disease virus (FMDV. The recombinant BaMV plasmid (pBVP1 was constructed by replacing DNA encoding the 35 N-terminal amino acid residues of the BaMV coat protein with that encoding 37 amino acid residues (T128-N164 of FMDV VP1. Results The pBVP1 was able to infect host plants and to generate a chimeric virion BVP1 expressing VP1 epitopes in its coat protein. Inoculation of swine with BVP1 virions resulted in the production of anti-FMDV neutralizing antibodies. Real-time PCR analysis of peripheral blood mononuclear cells from the BVP1-immunized swine revealed that they produced VP1-specific IFN-γ. Furthermore, all BVP1-immunized swine were protected against FMDV challenge. Conclusion Chimeric BaMV virions that express partial sequence of FMDV VP1 can effectively induce not only humoral and cell-mediated immune responses but also full protection against FMDV in target animals. This BaMV-based vector technology may be applied to other vaccines that require correct expression of antigens on chimeric viral particles.

  13. HIV-1 tropism testing and clinical management of CCR5 antagonists: Quebec review and recommendations.

    Science.gov (United States)

    Tremblay, Cécile; Hardy, Isabelle; Lalonde, Richard; Trottier, Benoit; Tsarevsky, Irina; Vézina, Louis-Philippe; Roger, Michel; Wainberg, Mark; Baril, Jean-Guy

    2013-01-01

    HIV-1 tropism assays play a crucial role in determining the response to CCR5 receptor antagonists. Initially, phenotypic tests were used, but limited access to these tests prompted the development of alternative strategies. Recently, genotyping tropism has been validated using a Canadian technology in clinical trials investigating the use of maraviroc in both experienced and treatment-naive patients. The present guidelines review the evidence supporting the use of genotypic assays and provide recommendations regarding tropism testing in daily clinical management.

  14. Relationship between white spot syndrome virus (WSSV) loads and characterizations of water quality in Litopenaeus vannamei culture ponds during the tropical storm.

    Science.gov (United States)

    Zhang, J S; Li, Z J; Wen, G L; Wang, Y L; Luo, L; Zhang, H J; Dong, H B

    2016-01-01

    An in-situ experiment was conducted to investigate the effect of tropical storm on the white spot syndrome virus (WSSV) loads in Litopenaeus vannamei rearing ponds. White spot syndrome virus loads, heterotrophic bacteria, Vibrio and water quality (including temperature, dissolved oxygen (DO), salinity, pH, NH 4 -N, and NO 2 -N) were continually monitored through one tropical storm. The WSSV loads decreased when tropical storm made landfall, and substantially increased when typhoon passed. The variation of WSSV loads was correlated with DO, temperature, heterotrophic bacteria count, and ammonia-N concentrations. These results suggested that maintaining high level DO and promoting heterotrophic bacteria growth in the shrimp ponds might prevent the diseases' outbreak after the landfall of tropical storm.

  15. Chimeric classical swine fever (CSF)-Japanese encephalitis (JE) viral replicon as a non-transmissible vaccine candidate against CSF and JE infections.

    Science.gov (United States)

    Yang, Zhenhua; Wu, Rui; Li, Robert W; Li, Ling; Xiong, Zhongliang; Zhao, Haizhong; Guo, Deyin; Pan, Zishu

    2012-04-01

    A trans-complemented chimeric CSF-JE virus replicon was constructed using an infectious cDNA clone of the CSF virus (CSFV) Alfort/187 strain. The CSFV E2 gene was deleted, and a fragment containing the region encoding a truncated envelope protein (tE, amino acid 292-402, domain III) of JE virus (JEV) was inserted into the resultant plasmid, pA187delE2, to generate the recombinant cDNA clone pA187delE2/JEV-tE. Porcine kidney 15 (PK15) cells that constitutively express the CSFV E2p7 proteins were then transfected with in vitro-transcribed RNA from pA187delE2/JEV-tE. As a result, the chimeric CSF-JE virus replicon particle (VRP), rv187delE2/JEV-tE, was rescued. In a mouse model, immunization with the chimeric CSF-JE VRP induced strong production of JEV-specific antibody and conferred protection against a lethal JEV challenge. Pigs immunized with CSF-JE VRP displayed strong anti-CSFV and anti-JEV antibody responses and protection against CSFV and JEV challenge infections. Our evidence suggests that E2-complemented CSF-JE VRP not only has potential as a live-attenuated non-transmissible vaccine candidate against CSF and JE but also serves as a potential DIVA (Differentiating Infected from Vaccinated Animals) vaccine for CSF in pigs. Together, our data suggest that the non-transmissible chimeric VRP expressing foreign antigenic proteins may represent a promising strategy for bivalent DIVA vaccine design. Copyright © 2012 Elsevier B.V. All rights reserved.

  16. An adenine-to-guanine nucleotide change in the IRES SL-IV domain of picornavirus/hepatitis C chimeric viruses leads to a nonviable phenotype

    International Nuclear Information System (INIS)

    McKnight, Kevin L.; Sandefur, Stephanie; Phipps, Krista M.; Heinz, Beverly A.

    2003-01-01

    The inability for the internal ribosomal entry site (IRES) of hepatitis C virus (HCV) to be readily studied in the context of viral replication has been circumvented by constructing chimeras such as with poliovirus (PV), in which translation of the genome polyprotein is under control of the HCV IRES. During our attempts to configure the PV/HCV chimera for our drug discovery efforts, we discovered that an adenine- (A) to-guanine (G) change at nt 350 in domain IV of the HCV IRES resulted in a nonviable phenotype. Similarly, a mengovirus (MV)/HCV chimera using the same configuration with a G at nt 350 (G-350) was found to be nonviable. In contrast, a bovine viral diarrhea virus (BVDV)/HCV chimera remained viable with G-350 in the HCV IRES insert. Second-site, resuscitating mutations were identified from the G-350 PV/HCV and MV/HCV viruses after blind passaging. For both viruses, the resuscitating mutations involved destabilization of domain IV in the HCV IRES. The nonviability of G-350 in the picornavirus/HCV chimeric background might be linked to translation efficiency as indicated by analyses with dual reporter and PV/HCV replicon constructs

  17. HIV-1 Tropism Testing and Clinical Management of CCR5 Antagonists: Quebec Review and Recommendations

    Directory of Open Access Journals (Sweden)

    Cécile Tremblay

    2013-01-01

    Full Text Available HIV-1 tropism assays play a crucial role in determining the response to CCR5 receptor antagonists. Initially, phenotypic tests were used, but limited access to these tests prompted the development of alternative strategies. Recently, genotyping tropism has been validated using a Canadian technology in clinical trials investigating the use of maraviroc in both experienced and treatment-naive patients. The present guidelines review the evidence supporting the use of genotypic assays and provide recommendations regarding tropism testing in daily clinical management.

  18. A mosaic adenovirus possessing serotype Ad5 and serotype Ad3 knobs exhibits expanded tropism

    International Nuclear Information System (INIS)

    Takayama, Koichi; Reynolds, Paul N.; Short, Joshua J.; Kawakami, Yosuke; Adachi, Yasuo; Glasgow, Joel N.; Rots, Marianne G.; Krasnykh, Victor; Douglas, Joanne T.; Curiel, David T.

    2003-01-01

    The efficiency of cancer gene therapy with recombinant adenoviruses based on serotype 5 (Ad5) has been limited partly because of variable, and often low, expression by human primary cancer cells of the primary cellular-receptor which recognizes the knob domain of the fiber protein, the coxsackie and adenovirus receptor (CAR). As a means of circumventing CAR deficiency, Ad vectors have been retargeted by utilizing chimeric fibers possessing knob domains of alternate Ad serotypes. We have reported that ovarian cancer cells possess a primary receptor for Ad3 to which the Ad3 knob binds independently of the CAR-Ad5 knob interaction. Furthermore, an Ad5-based chimeric vector, designated Ad5/3, containing a chimeric fiber proteins possessing the Ad3 knob, demonstrates CAR-independent tropism by virtue of targeting the Ad3 receptor. Based on these findings, we hypothesized that a mosaic virus possessing both the Ad5 knob and the Ad3 knob on the same virion could utilize either primary receptor, resulting in expanded tropism. In this study, we generated a dual-knob mosaic virus by coinfection of 293 cells with Ad5-based and Ad5/3-based vectors. Characterization of the resultant virions confirmed the incorporation of both Ad5 and Ad3 knobs in the same particle. Furthermore, this mosaic virus was able to utilize either receptor, CAR and the Ad3 receptor, for virus attachment to cells. Enhanced Ad infectivity with the mosaic virus was shown in a panel of cell lines, with receptor profiles ranging from CAR-dominant to Ad3 receptor-dominant. Thus, this mosaic virus strategy may offer the potential to improve Ad-based gene therapy approaches by infectivity enhancement and tropism expansion

  19. CCR2 V64I polymorphism in rifampicin resistant tuberculosis patients in Moewardi General Hospital Surakarta, Indonesia

    Science.gov (United States)

    Marwoto; Agung Prasetyo, Afiono; Reviono; Suradi

    2018-05-01

    CC chemokine receptor-2 (CCR2) play important roles in inflammation. The CCR2 V64I polymorphism already reported associated with many diseases; however, the association of CCR2 V64I polymorphism with tuberculosis is still unknown. Also, there is no report about the presentation of CCR2 V64I polymorphisms in Indonesian tuberculosis patients with rifampicin-mono resistant status has ever been published, to the best of our knowledge. This study evaluated the presence of CCR2 V64I polymorphisms in Javanese rifampicin-mono resistant tuberculosis patients. In an ongoing molecular epidemiology study of human genomic polymorphisms and infection, 51 Javanese rifampicin-mono resistant tuberculosis patients in Dr. Moewardi General Hospital in Surakarta were enrolled in the study. The blood samples were aliquoted and fractionated. The nucleic acids were extracted from all blood samples and subjected to the CCR2 V64I polymorphisms detection by a polymerase chain reaction-sequence-specific primer (PCR-SSP) technique. PCR products were analyzed in 3% agarose. CCR2 64V and 64I homozygote were found in 23.5% (12/51) and 23.5% (12/51) blood samples, respectively. The CCR2 VI genotype was found in 52.9% (27/51) blood samples. The CCR2 VI genotype was found predominant in Javanese rifampicin-mono resistant tuberculosis patients and may have anassociation with the clinical progression.

  20. Suppression of CCR impacts metabolite profile and cell wall composition in Pinus radiata tracheary elements.

    Science.gov (United States)

    Wagner, Armin; Tobimatsu, Yuki; Goeminne, Geert; Phillips, Lorelle; Flint, Heather; Steward, Diane; Torr, Kirk; Donaldson, Lloyd; Boerjan, Wout; Ralph, John

    2013-01-01

    Suppression of the lignin-related gene cinnamoyl-CoA reductase (CCR) in the Pinus radiata tracheary element (TE) system impacted both the metabolite profile and the cell wall matrix in CCR-RNAi lines. UPLC-MS/MS-based metabolite profiling identified elevated levels of p-coumaroyl hexose, caffeic acid hexoside and ferulic acid hexoside in CCR-RNAi lines, indicating a redirection of metabolite flow within phenylpropanoid metabolism. Dilignols derived from coniferyl alcohol such as G(8-5)G, G(8-O-4)G and isodihydrodehydrodiconiferyl alcohol (IDDDC) were substantially depleted, providing evidence for CCR's involvement in coniferyl alcohol biosynthesis. Severe CCR suppression almost halved lignin content in TEs based on a depletion of both H-type and G-type lignin, providing evidence for CCR's involvement in the biosynthesis of both lignin types. 2D-NMR studies revealed minor changes in the H:G-ratio and consequently a largely unchanged interunit linkage distribution in the lignin polymer. However, unusual cell wall components including ferulate and unsaturated fatty acids were identified in TEs by thioacidolysis, pyrolysis-GC/MS and/or 2D-NMR in CCR-RNAi lines, providing new insights into the consequences of CCR suppression in pine. Interestingly, CCR suppression substantially promoted pyrolytic breakdown of cell wall polysaccharides, a phenotype most likely caused by the incorporation of acidic compounds into the cell wall matrix in CCR-RNAi lines.

  1. CXCR4-using HIV variants in a cohort of Black men who have sex with men: HIV Prevention Trials Network 061.

    Science.gov (United States)

    Chen, Iris; Huang, Wei; Connor, Matthew B; Frantzell, Arne; Cummings, Vanessa; Beauchamp, Geetha G; Griffith, Sam; Fields, Sheldon D; Scott, Hyman M; Shoptaw, Steven; Del Rio, Carlos; Magnus, Manya; Mannheimer, Sharon; Tieu, Hong-Van; Wheeler, Darrell P; Mayer, Kenneth H; Koblin, Beryl A; Eshleman, Susan H

    2016-07-01

    To evaluate factors associated with HIV tropism among Black men who have sex with men (MSM) in the United States enrolled in a clinical study (HIV Prevention Trials Network 061). HIV tropism was analyzed using a phenotypic assay (Trofile assay, Monogram Biosciences). Samples were analyzed from 43 men who were HIV infected at enrollment and reported either exclusive insertive intercourse or exclusive receptive intercourse; samples were also analyzed from 20 men who were HIV uninfected at enrollment and seroconverted during the study. Clonal analysis of individual viral variants was performed for seroconverters who had dual/mixed (DM) viruses. DM viruses were detected in samples from 11 (26%) of the 43 HIV-infected men analyzed at the enrollment visit; HIV tropism did not differ between those reporting exclusive insertive vs receptive intercourse. DM viruses were also detected in five (25%) of the 20 seroconverters. DM viruses were associated with lower CD4 cell counts. Seroconverters with DM viruses had dual-tropic viruses only or mixed populations of CCR5- and dual-tropic viruses. DM viruses were frequently detected among Black MSM in this study, including seroconverters. Further studies are needed to understand factors driving transmission and selection of CXCR4- and dual-tropic viruses among Black MSM.

  2. CCR2-V64I genetic polymorphism: a possible involvement in HER2+ breast cancer.

    Science.gov (United States)

    Banin-Hirata, Bruna Karina; Losi-Guembarovski, Roberta; Oda, Julie Massayo Maeda; de Oliveira, Carlos Eduardo Coral; Campos, Clodoaldo Zago; Mazzuco, Tânia Longo; Borelli, Sueli Donizete; Ceribelli, Jesus Roberto; Watanabe, Maria Angelica Ehara

    2016-05-01

    Many tumor cells express chemokines and chemokine receptors, and these molecules can affect both tumor progression and anti-tumor immune response. Genetic polymorphisms of some chemokine receptors were found to be closely related to malignant tumors, especially in metastasis process, including breast cancer (BC). Considering this, it was investigated a possible role for CCR2-V64I (C-C chemokine receptor 2) and CCR5-Δ32 (C-C chemokine receptor 5) genetic variants in BC context. Patients were divided into subgroups according to immunohistochemical profile of estrogen (ER) and progesterone (PR) receptors and the human epidermal growth factor receptor 2 (HER2) overexpression. No significant associations were found in relation to susceptibility (CCR2-V64I: OR 1.32; 95 % CI 0.57-3.06; CCR5-∆32: OR 1.04; 95 % CI 0.60-1.81), clinical outcome (tumor size, lymph nodes commitment and/or distant metastasis, TNM staging and nuclear grade) or therapeutic response (recurrence and survival). However, it was found a significant correlation between CCR2-V64I allelic variant and HER2 immunohistochemical positive samples (p = 0.026). All in all, we demonstrate, for the first time, a positive correlation between CCR2 receptor gene polymorphism and a subgroup of BC related to poor prognosis, which deserves further investigation in larger samples for validation.

  3. Fabrication and characterization of gold nano-wires templated on virus-like arrays of tobacco mosaic virus coat proteins

    International Nuclear Information System (INIS)

    Wnęk, M; Stockley, P G; Górzny, M Ł; Evans, S D; Ward, M B; Brydson, R; Wälti, C; Davies, A G

    2013-01-01

    The rod-shaped plant virus tobacco mosaic virus (TMV) is widely used as a nano-fabrication template, and chimeric peptide expression on its major coat protein has extended its potential applications. Here we describe a simple bacterial expression system for production and rapid purification of recombinant chimeric TMV coat protein carrying C-terminal peptide tags. These proteins do not bind TMV RNA or form disks at pH 7. However, they retain the ability to self-assemble into virus-like arrays at acidic pH. C-terminal peptide tags in such arrays are exposed on the protein surface, allowing interaction with target species. We have utilized a C-terminal His-tag to create virus coat protein-templated nano-rods able to bind gold nanoparticles uniformly. These can be transformed into gold nano-wires by deposition of additional gold atoms from solution, followed by thermal annealing. The resistivity of a typical annealed wire created by this approach is significantly less than values reported for other nano-wires made using different bio-templates. This expression construct is therefore a useful additional tool for the creation of chimeric TMV-like nano-rods for bio-templating. (paper)

  4. Fabrication and characterization of gold nano-wires templated on virus-like arrays of tobacco mosaic virus coat proteins

    Science.gov (United States)

    Wnęk, M.; Górzny, M. Ł.; Ward, M. B.; Wälti, C.; Davies, A. G.; Brydson, R.; Evans, S. D.; Stockley, P. G.

    2013-01-01

    The rod-shaped plant virus tobacco mosaic virus (TMV) is widely used as a nano-fabrication template, and chimeric peptide expression on its major coat protein has extended its potential applications. Here we describe a simple bacterial expression system for production and rapid purification of recombinant chimeric TMV coat protein carrying C-terminal peptide tags. These proteins do not bind TMV RNA or form disks at pH 7. However, they retain the ability to self-assemble into virus-like arrays at acidic pH. C-terminal peptide tags in such arrays are exposed on the protein surface, allowing interaction with target species. We have utilized a C-terminal His-tag to create virus coat protein-templated nano-rods able to bind gold nanoparticles uniformly. These can be transformed into gold nano-wires by deposition of additional gold atoms from solution, followed by thermal annealing. The resistivity of a typical annealed wire created by this approach is significantly less than values reported for other nano-wires made using different bio-templates. This expression construct is therefore a useful additional tool for the creation of chimeric TMV-like nano-rods for bio-templating.

  5. Preclinical discovery and development of maraviroc for the treatment of HIV.

    Science.gov (United States)

    Veljkovic, Nevena; Vucicevic, Jelica; Tassini, Sabrina; Glisic, Sanja; Veljkovic, Veljko; Radi, Marco

    2015-06-01

    Maraviroc is a first-in-class antiretroviral (ARV) drug acting on a host cell target (CCR5), which blocks the entry of the HIV virus into the cell. Maraviroc is currently indicated for combination ARV treatment in adults infected only with CCR5-tropic HIV-1. This drug discovery case history focuses on the key studies that led to the discovery and approval of maraviroc, as well as on post-launch clinical reports. The article is based on the data reported in published preclinical and clinical studies, conference posters and on drug package data. The profound understanding of HIV's entry mechanisms has provided a strong biological rationale for targeting the chemokine receptor CCR5. The CCR5-antagonist mariviroc, with its unique mode of action and excellent safety profile, is an important therapeutic option for HIV patients. In general, the authors believe that targeting host factors is a useful approach for combating new and re-emerging transmissible diseases, as well as pathogens that easily become resistant to common antiviral drugs. Maraviroc, offering a potent and safe cellular receptor-mediated pharmacological response to HIV, has paved the way for the development of a new generation of host-targeting antivirals.

  6. Role of the frequency of blood CD4{sup +} CXCR5{sup +} CCR6{sup +} T cells in autoimmunity in patients with Sjoegren's syndrome

    Energy Technology Data Exchange (ETDEWEB)

    Li, Xue-yi; Wu, Zhen-biao; Ding, Jin; Zheng, Zhao-hui [Department of Clinical Immunology, State key Discipline of Cell Biology, Xi-jing Hospital, Fourth Military Medical University, Shaanxi Province (China); Li, Xiao-yan [Department of Endocrine and Metabolic Diseases, Shaanxi Provincial People' s Hospital, Xi' an, Shaanxi Province (China); Chen, Li-na [Department of Clinical Immunology, State key Discipline of Cell Biology, Xi-jing Hospital, Fourth Military Medical University, Shaanxi Province (China); Zhu, Ping, E-mail: zhuping@fmmu.edu.cn [Department of Clinical Immunology, State key Discipline of Cell Biology, Xi-jing Hospital, Fourth Military Medical University, Shaanxi Province (China)

    2012-06-01

    Highlights: Black-Right-Pointing-Pointer The frequency of CD4{sup +} CXCR5{sup +} CCR6{sup +} T cells increased in pSS patients and positively correlated with autoantibodies in the blood. Black-Right-Pointing-Pointer CD4{sup +} CXCR5{sup +} CCR6{sup +} T cells in blood invariably coexpressed PD-1, ICOS, CD40L, Bcl-6 and secreted IL-21 after stimulated by PHA. Black-Right-Pointing-Pointer CD4{sup +} CXCR5{sup +} CCR6{sup +} Tfh cells in blood may be suitable biomarkers for the evaluation of the active immune stage of pSS patients. -- Abstract: The blood CD4{sup +} CXCR5{sup +} T cells, known as 'circulating' Tfh, have been shown to efficiently induce naieve B cells to produce immunoglobulin. They play an important role in certain autoimmune diseases. In the present study, we show for the first time that the frequency of CD4{sup +} CXCR5{sup +} T cells is increased in pSS patients and positively correlated with autoantibodies in the blood. The concentration of Th17-like subsets (CD4{sup +} CXCR5{sup +} CCR6{sup +}) in pSS patients was found to be significantly higher than in healthy controls. Functional assays showed that activated Th17-like subtypes in the blood display the key features of Tfh cells, including invariably coexpressed PD-1, ICOS, CD40L and IL-21. Th17 subsets were found to highly express Bcl-6 protein and Th1 and Th2 were not. Bcl-6 is believed to be a master transforming factor for Tfh cell differentiation and facilitate B cell proliferation and somatic hypermutation within the germinal center. These data indicate that Th17 subsets of CD4{sup +} CXCR5{sup +} T cells in the blood may participate in the antibody-related immune responses and that high frequency of CD4{sup +} CXCR5{sup +} CCR6{sup +} Tfh cells in blood may be suitable biomarkers for the evaluation of the active immune stage of pSS patients. It might provide insights into the pathogenesis and perhaps help researchers identify novel therapeutic targets for pSS.

  7. Chimeric Amino Acid Rearrangements as Immune Targets in Prostate Cancer

    Science.gov (United States)

    2016-05-01

    COVERED 4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER Chimeric Amino Acid Rearrangements as Immune Targets in Prostate Cancer 5b. GRANT NUMBER W81XWH...that result from gene rearrangements given their high frequency relative to somatic point mutations. Gene rearrangements can yield novel chimeric

  8. Production of a Recombinant Dengue Virus 2 NS5 Protein and Potential Use as a Vaccine Antigen.

    Science.gov (United States)

    Alves, Rúbens Prince Dos Santos; Pereira, Lennon Ramos; Fabris, Denicar Lina Nascimento; Salvador, Felipe Scassi; Santos, Robert Andreata; Zanotto, Paolo Marinho de Andrade; Romano, Camila Malta; Amorim, Jaime Henrique; Ferreira, Luís Carlos de Souza

    2016-06-01

    Dengue fever is caused by any of the four known dengue virus serotypes (DENV1 to DENV4) that affect millions of people worldwide, causing a significant number of deaths. There are vaccines based on chimeric viruses, but they still are not in clinical use. Anti-DENV vaccine strategies based on nonstructural proteins are promising alternatives to those based on whole virus or structural proteins. The DENV nonstructural protein 5 (NS5) is the main target of anti-DENV T cell-based immune responses in humans. In this study, we purified a soluble recombinant form of DENV2 NS5 expressed in Escherichia coli at large amounts and high purity after optimization of expression conditions and purification steps. The purified DENV2 NS5 was recognized by serum from DENV1-, DENV2-, DENV3-, or DENV4-infected patients in an epitope-conformation-dependent manner. In addition, immunization of BALB/c mice with NS5 induced high levels of NS5-specific antibodies and expansion of gamma interferon- and tumor necrosis factor alpha-producing T cells. Moreover, mice immunized with purified NS5 were partially protected from lethal challenges with the DENV2 NGC strain and with a clinical isolate (JHA1). These results indicate that the recombinant NS5 protein preserves immunological determinants of the native protein and is a promising vaccine antigen capable of inducing protective immune responses. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  9. Generating chimeric mice from embryonic stem cells via vial coculturing or hypertonic microinjection.

    Science.gov (United States)

    Lee, Kun-Hsiung

    2014-01-01

    The generation of a fertile embryonic stem cell (ESC)-derived or F0 (100 % coat color chimerism) mice is the final criterion in proving that the ESC is truly pluripotent. Many methods have been developed to produce chimeric mice. To date, the most popular methods for generating chimeric embryos is well sandwich aggregation between zona pellucida (ZP) removed (denuded) 2.5-day post-coitum (dpc) embryos and ESC clumps, or direct microinjection of ESCs into the cavity (blastocoel) of 3.5-dpc blastocysts. However, due to systemic limitations and the disadvantages of conventional microinjection, aggregation, and coculturing, two novel methods (vial coculturing and hypertonic microinjection) were developed in recent years at my laboratory.Coculturing 2.5-dpc denuded embryos with ESCs in 1.7-mL vials for ~3 h generates chimeras that have significantly high levels of chimerism (including 100 % coat color chimerism) and germline transmission. This method has significantly fewer instrumental and technological limitations than existing methods, and is an efficient, simple, inexpensive, and reproducible method for "mass production" of chimeric embryos. For laboratories without a microinjection system, this is the method of choice for generating chimeric embryos. Microinjecting ESCs into a subzonal space of 2.5-dpc embryos can generate germline-transmitted chimeras including 100 % coat color chimerism. However, this method is adopted rarely due to the very small and tight space between ZP and blastomeres. Using a laser pulse or Piezo-driven instrument/device to help introduce ESCs into the subzonal space of 2.5-dpc embryos demonstrates the superior efficiency in generating ESC-derived (F0) chimeras. Unfortunately, due to the need for an expensive instrument/device and extra fine skill, not many studies have used either method. Recently, ESCs injected into the large subzonal space of 2.5-dpc embryos in an injection medium containing 0.2-0.3 M sucrose very efficiently generated

  10. Replacement of Murine Leukemia Virus Readthrough Mechanism by Human Immunodeficiency Virus Frameshift Allows Synthesis of Viral Proteins and Virus Replication

    Science.gov (United States)

    Brunelle, Marie-Noëlle; Brakier-Gingras, Léa; Lemay, Guy

    2003-01-01

    Retroviruses use unusual recoding strategies to synthesize the Gag-Pol polyprotein precursor of viral enzymes. In human immunodeficiency virus, ribosomes translating full-length viral RNA can shift back by 1 nucleotide at a specific site defined by the presence of both a slippery sequence and a downstream stimulatory element made of an extensive secondary structure. This so-called frameshift mechanism could become a target for the development of novel antiviral strategies. A different recoding strategy is used by other retroviruses, such as murine leukemia viruses, to synthesize the Gag-Pol precursor; in this case, a stop codon is suppressed in a readthrough process, again due to the presence of a specific structure adopted by the mRNA. Development of antiframeshift agents will greatly benefit from the availability of a simple animal and virus model. For this purpose, the murine leukemia virus readthrough region was rendered inactive by mutagenesis and the frameshift region of human immunodeficiency virus was inserted to generate a chimeric provirus. This substitution of readthrough by frameshift allows the synthesis of viral proteins, and the chimeric provirus sequence was found to generate infectious viruses. This system could be a most interesting alternative to study ribosomal frameshift in the context of a virus amenable to the use of a simple animal model. PMID:12584361

  11. The chimeric transcript RUNX1-GLRX5: a biomarker for good postoperative prognosis in Stage IA non-small-cell lung cancer.

    Science.gov (United States)

    Ishikawa, Rie; Amano, Yosuke; Kawakami, Masanori; Sunohara, Mitsuhiro; Watanabe, Kousuke; Kage, Hidenori; Ohishi, Nobuya; Yatomi, Yutaka; Nakajima, Jun; Fukayama, Masashi; Nagase, Takahide; Takai, Daiya

    2016-02-01

    Stage IA non-small-cell lung cancer cases have been recognized as having a low risk of relapse; however, occasionally, relapse may occur. To predict clinical outcome in Stage IA non-small-cell lung cancer patients, we searched for chimeric transcripts that can be used as biomarkers and identified a novel chimeric transcript, RUNX1-GLRX5, comprising RUNX1, a transcription factor, and GLRX5. This chimera was detected in approximately half of the investigated Stage IA non-small-cell lung cancer patients (44/104 cases, 42.3%). Although there was no significant difference in the overall survival rate between RUNX1-GLRX5-positive and -negative cases (P = 0.088), a significantly lower relapse rate was observed in the RUNX1-GLRX5-positive cases (P = 0.039), indicating that this chimera can be used as a biomarker for good prognosis in Stage IA patients. Detection of the RUNX1-GLRX5 chimeric transcript may therefore be useful for the determination of a postoperative treatment plan for Stage IA non-small-cell lung cancer patients. © The Author 2015. Published by Oxford University Press.

  12. Custom-engineered chimeric foot-and-mouth disease vaccine elicits protective immune responses in pigs

    Science.gov (United States)

    Chimeric foot-and-mouth disease viruses (FMDV) of which the antigenic properties can be readily manipulated is a potentially powerful approach in the control of foot-and-mouth disease (FMD) in sub-Saharan Africa. FMD vaccine application is complicated by the extensive variability of the South Africa...

  13. Highly pathogenic influenza A(H5N1 virus survival in complex artificial aquatic biotopes.

    Directory of Open Access Journals (Sweden)

    Viseth Srey Horm

    Full Text Available BACKGROUND: Very little is known regarding the persistence of Highly Pathogenic Avian Influenza (HPAI H5N1 viruses in aquatic environments in tropical countries, although environmental materials have been suggested to play a role as reservoirs and sources of transmission for H5N1 viruses. METHODOLOGY/PRINCIPAL FINDINGS: The survival of HPAI H5N1 viruses in experimental aquatic biotopes (water, mud, aquatic flora and fauna relevant to field conditions in Cambodia was investigated. Artificial aquatic biotopes, including simple ones containing only mud and water, and complex biotopes involving the presence of aquatic flora and fauna, were set up. They were experimentally contaminated with H5N1 virus. The persistence of HPAI H5N1 virus (local avian and human isolates was determined by virus isolation in embryonated chicken eggs and by real-time reverse-polymerase chain reaction. Persistence of infectious virus did not exceed 4 days, and was only identified in rain water. No infectious virus particles were detected in pond and lake water or mud even when high inoculum doses were used. However, viral RNA persisted up to 20 days in rain water and 7 days in pond or lake water. Viral RNA was also detected in mud samples, up to 14 days post-contamination in several cases. Infectious virus and viral RNA was detected in few cases in the aquatic fauna and flora, especially in bivalves and labyrinth fish, although these organisms seemed to be mostly passive carriers of the virus rather than host allowing virus replication. CONCLUSIONS/SIGNIFICANCE: Although several factors for the survival and persistence of HPAI viruses in the environment are still to be elucidated, and are particularly hard to control in laboratory conditions, our results, along with previous data, support the idea that environmental surveillance is of major relevance for avian influenza control programs.

  14. Acid Stability of the Hemagglutinin Protein Regulates H5N1 Influenza Virus Pathogenicity

    Energy Technology Data Exchange (ETDEWEB)

    DuBois, Rebecca M.; Zaraket, Hassan; Reddivari, Muralidhar; Heath, Richard J.; White, Stephen W.; Russell, Charles J. (Tennessee-HSC); (SJCH)

    2012-12-10

    Highly pathogenic avian influenza viruses of the H5N1 subtype continue to threaten agriculture and human health. Here, we use biochemistry and x-ray crystallography to reveal how amino-acid variations in the hemagglutinin (HA) protein contribute to the pathogenicity of H5N1 influenza virus in chickens. HA proteins from highly pathogenic (HP) A/chicken/Hong Kong/YU562/2001 and moderately pathogenic (MP) A/goose/Hong Kong/437-10/1999 isolates of H5N1 were found to be expressed and cleaved in similar amounts, and both proteins had similar receptor-binding properties. However, amino-acid variations at positions 104 and 115 in the vestigial esterase sub-domain of the HA1 receptor-binding domain (RBD) were found to modulate the pH of HA activation such that the HP and MP HA proteins are activated for membrane fusion at pH 5.7 and 5.3, respectively. In general, an increase in H5N1 pathogenicity in chickens was found to correlate with an increase in the pH of HA activation for mutant and chimeric HA proteins in the observed range of pH 5.2 to 6.0. We determined a crystal structure of the MP HA protein at 2.50 {angstrom} resolution and two structures of HP HA at 2.95 and 3.10 {angstrom} resolution. Residues 104 and 115 that modulate the acid stability of the HA protein are situated at the N- and C-termini of the 110-helix in the vestigial esterase sub-domain, which interacts with the B loop of the HA2 stalk domain. Interactions between the 110-helix and the stalk domain appear to be important in regulating HA protein acid stability, which in turn modulates influenza virus replication and pathogenesis. Overall, an optimal activation pH of the HA protein is found to be necessary for high pathogenicity by H5N1 influenza virus in avian species.

  15. Downregulation of CCR1 inhibits human hepatocellular carcinoma cell invasion

    International Nuclear Information System (INIS)

    Wu Xiaofeng; Fan Jia; Wang Xiaoying; Zhou Jian; Qiu Shuangjian; Yu Yao; Liu Yinkun; Tang Zhaoyou

    2007-01-01

    CC chemokine receptor 1 (CCR1) has an important role in the recruitment of leukocytes to the site of inflammation. The migration and metastasis of tumor cells shares many similarities with leukocyte trafficking, which is mainly regulated by chemokine receptor-ligand interactions. CCR1 is highly expressed in hepatocellular carcinoma (HCC) cells and tissues with unknown functions. In this study, we silenced CCR1 expression in the human HCC cell line HCCLM3 using artificial microRNA (miRNA)-mediated RNA interference (RNAi) and examined the invasiveness and proliferation of CCR1-silenced HCCLM3 cells and the matrix metalloproteinase (MMP) activity. The miRNA-mediated knockdown expression of CCR1 significantly inhibited the invasive ability of HCCLM3 cells, but had only a minor effect on the cellular proliferation rate. Moreover, CCR1 knockdown significantly reduced the secretion of MMP-2. Together, these findings indicate that CCR1 has an important role in HCCLM3 invasion and that CCR1 might be a new target of HCC treatment

  16. Cloning and expression analysis of cinnamoyl-CoA reductase (CCR) genes in sorghum.

    Science.gov (United States)

    Li, Jieqin; Fan, Feifei; Wang, Lihua; Zhan, Qiuwen; Wu, Peijin; Du, Junli; Yang, Xiaocui; Liu, Yanlong

    2016-01-01

    Cinnamoyl-CoA reductase (CCR) is the first enzyme in the monolignol-specific branch of the lignin biosynthetic pathway. In this research, three sorghum CCR genes including SbCCR1, SbCCR2-1 and SbCCR2-2 were cloned and characterized. Analyses of the structure and phylogeny of the three CCR genes showed evolutionary conservation of the functional domains and divergence of function. Transient expression assays in Nicotiana benthamiana leaves demonstrated that the three CCR proteins were localized in the cytoplasm. The expression analysis showed that the three CCR genes were induced by drought. But in 48 h, the expression levels of SbCCR1 and SbCCR2-2 did not differ between CK and the drought treatment; while the expression level of SbCCR2-1 in the drought treatment was higher than in CK. The expression of the SbCCR1 and SbCCR2-1 genes was not induced by sorghum aphid [Melanaphis sacchari (Zehntner)] attack, but SbCCR2-2 was significantly induced by sorghum aphid attack. It is suggested that SbCCR2-2 is involved in the process of pest defense. Absolute quantitative real-time PCR revealed that the three CCR genes were mainly expressed in lignin deposition organs. The gene copy number of SbCCR1 was significantly higher than those of SbCCR2-1 and SbCCR2-2 in the tested tissues, especially in stem. The results provide new insight into the functions of the three CCR genes in sorghum.

  17. Chimeric polyomavirus-derived virus-like particles: the immunogenicity of an inserted peptide applied without adjuvant to mice depends on its insertion site and its flanking linker sequence

    OpenAIRE

    Lawatscheck, R.; Aleksaite, E.; Schenk, J.A.; Micheel, B.; Jandrig, B.; Holland, G.; Sasnauskas, K.; Gedvilaite, A.; Ulrich, R.G.

    2007-01-01

    We inserted the sequence of the carcinoembryonic antigen-derived T cell epitope CAP-1-6D (CEA) into different positions of the hamster polyomavirus major capsid protein VP1. Independently from additional flanking linkers, yeast-expressed VP1 proteins harboring the CEA insertion between VP1 amino acid residues 80 and 89 (site 1) or 288 and 295 (site 4) or simultaneously at both positions assembled to chimeric virus-like particles (VLPs). BALB/c mice immunized with adjuvant-free VLPs developed ...

  18. Parainfluenza virus infections in a tropical city: clinical and epidemiological aspects

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    Mariana Mota Moura Fé

    Full Text Available Little information on the epidemiology and clinical characteristics of human parainfluenza virus (HPIV infections, especially in children from tropical countries, has been published. The aim of this study was to determine the frequency of HPIV infections in children attended at a large hospital in Fortaleza in Northeast Brazil, and describe seasonal patterns, clinical and epidemiological characteristics of these infections. From January 2001 to December 2006, a total of 3070 nasopharyngeal aspirates collected from children were screened by indirect immunofluorescence for human parainfluenza viruses 1, 2, and 3 (HPIV-1, 2 and 3 and other respiratory viruses. Viral antigens were identified in 933 samples and HPIV in 117. The frequency of HPIV-3, HPIV-1 and HPIV-2 was of 83.76%, 11.96% and 4.27%, respectively. Only HPIV-3 showed a seasonal occurrence, with most cases observed from September to November, and with an inverse relationship to the rainy season. Most HPIV-3 infections seen in outpatients were diagnosed as upper respiratory tract infections.

  19. Associations of fractalkine receptor (CX3CR1) and CCR5 gene variants with hypertension, diabetes and atherosclerosis in chronic renal failure patients undergoing hemodialysis.

    Science.gov (United States)

    Bagci, Binnur; Bagci, Gokhan; Huzmeli, Can; Sezgin, Ilhan; Ozdemir, Ozturk

    2016-07-01

    We aimed to investigate the associations of fractalkine receptor (CX3CR1) V249I, T280M and CCR5-59029 A/G gene polymorphisms in chronic renal failure (CRF) subjects undergoing hemodialysis and to evaluate possible associations of these polymorphisms with hypertension (HT), diabetes mellitus (DM) and atherosclerosis (AS). A total of 225 CRF subjects undergoing hemodialysis and 201 healthy controls were enrolled in the study. CRF subjects were divided into three major subgroups according to comorbidities including HT (n = 127), DM (n = 65) and AS (n = 33). Genotyping was done using polymerase chain reaction-restriction fragment length polymorphism method. The II genotype and I allele frequencies of CX3CR1 V249I polymorphism were found significantly more frequent in CRF subjects, CRF subjects with DM and CRF subjects with AS compared with controls (p < 0.05 for all comparisons). G allele frequency of CCR5 polymorphism was found significantly more prevalent in CRF subjects with DM than that of controls. Further, GG genotype and G allele frequencies of CCR5 polymorphism were significantly more prevalent in CRF subjects with AS compared with controls (p < 0.05). We also explored these polymorphisms among CRF subjects with and without following comorbidities: HT, DM, AS. We found significant association between CRF subjects with HT and without HT in terms of genotype and allele frequencies of V249I polymorphism (p < 0.05). CX3CR1 T280M polymorphism was not found significantly different in none of the comparisons. These data demonstrate possible associations between CX3CR1 V249I and CCR5-59029 A/G polymorphisms and/or HT, DM and AS in CRF subjects.

  20. Chimeric rabies glycoprotein with a transmembrane domain and cytoplasmic tail from Newcastle disease virus fusion protein incorporates into the Newcastle disease virion at reduced levels.

    Science.gov (United States)

    Yu, Gui Mei; Zu, Shu Long; Zhou, Wei Wei; Wang, Xi Jun; Shuai, Lei; Wang, Xue Lian; Ge, Jin Ying; Bu, Zhi Gao

    2017-08-31

    Rabies remains an important worldwide health problem. Newcastle disease virus (NDV) was developed as a vaccine vector in animals by using a reverse genetics approach. Previously, our group generated a recombinant NDV (LaSota strain) expressing the complete rabies virus G protein (RVG), named rL-RVG. In this study, we constructed the variant rL-RVGTM, which expresses a chimeric rabies virus G protein (RVGTM) containing the ectodomain of RVG and the transmembrane domain (TM) and a cytoplasmic tail (CT) from the NDV fusion glycoprotein to study the function of RVG's TM and CT. The RVGTM did not detectably incorporate into NDV virions, though it was abundantly expressed at the surface of infected BHK-21 cells. Both rL-RVG and rL-RVGTM induced similar levels of NDV virus-neutralizing antibody (VNA) after initial and secondary vaccination in mice, whereas rabies VNA induction by rL-RVGTM was markedly lower than that induced by rL-RVG. Though rL-RVG could spread from cell to cell like that in rabies virus, rL-RVGTM lost this ability and spread in a manner similar to the parental NDV. Our data suggest that the TM and CT of RVG are essential for its incorporation into NDV virions and for spreading of the recombinant virus from the initially infected cells to surrounding cells.

  1. In silico prediction of monovalent and chimeric tetravalent vaccines for prevention and treatment of dengue fever.

    Science.gov (United States)

    Vijayakumar, Subramaniyan; Ramesh, Venkatachalam; Prabhu, Srinivasan; Manogar, Palani

    2017-11-01

    Reverse vaccinology method was used to predict the monovalent peptide vaccine candidate to produce antibodies for therapeutic purpose and to predict tetravalent vaccine candidate to act as a common vaccine to cover all the fever dengue virus serotypes. Envelope (E)-proteins of DENV-1-4 serotypes were used for vaccine prediction using NCBI, Uniprot/Swissprot, Swiss-prot viewer, VaxiJen V2.0, TMHMM, BCPREDS, Propred-1, Propred and MHC Pred,. E-proteins of DENV-1-4 serotypes were identified as antigen from which T cell epitopes, through B cell epitopes, were predicted to act as peptide vaccine candidates. Each selected T cell epitope of E-protein was confirmed to act as vaccine and to induce complementary antibody against particular serotype of dengue virus. Chimeric tetravalent vaccine was formed by the conjugation of four vaccines, each from four dengue serotypes to act as a common vaccine candidate for all the four dengue serotypes. It can be justifiably concluded that the monovalent 9-mer T cell epitope for each DENV serotypes can be used to produce specific antibody agaomst dengue virus and a chimeric common tetravalent vaccine candidate to yield a comparative vaccine to cover any of the four dengue virus serotype. This vaccine is expected to act as highly immunogenic against preventing dengue fever.

  2. Development of a mouse-feline chimeric antibody against feline tumor necrosis factor-alpha

    Science.gov (United States)

    DOKI, Tomoyoshi; TAKANO, Tomomi; HOHDATSU, Tsutomu

    2016-01-01

    Feline infectious peritonitis (FIP) is a fatal inflammatory disease caused by FIP virus infection. Feline tumor necrosis factor (fTNF)-alpha is closely involved in the aggravation of FIP pathology. We previously described the preparation of neutralizing mouse anti-fTNF-alpha monoclonal antibody (mAb 2–4) and clarified its role in the clinical condition of cats with FIP using in vitro systems. However, administration of mouse mAb 2–4 to cat may lead to a production of feline anti-mouse antibodies. In the present study, we prepared a mouse-feline chimeric mAb (chimeric mAb 2–4) by fusing the variable region of mouse mAb 2–4 to the constant region of feline antibody. The chimeric mAb 2–4 was confirmed to have fTNF-alpha neutralization activity. Purified mouse mAb 2–4 and chimeric mAb 2–4 were repeatedly administered to cats, and the changes in the ability to induce feline anti-mouse antibody response were investigated. In the serum of cats treated with mouse mAb 2–4, feline anti-mouse antibody production was induced, and the fTNF-alpha neutralization effect of mouse mAb 2–4 was reduced. In contrast, in cats treated with chimeric mAb 2–4, the feline anti-mouse antibody response was decreased compared to that of mouse mAb 2–4-treated cats. PMID:27264736

  3. Construction of a hepatitis B virus neutralizing chimeric monoclonal antibody recognizing escape mutants of the viral surface antigen (HBsAg).

    Science.gov (United States)

    Golsaz-Shirazi, Forough; Amiri, Mohammad Mehdi; Farid, Samira; Bahadori, Motahareh; Bohne, Felix; Altstetter, Sebastian; Wolff, Lisa; Kazemi, Tohid; Khoshnoodi, Jalal; Hojjat-Farsangi, Mohammad; Chudy, Michael; Jeddi-Tehrani, Mahmood; Protzer, Ulrike; Shokri, Fazel

    2017-08-01

    Hepatitis B virus (HBV) infection is a global burden on the health-care system and is considered as the tenth leading cause of death in the world. Over 248 million patients are currently suffering from chronic HBV infection worldwide and annual mortality rate of this infection is 686000. The "a" determinant is a hydrophilic region present in all antigenic subtypes of hepatitis B surface antigen (HBsAg), and antibodies against this region can neutralize the virus and are protective against all subtypes. We have recently generated a murine anti-HBs monoclonal antibody (4G4), which can neutralize HBV infection in HepaRG cells and recognize most of the escape mutant forms of HBsAg. Here, we describe the production and characterization of the chimeric human-murine antibody 4G4 (c-4G4). Variable region genes of heavy and light chains of the m-4G4 were cloned and fused to constant regions of human kappa and IgG1 by splice overlap extension (SOE) PCR. The chimeric antibody was expressed in Chinese Hamster Ovary (CHO)-K1 cells and purified from culture supernatant. Competition ELISA proved that both antibodies bind the same epitope within HBsAg. Antigen-binding studies using ELISA and Western blot showed that c-4G4 has retained the affinity and specificity of the parental murine antibody, and displayed a similar pattern of reactivity to 13 escape mutant forms of HBsAg. Both, the parental and c-4G4 showed a comparably high HBV neutralization capacity in cell culture even at the lowest concentration (0.6μg/ml). Due to the ability of c-4G4 to recognize most of the sub-genotypes and escape mutants of HBsAg, this antibody either alone or in combination with other anti-HBs antibodies could be considered as a potent alternative for Hepatitis B immune globulin (HBIG) as an HBV infection prophylactic or for passive immunotherapy against HBV infection. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. Interleukin 4 increases CCR9 expression and homing of lymphocytes to gut-associated lymphoid tissue in chickens.

    Science.gov (United States)

    Annamalai, Thavamathi; Selvaraj, Ramesh K

    2012-01-15

    The effects of in vitro and in vivo IL-4 supplementation on thymocyte and splenocyte CCR9 mRNA amount and migration were studied. Thymocytes, splenocytes, splenocytes+thymocytes (2:1), and splenocytes+bursocyte cells (2:1) were supplemented with either 0 or 5 ng/ml IL-4 for 5d. CCR9 mRNA was undetectable in all experimental groups supplemented with 0 ng/ml IL-4. IL-4 treatment (5 ng/ml) upregulated (P=0.01) CCR9 mRNA only in the splenocyte+thymocyte cell culture. IL-4-mediated CCR9 mRNA induction in the splenocyte+thymocyte cell culture was dependent on the in vitro dose of IL-4 supplementation. IL-4-treated splenocyte+thymocyte cells when injected in vivo preferentially migrated to cecal tonsils. In vivo supplementation of IL-4 was achieved through in ovo injection of recombinant chicken IL-4 plasmid. Cecal tonsils in chicks hatched from IL-4-plasmid-injected eggs weighed more, had a higher amount of CCR9 mRNA, and had a higher percentage of CD8(+) cells than cecal tonsils from chicks hatched from PBS-injected eggs. It could be concluded that IL-4 induces CCR9 mRNA in thymocytes and splenocytes and directs the migration of cells to gut-associated lymphoid tissue. Copyright © 2011 Elsevier B.V. All rights reserved.

  5. Generation of Novel Chimeric Mice with Humanized Livers by Using Hemizygous cDNA-uPA/SCID Mice.

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    Chise Tateno

    Full Text Available We have used homozygous albumin enhancer/promoter-driven urokinase-type plasminogen activator/severe combined immunodeficient (uPA/SCID mice as hosts for chimeric mice with humanized livers. However, uPA/SCID mice show four disadvantages: the human hepatocytes (h-heps replacement index in mouse liver is decreased due to deletion of uPA transgene by homologous recombination, kidney disorders are likely to develop, body size is small, and hemizygotes cannot be used as hosts as more frequent homologous recombination than homozygotes. To solve these disadvantages, we have established a novel host strain that has a transgene containing albumin promoter/enhancer and urokinase-type plasminogen activator cDNA and has a SCID background (cDNA-uPA/SCID. We applied the embryonic stem cell technique to simultaneously generate a number of transgenic lines, and found the line with the most appropriate levels of uPA expression-not detrimental but with a sufficiently damaged liver. We transplanted h-heps into homozygous and hemizygous cDNA-uPA/SCID mice via the spleen, and monitored their human albumin (h-alb levels and body weight. Blood h-alb levels and body weight gradually increased in the hemizygous cDNA-uPA/SCID mice and were maintained until they were approximately 30 weeks old. By contrast, blood h-alb levels and body weight in uPA/SCID chimeric mice decreased from 16 weeks of age onwards. A similar decrease in body weight was observed in the homozygous cDNA-uPA/SCID genotype, but h-alb levels were maintained until they were approximately 30 weeks old. Microarray analyses revealed identical h-heps gene expression profiles in homozygous and hemizygous cDNA-uPA/SCID mice were identical to that observed in the uPA/SCID mice. Furthermore, like uPA/SCID chimeric mice, homozygous and hemizygous cDNA-uPA/SCID chimeric mice were successfully infected with hepatitis B virus and C virus. These results indicate that hemizygous cDNA-uPA/SCID mice may be novel and

  6. Generation of Novel Chimeric Mice with Humanized Livers by Using Hemizygous cDNA-uPA/SCID Mice.

    Science.gov (United States)

    Tateno, Chise; Kawase, Yosuke; Tobita, Yoshimi; Hamamura, Satoko; Ohshita, Hiroki; Yokomichi, Hiroshi; Sanada, Harumi; Kakuni, Masakazu; Shiota, Akira; Kojima, Yuha; Ishida, Yuji; Shitara, Hiroshi; Wada, Naoko A; Tateishi, Hiromi; Sudoh, Masayuki; Nagatsuka, Shin-Ichiro; Jishage, Kou-Ichi; Kohara, Michinori

    2015-01-01

    We have used homozygous albumin enhancer/promoter-driven urokinase-type plasminogen activator/severe combined immunodeficient (uPA/SCID) mice as hosts for chimeric mice with humanized livers. However, uPA/SCID mice show four disadvantages: the human hepatocytes (h-heps) replacement index in mouse liver is decreased due to deletion of uPA transgene by homologous recombination, kidney disorders are likely to develop, body size is small, and hemizygotes cannot be used as hosts as more frequent homologous recombination than homozygotes. To solve these disadvantages, we have established a novel host strain that has a transgene containing albumin promoter/enhancer and urokinase-type plasminogen activator cDNA and has a SCID background (cDNA-uPA/SCID). We applied the embryonic stem cell technique to simultaneously generate a number of transgenic lines, and found the line with the most appropriate levels of uPA expression-not detrimental but with a sufficiently damaged liver. We transplanted h-heps into homozygous and hemizygous cDNA-uPA/SCID mice via the spleen, and monitored their human albumin (h-alb) levels and body weight. Blood h-alb levels and body weight gradually increased in the hemizygous cDNA-uPA/SCID mice and were maintained until they were approximately 30 weeks old. By contrast, blood h-alb levels and body weight in uPA/SCID chimeric mice decreased from 16 weeks of age onwards. A similar decrease in body weight was observed in the homozygous cDNA-uPA/SCID genotype, but h-alb levels were maintained until they were approximately 30 weeks old. Microarray analyses revealed identical h-heps gene expression profiles in homozygous and hemizygous cDNA-uPA/SCID mice were identical to that observed in the uPA/SCID mice. Furthermore, like uPA/SCID chimeric mice, homozygous and hemizygous cDNA-uPA/SCID chimeric mice were successfully infected with hepatitis B virus and C virus. These results indicate that hemizygous cDNA-uPA/SCID mice may be novel and useful hosts for

  7. Tumor hypoxia modulates podoplanin/CCL21 interactions in CCR7+ NK cell recruitment and CCR7+ tumor cell mobilization.

    Science.gov (United States)

    Tejchman, Anna; Lamerant-Fayel, Nathalie; Jacquinet, Jean-Claude; Bielawska-Pohl, Aleksandra; Mleczko-Sanecka, Katarzyna; Grillon, Catherine; Chouaib, Salem; Ugorski, Maciej; Kieda, Claudine

    2017-05-09

    Podoplanin (PDPN), an O-glycosylated, transmembrane, mucin-type glycoprotein, is expressed by cancer associated fibroblasts (CAFs). In malignant transformation, PDPN is subjected to changes and its role is yet to be established. Here we show that it is involved in modulating the activity of the CCL21/CCR7 chemokine/receptor axis in a hypoxia-dependent manner. In the present model, breast cancer MDA-MB-231 cells and NKL3 cells express the surface CCR7 receptor for CCL21 chemokine which is a potent chemoattractant able to bind to PDPN. The impact of the CCL21/CCR7 axis in the molecular mechanism of the adhesion of NKL3 cells and of MDA-MB-231 breast cancer cells was reduced in a hypoxic tumor environment. In addition to its known effect on migration, CCL21/CCR7 interaction was shown to allow NK cell adhesion to endothelial cells (ECs) and its reduction by hypoxia. A PDPN expressing model of CAFs made it possible to demonstrate the same CCL21/CCR7 axis involvement in the tumor cells to CAFs recognition mechanism through PDPN binding of CCL21. PDPN was induced by hypoxia and its overexpression undergoes a reduction of adhesion, making it an anti-adhesion molecule in the absence of CCL21, in the tumor. CCL21/CCR7 modulated NK cells/ECs and MDA-MB-231 cells/CAF PDPN-dependent interactions were further shown to be linked to hypoxia-dependent microRNAs as miRs: miR-210 and specifically miR-21, miR-29b which influence PDPN expression.

  8. Screening of chemokine receptor CCR4 antagonists by capillary zone electrophoresis

    Directory of Open Access Journals (Sweden)

    Zhe Sun

    2011-11-01

    Full Text Available CC chemokine receptor 4 (CCR4 is a kind of G-protein-coupled receptor, which plays a pivotal role in allergic inflammation. The interaction between 2-(2-(4-chloro-phenyl-5-{[(naphthalen-1-ylmethyl-carbamoyl]-methyl}-4-oxo-thiazolidin-3-yl-N-(3-morpholin-4-yl-propyl-acetamide (S009 and the N-terminal extracellular tail (ML40 of CCR4 has been validated to be high affinity by capillary zone electrophoresis (CZE. The S009 is a known CCR4 antagonist. Now, a series of new thiourea derivatives have been synthesized. Compared with positive control S009, they were screened using ML40 as target by CZE to find some new drugs for allergic inflammation diseases. The synthesized compounds XJH-5, XJH-4, XJH-17 and XJH-1 displayed the interaction with ML40, but XJH-9, XJH-10, XJH-11, XJH-12, XJH-13, XJH-14, XJH-3, XJH-8, XJH-6, XJH-7, XJH-15, XJH-16 and XJH-2 did not bind to ML40. Both qualification and quantification characterizations of the binding were determined. The affinity of the four compounds was valued by the binding constant, which was similar with the results of chemotactic experiments. The established CEZ method is capable of sensitive and fast screening for a series of lactam analogs in the drug discovery for allergic inflammation diseases. Keywords: Capillary zone electrophoresis, CCR4 antagonist, 2-(2-(4-chloro-phenyl-5-{[(naphthalen-1-ylmethyl-carbamoyl]-methyl}-4-oxo-thiazolidin-3-yl-N-(3-morpholin-4-yl-propyl-acetamide, Interactions, Structural modification

  9. Porcine induced pluripotent stem cells produce chimeric offspring.

    Science.gov (United States)

    West, Franklin D; Terlouw, Steve L; Kwon, Dae Jin; Mumaw, Jennifer L; Dhara, Sujoy K; Hasneen, Kowser; Dobrinsky, John R; Stice, Steven L

    2010-08-01

    Ethical and moral issues rule out the use of human induced pluripotent stem cells (iPSCs) in chimera studies that would determine the full extent of their reprogrammed state, instead relying on less rigorous assays such as teratoma formation and differentiated cell types. To date, only mouse iPSC lines are known to be truly pluripotent. However, initial mouse iPSC lines failed to form chimeric offspring, but did generate teratomas and differentiated embryoid bodies, and thus these specific iPSC lines were not completely reprogrammed or truly pluripotent. Therefore, there is a need to address whether the reprogramming factors and process used eventually to generate chimeric mice are universal and sufficient to generate reprogrammed iPSC that contribute to chimeric offspring in additional species. Here we show that porcine mesenchymal stem cells transduced with 6 human reprogramming factors (POU5F1, SOX2, NANOG, KLF4, LIN28, and C-MYC) injected into preimplantation-stage embryos contributed to multiple tissue types spanning all 3 germ layers in 8 of 10 fetuses. The chimerism rate was high, 85.3% or 29 of 34 live offspring were chimeras based on skin and tail biopsies harvested from 2- to 5-day-old pigs. The creation of pluripotent porcine iPSCs capable of generating chimeric offspring introduces numerous opportunities to study the facets significantly affecting cell therapies, genetic engineering, and other aspects of stem cell and developmental biology.

  10. Functionality of Chimeric E2 Glycoproteins of BVDV and CSFV in Virus Replication

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    H.G.P. van Gennip

    2008-01-01

    Full Text Available An intriguing difference between the E2 glycoprotein of CSFV and the other groups of pestiviruses (nonCSFV is a lack of two cysteine residues on positions cysteine 751 and 798. Other groups of pestivirus are not restricted to one species as swine, whereas CSFV is restricted to swine and wild boar. We constructed chimeric CSFV/BVDV E2 genes based on a 2D model of E2 proposed by van Rijn et al. (van Rijn et al. 1994, J Virol 68, 3934–42 and confirmed their expression by immunostaining of plasmid-transfected SK6 cells. No equivalents for the antigenic units B/C and A were found on E2 of BVDVII. This indicates major structural differences in E2. However, the immunodominant BVDVII domain A, containing epitopes with essential amino acids between position 760–764, showed to be dependent on the presence of the region defined by amino acids 684 to 796. As for the A domain of CSFV, the BVDVII A-like domain seemed to function as a separate unit. These combined domains in E2 proved to be the only combination which was functional in viral background of CSFV C-strain. The fitness of this virus (vfl c36BVDVII 684–796 seemed to be reduced compared to vfl c9 (with the complete antigenic region of BVDVII.

  11. Functionality of Chimeric E2 Glycoproteins of BVDV and CSFV in Virus Replication

    Directory of Open Access Journals (Sweden)

    H.G.P. Van Gennip

    2008-01-01

    Full Text Available An intriguing difference between the E2 glycoprotein of CSFV and the other groups of pestiviruses (nonCSFV is a lack of two cysteine residues on positions cysteine 751 and 798. Other groups of pestivirus are not restricted to one species as swine, whereas CSFV is restricted to swine and wild boar. We constructed chimeric CSFV/BVDV E2 genes based on a 2D model of E2 proposed by van Rijn et al. (van Rijn et al. 1994, J Virol 68, 3934–42 and confirmed their expression by immunostaining of plasmid-transfected SK6 cells. No equivalents for the antigenic units B/C and A were found on E2 of BVDVII. This indicates major structural differences in E2. However, the immunodominant BVDVII domain A, containing epitopes with essential amino acids between position 760–764, showed to be dependent on the presence of the region defined by amino acids 684 to 796. As for the A domain of CSFV, the BVDVII A-like domain seemed to function as a separate unit. These combined domains in E2 proved to be the only combination which was functional in viral background of CSFV C-strain. The fitness of this virus (vflc36 BVDVII 684–796 seemed to be reduced compared to vflc9 (with the complete antigenic region of BVDVII.

  12. Poinsettia latent virus is not a cryptic virus, but a natural polerovirus-sobemovirus hybrid

    International Nuclear Information System (INIS)

    Siepen, Marc aus dem; Pohl, Jens O.; Koo, Bong-Jin; Wege, Christina; Jeske, Holger

    2005-01-01

    The biochemical and genetic features of Poinsettia latent virus (PnLV, formerly named Poinsettia cryptic virus), which is spread worldwide in commercial cultivars of Euphorbia pulcherrima without inducing symptoms, have been determined using virus-purification, immunological techniques, electron microscopy, cloning, and sequencing. PnLV was found to be a chimeric virus with one 4652 bases, plus strand RNA showing a close relationship to poleroviruses within the first three quarters of its genome but to sobemoviruses in the last quarter. Thus, we propose to classify this virus as 'polemovirus'. Similarities of protein and nucleic acid sequences at the 5' and extreme 3' end of its RNA suggest a replication mode like that of poleroviruses, whereas the coat protein sequence is closely related to that of sobemoviruses. Consistent with these results, PnLV forms stable icosahedra of 34 nm in diameter. The consequences for the taxonomy of PnLV and for gardeners' practice are discussed

  13. Production of Japanese Encephalitis Virus Antigens in Plants Using Bamboo Mosaic Virus-Based Vector

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    Tsung-Hsien Chen

    2017-05-01

    Full Text Available Japanese encephalitis virus (JEV is among the major threats to public health in Asia. For disease control and prevention, the efficient production of safe and effective vaccines against JEV is in urgent need. In this study, we produced a plant-made JEV vaccine candidate using a chimeric virus particle (CVP strategy based on bamboo mosaic virus (BaMV for epitope presentation. The chimeric virus, designated BJ2A, was constructed by fusing JEV envelope protein domain III (EDIII at the N-terminus of BaMV coat protein, with an insertion of the foot-and-mouth disease virus 2A peptide to facilitate the production of both unfused and epitope-presenting for efficient assembly of the CVP vaccine candidate. The strategy allowed stable maintenance of the fusion construct over long-term serial passages in plants. Immuno-electron microscopy examination and immunization assays revealed that BJ2A is able to present the EDIII epitope on the surface of the CVPs, which stimulated effective neutralizing antibodies against JEV infection in mice. This study demonstrates the efficient production of an effective CVP vaccine candidate against JEV in plants by the BaMV-based epitope presentation system.

  14. O'nyong nyong virus molecular determinants of unique vector specificity reside in non-structural protein 3.

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    Kali D Saxton-Shaw

    Full Text Available O'nyong nyong virus (ONNV and Chikungunya virus (CHIKV are two closely related alphaviruses with very different infection patterns in the mosquito, Anopheles gambiae. ONNV is the only alphavirus transmitted by anopheline mosquitoes, but specific molecular determinants of infection of this unique vector specificity remain unidentified. Fifteen distinct chimeric viruses were constructed to evaluate both structural and non-structural regions of the genome and infection patterns were determined through artificial infectious feeds in An. gambiae with each of these chimeras. Only one region, non-structural protein 3 (nsP3, was sufficient to up-regulate infection to rates similar to those seen with parental ONNV. When ONNV non-structural protein 3 (nsP3 replaced nsP3 from CHIKV virus in one of the chimeric viruses, infection rates in An. gambiae went from 0% to 63.5%. No other single gene or viral region addition was able to restore infection rates. Thus, we have shown that a non-structural genome element involved in viral replication is a major element involved in ONNV's unique vector specificity.

  15. Recurrent chimeric RNAs enriched in human prostate cancer identified by deep sequencing

    Science.gov (United States)

    Kannan, Kalpana; Wang, Liguo; Wang, Jianghua; Ittmann, Michael M.; Li, Wei; Yen, Laising

    2011-01-01

    Transcription-induced chimeric RNAs, possessing sequences from different genes, are expected to increase the proteomic diversity through chimeric proteins or altered regulation. Despite their importance, few studies have focused on chimeric RNAs especially regarding their presence/roles in human cancers. By deep sequencing the transcriptome of 20 human prostate cancer and 10 matched benign prostate tissues, we obtained 1.3 billion sequence reads, which led to the identification of 2,369 chimeric RNA candidates. Chimeric RNAs occurred in significantly higher frequency in cancer than in matched benign samples. Experimental investigation of a selected 46 set led to the confirmation of 32 chimeric RNAs, of which 27 were highly recurrent and previously undescribed in prostate cancer. Importantly, a subset of these chimeras was present in prostate cancer cell lines, but not detectable in primary human prostate epithelium cells, implying their associations with cancer. These chimeras contain discernable 5′ and 3′ splice sites at the RNA junction, indicating that their formation is mediated by splicing. Their presence is also largely independent of the expression of parental genes, suggesting that other factors are involved in their production and regulation. One chimera, TMEM79-SMG5, is highly differentially expressed in human cancer samples and therefore a potential biomarker. The prevalence of chimeric RNAs may allow the limited number of human genes to encode a substantially larger number of RNAs and proteins, forming an additional layer of cellular complexity. Together, our results suggest that chimeric RNAs are widespread, and increased chimeric RNA events could represent a unique class of molecular alteration in cancer. PMID:21571633

  16. Glycosylphosphatidylinositol-Anchored Anti-HIV scFv Efficiently Protects CD4 T Cells from HIV-1 Infection and Deletion in hu-PBL Mice

    Science.gov (United States)

    Ye, Chaobaihui; Wang, Weiming; Cheng, Liang; Li, Guangming; Wen, Michael; Wang, Qi; Zhang, Qing; Li, Dan

    2016-01-01

    ABSTRACT Despite success in viral inhibition and CD4 T cell recovery by highly active antiretroviral treatment (HAART), HIV-1 is still not curable due to the persistence of the HIV-1 reservoir during treatment. One patient with acute myeloid leukemia who received allogeneic hematopoietic stem cell transplantation from a homozygous CCR5 Δ32 donor has had no detectable viremia for 9 years after HAART cessation. This case has inspired a field of HIV-1 cure research focusing on engineering HIV-1 resistance in permissive cells. Here, we employed a glycosylphosphatidylinositol (GPI)-scFv X5 approach to confer resistance of human primary CD4 T cells to HIV-1. We showed that primary CD4 T cells expressing GPI-scFv X5 were resistant to CCR5 (R5)-, CXCR4 (X4)-, and dual-tropic HIV-1 and had a survival advantage compared to control cells ex vivo. In a hu-PBL mouse study, GPI-scFv X5-transduced CD4 T cells were selected in peripheral blood and lymphoid tissues upon HIV-1 infection. Finally, GPI-scFv X5-transduced CD4 T cells, after being cotransfused with HIV-infected cells, showed significantly reduced viral loads and viral RNA copy numbers relative to CD4 cells in hu-PBL mice compared to mice with GPI-scFv AB65-transduced CD4 T cells. We conclude that GPI-scFv X5-modified CD4 T cells could potentially be used as a genetic intervention against both R5- and X4-tropic HIV-1 infections. IMPORTANCE Blocking of HIV-1 entry is one of most promising approaches for therapy. Genetic disruption of the HIV-1 coreceptor CCR5 by nucleases in T cells is under 2 clinical trials and leads to reduced viremia in patients. However, the emergence of viruses using the CXCR4 coreceptor is a concern for therapies applying single-coreceptor disruption. Here, we report that HIV-1-permissive CD4 T cells engineered with GPI-scFv X5 are resistant to R5-, X4-, or dual-tropic virus infection ex vivo. In a preclinical study using hu-PBL mice, we show that CD4 T cells were protected and that GPI-scFv X5

  17. [Single nucleotide polymorphisms of HIV coreceptor CCR5 gene in Chinese Yi ethnic group and its association with HIV infection].

    Science.gov (United States)

    Ma, Li-ying; Hong, Kun-xue; Lu, Xiao-zhi; Qin, Guang-ming; Chen, Jian-ping; Chen, Kang-lin; Ruan, Yu-hua; Xing, Hui; Zhu, Jia-hong; Shao, Yi-ming

    2005-11-30

    To investigate the single nucleotide polymorphism (SNP) of HIV-1 coreceptor CCR5 gene in Chinese Yi ethnic group and the association between these SNPs and HIV/AIDS. Peripheral blood samples of 102 HIV negative persons of Chinese Yi nationality, 87 males amd 15 females, aged 23 (12-37), and 68 HIV carriers, 61 males and 7 females, aged 27 (17-51). The regulatory and structural regions of the HIV coreceptor CCR5 gene were amplified from the genomic DNA by nested PCR, each of the two regions was divided into three gene fragments which were overlapped. High throughput DHPLC was used for screening of unknown mutations in each gene fragment. The PCR products showing different peak traces from wild types in DHPLC were sequenced by forward and reverse primers respectively. The sequences were analyzed with the help of Sequence Navigator software to search for SNP loci. Statistical analysis by SPSS and PPAP softwares were made to study the association between these SNPs and HIV infection. Five SNPs (A77G, G316A, T532C, C921T, and G668A) and a AGA deletion of the 686-688 nucleotides were discovered in the coding region of this gene in Chinese Yi ethnic group. C921T mutation was a nonsense mutation, and the other SNPs (A77G, G316A, T532C, and G668A) are sense mutation, with the amino acid changes of K26R, G106R, C178R, and R223Q. Only the frequency of R223Q allelic gene was high (0.08) but those of the others were low (less than 0.01). There was no significant difference in the allele frequency between the HIV negative and HIV positive groups (all P > 0.05). Five SNP loci (T58934G, G59029A, T59353C, G59402A, and C59653T) were found in the regulatory region of CCR5 gene with high allelic frequencies of 0.1912-0.2941. Between the HIV negative and HIV positive groups, there were no differences in the SNP loc (all P > 0.05). Statistical analysis of the association between the linkage of mutation loci with HIV infection suggested a significant difference in the haplotype frequency

  18. Systemic MCP1/CCR2 blockade and leukocyte specific MCP1/CCR2 inhibition affect aortic aneurysm formation differently

    NARCIS (Netherlands)

    de Waard, Vivian; Bot, Ilze; de Jager, Saskia C. A.; Talib, Sara; Egashira, Kensuke; de Vries, Margreet R.; Quax, Paul H. A.; Biessen, Erik A. L.; van Berkel, Theo J. C.

    2010-01-01

    Objective: CCR2, the receptor for monocyte chemoattractant protein 1 (MCP1), is involved in atherosclerosis and abdominal aortic aneurysms (AAAs). Here, we explored the potential beneficial blockade of the MCP1/CCR2 pathway. Methods: We applied an AAA model in aging apolipoprotein E deficient mice

  19. Chimeric peptide-mediated siRNA transduction to inhibit HIV-1 infection.

    Science.gov (United States)

    Bivalkar-Mehla, Shalmali; Mehla, Rajeev; Chauhan, Ashok

    2017-04-01

    Persistent human immunodeficiency virus 1 (HIV-1) infection provokes immune activation and depletes CD4 +  lymphocytes, leading to acquired immunodeficiency syndrome. Uninterrupted administration of combination antiretroviral therapy (cART) in HIV-infected patients suppresses viral replication to below the detectable level and partially restores the immune system. However, cART-unresponsive residual HIV-1 infection and elusive transcriptionally silent but reactivatable viral reservoirs maintain a permanent viral DNA blue print. The virus rebounds within a few weeks after interruption of suppressive therapy. Adjunct gene therapy to control viral replication by ribonucleic acid interference (RNAi) is a post-transcriptional gene silencing strategy that could suppress residual HIV-1 burden and overcome viral resistance. Small interfering ribonucleic acids (siRNAs) are efficient transcriptional inhibitors, but need delivery systems to reach inside target cells. We investigated the potential of chimeric peptide (FP-PTD) to deliver specific siRNAs to HIV-1-susceptible and permissive cells. Chimeric FP-PTD peptide was designed with an RNA binding domain (PTD) to bind siRNA and a cell fusion peptide domain (FP) to enter cells. FP-PTD-siRNA complex entered and inhibited HIV-1 replication in susceptible cells, and could be a candidate for in vivo testing.

  20. Biased and Constitutive Signaling in the CC-Chemokine Receptor CCR5 by manipulating the Interface between Transmembrane Helix 6 and 7

    DEFF Research Database (Denmark)

    Steen, Anne; Thiele, Stefanie; Guo, Dong

    2013-01-01

    The equilibrium state of CCR5 is manipulated here toward either activation or inactivation by introduction of single amino acid substitutions in the transmembrane domains (TMs) 6 and 7. Insertion of a steric hindrance mutation in the center of TM7 (G286F in position VII:09/7.42) resulted in biase...

  1. Modulators of the human CCR5 receptor. Part 1: Discovery and initial SAR of 1-(3,3-diphenylpropyl)-piperidinyl amides and ureas.

    Science.gov (United States)

    Burrows, Jeremy N; Cumming, John G; Fillery, Shaun M; Hamlin, Gordon A; Hudson, Julian A; Jackson, Ruth J; McLaughlin, Sharon; Shaw, John S

    2005-01-03

    Investigation of weak screening hits led to the identification of N-alkyl-N-[1-(3,3-diphenylpropyl)piperidin-4-yl]-2-phenylacetamides and N-alkyl-N-[1-(3,3-diphenylpropyl)piperidin-4-yl]-N'-benzylureas as potent, selective ligands for the human CCR5 chemokine receptor.

  2. Impact of CCR5delta32 Host Genetic Background and Disease Progression on HIV-1 Intrahost Evolutionary Processes: Efficient Hypothesis Testing through Hierarchical Phylogenetic Models

    NARCIS (Netherlands)

    Edo-Matas, Diana; Lemey, Philippe; Tom, Jennifer A.; Serna-Bolea, Cèlia; van den Blink, Agnes E.; van 't Wout, Angélique B.; Schuitemaker, Hanneke; Suchard, Marc A.

    2011-01-01

    The interplay between C-C chemokine receptor type 5 (CCR5) host genetic background, disease progression, and intrahost HIV-1 evolutionary dynamics remains unclear because differences in viral evolution between hosts limit the ability to draw conclusions across hosts stratified into clinically

  3. Narcolepsy susceptibility gene CCR3 modulates sleep-wake patterns in mice.

    Directory of Open Access Journals (Sweden)

    Hiromi Toyoda

    Full Text Available Narcolepsy is caused by the loss of hypocretin (Hcrt neurons and is associated with multiple genetic and environmental factors. Although abnormalities in immunity are suggested to be involved in the etiology of narcolepsy, no decisive mechanism has been established. We previously reported chemokine (C-C motif receptor 3 (CCR3 as a novel susceptibility gene for narcolepsy. To understand the role of CCR3 in the development of narcolepsy, we investigated sleep-wake patterns of Ccr3 knockout (KO mice. Ccr3 KO mice exhibited fragmented sleep patterns in the light phase, whereas the overall sleep structure in the dark phase did not differ between Ccr3 KO mice and wild-type (WT littermates. Intraperitoneal injection of lipopolysaccharide (LPS promoted wakefulness and suppressed both REM and NREM sleep in the light phase in both Ccr3 KO and WT mice. Conversely, LPS suppressed wakefulness and promoted NREM sleep in the dark phase in both genotypes. After LPS administration, the proportion of time spent in wakefulness was higher, and the proportion of time spent in NREM sleep was lower in Ccr3 KO compared to WT mice only in the light phase. LPS-induced changes in sleep patterns were larger in Ccr3 KO compared to WT mice. Furthermore, we quantified the number of Hcrt neurons and found that Ccr3 KO mice had fewer Hcrt neurons in the lateral hypothalamus compared to WT mice. We found abnormalities in sleep patterns in the resting phase and in the number of Hcrt neurons in Ccr3 KO mice. These observations suggest a role for CCR3 in sleep-wake regulation in narcolepsy patients.

  4. Sequential immunization with V3 peptides from primary human immunodeficiency virus type 1 produces cross-neutralizing antibodies against primary isolates with a matching narrow-neutralization sequence motif.

    Science.gov (United States)

    Eda, Yasuyuki; Takizawa, Mari; Murakami, Toshio; Maeda, Hiroaki; Kimachi, Kazuhiko; Yonemura, Hiroshi; Koyanagi, Satoshi; Shiosaki, Kouichi; Higuchi, Hirofumi; Makizumi, Keiichi; Nakashima, Toshihiro; Osatomi, Kiyoshi; Tokiyoshi, Sachio; Matsushita, Shuzo; Yamamoto, Naoki; Honda, Mitsuo

    2006-06-01

    An antibody response capable of neutralizing not only homologous but also heterologous forms of the CXCR4-tropic human immunodeficiency virus type 1 (HIV-1) MNp and CCR5-tropic primary isolate HIV-1 JR-CSF was achieved through sequential immunization with a combination of synthetic peptides representing HIV-1 Env V3 sequences from field and laboratory HIV-1 clade B isolates. In contrast, repeated immunization with a single V3 peptide generated antibodies that neutralized only type-specific laboratory-adapted homologous viruses. To determine whether the cross-neutralization response could be attributed to a cross-reactive antibody in the immunized animals, we isolated a monoclonal antibody, C25, which neutralized the heterologous primary viruses of HIV-1 clade B. Furthermore, we generated a humanized monoclonal antibody, KD-247, by transferring the genes of the complementary determining region of C25 into genes of the human V region of the antibody. KD-247 bound with high affinity to the "PGR" motif within the HIV-1 Env V3 tip region, and, among the established reference antibodies, it most effectively neutralized primary HIV-1 field isolates possessing the matching neutralization sequence motif, suggesting its promise for clinical applications involving passive immunizations. These results demonstrate that sequential immunization with B-cell epitope peptides may contribute to a humoral immune-based HIV vaccine strategy. Indeed, they help lay the groundwork for the development of HIV-1 vaccine strategies that use sequential immunization with biologically relevant peptides to overcome difficulties associated with otherwise poorly immunogenic epitopes.

  5. Variations in CCR5, but not HFE, ELMO1, or SLC12A3, are associated with susceptibility to kidney disease in north Indian individuals with type 2 diabetes.

    Science.gov (United States)

    Yadav, Ashok K; Kumar, Vinod; Dutta, Pinaki; Bhansali, Anil; Jha, Vivekanand

    2014-11-01

    Diabetic nephropathy (DN), the leading cause of end-stage renal disease worldwide, may have a genetic component. In the present study, we investigated variations in a set of genes with susceptibility to DN in a north Indian population. Four genes (HFE, ELMO1, SLC12A3, and CCR5) were selected on the basis of reported association with type 2 diabetes and nephropathy. In all, 417 diabetic subjects (215 without kidney disease [DM] and 202 with DN) and 197 healthy controls (HC) were evaluated for variations in HFE (845 G>A and 187G>C), SLC12A3 (g.34372G>A), CCR5 (59029A>G), and ELMO1 (+9170 G>A). Polymorphism analysis was performed by polymerase chain reaction-restriction fragment length polymorphism and Taqman allele discrimination assays. Significant differences were found in genotype and allelic frequency in SLC12A3 (g.34372G>A) between diabetic subjects and HC (P A (AA+GA) genotype between diabetic subjects with and without nephropathy. However, the CCR5 59029AA genotype and A allele were significantly more frequent in diabetics compared with the HC (P = 0.01 and 0.03, respectively) and subjects with DN versus DM (P = 0.002 and 0.01, respectively). For ELMO1 (+9170 G>A), the GG genotype frequency was higher in the diabetic versus HC group. There were no differences in the frequency of HFE-845 G>A and HFE-187G>C among the groups. This study shows that the CCR5 AA genotype is over-represented in subjects with kidney disease due to type 2 diabetes. The CCR5 59029G>A and ELMO1 (+9170 G>A) loci are more frequent, and the SLC12A3 34372 AA genotype is associated with a reduced risk of diabetes. © 2014 Ruijin Hospital, Shanghai Jiaotong University School of Medicine and Wiley Publishing Asia Pty Ltd.

  6. Chimeric rhinoviruses displaying MPER epitopes elicit anti-HIV neutralizing responses.

    Directory of Open Access Journals (Sweden)

    Guohua Yi

    Full Text Available The development of an effective AIDS vaccine has been a formidable task, but remains a critical necessity. The well conserved membrane-proximal external region (MPER of the HIV-1 gp41 glycoprotein is one of the crucial targets for AIDS vaccine development, as it has the necessary attribute of being able to elicit antibodies capable of neutralizing diverse isolates of HIV.Guided by X-ray crystallography, molecular modeling, combinatorial chemistry, and powerful selection techniques, we designed and produced six combinatorial libraries of chimeric human rhinoviruses (HRV displaying the MPER epitopes corresponding to mAbs 2F5, 4E10, and/or Z13e1, connected to an immunogenic surface loop of HRV via linkers of varying lengths and sequences. Not all libraries led to viable chimeric viruses with the desired sequences, but the combinatorial approach allowed us to examine large numbers of MPER-displaying chimeras. Among the chimeras were five that elicited antibodies capable of significantly neutralizing HIV-1 pseudoviruses from at least three subtypes, in one case leading to neutralization of 10 pseudoviruses from all six subtypes tested.Optimization of these chimeras or closely related chimeras could conceivably lead to useful components of an effective AIDS vaccine. While the MPER of HIV may not be immunodominant in natural infection by HIV-1, its presence in a vaccine cocktail could provide critical breadth of protection.

  7. Effects of treatment with Maraviroc a CCR5 inhibitor on a human hepatic stellate cell line.

    Science.gov (United States)

    Coppola, Nicola; Perna, Angelica; Lucariello, Angela; Martini, Salvatore; Macera, Margherita; Carleo, Maria A; Guerra, Germano; Esposito, Vincenzo; De Luca, Antonio

    2018-08-01

    After an acute liver damage, tissue regeneration repairs lesions with degradation of deposed fibrotic material, while mechanisms of tissue restoration are persistently activated following several repeated injuries, inducing deposition of extracellular matrix. (ECM). Factors responsible for ECM remodeling have been identified in a pathway involving a family of zinc-dependent enzyme matrix metalloproteinases (MMPs), together with tissue inhibitor of metalloproteinases (TIMPs). Recent experimental models suggested a role of CCR5 receptor in the genesis of liver fibrosis. Drawing from these background we decided to evaluate the effects of the treatment with the CCR5 inhibitor Maraviroc on LX-2, a human hepatic stellate cell line (HSC). Treatment with Maraviroc resulted in a block in S phase of LX-2 cells with increased expression levels of cyclin D1 and p21 while the expression of p53 was reduced. Treatment with Maraviroc was also able to block the accumulation of fibrillar collagens and extracellular matrix proteins (ECM), as demonstrated by the decrease of specific markers as Collagen type I, α-SMA, and TGF-β1. In addition we observed a down regulation of both metalloproteins (MMP-2, MMP-9), used for the degradation of the extracellular matrix and their inhibitors (TIMP-1, TIMP-2). The identification of a compound that may modulate the dynamic of liver fibrosis could be crucial in all chronic liver diseases. Maraviroc could play an important role because, in addition to its own anti-HIV activity, it could reduce the release of pro-inflammatory citokynes implicated in liver fibrogenesis. © 2018 Wiley Periodicals, Inc.

  8. Expression analysis of cinnamoyl-CoA reductase (CCR) gene in developing seedlings of Leucaena leucocephala: a pulp yielding tree species.

    Science.gov (United States)

    Srivastava, Sameer; Gupta, Ranadheer K; Arha, Manish; Vishwakarma, Rishi K; Rawal, Shuban K; Kavi Kishor, P B; Khan, Bashir M

    2011-02-01

    Removal of lignin is a major hurdle for obtaining good quality pulp. Leucaena leucocephala (subabul) is extensively used in paper industry in India; therefore, as a first step to generate transgenic plants with low lignin content, cDNA and genomic clones of CCR gene were isolated and characterized. The cDNA encoding CCR (EC 1.2.1.44) was designated as Ll-CCR; the sequence analysis revealed an Open Reading Frame (ORF) of 1005 bp. Phylogenetic analysis showed that Ll-CCR sequence is highly homologous to CCRs from other dicot plants. The 2992 bp genomic clone of Leucaena CCR consists of 5 exons and 4 introns. The haploid genome of L. leucocephala contains two copies as revealed by DNA blot hybridization. Ll-CCR gene was over-expressed in Escherichia coli, which showed a molecular mass of approximately 38 kDa. Protein blot analysis revealed that Ll-CCR protein is expressed at higher levels in root and in stem, but undetectable in leaf tissues. Expression of CCR gene in Leucaena increased up to 15 d in case of roots and stem as revealed by QRT-PCR studies in 0-15 d old seedlings. ELISA based studies of extractable CCR protein corroborated with QRT-PCR data. CCR protein was immuno-cytolocalized around xylem tissue. Lignin estimation and expression studies of 5, 10 and 15 d old stem and root suggest that CCR expression correlates with quantity of lignin produced, which makes it a good target for antisense down regulation for producing designer species for paper industry. Copyright © 2010 Elsevier Masson SAS. All rights reserved.

  9. Comparison of chemokines (CCL-5 and SDF-1), chemokine receptors (CCR-5 and CXCR-4) and IL-6 levels in patients with different severities of depression.

    Science.gov (United States)

    Ogłodek, Ewa A; Szota, Anna; Just, Marek J; Moś, Danuta; Araszkiewicz, Aleksander

    2014-10-01

    Depression can be perceived as a psychoneuroimmunological disorder in which cytokines affecting the body's neurochemical and neuroendocrine functions play an important role. Among cytokines, chemokines participating in activation of the inflammatory response are considered to be crucial. 160 men and women were enrolled in the study. 120 of them were diagnosed with various types of depression. The mean age was 45.2 ± 4.5 years (range: 19-47 years). The control group consisted of 40 healthy individuals. The average age in this group was 42.4 ± 4.1 years. Plasma levels of chemokines and their receptors (CCL-5 - RANTES and CXCR-5, SDF-1 and CXCR-4), as well as of IL-6, were assessed by ELISA. There was an increase in SDF-1 and CCL-5 levels in women and men with different severities of depression, versus the control group. Also, an increase in the IL-6 levels, CXCR4 and CCR-5 receptors was observed in both women and men with all types of depression. Levels of SDF-1 and CCL-5 chemokines, as well as of CCR-5 and CXCR4 chemokine receptors, were higher in women than in men. The results of this study indicate the need for assessment of CCL-5 and SDF-1 chemokines levels, as they are likely markers of developing depression. Early measurement of these chemokines levels may be helpful in choosing the best pharmacotherapy. Copyright © 2014 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

  10. Studies of tolerance induction through mixed chimerism in cynomolgus monkeys. Method for detection of chimeric cells and effect of thymic irradiation on induction of tolerance

    International Nuclear Information System (INIS)

    Hoshino, Tomoaki; Kawai, Tatsuo; Ota, Kazuo

    1996-01-01

    To establish the method for the detection of chimerism in cynomologus monkeys, we tested cross reactivity of various anti-HLA monoclonal antibodies (mAb) to cynomolgus monkeys. In 29 mAb we tested, only three monoclonal anti-HLA antibodies crossreacted with lymphocytes of monkeys. With these mAb, chimeric cell can be detected up to 1% by flow cytometric analysis (study 1). Utilizing the method we developed in study 1, we applied the regimen that induces mixed chimerism and skin graft tolerance in mice to renal allotransplantation of cynomolgus monkey. Regimen A includes non-lethal dose of total body irradiation (TBI), administration of anti-thymocyte globulin (ATG) for 3 days, donor bone marrow infusion and 45 days course of cyclosporine (CYA) administration. We added 7 Gy of thymic irradiation on day-6 in regimen B and on day-1 in regimen C. Although all monkeys in regimen A and B consistently developed chimerism, they rejected kidney allografts soon after stopping CYA. In contrast, 4 monkeys out of 5 failed to develop chimerism in regimen C, but renal allograft tolerance was induced in one monkey who developed chimerism in regimen C. In conclusion, the induction of chimerism is considered necessary but not sufficient for tolerance induction. (author)

  11. Studies of tolerance induction through mixed chimerism in cynomolgus monkeys. Method for detection of chimeric cells and effect of thymic irradiation on induction of tolerance

    Energy Technology Data Exchange (ETDEWEB)

    Hoshino, Tomoaki; Kawai, Tatsuo; Ota, Kazuo [Tokyo Women`s Medical Coll. (Japan)

    1996-12-01

    To establish the method for the detection of chimerism in cynomologus monkeys, we tested cross reactivity of various anti-HLA monoclonal antibodies (mAb) to cynomolgus monkeys. In 29 mAb we tested, only three monoclonal anti-HLA antibodies crossreacted with lymphocytes of monkeys. With these mAb, chimeric cell can be detected up to 1% by flow cytometric analysis (study 1). Utilizing the method we developed in study 1, we applied the regimen that induces mixed chimerism and skin graft tolerance in mice to renal allotransplantation of cynomolgus monkey. Regimen A includes non-lethal dose of total body irradiation (TBI), administration of anti-thymocyte globulin (ATG) for 3 days, donor bone marrow infusion and 45 days course of cyclosporine (CYA) administration. We added 7 Gy of thymic irradiation on day-6 in regimen B and on day-1 in regimen C. Although all monkeys in regimen A and B consistently developed chimerism, they rejected kidney allografts soon after stopping CYA. In contrast, 4 monkeys out of 5 failed to develop chimerism in regimen C, but renal allograft tolerance was induced in one monkey who developed chimerism in regimen C. In conclusion, the induction of chimerism is considered necessary but not sufficient for tolerance induction. (author)

  12. Comparative study of enteric viruses, coliphages and indicator bacteria for evaluating water quality in a tropical high-altitude system.

    Science.gov (United States)

    Espinosa, Ana C; Arias, Carlos F; Sánchez-Colón, Salvador; Mazari-Hiriart, Marisa

    2009-10-27

    Bacteria used as indicators for pathogenic microorganisms in water are not considered adequate as enteric virus indicators. Surface water from a tropical high-altitude system located in Mexico City that receives rainwater, treated and non-treated wastewater used for irrigation, and groundwater used for drinking, was studied. The presence of enterovirus, rotavirus, astrovirus, coliphage, coliform bacteria, and enterococci was determined during annual cycles in 2001 and 2002. Enteric viruses in concentrated water samples were detected by reverse transcriptase-polymerase chain reaction (RT-PCR). Coliphages were detected using the double agar layer method. Bacteria analyses of the water samples were carried out by membrane filtration. The presence of viruses and bacteria in the water used for irrigation showed no relationship between current bacterial indicator detection and viral presence. Coliphages showed strong association with indicator bacteria and enterovirus, but weak association with other enteric viruses. Enterovirus and rotavirus showed significant seasonal differences in water used for irrigation, although this was not clear for astrovirus. Coliphages proved to be adequate faecal pollution indicators for the irrigation water studied. Viral presence in this tropical high-altitude system showed a similar trend to data previously reported for temperate zones.

  13. Effect of female genital schistosomiasis and anti-schistosomal treatment on monocytes, CD4+ T-cells and CCR5 expression in the female genital tract.

    Science.gov (United States)

    Kleppa, Elisabeth; Ramsuran, Veron; Zulu, Siphosenkosi; Karlsen, Gunn Hege; Bere, Alfred; Passmore, Jo-Ann S; Ndhlovu, Patricia; Lillebø, Kristine; Holmen, Sigve D; Onsrud, Mathias; Gundersen, Svein Gunnar; Taylor, Myra; Kjetland, Eyrun F; Ndung'u, Thumbi

    2014-01-01

    Schistosoma haematobium is a waterborne parasite that may cause female genital schistosomiasis (FGS), characterized by genital mucosal lesions. There is clinical and epidemiological evidence for a relationship between FGS and HIV. We investigated the impact of FGS on HIV target cell density and expression of the HIV co-receptor CCR5 in blood and cervical cytobrush samples. Furthermore we evaluated the effect of anti-schistosomal treatment on these cell populations. The study followed a case-control design with post treatment follow-up, nested in an on-going field study on FGS. Blood and cervical cytobrush samples were collected from FGS negative and positive women for flow cytometry analyses. Urine samples were investigated for schistosome ova by microscopy and polymerase chain reaction (PCR). FGS was associated with a higher frequency of CD14+ cells (monocytes) in blood (11.5% in FGS+ vs. 2.2% in FGS-, p = 0.042). Frequencies of CD4+ cells expressing CCR5 were higher in blood samples from FGS+ than from FGS- women (4.7% vs. 1.5%, p = 0.018). The CD14+ cell population decreased significantly in both compartments after anti-schistosomal treatment (p = 0.043). Although the frequency of CD4+ cells did not change after treatment, frequencies of CCR5 expression by CD4+ cells decreased significantly in both compartments (from 3.4% to 0.5% in blood, p = 0.036; and from 42.4% to 5.6% in genital samples, p = 0.025). The results support the hypothesis that FGS may increase the risk of HIV acquisition, not only through damage of the mucosal epithelial barrier, but also by affecting HIV target cell populations, and that anti-schistosomal treatment can modify this.

  14. Comparison of human immunodeficiency virus type 1 tropism profiles in clinical samples by the Trofile and MT-2 assays

    NARCIS (Netherlands)

    Coakley, Eoin; Reeves, Jacqueline D.; Huang, Wei; Mangas-Ruiz, Marga; Maurer, Irma; Harskamp, Agnes M.; Gupta, Soumi; Lie, Yolanda; Petropoulos, Christos J.; Schuitemaker, Hanneke; van 't Wout, Angélique B.

    2009-01-01

    The recent availability of CCR5 antagonists as anti-human immunodeficiency virus (anti-HIV) therapeutics has highlighted the need to accurately identify CXCR4-using variants in patient samples when use of this new drug class is considered. The Trofile assay (Monogram Biosciences) has become the

  15. CCR7 signaling pathway and retinal neovascularization

    Directory of Open Access Journals (Sweden)

    Lin-Hui Yuan

    2015-11-01

    Full Text Available Retinal neovascularization diseases are the major causes of blindness. C-C chemokine receptor type 7(CCR7can promote the expression of vascular endothelial growth factor(VEGFthrough the extracellular signal regulated kinase(ERKpathway, leading to vascular leakage, proliferation of vascular endothelial cell, neovascularization and etc. The detection of CCR7 can guide the diagnosis and treatments of retinal neovascularization diseases.

  16. Downregulation of mouse CCR3 by lentiviral shRNA inhibits proliferation and induces apoptosis of mouse eosinophils.

    Science.gov (United States)

    Zhu, Xin-Hua; Liao, Bing; Xu, Yi; Liu, Ke; Huang, Yun; Huang, Quan-Long; Liu, Yue-Hui

    2017-02-01

    RNA interference has been considered as an effective gene silencing method in basic and preclinical investigations. The aims of the present study were to construct a lentiviral vector expressing a short hairpin RNA (shRNA) targeting the murine CC chemokine receptor 3 (mCCR3), and to investigate its effects on the proliferation and apoptosis of mouse eosinophils. A recombinant lentiviral vector expressing four fragments of mouse CCR3 shRNA (pLVX‑mCCR3‑1+2+3+4‑shRNA) was constructed using subcloning techniques. This novel lentivirus was then packaged into 293T cells by co‑transduction with plasmids, including Baculo p35, pCMV R8.2 and VSV. The interference effects of the vector were verified using polymerase chain reaction (PCR) and western blot analyses. The effects of the interference on the proliferation and apoptosis of mouse eosinophils were investigated using 3‑(4,5‑dimethylthiazol‑2‑yl)‑5‑(3‑carboxymethoxyphenyl)‑2‑(4‑sulfophenyl)‑2H‑tetrazolium and terminal deoxynucleotidyl transferase dUTP nick end labeling methods, respectively. The results of the PCR and western blot analyses confirmed that the novel recombinant vector, pLVX‑mCCR3‑1+2+3+4‑shRNA, had high efficiency in inhibiting the mRNA and protein expression levels of mCCR3 in mouse eosinophils. The downregulation of mCCR3 significantly inhibited proliferation of the eosinophils. Furthermore, the present study found that the downregulation of mCCR3 significantly promoted apoptosis of the eosinophils. Therefore, the downregulation of mCCR3 led to the inhibition of proliferation and induction of apoptosis in mouse eosinophils. The predominant characteristics of allergic rhinitis are eosinophil infiltration and release of inflammatory mediators, which appear in a variety of clinical manifestations. The results of the present study indicate that mCCR3 silencing may serve as a putative approach for the treatment of allergic rhinitis.

  17. Neuraminidase and hemagglutinin matching patterns of a highly pathogenic avian and two pandemic H1N1 influenza A viruses.

    Directory of Open Access Journals (Sweden)

    Yonghui Zhang

    Full Text Available BACKGROUND: Influenza A virus displays strong reassortment characteristics, which enable it to achieve adaptation in human infection. Surveying the reassortment and virulence of novel viruses is important in the prevention and control of an influenza pandemic. Meanwhile, studying the mechanism of reassortment may accelerate the development of anti-influenza strategies. METHODOLOGY/PRINCIPAL FINDINGS: The hemagglutinin (HA and neuraminidase (NA matching patterns of two pandemic H1N1 viruses (the 1918 and current 2009 strains and a highly pathogenic avian influenza A virus (H5N1 were studied using a pseudotyped particle (pp system. Our data showed that four of the six chimeric HA/NA combinations could produce infectious pps, and that some of the chimeric pps had greater infectivity than did their ancestors, raising the possibility of reassortment among these viruses. The NA of H5N1 (A/Anhui/1/2005 could hardly reassort with the HAs of the two H1N1 viruses. Many biological characteristics of HA and NA, including infectivity, hemagglutinating ability, and NA activity, are dependent on their matching pattern. CONCLUSIONS/SIGNIFICANCE: Our data suggest the existence of an interaction between HA and NA, and the HA NA matching pattern is critical for valid viral reassortment.

  18. Chimeric enzymes with improved cellulase activities

    Science.gov (United States)

    Xu, Qi; Baker, John O; Himmel, Michael E

    2015-03-31

    Nucleic acid molecules encoding chimeric cellulase polypeptides that exhibit improved cellulase activities are disclosed herein. The chimeric cellulase polypeptides encoded by these nucleic acids and methods to produce the cellulases are also described, along with methods of using chimeric cellulases for the conversion of cellulose to sugars such as glucose.

  19. Characterization of emergent HIV resistance in treatment-naive subjects enrolled in a vicriviroc phase 2 trial.

    Science.gov (United States)

    McNicholas, Paul; Wei, Yi; Whitcomb, Jeannette; Greaves, Wayne; Black, Todd A; Tremblay, Cecile L; Strizki, Julie M

    2010-05-15

    Vicriviroc is a C-C motif chemokine receptor 5 (CCR5) antagonist that is in clinical development for the treatment of human immunodeficiency virus type 1 (HIV-1) infection. This study explored the molecular basis for the development of phenotypically resistant virus. HIV-1 RNA from treatment-naive subjects who experienced virological failure in a phase 2 dose-finding trial was evaluated for coreceptor usage and susceptibility. For viruses that exhibited reduced susceptibility to vicriviroc, envelope clones were phenotypically and genotypically characterized. Twenty-six vicriviroc-treated subjects experienced virological failure; for 24 the virus remained CCR5-tropic, and 2 had dual/X4 virus. Reduced susceptibility to vicriviroc, manifested as decreases in the maximum percent inhibition value (no increase in median inhibitory concentration), was detected in 4 of the 26 subjects who experienced virological failure. Clonal analysis of envelopes in samples from these 4 subjects revealed multiple sequence changes in gp160, principally within the variable domain 1/variable domain 2, variable domain 3, and variable domain 4 loops. However, no consistent pattern of mutations was observed across subjects. In this study, only a small proportion of treatment failures were associated with tropism changes or reduced susceptibility to vicriviroc. Genotypic analysis of cloned env sequences revealed no specific mutational pattern associated with reduced susceptibility to vicriviroc, although numerous changes were observed in the variable domain 3 loop and in other regions of gp160.

  20. CCR2 deficiency leads to increased eosinophils, alternative macrophage activation, and type 2 cytokine expression in adipose tissue.

    Science.gov (United States)

    Bolus, W Reid; Gutierrez, Dario A; Kennedy, Arion J; Anderson-Baucum, Emily K; Hasty, Alyssa H

    2015-10-01

    Adipose tissue (AT) inflammation during obesity is mediated by immune cells and closely correlates with systemic insulin resistance. In lean AT, eosinophils are present in low but significant numbers and capable of promoting alternative macrophage activation in an IL-4/IL-13-dependent manner. In WT mice, obesity causes the proportion of AT eosinophils to decline, concomitant with inflammation and classical activation of AT macrophages. In this study, we show that CCR2 deficiency leads to increased eosinophil accumulation in AT. Furthermore, in contrast to WT mice, the increase in eosinophils in CCR2(-/-) AT is sustained and even amplified during obesity. Interestingly, a significant portion of eosinophils is found in CLSs in AT of obese CCR2(-/-) mice, which is the first time eosinophils have been shown to localize to these inflammatory hot spots. CCR2(-/-) bone marrow precursors displayed increased expression of various key eosinophil genes during in vitro differentiation to eosinophils, suggesting a potentially altered eosinophil phenotype in the absence of CCR2. In addition, the proportion of eosinophils in AT positively correlated with local expression of Il5, a potent eosinophil stimulator. The increase in eosinophils in CCR2(-/-) mice was detected in all white fat pads analyzed and in the peritoneal cavity but not in bone marrow, blood, spleen, or liver. In AT of CCR2(-/-) mice, an increased eosinophil number positively correlated with M2-like macrophages, expression of the Treg marker Foxp3, and type 2 cytokines, Il4, Il5, and Il13. This is the first study to link CCR2 function with regulation of AT eosinophil accumulation. © Society for Leukocyte Biology.

  1. Immunization with a novel chimeric peptide representing B and T cell epitopes from HER2 extracellular domain (HER2 ECD) for breast cancer.

    Science.gov (United States)

    Mahdavi, Manijeh; Keyhanfar, Mehrnaz; Jafarian, Abbas; Mohabatkar, Hassan; Rabbani, Mohammad

    2014-12-01

    Because of direct stimulating immune system against disease, vaccination or active immunotherapy is preferable compared to passive immunotherapy. For this purpose, a newly designed chimeric peptide containing epitopes for both B and T cells from HER2 ECD subdomain III was proposed. To evaluate the effects of the active immunization, a discontinuous B cell epitope peptide was selected based on average antigenicity by bioinformatics analysis. The selected peptide was collinearly synthesized as a chimera with a T helper epitope from the protein sequence of measles virus fusion (208-302) using the GPSL linker. Three mice were immunized with the chimeric peptide. Reactive antibodies with HER2 protein in ELISA and immunofluorescence assays with no cross-reactivity were generated. The 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay indicated that the anti-peptide sera had inhibitory effects on proliferation of SK-BR-3 cells. Hence, the newly designed, discontinuous chimeric peptide representing B and T cell epitopes from subdomain III of HER2-ECD can form the basis for future vaccines design, where these data can be applied for monoclonal antibody production targeting the distinct epitope of HER2 receptor compared to the two broadly used anti-HER2 monoclonal antibodies, Herceptin and pertuzumab.

  2. HIV coreceptor phenotyping in the clinical setting.

    Science.gov (United States)

    Low, Andrew J; Swenson, Luke C; Harrigan, P Richard

    2008-01-01

    The introduction of CCR5 antagonists increases the options available for constructing antiretroviral regimens. However, this option is coupled with the caveat that patients should be tested for HIV coreceptor tropism prior to initiating CCR5 antagonist-based therapy. Failure to screen for CXCR4 usage increases the risk of using an ineffective drug, thus reducing the likelihood of viral suppression and increasing their risk for developing antiretroviral resistance. This review discusses current and future methods of determining HIV tropism, with a focus on their utility in the clinical setting for screening purposes. Some of these methods include recombinant phenotypic tests, such as the Monogram Trofile assay, as well as genotype-based predictors, heteroduplex tracking assays, and flow cytometry based methods. Currently, the best evidence supports the use of phenotypic methods, although other methods of screening for HIV coreceptor usage prior to the administration of CCR5 antagonists may reduce costs and increase turnaround time over phenotypic methods. The presence of low levels of X4 virus is a challenge to all assay methods, resulting in reduced sensitivity in clinical, patient-derived samples when compared to clonally derived samples. Gaining a better understanding of the output of these assays and correlating them with clinical progression and therapy response will provide some indication on how both genotype-based, and phenotypic assays for determining HIV coreceptor usage can be improved. In addition, leveraging new technologies capable of detecting low-level minority species may provide the most significant advances in ensuring that individuals with low levels of dual/mixed tropic virus are not inadvertently prescribed CCR5 antagonists.

  3. The Isolation of Novel Phage Display-Derived Human Recombinant Antibodies Against CCR5, the Major Co-Receptor of HIV

    OpenAIRE

    Shimoni, Moria; Herschhorn, Alon; Britan-Rosich, Yelena; Kotler, Moshe; Benhar, Itai; Hizi, Amnon

    2013-01-01

    Selecting for antibodies against specific cell-surface proteins is a difficult task due to many unrelated proteins that are expressed on the cell surface. Here, we describe a method to screen antibody-presenting phage libraries against native cell-surface proteins. We applied this method to isolate antibodies that selectively recognize CCR5, which is the major co-receptor for HIV entry (consequently, playing a pivotal role in HIV transmission and pathogenesis). We employed a phage screening s...

  4. Dissociation between peripheral blood chimerism and tolerance to hindlimb composite tissue transplants: preferential localization of chimerism in donor bone.

    Science.gov (United States)

    Rahhal, Dina N; Xu, Hong; Huang, Wei-Chao; Wu, Shengli; Wen, Yujie; Huang, Yiming; Ildstad, Suzanne T

    2009-09-27

    Mixed chimerism induces donor-specific tolerance to composite tissue allotransplants (CTAs). In the present studies, we used a nonmyeloablative conditioning approach to establish chimerism and promote CTA acceptance. Wistar Furth (RT1A(u)) rats were conditioned with 600 to 300 cGy total body irradiation (TBI, day-1), and 100 x 10(6) T-cell-depleted ACI (RT1A(abl)) bone marrow cells were transplanted on day 0, followed by a 11-day course of tacrolimus and one dose of antilymphocyte serum (day 10). Heterotopic osteomyocutaneous flap transplantation was performed 4 to 6 weeks after bone marrow transplantation. Mixed chimerism was initially achieved in almost all recipients, but long-term acceptance of CTA was only achieved in rats treated with 600 cGy TBI. When anti-alphabeta-T-cell receptor (TCR) monoclonal antibody (mAb) (day-3) was added into the regimens, donor chimerism was similar to recipients preconditioned without anti-alphabeta-TCR mAb. However, the long-term CTA survival was significantly improved in chimeras receiving more than or equal to 300 cGy TBI plus anti-alphabeta-TCR mAb. Higher levels of donor chimerism were associated with CTA acceptance. The majority of flap acceptors lost peripheral blood chimerism within 6 months. However, donor chimerism persisted in the transplanted bone at significantly higher levels compared with other hematopoietic compartments. The compartment donor chimerism may be responsible for the maintenance of tolerance to CTA. Long-term acceptors were tolerant to a donor skin graft challenge even in the absence of peripheral blood chimerism. Mixed chimerism established by nonmyeloablative conditioning induces long-term acceptance of CTA, which is associated with persistent chimerism preferentially in the transplanted donor bone.

  5. Memory CD4(+)CCR5(+) T cells are abundantly present in the gut of newborn infants to facilitate mother-to-child transmission of HIV-1

    NARCIS (Netherlands)

    Bunders, Madeleine J.; van der Loos, Chris M.; Klarenbeek, Paul L.; van Hamme, John L.; Boer, Kees; Wilde, Jim C. H.; de Vries, Niek; van Lier, Rene A. W.; Kootstra, Neeltje; Pals, Steven T.; Kuijpers, Taco W.

    2012-01-01

    Despite potential clinical importance, target cells for mother-to-child transmission of HIV-1 have not yet been identified. Cord blood-derived CD4(+) T cells are largely naive and do not express CCR5, the mandatory coreceptor for transmitted HIV-1 R5 strains in infants. In the present study, we

  6. Comparative study of enteric viruses, coliphages and indicator bacteria for evaluating water quality in a tropical high-altitude system

    Directory of Open Access Journals (Sweden)

    Mazari-Hiriart Marisa

    2009-10-01

    Full Text Available Abstract Background Bacteria used as indicators for pathogenic microorganisms in water are not considered adequate as enteric virus indicators. Surface water from a tropical high-altitude system located in Mexico City that receives rainwater, treated and non-treated wastewater used for irrigation, and groundwater used for drinking, was studied. Methods The presence of enterovirus, rotavirus, astrovirus, coliphage, coliform bacteria, and enterococci was determined during annual cycles in 2001 and 2002. Enteric viruses in concentrated water samples were detected by reverse transcriptase-polymerase chain reaction (RT-PCR. Coliphages were detected using the double agar layer method. Bacteria analyses of the water samples were carried out by membrane filtration. Results The presence of viruses and bacteria in the water used for irrigation showed no relationship between current bacterial indicator detection and viral presence. Coliphages showed strong association with indicator bacteria and enterovirus, but weak association with other enteric viruses. Enterovirus and rotavirus showed significant seasonal differences in water used for irrigation, although this was not clear for astrovirus. Conclusion Coliphages proved to be adequate faecal pollution indicators for the irrigation water studied. Viral presence in this tropical high-altitude system showed a similar trend to data previously reported for temperate zones.

  7. CCR6(+) Th cell populations distinguish ACPA positive from ACPA negative rheumatoid arthritis.

    Science.gov (United States)

    Paulissen, Sandra M J; van Hamburg, Jan Piet; Davelaar, Nadine; Vroman, Heleen; Hazes, Johanna M W; de Jong, Pascal H P; Lubberts, Erik

    2015-11-30

    Patients with rheumatoid arthritis (RA) can be separated into two major subpopulations based on the absence or presence of serum anti-citrullinated protein antibodies (ACPAs). The more severe disease course in ACPA(+) RA and differences in treatment outcome between these subpopulations suggest that ACPA(+) and ACPA(-) RA are different disease subsets. The identification of T-helper (Th) cells specifically recognizing citrullinated peptides, combined with the strong association between HLA-DRB1 and ACPA positivity, point toward a pathogenic role of Th cells in ACPA(+) RA. In this context we recently identified a potential pathogenic role for CCR6(+) Th cells in RA. Therefore, we examined whether Th cell population distributions differ by ACPA status. We performed a nested matched case-control study including 27 ACPA(+) and 27 ACPA(-) treatment-naive early RA patients matched for disease activity score in 44 joints, presence of rheumatoid factor, sex, age, duration of complaints and presence of erosions. CD4(+)CD45RO(+) (memory) Th cell distribution profiles from these patients were generated based on differential chemokine receptor expression and related with disease duration. ACPA status was not related to differences in total CD4(+) T cell or memory Th cell proportions. However, ACPA(+) patients had significantly higher proportions of Th cells expressing the chemokine receptors CCR6 and CXCR3. Similar proportions of CCR4(+) and CCR10(+) Th cells were found. Within the CCR6(+) cell population, four Th subpopulations were distinguished based on differential chemokine receptor expression: Th17 (CCR4(+)CCR10(-)), Th17.1 (CXCR3(+)), Th22 (CCR4(+)CCR10(+)) and CCR4/CXCR3 double-positive (DP) cells. In particular, higher proportions of Th22 (p = 0.02), Th17.1 (p = 0.03) and CCR4/CXCR3 DP (p = 0.01) cells were present in ACPA(+) patients. In contrast, ACPA status was not associated with differences in Th1 (CCR6(-)CXCR3(+); p = 0.90), Th2 (CCR6(-)CCR4(+); p = 0.27) and T

  8. A rhodanine derivative CCR-11 inhibits bacterial proliferation by inhibiting the assembly and GTPase activity of FtsZ.

    Science.gov (United States)

    Singh, Parminder; Jindal, Bhavya; Surolia, Avadhesha; Panda, Dulal

    2012-07-10

    A perturbation of FtsZ assembly dynamics has been shown to inhibit bacterial cytokinesis. In this study, the antibacterial activity of 151 rhodanine compounds was assayed using Bacillus subtilis cells. Of 151 compounds, eight strongly inhibited bacterial proliferation at 2 μM. Subsequently, we used the elongation of B. subtilis cells as a secondary screen to identify potential FtsZ-targeted antibacterial agents. We found that three compounds significantly increased bacterial cell length. One of the three compounds, namely, CCR-11 [(E)-2-thioxo-5-({[3-(trifluoromethyl)phenyl]furan-2-yl}methylene)thiazolidin-4-one], inhibited the assembly and GTPase activity of FtsZ in vitro. CCR-11 bound to FtsZ with a dissociation constant of 1.5 ± 0.3 μM. A docking analysis indicated that CCR-11 may bind to FtsZ in a cavity adjacent to the T7 loop and that short halogen-oxygen, H-bonding, and hydrophobic interactions might be important for the binding of CCR-11 with FtsZ. CCR-11 inhibited the proliferation of B. subtilis cells with a half-maximal inhibitory concentration (IC(50)) of 1.2 ± 0.2 μM and a minimal inhibitory concentration of 3 μM. It also potently inhibited proliferation of Mycobacterium smegmatis cells. Further, CCR-11 perturbed Z-ring formation in B. subtilis cells; however, it neither visibly affected nucleoid segregation nor altered the membrane integrity of the cells. CCR-11 inhibited HeLa cell proliferation with an IC(50) value of 18.1 ± 0.2 μM (∼15 × IC(50) of B. subtilis cell proliferation). The results suggested that CCR-11 inhibits bacterial cytokinesis by inhibiting FtsZ assembly, and it can be used as a lead molecule to develop FtsZ-targeted antibacterial agents.

  9. Inability of Kaplan radiation leukemia virus to replicate on mouse fibroblasts is conferred by its long terminal repeat

    International Nuclear Information System (INIS)

    Rassart, E.; Paquette, Y.; Jolicoeur, P.

    1988-01-01

    The molecularly cloned infectious Kaplan radiation leukemia virus has previously been shown to be unable to replicate on mouse fibroblasts. To map the viral sequences responsible for this, we constructed chimeric viral DNA genomes in vitro with parental cloned infectious viral DNAs from the nonfibrotropic (F-) BL/VL3 V-13 radiation leukemia virus and the fibrotropic (F+) endogenous BALB/c or Moloney murine leukemia viruses (MuLV). Infectious chimeric MuLVs, recovered after transfection of Ti-6 lymphocytes with these recombinant DNAs, were tested for capacity to replicate on mouse fibroblasts in vitro. We found that chimeric MuLVs harboring the long terminal repeat (LTR) of a fibrotropic MuLV replicated well on mouse fibroblasts. Conversely, chimeric MuLVs harboring the LTR of a nonfibrotropic MuLV were restricted on mouse fibroblasts. These results indicate that the LTR of BL/VL3 radiation leukemia virus harbors the primary determinant responsible for its inability to replicate on mouse fibroblasts in vitro. Our results also show that the primary determinant allowing F+ MuLVs (endogenous BALB/c and Moloney MuLVs) to replicate on mouse fibroblasts in vitro resides within the LTR

  10. MCP-1/CCR-2-double-deficiency severely impairs the migration of hematogenous inflammatory cells following transient cerebral ischemia in mice.

    Science.gov (United States)

    Schuette-Nuetgen, Katharina; Strecker, Jan-Kolja; Minnerup, Jens; Ringelstein, E Bernd; Schilling, Matthias

    2012-02-01

    Monocyte chemoattractant protein-1 (MCP-1) and its receptor CCR-2 are known to play a major role in inflammatory responses after cerebral ischemia. Mice deficient in either MCP-1 or CCR-2 have been reported to develop smaller infarct sizes and show decreased numbers of infiltrating inflammatory cells. In the present study we used green fluorescent protein (GFP) transgenic mice to investigate the effect of MCP-1/CCR-2-double deficiency on the recruitment of inflammatory cells in a model of both, mild and severe cerebral ischemia. We show that MCP-1/CCR-2-double deficiency virtually entirely abrogates the recruitment of hematogenous macrophages and significantly reduces neutrophil migration to the ischemic brain 4 and 7 days following focal cerebral ischemia. This argues for a predominant role of the MCP-1/CCR-2 axis in chemotaxis of monocytes despite a wide redundancy in the chemokine-receptor-system. Chemokine analysis revealed that even candidates known to be involved in monocyte and neutrophil recruitment like MIP-1α, CXCL-1, C5a, G-CSF and GM-CSF showed a reduced and delayed or even a lack of relevant compensatory response in MCP-1(-/-)/CCR-2(-/-)-mice. Solely, chemokine receptor 5 (CCR-5) increased early in both, but rose above wildtype levels at day 7 in MCP-1(-/-)/CCR-2(-/-)-animals, which might explain the higher number of activated microglial cells compared to control mice. Our study was, however, not powered to investigate infarct volumes. Further studies are needed to clarify whether these mechanisms of inflammatory cell recruitment might be essential for early infarct development and final infarct size and to evaluate potential therapeutic implications. Copyright © 2011 Elsevier Inc. All rights reserved.

  11. CCR Interns | Center for Cancer Research

    Science.gov (United States)

    The Cancer Research Interns (CRI) Summer Program was inaugurated in 2004 to provide an open door for students looking for an initial training opportunity. The goal is to enhance diversity within the CCR (Center for Cancer Research) training program and we have placed 338 students from 2004 to 2017, in labs and branches across the division.  The CCR and the Center for Cancer Training’s Office of Training and Education provide stipend support, some Service & Supply funds, and travel support for those students who meet the financial eligibility criteria (

  12. Novel chimeric virus-like particles vaccine displaying MERS-CoV receptor-binding domain induce specific humoral and cellular immune response in mice.

    Science.gov (United States)

    Wang, Chong; Zheng, Xuexing; Gai, Weiwei; Wong, Gary; Wang, Hualei; Jin, Hongli; Feng, Na; Zhao, Yongkun; Zhang, Weijiao; Li, Nan; Zhao, Guoxing; Li, Junfu; Yan, Jinghua; Gao, Yuwei; Hu, Guixue; Yang, Songtao; Xia, Xianzhu

    2017-04-01

    Middle East respiratory syndrome coronavirus (MERS-CoV) has continued spreading since its emergence in 2012 with a mortality rate of 35.6%, and is a potential pandemic threat. Prophylactics and therapies are urgently needed to address this public health problem. We report here the efficacy of a vaccine consisting of chimeric virus-like particles (VLP) expressing the receptor binding domain (RBD) of MERS-CoV. In this study, a fusion of the canine parvovirus (CPV) VP2 structural protein gene with the RBD of MERS-CoV can self-assemble into chimeric, spherical VLP (sVLP). sVLP retained certain parvovirus characteristics, such as the ability to agglutinate pig erythrocytes, and structural morphology similar to CPV virions. Immunization with sVLP induced RBD-specific humoral and cellular immune responses in mice. sVLP-specific antisera from these animals were able to prevent pseudotyped MERS-CoV entry into susceptible cells, with neutralizing antibody titers reaching 1: 320. IFN-γ, IL-4 and IL-2 secreting cells induced by the RBD were detected in the splenocytes of vaccinated mice by ELISpot. Furthermore, mice inoculated with sVLP or an adjuvanted sVLP vaccine elicited T-helper 1 (Th1) and T-helper 2 (Th2) cell-mediated immunity. Our study demonstrates that sVLP displaying the RBD of MERS-CoV are promising prophylactic candidates against MERS-CoV in a potential outbreak situation. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. A Respiratory Syncytial Virus Vaccine Vectored by a Stable Chimeric and Replication-Deficient Sendai Virus Protects Mice without Inducing Enhanced Disease.

    Science.gov (United States)

    Wiegand, Marian Alexander; Gori-Savellini, Gianni; Gandolfo, Claudia; Papa, Guido; Kaufmann, Christine; Felder, Eva; Ginori, Alessandro; Disanto, Maria Giulia; Spina, Donatella; Cusi, Maria Grazia

    2017-05-15

    Respiratory syncytial virus (RSV) is a major cause of severe respiratory infections in children and elderly people, and no marketed vaccine exists. In this study, we generated and analyzed a subunit vaccine against RSV based on a novel genome replication-deficient Sendai virus (SeV) vector. We inserted the RSV F protein, known to be a genetically stable antigen, into our vector in a specific way to optimize the vaccine features. By exchanging the ectodomain of the SeV F protein for its counterpart from RSV, we created a chimeric vectored vaccine that contains the RSV F protein as an essential structural component. In this way, the antigen is actively expressed on the surfaces of vaccine particles in its prefusion conformation, and as recently reported for other vectored vaccines, the occurrence of silencing mutations of the transgene in the vaccine genome can be prevented. In addition, its active gene expression contributes to further stimulation of the immune response. In order to understand the best route of immunization, we compared vaccine efficacies after intranasal (i.n.) or intramuscular (i.m.) immunization of BALB/c mice. Via both routes, substantial RSV-specific immune responses were induced, consisting of serum IgG and neutralizing antibodies, as well as cytotoxic T cells. Moreover, i.n. immunization was also able to stimulate specific mucosal IgA in the upper and lower respiratory tract. In virus challenge experiments, animals were protected against RSV infection after both i.n. and i.m. immunization without inducing vaccine-enhanced disease. Above all, the replication-deficient SeV appeared to be safe and well tolerated. IMPORTANCE Respiratory syncytial virus (RSV) is a major cause of respiratory diseases in young children and elderly people worldwide. There is a great demand for a licensed vaccine. Promising existing vaccine approaches based on live-attenuated vaccines or viral vectors have suffered from unforeseen drawbacks related to immunogenicity

  14. Association between the CCR5 32-bp deletion allele and late onset of schizophrenia

    DEFF Research Database (Denmark)

    Rasmussen, H.B.; Timm, S.; Wang, A.G.

    2006-01-01

    OBJECTIVE: The 32-bp deletion allele in chemokine receptor CCR5 has been associated with several immune-mediated diseases and might be implicated in schizophrenia as well. METHOD: The authors genotyped DNA samples from 268 schizophrenia patients and 323 healthy subjects. Age at first admission...... to a psychiatric hospital department served as a measure of disease onset. RESULTS: Patients and comparison subjects differed marginally in their genotype distribution, with a slightly higher frequency of the deletion allele seen in the patients. The authors found the deletion allele to be associated with higher......-onset schizophrenia) and healthy subjects differed significantly. This was reflected in an increased frequency of the deletion allele in the patient subgroup. Patients with ages at first admission below and above 40 years significantly differed in distribution of genotypes and alleles, with an overrepresentation...

  15. Theoretical design of a new chimeric protein for the treatment of breast cancer

    Science.gov (United States)

    Soleimani, Meysam; Mahnam, Karim; Mirmohammad-Sadeghi, Hamid; Sadeghi-Aliabadi, Hojjat; Jahanian-Najafabadi, Ali

    2016-01-01

    p28 and NRC peptides are two anticancer peptides with various mechanisms have shown to be effective against breast cancer. Therefore, it seems that construction of a chimeric protein containing the two peptides might cause synergistic cytotoxic effects. However, since the two peptides bear opposite charges, production of a chimeric protein in which the two moieties do not intervene each other is difficult. In this study, our goal was to find a suitable peptide linker for the new chimeric protein in a manner that none of the peptides intervene the other’s function. We selected some linkers with different characteristics and lengths and created a small library of the chimeric proteins harboring these linkers. Homology modeling and molecular dynamic simulation revealed that (PA)5P and (EAAAK)3 linkers can separate the p28 and NRC peptides effectively. Thus, the chimeric protein linked with (PA)5P or (EAAAK)3 linkers might show synergistic and stronger anticancer effects than the separate peptide moieties because they could exert their cytotoxic effects freely which is not influenced by the other part. PMID:27499788

  16. CCR2 mediates Helicobacter pylori-induced immune tolerance and contributes to mucosal homeostasis.

    Science.gov (United States)

    Sun, Xia; Zhang, Min; El-Zaatari, Mohamad; Huffnagle, Gray B; Kao, John Y

    2017-04-01

    We previously demonstrated that H. pylori infection leads to increased induction of regulatory T cells in local and systemic immune compartments. Here, we investigate the role of CCR2 in the tolerogenic programing of dendritic cells in a mouse model of H. pylori infection. CCR2 deficient (CCR2KO) mice and wild-type (Wt) mice infected with H. pylori SS1 strain were analyzed by qPCR and FACS analysis. In vitro, bone marrow-derived DC on day 6 from CCR2KO and Wt mice cocultured with or without H. pylori were examined to determine the impact of CCR2 signaling on dendritic cells function by qPCR, ELISA, and FACS analyses. Acute H. pylori infection was associated with a threefold increase in CCR2 mRNA expression in the gastric mucosa. H. pylori-infected CCR2KO mice exhibited a higher degree of mucosal inflammation, that is, increased gastritis scores and pro-inflammatory cytokine mRNA levels, but lower degree of H. pylori gastric colonization compared to infected Wt mice. Peripheral H. pylori-specific immune response measured in the CCR2KO spleen was characterized by a higher Th17 response and a lower Treg response. In vitro, CCR2KO bone marrow-derived DC was less mature and shown a lower Treg/Th17 ratio. Moreover, blockade of CCR2 signaling by MCP-1 neutralizing antibody inhibited H. pylori-stimulated bone marrow-derived DC maturation. Our results indicate that CCR2 plays an essential role in H. pylori-induced immune tolerance and shed light on a novel mechanism of CCR2-dependent DC Treg induction, which appears to be important in maintaining mucosal homeostasis during H. pylori infection. © 2016 John Wiley & Sons Ltd.

  17. Identification and analysis of pig chimeric mRNAs using RNA sequencing data

    Science.gov (United States)

    2012-01-01

    Background Gene fusion is ubiquitous over the course of evolution. It is expected to increase the diversity and complexity of transcriptomes and proteomes through chimeric sequence segments or altered regulation. However, chimeric mRNAs in pigs remain unclear. Here we identified some chimeric mRNAs in pigs and analyzed the expression of them across individuals and breeds using RNA-sequencing data. Results The present study identified 669 putative chimeric mRNAs in pigs, of which 251 chimeric candidates were detected in a set of RNA-sequencing data. The 618 candidates had clear trans-splicing sites, 537 of which obeyed the canonical GU-AG splice rule. Only two putative pig chimera variants whose fusion junction was overlapped with that of a known human chimeric mRNA were found. A set of unique chimeric events were considered middle variances in the expression across individuals and breeds, and revealed non-significant variance between sexes. Furthermore, the genomic region of the 5′ partner gene shares a similar DNA sequence with that of the 3′ partner gene for 458 putative chimeric mRNAs. The 81 of those shared DNA sequences significantly matched the known DNA-binding motifs in the JASPAR CORE database. Four DNA motifs shared in parental genomic regions had significant similarity with known human CTCF binding sites. Conclusions The present study provided detailed information on some pig chimeric mRNAs. We proposed a model that trans-acting factors, such as CTCF, induced the spatial organisation of parental genes to the same transcriptional factory so that parental genes were coordinatively transcribed to give birth to chimeric mRNAs. PMID:22925561

  18. Identification and analysis of pig chimeric mRNAs using RNA sequencing data

    Directory of Open Access Journals (Sweden)

    Ma Lei

    2012-08-01

    Full Text Available Abstract Background Gene fusion is ubiquitous over the course of evolution. It is expected to increase the diversity and complexity of transcriptomes and proteomes through chimeric sequence segments or altered regulation. However, chimeric mRNAs in pigs remain unclear. Here we identified some chimeric mRNAs in pigs and analyzed the expression of them across individuals and breeds using RNA-sequencing data. Results The present study identified 669 putative chimeric mRNAs in pigs, of which 251 chimeric candidates were detected in a set of RNA-sequencing data. The 618 candidates had clear trans-splicing sites, 537 of which obeyed the canonical GU-AG splice rule. Only two putative pig chimera variants whose fusion junction was overlapped with that of a known human chimeric mRNA were found. A set of unique chimeric events were considered middle variances in the expression across individuals and breeds, and revealed non-significant variance between sexes. Furthermore, the genomic region of the 5′ partner gene shares a similar DNA sequence with that of the 3′ partner gene for 458 putative chimeric mRNAs. The 81 of those shared DNA sequences significantly matched the known DNA-binding motifs in the JASPAR CORE database. Four DNA motifs shared in parental genomic regions had significant similarity with known human CTCF binding sites. Conclusions The present study provided detailed information on some pig chimeric mRNAs. We proposed a model that trans-acting factors, such as CTCF, induced the spatial organisation of parental genes to the same transcriptional factory so that parental genes were coordinatively transcribed to give birth to chimeric mRNAs.

  19. Attenuation and immunogenicity of recombinant yellow fever 17D-dengue type 2 virus for rhesus monkeys

    Directory of Open Access Journals (Sweden)

    Galler R.

    2005-01-01

    Full Text Available A chimeric yellow fever (YF-dengue serotype 2 (dengue 2 virus was constructed by replacing the premembrane and envelope genes of the YF 17D virus with those from dengue 2 virus strains of Southeast Asian genotype. The virus grew to high titers in Vero cells and, after passage 2, was used for immunogenicity and attenuation studies in rhesus monkeys. Subcutaneous immunization of naive rhesus monkeys with the 17D-D2 chimeric virus induced a neutralizing antibody response associated with the protection of 6 of 7 monkeys against viremia by wild-type dengue 2 virus. Neutralizing antibody titers to dengue 2 were significantly lower in YF-immune animals than in YF-naive monkeys and protection against challenge with wild-type dengue 2 virus was observed in only 2 of 11 YF-immune monkeys. An anamnestic response to dengue 2, indicated by a sharp increase of neutralizing antibody titers, was observed in the majority of the monkeys after challenge with wild-type virus. Virus attenuation was demonstrated using the standard monkey neurovirulence test. The 17D-D2 chimera caused significantly fewer histological lesions than the YF 17DD virus. The attenuated phenotype could also be inferred from the limited viremias compared to the YF 17DD vaccine. Overall, these results provide further support for the use of chimeric viruses for the development of a new live tetravalent dengue vaccine.

  20. CCR presentations at AACR - 2018 | Center for Cancer Research

    Science.gov (United States)

    CCR presentations at AACR Several CCR scientists will present their research at the AACR Annual Meeting in Chicago, IL, between April 14-18, 2018. Selected oral presentations are listed below. A full list of abstracts can be found on the AACR website.

  1. Liver transplantation : chimerism, complications and matrix metalloproteinases

    NARCIS (Netherlands)

    Hove, Willem Rogier ten

    2011-01-01

    Chimerism after orthotopic liver transplantation (OLT) is the main focus of the studies described in this thesis. The first study showed that chimerism of different cell lineages within the liver graft does occur after OLT. Subsequently, in allogeneic blood stem cell recipients, chimerism was

  2. A rare subset of skin-tropic regulatory T cells expressing Il10/Gzmb inhibits the cutaneous immune response.

    Science.gov (United States)

    Ikebuchi, Ryoyo; Teraguchi, Shunsuke; Vandenbon, Alexis; Honda, Tetsuya; Shand, Francis H W; Nakanishi, Yasutaka; Watanabe, Takeshi; Tomura, Michio

    2016-10-19

    Foxp3 + regulatory T cells (Tregs) migrating from the skin to the draining lymph node (dLN) have a strong immunosuppressive effect on the cutaneous immune response. However, the subpopulations responsible for their inhibitory function remain unclear. We investigated single-cell gene expression heterogeneity in Tregs from the dLN of inflamed skin in a contact hypersensitivity model. The immunosuppressive genes Ctla4 and Tgfb1 were expressed in the majority of Tregs. Although Il10-expressing Tregs were rare, unexpectedly, the majority of Il10-expressing Tregs co-expressed Gzmb and displayed Th1-skewing. Single-cell profiling revealed that CD43 + CCR5 + Tregs represented the main subset within the Il10/Gzmb-expressing cell population in the dLN. Moreover, CD43 + CCR5 + CXCR3 - Tregs expressed skin-tropic chemokine receptors, were preferentially retained in inflamed skin and downregulated the cutaneous immune response. The identification of a rare Treg subset co-expressing multiple immunosuppressive molecules and having tissue-remaining capacity offers a novel strategy for the control of skin inflammatory responses.

  3. Characterization of monoclonal antibodies that specifically recognize the palm subdomain of hepatitis C virus nonstructural protein 5B polymerase.

    Science.gov (United States)

    Ingravallo, P; Lahser, F; Xia, E; Sodowich, B; Lai, V C; Hong, Z; Zhong, W

    2001-06-01

    The nonstructural protein 5B (NS5B) of hepatitis C virus (HCV) is an RNA-dependent RNA polymerase (RdRp) which plays an essential role in viral RNA replication. Antibodies that specifically recognize NS5B will have utilities in monitoring NS5B production and subcellular localization, as well as in structure-function studies. In this report, three mouse monoclonal antibodies (mAbs), 16A9C9, 16D9A4 and 20A12C7, against a recombinant NS5B protein (genotype 1a, H-77 strain) were produced. These mAbs specifically recognize HCV NS5B, but not RdRps of polivirus (PV), bovine viral diarrhea virus (BVDV) or GB virus B (GBV-B). The mAbs can readily detect NS5B in cellular lysates of human osteosarcoma Saos2 cells constitutively expressing the nonstructural region of HCV (NS3-NS4A-NS4B-NS5A-NS5B). NS5B proteins of different HCV genotypes/subtypes (1a, 1b, 2a, 2c, 5a) showed varied affinity for these mAbs. Interestingly, the epitopes for the mAbs were mapped to the palm subdomain (amino acid 188-370) of the HCV RdRp as determined by immunoblotting analysis of a panel of HCV/GBV-B chimeric NS5B proteins. The binding site was mapped between amino acid 231 and 267 of NS5B for 16A9C9, and between 282 and 372 for 16D9A4 and 20A12C7. Furthermore, these mAbs showed no inhibitory effect on the NS5B polymerase activity in vitro.

  4. Heterologous prime-boost strategy in non-human primates combining the infective dengue virus and a recombinant protein in a formulation suitable for human use.

    Science.gov (United States)

    Valdés, Iris; Hermida, Lisset; Gil, Lázaro; Lazo, Laura; Castro, Jorge; Martín, Jorge; Bernardo, Lídice; López, Carlos; Niebla, Olivia; Menéndez, Tamara; Romero, Yaremis; Sánchez, Jorge; Guzmán, María G; Guillén, Gerardo

    2010-05-01

    The aim of the present work was to test the concept of the heterologous prime-boost strategy combining an infective dengue virus with a recombinant chimeric protein carrying domain III of the envelope protein. Two studies in monkeys, combining recombinant protein PD5 (domain III of the envelope protein from dengue-2 virus, fused to the protein carrier P64k) and the infective dengue virus in the same immunization schedules were carried out. Humoral and cell-mediated immunity were evaluated. In the first study, monkeys received four doses of the protein PD5 and were subsequently infected with one dose of dengue virus. Antibody response measured after virus inoculation was significantly higher compared to that in non-primed monkeys and comparable to that elicited after two doses of infective virus. In a second study, monkeys were infected with one dose of the virus and subsequently boosted with one dose of the recombinant protein, reaching high levels of neutralizing antibodies, which were still detectable 14 months after the last immunization. In addition, the cellular immune response was also recalled. The results obtained in the present work support the approach of heterologous prime-boosting, in either order prime or boost, combining the chimeric protein PD5 (formulated in alum-CPS-A) and an infective dengue virus. The latter could potentially be replaced by an attenuated vaccine candidate. Copyright 2009 International Society for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

  5. Relation of circulating concentrations of chemokine receptor CCR5 ligands to C-peptide, proinsulin and HbA1c and disease progression in type 1 diabetes

    DEFF Research Database (Denmark)

    Pfleger, C; Kaas, A; Hansen, L

    2008-01-01

    Th1 related chemokines CCL3 and CCL5 and Th2 related CCL4 as ligands of the receptor CCR5 contribute to disease development in animal models of type 1 diabetes. In humans, no data are available addressing the role of these chemokines regarding disease progression and remission. We investigated lo...

  6. Vif of Feline Immunodeficiency Virus from Domestic Cats Protects against APOBEC3 Restriction Factors from Many Felids▿

    Science.gov (United States)

    Zielonka, Jörg; Marino, Daniela; Hofmann, Henning; Yuhki, Naoya; Löchelt, Martin; Münk, Carsten

    2010-01-01

    To get more insight into the role of APOBEC3 (A3) cytidine deaminases in the species-specific restriction of feline immunodeficiency virus (FIV) of the domestic cat, we tested the A3 proteins present in big cats (puma, lion, tiger, and lynx). These A3 proteins were analyzed for expression and sensitivity to the Vif protein of FIV. While A3Z3s and A3Z2-Z3s inhibited Δvif FIV, felid A3Z2s did not show any antiviral activity against Δvif FIV or wild-type (wt) FIV. All felid A3Z3s and A3Z2-Z3s were sensitive to Vif of the domestic cat FIV. Vif also induced depletion of felid A3Z2s. Tiger A3s showed a moderate degree of resistance against the Vif-mediated counter defense. These findings may imply that the A3 restriction system does not play a major role to prevent domestic cat FIV transmission to other Felidae. In contrast to the sensitive felid A3s, many nonfelid A3s actively restricted wt FIV replication. To test whether VifFIV can protect also the distantly related human immunodeficiency virus type 1 (HIV-1), a chimeric HIV-1.VifFIV was constructed. This HIV-1.VifFIV was replication competent in nonpermissive feline cells expressing human CD4/CCR5 that did not support the replication of wt HIV-1. We conclude that the replication of HIV-1 in some feline cells is inhibited only by feline A3 restriction factors and the absence of the appropriate receptor or coreceptor. PMID:20444897

  7. Vif of feline immunodeficiency virus from domestic cats protects against APOBEC3 restriction factors from many felids.

    Science.gov (United States)

    Zielonka, Jörg; Marino, Daniela; Hofmann, Henning; Yuhki, Naoya; Löchelt, Martin; Münk, Carsten

    2010-07-01

    To get more insight into the role of APOBEC3 (A3) cytidine deaminases in the species-specific restriction of feline immunodeficiency virus (FIV) of the domestic cat, we tested the A3 proteins present in big cats (puma, lion, tiger, and lynx). These A3 proteins were analyzed for expression and sensitivity to the Vif protein of FIV. While A3Z3s and A3Z2-Z3s inhibited Deltavif FIV, felid A3Z2s did not show any antiviral activity against Deltavif FIV or wild-type (wt) FIV. All felid A3Z3s and A3Z2-Z3s were sensitive to Vif of the domestic cat FIV. Vif also induced depletion of felid A3Z2s. Tiger A3s showed a moderate degree of resistance against the Vif-mediated counter defense. These findings may imply that the A3 restriction system does not play a major role to prevent domestic cat FIV transmission to other Felidae. In contrast to the sensitive felid A3s, many nonfelid A3s actively restricted wt FIV replication. To test whether Vif(FIV) can protect also the distantly related human immunodeficiency virus type 1 (HIV-1), a chimeric HIV-1.Vif(FIV) was constructed. This HIV-1.Vif(FIV) was replication competent in nonpermissive feline cells expressing human CD4/CCR5 that did not support the replication of wt HIV-1. We conclude that the replication of HIV-1 in some feline cells is inhibited only by feline A3 restriction factors and the absence of the appropriate receptor or coreceptor.

  8. Molecular interaction of a potent nonpeptide agonist with the chemokine receptor CCR8

    DEFF Research Database (Denmark)

    Jensen, Pia C; Nygaard, Rie; Thiele, Stefanie

    2007-01-01

    Most nonpeptide antagonists for CC-chemokine receptors share a common pharmacophore with a centrally located, positively charged amine that interacts with the highly conserved glutamic acid (Glu) located in position 6 of transmembrane helix VII (VII:06). We present a novel CCR8 nonpeptide agonist......, 8-[3-(2-methoxyphenoxy)benzyl]-1-phenethyl-1,3,8-triaza-spiro[4.5]decan-4-one (LMD-009), that also contains a centrally located, positively charged amine. LMD-009 selectively stimulated CCR8 among the 20 identified human chemokine receptors. It mediated chemotaxis, inositol phosphate accumulation......-binding pockets of CCR8 uncovered that the binding of LMD-009 and of four analogs [2-(1-(3-(2-methoxyphenoxy)benzyl)-4-hydroxypiperidin-4-yl)benzoic acid (LMD-584), N-ethyl-2-4-methoxybenzenesulfonamide (LMD-902), N-(1-(3-(2-methoxyphenoxy)benzyl)piperidin-4-yl)-2-phenyl-4-(pyrrolidin-1yl)butanamide (LMD-268...

  9. Anti-HIV Effect of Liposomes Bearing CXCR4 Receptor Antagonist ...

    African Journals Online (AJOL)

    Keywords: Antagonist, CXCR4, Liposomes, Receptor, Inflammation, HIV. Tropical Journal of ... receptors and inhibits HIV-1 entry mediated through CCR3, CCR5, and ..... circulation, facilitating HIV-targeted drug delivery. By tissue distribution ...

  10. Structural basis for the development of avian virus capsids that display influenza virus proteins and induce protective immunity.

    Science.gov (United States)

    Pascual, Elena; Mata, Carlos P; Gómez-Blanco, Josué; Moreno, Noelia; Bárcena, Juan; Blanco, Esther; Rodríguez-Frandsen, Ariel; Nieto, Amelia; Carrascosa, José L; Castón, José R

    2015-03-01

    Bioengineering of viruses and virus-like particles (VLPs) is a well-established approach in the development of new and improved vaccines against viral and bacterial pathogens. We report here that the capsid of a major avian pathogen, infectious bursal disease virus (IBDV), can accommodate heterologous proteins to induce protective immunity. The structural units of the ~70-nm-diameter T=13 IBDV capsid are trimers of VP2, which is made as a precursor (pVP2). The pVP2 C-terminal domain has an amphipathic α helix that controls VP2 polymorphism. In the absence of the VP3 scaffolding protein, 466-residue pVP2 intermediates bearing this α helix assemble into genuine VLPs only when expressed with an N-terminal His6 tag (the HT-VP2-466 protein). HT-VP2-466 capsids are optimal for protein insertion, as they are large enough (cargo space, ~78,000 nm(3)) and are assembled from a single protein. We explored HT-VP2-466-based chimeric capsids initially using enhanced green fluorescent protein (EGFP). The VLP assembly yield was efficient when we coexpressed EGFP-HT-VP2-466 and HT-VP2-466 from two recombinant baculoviruses. The native EGFP structure (~240 copies/virion) was successfully inserted in a functional form, as VLPs were fluorescent, and three-dimensional cryo-electron microscopy showed that the EGFP molecules incorporated at the inner capsid surface. Immunization of mice with purified EGFP-VLPs elicited anti-EGFP antibodies. We also inserted hemagglutinin (HA) and matrix (M2) protein epitopes derived from the mouse-adapted A/PR/8/34 influenza virus and engineered several HA- and M2-derived chimeric capsids. Mice immunized with VLPs containing the HA stalk, an M2 fragment, or both antigens developed full protection against viral challenge. Virus-like particles (VLPs) are multimeric protein cages that mimic the infectious virus capsid and are potential candidates as nonliving vaccines that induce long-lasting protection. Chimeric VLPs can display or include foreign

  11. Design and validation of new genotypic tools for easy and reliable estimation of HIV tropism before using CCR5 antagonists.

    Science.gov (United States)

    Poveda, Eva; Seclén, Eduardo; González, María del Mar; García, Federico; Chueca, Natalia; Aguilera, Antonio; Rodríguez, Jose Javier; González-Lahoz, Juan; Soriano, Vincent

    2009-05-01

    Genotypic tools may allow easier and less expensive estimation of HIV tropism before prescription of CCR5 antagonists compared with the Trofile assay (Monogram Biosciences, South San Francisco, CA, USA). Paired genotypic and Trofile results were compared in plasma samples derived from the maraviroc expanded access programme (EAP) in Europe. A new genotypic approach was built to improve the sensitivity to detect X4 variants based on an optimization of the webPSSM algorithm. Then, the new tool was validated in specimens from patients included in the ALLEGRO trial, a multicentre study conducted in Spain to assess the prevalence of R5 variants in treatment-experienced HIV patients. A total of 266 specimens from the maraviroc EAP were tested. Overall geno/pheno concordance was above 72%. A high specificity was generally seen for the detection of X4 variants using genotypic tools (ranging from 58% to 95%), while sensitivity was low (ranging from 31% to 76%). The PSSM score was then optimized to enhance the sensitivity to detect X4 variants changing the original threshold for R5 categorization. The new PSSM algorithms, PSSM(X4R5-8) and PSSM(SINSI-6.4), considered as X4 all V3 scoring values above -8 or -6.4, respectively, increasing the sensitivity to detect X4 variants up to 80%. The new algorithms were then validated in 148 specimens derived from patients included in the ALLEGRO trial. The sensitivity/specificity to detect X4 variants was 93%/69% for PSSM(X4R5-8) and 93%/70% for PSSM(SINSI-6.4). PSSM(X4R5-8) and PSSM(SINSI-6.4) may confidently assist therapeutic decisions for using CCR5 antagonists in HIV patients, providing an easier and rapid estimation of tropism in clinical samples.

  12. A Bacteriophage-Related Chimeric Marine Virus Infecting Abalone

    Science.gov (United States)

    Zhuang, Jun; Cai, Guiqin; Lin, Qiying; Wu, Zujian; Xie, Lianhui

    2010-01-01

    Marine viruses shape microbial communities with the most genetic diversity in the sea by multiple genetic exchanges and infect multiple marine organisms. Here we provide proof from experimental infection that abalone shriveling syndrome-associated virus (AbSV) can cause abalone shriveling syndrome. This malady produces histological necrosis and abnormally modified macromolecules (hemocyanin and ferritin). The AbSV genome is a 34.952-kilobase circular double-stranded DNA, containing putative genes with similarity to bacteriophages, eukaryotic viruses, bacteria and endosymbionts. Of the 28 predicted open reading frames (ORFs), eight ORF-encoded proteins have identifiable functional homologues. The 4 ORF products correspond to a predicted terminase large subunit and an endonuclease in bacteriophage, and both an integrase and an exonuclease from bacteria. The other four proteins are homologous to an endosymbiont-derived helicase, primase, single-stranded binding (SSB) protein, and thymidylate kinase, individually. Additionally, AbSV exhibits a common gene arrangement similar to the majority of bacteriophages. Unique to AbSV, the viral genome also contains genes associated with bacterial outer membrane proteins and may lack the structural protein-encoding ORFs. Genomic characterization of AbSV indicates that it may represent a transitional form of microbial evolution from viruses to bacteria. PMID:21079776

  13. A bacteriophage-related chimeric marine virus infecting abalone.

    Directory of Open Access Journals (Sweden)

    Jun Zhuang

    Full Text Available Marine viruses shape microbial communities with the most genetic diversity in the sea by multiple genetic exchanges and infect multiple marine organisms. Here we provide proof from experimental infection that abalone shriveling syndrome-associated virus (AbSV can cause abalone shriveling syndrome. This malady produces histological necrosis and abnormally modified macromolecules (hemocyanin and ferritin. The AbSV genome is a 34.952-kilobase circular double-stranded DNA, containing putative genes with similarity to bacteriophages, eukaryotic viruses, bacteria and endosymbionts. Of the 28 predicted open reading frames (ORFs, eight ORF-encoded proteins have identifiable functional homologues. The 4 ORF products correspond to a predicted terminase large subunit and an endonuclease in bacteriophage, and both an integrase and an exonuclease from bacteria. The other four proteins are homologous to an endosymbiont-derived helicase, primase, single-stranded binding (SSB protein, and thymidylate kinase, individually. Additionally, AbSV exhibits a common gene arrangement similar to the majority of bacteriophages. Unique to AbSV, the viral genome also contains genes associated with bacterial outer membrane proteins and may lack the structural protein-encoding ORFs. Genomic characterization of AbSV indicates that it may represent a transitional form of microbial evolution from viruses to bacteria.

  14. Sensitization with vaccinia virus encoding H5N1 hemagglutinin restores immune potential against H5N1 influenza virus.

    Science.gov (United States)

    Yasui, Fumihiko; Itoh, Yasushi; Ikejiri, Ai; Kitabatake, Masahiro; Sakaguchi, Nobuo; Munekata, Keisuke; Shichinohe, Shintaro; Hayashi, Yukiko; Ishigaki, Hirohito; Nakayama, Misako; Sakoda, Yoshihiro; Kida, Hiroshi; Ogasawara, Kazumasa; Kohara, Michinori

    2016-11-28

    H5N1 highly pathogenic avian influenza (H5N1 HPAI) virus causes elevated mortality compared with seasonal influenza viruses like H1N1 pandemic influenza (H1N1 pdm) virus. We identified a mechanism associated with the severe symptoms seen with H5N1 HPAI virus infection. H5N1 HPAI virus infection induced a decrease of dendritic cell number in the splenic extrafollicular T-cell zone and impaired formation of the outer layers of B-cell follicles, resulting in insufficient levels of antibody production after infection. However, in animals vaccinated with a live recombinant vaccinia virus expressing the H5 hemagglutinin, infection with H5N1 HPAI virus induced parafollicular dendritic cell accumulation and efficient antibody production. These results indicate that a recombinant vaccinia encoding H5 hemagglutinin gene does not impair dendritic cell recruitment and can be a useful vaccine candidate.

  15. Activation/proliferation and apoptosis of bystander goat lymphocytes induced by a macrophage-tropic chimeric caprine arthritis encephalitis virus expressing SIV Nef

    International Nuclear Information System (INIS)

    Bouzar, Baya Amel; Rea, Angela; Hoc-Villet, Stephanie; Garnier, Celine; Guiguen, Francois; Jin Yuhuai; Narayan, Opendra; Chebloune, Yahia

    2007-01-01

    Caprine arthritis encephalitis virus (CAEV) is the natural lentivirus of goats, well known for its tropism for macrophages and its inability to cause infection in lymphocytes. The viral genome lacks nef, tat, vpu and vpx coding sequences. To test the hypothesis that when nef is expressed by the viral genome, the virus became toxic for lymphocytes during replication in macrophages, we inserted the SIVsmm PBj14 nef coding sequences into the genome of CAEV thereby generating CAEV-nef. This recombinant virus is not infectious for lymphocytes but is fully replication competent in goat macrophages in which it constitutively expresses the SIV Nef. We found that goat lymphocytes cocultured with CAEV-nef-infected macrophages became activated, showing increased expression of the interleukin-2 receptor (IL-2R). Activation correlated with increased proliferation of the cells. Interestingly, a dual effect in terms of apoptosis regulation was observed in exposed goat lymphocytes. Nef was found first to induce a protection of lymphocytes from apoptosis during the first few days following exposure to infected macrophages, but later it induced increased apoptosis in the activated lymphocytes. This new recombinant virus provides a model to study the functions of Nef in the context of infection of macrophages, but in absence of infection of T lymphocytes and brings new insights into the biological effects of Nef on lymphocytes

  16. Generation of representative primary virus isolates from blood plasma after isolation of HIV-1 with CD44 MicroBeads

    NARCIS (Netherlands)

    Cornelissen, M; Heeregrave, E.J.; Zorgdrager, F.; Pollakis, G.; Paxton, W.A.; van der Kuyl, A.C.

    2010-01-01

    Infection of cell cultures with cell-free virus isolated from HIV-infected patients is notoriously difficult and results in a loss of viral variation. Here, we describe viral sequences from PBMC, U87.CD4.CCR5 and U87.CD4.CXCR4 cell cultures and compare them to those from blood plasma from 12

  17. Establishment of donor Chimerism Using Allogeneic Bone Marrow with AMP Cell Co-infusion

    Science.gov (United States)

    2017-09-01

    AWARD NUMBER: W81XWH-15-1-0234 TITLE: Establishment of donor Chimerism Using Allogeneic Bone Marrow with AMP Cell Co-infusion PRINCIPAL...14/2017 4. TITLE AND SUBTITLE Establishment of donor Chimerism Using Allogeneic Bone Marrow with AMP Cell Co-infusion 5a. CONTRACT NUMBER 5b. GRANT...tolerance induction of all types of allografts. In this study, we investigate whether co-infusion of amnion- derived multipotent progenitor (AMP) cells

  18. Analysis of the relationship between single nucleotide polymorphism of the CD209, IL-10, IL-28 and CCR5 D32 genes with the human predisposition to developing tick-borne encephalitis

    Directory of Open Access Journals (Sweden)

    Piotr Czupryna

    2017-01-01

    Full Text Available Introduction: It is known that in the pathogenesis of tick-borne encephalitis (TBE various molecules play a significant role. The most prominent factors include IL-10, IL-28B, CD-209 and CCR5. It is reasonable to search for genetic predispositions to the development of various clinical forms of TBE related to the genetic variation of IL-10, IL-28B, CD-209 and CCR5. In this study we aimed to search for the relationship between single nucleotide polymorphism in the promoter region of the CD209, IL-10, IL-28 and 32 base pair deletion in CCR5 coding region (Δ 32 with the human predisposition to development of various clinical presentations of TBE. We tried to assess the relation between the presence of particular alleles and genotypes with laboratory and clinical parameters. Material/Methods 59 patients with TBE and 57 people, bitten by a tick who never developed TBE (Polish cohort, were included in the study. To assess the distribution of single nucleotide polymorphisms, TaqMan SNP genotyping assays were used for IL10: rs1800872 and rs1800896, for CD 209 rs4804803 and rs2287886, rs12979860 for IL 28B SNPs according to the manufacturer’s protocol using real-time PCR technology on the StepOne thermal cycler. Results Comparison between TBE patients and CG showed that in SNP rs2287886 CD 209 AG heterozygotes were more frequent in the TBE group, while homozygotes GG were more frequent in the CG group. Conclusions SNP rs2287886 CD 209 AG heterozygotes predispose humans to develop TBE. Single nucleotide polymorphism in the promoter region of the CD209, IL-10, IL-28 and CCR5 D32 genes does not correlate with the severity of TBE.

  19. Chimeric HCMV/HSV-1 and Δγ134.5 oncolytic herpes simplex virus elicit immune mediated antigliomal effect and antitumor memory

    Directory of Open Access Journals (Sweden)

    Mohammed G. Ghonime

    2018-02-01

    Full Text Available Malignant gliomas are the most common primary brain tumor and are characterized by rapid and highly invasive growth. Because of their poor prognosis, new therapeutic strategies are needed. Oncolytic virotherapy (OV is a promising strategy for treating cancer that incorporates both direct viral replication mediated and immune mediated mechanisms to kill tumor cells. C134 is a next generation Δγ134.5 oHSV-1 with improved intratumoral viral replication. It remains safe in the CNS environment by inducing early IFN signaling which restricts its replication in non-malignant cells. We sought to identify how C134 performed in an immunocompetent tumor model that restricts its replication advantage over first generation viruses. To achieve this we identified tumors that have intact IFN signaling responses that restrict C134 and first generation virus replication similarly. Our results show that both viruses elicit a T cell mediated anti-tumor effect and improved animal survival but that subtle difference exist between the viruses effect on median survival despite equivalent in vivo viral replication. To further investigate this we examined the anti-tumor activity in immunodeficient mice and in syngeneic models with re-challenge. These studies show that the T cell response is integral to C134 replication independent anti-tumor response and that OV therapy elicits a durable and circulating anti-tumor memory. The studies also show that repeated intratumoral administration can extend both OV anti-tumor effects and induce durable anti-tumor memory that is superior to tumor antigen exposure alone.

  20. Transmission of chimeric HIV by mating in conventional mice: prevention by pre-exposure antiretroviral therapy and reduced susceptibility during estrus

    Directory of Open Access Journals (Sweden)

    Eran Hadas

    2013-09-01

    Heterosexual transmission accounts for the majority of new human immunodeficiency virus (HIV cases worldwide. The current approach to investigate HIV heterosexual transmission in animals involves application of virus stock to the vaginal surface, a method that does not reproduce the physiological conditions of vaginal intercourse that influence the rate of transmission. We have previously described efficient infection of conventional mice using EcoHIV/NL4-3 and EcoHIV/NDK, chimeric HIV molecular clones constructed to express all HIV structural and regulatory genes except envelope, which is replaced by a rodent-tropic envelope gene. Here we investigated whether EcoHIV/NDK-infected male mice transmit virus to females during coitus, and the sensitivity of this transmission to HIV pre-exposure prophylaxis and the estrus state. Our general approach was to allow mating between EcoHIV/NDK-infected male mice and uninfected females for 1–7 nights. At 1–6 weeks after mating, mice were euthanized and virus burdens were measured by quantitative PCR (qPCR amplification of HIV RNA or DNA in peritoneal macrophages, inguinal lymph node cells, spleen cells or vas deferens, or by ELISA for antibodies to HIV Gag. We found that 70–100% of female mice mated to EcoHIV/NDK-infected males acquired infection. Pericoital treatment of females with either 2′,3′-dideoxcytidine (ddC or tenofovir largely prevented their EcoHIV/NDK infection by mating (P<0.05 and P<0.003, respectively. In males, T cells were dispensable for virus transmission. The rate of EcoHIV/NDK sexual transmission to females in estrus declined sharply (P=0.003 but their infection by injection was unaffected, indicating that the local environment in the female reproductive tract influences susceptibility to HIV. We conclude that this system of EcoHIV/NDK transmission during mouse mating reproduces key features of heterosexual transmission of HIV in humans and can be used to investigate its biology and control.

  1. Proof of activity with AMD11070, an orally bioavailable inhibitor of CXCR4-tropic HIV type 1.

    Science.gov (United States)

    Moyle, Graeme; DeJesus, Edwin; Boffito, Marta; Wong, Rebecca S; Gibney, Colleen; Badel, Karin; MacFarland, Ron; Calandra, Gary; Bridger, Gary; Becker, Stephen

    2009-03-15

    The X4 Antagonist Concept Trial investigates the safety and antiviral activity of AMD11070, a potent inhibitor of X4-tropic human immunodeficiency virus (HIV) in vitro in HIV-infected patients harboring X4-tropic virus. Patients enrolled in the study had an X4 virus population 2000 relative luminescence units (rlu; by the Monogram Trofile Assay) and an HIV-1 RNA level 5000 copies/mL. Patients received AMD11070 monotherapy for 10 days. Coreceptor tropism, plasma HIV-1 RNA level, and CD4 cell count were measured at study entry, on day 5, and on day 10. Daily predose and serial samples on the last day of treatment were obtained for determination of plasma AMD11070 concentration. Ten patients were given AMD11070 monotherapy (200 mg to 8 patients and 100 mg to 2 patients) twice daily for 10 days. The median baseline CD4 cell count was 160 cells/mm(3), and the median HIV-1 RNA level was 91,447 copies/mL. Four of 9 evaluable patients achieved a reduction in X4 virus population of >or= rlu. The median change in X4 virus population at the end of treatment was -0.22 log(10) rlu (range, -1.90 to 0.23 log(10) rlu). Three of 4 patients who responded to therapy showed a tropism shift from dual- or mixed-tropic viruses to exclusively R5 virus by day 10. There were no drug-related serious adverse events, adverse events of greater than grade 2, or laboratory abnormalities. These results demonstrate the activity of AMD11070, the first oral CXCR4 antagonist, against X4-tropic HIV-1. The drug was well tolerated, with no serious safety concerns. AMD11070 is on clinical hold because of histologic changes to the liver observed in long-term animal studies; additional preclinical safety assessments are pending.

  2. Nonproductive human immunodeficiency virus type 1 infection of human fetal astrocytes: independence from CD4 and major chemokine receptors.

    Science.gov (United States)

    Sabri, F; Tresoldi, E; Di Stefano, M; Polo, S; Monaco, M C; Verani, A; Fiore, J R; Lusso, P; Major, E; Chiodi, F; Scarlatti, G

    1999-11-25

    Human immunodeficiency virus type 1 (HIV-1) infection of the brain is associated with neurological manifestations both in adults and in children. The primary target for HIV-1 infection in the brain is the microglia, but astrocytes can also be infected. We tested 26 primary HIV-1 isolates for their capacity to infect human fetal astrocytes in culture. Eight of these isolates, independent of their biological phenotype and chemokine receptor usage, were able to infect astrocytes. Although no sustained viral replication could be demonstrated, the virus was recovered by coculture with receptive cells such as macrophages or on stimulation with interleukin-1beta. To gain knowledge into the molecular events that regulate attachment and penetration of HIV-1 in astrocytes, we investigated the expression of several chemokine receptors. Fluorocytometry and calcium-mobilization assay did not provide evidence of expression of any of the major HIV-1 coreceptors, including CXCR4, CCR5, CCR3, and CCR2b, as well as the CD4 molecule on the cell surface of human fetal astrocytes. However, mRNA transcripts for CXCR4, CCR5, Bonzo/STRL33/TYMSTR, and APJ were detected by RT-PCR. Furthermore, infection of astrocytes by HIV-1 isolates with different chemokine receptor usage was not inhibited by the chemokines SDF-1beta, RANTES, MIP-1beta, or MCP-1 or by antibodies directed against the third variable region or the CD4 binding site of gp120. These data show that astrocytes can be infected by primary HIV-1 isolates via a mechanism independent of CD4 or major chemokine receptors. Furthermore, astrocytes are potential carriers of latent HIV-1 and on activation may be implicated in spreading the infection to other neighbouring cells, such as microglia or macrophages. Copyright 1999 Academic Press.

  3. Chimeric immune receptors (CIRs) specific to JC virus for immunotherapy in progressive multifocal leukoencephalopathy (PML)

    NARCIS (Netherlands)

    W. Yang; E.L. Beaudoin; L. Lu; R.A. Du Pasquier (Renaud); M.J. Kuroda; R.A. Willemsen (Ralph); I.J. Koralnik; R.P. Junghans

    2007-01-01

    textabstractProgressive multifocal leukoencephalopathy (PML) is a deadly brain disease caused by the polyomavirus JC (JCV). The aim of this study is to develop 'designer T cells' armed with anti-JCV TCR-based chimeric immune receptors (CIRs) by gene modification for PML immunotherapy. Two T cell

  4. The ins and outs of ligand binding to CCR2

    NARCIS (Netherlands)

    Zweemer, Annelien Jacomina Maria

    2014-01-01

    This thesis provides novel insights in the molecular mechanism of action of antagonists for the chemokine receptor CCR2. CCR2 belongs to the protein family of G protein-coupled receptors (GPCRs). It is involved in several inflammatory diseases and therefore many small molecule antagonists targeting

  5. Contemporary avian influenza A virus subtype H1, H6, H7, H10, and H15 hemagglutinin genes encode a mammalian virulence factor similar to the 1918 pandemic virus H1 hemagglutinin.

    Science.gov (United States)

    Qi, Li; Pujanauski, Lindsey M; Davis, A Sally; Schwartzman, Louis M; Chertow, Daniel S; Baxter, David; Scherler, Kelsey; Hartshorn, Kevan L; Slemons, Richard D; Walters, Kathie-Anne; Kash, John C; Taubenberger, Jeffery K

    2014-11-18

    Zoonotic avian influenza virus infections may lead to epidemics or pandemics. The 1918 pandemic influenza virus has an avian influenza virus-like genome, and its H1 hemagglutinin was identified as a key mammalian virulence factor. A chimeric 1918 virus expressing a contemporary avian H1 hemagglutinin, however, displayed murine pathogenicity indistinguishable from that of the 1918 virus. Here, isogenic chimeric avian influenza viruses were constructed on an avian influenza virus backbone, differing only by hemagglutinin subtype expressed. Viruses expressing the avian H1, H6, H7, H10, and H15 subtypes were pathogenic in mice and cytopathic in normal human bronchial epithelial cells, in contrast to H2-, H3-, H5-, H9-, H11-, H13-, H14-, and H16-expressing viruses. Mouse pathogenicity was associated with pulmonary macrophage and neutrophil recruitment. These data suggest that avian influenza virus hemagglutinins H1, H6, H7, H10, and H15 contain inherent mammalian virulence factors and likely share a key virulence property of the 1918 virus. Consequently, zoonotic infections with avian influenza viruses bearing one of these hemagglutinins may cause enhanced disease in mammals. Influenza viruses from birds can cause outbreaks in humans and may contribute to the development of pandemics. The 1918 pandemic influenza virus has an avian influenza virus-like genome, and its main surface protein, an H1 subtype hemagglutinin, was identified as a key mammalian virulence factor. In a previous study, a 1918 virus expressing an avian H1 gene was as virulent in mice as the reconstructed 1918 virus. Here, a set of avian influenza viruses was constructed, differing only by hemagglutinin subtype. Viruses with the avian H1, H6, H7, H10, and H15 subtypes caused severe disease in mice and damaged human lung cells. Consequently, infections with avian influenza viruses bearing one of these hemagglutinins may cause enhanced disease in mammals, and therefore surveillance for human infections

  6. Study progress of CCR3 in wet age-related macular degeneration

    Directory of Open Access Journals (Sweden)

    Xian-Wei Wu

    2014-03-01

    Full Text Available According to the study, chemokine receptor 3(CCR3in the eye is mainly distributed in retinal pigment epithelial cells, and also expressed in the choroidal vascular endothelial cells(CECs. The specificity of CCR3's high expression in wet age-related macular degeneration(AMDwas found, and it is proved that in wet-AMD patients, it plays an important role in the formation of choroidal neovascularization(CNV. In this paper, the structure, function, the problem of current research and the future direction of CCR3 were summarized. It is believed that with the further research on CCR3, it will not only help us to find a new method of wet-AMD diagnosis and treatment, but also may provide an important reference for other CNV disease research and new anti-CNV drugs.

  7. Structure and dynamics of the gp120 V3 loop that confers noncompetitive resistance in R5 HIV-1(JR-FL to maraviroc.

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    Yuzhe Yuan

    Full Text Available Maraviroc, an (HIV-1 entry inhibitor, binds to CCR5 and efficiently prevents R5 human immunodeficiency virus type 1 (HIV-1 from using CCR5 as a coreceptor for entry into CD4(+ cells. However, HIV-1 can elude maraviroc by using the drug-bound form of CCR5 as a coreceptor. This property is known as noncompetitive resistance. HIV-1(V3-M5 derived from HIV-1(JR-FLan is a noncompetitive-resistant virus that contains five mutations (I304V/F312W/T314A/E317D/I318V in the gp120 V3 loop alone. To obtain genetic and structural insights into maraviroc resistance in HIV-1, we performed here mutagenesis and computer-assisted structural study. A series of site-directed mutagenesis experiments demonstrated that combinations of V3 mutations are required for HIV-1(JR-FLan to replicate in the presence of 1 µM maraviroc, and that a T199K mutation in the C2 region increases viral fitness in combination with V3 mutations. Molecular dynamic (MD simulations of the gp120 outer domain V3 loop with or without the five mutations showed that the V3 mutations induced (i changes in V3 configuration on the gp120 outer domain, (ii reduction of an anti-parallel β-sheet in the V3 stem region, (iii reduction in fluctuations of the V3 tip and stem regions, and (iv a shift of the fluctuation site at the V3 base region. These results suggest that the HIV-1 gp120 V3 mutations that confer maraviroc resistance alter structure and dynamics of the V3 loop on the gp120 outer domain, and enable interactions between gp120 and the drug-bound form of CCR5.

  8. Chemokine receptor Ccr1 drives neutrophil-mediated kidney immunopathology and mortality in invasive candidiasis.

    Directory of Open Access Journals (Sweden)

    Michail S Lionakis

    Full Text Available Invasive candidiasis is the 4(th leading cause of nosocomial bloodstream infection in the US with mortality that exceeds 40% despite administration of antifungal therapy; neutropenia is a major risk factor for poor outcome after invasive candidiasis. In a fatal mouse model of invasive candidiasis that mimics human bloodstream-derived invasive candidiasis, the most highly infected organ is the kidney and neutrophils are the major cellular mediators of host defense; however, factors regulating neutrophil recruitment have not been previously defined. Here we show that mice lacking chemokine receptor Ccr1, which is widely expressed on leukocytes, had selectively impaired accumulation of neutrophils in the kidney limited to the late phase of the time course of the model; surprisingly, this was associated with improved renal function and survival without affecting tissue fungal burden. Consistent with this, neutrophils from wild-type mice in blood and kidney switched from Ccr1(lo to Ccr1(high at late time-points post-infection, when Ccr1 ligands were produced at high levels in the kidney and were chemotactic for kidney neutrophils ex vivo. Further, when a 1∶1 mixture of Ccr1(+/+ and Ccr1(-/- donor neutrophils was adoptively transferred intravenously into Candida-infected Ccr1(+/+ recipient mice, neutrophil trafficking into the kidney was significantly skewed toward Ccr1(+/+ cells. Thus, neutrophil Ccr1 amplifies late renal immunopathology and increases mortality in invasive candidiasis by mediating excessive recruitment of neutrophils from the blood to the target organ.

  9. Dengue virus in sub-tropical northern and central Viet Nam: population immunity and climate shape patterns of viral invasion and maintenance.

    Directory of Open Access Journals (Sweden)

    Maia A Rabaa

    Full Text Available Dengue virus transmission occurs in both epidemic and endemic cycles across tropical and sub-tropical regions of the world. Incidence is particularly high in much of Southeast Asia, where hyperendemic transmission plagues both urban and rural populations. However, endemicity has not been established in some areas with climates that may not support year-round viral transmission. An understanding of how dengue viruses (DENV enter these environments and whether the viruses persist in inapparent local transmission cycles is central to understanding how dengue emerges in areas at the margins of endemic transmission. Dengue is highly endemic in tropical southern Vietnam, while increasingly large seasonal epidemics have occurred in northern Viet Nam over the last decade. We have investigated the spread of DENV-1 throughout Vietnam to determine the routes by which the virus enters northern and central regions of the country. Phylogeographic analysis of 1,765 envelope (E gene sequences from Southeast Asia revealed frequent movement of DENV between neighboring human populations and strong local clustering of viral lineages. Long-distance migration of DENV between human population centers also occurred regularly and on short time-scales, indicating human-mediated viral invasion into northern Vietnam. Human populations in southern Vietnam were found to be the primary source of DENV circulating throughout the country, while central and northern Vietnam acted as sink populations, likely due to reduced connectedness to other populations in the case of the central regions and to the influence of temperature variability on DENV replication and vector survival and competence in the north. Finally, phylogeographic analyses suggested that viral movement follows a gravity model and indicates that population immunity and physical and economic connections between populations may play important roles in shaping patterns of DENV transmission.

  10. Secretory overexpression and isotopic labeling of the chimeric relaxin family peptide R3/I5 in Pichia pastoris.

    Science.gov (United States)

    Guo, Yu-Qi; Wu, Qing-Ping; Shao, Xiao-Xia; Shen, Ting; Liu, Ya-Li; Xu, Zeng-Guang; Guo, Zhan-Yun

    2015-06-01

    Relaxin family peptides are a group of peptide hormones with divergent biological functions. Mature relaxin family peptides are typically composed of two polypeptide chains with three disulfide linkages, rendering their preparation a challenging task. In the present study, we established an efficient approach for preparation of the chimeric relaxin family peptide R3/I5 through secretory overexpression in Pichia pastoris and in vitro enzymatic maturation. A designed single-chain R3/I5 precursor containing the B-chain of human relaxin-3 and the A-chain of human INSL5 was overexpressed in PichiaPink strain 1 by high-density fermentation in a two-liter fermenter, and approximately 200 mg of purified precursor was obtained from one liter of the fermentation supernatant. We also developed an economical approach for preparation of the uniformly (15)N-labeled R3/I5 precursor by culturing in shaking flasks, and approximately 15 mg of purified (15)N-labeled precursor was obtained from one liter of the culture supernatant. After purification by cation ion-exchange chromatography and reverse-phase high performance liquid chromatography, the R3/I5 precursor was converted to the mature two-chain form by sequential treatment with endoproteinase Lys-C and carboxypeptidase B. The mature R3/I5 peptide had an α-helix-dominated conformation and retained full receptor-binding and receptor activation activities. Thus, Pichia overexpression was an efficient approach for sample preparation and isotopic labeling of the chimeric R3/I5 peptide. This approach could also be extended to the preparation of other relaxin family peptides in future studies.

  11. The Not5 subunit of the ccr4-not complex connects transcription and translation.

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    Zoltan Villanyi

    2014-10-01

    Full Text Available Recent studies have suggested that a sub-complex of RNA polymerase II composed of Rpb4 and Rpb7 couples the nuclear and cytoplasmic stages of gene expression by associating with newly made mRNAs in the nucleus, and contributing to their translation and degradation in the cytoplasm. Here we show by yeast two hybrid and co-immunoprecipitation experiments, followed by ribosome fractionation and fluorescent microscopy, that a subunit of the Ccr4-Not complex, Not5, is essential in the nucleus for the cytoplasmic functions of Rpb4. Not5 interacts with Rpb4; it is required for the presence of Rpb4 in polysomes, for interaction of Rpb4 with the translation initiation factor eIF3 and for association of Rpb4 with mRNAs. We find that Rpb7 presence in the cytoplasm and polysomes is much less significant than that of Rpb4, and that it does not depend upon Not5. Hence Not5-dependence unlinks the cytoplasmic functions of Rpb4 and Rpb7. We additionally determine with RNA immunoprecipitation and native gel analysis that Not5 is needed in the cytoplasm for the co-translational assembly of RNA polymerase II. This stems from the importance of Not5 for the association of the R2TP Hsp90 co-chaperone with polysomes translating RPB1 mRNA to protect newly synthesized Rpb1 from aggregation. Hence taken together our results show that Not5 interconnects translation and transcription.

  12. [Novel echogenic needle for ultrasound-guided peripheral nerve block "Hakko type CCR"].

    Science.gov (United States)

    Takayama, Wataru; Yasumura, Rie; Kaneko, Takehiko; Kobayashi, Yoshiro; Kamada, Takaaki; Yoshikawa, Tamotsu; Aoyama, Yasuhiko

    2009-04-01

    A novel echogenic insulated nerve block needle (CCR-needle: Echogenic Needle Type CCR; Hakko, Japan) is commercially available since 2006 in Japan. This needle has three echogenic dimples, namely corner cube reflectors (CCR) on its tip. The CCR-needle will potentially provide a significant advantage for detecting the needle tip. In this report, we firstly evaluated this new disposable echogenic needle in simulation phantom, and demonstrated improved visibility of the needle tip. Afterwards, an interscalene brachial plexus block was performed on a male patient undergoing shoulder surgery. The needle insertion procedure was the "out of plane" ultrasound-guided technique using simultaneous electrical nerve stimulation. The surgery was successfully conducted without any complications.

  13. Incorporation of chimeric HIV-SIV-Env and modified HIV-Env proteins into HIV pseudovirions

    International Nuclear Information System (INIS)

    Devitt, Gerard; Emerson, Vanessa; Holtkotte, Denise; Pfeiffer, Tanya; Pisch, Thorsten; Bosch, Valerie

    2007-01-01

    Low level incorporation of the viral glycoprotein (Env) into human immunodeficiency virus (HIV) particles is a major drawback for vaccine strategies against HIV/AIDS in which HIV particles are used as immunogen. Within this study, we have examined two strategies aimed at achieving higher levels of Env incorporation into non-infectious pseudovirions (PVs). First, we have generated chimeric HIV/SIV Env proteins containing the truncated C-terminal tail region of simian immunodeficiency virus (SIV)mac239-Env767 stop , which mediates strongly increased incorporation of SIV-Env into SIV particles. In a second strategy, we have employed a truncated HIV-Env protein (Env-Tr752 N750K ) which we have previously demonstrated to be incorporated into HIV virions, generated in infected T-cells, to a higher level than that of Wt-HIV-Env. Although the chimeric HIV/SIV Env proteins were expressed at the cell surface and induced increased levels of cell-cell fusion in comparison to Wt-HIV-Env, they did not exhibit increased incorporation into either HIV-PVs or SIV-PVs. Only Env-Tr752 N750K exhibited significantly higher (threefold) levels of incorporation into HIV-PVs, an improvement, which, although not dramatic, is worthwhile for the large-scale preparation of non-infectious PVs for vaccine studies aimed at inducing Env humoral responses

  14. Protective and immunological behavior of chimeric yellow fever dengue vaccine.

    Science.gov (United States)

    Halstead, Scott B; Russell, Philip K

    2016-03-29

    Clinical observations from the third year of the Sanofi Pasteur chimeric yellow fever dengue tetravalent vaccine (CYD) trials document both protection and vaccination-enhanced dengue disease among vaccine recipients. Children who were 5 years-old or younger when vaccinated experienced a DENV disease resulting in hospitalization at 5 times the rate of controls. On closer inspection, hospitalized cases among vaccinated seropositives, those at highest risk to hospitalized disease accompanying a dengue virus (DENV) infection, were greatly reduced by vaccination. But, seronegative individuals of all ages after being vaccinated were only modestly protected from mild to moderate disease throughout the entire observation period despite developing neutralizing antibodies at high rates. Applying a simple epidemiological model to the data, vaccinated seronegative individuals of all ages were at increased risk of developing hospitalized disease during a subsequent wild type DENV infection. The etiology of disease in placebo and vaccinated children resulting in hospitalization during a DENV infection, while clinically similar are of different origin. The implications of the observed mixture of DENV protection and enhanced disease in CYD vaccinees are discussed. Copyright © 2016 Elsevier Ltd. All rights reserved.

  15. Antigenic properties of a transport-competent influenza HA/HIV Env chimeric protein

    International Nuclear Information System (INIS)

    Ye Ling; Sun Yuliang; Lin Jianguo; Bu Zhigao; Wu Qingyang; Jiang, Shibo; Steinhauer, David A.; Compans, Richard W.; Yang Chinglai

    2006-01-01

    The transmembrane subunit (gp41) of the HIV Env glycoprotein contains conserved neutralizing epitopes which are not well-exposed in wild-type HIV Env proteins. To enhance the exposure of these epitopes, a chimeric protein, HA/gp41, in which the gp41 of HIV-1 89.6 envelope protein was fused to the C-terminus of the HA1 subunit of the influenza HA protein, was constructed. Characterization of protein expression showed that the HA/gp41 chimeric proteins were expressed on cell surfaces and formed trimeric oligomers, as found in the HIV Env as well as influenza HA proteins. In addition, the HA/gp41 chimeric protein expressed on the cell surface can also be cleaved into 2 subunits by trypsin treatment, similar to the influenza HA. Moreover, the HA/gp41 chimeric protein was found to maintain a pre-fusion conformation. Interestingly, the HA/gp41 chimeric proteins on cell surfaces exhibited increased reactivity to monoclonal antibodies against the HIV Env gp41 subunit compared with the HIV-1 envelope protein, including the two broadly neutralizing monoclonal antibodies 2F5 and 4E10. Immunization of mice with a DNA vaccine expressing the HA/gp41 chimeric protein induced antibodies against the HIV gp41 protein and these antibodies exhibit neutralizing activity against infection by an HIV SF162 pseudovirus. These results demonstrate that the construction of such chimeric proteins can provide enhanced exposure of conserved epitopes in the HIV Env gp41 and may represent a novel vaccine design strategy for inducing broadly neutralizing antibodies against HIV

  16. Chemokine Receptor Ccr1 Drives Neutrophil-Mediated Kidney Immunopathology and Mortality in Invasive Candidiasis

    Science.gov (United States)

    Lionakis, Michail S.; Swamydas, Muthulekha; Wan, Wuzhou; Richard Lee, Chyi-Chia; Cohen, Jeffrey I.; Scheinberg, Phillip; Gao, Ji-Liang; Murphy, Philip M.

    2012-01-01

    Invasive candidiasis is the 4th leading cause of nosocomial bloodstream infection in the US with mortality that exceeds 40% despite administration of antifungal therapy; neutropenia is a major risk factor for poor outcome after invasive candidiasis. In a fatal mouse model of invasive candidiasis that mimics human bloodstream-derived invasive candidiasis, the most highly infected organ is the kidney and neutrophils are the major cellular mediators of host defense; however, factors regulating neutrophil recruitment have not been previously defined. Here we show that mice lacking chemokine receptor Ccr1, which is widely expressed on leukocytes, had selectively impaired accumulation of neutrophils in the kidney limited to the late phase of the time course of the model; surprisingly, this was associated with improved renal function and survival without affecting tissue fungal burden. Consistent with this, neutrophils from wild-type mice in blood and kidney switched from Ccr1lo to Ccr1high at late time-points post-infection, when Ccr1 ligands were produced at high levels in the kidney and were chemotactic for kidney neutrophils ex vivo. Further, when a 1∶1 mixture of Ccr1+/+ and Ccr1−/− donor neutrophils was adoptively transferred intravenously into Candida-infected Ccr1+/+ recipient mice, neutrophil trafficking into the kidney was significantly skewed toward Ccr1+/+ cells. Thus, neutrophil Ccr1 amplifies late renal immunopathology and increases mortality in invasive candidiasis by mediating excessive recruitment of neutrophils from the blood to the target organ. PMID:22916017

  17. Evidence for Anger Saliency during the Recognition of Chimeric Facial Expressions of Emotions in Underage Ebola Survivors

    Directory of Open Access Journals (Sweden)

    Martina Ardizzi

    2017-06-01

    Full Text Available One of the crucial features defining basic emotions and their prototypical facial expressions is their value for survival. Childhood traumatic experiences affect the effective recognition of facial expressions of negative emotions, normally allowing the recruitment of adequate behavioral responses to environmental threats. Specifically, anger becomes an extraordinarily salient stimulus unbalancing victims’ recognition of negative emotions. Despite the plethora of studies on this topic, to date, it is not clear whether this phenomenon reflects an overall response tendency toward anger recognition or a selective proneness to the salience of specific facial expressive cues of anger after trauma exposure. To address this issue, a group of underage Sierra Leonean Ebola virus disease survivors (mean age 15.40 years, SE 0.35; years of schooling 8.8 years, SE 0.46; 14 males and a control group (mean age 14.55, SE 0.30; years of schooling 8.07 years, SE 0.30, 15 males performed a forced-choice chimeric facial expressions recognition task. The chimeric facial expressions were obtained pairing upper and lower half faces of two different negative emotions (selected from anger, fear and sadness for a total of six different combinations. Overall, results showed that upper facial expressive cues were more salient than lower facial expressive cues. This priority was lost among Ebola virus disease survivors for the chimeric facial expressions of anger. In this case, differently from controls, Ebola virus disease survivors recognized anger regardless of the upper or lower position of the facial expressive cues of this emotion. The present results demonstrate that victims’ performance in the recognition of the facial expression of anger does not reflect an overall response tendency toward anger recognition, but rather the specific greater salience of facial expressive cues of anger. Furthermore, the present results show that traumatic experiences deeply modify

  18. Molecular requirements for inhibition of the chemokine receptor CCR8--probe-dependent allosteric interactions

    DEFF Research Database (Denmark)

    Rummel, Pia Cwarzko; Arfelt, K N; Baumann, L

    2012-01-01

    Here we present a novel series of CCR8 antagonists based on a naphthalene-sulfonamide structure. This structure differs from the predominant pharmacophore for most small-molecule CC-chemokine receptor antagonists, which in fact activate CCR8, suggesting that CCR8 inhibition requires alternative...

  19. CCR Magazines | Center for Cancer Research

    Science.gov (United States)

    The Center for Cancer Research (CCR) has two magazines, MILESTONES and LANDMARKS, that highlight our annual advances and top contributions to the understanding, detection, treatment and prevention of cancer over the years.

  20. Blocking spinal CCR2 with AZ889 reversed hyperalgesia in a model of neuropathic pain

    Directory of Open Access Journals (Sweden)

    Vaillancourt François

    2010-12-01

    Full Text Available Abstract Background The CCR2/CCL2 system has been identified as a regulator in the pathogenesis of neuropathy-induced pain. However, CCR2 target validation in analgesia and the mechanism underlying antinociception produced by CCR2 antagonists remains poorly understood. In this study, in vitro and in vivo pharmacological approaches using a novel CCR2 antagonist, AZ889, strengthened the hypothesis of a CCR2 contribution to neuropathic pain and provided confidence over the possibilities to treat neuropathic pain with CCR2 antagonists. Results We provided evidence that dorsal root ganglia (DRG cells harvested from CCI animals responded to stimulation by CCL2 with a concentration-dependent calcium rise involving PLC-dependent internal stores. This response was associated with an increase in evoked neuronal action potentials suggesting these cells were sensitive to CCR2 signalling. Importantly, treatment with AZ889 abolished CCL2-evoked excitation confirming that this activity is CCR2-mediated. Neuronal and non-neuronal cells in the spinal cord were also excited by CCL2 applications indicating an important role of spinal CCR2 in neuropathic pain. We next showed that in vivo spinal intrathecal injection of AZ889 produced dose-dependent analgesia in CCI rats. Additionally, application of AZ889 to the exposed spinal cord inhibited evoked neuronal activity and confirmed that CCR2-mediated analgesia involved predominantly the spinal cord. Furthermore, AZ889 abolished NMDA-dependent wind-up of spinal withdrawal reflex pathway in neuropathic animals giving insight into the spinal mechanism underlying the analgesic properties of AZ889. Conclusions Overall, this study strengthens the important role of CCR2 in neuropathic pain and highlights feasibility that interfering on this mechanism at the spinal level with a selective antagonist can provide new analgesia opportunities.

  1. Pulmonary CCR2+CD4+ T cells are immune regulatory and attenuate lung fibrosis development.

    Science.gov (United States)

    Milger, Katrin; Yu, Yingyan; Brudy, Eva; Irmler, Martin; Skapenko, Alla; Mayinger, Michael; Lehmann, Mareike; Beckers, Johannes; Reichenberger, Frank; Behr, Jürgen; Eickelberg, Oliver; Königshoff, Melanie; Krauss-Etschmann, Susanne

    2017-11-01

    Animal models have suggested that CCR2-dependent signalling contributes to the pathogenesis of pulmonary fibrosis, but global blockade of CCL2 failed to improve the clinical course of patients with lung fibrosis. However, as levels of CCR2 + CD4 + T cells in paediatric lung fibrosis had previously been found to be increased, correlating with clinical symptoms, we hypothesised that distinct CCR2 + cell populations might either increase or decrease disease pathogenesis depending on their subtype. To investigate the role of CCR2 + CD4 + T cells in experimental lung fibrosis and in patients with idiopathic pulmonary fibrosis and other fibrosis. Pulmonary CCR2 + CD4 + T cells were analysed using flow cytometry and mRNA profiling, followed by in silico pathway analysis, in vitro assays and adoptive transfer experiments. Frequencies of CCR2 + CD4 + T cells were increased in experimental fibrosis-specifically the CD62L - CD44 + effector memory T cell phenotype, displaying a distinct chemokine receptor profile. mRNA profiling of isolated CCR2 + CD4 + T cells from fibrotic lungs suggested immune regulatory functions, a finding that was confirmed in vitro using suppressor assays. Importantly, adoptive transfer of CCR2 + CD4 + T cells attenuated fibrosis development. The results were partly corroborated in patients with lung fibrosis, by showing higher percentages of Foxp3 + CD25 + cells within bronchoalveolar lavage fluid CCR2 + CD4 + T cells as compared with CCR2 - CD4 + T cells. Pulmonary CCR2 + CD4 + T cells are immunosuppressive, and could attenuate lung inflammation and fibrosis. Therapeutic strategies completely abrogating CCR2-dependent signalling will therefore also eliminate cell populations with protective roles in fibrotic lung disease. This emphasises the need for a detailed understanding of the functions of immune cell subsets in fibrotic lung disease. © Article author(s) (or their employer(s) unless otherwise stated in the text of the article) 2017. All rights

  2. Capsid coding sequences of foot-and-mouth disease viruses are determinants of pathogenicity in pigs.

    Science.gov (United States)

    Lohse, Louise; Jackson, Terry; Bøtner, Anette; Belsham, Graham J

    2012-05-24

    The surface exposed capsid proteins, VP1, VP2 and VP3, of foot-and-mouth disease virus (FMDV) determine its antigenicity and the ability of the virus to interact with host-cell receptors. Hence, modification of these structural proteins may alter the properties of the virus.In the present study we compared the pathogenicity of different FMDVs in young pigs. In total 32 pigs, 7-weeks-old, were exposed to virus, either by direct inoculation or through contact with inoculated pigs, using cell culture adapted (O1K B64), chimeric (O1K/A-TUR and O1K/O-UKG) or field strain (O-UKG/34/2001) viruses. The O1K B64 virus and the two chimeric viruses are identical to each other except for the capsid coding region.Animals exposed to O1K B64 did not exhibit signs of disease, while pigs exposed to each of the other viruses showed typical clinical signs of foot-and-mouth disease (FMD). All pigs infected with the O1K/O-UKG chimera or the field strain (O-UKG/34/2001) developed fulminant disease. Furthermore, 3 of 4 in-contact pigs exposed to the O1K/O-UKG virus died in the acute phase of infection, likely from myocardial infection. However, in the group exposed to the O1K/A-TUR chimeric virus, only 1 pig showed symptoms of disease within the time frame of the experiment (10 days). All pigs that developed clinical disease showed a high level of viral RNA in serum and infected pigs that survived the acute phase of infection developed a serotype specific antibody response. It is concluded that the capsid coding sequences are determinants of FMDV pathogenicity in pigs.

  3. Serum concentrations of chemokines (CCL-5 and CXCL-12), chemokine receptors (CCR-5 and CXCR-4), and IL-6 in patients with posttraumatic stress disorder and avoidant personality disorder.

    Science.gov (United States)

    Ogłodek, Ewa A; Szota, Anna M; Moś, Danuta M; Araszkiewicz, Aleksander; Szromek, Adam R

    2015-12-01

    Posttraumatic stress disorder (PTSD) can be perceived as a psychoneuroimmunological disorder in which cytokines affecting the neurochemical and neuroendocrine functions of the body play an important role. Among cytokines, chemokines participating in activation of the inflammatory response are considered to be crucial. 220 men and women were enrolled in the study. 180 of them constituted the study group. The studied groups consisted of: 60 patients with a diagnosed avoidant personality disorders (APD), 60 patients with a diagnosed APD and with PTSD and of 60 patients with PTSD but without a APD. There were 30 women and 30 men in each group of 60 subjects. The control group consisted of 40 healthy individuals. The plasma levels of chemokines and their receptors (CCL-5, CXCR-5, CXCL-12 and CXCR-4), as well as IL-6, were assessed by ELISA. There was an increase in the CXCL-12 and CCL-5 levels in women and men with the PTSD versus the control group. Also, increased levels of IL-6 and the receptors CXCR-4, CCR-5 were observed in women and men with PTSD. The levels of CXCL-12 and CCL-5 chemokines, as well as CCR-5 and CXCR4 receptors were higher in women than in men. The results of this study indicate a need for assessment of the CCL-5 and CXCL-12 chemokine levels, as they are likely markers of PTSD. Measurement of the concentrations of chemokines, chemokine receptors and IL-6 in women and men with PTSD along with concomittant APD may be useful for early detection of mental disorders. Copyright © 2015 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

  4. The chemokine receptor CCR2 maintains plasmacytoid dendritic cell homeostasis

    DEFF Research Database (Denmark)

    Cédile, Oriane; Østerby Jørgensen, Line; Frank, Ida

    2017-01-01

    Thymic dendritic cells (DC) play a role in central tolerance. Three thymic DC subtypes have been described: plasmacytoid DC (pDC) and two conventional DC (cDC), CD8α+ Sirpα- DC and Sirpα+ CD8α- cDC. Both pDC and Sirpα+ cDC can take up antigen in periphery and migrate into the thymus in response t...... by CCL2 or CCR2 deficiency. Although some thymic progenitors expressed CCR2, this did not include those that give rise to pDC. Based on these results, we propose that CCR2 is involved in pDC homeostasis but its ligand CCL2 does not play a major role....

  5. Characterization of Vesicular Stomatitis Virus Recombinants That Express and Incorporate High Levels of Hepatitis C Virus Glycoproteins

    OpenAIRE

    Buonocore, Linda; Blight, Keril J.; Rice, Charles M.; Rose, John K.

    2002-01-01

    We generated recombinant vesicular stomatitis viruses (VSV) expressing genes encoding hybrid proteins consisting of the extracellular domains of hepatitis C virus (HCV) glycoproteins fused at different positions to the transmembrane and cytoplasmic domains of the VSV G glycoprotein (E1G and E2G). We show that these chimeric proteins are transported to the cell surface and incorporated into VSV virions efficiently. We also generated VSV recombinants in which the gene encoding the VSV G protein...

  6. DISTRIBUTION OF CCR2-64I GENE AMONG THE TRIBES AND CASTE POPULATION OF VIDARBHA, INDIA.

    Directory of Open Access Journals (Sweden)

    Arvind B Chavhan

    2013-08-01

    Results: The genotyping for the CCR2-64I mutation among the selected tribe and a caste reveal that all of the tribes and a caste was found to be heterozygous for the CCR2-64I mutation. Among the tribes Gonds showed highest genotype frequency (29.28% and (11.76% for heterozygous (CCR2/64I and Homozygous (64I/64I respectively, having an allelic frequency (0.233. A pooled allelic frequencies of the wild-type allele CCR2 and CCR2 64I the variant were found to be 0.854 and 0.146, respectively. No significant deviations from the HWE were observed for tribes and a caste population for the CCR2- 64I mutant χ2=2.76. The study reports the presence of mutant CCR2- 64I gene in tribes and caste population from Vidarbha region.

  7. Study of brain atrophy using X-ray computed tomography. Measurement of CSF space-cranial cavity ratio (CCR)

    Energy Technology Data Exchange (ETDEWEB)

    Kawabata, Masayoshi

    1987-04-01

    Cerebrospinal fluid space-cranial cavity ratio (CCR) of 811 subjects with no brain damage were investigated using X-ray computed tomography. Brain volume of healthy adults aged 20 - 59 years was almost constant and decreased gradually after 60 years. CCR of men aged 20 - 49 years kept constant value and increased with aging after 50 years. CCR of women aged 20 - 59 years kept equal value and CCR increased with aging after 60 years. Brain atrophy with aging was investigated in this study also. In retrospective study, CCR of patients in any age diagnosed brain atrophy in daily CT reports were beyond the normal range of CCR of healthy subjects aged 20 - 49 years. In 48 patients with Parkinson's disease, almost of CCR of them were included within normal range of CCR in age-matched control.

  8. Expression pattern of Ccr2 and Cx3cr1 in inherited retinal degeneration.

    Science.gov (United States)

    Kohno, Hideo; Koso, Hideto; Okano, Kiichiro; Sundermeier, Thomas R; Saito, Saburo; Watanabe, Sumiko; Tsuneoka, Hiroshi; Sakai, Tsutomu

    2015-10-12

    Though accumulating evidence suggests that microglia, resident macrophages in the retina, and bone marrow-derived macrophages can cause retinal inflammation which accelerates photoreceptor cell death, the details of how these cells are activated during retinal degeneration (RD) remain uncertain. Therefore, it is important to clarify which cells play a dominant role in fueling retinal inflammation. However, distinguishing between microglia and macrophages is difficult using conventional techniques such as cell markers (e.g., Iba-1). Recently, two mouse models for visualizing chemokine receptors were established, Cx3cr1 (GFP/GFP) and Ccr2 (RFP/RFP) mice. As Cx3cr1 is expressed in microglia and Ccr2 is reportedly expressed in activated macrophages, these mice have the potential to distinguish microglia and macrophages, yielding novel information about the activation of these inflammatory cells and their individual roles in retinal inflammation. In this study, c-mer proto-oncogene tyrosine kinase (Mertk) (-/-) mice, which show photoreceptor cell death due to defective retinal pigment epithelium phagocytosis, were employed as an animal model of RD. Mertk (-/-) Cx3cr1 (GFP/+) Ccr2 (RFP/+) mice were established by breeding Mertk (-/-) , Cx3cr1 (GFP/GFP) , and Ccr2 (RFP/RFP) mice. The retinal morphology and pattern of inflammatory cell activation and invasion of Mertk (-/-) Cx3cr1 (GFP/+) Ccr2 (RFP/+) mice were evaluated using retina and retinal pigment epithelium (RPE) flat mounts, retinal sections, and flow cytometry. Four-week-old Mertk (-/-) Cx3cr1 (GFP/+) Ccr2 (RFP/+) mice showed Cx3cr1-GFP-positive microglia in the inner retina. Cx3cr1-GFP and Ccr2-RFP dual positive activated microglia were observed in the outer retina and subretinal space of 6- and 8-week-old animals. Ccr2-RFP single positive bone marrow-derived macrophages were observed to migrate into the retina of Mertk (-/-) Cx3cr1 (GFP/+) Ccr2 (RFP/+) mice. These invading cells were still observed in the

  9. Demonstration of Hepatitis C Virus RNA with In Situ Hybridization Employing a Locked Nucleic Acid Probe in Humanized Liver of Infected Chimeric Mice and in Needle-Biopsied Human Liver

    Directory of Open Access Journals (Sweden)

    Kazuya Shiogama

    2013-01-01

    Full Text Available Background. In situ hybridization (ISH with high sensitivity has been requested to demonstrate hepatitis C virus (HCV RNA in formalin-fixed, paraffin-embedded (FFPE sections of the liver. Methods. ISH employing a locked-nucleic-acid- (LNA-modified oligonucleotide probe and biotin-free catalyzed signal amplification system (CSAII was applied to HCV-RNA detection in the liver tissue. Nested reverse-transcription polymerase chain reaction (RT-PCR was performed for HCV genotyping using total RNA extracted from FFPE sections. The target tissues included FFPE tissue sections of humanized livers in HCV-infected chimeric mice (HCV genotypes 1a, 1b, and 2a and noninfected and of needle-biopsied livers from HCV-infected patients. Results. HCV-RNA was demonstrated with the ISH technique in HCV-infected liver tissues from both chimeric mice and 9 (82% of 11 patients with HCV infection. The HCV signals were sensitive to RNase. Nested RT-PCR confirmed the genotype in 8 (73% of 11 livers (type 1b: 6 lesions and type 2a: 2 lesions. HCV-RNA was not identified in chronic hepatitis B lesions, fatty liver, autoimmune hepatitis, and hepatocellular carcinoma. Conclusion. ISH using the LNA-modified oligonucleotide probe and CSAII was applicable to detecting HCV-RNA in routinely prepared FFPE liver specimens.

  10. Functional interaction between the N- and C-terminal domains of murine leukemia virus surface envelope protein

    International Nuclear Information System (INIS)

    Lu, C.-W.; Roth, Monica J.

    2003-01-01

    A series of murine leukemia viruses (MuLVs) with chimeric envelope proteins (Env) was generated to map functional interactions between the N- and the C-terminal domains of surface proteins (SU). All these chimeras have the 4070A amphotropic receptor-binding region flanked by various lengths of Moloney ecotropic N- and C-terminal Env. A charged residue, E49 (E16 on the mature protein), was identified at the N-terminals of Moloney MuLV SU that is important for the interaction with the C-terminal domain of the SU. The region that interacts with E49 was localized between junction 4 (R265 of M-MuLV Env) and junction 6 (L374 of M-MuLV Env) of SU. Sequencing the viable chimeric Env virus populations identified residues within the SU protein that improved the replication kinetics of the input chimeric Env viruses. Mutations in the C-domain of SU (G387E/R, L435I, L442P) were found to improve chimera IV4, which displayed a delayed onset of replication. The replication of AE6, containing a chimeric junction in the SU C-terminus, was improved by mutations in the N-domain (N40H, E80K), the proline-rich region (Q252R), or the transmembrane protein (L538N). Altogether, these observations provide insights into the structural elements required for Env function

  11. Immunogenicity evaluation of MS2 phage-mediated chimeric nanoparticle displaying an immunodominant B cell epitope of foot-and-mouth disease virus

    Directory of Open Access Journals (Sweden)

    Guoqiang Wang

    2018-05-01

    Full Text Available Foot-and-mouth disease (FMD is a highly contagious disease of cloven-hoofed animals that has caused tremendous economic losses worldwide. In this study, we designed a chimeric nanoparticles (CNPs vaccine that displays the predominant epitope of the serotype O foot-and-mouth disease virus (FMDV VP1 131-160 on the surface of MS2 phage. The recombinant protein was expressed in Escherichia Coli and can self-assemble into CNPs with diameter at 25–30 nm in vitro. A tandem repeat peptide epitopes (TRE was prepared as control. Mice were immunized with CNPs, TRE and commercialized synthetic peptide vaccines (PepVac, respectively. The ELISA results showed that CNPs stimulated a little higher specific antibody levels to PepVac, but was significantly higher than the TRE groups. Moreover, the results from specific IFN-γ responses and lymphocyte proliferation test indicated that CNP immunized mice exhibited significantly enhanced cellular immune response compared to TRE. These results suggested that the CNPs constructed in current study could be a potential alternative vaccine in future FMDV control.

  12. Property of hepatitis B virus replication in Tupaia belangeri hepatocytes

    International Nuclear Information System (INIS)

    Sanada, Takahiro; Tsukiyama-Kohara, Kyoko; Yamamoto, Naoki; Ezzikouri, Sayeh; Benjelloun, Soumaya; Murakami, Shuko; Tanaka, Yasuhito; Tateno, Chise; Kohara, Michinori

    2016-01-01

    The northern treeshrew (Tupaia belangeri) has been reported to be an effective candidate for animal infection model with hepatitis B virus (HBV). The objective of our study was to analyze the growth characteristics of HBV in tupaia hepatocytes and the host response to HBV infection. We established primary tupaia hepatocytes (3–6-week old tupaia) and infected them with HBV genotypes A, B and C, and all the genotypes proliferated as well as those in human primary hepatocytes (>10"5 copies/ml in culture supernatant). We next generated a chimeric mouse with tupaia liver by transplantation of tupaia primary hepatocytes to urokinase-type plasminogen activator cDNA (cDNA-uPA)/severe combined immunodeficient (SCID) mice and the replacement ratio with tupaia hepatocytes was found to be more than 95%. Infection of chimeric mice with HBV (genotypes B, C, and D) resulted in HBV-DNA level of 10"4-10"6 copies/ml after 8 weeks of infection, which were almost similar to that in humanized chimeric mouse. In contrast, serum HBV level in adult tupaia (1-year-old tupaia) was quite low (<10"3 copies/ml). Understanding the differences in the response to HBV infection in primary tupaia hepatocytes, chimeric mouse, and adult tupaia will contribute to elucidating the mechanism of persistent HBV infection and viral eradication. Thus, T. belangeri was found to be efficient for studying the host response to HBV infection, thereby providing novel insight into the pathogenesis of HBV. - Highlights: • Primary hepatocytes were established from tupaia that is a novel HBV infection model. • Tupaia primary hepatocytes were susceptible for HBV infection. • The immunodeficient chimeric mice with tupaia hepatocytes were established. • The chimeric mice with tupaia hepatocytes were susceptible for HBV infection.

  13. Property of hepatitis B virus replication in Tupaia belangeri hepatocytes

    Energy Technology Data Exchange (ETDEWEB)

    Sanada, Takahiro [Department of Microbiology and Cell Biology, Tokyo Metropolitan Institute of Medical Science, 2-1-6, Kamikitazawa, Setagaya-ku, Tokyo 156-8506 (Japan); Tsukiyama-Kohara, Kyoko, E-mail: kkohara@vet.kagoshima-u.ac.jp [Transboundary Animal Diseases Centre, Joint Faculty of Veterinary Medicine, Kagoshima University, 1-21-24, Korimoto, Kagoshima-city, Kagoshima 890-0065 (Japan); Laboratory of Animal Hygiene, Joint Faculty of Veterinary Medicine, Kagoshima University, 1-21-24, Korimoto, Kagoshima, Kagoshima 890-0065 (Japan); Yamamoto, Naoki [Department of Microbiology and Cell Biology, Tokyo Metropolitan Institute of Medical Science, 2-1-6, Kamikitazawa, Setagaya-ku, Tokyo 156-8506 (Japan); Ezzikouri, Sayeh; Benjelloun, Soumaya [Viral Hepatitis Laboratory, Virology Unit, Institut Pasteur du Maroc, 1, Louis Pasteur, Casablanca 20360 (Morocco); Murakami, Shuko; Tanaka, Yasuhito [Department of Virology and Liver Unit, Nagoya City University Graduate School of Medical Sciences, Kawasumi 1, Mizuho-ku, Nagoya, Aichi 467-8601 (Japan); Tateno, Chise [PhoenixBio Co. Ltd., 3-4-1, Kagamiyama, Higashi-Hiroshima, Hiroshima 739-0046 (Japan); Kohara, Michinori, E-mail: kohara-mc@igakuken.or.jp [Department of Microbiology and Cell Biology, Tokyo Metropolitan Institute of Medical Science, 2-1-6, Kamikitazawa, Setagaya-ku, Tokyo 156-8506 (Japan)

    2016-01-08

    The northern treeshrew (Tupaia belangeri) has been reported to be an effective candidate for animal infection model with hepatitis B virus (HBV). The objective of our study was to analyze the growth characteristics of HBV in tupaia hepatocytes and the host response to HBV infection. We established primary tupaia hepatocytes (3–6-week old tupaia) and infected them with HBV genotypes A, B and C, and all the genotypes proliferated as well as those in human primary hepatocytes (>10{sup 5} copies/ml in culture supernatant). We next generated a chimeric mouse with tupaia liver by transplantation of tupaia primary hepatocytes to urokinase-type plasminogen activator cDNA (cDNA-uPA)/severe combined immunodeficient (SCID) mice and the replacement ratio with tupaia hepatocytes was found to be more than 95%. Infection of chimeric mice with HBV (genotypes B, C, and D) resulted in HBV-DNA level of 10{sup 4}-10{sup 6} copies/ml after 8 weeks of infection, which were almost similar to that in humanized chimeric mouse. In contrast, serum HBV level in adult tupaia (1-year-old tupaia) was quite low (<10{sup 3} copies/ml). Understanding the differences in the response to HBV infection in primary tupaia hepatocytes, chimeric mouse, and adult tupaia will contribute to elucidating the mechanism of persistent HBV infection and viral eradication. Thus, T. belangeri was found to be efficient for studying the host response to HBV infection, thereby providing novel insight into the pathogenesis of HBV. - Highlights: • Primary hepatocytes were established from tupaia that is a novel HBV infection model. • Tupaia primary hepatocytes were susceptible for HBV infection. • The immunodeficient chimeric mice with tupaia hepatocytes were established. • The chimeric mice with tupaia hepatocytes were susceptible for HBV infection.

  14. Reversible Heat-Induced Inactivation of Chimeric β-Glucuronidase in Transgenic Plants1

    Science.gov (United States)

    Almoguera, Concepción; Rojas, Anabel; Jordano, Juan

    2002-01-01

    We compared the expression patterns in transgenic tobacco (Nicotiana tabacum) of two chimeric genes: a translational fusion to β-glucuronidase (GUS) and a transcriptional fusion, both with the same promoter and 5′-flanking sequences of Ha hsp17.7 G4, a small heat shock protein (sHSP) gene from sunflower (Helianthus annuus). We found that immediately after heat shock, the induced expression from the two fusions in seedlings was similar, considering chimeric mRNA or GUS protein accumulation. Surprisingly, we discovered that the chimeric GUS protein encoded by the translational fusion was mostly inactive in such conditions. We also found that this inactivation was fully reversible. Thus, after returning to control temperature, the GUS activity was fully recovered without substantial changes in GUS protein accumulation. In contrast, we did not find differences in the in vitro heat inactivation of the respective GUS proteins. Insolubilization of the chimeric GUS protein correlated with its inactivation, as indicated by immunoprecipitation analyses. The inclusion in another chimeric gene of the 21 amino-terminal amino acids from a different sHSP lead to a comparable reversible inactivation. That effect not only illustrates unexpected post-translational problems, but may also point to sequences involved in interactions specific to sHSPs and in vivo heat stress conditions. PMID:12011363

  15. Chemokine CCL2 and chemokine receptor CCR2 in early active multiple sclerosis

    DEFF Research Database (Denmark)

    Sørensen, Torben Lykke; Ransohoff, R M; Strieter, R M

    2004-01-01

    The chemokine monocyte chemoattractant protein (MCP)-1/CCL2 and its receptor CCR2 have been strongly implicated in disease pathogenesis in experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis (MS), whereas data on the CCL2-CCR2 axis are scarce in MS. We studied...... the expression of CCR2 on leukocytes in blood and cerebrospinal fluid (CSF) from patients with monosymptomatic optic neuritis and MS, and the concentration of CCL2 in the CSF from these patients. Results were compared with the results in non-inflammatory neurological controls and were correlated with other...... parameters (magnetic resonance imaging and CSF data). Our findings suggest a limited role for CCL2/CCR2 in early active MS....

  16. Analysis of proteins of mouse sarcoma pseudotype viruses: type-specific radioimmunoassays for ecotropic virus p30's

    International Nuclear Information System (INIS)

    Kennel, S.J.; Tennant, R.W.

    1979-01-01

    Murine sarcoma virus pseudotypes were prepared by infection of nonproducer cells (A1-2), which were transformed by the Gazdar strain of mouse sarcoma virus, with Gross (N-tropic), WN1802B (B-tropic), or Moloney (NB-tropic) viruses. The respective host range pseudotype sarcoma viruses were defined by the tritration characteristics on cells with the appropriate Fv-1 genotype. Proteins from virus progeny were analyzed by sodium dodecyl sulfate--polyacrylamide gel electrophoresis. Bands present in both the 65,000- and the 10,000- to 20,000-molecular-weight regions of the gel distinguished the pseudotype viruses from their respective helpers. Furthermore, two protein bands were noted in the p30 region of murine sarcoma virus (Gross), one corresponding to Gross virus p30, and another of slightly slower mobility. However, since the mobility of the putative sarcoma p30 is nearly indentical to that of WN1802B, its presence could not be established by sodium dodecyl sulfate--polyacrylamide gel electrophoresis. Type-specific radioimmunossays for Gross virus p30 and for WN1802B p30 were applied for analysis of pseudotype preparations, and among several ecotropic viruses tested, only the homologous virus scored in the respective assay. By use of these assays, pseudotype viruses were found to contain only 8 to 48% helper-specific p30's; the remainder is presumably derived from the sarcoma virus

  17. Prolonged Survival of Subcutaneous Allogeneic Islet Graft by Donor Chimerism without Immunosuppressive Treatment

    Directory of Open Access Journals (Sweden)

    Brend Ray-Sea Hsu

    2017-01-01

    Full Text Available The aim of this study was to investigate whether tolerance-induced protection of islets in the renal subcapsular space can also prevent subcutaneous allogeneic islets from being rejected. We used bone marrow stem cells from C57BL/6 (H2b mice to construct donor chimerism in conditioned diabetic BALB/c (H2d mice and investigated the effect of donor chimerism on engraftment and survival of subcutaneously transplanted allogeneic islets in streptozotocin-induced diabetic mice. We also studied the anti-inflammatory effect of mesenchymal stem cell on islet engraftment. Full but not low-grade or no donor chimerism was associated with successful engraftment of allogeneic islets and restoration of normoglycemia in the treated diabetic mice. The temporary hyperglycemia was 11 ± 1 versus 19 ± 5 days (p<0.05 for the mice with full donor chimerism with transplanted islets in the renal subcapsular space versus the subcutaneous space, respectively. Cotransplantation of mesenchymal stem cell did not enhance alloislet engraftment. Full multilineage donor chimerism was associated with a higher transient expansion of CD11b+ and Gr-1+ myeloid progenitor cells and effector memory CD4 and CD8 T cells. In conclusion, full donor chimerism protected both renal subcapsular and subcutaneous allogeneic islets in this rodent transplantation model.

  18. Control of Both Myeloid Cell Infiltration and Angiogenesis by CCR1 Promotes Liver Cancer Metastasis Development in Mice

    Directory of Open Access Journals (Sweden)

    Mathieu Paul Rodero

    2013-06-01

    Full Text Available Expression of the CC chemokine receptor 1 (CCR1 by tumor cells has been associated with protumoral activity; however, its role in nontumoral cells during tumor development remains elusive. Here, we investigated the role of CCR1 deletion on stromal and hematopoietic cells in a liver metastasis tumor model. Metastasis development was strongly impaired in CCR1-deficient mice compared to control mice and was associated with reduced liver monocyte infiltration. To decipher the role of myeloid cells, sublethally irradiated mice were reconstituted with CCR1-deficient bone marrow (BM and showed better survival rates than the control reconstituted mice. These results point toward the involvement of CCR1 myeloid cell infiltration in the promotion of tumor burden. In addition, survival rates were extended in CCR1-deficient mice receiving either control or CCR1-deficient BM, indicating that host CCR1 expression on nonhematopoietic cells also supports tumor growth. Finally, we found defective tumor-induced neoangiogenesis (in vitro and in vivo in CCR1-deficient mice. Overall, our results indicate that CCR1 expression by both hematopoietic and nonhematopoietic cells favors tumor aggressiveness. We propose CCR1 as a potential therapeutical target for liver metastasis therapy.

  19. Profile of State College and Career Readiness Assessments (CCR) Policy. Florida

    Science.gov (United States)

    Center on Education Policy, 2011

    2011-01-01

    This individual profile provides information on Florida's college and career readiness assessment policy. Some of the categories presented include: (1) CCR assessment policy; (2) Purpose; (3) Major changes in CCR assessment policy since the 2009-10 school year for financial reasons; (4) State financial support for students to take the CCR…

  20. Profile of State College and Career Readiness Assessments (CCR) Policy. Arkansas

    Science.gov (United States)

    Center on Education Policy, 2011

    2011-01-01

    This individual profile provides information on Arkansas' college and career readiness assessment policy. Some of the categories presented include: (1) CCR assessment policy; (2) Purpose; (3) Major changes in CCR assessment policy since the 2009-10 school year for financial reasons; (4) State financial support for students to take the CCR…

  1. Profile of State College and Career Readiness Assessments (CCR) Policy. Minnesota

    Science.gov (United States)

    Center on Education Policy, 2011

    2011-01-01

    This individual profile provides information on Minnesota's college and career readiness assessment policy. Some of the categories presented include: (1) CCR assessment policy; (2) Purpose; (3) Major changes in CCR assessment policy since the 2009-10 school year for financial reasons; (4) State financial support for students to take the CCR…

  2. Profile of State College and Career Readiness Assessments (CCR) Policy. Alaska

    Science.gov (United States)

    Center on Education Policy, 2011

    2011-01-01

    This individual profile provides information on Alaska's college and career readiness assessment policy. Some of the categories presented include: (1) CCR assessment policy; (2) Purpose; (3) Major changes in CCR assessment policy since the 2009-10 school year for financial reasons; (4) State financial support for students to take the CCR…

  3. Profile of State College and Career Readiness Assessments (CCR) Policy. Kentucky

    Science.gov (United States)

    Center on Education Policy, 2011

    2011-01-01

    This individual profile provides information on Kentucky's college and career readiness assessment policy. Some of the categories presented include: (1) CCR assessment policy; (2) Purpose; (3) Major changes in CCR assessment policy since the 2009-10 school year for financial reasons; (4) State financial support for students to take the CCR…

  4. Profile of State College and Career Readiness Assessments (CCR) Policy. Georgia

    Science.gov (United States)

    Center on Education Policy, 2011

    2011-01-01

    This individual profile provides information on Georgia's college and career readiness assessment policy. Some of the categories presented include: (1) CCR assessment policy; (2) Purpose; (3) Major changes in CCR assessment policy since the 2009-10 school year for financial reasons; (4) State financial support for students to take the CCR…

  5. Profile of State College and Career Readiness Assessments (CCR) Policy. Alabama

    Science.gov (United States)

    Center on Education Policy, 2011

    2011-01-01

    This individual profile provides information on Alabama's college and career readiness assessment policy. Some of the categories presented include: (1) CCR assessment policy; (2) Purpose; (3) Major changes in CCR assessment policy since the 2009-10 school year for financial reasons; (4) State financial support for students to take the CCR…

  6. Profile of State College and Career Readiness Assessments (CCR) Policy. Tennessee

    Science.gov (United States)

    Center on Education Policy, 2011

    2011-01-01

    This individual profile provides information on Tennessee's college and career readiness assessment policy. Some of the categories presented include: (1) CCR assessment policy; (2) Purpose; (3) Major changes in CCR assessment policy since the 2009-10 school year for financial reasons; (4) State financial support for students to take the CCR…

  7. Profile of State College and Career Readiness Assessments (CCR) Policy. California

    Science.gov (United States)

    Center on Education Policy, 2011

    2011-01-01

    This individual profile provides information on California's college and career readiness assessment policy. Some of the categories presented include: (1) CCR assessment policy; (2) Purpose; (3) Major changes in CCR assessment policy since the 2009-10 school year for financial reasons; (4) State financial support for students to take the CCR…

  8. Chimeric opioid peptides: Tools for identifying opioid receptor types

    International Nuclear Information System (INIS)

    Xie, G.; Miyajima, A.; Yokota, T.; Arai, K.; Goldstein, A.

    1990-01-01

    The authors synthesized several chimeric [125J-labelled] peptides in which the N-terminal nine residues of dynorphin-32, a peptide selective for the κ opioid receptor, were replaced by opioid peptides selective for other opioid receptor types. Each chimeric peptide retained the high affinity and type selectivity characteristic of its N-terminal sequence. The common C-terminal two-thirds of the chimeric peptides served as an epitope recognized by the same monoclonal antibody. When bound to receptors on a cell surface or membrane preparation, these peptides could still bind specifically to the monoclonal antibody. These chimeric peptides should be useful for isolating μ, δ, and κ opioid receptors and for identifying opioid receptors on transfected cells in expression cloning procedures. The general approach using chimeric peptides should be applicable to other peptide receptors

  9. A CCR2 macrophage endocytic pathway mediates extravascular fibrin clearance in vivo

    DEFF Research Database (Denmark)

    Motley, Michael P; Madsen, Daniel H; Jürgensen, Henrik J

    2016-01-01

    cellular endocytosis and lysosomal targeting, revealing a novel intracellular pathway for extravascular fibrin degradation. A C-C chemokine receptor type 2 (CCR2)-positive macrophage subpopulation constituted the majority of fibrin-uptaking cells. Consequently, cellular fibrin uptake was diminished...... by elimination of CCR2-expressing cells. The CCR2-positive macrophage subtype was different from collagen-internalizing M2-like macrophages. Cellular fibrin uptake was strictly dependent on plasminogen and plasminogen activator. Surprisingly, however, fibrin endocytosis was unimpeded by the absence of the fibrin...... subsets of macrophages employing distinct molecular pathways....

  10. Zika Virus Fact Sheet

    Science.gov (United States)

    ... is caused by a virus transmitted primarily by Aedes mosquitoes. People with Zika virus disease can have symptoms including mild fever, skin ... framework. Q&A: Zika virus and complication ... mosquito from the Aedes genus, mainly Aedes aegypti in tropical regions. Aedes ...

  11. Structural refinement and prediction of potential CCR2 antagonists through validated multi-QSAR modeling studies.

    Science.gov (United States)

    Amin, Sk Abdul; Adhikari, Nilanjan; Baidya, Sandip Kumar; Gayen, Shovanlal; Jha, Tarun

    2018-01-03

    Chemokines trigger numerous inflammatory responses and modulate the immune system. The interaction between monocyte chemoattractant protein-1 and chemokine receptor 2 (CCR2) may be the cause of atherosclerosis, obesity, and insulin resistance. However, CCR2 is also implicated in other inflammatory diseases such as rheumatoid arthritis, multiple sclerosis, asthma, and neuropathic pain. Therefore, there is a paramount importance of designing potent and selective CCR2 antagonists despite a number of drug candidates failed in clinical trials. In this article, 83 CCR2 antagonists by Jhonson and Jhonson Pharmaceuticals have been considered for robust validated multi-QSAR modeling studies to get an idea about the structural and pharmacophoric requirements for designing more potent CCR2 antagonists. All these QSAR models were validated and statistically reliable. Observations resulted from different modeling studies correlated and validated results of other ones. Finally, depending on these QSAR observations, some new molecules were proposed that may exhibit higher activity against CCR2.

  12. P2X1 Receptor Antagonists Inhibit HIV-1 Fusion by Blocking Virus-Coreceptor Interactions.

    Science.gov (United States)

    Giroud, Charline; Marin, Mariana; Hammonds, Jason; Spearman, Paul; Melikyan, Gregory B

    2015-09-01

    HIV-1 Env glycoprotein-mediated fusion is initiated upon sequential binding of Env to CD4 and the coreceptor CXCR4 or CCR5. Whereas these interactions are thought to be necessary and sufficient to promote HIV-1 fusion, other host factors can modulate this process. Previous studies reported potent inhibition of HIV-1 fusion by selective P2X1 receptor antagonists, including NF279, and suggested that these receptors play a role in HIV-1 entry. Here we investigated the mechanism of antiviral activity of NF279 and found that this compound does not inhibit HIV-1 fusion by preventing the activation of P2X1 channels but effectively blocks the binding of the virus to CXCR4 or CCR5. The notion of an off-target effect of NF279 on HIV-1 fusion is supported by the lack of detectable expression of P2X1 receptors in cells used in fusion experiments and by the fact that the addition of ATP or the enzymatic depletion of ATP in culture medium does not modulate viral fusion. Importantly, NF279 fails to inhibit HIV-1 fusion with cell lines and primary macrophages when added at an intermediate stage downstream of Env-CD4-coreceptor engagement. Conversely, in the presence of NF279, HIV-1 fusion is arrested downstream of CD4 binding but prior to coreceptor engagement. NF279 also antagonizes the signaling function of CCR5, CXCR4, and another chemokine receptor, as evidenced by the suppression of calcium responses elicited by specific ligands and by recombinant gp120. Collectively, our results demonstrate that NF279 is a dual HIV-1 coreceptor inhibitor that interferes with the functional engagement of CCR5 and CXCR4 by Env. Inhibition of P2X receptor activity suppresses HIV-1 fusion and replication, suggesting that P2X signaling is involved in HIV-1 entry. However, mechanistic experiments conducted in this study imply that P2X1 receptor is not expressed in target cells or involved in viral fusion. Instead, we found that inhibition of HIV-1 fusion by a specific P2X1 receptor antagonist, NF

  13. Benzimidazoles as benzamide replacements within cyclohexane-based CC chemokine receptor 2 (CCR2) antagonists.

    Science.gov (United States)

    Cherney, Robert J; Mo, Ruowei; Meyer, Dayton T; Pechulis, Anthony D; Guaciaro, Michael A; Lo, Yvonne C; Yang, Gengjie; Miller, Persymphonie B; Scherle, Peggy A; Zhao, Qihong; Cvijic, Mary Ellen; Barrish, Joel C; Decicco, Carl P; Carter, Percy H

    2012-10-01

    We describe the design, synthesis, and evaluation of benzimidazoles as benzamide replacements within a series of trisubstituted cyclohexane CCR2 antagonists. 7-Trifluoromethylbenzimidazoles displayed potent binding and functional antagonism of CCR2 while being selective over CCR3. These benzimidazoles were also incorporated into lactam-containing antagonists, thus completely eliminating the customary bis-amide. Copyright © 2012 Elsevier Ltd. All rights reserved.

  14. Newcastle disease virus-based H5 influenza vaccine protects chickens from lethal challenge with a highly pathogenic H5N2 avian influenza virus.

    Science.gov (United States)

    Ma, Jingjiao; Lee, Jinhwa; Liu, Haixia; Mena, Ignacio; Davis, A Sally; Sunwoo, Sun Young; Lang, Yuekun; Duff, Michael; Morozov, Igor; Li, Yuhao; Yang, Jianmei; García-Sastre, Adolfo; Richt, Juergen A; Ma, Wenjun

    2017-01-01

    Since December 2014, Eurasian-origin, highly pathogenic avian influenza H5 viruses including H5N1, H5N2, and H5N8 subtypes (called H5N x viruses), which belong to the H5 clade 2.3.4.4, have been detected in U.S. wild birds. Subsequently, highly pathogenic H5N2 and H5N8 viruses have caused outbreaks in U.S. domestic poultry. Vaccination is one of the most effective ways to control influenza outbreaks and protect animal and public health. Newcastle disease virus (NDV)-based influenza vaccines have been demonstrated to be efficacious and safe in poultry. Herein, we developed an NDV-based H5 vaccine (NDV-H5) that expresses a codon-optimized ectodomain of the hemagglutinin from the A/chicken/Iowa/04-20/2015 (H5N2) virus and evaluated its efficacy in chickens. Results showed that both live and inactivated NDV-H5 vaccines induced hemagglutinin inhibition antibody titers against the H5N2 virus in immunized chickens after prime and booster, and both NDV-H5 vaccines completely protected chickens from lethal challenge with the highly pathogenic H5N2 A/turkey/Minnesota/9845-4/2015 virus. No clinical signs and only minimal virus shedding was observed in both vaccinated groups. In contrast, all mock-vaccinated, H5N2-infected chickens shed virus and died within 5 days post challenge. Furthermore, one dose of the live NDV-H5 vaccine also provided protection of 90% chickens immunized by coarse spraying; after exposure to H5N2 challenge, sera from vaccinated surviving chickens neutralized both highly pathogenic H5N1 and H5N8 viruses. Taken together, our results suggest that the NDV-based H5 vaccine is able to protect chickens against intercontinental highly pathogenic H5N x viruses and can be used by mass application to protect the poultry industry.

  15. Recombinant Parainfluenza Virus 5 Expressing Hemagglutinin of Influenza A Virus H5N1 Protected Mice against Lethal Highly Pathogenic Avian Influenza Virus H5N1 Challenge

    Science.gov (United States)

    Li, Zhuo; Mooney, Alaina J.; Gabbard, Jon D.; Gao, Xiudan; Xu, Pei; Place, Ryan J.; Hogan, Robert J.; Tompkins, S. Mark

    2013-01-01

    A safe and effective vaccine is the best way to prevent large-scale highly pathogenic avian influenza virus (HPAI) H5N1 outbreaks in the human population. The current FDA-approved H5N1 vaccine has serious limitations. A more efficacious H5N1 vaccine is urgently needed. Parainfluenza virus 5 (PIV5), a paramyxovirus, is not known to cause any illness in humans. PIV5 is an attractive vaccine vector. In our studies, a single dose of a live recombinant PIV5 expressing a hemagglutinin (HA) gene of H5N1 (rPIV5-H5) from the H5N1 subtype provided sterilizing immunity against lethal doses of HPAI H5N1 infection in mice. Furthermore, we have examined the effect of insertion of H5N1 HA at different locations within the PIV5 genome on the efficacy of a PIV5-based vaccine. Interestingly, insertion of H5N1 HA between the leader sequence, the de facto promoter of PIV5, and the first viral gene, nucleoprotein (NP), did not lead to a viable virus. Insertion of H5N1 HA between NP and the next gene, V/phosphorprotein (V/P), led to a virus that was defective in growth. We have found that insertion of H5N1 HA at the junction between the small hydrophobic (SH) gene and the hemagglutinin-neuraminidase (HN) gene gave the best immunity against HPAI H5N1 challenge: a dose as low as 1,000 PFU was sufficient to protect against lethal HPAI H5N1 challenge in mice. The work suggests that recombinant PIV5 expressing H5N1 HA has great potential as an HPAI H5N1 vaccine. PMID:23077314

  16. Construction and growth properties of bovine herpesvirus type 5 recombinants defective in the glycoprotein E or thymidine kinase gene or both

    Directory of Open Access Journals (Sweden)

    M.C.S. Brum

    2010-02-01

    Full Text Available Bovine herpesvirus type 5 (BoHV-5 is an important pathogen of cattle in South America. We describe here the construction and characterization of deletion mutants defective in the glycoprotein E (gE or thymidine kinase (TK gene or both (gE/TK from a highly neurovirulent and well-characterized Brazilian BoHV-5 strain (SV507/99. A gE-deleted recombinant virus (BoHV-5 gE∆ was first generated in which the entire gE open reading frame was replaced with a chimeric green fluorescent protein gene. A TK-deleted recombinant virus (BoHV-5 TK∆ was then generated in which most of the TK open reading frame sequences were deleted and replaced with a chimeric β-galactosidase gene. Subsequently, using the BoHV-5 gE∆ virus as backbone, a double gene-deleted (TK plus gE BoHV-5 recombinant (BoHV-5 gE/TK∆ was generated. The deletion of the gE and TK genes was confirmed by immunoblotting and PCR, respectively. In Madin Darby bovine kidney (MDBK cells, the mutants lacking gE (BoHV-5 gE∆ and TK + gE (BoHV-5 gE/TK∆ produced small plaques while the TK-deleted BoHV-5 produced wild-type-sized plaques. The growth kinetics and virus yields in MDBK cells for all three recombinants (BoHV-5 gE∆, BoHV-5 TK∆ and BoHV-5 gE/TK∆ were similar to those of the parental virus. It is our belief that the dual gene-deleted recombinant (BoHV-5 gE/TK∆ produced on the background of a highly neurovirulent Brazilian BoHV-5 strain may have potential application in a vaccine against BoHV-5.

  17. In-silico guided discovery of novel CCR9 antagonists

    Science.gov (United States)

    Zhang, Xin; Cross, Jason B.; Romero, Jan; Heifetz, Alexander; Humphries, Eric; Hall, Katie; Wu, Yuchuan; Stucka, Sabrina; Zhang, Jing; Chandonnet, Haoqun; Lippa, Blaise; Ryan, M. Dominic; Baber, J. Christian

    2018-03-01

    Antagonism of CCR9 is a promising mechanism for treatment of inflammatory bowel disease, including ulcerative colitis and Crohn's disease. There is limited experimental data on CCR9 and its ligands, complicating efforts to identify new small molecule antagonists. We present here results of a successful virtual screening and rational hit-to-lead campaign that led to the discovery and initial optimization of novel CCR9 antagonists. This work uses a novel data fusion strategy to integrate the output of multiple computational tools, such as 2D similarity search, shape similarity, pharmacophore searching, and molecular docking, as well as the identification and incorporation of privileged chemokine fragments. The application of various ranking strategies, which combined consensus and parallel selection methods to achieve a balance of enrichment and novelty, resulted in 198 virtual screening hits in total, with an overall hit rate of 18%. Several hits were developed into early leads through targeted synthesis and purchase of analogs.

  18. Complementation studies with the novel "Bungowannah" virus provide new insights in the compatibility of pestivirus proteins.

    Science.gov (United States)

    Richter, Maria; Reimann, Ilona; Wegelt, Anne; Kirkland, Peter D; Beer, Martin

    2011-09-30

    In recent years several atypical pestiviruses have been described. Bungowannah virus is the most divergent virus in this group. Therefore, heterologous complementation was used to clarify the phylogenetic relationship and to analyze the exchangeability of genome regions encoding structural proteins. Using a BVDV type 1 backbone, chimeric constructs with substituted envelope proteins E(rns), E1 and E2, were investigated. While all constructs replicated autonomously, infectious high titer chimeric virus could only be observed after exchanging the complete E1-E2 encoding region. The complementation of E1 and E2 alone resulted only in replicons. Complementation of BVDV-E(rns) was only efficient if Bungowannah virus-E(rns) was expressed from a bicistronic construct. Our data provide new insights in the compatibility of pestivirus proteins and demonstrate that heterologous complementation could be useful to characterize new pestiviruses. Copyright © 2011 Elsevier Inc. All rights reserved.

  19. HIV type 1 chemokine receptor usage in mother-to-child transmission.

    Science.gov (United States)

    Salvatori, F; Scarlatti, G

    2001-07-01

    To investigate the role of the HIV-1 phenotype in mother-to-child HIV-1 transmission, we evaluated coreceptor usage and replication kinetics in chemokine receptor-expressing U87MG.CD4 cells of primary isolates from 32 HIV-1-infected mothers of Italian origin, none under preventive antiretroviral therapy, and from their infected infants. Five of 15 mothers of infected children and 2 of 17 mothers of uninfected children harbored viruses able to use CXCR4 as coreceptor. However, all isolates used CCR5, alone or in association with CXCR4. The replicative capacity in coreceptor-expressing cells of the viral isolates did not differ between the two groups of mothers. All mothers with an R5 virus transmitted a virus with the same coreceptor usage, whereas those four with a multitropic virus transmitted such a virus in one case. Although the presence of a mixed viral population was documented in the mothers, we did not observe transmission solely of X4 viruses. Interestingly, the only child infected with a multitropic virus carried a defective CCR5 allele. Analysis of the env V3 region of the provirus from this child revealed infection with multiple viral variants with a predominance of R5-type over X4-type sequences. These findings show that CCR5 usage of a viral isolate is not a discriminating risk factor for vertical transmission. Furthermore, X4 viruses can be transmitted to the newborn, although less frequently. In particular, we document the transmission of multiple viral variants with different coreceptor usage in a Delta32 CCR5 heterozygous child, and demonstrate that the heterozygous genotype per se does not contribute to the restriction of R5-type virus spread.

  20. Vaccination with Recombinant Parainfluenza Virus 5 Expressing Neuraminidase Protects against Homologous and Heterologous Influenza Virus Challenge.

    Science.gov (United States)

    Mooney, Alaina J; Gabbard, Jon D; Li, Zhuo; Dlugolenski, Daniel A; Johnson, Scott K; Tripp, Ralph A; He, Biao; Tompkins, S Mark

    2017-12-01

    Seasonal human influenza virus continues to cause morbidity and mortality annually, and highly pathogenic avian influenza (HPAI) viruses along with other emerging influenza viruses continue to pose pandemic threats. Vaccination is considered the most effective measure for controlling influenza; however, current strategies rely on a precise vaccine match with currently circulating virus strains for efficacy, requiring constant surveillance and regular development of matched vaccines. Current vaccines focus on eliciting specific antibody responses against the hemagglutinin (HA) surface glycoprotein; however, the diversity of HAs across species and antigenic drift of circulating strains enable the evasion of virus-inhibiting antibody responses, resulting in vaccine failure. The neuraminidase (NA) surface glycoprotein, while diverse, has a conserved enzymatic site and presents an appealing target for priming broadly effective antibody responses. Here we show that vaccination with parainfluenza virus 5 (PIV5), a promising live viral vector expressing NA from avian (H5N1) or pandemic (H1N1) influenza virus, elicited NA-specific antibody and T cell responses, which conferred protection against homologous and heterologous influenza virus challenges. Vaccination with PIV5-N1 NA provided cross-protection against challenge with a heterosubtypic (H3N2) virus. Experiments using antibody transfer indicate that antibodies to NA have an important role in protection. These findings indicate that PIV5 expressing NA may be effective as a broadly protective vaccine against seasonal influenza and emerging pandemic threats. IMPORTANCE Seasonal influenza viruses cause considerable morbidity and mortality annually, while emerging viruses pose potential pandemic threats. Currently licensed influenza virus vaccines rely on the antigenic match of hemagglutinin (HA) for vaccine strain selection, and most vaccines rely on HA inhibition titers to determine efficacy, despite the growing