Diverse fates of uracilated HIV-1 DNA during infection of myeloid lineage cells.

Science.gov (United States)

Hansen, Erik C; Ransom, Monica; Hesselberth, Jay R; Hosmane, Nina N; Capoferri, Adam A; Bruner, Katherine M; Pollack, Ross A; Zhang, Hao; Drummond, Michael Bradley; Siliciano, Janet M; Siliciano, Robert; Stivers, James T

2016-09-20

We report that a major subpopulation of monocyte-derived macrophages (MDMs) contains high levels of dUTP, which is incorporated into HIV-1 DNA during reverse transcription (U/A pairs), resulting in pre-integration restriction and post-integration mutagenesis. After entering the nucleus, uracilated viral DNA products are degraded by the uracil base excision repair (UBER) machinery with less than 1% of the uracilated DNA successfully integrating. Although uracilated proviral DNA showed few mutations, the viral genomic RNA was highly mutated, suggesting that errors occur during transcription. Viral DNA isolated from blood monocytes and alveolar macrophages (but not T cells) of drug-suppressed HIV-infected individuals also contained abundant uracils. The presence of viral uracils in short-lived monocytes suggests their recent infection through contact with virus producing cells in a tissue reservoir. These findings reveal new elements of a viral defense mechanism involving host UBER that may be relevant to the establishment and persistence of HIV-1 infection.

Biochemical characterization of cells transformed via transfection by feline sarcoma virus proviral DNA.

OpenAIRE

Rosenberg, Z F; Sahagan, B G; Snyder, H W; Worley, M B; Essex, M; Haseltine, W A

1981-01-01

Murine fibroblasts transformed by transfection with DNA from mink cells infected with the Snyder-Theilen strain of feline sarcoma virus and subgroup B feline leukemia virus were analyzed for the presence of integrated proviral DNA and the expression of feline leukemia virus- and feline sarcoma virus-specific proteins. The transformed murine cells harbored at least one intact feline sarcoma virus provirus, but did not contain feline leukemia virus provirus. The transformed murine cells express...

Diverse fates of uracilated HIV-1 DNA during infection of myeloid lineage cells

Science.gov (United States)

Hansen, Erik C; Ransom, Monica; Hesselberth, Jay R; Hosmane, Nina N; Capoferri, Adam A; Bruner, Katherine M; Pollack, Ross A; Zhang, Hao; Drummond, Michael Bradley; Siliciano, Janet M; Siliciano, Robert; Stivers, James T

2016-01-01

We report that a major subpopulation of monocyte-derived macrophages (MDMs) contains high levels of dUTP, which is incorporated into HIV-1 DNA during reverse transcription (U/A pairs), resulting in pre-integration restriction and post-integration mutagenesis. After entering the nucleus, uracilated viral DNA products are degraded by the uracil base excision repair (UBER) machinery with less than 1% of the uracilated DNA successfully integrating. Although uracilated proviral DNA showed few mutations, the viral genomic RNA was highly mutated, suggesting that errors occur during transcription. Viral DNA isolated from blood monocytes and alveolar macrophages (but not T cells) of drug-suppressed HIV-infected individuals also contained abundant uracils. The presence of viral uracils in short-lived monocytes suggests their recent infection through contact with virus producing cells in a tissue reservoir. These findings reveal new elements of a viral defense mechanism involving host UBER that may be relevant to the establishment and persistence of HIV-1 infection. DOI: http://dx.doi.org/10.7554/eLife.18447.001 PMID:27644592

A Combinatorial CRISPR-Cas9 Attack on HIV-1 DNA Extinguishes All Infectious Provirus in Infected T Cell Cultures

NARCIS (Netherlands)

Wang, Gang; Zhao, Na; Berkhout, Ben; Das, Atze T.

2016-01-01

Current drug therapies effectively suppress HIV-1 replication but do not inactivate the provirus that persists in latent reservoirs. Recent studies have found that the guide RNA (gRNA)-directed CRISPR/Cas9 system can be used for sequence-specific attack on this proviral DNA. Although potent

Influenza vaccination of HIV-1-positive and HIV-1-negative former intravenous drug users.

Science.gov (United States)

Amendola, A; Boschini, A; Colzani, D; Anselmi, G; Oltolina, A; Zucconi, R; Begnini, M; Besana, S; Tanzi, E; Zanetti, A R

2001-12-01

The immunogenicity of an anti-influenza vaccine was assessed in 409 former intravenous drug user volunteers and its effect on the levels of HIV-1 RNA, proviral DNA and on CD4+ lymphocyte counts in a subset HIV-1-positive subjects was measured. HIV-1-positive individuals (n = 72) were divided into three groups on the basis of their CD4+ lymphocyte counts, while the 337 HIV-1-negative participants were allocated into group four. Haemagglutination inhibiting (HI) responses varied from 45.8 to 70% in the HIV-1-positive subjects and were significantly higher in group four (80.7% responses to the H1N1 strain, 81.6% to the H3N2 strain, and 83% to the B strain). The percentage of subjects with HI protective antibody titres (> or = 1:40) increased significantly after vaccination, especially in HIV-1 uninfected subjects. Immunization caused no significant changes in CD4+ counts and in neither plasma HIV-1 RNA nor proviral DNA levels. Therefore, vaccination against influenza may benefit persons infected by HIV-1. Copyright 2001 Wiley-Liss, Inc.

EFFECT OF SHORT-TERM ART INTERRUPTION ON LEVELS OF INTEGRATED HIV DNA.

Science.gov (United States)

Strongin, Zachary; Sharaf, Radwa; VanBelzen, D Jake; Jacobson, Jeffrey M; Connick, Elizabeth; Volberding, Paul; Skiest, Daniel J; Gandhi, Rajesh T; Kuritzkes, Daniel R; O'Doherty, Una; Li, Jonathan Z

2018-03-28

Analytic treatment interruption (ATI) studies are required to evaluate strategies aimed at achieving ART-free HIV remission, but the impact of ATI on the viral reservoir remains unclear. We validated a DNA size selection-based assay for measuring levels of integrated HIV DNA and applied it to assess the effects of short-term ATI on the HIV reservoir. Samples from participants from four AIDS Clinical Trials Group (ACTG) ATI studies were assayed for integrated HIV DNA levels. Cryopreserved PBMCs were obtained for 12 participants with available samples pre-ATI and approximately 6 months after ART resumption. Four participants also had samples available during the ATI. The median duration of ATI was 12 weeks. Validation of the HIV Integrated DNA size-Exclusion (HIDE) assay was performed using samples spiked with unintegrated HIV DNA, HIV-infected cell lines, and participant PBMCs. The HIDE assay eliminated 99% of unintegrated HIV DNA species and strongly correlated with the established Alu- gag assay. For the majority of individuals, integrated DNA levels increased during ATI and subsequently declined upon ART resumption. There was no significant difference in levels of integrated HIV DNA between the pre- and post-ATI time points, with the median ratio of post:pre-ATI HIV DNA levels of 0.95. Using a new integrated HIV DNA assay, we found minimal change in the levels of integrated HIV DNA in participants who underwent an ATI followed by 6 months of ART. This suggests that short-term ATI can be conducted without a significant impact on levels of integrated proviral DNA in the peripheral blood. IMPORTANCE Interventions aimed at achieving sustained antiretroviral therapy (ART)-free HIV remission require treatment interruption trials to assess their efficacy. However, these trials are accompanied by safety concerns related to the expansion of the viral reservoir. We validated an assay that uses an automated DNA size-selection platform for quantifying levels of integrated

c-DNA of HIV-1 detection on spot of Buffy-Coat of leukocytes (DBCS

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Marco Rossi de Gasperis

2010-03-01

Full Text Available Introduction:The elective way for the diagnosis of HIV-1-infection in the window period and in children under the age of 16-18 months is to search virus integrated in leukocytes. Aim of the study was to assess the sensitivity and specificity of extraction from Buffy-Dried Coat Spot (DBCS in leukocyte to detect c-DNA with nested-PCR in HIV-1-infected individuals compared to Dried Blood Spot (DBS both extracted by automated instrument EZ1 (QIAGEN, Hilden, Germany. Both DBCS and both DBS were compared with those tests from whole blood by conventional DNA-extraction Methods: Five ml of whole blood from 50 HIV-infected individuals were collected. 40 μl of each sample were spotted on “FTA ELUTE Micro Card” (Whatman, Inc., Clifton, NJ, 200 μl were extracted according to the manual procedure (QIAGEN “QIAamp DNA minikit and the remaining sample was incubated at 37 °C for 120 minutes. Plasma was centrifuged at 1000 rcf/1g for 10 minutes at room temperature. Forty μl of the obtained buffy-coat was spotted. Both DBCS and both DBS were dried at room temperature for 24 hours.Two of 5 punch from each spot were extracted with TISSUE DNA kit (Biorobot EZ1 DSP “Qiagen” and eluted in 50 μl of buffer.The recovery of genomic DNA was measured amplifying the ß-globin gene by Real-Time “SybrGreen I”.The DNA was amplified for the “pol” gene of HIV-1 by nested PCR and revealed in “SYBR-green I”. Eight HIV-antibody-negative samples were used as internal control. Results:The experimental protocol adopted for the DBCS showed high sensitivity and specificity.The extracted DNA from DBS and DBCS was characterized by excellent quality and without any inhibitory agents. The amount of proviral DNA extracted from DBCS is similar to that obtained by conventional extraction, while the DBS test was significantly less sensitive. Conclusion:These preliminary data suggest that the amount of c-DNA obtained with DBS technique is often not enough for the

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.

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

Science.gov (United States)

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.

Quantification of integrated HIV DNA by repetitive-sampling Alu-HIV PCR on the basis of poisson statistics.

Science.gov (United States)

De Spiegelaere, Ward; Malatinkova, Eva; Lynch, Lindsay; Van Nieuwerburgh, Filip; Messiaen, Peter; O'Doherty, Una; Vandekerckhove, Linos

2014-06-01

Quantification of integrated proviral HIV DNA by repetitive-sampling Alu-HIV PCR is a candidate virological tool to monitor the HIV reservoir in patients. However, the experimental procedures and data analysis of the assay are complex and hinder its widespread use. Here, we provide an improved and simplified data analysis method by adopting binomial and Poisson statistics. A modified analysis method on the basis of Poisson statistics was used to analyze the binomial data of positive and negative reactions from a 42-replicate Alu-HIV PCR by use of dilutions of an integration standard and on samples of 57 HIV-infected patients. Results were compared with the quantitative output of the previously described Alu-HIV PCR method. Poisson-based quantification of the Alu-HIV PCR was linearly correlated with the standard dilution series, indicating that absolute quantification with the Poisson method is a valid alternative for data analysis of repetitive-sampling Alu-HIV PCR data. Quantitative outputs of patient samples assessed by the Poisson method correlated with the previously described Alu-HIV PCR analysis, indicating that this method is a valid alternative for quantifying integrated HIV DNA. Poisson-based analysis of the Alu-HIV PCR data enables absolute quantification without the need of a standard dilution curve. Implementation of the CI estimation permits improved qualitative analysis of the data and provides a statistical basis for the required minimal number of technical replicates. © 2014 The American Association for Clinical Chemistry.

High level of APOBEC3F/3G editing in HIV-2 DNA vif and pol sequences from antiretroviral-naive patients.

Science.gov (United States)

Bertine, Mélanie; Charpentier, Charlotte; Visseaux, Benoit; Storto, Alexandre; Collin, Gilles; Larrouy, Lucile; Damond, Florence; Matheron, Sophie; Brun-Vézinet, Françoise; Descamps, Diane

2015-04-24

In HIV-1, hypermutation introduced by APOBEC3F/3G cytidine deaminase activity leads to defective viruses. In-vivo impact of APOBEC3F/3G editing on HIV-2 sequences remains unknown. The objective of this study was to assess the level of APOBEC3F/3G editing in HIV-2-infected antiretroviral-naive patients. Direct sequencing of vif and pol regions was performed on HIV-2 proviral DNA from antiretroviral-naive patients included in the French Agence Nationale de Recherches sur le SIDA et les hépatites virales CO5 HIV-2 cohort. Hypermutated sequences were identified using Hypermut2.0 program. HIV-1 proviral sequences from Genbank were also assessed. Among 82 antiretroviral-naive HIV-2-infected patients assessed, 15 (28.8%) and five (16.7%) displayed Vif proviral defective sequences in HIV-2 groups A and B, respectively. A lower proportion of defective sequences was observed in protease-reverse transcriptase region. A higher median number of G-to-A mutations was observed in HIV-2 group B than in group A, both in Vif and protease-reverse transcriptase regions (P = 0.02 and P = 0.006, respectively). Compared with HIV-1 Vif sequences, a higher number of Vif defective sequences was observed in HIV-2 group A (P = 0.00001) and group B sequences (P = 0.013). We showed for the first time a high level of APOBEC3F/3G editing in HIV-2 sequences from antiretroviral-naive patients. Our study reported a group effect with a significantly higher level of APOBEC3F/3G editing in HIV-2 group B than in group A sequences.

Number and location of mouse mammary tumor virus proviral DNA in mouse DNA of normal tissue and of mammary tumors.

Science.gov (United States)

Groner, B; Hynes, N E

1980-01-01

The Southern DNA filter transfer technique was used to characterize the genomic location of the mouse mammary tumor proviral DNA in different inbred strains of mice. Two of the strains (C3H and CBA) arose from a cross of a Bagg albino (BALB/c) mouse and a DBA mouse. The mouse mammary tumor virus-containing restriction enzyme DNA fragments of these strains had similar patterns, suggesting that the proviruses of these mice are in similar genomic locations. Conversely, the pattern arising from the DNA of the GR mouse, a strain genetically unrelated to the others, appeared different, suggesting that its mouse mammary tumor proviruses are located in different genomic sites. The structure of another gene, that coding for beta-globin, was also compared. The mice strains which we studied can be categorized into two classes, expressing either one or two beta-globin proteins. The macroenvironment of the beta-globin gene appeared similar among the mice strains belonging to one genetic class. Female mice of the C3H strain exogenously transmit mouse mammary tumor virus via the milk, and their offspring have a high incidence of mammary tumor occurrence. DNA isolated from individual mammary tumors taken from C3H mice or from BALB/c mice foster nursed on C3H mothers was analyzed by the DNA filter transfer technique. Additional mouse mammary tumor virus-containing fragments were found in the DNA isolated from each mammary tumor. These proviral sequences were integrated into different genomic sites in each tumor. Images PMID:6245257

Detection and quantification of proviral HIV-1 184 M/V in circulating CD4(+) T cells of patients on HAART with a viremia less than 1000 copies/ml

DEFF Research Database (Denmark)

Mohey, Rajesh; Jørgensen, Anne Louise; Møller, Bjarne K

2005-01-01

and incorporation of resistant forms in the long-lived CD4+ T cellular DNA compartment is not clear. Objective To investigate the relationship between lamivudine associated mutant-type 184V and the wild-type 184M proviral forms in the circulating CD4+ T cells of patients and low-level viremia. Study design Cross-sectional......Background Highly active anti-retroviral therapy (HAART) effectively reduces HIV replication but does not completely hinder it. Sub-optimal therapy leads to HIV resistance to the drugs administered. However, the role of low-level viremia (viral-load less than 1000 copies/ml) on mutation genesis...... study of 50 patients on long-term HAART, with a viremia of less than 1000 copies/ml. Patients were stratified into three groups; on lamivudine, group I (viral load

Quantification of bovine leukemia virus proviral DNA using a low-cost real-time polymerase chain reaction.

Science.gov (United States)

Petersen, M I; Alvarez, I; Trono, K G; Jaworski, J P

2018-04-11

The detection of bovine leukemia virus (BLV) proviral DNA is an important tool to address whether an animal is infected with BLV. Compared with serological assays, real-time PCR accounts for greater sensitivity and can serve as a confirmatory test for the clarification of inconclusive or discordant serological test results. However, the high cost related to real-time PCR assays has limited their systematic inclusion in BLV surveillance and eradication programs. The aim of the present study was to validate a low-cost quantitative real-time PCR. Interestingly, by using SYBR Green detection dye, we were able to reduce the cost of a single reaction by a factor of 5 compared with most common assays based on the use of fluorogenic probes (i.e., TaqMan technology). This approach allowed a highly sensitive and specific detection and quantification of BLV proviral DNA from purified peripheral blood leukocytes and a milk matrix. Due to its simplicity and low cost, our in-house BLV SYBR quantitative real-time PCR might be used either as a screening or as a confirmatory test in BLV control programs. Copyright © 2018 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Cocaine modulates HIV-1 integration in primary CD4+ T cells: implications in HIV-1 pathogenesis in drug-abusing patients

Science.gov (United States)

Addai, Amma B.; Pandhare, Jui; Paromov, Victor; Mantri, Chinmay K.; Pratap, Siddharth; Dash, Chandravanu

2015-01-01

Epidemiologic studies suggest that cocaine abuse worsens HIV-1 disease progression. Increased viral load has been suggested to play a key role for the accelerated HIV disease among cocaine-abusing patients. The goal of this study was to investigate whether cocaine enhances proviral DNA integration as a mechanism to increase viral load. We infected CD4+ T cells that are the primary targets of HIV-1 in vivo and treated the cells with physiologically relevant concentrations of cocaine (1 µM–100 µM). Proviral DNA integration in the host genome was measured by nested qPCR. Our results illustrated that cocaine from 1 µM through 50 µM increased HIV-1 integration in CD4+ T cells in a dose-dependent manner. As integration can be modulated by several early postentry steps of HIV-1 infection, we examined the direct effects of cocaine on viral integration by in vitro integration assays by use of HIV-1 PICs. Our data illustrated that cocaine directly increases viral DNA integration. Furthermore, our MS analysis showed that cocaine is able to enter CD4+ T cells and localize to the nucleus-. In summary, our data provide strong evidence that cocaine can increase HIV-1 integration in CD4+ T cells. Therefore, we hypothesize that increased HIV-1 integration is a novel mechanism by which cocaine enhances viral load and worsens disease progression in drug-abusing HIV-1 patients. PMID:25691383

Targeted HIV-1 Latency Reversal Using CRISPR/Cas9-Derived Transcriptional Activator Systems.

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Julia K Bialek

Full Text Available CRISPR/Cas9 technology is currently considered the most advanced tool for targeted genome engineering. Its sequence-dependent specificity has been explored for locus-directed transcriptional modulation. Such modulation, in particular transcriptional activation, has been proposed as key approach to overcome silencing of dormant HIV provirus in latently infected cellular reservoirs. Currently available agents for provirus activation, so-called latency reversing agents (LRAs, act indirectly through cellular pathways to induce viral transcription. However, their clinical performance remains suboptimal, possibly because reservoirs have diverse cellular identities and/or proviral DNA is intractable to the induced pathways. We have explored two CRISPR/Cas9-derived activator systems as targeted approaches to induce dormant HIV-1 proviral DNA. These systems recruit multiple transcriptional activation domains to the HIV 5' long terminal repeat (LTR, for which we have identified an optimal target region within the LTR U3 sequence. Using this target region, we demonstrate transcriptional activation of proviral genomes via the synergistic activation mediator complex in various in culture model systems for HIV latency. Observed levels of induction are comparable or indeed higher than treatment with established LRAs. Importantly, activation is complete, leading to production of infective viral particles. Our data demonstrate that CRISPR/Cas9-derived technologies can be applied to counteract HIV latency and may therefore represent promising novel approaches in the quest for HIV elimination.

Vaccination of rhesus macaques with a vif-deleted simian immunodeficiency virus proviral DNA vaccine

International Nuclear Information System (INIS)

Sparger, Ellen E.; Dubie, Robert A.; Shacklett, Barbara L.; Cole, Kelly S.; Chang, W.L.; Luciw, Paul A.

2008-01-01

Studies in non-human primates, with simian immunodeficiency virus (SIV) and simian/human immunodeficiency virus (SHIV) have demonstrated that live-attenuated viral vaccines are highly effective; however these vaccine viruses maintain a low level of pathogenicity. Lentivirus attenuation associated with deletion of the viral vif gene carries a significantly reduced risk for pathogenicity, while retaining the potential for virus replication of low magnitude in the host. This report describes a vif-deleted simian immunodeficiency virus (SIV)mac239 provirus that was tested as an attenuated proviral DNA vaccine by inoculation of female rhesus macaques. SIV-specific interferon-γ enzyme-linked immunospot responses of low magnitude were observed after immunization with plasmid containing the vif-deleted SIV provirus. However, vaccinated animals displayed strong sustained virus-specific T cell proliferative responses and increasing antiviral antibody titers. These immune responses suggested either persistent vaccine plasmid expression or low level replication of vif-deleted SIV in the host. Immunized and unvaccinated macaques received a single high dose vaginal challenge with pathogenic SIVmac251. A transient suppression of challenge virus load and a greater median survival time was observed for vaccinated animals. However, virus loads for vaccinated and unvaccinated macaques were comparable by twenty weeks after challenge and overall survival curves for the two groups were not significantly different. Thus, a vif-deleted SIVmac239 proviral DNA vaccine is immunogenic and capable of inducing a transient suppression of pathogenic challenge virus, despite severe attenuation of the vaccine virus

Quantification of HTLV-I proviral load in experimentally infected rabbits

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Kindt Thomas J

2005-05-01

Full Text Available Abstract Background Levels of proviral load in HTLV-1 infected patients correlate with clinical outcome and are reasonably prognostic. Adaptation of proviral load measurement techniques is examined here for use in an experimental rabbit model of HTLV-1 infection. Initial efforts sought to correlate proviral load with route and dose of inoculation and with clinical outcome in this model. These methods contribute to our continuing goal of using the model to test treatments that alleviate virus infection. Results A real-time PCR assay was used to measure proviral load in blood and tissue samples from a series of rabbits infected using HTLV-1 inocula prepared as either cell-free virus particles, infected cells or blood, or by naked DNA injection. Proviral loads from asymptomatically infected rabbits showed levels corresponding to those reported for human patients with clinically silent HTLV-1 infections. Proviral load was comparably increased in 50% of experimentally infected rabbits that developed either spontaneous benign or malignant tumors while infected. Similarly elevated provirus was found in organs of rabbits with experimentally induced acute leukemia/lymphoma-like disease. Levels of provirus in organs taken at necropsy varied widely suggesting that reservoirs of infections exist in non-lymphoid organs not traditionally thought to be targets for HTLV-1. Conclusion Proviral load measurement is a valuable enhancement to the rabbit model for HTLV-1 infection providing a metric to monitor clinical status of the infected animals as well as a means for the testing of treatment to combat infection. In some cases proviral load in blood did not reflect organ proviral levels, revealing a limitation of this method for monitoring health status of HTLV-1 infected individuals.

CRISPR-Cas9 Can Inhibit HIV-1 Replication but NHEJ Repair Facilitates Virus Escape

NARCIS (Netherlands)

Wang, Gang; Zhao, Na; Berkhout, Ben; Das, Atze T.

2016-01-01

Several recent studies demonstrated that the clustered regularly interspaced short palindromic repeats (CRISPR)-associated endonuclease Cas9 can be used for guide RNA (gRNA)-directed, sequence-specific cleavage of HIV proviral DNA in infected cells. We here demonstrate profound inhibition of HIV-1

Accuracy Assessment and Analysis for GPT2

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YAO Yibin

2015-07-01

Full Text Available GPT(global pressure and temperature is a global empirical model usually used to provide temperature and pressure for the determination of tropospheric delay, there are some weakness to GPT, these have been improved with a new empirical model named GPT2, which not only improves the accuracy of temperature and pressure, but also provides specific humidity, water vapor pressure, mapping function coefficients and other tropospheric parameters, and no accuracy analysis of GPT2 has been made until now. In this paper high-precision meteorological data from ECWMF and NOAA were used to test and analyze the accuracy of temperature, pressure and water vapor pressure expressed by GPT2, testing results show that the mean Bias of temperature is -0.59℃, average RMS is 3.82℃; absolute value of average Bias of pressure and water vapor pressure are less than 1 mb, GPT2 pressure has average RMS of 7 mb, and water vapor pressure no more than 3 mb, accuracy is different in different latitudes, all of them have obvious seasonality. In conclusion, GPT2 model has high accuracy and stability on global scale.

Natural controlled HIV infection: Preserved HIV-specific immunity despite undetectable replication competent virus

International Nuclear Information System (INIS)

Kloosterboer, Nico; Groeneveld, Paul H.P.; Jansen, Christine A.; Vorst, Teun J.K. van der; Koning, Fransje; Winkel, Carel N.; Duits, Ashley J.; Miedema, Frank; Baarle, Debbie van; Rij, Ronald P. van; Brinkman, Kees; Schuitemaker, Hanneke

2005-01-01

Long-term non-progressive HIV infection, characterized by low but detectable viral load and stable CD4 counts in the absence of antiviral therapy, is observed in about 5% of HIV-infected patients. Here we identified four therapy naive individuals who are strongly seropositive for HIV-1 but who lack evidence of detectable HIV p24 antigen, plasma RNA, and proviral DNA in routine diagnostic testing. With an ultrasensitive PCR, we established that frequencies of pol proviral DNA sequences were as low as 0.2-0.5 copies/10 6 PBMC. HIV could not be isolated using up to 30 x 10 6 patient PBMC. One individual was heterozygous for CCR5 Δ32, but CCR5 expression on CD4 + T cells was normal to high in all four individuals. In vitro R5 and X4 HIV-1 susceptibility of CD8-depleted PBMC of all study subjects was significantly lower than the susceptibility of CD8-depleted PBMC of healthy blood donors. All individuals expressed protective HLA-B*58s alleles and showed evidence of HIV-specific cellular immunity either by staining with HLA-B*57 tetramers folded with an HIV RT or gag peptide or after stimulation with HIV-1 p24 gag, RT, or nef peptides in ELIspot analysis. HIV-specific CD4 + T helper cells were demonstrated by proliferation of CD4 + T cells and intracellular staining for IL-2 and IFNγ after stimulation with an HIV-gag peptide pool. Sera of all individuals showed antibody-mediated neutralization of both R5 and X4 HIV-1 variants. These data implicate that very low-level antigen exposure is sufficient for sustained HIV-specific immunity and suggest the possibility of a multi-factorial control of HIV infection

Rapid turnover of 2-LTR HIV-1 DNA during early stage of highly active antiretroviral therapy.

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Weijun Zhu

Full Text Available BACKGROUND: Despite prolonged treatment with highly active antiretroviral therapy (HAART, the infectious HIV-1 continues to replicate and resides latently in the resting memory CD4+ T lymphocytes, which blocks the eradication of HIV-1. The viral persistence of HIV-1 is mainly caused by its proviral DNA being either linear nonintegrated, circular nonintegrated, or integrated. Previous reports have largely focused on the dynamics of HIV-1 DNA from the samples collected with relatively long time intervals during the process of disease and HAART treatment, which may have missed the intricate changes during the intervals in early treatment. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we investigated the dynamics of HIV-1 DNA in patients during the early phase of HARRT treatment. Using optimized real time PCR, we observed significant changes in 2-LTR during the first 12-week of treatment, while total and integrated HIV-1 DNA remained stable. The doubling time and half-life of 2-LTR were not correlated with the baseline and the rate of changes in plasma viral load and various CD4+ T-cell populations. Longitudinal analyses on 2-LTR sequences and plasma lipopolysaccharide (LPS levels did not reveal any significant changes in the same treatment period. CONCLUSIONS/SIGNIFICANCE: Our study revealed the rapid changes in 2-LTR concentration in a relatively large number of patients during the early HAART treatment. The rapid changes indicate the rapid infusion and clearance of cells bearing 2-LTR in the peripheral blood. Those changes are not expected to be caused by the blocking of viral integration, as our study did not include the integrase inhibitor raltegravir. Our study helps better understand the dynamics of HIV-DNA and its potential role as a biomarker for the diseases and for the treatment efficacy of HAART.

Trans-activation of the 5' to 3' viral DNA strand transfer by nucleocapsid protein during reverse transcription of HIV1 RNA.

Science.gov (United States)

Darlix, J L; Vincent, A; Gabus, C; de Rocquigny, H; Roques, B

1993-08-01

Two DNA strand transfer reactions take place during reverse transcription of the retroviral genome. The first transfer, that of the minus-strand strong stop DNA from the 5' end of the viral RNA to the 3' end, has been studied in vitro with two RNAs mimicking the 5' and 3' regions of the HIV1 genome and with nucleocapsid protein, NCp7, and reverse transcriptase. The results show that NCp7 strongly activates the 5' to 3' DNA strand transfer during reverse transcription while a basic peptide resembling NCp7 is inactive. Activation of the first transfer by several NCp7 derived peptides and the influence of the terminal redundancies (R) present at the 5' and 3' ends of HIV1 RNA were also examined. The first transfer is optimal in the presence of intact NCp7 and necessitates R on both the 5' and 3' RNAs. Sequencing of full length viral DNA products reveals approximately 40% misincorporations at the first nucleotide beyond the transfer point. If such base misincorporations occur during proviral DNA synthesis with possible homologous recombinations it may well contribute to the high level of genetic variability of HIV.

The proviral genome of radiation leukemia virus (RadLV): molecular cloning, restriction analysis and integration sites in tumor cell DNA

International Nuclear Information System (INIS)

Janowski, M.; Merregaert, J.; Nuyten, J.M.; Maisin, J.R.

1984-01-01

An infectious clone of the linear, unintegrated RadLV provirus was obtained by insertion in the plasmid pBR322. Its restriction map was indistinguishable from that of the majority of the multiple proviral copies, which are found apparently at random sites in the DNA of RadLV-induced rat thymic lymphomas [fr

SUN2 Modulates HIV-1 Infection and Latency through Association with Lamin A/C To Maintain the Repressive Chromatin.

Science.gov (United States)

Sun, Wei-Wei; Jiao, Shi; Sun, Li; Zhou, Zhaocai; Jin, Xia; Wang, Jian-Hua

2018-05-01

The postintegrational latency of HIV-1 is characterized by reversible silencing of long terminal repeat (LTR)-driven transcription of the HIV genome. It is known that the formation of repressive chromatin at the 5'-LTR of HIV-1 proviral DNA impedes viral transcription by blocking the recruitment of positive transcription factors. How the repressive chromatin is formed and modulated during HIV-1 infection remains elusive. Elucidation of which chromatin reassembly factor mediates the reorganization of chromatin is likely to facilitate the understanding of the host's modulation of HIV-1 transcription and latency. Here we revealed that "Sad1 and UNC84 domain containing 2" (SUN2), an inner nuclear membrane protein, maintained the repressive chromatin and inhibited HIV LTR-driven transcription of proviral DNA through an association with lamin A/C. Specifically, lamin A/C tethered SUN2 to the nucleosomes 1 and 2 of the HIV-1 5'-LTR to block the initiation and elongation of HIV-1 transcription. SUN2 knockdown converted chromatin to an active form and thus enhanced the phosphorylation of RNA polymerase II and its recruitment to the 5'-LTR HIV-1 proviral DNA, leading to reactivation of HIV-1 from latency. Conversely, the exogenous factors such as tumor necrosis factor alpha (TNF-α) induced reactivation, and the replication of HIV-1 led to the disassociation between SUN2 and lamin A/C, suggesting that disruption of the association between SUN2 and lamin A/C to convert the repressive chromatin to the active form might be a prerequisite for the initiation of HIV-1 transcription and replication. Together, our findings indicate that SUN2 is a novel chromatin reassembly factor that helps to maintain chromatin in a repressive state and consequently inhibits HIV-1 transcription. IMPORTANCE Despite the successful use of scores of antiretroviral drugs, HIV latency poses a major impediment to virus eradication. Elucidation of the mechanism of latency facilitates the discovery of new

Clinical Outcomes of Virologically-Suppressed Patients with Pre-existing HIV-1 Drug Resistance Mutations Switching to Rilpivirine/Emtricitabine/Tenofovir Disoproxil Fumarate in the SPIRIT Study.

Science.gov (United States)

Porter, Danielle P; Toma, Jonathan; Tan, Yuping; Solberg, Owen; Cai, Suqin; Kulkarni, Rima; Andreatta, Kristen; Lie, Yolanda; Chuck, Susan K; Palella, Frank; Miller, Michael D; White, Kirsten L

2016-02-01

Antiretroviral regimen switching may be considered for HIV-1-infected, virologically-suppressed patients to enable treatment simplification or improve tolerability, but should be guided by knowledge of pre-existing drug resistance. The current study examined the impact of pre-existing drug resistance mutations on virologic outcomes among virologically-suppressed patients switching to Rilpivirine (RPV)/emtricitabine (FTC)/tenofovir disoproxil fumarate (TDF). SPIRIT was a phase 3b study evaluating the safety and efficacy of switching to RPV/FTC/TDF in virologically-suppressed HIV-1-infected patients. Pre-existing drug resistance at baseline was determined by proviral DNA genotyping for 51 RPV/FTC/TDF-treated patients with known mutations by historical RNA genotype and matched controls and compared with clinical outcome at Week 48. Drug resistance mutations in protease or reverse transcriptase were detected in 62.7% of patients by historical RNA genotype and in 68.6% by proviral DNA genotyping at baseline. Proviral DNA sequencing detected 89% of occurrences of NRTI and NNRTI resistance-associated mutations reported by historical genotype. Mutations potentially affecting RPV activity, including E138A/G/K/Q, Y181C, and H221Y, were detected in isolates from 11 patients by one or both assays. None of the patients with single mutants had virologic failure through Week 48. One patient with pre-existing Y181Y/C and M184I by proviral DNA genotyping experienced virologic failure. Nineteen patients with K103N present by historical genotype were confirmed by proviral DNA sequencing and 18/19 remained virologically-suppressed. Virologic success rates were high among virologically-suppressed patients with pre-existing NRTI and NNRTI resistance-associated mutations who switched to RPV/FTC/TDF in the SPIRIT study. While plasma RNA genotyping remains preferred, proviral DNA genotyping may provide additional value in virologically-suppressed patients for whom historical resistance

Structure and possible function of a G-quadruplex in the long terminal repeat of the proviral HIV-1 genome.

Science.gov (United States)

De Nicola, Beatrice; Lech, Christopher J; Heddi, Brahim; Regmi, Sagar; Frasson, Ilaria; Perrone, Rosalba; Richter, Sara N; Phan, Anh Tuân

2016-07-27

The long terminal repeat (LTR) of the proviral human immunodeficiency virus (HIV)-1 genome is integral to virus transcription and host cell infection. The guanine-rich U3 region within the LTR promoter, previously shown to form G-quadruplex structures, represents an attractive target to inhibit HIV transcription and replication. In this work, we report the structure of a biologically relevant G-quadruplex within the LTR promoter region of HIV-1. The guanine-rich sequence designated LTR-IV forms a well-defined structure in physiological cationic solution. The nuclear magnetic resonance (NMR) structure of this sequence reveals a parallel-stranded G-quadruplex containing a single-nucleotide thymine bulge, which participates in a conserved stacking interaction with a neighboring single-nucleotide adenine loop. Transcription analysis in a HIV-1 replication competent cell indicates that the LTR-IV region may act as a modulator of G-quadruplex formation in the LTR promoter. Consequently, the LTR-IV G-quadruplex structure presented within this work could represent a valuable target for the design of HIV therapeutics. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

The GPT methodology. New fields of application

International Nuclear Information System (INIS)

Gandini, A.; Gomit, J.M.; Abramytchev, V.

1996-01-01

The GPT (Generalized Perturbation Theory) methodology is described, and a new application is discussed. The results obtained for a simple model (zero dimension, six parameters of interest) show that the expressions obtained using the GPT methodology, lead to results close to those obtained through direct calculations. The GPT methodology is useful to be used for radioactive waste disposal problems. The potentiality of the method linked to zero dimension model can be extended to radionuclide migration problems with space description. (K.A.)

Development of GPT-based optimization algorithm

International Nuclear Information System (INIS)

White, J.R.; Chapman, D.M.; Biswas, D.

1985-01-01

The University of Lowell and Westinghouse Electric Corporation are involved in a joint effort to evaluate the potential benefits of generalized/depletion perturbation theory (GPT/DTP) methods for a variety of light water reactor (LWR) physics applications. One part of that work has focused on the development of a GPT-based optimization algorithm for the overall design, analysis, and optimization of LWR reload cores. The use of GPT sensitivity data in formulating the fuel management optimization problem is conceptually straightforward; it is the actual execution of the concept that is challenging. Thus, the purpose of this paper is to address some of the major difficulties, to outline our approach to these problems, and to present some illustrative examples of an efficient GTP-based optimization scheme

Sensitive non-radioactive detection of HIV-1

DEFF Research Database (Denmark)

Teglbjærg, Lars Stubbe; Nielsen, C; Hansen, J E

1992-01-01

This report describes the use of the polymerase chain reaction (PCR) for the non-radioactive detection of HIV-1 proviral genomic sequences in HIV-1 infected cells. We have developed a sensitive assay, using three different sets of nested primers and our results show that this method is superior...... to standard PCR for the detection of HIV-1 DNA. The assay described features the use of a simple and inexpensive sample preparation technique and a non-radioactive hybridization procedure for confirmation of results. To test the suitability of the assay for clinical purposes, we tested cell samples from 76...

Depletion GPT-free sensitivity analysis for reactor eigenvalue problems

International Nuclear Information System (INIS)

Kennedy, C.; Abdel-Khalik, H.

2013-01-01

This manuscript introduces a novel approach to solving depletion perturbation theory problems without the need to set up or solve the generalized perturbation theory (GPT) equations. The approach, hereinafter denoted generalized perturbation theory free (GPT-Free), constructs a reduced order model (ROM) using methods based in perturbation theory and computes response sensitivity profiles in a manner that is independent of the number or type of responses, allowing for an efficient computation of sensitivities when many responses are required. Moreover, the reduction error from using the ROM is quantified in the GPT-Free approach by means of a Wilks' order statistics error metric denoted the K-metric. Traditional GPT has been recognized as the most computationally efficient approach for performing sensitivity analyses of models with many input parameters, e.g. when forward sensitivity analyses are computationally intractable. However, most neutronics codes that can solve the fundamental (homogenous) adjoint eigenvalue problem do not have GPT capabilities unless envisioned during code development. The GPT-Free approach addresses this limitation by requiring only the ability to compute the fundamental adjoint. This manuscript demonstrates the GPT-Free approach for depletion reactor calculations performed in SCALE6 using the 7x7 UAM assembly model. A ROM is developed for the assembly over a time horizon of 990 days. The approach both calculates the reduction error over the lifetime of the simulation using the K-metric and benchmarks the obtained sensitivities using sample calculations. (authors)

Prevalence of HIV infection in seronegative high-risk individuals examined by virus isolation and PCR

DEFF Research Database (Denmark)

Nielsen, C; Teglbjærg, Lars Stubbe; Pedersen, C

1991-01-01

HIV seronegative individuals with high-risk behavior were tested for HIV infection by sensitive virus isolation techniques using T4 lymphocytes and monocyte/macrophages, and by detection of proviral DNA using PCR with three different sets of nested primers. No evidence of HIV infection was found...... among the 31 seronegative high-risk subjects, either by virus isolation of by PCR (97.5% confidence limits, 0-11). Our results indicate that ongoing HIV infection in seronegative persons at high risk of infection is a rare event....

Effect of analytical treatment interruption and reinitiation of antiretroviral therapy on HIV reservoirs and immunologic parameters in infected individuals.

Science.gov (United States)

Clarridge, Katherine E; Blazkova, Jana; Einkauf, Kevin; Petrone, Mary; Refsland, Eric W; Justement, J Shawn; Shi, Victoria; Huiting, Erin D; Seamon, Catherine A; Lee, Guinevere Q; Yu, Xu G; Moir, Susan; Sneller, Michael C; Lichterfeld, Mathias; Chun, Tae-Wook

2018-01-01

Therapeutic strategies aimed at achieving antiretroviral therapy (ART)-free HIV remission in infected individuals are under active investigation. Considering the vast majority of HIV-infected individuals experience plasma viral rebound upon cessation of therapy, clinical trials evaluating the efficacy of curative strategies would likely require inclusion of ART interruption. However, it is unclear what impact short-term analytical treatment interruption (ATI) and subsequent reinitiation of ART have on immunologic and virologic parameters of HIV-infected individuals. Here, we show a significant increase of HIV burden in the CD4+ T cells of infected individuals during ATI that was correlated with the level of plasma viral rebound. However, the size of the HIV reservoirs as well as immune parameters, including markers of exhaustion and activation, returned to pre-ATI levels 6-12 months after the study participants resumed ART. Of note, the proportions of near full-length, genome-intact and structurally defective HIV proviral DNA sequences were similar prior to ATI and following reinitiation of ART. In addition, there was no evidence of emergence of antiretroviral drug resistance mutations within intact HIV proviral DNA sequences following reinitiation of ART. These data demonstrate that short-term ATI does not necessarily lead to expansion of the persistent HIV reservoir nor irreparable damages to the immune system in the peripheral blood, warranting the inclusion of ATI in future clinical trials evaluating curative strategies.

N-terminally truncated POM121C inhibits HIV-1 replication.

Directory of Open Access Journals (Sweden)

Hideki Saito

Full Text Available Recent studies have identified host cell factors that regulate early stages of HIV-1 infection including viral cDNA synthesis and orientation of the HIV-1 capsid (CA core toward the nuclear envelope, but it remains unclear how viral DNA is imported through the nuclear pore and guided to the host chromosomal DNA. Here, we demonstrate that N-terminally truncated POM121C, a component of the nuclear pore complex, blocks HIV-1 infection. This truncated protein is predominantly localized in the cytoplasm, does not bind to CA, does not affect viral cDNA synthesis, reduces the formation of 2-LTR and diminished the amount of integrated proviral DNA. Studies with an HIV-1-murine leukemia virus (MLV chimeric virus carrying the MLV-derived Gag revealed that Gag is a determinant of this inhibition. Intriguingly, mutational studies have revealed that the blockade by N-terminally-truncated POM121C is closely linked to its binding to importin-β/karyopherin subunit beta 1 (KPNB1. These results indicate that N-terminally-truncated POM121C inhibits HIV-1 infection after completion of reverse transcription and before integration, and suggest an important role for KPNB1 in HIV-1 replication.

Comparison of digital PCR platforms and semi-nested qPCR as a tool to determine the size of the HIV reservoir

NARCIS (Netherlands)

Bosman, K. J.; Nijhuis, M.; van Ham, P. M.; Wensing, A. M. J.; Vervisch, K.; Vandekerckhove, L.; De Spiegelaere, W.

2015-01-01

HIV persists in latently infected cells of patients on antiretroviral therapy (ART). This persistent proviral DNA reservoir is an important predictor of viral rebound upon therapy failure or interruption and forms a major obstacle towards cure. Accurate quantification of the low levels of persisting

Molecular cloning of human T-cell lymphotrophic virus type I-like proviral genome from the peripheral lymphocyte DNA of a patient with chronic neurologic disorders

International Nuclear Information System (INIS)

Reddy, E.P.; Mettus, R.V.; DeFreitas, E.; Wroblewska, Z.; Cisco, M.; Koprowski, H.

1988-01-01

Human T-cell lymphotropic virus type 1 (HTLV-I), the etiologic agent of human T-cell leukemia, has recently been shown to be associated with neurologic disorders such as tropical spastic paraparesis, HTLV-associated myelopathy, and possibly with multiple sclerosis. In this communication, the authors have examined one specific case of neurologic disorder that can be classified as multiple sclerosis or tropical spastic paraparesis. The patient suffering from chronic neurologic disorder was found to contain antibodies to HTLV-I envelope and gag proteins in his serum and cerebrospinal fluid. Lymphocytes from peripheral blood and cerebrospinal fluid of the patient were shown to express viral RNA sequences by in situ hybridization. Southern blot analysis of the patient lymphocyte DNA revealed the presence of HTLV-I-related sequences. Blot-hybridization analysis of the RNA from fresh peripheral lymphocytes stimulated with interleukin 2 revealed the presence of abundant amounts of genomic viral RNA with little or no subgenomic RNA. They have clones the proviral genome from the DNA of the peripheral lymphocytes and determined its restriction map. This analysis shows that this proviral genome is very similar if not identical to that of the prototype HTLV-I genome

Resistance mutations and CTL epitopes in archived HIV-1 DNA of patients on antiviral treatment: toward a new concept of vaccine.

Directory of Open Access Journals (Sweden)

Jennifer Papuchon

Full Text Available Eleven patients responding successfully to first-line antiretroviral therapy (ART were investigated for proviral drug resistance mutations (DRMs in RT by ultra-deep pyrosequencing (UDPS. After molecular typing of the class I alleles A and B, the CTL epitopes in the Gag, Nef and Pol regions of the provirus were sequenced and compared to the reference HXB2 HIV-1 epitopes. They were then matched with the HLA alleles with determination of theoretical affinity (TA. For 3 patients, the results could be compared with an RNA sample of the circulating virus at initiation of therapy. Five out of 11 patients exhibited DRMs by UDPS. The issue is whether a therapeutic switch is relevant in these patients by taking into account the identity of the archived resistance mutations. When the archived CTL epitopes were determined on the basis of the HLA alleles, different patterns were observed. Some epitopes were identical to those reported for the reference with the same TA, while others were mutated with a decrease in TA. In 2 cases, an epitope was observed as a combination of subpopulations at entry and was retrieved as a single population with lower TA at success. With regard to immunological stimulation and given the variability of the archived CTL epitopes, we propose a new concept of curative vaccine based on identification of HIV-1 CTL epitopes after prior sequencing of proviral DNA and matching with HLA class I alleles.

Sensitive quantification of the HIV-1 reservoir in gut-associated lymphoid tissue.

Science.gov (United States)

Morón-López, Sara; Puertas, Maria C; Gálvez, Cristina; Navarro, Jordi; Carrasco, Anna; Esteve, Maria; Manyé, Josep; Crespo, Manel; Salgado, Maria; Martinez-Picado, Javier

2017-01-01

The implementation of successful strategies to achieve an HIV cure has become a priority in HIV research. However, the current location and size of HIV reservoirs is still unknown since there are limited tools to evaluate HIV latency in viral sanctuaries such as gut-associated lymphoid tissue (GALT). As reported in the so called "Boston Patients", despite undetectable levels of proviral HIV-1 DNA in blood and GALT, viral rebound happens in just few months after ART interruption. This fact might imply that current methods are not sensitive enough to detect residual reservoirs. Showing that, it is imperative to improve the detection and quantification of HIV-1 reservoir in tissue samples. Herein, we propose a novel non-enzymatic protocol for purification of Lamina Propria Leukocytes (LPL) from gut biopsies combined to viral HIV DNA (vDNA) quantification by droplet digital PCR (ddPCR) to improve the sensitivity and accuracy of viral reservoir measurements (LPL-vDNA assay). Endoscopic ileum biopsies were sampled from 12 HIV-1-infected cART-suppressed subjects. We performed a DTT/EDTA-based treatment for epithelial layer removal followed by non-enzymatic disruption of the tissue to obtain lamina propria cell suspension (LP). CD45+ cells were subsequently purified by flow sorting and vDNA was determined by ddPCR. vDNA quantification levels were significantly higher in purified LPLs (CD45+) than in bulk LPs (pgut-associated viral sanctuaries, which might be used to evaluate any proposed eradication strategy.

HIV shedding in cervico-vaginal secretions in pregnant women.

Science.gov (United States)

Gardella, Barbara; Roccio, Marianna; Maccabruni, Anna; Mariani, Bianca; Panzeri, Lucia; Zara, Francesca; Spinillo, Arsenio

2011-07-01

The purpose of this study was to evaluate the presence of HIV-1 in cervico-vaginal secretions of pregnant as compared to non-pregnant HIV-seropositive women. We compared 43 known HIV seropositive pregnant patients versus 241 age-matched (± 2 years) control non-pregnant HIV-seropositive subjects. In pregnant patients blood and cervico-vaginal samples were obtained during each trimester of pregnancy. In control subjects the same samples were obtained at enrolment. HIV-1 RNA was measured in plasma; proviral HIV-1 DNA, cell-associated and cell-free HIV-1 RNA in cervico-vaginal secretion by competitive polymerase chain reaction (cRT-PCR) and reverse transcriptase PCR. The genital shedding of HIV-DNA (22/43 as compared to 79/241, p = 0.02), and cell-free HIV-RNA detection (26/43 as compared to 72/241, p pregnant than in non pregnant women. Pregnancy correlated with a significant positive trend in the cervico-vaginal load of HIV-DNA (Spearman Rho= 0.149, p= 0.012), and cell-free HIV-RNA (Spearman Rho= 0.253, p HIV-RNA transcripts (Spearman Rho = 0.06, p= 0.31). After correction for potential confounders, first trimester pregnant women had increased rates of genital HIV- DNA (odds ratio = 1.94, 95% confidence interval = 1.01 3.78) and cell-free HIV-RNA (odds ratio = 4.07, 95% confidence interval = 1.97 8.41) detection compared to nonpregnant controls. The shedding of genital HIV was increased in pregnant compared to non pregnant subjects, even in patients with undetectable viremia. In this low-risk HIV-positive population the risks of vertical or horizontal transmissions should not be underestimated.

The prion protein has RNA binding and chaperoning properties characteristic of nucleocapsid protein NCP7 of HIV-1.

Science.gov (United States)

Gabus, C; Derrington, E; Leblanc, P; Chnaiderman, J; Dormont, D; Swietnicki, W; Morillas, M; Surewicz, W K; Marc, D; Nandi, P; Darlix, J L

2001-06-01

Transmissible spongiform encephalopathies are fatal neurodegenerative diseases associated with the accumulation of a protease-resistant form of the prion protein (PrP). Although PrP is conserved in vertebrates, its function remains to be identified. In vitro PrP binds large nucleic acids causing the formation of nucleoprotein complexes resembling human immunodeficiency virus type 1 (HIV-1) nucleocapsid-RNA complexes and in vivo MuLV replication accelerates the scrapie infectious process, suggesting possible interactions between retroviruses and PrP. Retroviruses, including HIV-1 encode a major nucleic acid binding protein (NC protein) found within the virus where 2000 NC protein molecules coat the dimeric genome. NC is required in virus assembly and infection to chaperone RNA dimerization and packaging and in proviral DNA synthesis by reverse transcriptase (RT). In HIV-1, 5'-leader RNA/NC interactions appear to control these viral processes. This prompted us to compare and contrast the interactions of human and ovine PrP and HIV-1 NCp7 with HIV-1 5'-leader RNA. Results show that PrP has properties characteristic of NCp7 with respect to viral RNA dimerization and proviral DNA synthesis by RT. The NC-like properties of huPrP map to the N-terminal region of huPrP. Interestingly, PrP localizes in the membrane and cytoplasm of PrP-expressing cells. These findings suggest that PrP is a multifunctional protein possibly participating in nucleic acid metabolism.

In vitro nuclear interactome of the HIV-1 Tat protein.

LENUS (Irish Health Repository)

Gautier, Virginie W

2009-01-01

BACKGROUND: One facet of the complexity underlying the biology of HIV-1 resides not only in its limited number of viral proteins, but in the extensive repertoire of cellular proteins they interact with and their higher-order assembly. HIV-1 encodes the regulatory protein Tat (86-101aa), which is essential for HIV-1 replication and primarily orchestrates HIV-1 provirus transcriptional regulation. Previous studies have demonstrated that Tat function is highly dependent on specific interactions with a range of cellular proteins. However they can only partially account for the intricate molecular mechanisms underlying the dynamics of proviral gene expression. To obtain a comprehensive nuclear interaction map of Tat in T-cells, we have designed a proteomic strategy based on affinity chromatography coupled with mass spectrometry. RESULTS: Our approach resulted in the identification of a total of 183 candidates as Tat nuclear partners, 90% of which have not been previously characterised. Subsequently we applied in silico analysis, to validate and characterise our dataset which revealed that the Tat nuclear interactome exhibits unique signature(s). First, motif composition analysis highlighted that our dataset is enriched for domains mediating protein, RNA and DNA interactions, and helicase and ATPase activities. Secondly, functional classification and network reconstruction clearly depicted Tat as a polyvalent protein adaptor and positioned Tat at the nexus of a densely interconnected interaction network involved in a range of biological processes which included gene expression regulation, RNA biogenesis, chromatin structure, chromosome organisation, DNA replication and nuclear architecture. CONCLUSION: We have completed the in vitro Tat nuclear interactome and have highlighted its modular network properties and particularly those involved in the coordination of gene expression by Tat. Ultimately, the highly specialised set of molecular interactions identified will

Inhibition of HIV Expression and Integration in Macrophages by Methylglyoxal-Bis-Guanylhydrazone.

Science.gov (United States)

Jin, Xia; McGrath, Michael S; Xu, Hua

2015-11-01

Macrophages are a target for infection with HIV and represent one of the viral reservoirs that are relatively resistant to current antiretroviral drugs. Here we demonstrate that methylglyoxal-bis-guanylhydrazone (MGBG), a polyamine analog and potent S-adenosylmethionine decarboxylase inhibitor, decreases HIV expression in monocytes and macrophages. MGBG is selectively concentrated by these cells through a mechanism consistent with active transport by the polyamine transporter. Using a macrophage-tropic reporter virus tagged with the enhanced green fluorescent protein, we demonstrate that MGBG decreases the frequency of HIV-infected cells. The effect is dose dependent and correlates with the production of HIV p24 in culture supernatants. This anti-HIV effect was further confirmed using three macrophage-tropic primary HIV isolates. Viral life cycle mapping studies show that MGBG inhibits HIV DNA integration into the cellular DNA in both monocytes and macrophages. Our work demonstrates for the first time the selective concentration of MGBG by monocytes/macrophages, leading to the inhibition of HIV-1 expression and a reduction in proviral load within macrophage cultures. These results suggest that MGBG may be useful in adjunctive macrophage-targeted therapy for HIV infection. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Design and implementation of an external quality assessment program for HIV viral load measurements using dried blood spots.

Science.gov (United States)

Prach, Lisa M; Puren, Adrian; Lippman, Sheri A; Carmona, Sergio; Stephenson, Sophie; Cutler, Ewalde; Barnhart, Scott; Liegler, Teri

2015-03-01

An external quality assurance program was developed for HIV-1 RNA viral load measurements taken from dried blood spots using a reference panel and field-collected specimens. The program demonstrated that accurate and reproducible quantitation can be obtained from field-collected specimens. Residual proviral DNA may confound interpretation in virologically suppressed subjects. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

APOBEC3G inhibits HIV-1 RNA elongation by inactivating the viral trans-activation response element.

Science.gov (United States)

Nowarski, Roni; Prabhu, Ponnandy; Kenig, Edan; Smith, Yoav; Britan-Rosich, Elena; Kotler, Moshe

2014-07-29

Deamination of cytidine residues in viral DNA is a major mechanism by which APOBEC3G (A3G) inhibits vif-deficient human immunodeficiency virus type 1 (HIV-1) replication. dC-to-dU transition following RNase-H activity leads to viral cDNA degradation, production of non-functional proteins, formation of undesired stop codons and decreased viral protein synthesis. Here, we demonstrate that A3G provides an additional layer of defense against HIV-1 infection dependent on inhibition of proviral transcription. HIV-1 transcription elongation is regulated by the trans-activation response (TAR) element, a short stem-loop RNA structure required for elongation factors binding. Vif-deficient HIV-1-infected cells accumulate short viral transcripts and produce lower amounts of full-length HIV-1 transcripts due to A3G deamination of the TAR apical loop cytidine, highlighting the requirement for TAR loop integrity in HIV-1 transcription. We further show that free single-stranded DNA (ssDNA) termini are not essential for A3G activity and a gap of CCC motif blocked with juxtaposed DNA or RNA on either or 3'+5' ends is sufficient for A3G deamination. These results identify A3G as an efficient mutator and that deamination of (-)SSDNA results in an early block of HIV-1 transcription. Copyright © 2014 Elsevier Ltd. All rights reserved.

DNA/MVA Vaccines for HIV/AIDS

Directory of Open Access Journals (Sweden)

Smita S. Iyer

2014-02-01

Full Text Available Since the initial proof-of-concept studies examining the ability of antigen-encoded plasmid DNA to serve as an immunogen, DNA vaccines have evolved as a clinically safe and effective platform for priming HIV-specific cellular and humoral responses in heterologous “prime-boost” vaccination regimens. Direct injection of plasmid DNA into the muscle induces T- and B-cell responses against foreign antigens. However, the insufficient magnitude of this response has led to the development of approaches for enhancing the immunogenicity of DNA vaccines. The last two decades have seen significant progress in the DNA-based vaccine platform with optimized plasmid constructs, improved delivery methods, such as electroporation, the use of molecular adjuvants and novel strategies combining DNA with viral vectors and subunit proteins. These innovations are paving the way for the clinical application of DNA-based HIV vaccines. Here, we review preclinical studies on the DNA-prime/modified vaccinia Ankara (MVA-boost vaccine modality for HIV. There is a great deal of interest in enhancing the immunogenicity of DNA by engineering DNA vaccines to co-express immune modulatory adjuvants. Some of these adjuvants have demonstrated encouraging results in preclinical and clinical studies, and these data will be examined, as well.

Transgenic Chinese hamster V79 cell lines which exhibit variable levels of gpt mutagenesis

International Nuclear Information System (INIS)

Klein, C.B.; Rossman, T.G.

1990-01-01

The Escherichia coli gpt gene coding for xanthine-guanine phosphoribosyl transferase has been stably transfected into HPRT - Chinese hamster V79 cells. Several gpt - cell lines have been established, which retain the sequence(s) even after long-term culture without selection for gpt. While spontaneous mutagenesis to gpt - occurs rather frequently for most cell lines, it cannot be correlated with either the number of plasmid integration sites or deletion of the plasmid sequence(s). One transgenic cell line (g12), which continuously maintains a low spontaneous mutation frequency was used in comparative mutagenesis studies with wild-type V79 cells (gpt vs. hprt). Alkylating agents such as N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and β-propiolactone (BPL) are shown to be equally toxic and mutagenic in both g12 and V79 cells. UV and X-rays are also equally toxic to both cell lines. The data presented here suggests that g12 cells may be useful to study mammalian mutagenesis by agents which yield limited response at the hprt locus

Filtration Isolation of Nucleic Acids: A Simple and Rapid DNA Extraction Method.

Science.gov (United States)

McFall, Sally M; Neto, Mário F; Reed, Jennifer L; Wagner, Robin L

2016-08-06

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad to extract cellular DNA from whole blood in less than 2 min. The blood specimen is treated with detergent, mixed briefly and applied by pipet to the separation membrane. The lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the separation membrane. The extracted DNA is retained on the membrane during a simple wash step wherein PCR inhibitors are wicked into the absorbent blotting pad. The membrane containing the entrapped DNA is then added to the PCR reaction without further purification. This simple method does not require laboratory equipment and can be easily implemented with inexpensive laboratory supplies. Here we describe a protocol for highly sensitive detection and quantitation of HIV-1 proviral DNA from 100 µl whole blood as a model for early infant diagnosis of HIV that could readily be adapted to other genetic targets.

Sensitive quantification of the HIV-1 reservoir in gut-associated lymphoid tissue.

Directory of Open Access Journals (Sweden)

Sara Morón-López

Full Text Available The implementation of successful strategies to achieve an HIV cure has become a priority in HIV research. However, the current location and size of HIV reservoirs is still unknown since there are limited tools to evaluate HIV latency in viral sanctuaries such as gut-associated lymphoid tissue (GALT. As reported in the so called "Boston Patients", despite undetectable levels of proviral HIV-1 DNA in blood and GALT, viral rebound happens in just few months after ART interruption. This fact might imply that current methods are not sensitive enough to detect residual reservoirs. Showing that, it is imperative to improve the detection and quantification of HIV-1 reservoir in tissue samples. Herein, we propose a novel non-enzymatic protocol for purification of Lamina Propria Leukocytes (LPL from gut biopsies combined to viral HIV DNA (vDNA quantification by droplet digital PCR (ddPCR to improve the sensitivity and accuracy of viral reservoir measurements (LPL-vDNA assay.Endoscopic ileum biopsies were sampled from 12 HIV-1-infected cART-suppressed subjects. We performed a DTT/EDTA-based treatment for epithelial layer removal followed by non-enzymatic disruption of the tissue to obtain lamina propria cell suspension (LP. CD45+ cells were subsequently purified by flow sorting and vDNA was determined by ddPCR.vDNA quantification levels were significantly higher in purified LPLs (CD45+ than in bulk LPs (p<0.01. The levels of vDNA were higher in ileum samples than in concurrent PBMC from the same individuals (p = 0.002. As a result of the increased sensitivity of this purification method, the Poisson 95% confidence intervals of the vDNA quantification data from LPLs were narrower than that from bulk LPs. Of note, vDNA was unambiguously quantified above the detection limit in 100% of LPL samples, while only in 58% of bulk LPs.We propose an innovative combined protocol for a more sensitive detection of the HIV reservoir in gut-associated viral sanctuaries

Formation of stable and functional HIV-1 nucleoprotein complexes in vitro.

Science.gov (United States)

Tanchou, V; Gabus, C; Rogemond, V; Darlix, J L

1995-10-06

HIV genomic RNA resides within the nucleocapsid, in the interior of the virus, which serves to protect the RNA against nuclease degradation and to promote its reverse transcription. To investigate the role of nucleocapsid protein (NCp7) in the stability and replication of genomic RNA within the nucleocapsid, we used NCp7, reverse transcriptase (RT) and RNAs representing the 5' and 3' regions of the genome to reconstitute functional HIV-1 nucleocapsids. The nucleoprotein complexes generated in vitro were found to be stable, which, according to biochemical and genetic data, probably results from the tight binding of NCp7 molecules to the RNA and strong NCp7/NCp7 interactions. The nucleoprotein complexes efficiently protected viral RNA against RNase degradation and, at the same time, promoted viral DNA synthesis by RT. DNA strand transfer from the 5' to the 3' RNA template was very efficient in nucleoprotein complexes formed in the presence of both RNAs, but not when the RNAs were in separate complexes. These results indicate that the in vitro reconstituted HIV-1 nucleoprotein complexes function like virion nucleocapsids and thus provide a way to study at the molecular level this viral substructure and the synthesis of proviral DNA, and to search for new anti-HIV agents.

Marcadores virológicos no convencionales en pacientes infectados con el virus de la inmunodeficiencia humana: ADN HIV-T, ADN HIV- 2LTR y ARN de HIV Non conventional virological markers in HIV-infected patients: T-HIV DNA, 2LTR-HIV DNA and HIV RNA

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Rosana Gariglio

2004-10-01

Full Text Available La terapia antirretroviral de alta eficacia (TAAE induce una reducción marcada y persistente de la viremia plasmática, contribuyendo a disminuir la mortalidad y morbilidad de los pacientes HIV-positivos. Así, la carga viral (CV es el método de referencia para evaluar la eficacia terapéutica. Sin embargo, aun en presencia de una TAAE eficiente no se ha logrado la erradicación viral. En este estudio analizamos la presencia del ADN total de HIV (ADN HIV-T, del ADN no integrado con 2LTR (ADN HIV-2LTR y del ARN de HIV, en un grupo de 55 pacientes HIV-positivos en distintos estadios clínicos, con y sin TAAE, mediante ensayos de PCR con revelado colorimétrico en microplaca, optimizados en nuestro laboratorio. La sensibilidad clínica del ARN del HIV fue evaluada con el bDNA, resultando del 74% y del 64%, respectivamente, con una concordancia del 85%. Este ensayo podría ser utilizado en el seguimiento de pacientes bajo TAAE. El ADN HIV-2LTR resultó positivo en el 54% aunque estuvo ausente en pacientes con elevada CV. Este marcador se consideraba un producto lábil y su presencia se asociaba a infección reciente. Sin embargo, actuales evidencias ponen en discusión su estabilidad por lo que su significado clínico debe ser reconsiderado. La ausencia del ADN HIV-2LTR en pacientes con CV detectable puede relacionarse con la heterogeneidad de la secuencia utilizada para su detección. El ADN HIV-T estuvo presente en el 100% de las muestras y resultaría relevante como marcador de remisión cuando se dispongan de terapias que efectivamente erradiquen la infección.Highly active antiretroviral therapy (HAART induces a persistent reduction of the plasmatic viremia, contributing to decrease mortality and morbidity of infected people with human immunodeficiency virus (HIV. Thus, viral load (VL is the reference method to evaluate therapy effectiveness. However, even in the presence of efficient HAART viral eradication was yet not achieved. In this

Peripheral blood mononuclear cells HIV DNA levels impact intermittently on neurocognition.

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Lucette A Cysique

Full Text Available To determine the contribution of peripheral blood mononuclear cells' (PBMCs HIV DNA levels to HIV-associated dementia (HAD and non-demented HIV-associated neurocognitive disorders (HAND in chronically HIV-infected adults with long-term viral suppression on combined antiretroviral treatment (cART.Eighty adults with chronic HIV infection on cART (>97% with plasma and CSF HIV RNA <50 copies/mL were enrolled into a prospective observational cohort and underwent assessments of neurocognition and pre-morbid cognitive ability at two visits 18 months apart. HIV DNA in PBMCs was measured by real-time PCR at the same time-points.At baseline, 46% had non-demented HAND; 7.5% had HAD. Neurocognitive decline occurred in 14% and was more likely in those with HAD (p<.03. Low pre-morbid cognitive ability was uniquely associated with HAD (p<.05. Log10 HIV DNA copies were stable between study visits (2.26 vs. 2.22 per 106 PBMC. Baseline HIV DNA levels were higher in those with lower pre-morbid cognitive ability (p<.04, and higher in those with no ART treatment during HIV infection 1st year (p = .03. Baseline HIV DNA was not associated with overall neurocognition. However, % ln HIV DNA change was associated with decline in semantic fluency in unadjusted and adjusted analyses (p = .01-.03, and motor-coordination (p = .02-.12 to a lesser extent.PBMC HIV DNA plays a role in HAD pathogenesis, and this is moderated by pre-morbid cognitive ability in the context of long-term viral suppression. While the HIV DNA levels in PBMC are not associated with current non-demented HAND, increasing HIV DNA levels were associated with a decline in neurocognitive functions associated with HAND progression.

Retroviral DNA Integration Directed by HIV Integration Protein in Vitro

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Bushman, Frederic D.; Fujiwara, Tamio; Craigie, Robert

1990-09-01

Efficient retroviral growth requires integration of a DNA copy of the viral RNA genome into a chromosome of the host. As a first step in analyzing the mechanism of integration of human immunodeficiency virus (HIV) DNA, a cell-free system was established that models the integration reaction. The in vitro system depends on the HIV integration (IN) protein, which was partially purified from insect cells engineered to express IN protein in large quantities. Integration was detected in a biological assay that scores the insertion of a linear DNA containing HIV terminal sequences into a λ DNA target. Some integration products generated in this assay contained five-base pair duplications of the target DNA at the recombination junctions, a characteristic of HIV integration in vivo; the remaining products contained aberrant junctional sequences that may have been produced in a variation of the normal reaction. These results indicate that HIV IN protein is the only viral protein required to insert model HIV DNA sequences into a target DNA in vitro.

Defective proviruses rapidly accumulate during acute HIV-1 infection

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Bruner, Katherine M.; Murray, Alexandra J.; Pollack, Ross A.; Soliman, Mary G.; Laskey, Sarah B.; Capoferri, Adam A.; Lai, Jun; Strain, Matthew C.; Lada, Steven M.; Hoh, Rebecca; Ho, Ya-Chi; Richman, Douglas D.; Deeks, Steven G.; Siliciano, Janet D.; Siliciano, Robert F.

2016-01-01

Although antiretroviral therapy (ART) suppresses viral replication to clinically undetectable levels, HIV-1 persists in CD4+ T cells in a latent form not targeted by the immune system or ART1–5. This latent reservoir is a major barrier to cure. Many individuals initiate ART during chronic infection, and in this setting, most proviruses are defective6. However, the dynamics of the accumulation and persistence of defective proviruses during acute HIV-1 infection are largely unknown. Here we show that defective proviruses accumulate rapidly within the first few weeks of infection to make up over 93% of all proviruses, regardless of how early ART is initiated. Using an unbiased method to amplify near full-length proviral genomes from HIV-1 infected adults treated at different stages of infection, we demonstrate that early ART initiation limits the size of the reservoir but does not profoundly impact the proviral landscape. This analysis allows us to revise our understanding of the composition of proviral populations and estimate the true reservoir size in individuals treated early vs. late in infection. Additionally, we demonstrate that common assays for measuring the reservoir do not correlate with reservoir size. These findings reveal hurdles that must be overcome to successfully analyze future HIV-1 cure strategies. PMID:27500724

Frequency of subtype B and F1 dual infection in HIV-1 positive, Brazilian men who have sex with men

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Soares de Oliveira Ana

2012-09-01

Full Text Available Abstract Background Because various HIV vaccination studies are in progress, it is important to understand how often inter- and intra-subtype co/superinfection occurs in different HIV-infected high-risk groups. This knowledge would aid in the development of future prevention programs. In this cross-sectional study, we report the frequency of subtype B and F1 co-infection in a clinical group of 41 recently HIV-1 infected men who have sex with men (MSM in São Paulo, Brazil. Methodology Proviral HIV-1 DNA was isolated from subject's peripheral blood polymorphonuclear leukocytes that were obtained at the time of enrollment. Each subject was known to be infected with a subtype B virus as determined in a previous study. A small fragment of the integrase gene (nucleotide 4255–4478 of HXB2 was amplified by nested polymerase chain reaction (PCR using subclade F1 specific primers. The PCR results were further confirmed by phylogenetic analysis. Viral load (VL data were extrapolated from the medical records of each patient. Results For the 41 samples from MSM who were recently infected with subtype B virus, it was possible to detect subclade F1 proviral DNA in five patients, which represents a co-infection rate of 12.2%. In subjects with dual infection, the median VL was 5.3 × 104 copies/ML, whereas in MSM that were infected with only subtype B virus the median VL was 3.8 × 104 copies/ML (p > 0.8. Conclusions This study indicated that subtype B and F1 co-infection occurs frequently within the HIV-positive MSM population as suggested by large number of BF1 recombinant viruses reported in Brazil. This finding will help us track the epidemic and provide support for the development of immunization strategies against the HIV.

Generalized perturbation theory (GPT) methods. A heuristic approach

International Nuclear Information System (INIS)

Gandini, A.

1987-01-01

Wigner first proposed a perturbation theory as early as 1945 to study fundamental quantities such as the reactivity worths of different materials. The first formulation, CPT, for conventional perturbation theory is based on universal quantum mechanics concepts. Since that early conception, significant contributions have been made to CPT, in particular, Soodak, who rendered a heuristic interpretation of the adjoint function, (referred to as the GPT method for generalized perturbation theory). The author illustrates the GPT methodology in a variety of linear and nonlinear domains encountered in nuclear reactor analysis. The author begins with the familiar linear neutron field and then generalizes the methodology to other linear and nonlinear fields, using heuristic arguments. The author believes that the inherent simplicity and elegance of the heuristic derivation, although intended here for reactor physics problems might be usefully adopted in collateral fields and includes such examples

Comparison of HTLV-I Proviral Load in Adult T Cell Leukemia/Lymphoma (ATL), HTLV-I-Associated Myelopathy (HAM-TSP) and Healthy Carriers.

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Akbarin, Mohammad Mehdi; Rahimi, Hossein; Hassannia, Tahereh; Shoja Razavi, Ghazaleh; Sabet, Faezeh; Shirdel, Abbas

2013-03-01

Human T Lymphocyte Virus Type one (HTLV-I) is a retrovirus that infects about 10-20 million people worldwide. Khorasan province in Iran is an endemic area. The majority of HTLV-I-infected individuals sustain healthy carriers but small proportion of infected population developed two progressive diseases: HAM/TSP and ATL. The proviral load could be a virological marker for disease monitoring, therefore in the present study HTLV-I proviral load has been evaluated in ATL and compared to HAM/TSP and healthy carriers. In this case series study, 47 HTLV-I infected individuals including 13 ATL, 23 HAM/TSP and 11 asymptomatic subjects were studied. Peripheral blood mononuclear cells (PBMCs) were investigated for presence of HTLV-I DNA provirus by PCR using LTR and Tax fragments. Then in infected subjects, HTLV-I proviral load was measured using real time PCR TaqMan method. The average age of patients in ATL was 52±8, in HAM/TSP 45.52±15.17 and in carrier's 38.65±14.9 years which differences were not statistically significant. The analysis of data showed a significant difference in mean WBC among study groups (ATL vs HAM/TSP and carriers P=0.0001). Moreover, mean HTLV-I proviral load was 11967.2 ± 5078, 409 ± 71.3 and 373.6 ± 143.3 in ATL, HAM/TSP and Healthy Carriers, respectively. The highest HTLV-I proviral load was measured in ATL group that had a significant correlation with WBC count (R=0.495, P=0.001). The proviral load variations between study groups was strongly significant (ATL vs carrier P=0.0001; ATL vs HAM/TSP P= 0.0001 and HAM/TSP vs carriers P< 0.05). Conclusion : The present study demonstrated that HTLV-I proviral load was higher in ATL group in comparison with HAM/TSP and healthy carriers. Therefore, HTLV-I proviral load is a prognostic factor for development of HTLV-I associated diseases and can be used as a monitoring marker for the efficiency of therapeutic regime.

Cell-associated HIV DNA measured early during infection has prognostic value independent of serum HIV RNA measured concomitantly

DEFF Research Database (Denmark)

Katzenstein, Terese L; Oliveri, Roberto S; Benfield, Thomas

2002-01-01

Using data from the Danish AIDS Cohort of HIV-infected homosexual men established in the 1980s, the prognostic value of early HIV DNA loads was evaluated. In addition to DNA measurements, concomitant serum HIV RNA levels, CD4 cell counts and CCR5 genotypes were determined. The patients were divided...... into 3 groups, according to whether their cell-associated HIV DNA load was or = 2,500 DNA copies/10(6) peripheral blood mononuclear cells. Clinical progression rates differed significantly between the groups (p value independent...... of serum HIV RNA (p value. Patients heterozygous for the CCR5 delta 32 allele had significantly lower HIV DNA loads than those homozygous for the normal allele (p

HIV-DNA Given with or without Intradermal Electroporation Is Safe and Highly Immunogenic in Healthy Swedish HIV-1 DNA/MVA Vaccinees: A Phase I Randomized Trial.

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Charlotta Nilsson

Full Text Available We compared safety and immunogenicity of intradermal (ID vaccination with and without electroporation (EP in a phase I randomized placebo-controlled trial of an HIV-DNA prime HIV-MVA boost vaccine in healthy Swedish volunteers.HIV-DNA plasmids encoding HIV-1 genes gp160 subtypes A, B and C; Rev B; Gag A and B and RTmut B were given ID at weeks 0, 6 and 12 in a dose of 0.6 mg. Twenty-five volunteers received vaccine using a needle-free device (ZetaJet with (n=16 or without (n=9 ID EP (Dermavax. Five volunteers were placebo recipients. Boosting with recombinant MVA-CMDR expressing HIV-1 Env, Gag, Pol of CRF01_AE (HIV-MVA or placebo was performed at weeks 24 and 40. Nine of the vaccinees received a subtype C CN54 gp140 protein boost together with HIV-MVA.The ID/EP delivery was very well tolerated. After three HIV-DNA immunizations, no statistically significant difference was seen in the IFN-γ ELISpot response rate to Gag between HIV-DNA ID/EP recipients (5/15, 33% and HIV-DNA ID recipients (1/7, 14%, p=0.6158. The first HIV-MVA or HIV-MVA+gp140 vaccination increased the IFN-γ ELISpot response rate to 18/19 (95%. CD4+ and/or CD8+ T cell responses to Gag or Env were demonstrable in 94% of vaccinees. A balanced CD4+ and CD8+ T cell response was noted, with 78% and 71% responders, respectively. IFN-γ and IL-2 dominated the CD4+ T cell response to Gag and Env. The CD8+ response to Gag was broader with expression of IFN-γ, IL-2, MIP-1β and/or CD107. No differences were seen between DNA vaccine groups. Binding antibodies were induced after the second HIV-MVA+/-gp140 in 93% of vaccinees to subtype C Env, with the highest titers among EP/gp140 recipients.Intradermal electroporation of HIV-DNA was well tolerated. Strong cell- and antibody-mediated immune responses were elicited by the HIV-DNA prime and HIV-MVA boosting regimen, with or without intradermal electroporation use.International Standard Randomised Controlled Trial Number (ISRCTN 60284968.

HIV-DNA Given with or without Intradermal Electroporation Is Safe and Highly Immunogenic in Healthy Swedish HIV-1 DNA/MVA Vaccinees: A Phase I Randomized Trial.

Science.gov (United States)

Nilsson, Charlotta; Hejdeman, Bo; Godoy-Ramirez, Karina; Tecleab, Teghesti; Scarlatti, Gabriella; Bråve, Andreas; Earl, Patricia L; Stout, Richard R; Robb, Merlin L; Shattock, Robin J; Biberfeld, Gunnel; Sandström, Eric; Wahren, Britta

2015-01-01

We compared safety and immunogenicity of intradermal (ID) vaccination with and without electroporation (EP) in a phase I randomized placebo-controlled trial of an HIV-DNA prime HIV-MVA boost vaccine in healthy Swedish volunteers. HIV-DNA plasmids encoding HIV-1 genes gp160 subtypes A, B and C; Rev B; Gag A and B and RTmut B were given ID at weeks 0, 6 and 12 in a dose of 0.6 mg. Twenty-five volunteers received vaccine using a needle-free device (ZetaJet) with (n=16) or without (n=9) ID EP (Dermavax). Five volunteers were placebo recipients. Boosting with recombinant MVA-CMDR expressing HIV-1 Env, Gag, Pol of CRF01_AE (HIV-MVA) or placebo was performed at weeks 24 and 40. Nine of the vaccinees received a subtype C CN54 gp140 protein boost together with HIV-MVA. The ID/EP delivery was very well tolerated. After three HIV-DNA immunizations, no statistically significant difference was seen in the IFN-γ ELISpot response rate to Gag between HIV-DNA ID/EP recipients (5/15, 33%) and HIV-DNA ID recipients (1/7, 14%, p=0.6158). The first HIV-MVA or HIV-MVA+gp140 vaccination increased the IFN-γ ELISpot response rate to 18/19 (95%). CD4+ and/or CD8+ T cell responses to Gag or Env were demonstrable in 94% of vaccinees. A balanced CD4+ and CD8+ T cell response was noted, with 78% and 71% responders, respectively. IFN-γ and IL-2 dominated the CD4+ T cell response to Gag and Env. The CD8+ response to Gag was broader with expression of IFN-γ, IL-2, MIP-1β and/or CD107. No differences were seen between DNA vaccine groups. Binding antibodies were induced after the second HIV-MVA+/-gp140 in 93% of vaccinees to subtype C Env, with the highest titers among EP/gp140 recipients. Intradermal electroporation of HIV-DNA was well tolerated. Strong cell- and antibody-mediated immune responses were elicited by the HIV-DNA prime and HIV-MVA boosting regimen, with or without intradermal electroporation use. International Standard Randomised Controlled Trial Number (ISRCTN) 60284968.

Interaction between HIV-1 Tat and DNA-PKcs modulates HIV transcription and class switch recombination.

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Zhang, Shi-Meng; Zhang, He; Yang, Tian-Yi; Ying, Tian-Yi; Yang, Pei-Xiang; Liu, Xiao-Dan; Tang, Sheng-Jian; Zhou, Ping-Kun

2014-01-01

HIV-1 tat targets a variety of host cell proteins to facilitate viral transcription and disrupts host cellular immunity by inducing lymphocyte apoptosis, but whether it influences humoral immunity remains unclear. Previously, our group demonstrated that tat depresses expression of DNA-PKcs, a critical component of the non-homologous end joining pathway (NHEJ) of DNA double-strand breaks repair, immunoglobulin class switch recombination (CSR) and V(D)J recombination, and sensitizes cells to ionizing radiation. In this study, we demonstrated that HIV-1 Tat down-regulates DNA-PKcs expression by directly binding to the core promoter sequence. In addition, Tat interacts with and activates the kinase activity of DNA-PKcs in a dose-dependent and DNA independent manner. Furthermore, Tat inhibits class switch recombination (CSR) at low concentrations (≤ 4 µg/ml) and stimulates CSR at high concentrations (≥ 8 µg/ml). On the other hand, low protein level and high kinase activity of DNA-PKcs promotes HIV-1 transcription, while high protein level and low kinase activity inhibit HIV-1 transcription. Co-immunoprecipitation results revealed that DNA-PKcs forms a large complex comprised of Cyclin T1, CDK9 and Tat via direct interacting with CDK9 and Tat but not Cyclin T1. Taken together, our results provide new clues that Tat regulates host humoral immunity via both transcriptional depression and kinase activation of DNA-PKcs. We also raise the possibility that inhibitors and interventions directed towards DNA-PKcs may inhibit HIV-1 transcription in AIDS patients.

Integrated and Total HIV-1 DNA Predict Ex Vivo Viral Outgrowth.

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Maja Kiselinova

2016-03-01

Full Text Available The persistence of a reservoir of latently infected CD4 T cells remains one of the major obstacles to cure HIV. Numerous strategies are being explored to eliminate this reservoir. To translate these efforts into clinical trials, there is a strong need for validated biomarkers that can monitor the reservoir over time in vivo. A comprehensive study was designed to evaluate and compare potential HIV-1 reservoir biomarkers. A cohort of 25 patients, treated with suppressive antiretroviral therapy was sampled at three time points, with median of 2.5 years (IQR: 2.4-2.6 between time point 1 and 2; and median of 31 days (IQR: 28-36 between time point 2 and 3. Patients were median of 6 years (IQR: 3-12 on ART, and plasma viral load (<50 copies/ml was suppressed for median of 4 years (IQR: 2-8. Total HIV-1 DNA, unspliced (us and multiply spliced HIV-1 RNA, and 2LTR circles were quantified by digital PCR in peripheral blood, at 3 time points. At the second time point, a viral outgrowth assay (VOA was performed, and integrated HIV-1 DNA and relative mRNA expression levels of HIV-1 restriction factors were quantified. No significant change was found for long- and short-term dynamics of all HIV-1 markers tested in peripheral blood. Integrated HIV-1 DNA was associated with total HIV-1 DNA (p<0.001, R² = 0.85, us HIV-1 RNA (p = 0.029, R² = 0.40, and VOA (p = 0.041, R2 = 0.44. Replication-competent virus was detected in 80% of patients by the VOA and it correlated with total HIV-1 DNA (p = 0.039, R² = 0.54. The mean quantification difference between Alu-PCR and VOA was 2.88 log10, and 2.23 log10 between total HIV-1 DNA and VOA. The levels of usHIV-1 RNA were inversely correlated with mRNA levels of several HIV-1 restriction factors (TRIM5α, SAMHD1, MX2, SLFN11, pSIP1. Our study reveals important correlations between the viral outgrowth and total and integrated HIV-1 DNA measures, suggesting that the total pool of HIV-1 DNA may predict the size of the

PrPC has nucleic acid chaperoning properties similar to the nucleocapsid protein of HIV-1.

Science.gov (United States)

Derrington, Edmund; Gabus, Caroline; Leblanc, Pascal; Chnaidermann, Jonas; Grave, Linda; Dormont, Dominique; Swietnicki, Wieslaw; Morillas, Manuel; Marck, Daniel; Nandi, Pradip; Darlix, Jean-Luc

2002-01-01

The function of the cellular prion protein (PrPC) remains obscure. Studies suggest that PrPC functions in several processes including signal transduction and Cu2+ metabolism. PrPC has also been established to bind nucleic acids. Therefore we investigated the properties of PrPC as a putative nucleic acid chaperone. Surprisingly, PrPC possesses all the nucleic acid chaperoning properties previously specific to retroviral nucleocapsid proteins. PrPC appears to be a molecular mimic of NCP7, the nucleocapsid protein of HIV-1. Thus PrPC, like NCP7, chaperones the annealing of tRNA(Lys) to the HIV-1 primer binding site, the initial step of retrovirus replication. PrPC also chaperones the two DNA strand transfers required for production of a complete proviral DNA with LTRs. Concerning the functions of NCP7 during budding, PrPC also mimices NCP7 by dimerizing the HIV-1 genomic RNA. These data are unprecedented because, although many cellular proteins have been identified as nucleic acid chaperones, none have the properties of retroviral nucleocapsid proteins.

Comparison of HIV DNA and RNA in gut-associated lymphoid tissue of HIV-infected controllers and noncontrollers.

Science.gov (United States)

Hatano, Hiroyu; Somsouk, Ma; Sinclair, Elizabeth; Harvill, Kara; Gilman, Lee; Cohen, Michelle; Hoh, Rebecca; Hunt, Peter W; Martin, Jeffrey N; Wong, Joseph K; Deeks, Steven G; Yukl, Steven A

2013-09-10

HIV-infected controllers have provided novel insights into mechanisms of viral control. We investigated the degree to which HIV DNA and RNA are present in gut-associated lymphoid tissue (GALT) of controllers. Cross-sectional cohort study. Colorectal biopsy pieces were obtained from five untreated noncontrollers, five ART-suppressed patients, and nine untreated controllers. Rectal HIV DNA was lower in controllers (median 496 copies/10(6) CD4 T cells) than in untreated noncontrollers (117483 copies/10(6) CD4+ T cells, P = 0.001) and ART-suppressed patients (6116 copies/10(6) CD4 T cells, P = 0.004). Similarly, rectal HIV RNA was lower in controllers (19 copies/10(6) CD4 T cells) than in noncontrollers (15210 copies/10(6) CD4+ T cells, P = 0.001) and ART-suppressed patients (1625 copies/10(6) CD4+ T cells, P = 0.0599). Rectal HIV RNA/DNA ratios were not statistically different between the three groups. Despite being able to maintain very low plasma HIV RNA levels in the absence of antiretroviral therapy (ART), HIV-infected controllers have readily measurable levels of HIV DNA and RNA in GALT. As expected, controllers had lower rectal HIV DNA and RNA compared with untreated noncontrollers and ART-suppressed individuals. Compared with the mechanisms of 'natural' viral control of controllers, long-term ART does not reduce the total HIV reservoir to the level of controllers.

HMGB1-dependent triggering of HIV-1 replication and persistence in dendritic cells as a consequence of NK-DC cross-talk.

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Héla Saïdi

Full Text Available HIV-1 has evolved ways to exploit DCs, thereby facilitating viral dissemination and allowing evasion of antiviral immunity. Recently, the fate of DCs has been found to be extremely dependent on the interaction with autologous NK cells, but the mechanisms by which NK-DC interaction controls viral infections remain unclear. Here, we investigate the impact of NK-DC cross-talk on maturation and functions of HIV-infected immature DCs.Immature DCs were derived from primary monocytes, cultured in the presence of IL-4 and GM-CSF. In some experiments, DCs were infected with R5-HIV-1(BaL or X4-HIV-1(NDK, and viral replication, proviral HIV-DNA and the frequency of infected DCs were measured. Autologous NK cells were sorted and either kept unstimulated in the presence of suboptimal concentration of IL-2, or activated by a combination of PHA and IL-2. The impact of 24 h NK-DC cross-talk on the fate of HIV-1-infected DCs was analyzed. We report that activated NK cells were required for the induction of maturation of DCs, whether uninfected or HIV-1-infected, and this process involved HMGB1. However, the cross-talk between HIV-1-infected DCs and activated NK cells was functionally defective, as demonstrated by the strong impairment of DCs to induce Th1 polarization of naïve CD4 T cells. This was associated with the defective production of IL-12 and IL-18 by infected DCs. Moreover, the crosstalk between activated NK cells and HIV-infected DCs resulted in a dramatic increase in viral replication and proviral DNA expression in DCs. HMGB1, produced both by NK cells and DCs, was found to play a pivotal role in this process, and inhibition of HMGB1 activity by glycyrrhizin, known to bind specifically to HMGB1, or blocking anti-HMGB1 antibodies, abrogated NK-dependent HIV-1 replication in DCs.These observations provide evidence for the crucial role of NK-DC cross-talk in promoting viral dissemination, and challenge the question of the in vivo involvement of HMGB1

DNA-mediated gene transfer into human diploid fibroblasts derived from normal and ataxia-telangiectasia donors: parameters for DNA transfer and properties of DNA transformants

International Nuclear Information System (INIS)

Debenham, P.G.; Webb, M.B.T.; Masson, W.K.; Cox, R.

1984-01-01

An investigation was made of the feasibility of DNA-mediated gene transfer into human diploid fibroblasts derived from patients with the radiation sensitive syndrome ataxia-telangiectasia (A-T) and from a normal donor. Although they are markedly different in their growth characteristics, both normal and A-T strains give similar frequencies for DNA transfer in a model system using the recombinant plasmid pSV2-gpt. pSV2-gpt DNA transformants arise with a frequency between 10 -5 and 10 -4 per viable cell. Analysis of such transformants, although possible, is severely handicapped by the limited clonal life span of diploid human cells. Despite these problems it may be concluded that diploid human fibroblasts are competent recipients for DNA-mediated gene transfer and the putative repair deficiency of A-T does not markedly effect the efficiency of this process. (author)

HIV-1 DNA predicts disease progression and post-treatment virological control

Science.gov (United States)

Williams, James P; Hurst, Jacob; Stöhr, Wolfgang; Robinson, Nicola; Brown, Helen; Fisher, Martin; Kinloch, Sabine; Cooper, David; Schechter, Mauro; Tambussi, Giuseppe; Fidler, Sarah; Carrington, Mary; Babiker, Abdel; Weber, Jonathan

2014-01-01

In HIV-1 infection, a population of latently infected cells facilitates viral persistence despite antiretroviral therapy (ART). With the aim of identifying individuals in whom ART might induce a period of viraemic control on stopping therapy, we hypothesised that quantification of the pool of latently infected cells in primary HIV-1 infection (PHI) would predict clinical progression and viral replication following ART. We measured HIV-1 DNA in a highly characterised randomised population of individuals with PHI. We explored associations between HIV-1 DNA and immunological and virological markers of clinical progression, including viral rebound in those interrupting therapy. In multivariable analyses, HIV-1 DNA was more predictive of disease progression than plasma viral load and, at treatment interruption, predicted time to plasma virus rebound. HIV-1 DNA may help identify individuals who could safely interrupt ART in future HIV-1 eradication trials. Clinical trial registration: ISRCTN76742797 and EudraCT2004-000446-20 DOI: http://dx.doi.org/10.7554/eLife.03821.001 PMID:25217531

DNA sequence changes in mutation induced by ultraviolet light in the gpt gene on the chromosome of Escherichia coli uvr+ und uvrA cells

International Nuclear Information System (INIS)

Sockett, H.; Romac, S.; Hutchinson, F.

1991-01-01

Sequence changes in mutations induced by ultraviolet light are reported for the chromosomal Escherichia coli gpt gene in almost isogenic E. coli uvr + and excision-deficient uvrA cells. Differences between the mutagenic spectra are ascribed to preferential removal of photoproducts in the transcribed strand by excision repair in uvr + cells. This conclusion is confirmed by analysis of published results for genes in both uvr + and uvr − cells, showing a similar selective removal of mutagenic products from the transcribed strand of the E. coli lacI gene and of the lambda phage cl repressor gene. Comparison of these data with published results for ultraviolet mutagenesis of gpt on a chromosome in Chinese hamster ovary cells showed that a mutagenic hot spot in mammalian cells is not present in E. coli; the possibility is suggested that the hot spot might arise from localized lack of excision repair. Otherwise, mutagenesis in hamster cells appeared similar to that in E. coli uvr + cells, except there appears to be a smaller fraction of single-base additions and deletions (frameshifts) in mammalian than in bacterial cells. Phenotypes of 6-thioguanine-resistant E. coli showed there is a gene (or genes) other than gpt involved in the utilization of thioguanine by bacteria

Sensitive Fluorescent Sensor for Recognition of HIV-1 dsDNA by Using Glucose Oxidase and Triplex DNA

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Yubin Li

2018-01-01

Full Text Available A sensitive fluorescent sensor for sequence-specific recognition of double-stranded DNA (dsDNA was developed on the surface of silver-coated glass slide (SCGS. Oligonucleotide-1 (Oligo-1 was designed to assemble on the surface of SCGS and act as capture DNA, and oligonucleotide-2 (Oligo-2 was designed as signal DNA. Upon addition of target HIV-1 dsDNA (Oligo-3•Oligo-4, signal DNA could bind on the surface of silver-coated glass because of the formation of C•GoC in parallel triplex DNA structure. Biotin-labeled glucose oxidase (biotin-GOx could bind to signal DNA through the specific interaction of biotin-streptavidin, thereby GOx was attached to the surface of SCGS, which was dependent on the concentration of target HIV-1 dsDNA. GOx could catalyze the oxidation of glucose and yield H2O2, and the HPPA can be oxidized into a fluorescent product in the presence of HRP. Therefore, the concentration of target HIV-1 dsDNA could be estimated with fluorescence intensity. Under the optimum conditions, the fluorescence intensity was proportional to the concentration of target HIV-1 dsDNA over the range of 10 pM to 1000 pM, the detection limit was 3 pM. Moreover, the sensor had good sequence selectivity and practicability and might be applied for the diagnosis of HIV disease in the future.

The HIV-1 transcriptional activator Tat has potent nucleic acid chaperoning activities in vitro.

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Kuciak, Monika; Gabus, Caroline; Ivanyi-Nagy, Roland; Semrad, Katharina; Storchak, Roman; Chaloin, Olivier; Muller, Sylviane; Mély, Yves; Darlix, Jean-Luc

2008-06-01

The human immunodeficiency virus type 1 (HIV-1) is a primate lentivirus that causes the acquired immunodeficiency syndrome (AIDS). In addition to the virion structural proteins and enzyme precursors, that are Gag, Env and Pol, HIV-1 encodes several regulatory proteins, notably a small nuclear transcriptional activator named Tat. The Tat protein is absolutely required for virus replication since it controls proviral DNA transcription to generate the full-length viral mRNA. Tat can also regulate mRNA capping and splicing and was recently found to interfere with the cellular mi- and siRNA machinery. Because of its extensive interplay with nucleic acids, and its basic and disordered nature we speculated that Tat had nucleic acid-chaperoning properties. This prompted us to examine in vitro the nucleic acid-chaperoning activities of Tat and Tat peptides made by chemical synthesis. Here we report that Tat has potent nucleic acid-chaperoning activities according to the standard DNA annealing, DNA and RNA strand exchange, RNA ribozyme cleavage and trans-splicing assays. The active Tat(44-61) peptide identified here corresponds to the smallest known sequence with DNA/RNA chaperoning properties.

Cell-associated HIV DNA measured early during infection has prognostic value independent of serum HIV RNA measured concomitantly

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Katzenstein, Terese L; Oliveri, Roberto S; Benfield, Thomas

2002-01-01

Using data from the Danish AIDS Cohort of HIV-infected homosexual men established in the 1980s, the prognostic value of early HIV DNA loads was evaluated. In addition to DNA measurements, concomitant serum HIV RNA levels, CD4 cell counts and CCR5 genotypes were determined. The patients were divided...... of serum HIV RNA (p normal allele (p

Sequence-specific activation of the DNA sensor cGAS by Y-form DNA structures as found in primary HIV-1 cDNA.

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Herzner, Anna-Maria; Hagmann, Cristina Amparo; Goldeck, Marion; Wolter, Steven; Kübler, Kirsten; Wittmann, Sabine; Gramberg, Thomas; Andreeva, Liudmila; Hopfner, Karl-Peter; Mertens, Christina; Zillinger, Thomas; Jin, Tengchuan; Xiao, Tsan Sam; Bartok, Eva; Coch, Christoph; Ackermann, Damian; Hornung, Veit; Ludwig, Janos; Barchet, Winfried; Hartmann, Gunther; Schlee, Martin

2015-10-01

Cytosolic DNA that emerges during infection with a retrovirus or DNA virus triggers antiviral type I interferon responses. So far, only double-stranded DNA (dsDNA) over 40 base pairs (bp) in length has been considered immunostimulatory. Here we found that unpaired DNA nucleotides flanking short base-paired DNA stretches, as in stem-loop structures of single-stranded DNA (ssDNA) derived from human immunodeficiency virus type 1 (HIV-1), activated the type I interferon-inducing DNA sensor cGAS in a sequence-dependent manner. DNA structures containing unpaired guanosines flanking short (12- to 20-bp) dsDNA (Y-form DNA) were highly stimulatory and specifically enhanced the enzymatic activity of cGAS. Furthermore, we found that primary HIV-1 reverse transcripts represented the predominant viral cytosolic DNA species during early infection of macrophages and that these ssDNAs were highly immunostimulatory. Collectively, our study identifies unpaired guanosines in Y-form DNA as a highly active, minimal cGAS recognition motif that enables detection of HIV-1 ssDNA.

Human macrophages support persistent transcription from unintegrated HIV-1 DNA

International Nuclear Information System (INIS)

Kelly, Jeremy; Beddall, Margaret H.; Yu Dongyang; Iyer, Subashini R.; Marsh, Jon W.; Wu Yuntao

2008-01-01

Retroviruses require integration of their RNA genomes for both stability and productive viral replication. In HIV infection of non-dividing, resting CD4 T cells, where integration is greatly impeded, the reverse transcribed HIV DNA has limited biological activity and a short half-life. In metabolically active and proliferating T cells, unintegrated DNA rapidly diminishes with cell division. HIV also infects the non-dividing but metabolically active macrophage population. In an in vitro examination of HIV infection of macrophages, we find that unintegrated viral DNA not only has an unusual stability, but also maintains biological activity. The unintegrated linear DNA, 1-LTR, and 2-LTR circles are stable for at least 30 days. Additionally, there is persistent viral gene transcription, which is selective and skewed towards viral early genes such as nef and tat with highly diminished rev and vif. One viral early gene product Nef was measurably synthesized. We also find that independent of integration, the HIV infection process in macrophages leads to generation of numerous chemokines

Variability of HIV-1 genomes among children and adolescents from Sao Paulo, Brazil.

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Sabri Saeed Sanabani

Full Text Available BACKGROUND: Genetic variability is a major feature of the human immunodeficiency virus type 1 (HIV-1 and considered the key factor to frustrating efforts to halt the virus epidemic. In this study, we aimed to investigate the genetic variability of HIV-1 strains among children and adolescents born from 1992 to 2009 in the state of Sao Paulo, Brazil. METHODOLOGY: Plasma and peripheral blood mononuclear cells (PBMC were collected from 51 HIV-1-positive children and adolescents on ART followed between September 1992 and July 2009. After extraction, the genetic materials were used in a polymerase chain reaction (PCR to amplify the viral near full length genomes (NFLGs from 5 overlapped fragments. NFLGs and partial amplicons were directly sequenced and data were phylogenetically inferred. RESULTS: Of the 51 samples studied, the NFLGs and partial fragments of HIV-1 from 42 PBMCs and 25 plasma were successfully subtyped. Results based on proviral DNA revealed that 22 (52.4% patients were infected with subtype B, 16 (38.1% were infected with BF1 mosaic variants and 4 (9.5% were infected with sub-subtype F1. All the BF1 recombinants were unique and distinct from any previously identified unique or circulating recombinant forms in South America. Evidence of dual infections was detected in 3 patients coinfected with the same or distinct HIV-1 subtypes. Ten of the 31 (32.2% and 12 of the 21 (57.1% subjects with recovered proviral and plasma, respectively, protease sequences were infected with major mutants resistant to protease inhibitors. The V3 sequences of 14 patients with available sequences from PBMC/or plasma were predicted to be R5-tropic virus except for two patients who harbored an X4 strain. CONCLUSIONS: The high proportion of HIV-1 BF1 recombinant, coinfection rate and vertical transmission in Brazil merits urgent attention and effective measures to reduce the transmission of HIV among spouses and sex partners.

Possible participation of oxidative stress in causation of cell proliferation and in vivo mutagenicity in kidneys of gpt delta rats treated with potassium bromate

International Nuclear Information System (INIS)

Umemura, Takashi; Tasaki, Masako; Kijima, Aki; Okamura, Toshiya; Inoue, Tomoki; Ishii, Yuji; Suzuki, Yuta; Masui, Norio; Nohmi, Takehiko; Nishikawa, Akiyoshi

2009-01-01

Clarifying the participation of oxidative stress among possible contributing factors in potassium bromate (KBrO 3 )-induced carcinogenesis is of importance from the perspective of human health protection. In the present study, utilizing the antioxidative effects of α-tocopherol (α-TP) or sodium ascorbic acid (SAA) to attenuate oxidative stress, alterations in bromodeoxyuridine labeling indices (BrdU-LIs) and reporter gene mutations in kidneys of male and female gpt delta rats given KBrO 3 were examined. Five male and female gpt delta rats in each group were given KBrO 3 at a concentration of 500 ppm in the drinking water for 9 weeks, with 1% of α-TP or SAA administered in the diet from 1 week prior to the KBrO 3 treatment until the end of the experiment. Increases in 8-hydroxydeoxyguanosine levels in kidney DNA of both sexes of rats given KBrO 3 were significantly inhibited by SAA, but not α-TP. While BrdU-LIs in the proximal tubules of female rats were also significantly reduced by SAA, those in the males and gpt mutant frequencies in kidney DNA of both sexes were not affected by SAA or α-TP. Immunohistochemical and Western blot analyses for α 2u -globulin strongly suggested that induction of cell proliferation observed in the males might primarily result from accumulation of this protein, independent of oxidative stress. The overall data indicated that while oxidative stress well correlates with induction of cell proliferation in females, its role in males and in generation of in vivo mutagenicity by KBrO 3 in both sexes is limited

Impact of HIV Type 1 DNA Levels on Spontaneous Disease Progression: A Meta-Analysis

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Tsiara, Chrissa G; Nikolopoulos, Georgios K; Bagos, Pantelis G

2012-01-01

Abstract Several studies have reported the prognostic strength of HIV-1 DNA with variable results however. The aims of the current study were to estimate more accurately the ability of HIV-1 DNA to predict progression of HIV-1 disease toward acquired immunodeficiency syndrome (AIDS) or death...... of primary studies indicated that HIV-1 DNA was a significantly better predictor than HIV-1 RNA of either AIDS alone (ratio of RRs=1.47, 95% CI: 1.05-2.07) or of combined (AIDS or death) progression outcomes (ratio of RRs=1.51, 95% CI: 1.11-2.05). HIV-1 DNA is a strong predictor of HIV-1 disease progression...

HIV-1-Specific Antibody Response and Function after DNA Prime and Recombinant Adenovirus 5 Boost HIV Vaccine in HIV-Infected Subjects.

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Johannes S Gach

Full Text Available Little is known about the humoral immune response against DNA prime-recombinant adenovirus 5 (rAd5 boost HIV vaccine among HIV-infected patients on long-term suppressive antiretroviral therapy (ART. Previous studies emphasized cellular immune responses; however, current research suggests both cellular and humoral responses are likely required for a successful therapeutic vaccine. Thus, we aimed to understand antibody response and function induced by vaccination of ART-treated HIV-1-infected patients with immune recovery. All subjects participated in EraMune 02, an open-label randomized clinical trial of ART intensification followed by a six plasmid DNA prime (envA, envB, envC, gagB, polB, nefB and rAd5 boost HIV vaccine with matching inserts. Antibody binding levels were determined with a recently developed microarray approach. We also analyzed neutralization efficiency and antibody-dependent cellular cytotoxicity (ADCC. We found that the DNA prime-rAd5 boost vaccine induced a significant cross-clade HIV-specific antibody response, which correlated with antibody neutralization efficiency. However, despite the increase in antibody binding levels, the vaccine did not significantly stimulate neutralization or ADCC responses. This finding was also reflected by a lack of change in total CD4+ cell associated HIV DNA in those who received the vaccine. Our results have important implications for further therapeutic vaccine design and administration, especially in HIV-1 infected patients, as boosting of preexisting antibody responses are unlikely to lead to clearance of latent proviruses in the HIV reservoir.

HIV sequence diversity during the early phase of infection is associated with HIV DNA reductions during antiretroviral therapy.

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Wang, Nidan; Li, Yijia; Han, Yang; Xie, Jing; Li, Taisheng

2017-06-01

The association between baseline human immunodeficiency virus (HIV) sequence diversity and HIV DNA decay after the initiation of antiretroviral therapy (ART) remains uncharacterized during the early stages of HIV infection. Samples were obtained from a cohort of 17 patients with early HIV infection (HIV-1 envelope (env) gene was amplified via single genome amplification (SGA) to determine the peripheral plasma HIV quasispecies. We categorized HIV quasispecies into two groups according to baseline viral sequence genetic distance, which was determined by the Poisson-Fitter tool. Total HIV DNA in peripheral blood mononuclear cells (PBMCs), viral load, and T cell subsets were measured prior to and after the initiation of ART. The median SGA sequence number was 17 (range 6-28). At baseline, we identified 7 patients with homogeneous viral populations (designated the Homogeneous group) and 10 patients with heterogeneous viral populations (designated the Heterogeneous group) based on SGA sequences. Both groups exhibited similar HIV DNA decay rates during the first 6 months of ART (P > 0.99), but the Homogenous group experienced more prominent decay than the Heterogeneous group after 6 months (P = 0.037). The Heterogeneous group had higher CD4 cell counts after ART initiation; however, both groups had comparable recovery in terms of CD4/CD8 ratios and CD8 T cell activation levels. Viral population homogeneity upon the initiation of ART is associated with a decrease in HIV DNA levels during ART. J. Med. Virol. 89:982-988, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Serum Glutamic-Oxaloacetic Transaminase (GOT) and Glutamic-Pyruvic Transaminase (GPT) Levels in Children and Adolescents with Intellectual Disabilities

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Lin, Jin-Ding; Lin, Pei-Ying; Chen, Li-Mei; Fang, Wen-Hui; Lin, Lan-Ping; Loh, Ching-Hui

2010-01-01

The elevated serum glutamic-oxaloacetic transaminase (GOT) and glutamic-pyruvic transaminase (GPT) rate among people with intellectual disabilities (ID) is unknown and have not been sufficiently studies. The present paper aims to provide the profile of GOT and GPT, and their associated relationship with other biochemical levels of children or…

Virological and immunological profiles among patients with undetectable viral load followed prospectively for 24 months

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Katzenstein, T.; Ullum, H.; Røge, B.T.

2003-01-01

OBJECTIVE: To quantify HIV-RNA in plasma, in lymphoid tissue and proviral DNA in peripheral blood mononuclear cells and to relate these to immunological markers among patients with plasma viral load counts of

Prevalence of XMRV Nucleic Acid and Antibody in HIV-1-Infected Men and in Men at Risk for HIV-1 Infection

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J. Spindler

2011-01-01

Full Text Available Xenotropic MLV-Related Virus (XMRV was recently reported to be associated with prostate cancer and chronic fatigue syndrome (CFS. Infection was also reported in 3.7% of healthy individuals. These highly reported frequencies of infection prompted concerns about the possibility of a new, widespread retroviral epidemic. The Multicenter AIDS Cohort Study (MACS provides an opportunity to assess the prevalence of XMRV infection and its association with HIV-1 infection among men who have sex with men. Reliable detection of XMRV infection requires the application of multiple diagnostic methods, including detection of human antibodies to XMRV and detection of XMRV nucleic acid. We, therefore, tested 332 patient plasma and PBMC samples obtained from recent visits in a subset of patients in the MACS cohort for XMRV antibodies using Abbott prototype ARCHITECT chemiluminescent immunoassays (CMIAs and for XMRV RNA and proviral DNA using a XMRV single-copy qPCR assay (X-SCA. Although 9 of 332 (2.7% samples showed low positive reactivity against a single antigen in the CMIA, none of these samples or matched controls were positive for plasma XMRV RNA or PBMC XMRV DNA by X-SCA. Thus, we found no evidence of XMRV infection among men in the MACS regardless of HIV-1 serostatus.

A Real Time PCR Platform for the Simultaneous Quantification of Total and Extrachromosomal HIV DNA Forms in Blood of HIV-1 Infected Patients

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Canovari, Benedetta; Scotti, Maddalena; Acetoso, Marcello; Valentini, Massimo; Petrelli, Enzo; Magnani, Mauro

2014-01-01

Background The quantitative measurement of various HIV-1 DNA forms including total, unintegrated and integrated provirus play an increasingly important role in HIV-1 infection monitoring and treatment-related research. We report the development and validation of a SYBR Green real time PCR (TotUFsys platform) for the simultaneous quantification of total and extrachromosomal HIV-1 DNA forms in patients. This innovative technique makes it possible to obtain both measurements in a single PCR run starting from frozen blood employing the same primers and standard curve. Moreover, due to identical amplification efficiency, it allows indirect estimation of integrated level. To specifically detect 2-LTR a qPCR method was also developed. Methodology/Findings Primers used for total HIV-1 DNA quantification spanning a highly conserved region were selected and found to detect all HIV-1 clades of group M and the unintegrated forms of the same. A total of 195 samples from HIV-1 patients in a wide range of clinical conditions were analyzed with a 100% success rate, even in patients with suppressed plasma viremia, regardless of CD4+ or therapy. No significant correlation was observed between the two current prognostic markers, CD4+ and plasma viremia, while a moderate or high inverse correlation was found between CD4+ and total HIV DNA, with strong values for unintegrated HIV DNA. Conclusions/Significance Taken together, the results support the use of HIV DNA as another tool, in addition to traditional assays, which can be used to estimate the state of viral infection, the risk of disease progression and to monitor the effects of ART. The TotUFsys platform allowed us to obtain a final result, expressed as the total and unintegrated HIV DNA copy number per microgram of DNA or 104 CD4+, for 12 patients within two working days. PMID:25364909

Lack of mutational hot spots during decitabine-mediated HIV-1 mutagenesis.

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Rawson, Jonathan M O; Landman, Sean R; Reilly, Cavan S; Bonnac, Laurent; Patterson, Steven E; Mansky, Louis M

2015-11-01

Decitabine has previously been shown to induce lethal mutagenesis of human immunodeficiency virus type 1 (HIV-1). However, the factors that determine the susceptibilities of individual sequence positions in HIV-1 to decitabine have not yet been defined. To investigate this, we performed Illumina high-throughput sequencing of multiple amplicons prepared from proviral DNA that was recovered from decitabine-treated cells infected with HIV-1. We found that decitabine induced an ≈4.1-fold increase in the total mutation frequency of HIV-1, primarily due to a striking ≈155-fold increase in the G-to-C transversion frequency. Intriguingly, decitabine also led to an ≈29-fold increase in the C-to-G transversion frequency. G-to-C frequencies varied substantially (up to ≈80-fold) depending upon sequence position, but surprisingly, mutational hot spots (defined as upper outliers within the mutation frequency distribution) were not observed. We further found that every single guanine position examined was significantly susceptible to the mutagenic effects of decitabine. Taken together, these observations demonstrate for the first time that decitabine-mediated HIV-1 mutagenesis is promiscuous and occurs in the absence of a clear bias for mutational hot spots. These data imply that decitabine-mediated G-to-C mutagenesis is a highly effective antiviral mechanism for extinguishing HIV-1 infectivity. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

HIV-1 Tat depresses DNA-PKCS expression and DNA repair, and sensitizes cells to ionizing radiation

International Nuclear Information System (INIS)

Sun Yi; Huang Yuechen; Xu Qinzhi; Wang Huiping; Bai Bei; Sui Jianli; Zhou Pingkun

2006-01-01

Purpose There is accumulating evidence that cancer patients with human immmunodeficiency virus-1/acquired immunodeficency syndrome (HIV-1/AIDS) have more severe tissue reactions and often develop cutaneous toxic effects when subjected to radiotherapy. Here we explored the effects of the HIV-1 Tat protein on cellular responses to ionizing radiation. Methods and Materials Two Tat-expressing cell lines, TT2 and TE671-Tat, were derived from human rhabdomyosarcoma cells by transfecting with the HIV-1 tat gene. Radiosensitivity was determined using colony-forming ability. Gene expression was assessed by cDNA microarray and immunohybridization. The Comet assay and γ-H2AX foci were use to detect DNA double-strand breaks (DSBs) and repair. Radiation-induced cell cycle changes were detected by flow cytometry. Results The radiosensitivity of TT2 and TE671-Tat cells was significantly increased as compared with parental TE671 cells or the control TE671-pCI cells. Tat also increased proliferation activity. The comet assay and γH2AX foci detection revealed a decreased capacity to repair radiation-induced DNA DSBs in Tat-expressing cells. Microarray assay demonstrated that the DNA repair gene DNA-PKcs, and cell cycle-related genes Cdc20, Cdc25C, KIF2C and CTS1 were downregulated in Tat-expressing cells. Depression of DNA-PKcs in Tat-expressing cells was further confirmed by RT-PCR and immuno-hybridization analysis. Tat-expressing cells exhibited a prolonged S phase arrest after 4 Gy γ-irradiation, and a noticeable delay in the initiation and elimination of radiation-induced G 2 /M arrest as compared with parental cells. In addition, the G 2 /M arrest was incomplete in TT2 cells. Moreover, HIV-1 Tat resulted in a constitutive overexpression of cyclin B1 protein. Conclusion HIV-1 Tat protein sensitizes cells to ionizing radiation via depressing DNA repair and dysregulating cell cycle checkpoints. These observations provide new insight into the increased tissue reactions of AIDS

Identification of Interleukin-27 (IL-27)/IL-27 Receptor Subunit Alpha as a Critical Immune Axis for In Vivo HIV Control.

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Ruiz-Riol, M; Berdnik, D; Llano, A; Mothe, B; Gálvez, C; Pérez-Álvarez, S; Oriol-Tordera, B; Olvera, A; Silva-Arrieta, S; Meulbroek, M; Pujol, F; Coll, J; Martinez-Picado, J; Ganoza, C; Sanchez, J; Gómez, G; Wyss-Coray, T; Brander, C

2017-08-15

Intact and broad immune cell effector functions and specific individual cytokines have been linked to HIV disease outcome, but their relative contribution to HIV control remains unclear. We asked whether the proteome of secreted cytokines and signaling factors in peripheral blood can be used to discover specific pathways critical for host viral control. A custom glass-based microarray, able to measure >600 plasma proteins involved in cell-to-cell communication, was used to measure plasma protein profiles in 96 HIV-infected, treatment-naive individuals with high (>50,000) or low (HIV RNA copies/ml) viral loads. Univariate and regression model analysis demonstrate that plasma levels of soluble interleukin-27 (IL-27) are significantly elevated in individuals with high plasma viremia ( P HIV-DNA copy numbers in peripheral blood mononuclear cells (PBMC) (Rho = 0.4011; P = 0.0027). Moreover, soluble IL-27 plasma levels are negatively associated with the breadth and magnitude of the total virus-specific T-cell responses and directly with plasma levels of molecules involved in Wnt/β-catenin signaling. In addition to IL-27, gene expression levels of the specific IL-27 receptor ( IL27RA ) in PBMC correlated directly with both plasma viral load (Rho = 0.3531; P = 0.0218) and the proviral copy number in the peripheral blood as an indirect measure of partial viral reservoir (Rho = 0.4580; P = 0.0030). These results were validated in unrelated cohorts of early infected subjects as well as subjects before and after initiation of antiretroviral treatment, and they identify IL-27 and its specific receptor as a critical immune axis for the antiviral immune response and as robust correlates of viral load and proviral reservoir size in PBMC. IMPORTANCE The detailed knowledge of immune mechanisms that contribute to HIV control is a prerequisite for the design of effective treatment strategies to achieve HIV cure. Cells communicate with each other by secreting signaling proteins, and

LEDGF/p75 Deficiency Increases Deletions at the HIV-1 cDNA Ends.

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Bueno, Murilo T D; Reyes, Daniel; Llano, Manuel

2017-09-15

Processing of unintegrated linear HIV-1 cDNA by the host DNA repair system results in its degradation and/or circularization. As a consequence, deficient viral cDNA integration generally leads to an increase in the levels of HIV-1 cDNA circles containing one or two long terminal repeats (LTRs). Intriguingly, impaired HIV-1 integration in LEDGF/p75-deficient cells does not result in a correspondent increase in viral cDNA circles. We postulate that increased degradation of unintegrated linear viral cDNA in cells lacking the lens epithelium-derived growth factor (LEDGF/p75) account for this inconsistency. To evaluate this hypothesis, we characterized the nucleotide sequence spanning 2-LTR junctions isolated from LEDGF/p75-deficient and control cells. LEDGF/p75 deficiency resulted in a significant increase in the frequency of 2-LTRs harboring large deletions. Of note, these deletions were dependent on the 3' processing activity of integrase and were not originated by aberrant reverse transcription. Our findings suggest a novel role of LEDGF/p75 in protecting the unintegrated 3' processed linear HIV-1 cDNA from exonucleolytic degradation.

Clonal expansion of genome-intact HIV-1 in functionally polarized Th1 CD4+ T cells.

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Lee, Guinevere Q; Orlova-Fink, Nina; Einkauf, Kevin; Chowdhury, Fatema Z; Sun, Xiaoming; Harrington, Sean; Kuo, Hsiao-Hsuan; Hua, Stephane; Chen, Hsiao-Rong; Ouyang, Zhengyu; Reddy, Kavidha; Dong, Krista; Ndung'u, Thumbi; Walker, Bruce D; Rosenberg, Eric S; Yu, Xu G; Lichterfeld, Mathias

2017-06-30

HIV-1 causes a chronic, incurable disease due to its persistence in CD4+ T cells that contain replication-competent provirus, but exhibit little or no active viral gene expression and effectively resist combination antiretroviral therapy (cART). These latently infected T cells represent an extremely small proportion of all circulating CD4+ T cells but possess a remarkable long-term stability and typically persist throughout life, for reasons that are not fully understood. Here we performed massive single-genome, near-full-length next-generation sequencing of HIV-1 DNA derived from unfractionated peripheral blood mononuclear cells, ex vivo-isolated CD4+ T cells, and subsets of functionally polarized memory CD4+ T cells. This approach identified multiple sets of independent, near-full-length proviral sequences from cART-treated individuals that were completely identical, consistent with clonal expansion of CD4+ T cells harboring intact HIV-1. Intact, near-full-genome HIV-1 DNA sequences that were derived from such clonally expanded CD4+ T cells constituted 62% of all analyzed genome-intact sequences in memory CD4 T cells, were preferentially observed in Th1-polarized cells, were longitudinally detected over a duration of up to 5 years, and were fully replication- and infection-competent. Together, these data suggest that clonal proliferation of Th1-polarized CD4+ T cells encoding for intact HIV-1 represents a driving force for stabilizing the pool of latently infected CD4+ T cells.

Low-level viremia and proviral DNA impede immune reconstitution in HIV-1-infected patients receiving highly active antiretroviral therapy

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Ostrowski, Sisse R; Katzenstein, Terese L; Thim, Per T.

2005-01-01

Immunological and virological consequences of low-level viremia in human immunodeficiency virus (HIV) type 1-infected patients receiving highly active antiretroviral therapy (HAART) remain to be determined....

Differentiating Immune Cell Targets in Gut-Associated Lymphoid Tissue for HIV Cure.

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Khan, Shahzada; Telwatte, Sushama; Trapecar, Martin; Yukl, Steven; Sanjabi, Shomyseh

2017-11-01

The single greatest challenge to an HIV cure is the persistence of latently infected cells containing inducible, replication-competent proviral genomes, which constitute only a small fraction of total or infected cells in the body. Although resting CD4 + T cells in the blood are a well-known source of viral rebound, more than 90% of the body's lymphocytes reside elsewhere. Many are in gut tissue, where HIV DNA levels per million CD4 + T cells are considerably higher than in the blood. Despite the significant contribution of gut tissue to viral replication and persistence, little is known about the cell types that support persistence of HIV in the gut; importantly, T cells in the gut have phenotypic, functional, and survival properties that are distinct from T cells in other tissues. The mechanisms by which latency is established and maintained will likely depend on the location and cytokine milieu surrounding the latently infected cells in each compartment. Therefore, successful HIV cure strategies require identification and characterization of the exact cell types that support viral persistence, particularly in the gut. In this review, we describe the seeding of the latent HIV reservoir in the gut mucosa; highlight the evidence for compartmentalization and depletion of T cells; summarize the immunologic consequences of HIV infection within the gut milieu; propose how the damaged gut environment may promote the latent HIV reservoir; and explore several immune cell targets in the gut and their place on the path toward HIV cure.

Progress in 3D Space-charge Calculations in the GPT Code

NARCIS (Netherlands)

Pöplau, G.; Rienen, van U.; Loos, de M.J.; Geer, van der S.B.

2004-01-01

The mesh-based 3D space-charge routine in the GPT (General Particle Tracer, Pulsar Physics) code scales linearly with the number of particles in terms of CPU time and allows a million particles to be tracked on a normal PC. The crucial ingredient of the routine is a non-equidistant multi-grid

The Alphabet Soup of HIV Reservoir Markers.

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Sharaf, Radwa R; Li, Jonathan Z

2017-04-01

Despite the success of antiretroviral therapy in suppressing HIV, life-long therapy is required to avoid HIV reactivation from long-lived viral reservoirs. Currently, there is intense interest in searching for therapeutic interventions that can purge the viral reservoir to achieve complete remission in HIV patients off antiretroviral therapy. The evaluation of such interventions relies on our ability to accurately and precisely measure the true size of the viral reservoir. In this review, we assess the most commonly used HIV reservoir assays, as a clear understanding of the strengths and weaknesses of each is vital for the accurate interpretation of results and for the development of improved assays. The quantification of intracellular or plasma HIV RNA or DNA levels remains the most commonly used tests for the characterization of the viral reservoir. While cost-effective and high-throughput, these assays are not able to differentiate between replication-competent or defective fractions or quantify the number of infected cells. Viral outgrowth assays provide a lower bound for the fraction of cells that can produce infectious virus, but these assays are laborious, expensive and substantially underestimate the potential reservoir of replication-competent provirus. Newer assays are now available that seek to overcome some of these problems, including full-length proviral sequencing, inducible HIV RNA assays, ultrasensitive p24 assays and murine adoptive transfer techniques. The development and evaluation of strategies for HIV remission rely upon our ability to accurately and precisely quantify the size of the remaining viral reservoir. At this time, all current HIV reservoir assays have drawbacks such that combinations of assays are generally needed to gain a more comprehensive view of the viral reservoir. The development of novel, rapid, high-throughput assays that can sensitively quantify the levels of the replication-competent HIV reservoir is still needed.

Human Immunodeficiency Virus-Type 1 LTR DNA contains an intrinsic gene producing antisense RNA and protein products

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Hsiao Chiu-Bin

2006-11-01

Full Text Available Abstract Background While viruses have long been shown to capitalize on their limited genomic size by utilizing both strands of DNA or complementary DNA/RNA intermediates to code for viral proteins, it has been assumed that human retroviruses have all their major proteins translated only from the plus or sense strand of RNA, despite their requirement for a dsDNA proviral intermediate. Several studies, however, have suggested the presence of antisense transcription for both HIV-1 and HTLV-1. More recently an antisense transcript responsible for the HTLV-1 bZIP factor (HBZ protein has been described. In this study we investigated the possibility of an antisense gene contained within the human immunodeficiency virus type 1 (HIV-1 long terminal repeat (LTR. Results Inspection of published sequences revealed a potential transcription initiator element (INR situated downstream of, and in reverse orientation to, the usual HIV-1 promoter and transcription start site. This antisense initiator (HIVaINR suggested the possibility of an antisense gene responsible for RNA and protein production. We show that antisense transcripts are generated, in vitro and in vivo, originating from the TAR DNA of the HIV-1 LTR. To test the possibility that protein(s could be translated from this novel HIV-1 antisense RNA, recombinant HIV antisense gene-FLAG vectors were designed. Recombinant protein(s were produced and isolated utilizing carboxy-terminal FLAG epitope (DYKDDDDK sequences. In addition, affinity-purified antisera to an internal peptide derived from the HIV antisense protein (HAP sequences identified HAPs from HIV+ human peripheral blood lymphocytes. Conclusion HIV-1 contains an antisense gene in the U3-R regions of the LTR responsible for both an antisense RNA transcript and proteins. This antisense transcript has tremendous potential for intrinsic RNA regulation because of its overlap with the beginning of all HIV-1 sense RNA transcripts by 25 nucleotides. The

HIV restriction by APOBEC3 in humanized mice.

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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.

Evaluation of a manual DNA extraction protocol and an isothermal amplification assay for detecting HIV-1 DNA from dried blood spots for use in resource-limited settings.

Science.gov (United States)

Jordan, Jeanne A; Ibe, Christine O; Moore, Miranda S; Host, Christel; Simon, Gary L

2012-05-01

In resource-limited settings (RLS) dried blood spots (DBS) are collected on infants and transported through provincial laboratories to a central facility where HIV-1 DNA PCR testing is performed using specialized equipment. Implementing a simpler approach not requiring such equipment or skilled personnel could allow the more numerous provincial laboratories to offer testing, improving turn-around-time to identify and treat infected infants sooner. Assess performances of a manual DNA extraction method and helicase-dependent amplification (HDA) assay for detecting HIV-1 DNA from DBS. 60 HIV-1 infected adults were enrolled, blood samples taken and DBS made. DBS extracts were assessed for DNA concentration and beta globin amplification using PCR and melt-curve analysis. These same extracts were then tested for HIV-1 DNA using HDA and compared to results generated by PCR and pyrosequencing. Finally, HDA limit of detection (LOD) studies were performed using DBS extracts prepared with known numbers of 8E5 cells. The manual extraction protocol consistently yielded high concentrations of amplifiable DNA from DBS. LOD assessment demonstrated HDA detected ∼470 copies/ml of HIV-1 DNA extracts in 4/4 replicates. No statistical difference was found using the McNemar's test when comparing HDA to PCR for detecting HIV-1 DNA from DBS. Using just a magnet, heat block and pipettes, the manual extraction protocol and HDA assay detected HIV-1 DNA from DBS at levels that would be useful for early infant diagnosis. Next steps will include assessing HDA for non-B HIV-1 subtypes recognition and comparison to Roche HIV-1 DNA v1.5 PCR assay. Copyright © 2012 Elsevier B.V. All rights reserved.

Using Resurrected Ancestral Proviral Proteins to Engineer Virus Resistance

Directory of Open Access Journals (Sweden)

Asunción Delgado

2017-05-01

Full Text Available Proviral factors are host proteins hijacked by viruses for processes essential for virus propagation such as cellular entry and replication. Pathogens and their hosts co-evolve. It follows that replacing a proviral factor with a functional ancestral form of the same protein could prevent viral propagation without fatally compromising organismal fitness. Here, we provide proof of concept of this notion. Thioredoxins serve as general oxidoreductases in all known cells. We report that several laboratory resurrections of Precambrian thioredoxins display substantial levels of functionality within Escherichia coli. Unlike E. coli thioredoxin, however, these ancestral thioredoxins are not efficiently recruited by the bacteriophage T7 for its replisome and therefore prevent phage propagation in E. coli. These results suggest an approach to the engineering of virus resistance. Diseases caused by viruses may have a devastating effect in agriculture. We discuss how the suggested approach could be applied to the engineering of plant virus resistance.

Quantification of viral DNA during HIV-1 infection: A review of relevant clinical uses and laboratory methods.

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Alidjinou, E K; Bocket, L; Hober, D

2015-02-01

Effective antiretroviral therapy usually leads to undetectable HIV-1 RNA in the plasma. However, the virus persists in some cells of infected patients as various DNA forms, both integrated and unintegrated. This reservoir represents the greatest challenge to the complete cure of HIV-1 infection and its characteristics highly impact the course of the disease. The quantification of HIV-1 DNA in blood samples constitutes currently the most practical approach to measure this residual infection. Real-time quantitative PCR (qPCR) is the most common method used for HIV-DNA quantification and many strategies have been developed to measure the different forms of HIV-1 DNA. In the literature, several "in-house" PCR methods have been used and there is a need for standardization to have comparable results. In addition, qPCR is limited for the precise quantification of low levels by background noise. Among new assays in development, digital PCR was shown to allow an accurate quantification of HIV-1 DNA. Total HIV-1 DNA is most commonly measured in clinical routine. The absolute quantification of proviruses and unintegrated forms is more often used for research purposes. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

The second chance story of HIV-1 DNA: Unintegrated? Not a problem!

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Wu, Yuntao

2008-07-09

Accumulation of high levels of unintegrated viral DNA is a common feature of retroviral infection. It was recently discovered that coinfection of cells with integrated and unintegrated HIV-1 can result in complementation, allowing viral replication in the absence of integration. This new mode of HIV-1 replication has numerous implications for the function of unintegrated viral DNA and its application as a therapeutic vector.

The second chance story of HIV-1 DNA: Unintegrated? Not a problem!

Directory of Open Access Journals (Sweden)

Wu Yuntao

2008-07-01

Full Text Available Abstract Accumulation of high levels of unintegrated viral DNA is a common feature of retroviral infection. It was recently discovered that coinfection of cells with integrated and unintegrated HIV-1 can result in complementation, allowing viral replication in the absence of integration. This new mode of HIV-1 replication has numerous implications for the function of unintegrated viral DNA and its application as a therapeutic vector.

VMF3/GPT3: refined discrete and empirical troposphere mapping functions

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Landskron, Daniel; Böhm, Johannes

2018-04-01

Incorrect modeling of troposphere delays is one of the major error sources for space geodetic techniques such as Global Navigation Satellite Systems (GNSS) or Very Long Baseline Interferometry (VLBI). Over the years, many approaches have been devised which aim at mapping the delay of radio waves from zenith direction down to the observed elevation angle, so-called mapping functions. This paper contains a new approach intended to refine the currently most important discrete mapping function, the Vienna Mapping Functions 1 (VMF1), which is successively referred to as Vienna Mapping Functions 3 (VMF3). It is designed in such a way as to eliminate shortcomings in the empirical coefficients b and c and in the tuning for the specific elevation angle of 3°. Ray-traced delays of the ray-tracer RADIATE serve as the basis for the calculation of new mapping function coefficients. Comparisons of modeled slant delays demonstrate the ability of VMF3 to approximate the underlying ray-traced delays more accurately than VMF1 does, in particular at low elevation angles. In other words, when requiring highest precision, VMF3 is to be preferable to VMF1. Aside from revising the discrete form of mapping functions, we also present a new empirical model named Global Pressure and Temperature 3 (GPT3) on a 5°× 5° as well as a 1°× 1° global grid, which is generally based on the same data. Its main components are hydrostatic and wet empirical mapping function coefficients derived from special averaging techniques of the respective (discrete) VMF3 data. In addition, GPT3 also contains a set of meteorological quantities which are adopted as they stand from their predecessor, Global Pressure and Temperature 2 wet. Thus, GPT3 represents a very comprehensive troposphere model which can be used for a series of geodetic as well as meteorological and climatological purposes and is fully consistent with VMF3.

Molecular cloning of a mouse DNA repair gene that complements the defect of group-A xeroderma pigmentosum

International Nuclear Information System (INIS)

Tanaka, K.; Satokata, I.; Ogita, Z.; Uchida, T.; Okada, Y.

1989-01-01

For isolation of the gene responsible for xeroderma pigmentosum (XP) complementation group A, plasmid pSV2gpt and genomic DNA from a mouse embryo were cotransfected into XP2OSSV cells, a group-A XP cell line. Two primary UV-resistant XP transfectants were isolated from about 1.6 X 10(5) pSV2gpt-transformed XP colonies. pSV2gpt and genomic DNA from the primary transfectants were again cotransfected into XP2OSSV cells and a secondary UV-resistant XP transfectant was obtained by screening about 4.8 X 10(5) pSV2gpt-transformed XP colonies. The secondary transfectant retained fewer mouse repetitive sequences. A mouse gene that complements the defect of XP2OSSV cells was cloned into an EMBL3 vector from the genome of a secondary transfectant. Transfections of the cloned DNA also conferred UV resistance on another group-A XP cell line but not on XP cell lines of group C, D, F, or G. Northern blot analysis of poly(A)+ RNA with a subfragment of cloned mouse DNA repair gene as the probe revealed that an approximately 1.0 kilobase mRNA was transcribed in the donor mouse embryo and secondary transfectant, and approximately 1.0- and approximately 1.3-kilobase mRNAs were transcribed in normal human cells, but none of these mRNAs was detected in three strains of group-A XP cells. These results suggest that the cloned DNA repair gene is specific for group-A XP and may be the mouse homologue of the group-A XP human gene

Extensive virologic and immunologic characterization in an HIV-infected individual following allogeneic stem cell transplant and analytic cessation of antiretroviral therapy: A case study.

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Cummins, Nathan W; Rizza, Stacey; Litzow, Mark R; Hua, Stephane; Lee, Guinevere Q; Einkauf, Kevin; Chun, Tae-Wook; Rhame, Frank; Baker, Jason V; Busch, Michael P; Chomont, Nicolas; Dean, Patrick G; Fromentin, Rémi; Haase, Ashley T; Hampton, Dylan; Keating, Sheila M; Lada, Steven M; Lee, Tzong-Hae; Natesampillai, Sekar; Richman, Douglas D; Schacker, Timothy W; Wietgrefe, Stephen; Yu, Xu G; Yao, Joseph D; Zeuli, John; Lichterfeld, Mathias; Badley, Andrew D

2017-11-01

Notwithstanding 1 documented case of HIV-1 cure following allogeneic stem cell transplantation (allo-SCT), several subsequent cases of allo-SCT in HIV-1 positive individuals have failed to cure HIV-1 infection. The aim of our study was to describe changes in the HIV reservoir in a single chronically HIV-infected patient on suppressive antiretroviral therapy who underwent allo-SCT for treatment of acute lymphoblastic leukemia. We prospectively collected peripheral blood mononuclear cells (PBMCs) by leukapheresis from a 55-year-old man with chronic HIV infection before and after allo-SCT to measure the size of the HIV-1 reservoir and characterize viral phylogeny and phenotypic changes in immune cells. At day 784 post-transplant, when HIV-1 was undetectable by multiple measures-including PCR measurements of both total and integrated HIV-1 DNA, replication-competent virus measurement by large cell input quantitative viral outgrowth assay, and in situ hybridization of colon tissue-the patient consented to an analytic treatment interruption (ATI) with frequent clinical monitoring. He remained aviremic off antiretroviral therapy until ATI day 288, when a low-level virus rebound of 60 HIV-1 copies/ml occurred, which increased to 1,640 HIV-1 copies/ml 5 days later, prompting reinitiation of ART. Rebounding plasma HIV-1 sequences were phylogenetically distinct from proviral HIV-1 DNA detected in circulating PBMCs before transplantation. The main limitations of this study are the insensitivity of reservoir measurements, and the fact that it describes a single case. allo-SCT led to a significant reduction in the size of the HIV-1 reservoir and a >9-month-long ART-free remission from HIV-1 replication. Phylogenetic analyses suggest that the origin of rebound virus was distinct from the viruses identified pre-transplant in the PBMCs.

Broad and potent immune responses to a low dose intradermal HIV-1 DNA boosted with HIV-1 recombinant MVA among healthy adults in Tanzania☆,☆☆

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Bakari, Muhammad; Aboud, Said; Nilsson, Charlotta; Francis, Joel; Buma, Deus; Moshiro, Candida; Aris, Eric A.; Lyamuya, Eligius F.; Janabi, Mohamed; Godoy-Ramirez, Karina; Joachim, Agricola; Polonis, Victoria R.; Bråve, Andreas; Earl, Patricia; Robb, Merlin; Marovich, Mary; Wahren, Britta; Pallangyo, Kisali; Biberfeld, Gunnel; Mhalu, Fred; Sandström, Eric

2016-01-01

Background We conducted a phase I/II randomized placebo-controlled trial with the aim of exploring whether priming with a low intradermal dose of a multiclade, multigene HIV-1 DNA vaccine could improve the immunogenicity of the same vaccine given intramuscularly prior to boosting with a heterologous HIV-1 MVA among healthy adults in Dar es Salaam, Tanzania. Methods Sixty HIV-uninfected volunteers were randomized to receive DNA plasmid vaccine 1 mg intradermally (id), n = 20, or 3.8 mg intramuscularly (im), n = 20, or placebo, n = 20, using a needle-free injection device. DNA plasmids encoding HIV-1 genes gp160 subtype A, B, C; rev B; p17/p24 gag A, B and Rtmut B were given at weeks 0, 4 and 12. Recombinant MVA (108 pfu) expressing HIV-1 Env, Gag, Pol of CRF01_AE or placebo was administered im at month 9 and 21. Results The vaccines were well tolerated. Two weeks after the third HIV-DNA injection, 22/38 (58%) vaccinees had IFN-γ ELISpot responses to Gag. Two weeks after the first HIV-MVA boost all 35 (100%) vaccinees responded to Gag and 31 (89%) to Env. Two to four weeks after the second HIV-MVA boost, 28/29 (97%) vaccinees had IFN-γ ELISpot responses, 27 (93%) to Gag and 23 (79%) to Env. The id-primed recipients had significantly higher responses to Env than im recipients. Intracellular cytokine staining for Gag-specific IFN-γ/IL-2 production showed both CD8+ and CD4+ T cell responses. All vaccinees had HIV-specific lymphoproliferative responses. All vaccinees reacted in diagnostic HIV serological tests and 26/29 (90%) had antibodies against gp160 after the second HIV-MVA boost. Furthermore, while all of 29 vaccinee sera were negative for neutralizing antibodies against clade B, C and CRF01 AE pseudoviruses in the TZM-bl neutralization assay, in a PBMC assay, the response rate ranged from 31% to 83% positives, depending upon the clade B or CRF01_AE virus tested. This vaccine approach is safe and highly immunogenic. Low dose, id HIV-DNA priming elicited higher

FISHing Out the Hidden Enemy: Advances in Detecting and Measuring Latent HIV-Infected Cells.

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Prasad, Vinayaka R; Kalpana, Ganjam V

2017-09-19

The indomitable aspect of HIV-1 infection is not that HIV-1 proviral DNA is integrated into host DNA but that it can also turn itself off, remaining invisible to drug or immune surveillance. Thus, the goals of eradication include ways to precisely excise HIV-1 DNA or wake up the silent HIV-1 provirus and eliminate the infected cells thus identified. Methods to identify and fish out the latently infected cells or to delineate their characteristics are being rapidly developed. In 2016, Baxter et al. (A. E. Baxter, J. Niessl, R. Fromentin, J. Richard, F. Porichis, R. Charlebois, M. Massanella, N. Brassard, N. Alsahafi, G. G. Delgado, J. P. Routy, B. D. Walker, A. Finzi, N. Chomont, and D. E. Kaufmann, Cell Host Microbe 20:368-380, 2016, https://doi.org/10.1016/j.chom.2016.07.015) and Martrus et al. (G. Martrus, A. Niehrs, R. Cornelis, A. Rechtien, W. García-Beltran, M. Lütgehetmann, C. Hoffmann, and M. Altfeld, J Virol 90:9018-9028, 2016, https://doi.org/10.1128/JVI.01448-16) reported using the fluorescence in situ hybridization-flow cytometry technique to identify and quantify cells expressing HIV-1 RNA and Gag protein, as well as bearing unique cell surface markers. In a recent article in mBio , Grau-Expósito et al. (J. Grau-Expósito, C. Serra-Peinado, L. Miguel, J. Navarro, A. Curran, J. Burgos, I. Ocaña, E. Ribera, A. Torrella, B. Planas, R. Badía, J. Castellví, V. Falcó, M. Crespo, and M. J. Buzon, mBio 8:e00876-17, 2017, https://doi.org/10.1128/mBio.00876-17) reported a similar method that they claim to be more sensitive. With these methods, researchers are one step closer to measuring latent reservoirs and eliminating critical barriers to HIV eradication. Copyright © 2017 Prasad and Kalpana.

Convergent evolution of SIV env after independent inoculation of rhesus macaques with infectious proviral DNA

International Nuclear Information System (INIS)

Buckley, Kathleen A.; Li Peilin; Khimani, Anis H.; Hofmann-Lehmann, Regina; Liska, Vladimir; Anderson, Daniel C.; McClure, Harold M.; Ruprecht, Ruth M.

2003-01-01

The env gene of three simian immunodeficiency virus (SIV) variants developed convergent mutations during disease progression in six rhesus macaques. The monkeys had been inoculated with supercoiled plasmids encoding infectious proviruses of SIVmac239 (a pathogenic, wild-type strain), SIVΔ3 (the live attenuated vaccine strain derived from SIVmac239), or SIVΔ3+ (a pathogenic progeny virus that had evolved from SIVΔ3). All six monkeys developed immunodeficiency and progressed to fatal disease. Although many divergent mutations arose in env among the different hosts, three regions consistently mutated in all monkeys studied; these similar mutations developed independently even though the animals had received only a single infectious molecular clone rather than standard viral inocula that contain viral quasispecies. Together, these data indicate that the env genes of SIVmac239, SIVΔ3, and SIVΔ3+, in the context of different proviral backbones, evolve similarly in different hosts during disease progression

Accurate episomal HIV 2-LTR circles quantification using optimized DNA isolation and droplet digital PCR.

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Malatinkova, Eva; Kiselinova, Maja; Bonczkowski, Pawel; Trypsteen, Wim; Messiaen, Peter; Vermeire, Jolien; Verhasselt, Bruno; Vervisch, Karen; Vandekerckhove, Linos; De Spiegelaere, Ward

2014-01-01

In HIV-infected patients on combination antiretroviral therapy (cART), the detection of episomal HIV 2-LTR circles is a potential marker for ongoing viral replication. Quantification of 2-LTR circles is based on quantitative PCR or more recently on digital PCR assessment, but is hampered due to its low abundance. Sample pre-PCR processing is a critical step for 2-LTR circles quantification, which has not yet been sufficiently evaluated in patient derived samples. We compared two sample processing procedures to more accurately quantify 2-LTR circles using droplet digital PCR (ddPCR). Episomal HIV 2-LTR circles were either isolated by genomic DNA isolation or by a modified plasmid DNA isolation, to separate the small episomal circular DNA from chromosomal DNA. This was performed in a dilution series of HIV-infected cells and HIV-1 infected patient derived samples (n=59). Samples for the plasmid DNA isolation method were spiked with an internal control plasmid. Genomic DNA isolation enables robust 2-LTR circles quantification. However, in the lower ranges of detection, PCR inhibition caused by high genomic DNA load substantially limits the amount of sample input and this impacts sensitivity and accuracy. Moreover, total genomic DNA isolation resulted in a lower recovery of 2-LTR templates per isolate, further reducing its sensitivity. The modified plasmid DNA isolation with a spiked reference for normalization was more accurate in these low ranges compared to genomic DNA isolation. A linear correlation of both methods was observed in the dilution series (R2=0.974) and in the patient derived samples with 2-LTR numbers above 10 copies per million peripheral blood mononuclear cells (PBMCs), (R2=0.671). Furthermore, Bland-Altman analysis revealed an average agreement between the methods within the 27 samples in which 2-LTR circles were detectable with both methods (bias: 0.3875±1.2657 log10). 2-LTR circles quantification in HIV-infected patients proved to be more

The mitochondrial DNA T16189C polymorphism and HIV-associated cardiomyopathy: a genotype-phenotype association study

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Poulton Joanna

2009-04-01

Full Text Available Abstract Background The mitochondrial DNA (mtDNA T16189C polymorphism, with a homopolymeric C-tract of 10–12 cytosines, is a putative genetic risk factor for idiopathic dilated cardiomyopathy in the African and British populations. We hypothesized that this variant may predispose to dilated cardiomyopathy in people who are infected with the human immunodeficiency virus (HIV. Methods A case-control study of 30 HIV-positive cases with dilated cardiomyopathy and 37 HIV-positive controls without dilated cardiomyopathy was conducted. The study was confined to persons of black African ancestry to minimize confounding of results by population admixture. HIV-positive patients with an echocardiographically confirmed diagnosis of dilated cardiomyopathy and HIV-positive controls with echocardiographically normal hearts were studied. Patients with secondary causes of cardiomyopathy (such as hypertension, diabetes, pregnancy, alcoholism, valvular heart disease, and opportunistic infection were excluded from the study. DNA samples were sequenced for the mtDNA T16189C polymorphism with a homopolymeric C-tract in the forward and reverse directions on an ABI3100 sequencer. Results The cases and controls were well matched for age (median 35 years versus 34 years, P = 0.93, gender (males 60% vs 53%, P = 0.54, and stage of HIV disease (mean CD4 T cell count 260.7/μL vs. 176/μL, P = 0.21. The mtDNA T16189C variant with a homopolymeric C-tract was detected at a frequency of 26.7% (8/30 in the HIV-associated cardiomyopathy cases and 13.5% (5/37 in the HIV-positive controls. There was no significant difference between cases and controls (Odds Ratio 2.33, 95% Confidence Interval 0.67–8.06, p = 0.11. Conclusion The mtDNA T16189C variant with a homopolymeric C-tract is not associated with dilated cardiomyopathy in black African people infected with HIV.

Using Resurrected Ancestral Proviral Proteins to Engineer Virus Resistance.

Science.gov (United States)

Delgado, Asunción; Arco, Rocio; Ibarra-Molero, Beatriz; Sanchez-Ruiz, Jose M

2017-05-09

Proviral factors are host proteins hijacked by viruses for processes essential for virus propagation such as cellular entry and replication. Pathogens and their hosts co-evolve. It follows that replacing a proviral factor with a functional ancestral form of the same protein could prevent viral propagation without fatally compromising organismal fitness. Here, we provide proof of concept of this notion. Thioredoxins serve as general oxidoreductases in all known cells. We report that several laboratory resurrections of Precambrian thioredoxins display substantial levels of functionality within Escherichia coli. Unlike E. coli thioredoxin, however, these ancestral thioredoxins are not efficiently recruited by the bacteriophage T7 for its replisome and therefore prevent phage propagation in E. coli. These results suggest an approach to the engineering of virus resistance. Diseases caused by viruses may have a devastating effect in agriculture. We discuss how the suggested approach could be applied to the engineering of plant virus resistance. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Cattle with the BoLA class II DRB3*0902 allele have significantly lower bovine leukemia proviral loads.

Science.gov (United States)

Hayashi, Takumi; Mekata, Hirohisa; Sekiguchi, Satoshi; Kirino, Yumi; Mitoma, Shuya; Honkawa, Kazuyuki; Horii, Yoichiro; Norimine, Junzo

2017-09-12

The bovine MHC (BoLA) class II DRB3 alleles are associated with polyclonal expansion of lymphocytes caused by bovine leukemia virus (BLV) infection in cattle. To examine whether the DRB3*0902 allele, one of the resistance-associated alleles, is associated with the proviral load, we measured BLV proviral load of BLV-infected cattle and clarified their DRB3 alleles. Fifty-seven animals with DRB3*0902 were identified out of 835 BLV-infected cattle and had significantly lower proviral load (Pclass II DRA/DRB3*0902 molecule plays an important immunological role in suppressing viral replication, resulting in resistance to the disease progression.

Repair of oxidative DNA base damage in the host genome influences the HIV integration site sequence preference.

Directory of Open Access Journals (Sweden)

Geoffrey R Bennett

Full Text Available Host base excision repair (BER proteins that repair oxidative damage enhance HIV infection. These proteins include the oxidative DNA damage glycosylases 8-oxo-guanine DNA glycosylase (OGG1 and mutY homolog (MYH as well as DNA polymerase beta (Polβ. While deletion of oxidative BER genes leads to decreased HIV infection and integration efficiency, the mechanism remains unknown. One hypothesis is that BER proteins repair the DNA gapped integration intermediate. An alternative hypothesis considers that the most common oxidative DNA base damages occur on guanines. The subtle consensus sequence preference at HIV integration sites includes multiple G:C base pairs surrounding the points of joining. These observations suggest a role for oxidative BER during integration targeting at the nucleotide level. We examined the hypothesis that BER repairs a gapped integration intermediate by measuring HIV infection efficiency in Polβ null cell lines complemented with active site point mutants of Polβ. A DNA synthesis defective mutant, but not a 5'dRP lyase mutant, rescued HIV infection efficiency to wild type levels; this suggested Polβ DNA synthesis activity is not necessary while 5'dRP lyase activity is required for efficient HIV infection. An alternate hypothesis that BER events in the host genome influence HIV integration site selection was examined by sequencing integration sites in OGG1 and MYH null cells. In the absence of these 8-oxo-guanine specific glycosylases the chromatin elements of HIV integration site selection remain the same as in wild type cells. However, the HIV integration site sequence preference at G:C base pairs is altered at several positions in OGG1 and MYH null cells. Inefficient HIV infection in the absence of oxidative BER proteins does not appear related to repair of the gapped integration intermediate; instead oxidative damage repair may participate in HIV integration site preference at the sequence level.

Mutations of mtDNA polymerase-γ and hyperlactataemia in the HIV ...

African Journals Online (AJOL)

Mutations of mtDNA polymerase-γ and hyperlactataemia in the HIV-infected Zulu population of South Africa. ... D B A Ojwach, C Aldous, P Kocheleff, B Sartorius ... of their capacity to impede human mitochondrial DNA polymerase-γ (POLG), ...

Photosynthetic acclimation of WS and WS-gpt2 in Arabidopsis thaliana under fluctuating natural light condition

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Pa'ee, Furzani; Johnson, Giles

2017-10-01

Photoacclimation is a process by which photosynthetic capacity is regulated in response to environmental adjustments in terms of light regime. Photoacclimation is essential in determining the photosynthetic capacity to optimize light use and to avoid potentially damaging effects. Previous work in our laboratory has identified a gene, gpt2 (At1g61800) that is essential for plants to acclimate to an increase and decrease of growth irradiance, separately. To investigate the photoacclimation ability towards fluctuating natural light condition in Arabidopsis thaliana, photosynthetic capacity was measured in plants of the accession Wassileskija (WS) and in plants lacking expression of the gene At1g61800 (WS-gpt2). The experiment was carried out over a time span from early Autumn to early Spring season in 2010-2011 and 2011-2012. The seedlings were grown in an unheated greenhouse in Manchester, UK without supplementary lighting. Gas exchange measurements and chlorophyll content estimation were performed on WS and WS-gpt2 and it showed that both sets of plants were able to acclimate to fluctuating natural light condition. Therefore, it is suggested that the mechanisms of acclimation in a separate growth light condition is mechanistically distinct than the mechanism under fluctuating natural light condition.

Integrase-independent HIV-1 infection is augmented under conditions of DNA damage and produces a viral reservoir

International Nuclear Information System (INIS)

Ebina, Hirotaka; Kanemura, Yuka; Suzuki, Yasutsugu; Urata, Kozue; Misawa, Naoko; Koyanagi, Yoshio

2012-01-01

HIV-1 possesses a viral protein, integrase (IN), which is necessary for its efficient integration in target cells. However, it has been reported that an IN-defective HIV strain is still capable of integration. Here, we assessed the ability of wild type (WT) HIV-1 to establish infection in the presence of IN inhibitors. We observed a low, yet clear infection of inhibitor-incubated cells infected with WT HIV which was identical to cells infected with IN-deficient HIV, D64A. Furthermore, the IN-independent integration could be enhanced by the pretreatment of cells with DNA-damaging agents suggesting that integration is mediated by a DNA repair system. Moreover, significantly faster viral replication kinetics with augmented viral DNA integration was observed after infection in irradiated cells treated with IN inhibitor compared to nonirradiated cells. Altogether, our results suggest that HIV DNA has integration potential in the presence of an IN inhibitor and may serve as a virus reservoir.

Integrase-independent HIV-1 infection is augmented under conditions of DNA damage and produces a viral reservoir

Energy Technology Data Exchange (ETDEWEB)

Ebina, Hirotaka, E-mail: hebina@virus.kyoto-u.ac.jp; Kanemura, Yuka; Suzuki, Yasutsugu; Urata, Kozue; Misawa, Naoko; Koyanagi, Yoshio

2012-05-25

HIV-1 possesses a viral protein, integrase (IN), which is necessary for its efficient integration in target cells. However, it has been reported that an IN-defective HIV strain is still capable of integration. Here, we assessed the ability of wild type (WT) HIV-1 to establish infection in the presence of IN inhibitors. We observed a low, yet clear infection of inhibitor-incubated cells infected with WT HIV which was identical to cells infected with IN-deficient HIV, D64A. Furthermore, the IN-independent integration could be enhanced by the pretreatment of cells with DNA-damaging agents suggesting that integration is mediated by a DNA repair system. Moreover, significantly faster viral replication kinetics with augmented viral DNA integration was observed after infection in irradiated cells treated with IN inhibitor compared to nonirradiated cells. Altogether, our results suggest that HIV DNA has integration potential in the presence of an IN inhibitor and may serve as a virus reservoir.

A multigrid based 3D space-charge routine in the tracking code GPT

NARCIS (Netherlands)

Pöplau, G.; Rienen, van U.; Loos, de M.J.; Geer, van der S.B.; Berz, M.; Makino, K.

2005-01-01

Fast calculation of3D non-linear space-charge fields is essential for the simulation ofhigh-brightness charged particle beams. We report on our development of a new 3D spacecharge routine in the General Particle Tracer (GPT) code. The model is based on a nonequidistant multigrid Poisson solver that

Radiation induced COX-2 expression and mutagenesis at non-targeted lung tissues of gpt delta transgenic mice

Science.gov (United States)

Chai, Y; Calaf, G M; Zhou, H; Ghandhi, S A; Elliston, C D; Wen, G; Nohmi, T; Amundson, S A; Hei, T K

2013-01-01

Background: Although radiation-induced bystander effects have been confirmed using a variety of endpoints, the mechanism(s) underlying these effects are not well understood, especially for in vivo study. Methods: A 1-cm2 area (1 cm × 1 cm) in the lower abdominal region of gpt delta transgenic mice was irradiated with 5 Gy of 300 keV X-rays, and changes in out-of-field lung and liver were observed. Results: Compared with sham-treated controls, the Spi− mutation frequency increased 2.4-fold in non-targeted lung tissues at 24 h after partial body irradiation (PBIR). Consistent with dramatic Cyclooxygenase 2 (COX-2) induction in the non-targeted bronchial epithelial cells, increasing levels of prostaglandin, together with 8-hydroxydeoxyguanosine, in the out-of-field lung tissues were observed after PBIR. In addition, DNA double-strand breaks and apoptosis were induced in bystander lung tissues after PBIR. Conclusion: The PBIR induces DNA damage and mutagenesis in non-targeted lung tissues, especially in bronchial epithelial cells, and COX-2 has an essential role in bystander mutagenesis. PMID:23321513

Three-Year Durability of Immune Responses Induced by HIV-DNA and HIV-Modified Vaccinia Virus Ankara and Effect of a Late HIV-Modified Vaccinia Virus Ankara Boost in Tanzanian Volunteers.

Science.gov (United States)

Joachim, Agricola; Munseri, Patricia J; Nilsson, Charlotta; Bakari, Muhammad; Aboud, Said; Lyamuya, Eligius F; Tecleab, Teghesti; Liakina, Valentina; Scarlatti, Gabriella; Robb, Merlin L; Earl, Patricia L; Moss, Bernard; Wahren, Britta; Mhalu, Fred; Ferrari, Guido; Sandstrom, Eric; Biberfeld, Gunnel

2017-08-01

We explored the duration of immune responses and the effect of a late third HIV-modified vaccinia virus Ankara (MVA) boost in HIV-DNA primed and HIV-MVA boosted Tanzanian volunteers. Twenty volunteers who had previously received three HIV-DNA and two HIV-MVA immunizations were given a third HIV-MVA immunization 3 years after the second HIV-MVA boost. At the time of the third HIV-MVA, 90% of the vaccinees had antibodies to HIV-1 subtype C gp140 (median titer 200) and 85% to subtype B gp160 (median titer 100). The majority of vaccinees had detectable antibody-dependent cellular cytotoxicity (ADCC)-mediating antibodies, 70% against CRF01_AE virus-infected cells (median titer 239) and 84% against CRF01_AE gp120-coated cells (median titer 499). A high proportion (74%) of vaccinees had IFN-γ ELISpot responses, 63% to Gag and 42% to Env, 3 years after the second HIV-MVA boost. After the third HIV-MVA, there was an increase in Env-binding antibodies and ADCC-mediating antibodies relative to the response seen at the time of the third HIV-MVA vaccination, p < .0001 and p < .05, respectively. The frequency of IFN-γ ELISpot responses increased to 95% against Gag or Env and 90% to both Gag and Env, p = .064 and p = .002, respectively. In conclusion, the HIV-DNA prime/HIV-MVA boost regimen elicited potent antibody and cellular immune responses with remarkable durability, and a third HIV-MVA immunization significantly boosted both antibody and cellular immune responses relative to the levels detected at the time of the third HIV-MVA, but not to higher levels than after the second HIV-MVA.

DNA base sequence changes induced by ultraviolet light mutagenesis of a gene on a chromosome in Chinese hamster ovary cells

Energy Technology Data Exchange (ETDEWEB)

Romac, S; Leong, P; Sockett, H; Hutchinson, F [Yale Univ., New Haven, CT (USA). Dept. of Molecular Biophysics and Biochemistry

1989-09-20

The DNA base sequence changes induced by mutagenesis with ultraviolet light have been determined in a gene on a chromosome of cultured Chinese hamster ovary (CHO) cells. The gene was the Excherichia coli gpt gene, of which a single copy was stably incorporated and expressed in the CHO cell genome. The cells were irradiated with ultraviolet light and gpt{sup -} colonies were selected by resistance to 6-thioguanine. The gpt gene was amplified from chromosomal DNA by use of the polymerase chain reaction (PCR) and the amplified DNA sequenced directly by the dideoxy method. Of the 58 sequenced mutants of independent origin 53 were base change mutations. Forty-one base substitutions were single base changes, ten had two adjacent (or tandem) base changes, and one had two base changes separated by a single base-pair. Only one mutant had a multiple base change mutation with two or more well separated base changes. In contrast much higher levels of such mutations were reported in ultraviolet mutagenesis of genes on a shuttle vector in primate cells. Two deletions of a single base-pair were observed and three deletions ranging from 6 to 37 base-pairs. The mutation spectrum in the gpt gene had similarities to the ultraviolet mutation spectra for several genes in prokaryotes, which suggests similarities in mutational mechanisms in prokaryotes and eukaryotes. (author).

CD32 is expressed on cells with transcriptionally active HIV but does not enrich for HIV DNA in resting T cells.

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Abdel-Mohsen, Mohamed; Kuri-Cervantes, Leticia; Grau-Exposito, Judith; Spivak, Adam M; Nell, Racheal A; Tomescu, Costin; Vadrevu, Surya Kumari; Giron, Leila B; Serra-Peinado, Carla; Genescà, Meritxell; Castellví, Josep; Wu, Guoxin; Del Rio Estrada, Perla M; González-Navarro, Mauricio; Lynn, Kenneth; King, Colin T; Vemula, Sai; Cox, Kara; Wan, Yanmin; Li, Qingsheng; Mounzer, Karam; Kostman, Jay; Frank, Ian; Paiardini, Mirko; Hazuda, Daria; Reyes-Terán, Gustavo; Richman, Douglas; Howell, Bonnie; Tebas, Pablo; Martinez-Picado, Javier; Planelles, Vicente; Buzon, Maria J; Betts, Michael R; Montaner, Luis J

2018-04-18

The persistence of HIV reservoirs, including latently infected, resting CD4 + T cells, is the major obstacle to cure HIV infection. CD32a expression was recently reported to mark CD4 + T cells harboring a replication-competent HIV reservoir during antiretroviral therapy (ART) suppression. We aimed to determine whether CD32 expression marks HIV latently or transcriptionally active infected CD4 + T cells. Using peripheral blood and lymphoid tissue of ART-treated HIV + or SIV + subjects, we found that most of the circulating memory CD32 + CD4 + T cells expressed markers of activation, including CD69, HLA-DR, CD25, CD38, and Ki67, and bore a T H 2 phenotype as defined by CXCR3, CCR4, and CCR6. CD32 expression did not selectively enrich for HIV- or SIV-infected CD4 + T cells in peripheral blood or lymphoid tissue; isolated CD32 + resting CD4 + T cells accounted for less than 3% of the total HIV DNA in CD4 + T cells. Cell-associated HIV DNA and RNA loads in CD4 + T cells positively correlated with the frequency of CD32 + CD69 + CD4 + T cells but not with CD32 expression on resting CD4 + T cells. Using RNA fluorescence in situ hybridization, CD32 coexpression with HIV RNA or p24 was detected after in vitro HIV infection (peripheral blood mononuclear cell and tissue) and in vivo within lymph node tissue from HIV-infected individuals. Together, these results indicate that CD32 is not a marker of resting CD4 + T cells or of enriched HIV DNA-positive cells after ART; rather, CD32 is predominately expressed on a subset of activated CD4 + T cells enriched for transcriptionally active HIV after long-term ART. Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

DNA damage repair machinery and HIV escape from innate immune sensing

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Christelle eBregnard

2014-04-01

Full Text Available Viruses have been long known to perturb cell cycle regulators and key players of the DNA damage response to benefit their life cycles. In the case of the human immunodeficiency virus (HIV, the viral auxiliary protein Vpr activates the structure-specific endonuclease SLX4 complex to promote escape from innate immune sensing and, as a side effect, induces replication stress in cycling cells and subsequent cell cycle arrest at the G2/M transition. This novel pathway subverted by HIV to prevent accumulation of viral reverse transcription by-products adds up to facilitating effects of major cellular exonucleases that degrade pathological DNA species. Within this review we discuss the impact of this finding on our understanding of the interplay between HIV replication and nucleic acid metabolism and its implications for cancer-related chronic inflammation.

Application of the heuristically based GPT theory to termohydraulic problems

International Nuclear Information System (INIS)

Alvim, A.C.M.

1988-01-01

Application of heuristically based generalized perturbation theory (GPT) to the thermohydraulic (generally nonlinear) field is here illustrated. After a short description of the general methodology, the (linear) equations governing the importance function relevant to a generic multichannel problem are derived, within the physical model adopted in the COBRA IV-I Code. These equations are put in a form which should benefit of the calculational scheme of the original COBRA Code in the sense that only minor changes of it (mostly implying physical constants and source terms redefinitions) should be necessary for their solutions. (author) [pt

Mutations in Ovis aries TMEM154 are associated with lower small ruminant lentivirus proviral concentration in one sheep flock.

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Alshanbari, F A; Mousel, M R; Reynolds, J O; Herrmann-Hoesing, L M; Highland, M A; Lewis, G S; White, S N

2014-08-01

Small ruminant lentivirus (SRLV), also called ovine progressive pneumonia virus or maedi-visna, is present in 24% of US sheep. Like human immunodeficiency virus, SRLV is a macrophage-tropic lentivirus that causes lifelong infection. The production impacts from SRLV are due to a range of disease symptoms, including pneumonia, arthritis, mastitis, body condition wasting and encephalitis. There is no cure and no effective vaccine for preventing SRLV infection. However, breed differences in prevalence and proviral concentration indicate a genetic basis for susceptibility to SRLV. Animals with high blood proviral concentration show increased tissue lesion severity, so proviral concentration represents a live animal test for control post-infection in terms of proviral replication and disease severity. Recently, it was found that sheep with two copies of TMEM154 haplotype 1 (encoding lysine at position 35) had lower odds of SRLV infection. In this study, we examined the relationship between SRLV control post-infection and variants in two genes, TMEM154 and CCR5, in four flocks containing 1403 SRLV-positive sheep. We found two copies of TMEM154 haplotype 1 were associated with lower SRLV proviral concentration in one flock (P < 0.02). This identified the same favorable diplotype for SRLV control post-infection as for odds of infection. However, frequencies of haplotypes 2 and 3 were too low in the other three flocks to test. The CCR5 promoter deletion did not have consistent association with SRLV proviral concentration. Future work in flocks with more balanced allele frequencies is needed to confirm or refute TMEM154 association with control of SRLV post-infection. Published 2014. This article is a U.S. Government work and is in the public domain in the USA. Animal Genetics published by John Wiley & Sons Ltd on behalf of Stichting International Foundation for Animal Genetics.

Sensitivity analyses by Generalized Perturbation Theory (GPT) methods applied to GUINEVERE and MYRRHA lead fast reactors

International Nuclear Information System (INIS)

Carta, M.; Bianchini, G.; Peluso, V.; Gandini, A.; Fabrizio, V.; Ricci, L.

2013-01-01

Conclusions: • GPT methodologies can provide a codified “key to reading” to the properties of some GUINEVERE integral characteristics respect to analogous characteristics in MYRRHA. • The present work has been focused on the analysis, by numerical simulations, of the representativeness, with respect to the MYRRHA situation assumed as reference system, of the behaviour in GUINEVERE of the spectral indexes F9/F5, F8/F5 and F7/F5 following a localized (core shell) or global (whole active core) lead (GUINEVERE) or lead-bismuth (MYRRHA) 20% density reduction. • The analysis, carried out by means of GPT methodologies implemented into the ERANOS French neutronic code, and using the US dispersion matrix BOLNA, provided encouraging results concerning the correlation for all the spectral indexes taken into account

Control of HIV-1 in Elite Suppressors despite Ongoing Replication and Evolution in Plasma Virus▿

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O'Connell, Karen A.; Brennan, Timothy P.; Bailey, Justin R.; Ray, Stuart C.; Siliciano, Robert F.; Blankson, Joel N.

2010-01-01

A subset of HIV-1-infected patients known as elite controllers or suppressors (ES) control the virus naturally. We have previously demonstrated sequence discordance between proviral and plasma gag clones in ES, much of which can be attributed to selective pressure from the host (J. R. Bailey, T. M. Williams, R. F. Siliciano, and J. N. Blankson, J. Exp. Med. 203:1357-1369, 2006). However, it is not clear whether ongoing viral replication continues in ES once the control of viremia has been established or whether selective pressure impacts this evolution. The cytotoxic T-lymphocyte (CTL) response in ES often targets Gag and frequently is superior to that of HIV-1 progressors, partially due to the HLA class I alleles B*57/5801 and B*27, which are overrepresented in ES. We therefore examined longitudinal plasma and proviral gag sequences from HLA-B*57/5801 and -B*27 ES. Despite the highly conserved nature of gag, we observed clear evidence of evolution in the plasma virus, largely due to synonymous substitutions. In contrast, evolution was rare in proviral clones, suggesting that ongoing replication in ES does not permit the significant reseeding of the latent reservoir. Interestingly, there was little continual evolution in CTL epitopes, and we detected de novo CTL responses to autologous viral mutants. Thus, some ES control viremia despite ongoing replication and evolution. PMID:20444904

CpG methylation controls reactivation of HIV from latency

Czech Academy of Sciences Publication Activity Database

Blažková, Jana; Trejbalová, Kateřina; Gondois-Rey, F.; Halfon, P.; Philibert, P.; Guiguen, A.; Verdin, E.; Olive, D.; Van Lint, C.; Hejnar, Jiří; Hirsch, I.

2009-01-01

Roč. 5, č. 8 (2009), e1000554-e1000554 E-ISSN 1553-7374 R&D Projects: GA ČR GA204/05/0939; GA ČR GP204/08/P616 Institutional research plan: CEZ:AV0Z50520514 Keywords : HIV-1 * proviral latency * CpG methylation * histone modifications * HAART * epigenetics Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 8.978, year: 2009

Safety and immunogenicity of an HIV-1 gag DNA vaccine with or without IL-12 and/or IL-15 plasmid cytokine adjuvant in healthy, HIV-1 uninfected adults.

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Spyros A Kalams

Full Text Available DNA vaccines are a promising approach to vaccination since they circumvent the problem of vector-induced immunity. DNA plasmid cytokine adjuvants have been shown to augment immune responses in small animals and in macaques.We performed two first in human HIV vaccine trials in the US, Brazil and Thailand of an RNA-optimized truncated HIV-1 gag gene (p37 DNA derived from strain HXB2 administered either alone or in combination with dose-escalation of IL-12 or IL-15 plasmid cytokine adjuvants. Vaccinations with both the HIV immunogen and cytokine adjuvant were generally well-tolerated and no significant vaccine-related adverse events were identified. A small number of subjects developed asymptomatic low titer antibodies to IL-12 or IL-15. Cellular immunogenicity following 3 and 4 vaccinations was poor, with response rates to gag of 4.9%/8.7% among vaccinees receiving gag DNA alone, 0%/11.5% among those receiving gag DNA+IL-15, and no responders among those receiving DNA+high dose (1500 ug IL-12 DNA. However, after three doses, 44.4% (4/9 of vaccinees receiving gag DNA and intermediate dose (500 ug of IL-12 DNA demonstrated a detectable cellular immune response.This combination of HIV gag DNA with plasmid cytokine adjuvants was well tolerated. There were minimal responses to HIV gag DNA alone, and no apparent augmentation with either IL-12 or IL-15 plasmid cytokine adjuvants. Despite the promise of DNA vaccines, newer formulations or methods of delivery will be required to increase their immunogenicity.Clinicaltrials.gov NCT00115960 NCT00111605.

Intracellular HIV-1 Gag localization is impaired by mutations in the nucleocapsid zinc fingers

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Muriaux Delphine

2007-08-01

Full Text Available Abstract Background The HIV-1 nucleocapsid protein (NC is formed of two CCHC zinc fingers flanked by highly basic regions. HIV-1 NC plays key roles in virus structure and replication via its nucleic acid binding and chaperoning properties. In fact, NC controls proviral DNA synthesis by reverse transcriptase (RT, gRNA dimerization and packaging, and virion assembly. Results We previously reported a role for the first NC zinc finger in virion structure and replication 1. To investigate the role of both NC zinc fingers in intracellular Gag trafficking, and in virion assembly, we generated series of NC zinc fingers mutations. Results show that all Zinc finger mutations have a negative impact on virion biogenesis and maturation and rendered defective the mutant viruses. The NC zinc finger mutations caused an intracellular accumulation of Gag, which was found either diffuse in the cytoplasm or at the plasma membrane but not associated with endosomal membranes as for wild type Gag. Evidences are also provided showing that the intracellular interactions between NC-mutated Gag and the gRNA were impaired. Conclusion These results show that Gag oligomerization mediated by gRNA-NC interactions is required for correct Gag trafficking, and assembly in HIV-1 producing cells and the release of infectious viruses.

Role of baseline HIV-1 DNA level in highly-experienced patients receiving raltegravir, etravirine and darunavir/ritonavir regimen (ANRS139 TRIO trial.

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Charlotte Charpentier

Full Text Available OBJECTIVE: In the ANRS 139 TRIO trial, the use of 3 new active drugs (raltegravir, etravirine, and darunavir/ritonavir, resulted in a potent and sustained inhibition of viral replication in multidrug-resistant treatment-experienced patients. The aim of this virological sub-study of the ANRS 139 TRIO trial was to assess: (i the evolution of HIV-1 DNA over the first year; and (ii the association between baseline HIV-1 DNA and virological outcome. METHODS: Among the 103 HIV-1-infected patients included in the ANRS-139 TRIO trial, HIV-1 DNA specimens were available for 92, 84, 88, and 83 patients at Week (W0, W12, W24, and W48, respectively. Quantification of total HIV-1 DNA was performed by using the commercial kit "Generic HIV DNA Cell" (Biocentric, Bandol, France. RESULTS: Baseline median HIV-1 DNA of patients displaying virological success (n= 61, viral blip (n= 20, and virological failure (n = 11 were 2.34 log(10 copies/10(6 PBMC (IQR= 2.15-2.66, 2.42 (IQR = 2.12-2.48, and 2.68 (IQR= 2.46-2.83, respectively. Although not statistically significant, patients exhibiting virological success or viral blip had a tendency to display lower baseline HIV-1 DNA than patients experiencing virological failure (P = 0.06. Median decrease of HIV-1 DNA between baseline and W48 was -0.13 log(10 copies/10(6 PBMC (IQR = -0.34 to +0.10, mainly explained by the evolution from W0 to W4. No more changes were observed in the W4-W48 period. CONCLUSIONS: In highly-experienced multidrug-resistant patients, HIV-1 DNA slightly decreased during the first month and then remained stable during the first year of highly potent antiretroviral regimen. In this population, baseline HIV-1 DNA might help to better predict the virological response and to tailor clinical therapeutic management as more aggressive therapeutic choices in patients with higher baseline HIV-1 DNA.

A comparative study of glutamate oxaloacetate transaminase (GOT) and glutamate pyruvate transaminase (GPT) levels in the saliva of diabetic and normal patients.

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Verma, M; Metgud, R; Madhusudan, A S; Verma, N; Saxena, M; Soni, A

2014-10-01

Diabetes has been reported to affect salivary glands adversely in humans and experimental models. Glutamate oxaloacetate transaminase (GOT), glutamate pyruvate transaminase (GPT) and lactate dehydrogenase (LDH) are salivary enzymes that also are widely distributed in animal tissues. We determined GOT and GPT levels in saliva samples of 100 type 1 and 30 type 2 diabetic patients using reflectance spectrophotometry and compared them to 30 age and sex matched healthy controls. Statistically significant differences were observed in the mean values of GOT and GPT in type 1 diabetics compared to type 2 and control groups. Significantly higher GOT levels were found in the 1-20 year age group of type 1 diabetics. Our findings suggest that salivary gland damage is due to the same immunological attack that affects pancreatic β cells and results in type 1 diabetes.

New Highly Sensitive Real-Time PCR Assay for HIV-2 Group A and Group B DNA Quantification.

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Bertine, Mélanie; Gueudin, Marie; Mélard, Adeline; Damond, Florence; Descamps, Diane; Matheron, Sophie; Collin, Fidéline; Rouzioux, Christine; Plantier, Jean-Christophe; Avettand-Fenoel, Véronique

2017-09-01

HIV-2 infection is characterized by a very low replication rate in most cases and low progression. This necessitates an approach to patient monitoring that differs from that for HIV-1 infection. Here, a new highly specific and sensitive method for HIV-2 DNA quantification was developed. The new test is based on quantitative real-time PCR targeting the long terminal repeat (LTR) and gag regions and using an internal control. Analytical performance was determined in three laboratories, and clinical performance was determined on blood samples from 63 patients infected with HIV-2 group A ( n = 35) or group B ( n = 28). The specificity was 100%. The 95% limit of detection was three copies/PCR and the limit of quantification was six copies/PCR. The within-run coefficients of variation were between 1.03% at 3.78 log 10 copies/PCR and 27.02% at 0.78 log 10 copies/PCR. The between-run coefficient of variation was 5.10%. Both manual and automated nucleic acid extraction methods were validated. HIV-2 DNA loads were detectable in blood cells from all 63 patients. When HIV-2 DNA was quantifiable, median loads were significantly higher in antiretroviral-treated than in naive patients and were similar for groups A and B. HIV-2 DNA load was correlated with HIV-2 RNA load ( r = 0.68; 95% confidence interval [CI], 0.4 to 0.8; P < 0.0001). Our data show that this new assay is highly sensitive and quantifies the two main HIV-2 groups, making it useful for the diagnosis of HIV-2 infection and for pathogenesis studies on HIV-2 reservoirs. Copyright © 2017 American Society for Microbiology.

HIV Persistence in Adipose Tissue Reservoirs.

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Couturier, Jacob; Lewis, Dorothy E

2018-02-01

The purpose of this review is to examine the evidence describing adipose tissue as a reservoir for HIV-1 and how this often expansive anatomic compartment contributes to HIV persistence. Memory CD4 T cells and macrophages, the major host cells for HIV, accumulate in adipose tissue during HIV/SIV infection of humans and rhesus macaques. Whereas HIV and SIV proviral DNA is detectable in CD4 T cells of multiple fat depots in virtually all infected humans and monkeys examined, viral RNA is less frequently detected, and infected macrophages may be less prevalent in adipose tissue. However, based on viral outgrowth assays, adipose-resident CD4 T cells are latently infected with virus that is replication-competent and infectious. Additionally, adipocytes interact with CD4 T cells and macrophages to promote immune cell activation and inflammation which may be supportive for HIV persistence. Antiviral effector cells, such as CD8 T cells and NK/NKT cells, are abundant in adipose tissue during HIV/SIV infection and typically exceed CD4 T cells, whereas B cells are largely absent from adipose tissue of humans and monkeys. Additionally, CD8 T cells in adipose tissue of HIV patients are activated and have a late differentiated phenotype, with unique TCR clonotypes of less diversity relative to blood CD8 T cells. With respect to the distribution of antiretroviral drugs in adipose tissue, data is limited, but there may be class-specific penetration of fat depots. The trafficking of infected immune cells within adipose tissues is a common event during HIV/SIV infection of humans and monkeys, but the virus may be mostly transcriptionally dormant. Viral replication may occur less in adipose tissue compared to other major reservoirs, such as lymphoid tissue, but replication competence and infectiousness of adipose latent virus are comparable to other tissues. Due to the ubiquitous nature of adipose tissue, inflammatory interactions among adipocytes and CD4 T cells and macrophages, and

Identification of Nevirapine-Resistant HIV-1 in the Latent Reservoir after Single-Dose Nevirapine to Prevent Mother-to-Child Transmission of HIV-1

Science.gov (United States)

Wind-Rotolo, Megan; Durand, Christine; Cranmer, Lisa; Reid, Alison; Martinson, Neil; Doherty, Meg; Jilek, Benjamin L.; Kagaayi, Joseph; Kizza, Allan; Pillay, Visva; Laeyendecker, Oliver; Reynolds, Steven J.; Eshleman, Susan H.; Lau, Bryan; Ray, Stuart C.; Siliciano, Janet D.; Quinn, Thomas C.; Siliciano, Robert F.

2009-01-01

Background Intrapartum single-dose nevirapine decreases mother-to-child transmission of human immunodeficiency virus type 1 (HIV-1) but promotes nevirapine resistance. Although resistant viruses fade to undetectable levels in plasma, they may persist as stably integrated proviruses within the latent reservoir in resting CD4+ T cells, potentially complicating future treatment. Methods Blood samples were collected from 60 women from South Africa and Uganda >6 months after they had received single-dose nevirapine. To selectively analyze the stable latent form of HIV-1, resting CD4+ T cells were isolated and activated in the presence of reverse-transcriptase inhibitors and integrase inhibitors, which allows for the specific isolation of viruses produced by cells with stably integrated proviral DNA. These viruses were then analyzed for nevirapine resistance. Results Although only a small number of latently infected cells were present in each blood sample (mean, 162 cells), nevirapine resistance mutations (K103N and G190A) were detected in the latent reservoir of 4 (8%) of 50 evaluable women. Conclusions A single dose of nevirapine can establish antiretroviral resistance within the latent reservoir. This results in a potentially lifelong risk of reemergence of nevirapine-resistant virus and highlights the need for strategies to prevent transmission that do not compromise successful future treatment. PMID:19338474

Comparative evaluation of Amplicor HIV-1 DNA test, version 1.5, by ...

African Journals Online (AJOL)

Human immunodeficiency virus (HIV) DNA polymerase chain reaction (PCR) test using venous blood sample has been used for many years in low resource settings for early infant diagnosis of HIV infection in children less than 18 months. The aim of this study was to evaluate and compare the performance characteristics of ...

Chromatin Regulation and the Histone Code in HIV Latency
.

Science.gov (United States)

Turner, Anne-Marie W; Margolis, David M

2017-06-01

The formation of a latent reservoir of Human Immunodeficiency Virus (HIV) infection hidden from immune clearance remains a significant obstacle to approaches to eradicate HIV infection. Towards an understanding of the mechanisms of HIV persistence, there is a growing body of work implicating epigenetic regulation of chromatin in establishment and maintenance of this latent reservoir. Here we discuss recent advances in the field of chromatin regulation, specifically in our understanding of the histone code, and how these discoveries relate to our current knowledge of the chromatin mechanisms linked to HIV transcriptional repression and the reversal of latency. We also examine mechanisms unexplored in the context of HIV latency and briefly discuss current therapies aimed at the induction of proviral expression within latently infected cells. We aim to emphasize that a greater understanding of the epigenetic mechanisms which govern HIV latency could lead to new therapeutic targets for latency reversal and clearance cure strategies.

FISHing Out the Hidden Enemy: Advances in Detecting and Measuring Latent HIV-Infected Cells

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Vinayaka R. Prasad

2017-09-01

Full Text Available The indomitable aspect of HIV-1 infection is not that HIV-1 proviral DNA is integrated into host DNA but that it can also turn itself off, remaining invisible to drug or immune surveillance. Thus, the goals of eradication include ways to precisely excise HIV-1 DNA or wake up the silent HIV-1 provirus and eliminate the infected cells thus identified. Methods to identify and fish out the latently infected cells or to delineate their characteristics are being rapidly developed. In 2016, Baxter et al. (A. E. Baxter, J. Niessl, R. Fromentin, J. Richard, F. Porichis, R. Charlebois, M. Massanella, N. Brassard, N. Alsahafi, G. G. Delgado, J. P. Routy, B. D. Walker, A. Finzi, N. Chomont, and D. E. Kaufmann, Cell Host Microbe 20:368–380, 2016, https://doi.org/10.1016/j.chom.2016.07.015 and Martrus et al. (G. Martrus, A. Niehrs, R. Cornelis, A. Rechtien, W. García-Beltran, M. Lutgehetmann, C. Hoffmann, and M. Altfeld, J Virol 90:9018–9028, 2016, https://doi.org/10.1128/JVI.01448-16 reported using the fluorescence in situ hybridization-flow cytometry technique to identify and quantify cells expressing HIV-1 RNA and Gag protein, as well as bearing unique cell surface markers. In a recent article in mBio, Grau-Expíósito et al. (J. Grau-Expíóósito, C. Serra-Peinado, L. Miguel, J. Navarro, A. Curran, J. Burgos, I. Ocaíóóña, E. Ribera, A. Torrella, B. Planas, R. Badíóóñía, J. Castellvíóóñíí, V. Falcíóóñííó, M. Crespo, and M. J. Buzon, mBio 8:e00876-17, 2017, https://doi.org/10.1128/mBio.00876-17 reported a similar method that they claim to be more sensitive. With these methods, researchers are one step closer to measuring latent reservoirs and eliminating critical barriers to HIV eradication.

Relationship between glutamate, GOT and GPT levels in maternal and fetal blood: a potential mechanism for fetal neuroprotection.

Science.gov (United States)

Zlotnik, Alexander; Tsesis, Svetlana; Gruenbaum, Benjamin Fredrick; Ohayon, Sharon; Gruenbaum, Shaun Evan; Boyko, Matthew; Sheiner, Eyal; Brotfain, Evgeny; Shapira, Yoram; Teichberg, Vivian Itzhak

2012-09-01

Excess glutamate in the brain is thought to be implicated in the pathophysiology of fetal anoxic brain injury, yet little is known about the mechanisms by which glutamate is regulated in the fetal brain. This study examines whether there are differences between maternal and fetal glutamate concentrations, and whether a correlation between them exists. 10 ml of venous blood was extracted from 87 full-term (>37 weeks gestation) pregnant women in active labor. Immediately after delivery of the neonate, 10 ml of blood from the umbilical artery and vein was extracted. Samples were analyzed for levels of glutamate, glutamate-oxaloacetate transaminase (GOT), and glutamate pyruvate transaminase (GPT). Fetal blood glutamate concentrations in both the umbilical artery and vein were found to be significantly higher than maternal blood (pGOT levels in the umbilical artery and vein were found to be significantly higher than maternal GOT levels (pGOT or GPT between the umbilical artery and vein. There was an association observed between glutamate levels in maternal blood and glutamate levels in both venous (R=0.32, pGOT, but not GPT levels. An association was observed between maternal and fetal blood glutamate levels. Copyright © 2012 Elsevier Ltd. All rights reserved.

Latent HIV reservoirs exhibit inherent resistance to elimination by CD8+ T cells.

Science.gov (United States)

Huang, Szu-Han; Ren, Yanqin; Thomas, Allison S; Chan, Dora; Mueller, Stefanie; Ward, Adam R; Patel, Shabnum; Bollard, Catherine M; Cruz, Conrad Russell; Karandish, Sara; Truong, Ronald; Macedo, Amanda B; Bosque, Alberto; Kovacs, Colin; Benko, Erika; Piechocka-Trocha, Alicja; Wong, Hing; Jeng, Emily; Nixon, Douglas F; Ho, Ya-Chi; Siliciano, Robert F; Walker, Bruce D; Jones, R Brad

2018-02-01

The presence of persistent, latent HIV reservoirs in CD4+ T cells obstructs current efforts to cure infection. The so-called kick-and-kill paradigm proposes to purge these reservoirs by combining latency-reversing agents with immune effectors such as cytotoxic T lymphocytes. Support for this approach is largely based on success in latency models, which do not fully reflect the makeup of latent reservoirs in individuals on long-term antiretroviral therapy (ART). Recent studies have shown that CD8+ T cells have the potential to recognize defective proviruses, which comprise the vast majority of all infected cells, and that the proviral landscape can be shaped over time due to in vivo clonal expansion of infected CD4+ T cells. Here, we have shown that treating CD4+ T cells from ART-treated individuals with combinations of potent latency-reversing agents and autologous CD8+ T cells consistently reduced cell-associated HIV DNA, but failed to deplete replication-competent virus. These CD8+ T cells recognized and potently eliminated CD4+ T cells that were newly infected with autologous reservoir virus, ruling out a role for both immune escape and CD8+ T cell dysfunction. Thus, our results suggest that cells harboring replication-competent HIV possess an inherent resistance to CD8+ T cells that may need to be addressed to cure infection.

HIV-DNA in the genital tract of women on long-term effective therapy is associated to residual viremia and previous AIDS-defining illnesses.

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Thierry Prazuck

Full Text Available OBJECTIVES: To assess the impact of long-term combined antiretroviral therapy (cART on HIV-RNA and HIV-DNA levels in cervicovaginal secretions of HIV-1-infected women with sustained undetectable plasma RNA viral load (PVL; to explore factors predictive of residual viral shedding; and to evaluate the risk of heterosexual transmission. METHODS: Women with undetectable PVL (6 months were included in this cross-sectional study. HIV-RNA and HIV-DNA were measured in blood and cervicovaginal lavage fluid (CVL. Women were systematically tested for genital infections. The risk of transmission to male partners during unprotected intercourse was estimated. RESULTS: Eighty-one women composed the study population: all had HIV-RNA <40 copies/mL in CVL. HIV-DNA was detectable in CVL of 29/78 patients (37%. There was a weak positive correlation between HIV-DNA levels in PBMCs and CVL (r = 0.20; p = 0.08. In multivariate analysis, two factors were associated with HIV-DNA detection in CVL: previous AIDS-defining illnesses (OR = 11; 95%CI = 2-61 and current residual viremia (20HIV-DNA detection in CVL. Twenty-eight percent of the women had unprotected intercourse with their regular HIV-seronegative male partner, for between 8 and 158 months. None of their male partners became infected, after a total of 14 000 exposures. CONCLUSION: In our experience, HIV-RNA was undetectable in the genital tract of women with sustained control of PVL on cART. HIV-DNA shedding persisted in about one third of cases, with no substantial evidence of residual infectiousness.

A new activity of anti-HIV and anti-tumor protein GAP31: DNA adenosine glycosidase - Structural and modeling insight into its functions

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Li, Hui-Guang [Department of Biochemistry, New York University School of Medicine, New York, NY 10016 (United States); Huang, Philip L. [American Biosciences, Boston, MA 02114 (United States); Zhang, Dawei; Sun, Yongtao [Department of Biochemistry, New York University School of Medicine, New York, NY 10016 (United States); Chen, Hao-Chia [Endocrinology and Reproduction Research Branch, National Institute of Child Health and Human Development, NIH, Bethesda, MD 20892 (United States); Zhang, John [Department of Chemistry, New York University, New York, NY 10003 (United States); Huang, Paul L. [Department of Medicine, Harvard Medical School and Massachusetts General Hospital, Boston, MA 02114 (United States); Kong, Xiang-Peng, E-mail: xiangpeng.kong@med.nyu.edu [Department of Biochemistry, New York University School of Medicine, New York, NY 10016 (United States); Lee-Huang, Sylvia, E-mail: sylvia.lee-huang@med.nyu.edu [Department of Biochemistry, New York University School of Medicine, New York, NY 10016 (United States)

2010-01-01

We report here the high-resolution atomic structures of GAP31 crystallized in the presence of HIV-LTR DNA oligonucleotides systematically designed to examine the adenosine glycosidase activity of this anti-HIV and anti-tumor plant protein. Structural analysis and molecular modeling lead to several novel findings. First, adenine is bound at the active site in the crystal structures of GAP31 to HIV-LTR duplex DNA with 5' overhanging adenosine ends, such as the 3'-processed HIV-LTR DNA but not to DNA duplex with blunt ends. Second, the active site pocket of GAP31 is ideally suited to accommodate the 5' overhanging adenosine of the 3'-processed HIV-LTR DNA and the active site residues are positioned to perform the adenosine glycosidase activity. Third, GAP31 also removes the 5'-end adenine from single-stranded HIV-LTR DNA oligonucleotide as well as any exposed adenosine, including that of single nucleotide dAMP but not from AMP. Fourth, GAP31 does not de-purinate guanosine from di-nucleotide GT. These results suggest that GAP31 has DNA adenosine glycosidase activity against accessible adenosine. This activity is distinct from the generally known RNA N-glycosidase activity toward the 28S rRNA. It may be an alternative function that contributes to the antiviral and anti-tumor activities of GAP31. These results provide molecular insights consistent with the anti-HIV mechanisms of GAP31 in its inhibition on the integration of viral DNA into the host genome by HIV-integrase as well as irreversible topological relaxation of the supercoiled viral DNA.

Creation of chimeric human/rabbit APOBEC1 with HIV-1 restriction and DNA mutation activities

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Ikeda, Terumasa; Ong, Eugene Boon Beng; Watanabe, Nobumoto; Sakaguchi, Nobuo; Maeda, Kazuhiko; Koito, Atsushi

2016-01-01

APOBEC1 (A1) proteins from lagomorphs and rodents have deaminase-dependent restriction activity against HIV-1, whereas human A1 exerts a negligible effect. To investigate these differences in the restriction of HIV-1 by A1 proteins, a series of chimeric proteins combining rabbit and human A1s was constructed. Homology models of the A1s indicated that their activities derive from functional domains that likely act in tandem through a dimeric interface. The C-terminal region containing the leucine-rich motif and the dimerization domains of rabbit A1 is important for its anti-HIV-1 activity. The A1 chimeras with strong anti-HIV-1 activity were incorporated into virions more efficiently than those without anti-HIV-1 activity, and exhibited potent DNA-mutator activity. Therefore, the C-terminal region of rabbit A1 is involved in both its packaging into the HIV-1 virion and its deamination activity against both viral cDNA and genomic RNA. This study identifies the novel molecular mechanism underlying the target specificity of A1.

Effect of ionizing radiation on DNA-mediated gene transfer efficiency

International Nuclear Information System (INIS)

Rubin, J.S.; Hall, E.J.; Hei, T.K.

1986-01-01

Ionizing radiation causes a number of molecular changes in cells including DNA damage and gene amplification. In this study the authors examined whether radiation can effect the efficiency of integration and expression of exogenous DNA sequences. They examined both 137 Cs γ rays and various monoenergetic neutron beams. This enabled them to test whether the LET or RBE of the radiation had any effect. Rat2 cells were transfected with various amounts of the bacterial plasmid pSV2-GPT along with carrier DNA for 24 hours

High discordance in blood and genital tract HIV-1 drug resistance in Indian women failing first-line therapy.

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Saravanan, Shanmugam; Gomathi, Selvamurthi; Delong, Allison; Kausalya, Bagavathi; Sivamalar, Sathasivam; Poongulali, Selvamuthu; Brooks, Katherine; Kumarasamy, Nagalingeswaran; Balakrishnan, Pachamuthu; Solomon, Sunil S; Cu-Uvin, Susan; Kantor, Rami

2018-05-24

Examine HIV-1 plasma viral load (PVL) and genital tract (GT) viral load (GVL) and drug resistance in India. At the YRG Centre for AIDS Research and Education, Chennai, we tested: PVL in women on first-line ART for ≥6 months; GVL when PVL >2000 copies/mL; and plasma, genital and proviral reverse transcriptase drug resistance when GVL >2000 copies/mL. Wilcoxon rank-sum and Fisher's exact tests were used to identify failure and resistance associations. Pearson correlations were calculated to evaluate PVL-GVL associations. Inter-compartmental resistance discordance was evaluated using generalized estimating equations. Of 200 women, 37% had detectable (>400 copies/mL) PVL and 31% had PVL >1000 copies/mL. Of women with detectable PVL, 74% had PVL >2000 copies/mL, of which 74% had detectable GVL. Higher PVL was associated with higher GVL. Paired plasma and genital sequences were available for 21 women; mean age of 34 years, median ART duration of 33 months, median CD4 count of 217 cells/mm3, median PVL of 5.4 log10 copies/mL and median GVL of 4.6 log10 copies/mL. Drug resistance was detected in 81%-91% of samples and 67%-76% of samples had dual-class resistance. Complete three-compartment concordance was seen in only 10% of women. GT-proviral discordance was significantly larger than plasma-proviral discordance. GT or proviral mutations discordant from plasma led to clinically relevant resistance in 24% and 30%, respectively. We identified high resistance and high inter-compartmental resistance discordance in Indian women, which might lead to unrecognized resistance transmission and re-emergence compromising treatment outcomes, particularly relevant to countries like India, where sexual HIV transmission is predominant.

The Latent Reservoir for HIV-1: How Immunologic Memory and Clonal Expansion Contribute to HIV-1 Persistence

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Murray, Alexandra J.; Kwon, Kyungyoon J.; Farber, Donna L.; Siliciano, Robert F.

2016-01-01

Combination antiretroviral therapy (ART) for HIV-1 infection reduces plasma virus levels to below the limit of detection of clinical assays. However, even with prolonged suppression of viral replication with ART, viremia rebounds rapidly after treatment interruption. Thus ART is not curative. The principal barrier to cure is a remarkably stable reservoir of latent HIV-1 in resting memory CD4+ T cells. Here we consider explanations for the remarkable stability of the latent reservoir. Stability does not appear to reflect replenishment from new infection events but rather normal physiologic processes that provide for immunologic memory. Of particular importance are proliferative processes that drive clonal expansion of infected cells. Recent evidence suggests that in some infected cells, proliferation is a consequence of proviral integration into host genes associated with cell growth. Efforts to cure HIV-1 infection by targeting the latent reservoir may need to consider the potential of latently infected cells to proliferate. PMID:27382129

A recoding method to improve the humoral immune response to an HIV DNA vaccine.

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Yaoxing Huang

Full Text Available This manuscript describes a novel strategy to improve HIV DNA vaccine design. Employing a new information theory based bioinformatic algorithm, we identify a set of nucleotide motifs which are common in the coding region of HIV, but are under-represented in genes that are highly expressed in the human genome. We hypothesize that these motifs contribute to the poor protein expression of gag, pol, and env genes from the c-DNAs of HIV clinical isolates. Using this approach and beginning with a codon optimized consensus gag gene, we recode the nucleotide sequence so as to remove these motifs without modifying the amino acid sequence. Transfecting the recoded DNA sequence into a human kidney cell line results in doubling the gag protein expression level compared to the codon optimized version. We then turn both sequences into DNA vaccines and compare induced antibody response in a murine model. Our sequence, which has the motifs removed, induces a five-fold increase in gag antibody response compared to the codon optimized vaccine.

Measuring replication competent HIV-1: advances and challenges in defining the latent reservoir.

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Wang, Zheng; Simonetti, Francesco R; Siliciano, Robert F; Laird, Gregory M

2018-02-13

Antiretroviral therapy cannot cure HIV-1 infection due to the persistence of a small number of latently infected cells harboring replication-competent proviruses. Measuring persistent HIV-1 is challenging, as it consists of a mosaic population of defective and intact proviruses that can shift from a state of latency to active HIV-1 transcription. Due to this complexity, most of the current assays detect multiple categories of persistent HIV-1, leading to an overestimate of the true size of the latent reservoir. Here, we review the development of the viral outgrowth assay, the gold-standard quantification of replication-competent proviruses, and discuss the insights provided by full-length HIV-1 genome sequencing methods, which allowed us to unravel the composition of the proviral landscape. In this review, we provide a dissection of what defines HIV-1 persistence and we examine the unmet needs to measure the efficacy of interventions aimed at eliminating the HIV-1 reservoir.

Randomized phase I trial HIV-CORE 003: Depletion of serum amyloid P component and immunogenicity of DNA vaccination against HIV-1.

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Borthwick, Nicola J; Lane, Thirusha; Moyo, Nathifa; Crook, Alison; Shim, Jung Min; Baines, Ian; Wee, Edmund G; Hawkins, Philip N; Gillmore, Julian D; Hanke, Tomáš; Pepys, Mark B

2018-01-01

The failure of DNA vaccination in humans, in contrast to its efficacy in some species, is unexplained. Observational and interventional experimental evidence suggests that DNA immunogenicity may be prevented by binding of human serum amyloid P component (SAP). SAP is the single normal DNA binding protein in human plasma. The drug (R)-1-[6-[(R)-2-carboxypyrrolidin-1-yl]-6-oxo-hexanoyl]pyrrolidine-2-carboxylic acid (CPHPC, miridesap), developed for treatment of systemic amyloidosis and Alzheimer's disease, depletes circulating SAP by 95-99%. The proof-of-concept HIV-CORE 003 clinical trial tested whether SAP depletion by CPHPC would enhance the immune response in human volunteers to DNA vaccination delivering the HIVconsv immunogen derived from conserved sub-protein regions of HIV-1. Human volunteers received 3 intramuscular immunizations with an experimental DNA vaccine (DDD) expressing HIV-1-derived immunogen HIVconsv, with or without prior depletion of SAP by CPHPC. All subjects were subsequently boosted by simian (chimpanzee) adenovirus (C)- and poxvirus MVA (M)-vectored vaccines delivering the same immunogen. After administration of each vaccine modality, the peak total magnitudes, kinetics, functionality and memory subsets of the T-cell responses to HIVconsv were thoroughly characterized. No differences were observed between the CPHPC treated and control groups in any of the multiple quantitative and qualitative parameters of the T-cell responses to HIVconsv, except that after SAP depletion, there was a statistically significantly greater breadth of T-cell specificities, that is the number of recognized epitopes, following the DDDC vaccination. The protocol used here for SAP depletion by CPHPC prior to DNA vaccination produced only a very modest suggestion of enhanced immunogenicity. Further studies will be required to determine whether SAP depletion might have a practical value in DNA vaccination for other plasmid backbones and/or immunogens. Clinicaltrials

Ex vivo activation of CD4+ T-cells from donors on suppressive ART can lead to sustained production of infectious HIV-1 from a subset of infected cells.

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John K Bui

2017-02-01

Full Text Available The fate of HIV-infected cells after reversal of proviral latency is not well characterized. Simonetti, et al. recently showed that CD4+ T-cells containing intact proviruses can clonally expand in vivo and produce low-level infectious viremia. We hypothesized that reversal of HIV latency by activation of CD4+ T-cells can lead to the expansion of a subset of virus-producing cells rather than their elimination. We established an ex vivo cell culture system involving stimulation of CD4+ T-cells from donors on suppressive antiretroviral therapy (ART with PMA/ionomycin (day 1-7, followed by rest (day 7-21, and then repeat stimulation (day 21-28, always in the presence of high concentrations of raltegravir and efavirenz to effectively block new cycles of viral replication. HIV DNA and virion RNA in the supernatant were quantified by qPCR. Single genome sequencing (SGS of p6-PR-RT was performed to genetically characterize proviruses and virion-associated genomic RNA. The replication-competence of the virions produced was determined by the viral outgrowth assay (VOA and SGS of co-culture supernatants from multiple time points. Experiments were performed with purified CD4+ T-cells from five consecutively recruited donors who had been on suppressive ART for > 2 years. In all experiments, HIV RNA levels in supernatant increased following initial stimulation, decreased or remained stable during the rest period, and increased again with repeat stimulation. HIV DNA levels did not show a consistent pattern of change. SGS of proviruses revealed diverse outcomes of infected cell populations, ranging from their apparent elimination to persistence and expansion. Importantly, a subset of infected cells expanded and produced infectious virus continuously after stimulation. These findings underscore the complexity of eliminating reservoirs of HIV-infected cells and highlight the need for new strategies to kill HIV-infected cells before they can proliferate.

Intradermal HIV-1 DNA Immunization Using Needle-Free Zetajet Injection Followed by HIV-Modified Vaccinia Virus Ankara Vaccination Is Safe and Immunogenic in Mozambican Young Adults: A Phase I Randomized Controlled Trial.

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Viegas, Edna Omar; Tembe, Nelson; Nilsson, Charlotta; Meggi, Bindiya; Maueia, Cremildo; Augusto, Orvalho; Stout, Richard; Scarlatti, Gabriella; Ferrari, Guido; Earl, Patricia L; Wahren, Britta; Andersson, Sören; Robb, Merlin L; Osman, Nafissa; Biberfeld, Gunnel; Jani, Ilesh; Sandström, Eric

2017-11-27

We assessed the safety and immunogenicity of HIV-DNA priming using Zetajet™, a needle-free device intradermally followed by intramuscular HIV-MVA boosts, in 24 healthy Mozambicans. Volunteers were randomized to receive three immunizations of 600 μg (n = 10; 2 × 0.1 ml) or 1,200 μg (n = 10; 2 × 0.2 ml) of HIV-DNA (3 mg/ml), followed by two boosts of 10 8 pfu HIV-MVA. Four subjects received placebo saline injections. Vaccines and injections were safe and well tolerated with no difference between the two priming groups. After three HIV-DNA immunizations, IFN-γ ELISpot responses to Gag were detected in 9/17 (53%) vaccinees, while none responded to Envelope (Env). After the first HIV-MVA, the overall response rate to Gag and/or Env increased to 14/15 (93%); 14/15 (93%) to Gag and 13/15 (87%) to Env. There were no significant differences between the immunization groups in frequency of response to Gag and Env or magnitude of Gag responses. Env responses were significantly higher in the higher dose group (median 420 vs. 157.5 SFC/million peripheral blood mononuclear cell, p = .014). HIV-specific antibodies to subtype C gp140 and subtype B gp160 were elicited in all vaccinees after the second HIV-MVA, without differences in titers between the groups. Neutralizing antibody responses were not detected. Two (13%) of 16 vaccinees, one in each of the priming groups, exhibited antibodies mediating antibody-dependent cellular cytotoxicity to CRF01_AE. In conclusion, HIV-DNA vaccine delivered intradermally in volumes of 0.1-0.2 ml using Zetajet was safe and well tolerated. Priming with the 1,200 μg dose of HIV-DNA generated higher magnitudes of ELISpot responses to Env.

Methamphetamine and HIV-Tat alter murine cardiac DNA methylation and gene expression

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Koczor, Christopher A., E-mail: ckoczor@emory.edu; Fields, Earl; Jedrzejczak, Mark J.; Jiao, Zhe; Ludaway, Tomika; Russ, Rodney; Shang, Joan; Torres, Rebecca A.; Lewis, William

2015-11-01

This study addresses the individual and combined effects of HIV-1 and methamphetamine (N-methyl-1-phenylpropan-2-amine, METH) on cardiac dysfunction in a transgenic mouse model of HIV/AIDS. METH is abused epidemically and is frequently associated with acquisition of HIV-1 infection or AIDS. We employed microarrays to identify mRNA differences in cardiac left ventricle (LV) gene expression following METH administration (10 d, 3 mg/kg/d, subcutaneously) in C57Bl/6 wild-type littermates (WT) and Tat-expressing transgenic (TG) mice. Arrays identified 880 differentially expressed genes (expression fold change > 1.5, p < 0.05) following METH exposure, Tat expression, or both. Using pathway enrichment analysis, mRNAs encoding polypeptides for calcium signaling and contractility were altered in the LV samples. Correlative DNA methylation analysis revealed significant LV DNA methylation changes following METH exposure and Tat expression. By combining these data sets, 38 gene promoters (27 related to METH, 11 related to Tat) exhibited differences by both methods of analysis. Among those, only the promoter for CACNA1C that encodes L-type calcium channel Cav1.2 displayed DNA methylation changes concordant with its gene expression change. Quantitative PCR verified that Cav1.2 LV mRNA abundance doubled following METH. Correlative immunoblots specific for Cav1.2 revealed a 3.5-fold increase in protein abundance in METH LVs. Data implicate Cav1.2 in calcium dysregulation and hypercontractility in the murine LV exposed to METH. They suggest a pathogenetic role for METH exposure to promote LV dysfunction that outweighs Tat-induced effects. - Highlights: • HIV-1 Tat and methamphetamine (METH) alter cardiac gene expression and epigenetics. • METH impacts gene expression or epigenetics more significantly than Tat expression. • METH alters cardiac mitochondrial function and calcium signaling independent of Tat. • METH alters DNA methylation, expression, and protein abundance of

Methamphetamine and HIV-Tat alter murine cardiac DNA methylation and gene expression

International Nuclear Information System (INIS)

Koczor, Christopher A.; Fields, Earl; Jedrzejczak, Mark J.; Jiao, Zhe; Ludaway, Tomika; Russ, Rodney; Shang, Joan; Torres, Rebecca A.; Lewis, William

2015-01-01

This study addresses the individual and combined effects of HIV-1 and methamphetamine (N-methyl-1-phenylpropan-2-amine, METH) on cardiac dysfunction in a transgenic mouse model of HIV/AIDS. METH is abused epidemically and is frequently associated with acquisition of HIV-1 infection or AIDS. We employed microarrays to identify mRNA differences in cardiac left ventricle (LV) gene expression following METH administration (10 d, 3 mg/kg/d, subcutaneously) in C57Bl/6 wild-type littermates (WT) and Tat-expressing transgenic (TG) mice. Arrays identified 880 differentially expressed genes (expression fold change > 1.5, p < 0.05) following METH exposure, Tat expression, or both. Using pathway enrichment analysis, mRNAs encoding polypeptides for calcium signaling and contractility were altered in the LV samples. Correlative DNA methylation analysis revealed significant LV DNA methylation changes following METH exposure and Tat expression. By combining these data sets, 38 gene promoters (27 related to METH, 11 related to Tat) exhibited differences by both methods of analysis. Among those, only the promoter for CACNA1C that encodes L-type calcium channel Cav1.2 displayed DNA methylation changes concordant with its gene expression change. Quantitative PCR verified that Cav1.2 LV mRNA abundance doubled following METH. Correlative immunoblots specific for Cav1.2 revealed a 3.5-fold increase in protein abundance in METH LVs. Data implicate Cav1.2 in calcium dysregulation and hypercontractility in the murine LV exposed to METH. They suggest a pathogenetic role for METH exposure to promote LV dysfunction that outweighs Tat-induced effects. - Highlights: • HIV-1 Tat and methamphetamine (METH) alter cardiac gene expression and epigenetics. • METH impacts gene expression or epigenetics more significantly than Tat expression. • METH alters cardiac mitochondrial function and calcium signaling independent of Tat. • METH alters DNA methylation, expression, and protein abundance of

HIV-1 and recombinant gp120 affect the survival and differentiation of human vessel wall-derived mesenchymal stem cells

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Pasquinelli Gianandrea

2011-05-01

Full Text Available Abstract Background HIV infection elicits the onset of a progressive immunodeficiency and also damages several other organs and tissues such as the CNS, kidney, heart, blood vessels, adipose tissue and bone. In particular, HIV infection has been related to an increased incidence of cardiovascular diseases and derangement in the structure of blood vessels in the absence of classical risk factors. The recent characterization of multipotent mesenchymal cells in the vascular wall, involved in regulating cellular homeostasis, suggests that these cells may be considered a target of HIV pathogenesis. This paper investigated the interaction between HIV-1 and vascular wall resident human mesenchymal stem cells (MSCs. Results MSCs were challenged with classical R5 and X4 HIV-1 laboratory strains demonstrating that these strains are able to enter and integrate their retro-transcribed proviral DNA in the host cell genome. Subsequent experiments indicated that HIV-1 strains and recombinant gp120 elicited a reliable increase in apoptosis in sub-confluent MSCs. Since vascular wall MSCs are multipotent cells that may be differentiated towards several cell lineages, we challenged HIV-1 strains and gp120 on MSCs differentiated to adipogenesis and endotheliogenesis. Our experiments showed that the adipogenesis is increased especially by upregulated PPARγ activity whereas the endothelial differentiation induced by VEGF treatment was impaired with a downregulation of endothelial markers such as vWF, Flt-1 and KDR expression. These viral effects in MSC survival and adipogenic or endothelial differentiation were tackled by CD4 blockade suggesting an important role of CD4/gp120 interaction in this context. Conclusions The HIV-related derangement of MSC survival and differentiation may suggest a direct role of HIV infection and gp120 in impaired vessel homeostasis and in genesis of vessel damage observed in HIV-infected patients.

Use of damaged plasmid to study DNA repair in X-ray sensitive (xrs) strains of Chinese hamster ovary (CHO) cells

International Nuclear Information System (INIS)

Smith-Ravin, J.; Jeggo, P.A.

1989-01-01

The effect of γ-irradiation of pSV2gpt DNA on its transfection frequency has been analysed using radiosensitive CHO xrs mutants showing a defect in double-strand break (dsb) rejoining. At low doses a sharp decrease in relative transfection frequency, i.e. transfection frequency of irradiated plasmid relative to untreated plasmid, as observed in xrs mutants compared with the parent line K1. Electrophoresis of irradiated plasmid DNA showed the decrease in transfection frequency in the xrs mutants correlated with the change of supercoiled molecules into open-circular forms. In the parent line CHO-K1, open-circular and supercoiled molecules have the same transfection frequency. The effect of linearization of pSV2gpt DNA by restriction enzymes on transfection frequency in xrs and wild-type strains was also examined. No difference in the relative transfection frequency between xrs and wild-type strains was detected. (author)

The Spleen Is an HIV-1 Sanctuary During Combined Antiretroviral Therapy.

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Nolan, David J; Rose, Rebecca; Rodriguez, Patricia H; Salemi, Marco; Singer, Elyse J; Lamers, Susanna L; McGrath, Michael S

2018-01-01

Combined antiretroviral therapy (cART) does not eradicate HIV, which persists for years and can re-establish replication if treatment is stopped. The current challenge is identifying those tissues harboring virus through cART. Here, we used HIV env-nef single genome sequencing and HIV gag droplet digital PCR (ddPCR) to survey 50 tissues from five subjects on cART with no detectable plasma viral load at death. The spleen most consistently contained multiple proviral and expressed sequences (4/5 participants). Spleen-derived HIV demonstrated two distinct phylogenetic patterns: multiple identical sequences, often from different tissues, as well as diverse viral sequences on long terminal branches. Our results suggested that ddPCR may overestimate the size of the tissue-based viral reservoir. The spleen, a lymphatic organ at the intersection of the immune and circulatory systems, may play a key role in viral persistence.

Class 1-Selective Histone Deacetylase (HDAC) Inhibitors Enhance HIV Latency Reversal while Preserving the Activity of HDAC Isoforms Necessary for Maximal HIV Gene Expression.

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Zaikos, Thomas D; Painter, Mark M; Sebastian Kettinger, Nadia T; Terry, Valeri H; Collins, Kathleen L

2018-03-15

Combinations of drugs that affect distinct mechanisms of HIV latency aim to induce robust latency reversal leading to cytopathicity and elimination of the persistent HIV reservoir. Thus far, attempts have focused on combinations of protein kinase C (PKC) agonists and pan-histone deacetylase inhibitors (HDIs) despite the knowledge that HIV gene expression is regulated by class 1 histone deacetylases. We hypothesized that class 1-selective HDIs would promote more robust HIV latency reversal in combination with a PKC agonist than pan-HDIs because they preserve the activity of proviral factors regulated by non-class 1 histone deacetylases. Here, we show that class 1-selective agents used alone or with the PKC agonist bryostatin-1 induced more HIV protein expression per infected cell. In addition, the combination of entinostat and bryostatin-1 induced viral outgrowth, whereas bryostatin-1 combinations with pan-HDIs did not. When class 1-selective HDIs were used in combination with pan-HDIs, the amount of viral protein expression and virus outgrowth resembled that of pan-HDIs alone, suggesting that pan-HDIs inhibit robust gene expression induced by class 1-selective HDIs. Consistent with this, pan-HDI-containing combinations reduced the activity of NF-κB and Hsp90, two cellular factors necessary for potent HIV protein expression, but did not significantly reduce overall cell viability. An assessment of viral clearance from in vitro cultures indicated that maximal protein expression induced by class 1-selective HDI treatment was crucial for reservoir clearance. These findings elucidate the limitations of current approaches and provide a path toward more effective strategies to eliminate the HIV reservoir. IMPORTANCE Despite effective antiretroviral therapy, HIV evades eradication in a latent form that is not affected by currently available drug regimens. Pharmacologic latency reversal that leads to death of cellular reservoirs has been proposed as a strategy for

The prion protein has DNA strand transfer properties similar to retroviral nucleocapsid protein.

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Gabus, C; Auxilien, S; Péchoux, C; Dormont, D; Swietnicki, W; Morillas, M; Surewicz, W; Nandi, P; Darlix, J L

2001-04-06

The transmissible spongiform encephalopathies are fatal neurodegenerative diseases that are associated with the accumulation of a protease-resistant form of the cellular prion protein (PrP). Although PrP is highly conserved and widely expressed in vertebrates, its function remains a matter of speculation. Indeed PrP null mice develop normally and are healthy. Recent results show that PrP binds to nucleic acids in vitro and is found associated with retroviral particles. Furthermore, in mice the scrapie infectious process appears to be accelerated by MuLV replication. These observations prompted us to further investigate the interaction between PrP and nucleic acids, and compare it with that of the retroviral nucleocapsid protein (NC). As the major nucleic acid-binding protein of the retroviral particle, NC protein is tightly associated with the genomic RNA in the virion nucleocapsid, where it chaperones proviral DNA synthesis by reverse transcriptase. Our results show that the human prion protein (huPrP) functionally resembles NCp7 of HIV-1. Both proteins form large nucleoprotein complexes upon binding to DNA. They accelerate the hybridization of complementary DNA strands and chaperone viral DNA synthesis during the minus and plus DNA strand transfers necessary to generate the long terminal repeats. The DNA-binding and strand transfer properties of huPrP appear to map to the N-terminal fragment comprising residues 23 to 144, whereas the C-terminal domain is inactive. These findings suggest that PrP could be involved in nucleic acid metabolism in vivo. Copyright 2001 Academic Press.

Mitochondrial DNA Haplogroup A Decreases the Risk of Drug Addiction but Conversely Increases the Risk of HIV-1 Infection in Chinese Addicts.

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Zhang, A-Mei; Hu, Qiu-Xiang; Liu, Feng-Liang; Bi, Rui; Yang, Bi-Qing; Zhang, Wen; Guo, Hao; Logan, Ian; Zheng, Yong-Tang; Yao, Yong-Gang

2016-08-01

Drug addiction is one of the most serious social problems in the world today and addicts are always at a high risk of acquiring HIV infection. Mitochondrial impairment has been reported in both drug addicts and in HIV patients undergoing treatment. In this study, we aimed to investigate whether mitochondrial DNA (mtDNA) haplogroup could affect the risk of drug addiction and HIV-1 infection in Chinese. We analyzed mtDNA sequence variations of 577 Chinese intravenous drug addicts (289 with HIV-1 infection and 288 without) and compared with 2 control populations (n = 362 and n = 850). We quantified the viral load in HIV-1-infected patients with and without haplogroup A status and investigated the potential effect of haplogroup A defining variants m.4824A > G and m.8794C > T on the cellular reactive oxygen species (ROS) levels by using an allotopic expression assay. mtDNA haplogroup A had a protective effect against drug addiction but appeared to confer an increased risk of HIV infection in addicts. HIV-1-infected addicts with haplogroup A had a trend for a higher viral load, although the mean viral load was similar between carriers of haplogroup A and those with other haplogroup. Hela cells overexpressing allele m.8794 T showed significantly decreased ROS levels as compared to cells with the allele m.8794C (P = 0.03). Our results suggested that mtDNA haplogroup A might protect against drug addiction but increase the risk of HIV-1 infection. The contradictory role of haplogroup A might be caused by an alteration in mitochondrial function due to a particular mtDNA ancestral variant.

NÍVEIS SÉRICOS DE TRANSAMINASE GLUTÂMICA-OXALACÉTICA (GOT E TRANSAMINASE GLUTÂMICA-PIRÚVICA (GPT EM BOVINOS DO ESTADO DE GOIÁS SERUM LEVELS OF GOT AND GPT IN BOVINES IN THE GOIÁS STATE

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Eduardo Cavalheiro Jardim

2007-09-01

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Foram analisados 162 hemossoros de bovinos mestiços zebu com a finalidade de obtenção dos níveis séricos da transaminase glutâmica-oxalacética e da transaminase glutâmica-pirúvica, de 18 bezerros(as, 31 novilhos(as e 83 vacas, procedentes de municípios do Estado de Goiás. Os valores encontrados foram os seguintes (URF: TGO, 77,5 +/- 27,3 (bezerros(as, 72,9 +/- 19,6 (novilhos(as e 81,8 +/- 28,4 (vacas; TGP, 29,5 +/- 8,1 (novilhos(as, 31,1 +/- 8.3 (novilhos(as e 34,1 +/- 10,6 (vacas. É muito difícil estandadizar condições de manejo, e os dados encontrados no presente trabalho confirmam que as transaminases são enzimas, dinâmicas, capazes de responderem rapidamente a um grande número de trocas fisiológicas. Posteriores trabalhos devem ser desenvolvidos buscando uniformizar os grupos de trabalho sob os diferentes pontos considerados no presente estudo.

With the objective of studying the serum levels of GPT and GOT, 162 blood serum from crossbred zebu were analyzed. The blood was taken from 48 calves, 31 steers and 83 cows from farms of counties in the State of Goiás. GOT serum levels (URF were as fo1lows: calves, 72.5 +/- 27.3, steers, 72.9 +/- 19.6 and cows 81.8 +/-28.4. For GPT serum levels (URF was found: calves, 29.5 +/- 8.1, steers, 31.1 +/- 8.3 and cows 34.1 +/- 10.6. Some differences were found in GOT and GPT, in comparison with other foreign reports in mature cows.

Prime-boost vaccination using DNA and whole inactivated virus vaccines provides limited protection against virulent feline immunodeficiency virus.

Science.gov (United States)

Dunham, Stephen P; Bruce, Jennifer; Klein, Dieter; Flynn, J Norman; Golder, Matthew C; MacDonald, Susan; Jarrett, Oswald; Neil, James C

2006-11-30

Protection against feline immunodeficiency virus (FIV) has been achieved using a variety of vaccines notably whole inactivated virus (WIV) and DNA. However protection against more virulent isolates, typical of those encountered in natural infections, has been difficult to achieve. In an attempt to improve protection against virulent FIV(GL8), we combined both DNA and WIV vaccines in a "prime-boost" approach. Thirty cats were divided into four groups receiving vaccinations and one unvaccinated control group. Following viral challenge, two vaccinated animals, one receiving DNA alone and one the prime-boost vaccine remained free of viraemia, whilst all controls became viraemic. Animals vaccinated with WIV showed apparent early enhancement of infection at 2 weeks post challenge (pc) with higher plasma viral RNA loads than control animals or cats immunised with DNA alone. Despite this, animals vaccinated with WIV or DNA alone showed significantly lower proviral loads in peripheral blood mononuclear cells and mesenteric lymph node cells, whilst those receiving the DNA-WIV prime-boost vaccine showed significantly lower proviral loads in PBMC, than control animals, at 35 weeks pc. Therefore both DNA and WIV vaccines conferred limited protection against viral challenge but the combination of WIV and DNA in a prime-boost approach appeared to offer no significant advantage over either vaccine alone.

Analysis of the influence of the demand rate on the accident rate of a plant equipped with a single protective channel by Generalized Perturbation Theory (GPT) methods

International Nuclear Information System (INIS)

Franca Walter, F.L.; Alvim, A.C.M.; Silva, F.C. da; Melo e Frutuoso, P.F.

1995-01-01

The application of the GPT methodology to a reliability engineering problem of great practical interest is discussed: that of the analysis of the influence of the demand rate on the accident rate of a process plant equipped with a single protective channel. This problem has been solved in the literature by traditional methods, that is, for each demand rate value the system of differential equations that governs the system behavior (derived from a Markovian reliability model) is solved and the resulting points are employed to generate the desired curve. This sensitivity analysis has been performed by means of a GPT approach in order to show how it could simplify the calculations. Although an analytical solution is available for the above equations, the application of the GPT approach needed the solution of the system for a few points (reference solutions) and the results agree very well with those published. (author). 9 refs, 4 figs

HIV-1 p24(gag derived conserved element DNA vaccine increases the breadth of immune response in mice.

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Viraj Kulkarni

Full Text Available Viral diversity is considered a major impediment to the development of an effective HIV-1 vaccine. Despite this diversity, certain protein segments are nearly invariant across the known HIV-1 Group M sequences. We developed immunogens based on the highly conserved elements from the p24(gag region according to two principles: the immunogen must (i include strictly conserved elements of the virus that cannot mutate readily, and (ii exclude both HIV regions capable of mutating without limiting virus viability, and also immunodominant epitopes located in variable regions. We engineered two HIV-1 p24(gag DNA immunogens that express 7 highly Conserved Elements (CE of 12-24 amino acids in length and differ by only 1 amino acid in each CE ('toggle site', together covering >99% of the HIV-1 Group M sequences. Altering intracellular trafficking of the immunogens changed protein localization, stability, and also the nature of elicited immune responses. Immunization of C57BL/6 mice with p55(gag DNA induced poor, CD4(+ mediated cellular responses, to only 2 of the 7 CE; in contrast, vaccination with p24CE DNA induced cross-clade reactive, robust T cell responses to 4 of the 7 CE. The responses were multifunctional and composed of both CD4(+ and CD8(+ T cells with mature cytotoxic phenotype. These findings provide a method to increase immune response to universally conserved Gag epitopes, using the p24CE immunogen. p24CE DNA vaccination induced humoral immune responses similar in magnitude to those induced by p55(gag, which recognize the virus encoded p24(gag protein. The inclusion of DNA immunogens composed of conserved elements is a promising vaccine strategy to induce broader immunity by CD4(+ and CD8(+ T cells to additional regions of Gag compared to vaccination with p55(gag DNA, achieving maximal cross-clade reactive cellular and humoral responses.

Thalassiolins A-C: new marine-derived inhibitors of HIV cDNA integrase.

Science.gov (United States)

Rowley, David C; Hansen, Mark S T; Rhodes, Denise; Sotriffer, Christoph A; Ni, Haihong; McCammon, J Andrew; Bushman, Frederic D; Fenical, William

2002-11-01

Human immunodeficiency virus (HIV) replication requires integration of viral cDNA into the host genome, a process mediated by the viral enzyme integrase. We describe a new series of HIV integrase inhibitors, thalassiolins A-C (1-3), isolated from the Caribbean sea grass Thalassia testudinum. The thalassiolins are distinguished from other flavones previously studied by the substitution of a sulfated beta-D-glucose at the 7-position, a substituent that imparts increased potency against integrase in biochemical assays. The most active of these molecules, thalassiolin A (1), displays in vitro inhibition of the integrase catalyzed strand transfer reaction (IC50=0.4 microM) and an antiviral IC50 of 30 microM. Molecular modeling studies indicate a favorable binding mode is probable at the catalytic core domain of HIV-1 integrase.

Critical involvement of the ATM-dependent DNA damage response in the apoptotic demise of HIV-1-elicited syncytia.

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Jean-Luc Perfettini

Full Text Available DNA damage can activate the oncosuppressor protein ataxia telangiectasia mutated (ATM, which phosphorylates the histone H2AX within characteristic DNA damage foci. Here, we show that ATM undergoes an activating phosphorylation in syncytia elicited by the envelope glycoprotein complex (Env of human immunodeficiency virus-1 (HIV-1 in vitro. This was accompanied by aggregation of ATM in discrete nuclear foci that also contained phospho-histone H2AX. DNA damage foci containing phosphorylated ATM and H2AX were detectable in syncytia present in the brain or lymph nodes from patients with HIV-1 infection, as well as in a fraction of blood leukocytes, correlating with viral status. Knockdown of ATM or of its obligate activating factor NBS1 (Nijmegen breakage syndrome 1 protein, as well as pharmacological inhibition of ATM with KU-55933, inhibited H2AX phosphorylation and prevented Env-elicited syncytia from undergoing apoptosis. ATM was found indispensable for the activation of MAP kinase p38, which catalyzes the activating phosphorylation of p53 on serine 46, thereby causing p53 dependent apoptosis. Both wild type HIV-1 and an HIV-1 mutant lacking integrase activity induced syncytial apoptosis, which could be suppressed by inhibiting ATM. HIV-1-infected T lymphoblasts from patients with inactivating ATM or NBS1 mutations also exhibited reduced syncytial apoptosis. Altogether these results indicate that apoptosis induced by a fusogenic HIV-1 Env follows a pro-apoptotic pathway involving the sequential activation of ATM, p38MAPK and p53.

Retroviral DNA--the silent winner: blood transfusion containing latent feline leukemia provirus causes infection and disease in naïve recipient cats.

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Nesina, Stefanie; Katrin Helfer-Hungerbuehler, A; Riond, Barbara; Boretti, Felicitas S; Willi, Barbara; Meli, Marina L; Grest, Paula; Hofmann-Lehmann, Regina

2015-12-21

The feline leukemia virus (FeLV) is a gamma-retrovirus of domestic cats that was discovered half a century ago. Cats that are infected with FeLV may develop a progressive infection resulting in persistent viremia, immunodeficiency, tumors, anemia and death. A significant number of cats mount a protective immune response that suppresses viremia; these cats develop a regressive infection characterized by the absence of viral replication and the presence of low levels of proviral DNA. The biological importance of these latter provirus carriers is largely unknown. Here, we demonstrate that ten cats that received a transfusion of blood from aviremic provirus carriers developed active FeLV infections, some with a progressive outcome and the development of fatal FeLV-associated disease. The infection outcome, disease spectrum and evolution into FeLV-C in one cat mirrored those of natural infection. Two cats developed persistent antigenemia; six cats were transiently antigenemic. Reactivation of infection occurred in some cats. One recipient developed non-regenerative anemia associated with FeLV-C, and four others developed a T-cell lymphoma, one with secondary lymphoblastic leukemia. Five of the ten recipient cats received provirus-positive aviremic blood, whereas the other five received provirus- and viral RNA-positive but aviremic blood. Notably, the cats that received blood containing only proviral DNA exhibited a later onset but graver outcome of FeLV infection than the cats that were transfused with blood containing proviral DNA and viral RNA. Leukocyte counts and cytokine analyses indicated that the immune system of the latter cats reacted quicker and more efficiently. Our results underline the biological and epidemiological relevance of FeLV provirus carriers and the risk of inadvertent FeLV transmission via blood transfusion and demonstrate the replication capacity of proviral DNA if uncontrolled by the immune system. Our results have implications not only for

Induction of HIV neutralizing antibodies against the MPER of the HIV envelope protein by HA/gp41 chimeric protein-based DNA and VLP vaccines.

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Ling Ye

Full Text Available Several conserved neutralizing epitopes have been identified in the HIV Env protein and among these, the MPER of gp41 has received great attention and is widely recognized as a promising target. However, little success has been achieved in eliciting MPER-specific HIV neutralizing antibodies by a number of different vaccine strategies. We investigated the ability of HA/gp41 chimeric protein-based vaccines, which were designed to enhance the exposure of the MPER in its native conformation, to induce MPER-specific HIV neutralizing antibodies. In characterization of the HA/gp41 chimeric protein, we found that by mutating an unpaired Cys residue (Cys-14 in its HA1 subunit to a Ser residue, the modified chimeric protein HA-C14S/gp41 showed increased reactivity to a conformation-sensitive monoclonal antibody against HA and formed more stable trimers in VLPs. On the other hand, HA-C14S/gp41 and HA/gp41 chimeric proteins expressed on the cell surfaces exhibited similar reactivity to monoclonal antibodies 2F5 and 4E10. Immunization of guinea pigs using the HA-C14S/gp41 DNA or VLP vaccines induced antibodies against the HIV gp41 as well as to a peptide corresponding to a segment of MPER at higher levels than immunization by standard HIV VLPs. Further, sera from vaccinated guinea pigs were found to exhibit HIV neutralizing activities. Moreover, sera from guinea pigs vaccinated by HA-C14S/gp41 DNA and VLP vaccines but not the standard HIV VLPs, were found to neutralize HIV pseudovirions containing a SIV-4E10 chimeric Env protein. The virus neutralization could be blocked by a MPER-specific peptide, thus demonstrating induction of MPER-specific HIV neutralizing antibodies by this novel vaccine strategy. These results show that induction of MPER-specific HIV neutralizing antibodies can be achieved through a rationally designed vaccine strategy.

Care of HIV-exposed and HIV-infected neonates

African Journals Online (AJOL)

However, further reduction in MTCT may be possible if newborns at high risk of acquiring HIV ... infants of breastfeeding mothers with newly diagnosed HIV infection, dual NVP/ .... birth HIV DNA PCR testing for HIV-exposed low birth weight.

Anti-HIV Antibody Responses and the HIV Reservoir Size during Antiretroviral Therapy.

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Sulggi A Lee

Full Text Available A major challenge to HIV eradication strategies is the lack of an accurate measurement of the total burden of replication-competent HIV (the "reservoir". We assessed the association of anti-HIV antibody responses and the estimated size of the reservoir during antiretroviral therapy (ART.We evaluated anti-HIV antibody profiles using luciferase immunoprecipitation systems (LIPS assay in relation to several blood-based HIV reservoir measures: total and 2-LTR DNA (rtPCR or droplet digital PCR; integrated DNA (Alu PCR; unspliced RNA (rtPCR, multiply-spliced RNA (TILDA, residual plasma HIV RNA (single copy PCR, and replication-competent virus (outgrowth assay. We also assessed total HIV DNA and RNA in gut-associated lymphoid tissue (rtPCR. Spearman correlations and linear regressions were performed using log-transformed blood- or tissue-based reservoir measurements as predictors and log-transformed antibody levels as outcome variables.Among 51 chronically HIV-infected ART-suppressed participants (median age = 57, nadir CD4+ count = 196 cells/mm3, ART duration = 9 years, the most statistically significant associations were between antibody responses to integrase and HIV RNA in gut-associated lymphoid tissue (1.17 fold-increase per two-fold RNA increase, P = 0.004 and between antibody responses to matrix and integrated HIV DNA in resting CD4+ T cells (0.35 fold-decrease per two-fold DNA increase, P = 0.003. However, these associations were not statistically significant after a stringent Bonferroni-adjustment of P<0.00045. Multivariate models including age and duration of ART did not markedly alter results.Our findings suggest that anti-HIV antibody responses may reflect the size of the HIV reservoir during chronic treated HIV disease, possibly via antigen recognition in reservoir sites. Larger, prospective studies are needed to validate the utility of antibody levels as a measure of the total body burden of HIV during treatment.

DNA restriction fragment length polymorphism analysis of Mycobacterium tuberculosis isolates from HIV-seropositive and HIV-seronegative patients in Kampala, Uganda

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Katabazi Fred A

2009-02-01

Full Text Available Abstract Background The identification and differentiation of strains of Mycobacterium tuberculosis by DNA fingerprinting has provided a better understanding of the epidemiology and tracing the transmission of tuberculosis. We set out to determine if there was a relationship between the risk of belonging to a group of tuberculosis patients with identical mycobacterial DNA fingerprint patterns and the HIV sero-status of the individuals in a high TB incidence peri-urban setting of Kampala, Uganda. Methods One hundred eighty three isolates of Mycobacterium tuberculosis from 80 HIV seropositive and 103 HIV seronegative patients were fingerprinted by standard IS6110-RFLP. Using the BioNumerics software, strains were considered to be clustered if at least one other patient had an isolate with identical RFLP pattern. Results One hundred and eighteen different fingerprint patterns were obtained from the 183 isolates. There were 34 clusters containing 54% (99/183 of the patients (average cluster size of 2.9, and a majority (96.2% of the strains possessed a high copy number (≥ 5 copies of the IS6110 element. When strains with P = 0.615, patients aged P = 0.100, and sex (aOR 1.12, 95%CI 0.60–2.06, P = 0.715. Conclusion The sample showed evidence of a high prevalence of recent transmission with a high average cluster size, but infection with an isolate with a fingerprint found to be part of a cluster was not associated with any demographic or clinical characteristics, including HIV status.

The proviral genome of radiation leukemia virus: Molecular cloning, nucleotide sequence of its long terminal repeat and integration in lymphoma cell DNA

International Nuclear Information System (INIS)

Janowski, M.; Merregaert, J.; Boniver, J.; Maisin, J.R.

1985-01-01

The proviral genome of a thymotropic and leukemogenic C57BL/Ka mouse retrovirus, RadLV/VL/sub 3/(T+L+), was cloned as a biologically active PstI insert in the bacterial plasmid pBR322. Its restriction map was compared to those, already known, of two nonthymotropic and nonleukemogenic viruses of the same mouse strain, the ecotropic BL/Ka(B) and the xenotropic constituent of the radiation leukemia virus complex (RadLV). Differences were observed in the pol gene and in the env gene. Moreover, the nucleotide sequence of the RadLV/VL/sub 3/(T+L+) long terminal repeat revealed the existence of two copies of a 42 bp long sequence, separated by 11 nucleotides and of which BL/Ka(B) possesses only one copy

Prostaglandin (PG) E3 synthesis elicted by adrenergic stimuli in guinea-pig trachea (GPT) is mediated primarily by B2 adrenergic receptors

International Nuclear Information System (INIS)

Nadel, G.L.; Malik, K.U.; Lew, D.B.

1990-01-01

The purpose of this study was to examine arachidonic acid (AA) metabolism and to characterize the type of adrenergic receptor (AR) involved in the production of the major metabolite of this fatty acid. [ 14 C]AA was incubated with GPT-rings and the radiolabelled products were extracted and separated by TLC method. The medium was also assayed for radiolabelled immunoreactive PG's (iPG's) and leukotrienes (LT) B4 and C4 by RIA or Enzyme immunoassay (EIA) after exposure to various AR agonists. [ 14 C]AA was incorporated into GPT-rings and metabolized mainly into iPGE2 and smaller amounts into PGF2α. Trace amounts of PGD2 and 6-keto-PGF1α but not LTB4 or LTC4 were detected by RIA and/or EIA. Incubation of GPT rings for 15 minutes with isoproterenol and salbutamol resulted in a significant increase of PGE2 synthesis (optimum conc: 10 -7 , 10 -7 M respectively). In contrast, dobutamine, norepinephrine, phenylnephrine and xylazine (up to 10 -6 M) did not significantly increase PGE2 production. Isoproterenol-induced iPGE2 production was inhibited by a selective β2 antagonist, butoxamine (70%: 10 -7 M, 91%: 10 -6 M) and somewhat reduced by β1 antagonists practolol and metoprolol (30-64%:10 -6 M). These data suggest that isoproterenol induced iPGE2 synthesis is primarily mediated via activation of β2 adrenergic receptor

Quantitative and molecular analyses of mutation in a pSV2gpt transformed CHO cell line

International Nuclear Information System (INIS)

Stankowski, L.F. Jr.; Tindall, K.R.; Hsie, A.W.

1983-01-01

Following NDA-mediated gene transfer we have isolated a cell line useful for studying gene mutation at the molecular level. This line, AS52, derived from a hypoxanthine-guanine phosphoribosyl transferase (HGPRT) deficient Chinese hamster ovary (CHO) cell line, carries a single copy of the E. coli xanthine-guanine phosphoribosyl transferase (XGPRT) gene (gpt) and exhibits a spontaneous mutant frequency of 20 TG/sup r/ mutants/10 6 clonable cells. As with HGPRT - mutants, XGPRT - mutants can be selected in 6-thioguanine. AS52 (XGPRT + ) and wild type CHO (HGPRT + ) cell exhibit almost identical cytotoxic responses to various agents. We observed significant differences in mutation induction by UV light and ethyl methanesulfonate (EMS). Ratios of XGPRT - to HGPRT - mutants induced per unit dose (J/m 2 for UV light and μg/ml for EMS) are 1.4 and 0.70, respectively. Preliminary Southern blot hybridization analyses has been performed on 30 XGPRT - AS52 mutants. A majority of spontaneous mutants have deletions ranging in size from 1 to 4 kilobases (9/19) to complete loss of gpt sequences (4/19); the remainder have no detectable (5/19) or only minor (1/19) alterations. 5/5 UV-induced and 5/6 EMS-induced mutants do not show a detectable change. Similar analyses are underway for mutations induced by x-irradiation and ICR 191 treatment

HIV DNA Is Frequently Present within Pathologic Tissues Evaluated at Autopsy from Combined Antiretroviral Therapy-Treated Patients with Undetectable Viral Loads.

Science.gov (United States)

Lamers, Susanna L; Rose, Rebecca; Maidji, Ekaterina; Agsalda-Garcia, Melissa; Nolan, David J; Fogel, Gary B; Salemi, Marco; Garcia, Debra L; Bracci, Paige; Yong, William; Commins, Deborah; Said, Jonathan; Khanlou, Negar; Hinkin, Charles H; Sueiras, Miguel Valdes; Mathisen, Glenn; Donovan, Suzanne; Shiramizu, Bruce; Stoddart, Cheryl A; McGrath, Michael S; Singer, Elyse J

2016-10-15

HIV infection treatment strategies have historically defined effectiveness through measuring patient plasma HIV RNA. While combined antiretroviral therapy (cART) can reduce plasma viral load (pVL) to undetectable levels, the degree that HIV is eliminated from other anatomical sites remains unclear. We investigated the HIV DNA levels in 229 varied autopsy tissues from 20 HIV-positive (HIV(+)) cART-treated study participants with low or undetectable plasma VL and cerebrospinal fluid (CSF) VL prior to death who were enrolled in the National Neurological AIDS Bank (NNAB) longitudinal study and autopsy cohort. Extensive medical histories were obtained for each participant. Autopsy specimens, including at least six brain and nonbrain tissues per participant, were reviewed by study pathologists. HIV DNA, measured in tissues by quantitative and droplet digital PCR, was identified in 48/87 brain tissues and 82/142 nonbrain tissues at levels >200 HIV copies/million cell equivalents. No participant was found to be completely free of tissue HIV. Parallel sequencing studies from some tissues recovered intact HIV DNA and RNA. Abnormal histological findings were identified in all participants, especially in brain, spleen, lung, lymph node, liver, aorta, and kidney. All brain tissues demonstrated some degree of pathology. Ninety-five percent of participants had some degree of atherosclerosis, and 75% of participants died with cancer. This study assists in characterizing the anatomical locations of HIV, in particular, macrophage-rich tissues, such as the central nervous system (CNS) and testis. Additional studies are needed to determine if the HIV recovered from tissues promotes the pathogenesis of inflammatory diseases, such as HIV-associated neurocognitive disorders, cancer, and atherosclerosis. It is well-known that combined antiretroviral therapy (cART) can reduce plasma HIV to undetectable levels; however, cART cannot completely clear HIV infection. An ongoing question is

c-DNA of HIV-1 detection on spot of Buffy-Coat of leukocytes (DBCS)

OpenAIRE

Marco Rossi de Gasperis; Maria Daniela Caione; Carlo Concato; Ersilia Fiscarelli; Nicola Di Pietro; Vittorio Salotti; Lorenza Putignani; Donato Menichella; Francesco Callea

2010-01-01

Introduction:The elective way for the diagnosis of HIV-1-infection in the window period and in children under the age of 16-18 months is to search virus integrated in leukocytes. Aim of the study was to assess the sensitivity and specificity of extraction from Buffy-Dried Coat Spot (DBCS) in leukocyte to detect c-DNA with nested-PCR in HIV-1-infected individuals compared to Dried Blood Spot (DBS) both extracted by automated instrument EZ1 (QIAGEN, Hilden, Germany). Both DBCS and both DBS were...

A zinc(II)-based two-dimensional MOF for sensitive and selective sensing of HIV-1 ds-DNA sequences

Energy Technology Data Exchange (ETDEWEB)

Zhao, Hai-Qing; Qiu, Gui-Hua; Liang, Zhen [Guangdong Provincial Key Laboratory of New Drug Screening, School of Pharmaceutical Sciences, Southern Medical University, Guangzhou 510515 (China); Li, Min-Min [The First Affiliated Hospital of Jinan University, Guangzhou 510515 (China); Sun, Bin; Qin, Liang; Yang, Shui-Ping; Chen, Wen-Hua [Guangdong Provincial Key Laboratory of New Drug Screening, School of Pharmaceutical Sciences, Southern Medical University, Guangzhou 510515 (China); Chen, Jin-Xiang, E-mail: jxchen@smu.edu.cn [Guangdong Provincial Key Laboratory of New Drug Screening, School of Pharmaceutical Sciences, Southern Medical University, Guangzhou 510515 (China)

2016-05-30

Coordination reaction of a known three-dimensional (3D) polymer precursor {Na_3[Na_9(Cbdcp)_6(H_2O)_1_8]}{sub n} (A, Cbdcp = N-(4-carboxybenzyl)-(3,5-dicarboxyl)pyridinium) with Zn(NO{sub 3}){sub 2}·6H{sub 2}O in H{sub 2}O or H{sub 2}O/DMF at 100 °C and in the presence of aspirin, 5-fluorouracil (5-FU) as modulators, trans-1,2-bis(4-pyridyl)ethylene (bpe) or 1,2-bis(4-pyridyl)ethane (bpea) as ancillary ligands afforded six novel Zn(II)-based metal-organic frameworks (MOFs), that is, {[Zn(Cbdcp)(H_2O)_3]·H_2O}{sub n} (1, 1D zigzag chain), {[Zn(HCbdcp)_2]·H_2O}{sub n} (2, 2D sheet), {[Zn(Cbdcp)(bpe)_1_/_2]·2H_2O}{sub n} (3, 3D polymer), {[Zn(Cbdcp)(bpe)_1_/_2]·2H_2O}{sub n} (4, 2D network), {[Zn(Cbdcp)(bpea)_1_/_2]·2H_2O}{sub n} (5, 3D polymer) and {[Zn(Cbdcp)(bpea)_1_/_2]·2H_2O}{sub n} (6, 2D network). Among them, compound 2 contains aromatic rings, positively charged pyridinium, Zn{sup 2+} cation centers and carboxylic acid groups lined up on the 2D sheet structure with a certain extended surface exposure. The unique structure of 2 facilitates effective association with carboxyfluorescein (FAM) labeled probe single stranded DNA (probe ss-DNA, delineates as P-DNA) to yield a P-DNA@2 system, and leads to fluorescence quenching of FAM via a photoinduced electron transfer process. The P-DNA@2 system is effective and reliable for the detection of human immunodeficiency virus 1 ds-DNA (HIV ds-DNA) sequences and capable of distinguishing complementary HIV ds-DNA from mismatched target sequences with the detection limit as low as 10 pM (S/N = 3). - Graphical abstract: Six water-stable zinc(II) zwitterionic carboxylate compounds with 1D chain, 2D and 3D networks were synthesized. Compound 2 can interact with the probe DNA through noncovalent bonds to form P-DNA@2 system. This system can be used as an effective, fluorescent sensing platform for the detection of HIV ds-DNA with the detection limit as low as 10 pM. - Highlights: • Six water-stable zinc

Field expansion of DNA polymerase chain reaction for early infant diagnosis of HIV-1: The Ethiopian experience

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Peter Fonjungo

2013-05-01

Full Text Available Background: Early diagnosis of infants infected with HIV (EID and early initiation of treatment significantly reduces the rate of disease progression and mortality. One of the challengesto identification of HIV-1-infected infants is availability and/or access to quality molecular laboratory facilities which perform molecular virologic assays suitable for accurate identificationof the HIV status of infants. Method: We conducted a joint site assessment and designed laboratories for the expansion of DNA polymerase chain reaction (PCR testing based on dried blood spot (DBS for EID insix regions of Ethiopia. Training of appropriate laboratory technologists and development of required documentation including standard operating procedures (SOPs was carried out. The impact of the expansion of EID laboratories was assessed by the number of tests performed as well as the turn-around time. Results: DNA PCR for EID was introduced in 2008 in six regions. From April 2006 to April 2008, a total of 2848 infants had been tested centrally at the Ethiopian Health and Nutrition Research Institute (EHNRI in Addis Ababa, and which was then the only laboratory with the capability to perform EID; 546 (19.2% of the samples were positive. By November 2010, EHNRI and the six laboratories had tested an additional 16 985 HIV-exposed infants, of which 1915 (11.3% were positive. The median turn-around time for test results was 14 days (range 14−21 days. Conclusion: Expansion of HIV DNA PCR testing facilities that can provide quality and reliable results is feasible in resource-limited settings. Regular supervision and monitoring for quality assurance of these laboratories is essential to maintain accuracy of testing.

Superior induction of T cell responses to conserved HIV-1 regions by electroporated alphavirus replicon DNA compared to that with conventional plasmid DNA vaccine.

Science.gov (United States)

Knudsen, Maria L; Mbewe-Mvula, Alice; Rosario, Maximillian; Johansson, Daniel X; Kakoulidou, Maria; Bridgeman, Anne; Reyes-Sandoval, Arturo; Nicosia, Alfredo; Ljungberg, Karl; Hanke, Tomás; Liljeström, Peter

2012-04-01

Vaccination using "naked" DNA is a highly attractive strategy for induction of pathogen-specific immune responses; however, it has been only weakly immunogenic in humans. Previously, we constructed DNA-launched Semliki Forest virus replicons (DREP), which stimulate pattern recognition receptors and induce augmented immune responses. Also, in vivo electroporation was shown to enhance immune responses induced by conventional DNA vaccines. Here, we combine these two approaches and show that in vivo electroporation increases CD8(+) T cell responses induced by DREP and consequently decreases the DNA dose required to induce a response. The vaccines used in this study encode the multiclade HIV-1 T cell immunogen HIVconsv, which is currently being evaluated in clinical trials. Using intradermal delivery followed by electroporation, the DREP.HIVconsv DNA dose could be reduced to as low as 3.2 ng to elicit frequencies of HIV-1-specific CD8(+) T cells comparable to those induced by 1 μg of a conventional pTH.HIVconsv DNA vaccine, representing a 625-fold molar reduction in dose. Responses induced by both DREP.HIVconsv and pTH.HIVconsv were further increased by heterologous vaccine boosts employing modified vaccinia virus Ankara MVA.HIVconsv and attenuated chimpanzee adenovirus ChAdV63.HIVconsv. Using the same HIVconsv vaccines, the mouse observations were supported by an at least 20-fold-lower dose of DNA vaccine in rhesus macaques. These data point toward a strategy for overcoming the low immunogenicity of DNA vaccines in humans and strongly support further development of the DREP vaccine platform for clinical evaluation.

Effective CD4+ T-cell restoration in gut-associated lymphoid tissue of HIV-infected patients is associated with enhanced Th17 cells and polyfunctional HIV-specific T-cell responses.

Science.gov (United States)

Macal, M; Sankaran, S; Chun, T-W; Reay, E; Flamm, J; Prindiville, T J; Dandekar, S

2008-11-01

Human immunodeficiency virus (HIV) infection leads to severe CD4+ T-cell depletion in gut-associated lymphoid tissue (GALT) that persists despite the initiation of highly active antiretroviral therapy (HAART). It is not known whether restoration of gut mucosal CD4+ T cells and their functions is feasible during therapy and how that relates to immune correlates and viral reservoirs. Intestinal biopsies and peripheral blood samples from HIV-infected patients who were either HAART naive or on long-term HAART were evaluated. Our data demonstrated that gut CD4+ T-cell restoration ranged from modest (50%), compared with uninfected controls. Despite persistent CD4+ T-cell proviral burden and residual immune activation in GALT during HAART, effective CD4+ T-cell restoration (>50%) was achieved, which was associated with enhanced Th17 CD4+ T-cell accumulation and polyfunctional anti-HIV cellular responses. Our findings suggest that a threshold of>50% CD4+ T-cell restoration may be sufficient for polyfunctional HIV-specific T cells with implications in the evaluation of vaccines and therapeutics.

Epigenetic regulation of HIV-1 latency: focus on polycomb group (PcG) proteins.

Science.gov (United States)

Khan, Sheraz; Iqbal, Mazhar; Tariq, Muhammad; Baig, Shahid M; Abbas, Wasim

2018-01-01

HIV-1 latency allows the virus to persist until reactivation, in a transcriptionally silent form in its cellular reservoirs despite the presence of effective cART. Such viral persistence represents a major barrier to HIV eradication since treatment interruption leads to rebound plasma viremia. Polycomb group (PcG) proteins have recently got a considerable attention in regulating HIV-1 post-integration latency as they are involved in the repression of proviral gene expression through the methylation of histones. This epigenetic regulation plays an important role in the establishment and maintenance of HIV-1 latency. In fact, PcG proteins act in complexes and modulate the epigenetic signatures of integrated HIV-1 promoter. Key role played by PcG proteins in the molecular control of HIV-1 latency has led to hypothesize that PcG proteins may represent a valuable target for future HIV-1 therapy in purging HIV-1 reservoirs. In this regard, various small molecules have been synthesized or explored to specifically block the epigenetic activity of PcG. In this review, we will highlight the possible therapeutic approaches to achieve either a functional or sterilizing cure of HIV-1 infection with special focus on histone methylation by PcG proteins together with current and novel pharmacological approaches to reactivate HIV-1 from latency that could ultimately lead towards a better clearance of viral latent reservoirs.

Immunization of neonatal mice with LAMP/p55 HIV gag DNA elicits robust immune responses that last to adulthood

International Nuclear Information System (INIS)

Ordonhez Rigato, Paula; Maciel, Milton; Goldoni, Adriana Leticia; Piubelli, Orlando; Alves de Brito, Cyro; Fusaro, Ana Elisa; Eurico de Alencar, Liciana Xavier; August, Thomas; Torres Azevedo Marques, Ernesto; Silva Duarte, Alberto Jose da; Sato, Maria Notomi

2010-01-01

Successful T cell priming in early postnatal life that can generate effective long-lasting responses until adulthood is critical in HIV vaccination strategies because it prevents early sexual initiation and breastfeeding transmission of HIV. A chimeric DNA vaccine encoding p55 HIV gag associated with lysosome-associated membrane protein 1 (LAMP-1; which drives the antigen to the MIIC compartment), has been used to enhance cellular and humoral antigen-specific responses in adult mice and macaques. Herein, we investigated LAMP-1/gag vaccine immunogenicity in the neonatal period in mice and its ability to generate long-lasting effects. Neonatal vaccination with chimeric LAMP/gag generated stronger Gag-specific immune responses, as measured by the breadth of the Gag peptide-specific IFN-γ, proliferative responsiveness, cytokine production and antibody production, all of which revealed activation of CD4+ T cells as well as the generation of a more robust CTL response compared to gag vaccine alone. To induce long-lived T and B cell memory responses, it was necessary to immunize neonates with the chimeric LAMP/gag DNA vaccine. The LAMP/gag DNA vaccine strategy could be particularly useful for generating an anti-HIV immune response in the early postnatal period capable of inducing long-term immunological memory.

Prostaglandin (PG) E3 synthesis elicted by adrenergic stimuli in guinea-pig trachea (GPT) is mediated primarily by B2 adrenergic receptors

Energy Technology Data Exchange (ETDEWEB)

Nadel, G.L.; Malik, K.U.; Lew, D.B. (Univ. of Tennessee, Memphis (United States))

1990-02-26

The purpose of this study was to examine arachidonic acid (AA) metabolism and to characterize the type of adrenergic receptor (AR) involved in the production of the major metabolite of this fatty acid. ({sup 14}C)AA was incubated with GPT-rings and the radiolabelled products were extracted and separated by TLC method. The medium was also assayed for radiolabelled immunoreactive PG's (iPG's) and leukotrienes (LT) B4 and C4 by RIA or Enzyme immunoassay (EIA) after exposure to various AR agonists. ({sup 14}C)AA was incorporated into GPT-rings and metabolized mainly into iPGE2 and smaller amounts into PGF2{alpha}. Trace amounts of PGD2 and 6-keto-PGF1{alpha} but not LTB4 or LTC4 were detected by RIA and/or EIA. Incubation of GPT rings for 15 minutes with isoproterenol and salbutamol resulted in a significant increase of PGE2 synthesis (optimum conc: 10{sup {minus}7}, 10{sup {minus}7}M respectively). In contrast, dobutamine, norepinephrine, phenylnephrine and xylazine (up to 10{sup {minus}6}M) did not significantly increase PGE2 production. Isoproterenol-induced iPGE2 production was inhibited by a selective {beta}2 antagonist, butoxamine (70%: 10{sup {minus}7}M, 91%: 10{sup {minus}6}M) and somewhat reduced by {beta}1 antagonists practolol and metoprolol (30-64%:10{sup {minus}6}M). These data suggest that isoproterenol induced iPGE2 synthesis is primarily mediated via activation of {beta}2 adrenergic receptor.

"cART intensification by the HIV-1 Tat B clade vaccine: progress to phase III efficacy studies".

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Cafaro, Aurelio; Sgadari, Cecilia; Picconi, Orietta; Tripiciano, Antonella; Moretti, Sonia; Francavilla, Vittorio; Pavone Cossut, Maria Rosaria; Buttò, Stefano; Cozzone, Giovanni; Ensoli, Fabrizio; Monini, Paolo; Ensoli, Barbara

2018-02-01

In spite of its success at suppressing HIV replication, combination antiretroviral therapy (cART) only partially reduces immune dysregulation and loss of immune functions. These cART-unmet needs appear to be due to persistent virus replication and cell-to-cell transmission in reservoirs, and are causes of increased patients' morbidity and mortality. Up to now, therapeutic interventions aimed at cART-intensification by attacking the virus reservoir have failed. Areas covered: We briefly review the rationale and clinical development of Tat therapeutic vaccine in cART-treated subjects in Italy and South Africa (SA). Vaccination with clade-B Tat induced cross-clade neutralizing antibodies, immune restoration, including CD4 + T cell increase particularly in low immunological responders, and reduction of proviral DNA. Phase III efficacy trials in SA are planned both in adult and pediatric populations. Expert commentary: We propose the Tat therapeutic vaccine as a pathogenesis-driven intervention that effectively intensifies cART and may lead to a functional cure and provide new perspectives for prevention and virus eradication strategies.

HIV-1 Env DNA vaccine plus protein boost delivered by EP expands B- and T-cell responses and neutralizing phenotype in vivo.

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Kar Muthumani

Full Text Available An effective HIV vaccine will most likely require the induction of strong T-cell responses, broadly neutralizing antibodies (bNAbs, and the elicitation of antibody-dependent cellular cytotoxicity (ADCC. Previously, we demonstrated the induction of strong HIV/SIV cellular immune responses in macaques and humans using synthetic consensus DNA immunogens delivered via adaptive electroporation (EP. However, the ability of this improved DNA approach to prime for relevant antibody responses has not been previously studied. Here, we investigate the immunogenicity of consensus DNA constructs encoding gp140 sequences from HIV-1 subtypes A, B, C and D in a DNA prime-protein boost vaccine regimen. Mice and guinea pigs were primed with single- and multi-clade DNA via EP and boosted with recombinant gp120 protein. Sera were analyzed for gp120 binding and induction of neutralizing antibody activity. Immunization with recombinant Env protein alone induced low-titer binding antibodies with limited neutralization breath. In contrast, the synthetic DNA prime-protein boost protocol induced significantly higher antibody binding titers. Furthermore, sera from DNA prime-protein boost groups were able to neutralize a broader range of viruses in a panel of tier 1 clade B viruses as well as multiple tier 1 clade A and clade C viruses. Further investigation of synthetic DNA prime plus adaptive EP plus protein boost appears warranted.

Synthetic AAV/CRISPR vectors for blocking HIV-1 expression in persistently infected astrocytes.

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Kunze, Christine; Börner, Kathleen; Kienle, Eike; Orschmann, Tanja; Rusha, Ejona; Schneider, Martha; Radivojkov-Blagojevic, Milena; Drukker, Micha; Desbordes, Sabrina; Grimm, Dirk; Brack-Werner, Ruth

2018-02-01

Astrocytes, the most abundant cells in the mammalian brain, perform key functions and are involved in several neurodegenerative diseases. The human immunodeficiency virus (HIV) can persist in astrocytes, contributing to the HIV burden and neurological dysfunctions in infected individuals. While a comprehensive approach to HIV cure must include the targeting of HIV-1 in astrocytes, dedicated tools for this purpose are still lacking. Here we report a novel Adeno-associated virus-based vector (AAV9P1) with a synthetic surface peptide for transduction of astrocytes. Analysis of AAV9P1 transduction efficiencies with single brain cell populations, including primary human brain cells, as well as human brain organoids demonstrated that AAV9P1 targeted terminally differentiated human astrocytes much more efficiently than neurons. We then investigated whether AAV9P1 can be used to deliver HIV-inhibitory genes to astrocytes. To this end we generated AAV9P1 vectors containing genes for HIV-1 proviral editing by CRISPR/Cas9. Latently HIV-1 infected astrocytes transduced with these vectors showed significantly diminished reactivation of proviruses, compared with untransduced cultures. Sequence analysis identified mutations/deletions in key HIV-1 transcriptional control regions. We conclude that AAV9P1 is a promising tool for gene delivery to astrocytes and may facilitate inactivation/destruction of persisting HIV-1 proviruses in astrocyte reservoirs. © 2017 Wiley Periodicals, Inc.

Development of 5 ' LTR DNA methylation of latent HIV-1 provirus in cell line models and in long-term-infected individuals

Czech Academy of Sciences Publication Activity Database

Trejbalová, Kateřina; Kovářová, Denisa; Blažková, Jana; Machala, L.; Jilich, D.; Weber, J.; Kučerová, Dana; Vencálek, O.; Hirsch, Ivan; Hejnar, Jiří

2016-01-01

Roč. 8, zima (2016), č. článku 19. ISSN 1868-7083 R&D Projects: GA ČR GAP304/12/1736 Institutional support: RVO:68378050 Keywords : HIV-1 * latent reservoir * DNA methylation * chromatin conformation * latent HIV-1 provirus reactivation * HIV-1-infected individuals Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.987, year: 2016

Size, Composition, and Evolution of HIV DNA Populations during Early Antiretroviral Therapy and Intensification with Maraviroc.

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Chaillon, Antoine; Gianella, Sara; Lada, Steven M; Perez-Santiago, Josué; Jordan, Parris; Ignacio, Caroline; Karris, Maile; Richman, Douglas D; Mehta, Sanjay R; Little, Susan J; Wertheim, Joel O; Smith, Davey M

2018-02-01

Residual viremia is common during antiretroviral therapy (ART) and could be caused by ongoing low-level virus replication or by release of viral particles from infected cells. ART intensification should impact ongoing viral propagation but not virion release. Eighteen acutely infected men were enrolled in a randomized controlled trial and monitored for a median of 107 weeks. Participants started ART with ( n = 9) or without ( n = 9) intensification with maraviroc (MVC) within 90 days of infection. Levels of HIV DNA and cell-free RNA were quantified by droplet digital PCR. Deep sequencing of C2-V3 env , gag , and pol (454 Roche) was performed on longitudinally collected plasma and peripheral blood mononuclear cell (PBMC) samples while on ART. Sequence data were analyzed for evidence of evolution by (i) molecular diversity analysis, (ii) nonparametric test for panmixia, and (iii) tip date randomization within a Bayesian framework. There was a longitudinal decay of HIV DNA after initiation of ART with no difference between MVC intensification groups (-0.08 ± 0.01 versus -0.09 ± 0.01 log 10 copies/week in MVC + versus MVC - groups; P = 0.62). All participants had low-level residual viremia (median, 2.8 RNA copies/ml). Across participants, medians of 56 (interquartile range [IQR], 36 to 74), 29 (IQR, 25 to 35), and 40 (IQR, 31 to 54) haplotypes were generated for env , gag , and pol regions, respectively. There was no clear evidence of viral evolution during ART and no difference in viral diversity or population structure from individuals with or without MVC intensification. Further efforts focusing on elucidating the mechanism(s) of viral persistence in various compartments using recent sequencing technologies are still needed, and potential low-level viral replication should always be considered in cure strategies. IMPORTANCE Residual viremia is common among HIV-infected people on ART. It remains controversial if this viremia is a consequence of propagating

Alphavirus replicon DNA expressing HIV antigens is an excellent prime for boosting with recombinant modified vaccinia Ankara (MVA or with HIV gp140 protein antigen.

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Maria L Knudsen

Full Text Available Vaccination with DNA is an attractive strategy for induction of pathogen-specific T cells and antibodies. Studies in humans have shown that DNA vaccines are safe, but their immunogenicity needs further improvement. As a step towards this goal, we have previously demonstrated that immunogenicity is increased with the use of an alphavirus DNA-launched replicon (DREP vector compared to conventional DNA vaccines. In this study, we investigated the effect of varying the dose and number of administrations of DREP when given as a prime prior to a heterologous boost with poxvirus vector (MVA and/or HIV gp140 protein formulated in glucopyranosyl lipid A (GLA-AF adjuvant. The DREP and MVA vaccine constructs encoded Env and a Gag-Pol-Nef fusion protein from HIV clade C. One to three administrations of 0.2 μg DREP induced lower HIV-specific T cell and IgG responses than the equivalent number of immunizations with 10 μg DREP. However, the two doses were equally efficient as a priming component in a heterologous prime-boost regimen. The magnitude of immune responses depended on the number of priming immunizations rather than the dose. A single low dose of DREP prior to a heterologous boost resulted in greatly increased immune responses compared to MVA or protein antigen alone, demonstrating that a mere 0.2 μg DREP was sufficient for priming immune responses. Following a DREP prime, T cell responses were expanded greatly by an MVA boost, and IgG responses were also expanded when boosted with protein antigen. When MVA and protein were administered simultaneously following multiple DREP primes, responses were slightly compromised compared to administering them sequentially. In conclusion, we have demonstrated efficient priming of HIV-specific T cell and IgG responses with a low dose of DREP, and shown that the priming effect depends on number of primes administered rather than dose.

Keeping your armour intact: how HIV-1 evades detection by the innate immune system: HIV-1 capsid controls detection of reverse transcription products by the cytosolic DNA sensor cGAS.

Science.gov (United States)

Maelfait, Jonathan; Seiradake, Elena; Rehwinkel, Jan

2014-07-01

HIV-1 infects dendritic cells (DCs) without triggering an effective innate antiviral immune response. As a consequence, the induction of adaptive immune responses controlling virus spread is limited. In a recent issue of Immunity, Lahaye and colleagues show that intricate interactions of HIV capsid with the cellular cofactor cyclophilin A (CypA) control infection and innate immune activation in DCs. Manipulation of HIV-1 capsid to increase its affinity for CypA results in reduced virus infectivity and facilitates access of the cytosolic DNA sensor cGAS to reverse transcribed DNA. This in turn induces a strong host response. Here, we discuss these findings in the context of recent developments in innate immunity and consider the implications for disease control and vaccine design. © 2014 The Authors. Bioessays published by WILEY Periodicals, Inc.

Inhibition of HIV-1 reverse transcriptase-catalyzed synthesis by intercalated DNA Benzo[a]Pyrene 7,8-Dihydrodiol-9,10-Epoxide adducts.

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Parvathi Chary

Full Text Available To aid in the characterization of the relationship of structure and function for human immunodeficiency virus type-1 reverse transcriptase (HIV-1 RT, this investigation utilized DNAs containing benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE-modified primers and templates as a probe of the architecture of this complex. BPDE lesions that differed in their stereochemistry around the C10 position were covalently linked to N (6-adenine and positioned in either the primer or template strand of a duplex template-primer. HIV-1 RT exhibited a stereoisomer-specific and strand-specific difference in replication when the BPDE-lesion was placed in the template versus the primer strand. When the C10 R-BPDE adduct was positioned in the primer strand in duplex DNA, 5 nucleotides from the 3΄ end of the primer terminus, HIV-1 RT could not fully replicate the template, producing truncated products; this block to further synthesis did not affect rates of dissociation or DNA binding affinity. Additionally, when the adducts were in the same relative position, but located in the template strand, similar truncated products were observed with both the C10 R and C10 S BPDE adducts. These data suggest that the presence of covalently-linked intercalative DNA adducts distant from the active site can lead to termination of DNA synthesis catalyzed by HIV-1 RT.

CTL escape mediated by proteasomal destruction of an HIV-1 cryptic epitope.

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Sylvain Cardinaud

2011-05-01

Full Text Available Cytotoxic CD8+ T cells (CTLs play a critical role in controlling viral infections. HIV-infected individuals develop CTL responses against epitopes derived from viral proteins, but also against cryptic epitopes encoded by viral alternative reading frames (ARF. We studied here the mechanisms of HIV-1 escape from CTLs targeting one such cryptic epitope, Q9VF, encoded by an HIVgag ARF and presented by HLA-B*07. Using PBMCs of HIV-infected patients, we first cloned and sequenced proviral DNA encoding for Q9VF. We identified several polymorphisms with a minority of proviruses encoding at position 5 an aspartic acid (Q9VF/5D and a majority encoding an asparagine (Q9VF/5N. We compared the prevalence of each variant in PBMCs of HLA-B*07+ and HLA-B*07- patients. Proviruses encoding Q9VF/5D were significantly less represented in HLA-B*07+ than in HLA-B*07- patients, suggesting that Q9FV/5D encoding viruses might be under selective pressure in HLA-B*07+ individuals. We thus analyzed ex vivo CTL responses directed against Q9VF/5D and Q9VF/5N. Around 16% of HLA-B*07+ patients exhibited CTL responses targeting Q9VF epitopes. The frequency and the magnitude of CTL responses induced with Q9VF/5D or Q9VF/5N peptides were almost equal indicating a possible cross-reactivity of the same CTLs on the two peptides. We then dissected the cellular mechanisms involved in the presentation of Q9VF variants. As expected, cells infected with HIV strains encoding for Q9VF/5D were recognized by Q9VF/5D-specific CTLs. In contrast, Q9VF/5N-encoding strains were neither recognized by Q9VF/5N- nor by Q9VF/5D-specific CTLs. Using in vitro proteasomal digestions and MS/MS analysis, we demonstrate that the 5N variation introduces a strong proteasomal cleavage site within the epitope, leading to a dramatic reduction of Q9VF epitope production. Our results strongly suggest that HIV-1 escapes CTL surveillance by introducing mutations leading to HIV ARF-epitope destruction by proteasomes.

A Phase I Double Blind, Placebo-Controlled, Randomized Study of the Safety and Immunogenicity of Electroporated HIV DNA with or without Interleukin 12 in Prime-Boost Combinations with an Ad35 HIV Vaccine in Healthy HIV-Seronegative African Adults.

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Juliet Mpendo

Full Text Available Strategies to enhance the immunogenicity of DNA vaccines in humans include i co-administration of molecular adjuvants, ii intramuscular administration followed by in vivo electroporation (IM/EP and/or iii boosting with a different vaccine. Combining these strategies provided protection of macaques challenged with SIV; this clinical trial was designed to mimic the vaccine regimen in the SIV study.Seventy five healthy, HIV-seronegative adults were enrolled into a phase 1, randomized, double-blind, placebo-controlled trial. Multi-antigenic HIV (HIVMAG plasmid DNA (pDNA vaccine alone or co-administered with pDNA encoding human Interleukin 12 (IL-12 (GENEVAX IL-12 given by IM/EP using the TriGrid Delivery System was tested in different prime-boost regimens with recombinant Ad35 HIV vaccine given IM.All local reactions but one were mild or moderate. Systemic reactions and unsolicited adverse events including laboratory abnormalities did not differ between vaccine and placebo recipients. No serious adverse events (SAEs were reported. T cell and antibody response rates after HIVMAG (x3 prime-Ad35 (x1 boost were independent of IL-12, while the magnitude of interferon gamma (IFN-γ ELISPOT responses was highest after HIVMAG (x3 without IL-12. The quality and phenotype of T cell responses shown by intracellular cytokine staining (ICS were similar between groups. Inhibition of HIV replication by autologous T cells was demonstrated after HIVMAG (x3 prime and was boosted after Ad35. HIV specific antibodies were detected only after Ad35 boost, although there was a priming effect with 3 doses of HIVMAG with or without IL-12. No anti-IL-12 antibodies were detected.The vaccines were safe, well tolerated and moderately immunogenic. Repeated administration IM/EP was well accepted. An adjuvant effect of co-administered plasmid IL-12 was not detected.ClinicalTrials.gov NCT01496989.

Development of 5' LTR DNA methylation of latent HIV-1 provirus in cell line models and in long-term-infected individuals

Czech Academy of Sciences Publication Activity Database

Trejbalová, K.; Kovářová, D.; Blažková, J.; Machala, L.; Jilich, D.; Weber, Jan; Kučerová, D.; Vencálek, O.; Hirsch, Ivan; Hejnar, J.

2016-01-01

Roč. 8, Feb 19 (2016), č. článku 19. ISSN 1868-7083 Institutional support: RVO:61388963 Keywords : HIV-1 * latent reservoir * DNA methylation * chromatin conformation * latent HIV-1 provirus reactivation * HIV-1-infected individuals Subject RIV: EE - Microbiology, Virology Impact factor: 4.987, year: 2016 http://clinicalepigeneticsjournal.biomedcentral.com/articles/10.1186/s13148-016-0185-6

Safety and immunogenicity of therapeutic DNA vaccination in individuals treated with antiretroviral therapy during acute/early HIV-1 infection.

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Eric S Rosenberg

2010-05-01

Full Text Available An effective therapeutic vaccine that could augment immune control of HIV-1 replication may abrogate or delay the need for antiretroviral therapy. AIDS Clinical Trials Group (ACTG A5187 was a phase I/II, randomized, placebo-controlled, double-blinded trial to evaluate the safety and immunogenicity of an HIV-1 DNA vaccine (VRC-HVDNA 009-00-VP in subjects treated with antiretroviral therapy during acute/early HIV-1 infection. (clinicaltrials.gov NCT00125099Twenty healthy HIV-1 infected subjects who were treated with antiretroviral therapy during acute/early HIV-1 infection and had HIV-1 RNA<50 copies/mL were randomized to receive either vaccine or placebo. The objectives of this study were to evaluate the safety and immunogenicity of the vaccine. Following vaccination, subjects interrupted antiretroviral treatment, and set-point HIV-1 viral loads and CD4 T cell counts were determined 17-23 weeks after treatment discontinuation.Twenty subjects received all scheduled vaccinations and discontinued antiretroviral therapy at week 30. No subject met a primary safety endpoint. No evidence of differences in immunogenicity were detected in subjects receiving vaccine versus placebo. There were also no significant differences in set-point HIV-1 viral loads or CD4 T cell counts following treatment discontinuation. Median set-point HIV-1 viral loads after treatment discontinuation in vaccine and placebo recipients were 3.5 and 3.7 log(10 HIV-1 RNA copies/mL, respectively.The HIV-1 DNA vaccine (VRC-HIVDNA 009-00-VP was safe but poorly immunogenic in subjects treated with antiretroviral therapy during acute/early HIV-1 infection. Viral set-points were similar between vaccine and placebo recipients following treatment interruption. However, median viral load set-points in both groups were lower than in historical controls, suggesting a possible role for antiretroviral therapy in persons with acute or early HIV-1 infection and supporting the safety of

Retroviral DNA integration: ASLV, HIV, and MLV show distinct target site preferences.

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Rick S Mitchell

2004-08-01

Full Text Available The completion of the human genome sequence has made possible genome-wide studies of retroviral DNA integration. Here we report an analysis of 3,127 integration site sequences from human cells. We compared retroviral vectors derived from human immunodeficiency virus (HIV, avian sarcoma-leukosis virus (ASLV, and murine leukemia virus (MLV. Effects of gene activity on integration targeting were assessed by transcriptional profiling of infected cells. Integration by HIV vectors, analyzed in two primary cell types and several cell lines, strongly favored active genes. An analysis of the effects of tissue-specific transcription showed that it resulted in tissue-specific integration targeting by HIV, though the effect was quantitatively modest. Chromosomal regions rich in expressed genes were favored for HIV integration, but these regions were found to be interleaved with unfavorable regions at CpG islands. MLV vectors showed a strong bias in favor of integration near transcription start sites, as reported previously. ASLV vectors showed only a weak preference for active genes and no preference for transcription start regions. Thus, each of the three retroviruses studied showed unique integration site preferences, suggesting that virus-specific binding of integration complexes to chromatin features likely guides site selection.

Relationship of HIV Reservoir Characteristics with Immune Status and Viral Rebound Kinetics in an HIV Therapeutic Vaccine Study

Science.gov (United States)

Li, Jonathan Z.; Heisey, Andrea; Ahmed, Hayat; Wang, Hongying; Zheng, Lu; Carrington, Mary; Wrin, Terri; Schooley, Robert T.; Lederman, Michael M.; Kuritzkes, Daniel R.

2014-01-01

Objectives To evaluate the impact of therapeutic HIV vaccination on the HIV reservoir, and assess the relationship of the viral reservoir with HIV-specific immune status and viral rebound kinetics. Design Retrospective analysis of ACTG A5197, a randomized, placebo-controlled trial of a therapeutic rAd5 HIV-1 gag vaccine. Methods Participants received vaccine/placebo at weeks 0, 4, and 26 prior to a 16-week analytic treatment interruption (ATI) at week 38. Cell-associated HIV-1 RNA and DNA (CA-RNA and CA-DNA) and HIV-1 residual viremia (RV) were quantified at weeks 0, 8, and 38. HIV-specific CD4+/CD8+ activity were assessed by an intracellular cytokine staining assay. Results At study entry, CA-RNA and CA-DNA levels were correlated inversely with the numbers of HIV-specific CD4+ interferon-γ-producing cells (CA-RNA: r = −0.23, P=0.03 and CA-DNA: r = −0.28, P<0.01, N=93). Therapeutic HIV vaccination induced HIV-specific CD4+ activity, but did not significantly affect levels of CA-RNA or CA-DNA. Vaccine recipients with undetectable RV at week 8 had higher frequencies of HIV-specific CD4+ and CD8+ interferon-γ-producing cells (undetectable versus detectable RV: 277 versus 161 CD4+ cells/106 lymphocytes, P=0.03 and 1326 versus 669 CD8+ cells/106 lymphocytes, P=0.04). Pre-ATI CA-RNA and CA-DNA were associated with post-ATI plasma HIV set point (CA-RNA: r = 0.51, P<0.01 and CA-DNA: r = 0.47, P<0.01). Conclusions Vaccine-induced T-cell responses were associated with a modest transient effect on RV, but more potent immune responses and/or combination treatment with latency-reversing agents are needed to reduce the HIV reservoir. HIV reservoir measures may act as biomarkers of post-ATI viral rebound kinetics. PMID:25254301

Impact of alemtuzumab on HIV persistence in an HIV-infected individual on antiretroviral therapy with Sezary syndrome.

Science.gov (United States)

Rasmussen, Thomas A; McMahon, James; Chang, J Judy; Symons, Jori; Roche, Michael; Dantanarayana, Ashanti; Okoye, Afam; Hiener, Bonnie; Palmer, Sarah; Lee, Wen Shi; Kent, Stephen J; Van Der Weyden, Carrie; Prince, H Miles; Cameron, Paul U; Lewin, Sharon R

2017-08-24

To study the effects of alemtuzumab on HIV persistence in an HIV-infected individual on antiretroviral therapy (ART) with Sezary syndrome, a rare malignancy of CD4 T cells. Case report. Blood was collected 30 and 18 months prior to presentation with Sezary syndrome, at the time of presentation and during alemtuzumab. T-cell subsets in malignant (CD7-CD26-TCR-VBeta2+) and nonmalignant cells were quantified by flow cytometry. HIV-DNA in total CD4 T cells, in sorted malignant and nonmalignant CD4 T cells, was quantified by PCR and clonal expansion of HIV-DNA assessed by full-length next-generation sequencing. HIV-hepatitis B virus coinfection was diagnosed and antiretroviral therapy initiated 4 years prior to presentation with Sezary syndrome and primary cutaneous anaplastic large cell lymphoma. The patient received alemtuzumab 10 mg three times per week for 4 weeks but died 6 weeks post alemtuzumab. HIV-DNA was detected in nonmalignant but not in malignant CD4 T cells, consistent with expansion of a noninfected CD4 T-cell clone. Full-length HIV-DNA sequencing demonstrated multiple defective viruses but no identical or expanded sequences. Alemtuzumab extensively depleted T cells, including more than 1 log reduction in total T cells and more than 3 log reduction in CD4 T cells. Finally, alemtuzumab decreased HIV-DNA in CD4 T cells by 57% but HIV-DNA remained detectable at low levels even after depletion of nearly all CD4 T cells. Alemtuzumab extensively depleted multiple T-cell subsets and decreased the frequency of but did not eliminate HIV-infected CD4 T cells. Studying the effects on HIV persistence following immune recovery in HIV-infected individuals who require alemtuzumab for malignancy or in animal studies may provide further insights into novel cure strategies.

Factors Leading to the Loss of Natural Elite Control of HIV-1 Infection.

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Pernas, María; Tarancón-Diez, Laura; Rodríguez-Gallego, Esther; Gómez, Josep; Prado, Julia G; Casado, Concepción; Dominguez-Molina, Beatriz; Olivares, Isabel; Coiras, Maite; León, Agathe; Rodriguez, Carmen; Benito, Jose Miguel; Rallón, Norma; Plana, Montserrat; Martinez-Madrid, Onofre; Dapena, Marta; Iribarren, Jose Antonio; Del Romero, Jorge; García, Felipe; Alcamí, José; Muñoz-Fernández, M Ángeles; Vidal, Francisco; Leal, Manuel; Lopez-Galindez, Cecilio; Ruiz-Mateos, Ezequiel

2017-12-06

HIV-1 elite controllers (EC) maintain undetectable viral load (VL) in the absence of antiretroviral treatment. However, these subjects have heterogeneous clinical outcomes including a proportion loosing HIV-1 control over time. In this work we compared, in a longitudinal design, transient EC, analyzed before and after the loss of virological control, versus persistent EC. The aim was to identify factors leading to the loss of natural virological control of HIV-1-infection with a longitudinal retrospective study design. Gag-specific T-cell response was assessed by in vitro intracellular poly-cytokine production quantified by flow cytometry. Viral diversity and sequence-dating were performed in proviral DNA by PCR amplification at limiting dilution in env and gag genes. The expression profile of 70 serum cytokines and chemokines was assessed by multiplex immunoassays. We identified transient EC as subjects with low Gag-specific T-cell polyfunctionality, high viral diversity and high proinflammatory cytokines levels before the loss of control. Gag-specific T-cell polyfunctionality was inversely associated with viral diversity in transient controllers before the loss of control (r=-0.8; p =0.02). RANTES was a potential biomarker of transient control. This study identified, virological and immunological factors including inflammatory biomarkers associated with two different phenotypes within EC. These results may allow a more accurate definition of EC, which could help in a better clinical management of these individuals and in the development of future curative approaches. IMPORTANCE There is a rare group of HIV-infected patients who have the extraordinary capacity to maintain undetectable viral load levels in the absence of antiretroviral treatment, the so called HIV-1 elite controllers (EC). However, there is a proportion within these subjects that eventually loses this capability. In this work we found differences in virological and immune factors including soluble

Optimization of HIV-1 Envelope DNA Vaccine Candidates within Three Different Animal Models, Guinea Pigs, Rabbits and Cynomolgus Macaques.

Science.gov (United States)

Borggren, Marie; Vinner, Lasse; Andresen, Betina Skovgaard; Grevstad, Berit; Repits, Johanna; Melchers, Mark; Elvang, Tara Laura; Sanders, Rogier W; Martinon, Frédéric; Dereuddre-Bosquet, Nathalie; Bowles, Emma Joanne; Stewart-Jones, Guillaume; Biswas, Priscilla; Scarlatti, Gabriella; Jansson, Marianne; Heyndrickx, Leo; Grand, Roger Le; Fomsgaard, Anders

2013-07-19

HIV-1 DNA vaccines have many advantageous features. Evaluation of HIV-1 vaccine candidates often starts in small animal models before macaque and human trials. Here, we selected and optimized DNA vaccine candidates through systematic testing in rabbits for the induction of broadly neutralizing antibodies (bNAb). We compared three different animal models: guinea pigs, rabbits and cynomolgus macaques. Envelope genes from the prototype isolate HIV-1 Bx08 and two elite neutralizers were included. Codon-optimized genes, encoded secreted gp140 or membrane bound gp150, were modified for expression of stabilized soluble trimer gene products, and delivered individually or mixed. Specific IgG after repeated i.d. inoculations with electroporation confirmed in vivo expression and immunogenicity. Evaluations of rabbits and guinea pigs displayed similar results. The superior DNA construct in rabbits was a trivalent mix of non-modified codon-optimized gp140 envelope genes. Despite NAb responses with some potency and breadth in guinea pigs and rabbits, the DNA vaccinated macaques displayed less bNAb activity. It was concluded that a trivalent mix of non-modified gp140 genes from rationally selected clinical isolates was, in this study, the best option to induce high and broad NAb in the rabbit model, but this optimization does not directly translate into similar responses in cynomolgus macaques.

Optimization of HIV-1 Envelope DNA Vaccine Candidates within Three Different Animal Models, Guinea Pigs, Rabbits and Cynomolgus Macaques

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Roger Le Grand

2013-07-01

Full Text Available HIV-1 DNA vaccines have many advantageous features. Evaluation of HIV-1 vaccine candidates often starts in small animal models before macaque and human trials. Here, we selected and optimized DNA vaccine candidates through systematic testing in rabbits for the induction of broadly neutralizing antibodies (bNAb. We compared three different animal models: guinea pigs, rabbits and cynomolgus macaques. Envelope genes from the prototype isolate HIV-1 Bx08 and two elite neutralizers were included. Codon-optimized genes, encoded secreted gp140 or membrane bound gp150, were modified for expression of stabilized soluble trimer gene products, and delivered individually or mixed. Specific IgG after repeated i.d. inoculations with electroporation confirmed in vivo expression and immunogenicity. Evaluations of rabbits and guinea pigs displayed similar results. The superior DNA construct in rabbits was a trivalent mix of non-modified codon-optimized gp140 envelope genes. Despite NAb responses with some potency and breadth in guinea pigs and rabbits, the DNA vaccinated macaques displayed less bNAb activity. It was concluded that a trivalent mix of non-modified gp140 genes from rationally selected clinical isolates was, in this study, the best option to induce high and broad NAb in the rabbit model, but this optimization does not directly translate into similar responses in cynomolgus macaques.

The Icsbp locus is a common proviral insertion site in mature B-cell lymphomas/plasmacytomas induced by exogenous murine leukemia virus

International Nuclear Information System (INIS)

Ma Shiliang; Sorensen, Annette Balle; Kunder, Sandra; Sorensen, Karina Dalsgaard; Quintanilla-Martinez, Leticia; Morris, David W.; Schmidt, Joerg; Pedersen, Finn Skou

2006-01-01

ICSBP (interferon consensus sequence binding protein)/IRF8 (interferon regulatory factor 8) is an interferon gamma-inducible transcription factor expressed predominantly in hematopoietic cells, and down-regulation of this factor has been observed in chronic myelogenous leukemia and acute myeloid leukemia in man. By screening about 1200 murine leukemia virus (MLV)-induced lymphomas, we found proviral insertions at the Icsbp locus in 14 tumors, 13 of which were mature B-cell lymphomas or plasmacytomas. Only one was a T-cell lymphoma, although such tumors constituted about half of the samples screened. This indicates that the Icsbp locus can play a specific role in the development of mature B-lineage malignancies. Two proviral insertions in the last Icsbp exon were found to act by a poly(A)-insertion mechanism. The remaining insertions were found within or outside Icsbp. Since our results showed expression of Icsbp RNA and protein in all end-stage tumor samples, a simple tumor suppressor function of ICSBP is not likely. Interestingly, proviral insertions at Icsbp have not been reported from previous extensive screenings of mature B-cell lymphomas induced by endogenous MLVs. We propose that ICSBP might be involved in an early modulation of an immune response to exogenous MLVs that might also play a role in proliferation of the mature B-cell lymphomas

LTR real-time PCR for HIV-1 DNA quantitation in blood cells for early diagnosis in infants born to seropositive mothers treated in HAART area (ANRS CO 01).

Science.gov (United States)

Avettand-Fènoël, Véronique; Chaix, Marie-Laure; Blanche, Stéphane; Burgard, Marianne; Floch, Corinne; Toure, Kadidia; Allemon, Marie-Christine; Warszawski, Josiane; Rouzioux, Christine

2009-02-01

HIV-1 diagnosis in babies born to seropositive mothers is one of the challenges of HIV epidemics in children. A simple, rapid protocol was developed for quantifying HIV-1 DNA in whole blood samples and was used in the ANRS French pediatric cohort in conditions of prevention of mother-to-child transmission. A quantitative HIV-1 DNA protocol (LTR real-time PCR) requiring small blood volumes was developed. First, analytical reproducibility was evaluated on 172 samples. Results obtained on blood cell pellets and Ficoll-Hypaque separated mononuclear cells were compared in 48 adult HIV-1 samples. Second, the protocol was applied to HIV-1 diagnosis in infants in parallel with plasma HIV-RNA quantitation. This prospective study was performed in children born between May 2005 and April 2007 included in the ANRS cohort. The assay showed good reproducibility. The 95% detection cut-off value was 6 copies/PCR, that is, 40 copies/10(6) leukocytes. HIV-DNA levels in whole blood were highly correlated with those obtained after Ficoll-Hypaque separation (r = 0.900, P mothers have received HAART. (c) 2008 Wiley-Liss, Inc.

Association of Sicca Syndrome with Proviral Load and Proinflammatory Cytokines in HTLV-1 Infection

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Clara Mônica Lima

2016-01-01

Full Text Available The Sjögren syndrome has been diagnosed in patients with HTLV-1 associated myelopathy and dry mouth and dry eyes are documented in HTLV-1 carriers. However the diagnosis of Sjögren syndrome in these subjects has been contested. In this cross-sectional study, we evaluated the role of immunological factors and proviral load, in sicca syndrome associated with HTLV-1 in patients without myelopathy. Subjects were recruited in the HTLV-1 Clinic, from 2009 to 2011. The proviral load and cytokine levels (IFN-γ, TNF-α, IL-5, and IL-10 were obtained from a database containing the values presented by the subjects at admission in the clinic. Of the 272 participants, 59 (21.7% had sicca syndrome and in all of them anti-Sjögren syndrome related antigen A (SSA and antigen B (SSB were negatives. The production of TNF-α and IFN-γ was higher in the group with sicca syndrome (P<0.05 than in HTLV-1 infected subjects without sicca syndrome. Our data indicates that patients with sicca syndrome associated with HTLV-1 do not have Sjögren syndrome. However the increased production of TNF-α and IFN-γ in this group of patients may contribute to the pathogenesis of sicca syndrome associated with HTLV-1.

Transcriptional regulation of latent feline immunodeficiency virus in peripheral CD4+ T-lymphocytes.

Science.gov (United States)

McDonnel, Samantha J; Sparger, Ellen E; Luciw, Paul A; Murphy, Brian G

2012-05-01

Feline immunodeficiency virus (FIV), the lentivirus of domestic cats responsible for feline AIDS, establishes a latent infection in peripheral blood CD4+ T-cells approximately eight months after experimental inoculation. In this study, cats experimentally infected with the FIV-C strain in the asymptomatic phase demonstrated an estimated viral load of 1 infected cell per approximately 10(3) CD4+ T-cells, with about 1 copy of viral DNA per cell. Approximately 1 in 10 proviral copies was capable of transcription in the asymptomatic phase. The latent FIV proviral promoter was associated with deacetylated, methylated histones, which is consistent with a condensed chromatin structure. In contrast, the transcriptionally active FIV promoter was associated with histone acetylation and demethylation. In addition, RNA polymerase II appeared to be paused on the latent viral promoter, and short promoter-proximal transcripts were detected. Our findings for the FIV promoter in infected cats are similar to results obtained in studies of human immunodeficiency virus (HIV)-1 latent proviruses in cell culture in vitro studies. Thus, the FIV/cat model may offer insights into in vivo mechanisms of HIV latency and provides a unique opportunity to test novel therapeutic interventions aimed at eradicating latent virus.

Dynamic interaction between STLV-1 proviral load and T-cell response during chronic infection and after immunosuppression in non-human primates.

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Sandrine Souquière

Full Text Available We used mandrills (Mandrillus sphinx naturally infected with simian T-cell leukemia virus type 1 (STLV-1 as a model for evaluating the influence of natural STLV-1 infection on the dynamics and evolution of the immune system during chronic infection. Furthermore, in order to evaluate the role of the immune system in controlling the infection during latency, we induced immunosuppression in the infected monkeys. We first showed that the STLV-1 proviral load was higher in males than in females and increased significantly with the duration of infection: mandrills infected for 10-6 years had a significantly higher proviral load than those infected for 2-4 years. Curiously, this observation was associated with a clear reduction in CD4+ T-cell number with age. We also found that the percentage of CD4(+ T cells co-expressing the activation marker HLA-DR and the mean percentage of CD25(+ in CD4(+ and CD8(+ T cells were significantly higher in infected than in uninfected animals. Furthermore, the STLV-1 proviral load correlated positively with T-cell activation but not with the frequency of T cells secreting interferon gamma in response to Tax peptides. Lastly, we showed that, during immunosuppression in infected monkeys, the percentages of CD8(+ T cells expressing HLA-DR(+ and of CD4(+ T cells expressing the proliferation marker Ki67 decreased significantly, although the percentage of CD8(+ T cells expressing HLA-DR(+ and Ki67 increased significantly by the end of treatment. Interestingly, the proviral load increased significantly after immunosuppression in the monkey with the highest load. Our study demonstrates that mandrills naturally infected with STLV-1 could be a suitable model for studying the relations between host and virus. Further studies are needed to determine whether the different compartments of the immune response during infection induce the long latency by controlling viral replication over time. Such studies would provide important

Prime-Boost Vaccination Using Chemokine-Fused gp120 DNA and HIV Envelope Peptides Activates Both Immediate and Long-Term Memory Cellular Responses in Rhesus Macaques

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Hong Qin

2010-01-01

Full Text Available HIV vaccine candidates with improved immunogenicity and induction of mucosal T-cell immunity are needed. A prime-boost strategy using a novel HIV glycoprotein 120 DNA vaccine was employed to immunize rhesus macaques. The DNA vaccine encoded a chimeric gp120 protein in fusion with monocyte chemoattractant protein-3, which was hypothesized to improve the ability of antigen-presenting cells to capture viral antigen through chemokine receptor-mediated endocytosis. DNA vaccination induced virus-reactive T cells in peripheral blood, detectable by T cell proliferation, INFγ ELISPOT and sustained IL-6 production, without humoral responses. With a peptide-cocktail vaccine containing a set of conserved polypeptides of HIV-1 envelope protein, given by nasogastric administration, primed T-cell immunity was significantly boosted. Surprisingly, long-term and peptide-specific mucosal memory T-cell immunity was detected in both vaccinated macaques after one year. Therefore, data from this investigation offer proof-of-principle for potential effectiveness of the prime-boost strategy with a chemokine-fused gp120 DNA and warrant further testing in the nonhuman primate models for developing as a potential HIV vaccine candidate in humans.

CD4+ and CD8+ T cell activation are associated with HIV DNA in resting CD4+ T cells.

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Leslie R Cockerham

Full Text Available The association between the host immune environment and the size of the HIV reservoir during effective antiretroviral therapy is not clear. Progress has also been limited by the lack of a well-accepted assay for quantifying HIV during therapy. We examined the association between multiple measurements of HIV and T cell activation (as defined by markers including CD38, HLA-DR, CCR5 and PD-1 in 30 antiretroviral-treated HIV-infected adults. We found a consistent association between the frequency of CD4+ and CD8+ T cells expressing HLA-DR and the frequency of resting CD4+ T cells containing HIV DNA. This study highlights the need to further examine this relationship and to better characterize the biology of markers commonly used in HIV studies. These results may also have implications for reactivation strategies.

Identification and functional analysis of a second RBF-2 binding site within the HIV-1 promoter

International Nuclear Information System (INIS)

Dahabieh, Matthew S.; Ooms, Marcel; Malcolm, Tom; Simon, Viviana; Sadowski, Ivan

2011-01-01

Transcription from the HIV-1 long terminal repeat (LTR) is mediated by numerous host transcription factors. In this study we characterized an E-box motif (RBE1) within the core promoter that was previously implicated in both transcriptional activation and repression. We show that RBE1 is a binding site for the RBF-2 transcription factor complex (USF1, USF2, and TFII-I), previously shown to bind an upstream viral element, RBE3. The RBE1 and RBE3 elements formed complexes of identical mobility and protein constituents in gel shift assays, both with Jurkat T-cell nuclear extracts and recombinant USF/TFII-I. Furthermore, both elements are regulators of HIV-1 expression; mutations in LTR-luciferase reporters and in HIV-1 molecular clones resulted in decreased transcription, virion production, and proviral expression in infected cells. Collectively, our data indicate that RBE1 is a bona fide RBF-2 binding site and that the RBE1 and RBE3 elements are necessary for mediating proper transcription from the HIV-1 LTR.

Heterologous prime-boost vaccination with DNA and MVA vaccines, expressing HIV-1 subtype C mosaic Gag virus-like particles, is highly immunogenic in mice.

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Ros Chapman

Full Text Available In an effort to make affordable vaccines suitable for the regions most affected by HIV-1, we have constructed stable vaccines that express an HIV-1 subtype C mosaic Gag immunogen (BCG-GagM, MVA-GagM and DNA-GagM. Mosaic immunogens have been designed to address the tremendous diversity of this virus. Here we have shown that GagM buds from cells infected and transfected with MVA-GagM and DNA-GagM respectively and forms virus-like particles. Previously we showed that a BCG-GagM prime MVA-GagM boost generated strong cellular immune responses in mice. In this study immune responses to the DNA-GagM and MVA-GagM vaccines were evaluated in homologous and heterologous prime-boost vaccinations. The DNA homologous prime boost vaccination elicited predominantly CD8+ T cells while the homologous MVA vaccination induced predominantly CD4+ T cells. A heterologous DNA-GagM prime MVA-GagM boost induced strong, more balanced Gag CD8+ and CD4+ T cell responses and that were predominantly of an effector memory phenotype. The immunogenicity of the mosaic Gag (GagM was compared to a naturally occurring subtype C Gag (GagN using a DNA homologous vaccination regimen. DNA-GagN expresses a natural Gag with a sequence that was closest to the consensus sequence of subtype C viruses sampled in South Africa. DNA-GagM homologous vaccination induced cumulative HIV-1 Gag-specific IFN-γ ELISPOT responses that were 6.5-fold higher than those induced by the DNA-GagN vaccination. Similarly, DNA-GagM vaccination generated 7-fold higher levels of cytokine-positive CD8+ T cells than DNA-GagN, indicating that this subtype C mosaic Gag elicits far more potent immune responses than a consensus-type Gag. Cells transfected and infected with DNA-GagM and MVA-GagM respectively, expressed high levels of GagM and produced budding virus-like particles. Our data indicates that a heterologous prime boost regimen using DNA and MVA vaccines expressing HIV-1 subtype C mosaic Gag is highly

Architecture and Assembly of HIV Integrase Multimers in the Absence of DNA Substrates*

Science.gov (United States)

Bojja, Ravi Shankar; Andrake, Mark D.; Merkel, George; Weigand, Steven; Dunbrack, Roland L.; Skalka, Anna Marie

2013-01-01

We have applied small angle x-ray scattering and protein cross-linking coupled with mass spectrometry to determine the architectures of full-length HIV integrase (IN) dimers in solution. By blocking interactions that stabilize either a core-core domain interface or N-terminal domain intermolecular contacts, we show that full-length HIV IN can form two dimer types. One is an expected dimer, characterized by interactions between two catalytic core domains. The other dimer is stabilized by interactions of the N-terminal domain of one monomer with the C-terminal domain and catalytic core domain of the second monomer as well as direct interactions between the two C-terminal domains. This organization is similar to the “reaching dimer” previously described for wild type ASV apoIN and resembles the inner, substrate binding dimer in the crystal structure of the PFV intasome. Results from our small angle x-ray scattering and modeling studies indicate that in the absence of its DNA substrate, the HIV IN tetramer assembles as two stacked reaching dimers that are stabilized by core-core interactions. These models of full-length HIV IN provide new insight into multimer assembly and suggest additional approaches for enzyme inhibition. PMID:23322775

Novel mucosal DNA-MVA HIV vaccination in which DNA-IL-12 plus Cholera Toxin B subunit (CTB) cooperates to enhance cellular systemic and mucosal genital tract immunity

OpenAIRE

Maeto, Cynthia Alejandra; Rodríguez, Ana María; Holgado, María Pía; Falivene, Juliana; Gherardi, Maria Magdalena

2017-01-01

Induction of local antiviral immune responses at the mucosal portal surfaces where HIV-1 and other viral pathogens are usually first encountered remains a primary goal for most vaccines against mucosally acquired viral infections. Exploring mucosal immunization regimes in order to find optimal vector combinations and also appropriate mucosal adjuvants in the HIV vaccine development is decisive. In this study we analyzed the interaction of DNA-IL-12 and cholera toxin B subunit (CTB) after thei...

Optimization of a multi-gene HIV-1 recombinant subtype CRF02AG DNA vaccine for expression of multiple immunogenic forms

International Nuclear Information System (INIS)

Ellenberger, Dennis; Li Bin; Smith, James; Yi Hong; Folks, Thomas; Robinson, Harriet; Butera, Salvatore

2004-01-01

We developed an AIDS vaccine for Western and West-Central Africa based on a DNA plasmid vector expressing HIV-1 recombinant subtype CRF02 A G gag, pol, and env genes. To optimize the production of noninfectious HIV-like particles (VLPs) and potentially improve the effectiveness of the vaccine, we generated four potential vaccine constructs: the parental (IC2) and three modifications (IC25, IC48, and IC90) containing mutations within the HIV protease. While the parental construct IC2 expressed aggregates of Gag proteins, the IC25 construct resulted in the production of immature VLPs (the core comprises unprocessed Pr 55Gag ). The remaining two constructs (IC48 and IC90) produced mature VLPs (the core comprises processed capsid p24) in addition to immature VLPs and aggregates of Gag proteins. VLPs incorporated significant levels of mature gp120 envelope glycoprotein. Importantly, the mature VLPs were fusion competent and entered coreceptor-specific target cells. The production of multiple antigenic forms, including fusion-competent VLPs, by candidate DNA vaccine constructs may provide immunologic advantages for induction of protective cellular and humoral responses against HIV-1 proteins

Structural and dynamic characterization of the upper part of the HIV-1 cTAR DNA hairpin

OpenAIRE

Zargarian, Loussin?; Kanevsky, Igor; Bazzi, Ali; Boynard, Jonathan; Chaminade, Fran?oise; Foss?, Philippe; Mauffret, Olivier

2009-01-01

First strand transfer is essential for HIV-1 reverse transcription. During this step, the TAR RNA hairpin anneals to the cTAR DNA hairpin; this annealing reaction is promoted by the nucleocapsid protein and involves an initial loop?loop interaction between the apical loops of TAR and cTAR. Using NMR and probing methods, we investigated the structural and dynamic properties of the top half of the cTAR DNA (mini-cTAR). We show that the upper stem located between the apical and the internal loop...

Chromosomal and regional localization of the loci for IGKC, IGGC, ALDB, HOXB, GPT, and PRNP in the American mink (Mustela vison): comparisons with human and mouse

DEFF Research Database (Denmark)

Khlebodarova, TM; Malchenko, Sergey; Matveeva, NM

1995-01-01

Chromosomal localization of the genes for gamma- and kappa-immunoglobulins (IGGC and IGKC, respectively), aldolase B (ALDB), prion protein (PRNP), homeo box B (HOXB), and glutamate pyruvate transaminase (GPT) were determined with the use of mink-rodent hybrid cells. Analysis of segregation...

Dendritic cell mediated delivery of plasmid DNA encoding LAMP/HIV-1 Gag fusion immunogen enhances T cell epitope responses in HLA DR4 transgenic mice.

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Gregory G Simon

2010-01-01

Full Text Available This report describes the identification and bioinformatics analysis of HLA-DR4-restricted HIV-1 Gag epitope peptides, and the application of dendritic cell mediated immunization of DNA plasmid constructs. BALB/c (H-2d and HLA-DR4 (DRA1*0101, DRB1*0401 transgenic mice were immunized with immature dendritic cells transfected by a recombinant DNA plasmid encoding the lysosome-associated membrane protein-1/HIV-1 Gag (pLAMP/gag chimera antigen. Three immunization protocols were compared: 1 primary subcutaneous immunization with 1x10(5 immature dendritic cells transfected by electroporation with the pLAMP/gag DNA plasmid, and a second subcutaneous immunization with the naked pLAMP/gag DNA plasmid; 2 primary immunization as above, and a second subcutaneous immunization with a pool of overlapping peptides spanning the HIV-1 Gag sequence; and 3 immunization twice by subcutaneous injection of the pLAMP/gag DNA plasmid. Primary immunization with pLAMP/gag-transfected dendritic cells elicited the greatest number of peptide specific T-cell responses, as measured by ex vivo IFN-gamma ELISpot assay, both in BALB/c and HLA-DR4 transgenic mice. The pLAMP/gag-transfected dendritic cells prime and naked DNA boost immunization protocol also resulted in an increased apparent avidity of peptide in the ELISpot assay. Strikingly, 20 of 25 peptide-specific T-cell responses in the HLA-DR4 transgenic mice contained sequences that corresponded, entirely or partially to 18 of the 19 human HLA-DR4 epitopes listed in the HIV molecular immunology database. Selection of the most conserved epitope peptides as vaccine targets was facilitated by analysis of their representation and variability in all reported sequences. These data provide a model system that demonstrates a the superiority of immunization with dendritic cells transfected with LAMP/gag plasmid DNA, as compared to naked DNA, b the value of HLA transgenic mice as a model system for the identification and evaluation

Use of human papillomavirus DNA, E6/E7 mRNA, and p16 immunocytochemistry to detect and predict anal high-grade squamous intraepithelial lesions in HIV-positive and HIV-negative men who have sex with men.

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Nittaya Phanuphak

Full Text Available Men who have sex with men (MSM are at high risk of having anal cancer. Anal high-grade squamous intraepithelial lesion (HSIL is the precursor of anal cancer. We explored the use of different biomarkers associated with human papillomavirus (HPV infection and HPV-mediated cell transformation to detect and predict HSIL among HIV-positive and HIV-negative MSM.A total of 123 HIV-positive and 123 HIV-negative MSM were enrolled and followed for 12 months. High-resolution anoscopy (HRA with biopsies were performed at every visit along with anal sample collection for cytology, high-risk HPV DNA genotyping, HPV E6/E7 mRNA, and p16 immunocytochemistry. Performance characteristics and area under the receiver operator characteristics curve were calculated for these biomarkers at baseline, and Cox regression compared the usefulness of these biomarkers in predicting incident HSIL. High-risk HPV DNA, E6/E7 mRNA, and p16 immunocytochemistry each identified 43-46% of MSM whose baseline test positivity would trigger HRA referral. E6/E7 mRNA had the highest sensitivity (64.7% and correctly classified the highest number of prevalent HSIL cases. With the exception of p16 immunochemistry, most tests showed significant increases in sensitivity but decreases specificity versus anal cytology, while the overall number of correctly classified cases was not significantly different. Baseline or persistent type 16 and/or 18 HPV DNA was the only test significantly predicting incident histologic HSIL within 12 months in models adjusted for HIV status and low-grade squamous intraepithelial lesions at baseline.Countries with a high HIV prevalence among MSM and limited HRA resources may consider using biomarkers to identify individuals at high risk of HSIL. E6/E7 mRNA had the highest sensitivity for prevalent HSIL detection regardless of HIV status, whereas type 16 and/or 18 HPV DNA performed best in predicting development of incident HSIL within 12 months.

Characterization of partial and near full-length genomes of HIV-1 strains sampled from recently infected individuals in São Paulo, Brazil.

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Sabri Saeed Sanabani

Full Text Available BACKGROUND: Genetic variability is a major feature of human immunodeficiency virus type 1 (HIV-1 and is considered the key factor frustrating efforts to halt the HIV epidemic. A proper understanding of HIV-1 genomic diversity is a fundamental prerequisite for proper epidemiology, genetic diagnosis, and successful drugs and vaccines design. Here, we report on the partial and near full-length genomic (NFLG variability of HIV-1 isolates from a well-characterized cohort of recently infected patients in São Paul, Brazil. METHODOLOGY: HIV-1 proviral DNA was extracted from the peripheral blood mononuclear cells of 113 participants. The NFLG and partial fragments were determined by overlapping nested PCR and direct sequencing. The data were phylogenetically analyzed. RESULTS: Of the 113 samples (90.3% male; median age 31 years; 79.6% homosexual men studied, 77 (68.1% NFLGs and 32 (29.3% partial fragments were successfully subtyped. Of the successfully subtyped sequences, 88 (80.7% were subtype B sequences, 12 (11% BF1 recombinants, 3 (2.8% subtype C sequences, 2 (1.8% BC recombinants and subclade F1 each, 1 (0.9% CRF02 AG, and 1 (0.9% CRF31 BC. Primary drug resistance mutations were observed in 14/101 (13.9% of samples, with 5.9% being resistant to protease inhibitors and nucleoside reverse transcriptase inhibitors (NRTI and 4.9% resistant to non-NRTIs. Predictions of viral tropism were determined for 86 individuals. X4 or X4 dual or mixed-tropic viruses (X4/DM were seen in 26 (30.2% of subjects. The proportion of X4 viruses in homosexuals was detected in 19/69 (27.5%. CONCLUSIONS: Our results confirm the existence of various HIV-1 subtypes circulating in São Paulo, and indicate that subtype B account for the majority of infections. Antiretroviral (ARV drug resistance is relatively common among recently infected patients. The proportion of X4 viruses in homosexuals was significantly higher than the proportion seen in other study populations.

In vivo electroporation enhances the immunogenicity of an HIV-1 DNA vaccine candidate in healthy volunteers.

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Sandhya Vasan

Full Text Available DNA-based vaccines have been safe but weakly immunogenic in humans to date.We sought to determine the safety, tolerability, and immunogenicity of ADVAX, a multigenic HIV-1 DNA vaccine candidate, injected intramuscularly by in vivo electroporation (EP in a Phase-1, double-blind, randomized placebo-controlled trial in healthy volunteers. Eight volunteers each received 0.2 mg, 1 mg, or 4 mg ADVAX or saline placebo via EP, or 4 mg ADVAX via standard intramuscular injection at weeks 0 and 8. A third vaccination was administered to eleven volunteers at week 36. EP was safe, well-tolerated and considered acceptable for a prophylactic vaccine. EP delivery of ADVAX increased the magnitude of HIV-1-specific cell mediated immunity by up to 70-fold over IM injection, as measured by gamma interferon ELISpot. The number of antigens to which the response was detected improved with EP and increasing dosage. Intracellular cytokine staining analysis of ELISpot responders revealed both CD4+ and CD8+ T cell responses, with co-secretion of multiple cytokines.This is the first demonstration in healthy volunteers that EP is safe, tolerable, and effective in improving the magnitude, breadth and durability of cellular immune responses to a DNA vaccine candidate.ClinicalTrials.gov NCT00545987.

Co-expression of HIV-1 virus-like particles and granulocyte-macrophage colony stimulating factor by GEO-D03 DNA vaccine

Science.gov (United States)

Hellerstein, Michael; Xu, Yongxian; Marino, Tracie; Lu, Shan; Yi, Hong; Wright, Elizabeth R.; Robinson, Harriet L.

2012-01-01

Here, we report on GEO-D03, a DNA vaccine that co-expresses non-infectious HIV-1 virus-like particles (VLPs) and the human cytokine, granulocyte-macrophage colony-stimulating factor (GM-CSF). The virus-like particles display the native gp160 form of the HIV-1 Envelope glycoprotein (Env) and are designed to elicit antibody against the natural form of Env on virus and virus-infected cells. The DNA-expressed HIV Gag, Pol and Env proteins also have the potential to elicit virus-specific CD4 and CD8 T cells. The purpose of the co-expressed GM-CSF is to target a cytokine that recruits, expands and differentiates macrophages and dendritic cells to the site of VLP expression. The GEO-D03 DNA vaccine is currently entered into human trials as a prime for a recombinant modified vaccinia Ankara (MVA) boost. In preclinical studies in macaques using an SIV prototype vaccine, this vaccination regimen elicited both anti-viral T cells and antibody, and provided 70% protection against acquisition during 12 weekly rectal exposures with a heterologous SIV. Higher avidity of the Env-specific Ab for the native form of the Env in the challenge virus correlated with lower likelihood of SIV infection. PMID:23111169

Mitochondrial DNA assessment in adipocytes and peripheral blood mononuclear cells of HIV-infected patients with lipodystrophy according to a validated case definition

NARCIS (Netherlands)

Casula, M.; van der Valk, M.; Wit, F. W.; Nievaard, M. A.; Reiss, P.

2007-01-01

BACKGROUND: Several studies have compared mitochondrial DNA (mtDNA) content in tissue from HIV-1-infected patients on highly active antiretroviral therapy with and without evidence of lipodystrophy, the diagnosis of which was based on subjective clinical assessment. OBJECTIVES: The aim of this study

Study of HIV-1 subtypes in serodiscordant couples attending an integrated counselling and testing centre in Mumbai using heteroduplex mobility analysis and DNA sequencing

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Mehta P

2010-01-01

Full Text Available Aims: To determine the prevalent subtypes of HIV-1 in serodiscordant couples. Setting: Integrated Counselling and Testing Centre (ICTC, Department of Microbiology. Study Design: Prospective pilot study. Participants: Thirty HIV-1 serodiscordant couples. Inclusion Criteria: a Documentation of HIV-1 infection in one partner and seronegative status in the other, current history of continued unprotected sexual activity within the partnership, demonstration that they have been in a partnership for at least 1 year and are not currently on highly active antiretroviral therapy HAART; b willingness of both partners to provide written informed consent including consent to continued couple counselling for 3 months. Materials and Methods: HIV-1 subtyping was carried out by heteroduplex mobility analysis (HMA by amplifying env region; and DNA sequencing by amplifying gag region. Results: HIV-1 env gene was amplified successfully in 10/30 samples; gag gene, in 25/30 samples; and both env and gag gene were amplified successfully in 5/30 samples. HIV-1 subtype C was detected from 21 samples; subtype B, from 7; and subtype A, from 2. Sample from 1 positive partner was detected as subtype C by env HMA and subtype B by gag sequencing. Conclusion: HIV-1 subtype C was found to be the predominant subtype of HIV-1 in serodiscordant couples attending our ICTC, followed by HIV-1 subtype B and HIV-1 subtype A, respectively. DNA sequencing was found to be the most reliable method for determining the subtypes of HIV-1.

Lamivudine monotherapy-based cART is efficacious for HBV treatment in HIV/HBV co-infection when baseline HBV DNA<20,000IU/ml

Science.gov (United States)

LI, Yijia; XIE, Jing; HAN, Yang; WANG, Huanling; ZHU, Ting; WANG, Nidan; LV, Wei; GUO, Fuping; QIU, Zhifeng; LI, Yanling; DU, Shanshan; SONG, Xiaojing; THIO, Chloe L; LI, Taisheng

2016-01-01

Background Although combination antiretroviral therapy (cART) including tenofovir (TDF)+lamivudine (3TC) or emtricitabine (FTC) is recommended for treatment of HIV/HBV co-infected patients, TDF is unavailable in some resource-limited areas. Some data suggest that 3TC monotherapy-based cART may be effective in patients with low pre-treatment HBV DNA. Methods Prospective study of 151 Chinese HIV/HBV co-infected subjects of whom 60 received 3TC-based cART and 91 received TDF+3TC-based cART. Factors associated with HBV DNA suppression at 24 and 48 weeks, including anti-HBV drugs, baseline HBV DNA, and baseline CD4 cell count, were evaluated overall and stratified by baseline HBV DNA using Poisson regression with a robust error variance. Results Baseline HBV DNA≥20,000 IU/ml was present in 48.3% and 44.0% of subjects in the 3TC and TDF groups, respectively (P=0.60). After 48 weeks of treatment, HBV DNA suppression rates were similar between these two groups (96.8% vs. 98.0% for 3TC and TDF+3TC, P>0.999) in subjects with baseline HBV DNAHBV DNA ≥20,000 IU/ml, TDF+3TC was associated with higher suppression rates (34.5% vs. 72.5% in 3TC and TDF+3TC groups, respectively, P=0.002). In stratified multivariate regression, TDF use (RR 1.98, P=0.010) and baseline HBV DNA (per 1 log increase in IU/ml, RR 0.74, PHBV DNA suppression only when baseline HBV DNA≥20,000IU/ml. Conclusion This study suggests that 3TC monotherapy-based cART is efficacious for HBV treatment through 48 weeks in HIV/HBV co-infection when baseline HBV DNA<20,000IU/ml. Studies with long-term follow-up are warranted to determine if this finding persists. PMID:26745828

Limited ability of DNA polymerase kappa to suppress benzo[a]pyrene-induced genotoxicity in vivo.

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Masumura, Kenichi; Toyoda-Hokaiwado, Naomi; Niimi, Naoko; Grúz, Petr; Wada, Naoko A; Takeiri, Akira; Jishage, Kou-Ichi; Mishima, Masayuki; Nohmi, Takehiko

2017-12-01

DNA polymerase kappa (Polk) is a specialized DNA polymerase involved in translesion DNA synthesis. To understand the protective roles against genotoxins in vivo, we established inactivated Polk knock-in gpt delta (inactivated Polk KI) mice that possessed reporter genes for mutations and expressed inactive Polk. In this study, we examined genotoxicity of benzo[a]pyrene (BP) to determine whether Polk actually suppressed BP-induced genotoxicity as predicted by biochemistry and in vitro cell culture studies. Seven-week-old inactivated Polk KI and wild-type (WT) mice were treated with BP at doses of 5, 15, or 50 mg/(kg·day) for three consecutive days by intragastric gavage, and mutations in the colon and micronucleus formation in the peripheral blood were examined. Surprisingly, no differences were observed in the frequencies of mutations and micronucleus formation at 5 or 50 mg/kg doses. Inactivated Polk KI mice exhibited approximately two times higher gpt mutant frequency than did WT mice only at the 15 mg/kg dose. The frequency of micronucleus formation was slightly higher in inactivated Polk KI than in WT mice at the same dose, but it was statistically insignificant. The results suggest that Polk has a limited ability to suppress BP-induced genotoxicity in the colon and bone marrow and also that the roles of specialized DNA polymerases in mutagenesis and carcinogenesis should be examined not only by in vitro assays but also by in vivo mouse studies. We also report the spontaneous mutagenesis in inactivated Polk KI mice at young and old ages. Environ. Mol. Mutagen. 58:644-653, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

An effective HIV-1 integrase inhibitor screening platform: Rationality validation of drug screening, conformational mobility and molecular recognition analysis for PFV integrase complex with viral DNA.

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Du, Wenyi; Zuo, Ke; Sun, Xin; Liu, Wei; Yan, Xiao; Liang, Li; Wan, Hua; Chen, Fengzheng; Hu, Jianping

2017-11-01

As an important target for the development of novel anti-AIDS drugs, HIV-1 integrase (IN) has been widely concerned. However, the lack of a complete accurate crystal structure of HIV-1 IN greatly blocks the discovery of novel inhibitors. In this work, an effective HIV-1 IN inhibitor screening platform, namely PFV IN, was filtered from all species of INs. Next, the 40.8% similarity with HIV-1 IN, as well as the high efficiency of virtual screening and the good agreement between calculated binding free energies and experimental ones all proved PFV IN is a promising screening platform for HIV-1 IN inhibitors. Then, the molecular recognition mechanism of PFV IN by its substrate viral DNA and six naphthyridine derivatives (NRDs) inhibitors was investigated through molecular docking, molecular dynamics simulations and water-mediated interactions analyses. The functional partition of NRDs IN inhibitors could be divided into hydrophobic and hydrophilic ones, and the Mg 2+ ions, water molecules and conserved DDE motif residues all interacted with the hydrophilic partition, while the bases in viral DNA and residues like Tyr212, Pro214 interacted with the hydrophobic one. Finally, the free energy landscape (FEL) and cluster analyses were performed to explore the molecular motion of PFV IN-DNA system. It is found that the association with NRDs inhibitors would obviously decrease the motion amplitude of PFV IN-DNA, which may be one of the most potential mechanisms of IN inhibitors. This work will provide a theoretical basis for the inhibitor design based on the structure of HIV-1 IN. Copyright © 2017 Elsevier Inc. All rights reserved.

Characterization and frequency of a newly identified HIV-1 BF1 intersubtype circulating recombinant form in São Paulo, Brazil

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Neto Walter

2010-04-01

Full Text Available Abstract Background HIV circulating recombinant forms (CRFs play an important role in the global and regional HIV epidemics, particularly in regions where multiple subtypes are circulating. To date, several (>40 CRFs are recognized worldwide with five currently circulating in Brazil. Here, we report the characterization of near full-length genome sequences (NFLG of six phylogenetically related HIV-1 BF1 intersubtype recombinants (five from this study and one from other published sequences representing CRF46_BF1. Methods Initially, we selected 36 samples from 888 adult patients residing in São Paulo who had previously been diagnosed as being infected with subclade F1 based on pol subgenomic fragment sequencing. Proviral DNA integrated in peripheral blood mononuclear cells (PBMC was amplified from the purified genomic DNA of all 36-blood samples by five overlapping PCR fragments followed by direct sequencing. Sequence data were obtained from the five fragments that showed identical genomic structure and phylogenetic trees were constructed and compared with previously published sequences. Genuine subclade F1 sequences and any other sequences that exhibited unique mosaic structures were omitted from further analysis Results Of the 36 samples analyzed, only six sequences, inferred from the pol region as subclade F1, displayed BF1 identical mosaic genomes with a single intersubtype breakpoint identified at the nef-U3 overlap (HXB2 position 9347-9365; LTR region. Five of these isolates formed a rigid cluster in phylogentic trees from different subclade F1 fragment regions, which we can now designate as CRF46_BF1. According to our estimate, the new CRF accounts for 0.56% of the HIV-1 circulating strains in São Paulo. Comparison with previously published sequences revealed an additional five isolates that share an identical mosaic structure with those reported in our study. Despite sharing a similar recombinant structure, only one sequence appeared to

Expanded breadth of the T-cell response to mosaic HIV-1 envelope DNA vaccination

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Korber, Bette [Los Alamos National Laboratory; Fischer, William [Los Alamos National Laboratory; Wallstrom, Timothy [Los Alamos National Laboratory

2009-01-01

An effective AIDS vaccine must control highly diverse circulating strains of HIV-1. Among HIV -I gene products, the envelope (Env) protein contains variable as well as conserved regions. In this report, an informatic approach to the design of T-cell vaccines directed to HIV -I Env M group global sequences was tested. Synthetic Env antigens were designed to express mosaics that maximize the inclusion of common potential Tcell epitope (PTE) 9-mers and minimize the inclusion of rare epitopes likely to elicit strain-specific responses. DNA vaccines were evaluated using intracellular cytokine staining (ICS) in inbred mice with a standardized panel of highly conserved 15-mer PTE peptides. I, 2 and 3 mosaic sets were developed that increased theoretical epitope coverage. The breadth and magnitude ofT-cell immunity stimulated by these vaccines were compared to natural strain Env's; additional comparisons were performed on mutant Env's, including gpl60 or gpl45 with or without V regions and gp41 deletions. Among them, the 2 or 3 mosaic Env sets elicited the optimal CD4 and CD8 responses. These responses were most evident in CD8 T cells; the 3 mosaic set elicited responses to an average of 8 peptide pools compared to 2 pools for a set of3 natural Env's. Synthetic mosaic HIV -I antigens can therefore induce T-cell responses with expanded breadth and may facilitate the development of effective T -cell-based HIV -1 vaccines.

Human nucleoporins promote HIV-1 docking at the nuclear pore, nuclear import and integration.

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Francesca Di Nunzio

Full Text Available The nuclear pore complex (NPC mediates nucleo-cytoplasmic transport of macromolecules and is an obligatory point of passage and functional bottleneck in the replication of some viruses. The Human Immunodeficiency Virus (HIV has evolved the required mechanisms for active nuclear import of its genome through the NPC. However the mechanisms by which the NPC allows or even assists HIV translocation are still unknown. We investigated the involvement of four key nucleoporins in HIV-1 docking, translocation, and integration: Nup358/RanBP2, Nup214/CAN, Nup98 and Nup153. Although all induce defects in infectivity when depleted, only Nup153 actually showed any evidence of participating in HIV-1 translocation through the nuclear pore. We show that Nup358/RanBP2 mediates docking of HIV-1 cores on NPC cytoplasmic filaments by interacting with the cores and that the C-terminus of Nup358/RanBP2 comprising a cyclophilin-homology domain contributes to binding. We also show that Nup214/CAN and Nup98 play no role in HIV-1 nuclear import per se: Nup214/CAN plays an indirect role in infectivity read-outs through its effect on mRNA export, while the reduction of expression of Nup98 shows a slight reduction in proviral integration. Our work shows the involvement of nucleoporins in diverse and functionally separable steps of HIV infection and nuclear import.

Structural determinants of HIV-1 nucleocapsid protein for cTAR DNA binding and destabilization, and correlation with inhibition of self-primed DNA synthesis.

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Beltz, Hervé; Clauss, Céline; Piémont, Etienne; Ficheux, Damien; Gorelick, Robert J; Roques, Bernard; Gabus, Caroline; Darlix, Jean-Luc; de Rocquigny, Hugues; Mély, Yves

2005-05-20

The nucleocapsid protein (NC) of human immunodeficiency virus type 1 (HIV-1) is formed of two highly conserved CCHC zinc fingers flanked by small basic domains. NC is required for the two obligatory strand transfers in viral DNA synthesis through its nucleic acid chaperoning properties. The first DNA strand transfer relies on NC's ability to bind and destabilize the secondary structure of complementary transactivation response region (cTAR) DNA, to inhibit self-priming, and to promote the annealing of cTAR to TAR RNA. To further investigate NC chaperone properties, our aim was to identify by fluorescence spectroscopy and gel electrophoresis, the NC structural determinants for cTAR binding and destabilization, and for the inhibition of self-primed DNA synthesis on a model system using a series of NC mutants and HIV-1 reverse transcriptase. NC destabilization and self-priming inhibition properties were found to be supported by the two fingers in their proper context and the basic (29)RAPRKKG(35) linker. The strict requirement of the native proximal finger suggests that its hydrophobic platform (Val13, Phe16, Thr24 and Ala25) is crucial for binding, destabilization and inhibition of self-priming. In contrast, only partial folding of the distal finger is required, probably for presenting the Trp37 residue in an appropriate orientation. Also, Trp37 and the hydrophobic residues of the proximal finger appear to be essential for the propagation of the melting from the cTAR ends up to the middle of the stem. Finally, both N-terminal and C-terminal basic domains contribute to cTAR binding but not to its destabilization.

Large Isoform of Mammalian Relative of DnaJ is a Major Determinant of Human Susceptibility to HIV-1 Infection

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Yu-Ping Chiang

2014-12-01

Full Text Available Individual differences in susceptibility to human immunodeficiency virus type 1 (HIV-1 infection have been of interest for decades. We aimed to determine the contribution of large isoform of Mammalian DnaJ (MRJ-L, a HIV-1 Vpr-interacting cellular protein, to this natural variation. Expression of MRJ-L in monocyte-derived macrophages was significantly higher in HIV-infected individuals (n = 31 than their uninfected counterparts (n = 27 (p = 0.009. Fifty male homosexual subjects (20 of them are HIV-1 positive were further recruited to examine the association between MRJ-L levels and occurrence of HIV infection. Bayesian multiple logistic regression revealed that playing a receptive role and increased levels of MRJ-L in macrophages were two risk factors for HIV-1 infection. A 1% rise in MRJ-L expression was associated with a 1.13 fold (95% CrI 1.06–1.29 increase in odds of contracting HIV-1 infection. Ex vivo experiments revealed that MRJ-L facilitated Vpr-dependent nuclear localization of virus. Infection of macrophage-tropic strain is a critical step in HIV-1 transmission. MRJ-L is a critical factor in this process; hence, subjects with higher macrophage MRJ-L levels are more vulnerable to HIV-1 infection.

Transcriptional Regulation of Latent Feline Immunodeficiency Virus in Peripheral CD4+ T-lymphocytes

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Brian G. Murphy

2012-05-01

Full Text Available Feline immunodeficiency virus (FIV, the lentivirus of domestic cats responsible for feline AIDS, establishes a latent infection in peripheral blood CD4+ T-cells approximately eight months after experimental inoculation. In this study, cats experimentally infected with the FIV-C strain in the asymptomatic phase demonstrated an estimated viral load of 1 infected cell per approximately 10³ CD4+ T-cells, with about 1 copy of viral DNA per cell. Approximately 1 in 10 proviral copies was capable of transcription in the asymptomatic phase. The latent FIV proviral promoter was associated with deacetylated, methylated histones, which is consistent with a condensed chromatin structure. In contrast, the transcriptionally active FIV promoter was associated with histone acetylation and demethylation. In addition, RNA polymerase II appeared to be paused on the latent viral promoter, and short promoter-proximal transcripts were detected. Our findings for the FIV promoter in infected cats are similar to results obtained in studies of human immunodeficiency virus (HIV-1 latent proviruses in cell culture in vitro studies. Thus, the FIV/cat model may offer insights into in vivo mechanisms of HIV latency and provides a unique opportunity to test novel therapeutic interventions aimed at eradicating latent virus.

Absence of A3Z3-Related Hypermutations in the env and vif Proviral Genes in FIV Naturally Infected Cats

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Lucía Cano-Ortiz

2018-05-01

Full Text Available Apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3 (APOBEC3; A3 proteins comprise an important family of restriction factors that produce hypermutations on proviral DNA and are able to limit virus replication. Vif, an accessory protein present in almost all lentiviruses, counteracts the antiviral A3 activity. Seven haplotypes of APOBEC3Z3 (A3Z3 were described in domestic cats (hap I–VII, and in-vitro studies have demonstrated that these proteins reduce infectivity of vif-defective feline immunodeficiency virus (FIV. Moreover, hap V is resistant to vif-mediated degradation. However, studies on the effect of A3Z3 in FIV-infected cats have not been developed. Here, the correlation between APOBEC A3Z3 haplotypes in domestic cats and the frequency of hypermutations in the FIV vif and env genes were assessed in a retrospective cohort study with 30 blood samples collected between 2012 and 2016 from naturally FIV-infected cats in Brazil. The vif and env sequences were analyzed and displayed low or undetectable levels of hypermutations, and could not be associated with any specific A3Z3 haplotype.

Microbial translocation is correlated with HIV evolution in HIV-HCV co-infected patients.

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Jean-Jacques Tudesq

Full Text Available Microbial translocation (MT is characterized by bacterial products passing into the blood through the gut barrier and is a key phenomenon in the pathophysiology of Human Immunodeficiency Virus (HIV infection. MT is also associated with liver damage in Hepatitis C Virus (HCV patients. The aim of the study was to assess MT in plasma of HIV-HCV co-infected patients. 16S rDNA (16 S Ribosomal DNA subunit marker and other markers of MT such as Lipopolysaccharide (LPS-binding protein (LBP, soluble CD14 (sCD14, intestinal fatty acid binding protein (I-FABP were used. Clinical, biological and immunological characteristics of the population were studied in order to correlate them with the intensity of the MT. We demonstrate that indirect markers of MT, LBP and CD14s, and a marker of intestinal permeability (I-FABP are significantly higher in HIV-HCV co-infected patients than in healthy controls (17.0 vs 2.6 μg/mL, p < 0.001; 1901.7 vs 1255.0 ng/mL, p = 0.018; 478.3 vs 248.1 pg/mL, p < 0.001, respectively, while a direct marker of MT (16S rDNA copies is not different between these two populations. However, plasma 16S rDNA was significantly higher in co-infected patients with long-standing HIV infections (RGM = 1.47 per 10 years, CI95% = [1.04:2.06], p = 0.03. Our findings show that in HIV-HCV co-infected patients, plasma 16S rDNA levels, directly reflecting MT, seem to be linked to the duration of HIV infection, while elevated levels of LBP and sCD14 reflect only a persistence of immune activation. The levels of these markers were not correlated with HCV evolution.

Are dinucleoside monophosphates relevant models for the study of DNA intrastrand cross-link lesions? The example of g[8-5m]T.

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Garrec, Julian; Dumont, Elise

2014-07-21

Oxidatively generated tandem lesions such as G[8-5m]T pose a potent threat to genome integrity. Direct experimental studies of the kinetics and thermodynamics of a specific lesion within DNA are very challenging, mostly due to the variety of products that can be formed in oxidative conditions. Dinucleoside monophosphates (DM) involving only the reactive nucleobases in water represent appealing alternative models on which most physical chemistry and structural techniques can be applied. However, it is not yet clear how relevant these models are. Here, we present QM/MM MD simulations of the cyclization step involved in the formation of G[8-5m]T from the guanine-thymine (GpT) DM in water, with the aim of comparing our results to our previous investigation of the same reaction in DNA ( Garrec , J. , Patel , C. , Rothlisberger , U. , and Dumont , E. ( 2012 ) J. Am. Chem. Soc. 134 , 2111 - 2119 ). We show that, despite the different levels of preorganization of the two systems, the corresponding reactions share many energetic and structural characteristics. The main difference lies in the angle between the G and T bases, which is slightly higher in the transition state (TS) and product of the reaction in water than in the reaction in DNA. This effect is due to the Watson-Crick H-bonds, which are absent in the {GpT+water} system and restrain the relative positioning of the reactive nucleobases in DNA. However, since the lesion is accommodated easily in the DNA macromolecule, the induced energetic penalty is relatively small. The high similarity between the two reactions strongly supports the use of GpT in water as a model of the corresponding reaction in DNA.

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

CRISPR/Cas9 Inhibits Multiple Steps of HIV-1 Infection.

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Yin, Lijuan; Hu, Siqi; Mei, Shan; Sun, Hong; Xu, Fengwen; Li, Jian; Zhu, Weijun; Liu, Xiaoman; Zhao, Fei; Zhang, Di; Cen, Shan; Liang, Chen; Guo, Fei

2018-05-09

CRISPR/Cas9 is an adaptive immune system where bacteria and archaea have evolved to resist the invading viruses and plasmid DNA by creating site-specific double-strand breaks in DNA. This study tested this gene editing system in inhibiting human immunodeficiency virus type 1 (HIV-1) infection by targeting the viral long terminal repeat and the gene coding sequences. Strong inhibition of HIV-1 infection by Cas9/gRNA was observed, which resulted not only from insertions and deletions (indels) that were introduced into viral DNA due to Cas9 cleavage, but also from the marked decrease in the levels of the late viral DNA products and the integrated viral DNA. This latter defect might have reflected the degradation of viral DNA that has not been immediately repaired after Cas9 cleavage. It was further observed that Cas9, when solely located in the cytoplasm, inhibits HIV-1 as strongly as the nuclear Cas9, except that the cytoplasmic Cas9 does not act on the integrated HIV-1 DNA and thus cannot be used to excise the latent provirus. Together, the results suggest that Cas9/gRNA is able to target and edit HIV-1 DNA both in the cytoplasm and in the nucleus. The inhibitory effect of Cas9 on HIV-1 is attributed to both the indels in viral DNA and the reduction in the levels of viral DNA.

Loss of long term protection with the inclusion of HIV pol to a DNA vaccine encoding gag.

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Garrod, Tamsin J; Gargett, Tessa; Yu, Wenbo; Major, Lee; Burrell, Christopher J; Wesselingh, Steven; Suhrbier, Andreas; Grubor-Bauk, Branka; Gowans, Eric J

2014-11-04

Traditional vaccine strategies that induce antibody responses have failed to protect against HIV infection in clinical trials, and thus cell-mediated immunity is now an additional criterion. Recent clinical trials that aimed to induce strong T cell responses failed to do so. Therefore, to enhance induction of protective T cell responses, it is crucial that the optimum antigen combination is chosen. Limited research has been performed into the number of antigens selected for an HIV vaccine. This study aimed to compare DNA vaccines encoding either a single HIV antigen or a combination of two antigens, using intradermal vaccination of C57BL/6 mice. Immune assays were performed on splenocytes, and in vivo protection was examined by challenge with a chimeric virus, EcoHIV, able to infect mouse but not human leukocytes, at 10 days (short term) and 60 days (long term) post final vaccination. At 60 days there was significantly lower frequency of induced antigen-specific CD8(+) T cells in the spleens of pCMVgag-pol-vaccinated mice compared with mice which received pCMVgag only. Most importantly, short term viral control of EcoHIV was similar for pCMVgag and pCMVgag-pol-vaccinated mice at day 10, but only the pCMVgag-vaccinated significantly controlled EcoHIV at day 60 compared with pCMV-vaccinated mice, showing that control was reduced with the inclusion of the HIV pol gene. Copyright © 2014 Elsevier B.V. All rights reserved.

Modulation of microtubule assembly by the HIV-1 Tat protein is strongly dependent on zinc binding to Tat

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Muller Sylviane

2008-07-01

Full Text Available Abstract Background During HIV-1 infection, the Tat protein plays a key role by transactivating the transcription of the HIV-1 proviral DNA. In addition, Tat induces apoptosis of non-infected T lymphocytes, leading to a massive loss of immune competence. This apoptosis is notably mediated by the interaction of Tat with microtubules, which are dynamic components essential for cell structure and division. Tat binds two Zn2+ ions through its conserved cysteine-rich region in vitro, but the role of zinc in the structure and properties of Tat is still controversial. Results To investigate the role of zinc, we first characterized Tat apo- and holo-forms by fluorescence correlation spectroscopy and time-resolved fluorescence spectroscopy. Both of the Tat forms are monomeric and poorly folded but differ by local conformational changes in the vicinity of the cysteine-rich region. The interaction of the two Tat forms with tubulin dimers and microtubules was monitored by analytical ultracentrifugation, turbidity measurements and electron microscopy. At 20°C, both of the Tat forms bind tubulin dimers, but only the holo-Tat was found to form discrete complexes. At 37°C, both forms promoted the nucleation and increased the elongation rates of tubulin assembly. However, only the holo-Tat increased the amount of microtubules, decreased the tubulin critical concentration, and stabilized the microtubules. In contrast, apo-Tat induced a large amount of tubulin aggregates. Conclusion Our data suggest that holo-Tat corresponds to the active form, responsible for the Tat-mediated apoptosis.

DNA vaccines expressing soluble CD4-envelope proteins fused to C3d elicit cross-reactive neutralizing antibodies to HIV-1

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Bower, Joseph F.; Green, Thomas D.; Ross, Ted M.

2004-01-01

DNA vaccines expressing the envelope (Env) of the human immunodeficiency virus type 1 (HIV-1) have been relatively ineffective at generating high-titer, long-lasting, neutralizing antibodies in a variety of animal models. In this study, DNA vaccines were constructed to express a fusion protein of the soluble human CD4 (sCD4) and the gp120 subunit of the HIV-1 envelope. To enhance the immunogenicity of the expressed fusion protein, three copies of the murine C3d (mC3d 3 ) were added to the carboxyl terminus of the complex. Monoclonal antibodies that recognize CD4-induced epitopes on gp120 efficiently bound to sCD4-gp120 or sCD4-gp120-mC3d 3 . In addition, both sCD4-gp120 and sCD4-gp120-mC3d 3 bound to cells expressing appropriate coreceptors in the absence of cell surface hCD4. Mice (BALB/c) vaccinated with DNA vaccines expressing either gp120-mC3d 3 or sCD4-gp120-mC3d 3 elicited antibodies that neutralized homologous virus infection. However, the use of sCD4-gp120-mC3d 3 -DNA elicited the highest titers of neutralizing antibodies that persisted after depletion of anti-hCD4 antibodies. Interestingly, only mice vaccinated with DNA expressing sCD4-gp120-mC3d 3 had antibodies that elicited cross-protective neutralizing antibodies. The fusion of sCD4 to the HIV-1 envelope exposes neutralizing epitopes that elicit broad protective immunity when the fusion complex is coupled with the molecular adjuvant, C3d

Multimeric scaffolds displaying the HIV-1 envelope MPER induce MPER-specific antibodies and cross-neutralizing antibodies when co-immunized with gp160 DNA.

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Shelly J Krebs

Full Text Available Developing a vaccine that overcomes the diversity of HIV-1 is likely to require a strategy that directs antibody (Ab responses toward conserved regions of the viral Envelope (Env. However, the generation of neutralizing Abs (NAbs targeting these regions through vaccination has proven to be difficult. One conserved region of particular interest is the membrane proximal external region (MPER of Env located within the gp41 ectodomain. In order to direct the immune response to this region, the MPER and gp41 ectodomain were expressed separately as N-terminal fusions to the E2 protein of Geobacillus stearothermophilus. The E2 protein acts as a scaffold by self-assembling into 60-mer particles, displaying up to 60 copies of the fused target on the surface. Rabbits were immunized with E2 particles displaying MPER and/or the gp41 ectodomain in conjunction with DNA encoding full-length gp160. Only vaccines including E2 particles displaying MPER elicited MPER-specific Ab responses. NAbs were elicited after two immunizations that largely targeted the V3 loop. To overcome V3 immunodominance in the DNA component, E2 particles displaying MPER were used in conjunction with gp160 DNA lacking hypervariable regions V2, V3, or combined V1V2V3. All rabbits had HIV binding Ab responses and NAbs following the second vaccination. Using HIV-2/HIV-1 MPER chimeric viruses as targets, NAbs were detected in 12/16 rabbits after three immunizations. Low levels of NAbs specific for Tier 1 and 2 viruses were observed in all groups. This study provides evidence that co-immunizing E2 particles displaying MPER and gp160 DNA can focus Ab responses toward conserved regions of Env.

Survey of feline leukemia virus and feline coronaviruses in captive neotropical wild felids from Southern Brazil.

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Guimaraes, Ana M S; Brandão, Paulo E; de Moraes, Wanderlei; Cubas, Zalmir S; Santos, Leonilda C; Villarreal, Laura Y B; Robes, Rogério R; Coelho, Fabiana M; Resende, Mauricio; Santos, Renata C F; Oliveira, Rosangela C; Yamaguti, Mauricio; Marques, Lucas M; Neto, Renata L; Buzinhani, Melissa; Marques, Regina; Messick, Joanne B; Biondo, Alexander W; Timenetsky, Jorge

2009-06-01

A total of 57 captive neotropical felids (one Leopardus geoffroyi, 14 Leopardus pardalis, 17 Leopardus wiedii, 22 Leopardus tigrinus, and three Puma yagouaroundi) from the Itaipu Binacional Wildlife Research Center (Refúgio Bela Vista, Southern Brazil) were anesthetized for blood collection. Feces samples were available for 44 animals, including one L. geoffroyi, eight L. pardalis, 14 L. wiedii, 20 L. tigrinus, and one P. yagouaroundi. Total DNA and RNA were extracted from blood and feces, respectively, using commercial kits. Blood DNA samples were evaluated by polymerase chain reaction (PCR) for feline leukemia virus (FeLV) proviral DNA, whereas reverse transcriptase-PCR was run on fecal samples for detection of coronavirus RNA. None of the samples were positive for coronaviruses. A male L. pardalis and a female L. tigrinus were positive for FeLV proviral DNA, and identities of PCR products were confirmed by sequencing. This is the first evidence of FeLV proviral DNA in these species in Southern Brazil.

HIV genetic information and clonal growth

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Based on an analysis of blood cells from five HIV-infected individuals, NCI researchers have identified more than 2,400 HIV DNA insertion sites. Analysis of these sites showed that there is extensive clonal expansion (growth) of HIV infected cells.

Therapeutic DNA vaccination of vertically HIV-infected children: report of the first pediatric randomised trial (PEDVAC).

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Palma, Paolo; Romiti, Maria Luisa; Montesano, Carla; Santilli, Veronica; Mora, Nadia; Aquilani, Angela; Dispinseri, Stefania; Tchidjou, Hyppolite K; Montano, Marco; Eriksson, Lars E; Baldassari, Stefania; Bernardi, Stefania; Scarlatti, Gabriella; Wahren, Britta; Rossi, Paolo

2013-01-01

Twenty vertically HIV-infected children, 6-16 years of age, with stable viral load control and CD4+ values above 400 cells/mm(3). Ten subjects continued their ongoing antiretroviral treatment (ART, Group A) and 10 were immunized with a HIV-DNA vaccine in addition to their previous therapy (ART and vaccine, Group B). The genetic vaccine represented HIV-1 subtypes A, B and C, encoded Env, Rev, Gag and RT and had no additional adjuvant. Immunizations took place at weeks 0, 4 and 12, with a boosting dose at week 36. Monitoring was performed until week 60 and extended to week 96. Safety data showed good tolerance of the vaccine. Adherence to ART remained high and persistent during the study and did not differ significantly between controls and vaccinees. Neither group experienced either virological failure or a decline of CD4+ counts from baseline. Higher HIV-specific cellular immune responses were noted transiently to Gag but not to other components of the vaccine. Lymphoproliferative responses to a virion antigen HIV-1 MN were higher in the vaccinees than in the controls (p = 0.047), whereas differences in reactivity to clade-specific Gag p24, RT or Env did not reach significance. Compared to baseline, the percentage of HIV-specific CD8+ lymphocytes releasing perforin in the Group B was higher after the vaccination schedule had been completed (p = 0.031). No increased CD8+ perforin levels were observed in control Group A. The present study demonstrates the feasibility, safety and moderate immunogenicity of genetic vaccination in vertically HIV-infected children, paving the way for amplified immunotherapeutic approaches in the pediatric population. clinicaltrialsregister.eu _2007-002359-18IT.

Therapeutic DNA vaccination of vertically HIV-infected children: report of the first pediatric randomised trial (PEDVAC.

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Paolo Palma

Full Text Available SUBJECTS: Twenty vertically HIV-infected children, 6-16 years of age, with stable viral load control and CD4+ values above 400 cells/mm(3. INTERVENTION: Ten subjects continued their ongoing antiretroviral treatment (ART, Group A and 10 were immunized with a HIV-DNA vaccine in addition to their previous therapy (ART and vaccine, Group B. The genetic vaccine represented HIV-1 subtypes A, B and C, encoded Env, Rev, Gag and RT and had no additional adjuvant. Immunizations took place at weeks 0, 4 and 12, with a boosting dose at week 36. Monitoring was performed until week 60 and extended to week 96. RESULTS: Safety data showed good tolerance of the vaccine. Adherence to ART remained high and persistent during the study and did not differ significantly between controls and vaccinees. Neither group experienced either virological failure or a decline of CD4+ counts from baseline. Higher HIV-specific cellular immune responses were noted transiently to Gag but not to other components of the vaccine. Lymphoproliferative responses to a virion antigen HIV-1 MN were higher in the vaccinees than in the controls (p = 0.047, whereas differences in reactivity to clade-specific Gag p24, RT or Env did not reach significance. Compared to baseline, the percentage of HIV-specific CD8+ lymphocytes releasing perforin in the Group B was higher after the vaccination schedule had been completed (p = 0.031. No increased CD8+ perforin levels were observed in control Group A. CONCLUSIONS: The present study demonstrates the feasibility, safety and moderate immunogenicity of genetic vaccination in vertically HIV-infected children, paving the way for amplified immunotherapeutic approaches in the pediatric population. TRIAL REGISTRATION: clinicaltrialsregister.eu _2007-002359-18IT.

Construction and characterisation of a full-length infectious molecular clone from a fast replicating, X4-tropic HIV-1 CRF02.AG primary isolate

International Nuclear Information System (INIS)

Tebit, Denis M.; Zekeng, Leopold; Kaptue, Lazare; Kraeusslich, Hans-Georg; Herchenroeder, Ottmar

2003-01-01

Based on our previous analysis of HIV-1 isolates from Cameroon, we constructed a full-length infectious molecular clone from a primary isolate belonging to the CRF02.AG group of recombinant viruses which dominate the HIV-epidemic in West and Central Africa. The virus derived by transfection of the proviral clone pBD6-15 replicated with similar efficiency compared to its parental isolate and used CXCR4 as coreceptor as well. Furthermore, HIV-1 BD6-15 exhibited similar replication properties and virus yield as the reference B-type HIV-1 strain NL4-3. Sequence analysis revealed open reading frames for all structural and accessory genes apart from vpr. Phylogenetic and bootscanning analyses confirmed that BD6-15 clusters with CRF02.AG recombinant strains from West and Central Africa with similar cross-over points as described for the CRF02.AG prototype strain lbNG. Thus, pBD6-15 represents the first non-subtype B infectious molecular clone of a fast replicating, high producer, X4-tropic primary HIV-1 isolate, which had only been briefly passaged in primary cells

During Stably Suppressive Antiretroviral Therapy Integrated HIV-1 DNA Load in Peripheral Blood is Associated with the Frequency of CD8 Cells Expressing HLA-DR/DP/DQ

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Alessandra Ruggiero

2015-09-01

Conclusions: The observed positive association between integrated HIV-1 DNA load and frequency of CD8+DR/DP/DQ+ cells indicates that a close correlation between HIV persistence and immune activation continues during consistently suppressive therapy. The inducers of the distinct activation profile warrant further investigation.

Prediction of HIV-1 coreceptor usage (tropism) by sequence analysis using a genotypic approach.

Science.gov (United States)

Sierra, Saleta; Kaiser, Rolf; Lübke, Nadine; Thielen, Alexander; Schuelter, Eugen; Heger, Eva; Däumer, Martin; Reuter, Stefan; Esser, Stefan; Fätkenheuer, Gerd; Pfister, Herbert; Oette, Mark; Lengauer, Thomas

2011-12-01

this video: Collection of a blood sample Isolation of the HIV RNA from the plasma and/or HIV proviral DNA from blood mononuclear cells Amplification of the env region Amplification of the V3 region Sequence reaction of the V3 amplicon Purification of the sequencing samples Sequencing the purified samples Sequence editing Sequencing data interpretation and tropism prediction.

Potential for false positive HIV test results with the serial rapid HIV testing algorithm.

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Baveewo, Steven; Kamya, Moses R; Mayanja-Kizza, Harriet; Fatch, Robin; Bangsberg, David R; Coates, Thomas; Hahn, Judith A; Wanyenze, Rhoda K

2012-03-19

Rapid HIV tests provide same-day results and are widely used in HIV testing programs in areas with limited personnel and laboratory infrastructure. The Uganda Ministry of Health currently recommends the serial rapid testing algorithm with Determine, STAT-PAK, and Uni-Gold for diagnosis of HIV infection. Using this algorithm, individuals who test positive on Determine, negative to STAT-PAK and positive to Uni-Gold are reported as HIV positive. We conducted further testing on this subgroup of samples using qualitative DNA PCR to assess the potential for false positive tests in this situation. Of the 3388 individuals who were tested, 984 were HIV positive on two consecutive tests, and 29 were considered positive by a tiebreaker (positive on Determine, negative on STAT-PAK, and positive on Uni-Gold). However, when the 29 samples were further tested using qualitative DNA PCR, 14 (48.2%) were HIV negative. Although this study was not primarily designed to assess the validity of rapid HIV tests and thus only a subset of the samples were retested, the findings show a potential for false positive HIV results in the subset of individuals who test positive when a tiebreaker test is used in serial testing. These findings highlight a need for confirmatory testing for this category of individuals.

The cytosolic exonuclease TREX1 inhibits the innate immune response to HIV-1

Science.gov (United States)

Yan, Nan; Regalado-Magdos, Ashton D.; Stiggelbout, Bart; Lee-Kirsch, Min Ae; Lieberman, Judy

2010-01-01

Viral infection triggers innate immune sensors to produce type I interferons (IFN). However, HIV infection of T cells and macrophages does not trip these alarms. How HIV avoids activating nucleic acid sensors is unknown. The cytosolic exonuclease TREX1 suppressed IFN triggered by HIV. In Trex1−/− mouse cells and human CD4+ T cells and macrophages in which TREX1 was inhibited by RNA interference, cytosolic HIV DNA accumulated, and HIV infection induced type I IFN that inhibited HIV replication and spreading. TREX1 bound to cytosolic HIV DNA and digested excess HIV DNA that would otherwise activate IFN expression via a TBK1, STING and IRF3 dependent pathway. HIV-stimulated IFN production in cells deficient in TREX1 did not involve known nucleic acid sensors. PMID:20871604

The Need for Cervical Cancer Control in HIV-Positive and HIV-Negative Women from Romania by Primary Prevention and by Early Detection Using Clinically Validated HPV/DNA Tests.

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Ramona Gabriela Ursu

Full Text Available In Romania, a country with no organized national surveillance program regarding cervical cancer, the early diagnosis of HPV (Human Papilloma Virus infections is a major requirement, especially in HIV-infected women. The objective of this study was to determine the HPV prevalence and type distribution in young HIV-positive women and to assess the difference in the risk factors for developing cervical cancer compared to those of HIV-negative women.We conducted one cross-sectional cohort study from June 2013-September 2014, including 1,032 women: 992 HIV- women who were 36.5 years old (limits: 17 ÷ 84 and 40 HIV + women who were 22.9 years old (limits: 17 ÷ 30 with iatrogenic HIV infected. We detected HPV types with the Linear Array HPV Genotyping test (Roche, Romania.DNA/HPV was detected in 18/40 (45% of the HIV+ patients and in 350/992 (35.2% of the HIV- patients (OR = 1.5, 95%CI 0.76÷2.96. After age adjustment, the overall HPV prevalence was 51.6% in HIV+ versus 63.2% in HIV- women aged under 25, and 22.2% in HPV+ versus 47.2% in HIV- women aged 25-34. We detect HIV being a risk factor for acquiring multiple HPV type infections (OR = 2.30, 95% CI 0.88÷5.97. The eight most common HPV types (high-risk, and low-risk for women below age 30, HIV+ / - were: HPV 16, 18, 31, 51, 58, 68, and 6 and 82 respectively. To assess the risk factors of HIV-positive women for acquiring HPV infection, we analyzed the CD4/μL, ARN/HIV copies/μL, the age group, the number of sexual partners, smoking, and the type of HPV infection (single versus multiple infections. We found that the number of sexual partners and smoking are statistically significant risk factors.Even though there are no significant differences regarding the prevalence of HPV infection in HIV + versus HIV - patients, multiple infections were more frequent in the first group. In our study group young HIV-infected patients under HAART therapy, high number of sexual partners (more than 3 and smoking

Elucidating the Burden of HIV in Tissues Using Multiplexed Immunofluorescence and In Situ Hybridization: Methods for the Single-Cell Phenotypic Characterization of Cells Harboring HIV In Situ.

Science.gov (United States)

Vasquez, Joshua J; Hussien, Rajaa; Aguilar-Rodriguez, Brandon; Junger, Henrik; Dobi, Dejan; Henrich, Timothy J; Thanh, Cassandra; Gibson, Erica; Hogan, Louise E; McCune, Joseph; Hunt, Peter W; Stoddart, Cheryl A; Laszik, Zoltan G

2018-02-01

Persistent tissue reservoirs of HIV present a major barrier to cure. Defining subsets of infected cells in tissues is a major focus of HIV cure research. Herein, we describe a novel multiplexed in situ hybridization (ISH) (RNAscope) protocol to detect HIV-DNA (vDNA) and HIV-RNA (vRNA) in formalin-fixed paraffin-embedded (FFPE) human tissues in combination with immunofluorescence (IF) phenotyping of the infected cells. We show that multiplexed IF and ISH (mIFISH) is suitable for quantitative assessment of HIV vRNA and vDNA and that multiparameter IF phenotyping allows precise identification of the cellular source of the ISH signal. We also provide semi-quantitative data on the impact of various tissue fixatives on the detectability of vDNA and vRNA with RNAscope technology. Finally, we describe methods to quantitate the ISH signal on whole-slide digital images and validation of the quantitative ISH data with quantitative real-time PCR for vRNA. It is our hope that this approach will provide insight into the biology of HIV tissue reservoirs and to inform strategies aimed at curing HIV.

Subtype C gp140 Vaccine Boosts Immune Responses Primed by the South African AIDS Vaccine Initiative DNA-C2 and MVA-C HIV Vaccines after More than a 2-Year Gap.

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Gray, Glenda E; Mayer, Kenneth H; Elizaga, Marnie L; Bekker, Linda-Gail; Allen, Mary; Morris, Lynn; Montefiori, David; De Rosa, Stephen C; Sato, Alicia; Gu, Niya; Tomaras, Georgia D; Tucker, Timothy; Barnett, Susan W; Mkhize, Nonhlanhla N; Shen, Xiaoying; Downing, Katrina; Williamson, Carolyn; Pensiero, Michael; Corey, Lawrence; Williamson, Anna-Lise

2016-06-01

A phase I safety and immunogenicity study investigated South African AIDS Vaccine Initiative (SAAVI) HIV-1 subtype C (HIV-1C) DNA vaccine encoding Gag-RT-Tat-Nef and gp150, boosted with modified vaccinia Ankara (MVA) expressing matched antigens. Following the finding of partial protective efficacy in the RV144 HIV vaccine efficacy trial, a protein boost with HIV-1 subtype C V2-deleted gp140 with MF59 was added to the regimen. A total of 48 participants (12 U.S. participants and 36 Republic of South Africa [RSA] participants) were randomized to receive 3 intramuscular (i.m.) doses of SAAVI DNA-C2 of 4 mg (months 0, 1, and 2) and 2 i.m. doses of SAAVI MVA-C of 1.45 × 10(9) PFU (months 4 and 5) (n = 40) or of a placebo (n = 8). Approximately 2 years after vaccination, 27 participants were rerandomized to receive gp140/MF59 at 100 μg or placebo, as 2 i.m. injections, 3 months apart. The vaccine regimen was safe and well tolerated. After the DNA-MVA regimen, CD4(+) T-cell and CD8(+) T-cell responses occurred in 74% and 32% of the participants, respectively. The protein boost increased CD4(+) T-cell responses to 87% of the subjects. All participants developed tier 1 HIV-1C neutralizing antibody responses as well as durable Env binding antibodies that recognized linear V3 and C5 peptides. The HIV-1 subtype C DNA-MVA vaccine regimen showed promising cellular immunogenicity. Boosting with gp140/MF59 enhanced levels of binding and neutralizing antibodies as well as CD4(+) T-cell responses to HIV-1 envelope. (This study has been registered at ClinicalTrials.gov under registration no. NCT00574600 and NCT01423825.). Copyright © 2016 Gray et al.

Torque Teno Virus in HIV-infected transgender in Surakarta, Indonesia

Science.gov (United States)

Hartono; Agung Prasetyo, Afiono; Fanani, Mohammad

2018-05-01

Torque Teno Virus (TTV) is a circular single-stranded DNA virus that may co-infected with human immunodeficiency virus (HIV), especially in the high-risk community e.g. the transgender performing high-riskbehavior. TTV shows an increased viremia in HIV patients and maybe influence the HIV clinical progression. Blood samples collected from transgender performing high-riskbehavior in Surakarta were tested by serological and molecular assays to detect the presence of HIV infection. The blood samples with HIV positive status were then tested by a nested polymerase chain reaction (PCR) to detect the presentation of TTV DNA. The amplified PCR products were molecularly cloned and subjected to sequence analysis. TTV DNA was detected in 40.0% HIV-positive samples. The molecular characterization revealed that the most prevalent was genogroup 3, followed by genogroup 2 and 1, respectively. TTV was detected in HIV-infected transgender performing high-riskbehavior in Surakarta with high infection rate.

Interferon-alpha mediates restriction of human immunodeficiency virus type-1 replication in primary human macrophages at an early stage of replication.

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Kelly M Cheney

2010-10-01

Full Text Available Type I interferons (IFNα and β are induced directly in response to viral infection, resulting in an antiviral state for the cell. In vitro studies have shown that IFNα is a potent inhibitor of viral replication; however, its role in HIV-1 infection is incompletely understood. In this study we describe the ability of IFNα to restrict HIV-1 infection in primary human macrophages in contrast to peripheral blood mononuclear cells and monocyte-derived dendritic cells. Inhibition to HIV-1 replication in cells pretreated with IFNα occurred at an early stage in the virus life cycle. Late viral events such as budding and subsequent rounds of infection were not affected by IFNα treatment. Analysis of early and late HIV-1 reverse transcripts and integrated proviral DNA confirmed an early post entry role for IFNα. First strand cDNA synthesis was slightly reduced but late and integrated products were severely depleted, suggesting that initiation or the nucleic acid intermediates of reverse transcription are targeted. The depletion of integrated provirus is disproportionally greater than that of viral cDNA synthesis suggesting the possibility of a least an additional later target. A role for either cellular protein APOBEC3G or tetherin in this IFNα mediated restriction has been excluded. Vpu, previously shown by others to rescue a viral budding restriction by tetherin, could not overcome this IFNα induced effect. Determining both the viral determinants and cellular proteins involved may lead to novel therapeutic approaches. Our results add to the understanding of HIV-1 restriction by IFNα.

Altered response hierarchy and increased T-cell breadth upon HIV-1 conserved element DNA vaccination in macaques.

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Viraj Kulkarni

Full Text Available HIV sequence diversity and potential decoy epitopes are hurdles in the development of an effective AIDS vaccine. A DNA vaccine candidate comprising of highly conserved p24(gag elements (CE induced robust immunity in all 10 vaccinated macaques, whereas full-length gag DNA vaccination elicited responses to these conserved elements in only 5 of 11 animals, targeting fewer CE per animal. Importantly, boosting CE-primed macaques with DNA expressing full-length p55(gag increased both magnitude of CE responses and breadth of Gag immunity, demonstrating alteration of the hierarchy of epitope recognition in the presence of pre-existing CE-specific responses. Inclusion of a conserved element immunogen provides a novel and effective strategy to broaden responses against highly diverse pathogens by avoiding decoy epitopes, while focusing responses to critical viral elements for which few escape pathways exist.

APOBEC3G inhibits elongation of HIV-1 reverse transcripts.

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Kate N Bishop

2008-12-01

Full Text Available APOBEC3G (A3G is a host cytidine deaminase that, in the absence of Vif, restricts HIV-1 replication and reduces the amount of viral DNA that accumulates in cells. Initial studies determined that A3G induces extensive mutation of nascent HIV-1 cDNA during reverse transcription. It has been proposed that this triggers the degradation of the viral DNA, but there is now mounting evidence that this mechanism may not be correct. Here, we use a natural endogenous reverse transcriptase assay to show that, in cell-free virus particles, A3G is able to inhibit HIV-1 cDNA accumulation not only in the absence of hypermutation but also without the apparent need for any target cell factors. We find that although reverse transcription initiates in the presence of A3G, elongation of the cDNA product is impeded. These data support the model that A3G reduces HIV-1 cDNA levels by inhibiting synthesis rather than by inducing degradation.

Telomere Length, Proviral Load and Neurologic Impairment in HTLV-1 and HTLV-2-Infected Subjects

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Benjamin Usadi

2016-08-01

Full Text Available Short or damaged telomeres have been implicated in degenerative conditions. We hypothesized that analysis of telomere length (TL in human T-cell lymphotropic virus (HTLV infection and HTLV-associated neuropathy might provide clues to the etiology of HTLV-associated disease and viral dynamics. A subset of 45 human T-cell lymphotropic virus type 1 (HTLV-1, 45 human T-cell lymphotropic virus type 2 (HTLV-2, and 45 seronegative subjects was selected from the larger HTLV Outcomes Study (HOST cohort, matched on age, sex and race/ethnicity. Telomere-to-single-copy gene (T/S ratio (a measure of TL and HTLV-1 and HTLV-2 proviral loads were measured in peripheral blood mononuclear cells (PBMCs using quantitative PCR (qPCR. Vibration sensation measured by tuning fork during neurologic examinations performed as part of the HOST study allowed for an assessment of peripheral neuropathy. TL was compared between groups using t-tests, linear and logistic regression. Mean T/S ratio was 1.02 ± 0.16 in HTLV-1, 1.03 ± 0.17 in HTLV-2 and 0.99 ± 0.18 in HTLV seronegative subjects (p = 0.322. TL was not associated with HTLV-1 or -2 proviral load. Shorter TL was significantly associated with impaired vibration sense in the HTLV-2 positive group only. Overall, we found no evidence that telomere length was affected by chronic HTLV-1 and HTLV-2 infection. That TL was only associated with peripheral neuropathy in the HTLV-2-positive group is intriguing, but should be interpreted cautiously. Studies with larger sample size and telomere length measurement in lymphocyte subsets may clarify the relationship between TL and HTLV-infection.

Potential for false positive HIV test results with the serial rapid HIV testing algorithm

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Baveewo Steven

2012-03-01

Full Text Available Abstract Background Rapid HIV tests provide same-day results and are widely used in HIV testing programs in areas with limited personnel and laboratory infrastructure. The Uganda Ministry of Health currently recommends the serial rapid testing algorithm with Determine, STAT-PAK, and Uni-Gold for diagnosis of HIV infection. Using this algorithm, individuals who test positive on Determine, negative to STAT-PAK and positive to Uni-Gold are reported as HIV positive. We conducted further testing on this subgroup of samples using qualitative DNA PCR to assess the potential for false positive tests in this situation. Results Of the 3388 individuals who were tested, 984 were HIV positive on two consecutive tests, and 29 were considered positive by a tiebreaker (positive on Determine, negative on STAT-PAK, and positive on Uni-Gold. However, when the 29 samples were further tested using qualitative DNA PCR, 14 (48.2% were HIV negative. Conclusion Although this study was not primarily designed to assess the validity of rapid HIV tests and thus only a subset of the samples were retested, the findings show a potential for false positive HIV results in the subset of individuals who test positive when a tiebreaker test is used in serial testing. These findings highlight a need for confirmatory testing for this category of individuals.

Therapeutic Vaccine Against HIV, Viral Variability, Cytotoxic T Lymphocyte Epitopes, and Genetics of Patients.

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Fleury, Herve; Tumiotto, Camille; Bellecave, Pantxika; Recordon-Pinson, Patricia

2018-01-01

The scientific and medical community is seeking to cure HIV. Several pathways have been or are being explored including therapeutic vaccination. Viroimmunological studies on primary infection as well as on elite controllers have demonstrated the importance of the cytotoxic CD8 response and have mainly oriented research on vaccine constructs toward this type of response. The results of these trials are clearly not commensurate with the hope placed in them. Might there be one or more uncontrolled variables? The genetics of patients need to be taken into consideration, especially their human lymphocyte antigen (HLA) alleles. There is a need to find a balance between the conservation of cytotoxic T lymphocyte (CTL) epitopes and presentation by HLA alleles. The pathway is a narrow one between adaptation of the virus to HLA I restriction and the definition of conserved proviral CTL epitopes presentable by HLA I alleles. It is likely that the genetics of patients will need to be considered for HIV-1 vaccine studies and that multidisciplinary collaboration will be essential in this field of infectious diseases.

Prevalence of human Papilloma Virus in HIV-positive and HIV-negative patients in the State of Bahia: a pilot study

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Conceição Queiroz

Full Text Available Human Papilloma Virus (HPV plays a central role in the development of cervical cancer. However, other coexisting factors, such as HIV infection, must be present for this to occur. We evaluated the prevalence of HPV in HIV-positive and HIV-negative patients in the city of Salvador , Bahia, Brazil, and determined the most prevalent types of HPV in these patients. Fifty-five cases were selected from among patients attending three institutions providing cervical pathology services in the city of Salvador. HIV testing (Elisa/WB, HPV-DNA testing by PCR, colposcopy, cytology and biopsy were carried out in all patients. The histopathological results were classified as follows: 11 cases were normal/negative for neoplasia, 15 were diagnosed as cervical intraepithelial neoplasia grade 1 (CIN 1, 10 were CIN 2, 15 cases were CIN 3 and there were four cases of invasive squamous cell carcinoma. Among the 55 patients studied, 43 tested positive for HPV-DNA and 20 for HIV. All HIV-positive patients were positive for HPV-DNA. The most prevalent types of HPV were HPV 16, 52, 58, 53, 54, 33 and 51, and there was little difference between the groups of HIV-positive and HIV-negative patients with respect to the type of HPV encountered. The HIV-positive patients were found to be infected with a greater number of types of HPV than the HIV-negative patients. This study corroborates the existence of regional variations in the distribution of certain types of HPV, which is probably due to the particular ethnic constitution found in this region of Brazil.

Prognostic evaluation of DNA index in HIV-HPV co-infected women cervical samples attending in reference centers for HIV-AIDS in Recife.

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Albert Eduardo Silva Martins

Full Text Available INTRODUCTION: Persistence of cervical infection caused by human papillomavirus (HPV types with high oncogenic risk may lead to cervical intraepithelial neoplasia (CIN. The aim of the present study was to evaluate whether, in HIV-positive women, the presence of aneuploidy in cervical cell samples is associated with presence and evolution of CIN. METHODS: The present study had two stages. In the first stage, comprising a cross-sectional study, the association between the presence of aneuploidy seen via flow cytometry and sociodemographic characteristics, habits and characteristics relating to HPV and HIV infection was analyzed. In the second stage, comprising a cohort study, it was investigated whether aneuploidy was predictive of CIN evolution. RESULTS: No association was observed between the presence of aneuploidy and HPV infection, or between its presence and alterations seen in oncotic cytological analysis. On the other hand, aneuploidy was associated with the presence of CIN (p = 0.030 in histological analysis and with nonuse of antiretroviral therapy (p = 0.001. Most of the HIV-positive women (234/272 presented normal CD4+ T lymphocyte counts (greater than 350 cells/mm3 and showed a greater aneuploidy regression rate (77.5% than a progression rate (23.9% over a follow-up of up to two years. CONCLUSION: Although there was an association between the presence of cervical tissue lesions and the DNA index, the latter was not predictive of progression of the cervical lesion. This suggests that progression of the cervical lesion to cancer in HIV-positive women may also be changed through improvement of the immunological state enabled by using antiretroviral therapy.

HIV testing during the neonatal period

African Journals Online (AJOL)

2015-04-24

Apr 24, 2015 ... transmission interventions on 6-week HIV polymerase ... and daily dose nevirapine (NVP) infant prophylaxis (Option B or B+) ... out other antiretrovirals, 32% of intrapartum-infected infants tested HIV DNA PCR negative.

The Oncolytic Virus MG1 Targets and Eliminates Cells Latently Infected With HIV-1: Implications for an HIV Cure.

Science.gov (United States)

Ranganath, Nischal; Sandstrom, Teslin S; Burke Schinkel, Stephanie C; Côté, Sandra C; Angel, Jonathan B

2018-02-14

Cells latently infected with human immunodeficiency virus (HIV) evade immune- and drug-mediated clearance. These cells harbor intracellular signaling defects, including impairment of the antiviral type I interferon response. Such defects have also been observed in several cancers and have been exploited for the development of therapeutic oncolytic viruses, including the recombinant Maraba virus (MG1). We therefore hypothesized that MG1 would infect and eliminate cells latently infected with HIV-1, while sparing healthy uninfected cells. Preferential infection and elimination by MG1 was first demonstrated in cell lines latently infected with HIV-1. Following this, a reduction in HIV-1 DNA and inducible HIV-1 replication was observed following MG1 infection of latently infected, resting CD4+ T cells generated using an in vitro model of latency. Last, MG1 infection resulted in a reduction in HIV-1 DNA and inducible HIV-1 replication in memory CD4+ T cells isolated from effectively treated, HIV-1-infected individuals. Our results therefore highlight a novel approach to eliminate the latent HIV-1 reservoir. © The Author(s) 2017. Published by Oxford University Press for the Infectious Diseases Society of America.

Hepatitis B Virus Vaccination in HIV-1-Infected Young Adults: A Tool to Reduce the Size of HIV-1 Reservoirs?

Science.gov (United States)

Bekele, Yonas; Graham, Rebecka Lantto; Soeria-Atmadja, Sandra; Nasi, Aikaterini; Zazzi, Maurizio; Vicenti, Ilaria; Naver, Lars; Nilsson, Anna; Chiodi, Francesca

2017-01-01

During anti-retroviral therapy (ART) HIV-1 persists in cellular reservoirs, mostly represented by CD4+ memory T cells. Several approaches are currently being undertaken to develop a cure for HIV-1 infection through elimination (or reduction) of these reservoirs. Few studies have so far been conducted to assess the possibility of reducing the size of HIV-1 reservoirs through vaccination in virologically controlled HIV-1-infected children. We recently conducted a vaccination study with a combined hepatitis A virus (HAV) and hepatitis B virus (HBV) vaccine in 22 HIV-1-infected children. We assessed the size of the virus reservoir, measured as total HIV-1 DNA copies in blood cells, pre- and postvaccination. In addition, we investigated by immunostaining whether the frequencies of CD4+ and CD8+ T cells and parameters of immune activation and proliferation on these cells were modulated by vaccination. At 1 month from the last vaccination dose, we found that 20 out of 22 children mounted a serological response to HBV; a majority of children had antibodies against HAV at baseline. The number of HIV-1 DNA copies in blood at 1 month postvaccination was reduced in comparison to baseline although this reduction was not statistically significant. A significant reduction of HIV-1 DNA copies in blood following vaccination was found in 12 children. The frequencies of CD4+ (naïve, effector memory) and CD8+ (central memory) T-cell subpopulations changed following vaccinations and a reduction in the activation and proliferation pattern of these cells was also noticed. Multivariate linear regression analysis revealed that the frequency of CD8+ effector memory T cells prior to vaccination was strongly predictive of the reduction of HIV-1 DNA copies in blood following vaccination of the 22 HIV-1-infected children. The results of this study suggest a beneficial effect of vaccination to reduce the size of virus reservoir in HIV-1-infected children receiving ART. A reduced frequency of

Hepatitis B Virus Vaccination in HIV-1-Infected Young Adults: A Tool to Reduce the Size of HIV-1 Reservoirs?

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Yonas Bekele

2018-01-01

Full Text Available During anti-retroviral therapy (ART HIV-1 persists in cellular reservoirs, mostly represented by CD4+ memory T cells. Several approaches are currently being undertaken to develop a cure for HIV-1 infection through elimination (or reduction of these reservoirs. Few studies have so far been conducted to assess the possibility of reducing the size of HIV-1 reservoirs through vaccination in virologically controlled HIV-1-infected children. We recently conducted a vaccination study with a combined hepatitis A virus (HAV and hepatitis B virus (HBV vaccine in 22 HIV-1-infected children. We assessed the size of the virus reservoir, measured as total HIV-1 DNA copies in blood cells, pre- and postvaccination. In addition, we investigated by immunostaining whether the frequencies of CD4+ and CD8+ T cells and parameters of immune activation and proliferation on these cells were modulated by vaccination. At 1 month from the last vaccination dose, we found that 20 out of 22 children mounted a serological response to HBV; a majority of children had antibodies against HAV at baseline. The number of HIV-1 DNA copies in blood at 1 month postvaccination was reduced in comparison to baseline although this reduction was not statistically significant. A significant reduction of HIV-1 DNA copies in blood following vaccination was found in 12 children. The frequencies of CD4+ (naïve, effector memory and CD8+ (central memory T-cell subpopulations changed following vaccinations and a reduction in the activation and proliferation pattern of these cells was also noticed. Multivariate linear regression analysis revealed that the frequency of CD8+ effector memory T cells prior to vaccination was strongly predictive of the reduction of HIV-1 DNA copies in blood following vaccination of the 22 HIV-1-infected children. The results of this study suggest a beneficial effect of vaccination to reduce the size of virus reservoir in HIV-1-infected children receiving ART. A reduced

Hepatitis B Virus Vaccination in HIV-1-Infected Young Adults: A Tool to Reduce the Size of HIV-1 Reservoirs?

Science.gov (United States)

Bekele, Yonas; Graham, Rebecka Lantto; Soeria-Atmadja, Sandra; Nasi, Aikaterini; Zazzi, Maurizio; Vicenti, Ilaria; Naver, Lars; Nilsson, Anna; Chiodi, Francesca

2018-01-01

During anti-retroviral therapy (ART) HIV-1 persists in cellular reservoirs, mostly represented by CD4+ memory T cells. Several approaches are currently being undertaken to develop a cure for HIV-1 infection through elimination (or reduction) of these reservoirs. Few studies have so far been conducted to assess the possibility of reducing the size of HIV-1 reservoirs through vaccination in virologically controlled HIV-1-infected children. We recently conducted a vaccination study with a combined hepatitis A virus (HAV) and hepatitis B virus (HBV) vaccine in 22 HIV-1-infected children. We assessed the size of the virus reservoir, measured as total HIV-1 DNA copies in blood cells, pre- and postvaccination. In addition, we investigated by immunostaining whether the frequencies of CD4+ and CD8+ T cells and parameters of immune activation and proliferation on these cells were modulated by vaccination. At 1 month from the last vaccination dose, we found that 20 out of 22 children mounted a serological response to HBV; a majority of children had antibodies against HAV at baseline. The number of HIV-1 DNA copies in blood at 1 month postvaccination was reduced in comparison to baseline although this reduction was not statistically significant. A significant reduction of HIV-1 DNA copies in blood following vaccination was found in 12 children. The frequencies of CD4+ (naïve, effector memory) and CD8+ (central memory) T-cell subpopulations changed following vaccinations and a reduction in the activation and proliferation pattern of these cells was also noticed. Multivariate linear regression analysis revealed that the frequency of CD8+ effector memory T cells prior to vaccination was strongly predictive of the reduction of HIV-1 DNA copies in blood following vaccination of the 22 HIV-1-infected children. The results of this study suggest a beneficial effect of vaccination to reduce the size of virus reservoir in HIV-1-infected children receiving ART. A reduced frequency of

Tight regulation of the Epstein-Barr virus setpoint: interindividual differences in Epstein-Barr virus DNA load are conserved after HIV infection

NARCIS (Netherlands)

Piriou, Erwan; van Dort, Karel; Otto, Sigrid; van Oers, Marinus H. J.; van Baarle, Debbie

2008-01-01

Healthy individuals carry a constant number of Epstein-Barr virus-infected B cells in the peripheral blood over time. Here, we show that interindividual differences in Epstein-Barr virus DNA levels are maintained after HIV infection, providing evidence for the existence of an individual Epstein-Barr

ANALYSIS OF HIV SUBTYPES AND CLINICAL STAGING OF HIV DISEASE/AIDS IN EAST JAVA

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Yulia Ismail

2012-04-01

Full Text Available Human Immunodeficiency Virus type 1 (HIV-1 known to cause Acquired Immune Deficiency Syndrome (AIDS disease are divided into several subtypes (A, B, C, D, F, G, H, J, K and Circulating Recombinant Form (CRF. Different characteristics of subtype of the virus and its interaction with the host can affect the severity of the disease. This study was to analyze HIV-1 subtypes circulating in HIV/AIDS patients from the East Java region descriptively and to analyze its relationship with clinical stadiums of HIV/AIDS. Information from this research was expected to complement the data of mocular epidemiology of HIV in Indonesia. This study utilited blood plasma from patients who had been tested to be HIV positive who sected treatment to or were reffered to the Intermediate Care Unit of Infectious Disease (UPIPI Dr. Soetomo Hospital Surabaya from various area representing the East Java regions. Plasma was separated from blood samples by centrifugation for use in the the molecular biology examination including RNA extraction, nested PCR using specific primer for HIV gp120 env gene region, DNA purifying, DNA sequencing, and homology and phylogenetic analysis. Based on the nucleotide sequence of the HIV gp120 env gene, it was found that the most dominant subtypes in East Java were in one group of Circulating Recombinant Form (CRF that is CRF01_AE, CRF33_01B and CRF34_01B which was also found in Southeast Asia. In the phylogenetic tree, most of HIV samples (30 samples are in the same branch with CRF01_AE, CRF33_01B and CRF34_01B, except for one sample (HIV40 which is in the same branch with subtype B. HIV subtypes are associated with clinical stadiums (disease severity since samples from different stages of HIV disease have the same subtype.

Early diagnosis of HIV in children below 18 months using DNA PCR test--assessment of the effectiveness of PMTCT interventions and challenges in early initiation of ART in a resource-limited setting.

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Gupta, Anil; Singh, Girraj; Kaushik, Pratap; Joshi, Bipin; Kalra, Kamlesh; Chakraborty, Sabyasachi

2013-04-01

This study was conducted to assess efficacy of the current Indian Prevention of Mother-to-Child Transmission (PMTCT) protocol in 217 HIV-exposed infants, and to assess challenges in the early initiation of antiretroviral treatment (ART) in 18 (8.3%) infants with HIV, as determined by the HIV-1 DNA polymerase chain reaction (PCR) test at ≥ 6 weeks to ART was initiated in 10 (55.6%) HIV-infected children at a mean ± standard deviation (SD) age of 10.45 ± 4.9 (range: 4-17.5) months; delay resulted in opportunistic infections in one-third of the children. A single-dose nevirapine PMTCT regimen should be replaced by a triple antiretroviral regimen; DBS DNA PCR-positive infants may be given ART, and simultaneously a whole blood specimen should be taken to determine whether ART should be continued.

Functional interactions of nucleocapsid protein of feline immunodeficiency virus and cellular prion protein with the viral RNA.

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Moscardini, Mila; Pistello, Mauro; Bendinelli, M; Ficheux, Damien; Miller, Jennifer T; Gabus, Caroline; Le Grice, Stuart F J; Surewicz, Witold K; Darlix, Jean-Luc

2002-04-19

All lentiviruses and oncoretroviruses examined so far encode a major nucleic-acid binding protein (nucleocapsid or NC* protein), approximately 2500 molecules of which coat the dimeric RNA genome. Studies on HIV-1 and MoMuLV using in vitro model systems and in vivo have shown that NC protein is required to chaperone viral RNA dimerization and packaging during virus assembly, and proviral DNA synthesis by reverse transcriptase (RT) during infection. The human cellular prion protein (PrP), thought to be the major component of the agent causing transmissible spongiform encephalopathies (TSE), was recently found to possess a strong affinity for nucleic acids and to exhibit chaperone properties very similar to HIV-1 NC protein in the HIV-1 context in vitro. Tight binding of PrP to nucleic acids is proposed to participate directly in the prion disease process. To extend our understanding of lentiviruses and of the unexpected nucleic acid chaperone properties of the human prion protein, we set up an in vitro system to investigate replication of the feline immunodeficiency virus (FIV), which is functionally and phylogenetically distant from HIV-1. The results show that in the FIV model system, NC protein chaperones viral RNA dimerization, primer tRNA(Lys,3) annealing to the genomic primer-binding site (PBS) and minus strand DNA synthesis by the homologous FIV RT. FIV NC protein is able to trigger specific viral DNA synthesis by inhibiting self-priming of reverse transcription. The human prion protein was found to mimic the properties of FIV NC with respect to primer tRNA annealing to the viral RNA and chaperoning minus strand DNA synthesis. Copyright 2002 Elsevier Science Ltd.

DNA-Directed alkylating agents. 7. Synthesis, DNA interaction, and antitumor activity of bis(hydroxymethyl)- and bis(carbamate)-substituted pyrrolizines and imidazoles.

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Atwell, G J; Fan, J Y; Tan, K; Denny, W A

1998-11-19

A series of bis(hydroxymethyl)-substituted imidazoles, thioimidazoles, and pyrrolizines and related bis(carbamates), linked to either 9-anilinoacridine (intercalating) or 4-(4-quinolinylamino)benzamide (minor groove binding) carriers, were synthesized and evaluated for sequence-specific DNA alkylation and cytotoxicity. The imidazole and thioimidazole analogues were prepared by initial synthesis of [(4-aminophenyl)alkyl]imidazole-, thioimidazole-, or pyrrolizine dicarboxylates, coupling of these with the desired carrier, and reduction to give the required bis(hydroxymethyl) alkylating moiety. The pyrrolizines were the most reactive alkylators, followed by the thioimidazoles, while the imidazoles were unreactive. The pyrrolizines and some of the thioimidazoles cross-linked DNA, as measured by agarose gel electrophoresis. Strand cleavage assays showed that none of the compounds reacted at purine N7 or N3 sites in the gpt region of the plasmid gpt2Eco, but the polymerase stop assay showed patterns of G-alkylation in C-rich regions. The corresponding thioimidazole bis(carbamates) were more selective than the bis(hydroxymethyl) pyrrolizines, with high-intensity bands at 5'-NCCN, 5'-NGCN and 5'-NCGN sequences in the PCR stopping assay ( indicates block sites). The data suggest that these targeted compounds, like the known thioimidazole bis(carbamate) carmethizole, alkylate exclusively at guanine residues via the 2-amino group, with little or no alkylation at N3 and N7 guanine or adenine sites. The cytotoxicities of the compounds correlated broadly with their reactivities, with the bis(hydroxymethyl)imidazoles being the least cytotoxic (IC50s >1 microM; P388 leukemia) and with the intercalator-linked analogues being more cytotoxic than the corresponding minor-groove-targeted ones. This was true also for the more reactive thioimidazole bis(carbamates) (IC50s 0.8 and 11 microM, respectively), but both were more active than the analogous "untargeted" carmethizole (IC50 20

IL-12 and GM-CSF in DNA/MVA immunizations against HIV-1 CRF12_BF Nef induced T-cell responses with an enhanced magnitude, breadth and quality.

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Ana María Rodríguez

Full Text Available In Argentina, the HIV epidemic is characterized by the co-circulation of subtype B and BF recombinant viral variants. Nef is an HIV protein highly variable among subtypes, making it a good tool to study the impact of HIV variability in the vaccine design setting. We have previously reported a specific cellular response against NefBF with low cross-reactivity to NefB in mice. The aim of this work was to analyze whether the co-administration of IL-12 and GM-CSF, using DNA and MVA vaccine vectors, could improve the final cellular response induced. Mice received three DNA priming doses of a plasmid that express NefBF plus DNAs expressing IL-12 and/or GM-CSF. Afterwards, all the groups were boosted with a MVAnefBF dose. The highest increase in the magnitude of the NefBF response, compared to that induced in the control was found in the IL-12 group. Importantly, a response with higher breadth was detected in groups which received IL-12 or GM-CSF, evidenced as an increased frequency of recognition of homologous (BF and heterologous (B Nef peptides, as well as a higher number of other Nef peptide pools representing different viral subtypes. However, these improvements were lost when both DNA cytokines were simultaneously administered, as the response was focused against the immunodominant peptide with a detrimental response towards subdominant epitopes. The pattern of cytokines secreted and the specific-T-cell proliferative capacity were improved in IL-12 and IL-12+GM-CSF groups. Importantly IL-12 generated a significant higher T-cell avidity against a B heterologous peptide.This study indicates that the incorporation of DNA expressing IL-12 in DNA/MVA schemes produced the best results in terms of improvements of T-cell-response key properties such as breadth, cross-reactivity and quality (avidity and pattern of cytokines secreted. These relevant results contribute to the design of strategies aimed to induce T-cell responses against HIV antigens with

Approaches to Preventative and Therapeutic HIV vaccines

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Gray, Glenda E.; Laher, Fatima; Lazarus, Erica; Ensoli, Barbara; Corey, Lawrence

2016-01-01

Novel strategies are being researched to discover vaccines to prevent and treat HIV-1. Nonefficacious preventative vaccine approaches include bivalent recombinant gp120 alone, HIV gene insertion into an Adenovirus 5 (Ad5) virus vector and the DNA prime/Ad5 boost vaccine regimen. However, the ALVAC-HIV prime/AIDSVAX® B/E gp120 boost regimen showed 31.2% efficacy at 3.5 years, and is being investigated as clade C constructs with an additional boost. Likewise, although multiple therapeutic vaccines have failed in the past, in a non-placebo controlled trial, a Tat vaccine demonstrated immune cell restoration, reduction of immune activation, and reduced HIV-1 DNA viral load. Monoclonal antibodies for passive immunization or treatment show promise, with VRC01 entering advanced clinical trials. PMID:26985884

Transfer of human genes conferring resistance to methylating mutagens, but not to UV irradiation and cross-linking agents, into Chinese hamster ovary cells

International Nuclear Information System (INIS)

Kaina, B.; Van Zeeland, A.A.; Backendorf, C.; Thielmann, H.W.; Van de Putte, P.

1987-01-01

Chinese hamster ovary cells were transfected by human DNA ligated to the bacterial gpt (xanthine-guanine-phosphoribosyltransferase) gene which was used either in its native form or after partial inactivation with methylnitrosourea. The gpt+ transfectants were screened for resistance to high doses of N-methyl-N'-nitro-N-nitrosoguanidine. Using this approach, we showed that Chinese hamster ovary cells can acquire N-methyl-N'-nitro-N-nitrosoguanidine resistance upon transfection with DNA from diploid human fibroblasts, that this resistance is transferable by secondary transfection and is specific for methylating mutagens, and that it is not caused by increased removal of O6-methylguanine, 3-methyladenine, and 7-methylguanine from DNA

Activation of HIV Transcription with Short-Course Vorinostat in HIV-Infected Patients on Suppressive Antiretroviral Therapy

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Solomon, Ajantha; Ghneim, Khader; Ahlers, Jeffrey; Cameron, Mark J.; Smith, Miranda Z.; Spelman, Tim; McMahon, James; Velayudham, Pushparaj; Brown, Gregor; Roney, Janine; Watson, Jo; Prince, Miles H.; Hoy, Jennifer F.; Chomont, Nicolas; Fromentin, Rémi; Procopio, Francesco A.; Zeidan, Joumana; Palmer, Sarah; Odevall, Lina; Johnstone, Ricky W.; Martin, Ben P.; Sinclair, Elizabeth; Deeks, Steven G.; Hazuda, Daria J.; Cameron, Paul U.; Sékaly, Rafick-Pierre; Lewin, Sharon R.

2014-01-01

Human immunodeficiency virus (HIV) persistence in latently infected resting memory CD4+ T-cells is the major barrier to HIV cure. Cellular histone deacetylases (HDACs) are important in maintaining HIV latency and histone deacetylase inhibitors (HDACi) may reverse latency by activating HIV transcription from latently infected CD4+ T-cells. We performed a single arm, open label, proof-of-concept study in which vorinostat, a pan-HDACi, was administered 400 mg orally once daily for 14 days to 20 HIV-infected individuals on suppressive antiretroviral therapy (ART). The primary endpoint was change in cell associated unspliced (CA-US) HIV RNA in total CD4+ T-cells from blood at day 14. The study is registered at ClinicalTrials.gov (NCT01365065). Vorinostat was safe and well tolerated and there were no dose modifications or study drug discontinuations. CA-US HIV RNA in blood increased significantly in 18/20 patients (90%) with a median fold change from baseline to peak value of 7.4 (IQR 3.4, 9.1). CA-US RNA was significantly elevated 8 hours post drug and remained elevated 70 days after last dose. Significant early changes in expression of genes associated with chromatin remodeling and activation of HIV transcription correlated with the magnitude of increased CA-US HIV RNA. There were no statistically significant changes in plasma HIV RNA, concentration of HIV DNA, integrated DNA, inducible virus in CD4+ T-cells or markers of T-cell activation. Vorinostat induced a significant and sustained increase in HIV transcription from latency in the majority of HIV-infected patients. However, additional interventions will be needed to efficiently induce virus production and ultimately eliminate latently infected cells. Trial Registration ClinicalTrials.gov NCT01365065 PMID:25393648

Activation of HIV transcription with short-course vorinostat in HIV-infected patients on suppressive antiretroviral therapy.

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Julian H Elliott

2014-10-01

Full Text Available Human immunodeficiency virus (HIV persistence in latently infected resting memory CD4+ T-cells is the major barrier to HIV cure. Cellular histone deacetylases (HDACs are important in maintaining HIV latency and histone deacetylase inhibitors (HDACi may reverse latency by activating HIV transcription from latently infected CD4+ T-cells. We performed a single arm, open label, proof-of-concept study in which vorinostat, a pan-HDACi, was administered 400 mg orally once daily for 14 days to 20 HIV-infected individuals on suppressive antiretroviral therapy (ART. The primary endpoint was change in cell associated unspliced (CA-US HIV RNA in total CD4+ T-cells from blood at day 14. The study is registered at ClinicalTrials.gov (NCT01365065. Vorinostat was safe and well tolerated and there were no dose modifications or study drug discontinuations. CA-US HIV RNA in blood increased significantly in 18/20 patients (90% with a median fold change from baseline to peak value of 7.4 (IQR 3.4, 9.1. CA-US RNA was significantly elevated 8 hours post drug and remained elevated 70 days after last dose. Significant early changes in expression of genes associated with chromatin remodeling and activation of HIV transcription correlated with the magnitude of increased CA-US HIV RNA. There were no statistically significant changes in plasma HIV RNA, concentration of HIV DNA, integrated DNA, inducible virus in CD4+ T-cells or markers of T-cell activation. Vorinostat induced a significant and sustained increase in HIV transcription from latency in the majority of HIV-infected patients. However, additional interventions will be needed to efficiently induce virus production and ultimately eliminate latently infected cells.ClinicalTrials.gov NCT01365065.

ATM supports gammaherpesvirus replication by attenuating type I interferon pathway.

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Darrah, Eric J; Stoltz, Kyle P; Ledwith, Mitchell; Tarakanova, Vera L

2017-10-01

Ataxia-Telangiectasia mutated (ATM) kinase participates in multiple networks, including DNA damage response, oxidative stress, and mitophagy. ATM also supports replication of diverse DNA and RNA viruses. Gammaherpesviruses are prevalent cancer-associated viruses that benefit from ATM expression during replication. This proviral role of ATM had been ascribed to its signaling within the DNA damage response network; other functions of ATM have not been considered. In this study increased type I interferon (IFN) responses were observed in ATM deficient gammaherpesvirus-infected macrophages. Using a mouse model that combines ATM and type I IFN receptor deficiencies we show that increased type I IFN response in the absence of ATM fully accounts for the proviral role of ATM during gammaherpesvirus replication. Further, increased type I IFN response rendered ATM deficient macrophages more susceptible to antiviral effects of type II IFN. This study identifies attenuation of type I IFN responses as the primary mechanism underlying proviral function of ATM during gammaherpesvirus infection. Copyright © 2017 Elsevier Inc. All rights reserved.

DBR1 siRNA inhibition of HIV-1 replication

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Naidu Yathi

2005-10-01

Full Text Available Abstract Background HIV-1 and all retroviruses are related to retroelements of simpler organisms such as the yeast Ty elements. Recent work has suggested that the yeast retroelement Ty1 replicates via an unexpected RNA lariat intermediate in cDNA synthesis. The putative genomic RNA lariat intermediate is formed by a 2'-5' phosphodiester bond, like that found in pre-mRNA intron lariats and it facilitates the minus-strand template switch during cDNA synthesis. We hypothesized that HIV-1 might also form a genomic RNA lariat and therefore that siRNA-mediated inhibition of expression of the human RNA lariat de-branching enzyme (DBR1 expression would specifically inhibit HIV-1 replication. Results We designed three short interfering RNA (siRNA molecules targeting DBR1, which were capable of reducing DBR1 mRNA expression by 80% and did not significantly affect cell viability. We assessed HIV-1 replication in the presence of DBR1 siRNA and found that DBR1 knockdown led to decreases in viral cDNA and protein production. These effects could be reversed by cotransfection of a DBR1 cDNA indicating that the inhibition of HIV-1 replication was a specific effect of DBR1 underexpression. Conclusion These data suggest that DBR1 function may be needed to debranch a putative HIV-1 genomic RNA lariat prior to completion of reverse transcription.

Protein carbonyl content: a novel biomarker for aging in HIV/AIDS patients.

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Kolgiri, Vaishali; Patil, Vinayak Wamanrao

The major complications of "treated" Human Immunodeficiency Virus (HIV) infection are cardiovascular disease, malignancy, renal disease, liver disease, bone disease, and perhaps neurological complications, which are phenomena of the normal aging process occurring at an earlier age in the HIV-infected population. The present study is aimed to explore protein carbonyl content as a biomarker for detecting oxidative DNA damage induced ART toxicity and/or accelerated aging in HIV/AIDS patients. To investigate the potential of carbonyl content as a biomarker for detecting oxidative Deoxyribonucleic acid (DNA) damage induced Antiretroviral Theraphy (ART) toxicity and/or accelerated aging in HIV/AIDS patients. In this case-control study a total 600 subjects were included. All subjects were randomly selected and grouped as HIV-negative (control group) (n=300), HIV-infected ART naive (n=100), HIV-infected on first line ART (n=100), and HIV-infected on second line ART (n=100). Seronegative control subjects were age- and sex-matched with the ART naive patients and the two other groups. Carbonyl protein was determined by the method described in Levine et al. DNA damage marker 8-OH-dG was determined using 8-hydroxy-2-deoxy Guanosine StressXpress ELA Kit by StressMarq Biosciences. Protein carbonyl content levels and oxidative DNA damage were significantly higher (paging in HIV/AIDS patients. Larger studies are warranted to elucidate the role of carbonyl content as a biomarker for premature aging in HIV/AIDS patients. Copyright © 2016 Sociedade Brasileira de Infectologia. Published by Elsevier Editora Ltda. All rights reserved.

Liver ultrastructural morphology and mitochondrial DNA levels in HIV/hepatitis C virus coinfection: no evidence of mitochondrial damage with highly active antiretroviral therapy.

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Matsukura, Motoi; Chu, Fanny F S; Au, May; Lu, Helen; Chen, Jennifer; Rietkerk, Sonja; Barrios, Rolando; Farley, John D; Montaner, Julio S; Montessori, Valentina C; Walker, David C; Côté, Hélène C F

2008-06-19

Liver mitochondrial toxicity is a concern, particularly in HIV/hepatitis C virus (HCV) coinfection. Liver biopsies from HIV/HCV co-infected patients, 14 ON-highly active antiretroviral therapy (HAART) and nine OFF-HAART, were assessed by electron microscopy quantitative morphometric analyses. Hepatocytes tended to be larger ON-HAART than OFF-HAART (P = 0.05), but mitochondrial volume, cristae density, lipid volume, mitochondrial DNA and RNA levels were similar. We found no evidence of increased mitochondrial toxicity in individuals currently on HAART, suggesting that concomitant HAART should not delay HCV therapy.

Current treatment of HIV/hepatitis B virus coinfection.

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Iser, David M; Sasadeusz, Joseph J

2008-05-01

Coinfection with HIV and hepatitis B virus (HBV) has become a significant global health problem. Liver disease is now one of the leading causes of morbidity and mortality in individuals with HIV, particularly those with viral hepatitis. There are a number of agents available with dual activity against HIV and HBV, and effective treatment depends on understanding the potential advantages and pitfalls in using these agents. There are a number of unresolved issues in the management of HIV/HBV coinfection. These include the role of liver biopsy, the significance of normal aminotransferase levels, serum HBV DNA threshold for treatment, treatment end-points, and the treatment of HBV when HIV does not yet require treatment. Treatment of HBV should be considered in individuals with HIV/HBV coinfection with evidence of significant fibrosis (>/=F2), or with elevated serum HBV DNA levels (>2000 IU/mL). Sustained suppression of serum HBV DNA to below the level of detection by the most sensitive available assay should be the goal of therapy, and, at present, treatment of HBV in HIV/HBV coinfection is lifelong. If antiretroviral therapy is required, then two agents with anti-HBV activity should be incorporated into the regimen. If antiretroviral therapy is not required, then the options are pegylated interferon, adefovir or the early introduction of antiretroviral therapy. Close monitoring is necessary to detect treatment failure or hepatic flares, such as immune reconstitution disease. Further studies of newer anti-HBV agents in individuals HIV/HBV coinfection may advance treatment of this important condition.

Identification of episomal human papillomavirus and other DNA viruses in cytological anal samples of HIV-uninfected men who have sex with men.

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Maria Gabriella Donà

Full Text Available To date, there have been only few studies that investigated integration of anal Human Papillomavirus (HPV. Most of them were conducted on HIV-infected individuals and mainly analyzed samples from high-grade lesions and invasive cancer. We aimed to investigate HPV physical status in HIV-negative men who have sex with men (MSM with a detectable anal HPV infection, irrespective of the presence of lesions. We also sought to explore the presence of other circular DNA viruses in the anal region. Study participants were attendees of an STI screening program, which were also screened for anal HPV infection and cytological abnormalities. HPV physical status was assessed using multiply-primed RCA. HPV16-positive samples were also analyzed using E2/E6 multiplex PCR, qRT-PCR and APOT assay. RCA and virus-specific PCR were employed to investigate the presence of other DNA viruses. Anal HPV infection was detected in 76.9% of the 230 MSM enrolled. The anal cytological reports were: 129 NILM, 37 ASC-US and 28 L-SIL (36 samples were inadequate for interpretation. HPV physical status was evaluated in the 109 anal specimens that harbored one or two different HPV genotypes. Integration was observed only in one HPV16-positive sample (0.9%, in which integrate-derived viral transcripts of type B were detected. Integration occurred in chromosome 14 q. In 22 of the 53 (41.5% mucosal HPV-negative samples, RCA restriction results would seem to indicate the presence of circular DNA viruses. Indeed, cutaneous HPV (4 samples, MCPyV (5 samples and TTV (4 samples were detected. In conclusion, anal HPV integration was rarely evidenced in HIV-uninfected MSM with no or mild anal cytological abnormalities, although the integration rate may have been underestimated because of the limitations of the employed assays. Other DNA viruses were detected in the anal samples of these individuals, although the significance of this occurrence needs to be assessed.

Absence of in vivo genotoxicity of 3-monochloropropane-1,2-diol and associated fatty acid esters in a 4-week comprehensive toxicity study using F344 gpt delta rats.

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Onami, Saeko; Cho, Young-Man; Toyoda, Takeshi; Horibata, Katsuyoshi; Ishii, Yuji; Umemura, Takashi; Honma, Masamitsu; Nohmi, Takehiko; Nishikawa, Akiyoshi; Ogawa, Kumiko

2014-07-01

3-Monochloropropane-1,2-diol (3-MCPD) is regarded as a rat renal and testicular carcinogen and has been classified as a possible human carcinogen (group 2B) by International Agency for Research on Cancer. This is potentially of great importance given that esters of this compound have recently found to be generated in many foods and food ingredients as a result of food processing. There have been a few reports about their toxicity, although we have recently found that the toxicity profile of 3-MCPD esters was similar to that of 3-MCPD in a rat 13-week repeated dose study, except for the acute renal toxicity seen in 3-MCPD-treated females. In the present study, to examine in vivo genotoxicity we administered equimolar doses of 3-MCPD or 3-MCPD fatty acid esters (palmitate diester, palmitate monoester and oleate diester) to 6-week-old male F344 gpt delta rats carrying a reporter transgene for 4 weeks by intragastric administration. In vivo micronucleus, Pig-a mutation and gpt assays were performed, as well as investigations of major toxicological parameters including histopathological features. As one result, the relative kidney weights of the 3-MCPD and all three ester groups were significantly increased compared with the vehicle control group. However, the frequency of micronucleated reticulocytes and Pig-a mutant red blood cells did not differ among groups. Moreover, no changes were observed in mutant frequencies of gpt and red/gam (Spi(-)) genes in the kidney and the testis of 3-MCPD and 3-MCPD-fatty-acid-esters-treated rats. In histopathological analyses, no treatment related changes were observed, except for decrease of eosinophilic bodies in the kidneys of all treated groups. These results suggest that 3-MCPD and its fatty acid esters are not in vivo genotoxins, although they may exert renal toxicity. © The Author 2014. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please e

CpG methylation controls reactivation of HIV from latency.

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Jana Blazkova

2009-08-01

Full Text Available DNA methylation of retroviral promoters and enhancers localized in the provirus 5' long terminal repeat (LTR is considered to be a mechanism of transcriptional suppression that allows retroviruses to evade host immune responses and antiretroviral drugs. However, the role of DNA methylation in the control of HIV-1 latency has never been unambiguously demonstrated, in contrast to the apparent importance of transcriptional interference and chromatin structure, and has never been studied in HIV-1-infected patients. Here, we show in an in vitro model of reactivable latency and in a latent reservoir of HIV-1-infected patients that CpG methylation of the HIV-1 5' LTR is an additional epigenetic restriction mechanism, which controls resistance of latent HIV-1 to reactivation signals and thus determines the stability of the HIV-1 latency. CpG methylation acts as a late event during establishment of HIV-1 latency and is not required for the initial provirus silencing. Indeed, the latent reservoir of some aviremic patients contained high proportions of the non-methylated 5' LTR. The latency controlled solely by transcriptional interference and by chromatin-dependent mechanisms in the absence of significant promoter DNA methylation tends to be leaky and easily reactivable. In the latent reservoir of HIV-1-infected individuals without detectable plasma viremia, we found HIV-1 promoters and enhancers to be hypermethylated and resistant to reactivation, as opposed to the hypomethylated 5' LTR in viremic patients. However, even dense methylation of the HIV-1 5'LTR did not confer complete resistance to reactivation of latent HIV-1 with some histone deacetylase inhibitors, protein kinase C agonists, TNF-alpha, and their combinations with 5-aza-2deoxycytidine: the densely methylated HIV-1 promoter was most efficiently reactivated in virtual absence of T cell activation by suberoylanilide hydroxamic acid. Tight but incomplete control of HIV-1 latency by Cp

HIV-1 Reservoir Association with Immune Activation

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Alejandro Vallejo

2015-09-01

Full Text Available In this issue of EBioMedicine, Ruggiero and colleagues describe immune activation biomarkers associated with the size of the HIV reservoir in a carefully designed cross-sectional study. The cohort consists of a homogeneous sample of HIV-1-infected patients with long-term plasma HIV-1 RNA suppression under antiretroviral treatment (ART. It is crucial to explore the potential utility of biomarkers that are easier (less labor intensive, less expensive to measure than integrated HIV DNA load, in order to quickly and accurately quantify cellular reservoirs of HIV.

Label-free super sandwich electrogenerated chemiluminescence biosensor for the determination of the HIV gene

International Nuclear Information System (INIS)

Ruan, Sanpeng; Li, Zhejian; Qi, Honglan; Gao, Qiang; Zhang, Chengxiao

2014-01-01

We describe a highly sensitive electrochemiluminescence (ECL) based method for the determination of the human immunodeficiency virus-1 (HIV-1) gene. A long-range self-assembled double strand DNA (ds-DNA) is used as a carrier, and the ruthenium complex Ru(phen) 3 2+ as an ECL indicator for signal amplification. The thiolated ss-DNA serving as a capture probe is firstly self-assembled on the surface of a gold electrode. After the target HIV-1 gene is completely hybridized with the capture probe, two previously hybridized auxiliary probes are hybridized with the target HIV-1 gene to form long-range super sandwich ds-DNA polymers on the surface of the electrode. Finally, the ECL indicator is intercalated into the super sandwich ds-DNA grooves. This results in a strongly increased ECL in tripropylamine solution because a large fraction of the intercalator is intercalated into super sandwich ds-DNA. The results showed that the increased ECL intensity is directly related to the logarithm of the concentration of the HIV-1 gene in the range from 0.1 pM to 0.1 nM, with a detection limit of 0.022 pM and using only 10 μL of analyte samples. The method can effectively discriminate target HIV-1 gene (a perfectly matched ss-DNA) from a 2-base mismatched ss-DNA. This work demonstrates that the high sensitivity and selectivity of an ECL DNA biosensor can be largely improved by using super sandwich ds-DNA along with ECL indicators. (author)

Sequential Immunization with gp140 Boosts Immune Responses Primed by Modified Vaccinia Ankara or DNA in HIV-Uninfected South African Participants.

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Gavin Churchyard

Full Text Available The safety and immunogenicity of SAAVI DNA-C2 (4 mg IM, SAAVI MVA-C (2.9 x 109 pfu IM and Novartis V2-deleted subtype C gp140 (100 mcg with MF59 adjuvant in various vaccination regimens was evaluated in HIV-uninfected adults in South Africa.Participants at three South African sites were randomized (1:1:1:1 to one of four vaccine regimens: MVA prime, sequential gp140 protein boost (M/M/P/P; concurrent MVA/gp140 (MP/MP; DNA prime, sequential MVA boost (D/D/M/M; DNA prime, concurrent MVA/gp140 boost (D/D/MP/MP or placebo. Peak HIV specific humoral and cellular responses were measured.184 participants were enrolled: 52% were female, all were Black/African, median age was 23 years (range, 18-42 years and 79% completed all vaccinations. 159 participants reported at least one adverse event, 92.5% were mild or moderate. Five, unrelated, serious adverse events were reported. The M/M/P/P and D/D/MP/MP regimens induced the strongest peak neutralizing and binding antibody responses and the greatest CD4+ T-cell responses to Env. All peak neutralizing and binding antibody responses decayed with time. The MVA, but not DNA, prime contributed to the humoral and cellular immune responses. The D/D/M/M regimen was poorly immunogenic overall but did induce modest CD4+ T-cell responses to Gag and Pol. CD8+ T-cell responses to any antigen were low for all regimens.The SAAVI DNA-C2, SAAVI MVA-C and Novartis gp140 with MF59 adjuvant in various combinations were safe and induced neutralizing and binding antibodies and cellular immune responses. Sequential immunization with gp140 boosted immune responses primed by MVA or DNA. The best overall immune responses were seen with the M/M/P/P regimen.ClinicalTrials.gov NCT01418235.

Herpes simplex virus type 2 (HSV-2) genital shedding in HSV-2-/HIV-1-co-infected women receiving effective combination antiretroviral therapy.

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Péré, Héléne; Rascanu, Aida; LeGoff, Jérome; Matta, Mathieu; Bois, Frédéric; Lortholary, Olivier; Leroy, Valériane; Launay, Odile; Bélec, Laurent

2016-03-01

The dynamics of genital shedding of HSV-2 DNA was assessed in HIV-1-infected women taking combination antiretroviral therapy (cART). HIV-1 RNA, HIV-1 DNA and HSV DNA loads were measured during 12-18 months using frozen plasma, PBMC and cervicovaginal lavage samples from 22 HIV-1-infected women, including 17 women naive for antiretroviral therapy initiating cART and 5 women with virological failure switching to a new regimen. Nineteen (86%) women were HSV-2-seropositive. Among HSV-2-/HIV-1-co-infected women, HIV-1 RNA loads showed a rapid fall from baseline after one month of cART, in parallel in paired plasma and cervicovaginal secretions. In contrast, HIV-1 DNA loads did not show significant variations from baseline up to 18 months of treatment in both systemic and genital compartments. HSV DNA was detected at least once in 12 (63%) of 19 women during follow up: HSV-2 shedding in the genital compartment was observed in 11% of cervicovaginal samples at baseline and in 16% after initiating or switching cART. Cervicovaginal HIV-1 RNA loads were strongly associated with plasma HIV-1 RNA loads over time, but not with cervicovaginal HSV DNA loads. Reactivation of genital HSV-2 replication frequently occurred despite effective cART in HSV-2-/HIV-1-co-infected women. Genital HSV-2 replication under cART does not influence cervicovaginal HIV-1 RNA or DNA shedding. © The Author(s) 2015.

Impact of Multi-Targeted Antiretroviral Treatment on Gut T Cell Depletion and HIV Reservoir Seeding during Acute HIV Infection

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Ananworanich, Jintanat; Schuetz, Alexandra; Vandergeeten, Claire; Sereti, Irini; de Souza, Mark; Rerknimitr, Rungsun; Dewar, Robin; Marovich, Mary; van Griensven, Frits; Sekaly, Rafick; Pinyakorn, Suteeraporn; Phanuphak, Nittaya; Trichavaroj, Rapee; Rutvisuttinunt, Wiriya; Chomchey, Nitiya; Paris, Robert; Peel, Sheila; Valcour, Victor; Maldarelli, Frank; Chomont, Nicolas; Michael, Nelson; Phanuphak, Praphan; Kim, Jerome H.

2012-01-01

Background Limited knowledge exists on early HIV events that may inform preventive and therapeutic strategies. This study aims to characterize the earliest immunologic and virologic HIV events following infection and investigates the usage of a novel therapeutic strategy. Methods and Findings We prospectively screened 24,430 subjects in Bangkok and identified 40 AHI individuals. Thirty Thais were enrolled (8 Fiebig I, 5 Fiebig II, 15 Fiebig III, 2 Fiebig IV) of whom 15 completed 24 weeks of megaHAART (tenofovir/emtricitabine/efavirenz/raltegravir/maraviroc). Sigmoid biopsies were completed in 24/30 at baseline and 13/15 at week 24. At baseline, the median age was 29 years and 83% were MSM. Most were symptomatic (87%), and were infected with R5-tropic (77%) CRF01_AE (70%). Median CD4 was 406 cells/mm3. HIV RNA was 5.5 log10 copies/ml. Median total blood HIV DNA was higher in Fiebig III (550 copy/106 PBMC) vs. Fiebig I (8 copy/106 PBMC) (p = 0.01) while the median %CD4+CCR5+ gut T cells was lower in Fiebig III (19%) vs. Fiebig I (59%) (p = 0.0008). After 24 weeks of megaHAART, HIV RNA levels of HIV DNA at week 0 predicted reservoir size at week 24 (pHIV DNA declined significantly and was undetectable in 3 of 15 in blood and 3 of 7 in gut. Frequency of CD4+CCR5+ gut T cells increased from 41% at baseline to 64% at week 24 (p>0.050); subjects with less than 40% at baseline had a significant increase in CD4+CCR5+ T cells from baseline to week 24 (14% vs. 71%, p = 0.02). Conclusions Gut T cell depletion and HIV reservoir seeding increases with progression of AHI. MegaHAART was associated with immune restoration and reduced reservoir size. Our findings could inform research on strategies to achieve HIV drug-free remission. PMID:22479485

Pathogenesis of oral FIV infection.

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Craig Miller

Full Text Available Feline immunodeficiency virus (FIV is the feline analogue of human immunodeficiency virus (HIV and features many hallmarks of HIV infection and pathogenesis, including the development of concurrent oral lesions. While HIV is typically transmitted via parenteral transmucosal contact, recent studies prove that oral transmission can occur, and that saliva from infected individuals contains significant amounts of HIV RNA and DNA. While it is accepted that FIV is primarily transmitted by biting, few studies have evaluated FIV oral infection kinetics and transmission mechanisms over the last 20 years. Modern quantitative analyses applied to natural FIV oral infection could significantly further our understanding of lentiviral oral disease and transmission. We therefore characterized FIV salivary viral kinetics and antibody secretions to more fully document oral viral pathogenesis. Our results demonstrate that: (i saliva of FIV-infected cats contains infectious virus particles, FIV viral RNA at levels equivalent to circulation, and lower but significant amounts of FIV proviral DNA; (ii the ratio of FIV RNA to DNA is significantly higher in saliva than in circulation; (iii FIV viral load in oral lymphoid tissues (tonsil, lymph nodes is significantly higher than mucosal tissues (buccal mucosa, salivary gland, tongue; (iv salivary IgG antibodies increase significantly over time in FIV-infected cats, while salivary IgA levels remain static; and, (v saliva from naïve Specific Pathogen Free cats inhibits FIV growth in vitro. Collectively, these results suggest that oral lymphoid tissues serve as a site for enhanced FIV replication, resulting in accumulation of FIV particles and FIV-infected cells in saliva. Failure to induce a virus-specific oral mucosal antibody response, and/or viral capability to overcome inhibitory components in saliva may perpetuate chronic oral cavity infection. Based upon these findings, we propose a model of oral FIV pathogenesis

Suppression of APOBEC3-mediated restriction of HIV-1 by Vif

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Yuqing eFeng

2014-08-01

Full Text Available The APOBEC3 restriction factors are a family of deoxycytidine deaminases that are able to suppress replication of viruses with a single-stranded DNA intermediate by inducing mutagenesis and functional inactivation of the virus. Of the seven human APOBEC3 enzymes, only APOBEC3-D, -F, -G, and -H appear relevant to restriction of HIV-1 in CD4+ T cells and will be the focus of this review. The restriction of HIV-1 occurs most potently in the absence of HIV-1 Vif that induces polyubiquitination and degradation of APOBEC3 enzymes through the proteasome pathway. To restrict HIV-1, APOBEC3 enzymes must be encapsidated into budding virions. Upon infection of the target cell during reverse transcription of the HIV-1 RNA into (-DNA APOBEC3 enzymes deaminate cytosines to forms uracils in single-stranded (- DNA regions. Upon replication of the (-DNA to (+DNA, the HIV-1 reverse transcriptase incorporates adenines opposite the uracils thereby inducing C/G to T/A mutations that can functionally inactivate HIV-1. APOBEC3G is the most studied APOBEC3 enzyme and it is known that Vif attempts to thwart APOBEC3 function not only by inducing its proteasomal degradation but by several degradation-independent mechanisms such as inhibiting APOBEC3G virion encapsidation, mRNA translation, and for those APOBEC3G molecules that still become virion encapsidated, Vif can inhibit APOBEC3G mutagenic activity. Although most Vif variants can induce efficient degradation of APOBEC3-D, -F, and -G, there appears to be differential sensitivity to Vif-mediated degradation for APOBEC3H. This review examines APOBEC3-mediated HIV restriction mechanisms, how Vif acts as a substrate receptor for a Cullin5 ubiquitin ligase complex to induce degradation of APOBEC3s, and the determinants and functional consequences of the APOBEC3 and Vif interaction from a biological and biochemical perspective.

Potent and reversible lentiviral vector restriction in murine induced pluripotent stem cells.

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Geis, Franziska K; Galla, Melanie; Hoffmann, Dirk; Kuehle, Johannes; Zychlinski, Daniela; Maetzig, Tobias; Schott, Juliane W; Schwarzer, Adrian; Goffinet, Christine; Goff, Stephen P; Schambach, Axel

2017-05-31

Retroviral vectors are derived from wild-type retroviruses, can be used to study retrovirus-host interactions and are effective tools in gene and cell therapy. However, numerous cell types are resistant or less permissive to retrovirus infection due to the presence of active defense mechanisms, or the absence of important cellular host co-factors. In contrast to multipotent stem cells, pluripotent stem cells (PSC) have potential to differentiate into all three germ layers. Much remains to be elucidated in the field of anti-viral immunity in stem cells, especially in PSC. In this study, we report that transduction with HIV-1-based, lentiviral vectors (LV) is impaired in murine PSC. Analyses of early retroviral events in induced pluripotent stem cells (iPSC) revealed that the restriction is independent of envelope choice and does not affect reverse transcription, but perturbs nuclear entry and proviral integration. Proteasomal inhibition by MG132 could not circumvent the restriction. However, prevention of cyclophilin A (CypA) binding to the HIV-1 capsid via use of either a CypA inhibitor (cyclosporine A) or CypA-independent capsid mutants improved transduction. In addition, application of higher vector doses also increased transduction. Our data revealed a CypA mediated restriction in iPSC, which was acquired during reprogramming, associated with pluripotency and relieved upon subsequent differentiation. We showed that murine PSC and iPSC are less susceptible to LV. The block observed in iPSC was CypA-dependent and resulted in reduced nuclear entry of viral DNA and proviral integration. Our study helps to improve transduction of murine pluripotent cells with HIV-1-based vectors and contributes to our understanding of retrovirus-host interactions in PSC.

Multi-spectroscopic and molecular docking studies on the interaction of darunavir, a HIV protease inhibitor with calf thymus DNA.

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Shi, Jie-Hua; Zhou, Kai-Li; Lou, Yan-Yue; Pan, Dong-Qi

2018-03-15

Molecular interaction of darunavir (DRV), a HIV protease inhibitor with calf thymus deoxyribonucleic acid (ct-DNA) was studied in physiological buffer (pH7.4) by multi-spectroscopic approaches hand in hand with viscosity measurements and molecular docking technique. The UV absorption and fluorescence results together revealed the formation of a DRV-ct-DNA complex having binding affinities of the order of 10 3 M -1 , which was more in keeping with the groove binding. The results that DRV bound to ct-DNA via groove binding mode was further evidenced by KI quenching studies, viscosity measurements, competitive binding investigations with EB and Rhodamine B and CD spectral analysis. The effect of ionic strength indicated the negligible involvement of electrostatic interaction between DRV and ct-DNA. The thermodynamic parameters regarding the binding interaction of DRV with ct-DNA in terms of enthalpy change (ΔH 0 ) and entropy change (ΔS 0 ) were -63.19kJ mol -1 and -141.92J mol -1 K -1 , indicating that hydrogen bonds and van der Waals forces played a predominant role in the binding process. Furthermore, molecular simulation studies suggested that DRV molecule was prone to bind in the A-T rich region of the minor groove of DNA. Copyright © 2017 Elsevier B.V. All rights reserved.

Dynamics of 103K/N and 184M/V HIV-1 drug resistant populations: relative comparison in plasma virus RNA versus CD45RO+T cell proviral DNA

DEFF Research Database (Denmark)

Jakobsen, Martin Roelsgaard; Tolstrup, M; Bertelsen, L

2007-01-01

on real-time PCR and amplification refractory mutation system (ARMS). RESULTS: The 103N and 184V mutations were not detected in patients with stable low viremia. Patients previously exposed to mono or dual therapy often carried minor viral populations of either one or both mutations in plasma. The viral......BACKGROUND: Viral populations defined by 103K/N and 184M/V as linked or single mutations in the HIV-1 reverse transcriptase gene were investigated in plasma samples and compared with previous findings in the CD45RO(+)T cell compartment. OBJECTIVE: To develop an ARMS assay for plasma virions...... an unequal distribution of linked-mutation populations in plasma and CD45RO(+)T cells. Furthermore, the linked 103N-184V mutation may be more fit than the single 184V mutation and this linked population emerges rapidly under inadequate drug pressure. Udgivelsesdato: 2007-Jul...

Detection of HIV-1 and Human Proteins in Urinary Extracellular Vesicles from HIV+ Patients

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Samuel I. Anyanwu

2018-01-01

Full Text Available Background. Extracellular vesicles (EVs are membrane bound, secreted by cells, and detected in bodily fluids, including urine, and contain proteins, RNA, and DNA. Our goal was to identify HIV and human proteins (HPs in urinary EVs from HIV+ patients and compare them to HIV− samples. Methods. Urine samples were collected from HIV+ (n=35 and HIV− (n=12 individuals. EVs were isolated by ultrafiltration and characterized using transmission electron microscopy, tandem mass spectrometry (LC/MS/MS, and nanoparticle tracking analysis (NTA. Western blots confirmed the presence of HIV proteins. Gene ontology (GO analysis was performed using FunRich and HIV Human Interaction database (HHID. Results. EVs from urine were 30–400 nm in size. More EVs were in HIV+ patients, P<0.05, by NTA. HIV+ samples had 14,475 HPs using LC/MS/MS, while only 111 were in HIV−. HPs in the EVs were of exosomal origin. LC/MS/MS showed all HIV+ samples contained at least one HIV protein. GO analysis showed differences in proteins between HIV+ and HIV− samples and more than 50% of the published HPs in the HHID interacted with EV HIV proteins. Conclusion. Differences in the proteomic profile of EVs from HIV+ versus HIV− samples were found. HIV and HPs in EVs could be used to detect infection and/or diagnose HIV disease syndromes.

HIV-1 vaccines

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Excler, Jean-Louis; Robb, Merlin L; Kim, Jerome H

2014-01-01

The development of a safe and effective preventive HIV-1 vaccine remains a public health priority. Despite scientific difficulties and disappointing results, HIV-1 vaccine clinical development has, for the first time, established proof-of-concept efficacy against HIV-1 acquisition and identified vaccine-associated immune correlates of risk. The correlate of risk analysis showed that IgG antibodies against the gp120 V2 loop correlated with decreased risk of HIV infection, while Env-specific IgA directly correlated with increased risk. The development of vaccine strategies such as improved envelope proteins formulated with potent adjuvants and DNA and vectors expressing mosaics, or conserved sequences, capable of eliciting greater breadth and depth of potentially relevant immune responses including neutralizing and non-neutralizing antibodies, CD4+ and CD8+ cell-mediated immune responses, mucosal immune responses, and immunological memory, is now proceeding quickly. Additional human efficacy trials combined with other prevention modalities along with sustained funding and international collaboration remain key to bring an HIV-1 vaccine to licensure. PMID:24637946

Similarities and differences in the nucleic acid chaperone activity of HIV-2 and HIV-1 nucleocapsid proteins in vitro.

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Pachulska-Wieczorek, Katarzyna; Stefaniak, Agnieszka K; Purzycka, Katarzyna J

2014-07-03

The nucleocapsid domain of Gag and mature nucleocapsid protein (NC) act as nucleic acid chaperones and facilitate folding of nucleic acids at critical steps of retroviral replication cycle. The basic N-terminus of HIV-1 NC protein was shown most important for the chaperone activity. The HIV-2 NC (NCp8) and HIV-1 NC (NCp7) proteins possess two highly conserved zinc fingers, flanked by basic residues. However, the NCp8 N-terminal domain is significantly shorter and contains less positively charged residues. This study characterizes previously unknown, nucleic acid chaperone activity of the HIV-2 NC protein. We have comparatively investigated the in vitro nucleic acid chaperone properties of the HIV-2 and HIV-1 NC proteins. Using substrates derived from the HIV-1 and HIV-2 genomes, we determined the ability of both proteins to chaperone nucleic acid aggregation, annealing and strand exchange in duplex structures. Both NC proteins displayed comparable, high annealing activity of HIV-1 TAR DNA and its complementary nucleic acid. Interesting differences between the two NC proteins were discovered when longer HIV substrates, particularly those derived from the HIV-2 genome, were used in chaperone assays. In contrast to NCp7, NCp8 weakly facilitates annealing of HIV-2 TAR RNA to its complementary TAR (-) DNA. NCp8 is also unable to efficiently stimulate tRNALys3 annealing to its respective HIV-2 PBS motif. Using truncated NCp8 peptide, we demonstrated that despite the fact that the N-terminus of NCp8 differs from that of NCp7, this domain is essential for NCp8 activity. Our data demonstrate that the HIV-2 NC protein displays reduced nucleic acid chaperone activity compared to that of HIV-1 NC. We found that NCp8 activity is limited by substrate length and stability to a greater degree than that of NCp7. This is especially interesting in light of the fact that the HIV-2 5'UTR is more structured than that of HIV-1. The reduced chaperone activity observed with NCp8 may

Newer Gene Editing Technologies toward HIV Gene Therapy

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Premlata Shankar

2013-11-01

Full Text Available Despite the great success of highly active antiretroviral therapy (HAART in ameliorating the course of HIV infection, alternative therapeutic approaches are being pursued because of practical problems associated with life-long therapy. The eradication of HIV in the so-called “Berlin patient” who received a bone marrow transplant from a CCR5-negative donor has rekindled interest in genome engineering strategies to achieve the same effect. Precise gene editing within the cells is now a realistic possibility with recent advances in understanding the DNA repair mechanisms, DNA interaction with transcription factors and bacterial defense mechanisms. Within the past few years, four novel technologies have emerged that can be engineered for recognition of specific DNA target sequences to enable site-specific gene editing: Homing Endonuclease, ZFN, TALEN, and CRISPR/Cas9 system. The most recent CRISPR/Cas9 system uses a short stretch of complementary RNA bound to Cas9 nuclease to recognize and cleave target DNA, as opposed to the previous technologies that use DNA binding motifs of either zinc finger proteins or transcription activator-like effector molecules fused to an endonuclease to mediate sequence-specific DNA cleavage. Unlike RNA interference, which requires the continued presence of effector moieties to maintain gene silencing, the newer technologies allow permanent disruption of the targeted gene after a single treatment. Here, we review the applications, limitations and future prospects of novel gene-editing strategies for use as HIV therapy.

Short communication: identification of a novel HIV type 1 subtype H/J recombinant in Canada with discordant HIV viral load (RNA) values in three different commercial assays.

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Kim, John E; Beckthold, Brenda; Chen, Zhaoxia; Mihowich, Jennifer; Malloch, Laurie; Gill, Michael John

2007-11-01

The presence of HIV-1 non-B subtypes is increasing worldwide. This poses challenges to commercial diagnostic and viral load (RNA) monitoring tests that are predominantly based on HIV-1 subtype B strains. Based on phylogenetic analysis of the gag, pol, and env gene regions, we describe the first HIV-1 H/J recombinant in Canada that presented divergent viral load values. DNA sequence analysis of the gag gene region further revealed that genetic diversity between this H/J recombinant and the primers and probes used in the bio-Merieux Nuclisens HIV-1 QT (Nuclisens) and Roche Amplicor Monitor HIV-1, v1.5 (Monitor) viral RNA assays can erroneously lead to undetectable viral load values. This observation appears to be more problematic in the Nuclisens assay. In light of increasing genetic diversity in HIV worldwide we recommend that DNA sequencing of HIV, especially in the gag gene region targeted by primers and probes used in molecular diagnostic and viral load tests, be incorporated into clinical monitoring practices.

Viruses & kidney disease: beyond HIV

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Waldman, Meryl; Marshall, Vickie; Whitby, Denise; Kopp, Jeffrey B.

2008-01-01

HIV-infected patients may acquire new viral co-infections; they may also experience the reactivation or worsening of existing viral infections, including active, smoldering, or latent infections. HIV-infected patients may be predisposed to these viral infections due to immunodeficiency or to risk factors common to HIV and other viruses. A number of these affect the kidney, either by direct infection or by deposition of immune complexes. In this review we discuss the renal manifestations and treatment of hepatitis C virus, BK virus, adenovirus, cytomegalovirus, and parvovirus B19 in patients with HIV disease. We also discuss an approach to the identification of new viral renal pathogens, using a viral gene chip to identify viral DNA or RNA. PMID:19013331

Suicidal function of DNA methylation in age-related genome disintegration.

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Mazin, Alexander L

2009-10-01

This article is dedicated to the 60th anniversary of 5-methylcytosine discovery in DNA. Cytosine methylation can affect genetic and epigenetic processes, works as a part of the genome-defense system and has mutagenic activity; however, the biological functions of this enzymatic modification are not well understood. This review will put forward the hypothesis that the host-defense role of DNA methylation in silencing and mutational destroying of retroviruses and other intragenomic parasites was extended during evolution to most host genes that have to be inactivated in differentiated somatic cells, where it acquired a new function in age-related self-destruction of the genome. The proposed model considers DNA methylation as the generator of 5mC>T transitions that induce 40-70% of all spontaneous somatic mutations of the multiple classes at CpG and CpNpG sites and flanking nucleotides in the p53, FIX, hprt, gpt human genes and some transgenes. The accumulation of 5mC-dependent mutations explains: global changes in the structure of the vertebrate genome throughout evolution; the loss of most 5mC from the DNA of various species over their lifespan and the Hayflick limit of normal cells; the polymorphism of methylation sites, including asymmetric mCpNpN sites; cyclical changes of methylation and demethylation in genes. The suicidal function of methylation may be a special genetic mechanism for increasing DNA damage and the programmed genome disintegration responsible for cell apoptosis and organism aging and death.

Identification of a methylated oligoribonucleotide as a potent inhibitor of HIV-1 reverse transcription complex.

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Grigorov, Boyan; Bocquin, Anne; Gabus, Caroline; Avilov, Sergey; Mély, Yves; Agopian, Audrey; Divita, Gilles; Gottikh, Marina; Witvrouw, Myriam; Darlix, Jean-Luc

2011-07-01

Upon HIV-1 infection of a target cell, the viral reverse transcriptase (RT) copies the genomic RNA to synthesize the viral DNA. The genomic RNA is within the incoming HIV-1 core where it is coated by molecules of nucleocapsid (NC) protein that chaperones the reverse transcription process. Indeed, the RT chaperoning properties of NC extend from the initiation of cDNA synthesis to completion of the viral DNA. New and effective drugs against HIV-1 continue to be required, which prompted us to search for compounds aimed at inhibiting NC protein. Here, we report that the NC chaperoning activity is extensively inhibited in vitro by small methylated oligoribonucleotides (mODN). These mODNs were delivered intracellularly using a cell-penetrating-peptide and found to impede HIV-1 replication in primary human cells at nanomolar concentrations. Extensive analysis showed that viral cDNA synthesis was severely impaired by mODNs. Partially resistant viruses with mutations in NC and RT emerged after months of passaging in cell culture. A HIV-1 molecular clone (NL4.3) bearing these mutations was found to replicate at high concentrations of mODN, albeit with a reduced fitness. Small, methylated ODNs such as mODN-11 appear to be a new type of highly potent inhibitor of HIV-1.

Virus evolution reveals an exclusive role for LEDGF/p75 in chromosomal tethering of HIV.

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Anneleen Hombrouck

2007-03-01

Full Text Available Retroviruses by definition insert their viral genome into the host cell chromosome. Although the key player of retroviral integration is viral integrase, a role for cellular cofactors has been proposed. Lentiviral integrases use the cellular protein LEDGF/p75 to tether the preintegration complex to the chromosome, although the existence of alternative host proteins substituting for the function of LEDGF/p75 in integration has been proposed. Truncation mutants of LEDGF/p75 lacking the chromosome attachment site strongly inhibit HIV replication by competition for the interaction with integrase. In an attempt to select HIV strains that can overcome the inhibition, we now have used T-cell lines that stably express a C-terminal fragment of LEDGF/p75. Despite resistance development, the affinity of integrase for LEDGF/p75 is reduced and replication kinetics in human primary T cells is impaired. Detection of the integrase mutations A128T and E170G at key positions in the LEDGF/p75-integrase interface provides in vivo evidence for previously reported crystallographic data. Moreover, the complementary inhibition by LEDGF/p75 knockdown and mutagenesis at the integrase-LEDGF/p75 interface points to the incapability of HIV to circumvent LEDGF/p75 function during proviral integration. Altogether, the data provide a striking example of the power of viral molecular evolution. The results underline the importance of the LEDGF/p75 HIV-1 interplay as target for innovative antiviral therapy. Moreover, the role of LEDGF/p75 in targeting integration will stimulate research on strategies to direct gene therapy vectors into safe landing sites.

Tools for Visualizing HIV in Cure Research.

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Niessl, Julia; Baxter, Amy E; Kaufmann, Daniel E

2018-02-01

The long-lived HIV reservoir remains a major obstacle for an HIV cure. Current techniques to analyze this reservoir are generally population-based. We highlight recent developments in methods visualizing HIV, which offer a different, complementary view, and provide indispensable information for cure strategy development. Recent advances in fluorescence in situ hybridization techniques enabled key developments in reservoir visualization. Flow cytometric detection of HIV mRNAs, concurrently with proteins, provides a high-throughput approach to study the reservoir on a single-cell level. On a tissue level, key spatial information can be obtained detecting viral RNA and DNA in situ by fluorescence microscopy. At total-body level, advancements in non-invasive immuno-positron emission tomography (PET) detection of HIV proteins may allow an encompassing view of HIV reservoir sites. HIV imaging approaches provide important, complementary information regarding the size, phenotype, and localization of the HIV reservoir. Visualizing the reservoir may contribute to the design, assessment, and monitoring of HIV cure strategies in vitro and in vivo.

Induction of DNA strand breaks by RSU-1069, a nitroimidazole-aziridine radiosensitizer

International Nuclear Information System (INIS)

Silver, A.R.J.; O'Neill, P.; Jenkins, T.C.

1985-01-01

[2- 14 C]-RSU-1069 [1-(2-nitro-1-imidazolyl)-3-(1-aziridino)-2-propanol], either as a parent or following radiation reduction, binds to calf thymus DNA in vitro. Radiation-reduced RSU-1069 binds to a greater extent and more rapidly than the parent compound. RSU-1137, a non-aziridino analogue of RSU-1069, binds following radiation reduction. Radiation-reduced misonidazole exhibits binding ratios a thousand-fold less than those of reduced RSU-1069. Both parent and reduced RSU-1069 cause single strand breaks (ssbs) in pSV2 gpt plasmid DNA with the reduced compound causing a greater number of breaks. Parent and reduced RSU-1137 and misonidazole do not cause ssbs. It is inferred that the aziridine moiety present in both parent and reduced RSU-1069 is required for ssb production. RSU-1069 reacts with inorganic phosphate probably via nucleophilic ring-opening of the aziridine fragment. Incubation of plasmid DNA with reduced RSU-1069 in the presence of either phosphate or deoxyribose-5-phosphate at concentrations greater than 0.35 mol dm -3 prevents strand breakage, whereas 1.2 mol dm -3 deoxyribose does not protect against strand breakage formation. It is proposed that the observed binding to DNA occurs via the aziridine and the reduced nitro group of RSU-1069 and that these two have different target sites. Binding to DNA via the reduced nitro group may serve to increase aziridine attack due to localization at or near its target. (author)

Increasing procaspase 8 expression using repurposed drugs to induce HIV infected cell death in ex vivo patient cells.

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Rahul Sampath

Full Text Available HIV persists because a reservoir of latently infected CD4 T cells do not express viral proteins and are indistinguishable from uninfected cells. One approach to HIV cure suggests that reactivating HIV will activate cytotoxic pathways; yet when tested in vivo, reactivating cells do not die sufficiently to reduce cell-associated HIV DNA levels. We recently showed that following reactivation from latency, HIV infected cells generate the HIV specific cytotoxic protein Casp8p41 which is produced by HIV protease cleaving procaspase 8. However, cell death is prevented, possibly due to low procaspase 8 expression. Here, we tested whether increasing procaspase 8 levels in CD4 T cells will produce more Casp8p41 following HIV reactivation, causing more reactivated cells to die. Screening 1277 FDA approved drugs identified 168 that increased procaspase 8 expression by at least 1.7-fold. Of these 30 were tested for anti-HIV effects in an acute HIVIIIb infection model, and 9 drugs at physiologic relevant levels significantly reduced cell-associated HIV DNA. Primary CD4 T cells from ART suppressed HIV patients were treated with one of these 9 drugs and reactivated with αCD3/αCD28. Four drugs significantly increased Casp8p41 levels following HIV reactivation, and decreased total cell associated HIV DNA levels (flurbiprofen: p = 0.014; doxycycline: p = 0.044; indomethacin: p = 0.025; bezafibrate: P = 0.018 without effecting the viability of uninfected cells. Thus procaspase 8 levels can be increased pharmacologically and, in the context of HIV reactivation, increase Casp8p41 causing death of reactivating cells and decreased HIV DNA levels. Future studies will be required to define the clinical utility of this or similar approaches.

Early initiation of highly active antiretroviral therapy fails to reverse immunovirological abnormalities in gut-associated lymphoid tissue induced by acute HIV infection.

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Tincati, Camilla; Biasin, Mara; Bandera, Alessandra; Violin, Michela; Marchetti, Giulia; Piacentini, Luca; Vago, Gian Luca; Balotta, Claudia; Moroni, Mauro; Franzetti, Fabio; Clerici, Mario; Gori, Andrea

2009-01-01

During the acute phase of HIV infection, large CD4+ T-cell depletion occurs in the gastrointestinal tract. The kinetics of CD4+ T-cell decrease and highly active antiretroviral therapy (HAART)-mediated immune reconstitution were evaluated. Rectosigmoid colonic (RSC) biopsies and blood samples of nine patients with acute HIV infection were collected. CD4+ T-cell count, HIV RNA, intracellular HIV DNA and messenger RNA cytokine expression were evaluated before and after 6 months of HAART. All nine patients presented symptomatic retroviral infection. Early HAART was associated with a sustained and comparable reduction of HIV RNA in plasma, peripheral blood mononuclear cells (PBMCs) and RSC biopsies. HIV DNA decreased in PBMCs, but was only marginally reduced in RSC biopsies. Comparisons between reduction rates of HIV DNA in these two compartments confirmed that HIV DNA clearance was less efficient in RSC biopsies compared with PBMCs. Assessment of immunological profiles in PBMCs and RSC biopsies showed that the T-helper (Th)1-like/Th2-like ratio was sharply decreased in RSC biopsies and increased in PBMCs throughout the study period. A persistent Th2-like profile was detected in RSC biopsies. Efficient clearing of HIV DNA observed in PBMCs correlated with the establishment of a more favourable Th1-like profile. A less efficient clearance of intracellular HIV DNA following early introduction of HAART is associated with persistent immunological impairment in gut-associated lymphoid tissue (GALT), which is reflected by the skewed expression of cytokines in this reservoir. The present study shows that early initiation of HAART, in the short-term, is not effective in containing the establishment of HIV infection and in reversing associated immunological GALT abnormalities.

Transfection of normal human and Chinese hamster DNA corrects diepoxybutane-induced chromosomal hypersensitivity of Fanconi anemia fibroblasts

International Nuclear Information System (INIS)

Shaham, M.; Adler, B.; Ganguly, S.; Chaganti, R.S.K.

1987-01-01

Cultured cells from individuals affected with Fanconi anemia (FA) exhibit spontaneous chromosome breakage and hypersensitivity to the cell killing and clastogenic effects of the difunctional alkylating agent diepoxybutane (DEB). The authors report here the correction of both of these DEB-hypersensitivity phenotypes of FA cells achieved by cotransfection of normal placental of Chinese hamster lung cell DNA and the plasmid pSV2-neo-SVgpt. Transfectants were selected for clonogenic survival after treatment with DEB at a dose of 5 μgml. At this dose of DEB, the clonogenicity of normal fibroblasts was reduced to 50% and that of FA fibroblasts was reduced to zero. DEB-resistant (DEB/sup r/) colonies selected in this system exhibited a normal response to DEB-induced chromosome breakage and resistance to repeated DEB treatment. The neo and gpt sequences were detected by Southern blot analysis of DNA from one of four DEB/sup r/ colonies independently derived from transfection of human DNA and one of three DEB/sup r/ colonies independently derived from transfection of Chinese hamster DNA. The results demonstrate that DNA sequences that complement the two hallmark cellular phenotypes (cellular and chromosomal hypersensitivity to alkylating agents) of FA are present in human as well as Chinese hamster DNA. The cloning of these genes using transfection strategies can be expected to enable molecular characterization of FA

Blood CXCR3+ CD4 T Cells Are Enriched in Inducible Replication Competent HIV in Aviremic Antiretroviral Therapy-Treated Individuals.

Science.gov (United States)

Banga, Riddhima; Procopio, Francesco A; Ruggiero, Alessandra; Noto, Alessandra; Ohmiti, Khalid; Cavassini, Matthias; Corpataux, Jean-Marc; Paxton, William A; Pollakis, Georgios; Perreau, Matthieu

2018-01-01

We recently demonstrated that lymph nodes (LNs) PD-1 + /T follicular helper (Tfh) cells from antiretroviral therapy (ART)-treated HIV-infected individuals were enriched in cells containing replication competent virus. However, the distribution of cells containing inducible replication competent virus has been only partially elucidated in blood memory CD4 T-cell populations including the Tfh cell counterpart circulating in blood (cTfh). In this context, we have investigated the distribution of (1) total HIV-infected cells and (2) cells containing replication competent and infectious virus within various blood and LN memory CD4 T-cell populations of conventional antiretroviral therapy (cART)-treated HIV-infected individuals. In the present study, we show that blood CXCR3-expressing memory CD4 T cells are enriched in cells containing inducible replication competent virus and contributed the most to the total pool of cells containing replication competent and infectious virus in blood. Interestingly, subsequent proviral sequence analysis did not indicate virus compartmentalization between blood and LN CD4 T-cell populations, suggesting dynamic interchanges between the two compartments. We then investigated whether the composition of blood HIV reservoir may reflect the polarization of LN CD4 T cells at the time of reservoir seeding and showed that LN PD-1 + CD4 T cells of viremic untreated HIV-infected individuals expressed significantly higher levels of CXCR3 as compared to CCR4 and/or CCR6, suggesting that blood CXCR3-expressing CD4 T cells may originate from LN PD-1 + CD4 T cells. Taken together, these results indicate that blood CXCR3-expressing CD4 T cells represent the major blood compartment containing inducible replication competent virus in treated aviremic HIV-infected individuals.

Viruses and kidney disease: beyond HIV.

Science.gov (United States)

Waldman, Meryl; Marshall, Vickie; Whitby, Denise; Kopp, Jeffrey B

2008-11-01

Human immunodeficiency virus (HIV)-infected patients may acquire new viral co-infections; they also may experience the reactivation or worsening of existing viral infections, including active, smoldering, or latent infections. HIV-infected patients may be predisposed to these viral infections owing to immunodeficiency or risk factors common to HIV and other viruses. A number of these affect the kidney, either by direct infection or by deposition of immune complexes. In this review we discuss the renal manifestations and treatment of hepatitis C virus, BK virus, adenovirus, cytomegalovirus, and parvovirus B19 in patients with HIV disease. We also discuss an approach to the identification of new viral renal pathogens, using a viral gene chip to identify viral DNA or RNA.

2012 HIV Diagnostics Conference: the molecular diagnostics perspective.

Science.gov (United States)

Branson, Bernard M; Pandori, Mark

2013-04-01

2012 HIV Diagnostic Conference Atlanta, GA, USA, 12-14 December 2012. This report highlights the presentations and discussions from the 2012 National HIV Diagnostic Conference held in Atlanta (GA, USA), on 12-14 December 2012. Reflecting changes in the evolving field of HIV diagnostics, the conference provided a forum for evaluating developments in molecular diagnostics and their role in HIV diagnosis. In 2010, the HIV Diagnostics Conference concluded with the proposal of a new diagnostic algorithm which included nucleic acid testing to resolve discordant screening and supplemental antibody test results. The 2012 meeting, picking up where the 2010 meeting left off, focused on scientific presentations that assessed this new algorithm and the role played by RNA testing and new developments in molecular diagnostics, including detection of total and integrated HIV-1 DNA, detection and quantification of HIV-2 RNA, and rapid formats for detection of HIV-1 RNA.

Detection of polyomavirus simian virus 40 tumor antigen DNA in AIDS-related systemic non-Hodgkin lymphoma

Science.gov (United States)

Vilchez, Regis A.; Lednicky, John A.; Halvorson, Steven J.; White, Zoe S.; Kozinetz, Claudia A.; Butel, Janet S.

2002-01-01

Systemic non-Hodgkin lymphoma (S-NHL) is a common malignancy during HIV infection, and it is hypothesized that infectious agents may be involved in the etiology. Epstein-Barr virus DNA is found in <40% of patients with AIDS-related S-NHL, suggesting that other oncogenic viruses, such as polyomaviruses, may play a role in pathogenesis. We analyzed AIDS-related S-NHL samples, NHL samples from HIV-negative patients, peripheral blood leukocytes from HIV-infected and -uninfected patients without NHL, and lymph nodes without tumors from HIV-infected patients. Specimens were examined by polymerase chain reaction analysis with use of primers specific for an N-terminal region of the oncoprotein large tumor antigen ( T-ag ) gene conserved among all three polyomaviruses (simian virus 40 [SV40], JC virus, and BK virus). Polyomavirus T-ag DNA sequences, proven to be SV40-specific, were detected more frequently in AIDS-related S-NHL samples (6 of 26) than in peripheral blood leukocytes from HIV-infected patients (6 of 26 vs. 0 of 69; p =.0001), NHL samples from HIV-negative patients (6 of 26 vs. 0 of 10; p =.09), or lymph nodes (6 of 26 vs. 0 of 7; p =.16). Sequences of C-terminal T-ag DNA from SV40 were amplified from two AIDS-related S-NHL samples. Epstein-Barr virus DNA sequences were detected in 38% (10 of 26) AIDS-related S-NHL samples, 50% (5 of 10) HIV-negative S-NHL samples, and 57% (4 of 7) lymph nodes. None of the S-NHL samples were positive for both Epstein-Barr virus DNA and SV40 DNA. Further studies of the possible role of SV40 in the pathogenesis of S-NHL are warranted.

T-cell tropism of simian T-cell leukaemia virus type 1 and cytokine profiles in relation to proviral load and immunological changes during chronic infection of naturally infected mandrills (Mandrillus sphinx).

Science.gov (United States)

Souquière, Sandrine; Mouinga-Ondeme, Augustin; Makuwa, Maria; Beggio, Paola; Radaelli, Antonia; De Giuli Morghen, Carlo; Mortreux, Franck; Kazanji, Mirdad

2009-08-01

Although a wide variety of non-human primates are susceptible to simian T-cell leukaemia virus type 1 (STLV-1), little is known about the virological or molecular determinants of natural STLV-1 infection. We determined STLV-1 virus tropism in vivo and its relation to the immune response by evaluating cytokine production and T-cell subsets in naturally infected and uninfected mandrills. With real-time PCR methods, we found that STLV-1 in mandrills infects both CD4(+) and CD8(+) T cells; however, proviral loads were significantly higher (P = 0.01) in CD4(+) than in CD8(+) cells (mean STLV-1 copies number per 100 cells (+/- SD) was 7.8 +/- 8 in CD4(+) T cells and 3.9 +/- 4.5 in CD8(+) T cells). After culture, STLV-1 provirus was detected in enriched CD4(+) but not in enriched CD8(+) T cells. After 6 months of culture, STLV-1-transformed cell lines expressing CD3(+), CD4(+) and HLADR(+) were established, and STLV-1 proteins and tax/rex mRNA were detected. In STLV-1 infected monkeys, there was a correlation between high proviral load and elevated levels of interleukin (IL)-2, IL-6, IL-10, interferon-gamma and tumour necrosis factor-alpha. The two monkeys with the highest STLV-1 proviral load had activated CD4(+)HLADR(+) and CD8(+)HLADR(+) T-cell subsets and a high percentage of CD25(+) in CD4(+) and CD8(+) T cells. Our study provides the first cellular, immunological and virological characterization of natural STLV-1 infection in mandrills and shows that they are an appropriate animal model for further physiopathological studies of the natural history of human T-cell leukaemia viruses.

Chromosomal locations of members of a family of novel endogenous human retroviral genomes

International Nuclear Information System (INIS)

Horn, T.M.; Huebner, K.; Croce, C.; Callahan, R.

1986-01-01

Human cellular DNA contains two distinguishable families of retroviral related sequences. One family shares extensive nucleotide sequence homology with infectious mammalian type C retroviral genomes. The other family contains major regions of homology with the pol genes of infectious type A and B and avian type C and D retroviral genomes. Analysis of the human recombinant clone HLM-2 has shown that the pol gene in the latter family is located within an endogenous proviral genome. The authors show that the proviral genome in HLM-2 and the related recombinant clone HLM-25 are located, respectively, on human chromosomes 1 and 5. Other related proviral genomes are located on chromosomes 7, 8, 11, 14, and 17

Detection of Cytomegalovirus DNA in Serum Correlates with Clinical Cytomegalovirus Retinitis in AIDS

DEFF Research Database (Denmark)

Hansen, K.K.; Ricksten, A.; Hofmann, B.

1994-01-01

The high sensitivity of nested polymerase chain reaction (PCR) offers the possibility of rapid detection of cytomegalovirus (CMV) DNA in serum. Five consecutive serum samples were examined from 52 human immunodeficiency virus (HIV)-seropositive patients (19 of whom had clinically presumed diagnosis...... became positive with the onset of clinical retinitis. In contrast, 29 of 33 HIV-seropositive subjects without clinical CMV chorioretinitis and matched with respect to age and CD4 T cell numbers were negative for CMV DNA in all 5 serum samples. Thus, the presence of CMV DNA in serum analyzed by PCR...... is a good predictive marker of CMV retinitis in HIV-seropositive subjects. A positive PCR results supports the clinical diagnosis and may be useful for monitoring response to antiviral treatment....

An "escape clock" for estimating the turnover of SIV DNA in resting CD4⁺ T cells.

Directory of Open Access Journals (Sweden)

Jeanette Reece

Full Text Available Persistence of HIV DNA presents a major barrier to the complete control of HIV infection under current therapies. Most studies suggest that cells with latently integrated HIV decay very slowly under therapy. However, it is much more difficult to study the turnover and persistence of HIV DNA during active infection. We have developed an "escape clock" approach for measuring the turnover of HIV DNA in resting CD4+ T cells. This approach studies the replacement of wild-type (WT SIV DNA present in early infection by CTL escape mutant (EM strains during later infection. Using a strain-specific real time PCR assay, we quantified the relative amounts of WT and EM strains in plasma SIV RNA and cellular SIV DNA. Thus we can track the formation and turnover of SIV DNA in sorted resting CD4+ T cells. We studied serial plasma and PBMC samples from 20 SIV-infected Mane-A*10 positive pigtail macaques that have a signature Gag CTL escape mutation. In animals with low viral load, WT virus laid down early in infection is extremely stable, and the decay of this WT species is very slow, consistent with findings in subjects on anti-retroviral medications. However, during active, high level infection, most SIV DNA in resting cells was turning over rapidly, suggesting a large pool of short-lived DNA produced by recent infection events. Our results suggest that, in order to reduce the formation of a stable population of SIV DNA, it will be important either to intervene very early or intervene during active replication.

HIV-1 Tat and AIDS-associated cancer: targeting the cellular anti-cancer barrier?

Directory of Open Access Journals (Sweden)

Daniel René

2008-05-01

Full Text Available Abstract The acquired immunodeficiency syndrome (AIDS is accompanied by a significant increase in the incidence of neoplasms. Several causative agents have been proposed for this phenomenon. These include immunodeficiency and oncogenic DNA viruses and the HIV-1 protein Tat. Cancer in general is closely linked to genomic instability and DNA repair mechanisms. The latter maintains genomic stability and serves as a cellular anti-cancer barrier. Defects in DNA repair pathway are associated with carcinogenesis. This review focuses on newly discovered connections of the HIV-1 protein Tat, as well as cellular co-factors of Tat, to double-strand break DNA repair. We propose that the Tat-induced DNA repair deficiencies may play a significant role in the development of AIDS-associated cancer.

HIV vaccines: new frontiers in vaccine development.

Science.gov (United States)

Duerr, Ann; Wasserheit, Judith N; Corey, Lawrence

2006-08-15

A human immunodeficiency virus (HIV) vaccine is the most promising and feasible strategy to prevent the events during acute infection that simultaneously set the course of the epidemic in the community and the course of the disease for the individual. Because safety concerns limit the use of live, attenuated HIV and inactivated HIV, a variety of alternate approaches is being investigated. Traditional antibody-mediated approaches using recombinant HIV envelope proteins have shown no efficacy in 2 phase III trials. Current HIV vaccine trials are focusing primarily on cytotoxic T lymphocyte-mediated products that use viral vectors, either alone or as boosts to DNA plasmids that contain viral genes. The most immunogenic of these products appear to be the recombinant adenovirus vector vaccines, 2 of which are now in advanced clinical development.

Multicolour probes for sequence-specific DNA detection based on graphene oxide.

Science.gov (United States)

Zhu, Qing; Xiang, Dongshan; Zhang, Cuiling; Ji, Xinghu; He, Zhike

2013-09-21

The bifunctionality of graphene oxide (GO) which can highly adsorb single-stranded DNA (ssDNA) and effectively quench the emission of organic dyes is reasonably utilized in a multiplexed DNA detection system, achieving sensitive and selective detection of HIV, VV and EV, respectively.

Detection of free circulating Epstein-Barr virus DNA in plasma of patients with Hodgkin’s disease

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Juliane Garcez Musacchio

Full Text Available CONTEXT AND OBJECTIVE: Free circulating Epstein-Barr virus (EBV DNA is often present in the plasma of Hodgkin’s disease patients. The aim here was to evaluate the prevalence of this finding, its correlation with the immunohistochemical expression of LMP-1 (latent membrane protein 1 and the influence of other clinical factors. DESIGN AND SETTING: Prospective study in two public tertiary institutions: Hematology Service, Universidade Federal do Rio de Janeiro, and Oncology Service, Instituto Nacional do Câncer, Rio de Janeiro. METHODS: A cohort of 30 patients with newly diagnosed Hodgkin’s disease was studied. The control group consisted of 13 healthy adult volunteers. EBV DNA was determined by conventional polymerase chain reaction (PCR. RESULTS: The median age was 28 years, and 16 patients were women. Advanced disease was present in 19 patients, and six were HIV-positive. EBV DNA was present in the plasma of 13 patients and one control (43% versus 8%, p = 0.03. EBV DNA prevalence was higher in HIV-positive patients (100% versus 29%, p = 0.0007 and those with advanced disease (63% versus 9%, p = 0.006. Among HIV-negative patients alone, EBV DNA prevalence remained higher in those with advanced disease. EBV DNA was found in 10/11 patients with LMP-1 expression in the lymph nodes, and in 3/19 without LMP-1 expression (kappa coefficient = 0.72. CONCLUSION: EBV DNA was present in 91% of patients with EBV-associated Hodgkin’s disease, and in all patients with HIV-associated Hodgkin’s disease. EBV DNA prevalence was higher in patients with advanced disease, irrespective of HIV status.

Low prevalence of liver disease but regional differences in HBV treatment characteristics mark HIV/HBV co-infection in a South African HIV clinical trial.

Directory of Open Access Journals (Sweden)

Prudence Ive

Full Text Available Hepatitis B virus (HBV infection is endemic in South Africa however, there is limited data on the degree of liver disease and geographic variation in HIV/HBV coinfected individuals. In this study, we analysed data from the CIPRA-SA 'Safeguard the household study' in order to assess baseline HBV characteristics in HIV/HBV co-infection participants prior to antiretroviral therapy (ART initiation.812 participants from two South African townships Soweto and Masiphumelele were enrolled in a randomized trial of ART (CIPRA-SA. Participants were tested for hepatitis B surface antigen (HBsAg, hepatitis B e antigen (HBeAg, and HBV DNA. FIB-4 scores were calculated at baseline.Forty-eight (5.9% were HBsAg positive, of whom 28 (58.3% were HBeAg positive. Of those with HBV, 29.8% had an HBV DNA<2000 IU/ml and ALT<40 IU/ml ; 83.0% had a FIB-4 score <1.45, consistent with absent or minimal liver disease. HBV prevalence was 8.5% in Masiphumelele compared to 3.8% in Soweto (relative risk 2.3; 95% CI: 1.3-4.0. More participants in Masiphumelele had HBeAg-negative disease (58% vs. 12%, p = 0.002 and HBV DNA levels ≤2000 IU/ml, (43% vs. 6% p<0.007.One third of HIV/HBV co-infected subjects had low HBV DNA levels and ALT while the majority had indicators of only mild liver disease. There were substantial regional differences in HBsAg and HbeAg prevalence in HIV/HBV co-infection between two regions in South Africa. This study highlights the absence of severe liver disease and the marked regional differences in HIV/HBV co-infection in South Africa and will inform treatment decisions in these populations.

Identification of a premature termination of DNA polymerization in ...

Indian Academy of Sciences (India)

2013-04-25

Apr 25, 2013 ... strands to the 5′ end of the template strands, forming double- stranded DNA with ... duplex stem is important for high processive polymerization. HIV-1 reverse ... KF has been used as a model to study DNA polymerase.

Evolutionary genomics and HIV restriction factors.

Science.gov (United States)

Pyndiah, Nitisha; Telenti, Amalio; Rausell, Antonio

2015-03-01

To provide updated insights into innate antiviral immunity and highlight prototypical evolutionary features of well characterized HIV restriction factors. Recently, a new HIV restriction factor, Myxovirus resistance 2, has been discovered and the region/residue responsible for its activity identified using an evolutionary approach. Furthermore, IFI16, an innate immunity protein known to sense several viruses, has been shown to contribute to the defense to HIV-1 by causing cell death upon sensing HIV-1 DNA. Restriction factors against HIV show characteristic signatures of positive selection. Different patterns of accelerated sequence evolution can distinguish antiviral strategies--offense or defence--as well as the level of specificity of the antiviral properties. Sequence analysis of primate orthologs of restriction factors serves to localize functional domains and sites responsible for antiviral action. We use recent discoveries to illustrate how evolutionary genomic analyses help identify new antiviral genes and their mechanisms of action.

KIR-HLA genotypes in HIV-infected patients lacking immunological recovery despite effective antiretroviral therapy.

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Alessandro Soria

Full Text Available BACKGROUND: In HIV-infected individuals, mechanisms underlying unsatisfactory immune recovery during effective combination antiretroviral therapy (cART have yet to be fully understood. We investigated whether polymorphism of genes encoding immune-regulating molecules, such as killer immunoglobulin-like receptors (KIR and their ligands class I human leukocyte antigen (HLA, could influence immunological response to cART. METHODS: KIR and HLA frequencies were analyzed in 154 HIV-infected and cART-treated patients with undetectable viral load divided into two groups: 'immunological non responders' (INR, N = 50, CD4(+ T-cell count 350/mm(3. Molecular KIR were typed using polymerase chain reaction-based genotyping. Comparisons were adjusted for baseline patient characteristics. RESULTS: The frequency of KIR2DL3 allele was significantly higher in FR than in INR (83.7% vs. 62%, P = 0.005. The functional compound genotype HLA-C1(+/KIR2DL3(+, even at multivariable analysis, when adjusted for nadir CD4(+ T-cell count, was associated with reduced risk of INR status: odds ratio (95% Confidence Intervals 0.34 (0.13-0.88, P = 0.03. CONCLUSIONS: Reduced presence of the inhibitory KIR2DL3 genotype detected in INR might provoke an imbalance in NK function, possibly leading to increased immune activation, impaired killing of latently infected cells, and higher proviral burden. These factors would hinder full immune recovery during therapy.

Subclinical herpesvirus shedding among HIV-1-infected men on antiretroviral therapy.

Science.gov (United States)

Agudelo-Hernandez, Arcadio; Chen, Yue; Bullotta, Arlene; Buchanan, William G; Klamar-Blain, Cynthia R; Borowski, Luann; Riddler, Sharon A; Rinaldo, Charles R; Macatangay, Bernard J C

2017-09-24

We evaluated the subclinical shedding of six different herpesviruses in antiretroviral drug-treated HIV-positive [HIV(+)] MSM, and determined how this is associated with markers of inflammation and immune activation. We obtained blood, semen, throat washing, urine, and stool from 15 antiretroviral-treated HIV-1-infected MSM with CD4 T-cell reconstitution, and 12 age-matched HIV-negative [HIV (-)] MSM from the Multicenter AIDS Cohort Study at four timepoints over 24 weeks to measure DNA levels of cytomegalovirus (CMV), Epstein-Barr virus (EBV), herpes simplex virus 1 and 2, human herpesvirus 6 (HHV6), and HHV8. T-cell activation and plasma levels of soluble markers of inflammation and activation were also measured at the corresponding timepoints. HIV(+) participants had a trend for higher total herpesvirus shedding rate. HIV(+) participants also had a significantly higher rate of shedding EBV and CMV compared with the HIV(-) group. Herpesvirus shedding was mostly seen in throat washings. In the HIV(+) group, herpesvirus shedding rate inversely correlated with plasma levels of interferon γ-induced protein 10 and soluble CD163. CMV DNA levels negatively correlated with levels of T-cell activation. There was a trend for a positive correlation between EBV shedding rate and plasma soluble CD14. HHV6 shedding rate negatively correlated with plasma levels of interleukin-6, soluble CD163, and interferon gamma-induced protein 10. Correlations were not observed among HIV(-) individuals. Among treated HIV-infected MSM, there are higher subclinical shedding rates of some herpesviruses that occur in different body compartments and negatively correlate with levels of inflammation and immune activation.

Analysis of HIV using a high resolution melting (HRM) diversity assay: automation of HRM data analysis enhances the utility of the assay for analysis of HIV incidence.

Science.gov (United States)

Cousins, Matthew M; Swan, David; Magaret, Craig A; Hoover, Donald R; Eshleman, Susan H

2012-01-01

HIV diversity may be a useful biomarker for discriminating between recent and non-recent HIV infection. The high resolution melting (HRM) diversity assay was developed to quantify HIV diversity in viral populations without sequencing. In this assay, HIV diversity is expressed as a single numeric HRM score that represents the width of a melting peak. HRM scores are highly associated with diversity measures obtained with next generation sequencing. In this report, a software package, the HRM Diversity Assay Analysis Tool (DivMelt), was developed to automate calculation of HRM scores from melting curve data. DivMelt uses computational algorithms to calculate HRM scores by identifying the start (T1) and end (T2) melting temperatures for a DNA sample and subtracting them (T2 - T1 =  HRM score). DivMelt contains many user-supplied analysis parameters to allow analyses to be tailored to different contexts. DivMelt analysis options were optimized to discriminate between recent and non-recent HIV infection and to maximize HRM score reproducibility. HRM scores calculated using DivMelt were compared to HRM scores obtained using a manual method that is based on visual inspection of DNA melting curves. HRM scores generated with DivMelt agreed with manually generated HRM scores obtained from the same DNA melting data. Optimal parameters for discriminating between recent and non-recent HIV infection were identified. DivMelt provided greater discrimination between recent and non-recent HIV infection than the manual method. DivMelt provides a rapid, accurate method of determining HRM scores from melting curve data, facilitating use of the HRM diversity assay for large-scale studies.

Potent nonnucleoside reverse transcriptase inhibitors target HIV-1 Gag-Pol.

Directory of Open Access Journals (Sweden)

Anna Figueiredo

2006-11-01

Full Text Available Nonnucleoside reverse transcriptase inhibitors (NNRTIs target HIV-1 reverse transcriptase (RT by binding to a pocket in RT that is close to, but distinct, from the DNA polymerase active site and prevent the synthesis of viral cDNA. NNRTIs, in particular, those that are potent inhibitors of RT polymerase activity, can also act as chemical enhancers of the enzyme's inter-subunit interactions. However, the consequences of this chemical enhancement effect on HIV-1 replication are not understood. Here, we show that the potent NNRTIs efavirenz, TMC120, and TMC125, but not nevirapine or delavirdine, inhibit the late stages of HIV-1 replication. These potent NNRTIs enhanced the intracellular processing of Gag and Gag-Pol polyproteins, and this was associated with a decrease in viral particle production from HIV-1-transfected cells. The increased polyprotein processing is consistent with premature activation of the HIV-1 protease by NNRTI-enhanced Gag-Pol multimerization through the embedded RT sequence. These findings support the view that Gag-Pol multimerization is an important step in viral assembly and demonstrate that regulation of Gag-Pol/Gag-Pol interactions is a novel target for small molecule inhibitors of HIV-1 production. Furthermore, these drugs can serve as useful probes to further understand processes involved in HIV-1 particle assembly and maturation.

Hairpin-induced tRNA-mediated (HITME) recombination in HIV-1

NARCIS (Netherlands)

Konstantinova, Pavlina; de Haan, Peter; Das, Atze T.; Berkhout, Ben

2006-01-01

Recombination due to template switching during reverse transcription is a major source of genetic variability in retroviruses. In the present study we forced a recombination event in human immunodeficiency virus type 1 (HIV-1) by electroporation of T cells with DNA from a molecular HIV-1 clone that

Cyclophilin B enhances HIV-1 infection.

Science.gov (United States)

DeBoer, Jason; Madson, Christian J; Belshan, Michael

2016-02-01

Cyclophilin B (CypB) is a member of the immunophilin family and intracellular chaperone. It predominantly localizes to the ER, but also contains a nuclear localization signal and is secreted from cells. CypB has been shown to interact with the Gag protein of human immunodeficiency type 1 (HIV-1). Several proteomic and genetic studies identified it as a potential factor involved in HIV replication. Herein, we show that over-expression of CypB enhances HIV infection by increasing nuclear import of viral DNA. This enhancement was unaffected by cyclosporine treatment and requires the N-terminus of the protein. The N-terminus contains an ER leader sequence, putative nuclear localization signal, and is required for secretion. Deletion of the N-terminus resulted in mislocalization from the ER and suppression of HIV infection. Passive transfer experiments showed that secreted CypB did not impact HIV infection. Combined, these experiments show that intracellular CypB modulates a pathway of HIV nuclear import. Copyright © 2015 Elsevier Inc. All rights reserved.

Epstein-Barr virus and human immunodeficiency virus serological responses and viral burdens in HIV-infected patients treated with HAART

Science.gov (United States)

O'Sullivan, Cathal E.; Peng, RongSheng; Cole, Kelly Stefano; Montelaro, Ronald C.; Sturgeon, Timothy; Jenson, Hal B.; Ling, Paul D.; Butel, J. S. (Principal Investigator)

2002-01-01

Epstein-Barr virus (EBV) associated non-Hodgkin lymphoma is recognized as a complication of human immunodeficiency virus (HIV) infection. Little is known regarding the influence of highly active antiretroviral therapy (HAART) on the biology of EBV in this population. To characterize the EBV- and HIV-specific serological responses together with EBV DNA levels in a cohort of HIV-infected adults treated with HAART, a study was conducted to compare EBV and HIV serologies and EBV DNA copy number (DNAemia) over a 12-month period after the commencement of HAART. All patients were seropositive for EBV at baseline. Approximately 50% of patients had detectable EBV DNA at baseline, and 27/30 had detectable EBV DNA at some point over the follow-up period of 1 year. Changes in EBV DNA copy number over time for any individual were unpredictable. Significant increases in the levels of Epstein-Barr nuclear antigen (EBNA) and Epstein-Barr early antigen (EA) antibodies were demonstrated in the 17 patients who had a good response to HAART. Of 29 patients with paired samples tested, four-fold or greater increases in titers were detected for EA in 12/29 (41%), for EBNA in 7/29 (24%), for VCA-IgG in 4/29 (14%); four-fold decreases in titers were detected in 2/29 (7%) for EA and 12/29 (41%) for EBNA. A significant decline in the titer of anti-HIV antibodies was also demonstrated. It was concluded that patients with advanced HIV infection who respond to HAART have an increase in their EBV specific antibodies and a decrease in their HIV-specific antibodies. For the cohort overall, there was a transient increase in EBV DNA levels that had declined by 12 months. Copyright 2002 Wiley-Liss, Inc.

Epidemiological and clinical characteristics of hepatitis B virus in HIV-infected patients in Guangdong, China.

Science.gov (United States)

Huang, S M; Cai, W P; Hu, F Y; Lan, Y; Liao, B L; Chen, Y P; Tang, X P

2016-09-01

This study investigated the epidemiological and clinical characteristics of hepatitis B virus (HBV) in HIV-infected adults at the time of antiretroviral therapy (ART) initiation in Guangdong province, China. A total of 2793 HIV-infected adults were enrolled between January 2004 and September 2011. Demographic data and laboratory parameters were collected, HBV-DNA levels were measured, and HBV genotypes were identified before ART initiation. The prevalence of hepatitis B surface antigen (HBsAg) in HIV-infected patients was 13.2%. A total of 266 HIV/HBV co-infected patients and 1469 HIV mono-infected patients were recruited. The median alanine aminotransferase and aspartate aminotransferase levels of HIV/HBV co-infected patients were higher than HIV mono-infected patients (32 U/L vs. 22 U/L, p HIV/HBV co-infected patients was lower than HIV mono-infected patients (59 cells/mm(3) vs. 141 cells/mm(3), p study indicates a high prevalence of HBsAg in HIV-infected adults in Guangdong. The level of CD4 cell count in HIV/HBV co-infected patients was much lower than HIV mono-infected patients, especially in patients who were HBeAg-positive and had a high level of HBV-DNA. The predominant HBV genotype in HIV/HBV co-infected patients is genotype B. © The Author(s) 2015.

Determination of HIV status of infants born to HIV-infected mothers: A review of the diagnostic methods with special focus on the applicability of p24 antigen testing in developing countries

DEFF Research Database (Denmark)

Wessman, Maria J; Theilgaard, Zahra Persson; Katzenstein, Terese L

2012-01-01

Abstract In 2009, 2.5 million children under the age of 15 y were living with human immunodeficiency virus/acquired immune deficiency syndrome (HIV/AIDS); 370,000 were diagnosed with HIV and 260,000 died due to AIDS. More than 90% of the children infected with HIV live in sub-Saharan Africa. Most...... children infected with HIV contract the infection in utero, during delivery, or via breast milk. This review outlines the current diagnostic methods to determine the HIV status of infants born to HIV-infected mothers. The HIV DNA and RNA polymerase chain reaction (PCR) tests are highly accurate...

Susceptibility Testing by Polymerase Chain Reaction DNA Quantitation: A Method to Measure Drug Resistance of Human Immunodeficiency Virus Type 1 Isolates

Science.gov (United States)

Eron, Joseph J.; Gorczyca, Paul; Kaplan, Joan C.; D'Aquila, Richard T.

1992-04-01

Polymerase chain reaction (PCR) DNA quantitation (PDQ) susceptibility testing rapidly and directly measures nucleoside sensitivity of human immunodeficiency virus type 1 (HIV-1) isolates. PCR is used to quantitate the amount of HIV-1 DNA synthesized after in vitro infection of peripheral blood mononuclear cells. The relative amounts of HIV-1 DNA in cell lysates from cultures maintained at different drug concentrations reflect drug inhibition of virus replication. The results of PDQ susceptibility testing of 2- or 3-day cultures are supported by assays measuring HIV-1 p24 antigen production in supernatants of 7- or 10-day cultures. DNA sequence analyses to identify mutations in the reverse transcriptase gene that cause resistance to 3'-azido-3'-deoxythymidine also support the PDQ results. With the PDQ method, both infectivity titration and susceptibility testing can be performed on supernatants from primary cultures of peripheral blood mononuclear cells. PDQ susceptibility testing should facilitate epidemiologic studies of the clinical significance of drug-resistant HIV-1 isolates.

Epstein-Barr virus DNA loads in adult human immunodeficiency virus type 1-infected patients receiving highly active antiretroviral therapy

Science.gov (United States)

Ling, Paul D.; Vilchez, Regis A.; Keitel, Wendy A.; Poston, David G.; Peng, Rong Sheng; White, Zoe S.; Visnegarwala, Fehmida; Lewis, Dorothy E.; Butel, Janet S.

2003-01-01

Patients with human immunodeficiency virus type 1 (HIV-1) infection are at high risk of developing Epstein-Barr virus (EBV)-associated lymphoma. However, little is known of the EBV DNA loads in patients receiving highly active antiretroviral therapy (HAART). Using a real-time quantitative polymerase chain reaction assay, we demonstrated that significantly more HIV-1-infected patients receiving HAART than HIV-1-uninfected volunteers had detectable EBV DNA in blood (57 [81%] of 70 vs. 11 [16%] of 68 patients; P=.001) and saliva (55 [79%] of 68 vs. 37 [54%] of 68 patients; P=.002). The mean EBV loads in blood and saliva samples were also higher in HIV-1-infected patients than in HIV-1-uninfected volunteers (P=.001). The frequency of EBV detection in blood was associated with lower CD4+ cell counts (P=.03) among HIV-1-infected individuals, although no differences were observed in the EBV DNA loads in blood or saliva samples in the HIV-1-infected group. Additional studies are needed to determine whether EBV-specific CD4+ and CD8+ cells play a role in the pathogenesis of EBV in HIV-1-infected patients receiving HAART.

ORIGINAL ARTICLES HBV/HIV co-infection: The dynamics of HBV ...

African Journals Online (AJOL)

B virus (HBV) exposure, and an estimated 400 million are chronically infected.1 ... transplantation, cancer and HIV/AIDS might induce reactivation of occult HBV with ... productive infection.7-9 Occult HBV infection refers to the presence of HBV DNA without ... CD4+ cell count of <200 cells/µl in patients infected with HIV.

EV02: a Phase I trial to compare the safety and immunogenicity of HIV DNA-C prime-NYVAC-C boost to NYVAC-C alone.

Science.gov (United States)

McCormack, Sheena; Stöhr, Wolfgang; Barber, Tristan; Bart, Pierre-Alexandre; Harari, Alexandre; Moog, Christiane; Ciuffreda, Donatella; Cellerai, Cristina; Cowen, Miranda; Gamboni, Romilda; Burnet, Séverine; Legg, Ken; Brodnicki, Elizabeth; Wolf, Hans; Wagner, Ralf; Heeney, Jonathan; Frachette, Marie-Joëlle; Tartaglia, Jim; Babiker, Abdel; Pantaleo, Giuseppe; Weber, Jonathan

2008-06-13

The aim of this randomised controlled trial was to see if the addition of 4 mg/ml DNA-C priming given by the intramuscular route at weeks 0 and 4 to NYVAC-C at weeks 20 and 24, safely increased the proportion of participants with HIV-specific T-cell responses measured by the interferon (IFN)-gamma ELISpot assay at weeks 26 and/or 28 compared to NYVAC-C alone. Although 2 individuals discontinued after the first DNA-C due to adverse events (1 vaso-vagal; 1 transient, asymptomatic elevation in alanine transaminase), the vaccines were well tolerated. Three others failed to complete the regimen (1 changed her mind; 2 lost to follow-up). Of the 35 that completed the regimen 90% (18/20) in the DNA-C group had ELISpot responses compared to 33% (5/15) that received NYVAC-C alone (p=0.001). Responses were to envelope in the majority (21/23). Of the 9 individuals with responses to envelope and other peptides, 8 were in the DNA-C group. These promising results suggest that DNA-C was an effective priming agent, that merits further investigation.

Cyclophilin B enhances HIV-1 infection

Energy Technology Data Exchange (ETDEWEB)

DeBoer, Jason; Madson, Christian J. [Department of Medical Microbiology and Immunology, Creighton University, Omaha, NE (United States); Belshan, Michael, E-mail: michaelbelshan@creighton.edu [Department of Medical Microbiology and Immunology, Creighton University, Omaha, NE (United States); The Nebraska Center for Virology, University of Nebraska, Lincoln, NE (United States)

2016-02-15

Cyclophilin B (CypB) is a member of the immunophilin family and intracellular chaperone. It predominantly localizes to the ER, but also contains a nuclear localization signal and is secreted from cells. CypB has been shown to interact with the Gag protein of human immunodeficiency type 1 (HIV-1). Several proteomic and genetic studies identified it as a potential factor involved in HIV replication. Herein, we show that over-expression of CypB enhances HIV infection by increasing nuclear import of viral DNA. This enhancement was unaffected by cyclosporine treatment and requires the N-terminus of the protein. The N-terminus contains an ER leader sequence, putative nuclear localization signal, and is required for secretion. Deletion of the N-terminus resulted in mislocalization from the ER and suppression of HIV infection. Passive transfer experiments showed that secreted CypB did not impact HIV infection. Combined, these experiments show that intracellular CypB modulates a pathway of HIV nuclear import. - Highlights: • CypB has been identified in several proteomic studies of HIV-1 infection. • CypB expression is upregulated in activated and infected T-cells. • Over-expression of CypB enhances HIV nuclear import and infection. • The N-terminus of CypB is necessary for these effects.

Cyclophilin B enhances HIV-1 infection

International Nuclear Information System (INIS)

DeBoer, Jason; Madson, Christian J.; Belshan, Michael

2016-01-01

Cyclophilin B (CypB) is a member of the immunophilin family and intracellular chaperone. It predominantly localizes to the ER, but also contains a nuclear localization signal and is secreted from cells. CypB has been shown to interact with the Gag protein of human immunodeficiency type 1 (HIV-1). Several proteomic and genetic studies identified it as a potential factor involved in HIV replication. Herein, we show that over-expression of CypB enhances HIV infection by increasing nuclear import of viral DNA. This enhancement was unaffected by cyclosporine treatment and requires the N-terminus of the protein. The N-terminus contains an ER leader sequence, putative nuclear localization signal, and is required for secretion. Deletion of the N-terminus resulted in mislocalization from the ER and suppression of HIV infection. Passive transfer experiments showed that secreted CypB did not impact HIV infection. Combined, these experiments show that intracellular CypB modulates a pathway of HIV nuclear import. - Highlights: • CypB has been identified in several proteomic studies of HIV-1 infection. • CypB expression is upregulated in activated and infected T-cells. • Over-expression of CypB enhances HIV nuclear import and infection. • The N-terminus of CypB is necessary for these effects.

Phosphate-methylated DNA aimed at HIV-1 RNA loops and integrated DNA inhibits viral infectivity

NARCIS (Netherlands)

Buck, H. M.; Koole, L. H.; van Genderen, M. H.; Smit, L.; Geelen, J. L.; Jurriaans, S.; Goudsmit, J.

1990-01-01

Phosphate-methylated DNA hybridizes strongly and specifically to natural DNA and RNA. Hybridization to single-stranded and double-stranded DNA leads to site-selective blocking of replication and transcription. Phosphate-methylated DNA was used to interrupt the life cycle of the human

DNA cytosine methylation in the bovine leukemia virus promoter is associated with latency in a lymphoma-derived B-cell line: potential involvement of direct inhibition of cAMP-responsive element (CRE)-binding protein/CRE modulator/activation transcription factor binding.

Science.gov (United States)

Pierard, Valérie; Guiguen, Allan; Colin, Laurence; Wijmeersch, Gaëlle; Vanhulle, Caroline; Van Driessche, Benoît; Dekoninck, Ann; Blazkova, Jana; Cardona, Christelle; Merimi, Makram; Vierendeel, Valérie; Calomme, Claire; Nguyên, Thi Liên-Anh; Nuttinck, Michèle; Twizere, Jean-Claude; Kettmann, Richard; Portetelle, Daniel; Burny, Arsène; Hirsch, Ivan; Rohr, Olivier; Van Lint, Carine

2010-06-18

Bovine leukemia virus (BLV) proviral latency represents a viral strategy to escape the host immune system and allow tumor development. Besides the previously demonstrated role of histone deacetylation in the epigenetic repression of BLV expression, we showed here that BLV promoter activity was induced by several DNA methylation inhibitors (such as 5-aza-2'-deoxycytidine) and that overexpressed DNMT1 and DNMT3A, but not DNMT3B, down-regulated BLV promoter activity. Importantly, cytosine hypermethylation in the 5'-long terminal repeat (LTR) U3 and R regions was associated with true latency in the lymphoma-derived B-cell line L267 but not with defective latency in YR2 cells. Moreover, the virus-encoded transactivator Tax(BLV) decreased DNA methyltransferase expression levels, which could explain the lower level of cytosine methylation observed in the L267(LTaxSN) 5'-LTR compared with the L267 5'-LTR. Interestingly, DNA methylation inhibitors and Tax(BLV) synergistically activated BLV promoter transcriptional activity in a cAMP-responsive element (CRE)-dependent manner. Mechanistically, methylation at the -154 or -129 CpG position (relative to the transcription start site) impaired in vitro binding of CRE-binding protein (CREB) transcription factors to their respective CRE sites. Methylation at -129 CpG alone was sufficient to decrease BLV promoter-driven reporter gene expression by 2-fold. We demonstrated in vivo the recruitment of CREB/CRE modulator (CREM) and to a lesser extent activating transcription factor-1 (ATF-1) to the hypomethylated CRE region of the YR2 5'-LTR, whereas we detected no CREB/CREM/ATF recruitment to the hypermethylated corresponding region in the L267 cells. Altogether, these findings suggest that site-specific DNA methylation of the BLV promoter represses viral transcription by directly inhibiting transcription factor binding, thereby contributing to true proviral latency.

Natural HIV-1 NEF accelerates virus replication in primary human lymphocytes

NARCIS (Netherlands)

de Ronde, A.; Klaver, B.; Keulen, W.; Smit, L.; Goudsmit, J.

1992-01-01

HIV-1 NEF genes were isolated directly from peripheral blood lymphocyte DNA of two HIV-1-infected individuals and cloned into an HXB-2-infectious molecular clone. The effect of NEF on virus production in T-cell lines and primary human lymphocytes was studied. Naturally occurring NEF accelerates

Novel mechanisms to inhibit HIV reservoir seeding using Jak inhibitors.

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Christina Gavegnano

2017-12-01

Full Text Available Despite advances in the treatment of HIV infection with ART, elucidating strategies to overcome HIV persistence, including blockade of viral reservoir establishment, maintenance, and expansion, remains a challenge. T cell homeostasis is a major driver of HIV persistence. Cytokines involved in regulating homeostasis of memory T cells, the major hub of the HIV reservoir, trigger the Jak-STAT pathway. We evaluated the ability of tofacitinib and ruxolitinib, two FDA-approved Jak inhibitors, to block seeding and maintenance of the HIV reservoir in vitro. We provide direct demonstration for involvement of the Jak-STAT pathway in HIV persistence in vivo, ex vivo, and in vitro; pSTAT5 strongly correlates with increased levels of integrated viral DNA in vivo, and in vitro Jak inhibitors reduce the frequency of CD4+ T cells harboring integrated HIV DNA. We show that Jak inhibitors block viral production from infected cells, inhibit γ-C receptor cytokine (IL-15-induced viral reactivation from latent stores thereby preventing transmission of infectious particles to bystander activated T cells. These results show that dysregulation of the Jak-STAT pathway is associated with viral persistence in vivo, and that Jak inhibitors target key events downstream of γ-C cytokine (IL-2, IL-7 and IL-15 ligation to their receptors, impacting the magnitude of the HIV reservoir in all memory CD4 T cell subsets in vitro and ex vivo. Jak inhibitors represent a therapeutic modality to prevent key events of T cell activation that regulate HIV persistence and together, specific, potent blockade of these events may be integrated to future curative strategies.

Cloning and characterization of human DNA repair genes

International Nuclear Information System (INIS)

Thompson, L.H.; Brookman, K.W.; Weber, C.A.; Salazar, E.P.; Stewart, S.A.; Carrano, A.V.

1987-01-01

The isolation of two addition human genes that give efficient restoration of the repair defects in other CHO mutant lines is reported. The gene designated ERCC2 (Excision Repair Complementing Chinese hamster) corrects mutant UV5 from complementation group 1. They recently cloned this gene by first constructing a secondary transformant in which the human gene was shown to have become physically linked to the bacterial gpt dominant-marker gene by cotransfer in calcium phosphate precipitates in the primary transfection. Transformants expressing both genes were recovered by selecting for resistance to both UV radiation and mycophenolic acid. Using similar methods, the human gene that corrects CHO mutant EM9 was isolated in cosmids and named XRCC1 (X-ray Repair Complementing Chinese hamster). In this case, transformants were recovered by selecting for resistance to CldUrd, which kills EM9 very efficiently. In both genomic and cosmid transformants, the XRCC1 gene restored resistance to the normal range. DNA repair was studied using the kinetics of strand-break rejoining, which was measured after exposure to 137 Cs γ-rays

Targeting HIV latency: pharmacologic strategies toward eradication

Science.gov (United States)

Xing, Sifei; Siliciano, Robert F.

2013-01-01

The latent reservoir for HIV-1 in resting CD4+ T cells remains a major barrier to HIV-1 eradication, even though highly active antiretroviral therapy (HAART) can successfully reduce plasma HIV-1 levels to below the detection limit of clinical assays and reverse disease progression. Proposed eradication strategies involve reactivation of this latent reservoir. Multiple mechanisms are believed to be involved in maintaining HIV-1 latency, mostly through suppression of transcription. These include cytoplasmic sequestration of host transcription factors and epigenetic modifications such as histone deacetylation, histone methylation and DNA methylation. Therefore, strategies targeting these mechanisms have been explored for reactivation of the latent reservoir. In this review, we discuss current pharmacological approaches toward eradication, focusing on small molecule latency-reversing agents, their mechanisms, advantages and limitations. PMID:23270785

Protein carbonyl content: a novel biomarker for aging in HIV/AIDS patients

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Vaishali Kolgiri

2017-01-01

Conclusions: Carbonyl content may has a role as a biomarker for detecting oxidative DNA damage induced ART toxicity and/or accelerated aging in HIV/AIDS patients. Larger studies are warranted to elucidate the role of carbonyl content as a biomarker for premature aging in HIV/AIDS patients.

Mechanisms in endogenous leukemia virus induction by radiation and chemicals

International Nuclear Information System (INIS)

Tennant, R.W.; Rascati, R.J.; Lavelle, G.C.

1976-01-01

A model of endogenous leukemia virus induction in AKR strain mouse cells based on two distinct types of alterations in cellular or proviral DNA is presented. The first type are non-repairable alterations, such as those caused by the incorporation of halogenated pyrimidines; the second type are repairable lesions, such as those caused by irradiation or certain other chemicals. The production of non-repairable lesions leads to the formation of a stable, proviral state which is dependent upon cell division for complete virus expression. A stable provirus intermediate state is not demonstrable in cells induced by treatments which cause repairable lesions, since replication of damaged or altered DNA must occur before the lesions are removed by repair synthesis. Experimental support for this model is presented

Unexpected dramatic increase in CD4+ cell count in a patient with AIDS after enfuvirtide treatment despite persistent viremia and resistance mutations.

Science.gov (United States)

Soria, Alessandro; Cavarelli, Mariangela; Sala, Stefania; Alessandrini, Anna Ida; Scarlatti, Gabriella; Lazzarin, Adriano; Castagna, Antonella

2008-06-01

An unexpected dramatic immune recovery was observed in a patient with full-blown AIDS receiving enfuvirtide-based antiretroviral therapy after multiple treatment failures. A complex interplay of viral and host factors, including the control of X4 viruses and proviral burden, may favor immune restoration with HIV neutralizing activity, despite persistent viremia.

Dolutegravir reshapes the genetic diversity of HIV-1 reservoirs.

Science.gov (United States)

Gantner, Pierre; Lee, Guinevere Q; Rey, David; Mesplede, Thibault; Partisani, Marialuisa; Cheneau, Christine; Beck-Wirth, Geneviève; Faller, Jean-Pierre; Mohseni-Zadeh, Mahsa; Martinot, Martin; Wainberg, Mark A; Fafi-Kremer, Samira

2018-04-01

Better understanding of the dynamics of HIV reservoirs under ART is a critical step to achieve a functional HIV cure. Our objective was to assess the genetic diversity of archived HIV-1 DNA over 48 weeks in blood cells of individuals starting treatment with a dolutegravir-based regimen. Eighty blood samples were prospectively and longitudinally collected from 20 individuals (NCT02557997) including: acutely (n = 5) and chronically (n = 5) infected treatment-naive individuals, as well as treatment-experienced individuals who switched to a dolutegravir-based regimen and were either virologically suppressed (n = 5) or had experienced treatment failure (n = 5). The integrase and V3 loop regions of HIV-1 DNA isolated from PBMCs were analysed by pyrosequencing at baseline and weeks 4, 24 and 48. HIV-1 genetic diversity was calculated using Shannon entropy. All individuals achieved or maintained viral suppression throughout the study. A low and stable genetic diversity of archived HIV quasispecies was observed in individuals starting treatment during acute infection. A dramatic reduction of the genetic diversity was observed at week 4 of treatment in the other individuals. In these patients and despite virological suppression, a recovery of the genetic diversity of the reservoirs was observed up to 48 weeks. Viral variants bearing dolutegravir resistance-associated substitutions at integrase position 50, 124, 230 or 263 were detected in five individuals (n = 5/20, 25%) from all groups except those who were ART-failing at baseline. None of these substitutions led to virological failure. These data demonstrate that the genetic diversity of the HIV-1 reservoir is reshaped following the initiation of a dolutegravir-based regimen and strongly suggest that HIV-1 can continue to replicate despite successful treatment.

Genital HSV Detection among HIV-1-Infected Pregnant Women in Labor

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Janna Patterson

2011-01-01

Full Text Available Objective. To compare genital HSV shedding among HIV-positive and HIV-negative women. Methods. Women with and without known HIV infection who delivered at the University of Washington Medical Center between 1989–1996 had HSV serologies done as part of clinical care. Genital swabs from HSV-2-seropositive women were evaluated by real-time quantitative HSV DNA PCR. Results. HSV-2 seroprevalence was 71% and 30% among 75 HIV-positive and 3051 HIV-negative women, respectively, (P<.001. HSV was detected at delivery in the genital tract of 30.8% of HIV-seropositive versus 9.5% of HIV-negative women (RR=3.2, 95% CI 1.6 to 6.5, P=.001. The number of virion copies shed per mL was similar (log 3.54 for HIV positive versus 3.90 for HIV negative, P=.99. Conclusions. Our study demonstrated that HIV-, HSV-2-coinfected women are more likely to shed HSV at delivery.

A Single HIV-1 Cluster and a Skewed Immune Homeostasis Drive the Early Spread of HIV among Resting CD4+ Cell Subsets within One Month Post-Infection

Science.gov (United States)

Avettand-Fenoël, Véronique; Nembot, Georges; Mélard, Adeline; Blanc, Catherine; Lascoux-Combe, Caroline; Slama, Laurence; Allegre, Thierry; Allavena, Clotilde; Yazdanpanah, Yazdan; Duvivier, Claudine; Katlama, Christine; Goujard, Cécile; Seksik, Bao Chau Phung; Leplatois, Anne; Molina, Jean-Michel; Meyer, Laurence; Autran, Brigitte; Rouzioux, Christine

2013-01-01

Optimizing therapeutic strategies for an HIV cure requires better understanding the characteristics of early HIV-1 spread among resting CD4+ cells within the first month of primary HIV-1 infection (PHI). We studied the immune distribution, diversity, and inducibility of total HIV-DNA among the following cell subsets: monocytes, peripheral blood activated and resting CD4 T cells, long-lived (naive [TN] and central-memory [TCM]) and short-lived (transitional-memory [TTM] and effector-memory cells [TEM]) resting CD4+T cells from 12 acutely-infected individuals recruited at a median 36 days from infection. Cells were sorted for total HIV-DNA quantification, phylogenetic analysis and inducibility, all studied in relation to activation status and cell signaling. One month post-infection, a single CCR5-restricted viral cluster was massively distributed in all resting CD4+ subsets from 88% subjects, while one subject showed a slight diversity. High levels of total HIV-DNA were measured among TN (median 3.4 log copies/million cells), although 10-fold less (p = 0.0005) than in equally infected TCM (4.5), TTM (4.7) and TEM (4.6) cells. CD3−CD4+ monocytes harbored a low viral burden (median 2.3 log copies/million cells), unlike equally infected resting and activated CD4+ T cells (4.5 log copies/million cells). The skewed repartition of resting CD4 subsets influenced their contribution to the pool of resting infected CD4+T cells, two thirds of which consisted of short-lived TTM and TEM subsets, whereas long-lived TN and TCM subsets contributed the balance. Each resting CD4 subset produced HIV in vitro after stimulation with anti-CD3/anti-CD28+IL-2 with kinetics and magnitude varying according to subset differentiation, while IL-7 preferentially induced virus production from long-lived resting TN cells. In conclusion, within a month of infection, a clonal HIV-1 cluster is massively distributed among resting CD4 T-cell subsets with a flexible inducibility, suggesting that

A single HIV-1 cluster and a skewed immune homeostasis drive the early spread of HIV among resting CD4+ cell subsets within one month post-infection.

Directory of Open Access Journals (Sweden)

Charline Bacchus

Full Text Available Optimizing therapeutic strategies for an HIV cure requires better understanding the characteristics of early HIV-1 spread among resting CD4+ cells within the first month of primary HIV-1 infection (PHI. We studied the immune distribution, diversity, and inducibility of total HIV-DNA among the following cell subsets: monocytes, peripheral blood activated and resting CD4 T cells, long-lived (naive [TN] and central-memory [TCM] and short-lived (transitional-memory [TTM] and effector-memory cells [TEM] resting CD4+T cells from 12 acutely-infected individuals recruited at a median 36 days from infection. Cells were sorted for total HIV-DNA quantification, phylogenetic analysis and inducibility, all studied in relation to activation status and cell signaling. One month post-infection, a single CCR5-restricted viral cluster was massively distributed in all resting CD4+ subsets from 88% subjects, while one subject showed a slight diversity. High levels of total HIV-DNA were measured among TN (median 3.4 log copies/million cells, although 10-fold less (p = 0.0005 than in equally infected TCM (4.5, TTM (4.7 and TEM (4.6 cells. CD3-CD4+ monocytes harbored a low viral burden (median 2.3 log copies/million cells, unlike equally infected resting and activated CD4+ T cells (4.5 log copies/million cells. The skewed repartition of resting CD4 subsets influenced their contribution to the pool of resting infected CD4+T cells, two thirds of which consisted of short-lived TTM and TEM subsets, whereas long-lived TN and TCM subsets contributed the balance. Each resting CD4 subset produced HIV in vitro after stimulation with anti-CD3/anti-CD28+IL-2 with kinetics and magnitude varying according to subset differentiation, while IL-7 preferentially induced virus production from long-lived resting TN cells. In conclusion, within a month of infection, a clonal HIV-1 cluster is massively distributed among resting CD4 T-cell subsets with a flexible inducibility

Diagnosing HIV “window phase” in routine laboratory: a teaching case

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Roberta Valentina Marotta

2011-12-01

Full Text Available Human immunodeficiency virus (HIV is the causative agent of acquired immunodeficiency syndrome (AIDS. It is a retrovirus that inserts itself into the host’s DNA and use the host – cell’s replication mechanisms for its own perpetuation. Two distinct species of HIV (HIV 1 and HIV 2 have been identified and they have a different global distribution. In this paper we describe a case of HIV early infection in an italian man of fourty years old. From this example we want to stress the importance to consider with high attention the validation of a negative index of HIV antibodies.We advise to follow this procedure that allows to avoid the risk to report a false negative.

Using Cellular Proteins to Reveal Mechanisms of HIV Infection | Center for Cancer Research

Science.gov (United States)

A vital step in HIV infection is the insertion of viral DNA into the genome of the host cell. In order for the insertion to occur, viral nucleic acid must be transported through the membrane that separates the main cellular compartment (the cytoplasm) from the nucleus, where the host DNA is located. Scientists are actively studying the mechanism used to transport viral DNA into the nucleus in the hopes of targeting this step with future anti-HIV treatments. Up to this point, researchers have identified some of the viral components that play a role in nuclear transport, but they have not determined how viral interactions with other molecules in the cell contribute to the process.

Mode of inhibition of HIV-1 Integrase by a C-terminal domain-specific monoclonal antibody*

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Merkel George

2006-06-01

Full Text Available Abstract Background To further our understanding of the structure and function of HIV-1 integrase (IN we developed and characterized a library of monoclonal antibodies (mAbs directed against this protein. One of these antibodies, mAb33, which is specific for the C-terminal domain, was found to inhibit HIV-1 IN processing activity in vitro; a corresponding Fv fragment was able to inhibit HIV-1 integration in vivo. Our subsequent studies, using heteronuclear nuclear magnetic resonance spectroscopy, identified six solvent accessible residues on the surface of the C-terminal domain that were immobilized upon binding of the antibody, which were proposed to comprise the epitope. Here we test this hypothesis by measuring the affinity of mAb33 to HIV-1 proteins that contain Ala substitutions in each of these positions. To gain additional insight into the mode of inhibition we also measured the DNA binding capacity and enzymatic activities of the Ala substituted proteins. Results We found that Ala substitution of any one of five of the putative epitope residues, F223, R224, Y226, I267, and I268, caused a decrease in the affinity of the mAb33 for HIV-1 IN, confirming the prediction from NMR data. Although IN derivatives with Ala substitutions in or near the mAb33 epitope exhibited decreased enzymatic activity, none of the epitope substitutions compromised DNA binding to full length HIV-1 IN, as measured by surface plasmon resonance spectroscopy. Two of these derivatives, IN (I276A and IN (I267A/I268A, exhibited both increased DNA binding affinity and uncharacteristic dissociation kinetics; these proteins also exhibited non-specific nuclease activity. Results from these investigations are discussed in the context of current models for how the C-terminal domain interacts with substrate DNA. Conclusion It is unlikely that inhibition of HIV-1 IN activity by mAb33 is caused by direct interaction with residues that are essential for substrate binding. Rather

Cyclic GMP-AMP Synthase is an Innate Immune Sensor of HIV and Other Retroviruses

OpenAIRE

Gao, Daxing; Wu, Jiaxi; Wu, You-Tong; Du, Fenghe; Aroh, Chukwuemika; Yan, Nan; Sun, Lijun; Chen, Zhijian J.

2013-01-01

Retroviruses, including HIV, can activate innate immune responses, but the host sensors for retroviruses are largely unknown. Here we show that HIV infection activates cyclic-GMP-AMP (cGAMP) synthase (cGAS) to produce cGAMP, which binds to and activates the adaptor protein STING to induce type-I interferons and other cytokines. Inhibitors of HIV reverse transcriptase, but not integrase, abrogated interferon-β induction by the virus, suggesting that the reverse transcribed HIV DNA triggers the...

Meticulous plasma isolation is essential to avoid false low-level viraemia in Roche Cobas HIV-1 viral load assays.

Science.gov (United States)

Mortier, Virginie; Vancoillie, Leen; Dauwe, Kenny; Staelens, Delfien; Demecheleer, Els; Schauvliege, Marlies; Dinakis, Sylvie; Van Maerken, Tom; Dessilly, Géraldine; Ruelle, Jean; Verhofstede, Chris

2017-10-24

Pre-analytical sample processing is often overlooked as a potential cause of inaccurate assay results. Here we demonstrate how plasma, extracted from standard EDTA-containing blood collection tubes, may contain traces of blood cells consequently resulting in a false low-level HIV-1 viral load when using Roche Cobas HIV-1 assays. The presence of human DNA in Roche Cobas 4800 RNA extracts and in RNA extracts from the Abbott HIV-1 RealTime assay was assessed by quantifying the human albumin gene by means of quantitative PCR. RNA was extracted from plasma samples before and after an additional centrifugation and tested for viral load and DNA contamination. The relation between total DNA content and viral load was defined. Elevated concentrations of genomic DNA were detected in 28 out of 100 Cobas 4800 extracts and were significantly more frequent in samples processed outside of the AIDS Reference Laboratory. An association between genomic DNA presence and spurious low-level viraemia results was demonstrated. Supplementary centrifugation of plasma before RNA extraction eliminated the contamination and the false viraemia. Plasma isolated from standard EDTA-containing blood collection tubes may contain traces of HIV DNA leading to false viral load results above the clinical cutoff. Supplementary centrifugation of plasma before viral load analysis may eliminate the occurrence of this spurious low-level viraemia.

Binding and thermodynamics of REV peptide-ctDNA interaction.

Science.gov (United States)

Upadhyay, Santosh Kumar

2017-03-01

The thermodynamics of DNA-ligand binding is important as it provides useful information to understand the details of binding processes. HIV-1 REV response element (RRE) located in the env coding region of the viral genome is reported to be well conserved across different HIV-1 isolates. In this study, the binding characteristics of Calf thymus DNA (ctDNA) and REV peptide from HIV-1 were investigated using spectroscopic (UV-visible, fluorescence, and circular dichroism (CD)) and isothermal titration calorimetric (ITC) techniques. Thermal stability and ligand binding properties of the ctDNA revealed that native ctDNA had a T m of 75.5 °C, whereas the ctDNA-REV peptide complex exhibited an incremental shift in the T m by 8 °C, indicating thermal stability of the complex. CD data indicated increased ellipticity due to large conformational changes in ctDNA molecule upon binding with REV peptide and two binding stoichiometric modes are apparent. The ctDNA experienced condensation due to large conformational changes in the presence of REV peptide and positive B→Ψ transition was observed at higher molar charge ratios. Fluorescence studies performed at several ligand concentrations revealed a gradual decrease in the fluorescence intensity of EtBr-bound ctDNA in response to increasing ligand concentrations. The fluorescence data further confirmed two stoichiometric modes of binding for ctDNA-REV peptide complex as previously observed with CD studies. The binding enthalpies were determined using ITC in the temperature range of 293 K-308 K. The ITC binding isotherm was exothermic at all temperatures examined, with low ΔH values indicating that the ctDNA-REV peptide interaction is driven largely by entropy. The heat capacity change (ΔC p ) was insignificant, an unusual finding in the area of DNA-peptide interaction studies. The variation in the values obtained for ΔH, ΔS, and ΔG with temperature further suggests that ctDNA-REV peptide interaction is entropically

Occult HBV infection in HIV-infected adults and evaluation of pooled NAT for HBV.

Science.gov (United States)

Dinesha, T R; Boobalan, J; Sivamalar, S; Subashini, D; Solomon, S S; Murugavel, K G; Balakrishnan, P; Smith, D M; Saravanan, S

2018-01-06

The study aimed to determine the prevalence of occult hepatitis B virus infection among HIV-infected persons and to evaluate the use of a pooling strategy to detect occult HBV infection in the setting of HIV infection. Five hundred and two HIV-positive individuals were tested for HBV, occult HBV and hepatitis C and D with serologic and nucleic acid testing (NAT). We also evaluated a pooled NAT strategy for screening occult HBV infection among the HIV-positive individuals. The prevalence of HBV infection among HIV-positive individuals was 32 (6.4%), and occult HBV prevalence was 10%. The pooling HBV NAT had a sensitivity of 66.7% and specificity of 100%, compared to HBV DNA NAT of individual samples. In conclusion, this study found a high prevalence of occult HBV infection among our HIV-infected population. We also demonstrated that pooled HBV NAT is highly specific, moderately sensitive and cost-effective. As conventional HBV viral load assays are expensive in resource-limited settings such as India, pooled HBV DNA NAT might be a good way for detecting occult HBV infection and will reduce HBV-associated complications. © 2018 John Wiley & Sons Ltd.

Enhancement of human immunodeficiency virus type 1-DNA vaccine potency through incorporation of T-helper 1 molecular adjuvants.

Science.gov (United States)

Calarota, Sandra A; Weiner, David B

2004-06-01

It is clear that the development of a safe and effective vaccine for human immunodeficiency virus type 1 (HIV-1) remains a crucial goal for controlling the acquired immunodeficiency syndrome epidemic. At present, it is not clear what arm of the immune response correlates with protection from HIV-1 infection or disease. Therefore, a strong cellular and humoral immune response will likely be needed to control this infection. Among different vaccine alternatives, DNA vaccines appeared more than a decade ago, demonstrating important qualities of inducing both humoral and cellular immune responses in animal models. However, after several years and various clinical studies in humans, supporting the safety of the HIV-DNA vaccine strategies, it has become clear that their potency should be improved. One way to modulate and enhance the immune responses induced by a DNA vaccine is by including genetic adjuvants such as cytokines, chemokines, or T-cell costimulatory molecules as part of the vaccine itself. Particularly, vaccine immunogenicity can be modulated by factors that attract professional antigen-presenting cells, provide additional costimulation, or enhance the uptake of plasmid DNA. This review focuses on developments in the coadministration of molecular adjuvants for the enhancement of HIV-1 DNA-vaccine potency.

Merkel cell polyomavirus IgG antibody levels are associated with progression to AIDS among HIV-infected individuals.