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Sample records for broadly neutralizing human

  1. Recognition determinants of broadly neutralizing human antibodies against dengue viruses.

    Science.gov (United States)

    Rouvinski, Alexander; Guardado-Calvo, Pablo; Barba-Spaeth, Giovanna; Duquerroy, Stéphane; Vaney, Marie-Christine; Kikuti, Carlos M; Navarro Sanchez, M Erika; Dejnirattisai, Wanwisa; Wongwiwat, Wiyada; Haouz, Ahmed; Girard-Blanc, Christine; Petres, Stéphane; Shepard, William E; Desprès, Philippe; Arenzana-Seisdedos, Fernando; Dussart, Philippe; Mongkolsapaya, Juthathip; Screaton, Gavin R; Rey, Félix A

    2015-04-02

    Dengue disease is caused by four different flavivirus serotypes, which infect 390 million people yearly with 25% symptomatic cases and for which no licensed vaccine is available. Recent phase III vaccine trials showed partial protection, and in particular no protection for dengue virus serotype 2 (refs 3, 4). Structural studies so far have characterized only epitopes recognized by serotype-specific human antibodies. We recently isolated human antibodies potently neutralizing all four dengue virus serotypes. Here we describe the X-ray structures of four of these broadly neutralizing antibodies in complex with the envelope glycoprotein E from dengue virus serotype 2, revealing that the recognition determinants are at a serotype-invariant site at the E-dimer interface, including the exposed main chain of the E fusion loop and the two conserved glycan chains. This 'E-dimer-dependent epitope' is also the binding site for the viral glycoprotein prM during virus maturation in the secretory pathway of the infected cell, explaining its conservation across serotypes and highlighting an Achilles' heel of the virus with respect to antibody neutralization. These findings will be instrumental for devising novel immunogens to protect simultaneously against all four serotypes of dengue virus.

  2. Broad neutralizing human monoclonal antibodies against influenza virus from vaccinated healthy donors

    Energy Technology Data Exchange (ETDEWEB)

    Kubota-Koketsu, Ritsuko; Mizuta, Hiroyuki [Department of Virology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 565-0871 (Japan); Oshita, Masatoshi; Ideno, Shoji [Osaka Research Laboratory, Benesis Corporation, Yodogawa-ku, Osaka 532-6505 (Japan); Yunoki, Mikihiro [Osaka Research Laboratory, Benesis Corporation, Yodogawa-ku, Osaka 532-6505 (Japan); Department of Virology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 565-0871 (Japan); Kuhara, Motoki [Ina Laboratory, Medical and Biological Laboratories Corporation, Ltd., Ina, Nagano 396-0002 (Japan); Yamamoto, Naomasa [Department of Biochemistry, School of Pharmaceutical Sciences, Ohu University, Koriyama, Fukushima 963-8611 (Japan); Okuno, Yoshinobu [Kanonji Institute, The Research Foundation for Microbial Diseases of Osaka University, Kanonji, Kagawa 768-0061 (Japan); Ikuta, Kazuyoshi, E-mail: ikuta@biken.osaka-u.ac.jp [Department of Virology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 565-0871 (Japan)

    2009-09-11

    Human monoclonal antibodies (HuMAbs) prepared from patients with viral infections could provide information on human epitopes important for the development of vaccines as well as potential therapeutic applications. Through the fusion of peripheral blood mononuclear cells from a total of five influenza-vaccinated volunteers, with newly developed murine-human chimera fusion partner cells, named SPYMEG, we obtained 10 hybridoma clones stably producing anti-influenza virus antibodies: one for influenza A H1N1, four for influenza A H3N2 and five for influenza B. Surprisingly, most of the HuMAbs showed broad reactivity within subtype and four (two for H3N2 and two for B) showed broad neutralizing ability. Importantly, epitope mapping revealed that the two broad neutralizing antibodies to H3N2 derived from different donors recognized the same epitope located underneath the receptor-binding site of the hemagglutinin globular region that is highly conserved among H3N2 strains.

  3. Structural and Functional Bases for Broad-Spectrum Neutralization of Avian and Human Influenza A Viruses

    OpenAIRE

    Sui, Jianhua; Hwang, William C; Perez, Sandra; Wei, Ge; Aird, Daniel; Chen, Li-Mei; Santelli, Eugenio; Stec, Boguslaw; Cadwell, Greg; Ali, Maryam; Wan, Hongquan; Murakami, Akikazu; Yammanuru, Anuradha; Han, Thomas; Cox, Nancy J

    2009-01-01

    Influenza virus remains a constant public health threat, owing to its ability to evade immune surveillance through rapid genetic drift and reassortment. Monoclonal antibody (mAb)-based immunotherapy is a promising strategy for disease control. Here we use a human Ab phage display library and H5 hemagglutinin (HA) ectodomain to select ten neutralizing mAbs (nAbs) with a remarkably broad range among Group 1 influenza viruses, including the H5N1 “bird flu” and the H1N1 “Spanish flu” strains. Not...

  4. HIV therapy by a combination of broadly neutralizing antibodies in humanized mice

    Science.gov (United States)

    Klein, Florian; Gruell, Henning; Scheid, Johannes F.; Bournazos, Stylianos; Mouquet, Hugo; Spatz, Linda A.; Diskin, Ron; Abadir, Alexander; Zang, Trinity; Dorner, Marcus; Billerbeck, Eva; Labitt, Rachael N.; Gaebler, Christian; Marcovecchio, Paola; Incesu, Reha-Baris; Eisenreich, Thomas R.; Bieniasz, Paul D.; Seaman, Michael S.; Bjorkman, Pamela J.; Ravetch, Jeffrey V.; Ploss, Alexander; Nussenzweig, Michel C.

    2013-01-01

    Summary Human antibodies to HIV-1 can neutralize a broad range of viral isolates in vitro and protect non-human primates against infection1,2. Previous work showed that antibodies exert selective pressure on the virus but escape variants emerge within a short period of time3,4. However, these experiments were performed before the recent discovery of more potent anti-HIV-1 antibodies and their improvement by structure-based design5-9. Here we re-examine passive antibody transfer as a therapeutic modality in HIV-1-infected humanized mice (hu-mice). Although HIV-1 can escape from antibody monotherapy, combinations of broadly neutralizing antibodies (bNAbs) can effectively control HIV-1 infection and suppress viral load to levels below detection. Moreover, in contrast to antiretroviral therapy (ART)10-12, the longer half-life of antibodies led to viremic control for an average of 60 days after cessation of therapy. Thus, combinations of potent monoclonal antibodies can effectively control HIV-1 replication in hu-mice, and should be re-examined as a therapeutic modality in HIV-1-infected individuals. PMID:23103874

  5. VLPs displaying a single L2 epitope induce broadly cross-neutralizing antibodies against human papillomavirus.

    Directory of Open Access Journals (Sweden)

    Ebenezer Tumban

    Full Text Available BACKGROUND: Virus-like Particles (VLPs display can be used to increase the immunogenicity of heterologous antigens. Here, we report the use of a bacteriophage MS2-based VLP display platform to develop a monovalent vaccine targeting a broadly neutralizing epitope in the minor capsid protein human papillomavirus (HPV that provides broad protection from diverse HPV types in a mouse pseudovirus infection model. METHODOLOGY/PRINCIPAL FINDINGS: Peptides spanning a previously described cross-neutralizing epitope from HPV type 16 were genetically inserted at the N-terminus of MS2 bacteriophage coat protein. Three of the four recombinant L2-coat proteins assembled into VLPs. L2-VLPs elicited high-titer anti-L2 antibodies in mice, similar to recombinant VLPs that we had previously made in which the L2 peptide was displayed on a surface-exposed loop on VLPs of a related bacteriophage, PP7. Somewhat surprisingly, L2-MS2 VLPs elicited antibodies that were much more broadly cross-reactive with L2 peptides from diverse HPV isolates than L2-PP7 VLPs. Similarly, mice immunized with L2-MS2 VLPs were protected from genital and cutaneous infection by highly diverse HPV pseudovirus types. CONCLUSION/SIGNIFICANCE: We show that peptides can be displayed in a highly immunogenic fashion at the N-terminus of MS2 coat protein VLPs. A VLP-based vaccine targeting HPV L2 elicits broadly cross-reactive and cross-protective antibodies to heterologous HPV types. L2-VLPs could serve as the basis of a broadly protective second generation HPV vaccine.

  6. Human broadly neutralizing antibodies to the envelope glycoprotein complex of hepatitis C virus

    DEFF Research Database (Denmark)

    Giang, Erick; Dorner, Marcus; Prentoe, Jannick C

    2012-01-01

    Abs recognizing five distinct antigenic regions on the virus envelope glycoprotein complex E1E2 from an HCV-immune phage-display antibody library by using an exhaustive-panning strategy. Many of these mAbs were broadly neutralizing. In particular, the mAb AR4A, recognizing a discontinuous epitope outside the CD81......bs on the E1E2 complex, has an exceptionally broad neutralizing activity toward diverse HCV genotypes and protects against heterologous HCV challenge in a small animal model. The mAb panel will be useful for the design and development of vaccine candidates to elicit broadly neutralizing antibodies...

  7. Broadly neutralizing human antibody that recognizes the receptor-binding pocket of influenza virus hemagglutinin

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    Whittle, James R.R.; Zhang, Ruijun; Khurana, Surender; King, Lisa R.; Manischewitz, Jody; Golding, Hana; Dormitzer, Philip R.; Haynes, Barton F.; Walter, Emmanuel B.; Moody, M. Anthony; Kepler, Thomas B.; Liao, Hua-Xin; Harrison, Stephen C. (Harvard-Med); (Novartis); (US-FDA); (Duke)

    2011-09-20

    Seasonal antigenic drift of circulating influenza virus leads to a requirement for frequent changes in vaccine composition, because exposure or vaccination elicits human antibodies with limited cross-neutralization of drifted strains. We describe a human monoclonal antibody, CH65, obtained by isolating rearranged heavy- and light-chain genes from sorted single plasma cells, coming from a subject immunized with the 2007 trivalent influenza vaccine. The crystal structure of a complex of the hemagglutinin (HA) from H1N1 strain A/Solomon Islands/3/2006 with the Fab of CH65 shows that the tip of the CH65 heavy-chain complementarity determining region 3 (CDR3) inserts into the receptor binding pocket on HA1, mimicking in many respects the interaction of the physiological receptor, sialic acid. CH65 neutralizes infectivity of 30 out of 36 H1N1 strains tested. The resistant strains have a single-residue insertion near the rim of the sialic-acid pocket. We conclude that broad neutralization of influenza virus can be achieved by antibodies with contacts that mimic those of the receptor.

  8. Signature biochemical properties of broadly cross-reactive HIV-1 neutralizing antibodies in human plasma.

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    Sajadi, Mohammad M; Lewis, George K; Seaman, Michael S; Guan, Yongjun; Redfield, Robert R; DeVico, Anthony L

    2012-05-01

    The common properties of broadly cross-reactive HIV-1 neutralization antibodies found in certain HIV-1-infected individuals holds significant value for understanding natural and vaccine-mediated anti-HIV immunity. Recent efforts have addressed this question by deriving neutralizing monoclonal anti-envelope antibodies from memory B cell pools of selected subjects. However, it has been more difficult to identify whether broadly neutralizing antibodies circulating in plasma possess shared characteristics among individuals. To address this question, we used affinity chromatography and isoelectric focusing to fractionate plasma immunoglobulin from 10 HIV-1-infected subjects (5 subjects with broad HIV-1 neutralizing activity and 5 controls). We find that plasma neutralizing activity typically partitions into at least two subsets of antibodies. Antibodies with restricted neutralization breadth have relatively neutral isoelectric points and preferentially bind to envelope monomers and trimers versus core antigens from which variable loops and other domains have been deleted. In comparison, broadly neutralizing antibodies account for a minor fraction of the total anti-envelope response. They are consistently distinguished by more basic isoelectric points and specificity for epitopes shared by monomeric gp120, gp120 core, or CD4-induced structures. Such biochemical properties might be exploited to reliably predict or produce broad anti-HIV immunity.

  9. Safety, pharmacokinetics and neutralization of the broadly neutralizing HIV-1 human monoclonal antibody VRC01 in healthy adults.

    Science.gov (United States)

    Ledgerwood, J E; Coates, E E; Yamshchikov, G; Saunders, J G; Holman, L; Enama, M E; DeZure, A; Lynch, R M; Gordon, I; Plummer, S; Hendel, C S; Pegu, A; Conan-Cibotti, M; Sitar, S; Bailer, R T; Narpala, S; McDermott, A; Louder, M; O'Dell, S; Mohan, S; Pandey, J P; Schwartz, R M; Hu, Z; Koup, R A; Capparelli, E; Mascola, J R; Graham, B S

    2015-12-01

    VRC-HIVMAB060-00-AB (VRC01) is a broadly neutralizing HIV-1 monoclonal antibody (mAb) isolated from the B cells of an HIV-infected patient. It is directed against the HIV-1 CD4 binding site and is capable of potently neutralizing the majority of diverse HIV-1 strains. This Phase I dose-escalation study in healthy adults was conducted at the National Institutes of Health (NIH) Clinical Center (Bethesda, MD, USA). Primary objectives were the safety, tolerability and pharmacokinetics (PK) of VRC01 intravenous (i.v.) infusion at 5, 20 or 40 mg/kg, given either once (20 mg/kg) or twice 28 days apart (all doses), and of subcutaneous (s.c.) delivery at 5 mg/kg compared to s.c. placebo given twice, 28 days apart. Cumulatively, 28 subjects received 43 VRC01 and nine received placebo administrations. There were no serious adverse events or dose-limiting toxicities. Mean 28-day serum trough concentrations after the first infusion were 35 and 57 μg/ml for groups infused with 20 mg/kg (n = 8) and 40 mg/kg (n = 5) doses, respectively. Mean 28-day trough concentrations after the second infusion were 56 and 89 μg/ml for the same two doses. Over the 5-40 mg/kg i.v. dose range (n = 18), the clearance was 0.016 l/h and terminal half-life was 15 days. After infusion VRC01 retained expected neutralizing activity in serum, and anti-VRC01 antibody responses were not detected. The human monoclonal antibody (mAb) VRC01 was well tolerated when delivered i.v. or s.c. The mAb demonstrated expected half-life and pharmacokinetics for a human immunoglobulin G. The safety and PK results support and inform VRC01 dosing schedules for planning HIV-1 prevention efficacy studies.

  10. Broad and potent HIV-1 neutralization by a human antibody that binds the gp41-120 interface

    OpenAIRE

    Huang, Jinghe; Kang, Byong H; Pancera, Marie; Lee, Jeong Hyun; Tong, Tommy; Feng, Yu; Georgiev, Ivelin S.; Chuang, Gwo-Yu; Druz, Aliaksandr; Doria-Rose, Nicole A.; Laub, Leo; Sliepen, Kwinten; van Gils, Marit J.; de la Peña, Alba Torrents; Derking, Ronald

    2014-01-01

    The isolation of human monoclonal antibodies (mAbs) is providing important insights regarding the specificities that underlie broad neutralization of HIV-1 (reviewed in 1 ). Here we report a broad and extremely potent HIV-specific mAb, termed 35O22, which binds novel HIV-1 envelope glycoprotein (Env) epitope. 35O22 neutralized 62% of 181 pseudoviruses with an IC50

  11. Viraemia suppressed in HIV-1-infected humans by broadly neutralizing antibody 3BNC117.

    Science.gov (United States)

    Caskey, Marina; Klein, Florian; Lorenzi, Julio C C; Seaman, Michael S; West, Anthony P; Buckley, Noreen; Kremer, Gisela; Nogueira, Lilian; Braunschweig, Malte; Scheid, Johannes F; Horwitz, Joshua A; Shimeliovich, Irina; Ben-Avraham, Sivan; Witmer-Pack, Maggi; Platten, Martin; Lehmann, Clara; Burke, Leah A; Hawthorne, Thomas; Gorelick, Robert J; Walker, Bruce D; Keler, Tibor; Gulick, Roy M; Fätkenheuer, Gerd; Schlesinger, Sarah J; Nussenzweig, Michel C

    2015-06-25

    HIV-1 immunotherapy with a combination of first generation monoclonal antibodies was largely ineffective in pre-clinical and clinical settings and was therefore abandoned. However, recently developed single-cell-based antibody cloning methods have uncovered a new generation of far more potent broadly neutralizing antibodies to HIV-1 (refs 4, 5). These antibodies can prevent infection and suppress viraemia in humanized mice and nonhuman primates, but their potential for human HIV-1 immunotherapy has not been evaluated. Here we report the results of a first-in-man dose escalation phase 1 clinical trial of 3BNC117, a potent human CD4 binding site antibody, in uninfected and HIV-1-infected individuals. 3BNC117 infusion was well tolerated and demonstrated favourable pharmacokinetics. A single 30 mg kg(-1) infusion of 3BNC117 reduced the viral load in HIV-1-infected individuals by 0.8-2.5 log10 and viraemia remained significantly reduced for 28 days. Emergence of resistant viral strains was variable, with some individuals remaining sensitive to 3BNC117 for a period of 28 days. We conclude that, as a single agent, 3BNC117 is safe and effective in reducing HIV-1 viraemia, and that immunotherapy should be explored as a new modality for HIV-1 prevention, therapy and cure.

  12. Structural Basis for Recognition of Human Enterovirus 71 by a Bivalent Broadly Neutralizing Monoclonal Antibody

    Science.gov (United States)

    Ku, Zhiqiang; Zuo, Teng; Kong, Liangliang; Zhang, Chao; Shi, Jinping; Liu, Qingwei; Chen, Tan; Zhang, Yingyi; Jiang, Wen; Zhang, Linqi; Huang, Zhong; Cong, Yao

    2016-01-01

    Enterovirus 71 (EV71) is the main pathogen responsible for hand, foot and mouth disease with severe neurological complications and even death in young children. We have recently identified a highly potent anti-EV71 neutralizing monoclonal antibody, termed D5. Here we investigated the structural basis for recognition of EV71 by the antibody D5. Four three-dimensional structures of EV71 particles in complex with IgG or Fab of D5 were reconstructed by cryo-electron microscopy (cryo-EM) single particle analysis all at subnanometer resolutions. The most critical EV71 mature virion-Fab structure was resolved to a resolution of 4.8 Å, which is rare in cryo-EM studies of virus-antibody complex so far. The structures reveal a bivalent binding pattern of D5 antibody across the icosahedral 2-fold axis on mature virion, suggesting that D5 binding may rigidify virions to prevent their conformational changes required for subsequent RNA release. Moreover, we also identified that the complementary determining region 3 (CDR3) of D5 heavy chain directly interacts with the extremely conserved VP1 GH-loop of EV71, which was validated by biochemical and virological assays. We further showed that D5 is indeed able to neutralize a variety of EV71 genotypes and strains. Moreover, D5 could potently confer protection in a mouse model of EV71 infection. Since the conserved VP1 GH-loop is involved in EV71 binding with its uncoating receptor, the scavenger receptor class B, member 2 (SCARB2), the broadly neutralizing ability of D5 might attribute to its inhibition of EV71 from binding SCARB2. Altogether, our results elucidate the structural basis for the binding and neutralization of EV71 by the broadly neutralizing antibody D5, thereby enhancing our understanding of antibody-based protection against EV71 infection. PMID:26938634

  13. Structural Basis for Recognition of Human Enterovirus 71 by a Bivalent Broadly Neutralizing Monoclonal Antibody.

    Science.gov (United States)

    Ye, Xiaohua; Fan, Chen; Ku, Zhiqiang; Zuo, Teng; Kong, Liangliang; Zhang, Chao; Shi, Jinping; Liu, Qingwei; Chen, Tan; Zhang, Yingyi; Jiang, Wen; Zhang, Linqi; Huang, Zhong; Cong, Yao

    2016-03-01

    Enterovirus 71 (EV71) is the main pathogen responsible for hand, foot and mouth disease with severe neurological complications and even death in young children. We have recently identified a highly potent anti-EV71 neutralizing monoclonal antibody, termed D5. Here we investigated the structural basis for recognition of EV71 by the antibody D5. Four three-dimensional structures of EV71 particles in complex with IgG or Fab of D5 were reconstructed by cryo-electron microscopy (cryo-EM) single particle analysis all at subnanometer resolutions. The most critical EV71 mature virion-Fab structure was resolved to a resolution of 4.8 Å, which is rare in cryo-EM studies of virus-antibody complex so far. The structures reveal a bivalent binding pattern of D5 antibody across the icosahedral 2-fold axis on mature virion, suggesting that D5 binding may rigidify virions to prevent their conformational changes required for subsequent RNA release. Moreover, we also identified that the complementary determining region 3 (CDR3) of D5 heavy chain directly interacts with the extremely conserved VP1 GH-loop of EV71, which was validated by biochemical and virological assays. We further showed that D5 is indeed able to neutralize a variety of EV71 genotypes and strains. Moreover, D5 could potently confer protection in a mouse model of EV71 infection. Since the conserved VP1 GH-loop is involved in EV71 binding with its uncoating receptor, the scavenger receptor class B, member 2 (SCARB2), the broadly neutralizing ability of D5 might attribute to its inhibition of EV71 from binding SCARB2. Altogether, our results elucidate the structural basis for the binding and neutralization of EV71 by the broadly neutralizing antibody D5, thereby enhancing our understanding of antibody-based protection against EV71 infection.

  14. Structural Basis for Recognition of Human Enterovirus 71 by a Bivalent Broadly Neutralizing Monoclonal Antibody.

    Directory of Open Access Journals (Sweden)

    Xiaohua Ye

    2016-03-01

    Full Text Available Enterovirus 71 (EV71 is the main pathogen responsible for hand, foot and mouth disease with severe neurological complications and even death in young children. We have recently identified a highly potent anti-EV71 neutralizing monoclonal antibody, termed D5. Here we investigated the structural basis for recognition of EV71 by the antibody D5. Four three-dimensional structures of EV71 particles in complex with IgG or Fab of D5 were reconstructed by cryo-electron microscopy (cryo-EM single particle analysis all at subnanometer resolutions. The most critical EV71 mature virion-Fab structure was resolved to a resolution of 4.8 Å, which is rare in cryo-EM studies of virus-antibody complex so far. The structures reveal a bivalent binding pattern of D5 antibody across the icosahedral 2-fold axis on mature virion, suggesting that D5 binding may rigidify virions to prevent their conformational changes required for subsequent RNA release. Moreover, we also identified that the complementary determining region 3 (CDR3 of D5 heavy chain directly interacts with the extremely conserved VP1 GH-loop of EV71, which was validated by biochemical and virological assays. We further showed that D5 is indeed able to neutralize a variety of EV71 genotypes and strains. Moreover, D5 could potently confer protection in a mouse model of EV71 infection. Since the conserved VP1 GH-loop is involved in EV71 binding with its uncoating receptor, the scavenger receptor class B, member 2 (SCARB2, the broadly neutralizing ability of D5 might attribute to its inhibition of EV71 from binding SCARB2. Altogether, our results elucidate the structural basis for the binding and neutralization of EV71 by the broadly neutralizing antibody D5, thereby enhancing our understanding of antibody-based protection against EV71 infection.

  15. Human Ig knockin mice to study the development and regulation of HIV-1 broadly neutralizing antibodies.

    Science.gov (United States)

    Verkoczy, Laurent; Alt, Frederick W; Tian, Ming

    2017-01-01

    A major challenge for HIV-1 vaccine research is developing a successful immunization approach for inducing broadly neutralizing antibodies (bnAbs). A key shortcoming in meeting this challenge has been the lack of animal models capable of identifying impediments limiting bnAb induction and ranking vaccine strategies for their ability to promote bnAb development. Since 2010, immunoglobulin knockin (KI) technology, involving inserting functional rearranged human variable exons into the mouse IgH and IgL loci has been used to express bnAbs in mice. This approach has allowed immune tolerance mechanisms limiting bnAb production to be elucidated and strategies to overcome such limitations to be evaluated. From these studies, along with the wealth of knowledge afforded by analyses of recombinant Ig-based bnAb structures, it became apparent that key functional features of bnAbs often are problematic for their elicitation in mice by classic vaccine paradigms, necessitating more iterative testing of new vaccine concepts. In this regard, bnAb KI models expressing deduced precursor V(D)J rearrangements of mature bnAbs or unrearranged germline V, D, J segments (that can be assembled into variable region exons that encode bnAb precursors), have been engineered to evaluate novel immunogens/regimens for effectiveness in driving bnAb responses. One promising approach emerging from such studies is the ability of sequentially administered, modified immunogens (designed to bind progressively more mature bnAb precursors) to initiate affinity maturation. Here, we review insights gained from bnAb KI studies regarding the regulation and induction of bnAbs, and discuss new Ig KI methodologies to manipulate the production and/or expression of bnAbs in vivo, to further facilitate vaccine-guided bnAb induction studies.

  16. Broadly neutralizing antibodies targeted to mucin-type carbohydrate epitopes of human immunodeficiency virus

    DEFF Research Database (Denmark)

    Hansen, J E; Nielsen, C; Arendrup, M

    1991-01-01

    The cancer-related mucin-type carbohydrate neoantigen Tn was found on gp160 and gp120 of human immunodeficiency virus (HIV). Immunoglobulin G (IgG) and IgM monoclonal antibodies (MAbs) against Tn neutralized infection with cell-free virus and blocked fusion between HIV-infected and uninfected cells......; this binding was inhibitable by pure Tn antigen, and indications were found that this inhibition occurred at a pre-entry step. Boosting the naturally occurring low-titer anti-Tn activity may be of prophylactic value, as suggested by the in vitro neutralization found in this study....

  17. Broadly neutralizing antibodies targeted to mucin-type carbohydrate epitopes of human immunodeficiency virus

    DEFF Research Database (Denmark)

    Hansen, J E; Nielsen, C; Arendrup, M;

    1991-01-01

    . This inhibition was found in infection of both lymphocytic cells and monocytoid cells. Viruses tested included six HIV-1 and five HIV-2 isolates propagated in different cells, as well as infectious plasma from AIDS patients. The antiviral effect of anti-Tn MAbs occurred by specific binding of the MAb to the virus......The cancer-related mucin-type carbohydrate neoantigen Tn was found on gp160 and gp120 of human immunodeficiency virus (HIV). Immunoglobulin G (IgG) and IgM monoclonal antibodies (MAbs) against Tn neutralized infection with cell-free virus and blocked fusion between HIV-infected and uninfected cells...

  18. Broadly Neutralizing Antibodies for HIV Eradication.

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    Stephenson, Kathryn E; Barouch, Dan H

    2016-02-01

    Passive transfer of antibodies has long been considered a potential treatment modality for infectious diseases, including HIV. Early efforts to use antibodies to suppress HIV replication, however, were largely unsuccessful, as the antibodies that were studied neutralized only a relatively narrow spectrum of viral strains and were not very potent. Recent advances have led to the discovery of a large portfolio of human monoclonal antibodies that are broadly neutralizing across many HIV-1 subtypes and are also substantially more potent. These antibodies target multiple different epitopes on the HIV envelope, thus allowing for the development of antibody combinations. In this review, we discuss the application of broadly neutralizing antibodies (bNAbs) for HIV treatment and HIV eradication strategies. We highlight bNAbs that target key epitopes, such as the CD4 binding site and the V2/V3-glycan-dependent sites, and we discuss several bNAbs that are currently in the clinical development pipeline.

  19. A broadly neutralizing human monoclonal antibody is effective against H7N9

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    Tharakaraman, Kannan; Subramanian, Vidya; Viswanathan, Karthik; Sloan, Susan; Yen, Hui-Ling; Barnard, Dale L.; Leung, Y. H. Connie; Szretter, Kristy J.; Koch, Tyree J.; Delaney, James C.; Babcock, Gregory J.; Wogan, Gerald N.; Sasisekharan, Ram; Shriver, Zachary

    2015-01-01

    Emerging strains of influenza represent a significant public health threat with potential pandemic consequences. Of particular concern are the recently emerged H7N9 strains which cause pneumonia with acute respiratory distress syndrome. Estimates are that nearly 80% of hospitalized patients with H7N9 have received intensive care unit support. VIS410, a human antibody, targets a unique conserved epitope on influenza A. We evaluated the efficacy of VIS410 for neutralization of group 2 influenza strains, including H3N2 and H7N9 strains in vitro and in vivo. VIS410, administered at 50 mg/kg, protected DBA mice infected with A/Anhui/2013 (H7N9), resulting in significant survival benefit upon single-dose (−24 h) or double-dose (−12 h, +48 h) administration (P H7N9) resulted in significant decreased lung viral load (P = 0.002) and decreased lung cytokine responses for nine of the 11 cytokines measured. Based on these results, we find that VIS410 may be effective either as monotherapy or combined with antivirals in treating H7N9 disease, as well as disease from other influenza strains. PMID:26283346

  20. Broad and potent HIV-1 neutralization by a human antibody that binds the gp41-gp120 interface

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Jinghe; Kang, Byong H.; Pancera, Marie; Lee, Jeong Hyun; Tong, Tommy; Feng, Yu; Imamichi, Hiromi; Georgiev, Ivelin S.; Chuang, Gwo-Yu; Druz, Aliaksandr; Doria-Rose, Nicole A.; Laub, Leo; Sliepen, Kwinten; van Gils, Marit J.; de la Peña, Alba Torrents; Derking, Ronald; Klasse, Per-Johan; Migueles, Stephen A.; Bailer, Robert T.; Alam, Munir; Pugach, Pavel; Haynes, Barton F.; Wyatt, Richard T.; Sanders, Rogier W.; Binley, James M.; Ward, Andrew B.; Mascola, John R.; Kwong, Peter D.; Connors, Mark [NIH

    2015-10-15

    The isolation of human monoclonal antibodies is providing important insights into the specificities that underlie broad neutralization of HIV-1 (reviewed in ref. 1). Here we report a broad and extremely potent HIV-specific monoclonal antibody, termed 35O22, which binds a novel HIV-1 envelope glycoprotein (Env) epitope. 35O22 neutralized 62% of 181 pseudoviruses with a half-maximum inhibitory concentration (IC50) <50 μg ml-1. The median IC50 of neutralized viruses was 0.033 μg ml-1, among the most potent thus far described. 35O22 did not bind monomeric forms of Env tested, but did bind the trimeric BG505 SOSIP.664. Mutagenesis and a reconstruction by negative-stain electron microscopy of the Fab in complex with trimer revealed that it bound to a conserved epitope, which stretched across gp120 and gp41. The specificity of 35O22 represents a novel site of vulnerability on HIV Env, which serum analysis indicates to be commonly elicited by natural infection. Binding to this new site of vulnerability may thus be an important complement to current monoclonal-antibody-based approaches to immunotherapies, prophylaxis and vaccine design.

  1. Broad-range neutralizing anti-influenza A human monoclonal antibodies: new perspectives in therapy and prophylaxis.

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    Clementi, Nicola; Criscuolo, Elena; Castelli, Matteo; Clementi, Massimo

    2012-10-01

    Broadly neutralizing monoclonal antibodies (mAbs) directed against different subtypes of influenza A viruses are novel tools for the potential development of effective anti-influenza prophylactic and therapeutic strategies. In both cases, the main candidates for passive transfer and new vaccine development are represented by protective mAbs directed against influenza hemagglutinin (HA). A large number of mAbs directed against influenza HA has been developed to date. However, even if they can be useful and contribute to develop new vaccinal strategies, only few of them can be a good candidate for human administration. In this review, we will describe the most relevant human mAb directed against influenza HA able to recognize highly divergent influenza isolates and possibly useful for human therapy and prophylaxis.

  2. Influenza virus antigenicity and broadly neutralizing epitopes.

    Science.gov (United States)

    Air, Gillian M

    2015-04-01

    A vaccine formulation that would be effective against all strains of influenza virus has long been a goal of vaccine developers, but antibodies after infection or vaccination were seen to be strain specific and there was little evidence of cross-reactive antibodies that neutralized across subtypes. Recently a number of broadly neutralizing monoclonal antibodies have been characterized. This review describes the different classes of broadly neutralizing antibodies and discusses the potential of their therapeutic use or for design of immunogens that induce a high proportion of broadly neutralizing antibodies.

  3. Variable epitope libraries: new vaccine immunogens capable of inducing broad human immunodeficiency virus type 1-neutralizing antibody response.

    Science.gov (United States)

    Charles-Niño, Claudia; Pedroza-Roldan, Cesar; Viveros, Monica; Gevorkian, Goar; Manoutcharian, Karen

    2011-07-18

    The extreme antigenic variability of human immunodeficiency virus (HIV) leads to immune escape of the virus, representing a major challenge in the design of effective vaccine. We have developed a novel concept for immunogen construction based on introduction of massive mutations within the epitopes targeting antigenically variable pathogens and diseases. Previously, we showed that these immunogens carrying large combinatorial libraries of mutated epitope variants, termed as variable epitope libraries (VELs), induce potent, broad and long lasting CD8+IFN-γ+ T-cell response. Moreover, we demonstrated that these T cells recognize more than 50% of heavily mutated variants (5 out of 10 amino acid positions were mutated in each epitope variant) of HIV-1 gp120 V3 loop-derived cytotoxic T lymphocyte epitope (RGPGRAFVTI) in mice. The constructed VELs had complexities of 10000 and 12500 individual members, generated as plasmid DNA or as M13 phage display combinatorial libraries, respectively, and with structural composition RGPGXAXXXX or XGXGXAXVXI, where X is any of 20 natural amino acids. Here, we demonstrated that sera from mice immunized with these VELs are capable of neutralizing 5 out of 10 viral isolates from Tier 2 reference panel of subtype B envelope clones, including HIV-1 isolates which are known to be resistant to neutralization by several potent monoclonal antibodies, described previously. These data indicate the feasibility of the application of immunogens based on VEL concept as an alternative approach for the development of molecular vaccines against antigenically variable pathogens.

  4. Administration of nucleoside-modified mRNA encoding broadly neutralizing antibody protects humanized mice from HIV-1 challenge

    Science.gov (United States)

    Pardi, Norbert; Secreto, Anthony J.; Shan, Xiaochuan; Debonera, Fotini; Glover, Joshua; Yi, Yanjie; Muramatsu, Hiromi; Ni, Houping; Mui, Barbara L.; Tam, Ying K.; Shaheen, Farida; Collman, Ronald G.; Karikó, Katalin; Danet-Desnoyers, Gwenn A.; Madden, Thomas D.; Hope, Michael J.; Weissman, Drew

    2017-01-01

    Monoclonal antibodies are one of the fastest growing classes of pharmaceutical products, however, their potential is limited by the high cost of development and manufacturing. Here we present a safe and cost-effective platform for in vivo expression of therapeutic antibodies using nucleoside-modified mRNA. To demonstrate feasibility and protective efficacy, nucleoside-modified mRNAs encoding the light and heavy chains of the broadly neutralizing anti-HIV-1 antibody VRC01 are generated and encapsulated into lipid nanoparticles. Systemic administration of 1.4 mg kg−1 of mRNA into mice results in ∼170 μg ml−1 VRC01 antibody concentrations in the plasma 24 h post injection. Weekly injections of 1 mg kg−1 of mRNA into immunodeficient mice maintain trough VRC01 levels above 40 μg ml−1. Most importantly, the translated antibody from a single injection of VRC01 mRNA protects humanized mice from intravenous HIV-1 challenge, demonstrating that nucleoside-modified mRNA represents a viable delivery platform for passive immunotherapy against HIV-1 with expansion to a variety of diseases. PMID:28251988

  5. Isolation and characterization of broadly neutralizing human monoclonal antibodies to the e1 glycoprotein of hepatitis C virus

    DEFF Research Database (Denmark)

    Meunier, Jean-Christophe; Russell, Rodney S.; Goossens, Vera

    2008-01-01

    The relative importance of humoral and cellular immunity in the prevention or clearance of hepatitis C virus (HCV) infection is poorly understood. However, there is considerable evidence that neutralizing antibodies are involved in disease control. Here we describe the detailed analysis of human...... monoclonal antibodies (MAbs) directed against HCV glycoprotein E1, which may have the potential to control HCV infection. We have identified two MAbs that can strongly neutralize HCV-pseudotyped particles (HCVpp) bearing the envelope glycoproteins of genotypes 1a, 1b, 4a, 5a, and 6a and less strongly...... neutralize HCVpp bearing the envelope glycoproteins of genotype 2a. Genotype 3a was not neutralized. The epitopes for both MAbs were mapped to the region encompassing amino acids 313 to 327. In addition, robust neutralization was also observed against cell culture-adapted viruses of genotypes 1a and 2a...

  6. Antiviral Therapy by HIV-1 Broadly Neutralizing and Inhibitory Antibodies

    Directory of Open Access Journals (Sweden)

    Zhiqing Zhang

    2016-11-01

    Full Text Available Human immunodeficiency virus type 1 (HIV-1 infection causes acquired immune deficiency syndrome (AIDS, a global epidemic for more than three decades. HIV-1 replication is primarily controlled through antiretroviral therapy (ART but this treatment does not cure HIV-1 infection. Furthermore, there is increasing viral resistance to ART, and side effects associated with long-term therapy. Consequently, there is a need of alternative candidates for HIV-1 prevention and therapy. Recent advances have discovered multiple broadly neutralizing antibodies against HIV-1. In this review, we describe the key epitopes on the HIV-1 Env protein and the reciprocal broadly neutralizing antibodies, and discuss the ongoing clinical trials of broadly neutralizing and inhibitory antibody therapy as well as antibody combinations, bispecific antibodies, and methods that improve therapeutic efficacy by combining broadly neutralizing antibodies (bNAbs with latency reversing agents. Compared with ART, HIV-1 therapeutics that incorporate these broadly neutralizing and inhibitory antibodies offer the advantage of decreasing virus load and clearing infected cells, which is a promising prospect in HIV-1 prevention and treatment.

  7. Antiviral Therapy by HIV-1 Broadly Neutralizing and Inhibitory Antibodies.

    Science.gov (United States)

    Zhang, Zhiqing; Li, Shaowei; Gu, Ying; Xia, Ningshao

    2016-11-18

    Human immunodeficiency virus type 1 (HIV-1) infection causes acquired immune deficiency syndrome (AIDS), a global epidemic for more than three decades. HIV-1 replication is primarily controlled through antiretroviral therapy (ART) but this treatment does not cure HIV-1 infection. Furthermore, there is increasing viral resistance to ART, and side effects associated with long-term therapy. Consequently, there is a need of alternative candidates for HIV-1 prevention and therapy. Recent advances have discovered multiple broadly neutralizing antibodies against HIV-1. In this review, we describe the key epitopes on the HIV-1 Env protein and the reciprocal broadly neutralizing antibodies, and discuss the ongoing clinical trials of broadly neutralizing and inhibitory antibody therapy as well as antibody combinations, bispecific antibodies, and methods that improve therapeutic efficacy by combining broadly neutralizing antibodies (bNAbs) with latency reversing agents. Compared with ART, HIV-1 therapeutics that incorporate these broadly neutralizing and inhibitory antibodies offer the advantage of decreasing virus load and clearing infected cells, which is a promising prospect in HIV-1 prevention and treatment.

  8. A hepatitis C virus (HCV) vaccine comprising envelope glycoproteins gpE1/gpE2 derived from a single isolate elicits broad cross-genotype neutralizing antibodies in humans

    DEFF Research Database (Denmark)

    Law, John Lok Man; Chen, Chao; Wong, Jason

    2013-01-01

    , an effective HCV vaccine which could elicit broadly cross-neutralizing antibodies has represented a major challenge. In this study, we tested for the presence of cross-neutralizing antibodies in human volunteers who were immunized with recombinant glycoproteins gpE1/gpE2 derived from a single HCV strain (HCV1...... of genotype 1a). Cross neutralization was tested in Huh-7.5 human hepatoma cell cultures using infectious recombinant HCV (HCVcc) expressing structural proteins of heterologous HCV strains from all known major genotypes, 1-7. Vaccination induced significant neutralizing antibodies against heterologous HCV...... genotype 1a virus which represents the most common genotype in North America. Of the 16 vaccinees tested, 3 were selected on the basis of strong 1a virus neutralization for testing of broad cross-neutralizing responses. At least 1 vaccinee was shown to elicit broad cross-neutralization against all HCV...

  9. Structural basis for penetration of the glycan shield of hepatitis C virus E2 glycoprotein by a broadly neutralizing human antibody.

    Science.gov (United States)

    Li, Yili; Pierce, Brian G; Wang, Qian; Keck, Zhen-Yong; Fuerst, Thomas R; Foung, Steven K H; Mariuzza, Roy A

    2015-04-17

    Hepatitis C virus (HCV) is a major cause of liver cirrhosis and hepatocellular carcinoma. A challenge for HCV vaccine development is to identify conserved epitopes able to elicit protective antibodies against this highly diverse virus. Glycan shielding is a mechanism by which HCV masks such epitopes on its E2 envelope glycoprotein. Antibodies to the E2 region comprising residues 412-423 (E2(412-423)) have broadly neutralizing activities. However, an adaptive mutation in this linear epitope, N417S, is associated with a glycosylation shift from Asn-417 to Asn-415 that enables HCV to escape neutralization by mAbs such as HCV1 and AP33. By contrast, the human mAb HC33.1 can neutralize virus bearing the N417S mutation. To understand how HC33.1 penetrates the glycan shield created by the glycosylation shift to Asn-415, we determined the structure of this broadly neutralizing mAb in complex with its E2(412-423) epitope to 2.0 Å resolution. The conformation of E2(412-423) bound to HC33.1 is distinct from the β-hairpin conformation of this peptide bound to HCV1 or AP33, because of disruption of the β-hairpin through interactions with the unusually long complementarity-determining region 3 of the HC33.1 heavy chain. Whereas Asn-415 is buried by HCV1 and AP33, it is solvent-exposed in the HC33.1-E2(412-423) complex, such that glycosylation of Asn-415 would not prevent antibody binding. Furthermore, our results highlight the structural flexibility of the E2(412-423) epitope, which may serve as an immune evasion strategy to impede induction of antibodies targeting this site by reducing its antigenicity.

  10. Cross-reactive broadly neutralizing antibodies: timing is everything

    OpenAIRE

    Euler, Zelda; Schuitemaker, Hanneke

    2012-01-01

    The recent surge of research into new broadly neutralizing antibodies in HIV-1 infection has recharged the field of HIV-1 vaccinology. In this review we discuss the currently known broadly neutralizing antibodies and focus on factors that may shape these antibodies in natural infection. We further discuss the role of these antibodies in the clinical course of the infection and consider immunological obstacles in inducing broadly neutralizing antibodies with a vaccine.

  11. Cross-reactive broadly neutralizing antibodies: timing is everything.

    Science.gov (United States)

    Euler, Zelda; Schuitemaker, Hanneke

    2012-01-01

    The recent surge of research into new broadly neutralizing antibodies in HIV-1 infection has recharged the field of HIV-1 vaccinology. In this review we discuss the currently known broadly neutralizing antibodies and focus on factors that may shape these antibodies in natural infection. We further discuss the role of these antibodies in the clinical course of the infection and consider immunological obstacles in inducing broadly neutralizing antibodies with a vaccine.

  12. The epitope and neutralization mechanism of AVFluIgG01, a broad-reactive human monoclonal antibody against H5N1 influenza virus.

    Directory of Open Access Journals (Sweden)

    Zhiliang Cao

    Full Text Available The continued spread of highly pathogenic avian influenza (HPAI H5N1 virus underscores the importance of effective antiviral approaches. AVFluIgG01 is a potent and broad-reactive H5N1-neutralizing human monoclonal antibody (mAb showing great potential for use either for therapeutic purposes or as a basis of vaccine development, but its antigenic epitope and neutralization mechanism have not been finely characterized. In this study, we first demonstrated that AVFluIgG01 targets a novel conformation-dependent epitope in the globular head region of H5N1 hemagglutinin (HA. By selecting mimotopes from a random peptide library in combination with computational algorithms and site-directed mutagenesis, the epitope was mapped to three conserved discontinuous sites (I-III that are located closely at the three-dimensional structure of HA. Further, we found that this HA1-specific human mAb can efficiently block both virus-receptor binding and post-attachment steps, while its Fab fragment exerts the post-attachment inhibition only. Consistently, AVFluIgG01 could inhibit HA-mediated cell-cell membrane fusion at a dose-dependent manner and block the acquisition of pH-induced protease sensitivity. These results suggest a neutralization mechanism of AVFluIgG01 by simultaneously blocking viral attachment to the receptors on host cells and interfering with HA conformational rearrangements associated with membrane fusion. The presented data provide critical information for developing novel antiviral therapeutics and vaccines against HPAI H5N1 virus.

  13. Immunization of rabbits with highly purified, soluble, trimeric human immunodeficiency virus type 1 envelope glycoprotein induces a vigorous B cell response and broadly cross-reactive neutralization.

    Directory of Open Access Journals (Sweden)

    Gerald V Quinnan

    Full Text Available Previously we described induction of cross-reactive HIV-1 neutralizing antibody responses in rabbits using a soluble HIV-1 gp140 envelope glycoprotein (Env in an adjuvant containing monophosphoryl lipid A (MPL and QS21 (AS02A. Here, we compared different forms of the same HIV-1 strain R2 Env for antigenic and biophysical characteristics, and in rabbits characterized the extent of B cell induction for specific antibody expression and secretion and neutralizing responses. The forms of this Env that were produced in and purified from stably transformed 293T cells included a primarily dimeric gp140, a trimeric gp140 appended to a GCN4 trimerization domain (gp140-GCN4, gp140-GCN4 with a 15 amino acid flexible linker between the gp120 and gp41 ectodomain (gp140-GCN4-L, also trimeric, and a gp140 with the flexible linker purified from cell culture supernatants as either dimer (gp140-L(D or monomer (gp140-L(M. Multimeric states of the Env proteins were assessed by native gel electrophoresis and analytical ultracentrifugation. The different forms of gp140 bound broadly cross-reactive neutralizing (BCN human monoclonal antibodies (mAbs similarly in ELISA and immunoprecipitation assays. All Envs bound CD4i mAbs in the presence and absence of sCD4, as reported for the R2 Env. Weak neutralization of some strains of HIV-1 was seen after two additional doses in AS02A. Rabbits that were given a seventh dose of gp140-GCN4-L developed BCN responses that were weak to moderate, similar to our previous report. The specificity of these responses did not appear similar to that of any of the known BCN human mAbs. Induction of spleen B cell and plasma cells producing immunoglobulins that bound trimeric gp140-GCN4-L was vigorous, based on ELISpot and flow cytometry analyses. The results demonstrate that highly purified gp140-GCN4-L trimer in adjuvant elicits BCN responses in rabbits accompanied by vigorous B cell induction.

  14. Development of Broadly Neutralizing Antibodies and Their Mapping by Monomeric gp120 in Human Immunodeficiency Virus Type 1-Infected Humans and Simian-Human Immunodeficiency Virus SHIVSF162P3N-Infected Macaques

    Science.gov (United States)

    Jia, Manxue; Lu, Hong; Markowitz, Martin; Cheng-Mayer, Cecilia

    2016-01-01

    ABSTRACT To improve our understanding of the similarities and differences between neutralizing antibodies elicited by simian-human immunodeficiency virus (SHIV)-infected rhesus macaques and human immunodeficiency virus type 1 (HIV-1)-infected humans, we examined the plasma of 13 viremic macaques infected with SHIVSF162P3N and 85 HIV-1-infected humans with known times of infection. We identified 5 macaques (38%) from 1 to 2 years postinfection (p.i.) with broadly neutralizing antibodies (bnAbs) against tier 2 HIV-1. In comparison, only 2 out of 42 (5%) human plasma samples collected in a similar time frame of 1 to 3 years p.i. exhibited comparable neutralizing breadths and potencies, with the number increasing to 7 out of 21 (30%) after 3 years p.i. Plasma mapping with monomeric gp120 identified only 2 out of 9 humans and 2 out of 4 macaques that contained gp120-reactive neutralizing antibodies, indicating distinct specificities in these plasma samples, with most of them recognizing the envelope trimer (including gp41) rather than the gp120 monomer. Indeed, a total of 20 gp120-directed monoclonal antibodies (MAbs) isolated from a human subject (AD358) and a Chinese rhesus macaque (GB40) displayed no or limited neutralizing activity against tier 2 strains. These isolated MAbs, mapped to the CD4-binding site, the V3 loop, the inner domain, and the C5 region of gp120, revealed genetic similarity between the human and macaque immunoglobulin genes used to encode some V3-directed MAbs. These results also support the use of envelope trimer probes for efficient isolation of HIV-1 bnAbs. IMPORTANCE HIV-1 vaccine research can benefit from understanding the development of broadly neutralizing antibodies (bnAbs) in rhesus macaques, commonly used to assess vaccine immunogenicity and efficacy. Here, we examined 85 HIV-1-infected humans and 13 SHIVSF162P3N-infected macaques for bnAbs and found that, similar to HIV-1-infected humans, bnAbs in SHIV-infected macaques are also rare

  15. Structural basis of hepatitis C virus neutralization by broadly neutralizing antibody HCV1

    Energy Technology Data Exchange (ETDEWEB)

    Kong, Leopold; Giang, Erick; Robbins, Justin B.; Stanfield, Robyn L.; Burton, Dennis R.; Wilson, Ian A.; Law, Mansun (Scripps)

    2012-10-29

    Hepatitis C virus (HCV) infects more than 2% of the global population and is a leading cause of liver cirrhosis, hepatocellular carcinoma, and end-stage liver diseases. Circulating HCV is genetically diverse, and therefore a broadly effective vaccine must target conserved T- and B-cell epitopes of the virus. Human mAb HCV1 has broad neutralizing activity against HCV isolates from at least four major genotypes and protects in the chimpanzee model from primary HCV challenge. The antibody targets a conserved antigenic site (residues 412-423) on the virus E2 envelope glycoprotein. Two crystal structures of HCV1 Fab in complex with an epitope peptide at 1.8-{angstrom} resolution reveal that the epitope is a {beta}-hairpin displaying a hydrophilic face and a hydrophobic face on opposing sides of the hairpin. The antibody predominantly interacts with E2 residues Leu{sup 413} and Trp{sup 420} on the hydrophobic face of the epitope, thus providing an explanation for how HCV isolates bearing mutations at Asn{sup 415} on the same binding face escape neutralization by this antibody. The results provide structural information for a neutralizing epitope on the HCV E2 glycoprotein and should help guide rational design of HCV immunogens to elicit similar broadly neutralizing antibodies through vaccination.

  16. Identification and specificity of broadly neutralizing antibodies against HIV

    OpenAIRE

    McCoy, Laura E.; Burton, Dennis R.

    2017-01-01

    Summary Beginning in 2009, studies of the humoral responses of HIV‐positive individuals have led to the identification of scores, if not hundreds, of antibodies that are both broadly reactive and potently neutralizing. This development has provided renewed impetus toward an HIV vaccine and led directly to the development of novel immunogens. Advances in identification of donors with the most potent and broad anti‐HIV serum neutralizing responses were crucial in this effort. Equally, developme...

  17. Broadly neutralizing antibodies: An approach to control HIV-1 infection.

    Science.gov (United States)

    Yaseen, Mahmoud Mohammad; Yaseen, Mohammad Mahmoud; Alqudah, Mohammad Ali

    2017-01-02

    Although available antiretroviral therapy (ART) has changed human immunodeficiency virus (HIV)-1 infection to a non-fatal chronic disease, the economic burden of lifelong therapy, severe adverse ART effects, daily ART adherence, and emergence of ART-resistant HIV-1 mutants require prospecting for alternative therapeutic modalities. Indeed, a growing body of evidence suggests that broadly neutralizing anti-HIV-1 antibodies (BNAbs) may offer one such feasible alternative. To evaluate their therapeutic potential in established HIV-1 infection, we sought to address recent advances in pre-clinical and clinical investigations in this area of HIV-1 research. In addition, we addressed the obstacles that may impede the success of such immunotherapeutic approach, suggested strategic solutions, and briefly compared this approach with the currently used ART to open new insights for potential future passive immunotherapy for HIV-1 infection.

  18. Fully human broadly neutralizing monoclonal antibodies against influenza A viruses generated from the memory B cells of a 2009 pandemic H1N1 influenza vaccine recipient

    Energy Technology Data Exchange (ETDEWEB)

    Hu, Weibin [Molecular Virus Unit, Key Laboratory of Molecular Virology and Immunology, Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai 200025 (China); Chen, Aizhong [Key Laboratory of Molecular Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031 (China); Miao, Yi [Shanghai Xuhui Central Hospital, Shanghai 200031 (China); Xia, Shengli [Center for Disease Control and Prevention of Henan Province, Zhengzhou 450016 (China); Ling, Zhiyang; Xu, Ke; Wang, Tongyan [Molecular Virus Unit, Key Laboratory of Molecular Virology and Immunology, Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai 200025 (China); Xu, Ying; Cui, Jun; Wu, Hongqiang; Hu, Guiyu; Tian, Lin; Wang, Lingling [Key Laboratory of Molecular Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031 (China); Shu, Yuelong [Chinese Center for Disease Control and Prevention, Beijing 102206 (China); Ma, Xiaowei [Hualan Biological Bacterin Company, Xinxiang 453003 (China); Xu, Bianli; Zhang, Jin [Center for Disease Control and Prevention of Henan Province, Zhengzhou 450016 (China); Lin, Xiaojun, E-mail: linxiaojun@hualan.com [Hualan Biological Bacterin Company, Xinxiang 453003 (China); Bian, Chao, E-mail: cbian@sibs.ac.cn [Key Laboratory of Molecular Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031 (China); Sun, Bing, E-mail: bsun@sibs.ac.cn [Molecular Virus Unit, Key Laboratory of Molecular Virology and Immunology, Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai 200025 (China); Key Laboratory of Molecular Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031 (China)

    2013-01-20

    Whether the 2009 pandemic H1N1 influenza vaccine can induce heterosubtypic cross-protective anti-hemagglutinin (HA) neutralizing antibodies is an important issue. We obtained a panel of fully human monoclonal antibodies from the memory B cells of a 2009 pandemic H1N1 influenza vaccine recipient. Most of the monoclonal antibodies targeted the HA protein but not the HA1 fragment. Among the analyzed antibodies, seven mAbs exhibited neutralizing activity against several influenza A viruses of different subtypes. The conserved linear epitope targeted by the neutralizing mAbs (FIEGGWTGMVDGWYGYHH) is part of the fusion peptide on HA2. Our work suggests that a heterosubtypic neutralizing antibody response primarily targeting the HA stem region exists in recipients of the 2009 pandemic H1N1 influenza vaccine. The HA stem region contains various conserved neutralizing epitopes with the fusion peptide as an important one. This work may aid in the design of a universal influenza A virus vaccine.

  19. A glycoconjugate antigen based on the recognition motif of a broadly neutralizing human immunodeficiency virus antibody, 2G12, is immunogenic but elicits antibodies unable to bind to the self glycans of gp120

    DEFF Research Database (Denmark)

    Astronomo, Rena D; Lee, Hing-Ken; Scanlan, Christopher N

    2008-01-01

    The glycan shield of human immunodeficiency virus type 1 (HIV-1) gp120 contributes to viral evasion from humoral immune responses. However, the shield is recognized by the HIV-1 broadly neutralizing antibody (Ab), 2G12, at a relatively conserved cluster of oligomannose glycans. The discovery of 2G......12 raises the possibility that a carbohydrate immunogen may be developed that could elicit 2G12-like neutralizing Abs and contribute to an AIDS vaccine. We have previously dissected the fine specificity of 2G12 and reported that the synthetic tetramannoside (Man(4)) that corresponds to the D1 arm...

  20. HIV-1 envelope glycoprotein immunogens to induce broadly neutralizing antibodies.

    Science.gov (United States)

    Sliepen, Kwinten; Sanders, Rogier W

    2016-01-01

    The long pursuit for a vaccine against human immunodeficiency virus 1 (HIV-1) has recently been boosted by a number of exciting developments. An HIV-1 subunit vaccine ideally should elicit potent broadly neutralizing antibodies (bNAbs), but raising bNAbs by vaccination has proved extremely difficult because of the characteristics of the HIV-1 envelope glycoprotein complex (Env). However, the isolation of bNAbs from HIV-1-infected patients demonstrates that the human humoral immune system is capable of making such antibodies. Therefore, a focus of HIV-1 vaccinology is the elicitation of bNAbs by engineered immunogens and by using vaccination strategies aimed at mimicking the bNAb maturation pathways in HIV-infected patients. Important clues can also be taken from the successful subunit vaccines against hepatitis B virus and human papillomavirus. Here, we review the different types of HIV-1 immunogens and vaccination strategies that are being explored in the search for an HIV-1 vaccine that induces bNAbs.

  1. Naturally selected hepatitis C virus polymorphisms confer broad neutralizing antibody resistance

    Science.gov (United States)

    Bailey, Justin R.; Wasilewski, Lisa N.; Snider, Anna E.; El-Diwany, Ramy; Osburn, William O.; Keck, Zhenyong; Foung, Steven K.H.; Ray, Stuart C.

    2014-01-01

    For hepatitis C virus (HCV) and other highly variable viruses, broadly neutralizing mAbs are an important guide for vaccine development. The development of resistance to anti-HCV mAbs is poorly understood, in part due to a lack of neutralization testing against diverse, representative panels of HCV variants. Here, we developed a neutralization panel expressing diverse, naturally occurring HCV envelopes (E1E2s) and used this panel to characterize neutralizing breadth and resistance mechanisms of 18 previously described broadly neutralizing anti-HCV human mAbs. The observed mAb resistance could not be attributed to polymorphisms in E1E2 at known mAb-binding residues. Additionally, hierarchical clustering analysis of neutralization resistance patterns revealed relationships between mAbs that were not predicted by prior epitope mapping, identifying 3 distinct neutralization clusters. Using this clustering analysis and envelope sequence data, we identified polymorphisms in E2 that confer resistance to multiple broadly neutralizing mAbs. These polymorphisms, which are not at mAb contact residues, also conferred resistance to neutralization by plasma from HCV-infected subjects. Together, our method of neutralization clustering with sequence analysis reveals that polymorphisms at noncontact residues may be a major immune evasion mechanism for HCV, facilitating viral persistence and presenting a challenge for HCV vaccine development. PMID:25500884

  2. Engineering broadly neutralizing antibodies for HIV prevention and therapy.

    Science.gov (United States)

    Hua, Casey K; Ackerman, Margaret E

    2016-08-01

    A combination of advances spanning from isolation to delivery of potent HIV-specific antibodies has begun to revolutionize understandings of antibody-mediated antiviral activity. As a result, the set of broadly neutralizing and highly protective antibodies has grown in number, diversity, potency, and breadth of viral recognition and neutralization. These antibodies are now being further enhanced by rational engineering of their anti-HIV activities and coupled to cutting edge gene delivery and strategies to optimize their pharmacokinetics and biodistribution. As a result, the prospects for clinical use of HIV-specific antibodies to treat, clear, and prevent HIV infection are gaining momentum. Here we discuss the diverse methods whereby antibodies are being optimized for neutralization potency and breadth, biodistribution, pharmacokinetics, and effector function with the aim of revolutionizing HIV treatment and prevention options.

  3. Molecular evolution of broadly neutralizing Llama antibodies to the CD4-binding site of HIV-1.

    Directory of Open Access Journals (Sweden)

    Laura E McCoy

    2014-12-01

    Full Text Available To date, no immunization of humans or animals has elicited broadly neutralizing sera able to prevent HIV-1 transmission; however, elicitation of broad and potent heavy chain only antibodies (HCAb has previously been reported in llamas. In this study, the anti-HIV immune responses in immunized llamas were studied via deep sequencing analysis using broadly neutralizing monoclonal HCAbs as a guides. Distinct neutralizing antibody lineages were identified in each animal, including two defined by novel antibodies (as variable regions called VHH identified by robotic screening of over 6000 clones. The combined application of five VHH against viruses from clades A, B, C and CRF_AG resulted in neutralization as potent as any of the VHH individually and a predicted 100% coverage with a median IC50 of 0.17 µg/ml for the panel of 60 viruses tested. Molecular analysis of the VHH repertoires of two sets of immunized animals showed that each neutralizing lineage was only observed following immunization, demonstrating that they were elicited de novo. Our results show that immunization can induce potent and broadly neutralizing antibodies in llamas with features similar to human antibodies and provide a framework to analyze the effectiveness of immunization protocols.

  4. Molecular Evolution of Broadly Neutralizing Llama Antibodies to the CD4-Binding Site of HIV-1

    Science.gov (United States)

    McCoy, Laura E.; Rutten, Lucy; Frampton, Dan; Anderson, Ian; Granger, Luke; Bashford-Rogers, Rachael; Dekkers, Gillian; Strokappe, Nika M.; Seaman, Michael S.; Koh, Willie; Grippo, Vanina; Kliche, Alexander; Verrips, Theo; Kellam, Paul; Fassati, Ariberto; Weiss, Robin A.

    2014-01-01

    To date, no immunization of humans or animals has elicited broadly neutralizing sera able to prevent HIV-1 transmission; however, elicitation of broad and potent heavy chain only antibodies (HCAb) has previously been reported in llamas. In this study, the anti-HIV immune responses in immunized llamas were studied via deep sequencing analysis using broadly neutralizing monoclonal HCAbs as a guides. Distinct neutralizing antibody lineages were identified in each animal, including two defined by novel antibodies (as variable regions called VHH) identified by robotic screening of over 6000 clones. The combined application of five VHH against viruses from clades A, B, C and CRF_AG resulted in neutralization as potent as any of the VHH individually and a predicted 100% coverage with a median IC50 of 0.17 µg/ml for the panel of 60 viruses tested. Molecular analysis of the VHH repertoires of two sets of immunized animals showed that each neutralizing lineage was only observed following immunization, demonstrating that they were elicited de novo. Our results show that immunization can induce potent and broadly neutralizing antibodies in llamas with features similar to human antibodies and provide a framework to analyze the effectiveness of immunization protocols. PMID:25522326

  5. Molecular evolution of broadly neutralizing Llama antibodies to the CD4-binding site of HIV-1.

    Science.gov (United States)

    McCoy, Laura E; Rutten, Lucy; Frampton, Dan; Anderson, Ian; Granger, Luke; Bashford-Rogers, Rachael; Dekkers, Gillian; Strokappe, Nika M; Seaman, Michael S; Koh, Willie; Grippo, Vanina; Kliche, Alexander; Verrips, Theo; Kellam, Paul; Fassati, Ariberto; Weiss, Robin A

    2014-12-01

    To date, no immunization of humans or animals has elicited broadly neutralizing sera able to prevent HIV-1 transmission; however, elicitation of broad and potent heavy chain only antibodies (HCAb) has previously been reported in llamas. In this study, the anti-HIV immune responses in immunized llamas were studied via deep sequencing analysis using broadly neutralizing monoclonal HCAbs as a guides. Distinct neutralizing antibody lineages were identified in each animal, including two defined by novel antibodies (as variable regions called VHH) identified by robotic screening of over 6000 clones. The combined application of five VHH against viruses from clades A, B, C and CRF_AG resulted in neutralization as potent as any of the VHH individually and a predicted 100% coverage with a median IC50 of 0.17 µg/ml for the panel of 60 viruses tested. Molecular analysis of the VHH repertoires of two sets of immunized animals showed that each neutralizing lineage was only observed following immunization, demonstrating that they were elicited de novo. Our results show that immunization can induce potent and broadly neutralizing antibodies in llamas with features similar to human antibodies and provide a framework to analyze the effectiveness of immunization protocols.

  6. Human monoclonal antibodies derived from a patient infected with 2009 pandemic influenza A virus broadly cross-neutralize group 1 influenza viruses.

    Science.gov (United States)

    Pan, Yang; Sasaki, Tadahiro; Kubota-Koketsu, Ritsuko; Inoue, Yuji; Yasugi, Mayo; Yamashita, Akifumi; Ramadhany, Ririn; Arai, Yasuha; Du, Anariwa; Boonsathorn, Naphatsawan; Ibrahim, Madiha S; Daidoji, Tomo; Nakaya, Takaaki; Ono, Ken-ichiro; Okuno, Yoshinobu; Ikuta, Kazuyoshi; Watanabe, Yohei

    2014-07-18

    Influenza viruses are a continuous threat to human public health because of their ability to evolve rapidly through genetic drift and reassortment. Three human monoclonal antibodies (HuMAbs) were generated in this study, 1H11, 2H5 and 5G2, and they cross-neutralize a diverse range of group 1 influenza A viruses, including seasonal H1N1, 2009 pandemic H1N1 (H1N1pdm) and avian H5N1 and H9N2. The three HuMAbs were prepared by fusing peripheral blood lymphocytes from an H1N1pdm-infected patient with a newly developed fusion partner cell line, SPYMEG. All the HuMAbs had little hemagglutination inhibition activity but had strong membrane-fusion inhibition activity against influenza viruses. A protease digestion assay showed the HuMAbs targeted commonly a short α-helix region in the stalk of the hemagglutinin. Furthermore, Ile45Phe and Glu47Gly double substitutions in the α-helix region made the HA unrecognizable by the HuMAbs. These two amino acid residues are highly conserved in the HAs of H1N1, H5N1 and H9N2 viruses. The HuMAbs reported here may be potential candidates for the development of therapeutic antibodies against group 1 influenza viruses.

  7. Human monoclonal antibodies derived from a patient infected with 2009 pandemic influenza A virus broadly cross-neutralize group 1 influenza viruses

    Energy Technology Data Exchange (ETDEWEB)

    Pan, Yang [Research Institute for Microbial Diseases, Osaka University, Suita, Osaka (Japan); Sasaki, Tadahiro [Research Institute for Microbial Diseases, Osaka University, Suita, Osaka (Japan); JST/JICA, Science and Technology Research Partnership for Sustainable Development (SATREPS), Tokyo (Japan); Kubota-Koketsu, Ritsuko [Research Institute for Microbial Diseases, Osaka University, Suita, Osaka (Japan); Kanonji Institute, The Research Foundation for Microbial Diseases of Osaka University, Kanonji, Kagawa (Japan); JST/JICA, Science and Technology Research Partnership for Sustainable Development (SATREPS), Tokyo (Japan); Inoue, Yuji [Research Institute for Microbial Diseases, Osaka University, Suita, Osaka (Japan); JST/JICA, Science and Technology Research Partnership for Sustainable Development (SATREPS), Tokyo (Japan); Yasugi, Mayo [Research Institute for Microbial Diseases, Osaka University, Suita, Osaka (Japan); Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Izumisano, Osaka (Japan); JST/JICA, Science and Technology Research Partnership for Sustainable Development (SATREPS), Tokyo (Japan); Yamashita, Akifumi; Ramadhany, Ririn; Arai, Yasuha [Research Institute for Microbial Diseases, Osaka University, Suita, Osaka (Japan); Du, Anariwa [Research Institute for Microbial Diseases, Osaka University, Suita, Osaka (Japan); JST/JICA, Science and Technology Research Partnership for Sustainable Development (SATREPS), Tokyo (Japan); Boonsathorn, Naphatsawan [Research Institute for Microbial Diseases, Osaka University, Suita, Osaka (Japan); Department of Medical Sciences, Ministry of Public Health, Muang, Nonthaburi (Thailand); JST/JICA, Science and Technology Research Partnership for Sustainable Development (SATREPS), Tokyo (Japan); Ibrahim, Madiha S. [Research Institute for Microbial Diseases, Osaka University, Suita, Osaka (Japan); Department of Microbiology and Immunology, Faculty of Veterinary Medicine, Damanhour University, Damanhour (Egypt); and others

    2014-07-18

    Highlights: • Influenza infection can elicit heterosubtypic antibodies to group 1 influenza virus. • Three human monoclonal antibodies were generated from an H1N1-infected patient. • The antibodies predominantly recognized α-helical stem of viral hemagglutinin (HA). • The antibodies inhibited HA structural activation during the fusion process. • The antibodies are potential candidates for future antibody therapy to influenza. - Abstract: Influenza viruses are a continuous threat to human public health because of their ability to evolve rapidly through genetic drift and reassortment. Three human monoclonal antibodies (HuMAbs) were generated in this study, 1H11, 2H5 and 5G2, and they cross-neutralize a diverse range of group 1 influenza A viruses, including seasonal H1N1, 2009 pandemic H1N1 (H1N1pdm) and avian H5N1 and H9N2. The three HuMAbs were prepared by fusing peripheral blood lymphocytes from an H1N1pdm-infected patient with a newly developed fusion partner cell line, SPYMEG. All the HuMAbs had little hemagglutination inhibition activity but had strong membrane-fusion inhibition activity against influenza viruses. A protease digestion assay showed the HuMAbs targeted commonly a short α-helix region in the stalk of the hemagglutinin. Furthermore, Ile45Phe and Glu47Gly double substitutions in the α-helix region made the HA unrecognizable by the HuMAbs. These two amino acid residues are highly conserved in the HAs of H1N1, H5N1 and H9N2 viruses. The HuMAbs reported here may be potential candidates for the development of therapeutic antibodies against group 1 influenza viruses.

  8. Broadly Neutralizing Alphavirus Antibodies Bind an Epitope on E2 and Inhibit Entry and Egress

    NARCIS (Netherlands)

    Fox, Julie M.; Long, Feng; Edeling, Melissa A.; Lin, Hueylie; van Duijl-Richter, Mareike K. S.; Fong, Rachel H.; Kahle, Kristen M.; Smit, Jolanda M.; Jin, Jing; Simmons, Graham; Doranz, Benjamin J.; Crowe, James E.; Fremont, Daved H.; Rossmann, Michael G.; Diamond, Michael S.

    2015-01-01

    We screened a panel of mouse and human monoclonal antibodies (MAbs) against chikungunya virus and identified several with inhibitory activity against multiple alphaviruses. Passive transfer of broadly neutralizing MAbs protected mice against infection by chikungunya, Mayaro, and O'nyong'nyong alphav

  9. Env-2dCD4 S60C complexes act as super immunogens and elicit potent, broadly neutralizing antibodies against clinically relevant human immunodeficiency virus type 1 (HIV-1).

    Science.gov (United States)

    Killick, Mark A; Grant, Michelle L; Cerutti, Nichole M; Capovilla, Alexio; Papathanasopoulos, Maria A

    2015-11-17

    The ability to induce a broadly neutralizing antibody (bNAb) response following vaccination is regarded as a crucial aspect in developing an effective vaccine against human immunodeficiency virus type 1 (HIV-1). The bNAbs target the HIV-1 envelope glycoprotein (Env) which is exposed on the virus surface, thereby preventing cell entry. To date, conventional vaccine approaches such as the use of Env-based immunogens have been unsuccessful. We expressed, purified, characterized and evaluated the immunogenicity of several unique HIV-1 subtype C Env immunogens in small animals. Here we report that vaccine immunogens based on Env liganded to a two domain CD4 variant, 2dCD4(S60C) are capable of consistently eliciting potent, broadly neutralizing antibody responses in New Zealand white rabbits against a panel of clinically relevant HIV-1 pseudoviruses. This was irrespective of the Env protein subtype and context. Importantly, depletion of the anti-CD4 antibodies appeared to abrogate the neutralization activity in the rabbit sera. Taken together, this data suggests that the Env-2dCD4(S60C) complexes described here are "super" immunogens, and potentially immunofocus antibody responses to a unique epitope spanning the 2dCD4(60C). Recent data from the two available anti-CD4 monoclonal antibodies, Ibalizumab and CD4-Ig (and bispecific variants thereof) have highlighted that the use of these broad and potent entry inhibitors could circumvent the need for a conventional vaccine targeting HIV-1. Overall, the ability of the unique Env-2dCD4(S60C) complexes to elicit potent bNAb responses has not been described previously, reinforcing that further investigation for their utility in preventing and controlling HIV-1/SIV infection is warranted.

  10. Novel Clostridium difficile Anti-Toxin (TcdA and TcdB Humanized Monoclonal Antibodies Demonstrate In Vitro Neutralization across a Broad Spectrum of Clinical Strains and In Vivo Potency in a Hamster Spore Challenge Model.

    Directory of Open Access Journals (Sweden)

    Hongyu Qiu

    Full Text Available Clostridium difficile (C. difficile infection (CDI is the main cause of nosocomial antibiotic-associated colitis and increased incidence of community-associated diarrhea in industrialized countries. At present, the primary treatment of CDI is antibiotic administration, which is effective but often associated with recurrence, especially in the elderly. Pathogenic strains produce enterotoxin, toxin A (TcdA, and cytotoxin, toxin B (TcdB, which are necessary for C. difficile induced diarrhea and gut pathological changes. Administration of anti-toxin antibodies provides an alternative approach to treat CDI, and has shown promising results in preclinical and clinical studies. In the current study, several humanized anti-TcdA and anti-TcdB monoclonal antibodies were generated and their protective potency was characterized in a hamster infection model. The humanized anti-TcdA (CANmAbA4 and anti-TcdB (CANmAbB4 and CANmAbB1 antibodies showed broad spectrum in vitro neutralization of toxins from clinical strains and neutralization in a mouse toxin challenge model. Moreover, co-administration of humanized antibodies (CANmAbA4 and CANmAbB4 cocktail provided a high level of protection in a dose dependent manner (85% versus 57% survival at day 22 for 50 mg/kg and 20 mg/kg doses, respectively in a hamster gastrointestinal infection (GI model. This study describes the protective effects conferred by novel neutralizing anti-toxin monoclonal antibodies against C. difficile toxins and their potential as therapeutic agents in treating CDI.

  11. Co-evolution of a broadly neutralizing HIV-1 antibody and founder virus.

    Science.gov (United States)

    Liao, Hua-Xin; Lynch, Rebecca; Zhou, Tongqing; Gao, Feng; Alam, S Munir; Boyd, Scott D; Fire, Andrew Z; Roskin, Krishna M; Schramm, Chaim A; Zhang, Zhenhai; Zhu, Jiang; Shapiro, Lawrence; Mullikin, James C; Gnanakaran, S; Hraber, Peter; Wiehe, Kevin; Kelsoe, Garnett; Yang, Guang; Xia, Shi-Mao; Montefiori, David C; Parks, Robert; Lloyd, Krissey E; Scearce, Richard M; Soderberg, Kelly A; Cohen, Myron; Kamanga, Gift; Louder, Mark K; Tran, Lillian M; Chen, Yue; Cai, Fangping; Chen, Sheri; Moquin, Stephanie; Du, Xiulian; Joyce, M Gordon; Srivatsan, Sanjay; Zhang, Baoshan; Zheng, Anqi; Shaw, George M; Hahn, Beatrice H; Kepler, Thomas B; Korber, Bette T M; Kwong, Peter D; Mascola, John R; Haynes, Barton F

    2013-04-25

    Current human immunodeficiency virus-1 (HIV-1) vaccines elicit strain-specific neutralizing antibodies. However, cross-reactive neutralizing antibodies arise in approximately 20% of HIV-1-infected individuals, and details of their generation could provide a blueprint for effective vaccination. Here we report the isolation, evolution and structure of a broadly neutralizing antibody from an African donor followed from the time of infection. The mature antibody, CH103, neutralized approximately 55% of HIV-1 isolates, and its co-crystal structure with the HIV-1 envelope protein gp120 revealed a new loop-based mechanism of CD4-binding-site recognition. Virus and antibody gene sequencing revealed concomitant virus evolution and antibody maturation. Notably, the unmutated common ancestor of the CH103 lineage avidly bound the transmitted/founder HIV-1 envelope glycoprotein, and evolution of antibody neutralization breadth was preceded by extensive viral diversification in and near the CH103 epitope. These data determine the viral and antibody evolution leading to induction of a lineage of HIV-1 broadly neutralizing antibodies, and provide insights into strategies to elicit similar antibodies by vaccination.

  12. Virological features associated with the development of broadly neutralizing antibodies to HIV-1.

    Science.gov (United States)

    Moore, Penny L; Williamson, Carolyn; Morris, Lynn

    2015-04-01

    The development of a preventative HIV-1 vaccine remains a global public health priority. This will likely require the elicitation of broadly neutralizing antibodies (bNAbs) able to block infection by diverse viral strains from across the world. Understanding the pathway to neutralization breadth in HIV-1 infected humans will provide insights into how bNAb lineages arise, a process that probably involves a combination of host and viral factors. Here, we focus on the role of viral characteristics and evolution in shaping bNAbs during HIV-1 infection, and describe how these findings may be translated into novel vaccine strategies.

  13. Structural Basis for Broad and Potent Neutralization of HIV-1 by Antibody VRC01

    Energy Technology Data Exchange (ETDEWEB)

    Zhou, Tongqing; Georgiev, Ivelin; Wu, Xueling; Yang, Zhi-Yong; Dai, Kaifan; Finzi, Andrés; Kwon, Young Do; Scheid, Johannes F.; Shi, Wei; Xu, Ling; Yang, Yongping; Zhu, Jiang; Nussenzweig, Michel C.; Sodroski, Joseph; Shapiro, Lawrence; Nabel, Gary J.; Mascola, John R.; Kwong, Peter D. (NIH); (Rockefeller); (DFCI)

    2010-08-26

    During HIV-1 infection, antibodies are generated against the region of the viral gp120 envelope glycoprotein that binds CD4, the primary receptor for HIV-1. Among these antibodies, VRC01 achieves broad neutralization of diverse viral strains. We determined the crystal structure of VRC01 in complex with a human immunodeficiency virus HIV-1 gp120 core. VRC01 partially mimics CD4 interaction with gp120. A shift from the CD4-defined orientation, however, focuses VRC01 onto the vulnerable site of initial CD4 attachment, allowing it to overcome the glycan and conformational masking that diminishes the neutralization potency of most CD4-binding-site antibodies. To achieve this recognition, VRC01 contacts gp120 mainly through immunoglobulin V-gene regions substantially altered from their genomic precursors. Partial receptor mimicry and extensive affinity maturation thus facilitate neutralization of HIV-1 by natural human antibodies.

  14. Broadly Neutralizing Anti-Influenza Virus Antibodies: Enhancement of Neutralizing Potency in Polyclonal Mixtures and IgA Backbones

    Science.gov (United States)

    He, Wenqian; Mullarkey, Caitlin E.; Duty, J. Andrew; Moran, Thomas M.; Palese, Peter

    2015-01-01

    ABSTRACT Current influenza virus vaccines rely upon the accurate prediction of circulating virus strains months in advance of the actual influenza season in order to allow time for vaccine manufacture. Unfortunately, mismatches occur frequently, and even when perfect matches are achieved, suboptimal vaccine efficacy leaves several high-risk populations vulnerable to infection. However, the recent discovery of broadly neutralizing antibodies that target the hemagglutinin (HA) stalk domain has renewed hope that the development of “universal” influenza virus vaccines may be within reach. Here, we examine the functions of influenza A virus hemagglutinin stalk-binding antibodies in an endogenous setting, i.e., as polyclonal preparations isolated from human sera. Relative to monoclonal antibodies that bind to the HA head domain, the neutralization potency of monoclonal stalk-binding antibodies was vastly inferior in vitro but was enhanced by several orders of magnitude in the polyclonal context. Furthermore, we demonstrated a surprising enhancement in IgA-mediated HA stalk neutralization relative to that achieved by antibodies of IgG isotypes. Mechanistically, this could be explained in two ways. Identical variable regions consistently neutralized virus more potently when in an IgA backbone compared to an IgG backbone. In addition, HA-specific memory B cells isolated from human peripheral blood were more likely to be stalk specific when secreting antibodies of IgA isotypes compared to those secreting IgG. Taken together, our data provide strong evidence that HA stalk-binding antibodies perform optimally when in a polyclonal context and that the targeted elicitation of HA stalk-specific IgA should be an important consideration during “universal” influenza virus vaccine design. IMPORTANCE Influenza viruses remain one of the most worrisome global public health threats due to their capacity to cause pandemics. While seasonal vaccines fail to protect against the

  15. Non-random escape pathways from a broadly neutralizing human monoclonal antibody map to a highly conserved region on the hepatitis C virus E2 glycoprotein encompassing amino acids 412-423.

    Directory of Open Access Journals (Sweden)

    Zhen-yong Keck

    2014-08-01

    Full Text Available A challenge for hepatitis C virus (HCV vaccine development is to define epitopes that are able to elicit protective antibodies against this highly diverse virus. The E2 glycoprotein region located at residues 412-423 is conserved and antibodies to 412-423 have broadly neutralizing activities. However, an adaptive mutation, N417S, is associated with a glycan shift in a variant that cannot be neutralized by a murine but by human monoclonal antibodies (HMAbs against 412-423. To determine whether HCV escapes from these antibodies, we analyzed variants that emerged when cell culture infectious HCV virions (HCVcc were passaged under increasing concentrations of a specific HMAb, HC33.1. Multiple nonrandom escape pathways were identified. Two pathways occurred in the context of an N-glycan shift mutation at N417T. At low antibody concentrations, substitutions of two residues outside of the epitope, N434D and K610R, led to variants having improved in vitro viral fitness and reduced sensitivity to HC33.1 binding and neutralization. At moderate concentrations, a S419N mutation occurred within 412-423 in escape variants that have greatly reduced sensitivity to HC33.1 but compromised viral fitness. Importantly, the variants generated from these pathways differed in their stability. N434D and K610R-associated variants were stable and became dominant as the virions were passaged. The S419N mutation reverted back to N419S when immune pressure was reduced by removing HC33.1. At high antibody concentrations, a mutation at L413I was observed in variants that were resistant to HC33.1 neutralization. Collectively, the combination of multiple escape pathways enabled the virus to persist under a wide range of antibody concentrations. Moreover, these findings pose a different challenge to vaccine development beyond the identification of highly conserved epitopes. It will be necessary for a vaccine to induce high potency antibodies that prevent the formation of escape

  16. Enhanced clearance of HIV-1-infected cells by broadly neutralizing antibodies against HIV-1 in vivo.

    Science.gov (United States)

    Lu, Ching-Lan; Murakowski, Dariusz K; Bournazos, Stylianos; Schoofs, Till; Sarkar, Debolina; Halper-Stromberg, Ariel; Horwitz, Joshua A; Nogueira, Lilian; Golijanin, Jovana; Gazumyan, Anna; Ravetch, Jeffrey V; Caskey, Marina; Chakraborty, Arup K; Nussenzweig, Michel C

    2016-05-20

    Antiretroviral drugs and antibodies limit HIV-1 infection by interfering with the viral life cycle. In addition, antibodies also have the potential to guide host immune effector cells to kill HIV-1-infected cells. Examination of the kinetics of HIV-1 suppression in infected individuals by passively administered 3BNC117, a broadly neutralizing antibody, suggested that the effects of the antibody are not limited to free viral clearance and blocking new infection but also include acceleration of infected cell clearance. Consistent with these observations, we find that broadly neutralizing antibodies can target CD4(+) T cells infected with patient viruses and can decrease their in vivo half-lives by a mechanism that requires Fcγ receptor engagement in a humanized mouse model. The results indicate that passive immunotherapy can accelerate elimination of HIV-1-infected cells.

  17. Stapled HIV-1 peptides recapitulate antigenic structures and engage broadly neutralizing antibodies.

    Science.gov (United States)

    Bird, Gregory H; Irimia, Adriana; Ofek, Gilad; Kwong, Peter D; Wilson, Ian A; Walensky, Loren D

    2014-12-01

    Hydrocarbon stapling can restore bioactive α-helical structure to natural peptides, yielding research tools and prototype therapeutics to dissect and target protein interactions. Here we explore the capacity of peptide stapling to generate high-fidelity, protease-resistant mimics of antigenic structures for vaccine development. HIV-1 has been refractory to vaccine technologies thus far, although select human antibodies can broadly neutralize HIV-1 by targeting sequences of the gp41 juxtamembrane fusion apparatus. To develop candidate HIV-1 immunogens, we generated and characterized stabilized α-helices of the membrane-proximal external region (SAH-MPER) of gp41. SAH-MPER peptides were remarkably protease resistant and bound to the broadly neutralizing 4E10 and 10E8 antibodies with high affinity, recapitulating the structure of the MPER epitope when differentially engaged by the two anti-HIV Fabs. Thus, stapled peptides may provide a new opportunity to develop chemically stabilized antigens for vaccination.

  18. Elicitation of broadly neutralizing influenza antibodies in animals with previous influenza exposure.

    Science.gov (United States)

    Wei, Chih-Jen; Yassine, Hadi M; McTamney, Patrick M; Gall, Jason G D; Whittle, James R R; Boyington, Jeffrey C; Nabel, Gary J

    2012-08-15

    The immune system responds to influenza infection by producing neutralizing antibodies to the viral surface protein, hemagglutinin (HA), which regularly changes its antigenic structure. Antibodies that target the highly conserved stem region of HA neutralize diverse influenza viruses and can be elicited through vaccination in animals and humans. Efforts to develop universal influenza vaccines have focused on strategies to elicit such antibodies; however, the concern has been raised that previous influenza immunity may abrogate the induction of such broadly protective antibodies. We show here that prime-boost immunization can induce broadly neutralizing antibody responses in influenza-immune mice and ferrets that were previously infected or vaccinated. HA stem-directed antibodies were elicited in mice primed with a DNA vaccine and boosted with inactivated vaccine from H1N1 A/New Caledonia/20/1999 (1999 NC) HA regardless of preexposure. Similarly, gene-based vaccination with replication-defective adenovirus 28 (rAd28) and 5 (rAd5) vectors encoding 1999 NC HA elicited stem-directed neutralizing antibodies and conferred protection against unmatched 1934 and 2007 H1N1 virus challenge in influenza-immune ferrets. Indeed, previous exposure to certain strains could enhance immunogenicity: The strongest HA stem-directed immune response was observed in ferrets previously infected with a divergent 1934 H1N1 virus. These findings suggest that broadly neutralizing antibodies against the conserved stem region of HA can be elicited through vaccination despite previous influenza exposure, which supports the feasibility of developing stem-directed universal influenza vaccines for humans.

  19. Hepatitis C virus resistance to broadly neutralizing antibodies measured using replication-competent virus and pseudoparticles.

    Science.gov (United States)

    Wasilewski, Lisa N; Ray, Stuart C; Bailey, Justin R

    2016-11-01

    A better understanding of natural variation in neutralization resistance and fitness of diverse hepatitis C virus (HCV) envelope (E1E2) variants will be critical to guide rational development of an HCV vaccine. This work has been hindered by inadequate genetic diversity in viral panels and by a lack of standardization of HCV entry assays. Neutralization assays generally use lentiviral pseudoparticles expressing HCV envelope proteins (HCVpp) or chimeric full-length viruses that are replication competent in cell culture (HCVcc). There have been few systematic comparisons of specific infectivities of E1E2-matched HCVcc and HCVpp, and to our knowledge, neutralization of E1E2-matched HCVpp and HCVcc has never been compared using a diverse panel of human broadly neutralizing monoclonal antibodies (bNAbs) targeting distinct epitopes. Here, we describe an efficient method for introduction of naturally occurring E1E2 genes into a full-length HCV genome, producing replication-competent chimeric HCVcc. We generated diverse panels of E1E2-matched HCVcc and HCVpp and measured the entry-mediating fitness of E1E2 variants using the two systems. We also compared neutralization of E1E2-matched HCVcc and HCVpp by a diverse panel of human bNAbs targeting epitopes across E1E2. We found no correlation between specific infectivities of E1E2-matched HCVcc versus HCVpp, but found a very strong positive correlation between relative neutralization resistance of these same E1E2-matched HCVcc and HCVpp variants. These results suggest that quantitative comparisons of neutralization resistance of E1E2 variants can be made with confidence using either HCVcc or HCVpp, allowing the use of either or both systems to maximize diversity of neutralization panels.

  20. Broadly Neutralizing Alphavirus Antibodies Bind an Epitope on E2 and Inhibit Entry and Egress.

    Science.gov (United States)

    Fox, Julie M; Long, Feng; Edeling, Melissa A; Lin, Hueylie; van Duijl-Richter, Mareike K S; Fong, Rachel H; Kahle, Kristen M; Smit, Jolanda M; Jin, Jing; Simmons, Graham; Doranz, Benjamin J; Crowe, James E; Fremont, Daved H; Rossmann, Michael G; Diamond, Michael S

    2015-11-19

    We screened a panel of mouse and human monoclonal antibodies (MAbs) against chikungunya virus and identified several with inhibitory activity against multiple alphaviruses. Passive transfer of broadly neutralizing MAbs protected mice against infection by chikungunya, Mayaro, and O'nyong'nyong alphaviruses. Using alanine-scanning mutagenesis, loss-of-function recombinant proteins and viruses, and multiple functional assays, we determined that broadly neutralizing MAbs block multiple steps in the viral lifecycle, including entry and egress, and bind to a conserved epitope on the B domain of the E2 glycoprotein. A 16 Å resolution cryo-electron microscopy structure of a Fab fragment bound to CHIKV E2 B domain provided an explanation for its neutralizing activity. Binding to the B domain was associated with repositioning of the A domain of E2 that enabled cross-linking of neighboring spikes. Our results suggest that B domain antigenic determinants could be targeted for vaccine or antibody therapeutic development against multiple alphaviruses of global concern.

  1. Roles of glycans in interactions between gp120 and HIV broadly neutralizing antibodies.

    Science.gov (United States)

    Qi, Yifei; Jo, Sunhwan; Im, Wonpil

    2016-03-01

    Many novel broadly neutralizing antibodies against human immunodeficiency virus (HIV) have been identified during the past decade, providing promising templates for the development of an effective HIV-1 vaccine. Structural studies reveal that the epitopes of some of these antibodies involve one or more crucial glycans, without which the binding is completely abolished. In this study, we have investigated the critical roles of glycans in interactions between HIV-1 gp120 and two broadly neutralizing antibodies PG9 (targeting V1/V2) and PGT128 (targeting V3) that are able to neutralize more than 70% of HIV-1 isolates. We have performed molecular dynamics simulations of a number of systems including antibody-gp120 complex with and without glycans, antibody, gp120 with and without glycans, and glycan-only systems. The simulation results show that the complex structures are stabilized by the glycans, and the multivalent interactions between the antibody and gp120 promote cooperativities to further enhance the binding. In the free gp120, the glycans increase the flexibility of the V1/V2 and V3 loops, which likely increases the entropy cost of the antibody recognition. However, the antibodies are able to bind the flexible interface by recognizing the preexisting glycan conformation, and penetrating the glycan shield with flexible complementarity determining region loops that sample the bound conformations occasionally.

  2. Broadly neutralizing antibodies developed by an HIV-positive elite neutralizer exact a replication fitness cost on the contemporaneous virus.

    Science.gov (United States)

    Sather, D Noah; Carbonetti, Sara; Kehayia, Jenny; Kraft, Zane; Mikell, Iliyana; Scheid, Johannes F; Klein, Florian; Stamatatos, Leonidas

    2012-12-01

    Approximately 1% of those infected with HIV-1 develop broad and potent serum cross-neutralizing antibody activities. It is unknown whether or not the development of such immune responses affects the replication of the contemporaneous autologous virus. Here, we defined a pathway of autologous viral escape from contemporaneous potent and broad serum neutralizing antibodies developed by an elite HIV-1-positive (HIV-1(+)) neutralizer. These antibodies potently neutralize diverse isolates from different clades and target primarily the CD4-binding site (CD4-BS) of the viral envelope glycoprotein. Viral escape required mutations in the viral envelope glycoprotein which limited the accessibility of the CD4-binding site to the autologous broadly neutralizing anti-CD4-BS antibodies but which allowed the virus to infect cells by utilizing CD4 receptors on their surface. The acquisition of neutralization resistance, however, resulted in reduced cell entry potential and slower viral replication kinetics. Our results indicate that in vivo escape from autologous broadly neutralizing antibodies exacts fitness costs to HIV-1.

  3. CD4 binding site broadly neutralizing antibody selection of HIV-1 escape mutants.

    Science.gov (United States)

    Dreja, Hanna; Pade, Corinna; Chen, Lei; McKnight, Áine

    2015-07-01

    All human immunodeficiency virus type-1 (HIV-1) viruses use CD4 to enter cells. Consequently, the viral envelope CD4-binding site (CD4bs) is relatively conserved, making it a logical neutralizing antibody target. It is important to understand how CD4-binding site variation allows for escape from neutralizing antibodies. Alanine scanning mutagenesis identifies residues in antigenic sites, whereas escape mutant selection identifies viable mutants. We selected HIV-1 to escape CD4bs neutralizing mAbs b12, A12 and HJ16. Viruses that escape from A12 and b12 remained susceptible to HJ16, VRC01 and J3, whilst six different viruses that escape HJ16 remained sensitive to A12, b12 and J3. In contrast, their sensitivity to VRC01 was variable. Triple HJ16/A12/b12-resistant virus proved that HIV-1 could escape multiple broadly neutralizing monoclonal antibodies, but still retain sensitivity to VRC01 and the llama-derived J3 nanobody. This antigenic variability may reflect that occurring in circulating viruses, so studies like this can predict immunologically relevant antigenic forms of the CD4bs for inclusion in HIV-1 vaccines.

  4. Broadly Neutralizing Antibodies against HIV-1 As a Novel Aspect of the Immune Response.

    Science.gov (United States)

    Shcherbakov, D N; Bakulina, A Y; Karpenko, L I; Ilyichev, A A

    2015-01-01

    The human immunodeficiency virus-1 (HIV-1) has the ability to evade the adaptive immune response due to high mutation rates. Soon after the discovery of HIV-1, it was originally proposed that neutralizing of antibodies to the virus occurs rarely or cannot be elicited at all. In the 1990s, there appeared reports that sera of select HIV-1-infected individuals contained antibodies capable of neutralizing different virus subtypes. Such antibodies were named broadly neutralizing antibodies (bNAbs). Since 2009, the development of new cell technologies has intensified research efforts directed at identifying new bNAbs with a neutralization potency of over 90% of primary HIV-1 isolates. These antibodies have unique characteristics which include high levels of somatic mutations and unusually long variable loops that penetrate through the glycan shield of HIV-1 Env to contact the protein surface. In this review, we will attempt to summarize the latest data on bNAbs against HIV-1 in terms of their interactions with the sites of vulnerability on HIV-1 glycoproteins.

  5. Pharmacokinetics and immunogenicity of broadly neutralizing HIV monoclonal antibodies in macaques.

    Science.gov (United States)

    Rosenberg, Yvonne; Sack, Markus; Montefiori, David; Labranche, Celia; Lewis, Mark; Urban, Lori; Mao, Lingjun; Fischer, Rainer; Jiang, Xiaoming

    2015-01-01

    The identification of highly potent broadly neutralizing antibodies (bnAbs) against HIV-1, and success in preventing SHIV infection following their passive administration, have increased the likelihood that immunotherapeutic strategies can be adopted to prevent and treat HIV-1 infection. However, while broad and potent neutralizing activity is an essential prerequisite, in vivo properties such as good circulatory stability and non-immunogenicity are equally critical for developing a human treatment. In the present study, glycoforms of the bnAbs 10-1074, NIH45-46G54W, 10E8, PGT121, PGT128, PGT145, PGT135, PG9, PG16, VRC01 and b12 were produced by Agrobacterium-mediated transient transfection of Nicotiana benthamiana and assessed following administration in rhesus macaques. The results indicate that (i) N-glycans within the VL domain impair plasma stability of plant-derived bnAbs and (ii) while PGT121 and b12 exhibit no immunogenicity in rhesus macaques after multiple injections, VRC01, 10-1074 and NIH45-46G54W elicit high titer anti-idiotypic antibodies following a second injection. These anti-idiotypic antibodies specifically bind the administered bnAb or a close family member, and inhibit the bnAb in neutralization assays. These findings suggest that specific mutations in certain bnAbs contribute to their immunogenicity and call attention to the prospect that these mutated bnAbs will be immunogenic in humans, potentially compromising their value for prophylaxis and therapy of HIV-1.

  6. Sequential Immunization Elicits Broadly Neutralizing Anti-HIV-1 Antibodies in Ig Knockin Mice.

    Science.gov (United States)

    Escolano, Amelia; Steichen, Jon M; Dosenovic, Pia; Kulp, Daniel W; Golijanin, Jovana; Sok, Devin; Freund, Natalia T; Gitlin, Alexander D; Oliveira, Thiago; Araki, Tatsuya; Lowe, Sarina; Chen, Spencer T; Heinemann, Jennifer; Yao, Kai-Hui; Georgeson, Erik; Saye-Francisco, Karen L; Gazumyan, Anna; Adachi, Yumiko; Kubitz, Michael; Burton, Dennis R; Schief, William R; Nussenzweig, Michel C

    2016-09-08

    A vaccine that elicits broadly neutralizing antibodies (bNAbs) against HIV-1 is likely to be protective, but this has not been achieved. To explore immunization regimens that might elicit bNAbs, we produced and immunized mice expressing the predicted germline PGT121, a bNAb specific for the V3-loop and surrounding glycans on the HIV-1 spike. Priming with an epitope-modified immunogen designed to activate germline antibody-expressing B cells, followed by ELISA-guided boosting with a sequence of directional immunogens, native-like trimers with decreasing epitope modification, elicited heterologous tier-2-neutralizing responses. In contrast, repeated immunization with the priming immunogen did not. Antibody cloning confirmed elicitation of high levels of somatic mutation and tier-2-neutralizing antibodies resembling the authentic human bNAb. Our data establish that sequential immunization with specifically designed immunogens can induce high levels of somatic mutation and shepherd antibody maturation to produce bNAbs from their inferred germline precursors.

  7. Ability to develop broadly neutralizing HIV-1 antibodies is not restricted by the germline Ig gene repertoire.

    Science.gov (United States)

    Scheepers, Cathrine; Shrestha, Ram K; Lambson, Bronwen E; Jackson, Katherine J L; Wright, Imogen A; Naicker, Dshanta; Goosen, Mark; Berrie, Leigh; Ismail, Arshad; Garrett, Nigel; Abdool Karim, Quarraisha; Abdool Karim, Salim S; Moore, Penny L; Travers, Simon A; Morris, Lynn

    2015-05-01

    The human Ig repertoire is vast, producing billions of unique Abs from a limited number of germline Ig genes. The IgH V region (IGHV) is central to Ag binding and consists of 48 functional genes. In this study, we analyzed whether HIV-1-infected individuals who develop broadly neutralizing Abs show a distinctive germline IGHV profile. Using both 454 and Illumina technologies, we sequenced the IGHV repertoire of 28 HIV-infected South African women from the Centre for the AIDS Programme of Research in South Africa (CAPRISA) 002 and 004 cohorts, 13 of whom developed broadly neutralizing Abs. Of the 259 IGHV alleles identified in this study, approximately half were not found in the International Immunogenetics Database (IMGT). This included 85 entirely novel alleles and 38 alleles that matched rearranged sequences in non-IMGT databases. Analysis of the rearranged H chain V region genes of mAbs isolated from seven of these women, as well as previously isolated broadly neutralizing Abs from other donors, provided evidence that at least eight novel or non-IMGT alleles contributed to functional Abs. Importantly, we found that, despite a wide range in the number of IGHV alleles in each individual, including alleles used by known broadly neutralizing Abs, there were no significant differences in germline IGHV repertoires between individuals who do and do not develop broadly neutralizing Abs. This study reports novel IGHV repertoires and highlights the importance of a fully comprehensive Ig database for germline gene usage prediction. Furthermore, these data suggest a lack of genetic bias in broadly neutralizing Ab development in HIV-1 infection, with positive implications for HIV vaccine design.

  8. Broadly neutralizing antibodies against human immunodeficiency virus type 1 (HIV-1) and rational design of HIV-1 vaccines%抗人类免疫缺陷病毒广谱中和抗体与疫苗设计

    Institute of Scientific and Technical Information of China (English)

    王若珂; 郭建影; 张林琦

    2013-01-01

    自发现人类免疫缺陷病毒1型(HIV-1)30年来,科学家们不断探索有效的 HIV-1疫苗,但至今效果不理想。由于“精英患者”的广谱中和血清能起到有效的抗病毒作用,研究人员希望通过对体液保护免疫机制的研究,推动疫苗的设计和优化,为尽早研发成功 HIV疫苗提供关键理论和技术支撑。近几年来,由于技术的突破和改进,从HIV感染者中分离获得广谱中和抗体的概率和数量大大提高。本文针对近几年来分离的有代表性的广谱中和抗体,根据其不同的识别位点分为四大类进行详细介绍,并总结了从研究广谱中和抗体中所获得的疫苗设计创新思路与启示。%Antibody response is a crucial host defense against human immunodeficiency virus type 1 (HIV-1) infection .In recent years , tremendous progress has been made in isolating and characterizing of broadly neutralizing monoclonal antibodies (bnmAbs ) from “elite neutralizers” who remain healthy despite of prolonged infection . These bnmAbs were found to target four major vulnerable sites on the envelope glycoprotein .Structural and functional characterization of these bnmAbs has provided critical foundation for our better understanding of protective antibody response in vivo .In the current review ,we summarize the characteristics of these bnmAbs and discuss on their implications for rational design of novel HIV-1 vaccines capable of inducing antibodies similar to those bnmAbs .

  9. The effects of somatic hypermutation on neutralization and binding in the PGT121 family of broadly neutralizing HIV antibodies.

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    Devin Sok

    Full Text Available Broadly neutralizing HIV antibodies (bnAbs are typically highly somatically mutated, raising doubts as to whether they can be elicited by vaccination. We used 454 sequencing and designed a novel phylogenetic method to model lineage evolution of the bnAbs PGT121-134 and found a positive correlation between the level of somatic hypermutation (SHM and the development of neutralization breadth and potency. Strikingly, putative intermediates were characterized that show approximately half the mutation level of PGT121-134 but were still capable of neutralizing roughly 40-80% of PGT121-134 sensitive viruses in a 74-virus panel at median titers between 15- and 3-fold higher than PGT121-134. Such antibodies with lower levels of SHM may be more amenable to elicitation through vaccination while still providing noteworthy coverage. Binding characterization indicated a preference of inferred intermediates for native Env binding over monomeric gp120, suggesting that the PGT121-134 lineage may have been selected for binding to native Env at some point during maturation. Analysis of glycan-dependent neutralization for inferred intermediates identified additional adjacent glycans that comprise the epitope and suggests changes in glycan dependency or recognition over the course of affinity maturation for this lineage. Finally, patterns of neutralization of inferred bnAb intermediates suggest hypotheses as to how SHM may lead to potent and broad HIV neutralization and provide important clues for immunogen design.

  10. Toward Effective HIV Vaccination INDUCTION OF BINARY EPITOPE REACTIVE ANTIBODIES WITH BROAD HIV NEUTRALIZING ACTIVITY

    Energy Technology Data Exchange (ETDEWEB)

    Nishiyama, Yasuhiro; Planque, Stephanie; Mitsuda, Yukie; Nitti, Giovanni; Taguchi, Hiroaki; Jin, Lei; Symersky, Jindrich; Boivin, Stephane; Sienczyk, Marcin; Salas, Maria; Hanson, Carl V.; Paul, Sudhir; (Texas-MED); (Viral Rickettsial)

    2009-11-23

    We describe murine monoclonal antibodies (mAbs) raised by immunization with an electrophilic gp120 analog (E-gp120) expressing the rare ability to neutralize genetically heterologous human immunodeficiency virus (HIV) strains. Unlike gp120, E-gp120 formed covalent oligomers. The reactivity of gp120 and E-gp120 with mAbs to reference neutralizing epitopes was markedly different, indicating their divergent structures. Epitope mapping with synthetic peptides and electrophilic peptide analogs indicated binary recognition of two distinct gp120 regions by anti-E-gp120 mAbs, the 421-433 and 288-306 peptide regions. Univalent Fab and single chain Fv fragments expressed the ability to recognize both peptides. X-ray crystallography of an anti-E-gp120 Fab fragment revealed two neighboring cavities, the typical antigen-binding cavity formed by the complementarity determining regions (CDRs) and another cavity dominated by antibody heavy chain variable (VH) domain framework (FR) residues. Substitution of the FR cavity VH Lys-19 residue by an Ala residue resulted in attenuated binding of the 421-433 region peptide probe. The CDRs and VH FR replacement/silent mutation ratios exceeded the ratio for a random mutation process, suggesting adaptive development of both putative binding sites. All mAbs studied were derived from VH1 family genes, suggesting biased recruitment of the V gene germ line repertoire by E-gp120. The conserved 421-433 region of gp120 is essential for HIV binding to host CD4 receptors. This region is recognized weakly by the FR of antibodies produced without exposure to HIV, but it usually fails to induce adaptive synthesis of neutralizing antibodies. We present models accounting for improved CD4-binding site recognition and broad HIV neutralizing activity of the mAbs, long sought goals in HIV vaccine development.

  11. A pilot study on an attenuated Chinese EIAV vaccine inducing broadly neutralizing antibodies.

    Science.gov (United States)

    Meng, Qinglai; Lin, Yuezhi; Ma, Jian; Ma, Yan; Zhao, Liping; Li, Shenwei; Liang, Hua; Zhou, Jianhua; Shen, Rongxian; Zhang, Xiaoyan; Shao, Yiming

    2011-08-01

    The attenuated Chinese equine infectious anemia virus (EIAV) vaccine has successfully protected millions of equine animals from EIA disease in China. In this pilot study, to determine whether this attenuated vaccine can induce broadly neutralizing antibodies, we immunized four horses with the attenuated Chinese vaccine strain EIAVFDDV and then observed the evolution of neutralizing antibodies against different EIAV strains. During the vaccination phase, all vaccinees rapidly developed high levels of neutralizing antibodies against the homologous vaccine strain (pLGFD3V), and 3 out of 4 horses showed a gradual increase in serum neutralizing activity against two relatively heterologous virulent variants of the challenge strain (pLGFD3Mu12V and DLV34). After challenge, the three horses that had developed high levels of neutralizing antibodies against pLGFD3Mu12V and DLV34 did not show signs of infection, which was demonstrated by immune suppression, while the one horse producing serum that could only neutralize pLGFD3V developed a febrile episode during the 8-month observation period. To assess whether the broadly neutralizing activity is associated with immune protection, sera drawn on the day of challenge from these four vaccinees and an additional four EIAVFDDV-vaccinated horses were analyzed for neutralizing antibodies against pLGFD3V, pLGFD3Mu12V and DLV34. Although there was no significant correlation between protection from infection and serum neutralizing activity against any of these three viral strains, protection from infection was observed to correlate better with serum neutralizing activity against the two heterologous virulent strains than against the homologous vaccine strain. These data indicate that EIAVFDDV induced broadly neutralizing antibodies, which might confer enhanced protection of vaccinees from infection by the challenge virus.

  12. Broadly-Reactive Neutralizing and Non-neutralizing Antibodies Directed against the H7 Influenza Virus Hemagglutinin Reveal Divergent Mechanisms of Protection.

    Science.gov (United States)

    Tan, Gene S; Leon, Paul E; Albrecht, Randy A; Margine, Irina; Hirsh, Ariana; Bahl, Justin; Krammer, Florian

    2016-04-01

    In the early spring of 2013, Chinese health authorities reported several cases of H7N9 influenza virus infections in humans. Since then the virus has established itself at the human-animal interface in Eastern China and continues to cause several hundred infections annually. In order to characterize the antibody response to the H7N9 virus we generated several mouse monoclonal antibodies against the hemagglutinin of the A/Shanghai/1/13 (H7N9) virus. Of particular note are two monoclonal antibodies, 1B2 and 1H5, that show broad reactivity to divergent H7 hemagglutinins. Monoclonal antibody 1B2 binds to viruses of the Eurasian and North American H7 lineages and monoclonal antibody 1H5 reacts broadly to virus isolates of the Eurasian lineage. Interestingly, 1B2 shows broad hemagglutination inhibiting and neutralizing activity, while 1H5 fails to inhibit hemagglutination and demonstrates no neutralizing activity in vitro. However, both monoclonal antibodies were highly protective in an in vivo passive transfer challenge model in mice, even at low doses. Experiments using mutant antibodies that lack the ability for Fc/Fc-receptor and Fc/complement interactions suggest that the protection provided by mAb 1H5 is, at least in part, mediated by the Fc-fragment of the mAb. These findings highlight that a protective response to a pathogen may not only be due to neutralizing antibodies, but can also be the result of highly efficacious non-neutralizing antibodies not readily detected by classical in vitro neutralization or hemagglutination inhibition assays. This is of interest because H7 influenza virus vaccines induce only low hemagglutination inhibiting antibody titers while eliciting robust antibody titers as measured by ELISA. Our data suggest that these binding but non-neutralizing antibodies contribute to protection in vivo.

  13. Broadly-Reactive Neutralizing and Non-neutralizing Antibodies Directed against the H7 Influenza Virus Hemagglutinin Reveal Divergent Mechanisms of Protection.

    Directory of Open Access Journals (Sweden)

    Gene S Tan

    2016-04-01

    Full Text Available In the early spring of 2013, Chinese health authorities reported several cases of H7N9 influenza virus infections in humans. Since then the virus has established itself at the human-animal interface in Eastern China and continues to cause several hundred infections annually. In order to characterize the antibody response to the H7N9 virus we generated several mouse monoclonal antibodies against the hemagglutinin of the A/Shanghai/1/13 (H7N9 virus. Of particular note are two monoclonal antibodies, 1B2 and 1H5, that show broad reactivity to divergent H7 hemagglutinins. Monoclonal antibody 1B2 binds to viruses of the Eurasian and North American H7 lineages and monoclonal antibody 1H5 reacts broadly to virus isolates of the Eurasian lineage. Interestingly, 1B2 shows broad hemagglutination inhibiting and neutralizing activity, while 1H5 fails to inhibit hemagglutination and demonstrates no neutralizing activity in vitro. However, both monoclonal antibodies were highly protective in an in vivo passive transfer challenge model in mice, even at low doses. Experiments using mutant antibodies that lack the ability for Fc/Fc-receptor and Fc/complement interactions suggest that the protection provided by mAb 1H5 is, at least in part, mediated by the Fc-fragment of the mAb. These findings highlight that a protective response to a pathogen may not only be due to neutralizing antibodies, but can also be the result of highly efficacious non-neutralizing antibodies not readily detected by classical in vitro neutralization or hemagglutination inhibition assays. This is of interest because H7 influenza virus vaccines induce only low hemagglutination inhibiting antibody titers while eliciting robust antibody titers as measured by ELISA. Our data suggest that these binding but non-neutralizing antibodies contribute to protection in vivo.

  14. Optimal immunization cocktails can promote induction of broadly neutralizing Abs against highly mutable pathogens.

    Science.gov (United States)

    Shaffer, J Scott; Moore, Penny L; Kardar, Mehran; Chakraborty, Arup K

    2016-10-24

    Strategies to elicit Abs that can neutralize diverse strains of a highly mutable pathogen are likely to result in a potent vaccine. Broadly neutralizing Abs (bnAbs) against HIV have been isolated from patients, proving that the human immune system can evolve them. Using computer simulations and theory, we study immunization with diverse mixtures of variant antigens (Ags). Our results show that particular choices for the number of variant Ags and the mutational distances separating them maximize the probability of inducing bnAbs. The variant Ags represent potentially conflicting selection forces that can frustrate the Darwinian evolutionary process of affinity maturation. An intermediate level of frustration maximizes the chance of evolving bnAbs. A simple model makes vivid the origin of this principle of optimal frustration. Our results, combined with past studies, suggest that an appropriately chosen permutation of immunization with an optimally designed mixture (using the principles that we describe) and sequential immunization with variant Ags that are separated by relatively large mutational distances may best promote the evolution of bnAbs.

  15. Broadly Neutralizing Antibody Responses in a Large Longitudinal Sub-Saharan HIV Primary Infection Cohort.

    Science.gov (United States)

    Landais, Elise; Huang, Xiayu; Havenar-Daughton, Colin; Murrell, Ben; Price, Matt A; Wickramasinghe, Lalinda; Ramos, Alejandra; Bian, Charoan B; Simek, Melissa; Allen, Susan; Karita, Etienne; Kilembe, William; Lakhi, Shabir; Inambao, Mubiana; Kamali, Anatoli; Sanders, Eduard J; Anzala, Omu; Edward, Vinodh; Bekker, Linda-Gail; Tang, Jianming; Gilmour, Jill; Kosakovsky-Pond, Sergei L; Phung, Pham; Wrin, Terri; Crotty, Shane; Godzik, Adam; Poignard, Pascal

    2016-01-01

    Broadly neutralizing antibodies (bnAbs) are thought to be a critical component of a protective HIV vaccine. However, designing vaccines immunogens able to elicit bnAbs has proven unsuccessful to date. Understanding the correlates and immunological mechanisms leading to the development of bnAb responses during natural HIV infection is thus critical to the design of a protective vaccine. The IAVI Protocol C program investigates a large longitudinal cohort of primary HIV-1 infection in Eastern and South Africa. Development of neutralization was evaluated in 439 donors using a 6 cross-clade pseudo-virus panel predictive of neutralization breadth on larger panels. About 15% of individuals developed bnAb responses, essentially between year 2 and year 4 of infection. Statistical analyses revealed no influence of gender, age or geographical origin on the development of neutralization breadth. However, cross-clade neutralization strongly correlated with high viral load as well as with low CD4 T cell counts, subtype-C infection and HLA-A*03(-) genotype. A correlation with high overall plasma IgG levels and anti-Env IgG binding titers was also found. The latter appeared not associated with higher affinity, suggesting a greater diversity of the anti-Env responses in broad neutralizers. Broadly neutralizing activity targeting glycan-dependent epitopes, largely the N332-glycan epitope region, was detected in nearly half of the broad neutralizers while CD4bs and gp41-MPER bnAb responses were only detected in very few individuals. Together the findings suggest that both viral and host factors are critical for the development of bnAbs and that the HIV Env N332-glycan supersite may be a favorable target for vaccine design.

  16. Broadly Neutralizing Antibody Responses in a Large Longitudinal Sub-Saharan HIV Primary Infection Cohort.

    Directory of Open Access Journals (Sweden)

    Elise Landais

    2016-01-01

    Full Text Available Broadly neutralizing antibodies (bnAbs are thought to be a critical component of a protective HIV vaccine. However, designing vaccines immunogens able to elicit bnAbs has proven unsuccessful to date. Understanding the correlates and immunological mechanisms leading to the development of bnAb responses during natural HIV infection is thus critical to the design of a protective vaccine. The IAVI Protocol C program investigates a large longitudinal cohort of primary HIV-1 infection in Eastern and South Africa. Development of neutralization was evaluated in 439 donors using a 6 cross-clade pseudo-virus panel predictive of neutralization breadth on larger panels. About 15% of individuals developed bnAb responses, essentially between year 2 and year 4 of infection. Statistical analyses revealed no influence of gender, age or geographical origin on the development of neutralization breadth. However, cross-clade neutralization strongly correlated with high viral load as well as with low CD4 T cell counts, subtype-C infection and HLA-A*03(- genotype. A correlation with high overall plasma IgG levels and anti-Env IgG binding titers was also found. The latter appeared not associated with higher affinity, suggesting a greater diversity of the anti-Env responses in broad neutralizers. Broadly neutralizing activity targeting glycan-dependent epitopes, largely the N332-glycan epitope region, was detected in nearly half of the broad neutralizers while CD4bs and gp41-MPER bnAb responses were only detected in very few individuals. Together the findings suggest that both viral and host factors are critical for the development of bnAbs and that the HIV Env N332-glycan supersite may be a favorable target for vaccine design.

  17. Vaccine Elicitation of High Mannose-Dependent Neutralizing Antibodies against the V3-Glycan Broadly Neutralizing Epitope in Nonhuman Primates.

    Science.gov (United States)

    Saunders, Kevin O; Nicely, Nathan I; Wiehe, Kevin; Bonsignori, Mattia; Meyerhoff, R Ryan; Parks, Robert; Walkowicz, William E; Aussedat, Baptiste; Wu, Nelson R; Cai, Fangping; Vohra, Yusuf; Park, Peter K; Eaton, Amanda; Go, Eden P; Sutherland, Laura L; Scearce, Richard M; Barouch, Dan H; Zhang, Ruijun; Von Holle, Tarra; Overman, R Glenn; Anasti, Kara; Sanders, Rogier W; Moody, M Anthony; Kepler, Thomas B; Korber, Bette; Desaire, Heather; Santra, Sampa; Letvin, Norman L; Nabel, Gary J; Montefiori, David C; Tomaras, Georgia D; Liao, Hua-Xin; Alam, S Munir; Danishefsky, Samuel J; Haynes, Barton F

    2017-02-28

    Induction of broadly neutralizing antibodies (bnAbs) that target HIV-1 envelope (Env) is a goal of HIV-1 vaccine development. A bnAb target is the Env third variable loop (V3)-glycan site. To determine whether immunization could induce antibodies to the V3-glycan bnAb binding site, we repetitively immunized macaques over a 4-year period with an Env expressing V3-high mannose glycans. Env immunizations elicited plasma antibodies that neutralized HIV-1 expressing only high-mannose glycans-a characteristic shared by early bnAb B cell lineage members. A rhesus recombinant monoclonal antibody from a vaccinated macaque bound to the V3-glycan site at the same amino acids as broadly neutralizing antibodies. A structure of the antibody bound to glycan revealed that the three variable heavy-chain complementarity-determining regions formed a cavity into which glycan could insert and neutralized multiple HIV-1 isolates with high-mannose glycans. Thus, HIV-1 Env vaccination induced mannose-dependent antibodies with characteristics of V3-glycan bnAb precursors.

  18. Trimerization of the HIV Transmembrane Domain in Lipid Bilayers Modulates Broadly Neutralizing Antibody Binding.

    Science.gov (United States)

    Reichart, Timothy M; Baksh, Michael M; Rhee, Jin-Kyu; Fiedler, Jason D; Sligar, Stephen G; Finn, M G; Zwick, Michael B; Dawson, Philip E

    2016-02-18

    The membrane-proximal external region (MPER) of HIV gp41 is an established target of antibodies that neutralize a broad range of HIV isolates. To evaluate the role of the transmembrane (TM) domain, synthetic MPER-derived peptides were incorporated into lipid nanoparticles using natural and designed TM domains, and antibody affinity was measured using immobilized and solution-based techniques. Peptides incorporating the native HIV TM domain exhibit significantly stronger interactions with neutralizing antibodies than peptides with a monomeric TM domain. Furthermore, a peptide with a trimeric, three-helix bundle TM domain recapitulates the binding profile of the native sequence. These studies suggest that neutralizing antibodies can bind the MPER when the TM domain is a three-helix bundle and this presentation could influence the binding of neutralizing antibodies to the virus. Lipid-bilayer presentation of viral antigens in Nanodiscs is a new platform for evaluating neutralizing antibodies.

  19. Two Escape Mechanisms of Influenza A Virus to a Broadly Neutralizing Stalk-Binding Antibody.

    Science.gov (United States)

    Chai, Ning; Swem, Lee R; Reichelt, Mike; Chen-Harris, Haiyin; Luis, Elizabeth; Park, Summer; Fouts, Ashley; Lupardus, Patrick; Wu, Thomas D; Li, Olga; McBride, Jacqueline; Lawrence, Michael; Xu, Min; Tan, Man-Wah

    2016-06-01

    Broadly neutralizing antibodies targeting the stalk region of influenza A virus (IAV) hemagglutinin (HA) are effective in blocking virus infection both in vitro and in vivo. The highly conserved epitopes recognized by these antibodies are critical for the membrane fusion function of HA and therefore less likely to be permissive for virus mutational escape. Here we report three resistant viruses of the A/Perth/16/2009 strain that were selected in the presence of a broadly neutralizing stalk-binding antibody. The three resistant viruses harbor three different mutations in the HA stalk: (1) Gln387Lys; (2) Asp391Tyr; (3) Asp391Gly. The Gln387Lys mutation completely abolishes binding of the antibody to the HA stalk epitope. The other two mutations, Asp391Tyr and Asp391Gly, do not affect antibody binding at neutral pH and only slightly reduce binding at low pH. Interestingly, they enhance the fusion ability of the HA, representing a novel mechanism that allows productive membrane fusion even in the presence of antibody and hence virus escape from antibody neutralization. Therefore, these mutations illustrate two different resistance mechanisms used by IAV to escape broadly neutralizing stalk-binding antibodies. Compared to the wild type virus, the resistant viruses release fewer progeny viral particles during replication and are more sensitive to Tamiflu, suggesting reduced viral fitness.

  20. HIV-1 broadly neutralizing antibody precursor B cells revealed by germline-targeting immunogen.

    Science.gov (United States)

    Jardine, Joseph G; Kulp, Daniel W; Havenar-Daughton, Colin; Sarkar, Anita; Briney, Bryan; Sok, Devin; Sesterhenn, Fabian; Ereño-Orbea, June; Kalyuzhniy, Oleksandr; Deresa, Isaiah; Hu, Xiaozhen; Spencer, Skye; Jones, Meaghan; Georgeson, Erik; Adachi, Yumiko; Kubitz, Michael; deCamp, Allan C; Julien, Jean-Philippe; Wilson, Ian A; Burton, Dennis R; Crotty, Shane; Schief, William R

    2016-03-25

    Induction of broadly neutralizing antibodies (bnAbs) is a major HIV vaccine goal. Germline-targeting immunogens aim to initiate bnAb induction by activating bnAb germline precursor B cells. Critical unmet challenges are to determine whether bnAb precursor naïve B cells bind germline-targeting immunogens and occur at sufficient frequency in humans for reliable vaccine responses. Using deep mutational scanning and multitarget optimization, we developed a germline-targeting immunogen (eOD-GT8) for diverse VRC01-class bnAbs. We then used the immunogen to isolate VRC01-class precursor naïve B cells from HIV-uninfected donors. Frequencies of true VRC01-class precursors, their structures, and their eOD-GT8 affinities support this immunogen as a candidate human vaccine prime. These methods could be applied to germline targeting for other classes of HIV bnAbs and for Abs to other pathogens.

  1. Broadly neutralizing anti-influenza antibodies require Fc receptor engagement for in vivo protection.

    Science.gov (United States)

    DiLillo, David J; Palese, Peter; Wilson, Patrick C; Ravetch, Jeffrey V

    2016-02-01

    In vivo protection by antimicrobial neutralizing Abs can require the contribution of effector functions mediated by Fc-Fcγ receptor (Fc-FcγR) interactions for optimal efficacy. In influenza, broadly neutralizing anti-hemagglutinin (anti-HA) stalk mAbs require Fc-FcγR interactions to mediate in vivo protection, but strain-specific anti-HA head mAbs do not. Whether this rule applies only to anti-stalk Abs or is applicable to any broadly neutralizing Ab (bNAb) against influenza is unknown. Here, we characterized the contribution of Fc-FcγR interactions during in vivo protection for a panel of 13 anti-HA mAbs, including bNAbs and non-neutralizing Abs, against both the stalk and head domains. All classes of broadly binding anti-HA mAbs required Fc-FcγR interactions to provide protection in vivo, including those mAbs that bind the HA head and those that do not neutralize virus in vitro. Further, a broadly neutralizing anti-neuraminidase (anti-NA) mAb also required FcγRs to provide protection in vivo, but a strain-specific anti-NA mAb did not. Thus, these findings suggest that the breadth of reactivity of anti-influenza Abs, regardless of their epitope, necessitates interactions with FcγRs on effector cell populations to mediate in vivo protection. These findings will guide the design of antiviral Ab therapeutics and inform vaccine design to elicit Abs with optimal binding properties and effector functions.

  2. Broadly Neutralizing Activity of Zika Virus-Immune Sera Identifies a Single Viral Serotype

    Directory of Open Access Journals (Sweden)

    Kimberly A. Dowd

    2016-08-01

    Full Text Available Recent epidemics of Zika virus (ZIKV have been associated with congenital malformation during pregnancy and Guillain-Barré syndrome. There are two ZIKV lineages (African and Asian that share >95% amino acid identity. Little is known regarding the ability of neutralizing antibodies elicited against one lineage to protect against the other. We investigated the breadth of the neutralizing antibody response following ZIKV infection by measuring the sensitivity of six ZIKV strains to neutralization by ZIKV-confirmed convalescent human serum or plasma samples. Contemporary Asian and early African ZIKV strains were similarly sensitive to neutralization regardless of the cellular source of virus. Furthermore, mouse immune serum generated after infection with African or Asian ZIKV strains was capable of neutralizing homologous and heterologous ZIKV strains equivalently. Because our study only defines a single ZIKV serotype, vaccine candidates eliciting robust neutralizing antibody responses should inhibit infection of both ZIKV lineages, including strains circulating in the Americas.

  3. Modeling neutralization kinetics of HIV by broadly neutralizing monoclonal antibodies in genital secretions coating the cervicovaginal mucosa.

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    Scott A McKinley

    Full Text Available Eliciting broadly neutralizing antibodies (bnAb in cervicovaginal mucus (CVM represents a promising "first line of defense" strategy to reduce vaginal HIV transmission. However, it remains unclear what levels of bnAb must be present in CVM to effectively reduce infection. We approached this complex question by modeling the dynamic tally of bnAb coverage on HIV. This analysis introduces a critical, timescale-dependent competition: to protect, bnAb must accumulate at sufficient stoichiometry to neutralize HIV faster than virions penetrate CVM and reach target cells. We developed a model that incorporates concentrations and diffusivities of HIV and bnAb in semen and CVM, kinetic rates for binding (kon and unbinding (koff of select bnAb, and physiologically relevant thicknesses of CVM and semen layers. Comprehensive model simulations lead to robust conclusions about neutralization kinetics in CVM. First, due to the limited time virions in semen need to penetrate CVM, substantially greater bnAb concentrations than in vitro estimates must be present in CVM to neutralize HIV. Second, the model predicts that bnAb with more rapid kon, almost independent of koff, should offer greater neutralization potency in vivo. These findings suggest the fastest arriving virions at target cells present the greatest likelihood of infection. It also implies the marked improvements in in vitro neutralization potency of many recently discovered bnAb may not translate to comparable reduction in the bnAb dose needed to confer protection against initial vaginal infections. Our modeling framework offers a valuable tool to gaining quantitative insights into the dynamics of mucosal immunity against HIV and other infectious diseases.

  4. Cooperation of B cell lineages in induction of HIV-1-broadly neutralizing antibodies.

    Science.gov (United States)

    Gao, Feng; Bonsignori, Mattia; Liao, Hua-Xin; Kumar, Amit; Xia, Shi-Mao; Lu, Xiaozhi; Cai, Fangping; Hwang, Kwan-Ki; Song, Hongshuo; Zhou, Tongqing; Lynch, Rebecca M; Alam, S Munir; Moody, M Anthony; Ferrari, Guido; Berrong, Mark; Kelsoe, Garnett; Shaw, George M; Hahn, Beatrice H; Montefiori, David C; Kamanga, Gift; Cohen, Myron S; Hraber, Peter; Kwong, Peter D; Korber, Bette T; Mascola, John R; Kepler, Thomas B; Haynes, Barton F

    2014-07-31

    Development of strategies for induction of HIV-1 broadly neutralizing antibodies (bnAbs) by vaccines is a priority. Determining the steps of bnAb induction in HIV-1-infected individuals who make bnAbs is a key strategy for immunogen design. Here, we study the B cell response in a bnAb-producing individual and report cooperation between two B cell lineages to drive bnAb development. We isolated a virus-neutralizing antibody lineage that targeted an envelope region (loop D) and selected virus escape mutants that resulted in both enhanced bnAb lineage envelope binding and escape mutant neutralization-traits associated with increased B cell antigen drive. Thus, in this individual, two B cell lineages cooperated to induce the development of bnAbs. Design of vaccine immunogens that simultaneously drive both helper and broadly neutralizing B cell lineages may be important for vaccine-induced recapitulation of events that transpire during the maturation of neutralizing antibodies in HIV-1-infected individuals.

  5. Influenza A HA's conserved epitopes and broadly neutralizing antibodies: a prediction method.

    Science.gov (United States)

    Ren, Jing; Ellis, John; Li, Jinyan

    2014-10-01

    A conserved epitope is an epitope retained by multiple strains of influenza as the key target of a broadly neutralizing antibody. Identification of conserved epitopes is of strong interest to help design broad-spectrum vaccines against influenza. Conservation score measures the evolutionary conservation of an amino acid position in a protein based on the phylogenetic relationships observed amongst homologous sequences. Here, Average Amino Acid Conservation Score (AAACS) is proposed as a method to identify HA's conserved epitopes. Our analysis shows that there is a clear distinction between conserved epitopes and nonconserved epitopes in terms of AAACS. This method also provides an excellent classification performance on an independent dataset. In contrast, alignment-based comparison methods do not work well for this problem, because conserved epitopes to the same broadly neutralizing antibody are usually not identical or similar. Location-based methods are not successful either, because conserved epitopes are located at both the less-conserved globular head (HA1) and the more-conserved stem (HA2). As a case study, two conserved epitopes on HA are predicted for the influenza A virus H7N9: One should match the broadly neutralizing antibodies CR9114 or FI6v3, while the other is new and requires validation by wet-lab experiments.

  6. Immunologic characteristics of HIV-infected individuals who make broadly neutralizing antibodies.

    Science.gov (United States)

    Borrow, Persephone; Moody, M Anthony

    2017-01-01

    Induction of broadly neutralizing antibodies (bnAbs) capable of inhibiting infection with diverse variants of human immunodeficiency virus type 1 (HIV-1) is a key, as-yet-unachieved goal of prophylactic HIV-1 vaccine strategies. However, some HIV-infected individuals develop bnAbs after approximately 2-4 years of infection, enabling analysis of features of these antibodies and the immunological environment that enables their induction. Distinct subsets of CD4(+) T cells play opposing roles in the regulation of humoral responses: T follicular helper (Tfh) cells support germinal center formation and provide help for affinity maturation and the development of memory B cells and plasma cells, while regulatory CD4(+) (Treg) cells including T follicular regulatory (Tfr) cells inhibit the germinal center reaction to limit autoantibody production. BnAbs exhibit high somatic mutation frequencies, long third heavy-chain complementarity determining regions, and/or autoreactivity, suggesting that bnAb generation is likely to be highly dependent on the activity of CD4(+) Tfh cells, and may be constrained by host tolerance controls. This review discusses what is known about the immunological environment during HIV-1 infection, in particular alterations in CD4(+) Tfh, Treg, and Tfr populations and autoantibody generation, and how this is related to bnAb development, and considers the implications for HIV-1 vaccine design.

  7. Broadly Neutralizing Anti-HIV Antibodies Prevent HIV Infection of Mucosal Tissue Ex Vivo.

    Science.gov (United States)

    Scott, Yanille M; Park, Seo Young; Dezzutti, Charlene S

    2016-02-01

    Broadly neutralizing monoclonal antibodies (nAbs) specific for HIV are being investigated for use in HIV prevention. Due to their ability to inhibit HIV attachment to and entry into target cells, nAbs may be suitable for use as topical HIV microbicides. As such, they would present an alternative intervention for individuals who may not benefit from using antiretroviral-based products for HIV prevention. We theorize that nAbs can inhibit viral transmission through mucosal tissue, thus reducing the incidence of HIV infection. The efficacy of the PG9, PG16, VRC01, and 4E10 antibodies was evaluated in an ex vivo human model of mucosal HIV transmission. nAbs reduced HIV transmission, causing 1.5- to 2-log10 reductions in HIV replication in ectocervical tissues and ≈3-log10 reductions in HIV replication in colonic tissues over 21 days. These antibodies demonstrated greater potency in colonic tissues, with a 50-fold higher dose being required to reduce transmission in ectocervical tissues. Importantly, nAbs retained their potency and reduced viral transmission in the presence of whole semen. No changes in tissue viability or immune activation were observed in colonic or ectocervical tissue after nAb exposure. Our data suggest that topically applied nAbs are safe and effective against HIV infection of mucosal tissue and support further development of nAbs as a topical microbicide that could be used for anal as well as vaginal protection.

  8. A Potent and Broad Neutralizing Antibody Recognizes and Penetrates the HIV Glycan Shield

    Energy Technology Data Exchange (ETDEWEB)

    Pejchal, Robert; Doores, Katie J.; Walker, Laura M.; Khayat, Reza; Huang, Po-Ssu; Wang, Sheng-Kai; Stanfield, Robyn L.; Julien, Jean-Philippe; Ramos, Alejandra; Crispin, Max; Depetris, Rafael; Katpally, Umesh; Marozsan, Andre; Cupo, Albert; Maloveste, Sebastien; Liu, Yan; McBride, Ryan; Ito, Yukishige; Sanders, Rogier W.; Ogohara, Cassandra; Paulson, James C.; Feizi, Ten; Scanlan, Christopher N.; Wong, Chi-Huey; Moore, John P.; Olson, William C.; Ward, Andrew B.; Poignard, Pascal; Schief, William R.; Burton, Dennis R.; Wilson, Ian A. (UWASH); (Progenics); (ICL); (Weill-Med); (NIH); (JSTA); (Scripps); (Oxford)

    2015-10-15

    The HIV envelope (Env) protein gp120 is protected from antibody recognition by a dense glycan shield. However, several of the recently identified PGT broadly neutralizing antibodies appear to interact directly with the HIV glycan coat. Crystal structures of antigen-binding fragments (Fabs) PGT 127 and 128 with Man{sub 9} at 1.65 and 1.29 angstrom resolution, respectively, and glycan binding data delineate a specific high mannose-binding site. Fab PGT 128 complexed with a fully glycosylated gp120 outer domain at 3.25 angstroms reveals that the antibody penetrates the glycan shield and recognizes two conserved glycans as well as a short {beta}-strand segment of the gp120 V3 loop, accounting for its high binding affinity and broad specificify. Furthermore, our data suggest that the high neutralization potency of PGT 127 and 128 immunoglobulin Gs may be mediated by cross-linking Env trimers on the viral surface.

  9. Broadly Neutralizing Antibodies Against HIV: New Insights to Inform Vaccine Design.

    Science.gov (United States)

    Sadanand, Saheli; Suscovich, Todd J; Alter, Galit

    2016-01-01

    HIV-1 poses immense immunological challenges to the humoral immune response because of its ability to shield itself and replicate and evolve rapidly. Although most currently licensed vaccines provide protection via the induction of antibodies (Abs) that can directly block infection ( 1 ), 30 years of HIV-1 vaccine research has failed to successfully elicit such Abs against globally relevant HIV strains. However, mounting evidence suggests that these broadly neutralizing antibodies (bNAbs) do emerge naturally in a significant fraction of infected subjects, albeit after years of infection, indicating that these responses can be selected naturally by the immune response but take long periods of time to evolve. We review the basic structural characteristics of broadly neutralizing antibodies and how they recognize the virus, and we discuss new vaccination strategies that aim to mimic natural evolution to guide B cells to produce protective Abs against HIV-1.

  10. A broadly flavivirus cross-neutralizing monoclonal antibody that recognizes a novel epitope within the fusion loop of E protein.

    Directory of Open Access Journals (Sweden)

    Yong-Qiang Deng

    Full Text Available Flaviviruses are a group of human pathogenic, enveloped RNA viruses that includes dengue (DENV, yellow fever (YFV, West Nile (WNV, and Japanese encephalitis (JEV viruses. Cross-reactive antibodies against Flavivirus have been described, but most of them are generally weakly neutralizing. In this study, a novel monoclonal antibody, designated mAb 2A10G6, was determined to have broad cross-reactivity with DENV 1-4, YFV, WNV, JEV, and TBEV. Phage-display biopanning and structure modeling mapped 2A10G6 to a new epitope within the highly conserved flavivirus fusion loop peptide, the (98DRXW(101 motif. Moreover, in vitro and in vivo experiments demonstrated that 2A10G6 potently neutralizes DENV 1-4, YFV, and WNV and confers protection from lethal challenge with DENV 1-4 and WNV in murine model. Furthermore, functional studies revealed that 2A10G6 blocks infection at a step after viral attachment. These results define a novel broadly flavivirus cross-reactive mAb with highly neutralizing activity that can be further developed as a therapeutic agent against severe flavivirus infections in humans.

  11. Honing a harder-hitting hammerhead improves broadly neutralizing antibody breadth and potency.

    Science.gov (United States)

    Lewis, George K

    2015-06-01

    While current HIV-1 therapies have greatly improved the quality and duration of life for infected individuals, a vaccine to prevent transmission of the virus is lacking. Broadly neutralizing monoclonal antibodies (bnmAbs) with the capacity to neutralize multiple HIV-1 variants have been isolated from HIV-1-infected individuals, and there has been a great effort to investigate how these bnmAbs arise, due their potential for HIV-1 vaccination. In this issue of the JCI, Willis and colleagues apply a computational approach to design variants of the bnmAb PG9 in an attempt to enhance potency and neutralization breadth. One of these variants was able to target multiple PG9-resistant strains, as the result of stabilization of the long heavy chain complementarity determining region 3 (HCDR3). The results of this study provide important insight and a unique approach to optimizing HIV-1 bnmABs.

  12. Immunologic Basis for Long HCDR3s in Broadly Neutralizing Antibodies against HIV-1

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    Lei eYu

    2014-06-01

    Full Text Available A large panel of potent broadly neutralizing antibodies (bnAbs against HIV-1 has been reported in recent years, raising hope for the design of an effective vaccine based on epitopes recognized by these protective antibodies. However, many of these bnAbs contain the feature of long heavy chain complementarity-determining region 3 (HCDR3 sequences, which is viewed as an obstacle to the development of an HIV-1 vaccine targeting long HCDR3 bnAb responses. This mini-review summarizes the current literature and discusses the different potential Immunologic mechanisms for generating long HCDR3, including D-D fusion, VH replacement, long N region addition and skewed D-J genes usage, among which potential VH replacement products appear to be significant contributors. VH replacement occurs through RAG-mediated secondary recombination and contributes to the diversified naïve B cell repertoire. During VH replacement, a short stretch of nucleotides from previously rearranged VH genes is left within the newly formed HCDR3, thus elongating its length. Accumulating evidence suggests that long HCDR3s are present at significant numbers in the human mature naïve B cell repertoire and are primarily generated by recombination during B cell development. These new observations indicate that long HCDR3s, though in low frequency, are a normal feature of human antibody naïve repertoire and they appear to be selected to target conserved epitopes located in deep regions of the HIV-1 envelope trimer during HIV-1 infection. Therefore, the presence of long HCDR3 sequences should not necessarily be viewed as an obstacle to the development of an HIV-1 vaccine that promotes bnAb responses.

  13. New connections: Cell to cell HIV-1 transmission, resistance to broadly neutralizing antibodies, and an envelope sorting motif.

    Science.gov (United States)

    Smith, S Abigail; Derdeyn, Cynthia A

    2017-03-01

    HIV-1 infection from cell to cell may provide an efficient mode of viral spread in vivo and could therefore present a significant challenge for preventative or therapeutic strategies based on broadly neutralizing antibodies. Indeed, Li et al show that the potency and magnitude of multiple HIV-1 broadly neutralizing antibody classes are decreased during cell to cell infection in a context dependent manner. A functional motif in gp41 appears to contribute to this differential susceptibility by modulating exposure of neutralization epitopes.

  14. Envelope deglycosylation enhances antigenicity of HIV-1 gp41 epitopes for both broad neutralizing antibodies and their unmutated ancestor antibodies.

    Directory of Open Access Journals (Sweden)

    Ben-Jiang Ma

    2011-09-01

    Full Text Available The HIV-1 gp41 envelope (Env membrane proximal external region (MPER is an important vaccine target that in rare subjects can elicit neutralizing antibodies. One mechanism proposed for rarity of MPER neutralizing antibody generation is lack of reverted unmutated ancestor (putative naive B cell receptor antibody reactivity with HIV-1 envelope. We have studied the effect of partial deglycosylation under non-denaturing (native conditions on gp140 Env antigenicity for MPER neutralizing antibodies and their reverted unmutated ancestor antibodies. We found that native deglycosylation of clade B JRFL gp140 as well as group M consensus gp140 Env CON-S selectively increased the reactivity of Env with the broad neutralizing human mAbs, 2F5 and 4E10. Whereas fully glycosylated gp140 Env either did not bind (JRFL, or weakly bound (CON-S, 2F5 and 4E10 reverted unmutated ancestors, natively deglycosylated JRFL and CON-S gp140 Envs did bind well to these putative mimics of naive B cell receptors. These data predict that partially deglycoslated Env would bind better than fully glycosylated Env to gp41-specific naïve B cells with improved immunogenicity. In this regard, immunization of rhesus macaques demonstrated enhanced immunogenicity of the 2F5 MPER epitope on deglyosylated JRFL gp140 compared to glycosylated JRFL gp140. Thus, the lack of 2F5 and 4E10 reverted unmutated ancestor binding to gp140 Env may not always be due to lack of unmutated ancestor antibody reactivity with gp41 peptide epitopes, but rather, may be due to glycan interference of binding of unmutated ancestor antibodies of broad neutralizing mAb to Env gp41.

  15. Specifically modified Env immunogens activate B-cell precursors of broadly neutralizing HIV-1 antibodies in transgenic mice.

    Science.gov (United States)

    McGuire, Andrew T; Gray, Matthew D; Dosenovic, Pia; Gitlin, Alexander D; Freund, Natalia T; Petersen, John; Correnti, Colin; Johnsen, William; Kegel, Robert; Stuart, Andrew B; Glenn, Jolene; Seaman, Michael S; Schief, William R; Strong, Roland K; Nussenzweig, Michel C; Stamatatos, Leonidas

    2016-02-24

    VRC01-class broadly neutralizing HIV-1 antibodies protect animals from experimental infection and could contribute to an effective vaccine response. Their predicted germline forms (gl) bind Env inefficiently, which may explain why they are not elicited by HIV-1 Env-immunization. Here we show that an optimized Env immunogen can engage multiple glVRC01-class antibodies. Furthermore, this immunogen activates naive B cells expressing the human germline heavy chain of 3BNC60, paired with endogenous mouse light chains in vivo. To address whether it activates B cells expressing the fully humanized gl3BNC60 B-cell receptor (BCR), we immunized mice carrying both the heavy and light chains of gl3BNC60. B cells expressing this BCR display an autoreactive phenotype and fail to respond efficiently to soluble forms of the optimized immunogen, unless it is highly multimerized. Thus, specifically designed Env immunogens can activate naive B cells expressing human BCRs corresponding to precursors of broadly neutralizing HIV-1 antibodies even when the B cells display an autoreactive phenotype.

  16. Elicitation of broadly neutralizing antibodies against HIV-1 – the germline/maturation hypothesis

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    Prabakaran ePonraj

    2014-08-01

    Full Text Available We have previously observed that all known broadly neutralizing antibodies (bnAbs against HIV-1 are highly divergent from their putative germline predecessors in contrast to bnAbs against henipaviruses and SARS coronavirus, which are much less divergent from their germline counterparts. We have hypothesized that because the germline antibodies are so different compared to the highly somatically mutated HIV-1 bnAbs they may not bind to the Env. This led us to the hypothesis that the immunogenicity of the highly conserved epitopes on the HIV-1 envelope glycoproteins (Envs is reduced or eliminated by their very weak or absent interactions with germline antibodies. Thus immune responses leading to elicitation of bnAbs may not be initiated and/or sustained; even if they are, the maturation pathways are so complex that prolonged periods of time may be required for elicitation of such bnAbs. In support of the hypothesis, our initial experiments showed that germline-like precursors of several bnAbs do not bind to their epitopes. Recently, a number of research groups working in the HIV vaccine field have obtained data supporting and further expanding the germline/maturation hypothesis. Vaccine immunogens that could bind putative germline antibody predecessors of known bnAbs were successfully generated. However, guiding the immune system through the exceptionally complex antibody maturation pathways in order to elicit those bnAbs remains a major challenge. Here, we summarize developments in the HIV-1 vaccine field based on the germline/maturation hypothesis including our recent data demonstrating germline-like VRC01 antibodies in a human cord blood IgM library.

  17. Targeting N-Glycan Cryptic Sugar Moieties for Broad-Spectrum Virus Neutralization: Progress in Identifying Conserved Molecular Targets in Viruses of Distinct Phylogenetic Origins

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    Denong Wang

    2015-03-01

    Full Text Available Identifying molecular targets for eliciting broadly virus-neutralizing antibodies is one of the key steps toward development of vaccines against emerging viral pathogens. Owing to genomic and somatic diversities among viral species, identifying protein targets for broad-spectrum virus neutralization is highly challenging even for the same virus, such as HIV-1. However, viruses rely on host glycosylation machineries to synthesize and express glycans and, thereby, may display common carbohydrate moieties. Thus, exploring glycan-binding profiles of broad-spectrum virus-neutralizing agents may provide key information to uncover the carbohydrate-based virus-neutralizing epitopes. In this study, we characterized two broadly HIV-neutralizing agents, human monoclonal antibody 2G12 and Galanthus nivalis lectin (GNA, for their viral targeting activities. Although these agents were known to be specific for oligomannosyl antigens, they differ strikingly in virus-binding activities. The former is HIV-1 specific; the latter is broadly reactive and is able to neutralize viruses of distinct phylogenetic origins, such as HIV-1, severe acute respiratory syndrome coronavirus (SARS-CoV, and human cytomegalovirus (HCMV. In carbohydrate microarray analyses, we explored the molecular basis underlying the striking differences in the spectrum of anti-virus activities of the two probes. Unlike 2G12, which is strictly specific for the high-density Man9GlcNAc2Asn (Man9-clusters, GNA recognizes a number of N-glycan cryptic sugar moieties. These include not only the known oligomannosyl antigens but also previously unrecognized tri-antennary or multi-valent GlcNAc-terminating N-glycan epitopes (Tri/m-Gn. These findings highlight the potential of N-glycan cryptic sugar moieties as conserved targets for broad-spectrum virus neutralization and suggest the GNA-model of glycan-binding warrants focused investigation.

  18. Optimal Combinations of Broadly Neutralizing Antibodies for Prevention and Treatment of HIV-1 Clade C Infection.

    Science.gov (United States)

    Wagh, Kshitij; Bhattacharya, Tanmoy; Williamson, Carolyn; Robles, Alex; Bayne, Madeleine; Garrity, Jetta; Rist, Michael; Rademeyer, Cecilia; Yoon, Hyejin; Lapedes, Alan; Gao, Hongmei; Greene, Kelli; Louder, Mark K; Kong, Rui; Karim, Salim Abdool; Burton, Dennis R; Barouch, Dan H; Nussenzweig, Michel C; Mascola, John R; Morris, Lynn; Montefiori, David C; Korber, Bette; Seaman, Michael S

    2016-03-01

    The identification of a new generation of potent broadly neutralizing HIV-1 antibodies (bnAbs) has generated substantial interest in their potential use for the prevention and/or treatment of HIV-1 infection. While combinations of bnAbs targeting distinct epitopes on the viral envelope (Env) will likely be required to overcome the extraordinary diversity of HIV-1, a key outstanding question is which bnAbs, and how many, will be needed to achieve optimal clinical benefit. We assessed the neutralizing activity of 15 bnAbs targeting four distinct epitopes of Env, including the CD4-binding site (CD4bs), the V1/V2-glycan region, the V3-glycan region, and the gp41 membrane proximal external region (MPER), against a panel of 200 acute/early clade C HIV-1 Env pseudoviruses. A mathematical model was developed that predicted neutralization by a subset of experimentally evaluated bnAb combinations with high accuracy. Using this model, we performed a comprehensive and systematic comparison of the predicted neutralizing activity of over 1,600 possible double, triple, and quadruple bnAb combinations. The most promising bnAb combinations were identified based not only on breadth and potency of neutralization, but also other relevant measures, such as the extent of complete neutralization and instantaneous inhibitory potential (IIP). By this set of criteria, triple and quadruple combinations of bnAbs were identified that were significantly more effective than the best double combinations, and further improved the probability of having multiple bnAbs simultaneously active against a given virus, a requirement that may be critical for countering escape in vivo. These results provide a rationale for advancing bnAb combinations with the best in vitro predictors of success into clinical trials for both the prevention and treatment of HIV-1 infection.

  19. A next-generation cleaved, soluble HIV-1 Env trimer, BG505 SOSIP.664 gp140, expresses multiple epitopes for broadly neutralizing but not non-neutralizing antibodies.

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    Rogier W Sanders

    2013-09-01

    Full Text Available A desirable but as yet unachieved property of a human immunodeficiency virus type 1 (HIV-1 vaccine candidate is the ability to induce broadly neutralizing antibodies (bNAbs. One approach to the problem is to create trimeric mimics of the native envelope glycoprotein (Env spike that expose as many bNAb epitopes as possible, while occluding those for non-neutralizing antibodies (non-NAbs. Here, we describe the design and properties of soluble, cleaved SOSIP.664 gp140 trimers based on the subtype A transmitted/founder strain, BG505. These trimers are highly stable, more so even than the corresponding gp120 monomer, as judged by differential scanning calorimetry. They are also homogenous and closely resemble native virus spikes when visualized by negative stain electron microscopy (EM. We used several techniques, including ELISA and surface plasmon resonance (SPR, to determine the relationship between the ability of monoclonal antibodies (MAbs to bind the soluble trimers and neutralize the corresponding virus. In general, the concordance was excellent, in that virtually all bNAbs against multiple neutralizing epitopes on HIV-1 Env were highly reactive with the BG505 SOSIP.664 gp140 trimers, including quaternary epitopes (CH01, PG9, PG16 and PGT145. Conversely, non-NAbs to the CD4-binding site, CD4-induced epitopes or gp41ECTO did not react with the trimers, even when their epitopes were present on simpler forms of Env (e.g. gp120 monomers or dissociated gp41 subunits. Three non-neutralizing MAbs to V3 epitopes did, however, react strongly with the trimers but only by ELISA, and not at all by SPR and to only a limited extent by EM. These new soluble trimers are useful for structural studies and are being assessed for their performance as immunogens.

  20. Peptide Paratope Mimics of the Broadly Neutralizing HIV-1 Antibody b12.

    Science.gov (United States)

    Haußner, Christina; Damm, Dominik; Nirschl, Sandra; Rohrhofer, Anette; Schmidt, Barbara; Eichler, Jutta

    2017-01-26

    The broadly neutralizing HIV-1 antibody b12 recognizes the CD4 binding site of the HIV-1 envelope glycoprotein gp120 and efficiently neutralizes HIV-1 infections in vitro and in vivo. Based on the 3D structure of a b12⋅gp120 complex, we have designed an assembled peptide (b12-M) that presents the parts of the three heavy-chain complementarity-determining regions (CDRs) of b12, which contain the contact sites of the antibody for gp120. This b12-mimetic peptide, as well as a truncated peptide presenting only two of the three heavy-chain CDRs of b12, were shown to recognize gp120 in a similar manner to b12, as well as to inhibit HIV-1 infection, demonstrating functional mimicry of b12 by the paratope mimetic peptides.

  1. Protection Against Clostridium difficile Infection With Broadly Neutralizing Antitoxin Monoclonal Antibodies

    OpenAIRE

    2012-01-01

    The spore-forming bacterium Clostridium difficile represents the principal cause of hospital-acquired diarrhea and pseudomembranous colitis worldwide. C. difficile infection (CDI) is mediated by 2 bacterial toxins, A and B; neutralizing these toxins with monoclonal antibodies (mAbs) provides a potential nonantibiotic strategy for combating the rising prevalence, severity, and recurrence of CDI. Novel antitoxin mAbs were generated in mice and were humanized. The humanized antitoxin A mAb PA-50...

  2. Host-Pathogen Coevolution and the Emergence of Broadly Neutralizing Antibodies in Chronic Infections.

    Science.gov (United States)

    Nourmohammad, Armita; Otwinowski, Jakub; Plotkin, Joshua B

    2016-07-01

    The vertebrate adaptive immune system provides a flexible and diverse set of molecules to neutralize pathogens. Yet, viruses such as HIV can cause chronic infections by evolving as quickly as the adaptive immune system, forming an evolutionary arms race. Here we introduce a mathematical framework to study the coevolutionary dynamics between antibodies and antigens within a host. We focus on changes in the binding interactions between the antibody and antigen populations, which result from the underlying stochastic evolution of genotype frequencies driven by mutation, selection, and drift. We identify the critical viral and immune parameters that determine the distribution of antibody-antigen binding affinities. We also identify definitive signatures of coevolution that measure the reciprocal response between antibodies and viruses, and we introduce experimentally measurable quantities that quantify the extent of adaptation during continual coevolution of the two opposing populations. Using this analytical framework, we infer rates of viral and immune adaptation based on time-shifted neutralization assays in two HIV-infected patients. Finally, we analyze competition between clonal lineages of antibodies and characterize the fate of a given lineage in terms of the state of the antibody and viral populations. In particular, we derive the conditions that favor the emergence of broadly neutralizing antibodies, which may have relevance to vaccine design against HIV.

  3. Host-Pathogen Coevolution and the Emergence of Broadly Neutralizing Antibodies in Chronic Infections.

    Directory of Open Access Journals (Sweden)

    Armita Nourmohammad

    2016-07-01

    Full Text Available The vertebrate adaptive immune system provides a flexible and diverse set of molecules to neutralize pathogens. Yet, viruses such as HIV can cause chronic infections by evolving as quickly as the adaptive immune system, forming an evolutionary arms race. Here we introduce a mathematical framework to study the coevolutionary dynamics between antibodies and antigens within a host. We focus on changes in the binding interactions between the antibody and antigen populations, which result from the underlying stochastic evolution of genotype frequencies driven by mutation, selection, and drift. We identify the critical viral and immune parameters that determine the distribution of antibody-antigen binding affinities. We also identify definitive signatures of coevolution that measure the reciprocal response between antibodies and viruses, and we introduce experimentally measurable quantities that quantify the extent of adaptation during continual coevolution of the two opposing populations. Using this analytical framework, we infer rates of viral and immune adaptation based on time-shifted neutralization assays in two HIV-infected patients. Finally, we analyze competition between clonal lineages of antibodies and characterize the fate of a given lineage in terms of the state of the antibody and viral populations. In particular, we derive the conditions that favor the emergence of broadly neutralizing antibodies, which may have relevance to vaccine design against HIV.

  4. Ontogeny-based immunogens for the induction of V2-directed HIV broadly neutralizing antibodies.

    Science.gov (United States)

    Moore, Penny L; Gorman, Jason; Doria-Rose, Nicole A; Morris, Lynn

    2017-01-01

    The development of a preventative HIV vaccine able to elicit broadly neutralizing antibodies (bNAbs) remains a major challenge. Antibodies that recognize the V2 region at the apex of the HIV envelope trimer are among the most common bNAb specificities during chronic infection and many exhibit remarkable breadth and potency. Understanding the developmental pathway of these antibodies has provided insights into their precursors, and the viral strains that engage them, as well as defined how such antibodies mature to acquire breadth. V2-apex bNAbs are derived from rare precursors with long anionic CDR H3s that are often deleted in the B cell repertoire. However, longitudinal studies suggest that once engaged, these precursors contain many of the structural elements required for neutralization, and can rapidly acquire breadth through moderate levels of somatic hypermutation in response to emerging viral variants. These commonalities in the precursors and mechanism of neutralization have enabled the identification of viral strains that show enhanced reactivity for V2 precursors from multiple donors, and may form the basis of germline targeting approaches. In parallel, new structural insights into the HIV trimer, the target of these quaternary antibodies, has created invaluable new opportunities for ontogeny-based immunogens designed to select for rare V2-bNAb precursors, and drive them toward breadth.

  5. New broadly reactive neutralizing antibodies against hepatitis B virus surface antigen.

    Science.gov (United States)

    Kucinskaite-Kodze, Indre; Pleckaityte, Milda; Bremer, Corinna M; Seiz, Pia L; Zilnyte, Milda; Bulavaite, Aiste; Mickiene, Gitana; Zvirblis, Gintautas; Sasnauskas, Kestutis; Glebe, Dieter; Zvirbliene, Aurelija

    2016-01-01

    Hepatitis B virus (HBV) surface antigen (HBsAg) is considered to be the most important target for the diagnosis and immune prophylaxis of HBV infection. HBsAg-specific monoclonal antibodies (MAbs) are extensively used for studying the complex structure of the HBsAg, mapping the neutralizing epitopes and development of HBV diagnostic tests. However, the efficiency of anti-HBV binding strongly depends on the epitope structure and MAb capability to recognize different HBV variants. In the current study, 9 MAbs against yeast-expressed HBsAg of ayw2 serotype were generated and 7 of them were shown to recognize a linear epitope comprising amino acid (aa) residues 119-GPCRTCT-125 within the main antigenic "a" determinant of HBsAg. One MAb of the highest affinity (clone HB1) was selected for detailed cross-reactivity studies, generation of recombinant single-chain antibody (scFv) and molecular modelling of antibody-epitope interaction. The importance of each aa residue within the identified MAb epitope was determined by alanine substitution study that revealed aa residues C(121), T(123), C(124) and T(125) as essential for binding. These aa residues are highly conserved among HBV variants. In contrast, alanine substitution of G119, P120 and R122 had no or minor influence on the reactivity with the MAb. Certain aa residues at position 122 (either R or K) define different HBV serotypes (either d or y), therefore, the affinity of the MAb HB1 for the epitope with R122K substitution was determined to evaluate its diagnostic potential. The MAb recognized both epitope variants with high affinity. Sequence alignment of the MAb epitope within different HBV strains demonstrated that the shortest peptide recognized by the MAb 121-CR(K)TCT-125 is identical among different human HBV genotypes (HBV A-F, H) and monkey HBV species (HBVCP, HBVGO, HBVGB, WMHBV). In line with these data, the MAb HB1 was cross-reactive in Western blot with a large panel of antigens derived from different HBV

  6. The HIV glycan shield as a target for broadly neutralizing antibodies.

    Science.gov (United States)

    Doores, Katie J

    2015-12-01

    The HIV envelope glycoprotein (Env) is the sole target for HIV broadly neutralizing antibodies (bnAbs). HIV Env is one of the most heavily glycosylated proteins known, with approximately half of its mass consisting of host-derived N-linked glycans. The high density of glycans creates a shield that impedes antibody recognition but, critically, some of the most potent and broadly active bnAbs have evolved to recognize epitopes formed by these glycans. Although the virus hijacks the host protein synthesis and glycosylation machinery to generate glycosylated HIV Env, studies have shown that HIV Env glycosylation diverges from that typically observed on host-derived glycoproteins. In particular, the high density of glycans leads to a nonself motif of underprocessed oligomannose-type glycans that forms the target of some of the most broad and potent HIV bnAbs. This review discusses the changing perception of the HIV glycan shield, and summarizes the protein-directed and cell-directed factors controlling HIV Env glycosylation that impact on HIV bnAb recognition and HIV vaccine design strategies.

  7. Identification of Broad-Genotype HPV L2 Neutralization Site for Pan-HPV Vaccine Development by a Cross-Neutralizing Antibody.

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    Daning Wang

    Full Text Available Human Papillomavirus (HPV, a non-enveloped, double-stranded DNA virus, is responsible for 5% of human cancers. The HPV capsid consists of major and minor structural proteins, L1 and L2. L1 proteins form an icosahedral shell with building blocks of the pentameric capsomere, and one L2 molecule extends outward from the central hole of the capsid. Thus, L2 is concealed within L1 and only becomes exposed when the capsid interacts with host cells. The low antigenic variation of L2 means that this protein could offer a target for the development of a pan-HPV vaccine. Toward this goal, here we describe an anti-L2 monoclonal antibody, 14H6, which broadly neutralizes at least 11 types of HPV, covering types 6, 11, 16, 18, 31, 33, 35, 45, 52, 58 and 59, in pseudovirion--based cell neutralization assay. The mAb 14H6 recognizes a minimal linear epitope located on amino acids 21 to 30 of the L2 protein. Alanine scanning mutagenesis and sequence alignment identified several conserved residues (Cys22, Lys23, Thr27, Cys28 and Pro29 that are involved in the 14H6 binding with L2. The epitope was grafted to several scaffolding proteins, including HPV16 L1 virus-like particles, HBV 149 core antigen and CRM197. The resultant chimeric constructs were expressed in Escherichia coli and purified with high efficiency. Immunization with these pan-HPV vaccine candidates elicited high titers of the L2-specific antibody in mice and conferred robust (3-log titers of cross-genotype neutralization, including against HPV11, 16, 18, 45, 52, 58 and 59. These findings will help in the development of an L2-based, pan-HPV vaccine.

  8. Broadly Neutralizing Antibody 8ANC195 Recognizes Closed and Open States of HIV-1 Env.

    Science.gov (United States)

    Scharf, Louise; Wang, Haoqing; Gao, Han; Chen, Songye; McDowall, Alasdair W; Bjorkman, Pamela J

    2015-09-10

    The HIV-1 envelope (Env) spike contains limited epitopes for broadly neutralizing antibodies (bNAbs); thus, most neutralizing antibodies are strain specific. The 8ANC195 epitope, defined by crystal and electron microscopy (EM) structures of bNAb 8ANC195 complexed with monomeric gp120 and trimeric Env, respectively, spans the gp120 and gp41 Env subunits. To investigate 8ANC195's gp41 epitope at higher resolution, we solved a 3.58 Å crystal structure of 8ANC195 complexed with fully glycosylated Env trimer, revealing 8ANC195 insertion into a glycan shield gap to contact gp120 and gp41 glycans and protein residues. To determine whether 8ANC195 recognizes the CD4-bound open Env conformation that leads to co-receptor binding and fusion, one of several known conformations of virion-associated Env, we solved EM structures of an Env/CD4/CD4-induced antibody/8ANC195 complex. 8ANC195 binding partially closed the CD4-bound trimer, confirming structural plasticity of Env by revealing a previously unseen conformation. 8ANC195's ability to bind different Env conformations suggests advantages for potential therapeutic applications.

  9. Broadly neutralizing epitopes in the Plasmodium vivax vaccine candidate Duffy Binding Protein.

    Science.gov (United States)

    Chen, Edwin; Salinas, Nichole D; Huang, Yining; Ntumngia, Francis; Plasencia, Manolo D; Gross, Michael L; Adams, John H; Tolia, Niraj Harish

    2016-05-31

    Plasmodium vivax Duffy Binding Protein (PvDBP) is the most promising vaccine candidate for P. vivax malaria. The polymorphic nature of PvDBP induces strain-specific immune responses, however, and the epitopes of broadly neutralizing antibodies are unknown. These features hamper the rational design of potent DBP-based vaccines and necessitate the identification of globally conserved epitopes. Using X-ray crystallography, small-angle X-ray scattering, hydrogen-deuterium exchange mass spectrometry, and mutational mapping, we have defined epitopes for three inhibitory mAbs (mAbs 2D10, 2H2, and 2C6) and one noninhibitory mAb (3D10) that engage DBP. These studies expand the currently known inhibitory epitope repertoire by establishing protective motifs in subdomain three outside the receptor-binding and dimerization residues of DBP, and introduce globally conserved protective targets. All of the epitopes are highly conserved among DBP alleles. The identification of broadly conserved epitopes of inhibitory antibodies provides critical motifs that should be retained in the next generation of potent vaccines for P. vivax malaria.

  10. N-terminal residues of an HIV-1 gp41 membrane-proximal external region antigen influence broadly neutralizing 2F5-like antibodies

    Institute of Scientific and Technical Information of China (English)

    Dezhi Li; Jie Liu; Li Zhang; Tianshu Xu; Junheng Chen; Liping Wang; Qi Zhao

    2015-01-01

    The Human immunodeficiency virus type 1(HIV-1) gp41 membrane proximal external region(MPER) is targeted by broadly neutralizing antibodies(e.g. 2F5, 4E10, Z13 e and m66.6), which makes this region a promising target for vaccine design. One strategy to elicit neutralizing antibodies against the MPER epitope is to design peptide immunogens mimicking neutralization structures. To probe 2F5-like neutralizing antibodies, two yeast-displayed antibody libraries from peripheral blood mononuclear cells from a HIV-1 patient were screened against the 2F5 epitope peptide SP62. Two 2F5-like antibodies were identified that specifically recognized SP62. However,these antibodies only weakly neutralized HIV-1 primary isolates. The epitopes recognized by these two 2F5-like antibodies include not only the 2F5 epitope(amino acids(aa) 662–667 in the MPER)but also several other residues(aa 652–655) locating at the N-terminus in SP62. Experimental results suggest that residues of SP62 adjacent to the 2F5 epitope influence the response of broadly neutralizing 2F5-like antibodies in vaccination. Our findings may aid the design of vaccine immunogens and development of therapeutics against HIV-1 infection.

  11. N-terminal residues of an HIV-1 gp41 membrane-proximal external region antigen influence broadly neutralizing 2F5-like antibodies.

    Science.gov (United States)

    Li, Dezhi; Liu, Jie; Zhang, Li; Xu, Tianshu; Chen, Junheng; Wang, Liping; Zhao, Qi

    2015-12-01

    The Human immunodeficiency virus type 1 (HIV-1) gp41 membrane proximal external region (MPER) is targeted by broadly neutralizing antibodies (e.g. 2F5, 4E10, Z13e and m66.6), which makes this region a promising target for vaccine design. One strategy to elicit neutralizing antibodies against the MPER epitope is to design peptide immunogens mimicking neutralization structures. To probe 2F5-like neutralizing antibodies, two yeast-displayed antibody libraries from peripheral blood mononuclear cells from a HIV-1 patient were screened against the 2F5 epitope peptide SP62. Two 2F5-like antibodies were identified that specifically recognized SP62. However, these antibodies only weakly neutralized HIV-1 primary isolates. The epitopes recognized by these two 2F5-like antibodies include not only the 2F5 epitope (amino acids (aa) 662-667 in the MPER) but also several other residues (aa 652-655) locating at the N-terminus in SP62. Experimental results suggest that residues of SP62 adjacent to the 2F5 epitope influence the response of broadly neutralizing 2F5-like antibodies in vaccination. Our findings may aid the design of vaccine immunogens and development of therapeutics against HIV-1 infection.

  12. A broadly neutralizing anti-influenza antibody reveals ongoing capacity of haemagglutinin-specific memory B cells to evolve

    Science.gov (United States)

    Fu, Ying; Zhang, Zhen; Sheehan, Jared; Avnir, Yuval; Ridenour, Callie; Sachnik, Thomas; Sun, Jiusong; Hossain, M. Jaber; Chen, Li-Mei; Zhu, Quan; Donis, Ruben O.; Marasco, Wayne A.

    2016-01-01

    Understanding the natural evolution and structural changes involved in broadly neutralizing antibody (bnAb) development holds great promise for improving the design of prophylactic influenza vaccines. Here we report an haemagglutinin (HA) stem-directed bnAb, 3I14, isolated from human memory B cells, that utilizes a heavy chain encoded by the IGHV3-30 germline gene. MAb 3I14 binds and neutralizes groups 1 and 2 influenza A viruses and protects mice from lethal challenge. Analysis of VH and VL germline back-mutants reveals binding to H3 and H1 but not H5, which supports the critical role of somatic hypermutation in broadening the bnAb response. Moreover, a single VLD94N mutation improves the affinity of 3I14 to H5 by nearly 10-fold. These data provide evidence that memory B cell evolution can expand the HA subtype specificity. Our results further suggest that establishing an optimized memory B cell pool should be an aim of ‘universal' influenza vaccine strategies. PMID:27619409

  13. Unique C2V3 sequence in HIV-1 envelope obtained from broadly neutralizing plasma of a slow progressing patient conferred enhanced virus neutralization.

    Directory of Open Access Journals (Sweden)

    Rajesh Ringe

    Full Text Available Broadly neutralizing antibodies to HIV-1 usually develops in chronic infections. Here, we examined the basis of enhanced sensitivity of an env clone amplified from cross neutralizing plasma of an antiretroviral naïve chronically infected Indian patient (ID50 >600-fold higher compared to other autologous env clones. The enhanced autologous neutralization of pseudotyped viruses expressing the sensitive envelope (Env was associated with increased sensitivity to reagents and monoclonal antibodies targeting distinct sites in Env. Chimeric viruses constructed by swapping fragments of sensitive Env into resistant Env backbone revealed that the presence of unique residues within C2V3 region of gp120 governed increased neutralization. The enhanced virus neutralization was also associated with low CD4 dependence as well as increased binding of Env trimers to IgG1b12 and CD4-IgG2 and was independent of gp120 shedding. Our data highlighted vulnerabilities in the Env obtained from cross neutralizing plasma associated with the exposure of discontinuous neutralizing epitopes and enhanced autologous neutralization. Such information may aid in Env-based vaccine immunogen design.

  14. Expression of HIV-1 broadly neutralizing antibodies mediated by recombinant adeno-associated virus 8 in vitro and in vivo.

    Science.gov (United States)

    Yu, Yongjiao; Fu, Lu; Jiang, Xiaoyu; Guan, Shanshan; Kuai, Ziyu; Kong, Wei; Shi, Yuhua; Shan, Yaming

    2016-12-01

    Despite unremitting efforts since the discovery of human immunodeficiency virus type 1 (HIV-1), an effective vaccine has not been generated. Viral vector-mediated transfer for expression of HIV-1 broadly neutralizing antibodies (BnAbs) is an attractive strategy. In this study, a recombinant adeno-associated virus 8 (rAAV8) vector was used to encode full-length antibodies against HIV-1 in 293T cells and Balb/c mice after gene transfer. The 10E8 or NIH45-46 BnAb was expressed from a single open reading frame by linking the heavy and light chains with a furin cleavage and a 2A self-processing peptide (F2A). The results showed that the BnAbs could be expressed in the 293T cell culture medium. A single intramuscular injection of rAAV8 led to long-term expression of BnAbs in Balb/c mice. The expressed antibodies in the supernatant of 293T cells and in Balb/c mice showed neutralization effects against HIV-1 pseudoviruses. Combined immunization of rAAV8 expressing 10E8 and rAAV8 expressing NIH45-46 in Balb/c mice could increase these neutralization effects on strains of HIV-1 sensitive to 10E8 or NIH45-46 antibody compared with a single injection of rAAV8 expressing either antibody alone. Therefore, the combined immunization may be a potential vaccine approach against HIV-1.

  15. Dose-response curve slope helps predict therapeutic potency and breadth of HIV broadly neutralizing antibodies.

    Science.gov (United States)

    Webb, Nicholas E; Montefiori, David C; Lee, Benhur

    2015-09-29

    A new generation of HIV broadly neutralizing antibodies (bnAbs) with remarkable potency, breadth and epitope diversity has rejuvenated interest in immunotherapeutic strategies. Potencies defined by in vitro IC50 and IC80 values (50 and 80% inhibitory concentrations) figure prominently into the selection of clinical candidates; however, much higher therapeutic levels will be required to reduce multiple logs of virus and impede escape. Here we predict bnAb potency at therapeutic levels by analysing dose-response curve slopes, and show that slope is independent of IC50/IC80 and specifically relates to bnAb epitope class. With few exceptions, CD4-binding site and V3-glycan bnAbs exhibit slopes >1, indicative of higher expected therapeutic effectiveness, whereas V2-glycan, gp41 membrane-proximal external region (MPER) and gp120-gp41 bnAbs exhibit less favourable slopes <1. Our results indicate that slope is one major predictor of both potency and breadth for bnAbs at clinically relevant concentrations, and may better coordinate the relationship between bnAb epitope structure and therapeutic expectations.

  16. Protection against Clostridium difficile infection with broadly neutralizing antitoxin monoclonal antibodies.

    Science.gov (United States)

    Marozsan, Andre J; Ma, Dangshe; Nagashima, Kirsten A; Kennedy, Brian J; Kang, Yun Kenneth; Arrigale, Robert R; Donovan, Gerald P; Magargal, Wells W; Maddon, Paul J; Olson, William C

    2012-09-01

    The spore-forming bacterium Clostridium difficile represents the principal cause of hospital-acquired diarrhea and pseudomembranous colitis worldwide. C. difficile infection (CDI) is mediated by 2 bacterial toxins, A and B; neutralizing these toxins with monoclonal antibodies (mAbs) provides a potential nonantibiotic strategy for combating the rising prevalence, severity, and recurrence of CDI. Novel antitoxin mAbs were generated in mice and were humanized. The humanized antitoxin A mAb PA-50 and antitoxin B mAb PA-41 have picomolar potencies in vitro and bind to novel regions of the respective toxins. In a hamster model for CDI, 95% of animals treated with a combination of humanized PA-50 and PA-41 showed long-term survival relative to 0% survival of animals treated with standard antibiotics or comparator mAbs. These humanized mAbs provide insight into C. difficile intoxication and hold promise as potential nonantibiotic agents for improving clinical management of CDI.

  17. All eyes on the next generation of HIV vaccines: strategies for inducing a broadly neutralizing antibody response.

    Science.gov (United States)

    Ahlers, Jeffrey D

    2014-04-01

    HIV-1 broadly neutralizing antibodies (BNAbs) develop after several years of infection through a recursive process of memory B cell adaptation and maturation against co-evolving virus quasispecies. Advances in single-cell sorting and memory B cell antibody cloning methods have identified many new HIV BNAbs targeting conserved epitopes on the HIV envelope (env) protein. 3D crystal structures and biophysical analyses of BNAbs bound to invariant virus structures expressed on monomeric gp120, epitope scaffolds, core structures, and native trimers have helped us to visualize unique binding interactions and paratope orientations that have been instrumental in guiding vaccine design. A paradigm shift in the approach to structure-based design of HIV-1 envelope immunogens came recently after several laboratories discovered that native viral envelopes or "env-structures" reverse-engineered to bind with high affinity to a handful of broadly neutralizing antibodies did not in fact bind the predicted germline precursors of these broadly neutralizing antibodies. A major challenge for HIV-1 B cell vaccine development moving forward is the design of new envelope immunogens that can trigger the selection and expansion of germline precursor and intermediate memory B cells to recapitulate B cell ontogenies associated with the maturation of a broadly neutralizing antibody response. Equally important for vaccine development is the identification of delivery systems, prime-boost strategies, and synergistic adjuvant combinations that can induce the magnitude and quality of antigen-specific T follicular helper (TFH) cell responses needed to drive somatic hypermutation (SHM) and B cell maturation against heterologous primary virus envelopes. Finding the combination of multi-protein envelope immunogens and immunization strategies that can evolve a potent broadly neutralizing antibody response portends to require a complex vaccine regimen that might be difficult to implement on any scale

  18. Broadly neutralizing antibodies against the rapidly evolving porcine reproductive and respiratory syndrome virus.

    Science.gov (United States)

    Robinson, Sally R; Li, Juan; Nelson, Eric A; Murtaugh, Michael P

    2015-05-04

    Neutralizing antibodies are a critical part of the immune armory for defense against viruses, and the mechanism by which many effective vaccines work to protect against viral infections. However, infections by rapidly evolving and genetically diverse viruses are often characterized by ineffective neutralizing antibody responses. Porcine reproductive and respiratory syndrome virus (PRRSV) is a highly genetically diverse RNA virus that causes PRRS, the most significant disease of pigs worldwide. The prevailing view of immunity to PRRSV is characterized by delayed and ineffectual production of neutralizing antibodies lacking cross-reactivity that is necessary for vaccine efficacy. Using an ELISA-based neutralizing assay developed to analyze PRRSV growth in porcine alveolar macrophages, the naturally permissive cell of PRRSV, we showed that sera from previously infected commercial sows had high levels of neutralizing activity against diverse PRRSV strains, including across distinct genotypes of PRRSV. Fifty percent cross-neutralization titers in excess of 1/1024 were observed. Neutralizing activity was dose-dependent and was maintained in the immunoglobulin fraction. Presence of high-titer, anti-PRRSV antibody activity that cross-neutralizes diverse strains of virus has prompted reevaluation of the role of neutralizing antibodies for cross-protection against PRRSV under field conditions. Understanding conditions that favor development of cross-neutralizing activity will be crucial for improved strategies to enhance cross-protection against PRRSV. More detailed studies are expected to elucidate mechanisms of neutralizing antibody production and maturation and to investigate conserved epitope targets of cross-neutralization in this rapidly evolving virus.

  19. Protective Effects of Broadly Neutralizing Immunoglobulin against Homologous and Heterologous Equine Infectious Anemia Virus Infection in Horses with Severe Combined Immunodeficiency▿

    Science.gov (United States)

    Taylor, Sandra D.; Leib, Steven R.; Wu, Wuwei; Nelson, Robert; Carpenter, Susan; Mealey, Robert H.

    2011-01-01

    Using the equine infectious anemia virus (EIAV) lentivirus model system, we previously demonstrated protective effects of broadly neutralizing immune plasma in young horses (foals) with severe combined immunodeficiency (SCID). However, in vivo selection of a neutralization-resistant envelope variant occurred. Here, we determined the protective effects of purified immunoglobulin with more potent broadly neutralizing activity. Overall, protection correlated with the breadth and potency of neutralizing activity in vitro. Four of five SCID foals were completely protected against homologous challenge, while partial protection occurred following heterologous challenge. These results support the inclusion of broadly neutralizing antibodies in lentivirus control strategies. PMID:21543497

  20. Protective effects of broadly neutralizing immunoglobulin against homologous and heterologous equine infectious anemia virus infection in horses with severe combined immunodeficiency.

    Science.gov (United States)

    Taylor, Sandra D; Leib, Steven R; Wu, Wuwei; Nelson, Robert; Carpenter, Susan; Mealey, Robert H

    2011-07-01

    Using the equine infectious anemia virus (EIAV) lentivirus model system, we previously demonstrated protective effects of broadly neutralizing immune plasma in young horses (foals) with severe combined immunodeficiency (SCID). However, in vivo selection of a neutralization-resistant envelope variant occurred. Here, we determined the protective effects of purified immunoglobulin with more potent broadly neutralizing activity. Overall, protection correlated with the breadth and potency of neutralizing activity in vitro. Four of five SCID foals were completely protected against homologous challenge, while partial protection occurred following heterologous challenge. These results support the inclusion of broadly neutralizing antibodies in lentivirus control strategies.

  1. Evolution of broadly cross-reactive HIV-1-neutralizing activity: therapy-associated decline, positive association with detectable viremia, and partial restoration of B-cell subpopulations.

    Science.gov (United States)

    Ferreira, Carolina B; Merino-Mansilla, Alberto; Llano, Anuska; Pérez, Ignacio; Crespo, Isabel; Llinas, Laia; Garcia, Felipe; Gatell, Jose M; Yuste, Eloisa; Sanchez-Merino, Victor

    2013-11-01

    Little is known about the stability of HIV-1 cross-neutralizing responses. Taking into account the fact that neutralization breadth has been positively associated with plasma viral load, there is no explanation for the presence of broadly neutralizing responses in a group of patients on treatment with undetectable viremia. In addition, the B-cell profile responsible for broadly cross-neutralizing responses is unknown. Here we studied the evolution of neutralizing responses and the B-cell subpopulation distribution in a group of patients with broadly cross-reactive HIV-1-neutralizing activity. We studied neutralization breadth evolution in a group of six previously identified broadly cross-neutralizing patients and six control patients during a 6-year period with a previously described minipanel of recombinant viruses from five different subtypes. B-cell subpopulation distribution during the study was also determined by multiparametric flow cytometry. Broadly cross-neutralizing activity was transient in four broad cross-neutralizers and stable, up to 4.6 years, in the other two. In four out of five broad cross-neutralizers who initiated treatment, a neutralization breadth loss occurred after viremia had been suppressed for as much as 20 months. B-cell subpopulation analyses revealed a significant increase in the frequency of naive B cells in broadly cross-reactive samples, compared with samples with less neutralization breadth (increased from 44% to 62%). We also observed a significant decrease in tissue-like and activated memory B cells (decreased from 19% to 12% and from 17% to 9%, respectively). Our data suggest that HIV-1 broadly cross-neutralizing activity is variable over time and associated with detectable viremia and partial B-cell restoration.

  2. Engineering HIV envelope protein to activate germline B cell receptors of broadly neutralizing anti-CD4 binding site antibodies.

    Science.gov (United States)

    McGuire, Andrew T; Hoot, Sam; Dreyer, Anita M; Lippy, Adriana; Stuart, Andrew; Cohen, Kristen W; Jardine, Joseph; Menis, Sergey; Scheid, Johannes F; West, Anthony P; Schief, William R; Stamatatos, Leonidas

    2013-04-08

    Broadly neutralizing antibodies (bnAbs) against HIV are believed to be a critical component of the protective responses elicited by an effective HIV vaccine. Neutralizing antibodies against the evolutionarily conserved CD4-binding site (CD4-BS) on the HIV envelope glycoprotein (Env) are capable of inhibiting infection of diverse HIV strains, and have been isolated from HIV-infected individuals. Despite the presence of anti-CD4-BS broadly neutralizing antibody (bnAb) epitopes on recombinant Env, Env immunization has so far failed to elicit such antibodies. Here, we show that Env immunogens fail to engage the germline-reverted forms of known bnAbs that target the CD4-BS. However, we found that the elimination of a conserved glycosylation site located in Loop D and two glycosylation sites located in variable region 5 of Env allows Env-binding to, and activation of, B cells expressing the germline-reverted BCRs of two potent broadly neutralizing antibodies, VRC01 and NIH45-46. Our results offer a possible explanation as to why Env immunogens have been ineffective in stimulating the production of such bNAbs. Importantly, they provide key information as to how such immunogens can be engineered to initiate the process of antibody-affinity maturation against one of the most conserved Env regions.

  3. Application of broad-spectrum humanized neutralizing monoclonal antibodies to influenza A virus in prevention and treatment of influenza%广谱全人源化甲型流感病毒中和抗体在流感预防与治疗中的应用

    Institute of Scientific and Technical Information of China (English)

    高灵茜; 杨利

    2013-01-01

    针对多个亚型甲型流感病毒的广谱中和性单克隆抗体(monoclonalantibody,mAb)是极具潜力的新型而有效的流感防治手段.抗流感病毒血凝素(hemagglutinin,HA)的保护性mAb,无论通过主动免疫还是被动免疫,均具有很好的效果.迄今,已有不少甲型流感病毒广谱中和性mAb被开发出来.尽管这些mAb具备临床应用潜能以及作为研制新型疫苗(抗独特型抗体疫苗)的可能,但仅有几种HA mAb对人类流感防治真正有效.此文主要对能高效识别多个亚型HA的人源化甲型流感病毒mAb做一综述.%Broad-spectrum neutralizing monoclonal antibodies (mAbs) against different subtypes of influenza A viruses are potential tools to prevent and treat influenza.Protective mAbs to influenza hemagglutinin (HA) have good results by passive and active immunization.So far,a number of mAbs against influenza HA have been developed.But only few of them are effective for influenza prevention and treatment.In this article,the human mAbs which can recognize divergent influenza isolates are reviewed.

  4. HIV-1 clade C escapes broadly neutralizing autologous antibodies with N332 glycan specificity by distinct mechanisms.

    Science.gov (United States)

    Deshpande, Suprit; Patil, Shilpa; Kumar, Rajesh; Hermanus, Tandile; Murugavel, Kailapuri G; Srikrishnan, Aylur K; Solomon, Suniti; Morris, Lynn; Bhattacharya, Jayanta

    2016-08-30

    The glycan supersite centered on N332 in the V3 base of the HIV-1 envelope (Env) is a target for broadly neutralizing antibodies (bnAbs) such as PGT121 and PGT128. In this study, we examined the basis of resistance of HIV-1 clade C Envs obtained from broadly cross neutralizing (BCN) plasma of an Indian donor with N332 specificity. Pseudotyped viruses expressing autologous envs were found to be resistant to autologous BCN plasma as well as to PGT121 and PGT128 mAbs despite the majority of Envs containing an intact N332 residue. While resistance of one of the Envs to neutralization by autologous plasma antibodies with shorter V1 loop length was found to be correlated with a N332S mutation, resistance to neutralization of rest of the Envs was found to be associated with longer V1 loop length and acquisition of protective N-glycans. In summary, we show evidence of escape of circulating HIV-1 clade C in an individual from autologous BCN antibodies by three distinct mechanisms.

  5. Structure-guided alterations of the gp41-directed HIV-1 broadly neutralizing antibody 2F5 reveal new properties regarding its neutralizing function.

    Directory of Open Access Journals (Sweden)

    Javier Guenaga

    Full Text Available The broadly neutralizing HIV-1 antibody 2F5 recognizes an epitope in the gp41 membrane proximal external region (MPER. The MPER adopts a helical conformation as free peptide, as post-fusogenic forms of gp41, and when bound to the 4E10 monoclonal antibody (Mab. However, when bound to 2F5, the epitope is an extended-loop. The antibody-peptide structure reveals binding between the heavy and light chains with most the long, hydrophobic CDRH3 not contacting peptide. However, mutagenesis identifies this loop as critical for binding, neutralization and for putative hydrophobic membrane interactions. Here, we examined length requirements of the 2F5 CDRH3 and plasticity regarding binding and neutralization. We generated 2F5 variants possessing either longer or shorter CDRH3s and assessed function. The CDRH3 tolerated elongations and reductions up to four residues, displaying a range of binding affinities and retaining some neutralizing capacity. 2F5 antibody variants selective recognition of conformationally distinctive MPER probes suggests a new role for the CDRH3 loop in destabilizing the helical MPER. Binding and neutralization were enhanced by targeted tryptophan substitutions recapitulating fully the activities of the wild-type 2F5 antibody in a shorter CDRH3 variant. MPER alanine scanning revealed binding contacts of this variant downstream of the 2F5 core epitope, into the 4E10 epitope region. This variant displayed increased reactivity to cardiolipin-beta-2-glycoprotein. Tyrosine replacements maintained neutralization while eliminating cardiolipin-beta-2-glycoprotein interaction. The data suggest a new mechanism of action, important for vaccine design, in which the 2F5 CDRH3 contacts and destabilizes the MPER helix downstream of its core epitope to allow induction of the extended-loop conformation.

  6. Glycan shifting on hepatitis C virus (HCV) E2 glycoprotein is a mechanism for escape from broadly neutralizing antibodies.

    Science.gov (United States)

    Pantua, Homer; Diao, Jingyu; Ultsch, Mark; Hazen, Meredith; Mathieu, Mary; McCutcheon, Krista; Takeda, Kentaro; Date, Shailesh; Cheung, Tommy K; Phung, Qui; Hass, Phil; Arnott, David; Hongo, Jo-Anne; Matthews, David J; Brown, Alex; Patel, Arvind H; Kelley, Robert F; Eigenbrot, Charles; Kapadia, Sharookh B

    2013-06-12

    Hepatitis C virus (HCV) infection is a major cause of liver disease and hepatocellular carcinoma. Glycan shielding has been proposed to be a mechanism by which HCV masks broadly neutralizing epitopes on its viral glycoproteins. However, the role of altered glycosylation in HCV resistance to broadly neutralizing antibodies is not fully understood. Here, we have generated potent HCV neutralizing antibodies hu5B3.v3 and MRCT10.v362 that, similar to the previously described AP33 and HCV1, bind to a highly conserved linear epitope on E2. We utilize a combination of in vitro resistance selections using the cell culture infectious HCV and structural analyses to identify mechanisms of HCV resistance to hu5B3.v3 and MRCT10.v362. Ultra deep sequencing from in vitro HCV resistance selection studies identified resistance mutations at asparagine N417 (N417S, N417T and N417G) as early as 5days post treatment. Comparison of the glycosylation status of soluble versions of the E2 glycoprotein containing the respective resistance mutations revealed a glycosylation shift from N417 to N415 in the N417S and N417T E2 proteins. The N417G E2 variant was glycosylated neither at residue 415 nor at residue 417 and remained sensitive to MRCT10.v362. Structural analyses of the E2 epitope bound to hu5B3.v3 Fab and MRCT10.v362 Fab using X-ray crystallography confirmed that residue N415 is buried within the antibody-peptide interface. Thus, in addition to previously described mutations at N415 that abrogate the β-hairpin structure of this E2 linear epitope, we identify a second escape mechanism, termed glycan shifting, that decreases the efficacy of broadly neutralizing HCV antibodies.

  7. Expression of Human Immunodeficiency Virus Type 1 Neutralizing Antibody Fragments Using Human Vaginal Lactobacillus

    Science.gov (United States)

    Marcobal, Angela; Liu, Xiaowen; Zhang, Wenlei; Dimitrov, Antony S.; Jia, Letong; Lee, Peter P.; Fouts, Timothy R.; Parks, Thomas P.

    2016-01-01

    Abstract Eradication of human immunodeficiency virus type 1 (HIV-1) by vaccination with epitopes that produce broadly neutralizing antibodies is the ultimate goal for HIV prevention. However, generating appropriate immune responses has proven difficult. Expression of broadly neutralizing antibodies by vaginal colonizing lactobacilli provides an approach to passively target these antibodies to the mucosa. We tested the feasibility of expressing single-chain and single-domain antibodies (dAbs) in Lactobacillus to be used as a topical microbicide/live biotherapeutic. Lactobacilli provide an excellent platform to express anti-HIV proteins. Broadly neutralizing antibodies have been identified against epitopes on the HIV-1 envelope and have been made into active antibody fragments. We tested single-chain variable fragment m9 and dAb-m36 and its derivative m36.4 as prototype antibodies. We cloned and expressed the antibody fragments m9, m36, and m36.4 in Lactobacillus jensenii-1153 and tested the expression levels and functionality. We made a recombinant L. jensenii 1153-1128 that expresses dAb-m36.4. All antibody fragments m9, m36, and m36.4 were expressed by lactobacilli. However, we noted the smaller m36/m36.4 were expressed to higher levels, ≥3 μg/ml. All L. jensenii-expressed antibody fragments bound to gp120/CD4 complex; Lactobacillus-produced m36.4 inhibited HIV-1BaL in a neutralization assay. Using a TZM-bl assay, we characterized the breadth of neutralization of the m36.4. Delivery of dAbs by Lactobacillus could provide passive transfer of these antibodies to the mucosa and longevity at the site of HIV-1 transmission. PMID:26950606

  8. Rational Design and Characterization of the Novel, Broad and Potent Bispecific HIV-1 Neutralizing Antibody iMabm36

    Science.gov (United States)

    Sun, Ming; Pace, Craig S.; Yao, Xin; Yu, Faye; Padte, Neal N.; Huang, Yaoxing; Seaman, Michael S.; Li, Qihan; Ho, David D.

    2014-01-01

    While broadly neutralizing monoclonal antibodies (bNAbs) have always been considered potential therapeutic options for the prophylactic and treatment of HIV infection, their lack of breadth against all HIV variants has been one of the limiting factors. To provide sufficient neutralization breadth and potency against diverse viruses, including neutralization escape variants, strategies to combine different bNAbs have been explored recently. We rationally designed and engineered a novel bispecific HIV-1 neutralizing antibody (bibNAb), iMabm36, for high potency and breadth against HIV. iMabm36 is composed of the anti-CD4 Ab ibalizumab (iMab) linked to two copies of the single-domain Ab m36 which targets a highly conserved CD4-induced epitope. iMabm36 neutralizes a majority of a large, multi-clade panel of pseudoviruses (96%, n=118) at an IC50 concentration of less than 10 μg/mL, with 83% neutralized at an IC50 concentration of less than 0.1μg/ml. In addition, iMabm36 neutralizes six replication-competent transmitted-founder viruses to 100% inhibition at a concentration of less than 0.1μg/ml in a PBMC-based neutralizing assay. Mechanistically, improved antiviral activity of iMabm36 is dependent on both CD4 binding activity of iMab component and CD4i binding activity of the m36 component. After characterizing viral resistance to iMabm36 neutralization was due to mutations residing in the bridging sheet of gp120, an optimized m36 variant was engineered that, when fused to iMab, improved antiviral activity significantly. Together inter-dependency of this dual mechanism of action enables iMabm36 to potently inhibit HIV-1 entry. These results demonstrate that mechanistic-based design of bibNAbs could generate potential preventive and therapeutic candidates for HIV/AIDS. PMID:24853313

  9. Direct administration in the respiratory tract improves efficacy of broadly neutralizing anti-influenza virus monoclonal antibodies.

    Science.gov (United States)

    Leyva-Grado, Victor H; Tan, Gene S; Leon, Paul E; Yondola, Mark; Palese, Peter

    2015-07-01

    The emergence of influenza virus strains resistant to approved neuraminidase inhibitors and the time constrains after infection when these drugs can be effective constitute major drawbacks for this class of drugs. This highlights a critical need to discover new therapeutic agents that can be used for the treatment of influenza virus-infected patients. The use of broadly neutralizing anti-influenza monoclonal antibodies (MAbs) has been sought as an alternative immunotherapy against influenza infection. Here, we tested in mice previously characterized broadly neutralizing anti-hemagglutinin (HA) stalk MAbs prophylactically and therapeutically using different routes of administration. The efficacy of treatment against an influenza H1N1 pandemic virus challenge was compared between two systemic routes of administration, intraperitoneal (i.p.) and intravenous (i.v.), and two local routes, intranasal (i.n.) and aerosol (a.e.). The dose of MAb required for prophylactic protection was reduced by 10-fold in animals treated locally (i.n. or a.e.) compared with those treated systemically (i.p. or i.v.). Improved therapeutic protection was observed in animals treated i.n. on day 5 postinfection (60% survival) compared with those treated via the i.p. route (20% survival). An increase in therapeutic efficacy against other influenza virus subtypes (H5N1) was also observed when a local route of administration was used. Our findings demonstrate that local administration significantly decreases the amount of broadly neutralizing monoclonal antibody required for protection against influenza, which highlights the potential use of MAbs as a therapeutic agent for influenza-associated disease.

  10. Immune System Regulation in the Induction of Broadly Neutralizing HIV-1 Antibodies

    Directory of Open Access Journals (Sweden)

    Garnett Kelsoe

    2013-12-01

    Full Text Available In this brief review, we discuss immune tolerance as a factor that determines the magnitude and quality of serum antibody responses to HIV-1 infection and vaccination in the context of recent work. We propose that many conserved, neutralizing epitopes of HIV-1 are weakly immunogenic because they mimic host antigens. In consequence, B cells that strongly bind these determinants are removed by the physiological process of immune tolerance. This structural mimicry may represent a significant impediment to designing protective HIV-1 vaccines, but we note that several vaccine strategies may be able to mitigate this evolutionary adaptation of HIV and other microbial pathogens.

  11. A constant threat for HIV: Fc-engineering to enhance broadly neutralizing antibody activity for immunotherapy of the acquired immunodeficiency syndrome.

    Science.gov (United States)

    Nimmerjahn, Falk

    2015-08-01

    Passive immunotherapy with polyclonal or hyperimmune serum immunoglobulin G (IgG) preparations provides an efficient means of protecting immunocompromised patients from microbial infections. More recently, the use of passive immunotherapy to prevent or to treat established infections with the human immunodeficiency virus (HIV) has gained much attention, due to promising preclinical data obtained in monkey and humanized mouse in vivo model systems, demonstrating that the transfer of HIV-specific antibodies can not only prevent HIV infection, but also diminish virus load during chronic infection. Furthermore, an array of broadly neutralizing HIV-specific antibodies has become available and the importance of the IgG constant region as a critical modulator of broadly neutralizing activity has been demonstrated. The aim of this review is to summarize the most recent findings with regard to the molecular and cellular mechanisms responsible for antibody-mediated clearance of HIV infection, and to discuss how this may help to improve HIV therapy via optimizing Fcγ-receptor-dependent activities of HIV-specific antibodies.

  12. Crystallographic Identification of Lipid as an Integral Component of the Epitope of HIV Broadly Neutralizing Antibody 4E10.

    Science.gov (United States)

    Irimia, Adriana; Sarkar, Anita; Stanfield, Robyn L; Wilson, Ian A

    2016-01-19

    Numerous studies of the anti-HIV-1 envelope glycoprotein 41 (gp41) broadly neutralizing antibody 4E10 suggest that 4E10 also interacts with membrane lipids, but the antibody regions contacting lipids and its orientation with respect to the viral membrane are unknown. Vaccine immunogens capable of re-eliciting these membrane proximal external region (MPER)-like antibodies may require a lipid component to be successful. We performed a systematic crystallographic study of lipid binding to 4E10 to identify lipids bound by the antibody and the lipid-interacting regions. We identified phosphatidic acid, phosphatidylglycerol, and glycerol phosphate as specific ligands for 4E10 in the crystal structures. 4E10 used its CDRH1 loop to bind the lipid head groups, while its CDRH3 interacted with the hydrophobic lipid tails. Identification of the lipid binding sites on 4E10 may aid design of immunogens for vaccines that include a lipid component in addition to the MPER on gp41 for generation of broadly neutralizing antibodies.

  13. The location of the broad HI absorption in 3C305: clear evidence for a jet-accelerated neutral outflow

    CERN Document Server

    Morganti, R; Tadhunter, C N; Van Moorsel, G; Emonts, B

    2005-01-01

    We present high-spatial resolution 21-cm HI VLA observations of the radio galaxy 3C305 (z=0.041). These new high-resolution data show that the ~1000 km/s broad HI absorption, earlier detected in low-resolution WSRT observations, is occurring against the bright, eastern radio lobe, about 1.6 kpc from the nucleus. We use new optical spectra taken with the WHT to make a detailed comparison of the kinematics of the neutral hydrogen with that of the ionised gas. The striking similarity between the complex kinematics of the two gas phases suggests that both the ionised gas and the neutral gas are part of the same outflow. Earlier studies of the ionised gas had already found evidence for a strong interaction between the radio jet and the interstellar medium at the location of the eastern radio lobe. Our results show that the fast outflow produced by this interaction also contains a component of neutral atomic hydrogen. The most likely interpretation is that the radio jet ionises the ISM and accelerates it to the hig...

  14. Protection against lethal enterovirus 71 challenge in mice by a recombinant vaccine candidate containing a broadly cross-neutralizing epitope within the VP2 EF loop.

    Science.gov (United States)

    Xu, Longfa; He, Delei; Li, Zhiqun; Zheng, Jun; Yang, Lisheng; Yu, Miao; Yu, Hai; Chen, Yixin; Que, Yuqiong; Shih, James Wai Kuo; Liu, Gang; Zhang, Jun; Zhao, Qinjian; Cheng, Tong; Xia, Ningshao

    2014-01-01

    Human enterovirus 71 (EV71) is the main causative agent of hand, foot, and mouth disease (HFMD) and is associated with several severe neurological complications in the Asia-Pacific region. Here, we evaluated that while passive transfer of neutralizing monoclonal antibody (nMAb) against the VP2 protein protect against lethal EV71 infection in BALB/c mice. Protective nMAb were mapped to residues 141-155 of VP2 by peptide ELISA. High-resolution structural analysis showed that the epitope is part of the VP2 EF loop, which is the "puff" region that forms the "southern rim" of the canyon. Moreover, a three-dimensional structural characterization for the puff region with prior neutralizing epitopes and receptor-binding sites that can serve to inform vaccine strategies. Interestingly, using hepatitis B virus core protein (HBc) as a carrier, we demonstrated that the cross-neutralizing EV71 antibodies were induced, and the VP2 epitope immunized mice serum also conferred 100% in vivo passive protection. The mechanism of in vivo protection conferred by VP2 nMAb is in part attributed to the in vitro neutralizing titer and ability to bind authentic viral particles. Importantly, the anti-VP2(aa141-155) antibodies could inhibit the binding of human serum to EV71 virions showed that the VP2 epitope is immunodominant. Collectively, our results suggest that a broad-spectrum vaccine strategy targeting the high-affinity epitope of VP2 EF loop may elicits effective immune responses against EV71 infection.

  15. DNA prime and virus-like particle boost from a single H5N1 strain elicits broadly neutralizing antibody responses against head region of H5 hemagglutinin.

    Science.gov (United States)

    Wang, Guiqin; Zhou, Fan; Buchy, Philippe; Zuo, Teng; Hu, Hongxing; Liu, Jingjing; Song, Yufeng; Ding, Heng; Tsai, Cheguo; Chen, Ze; Zhang, Linqi; Deubel, Vincent; Zhou, Paul

    2014-03-01

    Since 1996, highly pathogenic avian influenza (HPAI) H5N1 virus has presented a persistent threat to public health. Its high degree of genetic diversity also poses enormous challenges in developing effective vaccines. To search for vaccine regimens that could elicit broadly neutralizing antibody responses against diverse HPAI H5N1 strains, in the present study we tested H5 hemagglutinin (HA) from an A/Thailand/1(KAN)-1/2004 strain in a heterologous prime-boost vaccination. We demonstrated that priming mice with DNA and boosting with virus-like particle induced antibody responses that cross-neutralize all reported clades and subclades of HPAI H5N1 viruses and protect mice from high lethal dose HPAI H5N1 challenge in both active and passive immunizations. Unexpectedly, cross-divergent H5 neutralizing antibodies are directed to the HA head and block both attachment and postattachment of virus entry. Thus, we conclude that as a promising pan-H5 vaccine candidate this prime-boost regimen could be further developed in ferrets and in humans.

  16. Neutralization resistance of hepatitis C virus can be overcome by recombinant human monoclonal antibodies

    DEFF Research Database (Denmark)

    Pedersen, Jannie L; Carlsen, Thomas H R; Prentoe, Jannick

    2013-01-01

    Immunotherapy and vaccine development for hepatitis C virus (HCV) will depend on broadly reactive neutralizing antibodies (NAbs). However, studies in infectious strain JFH1-based culture systems expressing patient-derived Core-NS2 proteins have suggested neutralization resistance for specific HCV...... strains, in particular, of genotype 2. To further examine this phenomenon, we developed a panel of HCV genotype 2 recombinants for testing of sensitivity to neutralization by chronic-phase patient sera and lead human monoclonal antibodies (HMAbs). The novel Core-NS2 recombinants, with patient......-derived genotype 2a (strain T9), 2b (strains DH8 and DH10), and 2c (strain S83) consensus sequences, were viable in Huh7.5 hepatoma cells without requirement for adaptive mutations, reaching HCV infectivity titers of 3.9-4.5 log10 focus-forming units per milliliter. In in vitro neutralization assays, we...

  17. Testing the Neutral Theory of Biodiversity with Human Microbiome Datasets.

    Science.gov (United States)

    Li, Lianwei; Ma, Zhanshan Sam

    2016-08-16

    The human microbiome project (HMP) has made it possible to test important ecological theories for arguably the most important ecosystem to human health-the human microbiome. Existing limited number of studies have reported conflicting evidence in the case of the neutral theory; the present study aims to comprehensively test the neutral theory with extensive HMP datasets covering all five major body sites inhabited by the human microbiome. Utilizing 7437 datasets of bacterial community samples, we discovered that only 49 communities (less than 1%) satisfied the neutral theory, and concluded that human microbial communities are not neutral in general. The 49 positive cases, although only a tiny minority, do demonstrate the existence of neutral processes. We realize that the traditional doctrine of microbial biogeography "Everything is everywhere, but the environment selects" first proposed by Baas-Becking resolves the apparent contradiction. The first part of Baas-Becking doctrine states that microbes are not dispersal-limited and therefore are neutral prone, and the second part reiterates that the freely dispersed microbes must endure selection by the environment. Therefore, in most cases, it is the host environment that ultimately shapes the community assembly and tip the human microbiome to niche regime.

  18. 9G4 autoreactivity is increased in HIV-infected patients and correlates with HIV broadly neutralizing serum activity.

    Directory of Open Access Journals (Sweden)

    James J Kobie

    Full Text Available The induction of a broadly neutralizing antibody (BNAb response against HIV-1 would be a desirable feature of a protective vaccine. Vaccine strategies thus far have failed to elicit broadly neutralizing antibody responses; however a minority of HIV-infected patients do develop circulating BNAbs, from which several potent broadly neutralizing monoclonal antibodies (mAbs have been isolated. The findings that several BNmAbs exhibit autoreactivity and that autoreactive serum antibodies are observed in some HIV patients have advanced the possibility that enforcement of self-tolerance may contribute to the rarity of BNAbs. To examine the possible breakdown of tolerance in HIV patients, we utilized the 9G4 anti-idiotype antibody system, enabling resolution of both autoreactive VH4-34 gene-expressing B cells and serum antibodies. Compared with healthy controls, HIV patients had significantly elevated 9G4+ serum IgG antibody concentrations and frequencies of 9G4+ B cells, a finding characteristic of systemic lupus erythematosus (SLE patients, both of which positively correlated with HIV viral load. Compared to the global 9G4-IgD--memory B cell population, the 9G4+IgD--memory fraction in HIV patients was dominated by isotype switched IgG+ B cells, but had a more prominent bias toward "IgM only" memory. HIV envelope reactivity was observed both in the 9G4+ serum antibody and 9G4+ B cell population. 9G4+ IgG serum antibody levels positively correlated (r = 0.403, p = 0.0019 with the serum HIV BNAbs. Interestingly, other serum autoantibodies commonly found in SLE (anti-dsDNA, ANA, anti-CL did not correlate with serum HIV BNAbs. 9G4-associated autoreactivity is preferentially expanded in chronic HIV infection as compared to other SLE autoreactivities. Therefore, the 9G4 system provides an effective tool to examine autoreactivity in HIV patients. Our results suggest that the development of HIV BNAbs is not merely a consequence of a general breakdown in

  19. Binding of inferred germline precursors of broadly neutralizing HIV-1 antibodies to native-like envelope trimers.

    Science.gov (United States)

    Sliepen, Kwinten; Medina-Ramírez, Max; Yasmeen, Anila; Moore, John P; Klasse, Per Johan; Sanders, Rogier W

    2015-12-01

    HIV-1 envelope glycoproteins (Env) and Env-based immunogens usually do not interact efficiently with the inferred germline precursors of known broadly neutralizing antibodies (bNAbs). This deficiency may be one reason why Env and Env-based immunogens are not efficient at inducing bNAbs. We evaluated the binding of 15 inferred germline precursors of bNAbs directed to different epitope clusters to three soluble native-like SOSIP.664 Env trimers. We found that native-like SOSIP.664 trimers bind to some inferred germline precursors of bNAbs, particularly ones involving the V1/V2 loops at the apex of the trimer. The data imply that native-like SOSIP.664 trimers will be an appropriate platform for structure-guided design improvements intended to create immunogens able to target the germline precursors of bNAbs.

  20. A pan-HPV vaccine based on bacteriophage PP7 VLPs displaying broadly cross-neutralizing epitopes from the HPV minor capsid protein, L2.

    Directory of Open Access Journals (Sweden)

    Ebenezer Tumban

    Full Text Available BACKGROUND: Current human papillomavirus (HPV vaccines that are based on virus-like particles (VLPs of the major capsid protein L1 largely elicit HPV type-specific antibody responses. In contrast, immunization with the HPV minor capsid protein L2 elicits antibodies that are broadly cross-neutralizing, suggesting that a vaccine targeting L2 could provide more comprehensive protection against infection by diverse HPV types. However, L2-based immunogens typically elicit much lower neutralizing antibody titers than L1 VLPs. We previously showed that a conserved broadly neutralizing epitope near the N-terminus of L2 is highly immunogenic when displayed on the surface of VLPs derived from the bacteriophage PP7. Here, we report the development of a panel of PP7 VLP-based vaccines targeting L2 that protect mice from infection with carcinogenic and non-carcinogenic HPV types that infect the genital tract and skin. METHODOLOGY/PRINCIPAL FINDINGS: L2 peptides from eight different HPV types were displayed on the surface of PP7 bacteriophage VLPs. These recombinant L2 VLPs, both individually and in combination, elicited high-titer anti-L2 IgG serum antibodies. Immunized mice were protected from high dose infection with HPV pseudovirus (PsV encapsidating a luciferase reporter. Mice immunized with 16L2 PP7 VLPs or 18L2 PP7 VLPs were nearly completely protected from both PsV16 and PsV18 challenge. Mice immunized with the mixture of eight L2 VLPs were strongly protected from genital challenge with PsVs representing eight diverse HPV types and cutaneous challenge with HPV5 PsV. CONCLUSION/SIGNIFICANCE: VLP-display of a cross-neutralizing HPV L2 epitope is an effective approach for inducing high-titer protective neutralizing antibodies and is capable of offering protection from a spectrum of HPVs associated with cervical cancer as well as genital and cutaneous warts.

  1. Mechanism of human antibody-mediated neutralization of Marburg virus.

    Science.gov (United States)

    Flyak, Andrew I; Ilinykh, Philipp A; Murin, Charles D; Garron, Tania; Shen, Xiaoli; Fusco, Marnie L; Hashiguchi, Takao; Bornholdt, Zachary A; Slaughter, James C; Sapparapu, Gopal; Klages, Curtis; Ksiazek, Thomas G; Ward, Andrew B; Saphire, Erica Ollmann; Bukreyev, Alexander; Crowe, James E

    2015-02-26

    The mechanisms by which neutralizing antibodies inhibit Marburg virus (MARV) are not known. We isolated a panel of neutralizing antibodies from a human MARV survivor that bind to MARV glycoprotein (GP) and compete for binding to a single major antigenic site. Remarkably, several of the antibodies also bind to Ebola virus (EBOV) GP. Single-particle EM structures of antibody-GP complexes reveal that all of the neutralizing antibodies bind to MARV GP at or near the predicted region of the receptor-binding site. The presence of the glycan cap or mucin-like domain blocks binding of neutralizing antibodies to EBOV GP, but not to MARV GP. The data suggest that MARV-neutralizing antibodies inhibit virus by binding to infectious virions at the exposed MARV receptor-binding site, revealing a mechanism of filovirus inhibition.

  2. Ontogeny of recognition specificity and functionality for the broadly neutralizing anti-HIV antibody 4E10.

    Directory of Open Access Journals (Sweden)

    Kathryn A K Finton

    2014-09-01

    Full Text Available The process of antibody ontogeny typically improves affinity, on-rate, and thermostability, narrows polyspecificity, and rigidifies the combining site to the conformer optimal for binding from the broader ensemble accessible to the precursor. However, many broadly-neutralizing anti-HIV antibodies incorporate unusual structural elements and recognition specificities or properties that often lead to autoreactivity. The ontogeny of 4E10, an autoreactive antibody with unexpected combining site flexibility, was delineated through structural and biophysical comparisons of the mature antibody with multiple potential precursors. 4E10 gained affinity primarily by off-rate enhancement through a small number of mutations to a highly conserved recognition surface. Controverting the conventional paradigm, the combining site gained flexibility and autoreactivity during ontogeny, while losing thermostability, though polyspecificity was unaffected. Details of the recognition mechanism, including inferred global effects due to 4E10 binding, suggest that neutralization by 4E10 may involve mechanisms beyond simply binding, also requiring the ability of the antibody to induce conformational changes distant from its binding site. 4E10 is, therefore, unlikely to be re-elicited by conventional vaccination strategies.

  3. Broadly Neutralizing Antibodies Display Potential for Prevention of HIV-1 Infection of Mucosal Tissue Superior to That of Nonneutralizing Antibodies.

    Science.gov (United States)

    Cheeseman, Hannah M; Olejniczak, Natalia J; Rogers, Paul M; Evans, Abbey B; King, Deborah F L; Ziprin, Paul; Liao, Hua-Xin; Haynes, Barton F; Shattock, Robin J

    2017-01-01

    Definition of the key parameters mediating effective antibody blocking of HIV-1 acquisition within mucosal tissue may prove critical to effective vaccine development and the prophylactic use of monoclonal antibodies. Although direct antibody-mediated neutralization is highly effective against cell-free virus, antibodies targeting different sites of envelope vulnerability may display differential activity against mucosal infection. Nonneutralizing antibodies (nnAbs) may also impact mucosal transmission events through Fc-gamma receptor (FcγR)-mediated inhibition. In this study, a panel of broadly neutralizing antibodies (bnAbs) and nnAbs, including those associated with protection in the RV144 vaccine trial, were screened for the ability to block HIV-1 acquisition and replication across a range of cellular and mucosal tissue models. Neutralization potency, as determined by the TZM-bl infection assay, did not fully predict activity in mucosal tissue. CD4-binding site (CD4bs)-specific bnAbs, in particular VRC01, were consistent in blocking HIV-1 infection across all cellular and tissue models. Membrane-proximal external region (MPER) (2F5) and outer domain glycan (2G12) bnAbs were also efficient in preventing infection of mucosal tissues, while the protective efficacy of bnAbs targeting V1-V2 glycans (PG9 and PG16) was more variable. In contrast, nnAbs alone and in combinations, while active in a range of cellular assays, were poorly protective against HIV-1 infection of mucosal tissues. These data suggest that tissue resident effector cell numbers and low FcγR expression may limit the potential of nnAbs to prevent establishment of the initial foci of infection. The solid protection provided by specific bnAbs clearly demonstrates their superior potential over that of nonneutralizing antibodies for preventing HIV-1 infection at the mucosal portals of infection.

  4. Extra-epitopic hepatitis C virus polymorphisms confer resistance to broadly neutralizing antibodies by modulating binding to scavenger receptor B1

    Science.gov (United States)

    El-Diwany, Ramy; Mankowski, Madeleine C.; Wasilewski, Lisa N.; Brady, Jillian K.; Snider, Anna E.; Osburn, William O.; Murrell, Ben; Ray, Stuart C.

    2017-01-01

    Broadly-neutralizing monoclonal antibodies (bNAbs) may guide vaccine development for highly variable viruses including hepatitis C virus (HCV), since they target conserved viral epitopes that could serve as vaccine antigens. However, HCV resistance to bNAbs could reduce the efficacy of a vaccine. HC33.4 and AR4A are two of the most potent anti-HCV human bNAbs characterized to date, binding to highly conserved epitopes near the amino- and carboxy-terminus of HCV envelope (E2) protein, respectively. Given their distinct epitopes, it was surprising that these bNAbs showed similar neutralization profiles across a panel of natural HCV isolates, suggesting that some viral polymorphisms may confer resistance to both bNAbs. To investigate this resistance, we developed a large, diverse panel of natural HCV envelope variants and a novel computational method to identify bNAb resistance polymorphisms in envelope proteins (E1 and E2). By measuring neutralization of a panel of HCV pseudoparticles by 10 μg/mL of each bNAb, we identified E1E2 variants with resistance to one or both bNAbs, despite 100% conservation of the AR4A binding epitope across the panel. We discovered polymorphisms outside of either binding epitope that modulate resistance to both bNAbs by altering E2 binding to the HCV co-receptor, scavenger receptor B1 (SR-B1). This study is focused on a mode of neutralization escape not addressed by conventional analysis of epitope conservation, highlighting the contribution of extra-epitopic polymorphisms to bNAb resistance and presenting a novel mechanism by which HCV might persist even in the face of an antibody response targeting multiple conserved epitopes. PMID:28235087

  5. Extra-epitopic hepatitis C virus polymorphisms confer resistance to broadly neutralizing antibodies by modulating binding to scavenger receptor B1.

    Science.gov (United States)

    El-Diwany, Ramy; Cohen, Valerie J; Mankowski, Madeleine C; Wasilewski, Lisa N; Brady, Jillian K; Snider, Anna E; Osburn, William O; Murrell, Ben; Ray, Stuart C; Bailey, Justin R

    2017-02-01

    Broadly-neutralizing monoclonal antibodies (bNAbs) may guide vaccine development for highly variable viruses including hepatitis C virus (HCV), since they target conserved viral epitopes that could serve as vaccine antigens. However, HCV resistance to bNAbs could reduce the efficacy of a vaccine. HC33.4 and AR4A are two of the most potent anti-HCV human bNAbs characterized to date, binding to highly conserved epitopes near the amino- and carboxy-terminus of HCV envelope (E2) protein, respectively. Given their distinct epitopes, it was surprising that these bNAbs showed similar neutralization profiles across a panel of natural HCV isolates, suggesting that some viral polymorphisms may confer resistance to both bNAbs. To investigate this resistance, we developed a large, diverse panel of natural HCV envelope variants and a novel computational method to identify bNAb resistance polymorphisms in envelope proteins (E1 and E2). By measuring neutralization of a panel of HCV pseudoparticles by 10 μg/mL of each bNAb, we identified E1E2 variants with resistance to one or both bNAbs, despite 100% conservation of the AR4A binding epitope across the panel. We discovered polymorphisms outside of either binding epitope that modulate resistance to both bNAbs by altering E2 binding to the HCV co-receptor, scavenger receptor B1 (SR-B1). This study is focused on a mode of neutralization escape not addressed by conventional analysis of epitope conservation, highlighting the contribution of extra-epitopic polymorphisms to bNAb resistance and presenting a novel mechanism by which HCV might persist even in the face of an antibody response targeting multiple conserved epitopes.

  6. sCD4-17b bifunctional protein: Extremely broad and potent neutralization of HIV-1 Env pseudotyped viruses from genetically diverse primary isolates

    Directory of Open Access Journals (Sweden)

    Dey Barna

    2010-02-01

    Full Text Available Abstract Background We previously described a potent recombinant HIV-1 neutralizing protein, sCD4-17b, composed of soluble CD4 attached via a flexible polypeptide linker to an SCFv of the 17b human monoclonal antibody directed against the highly conserved CD4-induced bridging sheet of gp120 involved in coreceptor binding. The sCD4 moiety of the bifunctional protein binds to gp120 on free virions, thereby enabling the 17b SCFv moiety to bind and block the gp120/coreceptor interaction required for entry. The previous studies using the MAGI-CCR5 assay system indicated that sCD4-17b (in concentrated cell culture medium, or partially purified potently neutralized several genetically diverse HIIV-1 primary isolates; however, at the concentrations tested it was ineffective against several other strains despite the conservation of binding sites for both CD4 and 17b. To address this puzzle, we designed variants of sCD4-17b with different linker lengths, and tested the neutralizing activities of the immunoaffinity purified proteins over a broader concentration range against a large number of genetically diverse HIV-1 primary isolates, using the TZM-bl Env pseudotype assay system. We also examined the sCD4-17b sensitivities of isogenic viruses generated from different producer cell types. Results We observed that immunoaffinity purified sCD4-17b effectively neutralized HIV-1 pseudotypes, including those from HIV-1 isolates previously found to be relatively insensitive in the MAGI-CCR5 assay. The potencies were equivalent for the original construct and a variant with a longer linker, as observed with both pseudotype particles and infectious virions; by contrast, a construct with a linker too short to enable simultaneous binding of the sCD4 and 17b SCFv moieties was much less effective. sCD4-17b displayed potent neutralizing activity against 100% of nearly 4 dozen HIV-1 primary isolates from diverse genetic subtypes (clades A, B, C, D, F, and circulating

  7. Straightforward selection of broadly neutralizing single-domain antibodies targeting the conserved CD4 and coreceptor binding sites of HIV-1 gp120.

    Science.gov (United States)

    Matz, Julie; Kessler, Pascal; Bouchet, Jérôme; Combes, Olivier; Ramos, Oscar Henrique Pereira; Barin, Francis; Baty, Daniel; Martin, Loïc; Benichou, Serge; Chames, Patrick

    2013-01-01

    Few broadly neutralizing antibodies targeting determinants of the HIV-1 surface envelope glycoprotein (gp120) involved in sequential binding to host CD4 and chemokine receptors have been characterized. While these epitopes show low diversity among various isolates, HIV-1 employs many strategies to evade humoral immune response toward these sensitive sites, including a carbohydrate shield, low accessibility to these buried cavities, and conformational masking. Using trimeric gp140, free or bound to a CD4 mimic, as immunogens in llamas, we selected a panel of broadly neutralizing single-domain antibodies (sdAbs) that bind to either the CD4 or the coreceptor binding site (CD4BS and CoRBS, respectively). When analyzed as monomers or as homo- or heteromultimers, the best sdAb candidates could not only neutralize viruses carrying subtype B envelopes, corresponding to the Env molecule used for immunization and selection, but were also efficient in neutralizing a broad panel of envelopes from subtypes A, C, G, CRF01_AE, and CRF02_AG, including tier 3 viruses. Interestingly, sdAb multimers exhibited a broader neutralizing activity spectrum than the parental sdAb monomers. The extreme stability and high recombinant production yield combined with their broad neutralization capacity make these sdAbs new potential microbicide candidates for HIV-1 transmission prevention.

  8. Broad epitope coverage of a human in vitro antibody library

    Science.gov (United States)

    Sivasubramanian, Arvind; Lynaugh, Heather; Yu, Yao; Miles, Adam; Eckman, Josh; Schutz, Kevin; Piffath, Crystal; Boland, Nadthakarn; Durand, Stéphanie; Boland, Todd; Vásquez, Maximiliano; Xu, Yingda; Abdiche, Yasmina

    2017-01-01

    ABSTRACT Successful discovery of therapeutic antibodies hinges on the identification of appropriate affinity binders targeting a diversity of molecular epitopes presented by the antigen. Antibody campaigns that yield such broad “epitope coverage” increase the likelihood of identifying candidates with the desired biological functions. Accordingly, epitope binning assays are employed in the early discovery stages to partition antibodies into epitope families or “bins” and prioritize leads for further characterization and optimization. The collaborative program described here, which used hen egg white lysozyme (HEL) as a model antigen, combined 3 key capabilities: 1) access to a diverse panel of antibodies selected from a human in vitro antibody library; 2) application of state-of-the-art high-throughput epitope binning; and 3) analysis and interpretation of the epitope binning data with reference to an exhaustive set of published antibody:HEL co-crystal structures. Binning experiments on a large merged panel of antibodies containing clones from the library and the literature revealed that the inferred epitopes for the library clones overlapped with, and extended beyond, the known structural epitopes. Our analysis revealed that nearly the entire solvent-exposed surface of HEL is antigenic, as has been proposed for protein antigens in general. The data further demonstrated that synthetic antibody repertoires provide as wide epitope coverage as those obtained from animal immunizations. The work highlights molecular insights contributed by increasingly higher-throughput binning methods and their broad utility to guide the discovery of therapeutic antibodies representing a diverse set of functional epitopes. PMID:27748644

  9. Broadly Neutralizing Antibodies Display Potential for Prevention of HIV-1 Infection of Mucosal Tissue Superior to That of Nonneutralizing Antibodies

    Science.gov (United States)

    Cheeseman, Hannah M.; Olejniczak, Natalia J.; Rogers, Paul M.; Evans, Abbey B.; King, Deborah F. L.; Ziprin, Paul; Liao, Hua-Xin; Haynes, Barton F.

    2016-01-01

    ABSTRACT Definition of the key parameters mediating effective antibody blocking of HIV-1 acquisition within mucosal tissue may prove critical to effective vaccine development and the prophylactic use of monoclonal antibodies. Although direct antibody-mediated neutralization is highly effective against cell-free virus, antibodies targeting different sites of envelope vulnerability may display differential activity against mucosal infection. Nonneutralizing antibodies (nnAbs) may also impact mucosal transmission events through Fc-gamma receptor (FcγR)-mediated inhibition. In this study, a panel of broadly neutralizing antibodies (bnAbs) and nnAbs, including those associated with protection in the RV144 vaccine trial, were screened for the ability to block HIV-1 acquisition and replication across a range of cellular and mucosal tissue models. Neutralization potency, as determined by the TZM-bl infection assay, did not fully predict activity in mucosal tissue. CD4-binding site (CD4bs)-specific bnAbs, in particular VRC01, were consistent in blocking HIV-1 infection across all cellular and tissue models. Membrane-proximal external region (MPER) (2F5) and outer domain glycan (2G12) bnAbs were also efficient in preventing infection of mucosal tissues, while the protective efficacy of bnAbs targeting V1-V2 glycans (PG9 and PG16) was more variable. In contrast, nnAbs alone and in combinations, while active in a range of cellular assays, were poorly protective against HIV-1 infection of mucosal tissues. These data suggest that tissue resident effector cell numbers and low FcγR expression may limit the potential of nnAbs to prevent establishment of the initial foci of infection. The solid protection provided by specific bnAbs clearly demonstrates their superior potential over that of nonneutralizing antibodies for preventing HIV-1 infection at the mucosal portals of infection. IMPORTANCE Key parameters mediating effective antibody blocking of HIV-1 acquisition within mucosal

  10. Structure of HIV-1 gp120 V1/V2 domain with broadly neutralizing antibody PG9

    Energy Technology Data Exchange (ETDEWEB)

    McLellan, Jason S.; Pancera, Marie; Carrico, Chris; Gorman, Jason; Julien, Jean-Philippe; Khayat, Reza; Louder, Robert; Pejchal, Robert; Sastry, Mallika; Dai, Kaifan; O’Dell, Sijy; Patel, Nikita; Shahzad-ul-Hussan, Syed; Yang, Yongping; Zhang, Baoshan; Zhou, Tongqing; Zhu, Jiang; Boyington, Jeffrey C.; Chuang, Gwo-Yu; Diwanji, Devan; Georgiev, Ivelin; Kwon, Young Do; Lee, Doyung; Louder, Mark K.; Moquin, Stephanie; Schmidt, Stephen D.; Yang, Zhi-Yong; Bonsignori, Mattia; Crump, John A.; Kapiga, Saidi H.; Sam, Noel E.; Haynes, Barton F.; Burton, Dennis R.; Koff, Wayne C.; Walker, Laura M.; Phogat, Sanjay; Wyatt, Richard; Orwenyo, Jared; Wang, Lai-Xi; Arthos, James; Bewley, Carole A.; Mascola, John R.; Nabel, Gary J.; Schief, William R.; Ward, Andrew B.; Wilson, Ian A.; Kwong, Peter D. (UWASH); (NIH); (Scripps); (Duke); (IAVI); (Maryland-MED)

    2012-12-13

    Variable regions 1 and 2 (V1/V2) of human immunodeficiency virus-1 (HIV-1) gp120 envelope glycoprotein are critical for viral evasion of antibody neutralization, and are themselves protected by extraordinary sequence diversity and N-linked glycosylation. Human antibodies such as PG9 nonetheless engage V1/V2 and neutralize 80% of HIV-1 isolates. Here we report the structure of V1/V2 in complex with PG9. V1/V2 forms a four-stranded {beta}-sheet domain, in which sequence diversity and glycosylation are largely segregated to strand-connecting loops. PG9 recognition involves electrostatic, sequence-independent and glycan interactions: the latter account for over half the interactive surface but are of sufficiently weak affinity to avoid autoreactivity. The structures of V1/V2-directed antibodies CH04 and PGT145 indicate that they share a common mode of glycan penetration by extended anionic loops. In addition to structurally defining V1/V2, the results thus identify a paradigm of antibody recognition for highly glycosylated antigens, which - with PG9 - involves a site of vulnerability comprising just two glycans and a strand.

  11. Broadly Neutralizing Hemagglutinin Stalk-Specific Antibodies Induce Potent Phagocytosis of Immune Complexes by Neutrophils in an Fc-Dependent Manner

    Directory of Open Access Journals (Sweden)

    Caitlin E. Mullarkey

    2016-10-01

    Full Text Available Broadly neutralizing antibodies that recognize the conserved hemagglutinin (HA stalk have emerged as exciting new biotherapeutic tools to combat seasonal and pandemic influenza viruses. Our general understanding of the mechanisms by which stalk-specific antibodies achieve protection is rapidly evolving. It has recently been demonstrated that broadly neutralizing HA stalk-specific IgG antibodies require Fc-Fcγ receptor (FcγR interactions for optimal protection in vivo. Here we examine the neutrophil effector functions induced by stalk-specific antibodies. As the most abundant subset of blood leukocytes, neutrophils represent a critical innate effector cell population and serve an instrumental role in orchestrating downstream adaptive responses to influenza virus infection. Yet, the interplay of HA stalk-specific IgG, Fc-FcγR engagement, and neutrophils has remained largely uncharacterized. Using an in vitro assay to detect the production of reactive oxygen species (ROS, we show that human and mouse monoclonal HA stalk-specific IgG antibodies are able to induce the production of ROS by neutrophils, while HA head-specific antibodies do not. Furthermore, our results indicate that the production of ROS is dependent on Fc receptor (FcR engagement and phagocytosis. We went on to assess the ability of monoclonal HA stalk-specific IgA antibodies to induce ROS. Consistent with our findings for monoclonal IgGs, only HA stalk-specific IgA antibodies elicited ROS production by neutrophils. This induction is dependent on the engagement of FcαR1. Taken together, our findings describe a novel FcR-dependent effector function induced by HA stalk-specific IgG and IgA antibodies, and importantly, our studies shed light on the mechanisms by which HA stalk-specific antibodies achieve protection.

  12. Broadly Neutralizing Hemagglutinin Stalk-Specific Antibodies Induce Potent Phagocytosis of Immune Complexes by Neutrophils in an Fc-Dependent Manner.

    Science.gov (United States)

    Mullarkey, Caitlin E; Bailey, Mark J; Golubeva, Diana A; Tan, Gene S; Nachbagauer, Raffael; He, Wenqian; Novakowski, Kyle E; Bowdish, Dawn M; Miller, Matthew S; Palese, Peter

    2016-10-04

    Broadly neutralizing antibodies that recognize the conserved hemagglutinin (HA) stalk have emerged as exciting new biotherapeutic tools to combat seasonal and pandemic influenza viruses. Our general understanding of the mechanisms by which stalk-specific antibodies achieve protection is rapidly evolving. It has recently been demonstrated that broadly neutralizing HA stalk-specific IgG antibodies require Fc-Fcγ receptor (FcγR) interactions for optimal protection in vivo Here we examine the neutrophil effector functions induced by stalk-specific antibodies. As the most abundant subset of blood leukocytes, neutrophils represent a critical innate effector cell population and serve an instrumental role in orchestrating downstream adaptive responses to influenza virus infection. Yet, the interplay of HA stalk-specific IgG, Fc-FcγR engagement, and neutrophils has remained largely uncharacterized. Using an in vitro assay to detect the production of reactive oxygen species (ROS), we show that human and mouse monoclonal HA stalk-specific IgG antibodies are able to induce the production of ROS by neutrophils, while HA head-specific antibodies do not. Furthermore, our results indicate that the production of ROS is dependent on Fc receptor (FcR) engagement and phagocytosis. We went on to assess the ability of monoclonal HA stalk-specific IgA antibodies to induce ROS. Consistent with our findings for monoclonal IgGs, only HA stalk-specific IgA antibodies elicited ROS production by neutrophils. This induction is dependent on the engagement of FcαR1. Taken together, our findings describe a novel FcR-dependent effector function induced by HA stalk-specific IgG and IgA antibodies, and importantly, our studies shed light on the mechanisms by which HA stalk-specific antibodies achieve protection.

  13. Transcriptomic insights into human brain evolution: acceleration, neutrality, heterochrony.

    Science.gov (United States)

    Somel, Mehmet; Rohlfs, Rori; Liu, Xiling

    2014-12-01

    Primate brain transcriptome comparisons within the last 12 years have yielded interesting but contradictory observations on how the transcriptome evolves, and its adaptive role in human cognitive evolution. Since the human-chimpanzee common ancestor, the human prefrontal cortex transcriptome seems to have evolved more than that of the chimpanzee. But at the same time, most expression differences among species, especially those observed in adults, appear as consequences of neutral evolution at cis-regulatory sites. Adaptive expression changes in the human brain may be rare events involving timing shifts, or heterochrony, in specific neurodevelopmental processes. Disentangling adaptive and neutral expression changes, and associating these with human-specific features of the brain require improved methods, comparisons across more species, and further work on comparative development.

  14. Drift of the HIV-1 envelope glycoprotein gp120 toward increased neutralization resistance over the course of the epidemic: a comprehensive study using the most potent and broadly neutralizing monoclonal antibodies.

    Science.gov (United States)

    Bouvin-Pley, M; Morgand, M; Meyer, L; Goujard, C; Moreau, A; Mouquet, H; Nussenzweig, M; Pace, C; Ho, D; Bjorkman, P J; Baty, D; Chames, P; Pancera, M; Kwong, P D; Poignard, P; Barin, F; Braibant, M

    2014-12-01

    Extending our previous analyses to the most recently described monoclonal broadly neutralizing antibodies (bNAbs), we confirmed a drift of HIV-1 clade B variants over 2 decades toward higher resistance to bNAbs targeting almost all the identified gp120-neutralizing epitopes. In contrast, the sensitivity to bNAbs targeting the gp41 membrane-proximal external region remained stable, suggesting a selective pressure on gp120 preferentially. Despite this evolution, selected combinations of bNAbs remain capable of neutralizing efficiently most of the circulating variants.

  15. Low frequency of broadly neutralizing HIV antibodies during chronic infection even in quaternary epitope targeting antibodies containing large numbers of somatic mutations.

    Science.gov (United States)

    Hicar, Mark D; Chen, Xuemin; Kalams, Spyros A; Sojar, Hakimuddin; Landucci, Gary; Forthal, Donald N; Spearman, Paul; Crowe, James E

    2016-02-01

    Neutralizing antibodies (Abs) are thought to be a critical component of an appropriate HIV vaccine response. It has been proposed that Abs recognizing conformationally dependent quaternary epitopes on the HIV envelope (Env) trimer may be necessary to neutralize diverse HIV strains. A number of recently described broadly neutralizing monoclonal Abs (mAbs) recognize complex and quaternary epitopes. Generally, many such Abs exhibit extensive numbers of somatic mutations and unique structural characteristics. We sought to characterize the native antibody (Ab) response against circulating HIV focusing on such conformational responses, without a prior selection based on neutralization. Using a capture system based on VLPs incorporating cleaved envelope protein, we identified a selection of B cells that produce quaternary epitope targeting Abs (QtAbs). Similar to a number of broadly neutralizing Abs, the Ab genes encoding these QtAbs showed extensive numbers of somatic mutations. However, when expressed as recombinant molecules, these Abs failed to neutralize virus or mediate ADCVI activity. Molecular analysis showed unusually high numbers of mutations in the Ab heavy chain framework 3 region of the variable genes. The analysis suggests that large numbers of somatic mutations occur in Ab genes encoding HIV Abs in chronically infected individuals in a non-directed, stochastic, manner.

  16. Identification and Characterization of a Broadly Cross-Reactive HIV-1 Human Monoclonal Antibody That Binds to Both gp120 and gp41

    NARCIS (Netherlands)

    Zhang, Mei-Yun; Yuan, Tingting; Li, Jingjing; Borges, Andrew Rosa; Watkins, Jennifer D.; Guenaga, Javier; Yang, Zheng; Wang, Yanping; Wilson, Richard; Li, Yuxing; Polonis, Victoria R.; Pincus, Seth H.; Ruprecht, Ruth M.; Dimitrov, Dimiter S.

    2012-01-01

    Identification of broadly cross-reactive HIV-1-neutralizing antibodies (bnAbs) may assist vaccine immunogen design. Here we report a novel human monoclonal antibody (mAb), designated m43, which co-targets the gp120 and gp41 subunits of the HIV-1 envelope glycoprotein (Env). M43 bound to recombinant

  17. B cells from knock-in mice expressing broadly neutralizing HIV antibody b12 carry an innocuous B cell receptor responsive to HIV vaccine candidates.

    Science.gov (United States)

    Ota, Takayuki; Doyle-Cooper, Colleen; Cooper, Anthony B; Doores, Katherine J; Aoki-Ota, Miyo; Le, Khoa; Schief, William R; Wyatt, Richard T; Burton, Dennis R; Nemazee, David

    2013-09-15

    Broadly neutralizing Abs against HIV protect from infection, but their routine elicitation by vaccination has not been achieved. To generate small animal models to test vaccine candidates, we have generated targeted transgenic ("knock-in") mice expressing, in the physiological Ig H and L chain loci, two well-studied broadly neutralizing Abs: 4E10, which interacts with the membrane proximal external region of gp41, and b12, which binds to the CD4 binding site on gp120. 4E10HL mice are described in the companion article (Doyle-Cooper et al., J. Immunol. 191: 3186-3191). In this article, we describe b12 mice. B cells in b12HL mice, in contrast to the case in 4E10 mice, were abundant and essentially monoclonal, retaining the b12 specificity. In cell culture, b12HL B cells responded avidly to HIV envelope gp140 trimers and to BCR ligands. Upon transfer to wild-type recipients, b12HL B cells responded robustly to vaccination with gp140 trimers. Vaccinated b12H mice, although generating abundant precursors and Abs with affinity for Env, were unable to rapidly generate neutralizing Abs, highlighting the importance of developing Ag forms that better focus responses to neutralizing epitopes. The b12HL and b12H mice should be useful in optimizing HIV vaccine candidates to elicit a neutralizing response while avoiding nonprotective specificities.

  18. Neutralization of Botulinum Neurotoxin Type E by a Humanized Antibody

    Directory of Open Access Journals (Sweden)

    Yağmur Derman

    2016-09-01

    Full Text Available Botulinum neurotoxins (BoNTs cause botulism and are the deadliest naturally-occurring substances known to humans. BoNTs have been classified as one of the category A agents by the Centers for Disease Control and Prevention, indicating their potential use as bioweapons. To counter bio-threat and naturally-occurring botulism cases, well-tolerated antibodies by humans that neutralize BoNTs are relevant. In our previous work, we showed the neutralizing potential of macaque (Macaca fascicularis-derived scFv-Fc (scFv-Fc ELC18 by in vitro endopeptidase immunoassay and ex vivo mouse phrenic nerve-hemidiaphragm assay by targeting the light chain of the botulinum neurotoxin type E (BoNT/E. In the present study, we germline-humanized scFv-Fc ELC18 into a full IgG hu8ELC18 to increase its immunotolerance by humans. We demonstrated the protection and prophylaxis capacity of hu8ELC18 against BoNT/E in a mouse model. A concentration of 2.5 ng/mouse of hu8ELC18 protected against 5 mouse lethal dose (MLD in a mouse protection assay and complete neutralization of 1 LD50 of pure BoNT/E toxin was achieved with 8 ng of hu8ELC18 in mouse paralysis assay. Furthermore, hu8ELC18 protected mice from 5 MLD if injected up to 14 days prior to intraperitoneal BoNT/E administration. This newly-developed humanized IgG is expected to have high tolerance in humans.

  19. Neutralization of Botulinum Neurotoxin Type E by a Humanized Antibody

    Science.gov (United States)

    Derman, Yağmur; Selby, Katja; Miethe, Sebastian; Frenzel, André; Liu, Yvonne; Rasetti-Escargueil, Christine; Avril, Arnaud; Pelat, Thibaut; Urbain, Remi; Fontayne, Alexandre; Thullier, Philippe; Sesardic, Dorothea; Lindström, Miia; Hust, Michael; Korkeala, Hannu

    2016-01-01

    Botulinum neurotoxins (BoNTs) cause botulism and are the deadliest naturally-occurring substances known to humans. BoNTs have been classified as one of the category A agents by the Centers for Disease Control and Prevention, indicating their potential use as bioweapons. To counter bio-threat and naturally-occurring botulism cases, well-tolerated antibodies by humans that neutralize BoNTs are relevant. In our previous work, we showed the neutralizing potential of macaque (Macaca fascicularis)-derived scFv-Fc (scFv-Fc ELC18) by in vitro endopeptidase immunoassay and ex vivo mouse phrenic nerve-hemidiaphragm assay by targeting the light chain of the botulinum neurotoxin type E (BoNT/E). In the present study, we germline-humanized scFv-Fc ELC18 into a full IgG hu8ELC18 to increase its immunotolerance by humans. We demonstrated the protection and prophylaxis capacity of hu8ELC18 against BoNT/E in a mouse model. A concentration of 2.5 ng/mouse of hu8ELC18 protected against 5 mouse lethal dose (MLD) in a mouse protection assay and complete neutralization of 1 LD50 of pure BoNT/E toxin was achieved with 8 ng of hu8ELC18 in mouse paralysis assay. Furthermore, hu8ELC18 protected mice from 5 MLD if injected up to 14 days prior to intraperitoneal BoNT/E administration. This newly-developed humanized IgG is expected to have high tolerance in humans. PMID:27626446

  20. Evolution of the Humoral Response during HCV Infection: Theories on the Origin of Broadly Neutralizing Antibodies and Implications for Vaccine Design.

    Science.gov (United States)

    Murira, Armstrong; Lapierre, Pascal; Lamarre, Alain

    2016-01-01

    Similar to human immunodeficiency virus (HIV)-1, vaccine-induced elicitation of broadly neutralizing (bNt) antibodies (Abs) is gaining traction as a key goal toward the eradication of the hepatitis C virus (HCV) pandemic. Previously, the significance of the Ab response against HCV was underappreciated given the prevailing evidence advancing the role of the cellular immune response in clearance and overall control of the infection. However, recent findings have driven growing interest in the humoral arm of the immune response and in particular the role of bNt responses due to their ability to confer protective immunity upon passive transfer in animal models. Nevertheless, the origin and development of bNt Abs is poorly understood and their occurrence is rare as well as delayed with emergence only observed in the chronic phase of infection. In this review, we characterize the interplay between the host immune response and HCV as it progresses from the acute to chronic phase of infection. In addition, we place these events in the context of current hypotheses on the origin of bNt Abs against the HIV-1, whose humoral immune response is better characterized. Based on the increasing significance of the humoral immune response against HCV, characterization of these events may be critical in understanding the development of the bNt responses and, thus, provide strategies toward effective vaccine design.

  1. Recombinant human Fab fragments neutralize human type 1 immunodeficiency virus in vitro.

    Science.gov (United States)

    Barbas, C F; Björling, E; Chiodi, F; Dunlop, N; Cababa, D; Jones, T M; Zebedee, S L; Persson, M A; Nara, P L; Norrby, E

    1992-01-01

    A panel of 20 recombinant Fab fragments reactive with the surface glycoprotein gp120 of human type 1 immunodeficiency virus (HIV-1) were examined for their ability to neutralize MN and IIIB strains of the virus. Neutralization was determined as the ability of the Fab fragments to inhibit infection as measured in both a p24 ELISA and a syncytium-formation assay. One group of closely sequence-related Fab fragments was found to neutralize virus in both assays with a 50% neutralization titer at approximately 1 micrograms/ml. Another Fab neutralized in the p24 ELISA but not in the syncytium assay. The other Fab fragments showed weak or no neutralizing ability. The results imply that virion aggregation or crosslinking of gp120 molecules on the virion surface is not an absolute requirement for HIV-1 neutralization. Further, all of the Fab fragments were shown to be competitive with soluble CD4 for binding to gp120 and yet few neutralized the virus effectively, implying that the mechanism of neutralization in this case may not involve receptor blocking. The observation of a preponderance of high-affinity Fab fragments with poor or no neutralizing ability could have implications for vaccine strategies. PMID:1384050

  2. Broad antibody mediated cross-neutralization and preclinical immunogenicity of new codon-optimized HIV-1 clade CRF02_AG and G primary isolates.

    Directory of Open Access Journals (Sweden)

    Simon M Agwale

    Full Text Available Creation of an effective vaccine for HIV has been an elusive goal of the scientific community for almost 30 years. Neutralizing antibodies are assumed to be pivotal to the success of a prophylactic vaccine but previous attempts to make an immunogen capable of generating neutralizing antibodies to primary "street strain" isolates have resulted in responses of very limited breadth and potency. The objective of the study was to determine the breadth and strength of neutralizing antibodies against autologous and heterologous primary isolates in a cohort of HIV-1 infected Nigerians and to characterize envelopes from subjects with particularly broad or strong immune responses for possible use as vaccine candidates in regions predominated by HIV-1 CRF02_AG and G subtypes. Envelope vectors from a panel of primary Nigerian isolates were constructed and tested with plasma/sera from the same cohort using the PhenoSense HIV neutralizing antibody assay (Monogram Biosciences Inc, USA to assess the breadth and potency of neutralizing antibodies. The immediate goal of this study was realized by the recognition of three broadly cross-neutralizing sera: (NG2-clade CRF02_AG, NG3-clade CRF02_AG and NG9- clade G. Based on these findings, envelope gp140 sequences from NG2 and NG9, complemented with a gag sequence (Clade G and consensus tat (CRF02_AG and G antigens have been codon-optimized, synthesized, cloned and evaluated in BALB/c mice. The intramuscular administration of these plasmid DNA constructs, followed by two booster DNA immunizations, induced substantial specific humoral response against all constructs and strong cellular responses against the gag and tat constructs. These preclinical findings provide a framework for the design of candidate vaccine for use in regions where the HIV-1 epidemic is driven by clades CRF02_AG and G.

  3. Development of a Coxsackievirus A16 neutralization assay based on pseudoviruses for measurement of neutralizing antibody titer in human serum.

    Science.gov (United States)

    Jin, Jun; Ma, Hongxia; Xu, Lin; An, Dong; Sun, Shiyang; Huang, Xueyong; Kong, Wei; Jiang, Chunlai

    2013-02-01

    Serum neutralizing antibody titers are indicative of protective immunity against Coxsackievirus A16 (CV-A16) and Enterovirus 71 (EV71), the two main etiological agents of hand, foot and mouth disease (HFMD), and provide the basis for evaluating vaccine efficacy. The current CV-A16 neutralization assay based on inhibition of cytopathic effects requires manual microscopic examination, which is time-consuming and labor-intensive. In this study, a high-throughput neutralization assay was developed by employing CV-A16 pseudoviruses expressing luciferase for detecting infectivity in rhabdomyosarcoma (RD) cells and measuring serum viral neutralizing antibodies. Without the need to use infectious CV-A16 strains, the neutralizing antibody titer against CV-A16 could be determined within 15h by measuring luciferase signals by this assay. The pseudovirus CV-A16 neutralization assay (pCNA) was validated by comparison with a conventional CV-A16 neutralization assay (cCNA) in testing 174 human serum samples collected from children (age <5 years). The neutralizing antibody titers determined by these two assays were well correlated (R(2)=0.7689). These results suggest that the pCNA can serve as a rapid and objective procedure for the measurement of neutralizing antibodies against CV-A16.

  4. Preexisting human antibodies neutralize recently emerged H7N9 influenza strains

    Science.gov (United States)

    Henry Dunand, Carole J.; Leon, Paul E.; Kaur, Kaval; Tan, Gene S.; Zheng, Nai-Ying; Andrews, Sarah; Huang, Min; Qu, Xinyan; Huang, Yunping; Salgado-Ferrer, Marlene; Ho, Irvin Y.; Taylor, William; Hai, Rong; Wrammert, Jens; Ahmed, Rafi; García-Sastre, Adolfo; Palese, Peter; Krammer, Florian; Wilson, Patrick C.

    2015-01-01

    The emergence and seasonal persistence of pathogenic H7N9 influenza viruses in China have raised concerns about the pandemic potential of this strain, which, if realized, would have a substantial effect on global health and economies. H7N9 viruses are able to bind to human sialic acid receptors and are also able to develop resistance to neuraminidase inhibitors without a loss in fitness. It is not clear whether prior exposure to circulating human influenza viruses or influenza vaccination confers immunity to H7N9 strains. Here, we demonstrate that 3 of 83 H3 HA-reactive monoclonal antibodies generated by individuals that had previously undergone influenza A virus vaccination were able to neutralize H7N9 viruses and protect mice against homologous challenge. The H7N9-neutralizing antibodies bound to the HA stalk domain but exhibited a difference in their breadth of reactivity to different H7 influenza subtypes. Mapping viral escape mutations suggested that these antibodies bind at least two different epitopes on the stalk region. Together, these results indicate that these broadly neutralizing antibodies may contribute to the development of therapies against H7N9 strains and may also be effective against pathogenic H7 strains that emerge in the future. PMID:25689254

  5. Tenascin-C is an innate broad-spectrum, HIV-1–neutralizing protein in breast milk

    Science.gov (United States)

    Fouda, Genevieve G.; Jaeger, Frederick H.; Amos, Joshua D.; Ho, Carrie; Kunz, Erika L.; Anasti, Kara; Stamper, Lisa W.; Liebl, Brooke E.; Barbas, Kimberly H.; Ohashi, Tomoo; Moseley, Martin Arthur; Liao, Hua-Xin; Erickson, Harold P.; Alam, S. Munir; Permar, Sallie R.

    2013-01-01

    Achieving an AIDS-free generation will require elimination of postnatal transmission of HIV-1 while maintaining the nutritional and immunologic benefits of breastfeeding for infants in developing regions. Maternal/infant antiretroviral prophylaxis can reduce postnatal HIV-1 transmission, yet toxicities and the development of drug-resistant viral strains may limit the effectiveness of this strategy. Interestingly, in the absence of antiretroviral prophylaxis, greater than 90% of infants exposed to HIV-1 via breastfeeding remain uninfected, despite daily mucosal exposure to the virus for up to 2 y. Moreover, milk of uninfected women inherently neutralizes HIV-1 and prevents virus transmission in animal models, yet the factor(s) responsible for this anti-HIV activity is not well-defined. In this report, we identify a primary HIV-1–neutralizing protein in breast milk, Tenascin-C (TNC). TNC is an extracellular matrix protein important in fetal development and wound healing, yet its antimicrobial properties have not previously been established. Purified TNC captured and neutralized multiclade chronic and transmitted/founder HIV-1 variants, and depletion of TNC abolished the HIV-1–neutralizing activity of milk. TNC bound the HIV-1 Envelope protein at a site that is induced upon engagement of its primary receptor, CD4, and is blocked by V3 loop- (19B and F39F) and chemokine coreceptor binding site-directed (17B) monoclonal antibodies. Our results demonstrate the ability of an innate mucosal host protein found in milk to neutralize HIV-1 via binding to the chemokine coreceptor site, potentially explaining why the majority of HIV-1–exposed breastfed infants are protected against mucosal HIV-1 transmission. PMID:24145401

  6. 人免疫缺陷病毒1型广谱中和抗体研究进展%Research Progress on HIV-1 Broadly Neutralizing Antibodies

    Institute of Scientific and Technical Information of China (English)

    范燕峰; 赵娟

    2015-01-01

    人类免疫缺陷病毒1型(HIV-1 )是获得性免疫缺陷综合征的主要致病因素. 目前为止依然缺乏能快速诱导出HIV特异性广谱中和抗体的有效疫苗. 一旦HIV-1潜伏在细胞内,则很难被彻底消除,如何活化潜伏感染的细胞进而进行药物治疗是摆在全世界科学家面前的又一难题. 而用药物控制HIV-1传播策略的贯彻落实也遇到了诸如经济、政策和社会等各方面的困难. 因此,新的预防策略的研究迫在眉睫.%Human immunodeficiency virus type 1 ( HIV-1 ) is the major pathogenic factor of acquired immunodeficiency disease ( AIDS ) .So far, there still has no effective vaccine developed that can rapidly induce broadly neutralizing antibodies.How to activate latently infected cells and then apply drug treatment is another problem faced by scientists around the world,because once the latent HIV-1 is in the cell,it is diffi-cult to be completely eliminated .Besides,the implementation of the strategy of controlling HIV-1 infection by anti-HIV drugs has encountered economic,policy,social and other difficulties.Therefore,researches on new prevention strategies are at urgent needs.

  7. Neutral Theory Predicts the Relative Abundance and Diversity of Genetic Elements in a Broad Array of Eukaryotic Genomes

    Science.gov (United States)

    Serra, François; Becher, Verónica; Dopazo, Hernán

    2013-01-01

    It is universally true in ecological communities, terrestrial or aquatic, temperate or tropical, that some species are very abundant, others are moderately common, and the majority are rare. Likewise, eukaryotic genomes also contain classes or “species” of genetic elements that vary greatly in abundance: DNA transposons, retrotransposons, satellite sequences, simple repeats and their less abundant functional sequences such as RNA or genes. Are the patterns of relative species abundance and diversity similar among ecological communities and genomes? Previous dynamical models of genomic diversity have focused on the selective forces shaping the abundance and diversity of transposable elements (TEs). However, ideally, models of genome dynamics should consider not only TEs, but also the diversity of all genetic classes or “species” populating eukaryotic genomes. Here, in an analysis of the diversity and abundance of genetic elements in >500 eukaryotic chromosomes, we show that the patterns are consistent with a neutral hypothesis of genome assembly in virtually all chromosomes tested. The distributions of relative abundance of genetic elements are quite precisely predicted by the dynamics of an ecological model for which the principle of functional equivalence is the main assumption. We hypothesize that at large temporal scales an overarching neutral or nearly neutral process governs the evolution of abundance and diversity of genetic elements in eukaryotic genomes. PMID:23798991

  8. Structure and function of broadly reactive antibody PG16 reveal an H3 subdomain that mediates potent neutralization of HIV-1

    Energy Technology Data Exchange (ETDEWEB)

    Pejchal, Robert; Walker, Laura M.; Stanfield, Robyn L.; Phogat, Sanjay K.; Koff, Wayne C.; Poignard, Pascal; Burton, Dennis R.; Wilson, Ian A. (Scripps); (IAVI)

    2010-11-15

    Development of an effective vaccine against HIV-1 will likely require elicitation of broad and potent neutralizing antibodies against the trimeric surface envelope glycoprotein (Env). Monoclonal antibodies (mAbs) PG9 and PG16 neutralize {approx}80% of HIV-1 isolates across all clades with extraordinary potency and target novel epitopes preferentially expressed on Env trimers. As these neutralization properties are ideal for a vaccine-elicited antibody response to HIV-1, their structural basis was investigated. The crystal structure of the antigen-binding fragment (Fab) of PG16 at 2.5 {angstrom} resolution revealed its unusually long, 28-residue, complementarity determining region (CDR) H3 forms a unique, stable subdomain that towers above the antibody surface. A 7-residue 'specificity loop' on the 'hammerhead' subdomain was identified that, when transplanted from PG16 to PG9 and vice versa, accounted for differences in the fine specificity and neutralization of these two mAbs. The PG16 electron density maps also revealed that a CDR H3 tyrosine was sulfated, which was confirmed for both PG9 (doubly) and PG16 (singly) by mass spectral analysis. We further showed that tyrosine sulfation plays a role in binding and neutralization. An N-linked glycan modification is observed in the variable light chain, but not required for antigen recognition. Further, the crystal structure of the PG9 light chain at 3.0 {angstrom} facilitated homology modeling to support the presence of these unusual features in PG9. Thus, PG9 and PG16 use unique structural features to mediate potent neutralization of HIV-1 that may be of utility in antibody engineering and for high-affinity recognition of a variety of therapeutic targets.

  9. The location of the broad HI absorption in 3C 305 : clear evidence for a jet-accelerated neutral outflow

    NARCIS (Netherlands)

    Morganti, R; Oosterloo, TA; Tadhunter, CN; van Moorsel, G; Emonts, B

    2005-01-01

    We present high-spatial resolution 21-cm HI VLA observations of the radio galaxy 3C 305 ( z = 0.041). These new high-resolution data show that the similar to 1000 km s(-1) broad H. absorption, earlier detected in low-resolution WSRT observations, is occurring against the bright, eastern radio lobe,

  10. [Neutralizing Monoclonal and Chimeric Antibodies to Human IFN-γ].

    Science.gov (United States)

    Larina, M V; Aliev, T K; Solopova, O N; Pozdnyakova, L P; Korobova, S V; Yakimov, S A; Sveshnikov, P G; Dolgikh, D A; Kirpichnikov, M P

    2015-01-01

    Autoiminune disorders are chronic diseases characterized by abnormal immune response directed against self-antigens that leads to tissue damage and violation of its normal functioning. Such diseases often result in disability or even death of patients. Nowadays a number of monoclonal antibodies to pro-inflammatory cytokines and their receptors are successfully used for the targeted treatment of autoimmune diseases. One of the perspective targets in autoimmune disease therapy is interferon gamma, a key cytokine in Th1 cells differentiation, activation of macrophages, and inflammation. In the present work, 5 monoclonal antibodies to human IFN-γ were obtained. For the development of potential therapeutic agent, we have performed neutralizing activity and affinity analysis of the antibodies. Based on the data obtained, the monoclonal antibody F1 was selected. This antibody has a dissociation constant 1.7 x 10(-9) M and IC90 = 8.9 ± 2.0 nM measured upon antibody inhibition of the IFN-γ-induced HLA-DR expression on the surface of U937 cells. We have constructed a bicistronic vector for the production of recombinant chimeric Fab fragment F1 chim in E. coli cells. The recombinant chimeric Fab fragment Fl chim neutralizes IFN-γ activity in vitro and has a dissociation constant 1.8 x 10(-9) M.

  11. Limited naturally occurring escape in broadly neutralizing antibody epitopes in hepatitis C glycoprotein E2 and constrained sequence usage in acute infection.

    Science.gov (United States)

    Rodrigo, Chaturaka; Walker, Melanie R; Leung, Preston; Eltahla, Auda A; Grebely, Jason; Dore, Gregory J; Applegate, Tanya; Page, Kimberly; Dwivedi, Sunita; Bruneau, Julie; Morris, Meghan D; Cox, Andrea L; Osburn, William; Kim, Arthur Y; Schinkel, Janke; Shoukry, Naglaa H; Lauer, Georg M; Maher, Lisa; Hellard, Margaret; Prins, Maria; Luciani, Fabio; Lloyd, Andrew R; Bull, Rowena A

    2017-04-01

    Broadly neutralizing antibodies have been associated with spontaneous clearance of the hepatitis C infection as well as viral persistence by immune escape. Further study of neutralizing antibody epitopes is needed to unravel pathways of resistance to virus neutralization, and to identify conserved regions for vaccine design. All reported broadly neutralizing antibody (BNAb) epitopes in the HCV Envelope (E2) glycoprotein were identified. The critical contact residues of these epitopes were mapped onto the linear E2 sequence. All publicly available E2 sequences were then downloaded and the contact residues within the BNAb epitopes were assessed for the level of conservation, as well as the frequency of occurrence of experimentally-proven resistance mutations. Epitopes were also compared between two sequence datasets obtained from samples collected at well-defined time points from acute (180days) infections, to identify any significant differences in residue usage. The contact residues for all BNAbs were contained within 3 linear regions of the E2 protein sequence. An analysis of 1749 full length E2 sequences from public databases showed that only 10 out of 29 experimentally-proven resistance mutations were present at a frequency >5%. Comparison of subtype 1a viral sequences obtained from samples collected during acute or chronic infection revealed significant differences at positions 610 and 655 with changes in residue (p<0.05), and at position 422 (p<0.001) with a significant difference in variability (entropy). The majority of experimentally-described escape variants do not occur frequently in nature. The observed differences between acute and chronically isolated sequences suggest constraints on residue usage early in infection.

  12. Broadly cross-reactive antibodies dominate the human B cell response against 2009 pandemic H1N1 influenza virus infection

    OpenAIRE

    2011-01-01

    The 2009 pandemic H1N1 influenza pandemic demonstrated the global health threat of reassortant influenza strains. Herein, we report a detailed analysis of plasmablast and monoclonal antibody responses induced by pandemic H1N1 infection in humans. Unlike antibodies elicited by annual influenza vaccinations, most neutralizing antibodies induced by pandemic H1N1 infection were broadly cross-reactive against epitopes in the hemagglutinin (HA) stalk and head domain of multiple influenza strains. T...

  13. A human monoclonal antibody with neutralizing activity against highly divergent influenza subtypes.

    Directory of Open Access Journals (Sweden)

    Nicola Clementi

    Full Text Available The interest in broad-range anti-influenza A monoclonal antibodies (mAbs has recently been strengthened by the identification of anti-hemagglutinin (HA mAbs endowed with heterosubtypic neutralizing activity to be used in the design of "universal" prophylactic or therapeutic tools. However, the majority of the single mAbs described to date do not bind and neutralize viral isolates belonging to highly divergent subtypes clustering into the two different HA-based influenza phylogenetic groups: the group 1 including, among others, subtypes H1, H2, H5 and H9 and the group 2 including, among others, H3 subtype. Here, we describe a human mAb, named PN-SIA28, capable of binding and neutralizing all tested isolates belonging to phylogenetic group 1, including H1N1, H2N2, H5N1 and H9N2 subtypes and several isolates belonging to group 2, including H3N2 isolates from the first period of the 1968 pandemic. Therefore, PN-SIA28 is capable of neutralizing isolates belonging to subtypes responsible of all the reported pandemics, as well as other subtypes with pandemic potential. The region recognized by PN-SIA28 has been identified on the stem region of HA and includes residues highly conserved among the different influenza subtypes. A deep characterization of PN-SIA28 features may represent a useful help in the improvement of available anti-influenza therapeutic strategies and can provide new tools for the development of universal vaccinal strategies.

  14. Characterization of VRC01, a potent and broadly neutralizing anti-HIV mAb, produced in transiently and stably transformed tobacco.

    Science.gov (United States)

    Teh, Audrey Y-H; Maresch, Daniel; Klein, Katja; Ma, Julian K-C

    2014-04-01

    The proposed clinical trial in Africa of VRC01, a potent broadly neutralizing antibody (bNAb) capable of neutralizing 91% of known HIV-1 isolates, raises concerns about testing a treatment which will be too expensive to be accessible by the most important target population, the poor in under-developed regions such as sub-Saharan Africa. Here, we report the expression of VRC01 in plants as an economic alternative to conventional mammalian-cell-based production platforms. The heavy and light chain genes of VRC01 were cloned onto a single vector, pTRAk.2, which was transformed into Nicotiana benthamiana or Nicotiana tabacum using transient and stable expression production systems respectively. VRC01 has been successfully expressed transiently in plants with expression level of approximately 80 mg antibody/kg; stable transgenic lines expressing up to 100 mg antibody/kg were also obtained. Plant-produced VRC01 from both systems showed a largely homogeneous N-glycosylation profile with a single dominant glycoform. The binding kinetics to gp120 IIIB (approximately 1 nM), neutralization of HIV-1 BaL or a panel of 10 VRC01-sensitive HIV-1 Env pseudoviruses of VRC01 produced in transient and stable plants were also consistent with VRC01 from HEK cells.

  15. Lipid interactions and angle of approach to the HIV-1 viral membrane of broadly neutralizing antibody 10E8: Insights for vaccine and therapeutic design

    Science.gov (United States)

    Irimia, Adriana; Sarkar, Anita; Schiffner, Torben; Tingle, Ryan; Adachi, Yumiko; Deller, Marc C.; Burton, Dennis R.

    2017-01-01

    Among broadly neutralizing antibodies to HIV, 10E8 exhibits greater neutralizing breadth than most. Consequently, this antibody is the focus of prophylactic/therapeutic development. The 10E8 epitope has been identified as the conserved membrane proximal external region (MPER) of gp41 subunit of the envelope (Env) viral glycoprotein and is a major vaccine target. However, the MPER is proximal to the viral membrane and may be laterally inserted into the membrane in the Env prefusion form. Nevertheless, 10E8 has not been reported to have significant lipid-binding reactivity. Here we report x-ray structures of lipid complexes with 10E8 and a scaffolded MPER construct and mutagenesis studies that provide evidence that the 10E8 epitope is composed of both MPER and lipid. 10E8 engages lipids through a specific lipid head group interaction site and a basic and polar surface on the light chain. In the model that we constructed, the MPER would then be essentially perpendicular to the virion membrane during 10E8 neutralization of HIV-1. As the viral membrane likely also plays a role in selecting for the germline antibody as well as size and residue composition of MPER antibody complementarity determining regions, the identification of lipid interaction sites and the MPER orientation with regard to the viral membrane surface during 10E8 engagement can be of great utility for immunogen and therapeutic design. PMID:28225819

  16. Structural basis for broad neutralization of HIV-1 through the molecular recognition of 10E8 helical epitope at the membrane interface

    Science.gov (United States)

    Rujas, Edurne; Caaveiro, Jose M. M.; Partida-Hanon, Angélica; Gulzar, Naveed; Morante, Koldo; Apellániz, Beatriz; García-Porras, Miguel; Bruix, Marta; Tsumoto, Kouhei; Scott, Jamie K.; Jiménez, M. Ángeles; Nieva, José L.

    2016-01-01

    The mechanism by which the HIV-1 MPER epitope is recognized by the potent neutralizing antibody 10E8 at membrane interfaces remains poorly understood. To solve this problem, we have optimized a 10E8 peptide epitope and analyzed the structure and binding activities of the antibody in membrane and membrane-like environments. The X-ray crystal structure of the Fab-peptide complex in detergents revealed for the first time that the epitope of 10E8 comprises a continuous helix spanning the gp41 MPER/transmembrane domain junction (MPER-N-TMD; Env residues 671–687). The MPER-N-TMD helix projects beyond the tip of the heavy-chain complementarity determining region 3 loop, indicating that the antibody sits parallel to the plane of the membrane in binding the native epitope. Biophysical, biochemical and mutational analyses demonstrated that strengthening the affinity of 10E8 for the TMD helix in a membrane environment, correlated with its neutralizing potency. Our research clarifies the molecular mechanisms underlying broad neutralization of HIV-1 by 10E8, and the structure of its natural epitope. The conclusions of our research will guide future vaccine-design strategies targeting MPER. PMID:27905530

  17. Mechanisms of Hemagglutinin Targeted Influenza Virus Neutralization

    NARCIS (Netherlands)

    Brandenburg, Boerries; Koudstaal, Wouter; Goudsmit, Jaap; Klaren, Vincent; Tang, Chan; Bujny, Miriam V.; Korse, Hans J.W.M.; Kwaks, Ted; Otterstrom, Jason J.; Juraszek, Jarek; Oijen, Antoine M. van; Vogels, Ronald; Friesen, Robert H.E.

    2013-01-01

    Human monoclonal antibodies have been identified which neutralize broad spectra of influenza A or B viruses. Here, we dissect the mechanisms by which such antibodies interfere with infectivity. We distinguish four mechanisms that link the conserved hemagglutinin (HA) epitopes of broadly neutralizing

  18. The location of the broad H i absorption in 3C 305: clear evidence for a jet-accelerated neutral outflow

    Science.gov (United States)

    Morganti, R.; Oosterloo, T. A.; Tadhunter, C. N.; van Moorsel, G.; Emonts, B.

    2005-08-01

    We present high-spatial resolution 21-cm H i VLA observations of the radio galaxy 3C 305 (z=0.041). These new high-resolution data show that the ˜ 1000 km s-1 broad H I absorption, earlier detected in low-resolution WSRT observations, is occurring against the bright, eastern radio lobe, about 1.6 kpc from the nucleus. We use new optical spectra taken with the WHT to make a detailed comparison of the kinematics of the neutral hydrogen with that of the ionised gas. The striking similarity between the complex kinematics of the two gas phases suggests that both the ionised gas and the neutral gas are part of the same outflow. Earlier studies of the ionised gas had already found evidence for a strong interaction between the radio jet and the interstellar medium at the location of the eastern radio lobe. Our results show that the fast outflow produced by this interaction also contains a component of neutral atomic hydrogen. The most likely interpretation is that the radio jet ionises the ISM and accelerates it to the high outflow velocities observed. Our observations demonstrate that, following this strong jet-cloud interaction, not all gas clouds are destroyed and that part of the gas can cool and become neutral. The mass outflow rate measured in 3C 305 is comparable, although at the lower end of the distribution, to that found in Ultra-Luminous IR galaxies. This suggests that AGN-driven outflows, and in particular jet-driven outflows, can have a similar impact on the evolution of a galaxy as starburst-driven superwinds.

  19. Segmenting the human genome based on states of neutral genetic divergence.

    Science.gov (United States)

    Kuruppumullage Don, Prabhani; Ananda, Guruprasad; Chiaromonte, Francesca; Makova, Kateryna D

    2013-09-03

    Many studies have demonstrated that divergence levels generated by different mutation types vary and covary across the human genome. To improve our still-incomplete understanding of the mechanistic basis of this phenomenon, we analyze several mutation types simultaneously, anchoring their variation to specific regions of the genome. Using hidden Markov models on insertion, deletion, nucleotide substitution, and microsatellite divergence estimates inferred from human-orangutan alignments of neutrally evolving genomic sequences, we segment the human genome into regions corresponding to different divergence states--each uniquely characterized by specific combinations of divergence levels. We then parsed the mutagenic contributions of various biochemical processes associating divergence states with a broad range of genomic landscape features. We find that high divergence states inhabit guanine- and cytosine (GC)-rich, highly recombining subtelomeric regions; low divergence states cover inner parts of autosomes; chromosome X forms its own state with lowest divergence; and a state of elevated microsatellite mutability is interspersed across the genome. These general trends are mirrored in human diversity data from the 1000 Genomes Project, and departures from them highlight the evolutionary history of primate chromosomes. We also find that genes and noncoding functional marks [annotations from the Encyclopedia of DNA Elements (ENCODE)] are concentrated in high divergence states. Our results provide a powerful tool for biomedical data analysis: segmentations can be used to screen personal genome variants--including those associated with cancer and other diseases--and to improve computational predictions of noncoding functional elements.

  20. Llama antibody fragments recognizing various epitopes of the CD4bs neutralize a broad range of HIV-1 subtypes A, B and C.

    Science.gov (United States)

    Strokappe, Nika; Szynol, Agnieszka; Aasa-Chapman, Marlèn; Gorlani, Andrea; Forsman Quigley, Anna; Hulsik, David Lutje; Chen, Lei; Weiss, Robin; de Haard, Hans; Verrips, Theo

    2012-01-01

    Many of the neutralising antibodies, isolated to date, display limited activities against the globally most prevalent HIV-1 subtypes A and C. Therefore, those subtypes are considered to be an important target for antibody-based therapy. Variable domains of llama heavy chain antibodies (VHH) have some superior properties compared with classical antibodies. Therefore we describe the application of trimeric forms of envelope proteins (Env), derived from HIV-1 of subtype A and B/C, for a prolonged immunization of two llamas. A panel of VHH, which interfere with CD4 binding to HIV-1 Env were selected with use of panning. The results of binding and competition assays to various Env, including a variant with a stabilized CD4-binding state (gp120(Ds2)), cross-competition experiments, maturation analysis and neutralisation assays, enabled us to classify the selected VHH into three groups. The VHH of group I were efficient mainly against viruses of subtype A, C and B'/C. The VHH of group II resemble the broadly neutralising antibody (bnmAb) b12, neutralizing mainly subtype B and C viruses, however some had a broader neutralisation profile. A representative of the third group, 2E7, had an even higher neutralization breadth, neutralizing 21 out of the 26 tested strains belonging to the A, A/G, B, B/C and C subtypes. To evaluate the contribution of certain amino acids to the potency of the VHH a small set of the mutants were constructed. Surprisingly this yielded one mutant with slightly improved neutralisation potency against 92UG37.A9 (subtype A) and 96ZM651.02 (subtype C). These findings and the well-known stability of VHH indicate the potential application of these VHH as anti-HIV-1 microbicides.

  1. Broadly neutralizing antibody PGT121 allosterically modulates CD4 binding via recognition of the HIV-1 gp120 V3 base and multiple surrounding glycans.

    Directory of Open Access Journals (Sweden)

    Jean-Philippe Julien

    Full Text Available New broad and potent neutralizing HIV-1 antibodies have recently been described that are largely dependent on the gp120 N332 glycan for Env recognition. Members of the PGT121 family of antibodies, isolated from an African donor, neutralize ∼70% of circulating isolates with a median IC50 less than 0.05 µg ml(-1. Here, we show that three family members, PGT121, PGT122 and PGT123, have very similar crystal structures. A long 24-residue HCDR3 divides the antibody binding site into two functional surfaces, consisting of an open face, formed by the heavy chain CDRs, and an elongated face, formed by LCDR1, LCDR3 and the tip of the HCDR3. Alanine scanning mutagenesis of the antibody paratope reveals a crucial role in neutralization for residues on the elongated face, whereas the open face, which accommodates a complex biantennary glycan in the PGT121 structure, appears to play a more secondary role. Negative-stain EM reconstructions of an engineered recombinant Env gp140 trimer (SOSIP.664 reveal that PGT122 interacts with the gp120 outer domain at a more vertical angle with respect to the top surface of the spike than the previously characterized antibody PGT128, which is also dependent on the N332 glycan. We then used ITC and FACS to demonstrate that the PGT121 antibodies inhibit CD4 binding to gp120 despite the epitope being distal from the CD4 binding site. Together, these structural, functional and biophysical results suggest that the PGT121 antibodies may interfere with Env receptor engagement by an allosteric mechanism in which key structural elements, such as the V3 base, the N332 oligomannose glycan and surrounding glycans, including a putative V1/V2 complex biantennary glycan, are conformationally constrained.

  2. Structural and Kinetic Studies of the Human Nudix Hydrolase MTH1 Reveal the Mechanism for Its Broad Substrate Specificity*

    Science.gov (United States)

    Waz, Shaimaa; Nakamura, Teruya; Hirata, Keisuke; Koga-Ogawa, Yukari; Chirifu, Mami; Arimori, Takao; Tamada, Taro; Ikemizu, Shinji; Nakabeppu, Yusaku; Yamagata, Yuriko

    2017-01-01

    The human MutT homolog 1 (hMTH1, human NUDT1) hydrolyzes oxidatively damaged nucleoside triphosphates and is the main enzyme responsible for nucleotide sanitization. hMTH1 recently has received attention as an anticancer target because hMTH1 blockade leads to accumulation of oxidized nucleotides in the cell, resulting in mutations and death of cancer cells. Unlike Escherichia coli MutT, which shows high substrate specificity for 8-oxoguanine nucleotides, hMTH1 has broad substrate specificity for oxidized nucleotides, including 8-oxo-dGTP and 2-oxo-dATP. However, the reason for this broad substrate specificity remains unclear. Here, we determined crystal structures of hMTH1 in complex with 8-oxo-dGTP or 2-oxo-dATP at neutral pH. These structures based on high quality data showed that the base moieties of two substrates are located on the similar but not the same position in the substrate binding pocket and adopt a different hydrogen-bonding pattern, and both triphosphate moieties bind to the hMTH1 Nudix motif (i.e. the hydrolase motif) similarly and align for the hydrolysis reaction. We also performed kinetic assays on the substrate-binding Asp-120 mutants (D120N and D120A), and determined their crystal structures in complex with the substrates. Analyses of bond lengths with high-resolution X-ray data and the relationship between the structure and enzymatic activity revealed that hMTH1 recognizes the different oxidized nucleotides via an exchange of the protonation state at two neighboring aspartate residues (Asp-119 and Asp-120) in its substrate binding pocket. To our knowledge, this mechanism of broad substrate recognition by enzymes has not been reported previously and may have relevance for anticancer drug development strategies targeting hMTH1. PMID:28035004

  3. Are we ignoring neutral and negative human-animal relationships in zoos?

    Science.gov (United States)

    Hosey, Geoff; Melfi, Vicky

    2015-01-01

    Human-animal interactions (HAI), which may lead to human-animal relationships (HAR), may be positive, neutral, or negative in nature. Zoo studies show that visitors may be stressful, may have no effect, or may be enriching. There is also evidence that good HARs set up between animals and their keepers can have positive effects on animal welfare. However, we need to know more about negative HARs, and as a first step we attempt to do this here by considering cases where animals attack people in the zoo. Due to the sensitivity and rarity of these events data appear sparse and unsystematically collected. Here, information available in the public domain about the circumstances of these attacks has been collated to test hypotheses about negative HAIs derived from a model of zoo HARs. The limited data presented here broadly support the zoo HAR model, and suggest that attacks usually happen in unusual circumstances, where there may be a failure by the animal to recognise the HAR, or where the relationship, if there is one, does not hold; and give some support to the prediction that exposure to many keepers may impair the development of a positive HAR. This study may provide useful information for the zoo community to proactively collect systematic standardised records, which will enable a fuller understanding of zoo HARs, upon which appropriate measures might be adopted to build better zoo HARs, which are likely to positively impact zoo animal welfare, and reduce these rare incidences further.

  4. Human monoclonal antibodies to a novel cluster of conformational epitopes on HCV E2 with resistance to neutralization escape in a genotype 2a isolate

    DEFF Research Database (Denmark)

    Keck, Zhen-yong; Xia, Jinming; Wang, Yong

    2012-01-01

    The majority of broadly neutralizing antibodies to hepatitis C virus (HCV) are against conformational epitopes on the E2 glycoprotein. Many of them recognize overlapping epitopes in a cluster, designated as antigenic domain B, that contains residues G530 and D535. To gain information on other...... regions that will be relevant for vaccine design, we employed yeast surface display of antibodies that bound to genotype 1a H77C E2 mutant proteins containing a substitution either at Y632A (to avoid selecting non-neutralizing antibodies) or D535A. A panel of nine human monoclonal antibodies (HMAbs......) was isolated and designated as HC-84-related antibodies. Each HMAb neutralized cell culture infectious HCV (HCVcc) with genotypes 1-6 envelope proteins with varying profiles, and each inhibited E2 binding to the viral receptor CD81. Five of these antibodies neutralized representative genotypes 1-6 HCVcc...

  5. Cooperativity in virus neutralization by human monoclonal antibodies to two adjacent regions located at the amino terminus of hepatitis C virus E2 glycoprotein

    DEFF Research Database (Denmark)

    Keck, Zhenyong; Wang, Wenyan; Wang, Yong

    2013-01-01

    polyclonal antibodies to aa 412 to 423 from HCV-infected individuals confirmed broad neutralization, conflicting findings have been reported on polyclonal antibodies to an adjacent region, aa 434 to 446, that may or may not interfere with neutralization by antibodies to aa 412 to 423. To define the interplay......A challenge for hepatitis C virus (HCV) vaccine development is defining conserved epitopes that induce protective antibodies against this highly diverse virus. An envelope glycoprotein (E2) segment located at amino acids (aa) 412 to 423 contains highly conserved neutralizing epitopes. While...... between these antibodies, we isolated human monoclonal antibodies (HMAbs) to aa 412 to 423, designated HC33-related HMAbs (HC33 HMAbs), and characterized their interactions with other HMAbs to aa 434 to 446. A subset of the HC33 HMAbs neutralized genotype 1 to 6 infectious cell culture-derived HCV virions...

  6. An analog of camptothecin inactive against Topoisomerase I is broadly neutralizing of HIV-1 through inhibition of Vif-dependent APOBEC3G degradation.

    Science.gov (United States)

    Bennett, Ryan P; Stewart, Ryan A; Hogan, Priscilla A; Ptak, Roger G; Mankowski, Marie K; Hartman, Tracy L; Buckheit, Robert W; Snyder, Beth A; Salter, Jason D; Morales, Guillermo A; Smith, Harold C

    2016-12-01

    Camptothecin (CPT) is a natural product discovered to be active against various cancers through its ability to inhibit Topoisomerase I (TOP1). CPT analogs also have anti-HIV-1 (HIV) activity that was previously shown to be independent of TOP1 inhibition. We show that a cancer inactive CPT analog (O2-16) inhibits HIV infection by disrupting multimerization of the HIV protein Vif. Antiviral activity depended on the expression of the cellular viral restriction factor APOBEC3G (A3G) that, in the absence of functional Vif, has the ability to hypermutate HIV proviral DNA during reverse transcription. Our studies demonstrate that O2-16 has low cytotoxicity and inhibits Vif-dependent A3G degradation, enabling A3G packaging into HIV viral particles that results in A3G signature hypermutations in viral genomes. This antiviral activity was A3G-dependent and broadly neutralizing against sixteen HIV clinical isolates from groups M (subtypes A-G), N, and O as well as seven single and multi-drug resistant strains of HIV. Molecular modeling predicted binding near the PPLP motif crucial for Vif multimerization and activity. O2-16 also was active in blocking Vif degradation of APOBEC3F (A3F). We propose that CPT analogs not active against TOP1 have novel therapeutic potential as Vif antagonists that enable A3G-dependent hypermutation of HIV.

  7. Comprehensive mapping of functional epitopes on dengue virus glycoprotein E DIII for binding to broadly neutralizing antibodies 4E11 and 4E5A by phage display.

    Science.gov (United States)

    Frei, Julia C; Kielian, Margaret; Lai, Jonathan R

    2015-11-01

    Here we investigated the binding of Dengue virus envelope glycoprotein domain III (DIII) by two broadly neutralizing antibodies (bNAbs), 4E11 and 4E5A. There are four serotypes of Dengue virus (DENV-1 to -4), whose DIII sequences vary by up to 49%. We used combinatorial alanine scanning mutagenesis, a phage display approach, to map functional epitopes (those residues that contribute most significantly to the energetics of antibody-antigen interaction) on these four serotypes. Our results showed that 4E11, which binds strongly to DENV-1, -2, and -3, and moderately to DENV-4, recognized a common conserved core functional epitope involving DIII residues K310, L/I387, L389, and W391. There were also unique recognition features for each serotype, suggesting that 4E11 has flexible recognition requirements. Similar scanning studies for the related bNAb 4E5A, which binds more tightly to DENV-4, identified broader functional epitopes on DENV-1. These results provide useful information for immunogen and therapeutic antibody design.

  8. Serrumab: a novel human single chain-fragment antibody with multiple scorpion toxin-neutralizing capacities.

    Science.gov (United States)

    Pucca, Manuela Berto; Cerni, Felipe Augusto; Peigneur, Steve; Arantes, Eliane Candiani; Tytgat, Jan; Barbosa, José Elpidio

    2014-01-01

    In Brazil, scorpion envenomation is an important public health problem. The yellow scorpion, Tityus serrulatus (Ts), is considered the most dangerous species in the country, being responsible for the most severe clinical cases of envenomation. Currently, the administration of serum produced in horses is recognized and used as a treatment for accidents with scorpions. However, horse herds' maintenance is costly and the antibodies are heterologous, which can cause anaphylaxis and Serum Sickness. In the present work, a human monoclonal fragment antibody, Serrumab, has been analysed. Toxin neutralizing effects of Serrumab were evaluated using a two-electrode voltage-clamp technique. The results show that Serrumab presented a high neutralizing effect against Ts β-toxins (Ts1, 43.2% and Ts2, 68.8%) and none or low neutralizing effect against α-toxins (Ts3, 0% and Ts5, 10%). Additional experiments demonstrated that Serrumab was also able to neutralize the action of toxins from other scorpion genus (Css II, 45.96% and Lqh III, 100%/β- and α-toxins, respectively). This work indicated that Serrumab is able to neutralize many toxins in Ts venom, and could being considered as a neutralizing antibody for formulating a human anti-scorpion serum in Brazil. Additionally, this work demonstrated that Serrumab could neutralize different toxins from distinct scorpion genus. All these results reinforce the idea that Serrumab is a scFv antibody with multiple neutralizing capacities and a promising candidate for inclusion in scorpion anti-venoms against different genera.

  9. Crystal structure of the Hendra virus attachment G glycoprotein bound to a potent cross-reactive neutralizing human monoclonal antibody.

    Directory of Open Access Journals (Sweden)

    Kai Xu

    Full Text Available The henipaviruses, represented by Hendra (HeV and Nipah (NiV viruses are highly pathogenic zoonotic paramyxoviruses with uniquely broad host tropisms responsible for repeated outbreaks in Australia, Southeast Asia, India and Bangladesh. The high morbidity and mortality rates associated with infection and lack of licensed antiviral therapies make the henipaviruses a potential biological threat to humans and livestock. Henipavirus entry is initiated by the attachment of the G envelope glycoprotein to host cell membrane receptors. Previously, henipavirus-neutralizing human monoclonal antibodies (hmAb have been isolated using the HeV-G glycoprotein and a human naïve antibody library. One cross-reactive and receptor-blocking hmAb (m102.4 was recently demonstrated to be an effective post-exposure therapy in two animal models of NiV and HeV infection, has been used in several people on a compassionate use basis, and is currently in development for use in humans. Here, we report the crystal structure of the complex of HeV-G with m102.3, an m102.4 derivative, and describe NiV and HeV escape mutants. This structure provides detailed insight into the mechanism of HeV and NiV neutralization by m102.4, and serves as a blueprint for further optimization of m102.4 as a therapeutic agent and for the development of entry inhibitors and vaccines.

  10. Crystal structure of the Hendra virus attachment G glycoprotein bound to a potent cross-reactive neutralizing human monoclonal antibody.

    Science.gov (United States)

    Xu, Kai; Rockx, Barry; Xie, Yihu; DeBuysscher, Blair L; Fusco, Deborah L; Zhu, Zhongyu; Chan, Yee-Peng; Xu, Yan; Luu, Truong; Cer, Regina Z; Feldmann, Heinz; Mokashi, Vishwesh; Dimitrov, Dimiter S; Bishop-Lilly, Kimberly A; Broder, Christopher C; Nikolov, Dimitar B

    2013-01-01

    The henipaviruses, represented by Hendra (HeV) and Nipah (NiV) viruses are highly pathogenic zoonotic paramyxoviruses with uniquely broad host tropisms responsible for repeated outbreaks in Australia, Southeast Asia, India and Bangladesh. The high morbidity and mortality rates associated with infection and lack of licensed antiviral therapies make the henipaviruses a potential biological threat to humans and livestock. Henipavirus entry is initiated by the attachment of the G envelope glycoprotein to host cell membrane receptors. Previously, henipavirus-neutralizing human monoclonal antibodies (hmAb) have been isolated using the HeV-G glycoprotein and a human naïve antibody library. One cross-reactive and receptor-blocking hmAb (m102.4) was recently demonstrated to be an effective post-exposure therapy in two animal models of NiV and HeV infection, has been used in several people on a compassionate use basis, and is currently in development for use in humans. Here, we report the crystal structure of the complex of HeV-G with m102.3, an m102.4 derivative, and describe NiV and HeV escape mutants. This structure provides detailed insight into the mechanism of HeV and NiV neutralization by m102.4, and serves as a blueprint for further optimization of m102.4 as a therapeutic agent and for the development of entry inhibitors and vaccines.

  11. Structural comparison of four different antibodies interacting with human papillomavirus 16 and mechanisms of neutralization

    Energy Technology Data Exchange (ETDEWEB)

    Guan, Jian [Department of Medicine, The Pennsylvania State University College of Medicine, 500 University Drive, Hershey, PA 17033 (United States); Bywaters, Stephanie M.; Brendle, Sarah A. [Department of Pathology, The Pennsylvania State University College of Medicine, 500 University Drive, Hershey, PA 17033 (United States); Lee, Hyunwook; Ashley, Robert E. [Department of Medicine, The Pennsylvania State University College of Medicine, 500 University Drive, Hershey, PA 17033 (United States); Makhov, Alexander M.; Conway, James F. [Department of Structural Biology, University of Pittsburgh School of Medicine, 3501 5th Ave, Pittsburgh, PA 15260 (United States); Christensen, Neil D. [Department of Pathology, The Pennsylvania State University College of Medicine, 500 University Drive, Hershey, PA 17033 (United States); Hafenstein, Susan, E-mail: shafenstein@hmc.psu.edu [Department of Medicine, The Pennsylvania State University College of Medicine, 500 University Drive, Hershey, PA 17033 (United States)

    2015-09-15

    Cryo-electron microscopy (cryo-EM) was used to solve the structures of human papillomavirus type 16 (HPV16) complexed with fragments of antibody (Fab) from three different neutralizing monoclonals (mAbs): H16.1A, H16.14J, and H263.A2. The structure-function analysis revealed predominantly monovalent binding of each Fab with capsid interactions that involved multiple loops from symmetry related copies of the major capsid protein. The residues identified in each Fab-virus interface map to a conformational groove on the surface of the capsomer. In addition to the known involvement of the FG and HI loops, the DE loop was also found to constitute the core of each epitope. Surprisingly, the epitope mapping also identified minor contributions by EF and BC loops. Complementary immunological assays included mAb and Fab neutralization. The specific binding characteristics of mAbs correlated with different neutralizing behaviors in pre- and post-attachment neutralization assays. - Highlights: • We present HPV16-Fab complexes from neutralizing mAbs: H16.1A, H16.14J, and H263.A2. • The structure-function analysis revealed predominantly monovalent binding of each mAb. • Capsid–Fab interactions involved multiple loops from symmetry related L1 proteins. • Besides the known FG and HI loops, epitope mapping also identified DE, EF, and BC loops. • Neutralizing assays complement the structures to show multiple neutralization mechanisms.

  12. The membrane-proximal external region of the human immunodeficiency virus type 1 envelope: dominant site of antibody neutralization and target for vaccine design.

    Science.gov (United States)

    Montero, Marinieve; van Houten, Nienke E; Wang, Xin; Scott, Jamie K

    2008-03-01

    Enormous efforts have been made to produce a protective vaccine against human immunodeficiency virus type 1; there has been little success. However, the identification of broadly neutralizing antibodies against epitopes on the highly conserved membrane-proximal external region (MPER) of the gp41 envelope protein has delineated this region as an attractive vaccine target. Furthermore, emerging structural information on the MPER has provided vaccine designers with new insights for building relevant immunogens. This review describes the current state of the field regarding (i) the structure and function of the gp41 MPER; (ii) the structure and binding mechanisms of the broadly neutralizing antibodies 2F5, 4E10, and Z13; and (iii) the development of an MPER-targeting vaccine. In addition, emerging approaches to vaccine design are presented.

  13. Asian dust storm particles induce a broad toxicological transcriptional program in human epidermal keratinocytes.

    Science.gov (United States)

    Choi, Hyun; Shin, Dong Wook; Kim, Wonnyon; Doh, Seong-Jae; Lee, Soo Hwan; Noh, Minsoo

    2011-01-15

    Exposure to airborne dust particles originated from seasonal Asian dust storms in Chinese and Mongolian deserts results in increased incidence of a range of diseases including asthma, contact dermatitis and conjunctivitis. The areas affected by Asian dust particles extend from East China to the west coast of North America. In order to study toxicological mechanisms in human skin, we evaluated the effects of dust particles collected during Asian dust storms (Asian dust particles) on gene expression in human epidermal keratinocytes (HEK). In HEK, exposure to Asian dust particles significantly increased gene expressions of cytochrome P450 1A1 (CYP1A1), CYP1A2, and CYP1B1, which is an indication of aryl hydrocarbon receptor (AHR) activation. In addition, Asian dust particles increased gene transcription of the cytokines IL-6, IL-8, and GM-CSF, which have broad pro-inflammatory and immunomodulatory properties. Asian dust particles significantly up-regulated expression of caspase 14 in HEK, suggesting that Asian dust particles directly affect keratinocyte differentiation. We also demonstrated that protein extract of pollen, a material frequently adsorbed onto Asian dust particles, potentially contributes to the increased transcription of IL-6, CYP1A1, CYP1A2, and CYP1B1. Taken together, these studies suggest that Asian dust particles can exert toxicological effects on human skin through the activation of the cellular detoxification system, the production of pro-inflammatory and immunomodulatory cytokines, and changes in the expression of proteins essential in normal epidermal differentiation.

  14. Complement-Dependent Lysis of Influenza A Virus-Infected Cells by Broadly Cross-Reactive Human Monoclonal Antibodies ▿

    Science.gov (United States)

    Terajima, Masanori; Cruz, John; Co, Mary Dawn T.; Lee, Jane-Hwei; Kaur, Kaval; Wilson, Patrick C.; Ennis, Francis A.

    2011-01-01

    We characterized human monoclonal antibodies (MAbs) cloned from influenza virus-infected patients and from influenza vaccine recipients by complement-dependent lysis (CDL) assay. Most MAbs active in CDL were neutralizing, but not all neutralizing MAbs can mediate CDL. Two of the three stalk-specific neutralizing MAbs tested were able to mediate CDL and were more cross-reactive to temporally distant H1N1 strains than the conventional hemagglutination-inhibiting and neutralizing MAbs. One of the stalk-specific MAbs was subtype cross-reactive to H1 and H2 hemagglutinins, suggesting a role for stalk-specific antibodies in protection against influenza illness, especially by a novel viral subtype which can cause pandemics. PMID:21994454

  15. Plasma pharmacokinetics and biological activity of a human immunodeficiency virus type 1 neutralizing human monoclonal antibody, F105, in cynomolgus monkeys.

    Science.gov (United States)

    Cavacini, L A; Power, J; Emes, C L; Mace, K; Treacy, G; Posner, M R

    1994-05-01

    The IgG1 kappa human monoclonal antibody (HMab), F105 reacts with a discontinuous epitope on the CD4 binding site (CD4BS) of human immunodeficiency virus type 1 (HIV-1)/gp120 and has broad neutralizing activity. F105 HMab (60 mg/kg bolus) was administered intravenously to four monkeys and serum was collected at intervals to determine pharmacokinetics in a primate model. Average serum F105 concentrations, as determined by enzyme-linked immunosorbent assay, were analyzed with MINSQ software using a two-compartment, first-order model. The half-life for the alpha phase of the distribution curve is 6.7 h and for the beta elimination phase, 9.6 days. The volume of distribution is 0.65 L/kg and the rate of clearance 2 ml/kg/h. Serum levels of 1.3-1.6 mg/ml of F105 were maintained for 24 h. When monkey serum from day 15 postdose was tested, total serum F105 was 230 +/- 79 micrograms/ml and was immunoreactive with cells infected with the MN and IIIB strains of HIV-1 as determined by flow cytometry. Binding activity was identical to that obtained with stock F105 HMab. Identical neutralizing activity between the injected and uninjected antibody was also observed. Thus, serum neutralizing titers (90%) of 1:2000 at peak and 1:30 at day 15 postdose for MN virus were observed. These data indicate that high in vivo levels of HMab F105 can be attained by single bolus administration with full retention of biological activity. Of importance, levels of antibody necessary for effective neutralization can be achieved and maintained.

  16. Neutral versus Emotional Human Stimuli Processing in Children with Pervasive Developmental Disorders not Otherwise Specified

    Science.gov (United States)

    Vannetzel, Leonard; Chaby, Laurence; Cautru, Fabienne; Cohen, David; Plaza, Monique

    2011-01-01

    Pervasive developmental disorder not otherwise specified (PDD-NOS) represents up to two-thirds of autism spectrum disorders; however, it is usually described in terms of the symptoms not shared by autism. The study explores processing of neutral and emotional human stimuli (by auditory, visual and multimodal channels) in children with PDD-NOS (n =…

  17. Human DDX3 protein is a valuable target to develop broad spectrum antiviral agents.

    Science.gov (United States)

    Brai, Annalaura; Fazi, Roberta; Tintori, Cristina; Zamperini, Claudio; Bugli, Francesca; Sanguinetti, Maurizio; Stigliano, Egidio; Esté, José; Badia, Roger; Franco, Sandra; Martinez, Miguel A; Martinez, Javier P; Meyerhans, Andreas; Saladini, Francesco; Zazzi, Maurizio; Garbelli, Anna; Maga, Giovanni; Botta, Maurizio

    2016-05-10

    Targeting a host factor essential for the replication of different viruses but not for the cells offers a higher genetic barrier to the development of resistance, may simplify therapy regimens for coinfections, and facilitates management of emerging viral diseases. DEAD-box polypeptide 3 (DDX3) is a human host factor required for the replication of several DNA and RNA viruses, including some of the most challenging human pathogens currently circulating, such as HIV-1, Hepatitis C virus, Dengue virus, and West Nile virus. Herein, we showed for the first time, to our knowledge, that the inhibition of DDX3 by a small molecule could be successfully exploited for the development of a broad spectrum antiviral agent. In addition to the multiple antiviral activities, hit compound 16d retained full activity against drug-resistant HIV-1 strains in the absence of cellular toxicity. Pharmacokinetics and toxicity studies in rats confirmed a good safety profile and bioavailability of 16d. Thus, DDX3 is here validated as a valuable therapeutic target.

  18. Tetanus Neurotoxin Neutralizing Antibodies Screened from a Human Immune scFv Antibody Phage Display Library.

    Science.gov (United States)

    Wang, Han; Yu, Rui; Fang, Ting; Yu, Ting; Chi, Xiangyang; Zhang, Xiaopeng; Liu, Shuling; Fu, Ling; Yu, Changming; Chen, Wei

    2016-09-11

    Tetanus neurotoxin (TeNT) produced by Clostridium tetani is one of the most poisonous protein substances. Neutralizing antibodies against TeNT can effectively prevent and cure toxicosis. Using purified Hc fragments of TeNT (TeNT-Hc) as an antigen, three specific neutralizing antibody clones recognizing different epitopes were selected from a human immune scFv antibody phage display library. The three antibodies (2-7G, 2-2D, and S-4-7H) can effectively inhibit the binding between TeNT-Hc and differentiated PC-12 cells in vitro. Moreover, 2-7G inhibited TeNT-Hc binding to the receptor via carbohydrate-binding sites of the W pocket while 2-2D and S-4-7H inhibited binding of the R pocket. Although no single mAb completely protected mice from the toxin, they could both prolong survival when challenged with 20 LD50s (50% of the lethal dose) of TeNT. When used together, the mAbs completely neutralized 1000 LD50s/mg Ab, indicating their high neutralizing potency in vivo. Antibodies recognizing different carbohydrate-binding pockets could have higher synergistic toxin neutralization activities than those that recognize the same pockets. These results could lead to further production of neutralizing antibody drugs against TeNT and indicate that using TeNT-Hc as an antigen for screening human antibodies for TeNT intoxication therapy from human immune antibody library was convenient and effective.

  19. Tetanus Neurotoxin Neutralizing Antibodies Screened from a Human Immune scFv Antibody Phage Display Library

    Science.gov (United States)

    Wang, Han; Yu, Rui; Fang, Ting; Yu, Ting; Chi, Xiangyang; Zhang, Xiaopeng; Liu, Shuling; Fu, Ling; Yu, Changming; Chen, Wei

    2016-01-01

    Tetanus neurotoxin (TeNT) produced by Clostridium tetani is one of the most poisonous protein substances. Neutralizing antibodies against TeNT can effectively prevent and cure toxicosis. Using purified Hc fragments of TeNT (TeNT-Hc) as an antigen, three specific neutralizing antibody clones recognizing different epitopes were selected from a human immune scFv antibody phage display library. The three antibodies (2-7G, 2-2D, and S-4-7H) can effectively inhibit the binding between TeNT-Hc and differentiated PC-12 cells in vitro. Moreover, 2-7G inhibited TeNT-Hc binding to the receptor via carbohydrate-binding sites of the W pocket while 2-2D and S-4-7H inhibited binding of the R pocket. Although no single mAb completely protected mice from the toxin, they could both prolong survival when challenged with 20 LD50s (50% of the lethal dose) of TeNT. When used together, the mAbs completely neutralized 1000 LD50s/mg Ab, indicating their high neutralizing potency in vivo. Antibodies recognizing different carbohydrate-binding pockets could have higher synergistic toxin neutralization activities than those that recognize the same pockets. These results could lead to further production of neutralizing antibody drugs against TeNT and indicate that using TeNT-Hc as an antigen for screening human antibodies for TeNT intoxication therapy from human immune antibody library was convenient and effective. PMID:27626445

  20. Tetanus Neurotoxin Neutralizing Antibodies Screened from a Human Immune scFv Antibody Phage Display Library

    Directory of Open Access Journals (Sweden)

    Han Wang

    2016-09-01

    Full Text Available Tetanus neurotoxin (TeNT produced by Clostridium tetani is one of the most poisonous protein substances. Neutralizing antibodies against TeNT can effectively prevent and cure toxicosis. Using purified Hc fragments of TeNT (TeNT-Hc as an antigen, three specific neutralizing antibody clones recognizing different epitopes were selected from a human immune scFv antibody phage display library. The three antibodies (2-7G, 2-2D, and S-4-7H can effectively inhibit the binding between TeNT-Hc and differentiated PC-12 cells in vitro. Moreover, 2-7G inhibited TeNT-Hc binding to the receptor via carbohydrate-binding sites of the W pocket while 2-2D and S-4-7H inhibited binding of the R pocket. Although no single mAb completely protected mice from the toxin, they could both prolong survival when challenged with 20 LD50s (50% of the lethal dose of TeNT. When used together, the mAbs completely neutralized 1000 LD50s/mg Ab, indicating their high neutralizing potency in vivo. Antibodies recognizing different carbohydrate-binding pockets could have higher synergistic toxin neutralization activities than those that recognize the same pockets. These results could lead to further production of neutralizing antibody drugs against TeNT and indicate that using TeNT-Hc as an antigen for screening human antibodies for TeNT intoxication therapy from human immune antibody library was convenient and effective.

  1. Reduced influenza viral neutralizing activity of natural human trimers of surfactant protein D

    DEFF Research Database (Denmark)

    Hartshorn, Kevan L; White, Mitchell R; Tecle, Tesfaldet

    2007-01-01

    BACKGROUND: Surfactant protein D (SP-D) plays important roles in innate host defense against influenza A virus (IAV) infection. Common human polymorphisms of SP-D have been found in many human populations and associated with increased risk of certain infections. We recently reported that the Thr...... human SP-D multimers as well as reduced hemagglutination inhibiting activity against several strains of IAV. Natural SP-D trimers also had different interactions with human neutrophil peptide defensins (HNPs) in viral neutralization assays as compared to multimeric SP-D. CONCLUSION: These studies......-D can be useful for dissecting out different functional properties of the protein....

  2. Most neutralizing human monoclonal antibodies target novel epitopes requiring both Lassa virus glycoprotein subunits

    Science.gov (United States)

    Robinson, James E.; Hastie, Kathryn M.; Cross, Robert W.; Yenni, Rachael E.; Elliott, Deborah H.; Rouelle, Julie A.; Kannadka, Chandrika B.; Smira, Ashley A.; Garry, Courtney E.; Bradley, Benjamin T.; Yu, Haini; Shaffer, Jeffrey G.; Boisen, Matt L.; Hartnett, Jessica N.; Zandonatti, Michelle A.; Rowland, Megan M.; Heinrich, Megan L.; Martínez-Sobrido, Luis; Cheng, Benson; de la Torre, Juan C.; Andersen, Kristian G.; Goba, Augustine; Momoh, Mambu; Fullah, Mohamed; Gbakie, Michael; Kanneh, Lansana; Koroma, Veronica J.; Fonnie, Richard; Jalloh, Simbirie C.; Kargbo, Brima; Vandi, Mohamed A.; Gbetuwa, Momoh; Ikponmwosa, Odia; Asogun, Danny A.; Okokhere, Peter O.; Follarin, Onikepe A.; Schieffelin, John S.; Pitts, Kelly R.; Geisbert, Joan B.; Kulakoski, Peter C.; Wilson, Russell B.; Happi, Christian T.; Sabeti, Pardis C.; Gevao, Sahr M.; Khan, S. Humarr; Grant, Donald S.; Geisbert, Thomas W.; Saphire, Erica Ollmann; Branco, Luis M.; Garry, Robert F.

    2016-01-01

    Lassa fever is a severe multisystem disease that often has haemorrhagic manifestations. The epitopes of the Lassa virus (LASV) surface glycoproteins recognized by naturally infected human hosts have not been identified or characterized. Here we have cloned 113 human monoclonal antibodies (mAbs) specific for LASV glycoproteins from memory B cells of Lassa fever survivors from West Africa. One-half bind the GP2 fusion subunit, one-fourth recognize the GP1 receptor-binding subunit and the remaining fourth are specific for the assembled glycoprotein complex, requiring both GP1 and GP2 subunits for recognition. Notably, of the 16 mAbs that neutralize LASV, 13 require the assembled glycoprotein complex for binding, while the remaining 3 require GP1 only. Compared with non-neutralizing mAbs, neutralizing mAbs have higher binding affinities and greater divergence from germline progenitors. Some mAbs potently neutralize all four LASV lineages. These insights from LASV human mAb characterization will guide strategies for immunotherapeutic development and vaccine design. PMID:27161536

  3. Neutralization of Diverse Human Cytomegalovirus Strains Conferred by Antibodies Targeting Viral gH/gL/pUL128-131 Pentameric Complex

    Science.gov (United States)

    Ha, Sha; Li, Fengsheng; Troutman, Matthew C.; Freed, Daniel C.; Tang, Aimin; Loughney, John W.; Wang, I-Ming; Vlasak, Josef; Nickle, David C.; Rustandi, Richard R.; Hamm, Melissa; DePhillips, Pete A.; Zhang, Ningyan; McLellan, Jason S.; Zhu, Hua; Adler, Stuart P.; McVoy, Michael A.; An, Zhiqiang

    2017-01-01

    ABSTRACT Human cytomegalovirus (HCMV) is the leading cause of congenital viral infection, and developing a prophylactic vaccine is of high priority to public health. We recently reported a replication-defective human cytomegalovirus with restored pentameric complex glycoprotein H (gH)/gL/pUL128-131 for prevention of congenital HCMV infection. While the quantity of vaccine-induced antibody responses can be measured in a viral neutralization assay, assessing the quality of such responses, including the ability of vaccine-induced antibodies to cross-neutralize the field strains of HCMV, remains a challenge. In this study, with a panel of neutralizing antibodies from three healthy human donors with natural HCMV infection or a vaccinated animal, we mapped eight sites on the dominant virus-neutralizing antigen—the pentameric complex of glycoprotein H (gH), gL, and pUL128, pUL130, and pUL131. By evaluating the site-specific antibodies in vaccine immune sera, we demonstrated that vaccination elicited functional antiviral antibodies to multiple neutralizing sites in rhesus macaques, with quality attributes comparable to those of CMV hyperimmune globulin. Furthermore, these immune sera showed antiviral activities against a panel of genetically distinct HCMV clinical isolates. These results highlighted the importance of understanding the quality of vaccine-induced antibody responses, which includes not only the neutralizing potency in key cell types but also the ability to protect against the genetically diverse field strains. IMPORTANCE HCMV is the leading cause of congenital viral infection, and development of a preventive vaccine is a high public health priority. To understand the strain coverage of vaccine-induced immune responses in comparison with natural immunity, we used a panel of broadly neutralizing antibodies to identify the immunogenic sites of a dominant viral antigen—the pentameric complex. We further demonstrated that following vaccination of a replication

  4. A neutralizing human monoclonal antibody protects against lethal disease in a new ferret model of acute nipah virus infection.

    Directory of Open Access Journals (Sweden)

    Katharine N Bossart

    2009-10-01

    Full Text Available Nipah virus is a broadly tropic and highly pathogenic zoonotic paramyxovirus in the genus Henipavirus whose natural reservoirs are several species of Pteropus fruit bats. Nipah virus has repeatedly caused outbreaks over the past decade associated with a severe and often fatal disease in humans and animals. Here, a new ferret model of Nipah virus pathogenesis is described where both respiratory and neurological disease are present in infected animals. Severe disease occurs with viral doses as low as 500 TCID(50 within 6 to 10 days following infection. The underlying pathology seen in the ferret closely resembles that seen in Nipah virus infected humans, characterized as a widespread multisystemic vasculitis, with virus replicating in highly vascular tissues including lung, spleen and brain, with recoverable virus from a variety of tissues. Using this ferret model a cross-reactive neutralizing human monoclonal antibody, m102.4, targeting the henipavirus G glycoprotein was evaluated in vivo as a potential therapeutic agent. All ferrets that received m102.4 ten hours following a high dose oral-nasal Nipah virus challenge were protected from disease while all controls died. This study is the first successful post-exposure passive antibody therapy for Nipah virus using a human monoclonal antibody.

  5. Cortisol variation in humans affects memory for emotionally laden and neutral information.

    Science.gov (United States)

    Abercrombie, Heather C; Kalin, Ned H; Thurow, Marchell E; Rosenkranz, Melissa A; Davidson, Richard J

    2003-06-01

    In a test of the effects of cortisol on emotional memory, 90 men were orally administered placebo or 20 or 40 mg cortisol and presented with emotionally arousing and neutral stimuli. On memory tests administered within 1 hr of stimulus presentation, cortisol elevations caused a reduction in the number of errors committed on free-recall tasks. Two evenings later, when cortisol levels were no longer manipulated, inverted-U quadratic trends were found for recognition memory tasks, reflecting memory facilitation in the 20-mg group for both negative and neutral information. Results suggest that the effects of cortisol on memory do not differ substantially for emotional and neutral information. The study provides evidence of beneficial effects of acute cortisol elevations on explicit memory in humans.

  6. Identification and characterization of a broadly cross-reactive HIV-1 human monoclonal antibody that binds to both gp120 and gp41.

    Directory of Open Access Journals (Sweden)

    Mei-Yun Zhang

    Full Text Available Identification of broadly cross-reactive HIV-1-neutralizing antibodies (bnAbs may assist vaccine immunogen design. Here we report a novel human monoclonal antibody (mAb, designated m43, which co-targets the gp120 and gp41 subunits of the HIV-1 envelope glycoprotein (Env. M43 bound to recombinant gp140 s from various primary isolates, to membrane-associated Envs on transfected cells and HIV-1 infected cells, as well as to recombinant gp120 s and gp41 fusion intermediate structures containing N-trimer structure, but did not bind to denatured recombinant gp140 s and the CD4 binding site (CD4bs mutant, gp120 D368R, suggesting that the m43 epitope is conformational and overlaps the CD4bs on gp120 and the N-trimer structure on gp41. M43 neutralized 34% of the HIV-1 primary isolates from different clades and all the SHIVs tested in assays based on infection of peripheral blood mononuclear cells (PBMCs by replication-competent virus, but was less potent in cell line-based pseudovirus assays. In contrast to CD4, m43 did not induce Env conformational changes upon binding leading to exposure of the coreceptor binding site, enhanced binding of mAbs 2F5 and 4E10 specific for the membrane proximal external region (MPER of gp41 Envs, or increased gp120 shedding. The overall modest neutralization activity of m43 is likely due to the limited binding of m43 to functional Envs which could be increased by antibody engineering if needed. M43 may represent a new class of bnAbs targeting conformational epitopes overlapping structures on both gp120 and gp41. Its novel epitope and possibly new mechanism(s of neutralization could helpdesign improved vaccine immunogens and candidate therapeutics.

  7. Increased infectivity in human cells and resistance to antibody-mediated neutralization by truncation of the SIV gp41 cytoplasmic tail

    Directory of Open Access Journals (Sweden)

    Takeo eKuwata

    2013-05-01

    Full Text Available The role of antibodies in protecting the host from human immunodeficiency virus type 1 (HIV-1 infection is of considerable interest, particularly because the RV144 trial results suggest that antibodies contribute to protection. Although infection of nonhuman primates with simian immunodeficiency virus (SIV is commonly used as an animal model of HIV-1 infection, the viral epitopes that elicit potent and broad neutralizing antibodies to SIV have not been identified. We isolated a monoclonal antibody (MAb B404 that potently and broadly neutralizes various SIV strains. B404 targets a conformational epitope comprising the V3 and V4 loops of Env that intensely exposed when Env binds CD4. B404-resistant variants were obtained by passaging viruses in the presence of increasing concentration of B404 in PM1/CCR5 cells. Genetic analysis revealed that the Q733stop mutation, which truncates the cytoplasmic tail of gp41, was the first major substitution in Env during passage. The maximal inhibition by B404 and other MAbs were significantly decreased against a recombinant virus with a gp41 truncation compared with the parental SIVmac316. This indicates that the gp41 truncation was associated with resistance to antibody-mediated neutralization. The infectivities of the recombinant virus with the gp41 truncation were 7900-fold, 1000-fold, and 140-fold higher than those of SIVmac316 in PM1, PM1/CCR5, and TZM-bl cells, respectively. Immunoblotting analysis revealed that the gp41 truncation enhanced the incorporation of Env into virions. The effect of the gp41 truncation on infectivity was not obvious in the HSC-F macaque cell line, although the resistance of viruses harboring the gp41 truncation to neutralization was maintained. These results suggest that viruses with a truncated gp41 cytoplasmic tail were selected by increased infectivity in human cells and by acquiring resistance to neutralizing antibody.

  8. Learning from the 2009 H1N1 pandemic: prospects for more broadly effective influenza vaccines

    Institute of Scientific and Technical Information of China (English)

    Ethan C. Settembre; Philip R. Dormitzer; Rino Rappuoli

    2011-01-01

    Calls to develop a universal influenza vaccine have increased in the wake of the 2009 H1 N1 influenza pandemic. This demand comes at a time when analyses of the human antibody repertoire, informed by structures of complexes between broadly neutralizing antibodies and influenza hemagglutinin, have revealed the target of a class of broadly neutralizing antibodies. Recent studies suggest a path forward to more broadly protective influenza vaccines.%@@ Calls to develop a universal influenza vaccine have increased in the wake of the 2009 H1 N1 influenza pandemic.This demand comes at a time when analyses of the human antibody repertoire, informed by structures of complexes between broadly neutralizing antibodies and influenza hemagglutinin, have revealed the target of a class of broadly neutralizing antibodies.Recent studies suggest a path forward to more broadly protective influenza vaccines.

  9. Recognition of influenza H3N2 variant virus by human neutralizing antibodies

    Science.gov (United States)

    Bangaru, Sandhya; Nieusma, Travis; Kose, Nurgun; Thornburg, Natalie J.; Kaplan, Bryan S.; King, Hannah G.; Singh, Vidisha; Lampley, Rebecca M.; Cisneros, Alberto; Edwards, Kathryn M.; Edupuganti, Srilatha; Lai, Lilin; Richt, Juergen A.; Webby, Richard J.; Ward, Andrew B.; Crowe, James E.

    2016-01-01

    Since 2011, over 300 human cases of infection, especially in exposed children, with the influenza A H3N2 variant (H3N2v) virus that circulates in swine in the US have been reported. The structural and genetic basis for the lack of protection against H3N2v induced by vaccines containing seasonal H3N2 antigens is poorly understood. We isolated 17 human monoclonal antibodies (mAbs) that neutralized H3N2v virus from subjects experimentally immunized with an H3N2v candidate vaccine. Six mAbs exhibited very potent neutralizing activity (IC50 < 200 ng/ml) against the H3N2v virus but not against current human H3N2 circulating strains. Fine epitope mapping and structural characterization of antigen-antibody complexes revealed that H3N2v specificity was attributable to amino acid polymorphisms in the 150-loop and the 190-helix antigenic sites on the hemagglutinin protein. H3N2v-specific antibodies also neutralized human H3N2 influenza strains naturally circulating between 1995 and 2005. These results reveal a high level of antigenic relatedness between the swine H3N2v virus and previously circulating human strains, consistent with the fact that early human H3 seasonal strains entered the porcine population in the 1990s and reentered the human population, where they had not been circulating, as H3N2v about a decade later. The data also explain the increased susceptibility to H3N2v viruses in young children, who lack prior exposure to human seasonal strains from the 1990s. PMID:27482543

  10. Generation, characterization and epitope mapping of two neutralizing and protective human recombinant antibodies against influenza A H5N1 viruses.

    Directory of Open Access Journals (Sweden)

    Lina Sun

    Full Text Available BACKGROUND: The development of new therapeutic targets and strategies to control highly pathogenic avian influenza (HPAI H5N1 virus infection in humans is urgently needed. Broadly cross-neutralizing recombinant human antibodies obtained from the survivors of H5N1 avian influenza provide an important role in immunotherapy for human H5N1 virus infection and definition of the critical epitopes for vaccine development. METHODOLOGY/PRINCIPAL FINDINGS: We have characterized two recombinant baculovirus-expressed human antibodies (rhAbs, AVFluIgG01 and AVFluIgG03, generated by screening a Fab antibody phage library derived from a patient recovered from infection with a highly pathogenic avian influenza A H5N1 clade 2.3 virus. AVFluIgG01 cross-neutralized the most of clade 0, clade 1, and clade 2 viruses tested, in contrast, AVFluIgG03 only neutralized clade 2 viruses. Passive immunization of mice with either AVFluIgG01 or AVFluIgG03 antibody resulted in protection from a lethal H5N1 clade 2.3 virus infection. Furthermore, through epitope mapping, we identify two distinct epitopes on H5 HA molecule recognized by these rhAbs and demonstrate their potential to protect against a lethal H5N1 virus infection in a mouse model. CONCLUSIONS/SIGNIFICANCE: Importantly, localization of the epitopes recognized by these two neutralizing and protective antibodies has provided, for the first time, insight into the human antibody responses to H5N1 viruses which contribute to the H5 immunity in the recovered patient. These results highlight the potential of a rhAbs treatment strategy for human H5N1 virus infection and provide new insight for the development of effective H5N1 pandemic vaccines.

  11. Human umbilical cord hyaluronate. Neutral sugar content and carbohydrate-protein linkage studies.

    Science.gov (United States)

    Varma, R; Varma, R S; Allen, W S; Wardi, A H

    1975-07-14

    Paper chromatography of neutral sugars and gas chromatography of their aldononitrile acetates indicated the presence of fucose, arabinose and a small amount of glucose in purified human umbilical cord hyaluronate. The molar ratios of serine, threonine and aspartic acid to neutral sugars were not unity, suggesting the non-involvement of the neutral sugars and the amino acids in a carbohydrate-protein linkage. The same was indicated by an increase in the percentage of the aforementioned amino acids and by the absence of sugar alditols in umbilical cord hyaluronate reduced eith NaBH4 -PdCl2, after alkali treatment. This reduction caused a decrease in the intrinsic viscosity and molecular wieght to about one-half and an appreciable decrease in the specific rota tion of hyaluronate, suggesting a separation of the two antiparallel chains o the double helical hyaluronate. The umbilical cord hyluronate containe contained bound silicon and it is possible that this bound silicon may cross-link the two chains at interspersed intervals through the uronic acid moiety and/or through neutral sugars.

  12. Reduced influenza viral neutralizing activity of natural human trimers of surfactant protein D

    Directory of Open Access Journals (Sweden)

    Sorensen Grith L

    2007-02-01

    Full Text Available Abstract Background Surfactant protein D (SP-D plays important roles in innate host defense against influenza A virus (IAV infection. Common human polymorphisms of SP-D have been found in many human populations and associated with increased risk of certain infections. We recently reported that the Thr/Thr 11 form of SP-D is associated with low serum levels and assembles predominantly as trimers as opposed to the more common multimeric forms of SP-D. Methods Preliminary experiments were done to establish the effects of different monoclonal antibodies against SP-D on ability of SP-D to bind to or neutralize the virus. We then purified natural human trimeric and multimeric forms of SP-D from amniotic fluid and tested ability of these preparations to bind to IAV, to inhibit infectivity and hemagglutination activity of IAV in vitro. Results In initial experiments mAbs directed against different areas on the CRD of SP-D were found to have differing effects on antiviral activity. Using an mAb that did not interfere with antiviral activity of SP-D, we confirm that natural SP-D trimers had reduced ability to bind to IAV. In addition, the trimers had reduced ability to neutralize IAV as compared to natural human SP-D multimers as well as reduced hemagglutination inhibiting activity against several strains of IAV. Natural SP-D trimers also had different interactions with human neutrophil peptide defensins (HNPs in viral neutralization assays as compared to multimeric SP-D. Conclusion These studies indicate that a common human polymorphic form of SP-D may modulate host defense against IAV and give impetus to clinical studies correlating this genotype with risk for IAV infection in susceptible groups. We also show that mAbs directed against different areas on the carbohydrate recognition domain of SP-D can be useful for dissecting out different functional properties of the protein.

  13. Why Does the Molecular Structure of Broadly Neutralizing Monoclonal Antibodies Isolated from Individuals Infected with HIV-1 not Inform the Rational Design of an HIV-1 Vaccine?

    Directory of Open Access Journals (Sweden)

    Marc H V Van Regenmortel

    2015-05-01

    Full Text Available It is commonly assumed that neutralizing Mabs that bind to the HIV-1 Env glycoprotein are more specific reagents than anti-HIV-1 polyclonal antisera and that knowledge of the structure of these Mabs facilitates the rational design of effective HIV-1 vaccine immunogens. However, after more than ten years of unsuccessful experimentation using the structure-based reverse vaccinology approach, it is now evident that it is not possible to infer from the structure of neutralizing Mabs which HIV immunogens induced their formation nor which vaccine immunogens will elicit similar Abs in an immunized host. The use of Mabs for developing an HIV-1 vaccine was counterproductive because it overlooked the fact that the apparent specificity of a Mab very much depends on the selection procedure used to obtain it and also did not take into account that an antibody is never monospecific for a single epitope but is always polyspecific for many epitopes. When the rationale of the proponents of the unsuccessful rational design strategy is analyzed, it appears that investigators who claim they are designing a vaccine immunogen are only improving the binding reactivity of a single epitope-paratope pair and are not actually designing an immunogen able to generate protective antibodies. The task of a designer consists in imagining what type of immunogen is likely to elicit a protective immune response but in the absence of knowledge regarding which features of the immune system are responsible for producing a functional neutralizing activity in antibodies, it is not feasible to intentionally optimize a potential immunogen candidate in order to obtain the desired outcome. The only available option is actually to test possible solutions by trial-and-error experiments until the preset goal is perhaps attained. Rational design and empirical approaches in HIV vaccine research should thus not be opposed as alternative options since empirical testing is an integral part of a so

  14. Neutralization of biological activity and inhibition of receptor binding by antibodies against human thrombopoietin.

    Science.gov (United States)

    Tahara, T; Kuwaki, T; Matsumoto, A; Morita, H; Watarai, H; Inagaki, Y; Ohashi, H; Ogami, K; Miyazaki, H; Kato, T

    1998-01-01

    Thrombopoietin (TPO) is a recently isolated cytokine that primarily regulates megakaryocytopoiesis and thrombopoiesis. We recently reported the development of a variety of antibodies (Abs) to synthetic peptides of human (h)TPO and to recombinant human TPO (rhTPO). In this study, we characterized the Abs and mapped immunologically distinct areas of the molecule. Among the five different antipeptide polyclonal Abs, only one, raised against synthetic peptide D8 to Q28, neutralized the TPO-dependent growth of FDCP-2 cells expressing human Mpl (FDCP-hMpl5 cells). One out of seven anti-rhTPO monoclonal Abs, designated as TN1, also showed neutralizing activity. TN1 was found to be specifically reactive with two proteolytic fragments, residues S1 to R117 and A60 to K122 of hTPO, indicating that the epitope(s) of TN1 is localized in residues A60 to R117 of the molecule. These two neutralizing Abs inhibited the binding of biotinylated rhTPO to FDCP-hMpl5 cells. On the other hand, the other Abs, which reacted with five polypeptides of S47 to D62, L108 to A126, N172 to A190, S262 to T284, and P306 to G332 of hTPO, did not show either the neutralizing activity or the ability to inhibit the binding of biotinylated rhTPO to the cell surface hMpl. These findings indicate that two regions, residues D8 to Q28 and A60 to R117 of hTPO, may contain the domains associated with its receptor, C-Mpl. These Abs characterized here are valuable for studying the structural analysis and the biological function of hTPO mediated by its receptor.

  15. Characterization of Two Human Monoclonal Antibodies Neutralizing Influenza A H7N9 Viruses

    Science.gov (United States)

    Wang, Jianmin; Chen, Zhe; Bao, Linlin; Zhang, Weijia; Xue, Ying; Pang, XingHuo; Zhang, Xi

    2015-01-01

    H7N9 was a cause of significant global health concern due to its severe infection and approximately 35% mortality in humans. By screening a Fab antibody phage library derived from patients who recovered from H7N9 infections, we characterized two human monoclonal antibodies (HuMAbs), HNIgGD5 and HNIgGH8. The epitope of these two antibodies was dependent on two residues in the receptor binding site at positions V186 and L226 of the hemagglutinin glycoprotein. Both antibodies possessed high neutralizing activity. PMID:26063436

  16. Generation and characterization of the human neutralizing antibody fragment Fab091 against rabies virus

    Institute of Scientific and Technical Information of China (English)

    Chen LI; Feng ZHANG; Hong LIN; Zhong-can WANG; Xin-jian LIU; Zhen-qing FENG; Jin ZHU; Xiao-hong GUAN

    2011-01-01

    Aim: To transform the human anti-rabies virus glycoprotein (anti-RABVG) single-chain variable fragment (scFv) into a Fab fragment and to analyze its immunological activity.Methods: The Fab gene was amplified using overlap PCR and inserted into the vector pComb3XSS. The recombinant vector was then transformed into E coli Top10F' for expression and purification. The purified Fab was characterized using SDS-PAGE, Western blotting,indirect ELISA, competitive ELISA, and the fluorescent antibody virus neutralization test (FAVN), respectively, and examined in a Kunming mouse challenge model in vivo.Results: A recombinant vector was constructed. The Fab was expressed in soluble form In E coll Top10F'. Specific binding of the Fab to rabies virus was confirmed by indirect ELISA and immunoprecipitation (IP). The neutralizing antibody titer of Fab was 10.26 IU/mL.The mouse group treated with both vaccine and human rabies immunoglobulin (HRIG)/Fab091 (32 IU/kg) showed protection against rabies, compared with the control group (P<0.05, Logrank test).Conclusion: The antibody fragment Fab was shown to be a neutralizing antibody against RABVG. It can be used together with other monoclonal antibodies for post-exposure prophylaxis of rabies virus in future studies.

  17. Cortisol has different effects on human memory for emotional and neutral stimuli.

    Science.gov (United States)

    Rimmele, Ulrike; Domes, Gregor; Mathiak, Klaus; Hautzinger, Martin

    2003-12-19

    Adrenal stress hormones are considered to play a role in memory enhancement of emotionally arousing events. To investigate the effects of cortisol on human emotional memory, subjects were administered hydrocortisone (25 mg) or placebo and presented with either an emotionally arousing or a neutral story. Memory for the story was tested 1 week later. In all memory tests, subjects who viewed the emotional story scored better for the emotionally arousing story parts, indicating that arousal enhances memory. In memory of details, cortisol showed an interaction with story valence but no main effect: cortisol enhanced memory for details of the neutral story version, but impaired memory for details of the emotionally arousing version. We thus confirm a non-linear interaction between cortisol and arousal on memory formation.

  18. Uptake of neutral alpha- and beta-amino acids by human proximal tubular cells

    DEFF Research Database (Denmark)

    Jessen, H; Røigaard, H; Jacobsen, Christian

    1996-01-01

    The transport characteristics of amino acids in primary cell cultures from the proximal tubule of human adults (AHKE cells) were examined, using alpha-aminoisobutyric acid (AIB) and beta-alanine as representatives of alpha- and beta-amino acids, respectively. The Na(+)-gradient dependent influx...... of AIB occurred by a single, saturable transport system, whereas the Na(+)-gradient dependent uptake data for beta-alanine could be described in terms of two-independent transport components as well as one-transport one-leak model with identical kinetic constants for the high-affinity system. Competition...... experiments revealed that all the neutral amino acids tested reduced the uptake of AIB, whereas there was no effect of taurine, L-aspartic acid, and L-arginine. By contrast, the influx of beta-alanine was only drastically reduced by beta-amino acids, whereas the inhibition by neutral alpha-amino acids...

  19. Human TNF cytokine neutralization with a vNAR from Heterodontus francisci shark

    Science.gov (United States)

    Camacho-Villegas, Tanya; Mata-Gonzalez, Teresa; Paniagua-Solis, Jorge; Sanchez, Edna; Licea, Alexei

    2013-01-01

    The therapeutic use of single domain antibodies (sdAbs) is a promising new approach because these small antibodies maintain antigen recognition and neutralization capacity, have thermal and chemical stability and have good solubility. In this study, using phage display technology, we isolated a variable domain of a IgNAR (vNAR) from a Heterodontus francisci shark immunized against the recombinant human cytokine TNFα (rhTNFα). One clone T43, which expresses the vNAR protein in the periplasmic space, was isolated from the fourth round of panning. T43 had the capacity to recognize rhTNF and neutralize it in vitro, indicating that T43 has potential as a therapeutic that can be used for diseases in which this pro-inflammatory cytokine needs to be controlled. PMID:23221782

  20. Structure of a Human Astrovirus Capsid-Antibody Complex and Mechanistic Insights into Virus Neutralization

    Energy Technology Data Exchange (ETDEWEB)

    Bogdanoff, Walter A.; Campos, Jocelyn; Perez, Edmundo I.; Yin, Lu; Alexander, David L.; DuBois, Rebecca M. (UCSC)

    2016-11-02

    ABSTRACT

    Human astroviruses (HAstVs) are a leading cause of viral diarrhea in young children, the immunocompromised, and the elderly. There are no vaccines or antiviral therapies against HAstV disease. Several lines of evidence point to the presence of protective antibodies in healthy adults as a mechanism governing protection against reinfection by HAstV. However, development of anti-HAstV therapies is hampered by the gap in knowledge of protective antibody epitopes on the HAstV capsid surface. Here, we report the structure of the HAstV capsid spike domain bound to the neutralizing monoclonal antibody PL-2. The antibody uses all six complementarity-determining regions to bind to a quaternary epitope on each side of the dimeric capsid spike. We provide evidence that the HAstV capsid spike is a receptor-binding domain and that the antibody neutralizes HAstV by blocking virus attachment to cells. We identify patches of conserved amino acids that overlap the antibody epitope and may comprise a receptor-binding site. Our studies provide a foundation for the development of therapies to prevent and treat HAstV diarrheal disease.

    IMPORTANCEHuman astroviruses (HAstVs) infect nearly every person in the world during childhood and cause diarrhea, vomiting, and fever. Despite the prevalence of this virus, little is known about how antibodies in healthy adults protect them against reinfection. Here, we determined the crystal structure of a complex of the HAstV capsid protein and a virus-neutralizing antibody. We show that the antibody binds to the outermost spike domain of the capsid, and we provide evidence that the antibody blocks virus attachment to human cells. Importantly, our findings suggest that a subunit-based vaccine focusing the immune system on the HAstV capsid spike domain could be effective in protecting children against HAstV disease.

  1. Research progress on HIV broadly neutralizing antibody 4E10%HIV广泛中和抗体4E10的研究进展

    Institute of Scientific and Technical Information of China (English)

    姜山; 谢青

    2009-01-01

    s by 4E10 in that conformation. The lipid binding ability may be necessary to its neutralizing activity. In this paper, the characteristic of 4E10 and its epitope and research on the meeha-nism of its epitope exposure is reviewed.%4E10是目前中和作用最广泛的HIV单克隆抗体.其抗原核心表位被证实为NWF(D/N)IT基序,但利用抗原表位氨基酸未能诱导产生类似4E10的中和抗体.研究发现,4E10可能是一种自身抗体而难以诱导产生,而且其表位氨基酸在自然状态大部分隐蔽于HIV包膜磷脂层中,不但难以诱导产生抗体,而且无法与4E10紧密结合.此文就4E10抗体的发现、自身抗体特性及其表位完全暴露机制方面的研究进展进行了综述.

  2. Host-Primed Ebola Virus GP Exposes a Hydrophobic NPC1 Receptor-Binding Pocket, Revealing a Target for Broadly Neutralizing Antibodies

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    Zachary A. Bornholdt

    2016-02-01

    Full Text Available The filovirus surface glycoprotein (GP mediates viral entry into host cells. Following viral internalization into endosomes, GP is cleaved by host cysteine proteases to expose a receptor-binding site (RBS that is otherwise hidden from immune surveillance. Here, we present the crystal structure of proteolytically cleaved Ebola virus GP to a resolution of 3.3 Å. We use this structure in conjunction with functional analysis of a large panel of pseudotyped viruses bearing mutant GP proteins to map the Ebola virus GP endosomal RBS at molecular resolution. Our studies indicate that binding of GP to its endosomal receptor Niemann-Pick C1 occurs in two distinct stages: the initial electrostatic interactions are followed by specific interactions with a hydrophobic trough that is exposed on the endosomally cleaved GP1 subunit. Finally, we demonstrate that monoclonal antibodies targeting the filovirus RBS neutralize all known filovirus GPs, making this conserved pocket a promising target for the development of panfilovirus therapeutics.

  3. Capillary electrophoresis separation of human milk neutral and acidic oligosaccharides derivatized with 2-aminoacridone.

    Science.gov (United States)

    Galeotti, Fabio; Coppa, Giovanni V; Zampini, Lucia; Maccari, Francesca; Galeazzi, Tiziana; Padella, Lucia; Santoro, Lucia; Gabrielli, Orazio; Volpi, Nicola

    2014-03-01

    Human milk is a unique fluid in glycobiology due to the presence of many free structurally complex oligosaccharides emerging as important dietary factors during early life and having many biological and protective functions. Methods that allow accurate profiling of oligosaccharide mixtures in this complex biological fluid with quantification of the four known genetically determined groups are welcomed. A high-voltage CE separation and detection at 254 nm of 17 neutral and acidic human milk oligosaccharide (HMO) standard along with lactose derivatized with 2-aminoacridone, using a BGE containing 20% methanol as an organic modifier and borate, able to form on-capillary anionic borate-polyol complexes, is reported. This CE approach was able to separate both neutral HMOs and acidic HMOs, with the sialic acid residue, also in the presence of lactose in high content. This method was applied to the four secretory groups individually extracted by a rapid and simple preparative step. LODs were found ranging from ∼50 to 700 fmol. We were able to measure HMO content also in the presence of excess fluorophore, or interference from proteins, peptides, salts, and other impurities normally present in this complex biological fluid. Overall, CE equipped with a UV detector is a common analytical approach and this simple CE separation offers high resolution and sensitivity for the differentiation of human milk samples related to genetic groups and days of lactation by considering that important changes in HMO content are a reflection of the lactation day.

  4. Production of neutralizing monoclonal antibody against human vascular endothelial growth factor receptor Ⅱ

    Institute of Scientific and Technical Information of China (English)

    Rong LI; Dong-sheng XIONG; Xiao-feng SHAO; Jia LIU; Yuan-fu XU; Yuan-sheng XU; Han-zhi LIU; Zhen-ping ZHU; Chun-zheng YANG

    2004-01-01

    AIM: To prepare neutralizing monoclonal antibody (mAb) against extracellular immunoglobulin (Ig)-like domainⅢ of vascular endothelial growth factor receptor KDR and study its biological activity. METHODS: Soluble KDR Ig domain Ⅲ (KDR-Ⅲ) fusion protein was expressed in E Coli and purified from the bacterial periplasmic extracts via an affinity chromatography. Monoclonal antibodies against KDR-Ⅲ were prepared by hybridoma technique. ELISA and FACS analysis were used to identify its specificity. Immunoprecipitation and [3H]-thymidine incorporation assay were also used to detect the activity of anti-KDR mAb blocking the phosphorylation of KDR tyrosine kinase receptor and the influence on vascular endothelial growth factor-induced mitogenesis of human endothelial ceils.RESULTS: A monoclonal antibody, Ycom1D3 (IgG1), was generated from a mouse immunized with the recombinant KDR-Ⅲ protein. Ycom1D3 bound specifically to both the soluble KDR-Ⅲ and the cell-surface expressed KDR. Ycom1D3 effectively blocked VEGF/KDR interaction and inhibited VEGF-stimulated KDR activation in human endothelial cells. Furthermore, the antibody efficiently neutralized VEGF-induced mitogenesis of human endothelial cells. CONCLUSION: Our results suggest that the anti-KDR mAb, Ycom1D3, has potential applications in the treatment of cancer and other diseases where pathological angiogenesis is involved.

  5. Stoichiometry of monoclonal antibody neutralization of T-cell line-adapted human immunodeficiency virus type 1

    DEFF Research Database (Denmark)

    Schønning, Kristian; Lund, O; Lund, O S;

    1999-01-01

    In order to study the stoichiometry of monoclonal antibody (MAb) neutralization of T-cell line-adapted human immunodeficiency virus type 1 (HIV-1) in antibody excess and under equilibrium conditions, we exploited the ability of HIV-1 to generate mixed oligomers when different env genes...... are coexpressed. By the coexpression of Env glycoproteins that either can or cannot bind a neutralizing MAb in an env transcomplementation assay, virions were generated in which the proportion of MAb binding sites could be regulated. As the proportion of MAb binding sites in Env chimeric virus increased, MAb...... neutralization gradually increased. Virus neutralization by virion aggregation was minimal, as MAb binding to HIV-1 Env did not interfere with an AMLV Env-mediated infection by HIV-1(AMLV/HIV-1) pseudotypes of CD4(-) HEK293 cells. MAb neutralization of chimeric virions could be described as a third...

  6. Both selective and neutral processes drive GC content evolution in the human genome

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    Cagliani Rachele

    2008-03-01

    Full Text Available Abstract Background Mammalian genomes consist of regions differing in GC content, referred to as isochores or GC-content domains. The scientific debate is still open as to whether such compositional heterogeneity is a selected or neutral trait. Results Here we analyze SNP allele frequencies, retrotransposon insertion polymorphisms (RIPs, as well as fixed substitutions accumulated in the human lineage since its divergence from chimpanzee to indicate that biased gene conversion (BGC has been playing a role in within-genome GC content variation. Yet, a distinct contribution to GC content evolution is accounted for by a selective process. Accordingly, we searched for independent evidences that GC content distribution does not conform to neutral expectations. Indeed, after correcting for possible biases, we show that intron GC content and size display isochore-specific correlations. Conclusion We consider that the more parsimonious explanation for our results is that GC content is subjected to the action of both weak selection and BGC in the human genome with features such as nucleosome positioning or chromatin conformation possibly representing the final target of selective processes. This view might reconcile previous contrasting findings and add some theoretical background to recent evidences suggesting that GC content domains display different behaviors with respect to highly regulated biological processes such as developmentally-stage related gene expression and programmed replication timing during neural stem cell differentiation.

  7. Germline V-genes sculpt the binding site of a family of antibodies neutralizing human cytomegalovirus

    Energy Technology Data Exchange (ETDEWEB)

    Thomson, Christy A.; Bryson, Steve; McLean, Gary R.; Creagh, A. Louise; Pai, Emil F.; Schrader, John W. (Toronto); (UBC)

    2008-10-17

    Immunoglobulin genes are generated somatically through specialized mechanisms resulting in a vast repertoire of antigen-binding sites. Despite the stochastic nature of these processes, the V-genes that encode most of the antigen-combining site are under positive evolutionary selection, raising the possibility that V-genes have been selected to encode key structural features of binding sites of protective antibodies against certain pathogens. Human, neutralizing antibodies to human cytomegalovirus that bind the AD-2S1 epitope on its gB envelope protein repeatedly use a pair of well-conserved, germline V-genes IGHV3-30 and IGKV3-11. Here, we present crystallographic, kinetic and thermodynamic analyses of the binding site of such an antibody and that of its primary immunoglobulin ancestor. These show that these germline V-genes encode key side chain contacts with the viral antigen and thereby dictate key structural features of the hypermutated, high-affinity neutralizing antibody. V-genes may thus encode an innate, protective immunological memory that targets vulnerable, invariant sites on multiple pathogens.

  8. Crystal structure and size-dependent neutralization properties of HK20, a human monoclonal antibody binding to the highly conserved heptad repeat 1 of gp41.

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    Charles Sabin

    Full Text Available The human monoclonal antibody (mAb HK20 neutralizes a broad spectrum of primary HIV-1 isolates by targeting the highly conserved heptad repeat 1 (HR1 of gp41, which is transiently exposed during HIV-1 entry. Here we present the crystal structure of the HK20 Fab in complex with a gp41 mimetic 5-Helix at 2.3 Å resolution. HK20 employs its heavy chain CDR H2 and H3 loops to bind into a conserved hydrophobic HR1 pocket that is occupied by HR2 residues in the gp41 post fusion conformation. Compared to the previously described HR1-specific mAb D5, HK20 approaches its epitope with a different angle which might favor epitope access and thus contribute to its higher neutralization breadth and potency. Comparison of the neutralization activities of HK20 IgG, Fab and scFv employing both single cycle and multiple cycle neutralization assays revealed much higher potencies for the smaller Fab and scFv over IgG, implying that the target site is difficult to access for complete antibodies. Nevertheless, two thirds of sera from HIV-1 infected individuals contain significant titers of HK20-inhibiting antibodies. The breadth of neutralization of primary isolates across all clades, the higher potencies for C-clade viruses and the targeting of a distinct site as compared to the fusion inhibitor T-20 demonstrate the potential of HK20 scFv as a therapeutic tool.

  9. The molecular basis for the broad substrate specificity of human sulfotransferase 1A1.

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    Ilana Berger

    Full Text Available Cytosolic sulfotransferases (SULTs are mammalian enzymes that detoxify a wide variety of chemicals through the addition of a sulfate group. Despite extensive research, the molecular basis for the broad specificity of SULTs is still not understood. Here, structural, protein engineering and kinetic approaches were employed to obtain deep understanding of the molecular basis for the broad specificity, catalytic activity and substrate inhibition of SULT1A1. We have determined five new structures of SULT1A1 in complex with different acceptors, and utilized a directed evolution approach to generate SULT1A1 mutants with enhanced thermostability and increased catalytic activity. We found that active site plasticity enables binding of different acceptors and identified dramatic structural changes in the SULT1A1 active site leading to the binding of a second acceptor molecule in a conserved yet non-productive manner. Our combined approach highlights the dominant role of SULT1A1 structural flexibility in controlling the specificity and activity of this enzyme.

  10. Two amino acid residues confer type specificity to a neutralizing, conformationally dependent epitope on human papillomavirus type 11.

    Science.gov (United States)

    Ludmerer, S W; Benincasa, D; Mark, G E

    1996-01-01

    Characterization of virus binding by neutralizing antibodies is important both in understanding early events in viral infectivity and in development of vaccines. Neutralizing monoclonal antibodies (MAbs) to human papillomavirus type 11 (HPV11) have been described, but mapping the binding site has been difficult because of the conformational nature of key type-specific neutralization epitopes on the L1 coat protein. We have determined those residues of the L1 protein of HPV11 which confer type specificity to the binding of HPV11-neutralizing MAbs. Binding of three HPV11-specific neutralizing MAbs could be redirected to HPV6 L1 virus-like particles in which as few as two substitutions of corresponding amino acid residues from HPV11 L1 have been made, thus demonstrating the importance of these residues to MAb binding through the transfer of a conformationally dependent epitope. In addition, a fourth neutralizing MAb could be distinguished from the other neutralizing MAbs in terms of the amino acid residues which affect binding, suggesting the possibility that it neutralizes HPV11 through a different mechanism. PMID:8676509

  11. Advance in identification and analysis of epitope specificities of broadly neutralizing anti-HIV-1 sera%抗HIV广谱中和血清表位特异性研究进展

    Institute of Scientific and Technical Information of China (English)

    胡新韬; 洪坤学; 邵一鸣

    2011-01-01

    HIT疫苗研究的巨大障碍在于目前设计的包含Env蛋白或表住的疲苗在临床前及临床试验中难于诱导产生广谱中和抗体.近来的研究表明一些HIV-1感染者血清中存在的抗体能够中和多种病毒株,而新的表位作图技术对这些广谱中和血清表位特异性的深入分析将对广谱中和抗体靶向的病毒表位提供新的线索.通过广谱中和血清表位特异性研究获得的信息将促进新的广谱中和单克隆抗体的分离和潜在新表住的鉴定,为合理设计新的疫苗免疫原提供关键信息.%One obstacle to the development of an effective HIV-1 vaccine has been the difficulty in inducing broadly neutralizing antibodies with protective functions. However, recent studies show that antibodies in the sera of some HIV-1 infected individuals can neutralize diverse HIV-1 isolates. Detailed analyses of these sera with recently developed sophistical mapping methods can provide new insights into the viral epitopes targeted by broadly reactive NAbs. The new information emerging from these mapping efforts may be conducive to sharpening efforts to isolate new bnmAbs and moreover, may provide crucial information for the rational design of novel vaccine candidates.

  12. Complete epitopes for vaccine design derived from a crystal structure of the broadly neutralizing antibodies PGT128 and 8ANC195 in complex with an HIV-1 Env trimer.

    Science.gov (United States)

    Kong, Leopold; Torrents de la Peña, Alba; Deller, Marc C; Garces, Fernando; Sliepen, Kwinten; Hua, Yuanzi; Stanfield, Robyn L; Sanders, Rogier W; Wilson, Ian A

    2015-10-01

    The HIV-1 envelope gp160 glycoprotein (Env) is a trimer of gp120 and gp41 heterodimers that mediates cell entry and is the primary target of the humoral immune response. Broadly neutralizing antibodies (bNAbs) to HIV-1 have revealed multiple epitopes or sites of vulnerability, but mapping of most of these sites is incomplete owing to a paucity of structural information on the full epitope in the context of the Env trimer. Here, a crystal structure of the soluble BG505 SOSIP gp140 trimer at 4.6 Å resolution with the bNAbs 8ANC195 and PGT128 reveals additional interactions in comparison to previous antibody-gp120 structures. For 8ANC195, in addition to previously documented interactions with gp120, a substantial interface with gp41 is now elucidated that includes extensive interactions with the N637 glycan. Surprisingly, removal of the N637 glycan did not impact 8ANC195 affinity, suggesting that the antibody has evolved to accommodate this glycan without loss of binding energy. PGT128 indirectly affects the N262 glycan by a domino effect, in which PGT128 binds to the N301 glycan, which in turn interacts with and repositions the N262 glycan, thereby illustrating the important role of neighboring glycans on epitope conformation and stability. Comparisons with other Env trimer and gp120 structures support an induced conformation for glycan N262, suggesting that the glycan shield is allosterically modified upon PGT128 binding. These complete epitopes of two broadly neutralizing antibodies on the Env trimer can now be exploited for HIV-1 vaccine design.

  13. Neutralization of botulinum neurotoxin by a human monoclonal antibody specific for the catalytic light chain.

    Directory of Open Access Journals (Sweden)

    Sharad P Adekar

    Full Text Available BACKGROUND: Botulinum neurotoxins (BoNT are a family of category A select bioterror agents and the most potent biological toxins known. Cloned antibody therapeutics hold considerable promise as BoNT therapeutics, but the therapeutic utility of antibodies that bind the BoNT light chain domain (LC, a metalloprotease that functions in the cytosol of cholinergic neurons, has not been thoroughly explored. METHODS AND FINDINGS: We used an optimized hybridoma method to clone a fully human antibody specific for the LC of serotype A BoNT (BoNT/A. The 4LCA antibody demonstrated potent in vivo neutralization when administered alone and collaborated with an antibody specific for the HC. In Neuro-2a neuroblastoma cells, the 4LCA antibody prevented the cleavage of the BoNT/A proteolytic target, SNAP-25. Unlike an antibody specific for the HC, the 4LCA antibody did not block entry of BoNT/A into cultured cells. Instead, it was taken up into synaptic vesicles along with BoNT/A. The 4LCA antibody also directly inhibited BoNT/A catalytic activity in vitro. CONCLUSIONS: An antibody specific for the BoNT/A LC can potently inhibit BoNT/A in vivo and in vitro, using mechanisms not previously associated with BoNT-neutralizing antibodies. Antibodies specific for BoNT LC may be valuable components of an antibody antidote for BoNT exposure.

  14. Krüppeling erythropoiesis: an unexpected broad spectrum of human red blood cell disorders due to KLF1 variants.

    Science.gov (United States)

    Perkins, Andrew; Xu, Xiangmin; Higgs, Douglas R; Patrinos, George P; Arnaud, Lionel; Bieker, James J; Philipsen, Sjaak

    2016-04-14

    Until recently our approach to analyzing human genetic diseases has been to accurately phenotype patients and sequence the genes known to be associated with those phenotypes; for example, in thalassemia, the globin loci are analyzed. Sequencing has become increasingly accessible, and thus a larger panel of genes can be analyzed and whole exome and/or whole genome sequencing can be used when no variants are found in the candidate genes. By using such approaches in patients with unexplained anemias, we have discovered that a broad range of hitherto unrelated human red cell disorders are caused by variants in KLF1, a master regulator of erythropoiesis, which were previously considered to be extremely rare causes of human genetic disease.

  15. Leptospirosis in animals and human contacts in Egypt: broad range surveillance

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    Ahmed Samir

    2015-06-01

    Full Text Available INTRODUCTION: Leptospirosis is a re-emerging zoonotic disease of humans and animals worldwide. The disease is caused by pathogenic species of the genus Leptospira. These organisms are maintained in nature via chronic renal infection of carrier animals, which excrete the organisms in their urine. Humans become infected through direct or indirect exposure to infected animals and their urine or through contact with contaminated water and soil. This study was conducted to investigate Leptospira infections as a re-emerging zoonosis that has been neglected in Egypt. METHODS: Samples from 1,250 animals (270 rats, 168 dogs, 625 cows, 26 buffaloes, 99 sheep, 14 horses, 26 donkeys and 22 camels, 175 human contacts and 45 water sources were collected from different governorates in Egypt. The samples were collected from different body sites and prepared for culture, PCR and the microscopic agglutination test (MAT. RESULTS: The isolation rates of Leptospira serovars were 6.9%, 11.3% and 1.1% for rats, dogs and cows, respectively, whereas the PCR results revealed respective detection rates of 24%, 11.3% and 1.1% for rats, dogs and cows. Neither the other examined animal species nor humans yielded positive results via these two techniques. Only six Leptospira serovars (Icterohaemorrhagiae, Pomona, Canicola, Grippotyphosa, Celledoni and Pyrogenes could be isolated from rats, dogs and cows. Moreover, the seroprevalence of leptospiral antibodies among the examined humans determined using MAT was 49.7%. CONCLUSIONS: The obtained results revealed that rats, dogs and cows were the most important animal reservoirs for leptospirosis in Egypt, and the high seroprevalence among human contacts highlights the public health implications of this neglected zoonosis.

  16. Broad blockade antibody responses in human volunteers after immunization with a multivalent norovirus VLP candidate vaccine: immunological analyses from a phase I clinical trial.

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    Lisa C Lindesmith

    2015-03-01

    Full Text Available Human noroviruses (NoVs are the primary cause of acute gastroenteritis and are characterized by antigenic variation between genogroups and genotypes and antigenic drift of strains within the predominant GII.4 genotype. In the context of this diversity, an effective NoV vaccine must elicit broadly protective immunity. We used an antibody (Ab binding blockade assay to measure the potential cross-strain protection provided by a multivalent NoV virus-like particle (VLP candidate vaccine in human volunteers.Sera from ten human volunteers immunized with a multivalent NoV VLP vaccine (genotypes GI.1/GII.4 were analyzed for IgG and Ab blockade of VLP interaction with carbohydrate ligand, a potential correlate of protective immunity to NoV infection and illness. Immunization resulted in rapid rises in IgG and blockade Ab titers against both vaccine components and additional VLPs representing diverse strains and genotypes not represented in the vaccine. Importantly, vaccination induced blockade Ab to two novel GII.4 strains not in circulation at the time of vaccination or sample collection. GII.4 cross-reactive blockade Ab titers were more potent than responses against non-GII.4 VLPs, suggesting that previous exposure history to this dominant circulating genotype may impact the vaccine Ab response. Further, antigenic cartography indicated that vaccination preferentially activated preexisting Ab responses to epitopes associated with GII.4.1997. Study interpretations may be limited by the relevance of the surrogate neutralization assay and the number of immunized participants evaluated.Vaccination with a multivalent NoV VLP vaccine induces a broadly blocking Ab response to multiple epitopes within vaccine and non-vaccine NoV strains and to novel antigenic variants not yet circulating at the time of vaccination. These data reveal new information about complex NoV immune responses to both natural exposure and to vaccination, and support the potential

  17. Widespread Epigenetic Abnormalities Suggest a Broad DNA Methylation Erasure Defect in Abnormal Human Sperm

    Science.gov (United States)

    Siegmund, Kimberly; Yang, Allen; Laird, Peter W.; Sokol, Rebecca Z.

    2007-01-01

    Background Male-factor infertility is a common condition, and etiology is unknown for a high proportion of cases. Abnormal epigenetic programming of the germline is proposed as a possible mechanism compromising spermatogenesis of some men currently diagnosed with idiopathic infertility. During germ cell maturation and gametogenesis, cells of the germ line undergo extensive epigenetic reprogramming. This process involves widespread erasure of somatic-like patterns of DNA methylation followed by establishment of sex-specific patterns by de novo DNA methylation. Incomplete reprogramming of the male germ line could, in theory, result in both altered sperm DNA methylation and compromised spermatogenesis. Methodology/Principal Finding We determined concentration, motility and morphology of sperm in semen samples collected by male members of couples attending an infertility clinic. Using MethyLight and Illumina assays we measured methylation of DNA isolated from purified sperm from the same samples. Methylation at numerous sequences was elevated in DNA from poor quality sperm. Conclusions This is the first report of a broad epigenetic defect associated with abnormal semen parameters. Our results suggest that the underlying mechanism for these epigenetic changes may be improper erasure of DNA methylation during epigenetic reprogramming of the male germ line. PMID:18074014

  18. Widespread epigenetic abnormalities suggest a broad DNA methylation erasure defect in abnormal human sperm.

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    Sahar Houshdaran

    Full Text Available BACKGROUND: Male-factor infertility is a common condition, and etiology is unknown for a high proportion of cases. Abnormal epigenetic programming of the germline is proposed as a possible mechanism compromising spermatogenesis of some men currently diagnosed with idiopathic infertility. During germ cell maturation and gametogenesis, cells of the germ line undergo extensive epigenetic reprogramming. This process involves widespread erasure of somatic-like patterns of DNA methylation followed by establishment of sex-specific patterns by de novo DNA methylation. Incomplete reprogramming of the male germ line could, in theory, result in both altered sperm DNA methylation and compromised spermatogenesis. METHODOLOGY/PRINCIPAL FINDING: We determined concentration, motility and morphology of sperm in semen samples collected by male members of couples attending an infertility clinic. Using MethyLight and Illumina assays we measured methylation of DNA isolated from purified sperm from the same samples. Methylation at numerous sequences was elevated in DNA from poor quality sperm. CONCLUSIONS: This is the first report of a broad epigenetic defect associated with abnormal semen parameters. Our results suggest that the underlying mechanism for these epigenetic changes may be improper erasure of DNA methylation during epigenetic reprogramming of the male germ line.

  19. Structural and molecular basis for Ebola virus neutralization by protective human antibodies.

    Science.gov (United States)

    Misasi, John; Gilman, Morgan S A; Kanekiyo, Masaru; Gui, Miao; Cagigi, Alberto; Mulangu, Sabue; Corti, Davide; Ledgerwood, Julie E; Lanzavecchia, Antonio; Cunningham, James; Muyembe-Tamfun, Jean Jacques; Baxa, Ulrich; Graham, Barney S; Xiang, Ye; Sullivan, Nancy J; McLellan, Jason S

    2016-03-18

    Ebola virus causes hemorrhagic fever with a high case fatality rate for which there is no approved therapy. Two human monoclonal antibodies, mAb100 and mAb114, in combination, protect nonhuman primates against all signs of Ebola virus disease, including viremia. Here, we demonstrate that mAb100 recognizes the base of the Ebola virus glycoprotein (GP) trimer, occludes access to the cathepsin-cleavage loop, and prevents the proteolytic cleavage of GP that is required for virus entry. We show that mAb114 interacts with the glycan cap and inner chalice of GP, remains associated after proteolytic removal of the glycan cap, and inhibits binding of cleaved GP to its receptor. These results define the basis of neutralization for two protective antibodies and may facilitate development of therapies and vaccines.

  20. Sociability and gazing toward humans in dogs and wolves: Simple behaviors with broad implications.

    Science.gov (United States)

    Bentosela, Mariana; Wynne, C D L; D'Orazio, M; Elgier, A; Udell, M A R

    2016-01-01

    Sociability, defined as the tendency to approach and interact with unfamiliar people, has been found to modulate some communicative responses in domestic dogs, including gaze behavior toward the human face. The objective of this study was to compare sociability and gaze behavior in pet domestic dogs and in human-socialized captive wolves in order to identify the relative influence of domestication and learning in the development of the dog-human bond. In Experiment 1, we assessed the approach behavior and social tendencies of dogs and wolves to a familiar and an unfamiliar person. In Experiment 2, we compared the animal's duration of gaze toward a person's face in the presence of food, which the animals could see but not access. Dogs showed higher levels of interspecific sociability than wolves in all conditions, including those where attention was unavailable. In addition, dogs gazed longer at the person's face than wolves in the presence of out-of-reach food. The potential contributions of domestication, associative learning, and experiences during ontogeny to prosocial behavior toward humans are discussed.

  1. Increase of a BLSS closure using mineralized human waste in plant cultivation on a neutral substrate

    Science.gov (United States)

    Gros, Jean-Bernard; Ushakova, Sofya; Tikhomirov, Alexander A.; Kudenko, Yurii; Lasseur, Christophe; Shikhov, V.; Anischenko, O.

    The purpose of this work was to study the full-scale potential use of human mineralized waste (feces and urine) as a source of mineral elements for plants cultivation in a Biological Life Support System. The plants which are potential candidates for a photosynthesizing link were grown on a neutral solution containing human mineralized waste. Spring wheat Triticum aestivum L., peas Pisum sativum L. Ambrosia cultivar and leaf lettuce Lactuca sativa L., Vitamin variety, were taken as the investigation objects. The plants were grown by hydroponics method on expanded clay aggregates in a vegetation chamber in constant environmental conditions. During the plants growth a definite amount of human mineralized waste was added daily in the nutrient solution. The nutrient solution was not changed during the entire vegetation period. Estimation of the plant needs in macro elements was based on a total biological productivity equal to 0.04 kg.day--1 .m-2 . As the plant requirements in potassium exceeded the potassium content in human waste, water extract of wheat straw containing the required potassium amount was added to the nutrient solution. Knop's solution was used in the control experiments. The experiment and control plants did not show significant differences in their photosynthetic apparatus state and productivity. A small decrease in total productivity of the experimental plants was observed which can result in some reduction of ˆ2 production in a BLSS. Most I probably it is due to the reduced nitrogen use. Therefore in a real BLSS after the mineralization of human feces and urine, it will be efficient to implement a more complete oxidation of nitrogencontaining compounds system, including nitrification. In this case the plants, prospective representatives of the BLSS photosynthesizing unit, could be cultivated on the solutions mainly based on human mineralized waste.

  2. Broad T-cell receptor repertoire in T-lymphocytes derived from human induced pluripotent stem cells.

    Directory of Open Access Journals (Sweden)

    Chia-Wei Chang

    Full Text Available Human induced pluripotent stem cells (hiPSCs have enormous potential for the treatment of inherited and acquired disorders. Recently, antigen-specific T lymphocytes derived from hiPSCs have been reported. However, T lymphocyte populations with broad T cell receptor (TCR diversity have not been generated. We report that hiPSCs derived from skin biopsy are capable of producing T lymphocyte populations with a broad TCR repertoire. In vitro T cell differentiation follows a similar developmental program as observed in vivo, indicated by sequential expression of CD7, intracellular CD3 and surface CD3. The γδ TCR locus is rearranged first and is followed by rearrangement of the αβ locus. Both γδ and αβ T cells display a diverse TCR repertoire. Upon activation, the cells express CD25, CD69, cytokines (TNF-α, IFN-γ, IL-2 and cytolytic proteins (Perforin and Granzyme-B. These results suggest that most, if not all, mechanisms required to generate functional T cells with a broad TCR repertoire are intact in our in vitro differentiation protocol. These data provide a foundation for production of patient-specific T cells for the treatment of acquired or inherited immune disorders and for cancer immunotherapy.

  3. Selection and characterization of a human neutralizing antibody to human fibroblast growth factor-2

    Energy Technology Data Exchange (ETDEWEB)

    Tao, Jun [Department of Immunology, School of Basic Medical Science, Southern Medical University, Guangzhou 510515 (China); Xiang, Jun-Jian, E-mail: txjj@jnu.edu.cn [Laboratory of Antibody Engineering, College of Life Sciences and Technologies, Jinan University, Guangzhou 510632 (China); Department of Immunology, School of Basic Medical Science, Southern Medical University, Guangzhou 510515 (China); Li, Dan [Department of Immunology, School of Basic Medical Science, Southern Medical University, Guangzhou 510515 (China); Deng, Ning; Wang, Hong; Gong, Yi-Ping [Laboratory of Antibody Engineering, College of Life Sciences and Technologies, Jinan University, Guangzhou 510632 (China)

    2010-04-09

    Compelling evidences suggest that fibroblast growth factor-2 (FGF-2) plays important roles in tumor growth, angiogenesis and metastasis. Molecules blocking the FGF-2 signaling have been proposed as anticancer agents. Through screening of a human scFv phage display library, we have isolated several human single-chain Fv fragments (scFvs) that bind to human FGF-2. After expression and purification in bacteria, one scFv, named 1A2, binds to FGF-2 with a high affinity and specificity, and completes with FGF-2 binding to its receptor. This 1A2 scFv was then cloned into the pIgG1 vector and expressed in 293T cells. The purified hIgG1-1A2 antibody showed a high binding affinity of 8 x 10{sup -9} M to rhFGF-2. In a set of vitro assays, it inhibited various biological activities of FGF-2 such as the proliferation, migration and tube formation of human umbilical vein endothelial cells. More importantly, hIgG1-1A2 antibody also efficiently blocked the growth while inducing apoptosis of glioma cells. For the first time, we generated a human anti-FGF-2 antibody with proven in vitro anti-tumor activity. It may therefore present a new therapeutic candidate for the treatment of cancers that are dependent on FGF-2 signaling for growth and survival.

  4. "Unconventional" Neutralizing Activity of Antibodies Against HIV

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Neutralizing antibodies are recognized to be one of the essential elements of the adaptive immune response that must be induced by an effective vaccine against HIV. However, only a limited number of antibodies have been identified to neutralize a broad range of primary isolates of HIV-1 and attempts to induce such antibodies by immunization were unsuccessful. The difficulties to generate such antibodies are mainly due to intrinsic properties of HIV-1 envelope spikes, such as high sequence diversity, heavy glycosylation, and inducible and transient nature of certain epitopes. In vitro neutralizing antibodies are identified using "conventional" neutralization assay which uses phytohemagglutinin (PHA)-stimulated human PBMCs as target cells. Thus, in essence the assay evaluates HIV-1 replication in CD4+ T cells. Recently, several laboratories including us demonstrated that some monoclonal antibodies and HIV-1-specific polyclonal IgG purified from patient sera, although they do not have neutralizing activity when tested by the "conventional" neutralization assay, do exhibit potent and broad neutralizing activity in "unconventional" ways. The neutralizing activity of these antibodies and IgG fractions is acquired through post-translational modifications, through opsonization of virus particles into macrophages and immature dendritic cells (iDCs), or through expression of antibodies on the surface of HIV-1-susceptible cells. This review will focus on recent findings of this area and point out their potential applications in the development of preventive strategies against HIV.

  5. Viral escape from HIV-1 neutralizing antibodies drives increased plasma neutralization breadth through sequential recognition of multiple epitopes and immunotypes.

    Directory of Open Access Journals (Sweden)

    Constantinos Kurt Wibmer

    2013-10-01

    Full Text Available Identifying the targets of broadly neutralizing antibodies to HIV-1 and understanding how these antibodies develop remain important goals in the quest to rationally develop an HIV-1 vaccine. We previously identified a participant in the CAPRISA Acute Infection Cohort (CAP257 whose plasma neutralized 84% of heterologous viruses. In this study we showed that breadth in CAP257 was largely due to the sequential, transient appearance of three distinct broadly neutralizing antibody specificities spanning the first 4.5 years of infection. The first specificity targeted an epitope in the V2 region of gp120 that was also recognized by strain-specific antibodies 7 weeks earlier. Specificity for the autologous virus was determined largely by a rare N167 antigenic variant of V2, with viral escape to the more common D167 immunotype coinciding with the development of the first wave of broadly neutralizing antibodies. Escape from these broadly neutralizing V2 antibodies through deletion of the glycan at N160 was associated with exposure of an epitope in the CD4 binding site that became the target for a second wave of broadly neutralizing antibodies. Neutralization by these CD4 binding site antibodies was almost entirely dependent on the glycan at position N276. Early viral escape mutations in the CD4 binding site drove an increase in wave two neutralization breadth, as this second wave of heterologous neutralization matured to recognize multiple immunotypes within this site. The third wave targeted a quaternary epitope that did not overlap any of the four known sites of vulnerability on the HIV-1 envelope and remains undefined. Altogether this study showed that the human immune system is capable of generating multiple broadly neutralizing antibodies in response to a constantly evolving viral population that exposes new targets as a consequence of escape from earlier neutralizing antibodies.

  6. Clinical regressions and broad immune activation following combination therapy targeting human NKT cells in myeloma.

    Science.gov (United States)

    Richter, Joshua; Neparidze, Natalia; Zhang, Lin; Nair, Shiny; Monesmith, Tamara; Sundaram, Ranjini; Miesowicz, Fred; Dhodapkar, Kavita M; Dhodapkar, Madhav V

    2013-01-17

    Natural killer T (iNKT) cells can help mediate immune surveillance against tumors in mice. Prior studies targeting human iNKT cells were limited to therapy of advanced cancer and led to only modest activation of innate immunity. Clinical myeloma is preceded by an asymptomatic precursor phase. Lenalidomide was shown to mediate antigen-specific costimulation of human iNKT cells. We treated 6 patients with asymptomatic myeloma with 3 cycles of combination of α-galactosylceramide-loaded monocyte-derived dendritic cells and low-dose lenalidomide. Therapy was well tolerated and led to reduction in tumor-associated monoclonal immunoglobulin in 3 of 4 patients with measurable disease. Combination therapy led to activation-induced decline in measurable iNKT cells and activation of NK cells with an increase in NKG2D and CD56 expression. Treatment also led to activation of monocytes with an increase in CD16 expression. Each cycle of therapy was associated with induction of eosinophilia as well as an increase in serum soluble IL2 receptor. Clinical responses correlated with pre-existing or treatment-induced antitumor T-cell immunity. These data demonstrate synergistic activation of several innate immune cells by this combination and the capacity to mediate tumor regression. Combination therapies targeting iNKT cells may be of benefit toward prevention of cancer in humans.

  7. Molecular signatures of hemagglutinin stem-directed heterosubtypic human neutralizing antibodies against influenza A viruses.

    Directory of Open Access Journals (Sweden)

    Yuval Avnir

    2014-05-01

    Full Text Available Recent studies have shown high usage of the IGHV1-69 germline immunoglobulin gene for influenza hemagglutinin stem-directed broadly-neutralizing antibodies (HV1-69-sBnAbs. Here we show that a major structural solution for these HV1-69-sBnAbs is achieved through a critical triad comprising two CDR-H2 loop anchor residues (a hydrophobic residue at position 53 (Ile or Met and Phe54, and CDR-H3-Tyr at positions 98±1; together with distinctive V-segment CDR amino acid substitutions that occur in positions sparse in AID/polymerase-η recognition motifs. A semi-synthetic IGHV1-69 phage-display library screen designed to investigate AID/polη restrictions resulted in the isolation of HV1-69-sBnAbs that featured a distinctive Ile52Ser mutation in the CDR-H2 loop, a universal CDR-H3 Tyr at position 98 or 99, and required as little as two additional substitutions for heterosubtypic neutralizing activity. The functional importance of the Ile52Ser mutation was confirmed by mutagenesis and by BCR studies. Structural modeling suggests that substitution of a small amino acid at position 52 (or 52a facilitates the insertion of CDR-H2 Phe54 and CDR-H3-Tyr into adjacent pockets on the stem. These results support the concept that activation and expansion of a defined subset of IGHV1-69-encoded B cells to produce potent HV1-69-sBnAbs does not necessarily require a heavily diversified V-segment acquired through recycling/reentry into the germinal center; rather, the incorporation of distinctive amino acid substitutions by Phase 2 long-patch error-prone repair of AID-induced mutations or by random non-AID SHM events may be sufficient. We propose that these routes of B cell maturation should be further investigated and exploited as a pathway for HV1-69-sBnAb elicitation by vaccination.

  8. Stoichiometry of monoclonal antibody neutralization of T-cell line-adapted human immunodeficiency virus type 1

    DEFF Research Database (Denmark)

    Schønning, Kristian; Lund, O; Lund, O S;

    1999-01-01

    In order to study the stoichiometry of monoclonal antibody (MAb) neutralization of T-cell line-adapted human immunodeficiency virus type 1 (HIV-1) in antibody excess and under equilibrium conditions, we exploited the ability of HIV-1 to generate mixed oligomers when different env genes are coexpr......In order to study the stoichiometry of monoclonal antibody (MAb) neutralization of T-cell line-adapted human immunodeficiency virus type 1 (HIV-1) in antibody excess and under equilibrium conditions, we exploited the ability of HIV-1 to generate mixed oligomers when different env genes...

  9. Antiviral Activity of Favipiravir (T-705) against a Broad Range of Paramyxoviruses In Vitro and against Human Metapneumovirus in Hamsters.

    Science.gov (United States)

    Jochmans, D; van Nieuwkoop, S; Smits, S L; Neyts, J; Fouchier, R A M; van den Hoogen, B G

    2016-08-01

    The clinical impact of infections with respiratory viruses belonging to the family Paramyxoviridae argues for the development of antiviral therapies with broad-spectrum activity. Favipiravir (T-705) has demonstrated potent antiviral activity against multiple RNA virus families and is presently in clinical evaluation for the treatment of influenza. Here we demonstrate in vitro activity of T-705 against the paramyxoviruses human metapneumovirus (HMPV), respiratory syncytial virus, human parainfluenza virus, measles virus, Newcastle disease virus, and avian metapneumovirus. In addition, we demonstrate activity against HMPV in hamsters. T-705 treatment inhibited replication of all paramyxoviruses tested in vitro, with 90% effective concentration (EC90) values of 8 to 40 μM. Treatment of HMPV-challenged hamsters with T-705 at 200 mg/kg of body weight/day resulted in 100% protection from infection of the lungs. In all treated and challenged animals, viral RNA remained detectable in the respiratory tract. The observation that T-705 treatment had a significant effect on infectious viral titers, with a limited effect on viral genome titers, is in agreement with its proposed mode of action of viral mutagenesis. However, next-generation sequencing of viral genomes isolated from treated and challenged hamsters did not reveal (hyper)mutation. Polymerase activity assays revealed a specific effect of T-705 on the activity of the HMPV polymerase. With the reported antiviral activity of T-705 against a broad range of RNA virus families, this small molecule is a promising broad-range antiviral drug candidate for limiting the viral burden of paramyxoviruses and for evaluation for treatment of infections with (re)emerging viruses, such as the henipaviruses.

  10. Mechanistic studies of flux variability of neutral and ionic permeants during constant current dc iontophoresis with human epidermal membrane.

    Science.gov (United States)

    Li, S Kevin; Higuchi, William I; Kochambilli, Rajan P; Zhu, Honggang

    2004-04-01

    Although constant current iontophoresis is supposed to provide constant transdermal transport, significant flux variability and/or time-dependent flux drifts are observed during iontophoresis with human skin in vitro and human studies in vivo. The objectives of the present study were to determine (a) the causes of flux variability in constant current dc transdermal iontophoresis and (b) the relationships of flux variabilities among permeants of different physicochemical properties. Changes in the human epidermal membrane (HEM) effective pore size and/or electroosmosis during constant current dc iontophoresis were examined. Tetraethylammonium ion (TEA), urea, and mannitol were the model permeants. For the neutral permeants, the results in the present study showed a significant increase of fluxes with time in a given experiment and large HEM sample-to-sample variability. Although both effective pore size and pore charge density variations contributed to the time-dependent flux drifts observed in electroosmotic transport, the significant flux drifts observed were found to be primarily a result of the time-dependent increase in effective pore charge density. For the ionic permeant, the observed flux variability was smaller than that of the neutral permeants and was believed to be primarily due to effective pore size alteration in HEM during iontophoresis as suggested in a previous study. The different extents of flux variability observed between neutral and ionic permeants are consistent with the different iontophoretically enhanced transport mechanisms for the neutral and ionic permeants (i.e. electroosmosis and electrophoresis, respectively). The results of the present study also demonstrate that flux variability of two neutral permeants are inter-related, so the flux of one neutral permeant can be predicted if the permeability coefficient of the other neutral permeant is known.

  11. Recent Progress toward Engineering HIV-1-Specific Neutralizing Monoclonal Antibodies

    OpenAIRE

    Ming Sun; Yue Li; Huiwen Zheng; Yiming Shao

    2016-01-01

    The recent discoveries of broadly potent neutralizing human monoclonal antibodies represent a new generation of antiretrovirals for the treatment and prophylaxis. Antibodies are generally considered more effective and safer and have been proved to provide passive protection against mucosal challenge in humanized mice and macaques. Several neutralizing Abs could protect animals against HIV-1 but are not effective when used in an established infected model for therapy. In order to overcome the ...

  12. SARS Patients-derived Human Recombinant Antibodies to S and M Proteins Efficiently Neutralize SARS-Coronavirus Infectivity

    Institute of Scientific and Technical Information of China (English)

    MI-FANG LIANG; KONG-XING WU; ZHAO-HUI XIONG; QI JIN; DE-XIN LI; RUN-LEI DU; JING-ZHI LIU; CHUAN LI; QUAN-FU ZHANG; LU-LU HAN; JIAN-SHI YU; SHU-MIN DUAN; XIAO-FANG WANG

    2005-01-01

    Objective To develop a specific SARS virus-targeted antibody preparation for emergent prophylaxis and treatment of SARS virus infection. Methods By using phage display technology, we constructed a naive antibody library from convalescent SARS patient lymphocytes. To obtain the neutralizing antibody to SARS virus surface proteins, the library panning procedure was performed on purified SARS virions and the specific Fab antibody clones were enriched by four rounds of repeated panning procedure and screened by highthroughput selection. The selected Fab antibodies expressed in the periplasma of E. Coli were soluble and further purified and tested for their binding properties and antiviral function to SARS virus. The functional Fab antibodies were converted to full human IgG antibodies with recombinant baculovirus/insect cell systems and their neutralizing activities were further determined. Results After four rounds of the panning, a number of SARS-CoV virus-targeted human recombinant Fab antibodies were isolated from the SARS patient antibody library. Most of these were identified to recognize both natural and recombinant SARS spike (S) proteins, two Fab antibodies were specific for the virus membrane (M) protein, only one bound to SARS-CoV nucleocapsid protein. The SARS-CoV S and M protein-targeted Fab or IgG antibodies showed significant neutralizing activities in cytopathic effect (CPE) inhibition neutralization test, these antibodies were able to completely neutralize the SARS virus and protect the Vero cells from CPE after virus infection. However, the N protein-targeted Fab or IgG antibodies failed to neutralize the virus. In addition, the SARS N protein-targeted human Fab antibody reacted with the denatured N proteins, whereas none of the S and M protein specific neutralizing antibodies did. These results suggested that the S and M protein-specific neutralizing antibodies could recognize conformational epitopes which might be involved in the binding of virions

  13. Humanization and characterization of an anti-ricin neutralization monoclonal antibody.

    Directory of Open Access Journals (Sweden)

    Wei-Gang Hu

    Full Text Available Ricin is regarded as a high terrorist risk for the public due to its high toxicity and ease of production. Currently, there is no therapeutic or vaccine available against ricin. D9, a murine monoclonal antibody developed previously in our laboratory, can strongly neutralize ricin and is therefore a good candidate for humanization. Humanization of D9 variable regions was achieved by a complementarity-determining region grafting approach. The humanized D9 (hD9 variable regions were further grafted onto human heavy and light chain constant regions to assemble the complete antibody gene. A foot-and-mouth-disease virus-derived 2A self-processing sequence was introduced between heavy and light chain DNA sequences to cleave the recombinant protein into a functional full-length antibody molecule from a single open reading frame driven by a single promoter in an adenoviral vector. After expression in mammalian cells and purification, the hD9 was demonstrated to have equimolar expression of the full-length antibody heavy and light chains. More importantly, the hD9 exhibited high affinity to ricin with K(D of 1.63 nM, comparable to its parental murine D9 (2.55 nM. In a mouse model, intraperitoneal (i.p. administration of hD9, at a low dose of 5 µg per mouse, 4 hours after the i.p. challenge with 5×LD50 ricin was found to rescue 100% of the mice. In addition, administered 6 hours post-challenge, hD9 could still rescue 50% of the mice. The hD9 has the potential to be used for prophylactic or therapeutic purposes against ricin poisoning.

  14. Carnosine and neocuproine as neutralizing agents for copper overload-induced damages in cultured human cells.

    Science.gov (United States)

    Arnal, Nathalie; de Alaniz, María J T; Marra, Carlos A

    2011-07-15

    Copper is dangerous when it is present in excess, mainly because it can participate in the Fenton reaction, which produces radical species. As a consequence of copper pollution, people are involuntarily exposed to a copper overload under sub-clinical and sub-symptomatological conditions, which may be very difficult to detect. Thus, we investigated (i) the possible use of the chelator molecules carnosine and neocuproine to prevent the Cu overload-induced damage on cellular lipids and proteins, as tested in human cell culture systems, and (ii) the differential response of these two chelating agents in relation to their protective action, and the type of copper ion involved in the process, by using two types of human cultured cells (HepG2 and A-549). Cu treatment clearly enhanced (pcomplexing agent for Cu(II), but also an effective antioxidant that can dismutate superoxide radicals, scavenge hydroxyl radicals and neutralize TBARS formation. Carnosine should be investigated in more detail in order to establish its putative utility as an agent to prevent copper-associated damages in biological systems.

  15. An expanded sequence context model broadly explains variability in polymorphism levels across the human genome

    Science.gov (United States)

    Aggarwala, Varun; Voight, Benjamin F.

    2016-01-01

    The rate of single nucleotide polymorphism varies substantially across the human genome and fundamentally influences evolution and incidence of genetic disease. Previous studies have only considered the immediate flanking nucleotides around a polymorphic site –the site’s trinucleotide sequence context– to study polymorph levels across the genome. Moreover, the impact of larger sequence contexts has not been fully clarified, even though context substantially influences rates of polymorphism. Using a new statistical framework and data from the 1000 Genomes Project, we demonstrate that a heptanucleotide context explains >81% of variability in substitution probabilities, revealing new mutation-promoting motifs at ApT dinucleotide, CAAT, and TACG sequences. Our approach also identifies previously undocumented variability in C-to-T substitutions at CpG sites, which is not immediately explained by differential methylation intensity. Using our model, we present informative substitution intolerance scores for genes and a new intolerance score for amino acids, and we demonstrate clinical use of the model in neuropsychiatric diseases. PMID:26878723

  16. Interactions between Lipids and Human Anti-HIV Antibody 4E10 Can Be Reduced without Ablating Neutralizing Activity

    NARCIS (Netherlands)

    Xu, Hengyu; Song, Likai; Kim, Mikyung; Holmes, Margaret A.; Kraft, Zane; Sellhorn, George; Reinherz, Ellis L.; Stamatatos, Leonidas; Strong, Roland K.

    2010-01-01

    Human 4E10 is one of the broadest-specificity, HIV-1-neutralizing monoclonal antibodies known, recognizing a membrane-proximal linear epitope on gp41. The lipid cross-reactivity of 4E10 has been alternately suggested either to contribute to the apparent rarity of 4E10-like antibody responses in HIV

  17. Three amino acid residues in the envelope of human immunodeficiency virus type 1 CRF07_BC regulate viral neutralization susceptibility to the human monoclonal neutralizing antibody IgG1b12

    Institute of Scientific and Technical Information of China (English)

    Jianhui; Nie; Juan; Zhao; Qingqing; Chen; Weijin; Huang; Youchun; Wang

    2014-01-01

    The CD4 binding site(CD4bs) of envelope glycoprotein(Env) is an important conserved target for anti-human immunodeficiency virus type 1(HIV-1) neutralizing antibodies. Neutralizing monoclonal antibodies IgG1 b12(b12) could recognize conformational epitopes that overlap the CD4 bs of Env. Different virus strains, even derived from the same individual, showed distinct neutralization susceptibility to b12. We examined the key amino acid residues affecting b12 neutralization susceptibility using single genome amplification and pseudovirus neutralization assay. Eleven amino acid residues were identified that affect the sensitivity of Env to b12. Through site-directed mutagenesis, an amino acid substitution at position 182 in the V2 region of Env was confirmed to play a key role in regulating the b12 neutralization susceptibility. The introduction of V182 L to a resistant strain enhanced its sensitivity to b12 more than twofold. Correspondingly, the introduction of L182 V to a sensitive strain reduced its sensitivity to b12 more than tenfold. Amino acid substitution at positions 267 and 346 could both enhance the sensitivity to b12 more than twofold. However, no additive effect was observed when the three site mutageneses were introduced into the same strain, and the sensitivity was equivalent to the single V182 L mutation. CRF07_BC is a major circulating recombinant form of HIV-1 prevalent in China. Our data may provide important information for understanding the molecular mechanism regulating the neutralization susceptibility of CRF07_BC viruses to b12 and may be helpful for a vaccine design targeting the CD4 bs epitopes.

  18. 流感病毒广谱中和抗体与广谱流感病毒疫苗%Broadly neutralizing antibodies against influenza virus andb roader influenza vaccine

    Institute of Scientific and Technical Information of China (English)

    于永利

    2013-01-01

    A series of breakthroughs have recently been made in discovering broadly neutralizing antibodies ( bnAb) against infleu nza virus, and the discoveries have spawned the research on developing broader influenza vaccine ( BIV) .The bnAb and BIV are being developed as new biologics for the prevention or treatment of infection caused by different types of influ-enza virues s that continuously undergo antigenic evolution in various hosts .%近年来,在流感病毒的广谱中和抗体( bnAb )研究领域中有了突破性的进展,这些进展力推了广谱流感疫苗( BIV)的研制。bnAb 和BIV可能成为有效预防并治疗多型、易变、宿主众多的流感病毒感染的新型生物制品。

  19. Neutralizing antibody responses in macaques induced by human immunodeficiency virus type 1 monovalent or trivalent envelope glycoproteins.

    Directory of Open Access Journals (Sweden)

    Gerald V Quinnan

    Full Text Available A major goal of efforts to develop a vaccine to prevent HIV-1 infection is induction of broadly cross-reactive neutralizing antibodies (bcnAb. In previous studies we have demonstrated induction of neutralizing antibodies that did cross-react among multiple primary and laboratory strains of HIV-1, but neutralized with limited potency. In the present study we tested the hypothesis that immunization with multiple HIV-1 envelope glycoproteins (Envs would result in a more potent and cross-reactive neutralizing response. One Env, CM243(N610Q, was selected on the basis of studies of the effects of single and multiple mutations of the four gp41 glycosylation sites. The other two Envs included R2 (subtype B and 14/00/4 (subtype F, both of which were obtained from donors with bcnAb. Rhesus monkeys were immunized using a prime boost regimen as in previous studies. Individual groups of monkeys were immunized with either one of the three Envs or all three. The single N610Q and N615Q mutations of CM243 Env did not disrupt protein secretion, processing into, or reactivity with mAbs, unlike other single or multiple deglycosylation mutations. In rabbit studies the N610Q mutation alone or in combination was associated with an enhanced neutralizing response against homologous and heterologous subtype E viruses. In the subsequent monkey study the response induced by the R2 Env regimen was equivalent to the trivalent regimen and superior to the other monovalent regimens against the virus panel used for testing. The 14/00/4 Env induced responses superior to CM243(N610Q. The results indicate that elimination of the glycosylation site near the gp41 loop results in enhanced immunogenicity, but that immunization of monkeys with these three distinct Envs was not more immunogenic than with one.

  20. A novel human recombinant antibody fragment capable of neutralizing Mexican scorpion toxins.

    Science.gov (United States)

    Riaño-Umbarila, Lidia; Olamendi-Portugal, Timoteo; Morelos-Juárez, Citlalli; Gurrola, Georgina B; Possani, Lourival D; Becerril, Baltazar

    2013-12-15

    Using phage display and directed evolution, our group has progressed in the construction of a second family of human single chain variable fragments (scFv) which bind to scorpion toxins dangerous to mammals. It was observed that scFv C1 only bound initially to toxin Cn2, which constitutes 6.8% of whole venom from the scorpion Centruroides noxius Hoffman. Only a few amino acid changes were necessary to extend its recognition to other similar toxins and without affecting the recognition for its primary antigen (Cn2 toxin). One variant of scFv C1 (scFv 202F) was selected after two cycles of directed evolution against Cll1 toxin, the second major toxic component from the venom of the Mexican scorpion Centruroides limpidus limpidus Karsh (0.5% of the whole venom). scFv 202F is also capable of recognizing Cn2 toxin. Despite not having the highest affinity for toxins Cll1 (KD = 25.1 × 10(-9) M) or Cn2 (KD = 8.1 × 10(-9) M), this antibody fragment neutralized one LD50 of each one of these toxins. Additionally, scFv 202F moderately recognized Cll2 toxin which constitutes 1.5% of the venom from C. limpidus. Based on our previous experience, we consider that these results are promising; consequently, we continue working on generating new optimized variants from scFv C1 that could be part of a recombinant scorpion anti-venom from human origin, that might reach the market in the near future.

  1. Human coronavirus EMC does not require the SARS-coronavirus receptor and maintains broad replicative capability in mammalian cell lines.

    Science.gov (United States)

    Müller, Marcel A; Raj, V Stalin; Muth, Doreen; Meyer, Benjamin; Kallies, Stephan; Smits, Saskia L; Wollny, Robert; Bestebroer, Theo M; Specht, Sabine; Suliman, Tasnim; Zimmermann, Katrin; Binger, Tabea; Eckerle, Isabella; Tschapka, Marco; Zaki, Ali M; Osterhaus, Albert D M E; Fouchier, Ron A M; Haagmans, Bart L; Drosten, Christian

    2012-12-11

    A new human coronavirus (hCoV-EMC) has emerged very recently in the Middle East. The clinical presentation resembled that of the severe acute respiratory syndrome (SARS) as encountered during the epidemic in 2002/2003. In both cases, acute renal failure was observed in humans. HCoV-EMC is a member of the same virus genus as SARS-CoV but constitutes a sister species. Here we investigated whether it might utilize angiotensin-converting enzyme 2 (ACE2), the SARS-CoV receptor. Knowledge of the receptor is highly critical because the restriction of the SARS receptor to deep compartments of the human respiratory tract limited the spread of SARS. In baby hamster kidney (BHK) cells, lentiviral transduction of human ACE2 (hACE2) conferred permissiveness and replication for SARS-CoV but not for hCoV-EMC. Monkey and human kidney cells (LLC-MK2, Vero, and 769-P) and swine kidney cells were permissive for both viruses, but only SARS-CoV infection could be blocked by anti-hACE2 antibody and could be neutralized by preincubation of virus with soluble ACE2. Our data show that ACE2 is neither necessary nor sufficient for hCoV-EMC replication. Moreover, hCoV-EMC, but not SARS-CoV, replicated in cell lines from Rousettus, Rhinolophus, Pipistrellus, Myotis, and Carollia bats, representing four major chiropteran families from both suborders. As human CoV normally cannot replicate in bat cells from different families, this suggests that hCoV-EMC might use a receptor molecule that is conserved in bats, pigs, and humans, implicating a low barrier against cross-host transmission. IMPORTANCE A new human coronavirus (hCoV) emerged recently in the Middle East. The disease resembled SARS (severe acute respiratory syndrome), causing a fatal epidemic in 2002/2003. Coronaviruses have a reservoir in bats and because this novel virus is related to SARS-CoV, we investigated whether it might replicate in bat cells and use the same receptor (angiotensin-converting enzyme 2 [ACE2]). This knowledge is

  2. Mass spectrometric detection of multiple extended series of neutral highly fucosylated N-acetyllactosamine oligosaccharides in human milk

    Science.gov (United States)

    Pfenninger, Anja; Chan, Shiu-Yung; Karas, Michael; Finke, Berndt; Stahl, Bernd; Costello, Catherine E.

    2008-12-01

    Complex mixtures of high-molecular weight fractions of pooled neutral human milk oligosaccharides (obtained via gel permeation chromatography) have been investigated. The subfractions were each permethylated and analyzed by high-resolution mass spectrometry, using matrix-assisted laser desorption/ionization (MALDI)-Fourier transform ion cyclotron resonance (FTICR) mass spectrometry, in order to investigate their oligosaccharide compositions. The obtained spectra reveal that human milk contains more complex neutral oligosaccharides than have been described previously; the data show that these oligosaccharides can be highly fucosylated, and that their poly-N-acetyllactosamine cores are substituted with up to 10 fucose residues on an oligosaccharide that has 7-N-acetyllactosamine units. This is the first report of the existence in human milk of this large range of highly fucosylated oligosaccharides which possess novel, potentially immunologically active structures.

  3. Heterologous prime-boost vaccination with MF59-adjuvanted H5 vaccines promotes antibody affinity maturation towards the hemagglutinin HA1 domain and broad H5N1 cross-clade neutralization.

    Directory of Open Access Journals (Sweden)

    Surender Khurana

    Full Text Available In an open label clinical study (2007, MF59-adjuvanted hemagglutinin (HA vaccine from H5N1-A/Vietnam/1194/2004 (clade 1 was administered to subjects previously vaccinated (primed with clade 0 H5N3 (A/duck/Singapore/97 vaccine at least 6 years earlier (in 1999 or 2001. The primed individuals responded rapidly and generated high neutralizing antibody titers against the H5N1-Vietnam strain within 7 days of a single booster vaccination. Furthermore, significant cross-neutralization titers were measured against H5N1 clade 0, 1, and 2 viruses. In the current study, the impact of MF59 adjuvant during heterologous priming on the quality of humoral polyclonal immune response in different vaccine arms were further evaluated using real time kinetics assay by surface plasmon resonance (SPR. Total anti-H5N1 HA1 polyclonal sera antibody binding from the heterologous prime-boost groups after a single MF59-H5N1 boost was significantly higher compared with sera from unprimed individuals that received two MF59-H5N1 vaccinations. The antigen-antibody complex dissociation rates (surrogate for antibody affinity of the polyclonal sera against HA1 of H5N1-A/Vietnam/1194/2004 from the MF59-H5N3 primed groups were significantly higher compared to sera from unadjuvanted primed groups or unprimed individuals that received two MF59-H5N1 vaccines. Furthermore, strong inverse correlations were observed between the antibody dissociation off-rates of the immune sera against HA1 (but not HA2 and the virus neutralization titers against H5 vaccine strains and heterologous H5N1 strains. These findings supports the use of oil-in-water-adjuvanted pandemic influenza vaccines to elicit long term memory B cells with high affinity BCR capable of responding to potential variant pandemic viruses likely to emerge and adapt to human transmissions.

  4. Neutralization of tier-2 viruses and epitope profiling of plasma antibodies from human immunodeficiency virus type 1 infected donors from India.

    Directory of Open Access Journals (Sweden)

    Raiees Andrabi

    Full Text Available Broadly cross neutralizing antibodies (NAbs are generated in a group of HIV-1 infected individuals during the natural infection, but little is known about their prevalence in patients infected with viral subtypes from different geographical regions. We tested here the neutralizing efficiency of plasma antibodies from 80 HIV-1 infected antiretroviral drug naive patients against a panel of subtype-B and C tier 2 viruses. We detected cross-neutralizing antibodies in approximately 19-27% of the plasma, however the subtype-C specific neutralization efficiency predominated (p = 0.004. The neutralizing activity was shown to be exclusively mediated by the immunoglobulin G (IgG fraction in the representative plasma samples. Epitope mapping of three, the most cross-neutralizing plasma (CNP AIIMS206, AIIMS239 and AIIMS249 with consensus-C overlapping envelope peptides revealed ten different binding specificities with only V3 and IDR being common. The V3 and IDR were highly antigenic regions but no correlation between their reciprocal Max50 binding titers and neutralization was observed. In addition, the neutralizing activity of CNP was not substantially reduced by V3 and gp41 peptides except a modest contribution of MPER peptide. The MPER was rarely recognized by plasma antibodies though antibody depletion and competition experiments demonstrated MPER dependent neutralization in two out of three CNP. Interestingly, the binding specificity of one of the CNP (AIIMS206 overlapped with broadly neutralizing mAb 2F5 epitope. Overall, the data suggest that, despite the low immunogenicity of HIV-1 MPER, the antibodies directed to this region may serve as crucial reagents for HIV-1 vaccine design.

  5. Neutralization of tier-2 viruses and epitope profiling of plasma antibodies from human immunodeficiency virus type 1 infected donors from India.

    Science.gov (United States)

    Andrabi, Raiees; Bala, Manju; Kumar, Rajesh; Wig, Naveet; Hazarika, Anjali; Luthra, Kalpana

    2012-01-01

    Broadly cross neutralizing antibodies (NAbs) are generated in a group of HIV-1 infected individuals during the natural infection, but little is known about their prevalence in patients infected with viral subtypes from different geographical regions. We tested here the neutralizing efficiency of plasma antibodies from 80 HIV-1 infected antiretroviral drug naive patients against a panel of subtype-B and C tier 2 viruses. We detected cross-neutralizing antibodies in approximately 19-27% of the plasma, however the subtype-C specific neutralization efficiency predominated (p = 0.004). The neutralizing activity was shown to be exclusively mediated by the immunoglobulin G (IgG) fraction in the representative plasma samples. Epitope mapping of three, the most cross-neutralizing plasma (CNP) AIIMS206, AIIMS239 and AIIMS249 with consensus-C overlapping envelope peptides revealed ten different binding specificities with only V3 and IDR being common. The V3 and IDR were highly antigenic regions but no correlation between their reciprocal Max50 binding titers and neutralization was observed. In addition, the neutralizing activity of CNP was not substantially reduced by V3 and gp41 peptides except a modest contribution of MPER peptide. The MPER was rarely recognized by plasma antibodies though antibody depletion and competition experiments demonstrated MPER dependent neutralization in two out of three CNP. Interestingly, the binding specificity of one of the CNP (AIIMS206) overlapped with broadly neutralizing mAb 2F5 epitope. Overall, the data suggest that, despite the low immunogenicity of HIV-1 MPER, the antibodies directed to this region may serve as crucial reagents for HIV-1 vaccine design.

  6. Structure of a human rhinovirus-bivalently bound antibody complex: implications for viral neutralization and antibody flexibility.

    OpenAIRE

    1993-01-01

    The structure of a neutralizing immunoglobulin (monoclonal antibody mAb17-IA), bound to human rhinovirus 14 (HRV14), has been determined by cryo-electron microscopy and image reconstruction. The antibody bound bivalently across icosahedral twofold axes of the virus, and there were no detectable conformational changes in the capsid. Thus, bivalently bound IgGs do not appear to cause gross deformations in the capsid. Differences between the electron density of the constant domains of the bound ...

  7. Potent Intratype Neutralizing Activity Distinguishes Human Immunodeficiency Virus Type 2 (HIV-2) from HIV-1

    OpenAIRE

    Özkaya Şahin, Gülşen; Holmgren, Birgitta; da Silva, Zacarias; Nielsen, Jens; Nowroozalizadeh, Salma; Esbjörnsson, Joakim; Månsson, Fredrik; Andersson, Sören; Norrgren, Hans; Aaby, Peter; Jansson, Marianne; Fenyö, Eva Maria

    2012-01-01

    HIV-2 has a lower pathogenicity and transmission rate than HIV-1. Neutralizing antibodies could be contributing to these observations. Here we explored side by side the potency and breadth of intratype and intertype neutralizing activity (NAc) in plasma of 20 HIV-1-, 20 HIV-2-, and 11 dually HIV-1/2 (HIV-D)-seropositive individuals from Guinea-Bissau, West Africa. Panels of primary isolates, five HIV-1 and five HIV-2 isolates, were tested in a plaque reduction assay using U87.CD4-CCR5 cells a...

  8. Rapid Generation of Human-Like Neutralizing Monoclonal Antibodies in Urgent Preparedness for Influenza Pandemics and Virulent Infectious Diseases.

    Directory of Open Access Journals (Sweden)

    Weixu Meng

    Full Text Available The outbreaks of emerging infectious diseases caused by pathogens such as SARS coronavirus, H5N1, H1N1, and recently H7N9 influenza viruses, have been associated with significant mortality and morbidity in humans. Neutralizing antibodies from individuals who have recovered from an infection confer therapeutic protection to others infected with the same pathogen. However, survivors may not always be available for providing plasma or for the cloning of monoclonal antibodies (mAbs.The genome and the immunoglobulin genes in rhesus macaques and humans are highly homologous; therefore, we investigated whether neutralizing mAbs that are highly homologous to those of humans (human-like could be generated. Using the H5N1 influenza virus as a model, we first immunized rhesus macaques with recombinant adenoviruses carrying a synthetic gene encoding hemagglutinin (HA. Following screening an antibody phage display library derived from the B cells of immunized monkeys, we cloned selected macaque immunoglobulin heavy chain and light chain variable regions into the human IgG constant region, which generated human-macaque chimeric mAbs exhibiting over 97% homology to human antibodies. Selected mAbs demonstrated potent neutralizing activities against three clades (0, 1, 2 of the H5N1 influenza viruses. The in vivo protection experiments demonstrated that the mAbs effectively protected the mice even when administered up to 3 days after infection with H5N1 influenza virus. In particular, mAb 4E6 demonstrated sub-picomolar binding affinity to HA and superior in vivo protection efficacy without the loss of body weight and obvious lung damage. The analysis of the 4E6 escape mutants demonstrated that the 4E6 antibody bound to a conserved epitope region containing two amino acids on the globular head of HA.Our study demonstrated the generation of neutralizing mAbs for potential application in humans in urgent preparedness against outbreaks of new influenza infections or

  9. Potent neutralizing serum immunoglobulin A (IgA) in human immunodeficiency virus type 2-exposed IgG-seronegative individuals

    DEFF Research Database (Denmark)

    Lizeng, Q; Nilsson, C; Sourial, S;

    2004-01-01

    Links Potent neutralizing serum immunoglobulin A (IgA) in human immunodeficiency virus type 2-exposed IgG-seronegative individuals.Lizeng Q, Nilsson C, Sourial S, Andersson S, Larsen O, Aaby P, Ehnlund M, Bjorling E. Research Center, South Hospital, Stockholm, Sweden. The mechanisms behind...... the resistance to human immunodeficiency virus type 2 (HIV-2) infection are still not fully understood. In the present study, we explored the HIV-2-specific humoral serum immunoglobulin A (IgA) immune response in HIV-2-exposed IgG-seronegative (EGSN) individuals. Serum samples from heterosexual EGSN individuals...

  10. Breadth of neutralization and synergy of clinically relevant human monoclonal antibodies against HCV genotypes 1a, 1b, 2a, 2b, 2c, and 3a

    DEFF Research Database (Denmark)

    Carlsen, Thomas H R; Pedersen, Jannie; Prentoe, Jannick C;

    2014-01-01

    UNLABELLED: Human monoclonal antibodies (HMAbs) with neutralizing capabilities constitute potential immune-based treatments or prophylaxis against hepatitis C virus (HCV). However, lack of cell culture-derived HCV (HCVcc) harboring authentic envelope proteins (E1/E2) has hindered neutralization...... synergism obtained when pooling the most potent HMAbs could have significant implications for developing novel strategies to treat and control HCV....

  11. A non-VH1-69 heterosubtypic neutralizing human monoclonal antibody protects mice against H1N1 and H5N1 viruses.

    Directory of Open Access Journals (Sweden)

    Donata De Marco

    Full Text Available Influenza viruses are among the most important human pathogens and are responsible for annual epidemics and sporadic, potentially devastating pandemics. The humoral immune response plays an important role in the defense against these viruses, providing protection mainly by producing antibodies directed against the hemagglutinin (HA glycoprotein. However, their high genetic variability allows the virus to evade the host immune response and the potential protection offered by seasonal vaccines. The emergence of resistance to antiviral drugs in recent years further limits the options available for the control of influenza. The development of alternative strategies for influenza prophylaxis and therapy is therefore urgently needed. In this study, we describe a human monoclonal antibody (PN-SIA49 that recognizes a highly conserved epitope located on the stem region of the HA and able to neutralize a broad spectrum of influenza viruses belonging to different subtypes (H1, H2 and H5. Furthermore, we describe its protective activity in mice after lethal challenge with H1N1 and H5N1 viruses suggesting a potential application in the treatment of influenza virus infections.

  12. Chikungunya virus neutralization antigens and direct cell-to-cell transmission are revealed by human antibody-escape mutants.

    Directory of Open Access Journals (Sweden)

    Chia Yin Lee

    2011-12-01

    Full Text Available Chikungunya virus (CHIKV is an alphavirus responsible for numerous epidemics throughout Africa and Asia, causing infectious arthritis and reportedly linked with fatal infections in newborns and elderly. Previous studies in animal models indicate that humoral immunity can protect against CHIKV infection, but despite the potential efficacy of B-cell-driven intervention strategies, there are no virus-specific vaccines or therapies currently available. In addition, CHIKV has been reported to elicit long-lasting virus-specific IgM in humans, and to establish long-term persistence in non-human primates, suggesting that the virus might evade immune defenses to establish chronic infections in man. However, the mechanisms of immune evasion potentially employed by CHIKV remain uncharacterized. We previously described two human monoclonal antibodies that potently neutralize CHIKV infection. In the current report, we have characterized CHIKV mutants that escape antibody-dependent neutralization to identify the CHIKV E2 domain B and fusion loop "groove" as the primary determinants of CHIKV interaction with these antibodies. Furthermore, for the first time, we have also demonstrated direct CHIKV cell-to-cell transmission, as a mechanism that involves the E2 domain A and that is associated with viral resistance to antibody-dependent neutralization. Identification of CHIKV sub-domains that are associated with human protective immunity, will pave the way for the development of CHIKV-specific sub-domain vaccination strategies. Moreover, the clear demonstration of CHIKV cell-to-cell transmission and its possible role in the establishment of CHIKV persistence, will also inform the development of future anti-viral interventions. These data shed new light on CHIKV-host interactions that will help to combat human CHIKV infection and inform future studies of CHIKV pathogenesis.

  13. Cerebral net exchange of large neutral amino acids after lipopolysaccharide infusion in healthy humans

    DEFF Research Database (Denmark)

    Berg, Ronan Mg; Taudorf, Sarah; Bailey, Damian M;

    2010-01-01

    Alterations in circulating large neutral amino acids (LNAAs), leading to a decrease in the plasma ratio between branched-chain and aromatic amino acids (BCAA/AAA ratio), may be involved in sepsis-associated encephalopathy. We hypothesised that a decrease in the BCAA/AAA ratio occurs along with a ...

  14. Computational prediction of neutralization epitopes targeted by human anti-V3 HIV monoclonal antibodies.

    Directory of Open Access Journals (Sweden)

    Evgeny Shmelkov

    Full Text Available The extreme diversity of HIV-1 strains presents a formidable challenge for HIV-1 vaccine design. Although antibodies (Abs can neutralize HIV-1 and potentially protect against infection, antibodies that target the immunogenic viral surface protein gp120 have widely variable and poorly predictable cross-strain reactivity. Here, we developed a novel computational approach, the Method of Dynamic Epitopes, for identification of neutralization epitopes targeted by anti-HIV-1 monoclonal antibodies (mAbs. Our data demonstrate that this approach, based purely on calculated energetics and 3D structural information, accurately predicts the presence of neutralization epitopes targeted by V3-specific mAbs 2219 and 447-52D in any HIV-1 strain. The method was used to calculate the range of conservation of these specific epitopes across all circulating HIV-1 viruses. Accurately identifying an Ab-targeted neutralization epitope in a virus by computational means enables easy prediction of the breadth of reactivity of specific mAbs across the diversity of thousands of different circulating HIV-1 variants and facilitates rational design and selection of immunogens mimicking specific mAb-targeted epitopes in a multivalent HIV-1 vaccine. The defined epitopes can also be used for the purpose of epitope-specific analyses of breakthrough sequences recorded in vaccine clinical trials. Thus, our study is a prototype for a valuable tool for rational HIV-1 vaccine design.

  15. Cerebral net exchange of large neutral amino acids after lipopolysaccharide infusion in healthy humans

    DEFF Research Database (Denmark)

    2010-01-01

    Alterations in circulating large neutral amino acids (LNAAs), leading to a decrease in the plasma ratio between branched-chain and aromatic amino acids (BCAA/AAA ratio), may be involved in sepsis-associated encephalopathy. We hypothesised that a decrease in the BCAA/AAA ratio occurs along...

  16. Classification of influenza A virus and its value for research of universal influenza vaccine and broad-spectrum neutralization antibody%甲型流感病毒分类与通用流感疫苗及广谱中和活性抗体的研究

    Institute of Scientific and Technical Information of China (English)

    蒋露芳; 居丽雯; 姜庆五

    2011-01-01

    Influenza is a great harm contagious disease for human. Vaccination is considered to be the most effective means to prevent influenza. For the problems of existing vaccine on timeliness and effectiveness,universal influenza vaccine,which can preclude all the influenza virus and induce duration protective immunity, has been a hot research. Meanwhile, the broad-spectrum neutralization antibody against all influenza A viruses is also the current focus. All these have made the request to explore applicable classification of influenza A virus.%流感是对人类危害极大的传染病,疫苗接种被认为是预防流感的最有效手段.为解决流感病毒变异导致现有疫卣时效性与有效性的问题,研制能够预防所有流感病毒毒株、可诱导持久保护性免疫的通用流感疫苗一直是流感研究的热点,同时,能中和所有甲型流感病毒的广谱中和活性抗体的研究也已成为目前关注的重点,因此对甲型流感病毒分类研究也提出了相应的要求.

  17. Human TNF cytokine neutralization with a vNAR from Heterodontus francisci shark: a potential therapeutic use.

    Science.gov (United States)

    Camacho-Villegas, Tanya; Mata-Gonzalez, Teresa; Paniagua-Solis, Jorge; Sanchez, Edna; Licea, Alexei

    2013-01-01

    The therapeutic use of single domain antibodies (sdAbs) is a promising new approach because these small antibodies maintain antigen recognition and neutralization capacity, have thermal and chemical stability and have good solubility. In this study, using phage display technology, we isolated a variable domain of a IgNAR (vNAR) from a Heterodontus francisci shark immunized against the recombinant human cytokine TNFα (rhTNFα). One clone T43, which expresses the vNAR protein in the periplasmic space, was isolated from the fourth round of panning. T43 had the capacity to recognize rhTNF and neutralize it in vitro, indicating that T43 has potential as a therapeutic that can be used for diseases in which this pro-inflammatory cytokine needs to be controlled.

  18. Broad-spectrum sunscreens prevent the secretion of proinflammatory cytokines in human keratinocytes exposed to ultraviolet A and phototoxic lomefloxacin

    Energy Technology Data Exchange (ETDEWEB)

    Reinhardt, P.; Cybulski, M. [Lasers and Electro-Optics Div., Consumer and Clinical Radiation Protection Bureau, Product Safety Program, Healthy Environments and Consumer Safety Branch, Health Canada, Ottawa, Ontario (Canada)], E-mail: pascale_reinhardt@hc-sc.gc.ca; Miller, S.M.; Ferrarotto, C.; Wilkins, R. [Radiobiology Div., Consumer and Clinical Radiation Protection Bureau, Product Safety Program, Healthy Environments and Consumer Safety Branch, Health Canada, Ottawa, Ontario (Canada); Deslauriers, Y. [Lasers and Electro-Optics Div., Consumer and Clinical Radiation Protection Bureau, Product Safety Program, Healthy Environments and Consumer Safety Branch, Health Canada, Ottawa, Ontario (Canada)

    2006-02-15

    The combination of phototoxic drugs and ultraviolet (UV) radiation can trigger the release of proinflammatory cytokines. The present study measured the ability of sunscreens to prevent cytokine secretion in human keratinocytes following cotreatment of these cells with a known photoreactive drug and UVA. Keratinocytes were treated for 1 h with increasing concentrations of lomefloxacin (LOM) or norfloxacin (NOR), exposed to 15 J/cm{sup 2} UVA, and incubated for 24 h. NOR, owing to the absence of a fluorine atom in position 8, was non-phototoxic and used as a negative control. Cell viability and the release of 3 cytokines were assessed, namely interleukin-1{alpha} (IL-1{alpha}), interleukin-6 (IL-6), and tumour necrosis factor-{alpha} (TNF-{alpha}). The measurement of these cytokines may be a useful tool for detecting photoreactive compounds. To measure their ability to prevent cytokine secretion, various sunscreens were inserted between the UVA source and the cells. Treatment with NOR, NOR plus UVA, or LOM had no effect on the cells. LOM plus UVA, however, had an effect on cell viability and on cytokine secretion. IL-1{alpha} levels increased with LOM concentration. The release of TNF-{alpha} and IL-6 followed the same pattern at lower concentrations of LOM but peaked at 15 {mu}mol/L and decreased at higher concentrations. Sunscreens protected the cells from the effects of LOM plus UVA, as cell viability and levels of cytokines remained the same as in the control cells. In conclusion, the application of broad-spectrum sunscreen by individuals exposed to UVA radiation may prevent phototoxic reactions initiated by drugs such as LOM. (author)

  19. Canine distemper virus neutralization activity is low in human serum and it is sensitive to an amino acid substitution in the hemagglutinin protein

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Xinsheng, E-mail: xzhang@iavi.org [AIDS Vaccine Design and Development Laboratory, International AIDS Vaccine Initiative (IAVI), Brooklyn, NY (United States); Molecular and Cellular Biology Program, State University of New York, Brooklyn, NY (United States); Wallace, Olivia L.; Domi, Arban; Wright, Kevin J.; Driscoll, Jonathan [AIDS Vaccine Design and Development Laboratory, International AIDS Vaccine Initiative (IAVI), Brooklyn, NY (United States); Anzala, Omu [Kenya AIDS Vaccine Initiative (KAVI)-Institute of Clinical Research, Nairobi (Kenya); Sanders, Eduard J. [Centre for Geographic Medicine Research, Kenya Medical Research Institute (KEMRI), Kilifi, Kenya & Centre for Clinical Vaccinology and Tropical Medicine, University of Oxford, Headington (United Kingdom); Kamali, Anatoli [MRC/UVRI Uganda Virus Research Unit on AIDS, Masaka and Entebbe (Uganda); Karita, Etienne [Projet San Francisco, Kigali (Rwanda); Allen, Susan [Department of Pathology and Laboratory Medicine, Emory University, Atlanta, GA (United States); Fast, Pat [Department of Medical Affairs, International AIDS Vaccine Initiative, NY, NY (United States); Gilmour, Jill [Human Immunology Laboratory, International AIDS Vaccine Initiative, London (United Kingdom); Price, Matt A. [Department of Medical Affairs, International AIDS Vaccine Initiative, NY, NY (United States); Department of Epidemiology and Biostatistics, University of California at San Francisco, San Francisco, CA (United States); Parks, Christopher L. [AIDS Vaccine Design and Development Laboratory, International AIDS Vaccine Initiative (IAVI), Brooklyn, NY (United States); Molecular and Cellular Biology Program, State University of New York, Brooklyn, NY (United States)

    2015-08-15

    Serum was analyzed from 146 healthy adult volunteers in eastern Africa to evaluate measles virus (MV) and canine distemper virus (CDV) neutralizing antibody (nAb) prevalence and potency. MV plaque reduction neutralization test (PRNT) results indicated that all sera were positive for MV nAbs. Furthermore, the 50% neutralizing dose (ND50) for the majority of sera corresponded to antibody titers induced by MV vaccination. CDV nAbs titers were low and generally were detected in sera with high MV nAb titers. A mutant CDV was generated that was less sensitive to neutralization by human serum. The mutant virus genome had 10 nucleotide substitutions, which coded for single amino acid substitutions in the fusion (F) and hemagglutinin (H) glycoproteins and two substitutions in the large polymerase (L) protein. The H substitution occurred in a conserved region involved in receptor interactions among morbilliviruses, implying that this region is a target for cross-reactive neutralizing antibodies. - Highlights: • Screened 146 serum samples for measles virus (MV) and canine distemper virus (CDV) neutralizing antibody (nAb). • MV nAb is prevalent in the sera. • CDV neutralizing activity is generally low or absent and when detected it is present in sera with high MV nAb titers. • A neutralization-resistant CDV mutant was isolated using human serum selection. • A mutation was identified in the receptor-binding region of CDV hemagglutinin protein that confers the neutralization resistance.

  20. Evaluation of three different formats of a neutralizing single chain human antibody against toxin Cn2: neutralization capacity versus thermodynamic stability.

    Science.gov (United States)

    Quintero-Hernández, Veronica; Del Pozo-Yauner, Luis; Pedraza-Escalona, Martha; Juárez-González, Victor R; Alcántara-Recillas, Israel; Possani, Lourival D; Becerril, Baltazar

    2012-04-30

    The single-chain antibody fragment (scFv) 6009F, obtained by directed evolution, neutralizes the effects of the Cn2 toxin, which is the major toxic component of Centruroides noxius scorpion venom. In this work we compared the neutralization capacity and the thermodynamic stability of scFv 6009F with those of two other derived formats: Fab 6009F and diabody 6009F. Additionally, the affinity constants to Cn2 toxin of the three recombinant antibody fragments were determined by means of BIAcore. We found a correlation between the thermodynamic stability of these antibody fragments with their neutralization capacity. The order of thermodynamic stability determined was Fab≫scFv>diabody. The Fab and scFv were capable of neutralizing the toxic effects of Cn2 and whole venom but the diabody was unable to fully neutralize intoxication. In silico analysis of the diabody format indicates that the reduction of stability and neutralization capacity could be explained by a less cooperative interface between the heavy and the light variable domains.

  1. Hansa: an automated method for discriminating disease and neutral human nsSNPs.

    Science.gov (United States)

    Acharya, Vishal; Nagarajaram, Hampapathalu A

    2012-02-01

    Variations are mostly due to nonsynonymous single nucleotide polymorphisms (nsSNPs), some of which are associated with certain diseases. Phenotypic effects of a large number of nsSNPs have not been characterized. Although several methods have been developed to predict the effects of nsSNPs as "disease" or "neutral," there is still a need for development of methods with improved prediction accuracies. We, therefore, developed a support vector machine (SVM) based method named Hansa which uses a novel set of discriminatory features to classify nsSNPs into disease (pathogenic) and benign (neutral) types. Validation studies on a benchmark dataset and further on an independent dataset of well-characterized known disease and neutral mutations show that Hansa outperforms the other known methods. For example, fivefold cross-validation studies using the benchmark HumVar dataset reveal that at the false positive rate (FPR) of 20% Hansa yields a true positive rate (TPR) of 82% that is about 10% higher than the best-known method. Hansa is available in the form of a web server at http://hansa.cdfd.org.in:8080.

  2. Protection of Macaques against pathogenic simian/human immunodeficiency virus 89.6PD by passive transfer of neutralizing antibodies.

    Science.gov (United States)

    Mascola, J R; Lewis, M G; Stiegler, G; Harris, D; VanCott, T C; Hayes, D; Louder, M K; Brown, C R; Sapan, C V; Frankel, S S; Lu, Y; Robb, M L; Katinger, H; Birx, D L

    1999-05-01

    The role of antibody in protection against human immunodeficiency virus (HIV-1) has been difficult to study in animal models because most primary HIV-1 strains do not infect nonhuman primates. Using a chimeric simian/human immunodeficiency virus (SHIV) based on the envelope of a primary isolate (HIV-89.6), we performed passive-transfer experiments in rhesus macaques to study the role of anti-envelope antibodies in protection. Based on prior in vitro data showing neutralization synergy by antibody combinations, we evaluated HIV immune globulin (HIVIG), and human monoclonal antibodies (MAbs) 2F5 and 2G12 given alone, compared with the double combination 2F5/2G12 and the triple combination HIVIG/2F5/2G12. Antibodies were administered 24 h prior to intravenous challenge with the pathogenic SHIV-89.6PD. Six control monkeys displayed high plasma viremia, rapid CD4(+)-cell decline, and clinical AIDS within 14 weeks. Of six animals given HIVIG/2F5/2G12, three were completely protected; the remaining three animals became SHIV infected but displayed reduced plasma viremia and near normal CD4(+)-cell counts. One of three monkeys given 2F5/2G12 exhibited only transient evidence of infection; the other two had marked reductions in viral load. All monkeys that received HIVIG, 2F5, or 2G12 alone became infected and developed high-level plasma viremia. However, compared to controls, monkeys that received HIVIG or MAb 2G12 displayed a less profound drop in CD4(+) T cells and a more benign clinical course. These data indicate a general correlation between in vitro neutralization and protection and suggest that a vaccine that elicits neutralizing antibody should have a protective effect against HIV-1 infection or disease.

  3. The response of human thermal sensation and its prediction to temperature step-change (cool-neutral-cool.

    Directory of Open Access Journals (Sweden)

    Xiuyuan Du

    Full Text Available This paper reports on studies of the effect of temperature step-change (between a cool and a neutral environment on human thermal sensation and skin temperature. Experiments with three temperature conditions were carried out in a climate chamber during the period in winter. Twelve subjects participated in the experiments simulating moving inside and outside of rooms or cabins with air conditioning. Skin temperatures and thermal sensation were recorded. Results showed overshoot and asymmetry of TSV due to the step-change. Skin temperature changed immediately when subjects entered a new environment. When moving into a neutral environment from cool, dynamic thermal sensation was in the thermal comfort zone and overshoot was not obvious. Air-conditioning in a transitional area should be considered to limit temperature difference to not more than 5°C to decrease the unacceptability of temperature step-change. The linear relationship between thermal sensation and skin temperature or gradient of skin temperature does not apply in a step-change environment. There is a significant linear correlation between TSV and Qloss in the transient environment. Heat loss from the human skin surface can be used to predict dynamic thermal sensation instead of the heat transfer of the whole human body.

  4. Crystal structure of a human rhinovirus neutralizing antibody complexed with a peptide derived from viral capsid protein VP2.

    OpenAIRE

    1994-01-01

    The three-dimensional structure of the complex between the Fab fragment of an anti-human rhinovirus neutralizing antibody (8F5) and a cross-reactive synthetic peptide from the viral capsid protein VP2 has been determined at 2.5 A resolution by crystallographic methods. The refinement is presently at an R factor of 0.18 and the antigen-binding site and viral peptide are well defined. The peptide antigen adopts a compact fold by two tight turns and interacts through hydrogen bonds, some with io...

  5. The first human epitope map of the alphaviral E1 and E2 proteins reveals a new E2 epitope with significant virus neutralizing activity.

    Directory of Open Access Journals (Sweden)

    Ann R Hunt

    Full Text Available BACKGROUND: Venezuelan equine encephalitis virus (VEEV is responsible for VEE epidemics that occur in South and Central America and the U.S. The VEEV envelope contains two glycoproteins E1 (mediates cell membrane fusion and E2 (binds receptor and elicits virus neutralizing antibodies. Previously we constructed E1 and E2 epitope maps using murine monoclonal antibodies (mMAbs. Six E2 epitopes (E2(c,d,e,f,g,h bound VEEV-neutralizing antibody and mapped to amino acids (aa 182-207. Nothing is known about the human antibody repertoire to VEEV or epitopes that engage human virus-neutralizing antibodies. There is no specific treatment for VEE; however virus-neutralizing mMAbs are potent protective and therapeutic agents for mice challenged with VEEV by either peripheral or aerosol routes. Therefore, fully human MAbs (hMAbs with virus-neutralizing activity should be useful for prevention or clinical treatment of human VEE. METHODS: We used phage-display to isolate VEEV-specific hFabs from human bone marrow donors. These hFabs were characterized by sequencing, specificity testing, VEEV subtype cross-reactivity using indirect ELISA, and in vitro virus neutralization capacity. One E2-specific neutralizing hFAb, F5n, was converted into IgG, and its binding site was identified using competitive ELISA with mMAbs and by preparing and sequencing antibody neutralization-escape variants. FINDINGS: Using 11 VEEV-reactive hFabs we constructed the first human epitope map for the alphaviral surface proteins E1 and E2. We identified an important neutralization-associated epitope unique to the human immune response, E2 aa115-119. Using a 9 A resolution cryo-electron microscopy map of the Sindbis virus E2 protein, we showed the probable surface location of this human VEEV epitope. CONCLUSIONS: The VEEV-neutralizing capacity of the hMAb F5 nIgG is similar to that exhibited by the humanized mMAb Hy4 IgG. The Hy4 IgG has been shown to limit VEEV infection in mice both

  6. Limited Neutrality

    DEFF Research Database (Denmark)

    Nielsen, Morten Ebbe Juul

    2006-01-01

    Article Concerning the prospect of a kind of limited neutrality in place of the standard liberal egalitarian "neutrality of justification."......Article Concerning the prospect of a kind of limited neutrality in place of the standard liberal egalitarian "neutrality of justification."...

  7. A gp41 MPER-specific Llama VHH Requires a Hydrophobic CDR3 for Neutralization but not for Antigen Recognition

    NARCIS (Netherlands)

    Hulsik, D.L.; Liu, Y.Y.; Strokappe, N.M.; Battella, S.; el Khattabi, M.; Bonvin, A.M.J.J.; Verrips, C.T.; Rutten, L.

    2013-01-01

    The membrane proximal external region (MPER) of the HIV-1 glycoprotein gp41 is targeted by the broadly neutralizing antibodies 2F5 and 4E10. To date, no immunization regimen in animals or humans has produced HIV-1 neutralizing MPER-specific antibodies. We immunized llamas with gp41-MPER proteoliposo

  8. Vector optimization and needle-free intradermal application of a broadly protective polyvalent influenza A DNA vaccine for pigs and humans

    DEFF Research Database (Denmark)

    Borggren, Marie; Nielsen, Jens; Bragstad, Karoline;

    2015-01-01

    such as the induction of cellular and humoral immunity, inherent safety and rapid production time. We have previously developed a DNA vaccine encoding selected influenza proteins of pandemic origin and demonstrated broad protective immune responses in ferrets and pigs. In this study, we evaluated our DNA vaccine......The threat posed by the 2009 pandemic H1N1 virus emphasized the need for new influenza A virus vaccines inducing a broad cross-protective immune response for use in both humans and pigs. An effective and broad influenza vaccine for pigs would greatly benefit the pork industry and contribute...... to public health by diminishing the risk of emerging highly pathogenic reassortants. Current inactivated protein vaccines against swine influenza produce only short-lived immunity and have no efficacy against heterologous strains. DNA vaccines are a potential alternative with advantages...

  9. Cross-neutralizing anti-HIV-1 human single chain variable fragments(scFvs) against CD4 binding site and N332 glycan identified from a recombinant phage library

    Science.gov (United States)

    Khan, Lubina; Kumar, Rajesh; Thiruvengadam, Ramachandran; Parray, Hilal Ahmad; Makhdoomi, Muzamil Ashraf; Kumar, Sanjeev; Aggarwal, Heena; Mohata, Madhav; Hussain, Abdul Wahid; Das, Raksha; Varadarajan, Raghavan; Bhattacharya, Jayanta; Vajpayee, Madhu; Murugavel, K. G.; Solomon, Suniti; Sinha, Subrata; Luthra, Kalpana

    2017-01-01

    More than 50% of HIV-1 infection globally is caused by subtype_C viruses. Majority of the broadly neutralizing antibodies (bnAbs) targeting HIV-1 have been isolated from non-subtype_C infected donors. Mapping the epitope specificities of bnAbs provides useful information for vaccine design. Recombinant antibody technology enables generation of a large repertoire of monoclonals with diverse specificities. We constructed a phage recombinant single chain variable fragment (scFv) library with a diversity of 7.8 × 108 clones, using a novel strategy of pooling peripheral blood mononuclear cells (PBMCs) of six select HIV-1 chronically infected Indian donors whose plasma antibodies exhibited potent cross neutralization efficiency. The library was panned and screened by phage ELISA using trimeric recombinant proteins to identify viral envelope specific clones. Three scFv monoclonals D11, C11 and 1F6 selected from the library cross neutralized subtypes A, B and C viruses at concentrations ranging from 0.09 μg/mL to 100 μg/mL. The D11 and 1F6 scFvs competed with mAbs b12 and VRC01 demonstrating CD4bs specificity, while C11 demonstrated N332 specificity. This is the first study to identify cross neutralizing scFv monoclonals with CD4bs and N332 glycan specificities from India. Cross neutralizing anti-HIV-1 human scFv monoclonals can be potential candidates for passive immunotherapy and for guiding immunogen design. PMID:28332627

  10. Structural basis of clade-specific HIV-1 neutralization by humanized anti-V3 monoclonal antibody KD-247.

    Science.gov (United States)

    Kirby, Karen A; Ong, Yee Tsuey; Hachiya, Atsuko; Laughlin, Thomas G; Chiang, Leslie A; Pan, Yun; Moran, Jennifer L; Marchand, Bruno; Singh, Kamalendra; Gallazzi, Fabio; Quinn, Thomas P; Yoshimura, Kazuhisa; Murakami, Toshio; Matsushita, Shuzo; Sarafianos, Stefan G

    2015-01-01

    Humanized monoclonal antibody KD-247 targets the Gly(312)-Pro(313)-Gly(314)-Arg(315) arch of the third hypervariable (V3) loop of the HIV-1 surface glycoprotein. It potently neutralizes many HIV-1 clade B isolates, but not of other clades. To understand the molecular basis of this specificity, we solved a high-resolution (1.55 Å) crystal structure of the KD-247 antigen binding fragment and examined the potential interactions with various V3 loop targets. Unlike most antibodies, KD-247 appears to interact with its target primarily through light chain residues. Several of these interactions involve Arg(315) of the V3 loop. To evaluate the role of light chain residues in the recognition of the V3 loop, we generated 20 variants of KD-247 single-chain variable fragments with mutations in the antigen-binding site. Purified proteins were assessed for V3 loop binding using AlphaScreen technology and for HIV-1 neutralization. Our data revealed that recognition of the clade-specificity defining residue Arg(315) of the V3 loop is based on a network of interactions that involve Tyr(L32), Tyr(L92), and Asn(L27d) that directly interact with Arg(315), thus elucidating the molecular interactions of KD-247 with its V3 loop target.

  11. Human symbionts inject and neutralize antibacterial toxins to persist in the gut.

    Science.gov (United States)

    Wexler, Aaron G; Bao, Yiqiao; Whitney, John C; Bobay, Louis-Marie; Xavier, Joao B; Schofield, Whitman B; Barry, Natasha A; Russell, Alistair B; Tran, Bao Q; Goo, Young Ah; Goodlett, David R; Ochman, Howard; Mougous, Joseph D; Goodman, Andrew L

    2016-03-29

    The human gut microbiome is a dynamic and densely populated microbial community that can provide important benefits to its host. Cooperation and competition for nutrients among its constituents only partially explain community composition and interpersonal variation. Notably, certain human-associated Bacteroidetes--one of two major phyla in the gut--also encode machinery for contact-dependent interbacterial antagonism, but its impact within gut microbial communities remains unknown. Here we report that prominent human gut symbionts persist in the gut through continuous attack on their immediate neighbors. Our analysis of just one of the hundreds of species in these communities reveals 12 candidate antibacterial effector loci that can exist in 32 combinations. Through the use of secretome studies, in vitro bacterial interaction assays and multiple mouse models, we uncover strain-specific effector/immunity repertoires that can predict interbacterial interactions in vitro and in vivo, and find that some of these strains avoid contact-dependent killing by accumulating immunity genes to effectors that they do not encode. Effector transmission rates in live animals can exceed 1 billion events per minute per gram of colonic contents, and multiphylum communities of human gut commensals can partially protect sensitive strains from these attacks. Together, these results suggest that gut microbes can determine their interactions through direct contact. An understanding of the strategies human gut symbionts have evolved to target other members of this community may provide new approaches for microbiome manipulation.

  12. High-throughput pseudovirion-based neutralization assay for analysis of natural and vaccine-induced antibodies against human papillomaviruses.

    Directory of Open Access Journals (Sweden)

    Peter Sehr

    Full Text Available A highly sensitive, automated, purely add-on, high-throughput pseudovirion-based neutralization assay (HT-PBNA with excellent repeatability and run-to-run reproducibility was developed for human papillomavirus types (HPV 16, 18, 31, 45, 52, 58 and bovine papillomavirus type 1. Preparation of 384 well assay plates with serially diluted sera and the actual cell-based assay are separated in time, therefore batches of up to one hundred assay plates can be processed sequentially. A mean coefficient of variation (CV of 13% was obtained for anti-HPV 16 and HPV 18 titers for a standard serum tested in a total of 58 repeats on individual plates in seven independent runs. Natural antibody response was analyzed in 35 sera from patients with HPV 16 DNA positive cervical intraepithelial neoplasia grade 2+ lesions. The new HT-PBNA is based on Gaussia luciferase with increased sensitivity compared to the previously described manual PBNA (manPBNA based on secreted alkaline phosphatase as reporter. Titers obtained with HT-PBNA were generally higher than titers obtained with the manPBNA. A good linear correlation (R(2 = 0.7 was found between HT-PBNA titers and anti-HPV 16 L1 antibody-levels determined by a Luminex bead-based GST-capture assay for these 35 sera and a Kappa-value of 0.72, with only 3 discordant sera in the low titer range. In addition to natural low titer antibody responses the high sensitivity of the HT-PBNA also allows detection of cross-neutralizing antibodies induced by commercial HPV L1-vaccines and experimental L2-vaccines. When analyzing the WHO international standards for HPV 16 and 18 we determined an analytical sensitivity of 0.864 and 1.105 mIU, respectively.

  13. Evolutionary anatomies of positions and types of disease-associated and neutral amino acid mutations in the human genome

    Directory of Open Access Journals (Sweden)

    Subramanian Sankar

    2006-12-01

    Full Text Available Abstract Background Amino acid mutations in a large number of human proteins are known to be associated with heritable genetic disease. These disease-associated mutations (DAMs are known to occur predominantly in positions essential to the structure and function of the proteins. Here, we examine how the relative perpetuation and conservation of amino acid positions modulate the genome-wide patterns of 8,627 human disease-associated mutations (DAMs reported in 541 genes. We compare these patterns with 5,308 non-synonymous Single Nucleotide Polymorphisms (nSNPs in 2,592 genes from primary SNP resources. Results The abundance of DAMs shows a negative relationship with the evolutionary rate of the amino acid positions harboring them. An opposite trend describes the distribution of nSNPs. DAMs are also preferentially found in the amino acid positions that are retained (or present in multiple vertebrate species, whereas the nSNPs are over-abundant in the positions that have been lost (or absent in the non-human vertebrates. These observations are consistent with the effect of purifying selection on natural variation, which also explains the existence of lower minor nSNP allele frequencies at highly-conserved amino acid positions. The biochemical severity of the inter-specific amino acid changes is also modulated by natural selection, with the fast-evolving positions containing more radical amino acid differences among species. Similarly, DAMs associated with early-onset diseases are more radical than those associated with the late-onset diseases. A small fraction of DAMs (10% overlap with the amino acid differences between species within the same position, but are biochemically the most conservative group of amino acid differences in our datasets. Overlapping DAMs are found disproportionately in fast-evolving amino acid positions, which, along with the conservative nature of the amino acid changes, may have allowed some of them to escape natural

  14. Structural basis for Marburg virus neutralization by a cross-reactive human antibody.

    Science.gov (United States)

    Hashiguchi, Takao; Fusco, Marnie L; Bornholdt, Zachary A; Lee, Jeffrey E; Flyak, Andrew I; Matsuoka, Rei; Kohda, Daisuke; Yanagi, Yusuke; Hammel, Michal; Crowe, James E; Saphire, Erica Ollmann

    2015-02-26

    The filoviruses, including Marburg and Ebola, express a single glycoprotein on their surface, termed GP, which is responsible for attachment and entry of target cells. Filovirus GPs differ by up to 70% in protein sequence, and no antibodies are yet described that cross-react among them. Here, we present the 3.6 Å crystal structure of Marburg virus GP in complex with a cross-reactive antibody from a human survivor, and a lower resolution structure of the antibody bound to Ebola virus GP. The antibody, MR78, recognizes a GP1 epitope conserved across the filovirus family, which likely represents the binding site of their NPC1 receptor. Indeed, MR78 blocks binding of the essential NPC1 domain C. These structures and additional small-angle X-ray scattering of mucin-containing MARV and EBOV GPs suggest why such antibodies were not previously elicited in studies of Ebola virus, and provide critical templates for development of immunotherapeutics and inhibitors of entry.

  15. Pyruvate neutralizes peritoneal dialysate cytotoxicity: maintained integrity and proliferation of cultured human mesothelial cells.

    Science.gov (United States)

    Brunkhorst, R; Mahiout, A

    1995-07-01

    Toxic effects of commercially available peritoneal dialysate (PD) fluid include damage to mesothelial cells (MC), causing a severely disturbed proliferation of cultured MC. We investigated the injury to the cell membrane (by release of lactate dehydrogenase, LDH), the proliferation (by cell counts and by 3H-thymidine incorporation), and optional the cytokine generation (by IL-1 receptor-antagonist production, IL-1 ra) of cultured human MC during the 48 hours after a 30 minute exposure to PD containing either 35 mmol/liter sodium lactate or sodium pyruvate. All solutions had a pH of 5.2 to 5.6 and were composed as standard PD. Glucose contents of 1.36 and 3.86 mmol/liter were tested. After exposure to the lactate-PD containing 1.36% glucose, LDH activity was increased by more than 30%, proliferation of MC was inhibited by more than 30%, and IL-1 ra production was reduced significantly when compared to pyruvate-PD and the control solution. After preincubation with 3.86% glucose containing PD, all negative effects became even more pronounced in the lactate group whereas the MC maintained their integrity, rate of proliferation and IL-1 ra release after pre-exposure to pyruvate containing PD. These results suggest that the acute toxic effects of commercially available PD on the integrity, proliferation and IL-1 ra production of MC can be avoided by the use of sodium pyruvate instead of sodium lactate.

  16. Crystal structure of human TWEAK in complex with the Fab fragment of a neutralizing antibody reveals insights into receptor binding.

    Directory of Open Access Journals (Sweden)

    Alfred Lammens

    Full Text Available The tumor necrosis factor-like weak inducer of apoptosis (TWEAK is a multifunctional cytokine playing a key role in tissue regeneration and remodeling. Dysregulation of TWEAK signaling is involved in various pathological processes like autoimmune diseases and cancer. The unique interaction with its cognate receptor Fn14 makes both ligand and receptor promising targets for novel therapeutics. To gain insights into this important signaling pathway, we determined the structure of soluble human TWEAK in complex with the Fab fragment of an antibody selected for inhibition of receptor binding. In the crystallized complex TWEAK is bound by three Fab fragments of the neutralizing antibody. Homology modeling shows that Fab binding overlaps with the putative Fn14 binding site of TWEAK. Docking of the Fn14 cysteine rich domain (CRD to that site generates a highly complementary interface with perfectly opposing charged and hydrophobic residues. Taken together the presented structure provides new insights into the biology of TWEAK and the TWEAK/Fn14 pathway, which will help to optimize the therapeutic strategy for treatment of related cancer types and autoimmune diseases.

  17. Structure, Aggregation, and Activity of a Covalent Insulin Dimer Formed During Storage of Neutral Formulation of Human Insulin.

    Science.gov (United States)

    Hjorth, Christian Fogt; Norrman, Mathias; Wahlund, Per-Olof; Benie, Andrew J; Petersen, Bent O; Jessen, Christian M; Pedersen, Thomas Å; Vestergaard, Kirsten; Steensgaard, Dorte B; Pedersen, Jan Skov; Naver, Helle; Hubálek, František; Poulsen, Christian; Otzen, Daniel

    2016-04-01

    A specific covalently linked dimeric species of insulin high molecular weight products (HMWPs), formed during prolonged incubation of a neutral pharmaceutical formulation of human insulin, were characterized in terms of tertiary structure, self-association, biological activity, and fibrillation properties. The dimer was formed by a covalent link between A21Asn and B29Lys. It was analyzed using static and dynamic light scattering and small-angle X-ray scattering to evaluate its self-association behavior. The tertiary structure was obtained using nuclear magnetic resonance and X-ray crystallography. The biological activity of HMWP was determined using 2 in vitro assays, and its influence on fibrillation was investigated using Thioflavin T assays. The dimer's tertiary structure was nearly identical to that of the noncovalent insulin dimer, and it was able to form hexamers in the presence of zinc. The dimer exhibited reduced propensity for self-association in the absence of zinc but significantly postponed the onset of fibrillation in insulin formulations. Consistent with its dimeric state, the tested species of HMWP showed little to no biological activity in the used assays. This study is the first detailed characterization of a specific type of human insulin HMWP formed during storage of a marketed pharmaceutical formulation. These results indicate that this specific type of HMWP is unlikely to antagonize the physical stability of the formulation, as HMWP retained a tertiary structure similar to the noncovalent dimer and participated in hexamer assembly in the presence of zinc. In addition, increasing amounts of HMWP reduce the rate of insulin fibrillation.

  18. Use of sensitive, broad-spectrum molecular assays and human airway epithelium cultures for detection of respiratory pathogens.

    Directory of Open Access Journals (Sweden)

    Krzysztof Pyrc

    Full Text Available Rapid and accurate detection and identification of viruses causing respiratory tract infections is important for patient care and disease control. Despite the fact that several assays are available, identification of an etiological agent is not possible in ~30% of patients suffering from respiratory tract diseases. Therefore, the aim of the current study was to develop a diagnostic set for the detection of respiratory viruses with sensitivity as low as 1-10 copies per reaction. Evaluation of the assay using a training clinical sample set showed that viral nucleic acids were identified in ~76% of cases. To improve assay performance and facilitate the identification of novel species or emerging strains, cultures of fully differentiated human airway epithelium were used to pre-amplify infectious viruses. This additional step resulted in the detection of pathogens in all samples tested. Based on these results it can be hypothesized that the lack of an etiological agent in some clinical samples, both reported previously and observed in the present study, may result not only from the presence of unknown viral species, but also from imperfections in the detection methods used.

  19. Use of Sensitive, Broad-Spectrum Molecular Assays and Human Airway Epithelium Cultures for Detection of Respiratory Pathogens

    Science.gov (United States)

    Wojcik, Krzysztof; Gawron, Katarzyna; Zeglen, Slawomir; Karolak, Wojciech; Wojarski, Jacek; Ochman, Marek; Hubalewska-Mazgaj, Magdalena; Bochenek, Grazyna; Sanak, Marek; Zembala, Marian; Szczeklik, Andrzej; Potempa, Jan

    2012-01-01

    Rapid and accurate detection and identification of viruses causing respiratory tract infections is important for patient care and disease control. Despite the fact that several assays are available, identification of an etiological agent is not possible in ∼30% of patients suffering from respiratory tract diseases. Therefore, the aim of the current study was to develop a diagnostic set for the detection of respiratory viruses with sensitivity as low as 1–10 copies per reaction. Evaluation of the assay using a training clinical sample set showed that viral nucleic acids were identified in ∼76% of cases. To improve assay performance and facilitate the identification of novel species or emerging strains, cultures of fully differentiated human airway epithelium were used to pre-amplify infectious viruses. This additional step resulted in the detection of pathogens in all samples tested. Based on these results it can be hypothesized that the lack of an etiological agent in some clinical samples, both reported previously and observed in the present study, may result not only from the presence of unknown viral species, but also from imperfections in the detection methods used. PMID:22403676

  20. Structural Analysis of Human and Macaque Monoclonal Antibodies 2909 and 2.5B: Implications for the Configuration of the Quaternary Neutralizing Epitope of HIV-1 gp120

    Energy Technology Data Exchange (ETDEWEB)

    B Spurrier; J Sampson; M Totrov; H Li; T ONeal; C Williams; J Robinson; M Gorny; S Zolla-Pazner; X Kong

    2011-12-31

    The quaternary neutralizing epitope (QNE) of HIV-1 gp120 is preferentially expressed on the trimeric envelope spikes of intact HIV virions, and QNE-specific monoclonal antibodies (mAbs) potently neutralize HIV-1. Here, we present the crystal structures of the Fabs of human mAb 2909 and macaque mAb 2.5B. Both mAbs have long beta hairpin CDR H3 regions >20 {angstrom} in length that are each situated at the center of their respective antigen-binding sites. Computational analysis showed that the paratopes include the whole CDR H3, while additional CDR residues form shallow binding pockets. Structural modeling suggests a way to understand the configuration of QNEs and the antigen-antibody interaction for QNE mAbs. Our data will be useful in designing immunogens that may elicit potent neutralizing QNE Abs.

  1. Staphylococcal Entertotoxins of the Enterotoxin Gene Cluster (egcSEs Induce Nitrous Oxide- and Cytokine Dependent Tumor Cell Apoptosis in a Broad Panel of Human Tumor Cells

    Directory of Open Access Journals (Sweden)

    David eTerman

    2013-08-01

    Full Text Available The egcSEs comprise five genetically linked staphylococcal enterotoxins, SEG, SEI, SElM, SElN and SElO and two pseudotoxins which constitute an operon present in up to 80% of Staphylococcus aureus isolates. A preparation containing theses proteins was recently used to treat advanced lung cancer with pleural effusion. We investigated the hypothesis that egcSEs induce nitrous oxide (NO and associated cytokine production and that these agents may be involved in tumoricidal effects against a broad panel of clinically relevant human tumor cells. Preliminary studies showed that egcSEs and SEA activated T cells (range: 11-25% in a concentration dependent manner. Peripheral blood mononuclear cells (PBMCs stimulated with equimolar quantities of egcSEs expressed NO synthase and generated robust levels of nitrite (range: 200-250 µM, a breakdown product of NO; this reaction was inhibited by NG-monomethyl-L-arginine (L-NMMA (0.3 mM, an NO synthase antagonist. Cell free supernatants (CSFs of all egcSE-stimulated PBMCs were also equally effective in inducing concentration dependent tumor cell apoptosis in a broad panel of human tumor cells. The latter effect was due in part to the generation of NO and TNF-α since it was significantly abolished by L-NMMA, anti-TNF-α antibodies respectively and a combination thereof. A hierarchy of tumor cell sensitivity to these CFSs was as follows: lung carcinoma>osteogenic sarcoma>melanoma>breast carcinoma>neuroblastoma. Notably, SEG induced robust activation of NO/TNFα-dependent tumor cell apoptosis comparable to the other egcSEs and SEA despite TNF-α and IFN-γ levels that were 2 and 8 fold lower respectively than the other egcSEs and SEA. Thus, egcSEs produced by S. aureus induce NO synthase and the increased NO formation together with TNF-α appear to contribute to egcSE-mediated apoptosis against a broad panel of human tumor cells.

  2. Novel, broad-spectrum anticonvulsants containing a sulfamide group: advancement of N-((benzo[b]thien-3-yl)methyl)sulfamide (JNJ-26990990) into human clinical studies.

    Science.gov (United States)

    Parker, Michael H; Smith-Swintosky, Virginia L; McComsey, David F; Huang, Yifang; Brenneman, Douglas; Klein, Brian; Malatynska, Ewa; White, H Steve; Milewski, Michael E; Herb, Mark; Finley, Michael F A; Liu, Yi; Lubin, Mary Lou; Qin, Ning; Iannucci, Robert; Leclercq, Laurent; Cuyckens, Filip; Reitz, Allen B; Maryanoff, Bruce E

    2009-12-10

    In seeking broad-spectrum anticonvulsants to treat epilepsy and other neurological disorders, we synthesized and tested a group of sulfamide derivatives (4a-k, 5), which led to the clinical development of 4a (JNJ-26990990). This compound exhibited excellent anticonvulsant activity in rodents against audiogenic, electrically induced, and chemically induced seizures, with very weak inhibition of human carbonic anhydrase-II (IC(50) = 110 microM). The pharmacological profile for 4a supports its potential in the treatment of multiple forms of epilepsy, including pharmacoresistant variants. Mechanistically, 4a inhibited voltage-gated Na(+) channels and N-type Ca(2+) channels but was not effective as a K(+) channel opener. The pharmacokinetics and metabolic properties of 4a are discussed.

  3. Broad tuning of the human bitter taste receptor hTAS2R46 to various sesquiterpene lactones, clerodane and labdane diterpenoids, strychnine, and denatonium.

    Science.gov (United States)

    Brockhoff, Anne; Behrens, Maik; Massarotti, Alberto; Appendino, Giovanni; Meyerhof, Wolfgang

    2007-07-25

    Sesquiterpene lactones are a major class of natural bitter compounds occurring in vegetables and culinary herbs as well as in aromatic and medicinal plants, where they often represent the main gustatory and pharmacologically active component. Investigations on sesquiterpene lactones have mainly focused on their bioactive potential rather than on their sensory properties. In the present study, we report about the stimulation of heterologously expressed human bitter taste receptors, hTAS2Rs, by the bitter sesquiterpene lactone herbolide D. A specific response to herbolide D was observed i.a. for hTAS2R46, a so far orphan bitter taste receptor without any known ligand. By further investigation of its agonist pattern, we characterized hTAS2R46 as a bitter receptor broadly tuned to sesquiterpene lactones and to clerodane and labdane diterpenoids as well as to the unrelated bitter substances strychnine and denatonium.

  4. Analysis of cross-reactive neutralizing antibodies in human HFMD serum with an EV71 pseudovirus-based assay.

    Science.gov (United States)

    Zhang, Huafei; An, Dong; Liu, Wei; Mao, Qunying; Jin, Jun; Xu, Lin; Sun, Shiyang; Jiang, Liping; Li, Xiaojun; Shao, Jie; Ma, Hongxia; Huang, Xueyong; Guo, Shijie; Chen, Haiying; Cheng, Tong; Yang, Lisheng; Su, Weiheng; Kong, Wei; Liang, Zhenglun; Jiang, Chunlai

    2014-01-01

    Hand, foot and mouth disease, associated with enterovirus 71 (EV71) infections, has recently become an important public health issue throughout the world. Serum neutralizing antibodies are major indicators of EV71 infection and protective immunity. However, the potential for cross-reactivity of neutralizing antibodies for different EV71 genotypes and subgenotypes is unclear. Here we measured the cross-reactive neutralizing antibody titers against EV71 of different genotypes or subgenotypes in sera collected from EV71-infected children and vaccine-inoculated children in a phase III clinical trial (ClinicalTrials.gov Identifier: NCT01636245) using a new pseudovirus-based neutralization assay. Antibodies induced by EV71-C4a were cross-reactive for different EV71 genotypes, demonstrating that C4a is a good candidate strain for an EV71 vaccine. Our study also demonstrated that this new assay is practical for analyses of clinical samples from epidemiological and vaccine studies.

  5. X-ray absorption and resonance raman spectroscopy of human myeloperoxidase at neutral and acid pH.

    Science.gov (United States)

    Yue, K T; Taylor, K L; Kinkade, J M; Sinclair, R B; Powers, L S

    1997-04-01

    Myeloperoxidase (MPO), an important enzyme in the oxygen-dependent host defense system of human polymorphonuclear leukocytes, utilizes hydrogen peroxide to catalyze the production of hypochlorous acid, an oxidizing bactericidal agent. While MPO shows significant sequence homology with other peroxidases and this homology is particularly striking among the active-site residues, MPO exhibits unusual spectral features and the unique ability to catalyze the oxidation of chloride ions. We have investigated the MPO active-site with X-ray absorption (XAS) and resonance Raman (RRS) spectroscopies at neutral pH and also at the physiological acidic pH (pH approximately 3) and have compared these results with those of horseradish peroxidase (HRP). At pH 7.5, XAS results show that the iron heme active site is 6-coordinate where the distal ligand is likely nitrogen or oxygen, but not sulfur. The heme is distorted compared to HRP, other peroxidases, and heme compounds, but at pH approximately 3, the distal ligand is lost and the heme is less distorted. RRS results under identical pH conditions show that the skeletal core-size sensitive modes and v3 are shifted to higher frequency at pH approximately 3 indicating a 6- to 5-coordination change of high spin ferric heme. In addition, a new band at 270 cm(-1) is observed at pH approximately 3 which is consistent with the loss of the sixth ligand. The higher symmetry of the heme at pH approximately 3 is reflected by a single v4 mode in the (RRS) spectrum. HRP also loses its loosely associated distal water at this pH, but little change in heme distortion is observed. This change suggests that loss of the distal ligand in MPO releases stress on the heme which may facilitate binding of chloride ion.

  6. HIV-1广谱中和抗体的研究进展和启示%Recent progress in the development of broadly neutralizing anti-HIV-1 antibodies

    Institute of Scientific and Technical Information of China (English)

    汪桦; 刘懿锋; 张林琦

    2011-01-01

    1983年,Barré-Sinoussi和Montagnier等[1]首次分离到引起获得性免疫缺陷综合征(acquired immune deficiency syndrome,AIDS)的病毒,随后国际病毒分类委员会将其命名为人类免疫缺陷病毒[2](human immunodeficiency virus,HIV).据世界卫生组织(World Health Organization,WHO)及联合国艾滋病规划署(The Joint United Nations Programme on HIV/AIDS,UNAIDS)的最新统计[3],全球范围内HIV的感染人数约为3 330万,仅2009年新增的HIV感染人数就达260万,死亡人数为180万.

  7. H5N1型禽流感病毒广谱中和单克隆抗体的筛选及其中和机制初步研究%Broad-spectrum Neutralizing Monoclonal Antibodies Against H5N1 Avian Influenza A Viruses and Primary Research on The Mechanism

    Institute of Scientific and Technical Information of China (English)

    张晓; 曾晓燕; 刘哲; 金秋; 徐言; 冯振卿; 焦永军

    2011-01-01

    Avian influenza is a highly contagious disease of birds caused by A influenza viruses. The circulation in humans by the highly pathogenic H5N1 avian flu in the past few years have caused most pandemics and have heightened fear that the next influenza pandemic is due. Antibodies could be used as an efficient anti-virus agent in clinical therapy. The full-length HA of the A/Jiangsu/1/2007(H5N 1) about 1.7 kb was amplified, subcloned to the pFastBac vector and recombinant bacmid DNA was selected. The recombinant HA was expressed and purified HA about 70 ku was used as the antigen to immunize Balb/c mice. The whole H5N1 virus was used to select 5 mono-antibodies (mAbs), and all of them were tested using microneutralization assays. 8G10D7, one of the antibodies, had broad neutralizing effect against clade 2 and clade 9 H5N1 avian influenza A viruses, and the IC50 was from 1 : 256 to I ' 64. When detected with 8G10D7, all 4 viruses showed 70 ku and 43 ku protein band,which confirms that the binding site of the scFv antibodies were located at the HA1 domain. The nucleuses of MDCK cells infected by 4 viruses were colored purple, and red around the nucleus. 8Gl 0D7 showed HI activity to the 4 viruses, the HI has a positive correlation with neutralization concentration IC50, which also further confirms that the binding site of the scFv antibodies were located at the HA1 domain. When the mAb 8Gl 0D7 was used for the study of prophylaxis and therapeutic effect on influenza A viruses infection in an embryonated chicken eggs model. It had a complete 100% protection effect on the H5N1 viruses in avian host in the prophylactic and therapeutic groups. The 100% preventive protection effect could be reached when challenged with H5N1 avian influenza A viruse in human host in the prophylactic groups, and there is also a 87.5% protection effect with H5N1 viruses in human host in the therapeutic groups. Thereby, the study suggests that the mAb 8G10D7 could be used in therapies to

  8. Construction and Identification of Antibodies 2F5 and 4E10 Target Gene Vectors with the Neutralization of Anti-HIV Broad Spectrum%含抗HIV广谱中和抗体2F5、4E10靶基因载体的构建及鉴定

    Institute of Scientific and Technical Information of China (English)

    王晶妍; 张煜; 王珂; 王建英

    2012-01-01

    目的:构建含HIV gp120,gp41序列中广谱中和抗体2F5,4E10作用靶基因的载体并进行鉴定,为后期重组载体表达产物诱导产生中和抗体及抗HIV亚单位疫苗的研究奠定基础.方法:根据NCBI中HIV gp120,gp41基因序列中可与2F5、4E10结合的区域设计引物并进行PCR反应,将PCR得到的目的片段插入到载体pET28a中,对重组载体进行PCR鉴定、酶切鉴定及DNA测序.结果:PCR鉴定、酶切鉴定及DNA测序结果证实重组载体构建成功.结论:成功构建了含HIV gp120,gp41序列中广谱中和抗体2F5,4E10作用靶基因的载体.%Objective Antibodies 2F5 and 4E10 target gene vectors with the neutralization of anti-HIV broad spectrum were constructed and identified. This will certainly lay the foundation for future research of neutralizing antibodies from recom-binanl vector products in expression and induction and anti-HIV subunit vaccine. Method Primers were designed and PCR was conducted according to the binding region of the HIV gp120, gp41 gene sequences in NCBI and the 2F5 , 4E10. Then the target fragments from PCR were inserted into the vector pET28a. In the end. PCR, restriction enzyme digestion and DNA sequencing were performed for the recombinant vectors. The results; PCR, restriction enzyme digestion and DNA sequencing confirmed that the recombinant vector were successfully constructed. The conclusion; The construction of vectors of broad-spectrum neutralizing antibodies 2F5, 4E10 target genes containing HIV gp120. gp41 gene sequences was successfully conducted in this paper.

  9. HLA-A2–Restricted Cytotoxic T Lymphocyte Epitopes from Human Heparanase as Novel Targets for Broad-Spectrum Tumor Immunotherapy

    Directory of Open Access Journals (Sweden)

    Ting Chen

    2008-09-01

    Full Text Available Peptide vaccination for cancer immunotherapy requires identification of peptide epitopes derived from antigenic proteins associated with tumors. Heparanase (Hpa is broadly expressed in various advanced tumors and seems to be an attractive new tumor-associated antigen. The present study was designed to predict and identify HLA-A2– restricted cytotoxic T lymphocyte (CTL epitopes in the protein of human Hpa. For this purpose, HLA-A2–restricted CTL epitopes were identified using the following four-step procedure: 1 a computer-based epitope prediction from the amino acid sequence of human Hpa, 2 a peptide-binding assay to determine the affinity of the predicted protein with the HLA-A2 molecule, 3 stimulation of the primary T-cell response against the predicted peptides in vitro, and 4 testing of the induced CTLs toward different kinds of carcinoma cells expressing Hpa antigens and/or HLA-A2. The results demonstrated that, of the tested peptides, effectors induced by peptides of human Hpa containing residues 525-533 (PAFSYSFFV, Hpa525, 277-285 (KMLKSFLKA, Hpa277, and 405-413 (WLSLLFKKL, Hpa405 could effectively lyse various tumor cell lines that were Hpa-positive and HLA-A2-matched. We also found that these peptide-specific CTLs could not lyse autologous lymphocytes with low Hpa activity. Further study revealed that Hpa525, Hpa277, and Hpa405 peptides increased the frequency of IFN-γ–producing T cells compared to a negative peptide. Our results suggest that Hpa525, Hpa277, and Hpa405 peptides are new HLA-A2–restricted CTL epitopes capable of inducing Hpa-specific CTLs in vitro. Because Hpa is expressed in most advanced malignant tumors, Hpa525, Hpa277, and Hpa405 peptide–based vaccines may be useful for the immunotherapy for patients with advanced tumors.

  10. A focus reduction neutralization assay for hepatitis C virus neutralizing antibodies

    Directory of Open Access Journals (Sweden)

    Wychowski Czeslaw

    2007-03-01

    Full Text Available Abstract Background/Aim The role of humoral immunity in hepatitis C virus (HCV infection is poorly understood. Nevertheless, there is increasing interest in characterizing the neutralizing antibodies in the serum of HCV-infected patients. Focus reduction assays have been widely used to evaluate neutralizing antibody responses against a range of non-cytopathic viruses. Based on the recent development of a HCV cell culture system using the genotype 2 JFH-1-strain, we developed a focus reduction assay for HCV-neutralizing antibodies. Methods The focus reduction assay was based on a standard microneutralization assay in which immunostained foci on tissue culture plates are counted. The neutralizing anti-HCV antibodies titers of purified serum immunoglobulin samples from seventy-seven individuals were determined using a 50% focus reduction neutralization assay. Each titer was determined as the log value of the reciprocal antibody dilution that reduced the number of viral foci by 50%. IgG antibodies were first purified from each serum in order to avoid the facilitating effect of HDL on HCV entry. Results The assay's cut-off using an ELISA and RNA HCV-negative samples was found to be 1.25 log, corresponding to a dilution of 1:18. The assay was compared with a commercial HCV ELISA and exhibited specificity and sensitivity values of 100% and 96.5%, respectively, and good reproducibility (with intra-assay and inter-assay coefficients of variation of 6.7% and 12.6%, respectively. The assay did not show any cross-reactivity with anti-HIV, anti-HBs or heterophile antibody-positive samples. The neutralizing antibodies titers were 2.13 log (1:134 for homologous samples from HCV genotype 2 infected patients harboring the same genotype as JFH-1 and 1.93 log (1:85 for heterologous samples from patients infected by genotypes other than type 2. These results confirm the presence of broadly cross-neutralizing antibodies already reported using the HCV pseudoparticles

  11. Hyperimmune antisera against synthetic peptides representing the glycoprotein of human immunodeficiency virus type 2 can mediate neutralization and antibody-dependent cytotoxic activity.

    Science.gov (United States)

    Björling, E; Broliden, K; Bernardi, D; Utter, G; Thorstensson, R; Chiodi, F; Norrby, E

    1991-01-01

    Twenty-five 13- to 35-amino-acid-long peptides representing regions of human immunodeficiency virus type 2 (HIV-2), strain SBL6669, envelope proteins were evaluated for their immunogenic activity in guinea pigs. The peptides were selected to provide homologous representation of sites in the HIV-1 envelope proteins that were previously documented to have a particular immunogenic importance. A number of the HIV-2 peptides were found to be capable of inducing strain SBL6669 neutralizing and antibody-dependent cellular cytotoxicity (ADCC) antibodies. Two overlapping peptides covering amino acids 311-337 representing the central and C-terminal part of the variable third (V3) region, terminology according to Modrow et al. [Modrow, S., Hahn, B., Shaw, G. M., Gallo, R. C., Wong-Staal, F. & Wolf, H. (1987) J. Virol. 61, 570-578], showed the most pronounced capacity to induce neutralizing antibodies. One of the peptides (amino acids 318-337) also induced antibodies mediating ADCC. Two additional regions in the large glycoprotein, gp125, containing linear sites reacting with neutralizing antibodies were identified (amino acids, 119-137 and 472-509). The transmembrane protein, gp36, of HIV-2 harbored two regions of importance for induction of neutralizing antibodies (amino acids 595-614 and 714-729). ADCC activity was induced by two additional gp125-specific peptides (amino acids 291-311 and 446-461). Thus, except for the single V3-specific site there was no correlation between linear immunogenic sites stimulating neutralizing antibody and ADCC activity. These findings pave the way for development of synthetic vaccines against HIV-2 and possibly also simian immunodeficiency virus infections. The capacity of such a product to induce protective immunity can be evaluated in macaque monkeys. Images PMID:2068087

  12. Anisotropy in broad component of H$\\alpha$ line in the magnetospheric device RT-1

    CERN Document Server

    Kawazura, Yohei; Yoshida, Zensho; Nishiura, Masaki; Nogami, Tomoaki; Kashyap, Ankur; Yano, Yoshihisa; Saitoh, Haruhiko; Yamasaki, Miyuri; Mushiake, Toshiki; Nakatsuka, Masataka

    2016-01-01

    Temperature anisotropy in broad component of H$\\alpha$ line was found in the ring trap 1 (RT-1) device by Doppler spectroscopy. Since hot hydrogen neutrals emitting a broad component are mainly produced by charge exchange between neutrals and protons, the anisotropy in the broad component is the evidence of proton temperature anisotropy generated by betatron acceleration.

  13. The neutralization sensitivity of viruses representing human immunodeficiency virus type 1 variants of diverse subtypes from early in infection is dependent on producer cell, as well as characteristics of the specific antibody and envelope variant.

    Science.gov (United States)

    Provine, Nicholas M; Cortez, Valerie; Chohan, Vrasha; Overbaugh, Julie

    2012-05-25

    Neutralization properties of human immunodeficiency virus (HIV-1) are often defined using pseudoviruses grown in transformed cells, which are not biologically relevant HIV-1 producer cells. Little information exists on how these viruses compare to viruses produced in primary lymphocytes, particularly for globally relevant HIV-1 strains. Therefore, replication-competent chimeras encoding envelope variants from the dominant HIV-1 subtypes (A, C, and D) obtained early after infection were generated and the neutralization properties explored. Pseudoviruses generated in 293T cells were the most sensitive to antibody neutralization. Replicating viruses generated in primary lymphocytes were most resistant to neutralization by plasma antibodies and most monoclonal antibodies (b12, 4E10, 2F5, VRC01). These differences were not associated with differences in envelope content. Surprisingly, the virus source did not impact neutralization sensitivity of most viruses to PG9. These findings suggest that producer cell type has a major effect on neutralization sensitivity, but in an antibody dependent manner.

  14. Local CD4 and CD8 T-cell reactivity to HSV-1 antigens documents broad viral protein expression and immune competence in latently infected human trigeminal ganglia.

    Directory of Open Access Journals (Sweden)

    Monique van Velzen

    2013-08-01

    Full Text Available Herpes simplex virus type 1 (HSV-1 infection results in lifelong chronic infection of trigeminal ganglion (TG neurons, also referred to as neuronal HSV-1 latency, with periodic reactivation leading to recrudescent herpetic disease in some persons. HSV-1 proteins are expressed in a temporally coordinated fashion during lytic infection, but their expression pattern during latent infection is largely unknown. Selective retention of HSV-1 reactive T-cells in human TG suggests their role in controlling reactivation by recognizing locally expressed HSV-1 proteins. We characterized the HSV-1 proteins recognized by virus-specific CD4 and CD8 T-cells recovered from human HSV-1-infected TG. T-cell clusters, consisting of both CD4 and CD8 T-cells, surrounded neurons and expressed mRNAs and proteins consistent with in situ antigen recognition and antiviral function. HSV-1 proteome-wide scans revealed that intra-TG T-cell responses included both CD4 and CD8 T-cells directed to one to three HSV-1 proteins per person. HSV-1 protein ICP6 was targeted by CD8 T-cells in 4 of 8 HLA-discordant donors. In situ tetramer staining demonstrated HSV-1-specific CD8 T-cells juxtaposed to TG neurons. Intra-TG retention of virus-specific CD4 T-cells, validated to the HSV-1 peptide level, implies trafficking of viral proteins from neurons to HLA class II-expressing non-neuronal cells for antigen presentation. The diversity of viral proteins targeted by TG T-cells across all kinetic and functional classes of viral proteins suggests broad HSV-1 protein expression, and viral antigen processing and presentation, in latently infected human TG. Collectively, the human TG represents an immunocompetent environment for both CD4 and CD8 T-cell recognition of HSV-1 proteins expressed during latent infection. HSV-1 proteins recognized by TG-resident T-cells, particularly ICP6 and VP16, are potential HSV-1 vaccine candidates.

  15. Mitochondrial genome sequencing in Mesolithic North East Europe Unearths a new sub-clade within the broadly distributed human haplogroup C1.

    Directory of Open Access Journals (Sweden)

    Clio Der Sarkissian

    Full Text Available The human mitochondrial haplogroup C1 has a broad global distribution but is extremely rare in Europe today. Recent ancient DNA evidence has demonstrated its presence in European Mesolithic individuals. Three individuals from the 7,500 year old Mesolithic site of Yuzhnyy Oleni Ostrov, Western Russia, could be assigned to haplogroup C1 based on mitochondrial hypervariable region I sequences. However, hypervariable region I data alone could not provide enough resolution to establish the phylogenetic relationship of these Mesolithic haplotypes with haplogroup C1 mitochondrial DNA sequences found today in populations of Europe, Asia and the Americas. In order to obtain high-resolution data and shed light on the origin of this European Mesolithic C1 haplotype, we target-enriched and sequenced the complete mitochondrial genome of one Yuzhnyy Oleni Ostrov C1 individual. The updated phylogeny of C1 haplogroups indicated that the Yuzhnyy Oleni Ostrov haplotype represents a new distinct clade, provisionally coined "C1f". We show that all three C1 carriers of Yuzhnyy Oleni Ostrov belong to this clade. No haplotype closely related to the C1f sequence could be found in the large current database of ancient and present-day mitochondrial genomes. Hence, we have discovered past human mitochondrial diversity that has not been observed in modern-day populations so far. The lack of positive matches in modern populations may be explained by under-sampling of rare modern C1 carriers or by demographic processes, population extinction or replacement, that may have impacted on populations of Northeast Europe since prehistoric times.

  16. Analysis of memory B cell responses and isolation of novel monoclonal antibodies with neutralizing breadth from HIV-1-infected individuals.

    Directory of Open Access Journals (Sweden)

    Davide Corti

    Full Text Available BACKGROUND: The isolation of human monoclonal antibodies (mAbs that neutralize a broad spectrum of primary HIV-1 isolates and the characterization of the human neutralizing antibody B cell response to HIV-1 infection are important goals that are central to the design of an effective antibody-based vaccine. METHODS AND FINDINGS: We immortalized IgG(+ memory B cells from individuals infected with diverse clades of HIV-1 and selected on the basis of plasma neutralization profiles that were cross-clade and relatively potent. Culture supernatants were screened using various recombinant forms of the envelope glycoproteins (Env in multiple parallel assays. We isolated 58 mAbs that were mapped to different Env surfaces, most of which showed neutralizing activity. One mAb in particular (HJ16 specific for a novel epitope proximal to the CD4 binding site on gp120 selectively neutralized a multi-clade panel of Tier-2 HIV-1 pseudoviruses, and demonstrated reactivity that was comparable in breadth, but distinct in neutralization specificity, to that of the other CD4 binding site-specific neutralizing mAb b12. A second mAb (HGN194 bound a conserved epitope in the V3 crown and neutralized all Tier-1 and a proportion of Tier-2 pseudoviruses tested, irrespective of clade. A third mAb (HK20 with broad neutralizing activity, particularly as a Fab fragment, recognized a highly conserved epitope in the HR-1 region of gp41, but showed striking assay-dependent selectivity in its activity. CONCLUSIONS: This study reveals that by using appropriate screening methods, a large proportion of memory B cells can be isolated that produce mAbs with HIV-1 neutralizing activity. Three of these mAbs show unusual breadth of neutralization and therefore add to the current panel of HIV-1 neutralizing antibodies with potential for passive protection and template-based vaccine design.

  17. Automated facial coding software outperforms people in recognizing neutral faces as neutral from standardized datasets

    NARCIS (Netherlands)

    Lewinski, P.

    2015-01-01

    Little is known about people’s accuracy of recognizing neutral faces as neutral. In this paper, I demonstrate the importance of knowing how well people recognize neutral faces. I contrasted human recognition scores of 100 typical, neutral front-up facial images with scores of an arguably objective j

  18. Structural Basis of Neutralization of the Major Toxic Component from the Scorpion Centruroides noxius Hoffmann by a Human-derived Single-chain Antibody Fragment*

    Science.gov (United States)

    Canul-Tec, Juan Carlos; Riaño-Umbarila, Lidia; Rudiño-Piñera, Enrique; Becerril, Baltazar; Possani, Lourival D.; Torres-Larios, Alfredo

    2011-01-01

    It has previously been reported that several single-chain antibody fragments of human origin (scFv) neutralize the effects of two different scorpion venoms through interactions with the primary toxins of Centruroides noxius Hoffmann (Cn2) and Centruroides suffusus suffusus (Css2). Here we present the crystal structure of the complex formed between one scFv (9004G) and the Cn2 toxin, determined in two crystal forms at 2.5 and 1.9 Å resolution. A 15-residue span of the toxin is recognized by the antibody through a cleft formed by residues from five of the complementarity-determining regions of the scFv. Analysis of the interface of the complex reveals three features. First, the epitope of toxin Cn2 overlaps with essential residues for the binding of β-toxins to its Na+ channel receptor site. Second, the putative recognition of Css2 involves mainly residues that are present in both Cn2 and Css2 toxins. Finally, the effect on the increase of affinity of previously reported key residues during the maturation process of different scFvs can be inferred from the structure. Taken together, these results provide the structural basis that explain the mechanism of the 9004G neutralizing activity and give insight into the process of directed evolution that gave rise to this family of neutralizing scFvs. PMID:21489992

  19. Structural Basis of Neutralization of the Major Toxic Component from the Scorpion Centruroides noxius Hoffmann by a Human-derived Single-chain Antibody Fragment

    Energy Technology Data Exchange (ETDEWEB)

    Canul-Tec, Juan Carlos; Riaño-Umbarila, Lidia; Rudiño-Piñera, Enrique; Becerril, Baltazar; Possani, Lourival D.; Torres-Larios, Alfredo (U. NAM)

    2011-08-09

    It has previously been reported that several single-chain antibody fragments of human origin (scFv) neutralize the effects of two different scorpion venoms through interactions with the primary toxins of Centruroides noxius Hoffmann (Cn2) and Centruroides suffusus suffusus (Css2). Here we present the crystal structure of the complex formed between one scFv (9004G) and the Cn2 toxin, determined in two crystal forms at 2.5 and 1.9 {angstrom} resolution. A 15-residue span of the toxin is recognized by the antibody through a cleft formed by residues from five of the complementarity-determining regions of the scFv. Analysis of the interface of the complex reveals three features. First, the epitope of toxin Cn2 overlaps with essential residues for the binding of {beta}-toxins to its Na+ channel receptor site. Second, the putative recognition of Css2 involves mainly residues that are present in both Cn2 and Css2 toxins. Finally, the effect on the increase of affinity of previously reported key residues during the maturation process of different scFvs can be inferred from the structure. Taken together, these results provide the structural basis that explain the mechanism of the 9004G neutralizing activity and give insight into the process of directed evolution that gave rise to this family of neutralizing scFvs.

  20. Structural basis of neutralization of the major toxic component from the scorpion Centruroides noxius Hoffmann by a human-derived single-chain antibody fragment.

    Science.gov (United States)

    Canul-Tec, Juan Carlos; Riaño-Umbarila, Lidia; Rudiño-Piñera, Enrique; Becerril, Baltazar; Possani, Lourival D; Torres-Larios, Alfredo

    2011-06-10

    It has previously been reported that several single-chain antibody fragments of human origin (scFv) neutralize the effects of two different scorpion venoms through interactions with the primary toxins of Centruroides noxius Hoffmann (Cn2) and Centruroides suffusus suffusus (Css2). Here we present the crystal structure of the complex formed between one scFv (9004G) and the Cn2 toxin, determined in two crystal forms at 2.5 and 1.9 Å resolution. A 15-residue span of the toxin is recognized by the antibody through a cleft formed by residues from five of the complementarity-determining regions of the scFv. Analysis of the interface of the complex reveals three features. First, the epitope of toxin Cn2 overlaps with essential residues for the binding of β-toxins to its Na(+) channel receptor site. Second, the putative recognition of Css2 involves mainly residues that are present in both Cn2 and Css2 toxins. Finally, the effect on the increase of affinity of previously reported key residues during the maturation process of different scFvs can be inferred from the structure. Taken together, these results provide the structural basis that explain the mechanism of the 9004G neutralizing activity and give insight into the process of directed evolution that gave rise to this family of neutralizing scFvs.

  1. Structural Basis of Neutralization of the Major Toxic Component from the Scorpion Centruroides noxius Hoffmann by a Human-derived Single-chain Antibody Fragment*

    OpenAIRE

    Canul-Tec, Juan Carlos; Riaño-Umbarila, Lidia; Rudiño-Piñera, Enrique; Becerril, Baltazar; Possani, Lourival D.; Torres-Larios, Alfredo

    2011-01-01

    It has previously been reported that several single-chain antibody fragments of human origin (scFv) neutralize the effects of two different scorpion venoms through interactions with the primary toxins of Centruroides noxius Hoffmann (Cn2) and Centruroides suffusus suffusus (Css2). Here we present the crystal structure of the complex formed between one scFv (9004G) and the Cn2 toxin, determined in two crystal forms at 2.5 and 1.9 Å resolution. A 15-residue span of the toxin is recognized by th...

  2. A humanized monoclonal antibody neutralizes yellow fever virus strain 17D-204 in vitro but does not protect a mouse model from disease.

    Science.gov (United States)

    Calvert, Amanda E; Dixon, Kandice L; Piper, Joseph; Bennett, Susan L; Thibodeaux, Brett A; Barrett, Alan D T; Roehrig, John T; Blair, Carol D

    2016-07-01

    The yellow fever virus (YFV) vaccine 17D-204 is considered safe and effective, yet rare severe adverse events (SAEs), some resulting in death, have been documented following vaccination. Individuals exhibiting post-vaccinal SAEs are ideal candidates for antiviral monoclonal antibody (MAb) therapy; the time until appearance of clinical signs post-exposure is usually short and patients are quickly hospitalized. We previously developed a murine-human chimeric monoclonal antibody (cMAb), 2C9-cIgG, reactive with both virulent YFV and 17D-204, and demonstrated its ability to prevent and treat YF disease in both AG129 mouse and hamster models of infection. To counteract possible selection of 17D-204 variants that escape neutralization by treatment with a single MAb (2C9-cIgG), we developed a second cMAb, 864-cIgG, for use in combination with 2C9-cIgG in post-vaccinal therapy. MAb 864-cIgG recognizes/neutralizes only YFV 17D-204 vaccine substrain and binds to domain III (DIII) of the viral envelope protein, which is different from the YFV type-specific binding site of 2C9-cIgG in DII. Although it neutralized 17D-204 in vitro, administration of 864-cIgG had no protective capacity in the interferon receptor-deficient AG129 mouse model of 17D-204 infection. The data presented here show that although DIII-specific 864-cIgG neutralizes virus infectivity in vitro, it does not have the ability to abrogate disease in vivo. Therefore, combination of 864-cIgG with 2C9-cIgG for treatment of YF vaccination SAEs does not appear to provide an improvement on 2C9-cIgG therapy alone.

  3. A human antibody to the CD4 binding site of gp120 capable of highly potent but sporadic cross clade neutralization of primary HIV-1.

    Directory of Open Access Journals (Sweden)

    Johannes S Gach

    Full Text Available Primary isolates of HIV-1 resist neutralization by most antibodies to the CD4 binding site (CD4bs on gp120 due to occlusion of this site on the trimeric spike. We describe 1F7, a human CD4bs monoclonal antibody that was found to be exceptionally potent against the HIV-1 primary isolate JR-FL. However, 1F7 failed to neutralize a patient-matched primary isolate, JR-CSF even though the two isolates differ by <10% in gp120 at the protein level. In an HIV-1 cross clade panel (n = 157, 1F7 exhibited moderate breadth, but occasionally achieved considerable potency. In binding experiments using monomeric gp120s of select resistant isolates and domain-swap chimeras between JR-FL and JR-CSF, recognition by 1F7 was limited by sequence polymorphisms involving at least the C2 region of Env. Putative N-linked glycosylation site (PNGS mutations, notably at position 197, allowed 1F7 to neutralize JR-CSF potently without improving binding to the cognate, monomeric gp120. In contrast, flow cytometry experiments using the same PNGS mutants revealed that 1F7 binding is enhanced on cognate trimeric Env. BN-PAGE mobility shift experiments revealed that 1F7 is sensitive to the diagnostic mutation D368R in the CD4 binding loop of gp120. Our data on 1F7 reinforce how exquisitely targeted CD4bs antibodies must be to achieve cross neutralization of two closely related primary isolates. High-resolution analyses of trimeric Env that show the orientation of glycans and polymorphic elements of the CD4bs that affect binding to antibodies like 1F7 are desirable to understand how to promote immunogenicity of more conserved elements of the CD4bs.

  4. Focused Evolution of HIV-1 Neutralizing Antibodies Revealed by Structures and Deep Sequencing

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Xueling; Zhou, Tongqing; Zhu, Jiang; Zhang, Baoshan; Georgiev, Ivelin; Wang, Charlene; Chen, Xuejun; Longo, Nancy S.; Louder, Mark; McKee, Krisha; O’Dell, Sijy; Perfetto, Stephen; Schmidt, Stephen D.; Shi, Wei; Wu, Lan; Yang, Yongping; Yang, Zhi-Yong; Yang, Zhongjia; Zhang, Zhenhai; Bonsignori, Mattia; Crump, John A.; Kapiga, Saidi H.; Sam, Noel E.; Haynes, Barton F.; Simek, Melissa; Burton, Dennis R.; Koff, Wayne C.; Doria-Rose, Nicole A.; Connors, Mark; Mullikin, James C.; Nabel, Gary J.; Roederer, Mario; Shapiro, Lawrence; Kwong, Peter D.; Mascola, John R. (Tumaini); (NIH); (Duke); (Kilimanjaro Repro.); (IAVI)

    2013-03-04

    Antibody VRC01 is a human immunoglobulin that neutralizes about 90% of HIV-1 isolates. To understand how such broadly neutralizing antibodies develop, we used x-ray crystallography and 454 pyrosequencing to characterize additional VRC01-like antibodies from HIV-1-infected individuals. Crystal structures revealed a convergent mode of binding for diverse antibodies to the same CD4-binding-site epitope. A functional genomics analysis of expressed heavy and light chains revealed common pathways of antibody-heavy chain maturation, confined to the IGHV1-2*02 lineage, involving dozens of somatic changes, and capable of pairing with different light chains. Broadly neutralizing HIV-1 immunity associated with VRC01-like antibodies thus involves the evolution of antibodies to a highly affinity-matured state required to recognize an invariant viral structure, with lineages defined from thousands of sequences providing a genetic roadmap of their development.

  5. The 2010 Broad Prize

    Science.gov (United States)

    Education Digest: Essential Readings Condensed for Quick Review, 2011

    2011-01-01

    A new data analysis, based on data collected as part of The Broad Prize process, provides insights into which large urban school districts in the United States are doing the best job of educating traditionally disadvantaged groups: African-American, Hispanics, and low-income students. Since 2002, The Eli and Edythe Broad Foundation has awarded The…

  6. Characterization of human single-chain antibodies against highly pathogenic avian influenza H5N1 viruses: mimotope and neutralizing activity.

    Science.gov (United States)

    Yang, Jiupian; Yoshida, Reiko; Kariya, Yuki; Zhang, Xu; Hashiguchi, Shuhei; Nakashima, Toshihiro; Suda, Yasuo; Takada, Ayato; Ito, Yuji; Sugimura, Kazuhisa

    2010-10-01

    The development of new therapeutic targets and strategies to control highly pathogenic avian influenza (HPAI) H5N1 virus infection in humans is urgently needed. Neutralizing recombinant human antibodies would provide important agents for immunotherapy on human H5N1 virus infection and definition of the critical mimotope for vaccine development. In this study, we have characterized an anti-H5-specific scFv clone, 3D1 from the human-scFv-displaying phage library. 3D1 blocked the binding of H5-Fc to MDCK cells in flow cytometry and neutralized H5N1 subtype influenza A viruses in a microneutralization assay. Employing a peptide-displaying phage library, Ph.D-12, the mimotope was determined to be at #128-131 and #204-211 of H5, which are silic acid-binding regions. In consistency with this result, 3D1 binds the recombinant sugar-binding domain (#50G-#272E) produced by a baculovirus vector. The 3D1 antibody employs the germline gene VH1-23. As this antibody is the first human anti-H5 scFv clearly defined on the sugar-binding epitope, it allows us to investigate the influence of amino acid substitutions in this region on the determination of the binding specificity to either sialic acid α2,6-galactose (SA α2,6Gal) or sialic acid α2,3-galactose (SA α2,3Gal) providing new insight for the development of effective H5N1 pandemic vaccines.

  7. Broadly protective influenza vaccines: Redirecting the antibody response through adjuvation

    NARCIS (Netherlands)

    Cox, F.

    2016-01-01

    Influenza virus infections are responsible for significant morbidity worldwide and current vaccines have limited coverage, therefore it remains a high priority to develop broadly protective vaccines. With the discovery of broadly neutralizing antibodies (bnAbs) against influenza these vaccines becam

  8. Neutralization of West Nile virus by cross-linking of its surface proteins with Fab fragments of the human monoclonal antibody CR4354

    Energy Technology Data Exchange (ETDEWEB)

    Kaufmann, Bärbel; Vogt, Matthew R.; Goudsmit, Jaap; Holdaway, Heather A.; Aksyuk, Anastasia A.; Chipman, Paul R.; Kuhn, Richard J.; Diamond, Michael S.; Rossmann, Michael G. (Purdue); (WU-MED); (Crucell)

    2010-11-15

    Many flaviviruses are significant human pathogens, with the humoral immune response playing an essential role in restricting infection and disease. CR4354, a human monoclonal antibody isolated from a patient, neutralizes West Nile virus (WNV) infection at a postattachment stage in the viral life-cycle. Here, we determined the structure of WNV complexed with Fab fragments of CR4354 using cryoelectron microscopy. The outer glycoprotein shell of a mature WNV particle is formed by 30 rafts of three homodimers of the viral surface protein E. CR4354 binds to a discontinuous epitope formed by protein segments from two neighboring E molecules, but does not cause any detectable structural disturbance on the viral surface. The epitope occurs at two independent positions within an icosahedral asymmetric unit, resulting in 120 binding sites on the viral surface. The cross-linking of the six E monomers within one raft by four CR4354 Fab fragments suggests that the antibody neutralizes WNV by blocking the pH-induced rearrangement of the E protein required for virus fusion with the endosomal membrane.

  9. Antibody function in neutralization and protection against HIV-1

    NARCIS (Netherlands)

    Hessell, A.J.

    2009-01-01

    The ability to induce neutralizing antibodies is generally thought to be of great importance for vaccine efficacy. In HIV-1 research this quality has been elusive as the HIV-1 virus has evolved multiple mechanisms to evade neutralizing antibodies. This thesis traces studies with four broadly neutral

  10. A neutralizing epitope of human papillomavirus type 11 is principally described by a continuous set of residues which overlap a distinct linear, surface-exposed epitope.

    Science.gov (United States)

    Ludmerer, S W; Benincasa, D; Mark, G E; Christensen, N D

    1997-01-01

    A panel of monoclonal antibodies (MAbs) which neutralize human papillomavirus type 11 (HPV11) in the athymic mouse xenograph neutralization assay and bind HPV11 virus-like particles (VLPs) has been described. We recently presented evidence that the Gly131-Tyr132 residues of the major capsid protein L1 confer type 11-specific binding. However, residues distally located on the primary L1 sequence also were shown to affect binding. This poses the question whether the epitope is principally centered in the region of Gly131-Tyr132 or, alternatively, is comprised of diversely located residues which come into proximity only upon proper assembly. We analyzed the result of numerous substitutions located between Tyr123 and Val142 of the HPV11 L1 sequence. We show that substitutions at five positions result in loss of binding for one or more of these MAbs by an enzyme-linked immunosorbent assay which measures antibody binding to VLPs. We demonstrate that binding of these MAbs is redirected to HPV16 VLPs which harbor eight type 11-like substitutions within the homologous region. Three of these substitutions did not affect binding when individually substituted in HPV11 but yet were still required to transfer binding to substituted HPV16 VLPs. The results demonstrate that the epitope for this class of neutralizing MAbs, although conformational and requiring VLP assembly for presentation, principally lies along a 20-residue stretch of the L1 major capsid protein. This targets the region for evaluation of the possibility of receptor binding and suggests possibilities for the design of peptide inhibitors of virus infectivity. PMID:9094659

  11. H5N1 whole-virus vaccine induces neutralizing antibodies in humans which are protective in a mouse passive transfer model.

    Directory of Open Access Journals (Sweden)

    M Keith Howard

    Full Text Available BACKGROUND: Vero cell culture-derived whole-virus H5N1 vaccines have been extensively tested in clinical trials and consistently demonstrated to be safe and immunogenic; however, clinical efficacy is difficult to evaluate in the absence of wide-spread human disease. A lethal mouse model has been utilized which allows investigation of the protective efficacy of active vaccination or passive transfer of vaccine induced sera following lethal H5N1 challenge. METHODS: We used passive transfer of immune sera to investigate antibody-mediated protection elicited by a Vero cell-derived, non-adjuvanted inactivated whole-virus H5N1 vaccine. Mice were injected intravenously with H5N1 vaccine-induced rodent or human immune sera and subsequently challenged with a lethal dose of wild-type H5N1 virus. RESULTS: Passive transfer of H5N1 vaccine-induced mouse, guinea pig and human immune sera provided dose-dependent protection of recipient mice against lethal challenge with wild-type H5N1 virus. Protective dose fifty values for serum H5N1 neutralizing antibody titers were calculated to be ≤1∶11 for all immune sera, independently of source species. CONCLUSIONS: These data underpin the confidence that the Vero cell culture-derived, whole-virus H5N1 vaccine will be effective in a pandemic situation and support the use of neutralizing serum antibody titers as a correlate of protection for H5N1 vaccines.

  12. Measuring Prevention More Broadly, An Empirical...

    Data.gov (United States)

    U.S. Department of Health & Human Services — Measuring Prevention More Broadly, An Empirical Assessment of CHIPRA Core Measures Differences in CHIP design and structure, across states and over time, may limit...

  13. Highly pathogenic avian influenza A(H5N1) mutants transmissible by air are susceptible to human and animal neutralizing antibodies.

    Science.gov (United States)

    Du, Lanying; Li, Ye; Zhao, Guangyu; Wang, Lili; Zou, Peng; Lu, Lu; Zhou, Yusen; Jiang, Shibo

    2013-10-15

    A laboratory-generated reassortant H5 hemagglutinin (HA)/influenza A(H1N1) strain containing 4 mutations in influenza A(H5N1) HA has become transmissible by air among mammals. Here, we constructed 15 influenza A(H5N1) pseudoviruses containing a single mutation or a combination of mutations and showed that the pseudoviruses were susceptible to neutralizing antibodies from patients with influenza A(H5N1) infection and from mice immunized with a vaccine containing the conserved HA1 sequence of influenza A(H5N1). These results indicate that antibodies in patients currently infected by influenza A(H5N1) and antibodies induced by vaccines containing conserved sequences in HA1 of wild-type influenza A(H5N1) are highly effective in cross-neutralizing future influenza A(H5N1) mutants with airborne transmissibility, suggesting that human influenza pandemics caused by these influenza A(H5N1) variants can be prevented.

  14. The Broad Foundations, 2006

    Science.gov (United States)

    Broad Foundation, 2006

    2006-01-01

    The mission of the Broad Foundations is to transform K-12 urban public education through better governance, management, labor relations and competition; make significant contributions to advance major scientific and medical research; foster public appreciation of contemporary art by increasing access for audiences worldwide; and lead and…

  15. Investigation of immunosuppressive properties of inactivated human immunodeficiency virus and possible neutralization of this effect by some patient sera

    DEFF Research Database (Denmark)

    Hofmann, B; Langhoff, E; Lindhardt, B O;

    1989-01-01

    Retroviral infections are accompanied by immunosuppression in a variety of species. For feline leukemia virus, the immunosuppression has been ascribed to the transmembrane envelope protein, p15E, which suppresses the proliferative responses of cat, mouse, and human lymphocytes. A similar suppress......Retroviral infections are accompanied by immunosuppression in a variety of species. For feline leukemia virus, the immunosuppression has been ascribed to the transmembrane envelope protein, p15E, which suppresses the proliferative responses of cat, mouse, and human lymphocytes. A similar...... suppressive effect has been shown for a lysate of human immunodeficiency virus (HIV), strain HTLV-IIIB. Here we determined that detergent-disrupted HTLV-IIIB lystate exerted a strong suppressive effect on PHA-stimulated lymphocytes. Preparations of whole virions, a lysate of a local HIV isolate grown on MP-6...... the proliferation of a range of hemopoietic cell lines from man and mouse including three EBV transformed CD4- and IL-2 receptor-negative B-cell lines. The lysate also suppressed the formation of human bone marrow colonies, whereas the lysate had only a slight or no effect on fibroblasts. The suppression...

  16. Tolevamer is not efficacious in the neutralization of cytotoxin in a human gut model of Clostridium difficile infection.

    Science.gov (United States)

    Baines, Simon D; Freeman, Jane; Wilcox, Mark H

    2009-05-01

    The efficacy of tolevamer, a nonantimicrobial styrene derivative toxin-binding agent, in treating simulated Clostridium difficile infection in an in vitro human gut model was investigated. Tolevamer reduced neither the duration nor magnitude of cytotoxin activity by C. difficile, reflecting poor efficacy observed in recent phase III clinical trials.

  17. Transintestinal Cholesterol Transport Is Active in Mice and Humans and Controls Ezetimibe-Induced Fecal Neutral Sterol Excretion

    NARCIS (Netherlands)

    Jakulj, Lily; van DIjk, Theo H.; de Boer, Jan Freark; Kootte, Ruud S; Schonewille, Marleen; Paalvast, Yared; Boer, Theo; Bloks, Vincent W; Boverhof, Renze; Nieuwdorp, Max; Beuers, Ulrich H W; Stroes, Erik S G; Groen, Albert K

    2016-01-01

    Except for conversion to bile salts, there is no major cholesterol degradation pathway in mammals. Efficient excretion from the body is therefore a crucial element in cholesterol homeostasis. Yet, the existence and importance of cholesterol degradation pathways in humans is a matter of debate. We qu

  18. Squalamine as a broad-spectrum systemic antiviral agent with therapeutic potential.

    Science.gov (United States)

    Zasloff, Michael; Adams, A Paige; Beckerman, Bernard; Campbell, Ann; Han, Ziying; Luijten, Erik; Meza, Isaura; Julander, Justin; Mishra, Abhijit; Qu, Wei; Taylor, John M; Weaver, Scott C; Wong, Gerard C L

    2011-09-20

    Antiviral compounds that increase the resistance of host tissues represent an attractive class of therapeutic. Here, we show that squalamine, a compound previously isolated from the tissues of the dogfish shark (Squalus acanthias) and the sea lamprey (Petromyzon marinus), exhibits broad-spectrum antiviral activity against human pathogens, which were studied in vitro as well as in vivo. Both RNA- and DNA-enveloped viruses are shown to be susceptible. The proposed mechanism involves the capacity of squalamine, a cationic amphipathic sterol, to neutralize the negative electrostatic surface charge of intracellular membranes in a way that renders the cell less effective in supporting viral replication. Because squalamine can be readily synthesized and has a known safety profile in man, we believe its potential as a broad-spectrum human antiviral agent should be explored.

  19. Potent and synergistic neutralization of human immunodeficiency virus (HIV) type 1 primary isolates by hyperimmune anti-HIV immunoglobulin combined with monoclonal antibodies 2F5 and 2G12.

    Science.gov (United States)

    Mascola, J R; Louder, M K; VanCott, T C; Sapan, C V; Lambert, J S; Muenz, L R; Bunow, B; Birx, D L; Robb, M L

    1997-10-01

    Three antibody reagents that neutralize primary human immunodeficiency virus type 1 (HIV-1) isolates were tested for magnitude and breadth of neutralization when used alone or in double or triple combinations. Hyperimmune anti-HIV immunoglobulin (HIVIG) is derived from the plasma of HIV-1-infected donors, and monoclonal antibodies (MAbs) 2F5 and 2G12 bind to distinct regions of the HIV-1 envelope glycoprotein. The antibodies were initially tested against a panel of 15 clade B HIV-1 isolates, using a single concentration that is achievable in vivo (HIVIG, 2,500 microg/ml; MAbs, 25 microg/ml). Individual antibody reagents neutralized many of the viruses tested, but antibody potency varied substantially among the viruses. The virus neutralization produced by double combinations of HIVIG plus 2F5 or 2G12, the two MAbs together, or the triple combination of HIVIG, 2F5, and 2G12 was generally equal to or greater than that predicted by the effect of individual antibodies. Overall, the triple combination displayed the greatest magnitude and breadth of neutralization. Synergistic neutralization was evaluated by analyzing data from dose-response curves of each individual antibody reagent compared to the triple combination and was demonstrated against each of four viruses tested. Therefore, combinations of polyclonal and monoclonal anti-HIV antibodies can produce additive or synergistic neutralization of primary HIV-1 isolates. Passive immunotherapy for treatment or prophylaxis of HIV-1 should consider mixtures of potent neutralizing antibody reagents to expand the magnitude and breadth of virus neutralization.

  20. Inhibition of human immunodeficiency virus 1 (HIV-1) and herpes simplex virus 1 (HSV-1) infectivity with a broad range of lectins

    DEFF Research Database (Denmark)

    Hansen, J E; Nielsen, C; Vestergaard, B F

    1991-01-01

    Five lectins with specificity for N- and O-linked oligosaccharides were examined for inhibition of HIV-1 and HSV-1 infectivity in vitro. HIV-1 isolate HTLVIIIB was preincubated with lectin and subsequently inoculated onto MT-4 cells. Lectins specific for N-linked oligosaccharides blocked HIV infe......-1 infection, the most potent inhibition was found with the lectin HPA. These results indicate that lectins may have a broad antiviral effect on enveloped viruses only limited by types of oligosaccharides present on individual viruses....

  1. Toxicological screening of human plasma by on-line SPE-HPLC-DAD: identification and quantification of acidic and neutral drugs.

    Science.gov (United States)

    Mut, Ludmila; Grobosch, Thomas; Binscheck-Domaß, Torsten; Frenzel, Wolfgang

    2016-03-01

    A multi-analyte screening method for the quantification of 50 acidic/neutral drugs in human plasma based on on-line solid-phase extraction (SPE)-HPLC with photodiode array detection (DAD) was developed, validated and applied for clinical investigation. Acetone and methanol for protein precipitation, three different SPE materials (two electro-neutral, one strong anion-exchange, one weak cation-exchange) for on-line extraction, five HPLC-columns [one C18 (GeminiNX), two phenyl-hexyl (Gemini C6 -Phenyl, Kinetex Phenyl-Hexyl) and two pentafluorophenyl (LunaPFP(2), KinetexPFP)] for analytical separation were tested. For sample pre-treatment, acetone in the ratio 1:2 (plasma:acetone) showed a better baseline and fewer matrix peaks in the chromatogram than methanol. Only the strong anion-exchanger SPE cartridge (StrataX-A, pH 6) allowed the extraction of salicylic acid. Analytical separation was carried out on a Gemini C6 -Phenyl column (150 × 4.6 mm, 3 µm) using gradient elution with acetonitrile-water 90:10 (v/v) and phosphate buffer (pH 2.3). Linear calibration curves with correlation coefficients r ≥ 0.9950/0.9910 were obtained for 46/four analytes. Additionally, this method allows the quantification of 23 analytes for therapeutic drug monitoring. Limits of quantitation ranged from 0.1 (amobarbital) to 23 mg/L (salicylic acid). Inter-/intra-day precisions of quality control samples (low/high) were better than 13% and accuracy (bias) ranged from -14 to 10%. A computer-assisted database was created for automated detection of 223 analytes of toxicological interests. Four cases of multi-drug intoxications are presented.

  2. A rapid immunization strategy with a live attenuated tetravalent dengue vaccine elicits protective neutralizing antibody responses in non-human primates

    Directory of Open Access Journals (Sweden)

    Yuping eAmbuel

    2014-06-01

    Full Text Available Dengue viruses (DENVs cause approximately 390 million cases of DENV infections annually and over 3 billion people worldwide are at risk of infection. No dengue vaccine is currently available nor is there an antiviral therapy for DENV infections. We have developed a tetravalent live-attenuated DENV vaccine (TDV that consists of a molecularly characterized attenuated DENV-2 strain (TDV-2 and three chimeric viruses containing the pre-membrane and envelope genes of DENV-1, -3 and -4 expressed in the context of the TDV-2 genome. To impact dengue vaccine delivery in endemic areas and immunize travelers, a simple and rapid immunization strategy (RIS is preferred. We investigated RIS consisting of two full vaccine doses being administered subcutaneously or intradermally on the initial vaccination visit (day 0 at two different anatomical locations with a needle-free disposable syringe jet injection (DSJI delivery devices (PharmaJet in non-human primates (NHP. This vaccination strategy resulted in efficient priming and induction of neutralizing antibody responses to all four DENV serotypes comparable to those elicited by the traditional prime and boost (two months later vaccination schedule. In addition, the vaccine induced CD4+ and CD8+ T cells producing IFN-γ, IL-2, and TNF-α, and targeting the DENV-2 NS1, NS3 and NS5 proteins. Moreover, vaccine-specific T cells were cross-reactive with the non-structural NS3 and NS5 proteins of DENV-4. When animals were challenged with DENV-2 they were protected with no detectable viremia, and exhibited sterilizing immunity (no increase of neutralizing titers post- challenge. RIS could decrease vaccination visits and provide quick immune response to all four DENV serotypes. This strategy could increase vaccination compliance and would be especially advantageous for travelers into endemic areas.

  3. A recombinant mimetics of the HIV-1 gp41 prehairpin fusion intermediate fused with human IgG Fc fragment elicits neutralizing antibody response in the vaccinated mice

    Energy Technology Data Exchange (ETDEWEB)

    Qi, Zhi; Pan, Chungen; Lu, Hong; Shui, Yuan [Lindsley F. Kimball Research Institute, New York Blood Center, New York, NY 10065 (United States); Li, Lin [Lindsley F. Kimball Research Institute, New York Blood Center, New York, NY 10065 (United States); School of Pharmaceutical Sciences, Southern Medical University, Guangzhou, Guangdong 510515 (China); Li, Xiaojuan; Xu, Xueqing; Liu, Shuwen [School of Pharmaceutical Sciences, Southern Medical University, Guangzhou, Guangdong 510515 (China); Jiang, Shibo, E-mail: sjiang@nybloodcenter.org [Lindsley F. Kimball Research Institute, New York Blood Center, New York, NY 10065 (United States); School of Pharmaceutical Sciences, Southern Medical University, Guangzhou, Guangdong 510515 (China)

    2010-07-30

    Research highlights: {yields} One recombinant mimetics of gp41 prehairpin fusion intermediate (PFI) consisting of gp41 N46 sequence, foldon and IgG Fc, designated N46FdFc, was expressed. {yields} N46FdFc-induced antibodies in mice that neutralized HIV-1 infection, inhibited PIE7 binding to PFI, blocked gp41 six-helix bundle formation, and suppressed HIV-1 mediated cell-cell fusion. {yields} These findings provide an important clue for developing recombinant gp41 PFI mimetics-based HIV vaccines. -- Abstract: HIV-1 gp41 prehairpin fusion intermediate (PFI) composed of three N-terminal heptad repeats (NHR) plays a crucial role in viral fusion and entry and represents an attractive target for anti-HIV therapeutics (e.g., enfuvirtide) and vaccines. In present study, we constructed and expressed two recombinant gp41 PFI mimetics, designated N46Fd and N46FdFc. N46Fd consists of N46 (residues 536-581) in gp41 NHR and foldon (Fd), a trimerization motif. N46FdFc is composed of N46Fd fused with human IgG Fc fragment as an immunoenhancer. We immunized mice with N46 peptide, N46Fd and N46FdFc, respectively, and found that only N46FdFc elicited neutralizing antibody response in mice against infection by HIV-1 strains IIIB (clade B, X4), 92US657 (clade B, R5), and 94UG103 (clade A, X4R5). Anti-N46FdFc antibodies inhibited PIE7 binding to PFI, blocked gp41 six-helix bundle formation, and suppressed HIV-1 mediated cell-cell fusion. These findings provide an important clue for developing recombinant gp41 PFI mimetics-based HIV vaccines.

  4. Humanized-single domain antibodies (VH/VHH) that bound specifically to Naja kaouthia phospholipase A2 and neutralized the enzymatic activity.

    Science.gov (United States)

    Chavanayarn, Charnwit; Thanongsaksrikul, Jeeraphong; Thueng-In, Kanyarat; Bangphoomi, Kunan; Sookrung, Nitat; Chaicumpa, Wanpen

    2012-07-01

    Naja kaouthia (monocled cobra) venom contains many isoforms of secreted phospholipase A2 (sPLA(2)). The PLA(2) exerts several pharmacologic and toxic effects in the snake bitten subject, dependent or independent on the enzymatic activity. N. kaouthia venom appeared in two protein profiles, P3 and P5, after fractionating the venom by ion exchange column chromatography. In this study, phage clones displaying humanized-camel single domain antibodies (VH/V(H)H) that bound specifically to the P3 and P5 were selected from a humanized-camel VH/V(H)H phage display library. Two phagemid transfected E. coli clones (P3-1 and P3-3) produced humanized-V(H)H, while another clone (P3-7) produced humanized-VH. At the optimal venom:antibody ratio, the VH/V(H)H purified from the E. coli homogenates neutralized PLA(2) enzyme activity comparable to the horse immune serum against the N. kaouthia holo-venom. Homology modeling and molecular docking revealed that the VH/V(H)H covered the areas around the PLA(2) catalytic groove and inserted their Complementarity Determining Regions (CDRs) into the enzymatic cleft. It is envisaged that the VH/V(H)H would ameliorate/abrogate the principal toxicity of the venom PLA(2) (membrane phospholipid catabolism leading to cellular and subcellular membrane damage which consequently causes hemolysis, hemorrhage, and dermo-/myo-necrosis), if they were used for passive immunotherapy of the cobra bitten victim. The speculation needs further investigations.

  5. Evaluation of coconut water neutralized by different agents on the viability of human fibroblasts: an in vitro study

    Directory of Open Access Journals (Sweden)

    Priscilla Barbosa Ferreira SOARES

    Full Text Available Abstract Objective This study evaluated four types of pH adjustment of the coconut water (CW on viability of human fibroblasts (HFF. Material and method Natural and industrialized CW were adjusted to pH 7.0 using: (1 Sodium Hidroxide (NaOH, (2 Sodium bicarbonate (NaHCO3, (3 Triethanolamine (C6H15NO3, (4 2-Amino-2-Methil-1-Propanol (C4H11NO. Fibroblasts were plated at 2×104/ well in 96 well plates and maintained in the CW solutions for 2 h and 4 h. Positive control was represented by HFF maintained in DMEM and the negative control by tap water. Cell viability was analyzed by MTT formazan method. Data were analyzed by 3-way ANOVA followed by Tukey’s and Dunnet’s test. Result There are no significant effect on the cell viability regarding type of CW, period of evaluation, and the interactions between CW and period of evaluation, CW and pH adjustment method, pH adjustment method and period of evaluation (p>0.05. Conclusion The product used for CW pH adjustment did not influenced HFF viability, thought there are a tendency of better performance in natural CW.

  6. Five birds, one stone: neutralization of α-hemolysin and 4 bi-component leukocidins of Staphylococcus aureus with a single human monoclonal antibody.

    Science.gov (United States)

    Rouha, Harald; Badarau, Adriana; Visram, Zehra C; Battles, Michael B; Prinz, Bianka; Magyarics, Zoltán; Nagy, Gábor; Mirkina, Irina; Stulik, Lukas; Zerbs, Manuel; Jägerhofer, Michaela; Maierhofer, Barbara; Teubenbacher, Astrid; Dolezilkova, Ivana; Gross, Karin; Banerjee, Srijib; Zauner, Gerhild; Malafa, Stefan; Zmajkovic, Jakub; Maier, Sabine; Mabry, Robert; Krauland, Eric; Wittrup, K Dane; Gerngross, Tillman U; Nagy, Eszter

    2015-01-01

    Staphylococcus aureus is a major human pathogen associated with high mortality. The emergence of antibiotic resistance and the inability of antibiotics to counteract bacterial cytotoxins involved in the pathogenesis of S. aureus call for novel therapeutic approaches, such as passive immunization with monoclonal antibodies (mAbs). The complexity of staphylococcal pathogenesis and past failures with single mAb products represent considerable barriers for antibody-based therapeutics. Over the past few years, efforts have focused on neutralizing α-hemolysin. Recent findings suggest that the concerted actions of several cytotoxins, including the bi-component leukocidins play important roles in staphylococcal pathogenesis. Therefore, we aimed to isolate mAbs that bind to multiple cytolysins by employing high diversity human IgG1 libraries presented on the surface of yeast cells. Here we describe cross-reactive antibodies with picomolar affinity for α-hemolysin and 4 different bi-component leukocidins that share only ∼26% overall amino acid sequence identity. The molecular basis of cross-reactivity is the recognition of a conformational epitope shared by α-hemolysin and F-components of gamma-hemolysin (HlgAB and HlgCB), LukED and LukSF (Panton-Valentine Leukocidin). The amino acids predicted to form the epitope are conserved and known to be important for cytotoxic activity. We found that a single cross-reactive antibody prevented lysis of human phagocytes, epithelial and red blood cells induced by α-hemolysin and leukocidins in vitro, and therefore had superior effectiveness compared to α-hemolysin specific antibodies to protect from the combined cytolytic effect of secreted S. aureus toxins. Such mAb afforded high levels of protection in murine models of pneumonia and sepsis.

  7. The Ability of an Oligomeric Human Immunodeficiency Virus Type 1 (HIV-1) Envelope Antigen To Elicit Neutralizing Antibodies against Primary HIV-1 Isolates Is Improved following Partial Deletion of the Second Hypervariable Region

    OpenAIRE

    Barnett, S. W.; Lu, S.; Srivastava, I.; Cherpelis, S.; Gettie, A.; Blanchard, J.; Wang, S.; Mboudjeka, I.; Leung, L; Lian, Y.; Fong, A.; Buckner, C.; Ly, A.; Hilt, S.; Ulmer, J.

    2001-01-01

    Partial deletion of the second hypervariable region from the envelope of the primary-like SF162 virus increases the exposure of certain neutralization epitopes and renders the virus, SF162ΔV2, highly susceptible to neutralization by clade B and non-clade B human immunodeficiency virus (HIV-positive) sera (L. Stamatatos and C. Cheng-Mayer, J. Virol. 78:7840–7845, 1998). This observation led us to propose that the modified, SF162ΔV2-derived envelope may elicit higher titers of cross-reactive ne...

  8. Broad spectrum of in vivo fitness of human immunodeficiency virus type 1subpopulations differing at reverse transcriptase codons 41 and 215

    NARCIS (Netherlands)

    Goudsmit, J.; Ronde, A. de; Rooij, E. de; Boer, R.J. de

    1997-01-01

    Viral populations in a human immunodeficiency virus type 1 (HIV-1)-infected individual behave as a quasispecies with a rated distribution of fitness variants. Fitness distributions in naturally occurring viral populations have been difficult to study due to the lack of markers for individual virus c

  9. "Krüppeling" erythropoiesis : An unexpected broad spectrum of human red blood cell disorders due to KLF1 variants unveiled by genomic sequencing

    NARCIS (Netherlands)

    A. Perkins (Andrew); X. Xu (Xiangmin); D.R. Higgs (Douglas); G.P. Patrinos (George); A. Lionel, A. (Arnaud); J. Bieker (James); J.N.J. Philipsen (Sjaak)

    2016-01-01

    textabstractUntil recently our approach to the analysis of human genetic diseases has been to accurately phenotype patients and sequence the genes known to be associated with those phenotypes; for example, analysing the globin loci in cases of thalassemia. As sequencing has become increasingly acces

  10. Increased titers of neutralizing antibodies after immunization with both envelope proteins of the porcine endogenous retroviruses (PERVs

    Directory of Open Access Journals (Sweden)

    Denner Joachim

    2012-11-01

    Full Text Available Abstract Despite enormous difficulties to induce antibodies neutralizing HIV-1, especially broadly neutralizing antibodies directed against the conserved membrane proximal external region (MPER of the transmembrane envelope protein, such antibodies can be easily induced in the case of gammaretroviruses, among them the porcine endogenous retroviruses (PERVs. In addition to neutralizing antibodies directed against the transmembrane envelope protein p15E, neutralizing antibodies were also induced by immunization with the surface envelope protein gp70. PERVs represent a special risk for xenotransplantation using pig tissues or organs since they are integrated in the genome of all pigs and infect human cells and a vaccine may protect from transmission to the recipient. To investigate the effect of simultaneous immunization with both proteins in detail, a study was performed in hamsters. Gp70 and p15E of PERV were produced in E. coli, purified and used for immunization. All animals developed binding antibodies against the antigens used for immunization. Sera from animals immunized with p15E recognized epitopes in the MPER and the fusion peptide proximal region (FPPR of p15E. One MPER epitope showed a sequence homology to an epitope in the MPER of gp41 of HIV-1 recognized by broadly neutralizing antibodies found in HIV infected individuals. Neutralizing antibodies were detected in all sera. Most importantly, sera from animals immunized with gp70 had a higher neutralizing activity when compared with the sera from animals immunized with p15E and sera from animals immunized with gp70 together with p15E had a higher neutralizing activity compared with sera from animals immunized with each antigen alone. These immunization studies are important for the development of vaccines against other retroviruses including the human immunodeficiency virus HIV-1.

  11. Identification of a human homologue of the vesicle-associated membrane protein (VAMP)-associated protein of 33 kDa (VAP-33): a broadly expressed protein that binds to VAMP.

    Science.gov (United States)

    Weir, M L; Klip, A; Trimble, W S

    1998-01-01

    We report the identification of a human homologue of the vesicle-associated membrane protein (VAMP)-associated protein (hVAP-33) that has been implicated in neuronal exocytosis in Aplysia californica. This hVAP-33 shared 50% amino acid identity with the A. californica form and had similar length, structural organization and VAMP-binding abilities. However, in contrast with the neuron-specific expression seen in A. californica, hVAP-33 was broadly expressed, suggesting possible roles in vesicle fusion in both neuronal and non-neuronal cells. PMID:9657962

  12. CRF01_AE-specific neutralizing activity observed in plasma derived from HIV-1-infected Thai patients residing in northern Thailand: comparison of neutralizing breadth and potency between plasma derived from rapid and slow progressors.

    Directory of Open Access Journals (Sweden)

    Sompong Sapsutthipas

    Full Text Available BACKGROUND: Development of a protective vaccine against human immunodeficiency virus type 1 (HIV-1 is an important subject in the field of medical sciences; however, it has not yet been achieved. Potent and broadly neutralizing antibodies are found in the plasma of some HIV-1-infected patients, whereas such antibody responses have failed to be induced by currently used vaccine antigens. In order to develop effective vaccine antigens, it is important to reveal the molecular mechanism of how strong humoral immune responses are induced in infected patients. As part of such studies, we examined the correlation between the anti-HIV-1 neutralizing antibody response and disease progression. METHODOLOGY/PRINCIPAL FINDINGS: We evaluated the anti-HIV-1 neutralizing activity of plasma derived from 33 rapid and 34 slow progressors residing in northern Thailand. The level of neutralizing activity varied considerably among plasmas, and no statistically significant differences in the potency and breadth of neutralizing activities were observed overall between plasma derived from rapid and slow progressors; however, plasma of 4 slow progressors showed neutralizing activity against all target viruses, whereas none of the plasma of rapid progressors showed such neutralizing activity. In addition, 21% and 9% of plasmas derived from slow and rapid progressors inhibited the replication of more than 80% of CRF01_AE Env-recombinant viruses tested, respectively. Neutralization of subtype B and C Env-recombinant viruses by the selected plasma was also examined; however, these plasma samples inhibited the replication of only a few viruses tested. CONCLUSIONS/SIGNIFICANCE: Although no statistically significant differences were observed in the potency and breadth of anti-HIV-1 neutralizing activities between plasma derived from rapid and slow progressors, several plasma samples derived from slow progressors neutralized CRF01_AE Env-recombinant viruses more frequently than

  13. Structural Characterization of Humanized Nanobodies with Neutralizing Activity against the Bordetella pertussis CyaA-Hemolysin: Implications for a Potential Epitope of Toxin-Protective Antigen.

    Science.gov (United States)

    Malik, Aijaz Ahmad; Imtong, Chompounoot; Sookrung, Nitat; Katzenmeier, Gerd; Chaicumpa, Wanpen; Angsuthanasombat, Chanan

    2016-04-01

    Previously, the 126-kDa CyaA-hemolysin (CyaA-Hly) fragment cloned from Bordetella pertussis--the causative agent of whooping cough--and functionally expressed in Escherichia coli was revealed as a key determinant for CyaA-mediated hemolysis against target erythrocytes. Here, phagemid-transfected E. coli clones producing nanobodies capable of binding to CyaA-Hly were selected from a humanized-camel VH/VHH phage-display library. Subsequently verified for binding activities by indirect ELISA and Western blotting, four CyaA-Hly-specific nanobodies were obtained and designated according to the presence/absence of VHH-hallmark amino acids as VHH2, VH5, VH18 and VHH37. In vitro neutralization assay revealed that all four ~17-kDa His-tagged VH/VHH nanobodies, in particular VHH37, which were over-expressed as inclusions and successfully unfolded-refolded, were able to effectively inhibit CyaA-Hly-mediated hemolysis. Phage-mimotope searching revealed that only peptides with sequence homologous to Linker 1 connecting Blocks I and II within the CyaA-RTX subdomain were able to bind to these four CyaA-Hly-specific nanobodies. Structural analysis of VHH37 via homology modeling and intermolecular docking confirmed that this humanized nanobody directly interacts with CyaA-RTX/Linker 1 through multiple hydrogen and ionic bonds. Altogether, our present data demonstrate that CyaA-RTX/Linker 1 could serve as a potential epitope of CyaA-protective antigen that may be useful for development of peptide-based pertussis vaccines. Additionally, such toxin-specific nanobodies have a potential for test-driven development of a ready-to-use therapeutic in passive immunization for mitigation of disease severity.

  14. Structural Analysis of Human and Macaque mAbs 2909 and 2.5B: Implications for the Configuration of the Quaternary Neutralizing Epitope of HIV-1 gp120

    Energy Technology Data Exchange (ETDEWEB)

    Spurrier, Brett; Sampson, Jared M.; Totrov, Maxim; Li, Huiguang; O; Neal, Timothy; Williams, Constance; Robinson, James; Gorny, Miroslaw K.; Zolla-Pazner, Susan; Kong, Xiang-Peng (Molsoft); (Tulane); (NYUSM)

    2011-08-25

    The quaternary neutralizing epitope (QNE) of HIV-1 gp120 is preferentially expressed on the trimeric envelope spikes of intact HIV virions, and QNE-specific monoclonal antibodies (mAbs) potently neutralize HIV-1. Here, we present the crystal structures of the Fabs of human mAb 2909 and macaque mAb 2.5B. Both mAbs have long beta hairpin CDR H3 regions >20 {angstrom} in length that are each situated at the center of their respective antigen-binding sites. Computational analysis showed that the paratopes include the whole CDR H3, while additional CDR residues form shallow binding pockets. Structural modeling suggests a way to understand the configuration of QNEs and the antigen-antibody interaction for QNE mAbs. Our data will be useful in designing immunogens that may elicit potent neutralizing QNE Abs.

  15. Neutral Buoyancy Laboratory (NBL)

    Data.gov (United States)

    Federal Laboratory Consortium — The Neutral Buoyancy Laboratory (NBL) is an astronaut training facility and neutral buoyancy pool operated by NASA and located at the Sonny Carter Training Facility,...

  16. Autologous HIV-1 neutralizing antibodies: emergence of neutralization-resistant escape virus and subsequent development of escape virus neutralizing antibodies

    DEFF Research Database (Denmark)

    Arendrup, M; Nielsen, C; Hansen, J E;

    1992-01-01

    The capacity of consecutive human sera to neutralize sequentially obtained autologous virus isolates was studied. HIV-1 was isolated three times over a 48-164-week period from three individuals immediately after seroconversion and from two individuals in later stages of infection. Development of ...... escape virus may be part of the explanation of the apparent failure of the immune system to control HIV infection.......The capacity of consecutive human sera to neutralize sequentially obtained autologous virus isolates was studied. HIV-1 was isolated three times over a 48-164-week period from three individuals immediately after seroconversion and from two individuals in later stages of infection. Development...... of neutralizing antibodies to the primary virus isolates was detected 13-45 weeks after seroconversion. Emergence of escape virus with reduced sensitivity to neutralization by autologous sera was demonstrated. The patients subsequently developed neutralizing antibodies against the escape virus but after a delay...

  17. Selection of affinity-improved neutralizing human scFv against HBV PreS1 from CDR3 VH/VL mutant library.

    Science.gov (United States)

    Chen, YanMin; Bai, Yin; Guo, XiaoChen; Wang, WenFei; Zheng, Qi; Wang, FuXiang; Sun, Dejun; Li, DeShan; Ren, GuiPing; Yin, JieChao

    2016-07-01

    A CDR3 mutant library was constructed from a previously isolated anti-HBV neutralizing Homo sapiens scFv-31 template by random mutant primers PCR. Then the library was displayed on the inner membrane surface in Escherichia coli periplasmic space. Seven scFv clones were isolated from the mutant library through three rounds of screening by flow cytometry. Competition ELISA assay indicates that isolated scFv fragments show more efficient binding ability to HBV PreS1 compared with parental scFv-31. HBV neutralization assay indicated that two clones (scFv-3 and 59) show higher neutralizing activity by blocking the HBV infection to Chang liver cells. Our method provides a new strategy for rapid screening of mutant antibody library for affinity-enhanced scFv clones and the neutralizing scFvs obtained from this study provide a potential alternative of Hepatitis B immune globulin.

  18. Antigenic analysis of divergent genotypes human Enterovirus 71 viruses by a panel of neutralizing monoclonal antibodies: current genotyping of EV71 does not reflect their antigenicity.

    Science.gov (United States)

    Chen, Yixin; Li, Chuan; He, Delei; Cheng, Tong; Ge, Shengxiang; Shih, James Wai-Kuo; Zhao, Qinjian; Chen, Pei-Jer; Zhang, Jun; Xia, Ningshao

    2013-01-01

    In recent year, Enterovirus 71 (EV71)-associated hand, foot and mouth disease (HFMD) has become an important public health issue in China. EV71 has been classified into genotypes A, B1-B5 and C1-C5. With such genetic diversity, whether the convalescent or recovery antibody responses can cross-protect infections from other genotypes remains a question. Understanding of the antigenicity of such diverse genetic EV71 isolates is crucial for the EV71 vaccine development. Here, a total of 186 clones anti-EV71 MAbs was generated and characterized with Western blot and cell-based neutralization assay. Forty neutralizing anti-EV71 MAbs were further used to analyze the antigenic properties of 18 recent EV71 isolates representing seven genotypes in neutralization assay. We found that most neutralizing anti-EV71 MAbs are specific to conformational epitopes. We also classified the 40 neutralizing anti-EV71 MAbs into two classes according to their reactivity patterns with 18 EV71 isolates. Class I MAb can neutralize all isolates, suggesting conserved epitopes are present among EV71. Class II MAb includes four subclasses (IIa-IId) and neutralizes only subgroups of EV71 strains. Conversely, 18 EV71 strains were grouped into antigenic types 1 and four antigenic subtypes (2.1-2.4). These results suggest that the current genotyping of EV71 does not reflect their antigenicity which may be important in the selection of EV71 vaccine strains. This panel of neutralizing anti-EV71 MAbs may be useful for the recognition of emerging antigenic variants of EV71 and vaccine development.

  19. Evidence for neutral-current diffractive neutral pion production from hydrogen in neutrino interactions on hydrocarbon

    CERN Document Server

    Wolcott, J; Altinok, O; Bercellie, A; Betancourt, M; Bodek, A; Bravar, A; Budd, H; Cai, T; Carneiro, M F; Chvojka, J; Devan, J; Dytman, S A; Diaz, G A; Eberly, B; Endress, E; Felix, J; Fields, L; Galindo, R; Gallagher, H; Golan, T; Gran, R; Harris, D A; Higuera, A; Hurtado, K; Kiveni, M; Kleykamp, J; Kordosky, M; Le, T; Maher, E; Manly, S; Mann, W A; Marshall, C M; Caicedo, D A Martinez; McFarland, K S; McGivern, C L; McGowan, A M; Messerly, B; Miller, J; Mislivec, A; Morfin, J G; Mousseau, J; Naples, D; Nelson, J K; Norrick, A; Nuruzzaman,; Paolone, V; Park, J; Patrick, C E; Perdue, G N; Rakotondravohitra, L; Ramirez, M A; Ray, H; Ren, L; Rimal, D; Rodrigues, P A; Ruterbories, D; Schellman, H; Schmitz, D W; Salinas, C J Solano; Sanchez, S F; Tagg, N; Tice, B G; Valencia, E; Walton, T; Wospakrik, M; Zhang, D

    2016-01-01

    The MINERvA experiment observes an excess of events containing electromagnetic showers relative to the expectation from Monte Carlo simulations in neutral-current neutrino interactions with mean beam energy of 4.5 GeV on a hydrocarbon target. The excess is characterized and found to be consistent with neutral-current neutral pion production with a broad energy distribution peaking at 7 GeV and a total cross section of 0.26 +- 0.02 (stat) +- 0.08 (sys) x 10^{-39} cm^{2}. The angular distribution, electromagnetic shower energy, and spatial distribution of the energy depositions of the excess are consistent with expectations from neutrino neutral-current diffractive neutral pion production from hydrogen in the hydrocarbon target. These data comprise the first direct experimental observation and constraint for a reaction that poses an important background process in neutrino oscillation experiments searching for muon neutrino to electron neutrino oscillations.

  20. Neutral theory and the species abundance distribution: recent developments and prospects for unifying niche and neutral perspectives.

    Science.gov (United States)

    Matthews, Thomas J; Whittaker, Robert J

    2014-06-01

    Published in 2001, The Unified Neutral Theory of Biodiversity and Biogeography (UNTB) emphasizes the importance of stochastic processes in ecological community structure, and has challenged the traditional niche-based view of ecology. While neutral models have since been applied to a broad range of ecological and macroecological phenomena, the majority of research relating to neutral theory has focused exclusively on the species abundance distribution (SAD). Here, we synthesize the large body of work on neutral theory in the context of the species abundance distribution, with a particular focus on integrating ideas from neutral theory with traditional niche theory. First, we summarize the basic tenets of neutral theory; both in general and in the context of SADs. Second, we explore the issues associated with neutral theory and the SAD, such as complications with fitting and model comparison, the underlying assumptions of neutral models, and the difficultly of linking pattern to process. Third, we highlight the advances in understanding of SADs that have resulted from neutral theory and models. Finally, we focus consideration on recent developments aimed at unifying neutral- and niche-based approaches to ecology, with a particular emphasis on what this means for SAD theory, embracing, for instance, ideas of emergent neutrality and stochastic niche theory. We put forward the argument that the prospect of the unification of niche and neutral perspectives represents one of the most promising future avenues of neutral theory research.

  1. Automated Facial Coding Software Outperforms People in Recognizing Neutral Faces as Neutral from Standardized Datasets

    Directory of Open Access Journals (Sweden)

    Peter eLewinski

    2015-09-01

    Full Text Available Little is known about people’s accuracy of recognizing neutral faces as neutral. In this paper, I demonstrate the importance of knowing how well people recognize neutral faces. I contrasted human recognition scores of 100 typical, neutral front-up facial images with scores of an arguably objective judge – automated facial coding (AFC software. I hypothesized that the software would outperform humans in recognizing neutral faces because of the inherently objective nature of computer algorithms. Results confirmed this hypothesis. I provided the first-ever evidence that computer software (90% was more accurate in recognizing neutral faces than people were (59%. I posited two theoretical mechanisms, i.e. smile-as-a-baseline and false recognition of emotion, as possible explanations for my findings.

  2. Automated facial coding software outperforms people in recognizing neutral faces as neutral from standardized datasets.

    Science.gov (United States)

    Lewinski, Peter

    2015-01-01

    Little is known about people's accuracy of recognizing neutral faces as neutral. In this paper, I demonstrate the importance of knowing how well people recognize neutral faces. I contrasted human recognition scores of 100 typical, neutral front-up facial images with scores of an arguably objective judge - automated facial coding (AFC) software. I hypothesized that the software would outperform humans in recognizing neutral faces because of the inherently objective nature of computer algorithms. Results confirmed this hypothesis. I provided the first-ever evidence that computer software (90%) was more accurate in recognizing neutral faces than people were (59%). I posited two theoretical mechanisms, i.e., smile-as-a-baseline and false recognition of emotion, as possible explanations for my findings.

  3. HIV-1广谱中和活性样本膜蛋白基因序列特征分析%Sequence analysis on envelope genes from human immunodeficiency virus type 1 samples with broadly neutralizing activities

    Institute of Scientific and Technical Information of China (English)

    2016-01-01

    目的 分析广谱中和活性感染者不同时间点HIV-1膜蛋白基因的序列特征.方法 应用单拷贝基因组扩增技术(Single genome amplification,SGA)扩增获得不同时间点样本的全长膜蛋白(Env)基因,分析Env基因可变区序列长度、糖基化位点数目、细胞嗜性及中和表位关键氨基酸的变异.结果 系统进化分析显示在5个不同时间点获得的98条全长Env基因为HIV-1 B'亚型毒株;毒株序列随时间推移基因距离增加,V1V2区序列长度变化大于V4区,V3区与V5区序列长度保持恒定;Env基因共享序列有27个潜在糖基化位点(PNGS),V1V2区的糖基化位点数目变化较大,MPER区次之,V3区数目恒定;部分毒株嗜性随时间推移发生了从CCR5到CXCR4的转变;Env基因与广谱中和活性相关的特征氨基酸位点的突变率在4.76%~100.00%间.结论 广谱中和活性感染者不同时间点的Env基因序列差异随时间推移而增加,中和单抗识别的关键位点氨基酸存在不同类型和不同程度的突变,表明广谱中和活性感染者体内毒株在免疫压力下持续进化.

  4. Crystal structure of a human rhinovirus that displays part of the HIV-1 V3 loop and induces neutralizing antibodies against HIV-1.

    Science.gov (United States)

    Ding, Jianping; Smith, Allen D; Geisler, Sheila C; Ma, Xuejun; Arnold, Gail Ferstandig; Arnold, Eddy

    2002-07-01

    We report the 2.7 A resolution structure of a chimeric rhinovirus, MN-III-2, that displays part of the HIV-1 gp120 V3 loop and elicits HIV-neutralizing antibodies. The V3 loop insert is dominated by two type I beta turns. The structures of two adjacent tripeptides resemble those of analogous segments in three Fab/V3 loop peptide complexes. Although two of the three corresponding antibodies bind and neutralize MN-III-2 well, only one of the three can bind without significant rearrangement. These results suggest that the V3 loop insert: (1) can share some local conformational similarity to V3 loop sequences presented on different structural frameworks; (2) must be able to adopt multiple conformations, even in a relatively constrained environment; and (3) may mimic the conformational variability of the epitope on HIV-1, increasing the likelihood of eliciting appropriate neutralizing immune responses.

  5. Potent neutralization of influenza A virus by a single-domain antibody blocking M2 ion channel protein.

    Directory of Open Access Journals (Sweden)

    Guowei Wei

    Full Text Available Influenza A virus poses serious health threat to humans. Neutralizing antibodies against the highly conserved M2 ion channel is thought to offer broad protection against influenza A viruses. Here, we screened synthetic Camel single-domain antibody (VHH libraries against native M2 ion channel protein. One of the isolated VHHs, M2-7A, specifically bound to M2-expressed cell membrane as well as influenza A virion, inhibited replication of both amantadine-sensitive and resistant influenza A viruses in vitro, and protected mice from a lethal influenza virus challenge. Moreover, M2-7A showed blocking activity for proton influx through M2 ion channel. These pieces of evidence collectively demonstrate for the first time that a neutralizing antibody against M2 with broad specificity is achievable, and M2-7A may have potential for cross protection against a number of variants and subtypes of influenza A viruses.

  6. Cross-neutralizing activity of human anti-V3 monoclonal antibodies derived from non-B clade HIV-1 infected individuals.

    Science.gov (United States)

    Andrabi, Raiees; Williams, Constance; Wang, Xiao-Hong; Li, Liuzhe; Choudhary, Alok K; Wig, Naveet; Biswas, Ashutosh; Luthra, Kalpana; Nadas, Arthur; Seaman, Michael S; Nyambi, Phillipe; Zolla-Pazner, Susan; Gorny, Miroslaw K

    2013-05-10

    One approach to the development of an HIV vaccine is to design a protein template which can present gp120 epitopes inducing cross-neutralizing antibodies. To select a V3 sequence for immunogen design, we compared the neutralizing activities of 18 anti-V3 monoclonal antibodies (mAbs) derived from Cameroonian and Indian individuals infected with clade AG and C, respectively. It was found that V3 mAbs from the Cameroonian patients were significantly more cross-neutralizing than those from India. Interestingly, superior neutralizing activity of Cameroonian mAbs was also observed among the nine VH5-51/VL lambda genes encoding V3 mAbs which mediate a similar mode of recognition. This correlated with higher relative binding affinity to a variety of gp120s and increased mutation rates in V3 mAbs from Cameroon. These results suggest that clade C V3 is probably weakly immunogenic and that the V3 sequence of CRF02_AG viruses can serve as a plausible template for vaccine immunogen design.

  7. Neutralizing activities of human immunoglobulin derived from donors in Japan against mosquito-borne flaviviruses, Japanese encephalitis virus, West Nile virus, and dengue virus

    Directory of Open Access Journals (Sweden)

    Yunoki M

    2016-07-01

    Full Text Available Mikihiro Yunoki,1-3 Takeshi Kurosu,2 Ritsuko Kubota Koketsu,2,4 Kazuo Takahashi,5 Yoshinobu Okuno,4 Kazuyoshi Ikuta2,4 1Research and Development Division, Japan Blood Products Organization, Tokyo, 2Department of Virology, Research Institute for Microbial Diseases, Osaka University, Osaka, 3Pathogenic Risk Evaluation, Graduate School of Veterinary Medicine, Rakuno Gakuen University, Hokkaido, 4Research and Development Division, The Research Foundation for Microbial Diseases of Osaka University, Kagawa, 5Osaka Prefectural Institute of Public Health, Osaka, Japan Abstract: Japanese encephalitis virus (JEV, West Nile virus (WNV, and dengue virus (DenV are causal agents of Japanese encephalitis, West Nile fever, and dengue fever, respectively. JEV is considered to be indigenized and widespread in Japan, whereas WNV and DenV are not indigenized in Japan. Globulin products seem to reflect the status of the donor population according to antivirus neutralization activity. However, the anti-JEV, -WNV, and -DenV neutralization activities of globulin products derived from donors in Japan have not been clarified. Furthermore, potential candidates for the development of an effective immunotherapeutic drug for encephalitis caused by JEV, WNV, or DenV have also not been identified. Therefore, the aim of this study was to determine the overall status of the donor population in Japan based on globulin products by evaluating anti-JEV, -WNV, and -DenV neutralizing activities of intravenous immunoglobulin. Overall, intravenous immunoglobulin products showed stable neutralizing activity against JEV but showed no or only weak activity against WNV or DenV. These results suggest that the epidemiological level against WNV and DenV in the donor population of Japan is still low, suggesting that these viruses are not yet indigenized. In addition, JEV vaccinations and/or infections in the donor population do not induce a cross-reactive antibody against WNV. Keywords

  8. Development of a Broad-Range 23S rDNA Real-Time PCR Assay for the Detection and Quantification of Pathogenic Bacteria in Human Whole Blood and Plasma Specimens

    Directory of Open Access Journals (Sweden)

    Paolo Gaibani

    2013-01-01

    Full Text Available Molecular methods are important tools in the diagnosis of bloodstream bacterial infections, in particular in patients treated with antimicrobial therapy, due to their quick turn-around time. Here we describe a new broad-range real-time PCR targeting the 23S rDNA gene and capable to detect as low as 10 plasmid copies per reaction of targeted bacterial 23S rDNA gene. Two commercially available DNA extraction kits were evaluated to assess their efficiency for the extraction of plasma and whole blood samples spiked with different amount of either Staphylococcus aureus or Escherichia coli, in order to find the optimal extraction method to be used. Manual QIAmp extraction method with enzyme pre-treatment resulted the most sensitive for detection of bacterial load. Sensitivity of this novel assay ranged between 10 and 103 CFU per PCR reaction for E. coli and S. aureus in human whole blood samples depending on the extraction methods used. Analysis of plasma samples showed a 10- to 100-fold reduction of bacterial 23S rDNA in comparison to the corresponding whole blood specimens, thus indicating that whole blood is the preferential sample type to be used in this real-time PCR protocol. Our results thus show that the 23S rDNA gene represents an optimal target for bacteria quantification in human whole blood.

  9. Neutralization of English Consonants

    Institute of Scientific and Technical Information of China (English)

    庞彬彬

    2011-01-01

    This paper gives a brief account of English consonant cluster's structure and phonetic features from the perspective of the definition and cause of neutralization of English consonants as well as their distinctive features and oppositions.It comes up with the final conclusion that neutralization exists in only thirteen English consonant clusters,among a large number of consonant clusters.

  10. Soluble Human Cytomegalovirus gH/gL/pUL128-131 Pentameric Complex, but Not gH/gL, Inhibits Viral Entry to Epithelial Cells and Presents Dominant Native Neutralizing Epitopes.

    Science.gov (United States)

    Loughney, John W; Rustandi, Richard R; Wang, Dai; Troutman, Matthew C; Dick, Lawrence W; Li, Guanghua; Liu, Zhong; Li, Fengsheng; Freed, Daniel C; Price, Colleen E; Hoang, Van M; Culp, Timothy D; DePhillips, Pete A; Fu, Tong-Ming; Ha, Sha

    2015-06-26

    Congenital infection of human cytomegalovirus (HCMV) is one of the leading causes of nongenetic birth defects, and development of a prophylactic vaccine against HCMV is of high priority for public health. The gH/gL/pUL128-131 pentameric complex mediates HCMV entry into endothelial and epithelial cells, and it is a major target for neutralizing antibody responses. To better understand the mechanism by which antibodies interact with the epitopes of the gH/gL/pUL128-131 pentameric complex resulting in viral neutralization, we expressed and purified soluble gH/gL/pUL128-131 pentameric complex and gH/gL from Chinese hamster ovary cells to >95% purity. The soluble gH/gL, which exists predominantly as (gH/gL)2 homodimer with a molecular mass of 220 kDa in solution, has a stoichiometry of 1:1 and a pI of 6.0-6.5. The pentameric complex has a molecular mass of 160 kDa, a stoichiometry of 1:1:1:1:1, and a pI of 7.4-8.1. The soluble pentameric complex, but not gH/gL, adsorbs 76% of neutralizing activities in HCMV human hyperimmune globulin, consistent with earlier reports that the most potent neutralizing epitopes for blocking epithelial infection are unique to the pentameric complex. Functionally, the soluble pentameric complex, but not gH/gL, blocks viral entry to epithelial cells in culture. Our results highlight the importance of the gH/gL/pUL128-131 pentameric complex in HCMV vaccine design and emphasize the necessity to monitor the integrity of the pentameric complex during the vaccine manufacturing process.

  11. Differences in Glycoprotein Complex Receptor Binding Site Accessibility Prompt Poor Cross-Reactivity of Neutralizing Antibodies between Closely Related Arenaviruses.

    Science.gov (United States)

    Brouillette, Rachel B; Phillips, Elisabeth K; Ayithan, Natarajan; Maury, Wendy

    2017-04-01

    The glycoprotein complex (GPC) of arenaviruses, composed of stable signal peptide, GP1, and GP2, is the only antigen correlated with antibody-mediated neutralization. However, despite strong cross-reactivity of convalescent antisera between related arenavirus species, weak or no cross-neutralization occurs. Two closely related clade B viruses, Machupo virus (MACV) and Junín virus (JUNV), have nearly identical overall GPC architecture and share a host receptor, transferrin receptor 1 (TfR1). Given structural and functional similarities of the GP1 receptor binding site (RBS) of these viruses and the recent demonstration that the RBS is an important target for neutralizing antibodies, it is not clear how these viruses avoid cross-neutralization. To address this, MACV/JUNV chimeric GPCs were assessed for interaction with a group of α-JUNV GPC monoclonal antibodies (MAbs) and mouse antisera against JUNV or MACV GPC. All six MAbs targeted GP1, with those that neutralized JUNV GPC-pseudovirions competing with each other for RBS binding. However, these MAbs were unable to bind to a chimeric GPC composed of JUNV GP1 containing a small disulfide bonded loop (loop 10) unique to MACV GPC, suggesting that this loop may block MAbs interaction with the GP1 RBS. Consistent with this loop causing interference, mouse anti-JUNV GPC antisera that solely neutralized pseudovirions bearing autologous GP1 provided enhanced neutralization of MACV GPC when this loop was removed. Our studies provide evidence that loop 10, which is unique to MACV GP1, is an important impediment to binding of neutralizing antibodies and contributes to the poor cross-neutralization of α-JUNV antisera against MACV.IMPORTANCE Multiple New World arenaviruses can cause severe disease in humans, and some geographic overlap exists among these viruses. A vaccine that protects against a broad range of New World arenaviruses is desirable for purposes of simplicity, cost, and broad protection against multiple National

  12. Antibody-targeted NY-ESO-1 to mannose receptor or DEC-205 in vitro elicits dual human CD8+ and CD4+ T cell responses with broad antigen specificity.

    Science.gov (United States)

    Tsuji, Takemasa; Matsuzaki, Junko; Kelly, Marcus P; Ramakrishna, Venky; Vitale, Laura; He, Li-Zhen; Keler, Tibor; Odunsi, Kunle; Old, Lloyd J; Ritter, Gerd; Gnjatic, Sacha

    2011-01-15

    Immunization of cancer patients with vaccines containing full-length tumor Ags aims to elicit specific Abs and both CD4(+) and CD8(+) T cells. Vaccination with protein Ags, however, often elicits only CD4(+) T cell responses without inducing Ag-specific CD8(+) T cells, as exogenous protein is primarily presented to CD4(+) T cells. Recent data revealed that Ab-mediated targeting of protein Ags to cell surface receptors on dendritic cells could enhance the induction of both CD4(+) and CD8(+) T cells. We investigated in this study if these observations were applicable to NY-ESO-1, a cancer-testis Ag widely used in clinical cancer vaccine trials. We generated two novel targeting proteins consisting of the full-length NY-ESO-1 fused to the C terminus of two human mAbs against the human mannose receptor and DEC-205, both internalizing molecules expressed on APC. These targeting proteins were evaluated for their ability to activate NY-ESO-1-specific human CD4(+) and CD8(+) T cells in vitro. Both targeted NY-ESO-1 proteins rapidly bound to their respective targets on APC. Whereas nontargeted and Ab-targeted NY-ESO-1 proteins similarly activated CD4(+) T cells, cross-presentation to CD8(+) T cells was only efficiently induced by targeted NY-ESO-1. In addition, both mannose receptor and DEC-205 targeting elicited specific CD4(+) and CD8(+) T cells from PBLs of cancer patients. Receptor-specific delivery of NY-ESO-1 to APC appears to be a promising vaccination strategy to efficiently generate integrated and broad Ag-specific immune responses against NY-ESO-1 in cancer patients.

  13. Broad-spectrum antiviral agents

    Directory of Open Access Journals (Sweden)

    Jun-Da eZhu

    2015-05-01

    Full Text Available Development of highly effective, broad-spectrum antiviral agents is the major objective shared by the fields of virology and pharmaceutics. Antiviral drug development has focused on targeting viral entry and replication, as well as modulating cellular defense system. High throughput screening of molecules, genetic engineering of peptides, and functional screening of agents have identified promising candidates for development of optimal broad-spectrum antiviral agents to intervene in viral infection and control viral epidemics. This review discusses current knowledge, prospective applications, opportunities, and challenges in the development of broad-spectrum antiviral agents.

  14. Human Papillomavirus neutralizing and cross-reactive antibodies induced in HIV-positive subjects after vaccination with quadrivalent and bivalent HPV vaccines

    DEFF Research Database (Denmark)

    Faust, Helena; Nielsen, Lars Toft; Sehr, Peter;

    2016-01-01

    Ninety-one HIV-infected individuals (61 men and 30 women) were randomized to vaccination either with quadrivalent (Gardasil™) or bivalent (Cervarix™) HPV vaccine. Neutralizing and specific HPV-binding serum antibodies were measured at baseline and 12 months after the first vaccine dose. Presence...... of neutralizing and binding antibodies had good agreement (average Kappa for HPV types 6, 11, 16, 18, 31, 33 and 45 was 0.65). At baseline, 88% of subjects had antibodies against at least one genital HPV. Following vaccination with Cervarix™, all subjects became seropositive for HPV16 and 18. After Gardasil......™ vaccination, 96% of subjects seroconverted for HPV16 and 73% for HPV18. Levels of HPV16-specific antibodies were 10IU in 85% of study subjects after vaccination. Antibodies against non-vaccine HPV types appeared after Gardasil...

  15. Glycoprotein Exchange Vectors Based on Vesicular Stomatitis Virus Allow Effective Boosting and Generation of Neutralizing Antibodies to a Primary Isolate of Human Immunodeficiency Virus Type 1

    OpenAIRE

    Rose, Nina F.; Roberts, Anjeanette; Buonocore, Linda; Rose, John K.

    2000-01-01

    Live recombinant vesicular stomatitis viruses (VSVs) expressing foreign antigens are highly effective vaccine vectors. However, these vectors induce high-titer neutralizing antibody directed at the single VSV glycoprotein (G), and this antibody alone can prevent reinfection and boosting with the same vector. To determine if efficient boosting could be achieved by changing the G protein of the vector, we have developed two new recombinant VSV vectors based on the VSV Indiana serotype but with ...

  16. Human Papillomavirus neutralizing and cross-reactive antibodies induced in HIV-positive subjects after vaccination with quadrivalent and bivalent HPV vaccines.

    Science.gov (United States)

    Faust, Helena; Toft, Lars; Sehr, Peter; Müller, Martin; Bonde, Jesper; Forslund, Ola; Østergaard, Lars; Tolstrup, Martin; Dillner, Joakim

    2016-03-18

    Ninety-one HIV-infected individuals (61 men and 30 women) were randomized to vaccination either with quadrivalent (Gardasil™) or bivalent (Cervarix™) HPV vaccine. Neutralizing and specific HPV-binding serum antibodies were measured at baseline and 12 months after the first vaccine dose. Presence of neutralizing and binding antibodies had good agreement (average Kappa for HPV types 6, 11, 16, 18, 31, 33 and 45 was 0.65). At baseline, 88% of subjects had antibodies against at least one genital HPV. Following vaccination with Cervarix™, all subjects became seropositive for HPV16 and 18. After Gardasil™ vaccination, 96% of subjects seroconverted for HPV16 and 73% for HPV18. Levels of HPV16-specific antibodies were vaccination but >10IU in 85% of study subjects after vaccination. Antibodies against non-vaccine HPV types appeared after Gardasil™ vaccination for >50% of vaccinated females for HPV 31, 35 and 73 and for >50% of Cervarix™-vaccinated females for HPV 31, 33, 35, 45, 56 and 58. Cross-reactivity with non-genital HPV types was also detected. In conclusion, HIV-infected subjects responded to HPV vaccination with induction of neutralizing antibodies against both vaccine and non-vaccine types.

  17. Broad resonances in transport theory

    CERN Document Server

    Leupold, S

    2003-01-01

    The extension of the transport theoretical framework to include states with a broad mass distribution is discussed. The proper life-time and cross sections for a state with an arbitrarily given invariant mass is discussed in detail. (author)

  18. Conformational Masking and Receptor-Dependent Unmasking of Highly Conserved Env Epitopes Recognized by Non-Neutralizing Antibodies That Mediate Potent ADCC against HIV-1

    Directory of Open Access Journals (Sweden)

    George K. Lewis

    2015-09-01

    Full Text Available The mechanism of antibody-mediated protection is a major focus of HIV-1 vaccine development and a significant issue in the control of viremia. Virus neutralization, Fc-mediated effector function, or both, are major mechanisms of antibody-mediated protection against HIV-1, although other mechanisms, such as virus aggregation, are known. The interplay between virus neutralization and Fc-mediated effector function in protection against HIV-1 is complex and only partially understood. Passive immunization studies using potent broadly neutralizing antibodies (bnAbs show that both neutralization and Fc-mediated effector function provides the widest dynamic range of protection; however, a vaccine to elicit these responses remains elusive. By contrast, active immunization studies in both humans and non-human primates using HIV-1 vaccine candidates suggest that weakly neutralizing or non-neutralizing antibodies can protect by Fc-mediated effector function, albeit with a much lower dynamic range seen for passive immunization with bnAbs. HIV-1 has evolved mechanisms to evade each type of antibody-mediated protection that must be countered by a successful AIDS vaccine. Overcoming the hurdles required to elicit bnAbs has become a major focus of HIV-1 vaccine development. Here, we discuss a less studied problem, the structural basis of protection (and its evasion by antibodies that protect only by potent Fc-mediated effector function.

  19. Scorpion-Toxin Mimics of CD4 in Complex with Human Immunodeficiency Virus gp120: Crystal Structures, Molecular Mimicry, and Neutralization Breadth

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Chih-chin; Stricher, Francois; Martin, Loic; Decker, Julie M.; Majeed, Shahzad; Barthe, Phillippe; Hendrickson, Wayne A.; Robinson, James; Roumestand, Christian; Sodroski, Joseph; Wyatt, Richard; Shaw, George M.; Vita, Claudio; Kwong, Peter D. (Havard-Med); (NIH); (UAB); (Columbia); (CEA Saclay); (Tulane); (Faculty of Pharmacy)

    2010-07-19

    The binding surface on CD4 for the HIV-1 gp120 envelope glycoprotein has been transplanted previously onto a scorpion-toxin scaffold. Here, we use X-ray crystallography to characterize atomic-level details of gp120 with this transplant, CD4M33. Despite known envelope flexibility, the conformation of gp120 induced by CD4M33 was so similar to that induced by CD4 that localized measures were required to distinguish ligand-induced differences from lattice variation. To investigate relationships between structure, function, and mimicry, an F23 analog of CD4M33 was devised. Structural and thermodynamic analyses showed F23 to be a better molecular mimic of CD4 than CD4M33. F23 also showed increased neutralization breadth, against diverse isolates of HIV-1, HIV-2, and SIVcpz. Our results lend insight into the stability of the CD4 bound conformation of gp120, define measures that quantify molecular mimicry as a function of evolutionary distance, and suggest how such evaluations might be useful in developing mimetic antagonists with increased neutralization breadth.

  20. A strategy for the generation of specific human antibodies by directed evolution and phage display. An example of a single-chain antibody fragment that neutralizes a major component of scorpion venom.

    Science.gov (United States)

    Riaño-Umbarila, Lidia; Juárez-González, Victor Rivelino; Olamendi-Portugal, Timoteo; Ortíz-León, Mauricio; Possani, Lourival Domingos; Becerril, Baltazar

    2005-05-01

    This study describes the construction of a library of single-chain antibody fragments (scFvs) from a single human donor by individual amplification of all heavy and light variable domains (1.1 x 10(8) recombinants). The library was panned using the phage display technique, which allowed selection of specific scFvs (3F and C1) capable of recognizing Cn2, the major toxic component of Centruroides noxius scorpion venom. The scFv 3F was matured in vitro by three cycles of directed evolution. The use of stringent conditions in the third cycle allowed the selection of several improved clones. The best scFv obtained (6009F) was improved in terms of its affinity by 446-fold, from 183 nm (3F) to 410 pm. This scFv 6009F was able to neutralize 2 LD(50) of Cn2 toxin when a 1 : 10 molar ratio of toxin-to-antibody fragment was used. It was also able to neutralize 2 LD(50) of the whole venom. These results pave the way for the future generation of recombinant human antivenoms.

  1. The Broad Autism Phenotype Questionnaire

    Science.gov (United States)

    Hurley, Robert S. E.; Losh, Molly; Parlier, Morgan; Reznick, J. Steven; Piven, Joseph

    2007-01-01

    The broad autism phenotype (BAP) is a set of personality and language characteristics that reflect the phenotypic expression of the genetic liability to autism, in non-autistic relatives of autistic individuals. These characteristics are milder but qualitatively similar to the defining features of autism. A new instrument designed to measure the…

  2. Correlation between dengue-specific neutralizing antibodies and serum avidity in primary and secondary dengue virus 3 natural infections in humans.

    Directory of Open Access Journals (Sweden)

    Andreas Puschnik

    Full Text Available Although heterotypic secondary infection with dengue virus (DENV is associated with severe disease, the majority of secondary infections are mild or asymptomatic. The mechanisms of antibody-mediated protection are poorly understood. In 2010, 108 DENV3-positive cases were enrolled in a pediatric hospital-based study in Managua, Nicaragua, with 61 primary and 47 secondary infections. We analyzed DENV-specific neutralization titers (NT50, IgM and IgG avidity, and antibody titer in serum samples collected during acute and convalescent phases and 3, 6, and 18 months post-infection. NT50 titers peaked at convalescence and decreased thereafter. IgG avidity to DENV3 significantly increased between convalescent and 3-month time-points in primary DENV infections and between the acute and convalescent phase in secondary DENV infections. While avidity to DENV2, a likely previous infecting serotype, was initially higher than avidity to DENV3 in secondary DENV infections, the opposite relation was observed 3-18 months post-infection. We found significant correlations between IgM avidity and NT50 in acute primary cases and between IgG avidity and NT50 in secondary DENV infections. In summary, our findings indicate that IgM antibodies likely play a role in early control of DENV infections. IgG serum avidity to DENV, analyzed for the first time in longitudinal samples, switches from targeting mainly cross-reactive serotype(s to the current infecting serotype over time. Finally, serum avidity correlates with neutralization capacity.

  3. Evolution of the HIV-1 Envelope Glycoprotein Genes and Neutralizing Antibody Response in an Individual with Broadly Cross Neutralizing Antibodies

    Science.gov (United States)

    2010-08-31

    global mY/AIDS epidemic .... .. ... ..... ...... ... ..... ........ ...... ...... .... ....... ........... . 4 my classification and genetic ...Pneumocystis carinni pneumonia (PCP) among previously healthy homosexual men in Los Angeles, CA. At the time of publication, two of the five men had...healthy homosexual men across the United States. Disturbingly, severe and aggressive cases of Kaposi’s sarcoma, normally a slowly progressive cancer

  4. CO2-Neutral Fuels

    Science.gov (United States)

    Goede, Adelbert; van de Sanden, Richard

    2016-06-01

    Mimicking the biogeochemical cycle of System Earth, synthetic hydrocarbon fuels are produced from recycled CO2 and H2O powered by renewable energy. Recapturing CO2 after use closes the carbon cycle, rendering the fuel cycle CO2 neutral. Non-equilibrium molecular CO2 vibrations are key to high energy efficiency.

  5. CO2-Neutral Fuels

    NARCIS (Netherlands)

    Goede, A.; van de Sanden, M. C. M.

    2016-01-01

    Mimicking the biogeochemical cycle of System Earth, synthetic hydrocarbon fuels are produced from recycled CO2 and H2O powered by renewable energy. Recapturing CO2 after use closes the carbon cycle, rendering the fuel cycle CO2 neutral. Non-equilibrium molecular CO2 vibrations are key to high energy

  6. Mod en neutral seksualitet!

    DEFF Research Database (Denmark)

    Leer, Jonatan

    2013-01-01

    Towards a Neutral Sexuality! or Roland Barthes as a Queer Thinker? This article argues that the work of Roland Barthes has interesting perspectives in common with the queer theory. This argument will be put forward by using his concept of ‘the neutral’ that Barthes defines as “that which outplays...

  7. Bleach Neutralizes Mold Allergens

    Science.gov (United States)

    Science Teacher, 2005

    2005-01-01

    Researchers at National Jewish Medical and Research Center have demonstrated that dilute bleach not only kills common household mold, but may also neutralize the mold allergens that cause most mold-related health complaints. The study, published in the Journal of Allergy and Clinical Immunology, is the first to test the effect on allergic…

  8. Broad Diphotons from Narrow States

    CERN Document Server

    An, Haipeng; Zhang, Yue

    2015-01-01

    ATLAS and CMS have each reported a modest diphoton excess consistent with the decay of a broad resonance at ~ 750 GeV. We show how this signal can arise in a weakly coupled theory comprised solely of narrow width particles. In particular, if the decaying particle is produced off-shell, then the associated diphoton resonance will have a broad, adjustable width. We present simplified models which explain the diphoton excess through the three-body decay of a scalar or fermion. Our minimal ultraviolet completion is a weakly coupled and renormalizable theory of a singlet scalar plus a heavy vector-like quark and lepton. The smoking gun of this mechanism is an asymmetric diphoton peak recoiling against missing transverse energy, jets, or leptons.

  9. Study on the use of an enzyme-linked immunosorbent assay in determining human antibodies to diphtheria toxin as compared with a reference toxin neutralization assay.

    Science.gov (United States)

    Skoura, L; Efstratiou, A; Tsakris, A; Pournaras, S; George, R C; Douboyas, J

    1999-07-01

    Serum samples from 156 Greek persons were assessed by an IgG-specific enzyme-linked immunosorbent assay (ELISA) and a reference tissue culture toxin-neutralization (TN) assay for the quantitation of diphtheria toxin antibodies. By the reference method, 7.7% of the persons were susceptible to diphtheria (antitoxin or = 0.1 IU/ ml), while the corresponding figures were 17.9, 36.5 and 45.5% when they were tested by the immunoassay. None of the samples been susceptible by the TN assay were found to have some protection when tested by ELISA. However, three (6.7%) of the 45 samples showing a basic protection with TN, were fully protective when titrated by the immunoassay. In addition, 31 (31.3%) of the 99 samples been fully protective by the bioassay, were found to be either basically protective or susceptible by means of the ELISA. Overall, validity features of the immunoassay were: sensitivity 68.7%, specificity 94.7%, positive predictive value 95.8% and negative predictive value 63.5%. The ELISA tested in our study could be used to determine diphtheria antitoxin in individuals needed a booster immunization (susceptible or basic protective samples), although it might falsely include in the above categories samples that are within the fully protective levels of antibodies.

  10. Crystal Structure of PG16 and Chimeric Dissection with Somatically Related PG9: Structure-Function Analysis of Two Quaternary-Specific Antibodies That Effectively Neutralize HIV-1

    Energy Technology Data Exchange (ETDEWEB)

    Pancera, Marie; McLellan, Jason S.; Wu, Xueling; Zhu, Jiang; Changela, Anita; Schmidt, Stephen D.; Yang, Yongping; Zhou, Tongqing; Phogat, Sanjay; Mascola, John R.; Kwong, Peter D. (NIH); (Aids Vaccine)

    2010-11-03

    HIV-1 resists neutralization by most antibodies. Two somatically related human antibodies, PG9 and PG16, however, each neutralize 70 to 80% of circulating HIV-1 isolates. Here we present the structure of the antigen-binding fragment of PG16 in monoclinic and orthorhombic lattices at 2.4 and 4.0 {angstrom}, respectively, and use a combination of structural analysis, paratope dissection, and neutralization assessment to determine the functional relevance of three unusual PG9/PG16 features: N-linked glycosylation, extensive affinity maturation, and a heavy chain-third complementarity-determining region (CDR H3) that is one of the longest observed in human antibodies. Glycosylation extended off the side of the light chain variable domain and was not required for neutralization. The CDR H3 formed an axe-shaped subdomain, which comprised 42% of the CDR surface, with the axe head looming {approx}20 {angstrom} above the other combining loops. Comprehensive sets of chimeric swaps between PG9 and PG16 of light chain, heavy chain, and CDR H3 were employed to decipher structure-function relationships. Chimeric swaps generally complemented functionally, with differences in PG9/PG16 neutralization related primarily to residue differences in CDR H3. Meanwhile, chimeric reversions to genomic V genes showed isolate-dependent effects, with affinity maturation playing a significant role in augmenting neutralization breadth (P = 0.036) and potency (P < 0.0001). The structural and functional details of extraordinary CDR H3 and extensive affinity maturation provide insights into the neutralization mechanism of and the elicitation pathway for broadly neutralizing antibodies like PG9 and PG16.

  11. Molecular clips and tweezers hosting neutral guests.

    Science.gov (United States)

    Hardouin-Lerouge, Marie; Hudhomme, Piétrick; Sallé, Marc

    2011-01-01

    Intense current interest in supramolecular chemistry is devoted to the construction of molecular assemblies displaying controlled molecular motion associated to recognition. On this ground, molecular clips and tweezers have focused an increasing attention. This tutorial review points out the recent advances in the construction of always more sophisticated molecular clips and tweezers, illustrating their remarkably broad structural variety and focusing on their binding ability towards neutral guests. A particular attention is brought to recent findings in dynamic molecular tweezers whose recognition ability can be regulated by external stimuli. Porphyrin-based systems will not be covered here as this very active field has been recently reviewed.

  12. Autonomous mine detection system (AMDS) neutralization payload module

    Science.gov (United States)

    Majerus, M.; Vanaman, R.; Wright, N.

    2010-04-01

    The Autonomous Mine Detection System (AMDS) program is developing a landmine and explosive hazards standoff detection, marking, and neutralization system for dismounted soldiers. The AMDS Capabilities Development Document (CDD) has identified the requirement to deploy three payload modules for small robotic platforms: mine detection and marking, explosives detection and marking, and neutralization. This paper addresses the neutralization payload module. There are a number of challenges that must be overcome for the neutralization payload module to be successfully integrated into AMDS. The neutralizer must meet stringent size, weight, and power (SWaP) requirements to be compatible with a small robot. The neutralizer must be effective against a broad threat, to include metal and plastic-cased Anti-Personnel (AP) and Anti-Tank (AT) landmines, explosive devices, and Unexploded Explosive Ordnance (UXO.) It must adapt to a variety of threat concealments, overburdens, and emplacement methods, to include soil, gravel, asphalt, and concrete. A unique neutralization technology is being investigated for adaptation to the AMDS Neutralization Module. This paper will describe review this technology and how the other two payload modules influence its design for minimizing SWaP. Recent modeling and experimental efforts will be included.

  13. Maturation Pathways of Cross-Reactive HIV-1 Neutralizing Antibodies

    Directory of Open Access Journals (Sweden)

    Dimiter S. Dimitrov

    2009-11-01

    Full Text Available Several human monoclonal antibodies (hmAbs and antibody fragments, including the best characterized in terms of structure-function b12 and Fab X5, exhibit relatively potent and broad HIV-1 neutralizing activity. However, the elicitation of b12 or b12-like antibodies in vivo by vaccine immunogens based on the HIV-1 envelope glycoprotein (Env has not been successful. B12 is highly divergent from the closest corresponding germline antibody while X5 is less divergent. We have hypothesized that the relatively high degree of specific somatic hypermutations may preclude binding of the HIV-1 envelope glycoprotein (Env to closest germline antibodies, and that identifying antibodies that are intermediates in the pathways to maturation could help design novel vaccine immunogens to guide the immune system for their enhanced elicitation. In support of this hypothesis we have previously found that a germline-like b12 (monovalent and bivalent scFv as an Fc fusion protein or IgG lacks measurable binding to an Env as measured by ELISA with a sensitivity in the μM range [1]; here we present evidence confirming and expanding these findings for a panel of Envs. In contrast, a germline-like scFv X5 bound Env with high (nM affinity. To begin to explore the maturation pathways of these antibodies we identified several possible b12 intermediate antibodies and tested their neutralizing activity. These intermediate antibodies neutralized only some HIV-1 isolates and with relatively weak potency. In contrast, germline-like scFv X5 neutralized a subset of the tested HIV-1 isolates with comparable efficiencies to that of the mature X5. These results could help explain the relatively high immunogenicity of the coreceptor binding site on gp120 and the abundance of CD4-induced (CD4i antibodies in HIV-1-infected patients (X5 is a CD4i antibody as well as the maturation pathway of X5. They also can help identify antigens that can bind specifically to b12 germline and

  14. Between detection and neutralization.

    Energy Technology Data Exchange (ETDEWEB)

    Snell, Mark Kamerer; Green, Mary Wilson; Adams, Douglas Glenn; Pritchard, Daniel Allison

    2005-08-01

    Security system analytical performance analysis is generally based on the probability of system effectiveness. The probability of effectiveness is a function of the probabilities of interruption and neutralization. Interruption occurs if the response forces are notified in sufficient time to engage the adversary. Neutralization occurs if the adversary attack is defeated after the security forces have actively engaged the adversary. Both depend upon communications of data. This paper explores details of embedded communications functions that are often assumed to be inconsequential. It is the intent of the authors to bring focus to an issue in security system modeling that, if not well understood, has the potential to be a deciding factor in the overall system failure or effectiveness.

  15. Ultracold Neutral Plasmas

    CERN Document Server

    Killian, T C; Gupta, P; Laha, S; Martinez, Y N; Mickelson, P G; Nagel, S B; Saenz, A D; Simien, C E; Killian, Thomas C.

    2005-01-01

    Ultracold neutral plasmas are formed by photoionizing laser-cooled atoms near the ionization threshold. Through the application of atomic physics techniques and diagnostics, these experiments stretch the boundaries of traditional neutral plasma physics. The electron temperature in these plasmas ranges from 1-1000 K and the ion temperature is around 1 K. The density can approach $10^{11}$ cm$^{-3}$. Fundamental interest stems from the possibility of creating strongly-coupled plasmas, but recombination, collective modes, and thermalization in these systems have also been studied. Optical absorption images of a strontium plasma, using the Sr$^+$ ${^2S_{1/2}} -> {^2P_{1/2}}$ transition at 422 nm, depict the density profile of the plasma, and probe kinetics on a 50 ns time-scale. The Doppler-broadened ion absorption spectrum measures the ion velocity distribution, which gives an accurate measure of the ion dynamics in the first microsecond after photoionization.

  16. Gargamelle: neutral current event

    CERN Multimedia

    1973-01-01

    This event shows real tracks of particles from the 1200 litre Gargamelle bubble chamber that ran on the PS from 1970 to 1976 and on the SPS from 1976 to 1979. In this image a neutrino passes close to a nucleon and reemerges as a neutrino. Such events are called neutral curent, as they are mediated by the Z0 boson which has no electric charge.

  17. Influenza B-cells protective epitope characterization: a passkey for the rational design of new broad-range anti-influenza vaccines.

    Science.gov (United States)

    Clementi, Nicola; Criscuolo, Elena; Castelli, Matteo; Mancini, Nicasio; Clementi, Massimo; Burioni, Roberto

    2012-11-14

    The emergence of new influenza strains causing pandemics represents a serious threat to human health. From 1918, four influenza pandemics occurred, caused by H1N1, H2N2 and H3N2 subtypes. Moreover, in 1997 a novel influenza avian strain belonging to the H5N1 subtype infected humans. Nowadays, even if its transmission is still circumscribed to avian species, the capability of the virus to infect humans directly from avian reservoirs can result in fatalities. Moreover, the risk that this or novel avian strains could adapt to inter-human transmission, the development of resistance to anti-viral drugs and the lack of an effective prevention are all incumbent problems for the world population. In this scenario, the identification of broadly neutralizing monoclonal antibodies (mAbs) directed against conserved regions shared among influenza isolates has raised hopes for the development of monoclonal antibody-based immunotherapy and "universal" anti-influenza vaccines.

  18. A Potential of an Anti-HTLV-I gp46 Neutralizing Monoclonal Antibody (LAT-27 for Passive Immunization against Both Horizontal and Mother-to-Child Vertical Infection with Human T Cell Leukemia Virus Type-I

    Directory of Open Access Journals (Sweden)

    Hideki Fujii

    2016-02-01

    Full Text Available Although the number of human T-cell leukemia virus type-I (HTLV-I-infected individuals in the world has been estimated at over 10 million, no prophylaxis vaccines against HTLV-I infection are available. In this study, we took a new approach for establishing the basis of protective vaccines against HTLV-I. We show here the potential of a passively administered HTLV-I neutralizing monoclonal antibody of rat origin (LAT-27 that recognizes epitopes consisting of the HTLV-I gp46 amino acids 191–196. LAT-27 completely blocked HTLV-I infection in vitro at a minimum concentration of 5 μg/mL. Neonatal rats born to mother rats pre-infused with LAT-27 were shown to have acquired a large quantity of LAT-27, and these newborns showed complete resistance against intraperitoneal infection with HTLV-I. On the other hand, when humanized immunodeficient mice were pre-infused intravenously with humanized LAT-27 (hu-LAT-27, all the mice completely resisted HTLV-I infection. These results indicate that hu-LAT-27 may have a potential for passive immunization against both horizontal and mother-to-child vertical infection with HTLV-I.

  19. Broad-spectrum antiviral therapeutics.

    Directory of Open Access Journals (Sweden)

    Todd H Rider

    Full Text Available Currently there are relatively few antiviral therapeutics, and most which do exist are highly pathogen-specific or have other disadvantages. We have developed a new broad-spectrum antiviral approach, dubbed Double-stranded RNA (dsRNA Activated Caspase Oligomerizer (DRACO that selectively induces apoptosis in cells containing viral dsRNA, rapidly killing infected cells without harming uninfected cells. We have created DRACOs and shown that they are nontoxic in 11 mammalian cell types and effective against 15 different viruses, including dengue flavivirus, Amapari and Tacaribe arenaviruses, Guama bunyavirus, and H1N1 influenza. We have also demonstrated that DRACOs can rescue mice challenged with H1N1 influenza. DRACOs have the potential to be effective therapeutics or prophylactics for numerous clinical and priority viruses, due to the broad-spectrum sensitivity of the dsRNA detection domain, the potent activity of the apoptosis induction domain, and the novel direct linkage between the two which viruses have never encountered.

  20. Binding of HIV-1 gp41-directed neutralizing and non-neutralizing fragment antibody binding domain (Fab and single chain variable fragment (ScFv antibodies to the ectodomain of gp41 in the pre-hairpin and six-helix bundle conformations.

    Directory of Open Access Journals (Sweden)

    John M Louis

    Full Text Available We previously reported a series of antibodies, in fragment antigen binding domain (Fab formats, selected from a human non-immune phage library, directed against the internal trimeric coiled-coil of the N-heptad repeat (N-HR of HIV-1 gp41. Broadly neutralizing antibodies from that series bind to both the fully exposed N-HR trimer, representing the pre-hairpin intermediate state of gp41, and to partially-exposed N-HR helices within the context of the gp41 six-helix bundle. While the affinities of the Fabs for pre-hairpin intermediate mimetics vary by only 2 to 20-fold between neutralizing and non-neutralizing antibodies, differences in inhibition of viral entry exceed three orders of magnitude. Here we compare the binding of neutralizing (8066 and non-neutralizing (8062 antibodies, differing in only four positions within the CDR-H2 binding loop, in Fab and single chain variable fragment (ScFv formats, to several pre-hairpin intermediate and six-helix bundle constructs of gp41. Residues 56 and 58 of the mini-antibodies are shown to be crucial for neutralization activity. There is a large differential (≥ 150-fold in binding affinity between neutralizing and non-neutralizing antibodies to the six-helix bundle of gp41 and binding to the six-helix bundle does not involve displacement of the outer C-terminal helices of the bundle. The binding stoichiometry is one six-helix bundle to one Fab or three ScFvs. We postulate that neutralization by the 8066 antibody is achieved by binding to a continuum of states along the fusion pathway from the pre-hairpin intermediate all the way to the formation of the six-helix bundle, but prior to irreversible fusion between viral and cellular membranes.

  1. Characterization of a Large Panel of Rabbit Monoclonal Antibodies against HIV-1 gp120 and Isolation of Novel Neutralizing Antibodies against the V3 Loop.

    Directory of Open Access Journals (Sweden)

    Yali Qin

    Full Text Available We recently reported the induction of potent, cross-clade neutralizing antibodies (nAbs against Human Immunodeficiency Virus type-1 (HIV-1 in rabbits using gp120 based on an M-group consensus sequence. To better characterize these antibodies, 93 hybridomas were generated, which represent the largest panel of monoclonal antibodies (mAbs ever generated from a vaccinated rabbit. The single most frequently recognized epitope of the isolated mAbs was at the very C-terminal end of the protein (APTKAKRRVVEREKR, followed by the V3 loop. A total of seven anti-V3 loop mAbs were isolated, two of which (10A3 and 10A37 exhibited neutralizing activity. In contrast to 10A3 and most other anti-V3 loop nAbs, 10A37 was atypical with its epitope positioned more towards the C-terminal half of the loop. To our knowledge, 10A37 is the most potent and broadly neutralizing anti-V3 loop mAb induced by vaccination. Interestingly, all seven anti-V3 loop mAbs competed with PGT121, suggesting a possibility that early induction of potent anti-V3 loop antibodies could prevent induction of more broadly neutralizing PGT121-like antibodies that target the conserved base of the V3 loop stem.

  2. Ghost imaging with broad distance

    Institute of Scientific and Technical Information of China (English)

    段德洋; 张路; 杜少将; 夏云杰

    2015-01-01

    We present a scheme that is able to achieve the ghost imaging with broad distance. The physical nature of our scheme is that the different wavelength beams are separated in free space by an optical media according to the slow light or dispersion principle. Meanwhile, the equality of the optical distance of the two light arms is not violated. The photon correlation is achieved by the rotating ground glass plate (RGGP) and spatial light modulator (SLM), respectively. Our work shows that a monochromic ghost image can be obtained in the case of RGGP. More importantly, the position (or distance) of the object can be ascertained by the color of the image. Thus, the imaging and ranging processes are combined as one process for the first time to the best of our knowledge. In the case of SLM, we can obtain a colored image regardless of where the object is.

  3. Neutral Beams from Blazar Jets

    Science.gov (United States)

    Atoyan, Armen M.; Dermer, Charles D.

    2003-03-01

    We treat the production of neutrons, photons, and neutrinos through photomeson interactions of relativistic protons with ambient photons in the compact inner jets of blazars. Internal synchrotron and external isotropic radiation due to scattered optical/UV accretion-disk radiation are considered as target photon fields. Protons are assumed to be accelerated to a maximum energy limited by the size scale and magnetic field of the jet, and by competing energy losses. We characterize the conditions when the photomeson interactions of ultrarelativistic protons become effective, and show that the presence of the external radiation field makes possible strong energy losses for protons with energies Ep>~1015 eV. Without this component, effective energy losses of protons begin at Ep>~1018 eV, and would rapidly disappear with expansion of the blob. We develop a model describing the production and escape of neutrons from a comoving spherical blob, which continue to interact with the ambient external radiation field on the parsec-scale broad-line region (BLR). Neutrons may carry ~10% of the overall energy of the accelerated protons with Ep>~1015 eV outside the BLR. Ultra-high-energy gamma rays produced by photomeson interaction of neutrons outside the blob can also escape the BLR. The escaping neutrons, gamma rays, and neutrinos form a collimated neutral beam with a characteristic opening angle θ~1/Γ, where Γ is the bulk Lorentz factor of the inner jet. Energy and momentum is deposited in the extended jet from the decay of neutrons at distances ld(En)~(En/1017eV) kpc, and through pair-production attenuation of gamma rays with energies Eγ>~1015 eV which propagate to ~10-100 kpc distances. In this scenario, neutral beams of ultra-high-energy gamma rays and neutrons can be the reason for straight extended jets, such as in Pictor A. Fluxes of neutrinos detectable with kilometer-scale neutrino telescopes are predicted from flat-spectrum radio quasars such as 3C 279.

  4. Ultracold neutral plasmas

    Science.gov (United States)

    Lyon, M.; Rolston, S. L.

    2017-01-01

    By photoionizing samples of laser-cooled atoms with laser light tuned just above the ionization limit, plasmas can be created with electron and ion temperatures below 10 K. These ultracold neutral plasmas have extended the temperature bounds of plasma physics by two orders of magnitude. Table-top experiments, using many of the tools from atomic physics, allow for the study of plasma phenomena in this new regime with independent control over the density and temperature of the plasma through the excitation process. Characteristic of these systems is an inhomogeneous density profile, inherited from the density distribution of the laser-cooled neutral atom sample. Most work has dealt with unconfined plasmas in vacuum, which expand outward at velocities of order 100 m/s, governed by electron pressure, and with lifetimes of order 100 μs, limited by stray electric fields. Using detection of charged particles and optical detection techniques, a wide variety of properties and phenomena have been observed, including expansion dynamics, collective excitations in both the electrons and ions, and collisional properties. Through three-body recombination collisions, the plasmas rapidly form Rydberg atoms, and clouds of cold Rydberg atoms have been observed to spontaneously avalanche ionize to form plasmas. Of particular interest is the possibility of the formation of strongly coupled plasmas, where Coulomb forces dominate thermal motion and correlations become important. The strongest impediment to strong coupling is disorder-induced heating, a process in which Coulomb energy from an initially disordered sample is converted into thermal energy. This restricts electrons to a weakly coupled regime and leaves the ions barely within the strongly coupled regime. This review will give an overview of the field of ultracold neutral plasmas, from its inception in 1999 to current work, including efforts to increase strong coupling and effects on plasma properties due to strong coupling.

  5. The Weak Neutral Current

    CERN Document Server

    Erler, Jens

    2013-01-01

    This is a review of electroweak precision physics with particular emphasis on low-energy precision measurements in the neutral current sector of the electroweak theory and includes future experimental prospects and the theoretical challenges one faces to interpret these observables. Within the minimal Standard Model they serve as determinations of the weak mixing angle which are competitive with and complementary to those obtained near the Z-resonance. In the context of new physics beyond the Standard Model these measurements are crucial to discriminate between models and to reduce the allowed parameter space within a given model. We illustrate this for the minimal supersymmetric Standard Model with or without R-parity.

  6. Humanized-Single Domain Antibodies (VH/VHH that Bound Specifically to Naja kaouthia Phospholipase A2 and Neutralized the Enzymatic Activity

    Directory of Open Access Journals (Sweden)

    Wanpen Chaicumpa

    2012-07-01

    Full Text Available Naja kaouthia (monocled cobra venom contains many isoforms of secreted phospholipase A2 (sPLA2. The PLA2 exerts several pharmacologic and toxic effects in the snake bitten subject, dependent or independent on the enzymatic activity. N. kaouthia venom appeared in two protein profiles, P3 and P5, after fractionating the venom by ion exchange column chromatography. In this study, phage clones displaying humanized-camel single domain antibodies (VH/VHH that bound specifically to the P3 and P5 were selected from a humanized-camel VH/VHH phage display library. Two phagemid transfected E. coli clones (P3-1 and P3-3 produced humanized-VHH, while another clone (P3-7 produced humanized-VH. At the optimal venom:antibody ratio, the VH/VHH purified from the E. coli homogenates neutralized PLA2 enzyme activity comparable to the horse immune serum against the N. kaouthia holo-venom. Homology modeling and molecular docking revealed that the VH/VHH covered the areas around the PLA2 catalytic groove and inserted their Complementarity Determining Regions (CDRs into the enzymatic cleft. It is envisaged that the VH/VHH would ameliorate/abrogate the principal toxicity of the venom PLA2 (membrane phospholipid catabolism leading to cellular and subcellular membrane damage which consequently causes hemolysis, hemorrhage, and dermo-/myo-necrosis, if they were used for passive immunotherapy of the cobra bitten victim. The speculation needs further investigations.

  7. HIV-1自然感染中的中和抗体反应%Neutralizing antibodies responses during natural HIV-1 infection

    Institute of Scientific and Technical Information of China (English)

    任彩云; 李妍; 凌虹

    2012-01-01

    中和抗体(Nab)可以防止I型人类免疫缺陷病毒(HIV-1)侵入靶细胞.HIV-1感染数周后即可诱导产生Nab,这些早期抗体只能特异性地中和自体病毒但不能中和异源性病毒.在一些慢性感染者体内则可以检测到可同时中和同源性和异源性病毒的广谱中和抗体(BNab).BNab的靶点通常位于包膜蛋白的保守区域.HIV-1 BNab的产生还受到病毒变异及结构遮盖等因素的限制,同时Nab的中和广度与病毒载量具有相关性.%Neutralizing antibodies can protect a host against the infection by human immunodeficiency virus type 1 (HIV-1).Neutralizing antibodies can be induced several weeks after infection.However,the antibodies induced in the early stage can neutralize only the autologous but not heterologous viruses.Nevertheless,broad neutralizing antibodies,which can neutralize both autologous and heterologous viruses,have been found in some chronic patients.Inaddition,broad neutralizing antibodies usually target the conserved regions of HIV-1 envelope glycoprotein.However,the envelope glycoprotein mutation and its structure shielding limit the induction of these antibodies.

  8. Structural Basis of HIV-1 Neutralization by Affinity Matured Fabs Directed against the Internal Trimeric Coiled-Coil of gp41

    Energy Technology Data Exchange (ETDEWEB)

    Gustchina, Elena; Li, Mi; Louis, John M.; Anderson, D.Eric; Lloyd, John; Frisch, Christian; Bewley, Carole A.; Gustchina, Alla; Wlodawer, Alexander; Clore, G.Marius (NIH); (NCI); (AbD Serotec)

    2010-12-03

    The conserved internal trimeric coiled-coil of the N-heptad repeat (N-HR) of HIV-1 gp41 is transiently exposed during the fusion process by forming a pre-hairpin intermediate, thus representing an attractive target for the design of fusion inhibitors and neutralizing antibodies. In previous studies we reported a series of broadly neutralizing mini-antibodies derived from a synthetic naive human combinatorial antibody library by panning against a mimetic of the trimeric N-HR coiled coil, followed by affinity maturation using targeted diversification of the CDR-H2 loop. Here we report crystal structures of the N-HR mimetic 5-Helix with two Fabs that represent the extremes of this series: Fab 8066 is broadly neutralizing across a wide panel of B and C type HIV-1 viruses, whereas Fab 8062 is non-neutralizing. The crystal structures reveal important differences in the conformations of the CDR-H2 loops in the complexes that propagate into other regions of the antigen-antibody interface, and suggest that both neutralization properties and affinity for the target can be attributed, at least in part, to the differences in the interactions of the CDR-H2 loops with the antigen. Furthermore, modeling of the complex of an N-HR trimer with three Fabs suggests that the CDR-H2 loop may be involved in close intermolecular contacts between neighboring antibody molecules, and that such contacts may hinder the formation of complexes between the N-HR trimer and more than one antibody molecule depending on the conformation of the bound CDR-H2 loop which is defined by its interactions with antigen. Comparison with the crystal structure of the complex of 5-Helix with another neutralizing monoclonal antibody known as D5, derived using an entirely different antibody library and panning procedure, reveals remarkable convergence in the optimal sequence and conformation of the CDR-H2 loop.

  9. The UV-Optical Albedo of Broad Emission Line Clouds

    CERN Document Server

    Korista, K T; Korista, Kirk; Ferland, Gary

    1997-01-01

    We explore the effective UV-optical albedos of a variety of types of broad emission line clouds, as well as their possible effects on the observed spectra of AGN. An important albedo source in moderately ionized ionization-bounded clouds is that due to neutral hydrogen: Rayleigh scattering of continuum photons off the extreme damping wings of Lya. The photons resulting from this scattering mechanism may contribute significantly to the Lya emission line, especially in the very broad wings. In addition, line photons emitted near 1200 Angstroms (e.g., N V 1240) that stream toward the neutral portion of the cloud may be reflected off this Rayleigh scattering mirror, so that they preferentially escape from the illuminated face. Inclusion of this effect can alter predicted emission line strengths and profiles. In more highly-ionized ionization-bounded clouds, Thompson scattering dominates the UV-optical albedo, but this albedo is lessened by the hydrogen gas opacity. These clouds are most reflective on the long wav...

  10. Broad Leaves in Strong Flow

    CERN Document Server

    Miller, Laura

    2013-01-01

    Flexible broad leaves are thought to reconfigure in the wind and water to reduce the drag forces that act upon them. Simple mathematical models of a flexible beam immersed in a two-dimensional flow will also exhibit this behavior. What is less understood is how the mechanical properties of a leaf in a three-dimensional flow will passively allow roll up into a cone shape and reduce both drag and vortex induced oscillations. In this fluid dynamics video, the flows around the leaves are compared with those of simplified sheets using 3D numerical simulations and physical models. For some reconfiguration shapes, large forces and oscillations due to strong vortex shedding are produced. In the actual leaf, a stable recirculation zone is formed within the wake of the reconfigured cone. In physical and numerical models that reconfigure into cones, a similar recirculation zone is observed with both rigid and flexible tethers. These results suggest that the three-dimensional cone structure in addition to flexibility is ...

  11. Organic neutralization agents for neutralization-reionization mass spectrometry.

    Science.gov (United States)

    Zhang, M Y; McLafferty, F W

    1992-02-01

    Porter has shown that excited neutrals of specified internal energies can be prepared by neutralization of an ion beam with metal vapors of low ionization potential (IP). For specific problems in neutralization-reionization mass spectrometry, a metal with the desired IP value may not be available, or it may present experimental problems such as a high vaporization temperature, instrument contamination, or detector instability. The use of organic neutralization agents such as tetra-p-anisylethylene (IP = 6.0 eV) can minimize these problems (although cross sections for neutralization with these are a factor of 5 lower than those with metals), and can provide a much wider range of IP values. Their utility is demonstrated in the neutralization of C4H4 (+•) and CH8 (+•) ions to produce C4H4 and C4H8 of selected internal energies. However, for CH4 (+•) neutralization, the CH4 neutrals formed have a much lower internal energy than predicted, indicating that electron transfer from the neutralization agent predominantly produces its ions in excited states.

  12. Spectroscopy of neutral radium

    Energy Technology Data Exchange (ETDEWEB)

    Mol, Aran; De, Subhadeep; Jungmann, Klaus; Wilschut, Hans; Willmann, Lorenz [KVI, University of Groningen, Groningen (Netherlands)

    2008-07-01

    The heavy alkaline earth atoms radium is uniquely sensitive towards parity and time reversal symmetry violations due to a large enhancement of an intrinsic permanent electric dipole moment of the nucleous or the electron. Furthermore, radium is sensitive to atomic parity violation and the nuclear anapole moment. To prepare such experiments spectroscopy of relevant atomic states need to be done. At a later stage we will build a neutral atom trap for radium. We have built an atomic beam of the short lived isotope {sup 225}Ra with a flux of several 10{sup 4} atoms/sec. We are preparing the laser spectroscopy using this beam setup. In the preparation for efficient laser cooling and trapping we have successfully trapped barium, which is similar in it's requirements for laser cooling. The techniques which we have developed with barium can be used to trap rare radium isotopes. We report on the progress of the experiments.

  13. 76 FR 34087 - Broad Stakeholder Survey

    Science.gov (United States)

    2011-06-10

    ... SECURITY Broad Stakeholder Survey AGENCY: National Protection and Programs Directorate, DHS. ACTION: 60-day... comments concerning the Broad Stakeholder Survey. DATES: Comments are encouraged and will be accepted until.... The Broad Stakeholder Survey is designed to gather stakeholder feedback on the effectiveness of...

  14. 78 FR 20119 - Broad Stakeholder Survey

    Science.gov (United States)

    2013-04-03

    ... SECURITY Broad Stakeholder Survey AGENCY: National Protection and Programs Directorate, DHS. ACTION: 30-day... soliciting comments concerning the Broad Stakeholder Survey. DHS previously published this ICR in the Federal... responders across the Nation. The Broad Stakeholder Survey is designed to gather stakeholder feedback on...

  15. 77 FR 50144 - Broad Stakeholder Survey

    Science.gov (United States)

    2012-08-20

    ... SECURITY Broad Stakeholder Survey AGENCY: National Protection and Programs Directorate, DHS. ACTION: 60-day... comments concerning the Broad Stakeholder Survey. DATES: Comments are encouraged and will be accepted until... across the Nation. The Broad Stakeholder Survey is designed to gather stakeholder feedback on...

  16. Strange neutral currents in nuclei

    CERN Document Server

    Ressell, M T; Aufderheide, M B; Bloom, S D; Resler, D A

    1995-01-01

    We examine the effects on the nuclear neutral current Gamow-Teller (GT) strength of a finite contribution from a polarized strange quark sea. We perform nuclear shell model calculations of the neutral current GT strength for a number of nuclei likely to be present during stellar core collapse. We compare the GT strength when a finite strange quark contribution is included to the strength without such a contribution. As an example, the process of neutral current nuclear de-excitation via \

  17. HIV-1 binding and neutralizing antibodies of injecting drug users

    Directory of Open Access Journals (Sweden)

    Ouverney E.P.

    2005-01-01

    Full Text Available Previous studies have demonstrated a stronger seroreactivity against some synthetic peptides responsible for inducing neutralizing antibodies in injecting drug users (IDU compared to that of individuals sexually infected with HIV-1 (S, but the effectiveness in terms of the neutralizing ability of these antibodies has not been evaluated. Our objective was to study the humoral immune response of IDU by determining the specificity of their antibodies and the presence of neutralizing antibodies. The neutralization capacity against the HIV-1 isolate MN (genotype B, the primary HIV-1 isolate 95BRRJ021 (genotype F, and the seroreactivity with peptides known to induce neutralizing antibodies, from the V2 and V3 loops of different HIV-1 subtypes, were analyzed. Seroreactivity indicates that IDU plasma are more likely to recognize a broader range of peptides than S plasma, with significantly higher titers, especially of V3 peptides. Similar neutralization frequencies of the MN isolate were observed in plasma of the IDU (16/47 and S (20/60 groups in the 1:10 dilution. The neutralization of the 95BRRJ021 isolate was more frequently observed for plasma from the S group (15/23 than from the IDU group (15/47, P = 0.0108. No correlation between neutralization and seroreactivity with the peptides tested was observed. These results suggest that an important factor responsible for the extensive and broad humoral immune response observed in IDU is their infection route. There was very little difference in neutralizing antibody response between the IDU and S groups despite their differences in seroreactivity and health status.

  18. Heterologous expression and characterization of recombinant Lactococcus lactis neutral endopeptidase (Neprilysin)

    NARCIS (Netherlands)

    Lian, W; Wu, D; Konings, W.N; Mierau, I; Hersh, L.B

    1996-01-01

    A neutral endopeptidase (NEP) from Lactococcus lactis has recently been cloned and shown to contain high sequence homology with the human neutral endopeptidase, endopeptidase 24.11 (I. Mierau et al., J. Bacteriol. 175, 2087-2096, 1993). The gene for the neutral endopeptidase from L. lactis was clone

  19. Detection of neutralizing antibody to human adenovirus type 5 in marmosets%狨猴血清中重组人5型腺病毒中和抗体滴度的测定

    Institute of Scientific and Technical Information of China (English)

    孙亚纯; 李婷婷; 王一琳; 张玲; 朱海; 黎诚耀

    2016-01-01

    Objective To construct a recombinant human adenovirus type 5 (Ad5) expressing luciferase and GFP reporter gene and detect neutralizing antibodies against adenovirus type 5 in common marmosets (Callithrix jacchus) to provide basic laboratory data for evaluating adenovirus vaccines. Methods Luciferase and GFP reporter genes from plasmid pHAGE-CMV-GFP were inserted into pDC315 to construct the recombinant adenovirus shutter plasmid pDC315-Luc-GFP. The shutter plasmid was co-transduced with pBHGlox(delta)E1,3Cre in 293A cell line to package the recombinant adenovirus rAd5/Luc/GFP. Three rounds of plaque formation experiment were performed to select the monoclonal adenovirus followed by purification with cesium chloride density gradient centrifugation and virus titration with TCID50 method. Chemiluminescence assay and flow cytometry were employed to detect the neutralizing antibody levels in 14 common marmosets. Results The shuttle plasmid pDC315-Luc-GFP was successfully constructed and the recombinant adenovirus rAd5/Luc/GFP was packaged with a the titer reaching 6.9×101 .5 PFU/mL. In the 14 marmosets, chemiluminescence assay identified 4 (28.6%) marmosets that were positive for Ad5-neutralizing antibodies, including 2 with a antibody titer of 1/16 and another 2 with a titer of 1/32;flow cytomery detected Ad5-neutralizing antibodies in 3 marmosets at the titer of 1/16. Conclusion Chemiluminescence assay is a simple, sensitive, and accurate modality for detecting Ad5-neutralizing antibodies. Common marmosets have a very low positivity rate for Ad5-neutralizing antibodies and are therefore promising models for studying adenovirus-based vaccines and therapies.%目的:构建表达荧光素酶与绿色荧光蛋白报告基因的重组人5型腺病毒rAd5/Luc/GFP,检测血清中和抗体最佳实验方法,分析小型灵长类动物普通棉耳狨猴体内腺病毒5型中和抗体水平,为评价腺病毒载体疫苗提供实验动物基础数据。方法利

  20. Constraining the Europa Neutral Torus

    Science.gov (United States)

    Smith, Howard T.; Mitchell, Donald; mauk, Barry; Johnson, Robert E.; clark, george

    2016-10-01

    "Neutral tori" consist of neutral particles that usually co-orbit along with their source forming a toroidal (or partial toroidal) feature around the planet. The distribution and composition of these features can often provide important, if not unique, insight into magnetospheric particles sources, mechanisms and dynamics. However, these features can often be difficult to directly detect. One innovative method for detecting neutral tori is by observing Energetic Neutral Atoms (ENAs) that are generally considered produced as a result of charge exchange interactions between charged and neutral particles.Mauk et al. (2003) reported the detection of a Europa neutral particle torus using ENA observations. The presence of a Europa torus has extremely large implications for upcoming missions to Jupiter as well as understanding possible activity at this moon and providing critical insight into what lies beneath the surface of this icy ocean world. However, ENAs can also be produced as a result of charge exchange interactions between two ionized particles and in that case cannot be used to infer the presence of neutral particle population. Thus, a detailed examination of all possible source interactions must be considered before one can confirm that likely original source population of these ENA images is actually a Europa neutral particle torus. For this talk, we examine the viability that the Mauk et al. (2003) observations were actually generated from a neutral torus emanating from Europa as opposed to charge particle interactions with plasma originating from Io. These results help constrain such a torus as well as Europa source processes.

  1. HIV-1 resistance to neutralizing antibodies: Determination of antibody concentrations leading to escape mutant evolution.

    Science.gov (United States)

    Magnus, Carsten; Reh, Lucia; Trkola, Alexandra

    2016-06-15

    Broadly neutralizing antibodies against human immunodeficiency virus type 1 (HIV-1) are considered vital components of novel therapeutics and blueprints for vaccine research. Yet escape to even the most potent of these antibodies is imminent in natural infection. Measures to define antibody efficacy and prevent mutant selection are thus urgently needed. Here, we derive a mathematical framework to predict the concentration ranges for which antibody escape variants can outcompete their viral ancestors, referred to as mutant selection window (MSW). When determining the MSW, we focus on the differential efficacy of neutralizing antibodies against HIV-1 in two canonical infection routes, free-virus infection and cell-cell transmission. The latter has proven highly effective in vitro suggesting its importance for both in vivo spread as well as for escaping targeted intervention strategies. We observed a range of MSW patterns that highlight the potential of mutants to arise in both transmission pathways and over wide concentration ranges. Most importantly, we found that only when the arising mutant has both, residual sensitivity to the neutralizing antibody and reduced infectivity compared to the parental virus, antibody dosing outside of the MSW to restrict mutant selection is possible. Emergence of mutants that provide complete escape and have no considerable fitness loss cannot be prevented by adjusting antibody doses. The latter may in part explain the ubiquitous resistance to neutralizing antibodies observed in natural infection and antibody treatment. Based on our findings, combinations of antibodies targeting different epitopes should be favored for antibody-based interventions as this may render complete resistance less likely to occur and also increase chances that multiple escapes result in severe fitness loss of the virus making longer-term antibody treatment more feasible.

  2. Implementation of design of experiments (DOE) in the development and validation of a cell-based bioassay for the detection of anti-drug neutralizing antibodies in human serum.

    Science.gov (United States)

    Chen, Xinyi C; Zhou, Lei; Gupta, Shalini; Civoli, Francesca

    2012-02-28

    The administration of biological therapeutics can potentially elicit the development of neutralizing antibodies (NAbs) to the therapeutic drug in patients, which could have a significant impact on drug efficacy and safety. A rigorous in vitro cell-based assay for the detection of NAbs is critical for the assessment of the immunogenicity profile of the therapeutic drug. Conatumumab is a fully human monoclonal agonist antibody directed against the extracellular domain of human TRAIL receptor 2 (TR-2). It is being investigated as a cancer treatment because it is able to induce apoptosis in sensitive tumor cells. This report demonstrates how statistically designed experiments could be employed effectively in different stages of a NAb bioassay life cycle in order to characterize, optimize and stabilize the assay with added benefit of resource efficiency. By combining the approach of design of experiments (DOE) with subject matter expertise and experience, we were able to understand thoroughly how assay parameters affect the performance of the assay individually and interactively, identify the key assay parameters, define assay operating ranges and finally achieve a robust and sensitive cell-based assay for the detection of NAbs to Conatumumab. With the goal of developing a cell-based bioassay that is highly optimized for sensitivity, specificity, precision, and robustness, we performed 2 DOE experiments for assay optimization and 1 DOE experiment to validate assay robustness. We evaluated key operating parameters of the assay such as cell number, percentage of serum matrix, concentration of the therapeutic drug, concentration of the cross-linker, length of various incubation steps, cell age, interval between cell subculture and bioassay time, and detection equipment.

  3. CO2-neutral fuels

    Directory of Open Access Journals (Sweden)

    Goede A. P. H.

    2015-01-01

    Full Text Available The need for storage of renewable energy (RE generated by photovoltaic, concentrated solar and wind arises from the fact that supply and demand are ill-matched both geographically and temporarily. This already causes problems of overcapacity and grid congestion in countries where the fraction of RE exceeds the 20% level. A system approach is needed, which focusses not only on the energy source, but includes conversion, storage, transport, distribution, use and, last but not least, the recycling of waste. Furthermore, there is a need for more flexibility in the energy system, rather than relying on electrification, integration with other energy systems, for example the gas network, would yield a system less vulnerable to failure and better adapted to requirements. For example, long-term large-scale storage of electrical energy is limited by capacity, yet needed to cover weekly to seasonal demand. This limitation can be overcome by coupling the electricity net to the gas system, considering the fact that the Dutch gas network alone has a storage capacity of 552 TWh, sufficient to cover the entire EU energy demand for over a month. This lecture explores energy storage in chemicals bonds. The focus is on chemicals other than hydrogen, taking advantage of the higher volumetric energy density of hydrocarbons, in this case methane, which has an approximate 3.5 times higher volumetric energy density. More importantly, it allows the ready use of existing gas infrastructure for energy storage, transport and distribution. Intermittent wind electricity generated is converted into synthetic methane, the Power to Gas (P2G scheme, by splitting feedstock CO2 and H2O into synthesis gas, a mixture of CO and H2. Syngas plays a central role in the synthesis of a range of hydrocarbon products, including methane, diesel and dimethyl ether. The splitting is accomplished by innovative means; plasmolysis and high-temperature solid oxygen electrolysis. A CO2-neutral fuel

  4. CO2-neutral fuels

    Science.gov (United States)

    Goede, A. P. H.

    2015-08-01

    The need for storage of renewable energy (RE) generated by photovoltaic, concentrated solar and wind arises from the fact that supply and demand are ill-matched both geographically and temporarily. This already causes problems of overcapacity and grid congestion in countries where the fraction of RE exceeds the 20% level. A system approach is needed, which focusses not only on the energy source, but includes conversion, storage, transport, distribution, use and, last but not least, the recycling of waste. Furthermore, there is a need for more flexibility in the energy system, rather than relying on electrification, integration with other energy systems, for example the gas network, would yield a system less vulnerable to failure and better adapted to requirements. For example, long-term large-scale storage of electrical energy is limited by capacity, yet needed to cover weekly to seasonal demand. This limitation can be overcome by coupling the electricity net to the gas system, considering the fact that the Dutch gas network alone has a storage capacity of 552 TWh, sufficient to cover the entire EU energy demand for over a month. This lecture explores energy storage in chemicals bonds. The focus is on chemicals other than hydrogen, taking advantage of the higher volumetric energy density of hydrocarbons, in this case methane, which has an approximate 3.5 times higher volumetric energy density. More importantly, it allows the ready use of existing gas infrastructure for energy storage, transport and distribution. Intermittent wind electricity generated is converted into synthetic methane, the Power to Gas (P2G) scheme, by splitting feedstock CO2 and H2O into synthesis gas, a mixture of CO and H2. Syngas plays a central role in the synthesis of a range of hydrocarbon products, including methane, diesel and dimethyl ether. The splitting is accomplished by innovative means; plasmolysis and high-temperature solid oxygen electrolysis. A CO2-neutral fuel cycle is

  5. Neutral evolution of mutational robustness.

    NARCIS (Netherlands)

    Nimwegen, E. van; Crutchfield, J.P.; Huynen, M.A.

    1999-01-01

    We introduce and analyze a general model of a population evolving over a network of selectively neutral genotypes. We show that the population's limit distribution on the neutral network is solely determined by the network topology and given by the principal eigenvector of the network's adjacency ma

  6. The merits of neutral theory

    NARCIS (Netherlands)

    Alonso, David; Etienne, Rampal S.; McKane, Alan J.

    2006-01-01

    Hubbell's neutral theory of biodiversity has challenged the classic niche-based view of ecological community structure. Although there have been many attempts to falsify Hubbell's theory, we argue that falsification should not lead to rejection, because there is more to the theory than neutrality al

  7. Application Neutrality and a Paradox of Side Payments

    CERN Document Server

    Altman, Eitan; Kesidis, George

    2010-01-01

    The ongoing debate over "net neutrality" covers a broad set of issues related to the regulation of public networks. This paper contains two separate contributions: (a) an extension of the quadratic-utility framework we proposed in a previous paper to study the impact of side payments in a system involving pluralities of access and content providers; and (b) a variation of this model to deal with the question of application neutrality. Our analysis of the generalized framework (a) reveals an interesting "paradox" that did not occur with monopolistic players: side payments handicap the providers who perceive them. Application neutrality (b) refers to price discrimination: ISPs charging consumers different fees depending on their use of the network (web surfing, VoIP, file sharing, etc.). We analyze the consequences of such discrimination for a simple two-application setting.

  8. Retrocyclins neutralize bacterial toxins by potentiating their unfolding.

    Science.gov (United States)

    Kudryashova, Elena; Seveau, Stephanie; Lu, Wuyuan; Kudryashov, Dmitri S

    2015-04-15

    Defensins are a class of immune peptides with a broad range of activities against bacterial, fungal and viral pathogens. Besides exerting direct anti-microbial activity via dis-organization of bacterial membranes, defensins are also able to neutralize various unrelated bacterial toxins. Recently, we have demonstrated that in the case of human α- and β-defensins, this later ability is achieved through exploiting toxins' marginal thermodynamic stability, i.e. defensins act as molecular anti-chaperones unfolding toxin molecules and exposing their hydrophobic regions and thus promoting toxin precipitation and inactivation [Kudryashova et al. (2014) Immunity 41, 709-721]. Retrocyclins (RCs) are humanized synthetic θ-defensin peptides that possess unique cyclic structure, differentiating them from α- and β-defensins. Importantly, RCs are more potent against some bacterial and viral pathogens and more stable than their linear counterparts. However, the mechanism of bacterial toxin inactivation by RCs is not known. In the present study, we demonstrate that RCs facilitate unfolding of bacterial toxins. Using differential scanning fluorimetry (DSF), limited proteolysis and collisional quenching of internal tryptophan fluorescence, we show that hydrophobic regions of toxins normally buried in the molecule interior become more exposed to solvents and accessible to proteolytic cleavage in the presence of RCs. The RC-induced unfolding of toxins led to their precipitation and abrogated activity. Toxin inactivation by RCs was strongly diminished under reducing conditions, but preserved at physiological salt and serum concentrations. Therefore, despite significant structural diversity, α-, β- and θ-defensins employ similar mechanisms of toxin inactivation, which may be shared by anti-microbial peptides from other families.

  9. A New Glycan-Dependent CD4-Binding Site Neutralizing Antibody Exerts Pressure on HIV-1 In Vivo.

    Directory of Open Access Journals (Sweden)

    Natalia T Freund

    2015-10-01

    Full Text Available The CD4 binding site (CD4bs on the envelope glycoprotein is a major site of vulnerability that is conserved among different HIV-1 isolates. Many broadly neutralizing antibodies (bNAbs to the CD4bs belong to the VRC01 class, sharing highly restricted origins, recognition mechanisms and viral escape pathways. We sought to isolate new anti-CD4bs bNAbs with different origins and mechanisms of action. Using a gp120 2CC core as bait, we isolated antibodies encoded by IGVH3-21 and IGVL3-1 genes with long CDRH3s that depend on the presence of the N-linked glycan at position-276 for activity. This binding mode is similar to the previously identified antibody HJ16, however the new antibodies identified herein are more potent and broad. The most potent variant, 179NC75, had a geometric mean IC80 value of 0.42 μg/ml against 120 Tier-2 HIV-1 pseudoviruses in the TZM.bl assay. Although this group of CD4bs glycan-dependent antibodies can be broadly and potently neutralizing in vitro, their in vivo activity has not been tested to date. Here, we report that 179NC75 is highly active when administered to HIV-1-infected humanized mice, where it selects for escape variants that lack a glycan site at position-276. The same glycan was absent from the virus isolated from the 179NC75 donor, implying that the antibody also exerts selection pressure in humans.

  10. The Antiparticles of Neutral Bosons

    CERN Document Server

    Perkins, Walton A

    2013-01-01

    With the advent of the ability to create and study antihydrogen, we think it is appropriate to consider the possibility that antiphotons might not be identical to photons. First of all, we will look at the experimental evidence concerning multiple neutral pions and multiple photons. Because of its internal structure, the neutral kaon is not identical to its antiparticle. We will consider internal structures for the neutral pion and photon for which the antiparticle differs from the particle. Interestingly, the antiphoton thus created from neutrinos does not interact with electrons because its neutrinos have the wrong helicity.

  11. Positional nystagmus showing neutral points.

    Science.gov (United States)

    Hiruma, Kiyoshi; Numata, Tsutomu

    2004-01-01

    We encountered patients who had their static direction-changing positional nystagmus canceled at about 20-30 degrees yaw head rotation from the supine position. This nystagmus was also canceled when the head was rotated 180 degrees from this position. We call these head positions neutral points. At the neutral points, the cupula of the horizontal semicircular canal of the affected ear is positioned vertical to the gravitational plane and no deflection of the cupula occurs. The positional nystagmus observed (except the neutral points) was thought to occur due to a "heavy cupula" or "light cupula", which may be determined by the specific gravity of its endolymph.

  12. Reactivation of Neutralized HIV-1 by Dendritic Cells Is Dependent on the Epitope Bound by the Antibody

    NARCIS (Netherlands)

    van Montfort, Thijs; Thomas, Adri A M; Krawczyk, Przemek M; Berkhout, Ben; Sanders, Rogier W; Paxton, William A

    2015-01-01

    Ab-neutralized HIV-1 can be captured by dendritic cells (DCs), which subsequently transfer infectious HIV-1 to susceptible CD4(+) T cells. In this study, we examined the capacity of early Abs, as well as recently identified broadly neutralizing Abs (bNAbs) targeting different envelope glycoprotein (

  13. Broad Prize: Do the Successes Spread?

    Science.gov (United States)

    Samuels, Christina A.

    2011-01-01

    When the Broad Prize for Urban Education was created in 2002, billionaire philanthropist Eli Broad said he hoped the awards, in addition to rewarding high-performing school districts, would foster healthy competition; boost the prestige of urban education, long viewed as dysfunctional; and showcase best practices. Over the 10 years the prize has…

  14. Transcytosis-blocking abs elicited by an oligomeric immunogen based on the membrane proximal region of HIV-1 gp41 target non-neutralizing epitopes.

    Science.gov (United States)

    Matoba, Nobuyuki; Griffin, Tagan A; Mittman, Michele; Doran, Jeffrey D; Alfsen, Annette; Montefiori, David C; Hanson, Carl V; Bomsel, Morgane; Mor, Tsafrir S

    2008-05-01

    CTB-MPR(649-684), a translational fusion protein consisting of cholera toxin B subunit (CTB) and residues 649 684 of gp41 membrane proximal region (MPR), is a candidate vaccine aimed at blocking early steps of HIV-1 mucosal transmission. Bacterially produced CTB MPR(649-684) was purified to homogeneity by two affinity chromatography steps. Similar to gp41 and derivatives thereof, the MPR domain can specifically and reversibly self-associate. The affinities of the broadly-neutralizing monoclonal Abs 4E10 and 2F5 to CTB MPR(649-684) were equivalent to their nanomolar affinities toward an MPR peptide. The fusion protein's affinity to GM1 ganglioside was comparable to that of native CTB. Rabbits immunized with CTB-MPR(649-684) raised only a modest level of anti-MPR(649-684) Abs. However, a prime-boost immunization with CTB-MPR(649-684) and a second MPR(649-684)-based immunogen elicited a more productive anti-MPR(649-684) antibody response. These Abs strongly blocked the epithelial transcytosis of a primary subtype B HIV-1 isolate in a human tight epithelial model, expanding our previously reported results using a clade D virus. The Abs recognized epitopes at the N-terminal portion of the MPR peptide, away from the 2F5 and 4E10 epitopes and were not effective in neutralizing infection of CD4+ cells. These results indicate distinct vulnerabilities of two separate interactions of HIV-1 with human cells - Abs against the C-terminal portion of the MPR can neutralize CD4+-dependent infection, while Abs targeting the MPR's N-terminal portion can effectively block galactosyl ceramide dependent transcytosis. We propose that Abs induced by MPR(649-684)-based immunogens may provide broad protective value independent of infection neutralization.

  15. Neutrality Versus Materiality: A Thermodynamic Theory of Neutral Surfaces

    Directory of Open Access Journals (Sweden)

    Rémi Tailleux

    2016-09-01

    Full Text Available In this paper, a theory for constructing quasi-neutral density variables γ directly in thermodynamic space is formulated, which is based on minimising the absolute value of a purely thermodynamic quantity J n . Physically, J n has a dual dynamic/thermodynamic interpretation as the quantity controlling the energy cost of adiabatic and isohaline parcel exchanges on material surfaces, as well as the dependence of in-situ density on spiciness, in a description of water masses based on γ, spiciness and pressure. Mathematically, minimising | J n | in thermodynamic space is showed to be equivalent to maximising neutrality in physical space. The physics of epineutral dispersion is also reviewed and discussed. It is argued, in particular, that epineutral dispersion is best understood as the aggregate effect of many individual non-neutral stirring events (being understood here as adiabatic and isohaline events with non-zero buoyancy, so that it is only the net displacement aggregated over many events that is approximately neutral. This new view resolves an apparent paradox between the focus in neutral density theory on zero-buoyancy motions and the overwhelming evidence that lateral dispersion in the ocean is primarily caused by non-zero buoyancy processes such as tides, residual currents and sheared internal waves. The efficiency by which a physical process contributes to lateral dispersion can be characterised by its energy signature, with those processes releasing available potential energy (negative energy cost being more efficient than purely neutral processes with zero energy cost. The latter mechanism occurs in the wedge of instability, and its source of energy is the coupling between baroclinicity, thermobaricity, and density compensated temperature/salinity anomalies. Such a mechanism, which can only exist in a salty ocean, is speculated to be important for dissipating spiciness anomalies and neutral helicity. The paper also discusses potential

  16. Neutral theory in community ecology

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    One of the central goals of community ecology is to understand the forces that maintain species diversity within communities. The traditional niche-assembly theory asserts that species live together in a community only when they differ from one another in resource uses. But this theory has some difficulties in explaining the diversity often observed in specie-rich communities such as tropical forests. As an alternative to the niche theory, Hubbell and other ecologists introduced a neutral model. Hubbell argues that the number of species in a community is controlled by species extinction and immigration or speciation of new species. Assuming that all individuals of all species in a trophically similar com-munity are ecologically equivalent, Hubbell's neutral theory predicts two important statistical distributions. One is the asymptotic log-series distribution for the metacommunities under point mutation speciation, and the other is the zero-sum multinomial distribution for both local communities under dispersal limitation and metacommunities under random fission speciation. Unlike the niche-assembly theory, the neutral theory takes similarity in species and individuals as a starting point for investigating species diversity. Based on the fundamental processes of birth, death, dispersal and spe-ciation, the neutral theory provided the first mechanistic explanation of species abundance distribution commonly observed in natural communities. Since the publication of the neutral theory, there has been much discussion about it, pro and con. In this paper, we summarize recent progress in the assumption, prediction and speciation mode of the neutral theory, including progress in the theory itself, tests about the assumption of the theory, prediction and speciation mode at the metacommunity level. We also suggest that the most important task in the future is to bridge the niche-assembly theory and the neutral theory, and to add species differences to the neutral theory and

  17. Vendor neutral archive in PACS

    Directory of Open Access Journals (Sweden)

    Tapesh Kumar Agarwal

    2012-01-01

    Full Text Available An archive is a location containing a collection of records, documents, or other materials of historical importance. An integral part of Picture Archiving and Communication System (PACS is archiving. When a hospital needs to migrate a PACS vendor, the complete earlier data need to be migrated in the format of the newly procured PACS. It is both time and money consuming. To address this issue, the new concept of vendor neutral archive (VNA has emerged. A VNA simply decouples the PACS and workstations at the archival layer. This is achieved by developing an application engine that receives, integrates, and transmits the data using the different syntax of a Digital Imaging and Communication in Medicine (DICOM format. Transferring the data belonging to the old PACS to a new one is performed by a process called migration of data. In VNA, a number of different data migration techniques are available to facilitate transfer from the old PACS to the new one, the choice depending on the speed of migration and the importance of data. The techniques include simple DICOM migration, prefetch-based DICOM migration, medium migration, and the expensive non-DICOM migration. "Vendor neutral" may not be a suitable term, and "architecture neutral," "PACS neutral," "content neutral," or "third-party neutral" are probably better and preferred terms. Notwithstanding this, the VNA acronym has come to stay in both the medical IT user terminology and in vendor nomenclature, and radiologists need to be aware of its impact in PACS across the globe.

  18. Heavy chain-only IgG2b llama antibody effects near-pan HIV-1 neutralization by recognizing a CD4-induced epitope that includes elements of coreceptor- and CD4-binding sites.

    Science.gov (United States)

    Acharya, Priyamvada; Luongo, Timothy S; Georgiev, Ivelin S; Matz, Julie; Schmidt, Stephen D; Louder, Mark K; Kessler, Pascal; Yang, Yongping; McKee, Krisha; O'Dell, Sijy; Chen, Lei; Baty, Daniel; Chames, Patrick; Martin, Loïc; Mascola, John R; Kwong, Peter D

    2013-09-01

    The conserved HIV-1 site of coreceptor binding is protected from antibody-directed neutralization by conformational and steric restrictions. While inaccessible to most human antibodies, the coreceptor site has been shown to be accessed by antibody fragments. In this study, we used X-ray crystallography, surface plasmon resonance, and pseudovirus neutralization to characterize the gp120-envelope glycoprotein recognition and HIV-1 neutralization of a heavy chain-only llama antibody, named JM4. We describe full-length IgG2b and IgG3 versions of JM4 that target the coreceptor-binding site and potently neutralize over 95% of circulating HIV-1 isolates. Contrary to established trends that show improved access to the coreceptor-binding region by smaller antibody fragments, the single-domain (VHH) version of JM4 neutralized less well than the full-length IgG2b version of JM4. The crystal structure at 2.1-Å resolution of VHH JM4 bound to HIV-1 YU2 gp120 stabilized in the CD4-bound state by the CD4-mimetic miniprotein, M48U1, revealed a JM4 epitope that combined regions of coreceptor recognition (including the gp120 bridging sheet, V3 loop, and β19 strand) with gp120 structural elements involved in recognition of CD4 such as the CD4-binding loop. The structure of JM4 with gp120 thus defines a novel CD4-induced site of vulnerability involving elements of both coreceptor- and CD4-binding sites. The potently neutralizing JM4 IgG2b antibody that targets this newly defined site of vulnerability adds to the expanding repertoire of broadly neutralizing antibodies that effectively neutralize HIV-1 and thereby potentially provides a new template for vaccine development and target for HIV-1 therapy.

  19. The broad spectrum revisited: Evidence from plant remains

    OpenAIRE

    Weiss, Ehud; Wetterstrom, Wilma; Nadel, Dani; Bar-Yosef, Ofer

    2004-01-01

    The beginning of agriculture is one of the most important developments in human history, with enormous consequences that paved the way for settled life and complex society. Much of the research on the origins of agriculture over the last 40 years has been guided by Flannery's [Flannery, K. V. (1969) in The Domestication and Exploitation of Plants and Animals, eds. Ucko, P. J. & Dimbleby, G. W. (Duckworth, London), pp. 73–100] “broad spectrum revolution” (BSR) hypothesis, which posits that the...

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

    Directory of Open Access Journals (Sweden)

    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.

  1. SYBR Green实时荧光PCR方法检测广谱人乳头瘤病毒的方法建立%Establishment of SYBR Green Real-time Fluorescence PCR Assay covering a broad range of Human Papillomavirus Genotypes

    Institute of Scientific and Technical Information of China (English)

    徐平; 章晓鹰; 张慧敏

    2011-01-01

    Objective To establish a novel Real-time fluorescence PCR assay for simultaneous detection of a broad range of HPV. Genotypes. Methods Instead of universal specific PCR primers and specific probe,degenerate PCR primers and SYBR Green stain were used in novel Real-time fluorescence PCR assay,positive rates were compared with the kits(13 high-risk and 6 low-risk)used in clinical diagnosis,meanwhile,detected rate were compared between SYBR Green Real-time Fluorescence PCR and degenerate PCR. Results Detected rate of SYBR Green Real-time fluorescence PCR was 69 percent among the 100 positive case from cervical swab samples detected by the kits,but the kits showed negative results for 40 positive case of cutaneous warts detected by methods of SYBR Green Real-time Fluorescence PCR. Positive rates of SYBR Green Real-time Fluorescence PCR for 90 cervical type and 60 cutaneous type consist with degenerate PCR(Kappa=0. 907). Conclusion Compared with the kits used in clinical, SYBR Green Real-time Fluorescence PCR covered a wide types of HPV genotypes including cutaneous types and cervical types. It also has high efficiency,quick and simple features than degenerate PCR. However,it is satisfactory for clinical and research.%目的 建立实时荧光PCR方法以检测广谱人乳头瘤病毒.方法 该实时荧光PCR方法利用了degenerate PCR引物与非特异性的SYBR Green染料替代通常使用的PCR特异性引物与探针,并与市售高危13型、低危6型试剂盒以及degenerate PCR方法进行比较.结果 对于用高危13型试剂盒及低危6型试剂盒检测的100例黏膜型人乳头瘤病毒阳性标本,用SYBR Green实时荧光PCR方法检出的阳性数为69例,敏感性为69%,相反,用SYBR Green实时荧光PCR方法检测出的40例皮肤型人乳头瘤病毒阳性标本,用高危13型试剂盒以及低危6型试剂盒检测均为阴性;对于用SYBR Green实时荧光PCR方法及degenerate PCR方法检测的90例黏膜型、60例皮肤型标本,其

  2. Broad-Leaved Forest Community Structure Features in Different Habitats without Human Disturbance%无人为干扰下不同生境阔叶林的群落结构特征

    Institute of Scientific and Technical Information of China (English)

    雷平; 郭洪兴; 邹志安; 邹思成; 兰文军; 徐新宇; 袁荣斌

    2014-01-01

    以江西武夷山国家级自然保护区核心区为无干扰环境,选取河岸带、内陆森林和林线3种生境阔叶林群落为研究对象。群落结构特征分析结果显示:1)不同生境类型的群落建群种不同,但群落各层主要优势种的重要值均呈差异不显著,物种组成上均以壳斗科、樟科、山茶科、山矾科、杜鹃花科、蔷薇科、冬青科等为常见,且种属数较多。2)立木总株数(乔灌木)林线群落高于其他生境群落,但不同生境类型的群落间乔木与灌木平均株数均呈差异不显著。3)群落各层物种的种数及各物种多样性指数均有河岸带>内陆森林>林线,乔木层、草本层与层间植物物种种数以及物种多样性Patrick、Gleason、Margalef和Shannon-Weiner指数,河岸带均与其他生境类型群落差异呈显著性;而灌木层物种种数及多样性Simpson和Pielou指数差异不呈显著性。4)散点图和线性回归得出林线群落活立木平均高度和平均胸径均高于其他生境类型的群落。%In core area of Jiangxi Wuyishan National Nature Reserve, as the non-disturbance condition, three habitats broad-leaved community of riparian zone, inland and forest line were selected for study aspects. Community structure characteristics analysis results showed that: 1) there were different constructive species of the community in different habitats, but the important value of the main dominant species in the communities’ layers has no significant difference. Species composition dominated by Fagaceae, Theaceae, Lauraceae, Symplocaceae, Ericaceae, Rosaceae, Aquifoliaceae with much of genera and species in all types of the habitats. 2) The total number of the standing timber (including trees and shrubs) in the forest line community was more than other communities, but the average number of the trees and shrubs in different habitats has no significant difference. 3) Community species and each

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

    Science.gov (United States)

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

    2005-11-01

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

  4. Post-learning psychosocial stress enhances consolidation of neutral stimuli.

    Science.gov (United States)

    Preuss, Diana; Wolf, Oliver T

    2009-10-01

    Post-learning stress has been reported to enhance memory consolidation in humans. This effect was observed in studies using physical stressors or an anticipatory speech task. In the present study 58 participants (28 females and 30 males) were exposed to a psychosocial stressor (Trier Social Stress Test) or a control condition following the presentation of neutral and emotionally arousing positive and negative pictures, which were accompanied by a brief narrative. The stressor induced a significant neuroendocrine stress response in men and women. In a 24h delayed free recall test the stress group showed an enhanced memory for neutral but not for emotionally arousing positive and negative items. Additionally, a significant correlation between the cortisol stress response and memory for neutral items was evident. Thus, in contrast to previous studies, post-learning stress primarily enhanced consolidation of neutral material. Several theoretical and methodological explanations for the observed effects are discussed.

  5. Broad Anti-Hepatitis C Virus (HCV) Antibody Responses Are Associated with Improved Clinical Disease Parameters in Chronic HCV Infection

    Science.gov (United States)

    Swann, Rachael E.; Cowton, Vanessa M.; Robinson, Mark W.; Cole, Sarah J.; Barclay, Stephen T.; Mills, Peter R.; Thomson, Emma C.; McLauchlan, John

    2016-01-01

    ABSTRACT During hepatitis C virus (HCV) infection, broadly neutralizing antibody (bNAb) responses targeting E1E2 envelope glycoproteins are generated in many individuals. It is unclear if these antibodies play a protective or a pathogenic role during chronic infection. In this study, we investigated whether bNAb responses in individuals with chronic infection were associated with differences in clinical presentation. Patient-derived purified serum IgG was used to assess the breadth of HCV E1E2 binding and the neutralization activity of HCV pseudoparticles. The binding and neutralization activity results for two panels bearing viral envelope proteins representing either an intergenotype or an intragenotype 1 group were compared. We found that the HCV load was negatively associated with strong cross-genotypic E1E2 binding (P = 0.03). Overall, we observed only a modest correlation between total E1E2 binding and neutralization ability. The breadth of intergenotype neutralization did not correlate with any clinical parameters; however, analysis of individuals with genotype 1 (gt1) HCV infection (n = 20), using an intragenotype pseudoparticle panel, found a strong association between neutralization breadth and reduced liver fibrosis (P = 0.006). A broad bNAb response in our cohort with chronic infection was associated with a single nucleotide polymorphism (SNP) in the HLA-DQB1 gene (P = 0.038), as previously reported in a cohort with acute disease. Furthermore, the bNAbs in these individuals targeted more than one region of E2-neutralizing epitopes, as assessed through cross-competition of patient bNAbs with well-characterized E2 antibodies. We conclude that the bNAb responses in patients with chronic gt1 infection are associated with lower rates of fibrosis and host genetics may play a role in the ability to raise such responses. IMPORTANCE Globally, there are 130 million to 150 million people with chronic HCV infection. Typically, the disease is progressive and is a

  6. Broad Balmer line emission and cosmic ray acceleration efficiency in supernova remnant shocks

    CERN Document Server

    Morlino, G; Bandiera, R; Amato, E

    2013-01-01

    Balmer emission may be a powerful diagnostic tool to test the paradigm of cosmic ray (CR) acceleration in young supernova remnant (SNR) shocks. The width of the broad Balmer line is a direct indicator of the downstream plasma temperature. In case of efficient particle acceleration an appreciable fraction of the total kinetic energy of the plasma is channeled into CRs, therefore the downstream temperature decreases and so does the broad Balmer line width. This width also depends on the level of thermal equilibration between ions and neutral hydrogen atoms in the downstream. Since in general in young SNR shocks only a few charge exchange (CE) reactions occur before ionization, equilibration between ions and neutrals is not reached, and a kinetic description of the neutrals is required in order to properly compute Balmer emission. We provide a method for the calculation of Balmer emission using a self-consistent description of the shock structure in the presence of neutrals and CRs. We use a recently developed s...

  7. Neutral B meson flavor tagging

    Science.gov (United States)

    Wilson, Robert J.

    2001-07-01

    We present an investigation of the use of net charge and kaon identification to tag the flavor of neutral B mesons. The net charge of the neutral B meson decay products is zero if all charged particles are used and slightly non-zero if only undiscriminated hadronic final states are used. The net charge of the kaons alone correctly tags the identity of the neutral meson in at least a third of all decays. We have parametrized the particle identification capability of several techniques, such as dE/dx in time projection chambers, LEP/SLC ring-imaging chambers and an enhanced BaBar DIRC. Using these parametrisations we compare the relative tagging power of each technique to that of an ideal detector.

  8. Neutral B meson flavor tagging

    CERN Document Server

    Wilson, R J

    2001-01-01

    We present an investigation of the use of net charge and kaon identification to tag the flavor of neutral B mesons. The net charge of the neutral B meson decay products is zero if all charged particles are used and slightly non-zero if only undiscriminated hadronic final states are used. The net charge of the kaons alone correctly tags the identity of the neutral meson in at least a third of all decays. We have parametrized the particle identification capability of several techniques, such as dE/dx in time projection chambers, LEP/SLC ring-imaging chambers and an enhanced BaBar DIRC. Using these parametrisations we compare the relative tagging power of each technique to that of an ideal detector. (8 refs).

  9. Neutral hydrogen in galactic fountains

    CERN Document Server

    Booth, C M

    2007-01-01

    Simulations of an isolated Milky Way-like galaxy, in which supernovae power a galactic fountain, reproduce the observed velocity and 21cm brightness statistics of galactic neutral hydrogen (HI). The simulated galaxy consists of a thin HI disk, similar in extent and brightness to that observed in the Milky Way, and extra-planar neutral gas at a range of velocities due to the galactic fountain. Mock observations of the neutral gas resemble the HI flux measurements from the Leiden-Argentine-Bonn (LAB) HI, survey, including a high-velocity tail which matches well with observations of high-velocity clouds. The simulated high-velocity clouds are typically found close to the galactic disk, with a typical line-of-sight distance of 13kpc from observers on the solar circle. The fountain efficiently cycles matter from the centre of the galaxy to its outskirts at a rate of around 0.5 M_sun/yr

  10. The unified neutral theory of biodiversity and biogeography at age ten.

    Science.gov (United States)

    Rosindell, James; Hubbell, Stephen P; Etienne, Rampal S

    2011-07-01

    A decade has now passed since Hubbell published The Unified Neutral Theory of Biodiversity and Biogeography. Neutral theory highlights the importance of dispersal limitation, speciation and ecological drift in the natural world and provides quantitative null models for assessing the role of adaptation and natural selection. Significant advances have been made in providing methods for understanding neutral predictions and comparing them with empirical data. In this review, we describe the current state-of-the-art techniques and ideas in neutral theory and how these are of relevance to ecology. The future of neutral theory is promising, but its concepts must be applied more broadly beyond the current focus on species-abundance distributions.

  11. Human peripheral blood lymphocytes from recently vaccinated individuals produce both type-specific and intertypic cross-reacting neutralizing antibody on in vitro stimulation with one type of poliovirus.

    NARCIS (Netherlands)

    F.G.C.M. Uytdehaag (Fons); H.G. Loggen; T. Logtenberg (Ton); R.A. Lichtveld (Rob); G. van Steenis (Bert); J.A.A.M. van Asten (Jack); A.D.M.E. Osterhaus (Albert)

    1985-01-01

    textabstractAn in vitro system of poliovirus-specific antibody production by peripheral blood B cells on stimulation by the virus has been developed. Virus-neutralizing antibodies in culture supernatant fluids, or virus-specific antibody-secreting cells (ASC) were detected by microneutralization ass

  12. 2010年中国人免疫球蛋白中EV71中和抗体效价的比较研究%Study of anti- EV71 neutralizing titers of human immunoglobulins made in China in 2010

    Institute of Scientific and Technical Information of China (English)

    毛群颖; 郭增兵; 李青; 梁争论; 李凤祥; 王军志

    2011-01-01

    Objective: To explore the anti - EV71 titer in human immunoglobulins produced by different blood product manufacturers in China in 2010. To provide specific drugs for hand,foot and mouth disease( HFMD) caused by EV71. Methods: 97 Lots of human immunoglobulins for intravenous injection (IVIG) produced by different manufacturers were tested for neutralizing antibodies (NTAb) to EV71 by microneutralization test with 2 different EV71 viruses, which isolated from mainland china in the past two years. The difference of anti - EV71 titer in IVIG has been compared. Results:The geometric mean titers (GMTs) of EV71 NTAb in 97 Lots IVIG is 1: 180 (1= 170 -1:191). The titers of EV71 NTAb in 12 Lots IVIG produced by 7 different manufacturers have been more than 1 ':256,and the IVIG of Lot 20100406 produced by S manufacturer has the highest titer (1:543). The A manufacturer had made the most Lot of IVIG (5 batches) with anti -EV71 titer higher than 1:512,that made the average of anti -EV71 from A manufacturer to be obviously higher than other 10 manufacturers (P <0. 05). Further analysis revealed that S manufacturer had tested the anti - EV71 titers in plasma of raw materials and selected the plasma with high titer of EV71 to produce this Lot IVIG; and A manufacturer selected the areas with high anti - EV71 levels in populations as a plasma source to feed and make IVIGs. Conclusion: This study shows that through controlling the neutralizing antibodies levels to EV71 of raw materials efficiently by the manufacturer, it is scientific and feasible to produce IVIG with high anti -EV71 titer.%目的:了解2010年我国生产的静注人免疫球蛋白(IVIG)中抗- EV71中和抗体效价的情况,为治疗EV71引起的手足口病( HFMD)提供特异性治疗药物.方法:采用经典微量细胞病变法,应用近2年分离自中国大陆的2株肠道病毒71型(EV71)病毒株,对我国不同血液制品厂家生产的97批IVIG进行抗- EV71中和效价

  13. Serum neutralizing activities from a Beijing homosexual male cohort infected with different subtypes of HIV-1 in China.

    Directory of Open Access Journals (Sweden)

    Mingshun Zhang

    Full Text Available Protective antibodies play a critical role in an effective HIV vaccine; however, eliciting antibodies to block infection by viruses from diverse genetic subtypes remains a major challenge. As the world's most populous country, China has been under the threat of at least three major subtypes of circulating HIV-1 viruses. Understanding the cross reactivity and specificities of serum antibody responses that mediate broad neutralization of the virus in HIV-1 infected Chinese patients will provide valuable information for the design of vaccines to prevent HIV-1 transmission in China. Sera from a cohort of homosexual men, who have been managed by a major HIV clinical center in Beijing, China, were analyzed for cross-sectional neutralizing activities against pseudotyped viruses expressing Env antigens of the major subtype viruses (AE, BC and B subtypes circulating in China. Neutralizing activities in infected patients' blood were most capable of neutralizing viruses in the homologous subtype; however, a subset of blood samples was able to achieve broad neutralizing activities across different subtypes. Such cross neutralizing activity took 1-2 years to develop and CD4 binding site antibodies were critical components in these blood samples. Our study confirmed the presence of broadly neutralizing sera in China's HIV-1 patient population. Understanding the specificity and breadth of these neutralizing activities can guide efforts for the development of HIV vaccines against major HIV-1 viruses in China.

  14. Broader HIV-1 neutralizing antibody responses induced by envelope glycoprotein mutants based on the EIAV attenuated vaccine

    Directory of Open Access Journals (Sweden)

    Liu Lianxing

    2010-09-01

    Full Text Available Abstract Background In order to induce a potent and cross-reactive neutralizing antibody (nAb, an effective envelope immunogen is crucial for many viral vaccines, including the vaccine for the human immunodeficiency virus (HIV. The Chinese equine infectious anemia virus (EIAV attenuated vaccine has controlled the epidemic of this virus after its vaccination in over 70 million equine animals during the last 3 decades in China. Data from our past studies demonstrate that the Env protein of this vaccine plays a pivotal role in protecting horses from both homologous and heterogeneous EIAV challenges. Therefore, the amino acid sequence information from the Chinese EIAV attenuated vaccine, in comparison with the parental wild-type EIAV strains, was applied to modify the corresponding region of the envelope glycoprotein of HIV-1 CN54. The direction of the mutations was made towards the amino acids conserved in the two EIAV vaccine strains, distinguishing them from the two wild-type strains. The purpose of the modification was to enhance the immunogenicity of the HIV Env. Results The induced nAb by the modified HIV Env neutralized HIV-1 B and B'/C viruses at the highest titer of 1:270. Further studies showed that a single amino acid change in the C1 region accounts for the substantial enhancement in induction of anti-HIV-1 neutralizing antibodies. Conclusions This study shows that an HIV envelope modified by the information of another lentivirus vaccine induces effective broadly neutralizing antibodies. A single amino acid mutation was found to increase the immunogenicity of the HIV Env.

  15. Enhancing exposure of HIV-1 neutralization epitopes through mutations in gp41.

    Directory of Open Access Journals (Sweden)

    Catherine A Blish

    2008-01-01

    Full Text Available BACKGROUND: The generation of broadly neutralizing antibodies is a priority in the design of vaccines against HIV-1. Unfortunately, most antibodies to HIV-1 are narrow in their specificity, and a basic understanding of how to develop antibodies with broad neutralizing activity is needed. Designing methods to target antibodies to conserved HIV-1 epitopes may allow for the generation of broadly neutralizing antibodies and aid the global fight against AIDS by providing new approaches to block HIV-1 infection. Using a naturally occurring HIV-1 Envelope (Env variant as a template, we sought to identify features of Env that would enhance exposure of conserved HIV-1 epitopes. METHODS AND FINDINGS: Within a cohort study of high-risk women in Mombasa, Kenya, we previously identified a subtype A HIV-1 Env variant in one participant that was unusually sensitive to neutralization. Using site-directed mutagenesis, the unusual neutralization sensitivity of this variant was mapped to two amino acid mutations within conserved sites in the transmembrane subunit (gp41 of the HIV-1 Env protein. These two mutations, when introduced into a neutralization-resistant variant from the same participant, resulted in 3- to >360-fold enhanced neutralization by monoclonal antibodies specific for conserved regions of both gp41 and the Env surface subunit, gp120, >780-fold enhanced neutralization by soluble CD4, and >35-fold enhanced neutralization by the antibodies found within a pool of plasmas from unrelated individuals. Enhanced neutralization sensitivity was not explained by differences in Env infectivity, Env concentration, Env shedding, or apparent differences in fusion kinetics. Furthermore, introduction of these mutations into unrelated viral Env sequences, including those from both another subtype A variant and a subtype B variant, resulted in enhanced neutralization susceptibility to gp41- and gp120-specific antibodies, and to plasma antibodies. This enhanced

  16. Fully Bayesian tests of neutrality using genealogical summary statistics

    Directory of Open Access Journals (Sweden)

    Drummond Alexei J

    2008-10-01

    Full Text Available Abstract Background Many data summary statistics have been developed to detect departures from neutral expectations of evolutionary models. However questions about the neutrality of the evolution of genetic loci within natural populations remain difficult to assess. One critical cause of this difficulty is that most methods for testing neutrality make simplifying assumptions simultaneously about the mutational model and the population size model. Consequentially, rejecting the null hypothesis of neutrality under these methods could result from violations of either or both assumptions, making interpretation troublesome. Results Here we harness posterior predictive simulation to exploit summary statistics of both the data and model parameters to test the goodness-of-fit of standard models of evolution. We apply the method to test the selective neutrality of molecular evolution in non-recombining gene genealogies and we demonstrate the utility of our method on four real data sets, identifying significant departures of neutrality in human influenza A virus, even after controlling for variation in population size. Conclusion Importantly, by employing a full model-based Bayesian analysis, our method separates the effects of demography from the effects of selection. The method also allows multiple summary statistics to be used in concert, thus potentially increasing sensitivity. Furthermore, our method remains useful in situations where analytical expectations and variances of summary statistics are not available. This aspect has great potential for the analysis of temporally spaced data, an expanding area previously ignored for limited availability of theory and methods.

  17. Giant Broad Line Regions in Dwarf Seyferts

    CERN Document Server

    Devereux, Nick

    2015-01-01

    High angular resolution spectroscopy obtained with the Hubble Space Telescope (HST) has revealed a remarkable population of galaxies hosting dwarf Seyfert nuclei with an unusually large broad-line region (BLR). These objects are remarkable for two reasons. Firstly, the size of the BLR can, in some cases, rival those seen in the most luminous quasars. Secondly, the size of the BLR is not correlated with the central continuum luminosity, an observation that distinguishes them from their reverberating counterparts. Collectively, these early results suggest that non-reverberating dwarf Seyferts are a heterogeneous group and not simply scaled versions of each other. Careful inspection reveals broad H Balmer emission lines with single peaks, double peaks, and a combination of the two, suggesting that the broad emission lines are produced in kinematically distinct regions centered on the black hole (BH). Because the gravitational field strength is already known for these objects, by virtue of knowing their BH mass, ...

  18. Modelling Demand for Broad Money in Australia

    OpenAIRE

    Abbas Valadkhani

    2002-01-01

    The existence of a stable demand for money is very important for the conduct of monetary policy. It is argued that previous work on the demand for money in Australia has not been very satisfactory in a number of ways. This paper examines the long- and short-run determinants of the demand for broad money employing the Johansen cointegration technique and a short-run dynamic model. Using quarterly data for the period 1976:3-2002:2, this paper finds, inter alia, that the demand for broad money i...

  19. A triclade DNA vaccine designed on the basis of a comprehensive serologic study elicits neutralizing antibody responses against all clades and subclades of highly pathogenic avian influenza H5N1 viruses.

    Science.gov (United States)

    Zhou, Fan; Wang, Guiqin; Buchy, Philippe; Cai, Zhipeng; Chen, Honglin; Chen, Zhiwei; Cheng, Genhong; Wan, Xiu-Feng; Deubel, Vincent; Zhou, Paul

    2012-06-01

    Because of their rapid evolution, genetic diversity, broad host range, ongoing circulation in birds, and potential human-to-human transmission, H5N1 influenza viruses remain a major global health concern. Their high degree of genetic diversity also poses enormous burdens and uncertainties in developing effective vaccines. To overcome this, we took a new approach, i.e., the development of immunogens based on a comprehensive serologic study. We constructed DNA plasmids encoding codon-optimized hemagglutinin (HA) from 17 representative strains covering all reported clades and subclades of highly pathogenic avian influenza H5N1 viruses. Using DNA plasmids, we generated the corresponding H5N1 pseudotypes and immune sera. We performed an across-the-board pseudotype-based neutralization assay and determined antigenic clusters by cartography. We then designed a triclade DNA vaccine and evaluated its immunogenicity and protection in mice. We report here that (sub)clades 0, 1, 3, 4, 5, 6, 7.1, and 9 were grouped into antigenic cluster 1, (sub)clades 2.1.3.2, 2.3.4, 2.4, 2.5, and 8 were grouped into another antigenic cluster, with subclade 2.2.1 loosely connected to it, and each of subclades 2.3.2.1 and 7.2 was by itself. Importantly, the triclade DNA vaccine encoding HAs of (sub)clades 0, 2.3.2.1, and 7.2 elicited broadly neutralizing antibody responses against all H5 clades and subclades and protected mice against high-lethal-dose heterologous H5N1 challenge. Thus, we conclude that broadly neutralizing antibodies against all H5 clades and subclades can indeed be elicited with immunogens on the basis of a comprehensive serologic study. Further evaluation and optimization of such an approach in ferrets and in humans is warranted.

  20. Instructed neutralization, spontaneous neutralization and prevented neutralization after an obsession-like thought.

    NARCIS (Netherlands)

    M. van den Hout; M. Kindt; T. Weiland; M. Peters

    2002-01-01

    Building on 2 earlier experiments (S. Rachman et al [1996] and M. A. van den Hout [2001]) the present study investigated the effects of neutralizing the consequences of an obsession-like thought in healthy participants. Just like in the earlier studies, writing out and thinking of such a thought gen

  1. Teaching the Broad, Interdisciplinary Impact of Evolution

    Science.gov (United States)

    Benson, David; Atlas, Pierre; Haberski, Raymond; Higgs, Jamie; Kiley, Patrick; Maxwell, Michael, Jr.; Mirola, William; Norton, Jamey

    2009-01-01

    As perhaps the most encompassing idea in biology, evolution has impacted not only science, but other academic disciplines as well. The broad, interdisciplinary impact of evolution was the theme of a course taught at Marian College, Indianapolis, Indiana in 2002, 2004, and 2006. Using a strategy that could be readily adopted at other institutions,…

  2. The GREGOR Broad-Band Imager

    Science.gov (United States)

    von der Lühe, O.; Volkmer, R.; Kentischer, T. J.; Geißler, R.

    2012-11-01

    The design and characteristics of the Broad-Band Imager (BBI) of GREGOR are described. BBI covers the visible spectral range with two cameras simultaneously for a large field and with critical sampling at 390 nm, and it includes a mode for observing the pupil in a Foucault configuration. Samples of first-light observations are shown.

  3. Boosting of HIV-1 neutralizing antibody responses by a distally related retroviral envelope protein

    DEFF Research Database (Denmark)

    Uchtenhagen, Hannes; Schiffner, Torben; Bowles, Emma

    2014-01-01

    Our knowledge of the binding sites for neutralizing Abs (NAb) that recognize a broad range of HIV-1 strains (bNAb) has substantially increased in recent years. However, gaps remain in our understanding of how to focus B cell responses to vulnerable conserved sites within the HIV-1 envelope glycop...

  4. A highly conserved neutralizing epitope on group 2 influenza A viruses

    NARCIS (Netherlands)

    Ekiert, D.C.; Friesen, R.H.E.; Bhanha, G.; Kwaks, T.; Jongeneelen, M.; Yu, W.; Ophorst,