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Sample records for systemic neutralizing antibodies

  1. A novel reporter system for neutralizing and enhancing antibody assay against dengue virus

    PubMed Central

    2014-01-01

    Background Dengue virus (DENV) still poses a global public health threat, and no vaccine or antiviral therapy is currently available. Antibody plays distinct roles in controlling DENV infections. Neutralizing antibody is protective against DENV infection, whereas sub-neutralizing concentration of antibody can increase DENV infection, termed antibody-dependent enhancement (ADE). Plaque-based assay represents the most widely accepted method measuring neutralizing or enhancing antibodies. Results In this study, a novel reporter virus-based system was developed for measuring neutralization and ADE activity. A stable Renilla luciferase reporter DENV (Luc-DENV) that can produce robust luciferase signals in BHK-21 and K562 cells were used to establish the assay and validated against traditional plaque-based assay. Luciferase value analysis using various known DENV-specific monoclonal antibodies showed good repeatability and a well linear correlation with conventional plaque-based assays. The newly developed assay was finally validated with clinical samples from infected animals and individuals. Conclusions This reporter virus-based assay for neutralizing and enhancing antibody evaluation is rapid, lower cost, and high throughput, and will be helpful for laboratory detection and epidemiological investigation for DENV antibodies. PMID:24548533

  2. Albumin-binding adenoviruses circumvent pre-existing neutralizing antibodies upon systemic delivery.

    PubMed

    Rojas, Luis Alfonso; Condezo, Gabriela N; Moreno, Rafael; Fajardo, Carlos Alberto; Arias-Badia, Marcel; San Martín, Carmen; Alemany, Ramon

    2016-09-10

    Recombinant adenoviruses are used as vaccines, gene therapy vectors, and oncolytic viruses. However, the efficacy of such therapies is limited by pre-existing neutralizing antibodies (NAbs), especially when the virus is administered systemically for a wider biodistribution or to reach multiple metastases. To protect adenovirus against NAbs we inserted an albumin-binding domain (ABD) in the main adenovirus capsid protein, the hexon. This domain binds serum albumin to shield the virus upon systemic administration. The ABD-modified adenoviruses bind human and mouse albumin and maintain the infectivity and replication capacity in presence of NAbs. In pre-immunized mice non-modified viruses are completely neutralized, whereas ABD-modified viruses preserve the ability to transduce target organs, induce oncolysis, or generate immune responses to expressed proteins. Our results indicate that albumin coating of the virus capsid represents an effective approach to evade pre-existing NAbs. This strategy has translational relevance in the use of adenovirus for gene therapy, cancer virotherapy, and vaccination. PMID:27388756

  3. Rapid High-Level Production of Functional HIV Broadly Neutralizing Monoclonal Antibodies in Transient Plant Expression Systems

    PubMed Central

    Rosenberg, Yvonne; Sack, Markus; Montefiori, David; Forthal, Donald; Mao, Lingjun; -Abanto, Segundo Hernandez; Urban, Lori; Landucci, Gary; Fischer, Rainer; Jiang, Xiaoming

    2013-01-01

    Passive immunotherapy using anti-HIV broadly neutralizing monoclonal antibodies (mAbs) has shown promise as an HIV treatment, reducing mother-to-child-transmission (MTCT) of simian/human immunodeficiency virus (SHIV) in non-human primates and decreasing viral rebound in patients who ceased receiving anti-viral drugs. In addition, a cocktail of potent mAbs may be useful as mucosal microbicides and provide an effective therapy for post-exposure prophylaxis. However, even highly neutralizing HIV mAbs used today may lose their effectiveness if resistance occurs, requiring the rapid production of new or engineered mAbs on an ongoing basis in order to counteract the viral resistance or the spread of a certain HIV-1 clade in a particular region or patient. Plant-based expression systems are fast, inexpensive and scalable and are becoming increasingly popular for the production of proteins and monoclonal antibodies. In the present study, Agrobacterium-mediated transient transfection of plants, utilizing two species of Nicotiana, have been tested to rapidly produce high levels of an HIV 89.6PΔ140env and several well-studied anti-HIV neutralizing monoclonal antibodies (b12, 2G12, 2F5, 4E10, m43, VRC01) or a single chain antibody construct (m9), for evaluation in cell-based viral inhibition assays. The protein-A purified plant-derived antibodies were intact, efficiently bound HIV envelope, and were equivalent to, or in one case better than, their counterparts produced in mammalian CHO or HEK-293 cells in both neutralization and antibody dependent viral inhibition assays. These data indicate that transient plant-based transient expression systems are very adaptable and could rapidly generate high levels of newly identified functional recombinant HIV neutralizing antibodies when required. In addition, they warrant detailed cost-benefit analysis of prolonged incubation in plants to further increase mAb production. PMID:23533588

  4. Sufficient virus-neutralizing antibody in the central nerve system improves the survival of rabid rats

    PubMed Central

    2012-01-01

    Background Rabies is known to be lethal in human. Treatment with passive immunity for the rabies is effective only when the patients have not shown the central nerve system (CNS) signs. The blood–brain barrier (BBB) is a complex functional barrier that may compromise the therapeutic development in neurological diseases. The goal of this study is to determine the change of BBB integrity and to assess the therapeutic possibility of enhancing BBB permeability combined with passive immunity in the late stage of rabies virus infection. Methods The integrity of BBB permeability in rats was measured by quantitative ELISA for total IgG and albumin levels in the cerebrospinal fluid (CSF) and by exogenously applying Evans blue as a tracer. Western blotting of occludin and ZO-1, two tight junction proteins, was used to assess the molecular change of BBB structure. The breakdown of BBB with hypertonic arabinose, recombinant tumor necrosis factor-alpha (rTNF-γ), and focused ultrasound (FUS) were used to compare the extent of BBB disruption with rabies virus infection. Specific humoral immunity was analyzed by immunofluorescent assay and rapid fluorescent focus inhibition test. Virus-neutralizing monoclonal antibody (mAb) 8-10E was administered to rats with hypertonic breakdown of BBB as a passive immunotherapy to prevent the death from rabies. Results The BBB permeability was altered on day 7 post-infection. Increased BBB permeability induced by rabies virus infection was observed primarily in the cerebellum and spinal cord. Occludin was significantly decreased in both the cerebral cortex and cerebellum. The rabies virus-specific antibody was not strongly elicited even in the presence of clinical signs. Disruption of BBB had no direct association with the lethal outcome of rabies. Passive immunotherapy with virus-neutralizing mAb 8-10E with the hypertonic breakdown of BBB prolonged the survival of rabies virus-infected rats. Conclusions We demonstrated that the BBB

  5. Enhanced HIV-1 neutralization by antibody heteroligation

    PubMed Central

    Mouquet, Hugo; Warncke, Malte; Scheid, Johannes F.; Seaman, Michael S.; Nussenzweig, Michel C.

    2012-01-01

    Passive transfer of broadly neutralizing human antibodies against HIV-1 protects macaques against infection. However, HIV-1 uses several strategies to escape antibody neutralization, including mutation of the gp160 viral surface spike, a glycan shield to block antibody access to the spike, and expression of a limited number of viral surface spikes, which interferes with bivalent antibody binding. The latter is thought to decrease antibody apparent affinity or avidity, thereby interfering with neutralizing activity. To test the idea that increasing apparent affinity might enhance neutralizing activity, we engineered bispecific anti–HIV-1 antibodies (BiAbs) that can bind bivalently by virtue of one scFv arm that binds to gp120 and a second arm to the gp41 subunit of gp160. The individual arms of the BiAbs preserved the binding specificities of the original anti-HIV IgG antibodies and together bound simultaneously to gp120 and gp41. Heterotypic bivalent binding enhanced neutralization compared with the parental antibodies. We conclude that antibody recognition and viral neutralization of HIV can be improved by heteroligation. PMID:22219363

  6. Broadly neutralizing antibodies against influenza viruses

    PubMed Central

    Laursen, Nick S.; Wilson, Ian A.

    2014-01-01

    Despite available antivirals and vaccines, influenza infections continue to be a major cause of mortality worldwide. Vaccination generally induces an effective, but strain-specific antibody response. As the virus continually evolves, new vaccines have to be administered almost annually when a novel strain becomes dominant. Furthermore, the sporadic emerging resistance to neuraminidase inhibitors among circulating strains suggests an urgent need for new therapeutic agents. Recently, several cross-reactive antibodies have been described, which neutralize an unprecedented spectrum of influenza viruses. These broadly neutralizing antibodies generally target conserved functional regions on the major influenza surface glycoprotein hemagglutinin (HA). The characterization of their neutralization breadth and epitopes on HA could stimulate the development of new antibody-based antivirals and broader influenza vaccines. PMID:23583287

  7. Human-like antibodies neutralizing Western equine encephalitis virus

    PubMed Central

    Hülseweh, Birgit; Rülker, Torsten; Pelat, Thibaut; Langermann, Claudia; Frenzel, Andrè; Schirrmann, Thomas; Dübel, Stefan; Thullier, Philippe; Hust, Michael

    2014-01-01

    This study describes the development of the first neutralizing antibodies against Western equine encephalitis virus (WEEV), a member of the genus Alphavirus. WEEV is transmitted by mosquitoes and can spread to the human central nervous system, causing symptoms ranging from mild febrile reactions to life-threatening encephalitis. WEEV has been classified as a biological warfare agent by the US Centers for Disease Control and Prevention. No anti-WEEV drugs are currently commercially available. Neutralizing antibodies are useful for the pre- and post-exposure treatment of WEEV infections. In this study, two immune antibody gene libraries were constructed from two macaques immunized with inactivated WEEV. Four antibodies were selected from these libraries and recloned as scFv-Fc, with a human Fc part. These antibodies bound WEEV specifically in ELISA with little or no cross-reaction with other alphaviruses. They were further analyzed by immunohistochemistry. All binders were suitable for the intracellular detection of WEEV particles. Neutralizing activity was determined in vitro. Three of the four antibodies were found to be neutralizing; about 1 ng/mL of the best antibody (ToR69–3A2) neutralized 50% of 5x104 TCID50/mL. Due to its human-like nature with a germinality index of 89% (VH) and 91% (VL), the ToR69–3A2 antibody is a promising candidate for future passive vaccine development. PMID:24518197

  8. Poliovirus neutralization epitopes: analysis and localization with neutralizing monoclonal antibodies.

    PubMed Central

    Emini, E A; Jameson, B A; Lewis, A J; Larsen, G R; Wimmer, E

    1982-01-01

    Two hybridomas (H3 and D3) secreting monoclonal neutralizing antibody to intact poliovirus type 1 (Mahoney strain) were established. Each antibody bound to a site qualitatively different from that to which the other antibody bound. The H3 site was located on intact virions and, to a lesser extent, on 80S naturally occurring empty capsids and 14S precursor subunits. The D3 site was found only on virions and empty capsids. Neither site was expressed on 80S heat-treated virions. The antibodies did not react with free denatured or undenatured viral structural proteins. Viral variants which were no longer capable of being neutralized by either one or the other antibody were obtained. Such variants arose during normal cell culture passage of wild-type virus and were present in the progeny viral population on the order of 10(-4) variant per wild-type virus PFU. Toluene-2,4-diisocyanate, a heterobifunctional covalent cross-linking reagent, was used to irreversibly bind the F(ab) fragments of the two antibodies to their respective binding sites. In this way, VP1 was identified as the structural protein containing both sites. PMID:6183443

  9. Human recombinant neutralizing antibodies against hantaan virus G2 protein.

    PubMed

    Koch, Joachim; Liang, Mifang; Queitsch, Iris; Kraus, Annette A; Bautz, Ekkehard K F

    2003-03-30

    Old world hantaviruses, causing hemorrhagic fever with renal syndrome (HFRS), still present a public health problem in Asia and Eastern Europe. The majority of cases has been recorded in China. The aim of our study was to generate human recombinant neutralizing antibodies to a hantavirus by phage display technology. To preserve the structural identity of viral protein, the panning procedure was performed on native Hantaan (HTN) (76-118) virus propagated in Vero-E6 cells. In total, five complete human recombinant IgG antibodies were produced in a baculovirus expression system. All of them were able to completely neutralize HTN, and Seoul (SEO) virus in a plaque reduction neutralization test (PRNT). Three of these antibodies could also completely neutralize Dobrava (DOB) virus but not Puumala (PUU) virus. All antibodies bind to Hantaan virus G2 protein localized in the virus envelope. The sequence areas within the HTN (76-118)-G2 protein detected by five selected antibodies were mapped using peptide scans. Two partial epitopes, 916-KVMATIDSF-924 and 954-LVTKDIDFD-963, were recognized, which presumably are of paramount importance for docking of the virus to host cell receptors. A consensus motif 916-KVXATIXSF-924 could be identified by mutational analysis. The neutralizing antibodies to the most widely distributed hantaviruses causing HFRS might be promising candidates for the development of an agent for prevention and treatment of HFRS in patients. PMID:12706090

  10. POLIOVIRUS TYPE 1: NEUTRALIZATION BY PAPAIN-DIGESTED ANTIBODIES.

    PubMed

    VOGT, A; KOPP, R; MAASS, G; REICH, L

    1964-09-25

    Papain-digested rabbit antibody (Porter's fractions I and II) can neutralize poliovirus. Neutralizing capacity after digestion ranged from 35 to 45 percent of that of the undigested antibody. No definite dissociation of the antibody fragments from the virus was observed after the reaction mixture had been diluted in a neutral medium. PMID:14175107

  11. Antibody neutralization of retargeted measles viruses.

    PubMed

    Lech, Patrycja J; Pappoe, Roland; Nakamura, Takafumi; Tobin, Gregory J; Nara, Peter L; Russell, Stephen J

    2014-04-01

    The measles virus (MV) vaccine lineage is a promising oncolytic but prior exposure to the measles vaccine or wild-type MV strains limits treatment utility due to the presence of anti-measles antibodies. MV entry can be redirected by displaying a polypeptide ligand on the Hemagglutinin (H) C-terminus. We hypothesized that retargeted MV would escape neutralization by monoclonal antibodies (mAbs) recognizing the H receptor-binding surface and be less susceptible to neutralization by human antisera. Using chimeric H proteins, with and without mutations that ablate MV receptor binding, we show that retargeted MVs escape mAbs that target the H receptor-binding surface by virtue of mutations that ablate infection via SLAM and CD46. However, C-terminally displayed domains do not mediate virus entry in the presence of human antibodies that bind to the underlying H domain. In conclusion, utility of retargeted oncolytic measles viruses does not extend to evasion of human serum neutralization. PMID:24725950

  12. Neutralizing antibodies to HIV-1 induced by immunization

    PubMed Central

    McCoy, Laura E.

    2013-01-01

    Most neutralizing antibodies act at the earliest steps of viral infection and block interaction of the virus with cellular receptors to prevent entry into host cells. The inability to induce neutralizing antibodies to HIV has been a major obstacle to HIV vaccine research since the early days of the epidemic. However, in the past three years, the definition of a neutralizing antibody against HIV has been revolutionized by the isolation of extremely broad and potent neutralizing antibodies from HIV-infected individuals. Considerable hurdles remain for inducing neutralizing antibodies to a protective level after immunization. Meanwhile, novel technologies to bypass the induction of antibodies are being explored to provide prophylactic antibody-based interventions. This review addresses the challenge of inducing HIV neutralizing antibodies upon immunization and considers notable recent advances in the field. A greater understanding of the successes and failures for inducing a neutralizing response upon immunization is required to accelerate the development of an effective HIV vaccine. PMID:23401570

  13. Maturation Pathways of Cross-Reactive HIV-1 Neutralizing Antibodies

    PubMed Central

    Xiao, Xiaodong; Chen, Weizao; Feng, Yang; Dimitrov, Dimiter S.

    2009-01-01

    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 intermediate antibodies

  14. INDUCTION OF NEUTRALIZING ANTIBODIES TO HENDRA AND NIPAH GLYCOPROTEINS USING A VENEZUELAN EQUINE ENCEPHALITIS VIRUS IN VIVO EXPESSION SYSTEM

    PubMed Central

    Defang, Gabriel N.; Khetawat, Dimple; Broder, Christopher C.; Quinnan, Gerald V.

    2010-01-01

    The emergence of Hendra Virus (HeV) and Nipah Virus (NiV) which can cause fatal infections in both animals and humans has triggered a search for an effective vaccine. Here, we have explored the potential for generating an effective humoral immune response to these zoonotic pathogens using an alphavirus-based vaccine platform. Groups of mice were immunized with Venezuelan equine encephalitis virus replicon particles (VRP) encoding the attachment or fusion glycoproteins of either HeV or NiV. We demonstrate the induction of highly potent cross-reactive neutralizing antibodies to both viruses using this approach. Preliminary study suggested early enhancement in the antibody response with use of a modified version of VRP. Overall, these data suggest that the use of an alphavirus-derived vaccine platform might serve as a viable approach for development of an effective vaccine against the henipaviruses. PMID:21050901

  15. Structure and Design of Broadly-Neutralizing Antibodies Against HIV

    PubMed Central

    Ryu, Seong Eon; Hendrickson, Wayne A.

    2012-01-01

    Since the discovery more than 30 years ago of human immunodeficiency virus (HIV) as the causative agent of the deadly disease, acquired immune deficiency disease (AIDS), there have been no efficient vaccines against the virus. For the infection of the virus, the HIV surface glycoprotein gp120 first recognizes the CD4 receptor on the target helper T-cell, which initiates HIV fusion with the target cell and, if unchecked, leads to destruction of the patient’s immune system. Despite the difficulty of developing appropriate immune responses in HIV-infected individuals, patient sera often contain antibodies that have broad neutralization activity, indicating the possibility of immunological treatment and prevention. Recently, through extensive structural studies of neutralizing antibodies of HIV in complex with gp120, the critical mechanisms of broad neutralization against HIV have been elucidated. Based on these discoveries, the structure-aided designs of antibodies and novel scaffolds were performed to create extremely potent neutralizing antibodies against HIV. These new discoveries and advances shed light on the road to development of efficient immunological therapies against AIDS. PMID:22736269

  16. Structural Basis of Human Parechovirus Neutralization by Human Monoclonal Antibodies

    PubMed Central

    Shakeel, Shabih; Westerhuis, Brenda M.; Ora, Ari; Koen, Gerrit; Bakker, Arjen Q.; Claassen, Yvonne; Wagner, Koen; Beaumont, Tim; Wolthers, Katja C.

    2015-01-01

    ABSTRACT Since it was first recognized in 2004 that human parechoviruses (HPeV) are a significant cause of central nervous system and neonatal sepsis, their clinical importance, primarily in children, has started to emerge. Intravenous immunoglobulin treatment is the only treatment available in such life-threatening cases and has given moderate success. Direct inhibition of parechovirus infection using monoclonal antibodies is a potential treatment. We have developed two neutralizing monoclonal antibodies against HPeV1 and HPeV2, namely, AM18 and AM28, which also cross-neutralize other viruses. Here, we present the mapping of their epitopes using peptide scanning, surface plasmon resonance, fluorescence-based thermal shift assays, electron cryomicroscopy, and image reconstruction. We determined by peptide scanning and surface plasmon resonance that AM18 recognizes a linear epitope motif including the arginine-glycine-aspartic acid on the C terminus of capsid protein VP1. This epitope is normally used by the virus to attach to host cell surface integrins during entry and is found in 3 other viruses that AM18 neutralizes. Therefore, AM18 is likely to cause virus neutralization by aggregation and by blocking integrin binding to the capsid. Further, we show by electron cryomicroscopy, three-dimensional reconstruction, and pseudoatomic model fitting that ordered RNA interacts with HPeV1 VP1 and VP3. AM28 recognizes quaternary epitopes on the capsid composed of VP0 and VP3 loops from neighboring pentamers, thereby increasing the RNA accessibility temperature for the virus-AM28 complex compared to the virus alone. Thus, inhibition of RNA uncoating probably contributes to neutralization by AM28. IMPORTANCE Human parechoviruses can cause mild infections to severe diseases in young children, such as neonatal sepsis, encephalitis, and cardiomyopathy. Intravenous immunoglobulin treatment is the only treatment available in such life-threatening cases. In order to develop more

  17. Determination of anti-adeno-associated virus vector neutralizing antibody titer with an in vitro reporter system.

    PubMed

    Meliani, Amine; Leborgne, Christian; Triffault, Sabrina; Jeanson-Leh, Laurence; Veron, Philippe; Mingozzi, Federico

    2015-04-01

    Adeno-associated virus (AAV) vectors are a platform of choice for in vivo gene transfer applications. However, neutralizing antibodies (NAb) to AAV can be found in humans and some animal species as a result of exposure to the wild-type virus, and high-titer NAb develop following AAV vector administration. In some conditions, anti-AAV NAb can block transduction with AAV vectors even when present at low titers, thus requiring prescreening before vector administration. Here we describe an improved in vitro, cell-based assay for the determination of NAb titer in serum or plasma samples. The assay is easy to setup and sensitive and, depending on the purpose, can be validated to support clinical development of gene therapy products based on AAV vectors. PMID:25819687

  18. Neutralizing Antibodies Against Interferon-Beta

    PubMed Central

    2008-01-01

    The development of neutralizing antibodies (NAbs) is a major problem in multiple sclerosis (MS) patients treated with interferon-beta (IFN-ß). Whereas binding antibodies (BAbs) can be demonstrated in the vast majority of patients, only a smaller proportion of patients develop NAbs. The principle in NAb in vitro assays is the utilization of cultured cell lines that are responsive to IFN-ß. The cytopathic effect (CPE) assay measures the capacity of NAbs to neutralize IFN- ß's protective effect on cells challenged with virus and the MxA induction assay measures the ability of NAbs to reduce the IFN-ß-induced expression of MxA, either at the mRNA or the protein level. A titer of >20 neutralizing units/ml traditionally defines NAb posi-tivity. NAbs in high titers completely abrogate the in vivo response to IFN-ß, whereas the effect of low and intermediate titers is unpredictable. As clinically important NAbs appear only after 9-18 months IFN- ß0 therapy, short-term studies of two years or less are unsuitable for evaluation of clinical NAb effects. All long-term trials of three years or more concordantly show evidence of a detrimental effect of NAbs on relapses, disease activity on MRI, or on disease progression. Persistent high titers of NAbs indicate an abrogation of the biological response and, hence, absence of therapeutic efficacy, and this observation should lead to a change of therapy. As low and medium titers are ambiguous treatment decisions in patients with low NAb titres should be guided by determination of in vivo mRNA MxA induction and clinical disease activity. PMID:21180570

  19. Incomplete Neutralization and Deviation from Sigmoidal Neutralization Curves for HIV Broadly Neutralizing Monoclonal Antibodies

    PubMed Central

    McCoy, Laura E.; Falkowska, Emilia; Doores, Katie J.; Le, Khoa; Sok, Devin; van Gils, Marit J.; Euler, Zelda; Burger, Judith A.; Seaman, Michael S.; Sanders, Rogier W.; Schuitemaker, Hanneke; Poignard, Pascal; Wrin, Terri; Burton, Dennis R.

    2015-01-01

    The broadly neutralizing HIV monoclonal antibodies (bnMAbs) PG9, PG16, PGT151, and PGT152 have been shown earlier to occasionally display an unusual virus neutralization profile with a non-sigmoidal slope and a plateau at <100% neutralization. In the current study, we were interested in determining the extent of non-sigmoidal slopes and plateaus at <100% for HIV bnMAbs more generally. Using both a 278 panel of pseudoviruses in a CD4 T-cell (U87.CCR5.CXCR4) assay and a panel of 117 viruses in the TZM-bl assay, we found that bnMAbs targeting many neutralizing epitopes of the spike had neutralization profiles for at least one virus that plateaued at <90%. Across both panels the bnMAbs targeting the V2 apex of Env and gp41 were most likely to show neutralization curves that plateaued <100%. Conversely, bnMAbs targeting the high-mannose patch epitopes were less likely to show such behavior. Two CD4 binding site (CD4bs) Abs also showed this behavior relatively infrequently. The phenomenon of incomplete neutralization was also observed in a large peripheral blood mononuclear cells (PBMC)-grown molecular virus clone panel derived from patient viral swarms. In addition, five bnMAbs were compared against an 18-virus panel of molecular clones produced in 293T cells and PBMCs and assayed in TZM-bl cells. Examples of plateaus <90% were seen with both types of virus production with no consistent patterns observed. In conclusion, incomplete neutralization and non-sigmoidal neutralization curves are possible for all HIV bnMAbs against a wide range of viruses produced and assayed in both cell lines and primary cells with implications for the use of antibodies in therapy and as tools for vaccine design. PMID:26267277

  20. Incomplete Neutralization and Deviation from Sigmoidal Neutralization Curves for HIV Broadly Neutralizing Monoclonal Antibodies.

    PubMed

    McCoy, Laura E; Falkowska, Emilia; Doores, Katie J; Le, Khoa; Sok, Devin; van Gils, Marit J; Euler, Zelda; Burger, Judith A; Seaman, Michael S; Sanders, Rogier W; Schuitemaker, Hanneke; Poignard, Pascal; Wrin, Terri; Burton, Dennis R

    2015-08-01

    The broadly neutralizing HIV monoclonal antibodies (bnMAbs) PG9, PG16, PGT151, and PGT152 have been shown earlier to occasionally display an unusual virus neutralization profile with a non-sigmoidal slope and a plateau at <100% neutralization. In the current study, we were interested in determining the extent of non-sigmoidal slopes and plateaus at <100% for HIV bnMAbs more generally. Using both a 278 panel of pseudoviruses in a CD4 T-cell (U87.CCR5.CXCR4) assay and a panel of 117 viruses in the TZM-bl assay, we found that bnMAbs targeting many neutralizing epitopes of the spike had neutralization profiles for at least one virus that plateaued at <90%. Across both panels the bnMAbs targeting the V2 apex of Env and gp41 were most likely to show neutralization curves that plateaued <100%. Conversely, bnMAbs targeting the high-mannose patch epitopes were less likely to show such behavior. Two CD4 binding site (CD4bs) Abs also showed this behavior relatively infrequently. The phenomenon of incomplete neutralization was also observed in a large peripheral blood mononuclear cells (PBMC)-grown molecular virus clone panel derived from patient viral swarms. In addition, five bnMAbs were compared against an 18-virus panel of molecular clones produced in 293T cells and PBMCs and assayed in TZM-bl cells. Examples of plateaus <90% were seen with both types of virus production with no consistent patterns observed. In conclusion, incomplete neutralization and non-sigmoidal neutralization curves are possible for all HIV bnMAbs against a wide range of viruses produced and assayed in both cell lines and primary cells with implications for the use of antibodies in therapy and as tools for vaccine design. PMID:26267277

  1. Macrophage Internal HIV-1 Is Protected from Neutralizing Antibodies

    PubMed Central

    Koppensteiner, Herwig; Banning, Carina; Schneider, Carola; Hohenberg, Heinrich

    2012-01-01

    In macrophages, HIV-1 accumulates in intracellular vesicles designated virus-containing compartments (VCCs). These might play an important role in the constitution of macrophages as viral reservoirs and allow HIV-1 to evade the immune system by sequestration in an internal niche, which is difficult to access from the exterior. However, until now, evidence of whether internal virus accumulations are protected from the host's humoral immune response is still lacking. In order to be able to study the formation and antibody accessibility of VCCs, we generated HIV-1 with green fluorescent protein (GFP)-tagged Gag replicating in primary macrophages. Live-cell observations revealed faint initial cytosolic Gag expression and subsequent large intracellular Gag accumulations which stayed stable over days. Taking advantage of the opportunity to study the accessibility of intracellular VCCs via the cell surface, we demonstrate that macrophage internal HIV-1-containing compartments cannot be targeted by neutralizing antibodies. Furthermore, HIV-1 was efficiently transferred from antibody-treated macrophages to T cells. Three-dimensional reconstruction of electron microscopic slices revealed that Gag accumulations correspond to viral particles within enclosed compartments and convoluted membranes. Thus, although some VCCs were connected to the plasma membrane, the complex membrane architecture of the HIV-1-containing compartment might shield viral particles from neutralizing antibodies. In sum, our study provides evidence that HIV-1 is sequestered into a macrophage internal membranous web, posing an obstacle for the elimination of this viral reservoir. PMID:22205742

  2. A game of numbers: the stoichiometry of antibody-mediated neutralization of flavivirus infection

    PubMed Central

    Pierson, Theodore C.; Diamond, Michael S.

    2016-01-01

    The humoral response contributes to the protection against viral pathogens. Although antibodies have the potential to inhibit viral infections via several mechanisms, an ability to neutralize viruses directly may be particularly important. Neutralizing antibody titers are commonly used as predictors of protection from infection, especially in the context of vaccine responses and immunity. Despite the simplicity of the concept, how antibody binding results in virus inactivation is incompletely understood despite decades of research. Flaviviruses have been an attractive system in which to seek a structural and quantitative understanding of how antibody interactions with virions modulate infection because of the contribution of antibodies to both protection and pathogenesis. This review will present a stoichiometric model of antibody-mediated neutralization of flaviviruses and discuss how these concepts can inform the development of vaccines and antibody-based therapeutics. PMID:25595803

  3. Human papillomavirus type 16 virus-like particles expressed in attenuated Salmonella typhimurium elicit mucosal and systemic neutralizing antibodies in mice.

    PubMed Central

    Nardelli-Haefliger, D; Roden, R B; Benyacoub, J; Sahli, R; Kraehenbuhl, J P; Schiller, J T; Lachat, P; Potts, A; De Grandi, P

    1997-01-01

    Attenuated strains of Salmonella are attractive live vaccine candidates for eliciting mucosal as well as systemic immune responses. The ability to induce immune responses in the reproductive tract may be critical for the effectiveness of a prophylactic vaccine against genital human papillomaviruses (HPV), which are important etiologic agents in the development of cervical cancer. To examine the potential of a live Salmonella-based vaccine to prevent genital HPV infection, the L1 major capsid protein from HPV type 16 (HPV16) was constitutively expressed in the PhoPc strain of Salmonella typhimurium. As demonstrated by electron microscopy, the L1 protein expressed in these bacteria assembled into virus-like particles (VLPs) that resemble authentic papillomavirus virions. This is the first demonstration that papillomavirus VLPs can self-assemble in prokaryotes. BALB/c mice were immunized with the HPV16 L1 recombinant PhoPc strain by the oral and nasal routes. Despite a low stability of the L1-expressing plasmid in vivo, a double nasal immunization was effective in inducing L1-specific serum antibodies that recognized mainly native, but not disassembled, VLPs. These antibodies effectively neutralized HPV16 pseudotyped virions in an in vitro infectivity assay. Conformationally dependent anti-VLP immunoglobulin A (IgA) and IgG were also detected in oral and vaginal secretions, indicating that potentially protective antibody responses were elicited at mucosal sites. Recombinant attenuated Salmonella expressing HPV capsids may represent a promising vaccine candidate against genital HPV infection. PMID:9234794

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

    PubMed

    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. PMID:26827912

  5. Neutralizing antibodies decrease the envelope fluidity of HIV-1

    SciTech Connect

    Harada, Shinji Monde, Kazuaki; Tanaka, Yuetsu; Kimura, Tetsuya; Maeda, Yosuke; Yusa, Keisuke

    2008-01-05

    For successful penetration of HIV-1, the formation of a fusion pore may be required in order to accumulate critical numbers of fusion-activated gp41 with the help of fluidization of the plasma membrane and viral envelope. An increase in temperature to 40 {sup o}C after viral adsorption at 25 {sup o}C enhanced the infectivity by 1.4-fold. The enhanced infectivity was inhibited by an anti-CXCR4 peptide, T140, and anti-V3 monoclonal antibodies (0.5{beta} and 694/98-D) by post-attachment neutralization, but not by non-neutralizing antibodies (670-30D and 246-D) specific for the C5 of gp120 and cluster I of gp41, respectively. Anti-HLA-II and an anti-HTLV-I gp46 antibody, LAT27, neutralized the molecule-carrying HIV-1{sub C-2(MT-2)}. The anti-V3 antibodies suppressed the fluidity of the HIV-1{sub C-2} envelope, whereas the non-neutralizing antibodies did not. The anti-HLA-II antibody decreased the envelope fluidity of HIV-1{sub C-2(MT-2)}, but not that of HIV-1{sub C-2}. Therefore, fluidity suppression by these antibodies represents an important neutralization mechanism, in addition to inhibition of viral attachment.

  6. Development of a drug delivery system for efficient alveolar delivery of a neutralizing monoclonal antibody to treat pulmonary intoxication to ricin.

    PubMed

    Respaud, Renaud; Marchand, Denis; Pelat, Thibaut; Tchou-Wong, Kam-Meng; Roy, Chad J; Parent, Christelle; Cabrera, Maria; Guillemain, Joël; Mac Loughlin, Ronan; Levacher, Eric; Fontayne, Alexandre; Douziech-Eyrolles, Laurence; Junqua-Moullet, Alexandra; Guilleminault, Laurent; Thullier, Philippe; Guillot-Combe, Emmanuelle; Vecellio, Laurent; Heuzé-Vourc'h, Nathalie

    2016-07-28

    The high toxicity of ricin and its ease of production have made it a major bioterrorism threat worldwide. There is however no efficient and approved treatment for poisoning by ricin inhalation, although there have been major improvements in diagnosis and therapeutic strategies. We describe the development of an anti-ricin neutralizing monoclonal antibody (IgG 43RCA-G1) and a device for its rapid and effective delivery into the lungs for an application in humans. The antibody is a full-length IgG and binds to the ricin A-chain subunit with a high affinity (KD=53pM). Local administration of the antibody into the respiratory tract of mice 6h after pulmonary ricin intoxication allowed the rescue of 100% of intoxicated animals. Specific operational constraints and aerosolization stresses, resulting in protein aggregation and loss of activity, were overcome by formulating the drug as a dry-powder that is solubilized extemporaneously in a stabilizing solution to be nebulized. Inhalation studies in mice showed that this formulation of IgG 43RCA-G1 did not induce pulmonary inflammation. A mesh nebulizer was customized to improve IgG 43RCA-G1 deposition into the alveolar region of human lungs, where ricin aerosol particles mostly accumulate. The drug delivery system also comprises a semi-automatic reconstitution system to facilitate its use and a specific holding chamber to maximize aerosol delivery deep into the lung. In vivo studies in monkeys showed that drug delivery with the device resulted in a high concentration of IgG 43RCA-G1 in the airways for at least 6h after local deposition, which is consistent with the therapeutic window and limited passage into the bloodstream. PMID:27173943

  7. Antibody Response to Hypervariable Region 1 Interferes with Broadly Neutralizing Antibodies to Hepatitis C Virus

    PubMed Central

    Keck, Zhen-yong; Girard-Blanc, Christine; Wang, Wenyan; Lau, Patrick; Zuiani, Adam; Rey, Felix A.; Krey, Thomas; Diamond, Michael S.

    2015-01-01

    ABSTRACT Hypervariable region 1 (HVR1) (amino acids [aa] 384 to 410) on the E2 glycoprotein of hepatitis C virus contributes to persistent infection by evolving escape mutations that attenuate binding of inhibitory antibodies and by blocking access of broadly neutralizing antibodies to their epitopes. A third proposed mechanism of immune antagonism is that poorly neutralizing antibodies binding to HVR1 interfere with binding of other superior neutralizing antibodies. Epitope mapping of human monoclonal antibodies (HMAbs) that bind to an adjacent, conserved domain on E2 encompassing aa 412 to 423 revealed two subsets, designated HC33 HMAbs. While both subsets have contact residues within aa 412 to 423, alanine-scanning mutagenesis suggested that one subset, which includes HC33.8, has an additional contact residue within HVR1. To test for interference of anti-HVR1 antibodies with binding of antibodies to aa 412 to 423 and other E2 determinants recognized by broadly neutralizing HMAbs, two murine MAbs against HVR1 (H77.16) and aa 412 to 423 (H77.39) were studied. As expected, H77.39 inhibited the binding of all HC33 HMAbs. Unexpectedly, H77.16 also inhibited the binding of both subsets of HC33 HMAbs. This inhibition also was observed against other broadly neutralizing HMAbs to epitopes outside aa 412 to 423. Combination antibody neutralization studies by the median-effect analysis method with H77.16 and broadly reactive HMAbs revealed antagonism between these antibodies. Structural studies demonstrated conformational flexibility in this antigenic region, which supports the possibility of anti-HVR1 antibodies hindering the binding of broadly neutralizing MAbs. These findings support the hypothesis that anti-HVR1 antibodies can interfere with a protective humoral response against HCV infection. IMPORTANCE HVR1 contributes to persistent infection by evolving mutations that escape from neutralizing antibodies to HVR1 and by shielding broadly neutralizing antibodies from

  8. Neutralizing antibodies in Borna disease virus-infected rats.

    PubMed Central

    Hatalski, C G; Kliche, S; Stitz, L; Lipkin, W I

    1995-01-01

    Borna disease is a neurologic syndrome caused by infection with a nonsegmented, negative-strand RNA virus, Borna disease virus. Infected animals have antibodies to two soluble viral proteins, p40 and p23, and a membrane-associated viral glycoprotein, gp18. We examined the time course for the development of neutralization activity and the expression of antibodies to individual viral proteins in sera of infected rats. The appearance of neutralizing activity correlated with the development of immunoreactivity to gp18, but not p40 or p23. Monospecific and monoclonal antibodies to native gp18 and recombinant nonglycosylated gp18 were also found to have neutralizing activity and to immunoprecipitate viral particles or subparticles. These findings suggest that gp18 is likely to be present on the surface of the viral particles and is likely to contain epitopes important for virus neutralization. PMID:7815538

  9. Higher Throughput Quantification of Neutralizing Antibody to Herpes Simplex Viruses

    PubMed Central

    Blevins, Tamara P.; Mitchell, Michelle C.; Korom, Maria; Wang, Hong; Yu, Yinyi; Morrison, Lynda A.; Belshe, Robert B.

    2015-01-01

    We report a rapid, higher throughput method for measuring neutralizing antibody to herpes simplex virus (HSV) in human sera. Clinical isolates and sera from the Herpevac Trial for Women were used in a colorimetric assay in which infection of tissue culture (lack of neutralization) was indicated by substrate metabolism by beta-galactosidase induced in the ELVIS cell line. The neutralization assay was optimized by addition of guinea pig complement, which particularly enhanced neutralizing antibody titers to HSV-2. Higher neutralizing antibody titers were also achieved using virus particles isolated from the supernatant of infected cells rather than lysate of infected cells as the source of virus. The effect of assay incubation time and incubation time with substrate were also optimized. We found that incubating with substrate until a standard optical density of 1.0 was reached permitted a better comparison among virus isolates, and achieved reliable measurement of neutralizing antibody activity. Interestingly, in contrast to results in the absence of complement, addition of complement allowed sera from HSV-2 gD-vaccinated subjects to neutralize HSV-1 and HSV-2 clinical and laboratory isolates with equal potency. PMID:26658766

  10. Higher Throughput Quantification of Neutralizing Antibody to Herpes Simplex Viruses.

    PubMed

    Blevins, Tamara P; Mitchell, Michelle C; Korom, Maria; Wang, Hong; Yu, Yinyi; Morrison, Lynda A; Belshe, Robert B

    2015-01-01

    We report a rapid, higher throughput method for measuring neutralizing antibody to herpes simplex virus (HSV) in human sera. Clinical isolates and sera from the Herpevac Trial for Women were used in a colorimetric assay in which infection of tissue culture (lack of neutralization) was indicated by substrate metabolism by beta-galactosidase induced in the ELVIS cell line. The neutralization assay was optimized by addition of guinea pig complement, which particularly enhanced neutralizing antibody titers to HSV-2. Higher neutralizing antibody titers were also achieved using virus particles isolated from the supernatant of infected cells rather than lysate of infected cells as the source of virus. The effect of assay incubation time and incubation time with substrate were also optimized. We found that incubating with substrate until a standard optical density of 1.0 was reached permitted a better comparison among virus isolates, and achieved reliable measurement of neutralizing antibody activity. Interestingly, in contrast to results in the absence of complement, addition of complement allowed sera from HSV-2 gD-vaccinated subjects to neutralize HSV-1 and HSV-2 clinical and laboratory isolates with equal potency. PMID:26658766

  11. Neutralizing and non-neutralizing monoclonal antibodies against dengue virus E protein derived from a naturally infected patient

    PubMed Central

    2010-01-01

    Background Antibodies produced in response to infection with any of the four serotypes of dengue virus generally provide homotypic immunity. However, prior infection or circulating maternal antibodies can also mediate a non-protective antibody response that can enhance the course of disease in a subsequent heterotypic infection. Naturally occurring human monoclonal antibodies can help us understand the protective and pathogenic roles of the humoral immune system in dengue virus infection. Results Epstein-Barr Virus (EBV) transformation of B cells isolated from the peripheral blood of a human subject with previous dengue infection was performed. B cell cultures were screened by ELISA for antibodies to dengue (DENV) envelope (E) protein. ELISA positive cultures were cloned by limiting dilution. Three IgG1 human monoclonal antibodies (HMAbs) were purified and their binding specificity to E protein was verified by ELISA and biolayer interferometry. Neutralization and enhancement assays were conducted in epithelial and macrophage-like cell lines, respectively. All three HMAbs bound to E from at least two of the four DENV serotypes, one of the HMAbs was neutralizing, and all were able to enhance DENV infection. Conclusions HMAbs against DENV can be successfully generated by EBV transformation of B cells from patients at least two years after naturally acquired DENV infections. These antibodies show different patterns of cross-reactivity, neutralizing, and enhancement activity. PMID:20132551

  12. Arenavirus Glycan Shield Promotes Neutralizing Antibody Evasion and Protracted Infection

    PubMed Central

    Malinge, Pauline; Magistrelli, Giovanni; Fischer, Nicolas; Sahin, Mehmet; Bergthaler, Andreas; Igonet, Sebastien; ter Meulen, Jan; Rigo, Dorothée; Meda, Paolo; Rabah, Nadia; Coutard, Bruno; Bowden, Thomas A.; Lambert, Paul-Henri; Siegrist, Claire-Anne; Pinschewer, Daniel D.

    2015-01-01

    Arenaviruses such as Lassa virus (LASV) can cause severe hemorrhagic fever in humans. As a major impediment to vaccine development, delayed and weak neutralizing antibody (nAb) responses represent a unifying characteristic of both natural infection and all vaccine candidates tested to date. To investigate the mechanisms underlying arenavirus nAb evasion we engineered several arenavirus envelope-chimeric viruses and glycan-deficient variants thereof. We performed neutralization tests with sera from experimentally infected mice and from LASV-convalescent human patients. NAb response kinetics in mice correlated inversely with the N-linked glycan density in the arenavirus envelope protein’s globular head. Additionally and most intriguingly, infection with fully glycosylated viruses elicited antibodies, which neutralized predominantly their glycan-deficient variants, both in mice and humans. Binding studies with monoclonal antibodies indicated that envelope glycans reduced nAb on-rate, occupancy and thereby counteracted virus neutralization. In infected mice, the envelope glycan shield promoted protracted viral infection by preventing its timely elimination by the ensuing antibody response. Thus, arenavirus envelope glycosylation impairs the protective efficacy rather than the induction of nAbs, and thereby prevents efficient antibody-mediated virus control. This immune evasion mechanism imposes limitations on antibody-based vaccination and convalescent serum therapy. PMID:26587982

  13. Arenavirus Glycan Shield Promotes Neutralizing Antibody Evasion and Protracted Infection.

    PubMed

    Sommerstein, Rami; Flatz, Lukas; Remy, Melissa M; Malinge, Pauline; Magistrelli, Giovanni; Fischer, Nicolas; Sahin, Mehmet; Bergthaler, Andreas; Igonet, Sebastien; Ter Meulen, Jan; Rigo, Dorothée; Meda, Paolo; Rabah, Nadia; Coutard, Bruno; Bowden, Thomas A; Lambert, Paul-Henri; Siegrist, Claire-Anne; Pinschewer, Daniel D

    2015-11-01

    Arenaviruses such as Lassa virus (LASV) can cause severe hemorrhagic fever in humans. As a major impediment to vaccine development, delayed and weak neutralizing antibody (nAb) responses represent a unifying characteristic of both natural infection and all vaccine candidates tested to date. To investigate the mechanisms underlying arenavirus nAb evasion we engineered several arenavirus envelope-chimeric viruses and glycan-deficient variants thereof. We performed neutralization tests with sera from experimentally infected mice and from LASV-convalescent human patients. NAb response kinetics in mice correlated inversely with the N-linked glycan density in the arenavirus envelope protein's globular head. Additionally and most intriguingly, infection with fully glycosylated viruses elicited antibodies, which neutralized predominantly their glycan-deficient variants, both in mice and humans. Binding studies with monoclonal antibodies indicated that envelope glycans reduced nAb on-rate, occupancy and thereby counteracted virus neutralization. In infected mice, the envelope glycan shield promoted protracted viral infection by preventing its timely elimination by the ensuing antibody response. Thus, arenavirus envelope glycosylation impairs the protective efficacy rather than the induction of nAbs, and thereby prevents efficient antibody-mediated virus control. This immune evasion mechanism imposes limitations on antibody-based vaccination and convalescent serum therapy. PMID:26587982

  14. Harnessing the protective potential of HIV-1 neutralizing antibodies

    PubMed Central

    Smith, S Abigail; Derdeyn, Cynthia A

    2016-01-01

    Recent biological, structural, and technical advances are converging within the HIV-1 vaccine field to harness the power of antibodies for prevention and therapy. Numerous monoclonal antibodies with broad neutralizing activity against diverse HIV-1 isolates have now been identified, revealing at least five sites of vulnerability on the envelope (Env) glycoproteins. While there are practical and technological barriers blocking a clear path from broadly neutralizing antibodies (bNAb) to a protective vaccine, this is not a dead end. Scientists are revisiting old approaches with new technology, cutting new trails through unexplored territory, and paving new roads in the hopes of preventing HIV-1 infection. Other promising avenues to capitalize on the power of bNAbs are also being pursued, such as passive antibody immunotherapy and gene therapy approaches. Moreover, non-neutralizing antibodies have inhibitory activities that could have protective potential, alone or in combination with bNAbs. With a new generation of bNAbs, and a clinical trial that associated antibodies with reduced acquisition, the field is closer than ever to developing strategies to use antibodies against HIV-1. PMID:26918160

  15. Characterization and epitope mapping of monoclonal antibodies raised against rat hepatitis E virus capsid protein: An evaluation of their neutralizing activity in a cell culture system.

    PubMed

    Kobayashi, Tominari; Takahashi, Masaharu; Tanggis; Mulyanto; Jirintai, Suljid; Nagashima, Shigeo; Nishizawa, Tsutomu; Okamoto, Hiroaki

    2016-07-01

    Hepatitis E virus (HEV) is the causative agent of acute hepatitis. Rat HEV is a recently discovered virus related to, but distinct from, human HEV. Since laboratory rats can be reproducibly infected with rat HEV and a cell culture system has been established for rat HEV, this virus may be used as a surrogate virus for human HEV, enabling studies on virus replication and mechanism of infection. However, monoclonal antibodies (MAbs) against rat HEV capsid (ORF2) protein are not available. In this study, 12 murine MAbs were generated against a recombinant ORF2 protein of rat HEV (rRatHEV-ORF2: amino acids 101-644) and were classified into at least six distinct groups by epitope mapping and a cross-reactivity analysis with human HEV ORF2 proteins. Two non-cross-reactive MAbs recognizing the protruding (P) domain detected both non-denatured and denatured rRatHEV-ORF2 protein and efficiently captured cell culture-produced rat HEV particles that had been treated with deoxycholate and trypsin, but not those without prior treatment. In addition, these two MAbs were able to efficiently neutralize replication of cell culture-generated rat HEV particles without lipid membranes (but not those with lipid membranes) in a cell culture system, similar to human HEV. PMID:26992654

  16. Serum virus neutralization assay for detection and quantitation of serum neutralizing antibodies to influenza A virus in swine

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The serum virus neutralization (SVN) assay is a serological test to detect the presence and magnitude of functional systemic antibodies that prevent infectivity of a virus. The SVN assay is a highly sensitive and specific test that may be applied to influenza A viruses (IAV) in swine to measure the ...

  17. Engineered Bispecific Antibodies with Exquisite HIV-1-Neutralizing Activity.

    PubMed

    Huang, Yaoxing; Yu, Jian; Lanzi, Anastasia; Yao, Xin; Andrews, Chasity D; Tsai, Lily; Gajjar, Mili R; Sun, Ming; Seaman, Michael S; Padte, Neal N; Ho, David D

    2016-06-16

    While the search for an efficacious HIV-1 vaccine remains elusive, emergence of a new generation of virus-neutralizing monoclonal antibodies (mAbs) has re-ignited the field of passive immunization for HIV-1 prevention. However, the plasticity of HIV-1 demands additional improvements to these mAbs to better ensure their clinical utility. Here, we report engineered bispecific antibodies that are the most potent and broad HIV-neutralizing antibodies to date. One bispecific antibody, 10E8V2.0/iMab, neutralized 118 HIV-1 pseudotyped viruses tested with a mean 50% inhibitory concentration (IC50) of 0.002 μg/mL. 10E8V2.0/iMab also potently neutralized 99% of viruses in a second panel of 200 HIV-1 isolates belonging to clade C, the dominant subtype accounting for ∼50% of new infections worldwide. Importantly, 10E8V2.0/iMab reduced virus load substantially in HIV-1-infected humanized mice and also provided complete protection when administered prior to virus challenge. These bispecific antibodies hold promise as novel prophylactic and/or therapeutic agents in the fight against HIV-1. PMID:27315479

  18. HIV-1 resistance to neutralizing antibodies: Determination of antibody concentrations leading to escape mutant evolution.

    PubMed

    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. PMID:26494166

  19. Anti-Semaphorin 3A neutralization monoclonal antibody prevents sepsis development in lipopolysaccharide-treated mice.

    PubMed

    Yamashita, Naoya; Jitsuki-Takahashi, Aoi; Ogawara, Miyuki; Ohkubo, Wataru; Araki, Tomomi; Hotta, Chie; Tamura, Tomohiko; Hashimoto, Shu-ichi; Yabuki, Takashi; Tsuji, Toru; Sasakura, Yukie; Okumura, Hiromi; Takaiwa, Aki; Koyama, Chika; Murakami, Koji; Goshima, Yoshio

    2015-09-01

    Semaphorin 3A (Sema3A), originally identified as a potent growth cone collapsing factor in developing sensory neurons, is now recognized as a key player in immune, cardiovascular, bone metabolism and neurological systems. Here we established an anti-Sema3A monoclonal antibody that neutralizes the effects of Sema3A both in vitro and in vivo. The anti-Sema3A neutralization chick IgM antibodies were screened by combining an autonomously diversifying library selection system and an in vitro growth cone collapse assay. We further developed function-blocking chick-mouse chimeric and humanized anti-Sema3A antibodies. We found that our anti-Sema3A antibodies were effective for improving the survival rate in lipopolysaccharide-induced sepsis in mice. Our antibody is a potential therapeutic agent that may prevent the onset of or alleviate symptoms of human diseases associated with Sema3A. PMID:25855660

  20. Redesigned HIV antibodies exhibit enhanced neutralizing potency and breadth.

    PubMed

    Willis, Jordan R; Sapparapu, Gopal; Murrell, Sasha; Julien, Jean-Philippe; Singh, Vidisha; King, Hannah G; Xia, Yan; Pickens, Jennifer A; LaBranche, Celia C; Slaughter, James C; Montefiori, David C; Wilson, Ian A; Meiler, Jens; Crowe, James E

    2015-06-01

    Several HIV envelope-targeting (Env-targeting) antibodies with broad and potent neutralizing activity have been identified and shown to have unusual features. Of these, the PG9 antibody has a long heavy chain complementarity determining region 3 (HCDR3) and possesses unique structural elements that interact with protein and glycan features of the HIV Env glycoprotein. Here, we used the Rosetta software suite to design variants of the PG9 antibody HCDR3 loop with the goal of identifying variants with increased potency and breadth of neutralization for diverse HIV strains. One variant, designated PG9_N100(F)Y, possessed increased potency and was able to neutralize a diverse set of PG9-resistant HIV strains, including those lacking the Env N160 glycan, which is critical for PG9 binding. An atomic resolution structure of the PG9_N100(F)Y fragment antigen binding (Fab) confirmed that the mutated residue retains the paratope surface when compared with WT PG9. Differential scanning calorimetry experiments revealed that the mutation caused a modest increase in thermodynamic stability of the Fab, a feature predicted by the computational model. Our findings suggest that thermodynamic stabilization of the long HCDR3 in its active conformation is responsible for the increased potency of PG9_N100(F)Y, and strategies aimed at stabilizing this region in other HIV antibodies could become an important approach to in silico optimization of antibodies. PMID:25985274

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

    PubMed

    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. PMID:26537503

  2. HIV Neutralizing Antibodies Induced by Native-like Envelope Trimers

    PubMed Central

    Sanders, Rogier W.; van Gils, Marit J.; Derking, Ronald; Sok, Devin; Ketas, Thomas J.; Burger, Judith A.; Ozorowski, Gabriel; Cupo, Albert; Simonich, Cassandra; Goo, Leslie; Arendt, Heather; Kim, Helen J.; Lee, Jeong Hyun; Pugach, Pavel; Williams, Melissa; Debnath, Gargi; Moldt, Brian; van Breemen, Mariëlle J.; Isik, Gözde; Medina-Ramírez, Max; Back, Jaap Willem; Koff, Wayne; Julien, Jean-Philippe; Rakasz, Eva G.; Seaman, Michael S.; Guttman, Miklos; Lee, Kelly K.; Klasse, Per Johan; LaBranche, Celia; Schief, William R.; Wilson, Ian A.; Overbaugh, Julie; Burton, Dennis R.; Ward, Andrew B.; Montefiori, David C.; Dean, Hansi; Moore, John P.

    2015-01-01

    A challenge for HIV-1 immunogen design is inducing neutralizing antibodies (NAbs) against neutralization-resistant (Tier-2) viruses that dominate human transmissions. We show that a soluble recombinant HIV-1 envelope glycoprotein trimer that adopts a native conformation (BG505 SOSIP.664) induced NAbs potently against the sequence-matched Tier-2 virus in rabbits and similar but weaker responses in macaques. The trimer also consistently induced cross-reactive NAbs against more sensitive (Tier-1) viruses. Tier-2 NAbs recognized conformational epitopes that differed between animals and in some cases overlapped with those recognized by broadly neutralizing antibodies (bNAbs), whereas Tier-1 responses targeted linear V3 epitopes. A second trimer, B41 SOSIP.664, also induced a strong autologous Tier-2 NAb response in rabbits. Thus, native-like trimers represent a promising starting point for developing HIV-1 vaccines aimed at inducing bNAbs. PMID:26089353

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

    PubMed

    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

  4. Mice with different susceptibility to tick-borne encephalitis virus infection show selective neutralizing antibody response and inflammatory reaction in the central nervous system

    PubMed Central

    2013-01-01

    Background The clinical course of tick-borne encephalitis (TBE), a disease caused by TBE virus, ranges from asymptomatic or mild influenza-like infection to severe debilitating encephalitis or encephalomyelitis. Despite the medical importance of this disease, some crucial steps in the development of encephalitis remain poorly understood. In particular, the basis of the disease severity is largely unknown. Methods TBE virus growth, neutralizing antibody response, key cytokine and chemokine mRNA production and changes in mRNA levels of cell surface markers of immunocompetent cells in brain were measured in mice with different susceptibilities to TBE virus infection. Results An animal model of TBE based on BALB/c-c-STS/A (CcS/Dem) recombinant congenic mouse strains showing different severities of the infection in relation to the host genetic background was developed. After subcutaneous inoculation of TBE virus, BALB/c mice showed medium susceptibility to the infection, STS mice were resistant, and CcS-11 mice were highly susceptible. The resistant STS mice showed lower and delayed viremia, lower virus production in the brain and low cytokine/chemokine mRNA production, but had a strong neutralizing antibody response. The most sensitive strain (CcS-11) failed in production of neutralizing antibodies, but exhibited strong cytokine/chemokine mRNA production in the brain. After intracerebral inoculation, all mouse strains were sensitive to the infection and had similar virus production in the brain, but STS mice survived significantly longer than CcS-11 mice. These two strains also differed in the expression of key cytokines/chemokines, particularly interferon gamma-induced protein 10 (IP-10/CXCL10) and monocyte chemotactic protein-1 (MCP-1/CCL2) in the brain. Conclusions Our data indicate that the genetic control is an important factor influencing the clinical course of TBE. High neutralizing antibody response might be crucial for preventing host fatality, but high

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

    PubMed

    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

    2015-04-01

    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. PMID:25581790

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

    PubMed Central

    Plotkin, Joshua B.

    2016-01-01

    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. PMID:27442127

  7. Pharmacokinetics and pharmacodynamics of VEGF-neutralizing antibodies

    PubMed Central

    2011-01-01

    Background Vascular endothelial growth factor (VEGF) is a potent regulator of angiogenesis, and its role in cancer biology has been widely studied. Many cancer therapies target angiogenesis, with a focus being on VEGF-mediated signaling such as antibodies to VEGF. However, it is difficult to predict the effects of VEGF-neutralizing agents. We have developed a whole-body model of VEGF kinetics and transport under pathological conditions (in the presence of breast tumor). The model includes two major VEGF isoforms VEGF121 and VEGF165, receptors VEGFR1, VEGFR2 and co-receptors Neuropilin-1 and Neuropilin-2. We have added receptors on parenchymal cells (muscle fibers and tumor cells), and incorporated experimental data for the cell surface density of receptors on the endothelial cells, myocytes, and tumor cells. The model is applied to investigate the action of VEGF-neutralizing agents (called "anti-VEGF") in the treatment of cancer. Results Through a sensitivity study, we examine how model parameters influence the level of free VEGF in the tumor, a measure of the response to VEGF-neutralizing drugs. We investigate the effects of systemic properties such as microvascular permeability and lymphatic flow, and of drug characteristics such as the clearance rate and binding affinity. We predict that increasing microvascular permeability in the tumor above 10-5 cm/s elicits the undesired effect of increasing tumor interstitial VEGF concentration beyond even the baseline level. We also examine the impact of the tumor microenvironment, including receptor expression and internalization, as well as VEGF secretion. We find that following anti-VEGF treatment, the concentration of free VEGF in the tumor can vary between 7 and 233 pM, with a dependence on both the density of VEGF receptors and co-receptors and the rate of neuropilin internalization on tumor cells. Finally, we predict that free VEGF in the tumor is reduced following anti-VEGF treatment when VEGF121 comprises at least

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

    SciTech Connect

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

    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.

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

    PubMed

    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. PMID:22896678

  10. Antibody-Mediated Neutralization of the Exotoxin Mycolactone, the Main Virulence Factor Produced by Mycobacterium ulcerans

    PubMed Central

    Gersbach, Philipp; Hug, Melanie N.; Bieri, Raphael; Bomio, Claudio; Li, Jun; Huber, Sylwia; Altmann, Karl-Heinz; Pluschke, Gerd

    2016-01-01

    Background Mycolactone, the macrolide exotoxin produced by Mycobacterium ulcerans, causes extensive tissue destruction by inducing apoptosis of host cells. In this study, we aimed at the production of antibodies that could neutralize the cytotoxic activities of mycolactone. Methodology/Principal Findings Using the B cell hybridoma technology, we generated a series of monoclonal antibodies with specificity for mycolactone from spleen cells of mice immunized with the protein conjugate of a truncated synthetic mycolactone derivative. L929 fibroblasts were used as a model system to investigate whether these antibodies can inhibit the biological effects of mycolactone. By measuring the metabolic activity of the fibroblasts, we found that anti-mycolactone mAbs can completely neutralize the cytotoxic activity of mycolactone. Conclusions/Significance The toxin neutralizing capacity of anti-mycolactone mAbs supports the concept of evaluating the macrolide toxin as vaccine target. PMID:27351976

  11. Heparanase-neutralizing antibodies attenuate lymphoma tumor growth and metastasis

    PubMed Central

    Weissmann, Marina; Arvatz, Gil; Horowitz, Netanel; Feld, Sari; Naroditsky, Inna; Zhang, Yi; Ng, Mary; Hammond, Edward; Nevo, Eviatar; Vlodavsky, Israel; Ilan, Neta

    2016-01-01

    Heparanase is an endoglycosidase that cleaves heparan sulfate side chains of proteoglycans, resulting in disassembly of the extracellular matrix underlying endothelial and epithelial cells and associating with enhanced cell invasion and metastasis. Heparanase expression is induced in carcinomas and sarcomas, often associating with enhanced tumor metastasis and poor prognosis. In contrast, the function of heparanase in hematological malignancies (except myeloma) was not investigated in depth. Here, we provide evidence that heparanase is expressed by human follicular and diffused non-Hodgkin's B-lymphomas, and that heparanase inhibitors restrain the growth of tumor xenografts produced by lymphoma cell lines. Furthermore, we describe, for the first time to our knowledge, the development and characterization of heparanase-neutralizing monoclonal antibodies that inhibit cell invasion and tumor metastasis, the hallmark of heparanase activity. Using luciferase-labeled Raji lymphoma cells, we show that the heparanase-neutralizing monoclonal antibodies profoundly inhibit tumor load in the mouse bones, associating with reduced cell proliferation and angiogenesis. Notably, we found that Raji cells lack intrinsic heparanase activity, but tumor xenografts produced by this cell line exhibit typical heparanase activity, likely contributed by host cells composing the tumor microenvironment. Thus, the neutralizing monoclonal antibodies attenuate lymphoma growth by targeting heparanase in the tumor microenvironment. PMID:26729870

  12. Development and characterization of human monoclonal antibodies that neutralize multiple TGFβ isoforms

    PubMed Central

    Bedinger, Daniel; Lao, Llewelyn; Khan, Shireen; Lee, Steve; Takeuchi, Toshihiko; Mirza, Amer M.

    2016-01-01

    ABSTRACT Transforming growth factor (TGF)β levels are elevated in, and drive the progression of, numerous disease states such as advanced metastatic cancer and systemic and ocular fibrosis. There are 3 main isoforms, TGFβ1, 2, and 3. As multiple TGFβ isoforms are involved in disease processes, maximal therapeutic efficacy may require neutralization of 2 or more of the TGFβ isoforms. Fully human antibody phage display libraries were used to discover a number of antibodies that bind and neutralize various combinations of TGFβ1, 2 or 3. The primary panning did not yield any uniformly potent pan-isoform neutralizing antibodies; therefore, an antibody that displayed potent TGFβ 1, 2 inhibition, but more modest affinity versus TGFβ3, was affinity matured by shuffling with a light chain sub-library and further screening. This process yielded a high affinity pan-isoform neutralizing clone. Antibodies were analyzed and compared by binding affinity, as well as receptor and epitope competition by surface plasmon resonance methods. The antibodies were also shown to neutralize TGFβ effects in vitro in 3 assays: 1) interleukin (IL)-4 induced HT-2 cell proliferation; 2) TGFβ-mediated IL-11 release by A549 cells; and 3) decreasing SMAD2 phosphorylation in Detroit 562 cells. The antibodies’ potency in these in vitro assays correlated well with their isoform-specific affinities. Furthermore, the ability of the affinity-matured clone to decrease tumor burden in a Detroit 562 xenograft study was superior to that of the parent clone. This affinity-matured antibody acts as a very potent inhibitor of all 3 main isoforms of TGFβ and may have utility for therapeutic intervention in human disease. PMID:26563652

  13. Robust Neutralizing Antibodies Elicited by HIV-1 JRFL Envelope Glycoprotein Trimers in Nonhuman Primates

    PubMed Central

    Feng, Yu; Sharma, Shailendra Kumar; McKee, Krisha; Karlsson Hedestam, Gunilla B.; LaBranche, Celia C.; Montefiori, David C.; Mascola, John R.

    2013-01-01

    Host cell-mediated proteolytic cleavage of the human immunodeficiency virus type 1 (HIV-1) gp160 precursor glycoprotein into gp120 and gp41 subunits is required to generate fusion-competent envelope glycoprotein (Env) spikes. The gp120-directed broadly neutralizing monoclonal antibodies (bNabs) isolated from HIV-infected individuals efficiently recognize fully cleaved JRFL Env spikes; however, nonneutralizing gp120-directed monoclonal antibodies isolated from infected or vaccinated subjects recognize only uncleaved JRFL spikes. Therefore, as an immunogen, cleaved spikes that selectively present desired neutralizing epitopes to B cells may elicit cross-reactive neutralizing antibodies. Accordingly, we inoculated nonhuman primates (NHPs) with plasmid DNA encoding transmembrane-anchored, cleaved JRFL Env or by electroporation (EP). Priming with DNA expressing soluble, uncleaved gp140 trimers was included as a comparative experimental group of NHPs. DNA inoculation was followed by boosts with soluble JRFL gp140 trimers, and control NHPs were inoculated with soluble JRFL protein trimers without DNA priming. In the TZM-bl assay, elicitation of neutralizing antibodies against HIV-1 tier 1 isolates was robust following the protein boost. Neutralization of tier 2 isolates was detected, but only in animals primed with plasmid DNA and boosted with trimeric protein. Using the more sensitive A3R5 assay, consistent neutralization of both clade B and C tier 2 isolates was detected from all regimens assessed in the current study, exceeding levels achieved by our previous vaccine regimens in primates. Together, these data suggest a potential advantage of B cell priming followed by a rest interval and protein boosting to present JRFL Env spikes to the immune system to better generate HIV-1 cross-clade neutralizing antibodies. PMID:24067980

  14. Mechanism of Neutralization by the Broadly Neutralizing HIV-1 Monoclonal Antibody VRC01▿†

    PubMed Central

    Li, Yuxing; O'Dell, Sijy; Walker, Laura M.; Wu, Xueling; Guenaga, Javier; Feng, Yu; Schmidt, Stephen D.; McKee, Krisha; Louder, Mark K.; Ledgerwood, Julie E.; Graham, Barney S.; Haynes, Barton F.; Burton, Dennis R.; Wyatt, Richard T.; Mascola, John R.

    2011-01-01

    The structure of VRC01 in complex with the HIV-1 gp120 core reveals that this broadly neutralizing CD4 binding site (CD4bs) antibody partially mimics the interaction of the primary virus receptor, CD4, with gp120. Here, we extended the investigation of the VRC01-gp120 core interaction to the biologically relevant viral spike to better understand the mechanism of VRC01-mediated neutralization and to define viral elements associated with neutralization resistance. In contrast to the interaction of CD4 or the CD4bs monoclonal antibody (MAb) b12 with the HIV-1 envelope glycoprotein (Env), occlusion of the VRC01 epitope by quaternary constraints was not a major factor limiting neutralization. Mutagenesis studies indicated that VRC01 contacts within the gp120 loop D, the CD4 binding loop, and the V5 region were necessary for optimal VRC01 neutralization, as suggested by the crystal structure. In contrast to interactions with the soluble gp120 monomer, VRC01 interaction with the native viral spike did not occur in a CD4-like manner; VRC01 did not induce gp120 shedding from the Env spike or enhance gp41 membrane proximal external region (MPER)-directed antibody binding to the Env spike. Finally, VRC01 did not display significant reactivity with human antigens, boding well for potential in vivo applications. The data indicate that VRC01 interacts with gp120 in the context of the functional spike in a manner distinct from that of CD4. It achieves potent neutralization by precisely targeting the CD4bs without requiring alterations of Env spike configuration and by avoiding steric constraints imposed by the quaternary structure of the functional Env spike. PMID:21715490

  15. Naturally enveloped AAV vectors for shielding neutralizing antibodies and robust gene delivery in vivo

    PubMed Central

    György, Bence; Fitzpatrick, Zachary; Crommentuijn, Matheus HW; Mu, Dakai; Maguire, Casey A.

    2014-01-01

    Recently adeno-associated virus (AAV) became the first clinically approved gene therapy product in the western world. To develop AAV for future clinical application in a widespread patient base, particularly in therapies which require intravenous (i.v.) administration of vector, the virus must be able to evade pre-existing antibodies to the wild type virus. Here we demonstrate that in mice, AAV vectors associated with extracellular vesicles (EVs) can evade human anti-AAV neutralizing antibodies. We observed different antibody evasion and gene transfer abilities with populations of EVs isolated by different centrifugal forces. EV-associated AAV vector (ev-AAV) was up to 136-fold more resistant over a range of neutralizing antibody concentrations relative to standard AAV vector in vitro. Importantly in mice, at a concentration of passively transferred human antibodies which decreased i.v. administered standard AAV transduction of brain by 80%, transduction of ev-AAV transduction was not reduced and was 4,000-fold higher. Finally, we show that expressing a brain targeting peptide on the EV surface allowed significant enhancement of transduction compared to untargeted ev-AAV. Using ev-AAV represents an effective, clinically relevant approach to evade human neutralizing anti-AAV antibodies after systemic administration of vector. PMID:24917028

  16. Structural basis for the antibody neutralization of Herpes simplex virus

    SciTech Connect

    Lee, Cheng-Chung; Lin, Li-Ling; Chan, Woan-Eng; Ko, Tzu-Ping; Lai, Jiann-Shiun; Wang, Andrew H.-J.

    2013-10-01

    The gD–E317-Fab complex crystal revealed the conformational epitope of human mAb E317 on HSV gD, providing a molecular basis for understanding the viral neutralization mechanism. Glycoprotein D (gD) of Herpes simplex virus (HSV) binds to a host cell surface receptor, which is required to trigger membrane fusion for virion entry into the host cell. gD has become a validated anti-HSV target for therapeutic antibody development. The highly inhibitory human monoclonal antibody E317 (mAb E317) was previously raised against HSV gD for viral neutralization. To understand the structural basis of antibody neutralization, crystals of the gD ectodomain bound to the E317 Fab domain were obtained. The structure of the complex reveals that E317 interacts with gD mainly through the heavy chain, which covers a large area for epitope recognition on gD, with a flexible N-terminal and C-terminal conformation. The epitope core structure maps to the external surface of gD, corresponding to the binding sites of two receptors, herpesvirus entry mediator (HVEM) and nectin-1, which mediate HSV infection. E317 directly recognizes the gD–nectin-1 interface and occludes the HVEM contact site of gD to block its binding to either receptor. The binding of E317 to gD also prohibits the formation of the N-terminal hairpin of gD for HVEM recognition. The major E317-binding site on gD overlaps with either the nectin-1-binding residues or the neutralizing antigenic sites identified thus far (Tyr38, Asp215, Arg222 and Phe223). The epitopes of gD for E317 binding are highly conserved between two types of human herpesvirus (HSV-1 and HSV-2). This study enables the virus-neutralizing epitopes to be correlated with the receptor-binding regions. The results further strengthen the previously demonstrated therapeutic and diagnostic potential of the E317 antibody.

  17. Anti-idiotypic antibodies induce neutralizing antibodies to bovine herpesvirus 1.

    PubMed Central

    Srikumaran, S; Onisk, D V; Borca, M V; Nataraj, C; Zamb, T J

    1990-01-01

    A neutralizing murine monoclonal antibody (mAb) of the IgG2a isotype (MM-113), specific for bovine herpesvirus 1 (BHV-1) glycoprotein gIV, was used to develop anti-idiotypic antibodies (anti-Id) in a calf. The bovine anti-Id were isolated from the serum of the immunized calf by affinity chromatography on an MM-113-Sepharose column, followed by repeated adsorption on a murine IgG2a column. The anti-Id thus obtained specifically reacted with MM-113, but not with isotype-matched controls. They also inhibited the binding of MM-113 to BHV-1 in a concentration-dependent manner. Mice immunized with the anti-Id produced neutralizing antibodies to BHV-1. The anti-Id bound to cells permissive to BHV-1 in a cell-binding radioimmunoassay (RIA). PMID:2165998

  18. Tailored Immunogens Direct Affinity Maturation toward HIV Neutralizing Antibodies.

    PubMed

    Briney, Bryan; Sok, Devin; Jardine, Joseph G; Kulp, Daniel W; Skog, Patrick; Menis, Sergey; Jacak, Ronald; Kalyuzhniy, Oleksandr; de Val, Natalia; Sesterhenn, Fabian; Le, Khoa M; Ramos, Alejandra; Jones, Meaghan; Saye-Francisco, Karen L; Blane, Tanya R; Spencer, Skye; Georgeson, Erik; Hu, Xiaozhen; Ozorowski, Gabriel; Adachi, Yumiko; Kubitz, Michael; Sarkar, Anita; Wilson, Ian A; Ward, Andrew B; Nemazee, David; Burton, Dennis R; Schief, William R

    2016-09-01

    Induction of broadly neutralizing antibodies (bnAbs) is a primary goal of HIV vaccine development. VRC01-class bnAbs are important vaccine leads because their precursor B cells targeted by an engineered priming immunogen are relatively common among humans. This priming immunogen has demonstrated the ability to initiate a bnAb response in animal models, but recall and maturation toward bnAb development has not been shown. Here, we report the development of boosting immunogens designed to guide the genetic and functional maturation of previously primed VRC01-class precursors. Boosting a transgenic mouse model expressing germline VRC01 heavy chains produced broad neutralization of near-native isolates (N276A) and weak neutralization of fully native HIV. Functional and genetic characteristics indicate that the boosted mAbs are consistent with partially mature VRC01-class antibodies and place them on a maturation trajectory that leads toward mature VRC01-class bnAbs. The results show how reductionist sequential immunization can guide maturation of HIV bnAb responses. PMID:27610570

  19. Broadly neutralizing antibodies abrogate established hepatitis C virus infection

    PubMed Central

    de Jong, Ype P.; Dorner, Marcus; Mommersteeg, Michiel C.; Xiao, Jing W.; Balazs, Alejandro B.; Robbins, Justin B.; Winer, Benjamin Y.; Gerges, Sherif; Vega, Kevin; Labitt, Rachael N.; Donovan, Bridget M.; Giang, Erick; Krishnan, Anuradha; Chiriboga, Luis; Charlton, Michael R.; Burton, Dennis R.; Baltimore, David; Law, Mansun; Rice, Charles M.; Ploss, Alexander

    2015-01-01

    Hepatitis C virus (HCV) establishes a chronic infection in the majority of exposed individuals and can cause cirrhosis and hepatocellular carcinoma. The role of antibodies directed against HCV in disease progression is poorly understood. Neutralizing antibodies (nAbs) can prevent HCV infection in vitro and in animal models. However, the effects of nAbs on an established HCV infection are unclear. Here, we demonstrate that three broadly nAbs, AR3A, AR3B and AR4A, delivered with adeno-associated viral (AAV) vectors can confer protection against viral challenge in humanized mice. Furthermore, we provide evidence that nAbs can abrogate an ongoing HCV infection in primary hepatocyte cultures and in a human liver chimeric mouse model. These results showcase a novel therapeutic approach to interfere with HCV infection exploiting a previously unappreciated need for HCV to continuously infect new hepatocytes in order to sustain chronicity. PMID:25232181

  20. Elimination of HIV-1-infected cells by broadly neutralizing antibodies

    PubMed Central

    Bruel, Timothée; Guivel-Benhassine, Florence; Amraoui, Sonia; Malbec, Marine; Richard, Léa; Bourdic, Katia; Donahue, Daniel Aaron; Lorin, Valérie; Casartelli, Nicoletta; Noël, Nicolas; Lambotte, Olivier; Mouquet, Hugo; Schwartz, Olivier

    2016-01-01

    The Fc region of HIV-1 Env-specific broadly neutralizing antibodies (bNAbs) is required for suppressing viraemia, through mechanisms which remain poorly understood. Here, we identify bNAbs that exert antibody-dependent cellular cytotoxicity (ADCC) in cell culture and kill HIV-1-infected lymphocytes through natural killer (NK) engagement. These antibodies target the CD4-binding site, the glycans/V3 and V1/V2 loops on gp120, or the gp41 moiety. The landscape of Env epitope exposure at the surface and the sensitivity of infected cells to ADCC vary considerably between viral strains. Efficient ADCC requires sustained cell surface binding of bNAbs to Env, and combining bNAbs allows a potent killing activity. Furthermore, reactivated infected cells from HIV-positive individuals expose heterogeneous Env epitope patterns, with levels that are often but not always sufficient to trigger killing by bNAbs. Our study delineates the parameters controlling ADCC activity of bNAbs, and supports the use of the most potent antibodies to clear the viral reservoir. PMID:26936020

  1. Elimination of HIV-1-infected cells by broadly neutralizing antibodies.

    PubMed

    Bruel, Timothée; Guivel-Benhassine, Florence; Amraoui, Sonia; Malbec, Marine; Richard, Léa; Bourdic, Katia; Donahue, Daniel Aaron; Lorin, Valérie; Casartelli, Nicoletta; Noël, Nicolas; Lambotte, Olivier; Mouquet, Hugo; Schwartz, Olivier

    2016-01-01

    The Fc region of HIV-1 Env-specific broadly neutralizing antibodies (bNAbs) is required for suppressing viraemia, through mechanisms which remain poorly understood. Here, we identify bNAbs that exert antibody-dependent cellular cytotoxicity (ADCC) in cell culture and kill HIV-1-infected lymphocytes through natural killer (NK) engagement. These antibodies target the CD4-binding site, the glycans/V3 and V1/V2 loops on gp120, or the gp41 moiety. The landscape of Env epitope exposure at the surface and the sensitivity of infected cells to ADCC vary considerably between viral strains. Efficient ADCC requires sustained cell surface binding of bNAbs to Env, and combining bNAbs allows a potent killing activity. Furthermore, reactivated infected cells from HIV-positive individuals expose heterogeneous Env epitope patterns, with levels that are often but not always sufficient to trigger killing by bNAbs. Our study delineates the parameters controlling ADCC activity of bNAbs, and supports the use of the most potent antibodies to clear the viral reservoir. PMID:26936020

  2. Defensins Potentiate a Neutralizing Antibody Response to Enteric Viral Infection

    PubMed Central

    Treuting, Piper M.; Bromme, Beth A.; Wilson, Sarah S.; Wiens, Mayim E.; Lu, Wuyuan; Ouellette, André J.; Spindler, Katherine R.; Parks, William C.; Smith, Jason G.

    2016-01-01

    α-defensins are abundant antimicrobial peptides with broad, potent antibacterial, antifungal, and antiviral activities in vitro. Although their contribution to host defense against bacteria in vivo has been demonstrated, comparable studies of their antiviral activity in vivo are lacking. Using a mouse model deficient in activated α-defensins in the small intestine, we show that Paneth cell α-defensins protect mice from oral infection by a pathogenic virus, mouse adenovirus 1 (MAdV-1). Survival differences between mouse genotypes are lost upon parenteral MAdV-1 infection, strongly implicating a role for intestinal defenses in attenuating pathogenesis. Although differences in α-defensin expression impact the composition of the ileal commensal bacterial population, depletion studies using broad-spectrum antibiotics revealed no effect of the microbiota on α-defensin-dependent viral pathogenesis. Moreover, despite the sensitivity of MAdV-1 infection to α-defensin neutralization in cell culture, we observed no barrier effect due to Paneth cell α-defensin activation on the kinetics and magnitude of MAdV-1 dissemination to the brain. Rather, a protective neutralizing antibody response was delayed in the absence of α-defensins. This effect was specific to oral viral infection, because antibody responses to parenteral or mucosal ovalbumin exposure were not affected by α-defensin deficiency. Thus, α-defensins play an important role as adjuvants in antiviral immunity in vivo that is distinct from their direct antiviral activity observed in cell culture. PMID:26933888

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

    PubMed

    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. PMID:23178532

  4. Development of a poliovirus neutralization test with poliovirus pseudovirus for measurement of neutralizing antibody titer in human serum.

    PubMed

    Arita, Minetaro; Iwai, Masae; Wakita, Takaji; Shimizu, Hiroyuki

    2011-11-01

    In the Global Polio Eradication Initiative, laboratory diagnosis plays a critical role by isolating and identifying poliovirus (PV) from the stool samples from acute flaccid paralysis (AFP) cases. In recent years, reestablishment of PV circulation in countries where PV was previously eliminated has occurred because of decreased herd immunity, possibly due to poor vaccination coverage. To monitor the vulnerability of countries to PV circulation, surveillance of neutralizing-antibody titers against PV in susceptible populations is essential in the end game of the polio eradication program. In this study, we have developed a PV neutralization test with type 1, 2, and 3 PV pseudoviruses to determine the neutralizing-antibody titer against PV in human serum samples. With this test, the neutralizing-antibody titer against PV could be determined within 2 days by automated interpretation of luciferase signals without using infectious PV strains. We validated the pseudovirus PV neutralization test with 131 human serum samples collected from a wide range of age groups (ages 1 to >60 years) by comparison with a conventional neutralization test. We found good correlation in the neutralizing-antibody titers determined by these tests. These results suggest that a pseudovirus PV neutralization test would serve as a safe and simple procedure for the measurement of the neutralizing-antibody titer against PV. PMID:21880850

  5. Progress on the induction of neutralizing antibodies against HIV-1

    PubMed Central

    Vaine, Michael; Lu, Shan; Wang, Shixia

    2013-01-01

    The Human Immunodeficiency Virus Type -1 (HIV-1), the causative agent of AIDS in humans, is one of the most catastrophic pandemics to affect human health care in the latter 20th century. The best hope of controlling this pandemic is the development of a successful prophylactic vaccine. However, to date, this goal has proven to be exceptionally elusive. The recent failure of an experimental AIDS vaccine in a phase IIb study named the STEP trial, intended to solely elicit cell mediated immune responses against HIV-1, has highlighted the need for a balanced immune response consisting of not only cellular immunity but also a broad and potent antibody response which can prevent the infection of HIV-1. This article will review the efforts being made up to this point to elicit such antibody responses, especially with regards to the use of a DNA prime-protein boost regimen which has been proven to be a highly effective platform for the induction of neutralizing antibodies in both animal and early phase human studies. PMID:19627166

  6. Antibody-mediated neutralization of African swine fever virus: myths and facts.

    PubMed

    Escribano, José M; Galindo, Inmaculada; Alonso, Covadonga

    2013-04-01

    Almost all viruses can be neutralized by antibodies. However, there is some controversy about antibody-mediated neutralization of African swine fever virus (ASFV) with sera from convalescent pigs and about the protective relevance of antibodies in experimentally vaccinated pigs. At present, there is no vaccine available for this highly lethal and economically relevant virus and all classical attempts to generate a vaccine have been unsuccessful. This failure has been attributed, in part, to what many authors describe as the absence of neutralizing antibodies. The findings of some studies clearly contradict the paradigm of the impossibility to neutralize ASFV by means of monoclonal or polyclonal antibodies. This review discusses scientific evidence of these types of antibodies in convalescent and experimentally immunized animals, the nature of their specificity, the neutralization-mediated mechanisms demonstrated, and the potential relevance of antibodies in protection. PMID:23159730

  7. Anti-tick-borne encephalitis (TBE) virus neutralizing antibodies dynamics in natural infections versus vaccination.

    PubMed

    Remoli, Maria Elena; Marchi, Antonella; Fortuna, Claudia; Benedetti, Eleonora; Minelli, Giada; Fiorentini, Cristiano; Mel, Rosanna; Venturi, Giulietta; Ciufolini, Maria Grazia

    2015-03-01

    Tick-borne encephalitis (TBE) virus infection elicits a life-long lasting protection. However, little is known about the neutralizing antibodies titres following natural infection. In this study, subjects with past TBE disease (n = 62) were analysed for the presence and titre of anti-TBE neutralizing antibodies, and compared with a vaccinated cohort (n = 101). Neutralizing antibody titres were higher in individuals with past TBE and did not show an age-dependent decrease when compared with vaccinees. PMID:25722483

  8. Neutralizing determinants defined by monoclonal antibodies on polypeptides specified by bovine herpesvirus 1.

    PubMed Central

    Collins, J K; Butcher, A C; Riegel, C A; McGrane, V; Blair, C D; Teramoto, Y A; Winston, S

    1984-01-01

    Monoclonal antibodies were used to study neutralizing determinants on polypeptides of bovine herpesvirus 1. Two of three monoclonal antibodies which recognized nonoverlapping epitopes on a glycoprotein of 82,000 daltons were found to neutralize. A second group of monoclonal antibodies that individually precipitated five viral glycopolypeptides ranging in size from 102,000 to 55,000 daltons also neutralized. Two monoclonal antibodies which were the most efficient in neutralization recognized a non-glycosylated protein of 115,000 daltons which was the major polypeptide on the virus. A fourth group of monoclonal antibodies precipitated a non-glycosylated polypeptide of 91,000 daltons and several smaller polypeptides, but these antibodies demonstrated only limited neutralizing activity. Images PMID:6208375

  9. A Neutralizing Antibody Assay Based on a Reporter of Antibody-Dependent Cell-Mediated Cytotoxicity.

    PubMed

    Wu, Yuling; Li, Jia J; Kim, Hyun Jun; Liu, Xu; Liu, Weiyi; Akhgar, Ahmad; Bowen, Michael A; Spitz, Susan; Jiang, Xu-Rong; Roskos, Lorin K; White, Wendy I

    2015-11-01

    Benralizumab is a humanized anti-IL5 receptor α (IL5Rα) monoclonal antibody (mAb) with enhanced (afucosylation) antibody-dependent cell-mediated cytotoxicity (ADCC) function. An ADCC reporter cell-based neutralizing antibody (NAb) assay was developed and characterized to detect NAb against benralizumab in human serum to support the clinical development of benralizumab. The optimal ratio of target cells to effector cells was 3:1. Neither parental benralizumab (fucosylated) nor benralizumab Fab resulted in ADCC activity, confirming the requirement for ADCC activity in the NAb assay. The serum tolerance of the cells was determined to be 2.5%. The cut point derived from normal and asthma serum samples was comparable. The effective range of benralizumab was determined, and 35 ng/mL [80% maximal effective concentration (EC80)] was chosen as the standard concentration to run in the assessment of NAb. An affinity purified goat anti-benralizumab polyclonal idiotype antibody preparation was shown to have NAb since it inhibited ADCC activity in a dose-dependent fashion. The low endogenous concentrations of IL5 and soluble IL5 receptor (sIL5R) did not demonstrate to interfere with the assay. The estimated assay sensitivities at the cut point were 1.02 and 1.10 μg/mL as determined by the surrogate neutralizing goat polyclonal and mouse monoclonal anti-drug antibody (ADA) controls, respectively. The assay can detect NAb (at 2.5 μg/mL) in the presence of 0.78 μg/mL benralizumab. The assay was not susceptible to non-specific matrix effects. This study provides an approach and feasibility of developing an ADCC cell-based NAb assay to support biopharmaceuticals with an ADCC function. PMID:26205082

  10. Potent neutralizing monoclonal antibodies against Ebola virus infection.

    PubMed

    Zhang, Qi; Gui, Miao; Niu, Xuefeng; He, Shihua; Wang, Ruoke; Feng, Yupeng; Kroeker, Andrea; Zuo, Yanan; Wang, Hua; Wang, Ying; Li, Jiade; Li, Chufang; Shi, Yi; Shi, Xuanling; Gao, George F; Xiang, Ye; Qiu, Xiangguo; Chen, Ling; Zhang, Linqi

    2016-01-01

    Ebola virus infections cause a deadly hemorrhagic disease for which no vaccines or therapeutics has received regulatory approval. Here we show isolation of three (Q206, Q314 and Q411) neutralizing monoclonal antibodies (mAbs) against the surface glycoprotein (GP) of Ebola virus identified in West Africa in 2014 through sequential immunization of Chinese rhesus macaques and antigen-specific single B cell sorting. These mAbs demonstrated potent neutralizing activities against both pseudo and live Ebola virus independent of complement. Biochemical, single particle EM, and mutagenesis analysis suggested Q206 and Q411 recognized novel epitopes in the head while Q314 targeted the glycan cap in the GP1 subunit. Q206 and Q411 appeared to influence GP binding to its receptor NPC1. Treatment with these mAbs provided partial but significant protection against disease in a mouse model of Ebola virus infection. These novel mAbs could serve as promising candidates for prophylactic and therapeutic interventions against Ebola virus infection. PMID:27181584

  11. Potent neutralizing monoclonal antibodies against Ebola virus infection

    PubMed Central

    Zhang, Qi; Gui, Miao; Niu, Xuefeng; He, Shihua; Wang, Ruoke; Feng, Yupeng; Kroeker, Andrea; Zuo, Yanan; Wang, Hua; Wang, Ying; Li, Jiade; Li, Chufang; Shi, Yi; Shi, Xuanling; Gao, George F.; Xiang, Ye; Qiu, Xiangguo; Chen, Ling; Zhang, Linqi

    2016-01-01

    Ebola virus infections cause a deadly hemorrhagic disease for which no vaccines or therapeutics has received regulatory approval. Here we show isolation of three (Q206, Q314 and Q411) neutralizing monoclonal antibodies (mAbs) against the surface glycoprotein (GP) of Ebola virus identified in West Africa in 2014 through sequential immunization of Chinese rhesus macaques and antigen-specific single B cell sorting. These mAbs demonstrated potent neutralizing activities against both pseudo and live Ebola virus independent of complement. Biochemical, single particle EM, and mutagenesis analysis suggested Q206 and Q411 recognized novel epitopes in the head while Q314 targeted the glycan cap in the GP1 subunit. Q206 and Q411 appeared to influence GP binding to its receptor NPC1. Treatment with these mAbs provided partial but significant protection against disease in a mouse model of Ebola virus infection. These novel mAbs could serve as promising candidates for prophylactic and therapeutic interventions against Ebola virus infection. PMID:27181584

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

    PubMed

    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

  13. Sub-Domains of Ricin’s B Subunit as Targets of Toxin Neutralizing and Non-Neutralizing Monoclonal Antibodies

    PubMed Central

    Yermakova, Anastasiya; Vance, David J.; Mantis, Nicholas J.

    2012-01-01

    The B subunit (RTB) of ricin toxin is a galactose (Gal)−/N-acetylgalactosamine (GalNac)-specific lectin that mediates attachment, entry, and intracellular trafficking of ricin in host cells. Structurally, RTB consists of two globular domains with identical folding topologies. Domains 1 and 2 are each comprised of three homologous sub-domains (α, β, γ) that likely arose by gene duplication from a primordial carbohydrate recognition domain (CRD), although only sub-domains 1α and 2γ retain functional lectin activity. As part of our ongoing effort to generate a comprehensive B cell epitope map of ricin, we report the characterization of three new RTB-specific monoclonal antibodies (mAbs). All three mAbs, JB4, B/J F9 and C/M A2, were initially identified based on their abilities to neutralize ricin in a Vero cell cytotoxicty assay and to partially (or completely) block ricin attachment to cell surfaces. However, only JB4 proved capable of neutralizing ricin in a macrophage apoptosis assay and in imparting passive immunity to mice in a model of systemic intoxication. Using a combination of techniques, including competitive ELISAs, pepscan analysis, differential reactivity by Western blot, as well as affinity enrichment of phage displayed peptides, we tentatively localized the epitopes recognized by the non-neutralizing mAbs B/J F9 and C/M A2 to sub-domains 2α and 2β, respectively. Furthermore, we propose that the epitope recognized by JB4 is within sub-domain 2γ, adjacent to RTB’s high affinity Gal/GalNAc CRD. These data suggest that recognition of RTB’s sub-domains 1α and 2γ are critical determinants of antibody neutralizing activity and protective immunity to ricin. PMID:22984492

  14. A Glycoprotein Subunit Vaccine Elicits a Strong Rift Valley Fever Virus Neutralizing Antibody Response in Sheep

    PubMed Central

    Lebedev, Maxim; McVey, D. Scott; Wilson, William; Morozov, Igor; Young, Alan

    2014-01-01

    Abstract Rift Valley fever virus (RVFV), a member of the Bunyaviridae family, is a mosquito-borne zoonotic pathogen that causes serious morbidity and mortality in livestock and humans. The recent spread of the virus beyond its traditional endemic boundaries in Africa to the Arabian Peninsula coupled with the presence of susceptible vectors in nonendemic countries has created increased interest in RVF vaccines. Subunit vaccines composed of specific virus proteins expressed in eukaryotic or prokaryotic expression systems are shown to elicit neutralizing antibodies in susceptible hosts. RVFV structural proteins, amino-terminus glycoprotein (Gn), and carboxyl-terminus glycoprotein (Gc), were expressed using a recombinant baculovirus expression system. The recombinant proteins were reconstituted as a GnGc subunit vaccine formulation and evaluated for immunogenicity in a target species, sheep. Six sheep were each immunized with a primary dose of 50 μg of each vaccine immunogen with the adjuvant montanide ISA25; at day 21, postvaccination, each animal received a second dose of the same vaccine. The vaccine induced a strong antibody response in all animals as determined by indirect enzyme-linked immunosorbent assay (ELISA). A plaque reduction neutralization test (PRNT80) showed the primary dose of the vaccine was sufficient to elicit potentially protective virus neutralizing antibody titers ranging from 40 to 160, and the second vaccine dose boosted the titer to more than 1280. Furthermore, all animals tested positive for neutralizing antibodies at day 328 postvaccination. ELISA analysis using the recombinant nucleocapsid protein as a negative marker antigen indicated that the vaccine candidate is DIVA (differentiating infected from vaccinated animals) compatible and represents a promising vaccine platform for RVFV infection in susceptible species. PMID:25325319

  15. A novel human anti-interleukin-1β neutralizing monoclonal antibody showing in vivo efficacy

    PubMed Central

    Goh, Angeline XH; Bertin-Maghit, Sebastien; Ping Yeo, Siok; Ho, Adrian; Derks, Heidi; Mortellaro, Alessandra; Wang, Cheng-I

    2014-01-01

    The pro-inflammatory cytokine interleukin (IL)-1β is a clinical target in many conditions involving dysregulation of the immune system; therapeutics that block IL-1β have been approved to treat diseases such as rheumatoid arthritis (RA), neonatal onset multisystem inflammatory diseases, cryopyrin-associated periodic syndromes, active systemic juvenile idiopathic arthritis. Here, we report the generation and engineering of a new fully human antibody that binds tightly to IL-1β with a neutralization potency more than 10 times higher than that of the marketed antibody canakinumab. After affinity maturation, the derived antibody shows a >30-fold increased affinity to human IL-1β compared with its parent antibody. This anti-human IL-1β IgG also cross-reacts with mouse and monkey IL-1β, hence facilitating preclinical development. In a number of mouse models, this antibody efficiently reduced or abolished signs of disease associated with IL-1β pathology. Due to its high affinity for the cytokine and its potency both in vitro and in vivo, we propose that this novel fully human anti-IL-1β monoclonal antibody is a promising therapeutic candidate and a potential alternative to the current therapeutic arsenal. PMID:24671001

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

    PubMed Central

    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; Kaminga, Gift; Louder, Mark K.; Tran, Lillan M.; Chen, Yue; Cai, Fangping; Chen, Sheri; Moquin, Stephanie; Du, Xiulian; Joyce, Gordon M.; 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.

    2013-01-01

    Current HIV-1 vaccines elicit strain-specific neutralizing antibodies. However, cross-reactive neutralizing antibodies arise in ~20% of HIV-1-infected individuals, and details of their generation could provide a roadmap for effective vaccination. Here we report the isolation, evolution and structure of a broadly neutralizing antibody from an African donor followed from time of infection. The mature antibody, CH103, neutralized ~55% of HIV-1 isolates, and its co-crystal structure with gp120 revealed a novel loop-based mechanism of CD4-binding site recognition. Virus and antibody gene sequencing revealed concomitant virus evolution and antibody maturation. Notably, the CH103-lineage unmutated common ancestor 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 elucidate 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 via vaccination. PMID:23552890

  17. Cross-neutralizing human anti-poliovirus antibodies bind the recognition site for cellular receptor

    PubMed Central

    Chen, Zhaochun; Fischer, Elizabeth R.; Kouiavskaia, Diana; Hansen, Bryan T.; Ludtke, Steven J.; Bidzhieva, Bella; Makiya, Michelle; Agulto, Liane; Purcell, Robert H.; Chumakov, Konstantin

    2013-01-01

    Most structural information about poliovirus interaction with neutralizing antibodies was obtained in the 1980s in studies of mouse monoclonal antibodies. Recently we have isolated a number of human/chimpanzee anti-poliovirus antibodies and demonstrated that one of them, MAb A12, could neutralize polioviruses of both serotypes 1 and 2. This communication presents data on isolation of an additional cross-neutralizing antibody (F12) and identification of a previously unknown epitope on the surface of poliovirus virions. Epitope mapping was performed by sequencing of antibody-resistant mutants and by cryo-EM of complexes of virions with Fab fragments. The results have demonstrated that both cross-neutralizing antibodies bind the site located at the bottom of the canyon surrounding the fivefold axis of symmetry that was previously shown to interact with cellular poliovirus receptor CD155. However, the same antibody binds to serotypes 1 and 2 through different specific interactions. It was also shown to interact with type 3 poliovirus, albeit with about 10-fold lower affinity, insufficient for effective neutralization. Antibody interaction with the binding site of the cellular receptor may explain its broad reactivity and suggest that further screening or antibody engineering could lead to a universal antibody capable of neutralizing all three serotypes of poliovirus. PMID:24277851

  18. Flexibility in Surface-Exposed Loops in a Virus Capsid Mediates Escape from Antibody Neutralization

    PubMed Central

    Kolawole, Abimbola O.; Li, Ming; Xia, Chunsheng; Fischer, Audrey E.; Giacobbi, Nicholas S.; Rippinger, Christine M.; Proescher, Jody B. G.; Wu, Susan K.; Bessling, Seneca L.; Gamez, Monica; Yu, Chenchen; Zhang, Rebecca; Mehoke, Thomas S.; Pipas, James M.; Wolfe, Joshua T.; Lin, Jeffrey S.; Feldman, Andrew B.

    2014-01-01

    ABSTRACT New human norovirus strains emerge every 2 to 3 years, partly due to mutations in the viral capsid that allow escape from antibody neutralization and herd immunity. To understand how noroviruses evolve antibody resistance, we investigated the structural basis for the escape of murine norovirus (MNV) from antibody neutralization. To identify specific residues in the MNV-1 protruding (P) domain of the capsid that play a role in escape from the neutralizing monoclonal antibody (MAb) A6.2, 22 recombinant MNVs were generated with amino acid substitutions in the A′B′ and E′F′ loops. Six mutations in the E′F′ loop (V378F, A382K, A382P, A382R, D385G, and L386F) mediated escape from MAb A6.2 neutralization. To elucidate underlying structural mechanisms for these results, the atomic structure of the A6.2 Fab was determined and fitted into the previously generated pseudoatomic model of the A6.2 Fab/MNV-1 virion complex. Previously, two distinct conformations, A and B, of the atomic structures of the MNV-1 P domain were identified due to flexibility in the two P domain loops. A superior stereochemical fit of the A6.2 Fab to the A conformation of the MNV P domain was observed. Structural analysis of our observed escape mutants indicates changes toward the less-preferred B conformation of the P domain. The shift in the structural equilibrium of the P domain toward the conformation with poor structural complementarity to the antibody strongly supports a unique mechanism for antibody escape that occurs via antigen flexibility instead of direct antibody-antigen binding. IMPORTANCE Human noroviruses cause the majority of all nonbacterial gastroenteritis worldwide. New epidemic strains arise in part by mutations in the viral capsid leading to escape from antibody neutralization. Herein, we identify a series of point mutations in a norovirus capsid that mediate escape from antibody neutralization and determine the structure of a neutralizing antibody. Fitting of

  19. Passive neutralizing antibody controls SHIV viremia and enhances B cell responses in infant macaques

    PubMed Central

    Ng, Cherie T.; Jaworski, J. Pablo; Jayaraman, Pushpa; Sutton, William F.; Delio, Patrick; Kuller, LaRene; Anderson, David; Landucci, Gary; Richardson, Barbra A.; Burton, Dennis R.; Forthal, Donald N.; Haigwood, Nancy L.

    2010-01-01

    Maternal HIV-1-specific antibodies are efficiently transferred to newborns; their role in disease control is unknown. We administered non-sterilizing levels of neutralizing IgG, including the human neutralizing monoclonal IgG1b12, to six newborn macaques before oral challenge with SHIVSF612P3. All rapidly developed neutralizing antibodies and had significantly reduced plasma viremia for 6 months. These studies support the use of neutralizing antibodies in enhancing B cell responses and viral control in perinatal settings. PMID:20890292

  20. Both Neutralizing and Non-Neutralizing Human H7N9 Influenza Vaccine-Induced Monoclonal Antibodies Confer Protection.

    PubMed

    Henry Dunand, Carole J; Leon, Paul E; Huang, Min; Choi, Angela; Chromikova, Veronika; Ho, Irvin Y; Tan, Gene S; Cruz, John; Hirsh, Ariana; Zheng, Nai-Ying; Mullarkey, Caitlin E; Ennis, Francis A; Terajima, Masanori; Treanor, John J; Topham, David J; Subbarao, Kanta; Palese, Peter; Krammer, Florian; Wilson, Patrick C

    2016-06-01

    Pathogenic H7N9 avian influenza viruses continue to represent a public health concern, and several candidate vaccines are currently being developed. It is vital to assess if protective antibodies are induced following vaccination and to characterize the diversity of epitopes targeted. Here we characterized the binding and functional properties of twelve H7-reactive human antibodies induced by a candidate A/Anhui/1/2013 (H7N9) vaccine. Both neutralizing and non-neutralizing antibodies protected mice in vivo during passive transfer challenge experiments. Mapping the H7 hemagglutinin antigenic sites by generating escape mutant variants against the neutralizing antibodies identified unique epitopes on the head and stalk domains. Further, the broadly cross-reactive non-neutralizing antibodies generated in this study were protective through Fc-mediated effector cell recruitment. These findings reveal important properties of vaccine-induced antibodies and provide a better understanding of the human monoclonal antibody response to influenza in the context of vaccines. PMID:27281570

  1. Strain-Specific V3 and CD4 Binding Site Autologous HIV-1 Neutralizing Antibodies Select Neutralization-Resistant Viruses

    SciTech Connect

    Moody, M.  Anthony; Gao, Feng; Gurley, Thaddeus  C.; Amos, Joshua  D.; Kumar, Amit; Hora, Bhavna; Marshall, Dawn  J.; Whitesides, John  F.; Xia, Shi-Mao; Parks, Robert; Lloyd, Krissey  E.; Hwang, Kwan-Ki; Lu, Xiaozhi; Bonsignori, Mattia; Finzi, Andrés; Vandergrift, Nathan  A.; Alam, S.  Munir; Ferrari, Guido; Shen, Xiaoying; Tomaras, Georgia  D.; Kamanga, Gift; Cohen, Myron  S.; Sam, Noel  E.; Kapiga, Saidi; Gray, Elin S.; Tumba, Nancy  L.; Morris, Lynn; Zolla-Pazner, Susan; Gorny, Miroslaw  K.; Mascola, John  R.; Hahn, Beatrice H.; Shaw, George  M.; Sodroski, Joseph  G.; Liao, Hua-Xin; Montefiori, David C.; Hraber, Peter T.; Korber, Bette T.; Haynes, Barton F.

    2015-09-09

    The third variable (V3) loop and the CD4 binding site (CD4bs) of the viral envelope are frequently targeted by neutralizing antibodies (nAbs) in HIV-1-infected individuals. In chronic infection, virus escape mutants repopulate the plasma and V3 and CD4bs nAbs emerge that can neutralize heterologous tier 1 easy-to-neutralize, but not tier 2 difficult-to-neutralize HIV-1 isolates. However, neutralization sensitivity of autologous plasma viruses to this type of nAb response has not been studied. We describe the development and evolution in vivo of antibodies distinguished by their target specificity for V3and CD4bs epitopes on autologous tier 2 viruses but not on heterologous tier 2 viruses. A surprisingly high fraction of autologous circulating viruses was sensitive to these antibodies. These findings demonstrate a role for V3 and CD4bs antibodies in constraining the native envelope trimer in vivo to a neutralization-resistant phenotype, explaining why HIV-1 transmission generally occurs by tier 2 neutralization-resistant viruses.

  2. Strain-Specific V3 and CD4 Binding Site Autologous HIV-1 Neutralizing Antibodies Select Neutralization-Resistant Viruses

    DOE PAGESBeta

    Moody, M.  Anthony; Gao, Feng; Gurley, Thaddeus  C.; Amos, Joshua  D.; Kumar, Amit; Hora, Bhavna; Marshall, Dawn  J.; Whitesides, John  F.; Xia, Shi-Mao; Parks, Robert; et al

    2015-09-09

    The third variable (V3) loop and the CD4 binding site (CD4bs) of the viral envelope are frequently targeted by neutralizing antibodies (nAbs) in HIV-1-infected individuals. In chronic infection, virus escape mutants repopulate the plasma and V3 and CD4bs nAbs emerge that can neutralize heterologous tier 1 easy-to-neutralize, but not tier 2 difficult-to-neutralize HIV-1 isolates. However, neutralization sensitivity of autologous plasma viruses to this type of nAb response has not been studied. We describe the development and evolution in vivo of antibodies distinguished by their target specificity for V3and CD4bs epitopes on autologous tier 2 viruses but not on heterologous tiermore » 2 viruses. A surprisingly high fraction of autologous circulating viruses was sensitive to these antibodies. These findings demonstrate a role for V3 and CD4bs antibodies in constraining the native envelope trimer in vivo to a neutralization-resistant phenotype, explaining why HIV-1 transmission generally occurs by tier 2 neutralization-resistant viruses.« less

  3. Antibodies to the neutral glycolipid asialo ganglio-N-tetraosylceramide: association with gynecologic cancers.

    PubMed

    Witkin, S S; Bongiovanni, A M; Birnbaum, S; Caputo, T; Ledger, W J

    1985-03-01

    As part of our efforts to define subpopulations at increased risk for gynecologic malignancies, sera from 145 women were obtained prior to diagnosis and analyzed for antibody to asialo ganglio-N-tetraosylceramide. This neutral glycolipid is present on the surface of thymocytes and natural killer cells, and asialo ganglio-N-tetraosylceramide antibody has been shown in animals to block natural killer cell activity and promote tumor cell proliferation. With the use of an enzyme-linked immunosorbent assay and with a value of 2 SD above the mean for healthy women designated as the boundary for a positive response, antibody to asialo ganglio-N-tetraosylceramide was detected in only one of 30 (3%) healthy women, none of 16 pregnant women, none of 18 women with benign masses, and two of 24 (8%) women with microbial infections. All of the above samples that contained antibodies were barely over the 2 SD limit. In marked contrast, 19 of 35 (54%) women with gynecologic malignancies had asialo ganglio-N-tetraosylceramide antibodies, with positive values ranging to greater than 10 SD above the control mean. Asialo ganglio-N-tetraosylceramide antibody was found in six of eight (75%) patients with cervical cancer, five of eight (63%) with endometrial cancer, and seven of 15 (47%) with ovarian cancer. Of the eight patients with Stage I gynecologic cancer at any site, five (62%) had asialo ganglio-N-tetraosylceramide antibodies. Four of 22 (18%) women with Hodgkin's disease also had antibodies, with values just exceeding 2 SD above control levels. The presence of these antibodies may contribute to an impaired immune surveillance system in these women and so increase their susceptibility to malignancy. PMID:3976767

  4. Bispecific Antibodies Targeting Different Epitopes on the HIV-1 Envelope Exhibit Broad and Potent Neutralization

    PubMed Central

    Asokan, M.; Rudicell, R. S.; Louder, M.; McKee, K.; O'Dell, S.; Stewart-Jones, G.; Wang, K.; Xu, L.; Chen, X.; Choe, M.; Chuang, G.; Georgiev, I. S.; Joyce, M. G.; Kirys, T.; Ko, S.; Pegu, A.; Shi, W.; Todd, J. P.; Yang, Z.; Bailer, R. T.; Rao, S.; Kwong, P. D.; Nabel, G. J.

    2015-01-01

    ABSTRACT The potency and breadth of the recently isolated neutralizing human monoclonal antibodies to HIV-1 have stimulated interest in their use to prevent or to treat HIV-1 infection. Due to the antigenically diverse nature of the HIV-1 envelope (Env), no single antibody is highly active against all viral strains. While the physical combination of two broadly neutralizing antibodies (bNAbs) can improve coverage against the majority of viruses, the clinical-grade manufacturing and testing of two independent antibody products are time and resource intensive. In this study, we constructed bispecific immunoglobulins (IgGs) composed of independent antigen-binding fragments with a common Fc region. We developed four different bispecific IgG variants that included antibodies targeting four major sites of HIV-1 neutralization. We show that these bispecific IgGs display features of both antibody specificities and, in some cases, display improved coverage over the individual parental antibodies. All four bispecific IgGs neutralized 94% to 97% of antigenically diverse viruses in a panel of 206 HIV-1 strains. Among the bispecific IgGs tested, VRC07 × PG9-16 displayed the most favorable neutralization profile. It was superior in breadth to either of the individual antibodies, neutralizing 97% of viruses with a median 50% inhibitory concentration (IC50) of 0.055 μg/ml. This bispecific IgG also demonstrated in vivo pharmacokinetic parameters comparable to those of the parental bNAbs when administered to rhesus macaques. These results suggest that IgG-based bispecific antibodies are promising candidates for the prevention and treatment of HIV-1 infection in humans. IMPORTANCE To prevent or treat HIV-1 infection, antibodies must potently neutralize nearly all strains of HIV-1. Thus, the physical combination of two or more antibodies may be needed to broaden neutralization coverage and diminish the possibility of viral resistance. A bispecific antibody that has two different

  5. Analysis of defined combinations of monoclonal antibodies in anthrax toxin neutralization assays and their synergistic action.

    PubMed

    Ngundi, Miriam M; Meade, Bruce D; Little, Stephen F; Quinn, Conrad P; Corbett, Cindi R; Brady, Rebecca A; Burns, Drusilla L

    2012-05-01

    Antibodies against the protective antigen (PA) component of anthrax toxin play an important role in protection against disease caused by Bacillus anthracis. In this study, we examined defined combinations of PA-specific monoclonal antibodies for their ability to neutralize anthrax toxin in cell culture assays. We observed additive, synergistic, and antagonistic effects of the antibodies depending on the specific antibody combination examined and the specific assay used. Synergistic toxin-neutralizing antibody interactions were examined in more detail. We found that one mechanism that can lead to antibody synergy is the bridging of PA monomers by one antibody, with resultant bivalent binding of the second antibody. These results may aid in optimal design of new vaccines and antibody therapies against anthrax. PMID:22441391

  6. Neutralizing antibodies to different proteins of African swine fever virus inhibit both virus attachment and internalization.

    PubMed Central

    Gómez-Puertas, P; Rodríguez, F; Oviedo, J M; Ramiro-Ibáñez, F; Ruiz-Gonzalvo, F; Alonso, C; Escribano, J M

    1996-01-01

    African swine fever virus induces in convalescent pigs antibodies that neutralized the virus before and after binding to susceptible cells, inhibiting both virus attachment and internalization. A further analysis of the neutralization mechanisms mediated by the different viral proteins showed that antibodies to proteins p72 and p54 are involved in the inhibition of a first step of the replication cycle related to virus attachment, while antibodies to protein p30 are implicated in the inhibition of virus internalization. PMID:8764090

  7. Focused Evolution of HIV-1 Neutralizing Antibodies Revealed by Structures and Deep Sequencing

    SciTech Connect

    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.

    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.

  8. Envelope Variants Circulating as Initial Neutralization Breadth Developed in Two HIV-Infected Subjects Stimulate Multiclade Neutralizing Antibodies in Rabbits

    PubMed Central

    Malherbe, Delphine C.; Pissani, Franco; Sather, D. Noah; Guo, Biwei; Pandey, Shilpi; Sutton, William F.; Stuart, Andrew B.; Robins, Harlan; Park, Byung; Krebs, Shelly J.; Schuman, Jason T.; Kalams, Spyros; Hessell, Ann J.

    2014-01-01

    ABSTRACT Identifying characteristics of the human immunodeficiency virus type 1 (HIV-1) envelope that are effective in generating broad, protective antibodies remains a hurdle to HIV vaccine design. Emerging evidence of the development of broad and potent neutralizing antibodies in HIV-infected subjects suggests that founder and subsequent progeny viruses may express unique antigenic motifs that contribute to this developmental pathway. We hypothesize that over the course of natural infection, B cells are programmed to develop broad antibodies by exposure to select populations of emerging envelope quasispecies variants. To test this hypothesis, we identified two unrelated subjects whose antibodies demonstrated increasing neutralization breadth against a panel of HIV-1 isolates over time. Full-length functional env genes were cloned longitudinally from these subjects from months after infection through 2.6 to 5.8 years of infection. Motifs associated with the development of breadth in published, cross-sectional studies were found in both subjects. We compared the immunogenicity of envelope vaccines derived from time points obtained during and after broadening of neutralization activity within these subjects. Rabbits were coimmunized four times with selected multiple gp160 DNAs and gp140-trimeric envelope proteins. The affinity of the polyclonal response increased as a function of boosting. The most rapid and persistent neutralization of multiclade tier 1 viruses was elicited by envelopes that were circulating in plasma at time points prior to the development of 50% neutralization breadth in both human subjects. The breadth elicited in rabbits was not improved by exposure to later envelope variants. These data have implications for vaccine development in describing a target time point to identify optimal envelope immunogens. IMPORTANCE Vaccine protection against viral infections correlates with the presence of neutralizing antibodies; thus, vaccine components capable

  9. Mucosal delivery of the human immunodeficiency virus-1 Tat protein in mice elicits systemic neutralizing antibodies, cytotoxic T lymphocytes and mucosal IgA.

    PubMed

    Marinaro, Mariarosaria; Riccomi, Antonella; Rappuoli, Rino; Pizza, Mariagrazia; Fiorelli, Valeria; Tripiciano, Antonella; Cafaro, Aurelio; Ensoli, Barbara; De Magistris, Maria Teresa

    2003-09-01

    Human immunodeficiency virus (HIV)-1 Tat protein induces protection in non-human primates upon systemic vaccination. In view of the design of mucosal vaccines against HIV-1 we studied the immune response to native Tat (aa 1-86) in mice following intranasal delivery of the protein with two mucosal adjuvants, Escherichia coli heat-labile enterotoxin (LT) and LT-R72, a non-toxic mutant of LT. Immunization with Tat and the two adjuvants induced in BALB/c but not in C57BL/6 mice high and persistent levels of serum IgG and secretory IgA in vaginal and intestinal fluids. Mice sera neutralized Tat and recognized two epitopes mapping in the regions 1-20 and 46-60. Furthermore, their splenocytes proliferated and secreted IFN-gamma and IL-6 in response to Tat. Finally, CTLs were also elicited and they recognized an epitope localized within aa 11-40 of Tat. PMID:12922133

  10. Preparation of an antibody that recognizes and neutralizes Dictyostelium differentiation-inducing factor-1.

    PubMed

    Kubohara, Yuzuru; Kikuchi, Haruhisa; Nakamura, Koji; Matsuo, Yusuke; Oshima, Yoshiteru

    2010-05-28

    In the development of the cellular slime mold Dictyostelium discoideum, the differentiation-inducing factor-1 (DIF-1; 1-(3,5-dichloro-2,6-dihydroxy-4-methoxyphenyl)hexan-1-one) plays an important role in the regulation of cell differentiation and chemotaxis; however, the cellular signaling systems involving DIF-1 remain to be elucidated. To obtain a probe for DIF-1, we synthesized a DIF derivative (DIF-1-NH(2); 6-amino-1-(3,5-dichloro-2,6-dihydroxy-4-methoxyphenyl)hexan-1-one), and prepared an anti-DIF-1 antibody using a DIF-1-NH(2)-conjugated macromolecule as the immunogen. A 100-fold dilution of the antibody bound to DIF-1-NH(2)-conjugated resin, and this binding was inhibited by co-addition of 20 microM DIF-1 or DIF-1-NH(2). In a monolayer culture of HM44 cells, a DIF-deficient D. discoideum strain, 0.5 nM exogenous DIF-1 induced stalk cell formation in approximately 60% of the cells; this induction was dose-dependently inhibited by the antibody (diluted 12.5- or 25-fold). Furthermore, this inhibition by the antibody was recovered by co-addition of 2.5 or10 nM DIF-1. The results indicate that the anti-DIF-1 antibody recognizes DIF-1 and neutralizes its function. PMID:20416278

  11. H7N9 influenza virus neutralizing antibodies that possess few somatic mutations

    PubMed Central

    Thornburg, Natalie J.; Zhang, Heng; Bangaru, Sandhya; Kose, Nurgun; Lampley, Rebecca M.; Bombardi, Robin G.; Yu, Yingchun; Graham, Stephen; Branchizio, Andre; Yoder, Sandra M.; Rock, Michael T.; Creech, C. Buddy; Edwards, Kathryn M.; Lee, David; Li, Sheng; Wilson, Ian A.; García-Sastre, Adolfo; Albrecht, Randy A.; Crowe, James E.

    2016-01-01

    Avian H7N9 influenza viruses are group 2 influenza A viruses that have been identified as the etiologic agent for a current major outbreak that began in China in 2013 and may pose a pandemic threat. Here, we examined the human H7-reactive antibody response in 75 recipients of a monovalent inactivated A/Shanghai/02/2013 H7N9 vaccine. After 2 doses of vaccine, the majority of donors had memory B cells that secreted IgGs specific for H7 HA, with dominant responses against single HA subtypes, although frequencies of H7-reactive B cells ranged widely between donors. We isolated 12 naturally occurring mAbs with low half-maximal effective concentrations for binding, 5 of which possessed neutralizing and HA-inhibiting activities. The 5 neutralizing mAbs exhibited narrow breadth of reactivity with influenza H7 strains. Epitope-mapping studies using neutralization escape mutant analysis, deuterium exchange mass spectrometry, and x-ray crystallography revealed that these neutralizing mAbs bind near the receptor-binding pocket on HA. All 5 neutralizing mAbs possessed low numbers of somatic mutations, suggesting the clones arose from naive B cells. The most potent mAb, H7.167, was tested as a prophylactic treatment in a mouse intranasal virus challenge study, and systemic administration of the mAb markedly reduced viral lung titers. PMID:26950424

  12. H7N9 influenza virus neutralizing antibodies that possess few somatic mutations.

    PubMed

    Thornburg, Natalie J; Zhang, Heng; Bangaru, Sandhya; Sapparapu, Gopal; Kose, Nurgun; Lampley, Rebecca M; Bombardi, Robin G; Yu, Yingchun; Graham, Stephen; Branchizio, Andre; Yoder, Sandra M; Rock, Michael T; Creech, C Buddy; Edwards, Kathryn M; Lee, David; Li, Sheng; Wilson, Ian A; García-Sastre, Adolfo; Albrecht, Randy A; Crowe, James E

    2016-04-01

    Avian H7N9 influenza viruses are group 2 influenza A viruses that have been identified as the etiologic agent for a current major outbreak that began in China in 2013 and may pose a pandemic threat. Here, we examined the human H7-reactive antibody response in 75 recipients of a monovalent inactivated A/Shanghai/02/2013 H7N9 vaccine. After 2 doses of vaccine, the majority of donors had memory B cells that secreted IgGs specific for H7 HA, with dominant responses against single HA subtypes, although frequencies of H7-reactive B cells ranged widely between donors. We isolated 12 naturally occurring mAbs with low half-maximal effective concentrations for binding, 5 of which possessed neutralizing and HA-inhibiting activities. The 5 neutralizing mAbs exhibited narrow breadth of reactivity with influenza H7 strains. Epitope-mapping studies using neutralization escape mutant analysis, deuterium exchange mass spectrometry, and x-ray crystallography revealed that these neutralizing mAbs bind near the receptor-binding pocket on HA. All 5 neutralizing mAbs possessed low numbers of somatic mutations, suggesting the clones arose from naive B cells. The most potent mAb, H7.167, was tested as a prophylactic treatment in a mouse intranasal virus challenge study, and systemic administration of the mAb markedly reduced viral lung titers. PMID:26950424

  13. Human Antibody Titers to Epstein-Barr Virus (EBV) gp350 Correlate with Neutralization of Infectivity Better than Antibody Titers to EBV gp42 Using a Rapid Flow Cytometry-Based EBV Neutralization Assay

    PubMed Central

    Sashihara, Junji; Burbelo, Peter D.; Savoldo, Barbara; Pierson, Theodore C.; Cohen, Jeffrey I.

    2009-01-01

    Measurement of neutralizing antibodies to Epstein-Barr virus (EBV) is important for evaluation of candidate vaccines. The current neutralization assay is based on antibody inhibition of EBV transformation of B cells and requires 6 weeks to perform. We developed a rapid, quantitative flow cytometry assay and show that neutralizing antibody titers measured by the new assay strongly correlate with antibody titers in the standard transformation-based assay. Antibodies to EBV gp350 and gp42 have been shown to block infection of B cells by EBV. Using new assays to quantify antibodies to these glycoproteins, we show for the first time that that human plasma contains high titers of antibody to gp42; these titers correlate with neutralization of EBV infectivity or transformation. Furthermore, we show that antibody titers to EBV gp350 correlate more strongly with neutralization than antibody titers to gp42. These assays should be useful in accessing antibody responses to candidate EBV vaccines. PMID:19584018

  14. Structural repertoire of HIV-1-neutralizing antibodies targeting the CD4 supersite in 14 donors

    PubMed Central

    Zhou, Tongqing; Lynch, Rebecca M.; Chen, Lei; Acharya, Priyamvada; Wu, Xueling; Doria-Rose, Nicole A.; Joyce, M. Gordon; Lingwood, Daniel; Soto, Cinque; Bailer, Robert T.; Ernandes, Michael J.; Kong, Rui; Longo, Nancy S.; Louder, Mark K.; McKee, Krisha; O’Dell, Sijy; Schmidt, Stephen D.; Tran, Lillian; Yang, Zhongjia; Druz, Aliaksandr; Luongo, Timothy S.; Moquin, Stephanie; Srivatsan, Sanjay; Yang, Yongping; Zhang, Baoshan; Zheng, Anqi; Pancera, Marie; Kirys, Tatsiana; Georgiev, Ivelin S.; Gindin, Tatyana; Peng, Hung-Pin; Yang, An-Suei; Mullikin, James C.; Gray, Matthew D.; Stamatatos, Leonidas; Burton, Dennis R.; Koff, Wayne C.; Cohen, Myron S.; Haynes, Barton F.; Casazza, Joseph P.; Connors, Mark; Corti, Davide; Lanzavecchia, Antonio; Sattentau, Quentin J.; Weiss, Robin A.; West, Anthony P.; Bjorkman, Pamela J.; Scheid, Johannes F.; Nussenzweig, Michel C.; Shapiro, Lawrence; Mascola, John R.; Kwong, Peter D.

    2015-01-01

    The site on the HIV-1 gp120 glycoprotein that binds the CD4 receptor is recognized by broadly reactive antibodies, several of which neutralize over 90% of HIV-1 strains. To understand how antibodies achieve such neutralization, we isolated CD4-binding-site (CD4bs) antibodies and analyzed 16 co-crystal structures –8 determined here– of CD4bs antibodies from 14 donors. The 16 antibodies segregated by recognition mode and developmental ontogeny into two types: CDR H3-dominated and VH-gene-restricted. Both could achieve greater than 80% neutralization breadth, and both could develop in the same donor. Although paratope chemistries differed, all 16 gp120-CD4bs antibody complexes showed geometric similarity, with antibody-neutralization breadth correlating with antibody-angle of approach relative to the most effective antibody of each type. The repertoire for effective recognition of the CD4 supersite thus comprises antibodies with distinct paratopes arrayed about two optimal geometric orientations, one achieved by CDR H3 ontogenies and the other achieved by VH-gene-restricted ontogenies. PMID:26004070

  15. Neutralizing monoclonal antibodies against listeriolysin: mapping of epitopes involved in pore formation.

    PubMed Central

    Darji, A; Niebuhr, K; Hense, M; Wehland, J; Chakraborty, T; Weiss, S

    1996-01-01

    Six different mouse monoclonal antibodies (MAbs) and a specific rabbit polygonal antibody were raised against listeriolysin. Four of the MAbs also recognized seeligeriolysin, and five cross-reacted with ivanolysin. The hemolytic activity could be neutralized by the polygonal antibody as well as by five of the MAbs. None of the neutralizing antibodies interfered with the binding of listeriolysin to the cellular membrane. The epitopes recognized by the MAbs were localized by using overlapping synthetic peptides between positions 59 and 279, a region hitherto not implicated in mediating hemolytic activity. PMID:8675351

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

    PubMed Central

    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

    2016-01-01

    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. PMID:27351973

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

    PubMed

    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. PMID:27351973

  18. NEP: web server for epitope prediction based on antibody neutralization of viral strains with diverse sequences.

    PubMed

    Chuang, Gwo-Yu; Liou, David; Kwong, Peter D; Georgiev, Ivelin S

    2014-07-01

    Delineation of the antigenic site, or epitope, recognized by an antibody can provide clues about functional vulnerabilities and resistance mechanisms, and can therefore guide antibody optimization and epitope-based vaccine design. Previously, we developed an algorithm for antibody-epitope prediction based on antibody neutralization of viral strains with diverse sequences and validated the algorithm on a set of broadly neutralizing HIV-1 antibodies. Here we describe the implementation of this algorithm, NEP (Neutralization-based Epitope Prediction), as a web-based server. The users must supply as input: (i) an alignment of antigen sequences of diverse viral strains; (ii) neutralization data for the antibody of interest against the same set of antigen sequences; and (iii) (optional) a structure of the unbound antigen, for enhanced prediction accuracy. The prediction results can be downloaded or viewed interactively on the antigen structure (if supplied) from the web browser using a JSmol applet. Since neutralization experiments are typically performed as one of the first steps in the characterization of an antibody to determine its breadth and potency, the NEP server can be used to predict antibody-epitope information at no additional experimental costs. NEP can be accessed on the internet at http://exon.niaid.nih.gov/nep. PMID:24782517

  19. Neutralization of Botulinum Neurotoxin by a Human Monoclonal Antibody Specific for the Catalytic Light Chain

    PubMed Central

    Adekar, Sharad P.; Takahashi, Tsuyoshi; Jones, R. Mark; Al-Saleem, Fetweh H.; Ancharski, Denise M.; Root, Michael J.; Kapadnis, B. P.; Simpson, Lance L.; Dessain, Scott K.

    2008-01-01

    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. PMID:18714390

  20. Disease-enhancing antibodies improve the efficacy of bacterial toxin-neutralizing antibodies

    PubMed Central

    Chow, Siu-Kei; Smith, Cameron; MacCarthy, Thomas; Pohl, Mary Ann; Bergman, Aviv; Casadevall, Arturo

    2013-01-01

    SUMMARY During infection, humoral immunity produces a polyclonal response with various immunoglobulins recognizing different epitopes within the microbe or toxin. Despite this diverse response, the biological activity of an antibody (Ab) is usually assessed by the action of a monoclonal population. We demonstrate that a combination of monoclonal antibodies (mAbs) that are individually disease-enhancing or neutralizing to Bacillus anthracis protective antigen (PA), a component of anthrax toxin, results in significantly augmented protection against the toxin. This boosted protection is Fc gamma receptor (FcγR)-dependent and involves the formation of stoichiometrically defined mAb-PA complexes that requires immunoglobulin bivalence and simultaneous interaction between PA and the two mAbs. The formation of these mAb-PA complexes inhibits PA oligomerization, resulting in protection. These data suggest that functional assessments of single Abs may inaccurately predict how the same Abs will operate in polyclonal preparations and imply that potentially therapeutic mAbs may be overlooked in single Ab screens. PMID:23601104

  1. Immunogenic peptides comprising a T-helper epitope and a B-cell neutralizing antibody epitope

    DOEpatents

    Haynes, Barton F.; Korber, Bette T.; De Lorimier, Robert M.

    2006-12-26

    The present invention relates, generally, to a polyvalent immunogen and, more particularly, to a method of inducing neutralizing antibodies against HIV and to a polyvalent immunogen suitable for use in such a method.

  2. Latency of Herpes Simplex Virus in Absence of Neutralizing Antibody: Model for Reactivation

    NASA Astrophysics Data System (ADS)

    Sekizawa, Tsuyoshi; Openshaw, Harry; Wohlenberg, Charles; Notkins, Abner Louis

    1980-11-01

    Mice inoculated with herpes simplex virus (type 1) by the lip or corneal route and then passively immunized with rabbit antibody to herpes simplex virus developed a latent infection in the trigeminal ganglia within 96 hours. Neutralizing antibody to herpes simplex virus was cleared from the circulation and could not be detected in most of these mice after 2 months. Examination of ganglia from the antibody-negative mice revealed latent virus in over 90 percent of the animals, indicating that serum neutralizing antibody is not necessary to maintain the latent state. When the lips or corneas of these mice were traumatized, viral reactivation occurred in up to 90 percent of the mice, as demonstrated by the appearance of neutralizing antibody. This study provides a model for identifying factors that trigger viral reactivation.

  3. Affinity Maturation to Improve Human Monoclonal Antibody Neutralization Potency and Breadth against Hepatitis C Virus*

    PubMed Central

    Wang, Yong; Keck, Zhen-yong; Saha, Anasuya; Xia, Jinming; Conrad, Fraser; Lou, Jianlong; Eckart, Michael; Marks, James D.; Foung, Steven K. H.

    2011-01-01

    A potent neutralizing antibody to a conserved hepatitis C virus (HCV) epitope might overcome its extreme variability, allowing immunotherapy. The human monoclonal antibody HC-1 recognizes a conformational epitope on the HCV E2 glycoprotein. Previous studies showed that HC-1 neutralizes most HCV genotypes but has modest potency. To improve neutralization, we affinity-matured HC-1 by constructing a library of yeast-displayed HC-1 single chain Fv (scFv) mutants, using for selection an E2 antigen from one of the poorly neutralized HCVpp. We developed an approach by parallel mutagenesis of the heavy chain variable (VH) and κ-chain variable (Vk) genes separately, then combining the optimized VH and Vk mutants. This resulted in the generation of HC-1-related scFv variants exhibiting improved affinities. The best scFv variant had a 92-fold improved affinity. After conversion to IgG1, some of the antibodies exhibited a 30-fold improvement in neutralization activity. Both surface plasmon resonance and solution kinetic exclusion analysis showed that the increase in affinity was largely due to a lowering of the dissociation rate constant, Koff. Neutralization against a panel of HCV pseudoparticles and infectious 2a HCV virus improved with the affinity-matured IgG1 antibodies. Interestingly, some of these antibodies neutralized a viral isolate that was not neutralized by wild-type HC-1. Moreover, propagating 2a HCVcc under the selective pressure of WT HC-1 or affinity-matured HC-1 antibodies yielded no viral escape mutants and, with the affinity-matured IgG1, needed 100-fold less antibody to achieve complete virus elimination. Taken together, these findings suggest that affinity-matured HC-1 antibodies are excellent candidates for therapeutic development. PMID:22002064

  4. Isolation and Chimerization of a Highly Neutralizing Antibody Conferring Passive Protection against Lethal Bacillus anthracis Infection

    PubMed Central

    Rosenfeld, Ronit; Marcus, Hadar; Ben-Arie, Einat; Lachmi, Bat-El; Mechaly, Adva; Reuveny, Shaul; Gat, Orit; Mazor, Ohad; Ordentlich, Arie

    2009-01-01

    Several studies have demonstrated that the passive transfer of protective antigen (PA)-neutralizing antibodies can protect animals against Bacillus anthracis infection. The standard protocol for the isolation of PA-neutralizing monoclonal antibodies is based upon a primary selection of the highest PA-binders by ELISA, and usually yields only few candidates antibodies. We demonstrated that by applying a PA-neutralization functionality-based screen as the primary criterion for positive clones, it was possible to isolate more than 100 PA-neutralizing antibodies, some of which exhibited no measurable anti-PA titers in ELISA. Among the large panel of neutralizing antibodies identified, mAb 29 demonstrated the most potent activity, and was therefore chimerized. The variable region genes of the mAb 29 were fused to human constant region genes, to form the chimeric 29 antibody (cAb 29). Guinea pigs were fully protected against infection by 40LD50 B. anthracis spores following two separate administrations with 10 mg/kg of cAb 29: the first administration was given before the challenge, and a second dose was administered on day 4 following exposure. Moreover, animals that survived the challenge and developed endogenous PA-neutralizing antibodies with neutralizing titers above 100 were fully protected against repeat challenges with 40LD50 of B. anthracis spores. The data presented here emphasize the importance of toxin neutralization-based screens for the efficient isolation of protective antibodies that were probably overlooked in the standard screening protocol. The protective activity of the chimeric cAb 29 demonstrated in this study suggest that it may serve as an effective immunotherapeutic agent against anthrax. PMID:19629185

  5. Developmental pathway for potent V1V2-directed HIV-neutralizing antibodies.

    PubMed

    Doria-Rose, Nicole A; Schramm, Chaim A; Gorman, Jason; Moore, Penny L; Bhiman, Jinal N; DeKosky, Brandon J; Ernandes, Michael J; Georgiev, Ivelin S; Kim, Helen J; Pancera, Marie; Staupe, Ryan P; Altae-Tran, Han R; Bailer, Robert T; Crooks, Ema T; Cupo, Albert; Druz, Aliaksandr; Garrett, Nigel J; Hoi, Kam H; Kong, Rui; Louder, Mark K; Longo, Nancy S; McKee, Krisha; Nonyane, Molati; O'Dell, Sijy; Roark, Ryan S; Rudicell, Rebecca S; Schmidt, Stephen D; Sheward, Daniel J; Soto, Cinque; Wibmer, Constantinos Kurt; Yang, Yongping; Zhang, Zhenhai; Mullikin, James C; Binley, James M; Sanders, Rogier W; Wilson, Ian A; Moore, John P; Ward, Andrew B; Georgiou, George; Williamson, Carolyn; Abdool Karim, Salim S; Morris, Lynn; Kwong, Peter D; Shapiro, Lawrence; Mascola, John R

    2014-05-01

    Antibodies capable of neutralizing HIV-1 often target variable regions 1 and 2 (V1V2) of the HIV-1 envelope, but the mechanism of their elicitation has been unclear. Here we define the developmental pathway by which such antibodies are generated and acquire the requisite molecular characteristics for neutralization. Twelve somatically related neutralizing antibodies (CAP256-VRC26.01-12) were isolated from donor CAP256 (from the Centre for the AIDS Programme of Research in South Africa (CAPRISA)); each antibody contained the protruding tyrosine-sulphated, anionic antigen-binding loop (complementarity-determining region (CDR) H3) characteristic of this category of antibodies. Their unmutated ancestor emerged between weeks 30-38 post-infection with a 35-residue CDR H3, and neutralized the virus that superinfected this individual 15 weeks after initial infection. Improved neutralization breadth and potency occurred by week 59 with modest affinity maturation, and was preceded by extensive diversification of the virus population. HIV-1 V1V2-directed neutralizing antibodies can thus develop relatively rapidly through initial selection of B cells with a long CDR H3, and limited subsequent somatic hypermutation. These data provide important insights relevant to HIV-1 vaccine development. PMID:24590074

  6. Inhibitory and neutral antibodies to Plasmodium falciparum MSP119 form ring structures with their antigen.

    PubMed

    Dekker, Carien; Uthaipibull, Chairat; Calder, Lesley J; Lock, Matthew; Grainger, Munira; Morgan, William D; Dodson, Guy G; Holder, Anthony A

    2004-09-01

    Blood-stage malaria vaccine candidates include surface proteins of the merozoite. Antibodies to these proteins may either block essential steps during invasion or render the merozoite susceptible to phagocytosis or complement-mediated degradation. Structural information on merozoite surface proteins complexed to antibodies provides crucial information for knowledge-based vaccine design. The major merozoite surface protein MSP1 is an abundant surface molecule in Plasmodium falciparum. Only a subset of antibodies against MSP119 inhibits invasion (inhibitory antibodies), whereas other antibodies binding to MSP119 have no effect on invasion (neutral antibodies). Here we report on the complex of MSP119 with both inhibitory monoclonal antibody 12.10 and neutral monoclonal antibody 2F10. The complexes were established using both whole IgG's and Fab fragments, and analysed by dynamic light scattering, electron microscopy and analytical ultra centrifugation. Specific ring structures were formed in the ternary complex with the two antibodies, providing direct evidence of non-overlapping epitopes on MSP119. Mutational studies also indicated that the epitopes of the inhibitory and neutral antibodies are spatially remote. PMID:15279960

  7. Broadly neutralizing human monoclonal JC polyomavirus VP1–specific antibodies as candidate therapeutics for progressive multifocal leukoencephalopathy

    PubMed Central

    Jelcic, Ivan; Combaluzier, Benoit; Jelcic, Ilijas; Faigle, Wolfgang; Senn, Luzia; Reinhart, Brenda J.; Ströh, Luisa; Nitsch, Roger M.; Stehle, Thilo; Sospedra, Mireia; Grimm, Jan; Martin, Roland

    2016-01-01

    In immunocompromised individuals, JC polyomavirus (JCPyV) may mutate and gain access to the central nervous system resulting in progressive multifocal leukoencephalopathy (PML), an often fatal opportunistic infection for which no treatments are currently available. Despite recent progress, the contribution of JCPyV-specific humoral immunity to controlling asymptomatic infection throughout life and to eliminating JCPyV from the brain is poorly understood. We examined antibody responses against JCPyV major capsid protein VP1 (viral protein 1) variants in the serum and cerebrospinal fluid (CSF) of healthy donors (HDs), JCPyV-positive multiple sclerosis patients treated with the anti-VLA-4 monoclonal antibody natalizumab (NAT), and patients with NAT-associated PML. Before and during PML, CSF antibody responses against JCPyV VP1 variants show “recognition holes”; however, upon immune reconstitution, CSF antibody titers rise, then recognize PML-associated JCPyV VP1 variants, and may be involved in elimination of the virus. We therefore reasoned that the memory B cell repertoire of individuals who recovered from PML could be a source for the molecular cloning of broadly neutralizing antibodies for passive immunization. We generated a series of memory B cell-derived JCPyV VP1-specific human monoclonal antibodies from HDs and a patient with NAT-associated PML-immune reconstitution inflammatory syndrome (IRIS). These antibodies exhibited diverse binding affinity, cross-reactivity with the closely related BK polyomavirus, recognition of PML-causing VP1 variants, and JCPyV neutralization. Almost all antibodies with exquisite specificity for JCPyV, neutralizing activity, recognition of all tested JCPyV PML variants, and high affinity were derived from one patient who had recovered from PML. These antibodies are promising drug candidates for the development of a treatment of PML. PMID:26400911

  8. Broadly neutralizing human monoclonal JC polyomavirus VP1-specific antibodies as candidate therapeutics for progressive multifocal leukoencephalopathy.

    PubMed

    Jelcic, Ivan; Combaluzier, Benoit; Jelcic, Ilijas; Faigle, Wolfgang; Senn, Luzia; Reinhart, Brenda J; Ströh, Luisa; Nitsch, Roger M; Stehle, Thilo; Sospedra, Mireia; Grimm, Jan; Martin, Roland

    2015-09-23

    In immunocompromised individuals, JC polyomavirus (JCPyV) may mutate and gain access to the central nervous system resulting in progressive multifocal leukoencephalopathy (PML), an often fatal opportunistic infection for which no treatments are currently available. Despite recent progress, the contribution of JCPyV-specific humoral immunity to controlling asymptomatic infection throughout life and to eliminating JCPyV from the brain is poorly understood. We examined antibody responses against JCPyV major capsid protein VP1 (viral protein 1) variants in the serum and cerebrospinal fluid (CSF) of healthy donors (HDs), JCPyV-positive multiple sclerosis patients treated with the anti-VLA-4 monoclonal antibody natalizumab (NAT), and patients with NAT-associated PML. Before and during PML, CSF antibody responses against JCPyV VP1 variants show "recognition holes"; however, upon immune reconstitution, CSF antibody titers rise, then recognize PML-associated JCPyV VP1 variants, and may be involved in elimination of the virus. We therefore reasoned that the memory B cell repertoire of individuals who recovered from PML could be a source for the molecular cloning of broadly neutralizing antibodies for passive immunization. We generated a series of memory B cell-derived JCPyV VP1-specific human monoclonal antibodies from HDs and a patient with NAT-associated PML-immune reconstitution inflammatory syndrome (IRIS). These antibodies exhibited diverse binding affinity, cross-reactivity with the closely related BK polyomavirus, recognition of PML-causing VP1 variants, and JCPyV neutralization. Almost all antibodies with exquisite specificity for JCPyV, neutralizing activity, recognition of all tested JCPyV PML variants, and high affinity were derived from one patient who had recovered from PML. These antibodies are promising drug candidates for the development of a treatment of PML. PMID:26400911

  9. The Bordetella Adenylate Cyclase Repeat-in-Toxin (RTX) Domain Is Immunodominant and Elicits Neutralizing Antibodies*

    PubMed Central

    Wang, Xianzhe; Maynard, Jennifer A.

    2015-01-01

    The adenylate cyclase toxin (ACT) is a multifunctional virulence factor secreted by Bordetella species. Upon interaction of its C-terminal hemolysin moiety with the cell surface receptor αMβ2 integrin, the N-terminal cyclase domain translocates into the host cell cytosol where it rapidly generates supraphysiological cAMP concentrations, which inhibit host cell anti-bacterial activities. Although ACT has been shown to induce protective immunity in mice, it is not included in any current acellular pertussis vaccines due to protein stability issues and a poor understanding of its role as a protective antigen. Here, we aimed to determine whether any single domain could recapitulate the antibody responses induced by the holo-toxin and to characterize the dominant neutralizing antibody response. We first immunized mice with ACT and screened antibody phage display libraries for binding to purified ACT. The vast majority of unique antibodies identified bound the C-terminal repeat-in-toxin (RTX) domain. Representative antibodies binding two nonoverlapping, neutralizing epitopes in the RTX domain prevented ACT association with J774A.1 macrophages and soluble αMβ2 integrin, suggesting that these antibodies inhibit the ACT-receptor interaction. Sera from mice immunized with the RTX domain showed similar neutralizing activity as ACT-immunized mice, indicating that this domain induced an antibody response similar to that induced by ACT. These data demonstrate that RTX can elicit neutralizing antibodies and suggest it may present an alternative to ACT. PMID:25505186

  10. Immune escape by human immunodeficiency virus type 1 from neutralizing antibodies: evidence for multiple pathways.

    PubMed Central

    Watkins, B A; Reitz, M S; Wilson, C A; Aldrich, K; Davis, A E; Robert-Guroff, M

    1993-01-01

    Sera from many HIV-1-infected individuals contain broadly reactive, specific neutralizing antibodies. Despite their broad reactivity, variant viruses, resistant to neutralization, can be selected in vitro in the presence of such antisera. We have previously shown that neutralization resistance of an escape mutant with an amino acid substitution in the transmembrane protein (A582T) occurs because of alteration of a conformational epitope that is recognized by neutralizing antibodies directed against the CD4 binding site. In this report we demonstrate that immune escape via a single-amino-acid substitution (A281V) within a conserved region of the envelope glycoprotein gp120 confers neutralization resistance against a broadly reactive neutralizing antiserum from a seropositive individual. We show this alteration affects V3 and additional regions unrelated to V3 or the CD4 binding site. Together with previous studies on escape mutants selected in vitro, our findings suggest that immune-selective pressure can arise by multiple pathways. PMID:7693973

  11. 9G4+ Antibodies Isolated from HIV-Infected Patients Neutralize HIV-1 and Have Distinct Autoreactivity Profiles

    PubMed Central

    Alcéna, Danielle C.; Kobie, James J.; Kaminski, Denise A.; Rosenberg, Alexander F.; Mattiacio, Jonelle L.; Brewer, Matthew; Dewhurst, Stephen; Dykes, Carrie; Jin, Xia; Keefer, Michael C.; Sanz, Ignacio

    2013-01-01

    Potent HIV-1 specific broadly neutralizing antibodies (BNA) are uncommon in HIV infected individuals, and have proven hard to elicit by vaccination. Several, isolated monoclonal BNA are polyreactive and also recognize self-antigens, suggesting a breach of immune tolerance in persons living with HIV (PLWH). Persons with systemic lupus erythematosus (SLE) often have elevated levels of autoreactive antibodies encoded by the VH4-34 heavy chain immunoglobulin gene whose protein product can be detected by the 9G4 rat monoclonal antibody. We have recently found that levels of these “9G4+” antibodies are also elevated in PLWH. However, the putative autoreactive nature of these antibodies and the relationship of such reactivities with HIV neutralization have not been investigated. We therefore examined the autoreactivity and HIV neutralization potential of 9G4+ antibodies from PLWH. Results show that 9G4+ antibodies from PLWH bound to recombinant HIV-1 envelope (Env) and neutralized viral infectivity in vitro, whereas 9G4+ antibodies from persons with SLE did not bind to Env and failed to neutralize viral infectivity. In addition, while 9G4+ antibodies from PLWH retained the canonical anti-i reactivity that mediates B cell binding, they did not display other autoreactivities common to SLE 9G4+ antibodies, such as binding to cardiolipin and DNA and had much lower reactivity with apoptotic cells. Taken together, these data indicate that the autoreactivity of 9G4+ antibodies from PLWH is distinct from that of SLE patients, and therefore, their expansion is not due to a general breakdown of B cell tolerance but is instead determined in a more disease-specific manner by self-antigens that become immunogenic in the context of, and possibly due to HIV infection. Further studies of 9G4+ B cells may shed light on the regulation of B cell tolerance and interface between the generation of specific autoreactivities and the induction of antiviral immunity in persons living with HIV

  12. Assessment of neutralizing and non-neutralizing antibody responses against Porcine circovirus 2 in vaccinated and non-vaccinated farmed pigs.

    PubMed

    Worsfold, Cristina Solis; Dardari, Rkia; Law, Sampson; Eschbaumer, Michael; Nourozieh, Narges; Marshall, Frank; Czub, Markus

    2015-09-01

    Vaccination is the most efficacious procedure to curtail Porcine circovirus 2 (PCV2)-associated diseases (PCVAD). Experimental studies indicate that PCV2 vaccine-induced virus-neutralizing antibodies play a major role in protection from PCVAD. However, the immune response to PCV2 vaccination of pigs on farms is less clear. Analysing groups of age-matched vaccinated and non-vaccinated farmed pigs, we found significantly increased levels of virus-neutralizing antibodies only in vaccinated pigs belonging to the age group with the highest risk for developing PCVAD. Serum levels of PCV2 genomes were not different between corresponding age groups. Levels of antibodies directed against a linear peptide from the PCV2 capsid protein correlated with those of virus-neutralizing antibodies and reached the highest levels in older, non-vaccinated animals, pointing towards an intense interaction between PCV2-infected cells and the immune system. In conclusion, current PCV2 vaccines are in need of improvement to induce stronger and more rapid immunity to prevent PCV2 infection. PMID:26033118

  13. Anthrax vaccine recipients lack antibody against the loop neutralizing determinant: a protective neutralizing epitope from Bacillus anthracis protective antigen

    PubMed Central

    Oscherwitz, Jon; Quinn, Conrad P.; Cease, Kemp B.

    2015-01-01

    Background Epitope-focused immunogens can elicit antibody against the loop neutralizing determinant (LND), a neutralizing epitope found within the 2β2-2β3 loop of protective antigen (PA), which can protect rabbits from high-dose inhalation challenge with Bacillus anthracis Ames strain. Interestingly, data suggests that this epitope is relatively immunosilent in rabbits and non-human primates immunized with full length PA. Methods To determine whether the LND is immunosilent among humans vaccinated with PA, we screened antisera from AVA- or placebo-vaccinees from a clinical trial for antibody reactive with the LND. Results AVA-vaccinee sera had significant PA-specific antibody compared to placebo-vaccinee sera; however, sera from the two cohorts were indistinguishable with regard to the frequency of individuals with antibody specific for the LND. Conclusions AVA-vaccinees have a low frequency of antibody reactive with the LND. As with rabbits and non-human primates, the elicitation of LND-specific antibody in humans appears to require immunization with an epitope-focused vaccine. PMID:25820066

  14. The Effects of Somatic Hypermutation on Neutralization and Binding in the PGT121 Family of Broadly Neutralizing HIV Antibodies

    PubMed Central

    Vigneault, Francois; Julien, Jean-Philippe; Briney, Bryan; Ramos, Alejandra; Saye, Karen F.; Le, Khoa; Mahan, Alison; Wang, Shenshen; Kardar, Mehran; Yaari, Gur; Walker, Laura M.; Simen, Birgitte B.; St. John, Elizabeth P.; Chan-Hui, Po-Ying; Swiderek, Kristine; Kleinstein, Stephen H.; Alter, Galit; Seaman, Michael S.; Chakraborty, Arup K.; Koller, Daphne; Wilson, Ian A.; Church, George M.; Burton, Dennis R.; Poignard, Pascal

    2013-01-01

    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. PMID:24278016

  15. Ergonomically neutral arm support system

    DOEpatents

    Siminovitch, Michael J; Chung, Jeffrey Y; Dellinges, Steven; Lafever, Robin E

    2005-08-02

    An ergonomic arm support system maintains a neutral position for the forearm. A mechanical support structure attached to a chair or other mounting structure supports the arms of a sitting or standing person. The system includes moving elements and tensioning elements to provide a dynamic balancing force against the forearms. The support structure is not fixed or locked in a rigid position, but is an active dynamic system that is maintained in equipoise by the continuous operation of the opposing forces. The support structure includes an armrest connected to a flexible linkage or articulated or pivoting assembly, which includes a tensioning element such as a spring. The pivoting assembly moves up and down, with the tensioning element providing the upward force that balances the downward force of the arm.

  16. Cross-clade neutralizing antibodies against HIV-1 induced in rabbits by focusing the immune response on a neutralizing epitope

    SciTech Connect

    Zolla-Pazner, Susan; Cohen, Sandra; Pinter, Abraham; Krachmarov, Chavdar; Wrin, Terri; Wang Shixia; Lu Shan

    2009-09-15

    Studies were performed to induce cross-clade neutralizing antibodies (Abs) by testing various combinations of prime and boost constructs that focus the immune response on structurally-conserved epitopes in the V3 loop of HIV-1 gp120. Rabbits were immunized with gp120 DNA containing a V3 loop characterized by the GPGR motif at its tip, and/or with gp120 DNA with a V3 loop carrying the GPGQ motif. Priming was followed by boosts with V3-fusion proteins (V3-FPs) carrying the V3 sequence from a subtype B virus (GPGR motif), and/or with V3 sequences from subtypes A and C (GPGQ motif). The broadest and most consistent neutralizing responses were generated when using a clade C gp120 DNA prime and with the V3{sub B}-FP boost. Immune sera displayed neutralizing activity in three assays against pseudoviruses and primary isolates from subtypes A, AG, B, C, and D. Polyclonal Abs in the immune rabbit sera neutralized viruses that were not neutralized by pools of human anti-V3 monoclonal Abs. Greater than 80% of the neutralizing Abs were specific for V3, showing that the immune response could be focused on a neutralizing epitope and that vaccine-induced anti-V3 Abs have cross-clade neutralizing activity.

  17. Antibody to gp41 MPER Alters Functional Properties of HIV-1 Env without Complete Neutralization

    PubMed Central

    Kim, Arthur S.; Leaman, Daniel P.; Zwick, Michael B.

    2014-01-01

    Human antibody 10E8 targets the conserved membrane proximal external region (MPER) of envelope glycoprotein (Env) subunit gp41 and neutralizes HIV-1 with exceptional potency. Remarkably, HIV-1 containing mutations that reportedly knockout 10E8 binding to linear MPER peptides are partially neutralized by 10E8, producing a local plateau in the dose response curve. Here, we found that virus partially neutralized by 10E8 becomes significantly less neutralization sensitive to various MPER antibodies and to soluble CD4 while becoming significantly more sensitive to antibodies and fusion inhibitors against the heptad repeats of gp41. Thus, 10E8 modulates sensitivity of Env to ligands both pre- and post-receptor engagement without complete neutralization. Partial neutralization by 10E8 was influenced at least in part by perturbing Env glycosylation. With unliganded Env, 10E8 bound with lower apparent affinity and lower subunit occupancy to MPER mutant compared to wild type trimers. However, 10E8 decreased functional stability of wild type Env while it had an opposite, stabilizing effect on MPER mutant Envs. Clade C isolates with natural MPER polymorphisms also showed partial neutralization by 10E8 with altered sensitivity to various gp41-targeted ligands. Our findings suggest a novel mechanism of virus neutralization by demonstrating how antibody binding to the base of a trimeric spike cross talks with adjacent subunits to modulate Env structure and function. The ability of an antibody to stabilize, destabilize, partially neutralize as well as alter neutralization sensitivity of a virion spike pre- and post-receptor engagement may have implications for immunotherapy and vaccine design. PMID:25058619

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

    PubMed

    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. PMID:26748387

  19. Isolation of Potent CGRP Neutralizing Antibodies Using Four Simple Assays.

    PubMed

    Neal, Frances; Arnold, Joanne; Rossant, Christine J; Podichetty, Sadhana; Lowne, David; Dobson, Claire; Wilkinson, Trevor; Colley, Caroline; Howes, Rob; Vaughan, Tristan J

    2016-01-01

    Calcitonin gene-related peptide (CGRP) is a small neuropeptide and a potent vasodilator that is widely associated with chronic pain and migraine. An antibody that inhibits CGRP function would be a potential therapeutic for treatment of these disorders. Here we describe the isolation of highly potent antibodies to CGRP from phage and ribosome display libraries and characterization of their epitope, species cross-reactivity, kinetics, and functional activity. Homogenous time-resolved fluorescence (HTRF) binding assays identified antibodies with the desired species cross-reactivity from naïve libraries, and HTRF epitope competition assays were used to characterize and group scFv by epitope. The functional inhibition of CGRP and species cross-reactivity of purified scFv and antibodies were subsequently confirmed using cAMP assays. We show that epitope competition assays could be used as a surrogate for functional cell-based assays during affinity maturation, in combination with scFv off-rate ranking by biolayer interferometry (BLI). This is the first time it has been shown that off-rate ranking can be predictive of functional activity for anti-CGRP antibodies. Here we demonstrate how, by using just four simple assays, diverse panels of antibodies to CGRP can be identified. These assay formats have potential utility in the identification of antibodies to other therapeutic targets. PMID:26450103

  20. The modification of fluorescent antibody virus neutralization (FAVN) test for the detection of antibodies to rabies virus.

    PubMed

    Hostnik, P

    2000-08-01

    The fluorescent antibody virus neutralization test (FAVN) for the detection of antibodies against rabies virus was modified by using a monoclonal anti-rabies antibodies and peroxidase anti-mouse conjugate instead of a fluorescent anti-rabies conjugate. The results were read on an automatic multi-channel spectrophotometer. A total of 182 serum samples from dogs were tested by both the original and modified FAVN methods and the results were compared. Good correlation was found between the two tests. Practically, the modified FAVN test was quicker and could be used for a larger number of samples. PMID:11014062

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

    PubMed Central

    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

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

    PubMed

    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. PMID:25522326

  3. Discovering neutralizing antibodies targeting the stem epitope of H1N1 influenza hemagglutinin with synthetic phage-displayed antibody libraries

    PubMed Central

    Tung, Chao-Ping; Chen, Ing-Chien; Yu, Chung-Ming; Peng, Hung-Pin; Jian, Jhih-Wei; Ma, Shiou-Hwa; Lee, Yu-Ching; Jan, Jia-Tsrong; Yang, An-Suei

    2015-01-01

    Broadly neutralizing antibodies developed from the IGHV1–69 germline gene are known to bind to the stem region of hemagglutinin in diverse influenza viruses but the sequence determinants for the antigen recognition, including neutralization potency and binding affinity, are not clearly understood. Such understanding could inform designs of synthetic antibody libraries targeting the stem epitope on hemagglutinin, leading to artificially designed antibodies that are functionally advantageous over antibodies from natural antibody repertoires. In this work, the sequence space of the complementarity determining regions of a broadly neutralizing antibody (F10) targeting the stem epitope on the hemagglutinin of a strain of H1N1 influenza virus was systematically explored; the elucidated antibody-hemagglutinin recognition principles were used to design a phage-displayed antibody library, which was then used to discover neutralizing antibodies against another strain of H1N1 virus. More than 1000 functional antibody candidates were selected from the antibody library and were shown to neutralize the corresponding strain of influenza virus with up to 7 folds higher potency comparing with the parent F10 antibody. The antibody library could be used to discover functionally effective antibodies against other H1N1 influenza viruses, supporting the notion that target-specific antibody libraries can be designed and constructed with systematic sequence-function information. PMID:26456860

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

    SciTech Connect

    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.

    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.

  5. Functional analysis of neutralizing antibodies against Clostridium perfringens epsilon-toxin.

    PubMed

    McClain, Mark S; Cover, Timothy L

    2007-04-01

    The Clostridium perfringens epsilon-toxin causes a severe, often fatal illness (enterotoxemia) characterized by cardiac, pulmonary, kidney, and brain edema. In this study, we examined the activities of two neutralizing monoclonal antibodies against the C. perfringens epsilon-toxin. Both antibodies inhibited epsilon-toxin cytotoxicity towards cultured MDCK cells and inhibited the ability of the toxin to form pores in the plasma membranes of cells, as shown by staining cells with the membrane-impermeant dye 7-aminoactinomycin D. Using an antibody competition enzyme-linked immunosorbent assay (ELISA), a peptide array, and analysis of mutant toxins, we mapped the epitope recognized by one of the neutralizing monoclonal antibodies to amino acids 134 to 145. The antibody competition ELISA and analysis of mutant toxins suggest that the second neutralizing monoclonal antibody also recognizes an epitope in close proximity to this region. The region comprised of amino acids 134 to 145 overlaps an amphipathic loop corresponding to the putative membrane insertion domain of the toxin. Identifying the epitopes recognized by these neutralizing antibodies constitutes an important first step in the development of therapeutic agents that could be used to counter the effects of the epsilon-toxin. PMID:17261609

  6. Anti–IL-6 neutralizing antibody modulates blood-brain barrier function in the ovine fetus

    PubMed Central

    Zhang, Jiyong; Sadowska, Grazyna B.; Chen, Xiaodi; Park, Seon Yeong; Kim, Jeong-Eun; Bodge, Courtney A.; Cummings, Erin; Lim, Yow-Pin; Makeyev, Oleksandr; Besio, Walter G.; Gaitanis, John; Banks, William A.; Stonestreet, Barbara S.

    2015-01-01

    Impaired blood-brain barrier function represents an important component of hypoxic-ischemic brain injury in the perinatal period. Proinflammatory cytokines could contribute to ischemia-related blood-brain barrier dysfunction. IL-6 increases vascular endothelial cell monolayer permeability in vitro. However, contributions of IL-6 to blood-brain barrier abnormalities have not been examined in the immature brain in vivo. We generated pharmacologic quantities of ovine-specific neutralizing anti-IL-6 mAbs and systemically infused mAbs into fetal sheep at 126 days of gestation after exposure to brain ischemia. Anti–IL-6 mAbs were measured by ELISA in fetal plasma, cerebral cortex, and cerebrospinal fluid, blood-brain barrier permeability was quantified using the blood-to-brain transfer constant in brain regions, and IL-6, tight junction proteins, and plasmalemma vesicle protein (PLVAP) were detected by Western immunoblot. Anti–IL-6 mAb infusions resulted in increases in mAb (P < 0.05) in plasma, brain parenchyma, and cerebrospinal fluid and decreases in brain IL-6 protein. Twenty-four hours after ischemia, anti–IL-6 mAb infusions attenuated ischemia-related increases in blood-brain barrier permeability and modulated tight junction and PLVAP protein expression in fetal brain. We conclude that inhibiting the effects of IL-6 protein with systemic infusions of neutralizing antibodies attenuates ischemia-related increases in blood-brain barrier permeability by inhibiting IL-6 and modulates tight junction proteins after ischemia.—Zhang, J., Sadowska, G. B., Chen, X., Park, S. Y., Kim, J.-E., Bodge, C. A., Cummings, E., Lim, Y.-P., Makeyev, O., Besio, W. G., Gaitanis, J., Banks, W. A., Stonestreet, B. S. Anti–IL-6 neutralizing antibody modulates blood-brain barrier function in the ovine fetus. PMID:25609424

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

    PubMed

    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. PMID:27199430

  8. A high-throughput neutralizing assay for antibodies and sera against hepatitis E virus

    PubMed Central

    Cai, Wei; Tang, Zi-Min; Wen, Gui-Ping; Wang, Si-Ling; Ji, Wen-Fang; Yang, Min; Ying, Dong; Zheng, Zi-Zheng; Xia, Ning-Shao

    2016-01-01

    Hepatitis E virus (HEV) is the aetiological agent of enterically transmitted hepatitis. The traditional methods for evaluating neutralizing antibody titres against HEV are real-time PCR and the immunofluorescence foci assay (IFA), which are poorly repeatable and operationally complicated, factors that limit their applicability to high-throughput assays. In this study, we developed a novel high-throughput neutralizing assay based on biotin-conjugated p239 (HEV recombinant capsid proteins, a.a. 368–606) and staining with allophycocyanin-conjugated streptavidin (streptavidin APC) to amplify the fluorescence signal. A linear regression analysis indicated that there was a high degree of correlation between IFA and the novel assay. Using this method, we quantitatively evaluated the neutralization of sera from HEV-infected and vaccinated macaques. The anti-HEV IgG level had good concordance with the neutralizing titres of macaque sera. However, the neutralization titres of the sera were also influenced by anti-HEV IgM responses. Further analysis also indicated that, although vaccination with HEV vaccine stimulated higher anti-HEV IgG and neutralization titres than infection with HEV in macaques, the proportions of neutralizing antibodies in the infected macaques’ sera were higher than in the vaccinated macaques with the same anti-HEV IgG levels. Thus, the infection more efficiently stimulated neutralizing antibody responses. PMID:27122081

  9. A quantitative test to estimate neutralizing antibodies to the hepatitis C virus: cytofluorimetric assessment of envelope glycoprotein 2 binding to target cells.

    PubMed Central

    Rosa, D; Campagnoli, S; Moretto, C; Guenzi, E; Cousens, L; Chin, M; Dong, C; Weiner, A J; Lau, J Y; Choo, Q L; Chien, D; Pileri, P; Houghton, M; Abrignani, S

    1996-01-01

    Hepatitis C virus (HCV) is a major cause of chronic hepatitis. The virus does not replicate efficiently in cell cultures, and it is therefore difficult to assess infection-neutralizing antibodies and to evaluate protective immunity in vitro. To study the binding of the HCV envelope to cell-surface receptors, we developed an assay to assess specific binding of recombinant envelope proteins to human cells and neutralization thereof. HCV recombinant envelope proteins expressed in various systems were incubated with human cells, and binding was assessed by flow cytometry using anti-envelope antibodies. Envelope glycoprotein 2 (E2) expressed in mammalian cells, but not in yeast or insect cells, binds human cells with high affinity (Kd approximately 10(-8) M). We then assessed antibodies able to neutralize E2 binding in the sera of both vaccinated and carrier chimpanzees, as well as in the sera of humans infected with various HCV genotypes. Vaccination with recombinant envelope proteins expressed in mammalian cells elicited high titers of neutralizing antibodies that correlated with protection from HCV challenge. HCV infection does not elicit neutralizing antibodies in most chimpanzees and humans, although low titers of neutralizing antibodies were detectable in a minority of infections. The ability to neutralize binding of E2 derived from the HCV-1 genotype was equally distributed among sera from patients infected with HCV genotypes 1, 2, and 3, demonstrating that binding of E2 is partly independent of E2 hypervariable regions. However, a mouse monoclonal antibody raised against the E2 hypervariable region 1 can partially neutralize binding of E2, indicating that at least two neutralizing epitopes, one of which is hypervariable, should exist on the E2 protein. The neutralization-of-binding assay described will be useful to study protective immunity to HCV infection and for vaccine development. PMID:8700831

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

    PubMed Central

    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

  11. Mechanisms of enhanced neutralization of botulinum neurotoxin by monoclonal antibodies conjugated to antibodies specific for the erythrocyte complement receptor.

    PubMed

    Sharma, Rashmi; Zhao, Huiwu; Al-Saleem, Fetweh H; Ubaid, Ahmed Syed; Puligedda, Rama Devudu; Segan, Andrew T; Lindorfer, Margaret A; Bermudez, Rodney; Elias, Md; Adekar, Sharad P; Simpson, Lance L; Taylor, Ronald P; Dessain, Scott K

    2014-02-01

    Immune complexes formed between monoclonal antibodies (mAbs) and toxins can neutralize toxicity in vivo by multiple mechanisms. Toxin sequestration and clearance by mAbs may be improved by enhancing their ability to bind to red blood cells (RBCs) through immune adherence. This can be achieved by converting the mAbs to heteropolymers (HPs), which are antigen-specific mAbs cross-linked to mAbs targeting the complement receptor (CR1), a protein that is expressed on the surface of RBCs in primates and mediates delivery of complement C3b-containing immune complexes to tissue macrophages. Conversion of mAbs to HPs has been shown to enhance clearance of multivalent antigens from the blood circulation, but the interaction of HPs with monovalent toxins has not been examined. Using botulinum neurotoxin (BoNT) as a model system, we studied the effect of conversion of a pair of BoNT-specific mAbs into HPs on toxin neutralization and handling in vivo. Two HPs given in combination had 166-fold greater potency than un-modified mAbs, neutralizing 5000 LD50 BoNT, when tested in transgenic mice expressing human CR1 on RBC membranes. Improvement required adherence of BoNT to the RBC in vivo and 2 HPs, rather than an HP+mAb pair. The HP pair bound BoNT to RBCs in the circulation for 2h, in comparison to BoNT-neutralizing anti-serum, which induced no detectable RBC binding. HP pairs exhibited enhanced uptake by peritoneal macrophages in vitro, compared to pairs of mAbs or mAb+HP pairs. In a post-exposure therapeutic model, HPs gave complete protection from a lethal BoNT dose up to 3h after toxin exposure. In a pre-exposure prophylaxis model, mice given HP up to 5 days prior to BoNT administration were fully protected from a lethal BoNT dose. These studies elucidate general mechanisms for the neutralization of toxins by HP pairs and demonstrate the potential utility of HPs as BoNT therapeutics. PMID:24184879

  12. Neutralizing antibody response in dogs and cats inoculated with commercial inactivated rabies vaccines.

    PubMed

    Shiraishi, Rikiya; Nishimura, Masaaki; Nakashima, Ryuji; Enta, Chiho; Hirayama, Norio

    2014-04-01

    In Japan, the import quarantine regulation against rabies has required from 2005 that dogs and cats should be inoculated with the rabies vaccine and that the neutralizing antibody titer should be confirmed to be at least 0.5 international units (IU)/ml. The fluorescent antibody virus neutralization (FAVN) test is used as an international standard method for serological testing for rabies. To achieve proper immunization of dogs and cats at the time of import and export, changes in the neutralizing antibody titer after inoculation of the rabies vaccine should be understood in detail. However, few reports have provided this information. In this study, we aimed to determine evaluated, such changes by using sera from experimental dogs and cats inoculated with the rabies vaccine, and we tested samples using the routine FAVN test. In both dogs and cats, proper, regular vaccination enabled the necessary titer of neutralizing antibodies to be maintained in the long term. However, inappropriate timing of blood sampling after vaccination could result in insufficient detected levels of neutralizing antibodies. PMID:24389741

  13. Neutralizing Antibody Response in Dogs and Cats Inoculated with Commercial Inactivated Rabies Vaccines

    PubMed Central

    SHIRAISHI, Rikiya; NISHIMURA, Masaaki; NAKASHIMA, Ryuji; ENTA, Chiho; HIRAYAMA, Norio

    2013-01-01

    ABSTRACT In Japan, the import quarantine regulation against rabies has required from 2005 that dogs and cats should be inoculated with the rabies vaccine and that the neutralizing antibody titer should be confirmed to be at least 0.5 international units (IU)/ml. The fluorescent antibody virus neutralization (FAVN) test is used as an international standard method for serological testing for rabies. To achieve proper immunization of dogs and cats at the time of import and export, changes in the neutralizing antibody titer after inoculation of the rabies vaccine should be understood in detail. However, few reports have provided this information. In this study, we aimed to determine evaluated, such changes by using sera from experimental dogs and cats inoculated with the rabies vaccine, and we tested samples using the routine FAVN test. In both dogs and cats, proper, regular vaccination enabled the necessary titer of neutralizing antibodies to be maintained in the long term. However, inappropriate timing of blood sampling after vaccination could result in insufficient detected levels of neutralizing antibodies. PMID:24389741

  14. Use of Monoclonal Antibodies in the Sensitive Detection and Neutralization of Botulinum Neurotoxin Serotype B

    PubMed Central

    Cheng, Luisa W.; Henderson, Thomas D.; Lam, Tina I.; Stanker, Larry H.

    2015-01-01

    Botulinum neurotoxins (BoNT) are some of nature’s most potent toxins. Due to potential food contamination, and bioterrorism concerns, the development of detection reagents, therapeutics and countermeasures are of urgent interest. Recently, we have developed a sensitive electrochemiluminescent (ECL) immunoassay for BoNT/B, using monoclonal antibodies (mAbs) MCS6-27 and anti-BoNT/B rabbit polyclonal antibodies as the capture and detector. The ECL assay detected as little as 1 pg/mL BoNT/B in the buffer matrix, surpassing the detection sensitivities of the gold standard mouse bioassays. The ECL assay also allowed detection of BoNT/B in sera matrices of up to 100% sera with negligible matrix effects. This highly-sensitive assay allowed the determination of the biological half-lives of BoNT/B holotoxin in vivo. We further tested the toxin neutralization potential of our monoclonal antibodies using the mouse systemic and oral intoxication models. A combination of mAbs protected mice in both pre- and post-exposure models to lethal doses of BoNT/B. MAbs were capable of increasing survival of animals when administered even 10 h post-intoxication in an oral model, suggesting a likely time for BoNT/B complexes to reach the blood stream. More sensitive detection assays and treatments against BoNT intoxication will greatly enhance efforts to combat botulism. PMID:26633496

  15. Use of Monoclonal Antibodies in the Sensitive Detection and Neutralization of Botulinum Neurotoxin Serotype B.

    PubMed

    Cheng, Luisa W; Henderson, Thomas D; Lam, Tina I; Stanker, Larry H

    2015-12-01

    Botulinum neurotoxins (BoNT) are some of nature's most potent toxins. Due to potential food contamination, and bioterrorism concerns, the development of detection reagents, therapeutics and countermeasures are of urgent interest. Recently, we have developed a sensitive electrochemiluminescent (ECL) immunoassay for BoNT/B, using monoclonal antibodies (mAbs) MCS6-27 and anti-BoNT/B rabbit polyclonal antibodies as the capture and detector. The ECL assay detected as little as 1 pg/mL BoNT/B in the buffer matrix, surpassing the detection sensitivities of the gold standard mouse bioassays. The ECL assay also allowed detection of BoNT/B in sera matrices of up to 100% sera with negligible matrix effects. This highly-sensitive assay allowed the determination of the biological half-lives of BoNT/B holotoxin in vivo. We further tested the toxin neutralization potential of our monoclonal antibodies using the mouse systemic and oral intoxication models. A combination of mAbs protected mice in both pre- and post-exposure models to lethal doses of BoNT/B. MAbs were capable of increasing survival of animals when administered even 10 h post-intoxication in an oral model, suggesting a likely time for BoNT/B complexes to reach the blood stream. More sensitive detection assays and treatments against BoNT intoxication will greatly enhance efforts to combat botulism. PMID:26633496

  16. Characterization of Neutralizing Antibodies and Identification of Neutralizing Epitope Mimics on the Clostridium botulinum Neurotoxin Type A

    PubMed Central

    Wu, Han-Chung; Yeh, Chia-Tsui; Huang, Yue-Ling; Tarn, Lih-Jeng; Lung, Chien-Cheng

    2001-01-01

    Clostridium botulinum neurotoxin type A (BTx-A) is known to inhibit the release of acetylcholine at the neuromuscular junctions and synapses and to cause neuroparalysis and death. In this study, we have identified two monoclonal antibodies, BT57-1 and BT150-3, which protect ICR mice against lethal doses of BTx-A challenge. The neutralizing activities for BT57-1 and BT150-3 were 103 and 104 times the 50% lethal dose, respectively. Using immunoblotting analysis, BT57-1 was recognized as a light chain and BT150-3 was recognized as a heavy chain of BTx-A. Also, applying the phage display method, we investigated the antibodies' neutralizing B-cell epitopes. These immunopositive phage clones displayed consensus motifs, Asp-Pro-Leu for BT57-1 and Cys-X-Asp-Cys for BT150. The synthetic peptide P4M (KGTFDPLQEPRT) corresponded to the phage-displayed peptide selected by BT57-1 and was able to bind the antibodies specifically. This peptide was also shown by competitive inhibition assay to be able to inhibit phage clone binding to BT57-1. Aspartic acid (D5) in P4M was crucial to the binding of P4M to BT57-1, since its binding activity dramatically decreased when it was changed to lysine (K5). Finally, immunizing mice with the selected phage clones elicited a specific humoral response against BTx-A. These results suggest that phage-displayed random-peptide libraries are useful in identifying the neutralizing epitopes of monoclonal antibodies. In the future, the identification of the neutralizing epitopes of BTx-A may provide important information for the identification of the BTx-A receptor and the design of a BTx-A vaccine. PMID:11425742

  17. Antibodies Targeting Novel Neutralizing Epitopes of Hepatitis C Virus Glycoprotein Preclude Genotype 2 Virus Infection.

    PubMed

    Deng, Kai; Liu, Ruyu; Rao, Huiying; Jiang, Dong; Wang, Jianghua; Xie, Xingwang; Wei, Lai

    2015-01-01

    Currently, there is no effective vaccine to prevent hepatitis C virus (HCV) infection, partly due to our insufficient understanding of the virus glycoprotein immunology. Most neutralizing antibodies (nAbs) were identified using glycoprotein immunogens, such as recombinant E1E2, HCV pseudoparticles or cell culture derived HCV. However, the fact that in the HCV acute infection phase, only a small proportion of patients are self-resolved accompanied with the emergence of nAbs, indicates the limited immunogenicity of glycoprotein itself to induce effective antibodies against a highly evolved virus. Secondly, in previous reports, the immunogen sequence was mostly the genotype of the 1a H77 strain. Rarely, other genotypes/subtypes have been studied, although theoretically one genotype/subtype immunogen is able to induce cross-genotype neutralizing antibodies. To overcome these drawbacks and find potential novel neutralizing epitopes, 57 overlapping peptides encompassing the full-length glycoprotein E1E2 of subtype 1b were synthesized to immunize BALB/c mice, and the neutralizing reactive of the induced antisera against HCVpp genotypes 1-6 was determined. We defined a domain comprising amino acids (aa) 192-221, 232-251, 262-281 and 292-331 of E1, and 421-543, 564-583, 594-618 and 634-673 of E2, as the neutralizing regions of HCV glycoprotein. Peptides PUHI26 (aa 444-463) and PUHI45 (aa 604-618)-induced antisera displayed the most potent broad neutralizing reactive. Two monoclonal antibodies recognizing the PUHI26 and PUHI45 epitopes efficiently precluded genotype 2 viral (HCVcc JFH and J6 strains) infection, but they did not neutralize other genotypes. Our study mapped a neutralizing epitope region of HCV glycoprotein using a novel immunization strategy, and identified two monoclonal antibodies effective in preventing genotype 2 virus infection. PMID:26406225

  18. Antibodies Targeting Novel Neutralizing Epitopes of Hepatitis C Virus Glycoprotein Preclude Genotype 2 Virus Infection

    PubMed Central

    Rao, Huiying; Jiang, Dong; Wang, Jianghua; Xie, Xingwang; Wei, Lai

    2015-01-01

    Currently, there is no effective vaccine to prevent hepatitis C virus (HCV) infection, partly due to our insufficient understanding of the virus glycoprotein immunology. Most neutralizing antibodies (nAbs) were identified using glycoprotein immunogens, such as recombinant E1E2, HCV pseudoparticles or cell culture derived HCV. However, the fact that in the HCV acute infection phase, only a small proportion of patients are self-resolved accompanied with the emergence of nAbs, indicates the limited immunogenicity of glycoprotein itself to induce effective antibodies against a highly evolved virus. Secondly, in previous reports, the immunogen sequence was mostly the genotype of the 1a H77 strain. Rarely, other genotypes/subtypes have been studied, although theoretically one genotype/subtype immunogen is able to induce cross-genotype neutralizing antibodies. To overcome these drawbacks and find potential novel neutralizing epitopes, 57 overlapping peptides encompassing the full-length glycoprotein E1E2 of subtype 1b were synthesized to immunize BALB/c mice, and the neutralizing reactive of the induced antisera against HCVpp genotypes 1–6 was determined. We defined a domain comprising amino acids (aa) 192–221, 232–251, 262–281 and 292–331 of E1, and 421–543, 564–583, 594–618 and 634–673 of E2, as the neutralizing regions of HCV glycoprotein. Peptides PUHI26 (aa 444–463) and PUHI45 (aa 604–618)-induced antisera displayed the most potent broad neutralizing reactive. Two monoclonal antibodies recognizing the PUHI26 and PUHI45 epitopes efficiently precluded genotype 2 viral (HCVcc JFH and J6 strains) infection, but they did not neutralize other genotypes. Our study mapped a neutralizing epitope region of HCV glycoprotein using a novel immunization strategy, and identified two monoclonal antibodies effective in preventing genotype 2 virus infection. PMID:26406225

  19. Recombinant sheep pox virus proteins elicit neutralizing antibodies

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The aim of this study was to evaluate the immunogenicity and neutralizing activity of bacterially-expressed sheep pox virus (SPPV) structural proteins as candidate subunit vaccines to control sheep pox disease. SPPV structural proteins were identified by sequence homology with proteins from vaccinia...

  20. Protective antiviral antibodies that lack neutralizing activity: precedents and evolution of concepts.

    PubMed

    Schmaljohn, Alan L

    2013-07-01

    Antibody-mediated resistance to viral disease is often attributed solely to neutralizing antibodies (NAbs) despite a body of evidence -- more than 30 years in the making -- to show that other populations of antibodies (protective non-neutralizing antibodies, PnNAbs) can also contribute and are sometimes pivotal in host resistance to viruses. Recently, interest in varieties of PnNAbs has been restored and elevated by an HIV vaccine trial in which virus-specific nNAbs have been highlighted as a positive correlate of immunity. Here, I briefly review some of the historical precedents with many viruses other than HIV, along with the emergence of data over the course of some four decades, pointing emphatically to the importance of subsets of antiviral antibodies that operate by mechanisms other than classical virus neutralization. Foremost among suspected mechanisms of protection by PnNAbs is antibody-dependent cellular cytotoxicty (ADCC), but additional mechanisms have sometimes been incriminated or not experimentally excluded. Examples are given for the diversity of proteins and cognate epitopes bound by PnNAbs. Some such epitopes are restricted to virus-infected cell surfaces or found on secreted proteins; others may be associated with virions but unavailable to antibodies during much of the viral cycle; these are epitopes variously described as cryptic, transitional, dynamic, or reversibly masked. PMID:24191933

  1. Neutralizing antibodies derived from the B cells of 1918 influenza pandemic survivors.

    PubMed

    Yu, Xiaocong; Tsibane, Tshidi; McGraw, Patricia A; House, Frances S; Keefer, Christopher J; Hicar, Mark D; Tumpey, Terrence M; Pappas, Claudia; Perrone, Lucy A; Martinez, Osvaldo; Stevens, James; Wilson, Ian A; Aguilar, Patricia V; Altschuler, Eric L; Basler, Christopher F; Crowe, James E

    2008-09-25

    Investigation of the human antibody response to influenza virus infection has been largely limited to serology, with relatively little analysis at the molecular level. The 1918 H1N1 influenza virus pandemic was the most severe of the modern era. Recent work has recovered the gene sequences of this unusual strain, so that the 1918 pandemic virus could be reconstituted to display its unique virulence phenotypes. However, little is known about adaptive immunity to this virus. We took advantage of the 1918 virus sequencing and the resultant production of recombinant 1918 haemagglutinin (HA) protein antigen to characterize at the clonal level neutralizing antibodies induced by natural exposure of survivors to the 1918 pandemic virus. Here we show that of the 32 individuals tested that were born in or before 1915, each showed seroreactivity with the 1918 virus, nearly 90 years after the pandemic. Seven of the eight donor samples tested had circulating B cells that secreted antibodies that bound the 1918 HA. We isolated B cells from subjects and generated five monoclonal antibodies that showed potent neutralizing activity against 1918 virus from three separate donors. These antibodies also cross-reacted with the genetically similar HA of a 1930 swine H1N1 influenza strain, but did not cross-react with HAs of more contemporary human influenza viruses. The antibody genes had an unusually high degree of somatic mutation. The antibodies bound to the 1918 HA protein with high affinity, had exceptional virus-neutralizing potency and protected mice from lethal infection. Isolation of viruses that escaped inhibition suggested that the antibodies recognize classical antigenic sites on the HA surface. Thus, these studies demonstrate that survivors of the 1918 influenza pandemic possess highly functional, virus-neutralizing antibodies to this uniquely virulent virus, and that humans can sustain circulating B memory cells to viruses for many decades after exposure-well into the tenth

  2. Temporal analysis of HIV envelope sequence evolution and antibody escape in a subtype A-infected individual with a broad neutralizing antibody response

    PubMed Central

    Bosch, Katherine A.; Rainwater, Stephanie; Jaoko, Walter; Overbaugh, Julie

    2010-01-01

    The origin of broadly neutralizing HIV-specific antibodies and their relation to HIV evolution are not well defined. Here we examined virus evolution and neutralizing antibody escape in a subtype A infected individual with a broad, cross subtype, antibody response. The majority of envelope variants isolated over the first ~ 5 years post-infection were poorly neutralized by contemporaneous plasma that neutralized variants from earlier in infection, consistent with a dynamic process of escape. The majority of variants could be neutralized by later plasma, suggesting these evolving variants may have contributed to the elicitation of new antibody responses. However, some variants from later in infection were recognized by plasma from earlier in infection, including one notably neutralization-sensitive variant that was sensitive due to a proline at position 199 in V2. These studies suggest a complex pattern of virus evolution in this individual with a broad NAb response, including persistence of neutralization-sensitive viruses. PMID:20034648

  3. Inhibition of HIV-1 Env-Mediated Cell-Cell Fusion by Lectins, Peptide T-20, and Neutralizing Antibodies

    PubMed Central

    Yee, Michael; Konopka, Krystyna; Balzarini, Jan; Düzgüneş, Nejat

    2011-01-01

    Background: Broadly cross-reactive, neutralizing human monoclonal antibodies, including 2F5, 2G12, 4E10 and IgG1 b12, can inhibit HIV-1 infection in vitro at very low concentrations. We examined the ability of these antibodies to inhibit cell-cell fusion between Clone69TRevEnv cells induced to express the viral envelope proteins, gp120/gp41 (Env), and highly CD4-positive SupT1 cells. The cells were loaded with green and red-orange cytoplasmic fluorophores, and fusion was monitored by fluorescence microscopy. Results: Cell-cell fusion was inhibited completely by the carbohydrate binding proteins (CBPs), Hippeastrum hybrid (Amaryllis) agglutinin (HHA), and Galanthus nivalis (Snowdrop) agglutinin (GNA), and by the peptide, T-20, at relatively low concentrations. Anti-gp120 and anti-gp41 antibodies, at concentrations much higher than those required for neutralization, were not particularly effective in inhibiting fusion. Monoclonal antibodies b12, m14 IgG and 2G12 had moderate inhibitory activity; the IC50 of 2G12 was about 80 µg/ml. Antibodies 4E10 and 2F5 had no inhibitory activity at the concentrations tested. Conclusions: These observations raise concerns about the ability of neutralizing antibodies to inhibit the spread of viral genetic material from infected cells to uninfected cells via cell-cell fusion. The interaction of gp120/gp41 with cell membrane CD4 may be different in cell-cell and virus-cell membrane fusion reactions, and may explain the differential effects of antibodies in these two systems. The fluorescence assay described here may be useful in high throughput screening of potential HIV fusion inhibitors. PMID:21660189

  4. Neutralizing Antibodies and Sin Nombre Virus RNA after Recovery from Hantavirus Cardiopulmonary Syndrome

    PubMed Central

    Ye, Chunyan; Prescott, Joseph; Nofchissey, Robert; Goade, Diane

    2004-01-01

    Patients who later have a mild course of hantavirus cardiopulmonary syndrome (HCPS) are more likely to exhibit a high titer of neutralizing antibodies against Sin Nombre virus (SNV), the etiologic agent of HCPS, at the time of hospital admission. Because administering plasma from patients who have recovered from HCPS to those in the early stages of disease may be an advantageous form of passive immunotherapy, we examined the neutralizing antibody titers of 21 patients who had recovered from SNV infection. Even 1,000 days after admission to the hospital, 6 of 10 patients had titers of 800 or higher, with one sample retaining a titer of 3,200 after more than 1,400 days. None of the convalescent-phase serum samples contained detectable viral RNA. These results confirm that patients retain high titers of neutralizing antibodies long after recovery from SNV infection. PMID:15109416

  5. Autophagy-associated dengue vesicles promote viral transmission avoiding antibody neutralization.

    PubMed

    Wu, Yan-Wei; Mettling, Clément; Wu, Shang-Rung; Yu, Chia-Yi; Perng, Guey-Chuen; Lin, Yee-Shin; Lin, Yea-Lih

    2016-01-01

    One of the major defense mechanisms against virus spread in vivo is the blocking of viral infectibility by neutralizing antibodies. We describe here the identification of infectious autophagy-associated dengue vesicles released from infected cells. These vesicles contain viral proteins E, NS1, prM/M, and viral RNA, as well as host lipid droplets and LC3-II, an autophagy marker. The viral RNA can be protected within the autophagic organelles since anti-dengue neutralizing antibodies do not have an effect on the vesicle-mediated transmission that is able to initiate a new round of infection in target cells. Importantly, such infectious vesicles were also detected in a patient serum. Our study suggests that autophagy machinery plays a new role in dengue virus transmission. This discovery explains the inefficiency of neutralizing antibody upon dengue infection as a potential immune evasion mechanism in vivo. PMID:27558165

  6. Autophagy-associated dengue vesicles promote viral transmission avoiding antibody neutralization

    PubMed Central

    Wu, Yan-Wei; Mettling, Clément; Wu, Shang-Rung; Yu, Chia-Yi; Perng, Guey-Chuen; Lin, Yee-Shin; Lin, Yea-Lih

    2016-01-01

    One of the major defense mechanisms against virus spread in vivo is the blocking of viral infectibility by neutralizing antibodies. We describe here the identification of infectious autophagy-associated dengue vesicles released from infected cells. These vesicles contain viral proteins E, NS1, prM/M, and viral RNA, as well as host lipid droplets and LC3-II, an autophagy marker. The viral RNA can be protected within the autophagic organelles since anti-dengue neutralizing antibodies do not have an effect on the vesicle-mediated transmission that is able to initiate a new round of infection in target cells. Importantly, such infectious vesicles were also detected in a patient serum. Our study suggests that autophagy machinery plays a new role in dengue virus transmission. This discovery explains the inefficiency of neutralizing antibody upon dengue infection as a potential immune evasion mechanism in vivo. PMID:27558165

  7. Depletion of interfering antibodies in chronic hepatitis C patients and vaccinated chimpanzees reveals broad cross-genotype neutralizing activity

    PubMed Central

    Zhang, Pei; Zhong, Lilin; Struble, Evi Budo; Watanabe, Hisayoshi; Kachko, Alla; Mihalik, Kathleen; Virata-Theimer, Maria Luisa; Alter, Harvey J.; Feinstone, Stephen; Major, Marian

    2009-01-01

    Using human immune globulins made from antihepatitis C virus (HCV)-positive plasma, we recently identified two antibody epitopes in the E2 protein at residues 412–426 (epitope I) and 434–446 (epitope II). Whereas epitope I is highly conserved among genotypes, epitope II varies. We discovered that epitope I was implicated in HCV neutralization whereas the binding of non-neutralizing antibody to epitope II disrupted virus neutralization mediated by antibody binding at epitope I. These findings suggested that, if this interfering mechanism operates in vivo during HCV infection, a neutralizing antibody against epitope I can be restrained by an interfering antibody, which may account for the persistence of HCV even in the presence of an abundance of neutralizing antibodies. We tested this hypothesis by affinity depletion and peptide-blocking of epitope-II-specific antibodies in plasma of a chronically HCV-infected patient and recombinant E1E2 vaccinated chimpanzees. We demonstrate that, by removing the restraints imposed by the interfering antibodies to epitope-II, neutralizing activity can be revealed in plasma that previously failed to neutralize viral stock in cell culture. Further, cross-genotype neutralization could be generated from monospecific plasma. Our studies contribute to understanding the mechanisms of antibody-mediated neutralization and interference and provide a practical approach to the development of more potent and broadly reactive hepatitis C immune globulins. PMID:19380744

  8. A survey for neutralizing antibodies to the three types of poliovirus among children in Maiduguri, Nigeria.

    PubMed

    Baba, M M; Haruna, B A; Ogunmola, O; Ambe, J P; Shidali, N N; Oderinde, B; Marcello, A; Talle, M

    2012-04-01

    The milestone in polio eradication program is to protect effectively children aged 0-5 years against the three serotypes of poliovirus. It became necessary to measure the level of neutralizing antibodies to the three poliovirus types in an endemic State in Nigeria. Neutralizing antibodies to the poliovirus types among children aged 0-5 years was estimated using micro neutralization assay. Of 129 children, 99 (76.8%), 95 (73.6%), and 95 (73.6%) had neutralizing antibodies with the geometric mean titer of 42.7, 31.3, and 33.2 for the poliovirus type 1, 2, and 3, respectively. Fifty-three percent of the children were protected against the three types of poliovirus. Combination of poliovirus types 1 and 2, 1 and 3, and 2 and 3 were neutralized by 62.8, 58.9, and 61.2% of the children studied, respectively. Only poliovirus type 1 induced antibody titres ≥1:1,024. The number of children with neutralizing antibodies after receiving three doses was significantly higher than those who received one or two doses of oral polio vaccine (P ≤ 0.05). However, those who received more than three doses of oral polio vaccine showed no significant difference in their antibody response. The existence of immunity gap poses a risk of re-emergence of the paralytic poliovirus. The existence of unimmunized and unprotected children along with high birth rate could impede the success of polio vaccination in Nigeria. Elimination of non-compliance to polio vaccine, promotion of health education and documented evidence of vaccination of each child with the parents may facilitate the success of polio eradication program in Nigeria. PMID:22337311

  9. Modeling neutralization of Shiga 2 toxin by A-and B-subunit-specific human monoclonal antibodies.

    PubMed

    Skakauskas, Vladas; Katauskis, Pranas

    2016-06-01

    A mathematical model for Shiga 2 toxin neutralization by A-and B-subunit-specific human monoclonal antibodies initially delivered in the extracellular domain is presented, taking into account toxin and antibodies interaction in the extracellular domain, diffusion of toxin, antibodies, and their reaction products toward the cell, the receptor-mediated toxin and complex composed of toxin and antibody to A-subunit internalization from the extracellular into the intracellular medium and excretion of this complex back to the extracellular environment via recycling endosomal carriers. The retrograde transport of the intact toxin to the endoplasmic reticulum and its anterograde movement back to the vicinity of the plasma membrane with its subsequent exocytotic removal to the extracellular space via the secretory vesicle pathway is also taken into account. The model is composed of a set of coupled PDEs. A mathematical model based on a system of ODEs for Shiga 2 toxin neutralization by antibodies in the absence of cell is also studied. Both PDE and ODE systems are solved numerically. Numerical results are illustrated by figures and discussed. PMID:27155978

  10. Phage Display-Derived Cross-Reactive Neutralizing Antibody against Enterovirus 71 and Coxsackievirus A16.

    PubMed

    Zhang, Xiao; Sun, Chunyun; Xiao, Xiangqian; Pang, Lin; Shen, Sisi; Zhang, Jie; Cen, Shan; Yang, Burton B; Huang, Yuming; Sheng, Wang; Zeng, Yi

    2016-01-01

    Enterovirus 71 (EV71) and coxsackievirus A16 (CVA16) are members of the Picornaviridae family and are considered the main causative agents of hand, foot and mouth disease (HFMD). In recent decades large HFMD outbreaks caused by EV71 and CVA16 have become significant public health concerns in the Asia-Pacific region. Vaccines and antiviral drugs are unavailable to prevent EV71 and CVA16 infection. In the current study, a chimeric antibody targeting a highly conserved peptide in the EV71 VP4 protein was isolated by using a phage display technique. The antibody showed cross-neutralizing capability against EV71 and CVA16 in vitro. The results suggest that this phage display-derived antibody will have great potential as a broad neutralizing antibody against EV71 and CVA16 after affinity maturation and humanization. PMID:26073737

  11. Virus mutation frequencies can be greatly underestimated by monoclonal antibody neutralization of virions.

    PubMed Central

    Holland, J J; de la Torre, J C; Steinhauer, D A; Clarke, D; Duarte, E; Domingo, E

    1989-01-01

    Monoclonal antibody-resistant mutants have been widely used to estimate virus mutation frequencies. We demonstrate that standard virion neutralization inevitably underestimates monoclonal antibody-resistant mutant genome frequencies of vesicular stomatitis virus, due to phenotypic masking-mixing when wild-type (wt) virions are present in thousandsfold greater numbers. We show that incorporation of antibody into the plaque overlay medium (after virus penetration at 37 degrees C) can provide accurate estimates of genome frequencies of neutral monoclonal antibody-resistant mutant viruses in wt clones. By using this method, we have observed two adjacent G----A base transition frequencies in the I3 epitope to be of the order of 10(-4) in a wt glycine codon. This appears to be slightly lower than the frequencies observed at other sites for total (viable and nonviable) virus genomes when using a direct sequence approach. Images PMID:2479770

  12. Role of neutralizing antibodies and T-cells in pathogenesis of herpes simplex virus infection in congenitally athymic mice.

    PubMed

    Kapoor, A K; Buckmaster, A; Nash, A A; Field, H J; Wildy, P

    1982-11-01

    Congenitally athymic nude mice were infected with 10(4) p.f.u. herpes simplex type 1 (strain SC16). Following the passive transfer of neutralizing monoclonal antibodies (AP7, AP8 and AP12) it was observed that AP7 alone reduced the virus infectivity in the nervous system; AP8 and AP12 failed to protect mice probably due to poor in vivo binding to the neutralization site on the virus. Latent ganglionic infection could be established in nude mice following adoptive transfer of optimum number (2 x 10(7) cells/mouse) of immune lymph node cells from day 7 herpes virus-infected hairy immunocompetent donor mice. Moreover, in some of the immune lymph node cell protected nudes, latency could be maintained even in complete absence of neutralizing antibodies. Results of ear-ablation experiments revealed that removal of primary source of infection after day 5 of infection reduced the amount of virus in the ganglia and spinal cord. Acute neurological infection was not detected following transfer of protective anti-gp-D neutralizing antibody (LP2) in combination with removal of infected pinna. These data suggest that continuous seeding of virus occurs in related ganglia via the axonal route from infected ear pinna. It appears that local T-cell-mediated immune mechanisms are involved in maintenance of latency. PMID:6984425

  13. Recombinant Sheep Pox Virus Proteins Elicit Neutralizing Antibodies

    PubMed Central

    Chervyakova, Olga V.; Zaitsev, Valentin L.; Iskakov, Bulat K.; Tailakova, Elmira T.; Strochkov, Vitaliy M.; Sultankulova, Kulyaisan T.; Sandybayev, Nurlan T.; Stanbekova, Gulshan E.; Beisenov, Daniyar K.; Abduraimov, Yergali O.; Mambetaliyev, Muratbay; Sansyzbay, Abylay R.; Kovalskaya, Natalia Y.; Nemchinov, Lev. G.; Hammond, Rosemarie W.

    2016-01-01

    The aim of this work was to evaluate the immunogenicity and neutralizing activity of sheep pox virus (SPPV; genus Capripoxvirus, family Poxviridae) structural proteins as candidate subunit vaccines to control sheep pox disease. SPPV structural proteins were identified by sequence homology with proteins of vaccinia virus (VACV) strain Copenhagen. Four SPPV proteins (SPPV-ORF 060, SPPV-ORF 095, SPPV-ORF 117, and SPPV-ORF 122), orthologs of immunodominant L1, A4, A27, and A33 VACV proteins, respectively, were produced in Escherichia coli. Western blot analysis revealed the antigenic and immunogenic properties of SPPV-060, SPPV-095, SPPV-117 and SPPV-122 proteins when injected with adjuvant into experimental rabbits. Virus-neutralizing activity against SPPV in lamb kidney cell culture was detected for polyclonal antisera raised to SPPV-060, SPPV-117, and SPPV-122 proteins. To our knowledge, this is the first report demonstrating the virus-neutralizing activities of antisera raised to SPPV-060, SPPV-117, and SPPV-122 proteins. PMID:27338444

  14. Recombinant Sheep Pox Virus Proteins Elicit Neutralizing Antibodies.

    PubMed

    Chervyakova, Olga V; Zaitsev, Valentin L; Iskakov, Bulat K; Tailakova, Elmira T; Strochkov, Vitaliy M; Sultankulova, Kulyaisan T; Sandybayev, Nurlan T; Stanbekova, Gulshan E; Beisenov, Daniyar K; Abduraimov, Yergali O; Mambetaliyev, Muratbay; Sansyzbay, Abylay R; Kovalskaya, Natalia Y; Nemchinov, Lev G; Hammond, Rosemarie W

    2016-01-01

    The aim of this work was to evaluate the immunogenicity and neutralizing activity of sheep pox virus (SPPV; genus Capripoxvirus, family Poxviridae) structural proteins as candidate subunit vaccines to control sheep pox disease. SPPV structural proteins were identified by sequence homology with proteins of vaccinia virus (VACV) strain Copenhagen. Four SPPV proteins (SPPV-ORF 060, SPPV-ORF 095, SPPV-ORF 117, and SPPV-ORF 122), orthologs of immunodominant L1, A4, A27, and A33 VACV proteins, respectively, were produced in Escherichia coli. Western blot analysis revealed the antigenic and immunogenic properties of SPPV-060, SPPV-095, SPPV-117 and SPPV-122 proteins when injected with adjuvant into experimental rabbits. Virus-neutralizing activity against SPPV in lamb kidney cell culture was detected for polyclonal antisera raised to SPPV-060, SPPV-117, and SPPV-122 proteins. To our knowledge, this is the first report demonstrating the virus-neutralizing activities of antisera raised to SPPV-060, SPPV-117, and SPPV-122 proteins. PMID:27338444

  15. A neutralizing scFv antibody against infectious bursal disease virus screened by flow cytometry.

    PubMed

    Zhou, Yao; Xie, Zhi-Gang

    2015-09-01

    Infectious bursal disease (IBD) is considered a vital viral disease that threatens the poultry industry worldwide. In this study, a recombinant single chain variable fragment (scFv) antibody library derived from chickens immunized with VP2 protein of infectious bursal disease virus (IBDV) was constructed. The library was subjected to three rounds of screening by flow cytometry (FCM) against VP2/IBDV through a bacteria display technology, resulting in the enrichment of scFvs. Three scFv clones with different fluorescence intensity were obtained by colony pick up at random. The obtained scFv antibodies were expressed and purified. Relative affinity assay showed the three clones had different sensitivity to VP2, in accordance with fluorescence activity cell sorting analysis (FACS). The potential use of the isolated IBDV-specific scFv antibodies was demonstrated by the successful application of these antibodies in Western blotting and ELISA assay. What's more, in vitro neutralization measurement showed that one of the three isolated antibodies possessed the neutralization function against IBDV. This study provides new strategies for screening of antibody library, and scFv antibodies isolated in this study may be utilized as lead candidates for further development of diagnostic or therapeutic antibodies for detection and treatment of IBDV infection. PMID:26003676

  16. Cross-Reactivity of Neutralizing Antibodies among Malignant Catarrhal Fever Viruses

    PubMed Central

    Taus, Naomi S.; Cunha, Cristina W.; Marquard, Jana; O’Toole, Donal; Li, Hong

    2015-01-01

    Some members of the gamma herpesvirus genus Macavirus are maintained in nature as subclinical infections in well-adapted ungulate hosts. Transmission of these viruses to poorly adapted hosts, such as American bison and cattle, can result in the frequently fatal disease malignant catarrhal fever (MCF). Based on phylogenetic analysis, the MCF viruses (MCFV) cluster into two subgroups corresponding to the reservoir hosts’ subfamilies: Alcelaphinae/Hippotraginae and Caprinae. Antibody cross-reactivity among MCFVs has been demonstrated using techniques such as enzyme linked immunosorbent and immunofluorescence assays. However, minimal information is available as to whether virus neutralizing antibodies generated against one MCFV cross react with other members of the genus. This study tested the neutralizing activity of serum and plasma from select MCFV-infected reservoir hosts against alcelaphine herpesvirus 1 (AlHV-1) and ovine herpesvirus 2 (OvHV-2). Neutralizing antibody activity against AlHV-1 was detected in samples from infected hosts in the Alcelaphinae and Hippotraginae subfamilies, but not from hosts in the Caprinae subfamily. OvHV-2 neutralizing activity was demonstrated in samples from goats (Caprinae) but not from wildebeest (Alcelaphinae). These results show that neutralizing antibody cross reactivity is present to MCFVs within a virus subgroup but not between subgroups. This information is important for diagnosing infection with MCFVs and in the development of vaccines against MCF. PMID:26658281

  17. A sensitive retroviral pseudotype assay for influenza H5N1‐neutralizing antibodies

    PubMed Central

    Temperton, Nigel J.; Hoschler, Katja; Major, Diane; Nicolson, Carolyn; Manvell, Ruth; Hien, Vo Minh; Ha, Do Quang; De Jong, Menno; Zambon, Maria; Takeuchi, Yasuhiro; Weiss, Robin A.

    2007-01-01

    Background  The World Health Organisation (WHO) recommended the development of simple, safe, sensitive and specific neutralization assays for avian influenza antibodies. We have used retroviral pseudotypes bearing influenza H5 hemagglutinin (HA) as safe, surrogate viruses for influenza neutralization assays which can be carried out at Biosafety Level 2. Results  Using our assay, sera from patients who had recovered from infection with influenza H5N1, and sera from animals experimentally immunized or infected with H5 tested positive for the presence of neutralizing antibodies to H5N1. Pseudotype neutralizing antibody titers were compared with titers obtained by hemagglutinin inhibition (HI) assays and microneutralization (MN) assays using live virus, and showed a high degree of correlation, sensitivity and specificity. Conclusions  The pseudotype neutralization assay is as sensitive as horse erythrocyte HI and MN for the detection of antibodies to H5N1. It is safer, and can be applied in a high‐throughput format for human and animal surveillance and for the evaluation of vaccines. PMID:19453415

  18. Isolation of HIV-1-Neutralizing Mucosal Monoclonal Antibodies from Human Colostrum

    PubMed Central

    Friedman, James; Alam, S. Munir; Shen, Xiaoying; Xia, Shi-Mao; Stewart, Shelley; Anasti, Kara; Pollara, Justin; Fouda, Genevieve G.; Yang, Guang; Kelsoe, Garnett; Ferrari, Guido; Tomaras, Georgia D.; Haynes, Barton F.; Liao, Hua-Xin

    2012-01-01

    Background Generation of potent anti-HIV antibody responses in mucosal compartments is a potential requirement of a transmission-blocking HIV vaccine. HIV-specific, functional antibody responses are present in breast milk, and these mucosal antibody responses may play a role in protection of the majority of HIV-exposed, breastfeeding infants. Therefore, characterization of HIV-specific antibodies produced by B cells in milk could guide the development of vaccines that elicit protective mucosal antibody responses. Methods We isolated B cells from colostrum of an HIV-infected lactating woman with a detectable neutralization response in milk and recombinantly produced and characterized the resulting HIV-1 Envelope (Env)-specific monoclonal antibodies (mAbs). Results The identified HIV-1 Env-specific colostrum mAbs, CH07 and CH08, represent two of the first mucosally-derived anti-HIV antibodies yet to be reported. Colostrum mAb CH07 is a highly-autoreactive, weakly-neutralizing gp140-specific mAb that binds to linear epitopes in the gp120 C5 region and gp41 fusion domain. In contrast, colostrum mAb CH08 is a nonpolyreactive CD4-inducible (CD4i) gp120-specific mAb with moderate breadth of neutralization. Conclusions These novel HIV-neutralizing mAbs isolated from a mucosal compartment provide insight into the ability of mucosal B cell populations to produce functional anti-HIV antibodies that may contribute to protection against virus acquisition at mucosal surfaces. PMID:22624058

  19. Protection of Humanized Mice From Repeated Intravaginal HIV Challenge by Passive Immunization: A Model for Studying the Efficacy of Neutralizing Antibodies In Vivo.

    PubMed

    Deruaz, Maud; Moldt, Brian; Le, Khoa M; Power, Karen A; Vrbanac, Vladimir D; Tanno, Serah; Ghebremichael, Musie S; Allen, Todd M; Tager, Andrew M; Burton, Dennis R; Luster, Andrew D

    2016-08-15

    Humanized mice reconstituted with a human immune system can be mucosally infected with human immunodeficiency virus (HIV), opening up the possibility of studying HIV transmission in a small-animal model. Here we report that passive immunization with the broadly neutralizing antibody b12 protected humanized mice against repetitive intravaginal infection in a dose-dependent manner. In addition, treatment with the antibody PGT126, which is more potent in vitro, was more efficacious in vivo and provided sterilizing protection. Our results demonstrate that humanized mice can be used as a small-animal model to study the efficacy and mechanism of broadly neutralizing antibody protection against HIV acquisition. PMID:27357340

  20. Increased Efficacy of HIV-1 Neutralization by Antibodies at Low CCR5 Surface Concentration

    PubMed Central

    Choudhry, Vidita; Zhang, Mei-Yun; Harris, Ilia; Sidorov, Igor A.; Vu, Bang; Dimitrov, Antony S.; Fouts, Timothy; Dimitrov, Dimiter S.

    2007-01-01

    It has been observed that some antibodies, including the CD4-induced (CD4i) antibody IgG X5 and the gp41-specific antibody IgG 2F5, exhibit higher neutralizing activity in PBMC-based assays than in cell line based assays (Binley et al., J. Virology, 2004, 78: 13232). It has been hypothesized that the lower CCR5 concentration on the surface of the CD4 T lymphocyte compared to that on cell lines used for the neutralization assays could be a contributing factor to the observed differences in neutralizing activity. To test this hypothesis and to further elucidate the contribution of CCR5 concentration differences on antibody neutralizing activity, we used a panel of HeLa cell lines with well-defined and differential surface concentrations of CCR5 and CD4 in a pseudovirus-based assay. We observed that the CCR5 cell surface concentration but not the CD4 concentration had a significant effect on the inhibitory activity of X5 and several other CD4i antibodies including 17b and m9, as well as that of the gp41-specifc antibodies 2F5 and 4E10 but not on that of the CD4 binding site antibody (CD4bs), b12. The 50% inhibitory concentration (IC50) decreased up to two orders of magnitude in cell lines with low CCR5 concentration corresponding to that in CD4 T cells used in PBMC-based assays (about 103 per cell) compared to cell lines with high CCR5 concentration (about 104 or more). Our results suggest that the CCR5 cell surface concentration could be a contributing factor to the high neutralizing activities of some antibodies in PBMC-based-assays but other factors could also play an important role. These findings could have implications for development of vaccine immunogens based on the epitopes of X5 and other CD4i antibodies, for elucidation of the mechanisms of HIV-1 neutralization by antibodies, and for design of novel therapeutic approaches. PMID:16904645

  1. IFNα kinoid vaccine-induced neutralizing antibodies prevent clinical manifestations in a lupus flare murine model

    PubMed Central

    Zagury, Daniel; Le Buanec, Hélène; Mathian, Alexis; Larcier, Patrick; Burnett, Roger; Amoura, Zahir; Emilie, Dominique; Peltre, Gabriel; Bensussan, Armand; Bizzini, Bernard; Gallo, Robert C.; Koutouzov, Sophie

    2009-01-01

    A major involvement of IFNα in the etiopathogenesis of systemic lupus erythematosus has been suggested by clinical observations, including the increase of serum levels of this cytokine in patients with active disease. Supporting this hypothesis, we have shown that expression of IFNα from a recombinant adenovirus (IFNα Adv) precipitates lupus manifestations in genetically susceptible New Zealand Black (NZB) × New Zealand White (NZW)F1 mice (NZB/W) but not in BALB/c mice. In the present investigation, we have prepared an IFNα immunogen, termed IFNα kinoid, which, appropriately adjuvanted, induces transient neutralizing antibodies (Abs) but no cellular immune response to the cytokine and without apparent side effects. Using this preparation, we also showed that, in kinoid-vaccinated NZB/W mice, lupus manifestations, including proteinuria, histological renal lesions, and death triggered by IFNα Adv challenge were delayed/prevented as long as an effective threshold of anti-IFNα inhibitory capacity was present in the serum. PMID:19279210

  2. Neutralizing antibodies are unable to inhibit direct viral cell-to-cell spread of human cytomegalovirus.

    PubMed

    Jacob, Christian L; Lamorte, Louie; Sepulveda, Eliud; Lorenz, Ivo C; Gauthier, Annick; Franti, Michael

    2013-09-01

    Infection with human cytomegalovirus (CMV) during pregnancy is the most common cause of congenital disorders, and can lead to severe life-long disabilities with associated high cost of care. Since there is no vaccine or effective treatment, current efforts are focused on identifying potent neutralizing antibodies. A panel of CMV monoclonal antibodies identified from patent applications, was synthesized and expressed in order to reproduce data from the literature showing that anti-glycoprotein B antibodies neutralized virus entry into all cell types and that anti-pentameric complex antibodies are highly potent in preventing virus entry into epithelial cells. It had not been established whether antibodies could prevent subsequent rounds of infection that are mediated primarily by direct cell-to-cell transmission. A thorough validation of a plaque reduction assay to monitor cell-to-cell spread led to the conclusion that neutralizing antibodies do not significantly inhibit plaque formation or reduce plaque size when they are added post-infection. PMID:23849792

  3. Neutralizing monoclonal antibodies recognize antigenic variants among isolates of infectious hematopoietic necrosis virus

    USGS Publications Warehouse

    Winton, J.R.; Arakawa, C.N.; Lannan, C.N.; Fryer, J.L.

    1988-01-01

    eutralizing monoclonal antibodies were developed against strains of infectious hematopoietic necrosis virus (IHNV) from steelhead trout Salmo gairdneri in the Deschutes River of Oregon, chinook salmon Oncorhynchus tshawytscha in the Sacramento River of California, and rainbow trout Salmo gairdneri reared in the Hagerman Valley of Idaho, USA. These antibodies were tested for neutralization of 12 IHNV isolates obtained from salmonids in Japan, Alaska, Washington, Oregon, California, and Idaho. The antibodies recognized antigenic variants among the isolates and could be used to separate the viruses into 4 groups. The members of each group tended to be related by geographic area rather than by source host species, virulence, or date of isolation.

  4. Engineering Venom’s Toxin-Neutralizing Antibody Fragments and Its Therapeutic Potential

    PubMed Central

    Alvarenga, Larissa M.; Zahid, Muhammad; di Tommaso, Anne; Juste, Matthieu O.; Aubrey, Nicolas; Billiald, Philippe; Muzard, Julien

    2014-01-01

    Serum therapy remains the only specific treatment against envenoming, but anti-venoms are still prepared by fragmentation of polyclonal antibodies isolated from hyper-immunized horse serum. Most of these anti-venoms are considered to be efficient, but their production is tedious, and their use may be associated with adverse effects. Recombinant antibodies and smaller functional units are now emerging as credible alternatives and constitute a source of still unexploited biomolecules capable of neutralizing venoms. This review will be a walk through the technologies that have recently been applied leading to novel antibody formats with better properties in terms of homogeneity, specific activity and possible safety. PMID:25153256

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

    PubMed Central

    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

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

    PubMed

    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. PMID:26938634

  7. Heterosubtypic neutralizing antibodies are produced by individuals immunized with a seasonal influenza vaccine

    PubMed Central

    Corti, Davide; Suguitan, Amorsolo L.; Pinna, Debora; Silacci, Chiara; Fernandez-Rodriguez, Blanca M.; Vanzetta, Fabrizia; Santos, Celia; Luke, Catherine J.; Torres-Velez, Fernando J.; Temperton, Nigel J.; Weiss, Robin A.; Sallusto, Federica; Subbarao, Kanta; Lanzavecchia, Antonio

    2010-01-01

    The target of neutralizing antibodies that protect against influenza virus infection is the viral protein HA. Genetic and antigenic variation in HA has been used to classify influenza viruses into subtypes (H1–H16). The neutralizing antibody response to influenza virus is thought to be specific for a few antigenically related isolates within a given subtype. However, while heterosubtypic antibodies capable of neutralizing multiple influenza virus subtypes have been recently isolated from phage display libraries, it is not known whether such antibodies are produced in the course of an immune response to influenza virus infection or vaccine. Here we report that, following vaccination with seasonal influenza vaccine containing H1 and H3 influenza virus subtypes, some individuals produce antibodies that cross-react with H5 HA. By immortalizing IgG-expressing B cells from 4 individuals, we isolated 20 heterosubtypic mAbs that bound and neutralized viruses belonging to several HA subtypes (H1, H2, H5, H6, and H9), including the pandemic A/California/07/09 H1N1 isolate. The mAbs used different VH genes and carried a high frequency of somatic mutations. With the exception of a mAb that bound to the HA globular head, all heterosubtypic mAbs bound to acid-sensitive epitopes in the HA stem region. Four mAbs were evaluated in vivo and protected mice from challenge with influenza viruses representative of different subtypes. These findings reveal that seasonal influenza vaccination can induce polyclonal heterosubtypic neutralizing antibodies that cross-react with the swine-origin pandemic H1N1 influenza virus and with the highly pathogenic H5N1 virus. PMID:20389023

  8. Characterization of novel neutralizing monoclonal antibodies specific to human neurturin.

    PubMed

    Hongo, J A; Tsai, S P; Moffat, B; Schroeder, K A; Jung, C; Chuntharapai, A; Lampe, P A; Johnson, E M; de Sauvage, F J; Armanini, M; Phillips, H; Devaux, B

    2000-08-01

    Neurturin (NTN) a structural and functional relative of glial cell line-derived neurotrophic factor, was originally identified based on its ability to support the survival of sympathetic neurons in culture. Similar to glial cell line-derived neurotrophic factor (GDNF), Neurturin has been shown to bind to a high affinity glycosylphosphatidylinositol (GPI)-linked receptor (GFRalpha2) and induce phosphorylation of the tyrosine kinase receptor Ret, resulting in the activation of the mitogen activated protein kinase (MAPK) signalling pathway. A panel of six novel murine monoclonal antibodies (MAbs) specific to human Neurturin has been developed and characterized. Four of the MAbs tested inhibit, to varying degrees, binding of NTN to the GPI-linked GFRalpha2 receptor. Three MAbs cross-react with the murine homolog. These antibodies have been shown to be useful reagents for Western blotting, immunohistochemistry, and also for the development of a sensitive, quantitative enzyme-linked immunosorbent assay (ELISA) for human NTN. Novel, specific MAbs with varying epitope specificities and blocking activity will be valuable tools for both the in vitro and in vivo characterization of NTN and its relationship to the GFRalpha2 and Ret receptors. PMID:11001403

  9. Prefusion F–specific antibodies determine the magnitude of RSV neutralizing activity in human sera

    PubMed Central

    Ngwuta, Joan O.; Chen, Man; Modjarrad, Kayvon; Joyce, M. Gordon; Kanekiyo, Masaru; Kumar, Azad; Yassine, Hadi M.; Moin, Syed M.; Killikelly, April M.; Chuang, Gwo-Yu; Druz, Aliaksandr; Georgiev, Ivelin S.; Rundlet, Emily J.; Sastry, Mallika; Stewart-Jones, Guillaume B. E.; Yang, Yongping; Zhang, Baoshan; Nason, Martha C.; Capella, Cristina; Peeples, Mark E.; Ledgerwood, Julie E.; McLellan, Jason S.; Kwong, Peter D.; Graham, Barney S.

    2015-01-01

    Respiratory syncytial virus (RSV) is estimated to claim more lives among infants <1 year old than any other single pathogen, except malaria, and poses a substantial global health burden. Viral entry is mediated by a type I fusion glycoprotein (F) that transitions from a metastable prefusion (pre-F) to a stable postfusion (post-F) trimer. A highly neutralization-sensitive epitope, antigenic site Ø, is found only on pre-F. We determined what fraction of neutralizing (NT) activity in human sera is dependent on antibodies specific for antigenic site Ø or other antigenic sites on F in healthy subjects from ages 7 to 93 years. Adsorption of individual sera with stabilized pre-F protein removed >90% of NT activity and depleted binding antibodies to both F conformations. In contrast, adsorption with post-F removed ~30% of NT activity, and binding antibodies to pre-F were retained. These findings were consistent across all age groups. Protein competition neutralization assays with pre-F mutants in which sites Ø or II were altered to knock out binding of antibodies to the corresponding sites showed that these sites accounted for ~35 and <10% of NT activity, respectively. Binding competition assays with monoclonal antibodies (mAbs) indicated that the amount of site Ø–specific antibodies correlated with NT activity, whereas the magnitude of binding competed by site II mAbs did not correlate with neutralization. Our results indicate that RSV NT activity in human sera is primarily derived from pre-F–specific antibodies, and therefore, inducing or boosting NT activity by vaccination will be facilitated by using pre-F antigens that preserve site Ø. PMID:26468324

  10. Dengue Virus (DENV) Neutralizing Antibody Kinetics in Children After Symptomatic Primary and Postprimary DENV Infection.

    PubMed

    Clapham, Hannah E; Rodriguez-Barraquer, Isabel; Azman, Andrew S; Althouse, Benjamin M; Salje, Henrik; Gibbons, Robert V; Rothman, Alan L; Jarman, Richard G; Nisalak, Ananda; Thaisomboonsuk, Butsaya; Kalayanarooj, Siripen; Nimmannitya, Suchitra; Vaughn, David W; Green, Sharone; Yoon, In-Kyu; Cummings, Derek A T

    2016-05-01

    The immune response to dengue virus (DENV) infection is complex and not fully understood. Using longitudinal data from 181 children with dengue in Thailand who were followed for up to 3 years, we describe neutralizing antibody kinetics following symptomatic DENV infection. We observed that antibody titers varied by serotype, homotypic vs heterotypic responses, and primary versus postprimary infections. The rates of change in antibody titers over time varied between primary and postprimary responses. For primary infections, titers increased from convalescence to 6 months. By comparing homotypic and heterotypic antibody titers, we saw an increase in type specificity from convalescence to 6 months for primary DENV3 infections but not primary DENV1 infections. In postprimary cases, there was a decrease in titers from convalescence up until 6 months after infection. Beginning 1 year after both primary and postprimary infections, there was evidence of increasing antibody titers, with greater increases in children with lower titers, suggesting that antibody titers were boosted due to infection and that higher levels of neutralizing antibody may be more likely to confer a sterilizing immune response. These findings may help to model virus transmission dynamics and provide baseline data to support the development of vaccines and therapeutics. PMID:26704615

  11. Neutralizing Antibody Response and Antibody-Dependent Cellular Cytotoxicity in HIV-1-Infected Individuals from Guinea-Bissau and Denmark.

    PubMed

    Borggren, Marie; Jensen, Sanne Skov; Heyndrickx, Leo; Palm, Angelica A; Gerstoft, Jan; Kronborg, Gitte; Hønge, Bo Langhoff; Jespersen, Sanne; da Silva, Zacarias José; Karlsson, Ingrid; Fomsgaard, Anders

    2016-05-01

    The development of therapeutic and prophylactic HIV vaccines for African countries is urgently needed, but the question of what immunogens to use needs to be answered. One approach is to include HIV envelope immunogens derived from HIV-positive individuals from a geographically concentrated epidemic with more limited viral genetic diversity for a region-based vaccine. To address if there is a basis for a regional selected antibody vaccine, we have screened two regionally separate cohorts from Guinea-Bissau and Denmark for neutralizing antibody activity and antibody-dependent cellular cytotoxicity (ADCC) against local and nonlocal circulating HIV-1 strains. The neutralizing activity did not demonstrate higher potential against local circulating strains according to geography and subtype determination, but the plasma from Danish individuals demonstrated significantly higher inhibitory activity than that from Guinea-Bissau individuals against both local and nonlocal virus strains. Interestingly, an opposite pattern was observed with ADCC activity, where Guinea-Bissau individual plasma demonstrated higher activity than Danish plasma and was specifically against the local circulating subtype. Thus, on basis of samples from these two cohorts, no local-specific neutralizing activity was detected, but a local ADCC response was identified in the Guinea-Bissau samples, suggesting potential use of regional immunogens for an ADCC-inducing vaccine. PMID:26621287

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

    PubMed Central

    Albrecht, Randy A.; Margine, Irina; Hirsh, Ariana; Bahl, Justin; Krammer, Florian

    2016-01-01

    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. PMID:27081859

  13. Hepatitis C Virus Hypervariable Region 1 Variants Presented on Hepatitis B Virus Capsid-Like Particles Induce Cross-Neutralizing Antibodies

    PubMed Central

    Bankwitz, Dorothea; Osburn, William; Viazov, Sergei; Brovko, Olena; Zekri, Abdel-Rahman; Khudyakov, Yury; Nassal, Michael; Pumpens, Paul; Pietschmann, Thomas; Timm, Jörg; Roggendorf, Michael; Walker, Andreas

    2014-01-01

    Hepatitis C virus (HCV) infection is still a serious global health burden. Despite improved therapeutic options, a preventative vaccine would be desirable especially in undeveloped countries. Traditionally, highly conserved epitopes are targets for antibody-based prophylactic vaccines. In HCV-infected patients, however, neutralizing antibodies are primarily directed against hypervariable region I (HVRI) in the envelope protein E2. HVRI is the most variable region of HCV, and this heterogeneity contributes to viral persistence and has thus far prevented the development of an effective HVRI-based vaccine. The primary goal of an antibody-based HCV vaccine should therefore be the induction of cross-reactive HVRI antibodies. In this study we approached this problem by presenting selected cross-reactive HVRI variants in a highly symmetric repeated array on capsid-like particles (CLPs). SplitCore CLPs, a novel particulate antigen presentation system derived from the HBV core protein, were used to deliberately manipulate the orientation of HVRI and therefore enable the presentation of conserved parts of HVRI. These HVRI-CLPs induced high titers of cross-reactive antibodies, including neutralizing antibodies. The combination of only four HVRI CLPs was sufficient to induce antibodies cross-reactive with 81 of 326 (24.8%) naturally occurring HVRI peptides. Most importantly, HVRI CLPs with AS03 as an adjuvant induced antibodies with a 10-fold increase in neutralizing capability. These antibodies were able to neutralize infectious HCVcc isolates and 4 of 19 (21%) patient-derived HCVpp isolates. Taken together, these results demonstrate that the induction of at least partially cross-neutralizing antibodies is possible. This approach might be useful for the development of a prophylactic HCV vaccine and should also be adaptable to other highly variable viruses. PMID:25014219

  14. Mechanisms of Ricin Toxin Neutralization Revealed through Engineered Homodimeric and Heterodimeric Camelid Antibodies.

    PubMed

    Herrera, Cristina; Tremblay, Jacqueline M; Shoemaker, Charles B; Mantis, Nicholas J

    2015-11-13

    Novel antibody constructs consisting of two or more different camelid heavy-chain only antibodies (VHHs) joined via peptide linkers have proven to have potent toxin-neutralizing activity in vivo against Shiga, botulinum, Clostridium difficile, anthrax, and ricin toxins. However, the mechanisms by which these so-called bispecific VHH heterodimers promote toxin neutralization remain poorly understood. In the current study we produced a new collection of ricin-specific VHH heterodimers, as well as VHH homodimers, and characterized them for their ability neutralize ricin in vitro and in vivo. We demonstrate that the VHH heterodimers, but not homodimers were able to completely protect mice against ricin challenge, even though the two classes of antibodies (heterodimers and homodimers) had virtually identical affinities for ricin holotoxin and similar IC50 values in a Vero cell cytotoxicity assay. The VHH heterodimers did differ from the homodimers in their ability to promote toxin aggregation in solution, as revealed through analytical ultracentrifugation. Moreover, the VHH heterodimers that were most effective at promoting ricin aggregation in solution were also the most effective at blocking ricin attachment to cell surfaces. Collectively, these data suggest that heterodimeric VHH-based neutralizing agents may function through the formation of antibody-toxin complexes that are impaired in their ability to access host cell receptors. PMID:26396190

  15. Development of human neutralizing monoclonal antibodies for prevention and therapy of MERS-CoV infections

    PubMed Central

    Ying, Tianlei; Li, Haoyang; Lu, Lu; Dimitrov, Dimiter S; Jiang, Shibo

    2014-01-01

    The recent Middle East respiratory syndrome coronavirus (MERS-CoV) outbreak poses a serious threat to public health. Here, we summarize recent advances in identifying human neutralizing monoclonal antibodies (mAbs) against MERS-CoV, describe their mechanisms of action, and analyze their potential for treatment of MERS-CoV infections. PMID:25456101

  16. A rapid and quantitative assay for measuring antibody-mediated neutralization of West Nile virus infection

    SciTech Connect

    Pierson, Theodore C. . E-mail: piersontc@mail.nih.gov; Sanchez, Melissa D.; Puffer, Bridget A.; Ahmed, Asim A.; Geiss, Brian J.; Valentine, Laura E.; Altamura, Louis A.; Diamond, Michael S.; Doms, Robert W. . E-mail: doms@mail.med.upenn.edu

    2006-03-01

    West Nile virus (WNV) is a neurotropic flavivirus within the Japanese encephalitis antigenic complex that is responsible for causing West Nile encephalitis in humans. The surface of WNV virions is covered by a highly ordered icosahedral array of envelope proteins that is responsible for mediating attachment and fusion with target cells. These envelope proteins are also primary targets for the generation of neutralizing antibodies in vivo. In this study, we describe a novel approach for measuring antibody-mediated neutralization of WNV infection using virus-like particles that measure infection as a function of reporter gene expression. These reporter virus particles (RVPs) are produced by complementation of a sub-genomic replicon with WNV structural proteins provided in trans using conventional DNA expression vectors. The precision and accuracy of this approach stem from an ability to measure the outcome of the interaction between antibody and viral antigens under conditions that satisfy the assumptions of the law of mass action as applied to virus neutralization. In addition to its quantitative strengths, this approach allows the production of WNV RVPs bearing the prM-E proteins of different WNV strains and mutants, offering considerable flexibility for the study of the humoral immune response to WNV in vitro. WNV RVPs are capable of only a single round of infection, can be used under BSL-2 conditions, and offer a rapid and quantitative approach for detecting virus entry and its inhibition by neutralizing antibody.

  17. Early short-term treatment with neutralizing human monoclonal antibodies halts SHIV infection in infant macaques.

    PubMed

    Hessell, Ann J; Jaworski, J Pablo; Epson, Erin; Matsuda, Kenta; Pandey, Shilpi; Kahl, Christoph; Reed, Jason; Sutton, William F; Hammond, Katherine B; Cheever, Tracy A; Barnette, Philip T; Legasse, Alfred W; Planer, Shannon; Stanton, Jeffrey J; Pegu, Amarendra; Chen, Xuejun; Wang, Keyun; Siess, Don; Burke, David; Park, Byung S; Axthelm, Michael K; Lewis, Anne; Hirsch, Vanessa M; Graham, Barney S; Mascola, John R; Sacha, Jonah B; Haigwood, Nancy L

    2016-04-01

    Prevention of mother-to-child transmission (MTCT) of HIV remains a major objective where antenatal care is not readily accessible. We tested HIV-1-specific human neutralizing monoclonal antibodies (NmAbs) as a post-exposure therapy in an infant macaque model for intrapartum MTCT. One-month-old rhesus macaques were inoculated orally with the simian-human immunodeficiency virus SHIVSF162P3. On days 1, 4, 7 and 10 after virus exposure, we injected animals subcutaneously with NmAbs and quantified systemic distribution of NmAbs in multiple tissues within 24 h after antibody administration. Replicating virus was found in multiple tissues by day 1 in animals that were not treated. All NmAb-treated macaques were free of virus in blood and tissues at 6 months after exposure. We detected no anti-SHIV T cell responses in blood or tissues at necropsy, and no virus emerged after CD8(+) T cell depletion. These results suggest that early passive immunotherapy can eliminate early viral foci and thereby prevent the establishment of viral reservoirs. PMID:26998834

  18. Induction of HIV Neutralizing Antibody Lineages in Mice with Diverse Precursor Repertoires.

    PubMed

    Tian, Ming; Cheng, Cheng; Chen, Xuejun; Duan, Hongying; Cheng, Hwei-Ling; Dao, Mai; Sheng, Zizhang; Kimble, Michael; Wang, Lingshu; Lin, Sherry; Schmidt, Stephen D; Du, Zhou; Joyce, M Gordon; Chen, Yiwei; DeKosky, Brandon J; Chen, Yimin; Normandin, Erica; Cantor, Elizabeth; Chen, Rita E; Doria-Rose, Nicole A; Zhang, Yi; Shi, Wei; Kong, Wing-Pui; Choe, Misook; Henry, Amy R; Laboune, Farida; Georgiev, Ivelin S; Huang, Pei-Yi; Jain, Suvi; McGuire, Andrew T; Georgeson, Eric; Menis, Sergey; Douek, Daniel C; Schief, William R; Stamatatos, Leonidas; Kwong, Peter D; Shapiro, Lawrence; Haynes, Barton F; Mascola, John R; Alt, Frederick W

    2016-09-01

    The design of immunogens that elicit broadly reactive neutralizing antibodies (bnAbs) has been a major obstacle to HIV-1 vaccine development. One approach to assess potential immunogens is to use mice expressing precursors of human bnAbs as vaccination models. The bnAbs of the VRC01-class derive from the IGHV1-2 immunoglobulin heavy chain and neutralize a wide spectrum of HIV-1 strains via targeting the CD4 binding site of the envelope glycoprotein gp120. We now describe a mouse vaccination model that allows a germline human IGHV1-2(∗)02 segment to undergo normal V(D)J recombination and, thereby, leads to the generation of peripheral B cells that express a highly diverse repertoire of VRC01-related receptors. When sequentially immunized with modified gp120 glycoproteins designed to engage VRC01 germline and intermediate antibodies, IGHV1-2(∗)02-rearranging mice, which also express a VRC01-antibody precursor light chain, can support the affinity maturation of VRC01 precursor antibodies into HIV-neutralizing antibody lineages. PMID:27610571

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

    PubMed

    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

    2016-09-01

    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. PMID:27610569

  20. Antibody-mediated neutralization of Ebola virus can occur by two distinct mechanisms

    SciTech Connect

    Shedlock, Devon J.; Bailey, Michael A.; Popernack, Paul M.; Cunningham, James M.; Burton, Dennis R.; Sullivan, Nancy J.

    2010-06-05

    Human Ebola virus causes severe hemorrhagic fever disease with high mortality and there is no vaccine or treatment. Antibodies in survivors occur early, are sustained, and can delay infection when transferred into nonhuman primates. Monoclonal antibodies (mAbs) from survivors exhibit potent neutralizing activity in vitro and are protective in rodents. To better understand targets and mechanisms of neutralization, we investigated a panel of mAbs shown previously to react with the envelope glycoprotein (GP). While one non-neutralizing mAb recognized a GP epitope in the nonessential mucin-like domain, the rest were specific for GP1, were neutralizing, and could be further distinguished by reactivity with secreted GP. We show that survivor antibodies, human KZ52 and monkey JP3K11, were specific for conformation-dependent epitopes comprising residues in GP1 and GP2 and that neutralization occurred by two distinct mechanisms; KZ52 inhibited cathepsin cleavage of GP whereas JP3K11 recognized the cleaved, fusion-active form of GP.

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

    PubMed

    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. PMID:26332605

  2. A novel antibody discovery platform identifies anti-influenza A broadly neutralizing antibodies from human memory B cells.

    PubMed

    Xiao, Xiaodong; Chen, Yan; Varkey, Reena; Kallewaard, Nicole; Koksal, Adem C; Zhu, Qing; Wu, Herren; Chowdhury, Partha S; Dall'Acqua, William F

    2016-07-01

    Monoclonal antibody isolation directly from circulating human B cells is a powerful tool to delineate humoral responses to pathological conditions and discover antibody therapeutics. We have developed a platform aimed at improving the efficiencies of B cell selection and V gene recovery. Here, memory B cells are activated and amplified using Epstein-Barr virus infection, co-cultured with CHO-muCD40L cells, and then assessed by functional screenings. An in vitro transcription and translation (IVTT) approach was used to analyze variable (V) genes recovered from each B cell sample and identify the relevant heavy/light chain pair(s). We achieved efficient amplification and activation of memory B cells, and eliminated the need to: 1) seed B cells at clonal level (≤1 cell/well) or perform limited dilution cloning; 2) immortalize B cells; or 3) assemble V genes into an IgG expression vector to confirm the relevant heavy/light chain pairing. Cross-reactive antibodies targeting a conserved epitope on influenza A hemagglutinin were successfully isolated from a healthy donor. In-depth analysis of the isolated antibodies suggested their potential uses as anti-influenza A antibody therapeutics and uncovered a distinct affinity maturation pathway. Importantly, our results showed that cognate heavy/light chain pairings contributed to both the expression level and binding abilities of our newly isolated VH1-69 family, influenza A neutralizing antibodies, contrasting with previous observations that light chains do not significantly contribute to the function of this group of antibodies. Our results further suggest the potential use of the IVTT as a powerful antibody developability assessment tool. PMID:27049174

  3. A potent and broad neutralizing antibody recognizes and penetrates the HIV glycan shield.

    PubMed

    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

    2011-11-25

    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(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 β-strand segment of the gp120 V3 loop, accounting for its high binding affinity and broad specificity. 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. PMID:21998254

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

    SciTech Connect

    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.

    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.

  5. HIV-1 VACCINES. HIV-1 neutralizing antibodies induced by native-like envelope trimers.

    PubMed

    Sanders, Rogier W; van Gils, Marit J; Derking, Ronald; Sok, Devin; Ketas, Thomas J; Burger, Judith A; Ozorowski, Gabriel; Cupo, Albert; Simonich, Cassandra; Goo, Leslie; Arendt, Heather; Kim, Helen J; Lee, Jeong Hyun; Pugach, Pavel; Williams, Melissa; Debnath, Gargi; Moldt, Brian; van Breemen, Mariëlle J; Isik, Gözde; Medina-Ramírez, Max; Back, Jaap Willem; Koff, Wayne C; Julien, Jean-Philippe; Rakasz, Eva G; Seaman, Michael S; Guttman, Miklos; Lee, Kelly K; Klasse, Per Johan; LaBranche, Celia; Schief, William R; Wilson, Ian A; Overbaugh, Julie; Burton, Dennis R; Ward, Andrew B; Montefiori, David C; Dean, Hansi; Moore, John P

    2015-07-10

    A challenge for HIV-1 immunogen design is the difficulty of inducing neutralizing antibodies (NAbs) against neutralization-resistant (tier 2) viruses that dominate human transmissions. We show that a soluble recombinant HIV-1 envelope glycoprotein trimer that adopts a native conformation, BG505 SOSIP.664, induced NAbs potently against the sequence-matched tier 2 virus in rabbits and similar but weaker responses in macaques. The trimer also consistently induced cross-reactive NAbs against more sensitive (tier 1) viruses. Tier 2 NAbs recognized conformational epitopes that differed between animals and in some cases overlapped with those recognized by broadly neutralizing antibodies (bNAbs), whereas tier 1 responses targeted linear V3 epitopes. A second trimer, B41 SOSIP.664, also induced a strong autologous tier 2 NAb response in rabbits. Thus, native-like trimers represent a promising starting point for the development of HIV-1 vaccines aimed at inducing bNAbs. PMID:26089353

  6. Induction of broadly neutralizing H1N1 influenza antibodies by vaccination.

    PubMed

    Wei, Chih-Jen; Boyington, Jeffrey C; McTamney, Patrick M; Kong, Wing-Pui; Pearce, Melissa B; Xu, Ling; Andersen, Hanne; Rao, Srinivas; Tumpey, Terrence M; Yang, Zhi-Yong; Nabel, Gary J

    2010-08-27

    The rapid dissemination of the 2009 pandemic influenza virus underscores the need for universal influenza vaccines that elicit protective immunity to diverse viral strains. Here, we show that vaccination with plasmid DNA encoding H1N1 influenza hemagglutinin (HA) and boosting with seasonal vaccine or replication-defective adenovirus 5 vector encoding HA stimulated the production of broadly neutralizing influenza antibodies. This prime/boost combination increased the neutralization of diverse H1N1 strains dating from 1934 to 2007 as compared to either component alone and conferred protection against divergent H1N1 viruses in mice and ferrets. These antibodies were directed to the conserved stem region of HA and were also elicited in nonhuman primates. Cross-neutralization of H1N1 subtypes elicited by this approach provides a basis for the development of a universal influenza vaccine for humans. PMID:20647428

  7. Escape from neutralization by the respiratory syncytial virus-specific neutralizing monoclonal antibody palivizumab is driven by changes in on-rate of binding to the fusion protein

    SciTech Connect

    Bates, John T.; Keefer, Christopher J.; Slaughter, James C.; Kulp, Daniel W.; Schief, William R.

    2014-04-15

    The role of binding kinetics in determining neutralizing potency for antiviral antibodies is poorly understood. While it is believed that increased steady-state affinity correlates positively with increased virus-neutralizing activity, the relationship between association or dissociation rate and neutralization potency is unclear. We investigated the effect of naturally-occurring antibody resistance mutations in the RSV F protein on the kinetics of binding to palivizumab. Escape from palivizumab-mediated neutralization of RSV occurred with reduced association rate (K{sub on}) for binding to RSV F protein, while alteration of dissociation rate (K{sub off}) did not significantly affect neutralizing activity. Interestingly, linkage of reduced K{sub on} with reduced potency mirrored the effect of increased K{sub on} found in a high-affinity enhanced potency palivizumab variant (motavizumab). These data suggest that association rate is the dominant factor driving neutralization potency for antibodies to RSV F protein antigenic site A and determines the potency of antibody somatic variants or efficiency of escape of viral glycoprotein variants. - Highlights: • The relationship of affinity to neutralization for virus antibodies is uncertain. • Palivizumab binds to RSV escape mutant fusion proteins, but with reduced affinity. • Association rate (K{sub on}) correlated well with the potency of neutralization.

  8. HIV-1 Antibody Neutralization Breadth Is Associated with Enhanced HIV-Specific CD4+ T Cell Responses

    PubMed Central

    Soghoian, Damien Z.; Lindqvist, Madelene; Ghebremichael, Musie; Donaghey, Faith; Carrington, Mary; Seaman, Michael S.; Kaufmann, Daniel E.; Walker, Bruce D.

    2015-01-01

    ABSTRACT Antigen-specific CD4+ T helper cell responses have long been recognized to be a critical component of effective vaccine immunity. CD4+ T cells are necessary to generate and maintain humoral immune responses by providing help to antigen-specific B cells for the production of antibodies. In HIV infection, CD4+ T cells are thought to be necessary for the induction of Env-specific broadly neutralizing antibodies. However, few studies have investigated the role of HIV-specific CD4+ T cells in association with HIV neutralizing antibody activity in vaccination or natural infection settings. Here, we conducted a comprehensive analysis of HIV-specific CD4+ T cell responses in a cohort of 34 untreated HIV-infected controllers matched for viral load, with and without neutralizing antibody breadth to a panel of viral strains. Our results show that the breadth and magnitude of Gag-specific CD4+ T cell responses were significantly higher in individuals with neutralizing antibodies than in those without neutralizing antibodies. The breadth of Gag-specific CD4+ T cell responses was positively correlated with the breadth of neutralizing antibody activity. Furthermore, the breadth and magnitude of gp41-specific, but not gp120-specific, CD4+ T cell responses were significantly elevated in individuals with neutralizing antibodies. Together, these data suggest that robust Gag-specific CD4+ T cells and, to a lesser extent, gp41-specific CD4+ T cells may provide important intermolecular help to Env-specific B cells that promote the generation or maintenance of Env-specific neutralizing antibodies. IMPORTANCE One of the earliest discoveries related to CD4+ T cell function was their provision of help to B cells in the development of antibody responses. Yet little is known about the role of CD4+ T helper responses in the setting of HIV infection, and no studies to date have evaluated the impact of HIV-specific CD4+ T cells on the generation of antibodies that can neutralize

  9. Neutralizing antibodies respond to a bivalent dengue DNA vaccine or/and a recombinant bivalent antigen.

    PubMed

    Zhang, Zhi-Shan; Weng, Yu-Wei; Huang, Hai-Long; Zhang, Jian-Ming; Yan, Yan-Sheng

    2015-02-01

    There is currently no effective vaccine to prevent dengue infection, despite the existence of multiple studies on potential methods of immunization. The aim of the present study was to explore the effect of DNA and/or recombinant protein on levels of neutralizing antibodies. For this purpose, envelope domain IIIs of dengue serotypes 1 and 2 (DEN-1/2)were spliced by a linker (Gly‑Gly‑Ser‑Gly‑Ser)3 and cloned into the prokaryotic expression plasmid pET30a (+) and eukaryotic vector pcDNA3.1 (+). The chimeric bivalent protein was expressed in Escherichia coli, and one‑step purification by high‑performance liquid chromatography was conducted. Protein expression levels of the DNA plasmid were tested in BHK‑21 cells by indirect immunofluorescent assay. In order to explore a more effective immunization strategy and to develop neutralizing antibodies against the two serotypes, mice were inoculated with recombinant bivalent protein, the DNA vaccine, or the two given simultaneously. Presence of the specific antibodies was tested by ELISA and the presence of the neutralizing antibodies was determined by plaque reduction neutralization test. Results of the analysis indicated that the use of a combination of DNA and protein induced significantly higher titers of neutralizing antibodies against either DEN‑1 or DEN‑2 (1:64.0 and 1:76.1, respectively) compared with the DNA (1:24.7 and 1:26.9, DEN‑1 and DEN‑2, respectively) or the recombinant protein (1:34.9 and 1:45.3 in DEN‑1 and DEN‑2, respectively). The present study demonstrated that the combination of recombinant protein and DNA as an immunization strategy may be an effective method for the development of a vaccine to prevent dengue virus infection. PMID:25371092

  10. A Generic Mechanism for Enhanced Cytokine Signaling via Cytokine-Neutralizing Antibodies

    PubMed Central

    Shulgin, Boris; Helmlinger, Gabriel; Kosinsky, Yuri

    2016-01-01

    Enhancement or inhibition of cytokine signaling and corresponding immune cells responses are critical factors in various disease treatments. Cytokine signaling may be inhibited by cytokine-neutralizing antibodies (CNAs), which prevents further activation of cytokine receptors. However, CNAs may result in enhanced—instead of inhibitory—cytokine signaling (an “agonistic effect”) in various in vitro and in vivo experiments. This may lead to lack of efficacy or adverse events for cytokine-inhibiting based medicines. Alternatively, cytokine-antibody complexes may produce stronger signaling vs. cytokine alone, thereby increasing the efficacy of stimulating cytokine-based drugs, at equal or lower cytokine doses. In this paper, the effect of cytokine signaling enhancement by a CNA was studied in a generic mathematical model of interleukin-4 (IL-4) driven T-cell proliferation. The occurrence of the agonistic effect depends upon the antibody-to-cytokine binding affinity and initial concentrations of antibody and cytokine. Model predictions were in agreement with experimental studies. When the cytokine receptor consists of multiple subunits with substantially differing affinities (e.g., IL-4 case), the choice of the receptor chain to be blocked by the antibody is critical, for the agonistic effect to appear. We propose a generic mechanism for the effect: initially, binding of the CNA to the cytokine reduces free cytokine concentration; yet, cytokine molecules bound within the cytokine-CNA complex—and released later and over time—are “rescued” from earlier clearance via cellular internalization. Hence, although free cytokine-dependent signalling may be less potent initially, it will also be more sustained over time; and given non-linear dynamics, it will lead ultimately to larger cellular effector responses, vs. the same amount of free cytokine in the absence of CNA. We suggest that the proposed mechanism is a generic property of {cytokine, CNA, receptor

  11. RSV neutralization by palivizumab, but not by monoclonal antibodies targeting other epitopes, is augmented by Fc gamma receptors.

    PubMed

    van Mechelen, Lenny; Luytjes, Willem; de Haan, Cornelis A M; Wicht, Oliver

    2016-08-01

    Palivizumab efficiently blocks respiratory syncytial virus (RSV) infection in vitro. However, virus neutralization assays generally omit Fc region-mediated effects. We investigated the neutralization activity of RSV-specific monoclonal antibodies on cells with Fc receptors. Subneutralizing concentrations of antibodies resulted in antibody-dependent enhancement of RSV infection in monocytic cells. Contrary to antibodies targeting other epitopes, the neutralization by palivizumab was augmented in cells with Fc receptors. This unrecognized characteristic of palivizumab may be relevant for its performance in vivo. PMID:27185625

  12. Hepatitis C Virus E1 and E2 Proteins Used as Separate Immunogens Induce Neutralizing Antibodies with Additive Properties

    PubMed Central

    Beaumont, Elodie; Roch, Emmanuelle; Chopin, Lucie; Roingeard, Philippe

    2016-01-01

    Various strategies involving the use of hepatitis C virus (HCV) E1 and E2 envelope glycoproteins as immunogens have been developed for prophylactic vaccination against HCV. However, the ideal mode of processing and presenting these immunogens for effective vaccination has yet to be determined. We used our recently described vaccine candidate based on full-length HCV E1 or E2 glycoproteins fused to the heterologous hepatitis B virus S envelope protein to compare the use of the E1 and E2 proteins as separate immunogens with their use as the E1E2 heterodimer, in terms of immunogenetic potential and the capacity to induce neutralizing antibodies. The specific anti-E1 and anti-E2 antibody responses induced in animals immunized with vaccine particles harboring the heterodimer were profoundly impaired with respect to those in animals immunized with particles harboring E1 and E2 separately. Moreover, the anti-E1 and anti-E2 antibodies had additive neutralizing properties that increase the cross-neutralization of heterologous strains of various HCV genotypes, highlighting the importance of including both E1 and E2 in the vaccine for an effective vaccination strategy. Our study has important implications for the optimization of HCV vaccination strategies based on HCV envelope proteins, regardless of the platform used to present these proteins to the immune system. PMID:26966906

  13. Recombinant Outer Capsid Glycoprotein (VP7) of Rotavirus Expressed in Insect Cells Induces Neutralizing Antibodies in Rabbits

    PubMed Central

    Khodabandehloo, M; Shahrabadi, M Shamsi; Keyvani, H; Bambai, B; Sadigh, ZA

    2012-01-01

    Background: Rotaviruses cause diarrhea in infants and young children worldwide. Rotavirus outer capsid protein, VP7 is major neutralizing antigen that is important component of subunit vaccine to prevent rotavirus infection. Many efforts have been done to produce recombinant VP7 that maintain native characteristics. We used baculovirus expression system to produce rotavirus VP7 protein and to study its immunogenicity. Methods: Simian rotavirus SA11 full-length VP7 ORF was cloned into a cloning plasmid and then the cloned gene was inserted into the linear DNA of baculovirus Autographa californica Nuclear Polyhedrosis Virus (AcNPV) downstream of the polyhedrin promoter by in vitro recombination reactions. The expressed VP7 in the insect cells was recognized by rabbit hyperimmune serum raised against SA11 rotavirus by Immunofluorescence and western blotting assays. Rabbits were immunized subcutaneously by cell extracts expressing VP7 protein. Results: Reactivity with anti-rotavirus antibody suggested that expressed VP7 protein had native antigenic determinants. Injection of recombinant VP7 in rabbits elicited the production of serum antibodies, which were able to recognize VP7 protein from SA11 rotavirus by Western blotting test and neutralized SA11 rotavirus in cell culture. Conclusion: Recombinant outer capsid glycoprotein (VP7) of rotavirus expressed in insect cells induces neutralizing antibodies in rabbits and may be a candidate of rotavirus vaccine. PMID:23113180

  14. Broadening the neutralizing capacity of a family of antibody fragments against different toxins from Mexican scorpions.

    PubMed

    Rodríguez-Rodríguez, Everardo Remi; Olamendi-Portugal, Timoteo; Serrano-Posada, Hugo; Arredondo-López, Jonathan Noé; Gómez-Ramírez, Ilse; Fernández-Taboada, Guillermo; Possani, Lourival D; Anguiano-Vega, Gerardo Alfonso; Riaño-Umbarila, Lidia; Becerril, Baltazar

    2016-09-01

    New approaches aimed at neutralizing the primary toxic components present in scorpion venoms, represent a promising alternative to the use of antivenoms of equine origin in humans. New potential therapeutics developed by these approaches correspond to neutralizing antibody fragments obtained by selection and maturation processes from libraries of human origin. The high sequence identity shared among scorpion toxins is associated with an important level of cross reactivity exhibited by these antibody fragments. We have exploited the cross reactivity showed by single chain variable antibody fragments (scFvs) of human origin to re-direct the neutralizing capacity toward various other scorpion toxins. As expected, during these evolving processes several variants derived from a parental scFv exhibited the capacity to simultaneously recognize and neutralize different toxins from Centruroides scorpion venoms. A sequence analyses of the cross reacting scFvs revealed that specific mutations are responsible for broadening their neutralizing capacity. In this work, we generated a set of new scFvs that resulted from the combinatorial insertion of these point mutations. These scFvs are potential candidates to be part of a novel recombinant antivenom of human origin that could confer protection against scorpion stings. A remarkable property of one of these new scFvs (ER-5) is its capacity to neutralize at least three different toxins and its complementary capacity to neutralize the whole venom from Centruroides suffusus in combination with a second scFv (LR), which binds to a different epitope shared by Centruroides scorpion toxins. PMID:27212628

  15. Structural Constraints of Vaccine-Induced Tier-2 Autologous HIV Neutralizing Antibodies Targeting the Receptor-Binding Site.

    PubMed

    Bradley, Todd; Fera, Daniela; Bhiman, Jinal; Eslamizar, Leila; Lu, Xiaozhi; Anasti, Kara; Zhang, Ruijung; Sutherland, Laura L; Scearce, Richard M; Bowman, Cindy M; Stolarchuk, Christina; Lloyd, Krissey E; Parks, Robert; Eaton, Amanda; Foulger, Andrew; Nie, Xiaoyan; Karim, Salim S Abdool; Barnett, Susan; Kelsoe, Garnett; Kepler, Thomas B; Alam, S Munir; Montefiori, David C; Moody, M Anthony; Liao, Hua-Xin; Morris, Lynn; Santra, Sampa; Harrison, Stephen C; Haynes, Barton F

    2016-01-01

    Antibodies that neutralize autologous transmitted/founder (TF) HIV occur in most HIV-infected individuals and can evolve to neutralization breadth. Autologous neutralizing antibodies (nAbs) against neutralization-resistant (Tier-2) viruses are rarely induced by vaccination. Whereas broadly neutralizing antibody (bnAb)-HIV-Envelope structures have been defined, the structures of autologous nAbs have not. Here, we show that immunization with TF mutant Envs gp140 oligomers induced high-titer, V5-dependent plasma neutralization for a Tier-2 autologous TF evolved mutant virus. Structural analysis of autologous nAb DH427 revealed binding to V5, demonstrating the source of narrow nAb specificity and explaining the failure to acquire breadth. Thus, oligomeric TF Envs can elicit autologous nAbs to Tier-2 HIVs, but induction of bnAbs will require targeting of precursors of B cell lineages that can mature to heterologous neutralization. PMID:26725118

  16. Neutralizing Antibodies Against Adeno-Associated Viral Capsids in Patients with mut Methylmalonic Acidemia.

    PubMed

    Harrington, Elizabeth A; Sloan, Jennifer L; Manoli, Irini; Chandler, Randy J; Schneider, Mark; McGuire, Peter J; Calcedo, Roberto; Wilson, James M; Venditti, Charles P

    2016-05-01

    Isolated methylmalonic acidemia (MMA), a group of autosomal recessive inborn errors of metabolism, is most commonly caused by complete (mut(0)) or partial (mut(-)) deficiency of the enzyme methylmalonyl-CoA mutase (MUT). The severe metabolic instability and increased mortality experienced by many affected individuals, especially those with mut(0) MMA, has led centers to use elective liver transplantation as a treatment for these patients. We have previously demonstrated the efficacy of systemic adeno-associated viral (AAV) gene delivery as a treatment for MMA in a murine model and therefore sought to survey AAV antibody titers against serotypes 2, 8, and 9 in a group of well-characterized MMA patients, accrued via a dedicated natural history study ( clinicaltrials.gov ID: NCT00078078). Plasma samples provided by 42 patients (8 mut(-) and 34 mut(0); 10 had received organ transplantation), who ranged in age between 2 and 31 years, were analyzed to examine AAV2 (n = 35), AAV8 (n = 41), and AAV9 (n = 42) antibody titers. In total, the seroprevalence of antibodies against AAV2, AAV8, or AAV9 was 20%, 22%, and 24%, respectively. We observed a lower-than-expected seropositivity rate (titers ≥1:20) in the pediatric MMA patients (2-18 years) for both AAV2 (p < 0.05) and AAV8 (p < 0.01) neutralizing antibodies (NAbs) compared with historical controls. Those with positive NAb titers were typically older than 18 years (p < 0.05 all serotypes) or had received solid organ transplantation (p < 0.01 AAV8, AAV9). The mut(0) patients who had not been transplanted (n = 24)-that is, the subset with the greatest need for improved treatments-represented the seronegative majority, with 21 out of 24 patients lacking Abs against all AAV capsids tested. The unexpected lack of NAbs against AAV in this patient population has encouraging implications for systemic gene delivery as a treatment for mut MMA. PMID:26790480

  17. De Novo Sequencing and Resurrection of a Human Astrovirus-Neutralizing Antibody

    PubMed Central

    2016-01-01

    Monoclonal antibody (mAb) therapeutics targeting cancer, autoimmune diseases, inflammatory diseases, and infectious diseases are growing exponentially. Although numerous panels of mAbs targeting infectious disease agents have been developed, their progression into clinically useful mAbs is often hindered by the lack of sequence information and/or loss of hybridoma cells that produce them. Here we combine the power of crystallography and mass spectrometry to determine the amino acid sequence and glycosylation modification of the Fab fragment of a potent human astrovirus-neutralizing mAb. We used this information to engineer a recombinant antibody single-chain variable fragment that has the same specificity as the parent monoclonal antibody to bind to the astrovirus capsid protein. This antibody can now potentially be developed as a therapeutic and diagnostic agent. PMID:27213181

  18. Preexisting human antibodies neutralize recently emerged H7N9 influenza strains

    PubMed Central

    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

  19. Vaccine-Induced Antibodies that Neutralize Group 1 and Group 2 Influenza A Viruses.

    PubMed

    Joyce, M Gordon; Wheatley, Adam K; Thomas, Paul V; Chuang, Gwo-Yu; Soto, Cinque; Bailer, Robert T; Druz, Aliaksandr; Georgiev, Ivelin S; Gillespie, Rebecca A; Kanekiyo, Masaru; Kong, Wing-Pui; Leung, Kwanyee; Narpala, Sandeep N; Prabhakaran, Madhu S; Yang, Eun Sung; Zhang, Baoshan; Zhang, Yi; Asokan, Mangaiarkarasi; Boyington, Jeffrey C; Bylund, Tatsiana; Darko, Sam; Lees, Christopher R; Ransier, Amy; Shen, Chen-Hsiang; Wang, Lingshu; Whittle, James R; Wu, Xueling; Yassine, Hadi M; Santos, Celia; Matsuoka, Yumiko; Tsybovsky, Yaroslav; Baxa, Ulrich; Mullikin, James C; Subbarao, Kanta; Douek, Daniel C; Graham, Barney S; Koup, Richard A; Ledgerwood, Julie E; Roederer, Mario; Shapiro, Lawrence; Kwong, Peter D; Mascola, John R; McDermott, Adrian B

    2016-07-28

    Antibodies capable of neutralizing divergent influenza A viruses could form the basis of a universal vaccine. Here, from subjects enrolled in an H5N1 DNA/MIV-prime-boost influenza vaccine trial, we sorted hemagglutinin cross-reactive memory B cells and identified three antibody classes, each capable of neutralizing diverse subtypes of group 1 and group 2 influenza A viruses. Co-crystal structures with hemagglutinin revealed that each class utilized characteristic germline genes and convergent sequence motifs to recognize overlapping epitopes in the hemagglutinin stem. All six analyzed subjects had sequences from at least one multidonor class, and-in half the subjects-multidonor-class sequences were recovered from >40% of cross-reactive B cells. By contrast, these multidonor-class sequences were rare in published antibody datasets. Vaccination with a divergent hemagglutinin can thus increase the frequency of B cells encoding broad influenza A-neutralizing antibodies. We propose the sequence signature-quantified prevalence of these B cells as a metric to guide universal influenza A immunization strategies. PMID:27453470

  20. Maturation Pathway from Germline to Broad HIV-1 Neutralizer of a CD4-Mimic Antibody.

    PubMed

    Bonsignori, Mattia; Zhou, Tongqing; Sheng, Zizhang; Chen, Lei; Gao, Feng; Joyce, M Gordon; Ozorowski, Gabriel; Chuang, Gwo-Yu; Schramm, Chaim A; Wiehe, Kevin; Alam, S Munir; Bradley, Todd; Gladden, Morgan A; Hwang, Kwan-Ki; Iyengar, Sheelah; Kumar, Amit; Lu, Xiaozhi; Luo, Kan; Mangiapani, Michael C; Parks, Robert J; Song, Hongshuo; Acharya, Priyamvada; Bailer, Robert T; Cao, Allen; Druz, Aliaksandr; Georgiev, Ivelin S; Kwon, Young D; Louder, Mark K; Zhang, Baoshan; Zheng, Anqi; Hill, Brenna J; Kong, Rui; Soto, Cinque; Mullikin, James C; Douek, Daniel C; Montefiori, David C; Moody, Michael A; Shaw, George M; Hahn, Beatrice H; Kelsoe, Garnett; Hraber, Peter T; Korber, Bette T; Boyd, Scott D; Fire, Andrew Z; Kepler, Thomas B; Shapiro, Lawrence; Ward, Andrew B; Mascola, John R; Liao, Hua-Xin; Kwong, Peter D; Haynes, Barton F

    2016-04-01

    Antibodies with ontogenies from VH1-2 or VH1-46-germline genes dominate the broadly neutralizing response against the CD4-binding site (CD4bs) on HIV-1. Here, we define with longitudinal sampling from time-of-infection the development of a VH1-46-derived antibody lineage that matured to neutralize 90% of HIV-1 isolates. Structures of lineage antibodies CH235 (week 41 from time-of-infection, 18% breadth), CH235.9 (week 152, 77%), and CH235.12 (week 323, 90%) demonstrated the maturing epitope to focus on the conformationally invariant portion of the CD4bs. Similarities between CH235 lineage and five unrelated CD4bs lineages in epitope focusing, length-of-time to develop breadth, and extraordinary level of somatic hypermutation suggested commonalities in maturation among all CD4bs antibodies. Fortunately, the required CH235-lineage hypermutation appeared substantially guided by the intrinsic mutability of the VH1-46 gene, which closely resembled VH1-2. We integrated our CH235-lineage findings with a second broadly neutralizing lineage and HIV-1 co-evolution to suggest a vaccination strategy for inducing both lineages. PMID:26949186

  1. Structure of HCMV glycoprotein B in the postfusion conformation bound to a neutralizing human antibody

    PubMed Central

    Chandramouli, Sumana; Ciferri, Claudio; Nikitin, Pavel A.; Caló, Stefano; Gerrein, Rachel; Balabanis, Kara; Monroe, James; Hebner, Christy; Lilja, Anders E.; Settembre, Ethan C.; Carfi, Andrea

    2015-01-01

    Human cytomegalovirus (HCMV) poses a significant threat to immunocompromised individuals and neonates infected in utero. Glycoprotein B (gB), the herpesvirus fusion protein, is a target for neutralizing antibodies and a vaccine candidate due to its indispensable role in infection. Here we show the crystal structure of the HCMV gB ectodomain bound to the Fab fragment of 1G2, a neutralizing human monoclonal antibody isolated from a seropositive subject. The gB/1G2 interaction is dominated by aromatic residues in the 1G2 heavy chain CDR3 protruding into a hydrophobic cleft in the gB antigenic domain 5 (AD-5). Structural analysis and comparison with HSV gB suggest the location of additional neutralizing antibody binding sites on HCMV gB. Finally, immunoprecipitation experiments reveal that 1G2 can bind to HCMV virion gB suggesting that its epitope is exposed and accessible on the virus surface. Our data will support the development of vaccines and therapeutic antibodies against HCMV infection. PMID:26365435

  2. Maturation Pathway from Germline to Broad HIV-1 Neutralizer of a CD4-Mimic Antibody

    DOE PAGESBeta

    Bonsignori, Mattia; Zhou, Tongqing; Sheng, Zizhang; Chen, Lei; Gao, Feng; Joyce, M.  Gordon; Ozorowski, Gabriel; Chuang, Gwo-Yu; Schramm, Chaim A.; Wiehe, Kevin; et al

    2016-04-01

    Here, we report that antibodies with ontogenies from VH1-2 or VH1-46-germline genes dominate the broadly neutralizing response against the CD4-binding site (CD4bs) on HIV-1. We define with longitudinal sampling from time-of-infection the development of a VH1-46-derived antibody lineage that matured to neutralize 90% of HIV-1 isolates. Structures of lineage antibodies CH235 (week 41 from time-of-infection, 18% breadth), CH235.9 (week 152, 77%), and CH235.12 (week 323, 90%) demonstrated the maturing epitope to focus on the conformationally invariant portion of the CD4bs. Similarities between CH235 lineage and five unrelated CD4bs lineages in epitope focusing, length-of-time to develop breadth, and extraordinary level ofmore » somatic hypermutation suggested commonalities in maturation among all CD4bs antibodies. Fortunately, the required CH235-lineage hypermutation appeared substantially guided by the intrinsic mutability of the VH1-46 gene, which closely resembled VH1-2. Lastly, we integrated our CH235-lineage findings with a second broadly neutralizing lineage and HIV-1 co-evolution to suggest a vaccination strategy for inducing both lineages.« less

  3. Identification of Epitopes for Neutralizing Antibodies Against H10N8 Avian Influenza Virus Hemagglutinin.

    PubMed

    Hu, Jin-Fang; Sun, Chun-Yun; Rao, Mu-Ding; Xie, Liang-Zhi

    2016-08-01

    Objective To develop neutralizing monoclonal antibodies (MAbs) against H10N8 avian influenza virus hemagglutinin and to identify the binding sites. Methods MAbs against hemagglutinin of H10N8 avian influenza virus were developed by genetic engineering. Neutralizing MAbs were screened by microneutralization assay,and then tested by enzyme-linked immunosorbent assay and Western blot to identity the binding sites.The homology modeling process was performed using Discovery Studio 3.5 software,while the binding epitopes were analyzed by BioEdit software. Results One MAb that could neutralize the H10N8 pseudovirus was obtained and characterized. Analysis about epitopes suggested that the antibody could bind to the HA1 region of hemagglutinin,while the epitopes on antigen were conserved in H10 subtypes.Conclusions One neutralizing antibody was obtained by this research.The MAb may potentially be further developed as a pre-clinical candidate to treat avian influenza H10N8 virus infection. PMID:27594152

  4. Differential Specificity and Immunogenicity of Adenovirus Type 5 Neutralizing Antibodies Elicited by Natural Infection or Immunization▿

    PubMed Central

    Cheng, Cheng; Gall, Jason G. D.; Nason, Martha; King, C. Richter; Koup, Richard A.; Roederer, Mario; McElrath, M. Juliana; Morgan, Cecilia A.; Churchyard, Gavin; Baden, Lindsey R.; Duerr, Ann C.; Keefer, Michael C.; Graham, Barney S.; Nabel, Gary J.

    2010-01-01

    A recent clinical trial of a T-cell-based AIDS vaccine delivered with recombinant adenovirus type 5 (rAd5) vectors showed no efficacy in lowering viral load and was associated with increased risk of human immunodeficiency virus type 1 (HIV-1) infection. Preexisting immunity to Ad5 in humans could therefore affect both immunogenicity and vaccine efficacy. We hypothesized that vaccine-induced immunity is differentially affected, depending on whether subjects were exposed to Ad5 by natural infection or by vaccination. Serum samples from vaccine trial subjects receiving a DNA/rAd5 AIDS vaccine with or without prior immunity to Ad5 were examined for the specificity of their Ad5 neutralizing antibodies and their effect on HIV-1 immune responses. Here, we report that rAd5 neutralizing antibodies were directed to different components of the virion, depending on whether they were elicited by natural infection or vaccination in HIV vaccine trial subjects. Neutralizing antibodies elicited by natural infection were directed largely to the Ad5 fiber, while exposure to rAd5 through vaccination elicited antibodies primarily to capsid proteins other than fiber. Notably, preexisting immunity to Ad5 fiber from natural infection significantly reduced the CD4 and CD8 cell responses to HIV Gag after DNA/rAd5 vaccination. The specificity of Ad5 neutralizing antibodies therefore differs depending on the route of exposure, and natural Ad5 infection compromises Ad5 vaccine-induced immunity to weak immunogens, such as HIV-1 Gag. These results have implications for future AIDS vaccine trials and the design of next-generation gene-based vaccine vectors. PMID:19846512

  5. Enhancing neutralizing antibody production by an interferon-inducing porcine reproductive and respiratory syndrome virus strain.

    PubMed

    Wang, Rong; Xiao, Yueqiang; Opriessnig, Tanja; Ding, Yi; Yu, Ying; Nan, Yuchen; Ma, Zexu; Halbur, Patrick G; Zhang, Yan-Jin

    2013-11-12

    Porcine reproductive and respiratory syndrome (PRRS) virus (PRRSV) continues to cause substantial economic losses to the global swine industry. PRRSV appears to inhibit synthesis of type I interferons (IFNs), such as IFN-α and -β, which are critical for the innate immunity and play an important role in the modulation of adaptive immunity. An atypical PRRSV strain, A2MC2, is able to induce type I IFNs in vitro. In this study, A2MC2 induction of neutralizing antibodies in vivo was compared with the Ingelvac PRRS modified live virus (MLV) vaccine strain and VR-2385 (a moderate virulent strain). Three-week-old pigs were exposed to these PRRSV strains via intranasal or intramuscular routes to also account for a possible effect of inoculation routes. The interferon-inducing A2MC2 resulted in earlier onset and significantly higher levels of PRRSV neutralizing antibodies than the MLV. In addition, the A2MC2-induced neutralizing antibodies were capable of neutralizing VR-2385, a heterologous strain. The pigs exposed via intranasal route had higher titers of neutralizing antibodies than those injected via intramuscular route. Macroscopic and microscopic lung lesions 14 days post-exposure indicated that A2MC2 had similar virulence in vivo as VR-2385. Pulmonary alveolar macrophages (PAMs) collected during the necropsy 14 days post-exposure in the A2MC2 group had higher level expression of IFN-γ than the MLV group. These results indicate that A2MC2 can be further explored for development of an improved vaccine against PRRS. PMID:24063978

  6. Serum Neutralization Assay Can Efficiently Replace Plaque Reduction Neutralization Test for Detection and Quantitation of West Nile Virus Antibodies in Human and Animal Serum Samples

    PubMed Central

    Di Gennaro, Annapia; Casaccia, Claudia; Conte, Annamaria; Monaco, Federica; Savini, Giovanni

    2014-01-01

    A serum neutralization assay (SN) was compared with the official plaque reduction neutralization test for the quantitation of West Nile virus antibodies. A total of 1,348 samples from equid sera and 38 from human sera were tested by these two methods. Statistically significant differences were not observed, thus supporting the use of SN for routine purposes. PMID:25100824

  7. Neutralization of measles virus wild-type isolates after immunization with a synthetic peptide vaccine which is not recognized by neutralizing passive antibodies.

    PubMed

    El Kasmi, K C; Fillon, S; Theisen, D M; Hartter, H; Brons, N H; Muller, C P

    2000-03-01

    The sequence H379-410 of the measles virus haemagglutinin (MV-H) protein forms a surface-exposed loop and contains three cysteine residues (Cys-381, Cys-386 and Cys-394) which are conserved among all measles isolates. It comprises the minimal sequential B cell epitope (BCE) (H386-400) of the neutralizing and protective MAb BH6 that neutralizes all wild-type viruses tested. The aim of this study was to design synthetic peptides which induce neutralizing antibodies against MV wild-type isolates. Peptides containing one or two copies of T cell epitopes (TCE) and BCEs of different lengths (H386-400, B(CC); H379-400, B(CCC)), in different combinations and orientations were produced and iteratively optimized for inducing neutralizing antibodies. Peptides with the shorter BCE induced sera that cross-reacted with MV but did not neutralize. The longer BCE containing the three cysteines (B(CCC)) and two homologous TCE were required for neutralization activity. These sera neutralized wild-type strains of different clades and geographic origins. Neutralizing serum was also obtained after immunization with human promiscuous TCEs. Furthermore B(CCC)-based peptides were fully immunogenic even in the presence of pre-existing MV-specific antibodies. The results suggest that subunit vaccines based on such peptides could potentially be used to actively protect infants against wild-type viruses irrespective of persisting maternal antibodies. PMID:10675410

  8. Neutralizing Monoclonal Antibodies Directed against Defined Linear Epitopes on Domain 4 of Anthrax Protective Antigen▿

    PubMed Central

    Kelly-Cirino, Cassandra D.; Mantis, Nicholas J.

    2009-01-01

    The anthrax protective antigen (PA) is the receptor-binding subunit common to lethal toxin (LT) and edema toxin (ET), which are responsible for the high mortality rates associated with inhalational Bacillus anthracis infection. Although recombinant PA (rPA) is likely to be an important constituent of any future anthrax vaccine, evaluation of the efficacies of the various candidate rPA vaccines is currently difficult, because the specific B-cell epitopes involved in toxin neutralization have not been completely defined. In this study, we describe the identification and characterization of two murine monoclonal immunoglobulin G1 antibodies (MAbs), 1-F1 and 2-B12, which recognize distinct linear neutralizing epitopes on domain 4 of PA. 1-F1 recognized a 12-mer peptide corresponding to residues 692 to 703; this epitope maps to a region of domain 4 known to interact with the anthrax toxin receptor CMG-2 and within a conformation-dependent epitope recognized by the well-characterized neutralizing MAb 14B7. As expected, 1-F1 blocked PA's ability to associate with CMG-2 in an in vitro solid-phase binding assay, and it protected murine macrophage cells from intoxication with LT. 2-B12 recognized a 12-mer peptide corresponding to residues 716 to 727, an epitope located immediately adjacent to the core 14B7 binding site and a stretch of amino acids not previously identified as a target of neutralizing antibodies. 2-B12 was as effective as 1-F1 in neutralizing LT in vitro, although it only partially inhibited PA binding to its receptor. Mice passively administered 1-F1 or 2-B12 were partially protected against a lethal challenge with LT. These results advance our fundamental understanding of the mechanisms by which antibodies neutralize anthrax toxin and may have future application in the evaluation of candidate rPA vaccines. PMID:19703971

  9. Broadly neutralizing antibodies that inhibit HIV-1 cell to cell transmission

    PubMed Central

    Malbec, Marine; Porrot, Françoise; Rua, Rejane; Horwitz, Joshua; Klein, Florian; Halper-Stromberg, Ari; Scheid, Johannes F.; Eden, Caroline; Mouquet, Hugo; Nussenzweig, Michel C.

    2013-01-01

    The neutralizing activity of anti–HIV-1 antibodies is typically measured in assays where cell-free virions enter reporter cell lines. However, HIV-1 cell to cell transmission is a major mechanism of viral spread, and the effect of the recently described broadly neutralizing antibodies (bNAbs) on this mode of transmission remains unknown. Here we identify a subset of bNAbs that inhibit both cell-free and cell-mediated infection in primary CD4+ lymphocytes. These antibodies target either the CD4-binding site (NIH45-46 and 3BNC60) or the glycan/V3 loop (10-1074 and PGT121) on HIV-1 gp120 and act at low concentrations by inhibiting multiple steps of viral cell to cell transmission. These antibodies accumulate at virological synapses and impair the clustering and fusion of infected and target cells and the transfer of viral material to uninfected T cells. In addition, they block viral cell to cell transmission to plasmacytoid DCs and thereby interfere with type-I IFN production. Thus, only a subset of bNAbs can efficiently prevent HIV-1 cell to cell transmission, and this property should be considered an important characteristic defining antibody potency for therapeutic or prophylactic antiviral strategies. PMID:24277152

  10. Epitope Mapping of Anti-Interleukin-13 Neutralizing Antibody CNTO607

    SciTech Connect

    Teplyakov, Alexey; Obmolova, Galina; Wu, Sheng-Jiun; Luo, Jinquan; Kang, James; O'Neil, Karyn; Gilliland, Gary L.

    2009-06-24

    CNTO607 is a neutralizing anti-interleukin-13 (IL-13) human monoclonal antibody obtained from a phage display library. To determine how this antibody inhibits the biological effect of IL-13, we determined the binding epitope by X-ray crystallography. The crystal structure of the complex between CNTO607 Fab and IL-13 reveals the antibody epitope at the surface formed by helices A and D of IL-13. This epitope overlaps with the IL-4Ralpha/IL-13Ralpha1 receptor-binding site, which explains the neutralizing effect of CNTO607. The extensive antibody interface covers an area of 1000 A(2), which is consistent with the high binding affinity. The key features of the interface are the charge and shape complementarity of the molecules that include two hydrophobic pockets on IL-13 that accommodate Phe32 [complementarity-determining region (CDR) L2] and Trp100a (CDR H3) and a number of salt bridges between basic residues of IL-13 and acidic residues of the antibody. Comparison with the structure of the free Fab shows that the CDR residues do not change their conformation upon complex formation, with the exception of two residues in CDR H3, Trp100a and Asp100b, which change rotamer conformations. To evaluate the relative contribution of the epitope residues to CNTO607 binding, we performed alanine-scanning mutagenesis of the A-D region of IL-13. This study confirmed the primary role of electrostatic interactions for antigen recognition.

  11. Long antibody HCDR3s from HIV-naïve donors presented on a PG9 neutralizing antibody background mediate HIV neutralization.

    PubMed

    Willis, Jordan R; Finn, Jessica A; Briney, Bryan; Sapparapu, Gopal; Singh, Vidisha; King, Hannah; LaBranche, Celia C; Montefiori, David C; Meiler, Jens; Crowe, James E

    2016-04-19

    Development of broadly neutralizing antibodies (bnAbs) against HIV-1 usually requires prolonged infection and induction of Abs with unusual features, such as long heavy-chain complementarity-determining region 3 (HCDR3) loops. Here we sought to determine whether the repertoires of HIV-1-naïve individuals contain Abs with long HCDR3 loops that could mediate HIV-1 neutralization. We interrogated at massive scale the structural properties of long Ab HCDR3 loops in HIV-1-naïve donors, searching for structured HCDR3s similar to those of the HIV-1 bnAb PG9. We determined the nucleotide sequences encoding 2.3 × 10(7)unique HCDR3 amino acid regions from 70 different HIV-1-naïve donors. Of the 26,917 HCDR3 loops with 30-amino acid length identified, we tested 30 for further study that were predicted to have PG9-like structure when chimerized onto PG9. Three of these 30 PG9 chimeras bound to the HIV-1 gp120 monomer, and two were neutralizing. In addition, we found 14 naturally occurring HCDR3 sequences that acquired the ability to bind to the HIV-1 gp120 monomer when adding 2- to 7-amino acid mutations via computational design. Of those 14 designed Abs, 8 neutralized HIV-1, with IC50values ranging from 0.7 to 98 µg/mL. These data suggest that the repertoire of HIV-1-naïve individuals contains rare B cells that encode HCDR3 loops that bind or neutralize HIV-1 when presented on a PG9 background with relatively few or no additional mutations. Long HCDR3 sequences are present in the HIV-naïve B-cell repertoire, suggesting that this class of bnAbs is a favorable target for rationally designed preventative vaccine efforts. PMID:27044078

  12. Development of human neutralizing antibody to ADAMTS4 (aggrecanase-1) and ADAMTS5 (aggrecanase-2).

    PubMed

    Shiraishi, Aya; Mochizuki, Satsuki; Miyakoshi, Akira; Kojoh, Kanehisa; Okada, Yasunori

    2016-01-01

    ADAMTS4 (aggrecanase-1) and ADAMTS5 (aggrecanase-2), members of the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) gene family, are considered to play a key role in aggrecan degradation of articular cartilage in human osteoarthritis. Here, we developed a neutralizing antibody to these aggrecanases by screening human combinatorial antibody library. Among the five candidate antibodies, one antibody was immunoreactive with both ADAMTS4 and ADAMTS5, showing no or negligible cross-reactivity with 10 different related metalloproteinases of the ADAMTS, ADAM (a disintegrin and metalloproteinase) and MMP (matrix metalloproteinase) gene families. This antibody almost completely and partially inhibited aggrecanase activity of ADAMTS4 and ADAMTS5, respectively. It also suppressed the aggrecanase activity derived from interleukin-1-stimulated osteoarthritic chondrocytes. These data demonstrate that the antibody is specific to ADAMTS4 and ADAMTS5 and inhibits their aggrecanase activity at molecular and cellular levels, and suggest that this antibody may be useful for treatment of pathological conditions such as osteoarthritis. PMID:26612259

  13. Antibodies with specificity to native gp120 and neutralization activity against primary human immunodeficiency virus type 1 isolates elicited by immunization with oligomeric gp160.

    PubMed Central

    VanCott, T C; Mascola, J R; Kaminski, R W; Kalyanaraman, V; Hallberg, P L; Burnett, P R; Ulrich, J T; Rechtman, D J; Birx, D L

    1997-01-01

    Current human immunodeficiency virus type 1 (HIV-1) envelope vaccine candidates elicit high antibody binding titers with neutralizing activity against T-cell line-adapted but not primary HIV-1 isolates. Serum antibodies from these human vaccine recipients were also found to be preferentially directed to linear epitopes within gp120 that are poorly exposed on native gp120. Systemic immunization of rabbits with an affinity-purified oligomeric gp160 protein formulated with either Alhydrogel or monophosphoryl lipid A-containing adjuvants resulted in the induction of high-titered serum antibodies that preferentially bound epitopes exposed on native forms of gp120 and gp160, recognized a restricted number of linear epitopes, efficiently bound heterologous strains of monomeric gp120 and cell surface-expressed oligomeric gp120/gp41, and neutralized several strains of T-cell line-adapted HIV-1. Additionally, those immune sera with the highest oligomeric gp160 antibody binding titers had neutralizing activity against some primary HIV-1 isolates, using phytohemagglutinin-stimulated peripheral blood mononuclear cell targets. Induction of an antibody response preferentially reactive with natively folded gp120/gp160 was dependent on the tertiary structure of the HIV-1 envelope immunogen as well as its adjuvant formulation, route of administration, and number of immunizations administered. These studies demonstrate the capacity of a soluble HIV-1 envelope glycoprotein vaccine to elicit an antibody response capable of neutralizing primary HIV-1 isolates. PMID:9151820

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

    SciTech Connect

    Guan, Jian; Bywaters, Stephanie M.; Brendle, Sarah A.; Lee, Hyunwook; Ashley, Robert E.; Makhov, Alexander M.; Conway, James F.; Christensen, Neil D.; Hafenstein, Susan

    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.

  15. Neutralization of Japanese Encephalitis Virus by heme-induced broadly reactive human monoclonal antibody

    PubMed Central

    Gupta, Nimesh; de Wispelaere, Mélissanne; Lecerf, Maxime; Kalia, Manjula; Scheel, Tobias; Vrati, Sudhanshu; Berek, Claudia; Kaveri, Srinivas V.; Desprès, Philippe; Lacroix-Desmazes, Sébastien; Dimitrov, Jordan D.

    2015-01-01

    Geographical expansion and re-emerging new genotypes of the Japanese encephalitis virus (JEV) require the development of novel therapeutic approaches. Here, we studied a non-conventional approach for antibody therapy and show that, upon exposure to heme, a fraction of natural human immunoglobulins acquires high-affinity reactivity with the antigenic domain-III of JEV E glycoprotein. These JEV-reactive antibodies exhibited neutralizing activity against recently dominant JEV genotypes. This study opens new therapeutic options for Japanese encephalitis. PMID:26542535

  16. The protective role of humoral neutralizing antibody in the NIH potency test for rabies vaccines.

    PubMed

    Wunderli, P S; Shaddock, J H; Schmid, D S; Miller, T J; Baer, G M

    1991-09-01

    Intraperitoneal vaccination of mice with rabies vaccine results in both dosage-dependent rabies virus neutralizing antibody titres and protection from lethal intracerebral (i.c.) challenge with fixed strain CVS rabies virus. Pre-exposure adoptive intravenous transfer of naive or immune cells did not significantly protect naive Balb/c mice from lethal i.c. CVS challenge, but immune serum and anti-rabies glycoprotein monoclonal antibodies (individually and in combination) did confer significant protection when administered before or up to 24 h after lethal i.c. rabies virus challenge. PMID:1950097

  17. A Novel Humanized Antibody Neutralizes H5N1 Influenza Virus via Two Different Mechanisms

    PubMed Central

    Tan, Yunrui; Ng, Qingyong; Jia, Qiang

    2015-01-01

    ABSTRACT Highly pathogenic avian influenza virus subtype H5N1 continues to be a severe threat to public health, as well as the poultry industry, because of its high lethality and antigenic drift rate. Neutralizing monoclonal antibodies (MAbs) can serve as a useful tool for preventing, treating, and detecting H5N1. In the present study, humanized H5 antibody 8A8 was developed from a murine H5 MAb. Both the humanized and mouse MAbs presented positive activity in hemagglutination inhibition (HI), virus neutralization, and immunofluorescence assays against a wide range of H5N1 strains. Interestingly, both human and murine 8A8 antibodies were able to detect H5 in Western blot assays under reducing conditions. Further, by sequencing of escape mutants, the conformational epitope of 8A8 was found to be located within the receptor binding domain (RBD) of H5. The linear epitope of 8A8 was identified by Western blotting of overlapping fragments and substitution mutant forms of HA1. Reverse genetic H5N1 strains with individual mutations in either the conformational or the linear epitope were generated and characterized in a series of assays, including HI, postattachment, and cell-cell fusion inhibition assays. The results indicate that for 8A8, virus neutralization mediated by RBD blocking relies on the conformational epitope while binding to the linear epitope contributes to the neutralization by inhibiting membrane fusion. Taken together, the results of this study show that a novel humanized H5 MAb binds to two types of epitopes on HA, leading to virus neutralization via two mechanisms. IMPORTANCE Recurrence of the highly pathogenic avian influenza virus subtype H5N1 in humans and poultry continues to be a serious public health concern. Preventive and therapeutic measures against influenza A viruses have received much interest in the context of global efforts to combat the current and future pandemics. Passive immune therapy is considered to be the most effective and

  18. Requirement for Fc Effector Mechanisms in the APOBEC3/Rfv3-Dependent Neutralizing Antibody Response

    PubMed Central

    Halemano, Kalani; Barrett, Bradley S.; Heilman, Karl J.; Morrison, Thomas E.

    2015-01-01

    Antiretroviral neutralizing antibody (NAb) responses are often evaluated in the absence of Fc-dependent immune effectors. In murine Friend retrovirus infection, Apobec3/Rfv3 promotes a potent polyclonal NAb response. Here, we show that the Apobec3/Rfv3-dependent NAb response correlated with virus-specific IgG2 titers and that the in vivo neutralization potency of Apobec3/Rfv3-resistant antisera was dependent on activating Fcγ receptors but not complement. The data strengthen retroviral vaccine strategies aimed at eliciting NAbs that activate specific Fcγ receptors. PMID:25589647

  19. Anti-IL-6 neutralizing antibody modulates blood-brain barrier function in the ovine fetus.

    PubMed

    Zhang, Jiyong; Sadowska, Grazyna B; Chen, Xiaodi; Park, Seon Yeong; Kim, Jeong-Eun; Bodge, Courtney A; Cummings, Erin; Lim, Yow-Pin; Makeyev, Oleksandr; Besio, Walter G; Gaitanis, John; Banks, William A; Stonestreet, Barbara S

    2015-05-01

    Impaired blood-brain barrier function represents an important component of hypoxic-ischemic brain injury in the perinatal period. Proinflammatory cytokines could contribute to ischemia-related blood-brain barrier dysfunction. IL-6 increases vascular endothelial cell monolayer permeability in vitro. However, contributions of IL-6 to blood-brain barrier abnormalities have not been examined in the immature brain in vivo. We generated pharmacologic quantities of ovine-specific neutralizing anti-IL-6 mAbs and systemically infused mAbs into fetal sheep at 126 days of gestation after exposure to brain ischemia. Anti-IL-6 mAbs were measured by ELISA in fetal plasma, cerebral cortex, and cerebrospinal fluid, blood-brain barrier permeability was quantified using the blood-to-brain transfer constant in brain regions, and IL-6, tight junction proteins, and plasmalemma vesicle protein (PLVAP) were detected by Western immunoblot. Anti-IL-6 mAb infusions resulted in increases in mAb (P < 0.05) in plasma, brain parenchyma, and cerebrospinal fluid and decreases in brain IL-6 protein. Twenty-four hours after ischemia, anti-IL-6 mAb infusions attenuated ischemia-related increases in blood-brain barrier permeability and modulated tight junction and PLVAP protein expression in fetal brain. We conclude that inhibiting the effects of IL-6 protein with systemic infusions of neutralizing antibodies attenuates ischemia-related increases in blood-brain barrier permeability by inhibiting IL-6 and modulates tight junction proteins after ischemia. PMID:25609424

  20. Production in yeast of pseudotype virus-like particles harboring functionally active antibody fragments neutralizing the cytolytic activity of vaginolysin

    PubMed Central

    2011-01-01

    Background Recombinant antibodies can be produced in different formats and different expression systems. Single chain variable fragments (scFvs) represent an attractive alternative to full-length antibodies and they can be easily produced in bacteria or yeast. However, the scFvs exhibit monovalent antigen-binding properties and short serum half-lives. The stability and avidity of the scFvs can be improved by their multimerization or fusion with IgG Fc domain. The aim of the current study was to investigate the possibilities to produce in yeast high-affinity scFv-Fc proteins neutralizing the cytolytic activity of vaginolysin (VLY), the main virulence factor of Gardnerella vaginalis. Results The scFv protein derived from hybridoma cell line producing high-affinity neutralizing antibodies against VLY was fused with human IgG1 Fc domain. Four different variants of anti-VLY scFv-Fc fusion proteins were constructed and produced in yeast Saccharomyces cerevisiae. The non-tagged scFv-Fc and hexahistidine-tagged scFv-Fc proteins were found predominantly as insoluble aggregates and therefore were not suitable for further purification and activity testing. The addition of yeast α-factor signal sequence did not support secretion of anti-VLY scFv-Fc but increased the amount of its intracellular soluble form. However, the purified protein showed a weak VLY-neutralizing capability. In contrast, the fusion of anti-VLY scFv-Fc molecules with hamster polyomavirus-derived VP2 protein and its co-expression with VP1 protein resulted in an effective production of pseudotype virus-like particles (VLPs) that exhibited strong VLY-binding activity. Recombinant scFv-Fc molecules displayed on the surface of VLPs neutralized VLY-mediated lysis of human erythrocytes and HeLa cells with high potency comparable to that of full-length antibody. Conclusions Recombinant scFv-Fc proteins were expressed in yeast with low efficiency. New approach to display the scFv-Fc molecules on the surface of

  1. Generation of recombinant antibody fragments with toxin-neutralizing potential in loxoscelism.

    PubMed

    Karim-Silva, Sabrina; Moura, Juliana de; Noiray, Magali; Minozzo, Joao Carlos; Aubrey, Nicolas; Alvarenga, Larissa M; Billiald, Philippe

    2016-08-01

    Loxosceles spider bites often lead to serious envenomings and no definite therapy has yet been established. In such a context, it is of interest to consider an antibody-based targeted therapy. We have previously prepared a murine monoclonal IgG (LiMab7) that binds to 32-35kDa components of Loxosceles intermedia venom and neutralizes the dermonecrotic activity of the venom. Here, we re-engineered LiMab7 into a recombinant diabody. The protein was produced in bacteria and then it was functionally characterized. It proved to be efficient at neutralizing sphingomyelinase and hemolytic activities of the crude venom despite the slightly altered binding kinetic constants and the limited stability of the dimeric configuration. This is the first report of a specific recombinant antibody for a next-generation of Loxosceles antivenoms. PMID:27288291

  2. Neutralizing antibodies to botulinum neurotoxin type A in aesthetic medicine: five case reports

    PubMed Central

    Torres, Sebastian; Hamilton, Mark; Sanches, Elena; Starovatova, Polina; Gubanova, Elena; Reshetnikova, Tatiana

    2014-01-01

    Botulinum neurotoxin injections are a valuable treatment modality for many therapeutic indications as well as in the aesthetic field for facial rejuvenation. As successful treatment requires repeated injections over a long period of time, secondary resistance to botulinum toxin preparations after repeated injections is an ongoing concern. We report five case studies in which neutralizing antibodies to botulinum toxin type A developed after injection for aesthetic use and resulted in secondary treatment failure. These results add to the growing number of reports in the literature for secondary treatment failure associated with high titers of neutralizing antibodies in the aesthetic field. Clinicians should be aware of this risk and implement injection protocols that minimize resistance development. PMID:24379687

  3. Use of the hemadsorption phenomenon for determining virus and neutralizing antibody titers of rabies.

    PubMed

    Minamoto, N; Kurata, K; Kaizuka, I; Sazawa, H

    1976-05-01

    Chicken embryo cells infected with the HEP Flury strain of rabies virus adapted to tissue culture produced a hemadsorption (HAD) phenomenon by using goose erthyrocytes. The optimal conditions for HAD included the incubation of cell cultures at 37C for 3 days after virus inoculation, the use of a 0.4% suspension of goose erythrocytes in phosphate buffer adjusted at pH 6.2, and adsorption of erythrocytes at 4C. This phenomenon was inhibited with anti-rabies serum. Virus titer obtained with the HAD technique was almost the same as with the fluorescent antibody technique or the intracerebral inoculation of suckling mice. Results of the neutralization test by using the HAD technique could be easily determined 3 days after inoculation of chicken embryo cells with the mixture of 100 mean tissue culture infective doses of virus and diluted serum. The neutralizing antibody titers coincided with those obtained in mice. PMID:57927

  4. Fusion peptide of HIV-1 as a site of vulnerability to neutralizing antibody.

    PubMed

    Kong, Rui; Xu, Kai; Zhou, Tongqing; Acharya, Priyamvada; Lemmin, Thomas; Liu, Kevin; Ozorowski, Gabriel; Soto, Cinque; Taft, Justin D; Bailer, Robert T; Cale, Evan M; Chen, Lei; Choi, Chang W; Chuang, Gwo-Yu; Doria-Rose, Nicole A; Druz, Aliaksandr; Georgiev, Ivelin S; Gorman, Jason; Huang, Jinghe; Joyce, M Gordon; Louder, Mark K; Ma, Xiaochu; McKee, Krisha; O'Dell, Sijy; Pancera, Marie; Yang, Yongping; Blanchard, Scott C; Mothes, Walther; Burton, Dennis R; Koff, Wayne C; Connors, Mark; Ward, Andrew B; Kwong, Peter D; Mascola, John R

    2016-05-13

    The HIV-1 fusion peptide, comprising 15 to 20 hydrophobic residues at the N terminus of the Env-gp41 subunit, is a critical component of the virus-cell entry machinery. Here, we report the identification of a neutralizing antibody, N123-VRC34.01, which targets the fusion peptide and blocks viral entry by inhibiting conformational changes in gp120 and gp41 subunits of Env required for entry. Crystal structures of N123-VRC34.01 liganded to the fusion peptide, and to the full Env trimer, revealed an epitope consisting of the N-terminal eight residues of the gp41 fusion peptide and glycan N88 of gp120, and molecular dynamics showed that the N-terminal portion of the fusion peptide can be solvent-exposed. These results reveal the fusion peptide to be a neutralizing antibody epitope and thus a target for vaccine design. PMID:27174988

  5. Systems immunology: Beyond antibody titers.

    PubMed

    Cao, Raquel; Mejias, Asuncion; Ramilo, Octavio

    2016-07-01

    Despite the evident success of currently available vaccines to prevent infectious diseases, we still lack a full understanding of the mechanisms by which vaccines induce protective immune responses. Systems immunology applies multifaceted analytical tools to better understand the immune responses to vaccines by deep characterization of the cellular components, regulatory pathways, antibody responses and immune gene profiles with the ultimate goal of identifying the complex cellular, genetic and regulatory factors and mechanisms that contribute to effective and protective immune responses. PMID:27180310

  6. Impact of Valency of a Glycoprotein B-Specific Monoclonal Antibody on Neutralization of Herpes Simplex Virus ▿

    PubMed Central

    Krawczyk, Adalbert; Krauss, Jürgen; Eis-Hübinger, Anna M.; Däumer, Martin P.; Schwarzenbacher, Robert; Dittmer, Ulf; Schneweis, Karl E.; Jäger, Dirk; Roggendorf, Michael; Arndt, Michaela A. E.

    2011-01-01

    Herpes simplex virus (HSV) glycoprotein B (gB) is an integral part of the multicomponent fusion system required for virus entry and cell-cell fusion. Here we investigated the mechanism of viral neutralization by the monoclonal antibody (MAb) 2c, which specifically recognizes the gB of HSV type 1 (HSV-1) and HSV-2. Binding of MAb 2c to a type-common discontinuous epitope of gB resulted in highly efficient neutralization of HSV at the postbinding/prefusion stage and completely abrogated the viral cell-to-cell spread in vitro. Mapping of the antigenic site recognized by MAb 2c to the recently solved crystal structure of the HSV-1 gB ectodomain revealed that its discontinuous epitope is only partially accessible within the observed multidomain trimer conformation of gB, likely representing its postfusion conformation. To investigate how MAb 2c may interact with gB during membrane fusion, we characterized the properties of monovalent (Fab and scFv) and bivalent [IgG and F(ab′)2] derivatives of MAb 2c. Our data show that the neutralization capacity of MAb 2c is dependent on cross-linkage of gB trimers. As a result, only bivalent derivatives of MAb 2c exhibited high neutralizing activity in vitro. Notably, bivalent MAb 2c not only was capable of preventing mucocutaneous disease in severely immunodeficient NOD/SCID mice upon vaginal HSV-1 challenge but also protected animals even with neuronal HSV infection. We also report for the first time that an anti-gB specific monoclonal antibody prevents HSV-1-induced encephalitis entirely independently from complement activation, antibody-dependent cellular cytotoxicity, and cellular immunity. This indicates the potential for further development of MAb 2c as an anti-HSV drug. PMID:21123390

  7. Human cell lines used in a micro neutralization test for measuring influenza-neutralizing antibodies.

    PubMed

    Mittelholzer, C M; Brokstad, K A; Pauksens, K; Jonsson, R; Brytting, M; Linde, A

    2006-04-01

    An in situ neutralization test (NT) including ELISA for the measurement of influenza antigen was developed and evaluated. Two human cell lines, fibroblasts (HS27) cells and salivary gland epithelial duct (HSG) cells, were compared with Madin-Darby Canine Kidney (MDCK) cells. The viral production in the human cell lines was lower than that for MDCK cells, which influenced the results of the assay in the HSG and HS27 cells. However, when lowering the infectious dose, the NT using HS27 cells gave a sensitive and stable assay with low background in the ELISA. The NT titres were very low when using HSG cells compared to MDCK cells. The HS27 NT was used to analyze the humoral response after an influenza A infection in patients from a placebo-controlled zanamivir study. We found no differences in NT titres between patients treated with zanamivir or placebo. The MDCK and HS27 NT gave higher titres and more pronounced titre differences than the gold standard haemagglutinin inhibition (HAI) assay. Compared to the HAI assay, the sensitive NT using HS27 cells also revealed heterologous NT-titre rises after influenza infection in the patients. PMID:16623925

  8. A rapid and quantitative assay for measuring neutralizing antibodies of Coxsackievirus B3.

    PubMed

    Chen, Pan; Wu, Xing; Mao, Qunying; Gao, Fan; Hao, Xiaotian; Bian, Lianlian; Zhu, Fengcai; Li, Wenhui; Xu, Miao; Liang, Zhenglun

    2016-06-01

    Coxsackievirus B3 (CVB3) infection has been found to account for an increasing proportion cases of hand, foot and mouth disease (HFMD) in recent epidemiology studies. CVB3 is a single stranded, non-enveloped RNA virus and the infection can cause prominent health threat to pre-school children. Here, by taking approaches of reverse genetics, we established a single-round infection system for CVB3. The pseudovirus was produced by sequential transfection of CVB3 capsid expresser plasmid and CVB3 replicon RNA bearing firefly luciferase as a reporter. The CVB3 pseudovirus system was used for quantifying neutralizing antibody (NtAb) levels of 720 human serum samples and showed superior specificity and sensitivity comparing traditional cytopathic effect (CPE) assay. Furthermore, we compared the seroprevalence of CVB3 NtAbs in pre-school children and healthy adults, and found that only 11.94% of pre-school children were NtAbs positive which suggested that most children were naive to CVB3 infection; while there is much higher positive rate in adults (60%) indicating that most adults have experienced CVB3 infection during childhood. This rapid and quantitative assay greatly facilitates evaluating the level of NtAbs against CVB3 in populations and will help to advance CVB3 vaccine development. PMID:26947399

  9. Mechanistic Study of Broadly Neutralizing Human Monoclonal Antibodies against Dengue Virus That Target the Fusion Loop

    PubMed Central

    Costin, Joshua M.; Zaitseva, Elena; Kahle, Kristen M.; Nicholson, Cindo O.; Rowe, Dawne K.; Graham, Amanda S.; Bazzone, Lindsey E.; Hogancamp, Greg; Figueroa Sierra, Marielys; Fong, Rachel H.; Yang, Sung-Tae; Lin, Li; Robinson, James E.; Doranz, Benjamin J.; Chernomordik, Leonid V.; Michael, Scott F.; Schieffelin, John S.

    2013-01-01

    There are no available vaccines for dengue, the most important mosquito-transmitted viral disease. Mechanistic studies with anti-dengue virus (DENV) human monoclonal antibodies (hMAbs) provide a rational approach to identify and characterize neutralizing epitopes on DENV structural proteins that can serve to inform vaccine strategies. Here, we report a class of hMAbs that is likely to be an important determinant in the human humoral response to DENV infection. In this study, we identified and characterized three broadly neutralizing anti-DENV hMAbs: 4.8A, D11C, and 1.6D. These antibodies were isolated from three different convalescent patients with distinct histories of DENV infection yet demonstrated remarkable similarities. All three hMAbs recognized the E glycoprotein with high affinity, neutralized all four serotypes of DENV, and mediated antibody-dependent enhancement of infection in Fc receptor-bearing cells at subneutralizing concentrations. The neutralization activities of these hMAbs correlated with a strong inhibition of virus-liposome and intracellular fusion, not virus-cell binding. We mapped epitopes of these antibodies to the highly conserved fusion loop region of E domain II. Mutations at fusion loop residues W101, L107, and/or G109 significantly reduced the binding of the hMAbs to E protein. The results show that hMAbs directed against the highly conserved E protein fusion loop block viral entry downstream of virus-cell binding by inhibiting E protein-mediated fusion. Characterization of hMAbs targeting this region may provide new insights into DENV vaccine and therapeutic strategies. PMID:23077306

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

    SciTech Connect

    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

    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. Monoclonal anti-idiotypes induce neutralizing antibodies to enterovirus 70 conformational epitopes.

    PubMed Central

    Wiley, J A; Hamel, J; Brodeur, B R

    1992-01-01

    Monoclonal antibodies (MAbs) directed against the prototype enterovirus 70 (EV-70) strain J670/71 were generated and characterized in order to produce anti-idiotypic MAbs (MAb2s) for use as surrogate immunogens. Western immunoblot and radioimmunoprecipitation assays suggested that all the MAbs recognize conformational epitopes on the virion surface. An EV-70-neutralizing antibody, MAb/ev-12 (MAb1), was selected for the production of MAb2s. Five MAb2s were selected for their capacities to inhibit the interaction of MAb/ev-12 with EV-70 in dot immunobinding inhibition and immunofluorescence assays. In addition, these five MAb2s inhibited virus neutralization mediated by MAb/ev-12, suggesting that they recognize paratope-associated idiotopes. In competition enzyme immunosorbent assays, none of the five MAb2s recognized other neutralizing and nonneutralizing EV-70-specific MAbs, demonstrating that the MAb2s were specific for private idiotopes. Immunization with each of the MAb2s was carried out for the production of anti-anti-idiotypic antibodies (Ab3). All five MAb2s induced an immune response. Moreover, results suggested that they share idiotopes, since MAb2-MAb/ev-12 binding could be inhibited by homologous as well as heterologous Ab3s. Ab3 sera were shown to possess antibodies capable of immunoprecipitating 35S-labeled viral proteins in the same manner as MAb/ev-12. Nine of 15 mice immunized with MAb2s demonstrated Ab3 neutralizing activity specific for the prototype EV-70 strain, J670/71. The potential application of MAb2s to serve as surrogate immunogens for conformational epitopes is substantiated by the results presented in this report. Images PMID:1382141

  12. A murine cytomegalovirus-neutralizing monoclonal antibody exhibits autoreactivity and induces tissue damage in vivo.

    PubMed Central

    Chapman, A J; Farrell, H E; Thomas, J A; Papadimitriou, J M; Garlepp, M J; Scalzo, A A; Shellam, G R

    1994-01-01

    The autoreactivity of murine cytomegalovirus (MCMV)-neutralizing monoclonal antibody (mAb) AC1 was examined in vitro and in vivo. Both mAb AC1 and a human antiserum reactive with U1-small nuclear ribonucleoprotein (U1-snRNP) stained uninfected mouse embryo fibroblasts (MEF) in a speckled nuclear pattern and reacted with 70,000 molecular weight (MW) MEF nuclear antigens by immunoblotting, suggesting that mAb AC1 cross-reacted with the 70,000 MW component of U1-snRNP. However, only mAb AC1 cross-reacted with an additional epithelial cytoplasmic autoantigen present in cultured HEp2 cells. On tissue sections from uninfected mice, mAb AC1 predominantly reacted with a component of central and peripheral nervous systems, although cross-reactivity with the stratum spinosum of the skin and the outer sheath of hair follicles was also observed. Immunoblotting revealed that mAb AC1 reacted with phosphorylated epitopes present on a 98,000 MW MCMV structural protein and the 200,000 MW mouse neurofilament protein (NFP). Treatment of uninfected mice with mAb AC1 resulted in a severe interstitial pneumonia with greatly thickened and congested alveolar septa. Severe oedema of the hypodermis and a mild mesangial proliferative glomerulonephritis were also observed. These results demonstrate that a mAb reacting with a MCMV structural phosphoprotein which can protect mice against the dissemination of MCMV, can also promote the development of autoimmune disease. Therefore, the production of such cross-reactive antibodies may be an important mechanism in the development of autoimmunity following viral infection. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:7515848

  13. Germlining of the HIV-1 broadly neutralizing antibody domain m36

    PubMed Central

    Chen, Weizao; Li, Wei; Ying, Tianlei; Wang, Yanping; Feng, Yang; Dimitrov, Dimiter S.

    2015-01-01

    Engineered antibody domains (eAds) have emerged as a novel class of HIV-1 inhibitors and are currently under preclinical testing as promising drug candidates for prevention and therapy of HIV-1 infection. Reverse mutation of antibodies to germline sequences (germlining) could not only identify less mutated variants with lower probability of immunogenicity and other improved properties but also help elucidate their mechanisms of action. In this study, we sequentially reverted the framework (FRs) and complementary determining regions (CDRs) of m36, a human antibody heavy chain variable domain-based eAd targeting the coreceptor binding site of the viral envelope glycoprotein gp120, back to germline sequences. Two types of amino acid mutations and one region in the antibody V segment were identified that are critical for HIV-1 neutralization. These include four mutations to acidic acid residues distributed in the CDR1 and CDR2, two mutations to hydrophobic residues in the FR3 and CDR3, and partial FR2 and FR3 sequences flanking the CDR2 that are derived from a different gene family. An m36 variant with all five mutations in the FRs reverted back to germline showed slightly increased neutralizing activity against two HIV-1 isolates tested. Another variant with seven of twelve mutations in the V segment reverted retained potency within three-fold of that of the mature antibody. These results, together with an analysis of m36-gp120-CD4 docking structures, could have implications for the further development of m36 as candidate therapeutics and elucidation of its mechanism of potent and broad HIV-1 neutralization. PMID:25676867

  14. Novel Ricin Subunit Antigens With Enhanced Capacity to Elicit Toxin-Neutralizing Antibody Responses in Mice.

    PubMed

    Wahome, Newton; Sully, Erin; Singer, Christopher; Thomas, Justin C; Hu, Lei; Joshi, Sangeeta B; Volkin, David B; Fang, Jianwen; Karanicolas, John; Jacobs, Donald J; Mantis, Nicholas J; Middaugh, C Russell

    2016-05-01

    RiVax is a candidate ricin toxin subunit vaccine antigen that has proven to be safe in human phase I clinical trials. In this study, we introduced double and triple cavity-filling point mutations into the RiVax antigen with the expectation that stability-enhancing modifications would have a beneficial effect on overall immunogenicity of the recombinant proteins. We demonstrate that 2 RiVax triple mutant derivatives, RB (V81L/C171L/V204I) and RC (V81I/C171L/V204I), when adsorbed to aluminum salts adjuvant and tested in a mouse prime-boost-boost regimen were 5- to 10-fold more effective than RiVax at eliciting toxin-neutralizing serum IgG antibody titers. Increased toxin neutralizing antibody values and seroconversion rates were evident at different antigen dosages and within 7 days after the first booster. Quantitative stability/flexibility relationships analysis revealed that the RB and RC mutations affect rigidification of regions spanning residues 98-103, which constitutes a known immunodominant neutralizing B-cell epitope. A more detailed understanding of the immunogenic nature of RB and RC may provide insight into the fundamental relationship between local protein stability and antibody reactivity. PMID:26987947

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

    SciTech Connect

    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.

    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.

  16. Age-specific incidence of neutralization antibodies of Herpes simplex virus.

    PubMed Central

    Terzin, A. L.; Masic, M. G.

    1976-01-01

    Sera of 1255 individuals from Novi Sad, varying in age from less than 1 month to 69 years, have been tested for neutralization antibodies to Herpes implex virus type 1. The eight newborns tested and 97% of the 507 adults were positive, with titres ranging from 1/4 to 1/256. The titres in newborns were significantly lower than the titres in adults. After birth the maternal antibodies declined rapidly and 94% of infants at the age of greater than 6 months and less than 2 years were negative. After the first year infants in Novi Sad start to acquire herpes-neutralizing antibodies actively, reaching a 50% incidence of positives between the 2nd and 3rd year of age. Age-specific incidence rates of herpes positives found in Novi Sad have been compared with those reported from Edinburgh, Freiburg i. Br. and Louisiana. Possible influences of several circumstances upon the incidence rate of positives detected by the neutralization test are discussed. PMID:185287

  17. Influenza Virus-Specific Neutralizing IgM Antibodies Persist for a Lifetime

    PubMed Central

    Skountzou, Ioanna; Satyabhama, Lakshmipriyadarshini; Stavropoulou, Anastasia; Ashraf, Zuhha; Esser, E. Stein; Vassilieva, Elena; Koutsonanos, Dimitrios; Compans, Richard

    2014-01-01

    Detection of immunoglobulin M (IgM) antibodies has long been used as an important diagnostic tool for identifying active viral infections, but their relevance in later stages has not been clearly defined in vivo. In this study, we followed the kinetics, longevity, and function of influenza virus-specific IgM antibodies for 2 years following sublethal infection of mice with live mouse-adapted A/PR/8/34 virus or immunization with formalin-inactivated virus. These groups mounted robust protective immune responses and survived lethal challenges with 50× 50% lethal dose (LD50) mouse-adapted A/PR/8/34 virus 600 days after the primary exposure. Surprisingly, the virus-specific IgM antibodies persisted along with IgG antibodies, and we found a significantly higher number of IgM-positive (IgM+) virus-specific plasma cells than IgG+ plasma cells that persisted for at least 9 months postexposure. The IgM antibodies were functional as they neutralized influenza virus in the presence of complement just as well as IgG antibodies did. PMID:25165027

  18. Antibody

    MedlinePlus

    An antibody is a protein produced by the body's immune system when it detects harmful substances, called antigens. Examples ... microorganisms (bacteria, fungi, parasites, and viruses) and chemicals. Antibodies may be produced when the immune system mistakenly ...

  19. Protective monotherapy against lethal Ebola virus infection by a potently neutralizing antibody.

    PubMed

    Corti, Davide; Misasi, John; Mulangu, Sabue; Stanley, Daphne A; Kanekiyo, Masaru; Wollen, Suzanne; Ploquin, Aurélie; Doria-Rose, Nicole A; Staupe, Ryan P; Bailey, Michael; Shi, Wei; Choe, Misook; Marcus, Hadar; Thompson, Emily A; Cagigi, Alberto; Silacci, Chiara; Fernandez-Rodriguez, Blanca; Perez, Laurent; Sallusto, Federica; Vanzetta, Fabrizia; Agatic, Gloria; Cameroni, Elisabetta; Kisalu, Neville; Gordon, Ingelise; Ledgerwood, Julie E; Mascola, John R; Graham, Barney S; Muyembe-Tamfun, Jean-Jacques; Trefry, John C; Lanzavecchia, Antonio; Sullivan, Nancy J

    2016-03-18

    Ebola virus disease in humans is highly lethal, with case fatality rates ranging from 25 to 90%. There is no licensed treatment or vaccine against the virus, underscoring the need for efficacious countermeasures. We ascertained that a human survivor of the 1995 Kikwit Ebola virus disease outbreak maintained circulating antibodies against the Ebola virus surface glycoprotein for more than a decade after infection. From this survivor we isolated monoclonal antibodies (mAbs) that neutralize recent and previous outbreak variants of Ebola virus and mediate antibody-dependent cell-mediated cytotoxicity in vitro. Strikingly, monotherapy with mAb114 protected macaques when given as late as 5 days after challenge. Treatment with a single human mAb suggests that a simplified therapeutic strategy for human Ebola infection may be possible. PMID:26917593

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

    SciTech Connect

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

    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.

  1. Cross-Reactive and Potent Neutralizing Antibody Responses in Human Survivors of Natural Ebolavirus Infection.

    PubMed

    Flyak, Andrew I; Shen, Xiaoli; Murin, Charles D; Turner, Hannah L; David, Joshua A; Fusco, Marnie L; Lampley, Rebecca; Kose, Nurgun; Ilinykh, Philipp A; Kuzmina, Natalia; Branchizio, Andre; King, Hannah; Brown, Leland; Bryan, Christopher; Davidson, Edgar; Doranz, Benjamin J; Slaughter, James C; Sapparapu, Gopal; Klages, Curtis; Ksiazek, Thomas G; Saphire, Erica Ollmann; Ward, Andrew B; Bukreyev, Alexander; Crowe, James E

    2016-01-28

    Recent studies have suggested that antibody-mediated protection against the Ebolaviruses may be achievable, but little is known about whether or not antibodies can confer cross-reactive protection against viruses belonging to diverse Ebolavirus species, such as Ebola virus (EBOV), Sudan virus (SUDV), and Bundibugyo virus (BDBV). We isolated a large panel of human monoclonal antibodies (mAbs) against BDBV glycoprotein (GP) using peripheral blood B cells from survivors of the 2007 BDBV outbreak in Uganda. We determined that a large proportion of mAbs with potent neutralizing activity against BDBV bind to the glycan cap and recognize diverse epitopes within this major antigenic site. We identified several glycan cap-specific mAbs that neutralized multiple ebolaviruses, including SUDV, and a cross-reactive mAb that completely protected guinea pigs from the lethal challenge with heterologous EBOV. Our results provide a roadmap to develop a single antibody-based treatment effective against multiple Ebolavirus infections. PMID:26806128

  2. The past, present and future of neutralizing antibodies for hepatitis C virus

    PubMed Central

    Ball, Jonathan K.; Tarr, Alexander W.; McKeating, Jane A.

    2014-01-01

    Hepatitis C virus (HCV) is a major cause of liver disease and hepatocellular carcinoma worldwide. HCV establishes a chronic infection in the majority of cases. However, some individuals clear the virus, demonstrating a protective role for the host immune response. Although new all-oral drug combinations may soon replace traditional ribavirin–interferon therapy, the emerging drug cocktails will be expensive and associated with side-effects and resistance, making a global vaccine an urgent priority. T cells are widely accepted to play an essential role in clearing acute HCV infection, whereas the role antibodies play in resolution and disease pathogenesis is less well understood. Recent studies have provided an insight into viral neutralizing determinants and the protective role of antibodies during infection. This review provides a historical perspective of the role neutralizing antibodies play in HCV infection and discusses the therapeutic benefits of antibody-based therapies. This article forms part of a symposium in Antiviral Research on “Hepatitis C: next steps toward global eradication.” PMID:24583033

  3. Development of neutralizing monoclonal antibodies for oncogenic human papillomavirus types 31, 33, 45, 52, and 58.

    PubMed

    Brown, Martha J; Seitz, Hanna; Towne, Victoria; Müller, Martin; Finnefrock, Adam C

    2014-04-01

    Human papillomavirus (HPV) is the etiological agent for all cervical cancers, a significant number of other anogenital cancers, and a growing number of head and neck cancers. Two licensed vaccines offer protection against the most prevalent oncogenic types, 16 and 18, responsible for approximately 70% of cervical cancer cases worldwide and one of these also offers protection against types 6 and 11, responsible for 90% of genital warts. The vaccines are comprised of recombinantly expressed major capsid proteins that self-assemble into virus-like particles (VLPs) and prevent infection by eliciting neutralizing antibodies. Adding the other frequently identified oncogenic types 31, 33, 45, 52, and 58 to a vaccine would increase the coverage against HPV-induced cancers to approximately 90%. We describe the generation and characterization of panels of monoclonal antibodies to these five additional oncogenic HPV types, and the selection of antibody pairs that were high affinity and type specific and recognized conformation-dependent neutralizing epitopes. Such characteristics make these antibodies useful tools for monitoring the production and potency of a prototype vaccine as well as monitoring vaccine-induced immune responses in the clinic. PMID:24574536

  4. Persistence of Neutralizing Antibody Against Dengue Virus 2 After 70 Years from Infection in Nagasaki.

    PubMed

    Ngwe Tun, Mya Myat; Muta, Yoshihito; Inoue, Shingo; Morita, Kouichi

    2016-01-01

    This study aimed to investigate the duration of humoral immune responses to dengue virus (DENV) infection in Japanese who experienced acute febrile illness with hemorrhagic manifestations 70 years ago, when an epidemic of dengue occurred in Nagasaki, Japan, from 1942 to 1944. A Japanese volunteer requested serological diagnosis of DENV infection in 2014 and donated blood sample to measure the antibody titer against DENV by antiflavi IgG indirect ELISA, focus reduction neutralization test, and plaque reduction neutralization test. The serum sample of the volunteer was positive in flavi IgG ELISA and it indicated primary infection. In the neutralization test, the highest neutralizing titer was ≥218 for DENV-2. We report here the existence of DENV-specific antibodies in the serum of a person after 70 years from infection. Published reports indicated that DENV-1 was responsible for the 1942-1944 outbreak in Nagasaki. However, our data suggested that DENV-2 also played a role in this Nagasaki dengue epidemic. PMID:27493841

  5. Persistence of Neutralizing Antibody Against Dengue Virus 2 After 70 Years from Infection in Nagasaki

    PubMed Central

    Ngwe Tun, Mya Myat; Muta, Yoshihito; Inoue, Shingo; Morita, Kouichi

    2016-01-01

    Abstract This study aimed to investigate the duration of humoral immune responses to dengue virus (DENV) infection in Japanese who experienced acute febrile illness with hemorrhagic manifestations 70 years ago, when an epidemic of dengue occurred in Nagasaki, Japan, from 1942 to 1944. A Japanese volunteer requested serological diagnosis of DENV infection in 2014 and donated blood sample to measure the antibody titer against DENV by antiflavi IgG indirect ELISA, focus reduction neutralization test, and plaque reduction neutralization test. The serum sample of the volunteer was positive in flavi IgG ELISA and it indicated primary infection. In the neutralization test, the highest neutralizing titer was ≥218 for DENV-2. We report here the existence of DENV-specific antibodies in the serum of a person after 70 years from infection. Published reports indicated that DENV-1 was responsible for the 1942–1944 outbreak in Nagasaki. However, our data suggested that DENV-2 also played a role in this Nagasaki dengue epidemic. PMID:27493841

  6. Virus-Neutralizing Monoclonal Antibody Expressed in Milk of Transgenic Mice Provides Full Protection against Virus-Induced Encephalitis

    PubMed Central

    Kolb, Andreas F.; Pewe, Lecia; Webster, John; Perlman, Stanley; Whitelaw, C. Bruce A.; Siddell, Stuart G.

    2001-01-01

    Neutralizing antibodies represent a major host defense mechanism against viral infections. In mammals, passive immunity is provided by neutralizing antibodies passed to the offspring via the placenta or the milk as immunoglobulin G and secreted immunoglobulin A. With the long-term goal of producing virus-resistant livestock, we have generated mice carrying transgenes that encode the light and heavy chains of an antibody that is able to neutralize the neurotropic JHM strain of murine hepatitis virus (MHV-JHM). MHV-JHM causes acute encephalitis and acute and chronic demyelination in susceptible strains of mice and rats. Transgene expression was targeted to the lactating mammary gland by using the ovine β-lactoglobulin promoter. Milk from these transgenic mice contained up to 0.7 mg of recombinant antibody/ml. In vitro analysis of milk derived from different transgenic lines revealed a linear correlation between antibody expression and virus-neutralizing activity, indicating that the recombinant antibody is the major determinant of MHV-JHM neutralization in murine milk. Offspring of transgenic and control mice were challenged with a lethal dose of MHV-JHM. Litters suckling nontransgenic dams succumbed to fatal encephalitis, whereas litters suckling transgenic dams were fully protected against challenge, irrespective of whether they were transgenic. This demonstrates that a single neutralizing antibody expressed in the milk of transgenic mice is sufficient to completely protect suckling offspring against MHV-JHM-induced encephalitis. PMID:11222704

  7. Investigating antibody neutralization of lyssaviruses using lentiviral pseudotypes: a cross-species comparison.

    PubMed

    Wright, Edward; Temperton, Nigel J; Marston, Denise A; McElhinney, Lorraine M; Fooks, Anthony R; Weiss, Robin A

    2008-09-01

    Cross-neutralization between rabies virus (RABV) and two European bat lyssaviruses (EBLV-1 and -2) was analysed using lentiviral pseudotypes as antigen vectors. Glycoprotein (G-protein) cDNA from RABV challenge virus standard-11 (CVS-11) and EBLV-1 and -2 were cloned and co-expressed with human immunodeficiency virus (HIV) or murine leukemia virus (MLV) gag-pol and packageable green fluorescent protein (GFP) or luciferase reporter genes in human cells. The harvested lentiviral (HIV) vector infected over 40% of baby hamster kidney (BHK) target cells, providing high-titre pseudotype stocks. Tests on blinded antibody-positive (n=15) and -negative (n=45) sera, predetermined by the fluorescent antibody virus neutralization (FAVN) test approved by the World Health Organization (WHO) and Office International des Epizooties (OIE), revealed that the CVS-11 pseudotype assay had 100% concordance with FAVN and strongly correlated with neutralization titres (r2=0.89). Cross-neutralization tests using sera from RABV-vaccinated humans and animals on pseudotypes with CVS-11, EBLV-1 and EBLV-2 envelopes showed that the relative neutralization titres correlated broadly with the degree of G-protein diversity. Pseudotypes have three major advantages over live-virus neutralization tests: (i) they can be handled in low-biohazard-level laboratories; (ii) the use of reporter genes such as GFP or beta-galactosidase will allow the assay to be undertaken at low cost in laboratories worldwide; (iii) each assay requires <10 microl serum. This robust microassay will improve our understanding of the protective humoral immunity that current rabies vaccines confer against emerging lyssaviruses, and will be applicable to surveillance studies, thus helping to control the spread of rabies. PMID:18753230

  8. Characterization and Formulation of Multiple Epitope-Specific Neutralizing Monoclonal Antibodies for Passive Immunization against Cryptosporidiosis

    PubMed Central

    Schaefer, Deborah A.; Auerbach-Dixon, Beth A.; Riggs, Michael W.

    2000-01-01

    The coccidian parasite Cryptosporidium parvum causes diarrhea in humans, calves, and other mammals. Neither immunization nor parasite-specific pharmaceuticals that are consistently effective against this organism are available. While polyclonal antibodies against whole C. parvum reduce infection, their efficacy and predictability are suboptimal. We hypothesized that passive immunization against cryptosporidiosis could be improved by using neutralizing monoclonal antibodies (MAbs) targeting functionally defined antigens on the infective stages. We previously reported that the apical complex and surface-exposed zoite antigens CSL, GP25-200, and P23 are critical in the infection process and are therefore rational targets. In the present study, a panel of 126 MAbs generated against affinity-purified CSL, GP25-200, and P23 was characterized to identify the most efficacious neutralizing MAb formulation targeting each antigen. To identify neutralizing MAbs, sporozoite infectivity following exposure to individual MAbs was assessed by enzyme-linked immunosorbent assay. Of 126 MAbs evaluated, 47 had neutralizing activity. These were then evaluated individually in oocyst-challenged neonatal mice, and 14 MAbs having highly significant efficacy were identified for further testing in formulations. Epitope specificity assays were performed to determine if candidate MAbs recognized the same or different epitopes. Formulations of two or three neutralizing MAbs, each recognizing distinct epitopes, were then evaluated. A formulation of MAbs 3E2 (anti-CSL [αCSL]), 3H2 (αGP25-200), and 1E10 (αP23) provided highly significant additive efficacy over that of either individual MAbs or combinations of two MAbs and reduced intestinal infection by 86 to 93%. These findings indicate that polyvalent neutralizing MAb formulations targeting epitopes on defined antigens may provide optimal passive immunization against cryptosporidiosis. PMID:10768951

  9. Effect on influenza hemagglutinin protein binding with neutralizing antibody using terahertz spectroscopy technology

    NASA Astrophysics Data System (ADS)

    Sun, Yiwen; Zhong, Junlan; Zuo, Jian; Zhang, Cunlin

    2014-11-01

    Terahertz spectroscopy is sensitive to probe several aspects of biological systems. We have reported the terahertz dielectric spectrum is able to identify the type of the charges in the hydrogen-bonded antibodies' networks in our previous work. Recently we demonstrate a highly sensitive THz-TDS method to monitor binding interaction of influenza hemagglutinin (HA) against its target antibody F10. The terahertz dielectric properties of HA was strongly affected by the presence of a specific antibody. Protein solution concentration or even molecular binding interaction can also affect the terahertz signal. This enables us to detect the specificity and sensitivity of antibody-antigen binding under THz radiation.

  10. Detection of Infertility-related Neutralizing Antibodies with a Cell-free Microfluidic Method

    NASA Astrophysics Data System (ADS)

    Eyer, Klaus; Root, Katharina; Verboket, Pascal E.; Dittrich, Petra S.

    2015-11-01

    The unwanted emergence of neutralizing antibodies (nAbs) against an endogenous or a therapeutic protein can result in deficiency diseases or therapy failure. Here, we developed a cell-free microfluidic method for the sensitive detection and quantification of nAbs in human serum that are associated with infertility. We used cell-derived vesicles containing the luteinizing hormone (LH)/choriogonadotropin receptor (LHHCGR) to detect nAbs against LH. The method exploits the entire cellular signal amplification mechanism, and facilitates the detection of as little as 0.44 nM of LH-nAb (Kd 1.5 nM) in human serum matrix within only 15 minutes. In addition, dose-response curves can be generated in less than 2 hours to evaluate the nAB concentration and dissociation constant. The developed system is devoid of problems associated with cell-based assays and we believe that this simple effect-directed analysis can be used in clinical environments, and is adaptable to other hormones or cytokines and their respective nAbs.

  11. Detection of Infertility-related Neutralizing Antibodies with a Cell-free Microfluidic Method

    PubMed Central

    Eyer, Klaus; Root, Katharina; Verboket, Pascal E.; Dittrich, Petra S.

    2015-01-01

    The unwanted emergence of neutralizing antibodies (nAbs) against an endogenous or a therapeutic protein can result in deficiency diseases or therapy failure. Here, we developed a cell-free microfluidic method for the sensitive detection and quantification of nAbs in human serum that are associated with infertility. We used cell-derived vesicles containing the luteinizing hormone (LH)/choriogonadotropin receptor (LHHCGR) to detect nAbs against LH. The method exploits the entire cellular signal amplification mechanism, and facilitates the detection of as little as 0.44 nM of LH-nAb (Kd 1.5 nM) in human serum matrix within only 15 minutes. In addition, dose-response curves can be generated in less than 2 hours to evaluate the nAB concentration and dissociation constant. The developed system is devoid of problems associated with cell-based assays and we believe that this simple effect-directed analysis can be used in clinical environments, and is adaptable to other hormones or cytokines and their respective nAbs. PMID:26585778

  12. Pseudovirion Particles Bearing Native HIV Envelope Trimers Facilitate a Novel Method for Generating Human Neutralizing Monoclonal Antibodies against HIV

    PubMed Central

    Hicar, Mark D.; Chen, Xuemin; Briney, Bryan; Hammonds, Jason; Wang, Jaang-Jiun; Kalams, Spyros; Spearman, Paul W.; Crowe, James E.

    2010-01-01

    Monomeric HIV envelope vaccines fail to elicit broadly neutralizing antibodies or to protect against infection. Neutralizing antibodies against HIV bind to native, functionally active Env trimers on the virion surface. Gag-Env pseudovirions recapitulate the native trimer, and could serve as an effective epitope presentation platform for study of the neutralizing antibody response in HIV-infected individuals. To address if pseudovirions can recapitulate native HIV virion epitope structures, we carefully characterized these particles, concentrating on the antigenic structure of the coreceptor binding site. By blue native gel shift assays, Gag-Env pseudovirions were shown to contain native trimers that were competent for binding to neutralizing monoclonal antibodies. In ELISA, pseudovirions exhibited increased binding of known CD4-induced antibodies following addition of CD4. Using flow cytometric analysis, fluorescently labeled pseudovirions specifically identified a subset of antigen-specific B cells in HIV-infected subjects. Interestingly, the sequence of one of these novel human antibodies, identified during cloning of single HIV-specific B cells and designated 2C6, exhibited homology to mAb 47e, a known anti-CD4-induced coreceptor binding site antibody. The secreted monoclonal antibody 2C6 did not bind monomeric gp120, but specifically bound envelope on pseudovirions. A recombinant form of the antibody 2C6 acted as a CD4-induced epitope-specific antibody in neutralization assays, yet did not bind monomeric gp120. These findings imply specificity against a quaternary epitope presented on the pseudovirion envelope spike. These data demonstrate that Gag-Env pseudovirions recapitulate CD4 and coreceptor binding pocket antigenic structures and can facilitate identification of B cell clones that secrete neutralizing antibodies. PMID:20531016

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

    PubMed Central

    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. PMID:26766578

  14. Analysis of Cross-Reactive Neutralizing Antibodies in Human HFMD Serum with an EV71 Pseudovirus-Based Assay

    PubMed Central

    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. PMID:24964084

  15. High-current plasma contactor neutralizer system

    NASA Technical Reports Server (NTRS)

    Beattie, J. R.; Williamson, W. S.; Matossian, J. N.; Vourgourakis, E. J.; Burch, J. L.

    1989-01-01

    A plasma-contactor neutralizer system is described, for the stabilizing the Orbiter's potential during flights of the Atmospheric Laboratory for Applications and Science missions. The plasma contactor neutralizer will include a Xe plasma source that can provide steady-state ion-emission currents of up to 1.5 A. The Orbiter's potential will be maintained near that of the surrounding space plasma during electron-beam accelerator firings through a combination of ion emission from the Xe plasma source and electron collection from the ambient space plasma. Configuration diagrams and block diagrams are presented along with the performance characteristics of the system.

  16. Developmental Pathway of the MPER-Directed HIV-1-Neutralizing Antibody 10E8

    PubMed Central

    Zhang, Baoshan; McKee, Krisha; Longo, Nancy S.; Yang, Yongping; Huang, Jinghe; Parks, Robert; Eudailey, Joshua; Lloyd, Krissey E.; Alam, S. Munir; Haynes, Barton F.; Mullikin, James C.; Connors, Mark; Mascola, John R.; Shapiro, Lawrence; Kwong, Peter D.

    2016-01-01

    Antibody 10E8 targets the membrane-proximal external region (MPER) of HIV-1 gp41, neutralizes >97% of HIV-1 isolates, and lacks the auto-reactivity often associated with MPER-directed antibodies. The developmental pathway of 10E8 might therefore serve as a promising template for vaccine design, but samples from time-of-infection—often used to infer the B cell record—are unavailable. In this study, we used crystallography, next-generation sequencing (NGS), and functional assessments to infer the 10E8 developmental pathway from a single time point. Mutational analysis indicated somatic hypermutation of the 2nd-heavy chain-complementarity determining region (CDR H2) to be critical for neutralization, and structures of 10E8 variants with V-gene regions reverted to genomic origin for heavy-and-light chains or heavy chain-only showed structural differences >2 Å relative to mature 10E8 in the CDR H2 and H3. To understand these developmental changes, we used bioinformatic sieving, maximum likelihood, and parsimony analyses of immunoglobulin transcripts to identify 10E8-lineage members, to infer the 10E8-unmutated common ancestor (UCA), and to calculate 10E8-developmental intermediates. We were assisted in this analysis by the preservation of a critical D-gene segment, which was unmutated in most 10E8-lineage sequences. UCA and early intermediates weakly bound a 26-residue-MPER peptide, whereas HIV-1 neutralization and epitope recognition in liposomes were only observed with late intermediates. Antibody 10E8 thus develops from a UCA with weak MPER affinity and substantial differences in CDR H2 and H3 from the mature 10E8; only after extensive somatic hypermutation do 10E8-lineage members gain recognition in the context of membrane and HIV-1 neutralization. PMID:27299673

  17. Antibody Conjugation Approach Enhances Breadth and Potency of Neutralization of Anti-HIV-1 Antibodies and CD4-IgG

    PubMed Central

    Gavrilyuk, Julia; Ban, Hitoshi; Uehara, Hisatoshi; Sirk, Shannon J.; Saye-Francisco, Karen; Cuevas, Angelica; Zablowsky, Elise; Oza, Avinash; Seaman, Michael S.; Burton, Dennis R.

    2013-01-01

    Broadly neutralizing antibodies PG9 and PG16 effectively neutralize 70 to 80% of circulating HIV-1 isolates. In this study, the neutralization abilities of PG9 and PG16 were further enhanced by bioconjugation with aplaviroc, a small-molecule inhibitor of virus entry into host cells. A novel air-stable diazonium hexafluorophosphate reagent that allows for rapid, tyrosine-selective functionalization of proteins and antibodies under mild conditions was used to prepare a series of aplaviroc-conjugated antibodies, including b12, 2G12, PG9, PG16, and CD4-IgG. The conjugated antibodies blocked HIV-1 entry through two mechanisms: by binding to the virus itself and by blocking the CCR5 receptor on host cells. Chemical modification did not significantly alter the potency of the parent antibodies against nonresistant HIV-1 strains. Conjugation did not alter the pharmacokinetics of a model IgG in blood. The PG9-aplaviroc conjugate was tested against a panel of 117 HIV-1 strains and was found to neutralize 100% of the viruses. PG9-aplaviroc conjugate IC50s were lower than those of PG9 in neutralization studies of 36 of the 117 HIV-1 strains. These results support this new approach to bispecific antibodies and offer a potential new strategy for combining HIV-1 therapies. PMID:23427154

  18. Antibody conjugation approach enhances breadth and potency of neutralization of anti-HIV-1 antibodies and CD4-IgG.

    PubMed

    Gavrilyuk, Julia; Ban, Hitoshi; Uehara, Hisatoshi; Sirk, Shannon J; Saye-Francisco, Karen; Cuevas, Angelica; Zablowsky, Elise; Oza, Avinash; Seaman, Michael S; Burton, Dennis R; Barbas, Carlos F

    2013-05-01

    Broadly neutralizing antibodies PG9 and PG16 effectively neutralize 70 to 80% of circulating HIV-1 isolates. In this study, the neutralization abilities of PG9 and PG16 were further enhanced by bioconjugation with aplaviroc, a small-molecule inhibitor of virus entry into host cells. A novel air-stable diazonium hexafluorophosphate reagent that allows for rapid, tyrosine-selective functionalization of proteins and antibodies under mild conditions was used to prepare a series of aplaviroc-conjugated antibodies, including b12, 2G12, PG9, PG16, and CD4-IgG. The conjugated antibodies blocked HIV-1 entry through two mechanisms: by binding to the virus itself and by blocking the CCR5 receptor on host cells. Chemical modification did not significantly alter the potency of the parent antibodies against nonresistant HIV-1 strains. Conjugation did not alter the pharmacokinetics of a model IgG in blood. The PG9-aplaviroc conjugate was tested against a panel of 117 HIV-1 strains and was found to neutralize 100% of the viruses. PG9-aplaviroc conjugate IC50s were lower than those of PG9 in neutralization studies of 36 of the 117 HIV-1 strains. These results support this new approach to bispecific antibodies and offer a potential new strategy for combining HIV-1 therapies. PMID:23427154

  19. 3BNC117 a Broadly Neutralizing Antibody Suppresses Viremia in HIV-1-Infected Humans

    PubMed Central

    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-Shulman, 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.

    2016-01-01

    HIV-1 immunotherapy with a combination of first generation monoclonal antibodies was largely ineffective in pre-clinical and clinical settings and was therefore abandoned1–3. However, recently developed single cell based antibody cloning methods have uncovered a new generation of far more potent broadly neutralizing antibodies (bNAbs) to HIV-14,5. These antibodies can prevent infection and suppress viremia in humanized mice (hu-mice) and nonhuman primates, but their potential for human HIV-1 immunotherapy has not been evaluated6–10. Here we report the results of a first-in-man dose escalation phase 1 clinical trial of 3BNC117, a potent human CD4 binding site antibody11, in uninfected and HIV-1-infected individuals. 3BNC117 infusion was well tolerated and demonstrated favorable pharmacokinetics. A single 30 mg/kg infusion of 3BNC117 reduced the viral load (VL) in HIV-1-infected individuals by 0.8 – 2.5 log10 and viremia 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 viremia, and that immunotherapy should be explored as a new modality for HIV-1 prevention, therapy, and cure. PMID:25855300

  20. Opportunities to exploit non-neutralizing HIV-specific antibody activity

    PubMed Central

    Ackerman, Margaret E.; Alter, Galit

    2016-01-01

    Antibodies act as a nexus between innate and adaptive immunity: they provide a means to engage a spectrum of innate immune effector cells in order to clear viral particles and infected cells, and prime antigen presentation. This functional landscape is remarkably complex, and depends on antibody isotype, subclass, and glycosylation; the expression levels and patterns of a suite of Fc receptors with both complementary and opposing activities; and a host of innate immune cells capable of differential responses to opsonized particles and present at different sites. In vivo, even neutralizing antibodies rely on their ability to act as molecular beacons and recruit innate immune effector cells in order to provide protection, and results from both human and macaque studies have implicated these effector functions in vaccine-mediated protection. Thus, while enhancing effector function is a tractable handle for potentiating antibody-mediated protection from HIV infection, success will depend critically on leveraging understanding of the means by which antibodies with specific functional profiles could be elicited, which effector functions could provide optimal protection, and perhaps most critically, how to efficiently recruit the innate effector cells present at sites of infection. PMID:24191934

  1. Potent and broad neutralization of HIV-1 by a llama antibody elicited by immunization

    PubMed Central

    McCoy, Laura E.; Quigley, Anna Forsman; Strokappe, Nika M.; Bulmer-Thomas, Bianca; Seaman, Michael S.; Mortier, Daniella; Rutten, Lucy; Chander, Nikita; Edwards, Carolyn J.; Ketteler, Robin; Davis, David; Verrips, Theo

    2012-01-01

    Llamas (Lama glama) naturally produce heavy chain–only antibodies (Abs) in addition to conventional Abs. The variable regions (VHH) in these heavy chain–only Abs demonstrate comparable affinity and specificity for antigens to conventional immunoglobulins despite their much smaller size. To date, immunizations in humans and animal models have yielded only Abs with limited ability to neutralize HIV-1. In this study, a VHH phagemid library generated from a llama that was multiply immunized with recombinant trimeric HIV-1 envelope proteins (Envs) was screened directly for HIV-1 neutralization. One VHH, L8CJ3 (J3), neutralized 96 of 100 tested HIV-1 strains, encompassing subtypes A, B, C, D, BC, AE, AG, AC, ACD, CD, and G. J3 also potently neutralized chimeric simian-HIV strains with HIV subtypes B and C Env. The sequence of J3 is highly divergent from previous anti–HIV-1 VHH and its own germline sequence. J3 achieves broad and potent neutralization of HIV-1 via interaction with the CD4-binding site of HIV-1 Env. This study may represent a new benchmark for immunogens to be included in B cell–based vaccines and supports the development of VHH as anti–HIV-1 microbicides. PMID:22641382

  2. TPX Neutral Beam Injection System design

    SciTech Connect

    von Halle, A.; Bowen, O.N.; Edwards, J.W.

    1993-11-01

    The existing Tokamak Fusion Test Reactor Neutral Beam system is proposed to be modified for long pulse operation on the Tokamak Physics Experiment (TPX). Day one of TPX will call for one TFTR beamline modified for 1000 second pulse lengths oriented co-directional to the plasma current. The system design will be capable of accommodating an additional co-directional and a single counter directional beamline. For the TPX conceptual design, every attempt was made to use existing Neutral Beam hardware, plant facilities, auxiliary systems, service infrastructure, and control systems. This paper describes the moderate modifications required to the power systems, the ion sources, and the beam impinged surfaces of the ion dumps, the calorimeters, the various beam scrapers, and the neutralizers. Also described are the minimal modifications required to the vacuum, cryogenic, and gas systems and the major modification of replacing the beamline-torus duct in its entirety. Operational considerations for Neutral Beam subsystems over 1000 second pulse lengths will be explored including proposed operating scenarios for full steady state operation.

  3. Characterization of neutralizing monoclonal antibodies directed against tetanus toxin fragment C.

    PubMed

    Yousefi, Mehdi; Tahmasebi, Fathollah; Younesi, Vahid; Razavi, Alireza; Khoshnoodi, Jalal; Bayat, Ali Ahmad; Abbasi, Ebrahim; Rabbani, Hodjatallah; Jeddi-Tehrani, Mahmood; Shokri, Fazel

    2014-01-01

    Clostridium tetani causes a life-threatening infectious disease by production of tetanus neurotoxin (TeNT), a 150 kDa molecule composed of light (LC) and heavy chain (HC) polypeptides. The TeNT HC contains an N-terminal domain critical for LC translocation and a C-terminal toxin receptor-binding domain known as fragment C. Despite extensive investigations on epitope specificity of anti-TeNT antibodies, the immunodominant neutralizing epitopes of the toxin are poorly defined. This study describes the generation and characterization of four monoclonal antibodies (MAb) specific for TeNT. The characteristics of each MAb were explored in terms of isotype, specificity, affinity, and immuno-globulin heavy chain variable region (IGHV) gene usage using ELISA, Western blotting, and sequencing techniques. The toxin neutralizing activity of the MAbs was also investigated using the in vitro GT1b neutralizing assay. The data demonstrated that all MAbs bind to tetanus toxin and toxoid. Sub-fragments binding analysis showed that two MAbs react with fragment C, one with both fragment C and LC, and one with LC. Only the two fragment C-specific MAbs were able to neutralize the toxin. Sequencing of the expressed VH and VL genes revealed rearrangements of various VH and VL gene segments in all hybridoma clones. Clonality of the hybridomas was also confirmed by a competition assay that showed recognition of distinct epitopes by these MAbs. The results suggest the importance of TeNT fragment C in terms of immunogenicity and toxin neutralization activity. PMID:23369087

  4. Development of human-like scFv-Fc antibodies neutralizing Botulinum toxin serotype B

    PubMed Central

    Rasetti-Escargueil, Christine; Avril, Arnaud; Chahboun, Siham; Tierney, Rob; Bak, Nicola; Miethe, Sebastian; Mazuet, Christelle; Popoff, Michel R; Thullier, Philippe; Hust, Michael; Pelat, Thibaut; Sesardic, Dorothea

    2015-01-01

    Botulinum neurotoxins (BoNTs) are responsible for human botulism, a life-threatening disease characterized by flaccid muscle paralysis that occurs naturally by food poisoning or colonization of the gastrointestinal tract by BoNT-producing clostridia. BoNTs have been classified as category A agents by the Centers for Disease Control and Prevention. To date, 7 subtypes of BoNT/B were identified showing that subtypes B1 (16 strains) and B2 (32 strains) constitute the vast majority of BoNT/B strains. Neutralizing antibodies are required for the development of anti-botulism drugs to deal with the potential risk. In this study, macaques (Macaca fascicularis) were immunized with recombinant light chain (LC) or heavy chain (HC) of BoNT/B2, followed by the construction of 2 hyper-immune phage display libraries. The best single-chain variable fragments (scFvs) isolated from each library were selected according to their affinities and cross reactivity with BoNT/B1 toxin subtype. These scFvs against LC and HC were further analyzed by assessing the inhibition of in vitro endopeptidase activity of BoNT/B1 and B2 and neutralization of BoNT/B1 and B2 toxin-induced paralysis in the mouse ex vivo phrenic nerve assay. The antibodies B2–7 (against HC) and BLC3 (against LC) were produced as scFv-Fc, and, when tested individually, neutralized BoNT/B1 and BoNT/B2 in a mouse ex vivo phrenic nerve assay. Whereas only scFv-Fc BLC3 alone protected mice against BoNT/B2-induced paralysis in vivo, when B2–7 and BLC3 were combined they exhibited potent synergistic protection. The present study provided an opportunity to assess the extent of antibody-mediated neutralization of BoNT/B1 and BoNT/B2 subtypes in ex vivo and in vitro assays, and to confirm the benefit of the synergistic effect of antibodies targeting the 2 distinct functional domains of the toxin in vivo. Notably, the framework regions of the most promising antibodies (B2–7 and BLC3) are close to the human germline sequences

  5. Development of human-like scFv-Fc antibodies neutralizing Botulinum toxin serotype B.

    PubMed

    Rasetti-Escargueil, Christine; Avril, Arnaud; Chahboun, Siham; Tierney, Rob; Bak, Nicola; Miethe, Sebastian; Mazuet, Christelle; Popoff, Michel R; Thullier, Philippe; Hust, Michael; Pelat, Thibaut; Sesardic, Dorothea

    2015-01-01

    Botulinum neurotoxins (BoNTs) are responsible for human botulism, a life-threatening disease characterized by flaccid muscle paralysis that occurs naturally by food poisoning or colonization of the gastrointestinal tract by BoNT-producing clostridia. BoNTs have been classified as category A agents by the Centers for Disease Control and Prevention. To date, 7 subtypes of BoNT/B were identified showing that subtypes B1 (16 strains) and B2 (32 strains) constitute the vast majority of BoNT/B strains. Neutralizing antibodies are required for the development of anti-botulism drugs to deal with the potential risk. In this study, macaques (Macaca fascicularis) were immunized with recombinant light chain (LC) or heavy chain (HC) of BoNT/B2, followed by the construction of 2 hyper-immune phage display libraries. The best single-chain variable fragments (scFvs) isolated from each library were selected according to their affinities and cross reactivity with BoNT/B1 toxin subtype. These scFvs against LC and HC were further analyzed by assessing the inhibition of in vitro endopeptidase activity of BoNT/B1 and B2 and neutralization of BoNT/B1 and B2 toxin-induced paralysis in the mouse ex vivo phrenic nerve assay. The antibodies B2-7 (against HC) and BLC3 (against LC) were produced as scFv-Fc, and, when tested individually, neutralized BoNT/B1 and BoNT/B2 in a mouse ex vivo phrenic nerve assay. Whereas only scFv-Fc BLC3 alone protected mice against BoNT/B2-induced paralysis in vivo, when B2-7 and BLC3 were combined they exhibited potent synergistic protection. The present study provided an opportunity to assess the extent of antibody-mediated neutralization of BoNT/B1 and BoNT/B2 subtypes in ex vivo and in vitro assays, and to confirm the benefit of the synergistic effect of antibodies targeting the 2 distinct functional domains of the toxin in vivo. Notably, the framework regions of the most promising antibodies (B2-7 and BLC3) are close to the human germline sequences, which

  6. Production and characterization of neutralizing and nonneutralizing monoclonal antibodies against listeriolysin O.

    PubMed

    Nato, F; Reich, K; Lhopital, S; Rouyre, S; Geoffroy, C; Mazie, J C; Cossart, P

    1991-12-01

    Listeriolysin O (LLO) is a thiol-activated toxin secreted by the facultative intracellular pathogen Listeria monocytogenes. LLO is essential for the survival of the bacterium in the infected cell because it promotes lysis of the phagosome membrane and escape of the bacterium into the cytosol. LLO was used as an antigen for the production of nine monoclonal antibodies (MAbs) in mice. Three of these could inhibit the hemolytic activity of LLO. One of them inhibited binding of LLO to erythrocyte membranes. The two other antibodies blocked the activity of LLO at a step subsequent to membrane binding. Only two of the nine MAbs recognized three other purified SH-activated toxins, streptolysin O, alveolysin, and pneumolysin. Western blot (immunoblot) analysis of culture supernatants of Listeria ivanovii and Listeria seeligeri, two hemolytic species of the genus Listeria, revealed that two MAbs recognized ivanolysin and seeligerolysin. The latter was also recognized by two other MAbs, including one of the neutralizing antibodies. MAbs raised against a peptide, ECTG LAWEWWR, present in all thiol-activated toxins sequenced to date, recognized all toxins and were not neutralizing. Taken together, these results demonstrate the existence of regions important for hemolytic activity that are unique to hemolysins of the genus Listeria and show that regions outside the conserved peptide are important for activity of LLO. PMID:1937824

  7. Antibodies to a conformational epitope on gp41 neutralize HIV-1 by destabilizing the Env spike

    PubMed Central

    Lee, Jeong Hyun; Leaman, Daniel P.; Kim, Arthur S.; Torrents de la Peña, Alba; Sliepen, Kwinten; Yasmeen, Anila; Derking, Ronald; Ramos, Alejandra; de Taeye, Steven W.; Ozorowski, Gabriel; Klein, Florian; Burton, Dennis R.; Nussenzweig, Michel C.; Poignard, Pascal; Moore, John P.; Klasse, Per Johan; Sanders, Rogier W.; Zwick, Michael B.; Wilson, Ian A.; Ward, Andrew B.

    2015-01-01

    The recent identification of three broadly neutralizing antibodies (bnAbs) against gp120–gp41 interface epitopes has expanded the targetable surface on the HIV-1 envelope glycoprotein (Env) trimer. By using biochemical, biophysical and computational methods, we map the previously unknown trimer epitopes of two related antibodies, 3BC315 and 3BC176. A cryo-EM reconstruction of a soluble Env trimer bound to 3BC315 Fab at 9.3 Å resolution reveals that the antibody binds between two gp41 protomers, and neutralizes the virus by accelerating trimer decay. In contrast, bnAb 35O22 binding to a partially overlapping quaternary epitope at the gp120–gp41 interface does not induce decay. A conserved gp41-proximal glycan at N88 was also shown to play a role in the binding kinetics of 3BC176 and 3BC315. Finally, our data suggest that the dynamic structure of the Env trimer influences exposure of bnAb epitopes. PMID:26404402

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

    SciTech Connect

    Kubota-Koketsu, Ritsuko; Mizuta, Hiroyuki; Oshita, Masatoshi; Ideno, Shoji; Yunoki, Mikihiro; Kuhara, Motoki; Yamamoto, Naomasa; Okuno, Yoshinobu; Ikuta, Kazuyoshi

    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.

  9. Structural basis of potent Zika-dengue virus antibody cross-neutralization.

    PubMed

    Barba-Spaeth, Giovanna; Dejnirattisai, Wanwisa; Rouvinski, Alexander; Vaney, Marie-Christine; Medits, Iris; Sharma, Arvind; Simon-Lorière, Etienne; Sakuntabhai, Anavaj; Cao-Lormeau, Van-Mai; Haouz, Ahmed; England, Patrick; Stiasny, Karin; Mongkolsapaya, Juthathip; Heinz, Franz X; Screaton, Gavin R; Rey, Félix A

    2016-08-01

    Zika virus is a member of the Flavivirus genus that had not been associated with severe disease in humans until the recent outbreaks, when it was linked to microcephaly in newborns in Brazil and to Guillain-Barré syndrome in adults in French Polynesia. Zika virus is related to dengue virus, and here we report that a subset of antibodies targeting a conformational epitope isolated from patients with dengue virus also potently neutralize Zika virus. The crystal structure of two of these antibodies in complex with the envelope protein of Zika virus reveals the details of a conserved epitope, which is also the site of interaction of the envelope protein dimer with the precursor membrane (prM) protein during virus maturation. Comparison of the Zika and dengue virus immunocomplexes provides a lead for rational, epitope-focused design of a universal vaccine capable of eliciting potent cross-neutralizing antibodies to protect simultaneously against both Zika and dengue virus infections. PMID:27338953

  10. Antibodies to a conformational epitope on gp41 neutralize HIV-1 by destabilizing the Env spike.

    PubMed

    Lee, Jeong Hyun; Leaman, Daniel P; Kim, Arthur S; Torrents de la Peña, Alba; Sliepen, Kwinten; Yasmeen, Anila; Derking, Ronald; Ramos, Alejandra; de Taeye, Steven W; Ozorowski, Gabriel; Klein, Florian; Burton, Dennis R; Nussenzweig, Michel C; Poignard, Pascal; Moore, John P; Klasse, Per Johan; Sanders, Rogier W; Zwick, Michael B; Wilson, Ian A; Ward, Andrew B

    2015-01-01

    The recent identification of three broadly neutralizing antibodies (bnAbs) against gp120-gp41 interface epitopes has expanded the targetable surface on the HIV-1 envelope glycoprotein (Env) trimer. By using biochemical, biophysical and computational methods, we map the previously unknown trimer epitopes of two related antibodies, 3BC315 and 3BC176. A cryo-EM reconstruction of a soluble Env trimer bound to 3BC315 Fab at 9.3 Å resolution reveals that the antibody binds between two gp41 protomers, and neutralizes the virus by accelerating trimer decay. In contrast, bnAb 35O22 binding to a partially overlapping quaternary epitope at the gp120-gp41 interface does not induce decay. A conserved gp41-proximal glycan at N88 was also shown to play a role in the binding kinetics of 3BC176 and 3BC315. Finally, our data suggest that the dynamic structure of the Env trimer influences exposure of bnAb epitopes. PMID:26404402

  11. Antibodies to a conformational epitope on gp41 neutralize HIV-1 by destabilizing the Env spike

    NASA Astrophysics Data System (ADS)

    Lee, Jeong Hyun; Leaman, Daniel P.; Kim, Arthur S.; Torrents de La Peña, Alba; Sliepen, Kwinten; Yasmeen, Anila; Derking, Ronald; Ramos, Alejandra; de Taeye, Steven W.; Ozorowski, Gabriel; Klein, Florian; Burton, Dennis R.; Nussenzweig, Michel C.; Poignard, Pascal; Moore, John P.; Klasse, Per Johan; Sanders, Rogier W.; Zwick, Michael B.; Wilson, Ian A.; Ward, Andrew B.

    2015-09-01

    The recent identification of three broadly neutralizing antibodies (bnAbs) against gp120-gp41 interface epitopes has expanded the targetable surface on the HIV-1 envelope glycoprotein (Env) trimer. By using biochemical, biophysical and computational methods, we map the previously unknown trimer epitopes of two related antibodies, 3BC315 and 3BC176. A cryo-EM reconstruction of a soluble Env trimer bound to 3BC315 Fab at 9.3 Å resolution reveals that the antibody binds between two gp41 protomers, and neutralizes the virus by accelerating trimer decay. In contrast, bnAb 35O22 binding to a partially overlapping quaternary epitope at the gp120-gp41 interface does not induce decay. A conserved gp41-proximal glycan at N88 was also shown to play a role in the binding kinetics of 3BC176 and 3BC315. Finally, our data suggest that the dynamic structure of the Env trimer influences exposure of bnAb epitopes.

  12. Protective efficacy of neutralizing monoclonal antibodies in a nonhuman primate model of Ebola hemorrhagic fever.

    PubMed

    Marzi, Andrea; Yoshida, Reiko; Miyamoto, Hiroko; Ishijima, Mari; Suzuki, Yasuhiko; Higuchi, Megumi; Matsuyama, Yukie; Igarashi, Manabu; Nakayama, Eri; Kuroda, Makoto; Saijo, Masayuki; Feldmann, Friederike; Brining, Douglas; Feldmann, Heinz; Takada, Ayato

    2012-01-01

    Ebola virus (EBOV) is the causative agent of severe hemorrhagic fever in primates, with human case fatality rates up to 90%. Today, there is neither a licensed vaccine nor a treatment available for Ebola hemorrhagic fever (EHF). Single monoclonal antibodies (MAbs) specific for Zaire ebolavirus (ZEBOV) have been successfully used in passive immunization experiments in rodent models, but have failed to protect nonhuman primates from lethal disease. In this study, we used two clones of human-mouse chimeric MAbs (ch133 and ch226) with strong neutralizing activity against ZEBOV and evaluated their protective potential in a rhesus macaque model of EHF. Reduced viral loads and partial protection were observed in animals given MAbs ch133 and ch226 combined intravenously at 24 hours before and 24 and 72 hours after challenge. MAbs circulated in the blood of a surviving animal until virus-induced IgG responses were detected. In contrast, serum MAb concentrations decreased to undetectable levels at terminal stages of disease in animals that succumbed to infection, indicating substantial consumption of these antibodies due to virus replication. Accordingly, the rapid decrease of serum MAbs was clearly associated with increased viremia in non-survivors. Our results indicate that EBOV neutralizing antibodies, particularly in combination with other therapeutic strategies, might be beneficial in reducing viral loads and prolonging disease progression during EHF. PMID:22558378

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

    PubMed

    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. PMID:26553503

  14. Recombinant Encephalomyocarditis Viruses Elicit Neutralizing Antibodies against PRRSV and CSFV in Mice

    PubMed Central

    Zhu, Shu; Guo, Xin; Keyes, Lisa R.; Yang, Hanchun; Ge, Xinna

    2015-01-01

    Encephalomyocarditis virus (EMCV) is capable of infecting a wide range of species and the infection can cause myocarditis and reproductive failure in pigs as well as febrile illness in human beings. In this study, we introduced the entire ORF5 of the porcine reproductive and respiratory syndrome virus (PRRSV) or the neutralization epitope regions in the E2 gene of the classical swine fever virus (CSFV), into the genome of a stably attenuated EMCV strain, T1100I. The resultant viable recombinant viruses, CvBJC3m/I-ΔGP5 and CvBJC3m/I-E2, respectively expressed partial PRRSV envelope protein GP5 or CSFV neutralization epitope A1A2 along with EMCV proteins. These heterologous proteins fused to the N-terminal of the nonstructural leader protein could be recognized by anti-GP5 or anti-E2 antibody. We also tested the immunogenicity of these fusion proteins by immunizing BALB/c mice with the recombinant viruses. The immunized animals elicited neutralizing antibodies against PRRSV and CSFV. Our results suggest that EMCV can be engineered as an expression vector and serve as a tool in the development of novel live vaccines in various animal species. PMID:26076449

  15. Human Antibody Neutralizes Severe Fever with Thrombocytopenia Syndrome Virus, an Emerging Hemorrhagic Fever Virus

    PubMed Central

    Guo, Xiling; Zhang, Li; Zhang, Wenshuai; Chi, Ying; Zeng, Xiaoyan; Li, Xian; Qi, Xian; Jin, Qiu; Zhang, Xiao; Huang, Mingming; Wang, Hua; Chen, Yin; Bao, Changjun; Hu, Jianli; Liang, Shuyi; Bao, Lin; Wu, Tao

    2013-01-01

    Severe fever with thrombocytopenia syndrome virus (SFTSV), a newly discovered member of the Bunyaviridae family, is the causative agent of an emerging hemorrhagic fever, SFTS, in China. Currently, there are no vaccines or effective therapies against SFTS. In this study, a combinatorial human antibody library was constructed from the peripheral lymphocytes of 5 patients who had recovered from SFTS. The library was screened against purified virions for the production of single-chain variable-region fragments (ScFv). Of the 6 positive clones, one clone (monoclonal antibody [MAb] 4-5) showed neutralizing activity against SFTSV infection in Vero cells. MAb 4-5 was found to effectively neutralize all of the clinical isolates of SFTSV tested, which were isolated from patients in China from 2010 to 2012. MAb 4-5 was found to bind a linear epitope in the ectodomain of glycoprotein Gn. Its neutralizing activity is attributed to blockage of the interactions between the Gn protein and the cellular receptor, indicating that inhibition of virus-cell attachment is its main mechanism. These data suggest that MAb 4-5 can be used as a promising candidate molecule for immunotherapy against SFTSV infection. PMID:23863504

  16. Hypervariable antigenic region 1 of classical swine fever virus E2 protein impacts antibody neutralization.

    PubMed

    Liao, Xun; Wang, Zuohuan; Cao, Tong; Tong, Chao; Geng, Shichao; Gu, Yuanxing; Zhou, Yingshan; Li, Xiaoliang; Fang, Weihuan

    2016-07-19

    Envelope glycoprotein E2 of classical swine fever virus (CSFV) is the major antigen that induces neutralizing antibodies and confers protection against CSFV infection. There are three hypervariable antigenic regions (HAR1, HAR2 and HAR3) of E2 that are different between the group 1 vaccine C-strain and group 2 clinical isolates. This study was aimed to characterize the antigenic epitope region recognized by monoclonal antibody 4F4 (mAb-4F4) that is present in the group 2 field isolate HZ1-08, but not in the C-strain, and examine its impact on neutralization titers when antisera from different recombinant viruses were cross-examined. Indirect ELISA with C-strain E2-based chimeric proteins carrying the three HAR regions showed that the mAb-4F4 bound to HAR1 from HZ1-08 E2, but not to HAR2 or HAR3, indicating that the specific epitope is located in the HAR1 region. Of the 6 major residues differences between C-strain and field isolates, Glu713 in the HAR1 region of strain HZ1-08 is critical for mAb-4F4 binding either at the recombinant protein level or using intact recombinant viruses carrying single mutations. C-strain-based recombinant viruses carrying the most antigenic part of E2 or HAR1 from strain HZ1-08 remained non-pathogenic to pigs and induced good antibody responses. By cross-neutralization assay, we observed that the anti-C-strain serum lost most of its neutralization capacity to RecC-HZ-E2 and QZ-14 (subgroup 2.1d field isolate in 2014), and vice versa. More importantly, the RecC-HAR1 virus remained competent in neutralizing ReC-HZ-E2 and QZ-14 strains without compromising the neutralization capability to the recombinant C-strain. Thus, we propose that chimeric C-strain carrying the HAR1 region of field isolates is a good vaccine candidate for classical swine fever. PMID:27317266

  17. Development of a high-throughput β-Gal-based neutralization assay for quantitation of herpes simplex virus-neutralizing antibodies in human samples.

    PubMed

    Baccari, Amy; Cooney, Michael; Blevins, Tamara P; Morrison, Lynda A; Larson, Shane; Skoberne, Mojca; Belshe, Robert B; Flechtner, Jessica B; Long, Deborah

    2016-07-19

    Measurement of neutralizing antibodies against herpes simplex virus (HSV) is important for evaluation of candidate vaccines. The established plaque-reduction neutralization assay is time consuming, labor intensive, and difficult to validate and transfer. Here, we describe the characterization of a HSV-neutralization assay based on the expression of a reporter gene, β-galactosidase (β-Gal). Using previously constructed HSV-β-Gal recombinant viruses, HSV-2/Gal and HSV-1/tk12, we developed a colorimetric β-Gal-based neutralization assay that is sensitive and highly reproducible, and performed in less than 48h. HSV-1 and HSV-2 neutralizing titers measured by the β-Gal-based neutralization assay were equivalent to those obtained by a plaque reduction neutralization assay. Intra- and inter-assay precision studies demonstrated that the β-Gal-based assay was repeatable and yielded low and acceptable variation. In addition, comparison of HSV-2 neutralizing antibody (NAb) titers measured in two independent laboratories by two unique β-Gal-based assays showed a highly significant correlation (r=0.9499, p<0.0001) between the two assays. The new assay will serve as an important tool both for preclinical and clinical trials of new HSV vaccines. PMID:27265458

  18. Cellular impedance measurement as a new tool for poxvirus titration, antibody neutralization testing and evaluation of antiviral substances

    SciTech Connect

    Witkowski, Peter T.; Schuenadel, Livia; Wiethaus, Julia; Bourquain, Daniel R.; Kurth, Andreas; Nitsche, Andreas

    2010-10-08

    Research highlights: {yields} Real-time data acquisition by RT-CES requires low operative effort. {yields} Time to result is reduced by using RT-CES instead of conventional methods. {yields} RT-CES enables quantification of virus titers in unknown samples. {yields} RT-CES is a useful tool for high-throughput characterization of antiviral agents. {yields} An RT-CES-based virus neutralization test was established. -- Abstract: Impedance-based biosensing known as real-time cell electronic sensing (RT-CES) belongs to an emerging technology for analyzing the status of cells in vitro. In the present study protocols were developed for an RT-CES-based system (xCELLigence{sup TM}, Roche Applied Science, ACEA Biosciences Inc.) to supplement conventional techniques in pox virology. First, proliferation of cells susceptible to orthopoxviruses was monitored. For virus titration cells were infected with vaccinia virus and cell status, represented by the dimensionless impedance-based cell index (CI), was monitored. A virus-dose dependent decrease in electrical impedance could be shown. Calculation of calibration curves at a suitable CI covering a dynamic range of 4 log enabled the quantification of virus titers in unknown samples. Similarly, antiviral effects could be determined as shown for anti-poxviral agents ST-246 and Cidofovir. Published values for the in vitro concentration that inhibited virus replication by 50% (IC{sub 50}) could be confirmed while cytotoxicity in effective concentrations was excluded in long-term incubation experiments. Finally, an RT-CES-based virus neutralization test was established. Various poxvirus-specific antibodies were examined for their neutralizing activity and a calculation mode for the neutralizing antibody titer was introduced. In summary, the presented RT-CES-based methods outmatch end-point assays by observing the cell population throughout the entire experiment while workload and time to result are reduced.

  19. Ability to develop broadly neutralizing HIV-1 antibodies is not restricted by the germline immunoglobulin gene repertoire1

    PubMed Central

    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; Karim, Quarraisha Abdool; Karim, Salim S. Abdool; Moore, Penny L.; Travers, Simon A.; Morris, Lynn

    2015-01-01

    The human immunoglobulin repertoire is vast, producing billions of unique antibodies from a limited number of germline immunoglobulin genes. The immunoglobulin heavy chain variable region (IGHV) is central to antigen binding and is comprised of 48 functional genes. Here we analyzed whether HIV-1 infected individuals who develop broadly neutralizing antibodies 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 Center for the AIDS Programme of Research in South African (CAPRISA) 002 and 004 cohorts, 13 of whom developed broadly neutralizing antibodies. 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 monoclonal antibodies isolated from 7 of the CAPRISA women and previously isolated broadly neutralizing antibodies from other donors provided evidence that at least 8 novel or non-IMGT alleles contributed to functional antibodies. Importantly, we found that despite a wide range in the number of IGHV alleles in each individual, including alleles used by known broadly neutralizing antibodies, there were no significant differences in germline IGHV repertoires between individuals who do and do not develop broadly neutralizing antibodies. This study reports novel IGHV repertoires and highlights the importance of a fully comprehensive immunoglobulin database for germline gene usage prediction. Furthermore, these data suggest a lack of genetic bias in broadly neutralizing antibody development in HIV-1 infection, with implications for HIV vaccine design. PMID:25825450

  20. A Diverse Panel of Hepatitis C Virus Glycoproteins for Use in Vaccine Research Reveals Extremes of Monoclonal Antibody Neutralization Resistance

    PubMed Central

    Urbanowicz, Richard A.; McClure, C. Patrick; Brown, Richard J. P.; Tsoleridis, Theocharis; Persson, Mats A. A.; Krey, Thomas; Irving, William L.; Tarr, Alexander W.

    2015-01-01

    ABSTRACT Despite significant advances in the treatment of hepatitis C virus (HCV) infection, the need to develop preventative vaccines remains. Identification of the best vaccine candidates and evaluation of their performance in preclinical and clinical development will require appropriate neutralization assays utilizing diverse HCV isolates. We aimed to generate and characterize a panel of HCV E1E2 glycoproteins suitable for subsequent use in vaccine and therapeutic antibody testing. Full-length E1E2 clones were PCR amplified from patient-derived serum samples, cloned into an expression vector, and used to generate viral pseudoparticles (HCVpp). In addition, some of these clones were used to generate cell culture infectious (HCVcc) clones. The infectivity and neutralization sensitivity of these viruses were then determined. Bioinformatic and HCVpp infectivity screening of approximately 900 E1E2 clones resulted in the assembly of a panel of 78 functional E1E2 proteins representing distinct HCV genotypes and different stages of infection. These HCV glycoproteins differed markedly in their sensitivity to neutralizing antibodies. We used this panel to predict antibody efficacy against circulating HCV strains, highlighting the likely reason why some monoclonal antibodies failed in previous clinical trials. This study provides the first objective categorization of cross-genotype patient-derived HCV E1E2 clones according to their sensitivity to antibody neutralization. It has shown that HCV isolates have clearly distinguishable neutralization-sensitive, -resistant, or -intermediate phenotypes, which are independent of genotype. The panel provides a systematic means for characterization of the neutralizing response elicited by candidate vaccines and for defining the therapeutic potential of monoclonal antibodies. IMPORTANCE Hepatitis C virus (HCV) has a global burden of more than 170 million people, many of whom cannot attain the new, expensive, direct-acting antiviral

  1. Pharmacokinetics and Immunogenicity of Broadly Neutralizing HIV Monoclonal Antibodies in Macaques

    PubMed Central

    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. PMID:25807114

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

    PubMed Central

    Bangaru, Sandhya; Nieusma, Travis; Kose, Nurgun; Thornburg, Natalie J.; Finn, Jessica A.; Kaplan, Bryan S.; King, Hannah G.; Singh, Vidisha; Lampley, Rebecca M.; Sapparapu, Gopal; Cisneros, Alberto; Edwards, Kathryn M.; Slaughter, James C.; 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

  3. Engineering the vaccinia virus L1 protein for increased neutralizing antibody response after DNA immunization

    PubMed Central

    Shinoda, Kaori; Wyatt, Linda S; Irvine, Kari R; Moss, Bernard

    2009-01-01

    Background The licensed smallpox vaccine, comprised of infectious vaccinia virus, has associated adverse effects, particularly for immunocompromised individuals. Therefore, safer DNA and protein vaccines are being investigated. The L1 protein, a component of the mature virion membrane that is conserved in all sequenced poxviruses, is required for vaccinia virus entry into host cells and is a target for neutralizing antibody. When expressed by vaccinia virus, the unglycosylated, myristoylated L1 protein attaches to the viral membrane via a C-terminal transmembrane anchor without traversing the secretory pathway. The purpose of the present study was to investigate modifications of the gene expressing the L1 protein that would increase immunogenicity in mice when delivered by a gene gun. Results The L1 gene was codon modified for optimal expression in mammalian cells and potential N-glycosylation sites removed. Addition of a signal sequence to the N-terminus of L1 increased cell surface expression as shown by confocal microscopy and flow cytometry of transfected cells. Removal of the transmembrane domain led to secretion of L1 into the medium. Induction of binding and neutralizing antibodies in mice was enhanced by gene gun delivery of L1 containing the signal sequence with or without the transmembrane domain. Each L1 construct partially protected mice against weight loss caused by intranasal administration of vaccinia virus. Conclusion Modifications of the vaccinia virus L1 gene including codon optimization and addition of a signal sequence with or without deletion of the transmembrane domain can enhance the neutralizing antibody response of a DNA vaccine. PMID:19257896

  4. Minimally Mutated HIV-1 Broadly Neutralizing Antibodies to Guide Reductionist Vaccine Design

    PubMed Central

    Sarkar, Anita; Liang, Chi-Hui; Scherer, Erin A.; Henry Dunand, Carole J.; Adachi, Yumiko; Diwanji, Devan; Jones, Meaghan; Kalyuzhniy, Oleksandr; Kubitz, Michael; Spencer, Skye; Pauthner, Matthias; Saye-Francisco, Karen L.; Sesterhenn, Fabian; Wilson, Patrick C.; Galloway, Denise M.; Stanfield, Robyn L.; Wilson, Ian A.; Burton, Dennis R.; Schief, William R.

    2016-01-01

    An optimal HIV vaccine should induce broadly neutralizing antibodies (bnAbs) that neutralize diverse viral strains and subtypes. However, potent bnAbs develop in only a small fraction of HIV-infected individuals, all contain rare features such as extensive mutation, insertions, deletions, and/or long complementarity-determining regions, and some are polyreactive, casting doubt on whether bnAbs to HIV can be reliably induced by vaccination. We engineered two potent VRC01-class bnAbs that minimized rare features. According to a quantitative features frequency analysis, the set of features for one of these minimally mutated bnAbs compared favorably with all 68 HIV bnAbs analyzed and was similar to antibodies elicited by common vaccines. This same minimally mutated bnAb lacked polyreactivity in four different assays. We then divided the minimal mutations into spatial clusters and dissected the epitope components interacting with those clusters, by mutational and crystallographic analyses coupled with neutralization assays. Finally, by synthesizing available data, we developed a working-concept boosting strategy to select the mutation clusters in a logical order following a germline-targeting prime. We have thus developed potent HIV bnAbs that may be more tractable vaccine goals compared to existing bnAbs, and we have proposed a strategy to elicit them. This reductionist approach to vaccine design, guided by antibody and antigen structure, could be applied to design candidate vaccines for other HIV bnAbs or protective Abs against other pathogens. PMID:27560183

  5. Minimally Mutated HIV-1 Broadly Neutralizing Antibodies to Guide Reductionist Vaccine Design.

    PubMed

    Jardine, Joseph G; Sok, Devin; Julien, Jean-Philippe; Briney, Bryan; Sarkar, Anita; Liang, Chi-Hui; Scherer, Erin A; Henry Dunand, Carole J; Adachi, Yumiko; Diwanji, Devan; Hsueh, Jessica; Jones, Meaghan; Kalyuzhniy, Oleksandr; Kubitz, Michael; Spencer, Skye; Pauthner, Matthias; Saye-Francisco, Karen L; Sesterhenn, Fabian; Wilson, Patrick C; Galloway, Denise M; Stanfield, Robyn L; Wilson, Ian A; Burton, Dennis R; Schief, William R

    2016-08-01

    An optimal HIV vaccine should induce broadly neutralizing antibodies (bnAbs) that neutralize diverse viral strains and subtypes. However, potent bnAbs develop in only a small fraction of HIV-infected individuals, all contain rare features such as extensive mutation, insertions, deletions, and/or long complementarity-determining regions, and some are polyreactive, casting doubt on whether bnAbs to HIV can be reliably induced by vaccination. We engineered two potent VRC01-class bnAbs that minimized rare features. According to a quantitative features frequency analysis, the set of features for one of these minimally mutated bnAbs compared favorably with all 68 HIV bnAbs analyzed and was similar to antibodies elicited by common vaccines. This same minimally mutated bnAb lacked polyreactivity in four different assays. We then divided the minimal mutations into spatial clusters and dissected the epitope components interacting with those clusters, by mutational and crystallographic analyses coupled with neutralization assays. Finally, by synthesizing available data, we developed a working-concept boosting strategy to select the mutation clusters in a logical order following a germline-targeting prime. We have thus developed potent HIV bnAbs that may be more tractable vaccine goals compared to existing bnAbs, and we have proposed a strategy to elicit them. This reductionist approach to vaccine design, guided by antibody and antigen structure, could be applied to design candidate vaccines for other HIV bnAbs or protective Abs against other pathogens. PMID:27560183

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

    PubMed

    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. PMID:26917592

  7. Priming a broadly neutralizing antibody response to HIV-1 using a germline-targeting immunogen

    PubMed Central

    Jardine, Joseph G.; Ota, Takayuki; Sok, Devin; Pauthner, Matthias; Kulp, Daniel W.; Kalyuzhniy, Oleksandr; Skog, Patrick D.; Thinnes, Theresa C.; Bhullar, Deepika; Briney, Bryan; Menis, Sergey; Jones, Meaghan; Kubitz, Mike; Spencer, Skye; Adachi, Yumiko; Burton, Dennis R.; Schief, William R.; Nemazee, David

    2015-01-01

    A major goal of HIV-1 vaccine research is the design of immunogens capable of inducing broadly neutralizing antibodies (bnAbs) that bind to the viral envelope glycoprotein (Env). Poor binding of Env to unmutated precursors of bnAbs, including those of the VRC01-class, appears to be a major problem for bnAb induction. We engineered an immunogen that binds to VRC01-class bnAb precursors and immunized knock-in mice expressing germline-reverted VRC01 heavy chains. Induced antibodies showed characteristics of VRC01-class bnAbs, including a short light chain complementarity determining region 3 (CDRL3) and mutations that favored binding to near-native HIV-1 gp120 constructs. In contrast, native-like immunogens failed to activate VRC01-class precursors. The results suggest that rational epitope design can prime rare B cell precursors for affinity maturation to desired targets. PMID:26089355

  8. Apoptosis induced by islet amyloid polypeptide soluble oligomers is neutralized by diabetes-associated specific antibodies

    PubMed Central

    Bram, Yaron; Frydman-Marom, Anat; Yanai, Inbal; Gilead, Sharon; Shaltiel-Karyo, Ronit; Amdursky, Nadav; Gazit, Ehud

    2014-01-01

    Soluble oligomeric assemblies of amyloidal proteins appear to act as major pathological agents in several degenerative disorders. Isolation and characterization of these oligomers is a pivotal step towards determination of their pathological relevance. Here we describe the isolation of Type 2 diabetes-associated islet amyloid polypeptide soluble cytotoxic oligomers; these oligomers induced apoptosis in cultured pancreatic cells, permeated model lipid vesicles and interacted with cell membranes following complete internalization. Moreover, antibodies which specifically recognized these assemblies, but not monomers or amyloid fibrils, were exclusively identified in diabetic patients and were shown to neutralize the apoptotic effect induced by these oligomers. Our findings support the notion that human IAPP peptide can form highly toxic oligomers. The presence of antibodies identified in the serum of diabetic patients confirms the pathological relevance of the oligomers. In addition, the newly identified structural epitopes may also provide new mechanistic insights and a molecular target for future therapy. PMID:24589570

  9. Prevalence of neutralizing antibodies to West Nile virus in Eleonora's Falcons in the Canary Islands.

    PubMed

    Gangoso, Laura; Grande, Juan Manuel; Llorente, Francisco; Jiménez-Clavero, Miguel Ángel; Pérez, Jesús M; Figuerola, Jordi

    2010-10-01

    Birds are the major amplifying host for West Nile virus (WNV), a flavivirus that may affect humans and transmitted by bloodsucking vectors. Eleonora's Falcons (Falco eleonorae) migrate to the Canary Islands annually from WNV-endemic regions. To investigate the possible role of Eleonora's Falcons in the circulation of WNV, we measured WNV-specific antibodies in 81 falcons captured in 2006. None of the nestlings but 14.8% of the adults had WNV-neutralizing antibodies. RT-PCR did not detect flaviviruses in nonculicine ectoparasites (n=231) of the falcons. These findings suggest that WNV infection did not occur locally, but rather on the wintering grounds or during migration. PMID:20966288

  10. Binding of a neutralizing antibody to dengue virus alters the arrangement of surface glycoproteins

    SciTech Connect

    Lok, Shee-Mei; Kostyuchenko, Victor; Nybakken, Grant E.; Holdaway, Heather A.; Battisti, Anthony J.; Sukupolvi-Petty, Soila; Sedlak, Dagmar; Fremont, Daved H.; Chipman, Paul R.; Roehrig, John T.; Diamond, Michael S.; Kuhn, Richard J.; Rossmann, Michael G.

    2008-04-02

    The monoclonal antibody 1A1D-2 has been shown to strongly neutralize dengue virus serotypes 1, 2 and 3, primarily by inhibiting attachment to host cells. A crystal structure of its antigen binding fragment (Fab) complexed with domain III of the viral envelope glycoprotein, E, showed that the epitope would be partially occluded in the known structure of the mature dengue virus. Nevertheless, antibody could bind to the virus at 37 degrees C, suggesting that the virus is in dynamic motion making hidden epitopes briefly available. A cryo-electron microscope image reconstruction of the virus:Fab complex showed large changes in the organization of the E protein that exposed the epitopes on two of the three E molecules in each of the 60 icosahedral asymmetric units of the virus. The changes in the structure of the viral surface are presumably responsible for inhibiting attachment to cells.

  11. Structural Insights into the Neutralization Properties of the Fully Human, Anti-interferon Monoclonal Antibody Sifalimumab.

    PubMed

    Oganesyan, Vaheh; Peng, Li; Woods, Robert M; Wu, Herren; Dall'Acqua, William F

    2015-06-12

    We report the three-dimensional structure of human interferon α-2A (IFN-α2A) bound to the Fab fragment of a therapeutic monoclonal antibody (sifalimumab; IgG1/κ). The structure of the corresponding complex was solved at a resolution of 3.0 Å using molecular replacement and constitutes the first reported structure of a human type I IFN bound to a therapeutic antibody. This study revealed the major contribution made by the first complementarity-determining region in each of sifalimumab light and heavy chains. These data also provided the molecular basis for sifalimumab mechanism of action. We propose that its interferon-neutralizing properties are the result of direct competition for IFN-α2A binding to the IFN receptor subunit 1 (IFNAR1) and do not involve inhibiting IFN-α2A binding to the IFN receptor subunit 2 (IFNAR2). PMID:25925951

  12. Protein A-neutralizing monoclonal antibody protects neonatal mice against Staphylococcus aureus.

    PubMed

    Thammavongsa, Vilasack; Rauch, Sabine; Kim, Hwan Keun; Missiakas, Dominique M; Schneewind, Olaf

    2015-01-15

    Staphylococcus aureus is a cause of sepsis and meningitis in very-low-birth-weight (VLBW) infants. Clinical trials with S. aureus specific antibodies failed to protect VLBW neonates, which may be due to the immune evasive attributes of staphylococcal protein A (SpA). Here we show that mouse monoclonal antibody SpAKKAA-mAb 3F6, which neutralizes the immunoglobulin Fcγ-binding and B cell receptor crosslinking attributes of SpA, protects neonatal mice against S. aureus sepsis and raises protective immunity against subsequent staphylococcal infection. We developed a humanized SpAKKAA-mAb that protects neonatal mice against S. aureus sepsis and may therefore be subjected to clinical testing in VLBW neonates. PMID:25488332

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

    PubMed Central

    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.

    2016-01-01

    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. PMID:27028935

  14. Optimal combinations of broadly neutralizing antibodies for prevention and treatments of HIV-1 clade C infection

    DOE PAGESBeta

    Wagh, Kshitij; Bhattacharya, Tanmoy; Williamson, Carolyn; Robles, Alex; Bayne, Madeleine; Garrity, Jetta; Rist, Michael; Rademeyer, Cecilia; Yoon, Hyejin; Lapedes, Alan Scott; et al

    2016-03-30

    In this study, 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 themore » 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.« less

  15. Isolation and characterization of human neutralizing antibodies to rabies virus derived from a recombinant immune antibody library.

    PubMed

    Houimel, Mehdi; Dellagi, Koussay

    2009-11-01

    A human immune Fab library was constructed using RNAs from peripheral blood lymphocytes obtained from rabies virus hyperimmune volunteers on phagemid vector. The size of the constructed Fab library was 2 x 10(7) Escherichia coli transformants. After four rounds of panning on whole inactivated rabies virus (PV-11), phage clones displaying rabies virus-specific human Fab were selected. The specificity of soluble Fab antibody fragments, derived from positive phage clones was verified by ELISA. Among 20 specific Fab clones, the genetic sequence of 6 of them (FabRV01, FabRV02, FabRV03, FabRV04, FabRV05, and FabRV06) was analyzed. The variable heavy (VH) and variable light (VL) domains were found to share 90% and 93% homology with sequences encoded by the corresponding human germline genes, respectively. The soluble Fab fragments, expressed in Escherichia coli were purified by a single step Nickel-NTA affinity chromatography via a hexa-histidine tag and their binding specificities to rabies virus were confirmed. Three of the Fab antibodies, FabRV01, FabRV02 and FabRV03, showed binding characteristics to rabies virus glycoprotein antigenic site III with affinities in the K(D) range 7 x 10(-9) to 5 x 10(-8)M. The Fab fragments showed dose-dependent neutralization properties for the challenge virus standard (CVS-11). PMID:19559727

  16. Human monoclonal antibodies targeting the haemagglutinin glycoprotein can neutralize H7N9 influenza virus.

    PubMed

    Chen, Zhe; Wang, Jianmin; Bao, Linlin; Guo, Li; Zhang, Weijia; Xue, Ying; Zhou, Hongli; Xiao, Yan; Wang, Jianwei; Wu, Fan; Deng, Ying; Qin, Chuan; Jin, Qi

    2015-01-01

    The recently identified avian-originated influenza H7N9 virus causes severe pulmonary disease and may lead to death in humans. Currently, treatment options for the prevention and control of fatal H7N9 infections in humans remain limited. Here we characterize two human monoclonal antibodies (HuMAbs), HNIgGA6 and HNIgGB5, by screening a Fab antibody phage library derived from patients who recovered from H7N9 infection. Both antibodies exhibit high neutralizing activity against H7N9 virus in cells. Two amino acids in the receptor-binding site, 186V and 226L, are crucial for the binding of these two HuMAbs to viral haemagglutinin antigens. Prophylaxis with HNIgGA6 and HNIgGB5 confers significant immunity against H7N9 virus in a mouse model and significantly reduces the pulmonary virus titre. When administered post infection, therapeutic doses of the HuMAbs also provide robust protection against lethality. These antibodies might represent a potential alternative or adjunct to H7N9 pandemic interventions. PMID:25819694

  17. Broadly Neutralizing Antibodies to HIV and Their Role in Vaccine Design.

    PubMed

    Burton, Dennis R; Hangartner, Lars

    2016-05-20

    HIV employs multiple means to evade the humoral immune response, particularly the elicitation of and recognition by broadly neutralizing antibodies (bnAbs). Such antibodies can act antivirally against a wide spectrum of viruses by targeting relatively conserved regions on the surface HIV envelope trimer spike. Elicitation of and recognition by bnAbs are hindered by the arrangement of spikes on virions and the relatively difficult access to bnAb epitopes on spikes, including the proximity of variable regions and a high density of glycans. Yet, in a small proportion of HIV-infected individuals, potent bnAb responses do develop, and isolation of the corresponding monoclonal antibodies has been facilitated by identification of favorable donors with potent bnAb sera and by development of improved methods for human antibody generation. Molecular studies of recombinant Env trimers, alone and in interaction with bnAbs, are providing new insights that are fueling the development and testing of promising immunogens aimed at the elicitation of bnAbs. PMID:27168247

  18. Cross-neutralization of influenza A viruses mediated by a single antibody loop.

    PubMed

    Ekiert, Damian C; Kashyap, Arun K; Steel, John; Rubrum, Adam; Bhabha, Gira; Khayat, Reza; Lee, Jeong Hyun; Dillon, Michael A; O'Neil, Ryann E; Faynboym, Aleksandr M; Horowitz, Michael; Horowitz, Lawrence; Ward, Andrew B; Palese, Peter; Webby, Richard; Lerner, Richard A; Bhatt, Ramesh R; Wilson, Ian A

    2012-09-27

    Immune recognition of protein antigens relies on the combined interaction of multiple antibody loops, which provide a fairly large footprint and constrain the size and shape of protein surfaces that can be targeted. Single protein loops can mediate extremely high-affinity binding, but it is unclear whether such a mechanism is available to antibodies. Here we report the isolation and characterization of an antibody called C05, which neutralizes strains from multiple subtypes of influenza A virus, including H1, H2 and H3. X-ray and electron microscopy structures show that C05 recognizes conserved elements of the receptor-binding site on the haemagglutinin surface glycoprotein. Recognition of the haemagglutinin receptor-binding site is dominated by a single heavy-chain complementarity-determining region 3 loop, with minor contacts from heavy-chain complementarity-determining region 1, and is sufficient to achieve nanomolar binding with a minimal footprint. Thus, binding predominantly with a single loop can allow antibodies to target small, conserved functional sites on otherwise hypervariable antigens. PMID:22982990

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

    PubMed Central

    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

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

    PubMed

    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

  1. Eliciting neutralizing antibodies with gp120 outer domain constructs based on M-group consensus sequence.

    PubMed

    Qin, Yali; Banasik, Marisa; Kim, SoonJeung; Penn-Nicholson, Adam; Habte, Habtom H; LaBranche, Celia; Montefiori, David C; Wang, Chong; Cho, Michael W

    2014-08-01

    One strategy being evaluated for HIV-1 vaccine development is focusing immune responses towards neutralizing epitopes on the gp120 outer domain (OD) by removing the immunodominant, but non-neutralizing, inner domain. Previous OD constructs have not elicited strong neutralizing antibodies (nAbs). We constructed two immunogens, a monomeric gp120-OD and a trimeric gp120-OD×3, based on an M group consensus sequence (MCON6). Their biochemical and immunological properties were compared with intact gp120. Results indicated better preservation of critical neutralizing epitopes on gp120-OD×3. In contrast to previous studies, our immunogens induced potent, cross-reactive nAbs in rabbits. Although nAbs primarily targeted Tier 1 viruses, they exhibited significant breadth. Epitope mapping analyses indicated that nAbs primarily targeted conserved V3 loop elements. Although the potency and breadth of nAbs were similar for all three immunogens, nAb induction kinetics indicated that gp120-OD×3 was superior to gp120-OD, suggesting that gp120-OD×3 is a promising prototype for further gp120 OD-based immunogen development. PMID:25046154

  2. Eliciting Neutralizing Antibodies with gp120 Outer Domain Constructs Based on M-Group Consensus Sequence

    PubMed Central

    Qin, Yali; Banasik, Marisa; Kim, SoonJeung; Penn-Nicholson, Adam; Habte, Habtom H; Labranche, Celia; Montefiori, David C; Wang, Chong; Cho, Michael W

    2014-01-01

    One strategy being evaluated for HIV-1 vaccine development is focusing immune responses towards neutralizing epitopes on the gp120 outer domain (OD) by removing the immunodominant, but non-neutralizing, inner domain. Previous OD constructs have not elicited strong neutralizing antibodies (nAbs). We constructed two immunogens, a monomeric gp120-OD and a trimeric gp120-OD×3, based on an M group consensus sequence (MCON6). Their biochemical and immunological properties were compared with intact gp120. Results indicated better preservation of critical neutralizing epitopes on gp120-OD×3. In contrast to previous studies, our immunogens induced potent, cross-reactive nAbs in rabbits. Although nAbs primarily targeted Tier 1 viruses, they exhibited significant breadth. Epitope mapping analyses indicated that nAbs primarily targeted conserved V3 loop elements. Although the potency and breadth of nAbs were similar for all three immunogens, nAb induction kinetics indicated that gp120-OD×3 was superior to gp120-OD, suggesting that gp120-OD×3 is a promising prototype for further gp120 OD-based immunogen development. PMID:25046154

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

    PubMed

    Guan, Jian; Bywaters, Stephanie M; Brendle, Sarah A; Lee, Hyunwook; Ashley, Robert E; Makhov, Alexander M; Conway, James F; Christensen, Neil D; Hafenstein, Susan

    2015-09-01

    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. PMID:25996608

  4. Protective role of magnesium in the neutralization by antibodies of Chlamydia trachomatis infectivity.

    PubMed Central

    Peterson, E M; Zhong, G M; Carlson, E; de la Maza, L M

    1988-01-01

    Neutralization of the infectivity of Chlamydia trachomatis was assessed by using polyclonal antisera and monoclonal antibodies (MAbs). Polyclonal antisera and a species-reactive MAb as well as a subspecies-specific MAb, both of which were directed toward the major outer membrane protein of C. trachomatis, reduced the number of chlamydial inclusion-forming units in an in vitro assay. Neutralization was dependent on the presence of complement. The species-specific MAb reacted with all 15 serovars by a microimmunofluorescence assay and a dot blot enzyme-linked immunosorbent assay with heat-treated elementary bodies. On the other hand, this same MAb reacted with all serovars, except those in the C complex, by the dot blot enzyme-linked immunosorbent assay with viable organisms and neutralized in vitro all 10 serovars tested, except those in the C complex. When neutralization assays were performed in a solution containing Mg2+, neutralization by both polyclonal antisera and MAbs was significantly reduced. A dose response to Mg2+ supplied as MgSO4 revealed that all concentrations tested from 50 to 800 microM had some effect. Concentrations of greater than or equal to 400 microM MgSO4 completely abolished neutralization at the lowest dilution of polyclonal antisera and species-reactive MAb tested. Although Mg2+ also blocked the neutralization effect of the subspecies-specific MAb, this neutralization was not as complete as that observed with the species-reactive MAb. Addition of Mg2+ to the assay over the initial 45 min of incubation of C. trachomatis with MAb and complement showed that the organisms could be rescued to some extent over the first 30 min of incubation, after which time neutralization of infectivity could not be reversed. C. trachomatis treated with Mg2+, the species-reactive MAb, and complement were lethal to mice in an in vivo toxicity and infectivity assay, whereas mice injected with organisms incubated with the same MAb and complement without Mg2

  5. Subgroup J avian leukosis virus neutralizing antibody escape variants contribute to viral persistence in meat-type chickens.

    PubMed

    Pandiri, A R; Mays, J K; Silva, R F; Hunt, H D; Reed, W M; Fadly, A M

    2010-06-01

    We have previously demonstrated a high incidence of chickens with persistent viremia even in the presence of neutralizing antibodies (V+A+) against the inoculated parental virus in commercial meat-type chickens inoculated at hatch with subgroup J avian leukosis virus (ALV J) field isolates. In this study, we used an ALV J molecular clone, ADOL pR5-4, to determine the role of neutralizing antibody (NAb) escape mutants in maintaining a high incidence of viral persistence, namely, V+A+ infection profile in commercial meat-type chickens. Chickens were housed as a flock in a pen or housed in isolation in solitary Horsfall-Bauer units for testing for NAb escape variants. The emergence of NAb escape variants was evaluated by sequential autologous virus neutralization (VN) (between virus and antibody from the same sampling period) and heterologous VN (between virus and antibody from preceding and succeeding sampling periods). Sequential virus isolates and corresponding antisera from 18 chickens were examined by VN matrix. In all chickens, autologous virus isolates were not neutralized by corresponding antisera. However, some of these resilient autologous virus isolates were neutralized by antibodies from subsequent sampling intervals. Nucleotide sequence analysis of consecutive isolates from three individually housed chickens with V+A+ infection profile revealed distinct changes within the envelope region, suggesting viral evolution to escape the host immune response. These results demonstrate that the emergence of antibody escape variants in commercial meat-type chickens contributes to ALV J persistence. PMID:20608529

  6. Viral variants that initiate and drive maturation of V1V2-directed HIV-1 broadly neutralizing antibodies

    PubMed Central

    Bhiman, Jinal N.; Anthony, Colin; Doria-Rose, Nicole A.; Karimanzira, Owen; Schramm, Chaim A.; Khoza, Thandeka; Kitchin, Dale; Botha, Gordon; Gorman, Jason; Garrett, Nigel J.; Abdool Karim, Salim S.; Shapiro, Lawrence; Williamson, Carolyn; Kwong, Peter D.; Mascola, John R.; Morris, Lynn; Moore, Penny L.

    2015-01-01

    The elicitation of broadly neutralizing antibodies (bNAbs) is likely to be essential for a preventative HIV-1 vaccine, but this has not yet been achieved by immunization. In contrast some HIV-1-infected individuals naturally mount bNAb responses during chronic infection, suggesting that years of maturation are required for breadth1-6. Recent studies have shown that viral diversification precedes the emergence of bNAbs but the significance of this observation is unknown7,8. Here, we delineate the key viral events that drove neutralization breadth within the CAP256-VRC26 family of 33 monoclonal antibodies (mAbs) isolated from a superinfected individual. First, we identified minority viral variants that were distinct from both transmitted/founder (T/F) viruses and efficiently engaged the bNAb precursor, termed bNAb-initiating envelopes. Second, deep sequencing revealed a pool of diverse epitope variants (immunotypes) that were preferentially neutralized by broader members of the antibody lineage. In contrast, a “dead-end” antibody sublineage unable to neutralize these immunotypes showed limited evolution and failed to develop breadth. Thus, early viral escape at key antibody-virus contact sites selects for sublineages that can tolerate these changes, providing a new mechanism for the generation of neutralization breadth within a developing antibody lineage. PMID:26457756

  7. Preexisting Neutralizing Antibody Responses Distinguish Clinically Inapparent and Apparent Dengue Virus Infections in a Sri Lankan Pediatric Cohort

    PubMed Central

    Corbett, Kizzmekia S.; Katzelnick, Leah; Tissera, Hasitha; Amerasinghe, Ananda; de Silva, Aruna Dharshan; de Silva, Aravinda M.

    2015-01-01

    Dengue viruses (DENVs) are mosquito-borne flaviviruses that infect humans. The clinical presentation of DENV infection ranges from inapparent infection to dengue hemorrhagic fever and dengue shock syndrome. We analyzed samples from a pediatric dengue cohort study in Sri Lanka to explore whether antibody responses differentiated clinically apparent infections from clinically inapparent infections. In DENV-naive individuals exposed to primary DENV infections, we observed no difference in the quantity or quality of acquired antibodies between inapparent and apparent infections. Children who experienced primary infections had broad, serotype–cross-neutralizing antibody responses that narrowed in breadth to a single serotype over a 12-month period after infection. In DENV immune children who were experiencing a repeat infection, we observed a strong association between preexisting neutralizing antibodies and clinical outcome. Notably, children with preexisting monospecific neutralizing antibody responses were more likely to develop fever than children with cross-neutralizing responses. Preexisting DENV neutralizing antibodies are correlated with protection from dengue disease. PMID:25336728

  8. A High-Affinity Native Human Antibody Neutralizes Human Cytomegalovirus Infection of Diverse Cell Types

    PubMed Central

    Liu, Keyi; Park, Minha; DeChene, Neal; Stephenson, Robert; Tenorio, Edgar; Ellsworth, Stote L.; Tabata, Takako; Petitt, Matthew; Tsuge, Mitsuru; Fang-Hoover, June; Adler, Stuart P.; Cui, Xiaohong; McVoy, Michael A.; Pereira, Lenore

    2014-01-01

    Human cytomegalovirus (HCMV) is the most common infection causing poor outcomes among transplant recipients. Maternal infection and transplacental transmission are major causes of permanent birth defects. Although no active vaccines to prevent HCMV infection have been approved, passive immunization with HCMV-specific immunoglobulin has shown promise in the treatment of both transplant and congenital indications. Antibodies targeting the viral glycoprotein B (gB) surface protein are known to neutralize HCMV infectivity, with high-affinity binding being a desirable trait, both to compete with low-affinity antibodies that promote the transmission of virus across the placenta and to displace nonneutralizing antibodies binding nearby epitopes. Using a miniaturized screening technology to characterize secreted IgG from single human B lymphocytes, 30 antibodies directed against gB were previously cloned. The most potent clone, TRL345, is described here. Its measured affinity was 1 pM for the highly conserved site I of the AD-2 epitope of gB. Strain-independent neutralization was confirmed for 15 primary HCMV clinical isolates. TRL345 prevented HCMV infection of placental fibroblasts, smooth muscle cells, endothelial cells, and epithelial cells, and it inhibited postinfection HCMV spread in epithelial cells. The potential utility for preventing congenital transmission is supported by the blockage of HCMV infection of placental cell types central to virus transmission to the fetus, including differentiating cytotrophoblasts, trophoblast progenitor cells, and placental fibroblasts. Further, TRL345 was effective at controlling an ex vivo infection of human placental anchoring villi. TRL345 has been utilized on a commercial scale and is a candidate for clinical evaluation. PMID:25534746

  9. Evaluating the synergistic neutralizing effect of anti-botulinum oligoclonal antibody preparations.

    PubMed

    Diamant, Eran; Lachmi, Bat-El; Keren, Adi; Barnea, Ada; Marcus, Hadar; Cohen, Shoshana; David, Alon Ben; Zichel, Ran

    2014-01-01

    Botulinum neurotoxins (BoNT) are considered some of the most lethal known substances. There are seven botulinum serotypes, of which types A, B and E cause most human botulism cases. Anti-botulinum polyclonal antibodies (PAbs) are currently used for both detection and treatment of the disease. However, significant improvements in immunoassay specificity and treatment safety may be made using monoclonal antibodies (MAbs). In this study, we present an approach for the simultaneous generation of highly specific and neutralizing MAbs against botulinum serotypes A, B, and E in a single process. The approach relies on immunization of mice with a trivalent mixture of recombinant C-terminal fragment (Hc) of each of the three neurotoxins, followed by a parallel differential robotic hybridoma screening. This strategy enabled the cloning of seven to nine MAbs against each serotype. The majority of the MAbs possessed higher anti-botulinum ELISA titers than anti-botulinum PAbs and had up to five orders of magnitude greater specificity. When tested for their potency in mice, neutralizing MAbs were obtained for all three serotypes and protected against toxin doses of 10 MsLD50-500 MsLD50. A strong synergistic effect of up to 400-fold enhancement in the neutralizing activity was observed when serotype-specific MAbs were combined. Furthermore, the highly protective oligoclonal combinations were as potent as a horse-derived PAb pharmaceutical preparation. Interestingly, MAbs that failed to demonstrate individual neutralizing activity were observed to make a significant contribution to the synergistic effect in the oligoclonal preparation. Together, the trivalent immunization strategy and differential screening approach enabled us to generate highly specific MAbs against each of the A, B, and E BoNTs. These new MAbs may possess diagnostic and therapeutic potential. PMID:24475231

  10. Evaluating the Synergistic Neutralizing Effect of Anti-Botulinum Oligoclonal Antibody Preparations

    PubMed Central

    Diamant, Eran; Lachmi, Bat-El; Keren, Adi; Barnea, Ada; Marcus, Hadar; Cohen, Shoshana; David, Alon Ben; Zichel, Ran

    2014-01-01

    Botulinum neurotoxins (BoNT) are considered some of the most lethal known substances. There are seven botulinum serotypes, of which types A, B and E cause most human botulism cases. Anti-botulinum polyclonal antibodies (PAbs) are currently used for both detection and treatment of the disease. However, significant improvements in immunoassay specificity and treatment safety may be made using monoclonal antibodies (MAbs). In this study, we present an approach for the simultaneous generation of highly specific and neutralizing MAbs against botulinum serotypes A, B, and E in a single process. The approach relies on immunization of mice with a trivalent mixture of recombinant C-terminal fragment (Hc) of each of the three neurotoxins, followed by a parallel differential robotic hybridoma screening. This strategy enabled the cloning of seven to nine MAbs against each serotype. The majority of the MAbs possessed higher anti-botulinum ELISA titers than anti-botulinum PAbs and had up to five orders of magnitude greater specificity. When tested for their potency in mice, neutralizing MAbs were obtained for all three serotypes and protected against toxin doses of 10 MsLD50–500 MsLD50. A strong synergistic effect of up to 400-fold enhancement in the neutralizing activity was observed when serotype-specific MAbs were combined. Furthermore, the highly protective oligoclonal combinations were as potent as a horse-derived PAb pharmaceutical preparation. Interestingly, MAbs that failed to demonstrate individual neutralizing activity were observed to make a significant contribution to the synergistic effect in the oligoclonal preparation. Together, the trivalent immunization strategy and differential screening approach enabled us to generate highly specific MAbs against each of the A, B, and E BoNTs. These new MAbs may possess diagnostic and therapeutic potential. PMID:24475231

  11. Neutralization of Clostridium difficile Toxin B Mediated by Engineered Lactobacilli That Produce Single-Domain Antibodies

    PubMed Central

    Andersen, Kasper Krogh; Strokappe, Nika M.; Hultberg, Anna; Truusalu, Kai; Smidt, Imbi; Mikelsaar, Raik-Hiio; Mikelsaar, Marika; Verrips, Theo; Hammarström, Lennart

    2015-01-01

    Clostridium difficile is the primary cause of nosocomial antibiotic-associated diarrhea in the Western world. The major virulence factors of C. difficile are two exotoxins, toxin A (TcdA) and toxin B (TcdB), which cause extensive colonic inflammation and epithelial damage manifested by episodes of diarrhea. In this study, we explored the basis for an oral antitoxin strategy based on engineered Lactobacillus strains expressing TcdB-neutralizing antibody fragments in the gastrointestinal tract. Variable domain of heavy chain-only (VHH) antibodies were raised in llamas by immunization with the complete TcdB toxin. Four unique VHH fragments neutralizing TcdB in vitro were isolated. When these VHH fragments were expressed in either secreted or cell wall-anchored form in Lactobacillus paracasei BL23, they were able to neutralize the cytotoxic effect of the toxin in an in vitro cell-based assay. Prophylactic treatment with a combination of two strains of engineered L. paracasei BL23 expressing two neutralizing anti-TcdB VHH fragments (VHH-B2 and VHH-G3) delayed killing in a hamster protection model where the animals were challenged with spores of a TcdA− TcdB+ strain of C. difficile (P < 0.05). Half of the hamsters in the treated group survived until the termination of the experiment at day 5 and showed either no damage or limited inflammation of the colonic mucosa despite having been colonized with C. difficile for up to 4 days. The protective effect in the hamster model suggests that the strategy could be explored as a supplement to existing therapies for patients. PMID:26573738

  12. Neutralization of Clostridium difficile Toxin B Mediated by Engineered Lactobacilli That Produce Single-Domain Antibodies.

    PubMed

    Andersen, Kasper Krogh; Strokappe, Nika M; Hultberg, Anna; Truusalu, Kai; Smidt, Imbi; Mikelsaar, Raik-Hiio; Mikelsaar, Marika; Verrips, Theo; Hammarström, Lennart; Marcotte, Harold

    2016-02-01

    Clostridium difficile is the primary cause of nosocomial antibiotic-associated diarrhea in the Western world. The major virulence factors of C. difficile are two exotoxins, toxin A (TcdA) and toxin B (TcdB), which cause extensive colonic inflammation and epithelial damage manifested by episodes of diarrhea. In this study, we explored the basis for an oral antitoxin strategy based on engineered Lactobacillus strains expressing TcdB-neutralizing antibody fragments in the gastrointestinal tract. Variable domain of heavy chain-only (VHH) antibodies were raised in llamas by immunization with the complete TcdB toxin. Four unique VHH fragments neutralizing TcdB in vitro were isolated. When these VHH fragments were expressed in either secreted or cell wall-anchored form in Lactobacillus paracasei BL23, they were able to neutralize the cytotoxic effect of the toxin in an in vitro cell-based assay. Prophylactic treatment with a combination of two strains of engineered L. paracasei BL23 expressing two neutralizing anti-TcdB VHH fragments (VHH-B2 and VHH-G3) delayed killing in a hamster protection model where the animals were challenged with spores of a TcdA(-) TcdB(+) strain of C. difficile (P < 0.05). Half of the hamsters in the treated group survived until the termination of the experiment at day 5 and showed either no damage or limited inflammation of the colonic mucosa despite having been colonized with C. difficile for up to 4 days. The protective effect in the hamster model suggests that the strategy could be explored as a supplement to existing therapies for patients. PMID:26573738

  13. Passive antibody therapy of Lassa fever in cynomolgus monkeys: importance of neutralizing antibody and Lassa virus strain.

    PubMed Central

    Jahrling, P B; Peters, C J

    1984-01-01

    Lassa virus-infected cynomolgus monkeys were passively immunized with immune plasma of primate or human origin to gain insight into criteria for plasma selection and administration to human Lassa fever patients. Protective efficacy was correlated with neutralizing antibody concentrations, expressed as a log10 neutralization index (LNI). Convalescent Lassa-immune monkey plasma was titrated for protective efficacy in monkeys by intravenous inoculation with dilutions of plasma on the day of subcutaneous Lassa virus inoculation (day 0) and again on days 3 and 6. Monkeys that received undiluted plasma (LNI = 4.1) (1 ml/kg per treatment) survived a lethal viral dose, whereas those given a 1:3 dilution (LNI = 2.6) of this same plasma (1 ml/kg per treatment) died. Protection was restored when the volume of the 1:3 plasma dilution was increased to 3 ml/kg per treatment. Plasma diluted 1:9 or more (LNI = 1.5 or less) delayed onset and suppressed the magnitude of viremia but failed to confer protection at 3 ml/kg per treatment. Immunological enhancement, defined as increased viremia or accelerated death, did not occur following inadequate treatment. Human convalescent plasma also protected recipient monkeys; reductions in mortality and viremia were accurately predicted by the LNI of the plasma. Plasma of Liberian origin neutralized a Liberian Lassa strain more effectively than a Sierra Leone strain in vitro (LNI = 2.8 and 1.6, respectively) and protected monkeys more effectively against the Liberian strain. Geographic origin is thus a factor in the selection of optimal plasma for treatment of human Lassa fever, since geographically matched plasma is more likely to contain adequate LNI titers against homologous Lassa virus strains.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:6715049

  14. Localization of neutralizing regions of the envelope gene of feline leukemia virus by using anti-synthetic peptide antibodies.

    PubMed Central

    Elder, J H; McGee, J S; Munson, M; Houghten, R A; Kloetzer, W; Bittle, J L; Grant, C K

    1987-01-01

    We synthesized 27 synthetic peptides corresponding to approximately 80% of the sequences encoding gp70 and p15E of Gardner-Arnstein feline leukemia virus (FeLV) subtype B. The peptides were conjugated to keyhole limpet hemocyanin and injected into rabbits for preparation of antipeptide antisera. These sera were then tested for their ability to neutralize a broad range of FeLV isolates in vitro. Eight peptides elicited neutralizing responses against subtype B isolates. Five of these peptides corresponded to sequences of gp70 and three to p15E. The ability of these antipeptide antisera to neutralize FeLV subtypes A and C varied. In certain circumstances, failure to neutralize a particular isolate corresponded to sequence changes within the corresponding peptide region. However, four antibodies which preferentially neutralized the subtype B viruses were directed to epitopes in common with Sarma subtype C virus. These results suggest that distal changes in certain subtypes (possibly glycosylation differences) alter the availability of certain epitopes in one virus isolate relative to another. We prepared a "nest" of overlapping peptides corresponding to one of the neutralizing regions of gp70 and performed slot blot analyses with both antipeptide antibodies and a monoclonal antibody which recognized this epitope. We were able to define a five-amino-acid sequence required for reactivity. Comparisons were made between an anti-synthetic peptide antibody and a monoclonal antibody reactive to this epitope for the ability to bind both peptide and virus, as well as to neutralize virus in vitro. Both the anti-synthetic peptide and the monoclonal antibodies bound peptide and virus to high titers. However, the monoclonal antibody had a 4-fold-higher titer against virus and a 10-fold-higher neutralizing titer than did the anti-synthetic peptide antibody. Competition assays were performed with these two antibodies adjusted to equivalent antivirus titers against intact virions

  15. Neutralizing human monoclonal antibodies to conformational epitopes of human T-cell lymphotropic virus type 1 and 2 gp46.

    PubMed Central

    Hadlock, K G; Rowe, J; Perkins, S; Bradshaw, P; Song, G Y; Cheng, C; Yang, J; Gascon, R; Halmos, J; Rehman, S M; McGrath, M S; Foung, S K

    1997-01-01

    Ten human monoclonal antibodies derived from peripheral B cells of a patient with human T-cell lymphotropic virus (HTLV)-associated myelopathy are described. One monoclonal antibody recognized a linear epitope within the carboxy-terminal 43 amino acids of HTLV gp21, and two monoclonal antibodies recognized linear epitopes within HTLV type 1 (HTLV-1) gp46. The remaining seven monoclonal antibodies recognized denaturation-sensitive epitopes within HTLV-1 gp46 that were expressed on the surfaces of infected cells. Two of these antibodies also bound to viable HTLV-2 infected cells and immunoprecipitated HTLV-2 gp46. Virus neutralization was determined by syncytium inhibition assays. Eight monoclonal antibodies, including all seven that recognized denaturation-sensitive epitopes within HTLV-1 gp46, possessed significant virus neutralization activity. By competitive inhibition analysis it was determined that these antibodies recognized at least four distinct conformational epitopes within HTLV-1 gp46. These findings indicate the importance of conformational epitopes within HTLV-1 gp46 in mediating a neutralizing antibody response to HTLV infection. PMID:9223472

  16. Global Shape and Ligand Binding Efficiency of the HIV-1-neutralizing Antibodies Differ from Those of Antibodies That Cannot Neutralize HIV-1*

    PubMed Central

    Solanki, Ashish K.; Rathore, Yogendra S.; Badmalia, Maulik D.; Dhoke, Reema R.; Nath, Samir K.; Nihalani, Deepak; Ashish

    2014-01-01

    Asymmetric disposition of Fab arms in the structures solved for the broadly neutralizing monoclonal antibody (nmAb) IgG1 b12 raised the question of whether the unusual shape observed for b12 is common for all IgG1 mAbs or if there is a difference in the overall shape of nmAbs versus non-nmAbs. We compared small angle x-ray scattering (SAXS) data-based models and limited proteolysis profiles of some IgG1 mAbs known to be having and lacking HIV-1 neutralizing potency. In non-nmAbs, the Fab arms were found to be symmetrically disposed in space relative to central Fc, but in most nmAbs, the Fab arms were asymmetrically disposed, as seen for IgG1 b12. The only exceptions were 2G12 and 4E10, where both Fab arms were closed above Fc, suggesting some Fab-Fc and/or Fab-Fab interaction in the nmAbs that constrained extension of the Fab-Fc linker. Interestingly, these observations were correlated with differential proteolysis profiles of the mAbs by papain. Under conditions when papain could cut both Fab arms of non-nmAbs, only one Fab arm could be removed from neutralizing ones (except for 2G12 and 4E10). Chromatography and small angle x-ray scattering results of papain-digested products revealed that 1) the Fab-Fc or Fab-Fab interactions in unliganded mAbs are retained in digested products, and 2) whereas anti-gp120 non-nmAbs could bind two gp120 molecules, nmAbs could bind only one gp120. Additional experiments showed that except for 2G12 and 4E10, unopen shapes of nmAbs remain uninfluenced by ionic strength but can be reversibly opened by low pH of buffer accompanied by loss of ligand binding ability. PMID:25331945

  17. Neutralization of Clostridium difficile toxin A with single-domain antibodies targeting the cell receptor binding domain.

    PubMed

    Hussack, Greg; Arbabi-Ghahroudi, Mehdi; van Faassen, Henk; Songer, J Glenn; Ng, Kenneth K-S; MacKenzie, Roger; Tanha, Jamshid

    2011-03-18

    Clostridium difficile is a leading cause of nosocomial infection in North America and a considerable challenge to healthcare professionals in hospitals and nursing homes. The gram-positive bacterium produces two high molecular weight exotoxins, toxin A (TcdA) and toxin B (TcdB), which are the major virulence factors responsible for C. difficile-associated disease and are targets for C. difficile-associated disease therapy. Here, recombinant single-domain antibody fragments (V(H)Hs), which specifically target the cell receptor binding domains of TcdA or TcdB, were isolated from an immune llama phage display library and characterized. Four V(H)Hs (A4.2, A5.1, A20.1, and A26.8), all shown to recognize conformational epitopes, were potent neutralizers of the cytopathic effects of toxin A on fibroblast cells in an in vitro assay. The neutralizing potency was further enhanced when V(H)Hs were administered in paired or triplet combinations at the same overall V(H)H concentration, suggesting recognition of nonoverlapping TcdA epitopes. Biacore epitope mapping experiments revealed that some synergistic combinations consisted of V(H)Hs recognizing overlapping epitopes, an indication that factors other than mere epitope blocking are responsible for the increased neutralization. Further binding assays revealed TcdA-specific V(H)Hs neutralized toxin A by binding to sites other than the carbohydrate binding pocket of the toxin. With favorable characteristics such as high production yield, potent toxin neutralization, and intrinsic stability, these V(H)Hs are attractive systemic therapeutics but are more so as oral therapeutics in the destabilizing environment of the gastrointestinal tract. PMID:21216961

  18. Seroprevalence of neutralizing antibodies against dengue virus in two localities in the state of Morelos, Mexico.

    PubMed

    Amaya-Larios, Irma Y; Martínez-Vega, Ruth Aralí; Mayer, Sandra V; Galeana-Hernández, Marisol; Comas-García, Andreu; Sepúlveda-Salinas, Karla J; Falcón-Lezama, Jorge A; Vasilakis, Nikos; Ramos-Castañeda, José

    2014-11-01

    Humoral immune response against dengue virus (DENV) is an important component in dengue-endemic transmission. We conducted a cross-sectional nested cohort study to determine the seroprevalence and frequency of neutralizing antibodies against DENV serotypes in two endemic localities in the state of Morelos, Mexico. The cohort participants (N = 1,196) were screened to determine previous exposure to DENV. Overall seroprevalence was 76.6% (95% confidence interval [95% CI] = 73.6-79.2), and prevalence of neutralizing antibodies in the 5- to 9-year-old group was 82.5% (95% CI = 67.2-92.7), 45% (95% CI = 29.3-61.5), and 65% (95% CI = 48.3-79.4) for DENV-1, DENV-2, and DENV-3, respectively. For participants older than 10 years, the observed seroprevalence was above 60% for each serotype, except DENV-4 in the 10- to 25-year-old group (42.9%); 81% of humoral responses were multitypic. The outcomes of our study contribute to understanding the immune component of dengue transmission and provide focal information for the evaluation of vaccine candidates under development. PMID:25294613

  19. Seroprevalence of Neutralizing Antibodies Against Dengue Virus in Two Localities in the State of Morelos, Mexico

    PubMed Central

    Amaya-Larios, Irma Y.; Martínez-Vega, Ruth Aralí; Mayer, Sandra V.; Galeana-Hernández, Marisol; Comas-García, Andreu; Sepúlveda-Salinas, Karla J.; Falcón-Lezama, Jorge A.; Vasilakis, Nikos; Ramos-Castañeda, José

    2014-01-01

    Humoral immune response against dengue virus (DENV) is an important component in dengue-endemic transmission. We conducted a cross-sectional nested cohort study to determine the seroprevalence and frequency of neutralizing antibodies against DENV serotypes in two endemic localities in the state of Morelos, Mexico. The cohort participants (N = 1,196) were screened to determine previous exposure to DENV. Overall seroprevalence was 76.6% (95% confidence interval [95% CI] = 73.6–79.2), and prevalence of neutralizing antibodies in the 5- to 9-year-old group was 82.5% (95% CI = 67.2–92.7), 45% (95% CI = 29.3–61.5), and 65% (95% CI = 48.3–79.4) for DENV-1, DENV-2, and DENV-3, respectively. For participants older than 10 years, the observed seroprevalence was above 60% for each serotype, except DENV-4 in the 10- to 25-year-old group (42.9%); 81% of humoral responses were multitypic. The outcomes of our study contribute to understanding the immune component of dengue transmission and provide focal information for the evaluation of vaccine candidates under development. PMID:25294613

  20. Conformation-Dependent High-Affinity Potent Ricin-Neutralizing Monoclonal Antibodies

    PubMed Central

    Hu, Wei-Gang; Yin, Junfei; Chau, Damon; Hu, Charles Chen; Lillico, Dustin; Yu, Justin; Negrych, Laurel M.; Cherwonogrodzky, John W.

    2013-01-01

    Ricin is a potential biothreat agent with no approved antidote available for ricin poisoning. The aim of this study was to develop potent antibody-based antiricin antidotes. Four strong ricin resistant hybridoma clones secreting antiricin monoclonal antibodies (mAbs) were developed. All four mAbs are bound to conformational epitopes of ricin toxin B (RTB) with high affinity (KD values from 2.55 to 36.27 nM). RTB not only triggers cellular uptake of ricin, but also facilitates transport of the ricin toxin A (RTA) from the endoplasmic reticulum to the cytosol, where RTA exerts its toxic activity. The four mAbs were found to have potent ricin-neutralizing capacities and synergistic effects among them as determined by an in vitro neutralization assay. In vivo protection assay demonstrated that all four mAbs had strong efficacy against ricin challenges. D9 was found to be exceptionally effective. Intraperitoneal (i.p.) administration of D9, at a dose of 5 μg, 6 weeks before or 6 hours after an i.p. challenge with 5 × LD50 of ricin was able to protect or rescue 100% of the mice, indicating that mAb D9 is an excellent candidate to be developed as a potent antidote against ricin poisoning for both prophylactic and therapeutic purposes. PMID:23484120

  1. Neutralizing Monoclonal Antibodies against Disparate Epitopes on Ricin Toxin's Enzymatic Subunit Interfere with Intracellular Toxin Transport.

    PubMed

    Yermakova, Anastasiya; Klokk, Tove Irene; O'Hara, Joanne M; Cole, Richard; Sandvig, Kirsten; Mantis, Nicholas J

    2016-01-01

    Ricin is a member of the A-B family of bacterial and plant toxins that exploit retrograde trafficking to the Golgi apparatus and endoplasmic reticulum (ER) as a means to deliver their cytotoxic enzymatic subunits into the cytoplasm of mammalian cells. In this study we demonstrate that R70 and SyH7, two well-characterized monoclonal antibodies (mAbs) directed against distinct epitopes on the surface of ricin's enzymatic subunit (RTA), interfere with toxin transport from the plasma membrane to the trans Golgi network. Toxin-mAb complexes formed on the cell surface delayed ricin's egress from EEA-1(+) and Rab7(+) vesicles and enhanced toxin accumulation in LAMP-1(+) vesicles, suggesting the complexes were destined for degradation in lysosomes. Three other RTA-specific neutralizing mAbs against different epitopes were similar to R70 and SyH7 in terms of their effects on ricin retrograde transport. We conclude that interference with toxin retrograde transport may be a hallmark of toxin-neutralizing antibodies directed against disparate epitopes on RTA. PMID:26949061

  2. Multiplex Evaluation of Influenza Neutralizing Antibodies with Potential Applicability to In-Field Serological Studies

    PubMed Central

    Terregino, Calogero; Rahman, Rafat; Cattoli, Giovanni

    2014-01-01

    The increased number of outbreaks of H5 and H7 LPAI and HPAI viruses in poultry has major public and animal health implications. The continuous rapid evolution of these subtypes and the emergence of new variants influence the ability to undertake effective surveillance. Retroviral pseudotypes bearing influenza haemagglutinin (HA) and neuraminidase (NA) envelope glycoproteins represent a flexible platform for sensitive, readily standardized influenza serological assays. We describe a multiplex assay for the study of neutralizing antibodies that are directed against both influenza H5 and H7 HA. This assay permits the measurement of neutralizing antibody responses against two antigenically distinct HAs in the same serum/plasma sample thus increasing the amount and quality of serological data that can be acquired from valuable sera. Sera obtained from chickens vaccinated with a monovalent H5N2 vaccine, chickens vaccinated with a bivalent H7N1/H5N9 vaccine, or turkeys naturally infected with an H7N3 virus were evaluated in this assay and the results correlated strongly with data obtained by HI assay. We show that pseudotypes are highly stable under basic cold-chain storage conditions and following multiple rounds of freeze-thaw. We propose that this robust assay may have practical utility for in-field serosurveillance and vaccine studies in resource-limited regions worldwide. PMID:25101305

  3. Multiplex evaluation of influenza neutralizing antibodies with potential applicability to in-field serological studies.

    PubMed

    Molesti, Eleonora; Wright, Edward; Terregino, Calogero; Rahman, Rafat; Cattoli, Giovanni; Temperton, Nigel J

    2014-01-01

    The increased number of outbreaks of H5 and H7 LPAI and HPAI viruses in poultry has major public and animal health implications. The continuous rapid evolution of these subtypes and the emergence of new variants influence the ability to undertake effective surveillance. Retroviral pseudotypes bearing influenza haemagglutinin (HA) and neuraminidase (NA) envelope glycoproteins represent a flexible platform for sensitive, readily standardized influenza serological assays. We describe a multiplex assay for the study of neutralizing antibodies that are directed against both influenza H5 and H7 HA. This assay permits the measurement of neutralizing antibody responses against two antigenically distinct HAs in the same serum/plasma sample thus increasing the amount and quality of serological data that can be acquired from valuable sera. Sera obtained from chickens vaccinated with a monovalent H5N2 vaccine, chickens vaccinated with a bivalent H7N1/H5N9 vaccine, or turkeys naturally infected with an H7N3 virus were evaluated in this assay and the results correlated strongly with data obtained by HI assay. We show that pseudotypes are highly stable under basic cold-chain storage conditions and following multiple rounds of freeze-thaw. We propose that this robust assay may have practical utility for in-field serosurveillance and vaccine studies in resource-limited regions worldwide. PMID:25101305

  4. West Nile virus-neutralizing antibodies in wild birds from southern Spain.

    PubMed

    Ferraguti, M; LA Puente, J Martínez-DE; Soriguer, R; Llorente, F; Jiménez-Clavero, M Á; Figuerola, J

    2016-07-01

    West Nile virus (WNV) is an emerging vector-borne arbovirus with a zoonotic life-cycle whose main reservoir hosts are birds. In humans and horses, WNV infections rarely result in clinical disease but on occasions - depending on factors such as climatic conditions, insect communities and background immunity levels in local populations - they can lead to outbreaks that threaten public and animal health. We tested for the presence of WNV antibodies in 149 birds belonging to 32 different species. Samples were first tested using a bird-specific ELISA kit and then both positive and doubtful results were confirmed by neutralization tests using WNV and Usutu virus. WNV antibodies were confirmed in a resident Sylvia melanocephala juvenile, supporting the idea of local transmission of WNV in southern Spain in 2013. In addition, the serum from an adult blackbird (Turdus merula) showed neutralization of both WNV and Usutu virus. We discuss our results in light of the occurrence of WNV on horse farms in southern Spain in 2013. PMID:26846720

  5. Conformation-dependent high-affinity potent ricin-neutralizing monoclonal antibodies.

    PubMed

    Hu, Wei-Gang; Yin, Junfei; Chau, Damon; Hu, Charles Chen; Lillico, Dustin; Yu, Justin; Negrych, Laurel M; Cherwonogrodzky, John W

    2013-01-01

    Ricin is a potential biothreat agent with no approved antidote available for ricin poisoning. The aim of this study was to develop potent antibody-based antiricin antidotes. Four strong ricin resistant hybridoma clones secreting antiricin monoclonal antibodies (mAbs) were developed. All four mAbs are bound to conformational epitopes of ricin toxin B (RTB) with high affinity (KD values from 2.55 to 36.27 nM). RTB not only triggers cellular uptake of ricin, but also facilitates transport of the ricin toxin A (RTA) from the endoplasmic reticulum to the cytosol, where RTA exerts its toxic activity. The four mAbs were found to have potent ricin-neutralizing capacities and synergistic effects among them as determined by an in vitro neutralization assay. In vivo protection assay demonstrated that all four mAbs had strong efficacy against ricin challenges. D9 was found to be exceptionally effective. Intraperitoneal (i.p.) administration of D9, at a dose of 5 μ g, 6 weeks before or 6 hours after an i.p. challenge with 5 × LD50 of ricin was able to protect or rescue 100% of the mice, indicating that mAb D9 is an excellent candidate to be developed as a potent antidote against ricin poisoning for both prophylactic and therapeutic purposes. PMID:23484120

  6. Neutralizing antibodies to African swine fever virus proteins p30, p54, and p72 are not sufficient for antibody-mediated protection.

    PubMed

    Neilan, J G; Zsak, L; Lu, Z; Burrage, T G; Kutish, G F; Rock, D L

    2004-02-20

    Although antibody-mediated immune mechanisms have been shown to be important in immunity to ASF, it remains unclear what role virus neutralizing antibodies play in the protective response. Virus neutralizing epitopes have been identified on three viral proteins, p30, p54, and p72. To evaluate the role(s) of these proteins in protective immunity, pigs were immunized with baculovirus-expressed p30, p54, p72, and p22 from the pathogenic African swine fever virus (ASFV) isolate Pr4. ASFV specific neutralizing antibodies were detected in test group animals. Following immunization, animals were challenged with 10(4) TCID(50) of Pr4 virus. In comparison to the control group, test group animals exhibited a 2-day delay to onset of clinical disease and reduced viremia levels at 2 days postinfection (DPI); however, by 4 DPI, there was no significant difference between the two groups and all animals in both groups died between 7 and 10 DPI. These results indicate that neutralizing antibodies to these ASFV proteins are not sufficient for antibody-mediated protection. PMID:14980493

  7. Characterization of a Large Panel of Rabbit Monoclonal Antibodies against HIV-1 gp120 and Isolation of Novel Neutralizing Antibodies against the V3 Loop.

    PubMed

    Qin, Yali; Banerjee, Saikat; Agrawal, Aditi; Shi, Heliang; Banasik, Marisa; Lin, Feng; Rohl, Kari; LaBranche, Celia; Montefiori, David C; Cho, Michael W

    2015-01-01

    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. PMID:26039641

  8. Development of Germline-Humanized Antibodies Neutralizing Botulinum Neurotoxin A and B.

    PubMed

    Miethe, Sebastian; Mazuet, Christelle; Liu, Yvonne; Tierney, Robert; Rasetti-Escargueil, Christine; Avril, Arnaud; Frenzel, André; Thullier, Philippe; Pelat, Thibaut; Urbain, Remi; Fontayne, Alexandre; Sesardic, Dorothea; Hust, Michael; Popoff, Michel Robert

    2016-01-01

    Botulinum neurotoxins (BoNTs) are counted among the most toxic substances known and are responsible for human botulism, a life-threatening disease characterized by flaccid muscle paralysis that occurs naturally by food poisoning or colonization of the gastrointestinal tract by BoNT-producing clostridia. To date, 7 serologically distinct serotypes of BoNT (serotype A-G) are known. Due to the high toxicity of BoNTs the Centers for Disease Control and Prevention (CDC) have classified BoNTs as category A agent, including the six biological agents with the highest potential risk of use as bioweapons. Well tolerated antibodies neutralizing BoNTs are required to deal with the potential risk. In a previous work, we described the development of scFv and scFv-Fc (Yumab) from macaque origin (Macaca fascicularis) neutralizing BoNT/A and B by targeting the heavy and light chain of each serotype. In the present study, we humanized the macaque antibodies SEM120-IIIC1 (anti-BoNT/A light chain), A1HC38 (anti-BoNT/A heavy chain), BLC3 (anti-BoNT/B light chain) and B2-7 (anti-BoNT/B heavy chain) by germline-humanization to obtain a better potential immunotolerance in humans. We increased the Germinality Index (GI) of SEM120-IIIC1 to 94.5%, for A1HC38, to 95% for BLC3 and to 94.4% for B2-7. Furthermore, the neutralization efficacies of the germline-humanized antibodies were analyzed in lethal and non-lethal in vivo mouse assays as full IgG. The germline-humanized IgGs hu8SEM120-IIIC1, hu8A1HC38, hu8BLC3 and hu8B2-7 were protective in vivo, when anti-heavy and anti-light chain antibodies were combined. The synergistic effect and high humanness of the selected IgGs makes them promising lead candidates for further clinical development. PMID:27560688

  9. Development of Germline-Humanized Antibodies Neutralizing Botulinum Neurotoxin A and B

    PubMed Central

    Liu, Yvonne; Tierney, Robert; Rasetti-Escargueil, Christine; Avril, Arnaud; Frenzel, André; Thullier, Philippe; Pelat, Thibaut; Urbain, Remi; Fontayne, Alexandre; Sesardic, Dorothea; Hust, Michael; Popoff, Michel Robert

    2016-01-01

    Botulinum neurotoxins (BoNTs) are counted among the most toxic substances known and are responsible for human botulism, a life-threatening disease characterized by flaccid muscle paralysis that occurs naturally by food poisoning or colonization of the gastrointestinal tract by BoNT-producing clostridia. To date, 7 serologically distinct serotypes of BoNT (serotype A-G) are known. Due to the high toxicity of BoNTs the Centers for Disease Control and Prevention (CDC) have classified BoNTs as category A agent, including the six biological agents with the highest potential risk of use as bioweapons. Well tolerated antibodies neutralizing BoNTs are required to deal with the potential risk. In a previous work, we described the development of scFv and scFv-Fc (Yumab) from macaque origin (Macaca fascicularis) neutralizing BoNT/A and B by targeting the heavy and light chain of each serotype. In the present study, we humanized the macaque antibodies SEM120-IIIC1 (anti-BoNT/A light chain), A1HC38 (anti-BoNT/A heavy chain), BLC3 (anti-BoNT/B light chain) and B2-7 (anti-BoNT/B heavy chain) by germline-humanization to obtain a better potential immunotolerance in humans. We increased the Germinality Index (GI) of SEM120-IIIC1 to 94.5%, for A1HC38, to 95% for BLC3 and to 94.4% for B2-7. Furthermore, the neutralization efficacies of the germline-humanized antibodies were analyzed in lethal and non-lethal in vivo mouse assays as full IgG. The germline-humanized IgGs hu8SEM120-IIIC1, hu8A1HC38, hu8BLC3 and hu8B2-7 were protective in vivo, when anti-heavy and anti-light chain antibodies were combined. The synergistic effect and high humanness of the selected IgGs makes them promising lead candidates for further clinical development. PMID:27560688

  10. Characterization of a Broadly Neutralizing Monoclonal Antibody That Targets the Fusion Domain of Group 2 Influenza A Virus Hemagglutinin

    PubMed Central

    Tan, Gene S.; Lee, Peter S.; Hoffman, Ryan M. B.; Mazel-Sanchez, Beryl; Krammer, Florian; Leon, Paul E.; Ward, Andrew B.; Wilson, Ian A.

    2014-01-01

    ABSTRACT Due to continuous changes to its antigenic regions, influenza viruses can evade immune detection and cause a significant amount of morbidity and mortality around the world. Influenza vaccinations can protect against disease but must be annually reformulated to match the current circulating strains. In the development of a broad-spectrum influenza vaccine, the elucidation of conserved epitopes is paramount. To this end, we designed an immunization strategy in mice to boost the humoral response against conserved regions of the hemagglutinin (HA) glycoprotein. Of note, generation and identification of broadly neutralizing antibodies that target group 2 HAs are rare and thus far have yielded only a few monoclonal antibodies (MAbs). Here, we demonstrate that mouse MAb 9H10 has broad and potent in vitro neutralizing activity against H3 and H10 group 2 influenza A subtypes. In the mouse model, MAb 9H10 protects mice against two divergent mouse-adapted H3N2 strains, in both pre- and postexposure administration regimens. In vitro and cell-free assays suggest that MAb 9H10 inhibits viral replication by blocking HA-dependent fusion of the viral and endosomal membranes early in the replication cycle and by disrupting viral particle egress in the late stage of infection. Interestingly, electron microscopy reconstructions of MAb 9H10 bound to the HA reveal that it binds a similar binding footprint to MAbs CR8020 and CR8043. IMPORTANCE The influenza hemagglutinin is the major antigenic target of the humoral immune response. However, due to continuous antigenic changes that occur on the surface of this glycoprotein, influenza viruses can escape the immune system and cause significant disease to the host. Toward the development of broad-spectrum therapeutics and vaccines against influenza virus, elucidation of conserved regions of influenza viruses is crucial. Thus, defining these types of epitopes through the generation and characterization of broadly neutralizing

  11. Modification of Ad5 Hexon Hypervariable Regions Circumvents Pre-Existing Ad5 Neutralizing Antibodies and Induces Protective Immune Responses

    PubMed Central

    Bruder, Joseph T.; Semenova, Elena; Chen, Ping; Limbach, Keith; Patterson, Noelle B.; Stefaniak, Maureen E.; Konovalova, Svetlana; Thomas, Charlie; Hamilton, Melissa; King, C. Richter; Richie, Thomas L.; Doolan, Denise L.

    2012-01-01

    The development of an effective malaria vaccine is a high global health priority. Vaccine vectors based on adenovirus type 5 are capable of generating robust and protective T cell and antibody responses in animal models and are currently being evaluated in clinical trials for HIV and malaria. They appear to be more effective in terms of inducing antigen-specific immune responses as compared with non-Ad5 serotype vectors. However, the high prevalence of neutralizing antibodies to Ad5 in the human population, particularly in the developing world, has the potential to limit the effectiveness of Ad5-based vaccines. We have generated novel Ad5-based vectors that precisely replace the hexon hypervariable regions with those derived from Ad43, a subgroup D serotype with low prevalence of neutralizing antibody in humans. We have demonstrated that these hexon-modified adenovectors are not neutralized efficiently by Ad5 neutralizing antibodies in vitro using sera from mice, rabbits and human volunteers. We have also generated hexon-modified adenovectors that express a rodent malaria parasite antigen, PyCSP, and demonstrated that they are as immunogenic as an unmodified vector. Furthermore, in contrast to the unmodified vector, the hexon-modified adenovectors induced robust T cell responses in mice with high levels of Ad5 neutralizing antibody. We also show that the hexon-modified vector can be combined with unmodified Ad5 vector in prime-boost regimens to induce protective responses in mice. Our data establish that these hexon-modified vectors are highly immunogenic even in the presence of pre-existing anti-adenovirus antibodies. These hexon-modified adenovectors may have advantages in sub-Saharan Africa where there is a high prevalence of Ad5 neutralizing antibody in the population. PMID:22496772

  12. Pichia pastoris-expressed dengue 3 envelope-based virus-like particles elicit predominantly domain III-focused high titer neutralizing antibodies

    PubMed Central

    Tripathi, Lav; Mani, Shailendra; Raut, Rajendra; Poddar, Ankur; Tyagi, Poornima; Arora, Upasana; de Silva, Aravinda; Swaminathan, Sathyamangalam; Khanna, Navin

    2015-01-01

    Dengue poses a serious public health risk to nearly half the global population. It causes ~400 million infections annually and is considered to be one of the fastest spreading vector-borne diseases. Four distinct serotypes of dengue viruses (DENV-1, -2, -3, and -4) cause dengue disease, which may be either mild or extremely severe. Antibody-dependent enhancement (ADE), by pre-existing cross-reactive antibodies, is considered to be the major mechanism underlying severe disease. This mandates that a preventive vaccine must confer simultaneous and durable immunity to each of the four prevalent DENV serotypes. Recently, we used Pichia pastoris, to express recombinant DENV-2 E ectodomain, and found that it assembled into virus-like particles (VLPs), in the absence of prM, implicated in the elicitation of ADE-mediating antibodies. These VLPs elicited predominantly type-specific neutralizing antibodies that conferred significant protection against lethal DENV-2 challenge, in a mouse model. The current work is an extension of this approach to develop prM-lacking DENV-3 E VLPs. Our data reveal that P. pastoris-produced DENV-3 E VLPs not only preserve the antigenic integrity of the major neutralizing epitopes, but also elicit potent DENV-3 virus-neutralizing antibodies. Further, these neutralizing antibodies appear to be exclusively directed toward domain III of the DENV-3 E VLPs. Significantly, they also lack discernible ADE potential toward heterotypic DENVs. Taken together with the high productivity of the P. pastoris expression system, this approach could potentially pave the way toward developing a DENV E-based, inexpensive, safe, and efficacious tetravalent sub-unit vaccine, for use in resource-poor dengue endemic countries. PMID:26441930

  13. Asymmetric recognition of the HIV-1 trimer by broadly neutralizing antibody PG9.

    PubMed

    Julien, Jean-Philippe; Lee, Jeong Hyun; Cupo, Albert; Murin, Charles D; Derking, Ronald; Hoffenberg, Simon; Caulfield, Michael J; King, C Richter; Marozsan, Andre J; Klasse, Per Johan; Sanders, Rogier W; Moore, John P; Wilson, Ian A; Ward, Andrew B

    2013-03-12

    PG9 is the founder member of an expanding family of glycan-dependent human antibodies that preferentially bind the HIV (HIV-1) envelope (Env) glycoprotein (gp) trimer and broadly neutralize the virus. Here, we show that a soluble SOSIP.664 gp140 trimer constructed from the Clade A BG505 sequence binds PG9 with high affinity (∼11 nM), enabling structural and biophysical characterizations of the PG9:Env trimer complex. The BG505 SOSIP.664 gp140 trimer is remarkably stable as assessed by electron microscopy (EM) and differential scanning calorimetry. EM, small angle X-ray scattering, size exclusion chromatography with inline multiangle light scattering and isothermal titration calorimetry all indicate that only a single PG9 fragment antigen-binding (Fab) binds to the Env trimer. An ∼18 Å EM reconstruction demonstrates that PG9 recognizes the trimer asymmetrically at its apex via contact with two of the three gp120 protomers, possibly contributing to its reported preference for a quaternary epitope. Molecular modeling and isothermal titration calorimetry binding experiments with an engineered PG9 mutant suggest that, in addition to the N156 and N160 glycan interactions observed in crystal structures of PG9 with a scaffolded V1/V2 domain, PG9 makes secondary interactions with an N160 glycan from an adjacent gp120 protomer in the antibody-trimer complex. Together, these structural and biophysical findings should facilitate the design of HIV-1 immunogens that possess all elements of the quaternary PG9 epitope required to induce broadly neutralizing antibodies against this region. PMID:23426631

  14. Characterization of Poliovirus Neutralization Escape Mutants of Single-Domain Antibody Fragments (VHHs)

    PubMed Central

    Schotte, Lise; Thys, Bert; Strauss, Mike; Filman, David J.; Rombaut, Bart

    2015-01-01

    To complete the eradication of poliovirus and to protect unvaccinated people subsequently, the development of one or more antiviral drugs will be necessary. A set of five single-domain antibody fragments (variable parts of the heavy chain of a heavy-chain antibody [VHHs]) with an in vitro neutralizing activity against poliovirus type 1 was developed previously (B. Thys, L. Schotte, S. Muyldermans, U. Wernery, G. Hassanzadeh-Ghassabeh, and B. Rombaut, Antiviral Res 87:257–264, 2010, http://dx.doi.org/10.1016/j.antiviral.2010.05.012), and their mechanisms of action have been studied (L. Schotte, M. Strauss, B. Thys, H. Halewyck, D. J. Filman, M. Bostina, J. M. Hogle, and B. Rombaut, J Virol 88:4403–4413, 2014, http://dx.doi.org/10.1128/JVI.03402-13). In this study, neutralization escape mutants were selected for each VHH. Sequencing of the P1 region of the genome showed that amino acid substitutions are found in the four viral proteins of the capsid and that they are located both in proximity to the binding sites of the VHHs and in regions further away from the canyon and hidden beneath the surface. Characterization of the mutants demonstrated that they have single-cycle replication kinetics that are similar to those of their parental strain and that they are all drug (VHH) independent. Their resistant phenotypes are stable, as they do not regain full susceptibility to the VHH after passage over HeLa cells in the absence of VHH. They are all at least as stable as the parental strain against heat inactivation at 44°C, and three of them are even significantly (P < 0.05) more resistant to heat inactivation. The resistant variants all still can be neutralized by at least two other VHHs and retain full susceptibility to pirodavir and 35-1F4. PMID:26014941

  15. Development of a High-Throughput Assay To Measure the Neutralization Capability of Anti-Cytomegalovirus Antibodies

    PubMed Central

    Gardner, Thomas J.; Bolovan-Fritts, Cynthia; Teng, Melissa W.; Redmann, Veronika; Kraus, Thomas A.; Sperling, Rhoda; Moran, Thomas; Britt, William; Weinberger, Leor S.

    2013-01-01

    Infection by human cytomegalovirus (CMV) elicits a strong humoral immune response and robust anti-CMV antibody production. Diagnosis of virus infection can be carried out by using a variety of serological assays; however, quantification of serum antibodies against CMV may not present an accurate measure of a patient's ability to control a virus infection. CMV strains that express green fluorescent protein (GFP) fusion proteins can be used as screening tools for evaluating characteristics of CMV infection in vitro. In this study, we employed a CMV virus strain, AD169, that ectopically expresses a yellow fluorescent protein (YFP) fused to the immediate-early 2 (IE2) protein product (AD169IE2-YFP) to quantify a CMV infection in human cells. We created a high-throughput cell-based assay that requires minimal amounts of material and provides a platform for rapid analysis of the initial phase of virus infection, including virus attachment, fusion, and immediate-early viral gene expression. The AD169IE2-YFP cell infection system was utilized to develop a neutralization assay with a monoclonal antibody against the viral surface glycoprotein gH. The high-throughput assay was extended to measure the neutralization capacity of serum from CMV-positive subjects. These findings describe a sensitive and specific assay for the quantification of a key immunological response that plays a role in limiting CMV dissemination and transmission. Collectively, we have demonstrated that a robust high-throughput infection assay can analyze the early steps of the CMV life cycle and quantify the potency of biological reagents to attenuate a virus infection. PMID:23389931

  16. Enhanced Potency of a Broadly Neutralizing HIV-1 Antibody In Vitro Improves Protection against Lentiviral Infection In Vivo

    PubMed Central

    Rudicell, Rebecca S.; Kwon, Young Do; Ko, Sung-Youl; Pegu, Amarendra; Louder, Mark K.; Georgiev, Ivelin S.; Wu, Xueling; Zhu, Jiang; Boyington, Jeffrey C.; Chen, Xuejun; Shi, Wei; Yang, Zhi-yong; Doria-Rose, Nicole A.; McKee, Krisha; O'Dell, Sijy; Schmidt, Stephen D.; Chuang, Gwo-Yu; Druz, Aliaksandr; Soto, Cinque; Yang, Yongping; Zhang, Baoshan; Zhou, Tongqing; Todd, John-Paul; Lloyd, Krissey E.; Eudailey, Joshua; Roberts, Kyle E.; Donald, Bruce R.; Bailer, Robert T.; Ledgerwood, Julie; Mullikin, James C.; Shapiro, Lawrence; Koup, Richard A.; Graham, Barney S.; Nason, Martha C.; Connors, Mark; Haynes, Barton F.; Rao, Srinivas S.; Roederer, Mario; Kwong, Peter D.

    2014-01-01

    ABSTRACT Over the past 5 years, a new generation of highly potent and broadly neutralizing HIV-1 antibodies has been identified. These antibodies can protect against lentiviral infection in nonhuman primates (NHPs), suggesting that passive antibody transfer would prevent HIV-1 transmission in humans. To increase the protective efficacy of such monoclonal antibodies, we employed next-generation sequencing, computational bioinformatics, and structure-guided design to enhance the neutralization potency and breadth of VRC01, an antibody that targets the CD4 binding site of the HIV-1 envelope. One variant, VRC07-523, was 5- to 8-fold more potent than VRC01, neutralized 96% of viruses tested, and displayed minimal autoreactivity. To compare its protective efficacy to that of VRC01 in vivo, we performed a series of simian-human immunodeficiency virus (SHIV) challenge experiments in nonhuman primates and calculated the doses of VRC07-523 and VRC01 that provide 50% protection (EC50). VRC07-523 prevented infection in NHPs at a 5-fold lower concentration than VRC01. These results suggest that increased neutralization potency in vitro correlates with improved protection against infection in vivo, documenting the improved functional efficacy of VRC07-523 and its potential clinical relevance for protecting against HIV-1 infection in humans. IMPORTANCE In the absence of an effective HIV-1 vaccine, alternative strategies are needed to block HIV-1 transmission. Direct administration of HIV-1-neutralizing antibodies may be able to prevent HIV-1 infections in humans. This approach could be especially useful in individuals at high risk for contracting HIV-1 and could be used together with antiretroviral drugs to prevent infection. To optimize the chance of success, such antibodies can be modified to improve their potency, breadth, and in vivo half-life. Here, knowledge of the structure of a potent neutralizing antibody, VRC01, that targets the CD4-binding site of the HIV-1 envelope

  17. Generation and Characterization of Neutralizing Human Monoclonal Antibodies against Human Immunodeficiency Virus Type 1 Tat Antigen

    PubMed Central

    Moreau, Emmanuel; Hoebeke, Johan; Zagury, Daniel; Muller, Sylviane; Desgranges, Claude

    2004-01-01

    The human immunodeficiency virus Tat regulatory protein is essential for virus replication and pathogenesis. From human peripheral blood mononuclear cells of three Tat toxoid-immunized volunteers, we isolated five Tat-specific human monoclonal antibodies (HMAbs): two full-length immunoglobulin G (IgG) antibodies and three single-chain fragment-variable (scFv) antibodies. The two IgGs were mapped to distinct epitopes within the basic region of Tat, and the three scFvs were mapped to the N-terminal domain of Tat. The three scFvs were highly reactive with recombinant Tat in Western blotting or immunoprecipitation, but results were in contrast to those for the two IgGs, which are sensitive to a particular folding of the protein. In transactivation assays, scFvs were able to inhibit both active recombinant Tat and native Tat secreted by a transfected CEM cell line while IgGs neutralized only native Tat. These HMAbs were able to reduce viral p24 production in human immunodeficiency virus type 1 strain IIIB chronically infected cell lines in a dose-dependent manner. PMID:15016898

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

    PubMed Central

    Scott, Yanille M.; Park, Seo Young

    2015-01-01

    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. PMID:26596954

  19. Efficacy of broadly neutralizing monoclonal antibody PG16 in HIV-infected humanized mice.

    PubMed

    Stoddart, Cheryl A; Galkina, Sofiya A; Joshi, Pheroze; Kosikova, Galina; Long, Brian R; Maidji, Ekaterina; Moreno, Mary E; Rivera, Jose M; Sanford, Ukina R; Sloan, Barbara; Cieplak, Witold; Wrin, Terri; Chan-Hui, Po-Ying

    2014-08-01

    Highly potent broadly neutralizing human monoclonal antibodies hold promise for HIV prophylaxis and treatment. We used the SCID-hu Thy/Liv and BLT humanized mouse models to study the efficacy of these antibodies, primarily PG16, against HIV-1 clades A, B, and C. PG16 targets a conserved epitope in the V1/V2 region of gp120 common to 70-80% of HIV-1 isolates from multiple clades and has extremely potent in vitro activity against HIVJR-CSF. PG16 was highly efficacious in SCID-hu mice as a single intraperitoneal administration the day before inoculation of R5-tropic HIV directly into their Thy/Liv implants and demonstrated even greater efficacy if PG16 administration was continued after Thy/Liv implant HIV inoculation. However, PG16 as monotherapy had no activity in humanized mice with established R5-tropic HIV infection. These results provide evidence of tissue penetration of the antibodies, which could aid in their ability to prevent infection if virus crosses the mucosal barrier. PMID:24971704

  20. Efficacy of Broadly Neutralizing Monoclonal Antibody PG16 in HIV-infected Humanized Mice

    PubMed Central

    Stoddart, Cheryl A.; Galkina, Sofiya A.; Joshi, Pheroze; Kosikova, Galina; Long, Brian R.; Maidji, Ekaterina; Moreno, Mary E.; Rivera, Jose M.; Sanford, Ukina R.; Sloan, Barbara; Cieplak, Witold; Wrin, Terri; Chan-Hui, Po-Ying

    2014-01-01

    Highly potent broadly neutralizing human monoclonal antibodies hold promise for HIV prophylaxis and treatment. We used the SCID-hu Thy/Liv and BLT humanized mouse models to study the efficacy of these antibodies, primarily PG16, against HIV-1 clade A, B, and C. PG16 targets a conserved epitope in the V1/V2 region of gp120 common to 70–80% of HIV-1 isolates from multiple clades and has extremely potent in vitro activity against HIVJR-CSF. PG16 was highly efficacious in SCID-hu mice as a single intraperitoneal administration the day before inoculation of R5-tropic HIV-1 directly into their Thy/Liv implants and demonstrated even greater efficacy if PG16 administration was continued after Thy/Liv implant HIV-1 infection. However, PG16 as monotherapy had no activity in humanized mice with established R5-tropic HIV-1 infection. These results provide evidence of tissue penetration of the antibodies, which could aid in their ability to prevent infection if virus crosses the mucosal barrier. PMID:24971704

  1. Development and characterization of neutralizing monoclonal antibodies against canine distemper virus hemagglutinin protein.

    PubMed

    Bi, Zhenwei; Xia, Xingxia; Wang, Yongshan; Mei, Yongjie

    2015-04-01

    Canine distemper virus (CDV) causes a serious multisystemic disease in dogs and other carnivora. Hemagglutinin (H) protein-specific antibodies are mainly responsible for protective immunity against CDV infection. In the present study, six neutralizing MAbs to the H protein of CDV were newly obtained and characterized by immunizing BALB/c mice with a recent Chinese field isolate. Competitive binding inhibition assay revealed that they recognized four distinct antigenic regions of the H protein. Immunofluorescence assay and western blotting showed that all MAbs recognize the conformational rather than the linear epitopes of the H protein. Furthermore, in immunofluorescence and virus neutralization assays, two of the MAbs were found to react only with the recent Chinese field isolate and not with older CDV strains, including vaccine strain Onderstepoort, indicating there are neutralization-related antigenic variations between the recent Chinese field isolate and the older CDV strains examined in this study. The newly established MAbs are useful for differentiating the expanding CDV strains and could be used in immunotherapy and immunodiagnosis against infection with CDV. PMID:25644427

  2. A Trimeric, V2-Deleted HIV-1 Envelope Glycoprotein Vaccine Elicits Potent Neutralizing Antibodies but Limited Breadth of Neutralization in Human Volunteers

    PubMed Central

    Lally, Michelle A.; Elizaga, Marnie; Montefiori, David; Tomaras, Georgia D.; McElrath, M. Juliana; Hural, John; De Rosa, Stephen C.; Sato, Alicia; Huang, Yunda; Frey, Sharon E.; Sato, Paul; Donnelly, John; Barnett, Susan; Corey, Lawrence J.

    2011-01-01

    Background. A key missing element in the development of a successful human immunodeficiency virus (HIV) vaccine is an immunogen that can generate broadly cross-neutralizing antibodies against primary isolates of the virus. Methods. This phase 1 clinical trial employed a DNA prime and subunit envelope protein boost in an attempt to generate cellular and humoral immune responses that might be desirable in a protective HIV vaccine. Priming was performed via intramuscular injection with gag and env DNA adsorbed to polylactide coglycolide microspheres, followed by boosting with a recombinant trimeric envelope (Env) glycoprotein delivered in MF59 adjuvant. Results. The DNA prime and protein boost were generally safe and well-tolerated. Env-specific CD4+ cellular responses were generated that were predominantly detected after Env protein boosting. Neutralizing antibody responses against the homologous SF162 viral isolate were remarkably strong and were present in the majority of vaccine recipients, including a strong response against CD4-induced epitopes on gp120. Despite the promising potency of this vaccine approach, neutralization breadth against heterologous tier 2 strains of HIV-1 was minimal. Conclusions. Potent neutralization against neutralization-sensitive strains of HIV is achievable in humans through a DNA prime, recombinant oligomeric Env protein boost regimen. Eliciting substantial breadth of neutralization remains an elusive goal.  Clinical Trials Registration. NCT00073216. PMID:21451004

  3. Neutral Beam Power System for TPX

    SciTech Connect

    Ramakrishnan, S.; Bowen, O.N.; O`Conner, T.; Edwards, J.; Fromm, N.; Hatcher, R.; Newman, R.; Rossi, G.; Stevenson, T.; von Halle, A.

    1993-11-01

    The Tokamak Physics Experiment (TPX) will utilize to the maximum extent the existing Tokamak Fusion Test Reactor (TFTR) equipment and facilities. This is particularly true for the TFTR Neutral Beam (NB) system. Most of the NB hardware, plant facilities, auxiliary sub-systems, power systems, service infrastructure, and control systems can be used as is. The major changes in the NB hardware are driven by the new operating duty cycle. The TFTR Neutral Beam was designed for operation of the Sources for 2 seconds every 150 seconds. The TPX requires operation for 1000 seconds every 4500 seconds. During the Conceptual Design Phase of TPX every component of the TFTR NB Electrical Power System was analyzed to verify whether the equipment can meet the new operational requirements with our without modifications. The Power System converts 13.8 kV prime power to controlled pulsed power required at the NB sources. The major equipment involved are circuit breakers, auto and rectifier transformers surge suppression components, power tetrodes, HV Decks, and HVDC power transmission to sources. Thermal models were developed for the power transformers to simulate the new operational requirements. Heat runs were conducted for the power tetrodes to verify capability. Other components were analyzed to verify their thermal limitations. This paper describes the details of the evaluation and redesign of the electrical power system components to meet the TPX operational requirements.

  4. Detailed Atomic Structure of Neutral and Near-Neutral Systems

    SciTech Connect

    Oliver, Paul; Hibbert, Alan

    2011-05-11

    This paper highlights the issues which need to be addressed in undertaking accurate calculations of multi-electron atoms and ions, particularly at or near the neutral end of an isoelectronic sequence. We illustrate the processes through two calculations--of transitions in Cl I and Sn II--and discuss the convergence of our results as well as updating previous work. In particular, in the case of Cl I, we propose new identifications of the levels involved in certain transitions which are important in determining the abundance of chlorine in the inter-stellar medium (ISM), while in singly ionised tin, our calculations suggest a re-evaluation of the the abundance of tin in the ISM. We also confirm recent identification of Sn II lines seen in tokamak plasmas.

  5. Polysaccharide mimicry of the epitope of the broadly neutralizing anti-HIV antibody, 2G12, induces enhanced antibody responses to self oligomannose glycans

    PubMed Central

    Dunlop, D Cameron; Bonomelli, Camille; Mansab, Fatma; Vasiljevic, Snezana; Doores, Katie J; Wormald, Mark R; Palma, Angelina S; Feizi, Ten; Harvey, David J; Dwek, Raymond A; Crispin, Max; Scanlan, Christopher N

    2010-01-01

    Immunologically, “self” carbohydrates protect the HIV-1 surface glycoprotein, gp120, from antibody recognition. However, one broadly neutralizing antibody, 2G12, neutralizes primary viral isolates by direct recognition of Manα1→2Man motifs formed by the host-derived oligomannose glycans of the viral envelope. Immunogens, capable of eliciting antibodies of similar specificity to 2G12, are therefore candidates for HIV/AIDS vaccine development. In this context, it is known that the yeast mannan polysaccharides exhibit significant antigenic mimicry with the glycans of HIV-1. Here, we report that modulation of yeast polysaccharide biosynthesis directly controls the molecular specificity of cross-reactive antibodies to self oligomannose glycans. Saccharomyces cerevisiae mannans are typically terminated by α1→3-linked mannoses that cap a Manα1→2Man motif that otherwise closely resembles the part of the oligomannose epitope recognized by 2G12. Immunization with S. cerevisiae deficient for the α1→3 mannosyltransferase gene (ΔMnn1), but not with wild-type S. cerevisiae, reproducibly elicited antibodies to the self oligomannose glycans. Carbohydrate microarray analysis of ΔMnn1 immune sera revealed fine carbohydrate specificity to Manα1→2Man units, closely matching that of 2G12. These specificities were further corroborated by enzyme-linked immunosorbent assay with chemically defined glycoforms of gp120. These antibodies exhibited remarkable similarity in the carbohydrate specificity to 2G12 and displayed statistically significant, albeit extremely weak, neutralization of HIV-1 compared to control immune sera. These data confirm the Manα1→2Man motif as the primary carbohydrate neutralization determinant of HIV-1 and show that the genetic modulation of microbial polysaccharides is a route towards immunogens capable of eliciting antibody responses to the glycans of HIV-1. PMID:20181792

  6. A Human Monoclonal Antibody with Neutralizing Activity against Highly Divergent Influenza Subtypes

    PubMed Central

    Solforosi, Laura; Moreno, Guisella J.; Gubareva, Larisa V.; Mishin, Vasiliy; Di Pietro, Andrea; Vicenzi, Elisa; Siccardi, Antonio G.; Clementi, Massimo; Burioni, Roberto

    2011-01-01

    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. PMID:22162996

  7. HPV16/18 L1 VLP Vaccine Induces Cross-Neutralizing Antibodies that May Mediate Cross-Protection

    PubMed Central

    Kemp, Troy J.; Hildesheim, Allan; Safaeian, Mahboobeh; Dauner, Joseph G.; Pan, Yuanji; Porras, Carolina; Schiller, John T.; Lowy, Douglas R.; Herrero, Rolando; Pinto, Ligia A.

    2011-01-01

    Human papillomavirus (HPV) L1 VLP-based vaccines are protective against HPV vaccine-related types; however, the correlates of protection have not been defined. We observed that vaccination with Cervarix™ induced cross-neutralizing antibodies for HPV types for which evidence of vaccine efficacy has been demonstrated (HPV31/45) but not for other types (HPV52/58). In addition, HPV31/45 cross-neutralizing titers showed a significant increase with number of doses (HPV31, p<0.001; HPV45, p<0.001) and correlated with HPV16/18 neutralizing titers, respectively. These findings raise the possibility that cross-neutralizing antibodies are effectors of cross-protection observed for the HPV16/18 vaccine. PMID:21241731

  8. Autologous antibody response against the principal neutralizing domain of human immunodeficiency virus type 1 isolated from infected humans.

    PubMed Central

    Holmbäck, K; Kusk, P; Hulgaard, E F; Bugge, T H; Scheibel, E; Lindhardt, B O

    1993-01-01

    High titers of neutralizing antibodies in human immunodeficiency virus type 1 (HIV-1) infection are directed primarily against the third hypervariable domain (V3) of the virion envelope glycoprotein gp120. This region has been designated the principal neutralizing domain of HIV-1. Because the frequency and significance of autologous V3 antibodies in natural infection are not fully clarified, we have cloned, sequenced, and expressed the V3 domain from virus of HIV-1-infected patients to test the autologous and heterologous V3 antibody response. The resulting recombinant Escherichia coli V3 fusion proteins reacted strongly with both autologous and heterologous patient antibodies in Western blots. Thirty-one different V3 fragments were cloned from 24 hemophiliac patients with different immunological and clinical statuses. Antibody reactivity against the autologous V3 fusion proteins was detected in all serum samples except one; moreover, all serum samples contained antibody reactivity against a vast majority of heterologous fusion proteins despite significant amino acid variability in V3. The results suggest that V3 antibodies are highly prevalent; further, we find no association between the stage of the HIV-1 infection and the presence of V3 antibodies. Images PMID:8437232

  9. Development of a Triple-Color Pseudovirion-Based Assay to Detect Neutralizing Antibodies against Human Papillomavirus

    PubMed Central

    Nie, Jianhui; Liu, Yangyang; Huang, Weijin; Wang, Youchun

    2016-01-01

    Pseudovirion-based neutralization assay is considered the gold standard method for evaluating the immune response to human papillomavirus (HPV) vaccines. In this study, we developed a multicolor neutralization assay to simultaneously detect the neutralizing antibodies against different HPV types. FluoroSpot was used to interpret the fluorescent protein expression instead of flow cytometry. The results of FluoroSpot and flow cytometry showed good consistency, with R2 > 0.98 for the log-transformed IC50 values. Regardless of the reporter color, the single-, dual-, and triple-color neutralization assays reported identical results for the same samples. In low-titer samples from naturally HPV-infected individuals, there was strong agreement between the single- and triple-color assays, with kappa scores of 0.92, 0.89, and 0.96 for HPV16, HPV18, and HPV58, respectively. Good reproducibility was observed for the triple-color assay, with coefficients of variation of 2.0%–41.5% within the assays and 8.3%–36.2% between the assays. Three triple-color systems, HPV16-18-58, HPV6-33-45, and HPV11-31-52, were developed that could evaluate the immunogenicity of a nonavalent vaccine in three rounds of the assay. With the advantages of an easy-to-use procedure and less sample consumption, the multiple-color assay is more suitable than classical assays for large sero-epidemiological studies and clinical trials and is more amenable to automation. PMID:27120611

  10. Development of a Triple-Color Pseudovirion-Based Assay to Detect Neutralizing Antibodies against Human Papillomavirus.

    PubMed

    Nie, Jianhui; Liu, Yangyang; Huang, Weijin; Wang, Youchun

    2016-04-01

    Pseudovirion-based neutralization assay is considered the gold standard method for evaluating the immune response to human papillomavirus (HPV) vaccines. In this study, we developed a multicolor neutralization assay to simultaneously detect the neutralizing antibodies against different HPV types. FluoroSpot was used to interpret the fluorescent protein expression instead of flow cytometry. The results of FluoroSpot and flow cytometry showed good consistency, with R² > 0.98 for the log-transformed IC50 values. Regardless of the reporter color, the single-, dual-, and triple-color neutralization assays reported identical results for the same samples. In low-titer samples from naturally HPV-infected individuals, there was strong agreement between the single- and triple-color assays, with kappa scores of 0.92, 0.89, and 0.96 for HPV16, HPV18, and HPV58, respectively. Good reproducibility was observed for the triple-color assay, with coefficients of variation of 2.0%-41.5% within the assays and 8.3%-36.2% between the assays. Three triple-color systems, HPV16-18-58, HPV6-33-45, and HPV11-31-52, were developed that could evaluate the immunogenicity of a nonavalent vaccine in three rounds of the assay. With the advantages of an easy-to-use procedure and less sample consumption, the multiple-color assay is more suitable than classical assays for large sero-epidemiological studies and clinical trials and is more amenable to automation. PMID:27120611

  11. Structure of a Major Antigenic Site on the Respiratory Syncytial Virus Fusion Glycoprotein in Complex with Neutralizing Antibody 101F

    SciTech Connect

    McLellan, Jason S.; Chen, Man; Chang, Jung-San; Yang, Yongping; Kim, Albert; Graham, Barney S.; Kwong, Peter D.

    2010-11-19

    Respiratory syncytial virus (RSV) is a major cause of pneumonia and bronchiolitis in infants and elderly people. Currently there is no effective vaccine against RSV, but passive prophylaxis with neutralizing antibodies reduces hospitalizations. To investigate the mechanism of antibody-mediated RSV neutralization, we undertook structure-function studies of monoclonal antibody 101F, which binds a linear epitope in the RSV fusion glycoprotein. Crystal structures of the 101F antigen-binding fragment in complex with peptides from the fusion glycoprotein defined both the extent of the linear epitope and the interactions of residues that are mutated in antibody escape variants. The structure allowed for modeling of 101F in complex with trimers of the fusion glycoprotein, and the resulting models suggested that 101F may contact additional surfaces located outside the linear epitope. This hypothesis was supported by surface plasmon resonance experiments that demonstrated 101F bound the peptide epitope {approx}16,000-fold more weakly than the fusion glycoprotein. The modeling also showed no substantial clashes between 101F and the fusion glycoprotein in either the pre- or postfusion state, and cell-based assays indicated that 101F neutralization was not associated with blocking virus attachment. Collectively, these results provide a structural basis for RSV neutralization by antibodies that target a major antigenic site on the fusion glycoprotein.

  12. Enveloped Virus-Like Particle Expression of Human Cytomegalovirus Glycoprotein B Antigen Induces Antibodies with Potent and Broad Neutralizing Activity

    PubMed Central

    Kirchmeier, Marc; Fluckiger, Anne-Catherine; Soare, Catalina; Bozic, Jasminka; Ontsouka, Barthelemy; Ahmed, Tanvir; Diress, Abebaw; Pereira, Lenore; Schödel, Florian; Plotkin, Stanley; Dalba, Charlotte; Klatzmann, David

    2014-01-01

    A prophylactic vaccine to prevent the congenital transmission of human cytomegalovirus (HCMV) in newborns and to reduce life-threatening disease in immunosuppressed recipients of HCMV-infected solid organ transplants is highly desirable. Neutralizing antibodies against HCMV confer significant protection against infection, and glycoprotein B (gB) is a major target of such neutralizing antibodies. However, one shortcoming of past HCMV vaccines may have been their failure to induce high-titer persistent neutralizing antibody responses that prevent the infection of epithelial cells. We used enveloped virus-like particles (eVLPs), in which particles were produced in cells after the expression of murine leukemia virus (MLV) viral matrix protein Gag, to express either full-length CMV gB (gB eVLPs) or the full extracellular domain of CMV gB fused with the transmembrane and cytoplasmic domains from vesicular stomatitis virus (VSV)-G protein (gB-G eVLPs). gB-G-expressing eVLPs induced potent neutralizing antibodies in mice with a much greater propensity toward epithelial cell-neutralizing activity than that induced with soluble recombinant gB protein. An analysis of gB antibody binding titers and T-helper cell responses demonstrated that high neutralizing antibody titers were not simply due to enhanced immunogenicity of the gB-G eVLPs. The cells transiently transfected with gB-G but not gB plasmid formed syncytia, consistent with a prefusion gB conformation like those of infected cells and viral particles. Two of the five gB-G eVLP-induced monoclonal antibodies we examined in detail had neutralizing activities, one of which possessed particularly potent epithelial cell-neutralizing activity. These data differentiate gB-G eVLPs from gB antigens used in the past and support their use in a CMV vaccine candidate with improved neutralizing activity against epithelial cell infection. PMID:24334684

  13. Enveloped virus-like particle expression of human cytomegalovirus glycoprotein B antigen induces antibodies with potent and broad neutralizing activity.

    PubMed

    Kirchmeier, Marc; Fluckiger, Anne-Catherine; Soare, Catalina; Bozic, Jasminka; Ontsouka, Barthelemy; Ahmed, Tanvir; Diress, Abebaw; Pereira, Lenore; Schödel, Florian; Plotkin, Stanley; Dalba, Charlotte; Klatzmann, David; Anderson, David E

    2014-02-01

    A prophylactic vaccine to prevent the congenital transmission of human cytomegalovirus (HCMV) in newborns and to reduce life-threatening disease in immunosuppressed recipients of HCMV-infected solid organ transplants is highly desirable. Neutralizing antibodies against HCMV confer significant protection against infection, and glycoprotein B (gB) is a major target of such neutralizing antibodies. However, one shortcoming of past HCMV vaccines may have been their failure to induce high-titer persistent neutralizing antibody responses that prevent the infection of epithelial cells. We used enveloped virus-like particles (eVLPs), in which particles were produced in cells after the expression of murine leukemia virus (MLV) viral matrix protein Gag, to express either full-length CMV gB (gB eVLPs) or the full extracellular domain of CMV gB fused with the transmembrane and cytoplasmic domains from vesicular stomatitis virus (VSV)-G protein (gB-G eVLPs). gB-G-expressing eVLPs induced potent neutralizing antibodies in mice with a much greater propensity toward epithelial cell-neutralizing activity than that induced with soluble recombinant gB protein. An analysis of gB antibody binding titers and T-helper cell responses demonstrated that high neutralizing antibody titers were not simply due to enhanced immunogenicity of the gB-G eVLPs. The cells transiently transfected with gB-G but not gB plasmid formed syncytia, consistent with a prefusion gB conformation like those of infected cells and viral particles. Two of the five gB-G eVLP-induced monoclonal antibodies we examined in detail had neutralizing activities, one of which possessed particularly potent epithelial cell-neutralizing activity. These data differentiate gB-G eVLPs from gB antigens used in the past and support their use in a CMV vaccine candidate with improved neutralizing activity against epithelial cell infection. PMID:24334684

  14. Human domain antibodies to conserved sterically restricted regions on gp120 as exceptionally potent cross-reactive HIV-1 neutralizers

    PubMed Central

    Chen, Weizao; Zhu, Zhongyu; Feng, Yang; Dimitrov, Dimiter S.

    2008-01-01

    The antibody access to some conserved structures on the HIV-1 envelope glycoprotein (Env) is sterically restricted. We have hypothesized that the smallest independently folded antibody fragments (domains) could exhibit exceptionally potent and broadly cross-reactive neutralizing activity by targeting hidden conserved epitopes that are not accessible by larger antibodies. To test this hypothesis, we constructed a large (size 2.5 × 1010), highly diversified library of human antibody variable domains (domain antibodies) and used it for selection of binders to conserved Env structures by panning sequentially against Envs from different isolates. The highest affinity binder, m36, neutralized all tested HIV-1 isolates from clades A– D with an activity on average higher than that of C34, a peptide similar to the fusion inhibitor T20, which is in clinical use, and that of m9, which exhibits a neutralizing activity superior to known potent cross-reactive antibodies. Large-size fusion proteins of m36 exhibited diminished neutralizing activity but preincubation of virions with soluble CD4 restored it, suggesting that m36 epitope is sterically restricted and induced by CD4 (CD4i). M36 bound to gp120-CD4 complexes better than to gp120 alone and competed with CD4i antibodies. M36 is the only reported representative of a promising class of potent, broadly cross-reactive HIV-1 inhibitors based on human domain antibodies. It has potential for prevention and therapy and as an agent for exploration of the closely guarded conserved Env structures with implications for design of small molecule inhibitors and elucidation of mechanisms of virus entry and evasion of immune responses. PMID:18957538

  15. TFTR neutral-beam power system

    SciTech Connect

    Winje, R.A.

    1982-10-01

    The TFTR Neutral Beam Power System (NBPS) consists of the accelerator grid power supply and the auxiliary power supplies required to operate the TFTR 120-keV ion sources. The current configuration of the NBPS including the 11-MVA accelerator grid power supply and the Arc and Filament power supplies isolated for operation at accelerator grid voltages up to 120 kV, is described. The prototype NBPS has been assembled at the Princeton Plasma Physics Laboratory and has been operated. The results of the initial operation and the description and resolution of some of the technical problems encountered during the commissioning tests are presented.

  16. Neutralization of venom-induced hemorrhage by equine antibodies raised by immunization with a plasmid encoding a novel P-II metalloproteinase from the lancehead pitviper Bothrops asper.

    PubMed

    Arce-Estrada, Viviana; Azofeifa-Cordero, Gabriela; Estrada, Ricardo; Alape-Girón, Alberto; Flores-Díaz, Marietta

    2009-01-14

    In this work, the cDNA encoding a novel P-II type metalloproteinase from Bothrops asper venom glands was cloned, sequenced and used for DNA immunization of animals with accelerated DNA-coated tungsten microparticles and the helius Gene Gun system. Specific antibodies against B. asper venom antigens were induced in mice co-immunized with the plasmid encoding the P-II metalloproteinase together with an expression plasmid encoding the murine IL-2. Similarly, specific antibodies against B. asper venom antigens were also induced in a horse co-immunized with the plasmid encoding the P-II metalloproteinase, together with a plasmid encoding the equine IL-6. The equine antibodies induced by immunization with the P-II metalloproteinase encoding plasmid cross react with several proteins of B. asper, Crotalus durissus durissus, and Lachesis stenophrys venoms in western blot, demonstrating antigenic similarity between the cloned metalloproteinase and other metalloproteinases present in these venoms. Furthermore, the equine antibodies induced by immunization with the P-II metalloproteinase encoding plasmid completely neutralized the hemorrhagic activity of the whole B. asper venom and partially the hemorrhagic activity of C. durissus durissus venom. The neutralizing ability of the produced antibodies raises, for the first time, the possibility of developing therapeutic antivenoms in horses by DNA immunization using tungsten microparticles. PMID:19013207

  17. In vivo neutralization of α-cobratoxin with high-affinity llama single-domain antibodies (VHHs) and a VHH-Fc antibody.

    PubMed

    Richard, Gabrielle; Meyers, Ashley J; McLean, Michael D; Arbabi-Ghahroudi, Mehdi; MacKenzie, Roger; Hall, J Christopher

    2013-01-01

    Small recombinant antibody fragments (e.g. scFvs and VHHs), which are highly tissue permeable, are being investigated for antivenom production as conventional antivenoms consisting of IgG or F(ab')2 antibody fragments do not effectively neutralize venom toxins located in deep tissues. However, antivenoms composed entirely of small antibody fragments may have poor therapeutic efficacy due to their short serum half-lives. To increase serum persistence and maintain tissue penetration, we prepared low and high molecular mass antivenom antibodies. Four llama VHHs were isolated from an immune VHH-displayed phage library and were shown to have high affinity, in the low nM range, for α-cobratoxin (α-Cbtx), the most lethal component of Naja kaouthia venom. Subsequently, our highest affinity VHH (C2) was fused to a human Fc fragment to create a VHH2-Fc antibody that would offer prolonged serum persistence. After in planta (Nicotiana benthamiana) expression and purification, we show that our VHH2-Fc antibody retained high affinity binding to α-Cbtx. Mouse α-Cbtx challenge studies showed that our highest affinity VHHs (C2 and C20) and the VHH2-Fc antibody effectively neutralized lethality induced by α-Cbtx at an antibody:toxin molar ratio as low as ca. 0.75×:1. Further research towards the development of an antivenom therapeutic involving these anti-α-Cbtx VHHs and VHH2-Fc antibody molecules should involve testing them as a combination, to determine whether they maintain tissue penetration capability and low immunogenicity, and whether they exhibit improved serum persistence and therapeutic efficacy. PMID:23894495

  18. In Vivo Neutralization of α-Cobratoxin with High-Affinity Llama Single-Domain Antibodies (VHHs) and a VHH-Fc Antibody

    PubMed Central

    Richard, Gabrielle; Meyers, Ashley J.; McLean, Michael D.; Arbabi-Ghahroudi, Mehdi; MacKenzie, Roger; Hall, J. Christopher

    2013-01-01

    Small recombinant antibody fragments (e.g. scFvs and VHHs), which are highly tissue permeable, are being investigated for antivenom production as conventional antivenoms consisting of IgG or F(ab’)2 antibody fragments do not effectively neutralize venom toxins located in deep tissues. However, antivenoms composed entirely of small antibody fragments may have poor therapeutic efficacy due to their short serum half-lives. To increase serum persistence and maintain tissue penetration, we prepared low and high molecular mass antivenom antibodies. Four llama VHHs were isolated from an immune VHH-displayed phage library and were shown to have high affinity, in the low nM range, for α-cobratoxin (α–Cbtx), the most lethal component of Naja kaouthia venom. Subsequently, our highest affinity VHH (C2) was fused to a human Fc fragment to create a VHH2-Fc antibody that would offer prolonged serum persistence. After in planta (Nicotiana benthamiana) expression and purification, we show that our VHH2-Fc antibody retained high affinity binding to α–Cbtx. Mouse α–Cbtx challenge studies showed that our highest affinity VHHs (C2 and C20) and the VHH2-Fc antibody effectively neutralized lethality induced by α–Cbtx at an antibody:toxin molar ratio as low as ca. 0.75×:1. Further research towards the development of an antivenom therapeutic involving these anti-α-Cbtx VHHs and VHH2-Fc antibody molecules should involve testing them as a combination, to determine whether they maintain tissue penetration capability and low immunogenicity, and whether they exhibit improved serum persistence and therapeutic efficacy. PMID:23894495

  19. Mechanism for neutralizing activity by the anti-CMV gH/gL monoclonal antibody MSL-109

    PubMed Central

    Fouts, Ashley E.; Comps-Agrar, Laëtitia; Stengel, Katharina F.; Ellerman, Diego; Schoeffler, Allyn J.; Warming, Søren; Eaton, Dan L.; Feierbach, Becket

    2014-01-01

    Cytomegalovirus (CMV) is a widespread opportunistic pathogen that causes birth defects when transmitted transplacentally and severe systemic illness in immunocompromised individuals. MSL-109, a human monoclonal IgG isolated from a CMV seropositive individual, binds to the essential CMV entry glycoprotein H (gH) and prevents infection of cells. Here, we suggest a mechanism for neutralization activity by MSL-109. We define a genetic basis for resistance to MSL-109 and have generated a structural model of gH that reveals the epitope of this neutralizing antibody. Using surface-based, time-resolved FRET, we demonstrate that gH/gL interacts with glycoprotein B (gB). Additionally, we detect homodimers of soluble gH/gL heterodimers and confirm this novel oligomeric assembly on full-length gH/gL expressed on the cell surface. We show that MSL-109 perturbs the dimerization of gH/gL:gH/gL, suggesting that dimerization of gH/gL may be required for infectivity. gH/gL homodimerization may be conserved between alpha- and betaherpesviruses, because both CMV and HSV gH/gL demonstrate self-association in the FRET system. This study provides evidence for a novel mechanism of action for MSL-109 and reveals a previously undescribed aspect of viral entry that may be susceptible to therapeutic intervention. PMID:24843144

  20. Neutralization and Binding Profile of Monoclonal Antibodies Generated Against Influenza A H1N1 Viruses.

    PubMed

    Shembekar, Nachiket; Mallajosyula, Vamsee V Aditya; Malik, Ankita; Saini, Ashok; Varadarajan, Raghavan; Gupta, Satish Kumar

    2016-08-01

    Monoclonal antibodies (MAbs) provide scope for the development of better therapeutics and diagnostic tools. Herein, we describe the binding and neutralization profile(s) for a panel of murine MAbs generated against influenza A H1N1 viruses elicited by immunization with pandemic H1 recombinant hemagglutinin (rHA)/whole virus or seasonal H1 rHA. Neutralizing MAbs, MA-2070 and MA-M, were obtained after pandemic A/California/07/2009 (H1N1) virus/rHA immunization(s). Both MAbs reacted specifically with rHA from A/California/07/2009 and A/England/195/2009 in ELISA. MA-2070 bound rHA of A/California/07/2009 with high affinity (KD = 51.36 ± 9.20 nM) and exhibited potent in vitro neutralization (IC50 = 2.50 μg/mL). MA-2070 bound within the stem domain of HA. MA-M exhibited both hemagglutination inhibition (HI, 1.50 μg/mL) and in vitro neutralization (IC50 = 0.66 μg/mL) activity against the pandemic A/California/07/2009 virus and showed higher binding affinity (KD = 9.80 ± 0.67 nM) than MA-2070. MAb, MA-H generated against the seasonal A/Solomon Islands/03/2006 (H1N1) rHA binds within the head domain and bound the seasonal H1N1 (A/Solomon Islands/03/2006 and A/New Caledonia/20/1990) rHAs with high affinity (KD; 0.72-8.23 nM). MA-H showed high HI (2.50 μg/mL) and in vitro neutralization (IC50 = 2.61 μg/mL) activity against the A/Solomon Islands/03/2006 virus. All 3 MAbs failed to react in ELISA with rHA from various strains of H2N2, H3N2, H5N1, H7N9, and influenza virus B, suggesting their specificity for either pandemic or seasonal H1N1 influenza virus. The MAbs reported here may be useful in developing diagnostic assays. PMID:27463230

  1. Neutralizing antibodies against feline herpesvirus type 1 in captive wild felids of Brazil.

    PubMed

    Ruthner Batista, Helena Beatriz de Carvalho; Kindlein Vicentini, Franco; Franco, Ana Cláudia; Rosado Spilki, Fernando; Ramos Silva, Jean Carlos; Adania, Cristina Harumi; Roehe, Paulo Michel

    2005-09-01

    Feline herpesvirus type 1 infection affects domestic cats, causing mainly upper respiratory tract diseases. Although this infection has been described in captive and free-ranging wild felids from Europe, Asia, North America, and Africa, no information is available on its occurrence among wild felids of Brazil. In this study, 250 serum samples of six species of Brazilian captive wild felids (Leopardus tigrinus, Leopardus wiedii, Herpailurus yaguarondi, Puma concolor, Leopardus pardalis, and Panthera onca) were examined for neutralizing antibodies to feline herpesvirus type 1. Positive sera were found in 72% of L. tigrinus samples, 15% of L. wiedii, 6% of L. pardalis, 8% of H. yaguarondi, 18% of P. concolor, and 14% of P. onca. The relatively low percentages of seropositivity and low antibody titers found among the last five species suggest that feline herpesvirus type 1 does not circulate extensively among these animals. Nevertheless, quarantine, serologic screening, and vaccination of newly introduced felids is recommended in zoos in order to prevent virus transmission and outbreaks of the disease among wild felids kept in captivity. PMID:17312763

  2. A Novel Chimeric Anti-PA Neutralizing Antibody for Postexposure Prophylaxis and Treatment of Anthrax

    PubMed Central

    Xiong, Siping; Tang, Qi; Liang, Xudong; Zhou, Tingting; Yang, Jin; Liu, Peng; Chen, Ya; Wang, Changjun; Feng, Zhenqing; Zhu, Jin

    2015-01-01

    Anthrax is a highly lethal infectious disease caused by the bacterium Bacillus anthracis, and the associated shock is closely related to the lethal toxin (LeTx) produced by the bacterium. The central role played by the 63 kDa protective antigen (PA63) region of LeTx in the pathophysiology of anthrax makes it an excellent therapeutic target. In the present study, a human/murine chimeric IgG mAb, hmPA6, was developed by inserting murine antibody variable regions into human constant regions using antibody engineering technology. hmPA6 expressed in 293F cells could neutralize LeTx both in vitro and in vivo. At a dose of 0.3 mg/kg, it could protect all tested rats from a lethal dose of LeTx. Even administration of 0.6 mg/kg hmPA6 48 h before LeTx challenge protected all tested rats. The results indicate that hmPA6 is a potential candidate for clinical application in anthrax treatment. PMID:26134518

  3. Effects of homologous and heterologous antiserum on neutralizing-antibody response to rabies vaccine*

    PubMed Central

    Archer, B. G.; Dierks, R. E.

    1968-01-01

    Heterologous antirabies serum is commonly used in the treatment of persons exposed to rabies. However, the high incidence of serum sickness which accompanies its use has prompted work to develop a homologous human product. As human antirabies serum is expensive and difficult to obtain in large quantities, a series of experiments was done on guinea-pigs to test the effects of homologous and heterologous antirabies serum. Similar amounts of homologous and heterologous antisera administered to guinea-pigs produced similar circulating neutralization titres one day later. The homologous antibody titres, however, decreased more slowly than the heterologous antibody titres. When homologous antiserum was given, followed by duck-embryo rabies vaccine, an apparent response to the vaccine was suppressed or delayed longer than when heterologous antiserum and vaccine were administered. However, when homologous antiserum was given with suckling-mouse-brain vaccine, of a much higher potency, the response to vaccine was apparent in the presence of a passive titre of 1:120. If a similar relationship is seen in man with the use of a homologous antirabies product, it will be essential to use high potency vaccines or alter the established vaccination schedules in order to overcome the inherent interference problems. PMID:5303907

  4. Boosting of HIV-1 Neutralizing Antibody Responses by a Distally Related Retroviral Envelope Protein

    PubMed Central

    Uchtenhagen, Hannes; Schiffner, Torben; Bowles, Emma; Heyndrickx, Leo; LaBranche, Celia; Applequist, Steven E.; Jansson, Marianne; De Silva, Thushan; Back, Jaap Willem; Achour, Adnane; Scarlatti, Gabriella; Fomsgaard, Anders; Montefiori, David; Stewart-Jones, Guillaume; Spetz, Anna-Lena

    2014-01-01

    Our knowledge of the binding sites for neutralizing antibodies (NAbs) 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 glycoprotein (Env). Here we report an immunization strategy composed of a trivalent HIV-1 (clade B envs) DNA prime, followed by a SIVmac239 gp140 Env protein boost that aimed to focus the immune response to structurally conserved parts of the HIV-1 and SIV Envs. Heterologous NAb titres, primarily to tier 1 HIV-1 isolates, elicited during the trivalent HIV-1 env prime, were significantly increased by the SIVmac239 gp140 protein boost in rabbits. Epitope mapping of antibody binding reactivity revealed preferential recognition of the C1, C2, V2, V3 and V5 regions. These results provide a proof of concept that a distally related retroviral SIV Env protein boost can increase pre-existing NAb responses against HIV-1. PMID:24829409

  5. Recombinant bovine herpesvirus-1 expressing p23 protein of Cryptosporidium parvum induces neutralizing antibodies in rabbits.

    PubMed

    Takashima, Yasuhiro; Xuan, Xuenan; Kimata, Isao; Iseki, Motohiro; Kodama, Yoshikatsu; Nagane, Noriko; Nagasawa, Hideyuki; Matsumoto, Yasunobu; Mikami, Takeshi; Otsuka, Haruki

    2003-04-01

    In order to develop a vaccine against cryptosporidiosis in cattle, we constructed a recombinant bovine herpesvirus-1 (BHV-1) expressing an immunodominant surface protein, p23, of Cryptosporidium parvum sporozoites. In the recombinant virus, the p23 gene under the control of a CAG promoter and a gene coding for an enhanced green fluorescent protein were integrated into the gG gene of BHV-1. Despite a low frequency of homologous recombination, cloning of the recombinants was easy because of the specific fluorescence of the plaques formed by recombinants. These plaques were among the plaques of the nonfluorescent parental virus. All clones selected for fluorescence also contained the p23 gene. In MDBK cells infected with the recombinant BHV-1, the antibody against the p23 protein recognized the p23 protein as an approximately 23-kDa specific band in Western blotting analysis. Rabbits immunized with the recombinant produced IgG against the p23 protein. It was also demonstrated that the sera of immunized rabbits reduced infection of C. parvum sporozoites in HCT-8 cells. The serum of an immunized rabbit reduced infection compared with the normal rabbit serum control. These results indicate that the recombinant BHV-1 induces neutralizing antibodies in rabbits. PMID:12760641

  6. Anti-Echis carinatus venom antibodies from chicken egg yolk: isolation, purification and neutralization efficacy.

    PubMed

    Paul, K; Manjula, J; Deepa, E P; Selvanayagam, Z E; Ganesh, K A; Subba Rao, P V

    2007-12-01

    High titer antibodies (IgY) were raised in egg yolk of white leghorn chicken (Gallus gallus domesticus) by immunizing with the venom of Echis carinatus (Saw scaled viper or carpet viper), an Indian venomous snake belonging to the family Viperidae. The anti-snake venom antibodies (antivenom) were isolated from egg yolk by the water dilution method, enriched by 19% sodium sulfate precipitation and purified by immunoaffinity chromatography. A single, electrophoretically pure IgY band of 180-200 kDa was obtained on SDS-PAGE. Immunoblot analysis revealed not only the specific binding of the antivenom but also dose-dependent blocking of antivenom by venom proteins. In neutralization studies, a preincubated mixture of both affinity-purified (50 mg/kg body weight) as well as partially purified (210 mg/kg body weight) anti-E. carinatus IgY with 2 LD(50) dose of E. carinatus venom (2 x 6.65 mg/kg body weight) gave 100% protection in mice when administered subcutaneously. PMID:17681579

  7. Inhibition of metastasis of rhabdomyosarcoma by a novel neutralizing antibody to CXC chemokine receptor-4

    PubMed Central

    Kashima, Kenji; Watanabe, Miho; Sato, Yasuko; Hata, Junichi; Ishii, Nobuya; Aoki, Yuko

    2014-01-01

    Rhabdomyosarcoma is the most common soft tissue sarcoma affecting children, and the overall cure rate of children with metastatic disease remains below 30%. The CXC chemokine receptor-4 (CXCR4)/stromal cell-derived factor-1 (SDF1) axis has been implicated in the promotion of metastatic potential in several tumors. In this study, we developed a novel anti-CXCR4 mAb, CF172, and investigated its antimetastatic activity against rhabdomyosarcoma cells in vitro and in vivo, to evaluate its potential as a therapeutic antibody to treat rhabdomyosarcoma. The CF172 molecule showed a specific binding reactivity against human CXCR4, as well as a specific neutralizing activity against CXCR4/SDF1 signal transduction. Using CF172, we determined that SJCRH30 rhabdomyosarcoma cells expressed high levels of CXCR4. In addition, CF172 was found to inhibit the SDF1-induced migration activity of SJCRH30 cells in vitro. Using xenograft models of SJCRH30 cells, we carried out in vivo efficacy studies for peritoneal and lymph node metastasis, which were clinically observed in rhabdomyosarcoma. These studies indicated that CF172 significantly decreased both types of metastasis of SJCRH30. In conclusion, we found that a novel anti-CXCR4 mAb, CF172, with specific reactivity against human CXCR4, prevented peritoneal metastasis and lymph node metastasis of rhabdomyosarcoma in animal models. These results suggest that CF172 is a potential antimetastasis therapeutic antibody for rhabdomyosarcoma treatment. PMID:25154453

  8. Structure of adeno-associated virus-2 in complex with neutralizing monoclonal antibody A20

    SciTech Connect

    McCraw, Dustin M.; O'Donnell, Jason K.; Taylor, Kenneth A.; Stagg, Scott M.; Chapman, Michael S.

    2012-09-15

    The use of adeno-associated virus (AAV) as a gene therapy vector is limited by the host neutralizing immune response. The cryo-electron microscopy (EM) structure at 8.5 A resolution is determined for a complex of AAV-2 with the Fab' fragment of monoclonal antibody (MAb) A20, the most extensively characterized AAV MAb. The binding footprint is determined through fitting the cryo-EM reconstruction with a homology model following sequencing of the variable domain, and provides a structural basis for integrating diverse prior epitope mappings. The footprint extends from the previously implicated plateau to the side of the spike, and into the conserved canyon, covering a larger area than anticipated. Comparison with structures of binding and non-binding serotypes indicates that recognition depends on a combination of subtle serotype-specific features. Separation of the neutralizing epitope from the heparan sulfate cell attachment site encourages attempts to develop immune-resistant vectors that can still bind to target cells.

  9. AAV Natural Infection Induces Broad Cross-Neutralizing Antibody Responses to Multiple AAV Serotypes in Chimpanzees.

    PubMed

    Calcedo, Roberto; Wilson, James M

    2016-06-01

    Cross-sectional studies of primates have revealed that natural neutralizing antibody (NAb) responses to adeno-associated viruses (AAV) span multiple serotypes. This differs from the phenotype of the NAb response to an AAV vector delivered to seronegative nonhuman primates that is typically restricted to the administered AAV serotype. To better understand the mechanism by which natural AAV infections result in broad NAb responses, we conducted a longitudinal study spanning 10 years in which we evaluated serum-circulating AAV NAb levels in captive-housed chimpanzees. In a cohort of 25 chimpanzees we identified 3 distinct groups of animals: those that never seroconverted to AAV (naïve), those that were persistently seropositive (chronic), and those that seroconverted during the 10-year period (acute). For the chronic group we found a broad seroresponse characterized by NAbs reacting to multiple AAV serotypes. A similar cross-neutralization pattern of NAbs was observed in the acute group. These data support our hypothesis that a single natural infection with AAV induces a broadly cross-reactive NAb response to multiple AAV serotypes. PMID:27314914

  10. Prediction of optimal peptide mixtures to induce broadly neutralizing antibodies to human immunodeficiency virus type 1.

    PubMed Central

    Holley, L H; Goudsmit, J; Karplus, M

    1991-01-01

    Sequences of the principal neutralizing determinant (PND) of the external envelope protein, gp120, from 245 isolates of human immunodeficiency virus type 1 are analyzed. The minimal set of peptides that would elicit antibodies to neutralize a majority of U.S. and European isolates of human immunodeficiency virus type 1 is determined with the assumption that peptides of a given length including the central Gly-Pro-Gly triad are required. In spite of the hypervariability of the PND, 90% of these 245 sequences include peptides from a set of 7 pentapeptides, 13 hexapeptides, or 17 heptapeptides. Tests of these peptide sets on 78 additional PND sequences show that 95% are covered by the 7 pentapeptides, 94% by the 13 hexapeptides, and 86% by the 17 heptapeptides. To anticipate variants not yet observed, single amino acid mutation frequencies from the 245 isolates are used to calculate an expanded set of the 10,000 most probable PND sequences. These sequences cover 86% of the total distribution expected for the central portion of the PND. Peptide lists derived from this expanded set when tested on the 78 additional sequences show that 7 pentapeptides cover 95%, 13 hexapeptides cover 94%, and 17 heptapeptides cover 94%. These results suggest that peptide cocktails of limited size with the potential to cover a large fraction of PND sequence variation may be feasible vaccine candidates. PMID:1862103

  11. HIV-1 gp41 Core with Exposed Membrane-Proximal External Region Inducing Broad HIV-1 Neutralizing Antibodies

    PubMed Central

    Zhou, Leilei; Xu, Liling; Jiang, Shibo; Chen, Ying-hua

    2011-01-01

    The membrane-proximal external region (MPER) of the HIV-1 gp41 consists of epitopes for the broadly cross-neutralizing monoclonal antibodies 2F5 and 4E10. However, antigens containing the linear sequence of these epitopes are unable to elicit potent and broad neutralizing antibody responses in vaccinated hosts, possibly because of inappropriate conformation of these epitopes. Here we designed a recombinant antigen, designated NCM, which comprises the N- and C-terminal heptad repeats that can form a six-helix bundle (6HB) core and the MPER domain of gp41. Two mutations (T569A and I675V) previously reported to expose the neutralization epitopes were introduced into NCM to generate mutants named NCM(TA), NCM(IV), and NCM(TAIV). Our results showed that NCM and its mutants could react with antibodies specific for 6HB and MPER of gp41, suggesting that these antigens are in the form of a trimer of heterodimer (i.e., 6HB) with three exposed MPER tails. Antigen with double mutations, NCM(TAIV), elicited much stronger antibody response in rabbits than immunogens with single mutation, NCM(TA) and NCM(IV), or no mutation, NCM. The purified MPER-specific antibodies induced by NCM(TAIV) exhibited broad neutralizing activity, while the purified 6HB-specific antibodies showed no detectable neutralizing activity. Our recombinant antigen design supported by an investigation of its underlying molecular mechanisms provides a strong scientific platform for the discovery of a gp41 MPER-based AIDS vaccine. PMID:21483871

  12. Trimeric Glycosylphosphatidylinositol-Anchored HCDR3 of Broadly Neutralizing Antibody PG16 Is a Potent HIV-1 Entry Inhibitor

    PubMed Central

    Liu, Lihong; Wang, Weiming; Yang, Lifei; Ren, Huanhuan; Kimata, Jason T.

    2013-01-01

    PG9 and PG16 are two quaternary-structure-specific broadly neutralizing antibodies with unique HCDR3 subdomains. Previously, we showed that glycosylphosphatidylinositol (GPI)-anchored HCDR3 subdomains (GPI-HCDR3) can be targeted to lipid rafts of the plasma membrane, bind to the epitope recognized by HCDR3 of PG16, and neutralize diverse HIV-1 isolates. In this study, we further developed trimeric GPI-HCDR3s and demonstrated that trimeric GPI-HCDR3 (PG16) dramatically improves anti-HIV-1 neutralization, suggesting that a stoichiometry of recognition of 3 or 2 HCDR3 molecules (PG16) to 1 viral spike is possible. PMID:23152526

  13. Isolation of a human-like antibody fragment (scFv) that neutralizes ricin biological activity

    PubMed Central

    Pelat, Thibaut; Hust, Michael; Hale, Martha; Lefranc, Marie-Paule; Dübel, Stefan; Thullier, Philippe

    2009-01-01

    Background Ricin is a lethal toxin that inhibits protein synthesis. It is easily extracted from a ubiquitously grown plant, Ricinus communis, and thus readily available for use as a bioweapon (BW). Anti-ricin antibodies provide the only known therapeutic against ricin intoxication. Results In this study, after immunizing a non-human primate (Macaca fascicularis) with the ricin chain A (RTA), a phage-displayed immune library was built (2 × 108 clones), that included the λ light chain fragment. The library was screened against ricin, and specific binders were sequenced and further analyzed. The best clone, 43RCA, was isolated using a new, stringent neutralization test. 43RCA had a high, picomolar affinity (41 pM) and neutralized ricin efficiently (IC50 = 23 ± 3 ng/ml, corresponding to a [scFv]/[ricin] molar ratio of 4). The neutralization capacity of 43RCA compared favourably with that of polyclonal anti-deglycosylated A chain (anti-dgRCA) IgGs, obtained from hyperimmune mouse serum, which were more efficient than any monoclonal at our disposal. The 43RCA sequence is very similar to that for human IgG germline genes, with 162 of 180 identical amino acids for the VH and VL (90% sequence identity). Conclusion Results of the characterization studies, and the high degree of identity with human germline genes, altogether make this anti-ricin scFv, or an IgG derived from it, a likely candidate for use in humans to minimize effects caused by ricin intoxication. PMID:19563687

  14. Structural basis for the neutralization of hepatitis E virus by a cross-genotype antibody

    PubMed Central

    Gu, Ying; Tang, Xuhua; Zhang, Xiao; Song, Cuiling; Zheng, Minghua; Wang, Kaihang; Zhang, Jun; Ng, Mun-Hon; Hew, Choy-Leong; Li, Shaowei; Xia, Ningshao; Sivaraman, J.

    2015-01-01

    Hepatitis E virus (HEV), a non-enveloped, positive-sense, single-stranded RNA virus, is a major cause of enteric hepatitis. Classified into the family Hepeviridae, HEV comprises four genotypes (genotypes 1-4), which belong to a single serotype. We describe a monoclonal antibody (mAb), 8G12, which equally recognizes all four genotypes of HEV, with ∼2.53–3.45 nM binding affinity. The mAb 8G12 has a protective, neutralizing capacity, which can significantly block virus infection in host cells. Animal studies with genotypes 1, 3 and 4 confirmed the cross-genotype neutralizing capacity of 8G12 and its effective prevention of hepatitis E disease. The complex crystal structures of 8G12 with the HEV E2s domain (the most protruded region of the virus capsid) of the abundant genotypes 1 and 4 were determined at 4.0 and 2.3 Å resolution, respectively. These structures revealed that 8G12 recognizes both genotypes through the epitopes in the E2s dimerization region. Structure-based mutagenesis and cell-model assays with virus-like particles identified several conserved residues (Glu549, Lys554 and Gly591) that are essential for 8G12 neutralization. Moreover, the epitope of 8G12 is identified as a key epitope involved in virus-host interactions. These findings will help develop a common strategy for the prevention of the most abundant form of HEV infection. PMID:25793314

  15. Development of an Enzyme-Linked Immunosorbent Spot Assay To Measure Serum-Neutralizing Antibodies against Coxsackievirus B3

    PubMed Central

    Yang, Lisheng; He, Delei; Tang, Min; Li, Zhiqun; Liu, Che; Xu, Longfa; Chen, Yixin; Du, Hailian; Zhao, Qinjian; Zhang, Jun; Xia, Ningshao

    2014-01-01

    Coxsackievirus B3 (CVB3) is the most common pathogen that induces acute and chronic viral myocarditis in children. The cytopathic effect (CPE)-based neutralization test (Nt-CPE) and the plaque reduction neutralization test (PRNT) are the most common methods for measuring neutralizing antibody titers against CVB3 in blood serum samples. However, these two methods are inefficient for CVB3 vaccine clinical trials, which require the testing of a large number of serum specimens. In this study, we developed an efficient neutralization test based on the enzyme-linked immunospot (Nt-ELISPOT) assay for measuring CVB3-neutralizing antibodies. This modified ELISPOT assay was based on the use of a monoclonal antibody against the viral capsid protein VP1 to detect the cells that are infected with CVB3, which, after immunoperoxidase staining, are counted as spots using an automated ELISPOT analyzer. Using the modified ELISPOT assay, we characterized the infection kinetics of CVB3 and divided the infection process of CVB3 on a cluster of cells into four phases. The stability of the Nt-ELISPOT was then evaluated. We found that over a wide range of infectious doses (102 to 106.5× 50% tissue culture infectious dose [TCID50] per well), the neutralizing titers of the sera were steady as long as they were tested during the log phase or the first half of the stationary phase of growth of the spots. We successfully shortened the testing period from 7 days to approximately 20 h. We also found that there was a good correlation (R2 = 0.9462) between the Nt-ELISPOT and the Nt-CPE assays. Overall, the Nt-ELISPOT assay is a reliable and efficient method for measuring neutralizing antibodies in serum. PMID:24391137

  16. Universal antibodies against the highly conserved influenza fusion peptide cross-neutralize several subtypes of influenza A virus

    SciTech Connect

    Hashem, Anwar M.; Van Domselaar, Gary; Li, Changgui; Wang, Junzhi; She, Yi-Min; Cyr, Terry D.; Sui, Jianhua; He, Runtao; Marasco, Wayne A.; Li, Xuguang

    2010-12-10

    Research highlights: {yields} The fusion peptide is the only universally conserved epitope in all influenza viral hemagglutinins. {yields} Anti-fusion peptide antibodies are universal antibodies that cross-react with all influenza HA subtypes. {yields} The universal antibodies cross-neutralize different influenza A subtypes. {yields} The universal antibodies inhibit the fusion process between the viruses and the target cells. -- Abstract: The fusion peptide of influenza viral hemagglutinin plays a critical role in virus entry by facilitating membrane fusion between the virus and target cells. As the fusion peptide is the only universally conserved epitope in all influenza A and B viruses, it could be an attractive target for vaccine-induced immune responses. We previously reported that antibodies targeting the first 14 amino acids of the N-terminus of the fusion peptide could bind to virtually all influenza virus strains and quantify hemagglutinins in vaccines produced in embryonated eggs. Here we demonstrate that these universal antibodies bind to the viral hemagglutinins in native conformation presented in infected mammalian cell cultures and neutralize multiple subtypes of virus by inhibiting the pH-dependant fusion of viral and cellular membranes. These results suggest that this unique, highly-conserved linear sequence in viral hemagglutinin is exposed sufficiently to be attacked by the antibodies during the course of infection and merits further investigation because of potential importance in the protection against diverse strains of influenza viruses.

  17. Immunological Characterization and Neutralizing Ability of Monoclonal Antibodies Directed Against Botulinum Neurotoxin Type H

    PubMed Central

    Fan, Yongfeng; Barash, Jason R.; Lou, Jianlong; Conrad, Fraser; Marks, James D.; Arnon, Stephen S.

    2016-01-01

    Background. Only Clostridium botulinum strain IBCA10-7060 produces the recently described novel botulinum neurotoxin type H (BoNT/H). BoNT/H (N-terminal two-thirds most homologous to BoNT/F and C-terminal one-third most homologous to BoNT/A) requires antitoxin to toxin ratios ≥1190:1 for neutralization by existing antitoxins. Hence, more potent and safer antitoxins against BoNT/H are needed. Methods. We therefore evaluated our existing monoclonal antibodies (mAbs) to BoNT/A and BoNT/F for BoNT/H binding, created yeast-displayed mutants to select for higher-affinity-binding mAbs by using flow cytometry, and evaluated the mAbs' ability to neutralize BoNT/H in the standard mouse bioassay. Results. Anti-BoNT/A HCC-binding mAbs RAZ1 and CR2 bound BoNT/H with high affinity. However, only 1 of 6 BoNT/F mAbs (4E17.2A) bound BoNT/H but with an affinity >800-fold lower (equilibrium dissociation binding constant [KD] = 7.56 × 10−8 M) than its BoNT/F affinity (KD = 9.1 × 10−11 M), indicating that the N-terminal two-thirds of BoNT/H is immunologically unique. The affinity of 4E17.2A for BoNT/H was increased >500-fold to KD = 1.48 × 10−10 M (mAb 4E17.2D). A combination of mAbs RAZ1, CR2, and 4E17.2D completely protected mice challenged with 280 mouse median lethal doses of BoNT/H at a mAb dose as low as 5 µg of total antibody. Conclusions. This 3-mAb combination potently neutralized BoNT/H and represents a potential human antitoxin that could be developed for the prevention and treatment of type H botulism. PMID:26936913

  18. Dengue Virus Neutralizing Antibody Levels Associated with Protection from Infection in Thai Cluster Studies

    PubMed Central

    Buddhari, Darunee; Aldstadt, Jared; Endy, Timothy P.; Srikiatkhachorn, Anon; Thaisomboonsuk, Butsaya; Klungthong, Chonticha; Nisalak, Ananda; Khuntirat, Benjawan; Jarman, Richard G.; Fernandez, Stefan; Thomas, Stephen J.; Scott, Thomas W.; Rothman, Alan L.; Yoon, In-Kyu

    2014-01-01

    Background Long-term homologous and temporary heterologous protection from dengue virus (DENV) infection may be mediated by neutralizing antibodies. However, neutralizing antibody titers (NTs) have not been clearly associated with protection from infection. Methodology/Principal Findings Data from two geographic cluster studies conducted in Kamphaeng Phet, Thailand were used for this analysis. In the first study (2004–2007), cluster investigations of 100-meter radius were triggered by DENV-infected index cases from a concurrent prospective cohort. Subjects between 6 months and 15 years old were evaluated for DENV infection at days 0 and 15 by DENV PCR and IgM ELISA. In the second study (2009–2012), clusters of 200-meter radius were triggered by DENV-infected index cases admitted to the provincial hospital. Subjects of any age ≥6 months were evaluated for DENV infection at days 0 and 14. In both studies, subjects who were DENV PCR positive at day 14/15 were considered to have been “susceptible” on day 0. Comparison subjects from houses in which someone had documented DENV infection, but the subject remained DENV negative at days 0 and 14/15, were considered “non-susceptible.” Day 0 samples were presumed to be from just before virus exposure, and underwent plaque reduction neutralization testing (PRNT). Seventeen “susceptible” (six DENV-1, five DENV-2, and six DENV-4), and 32 “non-susceptible” (13 exposed to DENV-1, 10 DENV-2, and 9 DENV-4) subjects were evaluated. Comparing subjects exposed to the same serotype, receiver operating characteristic (ROC) curves identified homotypic PRNT titers of 11, 323 and 16 for DENV-1, -2 and -4, respectively, to differentiate “susceptible” from “non-susceptible” subjects. Conclusions/Significance PRNT titers were associated with protection from infection by DENV-1, -2 and -4. Protective NTs appeared to be serotype-dependent and may be higher for DENV-2 than other serotypes. These findings are relevant

  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

    PubMed Central

    Sanders, Rogier W.; Derking, Ronald; Cupo, Albert; Julien, Jean-Philippe; Yasmeen, Anila; de Val, Natalia; Kim, Helen J.; Blattner, Claudia; de la Peña, Alba Torrents; Korzun, Jacob; Golabek, Michael; de los Reyes, Kevin; Ketas, Thomas J.; van Gils, Marit J.; King, C. Richter; Wilson, Ian A.; Ward, Andrew B.; Klasse, P. J.; Moore, John P.

    2013-01-01

    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. PMID:24068931

  20. A bispecific antibody effectively neutralizes all four serotypes of dengue virus by simultaneous blocking virus attachment and fusion.

    PubMed

    Shi, Xin; Deng, Yongqiang; Wang, Huajing; Ji, Guanghui; Tan, Wenlong; Jiang, Tao; Li, Xiaofeng; Zhao, Hui; Xia, Tian; Meng, Yanchun; Wang, Chao; Yu, Xiaojie; Yang, Yang; Li, Bohua; Qin, E-De; Dai, Jianxin; Qin, Cheng-Feng; Guo, Yajun

    2016-01-01

    Although dengue virus (DENV) infection severely threatens the health of humans, no specific antiviral drugs are currently approved for clinical use against DENV infection. Attachment and fusion are 2 critical steps for the flavivirus infection, and the corresponding functional epitopes are located at E protein domain III (E-DIII) and domain II (E-DII), respectively. Here, we constructed a bispecific antibody (DVD-1A1D-2A10) based on the 2 well-characterized anti-DENV monoclonal antibodies 1A1D-2 (1A1D) and 2A10G6 (2A10). The 1A1D antibody binds E-DIII and can block the virus attaching to the cell surface, while the 2A10 antibody binds E-DII and is able to prevent the virus from fusing with the endosomal membrane. Our data showed that DVD-1A1D-2A10 retained the antigen-binding activity of both parental antibodies. Importantly, it was demonstrated to be significantly more effective at neutralizing DENV than its parental antibodies both in vitro and in vivo, even better than the combination of them. To eliminate the potential antibody-dependent enhancement (ADE) effect, this bispecific antibody was successfully engineered to prevent Fc-γ-R interaction. Overall, we generated a bispecific anti-DENV antibody targeting both attachment and fusion stages, and this bispecific antibody broadly neutralized all 4 serotypes of DENV without risk of ADE, suggesting that it has great potential as a novel antiviral strategy against DENV. PMID:26905804

  1. Subgroup J Avian Leukosis Virus Neutralizing Antibody Escape Variants Contribute to Viral Persistence in Meat-Type Chickens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We have previously demonstrated a high incidence of chickens with persistent viremia even in the presence of neutralizing antibodies (NAb) against the inoculated parental virus (V+A+) in commercial meat-type chickens inoculated at hatch with Subgroup J avian leukosis virus (ALV J) field isolates. I...

  2. Neutralizing Antibodies against IFN-[Beta] in Multiple Sclerosis: Antagonization of IFN-[Beta] Mediated Suppression of MMPs

    ERIC Educational Resources Information Center

    Gilli, Francesca; Bertolotto, Antonio; Sala, Arianna; Hoffmann, Francine; Capobianco, Marco; Malucchi, Simona; Glass, Tracy; Kappos, Ludwig; Lindberg, Raija L. P.; Leppert, David

    2004-01-01

    Neutralizing antibodies (NAb) against interferon-[Beta] (IFN-Beta) develop in about a third of treated multiple sclerosis patients and are believed to reduce therapeutic efficacy of IFN-[Beta] on clinical and MRI measures. The expression of the interferon acute-response protein, myxovirus resistance protein A (MxA) is a sensitive measure of the…

  3. Charge neutralization apparatus for ion implantation system

    DOEpatents

    Leung, Ka-Ngo; Kunkel, Wulf B.; Williams, Malcom D.; McKenna, Charles M.

    1992-01-01

    Methods and apparatus for neutralization of a workpiece such as a semiconductor wafer in a system wherein a beam of positive ions is applied to the workpiece. The apparatus includes an electron source for generating an electron beam and a magnetic assembly for generating a magnetic field for guiding the electron beam to the workpiece. The electron beam path preferably includes a first section between the electron source and the ion beam and a second section which is coincident with the ion beam. The magnetic assembly generates an axial component of magnetic field along the electron beam path. The magnetic assembly also generates a transverse component of the magnetic field in an elbow region between the first and second sections of the electron beam path. The electron source preferably includes a large area lanthanum hexaboride cathode and an extraction grid positioned in close proximity to the cathode. The apparatus provides a high current, low energy electron beam for neutralizing charge buildup on the workpiece.

  4. Virus-neutralizing antibody response of mice to consecutive infection with human and avian influenza A viruses.

    PubMed

    Janulíková, J; Stropkovská, A; Bobišová, Z; Košík, I; Mucha, V; Kostolanský, F; Varečková, E

    2015-06-01

    In this work we simulated in a mouse model a naturally occurring situation of humans, who overcame an infection with epidemic strains of influenza A, and were subsequently exposed to avian influenza A viruses (IAV). The antibody response to avian IAV in mice previously infected with human IAV was analyzed. We used two avian IAV (A/Duck/Czechoslovakia/1956 (H4N6) and the attenuated virus rA/Viet Nam/1203-2004 (H5N1)) as well as two human IAV isolates (virus A/Mississippi/1/1985 (H3N2) of medium virulence and A/Puerto Rico/8/1934 (H1N1) of high virulence). Two repeated doses of IAV of H4 or of H5 virus elicited virus-specific neutralizing antibodies in mice. Exposure of animals previously infected with human IAV (of H3 or H1 subtype) to IAV of H4 subtype led to the production of antibodies neutralizing H4 virus in a level comparable with the level of antibodies against the human IAV used for primary infection. In contrast, no measurable levels of virus-neutralizing (VN) antibodies specific to H5 virus were detected in mice infected with H5 virus following a previous infection with human IAV. In both cases the secondary infection with avian IAV led to a significant increase of the titer of VN antibodies specific to the corresponding human virus used for primary infection. Moreover, cross-reactive HA2-specific antibodies were also induced by sequential infection. By virtue of these results we suggest that the differences in the ability of avian IAV to induce specific antibodies inhibiting virus replication after previous infection of mice with human viruses can have an impact on the interspecies transmission and spread of avian IAV in the human population. PMID:26104333

  5. Intranasal Delivery of Recombinant Parvovirus-Like Particles Elicits Cytotoxic T-Cell and Neutralizing Antibody Responses

    PubMed Central

    Sedlik, C.; Dridi, A.; Deriaud, E.; Saron, M. F.; Rueda, P.; Sarraseca, J.; Casal, J. I.; Leclerc, C.

    1999-01-01

    We previously demonstrated that chimeric porcine parvovirus-like particles (PPV:VLP) carrying heterologous epitopes, when injected intraperitoneally into mice without adjuvant, activate strong CD4+ and CD8+ T-cell responses specific for the foreign epitopes. In the present study, we investigated the immunogenicity of PPV:VLP carrying a CD8+ T-cell epitope from the lymphocytic choriomeningitis virus (LCMV) administered by mucosal routes. Mice immunized intranasally with recombinant PPV:VLP, in the absence of adjuvant, developed high levels of PPV-specific immunoglobulin G (IgG) and/or IgA in their serum, as well as in mucosal sites such as the bronchoalveolar and intestinal fluids. Antibodies in sera from mice immunized parenterally or intranasally with PPV:VLP were strongly neutralizing in vitro. Intranasal immunization with PPV:VLP carrying the LCMV CD8+ T-cell epitope also elicited a strong peptide-specific cytotoxic-T-cell (CTL) response. In contrast, mice orally immunized with recombinant PPV:VLP did not develop any antibody or CTL responses. We also showed that mice primed with PPV:VLP are still able to develop strong CTL responses after subsequent immunization with chimeric PPV:VLP carrying a foreign CD8+ T-cell epitope. These results highlight the attractive potential of PPV:VLP as a safe, nonreplicating antigen carrier to stimulate systemic and mucosal immunity after nasal administration. PMID:10074120

  6. Intranasal delivery of recombinant parvovirus-like particles elicits cytotoxic T-cell and neutralizing antibody responses.

    PubMed

    Sedlik, C; Dridi, A; Deriaud, E; Saron, M F; Rueda, P; Sarraseca, J; Casal, J I; Leclerc, C

    1999-04-01

    We previously demonstrated that chimeric porcine parvovirus-like particles (PPV:VLP) carrying heterologous epitopes, when injected intraperitoneally into mice without adjuvant, activate strong CD4(+) and CD8(+) T-cell responses specific for the foreign epitopes. In the present study, we investigated the immunogenicity of PPV:VLP carrying a CD8(+) T-cell epitope from the lymphocytic choriomeningitis virus (LCMV) administered by mucosal routes. Mice immunized intranasally with recombinant PPV:VLP, in the absence of adjuvant, developed high levels of PPV-specific immunoglobulin G (IgG) and/or IgA in their serum, as well as in mucosal sites such as the bronchoalveolar and intestinal fluids. Antibodies in sera from mice immunized parenterally or intranasally with PPV:VLP were strongly neutralizing in vitro. Intranasal immunization with PPV:VLP carrying the LCMV CD8(+) T-cell epitope also elicited a strong peptide-specific cytotoxic-T-cell (CTL) response. In contrast, mice orally immunized with recombinant PPV:VLP did not develop any antibody or CTL responses. We also showed that mice primed with PPV:VLP are still able to develop strong CTL responses after subsequent immunization with chimeric PPV:VLP carrying a foreign CD8(+) T-cell epitope. These results highlight the attractive potential of PPV:VLP as a safe, nonreplicating antigen carrier to stimulate systemic and mucosal immunity after nasal administration. PMID:10074120

  7. Induction of neutralizing antibodies by a tobacco chloroplast-derived vaccine based on a B cell epitope from canine parvovirus

    SciTech Connect

    Molina, Andrea; Veramendi, Jon . E-mail: jon@unavarra.es; Hervas-Stubbs, Sandra

    2005-11-25

    The 2L21 epitope of the VP2 protein from the canine parvovirus (CPV), fused to the cholera toxin B subunit (CTB-2L21), was expressed in transgenic tobacco chloroplasts. Mice and rabbits that received protein-enriched leaf extracts by parenteral route produced high titers of anti-2L21 antibodies able to recognize the VP2 protein. Rabbit sera were able to neutralize CPV in an in vitro infection assay with an efficacy similar to the anti-2L21 neutralizing monoclonal antibody 3C9. Anti-2L21 IgG and seric IgA antibodies were elicited when mice were gavaged with a suspension of pulverized tissues from CTB-2L21 transformed plants. Combined immunization (a single parenteral injection followed by oral boosters) shows that oral boosters help to maintain the anti-2L21 IgG response induced after a single injection, whereas parenteral administration of the antigen primes the subsequent oral boosters by promoting the induction of anti-2L21 seric IgA antibodies. Despite the induced humoral response, antibodies elicited by oral delivery did not show neutralizing capacity in the in vitro assay. The high yield of the fusion protein permits the preparation of a high number of vaccine doses from a single plant and makes feasible the oral vaccination using a small amount of crude plant material. However, a big effort has still to be done to enhance the protective efficacy of subunit vaccines by the oral route.

  8. Rapid assessment of antibody-induced ricin neutralization by employing a novel functional cell-based assay.

    PubMed

    Gal, Yoav; Alcalay, Ron; Sabo, Tamar; Noy-Porat, Tal; Epstein, Eyal; Kronman, Chanoch; Mazor, Ohad

    2015-09-01

    Ricin is one of the most potent and lethal toxins known against which there is no available antidote. Currently, the most promising countermeasures against the toxin are based on neutralizing antibodies elicited by active vaccination or administered passively. A cell-based assay is widely applied for the primary screening and evaluation of anti-ricin antibodies, yet such assays are usually time-consuming (18-72 h). Here, we report of a novel assay to monitor ricin activity, based on HeLa cells that stably express the rapidly-degraded ubiquitin-luciferase (Ub-FL, half-life of 2 min). Ricin-induced arrest of protein synthesis could be quantified within 3 to 6h post intoxication (IC90 of 300 and 100 ng/ml, respectively). Furthermore, by stabilizing the intracellular levels of Ub-FL in the last hour of the assay, a 3-fold increase in the assay sensitivity was attained. We applied this assay to monitor the efficacy of a ricin holotoxin-based vaccine by measuring the formation of neutralizing antibodies throughout the immunization course. The potency of anti-ricin monoclonal antibodies (directed to either subunit of the toxin) could also be easily and accurately measured in this assay format. Owing to its simplicity, this assay may be implemented for high-throughput screening of ricin-neutralizing antibodies and for identification of small-molecule inhibitors of the toxin, as well as other ribosome-inactivating toxins. PMID:26003675

  9. Cooperativity Between CD8+ T Cells, Non-Neutralizing Antibodies, and Alveolar Macrophages Is Important for Heterosubtypic Influenza Virus Immunity

    PubMed Central

    Laidlaw, Brian J.; Decman, Vilma; Ali, Mohammed-Alkhatim A.; Abt, Michael C.; Wolf, Amaya I.; Monticelli, Laurel A.; Mozdzanowska, Krystyna; Angelosanto, Jill M.; Artis, David; Erikson, Jan; Wherry, E. John

    2013-01-01

    Seasonal epidemics of influenza virus result in ∼36,000 deaths annually in the United States. Current vaccines against influenza virus elicit an antibody response specific for the envelope glycoproteins. However, high mutation rates result in the emergence of new viral serotypes, which elude neutralization by preexisting antibodies. T lymphocytes have been reported to be capable of mediating heterosubtypic protection through recognition of internal, more conserved, influenza virus proteins. Here, we demonstrate using a recombinant influenza virus expressing the LCMV GP33-41 epitope that influenza virus-specific CD8+ T cells and virus-specific non-neutralizing antibodies each are relatively ineffective at conferring heterosubtypic protective immunity alone. However, when combined virus-specific CD8 T cells and non-neutralizing antibodies cooperatively elicit robust protective immunity. This synergistic improvement in protective immunity is dependent, at least in part, on alveolar macrophages and/or other lung phagocytes. Overall, our studies suggest that an influenza vaccine capable of eliciting both CD8+ T cells and antibodies specific for highly conserved influenza proteins may be able to provide heterosubtypic protection in humans, and act as the basis for a potential “universal” vaccine. PMID:23516357

  10. Cooperativity between CD8+ T cells, non-neutralizing antibodies, and alveolar macrophages is important for heterosubtypic influenza virus immunity.

    PubMed

    Laidlaw, Brian J; Decman, Vilma; Ali, Mohammed-Alkhatim A; Abt, Michael C; Wolf, Amaya I; Monticelli, Laurel A; Mozdzanowska, Krystyna; Angelosanto, Jill M; Artis, David; Erikson, Jan; Wherry, E John

    2013-03-01

    Seasonal epidemics of influenza virus result in ∼36,000 deaths annually in the United States. Current vaccines against influenza virus elicit an antibody response specific for the envelope glycoproteins. However, high mutation rates result in the emergence of new viral serotypes, which elude neutralization by preexisting antibodies. T lymphocytes have been reported to be capable of mediating heterosubtypic protection through recognition of internal, more conserved, influenza virus proteins. Here, we demonstrate using a recombinant influenza virus expressing the LCMV GP33-41 epitope that influenza virus-specific CD8+ T cells and virus-specific non-neutralizing antibodies each are relatively ineffective at conferring heterosubtypic protective immunity alone. However, when combined virus-specific CD8 T cells and non-neutralizing antibodies cooperatively elicit robust protective immunity. This synergistic improvement in protective immunity is dependent, at least in part, on alveolar macrophages and/or other lung phagocytes. Overall, our studies suggest that an influenza vaccine capable of eliciting both CD8+ T cells and antibodies specific for highly conserved influenza proteins may be able to provide heterosubtypic protection in humans, and act as the basis for a potential "universal" vaccine. PMID:23516357

  11. Limited or no protection by weakly or nonneutralizing antibodies against vaginal SHIV challenge of macaques compared with a strongly neutralizing antibody

    PubMed Central

    Burton, Dennis R.; Hessell, Ann J.; Keele, Brandon F.; Klasse, Per Johan; Ketas, Thomas A.; Moldt, Brian; Dunlop, D. Cameron; Poignard, Pascal; Doyle, Lara A.; Cavacini, Lisa; Veazey, Ronald S.; Moore, John P.

    2011-01-01

    To guide vaccine design, we assessed whether human monoclonal antibodies (MAbs) b12 and b6 against the CD4 binding site (CD4bs) on HIV-1 gp120 and F240 against an immundominant epitope on gp41 could prevent vaginal transmission of simian HIV (SHIV)-162P4 to macaques. The two anti-gp120 MAbs have similar monomeric gp120-binding properties, measured in vitro, but b12 is strongly neutralizing and b6 is not. F240 is nonneutralizing. Applied vaginally at a high dose, the strongly neutralizing MAb b12 provided sterilizing immunity in seven of seven animals, b6 in zero of five animals, and F240 in two of five animals. Compared with control animals, the protection by b12 achieved statistical significance, whereas that caused by F240 did not. For two of three unprotected F240-treated animals there was a trend toward lowered viremia. The potential protective effect of F240 may relate to the relatively strong ability of this antibody to capture infectious virions. Additional passive transfer experiments also indicated that the ability of the administered anti-gp120 MAbs to neutralize the challenge virus was a critical influence on protection. Furthermore, when data from all of the experiments were combined, there was a significant increase in the number of founder viruses establishing infection in animals receiving MAb b6, compared with other nonprotected macaques. Thus, a gp120-binding, weakly neutralizing MAb to the CD4bs was, at best, completely ineffective at protection. A nonneutralizing antibody to gp41 may have a limited capacity to protect, but the results suggest that the central focus of HIV-1 vaccine research should be on the induction of potently neutralizing antibodies. PMID:21690411

  12. The Neutralizing Capacity of Antibodies Elicited by Parainfluenza Virus Infection of African Green Monkeys is Dependent on Complement

    PubMed Central

    Mayer, Anne E.; Johnson, John B.; Parks, Griffith D.

    2014-01-01

    The African Green Monkey (AGM) model was used to analyze the role of complement in neutralization of parainfluenza virus. Parainfluenza virus 5 (PIV5) and human parainfluenza virus type 2 were effectively neutralized in vitro by naïve AGM sera, but neutralizing capacity was lost by heat-inactivation. The mechanism of neutralization involved formation of massive aggregates, with no evidence of virion lysis. Following inoculation of the respiratory tract with a PIV5 vector expressing HIV gp160, AGM produced high levels of serum and tracheal antibodies against gp120 and the viral F and HN proteins. However, in the absence of complement these anti-PIV5 antibodies had very poor neutralizing capacity. Virions showed extensive deposition of IgG and C1q with post- but not pre-immune sera. These results highlight the importance of complement in the initial antibody response to parainfluenza viruses, with implications for understanding infant immune responses and design of vaccine strategies for these pediatric pathogens. PMID:25010267

  13. RELATIVE CONCENTRATIONS OF SERUM NEUTRALIZING ANTIBODY TO VP3 AND VP7 PROTEINS IN ADULTS INFECTED WITH A HUMAN ROTAVIRUS (JOURNAL VERSION)

    EPA Science Inventory

    Two outer capsid rotavirus proteins, VP3 and VP7, have been found to elicit neutralizing antibody production, but the immunogenicity of these proteins during human rotavirus infection has not been determined. The relative amounts of serum neutralizing antibody against the VP3 and...

  14. Potent Neutralization of Influenza A Virus by a Single-Domain Antibody Blocking M2 Ion Channel Protein

    PubMed Central

    Wei, Guowei; Meng, Weixu; Guo, Haijiang; Pan, Weiqi; Liu, Jinsong; Peng, Tao; Chen, Ling; Chen, Chang-You

    2011-01-01

    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. PMID:22164266

  15. Holes in the Glycan Shield of the Native HIV Envelope Are a Target of Trimer-Elicited Neutralizing Antibodies.

    PubMed

    McCoy, Laura E; van Gils, Marit J; Ozorowski, Gabriel; Messmer, Terrence; Briney, Bryan; Voss, James E; Kulp, Daniel W; Macauley, Matthew S; Sok, Devin; Pauthner, Matthias; Menis, Sergey; Cottrell, Christopher A; Torres, Jonathan L; Hsueh, Jessica; Schief, William R; Wilson, Ian A; Ward, Andrew B; Sanders, Rogier W; Burton, Dennis R

    2016-08-30

    A major advance in the search for an HIV vaccine has been the development of a near-native Envelope trimer (BG505 SOSIP.664) that can induce robust autologous Tier 2 neutralization. Here, potently neutralizing monoclonal antibodies (nAbs) from rabbits immunized with BG505 SOSIP.664 are shown to recognize an immunodominant region of gp120 centered on residue 241. Residue 241 occupies a hole in the glycan defenses of the BG505 isolate, with fewer than 3% of global isolates lacking a glycan site at this position. However, at least one conserved glycan site is missing in 89% of viruses, suggesting the presence of glycan holes in most HIV isolates. Serum evidence is consistent with targeting of holes in natural infection. The immunogenic nature of breaches in the glycan shield has been under-appreciated in previous attempts to understand autologous neutralizing antibody responses and has important potential consequences for HIV vaccine design. PMID:27545891

  16. Typing of cytopathic and noncytopathic bovine viral diarrhea virus reference and Canadian field strains using a neutralizing monoclonal antibody.

    PubMed Central

    Magar, R; Minocha, H C; Montpetit, C; Carman, P S; Lecomte, J

    1988-01-01

    Cytopathic and noncytopathic reference strains as well as Canadian field isolates of bovine viral diarrhea virus were analyzed by neutralization and immunofluorescence tests using a bovine viral diarrhea virus-specific neutralizing monoclonal antibody. Results on reference strains indicated three major antigenic groups: I) NADL-like, II) New York 1-like and III) Oregon C24V-like. Field isolates could be segregated into groups I and II and none could be typed into the group III. It appears that most bovine viral diarrhea virus strains share a common antigen which carries a major neutralization epitope. These characteristics would make this monoclonal antibody a useful reagent for taxonomic and epizootiological studies. Images Fig. 1. PMID:2450629

  17. Mucosal Immunization with Surface-Displayed Severe Acute Respiratory Syndrome Coronavirus Spike Protein on Lactobacillus casei Induces Neutralizing Antibodies in Mice

    PubMed Central

    Lee, Jong-Soo; Poo, Haryoung; Han, Dong P.; Hong, Seung-Pyo; Kim, Kwang; Cho, Michael W.; Kim, Eun; Sung, Moon-Hee; Kim, Chul-Joong

    2006-01-01

    Induction of mucosal immunity may be important for preventing SARS-CoV infections. For safe and effective delivery of viral antigens to the mucosal immune system, we have developed a novel surface antigen display system for lactic acid bacteria using the poly-γ-glutamic acid synthetase A protein (PgsA) of Bacillus subtilis as an anchoring matrix. Recombinant fusion proteins comprised of PgsA and the Spike (S) protein segments SA (residues 2 to 114) and SB (residues 264 to 596) were stably expressed in Lactobacillus casei. Surface localization of the fusion protein was verified by cellular fractionation analyses, immunofluorescence microscopy, and flow cytometry. Oral and nasal inoculations of recombinant L. casei into mice resulted in high levels of serum immunoglobulin G (IgG) and mucosal IgA, as demonstrated by enzyme-linked immunosorbent assays using S protein peptides. More importantly, these antibodies exhibited potent neutralizing activities against severe acute respiratory syndrome (SARS) pseudoviruses. Orally immunized mice mounted a greater neutralizing-antibody response than those immunized intranasally. Three new neutralizing epitopes were identified on the S protein using a peptide neutralization interference assay (residues 291 to 308, 520 to 529, and 564 to 581). These results indicate that mucosal immunization with recombinant L. casei expressing SARS-associated coronavirus S protein on its surface provides an effective means for eliciting protective immune response against the virus. PMID:16571824

  18. Cross-Reactive HIV-1 Neutralizing Monoclonal Antibodies Selected by Screening of an Immune Human Phage Library against an Envelope Glycoprotein (gp140) Isolated from a Patient (R2) with Broadly HIV-1 Neutralizing Antibodies

    PubMed Central

    Choudhry, Vidita; Zhang, Mei-Yun; Sidorov, Igor A.; Louise, John M.; Harris, Ilia; Dimitrov, Antony S.; Bouma, Peter; Cham, Fatim; Choudhary, Anil; Rybak, Susanna M.; Fouts, Timothy; Montefiori, David C.; Broder, Christopher C.; Quinnan, Gerald V.; Dimitrov, Dimiter S.

    2009-01-01

    Elicitation of broadly cross-reactive neutralizing antibodies (bcnAbs) in HIV infections is rare. To test the hypothesis that such antibodies could be elicited by HIV envelope glycoproteins (Envs) with unusual immunogenic properties and to identify novel bcnAbs, we used a soluble Env ectodomain (gp140) from a donor (R2) with high level of bcnAbs as an antigen for panning of an immune phage-displayed antibody library. The panning with the R2 Env resulted in significantly higher number of cross-reactive antibody clones than by using Envs from two other isolates (89.6 and IIIB). Two of the identified human monoclonal antibodies (hmAbs), m22 and m24, had sequences, neutralizing and binding activities similar or identical to those of the gp120-specific bcnAbs m18 and m14. The use of the R2 Env but not other Envs for panning resulted in the identification of a novel gp41-specific hmAb, m46. For several of the tested HIV-1 primary isolates its potency on molar basis was comparable to that of T20. It inhibited entry of primary isolates from different clades with an increased activity for cell lines with low CCR5 surface concentrations. The m46 neutralizing activity against a panel of clade C isolates was significantly higher in an assay based on peripheral blood mononuclear cells (4 out of 5 isolates were neutralized with an IC50 in the range from 1.5 to 25 μg/ml) than in an assay based on a cell line with relatively high concentration of cell-surface associated CCR5. In contrast to 2F5 and Z13, this antibody did not bind to denatured gp140 and gp41-derived peptides indicating a conformational nature of its epitope. It bound to a 5-helix bundle but not to N-heptad repeat coiled coils and a 6-helix bundle construct indicating contribution of both gp41 heptad repeats to its epitope and to a possible mechanism of neutralization. These results indicate that the R2 Env may contain unique exposed conserved epitopes that could contribute to its ability to elicit broadly cross

  19. Development of a Blocking ELISA for Detection of Serum Neutralizing Antibodies against Newly Emerged Duck Tembusu Virus

    PubMed Central

    Li, Xuesong; Li, Guoxin; Teng, Qiaoyang; Yu, Lei; Wu, Xiaogang; Li, Zejun

    2012-01-01

    Background Since April 2010, domesticated ducks in China have been suffering from an emerging infectious disease characterized by retarded growth, high fever, loss of appetite, decline in egg production, and death. The causative agent was identified as a duck Tembusu virus (DTMUV), a member of the Ntaya virus (NTAV) group within the genus Flavivirus, family Flaviviridae. DTMUV is highly contagious and spreads rapidly in many species of ducks. More than 10 million shelducks have been infected and approximately 1 million died in 2010. The disease remains a constant threat to the duck industry; however, it is not known whether DTMUV can infect humans or other mammalians, despite the fact that the virus has spread widely in southeast China, one of the most densely populated areas in the world. The lack of reliable methods to detect the serum antibodies against DTMUV has limited our ability to conduct epidemiological investigations in various natural hosts and to evaluate the efficiency of vaccines to DTMUV. Methodology/Principal Findings A neutralizing monoclonal antibody (mAb) 1F5 binding specifically to the E protein was developed. Based on the mAb, a blocking enzyme-linked immunosorbent assay (ELISA) was developed for the detection of neutralizing antibodies against DTMUV. The average value of percent inhibition (PI) of 350 duck serum samples obtained from DTMUV-free farms was 1.0% ±5.8% (mean ± SD). The selected cut-off PI values for negative and positive sera were 12.6% (mean +2SD) and 18.4% (mean +3SD), respectively. When compared with a serum neutralizing antibody test (SNT) using chicken embryonated eggs, the rate of coincidence was 70.6% between the blocking ELISA and SNT, based on the titration of 20 duck DTMUV-positive serum samples. Conclusions/Significance The blocking ELISA based on a neutralizing mAb allowed rapid, sensitive, and specific detection of neutralization-related antibodies against DTMUV. PMID:23300851

  20. Prevalence of Neutralizing Antibodies to Japanese Encephalitis Virus among High-Risk Age Groups in South Korea, 2010

    PubMed Central

    Ju, Young Ran; Han, Myung Guk; Lee, Won-Ja; Jeong, Young Eui

    2016-01-01

    After an extensive vaccination policy, Japanese encephalitis (JE) was nearly eliminated since the mid-1980s in South Korea. Vaccination in children shifted the affected age of JE patients from children to adults. However, an abrupt increase in JE cases occurred in 2010, and this trend has continued. The present study aimed to investigate the prevalence of neutralizing antibodies to the JE virus (JEV) among high-risk age groups (≥40 years) in South Korea. A plaque reduction neutralization test was conducted to evaluate the prevalence of neutralizing antibodies to JEV in 945 subjects within four age groups (30–39, 40–49, 50–59, and 60–69 years) in 10 provinces. Of the 945 enrolled subjects, 927 (98.1%) exhibited antibodies against JEV. No significant differences were found in the prevalence of neutralizing antibodies according to sex, age, or occupation. However, there were significant differences in the plaque reduction rate according to age and occupation; oldest age group had a higher reduction rate, and subjects who were employed in agriculture or forestry also had a higher value than the other occupations. We also found that three provinces (Gangwon, Jeonnam, and Gyeongnam) had a relatively lower plaque reduction rate than the other locations. In addition, enzyme-linked immunosorbent assays were conducted to determine recent viral infections and 12 (2.2%) subjects were found to have been recently infected by the virus. In conclusion, the present study clearly indicated that the prevalence of neutralizing antibodies has been maintained at very high levels among adult age groups owing to vaccination or natural infections, or both. In the future, serosurveillance should be conducted periodically using more representative samples to better understand the population-level immunity to JE in South Korea. PMID:26807709

  1. Capacity of Broadly Neutralizing Antibodies to Inhibit HIV-1 Cell-Cell Transmission Is Strain- and Epitope-Dependent

    PubMed Central

    Reh, Lucia; Magnus, Carsten; Schanz, Merle; Weber, Jacqueline; Uhr, Therese; Rusert, Peter; Trkola, Alexandra

    2015-01-01

    An increasing number of broadly neutralizing antibodies (bnAbs) are considered leads for HIV-1 vaccine development and novel therapeutics. Here, we systematically explored the capacity of bnAbs to neutralize HIV-1 prior to and post-CD4 engagement and to block HIV-1 cell-cell transmission. Cell-cell spread is known to promote a highly efficient infection with HIV-1 which can inflict dramatic losses in neutralization potency compared to free virus infection. Selection of bnAbs that are capable of suppressing HIV irrespective of the transmission mode therefore needs to be considered to ascertain their in vivo activity in therapeutic use and vaccines. Employing assay systems that allow for unambiguous discrimination between free virus and cell-cell transmission to T cells, we probed a panel of 16 bnAbs for their activity against 11 viruses from subtypes A, B and C during both transmission modes. Over a wide range of bnAb-virus combinations tested, inhibitory activity against HIV-1 cell-cell transmission was strongly decreased compared to free virus transmission. Activity loss varied considerably between virus strains and was inversely associated with neutralization of free virus spread for V1V2- and V3-directed bnAbs. In rare bnAb-virus combinations, inhibition for both transmission modes was comparable but no bnAb potently blocked cell-cell transmission across all probed virus strains. Mathematical analysis indicated an increased probability of bnAb resistance mutations to arise in cell-cell rather than free virus spread, further highlighting the need to block this pathway. Importantly, the capacity to efficiently neutralize prior to CD4 engagement correlated with the inhibition efficacy against free virus but not cell-cell transmitted virus. Pre-CD4 attachment activity proved strongest amongst CD4bs bnAbs and varied substantially for V3 and V1V2 loop bnAbs in a strain-dependent manner. In summary, bnAb activity against divergent viruses varied depending on the

  2. The molecular mode of action and species specificity of canakinumab, a human monoclonal antibody neutralizing IL-1β

    PubMed Central

    Rondeau, Jean-Michel; Ramage, Paul; Zurini, Mauro; Gram, Hermann

    2015-01-01

    Interleukin-1β (IL-1β) plays a key role in autoinflammatory diseases, such as systemic juvenile idiopathic arthritis (sJIA) or cryopyrin-associated periodic syndrome (CAPS). Canakinumab, a human monoclonal anti-IL-1β antibody, was recently approved for human use under the brand name Ilaris®. Canakinumab does not cross-react with IL-1β from mouse, rat, rabbit, or macaques. The crystal structure of the canakinumab Fab bound to human IL-1β was determined in an attempt to rationalize the species specificity. The X-ray analysis reveals a complex surface epitope with an intricate network of well-ordered water molecules at the antibody-antigen interface. The canakinumab paratope is largely pre-organized, as demonstrated by the structure determination of the free Fab. Glu 64 of human IL-1β is a pivotal epitope residue explaining the exquisite species specificity of canakinumab. We identified marmoset as the only non-human primate species that carries Glu 64 in its IL-1β and demonstrates full cross-reactivity of canakinumab, thereby enabling toxicological studies in this species. As demonstrated by the X-ray structure of the complex with IL-1β, canakinumab binds IL-1β on the opposite side with respect to the IL-1RAcP binding site, and in an approximately orthogonal orientation with respect to IL-1RI. However, the antibody and IL-1RI binding sites slightly overlap and the VH region of canakinumab would sterically interfere with the D1 domain of IL-1RI, as shown by a structural overlay with the IL-1β:IL-1RI complex. Therefore, direct competition with IL-1RI for IL-1β binding is the molecular mechanism of neutralization by canakinumab, which is also confirmed by competition assays with recombinant IL-1RI and IL-1RII. PMID:26284424

  3. Structural basis for diverse N-glycan recognition by HIV-1-neutralizing V1-V2-directed antibody PG16

    SciTech Connect

    Pancera, Marie; Shahzad-ul-Hussan, Syed; Doria-Rose, Nicole A.; McLellan, Jason S.; Bailer, Robert T.; Dai, Kaifan; Loesgen, Sandra; Louder, Mark K.; Staupe, Ryan P.; Yang, Yongping; Zhang, Baoshan; Parks, Robert; Eudailey, Joshua; Lloyd, Krissey E.; Blinn, Julie; Alam, S. Munir; Haynes, Barton F.; Amin, Mohammed N.; Wang, Lai-Xi; Burton, Dennis R.; Koff, Wayne C.; Nabel, Gary J.; Mascola, John R.; Bewley, Carole A; Kwong, Peter D.

    2013-08-05

    HIV-1 uses a diverse N-linked-glycan shield to evade recognition by antibody. Select human antibodies, such as the clonally related PG9 and PG16, recognize glycopeptide epitopes in the HIV-1 V1–V2 region and penetrate this shield, but their ability to accommodate diverse glycans is unclear. Here we report the structure of antibody PG16 bound to a scaffolded V1–V2, showing an epitope comprising both high mannose–type and complex-type N-linked glycans. We combined structure, NMR and mutagenesis analyses to characterize glycan recognition by PG9 and PG16. Three PG16-specific residues, arginine, serine and histidine (RSH), were critical for binding sialic acid on complex-type glycans, and introduction of these residues into PG9 produced a chimeric antibody with enhanced HIV-1 neutralization. Although HIV-1–glycan diversity facilitates evasion, antibody somatic diversity can overcome this and can provide clues to guide the design of modified antibodies with enhanced neutralization.

  4. G glycoprotein amino acid residues required for human monoclonal antibody RAB1 neutralization are conserved in rabies virus street isolates.

    PubMed

    Wang, Yang; Rowley, Kirk J; Booth, Brian J; Sloan, Susan E; Ambrosino, Donna M; Babcock, Gregory J

    2011-08-01

    Replacement of polyclonal anti-rabies immunoglobulin (RIG) used in rabies post-exposure prophylaxis (PEP) with a monoclonal antibody will eliminate cost and availability constraints that currently exist using RIG in the developing world. The human monoclonal antibody RAB1 has been shown to neutralize all rabies street isolates tested; however for the laboratory-adapted fixed strain, CVS-11, mutation in the G glycoprotein of amino acid 336 from asparagine (N) to aspartic acid (D) resulted in resistance to neutralization. Interestingly, this same mutation in the G glycoprotein of a second laboratory-adapted fixed strain (ERA) did not confer resistance to RAB1 neutralization. Using cell surface staining and lentivirus pseudotyped with rabies virus G glycoprotein (RABVpp), we identified an amino acid alteration in CVS-11 (K346), not present in ERA (R346), which was required in combination with D336 to confer resistance to RAB1. A complete analysis of G glycoprotein sequences from GenBank demonstrated that no identified rabies isolates contain the necessary combination of G glycoprotein mutations for resistance to RAB1 neutralization, consistent with the broad neutralization of RAB1 observed in direct viral neutralization experiments with street isolates. All combinations of amino acids 336 and 346 reported in the sequence database were engineered into the ERA G glycoprotein and RAB1 was able to neutralize RABVpp bearing ERA G glycoprotein containing all known combinations at these critical residues. These data demonstrate that RAB1 has the capacity to neutralize all identified rabies isolates and a minimum of two distinct mutations in the G glycoprotein are required for abrogation of RAB1 neutralization. PMID:21693135

  5. Human immunodeficiency virus type 1 neutralization epitope with conserved architecture elicits early type-specific antibodies in experimentally infected chimpanzees.

    PubMed Central

    Goudsmit, J; Debouck, C; Meloen, R H; Smit, L; Bakker, M; Asher, D M; Wolff, A V; Gibbs, C J; Gajdusek, D C

    1988-01-01

    Chimpanzees are susceptible to infection by divergent strains of human immunodeficiency virus type 1 (HIV-1), none of which cause clinical or immunological abnormalities. Chimpanzees were inoculated with one of four strains of HIV-1: human T-lymphotropic virus (HTLV) type IIIB, lymphadenopathy virus (LAV) type 1, HTLV type IIIRF, or an isolate from the brain of a patient with acquired immunodeficiency syndrome. Within 6 months after inoculation with the closely related strains HTLV-IIIB or LAV-1, six chimpanzees developed serum antibodies to the C-terminal half (amino acids 288-467) of the HTLV-IIIB external envelope glycoprotein gp120. Sera from five of those chimpanzees had HTLV-IIIB cell-fusion-inhibiting antibody titers greater than or equal to 20 at that time, indicating that they neutralized the infecting strain of HIV-1 in vitro. No antibodies to the carboxyl terminus of HTLV-IIIB gp120 were observed in sera of chimpanzees inoculated with HTLV-IIIRF or with the brain-tissue strain, and those sera did not neutralize HTLV-IIIB. A rabbit immunized with the C-terminal portion of gp120 acquired neutralizing antibodies that bound to four domains of the HTLV-IIIB external envelope as analyzed by reactivity to 536 overlapping nonapeptides of gp120. One of these domains in the variable region V3, with the amino acid sequence IRIQRGPGRAFVTIG (amino acids 307-321), bound to all chimpanzee sera that neutralized HTLV-IIIB but not to the serum of the HTLV-IIIRF-inoculated chimpanzee that did not neutralize HTLV-IIIB. The HTLV-IIIRF sequence at the same location, ITKGPGRVIYA, was recognized by the serum of the HTLV-IIIRF-inoculated chimpanzee but not by any sera of the HTLV-IIIB-inoculated or LAV-1-inoculated chimpanzees. The HTLV-IIIB residues RIQR and AFV and the HTLV-IIIRF residues lysine and VIYA, flanking a highly conserved beta-turn (GPGR), appear to be critical for antibody binding and subsequent type-specific virus neutralization. This neutralization epitope

  6. Identification and Characterization of a New Cross-Reactive Human Immunodeficiency Virus Type 1-Neutralizing Human Monoclonal Antibody

    PubMed Central

    Zhang, Mei-Yun; Xiao, Xiaodong; Sidorov, Igor A.; Choudhry, Vidita; Cham, Fatim; Zhang, Peng Fei; Bouma, Peter; Zwick, Michael; Choudhary, Anil; Montefiori, David C.; Broder, Christopher C.; Burton, Dennis R.; Quinnan, Gerald V.; Dimitrov, Dimiter S.

    2004-01-01

    The identification and characterization of new human monoclonal antibodies (hMAbs) able to neutralize primary human immunodeficiency virus type 1 (HIV-1) isolates from different subtypes may help in our understanding of the mechanisms of virus entry and neutralization and in the development of entry inhibitors and vaccines. For enhanced selection of broadly cross-reactive antibodies, soluble HIV-1 envelope glycoproteins (Envs proteins) from two isolates complexed with two-domain soluble CD4 (sCD4) were alternated during panning of a phage-displayed human antibody library; these two Env proteins (89.6 and IIIB gp140s), and one additional Env (JR-FL gp120) alone and complexed with sCD4 were used for screening. An antibody with relatively long HCDR3 (17 residues), designated m14, was identified that bound to all antigens and neutralized heterologous HIV-1 isolates in multiple assay formats. Fab m14 potently neutralized selected well-characterized subtype B isolates, including JRCSF, 89.6, IIIB, and Yu2. Immunoglobulin G1 (IgG1) m14 was more potent than Fab m14 and neutralized 7 of 10 other clade B isolates; notably, although the potency was on average significantly lower than that of IgG1 b12, IgG1 m14 neutralized two of the isolates with significantly lower 50% inhibitory concentrations than did IgG1 b12. IgG1 m14 neutralized four of four selected clade C isolates with potency higher than that of IgG1 b12. It also neutralized 7 of 17 clade C isolates from southern Africa that were difficult to neutralize with other hMAbs and sCD4. IgG1 m14 neutralized four of seven primary HIV-1 isolates from other clades (A, D, E, and F) much more efficiently than did IgG1 b12; for the other three isolates, IgG b12 was much more potent. Fab m14 bound with high (nanomolar range) affinity to gp120 and gp140 from various isolates; its binding was reduced by soluble CD4 and antibodies recognizing the CD4 binding site (CD4bs) on gp120, and its footprint as defined by alanine

  7. Induction of neutralizing antibodies in rhesus macaques using V3 mimotope peptides.

    PubMed

    Hessell, Ann J; McBurney, Sean; Pandey, Shilpi; Sutton, William; Liu, Lily; Li, Liuzhe; Totrov, Maxim; Zolla-Pazner, Susan; Haigwood, Nancy L; Gorny, Miroslaw K

    2016-05-23

    RV144 vaccinees with low HIV-1 Envelope-specific IgA antibodies (Abs) also had Abs directed to the hypervariable region 3 (V3) that inversely correlated with infection risk. Thus, anti-V3 HIV-1 Abs may contribute to protection from HIV-1 infection. The V3 region contains two dominant clusters of epitopes; one is preferentially recognized by mAbs encoded by VH5-51 and VL lambda genes, while the second one is recognized by mAbs encoded by other VH genes. We designed a study in rhesus macaques to induce anti-V3 Abs specific to each of these two dominant clusters of V3 epitopes to test whether the usage of the VH5-51 gene results in different characteristics of antibodies. The two C4-V3 immunogens used for immunization were each comprised of a fusion of the C4 peptide containing the T cell epitope and a V3 mimotope peptide mimicking the V3 epitope. The C4-447 peptide was designed to target B cells with several VH1-VH4 genes, the C4-VH5-51 peptide was designed to specifically target B cells with the VH5-51 gene. Six animals in two groups were immunized five times with these two immunogens, and screening of 10 sequential plasma samples post immunization demonstrated that C4-447 induced higher titers of plasma anti-V3 Abs and significantly more potent neutralizing activities against tier 1 and some tier 2 pseudoviruses than C4-VH5-51. Levels of anti-V3 Abs in buccal secretions were significantly higher in sequential samples derived from C4-447- than from C4-VH5-51-immunized animals. The titers of anti-V3 Abs in plasma strongly correlated with their levels in mucosal secretions. The results show that high titers of vaccine-induced anti-V3 Abs in plasma determine the potency and breadth of neutralization, as well as the rate of transduction of Abs to mucosal tissues, where they can play a role in preventing HIV-1 infection. PMID:27102818

  8. The Enterovirus 71 Procapsid Binds Neutralizing Antibodies and Rescues Virus Infection In Vitro

    PubMed Central

    Shingler, Kristin L.; Cifuente, Javier O.; Ashley, Robert E.; Makhov, Alexander M.; Conway, James F.

    2014-01-01

    ABSTRACT Enterovirus 71 (EV71) is responsible for seasonal outbreaks of hand, foot, and mouth disease in the Asia-Pacific region. The virus has the capability to cause severe disease and death, especially in young children. Although several vaccines are currently in clinical trials, no vaccines or therapeutics have been approved for use. Previous structural studies have revealed that two antigenically distinct capsid forms are produced in EV71-infected cells: an expanded empty capsid, sometimes called a procapsid, and the infectious virus. Specifically, an immunodominant epitope of EV71 that maps to the virus canyon is structurally different in the procapsid and virus. This structure-function study shows that the procapsid can sequester antibodies, thus enhancing EV71 infection in vitro. The results presented here suggest that, due to conformational differences between the EV71 procapsid and virus, the presence of the procapsid in natural virus infections should be considered in the future design of vaccines or therapeutics. IMPORTANCE In a picornavirus infection, both an infectious and a noninfectious empty capsid, sometimes referred to as a procapsid, are produced. It was novel to discover that the procapsid form of EV71 was expanded and antigenically distinct from the infectious virus. Previously, it had been supposed that this empty capsid was an off-pathway dead end or at best served for storage of pentameric subunits, which was later shown to be unlikely. It remains unexplained why picornaviruses evolutionarily conserve the wasteful production of so much noninfectious capsid. Here, we demonstrate that the EV71 procapsid has different antigenic properties than the infectious virus. Thus, the procapsid has the capacity to sequester neutralizing antibody and protect the virus, promoting or restoring a successful infection in vitro. This important observation should be considered in the future design and development of vaccines and therapeutics. PMID:25428877

  9. Potent neutralizing anti-CD1d antibody reduces lung cytokine release in primate asthma model

    PubMed Central

    Nambiar, Jonathan; Clarke, Adam W; Shim, Doris; Mabon, David; Tian, Chen; Windloch, Karolina; Buhmann, Chris; Corazon, Beau; Lindgren, Matilda; Pollard, Matthew; Domagala, Teresa; Poulton, Lynn; Doyle, Anthony G

    2015-01-01

    CD1d is a receptor on antigen-presenting cells involved in triggering cell populations, particularly natural killer T (NKT) cells, to release high levels of cytokines. NKT cells are implicated in asthma pathology and blockade of the CD1d/NKT cell pathway may have therapeutic potential. We developed a potent anti-human CD1d antibody (NIB.2) that possesses high affinity for human and cynomolgus macaque CD1d (KD ∼100 pM) and strong neutralizing activity in human primary cell-based assays (IC50 typically <100 pM). By epitope mapping experiments, we showed that NIB.2 binds to CD1d in close proximity to the interface of CD1d and the Type 1 NKT cell receptor β-chain. Together with data showing that NIB.2 inhibited stimulation via CD1d loaded with different glycolipids, this supports a mechanism whereby NIB.2 inhibits NKT cell activation by inhibiting Type 1 NKT cell receptor β-chain interactions with CD1d, independent of the lipid antigen in the CD1d antigen-binding cleft. The strong in vitro potency of NIB.2 was reflected in vivo in an Ascaris suum cynomolgus macaque asthma model. Compared with vehicle control, NIB.2 treatment significantly reduced bronchoalveolar lavage (BAL) levels of Ascaris-induced cytokines IL-5, IL-8 and IL-1 receptor antagonist, and significantly reduced baseline levels of GM-CSF, IL-6, IL-15, IL-12/23p40, MIP-1α, MIP-1β, and VEGF. At a cellular population level NIB.2 also reduced numbers of BAL lymphocytes and macrophages, and blood eosinophils and basophils. We demonstrate that anti-CD1d antibody blockade of the CD1d/NKT pathway modulates inflammatory parameters in vivo in a primate inflammation model, with therapeutic potential for diseases where the local cytokine milieu is critical. PMID:25751125

  10. Neutralizing antibody titers against dengue virus correlate with protection from symptomatic infection in a longitudinal cohort.

    PubMed

    Katzelnick, Leah C; Montoya, Magelda; Gresh, Lionel; Balmaseda, Angel; Harris, Eva

    2016-01-19

    The four dengue virus serotypes (DENV1-4) are mosquito-borne flaviviruses that infect ∼ 390 million people annually; up to 100 million infections are symptomatic, and 500,000 cases progress to severe disease. Exposure to a heterologous DENV serotype, the specific infecting DENV strains, and the interval of time between infections, as well as age, ethnicity, genetic polymorphisms, and comorbidities of the host, are all risk factors for severe dengue. In contrast, neutralizing antibodies (NAbs) are thought to provide long-lived protection against symptomatic infection and severe dengue. The objective of dengue vaccines is to provide balanced protection against all DENV serotypes simultaneously. However, the association between homotypic and heterotypic NAb titers and protection against symptomatic infection remains poorly understood. Here, we demonstrate that the titer of preinfection cross-reactive NAbs correlates with reduced likelihood of symptomatic secondary infection in a longitudinal pediatric dengue cohort in Nicaragua. The protective effect of NAb titers on infection outcome remained significant when controlled for age, number of years between infections, and epidemic force, as well as with relaxed or more stringent criteria for defining inapparent DENV infections. Further, individuals with higher NAb titers immediately after primary infection had delayed symptomatic infections compared with those with lower titers. However, overall NAb titers increased modestly in magnitude and remained serotype cross-reactive in the years between infections, possibly due to reexposure. These findings establish that anti-DENV NAb titers correlate with reduced probability of symptomatic DENV infection and provide insights into longitudinal characteristics of antibody-mediated immunity to DENV in an endemic setting. PMID:26729879

  11. Targeting the vaccinia virus L1 protein to the cell surface enhances production of neutralizing antibodies.

    PubMed

    Golden, Joseph W; Josleyn, Matthew D; Hooper, Jay W

    2008-06-25

    The current live-orthopoxvirus vaccine is associated with minor to serious adverse affects, and is contraindicated for use in a significant portion of the population. As an alternative vaccine, we have previously shown that a DNA subunit vaccine (4pox) based on four orthopoxvirus immunogens (L1R, B5R, A27L and A33R) can produce protective immunity against lethal orthopoxvirus challenges in mice and nonhuman primates. Because antibodies are critical for protection against secondary orthopoxvirus infections, we are now interested in strategies that will enhance the humoral immune response against vaccine targets. Here, we tested the immunogenicity of an L1R construct to which a tissue plasminogen activator signal sequence was placed in frame with the full-length L1R gene. The tPA-L1R construct produced a more robust neutralizing antibody response in vaccinated mice when the DNA vaccine was administered by gene-gun as a prime/single boost. When the tPA-L1R construct was substituted for the unmodified L1R gene in the 4pox vaccine, given as a prime and single boost, animals were better protected from lethal challenge with vaccinia virus (VACV). These findings indicate that adding a tPA-leader sequence can enhance the immunogenicity of the L1R gene when given as a DNA vaccine. Furthermore, our results demonstrate that a DNA-based vaccine is capable of establishing protection from lethal orthopoxvirus challenges when administered as a prime and single boost without requiring adjuvant. PMID:18485547

  12. Neutralizing antibody titers against dengue virus correlate with protection from symptomatic infection in a longitudinal cohort

    PubMed Central

    Katzelnick, Leah C.; Montoya, Magelda; Gresh, Lionel; Balmaseda, Angel; Harris, Eva

    2016-01-01

    The four dengue virus serotypes (DENV1–4) are mosquito-borne flaviviruses that infect ∼390 million people annually; up to 100 million infections are symptomatic, and 500,000 cases progress to severe disease. Exposure to a heterologous DENV serotype, the specific infecting DENV strains, and the interval of time between infections, as well as age, ethnicity, genetic polymorphisms, and comorbidities of the host, are all risk factors for severe dengue. In contrast, neutralizing antibodies (NAbs) are thought to provide long-lived protection against symptomatic infection and severe dengue. The objective of dengue vaccines is to provide balanced protection against all DENV serotypes simultaneously. However, the association between homotypic and heterotypic NAb titers and protection against symptomatic infection remains poorly understood. Here, we demonstrate that the titer of preinfection cross-reactive NAbs correlates with reduced likelihood of symptomatic secondary infection in a longitudinal pediatric dengue cohort in Nicaragua. The protective effect of NAb titers on infection outcome remained significant when controlled for age, number of years between infections, and epidemic force, as well as with relaxed or more stringent criteria for defining inapparent DENV infections. Further, individuals with higher NAb titers immediately after primary infection had delayed symptomatic infections compared with those with lower titers. However, overall NAb titers increased modestly in magnitude and remained serotype cross-reactive in the years between infections, possibly due to reexposure. These findings establish that anti-DENV NAb titers correlate with reduced probability of symptomatic DENV infection and provide insights into longitudinal characteristics of antibody-mediated immunity to DENV in an endemic setting. PMID:26729879

  13. An autoreactive antibody from an SLE/HIV-1 individual broadly neutralizes HIV-1

    PubMed Central

    Bonsignori, Mattia; Wiehe, Kevin; Grimm, Sebastian K.; Lynch, Rebecca; Yang, Guang; Kozink, Daniel M.; Perrin, Florence; Cooper, Abby J.; Hwang, Kwan-Ki; Chen, Xi; Liu, Mengfei; McKee, Krisha; Parks, Robert J.; Eudailey, Joshua; Wang, Minyue; Clowse, Megan; Criscione-Schreiber, Lisa G.; Moody, M. Anthony; Ackerman, Margaret E.; Boyd, Scott D.; Gao, Feng; Kelsoe, Garnett; Verkoczy, Laurent; Tomaras, Georgia D.; Liao, Hua-Xin; Kepler, Thomas B.; Montefiori, David C.; Mascola, John R.; Haynes, Barton F.

    2014-01-01

    Broadly HIV-1–neutralizing antibodies (BnAbs) display one or more unusual traits, including a long heavy chain complementarity-determining region 3 (HCDR3), polyreactivity, and high levels of somatic mutations. These shared characteristics suggest that BnAb development might be limited by immune tolerance controls. It has been postulated that HIV-1–infected individuals with autoimmune disease and defective immune tolerance mechanisms may produce BnAbs more readily than those without autoimmune diseases. In this study, we identified an HIV-1–infected individual with SLE who exhibited controlled viral load (<5,000 copies/ml) in the absence of controlling HLA phenotypes and developed plasma HIV-1 neutralization breadth. We collected memory B cells from this individual and isolated a BnAb, CH98, that targets the CD4 binding site (CD4bs) of HIV-1 envelope glycoprotein 120 (gp120). CH98 bound to human antigens including dsDNA, which is specifically associated with SLE. Anti-dsDNA reactivity was also present in the patient’s plasma. CH98 had a mutation frequency of 25% and 15% nt somatic mutations in the heavy and light chain variable domains, respectively, a long HCDR3, and a deletion in the light chain CDR1. The occurrence of anti-dsDNA reactivity by a HIV-1 CD4bs BnAb in an individual with SLE raises the possibility that some BnAbs and SLE-associated autoantibodies arise from similar pools of B cells. PMID:24614107

  14. Comparison of agglutinating and neutralizing antibodies to serovar hardjo in sows immunized with two commercial whole culture polivalent anti-leptospira bacterins.

    PubMed

    Soto, Francisco Rafael Martins; Pinheiro, Sônia Regina; Morais, Zenaide Maria; Gonçales, Amane Paldês; de Azevedo, Sérgio Santos; Bernardi, Fernanda; Camargo, Sebastião Rodrigues; Vasconcellos, Silvio Arruda

    2008-07-01

    It was performed the comparison of the intensity and duration of agglutinating and neutralizing antibodies to serovar Hardjo in swines vaccinated with two commercial anti-leptospira bacterins. Sows no reactive to 24 Leptospira sp serovars in the microscopic agglutination test (MAT) were divided in three groups: Group A (n=08): received two vaccine A doses with 30 days interval, Group B (n=08) two vaccine B doses with 30 days interval and Group C (n=08): control no vaccinated against leptospirosis.Blood samples were collected each 30 days during six months following the first vaccination. The sera were tested by MAT and growth inhibition test (GIT) to serovar Hardjo in order to evaluate respectively agglutinating and neutralizing antibodies. It was found that neutralizing antibodies persisted for a longer time than the agglutinating ones and that the absence of agglutinating antibodies does not means in the absence of the neutralizing. The peaks of agglutinating antibodies was obtained at least 30 days earlier than that produced by neutralizing. The duration of both kinds of antibodies measured differed between the two bacterines tested. The period for inducing neutralizing antibodies against serovar Hardjo indicated that gilts must be immunized with two doses of whole culture anti-leptospira bacterines applied 30 days each other at least 90 days before the first mating. For the maintenance of hight levels of neutralizing antibodies the revaccinations must be performed every six months after the first vaccination. PMID:24031250

  15. Comparison of agglutinating and neutralizing antibodies to serovar hardjo in sows immunized with two commercial whole culture polivalent anti-leptospira bacterins

    PubMed Central

    Soto, Francisco Rafael Martins; Pinheiro, Sônia Regina; Morais, Zenaide Maria; Gonçales, Amane Paldês; de Azevedo, Sérgio Santos; Bernardi, Fernanda; Camargo, Sebastião Rodrigues; Vasconcellos, Silvio Arruda

    2008-01-01

    It was performed the comparison of the intensity and duration of agglutinating and neutralizing antibodies to serovar Hardjo in swines vaccinated with two commercial anti-leptospira bacterins. Sows no reactive to 24 Leptospira sp serovars in the microscopic agglutination test (MAT) were divided in three groups: Group A (n=08): received two vaccine A doses with 30 days interval, Group B (n=08) two vaccine B doses with 30 days interval and Group C (n=08): control no vaccinated against leptospirosis.Blood samples were collected each 30 days during six months following the first vaccination. The sera were tested by MAT and growth inhibition test (GIT) to serovar Hardjo in order to evaluate respectively agglutinating and neutralizing antibodies. It was found that neutralizing antibodies persisted for a longer time than the agglutinating ones and that the absence of agglutinating antibodies does not means in the absence of the neutralizing. The peaks of agglutinating antibodies was obtained at least 30 days earlier than that produced by neutralizing. The duration of both kinds of antibodies measured differed between the two bacterines tested. The period for inducing neutralizing antibodies against serovar Hardjo indicated that gilts must be immunized with two doses of whole culture anti-leptospira bacterines applied 30 days each other at least 90 days before the first mating. For the maintenance of hight levels of neutralizing antibodies the revaccinations must be performed every six months after the first vaccination. PMID:24031250

  16. Comprehensive Cross-Clade Neutralization Analysis of a Panel of Anti-Human Immunodeficiency Virus Type 1 Monoclonal Antibodies

    PubMed Central

    Binley, James M.; Wrin, Terri; Korber, Bette; Zwick, Michael B.; Wang, Meng; Chappey, Colombe; Stiegler, Gabriela; Kunert, Renate; Zolla-Pazner, Susan; Katinger, Hermann; Petropoulos, Christos J.; Burton, Dennis R.

    2004-01-01

    Broadly neutralizing monoclonal antibodies (MAbs) are potentially important tools in human immunodeficiency virus type 1 (HIV-1) vaccine design. A few rare MAbs have been intensively studied, but we still have a limited appreciation of their neutralization breadth. Using a pseudovirus assay, we evaluated MAbs from clade B-infected donors and a clade B HIV+ plasma against 93 viruses from diverse backgrounds. Anti-gp120 MAbs exhibited greater activity against clade B than non-B viruses, whereas anti-gp41 MAbs exhibited broad interclade activity. Unexpectedly, MAb 4E10 (directed against the C terminus of the gp41 ectodomain) neutralized all 90 viruses with moderate potency. MAb 2F5 (directed against an epitope adjacent to that of 4E10) neutralized 67% of isolates, but none from clade C. Anti-gp120 MAb b12 (directed against an epitope overlapping the CD4 binding site) neutralized 50% of viruses, including some from almost every clade. 2G12 (directed against a high-mannose epitope on gp120) neutralized 41% of the viruses, but none from clades C or E. MAbs to the gp120 V3 loop, including 447-52D, neutralized a subset of clade B viruses (up to 45%) but infrequently neutralized other clades (≤7%). MAbs b6 (directed against the CD4 binding site) and X5 (directed against a CD4-induced epitope of gp120) neutralized only sensitive primary clade B viruses. The HIV+ plasma neutralized 70% of the viruses, including some from all major clades. Further analysis revealed five neutralizing immunotypes that were somewhat associated with clades. As well as the significance for vaccine design, our data have implications for passive-immunization studies in countries where clade C viruses are common, given that only MAbs b12 and 4E10 were effective against viruses from this clade. PMID:15542675

  17. Neutralization of influenza A viruses by insertion of a single antibody loop into the receptor binding site

    PubMed Central

    Ekiert, Damian C.; Kashyap, Arun K.; Steel, John; Rubrum, Adam; Bhabha, Gira; Khayat, Reza; Lee, Jeong Hyun; Dillon, Michael A.; O’Neil, Ryann E.; Faynboym, Aleksandr M.; Horowitz, Michael; Horowitz, Lawrence; Ward, Andrew B.; Palese, Peter; Webby, Richard; Lerner, Richard A.; Bhatt, Ramesh R.; Wilson, Ian A.

    2012-01-01

    Summary Immune recognition of protein antigens relies upon the combined interaction of multiple antibody loops, which provides a fairly large footprint and constrains the size and shape of protein surfaces that can be targeted. Single protein loops can mediate extremely high affinity binding, but it is unclear whether such a mechanism is available to antibodies. Here we report the isolation and characterization of antibody C05 that neutralizes strains from multiple subtypes of influenza A viruses, including H1, H2, and H3. Crystal and EM structures show that C5 recognizes conserved elements of the receptor binding site on the hemagglutinin (HA) surface glycoprotein. Recognition of the HA receptor binding site is dominated by a single HCDR3 loop, with minor contacts from HCDR1, and is sufficient to achieve nanomolar binding with a minimal footprint. Thus, binding predominantly with a single loop can allow antibodies to target small, conserved, functional sites on otherwise hypervariable antigens. PMID:22982990

  18. Crystal structure of the HIV neutralizing antibody 2G12 in complex with a bacterial oligosaccharide analog of mammalian oligomannose

    PubMed Central

    Stanfield, Robyn L; De Castro, Cristina; Marzaioli, Alberto M; Wilson, Ian A; Pantophlet, Ralph

    2015-01-01

    Human immunodeficiency virus-1 (HIV-1) is a major public health threat that continues to infect millions of people worldwide each year. A prophylactic vaccine remains the most cost-effective way of globally reducing and eliminating the spread of the virus. The HIV envelope spike, which is the target of many vaccine design efforts, is densely mantled with carbohydrate and several potent broadly neutralizing antibodies to HIV-1 recognize carbohydrate on the envelope spike as a major part of their epitope. However, immunizing with recombinant forms of the envelope glycoprotein does not typically elicit anti-carbohydrate antibodies. Thus, studies of alternative antigens that may serve as a starting point for carbohydrate-based immunogens are of interest. Here, we present the crystal structure of one such anti-carbohydrate HIV neutralizing antibody (2G12) in complex with the carbohydrate backbone of the lipooligosaccharide from Rhizobium radiobacter strain Rv3, which exhibits a chemical structure that naturally mimics the core high-mannose carbohydrate epitope of 2G12 on HIV-1 gp120. The structure described here provides molecular evidence of the structural homology between the Rv3 oligosaccharide and highly abundant carbohydrates on the surface of HIV-1 and raises the potential for the design of novel glycoconjugates that may find utility in efforts to develop immunogens for eliciting carbohydrate-specific neutralizing antibodies to HIV. PMID:25380763

  19. Neutralizing antibody and functional mapping of Bacillus anthracis protective antigen-The first step toward a rationally designed anthrax vaccine.

    PubMed

    McComb, Ryan C; Martchenko, Mikhail

    2016-01-01

    Anthrax is defined by the Centers for Disease Control and Prevention as a Category A pathogen for its potential use as a bioweapon. Current prevention treatments include Anthrax Vaccine Adsorbed (AVA). AVA is an undefined formulation of Bacillus anthracis culture supernatant adsorbed to aluminum hydroxide. It has an onerous vaccination schedule, is slow and cumbersome to produce and is slightly reactogenic. Next-generation vaccines are focused on producing recombinant forms of anthrax toxin in a well-defined formulation but these vaccines have been shown to lose potency as they are stored. In addition, studies have shown that a proportion of the antibody response against these vaccines is focused on non-functional, non-neutralizing regions of the anthrax toxin while some essential functional regions are shielded from eliciting an antibody response. Rational vaccinology is a developing field that focuses on designing vaccine antigens based on structural information provided by neutralizing antibody epitope mapping, crystal structure analysis, and functional mapping through amino acid mutations. This information provides an opportunity to design antigens that target only functionally important and conserved regions of a pathogen in order to make a more optimal vaccine product. This review provides an overview of the literature related to functional and neutralizing antibody epitope mapping of the Protective Antigen (PA) component of anthrax toxin. PMID:26611201

  20. Structural basis for immunization with postfusion respiratory syncytial virus fusion F glycoprotein (RSV F) to elicit high neutralizing antibody titers

    SciTech Connect

    Swanson, Kurt A.; Settembre, Ethan C.; Shaw, Christine A.; Dey, Antu K.; Rappuoli, Rino; Mandl, Christian W.; Dormitzer, Philip R.; Carfi, Andrea

    2012-02-07

    Respiratory syncytial virus (RSV), the main cause of infant bronchiolitis, remains a major unmet vaccine need despite more than 40 years of vaccine research. Vaccine candidates based on a chief RSV neutralization antigen, the fusion (F) glycoprotein, have foundered due to problems with stability, purity, reproducibility, and potency. Crystal structures of related parainfluenza F glycoproteins have revealed a large conformational change between the prefusion and postfusion states, suggesting that postfusion F antigens might not efficiently elicit neutralizing antibodies. We have generated a homogeneous, stable, and reproducible postfusion RSV F immunogen that elicits high titers of neutralizing antibodies in immunized animals. The 3.2-{angstrom} X-ray crystal structure of this substantially complete RSV F reveals important differences from homology-based structural models. Specifically, the RSV F crystal structure demonstrates the exposure of key neutralizing antibody binding sites on the surface of the postfusion RSV F trimer. This unanticipated structural feature explains the engineered RSV F antigen's efficiency as an immunogen. This work illustrates how structural-based antigen design can guide the rational optimization of candidate vaccine antigens.

  1. Structural Basis of Differential Neutralization of DENV-1 Genotypes by an Antibody that Recognizes a Cryptic Epitope

    PubMed Central

    Austin, S. Kyle; Dowd, Kimberly A.; Shrestha, Bimmi; Nelson, Christopher A.; Edeling, Melissa A.; Johnson, Syd; Pierson, Theodore C.; Diamond, Michael S.; Fremont, Daved H.

    2012-01-01

    We previously developed a panel of neutralizing monoclonal antibodies against Dengue virus (DENV)-1, of which few exhibited inhibitory activity against all DENV-1 genotypes. This finding is consistent with reports observing variable neutralization of different DENV strains and genotypes using serum from individuals that experienced natural infection or immunization. Herein, we describe the crystal structures of DENV1-E111 bound to a novel CC′ loop epitope on domain III (DIII) of the E protein from two different DENV-1 genotypes. Docking of our structure onto the available cryo-electron microscopy models of DENV virions revealed that the DENV1-E111 epitope was inaccessible, suggesting that this antibody recognizes an uncharacterized virus conformation. While the affinity of binding between DENV1-E111 and DIII varied by genotype, we observed limited correlation with inhibitory activity. Instead, our results support the conclusion that potent neutralization depends on genotype-dependent exposure of the CC′ loop epitope. These findings establish new structural complexity of the DENV virion, which may be relevant for the choice of DENV strain for induction or analysis of neutralizing antibodies in the context of vaccine development. PMID:23055922

  2. Protection Against H7 Subtype Influenza Virus Infection in Mice by Passive Transfer of Neutralizing Monoclonal Antibody.

    PubMed

    Zhang, Zhuo; Liu, Ming; Zheng, Shimin

    2015-10-01

    H7 subtype influenza viruses pose serious threats to both the poultry industry and public health. Recent human infections of avian H7N9 influenza viruses with substantial morbidity and mortality have raised concerns about this virus becoming a potential pandemic pathogen. Neutralizing antibodies have been proven to be highly effective in blocking influenza virus infections. In this study, in order to develop an antibody-based immunoprophylaxis against H7 subtype influenza virus, we first generated a neutralizing monoclonal antibody (MAb) by using a pseudotyped lentiviral vector carrying the hemagglutinin protein of H7 subtype influenza virus. In vitro studies demonstrated that this neutralizing MAb completely inhibited the infection of an H7 subtype influenza virus to cells. The protective efficacy of this MAb was then further tested in a mouse model. It was shown that passive immunization of this MAb protected mice from local virus challenge. Results of the current study lay a foundation for the development of neutralizing MAb-mediated prophylactic strategies to combat human H7 influenza virus infections. PMID:26492625

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

    PubMed

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

    2016-01-01

    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. PMID:27576440

  4. Functional characterization of the recombinant HIV-neutralizing monoclonal antibody 2F5 produced in maize seeds.

    PubMed

    Sabalza, M; Madeira, L; van Dolleweerd, C; Ma, J K; Capell, T; Christou, P

    2012-11-01

    Monoclonal antibodies (mAbs) that neutralize human immunodeficiency virus (HIV) can be used as microbicides to help prevent the spread of HIV in human populations. As an industry standard, HIV-neutralizing mAbs are produced as recombinant proteins in mammalian cells, but the high manufacturing costs and limited capacity reduce the ability of target populations in developing countries to gain access to these potentially life-saving medicines. Plants offer a more cost-effective and deployable production platform because they can be grown inexpensively and on a large scale in the region where the products are required. Here we show that the maize-derived HIV-neutralizing mAb 2F5 is assembled correctly in planta and binds to its antigen with the same affinity as 2F5 produced in mammalian cells. Although 2F5 has been produced at high levels in non-plant platforms, the yield in maize seeds is lower than previously achieved with another HIV-neutralizing mAb, 2G12. This suggests that the intrinsic properties of the antibody (e.g. sensitivity to specific proteases) and the environment provided by the production host (e.g. the relative abundance of different proteases, potential transgene silencing) may combine to limit the accumulation of some antibodies on a case-by-case basis. PMID:22965278

  5. Induction of neutralizing antibodies against Tityus serrulatus scorpion toxins by immunization with a mixture of defined synthetic epitopes.

    PubMed

    Alvarenga, L M; Diniz, C R; Granier, C; Chávez-Olórtegui, C

    2002-01-01

    We have used the Spot method of multiple peptide synthesis to prepare sets of immobilized overlapping peptides of uniform size (15 mer), covering the complete amino acid sequences of TsNTxP a non-toxic and immunogenic protein and TsIV, an alpha-type toxin that is the major lethal component of the venom of scorpion Tityus serrulatus. Anti-TsNTxP antibodies binding to peptides, revealed three antigenic regions, one in the N-terminal, the second in the central part and the other in the C-terminal part of TsNTxP. One peptide epitope in the C-terminal part of TsIV was identified with anti-TsIV neutralizing rabbit antibodies. Anti-peptide antibodies were raised against these four peptides all together covalently coupled to keyhole limpet hemocyanin (KLH) and found to neutralize in vitro the toxic effects of the T. serrulatus venom. Quantities of venom equivalent to 13.5 LD(50) were effectively neutralized by 1ml of the anti-peptide serum. The antigenic specificities of the anti-peptides were compared by an indirect enzyme-linked immunosorbent assay (ELISA) using synthetic peptides and crude venoms from T. serrulatus, T. bahiensis, T. cambridgei, T. stigmurus, Androctonus autralis Hector and Centruroides sculpturatus to coat the microtitration plates. The anti-peptide antibodies had a comparable high reactivity with the crude venom of T. serrulatus, moderate binding to T. bahiensis, T. cambridgei, T. stigmurus and Centruroides sculpturatus venoms but were unable to recognize the venom of Androctonus autralis Hector. These results show that by using peptides derived from the sequence of scorpion toxins, the generation of anti-peptide antibodies able to neutralize the cognate venom appears to be an alternative strategy for the easy preparation of antivenoms. PMID:11602284

  6. Structural Studies of Chikungunya Virus-Like Particles Complexed with Human Antibodies: Neutralization and Cell-to-Cell Transmission

    PubMed Central

    Mangala Prasad, Vidya; Wang, Cheng-I; Akahata, Wataru; Ng, Lisa F. P.

    2015-01-01

    ABSTRACT Chikungunya virus is a positive-stranded RNA alphavirus. Structures of chikungunya virus-like particles in complex with strongly neutralizing antibody Fab fragments (8B10 and 5F10) were determined using cryo-electron microscopy and X-ray crystallography. By fitting the crystallographically determined structures of these Fab fragments into the cryo-electron density maps, we show that Fab fragments of antibody 8B10 extend radially from the viral surface and block receptor binding on the E2 glycoprotein. In contrast, Fab fragments of antibody 5F10 bind the tip of the E2 B domain and lie tangentially on the viral surface. Fab 5F10 fixes the B domain rigidly to the surface of the virus, blocking exposure of the fusion loop on glycoprotein E1 and therefore preventing the virus from becoming fusogenic. Although Fab 5F10 can neutralize the wild-type virus, it can also bind to a mutant virus without inhibiting fusion or attachment. Although the mutant virus is no longer able to propagate by extracellular budding, it can, however, enter the next cell by traveling through junctional complexes without being intercepted by a neutralizing antibody to the wild-type virus, thus clarifying how cell-to-cell transmission can occur. IMPORTANCE Alphaviral infections are transmitted mainly by mosquitoes. Chikungunya virus (CHIKV), which belongs to the Alphavirus genus, has a wide distribution in the Old World that has expanded in recent years into the Americas. There are currently no vaccines or drugs against alphaviral infections. Therefore, a better understanding of CHIKV and its associated neutralizing antibodies will aid in the development of effective treatments. PMID:26537684

  7. Motif-Optimized Subtype A HIV Envelope-based DNA Vaccines Rapidly Elicit Neutralizing Antibodies When Delivered Sequentially

    PubMed Central

    Pissani, Franco; Malherbe, Delphine C.; Robins, Harlan; DeFilippis, Victor R.; Park, Byung; Sellhorn, George; Stamatatos, Leonidas; Overbaugh, Julie; Haigwood, Nancy L.

    2012-01-01

    HIV-1 infection results in the development of a diverging quasispecies unique to each infected individual. Envelope (Env)-specific neutralizing antibodies (NAbs) typically develop over months to years after infection and initially are limited to the infecting virus. In some subjects, antibody responses develop that neutralize heterologous isolates (HNAbs), a phenomenon termed broadening of the NAb response. Studies of co-crystalized antibodies and proteins have facilitated the identification of some targets of broadly neutralizing monoclonal antibodies (NmAbs) capable of neutralizing many or most heterologous viruses; however, the ontogeny of these antibodies in vivo remains elusive. We hypothesize that Env protein escape variants stimulate broad NAb development in vivo and could generate such NAbs when used as immunogens. Here we test this hypothesis in rabbits using HIV Env vaccines featuring: (1) use of individual quasispecies env variants derived from an HIV-1 subtype A-infected subject exhibiting high levels of NAbs within the first year of infection that increased and broadened with time; (2) motif optimization of envs to enhance in vivo expression of DNA formulated as vaccines; and (3) a combined DNA plus protein boosting regimen. Vaccines consisted of multiple env variants delivered sequentially and a simpler regimen that utilized only the least and most divergent clones. The simpler regimen was as effective as the more complex approach in generating modest HNAbs and was more efficient when modified, motif-optimized DNA was used in combination with trimeric gp140 protein. This is a rationally designed strategy that facilitates future vaccine design by addressing the difficult problem of generating HNAbs to HIV by empirically testing the immunogenicity of naturally occurring quasispecies env variants. PMID:22749601

  8. High Concentrations of Measles Neutralizing Antibodies and High-Avidity Measles IgG Accurately Identify Measles Reinfection Cases.

    PubMed

    Sowers, Sun B; Rota, Jennifer S; Hickman, Carole J; Mercader, Sara; Redd, Susan; McNall, Rebecca J; Williams, Nobia; McGrew, Marcia; Walls, M Laura; Rota, Paul A; Bellini, William J

    2016-08-01

    In the United States, approximately 9% of the measles cases reported from 2012 to 2014 occurred in vaccinated individuals. Laboratory confirmation of measles in vaccinated individuals is challenging since IgM assays can give inconclusive results. Although a positive reverse transcription (RT)-PCR assay result from an appropriately timed specimen can provide confirmation, negative results may not rule out a highly suspicious case. Detection of high-avidity measles IgG in serum samples provides laboratory evidence of a past immunologic response to measles from natural infection or immunization. High concentrations of measles neutralizing antibody have been observed by plaque reduction neutralization (PRN) assays among confirmed measles cases with high-avidity IgG, referred to here as reinfection cases (RICs). In this study, we evaluated the utility of measuring levels of measles neutralizing antibody to distinguish RICs from noncases by receiver operating characteristic curve analysis. Single and paired serum samples with high-avidity measles IgG from suspected measles cases submitted to the CDC for routine surveillance were used for the analysis. The RICs were confirmed by a 4-fold rise in PRN titer or by RT-quantitative PCR (RT-qPCR) assay, while the noncases were negative by both assays. Discrimination accuracy was high with serum samples collected ≥3 days after rash onset (area under the curve, 0.953; 95% confidence interval [CI], 0.854 to 0.993). Measles neutralizing antibody concentrations of ≥40,000 mIU/ml identified RICs with 90% sensitivity (95% CI, 74 to 98%) and 100% specificity (95% CI, 82 to 100%). Therefore, when serological or RT-qPCR results are unavailable or inconclusive, suspected measles cases with high-avidity measles IgG can be confirmed as RICs by measles neutralizing antibody concentrations of ≥40,000 mIU/ml. PMID:27335386

  9. High Concentrations of Measles Neutralizing Antibodies and High-Avidity Measles IgG Accurately Identify Measles Reinfection Cases

    PubMed Central

    Rota, Jennifer S.; Hickman, Carole J.; Mercader, Sara; Redd, Susan; McNall, Rebecca J.; Williams, Nobia; McGrew, Marcia; Walls, M. Laura; Rota, Paul A.; Bellini, William J.

    2016-01-01

    In the United States, approximately 9% of the measles cases reported from 2012 to 2014 occurred in vaccinated individuals. Laboratory confirmation of measles in vaccinated individuals is challenging since IgM assays can give inconclusive results. Although a positive reverse transcription (RT)-PCR assay result from an appropriately timed specimen can provide confirmation, negative results may not rule out a highly suspicious case. Detection of high-avidity measles IgG in serum samples provides laboratory evidence of a past immunologic response to measles from natural infection or immunization. High concentrations of measles neutralizing antibody have been observed by plaque reduction neutralization (PRN) assays among confirmed measles cases with high-avidity IgG, referred to here as reinfection cases (RICs). In this study, we evaluated the utility of measuring levels of measles neutralizing antibody to distinguish RICs from noncases by receiver operating characteristic curve analysis. Single and paired serum samples with high-avidity measles IgG from suspected measles cases submitted to the CDC for routine surveillance were used for the analysis. The RICs were confirmed by a 4-fold rise in PRN titer or by RT-quantitative PCR (RT-qPCR) assay, while the noncases were negative by both assays. Discrimination accuracy was high with serum samples collected ≥3 days after rash onset (area under the curve, 0.953; 95% confidence interval [CI], 0.854 to 0.993). Measles neutralizing antibody concentrations of ≥40,000 mIU/ml identified RICs with 90% sensitivity (95% CI, 74 to 98%) and 100% specificity (95% CI, 82 to 100%). Therefore, when serological or RT-qPCR results are unavailable or inconclusive, suspected measles cases with high-avidity measles IgG can be confirmed as RICs by measles neutralizing antibody concentrations of ≥40,000 mIU/ml. PMID:27335386

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

    PubMed

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

    2015-01-01

    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. PMID:26416571

  11. Immunoglobulin Gene Insertions and Deletions in the Affinity Maturation of HIV-1 Broadly Reactive Neutralizing Antibodies

    PubMed Central

    Kepler, Thomas B.; Liao, Hua-Xin; Alam, S. Munir; Bhaskarabhatla, Rekha; Zhang, Ruijun; Stewart, Shelley; Anasti, Kara; Kelsoe, Garnett; Parks, Robert; Lloyd, Krissey E.; Stolarchuk, Christina; Pritchett, Jamie; Solomon, Erika; Friberg, Emma; Morris, Lynn; Karim, Salim S. Abdool; Cohen, Myron S.; Walter, Emmanuel; Moody, M. Anthony; Wu, Xueling; Altae-Tran, Han R.; Georgiev, Ivelin S.; Kwong, Peter D.; Boyd, Scott D.; Fire, Andrew Z.; Mascola, John R.; Haynes, Barton F.

    2014-01-01

    Summary Induction of HIV-1 broad neutralizing antibodies (bnAbs) is a goal of HIV-1 vaccine development but has remained challenging partially due to unusual traits of bnAbs, including high somatic hypermutation (SHM) frequencies and in-frame insertions and deletions (indels). Here we examined the propensity and functional requirement for indels within HIV-1 bnAbs. High-throughput sequencing of the immunoglobulin (Ig) VHDJH genes in HIV-1 infected and uninfected individuals revealed that the indel frequency was elevated among HIV-1-infected subjects, with no unique properties attributable to bnAb-producing individuals. This increased indel occurrence depended only on the frequency of SHM point-mutations. Indel-encoded regions were generally proximal to antigen binding sites. Additionally, reconstruction of a HIV-1 CD4-binding site bnAb clonal lineage revealed that a large compound VHDJH indel was required for bnAb activity. Thus, vaccine development should focus on designing regimens targeted at sustained activation of bnAb lineages to achieve the required SHM and indel events. PMID:25211073

  12. Neutralizing antibodies against adeno-associated virus examined prospectively in pediatric patients with hemophilia.

    PubMed

    Li, C; Narkbunnam, N; Samulski, R J; Asokan, A; Hu, G; Jacobson, L J; Manco-Johnson, M J; Monahan, P E

    2012-03-01

    Recombinant adeno-associated virus (rAAV) is a promising gene delivery vector and has recently been used in patients with hemophilia. One limitation of AAV application is that most humans have experienced wild-type AAV serotype 2 exposure, which frequently generates neutralizing antibodies (NAbs) that may inhibit rAAV2 vector transduction. Employing alternative serotypes of rAAV vectors may circumvent this problem. We investigated the development of NAbs in early childhood by examining sera gathered prospectively from 62 children with hemophilia A, participating in a multi-institutional hemophilia clinical trial (the Joint Outcome Study). Clinical applications in hemophilia therapy have been suggested for serotypes AAV2, AAV5 and AAV8, therefore NAbs against these serotypes were serially assayed over a median follow-up of 4 years. NAbs prevalence increased during early childhood for all serotypes. NAbs against AAV2 (43.5%) were observed more frequently and at higher titers compared with both AAV5 (25.8%) and AAV8 (22.6%). NAbs against AAV5 or AAV8 were rarely observed in the absence of co-prevalent and higher titer AAV2 NAbs, suggesting that NAbs to AAV5 and AAV8 were detected following AAV2 exposure due to partial cross-reactivity of AAV2-directed NAbs. The results may guide rational design of clinical trials using alternative AAV serotypes and suggest that younger patients who are given AAV gene therapy will benefit from the lower prevalence of NAbs. PMID:21697954

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

    PubMed Central

    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, W