WorldWideScience

Sample records for neutralizing antibody specificities

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

    OpenAIRE

    Ming Sun; Yue Li; Huiwen Zheng; Yiming Shao

    2016-01-01

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

  2. Identification and specificity of broadly neutralizing antibodies against HIV

    OpenAIRE

    McCoy, Laura E.; Burton, Dennis R.

    2017-01-01

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

  3. In vitro neutralization of prions with PrP(Sc)-specific antibodies.

    Science.gov (United States)

    Taschuk, Ryan; Van der Merwe, Jacques; Marciniuk, Kristen; Potter, Andrew; Cashman, Neil; Griebel, Philip; Napper, Scott

    2015-01-01

    Prion diseases reflect the misfolding of a self-protein (PrP(C)) into an infectious, pathological isomer (PrP(Sc)). By targeting epitopes uniquely exposed by misfolding, our group developed PrP(Sc)-specific vaccines to 3 disease specific epitopes (DSEs). Here, antibodies induced by individual DSE vaccines are evaluated for their capacity to neutralize prions in vitro. For both purified antibodies and immunoreactive sera, the PrP(Sc)-specific antibodies were equally effective in neutralizing prions. Further, there was no significant increase in neutralizing activity when multiple DSEs were targeted within an assay. At a low antibody concentration, the PrP(Sc)-specific antibodies matched the neutralization achieved by an antibody that may act via both PrP(C) and PrP(Sc). At higher doses, however, this pan-specific antibody was more effective, potentially due to a combined deactivation of PrP(Sc) and depletion of PrP(C).

  4. Mechanisms of equine infectious anemia virus escape from neutralizing antibody responses define epitope specificity.

    Science.gov (United States)

    Sponseller, Brett A; Clark, Sandra K; Friedrich, Rachel A

    2012-08-01

    Determining mechanisms of viral escape to particular epitopes recognized by virus-neutralizing antibody can facilitate characterization of host-neutralizing antibody responses as type- versus group-specific, and provides necessary information for vaccine development. Our study reveals that a single N-glycan located in the 5' region of the Wyoming wild-type equine infectious anemia virus (EIAV) principal neutralizing domain (PND) accounts for the differences in neutralization phenotype observed between PND variants, while variations in charged amino acids within the PND do not appear to play a key role in viral escape. Site-directed mutagenesis and peptide mapping of a conserved epitope to neutralizing antibody in the 3' region of the PND showed rapid selective pressure for acquisition of a 5' PND N-glycan responsible for defining the specificity of the neutralizing-antibody response.

  5. Relative contributions of measles virus hemagglutinin- and fusion protein- specific serum antibodies to virus neutralization.

    NARCIS (Netherlands)

    R.L. de Swart (Rik); S. Yüksel (Selma); A.D.M.E. Osterhaus (Albert)

    2005-01-01

    textabstractThe relative contribution of measles virus hemagglutinin (H)- or fusion protein (F)-specific antibodies to virus neutralization (VN) has not been demonstrated. We have depleted these specific antibodies from sera collected from young adults, who had been vaccinated during childhood, by

  6. Relative contributions of measles virus hemagglutinin- and fusion protein- specific serum antibodies to virus neutralization.

    NARCIS (Netherlands)

    R.L. de Swart (Rik); S. Yüksel (Selma); A.D.M.E. Osterhaus (Albert)

    2005-01-01

    textabstractThe relative contribution of measles virus hemagglutinin (H)- or fusion protein (F)-specific antibodies to virus neutralization (VN) has not been demonstrated. We have depleted these specific antibodies from sera collected from young adults, who had been vaccinated during childhood, by p

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

    Directory of Open Access Journals (Sweden)

    Sharad P Adekar

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

  8. Radioimmunoassay for detection of VP1 specific neutralizing antibodies of foot and mouse disease virus

    Energy Technology Data Exchange (ETDEWEB)

    Patzer, E.J.; Jackson, M.L. (Genentech, Inc., South San Francisco CA (USA)); Moore, D.M. (U.S. Department of Agriculture, Plum Island Animal Disease Center, Greenport, NY (USA))

    1985-01-01

    A solid-phase radioimmunoassay was developed for the detection of antibodies against a specific region of the VP1 protein of the A24 and O1 serotypes of foot and mouth disease virus. The antibody titers from the radioimmunoassay showed a positive correlation with neutralizing antibody titers determined by a mouse protection assay. The specificity of the assay resides in the peptide used as antigen. The assay is rapid, reproducible and does not require the use of whole virions.

  9. Strain-specific V3 and CD4 binding site autologous HIV-1 neutralizing antibodies select neutralization-resistant viruses

    Science.gov (United States)

    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; Shaw, George M.; Sodroski, Joseph G.; Liao, Hua-Xin; Montefiori, David C.; Hraber, Peter T.; Korber, Bette T.; Haynes, Barton F.

    2015-01-01

    Summary The third variable (V3) loop and the CD4 binding site (CD4bs) of the HIV-1 envelope are frequently targeted by neutralizing antibodies (nAbs) in infected individuals. In chronic infection, HIV-1 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. PMID:26355218

  10. Induction of epitope-specific neutralizing antibodies against West Nile virus.

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    Oliphant, Theodore; Nybakken, Grant E; Austin, S Kyle; Xu, Qing; Bramson, Jonathan; Loeb, Mark; Throsby, Mark; Fremont, Daved H; Pierson, Theodore C; Diamond, Michael S

    2007-11-01

    Previous studies have established that an epitope on the lateral ridge of domain III (DIII-lr) of West Nile virus (WNV) envelope (E) protein is recognized by strongly neutralizing type-specific antibodies. In contrast, an epitope against the fusion loop in domain II (DII-fl) is recognized by flavivirus cross-reactive antibodies with less neutralizing potential. Using gain- and loss-of-function E proteins and wild-type and variant WNV reporter virus particles, we evaluated the expression pattern and activity of antibodies against the DIII-lr and DII-fl epitopes in mouse and human serum after WNV infection. In mice, immunoglobulin M (IgM) antibodies to the DIII-lr epitope were detected at low levels at day 6 after infection. However, compared to IgG responses against other epitopes in DI and DII, which were readily detected at day 8, the development of IgG against DIII-lr epitope was delayed and did not appear consistently until day 15. This late time point is notable since almost all death after WNV infection in mice occurs by day 12. Nonetheless, at later time points, DIII-lr antibodies accumulated and comprised a significant fraction of the DIII-specific IgG response. In sera from infected humans, DIII-lr antibodies were detected at low levels and did not correlate with clinical outcome. In contrast, antibodies to the DII-fl were detected in all human serum samples and encompassed a significant percentage of the anti-E protein response. Our experiments suggest that the highly neutralizing DIII-lr IgG antibodies have little significant role in primary infection and that the antibody response of humans may be skewed toward the induction of cross-reactive, less-neutralizing antibodies.

  11. Induction of Epitope-Specific Neutralizing Antibodies against West Nile Virus▿

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    Oliphant, Theodore; Nybakken, Grant E.; Austin, S. Kyle; Xu, Qing; Bramson, Jonathan; Loeb, Mark; Throsby, Mark; Fremont, Daved H.; Pierson, Theodore C.; Diamond, Michael S.

    2007-01-01

    Previous studies have established that an epitope on the lateral ridge of domain III (DIII-lr) of West Nile virus (WNV) envelope (E) protein is recognized by strongly neutralizing type-specific antibodies. In contrast, an epitope against the fusion loop in domain II (DII-fl) is recognized by flavivirus cross-reactive antibodies with less neutralizing potential. Using gain- and loss-of-function E proteins and wild-type and variant WNV reporter virus particles, we evaluated the expression pattern and activity of antibodies against the DIII-lr and DII-fl epitopes in mouse and human serum after WNV infection. In mice, immunoglobulin M (IgM) antibodies to the DIII-lr epitope were detected at low levels at day 6 after infection. However, compared to IgG responses against other epitopes in DI and DII, which were readily detected at day 8, the development of IgG against DIII-lr epitope was delayed and did not appear consistently until day 15. This late time point is notable since almost all death after WNV infection in mice occurs by day 12. Nonetheless, at later time points, DIII-lr antibodies accumulated and comprised a significant fraction of the DIII-specific IgG response. In sera from infected humans, DIII-lr antibodies were detected at low levels and did not correlate with clinical outcome. In contrast, antibodies to the DII-fl were detected in all human serum samples and encompassed a significant percentage of the anti-E protein response. Our experiments suggest that the highly neutralizing DIII-lr IgG antibodies have little significant role in primary infection and that the antibody response of humans may be skewed toward the induction of cross-reactive, less-neutralizing antibodies. PMID:17715236

  12. Structural basis of selectivity and neutralizing activity of a TGFα/epiregulin specific antibody.

    Science.gov (United States)

    Boyles, Jeffrey S; Atwell, Shane; Druzina, Zhanna; Heuer, Josef G; Witcher, Derrick R

    2016-11-01

    Recent studies have implicated a role of the epidermal growth factor receptor (EGFR) pathway in kidney disease. Skin toxicity associated with therapeutics which completely block the EGFR pathway precludes their use in chronic dosing. Therefore, we developed antibodies which specifically neutralize the EGFR ligands TGFα (transforming growth factor-alpha) and epiregulin but not EGF (epidermal growth factor), amphiregulin, betacellulin, HB-EGF (heparin-binding epidermal growth factor), or epigen. The epitope of one such neutralizing antibody, LY3016859, was characterized in detail to elucidate the structural basis for ligand specificity. Here we report a crystal structure of the LY3016859 Fab fragment in complex with soluble human TGFα. Our data demonstrate a conformational epitope located primarily within the C-terminal subdomain of the ligand. In addition, point mutagenesis experiments were used to highlight specific amino acids which are critical for both antigen binding and neutralization, most notably Ala(41) , Glu(44) , and His(45) . These results illustrate the structural basis for the ligand specificity/selectivity of LY3016859 and could also provide insight into further engineering to alter specificity and/or affinity of LY3016859. © 2016 The Protein Society.

  13. Type- and Subcomplex-Specific Neutralizing Antibodies against Domain III of Dengue Virus Type 2 Envelope Protein Recognize Adjacent Epitopes▿

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    Sukupolvi-Petty, Soila; Austin, S. Kyle; Purtha, Whitney E.; Oliphant, Theodore; Nybakken, Grant E.; Schlesinger, Jacob J.; Roehrig, John T.; Gromowski, Gregory D.; Barrett, Alan D.; Fremont, Daved H.; Diamond, Michael S.

    2007-01-01

    Neutralization of flaviviruses in vivo correlates with the development of an antibody response against the viral envelope (E) protein. Previous studies demonstrated that monoclonal antibodies (MAbs) against an epitope on the lateral ridge of domain III (DIII) of the West Nile virus (WNV) E protein strongly protect against infection in animals. Based on X-ray crystallography and sequence analysis, an analogous type-specific neutralizing epitope for individual serotypes of the related flavivirus dengue virus (DENV) was hypothesized. Using yeast surface display of DIII variants, we defined contact residues of a panel of type-specific, subcomplex-specific, and cross-reactive MAbs that recognize DIII of DENV type 2 (DENV-2) and have different neutralizing potentials. Type-specific MAbs with neutralizing activity against DENV-2 localized to a sequence-unique epitope on the lateral ridge of DIII, centered at the FG loop near residues E383 and P384, analogous in position to that observed with WNV-specific strongly neutralizing MAbs. Subcomplex-specific MAbs that bound some but not all DENV serotypes and neutralized DENV-2 infection recognized an adjacent epitope centered on the connecting A strand of DIII at residues K305, K307, and K310. In contrast, several MAbs that had poor neutralizing activity against DENV-2 and cross-reacted with all DENV serotypes and other flaviviruses recognized an epitope with residues in the AB loop of DIII, a conserved region that is predicted to have limited accessibility on the mature virion. Overall, our experiments define adjacent and structurally distinct epitopes on DIII of DENV-2 which elicit type-specific, subcomplex-specific, and cross-reactive antibodies with different neutralizing potentials. PMID:17881453

  14. Type- and subcomplex-specific neutralizing antibodies against domain III of dengue virus type 2 envelope protein recognize adjacent epitopes.

    Science.gov (United States)

    Sukupolvi-Petty, Soila; Austin, S Kyle; Purtha, Whitney E; Oliphant, Theodore; Nybakken, Grant E; Schlesinger, Jacob J; Roehrig, John T; Gromowski, Gregory D; Barrett, Alan D; Fremont, Daved H; Diamond, Michael S

    2007-12-01

    Neutralization of flaviviruses in vivo correlates with the development of an antibody response against the viral envelope (E) protein. Previous studies demonstrated that monoclonal antibodies (MAbs) against an epitope on the lateral ridge of domain III (DIII) of the West Nile virus (WNV) E protein strongly protect against infection in animals. Based on X-ray crystallography and sequence analysis, an analogous type-specific neutralizing epitope for individual serotypes of the related flavivirus dengue virus (DENV) was hypothesized. Using yeast surface display of DIII variants, we defined contact residues of a panel of type-specific, subcomplex-specific, and cross-reactive MAbs that recognize DIII of DENV type 2 (DENV-2) and have different neutralizing potentials. Type-specific MAbs with neutralizing activity against DENV-2 localized to a sequence-unique epitope on the lateral ridge of DIII, centered at the FG loop near residues E383 and P384, analogous in position to that observed with WNV-specific strongly neutralizing MAbs. Subcomplex-specific MAbs that bound some but not all DENV serotypes and neutralized DENV-2 infection recognized an adjacent epitope centered on the connecting A strand of DIII at residues K305, K307, and K310. In contrast, several MAbs that had poor neutralizing activity against DENV-2 and cross-reacted with all DENV serotypes and other flaviviruses recognized an epitope with residues in the AB loop of DIII, a conserved region that is predicted to have limited accessibility on the mature virion. Overall, our experiments define adjacent and structurally distinct epitopes on DIII of DENV-2 which elicit type-specific, subcomplex-specific, and cross-reactive antibodies with different neutralizing potentials.

  15. Kinetics of Epstein-Barr Virus (EBV) Neutralizing and Virus-Specific Antibodies after Primary Infection with EBV.

    Science.gov (United States)

    Bu, Wei; Hayes, Gregory M; Liu, Hui; Gemmell, Lorraine; Schmeling, David O; Radecki, Pierce; Aguilar, Fiona; Burbelo, Peter D; Woo, Jennifer; Balfour, Henry H; Cohen, Jeffrey I

    2016-04-01

    Prospective studies of antibodies to multiple Epstein-Barr virus (EBV) proteins and EBV neutralizing antibodies in the same individuals before, during, and after primary EBV infection have not been reported. We studied antibody responses to EBV in college students who acquired primary EBV infection during prospective surveillance and correlated the kinetics of antibody response with the severity of disease. Neutralizing antibodies and enzyme-linked immunosorbent assay (ELISA) antibodies to gp350, the major target of neutralizing antibody, reached peak levels at medians of 179 and 333 days after the onset of symptoms of infectious mononucleosis, respectively. No clear correlation was found between the severity of the symptoms of infectious mononucleosis and the peak levels of antibody to individual viral proteins or to neutralizing antibody. In summary, we found that titers of neutralizing antibody and antibodies to multiple EBV proteins increase over many months after primary infection with EBV.

  16. Precisely Molded Nanoparticle Displaying DENV-E Proteins Induces Robust Serotype-Specific Neutralizing Antibody Responses.

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    Stefan W Metz

    2016-10-01

    Full Text Available Dengue virus (DENV is the causative agent of dengue fever and dengue hemorrhagic fever. The virus is endemic in over 120 countries, causing over 350 million infections per year. Dengue vaccine development is challenging because of the need to induce simultaneous protection against four antigenically distinct DENV serotypes and evidence that, under some conditions, vaccination can enhance disease due to specific immunity to the virus. While several live-attenuated tetravalent dengue virus vaccines display partial efficacy, it has been challenging to induce balanced protective immunity to all 4 serotypes. Instead of using whole-virus formulations, we are exploring the potentials for a particulate subunit vaccine, based on DENV E-protein displayed on nanoparticles that have been precisely molded using Particle Replication in Non-wetting Template (PRINT technology. Here we describe immunization studies with a DENV2-nanoparticle vaccine candidate. The ectodomain of DENV2-E protein was expressed as a secreted recombinant protein (sRecE, purified and adsorbed to poly (lactic-co-glycolic acid (PLGA nanoparticles of different sizes and shape. We show that PRINT nanoparticle adsorbed sRecE without any adjuvant induces higher IgG titers and a more potent DENV2-specific neutralizing antibody response compared to the soluble sRecE protein alone. Antigen trafficking indicate that PRINT nanoparticle display of sRecE prolongs the bio-availability of the antigen in the draining lymph nodes by creating an antigen depot. Our results demonstrate that PRINT nanoparticles are a promising platform for delivering subunit vaccines against flaviviruses such as dengue and Zika.

  17. Precisely Molded Nanoparticle Displaying DENV-E Proteins Induces Robust Serotype-Specific Neutralizing Antibody Responses

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    Hoekstra, Gabriel; Yi, Xianwen; Stone, Michelle; Horvath, Katie; Miley, Michael J.; DeSimone, Joseph; Luft, Chris J.; de Silva, Aravinda M.

    2016-01-01

    Dengue virus (DENV) is the causative agent of dengue fever and dengue hemorrhagic fever. The virus is endemic in over 120 countries, causing over 350 million infections per year. Dengue vaccine development is challenging because of the need to induce simultaneous protection against four antigenically distinct DENV serotypes and evidence that, under some conditions, vaccination can enhance disease due to specific immunity to the virus. While several live-attenuated tetravalent dengue virus vaccines display partial efficacy, it has been challenging to induce balanced protective immunity to all 4 serotypes. Instead of using whole-virus formulations, we are exploring the potentials for a particulate subunit vaccine, based on DENV E-protein displayed on nanoparticles that have been precisely molded using Particle Replication in Non-wetting Template (PRINT) technology. Here we describe immunization studies with a DENV2-nanoparticle vaccine candidate. The ectodomain of DENV2-E protein was expressed as a secreted recombinant protein (sRecE), purified and adsorbed to poly (lactic-co-glycolic acid) (PLGA) nanoparticles of different sizes and shape. We show that PRINT nanoparticle adsorbed sRecE without any adjuvant induces higher IgG titers and a more potent DENV2-specific neutralizing antibody response compared to the soluble sRecE protein alone. Antigen trafficking indicate that PRINT nanoparticle display of sRecE prolongs the bio-availability of the antigen in the draining lymph nodes by creating an antigen depot. Our results demonstrate that PRINT nanoparticles are a promising platform for delivering subunit vaccines against flaviviruses such as dengue and Zika. PMID:27764114

  18. A neutralization test for specific detection of Nipah virus antibodies using pseudotyped vesicular stomatitis virus expressing green fluorescent protein.

    Science.gov (United States)

    Kaku, Yoshihiro; Noguchi, Akira; Marsh, Glenn A; McEachern, Jennifer A; Okutani, Akiko; Hotta, Kozue; Bazartseren, Boldbaatar; Fukushi, Shuetsu; Broder, Christopher C; Yamada, Akio; Inoue, Satoshi; Wang, Lin-Fa

    2009-09-01

    Nipah virus (NiV) is a new zoonotic paramyxovirus that emerged in 1998 and is now classified in the genus Henipavirus along with the closely related Hendra virus (HeV). NiV is highly pathogenic in several vertebrate species including humans, and the lack of available vaccines or specific treatment restricts it to biosafety level 4 (BSL4) containment. A serum neutralization test was developed for measuring NiV neutralizing antibodies under BSL2 conditions using a recombinant vesicular stomatitis virus (VSV) expressing green fluorescent protein (GFP) and bearing the F and G proteins of NiV (VSV-NiV-GFP). The neutralization titers were obtained by counting GFP-expressing cells or by measuring fluorescence. The performance of this new assay was compared against the conventional test using live NiV with panels of sera from several mammalian species, including sera from NiV outbreaks, experimental infections, as well as HeV-specific sera. The results obtained with the VSV-NiV-GFP based test correlated with those obtained using live NiV. Using a 50% reduction in VSV-NiV-GFP infected cells as the cut-off for neutralization, this new assay demonstrated its potential as an effective tool for detecting NiV neutralizing antibodies under BSL2 containment with greater speed, sensitivity and safety as compared to the conventional NiV serum neutralization test.

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

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

    2016-08-30

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

  20. Relative contribution of dengue prM- and E-specific polyclonal antibodies to neutralization and enhancement.

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    Rodpothong, P; Boonarkart, Ch; Ruangrung, K; Onsirisakul, N; Kanistanon, D; Auewarakul, P

    Viral surface proteins, premembrane protein (prM) and envelope (E) protein have been shown to induce a production of antibodies that are involved in both enhancement and neutralization. To explore the feasibility of modifying the relative immune responses to prM and E proteins, four DNA constructs were created and administered into groups of Balb/c mice; pPW01 contains prM and E genes of DENV1, pPW02 contains prM and E genes of DENV2, pPW03 contains DENV1 prM and DENV2 E, and pPW04 contains DENV2 prM and DENV1 E. Exchange of either prM or E from a heterologous serotype does not appear to have an effect on the immunogenicity of the proteins. We have proved that the chimeric pPW03 and pPW04 constructs can produce humoral response in mice. Immunized sera were subjected to neutralization and enhancement assays against DENV2. The results showed that only serotype-specific anti-E antibodies conferred protective function, while the cross-reactive anti-E and anti-prM enhanced infection. In addition, the enhancement of DENV2 infection exhibited a serotype-preference for anti-E antibodies while such response was not observed with anti-prM, reflecting a degree of structural conservation of prM. Taken together, neutralization and enhancement appeared to occur at the same time during the course of infection. Successful prevention of severe symptoms of DENV infection depends on the ability to induce high levels of neutralizing antibodies to subdue the effect of enhancing antibodies.

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

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    Kathryn A K Finton

    2014-09-01

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

  2. "Unconventional" Neutralizing Activity of Antibodies Against HIV

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

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

  3. Plasmodium falciparum synthetic LbL microparticle vaccine elicits protective neutralizing antibody and parasite-specific cellular immune responses.

    Science.gov (United States)

    Powell, Thomas J; Tang, Jie; Derome, Mary E; Mitchell, Robert A; Jacobs, Andrea; Deng, Yanhong; Palath, Naveen; Cardenas, Edwin; Boyd, James G; Nardin, Elizabeth

    2013-04-01

    Epitopes of the circumsporozoite (CS) protein of Plasmodium falciparum, the most pathogenic species of the malaria parasite, have been shown to elicit protective immunity in experimental animals and human volunteers. The mechanisms of immunity include parasite-neutralizing antibodies that can inhibit parasite motility in the skin at the site of infection and in the bloodstream during transit to the hepatocyte host cell and also block interaction with host cell receptors on hepatocytes. In addition, specific CD4+ and CD8+ cellular mechanisms target the intracellular hepatic forms, thus preventing release of erythrocytic stage parasites from the infected hepatocyte and the ensuing blood stage cycle responsible for clinical disease. An innovative method for producing particle vaccines, layer-by-layer (LbL) fabrication of polypeptide films on solid CaCO3 cores, was used to produce synthetic malaria vaccines containing a tri-epitope CS peptide T1BT comprising the antibody epitope of the CS repeat region (B) and two T-cell epitopes, the highly conserved T1 epitope and the universal epitope T. Mice immunized with microparticles loaded with T1BT peptide developed parasite-neutralizing antibodies and malaria-specific T-cell responses including cytotoxic effector T-cells. Protection from liver stage infection following challenge with live sporozoites from infected mosquitoes correlated with neutralizing antibody levels. Although some immunized mice with low or undetectable neutralizing antibodies were also protected, depletion of T-cells prior to challenge resulted in the majority of mice remaining resistant to challenge. In addition, mice immunized with microparticles bearing only T-cell epitopes were not protected, demonstrating that cellular immunity alone was not sufficient for protective immunity. Although the microparticles without adjuvant were immunogenic and protective, a simple modification with the lipopeptide TLR2 agonist Pam3Cys increased the potency and

  4. Variant-specific monoclonal and group-specific polyclonal human immunodeficiency virus type 1 neutralizing antibodies raised with synthetic peptides from the gp120 third variable domain.

    Science.gov (United States)

    Laman, J D; Schellekens, M M; Abacioglu, Y H; Lewis, G K; Tersmette, M; Fouchier, R A; Langedijk, J P; Claassen, E; Boersma, W J

    1992-03-01

    The third variable (V3) domain of the human immunodeficiency virus type 1 (HIV-1) external membrane glycoprotein gp120 is of crucial importance in eliciting neutralizing antibodies in infected persons. Polyclonal (PAb) and monoclonal (MAb) antibodies directed against selected epitopes in the V3 domain are valuable tools for analysis of the involvement of such sequences in neutralization and for definition of the relation between amino acid variability and immunological cross-reactions. The aim of this study was to obtain such site-specific antibodies. By using synthetic peptides derived from the V3 domain, a group-specific neutralizing PAb, two high-affinity HIV-1 IIIB neutralizing MAb, and two nonneutralizing MAb were raised. A 15-amino-acid peptide overlapping the tip of the V3 domain of HIV-1 MN was used to produce a rabbit PAb (W0/07). This PAb inhibited syncytium formation induced by HIV-1 IIIB and four field isolates. A similar IIIB-derived peptide was used to generate two murine immunoglobulin G1 (IgG1) MAb (IIIB-V3-13 and IIIB-V3-34). Pepscan analysis mapped the binding site of IIIB-V3-34 to the sequence IRIQRGPGR. The Kds of IIIB-V3-13 and IIIB-V3-34 for gp120 were 6.8 x 10(-11) and 1.6 x 10(-10) M, respectively. These MAb neutralized IIIB but not MN and inhibited syncytium formation induced by IIIB. They are applicable in enzyme-linked immunosorbent assays, immunocytochemistry, and flow cytometry. A peptide covering the left base of the V3 domain was used to generate two murine IgG1 MAb (IIIB-V3-21 and IIIB-V3-26). The binding site of IIIB-V3-21 was mapped to the sequence INCTRPN. These MAb did not neutralize HIV-1 and did not inhibit syncytium formation. This study supports the notion that HIV-1 neutralizing antibodies suitable for multiassay performance can be obtained with synthetic peptides and that high-affinity MAb can be generated. Such site-specific antibodies are useful reagents in the analysis of HIV-1 neutralization. In addition, the cross-neutralization

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

    Science.gov (United States)

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

    2016-02-24

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

  6. 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

    Energy Technology Data Exchange (ETDEWEB)

    Bates, John T. [The Vanderbilt Vaccine Center, Departments of Microbiology, and Immunology, Vanderbilt University Medical Center, Nashville, TN (United States); Keefer, Christopher J. [The Vanderbilt Vaccine Center, Departments of Pediatrics, Microbiology, and Immunology, Vanderbilt University Medical Center, Nashville, TN (United States); Slaughter, James C. [The Vanderbilt Vaccine Center, Departments of Biostatistics and Pathology, Microbiology, and Immunology, Vanderbilt University Medical Center, Nashville, TN (United States); Kulp, Daniel W. [IAVI Neutralizing Antibody Center and Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, CA (United States); Schief, William R. [IAVI Neutralizing Antibody Center and Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, CA (United States); Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery, The Scripps Research Institute, La Jolla, CA (United States); Crowe, James E., E-mail: james.crowe@vanderbilt.edu [The Vanderbilt Vaccine Center, Departments of Microbiology, and Immunology, Vanderbilt University Medical Center, Nashville, TN (United States); The Vanderbilt Vaccine Center, Departments of Pediatrics, Microbiology, and Immunology, Vanderbilt University Medical Center, Nashville, TN (United States)

    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.

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

    Science.gov (United States)

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

    2015-01-01

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

  8. Sensitive neutralization test for rubella antibody.

    Science.gov (United States)

    Sato, H; Albrecht, P; Krugman, S; Ennis, F A

    1979-01-01

    A modified rubella virus plaque neutralization test for measuring rubella antibody was developed based on the potentiation of the virus-antibody complex by heterologous anti-immunoglobulin. The test is highly sensitive, yielding titers on the average 50 to 100 times higher than the haemagglutination inhibition test or the conventional plaque neutralization test. The sensitivity of this enhanced neutralization test is somewhat limited by the existence of a prozone phenomenon which precludes testing of low-titered sera below a dilution of 1:16. No prozone effect was observed with cerebrospinal fluids. The specificity of the enhanced neutralization test was determined by seroconversion of individuals receiving rubella vaccine. Although the rubella hemagglutination inhibition test remains the test of choice in routine diagnostic and surveillance work, the enhanced rubella neutralization test is particularly useful in monitoring low-level antibody in the cerebrospinal fluid in patients with neurological disorders and in certain instances of vaccine failure. PMID:107192

  9. HIV-1 specific antibody titers and neutralization among chronically infected patients on long-term suppressive antiretroviral therapy (ART: a cross-sectional study.

    Directory of Open Access Journals (Sweden)

    Johannes S Gach

    Full Text Available The majority of potent and broadly neutralizing antibodies against HIV-1 have been isolated from untreated patients with acute or chronic infection. To assess the extent of HIV-1 specific antibody response and neutralization after many years of virologic suppression from potent combination ART, we examined antibody binding titers and neutralization of 51 patients with chronic HIV-1 infection on suppressive ART for at least three years. In this cross-sectional analysis, we found high antibody titers against gp120, gp41, and the membrane proximal external region (MPER in 59%, 43%, and 27% of patients, respectively. We observed significantly higher endpoint binding titers for gp120 and gp41 for patients with >10 compared to ≤ 10 years of detectable HIV RNA. Additionally, we observed higher median gp120 and gp41 antibody titers in patients with HIV RNA 10 years of detectable HIV RNA (8/20 [40.0%] versus 3/31 [9.7%] for ≤ 10 years, p = 0.02 and a trend toward greater neutralization in patients with ≤ 5 years of HIV RNA 5 years, p = 0.08. All patients with neutralizing activity mediated successful phagocytosis of VLPs by THP-1 cells after antibody opsonization. Our findings of highly specific antibodies to several structural epitopes of HIV-1 with antibody effector functions and neutralizing activity after long-term suppressive ART, suggest continuous antigenic stimulation and evolution of HIV-specific antibody response occurs before and after suppression with ART. These patients, particularly those with slower HIV progression and more time with detectable viremia prior to initiation of suppressive ART, are a promising population to identify and further study functional antibodies against HIV-1.

  10. Peste des petits ruminants virus-like particles induce both complete virus-specific antibodies and virus neutralizing antibodies in mice.

    Science.gov (United States)

    Liu, Fuxiao; Wu, Xiaodong; Zou, Yanli; Li, Lin; Wang, Zhiliang

    2015-03-01

    Peste des petits ruminants virus (PPRV), an etiological agent of peste des petits ruminants (PPR), is classified into the genus Morbillivirus in the family Paramyxoviridae. In a previous study, a recombinant baculovirus has been constructed to co-express the PPRV matrix (M), haemagglutinin (H) and nucleocapsid (N) proteins in insect cells, causing budding of PPR virus-like particles (VLPs) from insect cell membranes by viewing of ultrathin section with a transmission electron microscope. In this follow-up study, these PPR VLPs were purified by sucrose density gradient centrifugation for immunizing mice twice. Three weeks post-primary immunization and 2 weeks post-secondary immunization, all serum samples were obtained and subsequently subjected to indirect ELISA detection on complete virus-specific antibodies. In addition, all serum samples, which were collected 2 weeks post-secondary immunization, were used for virus neutralization test on PPRV neutralizing antibodies. The results showed that the purified PPR VLPs induced both types of antibodies mentioned above in mice, indicating a given potential of VLP-based vaccine candidate against PPR. Copyright © 2014 Elsevier B.V. All rights reserved.

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Science.gov (United States)

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

    2016-10-04

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

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

    Directory of Open Access Journals (Sweden)

    Caitlin E. Mullarkey

    2016-10-01

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

  14. Variant-specific monoclonal and group-specific polyclonal human immunodeficiency virus type 1 neutralizing antibodies raised with synthetic peptides from the gp120 third variable domain.

    OpenAIRE

    Laman, J. D.; Schellekens, M M; Abacioglu, Y H; Lewis, G K; Tersmette, M; Fouchier, R A; Langedijk, J. P.; Claassen, E.; Boersma, W J

    1992-01-01

    The third variable (V3) domain of the human immunodeficiency virus type 1 (HIV-1) external membrane glycoprotein gp120 is of crucial importance in eliciting neutralizing antibodies in infected persons. Polyclonal (PAb) and monoclonal (MAb) antibodies directed against selected epitopes in the V3 domain are valuable tools for analysis of the involvement of such sequences in neutralization and for definition of the relation between amino acid variability and immunological cross-reactions. The ai...

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

    Directory of Open Access Journals (Sweden)

    Shelly J Krebs

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

  16. Human peripheral blood lymphocytes from recently vaccinated individuals produce both type-specific and intertypic cross-reacting neutralizing antibody on in vitro stimulation with one type of poliovirus.

    NARCIS (Netherlands)

    F.G.C.M. Uytdehaag (Fons); H.G. Loggen; T. Logtenberg (Ton); R.A. Lichtveld (Rob); G. van Steenis (Bert); J.A.A.M. van Asten (Jack); A.D.M.E. Osterhaus (Albert)

    1985-01-01

    textabstractAn in vitro system of poliovirus-specific antibody production by peripheral blood B cells on stimulation by the virus has been developed. Virus-neutralizing antibodies in culture supernatant fluids, or virus-specific antibody-secreting cells (ASC) were detected by microneutralization ass

  17. Human peripheral blood lymphocytes from recently vaccinated individuals produce both type-specific and intertypic cross-reacting neutralizing antibody on in vitro stimulation with one type of poliovirus.

    NARCIS (Netherlands)

    F.G.C.M. Uytdehaag (Fons); H.G. Loggen; T. Logtenberg (Ton); R.A. Lichtveld (Rob); G. van Steenis (Bert); J.A.A.M. van Asten (Jack); A.D.M.E. Osterhaus (Albert)

    1985-01-01

    textabstractAn in vitro system of poliovirus-specific antibody production by peripheral blood B cells on stimulation by the virus has been developed. Virus-neutralizing antibodies in culture supernatant fluids, or virus-specific antibody-secreting cells (ASC) were detected by microneutralization ass

  18. Neutralizing Antibody Response after Intramuscular Purified Vero Cell Rabies Vaccination (PVRV in Iranian Patients with Specific Medical Conditions.

    Directory of Open Access Journals (Sweden)

    Pooneh Rahimi

    Full Text Available Post exposure prophylaxis using one of the WHO-approved vaccines is the method of choice for preventing rabies. Abnormal immune function in patients with some specific medical conditions, such as pregnancy, chronic hepatitis B virus infection, different types of cancers like lymphoma, diabetes I and II, corticosteroid consumption by patients with rheumatoid arthritis and lupus erythematosus, could impair the immunologic response to various vaccines. The immune response to rabies vaccination has never been examined in patients with any of these described medical conditions. This study purposed to evaluate the neutralyzing antibody response after vaccination with purified Vero cell rabies vaccine (PVRV according to the WHO-recommended Post-Exposure Prophylaxis (PEP "ESSEN" regimen.Thirty healthy volunteers and 50 volunteers with different medical conditions who were exposed to a suspected rabid animal in the 2nd or 3rd category of exposure received 5 doses of PVRV under the ESSEN protocol. Three blood samples were collected on days 0 (before the first dose, 14, and 35. The anti-rabies antibody titer was measured using the Rapid Fluorescent Foci Inhibition Test (RFFIT and an ELISA Bio-Rad, Platelia, Rabies II kit.All subjects reached NAb titers above 0.5 IU/ml by day 14 after vaccination. On day 35 (1 week after receiving the last rabies vaccine, anti-rabies antibodies were in the protective level (>0.5 IU/ml in both groups. There was no statistically significant difference in anti-rabies antibody response due to the type of exposure (category 2 or 3, and successful seroconversion was confirmed in both groups.In conclusion, the ESSEN protocol using the PVRV vaccine is sufficient for rabies prophylaxis in patients with specific medical conditions.

  19. Broadly Neutralizing Antibodies for HIV Eradication.

    Science.gov (United States)

    Stephenson, Kathryn E; Barouch, Dan H

    2016-02-01

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

  20. Functional characterization of two scFv-Fc antibodies from an HIV controller selected on soluble HIV-1 Env complexes: a neutralizing V3- and a trimer-specific gp41 antibody.

    Directory of Open Access Journals (Sweden)

    Maria Trott

    Full Text Available HIV neutralizing antibodies (nAbs represent an important tool in view of prophylactic and therapeutic applications for HIV-1 infection. Patients chronically infected by HIV-1 represent a valuable source for nAbs. HIV controllers, including long-term non-progressors (LTNP and elite controllers (EC, represent an interesting subgroup in this regard, as here nAbs can develop over time in a rather healthy immune system and in the absence of any therapeutic selection pressure. In this study, we characterized two particular antibodies that were selected as scFv antibody fragments from a phage immune library generated from an LTNP with HIV neutralizing antibodies in his plasma. The phage library was screened on recombinant soluble gp140 envelope (Env proteins. Sequencing the selected peptide inserts revealed two major classes of antibody sequences. Binding analysis of the corresponding scFv-Fc derivatives to various trimeric and monomeric Env constructs as well as to peptide arrays showed that one class, represented by monoclonal antibody (mAb A2, specifically recognizes an epitope localized in the pocket binding domain of the C heptad repeat (CHR in the ectodomain of gp41, but only in the trimeric context. Thus, this antibody represents an interesting tool for trimer identification. MAb A7, representing the second class, binds to structural elements of the third variable loop V3 and neutralizes tier 1 and tier 2 HIV-1 isolates of different subtypes with matching critical amino acids in the linear epitope sequence. In conclusion, HIV controllers are a valuable source for the selection of functionally interesting antibodies that can be selected on soluble gp140 proteins with properties from the native envelope spike.

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

    Science.gov (United States)

    Hua, Casey K; Ackerman, Margaret E

    2016-08-01

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

  2. A Single 17D Yellow Fever Vaccination Provides Lifelong Immunity; Characterization of Yellow-Fever-Specific Neutralizing Antibody and T-Cell Responses after Vaccination.

    Directory of Open Access Journals (Sweden)

    Rosanne W Wieten

    Full Text Available Prompted by recent amendments of Yellow Fever (YF vaccination guidelines from boost to single vaccination strategy and the paucity of clinical data to support this adjustment, we used the profile of the YF-specific CD8+ T-cell subset profiles after primary vaccination and neutralizing antibodies as a proxy for potentially longer lasting immunity.PBMCs and serum were collected in six individuals on days 0, 3, 5, 12, 28 and 180, and in 99 individuals >10 years after YF-vaccination. Phenotypic characteristics of YF- tetramer+ CD8+ T-cells were determined using class I tetramers. Antibody responses were measured using a standardized plaque reduction neutralization test (PRNT. Also, characteristics of YF-tetramer positive CD8+ T-cells were compared between individuals who had received a primary- and a booster vaccination. YF-tetramer+ CD8+ T-cells were detectable on day 12 (median tetramer+ cells as percentage of CD8+ T-cells 0.2%, range 0.07-3.1%. On day 180, these cells were still present (median 0.06%, range 0.02-0.78%. The phenotype of YF-tetramer positive CD8+ T-cells shifted from acute phase effector cells on day 12, to late differentiated or effector memory phenotype (CD45RA-/+CD27- on day 28. Two subsets of YF-tetramer positive T-cells (CD45RA+CD27- and CD45RA+CD27+ persisted until day 180. Within all phenotypic subsets, the T-bet: Eomes ratio tended to be high on day 28 after vaccination and shifted towards predominant Eomes expression on day 180 (median 6.0 (day 28 vs. 2.2 (day 180 p = 0.0625, suggestive of imprinting compatible with long-lived memory properties. YF-tetramer positive CD8+ T-cells were detectable up to 18 years post vaccination, YF-specific antibodies were detectable up to 40 years after single vaccination. Booster vaccination did not increase titers of YF-specific antibodies (mean 12.5 vs. 13.1, p = 0.583, nor induce frequencies or alter phenotypes of YF-tetramer+ CD8+ T-cells.The presence of a functionally competent YF-specific

  3. The role of antibodies specific for toxic sPLA2s and haemorrhagins in neutralizing potential of antisera raised against Vipera ammodytes ammodytes venom.

    Science.gov (United States)

    Halassy, Beata; Habjanec, Lidija; Brgles, Marija; Balija, Maja Lang; Leonardi, Adrijana; Kovacic, Lidija; Prijatelj, Petra; Tomasić, Jelka; Krizaj, Igor

    2008-08-01

    The contribution of antibodies directed against the two main toxic groups of proteins in the Vipera ammodytes ammodytes venom, haemorrhagic metalloproteinases (H) and neurotoxic sPLA2s (Atxs), to the overall protective efficacy of the whole venom antisera was investigated. Using ELISA assays we established a high correlation between the protective efficacy of the whole venom antisera in mice and their anti-Atxs antibody content. As the haemorrhage is the prevailing toxic effect of the venom in human, the lack of correlation also with anti-H IgG content exposed that the mouse model might not be optimal to evaluate the neutralizing potential of the venom-specific antisera for human therapy. We further revealed that Atxs and structurally very similar but non-toxic AtnI2 from the venom are not immuno cross-reactive.

  4. The breadth and titer of maternal HIV-1-specific heterologous neutralizing antibodies are not associated with a lower rate of mother-to-child transmission of HIV-1.

    Science.gov (United States)

    Chaillon, Antoine; Wack, Thierry; Braibant, Martine; Mandelbrot, Laurent; Blanche, Stéphane; Warszawski, Josiane; Barin, Francis

    2012-10-01

    It has been hypothesized that neutralizing antibodies (NAbs) should have broad specificity to be effective in protection against diverse HIV-1 variants. The mother-to-child transmission model of HIV-1 provides the opportunity to examine whether the breadth of maternal NAbs is associated with protection of infants from infection. Samples were obtained at delivery from 57 transmitting mothers (T) matched with 57 nontransmitting mothers (NT) enrolled in the multicenter French perinatal cohort (ANRS EPF CO1) between 1990 and 1996. Sixty-eight (59.6%) and 46 (40.4%) women were infected by B and non-B viruses, respectively. Neutralization assays were carried out with TZM-bl cells, using a panel of 10 primary isolates of 6 clades (A, B, C, F, CRF01_AE, and CRF02_AG), selected for their moderate or low sensitivity to neutralization. Neutralization breadths were not statistically different between T and NT mothers. However, a few statistically significant differences were observed, with higher frequencies or titers of NAbs toward several individual strains for NT mothers when the clade B-infected or non-clade B-infected mothers were analyzed separately. Our study confirms that the breadth of maternal NAbs is not associated with protection of infants from infection.

  5. The Breadth and Titer of Maternal HIV-1-Specific Heterologous Neutralizing Antibodies Are Not Associated with a Lower Rate of Mother-to-Child Transmission of HIV-1

    Science.gov (United States)

    Chaillon, Antoine; Wack, Thierry; Braibant, Martine; Mandelbrot, Laurent; Blanche, Stéphane; Warszawski, Josiane

    2012-01-01

    It has been hypothesized that neutralizing antibodies (NAbs) should have broad specificity to be effective in protection against diverse HIV-1 variants. The mother-to-child transmission model of HIV-1 provides the opportunity to examine whether the breadth of maternal NAbs is associated with protection of infants from infection. Samples were obtained at delivery from 57 transmitting mothers (T) matched with 57 nontransmitting mothers (NT) enrolled in the multicenter French perinatal cohort (ANRS EPF CO1) between 1990 and 1996. Sixty-eight (59.6%) and 46 (40.4%) women were infected by B and non-B viruses, respectively. Neutralization assays were carried out with TZM-bl cells, using a panel of 10 primary isolates of 6 clades (A, B, C, F, CRF01_AE, and CRF02_AG), selected for their moderate or low sensitivity to neutralization. Neutralization breadths were not statistically different between T and NT mothers. However, a few statistically significant differences were observed, with higher frequencies or titers of NAbs toward several individual strains for NT mothers when the clade B-infected or non-clade B-infected mothers were analyzed separately. Our study confirms that the breadth of maternal NAbs is not associated with protection of infants from infection. PMID:22811522

  6. The N276 glycosylation site is required for HIV-1 neutralization by the CD4 binding site specific HJ16 monoclonal antibody.

    Directory of Open Access Journals (Sweden)

    Sunita S Balla-Jhagjhoorsingh

    Full Text Available Immunogen design for HIV-1 vaccines could be based on epitope identification of naturally occurring neutralizing antibodies in infected patients. A tier 2 neutralizing monoclonal antibody (mAb, HJ16 recognizes a new epitope in the CD4 binding site (CD4bs region that only partially overlaps with the b12 epitope. We aimed to identify the critical binding site by resistance induction in a sensitive primary CRF02_AG strain. In four independent dose-escalation studies, the N276D mutation was consistently the only alteration found and it was confirmed to be responsible for resistance to HJ16 by site-directed mutagenesis in envelopes (envs of the homologous CRF02_AG, as well as of a subtype A and a subtype C primary isolate. This mutation removes an N-linked glycosylation site. The effect of N276D was very selective, as it failed to confer resistance to a range of other entry inhibitors. Remarkably, sensitivity to the CD4bs VRC01 and VRC03 mAbs was increased in the N276D mutated viruses. These data indicate that binding of the CD4bs specific HJ16 mAb critically depends on the interaction with the N276-glycan, thus indicating that HJ16 is the first glycan dependent CD4bs-specific mAb.

  7. Neutralizing antibodies in hepatitis C virus infection

    Institute of Scientific and Technical Information of China (English)

    Mirjam B Zeisel; Samira Fafi-Kremer; Isabel Fofana; Heidi Barth; Fran(c)oise Stoll-Keller; Michel Doffo(e)l; Thomas F Baumert

    2007-01-01

    Hepatitis C virus (HCV) is a major cause of hepatitis world-wide. The majority of infected individuals develop chronic hepatitis which can then progress to liver cirrhosis and hepatocellular carcinoma. Spontaneous viral clearance occurs in about 20%-30% of acutely infected individuals and results in resolution of infection without sequaelae. Both viral and host factors appear to play an important role for resolution of acute infection. A large body of evidence suggests that a strong, multispecific and long-lasting cellular immune response appears to be important for control of viral infection in acute hepatitis C. Due too the lack of convenient neutralization assays,the impact of neutralizing responses for control of viral infection had been less defined. In recent years, the development of robust tissue culture model systems for HCV entry and infection has finally allowed study of antibody-mediated neutralization and to gain further insights into viral targets of host neutralizing responses.In addition, detailed analysis of antibody-mediated neutralization in individual patients as well as cohorts with well defined viral isolates has enabled the study of neutralizing responses in the course of HCV infection and characterization of the impact of neutralizing antibodies for control of viral infection. This review will summarize recent progress in the understanding of the molecular mechanisms of antibody-mediated neutralization and its impact for HCV pathogenesis.(C) 2007 The WJG Press. All rights reserved.

  8. A novel hemagglutinin protein produced in bacteria protects chickens against H5N1 highly pathogenic avian influenza viruses by inducing H5 subtype-specific neutralizing antibodies

    Science.gov (United States)

    Sączyńska, Violetta; Romanik, Agnieszka; Florys, Katarzyna; Cecuda-Adamczewska, Violetta; Kęsik-Brodacka, Małgorzata; Śmietanka, Krzysztof; Olszewska, Monika; Domańska-Blicharz, Katarzyna; Minta, Zenon; Szewczyk, Bogusław; Płucienniczak, Grażyna; Płucienniczak, Andrzej

    2017-01-01

    The highly pathogenic (HP) H5N1 avian influenza viruses (AIVs) cause a mortality rate of up to 100% in infected chickens and pose a permanent pandemic threat. Attempts to obtain effective vaccines against H5N1 HPAIVs have focused on hemagglutinin (HA), an immunodominant viral antigen capable of eliciting neutralizing antibodies. The vast majority of vaccine projects have been performed using eukaryotic expression systems. In contrast, we used a bacterial expression system to produce vaccine HA protein (bacterial HA) according to our own design. The HA protein with the sequence of the H5N1 HPAIV strain was efficiently expressed in Escherichia coli, recovered in the form of inclusion bodies and refolded by dilution between two chromatographic purification steps. Antigenicity studies showed that the resulting antigen, referred to as rH5-E. coli, preserves conformational epitopes targeted by antibodies specific for H5-subtype HAs, inhibiting hemagglutination and/or neutralizing influenza viruses in vitro. The proper conformation of this protein and its ability to form functional oligomers were confirmed by a hemagglutination test. Consistent with the biochemical characteristics, prime-boost immunizations with adjuvanted rH5-E. coli protected 100% and 70% of specific pathogen-free, layer-type chickens against challenge with homologous and heterologous H5N1 HPAIVs, respectively. The observed protection was related to the positivity in the FluAC H5 test (IDVet) but not to hemagglutination-inhibiting antibody titers. Due to full protection, the effective contact transmission of the homologous challenge virus did not occur. Survivors from both challenges did not or only transiently shed the viruses, as established by viral RNA detection in oropharyngeal and cloacal swabs. Our results demonstrate that vaccination with rH5-E. coli could confer control of H5N1 HPAIV infection and transmission rates in chicken flocks, accompanied by reduced virus shedding. Moreover, the role of

  9. Correlation between dengue-specific neutralizing antibodies and serum avidity in primary and secondary dengue virus 3 natural infections in humans.

    Directory of Open Access Journals (Sweden)

    Andreas Puschnik

    Full Text Available Although heterotypic secondary infection with dengue virus (DENV is associated with severe disease, the majority of secondary infections are mild or asymptomatic. The mechanisms of antibody-mediated protection are poorly understood. In 2010, 108 DENV3-positive cases were enrolled in a pediatric hospital-based study in Managua, Nicaragua, with 61 primary and 47 secondary infections. We analyzed DENV-specific neutralization titers (NT50, IgM and IgG avidity, and antibody titer in serum samples collected during acute and convalescent phases and 3, 6, and 18 months post-infection. NT50 titers peaked at convalescence and decreased thereafter. IgG avidity to DENV3 significantly increased between convalescent and 3-month time-points in primary DENV infections and between the acute and convalescent phase in secondary DENV infections. While avidity to DENV2, a likely previous infecting serotype, was initially higher than avidity to DENV3 in secondary DENV infections, the opposite relation was observed 3-18 months post-infection. We found significant correlations between IgM avidity and NT50 in acute primary cases and between IgG avidity and NT50 in secondary DENV infections. In summary, our findings indicate that IgM antibodies likely play a role in early control of DENV infections. IgG serum avidity to DENV, analyzed for the first time in longitudinal samples, switches from targeting mainly cross-reactive serotype(s to the current infecting serotype over time. Finally, serum avidity correlates with neutralization capacity.

  10. A focus reduction neutralization assay for hepatitis C virus neutralizing antibodies

    Directory of Open Access Journals (Sweden)

    Wychowski Czeslaw

    2007-03-01

    Full Text Available Abstract Background/Aim The role of humoral immunity in hepatitis C virus (HCV infection is poorly understood. Nevertheless, there is increasing interest in characterizing the neutralizing antibodies in the serum of HCV-infected patients. Focus reduction assays have been widely used to evaluate neutralizing antibody responses against a range of non-cytopathic viruses. Based on the recent development of a HCV cell culture system using the genotype 2 JFH-1-strain, we developed a focus reduction assay for HCV-neutralizing antibodies. Methods The focus reduction assay was based on a standard microneutralization assay in which immunostained foci on tissue culture plates are counted. The neutralizing anti-HCV antibodies titers of purified serum immunoglobulin samples from seventy-seven individuals were determined using a 50% focus reduction neutralization assay. Each titer was determined as the log value of the reciprocal antibody dilution that reduced the number of viral foci by 50%. IgG antibodies were first purified from each serum in order to avoid the facilitating effect of HDL on HCV entry. Results The assay's cut-off using an ELISA and RNA HCV-negative samples was found to be 1.25 log, corresponding to a dilution of 1:18. The assay was compared with a commercial HCV ELISA and exhibited specificity and sensitivity values of 100% and 96.5%, respectively, and good reproducibility (with intra-assay and inter-assay coefficients of variation of 6.7% and 12.6%, respectively. The assay did not show any cross-reactivity with anti-HIV, anti-HBs or heterophile antibody-positive samples. The neutralizing antibodies titers were 2.13 log (1:134 for homologous samples from HCV genotype 2 infected patients harboring the same genotype as JFH-1 and 1.93 log (1:85 for heterologous samples from patients infected by genotypes other than type 2. These results confirm the presence of broadly cross-neutralizing antibodies already reported using the HCV pseudoparticles

  11. Neutralizing antibodies to Haemophilus ducreyi cytotoxin.

    OpenAIRE

    Lagergård, T; Purvén, M

    1993-01-01

    Neutralizing antibodies against cytotoxin produced by Haemophilus ducreyi bacteria were studied in rabbits by an assay employing HEp-2 cells and diluted crude cytotoxin preparations from the organism. Antisera to 12 different H. ducreyi strains were prepared by immunization of rabbits with bacterial sonicates combined with Freund's adjuvant. The antibody response during infection with H. ducreyi was studied in two groups of rabbits which were infected with five live strains by either single o...

  12. Crystal Structure of PG16 and Chimeric Dissection with Somatically Related PG9: Structure-Function Analysis of Two Quaternary-Specific Antibodies That Effectively Neutralize HIV-1

    Energy Technology Data Exchange (ETDEWEB)

    Pancera, Marie; McLellan, Jason S.; Wu, Xueling; Zhu, Jiang; Changela, Anita; Schmidt, Stephen D.; Yang, Yongping; Zhou, Tongqing; Phogat, Sanjay; Mascola, John R.; Kwong, Peter D. (NIH); (Aids Vaccine)

    2010-11-03

    HIV-1 resists neutralization by most antibodies. Two somatically related human antibodies, PG9 and PG16, however, each neutralize 70 to 80% of circulating HIV-1 isolates. Here we present the structure of the antigen-binding fragment of PG16 in monoclinic and orthorhombic lattices at 2.4 and 4.0 {angstrom}, respectively, and use a combination of structural analysis, paratope dissection, and neutralization assessment to determine the functional relevance of three unusual PG9/PG16 features: N-linked glycosylation, extensive affinity maturation, and a heavy chain-third complementarity-determining region (CDR H3) that is one of the longest observed in human antibodies. Glycosylation extended off the side of the light chain variable domain and was not required for neutralization. The CDR H3 formed an axe-shaped subdomain, which comprised 42% of the CDR surface, with the axe head looming {approx}20 {angstrom} above the other combining loops. Comprehensive sets of chimeric swaps between PG9 and PG16 of light chain, heavy chain, and CDR H3 were employed to decipher structure-function relationships. Chimeric swaps generally complemented functionally, with differences in PG9/PG16 neutralization related primarily to residue differences in CDR H3. Meanwhile, chimeric reversions to genomic V genes showed isolate-dependent effects, with affinity maturation playing a significant role in augmenting neutralization breadth (P = 0.036) and potency (P < 0.0001). The structural and functional details of extraordinary CDR H3 and extensive affinity maturation provide insights into the neutralization mechanism of and the elicitation pathway for broadly neutralizing antibodies like PG9 and PG16.

  13. Combination effect on HIV infection in vitro of soluble CD4 and HIV-neutralizing antibodies

    DEFF Research Database (Denmark)

    Hansen, J E; Sørensen, A M; Olofsson, S;

    1994-01-01

    In combination with HIV gp120 V3-loop antibody, two carbohydrate specific neutralizing antibodies (83D4 and 2G12) had a synergistic neutralizing effect on HIV infection. However, sCD4 and an antibody which blocks gp 120/CD4 binding (1B1) both displayed antagonism.......In combination with HIV gp120 V3-loop antibody, two carbohydrate specific neutralizing antibodies (83D4 and 2G12) had a synergistic neutralizing effect on HIV infection. However, sCD4 and an antibody which blocks gp 120/CD4 binding (1B1) both displayed antagonism....

  14. HIV-1 binding and neutralizing antibodies of injecting drug users

    Directory of Open Access Journals (Sweden)

    Ouverney E.P.

    2005-01-01

    Full Text Available Previous studies have demonstrated a stronger seroreactivity against some synthetic peptides responsible for inducing neutralizing antibodies in injecting drug users (IDU compared to that of individuals sexually infected with HIV-1 (S, but the effectiveness in terms of the neutralizing ability of these antibodies has not been evaluated. Our objective was to study the humoral immune response of IDU by determining the specificity of their antibodies and the presence of neutralizing antibodies. The neutralization capacity against the HIV-1 isolate MN (genotype B, the primary HIV-1 isolate 95BRRJ021 (genotype F, and the seroreactivity with peptides known to induce neutralizing antibodies, from the V2 and V3 loops of different HIV-1 subtypes, were analyzed. Seroreactivity indicates that IDU plasma are more likely to recognize a broader range of peptides than S plasma, with significantly higher titers, especially of V3 peptides. Similar neutralization frequencies of the MN isolate were observed in plasma of the IDU (16/47 and S (20/60 groups in the 1:10 dilution. The neutralization of the 95BRRJ021 isolate was more frequently observed for plasma from the S group (15/23 than from the IDU group (15/47, P = 0.0108. No correlation between neutralization and seroreactivity with the peptides tested was observed. These results suggest that an important factor responsible for the extensive and broad humoral immune response observed in IDU is their infection route. There was very little difference in neutralizing antibody response between the IDU and S groups despite their differences in seroreactivity and health status.

  15. A strategy for the generation of specific human antibodies by directed evolution and phage display. An example of a single-chain antibody fragment that neutralizes a major component of scorpion venom.

    Science.gov (United States)

    Riaño-Umbarila, Lidia; Juárez-González, Victor Rivelino; Olamendi-Portugal, Timoteo; Ortíz-León, Mauricio; Possani, Lourival Domingos; Becerril, Baltazar

    2005-05-01

    This study describes the construction of a library of single-chain antibody fragments (scFvs) from a single human donor by individual amplification of all heavy and light variable domains (1.1 x 10(8) recombinants). The library was panned using the phage display technique, which allowed selection of specific scFvs (3F and C1) capable of recognizing Cn2, the major toxic component of Centruroides noxius scorpion venom. The scFv 3F was matured in vitro by three cycles of directed evolution. The use of stringent conditions in the third cycle allowed the selection of several improved clones. The best scFv obtained (6009F) was improved in terms of its affinity by 446-fold, from 183 nm (3F) to 410 pm. This scFv 6009F was able to neutralize 2 LD(50) of Cn2 toxin when a 1 : 10 molar ratio of toxin-to-antibody fragment was used. It was also able to neutralize 2 LD(50) of the whole venom. These results pave the way for the future generation of recombinant human antivenoms.

  16. Humanized-single domain antibodies (VH/VHH) that bound specifically to Naja kaouthia phospholipase A2 and neutralized the enzymatic activity.

    Science.gov (United States)

    Chavanayarn, Charnwit; Thanongsaksrikul, Jeeraphong; Thueng-In, Kanyarat; Bangphoomi, Kunan; Sookrung, Nitat; Chaicumpa, Wanpen

    2012-07-01

    Naja kaouthia (monocled cobra) venom contains many isoforms of secreted phospholipase A2 (sPLA(2)). The PLA(2) exerts several pharmacologic and toxic effects in the snake bitten subject, dependent or independent on the enzymatic activity. N. kaouthia venom appeared in two protein profiles, P3 and P5, after fractionating the venom by ion exchange column chromatography. In this study, phage clones displaying humanized-camel single domain antibodies (VH/V(H)H) that bound specifically to the P3 and P5 were selected from a humanized-camel VH/V(H)H phage display library. Two phagemid transfected E. coli clones (P3-1 and P3-3) produced humanized-V(H)H, while another clone (P3-7) produced humanized-VH. At the optimal venom:antibody ratio, the VH/V(H)H purified from the E. coli homogenates neutralized PLA(2) enzyme activity comparable to the horse immune serum against the N. kaouthia holo-venom. Homology modeling and molecular docking revealed that the VH/V(H)H covered the areas around the PLA(2) catalytic groove and inserted their Complementarity Determining Regions (CDRs) into the enzymatic cleft. It is envisaged that the VH/V(H)H would ameliorate/abrogate the principal toxicity of the venom PLA(2) (membrane phospholipid catabolism leading to cellular and subcellular membrane damage which consequently causes hemolysis, hemorrhage, and dermo-/myo-necrosis), if they were used for passive immunotherapy of the cobra bitten victim. The speculation needs further investigations.

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

    Directory of Open Access Journals (Sweden)

    Constantinos Kurt Wibmer

    2013-10-01

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

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

    Directory of Open Access Journals (Sweden)

    Dimiter S. Dimitrov

    2009-11-01

    Full Text Available Several human monoclonal antibodies (hmAbs and antibody fragments, including the best characterized in terms of structure-function b12 and Fab X5, exhibit relatively potent and broad HIV-1 neutralizing activity. However, the elicitation of b12 or b12-like antibodies in vivo by vaccine immunogens based on the HIV-1 envelope glycoprotein (Env has not been successful. B12 is highly divergent from the closest corresponding germline antibody while X5 is less divergent. We have hypothesized that the relatively high degree of specific somatic hypermutations may preclude binding of the HIV-1 envelope glycoprotein (Env to closest germline antibodies, and that identifying antibodies that are intermediates in the pathways to maturation could help design novel vaccine immunogens to guide the immune system for their enhanced elicitation. In support of this hypothesis we have previously found that a germline-like b12 (monovalent and bivalent scFv as an Fc fusion protein or IgG lacks measurable binding to an Env as measured by ELISA with a sensitivity in the μM range [1]; here we present evidence confirming and expanding these findings for a panel of Envs. In contrast, a germline-like scFv X5 bound Env with high (nM affinity. To begin to explore the maturation pathways of these antibodies we identified several possible b12 intermediate antibodies and tested their neutralizing activity. These intermediate antibodies neutralized only some HIV-1 isolates and with relatively weak potency. In contrast, germline-like scFv X5 neutralized a subset of the tested HIV-1 isolates with comparable efficiencies to that of the mature X5. These results could help explain the relatively high immunogenicity of the coreceptor binding site on gp120 and the abundance of CD4-induced (CD4i antibodies in HIV-1-infected patients (X5 is a CD4i antibody as well as the maturation pathway of X5. They also can help identify antigens that can bind specifically to b12 germline and

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

    OpenAIRE

    Euler, Zelda; Schuitemaker, Hanneke

    2012-01-01

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

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

    Science.gov (United States)

    Euler, Zelda; Schuitemaker, Hanneke

    2012-01-01

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

  1. Autologous HIV-1 neutralizing antibodies: emergence of neutralization-resistant escape virus and subsequent development of escape virus neutralizing antibodies

    DEFF Research Database (Denmark)

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

    1992-01-01

    The capacity of consecutive human sera to neutralize sequentially obtained autologous virus isolates was studied. HIV-1 was isolated three times over a 48-164-week period from three individuals immediately after seroconversion and from two individuals in later stages of infection. Development of ...... escape virus may be part of the explanation of the apparent failure of the immune system to control HIV infection.......The capacity of consecutive human sera to neutralize sequentially obtained autologous virus isolates was studied. HIV-1 was isolated three times over a 48-164-week period from three individuals immediately after seroconversion and from two individuals in later stages of infection. Development...... of neutralizing antibodies to the primary virus isolates was detected 13-45 weeks after seroconversion. Emergence of escape virus with reduced sensitivity to neutralization by autologous sera was demonstrated. The patients subsequently developed neutralizing antibodies against the escape virus but after a delay...

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

    Science.gov (United States)

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

    2016-09-11

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

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Han Wang

    2016-09-01

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

  5. Antibody function in neutralization and protection against HIV-1

    NARCIS (Netherlands)

    Hessell, A.J.

    2009-01-01

    The ability to induce neutralizing antibodies is generally thought to be of great importance for vaccine efficacy. In HIV-1 research this quality has been elusive as the HIV-1 virus has evolved multiple mechanisms to evade neutralizing antibodies. This thesis traces studies with four broadly neutral

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

    Science.gov (United States)

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

    2015-04-02

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

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

    Science.gov (United States)

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

    2015-02-26

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

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

    Directory of Open Access Journals (Sweden)

    Laura E McCoy

    2015-08-01

    Full Text Available 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.

  9. Approaches to the induction of HIV broadly neutralizing antibodies.

    Science.gov (United States)

    Moore, Penny L; Williamson, Carolyn

    2016-11-01

    A vaccine that elicits antibody responses that can neutralize the diversity of HIV clades has not yet been achieved, and is a major focus of HIV vaccine research. Here, we provide an update on the barriers to eliciting such antibodies, and how advances in immunogen design may circumvent these roadblocks, focusing on data published in the last year. Studies of how broadly neutralizing antibodies (bNAbs) develop in HIV-infected donors continue to produce key insights, suggesting that for some viral targets there are common pathways to developing breadth. Germline-targeting strategies, that aim to recruit rare precursors of bNAbs, have shown promise in immunogenicity studies, and structural biology has led to advances in immunogen design. Mapping of strain-specific tier 2 vaccine responses has highlighted the challenges that remain in driving antibodies toward breadth. Elucidation of the HIV envelope structure, together with an understanding of how bNAbs emerge in vivo has guided the design of new immunogens and vaccine strategies that show promise for eliciting protective antibodies.

  10. The neutralization sensitivity of viruses representing human immunodeficiency virus type 1 variants of diverse subtypes from early in infection is dependent on producer cell, as well as characteristics of the specific antibody and envelope variant.

    Science.gov (United States)

    Provine, Nicholas M; Cortez, Valerie; Chohan, Vrasha; Overbaugh, Julie

    2012-05-25

    Neutralization properties of human immunodeficiency virus (HIV-1) are often defined using pseudoviruses grown in transformed cells, which are not biologically relevant HIV-1 producer cells. Little information exists on how these viruses compare to viruses produced in primary lymphocytes, particularly for globally relevant HIV-1 strains. Therefore, replication-competent chimeras encoding envelope variants from the dominant HIV-1 subtypes (A, C, and D) obtained early after infection were generated and the neutralization properties explored. Pseudoviruses generated in 293T cells were the most sensitive to antibody neutralization. Replicating viruses generated in primary lymphocytes were most resistant to neutralization by plasma antibodies and most monoclonal antibodies (b12, 4E10, 2F5, VRC01). These differences were not associated with differences in envelope content. Surprisingly, the virus source did not impact neutralization sensitivity of most viruses to PG9. These findings suggest that producer cell type has a major effect on neutralization sensitivity, but in an antibody dependent manner.

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

    Science.gov (United States)

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

    2013-04-25

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

  12. The neutralization of interferons by antibody. I. Quantitative and theoretical analyses of the neutralization reaction in different bioassay systems.

    Science.gov (United States)

    Grossberg, S E; Kawade, Y; Kohase, M; Yokoyama, H; Finter, N

    2001-09-01

    The highly specific ability of antibodies to inhibit the biologic activity of cytokines or other therapeutic proteins is widely used in research and a subject of increasing clinical importance. The need exists for a standardized approach to the reporting of neutralizing antibody potency soundly based on theoretical and practical considerations and tested by experimental data. Pursuant to the original studies of Kawade on the theoretical and functional aspects of neutralization of interferons (IFN), experimental data were obtained by different laboratories employing varied methodology to address two hypotheses concerning the nature of IFN neutralization reactions, based on a derived formula that allows expression of neutralizing power as the reduction of 10 laboratory units (LU)/ml to 1 LU/ml, the end point of most bioassays. Two hypotheses are posed: (1) antibody acts to neutralize a fixed amount of biologically active IFN molecules, or (2) antibody reduces IFN activity in a set ratio of added/residual biologically active IFN. The first, or fixed amount, hypothesis relates to the reactivity of high-affinity antibodies neutralizing equimolar amounts of antigen, whereas the second, or constant proportion, hypothesis postulates a reduction in the ratio of total added IFN to residual active IFN molecules, such as a low-affinity antibody might exhibit. Analyses of data of the neutralization of IFN-alpha and IFN-beta are presented, employing human polyclonal antibodies and murine monoclonal antibodies (mAb). The theoretical constructs of Kawade are extended in the Appendix and correlated with new experimental data in the text. The data clearly indicate that the low-antibody affinity, constant proportion hypothesis, rather than the high-antibody affinity, fixed amount hypothesis, is applicable, if the bioassay is sensitive to IFN. The findings presented here and in the following paper (pp. 743-755, this issue) taken together provide the basis for a standardized method of

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

    Science.gov (United States)

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

    2015-01-01

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

  14. HIV-2 neutralization by intact V3-specific Fab fragments

    Directory of Open Access Journals (Sweden)

    Sourial Samer

    2008-08-01

    Full Text Available Abstract The V3 region of both HIV-1 gp120 and HIV-2 gp125 surface glycoprotein has been described as a target for neutralizing antibodies. In this study a conformation-sensitive (3C4 and a linear site-specific (7C8 anti-HIV-2 V3 monoclonal antibody (mAb were characterized. The neutralization capacity of the purified mAbs and their respective papain-generated Fab fragments was analyzed. The Fabs were further characterized by sequence analysis. Our results demonstrate that neither purified mAbs were capable of neutralizing HIV-2, while intact Fab fragments from both mAbs blocked in vitro infection of HIV-2 isolates. Moreover, the conformation sensitive 3C4 Fab neutralized both subtype A and B HIV-2 isolates and SIVsm. Sequence analysis of the hypervariable regions of 3C4 Fab and 7C8 Fab revealed that the third CDR of the heavy chain (CDRH3 of the antibodies was not as long as many of the previously characterized neutralizing antibodies. Our findings suggest that whole 7C8 and 3C4 mAbs are sterically hindered from neutralizing HIV-2, whereas the smaller size of Fab fragments enables access to the V3 region on the virion surface.

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

    Science.gov (United States)

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

    2012-05-01

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

  16. Elicitation of structure-specific antibodies by epitope scaffolds

    OpenAIRE

    2010-01-01

    Elicitation of antibodies against targets that are immunorecessive, cryptic, or transient in their native context has been a challenge for vaccine design. Here we demonstrate the elicitation of structure-specific antibodies against the HIV-1 gp41 epitope of the broadly neutralizing antibody 2F5. This conformationally flexible region of gp41 assumes mostly helical conformations but adopts a kinked, extended structure when bound by antibody 2F5. Computational techniques were employed to transpl...

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

    Directory of Open Access Journals (Sweden)

    Zhiqing Zhang

    2016-11-01

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

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

    Science.gov (United States)

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

    2016-11-18

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

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

    Directory of Open Access Journals (Sweden)

    Tamara P Blevins

    Full Text Available 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.

  20. Neutralizing Antibodies and Pathogenesis of Hepatitis C Virus Infection

    Directory of Open Access Journals (Sweden)

    Françoise Stoll-Keller

    2012-10-01

    Full Text Available Hepatitis C virus (HCV infection is a major cause of chronic liver disease worldwide. The interplay between the virus and host innate and adaptive immune responses determines the outcome of infection. There is increasing evidence that host neutralizing responses play a relevant role in the resulting pathogenesis. Furthermore, viral evasion from host neutralizing antibodies has been revealed to be an important contributor in leading both to viral persistence in acute liver graft infection following liver transplantation, and to chronic viral infection. The development of novel model systems to study HCV entry and neutralization has allowed a detailed understanding of the molecular mechanisms of virus-host interactions during antibody-mediated neutralization. The understanding of these mechanisms will ultimately contribute to the development of novel antiviral preventive strategies for liver graft infection and an urgently needed vaccine. This review summarizes recent concepts of the role of neutralizing antibodies in viral clearance and protection, and highlights consequences of viral escape from neutralizing antibodies in the pathogenesis of HCV infection.

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

    Directory of Open Access Journals (Sweden)

    Rami Sommerstein

    2015-11-01

    Full Text Available 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.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  4. Mapping Polyclonal HIV-1 Antibody Responses via Next-Generation Neutralization Fingerprinting

    OpenAIRE

    Doria-Rose, Nicole A.; Altae-Tran, Han R.; Roark, Ryan S.; Schmidt, Stephen D.; Sutton, Matthew S.; Louder, Mark K.; Chuang, Gwo-Yu; Bailer, Robert T.; Cortez, Valerie; Kong, Rui; McKee, Krisha; O’Dell, Sijy; Wang, Felicia; Abdool Karim, Salim S.; Binley, James M.

    2017-01-01

    Computational neutralization fingerprinting, NFP, is an efficient and accurate method for predicting the epitope specificities of polyclonal antibody responses to HIV-1 infection. Here, we present next-generation NFP algorithms that substantially improve prediction accuracy for individual donors and enable serologic analysis for entire cohorts. Specifically, we developed algorithms for: (a) selection of optimized virus neutralization panels for NFP analysis, (b) estimation of NFP prediction c...

  5. Lactogenic immunity in transgenic mice producing recombinant antibodies neutralizing coronavirus.

    Science.gov (United States)

    Castilla, J; Sola, I; Pintado, B; Sánchez-Morgado, J M; Enjuanes, L

    1998-01-01

    Protection against coronavirus infections can be provided by the oral administration of virus neutralizing antibodies. To provide lactogenic immunity, eighteen lines of transgenic mice secreting a recombinant IgG1 monoclonal antibody (rIgG1) and ten lines of transgenic mice secreting recombinant IgA monoclonal antibodies (rIgA) neutralizing transmissible gastroenteritis coronavirus (TGEV) into the milk were generated. Genes encoding the light and heavy chains of monoclonal antibody (MAb) 6A.C3 were expressed under the control of regulatory sequences derived from the mouse genomic DNA encoding the whey acidic protein (WAP) and beta-lactoglobulin (BLG), which are highly abundant milk proteins. The MAb 6A.C3 binds to a highly conserved epitope present in coronaviruses of several species. This MAb does not allow the selection of neutralization escaping virus mutants. The antibody was expressed in the milk of transgenic mice with titers of one million as determined by RIA, and neutralized TGEV infectivity by one million fold corresponding to immunoglobulin concentrations of 5 to 6 mg per ml. Matrix attachment regions (MAR) sequences were not essential for rIgG1 transgene expression, but co-microinjection of MAR and antibody genes led to a twenty to ten thousand-fold increase in the antibody titer in 50% of the rIgG1 transgenic animals generated. Co-microinjection of the genomic BLG gene with rIgA light and heavy chain genes led to the generation of transgenic mice carrying the three transgenes. The highest antibody titers were produced by transgenic mice that had integrated the antibody and BLG genes, although the number of transgenic animals generated does not allow a definitive conclusion on the enhancing effect of BLG co-integration. Antibody expression levels were transgene copy number independent and integration site dependent. The generation of transgenic animals producing virus neutralizing antibodies in the milk could be a general approach to provide protection

  6. Lack of Durable Cross-Neutralizing Antibodies Against Zika Virus from Dengue Virus Infection.

    Science.gov (United States)

    Collins, Matthew H; McGowan, Eileen; Jadi, Ramesh; Young, Ellen; Lopez, Cesar A; Baric, Ralph S; Lazear, Helen M; de Silva, Aravinda M

    2017-05-01

    Cross-reactive antibodies elicited by dengue virus (DENV) infection might affect Zika virus infection and confound serologic tests. Recent data demonstrate neutralization of Zika virus by monoclonal antibodies or human serum collected early after DENV infection. Whether this finding is true in late DENV convalescence (>6 months after infection) is unknown. We studied late convalescent serum samples from persons with prior DENV or Zika virus exposure. Despite extensive cross-reactivity in IgG binding, Zika virus neutralization was not observed among primary DENV infections. We observed low-frequency (23%) Zika virus cross-neutralization in repeat DENV infections. DENV-immune persons who had Zika virus as a secondary infection had distinct populations of antibodies that neutralized DENVs and Zika virus, as shown by DENV-reactive antibody depletion experiments. These data suggest that most DENV infections do not induce durable, high-level Zika virus cross-neutralizing antibodies. Zika virus-specific antibody populations develop after Zika virus infection irrespective of prior DENV immunity.

  7. Enhanced neutralization of HIV by antibodies displayed on the S-layer of Caulobacter crescentus.

    Science.gov (United States)

    Duval, Mark; Lewis, Christopher J; Nomellini, John F; Horwitz, Marc S; Smit, John; Cavacini, Lisa A

    2011-12-01

    Innovative methods of prevention are needed to stop the more than two million new HIV-1 infections annually, particularly in women. Local application of anti-HIV antibodies has been shown to be effective at preventing infection in nonhuman primates; however, the concentrations needed are cost prohibitive. Display of antibodies on a particulate platform will likely prolong effectiveness of these anti-HIV agents and lower the cost of goods. Here, we demonstrate that the bacterium Caulobacter crescentus and its highly expressed surface-layer (S-layer) protein can provide this antibody display platform. Caulobacters displaying protein G, alone or with CD4 codisplay, successfully captured HIV-1-specific antibodies and demonstrated functional neutralization. Compared to soluble antibodies, a neutralizing anti-HIV antibody displayed on Caulobacter was as effective or more effective at neutralizing diverse HIV-1 isolates. Moreover, when an antibody reactive with an epitope induced by CD4 binding (CD4i) was codisplayed with CD4, there was significant enhancement in HIV-1 neutralization. These results suggest that caulobacters displaying anti-HIV antibodies offer a distinct improvement in the use of antibodies as microbicides. Furthermore, these reagents can specifically evaluate anti-HIV antibodies in concert with other HIV-1 blocking agents to assess the most suitable tools for conversion to scFvs, allowing for direct display within the S-layer protein and further reducing cost of goods. In summary, C. crescentus, which can be easily produced and chemically stabilized at low cost, is well suited for engineering as an effective platform, offering an inexpensive way to produce and deliver HIV-1-specific microbicides.

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

    Institute of Scientific and Technical Information of China (English)

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

    2005-01-01

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

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

    Science.gov (United States)

    Magnus, Carsten; Reh, Lucia; Trkola, Alexandra

    2016-06-15

    Broadly neutralizing antibodies against human immunodeficiency virus type 1 (HIV-1) are considered vital components of novel therapeutics and blueprints for vaccine research. Yet escape to even the most potent of these antibodies is imminent in natural infection. Measures to define antibody efficacy and prevent mutant selection are thus urgently needed. Here, we derive a mathematical framework to predict the concentration ranges for which antibody escape variants can outcompete their viral ancestors, referred to as mutant selection window (MSW). When determining the MSW, we focus on the differential efficacy of neutralizing antibodies against HIV-1 in two canonical infection routes, free-virus infection and cell-cell transmission. The latter has proven highly effective in vitro suggesting its importance for both in vivo spread as well as for escaping targeted intervention strategies. We observed a range of MSW patterns that highlight the potential of mutants to arise in both transmission pathways and over wide concentration ranges. Most importantly, we found that only when the arising mutant has both, residual sensitivity to the neutralizing antibody and reduced infectivity compared to the parental virus, antibody dosing outside of the MSW to restrict mutant selection is possible. Emergence of mutants that provide complete escape and have no considerable fitness loss cannot be prevented by adjusting antibody doses. The latter may in part explain the ubiquitous resistance to neutralizing antibodies observed in natural infection and antibody treatment. Based on our findings, combinations of antibodies targeting different epitopes should be favored for antibody-based interventions as this may render complete resistance less likely to occur and also increase chances that multiple escapes result in severe fitness loss of the virus making longer-term antibody treatment more feasible. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

  10. Short Communication: Neutralizing Antibodies in HIV-1-Infected Brazilian Individuals

    Science.gov (United States)

    Morgado, Mariza Gonçalvez; Côrtes, Fernanda Heloise; Guimarães, Monick Lindermeyer; Mendonça-Lima, Leila; Pilotto, Jose Henrique; Grinsztejn, Beatriz; Veloso, Valdiléa Gonçalves; Bongertz, Vera

    2013-01-01

    Abstract Tests for the detection of the humoral immune response to HIV-1 have to be standardized and established, demanding regional efforts. For this purpose the neutralizing antibody (NAb) assay for HIV-1 in TZM-bl cells was introduced in Brazil. Twenty plasma samples from HIV-1-infected individuals were assayed: 10 progressors and 10 long-term nonprogressors. These were tested against eight env-pseudotyped viruses (psVs) in the TZM-bl NAb assay and against HIV-1 strain HTLV/IIIB (HIV-1 IIIB) in primary lymphocytes. Forty-four percent of the samples showed neutralizing titers for psVs and 55% for HIV-1 IIIB. Plasma from progressors showed a broader neutralization and a higher potency. The introduction of these reference reagents encourages the participation of Brazil in future comparative assessments of anti-HIV-1 antibodies. PMID:23145941

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

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    Ben-Jiang Ma

    2011-09-01

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

  12. Validation of cross-genotype neutralization by hepatitis B virus-specific monoclonal antibodies by in vitro and in vivo infection.

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    Hamada-Tsutsumi, Susumu; Iio, Etsuko; Watanabe, Tsunamasa; Murakami, Shuko; Isogawa, Masanori; Iijima, Sayuki; Inoue, Takako; Matsunami, Kayoko; Tajiri, Kazuto; Ozawa, Tatsuhiko; Kishi, Hiroyuki; Muraguchi, Atsushi; Joh, Takashi; Tanaka, Yasuhito

    2015-01-01

    Vaccines based on hepatitis B virus (HBV) genotype A have been used worldwide for immunoprophylaxis and are thought to prevent infections by non-A HBV strains effectively, whereas, vaccines generated from genotype C have been used in several Asian countries, including Japan and Korea, where HBV genotype C is prevalent. However, acute hepatitis B caused by HBV genotype A infection has been increasing in Japan and little is known about the efficacy of immunization with genotype C-based vaccines against non-C infection. We have isolated human monoclonal antibodies (mAbs) from individuals who were immunized with the genotype C-based vaccine. In this study, the efficacies of these two mAbs, HB0116 and HB0478, were analyzed using in vivo and in vitro models of HBV infection. Intravenous inoculation of HBV genotype C into chimeric mice with human hepatocytes resulted in the establishment of HBV infection after five weeks, whereas preincubation of the inocula with HB0116 or HB0478 protected chimeric mice from genotype C infection completely. Interestingly, both HB0116 and HB0478 were found to block completely genotype A infection. Moreover, infection by a genotype C strain with an immune escape substitution of amino acid 145 in the hepatitis B surface protein was also completely inhibited by incubation with HB0478. Finally, in vitro analysis of dose dependency revealed that the amounts of HB0478 required for complete protection against genotype C and genotype A infection were 5.5 mIU and 55 mIU, respectively. These results suggested that genotype C-based vaccines have ability to induce cross-genotype immunity against HBV infection.

  13. Mapping Polyclonal HIV-1 Antibody Responses via Next-Generation Neutralization Fingerprinting.

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    Doria-Rose, Nicole A; Altae-Tran, Han R; Roark, Ryan S; Schmidt, Stephen D; Sutton, Matthew S; Louder, Mark K; Chuang, Gwo-Yu; Bailer, Robert T; Cortez, Valerie; Kong, Rui; McKee, Krisha; O'Dell, Sijy; Wang, Felicia; Abdool Karim, Salim S; Binley, James M; Connors, Mark; Haynes, Barton F; Martin, Malcolm A; Montefiori, David C; Morris, Lynn; Overbaugh, Julie; Kwong, Peter D; Mascola, John R; Georgiev, Ivelin S

    2017-01-01

    Computational neutralization fingerprinting, NFP, is an efficient and accurate method for predicting the epitope specificities of polyclonal antibody responses to HIV-1 infection. Here, we present next-generation NFP algorithms that substantially improve prediction accuracy for individual donors and enable serologic analysis for entire cohorts. Specifically, we developed algorithms for: (a) selection of optimized virus neutralization panels for NFP analysis, (b) estimation of NFP prediction confidence for each serum sample, and (c) identification of sera with potentially novel epitope specificities. At the individual donor level, the next-generation NFP algorithms particularly improved the ability to detect multiple epitope specificities in a sample, as confirmed both for computationally simulated polyclonal sera and for samples from HIV-infected donors. Specifically, the next-generation NFP algorithms detected multiple specificities in twice as many samples of simulated sera. Further, unlike the first-generation NFP, the new algorithms were able to detect both of the previously confirmed antibody specificities, VRC01-like and PG9-like, in donor CHAVI 0219. At the cohort level, analysis of ~150 broadly neutralizing HIV-infected donor samples suggested a potential connection between clade of infection and types of elicited epitope specificities. Most notably, while 10E8-like antibodies were observed in infections from different clades, an enrichment of such antibodies was predicted for clade B samples. Ultimately, such large-scale analyses of antibody responses to HIV-1 infection can help guide the design of epitope-specific vaccines that are tailored to take into account the prevalence of infecting clades within a specific geographic region. Overall, the next-generation NFP technology will be an important tool for the analysis of broadly neutralizing polyclonal antibody responses against HIV-1.

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

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

    2015-01-01

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

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

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    Larissa M. Alvarenga

    2014-08-01

    Full Text Available 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.

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

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

    2012-10-29

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

  17. JC polyomavirus mutants escape antibody-mediated neutralization.

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    Ray, Upasana; Cinque, Paola; Gerevini, Simonetta; Longo, Valeria; Lazzarin, Adriano; Schippling, Sven; Martin, Roland; Buck, Christopher B; Pastrana, Diana V

    2015-09-23

    JC polyomavirus (JCV) persistently infects the urinary tract of most adults. Under conditions of immune impairment, JCV causes an opportunistic brain disease, progressive multifocal leukoencephalopathy (PML). JCV strains found in the cerebrospinal fluid of PML patients contain distinctive mutations in surface loops of the major capsid protein, VP1. We hypothesized that VP1 mutations might allow the virus to evade antibody-mediated neutralization. Consistent with this hypothesis, neutralization serology revealed that plasma samples from PML patients neutralized wild-type JCV strains but failed to neutralize patient-cognate PML-mutant JCV strains. This contrasted with serological results for healthy individuals, most of whom robustly cross-neutralized all tested JCV variants. Mice administered a JCV virus-like particle (VLP) vaccine initially showed neutralizing "blind spots" (akin to those observed in PML patients) that closed after booster immunization. A PML patient administered an experimental JCV VLP vaccine likewise showed markedly increased neutralizing titer against her cognate PML-mutant JCV. The results indicate that deficient humoral immunity is a common aspect of PML pathogenesis and that vaccination may overcome this humoral deficiency. Thus, vaccination with JCV VLPs might prevent the development of PML.

  18. Antibodies against Marinobacter algicola and Salmonella typhimurium flagellins do not cross-neutralize TLR5 activation.

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    Raul Terron-Exposito

    Full Text Available Flagellins evoke strong innate and adaptive immune responses. These proteins may play a key role as radioprotectors, exert antitumoral activity in certain types of tumor and reduce graft-versus-host disease in allogeneic hematopoietic stem cell transplant recipients. Notwithstanding, flagellins are highly immunogenic, and repeated use leads to their neutralization by systemic antibodies. This neutralization is not prevented by using functional deleted flagellins. These observations led us to explore the possibility of preventing initial neutralization by means of another functional flagellin that does not belong to common pathogenic bacteria but that has the capacity to activate TLR5. Here we characterized the functional capacity of the two-phase Marinobacter algicola (MA-derived flagellins (F and FR as systemic and mucosal adjuvants and compared their performance with that of Salmonella typhimurium (STF flagellins (FljB and FliC. We also report for the first time on the in vitro and in vivo capacity of various flagellins to trigger TLR5 activation in the presence of species-specific anti-flagellin antibodies, the cross-neutralization mediated by these antibodies, and the sequential use of these flagellins for TLR5 activation. Our results showed that MA flagellins behave in a similar way to STF ones, inducing pro-inflammatory cytokines (IL8, CCL20, CCL2 and evoking a strong in vivo antibody response against a model epitope. More importantly, MA flagellins were fully functional, in vitro or in vivo, in the presence of a high concentration of neutralizing anti-flagellin STF antibodies, and STF flagellin was not inhibited by neutralizing anti-flagellin MA antibodies. The use of active flagellins from distinct bacteria could be a useful approach to prevent systemic neutralization of this group of adjuvants and to facilitate the rational design of flagellin-based vaccines and/or other therapeutic treatments (against ischemia, acute renal failure

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

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    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.

  20. Novel neutralizing monoclonal antibodies protect rodents against lethal filovirus challenges

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    Caleb D. Marceau

    2014-01-01

    Full Text Available Filoviruses are the causative agents of lethal hemorrhagic fever in human and non-human primates (NHP. The family of Filoviridae is composed of three genera, Ebolavirus, Marburgvirus and Cuevavirus. There are currently no approved vaccines or antiviral therapeutics for the treatment of filovirus infections in humans. Passive transfer of neutralizing antibodies targeting the Ebola virus (EBOV glycoprotein (GP has proven effective in protecting mice, guinea pigs and NHP from lethal challenges with EBOV. In this study, we generated two neutralizing monoclonal antibodies (MAbs, termed S9 and M4 that recognize the GP of EBOV or multiple strains of Marburg virus (MARV, respectively. We characterized the putative binding site of S9 as a linear epitope on the glycan cap of the GP1 subunit of the EBOV-GP. The M4 antibody recognizes an unknown conformational epitope on MARV-GP. Additionally, we demonstrated the post-exposure protection potential of these antibodies in both the mouse and guinea pig models of filovirus infection. These data indicate that MAbs S9 and M4 would be good candidates for inclusion in an antibody cocktail for the treatment of filovirus infections.

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

    Science.gov (United States)

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

    2016-01-01

    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.

  2. Humanized-Single Domain Antibodies (VH/VHH that Bound Specifically to Naja kaouthia Phospholipase A2 and Neutralized the Enzymatic Activity

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    Wanpen Chaicumpa

    2012-07-01

    Full Text Available Naja kaouthia (monocled cobra venom contains many isoforms of secreted phospholipase A2 (sPLA2. The PLA2 exerts several pharmacologic and toxic effects in the snake bitten subject, dependent or independent on the enzymatic activity. N. kaouthia venom appeared in two protein profiles, P3 and P5, after fractionating the venom by ion exchange column chromatography. In this study, phage clones displaying humanized-camel single domain antibodies (VH/VHH that bound specifically to the P3 and P5 were selected from a humanized-camel VH/VHH phage display library. Two phagemid transfected E. coli clones (P3-1 and P3-3 produced humanized-VHH, while another clone (P3-7 produced humanized-VH. At the optimal venom:antibody ratio, the VH/VHH purified from the E. coli homogenates neutralized PLA2 enzyme activity comparable to the horse immune serum against the N. kaouthia holo-venom. Homology modeling and molecular docking revealed that the VH/VHH covered the areas around the PLA2 catalytic groove and inserted their Complementarity Determining Regions (CDRs into the enzymatic cleft. It is envisaged that the VH/VHH would ameliorate/abrogate the principal toxicity of the venom PLA2 (membrane phospholipid catabolism leading to cellular and subcellular membrane damage which consequently causes hemolysis, hemorrhage, and dermo-/myo-necrosis, if they were used for passive immunotherapy of the cobra bitten victim. The speculation needs further investigations.

  3. Isolation of potent neutralizing antibodies from a survivor of the 2014 Ebola virus outbreak.

    Science.gov (United States)

    Bornholdt, Zachary A; Turner, Hannah L; Murin, Charles D; Li, Wen; Sok, Devin; Souders, Colby A; Piper, Ashley E; Goff, Arthur; Shamblin, Joshua D; Wollen, Suzanne E; Sprague, Thomas R; Fusco, Marnie L; Pommert, Kathleen B J; Cavacini, Lisa A; Smith, Heidi L; Klempner, Mark; Reimann, Keith A; Krauland, Eric; Gerngross, Tillman U; Wittrup, Karl D; Saphire, Erica Ollmann; Burton, Dennis R; Glass, Pamela J; Ward, Andrew B; Walker, Laura M

    2016-03-01

    Antibodies targeting the Ebola virus surface glycoprotein (EBOV GP) are implicated in protection against lethal disease, but the characteristics of the human antibody response to EBOV GP remain poorly understood. We isolated and characterized 349 GP-specific monoclonal antibodies (mAbs) from the peripheral B cells of a convalescent donor who survived the 2014 EBOV Zaire outbreak. Remarkably, 77% of the mAbs neutralize live EBOV, and several mAbs exhibit unprecedented potency. Structures of selected mAbs in complex with GP reveal a site of vulnerability located in the GP stalk region proximal to the viral membrane. Neutralizing antibodies targeting this site show potent therapeutic efficacy against lethal EBOV challenge in mice. The results provide a framework for the design of new EBOV vaccine candidates and immunotherapies.

  4. Neutralizing and IgG antibodies against simian virus 40 in healthy pregnant women in Italy.

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    Manola Comar

    Full Text Available Polyomavirus simian virus 40 (SV40 sequences have been detected in various human specimens and SV40 antibodies have been found in human sera from both healthy individuals and cancer patients. This study analyzed serum samples from healthy pregnant women as well as cord blood samples to determine the prevalence of SV40 antibodies in pregnancy.Serum samples were collected at the time of delivery from two groups of pregnant women as well as cord bloods from one group. The women were born between 1967 and 1993. Samples were assayed by two different serological methods, one group by neutralization of viral infectivity and the other by indirect ELISA employing specific SV40 mimotopes as antigens. Viral DNA assays by real-time polymerase chain reaction were carried out on blood samples.Neutralization and ELISA tests indicated that the pregnant women were SV40 antibody-positive with overall prevalences of 10.6% (13/123 and 12.7% (14/110, respectively. SV40 neutralizing antibodies were detected in a low number of cord blood samples. Antibody titers were generally low. No viral DNA was detected in either maternal or cord bloods.SV40-specific serum antibodies were detected in pregnant women at the time of delivery and in cord bloods. There was no evidence of transplacental transmission of SV40. These data indicate that SV40 is circulating at a low prevalence in the northern Italian population long after the use of contaminated vaccines.

  5. Enhanced neutralization potency of botulinum neurotoxin antibodies using a red blood cell-targeting fusion protein.

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    Sharad P Adekar

    Full Text Available Botulinum neurotoxin (BoNT potently inhibits cholinergic signaling at the neuromuscular junction. The ideal countermeasures for BoNT exposure are monoclonal antibodies or BoNT antisera, which form BoNT-containing immune complexes that are rapidly cleared from the general circulation. Clearance of opsonized toxins may involve complement receptor-mediated immunoadherence to red blood cells (RBC in primates or to platelets in rodents. Methods of enhancing immunoadherence of BoNT-specific antibodies may increase their potency in vivo. We designed a novel fusion protein (FP to link biotinylated molecules to glycophorin A (GPA on the RBC surface. The FP consists of an scFv specific for murine GPA fused to streptavidin. FP:mAb:BoNT complexes bound specifically to the RBC surface in vitro. In a mouse model of BoNT neutralization, the FP increased the potency of single and double antibody combinations in BoNT neutralization. A combination of two antibodies with the FP gave complete neutralization of 5,000 LD50 BoNT in mice. Neutralization in vivo was dependent on biotinylation of both antibodies and correlated with a reduction of plasma BoNT levels. In a post-exposure model of intoxication, FP:mAb complexes gave complete protection from a lethal BoNT/A1 dose when administered within 2 hours of toxin exposure. In a pre-exposure prophylaxis model, mice were fully protected for 72 hours following administration of the FP:mAb complex. These results demonstrate that RBC-targeted immunoadherence through the FP is a potent enhancer of BoNT neutralization by antibodies in vivo.

  6. Apolipoprotein E Mediates Evasion From Hepatitis C Virus Neutralizing Antibodies.

    Science.gov (United States)

    Fauvelle, Catherine; Felmlee, Daniel J; Crouchet, Emilie; Lee, JiYoung; Heydmann, Laura; Lefèvre, Mathieu; Magri, Andrea; Hiet, Marie-Sophie; Fofana, Isabel; Habersetzer, François; Foung, Steven K H; Milne, Ross; Patel, Arvind H; Vercauteren, Koen; Meuleman, Philip; Zeisel, Mirjam B; Bartenschlager, Ralf; Schuster, Catherine; Baumert, Thomas F

    2016-01-01

    Efforts to develop an effective vaccine against hepatitis C virus (HCV) have been hindered by the propensity of the virus to evade host immune responses. HCV particles in serum and in cell culture associate with lipoproteins, which contribute to viral entry. Lipoprotein association has also been proposed to mediate viral evasion of the humoral immune response, though the mechanisms are poorly defined. We used small interfering RNAs to reduce levels of apolipoprotein E (apoE) in cell culture-derived HCV-producing Huh7.5-derived hepatoma cells and confirmed its depletion by immunoblot analyses of purified viral particles. Before infection of naïve hepatoma cells, we exposed cell culture-derived HCV strains of different genotypes, subtypes, and variants to serum and polyclonal and monoclonal antibodies isolated from patients with chronic HCV infection. We analyzed the interaction of apoE with viral envelope glycoprotein E2 and HCV virions by immunoprecipitation. Through loss-of-function studies on patient-derived HCV variants of several genotypes and subtypes, we found that the HCV particle apoE allows the virus to avoid neutralization by patient-derived antibodies. Functional studies with human monoclonal antiviral antibodies showed that conformational epitopes of envelope glycoprotein E2 domains B and C were exposed after depletion of apoE. The level and conformation of virion-associated apoE affected the ability of the virus to escape neutralization by antibodies. In cell-infection studies, we found that HCV-associated apoE helps the virus avoid neutralization by antibodies against HCV isolated from chronically infected patients. This method of immune evasion poses a challenge for the development of HCV vaccines. Copyright © 2016 AGA Institute. Published by Elsevier Inc. All rights reserved.

  7. Lack of protection following passive transfer of polyclonal highly functional low-dose non-neutralizing antibodies.

    Science.gov (United States)

    Dugast, Anne-Sophie; Chan, Ying; Hoffner, Michelle; Licht, Anna; Nkolola, Joseph; Li, Hualin; Streeck, Hendrik; Suscovich, Todd J; Ghebremichael, Musie; Ackerman, Margaret E; Barouch, Dan H; Alter, Galit

    2014-01-01

    Recent immune correlates analysis from the RV144 vaccine trial has renewed interest in the role of non-neutralizing antibodies in mediating protection from infection. While neutralizing antibodies have proven difficult to induce through vaccination, extra-neutralizing antibodies, such as those that mediate antibody-dependent cellular cytotoxicity (ADCC), are associated with long-term control of infection. However, while several non-neutralizing monoclonal antibodies have been tested for their protective efficacy in vivo, no studies to date have tested the protective activity of naturally produced polyclonal antibodies from individuals harboring potent ADCC activity. Because ADCC-inducing antibodies are highly enriched in elite controllers (EC), we passively transferred highly functional non-neutralizing polyclonal antibodies, purified from an EC, to assess the potential impact of polyclonal non-neutralizing antibodies on a stringent SHIV-SF162P3 challenge in rhesus monkeys. Passive transfer of a low-dose of ADCC inducing antibodies did not protect from infection following SHIV-SF162P3 challenge. Passively administered antibody titers and gp120-specific, but not gp41-specific, ADCC and antibody induced phagocytosis (ADCP) were detected in the majority of the monkeys, but did not correlate with post infection viral control. Thus these data raise the possibility that gp120-specific ADCC activity alone may not be sufficient to control viremia post infection but that other specificities or Fc-effector profiles, alone or in combination, may have an impact on viral control and should be tested in future passive transfer experiments.

  8. Monoclonal antibodies neutralizing the haemolytic activity of box jellyfish (Chironex fleckeri) tentacle extracts.

    Science.gov (United States)

    Collins, S P; Comis, A; Marshall, M; Hartwick, R F; Howden, M E

    1993-09-01

    1. Three monoclonal antibodies have been produced which neutralize in vitro the haemolytic activity present in tentacle extracts of the box jellyfish (Chironex fleckeri). 2. Two of these monoclonal antibodies bound specifically to a component of relative molecular mass 50,000 in tentacle extract on Western blots. 3. This binding only occurred when the extracts were electrophoresed under non-reducing conditions. 4. The third monoclonal antibody did not display binding to Western blots of tentacle extract under any of our experimental conditions.

  9. Selection of peptide mimics of HIV-1 epitope recognized by neutralizing antibody VRC01.

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    Anton N Chikaev

    Full Text Available The ability to induce anti-HIV-1 antibodies that can neutralize a broad spectrum of viral isolates from different subtypes seems to be a key requirement for development of an effective HIV-1 vaccine. The epitopes recognized by the most potent broadly neutralizing antibodies that have been characterized are largely discontinuous. Mimetics of such conformational epitopes could be potentially used as components of a synthetic immunogen that can elicit neutralizing antibodies. Here we used phage display technology to identify peptide motifs that mimic the epitope recognized by monoclonal antibody VRC01, which is able to neutralize up to 91% of circulating primary isolates. Three rounds of biopanning were performed against 2 different phage peptide libraries for this purpose. The binding specificity of selected phage clones to monoclonal antibody VRC01 was estimated using dot blot analysis. The putative peptide mimics exposed on the surface of selected phages were analyzed for conformational and linear homology to the surface of HIV-1 gp120 fragment using computational analysis. Corresponding peptides were synthesized and checked for their ability to interfere with neutralization activity of VRC01 in a competitive inhibition assay. One of the most common peptides selected from 12-mer phage library was found to partially mimic a CD4-binding loop fragment, whereas none of the circular C7C-mer peptides was able to mimic any HIV-1 domains. However, peptides identified from both the 12-mer and C7C-mer peptide libraries showed rescue of HIV-1 infectivity in the competitive inhibition assay. The identification of epitope mimics may lead to novel immunogens capable of inducing broadly reactive neutralizing antibodies.

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

    Science.gov (United States)

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

    2017-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Evgeny Shmelkov

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

  12. Antigen-Specific Antibody Glycosylation Is Regulated via Vaccination.

    Science.gov (United States)

    Mahan, Alison E; Jennewein, Madeleine F; Suscovich, Todd; Dionne, Kendall; Tedesco, Jacquelynne; Chung, Amy W; Streeck, Hendrik; Pau, Maria; Schuitemaker, Hanneke; Francis, Don; Fast, Patricia; Laufer, Dagna; Walker, Bruce D; Baden, Lindsey; Barouch, Dan H; Alter, Galit

    2016-03-01

    Antibody effector functions, such as antibody-dependent cellular cytotoxicity, complement deposition, and antibody-dependent phagocytosis, play a critical role in immunity against multiple pathogens, particularly in the absence of neutralizing activity. Two modifications to the IgG constant domain (Fc domain) regulate antibody functionality: changes in antibody subclass and changes in a single N-linked glycan located in the CH2 domain of the IgG Fc. Together, these modifications provide a specific set of instructions to the innate immune system to direct the elimination of antibody-bound antigens. While it is clear that subclass selection is actively regulated during the course of natural infection, it is unclear whether antibody glycosylation can be tuned, in a signal-specific or pathogen-specific manner. Here, we show that antibody glycosylation is determined in an antigen- and pathogen-specific manner during HIV infection. Moreover, while dramatic differences exist in bulk IgG glycosylation among individuals in distinct geographical locations, immunization is able to overcome these differences and elicit antigen-specific antibodies with similar antibody glycosylation patterns. Additionally, distinct vaccine regimens induced different antigen-specific IgG glycosylation profiles, suggesting that antibody glycosylation is not only programmable but can be manipulated via the delivery of distinct inflammatory signals during B cell priming. These data strongly suggest that the immune system naturally drives antibody glycosylation in an antigen-specific manner and highlights a promising means by which next-generation therapeutics and vaccines can harness the antiviral activity of the innate immune system via directed alterations in antibody glycosylation in vivo.  .

  13. Antigen-Specific Antibody Glycosylation Is Regulated via Vaccination.

    Directory of Open Access Journals (Sweden)

    Alison E Mahan

    2016-03-01

    Full Text Available Antibody effector functions, such as antibody-dependent cellular cytotoxicity, complement deposition, and antibody-dependent phagocytosis, play a critical role in immunity against multiple pathogens, particularly in the absence of neutralizing activity. Two modifications to the IgG constant domain (Fc domain regulate antibody functionality: changes in antibody subclass and changes in a single N-linked glycan located in the CH2 domain of the IgG Fc. Together, these modifications provide a specific set of instructions to the innate immune system to direct the elimination of antibody-bound antigens. While it is clear that subclass selection is actively regulated during the course of natural infection, it is unclear whether antibody glycosylation can be tuned, in a signal-specific or pathogen-specific manner. Here, we show that antibody glycosylation is determined in an antigen- and pathogen-specific manner during HIV infection. Moreover, while dramatic differences exist in bulk IgG glycosylation among individuals in distinct geographical locations, immunization is able to overcome these differences and elicit antigen-specific antibodies with similar antibody glycosylation patterns. Additionally, distinct vaccine regimens induced different antigen-specific IgG glycosylation profiles, suggesting that antibody glycosylation is not only programmable but can be manipulated via the delivery of distinct inflammatory signals during B cell priming. These data strongly suggest that the immune system naturally drives antibody glycosylation in an antigen-specific manner and highlights a promising means by which next-generation therapeutics and vaccines can harness the antiviral activity of the innate immune system via directed alterations in antibody glycosylation in vivo.  .

  14. Identification of CD4-Binding Site Dependent Plasma Neutralizing Antibodies in an HIV-1 Infected Indian Individual.

    Science.gov (United States)

    Khan, Lubina; Makhdoomi, Muzamil Ashraf; Kumar, Sanjeev; Nair, Ambili; Andrabi, Raiees; Clark, Brenda E; Auyeung, Kate; Bhattacharya, Jayanta; Vajpayee, Madhu; Wig, Naveet; Pantophlet, Ralph; Luthra, Kalpana

    2015-01-01

    Dissecting antibody specificities in the plasma of HIV-1 infected individuals that develop broadly neutralizing antibodies (bNAbs) is likely to provide useful information for refining target epitopes for vaccine design. Several studies have reported CD4-binding site (CD4bs) antibodies as neutralization determinants in the plasma of subtype B-infected individuals; however there is little information on the prevalence of CD4bs specificities in HIV-infected individuals in India. Here, we report on the presence of CD4bs antibodies and their contribution to virus neutralization in the plasma from a cohort of HIV-1 infected Indian individuals. Plasma from 11 of the 140 HIV-1 infected individuals (7.9%) studied here exhibited cross-neutralization activity against a panel of subtype B and C viruses. Analyses of these 11 plasma samples for the presence of CD4bs antibodies using two CD4bs-selective probes (antigenically resurfaced HXB2gp120 core protein RSC3 and hyperglycosylated JRFLgp120 mutant ΔN2mCHO) revealed that five (AIIMS 617, 619, 627, 642, 660) contained RSC3-reactive plasma antibodies and only one (AIIMS 660) contained ΔN2mCHO-reactive antibodies. Plasma antibody depletion and competition experiments confirmed that the neutralizing activity in the AIIMS 660 plasma was dependent on CD4bs antibodies. To the best of our knowledge, this is the first study to report specifically on the presence of CD4bs antibodies in the plasma of a cohort of HIV-1 infected Indian donors. The identification of CD4bs dependent neutralizing antibodies in an HIV-1 infected Indian donor is a salient finding of this study and is supportive of ongoing efforts to induce similar antibodies by immunization.

  15. Identification of CD4-Binding Site Dependent Plasma Neutralizing Antibodies in an HIV-1 Infected Indian Individual.

    Directory of Open Access Journals (Sweden)

    Lubina Khan

    Full Text Available Dissecting antibody specificities in the plasma of HIV-1 infected individuals that develop broadly neutralizing antibodies (bNAbs is likely to provide useful information for refining target epitopes for vaccine design. Several studies have reported CD4-binding site (CD4bs antibodies as neutralization determinants in the plasma of subtype B-infected individuals; however there is little information on the prevalence of CD4bs specificities in HIV-infected individuals in India. Here, we report on the presence of CD4bs antibodies and their contribution to virus neutralization in the plasma from a cohort of HIV-1 infected Indian individuals. Plasma from 11 of the 140 HIV-1 infected individuals (7.9% studied here exhibited cross-neutralization activity against a panel of subtype B and C viruses. Analyses of these 11 plasma samples for the presence of CD4bs antibodies using two CD4bs-selective probes (antigenically resurfaced HXB2gp120 core protein RSC3 and hyperglycosylated JRFLgp120 mutant ΔN2mCHO revealed that five (AIIMS 617, 619, 627, 642, 660 contained RSC3-reactive plasma antibodies and only one (AIIMS 660 contained ΔN2mCHO-reactive antibodies. Plasma antibody depletion and competition experiments confirmed that the neutralizing activity in the AIIMS 660 plasma was dependent on CD4bs antibodies. To the best of our knowledge, this is the first study to report specifically on the presence of CD4bs antibodies in the plasma of a cohort of HIV-1 infected Indian donors. The identification of CD4bs dependent neutralizing antibodies in an HIV-1 infected Indian donor is a salient finding of this study and is supportive of ongoing efforts to induce similar antibodies by immunization.

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

    Science.gov (United States)

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

    2016-09-08

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

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

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Cheng-Chung; Lin, Li-Ling [Academia Sinica, Taipei 115, Taiwan (China); Academia Sinica, Taipei 115, Taiwan (China); Chan, Woan-Eng [Development Center for Biotechnology, New Taipei City 221, Taiwan (China); Ko, Tzu-Ping [Academia Sinica, Taipei 115, Taiwan (China); Academia Sinica, Taipei 115, Taiwan (China); Lai, Jiann-Shiun [Development Center for Biotechnology, New Taipei City 221, Taiwan (China); Ministry of Economic Affairs, Taipei 100, Taiwan (China); Wang, Andrew H.-J., E-mail: ahjwang@gate.sinica.edu.tw [Academia Sinica, Taipei 115, Taiwan (China); Academia Sinica, Taipei 115, Taiwan (China); Taipei Medical University, Taipei 110, Taiwan (China)

    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.

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

    Science.gov (United States)

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2015-10-15

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

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

    NARCIS (Netherlands)

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

    2010-01-01

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

  1. Serological survey on canine coronavirus antibodies in giant pandas by virus neutralization test

    Institute of Scientific and Technical Information of China (English)

    QIAOJun; XIAXian-zhu; YANGSong-tao; LIDe-sheng; HUGui-xue; GAOYu-wei; SUNHe-ting; ZHAOZhong-pen; XlEZhi-jing; YANFang; HEWen-qi; HUANGGen

    2004-01-01

    In order to survey the infectious situation of canine coronavirus (CCV) in giant panda population, a virus neutralization test detecting specific antibodies against CCV in giant panda's sera was established by using two-fold dilutions of serum and 100 TCID50 of the virus. The 62 sera samples of giant pandas, which were gathered from zoos and reserve region of Sichuan Province, China were detected. The neutralization antibody titer of 1:4 was recognized as the positive criterion, 8 sera samples were detected to be positive, and the positive rate was 12.9%. The titers of neutralizing antibody ranged from 1:8 to 1:32. It was the first comprehensive investigation on neutralization antibodies against CCV in giant panda population in China. The results of study showed that the infection of CCV in giant panda population was universal, which has posed a threat to the health of giant panda. Therefore, it is incumbent on us to study safe and effective vaccines to protect giant panda against CCV infection.

  2. Autologous HIV-1 neutralizing antibodies: emergence of neutralization-resistant escape virus and subsequent development of escape virus neutralizing antibodies

    DEFF Research Database (Denmark)

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

    1992-01-01

    The capacity of consecutive human sera to neutralize sequentially obtained autologous virus isolates was studied. HIV-1 was isolated three times over a 48-164-week period from three individuals immediately after seroconversion and from two individuals in later stages of infection. Development of ...... escape virus may be part of the explanation of the apparent failure of the immune system to control HIV infection.......The capacity of consecutive human sera to neutralize sequentially obtained autologous virus isolates was studied. HIV-1 was isolated three times over a 48-164-week period from three individuals immediately after seroconversion and from two individuals in later stages of infection. Development...

  3. Dengue reporter virus particles for measuring neutralizing antibodies against each of the four dengue serotypes.

    Science.gov (United States)

    Mattia, Kimberly; Puffer, Bridget A; Williams, Katherine L; Gonzalez, Ritela; Murray, Meredith; Sluzas, Emily; Pagano, Dan; Ajith, Sandya; Bower, Megan; Berdougo, Eli; Harris, Eva; Doranz, Benjamin J

    2011-01-01

    The lack of reliable, high-throughput tools for characterizing anti-dengue virus (DENV) antibodies in large numbers of serum samples has been an obstacle in understanding the impact of neutralizing antibodies on disease progression and vaccine efficacy. A reporter system using pseudoinfectious DENV reporter virus particles (RVPs) was previously developed by others to facilitate the genetic manipulation and biological characterization of DENV virions. In the current study, we demonstrate the diagnostic utility of DENV RVPs for measuring neutralizing antibodies in human serum samples against all four DENV serotypes, with attention to the suitability of DENV RVPs for large-scale, long-term studies. DENV RVPs used against human sera yielded serotype-specific responses and reproducible neutralization titers that were in statistical agreement with Plaque Reduction Neutralization Test (PRNT) results. DENV RVPs were also used to measure neutralization titers against the four DENV serotypes in a panel of human sera from a clinical study of dengue patients. The high-throughput capability, stability, rapidity, and reproducibility of assays using DENV RVPs offer advantages for detecting immune responses that can be applied to large-scale clinical studies of DENV infection and vaccination.

  4. Dengue reporter virus particles for measuring neutralizing antibodies against each of the four dengue serotypes.

    Directory of Open Access Journals (Sweden)

    Kimberly Mattia

    Full Text Available The lack of reliable, high-throughput tools for characterizing anti-dengue virus (DENV antibodies in large numbers of serum samples has been an obstacle in understanding the impact of neutralizing antibodies on disease progression and vaccine efficacy. A reporter system using pseudoinfectious DENV reporter virus particles (RVPs was previously developed by others to facilitate the genetic manipulation and biological characterization of DENV virions. In the current study, we demonstrate the diagnostic utility of DENV RVPs for measuring neutralizing antibodies in human serum samples against all four DENV serotypes, with attention to the suitability of DENV RVPs for large-scale, long-term studies. DENV RVPs used against human sera yielded serotype-specific responses and reproducible neutralization titers that were in statistical agreement with Plaque Reduction Neutralization Test (PRNT results. DENV RVPs were also used to measure neutralization titers against the four DENV serotypes in a panel of human sera from a clinical study of dengue patients. The high-throughput capability, stability, rapidity, and reproducibility of assays using DENV RVPs offer advantages for detecting immune responses that can be applied to large-scale clinical studies of DENV infection and vaccination.

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Science.gov (United States)

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

    2017-01-01

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

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

    Institute of Scientific and Technical Information of China (English)

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

    2011-01-01

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

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

    NARCIS (Netherlands)

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

    2013-01-01

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

  9. Natural strain variation and antibody neutralization of dengue serotype 3 viruses.

    Directory of Open Access Journals (Sweden)

    Wahala M P B Wahala

    2010-03-01

    Full Text Available Dengue viruses (DENVs are emerging, mosquito-borne flaviviruses which cause dengue fever and dengue hemorrhagic fever. The DENV complex consists of 4 serotypes designated DENV1-DENV4. Following natural infection with DENV, individuals develop serotype specific, neutralizing antibody responses. Monoclonal antibodies (MAbs have been used to map neutralizing epitopes on dengue and other flaviviruses. Most serotype-specific, neutralizing MAbs bind to the lateral ridge of domain III of E protein (EDIII. It has been widely assumed that the EDIII lateral ridge epitope is conserved within each DENV serotype and a good target for vaccines. Using phylogenetic methods, we compared the amino acid sequence of 175 E proteins representing the different genotypes of DENV3 and identified a panel of surface exposed amino acids, including residues in EDIII, that are highly variant across the four DENV3 genotypes. The variable amino acids include six residues at the lateral ridge of EDIII. We used a panel of DENV3 mouse MAbs to assess the functional significance of naturally occurring amino acid variation. From the panel of antibodies, we identified three neutralizing MAbs that bound to EDIII of DENV3. Recombinant proteins and naturally occurring variant viruses were used to map the binding sites of the three MAbs. The three MAbs bound to overlapping but distinct epitopes on EDIII. Our empirical studies clearly demonstrate that the antibody binding and neutralization capacity of two MAbs was strongly influenced by naturally occurring mutations in DENV3. Our data demonstrate that the lateral ridge "type specific" epitope is not conserved between strains of DENV3. This variability should be considered when designing and evaluating DENV vaccines, especially those targeting EDIII.

  10. HIV-1 subtype C superinfected individuals mount low autologous neutralizing antibody responses prior to intrasubtype superinfection

    Directory of Open Access Journals (Sweden)

    Basu Debby

    2012-09-01

    Full Text Available Abstract Background The potential role of antibodies in protection against intra-subtype HIV-1 superinfection remains to be understood. We compared the early neutralizing antibody (NAb responses in three individuals, who were superinfected within one year of primary infection, to ten matched non-superinfected controls from a Zambian cohort of subtype C transmission cases. Sequence analysis of single genome amplified full-length envs from a previous study showed limited diversification in the individuals who became superinfected with the same HIV-1 subtype within year one post-seroconversion. We hypothesized that this reflected a blunted NAb response, which may have made these individuals more susceptible to superinfection. Results Neutralization assays showed that autologous plasma NAb responses to the earliest, and in some cases transmitted/founder, virus were delayed and had low to undetectable titers in all three superinfected individuals prior to superinfection. In contrast, NAbs with a median IC50 titer of 1896 were detected as early as three months post-seroconversion in non-superinfected controls. Early plasma NAbs in all subjects showed limited but variable levels of heterologous neutralization breadth. Superinfected individuals also exhibited a trend toward lower levels of gp120- and V1V2-specific IgG binding antibodies but higher gp120-specific plasma IgA binding antibodies. Conclusions These data suggest that the lack of development of IgG antibodies, as reflected in autologous NAbs as well as gp120 and V1V2 binding antibodies to the primary infection virus, combined with potentially competing, non-protective IgA antibodies, may increase susceptibility to superinfection in the context of settings where a single HIV-1 subtype predominates.

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

    Science.gov (United States)

    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.

  12. Analysis of memory B cell responses and isolation of novel monoclonal antibodies with neutralizing breadth from HIV-1-infected individuals.

    Directory of Open Access Journals (Sweden)

    Davide Corti

    Full Text Available BACKGROUND: The isolation of human monoclonal antibodies (mAbs that neutralize a broad spectrum of primary HIV-1 isolates and the characterization of the human neutralizing antibody B cell response to HIV-1 infection are important goals that are central to the design of an effective antibody-based vaccine. METHODS AND FINDINGS: We immortalized IgG(+ memory B cells from individuals infected with diverse clades of HIV-1 and selected on the basis of plasma neutralization profiles that were cross-clade and relatively potent. Culture supernatants were screened using various recombinant forms of the envelope glycoproteins (Env in multiple parallel assays. We isolated 58 mAbs that were mapped to different Env surfaces, most of which showed neutralizing activity. One mAb in particular (HJ16 specific for a novel epitope proximal to the CD4 binding site on gp120 selectively neutralized a multi-clade panel of Tier-2 HIV-1 pseudoviruses, and demonstrated reactivity that was comparable in breadth, but distinct in neutralization specificity, to that of the other CD4 binding site-specific neutralizing mAb b12. A second mAb (HGN194 bound a conserved epitope in the V3 crown and neutralized all Tier-1 and a proportion of Tier-2 pseudoviruses tested, irrespective of clade. A third mAb (HK20 with broad neutralizing activity, particularly as a Fab fragment, recognized a highly conserved epitope in the HR-1 region of gp41, but showed striking assay-dependent selectivity in its activity. CONCLUSIONS: This study reveals that by using appropriate screening methods, a large proportion of memory B cells can be isolated that produce mAbs with HIV-1 neutralizing activity. Three of these mAbs show unusual breadth of neutralization and therefore add to the current panel of HIV-1 neutralizing antibodies with potential for passive protection and template-based vaccine design.

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

    Science.gov (United States)

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

    2013-02-01

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

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

    Science.gov (United States)

    Bogdanoff, Walter A; Morgenstern, David; Bern, Marshall; Ueberheide, Beatrix M; Sanchez-Fauquier, Alicia; DuBois, Rebecca M

    2016-05-13

    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.

  15. Human Monoclonal Antibodies Broadly Neutralizing against Influenza B Virus

    Science.gov (United States)

    Yasugi, Mayo; Kubota-Koketsu, Ritsuko; Yamashita, Akifumi; Kawashita, Norihito; Du, Anariwa; Sasaki, Tadahiro; Nishimura, Mitsuhiro; Misaki, Ryo; Kuhara, Motoki; Boonsathorn, Naphatsawan; Fujiyama, Kazuhito; Okuno, Yoshinobu; Nakaya, Takaaki; Ikuta, Kazuyoshi

    2013-01-01

    Influenza virus has the ability to evade host immune surveillance through rapid viral genetic drift and reassortment; therefore, it remains a continuous public health threat. The development of vaccines producing broadly reactive antibodies, as well as therapeutic strategies using human neutralizing monoclonal antibodies (HuMAbs) with global reactivity, has been gathering great interest recently. Here, three hybridoma clones producing HuMAbs against influenza B virus, designated 5A7, 3A2 and 10C4, were prepared using peripheral lymphocytes from vaccinated volunteers, and were investigated for broad cross-reactive neutralizing activity. Of these HuMAbs, 3A2 and 10C4, which recognize the readily mutable 190-helix region near the receptor binding site in the hemagglutinin (HA) protein, react only with the Yamagata lineage of influenza B virus. By contrast, HuMAb 5A7 broadly neutralizes influenza B strains that were isolated from 1985 to 2006, belonging to both Yamagata and Victoria lineages. Epitope mapping revealed that 5A7 recognizes 316G, 318C and 321W near the C terminal of HA1, a highly conserved region in influenza B virus. Indeed, no mutations in the amino acid residues of the epitope region were induced, even after the virus was passaged ten times in the presence of HuMAb 5A7. Moreover, 5A7 showed significant therapeutic efficacy in mice, even when it was administered 72 hours post-infection. These results indicate that 5A7 is a promising candidate for developing therapeutics, and provide insight for the development of a universal vaccine against influenza B virus. PMID:23408886

  16. Sub-domains of ricin's B subunit as targets of toxin neutralizing and non-neutralizing monoclonal antibodies.

    Directory of Open Access Journals (Sweden)

    Anastasiya Yermakova

    Full Text Available 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 cytotoxicity 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.

  17. Hepatitis C virus (HCV infection may elicit neutralizing antibodies targeting epitopes conserved in all viral genotypes.

    Directory of Open Access Journals (Sweden)

    Nicasio Mancini

    Full Text Available Anti-hepatitis C virus (HCV cross-neutralizing human monoclonal antibodies, directed against conserved epitopes on surface E2 glycoprotein, are central tools for understanding virus-host interplay, and for planning strategies for prevention and treatment of this infection. Recently, we developed a research aimed at identifying these antibody specificities. The characteristics of one of these antibodies (Fab e20 were addressed in this study. Firstly, using immunofluorescence and FACS analysis of cells expressing envelope HCV glycoproteins, Fab e20 was able to recognize all HCV genotypes. Secondly, competition assays with a panel of mouse and rat monoclonals, and alanine scanning mutagenesis analyses located the e20 epitope within the CD81 binding site, documenting that three highly conserved HCV/E2 residues (W529, G530 and D535 are critical for e20 binding. Finally, a strong neutralizing activity against HCV pseudoparticles (HCVpp incorporating envelope glycoproteins of genotypes 1a, 1b, 2a, 2b and 4, and against the cell culture-grown (HCVcc JFH1 strain, was observed. The data highlight that neutralizing antibodies against HCV epitopes present in all HCV genotypes are elicited during natural infection. Their availability may open new avenues to the understanding of HCV persistence and to the development of strategies for the immune control of this infection.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2015-09-15

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

  19. Intranasal Administration of Antibody-Bound Respiratory Syncytial Virus Particles Efficiently Primes Virus-Specific Immune Responses in Mice

    NARCIS (Netherlands)

    Kruijsen, Debby; Einarsdottir, Helga K.; Schijf, Marcel A.; Coenjaerts, Frank E.; van der Schoot, Ellen C.; Vidarsson, Gestur; van Bleek, Grada M.

    2013-01-01

    Infants are protected from a severe respiratory syncytial virus (RSV) infection in the first months of life by maternal antibodies or by prophylactically administered neutralizing antibodies. Efforts are under way to produce RSV-specific antibodies with increased neutralizing capacity compared to th

  20. Monoclonal Antibodies Follow Distinct Aggregation Pathways During Production-Relevant Acidic Incubation and Neutralization

    DEFF Research Database (Denmark)

    Pedersen, Thomas Skamris; Tian, Xinsheng; Thorolfsson, Matthias

    2016-01-01

    PURPOSE: Aggregation aspects of therapeutic monoclonal antibodies (mAbs) are of common concern to the pharmaceutical industry. Low pH treatment is applied during affinity purification and to inactivate endogenous retroviruses, directing interest to the mechanisms of acid-induced antibody...... aggregation. METHODS: We characterized the oligomerization kinetics at pH 3.3, as well as the reversibility upon neutralization, of three model mAbs with identical variable regions, representative of IgG1, IgG2 and IgG4 respectively. We applied size-exclusion high performance liquid chromatography......H IgG1 retained monomeric, whereas IgG2 and IgG4 exhibited two-phase oligomerization processes. After neutralization, IgG2 oligomers partially reverted to the monomeric state, while on the contrary, IgG4 oligomers tended to aggregate. Subclass-specific aggregation-prone motifs on the Fc fragments were...

  1. In vivo emergence of HIV-1 highly sensitive to neutralizing antibodies.

    Directory of Open Access Journals (Sweden)

    Marlén M I Aasa-Chapman

    Full Text Available BACKGROUND: The rapid and continual viral escape from neutralizing antibodies is well documented in HIV-1 infection. Here we report in vivo emergence of viruses with heightened sensitivity to neutralizing antibodies, sometimes paralleling the development of neutralization escape. METHODOLOGY/PRINCIPAL FINDINGS: Sequential viral envs were amplified from seven HIV-1 infected men monitored from seroconversion up to 5 years after infection. Env-recombinant infectious molecular clones were generated and tested for coreceptor use, macrophage tropism and neutralization sensitivity to homologous and heterologous serum, soluble CD4 and monoclonal antibodies IgG1b12, 2G12 and 17b. We found that HIV-1 evolves sensitivity to contemporaneous neutralizing antibodies during infection. Neutralization sensitive viruses grow out even when potent autologous neutralizing antibodies are present in patient serum. Increased sensitivity to neutralization was associated with susceptibility of the CD4 binding site or epitopes induced after CD4 binding, and mediated by complex envelope determinants including V3 and V4 residues. The development of neutralization sensitive viruses occurred without clinical progression, coreceptor switch or change in tropism for primary macrophages. CONCLUSIONS: We propose that an interplay of selective forces for greater virus replication efficiency without the need to resist neutralizing antibodies in a compartment protected from immune surveillance may explain the temporal course described here for the in vivo emergence of HIV-1 isolates with high sensitivity to neutralizing antibodies.

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

    Science.gov (United States)

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

    2017-01-02

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

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

    OpenAIRE

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

    2014-01-01

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

  4. Anthrax vaccination induced anti-lethal factor IgG: fine specificity and neutralizing capacity.

    Science.gov (United States)

    Crowe, Sherry R; Garman, Lori; Engler, Renata J M; Farris, A Darise; Ballard, Jimmy D; Harley, John B; James, Judith A

    2011-05-09

    The efficacy biomarker of the currently licensed anthrax vaccine (AVA) is based on quantity and neutralizing capacity of anti-protective antigen (anti-PA) antibodies. However, animal studies have demonstrated that antibodies to lethal factor (LF) can provide protection against in vivo bacterial spore challenges. Improved understanding of the fine specificities of humoral immune responses that provide optimum neutralization capacity may enhance the efficacy of future passive immune globulin preparations to treat and prevent inhalation anthrax morbidity and mortality. This study (n=1000) was designed to identify AVA vaccinated individuals who generate neutralizing antibodies and to determine what specificities correlate with protection. The number of vaccine doses, years post vaccination, and PA titer were associated with in vitro neutralization, reinforcing previous reports. In addition, African American individuals had lower serologic neutralizing activity than European Americans, suggesting a genetic role in the generation of these neutralizing antibodies. Of the vaccinated individuals, only 69 (6.9%) had moderate levels of anti-LF IgG compared to 244 (24.4%) with low and 687 (68.7%) with extremely low levels of IgG antibodies to LF. Using overlapping decapeptide analysis, we identified six common LF antigenic regions targeted by those individuals with moderate levels of antibodies to LF and high in vitro toxin neutralizing activity. Affinity purified antibodies directed against antigenic epitopes within the PA binding and ADP-ribotransferase-like domains of LF were able to protect mice against lethal toxin challenge. Findings from these studies have important implications for vaccine design and immunotherapeutic development.

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

    Science.gov (United States)

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

    2012-12-01

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

  6. Seroepidemiology of Human Papillomavirus 16 (HPV16) L2 and Generation of L2-Specific Human Chimeric Monoclonal Antibodies

    National Research Council Canada - National Science Library

    Wang, Joshua W; Jagu, Subhashini; Wu, Wai-Hong; Viscidi, Raphael P; Macgregor-Das, Anne; Fogel, Jessica M; Kwak, Kihyuck; Daayana, Sai; Kitchener, Henry; Stern, Peter L; Gravitt, Patti E; Trimble, Cornelia L; Roden, Richard B S

    2015-01-01

    Presently, the seroprevalence of human papillomavirus (HPV) minor capsid antigen L2-reactive antibody is not well understood, and no serologic standard exists for L2-specific neutralizing antibodies...

  7. Hepatitis C virus vaccine candidates inducing protective neutralizing antibodies.

    Science.gov (United States)

    Fauvelle, Catherine; Colpitts, Che C; Keck, Zhen-Yong; Pierce, Brian G; Foung, Steven K H; Baumert, Thomas F

    2016-12-01

    With more than 150 million chronically infected people, hepatitis C virus (HCV) remains a substantial global health burden. Direct-acting antivirals have dramatically improved viral cure. However, limited access to therapy, late stage detection of infection and re-infection following cure illustrate the need for a vaccine for global control of infection. Vaccines with induction of neutralizing antibodies (nAbs) have been shown to protect successfully against infections by multiple viruses and are currently developed for HCV. Areas covered: Here we review the progress towards the development of vaccines aiming to confer protection against chronic HCV infection by inducing broadly nAbs. The understanding or viral immune evasion in infected patients, the development of novel model systems and the recent structural characterization of viral envelope glycoprotein E2 has markedly advanced our understanding of the molecular mechanisms of virus neutralization with the concomitant development of several vaccine candidates. Expert commentary: While HCV vaccine development remains challenged by the high viral diversity and immune evasion, marked progress in HCV research has advanced vaccine design. Several vaccine candidates have shown robust induction of nAbs in animal models and humans. Randomized clinical trials are the next step to assess their clinical efficacy for protection against chronic infection.

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Yağmur Derman

    2016-09-01

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

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

    Science.gov (United States)

    Sliepen, Kwinten; Sanders, Rogier W

    2016-01-01

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

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

    Institute of Scientific and Technical Information of China (English)

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

    2004-01-01

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

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

    Science.gov (United States)

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

    2016-02-18

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

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

    Science.gov (United States)

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

    2016-11-01

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

  14. A novel universal neutralizing monoclonal antibody against enterovirus 71 that targets the highly conserved "knob" region of VP3 protein.

    Directory of Open Access Journals (Sweden)

    Tanja K Kiener

    Full Text Available Hand, foot and mouth disease caused by enterovirus 71(EV71 leads to the majority of neurological complications and death in young children. While putative inactivated vaccines are only now undergoing clinical trials, no specific treatment options exist yet. Ideally, EV71 specific intravenous immunoglobulins could be developed for targeted treatment of severe cases. To date, only a single universally neutralizing monoclonal antibody against a conserved linear epitope of VP1 has been identified. Other enteroviruses have been shown to possess major conformational neutralizing epitopes on both the VP2 and VP3 capsid proteins. Hence, we attempted to isolate such neutralizing antibodies against conformational epitopes for their potential in the treatment of infection as well as differential diagnosis and vaccine optimization. Here we describe a universal neutralizing monoclonal antibody that recognizes a conserved conformational epitope of EV71 which was mapped using escape mutants. Eight escape mutants from different subgenogroups (A, B2, B4, C2, C4 were rescued; they harbored three essential mutations either at amino acid positions 59, 62 or 67 of the VP3 protein which are all situated in the "knob" region. The escape mutant phenotype could be mimicked by incorporating these mutations into reverse genetically engineered viruses showing that P59L, A62D, A62P and E67D abolish both monoclonal antibody binding and neutralization activity. This is the first conformational neutralization epitope mapped on VP3 for EV71.

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

    Science.gov (United States)

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

    2017-02-28

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

  16. Interferon β-1b-neutralizing antibodies 5 years after clinically isolated syndrome

    DEFF Research Database (Denmark)

    Hartung, H-P; Freedman, M S; Polman, C H

    2011-01-01

    To determine the frequency and consequences of neutralizing antibodies (NAbs) in patients with a first event suggestive of multiple sclerosis (MS) treated with interferon β-1b (IFNβ-1b).......To determine the frequency and consequences of neutralizing antibodies (NAbs) in patients with a first event suggestive of multiple sclerosis (MS) treated with interferon β-1b (IFNβ-1b)....

  17. Development and characterization of mouse monoclonal antibodies specific for chicken interleukin 18

    Science.gov (United States)

    Four mouse monoclonal antibodies (mAbs) which are specific for chicken interleukin 18 (chIL18) were produced and characterized by enzyme-linked immunosorbent assay (ELISA), Western blotting, quantitative real-time PCR and neutralization assays. Monoclonal antibodies specific for chIL18 identified a ...

  18. Structure and specificity of lamprey monoclonal antibodies

    OpenAIRE

    Herrin, Brantley R.; Alder, Matthew N; Roux, Kenneth H.; Sina, Christina; Ehrhardt, Götz R. A.; Boydston, Jeremy A.; Turnbough, Charles L.; Cooper, Max D.

    2008-01-01

    Adaptive immunity in jawless vertebrates (lamprey and hagfish) is mediated by lymphocytes that undergo combinatorial assembly of leucine-rich repeat (LRR) gene segments to create a diverse repertoire of variable lymphocyte receptor (VLR) genes. Immunization with particulate antigens induces VLR-B-bearing lymphocytes to secrete antigen-specific VLR-B antibodies. Here, we describe the production of recombinant VLR-B antibodies specific for BclA, a major coat protein of Bacillus anthracis spores...

  19. Neutralizing antibody blocks adenovirus infection by arresting microtubule-dependent cytoplasmic transport.

    Science.gov (United States)

    Smith, Jason G; Cassany, Aurelia; Gerace, Larry; Ralston, Robert; Nemerow, Glen R

    2008-07-01

    Neutralizing antibodies are commonly elicited by viral infection. Most antibodies that have been characterized block early stages of virus entry that occur before membrane penetration, whereas inhibition of late stages in entry that occurs after membrane penetration has been poorly characterized. Here we provide evidence that the neutralizing antihexon monoclonal antibody 9C12 inhibits adenovirus infection by blocking microtubule-dependent translocation of the virus to the microtubule-organizing center following endosome penetration. These studies identify a previously undescribed mechanism by which neutralizing antibodies block virus infection, a situation that may be relevant for other nonenveloped viruses that use microtubule-dependent transport during cell entry.

  20. Neutralizing Antibody Blocks Adenovirus Infection by Arresting Microtubule-Dependent Cytoplasmic Transport▿

    Science.gov (United States)

    Smith, Jason G.; Cassany, Aurelia; Gerace, Larry; Ralston, Robert; Nemerow, Glen R.

    2008-01-01

    Neutralizing antibodies are commonly elicited by viral infection. Most antibodies that have been characterized block early stages of virus entry that occur before membrane penetration, whereas inhibition of late stages in entry that occurs after membrane penetration has been poorly characterized. Here we provide evidence that the neutralizing antihexon monoclonal antibody 9C12 inhibits adenovirus infection by blocking microtubule-dependent translocation of the virus to the microtubule-organizing center following endosome penetration. These studies identify a previously undescribed mechanism by which neutralizing antibodies block virus infection, a situation that may be relevant for other nonenveloped viruses that use microtubule-dependent transport during cell entry. PMID:18448546

  1. Structural bases of coronavirus attachment to host aminopeptidase N and its inhibition by neutralizing antibodies.

    Directory of Open Access Journals (Sweden)

    Juan Reguera

    Full Text Available The coronaviruses (CoVs are enveloped viruses of animals and humans associated mostly with enteric and respiratory diseases, such as the severe acute respiratory syndrome and 10-20% of all common colds. A subset of CoVs uses the cell surface aminopeptidase N (APN, a membrane-bound metalloprotease, as a cell entry receptor. In these viruses, the envelope spike glycoprotein (S mediates the attachment of the virus particles to APN and subsequent cell entry, which can be blocked by neutralizing antibodies. Here we describe the crystal structures of the receptor-binding domains (RBDs of two closely related CoV strains, transmissible gastroenteritis virus (TGEV and porcine respiratory CoV (PRCV, in complex with their receptor, porcine APN (pAPN, or with a neutralizing antibody. The data provide detailed information on the architecture of the dimeric pAPN ectodomain and its interaction with the CoV S. We show that a protruding receptor-binding edge in the S determines virus-binding specificity for recessed glycan-containing surfaces in the membrane-distal region of the pAPN ectodomain. Comparison of the RBDs of TGEV and PRCV to those of other related CoVs, suggests that the conformation of the S receptor-binding region determines cell entry receptor specificity. Moreover, the receptor-binding edge is a major antigenic determinant in the TGEV envelope S that is targeted by neutralizing antibodies. Our results provide a compelling view on CoV cell entry and immune neutralization, and may aid the design of antivirals or CoV vaccines. APN is also considered a target for cancer therapy and its structure, reported here, could facilitate the development of anti-cancer drugs.

  2. Neutralizing antibodies induced by recombinant virus-like particles of enterovirus 71 genotype C4 inhibit infection at pre- and post-attachment steps.

    Directory of Open Access Journals (Sweden)

    Zhiqiang Ku

    Full Text Available BACKGROUND: Enterovirus 71 (EV71 is a major causative agent of hand, foot and mouth disease, which has been prevalent in Asia-Pacific regions, causing significant morbidity and mortality in young children. Antibodies elicited by experimental EV71 vaccines could neutralize infection in vitro and passively protect animal models from lethal challenge, indicating that neutralizing antibodies play an essential role in protection. However, how neutralizing antibodies inhibit infection in vitro remains unclear. METHODS/FINDINGS: In the present study, we explored the mechanisms of neutralization by antibodies against EV71 virus-like particles (VLPs. Recombinant VLPs of EV71 genotype C4 were produced in insect cells using baculovirus vectors. Immunization with the VLPs elicited a high-titer, EV71-specific antibody response in mice. Anti-VLP mouse sera potently neutralized EV71 infection in vitro. The neutralizing antibodies in the anti-VLP mouse sera were found to target mainly an extremely conserved epitope (FGEHKQEKDLEYGAC located at the GH loop of the VP1 protein. The neutralizing anti-VLP antisera were able to inhibit virus binding to target cells efficiently. In addition, post-attachment treatment of virus-bound cells with the anti-VLP antisera also neutralized virus infection, although the antibody concentration required was higher than that of the pre-attachment treatment. CONCLUSIONS: Collectively, our findings represent a valuable addition to the understanding of mechanisms of EV71 neutralization and have strong implications for EV71 vaccine development.

  3. Purification and identification of Haemophilus ducreyi cytotoxin by use of a neutralizing monoclonal antibody.

    OpenAIRE

    Purvén, M; Frisk, A.; Lönnroth, I.; Lagergard, T

    1997-01-01

    Haemophilus ducreyi produces a cytotoxin responsible for the killing of cultured human epithelial cells. Cytotoxin-neutralizing antibodies were detected in the majority of sera from patients with culture-proven chancroid, and a significantly higher level of such antibodies in patients than in blood donors was noted both in areas where the disease is endemic and those where it is not. We produced neutralizing monoclonal antibodies (MAbs) in mice with a crude osmotic preparation of the cytotoxi...

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

    Science.gov (United States)

    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

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

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Xueling; Zhou, Tongqing; Zhu, Jiang; Zhang, Baoshan; Georgiev, Ivelin; Wang, Charlene; Chen, Xuejun; Longo, Nancy S.; Louder, Mark; McKee, Krisha; O’Dell, Sijy; Perfetto, Stephen; Schmidt, Stephen D.; Shi, Wei; Wu, Lan; Yang, Yongping; Yang, Zhi-Yong; Yang, Zhongjia; Zhang, Zhenhai; Bonsignori, Mattia; Crump, John A.; Kapiga, Saidi H.; Sam, Noel E.; Haynes, Barton F.; Simek, Melissa; Burton, Dennis R.; Koff, Wayne C.; Doria-Rose, Nicole A.; Connors, Mark; Mullikin, James C.; Nabel, Gary J.; Roederer, Mario; Shapiro, Lawrence; Kwong, Peter D.; Mascola, John R. (Tumaini); (NIH); (Duke); (Kilimanjaro Repro.); (IAVI)

    2013-03-04

    Antibody VRC01 is a human immunoglobulin that neutralizes about 90% of HIV-1 isolates. To understand how such broadly neutralizing antibodies develop, we used x-ray crystallography and 454 pyrosequencing to characterize additional VRC01-like antibodies from HIV-1-infected individuals. Crystal structures revealed a convergent mode of binding for diverse antibodies to the same CD4-binding-site epitope. A functional genomics analysis of expressed heavy and light chains revealed common pathways of antibody-heavy chain maturation, confined to the IGHV1-2*02 lineage, involving dozens of somatic changes, and capable of pairing with different light chains. Broadly neutralizing HIV-1 immunity associated with VRC01-like antibodies thus involves the evolution of antibodies to a highly affinity-matured state required to recognize an invariant viral structure, with lineages defined from thousands of sequences providing a genetic roadmap of their development.

  6. Neutralizing antibody fails to impact the course of Ebola virus infection in monkeys.

    Directory of Open Access Journals (Sweden)

    Wendelien B Oswald

    2007-01-01

    Full Text Available Prophylaxis with high doses of neutralizing antibody typically offers protection against challenge with viruses producing acute infections. In this study, we have investigated the ability of the neutralizing human monoclonal antibody, KZ52, to protect against Ebola virus in rhesus macaques. This antibody was previously shown to fully protect guinea pigs from infection. Four rhesus macaques were given 50 mg/kg of neutralizing human monoclonal antibody KZ52 intravenously 1 d before challenge with 1,000 plaque-forming units of Ebola virus, followed by a second dose of 50 mg/kg antibody 4 d after challenge. A control animal was exposed to virus in the absence of antibody treatment. Passive transfer of the neutralizing human monoclonal antibody not only failed to protect macaques against challenge with Ebola virus but also had a minimal effect on the explosive viral replication following infection. We show that the inability of antibody to impact infection was not due to neutralization escape. It appears that Ebola virus has a mechanism of infection propagation in vivo in macaques that is uniquely insensitive even to high concentrations of neutralizing antibody.

  7. Somatic populations of PGT135-137 HIV-1-neutralizing antibodies identified by 454 pyrosequencing and bioinformatics

    Directory of Open Access Journals (Sweden)

    Jiang eZhu

    2012-09-01

    Full Text Available Select HIV-1-infected individuals develop sera capable of neutralizing diverse viral strains. The molecular basis of this neutralization is currently being deciphered by the isolation of HIV-1-neutralizing antibodies. In one infected donor, three neutralizing antibodies, PGT135-137, were identified by assessment of neutralization from individually sorted B cells and found to recognize an epitope containing an N-linked glycan at residue 332 on HIV-1 gp120. Here we use deep sequencing and bioinformatics methods to interrogate the B cell record of this donor to gain a more complete understanding of the humoral immune response. PGT135-137-gene family-specific primers were used to amplify heavy and light chain-variable domain sequences. 454 pyrosequencing produced 141,298 heavy-chain sequences of IGHV4-39 origin and 87,229 light-chain sequences of IGKV3-15 origin. A number of heavy and light chain sequences of ~90% identity to PGT137, several to PGT136, and none of high identity to PGT135 were identified. After expansion of these sequences to include close phylogenetic relatives, a total of 202 heavy-chain sequences and 72 light-chain sequences were identified. These sequences were clustered into populations of 95% identity comprising 15 for heavy chain and 10 for light chain, and a select sequence from each population was synthesized and reconstituted with a PGT137-partner chain. Reconstituted antibodies showed varied neutralization phenotypes for HIV-1 clade A and D isolates. Sequence diversity of the antibody population represented by these tested sequences was notably higher than observed with a 454 pyrosequencing-control analysis on 10 antibodies of defined sequence, suggesting that this diversity results primarily from somatic maturation. Our results thus provide an example of how pathogens like HIV-1 are opposed by a varied humoral immune response, derived from intrinsic mechanisms of antibody development, and embodied by somatic populations

  8. Cooperativity between CD8+ T cells, non-neutralizing antibodies, and alveolar macrophages is important for heterosubtypic influenza virus immunity.

    Directory of Open Access Journals (Sweden)

    Brian J Laidlaw

    2013-03-01

    Full Text Available 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.

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

    DEFF Research Database (Denmark)

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

    1991-01-01

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

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

    Science.gov (United States)

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

    2016-01-01

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

  11. Virus-neutralizing antibody response of mice to consecutive infection with human and avian influenza A viruses.

    Science.gov (United States)

    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.

  12. Induction of neutralizing antibodies in mice immunized with scorpion toxins detoxified by liposomal entrapment

    Directory of Open Access Journals (Sweden)

    S.G. Fonseca

    1997-07-01

    Full Text Available The possibility of producing neutralizing antibodies against the lethal effects of scorpion toxins was evaluated in the mouse model by immunization with an immunogen devoid of toxicity. A toxic fraction (5 mg from the venom of the scorpion Tityus serrulatus was entrapped in sphingomyelin-cholesterol liposomes. The liposomes were treated for 1 h at 37oC with a 1% (w/w trypsin solution in 0.2 M sodium carbonate buffer, pH 8.3. This treatment led to a strong reduction in venom toxicity. Immunization was performed as follows: mice were injected sc with 20 µg of the liposome-entrapped toxic fraction on days 1 and 21 and a final injection (20 µg was administered ip on day 36. After injection of the immunogen, all mice developed an IgG response which was shown to be specific for the toxic antigen. The antibodies were measured 10 days after the end of the immunization protocol. In an in vitro neutralization assay we observed that pre-incubation of a lethal dose of the toxic fraction with immune serum strongly reduced its toxicity. In vivo protection assays showed that mice with anti-toxin antibodies could resist the challenge with the toxic fraction, which killed, 30 min after injection, all non-immune control mice

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

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

    2015-09-10

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

  15. Rabies neutralizing antibody detection by indirect immunperoxidase serum neutralization assay performed on chicken embryo related cell line

    Directory of Open Access Journals (Sweden)

    Tereza Cristina Cardoso

    2004-08-01

    Full Text Available The aim of this study was to evaluate the indirect immunoperoxidase virus neutralization (IPVN and mouse neutralization test (MNT to detect antibodies against rabies virus from vaccinated dogs and cattle. The IPVN was set up for the ability to measure 0.5 International Units/ml (IU of antibody required by the World Health Organization and the Office International des Epizooties as the minimum response for proof of rabies immunization. IPVN was developed and standardized in chicken embryo related (CER cell line when 141 dog and 110 cattle sera were applied by serial five-fold dilutions (1:5, 1:25, 1:125 as well as the positive and negative reference controls, all added in four adjacent wells, of 96-well microplates. A 50 µl amount of CVS32 strain dilution containing 50-200 TCID50/ml was mixed to each serum dilution, and after 90 min 50 µl of 3 x 10(5 cells/mlcell suspension added to each well. After five days of incubation, the monolayers were fixed and the IPVN test performed. The correlation coefficient between the MNT and IPVN performed in CER cells was r = 0.9949 for dog sera (n = 100 and r = 0.9307 for cattle sera (n = 99, as well as good specificity (94.7%, sensitivity (87.5%, and agreement (96.6% were also obtained. IPVN technique can adequately identify vaccinated and unvaccinated animals, even from low-responding vaccinated animals, with the advantage of low cost and faster then MNT standard test.

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

    Science.gov (United States)

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

    2015-01-01

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

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Science.gov (United States)

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

    2015-05-04

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

  19. Early low-titer neutralizing antibodies impede HIV-1 replication and select for virus escape.

    Directory of Open Access Journals (Sweden)

    Katharine J Bar

    Full Text Available Single genome sequencing of early HIV-1 genomes provides a sensitive, dynamic assessment of virus evolution and insight into the earliest anti-viral immune responses in vivo. By using this approach, together with deep sequencing, site-directed mutagenesis, antibody adsorptions and virus-entry assays, we found evidence in three subjects of neutralizing antibody (Nab responses as early as 2 weeks post-seroconversion, with Nab titers as low as 1∶20 to 1∶50 (IC(50 selecting for virus escape. In each of the subjects, Nabs targeted different regions of the HIV-1 envelope (Env in a strain-specific, conformationally sensitive manner. In subject CH40, virus escape was first mediated by mutations in the V1 region of the Env, followed by V3. HIV-1 specific monoclonal antibodies from this subject mapped to an immunodominant region at the base of V3 and exhibited neutralizing patterns indistinguishable from polyclonal antibody responses, indicating V1-V3 interactions within the Env trimer. In subject CH77, escape mutations mapped to the V2 region of Env, several of which selected for alterations of glycosylation. And in subject CH58, escape mutations mapped to the Env outer domain. In all three subjects, initial Nab recognition was followed by sequential rounds of virus escape and Nab elicitation, with Nab escape variants exhibiting variable costs to replication fitness. Although delayed in comparison with autologous CD8 T-cell responses, our findings show that Nabs appear earlier in HIV-1 infection than previously recognized, target diverse sites on HIV-1 Env, and impede virus replication at surprisingly low titers. The unexpected in vivo sensitivity of early transmitted/founder virus to Nabs raises the possibility that similarly low concentrations of vaccine-induced Nabs could impair virus acquisition in natural HIV-1 transmission, where the risk of infection is low and the number of viruses responsible for transmission and productive clinical

  20. Potent neutralization of influenza A virus by a single-domain antibody blocking M2 ion channel protein.

    Directory of Open Access Journals (Sweden)

    Guowei Wei

    Full Text Available Influenza A virus poses serious health threat to humans. Neutralizing antibodies against the highly conserved M2 ion channel is thought to offer broad protection against influenza A viruses. Here, we screened synthetic Camel single-domain antibody (VHH libraries against native M2 ion channel protein. One of the isolated VHHs, M2-7A, specifically bound to M2-expressed cell membrane as well as influenza A virion, inhibited replication of both amantadine-sensitive and resistant influenza A viruses in vitro, and protected mice from a lethal influenza virus challenge. Moreover, M2-7A showed blocking activity for proton influx through M2 ion channel. These pieces of evidence collectively demonstrate for the first time that a neutralizing antibody against M2 with broad specificity is achievable, and M2-7A may have potential for cross protection against a number of variants and subtypes of influenza A viruses.

  1. Early detection of neutralizing antibodies to interferon-beta in multiple sclerosis patients

    DEFF Research Database (Denmark)

    Hegen, H; Millonig, A; Bertolotto, A

    2014-01-01

    BACKGROUND: Neutralizing antibodies (NAb) affect efficacy of interferon-beta (IFN-b) treatment in multiple sclerosis (MS) patients. NAbs evolve in up to 44% of treated patients, usually between 6-18 months on therapy. OBJECTIVES: To investigate whether early binding antibody (BAb) titers or diffe......BACKGROUND: Neutralizing antibodies (NAb) affect efficacy of interferon-beta (IFN-b) treatment in multiple sclerosis (MS) patients. NAbs evolve in up to 44% of treated patients, usually between 6-18 months on therapy. OBJECTIVES: To investigate whether early binding antibody (BAb) titers...

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

    Science.gov (United States)

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

    2011-08-01

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

  3. Specificity, cross-reactivity, and function of antibodies elicited by Zika virus infection.

    Science.gov (United States)

    Stettler, Karin; Beltramello, Martina; Espinosa, Diego A; Graham, Victoria; Cassotta, Antonino; Bianchi, Siro; Vanzetta, Fabrizia; Minola, Andrea; Jaconi, Stefano; Mele, Federico; Foglierini, Mathilde; Pedotti, Mattia; Simonelli, Luca; Dowall, Stuart; Atkinson, Barry; Percivalle, Elena; Simmons, Cameron P; Varani, Luca; Blum, Johannes; Baldanti, Fausto; Cameroni, Elisabetta; Hewson, Roger; Harris, Eva; Lanzavecchia, Antonio; Sallusto, Federica; Corti, Davide

    2016-08-19

    Zika virus (ZIKV), a mosquito-borne flavivirus with homology to Dengue virus (DENV), has become a public health emergency. By characterizing memory lymphocytes from ZIKV-infected patients, we dissected ZIKV-specific and DENV-cross-reactive immune responses. Antibodies to nonstructural protein 1 (NS1) were largely ZIKV-specific and were used to develop a serological diagnostic tool. In contrast, antibodies against E protein domain I/II (EDI/II) were cross-reactive and, although poorly neutralizing, potently enhanced ZIKV and DENV infection in vitro and lethally enhanced DENV disease in mice. Memory T cells against NS1 or E proteins were poorly cross-reactive, even in donors preexposed to DENV. The most potent neutralizing antibodies were ZIKV-specific and targeted EDIII or quaternary epitopes on infectious virus. An EDIII-specific antibody protected mice from lethal ZIKV infection, illustrating the potential for antibody-based therapy.

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

    Science.gov (United States)

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

    2016-06-01

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

  5. Respiratory syncytial virus neutralizing antibodies in cord blood, respiratory syncytial virus hospitalization, and recurrent wheeze

    DEFF Research Database (Denmark)

    Stensballe, Lone Graff; Ravn, Henrik; Kristensen, Kim

    2008-01-01

    BACKGROUND: Respiratory syncytial virus (RSV) hospitalization is associated with wheeze. OBJECTIVE: To examine the influence of maternally derived RSV neutralizing antibodies in cord blood on RSV hospitalization and recurrent wheeze in infancy. METHODS: Among children from the Danish National Bir...

  6. Characterization of a Novel Neutralizing Monoclonal Antibody Against Ebola Virus GP.

    Science.gov (United States)

    Reynard, Olivier; Volchkov, Viktor E

    2015-10-01

    Ebola virus is the etiological agent of a severe hemorrhagic fever with a high mortality rate. As the only protein exposed on the surface of viral particles, the spike glycoprotein GP is the unique target for neutralizing monoclonal antibodies. In this study, we demonstrate the strong neutralization capacity of the monoclonal antibody #3327 and characterize its activity. GP residues that are required for recognition and neutralization were found to be located both in the internal fusion loop and in the receptor-binding domain. Analysis of Ebola virus entry in the presence of #3327 allows us to hypothesize that this antibody binds to the virus particle before internalization and endosomal processing of GP and likely prevents the final viral fusion step. Importantly, #3327 is able to block entry of virions bearing GP that contain the Q508 escape mutation common to a number of virus-neutralizing antibodies, and therefore provides future perspectives for treatment strategies against Ebola virus infection.

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

    Institute of Scientific and Technical Information of China (English)

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

    2015-01-01

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

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

    Science.gov (United States)

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

    2015-12-01

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

  9. Immunization routes in cattle impact the levels and neutralizing capacity of antibodies induced against S. aureus immune evasion proteins.

    Science.gov (United States)

    Boerhout, Eveline; Vrieling, Manouk; Benedictus, Lindert; Daemen, Ineke; Ravesloot, Lars; Rutten, Victor; Nuijten, Piet; van Strijp, Jos; Koets, Ad; Eisenberg, Susanne

    2015-09-28

    Vaccines against S. aureus bovine mastitis are scarce and show limited protection only. All currently available vaccines are applied via the parenteral (usually intramuscular) route. It is unknown, however, whether this route is the most suitable to specifically increase intramammary immunity to combat S. aureus at the site of infection. Hence, in the present study, immunization via mucosal (intranasal; IN), intramuscular (triangle of the neck; IM), intramammary (IMM) and subcutaneous (suspensory ligament; SC) routes were analyzed for their effects on the quantity of the antibody responses in serum and milk as well as the neutralizing capacity of the antibodies within serum. The experimental vaccine comprised the recombinant S. aureus immune evasion proteins extracellular fibrinogen-binding protein (Efb) and the leukotoxin subunit LukM in an oil-in-water adjuvant combined with a hydrogel and alginate. The highest titer increases for both Efb and LukM specific IgG1 and IgG2 antibody levels in serum and milk were observed following SC/SC immunizations. Furthermore, the harmful effects of Efb and leukotoxin LukMF' on host-defense were neutralized by serum antibodies in a route-dependent manner. SC/SC immunization resulted in a significant increase in the neutralizing capacity of serum antibodies towards Efb and LukMF', shown by increased phagocytosis of S. aureus and increased viability of bovine leukocytes. Therefore, a SC immunization route should be considered when aiming to optimize humoral immunity against S. aureus mastitis in cattle.

  10. Appearance and disappearance of neutralizing antibodies during interferon-beta therapy

    DEFF Research Database (Denmark)

    Sorensen, P Soelberg; Koch-Henriksen, Nils; Ross, C;

    2005-01-01

    Neutralizing antibodies (NABs) occur frequently in patients receiving interferon (IFN)-beta for multiple sclerosis (MS), but it is unclear whether occurrence of NABs is predictive for the persistence of NABs during continued IFN-beta therapy.......Neutralizing antibodies (NABs) occur frequently in patients receiving interferon (IFN)-beta for multiple sclerosis (MS), but it is unclear whether occurrence of NABs is predictive for the persistence of NABs during continued IFN-beta therapy....

  11. Chemokine receptor specific antibodies in cancer immunotherapy: achievements and challenges

    Directory of Open Access Journals (Sweden)

    Maria eVela

    2015-01-01

    Full Text Available The 1990s brought a burst of information regarding the structure, expression pattern, and role in leukocyte migration and adhesion of chemokines and their receptors. At that time, the FDA approved the first therapeutic antibodies for cancer treatment. A few years later it was reported that the chemokine receptors CXCR4 and CCR7 were involved on directing metastases to liver, lung, bone marrow or lymph nodes, and the over-expression of CCR4, CCR6 and CCR9 by certain tumors. The possibility of inhibiting the interaction of chemokine receptors present on the surface of tumor cells with their ligands emerged as a new therapeutic approach. Therefore, many research groups and companies began to develop small-molecule antagonists and specific antibodies, aiming to neutralize signaling from these receptors. Despite great expectations, so far, only one anti-chemokine receptor antibody has been approved for its clinical use, mogamulizumab, an anti-CCR4 antibody, granted in Japan to treat refractory adult T cell leukemia and lymphoma. Here we review the main achievements obtained with anti-chemokine receptor antibodies for cancer immunotherapy, including discovery and clinical studies, proposed mechanisms of action and therapeutic applications.

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

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    Devin Sok

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

  13. Molecular aspects of antibody-antigen interactions : size reduction of a herpes simplex virus neutralizing antibody and its antigen

    NARCIS (Netherlands)

    Schellekens, Gerardus Antonius

    1996-01-01

    Antibody molecules, produced as a response against foreign substances, interact with their antigen in a very specific manner. Antibodies with a predetermined specificity (monoclonal antibodies) can be produced and are widely used in medicine and science as indicator molecules. Genetic engineering of

  14. Identification of a CD4-Binding-Site Antibody to HIV that Evolved Near-Pan Neutralization Breadth

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Jinghe; Kang, Byong H.; Ishida, Elise; Zhou, Tongqing; Griesman, Trevor; Sheng, Zizhang; Wu, Fan; Doria-Rose, Nicole A.; Zhang, Baoshan; McKee, Krisha; O’Dell, Sijy; Chuang, Gwo-Yu; Druz, Aliaksandr; Georgiev, Ivelin S.; Schramm, Chaim A.; Zheng, Anqi; Joyce, M.  Gordon; Asokan, Mangaiarkarasi; Ransier, Amy; Darko, Sam; Migueles, Stephen A.; Bailer, Robert T.; Louder, Mark K.; Alam, S.  Munir; Parks, Robert; Kelsoe, Garnett; Von Holle, Tarra; Haynes, Barton F.; Douek, Daniel C.; Hirsch, Vanessa; Seaman, Michael S.; Shapiro, Lawrence; Mascola, John R.; Kwong, Peter D.; Connors, Mark

    2016-11-01

    Detailed studies of the broadly neutralizing antibodies (bNAbs) that underlie the best available examples of the humoral immune response to HIV are providing important information for the development of therapies and prophylaxis for HIV-1 infection. Here, we report a CD4-binding site (CD4bs) antibody, named N6, that potently neutralized 98% of HIV-1 isolates, including 16 of 20 that were resistant to other members of its class. N6 evolved a mode of recognition such that its binding was not impacted by the loss of individual contacts across the immunoglobulin heavy chain. In addition, structural analysis revealed that the orientation of N6 permitted it to avoid steric clashes with glycans, which is a common mechanism of resistance. Thus, an HIV-1-specific bNAb can achieve potent, near-pan neutralization of HIV-1, making it an attractive candidate for use in therapy and prophylaxis.

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

    Science.gov (United States)

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

    2014-01-01

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

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Science.gov (United States)

    Ahlers, Jeffrey D

    2014-04-01

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

  18. A Therapeutic Antibody against West Nile Virus Neutralizes Infection by Blocking Fusion within Endosomes

    NARCIS (Netherlands)

    Thompson, Bruce S.; Moesker, Bastiaan; Smit, Jolanda M.; Wilschut, Jan; Diamond, Michael S.; Fremont, Daved H.

    2009-01-01

    Defining the precise cellular mechanisms of neutralization by potently inhibitory antibodies is important for understanding how the immune system successfully limits viral infections. We recently described a potently inhibitory monoclonal antibody (MAb E16) against the envelope (E) protein of West N

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

    Science.gov (United States)

    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

  20. A Therapeutic Antibody against West Nile Virus Neutralizes Infection by Blocking Fusion within Endosomes

    NARCIS (Netherlands)

    Thompson, Bruce S.; Moesker, Bastiaan; Smit, Jolanda M.; Wilschut, Jan; Diamond, Michael S.; Fremont, Daved H.

    Defining the precise cellular mechanisms of neutralization by potently inhibitory antibodies is important for understanding how the immune system successfully limits viral infections. We recently described a potently inhibitory monoclonal antibody (MAb E16) against the envelope (E) protein of West

  1. Protective Monotherapy Against Lethal Ebola Virus Infection by a Potently Neutralizing Antibody

    Science.gov (United States)

    2016-07-11

    These mAbs bind and neutralize recent and 53   previous outbreak strains of Ebola virus, and mediate antibody-dependent cell-54   mediated...dilution. 61   62   Antibody purification , labeling, genetic analysis, and reversion to germline. The 63   usage of VH and VL gene segments was

  2. A Rapid Zika Diagnostic Assay to Measure Neutralizing Antibodies in Patients

    Directory of Open Access Journals (Sweden)

    Chao Shan

    2017-03-01

    Full Text Available The potential association of microcephaly and other congenital abnormalities with Zika virus (ZIKV infection during pregnancy underlines the critical need for a rapid and accurate diagnosis. Due to the short duration of ZIKV viremia in infected patients, a serologic assay that detects antibody responses to viral infection plays an essential role in diagnosing patient specimens. The current serologic diagnosis of ZIKV infection relies heavily on the labor-intensive Plaque Reduction Neutralization Test (PRNT that requires more than one-week turnaround time and represents a major bottleneck for patient diagnosis. To overcome this limitation, we have developed a high-throughput assay for ZIKV and dengue virus (DENV diagnosis that can attain the “gold standard” of the current PRNT assay. The new assay is homogeneous and utilizes luciferase viruses to quantify the neutralizing antibody titers in a 96-well format. Using 91 human specimens, we showed that the reporter diagnostic assay has a higher dynamic range and maintains the relative specificity of the traditional PRNT assay. Besides the improvement of assay throughput, the reporter virus technology has also shortened the turnaround time to less than two days. Collectively, our results suggest that, along with the viral RT-PCR assay, the reporter virus-based serologic assay could be potentially used as the first-line test for clinical diagnosis of ZIKV infection as well as for vaccine clinical trials.

  3. Antibody to gp41 MPER alters functional properties of HIV-1 Env without complete neutralization.

    Directory of Open Access Journals (Sweden)

    Arthur S Kim

    2014-07-01

    Full Text Available 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.

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

    Directory of Open Access Journals (Sweden)

    Raiees Andrabi

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

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

    Science.gov (United States)

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

    2012-01-01

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

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

    OpenAIRE

    1993-01-01

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

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

    Science.gov (United States)

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

    2014-12-01

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

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

    Directory of Open Access Journals (Sweden)

    Laura E McCoy

    2014-12-01

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

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

    Science.gov (United States)

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

    2014-01-01

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

  10. Development and characterization of neutralizing monoclonal antibodies against canine distemper virus hemagglutinin protein.

    Science.gov (United States)

    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.

  11. Antibodies to the buried N terminus of rhinovirus VP4 exhibit cross-serotypic neutralization.

    Science.gov (United States)

    Katpally, Umesh; Fu, Tong-Ming; Freed, Daniel C; Casimiro, Danilo R; Smith, Thomas J

    2009-07-01

    Development of a vaccine for the common cold has been thwarted by the fact that there are more than 100 serotypes of human rhinovirus (HRV). We previously demonstrated that the HRV14 capsid is dynamic and transiently displays the buried N termini of viral protein 1 (VP1) and VP4. Here, further evidence for this "breathing" phenomenon is presented, using antibodies to several peptides representing the N terminus of VP4. The antibodies form stable complexes with intact HRV14 virions and neutralize infectivity. Since this region of VP4 is highly conserved among all of the rhinoviruses, antiviral activity by these anti-VP4 antibodies is cross-serotypic. The antibodies inhibit HRV16 infectivity in a temperature- and time-dependent manner consistent with the breathing behavior. Monoclonal and polyclonal antibodies raised against the 30-residue peptide do not react with peptides shorter than 24 residues, suggesting that these peptides are adopting three-dimensional conformations that are highly dependent upon the length of the peptide. Furthermore, there is evidence that the N termini of VP4 are interacting with each other upon extrusion from the capsid. A Ser5Cys mutation in VP4 yields an infectious virus that forms cysteine cross-links in VP4 when the virus is incubated at room temperature but not at 4 degrees C. The fact that all of the VP4s are involved in this cross-linking process strongly suggests that VP4 forms specific oligomers upon extrusion. Together these results suggest that it may be possible to develop a pan-serotypic peptide vaccine to HRV, but its design will likely require details about the oligomeric structure of the exposed termini.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2010-08-26

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

  13. trans-Sialidase neutralizing antibody detection in Trypanosoma cruzi-infected domestic reservoirs.

    Science.gov (United States)

    Sartor, Paula A; Cardinal, Martha V; Orozco, Marcela M; Gürtler, Ricardo E; Leguizamón, M Susana

    2011-06-01

    The detection of Trypanosoma cruzi infection in domestic dogs and cats is relevant to evaluating human transmission risks and the effectiveness of insecticide spraying campaigns. However, the serological assays routinely used are associated with cross-reactivity in sera from mammals infected with Leishmania spp. We used a trans-sialidase inhibition assay (TIA) for T. cruzi diagnosis in serum samples from 199 dogs and 57 cats from areas where these types of infections are endemic. TIA is based on the antibody neutralization of recombinant trans-sialidase, an enzyme that is not detected in the coendemic Leishmania species or Trypanosoma rangeli parasites. T. cruzi infection was also evaluated by conventional serology (CS) (indirect immunofluorescence, indirect hemagglutination, enzyme-linked immunosorbent assay, and immunochromatographic dipstick test) and xenodiagnosis. Sera from 30 dogs and 15 cats from areas where these organisms are not endemic and 5 dogs with visceral leishmaniasis were found to be nonreactive by TIA and CS. Samples from dogs and cats demonstrated 91 and 95% copositivities between TIA and CS, whereas the conegativities were 98 and 97%, respectively. Sera from xenodiagnosis-positive dogs and cats also reacted by TIA (copositivities of 97 and 83%, respectively). TIA was reactive in three CS-negative samples and was able to resolve results in two cat serum samples that were CS inconclusive. Our study is the first to describe the development of trans-sialidase neutralizing antibodies in naturally infected dogs and cats. High CS conegativity and the absence of trans-sialidase neutralization in dog sera from areas where leishmaniasis is not endemic and from dogs with visceral leishmaniasis support TIA specificity. The TIA may be a useful tool for T. cruzi detection in the main domestic reservoirs.

  14. Measurement of neutralizing serum antibodies of patients vaccinated with human papillomavirus L1 or L2-based immunogens using furin-cleaved HPV Pseudovirions.

    Directory of Open Access Journals (Sweden)

    Joshua W Wang

    Full Text Available Antibodies specific for neutralizing epitopes in either Human papillomavirus (HPV capsid protein L1 or L2 can mediate protection from viral challenge and thus their accurate and sensitive measurement at high throughput is likely informative for monitoring response to prophylactic vaccination. Here we compare measurement of L1 and L2-specific neutralizing antibodies in human sera using the standard Pseudovirion-Based Neutralization Assay (L1-PBNA with the newer Furin-Cleaved Pseudovirion-Based Neutralization Assay (FC-PBNA, a modification of the L1-PBNA intended to improve sensitivity towards L2-specific neutralizing antibodies without compromising assay of L1-specific responses. For detection of L1-specific neutralizing antibodies in human sera, the FC- PBNA and L1-PBNA assays showed similar sensitivity and a high level of correlation using WHO standard sera (n = 2, and sera from patients vaccinated with Gardasil (n = 30 or an experimental human papillomavirus type 16 (HPV16 L1 VLP vaccine (n = 70. The detection of L1-specific cross-neutralizing antibodies in these sera using pseudovirions of types phylogenetically-related to those targeted by the L1 virus-like particle (VLP vaccines was also consistent between the two assays. However, for sera from patients (n = 17 vaccinated with an L2-based immunogen (TA-CIN, the FC-PBNA was more sensitive than the L1-PBNA in detecting L2-specific neutralizing antibodies. Further, the neutralizing antibody titers measured with the FC-PBNA correlated with those determined with the L2-PBNA, another modification of the L1-PBNA that spacio-temporally separates primary and secondary receptor engagement, as well as the protective titers measured using passive transfer studies in the murine genital-challenge model. In sum, the FC-PBNA provided sensitive measurement for both L1 VLP and L2-specific neutralizing antibody in human sera. Vaccination with TA-CIN elicits weak cross-protective antibody in a

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

    Science.gov (United States)

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

    2016-02-01

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

  16. Induction and characterization of monoclonal anti-idiotypic antibodies reactive with idiotopes of canine parvovirus neutralizing monoclonal antibodies.

    NARCIS (Netherlands)

    G.F. Rimmelzwaan (Guus); J. van Es (Johan); G.A. Drost; F.G.C.M. Uytdehaag (Fons); A.D.M.E. Osterhaus (Albert)

    1991-01-01

    textabstractMonoclonal anti-idiotypic (anti-Id) antibodies (Ab2) were generated against idiotypes (Id) of canine parvovirus (CPV) specific monoclonal antibodies (MoAbs). The binding of most of these anti-Id antibodies to their corresponding Id could be inhibited by antigen, thus classifying these an

  17. Neutralization of Virus Infectivity by Antibodies: Old Problems in New Perspectives

    Directory of Open Access Journals (Sweden)

    P. J. Klasse

    2014-01-01

    Full Text Available Neutralizing antibodies (NAbs can be both sufficient and necessary for protection against viral infections, although they sometimes act in concert with cellular immunity. Successful vaccines against viruses induce NAbs but vaccine candidates against some major viral pathogens, including HIV-1, have failed to induce potent and effective such responses. Theories of how antibodies neutralize virus infectivity have been formulated and experimentally tested since the 1930s; and controversies about the mechanistic and quantitative bases for neutralization have continually arisen. Soluble versions of native oligomeric viral proteins that mimic the functional targets of neutralizing antibodies now allow the measurement of the relevant affinities of NAbs. Thereby the neutralizing occupancies on virions can be estimated and related to the potency of the NAbs. Furthermore, the kinetics and stoichiometry of NAb binding can be compared with neutralizing efficacy. Recently, the fundamental discovery that the intracellular factor TRIM21 determines the degree of neutralization of adenovirus has provided new mechanistic and quantitative insights. Since TRIM21 resides in the cytoplasm, it would not affect the neutralization of enveloped viruses, but its range of activity against naked viruses will be important to uncover. These developments bring together the old problems of virus neutralization—mechanism, stoichiometry, kinetics, and efficacy—from surprising new angles.

  18. Induction of multi-epitope specific antibodies against HIV-1 by multi-epitope vaccines

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Some neutralizing antibodies against HIV-1 envelope proteins were highly effective to inhibit the infection of different strains in vitro, and existed in the infected individuals with very low levels. We suggested multi-epitope-vaccine as a new strategy to increase levels of neutralizing antibodies and the abilities against HIV mutation in vivo. Two candidate multi-epitope-vaccines induced antibodies with predefined multi-epitope-specificity in rhesus macaque. These antibodies recognized corresponding neutralizing epitopes on epitope-peptides, gp41 peptides, V3 loop peptide, rsgp41 and rgp120. Besides, three candidate epitope-vaccines in combination (another kind of multi-epitopevaccines) showed similar potency to induce predefined multiple immune responses in rabbits. These results suggest that multi-epitope-vaccines may be a new strategy to induce multi-antiviral activities against HIV-1 infection and mutafions.

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

    Science.gov (United States)

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

    2016-05-20

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

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

    Science.gov (United States)

    Qi, Yifei; Jo, Sunhwan; Im, Wonpil

    2016-03-01

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

  1. Neutralizing antibodies in cats infected with feline immunodeficiency virus.

    NARCIS (Netherlands)

    F. Tozzini; D. Matteucci; P. Bandecchi; F. Baldinotti; C.H.J. Siebelink (Kees); A.D.M.E. Osterhaus (Albert); M. Bendinelli

    1993-01-01

    textabstractSera from cats experimentally infected with five isolates of feline immunodeficiency virus (FIV) from various geographical regions and from FIV enzyme-linked immunosorbent assay-seropositive field cats from four European countries neutralized the Petaluma strain of FIV (FIV-P), originall

  2. Validating Antibodies to the Cannabinoid CB2 Receptor: Antibody Sensitivity Is Not Evidence of Antibody Specificity.

    Science.gov (United States)

    Marchalant, Yannick; Brownjohn, Philip W; Bonnet, Amandine; Kleffmann, Torsten; Ashton, John C

    2014-06-01

    Antibody-based methods for the detection and quantification of membrane integral proteins, in particular, the G protein-coupled receptors (GPCRs), have been plagued with issues of primary antibody specificity. In this report, we investigate one of the most commonly utilized commercial antibodies for the cannabinoid CB2 receptor, a GPCR, using immunoblotting in combination with mass spectrometry. In this way, we were able to develop powerful negative and novel positive controls. By doing this, we are able to demonstrate that it is possible for an antibody to be sensitive for a protein of interest-in this case CB2-but still cross-react with other proteins and therefore lack specificity. Specifically, we were able to use western blotting combined with mass spectrometry to unequivocally identify CB2 protein in over-expressing cell lines. This shows that a common practice of validating antibodies with positive controls only is insufficient to ensure antibody reliability. In addition, our work is the first to develop a label-free method of protein detection using mass spectrometry that, with further refinement, could provide unequivocal identification of CB2 receptor protein in native tissues.

  3. Safe and Objective Assay of Enterovirus 71 Neutralizing Antibodies via Pseudovirus

    Institute of Scientific and Technical Information of China (English)

    JIN Jun; XU Lin; GUO Shi-jie; SUN Shi-yang; ZHANG Shu; ZHU Chang-lin; KONG Wei; JIANG Chun-lai

    2012-01-01

    Current serum neutralization assays based on the inhibition of the eytopathic effect(Nt-CPE) need to manipulate live viruses,which are time-consuming,labor-intensive,and have the potential exposure to infectious agents,so a safe and objective assay via pseudovirus for the fast and efficient detection of enterovirus 71(EV71 ) neutralizing antibodies was developed.First,we generated EV71 pseudovirus containing firefly luciferase gene in place of the capsid gene P1 in EV71 genome.Vero cells infected with 200 CCID50(50% cell culture infective dose) of EV71 pseudovirus for 24 h were found to have the best performance.Seval sera were measured by EV71 pseudoparticle neutralization assay(Nt-PPN) and the conventional serological method Nt-CPE.Neutralizing antibody titers measured by Nt-PPN and those obtained by Nt-CPE demonstrate a high correlation between the two methods.Overall,the PPN assay represents a valid alternative to conventional serological methods for the evaluation of EV71 neutralizing antibodies.This method can be used for detecting neutralizing antibodies of other picornaviruses,such as hepatitis A virus(HAV) and coxsackievirus 16(CVA16),and make it possible to determine whether there is cross-reactivity between EV71 and CVA16.

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

    Science.gov (United States)

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

    1998-01-01

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

  5. Antigenic characterization of Brazilian bovine viral diarrhea virus isolates by monoclonal antibodies and cross-neutralization

    Directory of Open Access Journals (Sweden)

    Botton S.A.

    1998-01-01

    Full Text Available Nineteen Brazilian isolates of bovine viral diarrhea virus (BVDV were characterized antigenically with a panel of 19 monoclonal antibodies (mAbs (Corapi WV, Donis RO and Dubovi EJ (1990 American Journal of Veterinary Research, 55: 1388-1394. Eight isolates were further characterized by cross-neutralization using sheep monospecific antisera. Analysis of mAb binding to viral antigens by indirect immunofluorescence revealed distinct patterns of reactivity among the native viruses. Local isolates differed from the prototype Singer strain in recognition by up to 14 mAbs. Only two mAbs - one to the non-structural protein NS23/p125 and another to the envelope glycoprotein E0/gp48 - recognized 100% of the isolates. No isolate was recognized by more than 14 mAbs and twelve viruses reacted with 10 or less mAbs. mAbs to the major envelope glycoprotein E2/gp53 revealed a particularly high degree of antigenic variability in this glycoprotein. Nine isolates (47.3% reacted with three or less of 10 E2/gp53 mAbs, and one isolate was not recognized by any of these mAbs. Virus-specific antisera to eight isolates plus three standard BVDV strains raised in lambs had virus-neutralizing titers ranging from 400 to 3200 against the homologous virus. Nonetheless, many antisera showed significantly reduced neutralizing activity when tested against heterologous viruses. Up to 128-fold differences in cross-neutralization titers were observed for some pairs of viruses. When the coefficient of antigenic similarity (R was calculated, 49 of 66 comparisons (74.24% between viruses resulted in R values that antigenically distinguish strains. Moreover, one isolate had R values suggesting that it belongs to a distinct serologic group. The marked antigenic diversity observed among Brazilian BVDV isolates should be considered when planning diagnostic and immunization strategies.

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

    Science.gov (United States)

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

    2015-01-01

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

  7. Limited impact of passive non-neutralizing antibody immunization in acute SIV infection on viremia control in rhesus macaques.

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    Taku Nakane

    Full Text Available BACKGROUND: Antiviral antibodies, especially those with neutralizing activity against the incoming strain, are potentially important immunological effectors to control human immunodeficiency virus (HIV infection. While neutralizing activity appears to be central in sterile protection against HIV infection, the entity of inhibitory mechanisms via HIV and simian immunodeficiency virus (SIV-specific antibodies remains elusive. The recent HIV vaccine trial RV144 and studies in nonhuman primate models have indicated controversial protective efficacy of HIV/SIV-specific non-neutralizing binding antibodies (non-NAbs. While reports on HIV-specific non-NAbs have demonstrated virus inhibitory activity in vitro, whether non-NAbs could also alter the pathogenic course of established SIV replication in vivo, likewise via neutralizing antibody (NAb administration, has been unclear. Here, we performed post-infection passive immunization of SIV-infected rhesus macaques with polyclonal SIV-specific, antibody-dependent cell-mediated viral inhibition (ADCVI-competent non-NAbs. METHODS AND FINDINGS: Ten lots of polyclonal immunoglobulin G (IgG were prepared from plasma of ten chronically SIVmac239-infected, NAb-negative rhesus macaques, respectively. Their binding capacity to whole SIVmac239 virions showed a propensity similar to ADCVI activity. A cocktail of three non-NAb lots showing high virion-binding capacity and ADCVI activity was administered to rhesus macaques at day 7 post-SIVmac239 challenge. This resulted in an infection course comparable with control animals, with no significant difference in set point plasma viral loads or immune parameters. CONCLUSIONS: Despite virus-specific suppressive activity of the non-NAbs having been observed in vitro, their passive immunization post-infection did not result in SIV control in vivo. Virion binding and ADCVI activity with lack of virus neutralizing activity were indicated to be insufficient for antibody

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

    Science.gov (United States)

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

    2012-08-15

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

  9. Structural basis for immunization with postfusion respiratory syncytial virus fusion F glycoprotein (RSV F) to elicit high neutralizing antibody titers

    Energy Technology Data Exchange (ETDEWEB)

    Swanson, Kurt A.; Settembre, Ethan C.; Shaw, Christine A.; Dey, Antu K.; Rappuoli, Rino; Mandl, Christian W.; Dormitzer, Philip R.; Carfi, Andrea (Novartis)

    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.

  10. Generation and characterization of neutralizing monoclonal antibodies against baculo-expressed HPV 16 VLPs.

    Science.gov (United States)

    Vidyasagar, P; Sridevi, V N; Rajan, S; Praveen, A; Srikanth, A; Abhinay, G; Siva Kumar, V; Verma, R R; Rajendra, L

    2014-03-01

    Human papillomavirus (HPV) is the well-known second most cause of cervical cancer in women worldwide. According to the WHO survey, 70% of the total cervical cancers are associated with types HPV 16 and 18. Presently used prophylactic vaccine for HPV contains mainly capsid protein of L1 virus like particles (VLPs). Correct folding of VLPs and display of neutralizing epitopes are the major constraint for VLP-based vaccines. Further, monoclonal antibodies (mAbs) play a vital role in developing therapeutics and diagnostics. mAbs are also useful for the demonstration of VLP conformation, virus typing and product process assessment as well. In the present study, we have explored the usefulness of mAbs generated against sf-9 expressed HPV 16 VLPs demonstrated as type-specific and conformational dependent against HPV 16 VLPs by ELISA. High affinity and high pseudovirion neutralization titer of mAbs indicated their potential for the development of prophylactic vaccines for HPV. Also, the type-specific and conformational reactivity of the mAbs to HPV 16 VLPs in sf-9 cells by immunofluorescence assay proved their diagnostic potential.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2015-10-15

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

  12. Tetravalent dengue DIIIC protein together with alum and ODN elicits a Th1 response and neutralizing antibodies in mice.

    Science.gov (United States)

    Zuest, Roland; Valdes, Iris; Skibinski, David; Lin, Yufang; Toh, Ying Xiu; Chan, Katherine; Hermida, Lisset; Connolly, John; Guillen, Gerardo; Fink, Katja

    2015-03-17

    Dengue disease is a global challenge for healthcare systems particularly during outbreaks, and millions of dollars are spent every year for vector control. An efficient and safe vaccine that is cost-effective could resolve the burden that dengue virus imposes on affected countries. We describe here the immunogenicity of a tetravalent formulation of a recombinant fusion protein consisting of E domain III and the capsid protein of dengue serotypes 1-4 (Tetra DIIIC). E domain III is an epitope for efficient neutralizing antibodies while the capsid protein contains T cell epitopes. Besides combining B and T cell epitopes, Tetra DIIIC is highly immunogenic due to its aggregate form and a two-component adjuvant. Following previous studies assessing the monovalent DIIIC formulations, we addressed here the quality and breadth of the T cell- and antibody response of Tetra DIIIC in mice. Tetra DIIIC induced a Th1-type response against all four DENV serotypes and dengue-specific antibodies were predominantly IgG1 and IgG2a and neutralizing, while the induction of neutralizing antibodies was dependent on IFN signaling. Importantly, the Th1 and IgG1/IgG2a profile of the DIIIC vaccine approach is similar to an efficient natural anti-dengue response.

  13. Identification of the two rotavirus genes determining neutralization specificities

    Energy Technology Data Exchange (ETDEWEB)

    Offit, P.A.; Blavat, G.

    1986-01-01

    Bovine rotavirus NCDV and simian rotavirus SA-11 represent two distinct rotavirus serotypes. A genetic approach was used to determine which viral gene segments segregated with serotype-specific viral neutralization. There were 16 reassortant rotarviruses derived by coinfection of MA-104 cells in vitro with the SA-11 and NCDV strains. The parental origin of reassortant rotavirus double-stranded RNA segments was determined by gene segment mobility in polyacrylamide gels and by hybridization with radioactively labeled parental viral transcripts. The authors found that two rotavirus gene segments found previously to code for outer capsid proteins vp3 and vp7 cosegreated with virus neutralization specificities.

  14. Recombinant Sheep Pox Virus Proteins Elicit Neutralizing Antibodies

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    Olga V. Chervyakova

    2016-06-01

    Full Text Available 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.

  15. Harnessing the protective potential of HIV-1 neutralizing antibodies [version 1; referees: 2 approved

    Directory of Open Access Journals (Sweden)

    S Abigail Smith

    2016-01-01

    Full Text Available 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.

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

    Science.gov (United States)

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

    2016-07-01

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

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

    Directory of Open Access Journals (Sweden)

    Armita Nourmohammad

    2016-07-01

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

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

    Science.gov (United States)

    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

  19. Human Papillomavirus neutralizing and cross-reactive antibodies induced in HIV-positive subjects after vaccination with quadrivalent and bivalent HPV vaccines

    DEFF Research Database (Denmark)

    Faust, Helena; Nielsen, Lars Toft; Sehr, Peter;

    2016-01-01

    Ninety-one HIV-infected individuals (61 men and 30 women) were randomized to vaccination either with quadrivalent (Gardasil™) or bivalent (Cervarix™) HPV vaccine. Neutralizing and specific HPV-binding serum antibodies were measured at baseline and 12 months after the first vaccine dose. Presence...... of neutralizing and binding antibodies had good agreement (average Kappa for HPV types 6, 11, 16, 18, 31, 33 and 45 was 0.65). At baseline, 88% of subjects had antibodies against at least one genital HPV. Following vaccination with Cervarix™, all subjects became seropositive for HPV16 and 18. After Gardasil......™ vaccination, 96% of subjects seroconverted for HPV16 and 73% for HPV18. Levels of HPV16-specific antibodies were 10IU in 85% of study subjects after vaccination. Antibodies against non-vaccine HPV types appeared after Gardasil...

  20. Neutralization and Binding Profile of Monoclonal Antibodies Generated Against Influenza A H1N1 Viruses.

    Science.gov (United States)

    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.

  1. Epitope specificity plays a critical role in regulating antibody-dependent cell-mediated cytotoxicity against influenza A virus.

    Science.gov (United States)

    He, Wenqian; Tan, Gene S; Mullarkey, Caitlin E; Lee, Amanda J; Lam, Mannie Man Wai; Krammer, Florian; Henry, Carole; Wilson, Patrick C; Ashkar, Ali A; Palese, Peter; Miller, Matthew S

    2016-10-18

    The generation of strain-specific neutralizing antibodies against influenza A virus is known to confer potent protection against homologous infections. The majority of these antibodies bind to the hemagglutinin (HA) head domain and function by blocking the receptor binding site, preventing infection of host cells. Recently, elicitation of broadly neutralizing antibodies which target the conserved HA stalk domain has become a promising "universal" influenza virus vaccine strategy. The ability of these antibodies to elicit Fc-dependent effector functions has emerged as an important mechanism through which protection is achieved in vivo. However, the way in which Fc-dependent effector functions are regulated by polyclonal influenza virus-binding antibody mixtures in vivo has never been defined. Here, we demonstrate that interactions among viral glycoprotein-binding antibodies of varying specificities regulate the magnitude of antibody-dependent cell-mediated cytotoxicity induction. We show that the mechanism responsible for this phenotype relies upon competition for binding to HA on the surface of infected cells and virus particles. Nonneutralizing antibodies were poor inducers and did not inhibit antibody-dependent cell-mediated cytotoxicity. Interestingly, anti-neuraminidase antibodies weakly induced antibody-dependent cell-mediated cytotoxicity and enhanced induction in the presence of HA stalk-binding antibodies in an additive manner. Our data demonstrate that antibody specificity plays an important role in the regulation of ADCC, and that cross-talk among antibodies of varying specificities determines the magnitude of Fc receptor-mediated effector functions.

  2. Mechanistic insights into the neutralization of cytotoxic abrin by the monoclonal antibody D6F10.

    Directory of Open Access Journals (Sweden)

    Shradha Bagaria

    Full Text Available Abrin, an A/B toxin obtained from the Abrus precatorius plant is extremely toxic and a potential bio-warfare agent. Till date there is no antidote or vaccine available against this toxin. The only known neutralizing monoclonal antibody against abrin, namely D6F10, has been shown to rescue the toxicity of abrin in cells as well as in mice. The present study focuses on mapping the epitopic region to understand the mechanism of neutralization of abrin by the antibody D6F10. Truncation and mutational analysis of abrin A chain revealed that the amino acids 74-123 of abrin A chain contain the core epitope and the residues Thr112, Gly114 and Arg118 are crucial for binding of the antibody. In silico analysis of the position of the mapped epitope indicated that it is present close to the active site cleft of abrin A chain. Thus, binding of the antibody near the active site blocks the enzymatic activity of abrin A chain, thereby rescuing inhibition of protein synthesis by the toxin in vitro. At 1∶10 molar concentration of abrin:antibody, the antibody D6F10 rescued cells from abrin-mediated inhibition of protein synthesis but did not prevent cell attachment of abrin. Further, internalization of the antibody bound to abrin was observed in cells by confocal microscopy. This is a novel finding which suggests that the antibody might function intracellularly and possibly explains the rescue of abrin's toxicity by the antibody in whole cells and animals. To our knowledge, this study is the first report on a neutralizing epitope for abrin and provides mechanistic insights into the poorly understood mode of action of anti-A chain antibodies against several toxins including ricin.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2016-11-02

    ABSTRACT

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

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

  4. Challenges to the development of vaccines to hepatitis C virus that elicit neutralizing antibodies.

    Directory of Open Access Journals (Sweden)

    Heidi Edelgard Drummer

    2014-07-01

    Full Text Available Despite 20 years of research, a vaccine to prevent hepatitis C virus (HCV infection has not been developed. A vaccine to prevent HCV will need to induce broadly reactive immunity able to prevent infection by the 7 genetically and antigenically distinct genotypes circulating world-wide. Hepatitis C virus encodes two surface exposed glycoproteins, E1 and E2 that function as a heterodimer to mediate viral entry. Neutralizing antibodies (NAbs to both E1 and E2 have been described with the major NAb target being E2. The function of E2 is to attach virions to host cells via cell surface receptors that include, but is not limited to, the tetraspanin CD81 and scavenger receptor B class I. However, E2 has developed a number of immune evasion strategies to limit the effectiveness of the NAb response and possibly limit the ability of the immune system to generate potent NAbs in natural infection. Hypervariable regions that shield the underlying core domain, subdominant neutralization epitopes and glycan shielding combine to make E2 a difficult target for the immune system. This review summarizes recent information on the role of neutralizing antibodies to prevent HCV infection, the targets of the neutralizing antibody response and structural information on glycoprotein E2 in complex with neutralizing antibodies. This new information should provide a framework for the rational design of new vaccine candidates that elicit highly potent broadly reactive NAbs to prevent HCV infection.

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

    Science.gov (United States)

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

    2016-01-19

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

  6. Automated pipeline for rapid production and screening of HIV-specific monoclonal antibodies using pichia pastoris.

    Science.gov (United States)

    Shah, Kartik A; Clark, John J; Goods, Brittany A; Politano, Timothy J; Mozdzierz, Nicholas J; Zimnisky, Ross M; Leeson, Rachel L; Love, J Christopher; Love, Kerry R

    2015-12-01

    Monoclonal antibodies (mAbs) that bind and neutralize human pathogens have great therapeutic potential. Advances in automated screening and liquid handling have resulted in the ability to discover antigen-specific antibodies either directly from human blood or from various combinatorial libraries (phage, bacteria, or yeast). There remain, however, bottlenecks in the cloning, expression and evaluation of such lead antibodies identified in primary screens that hinder high-throughput screening. As such, "hit-to-lead identification" remains both expensive and time-consuming. By combining the advantages of overlap extension PCR (OE-PCR) and a genetically stable yet easily manipulatable microbial expression host Pichia pastoris, we have developed an automated pipeline for the rapid production and screening of full-length antigen-specific mAbs. Here, we demonstrate the speed, feasibility and cost-effectiveness of our approach by generating several broadly neutralizing antibodies against human immunodeficiency virus (HIV).

  7. Interactions between HIV-1 Neutralizing Antibodies and Model Lipid Membranes imaged with AFM

    Science.gov (United States)

    Zauscher, Stefan; Hardy, Gregory; Alam, Munir; Shapter, Joseph

    2012-02-01

    Lipid membrane interactions with rare, broadly neutralizing antibodies (NAbs), 2F5 and 4E10, play a critical role in HIV-1 neutralization. Our research is motivated by recent immunization studies that have shown that induction of antibodies that avidly bind the gp41-MPER antigen is not sufficient for neutralization. Rather, it is required that antigen designs induce polyreactive antibodies that recognize MPER antigens as well as the viral lipid membrane. However, the mechanistic details of how membrane properties influence NAb-lipid and NAb-antigen interactions remain unknown. Furthermore, it is well established that the native viral membrane is heterogeneous, representing a mosaic of lipid rafts and protein clustering. However, the size, physical properties, and dynamics of these regions are poorly characterized and their potential roles in HIV-1 neutralization are also unknown. To understand how membrane properties contribute to 2F5/4E10 membrane interactions, we have engineered biomimetic supported lipid bilayers (SLBs) and use atomic force microscopy to visualize membrane domains, antigen clustering, and antibody-membrane interactions at sub-nanometer z-resolution. Our results show that localized binding of HIV-1 antigens and NAbs occur preferentially with the most fluid membrane domain. This supports the theory that NAbs may interact with regions of low lateral lipid forces that allow antibody insertion into the bilayer.

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

    Science.gov (United States)

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

    2015-01-01

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

  9. Correlation between anti-interferon-β binding and neutralizing antibodies in interferon-β-treated multiple sclerosis patients

    DEFF Research Database (Denmark)

    Jensen, P E H; Sellebjerg, F; Søndergaard, H B;

    2012-01-01

    Measurements of binding antibodies (BAbs), neutralizing antibodies (NAbs) and MX1 mRNA expression are used to analyse the immunological reactions in patients with MS treated with IFN-β. The correlations between these are yet not fully understood.......Measurements of binding antibodies (BAbs), neutralizing antibodies (NAbs) and MX1 mRNA expression are used to analyse the immunological reactions in patients with MS treated with IFN-β. The correlations between these are yet not fully understood....

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

    Science.gov (United States)

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

    2013-01-01

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

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

    Science.gov (United States)

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

    1996-01-01

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

  12. Sensitivity of HIV-1 to neutralization by antibodies against O-linked carbohydrate epitopes despite deletion of O-glycosylation signals in the V3 loop

    DEFF Research Database (Denmark)

    Hansen, J E; Jansson, B; Gram, G J

    1996-01-01

    It has been suggested that threonine or serine residues in the V3 loop of HIV-1 gp120 are glycosylated with the short-chain O-linked oligosaccharides Tn or sialosyl-Tn that function as epitopes for broadly neutralizing carbohydrate specific antibodies. In this study we examined whether mutation...... with deletions of O-glycosylation signals in the V3 loop displayed any decrease in sensitivity to anti-Tn or anti-sialosyl-Tn antibody. This indicates that these broadly specific neutralization epitopes are located outside the V3 loop of gp 120........ Additionally, one of these T-A mutants (T308A) also abrogated the signal for N-glycosylation at N306 inside the V3-loop. The mutant clones were compared with the wild type virus as to sensitivity to neutralization with monoclonal and polyclonal antibodies specific for the tip of the V3 loop of BRU or for the O...

  13. Structural and Functional Characterization of Anti-A33 Antibodies Reveal a Potent Cross-Species Orthopoxviruses Neutralizer.

    Directory of Open Access Journals (Sweden)

    Michael H Matho

    2015-09-01

    Full Text Available Vaccinia virus A33 is an extracellular enveloped virus (EEV-specific type II membrane glycoprotein that is essential for efficient EEV formation and long-range viral spread within the host. A33 is a target for neutralizing antibody responses against EEV. In this study, we produced seven murine anti-A33 monoclonal antibodies (MAbs by immunizing mice with live VACV, followed by boosting with the soluble A33 homodimeric ectodomain. Five A33 specific MAbs were capable of neutralizing EEV in the presence of complement. All MAbs bind to conformational epitopes on A33 but not to linear peptides. To identify the epitopes, we have adetermined the crystal structures of three representative neutralizing MAbs in complex with A33. We have further determined the binding kinetics for each of the three antibodies to wild-type A33, as well as to engineered A33 that contained single alanine substitutions within the epitopes of the three crystallized antibodies. While the Fab of both MAbs A2C7 and A20G2 binds to a single A33 subunit, the Fab from MAb A27D7 binds to both A33 subunits simultaneously. A27D7 binding is resistant to single alanine substitutions within the A33 epitope. A27D7 also demonstrated high-affinity binding with recombinant A33 protein that mimics other orthopoxvirus strains in the A27D7 epitope, such as ectromelia, monkeypox, and cowpox virus, suggesting that A27D7 is a potent cross-neutralizer. Finally, we confirmed that A27D7 protects mice against a lethal challenge with ectromelia virus.

  14. Neutralization Interfering Antibodies: A “Novel” Example of Humoral Immune Dysfunction Facilitating Viral Escape?

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    Roberto Burioni

    2012-09-01

    Full Text Available The immune response against some viral pathogens, in particular those causing chronic infections, is often ineffective notwithstanding a robust humoral neutralizing response. Several evasion mechanisms capable of subverting the activity of neutralizing antibodies (nAbs have been described. Among them, the elicitation of non-neutralizing and interfering Abs has been hypothesized. Recently, this evasion mechanism has acquired an increasing interest given its possible impact on novel nAb-based antiviral therapeutic and prophylactic approaches. In this review, we illustrate the mechanisms of Ab-mediated interference and the viral pathogens described in literature as able to adopt this “novel” evasion strategy.

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

    Directory of Open Access Journals (Sweden)

    Liao Pi-Hung

    2012-06-01

    Full Text Available Abstract 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

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

    Directory of Open Access Journals (Sweden)

    Scott A McKinley

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

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

    Science.gov (United States)

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

    2016-03-01

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

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

    Directory of Open Access Journals (Sweden)

    Xiaohua Ye

    2016-03-01

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

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

    Science.gov (United States)

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

    2015-12-01

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

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Science.gov (United States)

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

    2016-04-01

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

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

    Directory of Open Access Journals (Sweden)

    Gene S Tan

    2016-04-01

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

  3. MERS Coronavirus Neutralizing Antibodies in Camels, Eastern Africa, 1983-1997

    NARCIS (Netherlands)

    Müller, Marcel A; Corman, Victor Max; Jores, Joerg; Meyer, Benjamin; Younan, Mario; Liljander, Anne; Bosch, Berend-Jan; Lattwein, Erik; Hilali, Mosaad; Musa, Bakri E; Bornstein, Set; Drosten, Christian

    2014-01-01

    To analyze the distribution of Middle East respiratory syndrome coronavirus (MERS-CoV)-seropositive dromedary camels in eastern Africa, we tested 189 archived serum samples accumulated during the past 30 years. We identified MERS-CoV neutralizing antibodies in 81.0% of samples from the main camel-ex

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

    NARCIS (Netherlands)

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

    2015-01-01

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

  5. Stabilization of HIV-1 envelope glycoprotein trimers to induce neutralizing antibodies

    NARCIS (Netherlands)

    de Taeye, S.W.

    2017-01-01

    HIV-1 has evolved various tricks to prevent the development of a potent humoral immune response. The only target for neutralizing antibodies (NAbs) is the HIV-1 envelope glycoprotein (Env), which is the sole viral protein embedded in the viral membrane. It consists of three gp41 subunits and three g

  6. High Rates of Neutralizing Antibodies to Toscana and Sandfly Fever Sicilian Viruses in Livestock, Kosovo.

    Science.gov (United States)

    Ayhan, Nazli; Sherifi, Kurtesh; Taraku, Arber; Bërxholi, Kristaq; Charrel, Rémi N

    2017-06-01

    Toscana and sandfly fever Sicilian viruses (TOSV and SFSV, respectively), both transmitted by sand flies, are prominent human pathogens in the Old World. Of 1,086 serum samples collected from cattle and sheep during 2013 in various regions of Kosovo (Balkan Peninsula), 4.7% and 53.4% had neutralizing antibodies against TOSV and SFSV, respectively.

  7. Tetravalent neutralizing antibody response against four dengue serotypes by a single chimeric dengue envelope antigen.

    Science.gov (United States)

    Apt, Doris; Raviprakash, Kanakatte; Brinkman, Alice; Semyonov, Andrey; Yang, Shumin; Skinner, Craig; Diehl, Lori; Lyons, Richard; Porter, Kevin; Punnonen, Juha

    2006-01-16

    We employed DNA shuffling and screening technologies to develop a single recombinant dengue envelope (E) antigen capable of inducing neutralizing antibodies against all four antigenically distinct dengue serotypes. By DNA shuffling of codon-optimized dengue 1-4 E genes, we created a panel of novel chimeric clones expressing C-terminal truncated E antigens that combined epitopes from all four dengue serotypes. DNA vaccines encoding these novel chimeras induced multivalent T cell and neutralizing antibody responses against all four dengue serotypes in mice. By contrast, a mixture of four unshuffled, parental DNA vaccines failed to produce tetravalent neutralizing antibodies in mice. The neutralizing antibody titers for some of these antigens could be further improved by extending the sequences to express full-length pre-membrane and envelope proteins. The chimeric antigens also protected mice against a lethal dengue-2 virus challenge. These data demonstrate that DNA shuffling and associated screening can lead to the selection of multi-epitope antigens against closely related dengue virus serotypes and suggest a broad utility for these technologies in optimizing vaccine antigens.

  8. Neutralizing antibodies to hepatitis C virus in perinatally infected children followed up prospectively

    DEFF Research Database (Denmark)

    Meunier, Jean-Christophe; Bukh, Jens; Diaz, Giacomo

    2011-01-01

    Little is known about the presence and role of neutralizing antibodies (NtAbs) in perinatal hepatitis C virus (HCV) infection. Using HCV pseudoparticles, NtAbs were studied longitudinally in 12 HCV-infected children with or without evidence of acute hepatitis during the first year of life. Broadly...

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

    NARCIS (Netherlands)

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

    2015-01-01

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

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

    Science.gov (United States)

    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.

  11. Cross-reactive neutralizing antibody responses to enterovirus 71 infections in young children: implications for vaccine development.

    Directory of Open Access Journals (Sweden)

    Mei-Liang Huang

    Full Text Available BACKGROUND: Recently, enterovirus 71 (EV71 has caused life-threatening outbreaks involving neurological and cardiopulmonary complications in Asian children with unknown mechanism. EV71 has one single serotype but can be phylogenetically classified into 3 main genogroups (A, B and C and 11 genotypes (A, B1∼B5 and C1∼C5. In Taiwan, nationwide EV71 epidemics with different predominant genotypes occurred in 1998 (C2, 2000-2001 (B4, 2004-2005 (C4, and 2008 (B5. In this study, sera were collected to measure cross-reactive neutralizing antibody titers against different genotypes. METHODS: We collected historical sera from children who developed an EV71 infection in 1998, 2000, 2005, 2008, or 2010 and measured cross-reactive neutralizing antibody titers against all 11 EV71 genotypes. In addition, we aligned and compared the amino acid sequences of P1 proteins of the tested viruses. RESULTS: Serology data showed that children infected with genogroups B and C consistently have lower neutralizing antibody titers against genogroup A (>4-fold difference. The sequence comparisons revealed that five amino acid signatures (N143D in VP2; K18R, H116Y, D167E, and S275A in VP1 are specific for genogroup A and may be related to the observed antigenic variations. CONCLUSIONS: This study documented antigenic variations among different EV71 genogroups and identified potential immunodominant amino acid positions. Enterovirus surveillance and vaccine development should monitor these positions.

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

    Science.gov (United States)

    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. Antibodies against recombinant catalytic domain of lethal toxin of Clostridium sordellii neutralize lethal toxin toxicity in HeLa cells.

    Science.gov (United States)

    Arya, Preetika; Ponmariappan, S; Singh, Lokendra; Prasad, G B K S

    2013-02-01

    Lethal toxin of Clostridium sordellii (MLD 150 ng/kg) is one of the most potent Clostridial toxins and is responsible for most of the diseases including sudden death syndrome in cattle, sheep and toxic shock syndrome, necrotizing faciitis, neonatal omphalitis and gangrene in humans. Lethal toxin (TcsL) is a single chain protein of about 270 kDa. In the present study, 1.6 kb DNA fragment encoding for the catalytic domain of TcsL was PCR amplified, cloned in pQE30 UA vector and expressed in E. coli SG 13009. The expression of recombinant lethal toxin protein (rTcsL) was optimized and it was purified under native conditions using a single step Ni-NTA affinity chromatography. The purified recombinant protein was used for the production of polyclonal antibodies in mice and rabbit. The raised antibodies reacted specifically with the purified rTcsL and intact native lethal toxin on Western blot. The biological activity of the recombinant protein was tested in HeLa cells where it showed the cytotoxicity. Further, the polyclonal antibodies were used for in-vitro neutralization of purified rTcsL, acid precipitated C. sordellii and C. difficile native toxins in HeLa cells. Mice and rabbit anti-rTcsL sera effectively neutralized the cytotoxicity of rTcsL and C. sordellii native toxin but it did not neutralize the cytotoxicity of C. difficile toxin in HeLa cells.

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

    Directory of Open Access Journals (Sweden)

    Elodie Beaumont

    Full Text Available 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.

  15. Mathematical and experimental analyses of antibody transport in hollow-fiber-based specific antibody filters.

    Science.gov (United States)

    Hout, Mariah S; Federspiel, William J

    2003-01-01

    We are developing hollow fiber-based specific antibody filters (SAFs) that selectively remove antibodies of a given specificity directly from whole blood, without separation of the plasma and cellular blood components and with minimal removal of plasma proteins other than the targeted pathogenic antibodies. A principal goal of our research is to identify the primary mechanisms that control antibody transport within the SAF and to use this information to guide the choice of design and operational parameters that maximize the SAF-based antibody removal rate. In this study, we formulated a simple mathematical model of SAF-based antibody removal and performed in vitro antibody removal experiments to test key predictions of the model. Our model revealed three antibody transport regimes, defined by the magnitude of the Damköhler number Da (characteristic antibody-binding rate/characteristic antibody diffusion rate): reaction-limited (Da /= 10). For a given SAF geometry, blood flow rate, and antibody diffusivity, the highest antibody removal rate was predicted for diffusion-limited antibody transport. Additionally, for diffusion-limited antibody transport the predicted antibody removal rate was independent of the antibody-binding rate and hence was the same for any antibody-antigen system and for any patient within one antibody-antigen system. Using SAF prototypes containing immobilized bovine serum albumin (BSA), we measured anti-BSA removal rates consistent with transport in the intermediate regime (Da approximately 3). We concluded that initial SAF development work should focus on achieving diffusion-limited antibody transport by maximizing the SAF antibody-binding capacity (hence maximizing the characteristic antibody-binding rate). If diffusion-limited antibody transport is achieved, the antibody removal rate may be raised further by increasing the number and length of the SAF fibers and by increasing the blood flow rate through the SAF.

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

    Science.gov (United States)

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

    2014-07-31

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

  17. Potent dengue virus neutralization by a therapeutic antibody with low monovalent affinity requires bivalent engagement.

    Directory of Open Access Journals (Sweden)

    Melissa A Edeling

    2014-04-01

    Full Text Available We recently described our most potently neutralizing monoclonal antibody, E106, which protected against lethal Dengue virus type 1 (DENV-1 infection in mice. To further understand its functional properties, we determined the crystal structure of E106 Fab in complex with domain III (DIII of DENV-1 envelope (E protein to 2.45 Å resolution. Analysis of the complex revealed a small antibody-antigen interface with the epitope on DIII composed of nine residues along the lateral ridge and A-strand regions. Despite strong virus neutralizing activity of E106 IgG at picomolar concentrations, E106 Fab exhibited a ∼20,000-fold decrease in virus neutralization and bound isolated DIII, E, or viral particles with only a micromolar monovalent affinity. In comparison, E106 IgG bound DENV-1 virions with nanomolar avidity. The E106 epitope appears readily accessible on virions, as neutralization was largely temperature-independent. Collectively, our data suggest that E106 neutralizes DENV-1 infection through bivalent engagement of adjacent DIII subunits on a single virion. The isolation of anti-flavivirus antibodies that require bivalent binding to inhibit infection efficiently may be a rare event due to the unique icosahedral arrangement of envelope proteins on the virion surface.

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

    Directory of Open Access Journals (Sweden)

    Chia Yin Lee

    2011-12-01

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

  19. The change of the scFv into the Fab format improves the stability and in vivo toxin neutralization capacity of recombinant antibodies.

    Science.gov (United States)

    Quintero-Hernández, Veronica; Juárez-González, Victor R; Ortíz-León, Mauricio; Sánchez, Rosalba; Possani, Lourival D; Becerril, Baltazar

    2007-02-01

    The antigen-binding fragment (Fab) has been considered a more functionally stable version of recombinant antibodies than single chain antibody fragments (scFvs), however this intuitive consideration has not been sufficiently proven in vivo. This communication shows that three out of four specific scFvs against a scorpion toxin, with different affinities and stabilities, become neutralizing in vivo when expressed as Fabs, despite the fact that they are not neutralizing in the scFv format. A scFv fragment previously obtained from a neutralizing mouse antibody (BCF2) was used to produce three derived scFvs by directed evolution. Only one of them was neutralizing, however when expressed as Fab, all of them became neutralizing fragments in vivo. The initial scFvBCF2 (earlier used for directed evolution) was not neutralizing in the scFv format. After expressing it as Fab did not become a neutralizing fragment, but did reduce the intoxication symptoms of experimental mice. The stability of the four Fabs derived from their respective scFvs was improved when tested in the presence of guanidinium chloride. The in vitro stability of the Fab format has been shown earlier, but the physiological consequences of this stability are shown in this communication. The present results indicate that improved functional stability conferred by the Fab format can replace additional maturation steps, when the affinity and stability are close to the minimum necessary to be neutralizing.

  20. Characterization and neutralization of Nemopilema nomurai (Scyphozoa: Rhizostomeae) jellyfish venom using polyclonal antibody.

    Science.gov (United States)

    Kang, Changkeun; Han, Dae-Yong; Park, Kwang-Il; Pyo, Min-Jung; Heo, Yunwi; Lee, Hyunkyoung; Kim, Gon Sup; Kim, Euikyung

    2014-08-01

    Jellyfish stings have often caused serious health concerns for sea bathers especially in tropical waters. In the coastal areas of Korea, China and Japan, the blooming and stinging accidents of poisonous jellyfish species have recently increased, including Nemopilema nomurai. We have generated a polyclonal antibody against N. nomurai jellyfish venom (NnV) by the immunization of white rabbits with NnV antigen. In the present study, the antibody has been characterized for its neutralizing effect against NnV. At first, the presence of NnV polyclonal antibody has been confirmed from the immunized rabbit serum by Enzyme linked immunosorbent assay (ELISA). Then, the neutralizing activities of the polyclonal antibody have been investigated using cell-based toxicity test, hemolysis assay, and mice lethality test. When the polyclonal antibody was preincubated with NnV, it shows a high effectiveness in neutralizing the NnV toxicities in a concentration-dependent manner. Moreover, we explored proteomic analyses using 2-D SDS-PAGE and MALDI-TOF mass spectrometry to illustrate the molecular identities of the jellyfish venom. From this, 18 different protein families have been identified as jellyfish venom-derived proteins; the main findings of which are matrix metalloproteinase-14, astacin-like metalloprotease toxin 3 precursor. It is expected that the present results would have contributed to our understandings of the envenomation by N. nomurai, their treatment and some valuable knowledge on the pathological processes of the jellyfish stinging.

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Science.gov (United States)

    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.

  3. Repeated epitope in the recombinant epitope-peptide could enhance ELDKWA-epitope-specific antibody response

    Institute of Scientific and Technical Information of China (English)

    LIU Zuqiang; WANG Zuguang; CHEN Yinghua

    2005-01-01

    Based on the hypothesis suggested by us that epitope-vaccine may be a new strategy against HIV mutation, we have studied several neutralizing epitopes on HIV envelope proteins. However we do not know whether a repeated epitope in a recombinant epitope-peptide can enhance epitope-specific antibody response or not. ELDKWA-epitope (aa669-674) on the C-domain of HIV-1 gp41 is a neutralizing epitope defined by the monoclonal antibody (mAb) 2F5 with broad neutralizing activity. In this study, we designed and prepared a series of the recombinant epitope-peptides bearing 1, 4 and 8 copies of ELDKWA-epitope respectively. In the comparison of the antisera induced by the three recombinant antigens, an obviously increased titre of ELDKWA-epitope-specific antibody was observed in the case of four and eight repeated epitopes. In flow cytometry analysis, the epitope-specific antibodies in both antisera showed stronger activity to bind the transfected CHO-WT cells that stably express HIV-1 envelope glycoprotein on the cell surfaces. These experimental results indicated that repeated epitope in the recombinant epitope-peptide could enhance ELDKWA-epitope-specific antibody response, which could contribute to designing an effective recombinant epitope-vaccine.

  4. LabKey Server NAb: A tool for analyzing, visualizing and sharing results from neutralizing antibody assays

    Directory of Open Access Journals (Sweden)

    Gao Hongmei

    2011-05-01

    Full Text Available Abstract Background Multiple types of assays allow sensitive detection of virus-specific neutralizing antibodies. For example, the extent of antibody neutralization of HIV-1, SIV and SHIV can be measured in the TZM-bl cell line through the degree of luciferase reporter gene expression after infection. In the past, neutralization curves and titers for this standard assay have been calculated using an Excel macro. Updating all instances of such a macro with new techniques can be unwieldy and introduce non-uniformity across multi-lab teams. Using Excel also poses challenges in centrally storing, sharing and associating raw data files and results. Results We present LabKey Server's NAb tool for organizing, analyzing and securely sharing data, files and results for neutralizing antibody (NAb assays, including the luciferase-based TZM-bl NAb assay. The customizable tool supports high-throughput experiments and includes a graphical plate template designer, allowing researchers to quickly adapt calculations to new plate layouts. The tool calculates the percent neutralization for each serum dilution based on luminescence measurements, fits a range of neutralization curves to titration results and uses these curves to estimate the neutralizing antibody titers for benchmark dilutions. Results, curve visualizations and raw data files are stored in a database and shared through a secure, web-based interface. NAb results can be integrated with other data sources based on sample identifiers. It is simple to make results public after publication by updating folder security settings. Conclusions Standardized tools for analyzing, archiving and sharing assay results can improve the reproducibility, comparability and reliability of results obtained across many labs. LabKey Server and its NAb tool are freely available as open source software at http://www.labkey.com under the Apache 2.0 license. Many members of the HIV research community can also access the Lab

  5. LabKey Server NAb: A tool for analyzing, visualizing and sharing results from neutralizing antibody assays

    Science.gov (United States)

    2011-01-01

    Background Multiple types of assays allow sensitive detection of virus-specific neutralizing antibodies. For example, the extent of antibody neutralization of HIV-1, SIV and SHIV can be measured in the TZM-bl cell line through the degree of luciferase reporter gene expression after infection. In the past, neutralization curves and titers for this standard assay have been calculated using an Excel macro. Updating all instances of such a macro with new techniques can be unwieldy and introduce non-uniformity across multi-lab teams. Using Excel also poses challenges in centrally storing, sharing and associating raw data files and results. Results We present LabKey Server's NAb tool for organizing, analyzing and securely sharing data, files and results for neutralizing antibody (NAb) assays, including the luciferase-based TZM-bl NAb assay. The customizable tool supports high-throughput experiments and includes a graphical plate template designer, allowing researchers to quickly adapt calculations to new plate layouts. The tool calculates the percent neutralization for each serum dilution based on luminescence measurements, fits a range of neutralization curves to titration results and uses these curves to estimate the neutralizing antibody titers for benchmark dilutions. Results, curve visualizations and raw data files are stored in a database and shared through a secure, web-based interface. NAb results can be integrated with other data sources based on sample identifiers. It is simple to make results public after publication by updating folder security settings. Conclusions Standardized tools for analyzing, archiving and sharing assay results can improve the reproducibility, comparability and reliability of results obtained across many labs. LabKey Server and its NAb tool are freely available as open source software at http://www.labkey.com under the Apache 2.0 license. Many members of the HIV research community can also access the LabKey Server NAb tool without

  6. Persistence of neutralizing antibodies after discontinuation of IFNbeta therapy in patients with relapsing-remitting multiple sclerosis

    DEFF Research Database (Denmark)

    Petersen, Bodil; Bendtzen, Klaus; Koch-Henriksen, Nils;

    2006-01-01

    The main objective was to follow serum levels of neutralizing antibodies (NABs) against interferon-beta (IFNbeta) after discontinuation of IFNbeta therapy.......The main objective was to follow serum levels of neutralizing antibodies (NABs) against interferon-beta (IFNbeta) after discontinuation of IFNbeta therapy....

  7. Interaction between Shiga Toxin and Monoclonal Antibodies: Binding Characteristics and in Vitro Neutralizing Abilities

    Directory of Open Access Journals (Sweden)

    Letícia B. Rocha

    2012-09-01

    Full Text Available Monoclonal antibodies (MAbs have been employed either for diagnosis or treatment of infections caused by different pathogens. Specifically for Shiga toxin-producing Escherichia coli (STEC, numerous immunoassays have been developed for STEC diagnosis, showing variability in sensitivity and specificity when evaluated by reference laboratories, and no therapy or vaccines are currently approved. Thus, the aim of this work was the characterization of the interaction between MAbs against Stx1 and Stx2 toxins and their neutralizing abilities to enable their use as tools for diagnosis and therapy. The selected clones designated 3E2 (anti-Stx1 and 2E11 (anti-Stx2 were classified as IgG1. 3E2 recognized the B subunit of Stx1 with an affinity constant of 2.5 × 10−10 M, detected as little as 6.2 ng of Stx1 and was stable up to 50 ºC. In contrast, 2E11 recognized the A subunit of Stx2, was stable up to 70 ºC, had a high dissociation constant of  6.1 × 10−10 M, and detected as little as 12.5 ng of Stx2. Neutralization tests showed that 160 ng of 3E2 MAb inhibited 80% of Stx1 activity and 500 µg 2E11 MAb were required for 60% inhibition of Stx2 activity. These MAb amounts reversed 25 to 80% of the cytotoxicity triggered by different STEC isolates. In conclusion, these MAbs show suitable characteristics for their use in STEC diagnosis and encourage future studies to investigate their protective efficacy.

  8. Docking of B-cell epitope antigen to specific hepatitis B antibody

    Indian Academy of Sciences (India)

    R Rajkannan; E J Padma Malar

    2007-09-01

    The interaction of pres1 region of hepatitis B virus B-cell epitope antigen with specific hepatitis B neutralizing monoclonal antibody was examined by docking study. We modelled the 3D complex structure of B-cell epitope antigen residues CTTPAQGNSMFPSCCCTKPTDGNCY by homology modelling and docked it with the crystal structure of monoclonal antibody specific for the pres1 region of the hepatitis B virus. At the optimized docked conformation, the interactions between the amino acids of antigen and antibody were examined. It is found that the docked complex is stabilized by 59.3 kcal/mol. The stability of the docked antigen-antibody complex is due to hydrogen bonding and van der Waals interactions. The amino acids of the antigen and antibody responsible for the interaction were identified.

  9. Ability of vaccine strain induced antibodies to neutralize field isolates of caliciviruses from Swedish cats.

    Science.gov (United States)

    Wensman, Jonas Johansson; Samman, Ayman; Lindhe, Anna; Thibault, Jean-Christophe; Berndtsson, Louise Treiberg; Hosie, Margaret J

    2015-12-12

    Feline calicivirus (FCV) is a common cause of upper respiratory tract disease in cats worldwide. Its characteristically high mutation rate leads to escape from the humoral immune response induced by natural infection and/or vaccination and consequently vaccines are not always effective against field isolates. Thus, there is a need to continuously investigate the ability of FCV vaccine strain-induced antibodies to neutralize field isolates. Seventy-eight field isolates of FCV isolated during the years 2008-2012 from Swedish cats displaying clinical signs of upper respiratory tract disease were examined in this study. The field isolates were tested for cross-neutralization using a panel of eight anti-sera raised in four pairs of cats following infection with four vaccine strains (F9, 255, G1 and 431). The anti-sera raised against F9 and 255 neutralised 20.5 and 11.5 %, and 47.4 and 64.1 % of field isolates tested, respectively. The anti-sera against the more recently introduced vaccine strains G1 and 431 neutralized 33.3 and 55.1 % (strain G1) or 69.2 and 89.7 % (strain 431) of the field isolates with titres ≥5. [corrected]. Dual vaccine strains displayed a higher cross-neutralization. This study confirms previous observations that more recently introduced vaccine strains induce antibodies with a higher neutralizing capacity compared to vaccine strains that have been used extensively over a long period of time. This study also suggests that dual FCV vaccine strains might neutralize more field isolates compared to single vaccine strains. Vaccine strains should ideally be selected based on updated knowledge on the antigenic properties of field isolates in the local setting, and there is thus a need for continuously studying the evolution of FCV together with the neutralizing capacity of vaccine strain induced antibodies against field isolates at a national and/or regional level.

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

    Science.gov (United States)

    Smith, S Abigail; Derdeyn, Cynthia A

    2017-03-01

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

  11. Lipophilicity is a key factor to increase the antiviral activity of HIV neutralizing antibodies.

    Science.gov (United States)

    Augusto, Marcelo T; Hollmann, Axel; Troise, Fulvia; Veiga, Ana S; Pessi, Antonello; Santos, Nuno C

    2017-04-01

    The HIV broadly neutralizing antibody 2F5 targets the transiently exposed epitope in the membrane proximal external region (MPER) of HIV-1 gp41, by a two-step mechanism involving the viral membrane and this viral glycoprotein. It was recently shown that 2F5 conjugation with a cholesterol moiety outside of the antibody paratope substantially increases its antiviral activity. Additionally, the antiviral activity of D5, a human antibody that binds to the N-terminal heptad repeat (NHR) of gp41 and lacks membrane binding, was boosted by the same cholesterol conjugation. In this work, we evaluated the membrane affinity of both antibodies towards membranes of different compositions, using surface plasmon resonance. A correlation was found between membrane affinity and antiviral activity against HIV-1. We propose that the conjugation of cholesterol to 2F5 or D5 allows a higher degree of antibody pre-concentration at the viral membrane. This way, the antibodies become more available to bind efficiently to the gp41 epitope, blocking viral fusion faster than the unconjugated antibody. These results set up a relevant strategy to improve the rational design of therapeutic antibodies against HIV.

  12. Polyfunctional HIV-Specific Antibody Responses Are Associated with Spontaneous HIV Control.

    Directory of Open Access Journals (Sweden)

    Margaret E Ackerman

    2016-01-01

    Full Text Available Elite controllers (ECs represent a unique model of a functional cure for HIV-1 infection as these individuals develop HIV-specific immunity able to persistently suppress viremia. Because accumulating evidence suggests that HIV controllers generate antibodies with enhanced capacity to drive antibody-dependent cellular cytotoxicity (ADCC that may contribute to viral containment, we profiled an array of extra-neutralizing antibody effector functions across HIV-infected populations with varying degrees of viral control to define the characteristics of antibodies associated with spontaneous control. While neither the overall magnitude of antibody titer nor individual effector functions were increased in ECs, a more functionally coordinated innate immune-recruiting response was observed. Specifically, ECs demonstrated polyfunctional humoral immune responses able to coordinately recruit ADCC, other NK functions, monocyte and neutrophil phagocytosis, and complement. This functionally coordinated response was associated with qualitatively superior IgG3/IgG1 responses, whereas HIV-specific IgG2/IgG4 responses, prevalent among viremic subjects, were associated with poorer overall antibody activity. Rather than linking viral control to any single activity, this study highlights the critical nature of functionally coordinated antibodies in HIV control and associates this polyfunctionality with preferential induction of potent antibody subclasses, supporting coordinated antibody activity as a goal in strategies directed at an HIV-1 functional cure.

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

    Science.gov (United States)

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

    2015-07-01

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

  14. Preexisting human antibodies neutralize recently emerged H7N9 influenza strains

    Science.gov (United States)

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

    2015-01-01

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

  15. Structure of an N276-Dependent HIV-1 Neutralizing Antibody Targeting a Rare V5 Glycan Hole Adjacent to the CD4 Binding Site

    Energy Technology Data Exchange (ETDEWEB)

    Wibmer, Constantinos Kurt; Gorman, Jason; Anthony, Colin S.; Mkhize, Nonhlanhla N.; Druz, Aliaksandr; York, Talita; Schmidt, Stephen D.; Labuschagne, Phillip; Louder, Mark K.; Bailer, Robert T.; Karim, Salim S. Abdool; Mascola, John R.; Williamson, Carolyn; Moore, Penny L.; Kwong, Peter D.; Morris, Lynn (NHLS-South Africa); (NIH); (Witwatersrand); (KwaZulu-Natal)

    2016-08-31

    ABSTRACT

    All HIV-1-infected individuals develop strain-specific neutralizing antibodies to their infecting virus, which in some cases mature into broadly neutralizing antibodies. Defining the epitopes of strain-specific antibodies that overlap conserved sites of vulnerability might provide mechanistic insights into how broadly neutralizing antibodies arise. We previously described an HIV-1 clade C-infected donor, CAP257, who developed broadly neutralizing plasma antibodies targeting an N276 glycan-dependent epitope in the CD4 binding site. The initial CD4 binding site response potently neutralized the heterologous tier 2 clade B viral strain RHPA, which was used to design resurfaced gp120 antigens for single-B-cell sorting. Here we report the isolation and structural characterization of CAP257-RH1, an N276 glycan-dependent CD4 binding site antibody representative of the early CD4 binding site plasma response in donor CAP257. The cocrystal structure of CAP257-RH1 bound to RHPA gp120 revealed critical interactions with the N276 glycan, loop D, and V5, but not with aspartic acid 368, similarly to HJ16 and 179NC75. The CAP257-RH1 monoclonal antibody was derived from the immunoglobulin-variable IGHV3-33 and IGLV3-10 genes and neutralized RHPA but not the transmitted/founder virus from donor CAP257. Its narrow neutralization breadth was attributed to a binding angle that was incompatible with glycosylated V5 loops present in almost all HIV-1 strains, including the CAP257 transmitted/founder virus. Deep sequencing of autologous CAP257 viruses, however, revealed minority variants early in infection that lacked V5 glycans. These glycan-free V5 loops are unusual holes in the glycan shield that may have been necessary for initiating this N276 glycan-dependent CD4 binding site B-cell lineage.

    IMPORTANCEThe conserved CD4 binding site on gp120 is a major target for HIV-1 vaccine design, but key events in the elicitation and maturation of

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

    Science.gov (United States)

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

    2017-01-01

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

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

    Science.gov (United States)

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

    2015-04-01

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

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

    DEFF Research Database (Denmark)

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

    1991-01-01

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

  19. Assay for the specificity of monoclonal antibodies in crossed immunoelectrophoresis

    DEFF Research Database (Denmark)

    Skjødt, K; Schou, C; Koch, C

    1984-01-01

    A method is described based on crossed immunoelectrophoresis of a complex antigen mixture in agarose gel followed by incubation of the gel with the monoclonal antibody. The bound monoclonal antibody is detected by the use of a secondary enzyme-labelled antibody. Using this technique we have been...... I molecules. In other experiments using the same technique we demonstrated the reaction of a monoclonal antibody specific for chicken Ig light chains. Udgivelsesdato: 1984-Aug-3...

  20. Neutralizing antibody titers against dengue virus correlate with protection from symptomatic infection in a longitudinal cohort.

    Science.gov (United States)

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    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.

  2. How HIV-1 entry mechanism and broadly neutralizing antibodies guide structure-based vaccine design.

    Science.gov (United States)

    Pancera, Marie; Changela, Anita; Kwong, Peter D

    2017-05-01

    An HIV-1 vaccine that elicits broadly neutralizing antibodies (bNAbs) remains to be developed. Here, we review how knowledge of bNAbs and HIV-1 entry mechanism is guiding the structure-based design of vaccine immunogens and immunization regimens. Isolation of bNAbs from HIV-1-infected donors has led to an unprecedented understanding of the sites of vulnerability that these antibodies target on the HIV-1 envelope (Env) as well as of the immunological pathways that these antibody lineages follow to develop broad and potent neutralization. Sites of vulnerability, however, reside in the context of diverse Env conformations required for HIV-1 entry, including a prefusion-closed state, a single-CD4-bound intermediate, a three-CD4-bound intermediate, a prehairpin intermediate and postfusion states, and it is not always clear which structural state optimally presents a particular site of vulnerability in the vaccine context. Furthermore, detailed knowledge of immunological pathways has led to debate among vaccine developers as to how much of the natural antibody-developmental pathway immunogens should mimic, ranging from only the recognized epitope to multiple antigens from the antibody-virus coevolution process. A plethora of information on bNAbs is guiding HIV-1-vaccine development. We highlight consideration of the appropriate structural context from the HIV-1-entry mechanism and extraordinary progress with replicating template B-cell ontogenies.

  3. Mimotopes selected with a neutralizing antibody against urease B from Helicobacter pylori induce enzyme inhibitory antibodies in mice upon vaccination

    Directory of Open Access Journals (Sweden)

    Long Min

    2010-11-01

    Full Text Available Abstract Background Urease B is an important virulence factor that is required for Helicobacter pylori to colonise the gastric mucosa. Mouse monoclonal antibodies (mAbs that inhibit urease B enzymatic activity will be useful as vaccines for the prevention and treatment of H. pylori infection. Here, we produced murine mAbs against urease B that neutralize the enzyme's activity. We mapped their epitopes by phage display libraries and investigated the immunogenicity of the selected mimotopes in vivo. Results The urease B gene was obtained (GenBank accession No. DQ141576 and the recombinant pGEX-4T-1/UreaseB protein was expressed in Escherichia coli as a 92-kDa recombinant fusion protein with glutathione-S-transferase (GST. Five mAbs U001-U005 were produced by a hybridoma-based technique with urease B-GST as an immunogen. Only U001 could inhibit urease B enzymatic activity. Immunoscreening via phage display libraries revealed two different mimotopes of urease B protein; EXXXHDM from ph.D.12-library and EXXXHSM from ph.D.C7C that matched the urease B proteins at 347-353 aa. The antiserum induced by selected phage clones clearly recognised the urease B protein and inhibited its enzymatic activity, which indicated that the phagotope-induced immune responses were antigen specific. Conclusions The present work demonstrated that phage-displayed mimotopes were accessible to the mouse immune system and triggered a humoral response. The urease B mimotope could provide a novel and promising approach for the development of a vaccine for the diagnosis and treatment of H. pylori infection.

  4. Detoxified Haemophilus ducreyi cytolethal distending toxin and induction of toxin specific antibodies in the genital tract.

    Science.gov (United States)

    Lundqvist, Annika; Fernandez-Rodrigues, Julia; Ahlman, Karin; Lagergård, Teresa

    2010-08-16

    Haemophilus ducreyi causes genital ulceration (chancroid), a sexually transmitted infection and still an important factor which contributes to the spread of HIV in developing countries. The bacterium produces a cytolethal distending toxin (HdCDT) causing cell cycle arrest and apoptosis/necrosis of human cells and contributes to the aggravation of ulcers. The aim of the study was to induce toxin-neutralizing antibodies in the genital tract of mice. Repeated subcutaneous (sc) immunisations with 5-10microg active HdCDT induced low levels of serum anti-HdCDT IgG without neutralizing capacity. High levels of specific IgG1 antibodies in serum and genital tract were generated after sc immunisations with 10microg formaldehyde detoxified HdCDT toxoid alone and the addition of aluminium salts or RIBI (based on the lipid A moiety) as adjuvant further increased the level of serum antibodies. A high correlation was found between elevated levels of anti-HdCDT IgG in sera, the level of neutralizing activity and the antibody level in genital tract (r=0.8). Thus, induction of high antibody levels specific to HdCDT in the genital tissue can be achieved by parenteral immunisation with the toxoid. The HdCDT toxoid can be considered as a candidate component in vaccine against chancroid.

  5. Hyperimmune antisera against synthetic peptides representing the glycoprotein of human immunodeficiency virus type 2 can mediate neutralization and antibody-dependent cytotoxic activity.

    Science.gov (United States)

    Björling, E; Broliden, K; Bernardi, D; Utter, G; Thorstensson, R; Chiodi, F; Norrby, E

    1991-01-01

    Twenty-five 13- to 35-amino-acid-long peptides representing regions of human immunodeficiency virus type 2 (HIV-2), strain SBL6669, envelope proteins were evaluated for their immunogenic activity in guinea pigs. The peptides were selected to provide homologous representation of sites in the HIV-1 envelope proteins that were previously documented to have a particular immunogenic importance. A number of the HIV-2 peptides were found to be capable of inducing strain SBL6669 neutralizing and antibody-dependent cellular cytotoxicity (ADCC) antibodies. Two overlapping peptides covering amino acids 311-337 representing the central and C-terminal part of the variable third (V3) region, terminology according to Modrow et al. [Modrow, S., Hahn, B., Shaw, G. M., Gallo, R. C., Wong-Staal, F. & Wolf, H. (1987) J. Virol. 61, 570-578], showed the most pronounced capacity to induce neutralizing antibodies. One of the peptides (amino acids 318-337) also induced antibodies mediating ADCC. Two additional regions in the large glycoprotein, gp125, containing linear sites reacting with neutralizing antibodies were identified (amino acids, 119-137 and 472-509). The transmembrane protein, gp36, of HIV-2 harbored two regions of importance for induction of neutralizing antibodies (amino acids 595-614 and 714-729). ADCC activity was induced by two additional gp125-specific peptides (amino acids 291-311 and 446-461). Thus, except for the single V3-specific site there was no correlation between linear immunogenic sites stimulating neutralizing antibody and ADCC activity. These findings pave the way for development of synthetic vaccines against HIV-2 and possibly also simian immunodeficiency virus infections. The capacity of such a product to induce protective immunity can be evaluated in macaque monkeys. Images PMID:2068087

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

    Science.gov (United States)

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

    2015-09-29

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

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

    Science.gov (United States)

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

    2017-01-26

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

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

    Science.gov (United States)

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

    2014-12-01

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

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

    Directory of Open Access Journals (Sweden)

    Daning Wang

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

  10. Human Papillomavirus neutralizing and cross-reactive antibodies induced in HIV-positive subjects after vaccination with quadrivalent and bivalent HPV vaccines.

    Science.gov (United States)

    Faust, Helena; Toft, Lars; Sehr, Peter; Müller, Martin; Bonde, Jesper; Forslund, Ola; Østergaard, Lars; Tolstrup, Martin; Dillner, Joakim

    2016-03-18

    Ninety-one HIV-infected individuals (61 men and 30 women) were randomized to vaccination either with quadrivalent (Gardasil™) or bivalent (Cervarix™) HPV vaccine. Neutralizing and specific HPV-binding serum antibodies were measured at baseline and 12 months after the first vaccine dose. Presence of neutralizing and binding antibodies had good agreement (average Kappa for HPV types 6, 11, 16, 18, 31, 33 and 45 was 0.65). At baseline, 88% of subjects had antibodies against at least one genital HPV. Following vaccination with Cervarix™, all subjects became seropositive for HPV16 and 18. After Gardasil™ vaccination, 96% of subjects seroconverted for HPV16 and 73% for HPV18. Levels of HPV16-specific antibodies were vaccination but >10IU in 85% of study subjects after vaccination. Antibodies against non-vaccine HPV types appeared after Gardasil™ vaccination for >50% of vaccinated females for HPV 31, 35 and 73 and for >50% of Cervarix™-vaccinated females for HPV 31, 33, 35, 45, 56 and 58. Cross-reactivity with non-genital HPV types was also detected. In conclusion, HIV-infected subjects responded to HPV vaccination with induction of neutralizing antibodies against both vaccine and non-vaccine types.

  11. Characterization of methylsulfinylalkyl glucosinolate specific polyclonal antibodies

    DEFF Research Database (Denmark)

    Mirza, Nadia Muhammad Akram; Schulz, Alexander; Halkier, Barbara Ann

    2016-01-01

    Antibodies towards small molecules, like plant specialized metabolites, are valuable tools for developing quantitative and qualitative analytical techniques. Glucosinolates are the specialized metabolites characteristic of the Brassicales order. Here we describe the characterization of polyclonal...... rabbit antibodies raised against the 4-methylsulfinylbutyl glucosinolate, glucoraphanin that is one of the major glucosinolates in the model plant Arabidopsis thaliana (hereafter Arabidopsis). Analysis of the cross-reactivity of the antibodies against a number of glucosinolates demonstrated...... that it was highly selective for methionine-derived aliphatic glucosinolates with a methyl-sulfinyl group in the side chain. Use of crude plant extracts from Arabidopsis mutants with different glucosinolate profiles showed that the antibodies recognized aliphatic glucosinolates in a plant extract and did not cross...

  12. Benefits of fresh-frozen plasma as a replacement fluid to neutralize ABO antibodies.

    Science.gov (United States)

    Won, Dong Il; Ham, Ji Yeon; Kim, Chan Duck; Suh, Jang Soo; Kim, Byung Chang

    2015-10-01

    ABO-incompatible organ transplantation requires pre-transplant conditioning to reduce ABO antibody levels in the recipients. With respect to replacement fluids used in plasma exchange, we intended to verify whether fresh-frozen plasma (FFP) containing soluble ABO substance (SAS) is more effective than albumin solution in reducing ABO IgG antibody levels. Apheresis data were retrospectively studied for in vivo effects, and in vitro plasma mixing studies were prospectively performed. The amount of ABO IgG antibodies bound to red cells was measured as the mean fluorescence intensity (MFI) using flow cytometry. Neutralization of ABO antibodies in the recipientst plasma by an ABO-incompatible donornc plasma was measured using the inhibition assay principle. The MFI value of the unneutralized control tube was divided by that of the neutralized test tube (neutralizing-capacity index, NCI). The plasma exchange procedures replaced with group AB FFP showed a significantly greater decreased titer than those replaced with albumin (P = 0.010). The in vitro plasma mixing study simulating plasma exchange also produced consistent results. When the pooled group O plasma was neutralized for anti-A by individual group AB plasmas (AB-to-O, N = 30), the NCI was 12.8 ± 5.4 (6.5-29.5). When this group O plasma was neutralized by pooled group AB or A plasma, the repeatedly measured NCI (N = 5) of A-to-O (11.4 ± 1.4) was not significantly different from that of AB-to-O (9.7 ± 1.3, P = 0.074). ABO antibody levels are reduced more effectively by group AB FFP than by albumin. Either group AB or donor type (group A or B) FFP can be infused to group O recipients. FFP units with higher SAS levels can be selected from multiple available candidate units using our protocol for measuring the neutralizing-capacity. © 2014 Wiley Periodicals, Inc.

  13. Protection of adenovirus from neutralizing antibody by cationic PEG derivative ionically linked to adenovirus

    OpenAIRE

    Sun X; Zhang Z; Gong T; Zhao D.; Han J; Zeng Q

    2012-01-01

    Qin Zeng, Jianfeng Han, Dong Zhao, Tao Gong, Zhirong Zhang, Xun SunKey Laboratory of Drug Targeting and Drug Delivery Systems, Ministry of Education, West China School of Pharmacy, Sichuan University, Chengdu, People's Republic of ChinaBackground: The generation of anti-adenovirus neutralizing antibody (NAb) in humans severely restricts the utilization of recombinant adenovirus serotype 5 (Ad5) vectors in gene therapy for a wide range of clinical trials. To overcome this limitation, w...

  14. Vaccine-induced cross-genotype reactive neutralizing antibodies against hepatitis C virus

    DEFF Research Database (Denmark)

    Meunier, Jean-Christophe; Gottwein, Judith M; Houghton, Michael

    2011-01-01

    We detected cross-reactive neutralizing antibodies (NtAb) against hepatitis C virus (HCV) in chimpanzees vaccinated with HCV-1 (genotype 1a) recombinant E1/E2 envelope glycoproteins. Five vaccinated chimpanzees, protected following HCV-1 challenge, were initially studied using the heterologous H77......a, with limited reactivity against 2a and 3a. Our study provides encouragement for the development of a recombinant envelope-based vaccine against hepatitis C....

  15. Characterization of Hemolysin of Moraxella bovis Using a Hemolysis-Neutralizing Monoclonal Antibody

    OpenAIRE

    2000-01-01

    A concentrated bacterial culture supernatant from the hemolytic Moraxella bovis strain UQV 148NF was used to immunize mice and generate monoclonal antibodies (MAbs). One, MAb G3/D7, neutralized the hemolytic activity of M. bovis and recognized a 94-kDa protein by Western blot analysis in hemolytic M. bovis strains representing each of the different fimbrial serogroups. Exposure of corneal epithelial cells to M. bovis concentrated culture supernatants demonstrated a role for an exotoxin in the...

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2009-11-23

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

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

    OpenAIRE

    2012-01-01

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

  18. Comparison of enzyme-linked immunosorbent assays and virus neutralization test for detection of antibodies to avian pneumovirus.

    Science.gov (United States)

    Alkahalaf, A N; Halvorson, D A; Saif, Y M

    2002-01-01

    Two different whole-virus enzyme-linked immunosorbent assays (ELISAs), developed in Ohio (OH) with APV/Minnesota/turkey/2a/97 and in Minnesota (MN) with APV/Colorado/turkey/97, and the virus neutralization (VN) test were used to test 270 turkey serum samples from 27 Minnesota turkey flocks for avian pneumovirus (APV) antibodies. In addition, 77 turkey serum samples and 128 ostrich serum samples from Ohio were tested. None of the turkey samples from Ohio had antibodies to APV by the VN test and OH ELISA. The ostrich samples were only tested with the VN test and were all negative for antibodies to APV. For the Minnesota serum samples, 107, 115, and 120 were positive by the VN test, the OH ELISA, and the MN ELISA, respectively. The Kappa values of 0.938 and 0.825 showed excellent agreement between the VN test and the OH ELISA and the MN ELISA, respectively, for detection of antibodies to the APV. The OH ELISA and MN ELISA had sensitivities of 1.0 and 0.953, specificities of 0.950 and 0.889, and accuracies of 0.970 and 0.914, respectively. Our results indicate that the 3 methods are sensitive and specific for diagnosis of the APV infection.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2011-09-20

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

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

    Directory of Open Access Journals (Sweden)

    Torres S

    2013-12-01

    Full Text Available Sebastian Torres,1 Mark Hamilton,2 Elena Sanches,4 Polina Starovatova,3 Elena Gubanova,3 Tatiana Reshetnikova51Di Stefano Velona Clinic, Catania, Italy; 2Hamilton Face Clinic, Dublin, Ireland; 3Preventive Medicine Clinic "Vallex M", Moscow, Russia; 4EKLAN Co Ltd Medical Center for Aesthetic Correction, Moscow, Russia; 5Department of Dermatovenereology and Cosmetology, State Medical University, Novosibirsk, RussiaAbstract: 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.Keywords: aesthetic medicine, botulinum neurotoxin type A, neutralizing antibody, secondary treatment failure

  1. Cross neutralizing antibodies in hamsters vaccinated with leptospiral bacterins produced with three serovars of serogroup Sejroe

    Directory of Open Access Journals (Sweden)

    Rosana Tabata

    2002-09-01

    Full Text Available Three leptospiral bacterins, produced with different serovars of Serogroup Sejroe, namely the hardjo (bacterin A, wolffi (bacterin B and guaricura (bacterin C, were evaluated in male hamsters (Mesocricetus auratus by comparing the agglutinating and neutralizing antibodies titers using microscopic agglutination (MAT and in vitro growth inhibition (GIT tests. The immunization schedule was based on two 1.0 mL doses of non-diluted formalininactivated whole culture bacterin given through subcutaneous route with 10-day interval. The challenge was performed ten days after the second vaccine dose, when the animals were inoculated with 0.2 mL of non-inactivated cultures of each serovar through intraperitoneal route. On the 21st post-challenge day (PCD, all animals were bled and their sera were joined in pools (n=8 and tested by MAT and GIT. All vaccinated and control animals presented no clinical signs of leptospirosis after the challenge, but the serovar guaricura was isolated from the kidneys of control animals on the 21st PCD. The MAT results showed cross agglutinins between serovars hardjo and wolffi, and between wolffi and guaricura. The GIT results revealed the presence of cross neutralizing antibodies between serovars wolffi or guaricura against hardjo, wolffi and guaricura. It was found that the tested strain of serovar hardjo did not produce detectable levels of neutralizing antibodies, indicating its poor immunogenicity.

  2. An efficiently cleaved HIV-1 clade C Env selectively binds to neutralizing antibodies.

    Directory of Open Access Journals (Sweden)

    Saikat Boliar

    Full Text Available An ideal HIV-1 Env immunogen is expected to mimic the native trimeric conformation for inducing broadly neutralizing antibody responses. The native conformation is dependent on efficient cleavage of HIV-1 Env. The clade B isolate, JRFL Env is efficiently cleaved when expressed on the cell surface. Here, for the first time, we report the identification of a native clade C Env, 4-2.J41 that is naturally and efficiently cleaved on the cell surface as confirmed by its biochemical and antigenic characteristics. In addition to binding to several conformation-dependent neutralizing antibodies, 4-2.J41 Env binds efficiently to the cleavage-dependent antibody PGT151; thus validating its native cleaved conformation. In contrast, 4-2.J41 Env occludes non-neutralizing epitopes. The cytoplasmic-tail of 4-2.J41 Env plays an important role in maintaining its conformation. Furthermore, codon optimization of 4-2.J41 Env sequence significantly increases its expression while retaining its native conformation. Since clade C of HIV-1 is the prevalent subtype, identification and characterization of this efficiently cleaved Env would provide a platform for rational immunogen design.

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

    Science.gov (United States)

    Ren, Jing; Ellis, John; Li, Jinyan

    2014-10-01

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

  4. Mutations That Alter Use of Hepatitis C Virus Cell Entry Factors Mediate Escape From Neutralizing Antibodies

    Science.gov (United States)

    FAUVELLE, CATHERINE; ZAHID, MUHAMMAD NAUMAN; TUREK, MARINE; HEYDMANN, LAURA; CURY, KARINE; HAYER, JULIETTE; COMBET, CHRISTOPHE; COSSET, FRANÇOIS–LOÏC; PIETSCHMANN, THOMAS; HIET, MARIE–SOPHIE; BARTENSCHLAGER, RALF; HABERSETZER, FRANÇOIS; DOFFOËL, MICHEL; KECK, ZHEN–YONG; FOUNG, STEVEN K. H.; ZEISEL, MIRJAM B.; STOLL–KELLER, FRANÇOISE; BAUMERT, THOMAS F.

    2017-01-01

    BACKGROUND & AIMS The development of vaccines and other strategies to prevent hepatitis C virus (HCV) infection is limited by rapid viral evasion. HCV entry is the first step of infection; this process involves several viral and host factors and is targeted by host-neutralizing responses. Although the roles of host factors in HCV entry have been well characterized, their involvement in evasion of immune responses is poorly understood. We used acute infection of liver graft as a model to investigate the molecular mechanisms of viral evasion. METHODS We studied factors that contribute to evasion of host immune responses using patient-derived antibodies, HCV pseudoparticles, and cell culture–derived HCV that express viral envelopes from patients who have undergone liver transplantation. These viruses were used to infect hepatoma cell lines that express different levels of HCV entry factors. RESULTS By using reverse genetic analyses, we identified altered use of host-cell entry factors as a mechanism by which HCV evades host immune responses. Mutations that alter use of the CD81 receptor also allowed the virus to escape neutralizing antibodies. Kinetic studies showed that these mutations affect virus–antibody interactions during postbinding steps of the HCV entry process. Functional studies with a large panel of patient-derived antibodies showed that this mechanism mediates viral escape, leading to persistent infection in general. CONCLUSIONS We identified a mechanism by which HCV evades host immune responses, in which use of cell entry factors evolves with escape from neutralizing antibodies. These findings advance our understanding of the pathogenesis of HCV infection and might be used to develop antiviral strategies and vaccines. PMID:22503792

  5. Fusion Proteins Cpn10-Erns with Properties of Generating CSFV-Neutralized Antibodies

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    When pigs are infected with classical swine fever virus (CSFV), the antibody primarily targets the structural glycoprotein E rns of the virus. Previous investigations have demonstrated that E rns has low or no virus neutralizing capacity. In this study, candidate subunit marker vaccine, chaperonin 10(Cpn10)-Erns, which possess the property of generating neutralized antibodies against lethal challenge of virulent CSFV was developed. The gene of E rns was isolated from Hog cholera lapinized virus (HCLV)-infected spleen cells of rabbits via RT-PCR method and fused to the downstream region of the cpn10 gene; the products of recombinant fusion protein (cpn10-Erns) induced expression in Escherichia coli, and the products were purified by affinity chromatography. During the course of vaccination, the candidate vaccines cpn10-E rns were used for the immunization of guinea pigs, and they induced a strong antibody response against cpn10-Erns. The antibodies can be immobilized by coating inactivated CSFV particles, indicating that these antibodies can recognize CSFV. Neutralization assay was carried out on rabbits according to National Regulations on Veterinary Drug. The results clearly indicate that the typical fever of rabbits induced by the live attenuated HCLV could be inhibited by preincubation with the antisera (dilution 1:4) induced by cpn10-Erns, but not inhibited by preincubation with the antisera induced only by Erns. Analogous results were observed for the group of the rabbits immunized with cpn10-Erns, which were protected against the typical fever induced by the challenge with HCLV. The findings of this study formed the basis of a new means for developing subunit marker vaccine against CSFV.

  6. EL4 cell-based colorimetric toxin neutralization activity assays for determination of neutralizing anti-ricin antibodies.

    Science.gov (United States)

    Lindsey, Changhong Y; Brown, J Edward; Torabazar, Nahid R; Smith, Leonard A

    2013-01-01

    A recombinant ricin toxin A-chain 1-33/44-198 vaccine (RVEc), developed at the United States Army Medical Research Institute of Infectious Diseases as a vaccine candidate, is under investigation in a phase 1 clinical study. To effectively evaluate the immunogenicity of this ricin vaccine and to eliminate the use of radioactive material, an EL4 cell-based colorimetric toxin neutralization activity (TNA) assay using a CellTiter 96 AQueous One Solution Cell Proliferation Assay Reagent has been developed, optimized, and applied in the vaccine efficacy studies. The TNA assay measures the protective neutralizing anti-ricin antibodies in animal sera by determining the cell viability after ricin exposure in the assay system and comparing it to a purified mouse polyclonal antiricin IgG standard curve. The standard curve of the anti-ricin TNA assay closely fits a four-parameter logistic regression model. The unknown test sample concentration was expressed as microg/mL, but not the 50% effective concentration (EC50), which was determined by most TNA assays. The neutralizing endpoint titers, not the 50% effective dilution (ED50), of human specimens were measured with the TNA assay in support of the clinical study of the RVEc vaccine. The optimal amount of ricin toxin, EL4 cells, and concentration of standards used in the assay system was established to minimize false-negative and false-positive results of serum specimens from the nonclinical and clinical studies of RVEc. The testing conditions were adjusted to optimize assay performance. The colorimetric TNA assay replaced a radioactive TNA assay previously used in the ricin vaccine studies.

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

    Science.gov (United States)

    Lewis, George K

    2015-06-01

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

  8. Neutralizing monoclonal antibodies against hepatitis C virus E2 protein bind discontinuous epitopes and inhibit infection at a postattachment step

    DEFF Research Database (Denmark)

    Sabo, Michelle C; Luca, Vincent C; Prentoe, Jannick

    2011-01-01

    The E2 glycoprotein of hepatitis C virus (HCV) mediates viral attachment and entry into target hepatocytes and elicits neutralizing antibodies in infected patients. To characterize the structural and functional basis of HCV neutralization, we generated a novel panel of 78 monoclonal antibodies...... of the homologous HCV strain in cell culture. Two of these bound E2 proteins from strains representative of HCV genotypes 1 to 6, and one of these MAbs, H77.39, neutralized infection of strains from five of these genotypes. The three most potent neutralizing MAbs in our panel, H77.16, H77.39, and J6.36, inhibited...

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Elise Landais

    2016-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2008-10-17

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

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

    Science.gov (United States)

    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.

  13. Preparation of Polyclonal Antibodies Against Testis-specific Protease 50 and Characterization of Antibody Specificity

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Testis-specific protease 50 (TSP50) is a testis-specific oncogene, which is abnormally activated in most tested patients with breast cancer. This property makes it an attractive molecular marker and a promising target for the diagnosis and therapy of breast cancer. In order to obtain the protective and specific polyclonal antibodies for further research, TSP50 cDNA was amplified by RT-PCR from normal human testicular tissue, and inserted into eukaryotic expression vector pcDNA3.1. Rabbit anti-TSP50 polyclonal antibodies were prepared by means of intramuscular injection of pcDNA3.1-TSP50 into the rabbits. Titers of the anti-sera were measured by ELISA and Western blotting with the E. coli cell lysate containing the induced GST-TSP50 fusion protein as an antigen. In addition, we examined the expression of TSP50 in both breast cancer cell line MCF-7 and breast cancer tissue by immunofluorescent and immunohistochemistry analysis.

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

    DEFF Research Database (Denmark)

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

    1999-01-01

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

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

    DEFF Research Database (Denmark)

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

    1999-01-01

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

  16. Localization of epitopes recognized by monoclonal antibodies that neutralized the H3N2 influenza viruses in man

    Science.gov (United States)

    Okada, Jun; Ohshima, Nobuko; Kubota-Koketsu, Ritsuko; Iba, Yoshitaka; Ota, Sayuri; Takase, Wakana; Yoshikawa, Tetsushi; Ishikawa, Toyokazu; Asano, Yoshizo; Okuno, Yoshinobu; Kurosawa, Yoshikazu

    2011-01-01

    Through extensive isolation of neutralizing mAbs against H3N2 influenza viruses representing the in vivo repertoire in a human donor, we examined the relationships between antigenic drift of influenza virus and protective antibodies generated in an infected individual. The majority of mAbs isolated from a donor born in 1960 were divided into three major groups with distinct strain specificity: 1968–1973, 1977–1993 and 1997–2003. In the present study, we developed a new method that allowed us to comprehensively determine the location of epitopes recognized by many mAbs. Original haemagglutinins (HAs) of several strains and chimaeric variants, in which one of the seven sites (A, B1, B2, C1, C2, D or E) was replaced by some other strain-derived sequence, were artificially expressed on the cell surface. The binding activity of mAbs to the HAs was examined by flow cytometry. By using this method, we determined the location of epitopes recognized by 98 different mAbs. Clones that neutralize the 1968–1973 strains bind to site B2/D, A or A/B1. While sites C, E and B were recognized by clones that neutralized the 1977–1993 strains, the majority of these clones bind to site C. Clones that neutralize the 1997–2003 strains bind to site B, A/B1, A/B2 or E/C2. PMID:21068214

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2010-11-15

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

  18. Roles of HIV neutralizing antibodies in the control of HIV infection%HIV中和抗体在抗HIV感染中的作用

    Institute of Scientific and Technical Information of China (English)

    刘沐桑; 郑楠; 刘维达

    2011-01-01

    Neutralizing antibodies specific for HIV can bind to the virus and inhibit HIV entry into host cells.The targets of HIV-specific neutralizing antibodies are the sites associated with receptor binding or membrane fusion on the envelop proteins of HIV.Relevant studies have provided theoretical basis for developing anti-HIV medicines and microbicides.The neutralization is sensitive to the conformation of related proteins.On the other hand, HIV has a latent ability to escape from antibodies neutralization.Therefore, the rational design of antigen to induce broad-spectrum neutralizing antibodies is necessary for the development of successful vaccines.This paper introduces HIV invasion mechanism as well as the relationship of HIV neutralizing antibodies with entry-related crucial proteins,targets and anti-HIV vaccines.%HIV的中和抗体可以与HIV病毒特异结合并且阻断病毒侵入细胞的抗体.HIV中和抗体的靶位是病毒的膜蛋白上那些与细胞受体结合和膜融合有关的区域,抗体的结合与相关蛋白的构象紧密相关,目前的相关研究为抗HIV药物和杀微生物剂的研制提供了理论基础.HIV具有逃逸中和抗体的能力.因此,为设计有效的疫苗,需要对抗原进行理性设计,以便更有效的诱导广谱中和抗体.概述HIV的侵入机制,中和抗体与侵入关键蛋白、靶点以及抗HIV疫苗研究的关系和进展.

  19. Predefined GPGRAFY-Epitope-Specific Monoclonal Antibodies with Different Activities for Recognizing Native HIV-1 gp120

    Institute of Scientific and Technical Information of China (English)

    蓝灿辉; 田海军; 陈应华

    2004-01-01

    A seven-amino acid epitope GPGRAFY at the tip of the V3 loop in HIV-1 gp120 is the principal neutralizing epitope,and a subset of anti-V3 antibodies specific for this epitope shows a broad range of neutralizing activity.GPGRAFY-epitope-specific neutralizing antibodies were produced using predefined GPGRAFY-epitope-specific peptides instead of a natural or recombinant gp120 bearing this epitope.All six monoclonal antibodies (mAbs) could recognize the GPGRAFY-epitope on peptides and two of the antibodies,9D8 and 2D7,could recognize recombinant gp120 in enzymelinked immunosorkentassy (ELISA) assays.In the flow cytometry analysis,the mAbs 9D8 and 2D7 could bind to HIV-Env+ CHO-WT cells and the specific bindings could be inhibited by the GPGRAFY-epitope peptide,which suggests that these two mAbs could recognize the native envelope protein gp120 expressed on the cell membrane.However,in syncytium assays,none of the mAbs was capable of inhibiting HIV-Env-mediated cell membrane fusion.The different activities for recognizing native HIV-1 gp120 might be associated with different antibody affinities against the epitopes.The development of conformational mimics of the neutralization epitope in the gp120 V3 loop could elicit neutralizing mAbs with high affinity.

  20. Nasal Immunization Confers High Avidity Neutralizing Antibody Response and Immunity to Primary and Recurrent Genital Herpes in Guinea Pigs

    Science.gov (United States)

    Persson, Josefine; Zhang, Yuan; Olafsdottir, Thorunn A.; Thörn, Karolina; Cairns, Tina M.; Wegmann, Frank; Sattentau, Quentin J.; Eisenberg, Roselyn J.; Cohen, Gary H.; Harandi, Ali M.

    2016-01-01

    Genital herpes is one of the most prevalent sexually transmitted infections in both the developing and developed world. Following infection, individuals experience life-long latency associated with sporadic ulcerative outbreaks. Despite many efforts, no vaccine has yet been licensed for human use. Herein, we demonstrated that nasal immunization with an adjuvanted HSV-2 gD envelope protein mounts significant protection to primary infection as well as the establishment of latency and recurrent genital herpes in guinea pigs. Nasal immunization was shown to elicit specific T cell proliferative and IFN-γ responses as well as systemic and vaginal gD-specific IgG antibody (Ab) responses. Furthermore, systemic IgG Abs displayed potent HSV-2 neutralizing properties and high avidity. By employing a competitive surface plasmon resonance (SPR) analysis combined with a battery of known gD-specific neutralizing monoclonal Abs (MAbs), we showed that nasal immunization generated IgG Abs directed to two major discontinuous neutralizing epitopes of gD. These results highlight the potential of nasal immunization with an adjuvanted HSV-2 envelope protein for induction of protective immunity to primary and recurrent genital herpes. PMID:28082979

  1. Neutralizing antibodies against two HIV-1 strains in consecutively collected serum samples: cross neutralization and association to HIV-1 related disease

    DEFF Research Database (Denmark)

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

    1992-01-01

    97 sera collected during a 10-year period from 10 HIV-1 infected individuals were tested for neutralizing capacity against a virus isolate FICPH-22 obtained from a Danish AIDS patient, and the laboratory strain HTLV-IIIB. Three patterns of serum neutralizing activity were demonstrated: (a) patients...... developing high neutralizing activity against both HIV strains; (b) patients developing high neutralizing activity against the Danish virus isolate; and (c) patients developing only low titers of neutralizing antibodies (NA) against both HIV strains. The HTLV-IIIB strain was less sensitive to serum...... neutralization than the FICPH-22 isolate and the appearance of NA against HTLV-IIIB was typically lacking several years behind that against FICPH-22 indicating a broadening of the NA response over time. No difference in clinical outcome was observed comparing patients reaching high titers of NA and patients...

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

    Directory of Open Access Journals (Sweden)

    Cinque Soto

    Full Text Available 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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Witkowski, Peter T. [Robert Koch-Institut, Zentrum fuer Biologische Sicherheit 1, Nordufer 20, 13353 Berlin (Germany); Charite Universitaetsmedizin, CCM, Institut fuer Virologie, Helmut Ruska Haus, Chariteplatz 1, 10117 Berlin (Germany); Schuenadel, Livia, E-mail: SchuenadelL@rki.de [FU-Berlin, Fachbereich Biologie, Chemie, Pharmazie, Takustrasse 3, 14195 Berlin (Germany); Robert Koch-Institut, Zentrum fuer Biologische Sicherheit 1, Nordufer 20, 13353 Berlin (Germany); Wiethaus, Julia; Bourquain, Daniel R.; Kurth, Andreas; Nitsche, Andreas [Robert Koch-Institut, Zentrum fuer Biologische Sicherheit 1, Nordufer 20, 13353 Berlin (Germany)

    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.

  4. Antibody specific epitope prediction-emergence of a new paradigm.

    Science.gov (United States)

    Sela-Culang, Inbal; Ofran, Yanay; Peters, Bjoern

    2015-04-01

    The development of accurate tools for predicting B-cell epitopes is important but difficult. Traditional methods have examined which regions in an antigen are likely binding sites of an antibody. However, it is becoming increasingly clear that most antigen surface residues will be able to bind one or more of the myriad of possible antibodies. In recent years, new approaches have emerged for predicting an epitope for a specific antibody, utilizing information encoded in antibody sequence or structure. Applying such antibody-specific predictions to groups of antibodies in combination with easily obtainable experimental data improves the performance of epitope predictions. We expect that further advances of such tools will be possible with the integration of immunoglobulin repertoire sequencing data.

  5. Increased titers of neutralizing antibodies after immunization with both envelope proteins of the porcine endogenous retroviruses (PERVs

    Directory of Open Access Journals (Sweden)

    Denner Joachim

    2012-11-01

    Full Text Available Abstract Despite enormous difficulties to induce antibodies neutralizing HIV-1, especially broadly neutralizing antibodies directed against the conserved membrane proximal external region (MPER of the transmembrane envelope protein, such antibodies can be easily induced in the case of gammaretroviruses, among them the porcine endogenous retroviruses (PERVs. In addition to neutralizing antibodies directed against the transmembrane envelope protein p15E, neutralizing antibodies were also induced by immunization with the surface envelope protein gp70. PERVs represent a special risk for xenotransplantation using pig tissues or organs since they are integrated in the genome of all pigs and infect human cells and a vaccine may protect from transmission to the recipient. To investigate the effect of simultaneous immunization with both proteins in detail, a study was performed in hamsters. Gp70 and p15E of PERV were produced in E. coli, purified and used for immunization. All animals developed binding antibodies against the antigens used for immunization. Sera from animals immunized with p15E recognized epitopes in the MPER and the fusion peptide proximal region (FPPR of p15E. One MPER epitope showed a sequence homology to an epitope in the MPER of gp41 of HIV-1 recognized by broadly neutralizing antibodies found in HIV infected individuals. Neutralizing antibodies were detected in all sera. Most importantly, sera from animals immunized with gp70 had a higher neutralizing activity when compared with the sera from animals immunized with p15E and sera from animals immunized with gp70 together with p15E had a higher neutralizing activity compared with sera from animals immunized with each antigen alone. These immunization studies are important for the development of vaccines against other retroviruses including the human immunodeficiency virus HIV-1.

  6. Neutralizing antibody response in the patients with hand, foot and mouth disease to enterovirus 71 and its clinical implications

    Directory of Open Access Journals (Sweden)

    Zhu Liye

    2011-06-01

    Full Text Available Abstract Enterovirus 71 (EV71 has emerged as a significant pathogen causing large outbreaks in China for the past 3 years. Developing an EV71 vaccine is urgently needed to stop the spread of the disease; however, the adaptive immune response of humans to EV71 infection remains unclear. We examined the neutralizing antibody titers in HFMD patients and compared them to those of asymptomatic healthy children and young adults. We found that 80% of HFMD patients became positive for neutralizing antibodies against EV71 (GMT = 24.3 one day after the onset of illness. The antibody titers in the patients peaked two days (GMT = 79.5 after the illness appeared and were comparable to the level of adults (GMT = 45.2. Noticeably, the antibody response was not correlated with disease severity, suggesting that cellular immune response, besides neutralizing antibodies, could play critical role in controlling the outcome of EV71 infection in humans.

  7. Detection of canine distemper virus serum neutralizing antibodies in captive U.S. phocids.

    Science.gov (United States)

    Clancy, Meredith M; Gamble, Kathryn C; Travis, Dominic A

    2013-03-01

    Antibodies to morbilliviruses have been documented in free-ranging pinnipeds throughout populations in the Atlantic and Arctic Oceans, but not from the Pacific Ocean. As a symbolic geographic barrier between the exposed Atlantic and naive Pacific populations, the captive phocid population in North America had undocumented serologic status. In this study, canine distemper virus (CDV) serum neutralization assays were used to assess the prevalence of antibodies in this population with participation of 25 U.S. institutions from grey seals (Halichoerus grypus, n = 6) and harbor seals (Phoca vitulina, n = 108). Historic and environmental risk factors associated with the epidemiology of distemper virus were collected by survey. Based on antibodies to canine distemper virus, the prevalence of exposure in this population was 25.5%, with 28 seals (grey, n = 2; harbor, n = 26) demonstrating antibody titers > or = 1:16, and positive titers ranged from 1:4 to 1:1,536. By survey analysis, strong associations with seropositive status were identified for captive origin (P = 0.013) and movement among institutions (P = 0.024). Size of population has positive correlation with likelihood of seropositive seals at an institution (P = 0.020). However, no major husbandry or enclosure-based risk factors were identified in institutions with seropositive seals, and no interaction between individual or institutional risk factors was identified. Previously undocumented prior to this study, CDV antibodies were measured in harbor seals (n = 2) recently stranded from the Pacific coast.

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

    Science.gov (United States)

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

    2015-06-25

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

  9. Requirements specification for the Neutral Beam Injector on FAST

    Energy Technology Data Exchange (ETDEWEB)

    Baruzzo, M., E-mail: matteo.baruzzo@igi.cnr.it [Consorzio RFX, EURATOM-ENEA Association, Corso Stati Uniti 4, 35127 Padova (Italy); Bolzonella, T. [Consorzio RFX, EURATOM-ENEA Association, Corso Stati Uniti 4, 35127 Padova (Italy); Calabro, G.; Crisanti, F.; Cucchiaro, A. [Associazione Euratom/ENEA sulla Fusione, CP 65-00044 Frascati, Rome (Italy); Marcuzzi, D.; Rigato, W. [Consorzio RFX, EURATOM-ENEA Association, Corso Stati Uniti 4, 35127 Padova (Italy); Schneider, M. [CEA, IRFM, F-13108 Saint-Paul-lez-Durance (France); Sonato, P.; Valisa, M.; Zaccaria, P. [Consorzio RFX, EURATOM-ENEA Association, Corso Stati Uniti 4, 35127 Padova (Italy); Artaud, J.F.; Basiuk, V. [CEA, IRFM, F-13108 Saint-Paul-lez-Durance (France); Cardinali, A. [Associazione Euratom/ENEA sulla Fusione, CP 65-00044 Frascati, Rome (Italy); Imbeaux, F. [CEA, IRFM, F-13108 Saint-Paul-lez-Durance (France); Lauro Taroni, L. [Consorzio RFX, EURATOM-ENEA Association, Corso Stati Uniti 4, 35127 Padova (Italy); Marinucci, M. [Associazione Euratom/ENEA sulla Fusione, CP 65-00044 Frascati, Rome (Italy); Mantica, P. [Istituto di Fisica del Plasma ' P.Caldirola' , Associazione Euratom-ENEA-CNR, Milano (Italy); Zonca, F. [Associazione Euratom/ENEA sulla Fusione, CP 65-00044 Frascati, Rome (Italy)

    2011-10-15

    This paper discusses the scientific and technical requirements for a Neutral Beam Injection system on the FAST tokamak and describes a preliminary conceptual design of a suitable injector. FAST is being proposed as a European experiment in support to the operations on ITER and to the design of DEMO. The specific mission of this device is an integrated approach to a number of outstanding burning plasmas physics and operational issues with an emphasis on the impact of fast particles on turbulent transport. Such scientific requirements set a series of technical challenges regarding the injector and the coupling of the injector to the FAST main chamber that are addressed in the paper. A preliminary conceptual design of the injector is proposed which attempts to meet the stated requirements.

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

    Science.gov (United States)

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

    2015-11-19

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

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    Simon M Agwale

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

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

    Science.gov (United States)

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2009-09-11

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2011-12-31

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

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

    Science.gov (United States)

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

    2012-04-30

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

  17. Bi-specific antibody therapy for the treatment of cancer

    NARCIS (Netherlands)

    Withoff, S; Helfrich, W; de Leij, LFMH; Molema, G

    2001-01-01

    Bi-specific antibodies (BsAbs) combine immune cell activation with tumor cell recognition as a result of which tumor cells are killed by pre-defined effector cells. In this review, a brief introduction to monoclonal antibodies will precede a more in-depth presentation of the current status of BsAb t

  18. Monoclonal Antibodies Specific for Hippurate Hydrolase of Campylobacter jejuni

    OpenAIRE

    Steele, Marina; Gyles, Carlton; Chan, Voon Loong; Odumeru, Joseph

    2002-01-01

    Eleven monoclonal antibodies raised against recombinant Campylobacter jejuni hippurate hydrolase were tested for binding to lysates from 19 C. jejuni strains, 12 other Campylobacter strains, and 21 non-Campylobacter strains. Several monoclonal antibodies bound to C. jejuni but not to other Campylobacter species and may be useful in a species-specific immunoassay.

  19. Second generation of pseudotype-based serum neutralization assay for Nipah virus antibodies: sensitive and high-throughput analysis utilizing secreted alkaline phosphatase.

    Science.gov (United States)

    Kaku, Yoshihiro; Noguchi, Akira; Marsh, Glenn A; Barr, Jennifer A; Okutani, Akiko; Hotta, Kozue; Bazartseren, Boldbaatar; Fukushi, Shuetsu; Broder, Christopher C; Yamada, Akio; Inoue, Satoshi; Wang, Lin-Fa

    2012-01-01

    Nipah virus (NiV), Paramyxoviridae, Henipavirus, is classified as a biosafety level (BSL) 4 pathogen, along with the closely related Hendra virus (HeV). A novel serum neutralization test was developed for measuring NiV neutralizing antibodies under BSL2 conditions using a recombinant vesicular stomatitis virus (VSV) expressing secreted alkaline phosphatase (SEAP) and pseudotyped with NiV F/G proteins (VSV-NiV-SEAP). A unique characteristic of this novel assay is the ability to obtain neutralization titers by measuring SEAP activity in supernatant using a common ELISA plate reader. This confers a remarkable advantage over the first generation of NiV-pseudotypes expressing green fluorescent protein or luciferase, which require expensive and specific measuring equipment. Using panels of NiV- and HeV-specific sera from various species, the VSV-NiV-SEAP assay demonstrated neutralizing antibody status (positive/negative) consistent with that obtained by conventional live NiV test, and gave higher antibody titers than the latter. Additionally, when screening sixty-six fruit bat sera at one dilution, the VSV-NiV-SEAP assay produced identical results to the live NiV test and only required a very small amount (2μl) of sera. The results suggest that this novel VSV-NiV-SEAP assay is safe, useful for high-throughput screening of sera using an ELISA plate reader, and has high sensitivity and specificity.

  20. Glycan masking of hemagglutinin for adenovirus vector and recombinant protein immunizations elicits broadly neutralizing antibodies against H5N1 avian influenza viruses.

    Directory of Open Access Journals (Sweden)

    Shih-Chang Lin

    Full Text Available The highly pathogenic avian influenza (HPAI H5N1 virus, a known trigger of diseases in poultry and humans, is perceived as a serious threat to public health. There is a clear need for a broadly protective H5N1 vaccine or vaccines for inducing neutralizing antibodies against multiple clades/subclades. We constructed single, double, and triple mutants of glycan-masked hemagglutiinin (HA antigens at residues 83, 127 and 138 (i.e., g83, g127, g138, g83+g127, g127+g138, g83+g138 and g83+g127+g138, and then obtained their corresponding HA-expressing adenovirus vectors and recombinant HA proteins using a prime-boost immunization strategy. Our results indicate that the glycan-masked g127+g138 double mutant induced more potent HA-inhibition, virus neutralization antibodies, cross-clade protection against heterologous H5N1 clades, correlated with the enhanced bindings to the receptor binding sites and the highly conserved stem region of HA. The immune refocusing stem-specific antibodies elicited by the glycan-masked H5HA g127+g138 and g83+g127+g138 mutants overlapped with broadly neutralizing epitopes of the CR6261 monoclonal antibody that neutralizes most group 1 subtypes. These findings may provide useful information in the development of a broadly protective H5N1 influenza vaccine.

  1. The Effect of Induced Antibodies with Respect to Neutralization, Clearance Rate and Functional Activity in a Rabbit/Infliximab Model

    DEFF Research Database (Denmark)

    Henriksen, Maiken Lumby; Teisner, Ane; Kjeldsen, Jens

    2016-01-01

    and Methods: We addressed this issue in a rabbit model of treatment with the anti-tumor-necrosis factor alpha (TNF) antibody, infliximab (IFX). We developed an inhibition ELISA to selectively measure absolute concentrations of neutralizing antibodies and another ELISA for measuring the concentration...

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

    Science.gov (United States)

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

    2016-03-01

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

  3. Neutralization Analysis of a Chicken Single-Chain Variable Fragment Derived from an Immune Antibody Library Against Infectious Bronchitis Virus.

    Science.gov (United States)

    Lin, Yuan; Li, Benqiang; Ye, Jiaxin; Wang, Man; Wang, Jianhua; Zhang, Ying; Zhu, Jianguo

    2015-09-01

    Avian infectious bronchitis virus (IBV), which is prevalent in many countries causing severe economic loss to the poultry industry, causes infectious bronchitis (IB) in birds. Recombinant single-chain variable fragments (scFvs) have been proven to effectively inhibit many viruses, both in vitro and in vivo, and they could be a potential diagnostic and therapeutic reagent to control IB. In this study, six anti-IBV chicken scFvs, ZL.10, ZL.64, ZL.78, ZL.80, ZL.138, and ZL.256, were obtained by screening random clones from an immune antibody library. An analysis of nucleotide sequences revealed that they represented distinctive genetic sequences and greatly varied in complementarity-determining region three of the heavy chain. Neutralization tests showed that ZL.10, which bound the S1 protein in western blots, inhibited the formation of syncytia in Vero cells 48 h post IBV infection and decreased the transcriptional level of nucleoprotein mRNA to 17.2%, while the other five scFvs, including ZL.78 and ZL.256, that bound the N protein did not. In conclusion, the results suggested that specific and neutralizing chicken scFvs against IBV, which can be safe and economical antibody reagents, can be produced in vitro through prokaryotic expression.

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

    Directory of Open Access Journals (Sweden)

    Moss Bernard

    2009-03-01

    Full Text Available Abstract 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.

  5. Activated human nasal epithelial cells modulate specific antibody response against bacterial or viral antigens.

    Directory of Open Access Journals (Sweden)

    Chiou-Yueh Yeh

    Full Text Available Nasal mucosa is an immune responsive organ evidenced by eliciting both specific local secretory IgA and systemic IgG antibody responses with intra-nasal administration of antigens. Nevertheless, the role of nasal epithelial cells in modulating such responses is unclear. Human nasal epithelial cells (hNECs obtained from sinus mucosa of patients with chronic rhinosinusitis were cultured in vitro and firstly were stimulated by Lactococcus lactis bacterium-like particles (BLPs in order to examine their role on antibody production. Secondly, both antigens of immunodominant protein IDG60 from oral Streptococcus mutans and hemagglutinin (HA from influenza virus were tested to evaluate the specific antibody response. Stimulated hNECs by BLPs exhibited a significant increase in the production of interleukin-6 (IL-6, and thymic stromal lymphopoietin (TSLP. Conditioned medium of stimulated hNECs has effects on enhancing the proliferation of CD4+ T cells together with interferon-γ and IL-5 production, increasing the costimulatory molecules on dendritic cells and augmenting the production of IDG60 specific IgA, HA specific IgG, IgA by human peripheral blood lymphocytes. Such production of antigen specific IgG and IgA is significantly counteracted in the presence of IL-6 and TSLP neutralizing antibodies. In conclusion, properly stimulated hNECs may impart immuno-modulatory effects on the antigen-specific antibody response at least through the production of IL-6 and TSLP.

  6. Generation and characterization of novel stromal specific antibodies

    Institute of Scientific and Technical Information of China (English)

    2005-01-01

    Rheumatoid synovial fibroblasts were used as an immunogen to produce monoclonal antibodies selected for their reactivity with stromal cell antigens. Mice were immunised with low passage whole cell preparations and the subsequent hybridomas were screened by immunohistochemistry on rheumatoid synovium and tonsil sections. The aim was to identify those antibodies that recognised antigens that were restricted to stromal cells and were not expressed on CD45 positive leucocytes. A significant number of antibodies detected antigen that identified endothelial cells. These antibodies were further characterised to determine whether the vessels identified by these antibodies were vascular or lymphatic.From five fusions clones were identified with predominant reactivity with: 1) fibroblasts and endothelial cells; or 2)broad stromal elements (fibroblast, endothelium, epithelium, follicular dendritic cells). A fibroblast-specific antibody that did not also identify vessels was not generated. Examples of each reactivity pattern are discussed.

  7. Rational Design of CXCR4 Specific Antibodies with Elongated CDRs

    Science.gov (United States)

    2015-01-01

    The bovine antibody (BLV1H12) which has an ultralong heavy chain complementarity determining region 3 (CDRH3) provides a novel scaffold for antibody engineering. By substituting the extended CDRH3 of BLV1H12 with modified CXCR4 binding peptides that adopt a β-hairpin conformation, we generated antibodies specifically targeting the ligand binding pocket of CXCR4 receptor. These engineered antibodies selectively bind to CXCR4 expressing cells with binding affinities in the low nanomolar range. In addition, they inhibit SDF-1-dependent signal transduction and cell migration in a transwell assay. Finally, we also demonstrate that a similar strategy can be applied to other CDRs and show that a CDRH2-peptide fusion binds CXCR4 with a Kd of 0.9 nM. This work illustrates the versatility of scaffold-based antibody engineering and could greatly expand the antibody functional repertoire in the future. PMID:25041362

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

    Directory of Open Access Journals (Sweden)

    Javier Guenaga

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

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

    Science.gov (United States)

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

    2016-03-18

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

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

    Energy Technology Data Exchange (ETDEWEB)

    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. (Purdue); (WU-MED); (CDC)

    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. Induction of neutralizing antibodies in mice immunized with scorpion toxins detoxified by liposomal entrapment

    OpenAIRE

    1997-01-01

    The possibility of producing neutralizing antibodies against the lethal effects of scorpion toxins was evaluated in the mouse model by immunization with an immunogen devoid of toxicity. A toxic fraction (5 mg) from the venom of the scorpion Tityus serrulatus was entrapped in sphingomyelin-cholesterol liposomes. The liposomes were treated for 1 h at 37oC with a 1% (w/w) trypsin solution in 0.2 M sodium carbonate buffer, pH 8.3. This treatment led to a strong reduction in venom toxicity. Immuni...

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

    Directory of Open Access Journals (Sweden)

    Garnett Kelsoe

    2013-12-01

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

  13. Exploiting Cross-reactivity to Neutralize Two Different Scorpion Venoms with One Single Chain Antibody Fragment*

    OpenAIRE

    Riaño-Umbarila, Lidia; Contreras-Ferrat, Gabriel; Olamendi-Portugal, Timoteo; Morelos-Juárez, Citlalli; Corzo, Gerardo; Possani, Lourival D.; Becerril, Baltazar

    2010-01-01

    We report the optimization of a family of human single chain antibody fragments (scFv) for neutralizing two scorpion venoms. The parental scFv 3F recognizes the main toxins of Centruroides noxius Hoffmann (Cn2) and Centruroides suffusus suffusus (Css2), albeit with low affinity. This scFv was subjected to independent processes of directed evolution to improve its recognition toward Cn2 (Riaño-Umbarila, L., Juárez-González, V. R., Olamendi-Portugal, T., Ortíz-León, M., Possani, L. D., and Bece...

  14. Phospho-Specific Antibody Probes of Intermediate Filament Proteins.

    Science.gov (United States)

    Goto, Hidemasa; Tanaka, Hiroki; Kasahara, Kousuke; Inagaki, Masaki

    2016-01-01

    Intermediate filaments (IFs) form one of the major cytoskeletal systems in the cytoplasm or beneath the nuclear membrane. Accumulating data have suggested that IF protein phosphorylation dramatically changes IF structure/dynamics in cells. For the production of an antibody recognizing site-specific protein phosphorylation (a site- and phosphorylation state-specific antibody), we first employed a strategy to immunize animals with an in vitro-phosphorylated polypeptide or a phosphopeptide (corresponding to a phosphorylated residue and its surrounding sequence of amino acids), instead of a phosphorylated protein. Our established methodology not only improves the chance of obtaining a phospho-specific antibody but also has the advantage that one can predesign a targeted phosphorylation site. It is now applied to the production of an antibody recognizing other types of site-specific posttranslational modification, such as acetylation or methylation. The use of such an antibody in immunocytochemistry enables us to analyze spatiotemporal distribution of site-specific IF protein phosphorylation. The antibody is of great use to identify a protein kinase responsible for in vivo IF protein phosphorylation and to monitor intracellular kinase activities through IF protein phosphorylation. Here, we present an overview of our methodology and describe stepwise approaches for the antibody characterization. We also provide some examples of analyses for IF protein phosphorylation involved in mitosis and signal transduction.

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

    Directory of Open Access Journals (Sweden)

    Prabakaran ePonraj

    2014-08-01

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

  16. Nothing's perfect: the art of defining HLA-specific antibodies.

    Science.gov (United States)

    Middleton, D; Jones, J; Lowe, D

    2014-05-01

    The advent of solid phase assays and in particular the single antigen bead (SAB) assay, on the Luminex platform has led to previously unheralded levels of HLA-specific antibody characterisation. However, it soon became apparent that the detection of antibodies detected by these assays was less than perfect and that not all antibodies determined could be considered clinically relevant. Thus, the major challenges currently faced by HLA laboratories are to interpret the complex data provided by these assays and use this to devise a safe and practical algorithm for the definition of a clinically relevant HLA-specific antibody. Taking into consideration recent evidence and scientific opinion in this area we aim here to put forward the viewpoint of our laboratory in how best to manage the tricky problem of defining HLA-specific antibodies. By taking a balanced approach which is less reliant upon a single technique we propose that the aim should be to define antibodies to a level that does not discriminate against the highly sensitised patient, but also maintains clinical safety and efficacy. Knowing that not all of the antibodies detected by SAB are clinically relevant should lead to giving greater opportunity for patients with these antibodies having a crossmatch performed. In the future, more emphasis should be given to epitopes when interpreting the results of these assays. Copyright © 2014 Elsevier B.V. All rights reserved.

  17. Identification and evaluation of nextgeneration PTM-specific antibodies

    DEFF Research Database (Denmark)

    Persson, Nina Emilia

    Over the past 30 years, antibody-based therapies have been established as important tools fortreatment of cancer, autoimmune diseases, inflammatory diseases, organ transplantations andinfectious diseases. There are today over 47 FDA approved monoclonal antibody products fordiagnostics and therapy...... peptide and glycopeptide targets.Using phage display and the microarray screening assay a new anti-Tn, G2-D11 was identified.This antibody differs in specificity compared to other anti-Tn antibodies. It is specific for twoadjacent Tn-antigens and compared to other Tn-antigen binding antibodies...... the influenced by therest of the peptide backbone is limited. Indicating a high degree of carbohydrate epitopedominance and independence of the peptide backbone. With an impressive affinity (KDapp=1.3 x10-8 M) and no cross-reactivity to either STn epitope or blood group A antigens, the G2-D11differ from Tn...

  18. Passive antibody therapy of Lassa fever in cynomolgus monkeys: importance of neutralizing antibody and Lassa virus strain.

    Science.gov (United States)

    Jahrling, P B; Peters, C J

    1984-05-01

    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)

  19. Milk from Brazilian women presents secretory IgA antibodies and neutralizes rotavirus G9P[5].

    Science.gov (United States)

    Santos, Simone M R; Ferreira, Thalita L; Quintal, Virgínia S; Carbonare, Solange B; Tino-de-Franco, Milene

    2013-01-01

    To verify the presence of anti-rotavirus serotype G9P[5] SIgA and the virus neutralization capacity of milk samples from Brazilian women. SIgA antibody levels reactive to rotavirus G9 were determined in 30 maternal milk samples by enzyme-linked immunosorbent assay (ELISA) using purified virus suspensions. The samples' capacity to neutralize rotavirus G9P[5] was analyzed using the MA-104 cells neutralization assay. Great individual variations were observed regarding the SIgA levels and neutralization titers, but all samples showed some G9P[5] neutralizing ability. A highly significant positive correlation was observed between antibody levels and neutralization titers. The high correlation between anti-rotavirus antibody levels and neutralizing capacity of the milk samples suggests a possible protective role of these antibodies against infection. These results also support the encouragement of the breast-feeding practice. Copyright © 2013 Sociedade Brasileira de Pediatria. Published by Elsevier Editora Ltda. All rights reserved.

  20. CRF01_AE-specific neutralizing activity observed in plasma derived from HIV-1-infected Thai patients residing in northern Thailand: comparison of neutralizing breadth and potency between plasma derived from rapid and slow progressors.

    Directory of Open Access Journals (Sweden)

    Sompong Sapsutthipas

    Full Text Available BACKGROUND: Development of a protective vaccine against human immunodeficiency virus type 1 (HIV-1 is an important subject in the field of medical sciences; however, it has not yet been achieved. Potent and broadly neutralizing antibodies are found in the plasma of some HIV-1-infected patients, whereas such antibody responses have failed to be induced by currently used vaccine antigens. In order to develop effective vaccine antigens, it is important to reveal the molecular mechanism of how strong humoral immune responses are induced in infected patients. As part of such studies, we examined the correlation between the anti-HIV-1 neutralizing antibody response and disease progression. METHODOLOGY/PRINCIPAL FINDINGS: We evaluated the anti-HIV-1 neutralizing activity of plasma derived from 33 rapid and 34 slow progressors residing in northern Thailand. The level of neutralizing activity varied considerably among plasmas, and no statistically significant differences in the potency and breadth of neutralizing activities were observed overall between plasma derived from rapid and slow progressors; however, plasma of 4 slow progressors showed neutralizing activity against all target viruses, whereas none of the plasma of rapid progressors showed such neutralizing activity. In addition, 21% and 9% of plasmas derived from slow and rapid progressors inhibited the replication of more than 80% of CRF01_AE Env-recombinant viruses tested, respectively. Neutralization of subtype B and C Env-recombinant viruses by the selected plasma was also examined; however, these plasma samples inhibited the replication of only a few viruses tested. CONCLUSIONS/SIGNIFICANCE: Although no statistically significant differences were observed in the potency and breadth of anti-HIV-1 neutralizing activities between plasma derived from rapid and slow progressors, several plasma samples derived from slow progressors neutralized CRF01_AE Env-recombinant viruses more frequently than

  1. Select human anthrax protective antigen (PA) epitope-specific antibodies provide protection from lethal toxin challenge

    Science.gov (United States)

    Crowe, Sherry R.; Ash, Linda L.; Engler, Renata J. M.; Ballard, Jimmy D.; Harley, John B.; Farris, A. Darise; James, Judith A.

    2010-01-01

    Bacillus anthracis remains a serious bioterrorism concern, and the currently licensed vaccine remains an incomplete solution for population protection from inhalation anthrax and has been associated with concerns regarding efficacy and safety. Thus, understanding how to generate long lasting protective immunity with reduced immunizations or providing protection through post exposure immunotherapeutics are long sought goals. Through evaluation of a large military cohort, we characterized the levels of antibodies against protective antigen and found that over half of anthrax vaccinees had low levels of in vitro toxin neutralization capacity in their sera. Using solid phase epitope mapping and confirmatory assays, we identified several neutralization-associated humoral epitopes and demonstrated that select anti-peptide responses mediated protection in vitro. Finally, passively transferred antibodies specific for select epitopes provided protection in an in vivo lethal toxin mouse model. Identification of these antigenic regions has important implications for vaccine design and the development of directed immunotherapeutics. PMID:20533877

  2. Differences in Glycoprotein Complex Receptor Binding Site Accessibility Prompt Poor Cross-Reactivity of Neutralizing Antibodies between Closely Related Arenaviruses.

    Science.gov (United States)

    Brouillette, Rachel B; Phillips, Elisabeth K; Ayithan, Natarajan; Maury, Wendy

    2017-04-01

    The glycoprotein complex (GPC) of arenaviruses, composed of stable signal peptide, GP1, and GP2, is the only antigen correlated with antibody-mediated neutralization. However, despite strong cross-reactivity of convalescent antisera between related arenavirus species, weak or no cross-neutralization occurs. Two closely related clade B viruses, Machupo virus (MACV) and Junín virus (JUNV), have nearly identical overall GPC architecture and share a host receptor, transferrin receptor 1 (TfR1). Given structural and functional similarities of the GP1 receptor binding site (RBS) of these viruses and the recent demonstration that the RBS is an important target for neutralizing antibodies, it is not clear how these viruses avoid cross-neutralization. To address this, MACV/JUNV chimeric GPCs were assessed for interaction with a group of α-JUNV GPC monoclonal antibodies (MAbs) and mouse antisera against JUNV or MACV GPC. All six MAbs targeted GP1, with those that neutralized JUNV GPC-pseudovirions competing with each other for RBS binding. However, these MAbs were unable to bind to a chimeric GPC composed of JUNV GP1 containing a small disulfide bonded loop (loop 10) unique to MACV GPC, suggesting that this loop may block MAbs interaction with the GP1 RBS. Consistent with this loop causing interference, mouse anti-JUNV GPC antisera that solely neutralized pseudovirions bearing autologous GP1 provided enhanced neutralization of MACV GPC when this loop was removed. Our studies provide evidence that loop 10, which is unique to MACV GP1, is an important impediment to binding of neutralizing antibodies and contributes to the poor cross-neutralization of α-JUNV antisera against MACV.IMPORTANCE Multiple New World arenaviruses can cause severe disease in humans, and some geographic overlap exists among these viruses. A vaccine that protects against a broad range of New World arenaviruses is desirable for purposes of simplicity, cost, and broad protection against multiple National

  3. Behaviour of non-donor specific antibodies during rapid re-synthesis of donor specific HLA antibodies after antibody incompatible renal transplantation.

    Directory of Open Access Journals (Sweden)

    Nithya S Krishnan

    Full Text Available HLA directed antibodies play an important role in acute and chronic allograft rejection. During viral infection of a patient with HLA antibodies, the HLA antibody levels may rise even though there is no new immunization with antigen. However it is not known whether the converse occurs, and whether changes on non-donor specific antibodies are associated with any outcomes following HLA antibody incompatible renal transplantation.55 patients, 31 women and 24 men, who underwent HLAi renal transplant in our center from September 2005 to September 2010 were included in the studies. We analysed the data using two different approaches, based on; i DSA levels and ii rejection episode post transplant. HLA antibody levels were measured during the early post transplant period and corresponding CMV, VZV and Anti-HBs IgG antibody levels and blood group IgG, IgM and IgA antibodies were quantified.Despite a significant DSA antibody rise no significant non-donor specific HLA antibody, viral or blood group antibody rise was found. In rejection episode analyses, multiple logistic regression modelling showed that change in the DSA was significantly associated with rejection (p = 0.002, even when adjusted for other antibody levels. No other antibody levels were predictive of rejection. Increase in DSA from pre treatment to a post transplant peak of 1000 was equivalent to an increased chance of rejection with an odds ratio of 1.47 (1.08, 2.00.In spite of increases or decreases in the DSA levels, there were no changes in the viral or the blood group antibodies in these patients. Thus the DSA rise is specific in contrast to the viral, blood group or third party antibodies post transplantation. Increases in the DSA post transplant in comparison to pre-treatment are strongly associated with occurrence of rejection.

  4. Heterosubtype neutralizing responses to influenza A (H5N1 viruses are mediated by antibodies to virus haemagglutinin.

    Directory of Open Access Journals (Sweden)

    Jean-Michel Garcia

    Full Text Available BACKGROUND: It is increasingly clear that influenza A infection induces cross-subtype neutralizing antibodies that may potentially confer protection against zoonotic infections. It is unclear whether this is mediated by antibodies to the neuraminidase (NA or haemagglutinin (HA. We use pseudoviral particles (H5pp coated with H5 haemagglutinin but not N1 neuraminidase to address this question. In this study, we investigate whether cross-neutralizing antibodies in persons unexposed to H5N1 is reactive to the H5 haemagglutinin. METHODOLOGY/PRINCIPAL FINDINGS: We measured H5-neutralization antibody titers pre- and post-vaccination using the H5N1 micro-neutralization test (MN and H5pp tests in subjects given seasonal vaccines and in selected sera from European elderly volunteers in a H5N1 vaccine trial who had detectable pre-vaccination H5N1 MN antibody titers. We found detectable (titer > or = 20 H5N1 neutralizing antibodies in a minority of pre-seasonal vaccine sera and evidence of a serological response to H5N1 in others after seasonal influenza vaccination. There was excellent correlation in the antibody titers between the H5N1 MN and H5pp tests. Similar correlations were found between MN and H5pp in the pre-vaccine sera from the cohort of H5N1 vaccine trial recipients. CONCLUSIONS/SIGNIFICANCE: Heterosubtype neutralizing antibody to H5N1 in healthy volunteers unexposed to H5N1 is mediated by cross-reaction to the H5 haemagglutinin.

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

    DEFF Research Database (Denmark)

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

    2008-01-01

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

  6. Structural analyses at pseudo atomic resolution of Chikungunya virus and antibodies show mechanisms of neutralization.

    Science.gov (United States)

    Sun, Siyang; Xiang, Ye; Akahata, Wataru; Holdaway, Heather; Pal, Pankaj; Zhang, Xinzheng; Diamond, Michael S; Nabel, Gary J; Rossmann, Michael G

    2013-04-02

    A 5.3 Å resolution, cryo-electron microscopy (cryoEM) map of Chikungunya virus-like particles (VLPs) has been interpreted using the previously published crystal structure of the Chikungunya E1-E2 glycoprotein heterodimer. The heterodimer structure was divided into domains to obtain a good fit to the cryoEM density. Differences in the T = 4 quasi-equivalent heterodimer components show their adaptation to different environments. The spikes on the icosahedral 3-fold axes and those in general positions are significantly different, possibly representing different phases during initial generation of fusogenic E1 trimers. CryoEM maps of neutralizing Fab fragments complexed with VLPs have been interpreted using the crystal structures of the Fab fragments and the VLP structure. Based on these analyses the CHK-152 antibody was shown to stabilize the viral surface, hindering the exposure of the fusion-loop, likely neutralizing infection by blocking fusion. The CHK-9, m10 and m242 antibodies surround the receptor-attachment site, probably inhibiting infection by blocking cell attachment. DOI:http://dx.doi.org/10.7554/eLife.00435.001.

  7. Conformation-Dependent High-Affinity Potent Ricin-Neutralizing Monoclonal Antibodies

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    Wei-Gang Hu

    2013-01-01

    Full Text Available 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.

  8. Generation and characterization of monoclonal antibodies specific to Coenzyme A

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    Malanchuk O. M.

    2015-06-01

    Full Text Available Aim. Generation of monoclonal antibodies specific to Coenzyme A. Methods. Hybridoma technique. KLH carrier protein conjugated with CoA was used for immunization. Screening of positive clones was performed with BSA conjugated to CoA. Results. Monoclonal antibody that specifically recognizes CoA and CoA derivatives, but not its precursors ATP and cysteine has been generated. Conclusion. In this study, we describe for the first time the production and characterization of monoclonal antibodies against CoA. The monoclonal antibody 1F10 was shown to recognize specifically CoA in Western blotting, ELISA and immunoprecipitation. These properties make this antiboby a particularly valuable reagent for elucidating CoA function in health and disease.

  9. Pichia pastoris-expressed Dengue 3 Envelope-based Virus-like Particles Elicit Predominantly Domain III-Focused High Titer Neutralizing Antibodies

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    Lav eTripathi

    2015-09-01

    Full Text Available 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 towards domain III of the DENV-3 E VLPs. Significantly, they also lack discernible ADE potential towards heterotypic DENVs. Taken together with the high productivity of the P. pastoris expression system, this approach could potentially pave the way towards developing a DENV E-based, inexpensive, safe and efficacious tetravalent sub-unit vaccine, for use in resource-poor dengue endemic countries.

  10. Vaccine-Elicited Tier 2 HIV-1 Neutralizing Antibodies Bind to Quaternary Epitopes Involving Glycan-Deficient Patches Proximal to the CD4 Binding Site.

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    Ema T Crooks

    2015-05-01

    Full Text Available Eliciting broad tier 2 neutralizing antibodies (nAbs is a major goal of HIV-1 vaccine research. Here we investigated the ability of native, membrane-expressed JR-FL Env trimers to elicit nAbs. Unusually potent nAb titers developed in 2 of 8 rabbits immunized with virus-like particles (VLPs expressing trimers (trimer VLP sera and in 1 of 20 rabbits immunized with DNA expressing native Env trimer, followed by a protein boost (DNA trimer sera. All 3 sera neutralized via quaternary epitopes and exploited natural gaps in the glycan defenses of the second conserved region of JR-FL gp120. Specifically, trimer VLP sera took advantage of the unusual absence of a glycan at residue 197 (present in 98.7% of Envs. Intriguingly, removing the N197 glycan (with no loss of tier 2 phenotype rendered 50% or 16.7% (n = 18 of clade B tier 2 isolates sensitive to the two trimer VLP sera, showing broad neutralization via the surface masked by the N197 glycan. Neutralizing sera targeted epitopes that overlap with the CD4 binding site, consistent with the role of the N197 glycan in a putative "glycan fence" that limits access to this region. A bioinformatics analysis suggested shared features of one of the trimer VLP sera and monoclonal antibody PG9, consistent with its trimer-dependency. The neutralizing DNA trimer serum took advantage of the absence of a glycan at residue 230, also proximal to the CD4 binding site and suggesting an epitope similar to that of monoclonal antibody 8ANC195, albeit lacking tier 2 breadth. Taken together, our data show for the first time that strain-specific holes in the glycan fence can allow the development of tier 2 neutralizing antibodies to native spikes. Moreover, cross-neutralization can occur in the absence of protecting glycan. Overall, our observations provide new insights that may inform the future development of a neutralizing antibody vaccine.

  11. Protection of adenovirus from neutralizing antibody by cationic PEG derivative ionically linked to adenovirus

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    Sun X

    2012-02-01

    Full Text Available Qin Zeng, Jianfeng Han, Dong Zhao, Tao Gong, Zhirong Zhang, Xun SunKey Laboratory of Drug Targeting and Drug Delivery Systems, Ministry of Education, West China School of Pharmacy, Sichuan University, Chengdu, People's Republic of ChinaBackground: The generation of anti-adenovirus neutralizing antibody (NAb in humans severely restricts the utilization of recombinant adenovirus serotype 5 (Ad5 vectors in gene therapy for a wide range of clinical trials. To overcome this limitation, we ionically complexed Ad5 with a newly synthesized copolymer, which we called APC, making an adenovirus shielded from NAb.Methods: APC, a cationic polyethylene glycol derivative, was synthesized via two steps of ring-opening copolymerization of ethylene oxide and allyl glycidyl ether, followed by the addition of 2-mercaptoethylamine. The copolymer or the control PEI-2k was ionically complexed to anionic Ad5 in 5% glucose, and in vitro transduction assays were carried out in coxsackievirus and adenovirus receptor-positive cells (A549 and coxsackievirus and adenovirus receptor-negative cells (B16 and SKOV3. The physical properties and morphology of adenovirus alone or the complexes were investigated respectively by zeta potential, size distribution, and transmission electron microscopy image. Then cytotoxicity of APC was examined using 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide assays. Finally, the ability of APC to protect adenovirus from NAb was evaluated by transfection assays after a neutralizing effect.Results: APC was successfully synthesized and showed a low cytotoxicity. Positively charged Ad5/APC exhibited slightly increased diameter (130.2 ± 0.60 nm than naked Ad5 (115.6 ± 5.46 nm while Ad5/PEI-2k showed severe aggregation (1382 ± 79.9 nm. Ad5/APC achieved a gene transfection level as high as Ad5/PEI-2k in A549 or B16 cells, and significantly higher than Ad5/PEI-2k in SKOV3 cells. Most importantly, after the exposure to the neutralizing

  12. Early preservation of CXCR5+ PD-1+ helper T cells and B cell activation predict the breadth of neutralizing antibody responses in chronic HIV-1 infection.

    Science.gov (United States)

    Cohen, Kristen; Altfeld, Marcus; Alter, Galit; Stamatatos, Leonidas

    2014-11-01

    Much is known about the characteristics of broadly neutralizing antibodies (bNAbs) generated during HIV-1 infection, but little is known about immunological mechanisms responsible for their development in only a minority of those infected by HIV-1. By monitoring longitudinally a cohort of HIV-1-infected subjects, we observed that the preservation of CXCR5(+) CD4(+) T helper cell frequencies and activation status of B cells during the first year of infection correlates with the maximum breadth of plasma neutralizing antibody responses during chronic infection independently of viral load. Although, during the first year of infection, no differences were observed in the abilities of peripheral CXCR5(+) CD4(+) T helper cells to induce antibody secretion by autologous naive B cells, higher frequencies of class-switched antibodies were detected in cocultures of CXCR5(+) CD4(+) T and B cells from the subjects who later developed broadly neutralizing antibody responses than those who did not. Furthermore, B cells from the former subjects had higher expression of AICDA than B cells from the latter subjects, and transcript levels correlated with the frequency of CXCR5(+) CD4(+) T cells. Thus, the early preservation of CXCR5(+) CD4(+) T cells and B cell function are central to the development of bNAbs. Our study provides a possible explanation for their infrequent generation during HIV-1 infection. Broadly neutralizing antibodies are developed by HIV-1-infected subjects, but so far (and despite intensive efforts over the past 3 decades) they have not been elicited by immunization. Understanding how bNAbs are generated during natural HIV-1 infection and why only some HIV-1-infected subjects generate such antibodies will assist our efforts to elicit bNAbs by immunization. CXCR5(+) PD-1(+) CD4(+) T cells are critical for the development of high-affinity antigen-specific antibody responses. In our study, we found that the HIV-1-infected subjects who develop bNAbs have a higher

  13. Characterization of Epitope-Specific Anti-Respiratory Syncytial Virus (Anti-RSV) Antibody Responses after Natural Infection and after Vaccination with Formalin-Inactivated RSV.

    Science.gov (United States)

    Widjaja, Ivy; Wicht, Oliver; Luytjes, Willem; Leenhouts, Kees; Rottier, Peter J M; van Kuppeveld, Frank J M; Haijema, Bert Jan; de Haan, Cornelis A M

    2016-07-01

    Antibodies against the fusion (F) protein of respiratory syncytial virus (RSV) play an important role in the protective immune response to this important respiratory virus. Little is known, however, about antibody levels against multiple F-specific epitopes induced by infection or after vaccination against RSV, while this is important to guide the evaluation of (novel) vaccines. In this study, we analyzed antibody levels against RSV proteins and F-specific epitopes in human sera and in sera of vaccinated and experimentally infected cotton rats and the correlation thereof with virus neutralization. Analysis of human sera revealed substantial diversity in antibody levels against F-, G (attachment)-, and F-specific epitopes between individuals. The highest correlation with virus neutralization was observed for antibodies recognizing prefusion-specific antigenic site Ø. Nevertheless, our results indicate that high levels of antibodies targeting other parts of the F protein can also mediate a potent antiviral antibody response. In agreement, sera of experimentally infected cotton rats contained high neutralizing activity despite lacking antigenic site Ø-specific antibodies. Strikingly, vaccination with formalin-inactivated RSV (FI-RSV) exclusively resulted in the induction of poorly neutralizing antibodies against postfusion-specific antigenic site I, although antigenic sites I, II, and IV were efficiently displayed in FI-RSV. The apparent immunodominance of antigenic site I in FI-RSV likely explains the low levels of neutralizing antibodies upon vaccination and challenge and may play a role in the vaccination-induced enhancement of disease observed with such preparations. RSV is an importance cause of hospitalization of infants. The development of a vaccine against RSV has been hampered by the disastrous results obtained with FI-RSV vaccine preparations in the 1960s that resulted in vaccination-induced enhancement of disease. To get a better understanding of the

  14. Exploiting cross-reactivity to neutralize two different scorpion venoms with one single chain antibody fragment.

    Science.gov (United States)

    Riaño-Umbarila, Lidia; Contreras-Ferrat, Gabriel; Olamendi-Portugal, Timoteo; Morelos-Juárez, Citlalli; Corzo, Gerardo; Possani, Lourival D; Becerril, Baltazar

    2011-02-25

    We report the optimization of a family of human single chain antibody fragments (scFv) for neutralizing two scorpion venoms. The parental scFv 3F recognizes the main toxins of Centruroides noxius Hoffmann (Cn2) and Centruroides suffusus suffusus (Css2), albeit with low affinity. This scFv was subjected to independent processes of directed evolution to improve its recognition toward Cn2 (Riaño-Umbarila, L., Juárez-González, V. R., Olamendi-Portugal, T., Ortíz-León, M., Possani, L. D., and Becerril, B. (2005) FEBS J. 272, 2591-2601) and Css2 (this work). Each evolved variant showed strong cross-reactivity against several toxins, and was capable of neutralizing Cn2 and Css2. Furthermore, each variant neutralized the whole venoms of the above species. As far as we know, this is the first report of antibodies with such characteristics. Maturation processes revealed key residue changes to attain expression, stability, and affinity improvements as compared with the parental scFv. Combination of these changes resulted in the scFv LR, which is capable of rescuing mice from severe envenomation by 3 LD(50) of freshly prepared whole venom of C. noxius (7.5 μg/20 g of mouse) and C. suffusus (26.25 μg/20 g of mouse), with surviving rates between 90 and 100%. Our research is leading to the formulation of an antivenom consisting of a discrete number of human scFvs endowed with strong cross-reactivity and low immunogenicity.

  15. A three monoclonal antibody combination potently neutralizes multiple botulinum neurotoxin serotype F subtypes

    Science.gov (United States)

    Fan, Yongfeng; Garcia-Rodriguez, Consuelo; Lou, Jianlong; Wen, Weihua; Conrad, Fraser; Zhai, Wenwu; Smith, Theresa J.; Smith, Leonard A.

    2017-01-01

    Human botulism is primarily caused by botulinum neurotoxin (BoNT) serotypes A, B and E, with around 1% caused by serotype F (BoNT/F). BoNT/F comprises at least seven different subtypes with the amino acid sequence difference between subtypes as high as 36%. The sequence differences present a significant challenge for generating monoclonal antibodies (mAbs) that can bind, detect and neutralize all BoNT/F subtypes. We used repertoire cloning of immune mouse antibody variable (V) regions and yeast display to generate a panel of 33 lead single chain Fv (scFv) mAbs that bound one or more BoNT/F subtypes with a median equilibrium dissociation constant (KD) of 4.06 × 10−9 M. By diversifying the V-regions of the lead mAbs and selecting for cross reactivity we generated five mAbs that bound each of the seven subtypes. Three scFv binding non-overlapping epitopes were converted to IgG that had KD for the different BoNT/F subtypes ranging from 2.2×10−8 M to 1.47×10−12 pM. An equimolar combination of the mAbs was able to potently neutralize BoNT/F1, F2, F4 and F7 in the mouse neutralization assay. The mAbs have potential utility as diagnostics capable of recognizing the known BoNT/F subtypes and could be developed as antitoxins to prevent and treat type F botulism. PMID:28323873

  16. Exploiting Cross-reactivity to Neutralize Two Different Scorpion Venoms with One Single Chain Antibody Fragment*

    Science.gov (United States)

    Riaño-Umbarila, Lidia; Contreras-Ferrat, Gabriel; Olamendi-Portugal, Timoteo; Morelos-Juárez, Citlalli; Corzo, Gerardo; Possani, Lourival D.; Becerril, Baltazar

    2011-01-01

    We report the optimization of a family of human single chain antibody fragments (scFv) for neutralizing two scorpion venoms. The parental scFv 3F recognizes the main toxins of Centruroides noxius Hoffmann (Cn2) and Centruroides suffusus suffusus (Css2), albeit with low affinity. This scFv was subjected to independent processes of directed evolution to improve its recognition toward Cn2 (Riaño-Umbarila, L., Juárez-González, V. R., Olamendi-Portugal, T., Ortíz-León, M., Possani, L. D., and Becerril, B. (2005) FEBS J. 272, 2591–2601) and Css2 (this work). Each evolved variant showed strong cross-reactivity against several toxins, and was capable of neutralizing Cn2 and Css2. Furthermore, each variant neutralized the whole venoms of the above species. As far as we know, this is the first report of antibodies with such characteristics. Maturation processes revealed key residue changes to attain expression, stability, and affinity improvements as compared with the parental scFv. Combination of these changes resulted in the scFv LR, which is capable of rescuing mice from severe envenomation by 3 LD50 of freshly prepared whole venom of C. noxius (7.5 μg/20 g of mouse) and C. suffusus (26.25 μg/20 g of mouse), with surviving rates between 90 and 100%. Our research is leading to the formulation of an antivenom consisting of a discrete number of human scFvs endowed with strong cross-reactivity and low immunogenicity. PMID:21156801

  17. Dengue virus neutralizing antibody levels associated with protection from infection in thai cluster studies.

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    Darunee Buddhari

    2014-10-01

    Full Text Available 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.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.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 for both dengue epidemiology studies and vaccine development efforts.

  18. Characterization of hemolysin of Moraxella bovis using a hemolysis-neutralizing monoclonal antibody.

    Science.gov (United States)

    Billson, F M; Harbour, C; Michalski, W P; Tennent, J M; Egerton, J R; Hodgson, J L

    2000-06-01

    A concentrated bacterial culture supernatant from the hemolytic Moraxella bovis strain UQV 148NF was used to immunize mice and generate monoclonal antibodies (MAbs). One, MAb G3/D7, neutralized the hemolytic activity of M. bovis and recognized a 94-kDa protein by Western blot analysis in hemolytic M. bovis strains representing each of the different fimbrial serogroups. Exposure of corneal epithelial cells to M. bovis concentrated culture supernatants demonstrated a role for an exotoxin in the pathogenesis of infectious bovine keratoconjunctivitis, while neutralization of hemolytic and cytotoxic activities by MAb G3/D7 implies that these activities are related or have common epitopes. The action of M. bovis hemolysin was further characterized in sheep erythrocyte preparations with a binding step and Ca(2+) required for lysis to proceed, similar to the RTX family of bacterial exotoxins. Neutralization of lytic activity in vitro is evidence for the presence of M. bovis antigens, which may be capable of protecting cattle from the development of infectious bovine keratoconjunctivitis.

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

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    Ebenezer Tumban

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

  20. A camelid single-domain antibody neutralizes botulinum neurotoxin A by blocking host receptor binding

    Energy Technology Data Exchange (ETDEWEB)

    Yao, Guorui; Lam, Kwok-ho; Weisemann, Jasmin; Peng, Lisheng; Krez, Nadja; Perry, Kay; Shoemaker, Charles B.; Dong, Min; Rummel, Andreas; Jin, Rongsheng (BCH); (Cornell); (Tufts CTSI); (UCI); (MHH)

    2017-08-07

    Antibody treatment is currently the only available countermeasure for botulism, a fatal illness caused by flaccid paralysis of muscles due to botulinum neurotoxin (BoNT) intoxication. Among the seven major serotypes of BoNT/A-G, BoNT/A poses the most serious threat to humans because of its high potency and long duration of action. Prior to entering neurons and blocking neurotransmitter release, BoNT/A recognizes motoneurons via a dual-receptor binding process in which it engages both the neuron surface polysialoganglioside (PSG) and synaptic vesicle glycoprotein 2 (SV2). Previously, we identified a potent neutralizing antitoxin against BoNT/A1 termed ciA-C2, derived from a camelid heavy-chain-only antibody (VHH). In this study, we demonstrate that ciA-C2 prevents BoNT/A1 intoxication by inhibiting its binding to neuronal receptor SV2. Furthermore, we determined the crystal structure of ciA-C2 in complex with the receptor-binding domain of BoNT/A1 (HCA1) at 1.68 Å resolution. The structure revealed that ciA-C2 partially occupies the SV2-binding site on HCA1, causing direct interference of HCA1 interaction with both the N-glycan and peptide-moiety of SV2. Interestingly, this neutralization mechanism is similar to that of a monoclonal antibody in clinical trials, despite that ciA-C2 is more than 10-times smaller. Taken together, these results enlighten our understanding of BoNT/A1 interactions with its neuronal receptor, and further demonstrate that inhibiting toxin binding to the host receptor is an efficient countermeasure strategy.

  1. Radiometric cytolysis inhibition assay, a new rapid test for neutralizing antibodies to intact and trypsin-cleaved poliovirus

    Energy Technology Data Exchange (ETDEWEB)

    Hovi, T.; Roivainen, M.

    1989-04-01

    We have developed a new rapid test, the radiometric cytolysis inhibition assay (RACINA), for the determination of neutralizing poliovirus antibodies. HeLa cells prelabeled with /sup 51/Cr, (/sup 3/H)leucine, or, preferentially, with (/sup 3/H)uridine are used as sensitive quantitative indicators of residual infectious virus. Both suspensions and monolayer cultures of the indicator cells can be used. Neutralization of a fraction of a high-titer virus preparation can be scored after the first replication cycle at 8 to 10 h. By lowering the incubation temperature to 30/degree/C, the completion of the cytolysis due to the first replication cycle of poliovirus was delayed beyond 21 h. This makes it possible to use the RACINA, unlike the standard microneutralization assay, for measuring antibodies to trypsin-cleaved polioviruses. The RACINA was found to be as sensitive as and more reproducible than the standard microneutralization assay in the measurement of neutralizing poliovirus antibodies. The RACINA is a rapid and reliable test for neutralizing antibodies and in principle it may be applicable for quantitation of neutralizing antibodies to other cytolytic agents as well.

  2. Cellulose antibody films for highly specific evanescent wave immunosensors

    Science.gov (United States)

    Hartmann, Andreas; Bock, Daniel; Jaworek, Thomas; Kaul, Sepp; Schulze, Matthais; Tebbe, H.; Wegner, Gerhard; Seeger, Stefan

    1996-01-01

    For the production of recognition elements for evanescent wave immunosensors optical waveguides have to be coated with ultrathin stable antibody films. In the present work non amphiphilic alkylated cellulose and copolyglutamate films are tested as monolayer matrices for the antibody immobilization using the Langmuir-Blodgett technique. These films are transferred onto optical waveguides and serve as excellent matrices for the immobilization of antibodies in high density and specificity. In addition to the multi-step immobilization of immunoglobulin G(IgG) on photochemically crosslinked and oxidized polymer films, the direct one-step transfer of mixed antibody-polymer films is performed. Both planar waveguides and optical fibers are suitable substrates for the immobilization. The activity and specificity of immobilized antibodies is controlled by the enzyme-linked immunosorbent assay (ELISA) technique. As a result reduced non-specific interactions between antigens and the substrate surface are observed if cinnamoylbutyether-cellulose is used as the film matrix for the antibody immobilization. Using the evanescent wave senor (EWS) technology immunosensor assays are performed in order to determine both the non-specific adsorption of different coated polymethylmethacrylat (PMMA) fibers and the long-term stability of the antibody films. Specificities of one-step transferred IgG-cellulose films are drastically enhanced compared to IgG-copolyglutamate films. Cellulose IgG films are used in enzymatic sandwich assays using mucine as a clinical relevant antigen that is recognized by the antibodies BM2 and BM7. A mucine calibration measurement is recorded. So far the observed detection limit for mucine is about 8 ng/ml.

  3. Atypical and classical memory B cells produce Plasmodium falciparum neutralizing antibodies

    DEFF Research Database (Denmark)

    Muellenbeck, Matthias F; Ueberheide, Beatrix; Amulic, Borko

    2013-01-01

    Antibodies can protect from Plasmodium falciparum (Pf) infection and clinical malaria disease. However, in the absence of constant reexposure, serum immunoglobulin (Ig) levels rapidly decline and full protection from clinical symptoms is lost, suggesting that B cell memory is functionally impaire...... that constant immune activation rather than impaired memory function leads to the accumulation of AtM in malaria. Understanding the memory B cell response to natural Pf infection may be key to the development of a malaria vaccine that induces long-lived protection.......Antibodies can protect from Plasmodium falciparum (Pf) infection and clinical malaria disease. However, in the absence of constant reexposure, serum immunoglobulin (Ig) levels rapidly decline and full protection from clinical symptoms is lost, suggesting that B cell memory is functionally impaired....... We show at the single cell level that natural Pf infection induces the development of classical memory B cells (CM) and atypical memory B cells (AtM) that produce broadly neutralizing antibodies against blood stage Pf parasites. CM and AtM contribute to anti-Pf serum IgG production, but only AtM show...

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

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    Luisa W. Cheng

    2015-11-01

    Full Text Available 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.

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

    Science.gov (United States)

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

    2013-04-08

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

  6. Detection of specific IgE antibodies in parasite diseases

    Directory of Open Access Journals (Sweden)

    Souza-Atta M.L.B.

    1999-01-01

    Full Text Available Activation of Th1 or Th2 cells is associated with production of specific immunoglobulin isotypes, offering the opportunity to use antibody measurement for evaluation of T cell function. Schistosomiasis and visceral leishmaniasis are diseases associated with Th2 activation. However, an IgE response is not always detected in these patients. In the present study we evaluated specific IgE antibodies to S. mansoni and L. chagasi antigens by ELISA after depletion of serum IgG with protein G immobilized on Sepharose beads or RF-absorbent (purified sheep IgG antibodies anti-human IgG. In schistosomiasis patients, specific IgE to SWAP antigen was demonstrable in only 10 of 21 patients (48% (mean absorbance ± SD = 0.102 ± 0.195 when unabsorbed serum was used. Depletion of IgG with protein G increased the number of specific IgE-positive tests to 13 (62% and the use of RF-absorbent increased the number of positive results to 20 (95% (mean absorbances ± SD = 0.303 ± 0.455 and 0.374 ± 0.477, respectively. Specific IgE anti-L. chagasi antibodies were not detected in unabsorbed serum from visceral leishmaniasis patients. When IgG was depleted with protein G, IgE antibodies were detected in only 3 (11% of 27 patients, and the use of RF-absorbent permitted the detection of this isotype in all 27 visceral leishmaniasis sera tested (mean absorbance ± SD = 0.104 ± 0.03. These data show that the presence of IgG antibodies may prevent the detection of a specific IgE response in these parasite diseases. RF-absorbent, a reagent that blocks IgG-binding sites and also removes rheumatoid factor, was more efficient than protein G for the demonstration of specific IgE antibodies.

  7. Characterization of a Large Panel of Rabbit Monoclonal Antibodies against HIV-1 gp120 and Isolation of Novel Neutralizing Antibodies against the V3 Loop.

    Directory of Open Access Journals (Sweden)

    Yali Qin

    Full Text Available We recently reported the induction of potent, cross-clade neutralizing antibodies (nAbs against Human Immunodeficiency Virus type-1 (HIV-1 in rabbits using gp120 based on an M-group consensus sequence. To better characterize these antibodies, 93 hybridomas were generated, which represent the largest panel of monoclonal antibodies (mAbs ever generated from a vaccinated rabbit. The single most frequently recognized epitope of the isolated mAbs was at the very C-terminal end of the protein (APTKAKRRVVEREKR, followed by the V3 loop. A total of seven anti-V3 loop mAbs were isolated, two of which (10A3 and 10A37 exhibited neutralizing activity. In contrast to 10A3 and most other anti-V3 loop nAbs, 10A37 was atypical with its epitope positioned more towards the C-terminal half of the loop. To our knowledge, 10A37 is the most potent and broadly neutralizing anti-V3 loop mAb induced by vaccination. Interestingly, all seven anti-V3 loop mAbs competed with PGT121, suggesting a possibility that early induction of potent anti-V3 loop antibodies could prevent induction of more broadly neutralizing PGT121-like antibodies that target the conserved base of the V3 loop stem.

  8. Antigenic specificity of serum antibodies in mice fed soy protein

    DEFF Research Database (Denmark)

    Christensen, Hanne Risager; Bruun, S.W.; Frøkiær, Hanne

    2003-01-01

    ingesting soy protein. Methods: Blood from mice fed a soy-containing diet was analyzed using ELISA and immunoblot for antibody reactivity towards various soy protein fractions and pure soy proteins/subunits. Mice bred on a soy-free diet were used as controls. Results: The detectable antigenic specificity...... of the serum antibodies of soy-consuming mice comprised glycinin and beta-conglycinin. Immunoblots with soy protein extract demonstrated antibody reactivity towards both the basic and the acidic chains of glycinin and the beta-conglycinin subunits with an individual response pattern among mice. Moreover......Background: Soybean protein is used in a number of food products but unfortunately is also a common cause of food allergy. Upon ingestion of soy protein, healthy mice like other animals and humans generate a soy-specific antibody response in the absence of signs of illness. Not much is known about...

  9. A novel affinity purification method to isolate peptide specific antibodies

    DEFF Research Database (Denmark)

    Karlsen, Alan E; Lernmark, A; Kofod, Hans

    1990-01-01

    affinity-purify anti-peptide antibodies. To test our system, rabbits were immunized with model peptides representing sequences of the putative rabbit growth hormone receptor and several HLA-DQ beta-chain molecules. Polystyrene plastic beads were coated with peptides. Immune serum was incubated...... with the beads and after a wash step the bound antibodies were eluted in 1 M acetic acid. The eluted material was composed predominantly of intact immunoglobulin as evidenced by the presence of heavy and light chain bands in SDS-PAGE. The eluted antibodies were peptide specific in ELISA and bound only to intact......, antigenic protein in immunoblot analyses. The sequence-specific nature of the eluted antibodies was confirmed since binding to the antigenic proteins could be displaced by the immunizing but not by unrelated peptides....

  10. Neutralizing antibodies in patients with chronic hepatitis C, genotype 1, against a panel of genotype 1 culture viruses: lack of correlation to treatment outcome.

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    Jannie Pedersen

    Full Text Available The correlation of neutralizing antibodies to treatment outcome in patients with chronic hepatitis C virus (HCV infection has not been established. The aim of this study was to determine whether neutralizing antibodies could be used as an outcome predictor in patients with chronic HCV, genotype 1, infection treated with pegylated interferon-α and ribavirin. Thirty-nine patients with chronic hepatitis C, genotype 1a or 1b, with either sustained virologic response (n = 23 or non-sustained virologic response (n = 16 were enrolled. Samples taken prior to treatment were tested for their ability to neutralize 6 different HCV genotype 1 cell culture recombinants (1a: H77/JFH1, TN/JFH1, DH6/JFH1; 1b: J4/JFH1, DH1/JFH1, DH5/JFH1. The results were expressed as the highest dilution yielding 50% neutralization (NAb50-titer. We observed no genotype or subtype specific differences in NAb50-titers between patients with chronic HCV infection with and without sustained virologic response when tested against any of the included culture viruses. However, NAb50-titers varied significantly with a mean reciprocal NAb50-titer of 800 (range: 100-6400 against DH6/JFH1 compared to a mean NAb50-titer of 50 (range: <50-400 against all other included isolates. Subsequent studies demonstrated that the efficient neutralization of DH6/JFH1 could be linked to engineered adaptive mutations in the envelope-2 protein. In analysis of envelope 1 and 2 sequences of HCV, recovered from a subset of patients, we observed no apparent link between relatedness of patient sequences with culture viruses used and the corresponding neutralization results. In conclusion, pre-treatment levels of neutralizing antibodies against HCV genotype 1 isolates could not predict treatment outcome in patients with chronic HCV infection. High neutralization susceptibility of DH6/JFH1 could be correlated with adaptive envelope mutations previously highlighted as important for neutralization. Our

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

    Science.gov (United States)

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

    2016-02-01

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

  12. Cross-reactive antibodies in convalescent SARS patients' sera against the emerging novel human coronavirus EMC (2012) by both immunofluorescent and neutralizing antibody tests.

    Science.gov (United States)

    Chan, Kwok-Hung; Chan, Jasper Fuk-Woo; Tse, Herman; Chen, Honglin; Lau, Candy Choi-Yi; Cai, Jian-Piao; Tsang, Alan Ka-Lun; Xiao, Xincai; To, Kelvin Kai-Wang; Lau, Susanna Kar-Pui; Woo, Patrick Chiu-Yat; Zheng, Bo-Jiang; Wang, Ming; Yuen, Kwok-Yung

    2013-08-01

    A severe acute respiratory syndrome (SARS)-like disease due to a novel betacoronavirus, human coronavirus EMC (HCoV-EMC), has emerged recently. HCoV-EMC is phylogenetically closely related to Tylonycteris-bat-coronavirus-HKU4 and Pipistrellus-bat-coronavirus-HKU5 in Hong Kong. We conducted a seroprevalence study on archived sera from 94 game-food animal handlers at a wild life market, 28 SARS patients, and 152 healthy blood donors in Southern China to assess the zoonotic potential and evidence for intrusion of HCoV-EMC and related viruses into humans. Anti-HCoV-EMC and anti-SARS-CoV antibodies were detected using screening indirect immunofluorescence (IF) and confirmatory neutralizing antibody tests. Two (2.1%) animal handlers had IF antibody titer of ≥ 1:20 against both HCoV-EMC and SARS-CoV with neutralizing antibody titer of SARS patients had significant IF antibody titers with 7/28 (25%) having anti-HCoV-EMC neutralizing antibodies at low titers which significantly correlated with that of HCoV-OC43. Bioinformatics analysis demonstrated a significant B-cell epitope overlapping the heptad repeat-2 region of Spike protein. Virulence of SARS-CoV over other betacoronaviruses may boost cross-reactive neutralizing antibodies against other betacoronaviruses. Convalescent SARS sera may contain cross-reactive antibodies against other betacoronaviruses and confound seroprevalence study for HCoV-EMC. Copyright © 2013 The British Infection Association. Published by Elsevier Ltd. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    Nicola Clementi

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

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

    Science.gov (United States)

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

    2017-01-01

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

  15. Characterization of Antibodies for Grain-Specific Gluten Detection.

    Science.gov (United States)

    Sharma, Girdhari M; Rallabhandi, Prasad; Williams, Kristina M; Pahlavan, Autusa

    2016-03-01

    Gluten ingestion causes immunoglobulin E (IgE)-mediated allergy or celiac disease in sensitive individuals, and a strict gluten-free diet greatly limits food choices. Immunoassays such as enzyme-linked immunosorbent assay (ELISA) are used to quantify gluten to ensure labeling compliance of gluten-free foods. Anti-gluten antibodies may not exhibit equal affinity to gluten from wheat, rye, and barley. Moreover, because wheat gluten is commonly used as a calibrator in ELISA, accurate gluten quantitation from rye and barley contaminated foods may be compromised. Immunoassays utilizing grain-specific antibodies and calibrators may help improve gluten quantitation. In this study, polyclonal antibodies raised against gluten-containing grain-specific peptides were characterized for their immunoreactivity to gluten from different grain sources. Strong immunoreactivity to multiple gluten polypeptides from wheat, rye, and barley was observed in the range 34 to 43 kDa with anti-gliadin, 11 to 15 and 72 to 95 kDa with anti-secalin, and 30 to 43 kDa with anti-hordein peptide antibodies, respectively. Minimal or no cross-reactivity with gluten from other grains was observed among these antibodies. The anti-consensus peptide antibody raised against a repetitive amino acid sequence of proline and glutamine exhibited immunoreactivity to gluten from wheat, rye, barley, and oat. The antibodies exhibited similar immunoreactivity with most of the corresponding grain cultivars by ELISA. The high specificity and minimal cross-reactivity of grain-specific antibodies suggest their potential use in immunoassays for accurate gluten quantitation.

  16. Estimated secondary structure propensities within V1/V2 region of HIV gp120 are an important global antibody neutralization sensitivity determinant.

    Directory of Open Access Journals (Sweden)

    Maxim Totrov

    Full Text Available BACKGROUND: Neutralization sensitivity of HIV-1 virus to antibodies and anti-sera varies greatly between the isolates. Significant role of V1/V2 domain as a global neutralization sensitivity regulator has been suggested. Recent X-ray structures revealed presence of well-defined tertiary structure within this domain but also demonstrated partial disorder and conformational heterogeneity. METHODS: Correlations of neutralization sensitivity with the conformational propensities for beta-strand and alpha-helix formation over the entire folded V1/V2 domain as well as within sliding 5-residue window were investigated. Analysis was based on a set of neutralization data for 106 HIV isolates for which consistent neutralization sensitivity measurements against multiple pools of human immune sera have been previously reported. RESULTS: Significant correlation between beta-sheet formation propensity of the folded segments of V1/V2 domain and neutralization sensitivity was observed. Strongest correlation peaks localized to the beta-strands B and C. Correlation persisted when subsets of HIV isolates belonging to clades B, C and circulating recombinant form BC where analyzed individually or in combinations. CONCLUSIONS: Observed correlations suggest that stability of the beta-sheet structure and/or degree of structural disorder in the V1/V2 domain is an important determinant of the global neutralization sensitivity of HIV-1 virus. While specific mechanism is to yet to be investigated, plausible hypothesis is that less ordered V1/V2s may have stronger masking effect on various neutralizing epitopes, perhaps effectively occupying larger volume and thereby occluding antibody access.

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

    Directory of Open Access Journals (Sweden)

    Wei-Gang Hu

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

  18. Generation of group-specific antibodies against sulfonamides

    NARCIS (Netherlands)

    Cliquet, P.; Cox, E.; Haasnoot, W.; Schacht, E.; Goddeeris, B.M.

    2003-01-01

    To develop a sulfonamide-specific ELISA, different attempts were made to obtain monoclonal antibodies specific for the common structure of sulfonamides. In a first approach, sulfanilamide was linked to albumins using glutaraldehyde or a succinimide ester as cross-linker. A weak immune response or

  19. Dengue serotype cross-reactive, anti-E protein antibodies confound specific immune memory for one year after infection

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    Ying Xiu eToh

    2014-08-01

    Full Text Available Dengue virus has four serotypes and is endemic globally in tropical countries. Neither a specific treatment nor an approved vaccine is available, and correlates of protection are not established. The standard neutralization assay cannot differentiate between serotype-specific and serotype cross-reactive antibodies in patients early after infection, leading to an overestimation of the long-term serotype-specific protection of an antibody response. It is known that the cross-reactive response in patients is temporary but few studies have assessed kinetics and potential changes in serum antibody specificity over time. To better define the specificity of polyclonal antibodies during disease and after recovery, longitudinal samples from patients with primary or secondary DENV-2 infection were collected over a period of one year. We found that serotype cross-reactive antibodies peaked three weeks after infection and subsided within one year. Since secondary patients rapidly produced antibodies specific for the virus envelope (E protein, an E-specific ELISA was superior compared to a virus particle-specific ELISA to identify patients with secondary infections. Dengue infection triggered a massive activation and mobilization of both naïve and memory B cells possibly from lymphoid organs into the blood, providing an explanation for the surge of circulating plasmablasts and the increase in cross-reactive E protein-specific antibodies.

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

    Science.gov (United States)

    Nimmerjahn, Falk

    2015-08-01

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

  1. Mapping of a region of dengue virus type-2 glycoprotein required for binding by a neutralizing monoclonal antibody.

    Science.gov (United States)

    Trirawatanapong, T; Chandran, B; Putnak, R; Padmanabhan, R

    1992-07-15

    Envelope glycoprotein E of flaviviruses is exposed at the surface of the virion, and is responsible for eliciting a neutralizing antibody (Ab) response, as well as protective immunity in the host. In this report, we describe a method for the fine mapping of a linear sequence of the E protein of dengue virus type-2 (DEN-2), recognized by a type-specific and neutralizing monoclonal Ab (mAb), 3H5. First, an Escherichia coli expression vector containing a heat-inducible lambda pL promoter was used to synthesize several truncated, and near-full length E polypeptides. Reactivities of these polypeptides with polyclonal mouse hyperimmune sera, as well as the 3H5 mAb revealed the location of the 3H5-binding site to be within a region of 166 amino acids (aa) between aa 255 and 422. For fine mapping, a series of targeted deletions were made inframe within this region using the polymerase chain reaction (PCR). The hydrophilicity pattern of this region was used as a guide to systematically delete the regions encoding the various groups of surface aa residues within the context of a near-full-length E polypeptide by using PCR. The 3H5-binding site was thus precisely mapped to a region encoding 12 aa (between aa 386 and 397). A synthetic peptide containing this sequence was able to bind to the 3H5 mAb specifically, as shown by enzyme-linked immunosorbent assay. In addition, we show that rabbit Abs raised against the synthetic peptide of 12 aa were able to bind to the authentic E protein, and to neutralize DEN-2 virus in a plaque reduction assay.

  2. A novel variable antibody fragment dimerized by leucine zippers with enhanced neutralizing potency against rabies virus G protein compared to its corresponding single-chain variable antibody fragment.

    Science.gov (United States)

    Li, Zhuang; Cheng, Yue; Xi, Hualong; Gu, Tiejun; Yuan, Ruosen; Chen, Xiaoxu; Jiang, Chunlai; Kong, Wei; Wu, Yongge

    2015-12-01

    Fatal rabies can be prevented effectively by post-exposure prophylactic (PEP) with rabies immunoglobulin (RIG). Single-chain variable fragments (scFv), which are composed of a variable heavy chain (VH) and a variable light chain (VL) connected by a peptide linker, can potentially be used to replace RIG. However, in our previous study, a scFv (scFV57S) specific for the rabies virus (RV) G protein showed a lower neutralizing potency than that of its parent IgG due to lower stability and altered peptide assembly pattern. In monoclonal antibodies, the VH and VL interact non-covalently, while in scFvs the VH is connected covalently with the VL by the artificial linker. In this study, we constructed and expressed two peptides 57VL-JUN-HIS and 57VH-FOS-HA in Escherichia coli. The well-known Fos and Jun leucine zippers were utilized to dimerize VH and VL similarly to the IgG counterpart. The two peptides assembled to form zipFv57S in vitro. Due to the greater similarity in structure with IgG, the zipFv57S protein showed a higher binding ability and affinity resulting in notable improvement of in vitro neutralizing activity over its corresponding scFv. The zipFv57S protein was also found to be more stable and showed similar protective rate as RIG in mice challenged with a lethal dose of RV. Our results not only indicated zipFv57S as an ideal alternative for RIG in PEP but also offered a novel and efficient hetero-dimerization pattern of VH and VL leading to enhanced neutralizing potency.

  3. Elicitation of neutralizing antibodies directed against CD4-induced epitope(s using a CD4 mimetic cross-linked to a HIV-1 envelope glycoprotein.

    Directory of Open Access Journals (Sweden)

    Antu K Dey

    Full Text Available The identification of HIV-1 envelope glycoprotein (Env structures that can generate broadly neutralizing antibodies (BNAbs is pivotal to the development of a successful vaccine against HIV-1 aimed at eliciting effective humoral immune responses. To that end, the production of novel Env structure(s that might induce BNAbs by presentation of conserved epitopes, which are otherwise occluded, is critical. Here, we focus on a structure that stabilizes Env in a conformation representative of its primary (CD4 receptor-bound state, thereby exposing highly conserved "CD4 induced" (CD4i epitope(s known to be important for co-receptor binding and subsequent virus infection. A CD4-mimetic miniprotein, miniCD4 (M64U1-SH, was produced and covalently complexed to recombinant, trimeric gp140 envelope glycoprotein (gp140 using site-specific disulfide linkages. The resulting gp140-miniCD4 (gp140-S-S-M64U1 complex was recognized by CD4i antibodies and the HIV-1 co-receptor, CCR5. The gp140-miniCD4 complex elicited the highest titers of CD4i binding antibodies as well as enhanced neutralizing antibodies against Tier 1 viruses as compared to gp140 protein alone following immunization of rabbits. Neutralization against HIV-2(7312/V434M and additional serum mapping confirm the specific elicitation of antibodies directed to the CD4i epitope(s. These results demonstrate the utility of structure-based approach in improving immunogenic response against specific region, such as the CD4i epitope(s here, and its potential role in vaccine application.

  4. Nef decreases HIV-1 sensitivity to neutralizing antibodies that target the membrane-proximal external region of TMgp41.

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    Rachel P J Lai

    2011-12-01

    Full Text Available Primate lentivirus nef is required for sustained virus replication in vivo and accelerated progression to AIDS. While exploring the mechanism by which Nef increases the infectivity of cell-free virions, we investigated a functional link between Nef and Env. Since we failed to detect an effect of Nef on the quantity of virion-associated Env, we searched for qualitative changes by examining whether Nef alters HIV-1 sensitivity to agents that target distinct features of Env. Nef conferred as much as 50-fold resistance to 2F5 and 4E10, two potent neutralizing monoclonal antibodies (nAbs that target the membrane proximal external region (MPER of TMgp41. In contrast, Nef had no effect on HIV-1 neutralization by MPER-specific nAb Z13e1, by the peptide inhibitor T20, nor by a panel of nAbs and other reagents targeting gp120. Resistance to neutralization by 2F5 and 4E10 was observed with Nef from a diverse range of HIV-1 and SIV isolates, as well as with HIV-1 virions bearing Env from CCR5- and CXCR4-tropic viruses, clade B and C viruses, or primary isolates. Functional analysis of a panel of Nef mutants revealed that this activity requires Nef myristoylation but that it is genetically separable from other Nef functions such as the ability to enhance virus infectivity and to downregulate CD4. Glycosylated-Gag from MoMLV substituted for Nef in conferring resistance to 2F5 and 4E10, indicating that this activity is conserved in a retrovirus that does not encode Nef. Given the reported membrane-dependence of MPER-recognition by 2F5 and 4E10, in contrast to the membrane-independence of Z13e1, the data here is consistent with a model in which Nef alters MPER recognition in the context of the virion membrane. Indeed, Nef and Glycosylated-Gag decreased the efficiency of virion capture by 2F5 and 4E10, but not by other nAbs. These studies demonstrate that Nef protects lentiviruses from one of the most broadly-acting classes of neutralizing antibodies. This newly

  5. Interference of coronavirus infection by expression of immunoglobulin G (IgG) or IgA virus-neutralizing antibodies.

    OpenAIRE

    Castilla, J; Sola, I.; Enjuanes, L

    1997-01-01

    Immunoglobulin gene fragments encoding the variable modules of the heavy and light chains of a transmissible gastroenteritis coronavirus (TGEV)-neutralizing monoclonal antibody (MAb) have been cloned and sequenced. The selected MAb recognizes a highly conserved viral epitope and does not lead to the selection of neutralization escape mutants. The sequences of MAb 6A.C3 kappa and gamma 1 modules were identified as subgroup V and subgroup IIIC, respectively. The chimeric immunoglobulin genes en...

  6. Dengue virus antibody database: Systematically linking serotype-specificity with epitope mapping in dengue virus

    Science.gov (United States)

    Chaudhury, Sidhartha; Gromowski, Gregory D.; Ripoll, Daniel R.; Khavrutskii, Ilja V.; Desai, Valmik; Wallqvist, Anders

    2017-01-01

    Background A majority infections caused by dengue virus (DENV) are asymptomatic, but a higher incidence of severe illness, such as dengue hemorrhagic fever, is associated with secondary infections, suggesting that pre-existing immunity plays a central role in dengue pathogenesis. Primary infections are typically associated with a largely serotype-specific antibody response, while secondary infections show a shift to a broadly cross-reactive antibody response. Methods/Principal findings We hypothesized that the basis for the shift in serotype-specificity between primary and secondary infections can be found in a change in the antibody fine-specificity. To investigate the link between epitope- and serotype-specificity, we assembled the Dengue Virus Antibody Database, an online repository containing over 400 DENV-specific mAbs, each annotated with information on 1) its origin, including the immunogen, host immune history, and selection methods, 2) binding/neutralization data against all four DENV serotypes, and 3) epitope mapping at the domain or residue level to the DENV E protein. We combined epitope mapping and activity information to determine a residue-level index of epitope propensity and cross-reactivity and generated detailed composite epitope maps of primary and secondary antibody responses. We found differing patterns of epitope-specificity between primary and secondary infections, where secondary responses target a distinct subset of epitopes found in the primary response. We found that secondary infections were marked with an enhanced response to cross-reactive epitopes, such as the fusion-loop and E-dimer region, as well as increased cross-reactivity in what are typically more serotype-specific epitope regions, such as the domain I-II interface and domain III. Conclusions/Significance Our results support the theory that pre-existing cross-reactive memory B cells form the basis for the secondary antibody response, resulting in a broadening of the response

  7. Protection against Chlamydia trachomatis infection and upper genital tract pathological changes by vaccine-promoted neutralizing antibodies directed to the VD4 of the major outer membrane protein

    DEFF Research Database (Denmark)

    Olsen, Anja W.; Follmann, Frank; Erneholm, Karin Susanne;

    2015-01-01

    The VD4 region from the Chlamydia trachomatis major outer membrane protein contains important neutralizing B-cell epitopes of relevance for antibody-mediated protection against genital tract infection. We developed a multivalent vaccine construct based on VD4s and their surrounding constant...... segments from serovars D, E, and F. Adjuvanted with cationic liposomes, this construct promoted strong immune responses to serovar-specific epitopes, the conserved LNPTIAG epitope and neutralized serovars D, E, and F. Vaccinated mice were protected against challenge, with protection defined as reduced...... bacterial numbers in vagina and prevention of pathological changes in the upper genital tract. Adoptive transfer of serumand T-cell depletion experiments demonstrated a dominant role for antibodies and CD4+ T cells in the protective immune response. Integrating a multivalent VD4 construct into the sequence...

  8. Reporter gene assay for the quantification of the activity and neutralizing antibody response to TNFα antagonists

    DEFF Research Database (Denmark)

    Lallemand, Christophe; Kavrochorianou, Nadia; Steenholdt, Casper;

    2011-01-01

    relative to Renilla luciferase expression. Thus, results are independent of cell number or differences in cell viability, resulting in intra and inter assay coefficients of variation of 10% or less. Normalization of results relative to the expression of an internal standard also provides a means......A cell-based assay has been developed for the quantification of the activity of TNFa antagonists based on human erythroleukemic K562 cells transfected with a NF¿B regulated firefly luciferase reporter-gene construct. Both drug activity and anti-drug neutralizing antibodies can be quantified...... with a high degree of precision within 2h, and without interference from cytokines and other factors known to activate NF¿B. The assay cells also contain the Renilla luciferase reporter gene under the control of a constitutive promoter that allows TNFa-induced firefly luciferase activity to be normalized...

  9. Reporter gene assay for the quantification of the activity and neutralizing antibody response to TNFα antagonists

    DEFF Research Database (Denmark)

    Lallemand, Christophe; Kavrochorianou, Nadia; Steenholdt, Casper;

    2011-01-01

    relative to Renilla luciferase expression. Thus, results are independent of cell number or differences in cell viability, resulting in intra and inter assay coefficients of variation of 10% or less. Normalization of results relative to the expression of an internal standard also provides a means......A cell-based assay has been developed for the quantification of the activity of TNFα antagonists based on human erythroleukemic K562 cells transfected with a NFκB regulated firefly luciferase reporter-gene construct. Both drug activity and anti-drug neutralizing antibodies can be quantified...... with a high degree of precision within 2h, and without interference from cytokines and other factors known to activate NFκB. The assay cells also contain the Renilla luciferase reporter gene under the control of a constitutive promoter that allows TNFα-induced firefly luciferase activity to be normalized...

  10. Is the treatment effect of IFN-beta restored after the disappearance of neutralizing antibodies?

    DEFF Research Database (Denmark)

    Sorensen, P S; Koch-Henriksen, Nils; Flachs, Esben Meulengracht

    2008-01-01

    OBJECTIVE: To establish whether multiple sclerosis (MS) patients, who have lost the therapeutic effect of interferon-beta (IFN-beta) owing to neutralizing antibodies (NAbs) and subsequently revert from a NAb-positive to a NAb-negative state under continued IFN-beta-1b therapy, regain clinical...... effect after reversion. BACKGROUND: Several studies have shown that a significant proportion of patients treated with IFN-beta develop NAbs that hamper or abolish the therapeutic effect of IFN-beta. However, some patients, who become NAb-positive under treatment with IFN-beta-1b, may revert to a NAb......-positive. A patient was defined as NAb-positive after two consecutive blood tests separated by at least 6 months. Reversion to a NAb-negative state required at least two consecutive negative tests. To allow for the confounding effect of time we employed a mixed Poisson model. RESULTS: Patients who had been NAb...

  11. DNA vaccine molecular adjuvants SP-D-BAFF and SP-D-APRIL enhance anti-gp120 immune response and increase HIV-1 neutralizing antibody titers.

    Science.gov (United States)

    Gupta, Sachin; Clark, Emily S; Termini, James M; Boucher, Justin; Kanagavelu, Saravana; LeBranche, Celia C; Abraham, Sakhi; Montefiori, David C; Khan, Wasif N; Stone, Geoffrey W

    2015-04-01

    Broadly neutralizing antibodies (bNAbs) specific for conserved epitopes on the HIV-1 envelope (Env) are believed to be essential for protection against multiple HIV-1 clades. However, vaccines capable of stimulating the production of bNAbs remain a major challenge. Given that polyreactivity and autoreactivity are considered important characteristics of anti-HIV bNAbs, we designed an HIV vaccine incorporating the molecular adjuvants BAFF (B cell activating factor) and APRIL (a proliferation-inducing ligand) with the potential to facilitate the maturation of polyreactive and autoreactive B cells as well as to enhance the affinity and/or avidity of Env-specific antibodies. We designed recombinant DNA plasmids encoding soluble multitrimers of BAFF and APRIL using surfactant protein D as a scaffold, and we vaccinated mice with these molecular adjuvants using DNA and DNA-protein vaccination strategies. We found that immunization of mice with a DNA vaccine encoding BAFF or APRIL multitrimers, together with interleukin 12 (IL-12) and membrane-bound HIV-1 Env gp140, induced neutralizing antibodies against tier 1 and tier 2 (vaccine strain) viruses. The APRIL-containing vaccine was particularly effective at generating tier 2 neutralizing antibodies following a protein boost. These BAFF and APRIL effects coincided with an enhanced germinal center (GC) reaction, increased anti-gp120 antibody-secreting cells, and increased anti-gp120 functional avidity. Notably, BAFF and APRIL did not cause indiscriminate B cell expansion or an increase in total IgG. We propose that BAFF and APRIL multitrimers are promising molecular adjuvants for vaccines designed to induce bNAbs against HIV-1. Recent identification of antibodies that neutralize most HIV-1 strains has revived hopes and efforts to create novel vaccines that can effectively stimulate HIV-1 neutralizing antibodies. However, the multiple immune evasion properties of HIV have hampered these efforts. These include the instability of

  12. Structure of a Major Antigenic Site on the Respiratory Syncytial Virus Fusion Glycoprotein in Complex with Neutralizing Antibody 101F

    Energy Technology Data Exchange (ETDEWEB)

    McLellan, Jason S.; Chen, Man; Chang, Jung-San; Yang, Yongping; Kim, Albert; Graham, Barney S.; Kwong, Peter D. (NIH)

    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.

  13. HIV-1 superinfection in women broadens and strengthens the neutralizing antibody response.

    Directory of Open Access Journals (Sweden)

    Valerie Cortez

    Full Text Available Identifying naturally-occurring neutralizing antibodies (NAb that are cross-reactive against all global subtypes of HIV-1 is an important step toward the development of a vaccine. Establishing the host and viral determinants for eliciting such broadly NAbs is also critical for immunogen design. NAb breadth has previously been shown to be positively associated with viral diversity. Therefore, we hypothesized that superinfected individuals develop a broad NAb response as a result of increased antigenic stimulation by two distinct viruses. To test this hypothesis, plasma samples from 12 superinfected women each assigned to three singly infected women were tested against a panel of eight viruses representing four different HIV-1 subtypes at matched time points post-superinfection (~5 years post-initial infection. Here we show superinfected individuals develop significantly broader NAb responses post-superinfection when compared to singly infected individuals (RR = 1.68, CI: 1.23-2.30, p = 0.001. This was true even after controlling for NAb breadth developed prior to superinfection, contemporaneous CD4+ T cell count and viral load. Similarly, both unadjusted and adjusted analyses showed significantly greater potency in superinfected cases compared to controls. Notably, two superinfected individuals were able to neutralize variants from four different subtypes at plasma dilutions >1∶300, suggesting that their NAbs exhibit elite activity. Cross-subtype breadth was detected within a year of superinfection in both of these individuals, which was within 1.5 years of their initial infection. These data suggest that sequential infections lead to augmentation of the NAb response, a process that may provide insight into potential mechanisms that contribute to the development of antibody breadth. Therefore, a successful vaccination strategy that mimics superinfection may lead to the development of broad NAbs in immunized individuals.

  14. Applying antibody-sensitive hypervariable region 1-deleted hepatitis C virus to the study of escape pathways of neutralizing human monoclonal antibody AR5A

    DEFF Research Database (Denmark)

    Velazquez-Moctezuma, Rodrigo; Law, Mansun; Bukh, Jens

    2017-01-01

    Hepatitis C virus (HCV) is a major cause of end-stage liver diseases. With 3–4 million new HCV infections yearly, a vaccine is urgently needed. A better understanding of virus escape from neutralizing antibodies and their corresponding epitopes are important for this effort. However, for viral is...

  15. Development of an EGFRvIII specific recombinant antibody

    Directory of Open Access Journals (Sweden)

    Li Gordon

    2010-10-01

    Full Text Available Abstract Background EGF receptor variant III (EGFRvIII is the most common variant of the EGF receptor observed in human tumors. It results from the in frame deletion of exons 2-7 and the generation of a novel glycine residue at the junction of exons 1 and 8. This novel juxtaposition of amino acids within the extra-cellular domain of the EGF receptor creates a tumor specific and immunogenic epitope. EGFRvIII expression has been seen in many tumor types including glioblastoma multiforme (GBM, breast adenocarcinoma, non-small cell lung carcinoma, ovarian adenocarcinoma and prostate cancer, but has been rarely observed in normal tissue. Because this variant is tumor specific and highly immunogenic, it can be used for both a diagnostic marker as well as a target for immunotherapy. Unfortunately many of the monoclonal and polyclonal antibodies directed against EGFRvIII have cross reactivity to wild type EGFR or other non-specific proteins. Furthermore, a monoclonal antibody to EGFRvIII is not readily available to the scientific community. Results In this study, we have developed a recombinant antibody that is specific for EGFRvIII, has little cross reactivity for the wild type receptor, and which can be easily produced. We initially designed a recombinant antibody with two anti-EGFRvIII single chain Fv's linked together and a human IgG1 Fc component. To enhance the specificity of this antibody for EGFRvIII, we mutated tyrosine H59 of the CDRH2 domain and tyrosine H105 of the CDRH3 domain to phenylalanine for both the anti-EGFRvIII sequence inserts. This mutated recombinant antibody, called RAbDMvIII, specifically detects EGFRvIII expression in EGFRvIII expressing cell lines as well as in EGFRvIII expressing GBM primary tissue by western blot, immuno