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Humbert, Michael

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Humbert

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Michael

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Humbert, Michael
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    Live-virus exposure of vaccine-protected macaques alters the anti-HIV-1 antibody repertoire in the absence of viremia
    (BioMed Central, 2013) Bachler, Barbara C; Humbert, Michael; Lakhashe, Samir K; Rasmussen, Robert A; Ruprecht, Ruth Margrit
    Background: We addressed the question whether live-virus challenges could alter vaccine-induced antibody (Ab) responses in vaccinated rhesus macaques (RMs) that completely resisted repeated exposures to R5-tropic simian-human immunodeficiency viruses encoding heterologous HIV clade C envelopes (SHIV-Cs). Results: We examined the Ab responses in aviremic RMs that had been immunized with a multi-component protein vaccine (multimeric HIV-1 gp160, HIV-1 Tat and SIV Gag-Pol particles) and compared anti-Env plasma Ab titers before and after repeated live-virus exposures. Although no viremia was ever detected in these animals, they showed significant increases in anti-gp140 Ab titers after they had encountered live SHIVs. When we investigated the dynamics of anti-Env Ab titers during the immunization and challenge phases further, we detected the expected, vaccine-induced increases of Ab responses about two weeks after the last protein immunization. Remarkably, these titers kept rising during the repeated virus challenges, although no viremia resulted. In contrast, in vaccinated RMs that were not exposed to virus, anti-gp140 Ab titers declined after the peak seen two weeks after the last immunization. These data suggest boosting of pre-existing, vaccine-induced Ab responses as a consequence of repeated live-virus exposures. Next, we screened polyclonal plasma samples from two of the completely protected vaccinees by peptide phage display and designed a strategy that selects for recombinant phages recognized only by Abs present after – but not before – any SHIV challenge. With this “subtractive biopanning” approach, we isolated V3 mimotopes that were only recognized after the animals had been exposed to live virus. By detailed epitope mapping of such anti-V3 Ab responses, we showed that the challenges not only boosted pre-existing binding and neutralizing Ab titers, but also induced Abs targeting neo-antigens presented by the heterologous challenge virus. Conclusions: Anti-Env Ab responses induced by recombinant protein vaccination were altered by the multiple, live SHIV challenges in vaccinees that had no detectable viral loads. These data may have implications for the interpretation of “vaccine only” responses in clinical vaccine trials.
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    Isolation of Monoclonal Antibodies with Predetermined Conformational Epitope Specificity
    (Public Library of Science, 2012) Sholukh, Anton M.; Mukhtar, Muhammad Mahmood; Humbert, Michael; Essono, Sosthène S.; Watkins, Jennifer D.; Vyas, Hemant Kumar; Shanmuganathan, Vivekanandan; Hemashettar, Girish; Kahn, Maria; Hu, Shiu-Lok; Montefiori, David C.; Polonis, Victoria R.; Schur, Peter; Ruprecht, Ruth Margrit
    Existing technologies allow isolating antigen-specific monoclonal antibodies (mAbs) from B cells. We devised a direct approach to isolate mAbs with predetermined conformational epitope specificity, using epitope mimetics (mimotopes) that reflect the three-dimensional structure of given antigen subdomains. We performed differential biopanning using bacteriophages encoding random peptide libraries and polyclonal antibodies (Abs) that had been affinity-purified with either native or denatured antigen. This strategy yielded conformational mimotopes. We then generated mimotope-fluorescent protein fusions, which were used as baits to isolate single memory B cells from rhesus monkeys (RMs). To amplify RM immunoglobulin variable regions, we developed RM-specific PCR primers and generated chimeric simian-human mAbs with predicted epitope specificity. We established proof-of-concept of our strategy by isolating mAbs targeting the conformational V3 loop crown of HIV Env; the new mAbs cross-neutralized viruses of different clades. The novel technology allows isolating mAbs from RMs or other hosts given experimental immunogens or infectious agents.
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    Identification of Mimotopes with Diagnostic Potential for Trypanosoma brucei gambiense Variant Surface Glycoproteins Using Human Antibody Fractions
    (Public Library of Science, 2012) Van Nieuwenhove, Liesbeth; Büscher, Philippe; Balharbi, Fatima; Humbert, Michael; Dieltjens, Tessa; Guisez, Yves; Lejon, Veerle
    Background: At present, screening of the population at risk for gambiense human African trypanosomiasis (HAT) is based on detection of antibodies against native variant surface glycoproteins (VSGs) of Trypanosoma brucei (T.b.) gambiense. Drawbacks of these native VSGs include culture of infective T.b. gambiense trypanosomes in laboratory rodents, necessary for production, and the exposure of non-specific epitopes that may cause cross-reactions. We therefore aimed at identifying peptides that mimic epitopes, hence called “mimotopes,” specific to T.b. gambiense VSGs and that may replace the native proteins in antibody detection tests. Methodology/Principal Findings A Ph.D.-12 peptide phage display library was screened with polyclonal antibodies from patient sera, previously affinity purified on VSG LiTat 1.3 or LiTat 1.5. The peptide sequences were derived from the DNA sequence of the selected phages and synthesised as biotinylated peptides. Respectively, eighteen and twenty different mimotopes were identified for VSG LiTat 1.3 and LiTat 1.5, of which six and five were retained for assessment of their diagnostic performance. Based on alignment of the peptide sequences on the original protein sequence of VSG LiTat 1.3 and 1.5, three additional peptides were synthesised. We evaluated the diagnostic performance of the synthetic peptides in indirect ELISA with 102 sera from HAT patients and 102 endemic negative controls. All mimotopes had areas under the curve (AUCs) of ≥0.85, indicating their diagnostic potential. One peptide corresponding to the VSG LiTat 1.3 protein sequence also had an AUC of ≥0.85, while the peptide based on the sequence of VSG LiTat 1.5 had an AUC of only 0.79. Conclusions/Significance: We delivered the proof of principle that mimotopes for T.b. gambiense VSGs, with diagnostic potential, can be selected by phage display using polyclonal human antibodies.
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    Inducing Cross-Clade Neutralizing Antibodies against HIV-1 by Immunofocusing
    (Public Library of Science, 2008) Humbert, Michael; Rasmussen, Robert; Ong, Helena; Kaiser, Fabian M. P.; Hu, Shiu-Lok; Ruprecht, Ruth Margrit
    Background: Although vaccines are important in preventing viral infections by inducing neutralizing antibodies (nAbs), HIV-1 has proven to be a difficult target and escapes humoral immunity through various mechanisms. We sought to test whether HIV-1 Env mimics may serve as immunogens. Methodology/Principal Findings: Using random peptide phage display libraries, we identified the epitopes recognized by polyclonal antibodies of a rhesus monkey that had developed high-titer, broadly reactive nAbs after infection with a simian-human immunodeficiency virus (SHIV) encoding env of a recently transmitted HIV-1 clade C (HIV-C). Phage peptide inserts were analyzed for conformational and linear homology using computational analysis; some peptides mimicked various domains of the original HIV-C Env, such as conformational V3 loop epitopes and the conserved linear region of the gp120 C-terminus. Next, we devised a novel prime/boost strategy to test the immunogenicity of such phage-displayed peptides and primed mice only once with HIV-C gp160 DNA followed by boosting with mixtures of recombinant phages. Conclusions/Significance: This strategy, which was designed to focus the immune system on a few Env epitopes (immunofocusing), not only induced HIV-C gp160 binding antibodies and cross-clade nAbs, but also linked a conserved HIV Env region for the first time to the induction of nAbs: the C-terminus of gp120. The identification of conserved antigen mimics may lead to novel immunogens capable of inducing broadly reactive nAbs.
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    SHIV-1157i and Passaged Progeny Viruses Encoding R5 HIV-1 Clade C Env Cause AIDS in Rhesus Monkeys
    (BioMed Central, 2008) Song, Ruijiang; Ong, Helena; Sharma, Prachi; Chenine, Agnès L; Kramer, Victor G; Xu, Weidong; Else, James G; Novembre, Francis J; Strobert, Elizabeth; O'Neil, Shawn P; Humbert, Michael; Rasmussen, Robert; Siddappa, Nagadenahalli B.; Ruprecht, Ruth Margrit
    Background: Infection of nonhuman primates with simian immunodeficiency virus (SIV) or chimeric simian-human immunodeficiency virus (SHIV) strains is widely used to study lentiviral pathogenesis, antiviral immunity and the efficacy of AIDS vaccine candidates. SHIV challenges allow assessment of anti-HIV-1 envelope responses in primates. As such, SHIVs should mimic natural HIV-1 infection in humans and, to address the pandemic, encode HIV-1 Env components representing major viral subtypes worldwide. Results: We have developed a panel of clade C R5-tropic SHIVs based upon env of a Zambian pediatric isolate of HIV-1 clade C, the world's most prevalent HIV-1 subtype. The parental infectious proviral clone, SHIV-1157i, was rapidly passaged through five rhesus monkeys. After AIDS developed in the first animal at week 123 post-inoculation, infected blood was infused into a sixth monkey. Virus reisolated at this late stage was still exclusively R5 tropic and mucosally transmissible. Here we describe the long-term follow-up of this initial cohort of six monkeys. Two have remained non-progressors, whereas the other four gradually progressed to AIDS within 123–270 weeks post-exposure. Two progressors succumbed to opportunistic infections, including a case of SV40 encephalitis. Conclusion: These data document the disease progression induced by the first mucosally transmissible, pathogenic R5 non-clade B SHIV and suggest that SHIV-1157i-derived viruses, including the late-stage, highly replication-competent SHIV-1157ipd3N4 previously described (Song et al., 2006), display biological characteristics that mirror those of HIV-1 clade C and support their expanded use for AIDS vaccine studies in nonhuman primates.
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    An Anti-HIV-1 V3 Loop Antibody Fully Protects Cross-Clade and Elicits T-Cell Immunity in Macaques Mucosally Challenged with an R5 Clade C SHIV
    (Public Library of Science, 2011) Hemashettar, Girish; Wang, Wendy; Novembre, Francis J.; Villinger, Francois; Ibegbu, Chris; Patel, Kalpana; Corti, Davide; Agatic, Gloria; Vanzetta, Fabrizia; Bianchi, Siro; Heeney, Jonathan L.; Sallusto, Federica; Lanzavecchia, Antonio; Watkins, Jennifer D.; Siddappa, Nagadenahalli B.; Lakhashe, Samir; Humbert, Michael; Sholukh, Anton M.; Wong, Yin Ling; Yoon, John C.; Ruprecht, Ruth Margrit
    Neutralizing antibodies have been shown to protect macaques against SHIV challenge. However, genetically diverse HIV-1 clades have evolved, and a key question left unanswered is whether neutralizing antibodies can confer cross-clade protection in vivo. The novel human monoclonal antibody HGN194 was isolated from an individual infected with an HIV-1 clade AG recombinant circulating recombinant form (CRF). HGN194 targets an epitope in the third hypervariable loop (V3) of HIV-1 gp120 and neutralizes a range of relatively neutralization-sensitive and resistant viruses. We evaluated the potential of HGN194 to protect infant rhesus monkeys against a SHIV encoding a primary CCR5-tropic HIV-1 clade C envelope. After high-dose mucosal challenge, all untreated controls became highly viremic while all HGN194-treated animals (50 mg/kg) were completely protected. When HGN194 was given at 1 mg/kg, one out of two monkeys remained aviremic, whereas the other had delayed, lower peak viremia. Interestingly, all protected monkeys given high-dose HGN194 developed Gag-specific proliferative responses of both CD4+ and CD8+ T cells. To test whether generation of the latter involved cryptic infection, we ablated CD8+ cells after HGN194 clearance. No viremia was detected in any protected monkeys, thus ruling out virus reservoirs. Thus, induction of CD8 T-cell immunity may have resulted from transient “Hit and Run” infection or cross priming via Ag-Ab-mediated cross-presentation. Together, our data identified the HGN194 epitope as protective and provide proof-of-concept that this anti-V3 loop mAb can prevent infection with sterilizing immunity after challenge with virus of a different clade, implying that V3 is a potential vaccine target.