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Generation and Characterization of Monoclonal Antibodies against a Cyclic Variant of Hepatitis C Virus E2 Epitope 412-422.

ABSTRACT: UNLABELLED:The hepatitis C virus (HCV) E2 envelope glycoprotein is crucial for virus entry into hepatocytes. A conserved region of E2 encompassing amino acids 412 to 423 (epitope I) and containing Trp420, a residue critical for virus entry, is recognized by several broadly neutralizing antibodies. Peptides embodying this epitope I sequence adopt a ?-hairpin conformation when bound to neutralizing monoclonal antibodies (MAbs) AP33 and HCV1. We therefore generated new mouse MAbs that were able to bind to a cyclic peptide containing E2 residues 412 to 422 (C-epitope I) but not to the linear counterpart. These MAbs bound to purified E2 with affinities of about 50 nM, but they were unable to neutralize virus infection. Structural analysis of the complex between C-epitope I and one of our MAbs (C2) showed that the Trp420 side chain is largely buried in the combining site and that the Asn417 side chain, which is glycosylated in E2 and solvent exposed in other complexes, is slightly buried upon C2 binding. Also, the orientation of the cyclic peptide in the antibody-combining site is rotated by 180° compared to the orientations of the other complexes. All these structural features, however, do not explain the lack of neutralization activity. This is instead ascribed to the high degree of selectivity of the new MAbs for the cyclic epitope and to their inability to interact with the epitope in more flexible and extended conformations, which recent data suggest play a role in the mechanisms of neutralization escape. IMPORTANCE:Hepatitis C virus (HCV) remains a major health care burden, affecting almost 3% of the global population. The conserved epitope comprising residues 412 to 423 of the viral E2 glycoprotein is a valid vaccine candidate because antibodies recognizing this region exhibit potent neutralizing activity. This epitope adopts a ?-hairpin conformation when bound to neutralizing MAbs. We explored the potential of cyclic peptides mimicking this structure to elicit anti-HCV antibodies. MAbs that specifically recognize a cyclic variant of the epitope bind to soluble E2 with a lower affinity than other blocking antibodies and do not neutralize virus. The structure of the complex between one such MAb and the cyclic epitope, together with new structural data showing the linear peptide bound to neutralizing MAbs in extended conformations, suggests that the epitope displays a conformational flexibility that contributes to neutralization escape. Such features can be of major importance for the design of epitope-based anti-HCV vaccines.

SUBMITTER: Sandomenico A

PROVIDER: S-EPMC4794675 | biostudies-literature | 2016 Jan

REPOSITORIES: biostudies-literature

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Generation and Characterization of Monoclonal Antibodies against a Cyclic Variant of Hepatitis C Virus E2 Epitope 412-422.

Sandomenico Annamaria A Leonardi Antonio A Berisio Rita R Sanguigno Luca L Focà Giuseppina G Focà Annalia A Ruggiero Alessia A Doti Nunzianna N Muscariello Livio L Barone Daniela D Farina Claudio C Owsianka Ania A Vitagliano Luigi L Patel Arvind H AH Ruvo Menotti M

Journal of virology 20160127 7

<h4>Unlabelled</h4>The hepatitis C virus (HCV) E2 envelope glycoprotein is crucial for virus entry into hepatocytes. A conserved region of E2 encompassing amino acids 412 to 423 (epitope I) and containing Trp420, a residue critical for virus entry, is recognized by several broadly neutralizing antibodies. Peptides embodying this epitope I sequence adopt a β-hairpin conformation when bound to neutralizing monoclonal antibodies (MAbs) AP33 and HCV1. We therefore generated new mouse MAbs that were ...[more]

PMID: 26819303

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Project description:UnlabelledHepatitis C virus (HCV) neutralization occurring at the E2 region 412-426 (EP-I) could be enhanced when antibodies directed specifically to the E2 region 434-446 (EP-II) were removed from serum samples of persistently infected patients and vaccinated chimpanzees, a phenomenon of so-called antibody interference. Here, we show that this type of interference can be observed in individuals after immunization with recombinant E1E2 proteins. One hundred twelve blinded serum samples from a phase I, placebo-controlled, dose escalation trial using recombinant HCV E1E2 with MF59C.1 adjuvant in healthy HCV-negative adults were tested in enzyme-linked immunosorbent assay for binding reactivity to peptides representing the E2 regions 412-426 (EP-I) and 434-446 (EP-II). All samples were subsequently tested for neutralizing activity using cell-culture HCV 1a(H77)/2a chimera, HCV pseudotype particles (HCVpp) H77, and HCVpp HCV-1 after treatment to remove EP-II-specific antibodies or mock treatment with a control peptide. Among the 112 serum samples, we found 22 double positive (EP-I and EP-II), 6 EP-II positive only, 14 EP-I positive only, and 70 double negative. Depleting EP-II antibodies from double-positive serum samples increased 50% inhibitory dose (ID50) neutralizing antibody titers (up to 4.9-fold) in up to 72% of samples (P ≤ 0.0005), contrasting with ID50 neutralization titer increases in 2 of 70 double-negative samples (2.9%; P > 0.5). In addition, EP-I-specific antibody levels in serum samples showed a significant correlation with ID50 neutralization titers when EP-II antibodies were removed (P < 0.0003).ConclusionThese data show that antibodies to the region 434-446 are induced during immunization of individuals with recombinant E1E2 proteins, and that these antibodies can mask effective neutralizing activity from EP-I-specific antibodies. Elicitation of EP-II-specific antibodies with interfering capacity should be avoided in producing an effective cross-neutralizing vaccine aimed at the HCV envelope proteins.

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

Dataset Information

Generation and Characterization of Monoclonal Antibodies against a Cyclic Variant of Hepatitis C Virus E2 Epitope 412-422.

Publications

Generation and Characterization of Monoclonal Antibodies against a Cyclic Variant of Hepatitis C Virus E2 Epitope 412-422.

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