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

Abstract

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 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 β-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.

Structure-Based Design of Hepatitis C Virus Vaccines That Elicit Neutralizing Antibody Responses to a Conserved Epitope

“…Characterization of MAbs induced by C1 or C2 vaccination, via alanine scanning mutagenesis, affinity measurements, or crystallography, may yield information regarding the induced repertoire and possible molecular details underlying the observed difference between epitope I-specific binding and neutralization, for instance, MAb binding to portions of the epitope that are inaccessible in the context of E2 or the virion. Such characterization of C1 and C2-elicited MAbs, which was utilized recently for another cyclic epitope I design as noted above (45), could indicate the basis of neutralization in immunized sera from our constructs and inform second-generation designs of one or both of those antigens. Additionally, as glycosylation or mutation at N415 is associated with escape from HCV1 (25,51) and other MAbs (9), addition of a glycan at N415, in addition to N417, may induce antibodies capable of targeting this epitope with a binding footprint that does not include N415 while maintaining binding to the conserved residues L413 and W420.…”

“…The designs resulting from the metaserver methodology described in that study were larger and distinct from the cyclic designs we produced and tested in vivo. More recently, a study was reported featuring a cyclic epitope I construct with a different mode of disulfide cyclization than C1 or C2, including epitope residues 412 to 422 rather than 412 to 423 (C1) or 409 to 425 (C2) and no glycan at N417 (45). After mouse immunization with this antigen, several elicited MAbs were tested for epitope binding and neutralization, but no measurable neutralization was observed.…”

Structural flexibility at a major conserved antibody target on hepatitis C virus E2 antigen

“…Such alternate conformations appear to be critical for virus neutralization. A recent study by Sandomenico et al (52) showed that antibodies raised against cyclized AS412 peptides that adopt an alternative conformation were not neutralizing. From the present study, the CD81bs front layer resembles a highly flexible loop despite being held by two disulfide bonds and despite high sequence conservation, which is generally not associated with flexible loops in viral proteins.…”

Hepatitis C Virus Escape Studies of Human Antibody AR3A Reveal a High Barrier to Resistance and Novel Insights on Viral Antibody Evasion Mechanisms

“…Rational vaccine design, in which select epitopes are emphasized, has so far had limited success for HCV. One approach relies on the induction of NAbs against extended or cyclic versions of linear epitopes (59)(60)(61). These approaches cannot readily be adapted to AR3A, as it has a conformational, nonlinear epitope.…”