Human Monoclonal Antibodies to a Novel Cluster of Conformational Epitopes on HCV E2 with Resistance to Neutralization Escape in a Genotype 2a Isolate

Abstract

The majority of broadly neutralizing antibodies to hepatitis C virus (HCV) are against conformational epitopes on the E2 glycoprotein. Many of them recognize overlapping epitopes in a cluster, designated as antigenic domain B, that contains residues G530 and D535. To gain information on other regions that will be relevant for vaccine design, we employed yeast surface display of antibodies that bound to genotype 1a H77C E2 mutant proteins containing a substitution either at Y632A (to avoid selecting non-neutralizing antibodies) or D535A. A panel of nine human monoclonal antibodies (HMAbs) was isolated and designated as HC-84-related antibodies. Each HMAb neutralized cell culture infectious HCV (HCVcc) with genotypes 1–6 envelope proteins with varying profiles, and each inhibited E2 binding to the viral receptor CD81. Five of these antibodies neutralized representative genotypes 1–6 HCVcc. Epitope mapping identified a cluster of overlapping epitopes that included nine contact residues in two E2 regions encompassing aa418–446 and aa611–616. Effect on virus entry was measured using H77C HCV retroviral pseudoparticles, HCVpp, bearing an alanine substitution at each of the contact residues. Seven of ten mutant HCVpp showed over 90% reduction compared to wild-type HCVpp and two others showed approximately 80% reduction. Interestingly, four of these antibodies bound to a linear E2 synthetic peptide encompassing aa434–446. This region on E2 has been proposed to elicit non-neutralizing antibodies in humans that interfere with neutralizing antibodies directed at an adjacent E2 region from aa410–425. The isolation of four HC-84 HMAbs binding to the peptide, aa434–446, proves that some antibodies to this region are to highly conserved epitopes mediating broad virus neutralization. Indeed, when HCVcc were passaged in the presence of each of these antibodies, virus escape was not observed. Thus, the cluster of HC-84 epitopes, designated as antigenic domain D, is relevant for vaccine design for this highly diverse virus.

Native Folding of a Recombinant gpE1/gpE2 Heterodimer Vaccine Antigen from a Precursor Protein Fused with Fc IgG

“…5). Interestingly, we observed significant competition of vaccinated sera with HC84.26 but not HC33.4, which can recognize linear E2 epitopes within aa 433 to 447 and aa 409 to 423, respectively (44,45). The lack of significant competition with HC33.4 was somewhat surprising because previously characterized murine anti-HCV MAbs have tended to target linear E2 epitopes (53, 54) and we observed significant binding of HC33.4 to both GNA E1E2 and Fc-d E1E2 heterodimers (Fig.…”

“…HC33.4, HC84.26, and AR3B target the disparate regions of E2 that form the cluster of differentiation 81 (CD81) receptor binding site (CD81bs) and are capable of binding soluble E2. HC33.4 recog- nizes a linear epitope in E2 (aa 409 to 423) (15,16,44,45). AR3B and HC84.26 are directed to conformation-specific E2 epitopes.…”

“…Many discrete MAbs with broad cross-neutralizing activity have been isolated and characterized (10,14), including some that recognize overlapping regions of the E2 protein as well as residues in the CD81bs (45,52). In this study, we used discrete MAbs (HC33.4, HC84.26, and AR3B) that bind to various regions of the CD81bs to block the E2-CD81 interaction (16,44,45,52).…”

“…In this study, we used discrete MAbs (HC33.4, HC84.26, and AR3B) that bind to various regions of the CD81bs to block the E2-CD81 interaction (16,44,45,52). In addition, we used monoclonal AR4A and AR5A antibodies that target discrete epitopes outside the CD81bs and that do not interfere with CD81 binding (15).…”

“…Huh7.5 cells were propagated in Dulbecco's modified Eagle's medium (Thermo Fisher Scientific) containing 10% heat-inactivated fetal bovine serum (Omega Scientific, Tarzana, CA), 0.1 mM nonessential amino acids (Invitrogen), and PenStrep (Invitrogen). The MAb mouse anti-cluster of differentiation 81 (CD81) clone JS-81 (BD Biosciences, Franklin Lakes, NJ), mouse isotype control IgG1 (R&D Systems, Minneapolis, MN), anti-HCV MAbs (HC33.4, HC84.26, AR3B, AR4A, and AR5A), and human anti-HIV antibody B6 have been described previously (15,16,44,45). A4 and H52 were provided by Jean Dubuisson (62,63).…”