The E2 glycoprotein of hepatitis C virus (HCV) is the major target of broadly neutralizing antibodies (bNAbs) that are critical for the efficacy of a prophylactic HCV vaccine. We previously showed that a cell culture-derived, disulfide-linked high-molecular-weight (HMW) form of the E2 receptor-binding domain lacking three variable regions, D123-HMW, elicits broad neutralizing activity against the 8 genotypes of HCV. A limitation to the use of this antigen is that it is produced only at low yields and does not have a homogenous composition. Here, we employed a sequential reduction and oxidation strategy to efficiently refold two high-yielding monomeric E2 species, D123 and a disulfide-minimized version (D123A7), into disulfide-linked HMW-like species (Delta123r and Delta123A7r). These proteins exhibited normal reactivity to bNAbs with continuous epitopes on the neutralizing face of E2, but reduced reactivity to conformation-dependent bNAbs and non-neutralizing antibodies (non-NAbs) compared with the corresponding monomeric species. Delta123r and Delta123A7r recapitulated the immunogenic properties of cell culture-derived D123-HMW in guinea pigs. The refolded antigens elicited antibodies that neutralized homologous and heterologous HCV genotypes, blocked the interaction between E2 and its cellular receptor CD81, and targeted the AS412, AS434, and AR3 domains. Of note, antibodies directed to epitopes overlapping with those of non-NAbs were absent. The approach to E2 antigen engineering outlined here provides an avenue for the development of preventive HCV vaccine candidates that induce bNAbs at higher yield and lower cost.
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We are grateful to Shirley Taylor (CSL) for the
SEC-MALS analysis; Cara Fraser for animal studies; Mansun Law,
Steven Foung, Jean Dubuisson, Harry Greenberg, and Catherine Owczarek for the kind gift of antibodies; and Jens Bukh for providing chimeric HCVcc clones. We gratefully acknowledge the contribution to
this work of the Victorian Operational Infrastructure Support Program received by the Burnet Institute. Reagents will be provided upon
request under material transfer agreements.