There are 3-4 million new hepatitis C virus (HCV) infections yearly. The extensive inter-genotypic sequence diversity of envelope proteins E1 and E2 of HCV and shielding of important epitopes by hypervariable region 1 (HVR1) of E2 are believed to be major hindrances to developing universally protective HCV vaccines. Using cultured viruses expressing the E1/E2 complex of isolates H77 (genotype 1a), J6 (2a), or S52 (3a), with and without HVR1, we tested HVR1-mediated neutralization occlusion in vitro against a panel of 12 well-characterized human monoclonal antibodies (HMAbs) targeting diverse E1, E2, and E1/E2 epitopes. Surprisingly, HVR1-mediated protection was greatest for S52, followed by J6 and then H77. HCV pulldown experiments showed that this phenomenon was caused by epitope shielding. Moreover, by regression analysis of HMAb binding and neutralization titer of HCV we found a strong correlation for HVR1-deleted viruses, but not for parental viruses retaining HVR1. The inter-genotype neutralization sensitivity of the parental viruses to HMAbs AR2A, AR3A, AR4A, AR5A, HC84.26, and HC33.4 varied greatly (>24- to >130-fold differences in IC50-values). However, except for AR5A, these differences decreased to less than 6.0-fold when comparing the corresponding HVR1-deleted viruses. Importantly, this simplified pattern of neutralization sensitivity in the absence of HVR1 was also demonstrated in a panel of HVR1-deleted viruses of genotypes 1a, 2a, 2b, 3a, 5a, and 6a, although for all HMAbs, except AR4A, an outlier was observed. Finally, unique amino acid residues in HCV E2 could explain these outliers in the tested cases of AR5A and HC84.26. Conclusion: HVR1 adds complexity to HCV neutralization by shielding a diverse array of unexpectedly cross-genotype-conserved E1/E2 epitopes. Thus, an HVR1-deleted antigen could be a better HCV vaccine immunogen.

Author contributions: J.P. and J.B. designed research; J.P., R.V.-M., E.H.A., and A.G. performed research; R.W., M.L., and H.A. contributed new reagents/analytic tools; J.P., R.V.-M., E.H.A., A.G., and J.B. analyzed data; and J.P. and J.B wrote the paper.

Cumulative evidence supports a role for neutralizing antibodies contributing to spontaneous viral clearance during acute hepatitis C virus (HCV) infection. Information on the timing and specificity of the B cell response associated with clearance is crucial to inform vaccine design. From an individual who cleared three sequential HCV infections with genotypes 1b, 1a and 3a strains, respectively, we employed peripheral B cells to isolate and characterize neutralizing human monoclonal antibodies (HMAbs) to HCV after the genotype 1 infections. The majority of isolated antibodies, designated as HMAbs 212, target conformational epitopes on the envelope glycoprotein E2 and bound broadly to genotype 1–6 E1E2 proteins. Further, some of these antibodies showed neutralization potential against cultured genotype 1–6 viruses. Competition studies with defined broadly neutralizing HCV HMAbs to epitopes in distinct clusters, designated antigenic domains B, C, D and E, revealed that the selected HMAbs compete with B, C and D HMAbs, previously isolated from subjects with chronic HCV infections. Epitope mapping studies revealed domain B and C specificity of these HMAbs 212. Sequential serum samples from the studied subject inhibited the binding of HMAbs 212 to autologous E2 and blocked a representative domain D HMAb. The specificity of this antibody response appears similar to that observed during chronic infection, suggesting that the timing and affinity maturation of the antibody response are the critical determinants in successful and repeated viral clearance. While additional studies should be performed for individuals with clearance or persistence of HCV, our results define epitope determinants for antibody E2 targeting with important implications for the development of a B cell vaccine.