Identification of Novel Determinants of Neutralization Epitope Shielding for Hepatitis C Virus in Vitro ^†

Epitope shielding is suggested as an important mechanism mediating the escape of hepatitis C virus (HCV) from host-neutralizing antibodies (nAb). Knowledge of the determinants of epitope shielding facilitates rational HCV vaccine design.

To identify the determinants of epitope shielding, which might have a negative effect on viral fitness, we used an evolutionary approach passaging HCV in cell culture in the absence of nAb.

Culture infectious HCV recombinants with genotype 1–6 Core-NS2 were subjected to 16–53 passages in Huh7.5 cells until peak infectivity titers of ~6 log10 focus forming units/ml were achieved, signifying a 0.5–3 log10 titer increase compared to the original viruses. Per virus, next-generation sequencing revealed 1–22 dominant open reading frame substitutions, including up to 9 substitutions in the envelope proteins. Polyclonal passaged viruses and recombinants engineered with the identified substitutions showed increased viral fitness and up to four orders of magnitude increased sensitivity to human monoclonal antibodies (AR3A, AR4A) and polyclonal antibodies (C211) compared to the original viruses. For genotype 1a, this effect was mediated by envelope protein substitutions at positions 361, 417, and 532; for 2a, by a position-532-substitution supported by substitutions at positions 410 and 546; and for 3a, by a position-418-substitution. While position 418 and 532 map to glycosylation motifs, the identified position-418-substitution is not expected to affect glycosylation, and different amino acid changes at position 532, all expected to disturb glycosylation, showed largely different effects on neutralization sensitivity, suggesting a mechanism not directly involving changes in glycan occupancy.

1a, 2a, and 3a viruses with the identified envelope protein substitutions showed decreased dependency on HCV co-receptor SR-BI, decreased temperature stability, and decreased viral breathing compared to the original viruses, suggesting “open” envelope states with increased epitope accessibility.

Thus, we identified novel determinants of epitope shielding, mediating “closed” envelope states, resulting in a dramatic decrease in nAb sensitivity with implications for HCV vaccine design.