Hepatitis C virus escape studies for human antibody AR3A reveals a high barrier to resistance and novel insights on viral antibody evasion mechanisms.

Yearly, similar to 2 million people become hepatitis C virus (HCV) infected, resulting in an elevated lifetime risk for severe liver-related chronic illnesses. Characterizing epitopes of broadly neutralizing antibodies (NAbs), such as AR3A, is critical to guide vaccine development. Previously identified alanine substitutions that can reduce AR3A binding to expressed H77 envelope were introduced into chimeric cell culture-infectious HCV recombinants (HCVcc) H77(core-NS2)/JFH1. Substitutions G523A, G530A, and D535A greatly reduced fitness, and S424A, P525A, and N540A, although viable, conferred only low-level AR3A resistance. Using highly NAb-sensitive hypervariable region 1 (HVR1)-deleted HCVcc, H77/JFH1(Delta HVR1) and J6(core-NS2)/JFH1(Delta HVR1), we previously reported a low barrier to developing AR5A NAb resistance substitutions. Here, we cultured Huh7.5 cells infected with H77/JFH1, H77/JFH1(Delta HVR1), or J6/JFH1(Delta HVR1) with AR3A. We identified the resistance envelope substitutions M345T in H77/JFH1, L438S and F442Y in H77/JFH1(Delta HVR1), and D431G in J6/JFH1(Delta HVR1). M345T increased infectivity and conferred low-level AR3A resistance to H77/JFH1 but not H77/JFH1(Delta HVR1). L438S and F442Y conferred high-level AR3A resistance to H77/JFH1(Delta HVR1) but abrogated the infectivity of H77/JFH1. D431G conferred AR3A resistance to J6/JFH1(Delta HVR1) but not J6/JFH1. This was possibly because D431G conferred broadly increased neutralization sensitivity to J6/JFH1(D431G) but not J6/JFH1(Delta HVR1)/D431G while decreasing scavenger receptor class B type I coreceptor dependency. Common substitutions at positions 431 and 442 did not confer high-level resistance in other genotype 2a recombinants [JFH1 or T9(core-NS2)/JFH1]. Although the data indicate that AR3A has a high barrier to resistance, our approach permitted identification of low-level resistance substitutions. Also, the HVR1-dependent effects on AR3A resistance substitutions suggest a complex role of HVR1 in virus escape and receptor usage, with important implications for HCV vaccine development. IMPORTANCE Hepatitis C virus (HCV) is a leading cause of liver-related mortality, and limited treatment accessibility makes vaccine development a high priority. The vaccine-relevant cross-genotype-reactive antibody AR3A has shown high potency, but the ability of the virus to rapidly escape by mutating the AR3A epitope (barrier to resistance) remains unexplored. Here, we succeeded in inducing only low-level AR3A resistance, indicating a higher barrier to resistance than what we have previously reported for AR5A. Furthermore, we identify AR3A resistance substitutions that have hypervariable region 1 (HVR1)-dependent effects on HCV viability and on broad neutralization sensitivity. One of these substitutions increased envelope breathing and decreased scavenger receptor class B type I HCV coreceptor dependency, both in an HVR1-dependent fashion. Thus, we identify novel AR3A-specific resistance substitutions and the role of HVR1 in protecting HCV from AR3-targeting antibodies. These viral escape mechanisms should be taken into consideration in future HCV vaccine development.