In Vivo Determinants of Hepatitis C Virus Adaptation and Escape From Neutralizing Antibody AR5A

BACKGROUND & AIMS: Mechanisms of hepatitis C virus (HCV) adaptation and escape from broadly neutralizing antibodies (bNAbs) have been primarily studied in vitro. Here, we used a previously developed in vivo adapted J6/JFH1A876P virus and the highly bNAb sensitive hypervariable region 1 (HVR1) deleted variant, J6/JFH1A876P,ΔHVR1, to study adaptation and bNAb AR5A escape in the HCV-permissive human-liver chimeric mouse model.

METHODS: In vitro identified AR5A escape substitution, L665S, was introduced into J6/JFH1A876P with or without HVR1. The infection of human liver chimeric mice with these recombinants revealed adaptive mutations, and the potential mechanism of adaptation was extensively characterized in vitro. Finally, we tested the barrier to resistance of AR5A in vivo by challenging passively immunized animals with HVR1-deleted viruses, either with or without the AR5A escape substitution, L665S.

RESULTS: L665S was found to be an escape substitution in vivo. Furthermore, sequence analysis showed that the escape substitution L665S arose as early as 2 weeks post infection. At week 8, we also identified antibody escape substitutions as well as several potential in vivo adaptive substitutions in E2. For J6/JFH1A876P, S449P and M702L increased cell-free particle infection and broadly affected antibody sensitivity for virus with HVR1. For J6/JFH1A876P,ΔHVR1, N430D and M702L substitutions increased both cell-free particle mediated infection and cell-to-cell spread, whereas N430D also increased thermal stability at 37°C.

CONCLUSIONS: We show that L665S is an AR5A escape mutation in vivo, supporting the use of cost-effective vaccine escape studies in vitro. We also identify novel in vivo adaptive mutations and characterize their mechanism of action, thus facilitating interpretation of future HCV in vivo studies.