In vitro adaptation and characterization of attenuated hypervariable region 1 swap chimeras of hepatitis C virus

Author summary Hepatitis C virus (HCV) employs several mechanisms to avoid eradication by the host immune system. This includes the virus' ability to evade the B-cell response by occluding conserved neutralizing antibody (NAb) epitopes on the HCV envelope protein 1 (E1) and 2 (E2). An important component of HCV NAb evasion is the E2 motif, hypervariable region 1 (HVR1), which broadly regulates HCV neutralization sensitivity. As HVR1 displays a high level of structural flexibility, it has not been possible to infer HVR1 localization from solved structures of E2. Consequently, little is known about its interaction with the remaining part of the envelope proteins. In this study, we describe substitutions at conserved E1 and E2 positions both within and outside of HVR1 that appear critical for HVR1-specific E1/E2 function. We showed that these substitutions regulated protection of conserved neutralization epitopes and influenced receptor dependency of viral entry. Finally, testing the effect of these substitutions on NAb binding to extracted protein and in HCV immunoprecipitation we demonstrated the challenges of studying these phenomena outside the context of neutralization assays of infectious virus particles. Overall, this study contributes to our understanding of HVR1-mediated NAb protection which must be overcome in HCV vaccine strategies. Hepatitis C virus (HCV) chronically infects 70 million people worldwide with an estimated annual disease-related mortality of 400,000. A vaccine could prevent spread of this pervasive human pathogen, but has proven difficult to develop, partly due to neutralizing antibody evasion mechanisms that are inherent features of the virus envelope glycoproteins, E1 and E2. A central actor is the E2 motif, hypervariable region 1 (HVR1), which protects several non-overlapping neutralization epitopes through an incompletely understood mechanism. Here, we show that introducing different HVR1-isolate sequences into cell-culture infectious JFH1-based H77 (genotype 1a) and J4 (genotype 1b) Core-NS2 recombinants can lead to severe viral attenuation. Culture adaptation of attenuated HVR1-swapped recombinants permitted us to identify E1/E2 substitutions at conserved positions both within and outside HVR1 that increased the infectivity of attenuated HVR1-swapped recombinants but were not adaptive for original recombinants. H77 recombinants with HVR1 from multiple other isolates consistently acquired substitutions at position 348 in E1 and position 385 in HVR1 of E2. Interestingly, HVR1-swapped J4 recombinants primarily acquired other substitutions: F291I (E1), F438V (E2), F447L/V/I (E2) and V710L (E2), indicating a different adaptation pathway. For H77 recombinants, the adaptive E1/E2 substitutions increased sensitivity to the neutralizing monoclonal antibodies AR3A and AR4A, whereas for J4 recombinants, they increased sensitivity to AR3A, while having no effect on sensitivity to AR4A. To evaluate effects of the substitutions on AR3A and AR4A binding, we performed ELISAs on extracted E1/E2 protein and performed immunoprecipitation of relevant viruses. However, extracted E1/E2 protein and immunoprecipitation of HCV particles only reproduced the neutralization phenotypes of the J4 recombinants. Finally, we found that the HVR1-swap E1/E2 substitutions decrease virus entry dependency on co-receptor SR-BI. Our study identifies E1/E2 positions that could be critical for intra-complex HVR1 interactions while emphasizing the need for developing novel tools for molecular studies of E1/E2 interactions.

Automated summary

The study identifies critical E1/E2 substitutions for understanding HVR1-mediated NAb protection, emphasizing the need to overcome neutralizing antibody evasion mechanisms in developing effective HCV vaccine strategies.