Applying antibody-sensitive hypervariable region 1-deleted hepatitis C virus to the study of escape pathways of neutralizing human monoclonal antibody AR5A

Abstract

Hepatitis C virus (HCV) is a major cause of end-stage liver diseases. With 3–4 million new HCV infections yearly, a vaccine is urgently needed. A better understanding of virus escape from neutralizing antibodies and their corresponding epitopes are important for this effort. However, for viral isolates with high antibody resistance, or antibodies with moderate potency, it remains challenging to induce escape mutations in vitro. Here, as proof-of-concept, we used antibody-sensitive HVR1-deleted (ΔHVR1) viruses to generate escape mutants for a human monoclonal antibody, AR5A, targeting a rare cross-genotype conserved epitope. By analyzing the genotype 1a envelope proteins (E1/E2) of recovered Core-NS2 recombinant H77/JFH1ΔHVR1 and performing reverse genetic studies we found that resistance to AR5A was caused by substitution L665W, also conferring resistance to the parental H77/JFH1. The mutation did not induce viral fitness loss, but abrogated AR5A binding to HCV particles and intracellular E1/E2 complexes. Culturing J6/JFH1ΔHVR1 (genotype 2a), for which fitness was decreased by L665W, with AR5A generated AR5A-resistant viruses with the substitutions I345V, L665S, and S680T, which we introduced into J6/JFH1 and J6/JFH1ΔHVR1. I345V increased fitness but had no effect on AR5A resistance. L665S impaired fitness and decreased AR5A sensitivity, while S680T combined with L665S compensated for fitness loss and decreased AR5A sensitivity even further. Interestingly, S680T alone had no fitness effect but sensitized the virus to AR5A. Of note, H77/JFH1L665S was non-viable. The resistance mutations did not affect cell-to-cell spread or E1/E2 interactions. Finally, introducing L665W, identified in genotype 1, into genotypes 2–6 parental and HVR1-deleted variants (not available for genotype 4a) we observed diverse effects on viral fitness and a universally pronounced reduction in AR5A sensitivity. Thus, we were able to take advantage of the neutralization-sensitive HVR1-deleted viruses to rapidly generate escape viruses aiding our understanding of the divergent escape pathways used by HCV to evade AR5A.

Hypervariable region 1 and N-linked glycans of hepatitis C regulate virion neutralization by modulating envelope conformations

“…Antibodies against HCV receptors were anti-CD81 (BD Pharmingen, catalog no. JS81), anti-SR-BI C16-71 (49,50), and anti-LDLr 3D8 (38). Control antibodies for receptor blocking assays were the isotype antibody 553447 for CD81, the antibody D for SR-BI (49), and a previously described in-house-produced isotype-matched control for LDLr (38).…”

Hepatitis C Virus Escape Studies of Human Antibody AR3A Reveal a High Barrier to Resistance and Novel Insights on Viral Antibody Evasion Mechanisms

“…Three of these antibodies, AR3A, AR4A, and AR5A, target epitopes that are conserved across genotypes (21)(22)(23). However, high epitope conservation does not necessarily result in a high barrier to resistance, as we recently reported for AR5A, for which the virus rapidly acquired AR5A resistance substitutions when cultured with the antibody (24). AR3A has been shown to provide protection in vivo when tested in a mouse model (14,21), underscoring the importance of testing the barrier to resistance for this antibody.…”

“…Selection of virus escape mutants in HCV cell culture (HCVcc) has been shown to be an effective methodology to identify epitope-specific mutations in vitro, and some of these have been shown to have in vivo relevance (24)(25)(26)(27)(28)(29)(30). However, viral escape mutants are generally difficult to generate with HCVcc because of the inherently high antibody resistance of most HCV isolates.…”

“…However, viral escape mutants are generally difficult to generate with HCVcc because of the inherently high antibody resistance of most HCV isolates. In addition, we have shown that the high fitness of certain viruses, like core-NS2 recombinants J6/JFH1 and SA13/JFH1, permits the viruses to spread in culture, even at high concentrations of antibody, without developing resistance substitutions (24,31), possibly aided by high levels of cell-to-cell spread (32,33). Previously, we studied AR5A resistance by using novel HCVcc with a deletion of hypervariable region 1 (HVR1) (24).…”

“…In addition, we have shown that the high fitness of certain viruses, like core-NS2 recombinants J6/JFH1 and SA13/JFH1, permits the viruses to spread in culture, even at high concentrations of antibody, without developing resistance substitutions (24,31), possibly aided by high levels of cell-to-cell spread (32,33). Previously, we studied AR5A resistance by using novel HCVcc with a deletion of hypervariable region 1 (HVR1) (24). HVR1 had been shown to be dispensable for fitness in vitro and in vivo (16,(34)(35)(36), with a possible involvement in interactions with the two HCV entry coreceptors scavenger receptor class B type I (SR-BI) and low-density lipoprotein receptor (37,38).…”