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

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.

丙型肝炎病毒（Hepatitis C virus, HCV）是引发终末期肝病的主要病原体。每年新增300万至400万例HCV感染病例，因此开发相关疫苗的需求极为迫切。深入解析病毒逃逸中和抗体的机制及其对应表位，对疫苗研发工作至关重要。然而，针对高抗体抗性的病毒分离株或中等效力的抗体，体外诱导逃逸突变仍存在较大挑战。本研究作为概念验证，利用抗体敏感型高变区1（HVR1）缺失（ΔHVR1）病毒，成功为靶向一类罕见跨基因型保守表位的人类单克隆抗体AR5A制备了逃逸突变株。通过分析回收的Core-NS2重组H77/JFH1ΔHVR1的基因型1a包膜蛋白（E1/E2），并开展反向遗传学研究，我们发现AR5A抗性由L665W位点替换介导，该突变同时可使亲本株H77/JFH1获得AR5A抗性。此突变未造成病毒适合度损失，但可阻断AR5A与HCV颗粒及细胞内E1/E2复合物的结合。针对基因型2a的J6/JFH1ΔHVR1毒株，L665W替换会降低病毒适合度，我们通过AR5A筛选获得了携带I345V、L665S及S680T替换的AR5A抗性毒株，并将这些突变引入J6/JFH1及J6/JFH1ΔHVR1毒株中。其中，I345V可提升病毒适合度，但对AR5A抗性无影响；L665S会损害病毒适合度并降低其对AR5A的敏感性；而S680T与L665S联合可补偿适合度损失，并进一步降低病毒对AR5A的敏感性。值得注意的是，单独的S680T未对病毒适合度产生影响，反而可使病毒对AR5A更为敏感。此外，H77/JFH1L665S毒株无法存活。上述抗性突变未影响病毒的细胞间传播及E1/E2相互作用。最后，我们将基因型1中鉴定到的L665W替换引入基因型2~6的亲本株及HVR1缺失变体（基因型4a无可用毒株），观察到其对病毒适合度产生多样影响，且所有毒株的AR5A敏感性均出现显著普遍的降低。综上，本研究借助中和敏感型HVR1缺失病毒，可快速制备HCV逃逸毒株，助力解析HCV规避AR5A的多样化逃逸通路。