Abstract
Broadly-neutralizing monoclonal antibodies (bNAbs) may guide vaccine development for highly variable viruses including hepatitis C virus (HCV), since they target conserved viral epitopes that could serve as vaccine antigens. However, HCV resistance to bNAbs could reduce the efficacy of a vaccine. HC33.4 and AR4A are two of the most potent anti-HCV human bNAbs characterized to date, binding to highly conserved epitopes near the amino- and carboxy-terminus of HCV envelope (E2) protein, respectively. Given their distinct epitopes, it was surprising that these bNAbs showed similar neutralization profiles across a panel of natural HCV isolates, suggesting that some viral polymorphisms may confer resistance to both bNAbs. To investigate this resistance, we developed a large, diverse panel of natural HCV envelope variants and a novel computational method to identify bNAb resistance polymorphisms in envelope proteins (E1 and E2). By measuring neutralization of a panel of HCV pseudoparticles by 10 μg/mL of each bNAb, we identified E1E2 variants with resistance to one or both bNAbs, despite 100% conservation of the AR4A binding epitope across the panel. We discovered polymorphisms outside of either binding epitope that modulate resistance to both bNAbs by altering E2 binding to the HCV co-receptor, scavenger receptor B1 (SR-B1). This study is focused on a mode of neutralization escape not addressed by conventional analysis of epitope conservation, highlighting the contribution of extra-epitopic polymorphisms to bNAb resistance and presenting a novel mechanism by which HCV might persist even in the face of an antibody response targeting multiple conserved epitopes.
Highlights
Hepatitis C virus (HCV) infects over 170 million people worldwide [1] and kills more people in the United States annually than HIV [2]
No obvious polymorphisms mediating this effect were identified in a small panel of E1E2 variants with varying HC33.4 and AR4A sensitivities, so we developed a larger panel of more than 100 E1E2 variants as well as a statistical approach to identify natural polymorphisms that were associated with resistance to each Broadly neutralizing antibodies (bNAbs)
To construct a library of E1E2 genes to predict relationships between amino acid sequence and neutralization sensitivity, we cloned more than 700 naturally occurring HCV genotype 1 E1E2 genes. Of these cloned E1E2s, 113 produced HCV pseudoparticles (HCVpp) that were functional in repeated tests when co-transfected with an HIV NL4.3Δenv-Luc reporter genome, as previously described [24]
Summary
Hepatitis C virus (HCV) infects over 170 million people worldwide [1] and kills more people in the United States annually than HIV [2]. A major goal for the development of a prophylactic vaccine against HCV is stimulation of an immune response that is protective against a wide range of naturally occurring viral variants [8,9], which is a daunting challenge given the enormous genetic diversity of HCV [10,11,12,13,14,15,16,17,18]. Neutralizing antibodies (bNAbs) are a useful guide for vaccine development, since they bind to relatively conserved viral epitopes, prevent successful entry of diverse HCV isolates, and have been associated with spontaneous clearance of HCV [19]. BNAb resistance polymorphisms have been identified by various methods, including alanine-scanning mutagenesis, mapping of longitudinal sequence evolution in infected humans [22], and passage of replication competent virus (HCVcc) in vitro in the presence of bNAbs [21,23], but an efficient method to identify common naturally-occurring resistance polymorphisms in circulating E1E2 variants has not been available
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