There are 3‐4 million new hepatitis C virus (HCV) infections yearly. The extensive intergenotypic sequence diversity of envelope proteins E1 and E2 of HCV and shielding of important epitopes by hypervariable region 1 (HVR1) of E2 are believed to be major hindrances to developing universally protective HCV vaccines. Using cultured viruses expressing the E1/E2 complex of isolates H77 (genotype 1a), J6 (2a), or S52 (3a), with and without HVR1, we tested HVR1‐mediated neutralization occlusion in vitro against a panel of 12 well‐characterized human monoclonal antibodies (HMAbs) targeting diverse E1, E2, and E1/E2 epitopes. Surprisingly, HVR1‐mediated protection was greatest for S52, followed by J6 and then H77. HCV pulldown experiments showed that this phenomenon was caused by epitope shielding. Moreover, by regression analysis of HMAb binding and neutralization titer of HCV we found a strong correlation for HVR1‐deleted viruses but not for parental viruses retaining HVR1. The intergenotype neutralization sensitivity of the parental viruses to HMAb antigenic region (AR) 2A, AR3A, AR4A, AR5A, HC84.26, and HC33.4 varied greatly (>24‐fold to >130‐fold differences in 50% inhibitory concentration values). However, except for AR5A, these differences decreased to less than 6.0‐fold when comparing the corresponding HVR1‐deleted viruses. Importantly, this simplified pattern of neutralization sensitivity in the absence of HVR1 was also demonstrated in a panel of HVR1‐deleted viruses of genotypes 1a, 2a, 2b, 3a, 5a, and 6a, although for all HMAbs, except AR4A, an outlier was observed. Finally, unique amino acid residues in HCV E2 could explain these outliers in the tested cases of AR5A and HC84.26. Conclusion: HVR1 adds complexity to HCV neutralization by shielding a diverse array of unexpectedly cross‐genotype‐conserved E1/E2 epitopes. Thus, an HVR1‐deleted antigen could be a better HCV vaccine immunogen. (Hepatology 2016;64:1881‐1892)

Cumulative evidence supports a role for neutralizing antibodies contributing to spontaneous viral clearance during acute hepatitis C virus (HCV) infection. Information on the timing and specificity of the B cell response associated with clearance is crucial to inform vaccine design. From an individual who cleared three sequential HCV infections with genotypes 1b, 1a and 3a strains, respectively, we employed peripheral B cells to isolate and characterize neutralizing human monoclonal antibodies (HMAbs) to HCV after the genotype 1 infections. The majority of isolated antibodies, designated as HMAbs 212, target conformational epitopes on the envelope glycoprotein E2 and bound broadly to genotype 1–6 E1E2 proteins. Further, some of these antibodies showed neutralization potential against cultured genotype 1–6 viruses. Competition studies with defined broadly neutralizing HCV HMAbs to epitopes in distinct clusters, designated antigenic domains B, C, D and E, revealed that the selected HMAbs compete with B, C and D HMAbs, previously isolated from subjects with chronic HCV infections. Epitope mapping studies revealed domain B and C specificity of these HMAbs 212. Sequential serum samples from the studied subject inhibited the binding of HMAbs 212 to autologous E2 and blocked a representative domain D HMAb. The specificity of this antibody response appears similar to that observed during chronic infection, suggesting that the timing and affinity maturation of the antibody response are the critical determinants in successful and repeated viral clearance. While additional studies should be performed for individuals with clearance or persistence of HCV, our results define epitope determinants for antibody E2 targeting with important implications for the development of a B cell vaccine. Studies of hepatitis C virus (HCV) infected individuals spontaneously clearing their acute infections provide an opportunity to characterize the specificities of associated protective antibody responses. In an individual who repeatedly resolved three separate HCV infections with different HCV genotypes, the antibodies induced during these acute infection episodes are similar to those induced during chronic infection. Surprisingly, the earliest detected antibodies were directed against conformational HCV epitopes on the envelope glycoprotein E2 (including polyprotein residues 434–446) known to be targeted by broadly neutralizing antibodies. Taken together, the key B-cell determinants in spontaneous clearance are the timing and affinity maturation of broadly neutralizing antibody responses.

Significance: Hepatitis C virus (HCV) is a pervasive human pathogen for which a vaccine is urgently needed. Vaccine development relies on inducing neutralizing antibodies (NAbs), but HCV employs complex mechanisms to evade neutralization that remain incompletely understood. Here, we discover a unique interplay between two separate molecular features of HCV envelope protein E2, the hypervariable region 1 and N-linked glycans, that protect HCV from NAbs. Furthermore, we find that they share a mechanism in which they broadly modulate NAb epitope availability by influencing stability of closed and open envelope protein conformations (e.g., envelope breathing) and virus entry dependency on scavenger receptor BI. The apparent importance of structural dynamics in understanding HCV NAb evasion and entry have important implications for rational vaccine design.

To achieve global elimination of hepatitis C virus (HCV), an effective cross-genotype vaccine is needed. The HCV envelope glycoprotein E2 is the main target for neutralizing antibodies (nAbs), which aid in HCV clearance and protection. E2 is structurally flexible and functions in engaging host receptors. Many nAbs bind to the “neutralizing face” on E2, including several broadly nAbs encoded by the V_H1-69 germline gene family that bind to a similar conformation (A) of this face. Here, a previously unknown conformation (B) of the neutralizing face is revealed in crystal structures of two of four additional E2–V_H1-69 nAb complexes. In this conformation, the E2 front-layer region is displaced upon antibody binding, exposing residues in the back layer for direct antibody interaction. This E2 B structure may represent another conformational state in the viral entry process that is susceptible to antibody neutralization and thus provide a new target for rational vaccine development.

Objective: HCV is a major cause of chronic liver disease worldwide, but the role of neutralising antibodies (nAbs) in its natural history remains poorly defined. We analysed the in vivo role of hypervariable region 1 (HVR1) for HCV virion properties, including nAb susceptibility. Design: Analysis of HCV from human liver chimeric mice infected with cell-culture-derived prototype genotype 2a recombinant J6/JFH1 or HVR1-deleted variant J6/JFH1 _ΔHVR1 identified adaptive mutations, which were analysed by reverse genetics in Huh7.5 and CD81-deficient S29 cells. The increased in vivo genomic stability of the adapted viruses facilitated ex vivo density analysis by ultracentrifugation and in vivo neutralisation experiments addressing the role of HVR1. Results: In vivo, J6/JFH1 and J6/JFH1 _ΔHVR1 depended on single substitutions within amino acids 867–876 in non-structural protein, NS2. The identified A876P-substitution resulted in a 4.7-fold increase in genomic stability. In vitro, NS2 substitutions enhanced infectivity 5–10-fold by increasing virus assembly. Mouse-derived mJ6/JFH1 _A876P and mJ6/JFH1 _ΔHVR1/A876P viruses displayed similar heterogeneous densities of 1.02–1.1 g/mL. Human liver chimeric mice loaded with heterologous patient H (genotype 1a) immunoglobulin had partial protection against mJ6/JFH1 _A876P and complete protection against mJ6/JFH1 _ΔHVR1/A876P . Interestingly, we identified a putative escape mutation, D476G, in mJ6/JFH1 _A876P . This mutation in hypervariable region 2 conferred 6.6-fold resistance against H06 IgG in vitro. Conclusions: The A876P-substitution bridges in vitro and in vivo studies using J6/JFH1-based recombinants. We provide the first in vivo evidence that HVR1 protects cross-genotype conserved HCV neutralisation epitopes, which advocates the possibility of using HVR1-deleted viruses as vaccine antigens to boost broadly reactive protective nAb responses.

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/JFH1_ΔHVR1 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/JFH1_ΔHVR1 (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/JFH1_ΔHVR1. 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/JFH1_L665S 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. Worldwide hepatitis C virus (HCV) is one of the leading causes of chronic liver diseases, including cirrhosis and cancer. Treatment accessibility is limited and development of a preventive vaccine has proven difficult, partly due to the high mutation rate of the virus. Recent studies of HCV antibody neutralization resistance have revealed important information about escape pathways and barriers to escape for several clinically promising human monoclonal antibodies. However, due to the varying levels of antibody shielding between HCV isolates these studies have been mostly limited to a few neutralization-sensitive HCV isolates. Here, we took advantage of the fact that deletion of the hypervariable region 1 (HVR1) increased antibody sensitivity of HCV isolates by increasing the exposure of important epitopes, thus facilitating studies of antibody escape for neutralization resistant isolates. We identified escape mutations in the envelope glycoprotein E2, at amino acid position L665, which conferred antibody resistance in parental HCV viruses from genotypes 1–6. We found that antibody escape was associated with loss of binding to HCV particles and intracellular envelope protein complexes. We also identified escape substitutions at L665 that were isolate-specific. Thus, our data sheds new light on antibody resistance mechanisms across diverse HCV isolates.