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  • HCV has evolved mechanisms to


    HCV has evolved mechanisms to evade humoral immune responses including high functional flexibility and variability of immunogenic portions of its envelope proteins. The highest sequence variability occurs in the first 27 kras-pdeδ pathway of the N-terminus of E2, which is referred to as the hypervariable region 1 (HVR1), and which is dispensable for HCV infection in vitro.[10], [11] The HVR1 is immunogenic and most patients mount antibodies targeting this region. However, these antibodies rapidly select resistant viral variants. Deletion of the HVR1 renders HCV more susceptible to antibody neutralization, and it increases virus binding to soluble CD81, suggesting that this region occludes key neutralization epitopes and the viral CD81 binding site.[10], [11] HCV E1E2 heterodimers are also heavily glycosylated at multiple sites within both E1 and E2, with glycans modulating glycoprotein function and antibody neutralization.[14], [15], [16] Structural analyses of the E2 core domain show that the conserved CD81 binding site is surrounded by several glycosylation sites and that it overlaps with the epitopes of bNabs isolated from patients, such as HC1 and HC11.[17], [18], [19], [20] (Fig. 1). Strikingly, in vitro HCV is unable to escape antibody pressure by HC1 and HC11 suggesting that immune responses targeting these epitopes may confer robust protection.[21], [22]
    Materials and methods
    HCV cell culture neutralization assay For inhibition of HCV infection, 200 μl of a Huh-7.5 cell suspension (5 × 104 cells per ml) was seeded into each well of a 96-well plate 24 h prior to inoculation. Luciferase reporter viruses were mixed with serial dilutions of indicated serum/antibody concentrations and pre-incubated for 1 h. This mixture was used to inoculate cells for 4 h in triplicates per dilution. Thereafter, 170 µl of DMEM was added onto the cells. Viral infection was determined 48 or 72 h after infection by removing the supernatant and lysing cells by adding passive lysis buffer or water, before measuring relative light units using a 96-well plate reader (Berthold).
    Deletion of HVR1 and glycosylation sites enhances virus neutralization To characterize the accessibility of HCV antibody epitopes, we performed neutralization assays with a panel of bNAbs (Fig. 3A and C and Table S1). Moreover, we quantified viral neutralization by CD81-LEL (Fig. 3B and C and Table S1). Dose dependence of neutralization for selected viruses is displayed in Fig. 3C. Radar plots indicating the inhibitory concentration 90% (IC90) of different antibodies or the CD81-LEL against parental HCV and all mutant viruses are displayed in Fig. 3A and 3B. In Fig. 3A, each plot shows a specific virus and its sensitivity towards given antibodies, whereas Fig. 3B highlights the susceptibility of each virus to competition by CD81-LEL. In the context of parental Jc1, deletion of glycans at position N423 and N448 increased the susceptibility to all tested antibodies, whereas removal of glycosylation at residues N417, N534, and N649 had little effect (Fig. 3A upper panels). As expected, deletion of HVR1 enhanced neutralization by all antibodies tested. Combination of deletion of HVR1 with deletion of specific N-glycosylation sites modified neutralization by these antibodies, and the Jc1-ΔHVR1-N534A mutant exhibited an enhanced susceptibility to neutralization by HC1 and HC11 (Fig. 3A and 3C). Removal of glycosylation at the N423, N534, and N417 site in the context of WT Jc1 enhanced inhibition of infection by CD81-LEL, whereas mutation of the N448 and N649 sites had no effect (Fig. 3B). Deletion of HVR1 increased neutralization by CD81-LEL and this was further enhanced ca. 5–10-fold by addition of the N534 mutation. Therefore, combined deletion of HVR1 and inactivation of glycosylation site N534 enhanced antibody and CD81 binding to virus particles and it had the most drastic effect on neutralization by bNabs and soluble CD81.