The Molecular Basis of Antibody Mediated Neutralization of Hepatitis C Virus.

摘要

Hepatitis C virus (HCV) is positive strand, blood-borne, hepatotropic RNA virus that causes chronic infection in ∼170 million people worldwide and is the leading cause of liver transplantation in the United States. HCV entry and attachment is mediated by the envelope protein E2 through interaction with several cellular receptors including CD81, scavenger receptor B1 (SRB-1), claudin-1, and occludin, although the exact mechanism by which these receptors facilitate infection remains unclear, largely due to the absence of a structural model of E2. The production of neutralizing antibodies against E2 is thought to be important for controlling HCV infection, likely by blocking virus interaction with these receptors. To better understand the structural and molecular basis of antibody neutralization of HCV, which could be used to inform novel therapeutic or vaccine approaches, we generated a panel of 78 monoclonal antibodies (MAbs) against the E2 protein from HCV genotypes 1 and 2 and assessed their neutralizing activity in vitro. Using this approach and by performing mechanistic studies, we identified three neutralizing MAbs, H77.16, H77.39, and J6.36, that inhibit infection at a post-attachment step. Using a yeast display library of E2 protein variants, we mapped the critical binding residues of these MAbs to distinct regions of the E2 protein: H77.16 binds within the HVR1 and to a conserved CD81 binding region ∼125 amino acid residues C-terminal to the HVR1; H77.39 binds to conserved residues upstream of the hypervariable region (HVR1); and J6.36 binds to amino acid residues within HVR1 as well a site ∼150 amino acids C-terminal to HVR1. Receptor-binding inhibition studies using E2 demonstrated that H77.16 potently inhibits binding to SR-B1, H77.39 potently inhibits binding to SR-B1 and CD81, and J6.36 potently inhibits binding to SR-B1 and modestly inhibits binding to CD81. Further mechanistic studies demonstrated that MAb-mediated neutralization could be enhanced by increases in pre-incubation temperature and time and that these results were likely due to altered epitope exposure on the viral surface. Together, these data provide new insight into the mechanisms by which antibodies neutralize infection of HCV.