Department of Pathology and Immunology, Washington University School of Medicine, 660 South Euclid Ave., Box 8051, Saint Louis, MO 63110, USA.
J Virol. 2011 Jul;85(14):7005-19. doi: 10.1128/JVI.00586-11. Epub 2011 May 4.
The E2 glycoprotein of hepatitis C virus (HCV) mediates viral attachment and entry into target hepatocytes and elicits neutralizing antibodies in infected patients. To characterize the structural and functional basis of HCV neutralization, we generated a novel panel of 78 monoclonal antibodies (MAbs) against E2 proteins from genotype 1a and 2a HCV strains. Using high-throughput focus-forming reduction or luciferase-based neutralization assays with chimeric infectious HCV containing structural proteins from both genotypes, we defined eight MAbs that significantly inhibited infection of the homologous HCV strain in cell culture. Two of these bound E2 proteins from strains representative of HCV genotypes 1 to 6, and one of these MAbs, H77.39, neutralized infection of strains from five of these genotypes. The three most potent neutralizing MAbs in our panel, H77.16, H77.39, and J6.36, inhibited infection at an early postattachment step. Receptor binding studies demonstrated that H77.39 inhibited binding of soluble E2 protein to both CD81 and SR-B1, J6.36 blocked attachment to SR-B1 and modestly reduced binding to CD81, and H77.16 blocked attachment to SR-B1 only. Using yeast surface display, we localized epitopes for the neutralizing MAbs on the E2 protein. Two of the strongly inhibitory MAbs, H77.16 and J6.36, showed markedly reduced binding when amino acids within hypervariable region 1 (HVR1) and at sites ∼100 to 200 residues away were changed, suggesting binding to a discontinuous epitope. Collectively, these studies help to define the structural and functional complexity of antibodies against HCV E2 protein with neutralizing potential.
丙型肝炎病毒 (HCV) 的 E2 糖蛋白介导病毒附着和进入靶肝细胞，并在感染患者中引发中和抗体。为了表征 HCV 中和的结构和功能基础，我们针对 HCV 1a 和 2a 基因型的 E2 蛋白生成了一个新的 78 个单克隆抗体 (Mab) 面板。使用包含两种基因型结构蛋白的嵌合感染性 HCV 的高通量焦点形成减少或基于荧光素酶的中和测定，我们鉴定了 8 种显著抑制细胞培养中同源 HCV 株感染的 Mab。其中两种结合了代表 HCV 基因型 1 至 6 的 E2 蛋白，其中一种 Mab，H77.39，中和了来自其中五种基因型的株系的感染。我们小组中三种最有效的中和 Mab，H77.16、H77.39 和 J6.36，在附着后早期抑制感染。受体结合研究表明，H77.39 抑制可溶性 E2 蛋白与 CD81 和 SR-B1 的结合，J6.36 阻断与 SR-B1 的附着并适度降低与 CD81 的结合，而 H77.16 仅阻断与 SR-B1 的附着。使用酵母表面展示，我们将中和 Mab 的表位定位在 E2 蛋白上。两种强抑制性 Mab，H77.16 和 J6.36，当 HVR1 内和 100 至 200 个残基左右的位点氨基酸发生变化时，其结合明显减少，表明结合的是不连续表位。总的来说，这些研究有助于定义具有中和潜力的 HCV E2 蛋白抗体的结构和功能复杂性。
