Keck Zhen-yong, Li Sophia H, Xia Jinming, von Hahn Thomas, Balfe Peter, McKeating Jane A, Witteveldt Jeroen, Patel Arvind H, Alter Harvey, Rice Charles M, Foung Steven K H
Department of Pathology, Stanford University, Stanford, California 94305, USA.
J Virol. 2009 Jun;83(12):6149-60. doi: 10.1128/JVI.00248-09. Epub 2009 Mar 25.
Broadly neutralizing antibodies are commonly present in the sera of patients with chronic hepatitis C virus (HCV) infection. To elucidate possible mechanisms of virus escape from these antibodies, retrovirus particles pseudotyped with HCV glycoproteins (HCVpp) isolated from sequential samples collected over a 26-year period from a chronically infected patient, H, were used to characterize the neutralization potential and binding affinity of a panel of anti-HCV E2 human monoclonal antibodies (HMAbs). Moreover, AP33, a neutralizing murine monoclonal antibody (MAb) to a linear epitope in E2, was also tested against selected variants. The HMAbs used were previously shown to broadly neutralize HCV and to recognize a cluster of highly immunogenic overlapping epitopes, designated domain B, containing residues that are also critical for binding of viral E2 glycoprotein to CD81, a receptor essential for virus entry. Escape variants were observed at different time points with some of the HMAbs. Other HMAbs neutralized all variants except for the isolate 02.E10, obtained in 2002, which was also resistant to MAb AP33. The 02.E10 HCVpp that have reduced binding affinities for all antibodies and for CD81 also showed reduced infectivity. Comparison of the 02.E10 nucleotide sequence with that of the strain H-derived consensus variant, H77c, revealed the former to have two mutations in E2, S501N and V506A, located outside the known CD81 binding sites. Substitution A506V in 02.E10 HCVpp restored binding to CD81, but its antibody neutralization sensitivity was only partially restored. Double substitutions comprising N501S and A506V synergistically restored 02.E10 HCVpp infectivity. Other mutations that are not part of the antibody binding epitope in the context of N501S and A506V were able to completely restore neutralization sensitivity. These findings showed that some nonlinear overlapping epitopes are more essential than others for viral fitness and consequently are more invariant during earlier years of chronic infection. Further, the ability of the 02.E10 consensus variant to escape neutralization by the tested antibodies could be a new mechanism of virus escape from immune containment. Mutations that are outside receptor binding sites resulted in structural changes leading to complete escape from domain B neutralizing antibodies, while simultaneously compromising viral fitness by reducing binding to CD81.
广泛中和抗体通常存在于慢性丙型肝炎病毒（HCV）感染患者的血清中。为了阐明病毒逃避这些抗体的可能机制，研究人员使用从一名慢性感染患者H在26年期间收集的连续样本中分离出的、以HCV糖蛋白假型化的逆转录病毒颗粒（HCVpp），来表征一组抗HCV E2人单克隆抗体（HMAb）的中和潜力和结合亲和力。此外，还针对选定的变体测试了AP33，一种针对E2中线性表位的中和鼠单克隆抗体（MAb）。所使用的HMAb先前已被证明可广泛中和HCV，并识别一组高度免疫原性的重叠表位，称为结构域B，其包含的残基对于病毒E2糖蛋白与CD81（病毒进入所必需的受体）的结合也至关重要。在不同时间点观察到一些HMAb出现了逃逸变体。其他HMAb可中和所有变体，但2002年获得的分离株02.E10除外，该分离株也对MAb AP33耐药。对所有抗体和CD81的结合亲和力降低的02.E10 HCVpp，其感染性也降低。将02.E10核苷酸序列与源自菌株H的共有变体H77c的序列进行比较，发现前者在E2中有两个突变，即S501N和V506A，位于已知的CD81结合位点之外。02.E10 HCVpp中的A506V替换恢复了与CD81的结合，但其抗体中和敏感性仅部分恢复。包含N501S和A506V的双重替换协同恢复了02.E10 HCVpp的感染性。在N501S和A506V背景下，不属于抗体结合表位的其他突变能够完全恢复中和敏感性。这些发现表明，一些非线性重叠表位对病毒适应性比其他表位更重要，因此在慢性感染的早期年份中更不易发生变异。此外，02.E10共有变体逃避所测试抗体中和的能力可能是病毒逃避免疫抑制的一种新机制。位于受体结合位点之外的突变导致结构变化，从而完全逃避结构域B中和抗体的作用，同时通过降低与CD81的结合而损害病毒适应性。
