B Cell Immunity to Hepatitis C Virus

丙型肝炎病毒(丙型肝炎病毒)感染在大多数患者中持续多年，并导致慢性肝病 强大的免疫反应。虽然在体外还没有明确确立保护性免疫的相关性， 多条证据表明，CD4+和CD8+T细胞的反应，虽然对控制急性 感染，都不足以预防长期持续。与此同时，不断涌现的证据 支持病毒中和(VN)抗体的重要性，以及B细胞反应改变 感染病程。因此，一种有效的疫苗需要同时诱导强大的T细胞和B细胞 回应。为了设计一种疫苗免疫原，能够诱导广泛反应的VN抗体来对抗这种病毒 对于高度多样化的病毒，更好地定义丙型肝炎病毒的免疫原性决定簇是至关重要的。我们的 假设这些VN表位被组织在离散的簇(VN域)中，并且特定的域 要么直接参与与病毒共受体的连接或结合，要么参与构象变化 病毒成熟或进入所需的。我们还提出，某些重叠的VN表位很可能是 更保守，更不可能导致病毒逃逸的突变。这项提议把两个人结合在一起 高生产率和互补性的研究团队，努力测试这些假设并开发 关于丙型肝炎病毒VN表位结构的急需信息。多个人类的特征 方格实验室分离的抗丙型肝炎病毒的单抗(HMAb)显示存在 在E2上有多个免疫原域，其中至少有三个通过阻断E2与CD81的结合来介导VN，以及 丙型肝炎病毒进入的重要辅助受体。柠檬实验室的补充工作导致了这一发展 允许细胞培养的新型病毒，允许VN的体外特征和病毒逃脱VN 抗体。我们的直接目标是利用这些新工具，并确定广泛保守的、 丙型肝炎病毒包膜上的免疫原域，可诱导VN抗体。我们会确定哪个信封 蛋白质结构域涉及病毒进入的特定阶段，以及每个结构域中的哪些表位 广泛被VN抗体识别，维持逃逸突变的能力较差。这将是 通过进一步研究VN HMAb的广泛面板，生成新的VN HMAb，以及 利用细胞培养感染丙型肝炎病毒嵌合体的VN逃逸突变体的体外筛选和鉴定 与来自1a型H77c病毒的结构蛋白结合。这些新的逃逸突变体将被用作 用于选择额外HMAb的抗原。总体而言，这些研究将创建一个高分辨率、功能性 构象VN表位图，包括基因特异性和广泛性的主要结合位点 丙型肝炎病毒包膜上的VN抗体。从这些努力中获得的信息将为 合理的疫苗设计，并提供对抗体分子特异性的迫切需要的洞察 应该由免疫引起，否则将对免疫治疗有用

Hepatitis C virus (HCV) infection persists for years in most patients and leads to chronic liver disease despite robust immune responses. Although there are no clearly established in vitro correlates of protective immunity, multiple lines of evidence suggest that CD4+ and CD8+ T cell responses, while critical for controlling acute infection, are insufficient for prevention of long-term persistence. At the same time, emerging evidence supports the importance of virus neutralizing (Vn) antibodies, and the ability of B cell responses to modify the course of infection. Thus, an effective vaccine will need to induce both robust T cell as well as B cell responses. To design a vaccine immunogen capable of eliciting broadly reactive, Vn antibodies against this highly diverse virus, it is critically important to better define the immunogenic determinants of HCV. Our hypothesis is that these Vn epitopes are organized in discrete clusters (Vn domains) and that specific domains either participate directly in attachment or binding to viral co-receptors, or in the conformational changes required for viral maturation or entry. We also propose that certain overlapping Vn epitopes are likely to be more conserved and less capable of mutations leading to virus escape. This proposal brings together two highly productive and complementary research teams in an effort to test these hypotheses and develop critically needed information on the structure of the Vn epitopes of HCV. Characterization of multiple human monoclonal antibodies (HMAbs) to HCV isolated by the Foung laboratory has revealed the existence of multiple immunogenic domains on E2, at least three of which mediate Vn by blocking E2 binding to CD81, an essential co-receptor for HCV entry. Complementary work in the Lemon laboratory has led to the development of novel cell culture-permissive viruses allowing in vitro characterization of Vn and viral escape from Vn antibodies. Our immediate aims are to exploit these novel tools and to identify broadly conserved, immunogenic domains on the HCV envelope that elicit Vn antibodies. We will determine which envelope protein domains are involved in specific phases of virus entry, and which epitopes within each domain are recognized by broadly Vn antibodies and are less capable of sustaining escape mutations. This will be accomplished through further studies of an extensive panel of Vn HMAbs, generation of new Vn HMAbs, and the in vitro selection and characterization of Vn escape mutants using a cell culture-infectious HCV chimera with structural proteins derived from genotype 1a H77c virus. These novel escape mutants will be utilized as antigen for selection of additional HMAbs. Collectively, these studies will create a high-resolution, functional map of conformational Vn epitopes comprising the major binding sites of both genotype-specific and broadly Vn antibodies on the HCV envelope. The information gained from these efforts will provide the basis for rational vaccine design, and provide much needed insight into the molecular specificities of antibodies that should be elicited by immunization or that would be useful for immunotherapy