Characterization of HCV vaccine induced-neutralizing antibody response in non-human primates

项目总结 慢性丙型肝炎病毒感染是肝病和肝细胞癌的主要原因，但目前还没有疫苗 由于其极高的遗传多样性而针对丙型肝炎病毒开发的。尽管病毒的变异性很高，但30% 的丙型肝炎病毒感染者通过产生广谱中和抗体(BNAbs)来清除病毒感染 与丙型肝炎病毒糖蛋白E2受体结合部位的保守表位和构象表位的结合 跨越E1E2异源二聚体。如果我们能刺激这种丙型肝炎病毒bNAb的产生，同时保护 通过接种疫苗进行T细胞反应，可能会阻止丙型肝炎病毒的流行。 我们最近从不同的丙型肝炎病毒分离株中鉴定出几种与人类生殖系前体结合的E2蛋白 BNAbs，建议使用这些变体开发疫苗。然而，目前还不清楚哪一种会使 更有效的候选疫苗：基于E2或E1E2的免疫原。在这份提案中，我们的目标是评估 这两套候选疫苗以及在非人类灵长类动物中不同的免疫原输送方法 包括能够靶向E1E2中和表位的生殖系基因片段。这项工作被组织成 三大具体目标。目标1：开发免疫策略，以诱导针对保守的丙型肝炎病毒E1E2的bNAbs 表位。与Project 5合作，我们将在小鼠身上测试不同的免疫平台，评估两种平台， 抗E1E2构象表位中和抗体的研制(本项目)和多特异性T细胞 单元格响应(项目1)。接下来，在T细胞和抗体诱导方面，最好的候选疫苗 每一个都将在非人类灵长类动物身上进行测试，这些灵长类动物经常使用抗体基因片段的同源基因 被最有效的人类丙型肝炎病毒特异性bNAb所使用。目标2：生成雌二醇特异性的高分辨率结构 在非人类灵长类动物接种疫苗和清除人类自然的丙型肝炎病毒感染后产生的bNAbs。 除了分析目标1中免疫动物的血清反应外，我们还将生成高分辨率 疫苗诱导的bNAbs或从清除者体内分离的人抗体的晶体结构 感染(项目2)。我们将在免疫后对E2特异性猕猴B细胞中的抗体基因进行排序和测序 并表达来自这些序列的抗体。然后我们将确定疫苗诱导的bNAbs的结构 在与免疫原的复合体中，并将它们与结合E2蛋白的人bNAbs的结构进行比较(项目4) 目的阐明丙型肝炎病毒广泛中和的结构决定因素。目标#3：确定完整的结构- 在膜环境中使用冷冻电子断层扫描(CRYO-ET)研究长度为E1E2的异源二聚体。要扩大规模 对于来自目标2的bNAb-E2结构，我们将使用冷冻和亚断层图像平均来检测bNAb E1E2异源二聚体在其完整膜形式中的识别。综合起来，这些目标的结果将导致 诱导丙型肝炎病毒特异性bNAb和强T细胞的新型候选疫苗的综合表征 细胞反应，促进有效的丙型肝炎疫苗的开发。

PROJECT SUMMARY Chronic HCV infection is a leading cause of liver disease and hepatocellular carcinoma, yet no vaccine has been developed against HCV due to its extremely high genetic diversity. Despite the high variability of the virus, 30% of HCV-infected individuals clear the viral infection by developing broadly neutralizing antibodies (bNAbs) that bind to conserved epitopes in the receptor binding site of HCV glycoprotein E2 and conformational epitopes spanning the E1E2 heterodimer. If we could stimulate the production of such HCV bNAbs along with protective T cell responses by vaccination, a rising epidemic of HCV might be stopped. We recently identified several E2 proteins from different HCV isolates that bind to germline precursors of human bNAbs, suggesting vaccine development using these variants. However, it is not yet clear which would make a more effective vaccine candidate: E2-based or E1E2-based immunogens. In this proposal, we aim to evaluate both sets of vaccine candidates along with different methods of immunogen delivery in non-human primates that include germline gene segments capable of targeting E1E2 neutralizing epitopes. The work is organized into three major Specific Aims. Aim #1: Develop immunization strategies that elicit bNAbs to conserved HCV E1E2 epitopes. Working together with Project 5, we will test different immunization platforms in mice, evaluating both, the development of neutralizing antibodies to E1E2 conformational epitopes (this Project), and multi-specific T cell responses (Project 1). Next, the best vaccine candidates with respect to both T cell and antibody induction will each be tested in non-human primates, which utilize an ortholog of an antibody gene segment frequently used by the most potent human HCV-specific bNAbs. Aim #2: Generate high-resolution structures of E2-specific bNAbs elicited after vaccination in non-human primates and after clearance of natural HCV infection in humans. In addition to analyzing serum responses from immunized animals in Aim 1, we will generate high-resolution crystal structures of vaccine-induced bNAbs or human antibodies isolated from individuals who cleared their infection (Project 2). We will sort and sequence antibody genes in E2-specific macaque B cells after immunization and express antibodies from these sequences. We will then determine the structures of vaccine-induced bNAbs in complex with immunogens and compare them to structures of human bNAbs bound to E2 proteins (Project 4) to elucidate the structural determinants of broad neutralization of HCV. Aim #3: Determine a structure of the full- length E1E2 heterodimer in a membrane environment using cryo-electron tomography (cryo-ET). To expand upon bNAb-E2 structures from Aim 2, we will use cryo-ET with sub-tomogram averaging to examine bNAb recognition of the E1E2 heterodimer in its intact membrane form. Together, the results of these aims will lead to the comprehensive characterization of novel vaccine candidates that elicit HCV-specific bNAbs and strong T- cell responses, facilitating the development of an effective HCV vaccine.