Novel HCV vaccine antigens targeting conserved neutralizing epitopes

项目摘要 据估计，约3%的世界人口感染丙型肝炎病毒（HCV）。最近的研究 已经鉴定了HCV E1和E2糖蛋白上的许多中和表位，并证明了 广泛中和抗体（bnAb）如何与这些表位相互作用的原子结构。2.65英寸的水晶 HCV E2核心（E2 c）与bnAb（AR 3C）复合物的结构揭示了这种长期寻求的原子细节， 为HCV疫苗的合理设计提供了结构基础。在U19提案的项目2中，我们 将联合收割机结合丙型肝炎病毒结构生物学的最新发现和计算领域的尖端技术， B细胞库的蛋白质设计和下一代测序（NGS），以促进HCV免疫原设计 用于在疫苗接种中诱导bnAb。具体目标是：（1）开发针对表位的免疫原 和E2核心免疫原。与bnAb复合的E2和E1中和表位的晶体结构 是最近决定的。我们假设，呈现移植HCV的异源蛋白支架 表位可以诱导针对该表位的交叉中和抗体。我们还假设E2 c结构域可以 作为亚单位疫苗免疫原进行优化，以诱导针对保守的 表位呈现在E2表面上。基于E2 c的晶体结构，我们将缩短可变环， 工程化表面N-连接聚糖，并将空间填充突变引入前层区域以稳定 E2 c构象。成功设计和表达的HCV抗原将在 铁蛋白颗粒和噬菌体Qβ VLP。(2)使用NGS库分析来评估抗体应答， 小鼠和非人灵长类动物免疫。有希望的候选免疫原将首先在小鼠中进行测试。在 与法律实验室的合作，一系列丙型肝炎病毒特异性免疫测定将可用并执行 绘制血清抗体反应图一小组设计的免疫原，可以诱导交叉中和 在西南国家灵长类动物研究中心， 中心（SNPRC）。基于流式细胞术的单个B细胞分选和基于微流体的B细胞包封将 用于分离HCV中和抗体（nAb）。基于NGS的库分析将用于获得 免疫期间抗体应答的定量读数，并追踪HCV的谱系发展 nAbs。抗体库谱将用于指导免疫原优化并比较不同的免疫原。 免疫原候选物。因此，项目2中提议的研究将是对 来自项目1中诺华E1 E2疫苗的人样本分析、表位作图和检测，以及 共同组成丙型肝炎病毒抗体反应和疫苗抗原研究中心。

Project Summary It has been estimated that ~3% of the word population is infected with hepatitis C virus (HCV). Recent studies have identified a number of neutralizing epitopes on the HCV E1 and E2 glycoproteins and demonstrated with atomic structures how broadly neutralizing antibodies (bnAbs) interact with these epitopes. The 2.65Å crystal structure of HCV E2 core (E2c) in complex with a bnAb (AR3C) revealed atomic details of this long-sought target, providing a structural basis for rational design of HCV vaccines. In Project 2 of this U19 proposal, we will combine the latest findings in HCV structural biology and cutting-edge technologies in computational protein design and next-generation sequencing (NGS) of B-cell repertoire to facilitate HCV immunogen design for the induction of bnAbs in vaccination. The specific aims are: (1) to develop epitope-focused immunogens and E2 core-based immunogens. Crystal structures of E2 and E1 neutralizing epitopes in complex with bnAbs have been determined recently. We hypothesize that heterologous protein scaffolds presenting a grafted HCV epitope can induce cross-neutralizing antibodies to the epitope. We also hypothesize that the E2c domain can be optimized as a subunit vaccine immunogen to induce cross-neutralizing antibodies to the conserved epitopes presented on the E2 surface. Based on the crystal structure of E2c, we will shorten the variable loops, engineer the surface N-linked glycans, and introduce space-filling mutations to the front layer region to stabilize the E2c conformation. Successfully designed and expressed HCV antigens will be displayed multivalently on ferritin particle and bacteriophage Qβ VLP. (2) to use NGS repertoire profiling to assess antibody response in mouse and non-human primate immunization. Promising immunogen candidates will be first tested in mice. In collaboration with the Law lab, an array of HCV-specific immunological assays will be available and performed to map the serum antibody responses. A small set of designed immunogens that can induce cross-neutralizing antibodies in mice will be tested in non-human primates (NHPs) at the Southwest National Primate Research Center (SNPRC). Flow cytometry-based single B-cell sorting and microfluidics-based B-cell encapsulation will be utilized to isolate HCV-neutralizing antibodies (nAbs). NGS-based repertoire profiling will be used to obtain a quantitative readout of antibody response during immunization and to trace the lineage development of HCV nAbs. The antibody repertoire profiles will be used to guide immunogen optimization and to compare different immunogen candidates. The research proposed in Project 2 will therefore be complementary to and will benefit from the human sample analysis, epitope mapping, and testing of the Novartis E1E2 vaccine in Project 1, and together constitute the center for studying hepatitis C virus antibody responses and vaccine antigens.