Novel HCV vaccine antigens targeting conserved neutralizing epitopes

项目摘要 据估计，全世界约有3%的人口感染丙型肝炎病毒(HCV)。最新研究 已经确定了丙型肝炎病毒E1和E2糖蛋白上的一些中和表位，并用 原子结构如何广泛中和抗体(BNAbs)与这些表位相互作用。2.65？水晶 丙型肝炎病毒E2核心(E2C)与bNab(AR3C)形成的复合体的结构揭示了这一长期寻找的原子细节 为丙型肝炎疫苗的合理设计提供了结构基础。在这份U19建议书的项目2中，我们 将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细胞谱系的蛋白质设计和下一代测序(NGS)有助于丙型肝炎病毒免疫原的设计 用于在疫苗接种中诱导bNAbs。其具体目标是：(1)开发以表位为中心的免疫原 和基于E2核心的免疫原。BNAbs络合物中E2和E1中和表位的晶体结构 都是最近确定的。我们假设异源蛋白支架呈现移植的丙型肝炎病毒 表位可以诱导出针对表位的交叉中和抗体。我们还假设E2C域可以 优化作为亚单位疫苗免疫原诱导保守者产生交叉中和抗体 出现在E2表面的表位。基于E2C的晶体结构，我们将缩短可变环， 设计表面N-连接的多糖，并在前层区域引入空间填充突变以稳定 E2C构象。成功设计和表达的丙型肝炎病毒抗原将在 铁蛋白颗粒和噬菌体QβVLP。(2)使用NGS谱系分析评估日本血吸虫病患者的抗体应答 小鼠和非人类灵长类动物免疫。有希望的候选免疫原将首先在小鼠身上进行测试。在……里面 与法律实验室合作，将提供并执行一系列针对丙型肝炎病毒的免疫分析 绘制血清抗体反应图。一种可以诱导交叉中和的一小套设计免疫原 西南国家灵长类动物研究中心将在非人类灵长类动物身上测试小鼠的抗体 中心(SNPRC)。基于流式细胞术的单个B细胞分选和基于微流控的B细胞包埋将 用于分离丙型肝炎病毒中和抗体(NAB)。将使用基于NGS的曲目分析来获得 丙型肝炎病毒免疫过程中抗体反应的定量读数及其谱系演变 纳布斯。抗体谱谱将用于指导免疫原优化，并比较不同的 免疫基因候选人。因此，项目2中提议的研究将是补充和受益的 从项目1中诺华E1E2疫苗的人体样本分析、表位绘制和测试，以及 共同构成了研究丙型肝炎病毒抗体反应和疫苗抗原的中心。

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.