Novel HCV vaccine antigens and nanoparticles

项目摘要 丙型肝炎病毒感染1-2%的世界人口，对公众健康构成重大威胁。 最近的研究已经确定了一组广泛的中和抗体(BNAbs)及其遗传偏好。 不同的丙型肝炎病毒E2结构与bNAbs和非nabs的络合物提供了结构 为合理设计疫苗奠定了基础。在本P01提案的项目2中，我们将结合 丙型肝炎病毒抗原、纳米颗粒工程、体内评估和B细胞的下一代测序(NGS) 协助合理设计可引起bNab反应的丙型肝炎疫苗的曲目。其中的具体目标 本项目是：(1)开发针对多供体类抗体反应的疫苗抗原。这个 主要的病毒中和部位，E2中和面(E2 NF)，是众所周知的多供体类抗体靶点 丙型肝炎病毒。E2NF2在构象上是灵活的，被免疫显性抗体包围和保护 环状结构和N-糖链。我们将通过计算模拟来优化E2和Nf附近的抗原表面 以及突变扫描，以将E2 NF“锁定”到所需的中和构象。刚性的E2 nF，以及 为了改进蛋白质折叠和最大限度地减少免疫优势表位的诱骗效应，应该 提高疫苗抗原的免疫原性，从而诱导多供体类bNAb对保守者 中和表位。(2)研制基于E1E2的疫苗抗原。除了E2 NF，原生E1E2 复合体还存在其他保守的bNab表位。我们将把扫描诱变应用于 E1和E2胞外区，并设计新的E1和E2融合结构以提高稳定性和产量 可溶性E1E2复合体的作用于针对该复合体的四级结构的bNAbs的诱导。(3) 开发自组装抗原呈递纳米颗粒疫苗。基因工程的E2和E1E2 将复合体与不同纳米平台的支架蛋白进行融合，以确定最佳的平台 用于向免疫系统展示丙型肝炎病毒抗原的多聚体。根据我们的初步数据，这一战略 将大大提高疫苗抗原的免疫原性，并引发快速而有效的NAB反应。(4)至 评价候选疫苗在小鼠和非人体内的免疫原性和抗体反应 灵长类(NHP)猕猴模型。经过改造的E2、E1E2抗原和纳米颗粒将首先 在小鼠身上进行研究，以确认它们在体内的免疫原性以及它们诱导抗体的能力，这些抗体可以结合和 中和丙型肝炎病毒。在体外和小鼠体内具有最好性能的抗原和纳米粒将被研究 进一步使用NHP模型。我们最近的研究表明，恒河猴对E1E2免疫的反应是 方式高度类似于人类使用的方式，并开发出针对E2和NF的bNAbs，其基因相似于 人的bNAbs。在这里，我们将免疫NHP，鉴定bNAbs，并执行曲目NGS来评估选定的 抗原设计。项目2的结果将是一组特征良好的候选丙型肝炎病毒疫苗。

Project Summary Hepatitis C virus (HCV) infects 1-2% of the world population and poses a significant threat to public health. Recent studies have identified a panel of broadly neutralizing antibodies (bnAbs) and their genetic preferences. The crystal structures of various HCV E2 constructs in complex with bnAbs and non-nAbs provide a structural basis for rational vaccine design. In Project 2 of this P01 proposal, we will combine structural optimization of HCV antigens, nanoparticle engineering, in vivo assessment, and next-generation sequencing (NGS) of B-cell repertoires to assist in rational design of HCV vaccines that can elicit a bnAb response. The Specific Aims in this project are: (1) To develop vaccine antigens to target multidonor class antibody responses. The major virus neutralizing site, the E2 neutralizing face (E2 NF), is a well-known multidonor class antibody target on HCV. E2 NF is conformationally flexible and is surrounded and protected by immunodominant variable loops and N-glycans. The antigenic surface at and around E2 NF will be optimized by computational modeling and mutagenesis scanning to “lock” E2 NF into desired neutralizing conformations. A rigid E2 NF, together with modifications to improve protein folding and to minimize the decoy effect of immunodominant epitopes, should improve the immunogenicity of vaccine antigens and as a result elicit multidonor class bnAbs to the conserved neutralizing epitopes. (2) To develop E1E2-based vaccine antigens. In addition to E2 NF, the native E1E2 complex also present other conserved bnAb epitopes. We will apply scanning mutagenesis to the interface of E1 and E2 ectodomains and design novel E1 and E2 fusion constructs to improve the stability and production of the soluble E1E2 complex for the elicitation of bnAbs targeting the quaternary structure of the complex. (3) To develop self-assembling antigen-presenting nanoparticle vaccines. The engineered E2 and E1E2 complex will be fused with the scaffold proteins of different nanoparticle platforms to identify a platform optimal for the multimeric display of HCV antigens to the immune system. Based on our preliminary data, this strategy will greatly improve the immunogenicity of vaccine antigens and elicit a rapid and potent nAb response. (4) To evaluate immunogenicity and antibody responses of vaccine candidates in the mouse and non-human primate (NHP) rhesus macaque models. The engineered E2, E1E2 antigens and nanoparticles will first be studied in mice to confirm their immunogenicity in vivo and their ability to elicit antibodies that can bind and neutralize HCV. The antigens and nanoparticles with the best properties in vitro and in mice will be studied further using the NHP model. We have recently shown that rhesus macaques react to E1E2 immunization in a manner highly reminiscent to that used by humans and develop bnAbs against E2 NF with genetic similarity to human bnAbs. Here, we will immunize NHPs, identify bnAbs, and perform repertoire NGS to evaluate selected antigen designs. The outcome of Project 2 would be a set of well-characterized HCV vaccine candidates.