Novel HCV vaccine antigens and nanoparticles

项目摘要 丙型肝炎病毒（HCV）感染了世界人口的1-2%，对公共卫生构成重大威胁。 最近的研究已经确定了一组广泛中和抗体（bnAb）及其遗传偏好。 与bnAb和非nAb复合的各种HCV E2构建体的晶体结构提供了结构上的互补性。 合理疫苗设计的基础。在本P01建议书的项目2中，我们将结合联合收割机的结构优化， HCV抗原、纳米颗粒工程、体内评估和B细胞的下一代测序（NGS） 这有助于合理设计可引发bnAb应答的HCV疫苗。具体目标在 本研究的主要目的是：（1）开发针对多供体类抗体反应的疫苗抗原。的 主要的病毒中和位点，E2中和面（E2 NF），是众所周知的多供体类抗体靶标 关于HCV E2 NF在构象上是柔性的，并被免疫显性可变区包围和保护 环和N-聚糖。将通过计算建模优化E2 NF处及其周围的抗原表面 和诱变扫描以将E2 NF“锁定”成所需的中和构象。刚性E2 NF，以及 修饰以改善蛋白质折叠和最小化免疫显性表位的诱饵效应， 提高疫苗抗原的免疫原性，并因此引发针对保守的 中和表位(2)开发基于E1 E2的疫苗抗原。除了E2 NF，天然E1 E2 复合物还呈现其他保守的bnAb表位。我们将扫描诱变应用于 E1和E2胞外域，并设计新的E1和E2融合构建体以提高稳定性和产量 用于引发靶向复合物四级结构的bnAb。（三） 研制自组装抗原提呈纳米颗粒疫苗。E2和E1 E2 复合物将与不同纳米颗粒平台的支架蛋白融合，以确定最佳平台 用于HCV抗原向免疫系统的多聚体展示。根据我们的初步数据， 将大大提高疫苗抗原的免疫原性，并引发快速和有效的nAb应答。(4)到 评价候选疫苗在小鼠和非人中的免疫原性和抗体应答 灵长类（NHP）恒河猴模型。工程化的E2、E1 E2抗原和纳米颗粒将首先被 在小鼠中进行研究，以确认它们的体内免疫原性和它们引发能够结合和 中和HCV。将研究在体外和小鼠中具有最佳性能的抗原和纳米颗粒 进一步使用NHP模型。我们最近发现恒河猴对E1 E2免疫的反应是， 的方式，并开发针对E2 NF的bnAb，其与 人bnAb。在这里，我们将免疫NHP，鉴定bnAb，并进行NGS，以评估选择的 抗原设计项目2的结果将是一组充分表征的HCV候选疫苗。

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.