Structure-based immunogen design for hepatitis C virus

 描述(申请人提供)：据估计，世界上3%的人口感染丙型肝炎病毒(丙型肝炎病毒)，约1.8亿人是丙型肝炎病毒的慢性携带者。最近的研究已经确定了丙型肝炎病毒E1和E2蛋白上的一些中和表位，并用原子结构证明了广谱中和抗体(BNAbs)如何与这些表位相互作用。丙型肝炎病毒E2蛋白与bNab(AR3C)形成的复合体的2.65？分辨率结构揭示了这个长期寻找的靶点的原子细节，为合理的疫苗设计提供了结构基础。在R21的应用中，我们建议将丙型肝炎病毒结构生物学的最新发现与蛋白质设计和抗体谱下一代测序(NGS)方面的尖端技术相结合，以促进丙型肝炎病毒免疫原设计，以诱导疫苗接种中的bNAbs。其具体目标是：(1)开发以表位为中心的免疫原。最近确定了一个E1和两个E2中和表位的bNAbs复合体的结构。我们假设，通过将这些线性表位嫁接到蛋白质支架上，我们可以将这些表位以其bNab结合的构象呈现出来，以诱导交叉中和抗体。“表位-支架”抗原将通过使用自动元服务器方法的计算方法来设计，以识别各种蛋白质支架，以实现最佳表位嫁接。成功设计和表达的抗原将以多价方式(24个副本)展示在铁蛋白颗粒上。(2)设计和优化E2糖蛋白作为亚单位免疫原。我们推测，具有稳定构象的优化的E2蛋白可以作为一种免疫原来诱导针对呈现在E2表面的多个表位的交叉中和抗体。基于E2核心区(E2C)的晶体结构，我们将设计可变环以最小化免疫显性非中和抗体反应，重新设计表面N-连接的糖链，并在前层区域引入空间填充突变以稳定E2C暴露的中和面。有希望的候选抗原将在免疫实验中进行研究，抗体反应将通过一系列免疫学分析和基于NGS的抗体库分析进行监测。

 DESCRIPTION (provided by applicant): It has been estimated that 3% of the word population is infected with hepatitis C virus (HCV) and approximately 180 million individuals are chronic carriers of HCV. Recent studies have identified a number of neutralizing epitopes on the HCV E1 and E2 proteins and demonstrated with atomic structures how broadly neutralizing antibodies (bnAbs) interact with these epitopes. The 2.65Å-resolution structure of HCV E2 protein in complex with a bnAb (AR3C) revealed atomic details of this long-sought target, providing a structural basis for rational vaccine design. In this R21 application, we propose to combine the latest findings in HCV structural biology and cutting-edge technologies in protein design and next-generation sequencing (NGS) of antibody repertoires to facilitate HCV immunogen design for the induction of bnAbs in vaccination. The specific aims are: (1) to develop epitope-focused immunogens. The structures of an E1 and two E2 neutralizing epitopes in complex with bnAbs have been determined recently. We hypothesize that by grafting these linear epitopes onto protein scaffolds we can present these epitopes in their bnAb-bound conformations to induce cross-neutralizing antibodies. The "epitope-scaffold" antigens will be designed by a computational approach using an automated meta-server method to identify diverse protein scaffolds for optimal epitope grafting. Successfully designed and expressed antigens will be displayed in a multivalent manner (24 copies) on ferritin particle. (2) to engineer and optimize E2 glycoprotein as a subunit immunogen. We hypothesize that an optimized E2 protein with stabilized conformation can serve as an immunogen to elicit cross-neutralizing antibodies to multiple epitopes presented on the E2 surface. Based on the crystal structure of E2 core domain (E2c), we will engineer the variable loops to minimize immunodominant non-neutralizing antibody response, reengineer N- linked glycans on the surface, and introduce space-filling mutations in the front layer region to stabilize the exposed neutralizing face of E2c. Promising antigen candidates will be studied in immunization experiments and the antibody responses will be monitored by an array of immunological assays and by NGS-based antibody repertoire analysis.