Structure-based immunogen design for hepatitis C virus

 描述（由申请人提供）：据估计，世界人口的3%感染了丙型肝炎病毒（HCV），约1.8亿人是HCV的慢性携带者。最近的研究已经鉴定了HCV E1和E2蛋白上的许多中和表位，并用原子结构证明了广泛中和抗体（bnAb）如何与这些表位相互作用。HCV E2蛋白与bnAb（AR 3C）复合物的2.65 nm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。在这个R21应用中，我们建议将HCV结构生物学的最新发现与蛋白质设计和抗体库的下一代测序（NGS）的尖端技术相结合，以促进HCV免疫原设计，用于诱导疫苗接种中的bnAb。其具体目标是：（1）开发表位聚焦的免疫原。最近已经确定了与bnAb复合的E1和两个E2中和表位的结构。我们假设，通过将这些线性表位移植到蛋白质支架上，我们可以将这些表位以其bnAb结合构象呈现以诱导交叉中和抗体。“表位-支架”抗原将通过使用自动化元服务器方法的计算方法来设计，以鉴定用于最佳表位移植的不同蛋白质支架。成功设计和表达的抗原将以多价方式（24个拷贝）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。我们假设，具有稳定构象的优化的E2蛋白可以作为免疫原，以引发针对E2表面上呈现的多个表位的交叉中和抗体。基于E2核心结构域（E2 c）的晶体结构，我们将工程化可变环以最小化免疫显性非中和抗体应答，重新工程化表面上的N-连接聚糖，并在前层区域中引入空间填充突变以稳定E2 c的暴露的中和面。将在免疫实验中研究有希望的候选抗原，并通过一系列免疫测定和基于NGS的抗体库分析来监测抗体应答。

 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.