Design of immunologically intact soluble HCV E1E2 complexes using transmembrane-mimic scaffolds

摘要 丙型肝炎病毒（HCV）感染的全球负担为7100万，每年新增175万例 每年感染。在美国，由于注射吸毒，年轻人的HCV感染正在增加。一 尽管直接作用抗病毒药物（DAA）的开发取得了重大进展，但仍需要预防性疫苗。 治疗HCV感染。HCV包膜糖蛋白E1和E2形成异二聚体和更高的- 在本机病毒上订购程序集。该复合物是候选HCV疫苗中的关键抗原，并且包含 HCV抗体应答的靶点，但对其组装的细节知之甚少， 结构HCV糖蛋白E1和E2组装决定簇的详细信息将大大推进 我们对HCV结构和抗HCV免疫应答的了解，将使合理的疫苗设计成为可能 来设计稳定的E1 E2组装和表位呈递。各种研究表明，C- E1和E2的末端跨膜结构域对于E1 E2复合物组装是至关重要的，然而， 跨膜结构域阻碍了详细的结构研究、生物化学表征和疫苗 发展我们建议设计可溶性的E1 E2组件，其中包含功能性替代物， 促进天然寡聚化的跨膜结构域，类似于稳定其他跨膜结构域的成功努力。 跨膜病毒糖蛋白，如流感血凝素和RSV F。我们提出了一个迭代的， 跨学科方法设计可溶性天然E1 E2组装体。在目标1中，支架将选自 已知的低聚结构和确定的结构。计算建模和设计方法将是 用于优化支架和基于初始实验数据产生新的支架。在目标2中， 使用来自Aim 1的最佳支架的纯化的可溶性E1 E2复合物将使用哺乳动物细胞产生 表达系统。将使用与一组抗体的结合试验确认天然E1 E2组装 靶向E1、E2和E1 E2上的构象表位以及HCV辅助受体。生物物理分析将是 用于评估设计构建体的大小、寡聚化和其它性质。那些像本地人一样 抗原性将通过冷冻EM进行结构表征，体内免疫原性研究将用于 证实最高可溶性E1 E2设计引发稳健的中和抗体，并确定 结构、抗原性、生物物理性质和免疫原性。提供概念验证，我们最初的 设计表现出对靶向天然E1 E2上构象表位的多种人抗体的反应性 复杂.本工作将为E1 E2基因的合理设计和结构生物学研究建立一个平台。这些研究将 从而能够研究E1 E2复合物的组装、免疫系统的识别以及在病毒进入中的作用 在HCV的不同遗传环境中。此外，通过这项工作学到的原则将提供 进一步努力的框架，旨在合理设计E1 E2糖蛋白HCV疫苗。

ABSTRACT The global burden of hepatitis C virus (HCV) infection is at 71 million with an annual rate of 1.75 million new infections each year. In the US, HCV infection is increasing in young adults because of injection drug use. A preventive vaccine is needed in spite of major advances in the development of direct acting antivirals (DAAs) for the treatment of HCV infections. The HCV envelope glycoproteins E1 and E2 form a heterodimer and higher- order assemblies on the native virus. This complex is the key antigen in candidate HCV vaccines, and comprises the target of the antibody response to HCV, yet strikingly little is known about the details of its assembly and structure. Detailed information on HCV glycoprotein E1 and E2 assembly determinants would greatly advance our knowledge of HCV structure and the anti-HCV immune response, and would enable rational vaccine design to engineer stable E1E2 assembly and epitope presentation. Various studies have demonstrated that the C- terminal transmembrane domains of E1 and E2 are critical for E1E2 complex assembly, yet the presence of the transmembrane domains hinders detailed structural studies, biochemical characterization, and vaccine development. We propose to design soluble E1E2 assemblies containing functional replacements for the transmembrane domains which promote native oligomerization, analogous to successful efforts to stabilize other transmembrane viral glycoproteins such as influenza hemagglutinin and RSV F. We propose an iterative, interdisciplinary approach to design soluble native E1E2 assemblies. In Aim 1, scaffolds will be selected from known oligomeric structures and defined architectures. Computational modeling and design methods will be used both to optimize scaffolds and to generate novel scaffolds based on initial experimental data. In Aim 2, purified soluble E1E2 complexes using the best scaffolds from Aim 1 will be produced using mammalian cell expression systems. Native E1E2 assembly will be confirmed using binding assays to a panel of antibodies targeting conformational epitopes on E1, E2 and E1E2, as well as HCV coreceptors. Biophysical assays will be used to assess size, oligomerization, and other properties of designed constructs. Those with native-like antigenicity will be structurally characterized by cryo-EM, and an in vivo immunogenicity study will be used to confirm that top soluble E1E2 designs elicit robust neutralizing antibodies, and to determine correlates between structure, antigenicity, biophysical properties, and immunogenicity. Providing a proof of concept, one of our initial designs exhibits reactivity to multiple human antibodies targeting conformational epitopes on the native E1E2 complex. This work will establish a platform for E1E2 rational design and structural biology. These studies will thereby enable the study of E1E2 complex assembly, recognition by the immune system, and role in viral entry across the diverse genetic landscape of HCV. Moreover, the principles learned through this work will provide the framework for further efforts aimed at rational design of an E1E2 glycoprotein HCV vaccine.