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

使用跨膜模拟支架设计免疫学完整的可溶性 HCV E1E2 复合物

• 批准号: 10043041
• 负责人: Thomas R Fuerst
• 金额: $ 19.19万
• 依托单位: UNIV OF MARYLAND, COLLEGE PARK
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-06-01 至 2022-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10043041
• 关键词: Affinity; Antibodies; Antibody Response; Antigens; Antiviral Agents; Architecture; Area; Binding; Biochemical; Biological Assay; Biophysics; C-terminal; CD81 gene; Cells; Complex; Computer Models; Cryoelectron Microscopy; Data; Development; Engineering; Enzyme-Linked Immunosorbent Assay; Epitopes; Exhibits; Genetic; Glycoproteins; Goals; Hepatitis C; Hepatitis C Therapy; Hepatitis C Vaccine; Hepatitis C virus; Human; Hydrophobicity; Immune response; Immune system; Immunologics; Individual; Infection; Influenza Hemagglutinin; Knowledge; Length; Light; Liver diseases; Malignant neoplasm of liver; Mammalian Cell; Maryland; Measures; Mediating; Membrane; Methods; Molecular Conformation; Monoclonal Antibodies; Mus; Patients; Plant Roots; Population; Preparation; Preventive vaccine; Property; Proteins; Role; Serum; Structure; Surface; System; Testing; Transmembrane Domain; Universities; Vaccine Clinical Trial; Vaccine Design; Vaccines; Variant; Viral; Virion; Virus; Virus Assembly; Work; analytical method; anti-hepatitis C; base; biophysical properties; comparative; design; design and construction; human monoclonal antibodies; immunogenicity; in vivo; injection drug use; insight; interdisciplinary approach; model design; neutralizing antibody; novel; novel strategies; scaffold; structural biology; three dimensional structure; vaccine development; virus envelope; young adult

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.

抽象的 全球丙型肝炎病毒 (HCV) 感染负担为 7100 万人，每年新增 175 万人 每年都有感染。在美国，由于注射吸毒，丙型肝炎病毒感染在年轻人中不断增加。一个 尽管在直接作用抗病毒药物（DAA）的开发方面取得了重大进展，但仍需要预防性疫苗 HCV 感染的治疗。 HCV 包膜糖蛋白 E1 和 E2 形成异二聚体和更高- 在本地病毒上订购程序集。该复合物是候选 HCV 疫苗中的关键抗原，包含 抗体对 HCV 反应的目标，但对其组装和组装的细节却知之甚少。 结构。有关 HCV 糖蛋白 E1 和 E2 组装决定因素的详细信息将极大地推进 我们对 HCV 结构和抗 HCV 免疫反应的了解，将使合理的疫苗设计成为可能 设计稳定的 E1E2 组装和表位呈现。各种研究表明，C- E1 和 E2 的末端跨膜结构域对于 E1E2 复合物的组装至关重要，但 E1 和 E2 的存在 跨膜结构域阻碍了详细的结构研究、生化表征和疫苗 发展。我们建议设计可溶性 E1E2 组件，其中包含以下功能的替代品： 促进天然寡聚化的跨膜结构域，类似于稳定其他物质的成功努力 跨膜病毒糖蛋白，例如流感血凝素和 RSV F。我们提出了一种迭代的、 设计可溶性原生 E1E2 组件的跨学科方法。在目标 1 中，支架将选自 已知的寡聚结构和确定的架构。计算建模和设计方法将 用于优化支架并根据初始实验数据生成新型支架。在目标 2 中， 将使用哺乳动物细胞生产使用 Aim 1 中最佳支架的纯化可溶性 E1E2 复合物 表达系统。天然 E1E2 组装将通过一组抗体的结合测定来确认 靶向 E1、E2 和 E1E2 以及 HCV 辅助受体上的构象表位。生物物理测定将 用于评估设计结构的大小、寡聚化和其他特性。那些具有本土特色的人 抗原性将通过冷冻电镜进行结构表征，并且将使用体内免疫原性研究来确定 确认顶级可溶性 E1E2 设计可引发强大的中和抗体，并确定之间的相关性 结构、抗原性、生物物理特性和免疫原性。提供概念证明，这是我们最初的目标之一 设计表现出对多种针对天然 E1E2 构象表位的人类抗体的反应性 复杂的。这项工作将为E1E2合理设计和结构生物学建立平台。这些研究将 从而能够研究 E1E2 复合物的组装、免疫系统的识别以及在病毒进入中的作用 跨越 HCV 的不同遗传景观。此外，通过这项工作学到的原则将提供 进一步努力合理设计 E1E2 糖蛋白 HCV 疫苗的框架。