Evaluation of structural conservation and glycan-mediated host receptor engagement of pan-lineage Lassa fusion glycoproteins by cryo-EM

通过冷冻电镜评估全谱系拉沙融合糖蛋白的结构保守性和聚糖介导的宿主受体接合

• 批准号: 10536102
• 负责人: Hailee Rose Perrett
• 金额: $ 3.38万
• 依托单位: SCRIPPS RESEARCH INSTITUTE, THE
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-01 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10536102
• 关键词: Affect; Antibodies; Antibody Response; Antigens; Area; Arenavirus; Bacteria; Binding; Biological Assay; Cells; Cessation of life; Charge; Clinic; Complex; Cryoelectron Microscopy; Crystallization; Data; Development; Dystroglycan; Engineering; Ensure; Enzyme-Linked Immunosorbent Assay; Epitopes; Evaluation; Exposure to; Extracellular Domain; Family; Foundations; GP2 gene; GTPBP1 gene; Glycoproteins; Goals; Human; Hydrophobic Interactions; Immune response; Immunization; Immunology; Laboratories; Lassa Fever; Lassa virus; Literature; Mediating; Membrane; Mentors; Methods; Modification; Molecular Conformation; Nature; Oligosaccharides; Patients; Persons; Polysaccharides; Positioning Attribute; Process; Proteins; Protocols documentation; Protomer; Public Health; Reagent; Receptor Cell; Recombinants; Reporting; Research; Research Institute; Resolution; Resource Development; Scheme; Site; Structure; Surface; Temperature; Testing; Therapeutic; Time; Training; Vaccines; Viral; Work; World Health Organization; alpha Dystroglycan; biophysical properties; clinically relevant; cross reactivity; design; diagnostic tool; disulfide bond; emerging pathogen; extracellular; field study; improved; member; monomer; neutralizing antibody; next generation; particle; pre-clinical; receptor; receptor binding; response; scaffold; skills; structural biology; sugar; therapeutic development; tomography; vaccine candidate; vaccine development; vaccine evaluation; vaccine trial; virology

Project Summary/Abstract: Recognized as a priority emerging pathogen by the World Health Organization due to its potential to cause a public health crisis and the absence of efficacious therapeutics or vaccines,1 Lassa fever affects an estimated 300,000 people per year and results in approximately 5000 deaths.2 Lassa virus (LASV)—a member of the Arenaviridae family—presents heavily glycosylated envelope glycoprotein complexes (GPC) which are the focus of many preclinical vaccine studies. The trimeric conformation of GPC is necessary to induce almost all neutralizing antibody responses in immunization studies3; however, the solubilized ectodomain is inherently unstable and has proved recalcitrant to unbound structural studies in the past.4 Our recent work describes the development of a trimerization scaffold positioned at the membrane proximal region of GPC which stabilizes the protein in its trimeric form. This project will use this platform to describe native-like, representative GPCs from across LASV lineages and structurally explore their interactions with host cell receptors. I hypothesize the high-resolution LASV structures will show conserved epitopes, especially at their receptor-binding regions, which will be used to guide next-generation immunogen design and induce more robust and neutralizing humoral responses. I will test this hypothesis through three primary aims: 1) solving the representative high-resolution, apo structures of LASV GPC across lineages by single- particle cryo-EM, 2) identifying the key residues which facilitate binding with GPC host cell receptors, and 3) developing a suite of stable, soluble GPC trimers representative of clinically relevant LASV strains. I expect the results of these aims will demonstrate conserved GPC epitopes which facilitate neutralizing immune responses will be predominantly quaternary in nature, which has thus far been largely undescribed in the literature due to the instability of the GPC trimer. During this process, I will receive comprehensive training in structural biology, virology, and immunology from my interdisciplinary mentors. The additional professional development resources at the Scripps Research Institute and in the broader San Diego area will ensure I develop the skills needed for my long-term goal of directing a research lab in emerging pathogen vaccine development.

项目摘要/摘要： 由于其潜力 造成公共卫生危机和缺乏高效疗法或疫苗，1个LASSA热 估计每年30万人影响约5000人死亡。2Lassa病毒 （LASV） - Arenaviridae家族的成员 - 大量糖基化包膜糖蛋白 复合物（GPC）是许多临床前疫苗研究的重点。三聚体构象 在免疫研究中，GPC的几乎所有中和抗体反应都是必要的。 但是，可溶性外生域本质上是不稳定的，已证明是顽固的 4过去的结构研究。4我们最近的工作描述了修剪脚手架的发展 位于GPC的膜近端区域，该区域以三聚体形式稳定蛋白质。 该项目将使用此平台来描述来自LASV的类似本地的代表GPC 谱系和结构探索他们与宿主细胞接收器的相互作用。 我假设高分辨率LASV结构将显示保守的表位，尤其是在其 受体结合区域，该区域将用于指导下一代免疫原设计和影响 更健壮和中和的体液反应。我将通过三个主要目的检验这一假设： 1）通过单个谱系求解LASV GPC的代表性高分辨率结构 粒子冷冻-EM，2）识别关键保留，以促进与GPC宿主细胞接收器结合， 3）开发一套稳定的可溶性GPC三聚体代表临床相关的LASV 菌株。我希望这些目标的结果将证明哪些设施的保守GPC表位 中和免疫反应本质上将主要是第四纪，迄今为止 由于GPC三聚体的不稳定性，文献中基本上没有描述。 在此过程中，我将接受结构生物学，病毒学和 我的跨学科导师的免疫学。额外的专业发展资源 Scripps研究所和更广泛的圣地亚哥地区将确保我发展所需的技能 对于我指导新兴病原体疫苗开发中的研究实验室的长期目标。