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

项目概要/摘要： 世界卫生组织将其视为优先新兴病原体，因为其可能 导致公共卫生危机和缺乏有效的治疗方法或疫苗，1拉沙热 据估计，拉沙病毒每年影响30万人，造成约5 000人死亡。 （LASV）-沙粒病毒科的成员-呈现高度糖基化的包膜糖蛋白 凝胶渗透色谱（GPC）是许多临床前疫苗研究的焦点。三聚体构象 在免疫研究中，GPC是诱导几乎所有中和抗体应答所必需的3; 然而，溶解的胞外域是固有地不稳定的，并且已被证明是不稳定的， 我们最近的工作描述了三聚支架的发展， 位于GPC的膜近端区域，其使蛋白质以其三聚体形式稳定。 这个项目将使用这个平台来描述来自LASV的类似本地的、有代表性的GPC 谱系和结构探索它们与宿主细胞受体的相互作用。 我假设高分辨率的LASV结构将显示保守的表位，特别是在它们的 受体结合区，这将用于指导下一代免疫原的设计和诱导 更强的中和体液反应。我将通过三个主要目标来检验这个假设： 1)解决了代表性的高分辨率，载脂蛋白结构的LASV GPC跨谱系的单- 2）鉴定促进与GPC宿主细胞受体结合的关键残基， 和3）开发一套代表临床相关LASV的稳定、可溶的GPC三聚体 菌株我希望这些目标的结果将证明保守的GPC表位， 中和性免疫应答在性质上将主要是四级的，这迄今为止一直是 由于GPC三聚体的不稳定性，在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 免疫学方面的知识额外的专业发展资源， 斯克里普斯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 我的长期目标是指导一个研究实验室，研究新出现的病原体疫苗的开发。

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.