Biochemical and Biophysical Characterization of HIV Env Trimer Spikes using Nanod

描述(申请人提供)：人类免疫缺陷病毒(HIV)是获得性免疫缺陷综合症的病原体，这是一种破坏性的感染，已达到大流行的程度。HIV进入允许细胞的第一步也是先决条件是病毒糖蛋白gp120与细胞内CD4受体的特异性结合；这种相互作用触发了一系列有序的步骤，最终导致病毒包膜与细胞膜融合，并使核衣壳进入细胞内部。Gp120通过与三聚体gp41的非共价相互作用被拴在病毒的包膜上，三聚体gp41是一种介导膜融合事件的完整膜蛋白。三聚体gp120/gp41代表暴露在病毒表面的主要表位，各种三聚体gp140缺失结构和可溶性单体gp120结构作为免疫原被研究以诱导针对HIV的广谱中和抗体(BNtAbbs)。尽管作出了英勇的努力，但针对艾滋病毒初级分离株的bNtAbbs的诱导一直都没有成功。这导致了一个结论，即对HIV包膜糖蛋白(Env)尖峰结构及其与bNtAbs的相互作用的基本了解是未来疫苗开发的关键。不幸的是，这一目标由于缺乏体外系统而受挫，该系统允许以可溶的、生物相关的和功能性的脂质结合构象来研究环境三聚体。磷脂纳米盘提供了这样的系统。纳米盘是从参与人类胆固醇反向运输的高密度脂蛋白颗粒中衍生出来的；它们提供了稳定的模型膜，膜蛋白可以天然和功能的形式嵌入其中。它们在大小上均一，在组成上有定义，并提供了一个稳定的、可溶的和单分散的平台，可以接受严格的生化、生物物理和结构询问。我们建议利用纳米盘技术将HIV Env三聚体组装在定义的脂质双层中，并表征三聚体尖峰的结构和功能。这个应用程序代表了一个非常适合R21应用程序的“概念验证”建议，因为它代表了中等风险、高收益的建议。上述目标的成功完成将为研究环境病毒的结构和功能提供一个新的平台，并为未来利用这种创新的免疫原开发艾滋病毒疫苗的研究奠定基础。 与公共卫生相关：艾滋病毒感染已经达到大流行的程度，开发有效的疫苗将是遏制艾滋病的首要任务。以前的疫苗开发尝试一直令人失望，很大程度上是因为“合理设计”的免疫原未能模仿HIV包膜(Env)尖峰结构的自然呈现。我们建议利用纳米盘技术来组装和表征可溶脂双层中的Env刺激物。这项工作将为未来的研究提供基础，这些研究将利用这种创新的免疫原开发艾滋病毒疫苗。

DESCRIPTION (provided by applicant): The Human Immunodeficiency Virus (HIV) is the causative agent of Acquired Immunodeficiency Syndrome, a devastating infection that has reached pandemic proportions. The first and pre- requisite step for HIV entry into a permissive cell is specific binding of the viral glycoprotein gp120 to the cellular CD4 receptor; this interaction triggers an ordered series of steps that ultimately results in fusion of the viral envelope with the cellular membrane and entry of the nucleocapsid into the cell interior. Gp120 is tethered to the viral envelope via non-covalent interactions with trimeric gp41, an integral membrane protein that mediates membrane fusion events. Trimeric gp120/gp41 spikes represent the primary epitope exposed on the viral surface and a variety of trimeric "gp140" deletion constructs and soluble monomeric gp120 constructs have been studied as immunogens to elicit broadly neutralizing antibodies (bNtAbs) to HIV. Despite heroic efforts, induction of bNtAbs against primary isolates of HIV has been uniformly unsuccessful. This has led to the conclusion that a fundamental understanding of the HIV envelope glycoprotein (Env) spike structure and its interaction with bNtAbs is key to future vaccine development. Unfortunately, this goal has been frustrated by the lack of an in vitro system that allows the study of Env trimers in a soluble, biologically relevant, and functional lipid-bound conformation. Phospholipid nanodiscs provide such a system. Nanodiscs are derived from high-density lipoprotein particles involved in reverse cholesterol transport in humans; they provide stable model membranes into which membrane proteins can be embedded in a native and functional form. They are homogenous in size and defined in composition, and provide a stable, soluble, and mono-disperse platform that is amenable to rigorous biochemical, biophysical, and structural interrogation. We propose to utilize nanodisc technology to assemble HIV Env trimers in a defined lipid bilayer and to characterize the structure and function of the trimeric spikes. This application represents a "proof-of-concept" proposal that is ideally suited to an R21 application as it represents modest-risk, high yield proposal. Successful completion of the stated goals will provide a novel platform to study Env structure and function and provide the foundation for future studies that will utilize this innovative immunogen for HIV vaccine development. PUBLIC HEALTH RELEVANCE: HIV infection has reached pandemic proportions and the development of an effective vaccine will be paramount to containing AIDS. Previous attempts at vaccine development have been disappointing, in large part because the "rationally designed" immunogens have failed to mimic the natural presentation of the HIV envelope (Env) spike structure. We propose to utilize nanodisc technology to assemble and characterize Env spikes in a soluble lipid bilayer. This work will provide the foundation for future studies that will utilize this innovative immunogen for HIV vaccine development.