Biochemical and Biophysical Characterization of HIV Env Trimer Spikes using Nanod

描述（由申请人提供）：人类免疫缺陷病毒（HIV）是获得性免疫缺陷综合征的病原体，这是一种破坏性感染，已达到大流行的程度。HIV进入允许细胞的第一步和先决条件是病毒糖蛋白gp 120与细胞CD 4受体的特异性结合;这种相互作用引发了一系列有序的步骤，最终导致病毒包膜与细胞膜融合，核衣壳进入细胞内部。gp 120通过与三聚体gp 41（一种介导膜融合事件的整合膜蛋白）的非共价相互作用被束缚在病毒包膜上。三聚体gp 120/gp 41刺突代表暴露在病毒表面上的主要表位，并且已经研究了多种三聚体“gp 140”缺失构建体和可溶性单体gp 120构建体作为引发针对HIV的广泛中和抗体（bNtAb）的免疫原。尽管做出了巨大的努力，但针对HIV的主要分离株的bNtAb的诱导一直不成功。这导致了这样的结论，即对HIV包膜糖蛋白（Env）刺突结构及其与bNtAb的相互作用的基本理解是未来疫苗开发的关键。不幸的是，这一目标已被挫败的缺乏一个体外系统，允许研究的Env三聚体在一个可溶的，生物学相关的，和功能性的脂质结合构象。磷脂纳米盘提供了这样的系统。纳米盘来源于参与人体胆固醇逆向转运的高密度脂蛋白颗粒;它们提供了稳定的模型膜，膜蛋白可以以天然和功能性形式嵌入其中。它们大小均匀，组成明确，提供稳定、可溶和单分散的平台，可进行严格的生物化学、生物物理和结构研究。我们建议利用nanodisc技术在定义的脂质双层中组装HIV Env三聚体，并表征三聚体尖峰的结构和功能。该应用程序代表了一个“概念验证”方案，非常适合R21应用程序，因为它代表了中等风险、高收益的方案。上述目标的成功完成将为研究Env结构和功能提供一个新的平台，并为未来利用这种创新免疫原开发HIV疫苗的研究提供基础。 公共卫生相关性：艾滋病毒感染已达到流行病的程度，研制有效的疫苗对遏制艾滋病至关重要。以前的疫苗开发尝试令人失望，很大程度上是因为“合理设计”的免疫原未能模仿HIV包膜（Env）刺突结构的自然呈现。我们建议利用nanodisc技术来组装和表征可溶性脂质双层中的Env尖峰。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进行HIV疫苗的开发。

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.