Biochemical and Biophysical Characterization of HIV Env Trimer Spikes using Nanod

使用 Nanod 对 HIV 包膜三聚体尖峰进行生化和生物物理表征

• 批准号: 7929402
• 负责人: Carlos Enrique Catalano
• 金额: $ 22万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-01 至 2012-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7929402
• 关键词: Acquired Immunodeficiency Syndrome; Affect; Antibodies; Antigens; Binding; Binding Proteins; Biochemical; Biological; Biological Process; CD4 Antigens; Cardiolipins; Cells; Cellular Membrane; Covalent Interaction; Data; Development; Epitopes; Event; Foundations; Future; Glycoproteins; Goals; Golgi Apparatus; HIV; HIV Envelope Protein gp120; HIV vaccine; HIV-1; High Density Lipoproteins; Human; In Situ; In Vitro; Infection; Integral Membrane Protein; Length; Lipid Bilayers; Lipid Binding; Lipids; Measures; Mediating; Membrane; Membrane Fusion; Membrane Lipids; Membrane Proteins; Molecular Conformation; Mono-S; Nucleocapsid; Patients; Peptide Hydrolases; Phospholipids; Preparation; Proteins; Proteolysis; Reaction; Research Personnel; Series; Site; Structure; Surface; System; Technology; Vaccines; Viral; Virus Diseases; Work; design; experience; gp160; high risk; innovation; insight; membrane model; neutralizing antibody; neutralizing vaccine; novel; pandemic disease; particle; public health relevance; reverse cholesterol transport; vaccine development; virus envelope

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.

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症的病原体，这是一种毁灭性的感染，已达到大流行的程度。 HIV 进入许可细胞的第一步也是先决步骤是病毒糖蛋白 gp120 与细胞 CD4 受体特异性结合；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以及核衣壳进入细胞内部。 Gp120 通过与三聚体 gp41（一种介导膜融合事件的整合膜蛋白）的非共价相互作用而与病毒包膜相连。三聚体 gp120/gp41 刺突代表暴露在病毒表面的主要表位，多种三聚体“gp140”缺失构建体和可溶性单体 gp120 构建体已被研究作为免疫原来引发针对 HIV 的广泛中和抗体 (bNtAb)。尽管做出了巨大的努力，针对 HIV 原代分离株的 bNtAb 诱导始终不成功。由此得出的结论是，对 HIV 包膜糖蛋白 (Env) 刺突结构及其与 bNtAb 相互作用的基本了解是未来疫苗开发的关键。不幸的是，由于缺乏体外系统来研究可溶性、生物相关性和功能性脂质结合构象的 Env 三聚体，这一目标未能实现。磷脂纳米盘提供了这样的系统。纳米圆盘源自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的形式嵌入其中。它们尺寸均匀、成分明确，提供稳定、可溶和单分散的平台，适合严格的生化、生物物理和结构研究。我们建议利用纳米圆盘技术将 HIV Env 三聚体组装在限定的脂质双层中，并表征三聚体刺突的结构和功能。该应用程序代表了一个“概念验证”提案，非常适合 R21 应用程序，因为它代表了适度风险、高收益的提案。成功完成既定目标将为研究 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 疫苗的研究奠定基础。 公共卫生相关性：艾滋病毒感染已达到大流行的程度，开发有效的疫苗对于遏制艾滋病至关重要。先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。我们建议利用纳米圆盘技术来组装和表征可溶性脂质双层中的环境尖峰。这项工作将为未来利用这种创新免疫原开发艾滋病毒疫苗的研究奠定基础。