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征的病因，这是一种毁灭性的感染，已达到大流行比例。 HIV进入允许细胞的第一个和必要的步骤是病毒糖蛋白GP120与细胞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，该步骤最终导致病毒包膜与细胞膜融合，并将Nucleocapsid进入细胞内部。 GP120通过与Trimeric GP41的非共价相互作用（一种介导膜融合事件的积分膜蛋白）束缚在病毒包膜上。三聚体GP120/GP41尖峰代表暴露在病毒表面上的主要表位，并且已经研究了各种三聚体“ GP140”缺失构建体和可溶性单体GP120构建体，作为疫苗的疫苗，以引起广泛中和的抗体（BNTABS）至HIV。尽管努力了英勇的努力，但诱导BNTAB对艾滋病毒的主要孤立株的诱导却没有成功。这导致了一个结论，即对HIV包膜糖蛋白（ENV）尖峰结构的基本了解及其与BNTABS的相互作用是未来疫苗开发的关键。不幸的是，由于缺乏体外系统，该目标允许在可溶性，生物学相关和功能性脂质结合的构象中研究ENV三聚体。磷脂纳米盘提供了这样的系统。纳米盘源自参与人类反向胆固醇转运的高密度脂蛋白颗粒。它们提供稳定的模型膜，其中膜蛋白可以以天然和功能形式嵌入。它们的大小是同质的，并在组成中定义，并提供了一个稳定，可溶和单分散的平台，该平台可与严格的生化，生物物理和结构询问相提并论。我们建议利用纳米轴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“概念验证”提案，理想地适用于R21应用程序，因为它代表了适度的风险，高收益率建议。成功完成陈述的目标将为研究ENV结构和功能提供一个新颖的平台，并为将来的研究提供基础，以将这种创新的免疫原进行HIV疫苗开发。 公共卫生相关性：艾滋病毒感染已达到大流行比例，有效疫苗的开发将对含有艾滋病的含量至关重要。以前的疫苗开发尝试令人失望，这在很大程度上是因为“理性设计的”免疫原未能模仿HIV信封（ENG）尖峰结构的自然表现。我们建议利用纳米轴技术在可溶性脂质双层中组装和表征ENV尖峰。这项工作将为将来的研究提供基础，该研究将利用这种创新的免疫原进行HIV疫苗开发。