The role of the trimer association domain (TAD) in controlling the conformation of the HIV-1 envelope trimer and protection of the CD4 binding site

三聚体关联结构域 (TAD) 在控制 HIV-1 包膜三聚体构象和保护 CD4 结合位点中的作用

• 批准号: 9203655
• 负责人: PAUL R CLAPHAM
• 金额: $ 25.13万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-06-01 至 2018-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9203655
• 关键词: Affect; Amino Acid Substitution; Amino Acids; Antibodies; Antigens; Binding; Binding Sites; Biological Assay; Brain; Cataloging; Catalogs; Codon Nucleotides; Data; Development; Ensure; Environment; Epitopes; Equilibrium; Frequencies; Future; Glycoproteins; Growth; HIV; HIV Envelope Protein gp120; HIV vaccine; HIV-1; Human; Immune; Individual; Infection; Investigation; Libraries; Maps; Membrane; Modification; Molecular Conformation; Monoclonal Antibodies; Mutagenesis; Mutation; Neutralization Tests; Play; Randomized; Reporting; Resolution; Role; Series; Site; Structure; Surface; Testing; Tissues; V3 Loop; Vaccines; Viral; Virus; Virus Replication; base; deep sequencing; design; mutant; neutralizing antibody; neutralizing monoclonal antibodies; next generation; novel; novel vaccines; optimism; pressure; vaccine trial; virus envelope

HIV-1 envelope (Env) vaccines have failed to induce neutralizing antibodies (nabs) with strong activity against diverse HIV-1. However, the identification of potent, broad neutralizing monoclonal antibodies along with evidence that V2 and V3 loop antibodies contributed to protection in the RV144 vaccine trial, have increased optimism that an Env based vaccine is possible. However, it remains unclear how to present conserved Env epitopes on immunogens so that broadly active nabs are elicited. Native Env trimers in immune tissue are likely to be closed to protect critical sites from nabs. The trimer association domain (TAD) at the trimer apex along with residues in the CD4 binding loop region help maintain a closed conformation. We propose that understanding how TAD conformation is controlled will be critical for the development of trimeric Env immunogens that elicit broad and potent nabs. However, determinants that maintain a closed trimer and regulate TAD conformation are poorly understood. Here, we will use EMPIRIC (Exceedingly Meticulous and Parallel Investigation of Randomized Individual Codons), a novel saturation mutagenesis approach to identify residues in the V1V2 and V3 loops that regulate TAD conformations across clades. This approach is supported by strong preliminary data and will be used to identify mutant trimers that carry TADs with distinct conformations. Such trimers will represent novel immunogens for future studies. Aim 1. To identify amino acids in the TAD that influence viral replication using EMPIRIC. These amino acids are candidates for regulating TAD and trimer conformations: We will use EMPIRIC saturation mutagenesis to investigate V1, V2 and V3 loops of the TAD in clade A, B and C Envs. We will catalogue mutations in these Env regions that confer wild type or enhanced replication in PBMCs compared to wt Env+ virus, before analyzing their effects on Env and TAD conformation in aim 2. Aim 2. To characterize mutant Envs for modifications in TAD and trimer conformation: We will investigate how mutations identified in aim 1 impact on TAD and trimer conformation. We will use a novel trimer binding assay and Env+ pseudovirus neutralization tests to characterize the different Env mutants. A broad panel of monoclonal antibodies against the TAD, CD4bs and other Env regions as well as sCD4 will be tested to determine changes in TAD conformation and CD4bs exposure as well as effects on other Env sites. Our data will provide detailed information on TAD amino acids that regulate trimer conformation. We will create an Env structural map relating specific V1V2 abd V3 loop amino acids to (1) distinct conformations, (2) exposure of conserved and variable neutralization epitopes and (3) access for CD4. This information will help establish universal, cross clade rules for regulating TAD and trimer conformation that will be invaluable for design of next generation trimer immunogens aiming to elicit broad neutralizing antibodies.

HIV-1 包膜 (Env) 疫苗未能诱导具有强活性的中和抗体 (nab) 对抗多种 HIV-1。然而，有效的、广泛的中和单克隆抗体的鉴定 有证据表明 V2 和 V3 环抗体在 RV144 疫苗试验中有助于保护， 人们更加乐观地认为基于 Env 的疫苗是可能的。但目前尚不清楚如何呈现 免疫原上保守的 Env 表位，从而引发广泛活性的 nab。 免疫组织中的天然 Env 三聚体可能会被关闭，以保护关键位点免受抗体检测。三聚体 三聚体顶端的关联结构域 (TAD) 以及 CD4 结合环区域中的残基有助于维持 闭合构象。我们建议了解 TAD 构象是如何控制的对于 开发可引发广泛且有效的抗体的三聚体包膜免疫原。然而，决定因素 维持闭合三聚体和调节 TAD 构象目前还知之甚少。在这里，我们将使用 EMPIRIC （随机个体密码子的极其细致和平行的调查），一种新颖的饱和 诱变方法来识别 V1V2 和 V3 环中调节 TAD 构象的残基 进化枝。该方法得到了强有力的初步数据的支持，并将用于识别突变三聚体 携带具有不同构象的 TAD。这种三聚体将代表未来研究的新型免疫原。 目标 1. 使用 EMPIRIC 鉴定 TAD 中影响病毒复制的氨基酸。这些氨基 酸是调节 TAD 和三聚体构象的候选酸：我们将使用 EMPIRIC 饱和度 诱变以研究进化枝 A、B 和 C Envs 中 TAD 的 V1、V2 和 V3 环。我们将编目 与 wt Env+ 相比，这些 Env 区域中的突变赋予 PBMC 野生型或增强的复制能力 病毒，然后分析其对目标 2 中的 Env 和 TAD 构象的影响。 目标 2. 表征 TAD 和三聚体构象修饰的突变体 Env：我们将 研究目标 1 中发现的突变如何影响 TAD 和三聚体构象。我们将用小说 三聚体结合测定和 Env+ 假病毒中和测试来表征不同的 Env 突变体。一个 针对 TAD、CD4b 和其他 Env 区域以及 sCD4 的广泛单克隆抗体将 进行测试以确定 TAD 构象和 CD4bs 暴露的变化以及对其他 Env 位点的影响。 我们的数据将提供有关调节三聚体构象的 TAD 氨基酸的详细信息。我们 将创建将特定 V1V2 abd V3 环氨基酸与 (1) 不同构象相关联的 Env 结构图， (2) 暴露保守和可变的中和表位，以及 (3) 获取 CD4。该信息将 帮助建立通用的、跨分支的规则来调节 TAD 和三聚体构象，这对于 设计下一代三聚体免疫原，旨在引发广泛的中和抗体。