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

HIV-1包膜（Env）疫苗未能诱导出具有强活性的中和抗体（nab 对抗多种HIV-1然而，有效的、广泛中和的单克隆抗体的鉴定沿着 在RV144疫苗试验中，有证据表明V2和V3环抗体有助于保护， 越来越多的乐观认为，一个基于Env的疫苗是可能的。然而，目前还不清楚如何提出 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三聚体可能被封闭以保护关键位点免受nab的影响。三聚体 在三聚体顶端沿着与CD4结合环区域中的残基的结合结构域（CD4）有助于维持 一种封闭的构造。我们建议，了解如何控制构象将是至关重要的， 三聚体Env免疫原的开发，其引发广泛和有效的Nab。然而， 维持封闭三聚体和调节构象的方法知之甚少。在这里，我们将使用EMPIRIC （随机个体密码子的过度测量和平行研究），一种新的饱和度 诱变方法来鉴定V1V2和V3环中调节跨膜构象的残基。 进化枝这种方法得到了强有力的初步数据的支持，并将用于鉴定突变三聚体， 携带不同构象的TADs。这种三聚体将代表未来研究的新免疫原。 目标1。使用EMPIRIC鉴定影响病毒复制的氨基酸。这些氨基 酸是调节二聚体和三聚体构象的候选者：我们将使用EMPIRIC饱和度 诱变以研究进化枝A、B和C Envs中的V1、V2和V3环。我们将目录 与wt Env+相比，这些Env区域中的突变赋予PBMC中的野生型或增强的复制 病毒，然后分析它们对目标2中Env和Env构象的影响。 目标2.为了表征突变体Envs在双链和三聚体构象中的修饰：我们将 研究目标1中鉴定的突变如何影响双链和三聚体构象。我们会用一本小说 三聚体结合试验和Env+假病毒中和试验来表征不同的Env突变体。一 一组针对CD4b、CD4b和其它Env区域以及sCD4的单克隆抗体将被 测试以确定Env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三聚体氨基酸的详细信息。我们 将创建将特定的V1V2和V3环氨基酸与（1）不同构象相关联的Env结构图， (2)保守和可变中和表位的暴露和（3）CD4的接近。这些信息将 帮助建立通用的，交叉进化枝规则，以调节双链和三聚体构象，这将是非常宝贵的 设计下一代三聚体免疫原，旨在引发广泛的中和抗体。

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.