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)疫苗不能诱导具有很强活性的中和抗体(Nabs)。 对抗不同的HIV-1。然而，鉴定有效的、广泛的中和单抗沿着 有证据表明，在RV144疫苗试验中，V2和V3环抗体有助于保护 人们更加乐观地认为，基于环境病毒的疫苗是可能的。然而，目前仍不清楚如何呈现 在免疫原上保守的环境抗原表位，从而诱导出广泛活性的NAB。 免疫组织中的天然Env三聚体可能会关闭，以保护关键部位免受NAB的侵袭。三聚体 三聚体顶端的结合结构域(TAD)以及CD4结合环区的残基有助于维持 封闭的构象。我们认为，了解TAD构象是如何被控制的对于 三聚体环状病毒免疫原的开发，可诱导广泛和有效的NAB。然而，决定因素是 维持一个封闭的三聚体和调节TAD构象还知之甚少。在这里，我们将使用Empiric (对随机个体密码子的极其细致和平行的研究)，一种新的饱和 用突变方法鉴定调节TAD构象的V1V2和V3环中的残基 克拉兹。这一方法得到了强有力的初步数据的支持，并将用于识别 携带具有不同构象的TADS。这些三聚体将为未来的研究提供新的免疫原。 目的1.利用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物质：我们将使用经验饱和度 突变研究A、B和C分支环境中TAD的V1、V2和V3环。我们会编目的 与wt Env+相比，这些Env区的突变赋予了PBMC野生型或增强的复制能力 病毒，然后分析它们对AIM 2中Env和TAD构象的影响。 目标2.表征突变环境中TAD和三聚体构象的修饰：我们将 研究在AIM 1中发现的突变如何影响TAD和三聚体构象。我们将用一本小说 三聚体结合试验和Env+假病毒中和试验对不同的Env突变体进行鉴定。一个 针对TAD、CD4b和其他Env区以及sCD4的广泛的单抗将是 测试以确定TAD构象和CD4b暴露的变化以及对其他环境位点的影响。 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和V3环氨基酸与(1)不同构象相关联的环境结构图， (2)保守和可变中和表位的暴露；(3)CD4的可获得性。此信息将 帮助建立通用的跨分支规则来调节TAD和三聚体构象，这对 设计下一代三聚体免疫原，旨在诱导广泛的中和抗体。

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.