Stabalizing HIV-1 Trimers by Linking gp120 Subunits

一种有效的HIV-1疫苗可能需要引发具有广泛中和性的抗体（nAb）， 结合真实Env三聚体的独特能力。因此，合乎逻辑的是，这些三聚体可能能够引起 在疫苗环境中的nAbs。然而，除了三聚体之外，颗粒还携带非功能性Env， 控制Ab应答并抑制或延迟nAb的形成。我们将检验反三聚体 当不受非功能性Env的束缚时，对颗粒疫苗的应答得到改善。我们的具体目标是： 具体目标1：研究V1 V2和V3环突变对侧向三聚体稳定性的影响。一 消除VLP上不需要的Ab靶标的策略可以是横向稳定三聚体（即相邻的三聚体之间 gp 120/gp 41原聚体）通过分子间二硫键连接。为了帮助半胱氨酸的放置，我们将 进行真正Env三聚体的靶向丙氨酸扫描诱变。我们有初步证据表明 通过BN-PAGE筛选使得横向不稳定突变体的鉴定可行。现有数据表明 可变环相互作用可以稳定相邻的GP 120亚基。 具体目标2：通过引入gp 120间二硫键来横向稳定真实的三聚体。我们 将在V1 V2和V3环中进行成对的半胱氨酸取代，试图引入二硫桥，称为 “SOSVV”，侧重于目标1中确定的职位。为了测试V-V二硫键是否存在，我们将 评价三聚体对离子去污剂和还原剂的稳定性。非变性PAGE结合研究将用于 评估三聚体真实性，如nAb结合排他性所示。我们还将研究 SOSVV三聚体在感染中起作用以及表达为可溶性gp 140的SOSVV突变体的稳定性。 具体目的3：评价不受约束的真实三聚体在兔中引发nAb的能力。如果SOSVV 形成稳定的三聚体，没有非功能性Env污染，我们将在兔子中测试它们的免疫原性。如果 如果中和反应未能产生，我们将尝试更高剂量和超免疫。另一种可能性 将使用与IgG复合的VLP来增强nAb反应。应急免疫原要么是 可溶性SOSVV或VLP免疫原，其中非功能性靶标被物种匹配的IgG掩蔽。 结果将推动连续的免疫接种，以解决问题并扩大中和反应。 具体目的4：增强猕猴中对真实三聚体的抗体应答。R33阶段有3个 主要组成部分首先，我们将调整我们的免疫原用于猕猴。猕猴既提供了机会， 挑战例如，SHIV感染中可以产生广泛的nAb。然而，Env免疫原 可能与内源性灵长类动物CD 4结合，导致非中和性Ab特异性的激发。因此，我们认为， 我们将评估CD 4结合敲除三聚体。我们将对两组12只猕猴进行免疫， 第二组为异源SHIV。其次，我们将尝试通过以下方法提高兔中的nAb滴度和广度： 各种战略。第三，我们将努力提高VLP的生产、纯化和质量。

An effective HIV-1 vaccine will likely need to elicit broadly neutralizing antibodies (nAbs) that possess a unique ability to bind to authentic Env trimers. It is logical, therefore, that these trimers may be able to elicit nAbs in a vaccine setting. In addition to trimers, however, particles bear non-functional Env that appears to dominate Ab responses and dampen or delay nAb development. We will test the hypothesis that anti-trimer responses to particulate vaccines are improved when unfettered by non-functional Env. Our Specific Aims are: Specific Aim 1: To Investigate the effect of V1V2 and V3 loop mutations on lateral trimer stability. One strategy to eliminate undesirable Ab targets on VLPs may be to laterally stabilize trimers (i.e. between adjacent gp120/gp41 protomers) by an inter-molecular disulfide bridge. To assist in the placement of cysteines, we will perform targeted alanine scanning mutagenesis of authentic Env trimers. We have preliminary evidence that screening by BN-PAGE makes the identification of laterally unstable mutants feasible. Available data suggests that variable loop interactions may stabilize neighboring gp120 subunits. Specific Aim 2: To laterally stabilize authentic trimers by introducing an inter-gp120 disulfide bond. We will make pairs of cysteine substitutions in V1V2 and V3 loops to try to introduce a disulfide bridge, termed "SOSVV", focusing on positions identified in Aim 1. To test whether a V-V disulfide bridge is present, we will evaluate trimer stability to ionic detergents and reducing agents. Native PAGE binding studies will be used to assess trimer authenticity as indicated by nAb binding exclusivity. We will also examine the ability of the SOSVV trimers to function in infection and the stability of SOSVV mutants expressed as a soluble gp140. Specific Aim 3: To evaluate the ability of unfettered authentic trimers to elicit nAbs in rabbits. If SOSVV forms stable trimers with no non-functional Env contamination, we will test their immunogenicity in rabbits. If neutralizing responses fail to develop, we will try higher doses and hyperimmunization. Another possibility would be to use VLPs complexed with IgG to augment nAb responses. Contingent immunogens will either be soluble SOSVV or VLP immunogens in which non-functional targets are masked by species-matched IgG. Results will drive successive immunizations to solve problems and amplify neutralizing responses. Specific Aim 4: To augment antibody responses to authentic trimers in macaques. The R33 phase has 3 main components. First, we will adapt our immunogens for macaques. Macaques offer both opportunities and challenges. For example, broad nAbs can be generated in SHIV-infections. However, Env-based immunogens may engage endogenous primate CD4, leading to the elicitation of non-neutralizing Ab specificities. Therefore, we will evaluate CD4 binding knockout trimers. We will immunize two groups of 12 macaques and challenge the second group with a heterologous SHIV. Second, we will try to improve nAb titer and breadth in rabbits by various strategies. Third, we will try to improve the production, purification and quality of VLPs.