Stabilizing HIV-1 Trimers by Linking gp120 Subunits

一种有效的HIV-1疫苗可能需要诱导具有广泛中和能力的抗体 独特的能力，可以绑定到真正的环境三聚体。因此，这是合乎逻辑的，这些三聚体可能能够引发 疫苗接种环境中的nabs。然而，除了三聚体之外，粒子还带有似乎不起作用的环境 控制抗体反应，抑制或延缓NAB的发展。我们将检验反三聚体的假设 当不受非功能性环境的约束时，对颗粒疫苗的反应会得到改善。我们的具体目标是： 具体目的1：探讨V1V2和V3环突变对侧向三聚体稳定性的影响。一 消除VLP上不想要的Ab靶的策略可能是横向稳定三聚体(即在相邻的 Gp120/gp41原核)通过分子间二硫键连接。为了协助半胱氨酸的放置，我们将 对正宗的环境三聚体进行靶向丙氨酸扫描突变。我们有初步证据表明 BN-PAGE的筛选使侧向不稳定突变体的鉴定成为可能。现有数据表明 可变环相互作用可能稳定相邻的gp120亚基。 具体目标2：通过引入gp120间二硫键来横向稳定真正的三聚体。我们 将在V1V2和V3环中进行成对的半胱氨酸取代，试图引入一种称为二硫键的桥 “SOSVV”，重点放在目标1中确定的位置。为了测试是否存在V-V二硫键，我们将 评估三聚体对离子洗涤剂和还原剂的稳定性。本机页面绑定研究将用于 根据NAB结合排他性来评估三聚体的真实性。我们还将检查 SOSVV三聚体在感染中的作用以及表达为可溶性gp140的SOSVV突变体的稳定性。 具体目标3：评估不受约束的真三聚体在兔体内诱导NAB的能力。如果SOSVV 形成稳定的三聚体，没有非功能性的环境污染，我们将在兔身上测试它们的免疫原性。如果 中和反应不能发展，我们将尝试更高的剂量和过度免疫。另一种可能性 将是使用与免疫球蛋白复合的VLP来增强NAB反应。或有免疫原要么是 可溶性的SOSVV或VLP免疫原，其中的非功能靶点被物种匹配的免疫球蛋白掩盖。 结果将推动连续接种以解决问题并放大中和反应。 具体目标4：增强猕猴对正品三聚体的抗体反应。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以适应猕猴。猕猴提供了机会和机会 挑战。例如，在SHIV感染中可能会产生广泛的nabs。然而，基于环境的免疫原 可能与内源性灵长类CD4结合，导致非中和抗体特异性的激发。因此， 我们将评估CD4结合基因敲除三聚体。我们将为两组12只猕猴接种疫苗，并挑战 第二组具有异源的Shiv。第二，我们将通过以下方式努力提高兔的NAB效价和广度 各种策略。第三，努力提高VLP的产量、纯度和质量。

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.