Stabilizing HIV-1 Trimers by Linking gp120 Subunits

描述（由申请方提供）：有效的HIV-1疫苗可能需要引发具有独特能力的广泛中和抗体（nAb），以结合真实的Env三聚体。因此，合乎逻辑的是，这些三聚体可能能够在疫苗环境中引发nAb。然而，除了三聚体之外，颗粒还具有非功能性Env，其似乎主导Ab应答并抑制或延迟nAb发展。我们将检验这样的假设，即当不受非功能性Env的束缚时，对颗粒疫苗的抗三聚体应答得到改善。我们的具体目标是： 具体目的1：研究V1 V2和V3环突变对侧向三聚体稳定性的影响。消除VLP上不需要的Ab靶标的一种策略可以是通过分子间二硫键横向稳定三聚体（即相邻gp 120/gp 41原聚体之间）。为了帮助放置半胱氨酸，我们将对真正的Env三聚体进行靶向丙氨酸扫描诱变。我们有初步的证据表明，通过BN-PAGE筛选横向不稳定突变体的鉴定是可行的。现有的数据表明，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，以尝试引入被称为“SOSVV”的二硫桥，重点关注Aim 1中鉴定的位置。为了测试是否存在V-V二硫键，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三聚体在感染中发挥作用的能力和表达为可溶性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，我们将在家兔中测试其免疫原性。如果中和反应未能发展，我们将尝试更高的剂量和超免疫。另一种可能性是使用与IgG复合的VLP来增强nAb应答。偶然免疫原是可溶性SOSVV或VLP免疫原，其中非功能性靶点被物种匹配的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阶段有三个主要组成部分。首先，我们将调整我们的免疫原用于猕猴。猕猴带来了机遇和挑战。例如，广泛的nAb可以在SHIV感染中产生。然而，基于Env的免疫原可能与内源性灵长类动物CD 4结合，导致引发非中和性Ab特异性。因此，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，并用异源SHIV攻击第二组。其次，我们将尝试通过各种策略来提高兔中的nAb滴度和广度。第三，我们将努力提高VLP的生产、纯化和质量。公共卫生相关性：我们将研究HIV病毒样颗粒作为免疫原，在兔子和猕猴中引发中和抗体。对病毒样颗粒上的非功能性形式的Env的抗体应答可能抑制或延迟中和应答的出现。我们将研究在真实的三聚体中连接相邻的gp 120原聚体的可能性，作为消除非功能性Env和放大针对真实三聚体产生的中和反应的方法。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: We will investigate HIV virus-like particles as immunogens for eliciting neutralizing antibodies in rabbits and macaques. Antibody responses to non-functional forms of Env on virus-like particles may dampen or delay the emergence of neutralizing responses. We will investigate the possibility to link adjacent gp120 protomers within authentic trimers as a way to eliminate non-functional Env and amplify neutralizing responses generated against authentic trimers.