Stabilizing HIV-1 Trimers by Linking gp120 Subunits

描述(由申请人提供)：一种有效的HIV-1疫苗可能需要诱导具有与真实环境三聚体结合的独特能力的广谱中和抗体(NAB)。因此，这些三聚体可能能够在疫苗环境中引发nabs，这是合乎逻辑的。然而，除了三聚体之外，颗粒还携带非功能性的Env，它似乎主导抗体反应，抑制或延迟NAB的发育。我们将检验这样一种假设，即当不受非功能Env的约束时，对颗粒疫苗的反三聚体反应会得到改善。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。消除VLP上不受欢迎的Ab靶标的一种策略可能是通过分子间二硫键横向稳定三聚体(即相邻的gp120/gp41原核之间)。为了帮助半胱氨酸的放置，我们将对正宗的Env三聚体进行定向丙氨酸扫描突变。我们有初步证据表明，BN-PAGE筛选使鉴定横向不稳定突变是可行的。现有数据表明，可变环相互作用可能稳定邻近的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环上进行成对的半胱氨酸取代，试图引入一种名为“SOSVV”的二硫键，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。原生页面结合研究将用于评估NAB结合排他性所指示的三聚体的真实性。我们还将检查SOSVV三聚体在感染中发挥作用的能力，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，且没有非功能性环境污染，我们将在兔身上测试它们的免疫原性。如果中和反应不能形成，我们将尝试更高的剂量和过度免疫。另一种可能性是使用与免疫球蛋白复合的VLP来增强NAB反应。偶发免疫原将是可溶性的SOSVV或VLP免疫原，其中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相有3个主要成分。首先，我们将调整我们的免疫原以适应猕猴。猕猴既有机遇，也有挑战。例如，在SHIV感染中可能会产生广泛的nabs。然而，基于Env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。我们将免疫两组12只猕猴，并用异源SIV攻击第二组。其次，我们将尝试通过各种策略来提高兔NAB的滴度和广度。第三，努力提高VLP的产量、纯度和质量。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身上引发中和抗体。对病毒样颗粒上非功能性形式的Env的抗体反应可能会抑制或推迟中和反应的出现。我们将研究在真实的三聚体中连接相邻的gp120原核表达体的可能性，以此来消除非功能的Env，并放大针对真实的三聚体产生的中和反应。

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.