Stabalizing HIV-1 Trimers by Linking gp120 Subunits

通过连接 gp120 亚基稳定 HIV-1 三聚体

• 批准号: 8874841
• 负责人: JAMES M BINLEY
• 金额: $ 33.44万
• 依托单位: SAN DIEGO BIOMEDICAL RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-10 至 2016-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8874841
• 关键词: Address; Affect; Alanine; Animals; Antibody Formation; Antigens; Binding; Binding Sites; Clinical Research; Complex; Cysteine; Data; Detergents; Development; Disulfides; Dose; Epitopes; Evaluation; Exhibits; HIV; HIV Envelope Protein gp120; HIV-1; Human; Immune Sera; Immunization; Immunoglobulin G; In Vitro; Infection; Kinetics; Knock-out; Lateral; Link; Macaca; Masks; Membrane; Molecular; Mutagenesis; Mutation; Oryctolagus cuniculus; Outcome; Particulate; Phase; Positioning Attribute; Primates; Problem Solving; Production; Protomer; Recombinants; Reducing Agents; Reporting; Resistance; Scanning; Serum; Solutions; Son of Sevenless Proteins; Specificity; Surface; Testing; Transmembrane Domain; Ursidae Family; V3 Loop; Vaccines; Virus-like particle; base; design; disulfide bond; evidence base; experience; gp160; hyperimmunization; immunogenicity; improved; mutant; neutralizing antibody; particle; prototype; response; screening; simian human immunodeficiency virus; theories

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.

有效的 HIV-1 疫苗可能需要引发广泛中和抗体 (nAb)，该抗体具有 与真正的 Env 三聚体结合的独特能力。因此，这些三聚体可能能够引发 疫苗环境中的 nAb。然而，除了三聚体之外，颗粒还带有非功能性的包膜，这似乎 控制抗体反应并抑制或延迟 nAb 的发育。我们将检验抗三聚体的假设 当不受非功能性环境的限制时，对颗粒疫苗的反应会得到改善。我们的具体目标是： 具体目标 1：研究 V1V2 和 V3 环突变对侧向三聚体稳定性的影响。一 消除 VLP 上不需要的抗体靶标的策略可能是横向稳定三聚体（即相邻的三聚体之间） gp120/gp41 原体）通过分子间二硫桥形成。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。我们有初步证据表明 BN-PAGE 筛选使得横向不稳定突变体的鉴定成为可能。现有数据表明 可变环相互作用可以稳定邻近的 gp120 亚基。 具体目标 2：通过引入 gp120 间二硫键来横向稳定真实的三聚体。我们 将在 V1V2 和 V3 环中进行成对的半胱氨酸取代，以尝试引入二硫桥，称为 “SOSVV”，重点关注目标 1 中确定的位置。为了测试 V-V 二硫桥是否存在，我们将 评估三聚体对离子洗涤剂和还原剂的稳定性。天然 PAGE 结合研究将用于 根据 nAb 结合排他性评估三聚体的真实性。我们还将考察相关人员的能力 SOSVV 三聚体在感染中发挥作用，并以可溶性 gp140 表达 SOSVV 突变体的稳定性。 具体目标 3：评估不受约束的真实三聚体在兔子中引发 nAb 的能力。如果SOSVV 形成稳定的三聚体，没有非功能性包膜污染，我们将在兔子中测试它们的免疫原性。如果 中和反应未能发展，我们将尝试更高剂量和超免疫。另一种可能性 的方法是使用与 IgG 复合的 VLP 来增强 nAb 反应。偶然的免疫原可以是 可溶性 SOSVV 或 VLP 免疫原，其中非功能性靶标被物种匹配的 IgG 掩盖。 结果将推动连续免疫接种以解决问题并放大中和反应。 具体目标 4：增强猕猴对真实三聚体的抗体反应。 R33相有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 挑战。例如，在 SHIV 感染中可以产生广泛的 nAb。然而，基于 Env 的免疫原 可能会与内源性灵长类 CD4 结合，从而引发非中和性抗体特异性。所以， 我们将评估 CD4 结合敲除三聚体。我们将对两组 12 只猕猴进行免疫并进行挑战 第二组携带异源SHIV。其次，我们将尝试通过以下方法提高兔子的 nAb 滴度和广度： 各种策略。三是努力提高病毒颗粒的生产、纯化和质量。

项目成果

Virus-like Particles Identify an HIV V1V2 Apex-Binding Neutralizing Antibody that Lacks a Protruding Loop.

病毒样颗粒识别缺乏突出环的 HIV V1V2 顶端结合中和抗体。

• 发表时间: 2017-05-16
• 影响因子: 32.4
• 作者: Cale, Evan M;Gorman, Jason;Radakovich, Nathan A;Crooks, Ema T;Osawa, Keiko;Tong, Tommy;Li, Jiaqi;Nagarajan, Raju;Ozorowski, Gabriel;Ambrozak, David R;Asokan, Mangai;Bailer, Robert T;Bennici, Anthony K;Chen, Xuejun;Doria;Druz
• 通讯作者: Druz

Inhibition of the HIV-1 spike by single-PG9/16-antibody binding suggests a coordinated-activation model for its three protomeric units.

单 PG9/16 抗体结合对 HIV-1 刺突的抑制表明其三个原体单元存在协调激活模型。

• 发表时间: 2013-06
• 作者: Löving, Robin;Sjöberg, Mathilda;Wu, Shang;Binley, James M;Garoff, Henrik
• 通讯作者: Garoff, Henrik

M48U1 CD4 mimetic has a sustained inhibitory effect on cell-associated HIV-1 by attenuating virion infectivity through gp120 shedding.

M48U1 CD4 模拟物通过 gp120 脱落减弱病毒粒子的感染性，对细胞相关的 HIV-1 具有持续的抑制作用。

• 发表时间: 2013-02-01
• 影响因子: 3.3
• 作者: Selhorst, Philippe;Grupping, Katrijn;Tong, Tommy;Crooks, Ema T;Martin, Loïc;Vanham, Guido;Binley, James M;Ariën, Kevin K
• 通讯作者: Ariën, Kevin K