Evaluation of HIV-1 Env intracytoplasmic domain as an AIDS vaccine immunogen

产品说明：（申请人提供）：HIV-1包膜蛋白被认为是感染期间的主要免疫靶标，并且已经成为AIDS疫苗开发的主要焦点，以引起持久的广泛中和抗体应答。这些努力主要集中在gp 120和gp 41胞外域，基于gp 41胞浆内结构域（ICD）未暴露且对包膜免疫原性重要的假设。然而，我们实验室和其他人最近的研究清楚地表明，ICD是HIV-1包膜结构、功能和抗原性的主要决定因素，并且ICD可以是针对相对保守的蛋白质片段的中和抗体的有效靶点。我们假设HIV-1 gp 41 ICD的关键片段实际上位于病毒或细胞脂质双层的外部，并且这些片段是Env抗原性和免疫原性的重要决定因素，无论是通过与其他Env结构域的关联还是作为中和抗体应答的不同表位。我们进一步假设，ICD可以被改造以增强艾滋病疫苗产生最佳中和抗体应答的能力。在本申请中，我们提出在2个具体目的中检验该假设：（i）确定代表性进化枝B和进化枝C Env中ICD序列相对于病毒和细胞脂质双层的拓扑结构，和（ii）评估ICD作为Env抗原性和免疫原性的决定因素在包膜抗体反应性和中和方面的作用。结构研究将利用高分辨率膜蛋白拓扑图技术和抗体反应性测定来定义病毒和细胞相关Env背景下的ICD结构，以及含有与流感病毒HA包膜蛋白的胞外域融合的HIV-1 ICD的新型嵌合蛋白的背景下的ICD结构。免疫学研究将采用我们建立的DNA疫苗程序，用密码子优化的HIV-1全长截短Env或工程化HA-gp 41构建体对兔进行实验性免疫，以分离ICD免疫原性。预计这些研究的结果将首次定义HIV-1 ICD的拓扑结构，并表征其作为免疫原引发广泛中和抗体的潜力。该项目的长期目标是设计ICD以优化Env免疫原的能力，从而引发持久的广泛反应性中和抗体。敷设总结：该提案评估了HIV-1包膜的一个相对不具有特征的片段的结构，并研究了这些包膜序列作为病毒中和特性的决定因素的作用，以及其作为疫苗增强中和抗体产生的潜力。

DESCRIPTION: (provided by applicant): The HIV-1 envelope proteins are recognized as primary immune targets during infection and have been a major focus of AIDS vaccine development to elicit enduring broadly neutralizing antibody responses. These efforts have largely focused on the gp120 and gp41 ectodomains, based on the assumption that the gp41 intracytoplasmic domain (ICD) was not exposed and important for envelope immunogenicity. Recent studies from our lab and others, however, clearly indicate that the ICD is a major determinant of HIV-1 envelope structure, function, and antigenicity and that the ICD can be a potent target for neutralizing antibodies directed to relatively conserved protein segments. We hypothesize that critical segments of the ICD of HIV-1 gp41 are actually located on the exterior of the viral or cellular lipid bilayer and that these segments are important determinants of Env antigenic and immunogenic properties, both by association with other Env domains and as distinct epitopes for neutralizing antibody responses. We hypothesize further that the ICD can be engineered to enhance AIDS vaccine capacity to produce optimal neutralizing antibody responses. We propose in this application to test this hypothesis in 2 specific aims: (i) To determine the topology of ICD sequences in representative clade B and clade C Envs relative to the viral and cellular lipid bilayers, and (ii) To assess the role of the ICD as a determinant of Env antigenicity and immunogenicity with respect to envelope antibody reactivity and neutralization. The structural studies will utilize high resolution membrane protein topology mapping techniques and antibody reactivity assays to define ICD structure in the context of virus-and cell-associated Env and in the context of novel chimeric proteins containing the HIV-1 ICD fused to the ectodomain of influenza virus HA envelope protein. The immunologic studies will employ experimental immunization of rabbits using our established DNA vaccine procedures with codon optimized HIV-1 full length of truncated Envs or engineered HA-gp41 constructs to isolate ICD immunogenicity. It is anticipated that the results of these studies will for the first time define the topology of the HIV-1 ICD and characterize its potential as an immunogen to elicit broadly neutralizing antibodies. The long range goal of this project is to engineer the ICD to optimize the capacity of Env immunogens to elicit enduring broadly reactive neutralizing antibodies. Lay summary: This proposal evaluates the structure of a relatively uncharacterized segment of the HIV-1 envelope and examines the role of these envelope sequences as determinants of viral neutralization properties and for its potential as a vaccine to enhance the production of neutralizing antibodies.