Topology, Antigenicity, and Immunogenicity of the C-terminal Tail (CTT) of HIV-1

描述（由申请人提供）：HIV-1包膜蛋白被认为是感染期间的主要免疫靶点，并且一直是艾滋病疫苗开发的主要焦点，以引起持久的广泛中和抗体反应。这些研究主要集中在gp120和gp41外结构域上，基于gp41 c端尾（CTT）未暴露并且对包膜免疫原性很重要的假设。然而，我们实验室和其他人最近的研究清楚地表明，CTT是HIV-1包膜结构、功能和抗原性的主要决定因素，并且CTT可以成为中和针对相对保守蛋白片段的抗体的有效靶点。基于这些观察结果，我们假设HIV gp41 CTT相对于脂质双分子层的拓扑结构是动态的，并且CTT序列至少暂时位于病毒或细胞脂质双分子层的外部。我们进一步假设CTT序列是Env抗原和免疫原性的重要决定因素，它们与其他Env结构域的关联以及作为不同的免疫靶点，包括针对保守CTT序列的中和抗体。在本申请中，我们提出在两个特定目标中验证这一假设：(i)生成CTT序列相对于病毒或细胞膜的明确拓扑图谱，以及Gag关联或融合过程对该拓扑的影响；（ii）评估CTT作为Env免疫原性和抗原性决定因素的作用，特别是与中和抗体相关的作用。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确定病毒和细胞相关Env背景下的CTT结构，以及包含HIV-1 CTT融合到流感病毒HA包膜蛋白外结构域的新型嵌合蛋白。免疫学研究将采用已建立的腺病毒载体或VLP疫苗程序，使用密码子优化的HIV-1全长截断Envs或工程HA-CTT构建物，对兔进行实验性免疫，以分离病毒或细胞膜背景下的CTT免疫原性。预计这些研究的结果将首次定义HIV-1 CTT的拓扑结构，并描述其作为免疫原决定因素的作用，以及它作为免疫原引发保守序列的广泛中和抗体的潜力。

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 C-terminal tail (CTT) was not exposed and important for envelope immunogenicity. Recent studies from our lab and others, however, clearly indicate that the CTT is a major determinant of HIV-1 envelope structure, function, and antigenicity and that the CTT can be a potent target for neutralizing antibodies directed to relatively conserved protein segments. Based on these observations, we hypothesize that the topology of the HIV gp41 CTT relative to the lipid bilayer is dynamic, and that CTT sequences are, at least transiently, located on the exterior of the viral or cellular lipid bilayer. We postulate further that CTT sequences are important determinants of Env antigenic and immunogenic properties, both by their association with other Env domains and as distinct immunologic targets, including neutralizing antibodies directed to conserved CTT sequences. We propose in this application to test this hypothesis in two specific aims: (i), to produce a definitive topological map of the CTT sequences relative to viral or cellular membranes and the influence of Gag association or fusion process on this topology, and (ii) to assess the role of the CTT as a determinant of Env immunogenicity and antigenicity, especially as related to neutralizing antibodies. The structural studies will utilize high-resolution membrane protein topology mapping techniques and antibody reactivity assays to define CTT structure in the context of virus- and cell-associated Env and in the context of novel chimeric proteins containing the HIV-1 CTT fused to the ectodomain of influenza virus HA envelope protein. The immunologic studies will employ experimental immunization of rabbits using established adenovirus vector or VLP vaccine procedures with codon optimized HIV-1 full length of truncated Envs or the engineered HA-CTT constructs to isolate CTT immunogenicity in the context of viral or cellular membranes. It is anticipated that the results of these studies will for the first time define the topology of the HIV-1 CTT and characterize its role as an immunogenic determinant and its potential as an immunogen to elicit broadly neutralizing antibodies to conserved sequences.