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

描述(申请人提供)：HIV-1包膜蛋白在感染过程中被认为是主要的免疫目标，并一直是艾滋病疫苗开发的主要焦点，以诱导持久的广泛中和抗体反应。这些努力主要集中在gp120和gp41胞外结构域，基于gp41 C末端尾巴(CTT)不暴露和对包膜免疫原性重要的假设。然而，最近来自我们实验室和其他实验室的研究清楚地表明，CTT是HIV-1包膜结构、功能和抗原性的主要决定因素，并且CTT可以成为针对相对保守的蛋白片段的抗体的有效靶点。基于这些观察，我们假设HIV gp41 CTT相对于脂双层的拓扑是动态的，并且CTT序列至少瞬时地位于病毒或细胞脂双层的外部。我们进一步推测，CTT序列是Env抗原性和免疫原性的重要决定因素，既是因为它们与其他Env结构域有关，也是作为不同的免疫靶点，包括针对保守的CTT序列的中和抗体。在这一应用中，我们建议在两个特定的目标下检验这一假设：(I)产生相对于病毒或细胞膜的CTT序列的确定的拓扑图以及GAG结合或融合过程对这种拓扑图的影响，以及(Ii)评估CTT作为环境免疫原性和抗原性的决定因素的作用，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分析来确定病毒和细胞相关环境中的CTT结构，以及包含HIV-1 CTT与流感病毒HA包膜蛋白胞外区融合的新型嵌合蛋白。免疫学研究将使用已建立的腺病毒载体或VLP疫苗程序对兔进行实验性免疫，该疫苗程序带有密码子优化的HIV-1全长截短env或工程HA-CTT构建物，以在病毒或细胞膜的背景下分离CTT免疫原性。预计这些研究的结果将首次确定HIV-1 CTT的拓扑结构，并表征其作为免疫原性决定簇的作用及其作为免疫原诱导针对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.