Mapping novel subdominant B*5701 epitopes in conserved regions of the HIV proteom

描述（由申请人提供）：基于T细胞的HIV疫苗具有高度可变的基因组，是一项艰巨的挑战，因为它必须防止许多病毒变体。作为一种新的疫苗方法，我们建议将T细胞免疫应答集中在HIV蛋白质组的四个高度保守区域，这些区域可能由于功能或结构限制而保持不变。这是一种简单且可测试的疫苗方法，旨在克服艾滋病毒的变异性。HLA-B*57 I类分子（B*57）与更好的疾病预后相关。在这里，我们将在一项原理验证研究中描述B*57+供者对我们的T细胞疫苗成分的反应。特异性Aim 1将在HIV蛋白组的四个高度保守区域绘制新的B*57限制性表位。靶向几种蛋白质（Gag， Pol和Env）应该有助于减少病毒逃逸以实现长期控制。已知的显性表位发现在Gag区域将被反向遗传学消灭。来自健康供体的CD8+ T细胞将被慢病毒载体转导的自体dc引物来表达这些多肽。优化最小表位并与B*5701结合验证。特异性Aim 2将描述CD8+ T细胞的特征，通过从健康供体体外启动产生选择的表位。我们将评估i)各种体外CTL功能；ii)这些反应单独或联合抑制野生型或减毒型NL4-3病毒体外复制的能力；iii)表位特异性疫苗接种前TCR库储备。我们将重点关注共同的（公认的）表位，特别是在特异性目标3中发现的与更好的疾病控制有关的表位。特异性目标3将在B*57+患者中寻找对所有新表位的反应，以确定是否有任何与疾病的相对控制有关。它们将在体外直接监测或在同源表位肽的一次再刺激后监测，以测量召回T细胞反应。它们的功能谱将通过多色流式细胞术进一步评估，如果可能的话，将其与同一个体中其他已知B*57反应的同期反应进行比较。还将进行纵向研究，这比横断面研究更能提供信息，以进一步阐明有希望的反应。第二个目标是开发一种离体人体模型，有助于推进未来候选疫苗的临床试验。总之，这项研究应该为HIV特异性CD8+ T细胞的特征提供新的见解，这些细胞对HIV控制很重要，应该在疫苗诱导的T细胞中寻求质量，以及确定HIV蛋白的最保守部分是否有价值的候选疫苗。

DESCRIPTION (provided by applicant): A T cell-based vaccine to HIV with its highly variable genome is a formidable challenge, since it will have to protect against many viral variants. As a novel vaccine approach, we propose to focus the T cell immune response to four highly conserved regions of the HIV proteome, which may be invariant due to functional or structural constraints. This is a simple and testable vaccine approach designed to overcome HIV variability. The HLA-B*57 class I molecule (B*57) is linked with better disease outcome. Here we will characterize responses to our T cell vaccine components in B*57+ donors in a proof-of-principle study. Specific Aim 1 will map new B*57-restricted epitopes in the four highly conserved regions of the HIV proteome. Targeting several proteins (Gag, Pol and Env) should help reduce virus escape for long term control. Known dominant epitopes found in the Gag regions will be extinguished by reverse genetics. CD8+ T cells from healthy donors will be primed by autologous DCs transduced by lentiviral vectors to express these polypeptides. Optimal minimal epitopes will be mapped and binding to B*5701 verified. Specific Aim 2 will characterize the CD8+ T cells to selected epitopes generated by ex vivo priming from healthy donors. We will assess i) various in vitro CTL functions; ii) the ability of these responses, individually or in combination, to suppress the in vitro replication of wild type or attenuated variant NL4-3 viruses; and iii) the epitope-specific pre-vaccination TCR repertoire reserve. We will focus on shared (commonly recognized) epitopes and in particular, to those found linked to greater disease control in Specific Aim 3. Specific Aim 3 will seek responses to all novel epitopes in B*57+ patients, to determine whether any is linked to relative control of disease. They will be monitored ex vivo directly or after one re-stimulation with cognate epitopic peptides, to measure recall T cell responses. Their functional profiles will be further assessed by polychromatic flow cytometry and, if possible, compared to contemporaneous responses to other known B*57 responses within the same individuals. Longitudinal studies which are more informative than cross-sectional ones will also be performed to further elucidate promising responses. A secondary goal is to develop an ex vivo human model useful for advancing future vaccine candidates to clinical testing. In summary, this study should provide new insights into the characteristics of HIV-specific CD8+ T cells important for HIV control and the qualities that should be sought in vaccine-induced T cells as well as to determine whether the most conserved portions of HIV proteins are valuable vaccine candidates. PUBLIC HEALTH RELEVANCE A T cell-based vaccine to HIV with its highly variable genome is a formidable challenge, since it will have to protect against many viral variants. We propose a simple and testable vaccine approach designed to overcome HIV variability by focusing the T cell response to highly conserved regions of the HIV proteome. We expect to learn more about how responding T cells contribute to HIV control and will evaluate an ex vivo human vaccination model that may help advance future vaccine candidates to clinical testing.