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

描述（由申请人提供）：针对具有高度可变基因组的HIV的基于T细胞的疫苗是一项艰巨的挑战，因为它必须针对许多病毒变体提供保护。作为一种新的疫苗方法，我们建议将T细胞免疫反应集中在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不变。这是一种简单且可测试的疫苗方法，旨在克服HIV变异性。HLA-B*57 I类分子（B*57）与更好的疾病结局相关。在这里，我们将在原理验证研究中描述B*57+供体对我们的T细胞疫苗组分的应答。Specific Aim 1将在HIV蛋白质组的四个高度保守区域中绘制新的B*57限制性表位。靶向几种蛋白质（Gag，Pol和Env）应有助于减少病毒逃逸，以实现长期控制。在Gag区发现的已知显性表位将通过反向遗传学消除。来自健康供体的CD 8 + T细胞将由慢病毒载体转导的自体DC引发以表达这些多肽。将绘制最佳最小表位并验证与B*5701的结合。特异性目标2将针对通过离体引发从健康供体产生的选定表位来表征CD 8 + T细胞。我们将评估i）各种体外CTL功能; ii）这些应答单独或组合抑制野生型或减毒变体NL 4 -3病毒的体外复制的能力;和iii）表位特异性疫苗接种前TCR库储备。我们将专注于共享（公认）表位，特别是那些发现与特定目标3中更好的疾病控制有关的表位。特异性目标3将寻求对B*57+患者中所有新表位的应答，以确定是否有任何与疾病的相对控制相关。它们将直接离体监测或在用同源表位肽再刺激一次后监测，以测量回忆T细胞应答。将通过多色流式细胞术进一步评估其功能特征，如果可能，将其与同一个体内对其他已知B*57应答的同期应答进行比较。还将进行比横断面研究信息量更大的纵向研究，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类模型，用于推进未来的候选疫苗进行临床试验。总之，这项研究应该提供新的见解艾滋病毒特异性CD 8 + T细胞的特点，艾滋病毒控制和质量，应寻求疫苗诱导的T细胞，以及确定是否最保守的部分艾滋病毒蛋白是有价值的疫苗候选人。 针对基因组高度可变的HIV的基于T细胞的疫苗是一项艰巨的挑战，因为它必须保护免受许多病毒变体的侵害。我们提出了一个简单的和可测试的疫苗的方法，旨在克服艾滋病毒的变异性，通过集中T细胞反应的高度保守的区域的艾滋病毒蛋白质组。我们希望更多地了解反应性T细胞如何有助于艾滋病毒控制，并将评估一种离体人类疫苗接种模型，这可能有助于推动未来的候选疫苗进行临床试验。

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.