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

描述(由申请人提供)：一种基于T细胞的艾滋病毒疫苗及其高度可变的基因组是一个巨大的挑战，因为它将不得不预防许多病毒变种。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的区域，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，旨在克服艾滋病毒的变异性。人类白细胞抗原-B*57-I类分子(B*57)与较好的疾病预后有关。在这里，我们将在一项验证性研究中描述B*57捐赠者对我们的T细胞疫苗成分的反应。特定目标1将在HIV蛋白质组的四个高度保守的区域绘制新的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，从而实现长期控制。在Gag区域发现的已知优势表位将被反向遗传学消灭。来自健康供者的CD8T细胞将被慢病毒载体转导的自体DC激活，以表达这些多肽。将绘制最佳最小表位图，并验证其与B*5701的结合。特定目标2将描述CD8T细胞的特征，以选择来自健康捐赠者的体外启动所产生的表位。我们将评估i)各种体外CTL功能；ii)这些反应单独或结合在一起，抑制野生型或减毒变异NL4-3病毒的体外复制的能力；iii)抗原表位特异性的疫苗接种前TCR谱系储备。我们将重点关注共享的(公认的)表位，特别是那些在特定目标3中被发现与更好的疾病控制有关的表位。特定目标3将在B*57患者中寻找对所有新表位的反应，以确定是否有任何表位与疾病的相对控制有关。他们将在体外直接监测，或在一次用同源表位多肽重新刺激后进行监测，以测量Recall T细胞的反应。它们的功能图谱将通过多色流式细胞术进一步评估，如果可能，还将与同一个体中其他已知B*57反应的同期反应进行比较。还将进行比横断面研究更有信息量的纵向研究，以进一步阐明有希望的反应。第二个目标是开发一种体外人体模型，用于将未来的候选疫苗推向临床测试。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T细胞对HIV控制非常重要，以及疫苗诱导的T细胞应该寻找的品质，以及确定HIV蛋白最保守的部分是否是有价值的候选疫苗。 公共卫生相关性基于T细胞的HIV疫苗及其高度可变的基因组是一个巨大的挑战，因为它将不得不保护自己免受许多病毒变种的影响。我们提出了一种简单且可测试的疫苗方法，旨在通过将T细胞反应集中在HIV蛋白质组的高度保守区域来克服HIV的变异性。我们希望更多地了解应答T细胞如何对艾滋病毒控制做出贡献，并将评估一种体外人类疫苗接种模型，该模型可能有助于推动未来的候选疫苗进入临床测试。

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.