Optimizing Gag CTL Epitopes to Improve Immunogenicity

描述(由申请人提供)：旨在通过传递天然蛋白来提高细胞免疫的艾滋病毒疫苗尚未成功。我们的体外启动实验表明，HIV Gag的免疫优势的HLA-A2限制性SL9表位产生不稳定的帮助非依赖性的CTL反应，而亚优势的TV9则激发不能完全亲和成熟的CDS‘1“反应。通过探测SL9特异性CD8+T细胞的大型组合多肽文库，鉴定了SL9的激动剂p41肽，可诱导稳定的SL9交叉反应T细胞。TV9的激动剂TV9p6具有强烈的四聚体CTL活性。重要的是，在裂解病毒感染的靶细胞方面，它们比TV9-CTL更有效。我们假设，对SL9表位的数字显性反应积极地抑制了对亚显性多肽的潜在更有效的反应。尤其是TV9。我们认为，HIV Gag作为人类白细胞抗原A2*携带者的免疫原的有效性可以通过定义其CTL表位等级来提高，这将允许操纵这些T细胞应答。在这里，我们将评估T细胞对SL9和TV9的竞争，通过转导表达GAG的树突状细胞体外启动人T细胞。目的1将确定SL9-T细胞是否来自健康供者的幼稚或记忆/效应器前体，以了解过度激活的反应是SL9的固有特性还是由于先前存在的交叉反应记忆的动员。AIM 2将构建四个慢病毒载体，其中SL9或TV9区域通过点突变进行修饰。P1-wtGag将编码wtGag；pl-ASL9将包含一个点突变，以禁用SL9与人类白细胞抗原A2的结合；p1-p41将其SL9替换为p41；第四个载体将构建在Aim 3定义的最有效载体上，其中TV9将替换为TV9p6。目标3将确定CTL表位的层次是否决定了反应细胞抑制病毒复制的能力。它将测试数字显性的SL9反应是否主动抑制对TV9和可能其他次要表位的更有效的反应。T细胞将根据它们在体外抑制艾滋病毒的能力进行排名。反应的多样性将通过克隆型TCR测序来判断。目的4将确定当用替代病毒攻击HHD小鼠时，最有效的载体是否会产生保护性记忆CD8+。免疫优势主要是在带有模型病毒的小鼠身上进行的研究。我们的研究将试图理解为什么SL9和TV9反应似乎在控制艾滋病毒感染方面没有发挥重要作用。一种新的方法，结合研究SL9和TV9的TCR退行性和反向遗传学，将被测试以提高HIV Gag的免疫原性。

DESCRIPTION (provided by applicant): HIV vaccines designed to promote cellular immunity by delivering native proteins have not been successful. We showed by ex vivo priming that immunodominant HLA-A2-restricted SL9 epitope of HIV Gag produces an unstable help-independent CTL response, while the subdominant TV9 provokes a CDS'1" response unable to full affinity maturation. The agonist p41 peptide of SL9, identified by probing a large combinatorial peptide library with SL9-specific CD8+ T cells, elicits stable SL9-crossreactive T cells. The agonist TV9p6 of TV9 primes avid and focused tetramer* CTLs. Importantly, they are more effective than TV9-CTLs in lysing virus-infected target cells. We hypothesize that the numerically dominant response to the SL9 epitope actively suppresses potentially more efficacious responses to subdominant peptides. particularly TV9. We propose that the effectiveness of HIV Gag as an immunogen for HLA-A2* carriers can be improved by defining its CTL epitope hierarchy, which will allow manipulation these T cell responses. Here we will evaluate T cell competition for SL9 and TV9 by ex vivo priming of human T cells with DCs transduced to express Gag. Aim 1 will determine whether SL9-T cells are derived from naive or memory/effector precursors from healthy donors, to see whether the overactivated response is an intrinsic property of SL9 or due to mobilization of pre-existing crossreactive memory. Aim 2 will construct four lentiviral vectors in which the SL9- or TV9-regions are modified by point mutations. pL-wtGag will encode wtGag; pL-ASL9 will contain a point mutation to disable binding of SL9 to HLA-A2; pL-p41 will have its SL9 replaced by p41; and the 4th vector will build on the most effective vector defined by Aim 3, in which TV9 is replaced with TV9p6. Aim 3 will determine whether the hierarchy of CTL epitopes determines the ability of the responding cells to suppress viral replication. It will test whether numerically dominant SL9 response actively suppresses more efficacious responses to TV9 and possibly other minor epitopes. T cells will be ranked according to their ability to suppression HIV in vitro. Diversity of the response will be judged by clonotype TCR sequencing. Aim 4 will determine whether the most effective vector will generate protective memory CD8+ when challenged with a surrogate virus in HHD mice. Immunodominance has been studied primarily in mice with model viruses. Our studies will attempt to understand why SL9- and TV9-responses do not appear to play important roles in controlling HIV infections. A new approach, combining studies of the TCR degeneracy for SL9 and TV9 with reverse genetics, will be tested to improve the immunogenicity of HIV Gag.