Optimizing Gag CTL Epitopes to Improve Immunogenicity

描述（由申请人提供）：旨在通过递送天然蛋白质来促进细胞免疫的HIV疫苗尚未成功。我们通过离体引发表明，HIV Gag的免疫显性HLA-A2限制性SL 9表位产生不稳定的辅助非依赖性CTL应答，而亚显性TV 9引起不能完全亲和力成熟的CDS 1+应答。通过用SL 9特异性CD 8 + T细胞探测大的组合肽文库鉴定的SL 9的激动剂p41肽激发稳定的SL 9交叉反应性T细胞。TV 9的激动剂TV 9 p6引发亲合力和聚焦的四聚体 * CTL。重要的是，它们在裂解病毒感染的靶细胞方面比TV 9-CTL更有效。我们推测，对SL 9表位的数字显性反应积极抑制了对亚显性肽的潜在更有效的反应。特别是TV 9。我们提出，HIV Gag作为HLA-A2* 携带者的免疫原的有效性可以通过定义其CTL表位层次来提高，这将允许操纵这些T细胞应答。在这里，我们将通过用转导以表达Gag的DC离体引发人T细胞来评估T细胞对SL 9和TV 9的竞争。目的1将确定SL 9-T细胞是否来源于来自健康供体的幼稚或记忆/效应前体，以了解过度激活的反应是SL 9的内在特性还是由于预先存在的交叉反应性记忆的动员。目的2构建4个SL 9或TV 9区经点突变修饰的慢病毒载体。pL-wtGag将编码wtGag; pL-ASL 9将含有点突变以使SL 9不能与HLA-A2结合; pL-p41将使其SL 9被p41替换;并且第4个载体将建立在由Aim 3定义的最有效的载体上，其中TV 9被TV 9 p6替换。目的3将确定CTL表位的层次结构是否决定了应答细胞抑制病毒复制的能力。它将测试数字上占优势的SL 9应答是否主动抑制对TV 9和可能的其他次要表位的更有效应答。T细胞将根据其体外抑制HIV的能力进行排名。将通过克隆型TCR测序判断应答的多样性。目的4将确定当在HHD小鼠中用替代病毒攻击时，最有效的载体是否将产生保护性记忆CD 8+。免疫优势主要在小鼠模型病毒中进行了研究。我们的研究将试图了解为什么SL 9和TV 9反应似乎在控制HIV感染中没有发挥重要作用。一种新的方法，结合对SL 9和TV 9的TCR简并性的研究与反向遗传学，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.