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促进细胞免疫的艾滋病毒疫苗尚未成功。我们通过体外启动表明，HIV Gag的免疫显性hla - a2限制性SL9表位产生不稳定的不依赖于帮助的CTL反应，而亚显性TV9表位则引发无法完全亲和成熟的CDS'1"反应。SL9的激动剂p41肽，通过与SL9特异性CD8+ T细胞探测一个大的组合肽库来鉴定，诱导稳定的SL9交叉反应T细胞。TV9的激动剂TV9p6可激活活跃和聚焦的四聚体* 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对亚显性肽的潜在更有效的反应。尤其是TV9。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表位等级来提高，这将允许操纵这些T细胞反应。在这里，我们将评估T细胞对SL9和TV9的竞争，通过体外启动人T细胞与转导表达Gag的dc。目的1将确定SL9- t细胞是否来源于健康供体的原始或记忆/效应前体，以了解过度激活的反应是SL9的固有特性还是由于预先存在的交叉反应记忆的动员。目的2将构建四个慢病毒载体，其中SL9-或tv9 -区域被点突变修饰。pL-wtGag将编码wtGag；l - asl9将包含一个点突变，使SL9无法与HLA-A2结合；pL-p41的SL9会被p41取代；第四个向量将建立在Aim 3定义的最有效向量上，其中TV9被TV9p6取代。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SL9反应是否会主动抑制对TV9和其他次要表位的更有效的反应。T细胞将根据其体外抑制HIV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将确定在HHD小鼠中使用替代病毒攻击时，最有效的载体是否会产生保护性记忆CD8+。免疫优势主要是在小鼠模型病毒中研究的。我们的研究将试图理解为什么SL9和tv9反应在控制HIV感染中似乎没有发挥重要作用。将SL9和TV9的TCR退化研究与反向遗传学相结合的新方法将用于提高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.