Optimizing Gag CTL Epitopes to Improve Immunogenicity

优化 Gag CTL 表位以提高免疫原性

• 批准号: 7618862
• 负责人: JUNE KAN-MITCHELL
• 金额: $ 3.95万
• 依托单位: UNIVERSITY OF TEXAS EL PASO
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-05-15 至 2011-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7618862
• 关键词: Affinity; Agonist; Antigens; Binding; Biological Assay; CD8B1 gene; Cells; Cellular Immunity; Characteristics; Consensus Sequence; Cytolysis; Data; Effectiveness; Epitopes; Gagging; Glean; HIV; HIV Infections; HIV vaccine; HLA-A2 Antigen; Human; In Vitro; Infection; Infection Control; Lead; Lentivirus Vector; Mediating; Memory; Minor; Modeling; Mus; Peptide Library; Peptides; Physiologic pulse; Play; Point Mutation; Population; Positioning Attribute; Price; Property; Proteins; Protocols documentation; Pulse taking; Research Personnel; Role; SL9 peptide; T memory cell; T-Lymphocyte; Testing; Vaccine Design; Viral; Virus; Work; Yang; base; cellular transduction; combinatorial; cytokine; gag Gene Products; immunogenicity; improved; insight; novel strategies; positional cloning; programs; response; vector

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 naTve 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.

旨在通过递送天然蛋白来促进细胞免疫的HIV疫苗尚未 成功的。我们通过体内启动表明，免疫主导的HLA-A2限制性SL9 HIV GAG的SL9表位 产生不稳定的独立的CTL响应，而亚域TV9挑衅CDS'1” 响应无法完全亲和力成熟。 SL9的激动剂P41肽，通过探测大的 与SL9特异性CD8+ T细胞的组合肽库，引起稳定的SL9触发性T细胞。这 TV9 Primes的Agonist TV9P6狂热和聚焦的四聚体* CTL。重要的是，它们比 裂解病毒感染的靶细胞中的TV9-CTL。 我们假设对SL9表位的数值主导反应会主动抑制 对亚抑制肽的反应可能更有效。特别是TV9。我们建议 HIV GAG作为HLA-A2*载体的免疫原的有效性可以通过定义其CTL来提高 表位层次结构，这将允许操纵这些T细胞响应。在这里，我们将评估T细胞 通过转导的DC的人体T细胞对SL9和TV9的竞争。 AIM 1将确定SL9-T细胞是从NATVE或内存/效应器前体得出的 健康的捐助者，查看过度活化的响应是SL9的固有特性还是由于 动员预先存在的交叉反应记忆。 AIM 2将构建四个慢病毒向量 SL9或TV9区域通过点突变进行了修改。 PL-WTGAG将编码WTGAG； PL-ASL9将包含一个 点突变以禁用SL9与HLA-A2的结合； PL-P41将其SL9替换为P41；和第四 向量将基于AIM 3定义的最有效的向量，其中TV9被TV9P6取代。目标3意志 确定CTL表位的层次结构是否确定响应细胞抑制的能力 病毒复制。它将测试数值主导的SL9响应是否会主动抑制更多 对TV9以及其他次要表位的有效反应。 T细胞将根据其排名 在体外抑制HIV的能力。响应的多样性将通过Clonotype TCR测序来判断。 AIM 4将确定最有效的向量是否会产生保护性内存CD8+ 在HHD小鼠中受到替代病毒的挑战。 免疫主导主要在具有模型病毒的小鼠中研究。我们的研究将尝试 理解为什么SL9和TV9-Respons似乎在控制艾滋病毒感染中没有起着重要作用。 一种新方法，将SL9和TV9的TCR堕落性研究与反向遗传学结合在一起，将是 测试以改善HIV GAG的免疫原性。