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.

旨在通过传递天然蛋白来提高细胞免疫力的艾滋病毒疫苗尚未被 成功。我们通过体外启动表明，HIV Gag的免疫优势表位为人类白细胞抗原A2限制性的SL9表位 产生不稳定的非依赖帮助的CTL反应，而次要的TV9则激发CDS‘1 反应不能完全亲和成熟。SL9的激动剂p41多肽，通过探测一个大的 与SL9特异性CD8+T细胞结合的多肽文库，可诱导稳定的SL9交叉反应T细胞。这个 TV9的激动剂TV9p6渴望和聚焦的四聚体*CTL。重要的是，它们比 TV9-CTL在裂解病毒感染靶细胞中的作用 我们假设，对SL9表位的数字显性反应主动抑制 对亚优势肽的潜在更有效的反应。尤其是TV9。我们建议， 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来提高 表位层级，这将允许操纵这些T细胞反应。在这里我们将评估T细胞 转导表达GAG的DC体外启动人T细胞对SL9和TV9的竞争 目标1将确定SL9-T细胞是来自NATve还是来自记忆/效应器前体 健康供者，以了解过度激活的反应是SL9的固有特性还是由于 调动先前存在的交叉反应记忆。目标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改。Pl-wtGag将编码wtGag；pl-ASL9将包含 点突变以禁用SL9与人类白细胞抗原A2的结合；Pl-p41将其SL9替换为p41；以及第4 向量将建立在目标3定义的最有效的向量上，其中TV9被TV9p6取代。目标3将 确定CTL表位的层级是否决定了反应细胞抑制的能力 病毒复制。它将测试数字占主导地位的SL9反应是否会主动抑制更多 对TV9和可能其他次要表位的有效应答。T细胞将根据它们的 在体外抑制艾滋病毒的能力。反应的多样性将通过克隆型TCR测序来判断。 目标4将确定最有效的载体是否会在以下情况下产生保护性记忆CD8+ 在HHD小鼠中用代理病毒进行挑战。 免疫优势主要是在带有模型病毒的小鼠身上进行的研究。我们的研究将尝试 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，将SL9和TV9的TCR简并研究与反向遗传学相结合，将是 经检测可提高HIV Gag的免疫原性。