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.

设计用于通过递送天然蛋白质来促进细胞免疫的HIV疫苗还没有被应用。 成功我们通过体外致敏表明，HIV Gag的免疫显性HLA-A2限制性SL9表位 产生不稳定的辅助非依赖性CTL应答，而亚显性TV9引起CDS1 "。 反应不能完全亲和力成熟。SL9的激动剂p41肽，通过探测大的 与SL9特异性CD8 + T细胞的组合肽文库，洗脱稳定的SL9交叉反应性T细胞。的 TV9的激动剂TV9p6引发亲合力和聚焦的四聚体 * CTL。重要的是，它们比 TV9-裂解病毒感染的靶细胞中的CTL。 我们假设对SL9表位的数字显性反应积极抑制了对SL9表位的免疫应答。 对亚优势肽的潜在更有效的反应。特别是TV9。我们建议 HIV Gag作为HLA-A2 * 携带者免疫原的有效性可以通过定义其CTL来提高 表位层次，这将允许操纵这些T细胞反应。在这里，我们将评估T细胞 通过用转导以表达Gag的DC离体引发人T细胞来竞争SL9和TV9。 目的1将确定SL9-T细胞是否来源于naTve或记忆/效应前体， 健康供体，以确定过度激活的反应是否是SL9的固有特性或由于 激活了预先存在的交叉反应记忆Aim 2将构建四种慢病毒载体， SL9-或TV9-区通过点突变修饰。pL-wtGag将编码wtGag; pL-ASL9将含有 使SL9不能与HLA-A2结合的点突变; pL-p41将使其SL9被p41取代;以及第四个 载体将建立在目标3定义的最有效载体上，其中TV9被TV9p6取代。目标3将 确定CTL表位的层次是否决定了应答细胞抑制 病毒复制它将测试数字上占优势的SL9反应是否主动抑制更多的 对TV9和可能的其他次要表位的有效应答。T细胞将根据其 体外抑制艾滋病病毒的能力。将通过克隆型TCR测序判断应答的多样性。 目标4将确定最有效的载体是否会产生保护性记忆CD8+， 在HHD小鼠中用替代病毒攻击。 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SL9和TV9反应在控制艾滋病毒感染方面似乎没有发挥重要作用。 一种新的方法，结合研究的TCR简并性的SL9和TV9与反向遗传学，将是 测试以提高HIV Gag的免疫原性。