Immunogenic epitopes of rare HIV mutants as vaccines

描述（由申请人提供）：在洛斯阿拉莫斯HIV分子免疫学数据库中记录了大量的HIV突变体。有趣的是，常见的突变，即在30-40%的病毒分离株中发现的突变，代表了很少不同的序列，而许多不同的罕见突变仅在单个或极少数个体中发现。对这些事实的一种解释可能是，常见突变体代表弱的免疫原性序列变异，这些变异最成功地逃避了CTL识别的选择压力，而更多的免疫原性罕见突变体则没有。此外，罕见的CTL表位突变体免疫原性增强的主要基础可能是其非保守氨基酸取代的含量增加。我们建议探索罕见的HIV突变CTL表位比常见的更具有免疫原性的假设，并建立它们可能更有效地作为产生抗HIV CTL反应的疫苗的原理证明。为了验证这种方法，我们选择了三个HLA-A2限制性表位[Gag p17 77-85；Gag p24 19-27；RT 309-317]，因为对HLA-A2 CTL表位的细胞免疫反应是迄今为止研究得最充分的，HLA-A2超家族与自然抗感染有关。第一步，我们将合成这三个表位在Los Alamos数据库中列出的所有突变体（总共107个单独的肽）。免疫原性将在Aim 2中使用HLA-A2转基因小鼠模型进行评估。因此，将对动物进行免疫，生成CTL系，并对这些系的亲和力进行评估(1)通过ELISpot：免疫肽，天然配体和/或常见突变体；(2)对应答细胞进行胞内染色；(3)用启动肽、天然配体和常见突变体脉冲的T2靶细胞。这些研究的本质是证明这些肽突变体能够产生特异性的ctl来识别免疫肽，但更重要的是，它们也能识别相应的天然HIV-1表位和常见突变体。最后，将使用相同的模型系统确定经鉴定的大多数免疫原性肽突变体的“鸡尾酒”混合物诱导的CTL反应的广度（目的3）。在拟议的原理验证研究之后，我们预计将在I/II期临床试验中获得一系列候选肽的毒性和有效性，作为II期SBIR工作的一部分。此外，II期将包括鉴定A2和其他常见MHC I类单倍型的多个其他CTL表位突变体，以设计覆盖多个HIV表位的更完整的疫苗。

DESCRIPTION (provided by applicant): A very large number of HIV mutants have been recorded in the Los Alamos HIV Molecular Immunology Database. Interestingly, common mutations, i.e., found in 30-40% of viral isolates, represent few different sequences, while numerous different rare mutations are found only in single or very few individuals. One interpretation of these facts may be that the common mutants represent weak immunogenic sequence variations that have most successfully evaded the selective pressure of CTL recognition, and that more immunogenic rare mutants have not. Furthermore, the principal basis for enhanced immunogenicity of the rarer CTL epitope mutants may be their increased content of non-conservative amino acid substitutions. We propose to explore the hypothesis that the rarer mutant CTL epitopes of HIV are more immunogenic than the common ones, and to establish the proof-of-principal that they may be more effective as vaccines in generating anti-HIV CTL responses. To validate this approach we have selected three HLA-A2 restricted epitopes [Gag p17 77-85; Gag p24 19-27; RT 309-317] since the cellular immune responses to HLA-A2 CTL epitopes are by far the most well studied and the HLA-A2 superfamily has been associated with natural resistance to infection. In a first step, we will synthesize all mutants listed in the Los Alamos Database for these three epitopes (a total of 107 individual peptides). Immunogenicity will be assessed in Aim 2 using HLA-A2 transgenic mouse model. Thus, animals will be immunized, CTL lines generated, and the avidity of these lines will be assessed (1) by ELISpot on: immunizing peptide, the native ligand, and/or common mutants; (2) by intracellular staining for the responding cells; and (3) on T2 target cells pulsed with the priming peptide, the native ligand, and common mutants. The essence of these studies is to demonstrate that these peptide mutants are able to generate specific CTLs that recognize the immunizing peptide, but more importantly, they also recognize the corresponding natural HIV-1 epitope and common mutants. Finally, the breadth of CTL responses induced by a "cocktail" mixture of the most immunogenic peptide mutants identified will be determined using the same model system (Aim 3). Following the proposed proof-of-principle studies, we anticipate having available a series of peptide candidates for toxicity and efficacy in phase I/II clinical trials as part of a phase II SBIR effort. In addition, phase II will include the identification of multiple other CTL epitope mutants for A2 and other common MHC class I haplotypes for the design of a more complete vaccine covering multiple HIV epitopes.