Immunogenic epitopes of rare HIV mutants as vaccines

描述(由申请人提供)：在洛斯阿拉莫斯艾滋病毒分子免疫学数据库中记录了大量的艾滋病毒突变。有趣的是，常见的突变，即在30%-40%的病毒分离株中发现的，几乎没有不同的序列，而许多不同的罕见突变只在单个或极少数的个体中发现。对这些事实的一种解释可能是，常见的突变体代表了最成功地避开了CTL识别的选择压力的弱免疫原性序列变异，而更多的免疫原性罕见突变体则没有。此外，较罕见的CTL表位突变体增强免疫原性的主要基础可能是它们增加了非保守氨基酸替代的含量。我们建议探索这样一个假设，即HIV的罕见突变CTL表位比普通CTL表位具有更强的免疫原性，并建立了它们作为疫苗在产生抗HIV CTL反应方面可能更有效的原理证明。为了验证这一方法，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最充分的，并且HLA-A2超家族与自然感染抵抗力有关。在第一步中，我们将合成洛斯阿拉莫斯数据库中列出的这三个表位的所有突变体(总共107个单肽)。将使用人类白细胞抗原A2转基因小鼠模型来评估AIM 2的免疫原性。因此，动物将被免疫，产生CTL系，这些系的亲和力将通过(1)ELISpot：免疫肽、天然配体和/或普通突变体；(2)通过对反应细胞的细胞内染色；以及(3)对T2靶细胞脉冲启动肽、天然配体和普通突变体进行评估。这些研究的实质是证明这些多肽突变体能够产生识别免疫多肽的特异性CTL，但更重要的是，它们还识别相应的天然HIV-1表位和常见突变体。最后，将使用相同的模型系统(目标3)来确定被识别的最具免疫原性的多肽突变的“鸡尾酒”混合物所诱导的CTL反应的广度。在建议的原则证明研究之后，我们预计将在I/II期临床试验中提供一系列毒性和有效性候选多肽，作为II期SBIR努力的一部分。此外，第二阶段将包括鉴定A2和其他常见MHC I类单倍型的多个其他CTL表位突变，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.