HIV Epitope Specific T Cell Responses and Control of HIV Replication

描述（由申请人提供）：开发有效的艾滋病毒疫苗的一个主要障碍是我们对构成保护性艾滋病毒特异性免疫反应的关键成分缺乏了解。这一建议的目的是研究，比迄今为止所做的更全面，广泛的不同的HIV表位特异性T细胞功能在控制未经治疗的早期HIV感染患者的HIV复制中的潜在免疫保护作用。表位特异性T细胞功能将被测量包括CD4+和CD8+ T细胞IFN-g、TNF-a和IL-2应答（以及这些应答细胞的成熟状态）、CD4+和CD8+ T细胞增殖和CD8+ T细胞穿孔素脱粒。所有这些功能都可以通过跨相关HIV抗原蛋白的重叠肽刺激PMBC后的多参数流式细胞术来测量。我们建议从HIV Gag和Nef表位开始这项工作。然而，由于测量所有这些T细胞反应仅这些表位将需要大量的血液，我们将重点关注个体自身Gag/Nef序列中的肽基序，这些肽基序已知对该个体的单倍型具有hla限制。在对未经治疗的早期HIV感染患者建立良好的HIV复制控制之前获得的储存血浆样本中的自体Gag和Nef序列进行测序后，然后在这些患者失去控制的后续时间点再次进行测序，我们将扫描这些序列，寻找HLA单倍型合适的基序，然后将它们合成为9-和15-mer肽，分别用于I类和II类刺激。这些多肽随后将用于流式细胞术检测，以鉴定自体Gag/Nef表位特异性CD4+和CD8+ T细胞增殖、IFN-g、TNF-a和IL- 2反应（以及细胞因子阳性细胞的成熟状态）和CD8+ T细胞穿孔素脱粒反应。为了控制先天免疫反应的变化，这些变化也可能影响HIV复制的控制，我们还将在相同的PBMC样本中测量NK细胞和功能，浆细胞样细胞和髓样树突状细胞和调节性T细胞。了解这种具有重要临床意义的保守HIV蛋白（如Gag和Nef）的模式，可以提高对保护性HIV特异性免疫反应关键成分的理解，并提供对HIV表位特异性T细胞反应类型的深入了解，这可能有助于在未来的候选HIV疫苗试验中进行监测。

DESCRIPTION (provided by applicant): A major obstacle to the development of an effective HIV vaccine is our lack of understanding of the critical components that constitute a protective, HIV-specific immune response. The aim of this proposal is to investigate, more comprehensively than has been done to date, the potential immunoprotective roles of a broad spectrum of different HIV epitope-specific T cell functions in controlling HIV replication among untreated patients with early HIV infection. The epitope-specific T cell functions that will be measured include CD4+ and CD8+ T cell IFN-g, TNF-a and IL-2 responses (and the maturational state of these responding cells), CD4+ and CD8+ T cell proliferation and CD8+ T cell perforin degranulation. All these functions can be measured by multiparameter flow cytometry after stimulation of PMBC with overlapping peptides that span relevant HIV antigenic proteins. We propose to begin this work by focusing on HIV Gag and Nef epitopes. However, since the measurement of all these T cell responses just these epitopes would require prohibitively large quantities of blood, we will focus on peptide motifs within an individual's autologous Gag/Nef sequence that are known to be HLA-restricted for that individual's haplotype. After sequencing autologous Gag and Nef sequences from stored plasma samples obtained just before untreated patients with early HIV infection established good control of HIV replication, and then again from subsequent timepoints when these same patients have lost that control, we will scan the sequences for HLA haplotype-appropriate motifs and then synthesize them as 9- and 15-mer peptides for Class I and II stimulation, respectively. These peptides will then be used in flow cytometry assays to identify autologous Gag/Nef epitope-specific CD4+ and CD8+ T cell proliferation and IFN-g, TNF-a and IL- 2 responses (and the maturational state of the cytokine-positive cells) and CD8+ T cell perforin degranulation responses. To control for changes in innate immune responses that could also affect control of HIV replication, we will also measure NK cells and function, plasmacytoid and myeloid dendritic cells and regulatory T cells in the same PBMC specimens. Understanding this pattern for clinically important, conserved HIV proteins such as Gag and Nef should improve understanding of the critical components of a protective, HIV-specific immune response and provide insight into the type of HIV epitope-specific T cell responses that may be useful to monitor in future trials of candidate HIV vaccines.