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缺乏对构成保护性的、艾滋病毒特异性免疫反应的关键成分的了解。这项建议的目的是要比迄今所做的更全面地研究各种不同的HIV表位特异性T细胞功能在控制未经治疗的早期HIV感染患者中的HIV复制方面的潜在免疫保护作用。将测量的表位特异性T细胞功能包括CD4+和CD8+T细胞的干扰素-g、肿瘤坏死因子-α和IL-2反应(以及这些反应细胞的成熟状态)、CD4+和CD8+T细胞的增殖和CD8+T细胞的穿孔素脱颗粒。所有这些功能都可以用多参数流式细胞仪在重叠的跨过HIV抗原蛋白的多肽刺激PMBC后进行检测。我们建议从关注HIV Gag和Nef表位开始这项工作。然而，由于测量所有这些T细胞反应仅仅是这些表位就需要令人望而却步的大量血液，我们将重点关注个体自体Gag/Nef序列中已知对该个体的单倍型具有人类白细胞抗原限制性的多肽基序。在对存储的血浆样本中的自体Gag和Nef序列进行测序后，在未经治疗的早期HIV感染患者建立了对HIV复制的良好控制之前，然后从这些相同的患者失去控制的后续时间点再次进行测序，我们将扫描序列中适合HLA单倍型的基序，然后将它们合成为9和15聚肽，分别用于I类和II类刺激。这些多肽随后将用于流式细胞仪分析，以确定自体GAG/Nef表位特异性的CD4+和CD8+T细胞的增殖和干扰素-g、肿瘤坏死因子-α和IL-2的反应(以及细胞因子阳性细胞的成熟状态)和CD8+T细胞的穿孔素脱颗粒反应。为了控制可能影响HIV复制控制的先天免疫反应的变化，我们还将在相同的PBMC样本中检测NK细胞和功能、浆细胞样树突状细胞和髓系树突状细胞以及调节性T细胞。了解临床上重要的、保守的HIV蛋白(如Gag和Nef)的这种模式，将提高对保护性、HIV特异性免疫反应的关键成分的理解，并提供对HIV表位特异性T细胞反应类型的洞察，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.