HIV Epitope Specific T Cell Responses and Control of HIV Replication

描述（由申请人提供）：开发有效HIV疫苗的主要障碍是我们对构成保护性HIV特异性免疫应答的关键组分缺乏了解。这项建议的目的是调查，更全面地比迄今为止，潜在的免疫保护作用的广谱不同的HIV表位特异性T细胞功能在控制HIV复制未经治疗的早期HIV感染患者。将测量的表位特异性T细胞功能包括CD 4+和CD 8 + T细胞IFN-g、TNF-α和IL-2应答（以及这些应答细胞的成熟状态）、CD 4+和CD 8 + T细胞增殖和CD 8 + T细胞穿孔素脱粒。所有这些功能都可以在用跨越相关HIV抗原蛋白的重叠肽刺激PMBC后通过多参数流式细胞术测量。我们建议开始这项工作的重点是HIV Gag和Nef表位。然而，由于仅测量这些表位的所有这些T细胞应答将需要大量血液，因此我们将关注个体的自体Gag/Nef序列内的肽基序，已知其对于该个体的单倍型是HLA限制的。在对来自储存的血浆样品的自体Gag和Nef序列进行测序后，所述血浆样品是在具有早期HIV感染的未经治疗的患者建立HIV复制的良好控制之前获得的，然后再次从随后的时间点，当这些相同的患者失去该控制时，我们将扫描HLA单倍型适当基序的序列，然后分别将它们合成为用于I类和II类刺激的9聚体肽和15聚体肽。然后将这些肽用于流式细胞术测定，以鉴定自体Gag/Nef表位特异性CD 4+和CD 8 + T细胞增殖和IFN-g、TNF-α和IL- 2应答（以及精氨酸阳性细胞的成熟状态）和CD 8 + T细胞穿孔素脱粒应答。为了控制也可能影响HIV复制控制的先天免疫应答的变化，我们还将测量相同PBMC标本中的NK细胞和功能、浆细胞样和髓样树突状细胞以及调节性T细胞。了解这种模式的临床重要的，保守的HIV蛋白，如Gag和Nef应该提高理解的关键组成部分的保护性，HIV特异性免疫反应，并提供洞察类型的HIV表位特异性T细胞反应，可能是有用的监测在未来的试验候选HIV疫苗。

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.