Evaluating HIV expression and latency in blood and tissues at the single cell level

描述(由申请人提供)：在制定干预措施以降低接受抗逆转录病毒治疗的患者中艾滋病毒残留水平方面的进展受到阻碍，部分原因是缺乏敏感和可靠的分析方法来衡量功能储存库的大小并跟踪潜伏期逆转治疗后的变化。此外，潜伏着HIV的细胞特征的异质性及其相对较低的频率阻碍了对体内病毒潜伏的潜在分子机制进行表征的努力。我们建议将基于微流体乳液的单细胞检测系统调整为1)可以量化在静止状态和药物治疗后转录活性的单个细胞的检测方法，以及2)允许分离和浓缩含有HIV DNA的细胞的方法，用于个体细胞询问感染细胞对旨在逆转病毒潜伏期的药物治疗的反应。在这项建议的R21阶段，我们将开发一种基于微流体/乳剂平台的单细胞Taqman分析，作为从长期抑制ART受试者分离的CD4T细胞中功能储存库的衡量标准，验证其性能特征，并将其与通过终端稀释共培养获得的复制能力储存库大小的金标准衡量结果进行比较。同时，我们将优化聚合酶链式反应激活的细胞分类(PACS)方法，从携带艾滋病毒的细胞和未携带艾滋病毒的细胞中回收总核酸，并确定这些核酸适合于靶向基因表达研究，以定量分析单细胞对药物的反应，以逆转病毒潜伏期。在这项提案的R33阶段，我们将测试单细胞Taqman试验的能力 预测HIV持续存在的两个关键后果：治疗中断后病毒反弹的动力学，以及长期抑制ART期间异常的T细胞和巨噬细胞激活。前者是由于瑞士西班牙中断治疗研究提供了独特而有价值的样本而成为可能的。此外，我们将使用PACS系统从长期接受抗逆转录病毒治疗的患者的血液和GALT中鉴定和丰富HIV DNA+细胞，从单个细胞收集核酸，并进行有针对性的基因表达测量，以评估基础和潜伏期逆转治疗后病毒RNA的表达水平及其与T细胞刺激、免疫功能和抑制信号相关基因的相关性，以确定HIV潜伏期逆转或缺失的细胞相关性。

DESCRIPTION (provided by applicant): Progress in the development of interventions to reduce levels of residual HIV in patients on ART is hampered, in part, by the lack of sensitive and reliable assays that can measure the functional reservoir size and track changes following latency reversing treatments. In addition, the heterogeneity of cellular characteristics of cells with latent HIV and their relative low frequencies confounds efforts to characterize underlying molecular mechanisms of viral latency in vivo. We propose to adapt a microfluidic-emulsion based, single cell assay system as 1) an assay that can quantify individual cells that are transcriptionally active at rest and after pharmacologic treatment and 2) an approach that permits the isolation and enrichment of HIV DNA containing cells for individual-cell-interrogation of infected cell responses to pharmacologic treatments intended to reverse viral latency. During the R21 phase of this proposal, we will develop a single cell Taqman assay based on the microfluidics/emulsion platform as a measure of the functional reservoir in CD4 T-cells isolated from subjects on long term suppressive ART, validate its performance characteristics and compare it to results from the gold standard measure of replication-competent reservoir size by terminal dilution co-culture. Concurrently, we will optimize a PCR-activated cell sorting (PACS) approach to recover total nucleic acids from cells that harbor HIV and those that do not and establish that these nucleic acids are suitable for targeted gene expression studies that quantitatively analyze single cell responses to pharmacologic agents for reversing viral latency. During the R33 phase of this proposal, we will test the single cell Taqman assay for its ability to predict two key consequences of HIV persistence: kinetics of viral rebound following treatment interruption and abnormal T-cell and macrophage activation during long term suppressive ART. The former is made possible by the availability of a unique and valuable sample set from the Swiss Spanish Interruption of Treatment study. Further, we will use the PACS system to identify and enrich HIV DNA+ cells from blood and GALT of patients on long term ART, collect the nucleic acid from individual single cells and perform targeted gene expression measurements to assess the magnitude of basal and post latency-reversal treatment viral RNA expression and their correlation with genes associated with T-cell stimulation, immune function, and inhibitory signaling to define cellular correlates of reversal or lack of reversal of HIV latency.