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

描述（由申请人提供）：降低抗逆转录病毒治疗患者体内残留艾滋病毒水平的干预措施的进展受到阻碍，部分原因是缺乏敏感和可靠的检测方法，无法测量功能储存库的大小，并跟踪延迟逆转治疗后的变化。此外，潜伏HIV细胞的细胞特性的异质性及其相对较低的频率混淆了表征病毒在体内潜伏的潜在分子机制的努力。我们建议采用一种基于微流体-乳液的单细胞检测系统，作为1)一种可以量化静息状态和药物治疗后转录活性的单个细胞的检测方法；2)一种允许分离和富集含有HIV DNA的细胞的方法，用于检测受感染细胞对药物治疗的反应，以逆转病毒潜伏期。在本方案的R21阶段，我们将开发基于微流体/乳液平台的单细胞Taqman试验，作为长期抑制性ART受试者分离的CD4 t细胞功能库的测量，验证其性能特征，并将其与通过末端稀释共培养的复制能力库大小的金标准测量结果进行比较。同时，我们将优化pcr激活细胞分选（PACS）方法，从携带HIV和不携带HIV的细胞中恢复总核酸，并确定这些核酸适合用于靶向基因表达研究，定量分析单细胞对逆转病毒潜伏期药物的反应。在该提案的R33阶段，我们将测试单细胞Taqman试验的能力

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.