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

描述（由申请方提供）：降低ART患者残留HIV水平的干预措施的开发进展受到阻碍，部分原因是缺乏灵敏可靠的检测方法，无法测量功能性储库大小并跟踪潜伏期逆转治疗后的变化。此外，潜伏HIV细胞的细胞特征的异质性及其相对低的频率混淆了表征体内病毒潜伏的潜在分子机制的努力。我们建议采用基于微流体乳液的单细胞测定系统作为1）可以定量在静息时和药物治疗后具有转录活性的单个细胞的测定和2）允许分离和富集含有HIV DNA的细胞的方法，用于对受感染细胞对旨在逆转病毒潜伏期的药物治疗的反应进行单个细胞询问。在本提案的R21阶段，我们将开发基于微流体/乳液平台的单细胞Taqman测定法，作为从长期抑制性ART受试者中分离的CD 4 T细胞中功能性储库的测量，验证其性能特征，并将其与通过终末稀释共培养的复制能力储库大小的金标准测量结果进行比较。同时，我们将优化PCR激活的细胞分选（PACS）方法，以从携带HIV的细胞和不携带HIV的细胞中回收总核酸，并确定这些核酸适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的药物的反应。在本提案的R33阶段，我们将测试单细胞Taqman检测试剂盒的能力， 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和长期抑制性ART期间T细胞和巨噬细胞的异常激活。前者是通过瑞士西班牙治疗中断研究中独特而有价值的样本集的可用性而实现的。此外，我们将使用PACS系统从长期ART患者的血液和GALT中鉴定和富集HIV DNA+细胞，从单个单细胞中收集核酸，并进行靶向基因表达测量，以评估基础和潜伏逆转治疗后病毒RNA表达的幅度及其与T细胞刺激，免疫功能，和抑制性信号传导来定义逆转或缺乏逆转HIV潜伏期的细胞相关性。

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.