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

在单细胞水平评估血液和组织中的 HIV 表达和潜伏期

• 批准号: 9547754
• 负责人: Joseph K Wong
• 金额: $ 44.7万
• 依托单位: NORTHERN CALIFORNIA INSTITUTE/RES/EDU
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-01 至 2021-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9547754
• 关键词: Antigens; Antiviral Agents; Biological Assay; Blood; CD4 Positive T Lymphocytes; Cardiovascular system; Cell Count; Cell Cycle; Cell Death; Cell Separation; Cell Survival; Cells; Cellular Assay; Cellular biology; Characteristics; Clinical; Coculture Techniques; Consequences of HIV; DNA; Development; Effectiveness; Emulsions; Frequencies; Gene Expression; Gene Targeting; Genes; Genetic Transcription; Genome; Gold; Gut associated lymphoid tissue; HIV; HIV Infections; Health; Hepatic; Heterogeneity; Individual; Inflammation; Interruption; Kidney; Kinetics; Learning; Life; Macrophage Activation; Measurable; Measurement; Measures; Metabolic; Microfluidics; Molecular; Morbidity - disease rate; Nucleic Acids; Pathologic; Patients; Performance; Pharmacological Treatment; Pharmacology; Phase; Phenotype; Population; Population Heterogeneity; Proviruses; Reproducibility; Residual state; Rest; Sampling; Signal Transduction; Sorting - Cell Movement; Specificity; System; T-Cell Activation; T-Lymphocyte; Testing; Time; Tissue Sample; Tissues; Transcript; Variant; Viral; Viral reservoir; Virus; Virus Activation; Virus Latency; antiretroviral therapy; base; cohort; cost effective; experience; immune activation; immune function; immunopathology; in vivo; individual variation; inflammatory marker; interest; malignant neurologic neoplasms; mortality; new technology; novel; novel strategies; public health relevance; response; single cell analysis; standard measure; therapy development; tool; treatment trial; viral RNA; viral rebound; virology

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.

描述（由申请人提供）：开发干预措施的进展，以减少ART患者的残留HIV水平，部分原因是缺乏敏感和可靠的测定法，这些测定方法可以测量功能性储层大小和跟踪延迟后反转处理后的变化。另外，具有潜在艾滋病毒的细胞细胞特征的异质性及其相对低频率混淆了体内病毒潜伏期的潜在分子机制的努力。 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.在该提案的R21阶段中，我们将基于微流体/乳液平台开发单个细胞Taqman测定法，作为对长期抑制艺术中从受试者中分离出的CD4 T细胞中功能储层的量度，从而验证其性能特征，并将其比较其与终端限制的重复储备尺寸的金标准量相比，其结果与该量相比。同时，我们将优化一种PCR激活的细胞分选（PACS）方法，以从含有HIV的细胞和不具有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，这些核酸适用于定量分析单个细胞对药理剂的反应用于逆转病毒延迟的单个细胞反应。在此提案的R33阶段，我们将测试单细胞Taqman分析的能力 预测HIV持续性的两个关键后果：在长期抑制ART期间，治疗中断和异常T细胞和巨噬细胞激活后，病毒反弹的动力学。通过瑞士西班牙语中断治疗研究中的独特且有价值的样本，使前者成为可能。 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艾滋病毒潜伏期。