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

基本信息

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
项目类别：
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

项目摘要

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）：降低接受 ART 的患者体内残留 HIV 水平的干预措施的开发进展受到阻碍，部分原因是缺乏能够测量功能性储库大小并跟踪潜伏期逆转后变化的灵敏且可靠的检测方法治疗。此外，潜伏 HIV 细胞的细胞特征的异质性及其相对较低的频率使表征体内病毒潜伏的潜在分子机制的努力变得混乱。我们建议采用基于微流体乳液的单细胞测定系统，作为 1) 一种可以量化静息时和药物治疗后转录活性的单个细胞的测定方法；2) 一种允许分离和富集含有 HIV DNA 的细胞的方法用于单个细胞询问受感染细胞对旨在逆转病毒潜伏期的药物治疗的反应。在该提案的 R21 阶段，我们将开发一种基于微流体/乳液平台的单细胞 Taqman 测定，作为从长期抑制性 ART 受试者中分离出的 CD4 T 细胞功能库的测量，验证其性能特征并进行比较它是通过终端稀释共培养来测量具有复制能力的储库大小的黄金标准测量结果。同时，我们将优化 PCR 激活细胞分选 (PACS) 方法，从携带 HIV 的细胞和不携带 HIV 的细胞中回收总核酸，并确定这些核酸适合用于定量分析单细胞对 HIV 的反应的靶向基因表达研究。用于逆转病毒潜伏期的药物。在该提案的 R33 阶段，我们将测试单细胞 Taqman 检测的能力预测 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中断治疗研究提供的独特且有价值的样本集而成为可能的。此外，我们将使用 PACS 系统从长期 ART 患者的血液和 GALT 中识别和富集 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，以评估基础和后潜伏期的程度 -逆转治疗病毒RNA表达及其与T细胞刺激、免疫功能和抑制信号传导相关基因的相关性，以确定HIV潜伏期逆转或缺乏逆转的细胞相关性。