Ex Vivo Detection and Analysis of Non-inducible Latent HIV

非诱导性潜伏 HIV 的离体检测和分析

基本信息

批准号：
9046237
负责人：
JAMES Ivan MULLINS
金额：
$ 23.64万
依托单位：
UNIVERSITY OF WASHINGTON
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-12-01 至 2017-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9046237
关键词：
Abate Address Antigens Biological Assay CD4 Positive T Lymphocytes Cell Count Cell Density Cell Fraction Cell Lineage Cell Proliferation Cell Separation Cells Chromosomes Consensus Cytolysis DNA DNA Methylation Detection Development Environment Frequencies Gene Expression Genes Genetic Transcription Genome Gold HIV HIV Infections Immune system In Vitro Individual Interleukin-2 Laboratories Latent Virus Metabolism Methodology Methods Peripheral Blood Mononuclear Cell Phase Population Procedures Production Property Protocols documentation Proviruses RNA Reading Regimen Residual state Resources Rest Sampling Shock Signal Transduction Structure T-Lymphocyte Technology Testing Time Transcript Viral Viral Proteins Virus Virus Activation Virus Integration Virus Latency bisulfite sequencing in vivo integration site killings methylation pattern novel novel strategies prevent protein expression public health relevance single cell analysis standard measure viral DNA whole genome

项目摘要

DESCRIPTION (provided by applicant): No current methods are able to efficiently "shock" cells into producing virus from latent HIV reservoirs, a prerequisite for killing antigen positive cells by the immune system. Understanding the mechanisms that hinder the induction of proviruses out of latency is critical to latency reversal and to reservoir depletion. We propose to identify at least some of these mechanisms through development and implementation of a new approach to defining properties of latently infected cells. In the R21 phase of the proposed studies we will develop an experimental pipeline that will enable isolation and >1,000-fold expansion of cells harboring a single inducible or a non-inducible provirus at a purity of at least 1% (compared to ~0.001% in unfractionated PBMC populations). We will then define the structure of these proviruses by a multilocus qPCR screen followed by complete provirus sequencing to determine the potential for encoding infectious virus. This procedure will also reveal the proviral integration sites in host chromosomes and presence of episomal, circular viral DNA. In the R33 phase, we will distinguish between inducible and non-inducible proviruses using virus outgrowth assays and will attempt to use the expanded cell populations for isolation of pure, latently infected cells using PCR-activated cell sorting. In addition, we will further develop our new approach to reservoir characterization by performing multiple simultaneous downstream analyses of the aforementioned expanded cell lineages and purified cell populations harboring inducible and non-inducible proviruses. These will include transcriptional activity of the provirus and adjacent cellular genes, viral protein production and DNA methylation patterns. These studies are likely to reveal mechanisms preventing viral induction from latency. In addition, by expanding and identifying cell populations harboring individual inducible and non-inducible proviruses we will also provide a valuable resource for ex vivo testing means of inducing proviruses out of latency.

描述（由适用提供）：没有当前方法能够有效地“冲击”细胞从潜在的HIV储藏中产生病毒，这是免疫系统杀死抗原阳性细胞的前提。理解阻碍预期病毒诱导的机制对于潜伏期逆转和储层消耗至关重要。我们建议通过开发和实施一种新方法来定义潜在感染细胞的特性，至少确定其中一些机制。在提出的研究的R21阶段，我们将开发一种实验管道，该管道将使构成单个诱导型或不可诱导病毒的细胞的隔离和> 1,000倍膨胀 1％（在未分流的PBMC种群中为〜0.001％）。然后，我们将通过多端口qPCR屏幕来定义这些病毒的结构，然后进行完整的病毒测序，以确定编码传染病病毒的潜力。该过程还将揭示宿主染色体中的病毒整合位点以及伴有圆形病毒DNA的存在。在R33阶段，我们将使用病毒生长测定法区分可诱导和不可诱导的病毒，并尝试使用扩展的细胞群体使用PCR激活的细胞分类来隔离纯，潜在感染的细胞。此外，我们将通过对近似扩展的细胞谱系和具有可诱导和不可诱导的病毒的纯化细胞群进行多个简单的下游分析来进一步开发我们的新方法来保留表征。这些将包括病毒和相邻细胞基因的转录活性，病毒蛋白的产生和DNA甲基化模式。这些研究可能揭示了防止潜伏期诱导病毒的机制。此外，通过扩大和识别具有个体诱导和不可诱导性病毒的细胞群体，我们还将为离体测试手段提供诱导病毒降低的预科病毒的宝贵资源。