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

• 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
• 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

 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（由申请人提供）：目前没有方法能够有效地“冲击”细胞以从潜伏的HIV储存库中产生病毒，这是免疫系统杀死抗原阳性细胞的先决条件。了解阻碍原病毒从潜伏期中诱导出来的机制对于潜伏期逆转和储存库耗尽至关重要。我们建议 通过开发和实施一种新方法来定义潜伏感染细胞的特性，至少确定其中一些机制。在拟议研究的 R21 阶段，我们将开发一个实验管道，该管道将能够分离并扩增含有单一诱导型或非诱导型原病毒的细胞，其纯度至少为 1%（与未分级 PBMC 群体中的约 0.001% 相比）。然后，我们将通过多位点 qPCR 筛选来定义这些原病毒的结构，然后进行完整的原病毒测序，以确定编码感染性病毒的潜力。该程序还将揭示宿主染色体中的前病毒整合位点以及附加型环状病毒 DNA 的存在。在 R33 阶段，我们将使用病毒生长测定区分诱导型和非诱导型原病毒，并将尝试使用扩增的细胞群通过 PCR 激活细胞分选来分离纯的、潜伏感染的细胞。此外，我们将通过对上述扩增的细胞谱系和含有诱导型和非诱导型原病毒的纯化细胞群进行多个同步下游分析，进一步开发我们的储库表征新方法。这些包括原病毒和邻近细胞基因的转录活性、病毒蛋白的产生和 DNA 甲基化模式。这些研究可能揭示阻止病毒诱导潜伏的机制。此外，通过扩大和鉴定含有单个可诱导和不可诱导原病毒的细胞群，我们还将为从潜伏期诱导原病毒的离体测试方法提供宝贵的资源。