PrimeFlow RNA for detection of latently-infected CD4+ T cells

PrimeFlow RNA 用于检测潜伏感染的 CD4 T 细胞

• 批准号: 9137856
• 负责人: Fabio Romerio
• 金额: $ 26.25万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-06-17 至 2018-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9137856
• 关键词: Biological Assay; Biological Markers; Biology; CD4 Positive T Lymphocytes; Cell surface; Cells; Clinical; Clinical Trials; DNA; Detection; Enrollment; Face; Flow Cytometry; Frequencies; Future; Genes; Genetic Transcription; Goals; HIV; HIV-1; Human; Individual; Infection; Interruption; Lead; Length; Life; Measures; Methods; Proteins; Proviruses; RNA; RNA Splicing; Rest; Sampling; Sorting - Cell Movement; Stimulus; Techniques; Technology; Time; Transcript; Validation; Viral; Viral reservoir; Virion; Virus Replication; antiretroviral therapy; assay development; base; comparative; in vivo; memory CD4 T lymphocyte; public health relevance; research study; therapy development; tool; transcriptome

 DESCRIPTION (provided by applicant): The development of therapies to eliminate latently infected cells and achieve a functional cure requires standardized assays that reliably and reproducibly assess the size of the latent reservoir. Two approaches have so far been used to estimate the size of the latent reservoir. PCR-based assays are rapid, sensitive, and reproducible, but do not discriminate between replication competent and defective proviruses. By converse, viral outgrowth assays (VOA's) assess the frequency of cells harboring replication competent provirus, but are intensive, variable, and poorly sensitive. An assay that measures the frequency of latently infected cells in a sensitive and reproducible fashion, and that is amenable to a high-throughput, single-cell platform would represent a critical tool for measuring the viral reservoir in future clinical eradication studies. PrimeFlow RNA is a technology that measures RNA expression at the single-cell level by flow cytometry. We have taken advantage of this technique to develop a sensitive, specific, and reproducible assay to detect HIV-1 infected cells. We have developed three HIV-specific probe sets to detect single-spliced, multiple-spliced, and full-length HIV-1 transcripts. By combining two HIV-specific probe sets we identified and enriched infected cells to >90% purity from initial mixtures of 10-100 infected cell per 106 uninfected cells, similar to those observed in vivo. The goal of this application is to validate PrimeFlow RNA for the detection, enumeration, and phenotypic characterization of latently infected cells in clinical samples. This will be accomplished through two specific aims. I Specific Aim 1, we will validate this method by assessing the size of the latent reservoir after ex vivo viral reactivation with latency reversing agents and mitogenic stimuli. We will compare these estimates with the ones obtained by PCR and QVOA. In addition, we will perform longitudinal analyses measuring viral reactivation after in vivo administration of latency reversing agents to HIV-infected individuals enrolled in clinical trials. A significant benefit of PrimeFlow RNA is that it is a flow cytometry-based technology, thus allowing phenotypic characterization as well as sorting of the positive cells. In Specific Aim 2 will take advantage of these aspects. We will sort HIV-infected and uninfected cells from clinical samples by PrimeFlow RNA after ex vivo viral reactivation, which we will use to carry out comparative complete transcriptome analyses. In addition, we will com- bine PrimeFlow RNA with standard flow cytometry to perform comparative analyses of cell surface and intra- cellular markers in HIV-infected and uninfected cells from clinical samples after ex vivo viral reactivation. Therefore the studies proposed in this application will achieve two major goals. First, they will lead to develop a new sensitive technique to measure the size of the latent reservoir. Second, they will generate new critical information about the biology of latently infected cells obtained from clinical samples.

 描述（由申请人提供）：消除潜伏感染细胞并实现功能性治愈的疗法的开发需要能够可靠且可重复地评估潜伏病毒库大小的标准化测定。迄今为止，已使用两种方法来估计潜在储层的大小。基于 PCR 的检测快速、灵敏且可重复，但不能区分具有复制能力的原病毒和有缺陷的原病毒。相反，病毒生长测定（VOA）评估具有复制能力的原病毒的细胞的频率，但强度大、变化多且敏感性差。一种以灵敏且可重复的方式测量潜伏感染细胞频率的检测方法，并且适用于高通量、单细胞平台，将成为未来临床根除研究中测量病毒库的关键工具。 PrimeFlow RNA 是一种通过流式细胞术测量单细胞水平 RNA 表达的技术。我们利用这项技术开发了一种灵敏、特异且可重复的检测方法来检测 HIV-1 感染细胞。我们开发了三种 HIV 特异性探针组来检测单剪接、多剪接和全长 HIV-1 转录本。通过组合两个 HIV 特异性探针组，我们从每 106 个未感染细胞 10-100 个感染细胞的初始混合物中鉴定出感染细胞，并将其富集到纯度 > 90%，与体内观察到的情况类似。此应用的目标是验证 PrimeFlow RNA 用于临床样本中潜伏感染细胞的检测、计数和表型表征。这将通过两个具体目标来实现。 I 具体目标 1，我们将通过评估 ex 后潜在储库的大小来验证该方法 使用潜伏逆转剂和促有丝分裂刺激进行体内病毒再激活。我们将这些估计值与 PCR 和 QVOA 获得的估计值进行比较。此外，我们将进行纵向分析，测量对参加临床试验的艾滋病毒感染者体内施用潜伏期逆转剂后病毒的重新激活。 PrimeFlow RNA 的一个显着优点是它是一种基于流式细胞术的技术，因此可以进行表型表征以及阳性细胞的分选。在具体目标 2 中，将利用 这些方面。在离体病毒再激活后，我们将通过 PrimeFlow RNA 对临床样本中 HIV 感染和未感染的细胞进行分类，我们将用它来进行比较完整的转录组分析。此外，我们将 PrimeFlow RNA 与标准流式细胞仪结合起来，对离体病毒再激活后临床样本中感染 HIV 和未感染细胞的细胞表面和细胞内标记进行比较分析。因此，本申请中提出的研究将实现两个主要目标。首先，他们将开发一种新的灵敏技术来测量潜在储层的大小。其次，他们将生成有关从临床样本中获得的潜伏感染细胞的生物学的新的关键信息。