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

 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标准化测定，该测定可可靠且可重复地评估潜伏储库的大小。到目前为止，有两种方法被用来估计潜在储层的大小。基于PCR的测定是快速的、灵敏的和可重复的，但不能区分有复制能力的前病毒和有缺陷的前病毒。匡威，病毒生长测定（VOA）评估携带有复制能力的前病毒的细胞的频率，但是是密集的、可变的和低灵敏度的。以灵敏和可重复的方式测量潜伏感染细胞频率的测定法，并且适合于高通量单细胞平台，将代表未来临床根除研究中测量病毒储库的关键工具。PrimeFlow RNA是一种通过流式细胞术在单细胞水平上测量RNA表达的技术。我们利用这种技术开发了一种灵敏、特异和可重复的检测HIV-1感染细胞的方法。我们已经开发了三种HIV特异性探针组来检测单剪接、多剪接和全长HIV-1转录物。通过组合两种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并富集感染细胞至>90%纯度，类似于体内观察到的那些。本申请的目的是验证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。具体目标1，我们将通过评估勘探后潜在储层的大小来验证该方法。 用潜伏期逆转剂和促有丝分裂刺激进行体内病毒再活化。我们将这些估计与PCR和QVOA获得的估计进行比较。此外，我们将进行纵向分析，测量在体内给药后的潜伏期逆转剂的HIV感染者参加临床试验的病毒再激活。PrimeFlow RNA的一个显著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将利用 这些方面。我们将在离体病毒再激活后通过PrimeFlow RNA从临床样本中分选HIV感染和未感染的细胞，我们将使用这些细胞进行比较完整的转录组分析。此外，我们将PrimeFlow RNA与标准流式细胞术相结合，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志物进行比较分析。因此，本申请中提出的研究将实现两个主要目标。首先，它们将导致开发一种新的敏感技术来测量潜在储层的大小。其次，它们将产生关于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。

 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.