TILDA: A new sensitive and precise assay to measure the size of the latent HIV reservoir

描述（由申请人提供）：现在很清楚，单靠抗逆转录病毒疗法（ART）并不能根除艾滋病毒：即使经过超过15年的强化和持续治疗，除例外情况外，在停止抗逆转录病毒疗法后的几周内，病毒的传播仍会恢复。在病毒抑制的受试者中，只有百万分之一的静止CD4+ T细胞含有能够产生复制能力病毒的潜伏前病毒。定量携带潜伏HIV病毒的细胞对于评估消灭它们的策略至关重要，但这些细胞的低频率使得这一工作极具挑战性。随着越来越多的新颖和创新的治疗策略将在人体中进行测试，以减少潜伏库的大小，迫切需要开发一种强大的、精确的和临床试验可扩展的测定方法，以测量潜伏细胞感染携带诱导型HIV的细胞的频率。我们最近开发了一种检测方法来测量潜伏和诱导的HIV病毒库的大小，它将满足这些标准。这种新方法被命名为TILDA （Tat/rev诱导极限稀释法），在PMA/ iononomycin的最大细胞激活下，测量具有多重剪接的HIV RNA的细胞频率。重要的是，我们的检测不需要提取病毒核酸，这使得TILDA适合高通量研究。TILDA仅需10mL血液，重现性极好（变异系数= 0.2），覆盖储层尺寸的广泛动态范围（超过3 log），可在两天内完成。这项提议的目的是证明TILDA可以用来精确测量潜伏感染细胞在病毒抑制的受试者中持续存在的频率。我们将使用TILDA来测量36名接受抑制性抗逆转录病毒治疗的受试者的储存库大小，并将这些值与两个在该检测方面具有强大专业知识的实验室（Siliciano博士和Markowitz博士，合作者）的金标准方法（定量病毒生长测定法，Q-VOA）测量的值相关联。在Specific Aim 1中，我们将确定TILDA测量的库细胞频率是否与Q-VOA测量的库细胞频率相关。我们假设这些频率是相关的，但TILDA将提供对潜伏感染细胞池大小的更好估计。在特异性目标2中，我们将使用TILDA来评估在艾滋病毒感染的急性和慢性阶段开始抗逆转录病毒治疗的受试者中可以诱导产生艾滋病毒的具有整合艾滋病毒DNA的细胞的相对比例。我们预测，在慢性感染期间开始抗逆转录病毒治疗的个体中，大部分整合基因组将无法诱导。在特异性目标3中，我们将使用TILDA来测量不同记忆CD4 T细胞亚群中潜伏HIV库的大小，假设分化程度更高的亚群（效应记忆细胞）将携带更大比例的缺陷病毒。该项目的总体目标是证明TILDA可以在临床环境中普遍使用，以精确测量潜伏库的大小并评估根除策略的有效性。

DESCRIPTION (provided by applicant): It is now clear that antiretroviral therapy (ART) alone does not eradicate HIV: Even after more than 15 years of intensive and continuous therapy, the spread of the virus resumes within a few weeks upon cessation of ART in all but exceptional cases. In virally suppressed subjects, only about one in a million resting CD4+ T cells contain latent proviruses capable of producing replication-competent virus. Quantifying cells harboring latent HIV is critical to evaluating strategies to eliminate them, but the low frequency of these cells makes this extremely challenging. With an increasing number of novel and innovative therapeutic strategies that will be tested in humans to reduce the size of the latent reservoir, there is an urgent need to develop a robust, precise and clinical trial scalable assay that measures the frequency of latently cells infected cells carrying inducible HIV. We have recently developed an assay to measure the magnitude of the latent -and inducible- HIV reservoir, which will fulfill these criteria. This novel assay, named TILDA for Tat/rev Induced Limiting Dilution Assay, measures the frequency of cells with multiply spliced HIV RNA upon maximal cellular activation with PMA/ionomycin. Importantly, our assay does not require extraction of viral nucleic acids, which makes TILDA suitable for high throughput studies. TILDA requires only 10mL of blood, is extremely reproducible (coefficient of variation = 0.2), covers a wide dynamic range of reservoir size (over 3 logs) and can be completed in two days. The objective of this proposal is to demonstrate that TILDA can be used to precisely measure the frequency of latently infected cells that persist in virally suppressed subjects. We will use TILDA to measure the size of the reservoir in 36 subjects on suppressive ART and will correlate these values with those measured by the gold standard method (quantitative viral outgrowth assay, Q-VOA) in two laboratories with strong expertise in this assay (Dr. Siliciano and Dr. Markowitz, collaborators). In Specific Aim 1, we will determine if the frequency of reservoir cells measured by TILDA correlates with those measured by Q-VOA. We hypothesize that these frequencies will correlate, but that TILDA will provide a better estimate of the size of the pool of latently infectd cells. In Specific Aim 2, we will use TILDA to assess the relative proportion of cells with integrated HIV DNA that can be induced to produce HIV in subjects who started ART during the acute and the chronic stages of HIV infection. We predict that a large fraction of the integrated genomes will not be inducible in individuals who started ART during chronic infection. In Specific Aim 3, we will use TILDA to measure the size of the latent HIV reservoir in distinct memory CD4 T cell subsets, with the hypothesis that the more differentiated subsets (effector memory cells) will carry a larger proportion of defective viruses. The overarching goal of this project is to demonstrate that TILDA can be used universally in a clinical setting to precisely measure the size of the latent reservoir and assess the efficacy of eradication strategies.