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

TILDA：一种新的灵敏且精确的检测方法，用于测量潜在 HIV 病毒库的大小

• 批准号: 8880118
• 负责人: Nicolas Chomont
• 金额: $ 16.2万
• 依托单位: UNIVERSITY OF MONTREAL HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-01-01 至 2017-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8880118
• 关键词: Acetates; Acute; Anti-Retroviral Agents; Biological Assay; Blood; CD4 Positive T Lymphocytes; Cell Count; Cells; Chronic; Clinical; Clinical Trials; Cohort Studies; DNA; DNA biosynthesis; Defective Viruses; Frequencies; Genome; Goals; Gold; HIV; HIV Infections; Health; Human; Individual; Infection; Interruption; Ionomycin; Laboratories; Life; Measurement; Measures; Memory; Methods; Names; Nucleic Acids; Pharmaceutical Preparations; Procedures; Production; Proviruses; RNA; RNA Splicing; Relative (related person); Research Project Grants; Rest; Source; Staging; Surrogate Markers; T-Lymphocyte Subsets; Testing; Therapeutic; Time; Transcript; Variant; Viral; Viral Genome; Viremia; Virion; Virus; antiretroviral therapy; base; innovation; memory CD4 T lymphocyte; novel; particle; phorbol-12-myristate

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.

描述（由申请人提供）：现在很明显，仅抗逆转录病毒疗法（ART）并不能消除HIV：即使经过超过15年的强化和连续治疗后，除非例外情况以外，但在停止艺术的几周内，病毒的传播在几周内恢复。在病毒抑制的受试者中，只有一百万个静息CD4+ T细胞中只有大约1个包含能够产生复制能力病毒的潜在病毒。量化带有潜在艾滋病毒的细胞对于评估消除它们的策略至关重要，但是这些细胞的低频使这一挑战性极具挑战性。随着越来越多的新型和创新的治疗策略将在人类中进行测试以减少潜在储层的大小，因此迫切需要开发出强大，精确和临床试验的可伸缩测定法，以测量潜在的细胞感染可诱导HIV的细胞的频率。我们最近开发了一种测定法，以测量将符合这些标准的潜在 - HIV水库的大小。这个新的新测定法被称为TAT/REV诱导的限制稀释测定法，在用PMA/离子霉素最大细胞激活后测量了用剪接的HIV RNA的细胞频率。重要的是，我们的测定不需要提取病毒核酸，这使得蒂尔达适合于高通量研究。 Tilda仅需要10毫升的血液，非常可复制（变化系数= 0.2），覆盖了较大的储层大小的动态范围（超过3个原木），可以在两天内完成。该提案的目的是证明Tilda可用于精确测量受病毒抑制受试者持续存在的潜在感染细胞的频率。我们将使用蒂尔达（Tilda）在抑制艺术上的36个受试者中测量储层的大小，并将这些值与通过金标准方法（定量病毒出生测定，q-voa，q-voa）在该测定中具有强大专业知识（Siliciano博士和Markowitz，Markowitz博士，合作者）的值相关联。在特定的目标1中，我们将确定由Tilda测量的储层细胞的频率是否与Q-VOA测量的储层细胞相关。我们假设这些频率将相关，但是Tilda将更好地估计潜在感染细胞的大小。在特定的目标2中，我们将使用TILDA评估具有综合HIV DNA的细胞的相对比例，这些细胞可以在急性和HIV感染的慢性阶段开始启动ART的受试者中引起HIV。我们预测，在慢性感染期间开始使用ART的个体中，很大一部分综合基因组将无法诱导。在特定的目标3中，我们将使用Tilda在不同的记忆CD4 T细胞子集中测量潜在的HIV储存库的大小，并假设较为差异化的子集（效应存储器单元）将带有更大比例的有缺陷病毒。该项目的总体目标是证明可以在临床环境中普遍使用Tilda来精确衡量潜在储层的大小并评估消除策略的功效。