A Novel Assay to Measure the Magnitude of the Inducible Viral Reservoir in HIV-infected Individuals
A Novel Assay to Measure the Magnitude of the Inducible Viral Reservoir in HIV-infected Individuals
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DOI：
10.1016/j.ebiom.2015.06.019
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发表时间：
2015-08-01
期刊：
影响因子：
11.1
通讯作者：
Chomont, Nicolas
中科院分区：
文献类型：
--
作者：
Procopio, Francesco Andrea;Fromentin, Remi;Chomont, Nicolas
背景资料：量化潜伏感染的细胞对于评估旨在减少长寿病毒库大小的治疗策略的功效至关重要，但这些细胞的频率较低使得这一工作非常具有挑战性。方法：我们开发了TILDA（达特/rev诱导的有限稀释测定）以测量具有诱导的多重剪接的HIV RNA的细胞的频率，因为这些转录物通常在潜伏感染的细胞中不存在，但在病毒再活化时被诱导。TILDA需要不到一百万个细胞，不需要RNA提取，可以在两天内完成。在接受ART的受抑制个体中，我们发现TILDA估计的潜伏感染的CD 4 + T细胞的中位频率为24个细胞/百万，这是定量病毒生长测定测量的频率的48倍，并且比通过基于PCR的测定测量的携带病毒DNA的细胞的频率低6-27倍。TILDA测量与大多数HIV DNA检测密切相关。与感染晚期相比，在感染早期开始ART的受试者中，通过TILDA测量的潜伏性储库的大小较低（p = 0.011）。在未经治疗的HIV疾病，携带潜伏但诱导型HIV的CD 4+细胞的频率大大超过了积极生产细胞的频率，表明即使在没有ART的情况下，大多数感染细胞在转录上是沉默的。解释：我们的研究结果表明，TILDA是一种可重复的和敏感的方法来测量生产和潜伏感染细胞在临床环境中的频率。我们表明，潜伏水库代表了相当大的一部分，所有感染的细胞之前，ART initiation.Research的上下文：在这篇手稿中，我们描述了一种新的检测方法，测量潜伏的HIV水库，主要障碍，以消除艾滋病毒的大小的发展。这种新的检测方法，称为TILDA，用于达特/rev诱导的有限稀释检测，仅需要10 ml血液，不需要提取病毒核酸，具有高度可重复性，涵盖了宽的动态范围的储库大小，可以在两天内完成。因此，TILDA可能代表用于评估旨在减少潜伏HIV储库大小的治疗策略的功效的现有测定的替代方法。(C)2015年，作者。由爱思唯尔公司出版
Background: Quantifying latently infected cells is critical to evaluate the efficacy of therapeutic strategies aimed at reducing the size of the long-lived viral reservoir, but the low frequency of these cells makes this very challenging.Methods: We developed TILDA (Tat/rev Induced Limiting Dilution Assay) to measure the frequency of cells with inducible multiply-spliced HIV RNA, as these transcripts are usually absent in latently infected cells but induced upon viral reactivation. TILDA requires less than a million cells, does not require RNA extraction and can be completed in two days.Findings: In suppressed individuals on ART, we found the median frequency of latently infected CD4+ T cells as estimated by TILDA to be 24 cells/million, which was 48 times more than the frequency measured by the quantitative viral outgrowth assay, and 6-27 times less than the frequencies of cells harbouring viral DNA measured by PCR-based assays. TILDA measurements strongly correlated with most HIV DNA assays. The size of the latent reservoir measured by TILDA was lower in subjects who initiated ART during the early compared to late stage of infection (p = 0.011). In untreated HIV disease, the frequency of CD4+ cells carrying latent but inducible HIV largely exceeded the frequency of actively producing cells, demonstrating that the majority of infected cells are transcriptionally silent even in the absence of ART.Interpretations: Our results suggest that TILDA is a reproducible and sensitive approach to measure the frequency of productively and latently infected cells in clinical settings. We demonstrate that the latent reservoir represents a substantial fraction of all infected cells prior to ART initiation.Research in context: In this manuscript, we describe the development of a novel assay that measures the magnitude of the latent HIV reservoir, the main barrier to HIV eradication. This novel assay, termed TILDA for Tat/rev Induced Limiting Dilution Assay, requires only 10 ml of blood, does not necessitate extraction of viral nucleic acids, is highly reproducible, covers a wide dynamic range of reservoir sizes and can be completed in two days. As such, TILDA may represent an alternative to existing assays used to evaluate the efficacy of therapeutic strategies aimed at reducing the size of the latent HIV reservoir. (C) 2015 The Authors. Published by Elsevier B.V.