Intact proviral DNA assay analysis of large cohorts of people with HIV provides a benchmark for the frequency and composition of persistent proviral DNA
Intact proviral DNA assay analysis of large cohorts of people with HIV provides a benchmark for the frequency and composition of persistent proviral DNA
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DOI：
10.1073/pnas.2006816117
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发表时间：
2020-08-04
影响因子：
11.1
通讯作者：
Siliciano, Janet D.
中科院分区：
文献类型：
--
作者：
Simonetti, Francesco R.;White, Jennifer A.;Siliciano, Janet D.
一个可扩展的方法来量化完整的HIV-1前病毒是至关重要的基础研究和临床试验针对HIV-1的治愈。完整前病毒DNA检测（IPDA）是一种表征HIV-1储库的新方法，专注于独立于转录状态的单个前病毒的遗传完整性。它使用多重数字液滴PCR来区分和单独定量完整的前病毒，定义为缺乏明显的致命缺陷，如大缺失和APOBEC 3G介导的超突变，从大多数具有此类缺陷的前病毒。这种区别很重要，因为只有完整的前病毒才会在ART中断时引起病毒反弹。为了评估IPDA的性能并提供支持其实施的基准数据，我们分析了来自几个不同队列的400名接受ART的HIV-1（+）成人的外周血样本，这些样本代表了美国治疗HIV-1感染的稳健样本。我们提供了直接的定量证据，证明缺陷前病毒的数量大大超过完整前病毒（>12.5倍）。然而，完整的前病毒的存在频率（中位数，54/10（6）CD 4（+）T细胞）明显高于通过定量病毒生长试验检测到的前病毒，后者需要诱导和体外生长（类似于1/10（6）CD 4（+）T细胞）。仅在6.3%的个体中观察到由序列多态性引起的IPDA扩增子信号问题，并且该问题很明显并且容易与低前病毒频率区分开，这是IPDA优于在这种情况下产生假阴性结果的标准PCR测定的优点。这里提供的大型IPDA数据集给出了迄今为止HIV-1前病毒在ART上持续存在的最清晰的定量图片。
A scalable approach for quantifying intact HIV-1 proviruses is critical for basic research and clinical trials directed at HIV-1 cure. The intact proviral DNA assay (IPDA) is a novel approach to characterizing the HIV-1 reservoir, focusing on the genetic integrity of individual proviruses independent of transcriptional status. It uses multiplex digital droplet PCR to distinguish and separately quantify intact proviruses, defined by a lack of overt fatal defects such as large deletions and APOBEC3G-mediated hypermutation, from the majority of proviruses that have such defects. This distinction is important because only intact proviruses cause viral rebound on ART interruption. To evaluate IPDA performance and provide benchmark data to support its implementation, we analyzed peripheral blood samples from 400 HIV-1(+) adults on ART from several diverse cohorts, representing a robust sample of treated HIV-1 infection in the United States. We provide direct quantitative evidence that defective proviruses greatly outnumber intact proviruses (by >12.5 fold). However, intact proviruses are present at substantially higher frequencies (median, 54/10(6) CD4(+) T cells) than proviruses detected by the quantitative viral outgrowth assay, which requires induction and in vitro growth (similar to 1/10(6) CD4(+) T cells). IPDA amplicon signal issues resulting from sequence polymorphisms were observed in only 6.3% of individuals and were readily apparent and easily distinguished from low proviral frequency, an advantage of the IPDA over standard PCR assays which generate false-negative results in such situations. The large IPDA dataset provided here gives the clearest quantitative picture to date of HIV-1 proviral persistence on ART.