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, 54/10⁶ CD4⁺ T cells) than proviruses detected by the quantitative viral outgrowth assay, which requires induction and in vitro growth (∼1/10⁶ 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.

量化完整 HIV-1 原病毒的可扩展方法对于针对 HIV-1 治愈的基础研究和临床试验至关重要。完整原病毒 DNA 测定 (IPDA) 是一种表征 HIV-1 病毒库的新方法，重点关注独立于转录状态的个体原病毒的遗传完整性。它使用多重数字液滴 PCR 来区分和单独量化完整的原病毒，其定义为缺乏明显的致命缺陷，例如大缺失和 APOBEC3G 介导的超突变，与大多数具有此类缺陷的原病毒不同。这种区别很重要，因为只有完整的原病毒才会在 ART 中断时引起病毒反弹。为了评估 IPDA 的表现并提供基准数据来支持其实施，我们分析了来自多个不同队列的 400 名接受 ART 治疗的 HIV-1 ⁺成年人的外周血样本，这些样本代表了美国接受治疗的 HIV-1 感染的可靠样本。我们提供了直接的定量证据，表明有缺陷的原病毒的数量大大超过完整的原病毒（超过 12.5 倍）。然而，完整原病毒的存在频率（中位数，54/10 ⁶ CD4 ⁺ T 细胞）比通过定量病毒生长测定检测到的原病毒要高得多，定量病毒生长测定需要诱导和体外生长（∼1/10 ⁶ CD4 ⁺ 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，并且很容易与低原病毒频率区分开来，这是 IPDA 相对于标准 PCR 检测的一个优势，标准 PCR 检测在这种情况下会产生假阴性结果。这里提供的大型 IPDA 数据集提供了迄今为止最清晰的 ART 中 HIV-1 前病毒持续性的定量图景。