Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA, USA.
Retrovirology. 2018 Feb 17;15(1):22. doi: 10.1186/s12977-018-0396-3.
The identification of the most appropriate marker to measure reservoir size has been a great challenge for the HIV field. Quantitative viral outgrowth assay (QVOA), the reference standard to quantify the amount of replication-competent virus, has several limitations, as it is laborious, expensive, and unable to robustly reactivate every single integrated provirus. PCR-based assays have been developed as an easier, cheaper and less error-prone alternative to QVOA, but also have limitations. Historically, measuring integrated HIV DNA has provided insights about how reservoirs are formed and maintained. In the 1990s, measuring integrated HIV DNA was instrumental in understanding that a subset of resting CD4 T cells containing integrated HIV DNA were the major source of replication-competent virus. Follow-up studies have further characterized the phenotype of these cells containing integrated HIV DNA, as well as shown the correlation between the integration levels and clinical parameters, such as duration of infection, CD4 count and viral load. Integrated HIV DNA correlates with total HIV measures and with QVOA. The integration assay has several limitations. First, it largely overestimates the reservoir size, as both defective and replication-competent proviruses are detected. Since defective proviruses are the majority in patients on ART, it follows that the number of proviruses capable of reactivating and releasing new virions is significantly smaller than the number of integrated proviruses. Second, in patients on ART clonal expansion could theoretically lead to the preferential amplification of proviruses close to an Alu sequence though longitudinal studies have not captured this effect. Proviral sequencing combined with integration measures is probably the best estimate of reservoir size, but it is expensive, time-consuming and requires considerable bioinformatics expertise. All these reasons limit its use on a large scale. Herein, we review the utility of measuring HIV integration and suggest combining it with sequencing and total HIV measurements can provide insights that underlie reservoir maintenance.
确定最适合测量储存库大小的标志物一直是 HIV 领域的一大挑战。定量病毒扩增检测（QVOA）是量化复制能力病毒数量的参考标准，但存在一些局限性，因为它既费力、昂贵，又无法有效地重新激活每个整合的前病毒。基于 PCR 的检测已被开发为一种更简单、更便宜且出错风险更低的替代 QVOA 的方法，但也有局限性。从历史上看，测量整合的 HIV DNA 提供了有关储存库如何形成和维持的见解。在 20 世纪 90 年代，测量整合的 HIV DNA 对于理解含有整合 HIV DNA 的静止 CD4 T 细胞亚群是复制能力病毒的主要来源起到了重要作用。后续研究进一步描述了含有整合 HIV DNA 的这些细胞的表型，并显示了整合水平与临床参数（如感染持续时间、CD4 计数和病毒载量）之间的相关性。整合 HIV DNA 与总 HIV 测量值和 QVOA 相关。整合检测有几个局限性。首先，它在很大程度上高估了储存库的大小，因为检测到了有缺陷和复制能力的前病毒。由于有缺陷的前病毒在接受 ART 的患者中占多数，因此能够重新激活和释放新病毒的前病毒数量明显小于整合前病毒的数量。其次，在接受 ART 的患者中，理论上可以通过克隆扩增导致靠近 Alu 序列的前病毒优先扩增，但纵向研究并未捕获到这种效应。与整合测量相结合的前病毒测序可能是对储存库大小的最佳估计，但它昂贵、耗时且需要相当多的生物信息学专业知识。所有这些原因都限制了其大规模使用。在此，我们回顾了测量 HIV 整合的实用性，并建议将其与测序和总 HIV 测量相结合，可以提供对储存库维持的基础的深入了解。
