AIDS Research Institute IrsiCaixa, Badalona, Spain.
University of Vic-Central University of Catalonia (UVic-UCC), Vic, Spain.
mBio. 2021 Jun 29;12(3):e0056021. doi: 10.1128/mBio.00560-21. Epub 2021 Jun 22.
Improved assays are critical to the successful implementation of novel HIV-1 cure strategies, given the limited ability of currently available assays to quantify true effects on the viral reservoir. As interventions based on immune clearance target infected cells producing viral antigens, irrespective of whether the viruses generated are infectious or not, we developed a novel assay to identify viral protein production at the single-cell level. The novel viral protein spot (VIP-SPOT) assay, based on the enzyme-linked ImmunoSpot (ELISpot) approach, quantifies the frequency of CD4 T cells that produce HIV antigen upon stimulation. The performance of the VIP-SPOT assay was validated in samples from viremic ( = 18) and antiretroviral therapy (ART)-treated subjects ( = 35), and the results were compared with total and intact proviral DNA and plasma viremia. The size of the functional reservoir, measured by VIP-SPOT, correlates with total HIV-1 DNA and, more strongly, with intact proviruses. However, the frequency of HIV antigen-producing cells is 100-fold lower than that of intact proviruses, thus suggesting that most latently infected cells harboring full-length proviruses are not prone to reactivation. Furthermore, VIP-SPOT was useful for evaluating the efficacy of latency reversing agents (LRAs) in primary cells. VIP-SPOT is a novel tool for measuring the size of the functional HIV-1 reservoir in a rapid, sensitive, and precise manner. It might benefit the evaluation of cure strategies based on immune clearance, as these will specifically target this minor fraction of the viral reservoir, and might assist in the identification of novel therapeutic candidates that modulate viral latency. Current efforts aimed at finding a definitive cure for HIV-1 infection are hampered mainly by the persistence of a viral reservoir in latently infected cells. While complete viral eradication from the body remains elusive, finding a functional cure to enable control of viremia without the need for continuous treatment is a key goal. As the lower reservoir size increases the likelihood of controlling viremia, new therapeutic strategies aim to reduce the size of this viral reservoir. Evaluating the efficacy of these strategies requires a robust assay to measure the viral reservoir. Currently available options are subject to overestimation or underestimation of the productive reservoir. In order to overcome this limitation, we have developed a novel assay, viral protein spot (VIP-SPOT), to precisely quantify the frequency of infected cells that retain the ability to reactivate and produce viral proteins.
改进的检测方法对于成功实施新型 HIV-1 治愈策略至关重要，因为目前可用的检测方法在定量评估病毒储存库的真实效果方面能力有限。由于基于免疫清除的干预措施针对的是产生病毒抗原的感染细胞，而不管产生的病毒是否具有传染性，因此我们开发了一种新的检测方法，用于在单细胞水平上识别病毒蛋白的产生。新型病毒蛋白斑点（VIP-SPOT）检测方法基于酶联免疫斑点（ELISpot）方法，定量检测刺激后产生 HIV 抗原的 CD4 T 细胞的频率。该 VIP-SPOT 检测方法的性能在病毒血症（ = 18）和抗逆转录病毒治疗（ART）治疗的受试者（ = 35）的样本中得到了验证，并将结果与总和完整前病毒 DNA 以及血浆病毒血症进行了比较。通过 VIP-SPOT 测量的功能性储存库的大小与 HIV-1 DNA 总量相关，与完整前病毒的相关性更强。然而，产生 HIV 抗原的细胞的频率比完整前病毒低 100 倍，这表明大多数潜伏感染细胞携带全长前病毒并不容易重新激活。此外，VIP-SPOT 可用于评估潜伏逆转剂（LRAs）在原代细胞中的疗效。VIP-SPOT 是一种快速、敏感和精确测量功能性 HIV-1 储存库大小的新工具。它可能有助于评估基于免疫清除的治愈策略的效果，因为这些策略将专门针对病毒储存库的这一小部分，并且可能有助于鉴定调节病毒潜伏的新治疗候选物。目前，寻找根治 HIV-1 感染的方法主要受到潜伏感染细胞中病毒储存库持续存在的阻碍。虽然从体内完全清除病毒仍然难以实现，但找到一种功能性治愈方法，使人们能够在无需持续治疗的情况下控制病毒血症，是一个关键目标。由于储存库的大小越低，控制病毒血症的可能性越大，因此新的治疗策略旨在减小这个病毒储存库的大小。评估这些策略的疗效需要一种强大的检测方法来测量病毒储存库。目前可用的方法存在对产毒储存库的高估或低估。为了克服这一限制，我们开发了一种新的检测方法，即病毒蛋白斑点（VIP-SPOT），以精确定量保留重新激活和产生病毒蛋白能力的感染细胞的频率。
