Ex vivo activation of CD4+ T-cells from donors on suppressive ART can lead to sustained production of infectious HIV-1 from a subset of infected cells
Ex vivo activation of CD4+ T-cells from donors on suppressive ART can lead to sustained production of infectious HIV-1 from a subset of infected cells
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DOI：
10.1371/journal.ppat.1006230
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发表时间：
2017-02-01
期刊：
影响因子：
6.7
通讯作者：
Mellors, John W.
中科院分区：
文献类型：
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作者：
Bui, John K.;Halvas, Elias K.;Mellors, John W.
逆转前病毒潜伏期后HIV感染细胞的命运还没有很好的表征。Simonetti等人最近发现，含有完整前病毒的CD 4（+）T细胞可在体内克隆扩增并产生低水平的感染性病毒血症。我们假设通过激活CD 4（+）T细胞逆转HIV潜伏期可以导致一部分病毒产生细胞的扩增，而不是消除它们。我们建立了一个体外细胞培养系统，包括用PMA/离子霉素刺激来自接受抑制性抗逆转录病毒治疗（ART）的供体的CD 4（+）T细胞（第1-7天），然后休息（第7-21天），然后重复刺激（第21-28天），始终在高浓度的雷特格韦和依法韦仑存在下有效阻断新的病毒复制周期。通过qPCR定量上清液中的HIV DNA和病毒体RNA。进行p6-PR-RT的单基因组测序（SGS）以遗传表征前病毒和病毒体相关基因组RNA。通过多个时间点的共培养物上清液的病毒生长测定（VOA）和SGS测定产生的病毒体的复制能力。实验用来自5个连续招募的供体的纯化的CD 4（+）T细胞进行，这些供体已经接受抑制性ART> 2年。在所有实验中，初始刺激后上清液中的HIV RNA水平增加，在休息期间降低或保持稳定，并在重复刺激后再次增加。HIV DNA水平没有显示出一致的变化模式。前病毒的SGS揭示了感染细胞群体的不同结果，从明显的消除到持久性和扩增。重要的是，受感染细胞的子集在刺激后持续扩增并产生感染性病毒。这些发现强调了消除HIV感染细胞库的复杂性，并强调了在HIV感染细胞增殖之前杀死它们的新策略的必要性。
The fate of HIV-infected cells after reversal of proviral latency is not well characterized. Simonetti, et al. recently showed that CD4(+) T-cells containing intact proviruses can clonally expand in vivo and produce low-level infectious viremia. We hypothesized that reversal of HIV latency by activation of CD4(+) T-cells can lead to the expansion of a subset of virus-producing cells rather than their elimination. We established an ex vivo cell culture system involving stimulation of CD4(+) T-cells from donors on suppressive antiretroviral therapy (ART) with PMA/ionomycin (day 1-7), followed by rest (day 7-21), and then repeat stimulation (day 21-28), always in the presence of high concentrations of raltegravir and efavirenz to effectively block new cycles of viral replication. HIV DNA and virion RNA in the supernatant were quantified by qPCR. Single genome sequencing (SGS) of p6-PR-RT was performed to genetically characterize proviruses and virion-associated genomic RNA. The replication-competence of the virions produced was determined by the viral outgrowth assay (VOA) and SGS of co-culture supernatants from multiple time points. Experiments were performed with purified CD4(+) T-cells from five consecutively recruited donors who had been on suppressive ART for > 2 years. In all experiments, HIV RNA levels in supernatant increased following initial stimulation, decreased or remained stable during the rest period, and increased again with repeat stimulation. HIV DNA levels did not show a consistent pattern of change. SGS of proviruses revealed diverse outcomes of infected cell populations, ranging from their apparent elimination to persistence and expansion. Importantly, a subset of infected cells expanded and produced infectious virus continuously after stimulation. These findings underscore the complexity of eliminating reservoirs of HIV-infected cells and highlight the need for new strategies to kill HIV-infected cells before they can proliferate.