In adults starting antiretroviral therapy (ART) during acute infection, 2% of proviruses that persist on ART are genetically intact by sequence analysis. In contrast, a recent report in children treated early failed to detect sequence-intact proviruses. In another cohort of children treated early, we sought to detect and characterize proviral sequences after 6 to 9 years on suppressive ART. Peripheral blood mononuclear cells (PBMC) from perinatally infected children from the Children with HIV Early antiRetroviral (CHER) study were analyzed. Nearly full-length proviral amplification and sequencing (NFL-PAS) were performed at one time point after 6 to 9 years on ART. Amplicons with large internal deletions were excluded (<9 kb). All amplicons of ≥9 kb were sequenced and analyzed through a bioinformatic pipeline to detect indels, frameshifts, or hypermutations that would render them defective. In eight children who started ART at a median age of 5.4 months (range, 2.0 to 11.1 months), 733 single NFL-PAS amplicons were generated. Of these, 534 (72.9%) had large internal deletions, 174 (23.7%) had hypermutations, 15 (1.4%) had small internal deletions, 3 (1.0%) had deletions in the packaging signal/major splice donor site, and 7 (1.0%) were sequence intact. These 7 intact sequences were from three children who initiated ART after 2.3 months of age, one of whom had two identical intact sequences, suggestive of a cell clone harboring a replication-competent provirus. No intact proviruses were detected in four children who initiated ART before 2.3 months of age. Rare, intact proviruses can be detected in children who initiate ART after 2.3 months of age and are probably, as in adults, maintained by clonal expansion of cells infected before ART initiation.IMPORTANCE There are limited data about the proviral landscape in children exhibiting long-term suppression after early treatment, particularly in Sub-Saharan Africa where HIV-1 subtype C predominates. Investigating the sequence-intact reservoir could provide insight on the mechanisms by which intact proviruses persist and inform ongoing cure efforts. Through nearly full-length proviral amplification and sequencing (NFL-PAS), we generated 733 NFL-PAS amplicons from eight children. We showed that rare, genetically intact proviruses could be detected in children who initiated ART after 2.3 months of age. The frequency of intact proviruses was lower (P < 0.05) than that reported for HIV subtype B-infected adults treated during early HIV infection. We show that cells harboring genetically intact HIV proviruses are rare in children exhibiting long-term suppression after early treatment and may require the processing of a large number of cells to assess reservoir size. This points to the need for efficient methods to accurately quantify latent reservoirs, particularly in pediatric studies where sample availability is limited.

中文翻译：

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在生命的第一年开始抗逆转录病毒治疗后，七至九岁的儿童仍会感染完整的HIV原病毒。

在成年人中，在急性感染期间开始使用抗逆转录病毒疗法（ART），通过序列分析，在ART上持续存在的2％的原病毒在基因上是完整的。相反，最近关于早期治疗儿童的报告未能检测出序列完整的原病毒。在另一组接受早期治疗的儿童中，我们寻求在抑制性抗逆转录病毒治疗6至9年后检测并鉴定原病毒序列。分析了来自艾滋病毒早期抗逆转录病毒儿童研究的围产期感染儿童的外周血单个核细胞（PBMC）。在ART上6至9年后的某个时间点进行了近乎全长的前病毒扩增和测序（NFL-PAS）。内部大缺失的扩增子被排除（<9 kb）。对所有≥9kb的扩增子进行了测序，并通过生物信息学管道进行了分析，以检测插入缺失，移码，或会使他们有缺陷的超变。在八个中位年龄为5.4个月（范围为2.0至11.1个月）的儿童中，产生了733个NFL-PAS扩增子。其中，有534个（72.9％）具有较大的内部缺失，有174个（23.7％）具有超突变，有15个（1.4％）的内部缺失较小，在包装信号/主要剪接供体位点中有3个（1.0％）具有缺失，以及7个（1.0％）的序列是完整的。这7个完整序列来自于2.3个月大后开始抗逆转录病毒治疗的三个孩子，其中一个具有两个相同的完整序列，这表明细胞克隆具有复制能力的前病毒。在2.3个月龄之前开始接受抗逆转录病毒治疗的四名儿童中未检测到完整的原病毒。在2.3个月后开始接受抗逆转录病毒治疗的儿童中，可以检测到罕见的完整原病毒，而且可能与成年人一样，重要信息在早期治疗后表现出长期抑制作用的儿童中，特别是在HIV-1 C型亚型占主导地位的撒哈拉以南非洲地区，有关感染前儿童长期病毒抑制的前病毒景观数据有限。研究序列完整的储库可以提供完整的前病毒持续存在的机制的信息，并为正在进行的治疗工作提供信息。通过近乎全长的前病毒扩增和测序（NFL-PAS），我们从8个孩子中产生了733个NFL-PAS扩增子。我们表明，在2.3个月大后开始接受抗逆转录病毒治疗的儿童中，可以检测到罕见的遗传上完整的原病毒。完整的原病毒的频率低于在早期HIV感染期间接受治疗的HIV亚型B感染成年人的报告频率（P <0.05）。我们表明，在早期治疗后表现出长期抑制作用的儿童中，带有基因完整的HIV原病毒的细胞很少见，可能需要处理大量细胞以评估储库的大小。这表明需要有效的方法来准确地量化潜伏储层，特别是在样本可得性有限的儿科研究中。