Characterizing HIV-1 diversity, evolution, and integration sites in children initiating cART in early infection

 描述（由申请人提供）：尽管联合抗逆转录病毒治疗（cART）在儿童中取得了成功，但HIV仍具有显著的持久性，除了一个记录良好的病例，其中非常早期的治疗可能导致病毒根除。cART期间的HIV-1持续存在是由于CD4 + T细胞的长寿群体的感染。早期开始cART与成人和年龄较大的儿童中感染细胞数量较少以及病毒多样性有限相关[1，3]，但对婴儿期开始cART的儿童中HIV-1群体遗传学知之甚少。在病毒种群基本上是同质的情况下开始cART的婴儿可能具有非常有限的病毒多样性，并且没有证据表明随着时间的推移病毒进化。为了优化HIV治疗策略，需要更好地描述在感染后12小时开始接受cART的婴儿中HIV-1群体遗传学特征。最近的证据表明，一些HIV-1感染的CD4+细胞可能有增殖和生存优势时，HIV整合在宿主基因相关的生长调节。这种克隆性扩增是否出现在接受早期cART治疗的儿童中尚不清楚。为了填补这些关键的知识空白，我们建议调查艾滋病毒的多样性，使用单基因组测序（SGS）以及病毒整合位点，使用一种新的整合位点测定（ISA），在纵向样本（从 基线至治疗开始后7 - 11年），形成来自患有HIV的儿童早期治疗（CHER）研究的早期抗逆转录病毒治疗的HIV-1感染儿童的队列。在具有足够HIV-1多样性的患者中，我们还将在治疗期间和治疗后将整合在CD4+细胞中的病毒序列与血浆中循环的病毒联系起来，以揭示持续性和/或反弹性病毒血症的特异性前病毒来源。这是以前没有做到过的。我们希望能找到一些病毒多样性非常有限的儿童，他们的相同病毒序列增加（来自少数增殖的CD4+细胞群，每个细胞群都有相同的整合位点）。在这些患者中，我们将使用一种新的特异性整合位点的数字液滴PCR（ddPCR）检测，其中引物直接穿过宿主-前病毒连接，以研究具有特异性整合前病毒的CD4+细胞的扩增和下降。这些结果将提供关于HIV-1储存库的规模、动态和持久性的关键信息。这项研究将由南非斯泰伦博斯大学和美国国家癌症研究所合作进行。NCI开发的SGS、ISA和ddPCR技术以及分析ISA数据的生物信息学算法将转移到南非，在那里将进行大部分SGS和ISA检测。这项技术转让将增加SA的重要研究能力。目前还不存在的。这项合作将更好地为旨在控制或治愈HIV感染而无需持续cART的策略提供信息，例如使用足以代表持续HIV-1人群的HIV-1抗原进行治疗性疫苗接种。

 DESCRIPTION (provided by applicant): Despite the success of combination antiretroviral therapy (cART) in children, HIV is remarkably persistent, except in one well-documented case, in which very early therapy likely resulted in viral eradication. HIV-1 persistence during cART is due to infection of long-living populations of CD4+ T cells. Early initiation of cART has been associated with lower numbers of infected cells and with restricted viral diversity in adults and older children [1, 3], but little is known about HIV-1 populations genetics in children who initiatd cART during infancy. It is possible that infants who initiated cART when the viral population was largely homogeneous have very limited viral diversity and no evidence of viral evolution over time. Better characterization of HIV-1 population genetics in infants initiated on cART at differen times after infection is needed to optimize HIV curative strategies. Recent evidence suggests that some HIV-1 infected CD4+ cells may have a proliferation and survival advantage when HIV integrations are in host genes related to growth regulation. Whether such clonal expansions emerge in children initiated on early cART is unknown. To fill these critical knowledge gaps, we propose the investigation of HIV diversity, using single genome sequencing (SGS) as well as viral integration sites, using a novel integration sites assay (ISA), in longitudinal samples (from baseline to 7-11 years post therapy initiation) in 30 children form a cohort of early antiretrovira treated HIV-1 infected children from the Children with HIV Early treatment (CHER) study. In patients who have sufficient HIV-1 diversity we will also link viral sequences integrated in the CD4+ cells with virus circulating in the blood plasma during and after treatment to reveal the specific proviral sources of persistent and/or rebound viremia. This has not been accomplished before. We expect to find a few children with very limited viral diversity and who have an increase in identical viral sequences (emanating from a few proliferating CD4+ cells populations, each with an identical integration site). In these patients, we will use a novel digitl droplet PCR (ddPCR) assay of specific integration sites with primers directed across the host-proviral junction to study the expansion and decline of CD4+ cells having a specific integrated provirus. Such results will provide critical information on the size, dynamics, and persistence of the HIV-1 reservoir. This study will be conducted as collaboration between Stellenbosch University, South Africa and the National Cancer Institute, U.S. The SGS, ISA and ddPCR technologies and bioinformatic algorithms to analyse the ISA data, developed at the NCI, will be transferred to South Africa, where the majority of the SGS and ISA assays will be performed. This technology transfer will add important research capabilities in S.A. that currently do not exist. This collaboration will better inform strategies aimed at controlling or curing HIV infectio without continued cART, such as therapeutic vaccination with HIV-1 antigens that adequately represent persistent HIV-1 populations.