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

 描述(申请人提供)：尽管联合抗逆转录病毒疗法(CART)在儿童中取得了成功，但艾滋病毒仍然非常持久，除了一个有充分记录的病例，在该病例中，非常早期的治疗很可能导致病毒根除。HIV-1在CART期间的持续是由于长期存活的CD4T细胞群的感染。在成人和年龄较大的儿童中，早期启动CART与较少的感染细胞数量和有限的病毒多样性有关[1，3]，但对在婴儿时期启动CART的儿童中HIV-1群体遗传学知之甚少。在病毒种群基本相同的情况下启动CART的婴儿可能病毒多样性非常有限，而且没有证据表明病毒随着时间的推移而演变。为了优化HIV治疗策略，需要更好地描述感染后不同时间在CART上启动的婴儿中HIV-1群体遗传学的特征。最近的证据表明，当HIV整合到与生长调节相关的宿主基因中时，一些HIV-1感染的CD4细胞可能具有增殖和生存优势。这种克隆性扩张是否出现在早期CART开始的儿童中尚不清楚。为了填补这些关键的知识空白，我们建议使用单基因组测序(SGS)和病毒整合位点(ISA)，在纵向样本(来自 从儿童HIV早期治疗(CHER)研究中，30名儿童中的30名儿童组成了早期抗逆转录病毒治疗的HIV-1感染儿童队列。对于有足够HIV-1多样性的患者，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/或反跳性病毒血症的特定前病毒来源。这是以前从未有过的。我们预计将发现少数病毒多样性非常有限的儿童，他们的病毒序列相同(来自几个增殖的CD4细胞群体，每个细胞具有相同的整合位点)。在这些患者中，我们将使用一种新的数字液滴聚合酶链式反应(DdPCR)分析特定整合位点，并使用跨越宿主-前病毒连接点的引物来研究具有特定整合前病毒的CD4细胞的扩张和衰退。这些结果将提供有关HIV-1宿主的大小、动态和持久性的关键信息。这项研究将在南非斯泰伦博斯大学和美国国家癌症研究所的合作下进行。国家癌症研究所开发的SGS、ISA和ddPCR技术以及用于分析ISA数据的生物信息学算法将被转移到南非，在那里将进行大部分SGS和ISA分析。这项技术转让将为S.A.增加目前尚不存在的重要研究能力。这一合作将更好地为旨在控制或治疗艾滋病毒感染的战略提供信息，而不需要持续的CART，例如用足以代表持久艾滋病毒-1人群的艾滋病毒-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.