Host and viral genomic determinants of HIV latent reservoir size and characteristics in individuals with substance use disorders

项目摘要/摘要 使用由研究联合领导的活着的注射吸毒者队列(PWID)的独特资源 调查员格雷戈里·柯克和合作调查员拉菲克提供的丰富的储层生物学经验 Sekaly，Joel Blankson，Greg Laird和研究Pi Robert Siliciano，我们将调查海洛因的影响和 使用可卡因可以潜伏在CD4+T细胞中储存HIV-1，这是治愈的主要障碍。我们 假设通过影响炎症和免疫途径，活性海洛因和/或可卡因可能改变T细胞 动态平衡和HIV-1前体在存储细胞亚群中的分布，从而影响大小， 潜藏的周转率和诱导性。此外，我们假设过去的注射吸毒和 相关的非结构化治疗中断将永久性地影响潜伏期的大小和诱导性 水库。主动药物使用和过去药物使用遗留效应也可能影响宿主效应细胞的能力 在治疗干预期间清除受感染的细胞。这些假设将使用以下样本进行测试 全基因组前病毒测序获知的活体队列参与者和实验方法 更好地了解PWID中的储蓄者，并告知如何应用治疗策略 对这群人来说。我们将首先通过以下方式确定活跃的海洛因/可卡因的使用对潜伏水库的影响 用一种新的方法量化活跃吸毒者静息中的CD4+T细胞和T细胞亚群的储存库的大小 完整前病毒的高通量检测。这项分析是基于全基因组序列分析的 导致发现大多数前病毒(98%)是有缺陷的。因此，我们将使用一种新颖的数字 一种滴状聚合酶链式反应方法，选择性地检测完整的、有可能引起病毒的前病毒 抢篮板球。我们还将测量T细胞激活和潜伏期对潜伏期前病毒的诱导性 使用Sekaly博士和同事开发的Tilda试验的逆转试剂。此外，我们还将开展 HIV-1前病毒的全基因组测序以确定与HIV-1治愈相关的其他特性 包括细胞溶解T淋巴细胞(CTL)的复制能力、克隆性、周转率和逃逸突变 表位。这些研究将在正在积极使用和不再使用的PWID中进行 用来区分药物使用的积极影响和遗留影响。我们还将衡量 主动吸毒者自体CD8+CTL和NK细胞清除自体感染细胞 潜伏期逆转，并在不再积极使用直接区分的PWID中进行类似的研究 药物效应来自过去药物使用对效应细胞功能的遗留影响。总而言之，这些关于 蓄水池的大小、诱导性和组成，以及宿主效应细胞清除感染细胞的能力， 应为设计PWID的治疗策略提供关键信息，并指导纵向和 在第二阶段的资助中进行机械研究。

Project Summary/Abstract Using the unique resources of the ALIVE cohort of persons who inject drugs (PWID) led by study co- investigator Gregory Kirk and the extensive experience in reservoir biology provided by co-investigators Rafick Sekaly, Joel Blankson, Greg Laird, and study PI Robert Siliciano, we will investigate the effects of heroin and cocaine use on the latent reservoir for HIV-1 in CD4+ T cells, which is the major barrier to cure. We hypothesize that by affecting inflammatory and immune pathways, active heroin and/or cocaine may alter T cell homeostasis and the distribution of HIV-1 proviruses among memory cells subsets, thereby affecting the size, turnover, and inducibility of the latent reservoir. In addition, we hypothesize that past injection drug use and the associated unstructured treatment interruptions will permanently affect the size and inducibility of the latent reservoir. Active drug use and legacy effects of past drug use may also affect that ability of host effector cells to clear infected cells during curative interventions. These hypotheses will be tested using samples from ALIVE cohort participants and experimental approaches informed by full genome proviral sequencing in order to achieve a better understanding of the reservoir in PWID and inform how curative strategies can be applied to this population. We will first determine the effects of active heroin/cocaine use on the latent reservoir by quantifying the size of the reservoir in resting CD4+ T cells and T cell subsets in active drug users with a novel high-throughput assay for intact proviruses. This assay is based on full genome sequence analysis by the Siliciano lab that led to the discovery that most proviruses (98%) are defective. Thus we will use a novel digital droplet PCR assay that selectively detects proviruses that are intact and have the potential to cause viral rebound. We will also measure the inducibility of latent proviruses in response to T cell activation and latency reversing agents using the TILDA assay developed by Dr. Sekaly and colleages. In addition, we will carry out full genome sequencing of HIV-1 proviruses to define additional properties that are relevant to HIV-1 cure including replication capacity, clonality, turnover rates, and escape mutations in cytolytic T lymphocyte (CTL) epitopes. These studies will be carried out in PWID who are actively using as well as those who are no longer using in order to distinguish active and legacy effects of drug use. We will also measure the ability of autologous CD8+ CTL and NK cells from active drug users to eliminate autologous infected cells following latency reversal and carry out similar studies in PWID who are no longer actively using to distinguish direct drug effects from legacy effects of drug past drug use on effector cell function. Together these studies of the size, inducibility, and composition of the reservoir, and the ability of host effector cells to clear infected cells, should provide critical information for the design of cure strategies for PWID and guide longitudinal and mechanistic studies in the second phase of the grant.