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

项目总结/摘要 利用由研究合作领导的注射毒品者（PWID）的ALIVE队列的独特资源， 研究员格雷戈里·柯克和共同研究员拉菲克在油藏生物学方面的丰富经验 Sekaly，Joel Blankson，Greg Laird和研究PI Robert Siliciano，我们将调查海洛因的影响， 可卡因的使用对CD 4 + T细胞中HIV-1的潜在储存库有影响，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/或可卡因可以改变T细胞 体内平衡和HIV-1前病毒在记忆细胞亚群中的分布，从而影响大小， 转换和潜在储层的诱导。此外，我们假设过去注射毒品的使用和 相关的非结构化治疗中断将永久性地影响潜在的 水库活跃药物使用和过去药物使用的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感染细胞。这些假设将使用以下样本进行检验： 通过全基因组前病毒测序告知ALIVE队列参与者和实验方法， 更好地了解PWID中的储库，并告知如何应用治疗策略 对这个人群。我们将首先通过以下方式确定活跃的海洛因/可卡因使用对潜在储库的影响： 用一种新的方法定量活性药物使用者中静息CD 4 + T细胞和T细胞亚群的储库大小， 完整前病毒高通量测定。该测定法基于全基因组序列分析， Siliciano实验室发现大多数前病毒（98%）都有缺陷。因此，我们将使用一种新的数字 液滴PCR检测，选择性检测完整的前病毒，并有可能导致病毒 反弹我们还将测量潜伏性前病毒对T细胞活化和潜伏的反应的诱导作用。 逆转剂使用由Sekaly博士和同事开发的TILDA测定。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 包括复制能力、克隆性、转换率和溶细胞性T淋巴细胞（CTL）的逃逸突变 表位这些研究将在积极使用以及不再使用的PWID中进行 使用，以区分药物使用的积极和遗留影响。我们还将测量 来自活跃吸毒者的自体CD 8 + CTL和NK细胞，以消除 潜伏期逆转，并在PWID中进行类似的研究，这些PWID不再积极用于区分直接 药物效应来自药物既往使用对效应细胞功能的遗留效应。这些研究一起， 储库的大小、诱导物和组成，以及宿主效应细胞清除感染细胞的能力， 应提供关键信息的治疗策略的设计PWID和指导纵向和 机械研究在第二阶段的赠款。

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.