Mechanisms of HIV persistence in distinct memory CD4+ T cell subsets in blood and

描述（由申请人提供）：现在很清楚，单靠抗逆转录病毒疗法（ART）并不能根除艾滋病毒：即使经过超过15年的强化和持续治疗，除例外情况外，在停止抗逆转录病毒疗法后的几周内，病毒的传播仍会恢复。一小群潜伏感染的细胞，通常被称为HIV感染的“储存库”，为反弹病毒血症提供了一个长期的来源。由于缺乏关于携带病毒库的特定亚群和与维持潜伏期相关的机制的信息，艾滋病毒的根除受到了阻碍。我们之前已经证明，HIV持续存在于三种表现出不同功能和生存能力的记忆T细胞亚群中，即病毒抑制受试者血液中的中枢（TCM）过渡（TTM）和效应（TEM）记忆CD4+ T细胞。有趣的是，我们的初步数据表明，这些细胞亚群也确保HIV在组织储存库（如淋巴结、直肠、结肠和回肠末端）中的持久性。因此，迫切需要更好地表征这三种细胞HIV储存库以实现根除。该项目的目的是确定在病毒抑制的受试者中导致潜伏感染细胞的重新激活和随后消除的作用机制。我们假设在中医、中药和TEM中导致HIV潜伏的机制是不同的，并且这些储存库应该被不同类型的抗潜伏药物靶向。我们将首先确定在TCM， TTM和TEM细胞中负责HIV潜伏期的分子机制（Specific Aim 1）。我们将重点关注DNA的表观遗传修饰，通过甲基化和组蛋白去乙酰化，关键转录因子的有限可用性和新生小瓶转录物的低效延伸。我们还将使用RNA测序来鉴定与24名病毒抑制受试者的CD4+ T细胞亚群中潜伏库大小相关的新细胞途径。然后，我们将选择最有效的化合物组合，干扰这些机制，并允许消除所有细胞储存库（Specific Aim 2）。我们选择了在以前的研究中显示出有希望的效果和/或目前正在临床试验中测试的分子。由于靶向单个细胞储存库或涉及病毒潜伏期的单个机制很可能不会成功，因此我们将测试来自每种类型的先导化合物的组合破坏来自病毒抑制受试者的潜伏感染CD4+ T细胞的潜伏期的能力。利用佛罗里达VGTI最近开发的一种新型共培养试验，我们将评估这种抗潜伏期药物组合的再激活是否可以导致自体HIV特异性CTL消除储库细胞。该研究项目的成果是3-5种临床耐受药物的组合，可以逆转来自病毒抑制受试者的真正潜伏感染细胞的潜伏期。这些研究的结果将构成旨在根除艾滋病毒的临床试验设计的基础。

DESCRIPTION (provided by applicant): It is now clear that antiretroviral therapy (ART) alone does not eradicate HIV: Even after more than 15 years of intensive and continuous therapy, the spread of the virus resumes within a few weeks upon cessation of ART in all but exceptional cases. A small pool of latently infected cells, usually referred as the "reservoir" of HIV infectio, provides a long-lived source of rebound viremia. Eradication of HIV has been hampered by the lack of information regarding the specific subsets harboring the viral reservoir and the mechanisms associated with the maintenance of latency. We have previously demonstrated that HIV persists in three memory T cell subsets displaying distinct functional and survival capacities, namely central (TCM) transitional (TTM) and effector (TEM) memory CD4+ T cells in the blood of virally suppressed subjects. Interestingly, our preliminary data indicate that these cell subsets also ensure HIV persistence in tissue reservoirs such as the lymph nodes, rectum, colon and terminal ileum. Therefore, there is an urgent need to better characterize these three cellular HIV reservoirs to achieve their eradication. The objective of this project is to identify mechanisms of action that can lead to the reactivation and subsequent elimination of latently infected cells in virally suppressed subjects. We hypothesize that the mechanisms responsible for HIV latency in TCM, TTM and TEM are different, and that these reservoirs should be targeted by different classes of anti-latency agents. We will first identify the molecular mechanisms responsible for HIV latency in TCM, TTM and TEM cells (Specific Aim 1). We will focus on epigenetic modification of the DNA through methylation and histone deacetylation, limited availability of critical transcription factors and inefficient elongation of the nascent vial transcripts. We will also use RNA sequencing to identify novel cellular pathways that correlate with the magnitude of the latent reservoir in the CD4+ T cell subsets from 24 virally suppressed subjects. We will then select the most efficient combination of compounds that will interfere with these mechanisms and allow elimination of all cellular reservoirs (Specific Aim 2). We have selected molecules that have shown promising effects in previous studies and/or that are currently tested in clinical trials. As it is highly likely that targeting a single cellular reservir or a single mechanism involved in viral latency will not be successful, we will test the ability of combinations of lead compounds from each class to disrupt latency in latently infected CD4+ T cells from virally suppressed subjects. Using a novel co-culture assay recently developed at VGTI Florida, we will assess if reactivation with this combination of anti-latency drugs can lead to the elimination of reservoir cells by autologous HIV specific CTL. The deliverable of this research project is a combination of 3-5 clinically tolerable agents that can reverse latency in authentic latently infected cells from virally suppressed subjects. The results from these studies will constitute the basis for the design of a clinical trial aimed at eradicating HIV.