Understanding HIV latency reversal and clearance of infected cells in vivo

项目概要/摘要： 潜伏感染的CD 4 + T细胞被认为是HIV根除或功能性治愈的主要障碍， 这些细胞的病毒再活化可能有助于在这些细胞上观察到的器官炎症和损伤。 抗逆转录病毒疗法开发更有效的潜伏期逆转剂的主要障碍 （LRA）包括缺乏关于体内控制潜伏期和潜伏期逆转的机制的知识， 以及这些在体外潜伏期模型中重演的程度。之间缺乏一致意见 潜伏期模型和LRA人体试验的不完全成功表明，了解 为什么不同的LRA在体内起作用或不起作用我们开发了一种新的“转录谱分析”方法， 可以同时测量不同机制对可逆抑制的贡献程度， 体内HIV转录。通过将这种方法应用于ART抑制患者的细胞， 初步数据表明一个新的范例，其中延迟不是（如通常假设的）由于块， HIV转录起始时，对近端延伸的阻断比以前更大，更普遍 实现，并且HIV转录的主要可逆阻断是以前未识别的远端阻断， 转录/多聚腺苷酸化（完成）和阻断多剪接。此外，我们还有有趣的新内容 这些数据表明LRA可能选择性地作用于HIV转录的不同机制阻断剂。这 本研究将利用LRA治疗的人类临床试验样本（目的1和2），以更好地了解 他们如何逆转体内潜伏机制，并确定测试新药物的最佳模型， 体外（目的3）。在aim 1中，我们将把我们的转录谱分析方法应用于用 双硫仑、伏立诺他、帕比司他和罗米地辛。我们假设这些药物优先增加 HIV转录起始和延伸，但克服完成和剪接阻断的能力较低。 在aim 2中，我们将把我们的方法应用于临床试验的血液和肠道样本， Toll样受体（TLR）7和9的激动剂，以了解这些药物如何逆转潜伏期并导致死亡 感染的细胞。我们假设TLR激动剂可以克服后来对HIV转录的阻断， 增加完整的转录本（和HIV蛋白/抗原），这可能有助于内在细胞清除 防御或免疫杀伤。在目标3中，我们将比较体外潜伏期模型， 它们概括了潜伏期和对LRA的反应的体内机制。然后我们将选出最好的模型 并测试新的药物组合是否有能力增加完整/剪接的转录本并导致死亡 感染的细胞。这三个目标的结果应该提供关键的新见解， 现有的LRA逆转了体内潜伏期的不同机制（目的1和2）， 清除再活化细胞（目标2和3），在实验室中研究LRA的最佳系统（目标3），以及新的 可以导致更有效的潜伏期逆转和/或杀死感染细胞的组合（目标3）。

Project Summary/Abstract: Latently-infected CD4+ T cells are thought to be the main barrier to HIV eradication or functional cure, and viral reactivation from these cells likely contributes to the organ inflammation and damage observed on antiretroviral therapy. Major impediments to the development of more effective latency-reversing agents (LRAs) include the lack of knowledge about the mechanisms that govern latency and latency reversal in vivo, and the degree to which these are recapitulated by latency models in vitro. The lack of agreement between latency models and the incomplete success of human trials with LRAs suggest that it is critical to understand why different LRAs do or do not work in vivo. We have developed a new “transcription profiling” approach that can simultaneously measure the degree to which different mechanisms contribute to reversible inhibition of HIV transcription in vivo. By applying this approach to cells from ART-suppressed patients, we have generated preliminary data suggesting a new paradigm in which latency is not (as commonly assumed) due to a block to HIV transcriptional initiation, the block to proximal elongation is greater and more pervasive than previously realized, and the main reversible blocks to HIV transcription are a previously-unrecognized block to distal transcription/polyadenylation (completion) and a block to multiple-splicing. In addition, we have intriguing new data suggesting that LRAs may act selectively on the different mechanistic blocks to HIV transcription. This study will utilize samples from clinical trials of humans treated with LRAs (aims 1 and 2) to better understand how they reverse the mechanisms of latency in vivo and to identify the optimum model to test new agents in vitro (aim 3). In aim 1, we will apply our transcription profiling approach to samples from humans treated with disulfiram, vorinostat, panobinostat, and romidepsin. We hypothesize that these agents preferentially increase HIV transcriptional initiation and elongation but have less ability to overcome blocks to completion and splicing. In aim 2, we will apply our approach to blood and gut samples from clinical trials of humans treated with agonists of toll-like receptor (TLR) 7 and 9 to understand how these agents reverse latency and lead to death of infected cells in vivo. We hypothesize that TLR agonists can overcome later blocks to HIV transcription, increasing the completed transcripts (and HIV protein/antigen) that may facilitate clearance by intrinsic cell defenses or immune killing. In aim 3, we will compare in vitro models of latency based on the degree to which they recapitulate in vivo mechanisms of latency and responses to LRAs. We will then select the best model and test new combinations of agents for their ability to increase completed/spliced transcripts and lead to death of infected cells. The results from these 3 aims should provide critical new insights on the degree to which existing LRAs reverse the different mechanisms of latency in vivo (aims 1 and 2), the effects that correlate with clearance of reactivated cells (aims 2 and 3), the best system to study LRAs in the laboratory (aim 3), and new combinations that can lead to more effective latency reversal and/or killing of infected cells (aim 3).