Selection and Evolution of HIV-1 reservoir cells in blood and tissues

摘要 病毒库细胞是一组极小但高度持久的HIV-1感染的CD4T细胞 尽管接受了高效的抗逆转录病毒治疗，但仍坚持下去，并对病毒反弹一次负责 治疗中断了。了解和表征这些细胞的生理学可能对 任何成功针对这些细胞的努力都被证明是极其困难的，因为它们的比例很低 血液和组织中的丰度和相当大的异质性。此外，越来越多的证据表明 这表明病毒库的CD4T细胞随着时间的推移而动态进化，并受到选择的影响 这种机制有利于一些储集层细胞的长期存留，同时消除另一些储集层细胞。在这里，我们计划 利用分子单细胞和成像分析技术的最新进展，并建议 全面描述血液和组织中病毒库细胞的纵向进化。我们假设 持续抑制抗逆转录病毒治疗选择具有更深潜伏期特征的完整前病毒， 可能是免疫选择机制的结果，这种机制优先消除更容易感染的前病毒 重新激活的信号，而更深的潜伏期持续存在。这些研究将使用样本进行 一个独特的、前瞻性跟踪的HIV-1患者队列，这些患者被确认为急性感染，并开始 确诊后立即进行抗逆转录病毒治疗。在具体目标1中，我们将使用新颖的下一代 利用测序技术纵向定位完整和缺陷前病毒的染色体位置 从血液和组织中提取，并评估其在淋巴组织中的显微解剖位置。随后，我们将 表征血液中完整前病毒染色体附近的表观遗传染色质环境 和组织，使用下一代测序分析平台来评估染色质可及性、抑制性 或激活组蛋白修饰和DNA甲基化(特定目标2)。在具体目标3中，我们将执行 新的，功能性的单细胞分析同时分析前病毒序列，相应的 血液和组织中单个病毒感染细胞的整合位点和HIV-1RNA表达谱； 化验将使我们能够单独表征单个感染细胞编码的病毒基因表达模式 对于完整和有缺陷的前病毒，并能够检验持续抗逆转录病毒治疗是 与潜伏期更深、响应性更低的前病毒逐渐积累相关 对病毒重新激活的刺激。总之，这些研究有可能在以下方面取得重大进展 了解病毒储存库细胞的复杂性和纵向进化，可能有助于识别 可作为治疗靶点的残留HIV-1感染细胞的易感性和脆弱性。

Abstract Viral reservoir cells are an extremely small but highly durable population of HIV-1-infected CD4 T cells that persist despite treatment with highly-effective antiretroviral therapy and are responsible for viral rebound once treatment is interrupted. Understanding and characterizing the physiology of these cells will likely be critical for any effort to successfully target these cells but has turned out to be extremely difficult, due to their low fractional abundance and considerable heterogeneity in blood and tissues. Moreover, there is growing evidence suggesting that viral reservoir CD4 T cells are dynamically evolving over time, and subject to selection mechanisms that favor the long-term persistence of some reservoir cells, while eliminating others. Here, we plan to take advantage of recent progress in molecular single-cell and imaging analysis techniques and propose to comprehensively profile the longitudinal evolution of viral reservoir cells in blood and tissues. We hypothesize that continuous suppressive antiretroviral therapy selects for intact proviruses with features of deeper latency, likely as a result of immune selection mechanisms that preferentially eliminate proviruses more susceptible to reactivation signals, while proviruses in deeper latency persist. These studies will be conducted using samples of a unique, prospectively followed cohort of HIV-1-patients who were identified in acute infection and started antiretroviral treatment immediately after diagnosis. In Specific Aim 1, we will use novel next-generation sequencing technologies to longitudinally profile the chromosomal position of intact and defective proviruses from blood and tissues, and evaluate their microanatomical location in lymphoid tissues. Subsequently, we will characterize the epigenetic chromatin environment within chromosomal proximity to intact proviruses from blood and tissues, using a platform of next-generation sequencing assays to evaluate chromatin accessibility, inhibitory or activating histone modifications and DNA methylation (Specific Aim 2). In Specific Aim 3, we will perform novel, functional single-cell assays to simultaneously analyze the proviral sequence, the corresponding integration sites and HIV-1 RNA expression profile of single virally infected cells from blood and tissues; this assay will allow us to individually characterize the viral gene expression pattern of single infected cells encoding for intact and defective proviruses, and enable testing the hypothesis that continuous antiretroviral therapy is associated with progressive accumulation of proviruses with deeper levels of latency and lower responsiveness to viral reactivation stimuli. Together, these studies have the potential to provide significant advances in understanding the complexity and longitudinal evolution of viral reservoir cells and may allow to identify susceptibilities and vulnerabilities of residual HIV-1-infected cells that could be therapeutically targeted.