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

摘要 病毒储库细胞是一种非常小但高度持久的HIV-1感染的CD 4 T细胞群体， 尽管接受了高效的抗逆转录病毒治疗，但仍然存在，并导致病毒反弹一次 治疗中断。了解和表征这些细胞的生理学可能对 任何成功靶向这些细胞的努力，但由于它们的低分数， 丰富和相当大的异质性的血液和组织。此外，越来越多的证据表明， 这表明病毒储库CD 4 T细胞随时间动态进化，并受到选择， 有利于一些储库细胞长期存在的机制，同时消除其他细胞。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，并提出 全面描绘血液和组织中病毒储库细胞的纵向进化。我们假设 持续的抑制性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， 这可能是由于免疫选择机制优先消除更易感染的前病毒， 重新激活信号，而前病毒在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，前瞻性随访的HIV-1患者队列，这些患者被确定为急性感染， 诊断后立即进行抗逆转录病毒治疗。在具体目标1中，我们将使用新的下一代 测序技术纵向分析完整和缺陷前病毒的染色体位置 从血液和组织，并评估其在淋巴组织中的显微解剖位置。后续我们将 表征染色体接近血液中完整前病毒的表观遗传染色质环境 和组织，使用下一代测序测定的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（特异性目的2）。在第三部中，我们将 同时分析前病毒序列的新型功能性单细胞测定， 整合位点和来自血液和组织的单个病毒感染细胞的HIV-1 RNA表达谱;这 分析将使我们能够单独表征单个感染细胞的病毒基因表达模式， 完整和有缺陷的前病毒，并使测试的假设，连续抗逆转录病毒治疗是 与潜伏期更深、反应性更低的前病毒进行性积累有关 病毒激活刺激。总之，这些研究有可能在以下方面取得重大进展： 了解病毒储库细胞的复杂性和纵向进化， 可能成为治疗靶点的残留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.