HIV persistence in vivo

这个项目的目的是帮助阐明艾滋病毒如何在体内持续存在，从而帮助开发新的艾滋病毒治愈导向疗法。这项工作的重点是一小部分CD4T细胞，它们含有复制能力强的艾滋病毒，即使在有效的抗逆转录病毒疗法(ART)的背景下也能保持几乎稳定的水平。尽管这些细胞被认为是一个“潜伏库”，但维持它们数量的细胞过程和潜在的基因表达模式仍然没有完全确定。事实上，传染性病毒的持续细胞间传播、抗逆转录病毒治疗前被感染细胞的增殖以及被感染细胞的长期静止(“潜伏期”)仍有可能成为有效抗逆转录病毒疗法期间治愈的障碍。区分这些机制对体内治愈艾滋病毒的障碍的贡献将是至关重要的，因为旨在对抗每一种单独机制的干预措施可能无法影响甚至恶化其他机制。 不幸的是，从感染艾滋病毒的研究参与者的血液和组织样本中区分这些机制的贡献是极其具有挑战性的。一个主要原因是体内含有HIV的CD4T细胞通常缺乏病毒或宿主编码标记的表达。这阻止了对高纯度细胞群体的鉴定和分离，以进行可能揭示正在进行的病毒复制、细胞增殖或细胞潜伏期的转录、表观遗传学或蛋白质组特征的综合下游分析类型。在这个项目中，我们正在开发两种互补的方法来克服这一技术挑战：一种是反向方法，即通过流式细胞仪对表型CD4T细胞亚群进行分选，以确定下游作为病毒宿主的特征；另一种是正向方法，即在油包水乳剂中检测后，分离含有HIV基因组DNA的细胞进行全面分析。

The purpose of this project is to help elucidate how HIV persists in vivo and thus to aid the development of novel HIV cure-directed therapies. The focus of the work is a small population of CD4 T cells that contain replication-competent HIV and persist at nearly stable levels even in the setting of effective antiretroviral therapy (ART). Although these cells have been considered to represent a "latent reservoir," the cellular processes and underlying gene expression patterns that maintain their numbers remain incompletely defined. Indeed, it remains possible that the ongoing cell-to-cell spread of infectious virus, the proliferation of cells infected before institution of ART, and the long-term quiescence ("latency") of infected cells all present barriers to cure during effective ART. Distinguishing the contributions of these mechanisms to the barrier to HIV cure in vivo will be critical because interventions designed to antagonize each individual mechanism may fail to affect or even worsen the others. Unfortunately, distinguishing the contributions of these mechanisms in blood and tissue samples from HIV-infected study participants has been extremely challenging. A major reason for this is that the CD4 T cells that contain HIV in vivo often lack expression of virus- or host-encoded markers. This prevents identification and isolation of highly pure populations of cells for the types of comprehensive downstream analysis that might reveal transcriptomic, epigenetic, or proteomic signatures of ongoing virus replication, cellular proliferation, or cellular latency. In this project, we are developing two complementary approaches to overcoming this technical challenge: a reverse approach in which phenotypic CD4 T cell subsets are sorted by flow cytometry for downstream characterization as hosts for the virus, and a forward approach in which cells containing HIV genomic DNA are isolated for comprehensive analysis following detection in water-in-oil emulsions.