Multiomic strategies to assess HIV reservoir persistence

艾滋病毒携带者(PLWH)潜伏的HIV蓄水池的持久性仍然是HIV感染的关键障碍 解药。由于我们的深刻认识，许多减少和控制水库的研究只取得了有限的成功 缺乏对在抗逆转录病毒期间使艾滋病毒储存库持续存在的细胞机制的了解 治疗(ART)。本提案和RFA AI-22-025的目标是定义HIV的特征 启用细胞死亡抵抗并确定这些机制是否影响储存库 PLWH中的减少策略。以前的研究已经从病毒储存库的角度对病毒进行了检查 整合的前病毒，包括病毒的多样性、完整性和整合位点，但还不能直接 确定使病毒库永久化的潜在细胞死亡抵抗机制。同样，面临的挑战 体外静息感染细胞的鉴定和特征，包括稀有性、异质性和缺乏 定义表型标记，限制了我们确定HIV储存库是如何维持在 艺术。为了解决这些问题，我们开发了一种新的单细胞策略，通过整合 同时进行表观遗传和细胞表面分析的前病毒DNA(转座酶可访问的评估 细胞表面图谱和病毒比对的染色体测序，V-ASAPseq)。使用这一战略，我们 已经在单细胞水平上直接在抗逆转录病毒治疗的PLWH中描绘了艾滋病毒的储存库，发现了广泛的储存库 个人内部和个人之间的异质性，但直接共享监管特征的可能性 与细胞死亡抗性有关。在这里，我们将应用V-ASAPseq和V-TEAseq(转录组，表观基因组， 和表面简档)，以确定HIV储存库的细胞死亡抵抗机制。我们的中央 假说是，在抗逆转录病毒治疗和免疫治疗挑战之后，储存库随着时间的推移而持续存在 与有针对性的蜂窝功能相关联，其中一些是共享的，另一些是不同的 HIV CD4T细胞亚群。在目标1中，我们将确定细胞死亡中与水库相关的变化。 随着时间的推移，在解剖间隔之间和重新激活之后的抵抗特征。在目标2中，我们将 确定HIV细胞在体外驱动细胞死亡易感性的表观遗传和转录特征 以及人体临床试验中的水库靶向免疫疗法后的体内持久性。把这些放在一起 研究将确定静息状态下、在ART期间和在ART中的水库持久性和细胞死亡的目标特征。 以减少艾滋病毒宿主为最终目标的水库靶向免疫疗法的背景。

Persistence of the latent HIV reservoir in people living with HIV (PLWH) remains the critical barrier to an HIV cure. Numerous reservoir reduction and control studies have met with only limited success due to our profound lack of understanding of the cellular mechanisms that allow the HIV reservoir to persist during antiretroviral therapy (ART). The goals of this proposal and RFA AI-22-025 are to define the characteristics of the HIV reservoir enabling cell death resistance and to determine whether these mechanisms impact reservoir reduction strategies in PLWH. Previous studies have examined the viral reservoir from the aspect of the integrated provirus, including viral diversity, intactness, and integration site, but have not been able to directly define potential cell death resistance mechanisms that perpetuate the viral reservoir. Similarly, challenges in identifying and characterizing resting infected cells ex vivo, including rarity, heterogeneity, and absence of a defining phenotypic marker, have limited our ability to determine how the HIV reservoir is maintained under ART. To address these issues, we developed a novel single cell strategy to identify HIV+ cells via integrated proviral DNA with simultaneous epigenetic and cell surface profiling (Assayfor Transposase Accessible Chromatinsequencing with cell surface profiling and viral alignments,V-ASAPseq). Using this strategy, we have directly profiled the HIV reservoir in ART treated PLWH at the single cell level, finding extensive reservoir heterogeneity within and between individuals, but the potential for shared regulatory characteristics directly relating to cell death resistance. Here, we will apply V-ASAPseq and V-TEAseq (transcriptome, epigenome, and surface profiling) to define cell death resistance mechanisms of the HIV reservoir. Our central hypothesis is that reservoir persistence over time under ART and after immunotherapeutic challenge is associated with targetable cellular features, some of which are shared and others distinct between subpopulations of HIV+ CD4+ T cells. In Aim 1 we will determine reservoir-associated changes in cell death resistance signatures over time, between anatomical compartments and after reactivation. In Aim 2, we will determine the epigenetic and transcriptional features of HIV+ cells that drive cell death susceptibility in vitro and persistence in vivo after reservoir-targeting immunotherapies from human clinical trials. Together these studies will define targetable features of reservoir persistence and cell death both at rest, during ART, and in the context of reservoir-targeting immunotherapies with the ultimate goal of reducing the HIV reservoir.