Multiomic strategies to assess HIV reservoir persistence

评估 HIV 储存持久性的多组学策略

• 批准号: 10676525
• 负责人: Michael R Betts
• 金额: $ 81.01万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-04-01 至 2028-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10676525
• 关键词: Address; Aftercare; Anatomy; Apoptosis; Binding; Biological Assay; Blood; Blood specimen; CD4 Positive T Lymphocytes; Cell Death; Cell Survival; Cell surface; Cells; Characteristics; Chromatin; Clinical Trials; Combination immunotherapy; DNA; Data; Detection; Development; Epigenetic Process; Failure; Genes; Genetic; Genetic Transcription; Goals; HIV; HIV Seropositivity; HIV resistance; Heterogeneity; Human; Immunotherapeutic agent; Immunotherapy; In Vitro; Individual; Infection; Interferons; Interruption; Lymphoid Tissue; Modality; Modeling; Outcome; Persons; Predisposition; Property; Proteins; Provirus Integration; Proviruses; RNA; Resistance; Rest; Sampling; Surface; Surface Antigens; System; Testing; Time; Transcript; Transposase; Viral; Viral reservoir; XCL1 gene; antiretroviral therapy; chimeric antigen receptor T cells; epigenome; in vivo; inhibitor; insight; integration site; latent HIV reservoir; multimodality; multiple omics; novel; peripheral blood; phenotypic biomarker; resistance mechanism; single-cell RNA sequencing; success; transcription factor; transcriptome; transcriptomics

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.

HIV 感染者 (PLWH) 中潜伏 HIV 病毒库的持续存在仍然是 HIV 感染的关键障碍 治愈。由于我们深刻的认识，许多水库削减和控制研究只取得了有限的成功。 缺乏对抗逆转录病毒治疗期间艾滋病毒储存库持续存在的细胞机制的了解 治疗（ART）。该提案和 RFA AI-22-025 的目标是定义 HIV 的特征 储库能够抵抗细胞死亡并确定这些机制是否影响储库 PLWH 的减少策略。此前的研究已经从病毒库的角度进行了检查 整合原病毒，包括病毒多样性、完整性和整合位点，但还不能直接 定义使病毒库永久存在的潜在细胞死亡抵抗机制。同样，挑战 离体鉴定和表征静息感染细胞，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 储存库如何在以下条件下维持的能力： 艺术。为了解决这些问题，我们开发了一种新颖的单细胞策略，通过整合识别 HIV+ 细胞 原病毒 DNA 具有同时表观遗传和细胞表面分析（Assayfor Transposase Accessible） 通过细胞表面分析和病毒比对进行染色质测序，V-ASAPseq）。使用这个策略，我们 在单细胞水平上直接分析了接受 ART 治疗的 PLWH 中的 HIV 储存库，发现了广泛的储存库 个体内部和个体之间的异质性，但直接共享监管特征的潜力 与细胞死亡抵抗力有关。在这里，我们将应用 V-ASAPseq 和 V-TEAseq（转录组、表观基因组、 和表面分析）来定义 HIV 储存库的细胞死亡抵抗机制。我们的中央 假设是，在 ART 下和免疫治疗挑战后，随着时间的推移，储库的持久性是 与可靶向的细胞特征相关，其中一些是共享的，另一些是不同的 HIV+ CD4+ T 细胞亚群。在目标 1 中，我们将确定与储存库相关的细胞死亡变化 随着时间的推移，解剖区之间和重新激活后的阻力特征。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针对储库的免疫疗法后体内的持久性。一起这些 研究将确定静息时、ART 期间和在 以储存库为目标的免疫疗法的背景，其最终目标是减少艾滋病毒储存库。