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.

艾滋病毒感染者（PLWH）体内潜伏的艾滋病毒储存库的持续存在仍然是艾滋病毒感染的关键障碍。 疗方许多水库减少和控制研究只取得了有限的成功，由于我们深刻的认识， 缺乏对细胞机制的理解，使艾滋病毒水库持续抗逆转录病毒治疗期间 治疗（ART）。本提案和RFA AI-22-025的目标是定义艾滋病毒的特征， 以确定这些机制是否影响能够抵抗细胞死亡的储库， 减少艾滋病毒/艾滋病感染者的战略。以前的研究已经从病毒库的角度对病毒库进行了研究。 整合的前病毒，包括病毒多样性、完整性和整合位点，但还无法直接 定义潜在的细胞死亡抵抗机制，使病毒库永久化。同样， 鉴定和表征离体静息感染细胞，包括稀有性、异质性和不存在 定义表型标记，限制了我们确定HIV储存库如何在 条为了解决这些问题，我们开发了一种新的单细胞策略，通过整合识别HIV+细胞。 同时进行表观遗传学和细胞表面分析的前病毒DNA（转座酶活性测定 色谱测序与细胞表面分析和病毒比对，V-ASAPseq）。通过这种策略，我们 在单细胞水平上直接分析了ART治疗的PLWH中的HIV储库， 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）来确定HIV储库的细胞死亡抗性机制。我们的中央 假设是在ART下和免疫激发后随时间的储库持续性 与目标细胞特征相关，其中一些是共享的，另一些是不同的， HIV+ CD 4 + T细胞亚群。在目标1中，我们将确定与细胞死亡相关的细胞死亡变化 随着时间的推移，解剖室之间和重新激活后的电阻特征。在目标2中，我们将 确定体外驱动细胞死亡易感性的HIV+细胞的表观遗传和转录特征 以及在来自人类临床试验的靶向免疫疗法后的体内持久性。综合这些 研究将定义水库持久性和细胞死亡的目标特征，无论是在休息时，在ART期间， 靶向HIV的免疫疗法的最终目标是减少HIV储存库。