Viral ASAPseq definition of CD4+ T cell viral reservoirs

CD4 T 细胞病毒库的 Viral ASAPseq 定义

• 批准号: 10548385
• 负责人: Michael R Betts
• 金额: $ 24.38万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-06-03 至 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10548385
• 关键词: Address; Autologous; Bioinformatics; Biological Assay; CD4 Positive T Lymphocytes; Cell Line; Cell surface; Cells; Cellular Assay; Characteristics; Clonal Expansion; DNA; Data; Detection; Development; Disease remission; Epigenetic Process; Euchromatin; Genome; Genomic DNA; Genomics; Goals; HIV; Heterochromatin; Hyperactivity; In Vitro; Label; Methods; Modeling; Molecular; Nature; Persons; Phenotype; Population; Procedures; Proviruses; Regulation; Regulatory Element; Research; Resolution; Sampling; Sequence Alignment; Structure; Surface; Surface Antigens; Techniques; Testing; Time; Tn5 transposase; Transposase; Viral; Viral reservoir; Viremia; antiretroviral therapy; base; bioinformatics pipeline; deep sequencing; epigenome; in vivo; latent HIV reservoir; lymph nodes; memory CD4 T lymphocyte; multimodality; novel; novel strategies; peripheral blood; preservation; programs; single-cell RNA sequencing; viral DNA; viral detection

While combination antiretroviral therapy (ART) effectively controls HIV viremia in most people living with HIV (PLWH), broadly applicable strategies to induce viral remission have remained elusive due to HIV reservoir persistence. Substantial progress has been made in defining the virological characteristics of the proviral reservoir of latently infected cells during ART. However, significant gaps remain in our understanding of the infected CD4+ T cells that constitute the cellular HIV reservoir that limit our ability to develop effective strategies for HIV cure. We have developed a novel high throughput 10X Genomics single cell assay to directly identify HIV infected cells via the presence of integrated proviral DNA. Termed "viral ASAPseq" [Assay for Transposase Accessible Chomatin (ATAC) Surface Antigen Profile sequencing, ASAPseq, with viral alignment, viral ASAPseq], this assay allows detection of HIV proviral DNA within euchromatin in combination with 154-marker cell surface antigen labeling, yielding multimodal single cell resolution of HIV infected cells. This assay does not require ex vivo activation to detect the presence of integrated viral DNA, thereby preserving the native state of the infected cell. We have successfully piloted this procedure in several conditions, including in vitro infected primary CD4+ T cells, primary lymph node CD4+ T cells from viremic PLWH, and peripheral blood CD4+ T cells from ART treated PLWH. We have further developed bioinformatic strategies to identify proviral DNA using autologous viral sequence alignment, define the surface marker composition of HIV infected cells, and characterize the epigenetic structure of infected cells. Thus, we are poised for the first time to gain a direct understanding of HIV reservoir composition directly ex vivo. Here we will apply this new technique to test the hypothesis that HIV infected CD4+ T cells have a cell surface or epigenetic signature distinct from uninfected CD4+ T cells in ART treated PLWH. We will further pilot a new strategy, scGETseq, capable of simultaneously and separately sequencing euchromatin and heterochromatin to distinguish and profile integrated HIV provirus within the active and latent reservoir of ART treated PLWH. Together our studies have the potential to provide the first full understanding of the unmanipulated CD4+ T cell HIV reservoir directly ex vivo.

虽然联合抗逆转录病毒疗法(ART)有效地控制了大多数艾滋病毒感染者的艾滋病毒病毒血症 (PLWH)，由于艾滋病毒宿主，广泛适用的诱导病毒缓解的策略仍然难以捉摸 坚持不懈。在确定前病毒的病毒学特征方面取得了实质性进展 抗逆转录病毒治疗期间潜伏感染细胞的蓄水池。然而，我们对这一问题的理解仍然存在重大差距。 被感染的CD4+T细胞构成了细胞内HIV的储存库，限制了我们发展有效的能力 治疗艾滋病毒的策略。我们已经建立了一种新的高通量10X基因组单细胞分析方法来直接 通过整合前病毒DNA的存在来鉴定感染艾滋病毒的细胞。称为“病毒ASAPseq”[检测 转座酶可及性Chomatin(ATAC)表面抗原图谱测序，与病毒 Align，Virus ASAPseq]，这种检测方法可以联合检测常染色质中的艾滋病毒前病毒DNA 通过154个标记的细胞表面抗原标记，产生艾滋病毒感染细胞的多模式单细胞分辨。 这种分析不需要体外激活来检测整合的病毒DNA的存在，因此 保持受感染细胞的天然状态。我们已经在几个方面成功地试行了这一程序 条件，包括体外感染的原代CD4+T细胞，原代淋巴中的CD4+T细胞来自病毒鼠 经ART治疗的PLWH患者外周血中的CD4+T细胞。我们进一步开发了生物信息学 利用自体病毒序列比对识别前病毒DNA的策略，定义表面标记 HIV感染细胞的组成，并表征感染细胞的表观遗传结构。因此，我们是 首次准备直接在体外直接了解艾滋病毒储存库的组成。在这里我们 将应用这项新技术来测试HIV感染的CD4+T细胞具有细胞表面或 经ART治疗的PLWH中不同于未感染的CD4+T细胞的表观遗传学特征。我们将进一步试点新的 能够同时和分别对常染色质和异染色质进行测序的策略scGETseq 区分和描述ART治疗的PLWH的活跃和潜伏储存库中整合的HIV前病毒。 总之，我们的研究有可能提供第一个对未被操纵的CD4+T细胞的全面了解 HIV储存库直接进行体外实验。