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.

抗逆转录病毒疗法（ART）有效地控制了大多数艾滋病毒的患者 （plwh），由于艾滋病毒水库，诱导病毒缓解的广泛适用策略仍然难以捉摸 持久性。定义病毒的病毒学特征已经取得了重大进展 在艺术期间，潜在感染细胞的储层。但是，我们对 感染的CD4+ T细胞构成细胞HIV储存库，该储层限制了我们发展有效的能力 艾滋病毒治愈的策略。我们已经开发了一种新型的高吞吐量10x基因组学单细胞测定到直接 通过存在综合前病毒DNA来鉴定感染HIV感染的细胞。称为“病毒Asapseq” [ 转座酶可访问的凝结蛋白（ATAC）表面抗原轮廓测序，AsapseQ，病毒 对齐，病毒ASAPLEQ]，该测定允许在联合菌质素内检测HIV前病毒DNA 具有154标记的细胞表面抗原标记，产生了HIV感染细胞的多模式单细胞分辨率。 该测定不需要离体激活来检测积分病毒DNA的存在，从而 保留感染细胞的本地状态。我们已经成功地试用了几个程序 条件，包括体外感染的原发性CD4+ T细胞，病毒性淋巴结CD4+ T细胞的原代淋巴结CD4+ T细胞 PLWH和ART处理过的PLWH的外周血CD4+ T细胞。我们进一步发展了生物信息学 使用自体病毒序列比对鉴定病毒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 直接在离体中直接了解HIV储层组成的直接理解。我们在这里 将应用这种新技术来检验以下假设：HIV感染的CD4+ T细胞具有细胞表面或 在ART处理的PLWH中，与未感染的CD4+ T细胞不同的表观遗传学特征。我们将进一步驾驶新的 策略，scgetSeq，能够同时和分别测序白染色质和异染色质 在Art处理过的PLWH的活跃和潜在储层中区分和概况综合的HIV病毒。 我们的研究共同提供了对未操纵的CD4+ T细胞的首次完全理解 HIV水库直接在体内。