Identification of the HIV Reservoir in Lymph Nodes Using Single Cell RNA-Seq

使用单细胞 RNA-Seq 鉴定淋巴结中的 HIV 储库

• 批准号: 9548050
• 负责人: Alexander Sigal
• 金额: $ 14.77万
• 依托单位: KWAZULU-NATAL RESEARCH INSTITUTE TB-HIV
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-02-15 至 2023-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9548050
• 关键词: AIDS/HIV problem; Adherence; Africa South of the Sahara; Anatomy; Anti-Retroviral Agents; Antiretroviral resistance; Atlases; BLR1 gene; Biological Assay; Biology; CD3 Antigens; CD4 Positive T Lymphocytes; Cell Separation; Cell Therapy; Cell surface; Cells; Characteristics; Clinical; Collaborations; Collection; Complement; Computer Analysis; DNA; Dependence; Development; Elements; Epidemic; Expression Profiling; Frequencies; Funding; Gene Expression; Gene Expression Profiling; Genetic Transcription; Goals; HIV; HIV Genome; HIV Infections; Helper-Inducer T-Lymphocyte; Heterogeneity; High Prevalence; Hospitals; Human; ImmunoPET; Individual; Infection; Institutional Review Boards; Joints; Lead; Lymphocyte; Macaca; Maintenance; Messenger RNA; Operative Surgical Procedures; Participant; Patients; Penetration; Pharmaceutical Preparations; Phenotype; Plasma; Play; Prevalence; Production; Protocols documentation; Recording of previous events; Regimen; Reporting; Role; SIV; Seeds; Shock; Site; Source; South Africa; South African; T memory cell; T-Lymphocyte; T-Lymphocyte Subsets; Techniques; Technology; Testing; Tissues; Transcript; Viral; Viral reservoir; Virus; Virus Replication; antiretroviral therapy; base; cell type; cohort; density; experimental study; gene therapy; lymph nodes; mRNA Expression; macrophage; novel strategies; patient population; peripheral blood; replication therapy; response; single-cell RNA sequencing; therapy design; transcriptome sequencing

PROJECT SUMMARY HIV persistence in the face of antiretroviral therapy necessitates lifelong treatment of infected individuals. HIV infection persists within a reservoir of CD4+ T cells that harbor latent integrated virus. The HIV reservoir may also reside in other long lived cell types such as macrophages, or may be maintained by HIV ongoing replication in the face of antiretroviral drugs (ARVs) due to lowered antiretroviral penetration in some anatomical compartments, or decreased sensitivity to ARVs due to cell-to-cell HIV spread. It is possible that multiple mechanisms of HIV persistence may play a role. Several approaches are being tested to target the HIV reservoir for elimination, including “shock and kill” strategies, which reinitiate HIV gene expression and specifically eliminate infected cells following viral replication, and gene therapy approaches to excise the HIV genome. In addition, there are emerging strategies that recognize and kill infected cells directly. Absent from these approaches is a comprehensive understanding of the phenotypes and characteristics of the cellular source(s) of the HIV reservoir under antiretroviral therapy (ART). Lymph nodes may serve as a central reservoir for HIV persistence due to their high concentration of lymphocytes, extensive cellular interactions within the lymph node which may allow HIV spread between cells, and limited ARV penetration. Multiple CD4+ T cells subtypes have been proposed be the source of the HIV reservoir, but it is still unclear which cell subtype is most crucial to maintain it, and whether there is inter-patient heterogeneity in reservoir composition which depends on ART regimen or other clinical parameters. Furthermore, the composition of the HIV reservoir in Sub-Saharan Africa has rarely been investigated, despite the massively high prevalence of people living with HIV/AIDS in this region, as well as the extensive rollout of ART in at the epicenter of the HIV epidemic in South Africa and the consequent large and diverse ART suppressed patient population which can be studied to understand HIV persistence. In the proposed study, we aim to quantitatively determine the cell type(s) responsible for HIV production in the lymph nodes of ART-treated patients. We will use a novel approach utilizing single cell RNA-Seq to derive both the transcriptional profile of individual host cells and their intracellular viral transcript levels and viral sequences. This will enable us to deeply characterize which cell type(s) are infected, the number of viral transcripts per cell, and the transcriptional changes associated with infection. We will also determine the HIV reservoir size and composition in the lymph nodes of each ART suppressed individual by the established techniques of detecting HIV DNA copies and using viral outgrowth assays. We will test lymph nodes from a large number of study participants, enabled by a unique combination of very high infection prevalence and state of the art hospital facilities at the study's base in Durban. We will investigate common elements of the HIV reservoir across participants, and characterize reservoir heterogeneity.

项目摘要 面对抗逆转录病毒疗法的艾滋病毒持续性必要的终身治疗感染者。艾滋病病毒 感染持续存在于具有潜在综合病毒的CD4+ T细胞的储层中。艾滋病毒水库可能 也驻留在其他长期寿命的细胞类型中，例如巨噬细胞，也可以通过HIV持续维持 面对抗逆转录病毒药物（ARV）的复制，由于某些抗逆转录病毒渗透率降低 解剖区室，或由于细胞到细胞HIV扩散而对ARV的敏感性降低。有可能 艾滋病毒持久性的多种机制可能起作用。正在测试几种方法以针对 消除艾滋病毒水库，包括“冲击和杀死”策略，重新启动艾滋病毒基因的表达和 特异性消除了病毒复制后被感染的细胞，以及对HIV进行切除的基因治疗方法 基因组。此外，还有一些新兴策略可以直接识别和杀死受感染的细胞。缺席 这些方法是对细胞的表型和特征的全面理解 在抗逆转录病毒疗法（ART）下，HIV储层的来源。 淋巴结可以作为HIV持久性的中央储层，因为它们的高浓度 淋巴细胞，淋巴结内广泛的细胞相互作用，可能允许艾滋病毒在细胞之间传播， 并有限的ARV穿透。已经提出了多个CD4+ T细胞亚型是HIV的来源 储层，但仍不清楚哪种细胞亚型对维护最重要，以及是否有病人间 储层组成的异质性取决于艺术方案或其他临床参数。 此外，很少研究撒哈拉以南非洲的艾滋病毒水库的组成，dospite 该地区艾滋病毒/艾滋病的人群的高度流行率以及广泛的推出 在南非的艾滋病毒流行病和随之而来的大型艺术中的艺术 被抑制的患者人群可以研究以了解艾滋病毒的持久性。 在拟议的研究中，我们旨在定量确定负责HIV产生HIV的细胞类型 经过艺术治疗的患者的淋巴结。我们将使用一种新的方法利用单细胞RNA-seq来得出 单个宿主细胞及其细胞内病毒转录水平和病毒的转录谱 序列。这将使我们能够深刻地表征哪种细胞类型被感染，病毒次数 每个细胞的转录物以及与感染相关的转录变化。我们还将确定艾滋病毒 储层的大小和组成在每种艺术的淋巴结中都被已建立的个人抑制 检测HIV DNA拷贝并使用病毒出生分析的技术。我们将测试来自 大量的研究参与者，通过非常高的感染率和 在德班研究基地的最先进的医院设施。我们将研究 跨参与者的HIV水库，并表征了储层异质性。