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 艾滋病毒在抗逆转录病毒疗法下的持续存在需要对受感染者进行终身治疗。艾滋病毒 感染在携带潜伏整合病毒的CD 4 + T细胞库中持续存在。HIV储存库可能 也存在于其他长寿的细胞类型，如巨噬细胞，或可能由HIV持续维持 由于一些国家抗逆转录病毒药物的渗透率降低， 解剖区室，或由于细胞间HIV传播而降低对ARV的敏感性。有可能 艾滋病毒持续存在的多种机制可能发挥了作用。目前正在测试几种方法， 消除艾滋病毒储存库，包括“休克和杀死”战略，重新启动艾滋病毒基因表达， 在病毒复制后特异性地消除受感染的细胞，以及切除HIV的基因治疗方法。 基因组此外，还出现了直接识别和杀死感染细胞的新策略。缺席 这些方法是对细胞表型和特征的全面理解， 在抗逆转录病毒疗法（ART）下的艾滋病毒储存源。 淋巴结可能作为HIV持久性的中心储存库，因为它们的高浓度的 淋巴细胞，淋巴结内广泛的细胞相互作用，这可能允许HIV在细胞之间传播， 和有限的抗逆转录病毒药物渗透。多种CD 4 + T细胞亚型被认为是HIV的来源。 但目前还不清楚哪种细胞亚型对维持它最关键，以及是否存在患者间的 储层组成的异质性取决于ART方案或其他临床参数。 此外，撒哈拉以南非洲的艾滋病毒库的组成很少得到调查， 该地区艾滋病毒/艾滋病感染者的发病率很高， 抗逆转录病毒疗法在南非艾滋病毒流行的中心，以及随之而来的大规模和多样化的抗逆转录病毒疗法 受抑制的患者群体可以通过研究来了解艾滋病毒的持续性。 在拟议的研究中，我们的目标是定量确定负责HIV生产的细胞类型。 ART治疗患者的淋巴结。我们将使用一种新的方法，利用单细胞RNA-Seq， 单个宿主细胞的转录谱和它们的细胞内病毒转录物水平以及病毒 序列的这将使我们能够深入地表征哪些细胞类型被感染，病毒的数量， 每个细胞的转录本，以及与感染相关的转录变化。我们还将确定艾滋病毒 每个ART抑制个体的淋巴结中的储库大小和组成由已建立的 检测HIV DNA拷贝和使用病毒生长测定的技术。我们将检测一个人的淋巴结 大量的研究参与者，由于非常高的感染率和 位于德班的研究基地拥有最先进的医院设施。我们将调查的共同要素 艾滋病毒库跨参与者，并表征水库异质性。