Single Cell Characterization of Latent HIV-1 Reservoirs

潜在 HIV-1 储库的单细胞表征

• 批准号: 9324120
• 负责人: Utkan Demirci
• 金额: $ 37.86万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-08-02 至 2020-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9324120
• 关键词: Affect; Anatomy; Anti-Retroviral Agents; Biological Assay; CD4 Positive T Lymphocytes; Cell Count; Cells; Clinical; Coupled; Cytolysis; DNA; Data; Development; Effector Cell; Encapsulated; Fluorescence; Frequencies; Future; Genes; Genetic; Genetic Transcription; Genome; Genomic DNA; Genomics; Gut associated lymphoid tissue; HIV-1; Histone Deacetylase Inhibitor; Immune; Individual; Infection; Inguinal lymph node group; Intervention Trial; Lead; Lymphocyte; Lymphoid Tissue; Measures; Memory; Methods; Microfluidics; Mononuclear; Patients; Peripheral Blood Mononuclear Cell; Phenotype; Plasma; Proteins; Proviruses; RNA; RNA Splicing; Reaction; Sampling; Shock; Sorting - Cell Movement; Techniques; Technology; Tissues; Viral; Viral Genome; Viral reservoir; Viremia; Virus; Virus Latency; Work; antiretroviral therapy; base; cohort; genome integrity; genome sequencing; in vivo; innovation; insight; killings; lymph nodes; nanoDroplet; nanolitre scale; new technology; novel; novel strategies; peripheral blood; public health relevance; response; single cell analysis; viral DNA; whole genome

 DESCRIPTION (provided by applicant): A major obstacle to HIV-1 eradication is the existence of latently infected cells that persist despite antiretroviral therapy (ART). Current eradication strategies focus on the reactivation and clearance of infected cells with various latency reactivating agents (LRA). However, viral reactivation alone is insufficient to reduce HIV-1 DNA reservoirs, and the association between increased cell-associated HIV-1 RNA and the frequency of reactivated cells is poorly understood. It is also unknown how reactivation strategies and HIV-1 genome integrity affect the frequency or amplitude of HIV-1 transcription. As a result, we have developed and implemented a novel method that provides insights into HIV-1 persistence that are inaccessible though existing technologies. Individual cells are encapsulated into nanoliter-scale reaction droplets, followed by intra-droplet lysis and PCR amplification of unspliced (us) and multiply spliced (ms) HIV-1 RNA and downstream isolation and sequencing of genomic viral DNA. We have successfully applied this method to identify transcriptionally active CD4+ T cells from HIV-1-infected patients on suppressive ART with and without LRA stimulation. Our data suggest the frequency of infected cells may increase while total cell-associated levels decrease and vice versa. We also observed dichotomies between usRNA and msRNA responses to latent reservoir activation. These findings suggest that single-cell analysis will be crucial in providing insight into which cells and tissues are targets for eradication in "shock and kill" approaches. We propose to utilize our assay to perform high-throughput reservoir quantification and downstream HIV-1 genome characterization of cells isolated from latently-infected peripheral blood and organized lymphoid tissue obtained from early and late ART treated individuals. We hypothesize that effector cells will display higher frequencies and amplitudes of HIV-1 RNA reactivation than those with memory or regulatory phenotype. Early treated individuals are thought to have smaller reservoirs, observed preferentially in effector memory cells which may have the propensity to reactive HIV-1 more efficiently. Our aims are to: (1) determine HIV-1 RNA transcriptional activity in response to various HIV-1 latency reversing agents in single cells derived from peripheral blood and organized lymphoid tissues from suppressed patients, (2) determine the single-cell responses to ex vivo LRA reactivation in early and late antiretroviral treated individuals, and (3) define the relationship between single-cell HIV-1 genetic sequence integrity, and HIV-1 transcriptional activity. In future studies, we plan to apply our novel methods to characterize the in vivo responses to interventional trials of HDACi and other immune modulating therapies. We also plan to adapt our assay to directly measure viral outgrowth in individual cells.

 描述(申请人提供)：根除HIV-1的一个主要障碍是潜伏感染细胞的存在，尽管进行了抗逆转录病毒治疗(ART)，但这些细胞仍然存在。目前的根除策略侧重于用各种潜伏期再活化剂(LRA)重新激活和清除受感染的细胞。然而，病毒的重新激活本身不足以减少HIV-1DNA储存库，而且细胞相关的HIV-1RNA增加和重新激活的细胞频率之间的联系还不清楚。重新激活策略和HIV-1基因组完整性如何影响HIV-1转录的频率或幅度也是未知的。因此，我们开发并实施了一种新的方法，提供了通过现有技术无法获得的对HIV-1持久性的见解。单个细胞被包裹在纳升级的反应液滴中，随后液滴内裂解和未剪接(US)和多重剪接(MS)的HIV-1 RNA的PCR扩增，以及下游基因组病毒DNA的分离和测序。我们已经成功地应用这种方法从HIV-1感染患者中识别转录活性的CD4+T细胞，在有和没有LRA刺激的情况下进行抑制ART。我们的数据表明，感染细胞的频率可能增加，而与细胞相关的总水平下降，反之亦然。我们还观察到usRNA和msRNA对潜在储存库激活的反应之间的二分性。这些发现表明，单细胞分析对于洞察哪些细胞和组织是“休克和杀死”方法的根除目标至关重要。我们建议利用我们的方法对从潜伏感染的外周血中分离出来的细胞和从接受抗逆转录病毒治疗的早期和晚期患者获得的有组织的淋巴组织进行高通量水库量化和下游HIV-1基因组表征。我们假设效应细胞将表现出比那些具有记忆或调节表型的细胞更高的HIV-1RNA重新激活的频率和幅度。早期治疗的个体被认为有较小的储存库，优先在效应器记忆细胞中观察到，这些细胞可能有更有效地反应HIV-1的倾向。我们的目标是：(1)确定来自受抑制患者的外周血和有组织淋巴组织中的单个细胞对各种HIV-1潜伏期逆转剂反应的HIV-1 RNA转录活性；(2)确定抗逆转录病毒治疗早期和晚期患者单个细胞对体外LRA重新激活的反应，以及(3)确定单细胞HIV-1基因序列完整性与HIV-1转录活性之间的关系。在未来的研究中，我们计划应用我们的新方法来表征对HDACi和其他免疫调节疗法的介入试验的体内反应。我们还计划调整我们的检测方法，以直接测量病毒在单个细胞中的生长。