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（应用程序提供）：HIV-1辐射的主要障碍是存在持续存在目的地抗逆转录病毒疗法（ART）的潜在感染细胞。当前的辐射策略集中于具有各种潜伏期重新激活剂（LRA）的感染细胞的重新激活和清除。然而，仅病毒重新激活不足以减少HIV-1 DNA储存剂，并且对细胞相关的HIV-1 RNA与重新激活细胞的频率之间的关联鲜为人知。复仇策略和HIV-1基因组完整性如何影响HIV-1转录的频率或放大器，这也未知。结果，我们开发了一种新颖的方法，该方法为HIV-1持久性提供了洞察力，这些持久性是无法访问的，尽管现有技术无法访问。将单个细胞封装在纳米尺度反应液滴中，然后进行滴血内裂解和无膜片（US）和多剪接（MS）HIV-1 RNA的PCR扩增以及基因组病毒DNA的下游分离和测序。我们已经成功地应用了这种方法来鉴定来自HIV-1感染患者的转录活性CD4+ T细胞，并具有LRA刺激的抑制作用。我们的数据表明，感染细胞的频率可能会增加，而总细胞相关水平降低，反之亦然。我们还观察到USRNA和MSRNA对潜在储层激活的反应之间的二分法。这些发现表明，单细胞分析对于提供有关“冲击和杀伤”方法辐射的目标的洞察至关重要。我们建议利用我们的评估来进行高通量的储层定量和下游HIV-1基因组对从潜伏感染的外周血和有组织的淋巴组织分离的细胞，并从早期和晚期治疗的人获得的细胞。我们假设效应细胞比具有记忆或调节表型的效应细胞显示HIV-1 RNA重新激活的频率和放大器更高。人们认为，早期治疗的个体具有较小的储层，在效应记忆细胞中优先观察到，这可能有望更有效地反应性HIV-1。 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转录活动。在未来的研究中，我们计划采用我们的新方法来表征HDACI和其他免疫调节疗法介入的体内反应。我们还计划调整我们的评估，以直接测量单个细胞中的病毒生长。