Single Cell Characterization of Latent HIV-1 Reservoirs

潜在 HIV-1 储库的单细胞表征

基本信息

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
项目类别：
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

项目摘要

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（由申请人提供）：根除 HIV-1 的一个主要障碍是尽管有抗逆转录病毒治疗（ART），但潜伏感染细胞的存在仍然存在。目前的根除策略侧重于使用各种潜伏再激活剂（LRA）来重新激活和清除受感染细胞。然而，单独的病毒再激活不足以减少HIV-1 DNA储存，而且细胞相关的HIV-1 RNA增加和细胞再激活频率之间的关系也知之甚少。目前尚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。因此，我们开发并实施了一种新方法，可以深入了解 HIV-1 的持久性，这是现有技术无法实现的。被封装到纳升级反应液滴中，然后对未剪接（us）和多重剪接（ms）HIV-1 RNA进行液滴内裂解和PCR扩增，以及基因组病毒DNA的下游分离和测序。应用此方法来识别我们的数据表明，受感染细胞的频率可能会增加，而总细胞相关水平会降低，反之亦然。这些发现表明，单细胞分析对于了解哪些细胞和组织是“休克和杀死”方法中的根除目标至关重要。对从潜伏感染的外周血和从早期和晚期 ART 治疗个体获得的有组织淋巴组织中分离的细胞进行定量和下游 HIV-1 基因组表征，我们发现效应细胞将表现出比接受治疗的细胞更高的 HIV-1 RNA 再激活频率和幅度。早期个体被认为具有较小的储存库，优先在效应记忆细胞中观察到，这些细胞可能更有效地反应 HIV-1。 (2) 确定早期和晚期抗逆转录病毒治疗个体对离体 LRA 再激活的单细胞反应，以及 ( 3) 定义单细胞 HIV-1 基因序列完整性和 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 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单个细胞中的病毒生长。