Novel ISH Approaches to Quantify Replication Competent Reservoirs

量化复制能力储库的新 ISH 方法

基本信息

批准号：
10164715
负责人：
JACOB D ESTES
金额：
$ 71.57万
依托单位：
OREGON HEALTH & SCIENCE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-06-03 至 2024-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10164715
关键词：
Acute Adherence Anatomy Animals Antibodies Archives Bar Codes Biological Assay Biopsy Blood Blood Circulation CD4 Positive T Lymphocytes Cell Line Cells Chronic Clinical Clinical Trials Competence Complement Cryopreserved Tissue Cues DNA DNA Probes Data Environment Event Flow Cytometry Future Gastrointestinal tract structure Genes Genetic Transcription Goals HIV HIV Infections Human Image Immune In Situ Hybridization Individual Infection Inflammation Inflammatory Interruption Intervention Studies Lead Macaca mulatta Measurement Measures Microscopic Modeling Monitor Monkeys Neighborhoods Organ Pathology Pattern Pharmaceutical Preparations Phenotype Proteins Proviruses RNA RNA Probes Research Proposals Residual state Resistance Rest SIV Sampling Source Structure of germinal center of lymph node T-Lymphocyte Subsets Time Tissues Transcript Transcription Elongation Translations Viral Viral Genome Viral Proteins Viral reservoir Virion Virus Virus Diseases Virus Latency Virus Receptors Virus Replication acute infection adverse outcome antiretroviral therapy base cell type cost immune activation memory CD4 T lymphocyte next generation novel novel imaging technique novel strategies particle peripheral blood repository response side effect simian human immunodeficiency virus social stigma viral RNA viral rebound

项目摘要

PROJECT SUMMARY The major obstacle to an 'HIV cure' is the persistence of viral reservoirs (VR) harboring replication competent viral genomes that have the capacity to produce infectious virus. These VR persist for long periods of time, and even after years of suppressive ART, the systemic spread of virus resumes within a few weeks upon cessation of ART in all but exceptional cases. Effective cure strategies will need to dramatically reduce or eliminate VR through safe and scalable approaches. It is currently thought that the major VR are long-lived latently infected resting memory CD4+ T cells, which remain quiescent until they are stimulated by external cues to produce virus. In addition to the truly latent VR, emerging data shows that in individuals on suppressive ART a subset of VR transcribe viral RNA (vRNA+) at variable levels (termed ‘active VR’). In some individuals, this might lead to residual levels of HIV replication, particularly in tissue microenvironments where drug concentrations are suboptimal. Even without full viral replication, this residual expression of virus may have adverse consequences and contribute to chronic immune activation/inflammation and non-AIDS defining clinical events. Eradicating HIV will require targeting both the ‘latent’ and ‘active’ VR, however, our current understanding of HIV reservoirs comes mostly from studies performed in peripheral blood, but the blood contains only a small fraction of VR during ART. We reason that to maximize efficacy of ‘HIV cure’ strategies, we need to first better characterize both the tissue compartments and the cellular subsets from which infection might rebound in HIV-infected individuals after ART is interrupted. Thus, the overarching goals of this research proposal in response to FOA PA-17-305 "Imaging the Persistent HIV Reservoir" are to validate and apply novel microscopic and flow cytometric RNA and DNA in situ hybridization (ISH) platforms that allow multiparametric single-cell characterization of VR with various levels of residual transcription and/or translation. We will use these approaches in HIV-infected samples and a NHP model of SIV infection, which allows detailed studies of diverse tissues. In Aim 1, we will optimize our unique ISH platforms to identify and characterize ‘latent’ and ‘active’ VR with putatively intact viral genomes and distinguish VR with different levels of transcriptional and translational activities. We will define relationships between classical VR (e.g., PCR) and ISH-based measurements and determine key differences between VR measures in the blood compared to different tissue environments, as well as a comprehensive analysis of the “immune neighborhoods” and “inflammatory landscapes" in which VR reside. In Aim 2, we will perform an interventional study using infection with SIVmac239M barcoded virus and determine the relationship between novel ISH measures of VR in blood and tissues with time to viral rebound in SIV-infected RMs after ART cessation. We hypothesize that these ISH measures are more likely to predict time to viral rebound and the number of rebounding viruses upon ART interruption than conventional assays and believe that these powerful new approaches will be important in monitoring VR in future clinical trials.

项目概要 “治愈艾滋病毒”的主要障碍是具有复制能力的病毒库（VR）的持续存在有能力产生传染性病毒的病毒基因组会持续很长时间，并且即使经过多年的抑制性抗逆转录病毒治疗，病毒的系统性传播也会在停止后几周内恢复在除特殊情况外的所有情况下，有效的治疗策略都需要大幅减少或消除 VR。目前认为主要的 VR 都是长期潜伏感染。静息记忆 CD4+ T 细胞保持静止状态，直到受到外部信号刺激产生病毒。除了真正潜在的 VR 之外，新出现的数据表明，在接受抑制性 ART 的个体中，VR 的一个子集在不同水平上转录病毒 RNA (vRNA+)（称为“主动 VR”）。 HIV 复制的残留水平，特别是在药物浓度较低的组织微环境中即使没有完全的病毒复制，病毒的这种残留表达也可能产生不良后果。并有助于慢性免疫激活/炎症和非艾滋病定义的临床事件。需要同时针对“潜伏”和“活跃”VR，但是，我们目前对 HIV 病毒库的了解主要来自于外周血中进行的研究，但血液中仅含有一小部分 VR 我们认为，为了最大限度地发挥“艾滋病毒治疗”策略的功效，我们首先需要更好地描述特征。 HIV感染者中感染可能反弹的组织区室和细胞亚群因此，本研究提案的总体目标是响应 FOA。 PA-17-305“对持久性 HIV 病毒库进行成像”将验证和应用新型显微镜和流程细胞计数 RNA 和 DNA 原位杂交 (ISH) 平台，可实现多参数单细胞分析我们将使用这些来表征具有不同水平的残留转录和/或翻译的 VR。 HIV 感染样本的方法和 SIV 感染的 NHP 模型，可以对不同的样本进行详细研究在目标 1 中，我们将优化我们独特的 ISH 平台，以识别和表征“潜在”和“活跃”VR。具有假定完整的病毒基因组，并通过不同水平的转录和翻译来区分 VR 我们将定义经典 VR（例如 PCR）和基于 ISH 的测量之间的关系和确定血液中 VR 测量值与不同组织环境相比的主要差异，如以及对 VR 所涉及的“免疫社区”和“炎症景观”的全面分析在目标 2 中，我们将使用 SIVmac239M 条形码病毒感染进行干预研究。确定血液和组织中 VR 的新 ISH 测量值与病毒反弹时间之间的关系我们发现这些 ISH 测量更有可能预测时间。与传统检测相比，ART 中断后的病毒反弹和反弹病毒数量相信这些强大的新方法对于未来临床试验中的 VR 监测非常重要。