Novel ISH Approaches to Quantify Replication Competent Reservoirs

量化复制能力储库的新 ISH 方法

• 批准号: 10413135
• 负责人: JACOB D ESTES
• 金额: $ 72.5万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 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/10413135
• 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

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.

项目总结 HIV治愈的主要障碍是具有复制能力的病毒库(VR)的持久性 有能力产生传染性病毒的病毒基因组。这些虚拟现实持续了很长一段时间，并且 即使在多年的抑制艺术之后，病毒的系统性传播在停止后的几周内仍会恢复。 在除特殊情况外的所有情况下都是艺术。有效的治疗策略将需要显著减少或消除VR 通过安全和可扩展的方法。目前认为，主要的VR是长寿的潜伏感染 静止的记忆CD4+T细胞，它们保持静止，直到受到外部提示的刺激而产生病毒。 除了真正潜伏的VR，新兴数据显示，在个人对抑制性艺术的VR子集 转录病毒RNA(vRNA+)在不同水平(称为‘活性VR’)。在某些人中，这可能会导致 艾滋病毒复制的残留水平，特别是在药物浓度为 不太理想。即使没有充分的病毒复制，病毒的这种残留表达也可能产生不利的后果 并有助于慢性免疫激活/炎症和非艾滋病定义临床事件。根除艾滋病毒 然而，我们目前对艾滋病毒携带者的理解是，这需要同时针对潜在的和主动的VR 大部分来自于在外周血液中进行的研究，但血液中只含有一小部分VR 在艺术上。我们的理由是，为了最大限度地发挥‘HIV治愈’策略的效果，我们需要首先更好地描述 在HIV感染者中，感染可能反弹的组织间隔和细胞亚群 艺术之后的个人被打断了。因此，这项研究提案的首要目标是回应《全球行动纲领》 PA-17-305《持久HIV储存库成像》将验证和应用新型显微和流动技术 允许多参数单细胞的细胞学RNA和DNA原位杂交(ISH)平台 VR的特征与不同水平的残存转录和/或翻译。我们将使用这些 HIV感染样本的方法和SIV感染的NHP模型，该模型允许详细研究不同的 纸巾。在目标1中，我们将优化我们独特的ISH平台，以识别和表征“潜在的”和“主动的”虚拟现实 具有假定完整的病毒基因组，并区分具有不同转录和翻译水平的VR 活动。我们将定义经典VR(例如，PCR)和基于ISH的测量之间的关系，以及 确定血液中VR测量与不同组织环境相比的关键差异，如 以及综合分析VR所处的“免疫社区”和“炎性景观” 住下来。在目标2中，我们将使用感染SIVmac239M条形码病毒和 确定血液和组织中VR的新ISH测量与病毒反弹时间的关系 ART停药后感染SIV的RMS。我们假设这些类似的测量方法更有可能预测时间 在ART中断时病毒反弹和反弹病毒的数量 相信这些强大的新方法在未来的临床试验中将在监测VR方面发挥重要作用。