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.

项目摘要 “艾滋病毒治愈”的主要障碍是携带复制能力的病毒储层（VR）的持久性 具有产生传染病的能力的病毒基因组。这些VR长期存在，并且 即使经过多年的抑制作用，病毒的全身传播在停止后几周内恢复 除特殊情况以外的所有艺术。有效的治疗策略将需要大大减少或消除VR 通过安全可扩展的方法。目前认为主要的VR是长期延伸的潜在感染 静止的记忆CD4+ T细胞，直到被外部线索刺激以产生病毒为止。 除了真正的潜在VR外，新兴数据还表明，在抑制艺术的个体中，VR的子集 在可变水平（称为“活动VR”）处转录病毒RNA（VRNA+）。在某些人中，这可能会导致 HIV复制的残留水平，尤其是在药物浓度的组织微环境中 次优。即使没有完全的病毒复制，这种病毒的残留表达也可能会带来不利的后果 并有助于慢性免疫活化/炎症和定义临床事件的非AID。根除艾滋病毒 但是，将需要针对“潜在”和“主动” VR，但是，我们目前对HIV水库的理解 主要来自外周血进行的研究，但血液仅包含一小部分VR 在艺术期间。我们认为，为了最大程度地提高“艾滋病毒治愈”策略的效率，我们首先需要更好地表征 组织隔室和细胞子集可能会从中感染中反弹的HIV感染 艺术后的个人中断。这，这项研究提案的总体目标是响应FOA PA-17-305“成像持续的HIV储藏”将验证并应用新型显微镜和流动 细胞术RNA和DNA原位杂交（ISH）平台，允许多参数单细胞 具有各种剩余转录和/或翻译的VR表征。我们将使用这些 HIV感染的样品和SIV感染的NHP模型的方法，该模型允许对潜水员进行详细研究 组织。在AIM 1中，我们将优化我们唯一的ISH平台，以识别和表征“潜在”和“ Active” VR 具有预定的完整病毒基因组，并以不同水平的转录和翻译区分VR 活动。我们将定义经典VR（例如PCR）和基于ISH的测量和 与不同的组织环境相比，确定血液中VR测量之间的关键差异，因为 以及对VR的“免疫社区”和“炎症景观”的全面分析 居住。在AIM 2中，我们将使用SIVMAC239M条形码病毒的感染进行介入研究 确定血液和组织中VR的新型ISH测量与病毒反弹的时间之间的关系 SIV感染的RMS在戒烟后。我们假设这些ISH措施更有可能预测时间 与常规测定法相比，病毒反弹和艺术中断时的反弹病毒数量和 相信这些有力的新方法对于监测将来的临床试验中的VR将很重要。