Defining Mechanisms of Viral Persistence in Situ at the Single-Cell Level

在单细胞水平上定义病毒原位持续存在的机制

• 批准号: 10064126
• 负责人: JACOB D ESTES
• 金额: $ 80.01万
• 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: 2019-12-03 至 2024-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10064126
• 关键词: Adherence; Anatomy; Anti-Inflammatory Agents; Atlases; Autopsy; Biochemistry; Blood; Cells; Characteristics; Chronic; Clinical; Communities; Cues; Data; Data Set; Event; Gastrointestinal tract structure; Genetic Transcription; Goals; HIV; HIV Infections; Immune; Immunology; In Situ; In Situ Hybridization; Individual; Infection; Inflammation; Inflammatory; Interdisciplinary Study; Interruption; Lead; Lymphoid Tissue; Maps; Mass Spectrum Analysis; Modeling; Molecular; Molecular Analysis; Monkeys; Multiplexed Ion Beam Imaging; Neighborhoods; Organ; Pathology; Pathway interactions; Peripheral; Pharmaceutical Preparations; Phenotype; Proteins; Proteomics; RNA; Research Proposals; Residual state; Resistance; Resolution; Rest; SIV; Sampling; Scientist; Source; Spleen; Testing; Time; Tissues; Transcript; Viral; Viral Genome; Viral reservoir; Virion; Virus; Virus Diseases; Virus Latency; Virus Replication; adverse outcome; antiretroviral therapy; body system; cell type; cost; density; immune activation; improved; in situ sequencing; innovation; insight; laser capture microdissection; memory CD4 T lymphocyte; mesenteric lymph node; multiple omics; next generation; nonhuman primate; novel; novel strategies; peripheral blood; response; side effect; social stigma; spatiotemporal; transcriptome; transcriptome sequencing; viral RNA

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 cases, 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 RFA-AI-18-053 “Single-Cell Multi-Omics of HIV Persistence”, is to merge our innovative in situ hybridization (ISH) approaches to quantify and map VR at high resolution with multiple new cutting-edge multi-omics platforms to investigate mechanisms of VR persistence at the single-cell level while retaining critically important contextual insight into the cellular immune neighborhoods and inflammatory landscapes in which VR reside. In Aim 1, we will utilize our suite of novel next-generation ISH (RNAscope, DNAscope and BASEscope) platforms to quantify and generate “atlases” of ‘latent’ and ‘active’ VR longitudinally within tissue compartments (peripheral and mesenteric lymph nodes, spleen, GI tract) before and at different timepoints during ART ± anti-inflammatory adjunctive therapy. In Aim 2, we will perform an in-depth phenotypic analysis of VR and the cellular immune neighborhoods and inflammatory landscapes in which they reside within tissues (guided by our high-resolution in situ VR mapping outlined above) using Multiplexed Ion Beam Imaging (MIBI) proteomic analysis as well as unbiased SNaPP and nanoPOTs mass spectrometry approaches for spatiotemporal molecular analyses on samples obtained by LCM of immune neighborhoods and single cells, as well as on dissociated FACS sorted single cells. In Aim 3, we will perform in depth FISSEQ that combines the spatial context of RNA-FISH and the global transcriptome profiling of RNA-seq on tissue sections (as outlined above) but retained at the single-cell level.

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’)。在某些情况下，这可能会导致残留 艾滋病毒复制水平，特别是在药物浓度低于最佳水平的组织微环境中。 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和 有助于慢性免疫激活/炎症和非艾滋病定义临床事件。根除艾滋病毒将会 需要同时针对潜在的和主动的VR，然而，我们目前对HIV宿主的理解来自于 大部分是从外周血液中进行的研究，但在ART过程中，血液中只含有一小部分VR。 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这两个组织的特征 艾滋病病毒感染者在接受抗逆转录病毒治疗后感染可能反弹的隔室和细胞亚群 被打断了。因此，本研究提案的首要目标是响应RFA-AI-18-053“Single-Cell HIV持久性的多组学“，是融合我们创新的原位杂交(ISH)方法来量化 并使用多个新的尖端多组学平台以高分辨率绘制VR图，以研究机制 单细胞水平的VR持久性，同时保持对细胞的至关重要的上下文洞察力 VR居住的免疫社区和炎症性景观。在目标1中，我们将利用我们的套件 新一代ISH(RNAScope、DNA Scope和Base Scope)平台用于量化和生成“地图集” 在组织隔室(外周和肠系膜淋巴结， 在ART±抗炎辅助治疗前和不同时间点)。在目标2中， 我们将对VR以及细胞免疫邻域和炎症进行深入的表型分析 它们居住在组织内的景观(由我们上面概述的高分辨率原位VR地图指导) 使用多路离子束成像(MIBI)蛋白质组分析以及无偏SNAPP和纳米POTS质量 免疫激光共聚焦显微镜样品时空分子分析的光谱方法 邻域和单细胞，以及在分离的FAC上对单细胞进行分类。在目标3中，我们将在 结合了RNA-FISH的空间背景和RNA-SEQ的全球转录组图谱的深度FISSEQ 在组织切片上(如上所述)，但保留在单细胞水平。