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

项目摘要 “治愈艾滋病”的主要障碍是病毒储存库（VR）的持续存在， 病毒基因组具有产生传染性病毒的能力。这些VR会持续很长一段时间， 即使在多年的抑制性ART后，病毒的系统性传播在停止后的几周内恢复 在所有的情况下，除了特殊情况。有效的治疗策略需要大大减少或消除VR 通过安全和可扩展的方法。目前认为主要的VR都是长期潜伏感染的 静息记忆CD 4 + T细胞，其保持静止，直到它们被外部刺激刺激以产生病毒。 除了真正潜在的VR，新出现的数据显示，在接受抑制性ART的个体中，VR的一个子集 以可变水平转录病毒RNA（vRNA+）（称为“活性VR”）。在某些情况下，这可能会导致残留 HIV复制水平，特别是在药物浓度不理想的组织微环境中。 即使没有完全的病毒复制，病毒的这种残余表达也可能具有不利的后果， 导致慢性免疫激活/炎症和非AIDS定义的临床事件。消灭艾滋病毒将 需要同时瞄准“潜伏”和“活跃”的VR，然而，我们目前对HIV储存库的理解来自于 大多数来自在外周血中进行的研究，但血液在ART期间仅含有一小部分VR。 我们的理由是，为了最大限度地提高“艾滋病毒治愈”策略的功效，我们需要首先更好地表征组织和组织。 HIV感染者在ART后感染可能反弹的细胞亚群 被打断了因此，本研究提案的总体目标是响应RFA-AI-18-053“单细胞 多组学的艾滋病毒持久性”，是合并我们的创新原位杂交（ISH）的方法，以量化 并使用多个新的尖端多组学平台以高分辨率绘制VR，以研究机制 在单细胞水平上的VR持久性，同时保留对细胞的至关重要的上下文洞察力 免疫社区和炎症景观，其中VR居住。在目标1中，我们将利用 新一代ISH（RNAscope、DNAscope和BASescope）平台，用于量化和生成“图谱” 在组织隔室（外周和肠系膜淋巴结， 脾、胃肠道）。在目标2中， 我们将对VR和细胞免疫邻域以及炎症进行深入的表型分析， 它们在组织中的位置（由我们上面概述的高分辨率原位VR映射指导） 使用多路离子束成像（MIBI）蛋白质组学分析以及无偏SNaPP和nanoPOTs质量 用于免疫LCM获得的样品的时空分子分析的光谱方法 邻近细胞和单细胞，以及解离的FACS分选的单细胞。在目标3中，我们将在 深度FISSEQ结合了RNA-FISH的空间背景和RNA-seq的全局转录组分析 在组织切片上（如上所述），但保留在单细胞水平。

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.