In situ and digital spatial profiling of the active HIV reservoir in autopsy-derived tissues

项目摘要/摘要(项目2) 艾滋病毒反弹潜力的研究需要严格的病毒和免疫学特征直接在 艾滋病病毒携带者的组织(PWH)接受抗逆转录病毒治疗，并发症最少。因此，该项目利用 旧金山尸检后心脏性猝死的系统研究， 对威斯康星医院和未感染对照人群的银行样本和尸检数据进行的尸检研究 心脏猝死(SCD)。到目前为止，我们已经收集了大量的组织样本，包括大脑，多个 47例HIV感染者的淋巴结链、肝、脾、心、肺血管和其他感兴趣的组织 和经历过SCD的500名未感染的人。重要的是，约80%的HIV+SCD病例接受了抗逆转录病毒治疗 并突然死于非艾滋病毒(即心脏)原因。因此，艾滋病后SCD队列是独一无二的 组织艾滋病毒持久性研究的资源。这个高度创新的项目涉及原位杂交和 尖端基于组织的转录/蛋白质组纳米串数字空间剖析(DSP)，以明确定义 完整的和表达艾滋病毒的前病毒和生物标志物的储存库在组织和组织之间的差异 确定转录活性HIV储存库中的组织特异性差异如何与原位宿主细胞相关 基因和蛋白质的表达。我们的中心假设是感染的CD4T细胞和各种髓系 免疫特许组织环境中的细胞表达全长、完整的HIV RNA转录本和BE 能够在ART停药后迅速病毒复发。我们期望观察到宿主基因的低表达 抗病毒因子和促进生存因子在表达艾滋病毒转录物的细胞团中的高表达。 此外，我们假设被鉴定为 在项目3中病毒反弹的预测因素将在感兴趣的特定淋巴组织区域内可视化。 我们的目标是：1)衡量完整和有缺陷的前病毒和艾滋病毒转录本的总负担 体内不同器官和组织的全谱；2)比较不同组织的细胞负荷和 SCD患者在接受和不接受抗逆转录病毒治疗时自发转录HIV转录的细胞表型 死亡；以及3)确定艾滋病毒负担和残余转录活性对宿主细胞因子的原位影响 (特别是抗病毒限制和先天免疫途径)在组织驻留的淋巴和髓系细胞中 接受抗逆转录病毒治疗的SCD患者与未感染的对照组进行比较。因此，该项目将有能力确定 与项目1和3协同实现艾滋病毒治愈的治疗方法的目标。

Project Summary/Abstract (Project 2) The study of HIV rebound potential necessitates rigorous viral and immunological characterization directly within tissues in people with HIV (PWH) on ART with minimal comorbidities. As a result, this project leverages the longitudinal San Francisco POstmortemSystematic InvesTigation of Sudden Cardiac Death (POST SCD) Study, a postmortem study to bank samples and autopsy data on PWH and uninfected controls who were victims of sudden cardiac death (SCD). To date we have collected extensive tissue samples, including brain, multiple lymph node chains, liver, spleen, heart, pulmonary vasculature and other tissues of interest from47 HIV-infected and >500 uninfected individuals who experienced SCD. Importantly, ~80% of HIV+ SCD cases were on ART and died suddenly of non-HIV (i.e. cardiac) causes. As a result, the HIV POST SCD cohort is a one-of-kind resource for the study of tissue HIV persistence. This highly innovative project involves in situ hybridization and cutting-edge tissue-based transcriptomic/proteomic nanoString Digital Spatial Profiling (DSP) to clearly define how the reservoirs of intact and HIV-expressing proviruses and biomarker expression differ between tissues and determine how tissue-specific differences in the transcriptionally active HIV reservoir relate to in situ host cell gene and protein expression. Our central hypothesis is that infected CD4 T cells and various myeloid lineage cells within immune privileged histologic environments express full-length, intact HIV RNA transcripts and be capable of rapid viral recrudescence following ART withdrawal. We expect to observe lower expression of host antiviral factors and higher expression of pro-survival factors in cell clusters expressing HIV transcripts. Furthermore, we posit that transcriptional activity and immune states of reservoir cells that are identified as predictors of viral rebound in Project 3 will be visualized within specific lymphoid tissue regions of interest. Our aims are to: 1) measure the total burden of intact and defective proviruses and HIV transcripts across the full spectrum of different organs and tissues in vivo; 2) compare across tissues the cellular burden and phenotypes of cells that spontaneously transcribe HIV transcripts in SCD victims on and off ART at the time of death; and 3) determine the in situ impact of HIV burden and residual transcriptional activity on host cell factors (particularly antiviral restriction and innate immune pathways) in tissue-resident lymphoid and myeloid cells in SCD victims on ART compared to uninfected controls. As a result, this project will have the capacity to identify targets for therapeutic approaches to achieve HIV cure in synergy with Projects 1 & 3.