Project 2: Revealing Viral Population and Host Environment Dynamics of SIV Tissue Reservoir

项目概要/摘要 尽管联合抗逆转录病毒疗法 (cART) 在控制 HIV-1 感染方面取得了显著成功，但病毒 水库在处理过程中会无限期地持续存在。这些剩余的病毒种群构成了主要负担 有效治愈 HIV-1，因为当治疗失败或分析后，它们会导致病毒血症迅速反弹 治疗中断（ATI）。不幸的是，我们对 HIV-1 持续存在机制的理解还不够深入。 cART 是有限的，因为病毒库群体规模非常小，而且它是在多种病毒中建立的 易受影响的组织，无法评估。因此，这些困难使得大多数储层研究 不包括组织样本或研究组织库的非常有限的样本。因此，我们不是 能够正确研究这些病毒种群，但它们的主要特征仍然未知。了解 治疗期间这些组织储存库的性质和特性是设计成功的 HIV-1 治疗方法的关键 策略。由 Hope 实验室和 Villinger 博士组成的团队优化和完善了他们的 89Zr 标记 FAB2 探针可在受感染的猕猴中体内检测表达 SIV env 的细胞。使用这个 方法，他们生成了一个新的基于 PET-CT 的工作流程，用于识别和下游 组织中 SIV 感染灶的特征。这种更新的“信标引导系统”非常敏感 在 cART 期间或 ATI 后早期发现和表征受感染细胞的小病灶非常强大， 在任何可检测到的病毒血症之前。这些焦点代表了极其罕见的事件，对于理解这些事件至关重要 SIV 的生物学基础，但迄今为止，只能通过随机和/或有偏见的组织调查来发现。在 目前的提案，我们打算利用 Hope 实验室的能力来定位受感染猕猴中的 SIV 活性病毒血症位点 以及我们团队优化的补充基因组分析工作流程，以执行深入的时空分析 储存病毒种群动态研究。我们将使用系统发育、进化和系统解剖学 分析以研究组织储库的特征。我们还将评估生物过程的贡献 例如克隆扩增、残留病毒复制以及储存过程中的病毒产量爆发 cART 期间的持续性。此外，我们将分析以下基因的特征和转录谱： 使用我们的多种互补基因组技术，在组织中储存储存库的不同细胞类型。 我们将使用 PET-CT 引导的 RNAseq 和 PET-CT 引导的 SIV 阳性组织的空间转录组学，以及 分离特定类型的受感染细胞，以使用单细胞分析来分析其转录谱。由于 希望实验室的研究结果表明肥大细胞可能是储存库持久性的关键，我们计划专门 研究这些肥大细胞在组织储存库病毒群体动态中的作用。通过遵循这个 多维方法，我们将以前所未有的细节定义组织中细胞的主要特性 在治疗过程中存在病毒储存库，并且是治疗时病毒反弹的最初来源 打断了。

PROJECT SUMMARY/ABSTRACT Despite the remarkable success of the combination antiretroviral therapy (cART) to control HIV-1 infection, viral reservoirs persist indefinitely under treatment. These remaining viral populations constitute the principal burden for an effective HIV-1 cure, as they lead to a rapid rebound in viremia when treatment fails or after analytic treatment interruption (ATI). Unfortunately, our understanding of the mechanisms of HIV-1 persistence during cART is limited because the viral reservoir population size is very small and it is established within a wide variety of susceptible tissues, inaccessible to evaluation. Hence these difficulties, the majority of the reservoir studies do not include tissue samples or study a very limited sampling of the tissue reservoirs. Therefore, we are not able to properly study these viral populations and their main characteristics remain unknown. Understanding the nature and properties of these tissue reservoirs during treatment is key to design a successful HIV-1 cure strategy. The team comprised by the Hope lab and Dr. Villinger has optimized and refined their 89Zr-labelled FAB2 probe that allows for the in vivo detection of cells expressing SIV env in an infected macaque. Using this approach, they have generated a new PET-CT-based workflow for the identification and downstream characterization of foci of SIV infection in tissues. This updated “beacon-guided system” is exquisitely sensitive and it is extremely powerful to find and characterize small foci of infected cells during cART or early after ATI, prior to any detectable viremia. These foci represent extremely rare events that are key to the understanding of underlying SIV biology and yet, to date, were only found through random and/or biased survey of tissue. In the current proposal, we intend to use the Hope lab ability to localize SIV active viremia sites in infected macaques and complementary genomic analysis workflows optimized by our team, to perform in-depth spatio-temporal studies of the reservoir viral population dynamics. We will use phylogenetic, evolutionary, and phyloanatomy analysis to study the characteristics tissue reservoirs. We will also assess the contribution of biological processes such as clonal expansion, residual viral replication, and viral production bursts in the process of reservoir persistence during cART. Additionally, we will analyze the characteristics and transcriptional profiles of the different cell types that harbor the reservoir in tissues using our multiple complementary genomic techniques. We will use PET-CT-guided RNAseq and PET-CT-guided spatial transcriptomics from SIV positive tissues, and isolate specific types of infected cells to analyze their transcriptional profiles using single-cell analysis. Due to Hope lab’s findings indicating that mast cells could be key in the reservoir persistence, we plan to specifically study the role of these mast cells in the viral population dynamics of the tissue reservoirs. By following this multidimensional approach, we will define with unprecedented detail the main properties of the cells in tissues that harbor the reservoir during treatment and are the initial source of the rebounding virus when treatment is interrupted.