Novel ISH Approaches to Quantify Replication Competent Reservoirs

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”）。对某些人来说，这可能会导致 HIV复制的残留水平，特别是在药物浓度 次优即使没有完全的病毒复制，病毒的这种残余表达也可能产生不良后果 并导致慢性免疫激活/炎症和非AIDS定义的临床事件。消除艾滋病毒 将需要针对“潜伏”和“活跃”的虚拟现实，然而，我们目前对艾滋病毒储存库的理解 主要来自于对外周血的研究，但血液中只含有一小部分VR 我们的理由是，为了最大限度地提高“艾滋病毒治愈”策略的疗效，我们需要首先更好地表征 在HIV感染者中，感染可能反弹的组织区室和细胞亚群 ART中断后的患者。因此，响应FOA的本研究提案的总体目标 PA-17-305“持续性HIV储库成像”旨在验证和应用新型显微镜和流动 细胞计数RNA和DNA原位杂交（ISH）平台，允许多参数单细胞 在一些实施方案中，本发明提供了具有各种残余转录和/或翻译水平的VR的表征。我们将会用这些 方法在HIV感染的样本和SIV感染的NHP模型，它允许详细研究不同的 组织中在目标1中，我们将优化我们独特的ISH平台，以识别和表征“潜在”和“主动”VR 与完整的病毒基因组，区分VR与不同水平的转录和翻译， 活动我们将定义经典VR之间的关系（例如，PCR）和基于ISH的测量，以及 确定与不同组织环境相比，血液中VR测量值之间的关键差异， 以及对VR所处的“免疫社区”和“炎症景观”的全面分析， 居住。在目标2中，我们将使用SIVmac 239 M条形码病毒感染进行干预性研究， 确定血液和组织中VR的新ISH测量与病毒反弹时间之间的关系， 停止抗逆转录病毒治疗后SIV感染的RM。我们假设，这些ISH措施更有可能预测时间 病毒反弹和ART中断后反弹病毒的数量， 相信这些强大的新方法将在未来的临床试验中监测VR方面发挥重要作用。

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.