Whole body to single cell analysis of the HIV reservoir

摘要 虽然猴免疫缺陷病毒(SIV)感染猴子的有效ART迅速包含病毒复制到 在血浆中检测不到的水平，我们最近开发的病毒包膜定向免疫PET/CT成像技术 已经能够检测到持续SIV信号的大厅，即使在长时间的艺术之后。然而，这一决议 这项技术仅限于1-2毫米的范围，远离“细胞水平”，它仍有待完全适应。 并针对艾滋病毒的检测进行了优化。因此，我们在这里建议优化我们的免疫-PET/CT技术 在体内检测HIV env在猴/人类免疫缺陷病毒(Shiv)感染的背景下 正在接受抗逆转录病毒治疗(ART)的猕猴为未来将该技术转化为 人类诊所。然而，为了在细胞和分子水平上解决水库，我们将结合PET探针 在近红外范围内发射荧光部分。使用手持式荧光束近红外成像 系统，我们将能够识别器官和组织中的病毒库，并收集适当的活检组织和 尸检。对阳性组织和阴性组织的分析将采用共聚焦、流式细胞术和RNAseq 技术。这将使我们能够精确地剖析哪些细胞谱系继续支持残存 ART下的病毒复制，确定他们的环境，并确定继续复制是否 继发于局部ART水平较低、特异性激活和/或缺乏局部抗病毒机制。它还将 允许测试在免疫PET/CT信号阴性的部位是否存在完全潜伏的病毒库。 我们将首先优化免疫PE/CT检测HIV B、C和AE分支的方法，因为最佳探针是 预计这些分支之间会有所不同。然后，我们将重点介绍提供最高病毒载量集的SHIV 指出在体内抗逆转录病毒治疗下的储存库寿命，以及病毒载量来自的功能性储存库 将反弹后的艺术中断。免疫PET/CT结合病毒信号近红外图谱的应用 在这个模型中，将允许对SHIV储存库进行全身到单细胞分析，并进行组织比较 来自与病毒信号阴性的相关组织相关的持久性储存库。这也将允许 描绘这些相似组织之间的功能差异，为长寿提供机制线索 在活体内的SHV储存库。

Abstract While potent ART of Simian Immunodeficiency virus (SIV) infected monkeys rapidly contains viral replication to undetectable levels in plasma, our recently developed viral env directed immunoPET/CT imaging technology has been able to detect foyers of continued SIV signals even after prolonged ART. However, the resolution of the technique is restricted to the 1-2 mm range, far from the “cellular level”, and it remains to be fully adapted and optimized for the detection of HIV. Therefore, here we propose to optimize our immune-PET/CT technology to detect HIV env in vivo both in the context of simian/human immunodeficiency virus (SHIV) infection of macaques under antiretroviral therapy (ART) paving the way for a future translation of the technology to the human clinic. However, to address reservoirs at a cellular and molecular level, we will combine the PET probes with fluorescent moieties emitting in the near-infrared range. Using a Fluobeam, hand-held, near-IR imaging system, we will be able to identify viral reservoirs in organs and tissues and collect the appropriate biopsies and necropsies. Analysis of positive vs negative tissues will follow by confocal, flow cytometry and RNASeq technologies. This will allow us the ability to precisely dissect which cell lineages continue to support residual viral replication under ART, identify their environment, and determine whether the continued replication is secondary to lower local levels of ART, specific activation and/or lack of local antiviral mechanisms. It will also allow for testing whether fully latent viral reservoirs exist in sites that are negative for immunoPET/CT signals. We will first optimize the detection of HIV clades B, C and AE by immunoPE/CT since the optimal probes are anticipated to differ between these clades. We will then focus on the SHIV providing the highest viral load set points to address reservoir longevity under ART in vivo, as well as functional reservoirs from which viral loads will rebound post ART interruption. The use of immunoPET/CT combined with near-IR mapping of viral signal will allow for whole body to single cell analyses of the SHIV reservoir in this model, with comparison of tissues from persistent reservoirs relative to correlated tissues negative for viral signals. This will also permit the delineation of functional differences between these similar tissues, providing mechanistic clues for the longevity of SHIV reservoirs in vivo.