Whole body to single cell analysis of the HIV reservoir

摘要 虽然猴免疫缺陷病毒（SIV）感染猴的有效ART快速包含病毒复制， 我们最近开发的病毒包膜定向免疫PET/CT成像技术， 即使经过长时间的ART，也能够检测到持续SIV信号的福耶斯。然而， 该技术仅限于1-2 mm的范围，远离“细胞水平”，并且仍有待于完全适应 并优化用于检测艾滋病病毒。因此，我们建议优化我们的免疫PET/CT技术， 为了在猴/人类免疫缺陷病毒（SHIV）感染的情况下检测体内HIV env， 接受抗逆转录病毒治疗（ART）的猕猴，为该技术未来的转化铺平了道路。 人类诊所然而，为了在细胞和分子水平上解决储库问题，我们将联合收割机 其中荧光部分在近红外范围内发射。使用Fluobeam，手持式，近红外成像 系统，我们将能够识别器官和组织中的病毒储存库，并收集适当的活组织检查， 尸检将通过共聚焦、流式细胞术和RNASeq分析阳性与阴性组织 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留的细胞。 在ART下的病毒复制，确定它们的环境，并确定是否继续复制， 继发于较低的局部ART水平、特异性激活和/或缺乏局部抗病毒机制。它还将 允许检测在免疫PET/CT信号阴性的部位是否存在完全潜伏的病毒储库。 我们将首先通过免疫PE/CT优化HIV进化枝B、C和AE的检测，因为最佳探针是 在这些进化枝之间有所不同然后，我们将重点放在提供最高病毒载量集的SHIV上 在体内ART下解决储库寿命的要点，以及病毒载量的功能性储库 会在ART中断后反弹。使用免疫PET/CT结合病毒信号的近红外成像 将允许在该模型中对SHIV储库进行全身至单细胞分析，并比较组织 相对于病毒信号阴性的相关组织，来自持久性储库。这也将使 描绘这些相似组织之间的功能差异，为长寿提供机制线索 体内SIV储库的数量。

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.