Whole body to single cell analysis of the HIV reservoir

HIV 储存库的全身到单细胞分析

基本信息

批准号：
10335265
负责人：
PHILIP J SANTANGELO
金额：
$ 70.83万
依托单位：
UNIVERSITY OF LOUISIANA AT LAFAYETTE
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-03-01 至 2025-02-28
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10335265
关键词：
Accounting Acute Address Area Autopsy Biopsy Blood CD4 Positive T Lymphocytes Cell Lineage Cells Chronic Clinic Collection Confocal Microscopy Data Detection Environment Evaluation Flow Cytometry Future Gut Mucosa HIV HIV Infections Hand Human Imaging technology ImmunoPET Immunologic Deficiency Syndromes Interruption Ionomycin Isotopes Label Longevity Lymphoid Macaca Macaca mulatta Maintenance Maps Microscopic Modeling Molecular Monitor Monkeys Organ PET/CT scan Patients Peripheral Pharmaceutical Preparations Plasma Positron-Emission Tomography Production Residual state Resolution SIV Secondary to Signal Transduction Site Sorting - Cell Movement Source Spleen Stains Supporting Cell Suspensions T memory cell Techniques Technology Testing Time Tissues Translations Viral Viral Load result Viral reservoir Virulent Virus Virus Diseases Virus Latency Virus Replication X-Ray Computed Tomography acute infection antiretroviral therapy base chronic infection design imaging system in vivo instrument latent HIV reservoir lymph node biopsy lymph nodes nonhuman primate novel programs reproductive tract simian human immunodeficiency virus single cell analysis tool transcriptome sequencing viral rebound

项目摘要

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.

抽象的虽然对猿猴免疫缺陷病毒 (SIV) 感染的猴子进行有效的 ART 治疗，但可以迅速抑制病毒复制，从而我们最近开发的病毒环境定向免疫 PET/CT 成像技术在血浆中检测不到水平即使在长时间的 ART 之后，也能够检测到持续的 SIV 信号。该技术仅限于1-2毫米范围，距离“细胞水平”还很远，还有待充分适应并针对 HIV 检测进行了优化，因此，我们在此建议优化我们的免疫 PET/CT 技术。在猿猴/人类免疫缺陷病毒 (SHIV) 感染的情况下检测体内 HIV 环境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铺平了道路然而，为了在细胞和分子水平上解决储存库问题，我们将结合 PET 探针。具有在近红外范围内发射的荧光部分，使用手持式近红外成像 Fluobeam。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检和随后将通过共聚焦、流式细胞术和 RNASeq 进行尸检分析。这将使我们能够精确剖析哪些细胞谱系继续支持残留。 ART下的病毒复制，识别其环境，并确定是否继续复制继发于局部 ART 水平较低、特异性激活和/或局部抗病毒机制缺乏。允许测试免疫 PET/CT 信号阴性的位点是否存在完全潜伏的病毒库。我们将首先优化免疫PE/CT对HIV分支B、C和AE的检测，因为最佳探针是预计这些进化枝之间会有所不同，然后我们将重点关注提供最高病毒载量集的 SHIV。指出了体内 ART 下病毒库寿命的问题，以及病毒载量的功能性病毒库的问题 ART 中断后，使用免疫PET/CT 结合病毒信号的近红外图谱将会反弹。将允许对该模型中的 SHIV 储存库进行全身到单细胞分析，并进行组织比较从相对于相关组织的持久储存库中获取阴性病毒信号。描述这些相似组织之间的功能差异，为长寿提供机制线索 SHIV 体内储存库。