Identification of HIV latency biomarkers with a dual fluorescence reporter HIV

使用双荧光报告基因 HIV 鉴定 HIV 潜伏生物标志物

基本信息

批准号：
8892911
负责人：
Eric M. Verdin
金额：
$ 70.5万
依托单位：
J. DAVID GLADSTONE INSTITUTES
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-03-01 至 2020-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8892911
关键词：
Biological Biological Markers Biology CD4 Positive T Lymphocytes Cell Surface Proteins Cell surface Cells Cessation of life Characteristics Data Set Diagnostic Engineering Event Fluorescence Frequencies Gene Expression Gene Expression Profile Genes Genome HIV HIV-1 In Vitro Individual Infection Latent Virus Life Mass Spectrum Analysis MicroRNAs Modality Modeling Molecular Profiling Nature Pathway interactions Patients Phenotype Population Proteome Receptor Signaling Reporter Research Rest Shock Signal Transduction Systems Biology T-Cell Activation T-Cell Receptor T-Lymphocyte T-Lymphocyte Subsets Techniques Testing Therapeutic United States National Institutes of Health Validation Viral Virus Virus Latency antiretroviral therapy base in vivo interest killings memory CD4 T lymphocyte novel public health relevance single cell analysis small molecule tool transcriptome sequencing

项目摘要

DESCRIPTION (provided by applicant): ABSTRACT Viral latency has emerged as the main barrier to eradicating HIV-1 infection. The current theoretical paradigm for eliminating the viral reservoir is known as `Shock and Kill'-i.e. reactivation of latent virus using non- targeted small molecules. However, significant hurdles must be overcome for this approach to be successful. These include stochastic viral reactivation, avoidance of global T-cell activation, and limited cellular death upon viral reactivation. Thus, an alternative strategy for targeting HIV-1 latency would make use of biomarkers to identify and selectively target latently infected cells in vivo without the need for reactivation. Additionally, these biomarkers would be invaluable for in vitro research given that patient-derived, native-state latently infected cells could now be purified for study. Unfortunatel however, such biomarkers do not currently exist. Here, we propose a multi-pronged, systems-biology based strategy to identify latency biomarkers in primary CD4+ T-cells. We propose to use a novel dual-fluorescence reporter HIV-1 genome to identify, quantify, and purify latently infected cells early post infection (<4 days), and without reactivation. Infection of primary CD4+ T-cells with this virus yields a reversible state of HIV latency in ~30% of infection events, which is a much higher frequency than many other currently used primary cell latency models. This model's unique set of characteristics is fundamental to our proposal to use a powerful combination of phenotyping tools to characterize the cells that become latent after HIV-1 infection of primary CD4+ T-cells. These tools will include: 1. CyTOF single-cell analysis of cell-surface markers, T-cell signaling networks, and T-cell effector function. 2. Quantitative mass spectrometry based identification of cell surface proteins enriched on latently infected cells in comparison to productively infected cells; 3. RNAseq and microRNAseq analysis of the cellular transcriptome of latently infected cells in comparison to productively infected cells. Most importantly, we will also screen our list of putative biomarkers for their predictive utility. Thiswill be done by validation not only in cells infected in vitro with the dual fluorescence virus, but als ex vivo in CD4+ T-cells isolated from HIV infected patients. We believe that this multi-pronged validation approach is vital to identifying bona fide biomarkers, and is likely to yield targets tht are invaluable for ex vivo validation of novel HIV-1 latency modulating therapeutics.

描述（由申请人提供）：抽象病毒潜伏期已成为根除HIV-1感染的主要障碍。消除病毒储层的当前理论范例被称为“冲击和杀死”。使用非靶向的小分子重新激活潜在病毒。但是，要成功的方法必须克服重大障碍。这些包括随机病毒重新激活，避免全局T细胞激活以及病毒重新激活后的细胞死亡有限。这是针对HIV-1潜伏期的替代策略，将利用生物标志物在体内识别并选择性地靶向潜在感染的细胞，而无需重新激活。此外，鉴于现在可以纯化患者衍生的本地潜在感染细胞，这些生物标志物对于体外研究将是无价的。但是，不幸的是，此类生物标志物目前尚不存在。在这里，我们提出了一种基于系统生物学的策略，以识别主要CD4+ T细胞中的潜伏生物标志物。我们建议使用一种新型的双荧光报告基因组HIV-1基因组来识别，量化和净化潜在感染后感染后感染（<4天），而无需重新激活。在该病毒中感染原发性CD4+ T细胞在约30％的感染事件中产生可逆的HIV潜伏期状态比当前使用的许多原始细胞潜伏期模型的频率要高得多。该模型的独特特征集对于我们提出的建议使用表型工具的强大组合来表征主CD4+ T细胞HIV-1感染后成为潜在的细胞。这些工具将包括：1。细胞表面标记，T细胞信号网络和T细胞效应子功能的单细胞分析。 2。与有效感染的细胞相比，基于质谱的定量质谱鉴定富含潜在感染细胞的细胞表面蛋白； 3。与有效感染的细胞相比，RNASEQ和MicroRNASEQ分析了潜在感染细胞的细胞转录组。最重要的是，我们还将筛选我们的假定生物标志物的预测效用列表。这将不仅通过双重荧光病毒在体外感染的细胞中进行验证，还可以通过从HIV感染患者中分离出的CD4+ T细胞中的ALS Exvo。我们认为，这种多管齐下的验证方法对于识别真正的生物标志物至关重要，并且很可能产生目标对于新型HIV-1延迟调节疗法的离体验证非常宝贵。