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）：摘要病毒潜伏已成为根除 HIV-1 感染的主要障碍，目前消除病毒库的理论范式被称为“休克和杀灭”，即使用非靶向小分子重新激活潜伏病毒。为了使这种方法取得成功，必须克服这些问题，包括随机病毒再激活、避免全局 T 细胞激活以及病毒再激活时有限的细胞死亡。因此，需要另一种针对 HIV-1 潜伏期的策略。不幸的是，使用生物标志物来识别和选择性地靶向体内潜伏感染的细胞，而无需重新激活，这些生物标志物对于体外研究来说将是非常有价值的，因为现在可以纯化来自患者的天然状态的潜伏感染细胞以供研究。然而，目前尚不存在此类生物标志物。在此，我们提出了一种基于系统生物学的多管齐下的策略来识别原代 CD4+ T 细胞中的潜伏生物标志物。感染后早期（<4 天），并且无需重新激活，用该病毒感染原代 CD4+ T 细胞会在约 30% 的感染事件中产生可逆的 HIV 潜伏状态。比当前使用的许多其他原代细胞潜伏模型的频率要高得多，该模型的独特特征是我们建议使用强大的表型分析工具组合来表征原代 CD4+ T 感染 HIV-1 后变为潜伏的细胞的基础。这些工具将包括： 1. 细胞表面标记物、T 细胞信号网络和 T 细胞效应器功能的 CyTOF 单细胞分析 2. 基于细胞表面富集蛋白质的定量质谱分析。 3. 与有效感染细胞相比，对潜伏感染细胞的细胞转录组进行 RNAseq 和 microRNAseq 分析最重要的是，我们还将筛选假定的生物标志物列表，以了解其预测效用。我们相信，这种多管齐下的验证方法对于鉴定真实性至关重要。真实的生物标志物，并且可能产生对于新型 HIV-1 潜伏期调节疗法的体外验证非常有价值的目标。