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

• 批准号: 9903192
• 负责人: Eric M. Verdin
• 金额: $ 69.48万
• 依托单位: BUCK INSTITUTE FOR RESEARCH ON AGING
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-03-01 至 2022-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9903192
• 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; Expression Profiling; Fluorescence; Frequencies; Gene Expression; Genes; HIV; HIV Genome; HIV-1; In Vitro; Individual; Infection; Mass Spectrum Analysis; MicroRNAs; Modality; Modeling; Nature; Pathway interactions; Patients; Phenotype; Population; Primary Infection; 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; Viral reservoir; Virus; Virus Integration; Virus Latency; antiretroviral therapy; base; effector T cell; in vivo; interest; memory CD4 T lymphocyte; multimodality; novel; novel marker; public health relevance; reactivation from latency; single cell analysis; single-cell RNA sequencing; small molecule; tool; transcriptome; transcriptome sequencing

 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）： 病毒潜伏期已成为根除HIV-1感染的主要障碍。目前用于消除病毒储库的理论范例被称为“休克和杀死”，即使用非靶向小分子重新激活潜伏病毒。然而，这一方法要取得成功，必须克服重大障碍。这些包括随机病毒再活化，避免整体T细胞活化，以及病毒再活化后有限的细胞死亡。因此，靶向HIV-1潜伏期的替代策略将利用生物标志物在体内鉴定和选择性靶向潜伏感染的细胞，而不需要再活化。此外，这些生物标志物对于体外研究将是非常宝贵的，因为现在可以纯化患者来源的天然状态潜伏感染细胞用于研究。然而，不幸的是，这种生物标志物目前并不存在。在这里，我们提出了一个多管齐下的，基于系统生物学的策略，以确定在原代CD 4 + T细胞的潜伏生物标志物。我们建议使用一种新的双荧光报告HIV-1基因组来识别，定量和纯化感染后早期（<4天）潜伏感染的细胞，而无需重新激活。该病毒感染原代CD 4 + T细胞会在约30%的感染事件中产生可逆的HIV潜伏期状态，这 是比许多其它当前使用的主要小区等待时间模型高得多的频率。该模型的独特特征集是我们建议使用表型分析工具的强大组合来表征HIV-1感染原代CD 4 + T细胞后潜伏的细胞的基础。这些工具将包括：1。细胞表面标志物、T细胞信号传导网络和T细胞效应子功能的CyTOF单细胞分析。2.与生产性感染细胞相比，基于定量质谱的鉴定在潜伏感染细胞上富集的细胞表面蛋白; 3.与生产性感染细胞相比，潜伏感染细胞的细胞转录组的RNAseq和microRNAseq分析。最重要的是，我们还将筛选我们的推定生物标志物列表，以确定其预测效用。这将通过验证不仅在体外用双荧光病毒感染的细胞中进行，而且在从HIV感染患者分离的CD 4 + T细胞中离体进行。我们相信，这种多管齐下的验证方法对于鉴定真正的生物标志物至关重要，并且可能产生对于新型HIV-1潜伏期调节疗法的离体验证非常宝贵的靶点。