Targeting Latently Infected Primary Cells using Integrated Glycoproteomics

 描述(申请人提供)：HIV潜伏感染细胞在接受抑制性抗逆转录病毒治疗(ART)的HIV+患者中非常罕见。为了针对这些潜伏感染的细胞而不进行潜伏再激活，识别与潜伏感染相关的特定细胞表面靶点是至关重要的。来自CD2、PD-1、LAG-3和TIGIT等蛋白质研究的令人信服的报告表明，在体外，有一种独特的细胞表面蛋白质用于浓缩潜伏感染的细胞。这些蛋白在潜伏感染建立过程中的具体变化仍有待阐明。有趣的是，上述所有蛋白质都是糖基化的(糖蛋白)。据估计，有超过1,000种细胞表面糖蛋白形成复杂和动态的细胞表面表型。询问细胞表面糖蛋白组将最大限度地增加识别特定细胞表面糖蛋白的机会，以靶向潜伏感染的细胞。为了实现这一目标，我们开发了集成糖蛋白质组学，它可以使用化学-酶法分离糖蛋白来定量糖蛋白质组，然后使用最先进的质谱仪(MS)进行定量分析。我们的创新方法获得了细胞表面糖蛋白质组的直接读数。该工作流程与高通量筛选/验证分析相结合，使用基于平行反应监测的质谱仪(PRM-MS)来监测潜伏感染细胞的其他复杂的表面糖蛋白组。因此，我们建议在体外产生的潜伏感染原代细胞模型中筛选细胞表面糖蛋白，以在R21期使用糖蛋白组学和PRM-MS来揭示潜伏相关的细胞表面糖蛋白，并使用R33期的抗体在体外靶向潜伏感染细胞。在R21阶段，我们将致力于在建立体外产生的潜伏感染原代细胞模型(目标1)的过程中发现糖蛋白质组的动态变化。潜伏期相关的糖蛋白将使用平行反应监测质谱学(PRM-MS)进行验证，这是一种靶向质谱学技术，可以验证大量改变的糖蛋白(目标2)。这种分析方法具有很高的创新性，能够从数千种细胞表面糖蛋白中发现特定的糖蛋白变化，以针对潜伏感染的细胞。只有在达到明确的里程碑时，才会进行R33阶段。我们提出了三项研究，以确定体外潜伏感染细胞表面特定糖蛋白变化的作用(目标3)。(1)将确定糖蛋白与潜伏感染的相关性，以揭示潜伏相关糖蛋白。(2)将使用糖蛋白特异性抗体捕获潜伏感染细胞。(3)将选择一组糖蛋白，以提高针对潜伏感染细胞的特异性和/或覆盖率。本研究的总体目标是确定特定的细胞表面糖蛋白(S)，使其能够靶向罕见的潜伏感染细胞x活体，而不需要重新激活HIV基因的表达。

 DESCRIPTION (provided by applicant): HIV latently infected cells are very rare in HIV+ individuals on suppressive anti-retroviral therapy (ART). To target these latently infected cells without latency-reactivation, it is critical to identify specific cell surface targets associated wth the latent infection. Compelling reports from studying proteins such as CD2, PD-1, LAG-3 and TIGIT suggest that there is a distinct profile of cell surface proteins for enrichment of latently infected cells ex vivo. The specific changes of these proteins during the establishment of latent infection remain to be elucidated. Interestingly, all of the above proteins are glycosylated (glycoproteins). It is estimated that there are over 1,000 cell surface glycoproteins that form a complex and dynamic cell surface phenotype. Interrogating the cell surface glycoproteome will maximize the chances to identify the specific cell surface glycoproteins to target latently infecte cells. Toward this goal, we have developed integrated glycoproteomics that can quantify the glycoproteome using chemical-enzymatic approaches for glycoprotein isolation followed by quantitative analysis using state-of-the-art mass spectrometry (MS). Our innovative method acquires a direct readout from the cell surface glycoproteome. The workflow is coupled with high throughput screen/validation assays using parallel reaction monitoring based mass spectrometry (PRM-MS) to monitor the otherwise complex surface glycoproteome of latently infected cells. We therefore propose to screen the cell surface glycoproteins in an in vitro-generated latently infected primary cell model to reveal latency- associated cell surface glycoproteins using glycoproteomics and PRM-MS in the R21 phase and target latently infected cells ex vivo using antibodies in the R33 phase. In the R21 phase, we will aim to discover the dynamic change of the glycoproteome during establishment of an in vitro-generated latently infected primary cell model (aim 1). The latency-associated glycoproteins will be validated using parallel reaction monitoring mass spectrometry (PRM-MS), a targeted mass spectrometry technique that can validate a large number of changed glycoproteins (aim 2). The analytical methods are highly innovative and have the capability to uncover the specific glycoprotein changes from thousands of cell surface glycoproteins for targeting latently infected cells. The R33 phase will be undertaken only if the well-defined milestones are achieved. We propose three studies to determine the roles of specific glycoprotein changes on latently infected cell surface ex vivo (aim 3). (1) The correlation of glycoproteins to latent infection will be determine to uncover latency-associated glycoproteins. (2) Glycoprotein-specific antibody will be used to capture latently infected cells. (3) A panel of glycoproteins will be selected to improve specificiy and/or coverage of targeting latently infected cells. The overall goal of this study is to determin the specific cell surface glycoprotein(s), which enable targeting the rare latently infected cells x vivo without the need to reactivate HIV gene expression.