Targeting Latently Infected Primary Cells using Integrated Glycoproteomics

 描述（由申请方提供）：在接受抑制性抗逆转录病毒治疗（ART）的HIV阳性个体中，HIV潜伏感染细胞非常罕见。为了靶向这些潜伏感染的细胞而没有潜伏再活化，关键是鉴定与潜伏感染相关的特异性细胞表面靶标。来自研究蛋白质如CD 2、PD-1、LAG-3和TIGIT的令人信服的报告表明，存在用于离体富集潜伏感染细胞的细胞表面蛋白质的独特谱。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，所有上述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。研究细胞表面糖蛋白组将最大限度地提高鉴定靶向潜伏感染细胞的特异性细胞表面糖蛋白的机会。为了实现这一目标，我们已经开发了整合的糖蛋白组学，可以量化的糖蛋白组使用化学-酶的方法，糖蛋白分离，然后使用国家的最先进的质谱（MS）的定量分析。我们的创新方法从细胞表面糖蛋白组获得直接读数。该工作流程与使用基于平行反应监测的质谱法（PRM-MS）的高通量筛选/验证测定相结合，以监测潜伏感染细胞的复杂表面糖蛋白组。因此，我们提出在体外产生的潜伏感染的原代细胞模型中筛选细胞表面糖蛋白，以在R21期中使用糖蛋白组学和PRM-MS揭示潜伏相关的细胞表面糖蛋白，并在R33期中使用抗体离体靶向潜伏感染的细胞。 在R21阶段，我们的目标是发现糖蛋白组在建立体外产生的潜伏感染原代细胞模型期间的动态变化（目的1）。将使用平行反应监测质谱法（PRM-MS）对潜伏相关糖蛋白进行验证，PRM-MS是一种靶向质谱技术，可验证大量变化的糖蛋白（目的2）。该分析方法具有高度创新性，能够从数千种细胞表面糖蛋白中发现特异性糖蛋白变化，以靶向潜伏感染细胞。 R33阶段只有在达到明确的里程碑后才能进行。我们提出了三项研究，以确定特定的糖蛋白的变化对潜伏感染细胞表面离体的作用（目的3）。(1)将确定糖蛋白与潜伏感染的相关性以揭示潜伏相关的糖蛋白。(2)糖蛋白特异性抗体将用于捕获潜伏感染的细胞。(3)选择一组糖蛋白以提高靶向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异性细胞表面糖蛋白，其能够靶向罕见的潜伏感染细胞，而不需要重新激活HIV基因表达。

 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.