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Targeting Latently Infected Primary Cells using Integrated Glycoproteomics

使用集成糖蛋白组学靶向潜在感染的原代细胞

基本信息

批准号：
9050031
负责人：
Hui Zhang
金额：
$ 20.25万
依托单位：
JOHNS HOPKINS UNIVERSITY
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：
2015-12-01 至 2017-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9050031
关键词：
Anti-Retroviral Agents Antibodies Biological Assay Biotechnology CD4 Positive T Lymphocytes Cell Surface Proteins Cell model Cell surface Cells Chemicals Complex Coupled DNA Flow Cytometry Gene Expression Glycoproteins Goals HIV HIV Infections HIV-1 Half-Life Immune response In Vitro Individual Joints Life Cycle Stages Mass Spectrum Analysis Membrane Glycoproteins Methods Modeling Monitor Nature Patients Phase Phenotype Proteins Proteomics Protocols documentation Proviruses Publications Reaction Reporting Rest Role Shock Specificity Surface T-Lymphocyte Techniques Technology Testing Therapeutic Validation Viral Viral Cytopathogenic Effect Viral reservoir Virus Work analytical method antiretroviral therapy base cell transformation genetic approach glycoproteomics high throughput screening improved innovation killings latent infection memory CD4 T lymphocyte public health relevance reactivation from latency statistics

项目摘要

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.