Development of Highly Multiplex Antigen Specificity Assays

高度多重抗原特异性检测的开发

基本信息

批准号：
9271150
负责人：
STEPHEN J ELLEDGE
金额：
$ 39.14万
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：
2015-06-22 至 2020-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9271150
关键词：
2-Amino-1-Methyl-6-Phenylimidazo[4,5-b]pyridine Acute Address Alpha Cell Antibodies Antibody Repertoire Antigen-Presenting Cells Antigens Apoptosis Autoantigens Autoimmunity Autologous B-Lymphocytes Bacteriophages Benchmarking Biocompatible Materials Biological Assay Blood CD4 Positive T Lymphocytes CD8-Positive T-Lymphocytes CD8B1 gene Cells Complex Cytotoxic T-Lymphocytes DNA sequencing Data Set Databases Development Ensure Epidemiology Foundations Goals Growth Helper-Inducer T-Lymphocyte High-Throughput DNA Sequencing Human Immune Targeting Immune response Immune system Immunoassay Immunodominant Epitopes Immunologic Tests Immunoprecipitation Individual Infection Libraries Measurement Methods Molecular Chaperones Patients Peptides Performance Phenotype Positioning Attribute Procedures Process Proteins Proteome Publications Research Personnel Sampling Serologic tests Serological Serum Signal Transduction Solid Specificity Synthetic Antigens System T cell response Technology Testing Time Viral Viral Antibodies Virus Virus Diseases Work base cancer immunotherapy clinically actionable cost experimental study human virome interest invariant chain new technology next generation novel peptide I public health relevance response screening synthetic biology vaccine development

项目摘要

DESCRIPTION (provided by applicant): There is an increasing unmet need for immunoassays that yield a maximal amount of information from a minimal amount of patient sample. One way to address this challenge is to develop very highly multiplexed, sample sparing assays. This proposal presents new methods that integrate synthetic biology and next generation DNA sequencing ("NGS") to characterize the specificities of humoral and cellular immune responses using genetically encoded antigen libraries. The multi-PI project builds upon a solid foundation, which has been established in recent years, for developing highly multiplex, sample sparing immunoassays. The platforms described in this proposal will be developed and tested on a new antigen library that encompasses the proteomes of all viruses known to infect humans ("the human virome"). Proof-of-concept studies confirm both the quality of this library, and its utility or developing sample sparing, highly multiplex antigen specificity assays. The following three Specific Aims have been developed to broadly analyze human immune responses to viruses, using an absolute minimal amount of sample. Specific Aim 1. Development of minimal human virome serologic assays Two novel sample sparing serologic assays are proposed: 1) a 384-well, simplified bacteriophage-NGS based assay, and 2) a rapid cytometric Luminex bead array assay. These technologies will comprehensively characterize anti-viral antibodies at low cost, and require less than a single microliter of blood. Specific Aim 2. Development of a minimal antigen library screening assay for cytotoxic T lymphocytes A new platform for profiling CD8+ cytotoxic T lymphocyte (CTL) specificities has been devised. The system employs lentiviral delivery of a genetically encoded antigen library to present MHC I-peptide complexes on autologous antigen presenting cells, followed by phenotypic library enrichment and NGS analysis. Specific Aim 3. Development of a minimal antigen library screening assay for T helper cells A new platform for profiling CD4+ T helper (Th) cell specificities has been devised. The system employs lentiviral delivery of a genetically encoded antigen library to present MHC II-peptide complexes on autologous antigen presenting cells, followed by phenotypic library enrichment and NGS analysis.

描述（由申请人提供）：对于从最少量的患者样本中产生最大量的信息的免疫测定的需求不断增加，解决这一挑战的一种方法是开发高度多重的、样品节省的测定。整合合成生物学和下一代 DNA 测序 (NGS) 的新方法，利用基因编码的抗原库来表征体液和细胞免疫反应的特异性。多 PI 项目建立在已经建立的坚实基础之上。近年来，针对高度多重、节省样品的免疫测定，将在新的抗原库上开发和测试，该抗原库包含已知感染人类的所有病毒的蛋白质组（“正在发育的人类病毒组”）。概念研究证实了该文库的质量及其实用性或开发样本保留、高度多重抗原特异性测定，已开发出以下三个特定目标，以使用绝对最小量广泛分析人类对病毒的免疫反应。具体目标 1. 开发最少的人类病毒组血清学测定法提出了两种新颖的样品保留血清学测定法：1) 384 孔、基于简化噬菌体 NGS 的测定法，以及 2) 快速细胞计数 Luminex 珠阵列测定法。以低成本表征抗病毒抗体，并且需要不到一微升的血液。具体目标 2. 开发细胞毒性 T 的最小抗原库筛选测定。设计了一种用于分析 CD8+ 细胞毒性 T 淋巴细胞 (CTL) 特异性的新平台，该系统采用慢病毒传递基因编码抗原库，在自体抗原呈递细胞上呈递 MHC I 肽复合物，然后进行表型库富集和 NGS 分析。具体目标 3. 开发用于 T 辅助细胞筛选的最小抗原库一种用于分析 CD4+ T 辅助 (Th) 细胞特异性的新平台测定方法已问世。该系统采用慢病毒传递基因编码的抗原库，在自体抗原呈递细胞上呈递 MHC II 肽复合物，然后进行表型库富集和 NGS 分析。