Development of Highly Multiplex Antigen Specificity Assays

 描述(由申请人提供)：从最少量的患者样本中产生最大信息量的免疫分析的需求日益增长。解决这一挑战的一种方法是开发高度多元化的样品节约分析方法。这项建议提出了一种新的方法，它结合了合成生物学和下一代DNA测序(“NGS”)，以利用遗传编码的抗原库来表征体液和细胞免疫反应的特异性。多PI项目建立在最近几年建立的坚实基础上，用于开发高度多元化、节省样本的免疫分析。这项提议中描述的平台将在一个新的抗原库上开发和测试，该抗原库包含所有已知感染人类的病毒(“人类病毒体”)的蛋白质组。概念验证研究既证实了该文库的质量，也证实了它的实用性或开发了样本保留、高度多重的抗原特异性分析。为了广泛分析人类对病毒的免疫反应，已经开发了以下三个具体目标，使用绝对最少的样本量。具体目的1.微量人病毒血清学检测方法的发展提出了两种新的节省样本的血清学检测方法：1)基于384孔、简化的噬菌体-NGS的检测方法；2)快速细胞学Luminex微珠阵列检测方法。这些技术将以低成本全面鉴定抗病毒抗体，并且只需要不到一微升的血液。特定目的2.细胞毒性T淋巴细胞最小抗原库筛选试验的建立一个新的分析CD8细胞毒性T淋巴细胞(CTL)特异性的平台已经被设计出来。该系统采用慢病毒传递基因编码的抗原库，在自体抗原提呈细胞上呈现MHC I-肽复合体，然后进行表型文库浓缩和NGS分析。特定目的3.辅助性T细胞最小抗原库筛选试验的建立一种新的分析CD4T辅助性(Th)细胞特性的平台已经设计出来。该系统采用慢病毒传递基因编码的抗原库，在自体抗原提呈细胞上呈现MHC II-肽复合体，然后进行表型文库浓缩和NGS分析。

 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.