Development of Highly Multiplex Antigen Specificity Assays

 描述（由申请人提供）：对于从最少量的患者样本中获得最大信息量的免疫测定，存在越来越多的未满足的需求。解决这一挑战的一种方法是开发高度多重化的样品保留测定法。该提案提出了整合合成生物学和下一代DNA测序（“NGS”）以使用遗传编码的抗原文库表征体液和细胞免疫应答的特异性的新方法。多PI项目建立在近年来建立的坚实基础上，用于开发高度多重，样品保留的免疫测定。本提案中描述的平台将在一个新的抗原库上开发和测试，该抗原库包括已知感染人类的所有病毒的蛋白质组（“人类病毒组”）。概念验证研究证实了该文库的质量及其在开发样品保留、高度多重抗原特异性测定中的实用性。开发了以下三个特定目的，以使用绝对最小量的样品广泛分析人类对病毒的免疫反应。具体目标1。最小人类病毒组血清学测定的开发提出了两种新的样品保留血清学测定：1）384孔、简化的基于噬菌体-NGS的测定，和2）快速细胞计数Luminex珠阵列测定。这些技术将以低成本全面表征抗病毒抗体，并且需要不到一微升的血液。具体目标2。细胞毒性T淋巴细胞最小抗原库筛选方法的建立一种新的分析CD 8+细胞毒性T淋巴细胞（CTL）特异性的平台已经被设计出来。该系统采用遗传编码的抗原文库的慢病毒递送以在自体抗原呈递细胞上呈递MHC I-肽复合物，随后进行表型文库富集和NGS分析。具体目标3。辅助性T细胞最小抗原库筛选方法的建立设计了一种新的分析CD 4+辅助性T细胞（Th）特异性的平台。该系统采用基因编码的抗原文库的慢病毒递送，以在自体抗原呈递细胞上呈递MHC II-肽复合物，然后进行表型文库富集和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.