Immune Phenotyping Core

摘要 免疫表型核心（核心C）的目标是为不同项目提供工具和试剂 将用于表征疫苗接种和感染引起的免疫反应 冠状病毒、流感病毒和登革热病毒。核心将利用现有的最先进技术 Krammer 实验室建立的血清学技术，以及细胞因子和细胞因子的多重分析 PBMC 和人扁桃体的趋化因子血浆谱和 Cytek Aurora 光谱流式细胞术谱 组织培养 (HC) 目前在 Fernandez-Sesma 实验室使用和优化。以下目标是 建议：目标 1：表征针对冠状病毒、流感病毒和登革热的抗体反应 病毒疫苗接种和感染。核心将提供检测、试剂和方案来测量结合 项目 1 的针对 SARS-CoV-2 的功能性抗体反应和项目 2 的针对流感病毒的功能性抗体反应。 此外，经处理的人扁桃体组织培养物 (HC) 上清液中 IgM、IgG 和 IgA 的分泌 与不同的 SARS-CoV-2 疫苗类型（项目 1）相比，流感病毒疫苗和病毒（项目 2）将 进行评估。目标 2：表征细胞因子/趋化因子对冠状病毒、流感病毒的反应 以及登革热病毒疫苗接种和感染。 Core 将分析细胞因子和趋化因子的水平 在接种疫苗/感染个体的血浆中以及经处理的人扁桃体组织培养物的上清液中 项目 1、2 和 3 的不同疫苗。目标 3：表征细胞对冠状病毒的反应， 流感病毒和登革热病毒的疫苗接种和感染。细胞免疫特征分析 使用光谱流式细胞术检测一段时间内接种疫苗/感染个体的 PBMC。人类扁桃体 HC 将 还可以通过 Cytek Aurora 光谱流式细胞术进行分析，以捕获早期免疫特征和 与这些 HC 治疗后适应性免疫反应相对应的细胞群变化 不同的疫苗。我们将获得单细胞水平的高分辨率数据来解决最具挑战性的问题 细胞群，包括表达病毒抗原的细胞。 PBMC 也将受到补充 Genomics Core 通过 RNAseq 进行转录组学分析。这些工具将用于生成 代表对疫苗接种和/或感染的纵向免疫反应的免疫特征 研究参与者根据数据参加了观察性非干预性队列研究 管理和分析核心（核心 E）。使用这些免疫学技术获得的数据将 由数据管理和分析核心进行分析，比较它们在不同系统中使用的情况 这些项目以及与 Genomics Core（Core D）中获得的基因组数据相结合 相同的样品。 VIVA 项目和核心生成的所有数据，包括免疫表型 Core，将由数据分析核心存入ImmPort。

SUMMARY The goal of the Immune Phenotyping Core (Core C) is to provide tools and reagents to the different projects that will be used to characterize the immune responses induced by vaccinations and infections with coronaviruses, influenza viruses and dengue viruses. The Core will leverage existing state-of-the-art serological techniques established in the Krammer laboratory, as well as multiplex analysis of cytokine and chemokine plasma profile and Cytek Aurora Spectral Flow Cytometry profiling of PBMCs and human tonsillar histocultures (HC) currently used and optimized in the Fernandez-Sesma laboratory. The following aims re proposed: Aim 1: Characterization of antibody responses to coronavirus, influenza virus and dengue virus vaccination and infection. The Core will provide assays, reagents and protocols to measure binding and functional antibody responses against SARS-CoV-2 for Project 1 and influenza viruses for Project 2. Additionally, the secretion of IgM, IgG and IgA in the supernatant of human tonsil histocultures (HC) treated with the different SARS-CoV-2 vaccines types (Project 1), influenza virus vaccines and viruses (Project 2) will be assessed. Aim 2: Characterization of cytokine/chemokine responses to coronavirus, influenza virus and dengue virus vaccination and infection. The Core will analyze the levels of cytokines and chemokines in the plasma of vaccinated/infected individuals and the supernatant of human tonsil histocultures treated with different vaccines for Projects 1, 2 and 3. Aim 3: Characterization of cellular responses to coronavirus, influenza virus and dengue virus vaccination and infection. Analysis of the cellular immune profiles of PBMCs from vaccinated/infected individuals over time, using Spectral Flow cytometry. Human tonsillar HC will be also analyzed by Cytek Aurora Spectral Flow Cytometry in order to capture early immune signatures and changes in cell populations corresponding to adaptive immune responses in those HC after treatment with different vaccines. We will obtain high-resolution data at the single-cell level to resolve the most challenging cell populations including cells expressing viral antigens. PBMCs will also be subjected to a complementary transcriptomics analysis by RNAseq conducted by the Genomics Core. These tools will serve to generate immune signatures representative of the longitudinal immune responses to vaccination and/or infection in study participants enrolled in observational non-interventional cohort studies in coordination with the Data Management and Analysis Core (Core E). Data obtained using these immunological techniques will be analyzed by the Data management and Analysis Core comparing them across the different systems used in the projects as well as in combination with the genomic data obtained in the Genomics Core (Core D) from the same samples. All data generated by the VIVA Projects and Cores, including the Immune Phenotyping Core, will be deposited by the Data Analysis Core into ImmPort.