Immune phenotyping of human immune responses to dengue vaccination and challenge

项目3：项目总结 登革病毒(DENV)是在热带和亚热带地区感染人类的最流行的蚊媒病毒 世界各地。流行的DENV有4个血清型，即DENV1、DENV2、DENV3和DENV4 人类。了解登革热疫苗的免疫保护和免疫致病反应 对登革病毒(DENV)感染的反应对安全有效的登革热的发展至关重要 疫苗。评估同型和异型免疫中的先天和获得性免疫反应 对于登革热疫苗接种和感染的反应，我们将分析从两个临床试验中获得的样本 评估登革热减毒活疫苗TV003(LATV)或缺失DENV-2的三价混合物 TV003(分别为CIR287和CIR300)的组件，抵御DENV-2挑战。TV003诱导100% 预防病毒血症和皮疹在DENV-2攻击后，三价制剂仅诱导20% 预防DENV-2病毒血症。使用来自上述疫苗试验的可用样本，这些样本完全是 在血清学上，我们将分析在这些细胞中产生的PBMC的细胞免疫反应 随着时间的推移，由临床核心(核心B)提供的个人。具有多参数免疫学和遗传学 免疫表型核心(核心C)和遗传核心(核心D)执行的方法，我们将介绍 对四价和三价疫苗制剂的先天和获得性免疫反应(目标1)和 挑战rDEN2DENV30，一种作为疫苗成分被丢弃的减毒活DENV-2(目标2)。另外， 我们将使用CORE B提供的人类扁桃体组织培养(HC)来分析诱导的免疫反应 通过不同的登革热疫苗混合物(目标3)。扁桃体有一个明确的空间结构和 细胞微环境，并且可以很容易地解离用于单细胞分析。它们还显示出重要的 了解先天和获得性免疫反应的产生，如生发的形成 中锋。这些事实使它们成为比较细胞悬液分析方法(如Aurora Flow)的理想选择 采用组织成像方法的细胞术，如空间转录学(核心D)。对这一问题的理解 不同疫苗配方中赋予免疫原性的成分以及不同疫苗的贡献 疫苗效力的血清型将对有效疫苗的开发至关重要。我们的方法将 提供有关对疫苗接种和感染产生免疫反应的重要信息以及 保护性疫苗诱导的免疫特征与数据确定的保护性较弱的疫苗 管理和分析核心(核心E)。外周血单核细胞和外周血中不同细胞群的分析 在扁桃体中，HC将扩展我们关于产生先天性和获得性免疫反应的知识，并将 允许可视化生发中心的形成和其他重要特征的产生 保护性免疫反应。在这个项目中组建的团队在DENV生物学方面拥有适当的专业知识， 免疫学和疫苗开发。

PROJECT 3: PROJECT SUMMARY Dengue virus (DENV) is the most prevalent mosquito–borne virus infecting humans in tropical and subtropical areas of the world. There are 4 DENV serotypes, namely DENV1, DENV2, DENV3 and DENV4 circulating in humans. Understanding the immunoprotective and immunopathogenic responses to dengue vaccines and responses to dengue virus (DENV) infection are critical to the development of a safe and effective dengue vaccine. To assess the innate and adaptive immune responses contributing to homo- and heterotypic immune responses to dengue vaccination and infection, we will analyze samples obtained from two clinical trials which evaluated the live attenuated dengue vaccine TV003 (LATV) or a trivalent admixture missing the DENV-2 component of TV003 (CIR287 and CIR300 respectively), against DENV-2 challenge. TV003 induced 100% protection against viremia and rash following DENV-2 challenge, the trivalent formulation induced only 20% protection against DENV-2 viremia. Using available samples from vaccine trials described above that are fully characterized serologically, we will analyze the cellular immune responses in PBMCs generated in those individuals over time, provided by the clinical core (Core B). With multiparametric immunological and genetic approaches performed by the immune phenotyping core (Core C) and genetic core (Core D), we will profile the innate and adaptive immune responses to tetra- and trivalent vaccine formulations (Aim 1) and to challenge with rDEN230, a live attenuated DENV-2 discarded as vaccine component (Aim 2). Additionally, we will use human tonsillar histocultures (HC) provided by core B to analyze the immune responses induced by different admixtures of the dengue vaccines (Aim 3). Tonsils have a well-defined spatial architecture and cellular microenvironments and can be readily dissociated for single cell assays. They also show important features to understand the generation of innate and adaptive immune responses, like the formation of germinal centers. These facts make them ideal for comparing cell suspension profiling assays such as Aurora flow cytometry with tissue imaging approaches like spatial transcriptomics (Core D). The understanding of the elements in the different vaccine formulations that confer immunogenicity and the contribution of the different serotypes to vaccine efficacy will be crucial for the development of efficacious vaccines. Our approach will provide important information on the generation of immune responses to vaccination and infections and the immune signatures induced by protective vaccines versus less protective ones as determined by the data management and analysis core (Core E). The analyses of the different cell populations present in PBMCs and in the tonsil HC will expand our knowledge on generation of innate and adaptive immune responses and will allow for the visualization of germinal center formation and other features important for the generation of protective immune responses. The team assembled in this project has the right expertise in DENV biology, immunology, and vaccine development.