Immune phenotyping of human immune responses to dengue vaccination and challenge

项目3：项目概要 登革病毒（DENV）是热带和亚热带地区最常见的蚊媒病毒 世界上的一些地区。有4种DENV血清型，即DENV 1、DENV 2、DENV 3和DENV 4在欧洲流行。 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）感染的反应对于开发安全有效的登革疫苗至关重要。 疫苗评估先天性和适应性免疫应答对同型和异型免疫的影响， 对登革热疫苗接种和感染的反应，我们将分析从两项临床试验中获得的样本， 评估活减毒登革热疫苗TV 003（LATV）或缺失DENV-2的三价混合物 TV 003的组分（分别为CIR 287和CIR 300）对抗DENV-2攻击。TV 003诱导100% 针对DENV-2攻击后的病毒血症和皮疹的保护，三价制剂仅诱导了20%的 针对DENV-2病毒血症的保护。使用上述疫苗试验中完全可用的样本 特征的血清学，我们将分析PBMC中产生的细胞免疫反应， 由临床核心（核心B）提供。多参数免疫和遗传 通过免疫表型核心（核心C）和遗传核心（核心D）进行的方法，我们将分析 对四价和三价疫苗制剂的先天性和适应性免疫应答（Aim 1）以及对 用rDEN 2攻击，rDEN 2是作为疫苗组分丢弃的减毒活DENV-2（目的2）。此外，本发明还 我们将使用由核心B提供的人扁桃体组织培养物（HC）来分析诱导的免疫应答 通过不同的登革热疫苗混合物（目标3）。扁桃体具有明确的空间结构， 细胞微环境中，并且可以容易地解离用于单细胞测定。它们也显示出重要的 了解先天性和适应性免疫反应的产生，如germinal的形成 中心.这些事实使它们成为比较细胞悬浮液分析测定法（如Aurora flow）的理想选择。 细胞计数与组织成像方法如空间转录组学（Core D）。的理解 不同疫苗制剂中赋予免疫原性的元素以及不同免疫原性的贡献 血清型与疫苗效力的关系对于开发有效的疫苗至关重要。我们的方法将 提供关于疫苗接种和感染产生免疫反应的重要信息， 由保护性疫苗诱导的免疫特征与由数据确定的保护性较低的疫苗相比 管理和分析核心（Core E）。存在于PBMC中的不同细胞群的分析和 在扁桃体HC将扩大我们的知识产生的先天性和适应性免疫反应， 允许对生发中心的形成和其他对产生 保护性免疫反应这个项目中的团队在DENV生物学方面拥有正确的专业知识， 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.