Exploring anti-viral T cell responses using single cell technologies to determine effective, durable anti-viral immunity for strategies

为了防止突破性感染。背景病毒感染是一个主要的健康挑战。虽然病毒抗原特异性T细胞是控制和调节抗病毒免疫的主要因素，但强大的T细胞免疫可能是短暂的或无效的，从而导致突破性感染。我们提供两个独特且成熟的队列，使用不同的临床批准的抗病毒干预措施，以研究抗病毒T细胞在预防突破性病毒感染中的作用；一组医务人员接种了针对SARS-CoV-2的辉瑞基因疫苗(Pitch研究)，以及一组接受了CD45RA缺失加回半相合干细胞移植的原发免疫缺陷儿童(MICA研究)。目的发展一种单细胞方法来表征突破性感染期间抗病毒免疫反应的表型和特异性。探讨抗原特异性记忆T细胞在对新出现和循环的病毒病原体的保护性免疫中的作用。在这两个队列中，内部先前的分析表明，T细胞发挥了关键作用，但抗病毒免疫力的减弱，以及反复和新出现的病毒暴露对这些干预措施的持续成功构成了挑战。深入了解这些干预措施背后的表型和T细胞机制将对指导未来的SARS-CoV-2疫苗接种策略和CD45RA-addback免疫疗法的未来迭代至关重要。实验方法：学生将使用BD Aurora和CITE-SEQ进行单细胞高维表型鉴定。从这些已建立的人类队列的外周血液中分离出的PBMC，将在一段时间内进行回顾性分析，以表征突破性感染前后的抗病毒T细胞反应。基于人类白细胞抗原分型和病毒免疫优势模式的抗原特异性四聚体将被用于识别病毒特异性反应。内部建立的以R为基础的计算管道、高维数据探索和可视化技术，如FLOWSOM和tSNE，将用于识别和表征细胞群体。为了验证抗原特异性记忆T细胞在保护性免疫中起关键作用的假设，学生将在体外建立抗原特异性患者原代T细胞系。为了评估保护机制，学生将使用基于流动的方法测试原代T细胞系对新出现的关注变种(SARS-CoV-2)的多肽反应性。学生还将评估功能亲和力、多功能性和T细胞杀伤。这些数据将与常规收集的关于SIREN/PING队列中的医护人员的全面临床数据以及MICA研究一起进行统计评估。

to prevent break-through infection.BackgroundViral infections are a major health challenge. Whilst viral antigen-specific T-cells are a major contributor in controlling and regulating anti-viral immunity, robust T cell immunity can be short-lived or ineffective leading to breakthrough infection. We offer two unique and well established cohorts, with different clinically-approved anti-viral interventions, to study the role of anti-viral T-cells in preventing breakthrough viral infection; a cohort of health care workers vaccinated with Pfizer mRNA vaccine to SARS-Cov-2 (PITCH study) and a cohort of children with primary immunodeficiency undergoing haplo identical stem-cell transplant with CD45RA-depleted add-back (MICA study).ObjectivesTo develop a single-cell approach to characterise the phenotype and specificity of anti-viral immune responses during breakthrough infection.To investigate the role of antigen-specific memory T cells in protective immunity to emerging and circulating viral pathogens.Novelty/TimelinessThe project is novel in that it aims to understand better the protective mechanisms behind an approved novel immunotherapeutic intervention (CD45RA addback), and novel vaccination strategy (Pfizer mRNA vaccine). In both cohorts, T-cells have been indicated by in-house previous analysis to play a key role however waning of anti-viral immunity, and repeated and emerging viral exposure pose a challenge to the ongoing success of these interventions. A deeper understanding of the phenotype and T-cell mechanisms behind these interventions will be critical in informing future SARS-Cov-2 vaccination strategies and future iterations of CD45RA-addback immunotherapy.Experimental ApproachThe student will use BD Aurora and CITE-seq for single-cell high-dimensional phenotyping. PBMCs isolated from peripheral blood from these established human cohorts, will be analysed retrospectively over a timecourse to characterise anti-viral T cell responses before and after breakthrough infection. Antigen-specific tetramers based on HLA typing and viral immunodominance patterns will be used to identify viral-specific responses. In-house established R -based computational pipelines, high-dimension data exploration and visualization techniques, such as FLOWSOM and tSNE, will be used to identify and characterize cell populations. To test the hypothesis that antigen-specific memory T-cells play a key role in protective immunity, the student will create antigen-specific patient-based primary T cell lines ex vivo. To assess mechanisms of protection, the student will test primary T cell lines for peptide responsiveness to emerging variants of concern (SARS-Cov-2) using by flow based methods. The student will also assess functional avidity, polyfunctionality and T cell killing. These data will be statistically assessed alongside comprehensive clinical data collected routinely on healthcare workers in the SIREN/PITCH cohort, and MICA study.