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

以防止突破性感染。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贡献者，但强大的T细胞免疫可能是短暂的或无效的，导致突破性感染。我们提供了两个独特的和完善的队列，具有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;一组接种了辉瑞公司SARS-Cov-2 mRNA疫苗的医护人员（PITCH研究）和一组接受单倍相合干细胞移植（CD 45 RA耗竭的回加）的原发性免疫缺陷儿童。（云母研究）.目的建立一种单细胞方法来研究突破性感染过程中抗病毒免疫应答的表型和特异性.研究抗原特异性记忆T细胞在对新出现和循环病毒病原体的保护性免疫中的作用.新奇/及时性该项目是新颖的，因为它旨在更好地了解批准的新型免疫干预背后的保护机制（CD 45 RA加回）和新的疫苗接种策略（Pfizer mRNA疫苗）。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。更深入地了解这些干预措施背后的表型和T细胞机制将是至关重要的，为未来的SARS-Cov-2疫苗接种策略和CD 45 RA-addback免疫疗法的未来迭代提供信息。将在一段时间内回顾性分析从这些已建立的人类队列的外周血中分离的PBMC，以观察突破性感染前后的抗病毒T细胞应答。基于HLA分型和病毒免疫显性模式的抗原特异性四聚体将用于鉴定病毒特异性应答。内部建立的基于R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。为了验证抗原特异性记忆T细胞在保护性免疫中发挥关键作用的假设，学生将体外创建抗原特异性基于患者的原代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体（SARS-Cov-2）的肽反应性。学生还将评估功能性亲和力，多功能性和T细胞杀伤。这些数据将与SIREN/PITCH队列和云母研究中常规收集的医护人员综合临床数据一起进行统计学评估。

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.