Implementation of a qPCR-based assay for the quantification of SARS-CoV-2-specific T cells in immunocompromised patients

总结 免受病毒感染的长期保护是由体液和细胞免疫途径介导的。 多发性骨髓瘤（MM）是美国第二常见的血液恶性肿瘤， 通过克隆浆细胞的产生，导致免疫抑制和反复的细菌和病毒感染。 SARS-CoV-2特异性抗体定量目前被用作临床终点，以确定 针对COVID-19的免疫保护，这一信息在免疫功能低下的人中更为相关。 缺乏体液应答但受细胞免疫保护的个体（即MM患者）。 尽管迫切需要量化细胞免疫，但传统免疫学的复杂性和缺乏可扩展性， 检测抗原特异性T细胞的方法（例如ELISpot、流式细胞术）迄今为止阻碍了大规模研究。 为了解决这个问题，我们开发了一种基于直接qPCR的快速T细胞活化（dqTACT）测定， 用病毒肽（即免疫显性肽）库离体刺激全血样品 覆盖SARS-CoV-2刺突蛋白）， SARS-CoV-2抗原特异性T细胞和CXCL 10， 随后直接扩增IFNGβ或IL 2，其由 由单核细胞和中性粒细胞产生， , 响应 到 T细胞活化。 该提案的总体目标是开发和实施可用作替代的qPCR方法 以测量MM患者中抗原特异性T细胞的存在和功能。具体来说，我们假设 SARS-CoV-2特异性T细胞可能是既往感染和疫苗接种有效性的生物标志物 战略，补充量化的体液反应。 在UH 2阶段，我们将定义分析灵敏度和特异性，并建立临界值/阈值， 通过比较dqTACT检测试剂盒和 用于测量细胞免疫应答的金标准测定，例如流式细胞术和ELISpot。 在UH 3阶段，我们将测试在小鼠中对SARS-CoV-2的细胞免疫的存在和持久性。 恢复期和接种疫苗的骨髓瘤患者。注释良好的患者人群将用于定义 敏感性、特异性和对临床终点反应的阈值，例如， 对SARS-CoV-2的体液和细胞免疫。我们将估计疫苗接种者中标记物的流行率， 骨髓瘤患者。然后，我们将扩大我们的研究，并使用库存以及新鲜样本，从我们的大 在IRB批准的研究中招募的骨髓瘤/免疫功能低下人群和健康对照。 首要目标是使用dqTACT测定来测试由于自然感染或免疫缺陷而导致的T细胞的存在。 骨髓瘤患者接种疫苗，可能有助于全国范围的加强策略或被动抗体输注 支持保护这些弱势群体。 关键可交付成果是一种能够大规模快速量化T细胞免疫功能的检测方法。

SUMMARY Long-term protection from viral infections is mediated by both the humoral and cellular immune pathways. Multiple Myeloma (MM) is the second most common hematological malignancy in the US and is characterized by clonal plasma cell production, resulting in immune suppression and recurrent bacterial and viral infections. SARS-CoV-2-specific antibody quantification are currently being used as clinical endpoints to determine immune protection against COVID-19, and this information is even more relevant in immunocompromised individuals who lack a humoral response, but are protected by the cellular immunity (i.e. MM patients). Despite the urgent need to quantify cellular immunity, the complexity and lack of scalability of traditional methods (e.g. ELISpot, flow cytometry) to detect antigen-specific T cells has so far prevented large scale studies. To address this problem, we developed a direct qPCR-based rapid T cell activation (dqTACT) assay based on ex vivo stimulation of whole blood samples with a pool of viral peptides (i.e.immunodominant peptides covering SARS-CoV-2 Spike protein), SARS-CoV-2 antigen-specific T cells, and CXCL10, followed by direct amplification of IFNG 𝛾 or IL2 which are produced by which is produced by monocytes and neutrophils in , response to T cell activation. The overarching aim of this proposal is to develop and implement a qPCR method that can be used as a proxy to measure the presence and functionality of antigen specific T cells in MM patients. Specifically, we hypothesize that SARS-CoV-2 specific T cells might be a biomarker of previous infection and of efficacy of vaccination strategies, complementary to quantification of the humoral response. In the UH2 phase, we will define analytical sensitivity and specificity and establish cut-off/thresholds and appropriate positive and quality controls, accuracy and false result rate by comparing the dqTACT assay with the gold standard assays such as flow cytometry and ELISpot for measuring cellular immune responses. In the UH3 phase, we will test the presence and persistence of cellular immunity to SARS-CoV-2 in convalescent and vaccinated myeloma patients. Well annotated patient populations will be used to define sensitivity, specificity, and thresholds with response to clinical end-points, such as presence and persistence of humoral and cellular immunity to SARS-CoV-2. We will estimate the prevalence of the markers within vaccinated myeloma patients. We will then extend our studies and use banked as well as fresh samples from our large myeloma/immunocompromised population and healthy controls enrolled in IRB approved studies. The overarching goal is to use the dqTACT assay to test the presence of T cells due to natural infection or vaccination in myeloma patients, possibly aiding in nationwide booster strategies or passive antibody infusion support to protect these vulnerable population. Key deliverables an assay to rapidly quantify the functionality of T cellular immunity at scale.