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

实施基于 qPCR 的检测方法，对免疫功能低下患者的 SARS-CoV-2 特异性 T 细胞进行定量

• 批准号: 10580531
• 负责人: Ernesto Guccione
• 金额: $ 28.02万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-18 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10580531
• 关键词: 2019-nCoV; Address; Antibodies; Antibody Response; Bacterial Infections; Biological Assay; Biological Markers; Biotechnology; Blood; Blood specimen; Bypass; COVID-19; COVID-19 assay; COVID-19 complications; COVID-19 vaccination; CXCL10 gene; Cancer Patient; Cells; Cellular Immunity; Clinical; Cohort Studies; Data; Enrollment; Flow Cytometry; Goals; Hematologic Neoplasms; Hematology; Humoral Immunities; IL2 gene; Immune; Immune response; Immunity; Immunocompromised Host; Immunoglobulin G; Immunosuppression; Individual; Infection; Infusion procedures; Institutional Review Boards; Interferon Type II; Measures; Mediating; Methods; Modeling; Monitor; Multiple Myeloma; Nature; Oncology; Outcome; Pathway interactions; Patients; Peptides; Peripheral Blood Mononuclear Cell; Phase; Plasma Cells; Population; Prevalence; Production; Proxy; Publishing; Quality Control; RNA purification; Recommendation; Recurrence; Reporting; SARS-CoV-2 antigen; SARS-CoV-2 immunity; SARS-CoV-2 infection; SARS-CoV-2 spike protein; Sampling; Sensitivity and Specificity; Social Distance; Specificity; Steroids; T-Cell Activation; T-Lymphocyte; Testing; Time; Vaccinated; Vaccination; Vaccinee; Vaccines; Validation; Viral; Viral Antibodies; Virus Diseases; Vulnerable Populations; Whole Blood; Work; antigen detection; antigen-specific T cells; booster vaccine; cancer cell; chemotherapy; cohort; comorbidity; coronavirus disease; hypogammaglobulinemia; monocyte; neutralizing antibody; neutrophil; older patient; passive antibodies; patient population; prevent; prophylactic; response; targeted treatment; vaccination strategy

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.

概括 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，其特征是 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来确定 针对 COVID-19 的免疫保护，此信息在免疫功能低下的情况下更为相关 缺乏体液反应但受到细胞免疫保护的个体（即多发性骨髓瘤患者）。 尽管迫切需要量化细胞免疫，但传统方法的复杂性和缺乏可扩展性 迄今为止，检测抗原特异性 T 细胞的方法（例如 ELISpot、流式细胞术）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 细胞激活 (dqTACT) 检测方法。 用一组病毒肽（即免疫显性肽）对全血样本进行离体刺激 涵盖 SARS-CoV-2 刺突蛋白）， SARS-CoV-2 抗原特异性 T 细胞和 CXCL10， 然后直接扩增由以下物质产生的 IFNG 𝛾 或 IL2： 它是由单核细胞和中性粒细胞产生的 , 回复 到 T 细胞激活。 该提案的总体目标是开发和实施可用作代理的 qPCR 方法 测量 MM 患者中抗原特异性 T 细胞的存在和功能。 SARS-CoV-2 特异性 T 细胞可能是既往感染和疫苗接种效果的生物标志物 策略，补充体液反应的量化。 在 UH2 阶段，我们将定义分析灵敏度和特异性，并建立截止/阈值和 通过将 dqTACT 检测与 用于测量细胞免疫反应的金标准测定，例如流式细胞术和 ELISpot。 在 UH3 阶段，我们将测试 SARS-CoV-2 细胞免疫的存在和持续性 恢复期和疫苗接种骨髓瘤患者将用于定义患者群体。 敏感性、特异性和阈值与临床终点的反应，例如存在和持续性 我们将估计肺炎中标记物的流行率。 然后，我们将扩展我们的研究并使用我们大量的储存样本和新鲜样本。 骨髓瘤/免疫功能低下人群和健康对照参加 IRB 批准的研究。 总体目标是使用 dqTACT 检测来测试由于自然感染或 骨髓瘤患者接种疫苗，可能有助于全国范围内的加强策略或抗体被动输注 支持保护这些弱势群体。 关键交付成果是一种快速大规模量化 T 细胞免疫功能的检测方法。