Rapid, scalable assessment of SARS-CoV-2 cellular immunity by whole-blood PCR

Fast, high-throughput methods for measuring the level and duration of protective immune responses to SARS-CoV-2 are needed to anticipate the risk of breakthrough infections. Here we report the development of two quantitative PCR assays for SARS-CoV-2- specific T cell activation. The assays are rapid, internally normalized and probe-based: qTACT requires RNA extraction and dqTACT avoids sample preparation steps. Both assays rely on the quantification of CXCL10 messenger RNA, a chemokine whose expression is strongly correlated with activation of antigen-specific T cells. On restimulation of whole-blood cells with SARS-CoV-2 viral antigens, viral-specific T cells secrete IFN-γ, which stimulate monocytes to produce CXCL10. CXCL10 mRNA can thus serve as a proxy to quantify cellular immunity. Our assays may allow large-scale monitoring of the magnitude and duration of functional T cell immunity to SARS-CoV-2, thus helping to prioritize revaccination strategies in vulnerable populations. Total RNA was extracted from whole blood after overnight stimulation with a SARS-CoV-2 spike peptide pool (SpG) or DMSO control. The cohort consists of 11 naïve donors, 8 COVID-19 convalescent donors, and 16 vaccinated donors.

为预判突破性感染风险，目前亟需快速、高通量的检测方法，以评估机体针对严重急性呼吸综合征冠状病毒2（SARS-CoV-2）的保护性免疫应答水平与持续时长。本研究开发了两种用于检测SARS-CoV-2特异性T细胞活化的定量PCR（qPCR）检测方法。该两类检测方法具有快速、内归一化及基于探针的特点：qTACT需进行RNA提取，而dqTACT则无需样本制备步骤。两类检测方法均基于CXCL10信使RNA（mRNA）的定量检测——CXCL10是一种趋化因子，其表达水平与抗原特异性T细胞的活化程度显著相关。当使用SARS-CoV-2病毒抗原对全血细胞进行再刺激时，病毒特异性T细胞会分泌干扰素γ（IFN-γ），该细胞因子可刺激单核细胞产生CXCL10。因此，CXCL10 mRNA可作为替代标志物，用于量化机体的细胞免疫水平。本研究开发的检测方法可实现对SARS-CoV-2功能性T细胞免疫的强度与持续时长的大规模监测，从而有助于为脆弱人群优先制定加强免疫策略。实验过程中，将全血分别用SARS-CoV-2刺突肽库（SpG）或二甲基亚砜（DMSO）对照进行过夜刺激后，提取总RNA。该研究队列包含11名未感染过新冠且未接种疫苗的供者、8名新冠康复供者以及16名新冠疫苗接种供者。