Optimization and validation of flow cytometry method for quantification of SARS-CoV-2 antigen-reactive human memory T cells

Abstract

A proper and representative monitoring of SARS-CoV-2 herd immunity including a long-term health impact on recovered patients and vaccinated individuals is of great importance. For this, a monitoring campaign should assesses both humoral and T-cell immune arms. Upon that, analyzing antigen specific-cell activation and cellular phenotype are informative. We developed a flow cytometry method for detection of intracellular IFN-producing antigen-reactive T cells after exposure of human peripheral blood mononuclear cells (PBMC) to SARS-CoV-2 virus antigens. The method was validated according to the following characteristics: sensitivity, specificity, precision, and robustness. We used positive samples from donors recovered from COVID-19 and negative samples from donors who had no contact with COVID-19 patients and lacking antibodies to SARS-CoV-2. All samples were tested by laboratory methods. Peripheral blood mononuclear cells were isolated from donor blood by centrifugation in a Ficoll density gradient. Specific T cells were stimulated with S-protein as well as N, M, ORF3a, and ORF7a protein peptides to count IFN-producing T cells by flow cytometer. The data were statistically analyzed. The area limited by ROC-curve and false positive rate (AUC) for CD4 and CD8 cells was from 0.97 to 1.00. Precision was considered acceptable because the coefficient of variation for all PBCM did not exceed 20%. Robustness was confirmed for frozen and freshly prepared PBMC samples. The thresholds levels to recognize immune and non-immune samples were defined for CD4-positive T-cells (0.029%) and CD8-positive T-cells (0.0640.068%). Also, acceptance criteria for positive and negative controls were defined. Based on the validation, the suitability of the method Evaluation of antigen-reactive T cells that produce intracellular IFN in response to SARS-CoV-2 virus antigens by flow cytometry was confirmed. The method allows for reliable data that was used to characterize standard control samples for internal quality control of TigraTest SARS-CoV-2 kits.