Optimisation of SARS-CoV-2 peptide stimulation and measurement of cytokine output by intracellular flow cytometry and bio-Plex analysis

Abstract

Our study was conducted to optimise a peptide stimulation and an intracellular cytokine staining protocol, alongside Bio-Plex supernatant analysis, for use in patients who had previously contracted SARS-CoV-2 or received vaccination against this virus in a clinical laboratory setting. Peripheral Blood Mononuclear Cell extraction and cryopreservation allowed for cells to be stored long term and enhanced logistical processing of samples. Viability and functionality of cells were analysed by flow cytometric methodology using viability staining monoclonal antibodies conjugated to fluorochromes. Antibiotics and Benzonase Nuclease did not impact lymphocyte viability and so cell culture conditions were optimised in terms of retaining viability and functionality. Optimisation of peptide stimulation with Influenza and SARS-CoV-2 peptide pools was conducted through stimulation experiments assessing peptide concentration, peptide stimulation time and enrichment studies to increase precursor frequency. Cytokine output was measured by flow cytometry and Bio-Plex methodologies, with positive cytokine readings predominantly detected in the cell culture supernatant. Analysis of both intracellular and extracellular compartments allowed for detection of cytokines and established the retained cellular functionality post cryopreservation. These results also indicated that our peptide stimulation method can generate antigen-specific T lymphocytes upon exposure to SARS-CoV-2 peptide pools. Moreover, the measurement of specific cytokines could be applied to an array of conditions, such as chronic inflammatory diseases, but to also offer an alternative method of measuring vaccine responses. This platform is easily adaptable and can remain relevant alongside changing vaccine composition, thus ensuring its applicability to future vaccination programmes.