T cell responses
Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood by density gradient separation then frozen in liquid nitrogen until use. Thawed PBMCs were rested for 2 hours in 10% human AB serum supplemented RPMI medium. Next, the cells were stimulated with sterile ddH2O or 1 µg/mL overlapping peptide pools representing the WT SARS-CoV-2 S, N and M proteins (Miltenyi Biotec, Bergisch Gladbach, Germany) for 16 hours in the presence of 1 µg/mL anti-CD28 and anti-CD49d costimulatory antibodies (clones CD28.2 and 9F10, Biolegend, San Diego, USA). After 2 hours of stimulation, 10 µg/mL brefeldin A (Sigma, Kawasaki, Japan) was added.40 The cells were then washed and subjected to immunostaining using a fixable viability dye (eBioscience, Santa Clara, USA, 1:60) and antibodies against CD3 (HIT3a, 1:60), CD4 (OKT4, 1:60), CD8 (HIT8a, 1:60), IFN-γ (B27, 1:15) and IL-2 (MQ1-17H12, 1:15) antibodies (Biolegend, San Diego, USA). Data acquisition was carried out using flow cytometry (LSR II; BD Biosciences, Franklin Lakes, USA) and analyzed by Flowjo v10 software (BD, Ashland, USA). The antigen-specific IFN-γ+ or IL-2+ T cells were calculated by subtracting the background (sterile ddH2O) data, and presented as the percentage of CD4+ or CD8+ T cells.41 T cell response against a single peptide pool was considered positive when the frequency of cytokine-expressing cells was higher than or equal to 0.005% and the stimulation index was higher than 2; negative values were imputed as 0.0025%. Total T cell responses against S, N and M peptide pools were also added together, with a cut-off of 0.01%.