Mabs 1 d1k

96-well FluoroSpot plates were coated with anti-cytokine antibodies for IFNγ and IL-5 (mAbs 1-D1K and TRFK5, respectively; Mabtech, Stockholm, Sweden) at a concentration of 10 μg/mL. PBMCs were stimulated in triplicate at a density of 200x10³ cells/well with S MPs corresponding to each of the SARS-CoV-2 variants analyzed (1 μg/mL), PHA (1 μg/mL), and DMSO (0.1%), as positive and negative controls respectively. After 20 hours of incubation at 37°C, 5% CO₂, cells were discarded and plates were washed before the addition of cytokine antibodies (mAbs 7-B6-1-BAM and 5A10-WASP; Mabtech, Stockholm, Sweden). After a 2-hour incubation, plates were washed again with PBS/0.05% Tween20 and incubated for 1 hour with fluorophore-conjugated antibodies (Anti-BAM-490 and Anti-WASP-640). An AID iSPOT FluoroSpot reader (AIS-diagnostika, Germany) was used to count the fluorescent spots that resulted from cells secreting IFNγ and IL-5. Each peptide MP was considered positive compared to the DMSO negative control based on the following criteria: 20 or more spot forming cells (SFC) per 10⁶ PBMC after background subtraction, a stimulation index (S.I.) greater than 2, and a p value < 0.05 by either a Poisson or t test calculated between the triplicates of the MP and the relative negative control.

96-well FluoroSpot plates were coated with anti-cytokine antibodies for IFNγ and IL-5 (mAbs 1-D1K and TRFK5, respectively; Mabtech, Stockholm, Sweden) at a concentration of 10μg/mL. PBMCs were stimulated in triplicate at a density of 200×10³ cells/well with S MPs corresponding to each of the SAR-SCoV-2 variants analyzed (1μg/mL), PHA (1μg/mL), and DMSO (0.1%), as positive and negative controls respectively. After 20 hours of incubation at 37°C, 5% CO₂, cells were discarded and plates were washed before the addition of cytokine antibodies (mAbs 7-B6–1-BAM and 5A10-WASP; Mabtech, Stockholm, Sweden). After a 2-hour incubation, plates were washed again with PBS/0.05% Tween20 and incubated for 1 hour with fluorophore-conjugated antibodies (Anti-BAM-490 and Anti-WASP-640). An AID iSPOT FluoroSpot reader (AIS-diagnostika, Germany) was used to count the fluorescent spots that resulted from cells secreting IFNγ and IL-5. Each peptide MP was considered positive compared to the DMSO negative control based on the following criteria: 20 or more spot forming cells (SFC) per 10⁶ PBMC after subtraction, a stimulation index (S.I.) greater than 2, and a p value <0.05 by either a Poisson or T test calculated between the triplicates of the MP and the relative negative control.