Pre coated 96 well plates

Precoated 96-well plates were purchased from Mabtech and the assay was carried out according to the manufacturer’s instructions. Briefly, plates were washed with sterile PBS (Thermo Fisher Scientific) and blocked with R10F for at least 1 h. Then, 2.5 × 10⁵ (SARS-CoV-2 and influenza) or 2.5 × 10⁴ (CD3, positive control) PBMCs per well were stimulated in triplicates with overlapping peptide pools at a concentration of 0.7 µg ml⁻¹ for SARS-CoV-2 S1, S2 and nucleocapsid or 0.4 µg ml⁻¹ for membrane per individual peptide. Influenza vaccine (season 2020–2021; 1 µg ml⁻¹ per hemagglutinin; Vaxigrip Tetra Sanofi) and anti-CD3 antibody (1:1,000; Mabtech) were used as controls. In some cases (limited number of PBMCs), the ELISpot was run in duplicate. Negative control comprised equimolar amounts of DMSO. Plates were incubated for 20 h, plates developed and spots counted using the ImmunoSpot S6 Ultimate Reader equipped with the ImmunoSpot Software. The mean spot counts for the DMSO negative control were subtracted from the mean of the SARS-CoV-2 and influenza or CD3-stimulated cells. Data are displayed as SFCs per million of PBMCs. The cutoff response in the test cohort was determined using the mean from all individuals in the DMSO negative control + 2 s.d. A response >15 SFCs per 10⁶ PBMCs was considered positive.

IFN-γ responses were measured in pre-coated 96-well plates (Mabtech AB, Sweden) using 200,000 PBMC in AIM-V medium (Gibco) [31 ]. Positive (anti-CD3 or ConA) and negative controls (DMSO/AIM-V), A/California/07/09 whole virus, split virus antigen and peptide libraries (final concentration of 2μg/ml in DMSO/AIM-V) were added. Peptide assays were conducted for a subset of patients with available PBMCs (n = 16, 11 acute and 5 convalescent patients). The plates were read using a CTL S 6 Ultra V Immunospot analyzer (Cellular Technology Limited, OH, USA), and the results were plotted using GraphPad Prims 5 software (GraphPad Software, Inc., USA).

Venous blood samples were collected into two 4 mL tubes containing heparin. Sufficient PBMCs were separated by a Ficoll-Paque centrifugation technique and counted using an automated hematology analyzer. The final cell suspension was prepared at a density of 2.5×10⁵ cells/100 µL. The IFN-γ ELISpot assay used in the study is the positive control part of the T-SPOT.TB assay (Oxford Immunotec, London, United Kingdom). The ELISpot assay was initiated by adding 100 µL of suspension and 50 µL of positive control solution containing PHA (Mabtech, Stockholm, Sweden) to commercially available pre-coated 96-well plates (Mabtech). The plates were incubated in a humidified incubator at 37°C with 5% CO₂ for 18 h. The distinct dark-blue spots produced as a result of antigen stimulation were evaluated and counted by an ImmunoSpot® Analyzer (Cellular Technology Ltd., Cleveland, OH, United States). The completely-developed assay plates were archived for potential re-examination in case of anomalies. The numbers of spot-forming units (SFUs) in paired wells were reported per 2.5×10⁵ PBMCs.