ELISpot stripes containing PVDF membranes (MilliporeSigma MultiScreen HTS; Fisher Scientific, https://www.fishersci.com) were activated with 50 µL of 35% ethanol for 10 s and washed with distilled water. Plates were then coated for 3 h with 60 µL of monoclonal antibodies against IFN-γ (10 µg/mL of clone 1-D1K; Mabtech, https://www.mabtech.com). Thereafter, ELISpot plates were washed and then blocked with 150 µL AIM-V (Thermo Fisher Scientific, https://www.thermofisher.com). After 30 min at 37°C, AIM-V was discarded and duplicates of 250,000 peripheral blood mononuclear cells (PBMC) were grown in the presence or absence of either PepTivator SARS-CoV-2 protein S1/S2 or membrane (M) protein (600 pmol/mL of each peptide; Miltenyi Biotec, https://www.miltenyibiotec.com) or an S1 protein (4 µg/mL; Sino Biologic, https://www.sinobiological.com) in 150 µL of AIM-V. The peptide mix (PepTivator) of the S1/S2 protein covered the immunodominant domains, the peptide mix of the M protein the complete sequence of the glycoprotein. The S1 protein was a recombinant protein expressed in (human) HEK293 cells (Appendix Figure 1). After 19 h incubation at 37°C, the ELISpot plates were washed and captured IFN-γ was detected by incubation for 1 h with 50 µL of the alkaline phosphatase-conjugated monoclonal antibody against IFN-γ (clone 7-B6–1, Mabtech), diluted 1:200 with phosphate-buffered saline plus 0.5% bovine serum album. After further washing, 50 µL of nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl-phosphate was added; purple spots appeared within 7 min. Spot numbers were analyzed by an ELISpot reader (AID Fluorospot, Autoimmun Diagnostika GmbH, https://www.aid-diagnostika.com). Mean values of duplicate cell cultures were considered. We determined SARS-CoV-2–specific spots by spot increment, defined as stimulated minus nonstimulated values. Stimulated spot numbers >3-fold higher than negative (unstimulated) controls combined with an increment value of >3 to any of the 3 antigens were considered positive. Of note, the negative controls reached a mean value of 0.27 spots and an SD of 0.48 (Appendix).

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