Stimulation
A single 10ml sodium heparin vacutainer (BD) tube of blood was collected from each participant and processed in the laboratory within 12 hours of blood draw. Whole blood samples (1ml) were aliquoted into sterile T332 Micrewtubes (Simport Scientific, Saint-Mathieu-de-Beloeil, Canada) containing pre-aliquoted peptides. One tube contained the T cell mitogen, phytohaemagglutinin-L (Sigma-Aldrich, St. Louis, Missouri, USA; final concentration 50µg/ml) as a positive control, and another contained 50µl PBS (negative control). Samples were incubated at 37°C, 5% CO2, for 18-22 hours. Tubes were then centrifuged at 2000g for 3 minutes before harvesting ~200-300µl plasma from the top of each blood sample. Plasma samples were stored at -20°C for up to 6 weeks prior to analysis by ELISA or Luminex xMAP array.
ELISA for IFN-γ
IFN-γ protein was measured by a commercially available IFN-γ ELISA MAX Deluxe kit (BioLegend, San Diego, California, USA), following manufacturer’s instructions with a few modifications: an additional point on the standard curve (1000pg/ml); a 1-hour incubation for standards, samples and blanks; and a pre-read step (at 450nm with just the TMB substrate) to standardise development time of the assay. When standard 2 reached 0.1 OD, stop solution was added and the plate was read at 450nm. The amount of IFN-γ in each sample was analysed using the Gen5 software 8-point standard curve. To calculate the T cell response to SARS-CoV-2, the amount of IFN-γ in the control (PBS only) sample was subtracted from the corresponding value for the SARS-CoV-2 peptide stimulated sample and reported as pg/ml of plasma. In the absence of a response to the peptides, the amount of IFN-γ was calculated as below the lower limit of detection (LLOD). Therefore, a value of the lowest value on the standard curve was given for that sample (7.813pg/ml). Cut off for determining a positive response was set by Youden’s index (J ) (see Statistics below) which determined the optimal cut off value of >24.04pg/ml in the test for natural infection-induced responses, and >42.34pg/ml in the test for vaccine-induced responses.