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.