Apex software 1

T-cell response was evaluated at four weeks after first dose and at two weeks after the second vaccine dose using human interferon gamma (IFN-γ) ELISpot kit (Mabtech, Nacka Strand, Sweden) according to manufacturer instructions. Briefly, peripheral blood mononuclear cells (PBMCs) were counted using an automated cell counter (Sysmex XN-10™ Automated Hematology Analyzer). ELISpot plates were blocked for 30 min with 10% FCS containing RPMI media prior to the addition of 250,000 PBMCs/well. The stimulation solutions were S-peptide consisting of 100 peptides from spike protein, and NMO-peptide pools consisting of 101 peptides from nucleocapsid (N), membrane (M), open reading frame (ORF) 1, non-structural protein (nsp) 3, ORF-3a, ORF-7a, and ORF8 proteins. ELISpot plates were then incubated for 20 hours at 37°C and 5% CO2, washed and developed using a conjugated secondary antibody that bound to membrane-captured IFN-γ. The plates were read using IRIS (Mabtech) and spots were analyzed using Apex software 1.1 (Mabtech) and converted to spot-forming units (SFU) per million cells.

T-cell responses were assessed using human interferon-gamma (IFN-γ) ELISpot kits according to the manufacturers’ instruction (Mabtech AB, Nacka Strand, Sweden – as previously described (14 (link))). This entailed the use of two peptide pools : (1) an S-defined peptide pool (Mabtech) consisting of 100 peptides from spike (S) protein (purity of 90%); and (2) an NMO-defined peptide pool (Mabtech) consisting of 101 peptides from nucleocapsid (N), membrane (M), open reading frame (ORF) 1, non-structural protein (nsp) 3, ORF-3a, ORF-7a, and ORF-8 proteins (purity of 87%). The ELISpot plates were read using IRIS (Mabtech) and spots were analyzed using Apex software 1.1 (Mabtech) and converted to spot-forming units (SFU) per million cells. The cut-off for positive response was set as 20 SFU.

Cellular immunity was determined by IFN-γ ELISpot (Mabtech, Nacka Strand, Sweden) to ancestral strains on a subset of participants for each group (n = 20). Peripheral blood mononuclear cells (PBMCs) were counted and stimulated with S-peptides that consisted of 100 peptides from spike proteins, and nucleoprotein-membrane protein-open reading frame protein (NMO)-peptide pools, consisting of 101 peptides from nucleocapsid (n), membrane (M), open reading frame (ORF) 1, non-structural protein (nsp) 3, ORF-3a, ORF-7a, and ORF8 proteins. Negative controls contained only cell culture media, while positive controls contained an anti-cluster of differentiation 3 (CD3) at a dilution of 1:1000. ELISpot plates were then incubated for 20 h at 37 °C and 5% CO₂, washed and developed using a conjugated secondary antibody that was bound to membrane-captured IFN-γ. The plates were read using IRIS (Mabtech) and spots were analyzed using Apex software 1.1 (Mabtech) and converted to spot-forming units (SFU) per million cells.