ELISpot assay

The T-cell response to vaccination was analyzed using an IFNγ ELISpot assay. Peptide arrays of the HA protein of swine influenza virus strain Ohio/2011, Manitoba/2005, Texas/1998, and Colorado/1977 were synthesized by GenScript. These peptide arrays spanned the entire HA protein of each strain and consist of 17-mers with 10 amino acid overlap. Peptide arrays of the HA protein of human influenza virus strain Texas/1977, Mississippi/1985, and Aichi/1968 were also synthesized by GenScript and were 17-mers with 12 amino acid overlap. Potential immunogenic peptides were identified using a matrix of peptides pools, and the epitopes were confirmed using individual peptides. For ELISpot assays on mice splenocytes, 96-well polyvinylidene difluoride-backed plates (MultiScreen-IP, Millipore) were coated with 50 μl of anti-mouse IFN-γ mAb AN18 (5 µg/ ml; Mabtech) overnight at 4 °C before being washed and blocked with cRMPI 10% FBS for 1 hr at 37 °C. To re-stimulate splenocytes, single-cell suspension of mouse splenocytes was added to each well and an equal volume (50 μL) of peptide (5 μg/mL) was added to the splenocytes. These plates, containing splenocytes re-stimulated with peptide, were incubated overnight at 37 °C with 5% CO₂ to allow for IFNγ production. Plates were then washed 6× with PBS and incubated with 50 μL of biotinylated anti-mouse IFN-γ R4-6A2 mAb (1:1000 dilution; Mabtech) diluted in PBS with 1% FBS for 1 h at RT. Plates were washed 6✕ with PBS and incubated with 50 µl of streptavidin-alkaline phosphatase conjugate (1:1000 dilution; Mabtech) diluted in PBS 1% FBS. After 1 h at RT, the plates were washed 6✕ with PBS and developed by adding 100 µl of BCIP/NBT (Plus) alkaline phosphatase substrate (Thermo Fisher). Development was stopped by washing several times in dH₂O. The plates were air dried and spots were counted using an automated ELISpot plate reader (AID iSpot Reader Spectrum). Results are expressed as an SFC per 10⁶ splenocytes. Swine ELISpot assays on PBMCs were performed as described above, however, plates were coated with 50 μL of anti-porcine IFN-γ mAb pIFNγ-I (5 µg/ ml; Mabtech). After overnight incubation of the swine PBMCs with peptides to allow for re-stimulation and IFN-γ production, plates were incubated with 50 μL of biotinylated anti-porcine IFN-γ mAb P2C11 (1:1000 dilution; Mabtech). One pig in the epigraph group was excluded from the ELISpot analysis due to cell viability loss. The MHCI binding predictions were made on 3/5/2020 using the IEDB analysis resource Consensus tool²².