T-cell ELISpot Assay for Antigen-Specific Responses

Mouse T-cell ELISpot reagents were obtained from Mabtech. Antigen specific IFN-γ⁺ and IL-2⁺ T cells were quantified per manufacturer's instructions. Positive control wells were stimulated with 10 ng/mL PMA (Sigma-Aldrich) and 500 ng/mL ionomycin (Sigma-Aldrich). Test splenocyte wells were stimulated with the appropriate peptides at a concentration of 2.5 µg/mL as previously described (Suschak et al., 2021 (link)). Cells were incubated for 20 h at 37 °C in 5% CO₂. Positive spots were visualized on a CTL Imager and counting was performed with Immunospot software (Cellular Technology Ltd.). Splenocytes from vaccinated mice were stimulated with pooled 15-mer peptides (2 pools of 17 peptides) containing a 5-base overlap in previously identified T-cell dominant regions of the Afg09-2990 M-segment open reading frames (Mimotopes).