Fig 4. Peptide immunogenicity measured by IFNγ ELISpot.

(A) PBMC (2.5 x 10⁵) isolated at three different time points (42, 49, and 63 dpv) from pigs immunized with empty plasmid (Sham), epitope-driven DNA vaccine (PigMatrix-EDV) and commercial vaccine (FluSure) were restimulated with pooled peptides (All, Int, Ext-II, and Int-I) at 10 μg/mL and whole virus (WV). The number of epitope-specific IFNγ spot forming cells (SFC) induced by the pools were measured using ELISpot assays. “High responder pig” (FS-442) is marked with +. (B) To evaluate vaccine boost effect, IFNγ responses to pooled peptides were measured at three different time points. For A and B, SFC over background, adjusted to spots per 10⁶ of PBMC seeded, are represented with bars indicating means and error bars indicating standard deviation (SD). Pooled peptide responses showing statistical significance when compared to Sham are indicated: **p<0.01, *p<0.05. Significant statistical difference for PigMatrix-EDV between restimulations at 49 dpv is also shown. Same colors and shapes are used in both figures. (C) PBMC from pigs vaccinated with PigMatrix-EDV and FluSure were restimulated with individual class I peptides and (D) class II peptides one week after the second boost (49 dpv). For C and D, SFC over background per 10⁶ PBMC are shown. A response was considered positive if the number of spots was greater than or equal to 20 SFC over background per 10⁶ PBMCs (dashed line).