Peptide immunogenicity measured by IFNγ ELISpot.

<p>(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).</p

Comparison between prediction for prevalent and cohort-specific SLA alleles.

<p>Peptides were predicted to bind to a set of previously reported class I and class II SLA alleles prevalent in the U.S. swine population (prevalent). Based on low-resolution SLA-typing results, those alleles were not represented in the studied pigs. Prediction matrices were developed to predict binding potential of peptides to the most frequent SLA alleles found in the cohort (cohort-specific). (Top) Mean of significant Z-scores (above 1.64) over prevalent class I SLA alleles (SLA-1*0101, 1*0401, 2*0101, and 2*0401) and cohort-specific (SLA-1*0801, 1*1201, 1*1301, 2*0501, and 2*1201) are shown for each peptide. Peptides with a mean of significant Z-scores above 1.64 (dashed line) are considered potential binders. (Bottom) Cluster scores calculated for prevalent class II SLA alleles (DRB1*0101, 0201, 0401, and 0601) and cohort-specific alleles (DRB1*0402, 0602, 0701, and 1001) are shown for each peptide. Cluster scores above 10 (dashed line) are considered as potential binders. Based on the retrospective evaluation, peptides were classified in four categories (TN: true negatives, TP: true positives, FN: false negatives, and FP: false positives). AUC, Sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) are shown.</p

Class I and II predicted peptides.

<p>Peptides were selected based on predicted binding to class I and class II SLA alleles and conservation in IAV strains. The identity percentage between peptides and IAV strains is shown (100% dark gray, 99% - 85% gray, and <85% white). The Peptide ID is coded to the source protein. Sequences not available are marked with -.</p

Geometric mean reciprocal titers of HI antibodies to different virus in sera collected at 42 dpv.

<p>Geometric mean reciprocal titers of HI antibodies to different virus in sera collected at 42 dpv.</p

Experimental outline.

<p>Experimental outline.</p