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. 2012 Feb;80(2):815–831. doi: 10.1128/IAI.05526-11

Fig 8.

Fig 8

Antigen-specific stimulation of cytokine responses in spleen cells from mice vaccinated with RASV χ11021 harboring either Asd+/MurA+ lysis plasmid pYA3681 (vector control) or plasmids pYA4890, pYA4891, and pYA4892 (specifying synthesis of SopENt80-E2C/BlaSS-Ag85A294-BlaCT) or harboring plasmids pYA4893 and pYA4894 (specifying synthesis of OmpCSS-E2C/BlaSS-Ag85A294-BlaCT), or were immunized simultaneously with both χ11021(pYA4892) and χ11021(pYA4894). Ag85A-specific IFN-γ (A), TNF-α (B), IL-2 (C), and IL-4 (D) cytokine-forming lymphocytes were detected by ELISPOT assay. C57BL/6 mice were orally immunized with the Salmonella vaccine strains or BSG dosed at days 0, 21, and 49. Three weeks after the last immunization, spleen cells from three mice per group were harvested and pooled. Cells were restimulated for 40 h (for IFN-γ- and TNF-α-secreting cells) or 66 h (for IL-2- and IL-14-secreting cells) with 1 μg/well of recombinant Ag85A or medium for ELISPOT assays. The results are presented as the number of ELISPOTs per million lymphocytes minus background number of ELISPOTs from unpulsed mock controls. *, P < 0.05, and ***, P < 0.001, for comparison of BSG-dosed mice. (A) For Ag85A-specific IFN-γ secreting cells, *, P < 0.05 for comparison of mice immunized with either χ11021(pYA4890) or χ11021(pYA4891) and mice immunized with χ11021(pYA3681); **, P < 0.01 for comparison of mice immunized with both χ11021(pYA4892) and χ11021(pYA4894) versus mice immunized with χ11021(pYA4894); ***, P < 0.001 for comparison of mice immunized with both strains χ11021(pYA4892) and χ11021(pYA4894) versus mice immunized with χ11021(pYA3681). (B) For Ag85A-specific TNF-α-secreting cells; *, P < 0.05 for comparison of mice immunized with χ11021(pYA4890) and mice immunized with χ11021(pYA4892) or χ11021(pYA3681); **, P < 0.01 for comparison of mice immunized with both χ11021(pYA4892) and χ11021(pYA4894) versus mice immunized with χ11021(pYA4894) or χ11021(pYA3681). (C) For Ag85A-specific IL-2-secreting cells, **, P < 0.01 for comparison of mice immunized with both χ11021(pYA4892) and χ11021(pYA4894) versus mice immunized with χ11021(pYA4894). (D) For Ag85A-specific IL-4-secreting cells; *, P < 0.05 for comparison of mice immunized with χ11021(pYA3681) and mice immunized with χ11021(pYA4890) and P < 0.05 for comparison of mice immunized with χ11021(pYA4890) and mice immunized with χ11021(pYA4891); ***, P < 0.001 for comparison of mice immunized with both strains χ11021(pYA4892) and χ11021(pYA4894) versus mice immunized with χ1102(pYA4894). Error bars represent variations between triplicate wells. The statistical significance was calculated by one-way ANOVA and Tukey's posttest.