Fig. 6
Recombinant BCG vaccine expressing Ag85B and C5 peptide (BCG85C5) induces TLR-2-dependent protection against aerosol-induced tuberculosis of mice. a NIH short-term BCG vaccine induced protection mouse model. C57Bl/6 mice (4–6 weeks M/F) were vaccinated as indicated with BCG strains or left untreated (naive); followed by aerosol challenge with 100 CFU per mouse of virulent M. tuberculosis Erdman (Mtb) and killed for bacterial (CFU) counts of lungs and spleens and for T-ell assays. b, c Splenocytes on day 21 were stained using Ag85B-MHC-II tetramer (NIH tetramer core; Emory University) for CD4 T cells. The data averaged for three mice; t test. d Splenocytes were cultured with soluble antigens (5 µg/mL) as indicated for 18 h and IFN-γ ELISA. The data averaged for three mice per group (mean ± SD; *p < 0.01; **p < 0.0071; one-way ANOVA). e, f On day 60, organs were harvested for Mtb counts. BCG85C5 yields better protection than wt-BCG and BCG85 in reducing the load of Mtb in the lungs and spleens of wild-type mice. Protection generated by BCG85C5 is significantly reduced in TLR-2 knockout mice (*p < 0.0091; two-way ANOVA; n = 5 mice per group and vaccine). g, h Lung T cells of wild-type and TLR-2 knockout mice on day 60 (n = 3 per group) were analyzed with flow cytometry using tetramers specific for MHC-I type epitopes from ESAT-6, Mtb32a, PstS3, and TB10.4 antigens of Mtb (histograms illustrated in Supplementary Fig. S11) (see the Methods section). Cells were gated on CD8, intracellular IFN-γ and MHC class-I tetramers. BCG85C5 induces a stronger expansion of antigen-specific tetramer-positive CD8 T cells in wild-type mice compared to TLR-2 knockout mice (*p < 0.01; one-way ANOVA). i Purified CD8 T-cell pools from lungs of vaccinated or naive mice (n = 3 per group) were overlaid on Mtb infected macrophage monolayers followed by CFU counts at 72 h (p-values, vaccine groups vs. naive; one-way ANOVA). j, k Lung T cells of mice collected on day 60 (n = 3 per group) were stained using Ag85B-specific MHC-II tetramer and for multifunctional cytokine secreting CD4 T cells (MFCs) (*p < 0.01). P-values were determined using one- or two-way ANOVA with Dunnett’s post-test
