Fig. 7
BCG85C5 vaccine induces a robust protection against primary challenge of tuberculosis in mice through effector (TEM) and central memory (TCM) T cells. a NIH short-term protection (Fig. 6a) and a long-term protection (rechallenge) models are shown. Rechallenged mice were vaccinated, treated with isoniazid and rifampin (mix of 10 mg/kg dose each), rested and challenged with Mtb. The data for NIH and rechallenge models are shown in Fig. 7 and Fig. 8, respectively. Post-vaccine mice were killed on day 21, post challenge on day 60 and post rechallenge mice on day 120. Post vaccination Ag85B-specific T cell analysis in Supplementary Fig. S10. b, c CFU counts of Mtb in the lungs and spleens after primary challenge (day 60). BCG85C5 reduces Mtb CFUs (mean ± SD) of lungs and spleens better than BCG. P-values and log10 difference of CFUs indicated (footnote; n = 5 mice per group; two-way ANOVA). d Histogram analysis of T cells for memory markers using flow cytometry. e–g BCG85C5 induces both TEM and TCM T cells in the lungs after primary challenge. Vaccine groups with significant differences in memory populations are highlighted (*p < 0.02; **p < 0.01; ***p < 0.008; one-way ANOVA). BCG85C5, BCG85CFP and wt-BCG induce comparable numbers of TEM in lungs, spleens, and lymph nodes, while, BCG85C5 and BCG85CFP induce better TCM than wt-BCG in the target organ lungs (Fig. 7e; p boxes a–e). CD4/CD8 panels (Fig. 7e, f, g) indicate percent of cells calculated after estimating absolute numbers (n = 3 mice), which were then averaged. BCG85C5 increases absolute numbers of effector CD62L− CD4 and CD8 T cells in the lymph nodes compared with wt- BCG (vs. Fig. 7g; p boxes a, b) and effector CD62L− CD8 T cells in the spleens (vs. Fig. 7f). h BCG85C5 enhances levels of multifunctional cytokine secreting T cells of lungs (*p < 0.01; one-way ANOVA). i TB10.4 tetramer specific CD8 T cells of the lungs, spleens, and lymph nodes. Clockwise. TB10.4+ CD8 T cells in naive or vaccinated mice. Histogram of TB10.4+ CD8 T cells. Purified CD8 pools (n = 3) of naive or vaccinated group, overlaid in vitro on Mtb-infected macrophages followed by CFU counts. (*p < 0.009; one-way ANOVA). P-values determined using one- or two-way ANOVA with Dunnett’s post-test
