Bacillus Calmette–Guérin (BCG) is the only licensed vaccine against tuberculosis (TB); however, the temporal dynamics of post-vaccination immunity have not been fully established. We examined systemic and tissue-specific immune responses at 4, 6, and 8 weeks following BCG immunization in C57BL/6 mice. Serum cytokine analysis revealed progressive increases in IFN-γ and IL-2, with the highest concentrations observed at week 8. In contrast, ELISpot analysis indicated that IFN-γ-producing splenocytes peaked at week 4 and subsequently decreased, although their numbers remained significantly higher at weeks 6 and 8 compared with the unvaccinated controls. Lung-derived immune cells showed a modest increase in IFN-γ-producing cells at later time points; however, these differences did not reach statistical significance. Gene expression analysis revealed significant late-stage increases in IFN-γ and CXCL10 transcripts in lung tissue, which indicated enhanced local Th1-associated transcriptional activity. Histological examination revealed immune activation without marked tissue pathology, whereas immunohistochemical (IHC) analysis showed time-dependent changes in cytokine-positive cells in the spleen and lung. Following aerosol challenge with Mycobacterium tuberculosis (M.tb), mice vaccinated for 6 or 8 weeks exhibited reduced pulmonary bacterial burden, with the greatest reduction observed in the 8 weeks group at later post-challenge time points. Taken together, these results demonstrate that BCG-induced immunity exhibits distinct assay- and tissue-specific kinetics, characterized by an early splenic cellular response, followed by later systemic and pulmonary immune activation. This integrated temporal analysis provides useful insight for selecting assessment time points in preclinical BCG studies.

To Cite This Article: Choi EB, Yang YJ, Kim KW, Moon SY, Lim HJ, Lee YK, Ouh IO and Park KI, 2026. Temporal analysis of the immune response following single subcutaneous Bacillus Calmette–Guérin Vaccination in C57BL/6 mice. Pak Vet J, 46(8): 2004-2015. http://dx.doi.org/10.29261/pakvetj/2026.185