Time Course Characterization of Protective Immune Responses Following BCG Vaccination in BALB/c Mice

Background/Objectives: Tuberculosis (TB) remains a major global health challenge, requiring standardized animal models to evaluate vaccine-induced immune responses. This study characterized time-dependent immune responses following Bacillus Calmette–Guérin (BCG) vaccination in BALB/c mice. Methods: BALB/c mice were vaccinated with BCG, and the immune responses and protective efficacy were evaluated at 4, 6, and 8 weeks post-immunization. The cytokine expression in serum, lung, and spleen tissues was analyzed using ELISA, quantitative PCR, and immunohistochemistry. Protective efficacy was assessed via colony-forming unit (CFU) enumeration and the immunohistochemical detection of Mycobacterium TB after aerosol challenge. Results: The BCG vaccination induced time-dependent and tissue-specific cytokine responses. Pulmonary IL-1β and splenic IFN-γ levels were significantly increased four weeks post-vaccination. At 8 weeks, serum IL-2, pulmonary IL-2, and TNF-α were significantly increased, whereas no significant changes in cytokines were observed at 6 weeks. After the challenge, BCG-vaccinated mice exhibited reduced bacterial burdens compared with controls, but the differences among the 4-, 6-, and 8-week groups were modest. Conclusions: Immune responses became detectable starting four weeks after BCG vaccination, with temporal differences observed in cytokine expression. Week 8 may serve as a reference point for monitoring cytokine dynamics rather than as an optimal time for protection.