Mycobacterium bovis BCG-mediated protection against W-Beijing strains of Mycobacterium tuberculosis is diminished concomitant with the emergence of regulatory T cells

Abstract

Despite issues relating to variable efficacy in the past, the Mycobacterium bovis BCG vaccine remains the basis for new-generation recombinant vaccines currently in clinical trials. To date, vaccines have been tested mostly against laboratory strains and not against the newly emerging clinical strains. In this study, we evaluated the ability of BCG Pasteur to protect mice from aerosol infections with two highly virulent W-Beijing clinical strains, HN878 and SA161. In a conventional 30-day protection assay, BCG was highly protective against both strains, but by day 60 of the assay, this protection was diminished. Histological examination of the lungs of vaccinated animals showed reduced lung consolidation and smaller and more-organized granulomas in the vaccinated mice after 30 days, but in both cases, these tissues demonstrated worsening pathology over time. Effector T cell responses were increased in the vaccinated mice infected with HN878, but these diminished in number after day 30 of the infections concomitant with increased CD4(+) Foxp3(+) T cells in the lungs, draining lymph nodes, and the spleen. Given the concomitant decrease in effector immunity and continued expansion of regulatory Foxp3(+) cells observed here, it is reasonable to hypothesize that downregulation of effector immunity by these cells may be a serious impediment to the efficacy of BCG-based vaccines.

Fig. 1.

Course of infections in the lungs of control and BCG-vaccinated mice. Data show the bacterial load in the lungs of control (closed circles) and BCG-vaccinated (open circles) mice infected by aerosol with M. tuberculosis strain H37Rv, HN878, or SA161. CFU were determined by plating serial dilutions of organ homogenates on nutrient 7H11 agar and counting CFU after 3 weeks of incubation at 37°C. Results are expressed as the mean (n = 5) of the bacterial load in each group expressed as log₁₀ CFU (± standard error of the mean [SEM]). Data points indicated by asterisks were significantly different from results for saline controls. NS, not significant.

Fig. 2.

Histopathology of the lungs of mice infected with W-Beijing strain HN878. Representative photomicrographs of lung sections taken at days 30 and 60 from control or vaccinated mice infected with strain HN878. Hematoxylin and eosin staining. Size bars, 500 μm.

Fig. 3.

Histopathology of the lungs of mice infected with W-Beijing strain SA161. Representative photomicrographs of lung sections taken at days 20 through 90 from control or vaccinated mice infected with strain SA161. Hematoxylin and eosin staining. Size bars, 500 μm.

Fig. 4.

Kinetics of influx of CD4 effector T cells in mice infected with strains HN878 and SA161. Lung cells obtained from control (closed circles) and BCG-vaccinated (open circles) mice were analyzed by flow cytometry. Effector cells were defined as those that stained positive as CD4⁺ CD44^hi CD62L^lo IFN-γ⁺ cells. The data are expressed as the mean percentage of total CD4 cells in each organ ± SEM (n = 5 mice per group). Data points indicated by asterisks were significantly different from results for saline controls. NS, not significant.

Fig. 5.

Kinetics of influx of CD4⁺ Foxp3⁺ regulatory T cells in mice infected with strains HN878 and SA161. The total numbers of CD4⁺ Foxp3⁺ cells in the lungs of control (closed circles) and BCG-vaccinated (open circles) mice were analyzed by flow cytometry. The data are expressed as the mean percentage of total CD4 cells in each organ ± SEM (n = 5 mice per group). Data points indicated by asterisks were significantly different from results for saline controls. NS, not significant.

Fig. 6.

Kinetics of influx of CD4⁺ IL-17⁺ T cells in control (closed circles) and BCG-vaccinated (open circles) mice were analyzed by flow cytometry. The data are expressed as the mean percentage of total CD4 cells in each organ ± SEM (n = 5 mice per group). Data points indicated by asterisks were significantly different from results for saline controls. NS, not significant.

Fig. 7.

Kaplan-Meier analysis of survival of mice after infection with HN878 or SA161. Data show the time to death of control (closed circles) and BCG-vaccinated (open circles) mice (P < 0.0001 in both cases, Kaplan-Meier analysis).