Variable Virulence and Efficacy of BCG Vaccine Strains in Mice and Correlation With Genome Polymorphisms

Abstract

Bacille Calmette-Guérin (BCG), an attenuated strain of Mycobacterium bovis, is the only vaccine available for tuberculosis (TB) control. However, BCG is not an ideal vaccine and has two major limitations: BCG exhibits highly variable effectiveness against the development of TB both in pediatric and adult populations and can cause disseminated BCG disease in immunocompromised individuals. BCG comprises a number of substrains that are genetically distinct. Whether and how these genetic differences affect BCG efficacy remains largely unknown. In this study, we performed comparative analyses of the virulence and efficacy of 13 BCG strains, representing different genetic lineages, in SCID and BALB/c mice. Our results show that BCG strains of the DU2 group IV (BCG-Phipps, BCG-Frappier, BCG-Pasteur, and BCG-Tice) exhibit the highest levels of virulence, and BCG strains of the DU2 group II (BCG-Sweden, BCG-Birkhaug) are among the least virulent group. These distinct levels of virulence may be explained by strain-specific duplications and deletions of genomic DNA. There appears to be a general trend that more virulent BCG strains are also more effective in protection against Mycobacterium tuberculosis challenge. Our findings have important implications for current BCG vaccine programs and for future TB vaccine development.

Figure 1

Differential virulence of BCG strains in SCID mice. (a) Survival curves of SCID mice infected with 13 different BCG strains. The survival curves were plotted using the Kaplan–Meier method and differences between curves were analyzed using the log-rank test. BCG strains belonging to the same DU2 groups are highlighted in the same color. (b,c) CFUs in the lungs (b) and spleen (c) of SCID mice at 1 and 4 weeks after infection with 13 different BCG strains. The first bar of each strain is the data from week 1 and the second bar is the data from week 4. Data were plotted as mean ± SD (n = 4). BCG, Bacille Calmette–Guérin; CFU, colony-forming unit; PBS, phosphate-buffered saline.

Figure 2

Variable effectiveness of 13 BCG strains against M. tb challenge in BALB/c mice. (a,b) CFUs in the lungs (a) and spleen (b) at 4 weeks after M. tb challenge in mice immunized with 13 different BCG strains. Each data point represents one mouse and the data are plotted as mean ± SEM (n = 5). Data were analyzed by one-way analysis of variance and Tukey's multiple comparisons. Animal groups exhibiting statistically significant differences with the unvaccinated PBS control group are indicated as follows: *P < 0.05, **P < 0.01, ***P < 0.001. (c) CFUs in the lungs at 4 weeks after M. tb challenge in mice immunized with eight different BCG strains. In the second experiment, eight BCG strains representing four different DU2 groups were selected to repeat the experiment as in (a). Statistically significant differences between BCG groups and the unvaccinated PBS control group are indicated. *P < 0.05. BCG, Bacille Calmette–Guérin; CFU, colony-forming unit; PBS, phosphate-buffered saline.

Figure 3

Variable protective efficacy of BCG groups. (a) Comparison of effectiveness of BCG groups based on tandem duplication DU2. The data of Figure 2a were redrawn by combining BCG strains of the DU2 same group. One-way analysis of variance (ANOVA) and Bonferroni's multiple comparisons were performed for statistical analysis. **P < 0.01, ***P < 0.001. (b) Comparison of effectiveness of BCG strains based on virulence. The 13 BCG strains were divided into three major groups based on virulence: the most virulent group: BCG-Phipps, BCG-Pasteur, BCG-Tice, and BCG-Frappier; the intermediate virulent group: BCG-China, BCG-Russia, BCG-Moreau, and BCG-Danish; and the least virulent group: BCG-Japan, BCG-Birkhaug, BCG-Sweden, BCG-Glaxo, and BCG-Prague. The data of Figure 2a were redrawn by combining data of the same group. One-way ANOVA and Bonferroni's multiple comparisons were performed for statistical analysis. ***P < 0.001. BCG, Bacille Calmette–Guérin; CFU, colony-forming unit; PBS, phosphate-buffered saline.