Disruption of the SapM locus in Mycobacterium bovis BCG improves its protective efficacy as a vaccine against M. tuberculosis.
We studied the vaccine potential of BCG mutants deficient in the secreted acid phosphatase, SapM, or in the capping of the immunomodulatory ManLAM cell wall component with α-1,2-oligomannoside.Persistence of the SapM-mutated BCG in vivo resembled that of the parental BCG indicating that this mutation will likely not compromise the safety of the BCG vaccine.The SapM mutant BCG vaccine was more effective than the parental vaccine in inducing recruitment and activation of CD11c(+) MHC-II(int) CD40(int) dendritic cells (DCs) to the draining lymph nodes.
Affiliation: Unit for Medical Biotechnology, Department for Molecular Biomedical Research, Ghent, Belgium. email@example.com
- Acid Phosphatase/genetics*/immunology
- BCG Vaccine/administration & dosage/genetics/immunology*
- Bacterial Proteins/genetics*/immunology
- Mycobacterium bovis/enzymology*/genetics/immunology
- Mycobacterium tuberculosis/immunology*/physiology
- Sequence Deletion*
- Tuberculosis, Pulmonary/immunology/microbiology/prevention & control*
- Mice, Inbred BALB C
- Mice, Inbred C57BL
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fig01: Protective efficacy of M. bovis BCG WT versus mutant SapM::T (Mb3338) in BALB/c miceA,B. BALB/c mice were vaccinated subcutaneously with the parental or mutant M. bovis BCG strain. Three months post-vaccination, animals were intravenously infected with luminescent M. tuberculosis H37Rv. Mice were sacrificed 2, 4, and 8 weeks post-infection, and the number of bacteria in lungs (A) and spleens (B) was determined by luminometry. Individual mice were evaluated for RLU, and averages and error bars of +/− 1× standard deviation (s.d.) are shown. (One-way ANOVA, Bonferroni's multiple comparison test; ***p < 0.0001; **p < 0.0012; *p < 0.05; ns, not significant).C,D. Mortality of mice was monitored weekly for 11 months. The percentage of survival is shown. The mean survival time (MST) for unvaccinated mice was 23.5 weeks, and for mice vaccinated with BCG WT and SapM::T 26.5 weeks and 32 weeks, respectively (p = 0.0145; Kaplan–Meier, Bonferroni). We have included the 95% confidence intervals (CI) for the survival proportions in panel D.E,F. BALB/c mice were vaccinated subcutaneously with the parental or SapM mutant M. bovis BCG. After 3.5 months, animals were intratracheally infected with luminescent M. tuberculosis H37Rv. Mice were sacrificed 4 or 8 weeks post-infection and the number of bacteria in lung (E) and spleen (F) was determined by luminometry. Individual mice were evaluated for RLU, and averages and error bars of +/− 1× s.d. are shown. (One-way ANOVA, Bonferroni's multiple comparison test; ***p < 0.0001; **p < 0.0012; *p < 0.05; ns, not significant; ns#, not significant with 95% CI of difference from −0.14 to 0.946).G,H. Mortality was also monitored weekly for 17 months. The percentage of survival is shown. The MST for unvaccinated mice was 44 weeks, and for mice vaccinated with BCG WT and SapM::T, respectively, it was 48 and 56 weeks (p = 0.05; Kaplan–Meier, Bonferroni; Panel G). Panel H shows survival with indication of the 95% CI for the survival proportions. At the time-points beyond 50 weeks post-challenge, 95% CI on the survival proportions of SapM vaccinees do not overlap with survival proportions of the WT vaccinees, demonstrating a significant difference (p < 0.05).
Mice in the first group were sacrificed 2, 4 or 8 weeks post-infection, and the number of bacteria in spleen and lungs was determined by luminometry, a validated method which correlates very well with CFU counting (Bonay et al, 1999). Parental BCG and all three mutant strains provided significant protection against growth of M. tb: the number of bacteria in spleen and lung were 10-fold to 100-fold lower than in the unvaccinated mice infected with M. tb (Fig 1A and B and Suppl. Fig 1A and B). At 2 and 4 weeks post-infection, no significant differences in lung or spleen bacterial load was found between the mutant BCG vaccinated groups and the parental BCG vaccinees. However, 8 weeks post-infection, the number of bacteria in lungs of mice vaccinated with the SapM::T mutant was about four times lower than in mice vaccinated with parental BCG (p < 0.05; Fig 1A), but this was not the case for the ManLAM mutants (Suppl. Fig 1A and B). This difference was mainly due to a 10-fold higher bacterial amplification between weeks 4 and 8 in parental BCG vaccinated mice compared to a stable load in the SapM vaccinees.