Failure of the Mycobacterium bovis BCG vaccine: some species of environmental mycobacteria block multiplication of BCG and induction of protective immunity to tuberculosis

Abstract

The efficacy of Mycobacterium bovis bacillus Calmette-Guérin (BCG) vaccine against pulmonary tuberculosis (TB) varies enormously in different populations. The prevailing hypothesis attributes this variation to interactions between the vaccine and mycobacteria common in the environment, but the precise mechanism has so far not been clarified. Our study demonstrates that prior exposure to live environmental mycobacteria can result in a broad immune response that is recalled rapidly after BCG vaccination and controls the multiplication of the vaccine. In these sensitized mice, BCG elicits only a transient immune response with a low frequency of mycobacterium-specific cells and no protective immunity against TB. In contrast, the efficacy of TB subunit vaccines was unaffected by prior exposure to environmental mycobacteria. Six different isolates from soil and sputum samples from Karonga district in Northern Malawi (a region in which BCG vaccination has no effect against pulmonary TB) were investigated in the mouse model, and two strains of the Mycobacterium avium complex were found to block BCG activity completely.

FIG. 1.

BCG multiplication is inhibited in mice previously sensitized with environmental mycobacteria. (A) CBA/J mice. (B) C57BL/6J mice. The growth of BCG was compared in naive mice (open symbols) and in sensitized mice (solid symbols). The data shown are the means of BCG CFU ± standard errors. For both groups, five animals were sacrificed for each time point. The experiment was repeated twice with similar results.

FIG. 2.

Influence of previous sensitization with environmental mycobacteria on the BCG-specific immune responses. BCG was administered s.c., and the frequencies of IFN-γ-producing cells isolated from the draining lymph nodes (A) and the blood (B) in naive mice (open symbols) and sensitized mice (solid symbols) were detected by the ELISPOT assay postvaccination after in vitro stimulation with BCG-Ag. The data presented here represent the logarithmic mean of results obtained from lymph node cells from three individual mice per group ± standard errors. The responses in the blood were analyzed on cells pooled from three animals for each time-point. A pilot experiment conducted on weeks 2, 4, and 6 supported the overall difference in the response profiles of the two groups of animals.

FIG. 3.

Growth of isolates from Karonga, Malawi, in the mouse model. To evaluate the virulence of the isolates, mice were infected i.v. with M. fortuitum strains S160 (open circles), S78/2 (solid circles), and 2001 (solid triangles); M. chelonae strain 2015 (open triangles); and M. avium complex strains 1891 and 2011 (solid and open squares, respectively). The mycobacterial loads were determined in the liver, spleen, and lung at the time points indicated. M. chelonae and M. fortuitum were all below the level of detection from day 14 onwards. Data are given as means with standard errors (n = 4).

FIG. 4.

(A) Growth of BCG in sensitized and naive animals. Mice were infected s.c. with 2 × 10⁶ CFU of either M. fortuitum, M. chelonae, or M. avium or were left untreated. After chemotherapy, mice were infected with BCG Pasteur. BCG CFU in the spleen are shown as means with standard errors (n = 4). Statistically significant differences between sensitized (solid bars) and naive (open bars) mice are indicated: ∗, P < 0.05; ∗∗, P < 0.01, according to Student's t test. ND, not done. (B) Effects of sensitization on the IFN-γ response to BCG. Spleen cells were pooled from four individual mice sensitized with the environmental strains (shaded bars), naive mice inoculated with BCG (open bars), or sensitized mice inoculated with BCG (solid bars). IFN-γ production was assessed in the supernatants of spleen cell cultures stimulated in vitro with BCG Ag and are given as means with standard errors. Statistically significant effects of sensitization on the response to BCG infection are labeled: ∗, P < 0.05; ∗∗, P < 0.01, according to Student's t test.