Immunogenicity and protective efficacy of the Mycobacterium tuberculosis fadD26 mutant

Abstract

The Mycobacterium tuberculosis fadD26 mutant has impaired synthesis of phthiocerol dimycocerosates (DIM) and is attenuated in BALB/c mice. Survival analysis following direct intratracheal infection confirmed the attenuation: 60% survival at 4 months post-infection versus 100% mortality at 9 weeks post-infection with the wild-type strain. The fadD26 mutant induced less pneumonia and larger DTH reactions. It induced lower but progressive production of interferon (IFN)-gamma, interleukin (IL)-4 and tumour necrosis factor (TNF)-alpha. Used as a subcutaneous vaccine 60 days before intratracheal challenge with a hypervirulent strain of M. tuberculosis (Beijing code 9501000), the mutant induced a higher level of protection than did Bacille Calmette-Guérin (BCG). Seventy per cent of the mice vaccinated with the fadD26 mutant survived at 16 weeks after challenge compared to 30% of those vaccinated with BCG. Similarly, there was less tissue damage (pneumonia) and lower colony-forming units (CFU) in the mice vaccinated with the fadD26 mutant compared to the findings in mice vaccinated with BCG. These data suggest that DIM synthesis is important for the pathogenicity of M. tuberculosis, and that inactivation of DIM synthesis can increase the immunogenicity of live vaccines, and increase their ability to protect against tuberculosis.

Fig. 1

Survival, pathology, and bacillary loads during lung infection with the fadD26 mutant or its parental strain MT103 in BALB/c mice. (a) All the mice (n=20) died 9 weeks after infection with MT 103 (black squares), whereas mice infected with the mutant (white squares) had 60% survival (P < 0·05). (b) The size of granulomas and the percentage of the lung occupied by pneumonia (c) in mice infected with MT103 (black bars) and or the fadD26 mutant (white bars). (d) Comparison of colony-forming units (CFU)/lung in mice infected with MT103 (black bars) or the fadD26 mutant (white bars). CFU and morphometry data are from four different animals at each time-point in two different experiments. Asterisks represent statistical significance (P < 0·05).

Fig. 2

Representative histopathology of lungs from mice infected with the fadD26 mutant or with MT103, and from mice challenged with the Beijing strain after vaccination with the fadD26 mutant or with Bacille Calmette–Guérin (BCG). (a) Extensive pneumonia and small granulomas (arrowheads) 60 days after intratracheal infection with MT103. (b) Intermediate-sized pneumonic patches (asterisks) and granulomas (arrowheads) 120 days after infection with the fadD26 mutant. (c) 120 days after intratracheal challenge with Beijing strain, extensive pneumonia and small granulomas (arrowheads) were observed in mice vaccinated with BCG. (d) By contrast, 120 days after intratracheal challenge with the Beijing strain in mice vaccinated with the fadD26 mutant, there were smaller patches of pneumonia and bigger granulomas (arrowheads).

Fig. 3

Quantitative expression of mRNA encoding cytokines and nitric oxide synthase (iNOS) determined by real-time polymerase chain reaction (PCR) in lung homogenates from mice infected with the fadD26 mutant (white bars) or with its parental strain MT 103 (black bars). Bars represent the means and standard deviation from four different animals at each time point. Asterisks represent statistical significance (P < 0·05).

Fig. 4

Upper panel: delayed hypersensitivity responses (DTH) to soluble antigens of M. tuberculosis in BALB/c mice infected with the MT103 strain (black bars) or with the fadD26 mutant (white bars). Similar DTH response kinetics were produced by both strains; the peak of DTH in mice infected with fadD26 mutant strain was at day 120. Lower panel: interferon gamma [interferon (IFN)-γ] expression in spleen determined by real-time polymerase chain reaction (PCR).

Fig. 5

Survival, lung colony-forming units (CFU) and histopathology after intratracheal challenge with the Beijing strain in BALB/c mice vaccinated with the fadD26 mutant or with Bacille Calmette–Guérin (BCG). (a) Control non-vaccinated mice died after 6 weeks of infection (black squares), whereas 30% and 70% survival was observed 120 days after the challenge in mice vaccinated with BCG (white squares) and the fadD26 mutant (white circles), respectively. The P-values are indicated on the figure. (b) Mice vaccinated with the fadD26 mutant (white bars) developed significantly lower CFU than did BCG-vaccinated animals (black bars) at days 60 and 120 after challenge. (c) BCG vaccinated mice developed extensive pneumonia (black bars), whereas scarce pneumonic patches were observed in mice vaccinated with the fadD26 mutant (white bars). Asterisks represent statistical significance (P < 0·05). (d) Mice vaccinated with the fadD26 mutant (white bars) showed bigger granulomas than did BCG-vaccinated mice (black bars).