• Vaccine Name: attenuated M. bovis strain WAg539
• Target Pathogen: Mycobacterium tuberculosis
• Target Disease: Tuberculosis
• Vaccine Ontology ID: VO_0000543
• Type: Live, attenuated vaccine
• Antigen: M. bovis with gene mutations due to signature-tagged mutigenesis to create attenuated mutants. Ten genes were found to be deleted or interrupted in the 10.1-kb deletion in WAg539, including mmpL4, mmpS4, ufaA1, and sigK, as well as unknown genes (Collins et al., 2005).
• Preparation: M. bovis strain and its recombinants were cultured in Middlebrook 7H9 (Difco) liquid medium supplemented with albumin-dextrose complex (Difco), 0.05% Tween 80, and and 0.4% sodium pyruvate and on Middlebrook 7H11 (Difco) solid medium supplemented with albumin-dextrose complex and 0.4% sodium pyruvate. Recombinants were cultured on kanamycin-supplemented medium. M. bovis culture from pig spleens were homogenized, filtered, and further processed (Collins et al., 2005). Forty-five recombinants with unique tags were subcultured separately and pooled together. The forty-five M. bovis recombinants were subcultured separately, and 10^6 CFU of each pool was inoculated subcutaneously into the flank of each guinea pig. Splenic culture were extracted from sacrificed animals and used to isolate 500-2,000 colonies per spleen. Tags identified in both the inoculated and recovered pools were amplified by PCR and processed.
• Description: Signature tag morphogenesis was used to generate illegitimate recombinants of M. bovis. Several of the recombinant strains produced protection that was "at least as good as that provided by M. bovis BCG" in guinea pig models (Collins et al., 2005).

Guinea pig Response

• Host Strain: Dunkin-Hartley
• Vaccination Protocol: Groups of six guinea pigs were vaccinated with 10^5 CFU of one of the avirulent recombinant strains or BCG. Six animals were not vaccinated (control group) (Collins et al., 2005).
• Challenge Protocol: Guinea pigs were challenged by aerosol with 2 to 10 CFU of wild-type M. bovis eight weeks post-vaccination (Collins et al., 2005).
• Efficacy: Four mutants which did not induce any visible lesion were tested for vaccine efficacy. In the case of three mutants, animals were sacrificed 5 weeks post-challenge. The final mutant, WAg585 were sacrificed at 8 weeks post-challenge. Mutant WAg539 gave better, though not significantly different, protection than BCG vaccine, as well as the best vaccination of the four mutants (Collins et al., 2005).
• Description: Animals were sacrificed and autopsied 5 weeks after challenge to enable the following analyses: weight, gross pathology, mycobacterial culturing and enumeration in splenic and lung tissues, and statistical analysis of macroscopic lesion count and other quantifiable measurements (Collins et al., 2005).