Immunological evaluation of Recombinant Mycobacterium vaccae expressing Mycobacterium tuberculosis-specific antigens in mice

Tuberculosis (TB) is considered to be one of the leading causes of death from infectious diseases all over the world. The danger is coupled with the emergence of multi-drug resistant strains which renders treatment a real challenge. The Vaccine-BCG- used against TB is very disputable giving wide ranges of protection reaching as low as nil, in some parts of the world. Several M. tuberculosis-specific DNA regions including RD1 were found to be deleted from the vaccine strains. Deleted region RD1 encode proteins that are major targets for T cell recognition in humans and animals infected with M. tuberculosis and M. bovis, respectively. The aim of this study is to express open reading frames (ORFs) from RD1 region in M. vaccae and evaluate their ability to induce specific immune responses in mice. The appropriate ORFs were cloned in M. vaccae using the shuttle vectors pSMT3, and pDE22. Expression of the corresponding proteins was detected by western blotting using anti-hemagglutinin (HA) and/ or antigens- specific antibodies. Recombinant mycobacteria were injected in Balb/C mice, spleens were removed, and specific immunity to the recombinant antigens was assessed using standard spleenocytes culture and cytokines quantitation assays. This system of expression using shuttle vectors proved to be a promising system to identify candidate antigens to be used in diagnosis and to develop improved vaccine strains of BCG.