Resumo

Bovine tuberculosis, caused by Mycobacterium bovis, is a major zoonotic and economic problem in Latin America. As for other infectious diseases, the use of a vaccine would contribute for controlling the diseases. BCG is a live attenuated vaccine currently used in humans. The BCG vaccine improvement by overexpressing protective antigens is a promising approach for obtaining more effective vaccines against bovine tuberculosis. M. bovis antigen 85B (Ag85B) demonstrates strong immunogenicity and is a promising target for overexpression in BCG. An expression system was developed in our laboratory employing a BCG Pasteur auxotrophic for leucine (BCG ΔleuD) and a replicative vector (pUP410) that complements this mutation. This system allows the production of stable recombinant strains in vivo with high levels of protein expression. Thus, the aim of this study was to develop a recombinant vaccine using the BCG Pasteur ΔleuD overexpressing Ag85B, and to evaluate the immune response induced by the recombinant BCG in mice. Material and methods: An immunodominant portion of Ag85B encoding gene was amplified by PCR and subsequently cloned into pUP410 vector under control of M. leprae 18 kDa promoter. The recombinant vector was introduced into BCG ΔleuD by electroporation and protein expression was demonstrated by Western blot. Groups of mice were inoculated with 106 UFC of recombinant BGC ΔleuD/Ag85Bt or parental BGC (control) and their splenocytes were obtained. The mice immune response was evaluated by measuring cytokine mRNA levels after stimulating the cells with bovine PPD or Ag85B recombinant protein. REST software was employed for qPCR statistical analysis. Results and discussion: The recombinant vector pUP410::fbpBt was confirmed by PCR and restriction analysis, and Ag85B was readily detectable in rBCG ΔleuD lysates. After in vitro stimulation, spleen cells from animals inoculated with the rBCG produced high level of mRNA for Th-1 cytokines, immune response known to confer protection against bovine tuberculosis. Conclusions: We conclude that BCG ΔleuD overexpressing Ag85B can be evaluated as vaccine candidate in bovines.