Characterisation of alternative expression vectors for recombinant Bacillus Calmette-Guérin as live bacterial delivery systems

BACKGROUND: Bacillus Calmette-Guérin (BCG) is considered a promising live bacterial delivery system. However, several proposals for rBCG vaccines have not progressed, mainly due to the limitations of the available expression systems. OBJECTIVES: To obtain a set of mycobacterial vectors using a range of promoters with different strengths based on a standard backbone, previously shown to be stable. METHODS: Mycobacterial expression vectors based on the pLA71 vector as backbone, were obtained inserting different promoters (P(AN), P(αAg,) P(Hsp60), P(BlaF*) and P(L5)) and the green fluorescence protein (GFP) as reporter gene, to evaluate features such as their relative strengths, and the in vitro (inside macrophages) and in vivo stability. FINDINGS: The relative fluorescence observed with the different vectors showed increasing strength of the promoters: P(AN) was the weakest in both Mycobacterium smegmatis and BCG and P(BlaF*) was higher than P(Hsp60) in BCG. The relative fluorescence observed in a macrophage cell line showed that P(BlaF*) and P(Hsp60) were comparable. It was not possible to obtain strains transformed with the extrachromosomal expression vector containing the P(L5) in either species. MAIN CONCLUSION: We have obtained a set of potentially stable mycobacterial vectors with a arrange of expression levels, to be used in the development of rBCG vaccines.