Abstract
Little is known about the effect of different modes of expression of an antigen in rBCG on immune response. An appropriate wing of the immune system, with different degrees, is activated upon encounter with a foreign antigen. Knowledge of these responses is vital to the development of future recombinant vaccine. Various E. coli-mycobacterial species shuttle vector constructs were made using a combination of mycobacterial promoters and signal sequences. Thus enabling foreign antigens to be expressed cytoplasmically or secreted outside rBCG as native proteins or membrane-associated lipoproteins. A pivotal study using an E. coli beta-lactamase as a reporter gene is described for the evaluation of the strength of promoter and signal sequence constructs both in vitro and most importantly in vivo using the mouse macrophage cell line J-774. Expression of the diphtheria toxin fragment B as a foreign antigen was detected in vitro with all constructed plasmid vectors in rBCG using a western blot as a means of detection. It was observed that all hsp60 promoter-based constructs exhibited a high frequency with variable degree of plasmid DNA deletions Using three different rBCG substrains, the BCG Tokyo was found to be more stable (P<0.01) and exhibited less degree of deletion (P<0.001) compared to either BCG Moreau or BCG Pasteur. Sequence analysis of deleted plasmid DNA revealed a specific region common with nearly all plasmid deletions. Such a region of the DNA was found to correspond to the first transcriptional starting site of the hsp60 promoter. Furthermore no differences were observed in the level of expression among the three-rBCG substrains, retaining plasmid DNA, when detected by immunoblotting.