Plasmid instability when the <i>hsp</i>60 gene promoter is used to express the protective non-toxic fragment B of the diphtheria toxin in recombinant BCG

Abstract

The genetic modification of the live attenuated Mycobacterium bovis BCG to deliver a protective Corynebacterium diphtheriae antigen in vivo could be a safer and less costly alternative to the new and more expensive DTP vaccines available today, in particular to third world-countries. The stability of expression of heterologous antigens in BCG, however, is a major challenge to the use of live recombinant bacteria in vaccine development and appears to be dependent to a certain extent, on a genetic compatibility between the expression cassette within the plasmid construct and the mycobacterium host. In the quest for the best recombinant BCG transformant to express the dtb gene of C. diphtheriae we generated two new rBCG strains by transforming the Moreau substrain of BCG with the mycobacterial expression vectors pUS973 and pUS977, each one carrying a different promoter to drive the expression of the target antigen. After transformation recombinant BCG clones were selected on Middlebrook 7H10 kanamycin Agar plates, expanded in Middlebrook 7H9 kanamycin Broth and analyzed by agarose gel electrophoresis and immunoblotting. rBCGs transformed with the construct carrying the weak P AN promoter from M. paratuberculosis stably expressed the dtb gene. Conversely, rBCGs transformed with the construct carrying the strong mycobacterium hsp60 promoter were unstable and consequently unfit for the expression of the C. diphtheriae gene.

<i>Mycobacterium bovis</i> BCG as a Delivery System for the <i>dtb</i> Gene Antigen from Diphtheria Toxin

“…Despite the advances reached so far, a major limitation of the rBCG technology is the stability of the modified BCG [42]. In the present study, the promoter P AN of M. paratuberculosis [43] subunit of pertussis toxin [30].…”

“…Although the expression of DT antigens in different bacterial species has been previously done [8] [37]- [41], in BCG the expressed DT antigens came from the mutant CRM 197 [27] and the diphtheria toxin dtb gene of the PW8 vaccine strain [42]. It is well known that DTB is crucial for the adherence of diphtheria toxin to target cell receptors, a fundamental step in the internalization of the DT toxic fragment A.…”