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 PAN 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.
Highlights
Diphtheria, the classical disease caused by C. diphtheriae is an acute communicable infection of the upper respiratory tract that can be fatal [1,2]
We report our first results over the expression of the C. diphtheriae gene dtbPW8 by the M. bovis Bacillus of Calmette-Guérin (BCG) substrain Moreau used in Brazil to produce the vaccine against TB, modified by the transformation with distinct plasmid vectors, pUS973 and pUS977, to generate two new recombinant strains in which the expression of the target gene is driven by a strong or a weak (PAN) mycobacterium promoter
Our best results came out with the BCG transformed with the pUS977dtbPW8 construct in which the expression of DTB is driven by the PAN promoter, a regulatory sequence derived from M. paratuberculosis
Summary
Diphtheria, the classical disease caused by C. diphtheriae is an acute communicable infection of the upper respiratory tract that can be fatal [1,2]. The current toxoid based vaccine presents risks to vaccinees associated to the production process. These risks include incomplete inactivation and the presence of reactor and unknown proteins generated during the inactivation process. For this reason the immunization against diphtheria has to be done with three doses to minimize adverse effects that could take place with a full single dose [6,7]. The purification of the diphtheria toxoid would increase the cost of production considerably
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