Stable expression of Mycobacterium bovis antigen 85B in auxotrophic M. bovis bacillus Calmette-Guérin

Abstract

BACKGROUND Bovine tuberculosis (TB) is a zoonotic disease caused by Mycobacterium bovis, responsible for causing major losses in livestock. A cost effective alternative to control the disease could be herd vaccination. The bacillus Calmette-Guérin (BCG) vaccine has a limited efficacy against bovine TB, but can improved by over-expression of protective antigens. The M. bovis antigen 85B demonstrates ability to induce protective immune response against bovine TB in animal models. However, current systems for the construction of recombinant BCG expressing multiple copies of the gene result in strains of low genetic stability that rapidly lose the plasmid in vivo. Employing antibiotic resistance as selective markers, these systems also compromise vaccine safety. We previously reported the construction of a stable BCG expression system using auxotrophic complementation as a selectable marker.OBJECTIVES The fundamental aim of this study was to construct strains of M. bovis BCG Pasteur and the auxotrophic M. bovis BCG ΔleuD expressing Ag85B and determine their stability in vivo.METHODS Employing the auxotrophic system, we constructed rBCG strains that expressed M. bovis Ag85B and compared their stability with a conventional BCG strain in mice. Stability was measured in terms of bacterial growth on the selective medium and retention of antigen expression.FINDINGS The auxotrophic complementation system was highly stable after 18 weeks, even during in vivo growth, as the selective pressure and expression of antigen were maintained comparing to the conventional vector.MAIN CONCLUSION The Ag85B continuous expression within the host may generate a stronger and long-lasting immune response compared to conventional systems.

Anti-trypanosomal screening of Salvadoran flora

“…Linalool and E-nerolidol were reported as the major constituents of its essential oil [29,30], whereas a recent study revealed that the composition was variable and only E-nerolidol was detected as a common constituent among the eight cultivars from different places in Guatemala [31]. Although linalool has been reported to show anti-trypanosomal activity [32], nothing has been reported on anti-trypanosomal constituents of the MeOH extract of this species. Aerial parts of P. jaquemontianum (510 g) were extracted with MeOH at room temperature to give 83.5 g of the extract.…”

Computational identification of significant immunogenic epitopes of the putative outer membrane proteins from Mycobacterium tuberculosis

“…BCG is the currently available vaccine against TB. It expresses an Mtb immunodominant protein Antigen 85B (Ag85B) [ 23 ]. The antigen85 (Ag85) proteins comprise of Ag85A, Ag85B, and Ag85C.…”

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

“…(12,25) On the other hand, the level of expression of heterologous genes in mycobacteria is modulated mainly by the promoter, yet not solely. Although, there are several studies performed with a set of promoters and in different vectors as backbone, (20,26) investigations in the search for new and improved promoters continue to be performed. (25,27) We have extensive experience with the mycobacterial expression vector pLA71, containing the P BlaF* promoter, which we have used to express many different antigens from different pathogens.…”

“…(6) On the other hand, it has been shown that maintaining the plasmid based on antibiotic resistance does not determine that the antigen will be expressed over time. (20) Rizze et al (20) developed an auxotrophic mycobacterial system based on leuD complementation to express 85B and 85BT antigens (BCG ΔleuD-85B and BCG ΔleuD-85BT), which proved to be stable both for the presence of the plasmid and for the expression of these antigens over a long period of time after immunisation of mice. We have previously obtained a BCG strain auxotrophic for lysine, (BCG-Δ-lysA), complemented with a pLA71-based expression vector (7) which was shown to maintain expression of the antigen through several in vitro passages without antibiotic selection (results not shown).…”