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.

Fig. 1. : schematic outline of the process of constructing recombinant plasmids for Ag85B expression in Mycobacterium bovis bacillus Calmette-Guérin. The DNA fragments cloned into vector pUP410 were previously amplified by polymerase chain reaction (PCR) from M. bovis genomic DNA. (A) Cloning of the fbpB coding region and its endogenous promoter, resulting in pUP410::85B; (B) cloning of a portion of fbpB that encodes the mature protein. First, a portion of the fbpB coding region (120 to 978 bp) was cloned into vector pUS2000, yielding pUS2000::85BT. From the resulting recombinant vector, an 1149-bp amplicon containing the cloned fragment under control of an 18-kDa promoter from M. leprae was obtained by PCR and then cloned into pUP410, yielding pUP410::85BT. The kanamycin resistance gene was removed by digestion with the HindIII enzyme, and the digestion product was ligated using the T4 ligase enzyme.

Fig. 2. : western blot using a 6× His-tag monoclonal antibody or polyclonal anti-r85B antibody to confirm the identity of rAg85B. (A) rAg85B was overexpressed in Escherichia coli Star (DE3), purified using a nickel affinity column, and identified by 6× His-tag monoclonal antibody. Lane 1, Full-Range Rainbow Molecular Weight Protein Marker (GE); lane 2, purified rAg85B. (B) rAg85B was recognised by polyclonal anti-r85B antibody produced in mice. Lane 1, Full-Range Rainbow Molecular Weight Protein Marker (GE); lane 2, purified rAg85B after refolding; lane 3, purified rAg85B.

Fig. 3. : western blot employing polyclonal anti-r85B antibody to confirm r85B expression in Mycobacterium bovis bacillus Calmette-Guérin (BCG) strains. BCG ∆leuD expression profile. Lanes 1 and 2, whole-cell lysates and culture supernatant, respectively, of rBCG transformed with pUP410::85BT; lanes 3 and 4, whole-cell lysates and culture supernatant of rBCG transformed with pUP410::85B; lanes 5 and 6, whole-cell lysates and culture supernatant of wild-type BCG.

Fig. 4. : in vivo stability of rBCG transformed with vectors containing auxotrophic complementation. Mice were inoculated with bacillus Calmette-Guérin (BCG) ∆leuD-85B, BCG ∆leuD-85BT, BCG Pasteur-85B, or BCG Pasteur-85BT. Bacteria were recovered from spleens, eight and 18 weeks after inoculation and plated on selective and non-selective media. (A-B) Ratios of resistant versus total BCG colonies by spleen were calculated for each strain and construction tested. Vertical bars indicate standard deviation values, and significance was determined by paired t-test: ** Statistical significance, p < 0.01. (C) Western blot demonstrating r85B expression in BCG strains recovered from spleens: line 1, BCG Pasteur-85B; line 2, BCG ΔleuD-85B; line 3: BCG ΔleuD-85BT; lines 4 and 5, positive control strains BCG ΔleuD-85B and BCG ΔleuD-85BT, respectively, before inoculation.