Engineering probiotic bacteria to express tcdB antigen as an oral vaccine carrier against Clostridium difficile infection

Background
Clostridium (now known as Clostridioides) difficile (C. difficile) is a spore-forming, gram-positive organism that can pose serious public health complications. The elderly are especially vulnerable to C. difficile infections, which can be fatal. C. difficile strains cause symptomatic diseases via the release of two toxins; tcdA and tcdB, that induce inflammation and tissue damage. Vaccines targeting any of these toxins may offer an effective strategy against C. difficile infections.
Objective
This study aimed to use live probiotics as oral vaccines to express the C. difficile toxin B gene. Oral vaccination has many advantages over intramuscular injection, as it has higher compliance, feasibility, and simpler administration. In addition, oral vaccines can stimulate both mucosal and systemic immunity against the target antigen. Probiotic bacteria were chosen as they present ideal candidates for this goal in terms of safety and health promotion.
Materials and methods
We chose two probiotic strains: Lactobacillus gasseri ATCC 33323 in addition to Enterococcus faecium NM1015, which had previously been identified in our lab and is capable of suppressing C. difficile colonization. The C-terminal of the tcdB gene was amplified by polymerase chain reaction (PCR) from C. difficile, cloned, and transformed into an E. coli EZ strain. The tcdB fragment was digested with BamHI and XhoI (NEB, UK) and subcloned into the bile salt-inducible expression plasmid pLB210 (obtained from INRA, France). The transformation and electroporation procedures were employed to insert cloning and expression plasmids into the target bacteria. Colony PCR was used to confirm the engineering strains. Reverse transcription PCR was used to confirm the expression of the C. difficile tcdB fragment.
Results and conclusion
The expression vector ‘p210-tcdB’ was constructed, then introduced into the selected probiotic strains by electroporation and confirmed by colony PCR and plasmid extraction. The reverse transcription (RT)-PCR confirmed the expression of the gene by the engineered strains. No significant difference in the survival rate was observed between the engineered strains and their parental types at pH 2.00 and 1% oxygen. Moreover, the strains showed satisfactory plasmid stability for 210 generations. Future work will involve the in vivo evaluation of the engineered probiotic strains as oral vaccines against C. difficile using an animal model.