Oral immunization with engineered probiotics expressing tcdB protects mice against Clostridioides difficile infection

Clostridioides difficile infection (CDI) is a major global health concern driven by the virulence factors toxins A (TcdA) and B (TcdB). Vaccination targeting these toxins, particularly via the oral mucosal route, represents a promising preventive strategy. This study investigated the efficacy of genetically engineered probiotics Lactobacillus gasseri NM0323 and Enterococcus faecium NM0423 as oral vaccine carriers expressing the C-terminal domain of the tcdB gene. Mice were orally immunized, boosted, and subsequently challenged with C. difficile. Quantitative PCR analysis revealed a significant reduction in C. difficile load and markedly lower tcdA gene expression in the vaccinated group compared to control groups. Furthermore, histopathological and immunohistochemical analyses demonstrated preserved intestinal integrity and minimal Toxin A reactivity in immunized mice. Immunological assessment also showed that the vaccinated mice exhibited elevated levels of serum IgG, IgA, and IgM. These findings strongly support the potential of engineered L. gasseri and E. faecium as effective, live oral vaccine delivery platforms for CDI prevention and suggest their broader application against other enteric pathogens.