Oral vaccination with recombinant Lactobacillus casei expressing Malt of Aeromonas veronii and IFN-γ as molecular adjuvant

Aeromonas veronii is a pathogen of human-livestock-aquatic organisms that causes notable economic loss in the aquaculture industry and threatens public health security. Antibiotics are the first-line treatment against this pathogen. However, prolonged use of antibiotics can lead to bacterial resistance and disrupt ecological balance. Oral recombinant Lactobacillus casei vaccine is a potential alternative to antibiotic resistance in diverse pathogenic bacteria. In recent years, adjuvants have been found to be effective in enhancing the immunogenicity of recombinant subunit vaccines. IFN-γ, as a molecular adjuvant, enhances the host immune response to pathogens, reduces inflammatory responses, and enhances long-term protection by vaccines through multiple pathways. Therefore, crucian carp-derived IFN-γ showing high homologous adaptation with fish was used as a molecular adjuvant for subunit vaccines. On this basis, the highly immunogenic Malt gene from A. veronii was combined with IFN-γ to construct recombinant L. casei (Lc-pPG-Malt-IFN-γ, Lc-pPG-Malt, and Lc-pPG-IFN-γ). The immunological effects of the three recombinants were evaluated. Lc-pPG-Malt and Lc-pPG-Malt-IFN-γ showed higher stimulation of IgM antibodies. Among the three, Lc-pPG-Malt-IFN-γ showed the highest and significant upregulation of the activities of humoral immune response-related enzymes, expression of cellular immune response-related gene, phagocytic activity, and phagocytic index. All recombinants colonised well in the gut of crucian carp, with Lc-pPG-Malt-IFN-γ having the highest colonisation rate. Moreover, the Lc-pPG-Malt-IFN-γ group (64%) had a better survival rate than that of Lc-pPG-Malt (47%), Lc-pPG-IFN-γ, Lc-pPG (control), and phosphate-buffered saline (PBS; blank) groups with the virulent strain A. veronii challenge. In conclusion, subunit vaccines of recombinant L. casei with an antigenic conjugate molecular adjuvant (IFN-γ) were more effective in immunising crucian carp compared with recombinant L. casei vaccines with a single antigen stimulation.