Development and immune evaluation of a LAMP-targeted TolC II DNA vaccine delivered by mannosylated chitosan against Aeromonas veronii in crucian carp (Carassius auratus).

Crucian carp (Carassius auratus) is one of the major freshwater aquaculture species in China and is frequently threatened by motile Aeromonas septicemia (MAS), a major bacterial disease closely associated with Aeromonas veronii infection in crucian carp culture. To develop an effective DNA vaccine against A. veronii, an epitope-rich truncated antigen fragment, designated TolC II, was screened and selected. TolC II was inserted in place of the luminal domain of crucian carp lysosome-associated membrane protein (LAMP), and the resulting chimeric sequence was cloned into pcDNA3.1 (+) to construct the plasmid pcDNA-LolP, which encodes the fusion protein LolP. After in vitro expression was verified, pcDNA-LolP was encapsulated in mannosylated chitosan nanoparticles (MCs) to generate MCs-pcDNA-LolP. Crucian carp were then immunized by intramuscular injection to evaluate the immunogenicity and protective efficacy of this vaccine. The results showed that vaccination significantly increased serum-specific IgM levels and enhanced the activities of innate immune-related enzymes, including ACP and AKP. In addition, splenic MHC II transcription was significantly upregulated, which was consistent with enhanced MHC II-related antigen presentation. Challenge tests further showed that fish vaccinated with MCs-pcDNA-LolP achieved a relative percent survival (RPS) of 69.2%, which was higher than that of the other control groups. Collectively, these findings indicate that MCs-pcDNA-LolP provides effective immune protection and represents a promising DNA vaccine candidate against A. veronii infection in crucian carp.