Protection of Japanese flounder (Paralichthys olivaceus) against Vibrio anguillarum with a DNA vaccine containing the mutated zinc-metalloprotease gene

Vibrio anguillarum is one of the causative agents of vibriosis, a systemic disease of fish characterized by acute hemorrhagic septicemia. The extracellular zinc metalloprotease (EmpA) is a putative virulence factor involved in pathogenicity of V. anguillarum. Here we described the results of immunization against V. anguillarum with the plasmid expressing the mutated EmpA (m-EmpA7), which had no protelytic activity or cytotoxicity. In vitro protein expression of m-empA7 gene was determined by fluorescent microscopy and Western-blot after transfection of Chinese hamster ovary (CHO) and human embryonic kidney (HEK293T) cell lines. All three groups of fish immunized with a single intramuscular (i.m.) injection of different doses of the m-EmpA7 DNA vaccine showed significant serum antibody levels after vaccination, compared with the fish injected with the control eukaryotic expression vector pEGFP-N1 and PBS. In addition, fish receiving the DNA vaccine developed a protective response to a live V. anguillarum challenge 4 weeks post-inoculation, as demonstrated by increased survival of vaccinated fish over the control and by decreased histological alterations in vaccinated fish. Furthermore, humoral immune responses and protective effects were significantly increased at higher vaccine doses using a single intramuscularly injection route.