Oral Vaccine Against Ipnv Based On Antibiotic-Free Resistance Recombinant Lactobacillus Casei Expressing Ck6-Vp2 Fusion Protein

Infectious pancreatic necrosis (IPN) is a disease caused by the infectious pancreatic necrosis virus (IPNV). The condition is associated with highly fatal hemorrhage and putrescence of hepatopancreas in fry and juvenile salmonids, resulting in significant economic losses. The conventionally developed recombinant Lactobacillus casei oral vaccines against IPNV use antibiotic resistance as a selectable marker, which is linked to the development of antibiotic resistance, and this poses serious threats to biosafety. Herein, we developed a safe and effective oral vaccine against IPNV by replacing the chloramphenicol resistance (Cmr) marker in the L. casei expression system with an enhanced green fluorescent protein (eGFP) as a reporter gene. The rainbow trout chemokine (CK6) was used as a target molecule for antigen-presenting cells co-expressed with IPNV VP2. We constructed and evaluated the immunogenicity of the antibiotic-free selection marker pPG(F)-612-CK6-VP2-eGFP/L. casei 393. The levels of anti-IPNV-specific IgM and IgT of rainbow trouts immunized with pPG(F)-612-CK6-VP2-eGFP/L. casei 393 were significantly higher than those of phosphate-buffered saline (PBS) or pPG-612/L. casei 393 groups. Also, the candidate vaccine exhibited neutralizing effects against IPNV. The viral loads of IPNV in the pPG(F)-612-CK6-VP2eGFP/L. casei 393 group were significantly lower than those in the control groups. These results indicate that the antibiotic-free resistance genetically engineered L. casei oral vaccine can provide a safe and promising strategy for the development of recombinant L. casei vaccine against IPNV.