Immunogenicity and protective efficacy of Vibrio vulnificus outer membrane vesicles in zebrafish: Implications for fish immunization.

Outer membrane vesicles (OMVs) from Vibrio vulnificus were evaluated for immunogenicity, safety, and protection in a zebrafish model. OMVs were isolated at laboratory scale by differential centrifugation and sterile filtration. Vesicles were characterized by nanoparticle tracking analysis, transmission electron microscopy, and BCA protein assay. Zebrafish (Danio rerio) were immunized intraperitoneally with graded OMV doses and subsequently challenged with V. vulnificus. Safety was assessed by clinical observation, serum biochemical indices, and histopathology of target tissues. Protection was evaluated by survival analysis. Transcriptome profiling of immune-relevant organs, together with qRT-PCR validation, was used to delineate immune pathways. OMV immunization was well tolerated, with no apparent cytotoxicity or histopathological alterations, and liver enzyme activities comparable to controls. Immunized fish exhibited dose-dependent protection with significantly higher survival following bacterial challenge versus PBS controls. Transcriptomic analyses indicated enrichment of cytokine signaling, leukocyte activation, and antigen processing and presentation; upregulation of genes such as MHC class II, CXCL12, and GBP-3 was consistent with initiation of adaptive immune pathways. These results indicate that V. vulnificus OMVs can safely stimulate balanced immune responses and confer protection in zebrafish, supporting their potential as immunoprophylactic candidates in aquaculture. Practical considerations for scale-up and immersion/oral deployment are discussed.