Study on immune response and protective efficacy of Vibrio alginolyticus flagellin FlaD and FlaF subunit vaccine combined with Montanide™ ISA 763B VG adjuvant in pearl gentian grouper (♀ Epinephelus fuscoguttatus × ♂ Epinephelus lanceolatus)

Vibrio alginolyticus is a major mariculture pathogen that causes vibriosis and heavy losses in many high-value fish species, such as the pearl gentian grouper. To provide an antibiotic-sparing alternative, we evaluated the immunogenicity and protective efficacy of recombinant flagellin subunits rFlaD and rFlaF, single or formulated with the water-in-oil adjuvant Montanide™ ISA 763B VG for pearl gentian grouper. Recombinant proteins were expressed in Escherichia coli , then purified, and used for intraperitoneal vaccination. At 6-week post-vaccination, the relative percent survival was determined. Immune responses were assessed by measuring serum lysozyme, catalase, and superoxide dismutase activities, quantifying antigen-specific IgM titers via indirect ELISA, and analyzing the expression profiles of immune-related genes. Both antigens conferred protection with relative percent survival values of 62.80 % for rFlaD and 58.67 % for rFlaF, but increased to 79.33 % and 73.43 % in the adjuvant group, respectively. Vaccination elevated the serum lysozyme, catalase, and superoxide dismutase activities, peaking at 3- to 4-week post-vaccination. Antigen-specific serum IgM also rose significantly, with the adjuvanted rFlaD group reaching the highest titer of 1:1280. RT-qPCR showed up-regulation of the immune-related genes in spleen and head kidney, indicating the activation of both innate and adaptive pathways. Collectively, the rFlaD combined with the Montanide™ ISA 763B VG formulation demonstrates strong potential as a subunit vaccine. Its efficacy is augmented by the dual functionality of the flagellin subunit, which acts as both a protective antigen and an inherent immunostimulant, offering a sustainable approach to combat V. alginolyticus in pearl gentian grouper aquaculture. • The vaccine significantly induces the expression of regulatory cytokines. • The adjuvant significantly enhances the immune response. • The rFlaD + Montanide™ ISA 763B VG vaccine formulation is highly effective. • Vibrio alginolyticus flagella acts both as an antigen and as an adjuvant.