Genome-based screening and immunogenicity evaluation of outer membrane protein antigens for Vibrio harveyi subunit vaccine in pearl gentian grouper (Epinephelus lanceolatus × Epinephelus fuscoguttatus).

Vibrio harveyi, a major aquatic pathogen prevalent in tropical and subtropical waters, causes substantial economic losses in global mariculture. This study focused on developing subunit vaccines targeting the outer membrane proteins of a new highly virulent strain of V. harveyi, which are recognized as key protective antigens. We performed whole-genome sequencing of a highly virulent endemic strain, followed by systematic screening and identification of immunogenic OMPs. Two candidate antigens, OmpW and OmpK, were successfully expressed and purified using a prokaryotic expression system. To assess the protective efficacy of the vaccine, pearl gentian grouper (Epinephelus lanceolatus × Epinephelus fuscoguttatus) were immunized via intraperitoneal injection, followed by a challenge with a virulent strain of V. harveyi. Comprehensive genomic characterization of the pathogen indicated the presence of a wide array of virulence-associated genes, from which 17 antigenic proteins were selected as potential candidates for vaccine development. Compared to the GST control group, grouper immunized with recombinant OmpW or OmpK showed significantly higher serum antibody titers (P < 0.05) and substantial upregulation (2.15-48.6 fold) of key immune-related genes (MHCII and IL1β) in head kidney tissues. Notably, vaccinated fish exhibited a 38.7-50 % increase in serum lysozyme activity, confirming the activation of innate immune mechanisms. These findings collectively demonstrate that OmpW and OmpK provide comprehensive immune protection through synergistic enhancement of both adaptive (specific antibody production) and innate (lysozyme-mediated) immune responses. Histopathological examination confirmed the protective effects, showing significantly reduced inflammatory lesions and cellular necrosis in vital organs (head kidney, spleen, and liver) of immunized fish. Our results demonstrate that recombinant OmpW and OmpK proteins induce comprehensive immune protection through synergistic activation of both humoral and cellular immune pathways, establishing a foundation for developing effective V. harveyi subunit vaccines.