Design and Evaluation of Universal Multi‐Epitope Vaccines Against <i>Vibrio</i> Infections Using a Single/Multi‐Target Multi‐Derived Epitope Strategy

Vibrio vaccines play an important role in the prevention of Vibrio infections and in the sustainable development of the aquaculture industry. However, the genetic diversity and antigenic variation among Vibrio species make broad-spectrum protection a major challenge for conventional vaccine development, which often relies on inactivated whole cells or single-subunit antigens. To overcome this limitation, we developed a novel single/multi-target multi-derived epitope strategy, defined by two parameters: the number of target outer membrane proteins (OMPs) (single- or multi-target) and the incorporation of homologous epitope sequences from diverse Vibrio species (multi-derived). Four conserved epitopes, two immunodominant (K2, B3) and two cryptic (K7, U2), were identified from OMPs (OmpK, OmpU, LamB). Two multi-epitope vaccines were designed and constructed: EVK-10 (single-target multi-derived) and EVK-20 (multi-target multi-derived). Following prokaryotic expression and purification, the resulting vaccines were used to immunise zebrafish. Both vaccines subsequently induced substantial antibody responses, with the titers peaking at 21 days post-vaccination. Challenge trials demonstrated that both vaccines provided broad cross-immunoprotection against five heterologous Vibrio strains (V. parahaemolyticus, V. alginolyticus, V. harveyi, V. anguillarum, V. vulnificus). EVK-10 achieved relative percent survival (RPS) values of 59.3%, 67.9%, 53.3%, 36.0% and 45.8%, respectively, while EVK-20 showed superior RPS of 74.1%, 75.0%, 73.3%, 68.0% and 66.7%. In contrast, the single-derived rOmpK (V. parahaemolyticus derived) control induced strong protection only against closely related species (V. parahaemolyticus: 85.5%; V. alginolyticus: 82.1%) but lower protection against distantly related ones (V. anguillarum: 24.0%; V. vulnificus: 29.2%). These results demonstrate that the multi-derived strategy confers broader cross-protection than single-derived vaccines, and the multi-target approach (EVK-20) provides stronger and more redundant immunity than the single-target design (EVK-10). This study validates the single/multi-target multi-derived epitope strategy as an effective platform for developing universal vaccines against complex bacterial pathogens like Vibrio.