Recombinant turbot IFN-γ as a molecular adjuvant enhances the protective efficacy of an inactivated Aeromonas salmonicida vaccine.

The development of potent adjuvants is pivotal to improving the immunogenicity and protective efficacy of inactivated vaccines in aquaculture. This study evaluated the potential of recombinant turbot interferon-gamma (rIfn-γ) as a molecular adjuvant, when administered alone or in combination with aluminum hydroxide (Al), to enhance the efficacy of an inactivated Aeromonas salmonicida vaccine. Turbot were immunized with one of eight formulations: PBS (control), aluminum hydroxide (Al), rIfn-γ, rIfn-γ+Al, formalin-killed cells (FKC), FKC+Al, FKC+rIfn-γ, or FKC+rIfn-γ+Al. All formulations exhibited excellent safety profiles, with no mortality or adverse clinical signs observed in any experimental groups. Following challenge with A. salmonicida at 28 days post-vaccination, the FKC+rIfn-γ+Al group achieved the highest relative percent survival (RPS, 53.3%), significantly outperforming the FKC-alone group (30.0%), and surpassing the FKC+Al group (45.0%) and FKC+rIfn-γ group (46.7%). This group also elicited the strongest antigen-specific serum IgM response. Mechanistically, transcriptional analysis of immune-related genes in the spleen revealed that the rIfn-γ+Al composite adjuvant induced a robust, sequential immune activation cascade: early upregulation of pro-inflammatory cytokines (tnf-α and il-1β), followed by induction of Th1-polarizing cytokines (il-12 and ifn-γ) that bridge innate and adaptive immunity, concurrent with sustained upregulation of antigen presentation genes (mhc-i and mhc-ii), and culminating in late-stage activation of T-cell markers (cd4 and cd8). These findings demonstrate that rIfn-γ functions as an effective molecular adjuvant for the development of turbot vaccines. Moreover, this study establishes a novel cytokine-mineral salt composite adjuvant strategy, offering significant potential for the development of next-generation inactivated vaccines for aquaculture.