Plasmid-DNA Encoded gata3 and eomes Increase Antibody Response and Performance of an Inactivated Salmonid Alphavirus Subtype 3 Vaccine

Background: Pancreas disease, caused by salmonid alphavirus subtype 3 (SAV3), remains a significant threat to Atlantic salmon (Salmo salar L.) aquaculture in Norway despite the widespread use of inactivated vaccines. Molecular adjuvants represent a promising strategy to improve vaccine efficacy by enhancing immune responses. Methods: In this study, we evaluated the immunological and protective effects of two transcription factors, gata3 and eomes encoded by plasmid DNA adjuvants in combination with a low-dose inactivated SAV3 vaccine. Atlantic salmon were immunized with inactivated virus alone or in combination with either gata3- or eomes-encoding plasmids, followed by SAV3 challenge. Vaccine performance was assessed by growth, antibody response (ELISA and virus neutralization assay), and histopathological scoring of heart and pancreas. Results: The effects of gata3 and eomes were context-dependent: eomes reduced pancreas lesions at 4 weeks post-challenge, while both plasmid-adjuvant groups showed improved growth at 10 weeks post-challenge compared to the plasmid control. Although antibody responses were increased in all vaccinated groups at 6 weeks post-immunization, the plasmid control performed comparably to the GATA3 or EOMES groups, and no significant differences were detected in the virus neutralization test. Interestingly, the empty plasmids given to control fish exhibited immunostimulatory activity, warranting further investigation. In vitro and in vivo fluorescence microscopy confirmed expression of the transcription factors in transfected cells and at the injection site. Conclusions: Our results suggest that transcription factor-encoding plasmids can provide selective adjuvant effects, while also highlighting the potential that plasmid DNA can have as adjuvants in fish vaccine development.