N1MΨU-modified mRNA vaccines break the mold in fish by enhancing innate immune activation

Messenger RNA (mRNA) vaccines have revolutionized immunization strategies in mammals, with chemical modifications such as N1-methyl-pseudouridine (N1MΨU) enhancing stability, translation, and reducing innate immune activation. However, the immunological implications of such modifications in non-mammalian species, particularly teleost fish, remain unclear. In this study, we evaluated the innate immune responses elicited by four vaccine platforms: a DNA vaccine, a live attenuated viral hemorrhagic septicemia virus (VHSV), an unmodified mRNA, and an N1MΨU-modified mRNA. All four vaccines encode the G protein of VHSV (G_VHSV) in rainbow trout. Following intramuscular injection, both mRNA vaccine formats induced robust type I interferon (IFN) responses, comparable to those induced by the DNA and attenuated virus vaccines. Notably, the N1MΨU-modified mRNA vaccine did not suppress IFN induction, as observed in mammalian systems, but instead triggered distinct temporal transcriptional dynamics and stronger enrichment of autophagy, ubiquitination, and transcription-related pathways. These findings suggest that modified mRNA retains a strong immunostimulatory capacity in fish and may activate immune pathways differently than in mammals. Our results emphasize the need to reassess assumptions from mammalian vaccine design when translating mRNA vaccine strategies to non-mammalian vertebrates and highlight the potential for tailored mRNA vaccine development in aquaculture species.