A spleen-targeted mRNA nanovaccine boosts antiviral immunity via mTORC1-driven metabolic reprogramming in a fish model

Viral diseases continue to challenge the sustainability of aquaculture, contributing to large-scale mortality and persistent economic loss, while effective vaccines that can induce long-lasting adaptive immunity in fish are still lacking. Nervous necrosis virus (NNV), a neurotropic pathogen with a broad host range, is among the most damaging viruses in marine species and is therefore widely used as a model for investigating antiviral responses in teleost. In parallel, mRNA-based vaccines have emerged as a versatile platform for antigen delivery, yet their use in fish remains limited by suboptimal tissue targeting and an incomplete understanding of how metabolic pathways shape immune activation. In this study, we designed a spleen-targeted lipid nanoparticle mRNA vaccine encoding the NNV capsid protein and used the NNV infection model to explore the contribution of mechanistic target of rapamycin complex 1 (mTORC1) to vaccine-induced immunity. By adjusting lipid composition, we were able to shift in vivo expression from the liver toward the spleen, generating a stable formulation (LNP 18 -CP) that showed good biocompatibility and no detectable adverse effects in juvenile grouper. Immunization with LNP 18 -CP markedly improved survival following lethal challenge, reduced viral burdens in key tissues, and was accompanied by enhanced activation of antigen presentation pathways, expansion of T- and B-cell populations, and production of antigen-specific IgM and neutralizing antibodies. Metabolomic analysis of spleen tissue indicated broad metabolic remodeling after vaccination, particularly involving amino acid, lipid, and energy metabolism, with consistent enrichment of mTOR-related pathways. When mTORC1 activity was inhibited by rapamycin, lymphocyte responses and antigen-presenting functions were attenuated, viral replication increased, and overall protection declined. Conversely, improving intracellular delivery with L-leucyl-L-leucine methyl ester (LLME) further strengthened mTORC1 activation and adaptive immune responses, leading to enhanced antiviral efficacy. Together, these results support a model in which mTORC1-dependent immunometabolic reprogramming underlies the protective effect of this spleen-targeted mRNA nanovaccine, and they provide a practical framework for the development of similar strategies against viral diseases.