Erythrocyte-targeted recombinant subunit vaccine: A novel approach to improve viral vaccines in fish

Teleost erythrocytes or red blood cells (RBCs) are nucleated cells with an increasingly recognized role in antiviral defense. To explore the potential of RBCs as immune delivery-competent cells, we explored their response to nanostructured subunit vaccines (nanopellets [NPs]) based on viral antigens from viral hemorrhagic septicemia virus (VHSV) or infectious pancreatic necrosis virus (IPNV) functionalized with interferon-gamma (IFNγ). Additionally, we developed a synthetic RBC-targeting ligand added to the VHSV vaccine for cell-targeting. In ex vivo and in vivo assays, IFNγ enhanced NP uptake by RBCs and triggered an antiviral response via the upregulation of immune-related genes, including mx1-3, vig1, and ifit5. RNA sequencing analysis demonstrated activation by VHSV-IFNγ NP of classical immune pathways, including cytokine production, interferon signaling, and pattern recognition receptor signaling, whereas IPNV-IFNγ NP induced cellular stress responses, such as autophagy and NF-kappaB-related pathways. The addition of the RBC-targeting ligand to the VHSV construct further enriched IFNγ and cytokine signaling, galactoside-binding, endoplasmic reticulum stress, and antigen presentation pathways. Our findings support the innovative use of fish RBCs as a dual platform for immune stimulation and antigen dissemination, positioning them as a promising cellular target in next-generation vaccine strategies for aquaculture.