The high prevalence of infectious diseases has made fish vaccination a major ambition for the aquaculture sector. Even though vaccines are one of the most cost-effective means of reducing economic losses due to viral and bacterial infections, their poor immunogenicity is still a concern. Furthermore, species diversity and limited knowledge of the fish immune system are themselves challenges in developing new vaccines. Interestingly, the most abundant cell type in fish blood, the erythrocytes or red blood cells (RBCs), have been implicated in several immune-related functions, such as antiviral response, phagocytosis, or cytokine-mediated signaling, and can even halt viral hemorrhagic septicemia virus (VHSV) infection. As part of our contribution, our next step was to evaluate rainbow trout RBCs as cellular targets of a recombinant protein subunit vaccine (referred to as nanopellet, NP) against VHSV with an incorporated ligand to RBCs and interferon-gamma (IFNγ) as an adjuvant. Previously, we demonstrated that the ligand-targeted NPs were successfully internalized by rainbow trout RBCs and led to the upregulation of genes associated with the antiviral immune response in vitro. In this study, RNA-Seq-based transcriptome analysis revealed the activation of functional pathways related to endoplasmic reticulum stress- and mitochondrial stress-related pathways, as well as uptake, secretion, and transport of vesicles in RBCs after immunization with ligand-targeted NPs. Even more, we successfully isolated extracellular vesicles (EVs) derived from RBCs stimulated with ligand-targeted NPs and we characterized them by confocal and electron microscopy. Further study of the miRNA cargo in EVs from fish RBCs will be an essential step in identifying promising modulating molecules and biomarkers for the development of next-generation vaccines.
Acknowledgements: This work was supported by the Agencia Estatal de Investigacion, Spain (Retos Investigacion: RTI2018-096957-B-C22) and Universidad Miguel Hernandez VIPROY 2022 program.