Abstract:
Infectious pancreatic necrosis virus (IPNV) remains a critical challenge in salmonid aquaculture, with conventional vaccines often presenting limited immunogenicity and labor-intensive production (Rimstad 2014). Recognizing that fish nucleated red blood cells (RBCs) are not only the most abundant cells in circulation but also have emerged as pivotal immune actors—capable of antigen processing, presentation, and cytokine production—we developed a recombinant, modular nanostructured vaccine (nanopellets, NPs) to target this cell type as ideal carriers for systemic vaccine delivery (Nombela and Ortega-Villaizan 2018, Puente-Marin, Nombela et al. 2018). These NPs consist of the IPNV-VP2 capsid protein, either alone (IPNVNP) or fused to rainbow trout interferon gamma (IFNγ) to generate an adjuvanted construct (IPNV-IFNγNP), while a fluorescent iRFPNP served as a control. NP constructs were expressed in E. coli and characterized by electron microscopy, which confirmed their stable nanostructured assembly under extreme conditions relevant for aquaculture.
In vitro assays with purified rainbow trout RBCs demonstrated that IPNV-IFNγNP were efficiently internalized by RBCs in a dose- and time-dependent manner, outperforming non-adjuvanted counterparts. Intraperitoneal administration of IPNV-IFNγNP led to enhanced presence in blood as well as in primary immune organs such as the head kidney and spleen. RNA sequencing (RNA-seq) analysis identified over 1,800 differentially expressed genes in IPNV-IFNγNP-treated RBCs that were enriched in interferon signaling, vesicle trafficking, and mitochondrial pathways. Also, the transcriptomic profiling of RBCs from immunized fish revealed the robust upregulation of antiviral genes (e.g., mx, vig1, isg15, and ifit5).
These results underscore the potential of harnessing RBCs as systemic antigen carriers to enhance innate and adaptive immune responses. Further, NP-based subunit vaccine is a promising strategy to promote more sustainable and welfare-friendly aquaculture practices. Ongoing work will focus on functionalizing the NP-based platform with ligands targeting RBCs to improve immunization strategies.
Acknowledgements:
This work was supported by the Agencia Estatal de Investigación, Spain (Retos Investigación: RTI2018-096957-B-C22).
References:
Nombela, I. and M. D. M. Ortega-Villaizan (2018). "Nucleated red blood cells: Immune cell mediators of the antiviral response." PLoS Pathog 14(4): e1006910.
Puente-Marin, S., I. Nombela, V. Chico, S. Ciordia, M. C. Mena, J. Coll, L. Mercado and M. D. M. Ortega-Villaizan (2018). "Rainbow Trout Erythrocytes ex vivo Transfection With a DNA Vaccine Encoding VHSV Glycoprotein G Induces an Antiviral Immune Response." Front Immunol 9: 2477.
Rimstad, E. (2014). Vaccination against Infectious Pancreatic Necrosis. Fish Vaccination: 303-312.