Oral pcDNA3.1-VP4/VP56-FlaC DNA vaccine encapsulated by chitosan/sodium alginate nanoparticles confers remarkable protection against GCRV infection in grass carp

Grass carp hemorrhagic disease caused by grass carp reovirus (GCRV) results in huge losses to the grass carp industry. In previous studies, DNA vaccination provided an efficient strategy for combating viral infection, but biodegradation and adverse environment hindered its application. In this study, Chitosan/sodium alginate (CS/SA) nanoparticles (NPs) loading system was applied to develop the oral DNA nano vaccine CS/SA/VP4C NPs and the oral DNA nano vaccine CS/SA/VP56C NPs to enhance its application effect. The recombinant plasmids pcDNA3.1-VP4C and pcDNA3.1-VP56C were constructed to express GCRV major outer capsid protein VP4 fusing Aeromonas hydrophila flagellin C (VP4C) and GCRV fiber protein VP56 fusing A. hydrophila flagellin C (VP56C), respectively. The oral DNA nano vaccines were about 100 nm in size with a regular spherical structure observed by transmission electron microscope. In addition, the fusion proteins VP4C and VP56C could be efficiently expressed in both fathead minnow cells and grass carp intestine epithelial cells by indirect immunofluorescence and western blotting analysis. The CS/SA/VP4C NPs and CS/SA/VP56C NPs were orally vaccinated with grass carp to evaluate the immune protective effect. The results showed that CS/SA/VP4C + VP56C NPs group had the highest protective effect that the survival rate increased by 42% compared with the control group. In addition, CS/SA/VP4C + VP56C NPs treatment could rapidly up-regulate serum innate immune indexes (total superoxide dismutase, lysozyme and complement C3), and significantly increase the mRNA expression of IFN1, MHC-IIα, IL-1β, TNFα, IgM, and IgT. Meanwhile, CS/SA/VP4C + VP56C NPs treatment greatly inhibited the replication of GCRV in the hindgut, spleen and head kidney. These results suggested that oral administration of CS/SA/VP4C + VP56C NPs could effectively prevent GCRV infection by improving adaptive immunity and innate immunity. Therefore, our study provides a new strategy for the fish viral disease prevention, which also establishes a theoretical basis for developing multiple DNA vaccines in the aquaculture industry.