Oral Lactobacillus casei expressing VP56310-500 and adjuvant flagellin C delivered by alginate-chitosan microcapsules remarkably enhances the immune protection against GCRV infection in grass carp

Due to grass carp haemorrhagic disease caused by grass carp reovirus (GCRV), aquaculture industry suffers huge economic loss. Oral Lactobacillus casei vaccine, with its excellent safety and effectiveness, is a promising strategy to prevent infectious diseases. While, the oral vaccine encapsulated with alginate-chitosan (SA/CS) can further improve its immune function. In this study, recombinant L. casei (rLc) expressing multiple fragments of VP56 was constructed, which was a key outer capsid protein of GCRV. By mixing rLcs with fodder and fed grass carp, the corresponding anti-VP56 fragment sera was produced, and VP56310-500 was screened as optimal fragment of GCRV-II by adopting enzyme linked immunosorbent assay (ELISA). Furthermore, SA/CS was used as the encapsulating material of L. casei live vaccine expressing VP56310-500, thereby preparing oral microcapsule live vaccine SCL-56310-500. With good resistance to bile (0-0.4%), NaCl (0-0.8%), simulated intestinal fluid, and simulated gastric fluid (pH = 1.5-4.5). Also, it had the ability to maintain high viability after colonizing intestine of grass carp in 7 days. In addition, Flagellin C (FlaC) was adopted as an immune adjuvant, and rLc was con-structed to express VP56310-500-FlaC (56310-500C), thereby enhancing immune effect of oral live vaccine. After oral administration of encapsulated L. casei vaccine, its protective effect on GCRV-II infection was evaluated. As indicated by the results, SCL-56310-500C effectively up-regulated the activity of serum biochemical enzymes (including TSOD, lysozyme, and complement C3), and induced mRNA expression of important immune genes (including IL-1 beta, IFN1, MHC-II alpha, IgM, and IgT in immune tissues). Simultaneously, SCL-56310-500C greatly sup-pressed GCRV replication in head kidney, spleen, and intestine. Correspondingly, grass carp vaccinated orally with SCL-56310-500C have distinctly higher survival rate (58% versus 24% in control) against GCRV infection. These results illustrated that an oral probiotic vaccine SCL-56310-500C effective in GCRV infection prevention was developed. Thus, this study offered an inspiring strategy for the prevention of fish viral diseases.