Preparation And Application Of Oral Microencapsulated Vaccine Based On Grass Carp Reovirus (GCRV-Ⅱ) VP56 Protein

Grass carp is an important economic fish and occupies an important position in the freshwater aquaculture industry in China.Grass carp hemorrhagic disease caused by grass carp reovirus(GCRV)brings huge economic losses to the aquaculture industry every year.GCRV has three genotypes,among which GCRV-II is the most infectious and lethal,and the VP56 protein encoded by the GCRV-II S7 gene segment is highly immunogenic.Oral vaccines are an ideal method to prevent grass carp hemorrhagic disease in the aquaculture industry.However,easy antigenic degradation and low immunogenicity are the main drawbacks of oral vaccines.In this study,we selected the VP56 protein as the antigen,and the flagellin Fla B as the adjuvant for this vaccine.To overcome the difficulty that the antigen is easily degraded,we used sodium alginate as the delivery vehicle.Based on this,we prepared an oral microencapsulated vaccine based on grass carp reovirus(GCRV-II)VP56 protein and adjuvant Fla B delivered by sodium alginate,and evaluated its protective effect against GCRV-II infection in grass carp.This work was divided into three parts: antigen epitope screening,preparation of sodium alginate oral microcapsuled vaccine and in vivo testing.1.VP56-3 is the optimal antigenic epitope regionWe analyzed the structure of VP56 protein by bioinformatics software,divided VP56 into three fragments based on its hydrophilicity and antigenic epitopes and expressed the full length of GCRV-II VP56 gene and three potential antigenic epitope regions in Escherichia coli,and purified the recombinant protein by glutathione affinity columns,respectively.Analysis by enzyme-linked immunosorbent assay(ELISA)and neutralizing antibody assay revealed that the VP56-3 fragment had the strongest binding ability to the grass carp anti-GCRV-II serum,and the grass carp serum produced by this fragment could effectively inhibit the cell damage by GCRVII with the strongest effect,and the cell survival rate of the VP56-3 fragment group was still 20% under the dilution condition of 1:40.2.Oral microencapsulated vaccines are spherical and have good stability,slow release and low toxicityThe oral microcapsuled vaccine(SA-VP56-3/Fla B)was prepared by encapsulation method using sodium alginate as a carrier to encapsulate VP56-3 and adjuvant protein Fla B.The microcapsules were analyzed by dynamic light scattering experiments with a particle size of 1.24 ± 0.22 μm,a PDI of 0.38 ± 0.01 and a zeta potential of-18.60 ± 0.47 m V,which had good stability.The morphological characteristics were observed by transmission electron microscopy and scanning electron microscopy,and the microcapsules showed a more regular spherical structure with obvious folds and grooves on the surface.The in vitro release assay,in vivo fluorescence imaging system,hemolytic activity and cytotoxicity analysis revealed that SA-VP56-3/Fla B was able to resist the hydrolysis of grass carp intestinal protease,with good slow release and low toxicity.3.Oral microencapsulated vaccine enhances the body’s immune response and significantly improved protection against GCRV infection in grass carpWe orally inoculated grass carp with SA-VP56-3/Fla B and measured immunerelated parameters(serum neutralizing antibodies,specific antigen binding capacity of serum,serum enzyme activity(TSOD,LZM,C3),immune-related genes((Ig M,IFN1,MHC-II,CD8)in head kidney and spleen,Ig Z in hindgut)).The results showed that SA-VP56-3/Fla B significantly induced a strong immune response compared to the other groups,that the serum in the SA-VP56-3/Fla B group had neutralizing activity and was the most potent inhibitor of the virus,with a cell survival rate of 82% at a 1:1dilution,and that antibodies with neutralizing activity contributed to the host’s immune defense and inhibited virus transmission through the systemic circulation;SA VP56-3/Fla B group also had the strongest serum binding capacity to specific antigens and still effectively bound to the antigenic protein VP56-3 at a 1:2000 dilution.Analysis of serum enzyme activity revealed that the SA-VP56-3/Fla B group was able to increase the content and activity of TSOD,LZM,and C3,and the increase in the activity of these indicators may be due to an increase in the number of immune cells associated with the process or an increase in host immunity.Analysis of the expression trends of immune-related genes revealed that the SA-VP56-3/Fla B group was able to significantly upregulate the expression of Ig M,IFN1,MHC-II,CD8 in the head kidney and spleen,as well as Ig Z in the hindgut,inhibiting viral replication.After 2 weeks of GCRV challenge,the SA-VP56-3/Fla B group had the highest survival rate(56%)compared to the control group(10%)and the SA-VP56-3/Fla B group had the lowest tissue viral load in surviving grass carp.These results indicate that the SA-VP56-3/Fla B vaccine is effective in protecting grass carp from GCRV-II infection.In this study,a valuable potential antigenic epitope region of GCRV-II was screened as a candidate antigen and SA-VP56-3/Fla B oral microencapsulated vaccine was prepared using flagellin B(Fla B)as an adjuvant and sodium alginate as a delivery system.After immunization of grass carp,the immunization effect was evaluated using different indicators,and the vaccine was found to have good protection against grass carp with 56% protection,which indicated that SA-VP56-3/Fla B could be a candidate oral vaccine against GCRV-II infection in aquaculture.