The recombinant subunit vaccine encapsulated by alginate-chitosan microsphere enhances the immune effect against Micropterus salmoides rhabdovirus.

The disease caused by Micropterus salmoides rhabdovirus (MSRV) has brought substantial economic losses to the largemouth bass aquaculture industry in China. Vaccination was considered as a potential way to prevent and control this disease. As a kind of sustained and controlled release system, alginate and chitosan microspheres (SA-CS) are widely used in the development of oral vaccination for fish. Here, we prepared a king of alginate-chitosan composite microsphere to encapsulate the second segment of MSRV glycoprotein (G2 protein) and then evaluated the immune effect of the microsphere vaccine on largemouth bass. Largemouth bass were vaccinated via intragastric immunization by different treatments (PBS, SA-CS, G2 and SA-CS-G2). The results showed that a stronger immune response including serum antibody levels, immune-related physiological indexes (acid phosphatase, alkaline phosphatase, superoxide dismutase and total antioxidant capacity) and the expression of immune-related gene (IgM、IL-8、IL-1ß、CD4、TGF-ß、TNF-α) can be induced obviously with SA-CS-G2 groups compared with G2 groups when fish were vaccinated. Furthermore, fish were injected with a lethal dose of MSRV after immunization for 28 days, and the highest relative percentage survival (54.8%) was observed in SA-CS-G2 group (40 µg per fish), which is significantly higher than that of G2 group (25.8%). This study showed that alginate-chitosan microspheres as the vaccine carrier can effectively improve the immune effect of oral vaccination and induce better immune protection effect against MSRV infection.

Antigenic epitope screening and functional modification of mannose enhance the efficacy of largemouth bass virus subunit vaccines.

Major capsid protein (MCP) can be used as a subunit vaccine against largemouth bass virus (LMBV). However, subunit vaccines usually have low immunogenicity. Here, to identify the major immunogenicity determinant region of the MCP gene, we truncated the MCP of the LMBV gene into four parts (MCP-1, MCP-2, MCP-3 and MCP-4). Enzyme-linked immunosorbent assay (ELISA) was used to identify the antigenicity of these four truncated MCP proteins. Then, the highly antigenic truncated protein was modified with mannose and connected with functionalized single-walled carbon nanotubes (SWCNTs) as carriers. Largemouth basses were immunized by bath immersion, challenged with LMBV on the 28th day after immunization and evaluated for related immune indicators. The results indicated that the MCP-2 protein could induce a higher antibody titre than the other truncated MCP proteins. We found that the levels of immune-related genes (TNF-α, CD40, IgM, IFNγ and IL-10) in the spleen and kidney were significantly increased in the MCP-2 and MCP-2-Man groups. ELISA results showed that the antibody content in the serum increased significantly in the MCP-2 group 7 days post-vaccination and increased with days in all the vaccinated groups, with the highest observed on the 21st day. Notably, the MCP-2-Man vaccine (10 mg L-1 ) showed durability of immunoprotection efficacy that could protect largemouth basses from LMBV challenge, and the immune protection rate reached 78.94%. These results suggest that MCP-2 might be the major immunogenicity determinant region of LMBV and that the mannose-modified MCP-2 vaccine can induce stronger adaptive immune responses.

Protective immunity against spring viremia of carp virus by mannose modified chitosan loaded DNA vaccine.

Spring viremia of carp virus (SVCV) usually be considered as one of the serious in viral diseases of aquaculture, and DNA vaccine with novel delivery mechanism or adjuvant has proven to be a promising and effective strategy to control aquatic animal diseases. In this study, the mannose-modified chitosan, a carrier system for vaccine delivery, were used to developed a chitosan-encapsulated DNA vaccine (CS-M-G) against SVCV, then investigated immune response induced by the vaccine. Our results showed that CS-M-G was confirmed the spherical or elliptical with even distribution and ranging from approximately 50 to 150 nm in size, the expression of the antigen gene could still be detected after 21 d post vaccination. The CS-M-G induces the highest antibody levels in the 20 µg dose group which is about 3 times than naked plasmid group at 21 d post vaccination, and still hold a higher level than control group at 28 d post vaccination. On the side, strongest protection with relative percent survival of 62.1% in the 20 µg CS-M-G group, which could produce significantly higher enzyme activities and up-regulated expression of immune-associated genes than control group. Thus, our results indicate that DNA vaccine loaded with mannose-modified chitosan induces strong immune response and provided an effective protection against SVCV infection, may be helpful and extended for developing more aquatic animal vaccines in the future.