Evaluation Of Immune Efficacy For GCRV Vp6DNA Vaccine And Subunit Vaccine

Grass carp reovirus (GCRV), as a large family of double-stranded RNA (dsRNA) viruses, which is considered to be the most pathogenic aquareovirus, was first identified from a breakout of hemorrhagic disease affecting a vast majority (～85%) of fingerling and yearling grass carp from southern China. However, current vaccines include inactivated vaccine and attenuated vaccine. Inactivated vaccines can not enter the major histocompatibility complex (MHC) class I and can not effectively induce cytotoxic T cells (CTL) response, therefore, the immune effect is relatively not ideal and the immunity lasting is poor. The attenuated vaccines are high production costs, virulent atavism, poor security and have potential danger that come around to the pathogenic. Fish vaccines conventionally can be administered by soak and injection. Although the soak administration is simple, requires a lot of vaccine. However, the injection administration has certain specifications on the size of vaccinated fish, high operating strength, high technical requirements, the difficult of technology promotion and causing greater damage to fish in catching and injecting process. Therefore, more efficient and economic oral subunit/DNA combination vaccine which is secure, reliable, efficient multivalent and easy to use against grass carp hemorrhage is urgently desired in China.1. Evaluation of immune efficacy for GCRV vp6DNA vaccineTo evaluate the immune efficacy of grass carp reovirus (GCRV) vp6DNA vaccine, the baculovirus transfer vector pFastBacTMdual-derived DNA vaccine vector pFastBac-FA-VP6-ph-VP6was constructed, in which a vp6gene was controlled by baculovirus polyhedron (Ph) promoter, the another vp6expression cassette driven by the β-actin promoter of Megalobrama amblycephala was at the downstream of the baculovirus p10promoter. In vaccinated group the grass carp (60-120g in body weight and14-20cm in body length) were infected with10,30and60μg of the vaccine vector, respectively;30μg of pFastBacTMDual was injected in negative control group and0.4ml of sterile water was injected in unvaccinated control group. RT-PCR analyzed the vp6expression in vaccinated fish, as well as antibody titers in the blood were determined by indirect agglutination reaction on the14th,21st,28th,49th and70th days post-vaccination and immune efficacy was evaluated by the challenge of GCRV on the21st day post-vaccination. The results showed that the specific antibody could be detected and antibody level reached peak on the28th day after immunization in all vaccinated groups. The mortality of fish were0%,0%and5%respectively in vaccinated group injected with10,30and60μg of DNA vaccine, the mortalities were30%and100%respectively in negative control group and unvaccinated control group. All results suggested that the DNA vaccine had good immuno-protective efficacy to the challenge of GCRV.2. Prokaryotic expression VP6protein of GCRV and immune efficacy studiesIn order to study the immuno-protective efficacy of GCRV-VP6subunit vaccine, the recombinant expression vector pET28a(+)-VP6was constructed by cloning vp6gene of GCRV into the prokaryotic expression plasmid pET-28a(+). The results of SDS-PAGE and Western blotting showed that the molecular mass of the recombinant VP6protein was approximately43kDa. The recombinant protein reached20%of the total bacterial proteins, was mainly in the form of insoluble bodies. The grass carp(60～120g in body weight and14～20cm in body length) was immunized with500μg purified VP6protein by Ni2+affinity chromatography and antibody titers in the blood of fish were determined by means of indirect agglutination reaction on the14th,21st,28th,49th and70th days post-vaccination. The results showed that the specific antibody could be detected on the14th day, reached peak on the21st day and could be still detected on the70th day after immunization. The fish in vaccinated group and control group were challenged with GCRV on21st days after vaccine delivery and the relative survival rate in the vaccined group was100%. These results provided important academic foundation for research and development of GCRV genetic vaccine.3. Evaluation of immune efficacy for GCRV vp6subunit/DNA vaccine by oral administrationBaculovirus was adopted for high capacity production effectively and gene delivery into mammalian cells safely. To obtain an oral GCRV vp6subunit/DNA vaccine which is safe and reliable, efficient and convenient to use, in the study, a donor vector pFastBac-FA-VP6-ph-VP6containing two GCRV vp6genes, one driven by the Megalobrama amblycephala β-actin promoter and another controlled by baculovirus polyhedrin promoter was constructed to generate the recombinant baculovirus BacFish-VP6by Bac to Bac. From the hemolymph of5th instar silkworm inoculated with BacFish-VP6, a53kDa recombinant VP6protein could be detected and the peak expression was at120h post-inoculation with BacFish-VP6, accounting for5%of the total haemolymph protein with SDS-PAGE and Western blotting. And the infected pupae collected at120h post-inoculation with BacFish-VP6were used to make freeze-dried powder as an oral vaccine. When the grass carps were orally administrated with feed (plus2.5%starch) containing1%,5%and10%of the freeze-dried powder, their specific antibody against VP6could be detected and elevated in a dose-dependant manner, with the highest antibody level occurring at3-4weeks post-vaccination. Moreover, RT-PCR analysis showed that vp6transcripts could be detected in the vaccinated fish, and immunohistochemistry analysis showed that VP6protein was detected in the CIK cells infected with BacFish-VP6and the liver and kideny tissues of orally vaccinated fish. All the results suggested that vp6gene in the recombinant baculovirus could be successfully delivered into the fish cells and expressed correctly, stimulating specific immunereaction in fish. The initiatory findings suggested the powder of the silkworm pupae infected with BacFish-VP6could possess simultaneously the roles of subunit vaccine and DNA vaccine and can induce specific antibody in fish be used as an orally administered vaccine.