Genetic Engineering Vaccines Against Rabbit Hemorrhagic Disease Virus

[Objective] Rabbit hemorrhagic disease (RHD) is a highly contagious and lethal infection that affects both wild and domestic rabbits. Its etiological agent, the rabbit hemorrhagic disease virus (RHDV), is considered to be the single most economically important disease of rabbits worldwide. Vaccination is the main method to prevent and ccontrol RHD.Because the lack of suitable cell culture system for RHDV has hindered large-scale production of the virus as a source of vaccine antigens, commercially available vaccines are still produced from tissues collected from experimentally infected rabbits. However, this strategy raises serious concerns about biological safety, contaminant residues and animal welfare. It has become a focus to develop safer and more effective vaccines for control RHD. The VP60protein has been implicated as main protein antigen in virus diagnosis and vaccine design, although several recombinant VP60proteins have been obtained from insect cell lines or in Pichia pastoris, the scaling up of these sources for antigen enrichment purposes is both difficult and expensive. The goal of this study was to develop a new subunit vaccine expressing the dominant antigen regions of VP60capsid fused with universal Th cell epitope and a new suicidal DNA vaccine expressing the capsid protein VP60based on a Semliki Forest virus (SFV) replicon the alpha virus replicon, which has been shown to potentially induce high-level humeral and cell-mediated immunity against a variety of antigens and provide protection for animals, and to evaluate its immunogenicity and protective immunity in rabbits.[Methods](1) The AB-Th gene of RHDV was cloned into pET-30a by overlapping PCR after transformed into E.coil BL21use the IPTG to induce the expression.In order to evaluated the immunes effect of subunit vaccine, recombination protein AB-Th was purified and did animal immunity test to mate with FA, AB,PBS was to be control group, detect by MTT for peripheral blood lymphocytes proliferation and Enzyme-linked immunosorbent assay(ELISA). IFN-y and IL-4ELISA kits were used to detect the production of cytokines. To test whether this vaccine could induce a protective effect against a lethal RHDV infection, the rabbits were infected with a wild type RHDV strain, JX/CHA/97.(2) The VP60gene of RHDV was cloned by RT-PCR and inserted into Semliki Forest virus (SFV) replicon-based plasmid, namely suicidal plasmid, pSCAl.The recombinant plasmid was confirmed by restriction enzyme analysis and nucleic acid sequencing and named pSCAl/VP60.Then the recombinant plasmid was transfected into BHK-21cells by Lipofectamine PlusTM Reagent. The capsid proteins of VP60expressed in BHK-21cells were confirmed by RT-PCR, western blot and indirect immunofluorescence assay (IFA).To evaluate the immunogenicity of semliki forest virus replicon-based recombinant plasmid, the New Zealand White Rabbits were vaccinated intramuscularly with pSCAl/VP60,pSCA, PBS was to be control group,and the immune responses induced by suicidal DNA vaccine were detected by MTT for peripheral blood lymphocytes proliferation and Enzyme-linked immunosorbent assay. IFN-y and IL-4ELISA kits were used to detect the production of cytokines. To test whether this vaccine could induce a protective effect against a lethal RHDV infection, the rabbits were infected with a wild type RHDV strain, JX/CHA/97.[Results:](1) The prokaryotes expression vector pET30a/AB-Th carrying the dominant antigen regions of VP60and universal Th cell epitope, has been constructed successfully, and the fused protein AB-Th was well expressed in BL21cells. After purified, the recombinant protein was inoculated into rabbits. The results showed that the recombination protein could induce both high level specific antibody against RHDV and strong cellular immune response in rabbits. For example, lymphocyte proliferation assay indicated that the recombination protein could induce obvious and intense lymphocyte proliferative responses in experimental rabbits than the control group, Cytokine assays also suggested that the mean levels of IFN-y and IL-4were significantly higher in the rabbits inoculated with AB-Th than those of rabbits inoculated with PBS.(2)To demonstrate expression of the RHDV VP60proteins, transfected BHK-21cells were analyzed by IFA. Cells transfected with pSCA/VP60showed specific green fluorescence, but the negative control, which was transfected with the same amount of pSCAl, and non-transfected cells did not show any fluorescence emission. The in vitro expression of VP60was also detected by western blot analysis. The results showed that rabbit hyperimmune serum against VP60protein reacted with the60kDa protein in the lysates of DNA construct-transfected cells, which indicates that VP60could be expressed in BHK-21cells. To evaluate the immunogenicity of the recombinant replicon plasmid pSCA/VP60, it was injected into rabbits as described in the Methods. Blood was collected at week2after the first vaccination and week4after the second vaccination to test for the presence of anti-VP60antibodies. Total anti-VP60antibody response was determined by an indirect ELISA. The mean antibody level of the pSCA/VP60-vaccinated group was significantly higher (0.01<P<0.05) than those of the pSCAl and PBS control groups. After the booster immunization, the mean antibody level increased, but this increase was not statistically significant (P>0.05). To investigate cellular immunity responses induced by pSCA/VP60, we analyzed the lymphocyte proliferative responses of all rabbits at0,1and3weeks ppi. The results indicated that pSCA/VP60induced obvious and intense lymphocyte proliferative responses.ELISA kits were used to detect the production of IFN-y and IL-4in sera at weeks1,2,3and4post-immunization. The results show that the mean levels of IFN-y and IL-4were significantly higher in the rabbits inoculated with pSCA/VP60compared with those inoculated with PBS. To test whether this vaccine could induce a protective effect against a lethal RHDV infection, the rabbits were infected with a wildtype RHDV strain, JX/CHA/97,14days after the last immunization. The results showed that all rabbits from the control group displayed the clinical symptoms and typical lesions of the disease and died within48-72h. All vaccinated rabbits survived without any clinical signs of disease.[Conclusion] In conclusion, we have developed two kinds of new type engineering vaccines against RHD, which were proved to be promising vaccine candidates that facilitates the prevention of rabbit hemorrhagic disease caused by RHDV.