Development Of Novel Subunit Vaccine Against VP1 And VP2 Proteins Of Foot And Mouth Disease Virus

Foot-and-mouth disease(FMD)is caused by foot-and-mouth disease virus,which is one of the most important pathogens in pigs,cattle,and sheep.At present,the prevention and control of FMD is mainly carried out with inactivated vaccines.However,considering the bio-safety risk of live virus involved in production,it is necessary to develop safe and effective subunit vaccines.The most important structural proteins of foot and mouth disease are VP1 and VP2.These two proteins have antigenic epitopes eliciting neutralizing antibodies after immunization.Therefore,VP1/VP2 serves as subunit vaccine targets.In this study,attempts were performed to produce novel VP1/VP2 based subunit vaccines against FMD.1.Expression of surface displayed FMDV VP1/VP2 in baculovirus The major immunogenic genes VP1/VP2 of FMDV and the full-length GP64 gene of baculovirus were inserted in pFastBacHTB vector under pPH promoter to obtain the recombinant plasmids pFBH-VP1-GP64 and pFBH-VP2-GP64.The plasmids were transformed into DH10Bac respectively for transposition.Blue and white spots were screened to obtain recombinant bacmids rBacmid-VP1-GP64 and rBacmid-VP2-GP64.Bacmids were transfected to obtain recombinant baculoviruses rBac-VP1-GP64 and rBac-VP2-GP64.Recombinant antigens were collected from virus infected insect cells and characterized with a series tests,including Western blot and IFA.The results showed that VP1 and VP2 were effectively expressed in Sf9 cells,laying a foundation for the immunogenicity analysis of animal experiments.2.Experimental study on the immune efficacy of recombinant baculovirus rBac-VP1-GP64 and rBac-VP2-GP64Recombinant baculovirus was used to infect Sf9 cells,and culture samples were collected for purification and quantification.BALB/c mice and pigs were used as experimental animal models for immunogenicity evaluation.BALB/c mice were randomly divided into groups of 4 mice each.100/μg rBac-VP1-GP64/rBac-VP2-GP64 recombinant virus was inoculated subcutaneously,and serve negative control.Mouse serum samples were collected at 0,14,28,and 42 days after immunization for ELISA and indirect immunofluorescence(IFA)detection antibodies.ELISA results showed that compared with the control group,the antibody level in the immunized group increased continuously with the boost of the immunity and reached the highest level in 42 days.IFA results are consistent with the observation in ELISA.The results of challenge experiments on pigs immunized with recombinant virus showed that the morbidity rate in the control group was 100%at 3 days,while in the immunization group,60%of the pigs remain health until 10 days post challenge.The results showed that the recombinant virus produced a good immune response in mice and pigs after immunization,and provided effective protection against FMDV challenge.3.Soluble expression of FMDV major immunogenic genes VP1/VP2 in baculovirus expression systemThe main immunogenic genes VP1/VP2 of FMDV and maltose-binding protein(MBP)gene sequences inserted in pFastBacHTB vector under pPH promoter to obtain the recombinant plasmids pFBH-VP1-MBP and pFBH-VP2-MBP.The plasmids were transformed into DH10Bac respectively for transposition.After blue and white spot screening,recombinant bacmids rBacmid-VP1-MBP and rBacmid-VP2-MBP were obtained and then transfected into Sf9 cells to obtain recombinant baculoviruses rBac-VP1-MBP and rBac-VP2-MBP.The virus infected a large number of insect cells,and the culture supernatant were collected for purification and further analysis.The results showed that target protein was expressed in large amounts in a soluble form,which laid the foundation for the later immunogenicity analysis.4.Immunogenicity analysis of recombinant baculovirus rBac-VP1-MBP and rBac-VP2-MBPA large number of recombinant baculoviruses were used to infect Sf9 cells,and the culture supernatant were collected for purification and quantification.BALB/c mice were used as animal models for immunogenicity evaluation.BALB/c mice were randomly divided into groups of 4 mice each.100μg rBac-VP1-MBP or rBac-VP2-MBP recombinant protein was inoculated subcutaneously,and serve negative control.Mouse serum samples were collected at 0,14,28,and 42 days after immunization to detect specific antibody levels.The ELISA results showed that compared with the control group,the antibody level in the immune group increased continuously with the strengthening of the immunity,and reached the highest level in 42 days.The IFA results are consistent with the ELISA results.These results indicate that the recombinant protein could induce considerable immune response in mice.The above research results provide experimental materials for the development of FMD subunit vaccines,and also provide a basis for their prevention and control and diagnosis.