Generation And Immunogenicity Of Foot-and-mouthdisease Virus Structural Polyprotein P1 And Japanese Encephalitis Virus Envelope Protein Expressed In Transgenic Rice

Foot and mouth disease virus(FMDV) is the causative agent of a significant economic disease affecting meat and milk producing domestic animals.Comprehensive vaccination of all susceptible hosts,using inactivated virus as an immunogen,constitutes the basis of all sanitary plans for the control and eradication of the disease.However, because of the probability of virus dissemination during the production of inactivated virus,it is becoming more important to develop new approaches.Japanese encephalitis is a potentially lethal disease of the central nervous system caused by infection with Japanese encephalitis virus(JEV),which is transmitted to human beings by mosquitoes.Pigs are considered the main vertebrate host and represent an important amplifier and reservoir for JEV.However,both mouse brain-grown formalin-inactivated JEV vaccines for human and inactivated or attenuated JEV vaccines for swine still have many limitations in terms of its high cost of production,lack of long-term immunity,probability of virus dissemination and risk of allergic reaction.Transgenic plant "edible vaccines" have the advantages which include their lack of contamination with animal pathogens,low cost of production,and heat stability compairing with the traditional vaccines.Rice has been widely suggested to be a potential alternative as the vehicle delivering antigen.It would be an economical strategy to develop new vaccines against these two viruses that expressing the immunogenic proteins of FMDV or JEV in transgenic rice.So capsid precursor polypeptide(P1) of FMDV strain O/ES/2001 and envelope protein(E) of JEV strain SA14-14-2 were expressed in transgenic rice which were transformed by the Agrobacterium-mediated method.Immunogenicity of rice-derived P1 and E protein was evaluated in a mouse model.The main projects are:1.Analysis of the expression of P1 protein and E protein in transgenic plants.The P1 gene of FMDV strain O/ES/2001 and the E gene of JEV strain SA14-14-2 were subcloned into the vector pRTL2,respectively,under the control of the dual cauliflower mosaic virus(CaMV 35S) promoter,resulting in the recombinant plasmid pRTL2-P1/E.The CaMV 35S-P1/E expression cassette was released from pRTL2-P1/E and inserted into the multiple cloning site(MCS) of pCAMBIA1301 to generate pCAMRT-P1/E for transformation.The japonica rice was transformed by the Agrobacterium-mediated method,after Southern blot,Northern blot and Western blot analysis,the P1 gene and the E gene were all expressed specifically.The level of P1 protein expressed in the leaves of the plants ranged from 0.6 to 1.3μg/mg of total soluble protein(TSP),the level of E protein ranged from 1.1 to 1.9μg/mg TSP.2.Immunogenicity of rice-derived P1 proteinFemale adult BALB/c mice(5-8 weeks old) were intraperitoneally(i.p.) immunized with transformed leaf extracts which were emulsified in Incomplete Freund's Adjuvant (IFA).High levels of FMDV-speciifc IgG were detected in the sera of mine vaccined whti rice-derived P1 protein on the day 56 after the primer immunization.Meanwhile, the mice immunized with the rice-derived P1 protein developed an approximately equal titer(1:20.27) with mice received E.coli-derived P1 protein(1:22.4).All animals immunized intraperitoneally with rice-derived P1 protein,E.coli-derived P1 protein and FMDV oil adjuvant inactivated vaccine were able to clear FMDV upon subsequent infection.For oral immunogenicity,female adult BALB/c mice(5-8 weeks old) were fed with fresh transformed leaf extracts.The mice were then sacrificed and pieces of small intestine were collected and used in the in vitro fragment culture on day 36.The serum IgG antibody titer of mice fed with rice-derived P1 protein was significantly higher than the mice fed with E.coli-derived P1 protein.Moreover,IgA antibody titers were detected both in intestine wash and in vitro fragment culture of mice fed with rice-derived P1 protein.These results demonstrated that rice-derived P1 protein induced mucosal immunity.However,only 20%to 40%mice were able to clear FMDV upon subsequent infection.3.Immunogenicity of rice-derived E proteinProtection against JEV is mainly antibody dependent and virus-neutralizing antibodies alone are sufficient to impart protection.Intraperitoneally(i.p.) and orally immunized female adult BALB/c mice as described previously.High levels of JEV-speciifc IgG were detected in the sera of mine vaccined whti rice-derived E protein on the day 56 after the primer immunization.Meanwhile,the mice immunized with the rice-derived E protein developed a significantly higher titer(1:19.2) than mice received E. coli-derived E protein(1:11.2).The serum IgG antibody titer of mice fed with rice-derived E protein was significantly higher than mice ted with E.coli-derived E protein.Moreover,IgA antibody titers were detected both in intestine wash and in vitro fragment culture of mice fed with rice-derived E protein.These results demonstrated that rice-derived E protein induced mucosal immunity.These results demonstrate the potential of using transgenic rice-based expression systems as alternative bioreactors for FMDV and JEV subunit vaccine.In future researches,the rice seed-promoter could be used to achieve high levels of antigen expression and many oral adjuvants such as CT or LT could be used to modify and enhance immune responses.