Preliminary Application Evaluation Of Aqueous Adjuvant In Live-attenuated Vaccine Of Japanese Encephalitis Virus

In order to reduce immunization times and enhance immune effect,at present,live vaccine were diluted with inactivated vaccines using aqueous adjuvant in some pig farms in China,such as porcine reproductive and respiratory syndrome live vaccine diluted with porcine circovirus inactivated vaccine,porcine reproductive and respiratory syndrome live vaccine diluted with Mycoplasma inactivated vaccine,and Classical Swine Fever live vaccine diluted with Haemophilus parasuis inactivated vaccine.However,it is not clear whether water based vaccine adjuvant affects the immune effect of live vaccine and how to affect it.So we mixed water adjuvant(MONTANIDE^TMGel 01 ST,Carbomer,astragalus polysaccharide,levamisole and chitosan)with SCYA201201 strain of JEV attenuated strain as the research object to carry out the experiment,in order to clarify the effects of aqueous adjuvant vaccine immunogenicity and influence mechanism.In the first part,the aqueous adjuvant（MONTANIDE Gel 01 ST）was mixed with JEV（SCYA201201）with the best titer of JEV and the optimal concentration of aqueous adjuvant Gel 01 ST,then mice were immunized with the vaccine mixture,and some related immunological experiments were carried out,including detection test of immunological index,virus challenge test and the protection,cross protection test with different genotypes（genotype I and III）,dynamics of antibody responses in serum,and the effect of temperature to vaccine.Our results showed that the protection rate was 100%（10/10）when the optimal concentration of MONTANIDE Gel 01 ST adjuvant was 10%and the best titer of attenuated strain is 10^7.0PFU/mL,Compared with mice immunized with attenuated strain alone,these mice immunized adjuvant and JEV strains generated higher IgG1/IgG2a antibodies（p<0.01）and neutralizing antibodies（p<0.01）,and produced more lymphocyte proliferations（p<0.01）andcytokinecontents（especiallyInterferon-gamma）.MONTANIDE Gel 01 ST adjuvant can also enhance the cross protection rate of Japanese encephalitis virulent strains between genotypeⅠandⅢ（90%and 100%respectively）,and provide high antibody level（p<0.01）and protection rate of 80%（8/10）in mice on 5 days after immunization.After the JEV live vaccine was placed at room temperature（25℃）for a certain time,then it mixed with adjuvant could still provide more than 66.7%protection rate,which was higher than the attenuated strain.The results show that the MONTANIDE Gel 01 ST can be used for JEV vaccine,and enhances the immune effect of vaccine with improved immune indexes.In the second part,mice were immunized with vaccine mixed with the four kinds of adjuvants（Carbomer,astragalus polysaccharide,levamisole and chitosan）alone or combine,then the antibody level in serum was detected,and protective experiments were carried out to explore effect of water adjuvant on the SCYA201201 strain.The experimental results showed that the four kinds of adjuvants had no effect on vaccine immunogenicity when it was used alone,except Carbopol of 4mg/mL concentration had weak effect on antibody levels.However,when adjuvants were used by mixed form,antibody level and immune protection rate in experimental group were lower than that in immunizd with attenuated JEV strain alone.The results showed that the water adjuvant could not enhance the immune response to JEV live vaccine,and had the immunosuppressive effect.It indicated that,when the live vaccine was used in clinic,it was necessary to avoid the adverse effects of some drugs（or chemical complexes）on the immune effect of live vaccine.