Preparation Of New Adjuvants And The Efficacy Tests Of Subunit Adjuvant Vaccine

Vaccine immunization is now one of the most effective ways to prevent the animal diseases, and the study of animal vaccine has great socioeconomic importance. Adjuvant become part and parcel of most vaccine as an immune stimulator, and new adjuvants have good prospective with the development of animal vaccine market.The technology of adjuvant has been developed rapidly at home these years and the effect of many adjuvants applied to subunit vaccine has been proved, but there is less production for commercialization of adjuvants in our country, most of which rely on import for vaccine. Traditional white oil emulsion needs the energy consumption and results in the high cost for production.A series of oil adjuvants and a CpG adjuvant which was rich of swine specific CpG-ODN were made in this study to investigate effective, handy and low cost adjuvants for subunit vaccine.The perfect formulation of complex oil adjuvant was screened by means of HLB value and the best composition of micro-emulsion was defined according to the Ternary Phase Diagram.The complex oil adjuvant developed in this study and named COA contained safer oil phase and the emulsifiers with lower toxicity. The techniques for preparation of the complex oil adjuvant and the properties of it which could be emulsified by low speed were the same as Montanide ISA206made by Seppic. The formulation of W/O/W of the complex oil adjuvant obtained in this study could release antigen constantly, stimulate timely and give persistent immunity.Another mineral oil microemulsion adjuvant as stable self emulsified system (SSES) was developed in this study and named MOM, which could be emulsified easily when mixed with water phase. The W/O formulation and the nano-particles of it could stimulate stronger immune response in animals.The animal oil micro-emulsion was made in this study using the improved method of MF59adjuvant and named MFM. It was also the stable self emulsified system (SSES), had nano-particles and strong stability.These three oil adjuvants mentioned above which could meet the demand of an open and use adjuvant, were applicable to productive practice because of its easier emulsification and stronger stability.CpG-sw was an adjuvant rich of swine specific CpG-ODN, which was made by genetic engineering and the throughtput of CpG would be raised by cultivation of the bacteria carrying the DNA fragment.These preparations mentioned above and thymosin al as several typical adjuvants including emulsion, nucleic acids and protein adjuvants were mixed with subunit antigen, respectively to immune mice. In order to confirm effectiveness of them, the traditional mineral oil adjuvant was used as the positive control and the antigen alone was used as the negative control. After the confirmation, the best adjuvant for subunit vaccine from them was screened by in vivo experiment.The results indicated that mice in groups MOM, positive control and CpG-sw, the negative reaction of the whole body appeared, but disappeared after one or two days, when the other groups had no such a negative reaction. The negative reaction at the injection sites in groups MOM and positive control were hard to disappear and that in group MOM were smaller than positive control. It was found that the hydrophilicity of outer phase and the toxicity of oil itself both affected the adjuvant safety.The antibody level in all the adjuvant groups were significantly higher than that of the negative control and the titres in groups MOM, positive control and CpG-sw were the highest. Compared to the secretive activity of the negative control, the secretive activity of mice spleen cells in the groups CpG and Thymosin al was slightly increased, but the secretive activity in the groups of oil adjuvant was slightly decreased. The data showed that different adjuvants had different properties and immunologic mechanism because of their each advantage.