Preparation And Immunogenicity Of Chitosan Salt Stabilized Emulsion

Oil emulsion adjuvant has been paid much attention to because of its strong humoral immune effect.Currently,most of the oil emulsion adjuvants on the market are non-ionic surfactant-stabilized emulsions,with electrically neutral surface potential and poor ability to adsorb antigens,so they cannot effectively deliver antigens,thereby resulting in a poor cellular immune effect.Therefore,this paper proposes to construct a human vaccine（squalene）and veterinary vaccine（white oil）with emulsion stabilized by chitosan salt as an adjuvant,aiming to enhance the cellular immune response of antigens while maintaining a good humoral immune response.The specific research contents are as follows:（1）The emulsion was prepared to develop a human vaccine adjuvant by using squalene as an oil phase and imported chitosan hydrochloride as a surfactant.By optimizing the preparation parameters such as chitosan hydrochloride concentration,oil-water volume ratio,external water phase and ultrasonic conditions,the preparation conditions of the emulsion were determined as follows:the concentration of chitosan hydrochloride was 4 mg·m L^-1,the external water phase was deionized water,the oil-water volume ratio was 1:9,the ultrasonic frequency was 20%,and the ultrasonic time was 3 min.The prepared emulsion was named Chitosan hydrochloride polymer stabilized emulsion（CPSE）,with an average particle size of 1783±161 nm and excellent dispersion.Meanwhile,it can be stored at 4°C for 4 months with good stability.The surface potential of CPSE was 50.3±0.7 m V,which can effectively adsorb antigens through electrostatic interaction.Besides,it has a lot of advantages such as rapid adsorption,high adsorption capacity,and reduced antigen release rate.At the cellular level,CPSE can significantly increase the uptake of antigens by bone marrow derived dendritic cells（BMDCs）and enhance the expression of CD40 and CD86 molecules,thus promoting the activation of BMDCs.In the animal-level evaluation,ovalbumin（OVA）was used as the model antigen to verify the immune effect of CPSE as an adjuvant.The results revealed that CPSE could induce and produce high levels of Ig G antibody,significantly increase the number of spleen cells secreting IFN-γ,promote the expression of activated molecules such as CD69,CD107,and SIINFEKL-MHC I on the surface of CD8⁺T cells,and induce strong cytotoxic T lymphocyte response,so it had stronger immune memory ability.In conclusion,CPSE can effectively enhance humoral and cellular immune responses and is expected to achieve long-term immunity.（2）Taking white oil as the oil phase,the immune activation effect of three kinds of domestic chitosan salts（chitosan quaternary ammonium salt,chitosan lactate,and chitosan hydrochloride）as surfactants were compared to prepare emulsions for developing veterinary vaccine adjuvants.Then the three chitosan salts were prepared according to the emulsion preparation conditions in the squalene system and the obtained emulsions were characterized.The obtained three emulsions were chitosan quaternary stabilized emulsion（CQE）（particle size:1715±60 nm;potential:75.5±0.3 m V）,chitosan lactate stabilized emulsion（CLE）（particle size:819±30 nm;potential:47.1±0.7 m V）,and chitosan hydrochloride stabilized emulsion（CHE）（particle size:621±6 nm;potential:50.7±0.7 m V）.Their antigen loading rate was all above90%.At the cellular level,all three emulsions can effectively enhance the uptake of antigens by BMDCs.CQE can significantly increase the expression of CD40 and CD86 molecules on the surface of BMDCs and then promote the activation of BMDCs,while CHE can enhance the expression of SIINFEKL-MHCI on the surface of BMDCs and has the potential for antigen cross-presentation.The animal immune experiment was carried out with OVA as the model antigen,and the results showed that CQE could increase the antibody level and the number of spleen cells secreting IL-4,thereby significantly improving the humoral immune effect;CLE could moderately enhance the humoral and cellular immune effect,with the best enhancement effect;CHE could promote the expression of activated molecules such as CD107,CD69,and SIINFEKL-MHC I on the surface of CD8⁺T cells,thus producing a good cellular immune effect.