Design And Activity Evaluation Of Self-emulsifying Oil-in-water Adjuvants

Veterinary vaccines were used to reduce the occurrence of major diseases,and is of great significance to the development of animal husbandry.Choosing the suitable adjuvant might be highly effective with vaccines.Due to their low viscosity and toxicity,emulsions,especially oil-in-water emulsions,have become the focus of research in the veterinary vaccine manufacturing industry in recent years.However,the traditional preparation of emulsion is limited by the emulsifying equipment,rotor speed,emulsifying time.Therefore,it is difficult to meet the industrial production of veterinary vaccines.Notably,self-emulsifying oil-in-water emulsions are mainly based on self-emulsifying systems containing oil,surfactant and co-surfactant,which can be completed by simple mixing and stirring.Self-emulsification materials are presently mostly used to administer oral pharmaceuticals such antitumor,antiviral,and antibacterial drugs,although there are few reports on their use in vaccines.In this study,an oil-in-water adjuvant was created by simply combining the oil,surfactant and co-surfactant.The formula(X2-17)was mixed with the foot-and-mouth disease inactivated virus(FMDV)to prepare the vaccine.Firstly,we investigated the relationship between the physical and chemical properties of the X2-17 and the antibody level of stimulated animal immune response.Later,we employed mice as an animal model to track immunological effectiveness(antibody duration,antibody level,and antibody subtype),as well as clinical indicators and pathological sections,in order to determine their safety.Finally,we further explored the level of related cytokines,lymph node dendritic cells(DCs)activation and differentiation of spleen lymphocytes.Taken together,we established a theoretical basis for the use of X2-17 by tracing the immunological mechanism of X2-17.This research is mainly divided into two parts:(1)Formulation design and characterization of self-emulsifying oil-in-water nanoadjuvantThe purpose of this experiment is to screen out a stable self-emulsifying oil-in-water formulation.We chose for the suitable oil,surfactant and co-surfactant in the multi-component system,and the mass ratio of various components was adjusted.We also investigated for the self-emulsifying system,which the relationship of the pseudo-ternary phase diagram and self-emulsifying properties.The results indicated X2-17 formula(squalene:BRIJ 20:Transcutol P=0.3:0.5:0.2)was a good nanoadjuvant,and its physical and chemical properties were investigated.DLS and Zeta potential showed that the particle size is 25.16±0.21 nm with the dispersion coefficient of 0.057±0.023 and the potential is-2.81±0.17 m V.These results indicated that the nanoparticle size distribution is uniform and the composition is stable.Place X2-17 in a constant temperature incubator(37°C),and in the given time to monitor the changes of particle size,and potential.The emulsion has no stratification,demulsification,or turbidity on the days 1,14,28,and 60.The results suggested that the formula X2-17 can be stored stably for 60 days at 37°C.The oil-in-water droplets were visually detected under the transmission electron microscope.The particle of X2-17 were dispersion spheres with sizes of 20-30 nm.The relationship between the self-emulsifying ability of X2-17 and the dilution ratio was investigated also.The results shows that the particle size changes were small in the 1:3,1:4 and the 1:9 groups.A certain dose of X2-17 was injected into the mice by subcutaneous injection,the mice had a normal diet,soft coat,good spirits,and no adverse phenomena such as redness and swelling at the injection site.Further important histopathological sections have no obvious damage or lesions to important organs.Above results,the formula X2-17 can produce homogeneous,stable and safe nanoemulsion with small particle size,and has good tolerance to the animal body,which provides a test basis for the subsequent application of veterinary vaccines.(2)Immune evaluation and mechanism study of self-emulsifying oil-in-water nanoadjuvantThe purpose of this experiment was to evaluate the immune efficacy of formula X2-17 and explored the immune mechanism.The vaccine was prepared by mixing the inactivated FMDV with formula X2-17.We detected the FMDV-specific antibodies and subtypes in the serum of the mice after subcutaneously immunization,and tracked the duration and the immunization route also.The results showed that the level of FMDV-specific antibodies in the serum of mice of the X2-17+FMDV group was significantly higher than that of pure FMDV group within 12 weeks(p<0.05),which is no significant difference with that of the commercial ISA206+FMDV group(p>0.05).Next,we analyzed the FMDV antibody subtypes in the sera of mice after immunization,the X2-17+FMDV group significantly promoted the production of Ig G2a,and the high Ig G2a/Ig G1 value expressed X2-17 tended to the Th1 immune pathway.In addition,we explored the DCs of lymph nodes activation ex vivo,the proportions of CD80~+CD11c~+,CD86~+CD11c~+,MHC-II~+CD11c~+cells in the X2-17+FMDV group were the highest level,but there was no significant difference from the ISA206-adjuvant group(p>0.05),indicating that X2-17 may contribute to the maturation of DCs,which in turn is conducive to antigen presentation.Meanwhile,X2-17+FMDV group can significantly promote the differentiation of spleen T cells to CD4~+cells,but there was not significantly different from that of commercial ISA206.Notably,we found the levels of IL-2,IL-4,IL-6 and IFN-γsecreted by stimulated mice in X2-17+FMDV group were significantly higher than those of pure FMDV group(p<0.05),but there was no significantly different from that of commercial ISA206.In summary,the results of this study confirmed that X2-17 can significantly improve the body’s immune response to FMDV,and showed a similar immune-enhancing effect to ISA206,providing experimental data for practical applications of X2-17.