Research On Immunological Effect And Preparation Of Yeast Beta Glucan Nanoemulsion Adjuvant

Vaccine adjuvants is a class of substances which could non-specifically enhance immune response to antigen or change the type of immune response after combined with antigen,but have no immunogenicity themselves. Yeast β-glucan is a green, safe, non-toxic, natural source of immune polysaccharides whose intrinsic immunostimulatory effects can induce and strengthen humoral and cellular immunity of body, and can enhance immune response of body as immunostimulants. Nanoemulsion adjuvant is the emulsion that its droplet size is at nano-scale （20-500nm） range, fabricated by a designed nanotechnological method, possessing merits of stability and immunological effects of the vaccine, meanwhile it also can overcome shortages of those traditional mineral oil adjuvants being used. In this research, in order to utilize the synergistic effect of the yeast β-glucan combined with nanoemulsion carrier, we for the first time successfully obtain an oil in water（O/W） type yeast β-glucan nanoemulsion （CMG-NE） adjuvant via a carefully designed nanotechnology method and oil-water affinity balance technology. The quality, safety, and immunological effects of yeast β-glucan nanoemulsion adjuvant were evaluated, which provided a basis for late-stage clinical application research. The detailed results are as follows:1.Formula selection of yeast β-glucan nanoemulsion adjuvantIn our protocol, we designed various formulas of nanoemulsion adjuvant based on previous research references. Then we took these ways that judged nanoemulsion formation or not via aqueous titration method, investigated the stability of the nanoemulsion under different temperatures conditions, and observed whether have any clinical adverse reaction happened to the mice after inoculated with nanoemulsion in order to select a fitter formula. Based on these results, and with the help of pseudo-ternary phase diagrams of nanoemulsion systems, we investigated the main factors that affect the formation of nanoemulsion, and defined the optimal formula of nanoemulsion based on the size of O/W type nanoemulsion area. Through selecting and optimizing, the composition of the yeast beta-glucan nanoemulsion adjuvant are as follows:aqueous solution of yeast β-glucan as water phase, medical white oil（Marcol 52） as the oil phase, Span 80 and Tween 80 as the surfactant, PEG 400 as the cosurfactant, and the mixed ratio of Span 80 and Tween 80 is 1:3（mass ratio of Span 80/Tween 80）, the Km is 2:1 （mass ratio of （Span 80+Tween 80）/PEG 400）.2. Evaluation of physical properties of yeast β-glucan nanoemulsion adjuvantBase on components and proportion determined by the formula, yeast β-glucan nanoemulsion adjuvant was prepared successfully by low-energy emulsification method （phase inversion method）. The appearance, structure type, size distribution and micro-morphology, viscosity, stability of yeast β-glucan nanoemulsion adjuvant were studied, and the results showed that the yeast β-glucan nanoemulsion adjuvant had a appearance of light brown yellow and homogenous transparent, oil in water （O/W） type structure, average particle diameter of 74.31nm, normal distribution of the particle size that mainly distributed between 60nm and 120nm, viscosity of 5s/0.4mL. Even after being centrifugated at a high speed of 10000 r/min for 20min or being stored under 4℃ and 25℃ for one year period, the nanoemulsion remained its good stability as the original, no turbidity, demulsification, stratification occurred.3. Safety evaluation of the yeast β-glucan nanoemulsion adjuvant10 mice were respectively intraperitoneal injected with yeast P-glucan nanoemulsion adjuvant at 3 times,4 times,5 times normal dose to test the acute toxicity; The pyrogen of yeast β-glucan nanoemulsion adjuvant was test based on the methods in pyrogen test regulations for biological products; Health rabbits were injected with 1mL yeast β-glucan nanoemulsion adjuvant at quadriceps of hind limbs, the changes of local muscle tissue at injection site were observed after injection. The results showed that the LD₅0 of yeast β-glucan nanoemulsion adjuvant to mice was above 25 mL/Kg（about 25000mg/Kg） that belonged to nontoxic based on the classification standards of acute toxicity, and that pyrogen test of yeast β-glucan.nanoemulsion adjuvant conformed with regulations, and the yeast P-glucan nanoemulsion adjuvant was absorbed completely, no visible muscle irritation and adjuvant residue were observed, suggesting yeast β-glucan nanoemulsion adjuvant conformed with the safety requirements of current biological products.4. The research on immunological effect of Pseudorabies live vaccine with yeast P-glucan as adjuvantUsing pseudorabies live vaccine（HB-98 strain） as antigen, we prepared pseudorabies live vaccine with various concentrations（1%,2%,3%） of yeast β-glucan solution as diluents. Balb/C mice inoculated with Pseudorabies live vaccine for a dose of 10⁵5TCID50 /each, followed by booster immunization after 4 weeks, then indicators such as serum antibodies and spleen lymphocytes proliferation activity were analyzed at regular intervals. The results showed that the vaccine groups with yeast β-glucan could induce higher PRV ELISA antibodies than the vaccine group with special diluent, especially the vaccine group with 2% yeast β-glucan.42d after the first inoculation, the PRV ELISA antibodies reached the peak values, and we found no significant difference between the vaccine groups with yeast β-glucan and the vaccine group with special diluent. Meanwhile, the level of neutralizing antibody induced by vaccine with yeast β-glucan was slightly higher than the vaccine with special dilution. The results of lymphocytes proliferation activities showed that yeast β-glucan could enhance the activity of spleen lymphocytes proliferation of mice in a certain dose range.7d and 14d after booster immunization, the spleen lymphocytes proliferation activities of the vaccine groups with 1%and 2%yeast β-glucan were both higher than the vaccine group with special dilution, but the vaccine group with 3%yeast β-glucan was lower compared with the vaccine group with special dilution.21 d after booster immunization, there existed no significant activities of lymphocytes proliferation.5. The research on immunological effect of porcine circovirus type 2 inactivated vaccine with yeast P-glucan nanoemulsion as adjuvantUsing inactivated vaccine against porcine circovirus type 2（PCV-2） as a model antigen, we prepared PCV-2 inactivated vaccine with yeast β-glucan nanoemulsion adjuvant, and the quality of PCV-2 inactivated vaccine with yeast P-glucan nanoemulsion adjuvant was evaluated. The results have shown that PCV-2 inactivated vaccine with the yeast β-glucan nanoemulsion adjuvant have a appearance of transparent clarification, and was identified as oil-in-water（O/W） type, other properties such as the viscosity, stability, sterility test were all conformed with the regulations of vaccine products. 14-day old suckling piglets were inoculated with 2 times dose of PCV-2 vaccines with yeast β-glucan nanoemulsion adjuvant to evaluate its safety. The results showed that PCV-2 inactivated vaccine with yeast β-glucan nanoemulsion adjuvant had good safety without any adverse reactions taken place. And 14-day old suckling piglets inoculated with PCV-2 vaccines with nanoemulsion adjuvant containing different concentrations of yeast β-glucan, booster immunization after 14 days, bloods were regularly collected for PCV-2 antibody analysis. The results showed that 1% yeast β-glucan nanoemulsion adjuvant possessed well adjuvant effect and could induce high level PCV-2 antibodies which have no significant difference from oil adjuvant at different time points after immunization.