| Objective:The physicochemical properties and immunogenicity of tetravalent influenza vaccine liposome freeze-dried powder prepared by thin film dispersion method and freeze-thaw freeze-dried method were investigated.Influenza vaccine liposomes with different particle sizes were prepared by the optimal preparation method,and the physicochemical properties,immunogenicity and stability were compared to select the most suitable particle sizes.Methods:1.Comparison of the immunogenicity of the tetravalent influenza vaccine liposome freeze-dried powder prepared by thin-film dispersion method and freeze-thaw freeze-dried method:Kunming mice were divided into PBS group,film dispersion method group,freeze-thaw method group and vaccine stock solution group,and immunized abdominally.After 7,14 and 28 days of immunization,the spleen lymphocyte proliferation test(MTT)was conducted by taking SI value as index for cells immunogenicity atudy,and blood clotting inhibition of immune was taken humoral immunogenicity with antidody titer ratio of after and before mice immunization as index.2.Comparative study on the effect of particle size on the immunogenicity of influenza vaccine liposome lyophilized powder:Kunming mice were divided into PBS group,3.7μm group,2.0 μm group,1.0 μm group,0.45μm group and stock vaccine group,and were intraperitoneally immunized.After 7,14 and 28 days of immunization,the SI value was measured by MTT method to compare the cellular immunogenicity.Spleen lymphocytes were labeled with CD4+and CD8+antibodies,and the immunogenicity of CD4+/CD8+was studied.The humoral immunity was compared by detecting HI value.3.Study the influence of particle size on the stability of influenza vaccine liposome lyophilized powder:Varied size of Lyophilized powder and suspension(3.7μm,2.0μm,1.0μm and 0.45 μm)were prepared.The acceleration experiments were carried out at 4℃,25±2℃(60%±5%)and 40±2℃(75%±5%).Envelopment rate,HI and SI were used as indicators to compare the physical stability and biological stability.Results:1.The particle size of the liposome prepared by freeze-thaw freeze-drying method was 3.71μm,and the encapsulation efficiency was 94.48%.The liposome prepared by thin film dispersion method was 2.82 μm,and the encapsulation efficiency was 83.43%.at 7 days of immunization,the SI value of the thin-filmw and freeze-thaw groups were significantly different from that of other groups(P<0.05).At 14 and 28 days of immunization,the SI value of those two above groups were higher than that of vaccine stock solution group and PBS group,and there was no significant difference between two.The results of hemagglutination inhibition test showed that,at 7 and 28 days of immunization,the antibody titer ratio(before and after immunization)of the freeze-thaw group was significantly different from that of the vaccine stock solution group2.The results of MTT test showed that the SI value of 3.7μm group was higher than that of other groups at 7 days of immunization,which was significantly different from that of vaccine stock solution and PBS group(P<0.05).The stimulation index of 3.7μm group and 2.0μm group was higher than that of other groups at 14 days and 28 days of immunization,but no significant difference between two.The results of spleen lymphocyte surface labeling experiment showed that CD4+/CD8+of all immunization groups were higher than that of PBS group at 7,14 and 28 days of immunization(P<0.05).The liposomal adjuvant modified influenza vaccine group was higher than that of vaccine stock group,but no significant difference between two.The results of the hemagglutination inhibition test showed that the antibody titers ratio of the 3.7μm group were significantly higher than that of vaccine stock group at 3 and 7 days of immunization(P<0.05).At 14,28 and 42 days of immunization,the antibody titers ratio of the 3.7μm group were higher than those of the other groups.For all groups,HI decreased after 56 days of immunization,but all bigger than 4.3.At 4℃,25 ± 2℃(60%±5%)and 40±2℃(75%±5%),the encapsulation efficiency of the lyophilized liposomes in the groups of 3.7 μm,2.0 μm,1.0 μm and 0.45μm was higher than that of the suspension,and the encapsulation efficiency of the group of 3.7μm decreased less than that of the other groups,which is about 7.94%decreasion in 6 months.At 25 ±2℃(60%±5%)storage condition for two month,the HI values of varied size liposome groups were lower than those of vaccine stock solution group,but all greater than 4.After 15 days storage at 40±2 ℃(75%±5%),only the HI of 3.7um the rati group is higher than 4.Conclusion:1.the freeze-thaw method was employed as the preparation method of tetravalent influenza vaccine.2.The vaccine liposome of 3.7μm can produce good humoral and cellular immune responses after administration,and 3.7μm is selected as the optimal liposome particle size.3.At 25±2℃(60%±5%)storage for 2 months,all experimental size group liposome groups were effect in terms of humoral immune responses.And 3.7μm group liposome showes the humoral immune effectivness even after 40±2℃(75%±5%)storage for 15 days. |
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