The Research On Cellular Immunity Of Influenza Vaccine Lyophilized Liposome Produced By Freeze-thawing Method

We had discussed the stability of liposome prepared by Film-dispersion lyophilized method, and the effects of humoral, mucosal and cellular immunity have also been studied, which shows significantly improvement of immunogenicity. But the Film-dispersion lyophilized method, due to the temperature raising during preparation process, may not be suitable for live vaccine liposome preparation.Based on the Film-dispersion method which we did previously, we studied the freeze-thawing lyophilized method in order to suit the liposome preparation for live vaccine. The main influence factors were investigated in the preparation process of liposome suspension. Taking encapsulation rate which determined by Lowry method as index, we selected the best prescription for liposome suspension by the uniform design method. We analyzed the particle shape and size distribution of the liposome suspension and dry powder through microscopic observation. And by taking appearance and encapsulation rate as index, the stability research of these products was also conducted. In this study, the clean ICR mice were immunized by pulmonary vaccine delivery approach. MTT method was employed to measure the spleen cell proliferation,and flow cytometry was used to detemine the levels of CD4and CD8T-cell and the ratio of CD4/CD8. And the cellular immunity comparison study of freeze-thawing and Film-dispersion products were conducted.According to the optimized prescription, the encapsulation rate of the product was84.14%,the liposome particles were distributed evenly and in forms of round or oval, the mean size of the particles was at1.91μm.The physical stability study results of freeze-thawing lyophilized liposomes at different temperatures (4℃,25±℃癈,40±2℃)were:The encapsulation efficiency has no significant change during storage at4℃for180days, and25℃for120days.Furthermore, the whole process of freeze-thawing lyophilized liposomes preparation is under mild condition, which could meets the special requirements of live attenuated vaccine liposomes preparation. At the same dose, by pulmonary delivery, the higher cellular immunity levels,compared with non-liposome group mice delivered by intraperitoneal injection, can be induced from both product(Freeze-thawing lyophilized liposomes and Film-dispersion lyophilized liposomes)group mice. And there is no significant difference between two product group mice in terms of cellular immunity levels, which demonstrates that Freeze-thawing method is capable in producing attenuated live vaccine liposome.