The Preparation And Cellular Immune Study Of Rabies Vaccine Liposome Prepared By Freeze-Thaw-Drying Method

Rabies is a kind of acute infectious disease caused by the rabies virus infection, which features mainly on central nervous system damage and belongs to the disease coexist-ed between man and beast. Once the incidence occurs, mortality is nearly 100%. The current application of the rabies vaccination is an effective method for rabies prevention. Although the traditional inactivated rabies vaccines have the characteristics of simple manufacturing procedure, the problems, such as effective antigenic determinant group changing, immune response time shortening and requiring muti-immunization, may occur during inactivating process. Although aluminium adjuvant overcomes the above shortcomings to some extent, but many problems still existed, like frequent side effects, delayed antibodies producing. Therefore the research on adjuvants of safe and producing antibodies earlier is becoming hotspot of rabies vaccine study.In this study, liposome, produced by thin film dispersion joining with freeze-thaw-drying method, was employed as vaccine adjuvant. The rabies vaccine liposome products were produced based on the optimal process. And then the status of the particles, particle size distribution, and the encapsulation efficiency of the lyophilized powder were investigated. The vaccine liposome suspension and the lyophilized powder were put at the condition of 4℃,25±2℃ and 37±2℃, sampled at given points of time, and taken microscopic observation and measured encapsulation rate as index for physical stability test The encapsulation efficiency of liposome preparation was (85.29±2.43)%(n= 5). The imagine of liposome under Motic B5 digital electron microscope(Oil,100 x) is round or elliptical with uniformly particle distribution. The average particle size is about 3.89μm. There is no obvious decline in encapsulation efficiency under the storage condition of 25 ±2℃ and 4℃ at the 30th day, indicating a good stability of lyophilized liposome.Diluting original vaccine liquild by 4,6 and 8 times, producing liposome lyophilized powder, then vaccinating mice respectively to determine optimal diluting times product. 4 times diluting liposome was marked as the best judging by the higher cell stimulation index. Vaccinating intraperitoneal clean KM mice with original vaccine liquild, non-diluting vaccine liposome and optimal diluting vaccine liposome by three needle strategy, taking saline as positive control, investigating cellular immunogenic-city after 28days of immunization by the methods of MTT experiment testing for lymphocyte proliferation and flow cytometry for lymphocyte surface markers. The result shows that the original vaccine liquild group, non-diluting vaccine liposome group,4 times diluting vaccine liposome group can all stimulate the body to produce cellular immunity(P<0.05). As for lymphocyte proliferation, there is significant difference between the following groups:non-diluting vaccine liposome and original vaccine liquild(t= 11.483, P< 0.05); 4 times diluting vaccine liposome and original vaccine liquild(t= 15.931, P< 0.05); 4 times diluting vaccine liposome and non-diluting vaccine liposome(t= 8.537, P< 0.05),indicating that the non-diluting vaccine liposome and 4 times diluting vaccine liposome can both induce higher cellular immune level comparing with the original vaccine liquild, and 4 times diluting vaccine liposome is better than non-diluting vaccine liposome in terms of stimulating index value. The result of lymphocyte surface markers experiment shows a big difference of CD4+/CD8+ ratio between original vaccine liquild group, vaccine liposome group, and 4 times diluting vaccine liposome group, comparing with control group(P<0.05), indicating the original vaccine liquild, vaccine liposome and 4 times diluting vaccine liposome can both stimulate the body to produce cellular immune. CD4+/CD8+ ratio from vaccine liposome group is significantly higher than that of original vaccine liquild group(t= 11.288, P< 0.05). CD4+/CD8+ ratio from 4 times diluting vaccine liposome group is significantly higher than that of original vaccine liquild group(t= 9.067, P< 0.05). CD4+/CD8+ ratio from 4 times diluting vaccine liposome group is significantly higher than that of vaccine liposome group (t= 4.789, P< 0.05), the cellular immune level induced by rabies vaccine liposome and 4 times diluting vaccine liposome is obviously higher than that by original vaccine liquild, and 4 times diluting vaccine liposome is better than non-diluting vaccine liposome, also indicating vaccine liposome is capable to enhance the body’s immune cells.The above results show that the freeze-thaw-drying method is feasible for rabies vaccine liposome preparation.The resulted vaccine liposome can induce significantly higher cellular immune level comparing with original vaccine liquild.4 times diluting vaccine liposome is better than non-diluting vaccine liposome concerning the cellular immune level. Liposome as a vaccine adjuvant can not only enhance cellular immunity but also humoral immunity.