Preparation Of DDA-TDB Cationic Liposome-encapsulated Influenza Vaccine And Preliminary Evaluation Of Immune Responses Induced By Intranasal Immunization In Mice

Objective At present,there are many problems at the use of influenza vaccine,such as weak immunogenicity,less effectively mucosal responses,single immunization route,and poor patient's compliance.To solve these problems,this research preparaed the cationic DDA-TDB liposome as adjuvant for influenza vaccine,which would be capable of inducing both systemic and mucosal antibody responses to prevent influenza epidemic via intranasal immunization.The cationic DDA-TDB liposome-encapsulated influenza vaccines are prepared to provide valuable reference for the research and development of new vaccines.Method 1 Neutral DSPC-Chol liposomal vaccine,cationic DOTAP-DC-Chol liposomal vaccine and DDA-TDB liposomal vaccine were prepared by lipid-film hydration method.Physicochemical characteristics of liposomal vaccines such as particle diameter,zeta potential,encapsulation efficiency and antigen loading were used as indexes to optimize prescription and technics.The characteristics of the obtained liposome influenza vaccine were also evaluated.2 The uptake of different QDs-labled liposome formulations was evaluated by bone marrow dendritic cells(BMDCs)in vitro using Confocal Laser Scanning Microscopy(CLSM)and Flow Cytometer(FCM).The effect of different liposome formulations on dendritic cells(DCs)maturation was investigated via analysing expression of MHC-II molecules,as well as the co-stimulatory molecules CD80 and CD86 on the surface of DCs by using FCM.3 The effect of liposomes act as an adjuvant of vaccine inducing mucosal,systemic and cellular immune response was studied in C57BL/6 female mice.The titers of anti-H3N2 sIgA antibody in nasal tissue,IgG,IgG1,IgG2 a and IgG2 b antibody in serum,as well as levels of IFN-?,IL-2 and IL-5 in spleen via intranasal administration were assessed using enzyme-linked immunosorbent assay(ELISA).Result 1 At the condition that the antigen/lipid was 10:100 by weight,and the hydrated buffer was tris-buffer at pH 7.4,the obtained liposomal vaccines were prepared by lipid-film hydration method.The particle size of DSPC-Chol liposomal vaccine was 500.3±21.0nm,and its zeta potential was ?4.2±0.1mV,as well as its encapsulation efficiency was 17.8±1.9%.The particle size of DOTAP-DC-chol liposomal vaccine was 339.0±3.9nm,and its zeta potential was 51.3±2.0 mV,as well as its encapsulation efficiency was 46.2±1.1%.The particle size of DDA-TDB liposomal vaccine was 1770.0±70.7nm,and its zeta potential was 53.8±3.6mV,as well as its encapsulation efficiency was 46.2±1.1%.Their particle size and encapsulation efficiency of liposomal vaccine varied slighly within 21 days when storing at 4?,indicating liposomal vaccines were relatively stabile.2 The results of cellular uptake showed that 32.73±2.6%of BMDCs receiving DDA/TDB liposomes were positive for QDs,while only 12.23±1.0%and 4.9±1.8%of the cells from mice given DOTAP/DC-Chol and DSPC/Chol liposomes contained QDs fluorescence respectively in vitro.Furthermore,BMDCs treated with H3N2-encapsulated DDA-TDB liposome showed significant increase in expression of CD80,CD86 and MHCII.This result suggests that the efficient uptake of DDA-TDB liposome could induce the maturation of DCs and induce subsequent immune responses.3 The sIgA antibody titer induced by intranasal administration was significantly higher than intraperitoneal administration(p<0.01).After intranasal immunization,the IgG antibody titer induced by DDA-TDB liposomal vaccine was significantly different from the IgG antibody titer induced by H3N2 alone,DSPC-chol and DOTAP-DC-chol liposomal influenza vaccine(p<0.01).The subtype of IgG antibody titer induced by DDA-TDB liposomal influenza vaccine after intranasal immunization such as IgG1 and IgG2 b,which were higher than DSPC-Chol and DOTAP-DC-Chol(p<0.05).Furthermore,the group of DDA-TDB liposome influenza vaccine via intranasal immunization was higher than DOTAP-DC-Chol group,which was significant different(p<0.05).These results showed that the DDA-TDB liposome vaccine could enhance the humoral and mucosal immune response and improve the level of cellular immune response via intranasal immunization.Conclusion The DDA-TDB liposomal influenza vaccine prepared by lipid-film hydration method had relatively high encapsulation efficiency and stability.The results of cellular uptake for liposome formulations and the maturation of DCs suggested that the efficient uptake of DDA-TDB liposome could induce the maturation of DCs and induce subsequent immune responses.After intranasal immunization with DDA-TDB liposomal influenza vaccine,the anti-H3N2 sIgA and serum anti-H3N2,IgG,IgG1 and IgG2b in mice were higher than those of the other three groups,as well as the level of cytokine IFN-?was higher.These results showed that DDA-TDB liposome as an adjuvant for nasal mucosal vaccine can significantly improve the mucosal,humoral immunity and Th2 immune response,which is promising for potential applicability in novel carrier for vaccine delivery.