Evaluation Of DDA Complex Liposome-encapsulated Influenza Vaccine On Immune Responses By Intranasal Immunization And Its Mechanism

Objective Influenza is an acute respiratory infection disease caused by influenza virus,which is seriously harmful to human health,vaccination is still the most effective measure to prevent influenza.Nowadays,the most commonly used influenza vaccines are inactivated vaccines,however,they usually have many side effects and poor immunogenicity.In order to improve the safety and immunogenicity of vaccines,we select DDA cationic lipid as vaccine adjuvants to construct DDA complex liposome-encapsulated influenza vaccine,and hope to obtain higher immunity through intranasal immunization to prevent influenza effectively,providing a valuable reference for the development of novel vaccines.Methods 1 We select DDA cationic lipid as the main component of lipid membrane,neutral phospholipid DSPC as auxiliary lipid,and modified with TPGS and PEG respectively to construct DDA-DSPC,DDA-DSPC-TPGS and DDA-DSPC-PEG liposomal influenza vaccines by thin film dispersion method.The physicochemical characteristics such as encapsulation efficiency(EE)and loading efficiency(LE)were investigated.In addition,we determined the optimal formulation process of lyophilized liposome vaccines by screening of lyoprotectants and lyophilization conditions.2 We cultured mouse bone marrow dendritic cells(BMDCs)in vitro,and investigated the cytotoxity and cellular uptake of DDA complex liposome to dendritic cells.Additionally,MHCII,CD80 and CD86 were used as indicators to evaluate the degree of DC2.4 maturation by flow cytometry.3 C57BL/6 mice were used as animal models to test murine secretory IgA(sIgA),serumIgG1 and the levers of IFN-? and IL-4 by enzyme-linked immunosorbent assay(ELISA).Results 1 Liposomal influenza vaccines were prepared by lipid-film hydration method,the dispersion medium was Tris-buffer with pH7.4,and the obtained DDA-DSPC liposomal vaccine had a particle size of 685.0±17.8 nm,and zeta potential of 25.4±1.9 mV,EE 65.7±2.0%,LE6.2±0.2%.The DDA-DSPC-TPGS liposomal vaccine had a particle size of 337.3±2.4 nm,and zeta potential of 15.4±2.1 mV,EE 78.5±1.5%,LE 4.2±0.2%.The DDA-DSPC-PEG liposomal vaccine had a particle size of 591.4±19.3 nm,a zeta potential of 6.9±0.8 mV,an EE of 45.8±3.0%,and an LE of 3.0±0.3%.All liposomal vaccines were spherical or ellipsoidal.In addition,when the liposomal vaccine combining with 5% sucrose and 5% lactose according to mass ratio 1:1,and under the conditions of-80°C and 8 h,the liposomal vaccine had the best lyophilization effect.2 In vitro uptake experiments showed that the DDA-DSPC liposome group had the highest uptake rate after incubation with immature BMDCs or DC2.4 for two hours,and the free antigen FITC-BSA group had the lowest uptake rate.The results of DCs maturation experiments showed that all the DDA complex liposomes could significantly up-regulate the expression of co-stimulatory molecules CD86 and MHC-II on DCs(p<0.05),indicating that DDA complex liposomes can promote the maturation of DCs.3 The DDA-DSPC group significantly increased the IgA antibody titer in nasal mucus after immunization with nasal mucosa,the DDA-DSPC-TPGS group significantly increased serum IgG1 and levels of spleen cytokine IFN-?,however,all formulations failed to induce the expression of IL-4.Conclusion We prepared the DDA complex liposome influenza vaccines by lipid-film hydration method,and studied the lyophilization prescription and conditions of lyophilized liposome vaccines.The appearance of each liposome vaccine group was spherical or ellipsoidal.The results of in vitro uptake experiments showed that DDA complex liposomes could increase the uptake of DCs,especially the DDA-DSPC group,its uptake effect was significantly higherthan the other two groups.The results of DCs maturation experiments showed that DDA complex liposomes could up-regulate the expression levels of co-stimulatory molecules CD80,CD86 and MHC-II on DCs compared with the antigen group,indicating that DDA complex liposomes can stimulate the differentiation and maturation of DCs.Animal experiments showed that the three groups of DDA complex liposome influenza vaccine can induce higher levels of serum IgG1 antibody,nasal mucus IgA antibody and IFN-? after immunization with nasal mucosa,especially the DDA-DSPC-TPGS group,induced a higher level of IFN-? and IgG1,indicating that TPGS has certain immunomodulatory effect.