Objective To validate the enhanced immune responses when cationic liposomes DOTAP was used as the adju-vant in H5N1 influenza split vaccine (simplified as influenza split vaccine). Methods DOTAP cationic liposomes influ-enza vaccines were prepared by evaporating and hydrating of lipid films. A total of 13 groups of BALB/c mice were inocula-ted subcutaneously with 0. 1μg, 1. 0μg, 10. 0μg HA dose in influenza split vaccine containing DOTAP, Alum CPG-ODN as adjuvant, respictively, and influenza split vaccine alone as negative control or PBS as blank at 0, 21 day. Hemaggluti-nin inhibition (HI) antibody titers were measured at 21, 42 day, the specific IgG antibody and subclass antibody IgG1 and IgG2a were measured by ELISA at 42 day;and the single spleen cell was isolated and stimulated with antigen in vitro, then the released level of IL-2, IL-4, IFN-γ were measured by ELISA at 42 day. A total of 3 groups of BALB/c mice were inoculated subcutaneously by influenza split vaccines wsith 100, 300, 600 μg dose of DOTAP liposomes at 0, 21 day. HI anti-body titers were measured at 21, 42 day, the specific IgG anti-body were measured by ELISA. Results All cationic liposome preparations had a diameter in 300-400 nm with a postive charge, and reached more than 50% efficiency of encapsulation. Serology test indicated that the titers of HI antibody and specific antibody of DOTAP cationic liposomes influenza split vaccines were significantly higher than that of the influenza split vaccine without adjuvant, and had no a statistical difference with influenza split vaccine containing Alum or CPG-ODN. The higher level of IgG1 antibodies were induced by DOTAP cationic liposomes influenza split vaccines in compari-son of that of IgG2a antibodies. However, the antibody IgG2a titers were higher than that of the influenza split vaccine con-tainig Alum in 42 day after vaccination, and lower than that of influenza split vaccine containing CPG-ODN. The result of Multiplex cytokine analysis indicated that both of high level of Th1 cytokine IFN-γand Th2 cytokine IL-4 were secreted by spleen cells from DOTAP cationic liposomes influenza split vaccines group. The titers of HI antibody and specific antibody in high,medium and low dose of DOTAP groups had a remarkable dose-effect relationship. Conclusion DOTAP cationic liposomes as a adjuvant could significantly enhance the immunogenity of H5N1 influenza split vaccine, and it had a strong capability to boost the humoral immunityas well as with Alum or CPG-ODN vaccine. In addtion, DOTAP cationic liposomes could induce cell mediated immunity as a adjuvant.%目的：研究阳离子脂质体DOTAP佐剂对H5N1型流感病毒裂解疫苗免疫效果的影响。方法制备DOTAP阳离子脂质体流感病毒裂解疫苗(简称DOTAP流感裂解疫苗),检测其包封率。将BALB/c小鼠分为13组,分别用含0.1、1.0、10.0μg HA/只剂量以DOTAP、Al(OH)3、CPG-ODN为佐剂以及不含佐剂的流感裂解疫苗于0、21天皮下免疫,PBS作为对照组,用血凝抑制试验检测小鼠初次免疫后21、42天血清HI抗体滴度；用ELISA检测初次免疫后21、42天血清特异性IgG抗体、IgG1、IgG2a亚类抗体滴度,以及初次免疫后42天小鼠脾脏单个核细胞体外经抗原刺激后细胞因子IL-2、IL-4、IFN-γ的分泌水平。将BALB/c小鼠分为3组,分别用含不同DOTAP剂量(100、300、600μg /只)的DOTAP流感裂解疫苗于0、21天皮下免疫,检测初次免疫后21、42天小鼠血清HI抗体滴度和IgG抗体滴度。结果 DOTAP流感裂解疫苗粒径在300~400 nm,带正电荷,包封率在50%以上；DOTAP流感裂解疫苗诱导的HI抗体水平和特异性IgG抗体水平均高于流感裂解疫苗,而与铝佐剂和CpG-ODN佐剂间差异无统计学意义；DOTAP流感裂解疫苗产生的抗体仍以IgG1亚类抗体为主,免疫后42天诱导的IgG2a亚类抗体水平高于流感裂解疫苗和铝佐剂,低于CpG-ODN佐剂；DOTAP 流感裂解疫苗免疫后既分泌高水平Th1型细胞因子IFN-γ,同时也分泌高水平Th2型细胞因子IL-4；不同DOTAP剂量的DOTAP流感裂解疫苗免疫后,其HI抗体滴度和IgG抗体滴度在低、中、高剂量组之间存在明显的量效关系。结论 DOTAP作为H5N1型流感病毒裂解疫苗的佐剂可显著提高流感裂解疫苗的免疫原性,其对体液免疫应答的增强作用不低于铝佐剂和CpG-ODN佐剂,并具有诱导细胞免疫应答的能力。
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