Objective To evaluate the immuno-potentiating effects of CpG-ODN plus alum as a composite adjuvant on influenza split virion vaccine.Methods BALB/c mice were immunized with various amounts of 2009 H1N1 influenza split virion vaccine,alone or in combination with CpG-ODN,alum,or both (composite adjuvant).Antigen-specific humoral immune responses were evaluated by ELISA,hemagglutination inhibiting (HI) assay and neutralizing assay.Antigen-specific cellular immune responses were evaluated by ELISPOT assay,intracellular cytokine staining assay and in vivo CTL assay.Results Compared with the control group immunized with antigen alone,a single use of either adjuvant weakly enhanced the humoral immune responses,as indicated by the increase of antigen-specific IgG titers,HI titers and neutralizing titers by 3-6 folds,2-4 folds and 4-8 folds,respectively,after two immunizations.In contrast,the composite adjuvant induced more potent humoral immune responses; the antigen-specific IgG titers,HI titers and neutralizing titers were increased by 23-57 folds,9-20 folds and 16-64 folds,respectively.Consequently,the composite adjuvant achieved antigen-sparing by at least 16 folds.In addition,the composite adjuvant significantly enhanced the antigen-specific cellular immune responses,as revealed by the increase of IFN-γ-secreting CD4+ T cells and the enhancement of CTL activity in immunized mice.Conclusion CpG-ODN plus alum as a composite adjuvant can enhance the immunogenicity of influenza split virion vaccine and achieve the antigen-sparing effect.