Objective To explore the possibility of using plasmid containing muti-copy of CpG ODN as the adjuvant for therapeutic vaccine against hepatitis B. MethodsA plasmid pKO-CG6 containing six copies of D type CpG ODN was constructed. The plasmid and the carrier plasmid pKO were used to stimulate peripheral blood monouclear cells (PBMC) of healthy or HBV infected subjects; then the proliferation of PBMCs and secretion of IFN-γ and IL-12 were examined. Recombinant HBsAg combined with either of the plasmid was used to immunize BALB/c mice, and immune responses to HBsAg were assayed. ResultsBoth plasmid pKO-CG6 and carrier plasmid pKO not only effectively activated the proliferation response of PBMCs from healthy controls and HBV infected subjects in vitro, but also promoted the production of IFN-γ and IL-12; the immuno-stimulation activity of pKO-CG6 was greatly stronger than that of the carrier plasmid pKO. In vivo study showed that although vector pKO could also act as immunological adjuvant for HBsAg in mice, plasmid pKO-CG6 elicited much stronger immune responses to HBsAg, especially the cell-mediated response. ConclusionPlasmid containing multi-copy of CpG ODN can effectively activate PBMCs of HBV infected subjects and enhance the immune responses to HBsAg in mice.