Adjuvants are important components of recombinant protein vaccines which are often poorly immunogenic. For decades, the search for new vaccine adjuvants has been predominantly empirical. In addition, combinations of more than one adjuvant plus antigen have been systematically studied. Plasmid DNA containing additional oligodeoxynucleotides with unmethylated CpG motifs (CpG ODN) entrapped in liposomes has been used as an adjuvant for DNA vaccines and has shown powerful immunostimulatory

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... . In our study, the combination of plasmid DNA containing 16 additional CpG ODNs (pv-16CpG) and aluminum hydroxide (AL) or incomplete Freund's adjuvant (IFA) was used as an adjuvant for a hepatitis B surface antigen (HBsAg) vaccine to immunize C57BL/6J mice. ELISA and ELISPOT assays were used to analyze the immunological effects of the novel vaccine. A significant enhancement of the anti-HBs titer and seroconversion was observed when the CpG plasmid was combined with IFA, but not with AL. In addition, anti-HBs antibody isotype analysis revealed that the combination of CpG plasmid and IFA induced a strong HBsAg-specific IgG2a response. Moreover, the ELISPOT assays suggested that pv-16CpG suspended in IFA evoked a strong T helper 1 (Th1) immune response and high IFN-γ production. These results demonstrate that pv-16CpG suspended in IFA is able to induce cellular and humoral immune responses to HBsAg, and confirm its potential as an adjuvant for use in protein vaccines.