Objective: To study whether the immunostimulatory activities of DNA vaccine could be enhanced by incorporation of human CpG-oligodeoxynucleotide (ODN) into its plasmid backbone.
Methods: Various copies of human CpG2006 sequence with immonostimulating activity for both human beings and mice were transfected into plasmid. Mouse spleen cells were cultured together with different kinds of plasmid DNA and then the amounts of IgM and interferon gamma (IFN-gamma) in the supernatant were examined by antibody sandwich ELISA. HBsAg plus plasmid DNA, as adjuvant, was injected into the shank muscles of 6 BALB/c mice in experimental group and recombinant HBsAg plus Al (OH)(2) was injected into 6 mice as controls. Four weeks later, the anti-HBsAg, IgG1 and IgG2a in their blood were examined.
Results: A minimum plasmid named pMini was constructed, containing only kanamycin-resistant gene and pUC18 replication origin. By incorporating different amounts of CpG2006 into pMini plasmid, two recombinant plasmids, pCpG11 and pCpG26, with 9 and 16 copies of CpG2006 respectively, were derived. With the increase of number of CpG2006 copies, the ability of recombinant plasmid DNA to induce the secretion of IgM by cultured murine splenocytes increased accordingly in sequence of pCpG26 > pCpG11 > pMini, and its ability to induce the secretion of IFN-gamma by cultured murine splenocytes decreased instead in sequence of pCpG26 < pCpG11 > pMini. After the mice were immunized with recombinant HBsAg plus pMini and pCpG1, the anti-HBs total antibodies and IgG1 isotype in their blood remained unchanged (P > 0.05), but pCpG26 increased the levels of anti-HBs total antibodies and IgG1 isotype by 3 times (P < 0.001) and 2 times (P < 0.02) respectively. The three kinds of plasmid DNA increased the level of specific IgG2a isotype by 5 approximately 10 times at similar degrees (P > 0.05).
Conclusion: The immunostimulatory activities of plasmid DNA can be enhanced by incorporation of human CpG-ODN.