Priming of immune responses to hepatitis B surface antigen with minimal DNA expression constructs modified with a nuclear localization signal peptide.

Nuclear localization signal (NLS) peptides conjugated to DNA increase transfection efficiency in vitro. We tested in mice whether conjugation of NLS peptides to DNA vaccines enhances their immunogenicity after intramuscular injection or gene gun mediated intradermal delivery. We constructed the plasmid pMOK-HBsAY that contains a transcription unit encoding hepatitis B surface antigen (HBsAg) and bacterial sequences for amplification of plasmid DNA. From this plasmid we derived the minimal expression construct pMOK-HBsAY-MIDGE, a covalently closed linear DNA that contains only the HBsAg transcription unit. Both constructs stimulated similar (predominantly IgG1) antibody response to HBsAg after gene gun immunization. In contrast, pMOK-HBsAY plasmid DNA was more efficient than pMOK-HBsAY-MIDGE DNA in priming predominantly IgG2a antibody responses to HBsAg after intramuscular injection. Both constructs efficiently primed cytotoxic T lymphocyte responses after intramuscular immunization. When a NLS peptide was coupled to the pMOK-HBsAY-MIDGE DNA, HBsAg transfection efficiency in vitro and priming of antibody responses to HBsAg after intramuscular (but not gene gun mediated) injection was enhanced 10- to 15-fold. These data show: (a) MIDGE constructs can be used as DNA vaccines indicating that bacterial sequences are not essential cofactors; and (b) in intramuscular (but not gene gun mediated) delivery the immunogenicity of a MIDGE-based vaccine is enhanced by coupling NLS peptides to the vector DNA.