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 trans fection efficiency in vitro. We tested in mice whether conjugation of NLS p eptides to DNA vaccines enhances their immunogenicity after intramuscular i njection or gene gun mediated intradermal delivery. We constructed the plas mid pMOK-HBsAY that contains a transcription unit encoding hepatitis B surf ace antigen (HBsAg) and bacterial sequences for amplification of plasmid DN A. From this plasmid we derived the minimal expression construct pMOK-HBsAY -MIDGE, a covalently closed linear DNA that contains only the HBsAg transcr iption unit. Both constructs stimulated similar (predominantly IgG1) antibo dy response to HBsAg after gene gun immunization. In contrast, pMOK-HBsAY p lasmid DNA was more efficient than pMOK-HBsAY-MIDGE DNA in priming predomin antly IgG2a antibody responses to HBsAg after intramuscular injection. Both constructs efficiently primed cytotoxic T lymphocyte responses after intra muscular immunization. When a NLS peptide was coupled to the pMOK-HBsAY-MID GE DNA, HBsAg transfection efficiency in vitro and priming of antibody resp onses to HBsAg after intramuscular (but not gene gun mediated) injection wa s enhanced 10- to 15-fold. These data show: (a) MIDGE constructs can be use d as DNA vaccines indicating that bacterial sequences are not essential cof actors; and (b) in intramuscular (but not gene gun mediated) delivery the i mmunogenicity of a MIDGE-based vaccine is enhanced by coupling NLS peptides to the vector DNA.