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.

Composition and arrangement of genes define the strength of IRES-driven translation in bicistronic mRNAs.

In addition to the cap-dependent mechanism, eukaryotic initiation of translation can occur by a cap-independent mechanism which directs ribosomes to defined start codons enabled by internal ribosome entry site (IRES) elements. IRES elements from poliovirus and encephalomyocarditis virus are often used to construct bi- or oligocistronic expression vectors to co-express various genes from one mRNA. We found that while cap-dependent translation initiation from bicistronic mRNAs remains comparable to monocistronic expression, internal initiation mediated by these viral IRESs is often very inefficient. Expression of bicistronic expression vectors containing the hepatitis B virus core antigen (HBcAg) together with various cytokines in the second cistron of bicistronic mRNAs gave rise to very low levels of the tested cytokines. On the other hand, the HBcAg was well expressed when positioned in the second cistron. This suggests that the arrangement of cistrons in a bicistronic setting is crucial for IRES-dependent translation of the second cistron. A systematic examination of expression of reporter cistrons from bicistronic mRNAs with respect to position was carried out. Using the dual luciferase assay system we show that the composition of reading frames on a bicistronic mRNA and the order in which they are arranged define the strength of IRES-dependent translation. Although the cellular environment and the nature of the IRES element influence translation strength the dominant determinant is the nature and the arrangement of cistrons on the mRNA.

Molecular assessment of post-BMT chimerism using various biologic specimens and automated DNA sizing technology.

Highly polymorphic microsatellite markers provide useful genetic markers for detection of complete or mixed chimerism in patients after allogeneic BMT (allo-BMT). We report application of automated DNA sizing technology for detection of post-BMT chimerism using fresh peripheral blood, BM, or archival blood smears and various DNA isolation techniques. Donors' and recipients' DNA was amplified with fluorescent PCR primers specific for short tandem repeat (STR) marker loci: FGA, VWA, TH01, F13A1, D21S11. Chimerism was assessed in 14 recipients after allo-BMT. A complete chimerism was detected in 10 patients, in 3 patients we observed fluctuations of chimerism status, and mixed chimerism was assessed in 1 patient. We show that DNA from different types of biologic specimens (whole peripheral blood, BM suspension, archival blood smears), prepared according to the various isolation techniques (salting-out method, phenol chloroform extraction, Chelex procedure) and amplified with fluorescent PCR primers for microsatellite markers, enable identification of chimerism status following allo-BMT in children.

Efficient vaccination by intradermal or intramuscular inoculation of plasmid DNA expressing hepatitis B surface antigen under desmin promoter/enhancer control.

The small surface antigen of the hepatitis B virus (HB5Ag) was cloned into expression plasmid pCI under either a viral (CMV) promoter;enhancer sequence control (plasmid pCI/S), or a human desmin promoter/enhancer sequence control (plasmid pDes/S). Cells of different species and tissue origin transiently transfected in vitro with pCI/S or pDes/S plasmid DNA expressed readily detectable amounts of HBsAg, either intracellularly (precipitated from cell lysates), or as secreted products (detectable in ELISA). When these plasmids were used in DNA vaccination, both efficiently primed humoral and/or cellular immune responses to HBsAg after a single injection in Balb/c mice. Intramuscular injection of a high dose of DNA (100 rig/mouse) of both plasmids primed MHC-I-restricted cytotoxic T lymphocyte (CTL) responses and Thi serum antibody responses (IgGlIgG2a ratio O.4C0.7) of comparable magnitude in all vaccinated mice. Intradermal injection of low doses of (particle-coated) DNA (1 microgm/mouse) of both plasmids with the gene gun primed Th2 serum antibody responses (IgGl/IgG2a ratio > 100) but no CTL responses. The data indicate that antigens can be efficiently expressed under viral or eukaryotic promoter/enhancer control for immunogenic in vivo presentation, but that the technique, dose and/or route of DNA injection have a decisive role in determining the type of immune response elicited.

Polyvalent DNA vaccines with bidirectional promoters.

The hepatitis B surface antigen (HBsAg) and core antigen (HBcAg) were coexpressed from a synthetic bidirectional promoter with the tetracycline-inactivated transactivator (tTA). The function of this autoregulative system was evaluated following either transfer into established cell lines or intramuscular and intradermal injection of high or low doses of DNA into mice. We measured in vitro antigen expression and in vivo the induction of specific humoral and cellular immune responses. Successful regulation of antigen expression was observed in cultured cells. DNA vaccination with these constructs efficiently primed hepatitis B virus (HBV) specific immunity. However, immunogenic concentrations of the antigens were expressed even in the absence of the transactivator, indicating that low expression level is sufficient to prime an immune response. The bidirectional promoter allows coexpression of either both HBV antigens or a HBV antigen and enhanced green fluorescent protein leading to efficient priming of stable immunity against both antigens. This study demonstrates the potential of synthetic polyvalent plasmids in DNA vaccination.