Marcin Kwissa

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

Abstract 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.

Codelivery of a DNA vaccine and a protein vaccine with aluminum phosphate stimulates a potent and multivalent immune response

The study explores the possibility of efficiently codelivering DNA vaccines and protein-based vaccines by formulation with aluminum phosphate (AlPO4). When mixed with aluminum adjuvants, plasmid DNA bound tightly onto aluminum hydroxide [Al(OH)3] but not to AlPO4. Different doses of DNA vaccines formulated with AlPO4 [but not Al(OH)3] induced enhanced humoral responses and supported priming of MHC class I restricted cellular immunity. Different proteins mixed with the plasmid DNA vaccine in the AlPO4 formulation did not impair its immunogenicity. Coinjection of two different vaccine-relevant antigens in the same AlPO4 formulation, one as a DNA vaccine and the other as a recombinant protein, elicited polyvalent, humoral, and cellular immune responses to all antigens delivered. The isotype profiles of the induced humoral responses and the cytokine profiles of the specifically primed T cell responses indicated that the combined vaccines supported copriming of Th1- and Th2-biased as well as balanced responses. These findings indicate that the AlPO4 adjuvant, a widely accepted adjuvant in human vaccination practice, can be used to combine protein- and DNA-based vaccination to prime an enhanced and balanced specific immunity.

Breaking tolerance in hepatitis B surface antigen (HBsAg) transgenic mice by vaccination with cross‐reactive, natural HBsAg variants

Processing exogenous hepatitis B surface antigen (HBsAg) of the hepatitis B virus (HBV) generates the Kb‐binding S208–215 epitope 1; processing endogenous HBsAg generates theKb‐binding S190–197 epitope 2. Cross‐reactive CD8+ T cell responses were primed to epitope 1 but not epitope 2 when mice were immunized with natural HBsAgayw, orHBsAgadw2 variants differing within both epitopes by one or two residues. Expression of HBsAgayw from a transgene in the liver renders (HBs‐tg) mice tolerant to epitope 1 of HBsAgayw. CD8+ T cells specific for epitope 1 could be primed in HBs‐tg mice by HBsAgadw2; these specific CD8+ T cells cross‐reacted with epitope 1 processed from the transgene‐encoded HBsAgayw. The liver of vaccinated HBsAgayw transgenic mice showed severe histopathology and contained functional (IFNγ‐producing), cross‐reactive CD8+ T cells, and vaccinated HBs‐tg mice showed reduced antigenemia. Hence, vaccination with natural HBsAg variants from different HBV sero/genotypes can prime cross‐reactive, specific CD8+ T cellimmunity that breaks tolerance to HBsAg.

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.

Priming polyvalent immunity by DNA vaccines expressing chimeric antigens with a stress protein-capturing, viral J-domain

The N‐terminal domain of large tumor antigens (T‐Ag) of polyomaviruses forms a DnaJ‐like structure with a conserved J domain that associates with constitutively expressed stress protein heat shock protein (hsp)73. Mutant (but not wild‐type) SV40 T‐Ag show stable, ATP‐dependent binding to the stress protein hsp73 when expressed in cells from different vertebrate tissues. Intracellular T/hsp73 complexes accumulate to high steady‐state levels. From this observation, we designed a vector system that supports stable expression of a large variety of hsp73capturing, chimeric antigens containing an N‐terminal, T‐Ag‐derived domain, and different C‐terminal antigenic domains from unrelated antigens. Most antigenic domains tested could be stably expressed only in eukaryotic cells as fusion protein/hsp73 complexes. The N‐terminal 77 residues representing the J domain of T‐Ag were required for stable hsp73 binding and efficient expression of chimeric antigens. Hsp73‐bound chimeric antigens expressed by DNA vaccines showed strikingly enhanced immunogenicity evident in humoral (antibody) and cellular cytolytic T lymphocytes (CTL) responses. The described system supports efficient expression of chimeric, polyvalent antigens and their codelivery with hsp73 as a “natural adjuvant” for enhanced immunogenicity for T and B cells.

Selective Expression of Immunogenic, Virus-Like Particle-Derived Antibody-Binding Epitopes

The incorporation of linear and conformational antibody-binding epitopes into polyepitope, chimeric antigens with satisfactory immunogenicity is a challenge. We selectively expressed antigen fragments encoding the linear e2 epitope (C79–149) of hepatitis B virus (pre)core antigen (HBc/eAg) and the conformational ‘a’ epitope (S80–180) of hepatitis B surface antigen (HBsAg) in a novel system. The domains were expressed as chimeric antigens containing either heat shock protein (hsp)73-binding simian virus 40 large tumor antigen (e.g. T77) or non-hsp-binding (e.g. T60) sequences at their N-termini. We compared their type of expression with their immunogenicity for B cells (when delivered as a DNA vaccine). The type of expression investigated included their level of expression, the secretion or intracellular expression of the antigen and the stress protein (hsp)-associated versus nonassociated expression. The linear e2 epitope of HBc/eAg was efficiently expressed as an intracellular, hsp73-binding fusion protein, and efficiently primed an HBc/eAg-specific antibody response when delivered in this form. The conformational ‘a’ epitope of HBsAg most efficiently stimulated B cells as a secreted, non-hsp-associated fusion protein. These data demonstrate that different B cell-stimulating epitopes of vaccine-relevant viral antigens can be selectively isolated and expressed in suitable expression systems, but that the requirements that have to be fulfilled to obtain optimal immunogenicity differ strikingly between individual epitopes.