Minicircle: Next Generation DNA Vectors for Vaccination.

The use of DNA vaccines requires pharmaceutical grade DNA that causes the immunization on the basis of a nucleic acid sequence that encodes the protein to be vaccinated against. This nucleic acid sequence can be a circular or linear plasmid, preferably a double stranded one and should not contain any other and especially not any "toxic" sequences. Sequences that are not desirable to be part of the DNA drug can be those deriving from the (typically) bacterial amplification system to produce the DNA vaccine. These could be those portions of a plasmid that are only used for controlling the bacterial replication of the plasmid or those used to select for the plasmid during cloning or even worse during production. After initial approaches to avoid the presence of these sequences in DNA vaccine plasmids with "mini-plasmids," a significant improvement in product safety was obtained by use of minicircles-circular and ccc-supercoiled expression cassettes of the DNA vaccine. Initial results proofed their extremely high expression level and recent comparison of DNA vaccines based on either plasmid or minicircle DNA show successful vaccination against HBV in mice, as shown in this overview chapter.

Strict control of transgene expression in a mouse model for sensitive biological applications based on RMCE compatible ES cells.

Recombinant mouse strains that harbor tightly controlled transgene expression proved to be indispensible tools to elucidate gene function. Different strategies have been employed to achieve controlled induction of the transgene. However, many models are accompanied by a considerable level of basal expression in the non-induced state. Thereby, applications that request tight control of transgene expression, such as the expression of toxic genes and the investigation of immune response to neo antigens are excluded. We developed a new Cre/loxP-based strategy to achieve strict control of transgene expression. This strategy was combined with RMCE (recombinase mediated cassette exchange) that facilitates the targeting of genes into a tagged site in ES cells. The tightness of regulation was confirmed using luciferase as a reporter. The transgene was induced upon breeding these mice to effector animals harboring either the ubiquitous (ROSA26) or liver-specific (Albumin) expression of CreER(T2), and subsequent feeding with Tamoxifen. Making use of RMCE, luciferase was replaced by Ovalbumin antigen. Mice generated from these ES cells were mated with mice expressing liver-specific CreER(T2). The transgenic mice were examined for the establishment of an immune response. They were fully competent to establish an immune response upon hepatocyte specific OVA antigen expression as indicated by a massive liver damage upon Tamoxifen treatment and did not show OVA tolerance. Together, this proves that this strategy supports strict control of transgenes that is even compatible with highly sensitive biological readouts.

PsaA (pneumococcal surface adhesin A) and PspA (pneumococcal surface protein A) DNA vaccines induce humoral and cellular immune responses against Streptococcus pneumoniae.

Streptococcus pneumoniae is one of the most important human pathogens and improvement of the currently used polysaccharide vaccines is being pursued. We constructed DNA vaccine vectors containing either the full-length psaA (pneumococcal surface adhesin A) or a truncated pspA (pneumococcal surface protein A--pspA') gene. Both constructs showed transient expression of the antigens in vertebrate cells and induced significant antibody response to the pneumococcal antigens in BALB/c mice injected intramuscularly (i.m.). Fusion with an N-terminal cytoplasmatic SV40 T-antigen (CT-Ag), which was previously shown to stabilize poorly expressed antigens through association with Hsp73, also induced anti-PspA antibody response. The induction of antibodies with a low IgG1:IgG2a ratio and elevated gamma interferon (IFN-gamma) production by spleen cells elicited by DNA vaccination indicate preferential priming of Th1 immunity. Since induction of antibodies against both PsaA and PspA was previously shown to correlate with protection against fatal infection with S. pneumoniae and cell-mediated immune responses could contribute to protection, further evaluation of PsaA and PspA as antigens for a DNA vaccine against S. pneumoniae could be promising.

Multiple effects of codon usage optimization on expression and immunogenicity of DNA candidate vaccines encoding the human immunodeficiency virus type 1 Gag protein.

We have analyzed the influence of codon usage modifications on the expression levels and immunogenicity of DNA vaccines, encoding the human immunodeficiency virus type 1 (HIV-1) group-specific antigen (Gag). In the presence of Rev, an expression vector containing the wild-type (wt) gag gene flanked by essential cis-acting sites such as the 5'-untranslated region and 3'-Rev response element supported substantial Gag protein expression and secretion in human H1299 and monkey COS-7 cells. However, only weak Gag production was observed from the murine muscle cell line C2C12. In contrast, optimization of the Gag coding sequence to that of highly expressed mammalian genes (syngag) resulted in an obvious increase in the G+C content and a Rev-independent expression and secretion of Gag in all tested mammalian cell lines, including murine C2C12 muscle cells. Mice immunized intramuscularly with the syngag plasmid showed Th1-driven humoral and cellular responses that were substantially higher than those obtained after injection of the Rev-dependent wild-type (wt) gag vector system. In contrast, intradermal immunization of both wt gag and syngag vector systems with the particle gun induced a Th2-biased antibody response and no cytotoxic T lymphocytes. Deletion analysis demonstrated that the CpG motifs generated within syngag by codon optimization do not contribute significantly to the high immunogenicity of the syngag plasmid. Moreover, low doses of coadministered stimulatory phosphorothioate oligodeoxynucleotides (ODNs) had only a weak effect on antibody production, whereas at higher doses immunostimulatory and nonstimulatory ODNs showed a dose-dependent suppression of humoral responses. These results suggest that increased Gag expression, rather than modulation of CpG-driven vector immunity, is responsible for the enhanced immunogenicity of the syngag DNA vaccine.

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.

Modulation of gene-gun-mediated Th2 immunity to hepatitis B surface antigen by bacterial CpG motifs or IL-12.

Using different DNA vaccination techniques, we studied the IgG1/IgG2a antibody and MHC-I-restricted cytotoxic T lymphocyte (CTL) responses to the hepatitis B surface antigen (HBsAg) in mice. A single intramuscular injection of 100 microg HBsAg-encoding pCI/S plasmid DNA efficiently primed IgG2a antibody (IgG1/IgG2a ratio <0.3) and CTL responses to HBsAg (Th1 immunity). In contrast, a single intradermal injection of 1 microg of particle-coated pCI/S DNA with the gene-gun-primed IgG1 antibody responses to HBsAg (IgG1/IgG2a ratio >80) but there was no CTL response (Th2 immunity). Injection of immune-stimulating CpG-containing oligodeoxy-nucleotides (ODN) into the skin area used for gene-gun-mediated pCI/S DNA delivery shifted the polarization of the response towards Th1 immunity. A similar shift from Th2 to Th1 immunity was observed when the skin area used for gene-gun-mediated DNA transfer was conditioned by injection of recombinant IL-12. DNA vaccination can thus prime polarized immunity to HBsAg. The polarization of immunity is determined by the technique of plasmid DNA delivery as well as by conditions of the tissue into which DNA is inoculated. Th1 immunity to HBsAg (primed by injection of naked pCI/S DNA) dominated Th2 immunity (primed by gene-gun-mediated pCI/S DNA). In contrast, an established HBsAg-specific Th2 immunity was readily shifted towards Th1 immunity (including specific CTL priming) by an intramuscular boost injection of pCI/S DNA. These data contribute to the rational design of DNA vaccination strategies to efficiently prime anti-viral Th1 immune effector specificities using the gene gun.

Activating immunity in the liver. I. Liver dendritic cells (but not hepatocytes) are potent activators of IFN-gamma release by liver NKT cells.

A prominent subset of the hepatic innate immune system is alpha-galactosylceramide (alphaGalCer)-reactive, (CD4(+) and CD4(-)CD8(-)) CD1d-restricted NKT cells. We investigated in C57BL/6 (B6) mice which hepatic cell type stimulates hepatic NKT cell activation. Surface expression of CD1d but not CD40, CD80, or CD86 costimulator molecules was detected in hepatocytes. Pulsed in vitro or in vivo with alphaGalCer, hepatocytes triggered IL-4 release by liver NKT cells but required exogenous IL-12 to trigger IFN-gamma release by NKT cells. Liver dendritic cells (DC) isolated from nontreated mice showed low surface expression of MHC, CD1d, and CD40, CD80, or CD86 costimulator molecules that were strikingly up-regulated after alphaGalCer injection. Although liver CD11c(+) DC displayed lower CD1d surface expression than hepatocytes, they were potent stimulators of IFN-gamma and IL-4 release by liver NKT when pulsed with alphaGalCer in vitro or in vivo. Liver DC are thus potent stimulators of proinflammatory cytokine release by NKT cells, are activated themselves in the process of NKT cell activation, and express an activated phenotype after the NKT cell population is eliminated following alphaGalCer stimulation.

IL-12/IL-18-dependent IFN-gamma release by murine dendritic cells.

Dendritic cells (DC) develop in GM-CSF-stimulated cultures from murine bone marrow progenitors in serum-free (or low serum) medium. CD11c(+) myeloid DC from 7-day cultures stimulated with TNF-alpha, IFN-alpha, IFN-gamma, or LPS up-regulated surface expression of CD40 and CD86 costimulator and MHC class II molecules, did not up-regulate the low "spontaneous" release of IL-18, and did not release IFN-gamma. Stimulation of in vitro-generated DC with exogenous IL-12 and IL-18 (but not with IL-4 or LPS plus IL-18) induced IFN-gamma expression and release in 15-20% of the DC (detectable by FACS analyses or ELISA). Endogenous IL-12 p70 produced by DC in response to ligation of CD40 stimulated IFN-gamma release when exogenous IL-18 was supplied. In vivo-generated, splenic CD8alpha(+) and CD8alpha(-) DC (from immunocompetent and immunodeficient H-2(d) and H-2(b) mice) cultured with IL-12 and IL-18 released IFN-gamma. The presence of LPS during the stimulation of DC with IL-18 plus endogenous (CD40 ligation) or exogenous IL-12 did not affect their IFN-gamma release. In contrast, splenic DC pretreated in vitro or in vivo by LPS strikingly down-regulated IFN-gamma release in response to stimulation by IL-18 and (endogenous or exogenous) IL-12. Hence, DC are a source of early IFN-gamma generated in response to a cascade of cytokine- and/or cell-derived signals that can be positively and negatively regulated.

Revealing the potential of DNA-based vaccination: lessons learned from the hepatitis B virus surface antigen.

DNA-based vaccination is a novel technique to efficiently stimulate humoral (antibody) and cellular (T cell) immune responses to protein antigens. In DNA-based vaccination, immunogenic proteins are expressed in in vivo transfected cells of the vaccine recipients in their native conformation with correct posttranslational modifications from antigen-encoding expression plasmid DNA. This ensures the integrity of antibody-defined epitopes and supports the generation of protective (neutralizing) antibody titers. Plasmid DNA vaccination is furthermore an exceptionally potent strategy to stimulate CD8+ cytotoxic T lymphocyte (CTL) responses because antigenic peptides are efficiently generated by endogenous processing of intracellular protein antigens. These key features make DNA-based immunization an attractive strategy for prophylactic and therapeutic vaccination against extra- and intracellular pathogens. In this brief review, we summarize the current state of expression vector design, DNA delivery strategies, priming immune responses to intracellular or secreted antigens by DNA vaccines and unique advantages of DNA- versus recombinant protein-based vaccines using the hepatitis B surface antigen (HBsAg) as a model antigen.

Targeting murine immune responses to selected T cell- or antibody-defined determinants of the hepatitis B surface antigen by plasmid DNA vaccines encoding chimeric antigen.

By exchanging sequences from the middle-surface (MS) and small-surface (S) Ag of hepatitis B virus (HBV) with corresponding sequences of the MS Ag of woodchuck hepatitis virus, we constructed chimeric MS variants. Using these constructs as DNA vaccines in mice, we selectively primed highly specific (non-cross-reactive) Ab responses to pre-S2 of the HBV MS Ag and the "a" determinant of the HBV S Ag, as well as L(d)- or K(b)-restricted CTL responses to HBV S epitopes. In transgenic mice that constitutively express large amounts of HBV surface Ag in the liver we could successfully suppress serum antigenemia (but not Ag production in the liver) by adoptive transfer of anti-pre-S2 or anti-"a" immunity but not CTL immunity. DNA vaccines greatly facilitate construction of chimeric fusion Ags that efficiently prime specific, high-affinity Ab and CTL responses. Such vaccines, in which sequences of an Ag of interest are exchanged between different but related viruses, are interesting tools for focusing humoral or cellular immunity on selected antigenic determinants and elucidating their biological role.

DNA vaccination of mice with a plasmid encoding Puumala hantavirus nucleocapsid protein mimics the B-cell response induced by virus infection.

Inoculation of naked DNA has been applied for the development of prophylactic and therapeutic vaccines against different viral infections. To study the humoral immune response induced by DNA vaccination we cloned the entire nucleocapsid protein-encoding sequence of the Puumala hantavirus strain Vranica/Hällnäs into the CMV promoter-driven expression unit of the plasmid pcDNA3, generating pcDNA3-VR1. A single dose injection of 50 microg of plasmid DNA into each M. tibialis anterior of BALB/c mice induced a high-titered antibody response against the nucleocapsid protein as documented 6 and 11 weeks after immunisation. PEPSCAN analysis of a serum pool of the pcDNA3-VR1-vaccinated animals revealed antibodies reacting with epitopes covering the whole nucleocapsid protein. The epitope-specificity of the immune response induced by DNA vaccination seems to reflect the antibody response in experimentally virus-infected bank voles (the natural host of the Puumala virus) and humans. The data suggest that DNA vaccination could be used for the identification of highly immunogenic epitopes in viral proteins.

Monocyte inflammatory protein-1 alpha facilitates priming of CD8(+) T cell responses to exogenous viral antigen.

Dendritic cells (DC) derived from bone marrow precursors of BALB/c or C57BL/6 mice in low-serum cultures supplemented with granulocyte macrophage colony stimulating factor and Flt(3) ligand were pulsed in vitro with hepatitis B surface antigen (HBsAg) particles. DC processed exogenous HBsAg and presented its MHC class I-binding epitopes to cytotoxic T lymphocytes (CTL). This specific and restricted interaction of DC with CTL stimulated release of IFN-gamma and macrophage inflammatory protein (MIP)-1 alpha from the responding CTL. MIP-1 alpha enhanced the survival of DC in vitro but did not induce proliferation. Furthermore, co-delivery of MIP-1 alpha facilitated CTL priming in vivo to exogenous HBsAg in low responder C57BL/6 (H-2(b)) mice: a single injection of a low dose of HBsAg particles (without further adjuvants) successfully primed K(b)-restricted CTL responses to HBsAg only when the exogenous antigen was co-delivered with 100 ng MIP-1 alpha. These in vitro and in vivo data point to an important role of MIP-1 alpha in the DC-dependent priming of CTL to exogenous viral antigens.

DNA vaccines.

BACKGROUND AND OBJECTIVES: Humoral and cellular immune responses to protein antigens can be efficiently primed by nucleic acid or DNA vaccination. In DNA-based vaccination, immunogenic proteins are expressed with correct posttranslational modification, conformation or oligomerization; this ensures the integrity of epitopes that stimulate neutralizing antibody (B cell) responses. DNA (or RNA) immunization is exceptionally potent in stimulating T cell responses because antigenic peptides are efficiently generated in (endogenous or exogenous) processing pathways (without interference by viral proteins) from intracellular or extracellular protein antigens expressed after transient in vivo transfection. Both features are difficult to achieve with recombinant subunit vaccines produced in eukaryotic or prokaryotic expression systems. The current state of vector designs, strategies for delivery of DNA vaccines, priming humoral and cellular immune responses by DNA vaccines, experimental strategies facilitated by DNA vaccines, unique advantages of DNA vaccination, experience of DNA vaccination in preclinical animal models and clinical trials, and potential risks of DNA vaccination are discussed. Excellent reviews on DNA-based vaccination have been published recently [1-3].

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.

DNA vaccination using coexpression of cytokine genes with a bacterial gene encoding a 60-kDa heat shock protein.

Coexpression of cytokine genes together with antigen-encoding genes in DNA vaccination vectors can increase humoral and cellular immune responses and may steer them in a Th1 or Th2 direction. In this study, the modulatory effect of interleukin (IL)-2, IL-4, and interferon (IFN)-gamma coexpressed with the 60-kDa heat shock protein (Hsp60) of Yersinia enterocolitica O:8 (Y-Hsp60) was studied. DNA vaccination with gamma-hsp60 evoked specific humoral and cellular immune responses as well as reduction of the splenic bacterial load upon challenge with Y. enterocolitica in a mouse infection model. Coexpression of IL-2 or IFN-gamma enhanced Y. enterocolitica-specific total IgG (P < 0.05) and IgG2a antibody responses. Coexpression of IFN-gamma also improved the proliferative T cell responses upon stimulation with Y-Hsp60. A reduction of the splenic bacterial load as compared with the plasmid encoding Y-Hsp60 only was found for the IFN-gamma coexpressing vector. Thus, coexpression of cytokine genes such as IFN-gamma in DNA vaccination vectors might improve immunity and help to overcome the side effects of standard adjuvants.

Modulating specific priming of immune effector functions by DNA-based vaccination strategies.

We have used the hepatitis B virus system in mice to investigate under which conditions the different DNA delivery techniques used in DNA vaccination introduce a T2 or T1 bias into the responses they elicit. Priming mice by an intramuscular injection of 100 microg plasmid DNA stimulated long-lasting T1 immune responses that included specific activation of potent MHC-1-restricted CTL responses. In contrast, priming mice with an intradermal injection of a low dose of plasmid DNA (0.1-1 microg/mouse) stimulated long-lasting T2 immune responses but no CTL responses. This polarization (spectrum of immune effector specificities) of immune responses elicited by plasmid DNA vaccines is determined by different factors, e.g. (i) the vaccination technique used; (ii) the age of the immunized animal; (iii) the condition of the local tissue environment to which DNA is delivered; (iv) the co delivery of immune-modulating factors; and (v) the type of antigen encoded by the plasmid DNA vaccine. DNA vaccination offers the possibility of constructing endogenous, chimaeric antigens with enhanced, hsp-facilitated expression, enhanced immunogenicity for B cells, and alternative, TAP-independent processing for MHC-1 restricted peptide presentation.

Ongoing murine T1 or T2 immune responses to the hepatitis B surface antigen are excluded from the liver that expresses transgene-encoded hepatitis B surface antigen.

Different protein- or DNA-based vaccination techniques are available that prime potent humoral and cellular, T1 or T2 immune responses to the hepatitis B surface Ag (HBsAg) in mice. T1 and T2 are immune responses with isotype profile indicating Th1 and Th2 immunoregulation. We tested whether HBsAg-specific immune responses can be established in transgenic mice that express HBsAg in the liver (HBs-tg mice) using either these different vaccination techniques or an adoptive transfer system. HBsAg-specific responses could not be primed in HBs-tg mice with the established, potent vaccine delivery techniques. In contrast, adoptive transfers of T1- and T2-type HBsAg-immune spleen cells into congenic HBs-tg hosts (that were not conditioned by pretreatment) suppressed HBsAg antigenemia and gave rise to HBsAg-specific serum Ab titers. The establishment of continuously rising anti-HBsAg serum Ab levels with alternative isotype profiles (reflecting T1 or T2 polarization) in transplanted HBs-tg hosts required donor CD4+ T cell-dependent restimulation of adoptively transferred immune cells by transgene-derived HBsAg. Injections of HBsAg-specific Abs into HBs-tg mice did not establish stable humoral immunity. The expanding T1 or T2 immune responses to HBsAg in HBs-tg hosts did not suppress transgene-directed HBsAg expression in the liver and did not induce liver injury. In addition to priming functional antiviral effector cells, the conditioning of the liver microenvironment to enable delivery of antiviral effector functions to this organ are therefore critical for effective antiviral defense. A major challenge in the development of a therapeutic vaccine against chronic hepatitis B or C virus infection is thus the efficient targeting of specifically induced immune effector specificities to the liver.