A novel therapeutic hepatitis B vaccine induces cellular and humoral immune responses and breaks tolerance in hepatitis B virus (HBV) transgenic mice.

Therapeutic vaccines are currently being developed for chronic hepatitis B and C. As an alternative to long-term antiviral treatment or to support only partially effective therapy, they should activate the patient's immune system effectively to fight and finally control the virus. A paradigm of therapeutic vaccination is the potent induction of T-cell responses against key viral antigens - besides activation of a humoral immune response. We have evaluated the potential of a novel vaccine formulation comprising particulate hepatitis B surface (HBsAg) and core antigen (HBcAg), and the saponin-based ISCOMATRIX™ adjuvant for its ability to stimulate T and B cell responses in C57BL/6 mice and its ability to break tolerance in syngeneic HBV transgenic (HBVtg) mice. In C57BL/6 mice, the vaccine induced multifunctional HBsAg- and HBcAg-specific CD8+ T cells detected by staining for IFNγ, TNFα and IL-2, as well as high antibody titers against both antigens. Vaccination of HBVtg animals induced potent HBsAg- and HBcAg-specific CD8+ T-cell responses in spleens and HBcAg-specific CD8+ T-cell responses in livers as well as anti-HBs seroconversion two weeks post injection. Vaccination further reduced HBcAg expression in livers of HBVtg mice without causing liver damage. In summary, this study demonstrates therapeutic efficacy of a novel vaccine formulation in a mouse model of immunotolerant, chronic HBV infection.

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 K(b)-binding S(208-215) epitope 1; processing endogenous HBsAg generates the K(b)-binding S(190-197) epitope 2. Cross-reactive CD8(+) T cell responses were primed to epitope 1 but not epitope 2 when mice were immunized with natural HBsAg(ayw), or HBsAg(adw2) variants differing within both epitopes by one or two residues. Expression of HBsAg(ayw) from a transgene in the liver renders (HBs-tg) mice tolerant to epitope 1 of HBsAg(ayw). CD8(+) T cells specific for epitope 1 could be primed in HBs-tg mice by HBsAg(adw2); these specific CD8(+) T cells cross-reacted with epitope 1 processed from the transgene-encoded HBsAg(ayw). The liver of vaccinated HBsAg(ayw) transgenic mice showed severe histopathology and contained functional (IFNgamma-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 cell immunity that breaks tolerance to HBsAg.

Different immunogenicity of H-2 Kb-restricted epitopes in natural variants of the hepatitis B surface antigen.

The small hepatitis B surface antigen (HBsAg) of hepatitis B virus (HBV) has limited variability, but some serotypes and genotypes have been defined. Although no biological or pathogenetic differences could be traced to HBV serotypes, the clinical picture, response to treatment and long-term prognosis of HBV infection may vary with the HBV genotype, possibly due to differences in specific T cell recognition of HBV antigens from different genotypes. We analyzed murine CD8(+) T cell responses to two K(b)-restricted HBsAg epitopes primed by four different HBsAg variants using protein- and DNA-based vaccination protocols. The K(b)-binding S(208-215) epitope 1 is processed from exogenous but not endogenous HBsAg. Variants of epitope 1 differing at two positions within the epitope (ILSPFLPL in ayw/adr versus IVSPFIPL in adw2) efficiently primed cross-reactive CD8(+) T cell responses. In contrast, the exchange of an N-terminal flanking residue (S to N) completely eliminated the immunogenicity of epitope 1. The K(b)-binding S(190-197) epitope 2 is processed from endogenous but not exogenous HBsAg. A single-residue exchange within the epitope (VWLSVIWM in ayw/adr versus VWLSAIWM in adw2) completely eliminated the immunogenicity of epitope 2. Single, conservative residue exchanges can thus give rise to diverging CD8(+) T cell repertoires, suggesting an impressive complexity and flexibility of the CD8(+) T cell repertoire to antigen variants from viruses with limited diversity.

Intranasal immunization with recombinant antigens associated with new cationic particles induces strong mucosal as well as systemic antibody and CTL responses.

New cationic nanoparticles (SMBV) were evaluated for use as a nasal vaccine delivery system for two recombinant proteins: HBsAg and beta-galactosidase. Each protein was formulated with SMBV and intranasally administrated to non-anesthetized mice. In each model, the formulated protein induced high levels of specific serum IgG antibodies and cytotoxic T lymphocyte (CTL) responses. Moreover, specific IgA antibodies were found in nasal as well as in vaginal washes of intranasally immunized mice with the protein associated with SMBV. In contrast, no IgG or IgA antibodies and no CTL were detected in mice immunized with free protein. The detection of a CTL response and an increase in both IgG1 and IgG2a antibodies in serum suggest that SMBV amplifies both Th1 and Th2 responses without modifying the Th1/Th2 profile of the immune response induced by the natural protein. These data demonstrate the high potential of SMBV for use as a nasal delivery system for sub-unit vaccines.

Priming Th1 immunity to viral core particles is facilitated by trace amounts of RNA bound to its arginine-rich domain.

Particulate hepatitis B core Ag (C protein) (HBcAg) and soluble hepatitis B precore Ag (E protein) (HBeAg) of the hepatitis B virus share >70% of their amino acid sequence and most T and B cell-defined epitopes. When injected at low doses into mice, HBcAg particles prime Th1 immunity while HBeAg protein primes Th2 immunity. HBcAg contains 5-20 ng RNA/microg protein while nucleotide binding to HBeAg is not detectable. Deletion of the C-terminal arginine-rich domain of HBcAg generates HBcAg-144 or HBcAg-149 particles (in which >98% of RNA binding is lost) that prime Th2-biased immunity. HBcAg particles, but not truncated HBcAg-144 or -149 particles stimulate IL-12 p70 release by dendritic cells and IFN-gamma release by nonimmune spleen cells. The injection of HBeAg protein or HBcAg-149 particles into mice primes Th1 immunity only when high doses of RNA (i.e., 20-100 microg/mouse) are codelivered with the Ag. Particle-incorporated RNA has thus a 1000-fold higher potency as a Th1-inducing adjuvant than free RNA mixed to a protein Ag. Disrupting the particulate structure of HBcAg releases RNA and abolishes its Th1 immunity inducing potency. Using DNA vaccines delivered intradermally with the gene gun, inoculation of 1 microg HBcAg-encoding pCI/C plasmid DNA primes Th1 immunity while inoculation of 1 microg HBeAg-encoding pCI/E plasmid DNA or HBcAg-149-encoding pCI/C-149 plasmid DNA primes Th2 immunity. Expression data show eukaryotic RNA associated with HBcAg, but not HBeAg, expressed by the DNA vaccine. Hence, codelivery of an efficient, intrinsic adjuvant (i.e., nanogram amounts of prokaryotic or eukaryotic RNA bound to arginine-rich sequences) by HBcAg nucleocapsids facilitates priming of anti-viral Th1 immunity.