Polyvalent vaccination against hepatitis B surface and core antigen using a dicistronic expression plasmid.

Genes encoding the small (S) surface antigen (HBsAg) or the core (C) antigen (HBcAg) of hepatitis B virus (HBV) were cloned into the monocistronic expression vectors pCMV-1 or pCMV-2 under HCMV-IE promoter control. Coding fragments of these vectors were fused to generate a dicistronic expression construct pCMV/C-S in which the antigens HBcAg and HBsAg are coexpressed. Transient in vitro transfection studies demonstrated that HBcAg and HBsAg are coexpressed from this construct. Vaccination of mice of different H-2 haplotypes with mono- or dicistronic expression plasmids induced humoral and cellular immune responses to HBsAg and the HBcAg. In particular, intramuscular injection of 'naked' dicistronic plasmid DNA into mice elicited polyvalent humoral and cytotoxic T lymphocyte responses to HBsAg and HBcAg. The studies demonstrate that dicistronic expression plasmids are a novel way to construct a polyvalent vaccine against HBV that comprises HBsAg and HBcAg as immunogens.

DNA vaccination with plasmids encoding the intracellular (HBcAg) or secreted (HBeAg) form of the core protein of hepatitis B virus primes T cell responses to two overlapping Kb- and Kd-restricted epitopes.

Plasmid DNA encoding either the intracellular form HBcAg or the secreted form HBeAg of the core protein of hepatitis B virus (HBV) was injected into the muscle of H-2b, H-2d or F1b x d mice. Serum antibody responses and class I-restricted cytotoxic T lymphocyte (CTL) responses to HBcAg/ HBeAg were detected in all mice tested. Stable murine H-2b and H-2d transfectants that express either intracellular HBcAg were secreted HBeAg were constructed. With these cell lines we restimulated in vitro T cells primed in vivo and detected their specific cytolytic reactivity against naturally processed peptides. CD8+ CTL responses elicited by DNA vaccination with plasmids encoding HBcAg or HBeAg were specific for the (previously described) Kd-binding HBcAg93-100 peptide MGLKFRQL in H-2b mice or the (newly defined) Kd-binding HBcAg87-96 peptide SYVNTNMGL in H-2d mice. The overlapping epitopes span residues 87-100 of HBcAg, and are present on HBcAg and HBeAg. CTL responses were equally well elicited in vivo by injecting HBcAg- or HBeAg-expressing plasmid DNA, and CTL efficiently recognize in vitro HBcAg- and HBeAg-expressing transfectants. DNA vaccination of F1b x d mice with HBcAg- or HBeAg-expressing plasmid DNA primed CTL populations that recognized the Kb- or the Kd-restricted epitope. Both Kb- and Kd-binding peptides are thus generated from cytoplasmic/nuclear HBcAg and secreted HBeAg. These data make it unlikely that the appearance of HBeAg-negative variants during chronic HBV infection results from CTL-driven selection. DNA vaccination is an efficient technique to prime CTL responses against overlapping epitopes present on intracellular or secreted viral protein antigens.

Long-term expression of the hepatitis B virus core-e- and X-proteins does not cause pathologic changes in transgenic mice.

BACKGROUND/AIMS: Chronic infections with the human hepatitis B virus can result in liver cirrhosis and primary hepatocellular carcinoma. The reasons for these long-term effects are unclear. The aim of this study was to generate transgenic mice expressing the HBV X- and c/e-gene under authentic and foreign promoter control and to test whether the respective gene products can cause pathologic effects during the lifespan of a mouse. Moreover, the temporal and the tissue-specific regulation of the crucial HBV c/e-gene promoter was analyzed. METHODS: Eight transgenic mouse lines were generated. Four contained the c/e- and X-gene and two contained only the X-gene under authentic promoter control. Two lines expressed only the X-gene under control of the rat insulin promoter/enhancer. Gene expression was tested by protein and mRNA analyses. During an observation period of 2 years, mice were sacrificed and organs subjected to histologic examination. Mice expressing the X-gene in pancreatic beta cells were tested for the development of diabetes. RESULTS: In the liver, slight histopathologic alterations but no neoplastic changes could be observed in mice expressing the X-gene. Activity of the c/e-gene promoter/enhancer was age dependent and was not restricted to hepatocytes. CONCLUSION: No evidence was obtained that long-term expression of the HBV c/e- and X-gene products can cause neoplasia during the lifespan of a mouse.

DNA immunization induces antibody and cytotoxic T cell responses to hepatitis B core antigen in H-2b mice.

The serum Ab response and the class I-restricted CTL response of C57BL/6 (H-2b) mice to hepatitis B (pre)core Ag (HBcAg, HBeAg) was studied. Injection of HBcAg particles without adjuvants into mice efficiently primed serum Ab responses but not CTL response. We constructed the expression plasmids pCMV-1/c and pCMV-1/e in which expression of HBcAg or HBeAg was driven by cytomegalovirus immediate early region promoter sequences. Stable murine RBL5/C transfectant lines expressing HBcAg were established. Intramuscular DNA immunization with plasmid pCMV-1/c (encoding intracellularly expressed core Ag) or pCMV-1/e (encoding secreted precore Ag) efficiently primed specific serum Ab responses and CTL responses. The CTL response elicited in this system was mediated by CD4-CD8+ effector cells primed in vivo. The CTL recognized the HBcAg93-100 8-mer peptide MGLKFRQL in the context of Kb. Hence, DNA immunization with HBcAg/HBeAg-expressing plasmids, but not immunization with exogenous HBcAg particles, elicits a class I-restricted CTL response of defined epitope/restriction specificity in H-2b mice.