H J Schlicht

Efficient processing of an antigenic sequence for presentation by MHC class I molecules depends on its neighboring residues in the protein

Processing of endogenously synthesized proteins generates short peptides that are presented by MHC class I molecules to CD8 T lymphocytes. Here it is documented that not only the sequence of the presented peptide but also the residues by which it is flanked in the protein determine the efficiency of processing and presentation. This became evident when a viral sequence of proven antigenicity was inserted at different positions into an unrelated carrier protein. Not different peptides, but different amounts of the antigenic insert itself were retrieved by isolation of naturally processed peptides from cells expressing the different chimeric proteins. Low yield of antigenic peptide from an unfavorable integration site could be overcome by flanking the insert with oligo-alanine to space it from disruptive neighboring sequences. Notably, the degree of protection against lethal virus disease related directly to the amount of naturally processed antigenic peptide.

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.

Differential antigen-processing pathways of the hepatitis B virus e and core proteins.

BACKGROUND & AIMS Hepatitis B e antigen (HBeAg) and hepatitis B core antigen (HBcAg) seem to play different roles in the induction and regulation of the antiviral immune response, although the two antigens share all major CD4(+) T-cell epitopes, and these epitopes can be processed from both antigens via the exogenous antigen-presenting pathway. The aim of this study was to test the ability of antigen-presenting cells to present epitopes from endogenously synthesized HBcAg/HBeAg on HLA class II molecules. METHODS Lymphoblastoid cell lines infected with recombinant vaccinia viruses containing various HBcAg or HBeAg constructs and stable transfectants were tested for their ability to stimulate HBcAg/HBeAg-specific CD4(+) T-cell clones. RESULTS Only antigen-presenting cells infected with HBeAg constructs but not those infected with HBcAg constructs were able to stimulate HBcAg/HBeAg-specific CD4(+) T-cell clones. T-cell activation by HBeAg constructs was completely inhibited by brefeldin A but not affected by chloroquin. In contrast, T-cell activation by exogenous, recombinant HBcAg was inhibited by chloroquin but not by brefeldin A. CONCLUSIONS The findings indicate that processing and HLA class II-associated presentation of endogenously synthesized HBeAg in virus-infected cells, including hepatocytes, may occur. This mechanism may be involved in the regulation of the CD4(+) T-cell response to HBcAg/HBeAg.

Reactivated fulminant hepatitis B virus replication after bone marrow transplantation: clinical course and possible treatment with ganciclovir

A female chronic hepatitis B virus carrier (HBV-DNA negative) suffered from simultaneous hepatitis B virus and cytomegalovirus reactivation after in vivo T cell depletion preceding transplantation of an in vitro T cell depleted marrow graft for treatment of acute leukaemia. Interstitial pneumonia developing after bone marrow transplantation was successfully treated with ganciclovir (day 13 until day 46). The initially unnoticed extensive hepatitis B virus replication finally led to clinical hepatitis (day 85) and liver failure (day 96). Liver transplantation was performed, but the patient died from septicaemia. Retrospective analysis of hepatitis B virus DNA revealed that the HBV replication started immediately after T cell depletion and was completely suppressed during ganciclovir administration. Screening for HBV-DNA seems to be mandatory in comparable cases, and antiviral chemotherapy should be seriously considered.

Detection of the human hepatitis B virus X-protein in transgenic mice after radioactive labelling at a newly introduced phosphorylation site

Besides the three essential genes encoding the envelope, core and polymerase proteins, all mammalian hepadnaviruses examined to date contain a fourth gene which is referred to as the x-gene. This gene is believed to encode a transcriptional transactivator which positively regulates viral gene expression. Attempts to detect X-protein in vivo or in tissue culture lead to varying results. Whereas some groups could detect a protein of the expected size, other groups did not. To establish optimal conditions for the isolation of the human hepatitis B virus X-protein, we introduced a recognition site for protein kinase A into the x-gene. Upon phosphorylation with radioactive ATP, this modified X-protein can be detected with very high specificity and sensitivity. Tissue culture experiments showed that X-protein expressed from a cytomegalovirus-driven plasmid is not soluble in non-ionic detergent but rather has to be extracted from the cell pellet by boiling with SDS at a slightly alkaline pH. This method was then used to examine the organs of several transgenic mouse lines which expressed the modified x-gene under control of the authentic promoter. The data show that expression of the x-gene and subsequent biosynthesis of the X-protein is not tissue-specific but rather can occur in most organs.

Accumulation of 8-Hydroxy-2’- Deoxyguanosine Adducts in HBx Recombinant HepG2 Cells and HBx Transgenic Mice

Background/Aims: Transgenic mice overexpressing hepatitis B x protein (HBx) show an increased susceptibility to mutations if exposed to mutagens. Also involved in HBx signalling, reactive oxygen intermediates (ROI) can induce DNA adducts such as 8-hydroxy-2′-deoxyguanosine that can in turn lead to G/T transversion mutations. Therefore, we investigated whether HBx expression increases the level of the mutational precursor 8-hydroxy-2′-deoxyguanosine in hepatocellular DNA. Methods: 8-hydroxy-2′-deoxyguanosine concentrations of DNA hydrolysates of HBx protein expressing HepG2 cells and livers of HBx transgenic mouse lines were determined electrochemically after HPLC fractionation. Results: 8-hydroxy-2′-deoxyguanosine concentrations in genomic DNA of HBx protein expressing cell lines correlated with the factor of transactivation. The 8-hydroxy-2′-deoxyguanosine levels were reduced after incubation of HBx recombinant cell lines with 0.1 or 1 mM of the antioxidant N-acetylcysteine. Hepatic 8-hydroxy-2′-deoxyguanosine concentrations in DNA of old transgenic mice were significantly, i.e. twofold, (p < 0.01) increased as compared to those of old nontransgenic or young transgenic controls and of control mice expressing a second HBV transactivator (MHBst76). Conclusion: HBx expression results in elevated DNA adduct levels. This could reflect a direct inhibitory interaction of HBx with cellular repair mechanisms. Alternatively, this may be an effect of an increased generation of reactive oxygen intermediates through HBx.

Molecular basis of the diversity of hepatitis B virus core-gene products

All hepatitis B viruses examined to date code for at least two different core-gene products which are referred to as the c- and the e-protein. In the case of the human hepatitis B virus, they are known as the HBcAg and the HBeAg. Although these proteins share most of their primary amino acid sequence, they exhibit quite distinct properties. The e-protein is located in the cytoplasm and the nucleus of infected cells and very efficiently assembles into nucleocapsids. By contrast, the e-protein does not form particles. It enters the secretory pathway and is actively secreted by the cells. Here we describe the biosynthetic pathways by which the c- and e-proteins are expressed and summarize recent data from our laboratory showing that the antigenic and biophysical properties which distinguish the HBeAg from the HBcAg are primarily due to the 10 amino acid long portion of the HBeAg leader sequence that remains attached to the HBeAg after cleavage.