The hepatitis B virus X protein transactivates viral core gene expression in vivo.

The function of the X protein in the life cycle of mammalian hepadnaviruses is unclear. Based on tissue culture experiments it has been suggested that this protein represents a transcriptional transactivator which might be essential for the expression of the viral core gene. Here we have examined whether the activity of the human hepatitis B virus (HBV) core gene in vivo depends on X coexpression. To this end we compared core gene expression between four lineages of transgenic mice carrying the HBV core gene in cis arrangement with the X gene (cex lineage) and six lineages containing a modified construct in which the start codon of the X gene had been deleted (ce lineage). Whereas all cex lineages consistently exhibited a high-level hepatic core gene expression, the liver-specific core gene expression pattern of the ce lineages was heterogenous with four lineages virtually not expressing the core gene. This defect was due to a strongly reduced transcription since no core mRNA could be detected by Northern blotting. To test whether core gene expression could be restored by providing an intact X gene in trans, we crossbred mice of two lines which expressed no core mRNA or core protein with transgenic mice expressing the X-gene product under the transcriptional regulation of the liver-specific major-urinary-protein promoter/enhancer (MUP-X mice). The introduction of the MUP-X transgene induced core mRNA expression and core protein biosynthesis in the livers of the double-transgenic mice. This demonstrates that the X-gene product has the capacity to transactivate HBV core gene expression in vivo.

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.

The hepatitis B virus e antigen cannot pass the murine placenta efficiently and does not induce CTL immune tolerance in H-2b mice in utero.

The function of the secretory core gene product (HBeAg) of the human hepatitis B virus is unclear. It has been discussed that this protein may be passed from the mother to the fetus, where it might induce immunologic tolerance. Here we have examined this possibility with transgenic mice expressing high levels of HBeAg. Analysis of serum samples obtained from nontransgenic fetuses which developed in HBeAg-positive mothers showed no evidence that the HBeAg can pass the placenta. Moreover, direct examination of the HBeAg- and HBcAg-specific cytotoxic T-cell immune response of H-2b mice which developed in either transgenic or nontransgenic mothers revealed no indication that mice which could have been exposed to the HBeAg in utero become tolerant to HBV core gene products. From these data we conclude that the placenta represents an efficient barrier for HBeAg transfer and that the HBeAg does not tolerize cytotoxic T cells, at least in mice of the H-2b haplotype.

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.

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.

Uptake of duck hepatitis B virus into hepatocytes occurs by endocytosis but does not require passage of the virus through an acidic intracellular compartment.

The infectious entry pathway of duck hepatitis B virus (DHBV) was investigated with primary duck hepatocytes. Virus uptake was measured by a selective PCR technique which allows for the detection of a successful infection without the need for viral replication or gene expression. To test whether DHBV uptake occurs by endocytosis, the effects of energy depletion were analyzed. The requirement for an acidic intracellular pH was tested with the lysosomotropic agent ammonium chloride. The data show that energy depletion prevents the uptake of DHBV into primary hepatocytes whereas ammonium chloride has no effect. From these data, we conclude that DHBV is taken up by its host cells by endocytosis. However, in contrast to that of most other enveloped viruses, escape of DHBV from the endocytotic route does not depend on an acidic intracellular compartment.

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.

Hepatitis B virus nucleic acids associated with human peripheral blood mononuclear cells do not originate from replicating virus.

There have been numerous reports suggesting that human peripheral blood mononuclear cells (PBMCs) can be productively infected with human hepatitis B virus (HBV). We therefore examined whether the PBMCs can be used to establish an in vitro infection system for HBV. Freshly purified PBMCs were incubated with HBV with or without mitogen stimulation. Successful infection was tested using a newly developed PCR method that can differentiate between the relaxed circular (RC) DNA of the virus inoculum and the covalently closed circular (CCC) DNA which is formed only after successful virus entry. This method enables virus uptake to be proven even if the infection is abortive because there is no gene expression because of the lack of liver specific gene expression factors. All attempts to detect CCC DNA after incubation of PBMCs with HBV failed. On the contrary, CCC DNA could easily be detected in infected liver or after in vitro infection of primary human hepatocytes. Because this result appeared to be contradictory to the published data, we analyzed PMBCs isolated from infected patients. We could confirm that HBV DNA and RNA are associated with these cells. However, even after restimulation with mitogens, we could only detect RC DNA. Moreover, we could also demonstrate that viral RNA is present in free virus. Apparently, a certain amount of defective particles do not reverse transcribe the packaged pregenomic RNA. In summary we found no evidence that PMBCs can be infected with HBV and conclude that all previous observations can be explained by adsorbed virus.

Recombinant duck interferon: a new reagent for studying the mode of interferon action against hepatitis B virus.

Although interferon is widely used to treat chronic hepatitis B virus infections, its mode of action against hepadnaviruses is largely unknown. This deficit is due mainly to the lack of suitable model systems. The duck system could not be used because purified duck interferon was not available in sufficient quantities. We have now cloned a DNA fragment that contains an intronless gene for duck interferon. The primary translation product consists of 191 amino acids, the N-terminal 30 residues of which constitute a signal peptide. Mature duck interferon is 50% identical to the recently cloned chicken interferon. Sequence homology to mammalian interferons is marginal, but conservation of four cysteine residues and inducibility by virus indicate a distant relationship between duck interferon and mammalian type I interferons. Purified recombinant duck interferon from Escherichia coli is biologically active: it activates the interferon-inducible Mx gene, prevents cell destruction by cytolytic RNA viruses, and has a strong inhibitory effect on duck hepatitis B virus in cultured primary duck hepatocytes. This new reagent should help to define the interferon-sensitive step of the hepadnavirus life cycle. Furthermore, the duck system can now be used for systematic studies of the in vivo effectiveness of interferon in chronic hepatitis B virus infections.

Further characterization of 2 types of precore variant hepatitis B virus isolates from Hong Kong.

Two precore variants of hepatitis B virus occur in chronic carriers in the Chinese community in Hong Kong. One variant has a serine at amino acid (aa) 15, and the other has a stop codon at aa 28, which inhibits production of hepatitis B e (HBe) antigen (Ag). The serine 15 strain is shown here to produce antigenically normal amounts of HBeAg. These variants are mutually exclusive, probably due to sequence requirements for encapsidation, and are separate lineages. Sequential core sequences from patients with either variant revealed more aa substitutions in those who have the codon 28 change (P = .02), particularly T helper and B cell epitopes, implying different selection pressures. Most substitutions occurred around the time of selection of the stop codon, implying that complete loss of HBeAg, an immunomodulatory protein, is necessary for immune pressure on core epitopes. Serine 15 strains select small numbers of substitutions throughout the anti-HBe phase, probably because of persistent immunomodulation.

Breaking tolerance leads to autoantibody production but not autoimmune liver disease in hepatitis B virus envelope transgenic mice.

Hepatitis B virus (HBV) transgenic mice containing the HBV envelope open reading frame under the transcriptional control of the mouse albumin promoter express hepatitis B surface Ag (HBsAg) in all of their hepatocytes and secrete HBsAg (10 to 40 ng/ml) into the circulation. Because these transgenic mice show no signs of spontaneous liver cell injury or autoimmunity toward the viral (self-) Ag, we asked whether the state of self-tolerance could be reversed by the induction of an acute necroinflammatory liver disease or by immunization with HBV envelope proteins, with the aim of creating a transgenic model for chronic, immune-mediated hepatitis. Our studies indicate that repetitive administration of bacterial LPS, IFN-gamma, or HBsAg-specific CTL, all of which were previously shown to cause liver cell injury and inflammation, does not break tolerance at the T or B cell level, suggesting that the intrahepatic lymphomononuclear cell infiltrate induced by these agents consists of HBsAg-nonspecific cells. The adoptive transfer of HBsAg-primed nontransgenic CD4+ T cells into transgenic mice did not induce anti-HBs autoantibody production by transgenic B cells, even though transgenic B cells were fully responsive to immunization with HBsAg when appropriate T cell help was provided in a nontransgenic environment. Immunization of transgenic mice with purified HBsAg in CFA and repetitive infection with rHBV envelope vaccinia virus led to production of T cell-dependent anti-HBs autoantibodies that cleared HBsAg from the serum, but not to activation of HBsAg-specific CTL. We conclude that HBV envelope transgenic mice are largely tolerant to the transgene product at the T cell but not at the B cell level, and that the activation of an anti-HBs response was not sufficient to induce an autoimmune liver disease in this HBV envelope transgenic mouse model.

Induction in vitro of a primary human antiviral cytotoxic T cell response.

We report herein the successful priming of human anti-viral cytotoxic T cells (CTL) in vitro using two induction strategies based on the stimulation of peripheral blood mononuclear cells isolated from uninfected donors with synthetic viral peptides. The peptides used contain HLA-A2 binding motifs and have been identified as HLA-A2-restricted CTL epitopes in patients infected by the hepatitis B and C viruses. One approach uses repetitive long-term stimulation and the other uses bulk cultures containing large numbers of naive peripheral blood mononuclear cells. Both approaches successfully induce HLA-A2-restricted CTL specific for several viral epitopes. Some CTL recognize endogenously synthesized antigen on target cells infected with recombinant vaccinia virus expressing the corresponding viral proteins. This simple technique permits easy analysis of the primary human CTL repertoire, and may be exploitable for production of specific CTL effector cells for adoptive immunotherapy and dissection of the cellular and molecular requirements for priming of naive human CTL.

Human cytomegalovirus UL97 kinase confers ganciclovir susceptibility to recombinant vaccinia virus.

We analyzed whether the phosphotransferase encoded by the UL97 open reading frame of human cytomegalovirus (HCMV) alone is sufficient to confer ganciclovir (GCV) susceptibility to a foreign virus. Two vaccinia virus recombinants (T1 and A5) containing the UL97 open reading frames from a GCV-sensitive HCMV and from a GCV-resistant strain were constructed. T1 exhibited a GCV-sensitive phenotype in plaque reduction assays, whereas A5 did not. Moreover, T1-infected cell cultures showed a strongly increased incorporation of [14C]GCV triphosphate into macromolecular DNA, compared with recombinant A5 or vaccinia virus controls, which could be inhibited by the addition of guanosine. This shows that UL97 kinase is the only additional gene product required to make vaccinia virus susceptible to GCV, and guanosine seems to be one natural substrate for the enzyme. The system described here should be very helpful for fast and detailed functional analyses of UL97 mutations found in GCV-resistant HCMV isolates.

Class I-restricted cytotoxic T lymphocytes are directly cytopathic for their target cells in vivo.

Although CD8-positive, class I-restricted CTL play a critical role in viral clearance and immunopathology in many model systems, they have not been shown to directly kill their target cells in vivo at the single cell level. Using a hepatitis B surface Ag transgenic mouse model of class I-restricted, CTL-mediated liver disease, we now demonstrate that CD8-positive, Ld-restricted hepatitis B surface Ag-specific CTL bind and kill their target cells in vivo by triggering them to undergo degenerative cytologic changes compatible with apoptosis. Unexpectedly, the data also indicate that the pathologic consequences of this direct, Ag-specific CTL effect are much less severe than the cytodestructive, Ag-nonspecific inflammatory response that they induce when they are activated by Ag recognition.

Low frequency of cytotoxic liver-infiltrating T lymphocytes specific for endogenous processed surface and core proteins in chronic hepatitis B.

To investigate the role of hepatitis B virus (HBV)-specific CD8+ T cells in chronic hepatitis B, the lytic activity of peripheral blood mononuclear cells (PBMC) and liver-infiltrating T cell clones and cytotoxic T cell (CTL) lines stimulated by recombinant vaccinia virus-infected cells were analyzed. Autologous and allogeneic Epstein-Barr virus-transformed B cells infected with vaccinia vectors (VAC) that contain sequences of the surface (S), secretory core (E), cytoplasmatic core (C) VAC antigen of HBV, or the wild-type (WT) VAC served as target cells. ELISA and immunoblotting showed HBV antigen expression in infected cells. Neither PBMC nor C- or E-VAC-stimulated CTL lines showed specific lytic activity. However, S-VAC stimulated blood- and liver-derived CTL from 2 patients with hepatitis B lysed autologous S-VAC but not WT-VAC-infected target cells. One of 158 CD8+ T cell clones from 6 patients with active hepatitis B lysed autologous, but not allogeneic, S-VAC-infected targets. Thus, CTL are present among liver-infiltrating T cells in chronic hepatitis B that recognize endogenously processed hepatitis B surface antigen but not HBc or HBe antigen. The lack of core-specific CTL may contribute to failure in virus elimination in chronic inflammation.

Mechanisms of class I restricted immunopathology. A transgenic mouse model of fulminant hepatitis.

The molecular and cellular mechanisms responsible for cytotoxic T lymphocyte (CTL)-induced immunopathology are not well defined. Using a model in which hepatitis B surface antigen (HBsAg)-specific CTL cause an acute necroinflammatory liver disease in HBsAg transgenic mice, we demonstrate that class I-restricted disease pathogenesis is an orderly, multistep process that involves direct as well as indirect consequences of CTL activation. It begins (step 1) almost immediately as a direct antigen-specific CTL-target cell interaction that triggers the HBsAg-positive hepatocyte to undergo programmed cell death (apoptosis). It progresses (step 2) within hours to a focal inflammatory response in which antigen-nonspecific lymphocytes and neutrophils amplify the local cytopathic effect of the CTL. The most destructive pathogenetic function of the CTL, however, is to secrete interferon gamma when they encounter antigen in vivo, thereby activating the intrahepatic macrophage and inducing a delayed-type hypersensitivity response (step 3) that destroys the liver and kills the mouse. We propose that the principles illustrated in this study are generally applicable to other models of class I-restricted, CTL-induced immunopathology, and we suggest that they contribute to the immunopathogenesis of viral hepatitis during hepatitis B virus infection in humans.

Analysis of the earliest steps of hepadnavirus replication: genome repair after infectious entry into hepatocytes does not depend on viral polymerase activity.

Hepadnaviruses contain a relaxed circular DNA genome (RC-DNA) with discontinuities in both strands. Upon infectious entry into a host cell, this genome is converted into a covalently closed superhelical form (CCC-DNA), which later serves as the template for transcription. Here we examined whether the viral polymerase activity is required for this repair reaction. Primary hepatocytes prepared from embryonated duck eggs were infected with the duck hepatitis B virus. Conversion of the RC-DNA into the CCC-DNA was then analyzed by a newly developed polymerase chain reaction technique. This method allows the efficient discrimination between the two DNA forms and is sensitive enough to monitor repair of the infecting viral DNA in the absence of replication and amplification. Thus, we were able to monitor this process in the presence of a potent inhibitor of the viral polymerase, the nucleoside analog 2',3'-dideoxyguanosine. The data show that inhibition of the viral polymerase activity has no influence on genome repair, suggesting that this enzymatic function is not required for conversion of the RC-DNA into the CCC-DNA. Consequently, antiviral drugs blocking the polymerase activity cannot prevent the infectious entry of the virus into a host cell.