Helper-dependent adenoviral vector-mediated delivery of woodchuck-specific genes for alpha interferon (IFN-alpha) and IFN-gamma: IFN-alpha but not IFN-gamma reduces woodchuck hepatitis virus replication in chronic infection in vivo

Alpha interferon (IFN-alpha) and IFN-gamma are able to suppress hepadnavirus replication. The intrahepatic expression of high levels of IFN may enhance the antiviral activity. We investigated the effects of woodchuck-specific IFN-alpha (wIFN-alpha) and IFN-gamma(wIFN-gamma) on woodchuck hepatitis virus (WHV) replication in vivo by helper-dependent adenoviral (HD-Ad) vector-mediated gene transfer. The expression of biologically active IFNs was demonstrated in vitro after transduction of woodchuck cells with HD-Ad vectors encoding wIFN-alpha (HD-AdwIFN-alpha) or wIFN-gamma (HD-AdwIFN-gamma). The transduction efficacy of the HD-Ad vector in woodchuck liver in vivo was tested with a vector expressing green fluorescence protein (GFP). Immunohistochemical staining of liver samples on day 5 after injection showed expression of GFP in a high percentage of liver cells surrounding the central vein. The transduction of livers of WHV carriers in vivo with HD-AdwIFN-alpha or HD-AdwIFN-gamma induced levels of biologically active IFN, which could be measured in the sera of these animals. Expression of wIFN-alpha in the liver reduced intrahepatic WHV replication and WHV DNA in sera of about 1 log step in two of two woodchucks. Transduction with HD-AdwIFN-gamma, however, reduced WHV replicative intermediates only slightly in two of three animals, which was not accompanied with significant changes in the WHV DNA in sera. We demonstrated for the first time the successful HD-Ad vector-mediated transfer of genes for IFN-alpha and IFN-gamma in vivo and timely limited reduction of WHV replication by wIFN-alpha, but not by wIFN-gamma.     

Replication of naturally occurring woodchuck hepatitis virus deletion mutants in primary hepatocyte cultures and after transmission to naive woodchucks

Woodchuck hepatitis virus (WHV) mutants with core internal deletions (CID) occur naturally in chronically WHV-infected woodchucks, as do hepatitis B virus mutants in humans. We studied the replication of WHV deletion mutants in primary woodchuck hepatocyte cultures and in vivo after transmission to naive woodchucks. By screening 14 wild-caught, chronically WHV-infected woodchucks, two woodchucks, WH69 and WH70, were found to harbor WHV CID mutants. Consistent with previous results, WHV CID mutants from both animals had deletions of variable lengths (90 to 135 bp) within the middle of the WHV core gene. In woodchuck WH69, WHV CID mutants represented a predominant fraction of the viral population in sera, normal liver tissues, and to a lesser extent, in liver tumor tissues. In primary hepatocytes of WH69, the replication of wild-type WHV and CID mutants was maintained at least for 7 days. Although WHV CID mutants were predominant in fractions of cellular WHV replicative intermediates, mutant covalently closed circular DNAs (cccDNAs) appeared to be a small part of cccDNA-enriched fractions. Analysis of cccDNA-enriched fractions from liver tissues of other woodchucks confirmed that mutant cccDNA represents only a small fraction of the total cccDNA pool. Four naive woodchucks were inoculated with sera from woodchuck WH69 or WH70 containing WHV CID mutants. All four woodchucks developed viremia after 3 to 4 weeks postinoculation (p.i.). They developed anti-WHV core antigen (WHcAg) antibody, lymphoproliferative response to WHcAg, and anti-WHV surface antigen. Only wild-type WHV, but no CID mutant, was found in sera from these woodchucks. The WHV CID mutant was also not identified in liver tissue from one woodchuck sacrificed in week 7 p.i. Three remaining woodchucks cleared WHV. Thus, the presence of WHV CID mutants in the inocula did not significantly change the course of acute self-limiting WHV infection. Our results indicate that the replication of WHV CID mutants might require some specific selective conditions. Further investigations on WHV CID mutants will allow us to have more insight into hepadnavirus replication.     

Molecular characterization of woodchuck type I interferons and their expression by woodchuck peripheral blood lymphocytes

Interferon (IFN)-alpha and -beta are important antiviral mediators. IFN-alpha is widely used for the treatment of chronic hepatitis B. In our previous studies, a subtype of woodchuck IFN-alpha (wIFN-alpha) was characterized and has been shown to be active in suppressing the replication of woodchuck hepatitis virus (WHV) in vitro and vivo. Here, we refined the analysis of the IFN-alpha/beta system of the woodchuck and studied the expression of wIFN-alpha/beta in peripheral blood lymphocytes (PBLs) from na?ve and WHV-infected woodchucks. A number of wIFN-alpha genes were sequenced and could be classified into 10 subtypes and 3 pseudotypes. The biological activity of different subtypes of wIFN-alpha was demonstrated by their ability to protect woodchuck cells against encephalomyocarditis virus infection and to induce MxA expression in woodchuck cells. Additionally, a partial sequence of wIFN-beta was characterized. A subtyping method for wIFN-alpha based on restriction length polymorphism analysis was developed. Further, the expression of wIFN by woodchuck PBLs after stimulation with polyI/C was investigated. The maximal production of wIFN by woodchuck PBLs occurred within the first 48 h after addition poly I/C. The wIFN-alpha subtypes 1, 4, and 5 were found to be produced by poly I/C-stimulated woodchuck PBLs, indicating a selective expression of wIFN-alpha subtypes. PBLs from chronically WHV-infected woodchucks showed a reduced ability to produce wIFN when stimulated with poly I/C. The results suggest that woodchucks with chronic WHV infection have impaired immunological responses to poly I/C.     

Lipopolysaccharide-induced innate immune responses in primary hepatocytes downregulates woodchuck hepatitis virus replication via interferon-independent pathways

Our previous studies have shown that Toll-like receptor (TLR) ligands, Poly I:C and lipopolysaccharide (LPS), are able to activate non-parenchymal liver cells and trigger the production of interferon (IFN) to inhibit hepatitis B virus replication in vivo and in vitro . However, little is known about TLR-mediated cellular responses in primary hepatocytes. By the model of woodchuck hepatitis virus (WHV) infected primary woodchuck hepatocytes (PWHs), Poly I:C and LPS stimulation resulted in upregulation of cellular antiviral genes and relevant TLRs mRNA expression respectively. LPS stimulation led to a pronounced reduction of WHV replicative intermediates without a significant IFN induction. Poly I:C transfection resulted in the production of IFN and a highly increased expression of antiviral genes in PWHs and slight inhibitory effect on WHV replication. LPS could activate nuclear factor kappa B, MAPK and PI-3k/Akt pathways in PWHs. Further, inhibitors of MAPK-ERK and PI-3k/Akt pathways, but not that of IFN signalling pathway, were able to block the antiviral effect of LPS. These results indicate that IFN- independent pathways which activated by LPS are able to downregulate hepadnaviral replication in hepatocytes.     

Characterization of T-cell response to woodchuck hepatitis virus core protein and protection of woodchucks from infection by immunization with peptides containing a T-cell epitope.

Specific activation of T cells appears to be a prerequisite for viral clearance during hepatitis B virus (HBV) infection. The T-cell response to HBV core protein is essential in determining an acute or chronic outcome of HBV infection, but how this immune response contributes to the course of infection remains unclear. This is due to results obtained from humans, which are restricted to phenomenological observations occurring during the clinical onset after HBV infection. Thus, a useful animal model is needed. Characterization of the T-cell response to the core protein (WHcAg) of woodchuck hepatitis virus (WHV) in woodchucks contributes to the understanding of these mechanisms. Therefore, we investigated the response of woodchuck peripheral blood mononuclear cells (PBMCs) to WHcAg and WHcAg-derived peptides, using our 5-bromo-2'-deoxyuridine assay. We demonstrated WHcAg-specific proliferation of PBMCs and nylon wool-nonadherent cells from acutely WHV-infected woodchucks. Using a cross-reacting anti-human T-cell (CD3) antiserum, we identified nonadherent cells as woodchuck T cells. T-cell epitope mapping with overlapping peptides, covering the entire WHcAg, revealed T-cell responses of acutely WHV-infected woodchucks to peptide1-20, peptide100-119, and peptide112-131. Detailed epitope analysis in the WHcAg region from amino acids 97 to 140 showed that T cells especially recognized peptide97-110. Establishment of polyclonal T-cell lines with WHcAg or peptide97-110 revealed reciprocal stimulation by peptide97-110 or WHcAg, respectively. We vaccinated woodchucks with peptide97-110 or WHcAg to prove the importance of this immunodominant T-cell epitope. All woodchucks immunized with peptide97-110 or WHcAg were protected. Our results show that the cellular immune response to WHcAg or to one T-cell epitope protects woodchucks from WHV infection.     

Primary human hepatocytes are susceptible to infection by hepatitis delta virus assembled with envelope proteins of woodchuck hepatitis virus

Hepatitis B virus (HBV) and hepatitis delta virus (HDV) share the HBV envelope proteins. When woodchucks chronically infected with woodchuck hepatitis virus (WHV) are superinfected with HDV, they produce HDV with a WHV envelope, wHDV. Several lines of evidence are provided that wHDV infects not only cultured primary woodchuck hepatocytes (PWH) but also primary human hepatocytes (PHH). Surprisingly, HBV-enveloped HDV (hHDV) and wHDV infected PHH with comparable efficiencies; however, hHDV did not infect PWH. The basis for these host range specificities was investigated using as inhibitors peptides bearing species-specific pre-S (where S is the small envelope protein) sequences. It was found that pre-S1 contributed to the ability of wHDV to infect both PHH and PWH. In addition, the inability of hHDV to infect PWH was not overcome using a chimeric form of hHDV containing WHV S protein, again supporting the essential role of pre-S1 in infection of target cells. One interpretation of these data is that host range specificity of HDV is determined entirely by pre-S1 and that the WHV and HBV pre-S1 proteins recognize different receptors on PHH.     

Protection against woodchuck hepatitis virus (WHV) infection by gene gun coimmunization with WHV core and interleukin-12

Woodchuck hepatitis virus (WHV) and hepatitis B virus (HBV) are closely similar with respect to genomic organization, host antiviral responses, and pathobiology of the infection. T-cell immunity against viral nucleocapsid (HBcAg or WHcAg) has been shown to play a critical role in viral clearance and protection against infection. Here we show that vaccination of healthy woodchucks by gene gun bombardment with a plasmid coding for WHcAg (pCw) stimulates proliferation of WHcAg-specific T cells but that these cells do not produce significant levels of gamma interferon (IFN-gamma) upon antigen stimulation. In addition, animals vaccinated with pCw alone were not protected against WHV inoculation. In order to induce a Th1 cytokine response, another group of woodchucks was immunized with pCw together with another plasmid coding for woodchuck interleukin-12 (IL-12). These animals exhibited WHcAg-specific T-cell proliferation with high IFN-gamma production and were protected against challenge with WHV, showing no viremia or low-level transient viremia after WHV inoculation. In conclusion, gene gun immunization with WHV core generates a non-Th1 type of response which does not protect against experimental infection. However, steering the immune response to a Th1 cytokine profile by IL-12 coadministration achieves protective immunity. These data demonstrate a crucial role of Th1 responses in the control of hepadnavirus replication and suggest new approaches to inducing protection against HBV infection.     

Lymphoid cells in the spleens of woodchuck hepatitis virus-infected woodchucks are a site of active viral replication.

Lymphoid cells were purified from the spleens of 15 woodchucks and examined for the presence of woodchuck hepatitis virus (WHV). Lymphoid cells from the spleens of eight of eight chronically infected animals contained high levels of WHV RNA and DNA. A 100-fold lower level of WHV DNA was found in the spleen from one of five animals that had recovered from acute WHV infections 2 years before this analysis. No WHV nucleic acids were observed in either of two uninfected animals. WHV DNA patterns in the lymphoid cells from the spleens of the chronically infected animals, which included the presence of single-stranded DNA and RNA-DNA hybrid molecules, were identical to those observed in WHV-infected liver. WHV DNA in these cells was present in intact, 27-nm core particles which also contained the endogenous DNA polymerase activity. These results indicate that the spleen is a site of active WHV DNA replication and is most likely a major source of WHV-infected cells in the circulating lymphoid cell population.     

In vitro and in vivo infectivity and pathogenicity of the lymphoid cell-derived woodchuck hepatitis virus

Woodchuck hepatitis virus (WHV) and human hepatitis B virus are closely related, highly hepatotropic mammalian DNA viruses that also replicate in the lymphatic system. The infectivity and pathogenicity of hepadnaviruses propagating in lymphoid cells are under debate. In this study, hepato- and lymphotropism of WHV produced by naturally infected lymphoid cells was examined in specifically established woodchuck hepatocyte and lymphoid cell cultures and coculture systems, and virus pathogenicity was tested in susceptible animals. Applying PCR-based assays discriminating between the total pool of WHV genomes and covalently closed circular DNA (cccDNA), combined with enzymatic elimination of extracellular viral sequences potentially associated with the cell surface, our study documents that virus replicating in woodchuck lymphoid cells is infectious to homologous hepatocytes and lymphoid cells in vitro. The productive replication of WHV from lymphoid cells in cultured hepatocytes was evidenced by the appearance of virus-specific DNA, cccDNA, and antigens, transmissibility of the virus through multiple passages in hepatocyte cultures, and the ability of the passaged virus to infect virus-naive animals. The data also revealed that WHV from lymphoid cells can initiate classical acute viral hepatitis in susceptible animals, albeit small quantities (approximately 10(3) virions) caused immunovirologically undetectable (occult) WHV infection that engaged the lymphatic system but not the liver. Our results provide direct in vitro and in vivo evidence that lymphoid cells in the infected host support propagation of infectious hepadnavirus that has the potential to induce hepatitis. They also emphasize a principal role of the lymphatic system in the maintenance and dissemination of hepadnavirus infection, particularly when infection is induced by low virus doses.     

Molecular characterization of the type I IFN receptor in two woodchuck species and detection of its expression in liver samples from woodchucks infected with woodchuck hepatitis virus (WHV)

Type I interferons (IFN-α/β) serve as the first line of defense against viral infection and share the same type I IFN receptor (IFNAR) complex, which is composed of IFNAR1 and -2. The Eastern woodchuck (Marmota monax) and Chinese woodchuck (Marmota himalayana) are suitable for studying hepatitis B virus (HBV) infection. Here, the complete or partial sequences of the IFNARs of both species were obtained and analyzed. Small interference RNAs targeting wIFNAR1 and -2 specifically down-regulated the expression of wIFNAR1 and -2 and the IFN-stimulated gene MxA in a woodchuck cell line, respectively. IFNAR2 was significantly up-regulated in primary woodchuck hepatocytes stimulated with IFN-α or -γ. The expression of woodchuck IFNAR1 and -2 was decreased in woodchucks chronically infected with woodchuck hepatitis virus (WHV). These results are essential for studying type I IFN-related innate immunity and therapy in hepadnaviral infection in the woodchuck model.     

Inhibition of cellular proteasome activities enhances hepadnavirus replication in an HBX-dependent manner

The X protein (HBX) of the hepatitis B virus (HBV) is not essential for the HBV life cycle in vitro but is important for productive infection in vivo. Our previous study suggests that interaction of HBX with the proteasome complex may underlie the pleiotropic functions of HBX. With the woodchuck model, we demonstrated that the X-deficient mutants of woodchuck hepatitis virus (WHV) are not completely replication defective, possibly behaving like attenuated viruses. In the present study, we analyzed the effects of the proteasome inhibitors on the replication of wild-type and X-negative HBV and WHV. Recombinant adenoviruses or baculoviruses expressing replicating HBV or WHV genomes have been developed as a robust and convenient system to study viral replication in tissue culture. In cells infected with either the recombinant adenovirus-HBV or baculovirus-WHV, the replication level of the X-negative construct was about 10% of that of the wild-type virus. In the presence of proteasome inhibitors, the replication of the wild-type virus was not affected, while the replication of the X-negative virus of either HBV or WHV was enhanced and restored to the wild-type level. Our data suggest that HBX affects hepadnavirus replication through a proteasome-dependent pathway.     

Hepadnavirus infection of peripheral blood lymphocytes in vivo: woodchuck and chimpanzee models of viral hepatitis.

The peripheral blood lymphocytes (PBL) of five hepatitis B virus (HBV)-infected chimpanzees and 17 woodchuck hepatitis virus (WHV)-infected woodchucks were examined for the presence of viral DNA and RNA. HBV DNA was detected in the PBL of three of three chronically infected chimpanzees but in neither of two animals with acute HBV infection. WHV DNA was found in the PBL of 11 of 13 chronically infected woodchucks and in the PBL and bone marrow of 1 of 4 woodchucks with antibody to WHV surface antigen. Viral DNA in the PBL and bone marrow was episomal, primarily existing as multimers with some monomeric forms. Integrated HBV DNA was detected in the PBL of one chronically infected chimpanzee, but only for a brief period. Viral RNA was also detected in the PBL, although less frequently than was DNA. HBV RNA in chimpanzee PBL existed as 3.8- and 7.5-kilobase species, while 2.3- and 3.8-kilobase WHV RNA was found in woodchuck PBL. Subfractionation of PBL isolated from the chronically infected chimpanzees demonstrated that HBV DNA and RNA were located in B and T cells. No HBV DNA was detected in the macrophages. These results, along with the recent reports of HBV nucleic acids in the PBL of human patients, suggest that infection of PBL may be a general phenomenon associated with the pathology of hepadnaviruses.     

Chemoimmunotherapy of chronic hepatitis B virus infection in the woodchuck model overcomes immunologic tolerance and restores T-cell responses to pre-S and S regions of the viral envelope protein

Treatment of chronic hepatitis B virus (HBV) infection could combine potent antiviral drugs and therapeutic vaccines to overcome immunological tolerance and induce the recovery phenotype to protect against disease progression. Conventional vaccination of woodchucks chronically infected with the woodchuck hepatitis virus (WHV) elicited differential T-cell response profiles depending on whether or not carriers were treated with the potent antiviral drug clevudine (CLV), which significantly reduces viral and antigen loads. The differential T-cell responses defined both CLV-dependent and CLV-independent epitopes of the pre-S and S regions of the WHV envelope protein. Only combined treatment involving CLV and conventional vaccine therapeutically restored the T-cell response profile of chronic WHV carrier woodchucks to that seen in prophylactic vaccination and in recovery from acute WHV infection. The results have implications for mechanisms of immunological tolerance operating in chronic HBV infection and suggest that such combined chemoimmunotherapy may be useful for treatment of humans with chronic HBV infection.     

Protection of chimpanzees from type B hepatitis by immunization with woodchuck hepatitis virus surface antigen.

Two chimpanzees immunized with woodchuck hepatitis virus (WHV) surface antigen (WHsAg) developed antibodies cross-reactive with hepatitis B virus (HBV) surface antigen (HBsAg). After challenge with HBV, one animal was completely protected and the other experienced a subclinical infection, without evidence of liver disease. Three woodchucks immunized with HBsAg developed antibodies to HBsAg which did not cross-react with WHsAg. After challenge with WHV, all three woodchucks developed typical acute infections with associated hepatic lesions. Serological studies with the cross-reactive antibodies raised in chimpanzees suggested that the protective epitopes of WHsAg were related to the group a specificity of HBsAg. These studies indicated that cross-protective epitopes are shared by HBV and WHV; however, the humoral response to these epitopes can vary among species.     

The precore gene of the woodchuck hepatitis virus genome is not essential for viral replication in the natural host.

A number of naturally occurring hepatitis B virus mutants that cannot synthesize the virus precore protein have been identified. Such mutants have been associated with more severe forms of hepatitis, including fulminant hepatitis. The most common mutation observed is a substitution of G to A in the distal precore gene that converts a codon specifying Trp (TGG) to a termination codon (TAG). Using oligonucleotide-directed mutagenesis, we have produced the same point mutation in the precore gene of an infectious clone of woodchuck hepatitis virus (WHV). Transfection of mutant WHV DNA into the livers of adult woodchucks resulted in replication of the mutant in three of three susceptible animals. Levels of virus replication and transient elevations in liver enzymes in serum were similar to those of adult animals infected with wild-type WHV. Virions, found to possess mutant precore genes by polymerase chain reaction amplification and DNA sequencing, were recovered from the serum of one of the animals and inoculated subcutaneously into neonatal woodchucks. They produced infection in all five animals studied. The level of virus replication in neonatal animals infected with this mutant virus was comparable to that found in neonatal woodchucks infected with wild-type WHV, but none of five woodchucks infected with the precore mutant virus as neonates became chronic virus carriers. It was concluded that the precore gene of the WHV genome is not essential for virus replication in the natural host but may be important for chronic infection.     

Apparent helper-independent infection of woodchucks by hepatitis delta virus and subsequent rescue with woodchuck hepatitis virus.

Hepatitis delta virus (HDV) is a subviral agent of humans which is dependent upon hepatitis B virus as a helper for transmission. HDV can be experimentally transmitted to woodchucks by using woodchuck hepatitis virus (WHV) as the helper. We used this model system to study two types of HDV infections: those of animals already chronically infected with WHV and those of animals without any evidence of prior exposure to WHV. At 5 to 10 days after infection with HDV, liver biopsies of these two groups of animals indicated that around 1% of the hepatocytes were infected (HDV antigen positive). Moreover, similar amounts of replicative forms of HDV RNA were detected. In contrast, by 20 days postinfection, the two groups of animals were quite different in the extent of the HDV infection. The animals chronically infected with WHV showed spread of the infection within the liver and the release of high titers of HDV into the serum. In contrast, the animals not previously exposed to WHV showed a progressive reduction in liver involvement, and at no time up to 165 days postinfection could we detect HDV particles in the serum. However, if these animals were inoculated with a relatively high titer of WHV at either 7 or even 33 days after the HDV infection, HDV viremia was observed. Our data support the interpretation that in these animals, hepatocytes were initially infected in the absence of helper virus, HDV genome replication took place, and ultimately these replicating genomes were rescued by the secondary WHV infection. The observation that HDV can survive in the liver for at least 33 days in the absence of coinfecting helper virus may be relevant to the reemergence of HDV infection following liver transplantation.