Hepatic expression of the woodchuck hepatitis virus X-antigen during acute and chronic infection and detection of a woodchuck hepatitis virus X-antigen antibody response

Hepatic Expression of the Woodchuck Hepatitis Virus X-Antigen During Acute and Chronic Infection and Detection of a Woodchuck Hepatitis Virus X-Antigen Antibody Response JAMES R. JACOB,1 MARY A. ASCENZI,1 CAROL A. RONEKER,1 ILLIA A. TOSHKOV,1 PAUL J. COTE,2 JOHN L. GERIN,2 1 AND BUD C. TENNANT

The expression and localization of the woodchuck hepatitis virus X-antigen (WHxAg) was examined and compared with other markers of a woodchuck hepatitis virus (WHV) infection using rabbit antisera generated against recombinant WHxAg produced in bacteria. Cellular fractionation studies showed that WHxAg was localized to the soluble and cytoskeletal fractions of the cell when assayed by immunoprecipitation of [35S]met-cys labeled extracts derived from primary cultures of acute WHV-infected hepatocytes. Immunohistochemical examination of liver from chronic WHV-infected animals showed WHV core antigen (WHcAg) and WHxAg expression in non-neoplastic tissue. The WHxAg was found localized to the cytoplasm of infected cells, similar to WHcAg. WHxAg expression was diminished in the foci of altered hepatocytes and in hepatocellular adenomas but was found in only 1 of 11 hepatocellular carcinomas (HCC). Hepatic biopsies from woodchucks experimentally inoculated with WHV were examined during the acute phase of infection and during convalescence for WHcAg and WHxAg expression by immunohistochemistry. Concurrent expression of WHcAg and WHxAg was observed during the viremic phase of infection. The two antigens exhibited similar localization to the cell cytoplasm, similar distribution within the liver lobule, and similar patterns of clearance during convalescence. An immune response to WHxAg was documented in some woodchucks following acute WHV infection. These studies further define the woodchuck model of HBV infection and should allow for the investigation of the role of hepadnaviral X-antigen expression in the pathogenesis of chronic hepatitis and HCC. (HEPATOLOGY 1997;26:1607-1615.)

Abbreviations: WHxAg, woodchuck hepatitis virus X-antigen; WHV, woodchuck hepatitis virus; WHcAg, woodchuck hepatitis virus core antigen; HCC, hepatocellular carcinoma; HBV, hepatitis B virus; MBP, maltose binding protein; ELISA, enzyme linked immunosorbent assay; PBS, phosphate-buffered saline; PBS-Brij, phosphatebuffered saline with 0.4% Brij 35; SDS-PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; HE, hematoxylin and eosin; HBsAg, hepatitis B virus surface antigen; WHsAg, woodchuck hepatitis virus surface antigen; WHeAg, woodchuck hepatitis e antigen. From the 1Department of Clinical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY; and 2Division of Molecular Virology and Immunology, Georgetown University School of Medicine, Rockville, MD. Received November 26, 1997; accepted July 11, 1997. Supported by NOI-A1-35164 (BCT) and NO1-AI-45179 (JLG) from the U.S. Public Health Service. Address reprint requests to: James R. Jacob, Ph.D., Dept. Clinical Sciences, Cornell University, C2-015 VMC, Ithaca, NY 14853. Fax: (607) 253-3289. Copyright q 1997 by the American Association for the Study of Liver Diseases. 0270-9139/97/2606-0033$3.00/0

The human hepatitis B virus (HBV) is the prototype member of the family hepadnaviridae.1 The hepadnaviral genome consists of a partial, double-stranded DNA molecule and follows a replication scheme similar to that of retroviruses.2 Related animal viruses have been found in the ground squirrel, woodchuck, and Pekin duck.3,4 All hepadnaviruses exhibit similar genomic organization, encoding two structural proteins, the surface (envelope) and core (nucleocapsid) antigens, and a viral DNA polymerase (reverse transcriptase). Only the mammalian hepadnaviruses encode a fourth, nonstructural gene which is termed the X-antigen.4 Except for the avian hepadnavirus, chronic hepadnaviral infections, in their respective hosts, are associated with the development of hepatocellular carcinoma (HCC).5 The hepadnaviral X gene of the woodchuck hepatitis virus (WHV) consists of an open reading frame of 423 bp that encodes a 141 amino acid protein, with a calculated molecular weight of 17,851 da. One report has suggested the WHV X-antigen (WHxAg) is expressed from a 0.65 kb non-polyadenylated RNA transcript found in the nucleus of infected cells.6 This gene product was originally termed the X-antigen because its function was unknown. The X-antigen now has been shown to function as a transcriptional transactivator7 and is essential for replication of hepadnaviruses in vivo.8,9 It has been postulated that the hepadnaviral X-antigen has a mechanistic role in the pathogenesis of hepatic disease and contributes to the development of HCC. Several studies have described the HBV X-antigen (HBxAg) in the liver of chronic HBV carriers as well as the presence of HBxAg in relation to HCC.10-13 Additionally, serologic studies have shown an immune response to HBxAg, but the relationship of this antibody to disease has not been clearly established.14-17 An immune response to HBxAg has been found primarily in patients with chronic active hepatitis in which cell necrosis presumably would expose the X protein to immune response.11,14,15 Currently, the best animal model for the study of the pathogenesis of hepadnaviral infections is the woodchuck (Marmota monax ). The experimental WHV infection of woodchucks and the establishment of the chronic carrier state has allowed for the development of antiviral therapies against HBV.18 This also provides the only system to monitor the development of hepadnaviral-induced HCC in a laboratory controlled environment.19 The purpose of the studies described in this study is to investigate the expression of WHxAg in woodchucks that are experimentally infected

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with WHV, to correlate WHxAg expression with other known markers of hepadnaviral infection, to detect a woodchuck immune response to WHxAg, and to begin to assess the relationship between WHxAg expression and hepatic disease. MATERIALS AND METHODS Recombinant Plasmid Expressing WHxAg. To produce a source of WHxAg for these studies, a recombinant WHxAg (rWHxAg) was expressed in bacteria. A DNA fragment encompassing the WHxAg gene was generated by polymerase chain reaction (PCR). The sequence of the oligonucleotide primer pair corresponding to the 5* and 3* ends of the WHxAg was as follows: 5*-CTTAGGATCC ATGGCTGCTCGCCTGTGT-3* (coding strand) 5*-TTTCTGCAG GTTACAGAAGTCGCATGCA-3* (noncoding strand). The DNA template used for the reaction was plasmid DNA (pWH2) containing a dimer of cloned WHV.20 A unique restriction site (in bold and underlined) was included in the primer pair to facilitate unidirectional cloning into the pMal-c2 bacterial expression vector (New England BioLabs, Beverly, MA). Standard techniques were used to generate and clone the polymerase chain reaction products.21 Briefly, the ends of the PCR reaction products were blunted with Klenow enzyme in the presence of an excess of deoxynucleotide triphosphates. The 446-bp product of the reaction mixture was isolated through elution from agarose gel. The DNA fragments were digested with BamHI and PstI (Boehringer Mannheim, Indianapolis, IN) and ligated into the pMal-c2 vector, which had been digested with identical enzymes. The ligation mixture was used to transform E. coli (DH5a). Resultant colonies were screened to ascertain the presence of an insert by restriction digest of DNA extracted by alkaline lysis.21 Bacterial colonies that contained an insert were subcloned and grown to log phase, and recombinant protein expression was induced with 0.3 mmol/ L isopropylthio-b-galactoside. Induced bacterial cultures were lysed in sample buffer (25 mmol/L TRIS [pH 7], 2% sodium dodecyl sulfate, 2% beta-mercaptoethanol, and 10% glycerol), and proteins were separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) followed by Coomassie Blue staining. The pMal-c2 vector expresses recombinant antigens as a fusion protein with the maltose binding protein (MBP). A bacterial clone, designated pMal-XP117 , expressing an appropriately sized fusion protein was further analyzed by DNA sequencing using a malE specific primer (New England BioLabs) for the pMal-c2 vector, confirming an inframe bacterial:fusion protein (MBP:rWHxAg) junction. Recombinant WHxAg Purification. For the production of antisera and development of an enzyme-linked immunosorbent assay (ELISA), the rWHxAg was cleaved and purified from the MBP fusion protein. The MBP:rWHxAg fusion protein expressed in bacteria was purified by amylose column chromatography according to the manufacturer’s protocol (New England BioLabs). This purified MBP:rWHxAg fusion protein was denatured in the presence of 6 mol/L guanidine-HCl, followed by dialysis to a buffer of 20 mmol/ L TRIS (pH 8.0), 100 mmol/l NaCl, and 2 mmol/l CaCl2 . The rWHxAg was proteolytically cleaved from the MBP with factor Xa (0.2%), during overnight incubation at 47C. The rWHxAg was separated from the MBP and non-cleaved fusion protein by a second passage over an amylose column in which the rWHxAg flows through. The rWHxAg was further purified by ion exchange chromatography (Q-sepharose; Pharmacia Biotek, Piscataway, NJ) and was used for the immunization of rabbits. A portion of this rWHxAg preparation was purified by electroelution from 12% SDS-PAGE after electrophoresis and used for hyperimmunization of the rabbits. Rabbit Antisera to the rWHxAg. Rabbit antiserum was generated against the rWHxAg for use in immunoprecipitation and immunohistochemical staining of liver tissue from woodchucks and as a

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positive control in ELISAs. Protocols for the use of laboratory animals in these studies were approved by the Institutional Animal Care and Use Committee. Two rabbits were pre-bled and immunized by intramuscular injection of 1 mg of purified MBP:rWHxAg fusion protein in Freund’s complete adjuvant, followed by a second immunization 2 weeks later with 100 mg of purified MBP:rWHxAg fusion protein in Freund’s incomplete adjuvant. Rabbits were hyperimmunized with 1 mg of gel-purified rWHxAg administered in phosphate buffered saline and bled 10 days after each sequential hyperimmunization. Reactivity of the antisera to the rWHxAg was established by immunoblot assay with the following modifications22: when woodchuck serum was used as the primary antibody, a rabbit anti-woodchuck immunoglobulin was employed as the second antibody, followed by incubation with a horseradish peroxidase–linked goat anti-rabbit immunoglobulin (New England BioLabs). Antibody was blocked before immunoassay by pre-incubating the rabbit or woodchuck antisera with 100 mg of gel purified rWHxAg at 377C for 45 minutes. Tissue Culture of Primary Woodchuck Hepatocytes and Immunoprecipitation of WHV Antigens. Primary cultures of hepatocytes were estab-

lished to show the reactivity of our antisera to authentic viral produced WHxAg. Full-thickness hepatic biopsies were obtained from one 8- and two 14-week-old woodchucks that had been infected with WHV at birth, one 3-year old chronic WHV carrier, and from normal, uninfected control woodchucks of identical age. The immunoprecipitation of WHV antigens was performed as described previously23 using [35S]met-cys (ú800 Ci/mmol; ICN, Costa Mesa, CA) to label de novo synthesized proteins. Cellular components were separated by previously described detergent fractionation procedures.24,25 Each subcellular fraction was immunoprecipitated with rabbit anti-WHV surface antigen (anti-WHsAg), rabbit antirWHxAg, or with woodchuck serum found positive for antiWHsAg, anti-WHxAg and anti-WHV core antigen (anti-WHcAg). Immunoprecipitated proteins were separated by 12% SDS-PAGE and detected by autoradiography. Histopathology and Immunohistochemistry. To assess the relationship between WHV antigen expression and hepatic disease, hepatic tissue from 11 adult, chronic WHV-carrier and 2 control woodchucks were obtained at necropsy. In addition, hepatic tissue was obtained by percutaneous needle biopsy of the liver during the course of acute, self-limiting experimental WHV-infection in three adult woodchucks and in one negative control animal. These specimens were fixed in neutral phosphate-buffered formalin, embedded in paraffin, and processed, as required, for histopathological and immunohistochemical evaluation. For histopathology, liver tissue was sectioned, deparaffinized, rehydrated, and stained with hematoxylin and eosin (HE). Histopathologic changes in the liver of the 11 chronic WHV-carrier woodchucks and 2 control animals described earlier were evaluated using a semiquantitative, nonlinear grading system similar to guidelines previously described.26-28 Briefly, histologic features associated with the WHV infection, e.g., portal tract infiltration, intralobular changes, ductular proliferation, steatosis, fibrosis, and the presence of altered hepatic foci or neoplasia, were scored on a scale of 0 to 4 according to their severity and distribution. A final score was obtained by adding the individual scores of the corresponding features. For immunohistochemistry, liver tissue was sectioned (3 mm), deparaffinized, and rehydrated in PBS with 0.4% Brij 35 (PBS-Brij). Endogenous peroxidase was blocked by incubation of the sections in 1% H2O2 in methanol for 10 minutes followed by immunohistochemical staining with a peroxidase method (Zymed, South San Francisco, CA). Tissue sections were treated with 0.1% trypsin solution for 1 hour at 377C to render the membranes permeable and were then washed 6 times in PBS-Brij. Endogenous avidin/biotin binding was blocked by incubation in an excess of avidin for 10 minutes, followed by washes in PBS-Brij and the procedure repeated using biotin. The sections were incubated in non-immune goat

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serum for 10 minutes at room temperature to block nonspecific adsorption of the rabbit antiserum to the tissue. Tissue sections were next incubated with primary antibody, either with rabbit anti-WHcAg (C.Seeger, Fox Chase Cancer Research Institute, Philadelphia, PA), rabbit anti-rWHxAg, or pre-immune rabbit serum, for 1 hour at 377C. Primary antisera were pre-incubated with acetone-fixed rat liver powder (Cappel [Organon Teknika], Durham, NC) before immunohistochemical staining. Samples were washed 6 times in PBS-Brij and incubated for 10 minutes at room temperature with a biotinylated secondary antibody (goat anti-rabbit immunoglobulin). Sequential washing in PBS-Brij was followed by incubation with a streptavidin peroxidase conjugate or a streptavidin-alkaline phosphatase conjugate for 5 to 10 minutes at room temperature, followed by washes in PBS-Brij. Sections were incubated with a solution of chromogen, which was either 3-3*diaminobenzidine-tetrahydroxychloride-dihydrate for peroxidase reaction or 5-bromo-4 chloro-3-indoyl phosphate/nitroblue tetrazolium for alkaline phosphatase reaction, for 5 to 10 minutes at room temperature and washed in dH2O. Sections were counterstained with Gill’s hematoxylin or 0.1% Neutral Red for 3 minutes, air dried, dehydrated by sequential washes in 95% ethanol, 100% ethanol, and xylene, and then mounted on glass slides with permount. The evaluation of the WHcAg and WHxAg expression was based upon both the distribution and intensity of staining of woodchuck liver specimens following immunohistochemical procedures described earlier. The percentage of cells that stained positive was scored on a scale from 0 to 4, similar to that used in previously reported studies.29,30 Serology. We assessed the relationship between the hepatic expression of WHxAg and WHcAg and serum markers of an acute WHV infection. Serum from 11 adult, chronic WHV carriers with HCC and 2 control woodchucks were collected at necropsy. Serum was collected during the course of acute, self-limiting experimental WHV-infection in three adult animals and 1 control woodchuck, described earlier. Serologic tests for the detection of circulating WHsAg and for anti-WHcAg and anti-WHsAg antibodies were performed using standard ELISA assays.31 To determine the viremic phase of disease, serum WHV DNA was measured by slot blot hybridization using a WHV-specific probe incorporating fluorescein-11–deoxyuridine triphosphate (Amersham, Arlington Heights, IL) for chemiluminescent detection of signal on nylon membranes (GeneScreenPlus; NEN [Dupont], Boston MA). Enzyme-Linked Immunosorbent Assay. An ELISA was developed as a rapid and sensitive assay to identify a woodchuck immune response to the WHxAg. This ELISA is a modification of procedures described previously.31 Briefly, ELISA plates were coated with 100 ng of rWHxAg/well in 0.6 mol/L carbonate buffer (NaHCO3 / Na2CO3 , pH 9.6) overnight at 47C. The ELISA plates were next incubated with 2% bovine serum albumin in PBS (pH 7.2) for 1 hour at 377C. Woodchuck serum samples were diluted (1:20) in dilution buffer consisting of PBS containing 2% bovine serum albumin and 0.2% Tween-20, which were pre-incubated on bovine serum albumin-coated ELISA plates for 2 hours at room temperature. The rabbit anti-rWHxAg serum served as a positive control, with corresponding pre-immune serum as negative control. Serum samples were transferred to rWHxAg-coated ELISA plates and incubated overnight at 47C. After being washed 10 times with wash buffer consisting of PBS containing 0.2% Tween-80, bound antibodies were detected by incubation with biotinylated protein G (diluted 1:5000 in dilution buffer, Pierce, Rockford, IL) for 1 hour at room temperature. After being washed 10 times with wash buffer, ELISA plates were incubated with a streptavidin biotinylated horseradish peroxidase complex (diluted 1:1,000 in diluted buffer (Amersham, Arlington Heights, IL) for 1 hour at room temperature, followed by 10 washes with wash buffer. The enzyme reaction was carried out with 0.1% tetramethyl benzidine and 0.04% H2O2 in a solution of 0.025 mol/L citric acid and 0.05 mol/L sodium acetate (pH 6). The reaction was stopped with 0.2 mol/L H2SO4 and the absorbance

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was measured at 450 nm (EIA plate reader Model EL308, BioTek Instruments, Winooski, VT). The signal-to-noise ratio (P/N) was calculated for each serum sample with absorbance values (P) at time zero as background (N). A P/N ú3 was interpreted as a positive signal for antibody. The end-point titers for antibody to WHxAg in the sera of rabbit 3175 immunized with rWHxAg and in experimental WHV-infected woodchuck 4124 were 1:128,000 and 1:1,280, respectively. RESULTS De Novo Synthesis and Cellular Localization of WHxAg. Labeling and fractionation of hepatocyte cultures established from 8- and 14-week-old woodchucks infected at birth provided evidence of the de novo synthesis of WHxAg (Fig. 1A). WHxAg-related polypeptides were precipitated and found to be distributed in both the soluble and cytoskeletal fractions (MW 17.8 kd). The distribution of the WHxAg in the respective subcellular fractions was consistent with the localization observed immunohistochemically and described later. In the analysis of subcellular fractionations, two additional bands of 19 and 16 kd were found to co-precipitate with the 17.8 kd WHxAg from the soluble fraction (Fig. 1B, lane 1). To determine if these additional bands were related WHxAg gene products or other cellular products, a second immunoprecipitation experiment was performed. Rabbit anti-WHxAg was pre-incubated with unlabeled, normal hepatocyte extract followed by precipitation of WHxAg from the soluble fraction of [35S]met-cys labeled, WHV-infected hepatocyte extracts (Fig. 1B, lane 2). After autoradiography, the WHxAg was detected, but the two additional protein bands were not, indicating that they are normal cellular proteins. Similar labeling and fractionation procedures were performed on primary cultures of hepatocytes established from a chronic WHV-carrier woodchuck followed by immunoprecipitation with polyclonal rabbit anti-WHsAg serum (Fig. 1C). WHsAg related polypeptides (WHsAg, MW 23 and 27 kd; and WHsAgpre-S2 , MW 33 and 36 kd) were precipitated from the soluble and cytoskeletal fractions. Additionally, WHcAg (MW 21.5 kd), the intracellular form of Woodchuck hepatitis e antigen (MW 24 kd), and WHxAg related polypeptides were observed in the soluble fraction. These results may reflect the precipitation of membrane associated viral particles that were not denatured under the detergent conditions used during fractionation. Only WHsAg polypeptides were observed in the cytoskeletal fraction upon precipitation with the rabbit anti-WHsAg serum. Parallel immunoprecipitation experiments with the rabbit antisera described ealier were performed using cellular fractions derived from [35S]met-cys labeled, uninfected primary hepatocyte cultures. Polypeptides migrating at an appropriate molecular size for any of the known viral antigens were not detected (data not shown). Woodchuck serum containing antibodies to WHsAg, WHcAg, and WHxAg by ELISA was also used to precipitate the [35S]met-cys labeled polypeptides from the cellular fractions (Fig. 1D). WHsAg related polypeptides were precipitated from the soluble and cytoskeletal fraction, identical to precipitation results using rabbit anti-WHsAg. Polypeptides of appropriate molecular size for WHcAg, the intracellular form of Woodchuck hepatitis e antigen, and WHxAg were observed in the soluble fraction. WHcAg and WHxAg related polypeptides were observed in the cytoskeletal fraction, simi-

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FIG. 1. Cellular fractionation of woodchuck hepatocytes in primary tissue culture. Hepatocytes were isolated from woodchucks during the acute phase of WHV infection and were labeled with [35S]met-cys. Subcellular components were separated by detergent extraction to yield soluble (sol), cytoskeletal (cytskl), and nuclear (nuc) fractions of cellular components. (A) Each cellular fraction was immunoprecipitated with rabbit anti-WHxAg and separated by 12% SDS-PAGE, followed by autoradiography. Molecular weight markers are given in kd. (B) Rabbit anti-WHxAg was pre-incubated (0) without or (/) with unlabeled, normal hepatocyte extract; it was then used to precipitate WHxAg from the soluble fraction of [35S]met-cys labeled, WHV-infected hepatocyte extracts showing WHxAg migrating at 17.8 kd and normal cellular proteins at 19 and 16 kd. Cellular fractionation of [35S]met-cys labeled hepatocyte cultures from chronic WHV-infected animals precipitated with (C) rabbit anti-WHsAg or (D) woodchuck serum positive for antibodies to WHsAg, WHcAg, and WHxAg. Proteins demonstrating appropriate molecular size for the following viral antigens are noted; X- WHxAg, core- WHcAg, e- intracellular WHV e antigen, S- WHsAg, and S2 - WHsAg pre-S2 . (E) Immunoblot showing reactivity of rabbit and woodchuck serum toward the rWHxAg. Pre-immune rabbit and negative woodchuck sera (pre) do not bind rWHxAg. Rabbit and woodchuck sera positive for anti-WHxAg (/) bind rWHxAg. Pre-incubation of (/) sera with an excess of rWHxAg prevents immunoblot reactivity (blocked). Rightmost lane shows Coomassie Blue stain of rWHxAg that migrated at 17.8 kd.

c

lar to results obtained with rabbit anti-WHxAg and consistent with the localization of WHcAg by immunohistochemistry. Histopathology and Immunohistochemistry of WHV Infected Tissue. Sections of adjacent hepatic tissue obtained postmortem

from 11 chronic WHV carrier woodchucks were stained with HE for histopathology and were studied by immunohistochemistry for expression of both WHcAg and WHxAg. A typical well-differentiated HCC and adjacent non-neoplastic tissue from chronic WHV carriers stained with HE are shown in Fig. 2A. Staining of an adjacent section from the same block with pre-immune normal rabbit serum was negative (Fig. 2B). Staining of adjacent sections for WHcAg (Fig. 2C) and WHxAg (Fig. 2D) showed that both viral antigens localized to the cytoplasm of non-neoplastic hepatocytes and were absent in the HCC. Normal, uninfected woodchuck liver tissue examined, using identical reagents and procedures, stained negative. Additionally, pre-incubation of rabbit antisera with rWHxAg blocked anti-WHxAg staining but did not interfere with anti-WHcAg staining (data not shown). A total of 11 tumors classified as HCC and 9 tumors classified as hepatocellular adenomas were examined. In 8 of 11 HCCs, both WHcAg and WHxAg stains were negative. In 3 HCCs, all of which were of the well-differentiated (grade 1) type, individual cells scattered throughout the tumor and representing fewer than 1% of the total hepatocytes in the section stained positively for WHcAg. In one of the 3 that stained positively for WHcAg, rare, single hepatocytes stained faintly positive for WHxAg. The hepatocytes of hepatocellular adenomas stained more basophilically than did the surrounding non-neoplastic hepatocytes, and the latter were compressed by the expanding neoplasm. The trabeculae of the adenomas characteristically were one- or, often, two-cells thick. In 2 of 9 hepatocellular adenomas, WHcAg staining was less intense than in the surrounding non-tumorous tissue, but WHcAg was detectable in 75% of hepatocytes in one and in 25% of hepatocytes in the other. In the latter adenoma, WHxAg was not detected and in the other, fewer than 1% of the tumor cells stained faintly positive for WHxAg. In 6 of 9 adenomas, WHcAg expression was detected in 5% or fewer of the hepatocytes. In these, WHcAg was expressed in single cells scattered

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throughout the tumor section or in groups of a few hepatocytes located adjacent to blood vessels or near the periphery of the tumor. In 4 of 6 tumors that stained as weakly positive for WHcAg, 1% or fewer of the cells stained weakly positive for WHxAg and 2 of 6 stained negative for WHxAg. One hepatocellular adenoma was negative for both WHcAg and WHxAg expression. In sections of non-tumorous liver from the 11 chronic WHV carriers, 198 basophilic foci of altered hepatocytes, 149 clear cell foci, and 38 acidophilic (small cell) foci were

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identified and examined for expression of WHcAg and WHxAg. In the basophilic foci, the number of cells that stained positively for WHcAg and WHxAg and the intensity of staining for both antigens was remarkably less than that in surrounding hepatocytes. In clear cell foci, staining for WHcAg and WHxAg was either diminished diffusely or, in some cases, was negative for both antigens. Acidophilic foci of altered hepatocytes stained negatively for both WHcAg and WHxAg.

The hepatic expression of WHxAg also was examined during the course of acute, self-limiting experimental WHVinfection in 3 adult woodchucks and compared with serum markers of WHV infection. The hepatic expression of WHxAg and WHcAg was compared by immunohistochemical staining of adjacent sections of liver tissues obtained during the acute phase of infection (Fig. 3). The patterns of WHcAg and of WHxAg staining were nearly identical in adjacent biopsy sections, suggesting co-localization of these

FIG. 2. Immunohistochemical staining of woodchuck hepatic tissue from woodchuck with chronic WHV infection for WHcAg and WHxAg expression. Rabbit anti-sera generated against the rWHxAg and rWHcAg were used to localize viral antigens in the hepatocytes of chronic WHVcarrier woodchuck livers using the chromagen 3-3*-diaminobenzidinetetrahydroxychloride-dihydrate in a peroxidase reaction. Photomicrographs of liver tissue stained with (A) hematoxylin and eosin showing non-neoplastic [nn] tissue adjacent to tumor [T] tissue, (B) pre-immune rabbit sera, (C) rabbit anti-WHcAg, and (D) rabbit anti-WHxAg. (Original magnification 1250.)

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FIG. 3. Immunohistochemical staining of woodchuck hepatic tissue for WHcAg and WHxAg. The tissue was obtained by percutaneous needle biopsy of the liver during the course of acute, self-limiting experimental WHV-infection. Rabbit antiserum generated against the rWHxAg and rWHcAg was used to localize viral antigens in the hepatocytes using the chromogen 5bromo-4 chloro-3-indoyl phosphate/nitroblue tetrazolium in an alkaline phosphatase reaction. Photomicrographs of liver tissue showing centrilobular and midzonal expression of viral antigens at 4 weeks (left column), panlobular expression at 8 weeks (middle column), and expression in peripheral lobular regions at 10 weeks (right column) after infection. Top row shows tissue stained with pre-immune rabbit sera; middle row shows adjacent sections stained with rabbit anti-WHcAg; and bottom row shows adjacent sections stained with rabbit anti-WHxAg. (Original magnification 1100.)

viral antigens in acutely infected liver. The intensity of WHcAg was characteristically greater than that of WHxAg. In the biopsies of liver obtained initially 4 weeks after WHV inoculation, WHcAg and WHxAg expression appeared to be confined to centrilobular and midzonal hepatocytes (acinar zones 2 and 3). Panlobular expression of both antigens was observed in biopsies taken 8 weeks following inoculation, during the period of time that viremia was present. At 10 weeks post-inoculation, WHcAg and WHxAg were observed primarily in peripheral lobular (acinar zone 1) hepatocytes, and this distribution was observed until antigen expression no longer could be detected. A summary of results of hepatic immunohistochemistry

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during the course of acute, self-limiting experimental WHVinfection in three adult woodchucks are presented in Fig. 4 and are compared with serologic tests performed concurrently. In individual woodchucks, WHcAg and WHxAg in the liver appeared and were cleared at approximately the same time. Viremia (WHV DNA/WHsAg) was detected before WHcAg, WHxAg was detected in hepatocytes in all 3 woodchucks, and WHV DNA and WHsAg were cleared from the serum before WHcAg and WHxAg no longer could be detected from the liver. In two animals (4232, 4282), WHxAg was detected in the liver for 2 weeks after WHcAg was no longer detectable and in one (4291), WHcAg was detected one week after WHxAg was undetectable.

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ELISA for Anti-WHxAg of Serum. An ELISA was used to detect anti-WHxAg antibodies in serum during the course of acute, self-limiting experimental WHV-infection in 3 adult woodchucks, as described in Fig. 4, and these results are shown in Fig. 5. A negative control woodchuck consistently had P/N ° 1.5. In one woodchuck (4232), an anti-WHxAg response (P/N ú 4) was observed in serum collected at week 21 and remained test-positive (P/N ú 6) through week 24 postinoculation. Strong positive values (P/N ú4 ) were detected in serum collected from a second woodchuck (4291) beginning at week 9 and continuing post-inoculation to week 22, when the final sample was available. In a third woodchuck (4282), a positive signal (P/N ú 3) was observed at week 12, but thereafter the P/N remained below 3. The presence of antibody to WHxAg in the woodchuck serum, as determined by ELISA, was confirmed by immunoblot assay (Fig. 1E). Rabbit pre-immune serum served as a negative control and rabbit anti-WHxAg serum served as positive control. Pre-incubation of the rabbit anti-WHxAg containing serum with an excess of rWHxAg blocked the reactivity through immunoblot. Similarly, woodchuck serum that was found negative for anti-WHxAg by ELISA did not react in the immunoblot assay, whereas woodchuck serum that was found positive for anti-WHxAg by ELISA exhibited binding to the rWHxAg. Pre-incubation of this woodchuck anti-WHxAg positive serum with an excess of rWHxAg also blocked reactivity by immunoblot. Serum samples from 11 chronic WHV carriers obtained

FIG. 4. Serologic and immunohistochemistry profiles during the course of acute, self-limiting experimental WHV-infection in three adult woodchucks. Serum markers were assayed either by slot blot (WHV DNA) or ELISA (WHsAg, anti-WHsAg, anti-WHcAg, and anti-WHxAg). Hepatic expression of WHcAg and WHxAg was determined by immunohistochemistry. Serum and hepatic tissue samples from a normal uninfected woodchuck served as a negative control for experimental WHV-infected woodchucks (4232, 4282, and 4291).

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after development of HCC were screened for anti-WHxAg antibodies. Sera from 4 of 11 woodchucks were found to be positive for antibody to WHxAg by ELISA. Two of 4 had low WHxAg expression in the liver by immunohistochemical staining, but correlation between the presence of antibody to WHxAg and WHxAg expression in the liver was not evident. The 4 woodchucks positive for anti-WHxAg exhibited a moderate degree of portal hepatitis, similar to that in antiWHxAg negative woodchucks. DISCUSSION

WHxAg was detected immunohistochemically in hepatocytes during the course of acute, self-limiting WHV-infection in adult woodchucks and in chronic WHV-carriers. WHxAg was found primarily in the cytoplasm of non-neoplastic hepatocytes, and WHxAg was not detected in 10 of 11 HCCs and in the eleventh, less than 1% of cells expressed WHxAg. WHxAg seemed to be co-localized with WHcAg. These results are in general agreement with those of Feitelson et al.32 who demonstrated WHxAg to the cytoplasm of hepatocytes from woodchucks with naturally acquired WHV infection. Cellular fractionation of cultured hepatocytes demonstrated that WHxAg partitioned with the soluble and the cytoskeletal fractions. These results are in agreement with those of Dandri et al.33 who detected WHxAg in the cytoplasmic, but not in the nuclear, fraction of primary cultured woodchuck hepatocytes. Studies of the expression of recombinant HBxAg in HepG2 cells have shown HBxAg localized to the cytoplasm of the perinuclear region.25,34 Separation of subcellular components has shown HBxAg to be associated with the cytoskeletal and nuclear fractions.25 Recently, studies using confocal microscopy have shown that HBxAg compartmentalized primarily in the cytoplasm and to a minor degree in the nucleus.35 Discrepancies in results to date may be attributable to differences in the techniques used (recombinant vs. authentic viral replication) or the experimental systems studied (HBV vs. WHV). Altered hepatic cell foci exhibited varying degrees of diminished expression of WHxAg and WHcAg. Few woodchuck HCCs express WHxAg, suggesting WHxAg may not be essential in maintaining the transformed phenotype. WHcAg expression was observed in most hepatocellular adenomas, albeit at low levels. WHxAg expression was detected less frequently in adenomas. Recently, Dandri et al.33 have reported detecting WHxAg in woodchuck neoplasms and have indicated that tumors that replicate WHV also express WHxAg. During acute WHV infection, the intracellular expression of WHxAg co-localized with the expression of WHcAg. Although these two gene products are expressed from separate promoters, a viral enhancer upstream from the X-antigen gene affects WHc gene expression as well. WHcAg and WHxAg expression were found in the liver during the viremic phase of infection when viral DNA and WHsAg were detected in the serum. Nearly every hepatocyte seemed to be infected with WHV at the peak of viremia, as judged by the intensity and distribution of WHcAg expression, which are results similar to those described by Kajino et al.36 The hepatocytes of acinar zones 2 and 3 appeared to be the first to show WHcAg and WHxAg expression and during clearance of WHV infection, hepatocytes of acinar zone 1 were the last to have detectable antigens. Similar zonal distribution of

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in hepatocellular adenomas. In HCC, the detection of WHxAg or WHcAg expression was highly unusual. The availability of validated reagents for the analysis of WHxAg in the woodchuck model of HBV infection now makes possible further studies of the role of the X gene product in the pathogenesis of hepatic injury and hepatocarcinogenesis. REFERENCES

FIG. 5. ELISA to detect circulating woodchuck antibody to WHxAg. Serum collected during the course of acute, self-limiting experimental WHVinfection in three adult woodchucks was assayed at a 1:20 dilution. Following an overnight incubation on ELISA plates coated with rWHxAg, bound antibody was detected with a protein G horseradish peroxidase conjugate. The signal to noise ratio (P/N) was calculated with absorbance values at time zero as background.

WHcAg in acute, self-limited WHV infection has been observed previously.36 Clearance of WHxAg from the liver appeared to parallel that of WHcAg. Feitelson et al.32 reported a transient antibody response to WHxAg during the course of acute infection in one woodchuck. They suggested that circulating antibodies to WHxAg correlated with the absence of WHxAg in the liver and with a high degree of hepatitis.32 We did not observe such a relationship. Antibody to WHxAg was detected by ELISA in woodchucks during or following acute WHV infection. Characteristically, anti-WHxAg was detected after clearance of WHsAg. In chronic WHV carriers with HCC, antibody to WHxAg was detected in the serum of 4 of 11 woodchucks. A correlation between the presence of antibody to WHxAg with the degree of hepatitis or the level of hepatic WHxAg expression was not found. The rabbit antisera generated to the rWHxAg was able to detect authentic WHxAg by immunohistochemistry and in cultured hepatocytes by immunoprecipitation. In validating these reagents, it was shown that woodchuck sera found by ELISA to contain antibody to WHxAg can detect rWHxAg by immunoblot as can the rabbit antisera. Failure to detect WHxAg in tissue homogenates by conventional immunoblot indicates either that the WHxAg is present in too low a concentration or that the authentic antigen does not present recognizable epitopes under denaturing conditions. A recent study in which it was estimated there are 4 to 8 1 105 molecules of WHxAg per woodchuck hepatocyte provides support for the former ideal.33 There is convincing evidence that the X gene is required for hepadnaviral replication.8,9 WHxAg appeared to be coexpressed with WHcAg in both acute and chronic WHV infection, indicating that cytoplasmic expression of WHxAg, like WHcAg, can be considered a cellular marker of WHV replication. There was a remarkably reduced expression of WHcAg and WHxAg in pre-neoplastic hepatocytic foci and

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1. Gust ID, Burrell CJ, Coulepis AG, Robinson WS, Zuckerman AJ. Taxonomic classification of human hepatitis B virus. Intervirology 1986;25: 14-29. 2. Summers J, Mason WS. Replication of the genome of a hepatitis B-like virus by reverse transcription of an RNA intermediate. Cell 1982;29: 403-415. 3. Gerber MA, Thung SN. Biology of Disease: Molecular and cellular pathology of hepatitis B. Lab Invest 1985;52:572-590. 4. Schodel F, Sprengel R, Weimer T, Fernholz D, Scheider R, Will H. Animal hepatitis B viruses. Adv Viral Oncology 1989;8:73-102. 5. Robinson WS. Molecular events in the pathogenesis of hepadnavirusassociated hepatocellular carcinoma. Ann Rev Med 1994;45:297-323. 6. Kaneko S, Miller RH. X-region-specific transcript in mammalian hepatitis B virus-infected liver. J Virol 1988;62:3979-3984. 7. Rossner MT. Hepatitis B virus X-gene product: A promiscuous transcriptional activator. J Med Virol 1992;36:101-117. 8. Zoulim F, Saputelli J, Seeger C. Woodchuck hepatitis virus X protein is required for viral infection in vivo. J Virol 1994;68:2026-2030. 9. Chen HS, Kaneko S, Girones R, Anderson RW, Hornbuckle WE, Tennant BC, Cote PJ, et al. The woodchuck hepatitis virus X gene is important for establishment of virus infection in woodchucks. J Virol 1993;67:1218-1226. 10. Moriarty AM, Alexander H, Lerner RA. Antibodies to peptides detect new hepatitis B antigen: serological correlation with hepatocellular carcinoma. Science 1985;227:429-433. 11. Haruna Y, Hayashi N, Katayama K, Nobukabu Y, Kasahara A, Sasaki Y, Fusamoto H, et al. Expression of X protein and hepatitis B virus replication in chronic hepatitis. HEPATOLOGY 1991;13:417-421. 12. Wang W, London WY, Lega L, Feitelson MA. HBxAg in the liver from carrier patients with chronic hepatitis and cirrhosis. HEPATOLOGY 1991; 14:29-37. 13. Zhu M, London WT, Duan L-X, Feitelson MA. The value of hepatitis B x antigen as a prognostic marker in the development of hepatocellular carcinoma. Int J Cancer 1993;55:571-576. 14. Levrero M, Jean-Jean O, Balsano C, Will H, Perricaudet M. Hepatitis B virus (HBV) X gene expression in human cells and anti-HBx antibodies detection in chronic HBV infection. Virology 1990;174:299-304. 15. Levrero M, Stemler M, Pasquinelli C, Alberti A, Jean-Jean O, Franco A, Balsano C, et al. Significance of anti-HBx antibodies in hepatitis B virus infection. HEPATOLOGY 1991;13:143-149. 16. Jung M-C, Stemler M, Weimer T, Spengler U, Dohrmann J, Hoffmann R, Eichenlaub D, et al. Immune response of peripheral blood mononuclear cells to HBx-antigen of hepatitis B virus. HEPATOLOGY 1991;13: 637-643,. 17. Kay A, Dupont de Dinechin S, Vitvitski-Trepo L, Mandart E, Shamoon B-M, Galibert F. Recognition of the N-terminal, C-terminal, and interior portions of HBx by sera from patients with hepatitis. Br J Med Virol 1991;33:228-235. 18. Korba BE, Cote PJ, Tennant BC, Gerin JL. Woodchuck virus infection as a model for the development of antiviral therapies against HBV. In: Hollinger FB, Lemon SM, Margolis HS, eds. Viral hepatitis and liver disease. Baltimore: Williams and Wilkins, 1991:663-665. 19. Gerin JL, Cote PJ, Korba BE, Miller RH, Purcell RH, Tennant BC. Hepatitis B virus and liver cancer: the woodchuck as an experimental model of hepadnavirus-induced liver cancer. In: Hollinger FB, Lemon SM, Margolis HS, eds. Viral hepatitis and liver disease. Baltimore: Williams and Wilkins, 1991:556-559. 20. Cohen JI, Miller RH, Rosenblum B, Denniston K, Gerin JL, Purcell RH. Sequence comparison of woodchuck hepatitis virus replicative forms shows conservation of the genome. Virology 1988;162:12-20. 21. Sambrook J, Fritsch EF, Maniatis T. A laboratory manual for molecular cloning. Cold Spring Harbor Laboratory: Cold Spring Harbor Press, 1989. 22. Jacob JR, Tennant BC. Transformation of immortalized woodchuck hepatic cell lines with the c-Ha-ras proto-oncogene. Carcinogenesis 1996; 17:631-636.

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23. Jacob JR, Liu RH, Roneker CA, Noronha F, Hotchkiss JH, Tennant BC. Characterization and immortalization of woodchuck hepatocytes isolated from normal and hepadnavirus-infected woodchucks (Marmota monax). Exp Cell Res 1994;212:42-48. 24. Stamatos NM, Chakrabarti S, Moss B, Hare DJ. Expression of polyomavirus virion proteins by a vaccinia virus vector: association of VP1 and VP2 with the nuclear framework. J Virol 1987;61:516-525. 25. Schek N, Bartenschlager R, Kuhn C, Schaller H. Phosphorylation and rapid turnover of hepatitis B virus X-protein expressed in HepG2 cells from recombinant vaccinia virus. Oncogene 1991;6:1735-1744. 26. Knodell RG, Ishak KG, Black WC, Chen TS, Craig R, Kaplowitz N, Kiernan TW, et al. Formulation and application of a numerical scoring system for assessing histological activity in asymptomatic chronic active hepatitis. HEPATOLOGY 1981;1:431-435. 27. Ishak KG. Chronic hepatitis: morphology and nomenclature. Modern Path 1994;94:690-713. 28. Hytrioglou P, Thung SN, Gerber MA. Histological classification and quantitation of the severity of chronic hepatitis: keep it simple! Semin Liver Dis 1995;15:414-421. 29. Wu PC, Lau JYN, Lau S, Lau T, Lai CL. Relationship between intrahepatic expression of hepatitis B viral antigens and histology in Chinese patients with chronic hepatitis B virus infection. Clin Microbiol Infect Dis 1993;100:648-653. 30. Gish RG, Lau JYN, Brooks L, Fang JWS, Steady SL, Imperial JC, Garcia-

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Kennedy R, et al. Ganciclovir treatment of hepatitis B virus infection in liver transplant recipients. HEPATOLOGY 1996;23:1-6. Cote PJ, Roneker C, Cass K, Schodel F, Peterson D, Tennant BC, deNoronha F, et al. New enzyme immunoassays for the serologic detection of woodchuck hepatitis virus infection. Viral Immunol 1993;6:161-169. Feitelson MA, Lega L, Duan L-X, Clayton M. Characteristics of woodchuck hepatitis X-antigen in the livers and sera from infected animals. J Hepatol 1993;17(suppl):24S-34S. Dandri M, Schirmacher P, Rogler CE. Woodchuck hepatitis virus X protein is present in chronically infected woodchuck liver and woodchuck hepatocellular carcinomas which are permissive for viral replication. J Virol 1996;70:5246-5254. Siddiqui A, Jameel S, Mapoles J. Expression of the hepatitis B x gene in mammalian cells. Proc Natl Acad Sci U S A 1987; 84:25132517. Doria M, Klein N, Lucito R, Schneider RJ. The hepatitis B virus Hbx protein is a dual specificity cytoplasmic activator of Ras and nuclear activator of transcription factors. EMBO J 1995; 14:47474757. Kajino K, Jilbert AR, Saputelli J, Aldrich CE, Cullen J, Mason WS. Woodchuck hepatitis virus infections: very rapid recovery after a prolonged viremia and infection of virtually every hepatocyte. J Virol 1994; 68:5792-5803.

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