Correlation of hepatitis B virus DNA and antigens in the liver

GASTROENTEROLOGY 1987;92:192-6

LIVER

AND

BILIARY

TRACT

Correlation of Hepatitis B Virus DNA and Antigens in the Liver A Study in Chronic Liver Disease MASAO OMATA, OSAMU YOKOSUKA, FUMIO IMAZEKI, YOSHIMI ITO, JUNK0 MORI, KATSUO UCHIUMI, and KUNIO OKUDA First Department of Internal Medicine, Chiba University School of Medicine, Chiba, Japan

Hepatitis B virus deoxyribonucleic acid [DNA) and antigens /HBsAg and HBcAg) were studied in liver biopsy specimens from 105 HBsAg-positive patients with chronic liver diseases. Free or integrated viral DNA, or both, was detected in 83 of 105 [79%) patients, whereas HBsAg and HBcAg were demonstrated immunohistologically in 96 (91%) and 39 (37%), respectively. Of 60 patients with detectable free viral DNA, 38 (63%) were positive for HBcAg, whereas only 1 of 45 (2%) with either integrated viral DNA alone (n = 23) or no detectable viral DNA (n = 22) was positive for HBcAg (p < 0.001). Furthermore, the amount of HBcAg was positively correlated with the amount offree viral DNA in the liver tissue. In contrast, HBsAg was well expressed not only in the liver with free viral DNA, but also in the liver with integrated DNA. These data suggest that the synthesis of HBcAg is primarily directed by free viral DNA, whereas that of HBs Ag may be directed by free as well as integrated viral DNAs. The specific localization of hepatitis B virus (HBV)associated antigens in the liver and hepatocytes provides important clinicopathological clues to HBV infection in humans (l-3). Recent introduction of molecular biological techniques into the study of HBV infection has clarified molecular events related to H&V replication and liver diseases. One example is the demonstration by Southern blot hybridization Received July 22, 1985. Accepted June 12, 1986. Address requests for reprints to: Masao Omata, M.D., First Department of Medicine, Chiba University School of Medicine, Chiba, Japan (280). This study was supported in part by a grant from the Japanese Ministry cif Education (B-58480215, C-60570310, I-59010029), and grants from the Japanese Ministry of Health and Welfare and from the Japanese Medical Research Foundation. Q 1987 by the American Gastroenterological Association 0016-5085/87/$3.50

of integration of HBV deoxyribonucleic acid (DNA) into genomic DNA in hepatocelluar carcinoma (4-6) and in nonneoplastic liver disease (7-9). It has also been shown that the presence of HBV DNA in serum closely correlates with the presence of intrahepatic hepatitis B core antigen (HBcAg) (10,ll). Since the HBV-associated antigens including hepatitis B surface antigen (HBsAg) and HBcAg are gene products of HBV DNA, a study of viral antigens and DNA in the same liver may provide further insight into the relation of genes and their products in human liver disease. We report our study on viral DNA and antigens in the liver from 105 HBsAgseropositive patients with chronic liver disease.

Materials and Methods Patients Percutaneous liver biopsy specimens were taken from 105 patients with chronic liver disease who were seen at the First Department of Medicine, Chiba University. Two needle cores were taken from all patients with a Tru-cut biopsy needle. One core was processed for immunohistologic (2,3,12) and routine histologic examination, and the other was immediately frozen and stored at -8O’C. All these patients had been seropositive for HBsAg for at least 6 mo before biopsy. The ages of patients ranged from 17 to 79 yr (mean 34 yr). Eighty patients were men and 25 were women. Liver biopsy showed nonspecific change in 18, chronic persistent hepatitis in 32, chronic active hepatitis in 38, and liver cirrhosis in 17. Of the 105 patients, 61 were seropositive for hepatitis B e antigen (HBeAg), 32 were seropositive for hepatitis B e antibody (anti-HBe), 8 were negative for both HBeAg and anti-HBe, and 4 were not tested for HBeAg and anti-HBe.

Abbreviation used in this paper: mRNA, messenger ribonucleic acid.

Analysis

193

HBV DNA AND ANTIGENS IN THE LIVER

January 1987

Localization of Hepatitis B Surface Antigen and Hepatitis B Core Antigen in the Liver

of HBV DNA

Details of blot hybridization techniques have been described (13,14). In brief, a needle core of biopsy specimen was homogenized in a mixture of 0.5 ml of 10 mM

Tris-HCl (pH 7.4) and 10 mM ethylenediaminetetraacetic acid by a Dounce homogenizer. To this was added 0.5 ml of 0.2 M NaCl, 0.02 M Tris-HCl (pH 7.4), 2 mM ethylenediaminetetraacetic acid, 1 mgiml pronase, and 1% sodium dodecyl sulfate, and the mixture was incubated for 30 min at 37°C. Extraction of DNA was done twice with 1 ml of phenol/chloroform (l:l, vol/vol), and the aqueous phase was then precipitated and washed three times with absolute ethanol chilled to -20°C. The nucleic acid pellets were resuspended in 100 ~1 of a mixture of 5 mM Tris-HCl (pH 7.4) and 1 mM ethylenediaminetetraacetic acid. Ten micrograms of nucleic acid from liver was electrophoresed on 1.0% or 1.5% agarose gel (Seakam Co., Rockland, Me.) and transferred to nitrocellulose filter (Schleicher & Schuell, Dassel, F.R.G.) by the Southern blot technique (15).An HBV DNA insert in pBH-20 (16)was excised from the plasmid and labeled with 32P-dCTP by the nick translation method (17). The nitrocellulose paper was baked dry and hybridized with the radioactive probe. After washing, nitrocellulose paper was dried and autoradiographed with Kodak X-Omat film (Eastman Kodak Co., Rochester, N.Y.). Ten micrograms of nucleic acid extracted from liver was digested with 10 U of Hind III or Eco RI or both for 2 h at 37°C with a mixture of 0.02 M Tris-HCl (pH 7.5), 0.05 M NaCl, 0.01% Triton, and 1 mM dithiothreitol. With the restriction enzyme analysis, we classified the blot hybridization patterns into free (episomal), integrated, or both according to the previous reports (7-9). In principle, when radioactive signals migrating faster than 3.2-kb marker in 1.0% agarose gel or 4.0-kb marker in 1.5% agarose gel were detected with undigested and Hind III cut nucleic acids, the pattern was classified as free or episomal viral DNA type: when very high molecular weight signals with uncut nucleic acids and a high molecular weight smear or band with the enzyme digestion was detected, the pattern was classified as integrated form (7-g). For quantitation of HBV DNA in tissue, density of the autoradiograph, analyzed with a Sakura PDS densitometer (Sakura, Tokyo, Japan), was compared with that of stan-

Hepatitis B surface antigen and HBcAg were localized in the liver by the direct and indirect immunoperoxidase methods. Details of the procedures have already been described (2,3,12). The number of HBsAg- and HBcAgpositive hepatocytes was semiquantitatively graded as follows: minimal, ~5% of total hepatocytes had the antigen; moderate, 5%-20%; abundant, >20%.

Serum Hepatitis B Surface Hepatitis B e Antigen

Antigen

and

Serum HBsAg was assayed by reversed passive hemagglutination, and hepatitis B e antigen (HBeAg) by radioimmunoassay (Abbott Laboratories, North Chicago, Ill.).

Statistical

Analysis

Statistical analysis was performed by the 2 test or Student’s t-test.

Results HBV DNA and Antigens Using blot hybridization, HBV DNA was detected in 83 of 105 (79%) patients. Of the 83 patients, free viral DNA alone was found in 53, integrated forms were found in 23, and both free and integrated forms were found in 7. Demographic and laboratory data are correlated with viral DNA patterns in liver tissue in Table 1. Free viral DNA was mainly found in HBeAg-seropositive patients, whereas the integrated form was most frequently found in anti-HBepositive patients. Serum alanine aminotransferase in patients with free viral DNA was higher than that in

dards and the quantity of viral DNA was graded as follows: minimal, 550 pg/kg tissue nucleic acids; moderate, 50-200 pglpg tissue nucleic acids; abundant, >200 pglpg tissue nucleic acids.

the patients with the integrated form [p < 0.01). Hepatitis B surface antigen was detected in 96 of 105 (91%) patients and HBcAg was principally detected in the nuclei of hepatocytes in 39 of 105 (37%) by immunoperoxidase methods.

Table

in Relation

1. Demographic

HBV DNA Free Free + integrated Integrated Undetectable

and Laboratory

Data on 105 Patients

No.

Male (%I

Age (Y4

53

85

32 2 11"

7

57

23 22

65 72

to Four Viral Deoxyribonucleic

Acid

ALT

NSC

CPH

CAH

LC

HBeAg positive

HBeAg positive

162 " 204" 172 t 186

6

20

22

5

50

36 k 8

1

1

3

2

4

39 ?z 14 34 +- 11

49 240 38 '-'28

9 2

2 9

8 5

4 6

2 5

2 2 17 11

Patterns Both negative 0

1 3 4

ALT, alanine aminotransferase; CAH, chronic active hepatitis; CPH, chronic persistent hepatitis; DNA, deoxyribonucleic acid; HBeAg, hepatitis B e antigen; HBV. hepatitis B virus; LC, liver cirrhosis; NSC, nonspecific change; 0 Mean + standard deviation.

194

OMATA

ET AL.

Correlation

GASTROENTEROLOGY

of HBV DNA ahd Antigens

Tissue viral DNA patterns were correlated with the existence of viral antigens (Table 2). Hepatitis B surface antigen was expressed well in all four Rrouas with either free or intee;rated viral DNAs in liver-tissue (Table 2). In contr& HBcAg was demonstrated in the liver in 38 of 60 (63%) patients with free viral DNA (with or without integrated form), but in only 1 of 45 (2%) with integrated or undetectable viral DNA. The difference was statistically highly significant (p < 0.001). Correlation of Episomal I-IBV DNA and Hepatitis B Core Antigen As these data suggested that the synthesis of HBcAg is related to the presence of episomal viral DNA, we correlated the semiquantitative amount of DNA and this antigen. Hepatitic B core antigen was detected more often in the liver with more fre$ viral DNA (Table 3). Correlation of semiquantitatively graded episomal viral DNA and HBcAg revealed a statistically significant correlation between the two (p < O.OOl), namely the majority (12 of 68) with no or little episomal HBV DNA had only a small amount of HBcAg detected in the liver, whereas 27 of 37 with moderate to large amounts of episomal DNA had moderate to abundant amounts of HBcAg detected in the liver (2 = 19.3). However, no such correlation was noted between episomal HBV DNA and HBsAg.

Discussion There have been a number of studies af the localiztition of HBsAg and HBcAg in the liver besides ours (2,3,12). Recent introduction of molecular biological techniques has made a study of viral DNA in dlinical materials possible (7-9). Two previous studies by Brechot et al. (7) and by Kam et al. (9) reported that free HBV DNA in the liver and serum HBV DNA were found in all of their HBeAgseropositive patients, whereas integrated viral DNA was often found in HBeAg-seronegative patients (16 Table 2. Relationship Between Hepatitis B Virus Deoxyribonucleic Acid Patterns and Viral Antigens in 105 Patients HBV DNA Free Free + integrated Integrated Undetectable

No. 53 7 23 22

HBsAK 50 7 22 17

(94%) (100%) (96%) (77%)

HBcAg 36 2 0 1

(72%) (29%) (0%) (5%)O

DNA, deoxyribonucleic acid; HBcAg, hepatitis B core antigen; HBsAg, hepatitis B surface antigen; HBV, hepatitis B virus. a A 31-yr-old cirrhotic man with a cluster of HBcAg-positive hepatocytes in one nodule.

Vol. 92. No. 1

Table 3. Relationship Between Semiquantitatively Graded Free Viral Deoxyribonucleic Acid and Viral Antigen Positivity Rates in 105 Patients Amount of free HBV DNA

Abundant Moderate Minimal Negative

No. 15 22 23 45

HBsAg positive 15/15 21122 21123 39145

(100%) (96%] (91%) (87%)

HBcAg positive 11115 16/22 11123 1145

(73%) (73%) (48%) (Z%]

DNA, deoxyribonucleic acid; HBcAg, hepatitis B core antigen; HBsAg, hepatitis B surface antigen; HBV, hepatitis B virus.

of 30). Data of intrahepatic HBsAg and HBcAg were not available in these studies. Bonino et al. (10) studied intrahepatic HBV antigens and serum HBV markers in 30 Italian patients (40% of them were &infected), and stressed the correlation of intrahepatic HBcAg and serum HBV DNA and HBeAg. Hadziyannis et al. (11) studied intrahepatic antigens and serum markers in 34 patients with various chronic liver diseases, and indicated that the presence of serum HBV DNA and intrahepatic HBcAg was often associated with active liver disease even in patients with anti-HBe. Of 34 patients, data by blot hybridization of extracted DNA from the liver were presented in 9 patients, and showed that free viral DNA was found in 5 HBeAgpositive patients with intrahepatic HBcAg, and integrated form was found in 4 anti-HBe-positive patients without HBcAg (11). To extend these previous studies of viral replication and liver disease, and to focus more on DNA template and product relation, we systematically studied HBV gene (HBV DNA in the liver by blot hybridization) and HBV gene products in the liver (HBsAg and HBcAg) and serum (HBeAg) of 105 Japanese patients. Our study demonstrated that HBsAg was effectively expressed in the liver that has either free or integrated viral forms, suggesting that HBsAg is efficiently translated from free and integrated viral DNA. In contrast, HBcAg was very poorly expressed in the liver with integrated viral DNA. Thus, HBcAg synthesis, at least within the amounts detectable by our method, may not be directed well by integrated viral DNA. Two lines of evidence in vitro and in experimental animals seem to support our contention. One is the sequencing analysis of integrated viral DNA in malignant cells. The tissue culture cell line PLC/PRF/5, isolated from a human hepatocellular carcinoma, synthesizes HBsAg but not HBcAg (18,19). The cell line has been shown to contain only integrated HBV DNA (4-6,20). The reason for the selective expression of the HBsAg gene is not clearly understood.

HBV DNA AND ANTIGENS IN THE LIVER

January 1987

However, recent sequencing analysis of integrated viral DNA suggested that the HBsAg gene is intact, whereas the HBcAg gene is often deleted or rearranged (21-23). Thus, deletion of HBcAg gene in the integrated viral DNA may explain the impaired synthesis of HBcAg in the liver with integrated HBV DNA. The second line of evidence to support our hypothesis is the studies of virus-specific messenger ribonucleic acid (mRNA); one analyzed free viral DNA and its mRNA in duck HBV infection (24),and others analyzed integrated HBV DNA and its mRNA in PLC/PRF/5 (23,25). These studies demonstrated the following. (a) Free viral DNA produces a large mRNA (-3.5 kb) and smaller mRNAs (-2.3 and 2.1 kb), and can direct the synthesis of two antigens. (b) Integrated viral DNA produces mainly the smaller mRNA (-21S), and therefore can direct the synthesis of HBsAg alone. Recently, we studied HBV-specific RNA transcript in patients with chronic liver disease, and found that a 29s RNA transcript (larger) is predominantly seen in the liver with free viral DNA and a 21s RNA transcript (smaller) is predominantly seen in the liver with integrated viral forms (unpublished data). Thus, the data in animals and in vitro are consistent with our findings in chronic liver disease. However, structural analysis of integrated HBV DNA is yet to be done to see whether the preferential HBcAg gene deletion is present in the liver in nonneoplastic liver disease. Clinically, elevated serum alanine aminotransferase levels in patients with HBV DNA and HBeAg in serum are often normalized when HBV DNA becomes undetectable and HBeAg is seroconverted to anti-HBe. Our data indicate that the presence of free viral DNA is related to high serum levels of alanine aminotransferase, but no significant elevation of the enzyme was noted in patients with integrated viral DNA. How can this difference be explained? Our data suggest that the difference in the gene products between patients with integrated and free DNA is the presence of HBcAg and possibly HBeAg synthesis in the latter group. Hepatitis B surface antigen is synthesized to a significant extent in both groups. Recent molecular biological studies on HBeAg synthesis revealed that HBeAg might be translated from the “Pre-C gene” (-29 codons preceding C-gene), and that the HBeAg peptide is hydrophobic and often membrane-bound (LJy A et al. and Ou J et al., Cold Spring Harbor Conference of Molecular Biology of Hepatitis B Viruses). Thus, it is conceivable that membrane-bound HBeAg modifies the chemical properties of hepatocellular membrane to undergo hepatocellular degeneration and necrosis. In summary, we have shown that there are two types of viral gene expression, e.g., free viral DNA

195

and HBsAg and HBcAg as its products, and integrated viral DNA and HBsAg as its expression. Reduction of free viral DNA in the liver by antiviral treatment may lead to a biochemically quiescent liver disease, even though integrated viral DNA may continue to exist.

References 1 Gudat F, Bianchi L, Sonnabend W, et al. Pattern of core and surface expression in liver tissue reflects state of specific immune response in hepatitis B. Lab Invest 1975;32:1-9. 2 Omata M, Afroudakis A, Aschcavai M, et al. Comparison of serum hepatitis B surface antigen (HBsAg) and serum anticore with tissue HBsAg and hepatitis B core antigen (HBcAg). Gastroenterology 1978;75:1003-9. 3. Omata M, Aschcavai M, Liew CT, et al. Hepatocellular carcinoma in the U.S.A.. etiologic consideration. Localization of hepatitis B antigens. Gastroenterology 1979:76:279-87. 4. Chakraborty PR, Ruiz-Opazo N, Shouval D, et al. Identification of integrated hepatitis B virus DNA and expression of viral DNA in an HBsAg-producing human hepatocellular carcinoma cell line. Nature 1980; 286:531-3. 5. Brechot C, Pourcel C, Louise A, et al. Presence of integrated hepatitis B virus DNA sequences in cellular DNA of human hepatocellular carcinoma. Nature 1980;286:533-5. 6. Edman JC, Gray P. Valenzuela P, et al. Integration of hepatitis B virus sequences and their expression in a human hepatoma cell. Nature 1980;286:535-8. 7. Brechot C, Hadchouel M, Scott J, et al. Detection of hepatitis B virus DNA in liver and serum: a direct appraisal of the chronic carrier state. Lancet 1981;ii:765-8. 8. Shafritz D, Shouval D, Shermann HI, et al. Integration of hepatitis B virus DNA into the genome of the liver cells in chronic liver disease and hepatocellular carcinoma. N Engl J Med 1981;305:1067-73. 9. Kam W, Rall LB, Smuckler EA, et al. Hepatitis B viral DNA in liver and serum of asymptomatic carriers. Proc Nat1 Acad Sci USA 1982;79:7522-6. 10. Bonino F, Hoyer B, Nelson J, et al. Hepatitis B virus DNA in the sera of HBsAg carriers: a marker of active hepatitis B virus replication in the liver. Hepatology 1981;1:386-91. 11. Hadziyannis SJ, Liberman HM, Karvountzis GG, et al. Analysis of liver disease, nuclear HBcAg, viral replication, and hepatitis B virus DNA in liver and serum of HBeAg vs. anti-HBe positive carriers of hepatitis B virus. Hepatology 1983;3:656-62. 12. Omata M, Mori J, Yokosuka0, etal. Hepatitis

13

14.

15.

16.

B virus antigens in liver tissue in hepatocellular carcinoma and advanced liver disease-relationship to liver cell dysplasia. Liver 1982;ii: 125-32. Omata M, Yokosuka 0, Imazeki F, et al. Transmission of duck hepatitis B virus from Chinese ducks to Japanese ducklings: study of viral DNA in serum and tissue. Hepatology 1984; 4:603-7. Yokosuka 0, Omata M, Zhou YZ, et al. Duck hepatitis B virus DNA in liver and serum of Chinese ducks: integration of viral DNA in a hepatocellular carcinoma. Proc Nat1 Acad Sci USA 1985:82:5180-l. Southern EM. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 1975;98:503-17. Cummings IW, Browne JK, Salser WA, et al. Isolation, characterization and comparison of recombinant DNAs derived

196

OMATA ET AL.

from genomes of human hepatitis B virus and woodchuck hepatitis virus. Proc Nat1 Acad Sci USA 1980;77:1842-6. 17. Rigby PWJ, Dieckmann M, Rhodes C, et al. Labeling of deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J Mol Biol 1977; 113:237-51. of 18. Marion PL, Salazar FH, Alexander J, et al. Polypeptide hepatitis B virus surface antigen produced by a hepatoma cell line. J Virol 1979;32:796-802. 19. Skelly J, Copeland JA, Howard CR, et al. Hepatitis B surface antigen produced by an hepatoma cell line. Nature 1979; 282:617-a. 20. Marion PL, Salazar FH, Alexander JJ, et al. State of hepatitis B viral DNA in a human hepatoma cell line. J Virol 1980; 33:795-806. 21. Ziemer M, Garcia P, Shaul Y, et al. Sequence of hepatitis B

GASTROENTEROLOGY Vol. 92, No. 1

22.

23.

24.

25.

virus DNA incorporated into the genome of a human hepatoma cell line. J Virol 1985;53:885-92. Koike K, Kobayashi Y, Mizusawa H, et al. Rearrangement of the surface antigen gene of hepatitis B virus integration in the human hepatoma cell line. Nucleic Acids Res 1983;ll: 5391-402. Ou JH, Rutter WJ. Hybrid hepatitis B virus-host transcripts in a human hepatoma cell. Proc Nat1 Acad Sci USA 1985; 82:83-7. Bushcer M, Reiser W, Will H, et al. Transcription and the putative RNA pregenome of duck hepatitis B virus: implication for reverse transcription. Cell 1985;40:717-24. Yaginuma K, Kobayashi H, Yoshida E, et al. Direct evidence for the expression of integrated hepatitis B virus DNA in human hepatoma tissue. Gann 1984;75:743-6.