Journal of Hepatology, 1992; 14: 286-293 @ 1992 Elsevier Science Publishers B.V. All rights reserved. 0168-8278/92/$05.00

286 HEPAT 01007

Hepatitis B x antigen and polymerase ant hepatitis B carriers with or without hepat

dies in the seru delta virus infect10

Effects of interferon treatment Laura

Legal, A. Vierucci’, B.S. Blumberg’, Giorgio Saracco3, Mario Rizzetto3, Minghua Zhu2 and M.A. Feitelson2

‘Department of Pediatrics, Universityof Florence, Florence, Italy, ‘Fox Chase Cancer Center, Philadelphia, PA, United States of America and 3Departrnentof Gastroenterology, Ospedale Molinette, Turin, Italy

(Received 17 January 1991)

previous

work has shown that the hepatitis I3 x antigen (HBxAg) and antibodies directed against the polymerase

of

hepatitis B virus (anti-pol) are early markers of hepatitis B virus (HBV) replication in natural infections. The present study was carried out to test the hypothesis that the appearance of one or both of these markers signaled reactivation in chronic carriers with liver disease who were treated with a-interferon (IFN). The results show that HBV DNA decreased among the patients who responded to therapy, and that among these responders, neither HBxAg nor anti-pol became detectable in serum for 12 months after treatment, in contrast to controls. Hence, the loss of HBxAg and anti-pol correlate with decreased levels of HBV DNA in response to IFN therapy. However, different patterns of HBxAg and anti-pol were observed among a-IFN-treated HBV carrier patients who were also chronically infected with the hepatitis delta virus (HDV). The treatment of such patients often resulted in the loss of HDV RNA from serum and delta antigen from liver. Most of these patients had increased levels of HBV DNA in serum. HBxAg and/or anti-pol also became detectable in patients who lost markers of HDV, implying that the suppression of HDV by IFN is accompanied by the appearance of early markers of HBV reactivation in some of the treated patients,

The pathogenesis of HBV infection is variable. Most infected patients remain asymptomatic for long periods, while others develop acute hepatitis, chronic liver disease, and/or primary hepatocellular carcinoma (PHC) (l-3). In many patients, the appearance and persistence of liver disease during chronic infection correlates with serum markers of ongoing virus replication (4). However, there are many chronically infected patients with liver disease who have little or no serological evidence of virus replication (5,6). One factor contributing to the variability in the outcome of chronic HBV infection is superinfection with hepatitis delta virus (HDV) (7,8). Infection of chronic HBV carriers with HDV often results in an exacerbation of chronic hepatitis (g-10,11) which is often accompaCorrespondence:

nied by the development of HDV replication markers and the loss of HBV replication markers in both serum and liver (8,X2,13). After HDV superinfection, markers of HBV replication appear in some coinfected individuals, suggesting HBV reactivation (14-16). The fact that both agents are responsive to interferon (IFN) treatment (17-19), raises the possibility that IIW depression of HDV replication may result in HBV reactivation among some patients. In such individuals, the inhibition of HDV replication by IFN would remove the suppression on HBV replication, resulting in the reappearance of HBV in the blood. The genome of HEW consists of four open reading frames (20-22). One of these regions encode the hepatitis B surface antigen (HBsAg; or envelope) polypep-

Mark Feitelson, Ph.D., Department of Pathology and Cell Biology, Thomas Jefferson University, 1020 Locust Street,

Philadelphia, PA 19107, U.S.A.

EFFECTS OF IFN ON HEixAg AND ANTI-POL

tides of the virus, and another encodes the hepatitis B core antigen (HBcAg) polypeptide, which is the major constituent of the virus nucleocapsid. An HBcAg-related polypeptide is proteolytically cleaved and secreted from infected cells as the hepatitis B e antigen (I-IBeAg) (1,23). I-IBeAg in serum is associated with virus replication and high infectivity (24,25). A third region encodes the polymerase polypeptides which are required for replication of the virus nucleic acids (26). Finally, the X gene encodes one or more polypeptides which may regulate virus gene expression and replication (27-29). The X gene product(s) may also be important in the pathogenesis of chronic liver disease, mcluding I-IBV associated PHC (30-33). Previous work from this laboratory has shown that HBxAg is sometimes present in the serum and often accompanies both HEW and HBeAg during acute infection, suggesting a close association of HBxAg and virus replication (27,34). Antibodies against the polymerase of HBV (anti-pol) represent another marker of infection which may also be sensitive to the levels of virus replication (35-37). Both HBxAg and anti-pol decrease with decreasing evidence of virus replication during chronic infection (27,34-37). The fact that HBxAg and anti-pol may become detectable prior to HBeAg in the course of acute infection (27,35) implies that I-IBxAg and/or antipol might also be early serum markers of virus reactivation. If this is true, then one or both of these markers may be useful to monitor anti-viral chemotherapy. Hence, the patterns of HBxAg and anti-pol were dete;mined in patients with chronic I-IBV and HDV infections undergoing IF’N therapy to see whether either marker alone, or in combination, signaled I-IBV reactivation a year following the end of IFN therapy.

Patients

All patients recruited for this study had already been treated in Italy with a-IFN or were selected as controls, as previously described (38). Among the 58 patients studied, all were Italian adults who were HBV infected aud had chronic liver disease (Table 1). Twenty-one of the IPN-treated patients were HBeAg-positive HBV carriers. An additional 24 carriers were randomly chosen as untreated controls. All patients selected for IPN treatment had HBV DNA in serum detectable by dot hybridization and were positive for HBcAg in the liver (38). Nine carriers had chronic persistent hepatitis (CPH), and 12 had chronic active hepatitis (CAH). A blood sample and liver biopsy were obtained from each patient prior to

287 IFN treatment or placebo. IFN-treated patients were given intramuscular injections of 5 million units per m* three times per week for 6 months as described (38). Approx. 12 months after the end of therapy, a blood sample was obtained from all patients and a second liver biopsy from about half the patients in this study. a-II% treatment was also administered to 13 HDVinfected Italian patients who were HBeAg-negative HBsAg carriers with chronic liver disease (Table 1). Again, stored serum samples from pre-existing collections were used. All patients had detectable HDV RNA and antibodies against HDV (anti-HID) in serum but no evidence of HBV DNA by slot blot hybridization (see below). A biood sample was obtained from all patients just before IFN treatment (38) and 18 months later. Liver biopsies were obtained from all patients prior to therapy and from nine patients following therapy. Hepatitis B and D viral markers and alanine aminotramferase levels in serum

HBsAg, anti-HBs, anti-HBc, HBeAg, and anti-HBe were determined using standard commercially available assays (35) (Abbott Laboratories, North Chicago, IL). Anti-HD was measured using a commercially available assay (Abbott). Serum analine aminotransferase (ALT) levels were measured by standard techniques (35). Serum samples were screened for HBxAg by enzymelinked immunoabsorbant assay as previously described (27). Briefly, microtiter wells were coated with human serum for testing, followed by anti-HBx made in rabbits against HBxAg synthetic peptides, and finally by addition of an enzyme conjugated anti-immunoglobulin and substrate for color development. Positive values were scored as being greater than two standard deviations above the mean of the negative controls, which was a panel of human serum samples from uninfected individuals. The specificity of binding was determined by repeating the assay with normal rabbit serum in the place of peptide antibodies. Specificity was also shown by preincubation of the peptide antibodies with the immunizing peptides prior to assay. Verification of the solid-phase assay was carried out by performing immunoprecipitation of randomly chosen positive and negative serum samples using a monoclonal anti-x (39), followed by analysis of the samples by sodium dodecyl sulfate/ polyacrylamide gel electrophoresis (SDS/PAGE), and finally by Western blotting, using anti-HBx peptide antisera (34). The specificity of both the monoclonal and peptide antibodies used here have also been previously documented using recombinant DNA derived HBxAg polypeptides (27.30. in). A specific solid-phase assay was also used for detection

L.LEGA et al.

288

of antibodies against the polymerase of HBV (35). In these assays, polymerase synthetic peptides were used for coating microtiter wells. Test serum samples were then added to wells, followed by an enzyme conjugated anti-i~unoglobulin reagent and finally substrate for color development. Positive values were scored as being greater than two standard deviations above the mean of the negative controls, which was a panel of human serum samples from uninfected individuals. The specificity of binding was determined by repeating the assay in the absence of the coating antigens and by preincubation of the test serum samples with excess soluble synthetic peptides prior to assay (35). Verification of specificity in the anti-pol solid-phase assay was carried out by determining whether serum samples could immunoprecipitate a recombinant DNA derived polymerase polypeptide fragment . Briefly, recombinant polymerase polypeptide was made from the construct pET3a-HA, amplified in Echerischia coli stain BL21(DE3)pLysS. The fragment chosen for expression included the 3’ end of the polymerase gene from the full length clone of HBV adw, (21) which was amplified in the polymerase chain reaction (PCR) using primers spanning nucleotide positions 24362412 (MF04) and 631-660 (MFHP,). These primers were made in the oligonucleotide synthesis facility at the Fox Chase Cancer center. This region of the polymerase gene was chosen for expression because it carried immunodominant determinants recognized by most patients with an anti-pol response (35-37). The expression product (32 000 daltons) was identified by immunoprecipitation using appropriate anti-pol peptide antisera (35). Tritium-labeled polymerase polypeptide was then used for immunoprecipitation, followed by SDS/PAGE, and the results visualized by fluorography.

was also determined by dot and blot hybridization using the same sample sizes as for HBV DNA above and assayed by published procedures (38,41). All hybridizations were carried out on Zetabind membrane (CUNO, Inc., Meriden, CT) under stringent conditions (42). Hybridization sensitivity was such that 5 pg of viral DNA was clearly observed after 24 h of autoradiography. Liver sam&es

Needle biopsy samples were formalin fixed, paraffin embedded, stained with hematoxylin and eosin, and had been previously evaluated for liver pathology under code (38). Additional sections from each case had been stained for HBsAg and HBcAg using commercially available kits, and for HDV, as described (38). Statistical analysis p values were calculated using Fisher’s exact test for independence in a 2 x 2 table. The relationship between two compared characteristics were considered significant when p < 0.05.

Results and Discussion

The initial characteristics of the patients chosen for this study are presented above and in Table 1. All patients were adult HBV carriers who were persistently positive for HBsAg. Each patient also had chronic liver disease, which was diagnosed histologically as chronic hepatitis or cirrhosis. Twenty-one of thece carriers, who had no evidence of HDV infection, were treated with a-IFN. Another 13 HBV carriers with HDV infection were also treated with a-IFN. An additional 24 HBV catriers with chronic liver disease were left untreated

Nucleic acid determinations

HBV DNA was assayed in serum by dot blot hybridization using 1 ~1 of serum (27). Negative samples were assayed by slot blot hybridization using viral DNA extracted from 100 ~1 of serum. Probes were made by isolating full-length HBV DNA insert from a recombinant plasmid (21) and radiolabeling using [32P]TTP and randcm priming (27). Serum samples (50 ~1 each) which were negative by dot and slot blot hybridization were assayed for HBV DNA by PCR using primers MF03 (residues 1903-1929) and MF04 (residues 2436-2412) for amplification of the core region, followed by agarose gel electrophoresis and Ethidium bromide staining. For quantitiation of HBV DNA after PCR, Ethidium bromide stained gels were photographed with Kodak Royal Pan film (which has a transparent background) and the bands analyzed by a scanning densitometer. HDV RNA

TABLE 1

Initial characteristicsof patients entered into this study HBV carriers

Treated -HDV (n = 21)

Sex (male/female) Age (years) mean range ALT (II-I/liter) mean range Liver pathology CPH CAH cirrhosis

+HDV (R = 13)

Untreated controls (n = 24)

16/.5

10/3

2014

39 21-60

41 25-72

45 21-65

116 66-187

135 60-296

151 60-408

9 12 1”

0

12 4b

9 13 2

a This patient also had CAH. bThree of these four patients also had CAH .

EFFECTS

OF IFN ON HBxAg AND ANTI-POL

289

TABLE 2 HBV markers and liver pathology IFlP

HBsAg+

I. Treated A. responders (n = 11) before after B. nonresponders (n = 10) before after II. Untreated controls (n = 21) tin.,,. c -10. 18 mo.

in a-IFN-treated

HBe Ag+

HBx Ag+

and control patients without HDV infection antipo1+

antiH%e+

HBV DNA

Liver pathology

dot

slot

PCR

CPH

CAH

CIR

fibrosis

11 8

11 0

1 1

3 0

0 3

11 0

11 1

11 11

6 5

5 3

0 1

0 2

10 10

10 10

0 2

2 2

0 0

10 8

10 8

10 10

3 2

7 6

0 2

0 0

24 23

24 20

2 4

8 6

0 5

12 12

15 15

24 24

9

13 N.D.

2 N.D.

0 N.D.

N.D.b

aAmong IFN-treated patients, the term ‘before’ refers to the listed characteristics of the patients in each group prior to a-IFN therapy, the term ‘after’ refers to the listed characteristics of the patients in each group a year after the end of a-IFN therapy. bN.D. = not done.

(Tables 2 and 3). The results of the trial and changes in the classical HBV markers (HBsAg, HBeAg, anti-HBc, anti-HBe, anti-HBs, and ALT) among the patients without HDV have been previously reported (38). Among the 21 HBV carriers without HDV infection who underwent a-FN treatment, 11 responded to a-EN and the remaining 10 did not respond (Table 2). Favorable response to treatment was defined as loss of HBeAg from serum within the course of treatment or up to 1 year following treatment. Three of the responders lost HBsAg from serum (Table 2). Further, each of the 11

and

responding patients lost HBV DNA from serum (as measured by dot blot hybridization) and I-IBcAg from liver. Retesting the sera of the responders for HBV DNA by slot blot hybridization showed that one patient had detectable HBV DNA even though HBeAg disappeared (Table 2). Retesting sera by PCR after a-IFN treatment showed that all responder patients continued to have low levels of virus in their sera, although the levels were 3-6 logs lower after a-IFN compared to pretreatment levels (data not shown). The fact that only three responders seroconverted to anti-HBe (38), also suggests that com-

TABLE 3 HBV and liver pathology in a-IFN-treated

patients with chronic HDV infection”

Case’

HBsAg

H%xAg

Anti-p01

H%V DNAb (PCR)

HBY RNAd

HDAg (liver)

Pathology

1 2 3 4 5 6 7 8 9 10 11 12 13

+I+ +I+

-/-I-

+/+ +/-

+I+ +I+

+I+I-

+I

CAH CAH,

i-li-l+I+ +I+

-1-b -l-I-I+ -I-I-I-l-

+I+ +I-1+ -I-l-I+ -l-l-

+I+ +I+ +I++ -J+I+ +/++ +/+ _s_l-

-I-I-

+/+ i-i-

+I+ +I

+I+I+ d-l+I+I+ i-l+1-I-II-II-

+I +I+I +I+I+1-I+I+I+ +I -t-I-

+I+

+I+

-I-

+I+

-Ii-+

i-f-

-u-

+I+ +I+ +I+ +I+ +I+ +/+ i-l-k

cirrhosis CAIWfibrosis CAH CAH/mild CAH CAI-WAH CAIWcirrhosis CAIWmild CAH CAH, cirrhosis/CAH. CAW CAI-UCAH CAH/inactive CAH cir/inactive cirrhosis

cir

~AII H%V-infected patients were negative for H%eAg, positive for antibodies against delta (anti-HD), and negative for HBV DNA by dot hybr&ation both prior to and after lFN treatment. Ail patients were negative for HBcAg in liver both prior to and after IFN treatment. Twelve of the 13 patients were anti-HBeC prior to and after IFN treatment. bs Ymbols refer to the ethidium bromide staining intensity of the PCR amplified products folIOWing gel electrophoresis. where (-1 indicates no observable band, and where the band intensity increases from just visible (?). to easily visible (+), t0 intense (+ +). Q!Lantitation of these bands was carried out as described in Patients and Methods. ‘Each of the case numbers here corresponds with the PCR results in Fig. 2. dResults for HDV RNA are from slot blot hybridization experiments.

LLEGA

290 plete virus clearance was not achieved in most cases. Despite the fact that all patients maintained 10~~ kv& Of virus replication, an improvement in liver histology was observed in five. Among the patients who did not respond to IFN, all remained positive for HEsAg and m&g, and eight for HBV DNA (Table 2). The eight patients in the latter group also remained HBsAg+ in the liver after treatment. None of the patients in the nonresponding group seroconverted to anti-me; nor did any show improvement in liver pathology. Hence, a-IFN treatment results in changes in virological markers and liver pathology in some patients which indicate a beneficial outcome. In several nonresponder patients, however, there was discordance between HBeAg and viral DNA, indicating that the HBeAg status is not always a satisfactory marker for the persistence of virus in seru.n or response to anti-viral chemotherapy (Table 2). This discordance is most likely to be due to the presence of precore mutant viruses (43). All of the 11 a-IFNresponder patients became negative for HBV DNA by dot blot hybridization (Table 2). However, one was HBV DNA+ by slot blot hybridization, and this patient was also HBxAg+ both prior to and following therapy. These findings suggest that this individual responded poorly to therapy. Hence, the persistence of HBxAg following a-IFN therapy may he associated with relatively high levels of virus replication, as shown earlier in untreated patients (27,34), even though HBeAg became undetectable. None of the responder patients became HBxAg+ after treatment. In contrast, two of ten nonresponder patients became HBxAgpositive (20%), as did four of 24 (17%) untreated patients (Table 2). Five of the six patients who became HBxAg+ by ELISA over the course of 18 months also had HBV DNA in serum detectable after therapy. Verification of the presence of HBxAg in serum was carried out by immunoprecipitation with an anti-x monoclonal antibody, followed by SDS/PAGE, and finally by Western blotting using anti-x peptide antisera (27,34,39). Representative results are presented in Fig. 1. These findings imply that HBV reactivation in treated patients who responded to IFN was less likely to occur than in patients who were unresponsive to treatment or in untreated controls. These observations may reflect the repeating cycles Of Viral reactivation, each followed closely by an acute exacerbation of CAH, which occur in the course of natural infection (44,45). However, using HBxAg as the sole marker of reactivation, these results were not statistically significant. Further, there was no relationship between the HBxAg status in serum and liver pathology in treated cases or untreated controls, In summary, the results are consistent with HBV reactivation occurring in

B

A 123456

et al.

12345678

-

32 kDa

17kDa -

Ffg. 1. Verificationaf HE?xAg and anti-pol in serum from a-IF’N treated patients. (A) Serumsamplesscoring negative (lanes l-3) or positive (lanes 4-6) for HBxAg by ELISA were immunoprecipitated with a monoclianalanti-x antibody, as described (34,39). The precipitates were then analyzedby SDS/PAGE, followedby Western blotting using a mixture of anti-x peptide antibodies (27). (B) Serum samples scoring negative (lanes l-4) or positive (lanes 5-g) for anti-pol by E’LISA were tested to see whether they would immunoprecipitatean in vitro radiolabeled fragment of the polymerase polypeptide. The immunoprecipitation was carried out by mixing the tritiated polymerase polypeptide made in rabbit reticulocyte lysates with1the test serum, and collecting the precipitate with protein A from Staphylococcus aureus. The precipitate was then analyzed by SDS/PAGE and fluorography.

untreated patients and those unresponsive to IFN, but not in those responsive to therapy. However, it is clear that HBxAg, alone does not consistently or significantly distinguish bletween these groups. Previous observations from this laboratory have shown that anti-poll reflects ongoing virus replication (35,36), and that it often becomes undetectable after virus elimination (35,37), implying that this marker could be sensitive to reactivation following anti-viral chemotherapy. In the populations studied here, anti-pol was detected by ELISA in three responder patients (27%) prior to therapy, but was undetectable in all responder patients after therapy (Tabi’e 2). In contrast, anti-pol was detected among two no.nresponder patients after therapy and in 12 of 24 untreated controls within an l&month period of observation (Talble 2). These observations are consistent with the conclusion that successful IFN therapy results in the loss of anti-pol. However, the detection of anti-pol prior to treatment, as with HBxAg, provided no predictive value as to the outcome of therapy. As with HBxAg, the presence of anti-pol among nonresponder patients

EFFECTS

OF IFN ON HBxAg AND ANTI-POL

did not correlate with changes in liver pathology (Table 2). The presence of anti-pol was verified by testing whether human serum samples immunoprecipitated an in vitro radiolabeled polymerase polypeptide fragment as shown by SDS/PACE analysis (Fig. 1). The fact that anti-pol did not appear among patients who responded to therapy, and that HBV DNA levels did not increase in such individuals, suggests that no virus reactivation was occurring in responder patients 12 months after the end of therapy. However, using anti-pol as the sole marker of virus reactivation, the difference among responder and control groups was not statistically significant. To further test whether HBxAg and anti-pol together could be used to monitor the outcome of treatment, the detection of these markers prior to and after treatment were compared to the outcome of therapy. Prior to IFN treatent, there was no statistically significant difference in the frequency of both markers in patients who later responded to therapy compared to nonresponders or to untreated controls. However, when both markers of reactivation were considered together, the difference between responder and controls with regard to the distribution of both these combined markers after treatment was significant (p = 0.024). There was also a significant difference between the mean levels of V DNA in serum after (but not before) treatment in responders compared to nonresponders or untreated controls @ = 0.002; Table 2). These results suggest that the loss of HBxAg and anti-pol in responder patients closely correlated with successful IFN treatment. It is possible that additional studies with larger populations will identify HBxAg and anti-pol as markers useful in monitoring the outcome of anti-viral trials, at least with a-IFN. The presence of HDV in HBsAg carriers is often accompanied by diminished or undetectable markers of HBV replication in the serum and liver. Among the 13 HDV-infected patients treated with IFN, all had persistent HBsAg, HDV RNA, and anti-HD prior to therapy (Table 3). Each of these patients had undetectable HBV DNA by slot blot hybridization and were negative for HBeAg and HBxAg prior to treatment. However, 11 cases had detectable HBV DNA by PCR (Fig. 2). In addition, all patients had CAH and/or cirrhosis by liver biopsy. Immunohistochemical staining showed that all 13 patients were delta antigen-positive and HBcAg-negative in the liver, Hence, these patients were replicating HDV at high levels relative to HBV prior to treatment. Approx. 12 months after the end of treatment (38), all patients remained anti-HD-positive, but only four cases still had detectable HDV RNA in serum (Table 3). These results show that IFN treatment results in a decrease in HDV markers in both serum and liver. With regard to

291 123 G

rat10

8

ratlo:

A?

A-B

At3

567 --A B A B A B

2.7

0.20

16.6 neg0 83

0 031 12.1

(B.'A)

(B/A)

4

9

A-B

A-B

10 A-

11 A?

12 A?

13 AT

10.6

0.29

0.035

0.53

0.043

46 9

+

-

‘6?lg. 2. PCR results from HDV infected HBV carriers undergoing o-IFN therapy. Data presented are the Ethidium bromide stained bands from each patient (numbered as in Table 3) assayed prior to (A) and after (B) a-IFN treatment. Increased levels of HJ3V DNA in serum after IFN treatment are reflected by a B/A ratio of greater than 1.0, while decreased levels are reflected by a ratio less than 1.0. The ratios reported here are the avl-rage of three experiments; all of which yielded ratios within 10% for each patient.

HBV markers, two patients developed HBxAg after treatment. None of these patients acquired BeAg or detectable HBV DNA by slot blot hybridization. However, one of the two HBxAg+ patients demonstrated an increase in the levels of HBV DNA following treatment (Table 3, Fig. 2), which is consistent with reactivation. Among the seven patients who had detectable anti-pol prior to therapy, three lost this marker and the remaining four remained persistently positive. Two of the three patients who lost anti-pol after treatment also had decreased amounts of HBV DNA in serum by PCR (Fig. 2). Among the four cases who were persistently antipal+, HBV DNA remained detectable by PCR in all and actually increased in two cases (Table 3). Two other patients who developed anti-pol after IFN treatment demonstrated an increase in HBV DNA by PCR (Fig. 2). These results imply that anti-pol is a sensitive marker of HBV replication, and that it may signal ongoing HBV replication or reactivation following IFN therapy. In addition, there was an inverse relationship between anti-pol and HDV RNA in the serum of most patients after therapy, which again suggests that there is an inverse relationship between the levels of replication of these two agents. Exploratory data analysis showed this relationship to be statistically significant @ = 0.021), although larger numbers of patients need to be studied for a longer period of time after the end of therapy to verify these findings. These results suggest, however, that the disappearance of HDV markers after IFN therapy can result in the appearance of HBV markers (HBxAg and anti-pol) associated with reactivation. The patterns of HBxAg and anti-pol from HBV car-

L.LEGA

292

riers without HDV infection who responded to a-IFN treatment compared to controls support the hypothesis that virus reactivation does not appear to occur within a year after the end of a-IFN treatment, while one or both markers reappeared within the same time frame in many patients who were either unresponsive to IFN or untreated. In contrast, the patterns of HBxAg and anti-pol in patients with chronic HDV infection supports the hypothesis of an inverse relationship between HBV and HDV replication in many cases. Iu both populations, the patterns of HBxAg and anti-pol provide information about HBV reactivation which are not available by measuring other serological markers of infection, and in doing so may provide valuable information as to the course of natural infection and the course of anti-viral chemotherapy. Further studies with additional and larger populations will be needed to verify the utility of HBxAg

References 1 Tiollais P, Pourcel C, Dejean A. The hepatitis B virus. Nature 1985; 317: 489-95. 2 Blumberg BS, London WT. Hepatitis B virus: pathogenesis and prevention of Nrnary cancer of the liver. Cancer 1982; 50: 2657-65. 3 Hoofnagle JH, Sh,afritz DA, Popper H. Chronic type B hepatitis and the ‘healthy’ HBsAg carrier state. Hepatology 1987; 7: 758-63. 4 Baumgarten R. Virus replication in patients with chronic hepatitis B and cirrhosis. In: Zuckerman AJ, ed. Viral Hepatitis and Liver Disease. New York: Alan R. Liss, 1988; 195-7. 5 Alberti A.. Tremolada F, Fattovich G, Bortolotti F, Realdi G. Virus replication and liver disease in chronic hepatitis B virus infection. Dig Dis Sci 1983; 28: 962-6. 6 Nowoslawski A, Krawczynski K, Nazarewicz T, Slusarczyk J. Immunoparhological aspects of hepatitis type B. Am J Med Sci 1975; 270: 1229-39. 7 Rizzetto M. The delta agent. Hepatology 1982; 3: 729-37. 8 Hoofnagle JH. Type D (delta) hepatitis. J Am Med Assoc 1989; 261: 1321-5. 9 Kane1 GC, Govindarajan S, Peters RL. Chronic delta infection and liver biopsy changes in chronic active hepatitis B. Ann Intern Med 1984; 101: 51-4. 10 Colombo M, Cambieri R, Rumi MG, Ronchi G, del Ninno E, de Franchi R. Long-term delta superinfection in hepatitis B surface antigen carriers and its relationship to the course of chronic hepatitis. Gastroenterology 1983; 85: 235-9. 11 Wu J-C, Lee S-D, Tsay S-H, et al. Symptomatic anti-HBe positive chronic hepatitis B in Taiwan with special reference to persistent HBV replication and HDV superinfection. J Med Viral 1988; 25: 141-8. 12 Chen P-J, Chen D-S, Chen C-R, et al. 6 Infection in asymptomatic carriers of hepatitis B surface antigen: low prevalence of b activity and effective suppression of hepatitis B virus replication. Hepatology 1988; 8: 1121-4. 13 Hadziyannis SJ, Sherman M, Lieberman HM, Shafritz DA. Liver disease activity and hepatitis B virus replication in chronic delta antigen-positive hepatitis B virus carriers. Hepatology 1985;5:544-7. 14 Rizzetto M, Canese MG, Gerin GL, London WT, Sly DL, Purcell RH. Transmission of the hepatitis B virus-associated

et al.

and anti-pol as markers reflecting the course of natural infection and anti-viral chemotherapy.

Aclknowledgements The authors would wish to thank Dr. Edward Lustbader for carrying out the statistical analysis and Dr. W.T. London for critical reading of this manuscript. This work was supported by USPHS Grants CA48656, CA40737, RR-05895, and CA-06927 from the National Institutes of Health, by a Focused Giving Grant 830702 from Johnson and Johnson, and by an appropriation from the Commonwealth of Pennsylvania. Dr. Lega was a visiting scientist supported by the University of Florence, Florence, Italy. Dr. Minghua Zhu was a visiting scientist from the 4th Military Medical University, Xi’an, Shaanxi, China.

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Hepatitis B x antigen and polymerase antibodies in the serum of hepatitis B carriers with or without hepatitis delta virus infection. Effects of interferon treatment.

Previous work has shown that the hepatitis B x antigen (HBxAg) and antibodies directed against the polymerase of hepatitis B virus (anti-pol) are earl...
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