Journal of Hepatology, 1992; 16: 153-158
@1992 ElsevierScientificPublishers Ireland Ltd. All rights reserved. 0168-8278/92/$05.00 HEPAT01138
Intrahepatic expression of hepatitis B core and surface antigens in chronic hepatitis delta-virus infection Chia-Ming Chu and Yun-Fan Liaw Liver Unit, Chang Gung Memorial Hospital and Chang Gung Medical College, Taipei, Taiwan
(Received 18June 1991)
To evaluate the interference of hepatitis B virus (HBV) protein expression in the liver in chronic hepatitis delta-virus (HDV) infection, the intrahepatic expression of hepatitis B core and surface antigens (HBcAg and HBsAg) was studied in 36 HBsAg carriers who were seropositive for anti-HDV and in 36 anti-HDV negative controls 1-18 with serum hepatitis B e-antigen (HBeAg) and 18 with anti-HBe-I. Of 18 HBeAg-positive patients with anti-HDV, 12 had HDV antigen (HDAg) in the liver. HBcAg was positive in 66.7% (8/12) of the HBeAg-positive patients with HDAg in the liver, and in 94.4% (17/18) of controls (p = 0.14). The distribution of HBcAg was exclusively cytoplasmic in 75% (6/8) of HDV-infected patients, but was mixed nuclear and cytoplasmic in 70.6% (12/17) of the controls. The prevalence and quantitative expression of HBcAg in the nucleus, but not in the cytoplasm, were significantly decreased in chronic HDV infection. HBsAg was positive in 91.6% (11/12) of HBeAg-positive patients with HDV infection and in all controls. Membranous expression of HBsAg was detected less frequently in HDV-infected patients than in controls (7/12 vs. 17/18, p = 0.05), while the prevalence and quantitative expression of H BsAg in the cytoplasm showed little or no difference. HDAg was detected in all of the anti-HBe-positive patients with anti-HDV. Of these, none had HBcAg detectable in the liver, nor did controls, while HBsAg was detected exclusively in the cytoplasm in 94.4% (17/18). The prevalence and quantitative expression of HBsAg in the cytoplasm was not different for HDV-infected patients or controls. The results demonstrate: (1) decreased expression of nuclear HBcAg and membranous HBsAg in HBsAg carriers with chronic HDV infection, suggesting decreased levels of HBV replication and (2) that HDV has little or no effect on the cytoplasmic expression of HBsAg. The mechanisms regulating the expression of HBcAg and HBsAg in the liver in chronic HDV infection await further investigation. K e y words." Immunofluorescence; HBeAg; Anti-HBe; HDAg
It has been suggested that hepatitis delta virus (HDV) has a direct inhibitory effect on hepatitis B virus (HBV) replication. In acute HDV infection, a transient decrease in the expression of the HBV gene product (hepatitis B surface and core antigens, HBsAg and HBcAg) in liver as well as in serum has been described in chronic-HBVinfected chimpanzees and humans (1-3). In chronic HDV infection, most patients exhibit a low level of HBV replication (4,5). Furthermore, studies by Hadziyannis et al. (6) and Govindarajan et ai. (7) reveal that the prevalence of HBV-DNA in serum or HBcAg in the liver was significantly lower in patients with chronic HDV infection than in those without.
However, the results of a recent study have shown that a substantial proportion of hepatitis B e-antigen (HBeAg)-positive patients with chronic HDV infection have HBV-DNA detectable in serum at levels similar to HDV-negative controls. This suggests that HBV and HDV replication might coexist without mutual inhibition (8). Furthermore, a study by Farci et al. (9) has also demonstrated continuing high levels of serum HBVDNA in HBeAg-positive patients with chronic HDV infection, suggesting that HDV infection does not always inhibit HBV replication. Notably, most of the patients in Hadziyannis's (6) and Govindarajan's series (7) were positive for antibody to HBeAg (anti-HBe), and were
Correspondence to: Chia-MingChu, M.D., Liver Unit, Chang Gung Memorial Hospital, 199 Tung Hwa North Road,Taipei,Taiwan, 10591.
154 likely to be previously anti-HBe-positive, asymptomatic HBsAg carriers without active HBV replication (10). On the other hand, there are several possible etiologies for chronic liver disease in anti-H Be-positive patients (1 1,12), such as continued active HBV replication without serum HBeAg due to a point mutation of the precore region of the HBV genome (13), non-B viral superinfection or superimposed non-viral liver disease. Previous findings that the prevalence of HBV-DNA in serum or HBcAg in the liver is significantly lower in anti-HBe-positive patients with HDV infection than in patients without (6,7) might only reflect the different etiologies of chronic liver disease between these two categories of patients, although the possibility of HDV-induced inhibition of HBV replication cannot be excluded (14,15). Therefore, further studies to include more HBeAg-positive patients are of great importance. The distribution and quantitative expression of HBV gene products in the liver in chronic HDV infection has rarely been studied. To evaluate the interference of HBV protein expression in the liver by HDV infection, the intrahepatic expression of HBcAg and HBsAg was studied in 18 HBeAg-positive and 18 anti-HBe-positive patients with serum antibodies against HDV (anti-HDV), and the results were compared with anti-HDV negative controls. The anti-HDV negative controls for anti-HBepositive patients with HDV infection enrolled in the present study were asymptomatic HBsAg carriers with minimal histologic changes instead of those with chronic liver disease.
Materials and Methods
Patients The study material consisted of 4 groups of patients with clinicopathologically verified chronic type B hepati-
C.-M. CHU and Y.-F. LIAW tis: Group I, 18 HBeAg- and anti-HDV-positive patients with chronic active hepatitis (CAH); Group II, 18 HBeAg-positive but anti-HDV-negative patients with CAH; Group III, 18 anti-HBe- and anti-HDV-positive patients with CAH; and Group IV, 18 anti-HBe-positive and anti-HDV-negative patients with minimal histologic changes. All patients in the Groups I and III had been positive for anti-HDV for at least 6 months. Patients in Groups I and II were well matched for age, sex, histology and biochemical activity, while patients in the Groups III and IV were matched for age and sex. The histologic diagnosis of chronic hepatitis was made according to standard criteria . No patients were homosexuals or intravenous drug abusers, nor had they ever received antiviral or immunosuppressive therapy. Clinical and laboratory data for the patients studied are listed in Table 1.
Laboratory methods Serum AST and ALT were measured by sequential multiple autoanalysers. HBsAg, HBeAg, anti-HBe and anti-HDV were assayed using commercially available radioimmunoassay kits (Ausria-II, HBeAg-RIA, and anti-delta, Abbott Laboratories, Chicago, IL, U.S.A.). Detection of viral antigens in the liver Liver specimens were obtained by percutaneous needle biopsy with a Menghini needle. Fragments of specimens were snap-frozen in liquid-nitrogen-cooled isopentane and stored at - 7 0 ° C until use. Samples of the same biopsy specimens were also fixed in 10% formaldehyde and embedded in paraffin wax for routine histologic diagnosis. Five-micrometer cryostat sections were dried overnight at room temperature and fixed in carbon tetrachloride at 4°C for 10 min, followed by extensive
TABLE I Clinical and laboratory data of patients studied Group No. of cases Age" (yr) Sex (malel AST (IU/I)a 01< 40) ALT (IU/I)" 01< 40) I. HBeAg-positive CAH with serum anti-HDV 18 28 + 5 18 95 + 45 195+ 82 II. HBeAg-positive CAH without serum anti-HDV 18 29 + 7 18 99 + 46 198+ 72 111.Anti-HBe-positive CAH with serum anti-HDV 18 32+7 18 94+46 201 +91 IV. Anti-HBe-positive MHC without serum anti-HDV 18 33 + 8 18 28 + 15 44 + 20 ~Data were expressed as mean+ SD. HBeAg=hepatitis B e-antigen: anti-HDV=antibody against hepatitis delta virus: anti-HBe=antibody against HBeAg; CAH=chronic active hepatitis; MHC=minimal histologicchange.
HEPATOCYTEEXPRESSIONOF HBcAgAND HBsAgIN CHRONICHDV washing with phosphate-buffered saline (pH 7.2) before staining. HBcAg were detected by indirect immunofluorescence using rabbit anti-HBc (Dako Corporation, Santa Barbara, CA, U.S.A.), as described elsewhere (17). HBsAg was detected by indirect irnmunofluorescence using mouse monoclonal anti-HBs against the 'a' determinant of HBsAg (Institute of Immunology Co., Ltd., Tokyo, Japan), followed by fluorescein isothiocyanate (FITC)-conjugated rabbit anti-mouse immunoglobulin G (Jackson Immuno Research Laboratories, West Grove, PA, U.S.A.). HDV antigen (HDAg) was detected by direct immunofluorescence using FITC-labeled antiHDV, kindly supplied by Dr. Rizzetto, as reported before (18). The topographical relationship between HBcAg and HDAg distribution in the liver was evaluated by examining the same areas in serial sections.
Statistical analysis The significance of the difference between two means was assayed using the Student t-test, and that between two proportions was assayed using the chi-square test or Fisher's exact test. The difference in the number of hepatocytes positive for viral antigens between the two groups of patients was compared using the Wilcoxon rank sum test, and the difference between more than two groups of patients was compared using the KruskalWallis test (19).
HDAg was detected in the hepatocyte nuclei in 12 of 18 HBeAg-positive patients and in all of the 18 antiHBe-positive patients with serum anti-HDV, but in none of the 36 anti-HDV-negative controls. The distribution and quantitative expression of HBcAg and HBsAg in relation to the degree of hepatocyte display of HDAg in patients with serum anti-HDV are listed in Table 2. Of 18 HBeAg-positive patients with anti-HDV, 13 were HBcAg-positive and 17 were HBsAg-positive. The distribution and quantitative expression of HBcAg and HBsAg in patients with different degrees of HDAg display in the liver showed no significant difference. Examination of serial sections revealed that HBcAg and HDAg were mostly localized in separate areas of the specimens. None of the 18 anti-HBe-positive patients with serum anti-HDV were HBcAg-positive, while 17 had HBsAg detectable exclusively in the cytoplasm. The prevalence and quantitative expression of HBsAg in the cytoplasm of patients with different degrees of HDAg display in the liver also showed no significant difference.
The distribution and quantitative expression of HBcAg and HBsAg in 12 HBeAg-positive patients with HDAg in the liver and in 18 HBeAg-positive controls are summarized in Table 3. HBcAg was positive in 8 (66.7%) of 12 patients with HDV infection and in 17 (94.4%) of 18 controls (p=0.14 by Fistier's exact test). Among patients with HDV infection, the distribution of HBcAg was exclusively cytoplasmic in 75% (6/8) and mixed nuclear and cytoplasmic in 25% (2/8), whereas in controls it was mixed nuclear and cytoplasmic in 70.6% (I 2/17) and exclusively cytoplasmic in 29.4% (5/17). The prevalence and quantitative expression of HBcAg in the nucleus, but not in the cytoplasm, was significantly decreased in HDV infection. HBsAg was positive in 11 (91.6%) of 12 patients with HDV infection and in all of 18 controls. Diffuse membranous expression of HBsAg was noted less frequently in patients with chronic HDV infection than in controls (7/12 vs. 17/18, p=0.05, by Fisher's exact test), while the prevalence and quantitative expression of HBsAg in the cytoplasm showed little or no difference. The membranous expression of HBsAg correlated with the presence of HBcAg in the liver: i.e., 24 of 25 patients with HBcAg had membranous expression of HBsAg, whereas none of the 5 patients without HBcAg did so (p=0.0008 by Fisher's exact test). All patients who were seropositive for both HBeAg and anti-HDV with or without HBcAg in the liver were persistently HBeAg-positive on follow-up for at least 6 months. The distribution and quantitative expression of HBcAg and HBsAg in anti-HBe-positive patients with and without HDV infection are summarized in Table 4. None of the 18 anti-HBe-positive patients with chronic HDV infection had detectable HBcAg and membranous HBsAg in the liver, nor did the 18 anti-HDV-negative controls. The cytoplasmic expression of HBsAg in the liver also showed no quantitative difference between them.
In support of previous observations that 68.4-100% of HBeAg-positive patients with HDV superinfection were HBV-DNA-positive in serum [8,9-1, about 70% of the HBeAg-positive patients with chronic HDV infection in the present series had HBcAg detectable in liver. Although the expression of HBcAg in the liver was less prevalent in patients with chronic HDV infection than in controls, the difference did not reach statistical significance (Table 3). The observation that many HBeAg-
C.-M. CHU and Y.-F. LIAW
positive patients with chronic HDV infection had simultaneous expression of HBcAg and HDAg might suggest that in certain patients HBV and HDV replication can coexist without mutual inhibition. However, the prevalence of HBcAg showed a trend towards a reverse correlation with the degrees of HDAg display in the liver (Table 2), although the difference was not significant possibly due to the small number of patients. Further studies of a larger series of patients are mandatory. Furthermore, examination in serial sections revealed that HBcAg and HDAg were mostly distributed in separate areas of the specimens, suggesting that HBV and HDV rarely replicate in the same cells. Whether the localization of HBcAg and HDAg is mutually exclusive also needs further investigation by double immunostaining. Notably, in the present series all HBeAg-positive patients with chronic HDV infection and no detectable HBcAg in the liver using conventional immunohistochemical techniques had persistent HBeAg in the serum on follow-up for at least 6 months, while it has been shown that HBeAg-positive patients without HBcAg in the liver or HBV-DNA in the serum usually clear HBeAg from their serum quite quickly 1-20,21]. This would suggest, that the presence of low levels of viral replication in these patients cannot be completely excluded. Further studies of the more sensitive markers of HBV replication, such as serum HBV-DNA using the polymerase chain reaction, are required. Perhaps the most interesting finding in the present
study is that the distribution of HBcAg was exclusively cytoplasmic in the majority of patients with chronic HDV infection, while, as in previous observations (17,22,23), the nuclear expression of HBcAg in combination with cytoplasmic HBcAg occurred in most HBeAg-positive controls. Moreover, the quantitative expression of HBcAg was significantly decreased in chronic HDV infection (Table 3) in the nucleus, but not in the cytoplasm. The clinical implication of the decreased nuclear expression of HBcAg in chronic HDV infection remains unclear. It has been shown that the intrahepatic distribution of HBcAg in chronic HBV infection correlates closely with the levels of HBV replication, with the highest levels of serum HBV-DNA in patients with predominantly nuclear HBcAg, with the lowest levels in patients with predominantly cytoplasmic HBcAg, and with the middle levels in those with mixed nuclear and cytoplasmic HBcAg 1-24]. The decreased nuclear expression of HBcAg in chronic HDV infection might therefore suggest a decreased level of HBV replication. This hypothesis needs to be confirmed by a serial follow-up study of serum levels of HBV-DNA in patients with HDV infection and controls. Furthermore, studies in cultured cells have revealed that pre-core proteins can be transported into the nucleus after removal of its signal sequence, while core proteins remain exclusively in the cytoplasm. It has also been suggested that a possible function of the precore protein might be to transport daughter viral genomes from the cytosol to the nucleus, where they can
TABLE 2 Intrahepatic expression of HBcAg and HBsAg in relation to HDAg expression in anti-HDV-positive chronic type B hepatitis Viral antigens in liver HBcAg-positive Nuclear 0 I+ 2+ 3+ 4+ Cytoplasmic 0 1+
2+ 3+ 4+ H BsAg-positive Membranous Cytoplasmic 0
HBeAg-positive cases with liver HDAg
Anti-HBe-positive cases with liver HDAg
1+ ( n = 4 )
2 + ( n = 10)
5(83.3%) 2(33.3%) 4 2 0 0 0 5(83.3%) 1
3(75%) 1(25%) 3 0 I 0 0 3(75%) I
5(62.5%) 1(12.5%) 7 I 0 0 0 5(62.5%) 3
0(0%) 0(0%) 4 0 0 0 0 0(0%) 4
0(0%) 0(0%) 10 0 0 0 0 0(0%) 10
0(0%) 0(0%) 4 0 0 0 0 0(0%) 4
3 1 0 6(100%) 4(66.7%) 6(100%) 0
1 I 0 4(100%) 3(75%) 4(100%) 0
3 1 0 7(87.5%) 4(50%) 7(87.5%) l
0 0 0 3(75%) 0(0%) 3(75%) l
0 0 0 10(100%) 0(0%) 10(100%) 0
0 0 0 4(100%) 0(0%) 4(10%) 0
2+ 3+ 4+
3 1 0
2 0 0
2 1 0
0 2 1
3 3 3
1 2 1
HBcAg = hepatitis B core antigen; HBsAg= hepatitis B surface antigen; HDAg= hepatitis delta virus antigen. For other abbreviations, see Table 1. Scale of 0 to 4 +, corresponding to positivity in 0%, 1-10%, 11-25%, 26-50%, > 50% of total hepatocytes examined.
HEPATOCYTE EXPRESSION OF HBcAg A N D HBsAg IN C H R O N I C HDV
TABLE 3 lntrahepatic expression of HBcAg and HBsAg in HBeAg-positive patients with and without HDV superinfection Viral antigens in liver H BcAg-positive Nuclear 0 1+
2+ 3+ 4+ Cytoplasmic 0 I+ 2+ 3+ 4+ H BsAg-positive Membranous Citoplasmic 0 1+ 2+ 3+ 4+
with (n = 12)
without (n= 18)
8(66.6%) 2(16.7%) 10
17(94.4%) 12(66.7%) 6
1 0 0 8(66.6%) 4 2 4 2 0 11(91.6%) 7(58.3%) 1 I(91.6%) I 6 4 I 0
n.s. < 0.05
4 1 1 17(94.4%) I 3 9 3 2 18(100%) 17(94.4%) 181100%) 0 8 6 2 1
< 0.05 n.s.
n.s. n.s. 0.05 n.s.
n.s.=non-significant (p>0.05). For further explanation of abbreviations, see Tables I and 2. For explanation of scale of 0 to 4 + , see Table 2.
direct the synthesis of additional viral RNA and increase the number of progeny particles (25). Whether there is selective inhibition of pre-core gene expression and therefore a diminished expression of nuclear HBcAg and a diminished level of HBV replication in chronic HDV infection requires further study. In agreement with previous observations (9,11,26), none of the anti-HBe-positive patients with chronic HDV infection in the present series had detectable HBcAg in the liver. These findings might support the suggestion that most of these patients were likely to be previously anti-HBe-positive asymptomatic HBsAg carriers without active HBV replication (10), or they might be due to the suppression of HBV replication by HDV. With regard to the intrahepatic expression of HBsAg in chronic HDV infection, the present results have revealed that the membranous expression of HBsAg is
significantly suppressed compared to HDV-negative controis (Table 2). It has been recognized that in chronic HBV infection the membranous expression of HBsAg correlates closely with active HBV replication (17,22,23). The significant decrease in the membranous expression of HBsAg in chronic HDV infection might also suggest a decreased level of HBV replication. On the other hand, it has been suggested that the pre-S sequence of the S-gene might play a role in the membranous expression of HBsAg (27). It remains uncertain whether the diminished membranous expression of HBsAg in chronic HDV infection is due to the selective inhibition of preS gene expression by HDV. Further studies on the expression of pre-S antigens in the liver in chronic HDV infection are necessary. Another important finding in the present study was that compared to HDV-negative controls the cytoplasmic expression of HBsAg in chronic
TABLE 4 Intrahepatic expression of HBcAg and HBsAg in anti-HBe-positive patients with and without HDV superinfection Viral antigens in liver HBcAg-positive H BsAg-positive Membranous Cytoplasmic 0 I+ 2+ 3+ 4+
with (n = 18)
without (n = 18)
0(0%) 17(94.4%) 0(0%) 17(94.4%) 1
0(0%) 18(100%) 0(0%) 18(100%) 0
4 7 5
3 9 4
n.s. n.s. n.s. n.s. n.s.
n.s.--non-significant (p >0.05). For further explanation of abbreviations, see Tables 1 and 2. For explanation of scale of 0 to 4 + , see Table 2.
158 HDV infection showed little or no quantitative change (Tables 3 and 4). These findings may be compatible with the biologic characteristics of HDV. It appears that, since HDV requires the helper function of HBsAg to enter and exit from the hepatocytes (28), it does not interfere with the expression of the S-gene product in the liver. In summary, the present results demonstrate that in HBsAg carriers with chronic HDV infection the intrahepatic expression of nuclear HBcAg and membranous HBsAg is significantly inhibited. In contrast, HDV has little or no effect on the cytoplasmic expression of
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Acknowledgements This study was supported by a grant from the National Science Council of R.O.C. (NSC 78-0419-B182-09). The authors thank W.C. Shyu, C.C. Wang and C.H. Lee for their technical assistance, and M.H. Tsai and L.F. Yao for their secretarial assistance.
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