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Expression of preS1 and preS2 in the liver of chronic hepatitis B virus carriers
JournalofHepatology. 1988; 7:157-163
157
Elsevier HEP 00442
Expression of preS1 and preS2 in the liver of chronic hepatitis B virus carriers
A. Fraiese, P. Pontisso, D. Cavalletto, G. Fattovich and A. Alberti Institute of Clinical Medicine, Clinica Medica I1, University of Padua, Padua (Italy)
(Received 17 December 1987) (Accepted 21 April 1988)
Summary Using monoclonat antibodies we have studied the expression and distribution pattern of preS1 and preS2 proteins in the liver of 25 patients with chronic hepatitis B virus infection. Both preS1 and preS2 were detected in the liver of most cases, independently of viral replication and of the state of the viral genome in the liver. In fact, preS1 and preS2 were present in the liver of 14 out of 15 patients with H B V - D N A in the serum as well as of 7 out of 10 cases with no evidence of viral replication, including 2 with integrated H B V - D N A sequences in the liver and 5 patients with no evidence of liver damage. The pattern of distribution of preS proteins was identical to that of hepatitis B surface antigen, with exclusive cytoplasmic staining in cases without viral replication and cytoplasmic and membranous positivity in those with viral replication. These results clearly indicate that translation of preS proteins is independent of viral replication and is not necessarily associated with liver damage.
Introduction Three distinct polypeptides are expressed on the hepatitis B virus envelope, being encoded by the preS and S regions of the viral genome, which contains three independent domains [1,2]. The large HBsAg polypeptide (P39/GP42) is encoded by the combined preS1/preS2/S subregions, while the combined preS2/S subregions code for the middle protein (GP33/GP36) and the S gene alone for the major HBsAg (P24/GP27) polypeptide,
Early studies indicated that the preS sequences, particularly preS1, are abundant on viral particles in viremic sera, while their expression is trivial in sera of HBsAg carriers without evidence of viral replication [2,3]. Recent data, however, have not confirmed these associations [4]. The role of preS proteins in the biology of the virus has not yet been clarified, but it has been suggested that they could play a role in virus assembly [5] and attachment to hepatocytes [6]. It has been proposed that preS synthesis in the liver and/or its secretion
Correspondence: Don. A. Alberti, lstituto di Medicina Clinica, Clinica Medica II, Via Giustiniani 2, 35100 Padova, Italy.
0168-8278/88/$03.50 (~) 1988 Elsevier Science Publishers B.V. (Biomedical Division)
158 into the circulation could be linked to HBV replication. However, Thung et al. [7] have recently reported the presence of preS1 and preS2 in the liver of carriers without liver HBcAg_ To define further the expression of preS1 and preS2 in liver and serum in relation to viral replication, we have studied by immunofluorescence with monoclonal antibodies liver biopsies from HBsAg carriers who either were positive for serum HBVDNA and had replicative HBV-DNA forms in the liver, or were negative for serum HBV-DNA and had integrated HBV-DNA sequences in the liver.
Materials and Methods
Patients with chronic HBV infection Liver biopsies were obtained from 25 chronic HBsAg carriers with different serologic profiles and histologic diagnoses. Ten patients were HBeAg-positive and HBV-DNA-positive in serum with histologic evidence either of chronic persistent hepatitis (4 cases) or of chronic active hepatitis (6 cases). Five additional patients (all with chronic active hepatitis on liver biopsy) were anti-HBe-positive in serum but • had evidence of ongoing viral replication, showing HBV-DNA serum positivity. The remaining 10 cases were anti-HBe-positive and HBV-DNA-negative in serum at the time of this study, and included 5 patients who had seroconverted from HBeAg to antiHBe 3-5 years previously and showed normal liver enzymes and chronic persistent hepatitis on liver biopsy, and 5 patients who had normal liver histology and were known as 'healthy' HBsAg carriers with normal transaminase levels from the first observation. Liver biopsies from 10 patients with various liver disorders, unrelated to HBV infection, were used as controls.
A. FRAIESE et al. dization, as previously described in detail [8], and its concentration was expressed semiquantitatively in a range from (-) to (+ + + +).
Serum levels of preSl and preS2 The expression of preS1 and preS2 epitopes on viral particles in serum was investigated by solid-phase RIA, using monoclonal antibodies [2] kindly provided to us by Professor W. Gerlich_ The monoclonal antibodies Q 19/10 (anti-preS2) and Ma 18/7 (anti-preS1) were coated to a solid phase at 10ktg/ml in carbonate buffer. Sera were then incubated at a dilution of 1:1000 in PBS-Tween 20 and, after several washings, viral particles bound to solidphase anti-preS were revealed with radiolabelled anti-HBs (125I-anti-HBs, Sorin Biomedica, Saluggia, Italy). The negative range of the assay was defined with a panel of 25 normal sera from HBsAg-negative individuals and the results were expressed as P/N ratio.
Detection of preS1 and preS2 in the liver Cryostatic liver sections were incubated with monoclonal anti-preSl or anti-preS2 antibodies (0.5 ,ug/ml) for 45 min at room temperature. Parallel sections were studied for HBsAg using monoclonal antibody to the 'a' common determinant of HBsAg and for HBcAg using our standard fluorescein-conjugated anti-HBc antiserum [9]. After incubation with different monoclonal antibodies, sections were washed and incubated with fluorescein-conjugated anti-mouse Ig serum (Behring Institut, Behringwerke AG, Marburg, F.R.G.). When the expression of preS epitopes was to be compared with HBcAg expression in the same liver biopsy, sections were first incubated with fluorescein-conjugated anti-HBc, followed by monoclonal anti-preS and by a rhodamine-conjugated anti-mouse Ig serum.
Serologic assessment
State of HBV-DNA in the liver
All patients were tested for HBsAg, HBeAg and anti-HBe and for anti-HDV in serum using commercial kits (Abbott Laboratories, Chicago). HBV-DNA was measured in serum by spot hybri-
In order to investigate whether preS expression in the liver is related to a particular state of the viral genome (free or integrated), liver biopsies from 8 patients, positive for preS1 and for preS2 by immuno-
LIVER PRE-SI AND PRE-S2 IN HBV INFECTION fluorescence and including 4 HBeAg-positive and 4 anti-HBe-positive cases, were studied by the Southern blot technique, as described recently in detail
159 expression of preS antigens in the liver of HDV-infected patients will be reported elsewhere (manuscript in preparation).
[101.
Pattern of preS expression in the liver and relation to HBsAg and HBcAg Results
PreS antigens in the liver No staining was observed with the monoclonal anti-preS1 and anti-preS2 antibodies in liver biopsies from HBsAg-negative cases. The results of preS1 and preS2 testing of liver biopsies from HBsAg-positive cases are described in Table 1. All 10 HBeAg-positive and H B V - D N A positive cases, as well as 4 of the 5 anti-HBe-positive and HBV-DNA-positive cases, were found to be positive for preS1 and preS2 antigens in the liver. One case, which was anti-HBe-positive and H B V - D N A positive in serum, had no evidence of expression of preS antigens in th.e liver, being the only case in this group with no H B s A g in the liver. PreS1 and preS2 reactivities were detected also in the liver of 7 out of 10 anti-HBe-positive cases without signs of viral replication (HBV-DNA-negative in serum)_ There was again a clear relation with the expression of H B s A g in the hepatocytes, as preS1 and preS2 were detected in 7 out of 8 HBsAg-positive biopsies and were absent in both HBsAg-negative biopsies. None of the 25 patients with chronic H B V infection was anti-HDV-positive in serum or HDAg-positive in the liver by immunofluorescence. Data on the
The distribution patterns of immunofluorescence staining with anti-preS1 and anti-preS2 monoclonal antibodies were almost identical in individual biopsies and were also similar to the staining pattern obtained with anti-HBs monoclonal antibody, although there were significant variations in intensity of positivity (Table 1). All biopsies positive for preS contained both preS1 and preS2 and also stained for HBsAg. As mentioned, a single biopsy from a patient with no viral replication was HBsAg-positive while preS-negative. In cases with H B V - D N A in serum, either HBeAg-positive or anti-HBe-positive, both cytoplasmic and membranous preS and S staining was observed_ In 5 biopsies, preS2 staining was most prominent (Fig. 1), particularly at the cell periphery, while in 2 other biopsies the signal was stronger for preS1 (Fig. 2), and in 7. biopsies preS1, preS2 and S staining were similar in pattern and intensity. In patients who were HBV-DNA-negative in serum, cytoplasmic positivity for preSI, preS2 and HBsAg was observed without membranous staining of hepatocytes (Fig. 3). Here, preS2 staining was often predominant (4 out of 7 cases had a strong preS2 cytoplasmic signal with a much weaker preS1 positivity), while preS1 staining was always the faintest and was barely detectable in 2 cases.
TABLE I HBV ENVELOPE PROTEINS (HBsAg, PRE-SI, PRE-S2) IN THE LIVER OF PATIENTS WITH CHRONIC INFECTION, AND RELATION TO SEROLOGIC PROFILE Patterns of immunofluorescencestaining with monoclonal antibodies (No. cases) Serologic profile
HBsAg++ preSl++ preS2+ +
HBsAg++ preSl++ preS2+
HBsAg++ PreSl+ preS2+ +
HBsAg+ + preS1preS1-
HBsAgpreSlpreS2-
HBeAg-positive ( 10 cases) Anti-HBe-positive, HBV-DNA-positive (5 cases) Anti-HBe-positive. HBV-DNA negative (10 cases)
6 1 3
1 1
3 2
0 0
0 1
0
4
1
2
++ = strongly positive; + = weakly positive;- = negative_
160
A. F R A I E S E et al.
Fig. I. Detection of preS2 by indirect immunofluorescence in the liver of an HBeAg-positive carrier. Strong membranous staining is seen associated with cytoplasmic staining.
Fig. 2. PreSl in the liver of an HBeAg-positive carrier.
LIVER PRE-S1 AND PRE-S2 IN HBV INFECTION
161
Fig. 3. Cytoplasmic staining with anti-preS2 monoclonal antibody in the liver of a 'healthy" HBsAg carrier who had no evidence of viral replication and showed the presence of integrated HBV-DNA sequences in the liver.
H B c A g was detected by direct immunofluorescence in the liver of all HBeAg-positive or anti-HBepositive cases who were positive in serum for HBVD N A by spot hybridization. Five patients had a diffuse H B c A g positivity (more than 70% of hepatocytes were positive for H B c A g ) , while 10 cases had focal H B c A g positivity. In general, HBcAg-positive biopsies had a stronger preS1 and preS2 fluorescence compared to HBcAg-negative biopsies, but the only absolute difference was the membranous pattern of preS antigens, which was seen exclusively in HBcAgpositive biopsies. By double immunofluorescence, no definite association of H B c A g and of preS antigens within the same liver cell was observed_ In fact, HBcAg was only occasionally detected in the nuclei of liver cells with cytoplasmic preS and S while it was frequently seen in liver cells with membranous preS and S staining, particularly in those cases with a more diffuse positivity.
PreS expression in liver biopsies with free or integrated HB V-DNA sequences Eight liver biopsies positive for preS1 and preS2 by immunofluorescence were studied by Southern blot for H B V - D N A seq,uences, as previously described [10]. Four biopsies were from HBeAg-positive cases and showed the typical replication pattern with a 3.2 kb band and a smear of replicative intermediates below. The other 4 biopsies came from anti-HBe-positive, HBV-DNA-negative cases and did not contain free H B V - D N A sequences. Two biopsies contained integrated H B V - D N A sequences with a sharp 11 kb band after HindllI digestion in one case and evidence of ' r a n d o m ' integration in the other.
Discussion
This study clearly demonstrates that preS1 and
162 preS2 sequences are expressed and accumulate in the cytoplasm of hepatocytes in most H B s A g chronic carriers, independently of viral replication activity. These findings extend recent observations of Thung and co-workers [7] and clearly establish that preS polypeptides are certainly also present in the liver of anti-HBe-positive ~healthy' carriers with integrated H B V - D N A sequences and no evidence of viral replication activity or of liver damage. This observation is at variance with our previous results obtained in a limited series of biopsies with human anti-preS antisera, which were found to stain only liver sections from patients with ongoing viral replication [11]. These discrepancies may reflect different specificities of human vs. monoclonal antipreS antibodies_ PreS2 expression in most biopsies was much stronger than preS1 expression, both in the cytoplasm and at the liver cell periphery, particularly in anti-HBe-positive, H B V - D N A - n e g a t i v e carriers. Large amounts of preS1 reactivity were in fact detected only in a few biopsies from H B e A g - p o s i t i v e cases with ongoing viral replication, suggesting that translation of the large envelope protein is more closely related to viral replication than that of the middle and major H B s A g proteins. This would be in agreement with the data of Klinkert et al. [3] obtained using the Western blot technique when comparing liver biopsies from viremic and non-viremic carriers, and would also explain further the results obtained with human antisera which contained mainly the anti-preS1 specificity. No relation was seen between the pattern of expression of preS antigens in the liver and activity of hepatitis, although m e m b r a n e staining was seen
References 1 Tiollais P, Charnay P, Vyas GN. Biology of the hepatitis B virus. Science 1981 ; 213:406-411. 2 Heermann KH, Goldmann U, Schwartz W, et al. Large surface proteins of hepatitis B virus containing the pre-S sequence. J Virol 1984; 52: 396-402. 3 Klinkert M-Q, Theilmann L, Pfaff E, Schaller H. Pre-Sl antigens and antibodies early in the course of acute hepatitis B virus infection. J Virol 1986; May: 522-525.
A. FRAIESE et al. mainly in patients with viral replication, as previously described for H B s A g reactivity [12]. It has been suggested that preS polypeptides, particularly preS1, could accumulate in the liver in the absence of complete production of the virus, on the basis of the inhibitory effect of preS1 on H B s A g secretion seen in cell lines not supporting viral replication [13] and of results obtained in the transgenic mouse model [14]. H o w e v e r , we did not find extensive accumulation of preS1 in the cytoplasm of hepatocytes of patients without H B V - D N A in serum, and serum preS1 reactivity was detected in several of these cases, although levels were significantly lower than those found in viremic sera. In conclusion, p r e S l and preS2 are both expressed in the liver of H B s A g carriers, i n d e p e n d e n t l y of viral replication, and are also secreted in the circulation, the absolute level being lower in anti-HBe-positive, H B V - D N A - n e g a t i v e cases, probably as a consequence of a lower concentration of viral particles rather than a lower rate of secretion, These results, however, do not contradict the possibility that in some H B s A g carriers accumulation of viral envelope proteins in the cytoplasm of hepatocytes could d e t e r m i n e morphologic changes and cell degeneration, but this remains to be proven by longitudinal studies of these cases.
Acknowledgement The authors are deeply grateful to Prof. W. Gerlich for the monoclonal antibodies used in this study.
4 Pei-Sheng H, Carithers RL. Fiorenza V, Peterson DL. Quantitative studies of the hepatitis B viral pre-S protein: lack of correlation with the HBeAg status. J Virol Methods 1987; 16: 97-114. 5 Ganem D, Varmus HE. The molecular biology of the hepatitis B viruses. Annu Rev Biochem 1987: 56:651-693. 6 Neurath AR, Kent SBH, Strick N, Parker K. Identification and chemical synthesis of a host cell receptor binding site on hepatitis B virus. Cell 1986; 46: 429-436. 7 Thung SN. Gerber MA, Kasambalides E J, et al. Demon-
LIVER PRE-S1 AND PRE-S2 IN HBV INFECTION
8
9
10
11
stration of pre-S polypeptides of hepatitis B virus in infected livers. Hepatology 1986; 6: 1315-1318. Pontisso P, Bortolotti F, Schiavon E, et al. Serum hepatitis B virus DNA in acute hepatitis type B. Digestion 1986; 34: 46-50. Alberti A, Tremolada F, Bortolotti F, et al. Virus replication and liver disease in chronic hepatitis B virus infection. Dig Dis Sci 1983; 28: 962-966. Pontisso P, Chemello L, Fattovich G, et al. Relationship between HBcAg in serum and in liver and HBV replication in patients with HBsAg positive chronic liver disease. J Med Virol 1985; 17: 145-152. Alberti A, Pontisso P, Fraiese A, et al. The preS/anti-preS
163 systems in hepatitis B virus infection. In: Robinson W, Koike K, Will H, eds. Hepadna Viruses. New York: Alan Liss, Inc., 1987; 397-410. 12 Gudat F, Bianchi L, et al. Pattern of core and surface expression in liver tissue reflects state of specific immune response in hepatitis B. Lab Invest 1975; 32: 101-107. 13 McLachlan A, Milich DR, Raney AK, et al. Expression of hepatitis B virus surface and core antigens: influences of pre-S and precore sequences. J Virol 1987; 61: 683-692. 14 Chisari FV, Filippi P, McLachlan A, et al. Expression of hepatitis B virus large envelope polypeptide inhibits hepatitis B surface antigen secretion in transgenic mice. J Virol 1986; 60: 880-887.
157
Elsevier HEP 00442
Expression of preS1 and preS2 in the liver of chronic hepatitis B virus carriers
A. Fraiese, P. Pontisso, D. Cavalletto, G. Fattovich and A. Alberti Institute of Clinical Medicine, Clinica Medica I1, University of Padua, Padua (Italy)
(Received 17 December 1987) (Accepted 21 April 1988)
Summary Using monoclonat antibodies we have studied the expression and distribution pattern of preS1 and preS2 proteins in the liver of 25 patients with chronic hepatitis B virus infection. Both preS1 and preS2 were detected in the liver of most cases, independently of viral replication and of the state of the viral genome in the liver. In fact, preS1 and preS2 were present in the liver of 14 out of 15 patients with H B V - D N A in the serum as well as of 7 out of 10 cases with no evidence of viral replication, including 2 with integrated H B V - D N A sequences in the liver and 5 patients with no evidence of liver damage. The pattern of distribution of preS proteins was identical to that of hepatitis B surface antigen, with exclusive cytoplasmic staining in cases without viral replication and cytoplasmic and membranous positivity in those with viral replication. These results clearly indicate that translation of preS proteins is independent of viral replication and is not necessarily associated with liver damage.
Introduction Three distinct polypeptides are expressed on the hepatitis B virus envelope, being encoded by the preS and S regions of the viral genome, which contains three independent domains [1,2]. The large HBsAg polypeptide (P39/GP42) is encoded by the combined preS1/preS2/S subregions, while the combined preS2/S subregions code for the middle protein (GP33/GP36) and the S gene alone for the major HBsAg (P24/GP27) polypeptide,
Early studies indicated that the preS sequences, particularly preS1, are abundant on viral particles in viremic sera, while their expression is trivial in sera of HBsAg carriers without evidence of viral replication [2,3]. Recent data, however, have not confirmed these associations [4]. The role of preS proteins in the biology of the virus has not yet been clarified, but it has been suggested that they could play a role in virus assembly [5] and attachment to hepatocytes [6]. It has been proposed that preS synthesis in the liver and/or its secretion
Correspondence: Don. A. Alberti, lstituto di Medicina Clinica, Clinica Medica II, Via Giustiniani 2, 35100 Padova, Italy.
0168-8278/88/$03.50 (~) 1988 Elsevier Science Publishers B.V. (Biomedical Division)
158 into the circulation could be linked to HBV replication. However, Thung et al. [7] have recently reported the presence of preS1 and preS2 in the liver of carriers without liver HBcAg_ To define further the expression of preS1 and preS2 in liver and serum in relation to viral replication, we have studied by immunofluorescence with monoclonal antibodies liver biopsies from HBsAg carriers who either were positive for serum HBVDNA and had replicative HBV-DNA forms in the liver, or were negative for serum HBV-DNA and had integrated HBV-DNA sequences in the liver.
Materials and Methods
Patients with chronic HBV infection Liver biopsies were obtained from 25 chronic HBsAg carriers with different serologic profiles and histologic diagnoses. Ten patients were HBeAg-positive and HBV-DNA-positive in serum with histologic evidence either of chronic persistent hepatitis (4 cases) or of chronic active hepatitis (6 cases). Five additional patients (all with chronic active hepatitis on liver biopsy) were anti-HBe-positive in serum but • had evidence of ongoing viral replication, showing HBV-DNA serum positivity. The remaining 10 cases were anti-HBe-positive and HBV-DNA-negative in serum at the time of this study, and included 5 patients who had seroconverted from HBeAg to antiHBe 3-5 years previously and showed normal liver enzymes and chronic persistent hepatitis on liver biopsy, and 5 patients who had normal liver histology and were known as 'healthy' HBsAg carriers with normal transaminase levels from the first observation. Liver biopsies from 10 patients with various liver disorders, unrelated to HBV infection, were used as controls.
A. FRAIESE et al. dization, as previously described in detail [8], and its concentration was expressed semiquantitatively in a range from (-) to (+ + + +).
Serum levels of preSl and preS2 The expression of preS1 and preS2 epitopes on viral particles in serum was investigated by solid-phase RIA, using monoclonal antibodies [2] kindly provided to us by Professor W. Gerlich_ The monoclonal antibodies Q 19/10 (anti-preS2) and Ma 18/7 (anti-preS1) were coated to a solid phase at 10ktg/ml in carbonate buffer. Sera were then incubated at a dilution of 1:1000 in PBS-Tween 20 and, after several washings, viral particles bound to solidphase anti-preS were revealed with radiolabelled anti-HBs (125I-anti-HBs, Sorin Biomedica, Saluggia, Italy). The negative range of the assay was defined with a panel of 25 normal sera from HBsAg-negative individuals and the results were expressed as P/N ratio.
Detection of preS1 and preS2 in the liver Cryostatic liver sections were incubated with monoclonal anti-preSl or anti-preS2 antibodies (0.5 ,ug/ml) for 45 min at room temperature. Parallel sections were studied for HBsAg using monoclonal antibody to the 'a' common determinant of HBsAg and for HBcAg using our standard fluorescein-conjugated anti-HBc antiserum [9]. After incubation with different monoclonal antibodies, sections were washed and incubated with fluorescein-conjugated anti-mouse Ig serum (Behring Institut, Behringwerke AG, Marburg, F.R.G.). When the expression of preS epitopes was to be compared with HBcAg expression in the same liver biopsy, sections were first incubated with fluorescein-conjugated anti-HBc, followed by monoclonal anti-preS and by a rhodamine-conjugated anti-mouse Ig serum.
Serologic assessment
State of HBV-DNA in the liver
All patients were tested for HBsAg, HBeAg and anti-HBe and for anti-HDV in serum using commercial kits (Abbott Laboratories, Chicago). HBV-DNA was measured in serum by spot hybri-
In order to investigate whether preS expression in the liver is related to a particular state of the viral genome (free or integrated), liver biopsies from 8 patients, positive for preS1 and for preS2 by immuno-
LIVER PRE-SI AND PRE-S2 IN HBV INFECTION fluorescence and including 4 HBeAg-positive and 4 anti-HBe-positive cases, were studied by the Southern blot technique, as described recently in detail
159 expression of preS antigens in the liver of HDV-infected patients will be reported elsewhere (manuscript in preparation).
[101.
Pattern of preS expression in the liver and relation to HBsAg and HBcAg Results
PreS antigens in the liver No staining was observed with the monoclonal anti-preS1 and anti-preS2 antibodies in liver biopsies from HBsAg-negative cases. The results of preS1 and preS2 testing of liver biopsies from HBsAg-positive cases are described in Table 1. All 10 HBeAg-positive and H B V - D N A positive cases, as well as 4 of the 5 anti-HBe-positive and HBV-DNA-positive cases, were found to be positive for preS1 and preS2 antigens in the liver. One case, which was anti-HBe-positive and H B V - D N A positive in serum, had no evidence of expression of preS antigens in th.e liver, being the only case in this group with no H B s A g in the liver. PreS1 and preS2 reactivities were detected also in the liver of 7 out of 10 anti-HBe-positive cases without signs of viral replication (HBV-DNA-negative in serum)_ There was again a clear relation with the expression of H B s A g in the hepatocytes, as preS1 and preS2 were detected in 7 out of 8 HBsAg-positive biopsies and were absent in both HBsAg-negative biopsies. None of the 25 patients with chronic H B V infection was anti-HDV-positive in serum or HDAg-positive in the liver by immunofluorescence. Data on the
The distribution patterns of immunofluorescence staining with anti-preS1 and anti-preS2 monoclonal antibodies were almost identical in individual biopsies and were also similar to the staining pattern obtained with anti-HBs monoclonal antibody, although there were significant variations in intensity of positivity (Table 1). All biopsies positive for preS contained both preS1 and preS2 and also stained for HBsAg. As mentioned, a single biopsy from a patient with no viral replication was HBsAg-positive while preS-negative. In cases with H B V - D N A in serum, either HBeAg-positive or anti-HBe-positive, both cytoplasmic and membranous preS and S staining was observed_ In 5 biopsies, preS2 staining was most prominent (Fig. 1), particularly at the cell periphery, while in 2 other biopsies the signal was stronger for preS1 (Fig. 2), and in 7. biopsies preS1, preS2 and S staining were similar in pattern and intensity. In patients who were HBV-DNA-negative in serum, cytoplasmic positivity for preSI, preS2 and HBsAg was observed without membranous staining of hepatocytes (Fig. 3). Here, preS2 staining was often predominant (4 out of 7 cases had a strong preS2 cytoplasmic signal with a much weaker preS1 positivity), while preS1 staining was always the faintest and was barely detectable in 2 cases.
TABLE I HBV ENVELOPE PROTEINS (HBsAg, PRE-SI, PRE-S2) IN THE LIVER OF PATIENTS WITH CHRONIC INFECTION, AND RELATION TO SEROLOGIC PROFILE Patterns of immunofluorescencestaining with monoclonal antibodies (No. cases) Serologic profile
HBsAg++ preSl++ preS2+ +
HBsAg++ preSl++ preS2+
HBsAg++ PreSl+ preS2+ +
HBsAg+ + preS1preS1-
HBsAgpreSlpreS2-
HBeAg-positive ( 10 cases) Anti-HBe-positive, HBV-DNA-positive (5 cases) Anti-HBe-positive. HBV-DNA negative (10 cases)
6 1 3
1 1
3 2
0 0
0 1
0
4
1
2
++ = strongly positive; + = weakly positive;- = negative_
160
A. F R A I E S E et al.
Fig. I. Detection of preS2 by indirect immunofluorescence in the liver of an HBeAg-positive carrier. Strong membranous staining is seen associated with cytoplasmic staining.
Fig. 2. PreSl in the liver of an HBeAg-positive carrier.
LIVER PRE-S1 AND PRE-S2 IN HBV INFECTION
161
Fig. 3. Cytoplasmic staining with anti-preS2 monoclonal antibody in the liver of a 'healthy" HBsAg carrier who had no evidence of viral replication and showed the presence of integrated HBV-DNA sequences in the liver.
H B c A g was detected by direct immunofluorescence in the liver of all HBeAg-positive or anti-HBepositive cases who were positive in serum for HBVD N A by spot hybridization. Five patients had a diffuse H B c A g positivity (more than 70% of hepatocytes were positive for H B c A g ) , while 10 cases had focal H B c A g positivity. In general, HBcAg-positive biopsies had a stronger preS1 and preS2 fluorescence compared to HBcAg-negative biopsies, but the only absolute difference was the membranous pattern of preS antigens, which was seen exclusively in HBcAgpositive biopsies. By double immunofluorescence, no definite association of H B c A g and of preS antigens within the same liver cell was observed_ In fact, HBcAg was only occasionally detected in the nuclei of liver cells with cytoplasmic preS and S while it was frequently seen in liver cells with membranous preS and S staining, particularly in those cases with a more diffuse positivity.
PreS expression in liver biopsies with free or integrated HB V-DNA sequences Eight liver biopsies positive for preS1 and preS2 by immunofluorescence were studied by Southern blot for H B V - D N A seq,uences, as previously described [10]. Four biopsies were from HBeAg-positive cases and showed the typical replication pattern with a 3.2 kb band and a smear of replicative intermediates below. The other 4 biopsies came from anti-HBe-positive, HBV-DNA-negative cases and did not contain free H B V - D N A sequences. Two biopsies contained integrated H B V - D N A sequences with a sharp 11 kb band after HindllI digestion in one case and evidence of ' r a n d o m ' integration in the other.
Discussion
This study clearly demonstrates that preS1 and
162 preS2 sequences are expressed and accumulate in the cytoplasm of hepatocytes in most H B s A g chronic carriers, independently of viral replication activity. These findings extend recent observations of Thung and co-workers [7] and clearly establish that preS polypeptides are certainly also present in the liver of anti-HBe-positive ~healthy' carriers with integrated H B V - D N A sequences and no evidence of viral replication activity or of liver damage. This observation is at variance with our previous results obtained in a limited series of biopsies with human anti-preS antisera, which were found to stain only liver sections from patients with ongoing viral replication [11]. These discrepancies may reflect different specificities of human vs. monoclonal antipreS antibodies_ PreS2 expression in most biopsies was much stronger than preS1 expression, both in the cytoplasm and at the liver cell periphery, particularly in anti-HBe-positive, H B V - D N A - n e g a t i v e carriers. Large amounts of preS1 reactivity were in fact detected only in a few biopsies from H B e A g - p o s i t i v e cases with ongoing viral replication, suggesting that translation of the large envelope protein is more closely related to viral replication than that of the middle and major H B s A g proteins. This would be in agreement with the data of Klinkert et al. [3] obtained using the Western blot technique when comparing liver biopsies from viremic and non-viremic carriers, and would also explain further the results obtained with human antisera which contained mainly the anti-preS1 specificity. No relation was seen between the pattern of expression of preS antigens in the liver and activity of hepatitis, although m e m b r a n e staining was seen
References 1 Tiollais P, Charnay P, Vyas GN. Biology of the hepatitis B virus. Science 1981 ; 213:406-411. 2 Heermann KH, Goldmann U, Schwartz W, et al. Large surface proteins of hepatitis B virus containing the pre-S sequence. J Virol 1984; 52: 396-402. 3 Klinkert M-Q, Theilmann L, Pfaff E, Schaller H. Pre-Sl antigens and antibodies early in the course of acute hepatitis B virus infection. J Virol 1986; May: 522-525.
A. FRAIESE et al. mainly in patients with viral replication, as previously described for H B s A g reactivity [12]. It has been suggested that preS polypeptides, particularly preS1, could accumulate in the liver in the absence of complete production of the virus, on the basis of the inhibitory effect of preS1 on H B s A g secretion seen in cell lines not supporting viral replication [13] and of results obtained in the transgenic mouse model [14]. H o w e v e r , we did not find extensive accumulation of preS1 in the cytoplasm of hepatocytes of patients without H B V - D N A in serum, and serum preS1 reactivity was detected in several of these cases, although levels were significantly lower than those found in viremic sera. In conclusion, p r e S l and preS2 are both expressed in the liver of H B s A g carriers, i n d e p e n d e n t l y of viral replication, and are also secreted in the circulation, the absolute level being lower in anti-HBe-positive, H B V - D N A - n e g a t i v e cases, probably as a consequence of a lower concentration of viral particles rather than a lower rate of secretion, These results, however, do not contradict the possibility that in some H B s A g carriers accumulation of viral envelope proteins in the cytoplasm of hepatocytes could d e t e r m i n e morphologic changes and cell degeneration, but this remains to be proven by longitudinal studies of these cases.
Acknowledgement The authors are deeply grateful to Prof. W. Gerlich for the monoclonal antibodies used in this study.
4 Pei-Sheng H, Carithers RL. Fiorenza V, Peterson DL. Quantitative studies of the hepatitis B viral pre-S protein: lack of correlation with the HBeAg status. J Virol Methods 1987; 16: 97-114. 5 Ganem D, Varmus HE. The molecular biology of the hepatitis B viruses. Annu Rev Biochem 1987: 56:651-693. 6 Neurath AR, Kent SBH, Strick N, Parker K. Identification and chemical synthesis of a host cell receptor binding site on hepatitis B virus. Cell 1986; 46: 429-436. 7 Thung SN. Gerber MA, Kasambalides E J, et al. Demon-
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163 systems in hepatitis B virus infection. In: Robinson W, Koike K, Will H, eds. Hepadna Viruses. New York: Alan Liss, Inc., 1987; 397-410. 12 Gudat F, Bianchi L, et al. Pattern of core and surface expression in liver tissue reflects state of specific immune response in hepatitis B. Lab Invest 1975; 32: 101-107. 13 McLachlan A, Milich DR, Raney AK, et al. Expression of hepatitis B virus surface and core antigens: influences of pre-S and precore sequences. J Virol 1987; 61: 683-692. 14 Chisari FV, Filippi P, McLachlan A, et al. Expression of hepatitis B virus large envelope polypeptide inhibits hepatitis B surface antigen secretion in transgenic mice. J Virol 1986; 60: 880-887.