Expression of pre-S1 antigen and pre-S1 antibody in sera obtained from HBV infected patients

We investigated the expression of Pre-S1 antigen and Pre-S1 antibody using a synthetic peptide and the monoclonal antibody against it. Four patients with acute hepatitis B and 87 chronic HBsAg carriers were included in this study. There was a significant correlation between Pre-S1 antigen titers and HBsAg titers. Pre-S1 antigen showed higher titers in patients with active viral replication, positive for HBeAg, HBV-DNA or HBV-related DNA polymerase. The ratio of Pre-S1 antigen titers to HBsAg titers, however, had no significant relationship with those replicative markers. It was suggested that Pre-S1 antigen was expressed with an intimate relation to the expression of HBsAg and was not so useful as a new replicative marker. Pre-S1Ag titers/HBsAg titers tended to be high in patients with chronic active hepatitis and high serum GPT levels. This may be due to the release of overproduced intracellular Pre-S1 antigen. Pre-S1 antibody was detected only in few cases of chronic HBsAg carriers. This result shows that the immune tolerance to Pre-S1 region may play a role in the persistence of HBV infection.     

Prevalence and significance of hepatitis B virus (HBV) pre-S mutants in serum and liver at different replicative stages of chronic HBV infection

Several types of naturally occurring pre-S mutants in sera or liver tissues in patients with chronic hepatitis B virus (HBV) infection have been identified. To clarify the prevalence and significance of emergence of pre-S mutants, 140 sera and 18 resected livers from patients with HBV were studied. Replicative status was designated as high, intermediate, and low based on the HBV-DNA levels in serum or the expression of HBV antigens in liver. In vitro transfection and Western blot analysis were performed to characterize expression and secretion of HBsAg by the mutant constructs. Five major types (I to V) of pre-S deletion mutants in serum and liver and 2 types (VI and VII) in liver were identified. Pre-S mutant was 6.4% at high replicative phase, 13% at intermediate, and 37.5% at low or nonreplicative phases in serum. In livers, the same tendency existed: pre-S2 deletion mutants emerged and prevailed at a low replicative phase in hepatocytes that expressed a novel marginal pattern of HBsAg and usually clustered in groups. The deletion sequence of pre-S2 region coincides with human leukocyte antigen-restricted T- and B-cell epitopes. In vitro HBsAg was retained in the hepatocytes and synthesis and secretion of major surface antigen decreased for most of the pre-S mutants. Pre-S mutants prevailed with evolution of chronic HBV, probably under immune pressure. Emergence of pre-S mutants may account for the life-long persistence and discrepancy of HBsAg in serum and liver in HBV and may confer growth advantage in view of the clustering proliferation of hepatocytes harboring pre-S2 mutant.     

Expression of pre-S gene-encoded proteins in liver and serum during chronic hepatitis B virus infection in comparison to other markers of active virus replication

Pre-S gene-encoded proteins of the hepatitis B virus (HBV) were studied in the liver by immunofluorescence and in serum by radioimmunoassay in 30 patients with chronic HBV infection. The results were compared with molecular hybridization analysis of HBV-DNA in liver and serum, with serum hepatitis B e antigen/antibody (HBeAg/anti-HBe) status and with underlying liver histology. Pre-S peptides were detected in the serum of 11 patients, 10 of whom were positive for serum HBV-DNA and/or liver hepatitis B core antigen. Only 4 of these patients were HBeAg positive. The prevalence of serum pre-S among HBV replicating carriers was 59% (10/17) compared to only 8% (1/13) among those with non-replicating virus (P less than 0.01). All patients with circulating pre-S peptides had active liver disease. Anti-pre-S was detected in the serum of only 4 patients, 3 with integrated HBV-DNA. In contrast to serum findings, pre-S peptides were detected in the liver of all patients with histochemically demonstrable hepatitis B surface antigen (HBsAg), regardless of HBV replicative status. HBsAg carriers with integrated HBV-DNA had abundant cytoplasmic pre-S1 and pre-S2 localized in numerous ground-glass hepatocytes. It is concluded that pre-S peptides are usually displayed in the liver simultaneously with histochemically detectable HBsAg; they are secreted in the serum in association with high HBV replication and release of HBV particles, but in the absence of episomal HBV replication, pre-S peptides seem to be largely retained within hepatocytic membranes.     

Ultrastructural localization of Pre-S2 polypeptides in the liver tissues of patients with chronic hepatitis B virus infection

Pre-S2 polypeptides in the liver tissue were investigated by immunoperoxidase staining in 26 patients with chronic hepatitis B virus (HBV) infection positive for HBeAg. Pre-S2 polypeptides were detected in the liver of 25 patients, in 17 of whom Pre-S2 polypeptides were localized both on the hepatocyte membrane and in the cytoplasm, and in eight of whom only in the cytoplasm. The localization pattern of Pre-S2 polypeptides was not correlated with the histological findings but with the replicative status of HBV. In cases with a high level of DNA-polymerase in the serum or with both nuclear and cytoplasmic expression of HBcAg in the liver, Pre-S2 polypeptides were more frequently expressed both on the hepatocyte membrane and in the cytoplasm (P less than 0.05). Membranous expression of Pre-S2 polypeptides was speculated to be linked to active replication of HBV in the hepatocytes. Under immune electron microscopy, Pre-S2 polypeptides were observed on the plasma membrane, membranes and cisternae of endoplasmic reticulum (ER), or perinuclear space. Moreover, Pre-S2 polypeptides were detected on the tubular structures and the intracisternal particles about 40 nm in diameter considered to represent HBV. These findings suggest that the immunoreactions of Pre-S2 polypeptides in the liver tissue are similar to those of HBsAg, indicating that Pre-S2 polypeptides play an important role in the immunocharacteristics of HBsAg.     

乙型肝炎前S2抗原／抗体的检测与其病毒标志物的关系及临床意义

目的:探讨乙型肝炎患者前S_2抗原、前S_2抗体与乙型肝炎病毒标志物(HBV M)之间的关系及临床意义。方法:对176例乙型肝炎患者用ELISA法检测前S_2抗原、前S_2抗体及HBV M;用聚合酶链反应(PCR)法检测乙型肝炎病毒HBV-DNA。结果:急性肝炎、慢性肝炎、肝硬变患者前S_2抗原检出率分别为11.11％、73.73％、75%;前S_2抗体在上述患者中的检出率分别为77.78％、11.02％、5％。前S_2抗原在HBV-DNA、HBeAg阳性病例中的检出率明显高于阴性组(P<0.001)。前S_2抗体阳性病例中,HBV-DNA、HBeAg均为阴性。结论:前S_2抗原的检出意味着病毒有复制或有传染性。前S_2抗体的出现标志着病毒被清除,在慢性肝病中检出并不意味着病情稳定,同时发现该抗体可以在急性乙型肝炎的急性期及血清乙肝病毒标志物全部阴性的患者中检出。     

Quantitative analysis of pre-S1 and pre-S2 in relation to HBsAg expression

Sera from four patients with acute hepatitis B and 87 patients with chronic hepatitis B were examined quantitatively for pre-S1 and pre-S2 antigens by solid-phase enzyme immunoassays. Pre-S1 and pre-S2 antigens were detected in HBsAg-positive sera irrespective of the presence of viral replicative markers, and their titers correlated with those of HBsAg (r = 0.74, p less than 0.01; r = 0.74, p less than 0.01, respectively). Sera positive for HBeAg showed higher titers of pre-S1 (p less than 0.01) and pre-S2 (p less than 0.01) antigens than sera negative for HBeAg. The titers of pre-S1 and pre-S2 antigens also correlated with the levels of HBV-associated DNA polymerase activity (r = 0.51, p less than 0.01; r = 0.59, p less than 0.01, respectively) and HBV-DNA (r = 0.50, p less than 0.01; r = 0.46, p less than 0.01, respectively). However, the ratios between the titers of pre-S antigens and HBsAg had no significant relationships with those viral replicative markers. These findings suggest that the expression of pre-S antigens is intimately related to the expression of HBsAg and that they are not useful as markers of viral replication. The ratios between the titers of pre-S antigens and HBsAg tended to be high in patients with chronic active hepatitis and high aminotransferase levels. This finding may have been due to the hepatic release of pre-S antigens, over-production of which may have some relationship to liver injury.     

Detection of antibodies against pre-S1 proteins in sera of patients with hepatitis B virus (HBV) infection

Pre-S1 (large S) proteins are components of the envelope of HBV. The presence of pre-S1 proteins is correlated with viral replication. To test sera of patients with HBV infection for the presence of antibodies against pre-S1 proteins (anti-pre-S1), an E. coli extract containing a pre-S fusion protein covering the greater part of the pre-S1 region was subjected to Western blotting and probed with sera of patients. Anti-pre-S1 was present in the sera of all 4 patients with acute self-limited HBV infection and in the sera of 3 patients with a fulminant course. The antibodies could not be detected in the sera of 6 patients with acute HBV infection entering chronicity, in the sera from 10 HBsAg carriers or in the sera of 25 patients with chronic liver disease, positive for HBsAg and antibodies to hepatitis Delta virus. In an additional study, anti-pre-S1 could not be found in 11 sera from 12 patients with previous HBV infection, positive for anti-HBs and anti-HBc. Antibodies to pre-S1 proteins appear at the early stage of acute resolving HBV infection and seem to play a role in the elimination of the virus. The antibodies are absent in the sera of patients with acute HBV infection entering a chronic course and in the sera of chronic HBsAg-carriers.     

Elimination of hepatitis B virus surface antigen and appearance of neutralizing antibodies in chronically infected patients without viral clearance

Summary. Seroconversion from hepatitis B surface antigen (HBsAg) to antibodies against HBsAg (anti‐HBs) usually indicates resolution of hepatitis B virus (HBV) infection. Here, two HBV‐infected patients with seroconversion to anti‐HBs were found to be persistently positive for HBeAg and HBV DNA. Immunohistology of liver biopsies confirmed the expression of HBV proteins in the liver of one patient. The neutralizing ability of anti‐HBs in patient sera was demonstrated by blocking HBV infection of primary tupaia hepatocytes. Analysis of the HBsAg‐encoding region of HBV isolates from patients indicated the coexistence of heterogeneous HBV genomes in patients. The majority of recombinant variant HBsAg was reactive in HBsAg assays and was able to bind to anti‐HBs. Circulating immune complexes (CIC) of HBsAg in patient sera could be detected by polyethylene glycol precipitation and trypsin digestion. Thus, neutralizing anti‐HBs may appear in chronic HBV carriers for long periods but does not necessarily lead to complete viral clearance.     

Expression of hepatitis B virus (HBV) markers in chronic liver disease positive for antibody to hepatitis C virus (HCV)

We investigated expression of HBV markers in chronic liver disease positive for antibody to HCV (anti-HCV). Sera from 107 patients with chronic non-A, non-B liver disease, 65 HBs antigen carriers with chronic liver disease and 14 asymptomatic HBV carriers were tested for the presence of anti-HCV. Anti-HCV was detected in 83 (78%) patients with chronic non-A, non-B liver disease, irrespective of the past history of blood transfusion, and anti-HCV prevalence was similar in each category of chronic liver disease. Fifty-three (64%) out of these 83 sera positive for anti-HCV has also antibodies to HBV. Anti-HBc antibody was detected frequently in liver cirrhotics with hepatocellular carcinoma than in chronic persistent hepatitis, chronic active hepatitis and cirrhotics without hepatocellular carcinoma. In addition, titers of anti-HBc antibody were significantly higher in cirrhotics with hepatocellular carcinoma than in the other groups. On the other hand, anti-HCV was detected in 7 out of 65 patients with HBV-related liver disease. Four out of these 7 were patients with HBV-related hepatocellular carcinoma. Anti-HCV was detected in none of asymptomatic HBV carriers. These findings suggest that infection with both HBV and HCV is likely to cause more serious liver disease than infection with a single agent.     

Evaluation of viral replication in children with chronic hepatitis B with and without interferon treatment

BACKGROUND: In chronic infection with hepatitis virus B the fact that HBeAg becomes negative does not always mean suppression of viral replication. METHOD: HBV replication was assessed in 74 patients with chronic hepatitis or viral B cirrhosis, in whom diagnosis was made according to clinical, biological, and histological criteria. The patients were divided into two groups: group I (36 patients with interferon- therapy, 3 million U/m 2/ dose, 3 doses/week over a period of 4-6 months) and group II (control group of 38 patients who did not undergo interferon therapy). After a follow up period of 6 years in which patients underwent clinical, biochemical and serologic monitorization, HBV DNA was detected by the hybridization method on solid medium. RESULTS: During evolution the levels of transaminases became normal in both groups. The HBe Ag/Ab seroconversion rate at the end of the interferon therapy was 52.8% and the spontaneous HBe Ag/Ab seroconversion rate was 72.7% in group II after an average evolution of 6 years. HBs Ag/Ab seroconversion was not detected in any patient. Assessment of viral replication by HBV DNA testing at the end of the follow up period showed higher levels as compared to the HBeAg testing (69.4% vs. 25% in group I, 55.2% vs. 7.9% in group II). The absence of viral replication (HBV DNA negative) had similar rates in both groups (30.6% in group I vs. 44.8% in group II, p>0.9) and HBV DNA titers in the two groups were not significantly different at the end of the follow up period. In both groups, HBV DNA titers were significantly higher in patients with positive HBeAg. The concordance between the two viral markers was 100%. CONCLUSION: Because of the fluctuating evolution, long-term follow up and monitorization (including HBV DNA testing) of patients with chronic hepatitis B and of inactive HBsAg carriers are necessary.     

Long-term clinical impact of occult hepatitis B virus infection in chronic hepatitis B patients

BACKGROUND/AIMS: Long-term clinical outcomes of occult hepatitis B virus (HBV) infection were studied. METHODS: Fifteen chronic hepatitis B patients were monitored for a median of 4.4 years (range 0.9-15.3) after hepatitis B surface antigen (HBsAg) seroclearance. Serum HBV DNA was measured by real-time detection polymerase chain reaction. Thirteen patients underwent liver biopsies at the end of follow-up and liver histology was evaluated by Ishak score. Liver HBV DNA was also measured for 12 patients. RESULTS: At the end of follow-up, HBV viremia was absent in 13 (87%) patients, and antibody titers to hepatitis B core antigen showed an inverse correlation with time from HBsAg seroclearance (r=-0.554; P=0.0040). However, all patients retained liver HBV DNA and tested positive for the covalently closed circular HBV DNA replicative intermediate. The hepatic HBV DNA loads had no relation to liver histology. Paired biopsies from 11 patients disclosed that each necroinflammatory score significantly improved after HBsAg seroclearance. Amelioration of liver fibrosis was also evident in eight (73%) patients (P=0.0391 by signed rank test). CONCLUSIONS: A long-standing but strongly suppressed HBV infection may confer histological amelioration after HBsAg seroclearance.     

前S2抗原与HBVDNA及HBV血清标志物的关系分析

目的探讨乙型肝炎病毒前S2（Pre-S2）抗原与乙肝病毒血清标志物五项、乙肝病毒DNA之间的相关性及临床意义。方法用酶联免疫吸附试验对982例乙肝患者血清标志物和乙肝病毒Pre-S2抗原进行检测;并用荧光定量PCR法对其进行HBVDNA检测。对HBV血清标志物不同阳性血清学模式进行分组分析。结果在HBeAg阳性模式组中,乙肝病毒Pre-S2和HBVDNA的检出率高,平均为95.34%和97.8%。在HBeAg阴性模式组中,乙肝病毒Pre-S2和HBVDNA的检出率低,平均为39.7%和32.3%。在HBsAg、HBeAg均为阴性的模式中Pre-S2抗原为1.45%。结论 Pre-S2抗原与HBeAg和HBVDNA密切相关;Pre-S2抗原检测完善和补充了乙肝病毒血清标志物检测的不足。     

Hepatitis B virus (HBV) markers and HBV-DNA in serum and liver tissue of patients with acute exacerbation of chronic type B hepatitis

We analysed the serum samples and the liver biopsies of six consecutive chronic HBsAg/anti-HBe carriers admitted to hospital because of an episode of acute hepatitis. The six patients became positive for IgM anti-HBc and negative for HBeAg, hepatitis Delta virus (HDV) markers, IgM anti-hepatitis A virus (HAV), anti-cytomegalovirus (CMV) and anti-Epstein-Barr virus (EBV). Two patients showed positivity for hepatitis B virus (HBV)-DNA in serum obtained on admission, with no positivity in the subsequent weeks; the results of the other four patients were always negative for seric HBV-DNA. The Southern-blot analysis of the DNA extracted from the liver tissue of four subjects showed the presence of HBV-DNA in the form of replicative intermediates; focal positivity of HBcAg was detected in the liver of only one. The liver biopsies of the last two patients were negative for HBV-DNA and for HBcAg. The analysis of HBV-DNA in the liver extracts and the demonstration of an increase of the IgM anti-HBc titre at the time of the abrupt elevation of the aminotransferase levels seem to be the most useful tools in revealing HBV activation as a cause of acute hepatitis in chronic HBsAg carriers, overall when the phase of viremia is transient.     

Analysis of serum pre-S antigens in chronic hepatitis B virus infection in relation to the expression pattern of hepatitis B virus DNA in the liver.

Pre-S antigens have been analyzed in the serum of patients with chronic hepatitis B virus (HBV) infection according to the expression pattern of HBV DNA in the liver. Pre-S1 and pre-S2 have been identified (1) in all viremic cases with free replicative forms of viral DNA irrespective of the simultaneous detection of integrated sequences; (2) in 2 out of 3 patients with only integrated HBV DNA, and (3) in 19 patients who lacked viral DNA sequences detectable in the host genome. The amounts of hepatitis B surface and pre-S antigens were significantly higher in high-viremic versus low-viremic patients and correlated with the hepatocellular expression of HBV DNA. Conversely, the pre-S-to-hepatitis B surface antigen ratios were lower in the presence of viral DNA sequences in the liver. In summary, detection and level of pre-S antigens are closely related to the hepatocellular expression of viral DNA and seem to reflect reliably different stages of the virus life cycle during the course of HBV infection.     

Detection of hepatitis delta virus (HDV) markers in HBV carriers]

To investigate the prevalence and clinical features of hepatitis delta virus (HDV) superinfection in hepatitis B virus (HBV) carriers, antibody to hepatitis delta antigen (anti-HD) was determined in the sera of 328 HBV carriers in Japan. 1) Of the 328 HBV carriers, six (1.8%) were seropositive for anti-HD by enzyme linked immunosorbent assay. IgM-antibody to hepatitis delta antigen was detected in 2/6 patients with a high anti-HDV titer. None of the patients was positive for hepatitis dealt antigen. 2) HBV carriers with chronic liver disease had a greater frequency of seropositivity of anti-HD than asymptomatic HBV carriers. These data indicate that HDV superinfection may be an etiologic agent of chronic liver disease in HBV carriers.     

Hepatitis B virus DNA in chronic HBsAg carriers: correlation with HBeAg/anti-HBe status, anti-HD and liver histology.

Hepatitis B virus DNA was determined in the sera of 198 chronic hepatitis B surface antigen (HBsAg) carriers by the spot hybridization technique. The results were correlated with hepatitis Be antigen (HBeAg) and antibody (anti-HBe), delta antibody (anti-HD) and liver histology. All subjects had a liver biopsy. The prevalence of HBV DNA was 63% in HBeAg-positive subjects and 8.8% in anti-HBe positives. HBV DNA was not found more frequently in chronic HBsAg carriers who had histological evidence of liver disease than in carriers without such evidence. Anti-HD was detected in 48.5% of subjects, with an increasing trend (p less than 0.001) according to the severity of liver disease. Among patients with more severe liver disease (CAH and cirrhosis), HBV DNA and HBeAg were detected less frequently in anti-HD-positive than in anti-HD-negative subjects (7% vs. 42.3%, p less than 0.001 and 7% vs. 34.4%, p less than 0.005, respectively). These findings indicate that HDV infection jointly affects both HBeAg status and HBV DNA.     

Chronic evolution of acute hepatitis B: the significance of simultaneous infections with hepatitis C and D. Copenhagen Hepatitis Acuta Programme

Seventy-six of 77 consecutive patients with hepatitis B surface antigen (HBsAg)-positive acute hepatitis were reevaluated using anti-hepatitis C virus (HCV), anti-hepatitis D virus (HDV), and IgM anti-hepatitis B core (HBc) testing. Anti-HCV and/or anti-HDV was found in 32 patients (42%). The presence of these markers was significantly associated with intravenous drug abuse (p less than 10(-6). Sixty-nine patients were IgM anti-HBc-positive, of whom two (3%) (95% confidence limits, 1-12%) became chronic HBsAg carriers with histologically verified chronic liver disease; both were anti-HCV and anti-HDV-negative. Among the remaining 67 IgM anti-HBc-positive patients 8 had HBV and HDV co-infection, 3 had HBV and HCV co-infection, and 1 had HBV, HCV, and HDV co-infection. Twenty-two had evidence of preceding or past HCV infection; two developed chronic active hepatitis in spite of HBsAg clearance. Seven patients with IgM anti-HBc negative. One was a chronic HBsAg carrier with HDV superinfection. One had subclinical acute HBV infection and became a chronic HBsAg carrier. In a further two patients reactivation of replication in a chronic HBV infection could not be disregarded. Three patients could not be classified; all had acute recent onset of symptoms, cleared HBsAg within 6 months, but lacked IgM anti-HBc. It is concluded that HCV and HDV superinfections in HBV carriers mimicking acute HBV infection with chronic evolution are rarely encountered in the present population in spite of high frequency of both HCV and HDV markers.     

Anti-pre-S antibodies in different groups of patients with hepatitis B virus infection

The anti-pre-S antibody in the samples of sera from normal healthy persons and patients with different clinical types of liver diseases due to hepatitis B virus (HBV) infection was detected by a newly established enzyme-linked immunosorbent assay technique. This test is a blocking assay where anti-pre-S antibody in the patient's serum blocks subsequent addition of horse radish peroxidase-labelled polymerized human serum albumin (pHSA) to the pHSA-receptor site of HBsAg molecules fixed on a solid surface. Anti-pre-S activity was not detected in any from 95 healthy persons who were negative for all HBV-markers or from 105 healthy HBV carriers. In 12 sera from HBV vaccine recipients, anti-pre-S activity was noted in higher proportions compared with anti-HBs, after both the second and third doses of vaccine. Anti-pre-S activity was detected in small proportions of HBsAg positive sera from acute viral hepatitis (4.2%) and chronic active hepatitis (10%). In subacute viral hepatitis patients, the anti-pre-S antibody was totally absent. However, anti-pre-S activity was recorded in high proportions of HBsAg-positive sera from patients with cirrhosis of liver (57.2%) and fulminant hepatitis (41.6%). The anti-pre-S antibodies were assumed to be implicated in the clearance of HBV particles from circulation without causing tissue damage.     

慢性乙型肝炎病毒感染S区基因缺失及相关因素研究

目的 测定慢性乙型肝炎病毒(HBV)感染者HBV DNA全序列,分析S区基因缺失模式、频率及相关因素.方法 慢性HBV感染者59例,其中HBV携带7例,慢性肝炎31例,肝硬化10例,重型肝炎6例,原发性肝癌5例.结果 25.4%(15/59)慢性HBV感染者有s区基因缺失,未发现S基因缺失.Pre-S基因缺失均见于C基因型患者.Pre-S基因缺失患者中,20%(3/15)HBsAg、抗HBs共存,与无S区缺失者比较有明显差异(P<0.05).PreS基因缺失与病程(偏相关系数0.28,P=0.049)、抗病毒治疗(偏相关系数-0.451,P=0.036)有密切关系.结论 Pre-S基因缺失在基因C型、严重肝病及活动性HBV复制患者多见,可能与病程长及抗病毒治疗有关.Pre-S基因缺失可导致HBV免疫逃避或免疫治疗失败,可能是肝脏疾病发展的重要原因.