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.     

The effect of recombinant alpha-interferon treatment on serum levels of hepatitis B virus-encoded proteins in man

The effect of alpha-interferon treatment on serum levels of hepatitis B virus-encoded proteins was analyzed in eight patients with chronic type B hepatitis who participated in a pilot study of interferon therapy. Three individuals became HBsAg-negative, 4 lost HBeAg but remained HBsAg-positive and 1 remained positive for both HBsAg and HBeAg. Initiation of interferon treatment was rapidly followed by reduction or loss of hepatitis B virus DNA in the serum but by little immediate change in hepatitis B virus antigen levels. Changes in hepatitis B virus antigens were usually delayed. Loss of HBsAg from the serum was preceded by the sequential disappearance of pre-S-encoded proteins (pre-S1 and polymerized human serum albumin) and HBeAg. In patients who lost HBeAg but remained HBsAg-positive, serum levels of pre-S1 and polymerized human serum albumin usually, but did not always, decrease. The individual who remained HBsAg- and HBeAg-positive had unchanged serum levels of pre-S1, polymerized human serum albumin and HBsAg. These results suggest that alpha-interferon inhibits hepatitis B virus DNA replication but has little direct effect on synthesis of hepatitis B virus gene products.     

Assay of hepatitis B virus DNA by polymerase chain reaction and its relationship to pre-S- and S-encoded viral surface antigens

The polymerase chain reaction was evaluated as a diagnostic tool in 72 chronic hepatitis B virus carriers. Hepatitis B virus DNA was detectable in the serum of HBsAg-positive virus carriers using aliquots as small as 100 al. The detection limit for cloned hepatitis B virus DNA was 100 ag. Primer pairs for different regions of the HBV genome resulted in different sensitivity. Detection of the amplified hepatitis B virus DNA by Southern blotting and subsequent scintillation counting or densitometry allowed a semiquantitative assay. Using several primer pairs in parallel for optimal detection, all HBeAg-positive HBsAg carriers, 80% of HBe antibody-positive symptomatic HBsAg carriers and 57% of asymptomatic HBe antibody-positive HBsAg carriers were found to have hepatitis B virus DNA in the serum. During antiviral therapy hepatitis B virus DNA disappeared by the polymerase chain reaction assay in patients who became HBeAg negative, but polymerase chain reaction detected a relapse earlier than did the conventional dot blot. Pre-S antigens were assayed in serum and liver samples from most chronic carriers by enzyme-linked immunosorbent assay and/or immunoblot. Although most viremic carriers were strongly positive for pre-S1 and pre-S2 antigens, some hepatitis B virus DNA-positive HBsAg carriers did not have detectable pre-S antigens, and vice versa. Our data show that assay of hepatitis B virus DNA in the serum by polymerase chain reaction is by far more proficient than by dot blot and that it cannot be replaced by serological assays of HBeAg or pre-S antigen.     

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.     

乙型肝炎病毒表面抗原阳性患者前-S1抗原和前-S2抗原与HBV DNA和HBV-M相关性分析

目的检测乙型肝炎病毒患者乙型肝炎病毒前-S1抗原（HBV pre-S1-Ag）、前-S2抗原（HBV pre-S2-Ag）、乙型肝炎病毒DNA（HBV DNA）和乙型肝炎病毒E抗原（HBeAg）,探讨其相关性和临床应用价值。方法应用酶联免疫测定法（ELISA）分别检测HBV pre-S1-Ag、HBV pre-S2-Ag和乙型肝炎病毒标志物（HBV-M）,荧光定量聚合酶链反应法（FQ-PCR）检测HBV DNA,并对检测结果进行统计学分析。结果 HBsAg阳性者中,pre-S1-Ag、pre-S2-Ag、HBV DNA阳性者分别为594例、541例、629例,其阳性率分别为66.29%、60.38%、70.20%。HBeAg阳性组pre-S1-Ag、pre-S2-Ag、HBV DNA的阳性率分别为90.21%、74.46%、93.32%,显著高于HBeAg阴性组的45.28%、48.01%、49.89%,差异有统计学意义（P〈0.01）。随着HBV DNA载量的增高,pre-S1-Ag、pre-S2-Ag、HBeAg阳性率随之增高。结论 pre-S1-Ag、pre-S2-Ag与HBV DNA和HBeAg阳性检出率具有显著相关性。联合检测pre-S1-Ag、pre-S2-Ag、HBV DNA及HBV-M,有助于HBV早期诊断、疗效观察和预后判断。     

Expression of Pre-S2 antigen and antibody in patients with hepatitis B virus infection

We investigated the expression of Pre-S2 antigen (Ag) and antibody (Ab) in sera quantitatively using ELISA. Four patients with acute hepatitis B and 87 chronic HBV carriers were included in this study. We also investigated the expression of Pre-S2Ag in the liver tissues obtained from 26 chronic HBV carriers by direct fluorescent antibody method. There was a significant correlation between Pre-S2Ag titers and HBsAg titers in sera. Pre-S2Ag titers were higher in sera positive for HBeAg, HBV-related DNA polymerase or HBV-DNA than in sera negative for those markers. The ratio of Pre-S2Ag titers to HBsAg titers, however, was constant irrespective of virus replicative markers. Pre-S2Ag in the liver had almost same intracellular localization with HBsAg in the liver. It was suggested that Pre-S2Ag was expressed with an intimate relation to the expression of HBsAg and was not useful as a virus replicative marker. Pre-S2Ag titers/HBsAg titers tended to be high in patients with chronic active hepatitis and high serum GPT levels compared to patients with chronic inactive hepatitis. This may be explained by the release of Pre-S2Ag in the liver. In addition, all patients positive for Pre-S2Ag on the membrane of hepatocytes had chronic active hepatitis. The overproduction of Pre-S2-containing surface proteins in the liver may have some implication related to cell injury via the immune response to Pre-S2Ag etc. Pre-S2Ab was detected only in few cases of chronic HBV infected patients. The lack of immune response to Pre-S2 region may play a role in the persistence of HBV infection.     

Pre-S1 proteins in sera of patients positive for HBsAg and antibodies to hepatitis delta virus

Infection with the hepatitis Delta virus results in a reduction in hepatitis B virus replication. To study the question as to whether expression of large surface (pre-S1) protein is changed in patients with previous and chronic hepatitis Delta virus infection, sera of 25 HBsAg- and anti-HD-positive patients were analyzed by the Western blot technique using an antibody directed against a pre-S1 fusion protein. Pre-S1 proteins were present only in 3 of the 25 sera. This finding suggests that the expression of pre-S1 proteins and HBsAg is regulated independently, and that pre-S1 proteins are not necessarily required for the envelope of hepatitis Delta virus.     

A biphasic pattern of anti-pre-S responses in acute hepatitis B virus infection

The clinical relevance of the immune response to the translation products of the pre-S1 and pre-S2 regions of hepatitis B virus was examined by testing sequential serum samples from 17 patients with acute self-limited hepatitis B and from two patients in whom chronic liver disease developed. Anti-pre-S antibodies were determined by enzyme immunoassays based on the inhibition of binding of monoclonal antibodies to epitopes in the pre-S1 and pre-S2 sequence. In acute, self-limited infection, anti-pre-S antibodies appeared in a biphasic pattern. The early antibodies were detected at the time of clinical signs of acute disease when HBsAg and often HBeAg were present, but hepatitis B virus DNA was no longer detectable in serum. Anti-pre-S levels then fell, but subsequently reappeared as the late antibody during the recovery phase, after development of anti-HBe, but before anti-HBs. Anti-pre-S responses were detected in 15 of 17 patients who recovered (88.2%) and in both patients with acute hepatitis B virus infection evolving to chronic liver disease. Although the early antibodies to pre-S1 and pre-S2 proteins appeared at the time of decreasing levels of infectious virus in serum in cases of self-limited infection, these antibodies also were transiently or continuously present with high levels of serum hepatitis B virus DNA in patients in whom chronic hepatitis B infection developed. Thus the anti-pre-S response in acute hepatitis is not a prognostic marker for clinical resolution.(ABSTRACT TRUNCATED AT 250 WORDS)     

Clinical significance of pre-S mutations in patients with genotype C hepatitis B virus infection

We investigated the overall and site-specific prevalence of pre-S mutations and its clinical significance in patients with genotype C hepatitis B virus (HBV) infection. Three hundred subjects were included: 50 asymptomatic carriers (AC), 87 chronic hepatitis (CH), 91 liver cirrhosis (LC) and 72 hepatocellular carcinoma (HCC). Pre-S mutations were determined by nucleotide sequence analysis. Possible correlations between pre-S mutations and clinical/virological parameters were examined. Pre-S mutations were detected in 82 cases (27.3%); it was more frequently found in HCC (43.1%) and LC (35.2%) group than in the CH (20.7%) and AC (2.0%) group. Pre-S2 deletion was the most commonly found mutation (10.7%), followed by pre-S2 start codon mutation (9.7%), pre-S1-S2 deletion (3.0%) and both pre-S2 deletion and start codon mutation (2.7%). Pre-S2 deletion and pre-S2 start codon mutation were more frequently detected in advanced diseases (LC and HCC). Pre-S mutations were associated with older age and higher rates of positive HBV DNA (>/=0.5 pg/mL). Advanced disease and positive HBV DNA were shown to be independent predictors of pre-S mutations by logistic regression analysis. These findings suggest that pre-S mutations, especially pre-S2 deletions and pre-S2 start codon mutations, are common in patients with genotype C HBV infection and are associated with advanced liver disease and active viral replication.     

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.     

Pre-S Proteins in Chronic Hepatitis B Virus Infection: Markers of Active Viral Infection

The role of large (pre-S1) and middle (pre-S2) proteins of HBsAg in hepatitis B virus (HBV) infection is not fully known. Therefore, we studied the expression of pre-S proteins in the liver and serum of 26 patients with chronic HBV infection, using immunoperoxidase staining and enzyme immunoassay. Pre-S1 and pre-S2 proteins were detected in a large number of patients in both liver and serum, irrespective of the disease activity. Serial sections showed that most cells positive for HBsAg were also positive for pre-S proteins. The localization of pre-S2 and HBsAg was similar, with cytoplasmic and membranous stainings of hepatocytes, whereas pre-S1 was expressed exclusively in cytoplasm. Serum levels of HBsAg, pre-S1, and pre-S2 of DNA polymerase-positive cases were significantly higher than those of DNA polymerase-negative cases. Membranous display of pre-S2 on hepatocytes was observed more often in DNA polymerase-positive patients, and their serum pre-S2 levels were significantly higher than those without it. The predominant localization of cytoplasmic HBcAg usually was associated with active, ongoing hepatitis. Its expression and DNA polymerase activity were significantly correlated. These results indicate that pre-S proteins in serum and the membranous display of pre-S2 on hepatocytes of patients with chronic HBV infection refect active viral replication, but their expression does not correlate with disease activity.     

Expression and clinical significance of pre-S1 and S2 proteins of HBV in sera of patients with chronic liver disease

To assess the significance of pre-S proteins expression during chronic hepatitis B virus (HBV) infection, we developed a sensitive labeled avidin biotin ELISA to detect pre-S1 and pre-S2 proteins. In serum specimens from 80 patients with chronic liver disease, the frequency of pre-S1 and S2 proteins was 53.7% and 47.5%, respectively. Furthermore, it reached 87.8% and 77.6%, respectively, in cases with chronic HBV infection, indicating that pre-S proteins are usually expressed in sera with chronic HBV infection. We found that the expression of pre-S proteins is closely associated with HBV replicating markers, such as HBV DNA, HBcAg and HBeAg, in sera of patients with chronic HBV infection. Both pre-S1 and S2 proteins were often concurrently expressed in liver and serum with chronic HBV infection. However, the frequency was slightly higher in liver than in serum, suggesting that it may be clinically valuable to detect pre-S proteins in serum and liver simultaneously to determine the status of HBV infection. Our results also indicated that pre-S proteins expression in serum can serve as markers of HBV infection, but cannot be used to estimate the severity and activity of hepatic pathology.     

Search for hepatitis B virus DNA in sera from patients with acute type B or non-A, non-B hepatitis

One hundred and three single sera from adults hospitalized with acute type B (78) or non-A, non-B (25) hepatitis were tested for the presence of hepatitis B virus DNA (HBV DNA). All sera from patients with type B hepatitis were IgM anti-HBc-positive. These patients were classified as benign (47) or fulminant (31) hepatitis. The 25 acute non-A, non-B patients were also classified as benign (21) or fulminant (4) hepatitis and were negative for serologic markers of past HBV infection. Serum HBV DNA was detected with similar frequency in benign (38.5%) and fulminant (FH, 34.6%) HBsAg-positive cases. HBV DNA was not detected in either the 26 acute HBsAg-negative hepatitis B cases who were positive for anti-HBc and anti-HBs or the 25 acute non-A, non-B hepatitis cases. The absence of HBV DNA in 43.8% of benign hepatitis B patients who were positive for HBsAg and HBeAg could possibly be attributed to either low level replication of HBV that was not detectable by the [32P]HBV DNA probe or to a period of delayed clearance of free HBeAg following cessation of HBV replication. Emergence of anti-HBs in the presence of HBsAg did not always correspond to clearance of HBV in fulminant type B cases. However, in acute type B hepatitis, irrespectively of severity, disappearance of HBsAg and appearance of anti-HBs was accompanied by reduction of HBV replication to undetectable levels.     

Detection of hepatitis B virus core gene products in sera and liver of HBV-infected individuals

The C gene of hepatitis B virus (HBV) codes for at least two different proteins (p 21c and p 17e). To investigate the expression of C-gene-encoded proteins in vivo, serum and liver samples from HBsAg-positive patients as well as serial serum samples from an HBV-transfected chimpanzee were studied. Antibodies directed against bacterially synthesized C-fusion proteins were used in Western blots to test for the presence of p 21c and p 17e. In serial serum samples from the chimpanzee, p 21c and p 17e were detected concomitantly during the acute phase of the infection. When sera of patients with chronic HBV infection were studied, all sera containing p 17e were found to be positive also for p 21c. Sera positive for HBV DNA but negative for HBeAg were only positive for p 21c, indicating that HBeAg/p 17e is not an absolutely reliable marker for infectivity. In liver tissue specimens from 20 patients with HBV-related liver diseases, p 21c was detected in five cases, indicating viral replication. The p 17e antigen, however, was present only in low amounts in three of these five, suggesting that synthesis of p 21c and p 17e is not strictly coupled. C/Pol-gene-encoded fusion proteins were found in the liver tissue of only one patient with cirrhosis, supporting our previous finding that detectable levels of these proteins are expressed rarely.     

Association of pre-S deletion mutant of hepatitis B virus with risk of hepatocellular carcinoma

BACKGROUND: Pre-S deletion mutant of hepatitis B virus (HBV) affects the expression of middle and small surface proteins, resulting in intracellular accumulation of large surface protein. The correlation between pre-S deletion mutant and risk of hepatocellular carcinoma (HCC) in hepatitis B virus carriers remains unclear. METHODS: Using molecular assays, pre-S deletion mutant of HBV were determined in 266 patients with chronic HBV genotype B or C infection. They included 202 asymptomatic carriers and 64 HCC patients. RESULTS: The overall prevalence of pre-S deletion mutant was 16.5%. Hepatocellular carcinoma (odds ratio [OR], 3.23; 95% confidence interval [CI], 1.23-8.48, P = 0.02) and genotype C (OR, 3.19; 95%CI, 1.54-6.62, P = 0.002) were independently associated with the presence of pre-S deletion mutant. The prevalence of pre-S deletion mutant was comparable between HCC patients with genotype B and C infection. Nevertheless, in asymptomatic carriers, patients with genotype C infection were significantly associated with the presence of pre-S deletion mutant compared to those with genotype B infection (20.8% vs 7.2%, P = 0.007). Compared with age- and genotype B-matched asymptomatic carriers, young HCC patients (<50 years of age) had a significantly higher frequency of pre-S deletion (3.4% vs 20%, P = 0.04). CONCLUSIONS: Pre-S deletion mutant is more frequent in HBV carriers with genotype C infection, and those with pre-S deletion mutant may be associated with the development of HCC, irrespective of HBV genotype.     

Expression of Pre-S-encoded proteins in sera of individuals chronically infected with hepatitis D virus

The sera of 16 individuals chronically infected with the hepatitis D virus were analyzed for hepatitis B virus (HBV) markers. The majority of these patients had a non-replicative form of viral type B hepatitis as indicated by negative tests for HBeAg and HBV-DNA. Pre-S-encoded proteins were detected in 13/16 sera. Sera that were negative for polymerized serum albumin did also not contain pre-S1-encoded proteins. The presence of pre-S-encoded proteins is probably predominantly associated with 22-nm HBsAg forms present in large amounts in sera of individuals with chronic type D hepatitis.     

Detection of antibodies to proteins encoded by the X-region of hepatitis B virus (HBV) in sera of patients with chronic HBV infection: correlation with other HBV markers

The genome of hepatitis B virus (HBV) contains a fourth open reading frame (ORF), designated X-region. It was the aim of our study to test sera of patients with chronic HBV infection for the presence of antibodies reactive with a 17 kd gene product of the X-ORF. For this purpose, a 35S-X-ORF-encoded protein, synthesized in vitro, was applied as antigen for the detection of antibodies to HBx proteins in sera of 86 individuals. Antibodies reacting with a gene product of the X-ORF were present in 10 out of 24 HBsAg-positive patients with hepatocellular carcinoma (PLC) or liver cirrhosis and in one out of 8 HBsAg-carriers. In addition, the antibodies could also be detected in 6 out of 35 sera from patients with PLC or cirrhosis negative for HBsAg but positive for anti-HBc and anti-HBs. Antibodies to a gene product of the X-ORF can be detected in sera of patients with chronic HBV-related liver disease, independently of HBsAg and the HBeAg/anti-HBe system.