Adsorption properties of different hepatitis B virus related antigens (HBsAg, HBcAg, HBeAg) on octanohydrazide-Sepharose 4B

Chromatography of plasma containing hepatitis B virus and partially purified viral antigens on a hydrophobic gel derivative (octanohydrazide-Sepharose 4B) revealed that HbsAg and HbcAg were adsorbed to the gel in 0.75 mol/l ammonium bicarbonate and eluted by a detergent, Berol. HBeAg in a purified HBcAg preparation from human liver, but not HBeAg in plasma, was bound to the gel. Furthermore, HBeAg in the HBcAg preparation, but not HBeAg in plasma, lost its antigenic reactivity in the presence of Berol, indicating that the two HBeAgs were present in different molecular configurations. However, HBeAg could be released from HBV (HBcAg) and form a component which sedimented slowly and was immune-reactive in the presence of the detergent. The results contribute to knowledge of the interrelationship between hepatitis B-related antigens and indicate that chromatography on hydrophobic gel derivatives can be used not only for the purification (and removal) of HBsAg but also of HBcAg.     

Cryptic association of e antigen with different morphologic forms of hepatitis B surface antigen

When highly purified HBsAg particles, separated by rate zonal centrifugation into populations differing in predominant size, were tested for HBeAg, the e1 specificity was detected preferentially in association with particle fractions containing large filaments and Dane particles. These results were obtained both by agar gel diffusion and by radioimmunoassay for e antigen. The e antigen activity present in these fractions was potentiated by prior treatment of particles with Tween 80, suggesting cryptic localization of e1 specificity within or under the outer membrane. The HBeAg released by detergent treatment from a purified preparation composed predominantly of small-particle forms of HBsAg was separated by electrofocusing into a peak of nonparticulate e antigen in the pH range of 5.7--6.0. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed three major polypeptides in this preparation with approximate molecular weights of 25,000, 55,000, and 70,000. Furthermore, two additional peaks of e antigen activity were detected which migrated in association with HBsAg particles at isoelectric points of 4.4 and 5.5--5.6. The major portion of e antigen remained in association with particles after further purification by rate zonal centrifugation.     

Evaluation of a new radioimmunoassay for detecting hepatitis B e antigen and its antibody

A new commercially available radioimmunoassay (RIA) (Abbott-HBe^TM) was used for determination of hepatitis B e-antigen (HBeAg) and its antibody (anti-HBe). Serial serum samples from 20 transiently HBsAg-positive patients with acute hepatitis were tested. In nearly all patients HBeAg could be shown for a short period with subsequent development of antiHBe. From 24 chronic HBsAg carriers serial serum samples collected during several years were tested. In 18 of 19 initially HBeAg-positive patients the HBeAg was lost after 6 months to 6 years; in 14 anti-HBe developed. A correlation was seen between the seroconversion and normalization of elevated alanine transferase levels. From another 22 chronic HBsAg carriers single serum samples were assayed. These samples were selected because neither HBeAg nor anti-HBe could be detected by the immunodiffusion (ID) technique. They had previously been examined for HBV-associated DNA-polymerase activity. In 20 patients HBeAg or anti-HBe could be detected by RIA. Those who were DNA-polymerase negative had anti-HBe, and 3 of 4 who were DNA-polymerase positive had HBeAg. When compared to earlier results by ID in these materials a higher frequency of HBeAg and, in particular, anti-HBe was detected by the RIA. By this test, HBeAg or anti-HBe was found in nearly all patients. The usefulness of HBeAg/anti-HBe in the evaluation of infectivity and prognosis in hepatitis B has been limited by the low sensitivity of the earlier test systems. Thus, this new RIA is a valuable addition to the diagnostic tests for patients with hepatitis B.     

Hepatitis B virus genotypes and spontaneous hepatitis B e antigen seroconversion in Taiwanese hepatitis B carriers

Hepatitis B virus (HBV) is classified into eight genotypes (A-H), and genotype C is associated with more aggressive liver disease compared to genotype B. However, the mechanisms responsible for the clinical differences remain unclear. To test whether genotype C patients had with lower rates of spontaneous hepatitis B ge antigen (HBeAg) seroconversion than genotype B patients, stored serum samples from 146 Taiwanese adult HBeAg-positive hepatitis B carriers followed-up for a mean of 52 months (range, 12-120 months) were tested for HBV genotype by a molecular method. Genotype C patients were significantly older than genotype B patients (mean age, 37 +/- 12 vs. 29 +/- 10 years, P < 0.001). During the follow-up period, genotype C patients had a significantly lower rate of spontaneous HBeAg seroconversion than genotype B patients (27 vs. 47%, P < 0.025). Spontaneous HBeAg seroconversion occurred one decade later in genotype C patients compared with genotype B patients. Multivariate analyses identified age < or =35 years (odds ratio: 2.08; 95% confidence interval [CI], 1.07-4.0; P < 0.05), high baseline serum alanine aminotransferase level (odds ratio: 2.34; 95%CI, 1.39-4.09; P < 0.005), and HBV genotype B (odds ratio: 1.94; 95%CI, 1.03-3.63; P < 0.05) as independent factors associated with spontaneous HBeAg seroconversion. In conclusion, genotype C patients, compared to genotype B patients, have a delayed HBeAg seroconversion in the immune clearance phase of chronic HBV infection, which may contribute to a more progressive liver disease and more refractory to antiviral therapy.     

Nucleotide mutations associated with hepatitis B e antigen negativity

One hundred and forty four patients with chronic hepatitis B were tested to identify new mutations associated with hepatitis B e antigen (HBeAg) negativity, using a full genome sequence analysis. All the patients were Chinese and had hepatitis B virus infection of genotype C. Patients with none of the pre-core or core promoter mutations were significantly (P < 0.001) less common in the group with anti-HBe (13%) than in the group with HBeAg (56%). The complete nucleotide sequence was determined in four anti-HBe-positive patients who had neither pre-core nor core promoter mutations and in five HBeAg-positive patients who also had neither of these mutations (the groups were matched for age and sex). Six mutations were found to be significantly more common in the former group than in the latter: G529A (3/4 vs. 0/5), C934A (4/4 vs. 1/5), A1053G (4/4 vs. 1/5), G1915T/A (4/4 vs. 0/5), T2005C/A (4/4 vs. 0/5), and C3026T (3/4 vs. 0/5). Three of the six mutations were significantly more common in the four anti-HBe-positive patients who had neither pre-core nor core promoter mutations, compared to 11 HBeAg-positive patients who had pre-core and core promoter mutations, and also compared to 15 anti-HBe-positive patients who had pre-core and core promoter mutations, suggesting further the specificity of these mutations. Of the six mutations, two resulted in amino acid substitution in the polymerase protein, and one is located near the enhancer I region. The results suggest that the six newly discovered mutations are associated with HBeAg negativity.     

Genetic heterogeneity in the precore region of hepatitis B virus in hepatitis B e antigen-negative chronic hepatitis B Patients: Spontaneous seroconversion and interferon-induced seroconversion

To elucidate the relationship between the clinical severity of chronic liver disease and the precore mutations in hepatitis B e antigen (HBeAg)-nega-tive hepatitis B virus (HBV) carriers, mutations were investigated in the precore region of HBV DNA in 20 chronic hepatitis B patients who sero-converted either spontaneously or after the administration of α-interferon (IFN), and 5 asymptomatic carriers. The precore mutation with a stop codon at nucleotide 1896 was found in all patients, irrespective of the histology and in all asymptomatic carriers. The second mutation at nucleotide 1899 was found in 40% of cases studied but always followed by the first mutation at nucleotide 1896. The mixed viral infection of precore mutant and wild-type HBV virus was found in 40% of seroconverted cases after IFN treatment and in sera of HBV carriers obtained within a year after the spontaneous Seroconversion. These data suggest that the precore mutants prevail over wild-type HBV in all HBeAg-negative HBV carriers within several years after the sero-conversion, but their prevalence could not confine the clinical severity of chronic liver disease. © 1995 Wiley-Liss, Inc.     

Development and validation of a model for hepatitis B e antigen seroconversion in entecavir-treated patients with chronic hepatitis B

Achieving hepatitis B e antigen (HBeAg) seroconversion is a satisfactory endpoint during antiviral treatment for chronic hepatitis B (CHB). This study aimed to develop and validate a novel scoring system to predict HBeAg seroconversion during entecavir (ETV) treatment. A total of 526 patients with HBeAg-positive CHB treated with ETV for at least 1 year were randomly assigned to the training and validation cohorts. Baseline parameters including hepatitis B virus DNA, hepatitis B surface antigen (HBsAg), hepatitis B core antibody (HBcAb), and alanine aminotransferase level were quantified. Patients who achieved HBeAg seroconversion were compared with those without HBeAg seroconversion. A prediction model was established to predict HBeAg seroconversion during ETV treatment. After a median follow up of 2.67 years, 93 (36.0%) and 87 (32.5%) patients in the training and validation cohorts developed HBeAg seroconversion. A prediction score composed of age, HBsAg and HBcAb quantification was derived. Areas under receiver operating characteristic curve at 5 years of this prediction score were 0.70 and 0.72 in the training and validation cohorts. By using the dual cutoff values of 0.28 and 0.58, the model was endowed with high sensitivity and specificity to exclude or identify patients developing HBeAg seroconversion (90.3% sensitivity and 90.2% specificity in the training cohort as well as 92.8% sensitivity and 84.4% specificity in the validation cohort, respectively). A novel prediction score that uses baseline clinical variables was developed and validated. The score accurately estimates the probabilities of developing HBeAg seroconversion at 5-years ETV therapy in patients with CHB.     

Hepatitis B core antigen in serum during acute hepatitis B

Hepatitis B core antigen was measured in sera of patients with acute and chronic hepatitis B virus infection by a modified radioimmunoassay based on high molarity treatment of samples to avoid masking of antigen by homologous antibody. A good correlation between hepatitis B core antigen levels and serum HBV-DNA was observed in sera obtained during chronic infection. In contrast, acute phase sera were often HBcAg positive but HBV-DNA negative, particularly when obtained during maximum liver damage. Sequential studies in 5 patients with acute hepatitis B showed that HBcAg positivity persisted beside HBV-DNA clearance and was often enhanced at the time of maximum liver damage, suggesting release of antigen from infected hepatocytes undergoing immunolysis, even after termination of virus replication.     

Gender disparity in distribution of the major hydrophilic region variants of hepatitis B virus genotype C according to hepatitis B e antigen serostatus

Hepatitis B virus (HBV) e antigen (HBeAg) seroconversion during chronic HBV infection is known to play an important role in disease progression and patient response to antiviral agents. The aim of the present study was to analyze gender disparity in distribution of major hydrophilic region (MHR) variants according to HBeAg serostatus. Prevalence of MHR variants from 68 Korean patients with chronic hepatitis (31 HBeAg-positive and 37 HBeAg-negative) was examined in terms of HBeAg serostatus and sex by direct sequencing analysis of the MHR. Gender disparity was observed in the distribution of MHR variants according to HBeAg serostatus. In male patients, the prevalence of MHR variants was significantly higher in HBeAg negative patients than in HBeAg positive patients [58.8% (10/17 patients) vs. 14.3% (3/21 patients), P=0.004]. However, the same was not true in female patients [55.0% (11/20 patients) vs. 60.0% (6/10 patients), P=1.000)]. In addition, 2 mutation types (L110I and G145A) related to HBeAg serostatus were found. In conclusion, HBeAg seroconversion in male chronic patients infected with genotype C could lead to mutations of MHR, major target to host immune response, which might in turn contribute to HBV persistence and immune evasion.     

Detection and characterization of hepatitis B virus DNA in serum of HBe antigen-negative HBsAg carriers

Sera from 153 Israeli patients in various stages of hepatitis B virus (HBV) infection with undetectable hepatitis Be antigen (HBeAg) were studied for the presence of HBV DNA in the serum by molecular hybridization. HBV DNA was detected in 10 patients: 3 with acute hepatitis, 4 asymptomatic hepatitis B surface antigen (HBsAg) carriers, 1 with chronic active hepatitis, 1 with cirrhosis, and 1 with mixed cryoglobulinemia. HBV DNA was detected in 7 of 10 HBeAg-positive control samples tested. Hybridization analysis was used for quantitative comparison of HBV DNA levels in serum. HBV DNA levels, found in HBeAg-negative patients sometimes exceeded the levels found in HBeAg-positive patients. Restriction enzyme analysis of serum HBV DNA from four HBeAg-negative samples gave undistinguishable digestion patterns as compared to 3 HbeAg-positive samples. However, heterogeneity in HBV DNA restriction fragments was detected among HBV genomes in sera of HBeAg-positive samples. These data demonstrate that HBV DNA may be present in the serum at various stages of HBV infection, regardless of HBeAg detection. Failure to detect HBeAg in these patients does not necessarily reflect low serum levels of viral particles, or the occurrence of HBV genome variants.     

Prevalence of Williams e1 antigen in comparison with e2 antigen in hepatitis B antigen carriers and patients in hemodialysis unit

The prevalence of both e1 and e2 antigens in 1,158 sera of asymptomatic HBsAg carriers, carriers in hemodialysis units, and HBsAg-negative blood donors was examined. The detection rate of e1 antigen was as high as 80% in asymptomatic carriers, 95% in hemodialysis patients, and even 13.1% in HBsAg-negative donors. All of the e1 antigen-positive specimens in such HBsAg-negative sera were found to have both or either anti-HBs and anti-HBc, suggesting the past history of Hepatitis B virus (HBV) infection of the donors. In the HBsAg-positive serum, the detection rate of e2 antigen (17%) was lower than that of e1 (80%), and all sera having e2 antigen were positive for e1 antigen. The titers of HBsAg, HBcAg, and anti-HBc in e2 antigen-positive sera were higher than that of sera detecting only e1 antigen. The appearance of e1 antigen and e2 antigen in the course of post-transfusion hepatitis B was studied with five cases. Retrospective study showed that three of them each received one unit of HBsAg-positive blood, and the other two received HBsAg-negative blood but with high-titered anti-HBc. In four cases out of five, in which e2 antigen was detected during the course of infection, the initial detection of e2 antigen occurred at or just before the elevation of liver enzyme levels. On the other hand, e1 antigen was detected relatively early after transfusion, and the time of onset. Moreover, the detection period of e1 antigen persisted longer, even after the disappearance of HBsAg antigenemia. These two separate studies suggest that not only e2 antigen but also e1 antigen are associated with the infection of HBV, but they are distinct from each other; the e2 antigen may have the properties of a signal of the viral activity in the patient as suggested by many others, but e1 antigen does not seem to bear such diagnostic values.     

Prevalence of hepatitis B e antigen and its antibody as detected by radioimmunoassays

A solid-phase radioimmunoassay (RIA) for the detection of hepatitis B e antigen (HBeAg) and antibody (anti-HBe) was developed. The RIA was approximately 1,000-fold more sensitive than rheophoresis for HBeAg, and approximately 6,000-fold more sensitive than rheophoresis for anti-HBe. Generally, less than one-fifth of hepatitis B antigen (HBsAg)-positive sera from blood donors were positive for either HBeAg or anti-HBe by rheophoresis; in contrast, more than 90% of the samples were positive by the RIA method. The ratio of HBeAg to anti-HBe among HBsAg carriers varied in different geographic localities. Also, the presence of HBeAg correlated directly with the titer of HBsAg and the presence of Dane core particles. Anti-HBe was associated with lower titers of serum HBsAg.     

The immune response induced by hepatitis B virus principal antigens

Hepatitis B virus （HBV） infection occurs primarily in hepatocytes in the liver with release of infectious virions and non-infectious empty surface antigen particles into the bloodstream. HBV replication is non-cytopathic. Transient infections run a course of several months, and chronic infections are often life-long. Chronic infections can lead to liver failure with cirrhosis and hepatocellniar carcinoma. It is generally accepted that neutralizing anti-HBs antibodies plays a key role in recovery from HBV infection by containing the spread of infection in the infected host and facilitating the removal and destruction of viral particles. However, the immune response initiated by the T-cell response to viral antigens is also important for viral clearance and disease pathogenesis in HBV infection. The three structural forms of the viral proteins, the HBsAg, the particulate HBcAg, and the nonparticulate HBeAg, may preferentially elicit different Th cell subsets. The different IgG subclass profiles of anti-HBs, anti-HBc, and anti-HBe in different HBV infection status were revealed. Moreover, the different IgG subclass profiles in chronic carriers did not change with different ALT and AST levels and may reflect the difference between stimulating antigens, immune response, and the stages of viral disease and provide the basis for the use of vaccines and prophylactic treatments for individuals at high risk of human HBV infection. This review elucidates the detailed understanding of the immune responses induced during transient and persistent infection, and the development of immunotherapy and immunodiagnosis in patients with HBV infection, and possible means of reducing the liver damage.     

A novel assay for hepatitis B e markers

The evaluation of a novel assay that allows simultaneous testing for hepatitis B e antigen and its antibody in a single well is described. The results of routine application and sequential studies on patients with acute hepatitis B and chronic hepatitis B treated with interferon are presented. The specificity and sensitivity of the assay and its ability to be used to follow the response of a patient during the whole seroconversion episode has been evaluated. The assay proved to give useful information about the reactivity of the sample, especially in those patients who were changing their “e” status. J. Med. Virol. 52:280–285, 1997. © 1997 Wiley-Liss, Inc.     

Physicochemical heterogeneity of hepatitis B e antigen detected in asymptomatic carriers and carriers in a hemodialysis unit.

Physicochemical studies of hepatitis B e antigen (HBeAg) revealed a clear cut difference between e1 and e2 antigen. The e1 antigen was found to have a MW of Ca 150,000 and a pI of 6.4-7.2, whereas both the MW and pI of the e2 antigen were heterogeneous depending upon the source of serum. Sera obtained from asymptomatic carriers were characterized by low titers of HBs antigen, HBc antigen and DNA polymerase and contained e2 antigen of larger molecular weight (200,000-300,000) with a narrow distribution range and a pI of 4.8 to 5.2 (type 1). On the other hand, the sera from patients in a hemodialysis unit who were HBs antigen carriers and had high titers of HBs antigen, HBc antigen and DNA polymerase contained e2 antigen of heterogeneous distribution in MW (from 300,000 to 70,000) and pI (type 2 and 3). The e2 antigen obtained from the higher MW type 3 serum had lower isoelectric points (pI 4.5 to 5.2) as was the case with e2 antigen obtained from asymptomatic carriers whereas relatively wide range of isoelectric points (pI 5.1 to 8.2) was found with the lower molecular weight e2 antigen.     

Quantitation of hepatitis B viral DNA by solution hybridization: Comparison with DNA polymerase and hepatitis B e antigen during antiviral therapy

Serological markers of hepatitis B virus (HBV) replication were assessed in a randomized, controlled trial of prednisone withdrawal followed by α -interferon in the treatment of chronic hepatitis B. HBV DNA levels in more than 700 serial serum samples from 41 patients were determined by a sensitive and quantitative solution hybridization assay. Results were compared with HBV DNA polymerase (DNAp) activity and hepatitis B e antigen (HBeAg) in 21 untreated controls and 20 treated patients. Among treated patients, the mean pretherapy HBV DNA values were higher in nonresponders than in responders. During prednisone treatment, DNA levels increased an average of 2.1-fold in responders and 1.4-fold in nonresponders. During the 2-week rest interval between prednisone and interferon, DNA values fell an average of 57% in responders. In contrast, the mean DNA values in nonresponders did not change during the same interval. This early distinction between responders and nonresponders was not apparent from DNAp or HBeAg results. During interferon treatment, HBV DNA became undetectable in responders and remained negative during a 1-year follow-up. DNA in nonresponders declined to 14% of baseline during interferon treatment but increased to pretherapy levels after treatment. DNAp values generally paralleled HBV DNA values, but DNAp activity showed more variability and lower sensitivity than did the hybridization assay results. HBeAg values varied independently of HBV DNA and DNAp with a much delayed decline in responders. These results indicate that HBV DNA, when measured quantitatively by a sensitive solution hybridization assay, is an early predictor of the effects of antiviral agents on replication.     

The separation and analysis of hepatitis B e antigen.

A solid-phase radioimmunoassay has been used successfully for detecting hepatitis B e antigen in fractionated hepatitis B virus-containing serum. Ammonium sulphate precipitation followed by gel filtration through a column of Sepharose CL-6B resulted in two fractions of antigen-containing material with molecular weights of 220,000 and 130,000. The smaller of these two fractions was found to possess an average isoelectric point of 4.9 and consisted of two major polypeptide species with estimated molecular weights of 66,000 and 17,000 respectively. Affinity chromatography on Blue Sepharose showed that e antigen was not retained under conditions which bound serum albumin. These results are discussed in relation to the immunopathogenesis of hepatitis B.     

Secular trend of age-specific prevalence of hepatitis B surface and e antigenemia in pregnant women in Taiwan

To elucidate the impact of aging of hepatitis B carrier women on their viral replicative markers in a hepatitis B endemic area, all the parturients admitted to the Hospital were studied from 1985 to 2000. Serum hepatitis B surface (HBsAg) and hepatitis B e antigen (HBeAg) were tested by radioimmunoassay. Mann-Whitney U and Student's t-tests were used for statistical analysis. The results showed the yearly prevalence rate of HBsAg in pregnant women seemed stable with a mean of 12.0 +/- 1.1% during the period. The yearly positive rate of HBeAg among HBsAg-positive pregnant women varied between 30.4% and 42.6% from 1985 to 1992 and declined from 29.6% in 1993 to 18.1% in 2000. The mean ratio of HBeAg/HBsAg in carrier parturients was 24.7% [intraquantile range (IQR) 20.5-28.4] from 1993 to 2000, which was significantly lower than that of 32.4% (IQR 31.0-39.0) from 1985 to 1992 (P < 0.0001). The mean age of HBeAg-positive primiparas from 1993 to 2000 was 29.1 +/- 3.9 years and significantly higher than that of 28.0 +/- 3.7 years from 1985 to 1993 (P < 0.001), as well as in secundiparas 31.2 +/- 3.8 years vs. 30.1 +/- 3.4 years (P < 0.001) and in total parturients 30.3 +/- 4.2 years vs. 29.3 +/- 3.8 years (P < 0.001). Thus, no significant decrease of HBsAg carriage was observed in the past 16 years, whereas a decreased ratio of HBeAg/HBsAg was noted in carrier parturients in the past 8 years and the elderly HBeAg-positive parturients from 1993 to 2000 may be the cause.     

The conversion of hepatitis B core antigen synthesized in e coli into e antigen

The e antigen (HBeAg) of hepatitis B virus (HBV) is a polypeptide of 17–20,000 daltons closely associated with the core antigen (HBcAg) of Dane particles, from which it is released by a variety of disruptive procedures. HBeAg could be a unique component of HBV core particles or a derivative of HBcAg. To resolve this question immunodiffusion experiments were carried out with preparations of HBcAg synthesized in E coli carrying a recombinant plasmid from which the HBcAg, but no other HBV gene, was expressed. HBcAg was converted into HBeAg by proteolytic degradation under dissociating conditions, thus confirming at the molecular level that HBeAg is a component of HBcAg. This offers a new route to the detection of HBeAg and antibodies to the antigen.