Failure of antibody to e antigen to precipitate Dane particles containing DNA polymerase activity and hepatitis B core antigen

Serum samples of 67 asymptomatic carriers were tested for Dane particles by electron microscopy, and for e antigen by immunodiffusion, as well as for hepatitis B antigen-associated DNA polymerase activity, and four samples enriched with respect to Dane particles were selected. Hepatitis B antigen particles in them were separated from e antigen and concentrated by centrifugation, and the Dane-rich preparations were incubated with buffer, antibody to e antigen (anti-HBe) or antibody to hepatitis B surface antigen. After centrifugation, the supernatant was tested for DNA polymerase activity, and both supernatant and precipitate were tested for hepatitis B core antigen (HBcAg) by the immune adherence haemagglutination method after the coat of the Dane particles had been disrupted with NP-40 and 2-mercaptoethanol. It was found that anti-HBe did not precipitate Dane particles as measured by DNA polymerase activity and HBcAg. On the basis of the results obtained, it has been concluded that e antigen does not exist on the surface of hepatitis B virions.     

Hepatitis B antigen-associated deoxyribonucleic acid polymerase activity and e antigen/anti-e system.

Serum samples of 403 asymptomatic blood donors carrying hepatitis B surface antigen (HB5Ag) were concentrated threefold and tested for e antigen and antibody to e antigen (anti-e) by immunodiffusion. Hepatitis B antigen (HBAg)-associated deoxyribonucleic acid (DNA) polymerase activity was specifically determined by the difference in incorporation of [methyl-3H]thymidine 5' -triphosphate into DNA by an aliquot of centrifuged serum samples after it had been treated either with normal rabbit serum or with rabbit antibody to HBSAg. All of 58 serum samples containing e antigen revealed HBAg-associated DNA polymerase activity, whereas none of 96 samples containing anti-e did. In the remaining 249 samples in which neither e antigen nor anti-e was found, 62 showed specific DNA polymerase activity, although at lower levels than the samples containing e antigen.     

The proteins of hepatitis B Dane particle cores.

Although several studies have been done to analyze the peptides of purified 22-nm HbsAg particles, no information has been published about the peptides of the core of the Dane particle which bears the other hepatitis B viral antigen. HbcAg. Dane particles and Dane particle cores (produced by NP-40 treatment of Dane particles) were purified by equilibrium centrifugation in CsCl density gradients. Two populations of Dane particles were observed at densities 1.27 and 1.24 g/ml, respectively. The higher buoyant density Dane particles yielded exclusively cores of buoyant density 1.38 g/ml in CsCl, and the lower buoyant density Dane particles yielded two kinds of cores with buoyant densities of 1.38 and 1.325 g/ml, respectively. Only the higher density Dane particles and cores manifested endogenously primed DNA polymerase activity. The peptides of density 1.38 g/ml Dane particle cores purified by equilibrium CsCl density gradient centrifugation and HBcAg particles from HBV infected chimpanzee liver purified in the same way were analyzed by SDS-polyacrylamide gel electrophoresis. Both kinds of particles were found to consistently contain 3 Coomassie blue staining peptides with approximate molecular weights of 19,000, 70,000 and 80,000 daltons (designated P-19, P-70 and P-80 respectively). In addition, the HBcAg particles from infected liver regularly yielded a protein component with molecular weight greater than 200,000 daltons. This component was occasionally present in electrophoresis runs of core peptides from only one of two patients. Its irregular appearance after gel electrophoresis suggests it may have been an aggregate not completely dissociated under the conditions used. The lower density core component consistently contained P-19, P-70, and P-80, and infrequently additional minor peptides of uncertain origin. The irregular occurrence of the minor peptides in varying amounts suggests they were not intrinsic core proteins.     

Demonstration of hepatitis B e antigen (HBeAg) in association with intact Dane particles.

Mild detergent treatment (0.1% Sarkosyl-0.1% beta-mercaptoethanol) of Dane particle-rich fraction from human serum resulted in the release of core particles together with HBe antigen activity when examined by the reversed passive haemagglutination method. Furthermore, when the core particles isolated by the above procedure were exposed to stronger detergent (1% Sarkosyl-0.1% beta-mercaptoethanol), additional HBe antigen activity was released only from intact core particles with DNA polymerase activity and not from empty core particles.     

Expression of hepatitis B virus-specific markers in asymptomatic hepatitis B surface antigen carriers.

A study was undertaken to assess the state of hepatitis B virus infection in a group of asymptomatic hepatitis B surface antigen (HBsAg) carriers. This study confirmed that the presence of hepatitis B e antigen (HBeAg) in serum was closely associated with serum HBsAg-specific deoxyribonucleic acid polymerase activity, hepatitis B core antigen (HBcAg) in serum and liver cell nuclei, and a histological picture of chronic hepatitis. No HBsAg-specific deoxyribonucleic acid polymerase activity or HBcAg was detected in highly concentrated anti-HBe-positive sera. In addition, liver biopsy specimens from carriers with anti-HBe were negative for HbcAg by immunofluorescence, and the liver histology was either normal or revealed only fatty changes. These data indicate that the anti-HBe-positive sera contained either no Dane particles or, if present, at least a 500-fold-lower concentration of Dane particles than that found in HBeAg-positive sera.     

Failure to detect naturally occurring serum inhibitors of hepatitis B virus deoxyribonucleic acid polymerase.

Sera of patients with past or ongoing hepatitis -B virus infection were tested for the presence of inhibitors of hepatitis -B virus-specific deoxyribonucleic acid (DNA) polymerase activity. None of the sera tested, which included those from anti-hepatitis B surface- and anti-hepatitis B core antigen-positive hemophiliacs, anti-hepatitis Bc antigen-positive hepatitis B surface antigen carriers, patients with hepatitis B surface antigen-positive chronic active hepatitis, hepatitis B surface antigen-positive hemodialysis patients, tumor patients with minimal hepatitis, patients with acute type B, type A, and type non-A, non-B hepatitis and individuals with autoimmune phenomena, contained inhibitors of DNA polymerase activity. This implies that the DNA polymerase test is not affected when utilized to quantitate DNA-containing Dane particles. In addition, there is no evidence that inhibitors of DNA polymerase activity play some pathogenic role in the course of hepatitis B virus infection.     

Non-a, non-b hepatitis: identification of hepatitis-B-like virus particles in serum and liver

Hepatitis B virus-like particles including: small spheres and filaments 15--25 nm in diameter together with a 35--40 nm Dane particle-like virion have been identified in sera of patients with non-A, non-B hepatitis. In a coded serological study, such particles were detected transiently in 3/4 acute, and persistently in 7/8 chronic cases of non-A, non-B hepatitis with non-A, non-B antigenemia. Only 2/12 similar cases without non-A, non-B antigens (Ag) in serum had detectable particles but neither patients with drugs, or type A hepatitis, nor cases of obstructive jaundice. The particles did not express hepatitis B surface (HBs) or non-A, non-B Ag at their surface but were associated, in three patients, with significant endogenous DNA polymerase activity. Furthermore, particles similar to hepatitis B cores (BHc) and also associated with DNA polymerase activity were demonstrated by sucrose gradient ultracentrifugation of a liver homogenate obtained from a patient who had died of non-A, non-B hepatitis. The non-A, non-B hepatitis virion described here appears, therefore, as a hepatitis B-like virus. The exact kinship between these two agents is currently being investigated.     

Non-A,non-b hepatitis virus: identification of a core antigen-antibody system that cross reacts with hepatitis b core antigen and antibody

Using immunodiffusion, a major cross-reactivity had been previously demonstrated between hepatitis B(HBe/3 Ag) and the antigen reported in the serum of non-A, non-B hepatitis patients, therefore redesignated NANBe Ag. By direct immunofluorescence a new antigen associated with, but distinct from, NANBe Ag has now been identified in the liver of 14/26 patients with NANB chronic active hepatitis. The homologous antibody was detected in the serum of these 14 patients. Behaving like HBc Ag and cross reacting with it by immunofluorescence, the new antigen was termed NANBc Ag. Anti NANBc also became detectable in serial acute phase and convalescence sera from 5/5 NANB Ag-positive posttransfusion hepatitis cases. Further characterization of NANBe and NANBc antigens achieved by fractionation of a NANB virus-infected liver showed NANBc Ag to be expressed on 22–25 nm HB core-like particles containing DNA polymerase activity. Cross-reactivity between NANBc and HBc antigens was confirmed by immunodiffusion. Liver-derived NANBe Ag identical to serum NANBe Ag exhibited the same physical properties as HBe/3 Ag and could be similarly released by disruption of the non-A, non-B virus cores. These results indicate that hepatitis B and NANB virions not only share the same structure and DNA polymerase activity but are also antigenically related and belong to the same new class of DNA viruses.     

A modified technique for the detection of hepatitis B virus-specific DNA polymerase

A modified and improved technique for the detection of hepatitis B virus-specific DNA polymerase activity is described. DNA polymerase is released from Dane particles by mixing samples with the detergent Nonidet P-40 and β-mercaptoethanol. After incubation of pretreated samples with a reaction mixture containing tritiated thynudine-methyl-5'-triphosphate (³H-TTP), DNA is precipitated onto a trichloroacetic acid (TCA)-treated paper. Unincorporated ³H-TTP is then chromatographically eluted with a 5% TCA solution and precipitated counts are determined. A sample is considered positive for DNA polymerase if the incorporated counts are significantly higher than the counts of a group of negative control samples.The modifications include pretreatment of the paper with TCA, Chromatographic elution of unincorporated ³H-TTP with TCA solution, prefiltration of the sample through bacteriological filters, and use of sound statistical methods for evaluation of data. These changes have led to a highly reproducible, reliable and sensitive technique. The coefficient of variation of negative control samples from various test runs was in the range of 2.7–8.5%. A linear relationship between incorporated counts and DNA polymerase concentration was shown.A total of 419 serum samples from asymptomatic HBsAg-carrying blood donors were tested. Twenty-three (5.5%) of these were found to contain detectable DNA polymerase activity. All 23 samples also contained HBeAg.     

Morphological irregularities in Dane particle cores

Electron microscopy of hepatitis B antigen has revealed Dane particles with abnormal morphology. These aberrant Dane particles contain incomplete cores. They were seen in large numbers in the serum of an HBsAg-carrying renal dialysis patient and were less numerous but invariably present in other Dane particle-containing sera --12 from asymptomatic carriers, 2 chronic hepatitis patients and 1 patient with acute hepatitis. All 16 sera were shown to contain HBeAg.     

Quantitation of hepatitis B virus (HBV) core antigen in serum in the presence of antibodies to HBV core antigen: comparison with assays of serum HBV DNA, DNA polymerase, and HBV e antigen.

A quantitation procedure for hepatitis B core antigen (HBcAg) in serum without prior removal of antibodies to HBcAg is described. The virus nucleoprotein core was released from hepatitis B virus (HBV) particles by treatment with Nonidet P-40 detergent and allowed to form immune complexes with homologous antibodies to HBcAg present in the sera of HBV-infected individuals. After precipitation with 2.0% polyethylene glycol-1.5% Tween 20, the HBcAg immune complexes were dissociated by treatment with 3 M KSCN and then adsorbed onto polystyrene beads in the presence of the SCN- ions. Thereby, HBcAg and antibodies to HBcAg were linked independently of each other to the matrix, and the core antigen could be quantitated directly by incubation of the beads with 125I-labeled anti-HBc. Even in the presence of an excess of antibodies to HBcAg in the polyethylene glycol precipitates, HBcAg could be detected without appreciably affecting the sensitivity. The assay proved to be specific for core determinants and exhibited excellent reproducibility. The application of the HBcAg assay in 185 hepatitis B e antigen-positive sera revealed HBc antigenemia in 99% of the sera containing hepatitis B e antigen at titers of greater than or equal to 1:256 and 43% of the sera with lower hepatitis B e antigen levels. However, only in 6 of the 34 HBcAg-negative sera could HBV DNA be detected by blot hybridization. When correlated with HBV-associated DNA polymerase (DNAP) activity, HBc antigenemia was found in all DNAP-positive sera (n = 95) and in 39% of the hepatitis B e antigen-positive sera without detectable DNAP activity (n = 44). Of the DNAP-negative sera with HBc antigenemia, 94% contained HBV DNA, whereas in the absence of HBcAg, HBV DNA could be detected only in 3 of 27 DNAP-negative sera. With regard to sensitivity, the HBcAg assay appeared to be less sensitive than the hybridization technique, but more sensitive than the DNAP assay.     

Comparison of indicators for dane particles

Summary The Dane particle is — because of its characteristics — believed to be the complete hepatitis-Bvirus. Sera containing high numbers of Dane particles were shown to be highly infectious. In the present study we related the HBeAg-, the HBsAg- and the anti-HBc titer to the level of DNA polymerase activity measured in 20 fold Dane particle concentrates. The data obtained indicate that the HBeAg concentration gives a semiquantitative estimate on the number of circulating Dane particles. Mean DNA polymerase activity was found to increase with HBsAg concentration and is therefore also of value-if determined in a HBeAg positive serum- for quantitation of Dane particles. The anti-HBc titer was found to be unrelated to the number of circulating Dane particles.This study was supported by the Deutsche Forschungsgemeinschaft (He 994/2)     

Immunological studies in children with acute viral hepatitis.

Sera from 116 consecutive unselected cases of sporadic acute viral hepatitis in children were examined for hepatitis B antigen (HBAg), smooth-muscle autoantibodies (SMA), other autoantibodies and immunoglobulins, and skin tests were performed with dinitrochlorobenzene (DNCB). HBAg was detected in twenty-one and SMA in ninety-eight out of 116 sera that had been obtained during the 1st or 2nd week from the onset of jaundice. Hepatitis B antigen was present in seventeen out of the eighteen SMA negative patients (94-4%) and in only four out of the ninety-eight SMA-positive patients (4-1%). The presence of SMA was not related to the sex and age of the patients or to the serum bilirubin and transaminase levels. SMA did not persist for more than 6 weeks from the onset of jaundice in most of the cases. In twenty-eight out of forty-one sera which were tested the IgM level was found to be elevated during the acute phase of illness and within normal limits during the recovery stage. A negative correlation between the presence of SMA and the elevated serum IgM level and the presence of HB Ag in the same patients was observed. The DNCB skin test was found to be positive in all fifty-two patients who did not have HBAg in their serum and in twenty out of the twenty-one patients who had circulating HbAg. From these findings there appears to be no gross impairment of cell-mediated immunity in acute viral hepatitis, and hepatitis A is associated with SMA production and an increase in serum IgM levels, when compared to hepatitis associated with HBAg.     

Identification of additional antigenic sites on Dane particles and the tubular forms of hepatitis B surface antigen.

Additional antigenic sites, distinct from those present on spherical 20 nm diam. particles of hepatitis B surface antigen (HBsAg), are exposed on the surface of Dane particles and tubular forms of HBsAg. The immunological relationship of these sites to e-antigen, an antigen detected earlier in HBsAg-positive sera from patients with chronic hepatitis, cirrhosis or acute hepatitis but not in healthy HBsAg-carriers, was established by immune electron microscopy and affinity chromatography. These findings suggest that e-antigen may be potentially useful in active immunization against hepatitis B.     

Biochemical characterization of Australia antigen. Evidence for defective particles of hepatitis B virus.

Australia antigen exists in the sera of chronic carriers in several particulate forms, one of which may represent the virion of hepatitis B. This report describes the existence of subpopulations of these 43-nm particles, the Dane particles, on the basis of the staining properties of their internal cores and banding characteristics in cesium chloride (CsCl) density gradients. These data suggested that only a minor proportion of Dane particles contained an intact viral genome and represent the standard infectious virus of hepatitis B. The bulk of the Dane particles appeared to be deficient in viral nucleic acid and, as defective interfering particles, may specifically interfere with the growth of standard virus. Such defective interfering particles could thereby play a role in the persistence of HBV infection in man.     

Association of hepatitis B e-antigen (HBeAg) determinants with the core of Dane particles.

Immunoprecipitates obtained by reacting Dane particle cores with human antibodies to hepatitis B core antigen (HBcAg) were chromatographed on columns of Sepharose 4B CL using 3 M-NaSCN as eluant. An antigen having the size and immunological specificity of monomeric e-antigen (HBeAg) was separated from HBcAg by this method. Antisera from animals immunized with HBcAg or HBeAg reacted not only with the antigen used for immunization but also with HBeAg and HBcAg, respectively. This indicates that HBeAg determinants are associated with the core of Dane particles.     

Distribution of HBcAg in hepatitis B detected by immunoperoxidase staining with three different preparations of anti-HBc antibodies.

To evaluate the role of the expression of hepatitis B core antigen (HBcAg) in liver cell damage the immunoperoxidase staining pattern of cryostat liver biopsy specimens from 16 chronic carriers of hepatitis B surface antigen (HBsAg) was investigated using three different kinds of anti-HBc antibodies. Polyclonal antibody prepared from recombinant HBcAg seemed to be more sensitive in detecting HBcAg than did monoclonal antibody from the same antigen. The topographical distribution of HBcAg detected by these two antibodies was similar, showing a close correlation to the histological activity of disease. Furthermore, the predominant localisation of cytoplasmic HBcAg usually reflected an active and severe ongoing hepatitis. On the other hand, monoclonal antibody prepared from purified Dane particles resulted in the prominent cytoplasmic staining for HBcAg regardless of histological severity of the hepatitis. The quantitative expression and topographical distribution of HBcAg depended on the type of anti-HBc antibodies used.     

Expression of hepatitis B virus core and precore antigens in insect cells and characterization of a core-associated kinase activity

The hepatitis B virus core open reading frame with and without the precore domain was expressed in insect cells using a baculovirus expression system. Precore antigen was not properly processed in insect cells and was present in highly insoluble cytoplasmic aggregates. Core antigen without the precore domain formed core particles with a diameter of 28 nm that were secreted into the medium. Both core and precore antigens were phosphorylated in insect cells. The immune response in mice to both antigens yielded antibodies with a high degree of preferential reactivity for the homologous immunizing polypeptide. A kinase activity that phosphorylated core antigen was associated with highly purified core particles. The kinase activity resembled that previously demonstrated for core particles purified from the cytoplasm of infected hepatocytes and detergent-treated Dane particles. Partial resistance of the phosphate-label to phosphatase treatment suggested that some of the phosphorylated sites are in the interior of the particle. The presence of kinase activity in recombinant core particles demonstrated that this activity is not derived from another hepatitis B virus-encoded polypeptide, and the lack of a kinase consensus sequence in the core open reading frame suggests that the kinase is of cellular origin.     

Application of hepatitis B core particles produced by human primary hepatocellular carcinoma (PLC/342) propagated in nude mice to the determination of anti-HBc by passive hemagglutination

Human primary hepatocellular carcinoma (PLC/342), carried by nude mice, produces hepatitis B core particles as well as hepatitis B surface antigen particles. Core particles purified form PLC/342 tumors displayed epitopes of hepatitis B core antigen (HBcAg) but not epitopes of hepatitis B e antigen (HBeAg) on their surface, unlike core particles prepared from Dane particles, derived from plasma of asymptomatic carriers, that expressed epitopes of both HBcAg and HBeAg. Core particles obtained from PLC/342 tumors were applied to the determination of antibody to HBcAg (anti-HBc) by passive hemagglutination. The assay detected anti-HBc not only in individuals with persistent infection with hepatitis B virus and in those who had recovered from transient infection, but also in patients with acute type B hepatitis, indicating that it can detect anti-HBc of either IgG or IgM class. A liberal availability of core particles from tumors carried by nude mice, taken together with an easy applicability of the method, would make the passive hemagglutination for anti-HBc a valuable tool in clinical and epidemiological studies, especially in places where sophisticated methods are not feasible.