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Demonstration of hepatitis B e antigen in hepatitis B core particles obtained from the nucleus of hepatocytes infected with hepatitis B virus.

Liver tissue infected with hepatitis B virus was homogenized and nuclei were separated by centrifugation. Hepatitis B core particles were obtained from the nucleus by the digestion with pronase followed by ultracentrifugation in a sucrose density gradient. Hepatitis B core particles were then treated with sodium dodecyl sulphate and 2 mercaptoethanol and tested for hepatitis B e antigen (HBeAg) by the haemagglutination method. The antigenicity of HBeAg was clearly demonstrated in hepatitis B core particles so treated, although untreated core particles did not reveal any detectable HBeAg activity. The localization of HBeAg in hepatitis B core particles was further supported by the results of a fluorescent antibody technique. When a frozen section of the liver infected with hepatitis B virus was stained with the specific rabbit antibody against HBeAg labelled with fluorescent isothiocyanate, only nuclei of hepatocytes were stained, in a similar distribution to hepatitis B core antigen visualized by fluorescent antibody against hepatitis B core antigen in a nearby section.

Cell Nucleus

Hepatitis B e antigen and infectivity of hepatitis B virus.

For confirmation of the difference in the infectivity of hepatitis B surface antigen (HBS Ag)-positive serum according to differences in the e antigen system, four chimpanzees were inoculated with serum positive for hepatitis B e antigen (HBe Ag), and three chimpanzees were inoculated with serum positive for antibody to HBe Ag (anti-HBe). Since the infectivity titrations are not yet completed, the end infectivity titer of each serum is not known. All four chimpanzees given injections of 10(-1), 10(-4), or 10(-8) dilutions of HBe Ag-positive serum developed hepatitis B virus infection, whereas the one chimpanzee injected with undiluted anti-HBe-positive serum became infected, and other chimpanzees injected with diluted anti-HBe-positive sera did not. As judged from the length of the incubation period before appearance of HBS Ag in blood, there seemed to be a remarkable difference in infectivity between the HBe Ag-positive serum and the anti-HBe-positive serum; the former serum was 10(8) times more infectious than the latter.

Animals

A partial characterization of hepatitis B e antigen.

Partially purified hepatitis B e antigen (HBeAG) was prepared by ultracentrifugation, ammonium sulfate precipitation, and molecular sieve chromatography of sera obtained from asymptomatic carriers of hepatitis B surface antigen. The antigenic specificity of the HBeAG preparations was investigated further with affinity chromatography. The results indicated that HBeAG is distinct and separable from DNA polymerase activity. Columns coupled with either goat IgG prepared from antiserum to human IgG or antibody to HBeAg bound all detectable HBeAg and bound 31% and 100% of the IgG, respectively, from a partially purified HBeAg preparation. Rate zonal sucrose sedimentation and molecular sieve and ion-exchange chromatography indicated a variability in molecular weight and charge; this finding suggested a heterogenous population of immunoreactivities containing HBeAg. Our preliminary results suggest the existence of an HBeAg-IgG complex.

Carrier State

Duality of hepatitis B e antigen in serum of persons infected with hepatitis B virus: evidence for the nonidentity of e antigen with immunoglobulins.

Hepatitis B e antigen (HBeAg) is detected in the serum of some persons infected with hepatitis B virus. Owing to a close correlation of HBeAg and hepatitis B virus in the serum, it has been used as a practical indicator of infectivity. Two entities of HBeAg activity physicochemically different from each other were demonstrated in the serum of persons infected with hepatitis B virus. One was associated with a molecule that precipitated in 1.33 M ammonium sulfate solution, was larger than IgG, and had an electrophoretic mobility in the beta- to gamma-globulin regions and an isoelectric point of approximately pH 5.7. In contrast, the other HBeAg activity was associated with a molecule that was soluble in 1.33 M ammonium sulfate solution, was smaller than IgG, and had an electrophoretic mobility in the alpha-globulin region and an isoelectric point at pH 4.8. In spite of their marked physicochemical differences, a line of antigenic identity was clearly observed for them when they were tested against antibody to HBeAg by a them when they were tested against antibody to HBeAg by a double immunodiffusion method. The HBeAg activity associated with the large molecule was completely removed by an affinity column of anti-IgG, whereas the activity of the small molecule was not. These results indicate that, in the serum, HBeAg exists as a molecule smaller than IgG and also in association with IgG.

Electrophoresis, Disc

Hepatitis B e antigen and antibody activity in hepatitis B virus infection.

Groups of institutionalized subjects, volunteer blood donors with serologic evidence of asymptomatic hepatitis B virus infection, and patients with acute type B viral hepatitis were studied for the presence of hepatitis B e antigen and antibody in a sequential manner over a period of two to three years. HBeAg was detected in 9.5% of institutionalized residents and volunteer blood donors and in 24% of patients with acute type B viral hepatitis. HBeAg positive subjects frequently had persistently elevated pyruvic glutamic transaminase levels in the serum. Anti HBe activity was observed in 26 to 32% of subjects positive for hepatitis B surface antigen. Long-term follow-up indicated that HBsAg positive subjects with anti HBe did not eventually become seronegative for HBsAg.

Adolescent

Free "small" and IgG-associated "large" hepatitis B e antigen in the serum and glomerular capillary walls of two patients with membranous glomerulonephritis.

Nephrotic syndrome developed in two children who carried hepatitis B virus. Both their serums contained hepatitis B surface antigen and hepatitis B e antigen (HBeAg). Two physicochemically and immunologically different categories of HBeAg activity were identified in their serums--i.e., small molecular (free) and large molecular (associated with IgG). Their kidney-biopsy specimens disclosed pathologic changes typical of membranous glomerulonephritis. By a fluorescent-antibody technic, HBeAg was found to be deposited in diffuse granular fashion, along glomerular capillary walls together with IgG and beta1C, but no deposition of hepatitis B surface antigen was detected. The presence of HBeAg in association with IgG both in the serum and in the kidneys of these patients suggests that HBeAg caused membranous glomerulonephritis by inducing the formation and deposition of immune complexes.

Antigen-Antibody Complex

Hepatitis B e antigen, DNA polymerase activity, and infection of household contacts with hepatitis B virus.

To determine if the presence of hepatitis B e antigen (HBeAg) and elevated DNA polymerase activity in the serum of chronic HBeAg carriers indicate increased contagiousness in a household setting, the household contacts of 74 carriers were prospectively evaluated for serologic evidence of hepatitis B infection. Thirty of the HBsAg carriers had HBeAg and 44 had anti-HBe. Twenty-eight HBeAg-positive carriers regularly demonstrated elevated DNA polymerase when serially drawn serum samples were analyzed. None of the anti-HBE-positive carriers demonstrated elevation of DNA polymerase activity. Both household contacts of HBeAg-positive and anti-HBe-positive carriers demonstrated serologic evidence of hepatitis B infection (HBsAg, anti-HBs, and anti-HBc). However, infection was significantly more frequent among spouses and sexual partners of carriers who had either HBeAg (P less than 0.001) or elevated DNA polymerase activity (P less than 0.001). Thus, the data indicate that a particular subpopulation of spouses and sexual partners of hepatitis B carriers are at significantly greater risk for acquiring infection.

Adult

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.

Carrier State

Full and empty Dane particles in chronic hepatitis B virus infection: relation to hepatitis B e antigen and presence of liver damage.

Circulating complete and defective hepatitis B virus forms, as represented by full, DNA polymerase-positive and empty, DNA polymerase-negative Dane particles, respectively, were investigated in sera from patients with chronic hepatitis B virus infection and related to the presence of e antigen and antibody and to the histological findings on liver biopsy. Complete hepatitis B virus particles were detected in the serum of all patients postive for e antigen, their percentage ranging from 15 to 61% of the total Dane particle population. Although most of these cases had chronic persistent or chronic active hepatitis, complete viral particles were also found in serum of 3 healthy carriers of hepatitis B surface antigen who had e antigen. These results indicate that e antigen is a marker of active virus replication and support its association with infectivity. It is also associated with liver damage because production of complete virus is a feature of chronic hepatitis. In the presence of anti-e, detection of Dane particles in serum appeared to be related to the histological findings. Most of the healthy carriers had no Dane particles in serum, whereas 80% of the cases with chronic liver disease had circulating Dane particles. However, in contrast to the cases with e antigen, 98 to 100% of Dane particles in these cases appeared to be defective in nucleic acid material on electron microscopy after positive staining. All of the patients with chronic active hepatitis in this group had progressed to cirrhosis and it is possible that production of complete virus particles is reduced in the later stages of the illness.

Carrier State

Hepatitis B "e" antigen: an apparent association with lactate dehydrogenase isozyme-5.

Serums containing the "e" antigen of hepatitis B virus were subjected to electrophoresis in polyacrylamide gel. An extra band appeared in the lactate dehydrogenase isozyme pattern, but this band was undetectable in serums containing antibodies to the e antigenic determinant. Prior separation of the lactate dehydrogenase isozyme-5 fraction by chromatography of serum on minicolumns of diethylaminoethyl-Sephadex-A50 improved electrophoretic identification of the extra band. Neutralization with antibodies to the e antigen as well as by antibodies to the homologous d or y component of the hepatitis B surface antigen removed the extra band; antibodies to the lactate dehydrogenase isozyme-5 removed both the normal and the extra enzymatic band of isozyme-5. This feature of the e antigen provides an assay system for laboratory diagnosis of potential clinical usefulness and suggests its possible role in pathogenesis of hepatocellular injury.

Antibody Specificity

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.

Carrier State

Shift from free "small" hepatitis B e antigen to IgG-bound "large" form in the circulation of human beings and a chimpanzee acutely infected with hepatitis B virus.

Hepatitis B e antigen (HBeAg) occurs both free and in association with IgG in the circulation of individuals infected with hepatitis B virus (HBV). We determined free and IgG-bound HBeAg forms separately in the serum of humans and a chimpanzee acutely infected with HBV. In a patient with laboratory acquired HBV infection, the proportion of free HBeAg gradually decreased from 89% to 23%. A similar shift from free to IgG-bound form of HBeAg was invariably observed in 5 additional patients with acute Type B hepatitis. In a chimpanzee that had been experimentally inoculated with HBV, free HBeAg appeared first, then gradually and completely shifted to the IgG-bound form. Finally, IgG-bound HBeAg disappeared, and antibody to HBeAg was detectable in the serum. The determination of both free and IgG-bound forms of HBeAg may provide valuable information, because their ratio reflects the stage and prognosis of acute HBV infection.

Acute Disease

Quantitative correlation between the Dane particle-associated DNA polymerase and the hepatitis B e antigen.

In order to evaluate the potential infectivity of blood of hepatitis B patients, the Dane particle associated DNA polymerase was determined, which is a reliable marker for the presence of complete viral particles. Enzyme activities were compared with hepatitis B e antigen (HBeAg) titers determined by radioimmunoassay. Detectable DNA polymerase activity was only present in HBeAg positive blood, preferentially in samples with high antigen titers (1 : 1000 and above). These samples therefore have to be considered as highly infectious. However, blood with low HBeAg levels and free of detectable polymerase activity can still be infectious, since the polymerase reaction is rather insensitive compared to the radioimmunological HBeAg determination.

DNA-Directed DNA Polymerase

Application of microtiter solid-phase radioimmunoassay to the determination of hepatitis B e antigen.

Microtiter solid-phase radioimmunoassay (micro-SPRIA) was applied to the detection of hepatitis B e antigen (HBeAg) in the serum. Antibody against HBeAg to coat the microtiter plate and for radiolabeling was obtained from the serum of asymptomatic carriers of hepatitis B surface antigen by an affinity column of partially purified HBeAg. The micro-SPRIA was sensitive and could be performed without prior concentration of the test serum. HBeAg was detected in 21 (35%) out of 60 serum samples positive for hepatitis B surface antigen, all of which were negative by the conventional immunodiffusion method even after they had been concentrated threefold.

Binding Sites

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.

Antibodies, Viral