Hitoshi Ohori

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.

Antigenic conversion from HBcAg to HBeAg by degradation of hepatitis B core particles.

Highly purified and homogenous hepatitis B core particles were obtained from an autopsy liver. The core particles consisted mainly of an electrophoretically single major polypeptide with a molecular weight of 20,000 daltons. When antigenic conversion from hepatitis B core antigen (HBcAg) to hepatitis B e antigen was achieved by treating these core particles by sonication or by passing them through an anti-HBc IgG-conjugated Sepharose 4B column, no appreciable changes were found in the above protein composition. The same antigenic conversion was also achieved by centrifugation of core particles in CsCl, which revealed the process of morphological disintegration accompanied by antigenic conversion. These observations may support the hypothesis that HBcAg resides on a protein conformation which consists of a polypeptide sharing HBe antigenicity.

Immunological and morphological properties of HBeAg subtypes (HBeAg/1 and HBeAg/2) in hepatitis B virus core particles

A polypeptide of 21 500 mol. wt., structurally associated with hepatitis B virus core particles, was shown to have two kinds of HBeAg antigenicity (HBeAg/1 and HBeAg/2). This was revealed by transferring a single core peptide from polyacrylamide gels to nitrocellulose sheets (Western blotting), which reacted with anti-HBeAg/1 and anti-HBeAg/2. Selective discrimination of the two HBe antigens was achieved by radioimmunoassay (RIA). When highly purified core particles were incubated at 37 degrees C in a 0.1% SDS-0.1% 2-mercaptoethanol solution, only HBeAg/1 was released after 5 min incubation and the release of HBeAg/2 occurred only after prolonging incubation for 30 min. The course of degradation was also detected by CsCl density gradient centrifugation. These results indicate that HBeAg/1 is less closely associated with core particles than is HBeAg/2. Electron microscopy showed that the core particles from which HBeAg/1 was removed were more labile than the original preparation when incubated at 56 degrees C in aqueous solution, or at 37 degrees C in Sarkosyl solutions; when placed in 1 M-NaCl or -CsCl solution, the particles swelled to a larger diameter than untreated cores.

Molecular heterogeneity of hepatitis B virus e antigen in liver and serum.

Hepatitis B virus e antigen (HBeAg) derived from liver at autopsy or from the serum of asymptomatic carriers has been characterized. The liver-derived HBeAg consisted of two different molecules, one with a mol. wt. of 30 000 (monomer) and the other with a mol. wt. of 90 000 (trimer), in a ratio of 3:1. Both were free of IgG. The serum-derived HBeAgs were heterogeneous with mol. wt. of 30 000, 90 000, 240 000, 400 000 and 540 000. Among them, the so-called IgG-free HBeAgs consisted almost exclusively of the 30 000 and 90 000 molecular species, in a ratio of 1:9. The serum HBeAg of mol. wt. 90 000 was further differentiated into two molecular species, one trimer and the other associated with albumin. The large mol. wt. HBeAgs (240 000, 400 000 and 540 000) were associated with IgG in ratios of one molecule of HBeAg to one, two or three molecules of IgG respectively. The complete dissociation of the IgG molecule was not achieved by 5 M-urea treatment of such HBeAgs, suggesting that it was bound in an immune complex. A hypothetical model is proposed which describes the heterogeneity of the HBeAgs derived from both the liver and serum, and containing HBeAgs either in a free form or associated with serum IgG.

Comparison of the Antigenicity and Protein Composition of Hepatitis B Virus e Antigen Subtypes HBeAg/1 and HBeAg/2

Summary Hepatitis virus B e antigen subtypes HBeAg/1 and HBeAg/2 can be distinguished by a micro-Ouchterlony immunodiffusion assay and a solid-phase radioimmunoassay using serum IgG-associated HBeAgs and liver- and serum-derived IgG-free HBeAgs. The liver- and serum-derived HBeAg consisted of two different antigenicities, HBeAg/1 and HBeAg/2. Both the liver-derived HBeAg/1 and HBeAg/2 had mol. wt. of 30000 (monomer) and 90000 (trimer) and shared the same isoelectric point of 4.3 to 4.8. Approximately 80% of liver-derived HBeAg (mol. wt. 30000 and 90000) had HBeAg/1 activity and the remaining 20% was HBeAg/2. The serum-derived HBeAg/1 and HBeAg/2 were both associated with IgG to form heterogeneous moieties with apparent mol. wt. of 240000, 400000 and 540000 or were free, with mol. wt. 30000 (monomer) and 90000 (trimer). The serum HBeAg of mol. wt. 90000 was further differentiated into two molecular species, one trimer and the other associated with albumin. Large types of both HBeAg/1 and HBeAg/2 were recovered by isoelectrictric focusing at pH 5.5 to 7.5 (mol. wt. 240000), 5.7 to 8.0 (mol. wt. 400000) and 6.4 to 8.4 (mol. wt. 540000) respectively, while small types of both exhibited a narrower range of isoelectric points similar to liver-derived antigens. In most sera, antigenicity of half of the serum IgG-associated HBeAg was HBeAg/1 and the remaining 50% was HBeAg/2, whereas 80% of the free HBeAg had HBeAg/1 activity and the remaining 20% was HBeAg/2. These results indicate that HBeAg/1 and HBeAg/2, although antigenically distinct, are very similar physicochemically with respect to both mol. wt. and isoelectric point. Dissociation of the ‘large’ HBeAg/1 and HBeAg/2 moieties into three mol. wt. species (160000, 32000 and 16000) was achieved by 6-m-guanidine-HCl treatment followed by gel filtration through Sepharose 6B. The 160000 species was IgG, having both anti-HBeAg/1 and anti-HBeAg/2 activities. In addition, the lower mol. wt. polypeptides (32000 and 16000) exhibited both HBeAg/1 and HBeAg/2 activities. Therefore, we suggest that the IgG associated with the HBeAg molecule forms a true immune complex rather than a non-specific association between HBeAg and IgG. Furthermore, the 32000 mol. wt. polypeptide split into two species of 16000 mol. wt. when heated at 100 °C for 2 min. On the basis of these results, the 16000 mol. wt. polypeptide may be assumed to be the essential polypeptide bearing HBeAg/1 and HBeAg/2 activities in the liver and serum.

Intra- and extracellular distribution and immunochemical characterization of hepatitis B virus nucleocapsid proteins produced by a human hepatoma cell line transfected with cloned viral DNA.

Hepatitis B virus (HBV)-related antigens produced by the human hepatoma cell line (HB611 cell), which had been transfected with a cloned HBV DNA and established as a stable producer of HBV (T. Tsurimoto, A. Fujiyama, and K. Matsubara, 1987, Proc. Natl. Acad. Sci. USA 84, 444-448), were investigated immunochemically and morphologically. All HBV-related antigens, HBV surface (HBsAg), e (HBeAg), and core (HBcAg), were semiquantitatively examined by the respective reversed passive hemagglutination assay (RPHA). RPHAs for HBcAg and for HBeAg were characterized as reacting only to the core particles and to the free form of nucleocapsid proteins, respectively. The amounts of HBsAg and nucleocapsid protein in culture medium were roughly related to the number of viable cells. The amount of core particles was, instead, proportional to the number of dead cells. Relative amounts of HBsAg, core particles, and nucleocapsid proteins in culture medium, cell surface, and cell lysate were determined and it was found that HBsAg and nucleocapsid proteins were effectively secreted into culture medium but core particles were not. Molecular species of nucleocapsid proteins were identified to be p17 and p18 (HBeAg polypeptides) in the culture medium and HBeAg polypeptides and p21.5 (HBcAg polypeptide) in the cytosol fraction. The p21.5 was preferentially found in the nuclear fraction.

Circular dichrosim and biochemical properties of the hepatitis B virus core antigen

The hepatitis B virus (HBV) core antigen was purified by mild procedures, including hydroxyapatite column chromatography, with care taken to avoid the degradation of the particles. Circular dichroism (CD) of the HBV core particles in saline showed low intensities of negative ellipticities in the region dominated by amide bond absorption. Acid treatment of the particles induced a remarkable change in the CD spectrum, with the appearance of a positive extremum at about 208 nm. The amino acid composition and the COOH-terminal residue of the isolated core polypeptide (Mr 21,000-21,500) were shown to be essentially the same as those of the polypeptide deduced from the nucleotide sequences which had been proposed for the HBV core antigen by other laboratories. We failed to detect any NH2-terminal dansyl-derivatives from the core polypeptide by the dansyl-Edman method. We also showed by the method of fluorescein polarization that the core polypeptide conjugated with fluorescein isothiocyanate has an affinity for serum albumin. This may indicate a state of disassembled or non-assembled core polypeptide in sera.