Ei Yamada

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.

Hepatitis C virus core region: helper T cell epitopes recognized by BALB/c and C57BL/6 mice

In this study, we characterized the B cell and T cell responses to the hydrophilic portion of hepatitis C virus (HCV) core protein in two strains of mice and identified the respective antigen determinants. BALB/c (H-2d) and C57BL/6 (B6:H-2b) mice were immunized by a subcutaneous injection of recombinant HCV core protein together with Freunds complete adjuvant. The level of antibody production, as determined by ELISA, was consistently higher in BALB/c than in B6 mice. However, antibodies in sera from each strain bound to the N-terminal region of the core protein within amino acids 1 to 28 (MSTNPKPQRKIKRNTNRRPQDVKFPGGG), according to an experiment using non-overlapping peptides that covered the hydrophilic portion of HCV core protein. The T cell responses were also higher in BALB/c than in B6 mice with respect to the proliferative responses of the draining lymph node cells in vitro. By limiting dilution cultures of the draining lymph node cells in vitro repetitively stimulated with recombinant core protein, T cell clones were established from both strains of mice and characterized. The surface markers of these clones were Thy-1.2+, CD3+, TCR alpha beta+, CD4+ and CD8+. The proliferative responses were inhibited in the presence of anti-CD4 or anti-MHC class II monoclonal antibodies. The T cell lines in BALB/c mice recognized an epitope in HCV core at amino acids 72 to 91 (EGRAWAQPGYPWPLYGNEGL). The T cell lines in B6 mice recognized an epitope at amino acids 55 to 74 (RPQPRGRRQPIPKARQPEGR). Thus, mice with different MHC haplotypes recognized different non-overlapping T cell antigenic determinants of HCV core proteins.

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.

Isolation and purification of a non-A, non-B hepatitis-associated microtubular aggregates protein

Blood-borne type non-A, non-B (NANB) hepatitis-associated microtubular aggregates protein was isolated and partially sequenced. The microtubular aggregates were isolated from the hepatocytes of NANB-infected chimpanzees and were found to have a buoyant density in sucrose solution of 1.21 to 1.23 g/ml. A single protein, recognized by our anti-microtubular aggregates monoclonal antibodies, was found to have an Mr of 44,000 (p44). This p44 protein was not found in uninfected chimpanzees. We determined a partial amino acid sequence for p44, and showed that it has no homology to any known proteins.

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.

cloning, sequencing and expression in Escherichia coli of cDNA for a non-A, non-B hepatitis-associated microtubular aggregates protein

A 1.7 kb cDNA encoding a novel antigen (p44; apparent Mr 44K) associated with non-A, non-B (NANB) hepatitis, was isolated from the hepatic cDNA library of a chimpanzee infected with NANB hepatitis. The library was screened with a monoclonal antibody against this antigen. The cDNA cloned contained an open reading frame encoding a 444 amino acid protein with an Mr calculated to be 50,468. The cDNA hybridized to a 1.9 kb mRNA obtained from chimpanzee hepatocytes infected with either the NANB or hepatitis delta viruses. It hybridized weakly to mRNA from hepatitis B virus-infected hepatocytes, and not at all to mRNA from normal chimpanzee hepatocytes. Southern blot analysis revealed that p44 is a host protein in chimpanzees, and that an identical gene exists in the human genome.

Production of antibodies directed against microtubular aggregates in hepatocytes of chimpanzees with non-A, non-B hepatitis.

We have previously used Epstein-Barr virus transformation to established two clonal lymphoblastoid cell lines (48-1 and S-1) producing monoclonal antibodies against microtubular aggregates that appear in the hepatocytes of chimpanzees with non-A, non-B hepatitis (NANBH). To obtain additional antibodies directed against the same structure, the mouse hybridoma method was employed. Partially purified microtubular aggregates were prepared from liver homogenates of a chimpanzee with NANBH and used as the immunogen. Hybridoma cultures were first screened by radioimmunoassay against the partially purified antigen and secondly by immunofluorescence (IF) using liver sections from a chimpanzee with NANBH. Twenty-seven cultures exhibited positive IF reactions similar to those observed with the original antibodies, 48-1 and S-1, and were cloned by limiting dilution. The specificities of the monoclonal antibodies were tested by IF on liver biopsy specimens from chimpanzees with hepatitis A, B, D or NANBH and from normal chimpanzees. All the antibodies proved to be IgG. Immunoelectron microscopy revealed that all 27 antibodies bound to the same structure, the microtubular aggregates, in hepatocytes of chimpanzees with NANBH. To determine the size of the antigen polypeptide recognized by these antibodies, polyacrylamide gel electrophoresis and Western blot assays were performed. Nine of the 27 antibodies specifically reacted with a single polypeptide of Mr 44K (p44). The remaining 18 antibodies detected no antigen polypeptide on the filters. The anti-p44 antibodies were then tested using cross-competition assays with 125I-labelled antibodies, and were found to be classifiable into three groups. In addition, the results indicate that at least three distinct epitopes are located on p44: epitope A recognized by group 1, epitope B recognized by group 2 and epitope C recognized by group 3.

Glycoprotein of Hepatitis B Surface Antigen(HBsAg) Produced by PLC/PRF/5 Hepatoma Cells

Hepatitis B virus (HBV) shows pleomorphism of three different particulate forms: small spherical particles, tubular particles and Dane particles. They share a common antigenicity of hepatitis B surface antigen (HBsAg). Small spherical particles from pooled plasma of chronic carriers of HBV have been used for biochemical characterization of HBsAg. Recently Macnab et al (5) established a tissue culture cell line (PLCfPRFj5) which produces HBsAg in the forms of small spherical and tubular particles (I, 5). This cell line enabled us to study the biochemical growth characteristics of HBV. When the polypeptide components of HBsAg produced by PLCfPRFj5 cells were examined (2,6, 7, 15), four or five polypeptides with molecular weights ranging from 22,000 to 49,000 were identified (6, 7, 15). In the present study, we characterized the polypeptide components of HBsAg with special attention to their oligosaccharide moiety by using a cloned high-producer subline of PLCfPRFj5 cells, which produced approximately 6 p,g of HBsAg per ml after 6 days-incubation. Confluent monolayer cultures of PLCjPRF j5 cells in IO-cm Falcon culture dishes were incubated for 3 days with 5 p,Ci of L-[14C]leucine per ml (355.0 mCij mmol) in leucine-free minimum essential medium (Nissui Pharmaceutical Co., Tokyo) containing 5% fetal calf serum (Flow Laboratories, Stanmore, N.S.W., Australia). Similar cultures were incubated for 3 days with D-[l, 6-3H]glucosamine (39.6 Cijmmol), D-[l-3H]mannose (13.2 Cijmmol), D-[l-3H]galactose (14.2 Cij mmol) or L-[5,6-3H]fucose (65.0 Cijmmol) (10 p,Cijml each) in RPMI-1640 medium containing 5% dialyzed fetal calf serum. All radioisotopes were purchased from New England Nuclear, Boston, Mass. Culture fluid supernatant was layered onto 30% sucrose and centrifuged for 20 hr in a Hitachi RP42 rotor to recover the HBsAg in the pellets. The pellets were suspended in 0.02 M Tris-HCI, pH 7.4-0.14 M NaCI (TBS), antibody to HBsAg (anti-HBs, prepared in goats and purified in IgG form) was added, and the mixture was incubated at 37 C for I hr and 4 C overnight. An adequate amount of serum-derived HBsAg was added to the above mixture as a carrier and incubated at 37 C for I hr. The resulting immunoprecipitate was washed three times with cold TBS containing I % Triton X-100 and I % sodium deoxycholate and another three times with distilled water.