Hybrid hepatitis B virus nucleocapsid bearing an immunodominant region from hepatitis B virus surface antigen.

A hepatitis B core antigen (HBcAg) gene bearing the 39-amino-acid-long domain A of hepatitis B surface antigen (HBsAg) within the HBcAg immunodominant loop has been constructed and expressed in Escherichia coli. Chimeric capsids demonstrated HBs but not HBc antigenicity and elicited in mice B-cell and T-cell responses against native HBcAg and HBsAg.     

Neutralization of hepatitis B virus surface antigen with immunoglobulin preparations

The in vitro experiments with immunoglobulin and blood plasma containing hepatitis B virus (HBV) markers, revealed that the control of immunoglobulin preparations for the presence of hepatitis B virus surface antigen (HBsAg), mandatory for Russia, was not sufficiently informative. The neutralization of HBsAg with specific antibodies to the level, not determined by the EIA method, reached not less than 24 ng/ ml in 2 hours of incubation and not less than 49 ng/ml in 24 hours of incubation, which, when evaluated in 1 lU of anti-HBs, was 34.6 +/- 0.9 ng and 70.7 +/- 1.8 ng of HBsAg respectively. The process of the formation of immune complexes depended mainly on the time of incubation of experimental samples and on the antibody--antigen proportion in the system. The neutralization of viruses by antibodies had no influence on the capacity of the polymerase chain reaction to detect HBV DNA.     

Quantitative assay of hepatitis B surface antigen by using surface plasmon resonance biosensor

We performed a basic experiment for the rapid, on-line, real-time measurement of hepatitis B surface antigen using a surface plasmon resonance biosensor. We immobilized anti-HBsAg (hepatitis B surface antigen) polyclonal antibody, as a ligand, to the dextran layer on a CM5 chip surface that had previously been activated byN-hydroxysuccinimide. A sample solution containing HBsAg was fed through a microfluidic channel, and the reflecting angle change due to the mass increase from the binding was detected. The binding characteristics between HBsAg and its polyclonal antibody followed the typical monolayer adsorption isotherm. When the entire immobilized antibody had interacted, no additional, non-specific binding occurred, suggesting the immunoreaction was very specific. The bound antigen per unit mass of the antibody was independent of the immobilized ligand density. No significant steric hindrance was observed at an immobilization density of approximately 17.6 ng/mm². The relationship between the HBsAg concentration in the sample solution and the antigen bound to the ligand was linear up to ca. 40 μg/mL. This linearity was much higher than that of the ELISA method. It appeared the antigen-antibody binding increased as the immobilized ligand density increased. In summary, this study showed the potential of this SPR biosensor-based method as a rapid, simple and multi-sample on-line assay. Once properly validated, it may serve as a more efficient method for HBsAg quantification for replacing the ELISA.     

Membrane structure of the hepatitis B virus surface antigen particle

Expression of S protein, an envelope protein of hepatitis B virus, in the absence of other viral proteins, leads to the secretion of hepatitis B virus surface antigen (HBsAg) particles that are formed by budding from the endoplasmic reticulum membranes. The HBsAg particles produced by mouse fibroblast cells show a unique lipid composition, with 1,2-diacyl glycerophosphocholine being the dominant component. The lipid organization of the HBsAg particles was studied by measuring electron spin resonance (ESR) using various spin-labeled fatty acids, and the results were compared with a parallel study on HVJ (Sendai virus) and vesicles reconstituted with total lipids of the HBsAg particles (HBs-lipid vesicles). HVJ and the HBs-lipid vesicles showed typical ESR spectra of lipids arranged in a lipid bilayer structure. In contrast, the ESR spectra obtained with the HBsAg particles showed that the movement of lipids in the particle is severely restricted and a typical immobilized signal characteristic of tight lipid-protein interactions was also evident. Phosphatidylcholine (PC) in the HBsAg particles was not exchangeable by a PC-specific exchange protein purified from bovine liver, while phospholipase A(2) from Naja naja vemon was able to hydrolyze all the PC in the particles. These analyses suggest that the lipids in the HBsAg particles are not organized in a typical lipid bilayer structure, but are located at the surface of the particles and are in a highly immobilized state. Based on these observations we propose a unique lipid assembly and membrane structure model for HBsAg particles.     

Detection of hepatitis B surface antigen using passive hemagglutination

The passive hemagglutination test (Sero-Test CCB) for the detection of hepatitis B surface antigen (HBsAg) has been developed. The comparative study of the sensitivity of Sero-Test CCB, the passive hemagglutination test Hepanostikon (developed by Organon, the Netherlands) and the radioimmunoassay (with the use of an experimental assay kit provided by the Institute of Vaccines in Dessau, GDR) has been carried out by the determination of HBsAg in 100 coded sera from viral hepatitis patients and hepatitis virus carriers. Both passive hemagglutination tests (Sero-Test CCB and Hepanostikon) have yielded coinciding results (r = 0.90). The sensitivity of Sero-Test CCB has been found to exceed that achieved with the use of electrophoretic techniques 30-150 times, though it is 8 times lower than the sensitivity of the radioimmunoassay. The test kits Sero-Test CCB HBsAg are used for the examination of donor blood and for the survey of groups of persons subjected to a high risk of contacting hepatitis B infection in hemodialysis and transplantation centers, surgical wards, etc.     

Vaccine- and hepatitis B immune globulin-induced escape mutations of hepatitis B virus surface antigen

Hepatitis B virus surface antigen (HBsAg) vaccination has been shown to be effective in preventing hepatitis B virus (HBV) infection. The protection is based on the induction of anti-HBs antibodies against a major cluster of antigenic epitopes of HBsAg, defined as the 'a' determinant region of small HBsAg. Prophylaxis of recurrent HBV infection in patients who have undergone liver transplantation for hepatitis B-related end-stage liver disease is achieved by the administration of hepatitis B immune globulins (HBIg) derived from HBsAg-vaccinated subjects. The anti-HBs-mediated immune pressure on HBV, however, seems to go along with the emergence and/or selection of immune escape HBV mutants that enable viral persistence in spite of adequate antibody titers. These HBsAg escape mutants harbor single or double point mutations that may significantly alter the immunological characteristics of HBsAg. Most escape mutations that influence HBsAg recognition by anti-HBs antibodies are located in the second 'a' determinant loop. Notably, HBsAg with an arginine replacement for glycine at amino acid 145 is considered the quintessential immune escape mutant because it has been isolated consistently in clinical samples of HBIg-treated individuals and vaccinated infants of chronically infected mothers. Direct binding studies with monoclonal antibodies demonstrated a more dramatic impact of this mutation on anti-HBs antibody recognition, compared with other point mutations in this antigenic domain. The clinical and epidemiological significance of these emerging HBsAg mutants will be a matter of research for years to come, especially as data available so far document that these mutants are viable and infectious strains. Strategies for vaccination programs and posttransplantation prophylaxis of recurrent hepatitis need to be developed that may prevent immune escape mutant HBV from spreading and to prevent these strains from becoming dominant during the next decennia.     

Detection of hepatitis B surface antigen in isolated hepatocytes.

HBsAg was detected by indirect immunofluorescent method in liver biopsy specimens of 60 symptom-free HBsAg positive volunteers. An effort was made to separate from the material intact cells suitable for studying HBsAg localization in the liver cells.     

Structure and expression of human hepatitis B virus surface antigen

In view of the growing occurrence rate of the virus-induced hepatitis B and also of the special role played by this particular virus (HBV) in the application of recombinant genetic techniques to the study of complex biological systems, an attempt was made to survey the available evidence concerning the widely investigated and practically the most important part of the viral genome, viz. the gene coding for the surface antigen (HBsAg) and the protein itself. The possible antigenic structure of the protein was investigated using data on the primary structure of 11 cloned HBsAg gene variants and on the synthesis of peptides simulating its immunological properties. Special emphasis was placed on quantitative assessment of antigenicity and immunogenicity. Expression of the gene in homologous systems was studied using cultures of eukaryotic tissues: both as part of HBV nucleotide sequences incorporated into the chromosome and as part of extrachromosomal DNA. The latest findings on HBsAg gene expression in yeast and bacteria are reviewed.     

Expression of the hepatitis B virus surface antigen in mammalian cells using an Epstein-Barr-virus-derived vector

 The hepatitis B virus surface antigen (HBsAg) gene, under control of the inducible mouse metallothionein I gene promoter, was inserted in an expression vector based on the Epstein-Barr virus (EBV). This vector was introduced into human cells by DNA transfection and clones were selected for their resistance to hygromycin B. The recombinant EBV vector replicates efficiently as an episome in human cells and approximately six copies per cell were found in one clone of hygromycin-B-resistant cells. These cells produce high levels of HBsAg in the presence of metals. The protein is mainly found in the cell medium, suggesting that the HBsAg is secreted from the cells. Received: 25 February 1996 / Received revision: 21 June 1996 / Accepted: 15 July 1996     

Nonoverlapping antigenic sites of woodchuck hepatitis virus surface antigen and their cross-reactivity with ground squirrel hepatitis virus and hepatitis B virus surface antigens.

Five nonoverlapping antigenic sites (sites I through V) of woodchuck hepatitis virus surface antigen were identified with competitive binding assays involving monoclonal antibodies. Site I contributed to cross-reactions among surface antigens of hepatitis B-like viruses infecting woodchucks, ground squirrels, and humans. At least three distinct sites (sites I, II, and III) are responsible for cross-reactions between woodchuck and ground squirrel hepatitis virus surface antigens. Sites IV and V of woodchuck hepatitis virus surface antigen are not major cross-reactive sites, suggesting that these elicit virus-specific antibodies. There were no cross-reactions with duck hepatitis B virus surface antigen.     

Properties of monoclonal antibodies interacting with determinants of hepatitis B virus surface antigen

Fourteen hybridoma clones have been isolated producing the monoclonal antibodies to the surface antigen of the hepatitis B virus (HBsAg). Monoclonal antibodies have been shown to react in high titres with HBsAg in the reactions of PHA, PH and ELISA. The specificity of monoclonal antibodies to two antigenic determinants has been found by the competitive solid phase ELISA technique. Monoclonal antibodies from nine clones react with one determinant while monoclonal antibodies from the rest five clones react with the other nonoverlapping determinant.     

Overcoming T cell tolerance to the hepatitis B virus surface antigen in hepatitis B virus-transgenic mice

The sequence of the hepatitis B virus (HBV) major envelope (Env) protein (ayw subtype) was scanned for the presence of H-2(d,b) motifs. Following binding and immunogenicity testing, two new H-2(d)-restricted epitopes (Env.362 and Env.364) were identified. These epitopes induced CTLs capable of recognizing naturally processed HBV-Env, but were apparently generated with lower efficiency than the previously defined dominant Env.28 epitope. Next, HBV-transgenic mice that express all of the HBV proteins and produce fully infectious particles were immunized with a mixture of lipopeptides encompassing the Env.28, Env.362, and Env.364 epitopes. Significant CTL responses were obtained, but they had no effect on viral replication in the liver, nor did they induce an inflammatory liver disease. However, in adoptive transfer experiments, CTL lines generated from the HBV-transgenic mice following immunization were able to inhibit viral replication in vivo without causing hepatitis. This is in contrast to CTL lines derived from nontransgenic mice that displayed both antiviral and cytopathic effects, presumably because they displayed higher avidity for the viral epitopes than the transgenic CTLs. These results suggest that T cell tolerance to HBV can be broken with appropriate immunization but the magnitude and characteristics of the resultant T cell response are significantly different from the response in HBV-naive individuals since their antiviral potential is stronger than their cytotoxic potential. This has obvious implications for immunotherapy of chronic HBV infection.     

Partial delipidation improves the T-cell antigenicity of hepatitis B virus surface antigen

Hepatitis B virus surface antigen (HBsAg) is a complex macromolecular particle composed of glycoproteins and lipids. The latter, representing 25% of the particle mass, are of host origin and determine the solubility, stability, and, indirectly, B-cell immunogenicity of HBsAg. HBsAg is a T-cell-dependent immunogen that does not elicit a detectable humoral immune response in 5% of HBsAg vaccine recipients and in most subjects suffering from chronic hepatitis B. We investigated the influence of the lipid content on the antigenicity of the particle. Lipids were partially removed from HBsAg by treatment with beta-D-octyl glucoside and density centrifugation. Sham treatment consisted of density centrifugation of HBsAg only. We compared the in vitro proliferative responses of established T-cell lines and nonfractionated peripheral blood mononuclear cells (PBMC) from HBsAg vaccinees and chronic HBV patients when stimulated with partially delipidated HBsAg, untreated HBsAg, or sham-treated HBsAg. In all experiments, delipidated HBsAg turned out to be 10 to 100 times more antigenic than its untreated or sham-treated counterpart. Remarkably, PBMC from vaccine nonresponders or chronic HBV patients displayed a proliferative response towards delipidated HBsAg, whereas native HBsAg never induced a response. A series of control experiments demonstrated that this enhancement of T-cell antigenicity was HBsAg specific and directly linked to lipid extraction. Nonspecific adjuvant effects of any kind could be ruled out. In vivo evaluation in mice demonstrated that delipidated particles lose most of their B-cell antigenicity. However, when native and delipidated particles were mixed, these mixtures induced equal or slightly superior anti-HBs responses to those induced by the same quantity of native HBsAg alone. In conclusion, our data show that partial delipidation of HBsAg strikingly increases the T-cell antigenicity of this unique viral antigen.     

Protection of chimpanzees from type B hepatitis by immunization with woodchuck hepatitis virus surface antigen.

Two chimpanzees immunized with woodchuck hepatitis virus (WHV) surface antigen (WHsAg) developed antibodies cross-reactive with hepatitis B virus (HBV) surface antigen (HBsAg). After challenge with HBV, one animal was completely protected and the other experienced a subclinical infection, without evidence of liver disease. Three woodchucks immunized with HBsAg developed antibodies to HBsAg which did not cross-react with WHsAg. After challenge with WHV, all three woodchucks developed typical acute infections with associated hepatic lesions. Serological studies with the cross-reactive antibodies raised in chimpanzees suggested that the protective epitopes of WHsAg were related to the group a specificity of HBsAg. These studies indicated that cross-protective epitopes are shared by HBV and WHV; however, the humoral response to these epitopes can vary among species.     

Expression of the hepatitis B virus surface antigen in Drosophila S2 cells

Drosophila melanogaster S2 cells were transfected with a plasmid vector (pAcHBsAgHy) containing the S gene, coding for the hepatitis B virus surface antigen (HBsAg), under control of the constitutive drosophila actin promoter (pAc), and the hygromycin B (Hy) selection gene. The vector was introduced into Schneider 2 (S2) Drosophila cells by DNA transfection and a cell population (S2AcHBsAgHy) was selected by its resistance to hygromycin B. The pAcHBsAgHy vector integrated in transfected S2 cell genome and approximately 1,000 copies per cell were found in a higher HBsAg producer cell subpopulation. The HBsAg production varied in different subpopulations, but did not when a given subpopulation was cultivated in different culture flasks. Higher HBsAg expression was found in S2AcHBsAgHy cells cultivated in Insect Xpress medium (13.5 μg/1E7 cells) and SFX medium (7 μg/1E7 cells) in comparison to SF900II medium (0.6 μg/1E7 cells). An increase of HBsAg was observed in culture maintained under hygromycin selection pressure. Data presented in the paper show that S2AcHBsAgHy cells produce efficiently the HBsAg which is mainly found in the cell supernatant, suggesting that HBsAg is secreted from the cells. The data also show that our approach using the Drosophila expression system is suitable for the preparation of other viral protein preparation.     

Isoprenylation mediates direct protein-protein interactions between hepatitis large delta antigen and hepatitis B virus surface antigen.

Hepatitis delta antigen (HDAg) consists of two protein species of 195 and 214 amino acids, respectively, which are identical in sequence except that the large HDAg has additional 19 amino acids at its C terminus and is prenylated. Previous studies have shown that the large HDAg and the surface antigen of hepatitis B virus (HBsAg) together can form empty hepatitis delta virus (HDV) particles. To understand the molecular mechanism of HDV virion morphogenesis, we investigated the possible direct protein-protein interaction between HDAg and HBsAg. We constructed recombinant baculoviruses expressing the major form of HBsAg and various mutant HDAgs and used these proteins for far-Western protein binding assays. We demonstrated that HBsAg interacted specifically with the large HDAg but not with the small HDAg. Using mutant HDAgs which have defective or aberrant prenylation, we showed that this interaction required isoprenylates on the cysteine residue of the C terminus of the large HDAg. Isoprenylation alone, without the remainder of the C-terminal amino acids of the large HDAg, was insufficient to mediate interaction with HBsAg. This study demonstrates a novel role of prenylates in HDV virion assembly.