Hepatitis B Virus Surface Antigen Particles Research Articles

Infectivity of hepatitis B virus (HBV) surface antigen (HBsAg) positive plasma with undetectable HBV-DNA: Can HBsAg screening be discontinued in Egyptian blood donors?

HBV infectivity data were reviewed and the 50% infectious dose (ID50 ) was reassessed in different HBsAg positive infection stages enabling modelling of transfusion-transmitted (TT)-HBV infection risk if HBsAg donor screening was replaced by individual donation nucleic acid amplification technology (ID-NAT). Quantitative HBsAg and HBV-DNA assays were performed against international standards to compare the ratio between potential infectious HBV virions and subviral HBsAg particles in Egyptian HBsAg positive blood donors as well as in Japanese chimpanzee samples of known infectivity. HBV-DNA load below the quantification limit of detection was estimated against a reference standard by replicate NAT testing (n=25). Infectivity of chimpanzee samples collected during ramp-up and declining viremic phase were tested in a human liver chimeric mice (HLCM) model and compared with published infectivity data from different HBsAg positive infection stages. Lowest estimates of ID50 in HBsAg positive plasma were 3-6 HBV virions in chimpanzee studies. Infectivity decreased approximately 10-100-fold in the declining viremic phase using HLCM. In acute-phase samples, HBV to HBsAg particle ratios varied between 1:102 -104 but in HBsAg positive blood donors this particle ratio reached 1:106 -1012 when viral load was below 100 HBV-DNA copies/ml. Modelled TT-HBV risk of an HBsAg positive/ID-NAT nonreactive blood transfusion was estimated at 9%-46% for components containing 20-200ml of plasma assuming an ID50 of 316 (point estimate between 100 and 1000) virions. In the Egyptian setting, discontinuation of HBsAg donor screening and reliance on ID-NAT alone seems to be unsafe.

Targeting Hepatitis D.

New therapeutic strategies to treat chronic hepatitis D are directed to deprive the hepatitis D virus (HDV) of functions necessary to complete its life cycle that are provided by the hepatitis B virus (HBV) and by the host. Current options are (1) the block by the synthetic peptide Myrcludex B of HBV surface antigen (HBsAg) entry into cells through the inhibition of the sodium taurocholate cotransporting receptor; (2) the inhibition with lonafarnib of the farnesylation of the large HD antigen, required for virion assembly; (3) the presumed reduction by the nucleic acid polymer REP 2139 of the release of the HBsAg and subviral HBV particles necessary for HD virion morphogenesis. Lonafarnib and Myrcludex in monotherapy reduced serum HDV-RNA but did not reduce the HBsAg and HD viremia rebounded after therapy; they may provide additional efficacy to pegylated interferon alpha (Peg IFN-α) therapy. Treatment with REP-2139 in combination with Peg IFN-α induced a sustained clearance both of the HDV-RNA and HBsAg in 5 of 12 patients, providing the best interim results so far obtained in the therapy of chronic hepatitis D.

Characterization of the 3rd International Standard for hepatitis B virus surface antigen (HBsAg)

BackgroundHBsAg is the most important marker for laboratory diagnosis of HBV infection. Validation and quality control of HBsAg tests requires International Standards (IS). Recently the 2nd IS was replaced by the 3rd IS. Both IS are made from plasma-derived hepatitis B vaccines, but production and geographical origin are different. ObjectiveCharacterization of the HBsAg in the source material (SM) for the 3rd IS and comparison with the 2nd IS and native HBsAg. Study designThe SM was analyzed using solid-phase immunoassays, quantitative immune electrophoresis, ultracentrifugation, immunoblotting and HBV DNA sequencing. ResultsThe plasma-derived HBsAg of the SM originated from at least two different HBV strains, both of subgenotype (sgt) B4, typical for Vietnam. The HBsAg subtype was heterogeneous with ayw1 and adw2. The HBsAg concentration was 23,700 IU/ml as determined by solid-phase immunoassay; immune electrophoresis calibrated with sgt B2 revealed a concentration of 24,500 IU/ml while calibration with sgt D1 provided lower values. Proteins in the SM are heterogeneous in size containing only traces of preS. The protein subunits are partially cross-linked. ConclusionsThe antigenicity of the 3rd IS is suitable for HBsAg calibration in laboratory tests. In contrast to the 2nd IS, the 3rd IS is representative for a highly endemic region. Similar to the 2nd IS and different from native HBsAg, preS domains are depleted, protein subunits are partially cross-linked and the HBsAg particles are partially aggregated in the 3rd IS. The HBV subgenotype differences between the two IS may lead to variations in different quantitative assays.

Nanoscale topography of hepatitis B antigen particles by atomic force microscopy

Hepatitis B virus envelope is mainly composed of three forms of the same protein expressed from different start codons of the same open reading frame. The smaller form named S protein corresponds to the C-terminal common region and represents about 80% of the envelope proteins. It is mainly referred as hepatitis B virus surface antigen (HBsAg). Over expressed in the host cell, this protein can be produced as spherical and tubular self-organized particles. Highly immunogenic, these particles are used in licensed hepatitis B vaccines. In this study we have combined transmission electron microscopy and atomic force microscopy to determine the shape and size of HBsAg particles produced from the yeast Hansenula polymorpha. Tapping mode atomic force microscopy in liquid allows structural details of the surface to be delineated with a resolution in the nanometer range. Particles were decorated by closely packed spike-like structures protruding from particle surface. Protrusions appeared uniformly distributed at the surface and an average number of 75 protrusions per particle were calculated. Importantly, we demonstrated that proteins mainly contribute to the topography of the protrusions.

Hepatitis B virus surface antigen suppresses the activation of monocytes through interaction with a serum protein and a monocyte-specific receptor.

During hepatitis B virus (HBV) infection, high numbers of non-infectious HBV surface antigen (HBsAg) particles are present in circulation. It is shown here that recombinant HBsAg (rHBsAg) particles, which contain the S protein only, bind almost exclusively to monocytes. Attachment of rHBsAg to the THP-1 pre-monocytic cell line occurs upon 1,25-dihydroxyvitamin D3-induced differentiation. Binding to monocytes is enhanced by a heat-labile serum protein and is inhibited by Ca(2+)/Mg(2+), low pH and an HBsAg-specific monoclonal antibody. Furthermore, it is shown that rHBsAg suppresses lipopolysaccharide- and IL-2-induced production of cytokines. These results suggest the existence of a monocyte-specific receptor, the engagement of which by HBsAg suppresses the activity of these cells.

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.

Clinical course and consequences of hepatitis B infection

Hepatitis B virus (HBV) is a small enveloped virus containing partially double-stranded DNA. The DNA and HBV-specific DNA polymerase are surrounded by the HBV core antigen (HBcAg), which in turn is surrounded by a lipoprotein envelope containing the HBV surface antigen (HBsAg). Serum of HBV-infected patients contains complete virus particles, as well as non-infectious spherical or filamentous HBsAg particles. Acute hepatitis is characterized by the appearance of serum HBV markers, including HBsAg and IgM anti-HBc, which then disappear during convalescence. Persistence of HBsAg for more than 6 months indicates a carrier state. Chronic hepatitis develops in 90% of newborns who become infected, compared with 29–40% of children infected and 5–10% of adults infected. The immune status of the infected person also influences the development of chronic hepatitis. Chronic HBV infection can be diagnosed by serology (identification of HBsAg and HBV DNA), biochemistry (elevated aminotransferase levels) and liver biopsy. The last is important to assess the severity of disease, its stage and prognosis, and to exclude other hepatic diseases. The outcome of chronic HBV infection varies between individuals, with estimated 5-year survivals of 97% for chronic persistent hepatitis, 86% for chronic active hepatitis, and 55% for chronic active hepatitis with cirrhosis. Treatment with interferon α is effective in up to 40% of cases, but in view of the very large number of infected people worldwide, vaccination to prevent spread of the disease is a more cost-effective option.

Retention behavior of very large biomolecules in ion-exchange chromatography

Hepatitis B virus surface antigen (HBsAg) particles were efficiently adsorbed (retained) on a Sulfate–cellulose (S–C) bead column, and then desorbed with sodium chloride solutions (0.5–3.0 M). The HBsAg particles were not efficiently retained onto either sulfopropyl–agarose (SP–A) or quaternary amine–agarose (Q–A) at pH 4.5, 6 and 8. The size-exclusion curve showed that proteins of molecular mass higher than ca. 20 000 cannot penetrate into the pores of S–C beads. The dynamic binding capacity (DBC) values of lysozyme (ca. 7 mg/ml-gel) and of γ-globulin (ca. 3 mg/ml gel) for S–C did not depend on the flow velocity while the DBC of γ-globulin for SP–A decreased sharply with an increase in flow velocity. These results indicated that very large molecules are adsorbed only onto the surface of S–C, which resulted in fast adsorption–desorption rates although the equilibrium adsorption capacity is lower than conventional porous gel beads. Because of the rapid adsorption rate, the DBC values of γ-globulin for S–C at high flow-rate regions are similar to those for SP–A. Bovine serum albumin was not adsorbed onto S–C. As this can not be explained by a simple electrostatic interaction mechanism, molecular recognition of S–C might be different from the agarose-based ion-exchange beads.

Localization of a unique hepatitis B virus epitope sheds new light on the structure of hepatitis B virus surface antigen.

In a search for monoclonal antibodies (MAbs) that can bind hepatitis B virus surface antigen (HBsAg) with amino acid substitutions in the immune dominant 'a' region (escape mutants) we investigated the epitope recognition site of the human MAb 4-7B. Pepscan analysis and experiments with alanine substitution as well as substitutions known from nature pointed to residues 178-186 in the small S protein with the amino acid sequence PFVQWFVGL (key amino acids in bold) as the minimal epitope. Single amino acid substitutions at positions 122(R/K)(d/y), 134(Y/F), 145(G/R), 148(T/A) and 160(K/R)(w/r), representing 'a' region variants in recombinant HBsAg COS-I cells, did not influence binding of MAb 4-7B. Synthetic peptides (residues 175-189) including the 4-7B epitope sequence were able to evoke an anti-HBs response in rabbits. According to established polypeptide models, the 4-7B epitope region is located in the lipid layer of 20 nm HBsAg particles. The present results, however, suggest that residues 178-186 are exposed on the surface of the 20 nm particle. This may change our view of the structure of HBsAg.

Fine structure of hepatitis B virus surface antigen produced by recombinant yeast: comparison with HBsAg of human origin

The ultrastructure of hepatitis B virus surface antigen (HBsAg) particles produced by recombinant yeast cells was examined using high-resolution negative staining, and ice embedding, electron microscopy. With negative staining, the HBsAg particles were spherical to slightly ovoid with a mean diameter of 27.5 nm and consisted of many subunits each 4 nm in diameter. Subunits were marked with a minute central pore. With ice embedding, particles were mostly spherical to ovoid, with a mean diameter of 23.7 nm and a 7–8 nm thick cortex surrounding an electron translucent core. Human HBsAg particles, examined using the same methods, were smaller, apparently because of molecular differences in polypeptide structure.

Saccharomyces cerevisiae can release hepatitis B virus surface antigen (HBsAg) particles into the medium by its secretory apparatus

We constructed a plasmid that directs the synthesis and secretion of hepatitis B virus (HBV) surface antigen (HBsAg) particles by Saccharomyces cerevisiae. This plasmid contains a proteinase-resistant HBsAg M (M-P31c) gene fused at its 5'-terminus with a chicken-lysozyme signal peptide (C-SIG) gene, which is placed under the yeast GLD (glyceraldehyde-3-phosphate dehydrogenase gene) promoter. The products encoded by the "C-SIG+M-P31c" (LM-P31c) gene were synthesized and assembled themselves into HBsAg particles in yeast cells, and the particles were released into the medium along with poly-HSA (polymerized human serum albumin) binding activity. The HBsAg particles purified from the medium were very similar in density (1.19 g cm-3), size (19.2 +/- 0.8 nm in diameter) and shape (sphere) to human-plasma-derived HBsAg particles. When several sec (temperature-sensitive secretion-defective) mutants were used as host cells, the release of HBsAg particles into the medium was blocked at 37 degrees C but not at 25 degrees C, indicating that the HBsAg particles are exported through the normal yeast secretion pathway. To our knowledge, this is the first report that yeast cells are capable of secreting particles into the medium.

Heterogeneity of hepatitis B virus surface antigen in sera of patients with acute hepatitis B infection in relative content of the antigenic determinants a and preS2.

Using five ELISA variants we have analyzed the ratios of the antigenic determinants 'a' (a-D), preS2 (preS2-D) and the polymerized human serum albumin binding domains (PHSA-BD) in the hepatitis B virus surface antigen (HBsAg) particles contained in sera of three patients with acute viral hepatitis B. Quantitative relations between the a-D, preS2-D and PHSA-BD were shown to differ in these sera. Population of the HBsAg particles in each of the sera appeared heterogeneous in respect to the PHSA-BD activity. The HBsAg particles with high affinity PHSA-BD prevailed in one serum. An additional class of particles with low affinity to PHSA or complete lack of the PHSA-BD was present in the remaining sera. The HBsAg particles carrying the high affinity PHSA-BD were characterized by an elevated (and almost constant for the three sera) surface density of the preS2-D. The results have led us to the conclusion that the existence of the preS2-D on the surface of the HBsAg particles is necessary but insufficient for the high affinity binding of the HBsAg to PHSA. A model for the interaction between the preS2-D carrying HBsAg particles and PHSA is presented.

Processing of hepatitis B virus surface antigen expressed by recombinant Oka varicella vaccine virus.

We have constructed a recombinant Oka varicella vaccine virus expressing hepatitis B virus (HBV) surface antigen (HBsAg). HBsAg was synthesized as 26K and 30K proteins in infected cells and secreted into the culture supernatant as 30K and 35K proteins. Inhibitors and glycosidase treatments, and pulse-chase labelling experiments, revealed the glycosylation process of HBsAg. The latter was synthesized as a non-glycosylated 26K protein and subjected to N-linked glycosylation to form a 30K protein with high mannose glycans. Three species of dimers composed of 26K and 30K subunits were then formed with disulphide bonds. Both subunits of the dimers were further subjected to O-linked glycosylation and conversion from high mannose glycans to complex glycans followed by sialylation. Three species of dimers composed of 30K and 35K subunits were secreted into the culture supernatant as HBsAg particles. HBsAg was synthesized, glycosylated with both N- and O-linked glycans, sialylated, and then secreted into the culture supernatant within 1 h. These modifications of HBsAg by glycans might stabilize its structure and enhance its immunogenicity as a live HBV vaccine.

Production of hepatitis B virus by stably transfected monocytic cell line U-937: a model for extrahepatic hepatitis B virus replication.

Human monocytic cell line U-937 was transfected with the hepatitis B virus (HBV) genome. Transfected cells releasing HBV surface antigen (HBsAg) and HBV e antigen (HBeAg) into the culture supernatant were cloned and further characterized. The HBV genomes were present in these cells in both chromosomally integrated and episomal forms. Intracellular replicative intermediates of HBV DNA and HBV-specific RNA transcripts of 3.6 and 2.2 kb were also detected. Viral DNA was present in the culture supernatant in viral particles with the buoyant densities of mature HBV virions. Electron microscopy revealed the presence of 22-nm spherical HBsAg particles, and Western blot analysis detected pre-S1 and -S2 antigens in particles in the culture supernatant. HBV DNA in U-937 cells was found to be positively regulated by corticosteroids, interleukin-1 beta, and transforming growth factor-1 beta. The stably transfected cell clones can be used to study HBV replication and its regulation in cells of bone marrow origin and to investigate the influence of HBV on the function of cells of the immune system.

Lipid composition of hepatitis B virus surface antigen particles and the particle-producing human hepatoma cell lines.

More than 90% of lipids of hepatitis B virus surface antigen (HBsAg) particles produced by two human hepatoma cell lines (huGK-14 and PLC/PRF/5) were composed of phospholipids, with phosphatidylcholine being the dominant component, accounting for more than 80% of total membrane lipids. Analysis of subclass compositions of phospholipids of HBsAg particles and the host cell lines revealed that 1,2-diacyl glycerophosphocholine was preferentially incorporated into the membrane of the HBsAg particles, although both host cell lines contained extremely high concentrations (more than 60% of total phospholipids) of ether-linked phospholipids. Phospholipids of other hepatoma cell lines (HuH-7, Hep-G2, and huL-1) which were not associated with hepatitis B virus (HBV) infection, were composed mostly of 1,2-diacylglycerophospholipids. Activities of dihydroxyacetone-phosphate acyltransferase, which is known to be an obligatory enzyme in ether lipid biosynthesis, were found to be elevated by three- to fourfold in both huGK-14 and PLC/PRF/5 cells compared to those of other hepatoma cell lines. The results suggest a possible relationship between HBV-induced hepatocellular carcinogenesis and the drastic change in the metabolism of membrane phospholipids.

Expression of hepatitis B virus surface and core antigens: influences of pre-S and precore sequences.

Amphotropic retroviral expression systems were used to synthesize hepatitis B virus surface antigen (HBsAg) and core antigen. The vectors permitted establishment of cell lines which expressed antigen from either the retroviral long terminal repeat or the mouse metallothionein-I promoter. HBsAgs were synthesized containing no pre-S sequences, pre-S(2) sequences alone, or pre-S(1) plus pre-S(2) sequences. Inclusion of pre-S(2) sequences did not affect the secretion or density of HBsAg particles but did reduce their mass by approximately 30%. Addition of pre-S(1) sequences almost completely abolished secretion of HBsAg and resulted in its localization in an aqueous-nonextractable pre- or early-Golgi cellular compartment. HBsAg was localized to the cytoplasm of the cell. This localization was unaffected by the presence of pre-S sequences in the antigen. Cell lines synthesizing hepatitis B antigens from core DNA fragments, containing or not containing precore sequences, secreted hepatitis B e antigen. However, the absence of precore DNA sequences resulted in additional synthesis of hepatitis core antigen, which was predominantly nuclear in localization.

Genetically engineered diphtheria toxin fusion proteins carrying the hepatitis B surface antigen

Tripartite fusion proteins comprising the nontoxic mutant protein CRM228 of diphtheria toxin (DT), the hepatitis B virus surface antigen (HBsAg), and β-galactosidase were obtained by expression of hybrid genes from the p r promoter of bacteriophage λ and purification by affinity chromatography. The antigenicity and immunogenicity of the individual protein constituents were analyzed. A major neutralizing epitope of DT was inactivated by the HBsAg insertion into the DT B fragment. The fusion proteins elicited antibodies reactive with 22 nm HBsAg particles. This suggests a novel approach towards the use of DT mutants as irflmunogenic carriers of heterologous antigens.

Enzyme-linked immunosorbent assay for detection of receptors for polymerized human serum albumin on hepatitis B surface antigen particles

An enzyme-linked immunosorbent assay (ELISA) was developed to detect the polymerized human serum albumin (pHSA) receptor on hepatitis B virus surface antigen (HBsAg) particles. The receptor values on particles from HBeAg-positive patients were significantly higher than those from anti-HBe-positive and HBeAg-, anti-HBe-negative patients. In patients' sera with moderate to high receptor values, there was significant correlation between the values obtained in the ELISA and serum DNA-polymerase activity ( P < 0.005), but not with HBeAg titer. During a 1-year follow-up of 47 HBeAg-positive patients with chronic hepatitis, termination of active viral replication, as evidenced by seroconversion from HBeAg to anti-HBe-positive, was observed in 1 of 26 with high initial receptor values, 2 of 12 with moderately high values and 6 of 9 with low receptor values. The results suggest that the ELISA described, which detects pHSA receptors on HBsAg particles, might be of value as a prognostic test in HBeAg-positive chronic hepatitis type B.