Quantitative assessment of LPS-HBsAg interaction by introducing a novel application of immunoaffinity chromatography.

Lipopolysaccharide (LPS), as a stubborn contamination, should be monitored and kept in an acceptable level during the pharmaceutical production process. Recombinant hepatitis B surface antigen (r-HBsAg) is one of the recombinant biological products, which is probable to suffer from extrinsic endotoxin due to its long and complex production process. This research aims to assess the potential interaction between LPS and r-HBsAg by recruiting immunoaffinity chromatography (IAC) as a novel tool to quantify the interaction. Molecular modeling was performed on the HBsAg molecule to theoretically predict its potential binding and interaction sites. Then dynamic light scattering (DLS) analysis was implemented on HBsAg, LPS, and mixtures of them to reveal the interaction. The virus-like particle (VLP) structure of HBsAg and the ribbon-like structure of LPS were visualized by transmission electron microscopy (TEM). Finally, the interaction was quantified by applying various LPS/HBsAg ratios ranging from 1.67 to 120 EU/dose in the IAC. Consequently, the LPS/HBsAg ratios in the eluate were measured from 1.67 to a maximum of 92.5 EU/dose. The results indicated that 77 to 100% of total LPS interacted with HBsAg by an inverse relationship to the incubated LPS concentration. The findings implied that the introduced procedure is remarkably practical in the quantification of LPS interaction with a target recombinant protein.

A Highly Sensitive Detection System based on Proximity-dependent Hybridization with Computer-aided Affinity Maturation of a scFv Antibody.

The hepatitis B virus (HBV) infection is a critical health problem worldwide, and HBV preS1 is an important biomarker for monitoring HBV infection. Previously, we found that a murine monoclonal antibody, mAb-D8, targets the preS1 (aa91-107) fragment of HBV. To improve its performance, we prepared the single-chain variable region of mAb-D8 (scFvD8) and constructed the three-dimensional structure of the scFvD8-preS1 (aa91-107) complex by computer modelling. The affinity of scFvD8 was markedly increased by the introduction of mutations L96Tyr to Ser and H98Asp to Ser. Furthermore, a highly sensitive immunosensor was designed based on a proximity-dependent hybridization strategy in which the preS1 antigen competitively reacts with an antibody labelled with DNA, resulting in decreased proximity-dependent hybridization and increased electrochemical signal from the Fc fragment, which can be used for the quantisation of preS1. The results showed a wide detection range from 1 pM to 50 pM with a detection limit of 0.1 pM. The sensitivity and specificity of this immunosensor in clinical serum samples were 100% and 96%, respectively. This study provides a novel system based on proximity-dependent hybridization and the scFv antibody fragment for the rapid quantisation of antigens of interest with a high sensitivity.

A display of pH-sensitive fusogenic GALA peptide facilitates endosomal escape from a Bio-nanocapsule via an endocytic uptake pathway.

BACKGROUND: An affibody-displaying bio-nanocapsule (ZHER2-BNC) with a hepatocyte specificity derived from hepatitis B virus (HBV) was converted into an affibody, ZHER2, that recognizes HER2 receptors. This affibody was previously reported to be the result of the endocytosis-dependent specific uptake of proteins and siRNA into target cancer cells. To assist the endosomal escape of inclusions, a helper lipid with pH-sensitive fusogenic ability (1,2-dioleoyl-sn-glycero-3-phos phoethanolamine; DOPE) was conjugated with a ZHER2-BNC. FINDINGS: In this study, we displayed a pH-sensitive fusogenic GALA peptide on the surface of a particle in order to confer the ability of endosomal escape to a ZHER2-BNC. A GALA-displaying ZHER2-BNC purified from yeast uneventfully formed a particle structure. Furthermore, endosomal escape of the particle was facilitated after endocytic uptake and release of the inclusions to the cytoplasm without the cell toxicity. CONCLUSION: The genetic fusion of a GALA peptide to the virus-like particle confers the ability of endosomal escape.

A new cell culture system for infection with hepatitis B virus that fuses HepG2 cells with primary human hepatocytes.

Hepatitis B virus (HBV) infection exhibits a very narrow host range and shows a strong tropism for liver parenchymal cells, however none of the previously established experimental models can reproduce the natural process of HBV infection. In the present study, primary human hepatocytes were fused with HepG2 cells to establish the hybrid HepCHLine-4 cell line with high susceptibility to HBV. The HepCHLine-4 cells expressed HBV-specific antigen when co-incubated with HBV-positive serum from a hepatitis B patient. Post-infection, HBV relaxed circular DNA and covalently closed circular DNA were detected in HepCHLine-4 cells using a nested polymerase chain reaction, and HBV-specific particles were visualized by electron microscopy of the culture media of HepCHLine-4 cells. HepG2 cells were not susceptible to HBV infection under the same conditions. The HepCHLine-4 cells can be sub-cultured for > 12 months while maintaining susceptibility to HBV and may, therefore, be useful for studying HBV infection and the viral life cycle in human hepatocytes.

Expression of hepatitis B surface antigen in Saccharomyces cerevisiae utilizing glyceraldeyhyde-3-phosphate dehydrogenase promoter of Pichia pastoris.

An expression vector constructed from genes of Pichia pastoris was applied for heterologous gene expression in Saccharomyces cerevisiae. Recombinant hepatitis B surface antigen was synthesized by cloning hepatitis B virus 'S' gene under the control of glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter of Pichia pastoris in Saccharomyces cerevisiae. Hepatitis B surface antigen was constitutively expressed, was stable and exhibited approximately 20-22 nm particle formation. Stability and absence of toxicity to the host with the expression vector indicates the expression system can be applied for large-scale production.

Hepatitis B small surface antigen particles are octahedral.

The infectious component of hepatitis B (HB) virus (HBV), the Dane particle, has a diameter of approximately 44 nm and consists of a double-layered capsid particle enclosing a circular, incomplete double-stranded DNA genome. The outer capsid layer is formed from the HB surface antigen (HBsAg) and lipid, whereas the inner layer is formed from the HB core Ag assembled into an icosahedral structure. During chronic infection HBsAg is expressed in large excess as noninfectious quasispherical particles and tubules with approximately 22-nm diameter. Here, we report cryo-EM reconstructions of spherical HBsAg particles at approximately 12-A resolution. We show that the particles possess different diameters and have separated them into two predominant populations, both of which have octahedral symmetry. Despite their differing diameters, the two forms of the particle have the same mass and are built through conformational switching of the same building block, a dimer of HBsAg. We propose that this conformational switching, combined with interactions with the underlying core, leads to the formation of HBV Dane particles of different sizes, dictated by the symmetry of the icosahedral core.

Assembly, structure, and antigenic properties of virus-like particles rich in HIV-1 envelope gp120.

In order to improve the immunogenicity of HIV-1 envelope glycoproteins, we have fused gp120 to a carrier protein, hepatitis B surface antigen (HBsAg), which is capable of spontaneous assembly into virus-like particles. The HBsAg-gp120 hybrid proteins assembled efficiently into 20-30 nm particles. The particles resemble native HBsAg particles in size and density, consistent with a lipid composition of about 25% and a gp120 content of about 100 per particle. Particulate gp120 folds in its native conformation and is biologically active, as shown by high affinity binding of CD4. The particles express conformational determinants targeted by a panel of broadly cross-reactive neutralizing antibodies, and they show tight packing of gp120. Because the particles are lipoprotein micelles, an array of gp120 on their surface closely mimics gp120 on the surface of HIV-1 virions. These gp120-rich particles can enhance the quality, as well as quantity, of antibodies elicited by a gp120 vaccine.

Selective destabilization of acidic phospholipid bilayers performed by the hepatitis B virus fusion peptide.

A peptide corresponding to the N-terminal region of the S protein of hepatitis B virus (Met-Glu-Asn-Ile-Thr-Ser-Gly-Phe-Leu-Gly-Pro-Leu-Leu-Val-Leu-Gln) has been previously demonstrated to perform aggregation and destabilization of acidic liposome bilayers and to adopt a highly stable beta-sheet conformation in the presence of phospholipids. The changes in the lipid moiety produced by this peptide have been followed by fluorescence depolarization and electron microscopy. The later was employed to determine the size and shape of the peptide-vesicle complexes, showing the presence of highly aggregated and fused structures only when negatively charged liposomes were employed. 1,6-Diphenyl-1,3,5-hexatriene depolarization measurements showed that the interaction of the peptide with both negatively charged and zwitterionic liposomes was accompanied by a substantial reduction of the transition amplitude without affecting the temperature of the gel-to-liquid crystalline phase transition. These data are indicative of the peptide insertion inside the bilayer of both types of liposomes affecting the acyl chain order, though only the interaction with acidic phospholipids leads to aggregation and fusion. This preferential destabilization of the peptide towards negatively charged phospholipids can be ascribed to the electrostatic interactions between the peptide and the polar head groups, as monitored by 1-(4-(trimethylammoniumphenyl)-6-phenyl-1,3, 5-hexatriene fluorescence depolarization analysis.

Evaluation of a new recombinant DNA hepatitis B vaccine (Shanvac-B).

We assessed the efficacy and safety of Shanvac-B, a new recombinant hepatitis B vaccine developed in India. Eighty-one healthy volunteers (75 women, 6 men; aged 18-40 yr), negative for markers for hepatitis B and HIV, received 20 microg of the vaccine intramuscularly at 0, 1 and 2 months. Forty-three (53%) seroconverted at one month after dose 1; of these, 26% were seroprotected (anti-HBs> 10 mIU/mL). Seroprotection at one month after doses 2 and 3 was 99% and 100%, respectively. Geometric mean titres of anti-HBs in subjects who seroconverted were 11 (range 2-366), 266 (8-7469) and 2246 (102-23680) mIU/mL, respectively. One subject developed urticarial rash after the second dose; there was no other adverse event. We conclude that this vaccine is safe and efficacious, providing significant protection even after two doses.

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.

Comparison between hepatitis B surface antigen (HBsAg) particles derived from mammalian cells (CHO) and yeast cells (Hansenula polymorpha): composition, structure and immunogenicity.

The composition, structure and immunogenicity of hepatitis B surface antigen (HBsAg) particles derived from Chinese hamster ovary (CHO) cells and from cells of the yeast Hansenula polymorpha were compared. The particles were similar in size distribution (mean 20-33 nm), in shape (spherical), in gross composition (protein to lipid weight ratio of 60:40), and in types of lipids (phospholipids > > sterols = sterol esters = triacylglycerols). Differences related to genetic engineering and type of host cells were found in peptide and lipid compositions. CHO-HBsAg has three peptides: S, M and L, each in two forms of glycosylation, while the Hansenula-HBsAg has only the nonglycosylated S peptide. The electrical surface potential at the lipid/water interface of HBsAg derived from Hansenula is more negative than that of HBsAg derived from CHO, which was close to neutrality. Although the numbers of cysteine residues (all in the S peptides) are identical (14), 11 of them are free thiols in the CHO-HBsAg, compared with three to four in the Hansenula-HBsAg. The fact that 85% of the phospholipids are hydrolyzed by phospholipase C and that all the aminophospholipids react with trinitrobenzenesulfate suggests that the particles derived from both cell types are either leaky vesicles or have a lipoprotein-like structure. Subcutaneous injection into mice of fluorescein-isothiocyanate-labeled HBsAg particles from both sources resulted in their accumulation in the marginal sinus of lymph nodes. The humoral responses to subcutaneous injection into mice of CHO- and Hansenula-HBsAg were similar: however, the cytotoxic T lymphocyte response to CHO-HBsAg was lower.

[Preparation and study of recombinant strains of Venezuelan equine encephalomyelitis virus expressing the PreS2-S gene of the hepatitis B virus]. / Poluchenie i issledovanie rekombintnykh shtammo-v virusa venesuél'skogo éntsefalomielita loshadei, ékspressiruiushchikh gen preS2-S virusa gepatita B.

Venezuelan equine encephalomyelitis (VEE) virus-based vectors for expression of heterologous genes have been constructed using full-length cDNA copy of the interstrain recombinant virus (Trinidad Donkey and 230 strain). Gene cassettes carrying the subgenomic mRNA promoter of VEE virus and the preS2-S gene of hepatitis B virus (HBV) were inserted before or after the genes of structural viral proteins. Live virus stocks were obtained by transfection of chick embryo fibroblasts with in vitro transcribed full-length RNA. Insertions of gene cassettes before the structural region resulted in expression of HBsAg (VEHB-25 and VEHB-361 viruses), whereas insertions in the 3' region did not. Recombinant virus VEHB-25 expressed HBsAg during 5 passages in Vero cells. VEHB-25 stimulated immune response to HBsAg and was less virulent than the parental virus.

Characterization of the HBsAg particle lipid membrane.

HBsAg particles are highly immunogenic and have been shown to be a suitable support for the presentation of foreign epitopes. More information about the topology of the HBsAg protein is a prerequisite to any rational attempt to replace the region of this protein with foreign epitopes without modifying the assembly of the particles. This topology and, more precisely, the mode of interaction of the HBsAg protein with the lipid will depend on the lipid organization in the particle envelope. Nothing is known concerning the lipid organization of HBsAg particles. The only available information concerns their lipid composition. Phospholipase D hydrolysis of HBsAg particles was used here to determine whether the particles were surrounded with a lipid monolayer or bilayer. The lipid fluidity within the particle envelope was evaluated by fluorescence polarization measurements. The data strongly suggest that the HBsAg particle membrane is organized as a discontinuous rigid bilayer of lipids interacting with protein aggregates.

Reconstitution of hepatitis B surface antigen proteins into phospholipid vesicles.

Hepatitis B surface antigen (HBsAg), devoid of 75% of its total lipids has been reconstituted with several phospholipids by the detergent dialysis method, using the non-ionic detergent beta-D-octyl glucoside. Upon reconstitution with both neutral and acidic phospholipids, HBsAg particles had the same morphology and, as indicated by trypsin hydrolysis, the topology of the surface proteins was maintained. However, only negatively charged phospholipids were able to completely revert the conformational changes which had been induced by removal of the lipids. The helical content, as indicated by CD techniques, and the antigenic activity, as measured by binding to polyclonal antibodies, of HBsAg reconstituted with acidic phospholipids were practically identical to those of the native antigen. Cholesterol had no effect on the antigenic activity recovered by reconstitution with any of the phospholipids.

Post-translational alterations in transmembrane topology of the hepatitis B virus large envelope protein.

The preS domain at the N-terminus of the large envelope protein (LHBs) of the hepatitis B virus is involved in (i) envelopment of viral nucleocapsids and (ii) binding to the host cell. While the first function suggests a cytosolic location of the preS domain during virion assembly, the function as an attachment site requires its translocation across the lipid bilayer and final exposure on the virion surface. We compared the transmembrane topology of newly synthesized LHBs in the endoplasmic reticulum (ER) membrane with its topology in the envelope of secreted virions. Protease sensitivity and the absence of glycosylation suggest that the entire preS domain of newly synthesized LHBs remains at the cytosolic side of ER vesicles. However, virions secreted from transfected cell cultures or isolated from the blood of persistent virus carriers expose antibody binding sites and proteolytic cleavage sites of the preS domain at their surface in approximately half of the LHBs molecules. Thus, preS domains appear to be transported across the viral lipid barrier by a novel post-translational translocation mechanism to fulfil a dual function in virion assembly and attachment to the host cell.

Hepadnavirus infection requires interaction between the viral pre-S domain and a specific hepatocellular receptor.

To better define the molecules involved in the initial interaction between hepadnaviruses and hepatocytes, we performed binding and infectivity studies with the duck hepatitis B virus (DHBV) and cultured primary duck hepatocytes. In competition experiments with naturally occurring subviral particles containing DHBV surface proteins, these DNA-free particles were found to interfere with viral infectivity if used at sufficiently high concentrations. In direct binding saturation experiments with radiolabelled subviral particles, a biphasic titration curve containing a saturable component was obtained. Quantitative evaluation of both the binding and the infectivity data indicates that the duck hepatocyte presents about 10(4) high-affinity binding sites for viral and subviral particles. Binding to these productive sites may be preceded by reversible virus attachment to a large number of less specific, nonsaturable primary binding sites. To identify which of the viral envelope proteins is responsible for hepatocyte-specific attachment, subviral particles containing only one of the two DHBV surface proteins were produced in Saccharomyces cerevisiae. In infectivity competition experiments, only particles containing the large pre-S/S protein were found to markedly reduce the efficiency of DHBV infection, while particles containing the small S protein had only a minor effect. Similarly, physical binding of radiolabelled serum-derived subviral particles to primary duck hepatocytes was inhibited well only by the yeast-derived pre-S/S particles. Together, these results strongly support the notion that hepadnaviral infection is initiated by specific attachment of the pre-S domain of the large DHBV envelope protein to a limited number of hepatocellular binding sites.

Immunoelectron microscopic detection of the hepatitis B virus major surface protein in dilated perinuclear membranes of yeast cells.

The major surface protein of hepatitis B virus produced in Saccharomyces cerevisiae can be recovered from cell lysates in the form of 22-mm lipoprotein particles. Immunoelectron microscopy was applied to investigate site and time of particle assembly. Thin sections of yeast cells revealed that production of the S protein provoked a dilation of the endoplasmic reticulum. Dilated areas were specifically labeled with a polyclonal antibody raised against glutaraldehyde-treated yeast-derived HBsAg particles. In contrast to previous postulates of particle formation during cell lysis and extract preparation, these results suggest that particle formation in yeast occurs in the endoplasmic reticulum and that transport of particles along the secretion pathway is blocked.

The position of heterologous epitopes inserted in hepatitis B virus core particles determines their immunogenicity.

The nucleocapsid (HBcAg) of the hepatitis B virus (HBV) has been suggested as a carrier moiety for vaccine purposes. We investigated the influence of the position of the inserted epitope within hybrid HBcAg particles on antigenicity and immunogenicity. For this purpose, genes coding for neutralizing epitopes of the pre-S region of the HBV envelope proteins were inserted at the amino terminus, the amino terminus through a precore linker sequence, the truncated carboxy terminus, or an internal site of HBcAg by genetic engineering and were expressed in Escherichia coli. All purified hybrid HBc/pre-S polyproteins were particulate. Amino- and carboxy-terminal-modified hybrid HBc particles retained HBcAg antigenicity and immunogenicity. In contrast, insertion of a pre-S(1) sequence between HBcAg residues 75 and 83 abrogated recognition of HBcAg by 5 of 6 anti-HBc monoclonal antibodies and diminished recognition by human polyclonal anti-HBc. Predictably, HBcAg-specific immunogenicity was also reduced. With respect to the inserted epitopes, a pre-S(1) epitope linked to the amino terminus of HBcAg was not surface accessible and not immunogenic. A pre-S(1) epitope fused to the amino terminus through a precore linker sequence was surface accessible and highly immunogenic. A carboxy-terminal-fused pre-S(2) sequence was also surface accessible but weakly immunogenic. Insertion of a pre-S(1) epitope at the internal site resulted in the most efficient anti-pre-S(1) antibody response. Furthermore, immunization with hybrid HBc/pre-S particles exclusively primed T-helper cells specific for HBcAg and not the inserted epitope. These results indicate that the position of the inserted B-cell epitope within HBcAg is critical to its immunogenicity.