HBsAg Particles Research Articles

Hepatitis B virus surface antigen impairs myeloid dendritic cell function: a possible immune escape mechanism of hepatitis B virus

Chronic hepatitis B virus (HBV) infection is the result of an inadequate immune response towards the virus. Myeloid dendritic cells (mDC) of patients with chronic HBV are impaired in their maturation and function, resulting in more tolerogenic rather than immunogenic responses, which may contribute to viral persistence. The mechanism responsible for altered mDC function remains unclear. The HBV-infected patients display large amounts of HBV particles and viral proteins in their circulation, especially the surface antigen HBsAg, which allows multiple interactions between the virus, its viral proteins and DC. To assess whether HBV directly influences mDC function, the effects of HBV and HBsAg on human mDC maturation and function were investigated in vitro. As already described for internalization of HBV by DC, the present study shows that peripheral blood-derived mDC of healthy controls also actively take up HBsAg in a time-dependent manner. Cytokine-induced maturation in the presence of HBV or HBsAg resulted in a significantly more tolerogenic mDC phenotype as demonstrated by a diminished up-regulation of costimulatory molecules and a decreased T-cell stimulatory capacity, as assessed by T-cell proliferation and interferon-gamma production. In addition, the presence of HBV significantly reduced interleukin-12 production by mDC. These results show that both HBV particles and purified HBsAg have an immune modulatory capacity and may directly contribute to the dysfunction of mDC in patients with chronic HBV. The direct immune regulatory effect of HBV and circulating HBsAg particles on the function of DC can be considered as part of the mechanism by which HBV escapes immunity.

Maturation of Recombinant Hepatitis B Virus Surface Antigen Particles

The major surface antigen of Hepatitis B virus (HBsAg) is a cysteine-rich, lipid-bound protein with 226 amino acids. Recombinant HBsAg (rHBsAg) with associated lipids can self-assemble into 22-nm immunogenic spherical particles, which are used in licensed Hepatitis B vaccines. Little is known about the structural evolvement or maturation upon assembly beyond an elevated level of disulfide formation. In this paper, we further characterized the maturation of HBsAg particles with respect to their degree of cross-linking, morphological changes, and changes in conformational flexibility. The lipid-containing rHBsAg particles undergo KSCN- and heat-induced maturation by formation of additional intra- and inter-molecular disulfide bonds. Direct measurements with atomic force microscopy (AFM) revealed morphological changes upon maturation through KSCN-induced and heat-/storage-incurred oxidative refolding. Particle uniformity and regularity was greatly improved, and protrusions formed by the protein subunits were more prominent on the surface of the mature particles. Decreased conformational flexibility in the mature rHBsAg particles was demonstrated by millisecond-scale unfolding kinetics in the presence of an environment-sensitive conformation probe. Both the accessible hydrophobic cavities under native conditions and the changeable hydrophobic cavities upon denaturant-induced unfolding showed substantial decrease upon maturation of the rHBsAg particles. These changes in the structural properties may be critical for the antigenicity and immunogenicity of this widely-used vaccine component.

Improving recovery of recombinant hepatitis B virus surface antigen by ion-exchange chromatographic supports with low ligand density

Ion-exchange chromatography (IEC) with the lowest step recovery (<50%) is still a bottleneck of the essentially four-step chromatographic procedure for the purification of recombinant hepatitis B surface antigen (r-HBsAg) from transformed Chinese hamster ovary (CHO) cell line. The low recovery is mainly resulted from the change of the assemble structure of HBsAg particles such as disassociation or aggregation on the surface of the absorbent during binding and eluting procedures. To improve the chromatographic efficiency of IEC, the influence of ligand density from 0.041 to 0.130 mmol diethylaminoethyl (DEAE)/ml absorbent on IEC of r-HBsAg was investigated. The retention time of HBsAg particles depended highly on ligand density. Higher HBsAg recovery and purification factor (PF) were obtained from the absorbents with lower ligand density. Under the optimum chromatographic conditions, the HBsAg recovery was increased from about 44% to 66% and the PF was increased from 9 to 13 by decreasing the ligand density from 0.130 to 0.041 mmol DEAE/ml absorbent.

Low-dose adenovirus vaccine encoding chimeric hepatitis B virus surface antigen-human papillomavirus type 16 E7 proteins induces enhanced E7-specific antibody and cytotoxic T-cell responses.

Induction of effective immune responses may help prevent cancer progression. Tumor-specific antigens, such as those of human papillomaviruses involved in cervical cancer, are targets with limited intrinsic immunogenicity. Here we show that immunization with low doses (10(6) infectious units/dose) of a recombinant human adenovirus type 5 encoding a fusion of the E7 oncoprotein of human papillomavirus type 16 to the carboxyl terminus of the surface antigen of hepatitis B virus (HBsAg) induces remarkable E7-specific humoral and cellular immune responses. The HBsAg/E7 fusion protein assembled efficiently into virus-like particles, which stimulated antibody responses against both carrier and foreign antigens, and evoked antigen-specific kill of an indicator cell population in vivo. Antibody and T-cell responses were significantly higher than those induced by a control adenovirus vector expressing wild-type E7. Such responses were not affected by preexisting immunity against either HBsAg or adenovirus. These data demonstrate that the presence of E7 on HBsAg particles does not interfere with particle secretion, as it occurs with bigger proteins fused to the C terminus of HBsAg, and results in enhancement of CD8(+)-mediated T-cell responses to E7. Thus, fusion to HBsAg is a convenient strategy for developing cervical cancer therapeutic vaccines, since it enhances the immunogenicity of E7 while turning it into an innocuous secreted fusion protein.

519 Novel mechanism of HepeX-B antibodies in prolonged blocking of release of hepatitis B virus and HBsAg particles from infected cells indicated by viral dynamics in-vivo

519 Novel mechanism of HepeX-B antibodies in prolonged blocking of release of hepatitis B virus and HBsAg particles from infected cells indicated by viral dynamics in-vivo

In vitroresistance to interferon of hepatitis B virus with precore mutation

Chronic hepatitis B virus (HBV) infection is predominantly treated with interferon alpha (IFN-alpha), which results in an efficient reduction of the viral load only in 20-40% of treated patients. Mutations at HBV precore prevail in different clinical status of HBV infection. The roles of precore mutation in the progression of chronic hepatitis and interferon sensitivity are still unknown. The aim of this study was to explore if there was any relationship between HBV precore mutation and sensitivity to interferon in vitro. HBV replication-competent recombinant constructs with different patterns of precore mutations were developed. Then the recombinants were transiently transfected into hepatoma cell line (Huh7) by calcium phosphate transfection method. With or without IFN, viral products in culture medium were collected and quantified 3 d after transfection. We obtained 4 recombinant constructs by orientation-cloning 1.2-fold-overlength HBV genome into pUC18 vector via the EcoRI and Hind III and PCR mediated site-directed mutagenesis method. All the recombinants contained mutations within precore region. Huh7 cells transfected with recombinants secreted HBsAg and HBV particles into the cell culture medium, indicating that all the recombinants were replication-competent. By comparing the amount of HBV DNA in the medium, we found that HBV DNA in medium reflecting HBV replication efficiency was different in different recombinants. Recombinants containing precore mutation had fewer HBV DNAs in culture medium than wild type. This result showed that recombinants containing precore mutation had lower replication efficiency than wild type. HBV DNA was decreased in pUC18-HBV1.2-WT recombinants after IFN was added while others with precore mutations were not, indicating that HBV harboring precore mutation was less sensitive to IFN in cell culture system. These data indicate that HBV harboring precore mutation may be resistant to IFN in vitro.

Uptake of hepatitis B virus into choriocarcinoma cells in the presence of proinflammatory cytokine tumor necrosis factor-α

The aim of this study was to establish an in vitro experimental model that mimics the interaction of hepatitis B virus (HBV) and the trophoblastic barrier. The barrier was represented by choriocarcinoma cell line, JAR cells, which were incubated with HBV in the presence or absence of tumor necrosis factor-alpha (TNF-alpha). At 12-hour intervals after a 24-hour incubation, the infection efficiency was examined by enzyme-linked immunosorbent assay of culture supernatants, immunohistochemistry of cell slides, Western blotting of cell lysates, polymerase chain reaction amplification for viral DNA of infected cells, and transmission electron microscopy for HBsAg particles in infected cells. In the absence of TNF-alpha, many JAR cell colonies showed a negative or punctate positive pattern after incubation with HBV. But when JAR cells were pretreated with TNF-alpha before cocultured with HBV, the infection was greatly enhanced, whereas the differences observed between the various time points in the second series of experiments did not reach a statistically significant level. JAR cells can support HBV infection in the presence of TNF-alpha. Our in vitro system provides key basis on further study reflecting mechanism of in vivo HBV intrauterine infection and transfer.

Expression of hepatitis B surface antigen in tobacco cell suspension cultures

Hepatitis B virus ‘ s’ gene coding for surface antigen was cloned into plant transformation vectors pHER100 and pHBs100 with and without endoplasmic reticulum retention signal, respectively. Transformed tobacco cell lines were analyzed for the integration of the transgene by PCR and Southern blot hybridization. Expression levels as determined by ELISA showed maximum expression levels of 2 μg HBsAg gm −1 fresh weight and 10 ng mL −1 of spent medium in pHER100 transformed cells. Western blot analysis confirmed the presence of 24 kDa band specific to HBsAg in the transformed cells. HBsAg was expressed both as intracellular and secreted forms in pHER100 transformed cells. The buoyant density in CsCl of HBsAg derived from pHBs100 transformed tobacco cells was determined and found to be 1.095 g mL −1. HBsAg obtained from transformed tobacco cells is similar to the human serum derived one in buoyant density properties. This is the first report on the secretion of HBsAg particles by plant cells into the cell culture medium.

Role of transfection in the priming of cytotoxic T-cells by DNA-mediated immunization

DNA vaccination results in remarkably strong, broad-based immune responses to the encoded proteins and it is a simple and effective method of inducing cytotoxic T-lymphocyte (CTL) responses. Bone marrow-derived cells can take up and present exogenous antigenic protein liberated by transfected fibroblasts or myoblasts after the injection of such cells. In addition, dendritic cells can carry the injected plasmid DNA, supporting the hypothesis that dendritic cells can be directly transfected. It is, however, unclear from the current data what proportion of the cytotoxic immune response is initiated by the transfer of protein compared to that resulting from direct transfection of professional antigen presenting cells. This question is addressed here by using a matched series of plasmid DNA vectors expressing the wild-type or several mutant forms of HBsAg that are secretion-defective or severely truncated. The data indicate that neither HBsAg particle formation nor its secretion or liberation plays a significant role in the development of the cytotoxic immune response. The results argue that direct transfection of bone marrow-derived cells is the major, and possibly the only, mechanism used for priming of naive CTL precursors directed against the HBsAg.

Virus-like particles: a new family of delivery systems

The development of antiviral vaccines has almost exclusively been based on live attenuated vaccines up until now. However, the efficacy of HBsAg particles as an antiHBV vaccine has clearly demonstrated that protective antiviral immunity can be achieved by other strategies. Virus-like particles formed by structural proteins were proven to be highly immunogenic and capable of inducing protective immunity against various viral infections in preclinical studies.Clinical trials using virus-like particles confirmed their safety and immunogenicity. Moreover, chimeric virus-like particles carrying foreign peptidic sequences were shown to elicit potent B- and T-cell responses. Virus-like particles formed by a fusion protein between the HBsAg and the circumsporozoïte surface protein are safe and immunogenic in volunteers and induce a partial protection against natural Plasmodium falciparum infection.

48-mer synthetic peptide analogue of the hepatitis B virus "a" determinant induces an anti-HBs antibody response after a single injection.

An extended (48 amino acid) synthetic peptide analogue of the hepatitus B virus (HBV) S protein (HBsAg) 'a' determinant has been produced by using 9-fluorenylmethoxycarbonyl (fmoc) chemistry and a low substitution polystyrene resin as the solid phase support. This peptide (S121/48) elicited a sustained anti-peptide antibody response in BALB/c (H-2(d)) mice when immunised with Freund's complete adjuvant (FCA). Cross-reactive, anti-HBs antibodies were induced, directed against a significant proportion of the conformationally restrained epitope repertoire on the native HBsAg particles. Similar responses were obtained by injection of guinea pigs, a species known both to be exquisitely sensitive to HBsAg and to produce a wide range of B cell responses to HBsAg antigens. Taken together, these data show for the first time, that a synthetic peptide mimicking conformational epitopes can be produced by chemical synthesis and can be used to induce significant titres of anti-HBs antibodies after a single injection. This immunogen has considerable potential for incorporation into novel delivery systems, e.g., microspheres, thus offering the potential of a controlled release, single dose hepatitis B vaccine.

Purification and characterization of hepatitis B virus surface antigen particles produced in Drosophila Schneider-2 cells

The small surface antigen of hepatitis B virus (HBV) was produced in Drosophila melanogaster Schneider-2 (DS-2) cells transfected stably using an inducible Drosophila metallothionein promoter. Selected clonal DS-2 cell-lines expressed and secreted large quantities of HBsAg particles consisting exclusively of non-glycosylated 25 kDa proteins. HBsAg produced by DS-2 cells had physical and biochemical properties very similar to 22 nm particles derived from the human hepatoma cell-line PLC/PRF/5. DS-2 cell-derived HBsAg particles were purified near homogeneity by a strategy based on protein concentration, precipitation and ultracentrifugation. The resulting HBsAg product was <98% pure. A single immunisation of BALB/c mice with both DS-2 and yeast-cell derived purified HBsAg particles without adjuvants elicited a substantial humoral antibody and class-I restricted cytotoxic T lymphocyte (CTL) response. Adsorbtion of HBsAg particles to aluminium hydroxide resulted in increased levels of HBsAg-specific antibodies. However, CTLs were not elicited by HBsAg/Alum combinations. Thus, stably transfected DS-2 cells provide a useful source for the production of HBV subviral particles for diagnostic and research purposes as well as for novel vaccine development.

High level expression of hepatitis B virus surface antigen in stably transfected Drosophila Schneider-2 cells

Two transfer vector systems have been constructed for the generation of Drosophila melanogaster Schneider-2 (DS-2) cells transfected stably and used to express the small surface antigen of hepatitis B virus (HBsAg). One system is based on the cotransfection of an expression vector for the S gene under the control of an inducible Drosophila metallothionein (Mtn) promotor and a resistance plasmid which carries a selectable marker dihydrofolate reductase (dhfr) gene under the control of a Drosophila actin 5C distal promoter. The second system is based on the transfection of a single plasmid, which includes both expression units. Both vector systems were suitable for the generation of stably transfected DS-2 cell-lines secreting high levels of HBsAg. The quantities of HBsAg expression from polyclonal DS-2 cells correlated strictly with the concentration of the transfected S gene expression vector. Clonal cell-lines selected from the most efficient HBsAg producing polyclonal cell-populations were examined in more detail. All of the transfected S genes were found to be integrated and the copy numbers per genome varied extremely between 10 and 240. Furthermore, the levels of secreted HBsAg varied greatly between different clones and in best they reached up to 7 μg/ml under serum-free cell culture conditions. Thus, DS-2 cells transfected stably provide an alternative source for the production of HBsAg particles for diagnostic purposes and vaccine development.

Improved immunogenicity of HIV-1 epitopes in HBsAg chimeric DNA vaccine plasmids by structural mutations of HBsAg.

To improve the immunogenicity of epitopes from the envelope protein of HIV-1, we have developed gene gun-delivered subunit DNA vaccines by inserting the sequences encoding the V3 region into the hepatitis B virus (HBV) envelope gene, often called the surface antigen (HBsAg). We have examined the possibility of modifying the immune response to V3 by introducing modifications into the carrier HBsAg in gene gun DNA immunization of mice. In some plasmid constructions, the V3 sequence was introduced into the preS2 region of the HBsAg. Although this region is not present in all protein subunits of the HBsAg particles produced, abolishing the internal translational initiation site for the S protein had no effect on the immune response to V3. Expression of V3 at the N-terminal or C-terminal part of the HBsAg protein resulted in equal anti-V3 antibody and cytotoxic T-lymphocyte (CTL) responses. However, elimination of secretion by single amino-acid mutations in the HBsAg decreased the anti-HBsAg antibody response but enhanced the anti-V3 antibody response. In contrast, the CTL response to V3 was independent of the structural mutations but could be improved by a total deletion of the HBsAg sequence part. Thus, the immune response to heterologous epitopes can be altered by modifications in the carrier HBsAg protein. Modifications of the HBsAg carrier might interfere with the dominant immune response to the HBsAg epitopes, allowing better antibody induction to less immunogenic foreign epitopes. However, for induction of CTL responses, the expression of minimal epitopes may be advantageous.

Nucleic acid hepatitis B virus (HBV) vaccine: current status

Hepatitis B virus (HBV) infection is considered to be one of the diseases causing a serious public health problem throughout the world. In the South East, HBV infection remains the cause of morbidity and mortality in a significantly high number of cases of all age groups. Though a vaccine is already available to protect against this infection, the problems associated with dose schedule, number of doses, protection rendered, and response in different populations of existing vaccine, indicate a need of further improvement in current HBV vaccine. This review discusses an advance made in the development of a new generation of HBV vaccine with increased efficacy and better safety. Using the fact that non-replicating DNA plasmids encoding reporter genes can be internalised and encoded proteins expressed by muscle cells following injection into them, HBsAg was chosen as a model for DNA-mediated immunisation in mice. This device produced major histocompatibility complex (MHC)-restricted cytotoxic T-lymphocytes (CTL) and T-helper response against HBsAg in them. The lymphocytes involved in CTL were found to be CD3+, CD4 and CD8+. T-helper response was predominantly and exclusively of Th1 type. Intramuscular injection of plasmid vectors containing HBV envelope proteins induced antibodies to HBsAg particles having kinetic properties and specificity similar to those occurring during HBV infection in human beings. Both humoral response, as well as CTL, were found to depend on the amount and existence of the intramuscular reservoir of protein produced by muscle fibres. The titre of antibodies produced was recorded to be more than the protective level in the majority of cases. Like HBsAg, HBcAg vectors were also demonstrated to be immunogenic, inducing high titres of antibodies in large animals. Thus, all these studies, though still at an early stage in the development of this new generation of HBV vaccine, clearly demonstrate the possibility of a new HBV vaccine capable of overcoming the problems associated with current vaccines.

Definition of the Primary Structure of Hepatitis B Virus (HBV) pre-S Hepatocyte Binding Domain Using Random Peptide Libraries

The pre-S-specific monoclonal antibody MA 18/7 has been shown to inhibit the binding of HBV to HepG2 cells and liver membranes. This antibody can thus be used to identify the critical residues of the pre-S region involved in the hepatocyte-binding domain. Using overlapping 7-mer peptides representing the pre-S region of HBV, the epitope recognized by MA 18/7 was shown to contain sequences from both the pre-S1 and pre-S2 regions, thus indicating that the hepatocyte-binding domain is conformationally dependent. To further characterize the primary structure of the hepatocyte-binding domain on the pre-S protein, a phage-displayed 15-mer peptide library and a 8-mer solid phase peptide library were used to analyze the fine specificity of the monoclonal antibody MA 18/7. Several mimotopes were identified with the phage-displayed peptide library, the majority of which possess a central motif with at least three identical residues present within the native pre-S1 sequence. No significant consensus sequences were found when these mimotopes were compared to the pre-S2 sequence. Mimotopes identified using the solid-phase peptide library also contained a similar motif. All phage mimotopes and a single mimotope from the solid-phase peptide library competed with recombinant HBsAg particles containing the pre-S1 region for binding to MA 18/7. Mouse antisera raised against four mimotopes from the phage display library reacted with HBsAg particles containing pre-S sequences. The data show that the structure of the pre-S molecule around the conserved DPAF motif in the pre-S region may have a functional role in binding HBV to cellular receptors, and that the central motif identified in mimotopes of this region may offer a novel strategy target for the improvement of existing hepatitis B vaccines which, at present, are mostly devoid of pre-S specificities.

Abnormal growth induced by expression of HBsAg in the secretion pathway of S. cerevisiae pep4 mutants

The toxicity of HBsAg in the secretion pathway of pep4 strains can be progressively reduced in modified SD media containing lower concentrations of ammonium sulphate. A procedure, combining a reduction of ammonium sulphate concentration in SD media with the disruption of the PEP4 gene of the host strain, was developed to enrich transformants which are not inhibited by HBsAg expressed in the secretion pathway. Abnormal growth of these non-inhibited transformants is characterized by the enlargement of cell morphology, a transition to pseudohyphal-like growth in nitrogen-starved media, an increase in HBsAg particle production, and the enhancement of growth rate in liquid media. This suggests a new approach to overcoming the toxicity of heterologous protein in the yeast secretion pathway.

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.

Cytotoxic T lymphocyte activity to hepatitis B virus DNA-transfected HepG2 cells in patients with chronic hepatitis B

Cytotoxic T lymphocyte (CTL) activity for hepatitis B virus (HBV) DNA-transfected HepG2 cells (designated HB3-5), which secrete HBsAg, HBeAg and HBV particles, was investigated in 31 patients with chronic HBV infection (18 chronic hepatitis and 13 asymptomatic carriers). 51Cr-labeled HB3-5 with A2 as a major HLA class I antigen served as target cells and T cells from peripheral blood mononuclear cells as effector cells. The CTL activity was measured by a 51Cr release assay. Patients were divided into two groups, the A2 group bearing HLA-A2 and the non-A2 group not bearing HLA-A2. Chronic hepatitis patients in the A2 group showed increased HBV Ag-specific cytotoxicity compared with that seen in the non-A2 group (5.2 +/- 3.1% vs. 0.9 +/- 1.4%; means +/- SD, P < 0.01). In the A2 group with chronic hepatitis, the cytotoxicity was greater in anti-HBe positive patients than in HBeAg positive patients (8.6 +/- 1.9% vs. 3.4 +/- 2.0%, P < 0.01), and asymptomatic carriers showed less cytotoxicity (0.35 +/- 0.31%, P < 0.001) compared with chronic hepatitis patients. In the non-A2 group, HBV Ag-specific CTL activity was negligible in most patients and thus no differences were found among all patient groups. The HBV Ag-specific cytotoxicity was inhibited by antibodies to CD3, HLA class I and hepatitis B nucleocapsid antigens. Removal of CD8+ cells also resulted in marked decrease in the cytotoxicity. These findings indicate that HBV Ag-specific cytotoxicity reflects liver cell damage and HBeAg/anti-HBe status. Furthermore, our assay system appears to be useful to assess CTL response in patients with chronic HBV infection.

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.