A glycopeptide containing the group-specific antigenic determinant of hepatitis b surface antigen (HBsAg)

The treatment of purified 22 nm structures of HBsAg with pronase in the presence of SDS resulted in the appearance of light fragments which contain predominantly group-specific ‘a’ antigenic determinant. By size and mobility in SDS-polyacrylamide gel electrophoresis the isolated material corresponds to globular proteins with molecular weight of 12,000 (or less) daltons. The analysed structures and HBsAg had different isoelectric points and spectrum in UV light. The released material in contrast with HBsAg was inactivated during the treatment with neuramindase. The aggregates of the obtained glycopeptide turned out to be immunogenic for guinea pigs. The immune sera contained antibodies against ‘a’ and ‘y’ specificities of HBsAg but not against normal human plasma proteins. The possibility of using the minimal structures carrying protective antigenic determinants of HBsAg as a vaccine is discussed.     

HBsAg diagnostic kits in the detection of hepatitis B virus mutation within "a" determinant

The performance of currently available hepatitis B surface antigen (HBsAg) commercial kits was analyzed by using a panel of 212 well-characterized plasma donors all over the country and a panel of nine recombinant HBsAg mutants containing single point or combinations of mutations between amino acid residues 124 and 147 of the "a" determinant. HBsAg commercial kits in this study were machine-based immunoassays with a one-step sandwich ELISA method using either an automatic closed system or manual system. The sensitivity of all machine-based assays evaluated with 105 HBsAg plasma panels was 100% (95% CL = 95.6-99.9%), whereas the specificity with 107 HBsAg negative plasma ranged from 99.07% to 100% (95% CL = 94.2-99.9%). The relative performance of these kits to detect the hepatitis B virus (HBV) mutant panel members of the "a" determinant was found to differ. Interestingly, any commercial kits with monoclonal antibody capture and polyclonal antibody detection (mono/poly), but not mono/mono Ab capture and detection, could pick up the common HBsAg Gly145Arg mutant either solely or in combination with other mutations within the "a" determinant. New versions of HBsAg test kits should recognize multiple HBsAg epitopes in order to detect mutant HBsAg, together with providing good analytical sensitivity and specificity, because of the importance of these assays in HBV diagnosis and in protecting the safety of the blood supply.     

Impact of changes in the amino acid sequence of the "a" determinant on the diagnostic capacities of test systems for the detection of HBsAg

The diagnostic capacities of 4 commercial test systems were comparatively estimated for the detection of HBsAg, by applying a panel of samples with the established amino acid sequence of the "a" determinant of HBsAg. The Roche Elecsys HBsAg test system demonstrated the highest sensitivity - the maximum HBsAg concentrations were found in 19 of 31 cases. Escape mutations in the major hydrophilic region (MHR) of HBsAG (P120S, M133T) were responsible for differences in the sensitivity of 4 test systems by 10- to 40-fold. There were also samples that showed differences in the diagnostic capacities of the test systems to detect HBsAg, but without amino acid replacements in the area of the "a" determinant, which seems to be associated with amino acid replacements in other regions of HBsAg.     

Adsorption properties of different hepatitis B virus related antigens (HBsAg, HBcAg, HBeAg) on octanohydrazide-Sepharose 4B

Chromatography of plasma containing hepatitis B virus and partially purified viral antigens on a hydrophobic gel derivative (octanohydrazide-Sepharose 4B) revealed that HbsAg and HbcAg were adsorbed to the gel in 0.75 mol/l ammonium bicarbonate and eluted by a detergent, Berol. HBeAg in a purified HBcAg preparation from human liver, but not HBeAg in plasma, was bound to the gel. Furthermore, HBeAg in the HBcAg preparation, but not HBeAg in plasma, lost its antigenic reactivity in the presence of Berol, indicating that the two HBeAgs were present in different molecular configurations. However, HBeAg could be released from HBV (HBcAg) and form a component which sedimented slowly and was immune-reactive in the presence of the detergent. The results contribute to knowledge of the interrelationship between hepatitis B-related antigens and indicate that chromatography on hydrophobic gel derivatives can be used not only for the purification (and removal) of HBsAg but also of HBcAg.     

Polyalbumin receptors on hepatitis B virus and on 22 nm hepatitis B surface antigen (HBsAg)2 particles

Receptors for polymerized human serum albumin ( pHSA ) were studied by solid-phase radioimmunoassay on different hepatitis B surface antigen (HBsAg) particles subpopulations prepared both from hepatitis B e antigen (HBeAg) and from anti-HBe-positive sera. HBsAg particles in HBeAg-positive serum showed higher expression of the receptor compared with HBsAg particles from anti-HBe-positive serum. Analysis of different morphological forms of virus particles was performed after separation by density-gradient ultracentrifugation. Maximum receptor expression was detected in HBV particles containing fractions while the 22-nm HBsAg particles had significantly lower receptor activity. These observations support the hypothesis of a pathogenetic role of the pHSA receptor in mediating virus access to hepatocytes. Indeed, the higher pHSA binding activity on HBV particles could allow selective attachment of the infectious virion to liver cells that bear similar albumin receptors on their surface.     

Determination of the hepatitis B virus surface antigen (HBsAg) by immunoenzyme analysis

Studies aimed at the development of a variant of ELISA for the determination of hepatitis B virus surface antigen (HBsAg) showed the assay to be most effective when a modified periodate method of conjugation of highly purified horseradish peroxidase with the IgG-fraction of antiserum to HBsAg was used. With the resulting conjugates, the "sandwich"-test on polystyrene solid phase could detect HBsAg in concentrations up to 5 ng/ml which is several thousand-fold higher in sensitivity than counter immunoelectrophoresis, one order of magnitude more sensitive than the passive hemagglutination test, and comparable with the radioimmunoassay (RIA). The specificity of the method was confirmed by positive results of the neutralization test. By this method, HBsAg in the sera of hepatitis B patients was detected as frequently as by the RIA.     

Impact of the heterogenicity of amino acid sequence on the immunoreactivity of an antigenic epitopic complex localized within amino acids 1192-1456 of protein NS3 protein of hepatitis C virus

Recombinant proteins were used to study the effect of heterogenicity of the primary structure of NS3 protein of hepatitis C (HCV) on the immunoreactivity of a complex of antigenic epitopes located within the amino acid sequences 1192-1456. Six genes encoding for the above fragment NS3 from different genotypes were collected from synthetic oligonucleotides and expressed in E. coli cells, by using the polymerase chain reaction. The homology of amino acid sequences of antigens ranged from 78.4 to 92.2%. All the antigens showed a higher coefficient of their reactivity with antibodies in the sera samples from patients infected by HCV of a respective genotype; however, there was no strong genotype-specific immunoreactivity. The findings lead to the conclusion that the primary structure of antigens has an impact on their immunoreactivity. Selection of variants of the primary structures of antigens is essential in developing a diagnostic test.     

Improved detection of natural hepatitis B virus surface antigen (HBsAg) mutants by a new version of the VITROS HBsAg assay

The sensitivity of immunoassays for hepatitis B virus (HBV) surface antigen (HBsAg) detection may be hampered by the presence of mutants involving the major antigenic determinant of the protein. The performance of the VITROS HBsAg Assay has been shown to be affected by mutations comprising amino acid changes at residues 143, 144, and 145 of the HBsAg molecule. Sixty-seven serum samples from HBV carriers containing major populations of natural HBsAg mutants assayed previously by that assay were tested by the new VITROS HBsAg ES Assay. Samples displayed either single or multiple amino acid substitutions between positions 112 and 145 of the HBsAg, including changes in relevant residues such as 118-120, 125-127, and 143-145. Testing of undiluted samples by the current assay gave rise to false negative results in two samples displaying the single substitutions 145A and 145R, and in one additional sample displaying a dual mutation 118A + 145A. Unusually weak reactivity (<25 S/CO units) was, in addition, recorded in samples containing mutants 143L (2 samples) and 115N + 120Q + 131K + 144A (1 sample). Testing samples at the 1/40 dilution by the modified assay did not produce, in contrast, false negative results, and reactivity below 25 S/CO units was recorded only in three cases. These results confirm that the capability of immunoassays to detect the presence of natural HBsAg mutants in clinical samples may be improved significantly by introducing changes in their design, and show that such improvement has been achieved successfully with the new VITROS HBsAg ES Assay.     

Geographical variation in prevalence of hepatitis B virus DNA in HBsAg negative patients.

AIMS--To study the geographical variation of the prevalence of hepatitis B virus (HBV) DNA in hepatitis B surface antigen (HBsAg) negative subjects. METHODS--A nested polymerase chain reaction (PCR) assay was used to amplify the core region of HBV. The assay was able to detect 10 molecules of a full length HBV plasmid. RESULTS--When applied to HBsAg negative paraffin wax embedded liver samples from Italy, Hong Kong, and the United Kingdom, a geographical variation in the prevalence of HBV-DNA positivity was noted. Two of 18 (11%) of Italian samples and 2/29 (6.9%) of Hong Kong samples were positive for HBV-DNA while none of the 70 cases from the United Kingdom was positive by nested PCR. Contamination by plasmid DNA was excluded using a novel method based on heteroduplex formation. One HBV-DNA positive case had idiopathic chronic active hepatitis, but the diagnoses in the other three HBV-DNA positive cases did not suggest any aetiological connection between HBV-DNA positivity and liver pathology. CONCLUSIONS--HBV-DNA could be detected in the liver tissues of a proportion of HBsAg negative subjects. The prevalence of such cases is related to the endemic rate of a geographical region. The use of HBV PCR on paraffin wax embedded tissues will be valuable for future studies on the molecular epidemiology of HBV.     

Identification of functionally important amino acid residues in the mitochondria targeting sequence of hepatitis B virus X protein

Chronic hepatitis B virus (HBV) infection has been strongly associated with hepatocellular carcinoma (HCC) and the X protein (HBx) is thought to mediate the cellular changes associated with carcinogenesis. Recently, isolation of the hepatitis B virus integrants from HCC tissue by others have established the fact that the X gene is often truncated at its C-terminus. Expression of the GFP fusion proteins of HBx and its truncation mutants with a GFP tag in human liver cell-lines in this study revealed that the C-terminus of HBx is indispensable for its specific localization in the mitochondria. A crucial region of seven amino acids at the C-terminus has been mapped out in which the cysteine residue at position 115 serves as the most important residue for the subcellular localization. When cysteine 115 of HBx is mutated to alanine the mitochondria targeting property of HBx is abrogated.     

Dependence of antigenic properties of human and avian influenza A virus NP proteins on strain variations in the amino acid sequence]

Human and avian influenza A strains with a known amino acid sequence of NP protein were studied in radioimmunoprecipitation test with a panel of anti-NP monoclonal antibodies. Two of 7 MAbs (315 and IVE8) reacted with variable epitopes. One of the epitopes was present only in human strains, while the other in both human and avian strains, but absent in gull strains and in one human strain, A/Puerto Rico/8/34 (H1N1). The variations recognized by antibodies 315 and IVE8 correlated with amino acid substitutes in positions 16 and 353, respectively.     

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.     

Synthetic antigens representing the antigenic variation of human hepatitis C virus

Immune responses against hepatitis C virus (HCV) have been studied by numerous groups. However, details concerning the production of antibodies to antigenically variable epitopes remain to be elucidated. Since the sequences of the variable regions of several HCV proteins are different among the virus strains infecting patients, we decided to design peptide combinations that represent the theoretical maximum antigenic variation of each epitope to be used as capture antigens. We prepared six peptide mixtures (hypervariable epitope constructs; HECs) representing six different epitopes from structural and non-structural proteins of HCV from genotypes 1-6. Plasma from 300 HCV patients was tested to determine if their antibodies recognize the synthetic constructs. All the patients were chronically infected with diverse HCV genotypes and did not receive antiviral treatment. Antibodies to one or more of the HECs were detected in all of the HCV-infected individuals. Immunogenicity of the HCV HECs was also evaluated in outbred and inbred mice. Strong HEC-specific antibodies were produced, and cellular responses were also induced that were Th-1 rather than Th-2. Our results show that HCV HECs are both antigens that can be used to detect the broad cross-reactivity of antibodies from HCV-infected patients, and strong immunogens that can induce antigen-specific humoral and cellular immune responses in mice.     

Mutant hepatitis B virus surface antigens (HBsAg) are immunogenic but may have a changed specificity

Mutant hepatitis B virus with substitutions within the coding region for HBV surface antigen (HBsAg) has been found naturally in chronic carriers. It is therefore important to clarify whether the identified substitutions within the HBsAg have impact on the antigenicity and immunogenicity of HBsAg. A total of nine mutated HBV s-genes with single representative mutations were generated by site-directed mutagenesis and subcloned into an expression vector. The binding of polyclonal and monoclonal antibodies to these mutant HBsAg (mtHBsAg) was tested by immunofluorescence (IF) staining of cells transfected with the expression vectors. The amino acid (aa) substitutions like G145R, F134S, and C147W affected the binding of anti-HBs antibodies to corresponding mtHBsAg to different extents. The impact of aa substitutions G145R and F134S on the immunogenicity was accessed by genetic immunization of mice with vectors expressing middle HBsAg with the corresponding mutations. The immunized mice developed antibodies to recombinant HBsAg containing the HBV preS region and HBsAg-specific cytotoxic T-cell. However, the development of antibody response to wild-type small HBsAg was significantly impaired by the aa substitutions in HBsAg. Based on this fact, we further investigated whether the mtHBsAg with the aa substitution G145R is able to induce mutant-specific antibody responses. Strikingly, serum samples from mice immunized with mtHBsAg with G145R recognized plasma-derived mtHBsAg. Two mouse MAbs specific to mtHBsAg were generated. One MAb recognized mtHBsAg with G145R but not wild type and other mtHBsAg. We conclude that HBsAg with aa substitutions are immunogenic but may have a changed fine specificity.     

Effects of systematic variation of amino acid sequence on the mechanical properties of a self-assembling,oligopeptide biomaterial

In order to elucidate design principles for biocompatible materials that can be created by in situ transformation from self-assembling oligopeptides, we investigate a class of oligopeptides that can self-assemble in salt solutions to form three-dimensional matrices. This class of peptides possesses a repeated sequence of amino acid residues with the type: hydrophobic/negatively-charged/hydrophobic/positively-charged. We systematically vary three chief aspects of this sequence type: (1) the hydrophobic side chains; (2) the charged side chains; and (3) the number of repeats. Each of these has been previously shown to influence the self-assembly properties of these materials. Employing a rheometric assay we measure the shear modulus of gels created from variants of each of these aspects. First, we observe order-of-magnitude changes in shear moduli when we vary oligopeptide length, with biphasic dependence on length. This result may be due to competition between, in short oligopeptides, additional repeats either increasing the diameter of the filaments or increasing the area of interaction between individual molecules and, in large oligopeptides, additional repeats allowing the oligopeptides to fold back upon themselves and decrease their effective length. Second, no statistically significant difference is observed among the hydrophobic variants, suggesting that hydrophobicity and steric overlap are unlikely to play a significant role in filament mechanical properties. Finally, in variation of the charged side chains we observe a small difference in the shear moduli that, if significant, may mean that decreasing the energetic penalty for dehydrating the charged side chains can lead to a stiffer matrix. Overall, we demonstrate that it is possible to achieve order-of-magnitude changes in shear modulus by simple variations of oligopeptide length, while the residue substitutions affect only self-assembly properties. Thus, diverse aspects of these molecules can be designed rationally to yield desirable materials properties of different types.     

Sensitivities of four new commercial hepatitis B virus surface antigen (HBsAg) assays in detection of HBsAg mutant forms

Mutations in hepatitis B virus surface antigen (HBsAg) involving amino acid substitution within the immunodominant "a" determinant may affect the performance of commercial HBsAg assays. The performances of four HBsAg assays that recently received Conformité Européene marking, Advia Centaur HBsAg (Bayer), Monolisa HBsAg Ultra (Bio-Rad), Liaison HBsAg (Dia Sorin), and Vidas HBsAg Ultra (bioMérieux), were compared with that of the routinely used HBsAg assay AxSYM HBsAg V2 (Abbott). Assays were evaluated for (i) analytical sensitivity performance with a national reference HBsAg panel (including 10 samples with calibrated HBsAg concentrations from 0.04 to 2.24 ng/ml) and (ii) the detection of HBsAg mutants by studying a panel of 35 HBsAg mutants (23 collected from patients and 12 recombinant mutants). The limits of detection of these assays were <0.15 ng/ml (from 0.089 to 0.121 ng/ml). The sensitivity performances for mutant virus detection varied, ranging from 37.1% to 91.4%. The lack of detection of these mutants by commercial assays was probably due to the epitope recognition of the anti-HBs assay reagents in the capture phase and in the conjugates. The prevalence and clinical impact of HBsAg mutants are under investigation. However, the manufacturers must be vigilant in the design of the assays in order to reduce the risk of missing a broad range of described S gene mutants.     

Evaluation of two new automated assays for hepatitis B virus surface antigen (HBsAg) detection: IMMULITE HBsAg and IMMULITE 2000 HBsAg

In recent years the diagnostic industry has developed new automated immunoassays for the qualitative detection of hepatitis B virus (HBV) surface antigen (HBsAg) in serum and plasma samples that are performed on analyzers that permit a high-speed throughput, random access, and primary tube sampling. The aim of the present study was the evaluation of two new automated HBsAg screening assays, IMMULITE HBsAg and IMMULITE 2000 HBsAg, from Diagnostic Products Corporation. The new HBsAg assays were compared to well-established tests (Auszyme Monoclonal [overnight incubation, version B], IMx HBsAg, AxSYM HBsAg, and Prism HBsAg [all from Abbott] and Elecsys HBsAg [Roche Diagnostics]). In the evaluation were included seroconversion panels, sera from the acute and chronic phases of infection, dilution series of various HBsAg standards, HBV subtypes and S gene mutants. To challenge the specificity of the new assays, sera from HBsAg-negative blood donors, pregnant women, and dialysis and hospitalized patients and potentially cross-reactive samples were investigated. IMMULITE HBsAg and IMMULITE 2000 HBsAg, although not as sensitive as the Elecsys HBsAg assay, were equivalent to the AxSYM HBsAg assay and showed a higher sensitivity than the Auszyme Monoclonal B and IMx HBsAg systems for detection of acute infection in seroconversion panels. The specificities (100%) of both IMMULITE assays on unselected blood donors and potentially interfering samples were comparable to those of the alternative assays after repeated testing. In conclusion, the new IMMULITE HBsAg and IMMULITE 2000 HBsAg assays show a good sensitivity for HBsAg detection compared to other well-established tests. The specificity on repeatedly tested samples was equivalent to that of the alternative assays. The rapid turnaround time, primary tube sampling, and on-board dilution make it an interesting assay system for clinical laboratory diagnosis.     

A hepatitis B virus variant found in the sera of immunised children induces a conformational change in the HBsAg "a" determinant.

The emergence of variants in the outer envelope proteins of hepatitis B virus (HBV) are found among individuals vaccinated against HBV and asymptomatic carriers of the infection. For example, children in The Gambia vaccinated against hepatitis B may show serological evidence of breakthrough infections, particularly if anti-HBs antibodies induced by the vaccine are low in titre. A single-point mutation at nucleotide 421 of the S gene is associated with such breakthrough infections. In the present study, the antigenicity of variant HBV S protein expressed as HBsAg particles in a vaccinia virus expression system has been characterised using a panel of monoclonal antibodies directed against linear and conformational determinations of the S protein. A cellular ELISA procedure using expressed antigen in Vero cells revealed differences in reactivity using four of the six antibodies that had been raised against the adw subtype of HBV and recognise conformational epitopes in the a determinant. In two instances, an enhanced reactivity for the variant antigen was found, confirming that point mutations in the a determinant of the S protein between residues 139 and 147 may result in significant changes in conformation. These findings also demonstrate that there are distinct antigenic differences between the vaccine strains of HBsAg/ adw subtype and the predominant HBsAg subtype circulating in West Africa. The implications of this work are that serodiagnosis of HBV infections may be unreliable in populations where there is a possibility of variant HBV infections emerging in the face of increasing herd immunity to HBV as a result of vaccination, particularly using monoclonal antibody-based diagnostic tests. Such variants may play a role in the maintenance of HBV infections in endemic regions.