Differentiation of hepatitis B virus genotypes D and E by ELISA using monoclonal antibodies to epitopes on the preS2-region product

An enzyme-linked immunosorbent assay (ELISA) has been described for serological determination of hepatitis B virus genotypes, using monoclonal antibodies (mAb) against seven distinct epitopes (b, m, k, s, u, f and g) on the preS2-region products of hepatitis B surface antigen (HBsAg). The usefulness of this method for serological detection of genotype E, however, was theoretical, because no HBsAg samples of this genotype were included in the original test panel. Moreover, the predicted serotype of genotype E (bksufg) closely resembled that of genotype D (bksu, bksuf or bksug). Four HBsAg samples of genotype E were tested by the original described ELISA. The epitope g, predicted to be present in these samples by amino acid sequences, was not detected when HBsAg of genotype E was captured on a solid phase by mAb to the common determinant 'a' of HBsAg and then reacted with mAb to g (5156) labeled with horseradish peroxidase. However, the four examples of HBsAg of genotype E were captured by mAb 5156 to g on a solid phase; they were then detected by labeled mAb to the common determinant 'a'. Since epitopes f and g co-occurred on HBsAg of genotype E, HBsAg samples of this genotype were also detected, by 'sandwiching' them between immobilized mAb to g and labeled mAb to f. By contrast, HBsAg of genotype D in 90 sera was not reactive when sandwiched between mAb to f and g. Thus, this modified ELISA enables the serological determination of all six genotypes of HBsAg and, by inference, of hepatitis B virus.     

Failure of preexisting antibody against hepatitis B surface antigen to prevent subsequent hepatitis B infection.

We studied a patient who developed acute hepatitis B virus (HBV) infection despite the presence of preexisting antibody to the surface antigen of HBV (anti-HBs). Anti-HBs has been reported to consist primarily of antibody against the common a determinant of HBV. Antibody directed against this major determinant appears to confer protection against HBV, regardless of the subtype. Our patient was shown to have had preexisting anti-HBs of anti-d but not anti-a specificity. She subsequently developed non-A, non-B viral hepatitis followed by an episode of acute hepatitis B after exposure to HBV of the ayw subtype.     

A study of the antigenicity and immunogenicity of a new hepatitis B vaccine using a panel of monoclonal antibodies

The successful prevention of infection with hepatitis B virus (HBV) has been achieved by vaccination with purified hepatitis B surface antigen (HBsAg). The ability of a novel synthetic HBV envelope antigen vaccine (Hep B-3, Hepagene ™; Medeva), which contains part of the pre-S1 and the complete pre-S2 regions and the whole of the S region and was produced in a mammalian cell line, to induce antibodies required for a protective immune response is of importance. In this study, the use of a panel of monoclonal antibodies known to bind to epitopes within the common “a” determinant has demonstrated that the epitopes present on this new vaccine are comparable to those found with plasma-derived HBsAg. In addition, the epitope specificity of the antibodies induced by this vaccine was examined and shown to accord well with previous results obtained using both a plasma-derived vaccine and a recombinant vaccine prepared in yeast. J. Med. Virol. 54:1–6, 1998. © 1998 Wiley-Liss, Inc.     

Overlapping gene mutations of hepatitis B virus in a chronic hepatitis B patient with hepatitis B surface antigen loss during lamivudine therapy

Disappearance of hepatitis B surface antigens (HBsAg) in chronic hepatitis B usually indicates clearance of hepatitis B virus (HBV) infection. However, false HBsAg negativity with mutations in pre-S2 and 'a' determinant has been reported. It is also known that YMDD mutations decrease the production of HBV and escape detection of serum HBsAg. Here, we report overlapping gene mutations in a patient with HBsAg loss during the lamivudine therapy. After 36 months of lamivudine therapy in a 44-yrold Korean chronic hepatitis B patient, serum HBsAg turned negative while HBV DNA remained positive by a DNA probe method. Nucleotide sequence of serum HBV DNA was compared with the HBV genotype C subtype adr registered in NCBI AF 286594. Deletion of nucleotides 23 to 55 (amino acids 12 to 22) was identified in the pre-S2 region. Sequencing of the 'a' determinant revealed amino acid substitutions as I126S, T131N, M133T, and S136Y. Methionine of rtM204 in the P gene was substituted for isoleucine indicating YIDD mutation (rtM204I). We identified a HBV mutant composed of pre-S2 deletions and 'a' determinant substitutions with YMDD mutation. Our result suggests that false HBsAg negativity can be induced by combination of overlapping gene mutations during the lamivudine therapy.     

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.     

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.     

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.     

Neutralization of hepatitis B virus infectivity by a murine monoclonal antibody: an experimental study in the chimpanzee

Two study chimpanzees were inoculated intravenously with approximately 1,000 chimpanzee infectious doses of hepatitis B virus (HBV), one with subtype adr and one with subtype ayw, each previously incubated with 0.1 ml of a murine monoclonal antibody (IgG 1(K) class) directed against a single epitope on hepatitis B surface antigen common to most or all HBV. Two control chimpanzees received identical doses of HBV not incubated with the murine anti-HBs. Neither study chimpanzee developed HBV infection during 12 months of follow-up as judged by normal serum aminotransferase activity, normal liver biopsies, and negative serological tests for HBV-associated antigens and antibodies. In contrast, both control chimpanzees became infected by HBV as evidenced by elevated serum aminotransferase activity, liver biopsy changes characteristic of viral hepatitis, and the appearance of hepatitis B surface antigen (HBsAg) in their sera. Both study chimpanzees were shown to be fully susceptible to infection with these same HBV inocula when challenged 15 months after the initial inoculations at a time when passively administered anti-HBs was no longer detectable. Prior to challenge with HBV, one of the two study chimpanzees received a second injection of the same volume of the murine monoclonal anti-HBs. The survival of this anti-HBs in serum was reduced from six weeks (after the initial injection) to approximately two weeks.(ABSTRACT TRUNCATED AT 250 WORDS)     

Detection and significance of anti-HBc in the blood bank; preliminary results of a controlled prospective study

It has been suggested that post-transfusion hepatitis B (PTHB) may occur after transfusion with blood negative for hepatitis B surface antigen (HBsAg) but positive for antibody against hepatitis B core antigen (anti-HBc). We are currently conducting a controlled prospective study of recipients of such blood to investigate this possibility.Blood donors were routinely screened for HBsAg by radioimmunoassay (RIA) and those found negative were tested for anti-HBc by RIA. The HBsAg negative, anti-HBc positive donors were then tested for antibody directed against HBsAg by RIA, antibody directed against hepatitis B ‘e’ antigen by enzyme-immunoassay and for the liver enzymes SGOT and SGPT.Todate, follow-up has been completed in the recipients of 141 anti-HBc positive blood donors and in a control group of the recipients of 141 anti-HBc negative blood donors. All the recipients were bled repeatedly with intervals of 4–5 weeks up to 7 months post-transfusion. Currently, no clear-cut sero con version was observed in recipients of either group for any of the antibody markers of HBV infection. While none of the 141 recipients of anti-HBc negative blood became positive for HBsAg, recipients of anti-HBc positive blood acquired HBsAg and developed clinically manifest post-transfusion hepatitis B.     

Detection of hepatitis B surface antigen (HBsAg) in peripheral blood mononuclear cells of patients with acute and chronic active hepatitis B

Seventy five patients with acute and chronic active hepatitis (CAH) were studied by indirect immunofluorescence with monoclonal antibodies for the presence of hepatitis B surface antigen (HBsAg) on peripheral blood mononuclear cells (PBMC). The viral surface antigen was detected in the PBMC of all the patients with hepatitis B virus (HBV)-induced CAH and in acute patients with more than 2 months of evolution. No HBsAg was detected in the samples obtained from 12 normal controls or from 14 non-A, non-B CAH patients. Analysis of PBMC subsets revealed that HBsAg was present in non-T cells; dual fluorescence studies showed HBsAg on surface Ig-positive lymphocytes. The binding of anti-HBs monoclonal antibodies was higher than that of a goat anti-HBs serum, and the highest reactivity was observed with an antibody against the pre-S(2)-region sequence. Both HBsAg and hepatitis B core antigen (HBcAg) were also detected in lysates of PBMC by dot blot analysis.     

Acute hepatitis B viral infection in a patient with anti-HBs.

We report a patient with antibody to hepatitis B surface antigen (anti-HBs) but no antibodies to other hepatitis B virus components, who developed acute symptomatic type B hepatitis. The possible explanations for this unusual serological pattern are 1) the antibody-positive status, which developed against only a subdeterminant of hepatitis B surface antigen (HBsAg), arose naturally or as the result of cross-reaction with a variety of antigens; and 2) seroconversion to anti-HBs occurred in response to surface antigen of a mutant strain of hepatitis B virus (HBV). This anti-HBs positivity, in the absence of antibody to hepatitis B core antigen, does not provide natural immunization against HBV infection, and so is not protective. Individuals who are positive to anti-HBs antibody alone which is not elicited by HBV vaccine, should be vaccinated against possible HBV infection.     

Effect of variation in the common "a" determinant on the antigenicity of hepatitis B surface antigen

Antibody to the common "a" determinant of hepatitis B surface antigen (HBsAg) protects against infection with hepatitis B virus. A number of variant surface antigens with amino acid substitutions within the "a" determinant have been described in patients around the world. Both wild type and variant HBsAgs were expressed in the yeast Pichia pastoris and the antigens were semi-purified and quantitated. The effect on antigenicity of these changes was investigated in a quantitative fashion using four monoclonal antibodies known to bind to different epitopes within the common "a" determinant. The results suggest that amino acid substitution of T131I, K141E and G145R and insertion of 3 amino acids between residues 123 and 124 markedly affect the antigenic structure of HBsAg. These substitutions and insertions in the viral envelope may lead to evasion of the virus neutralizing antibody response and also to reduce efficiency of detection by immunoassays used for diagnosis and blood-bank screening.     

Stimulation of a memory B cell response does not require primed helper T cells

The use of universally immunogenic T cell epitopes, such as those identified in tetanus toxin or malaria circumsporozoite protein, could represent a major improvement in the development of synthetic vaccines. However, one limitation of this approach is the lack of T cell cross-reactivity between the vaccine and the pathogen. To determine whether the memory B cell response elicited by immunization with a synthetic peptide containing a B cell epitope linked to a T cell epitope can be restimulated by the same B cell epitope linked to different T cell epitope(s), we used a synthetic peptide which contains non-overlapping B and T cell determinants from hepatitis B surface antigen (HBsAg) of hepatitis B virus (HBV). The results of this study clearly show that primed T cells can increase the antibody response against a B cell epitope linked to the priming T cell determinant. However, the antibody response obtained was weaker than that obtained after two injections of the peptide containing both B and T cell epitopes, showing the important role played by memory B cells in secondary antibody responses. Moreover, a strong antibody response against the B cell epitope was elicited by boosting mice with the B cell epitope linked to a heterologous carrier, thus demonstrating that a strong B cell memory response can be revealed in the absence of primed T cells. These results therefore provide new important information for the design of synthetic or recombinant vaccines.     

Evaluation of murine monoclonal antibodies targeting different epitopes of the hepatitis B virus surface antigen by using immunological as well as molecular biology and biochemical approaches

The hepatitis B virus surface protein (HBsAg) displays the major B cells antigenic determinants that can induce protective immunity and prevent the hepatitis B virus (HBV) infection, a major health problem. A panel of murine monoclonal antibodies against the HBsAg (MAb anti-HBs), raised after mice immunization with a pool of plasma of hepatitis chronic carriers, has been established. Mainly using simple immunological tools such as enzyme-linked immunosorbent assay (ELISA) and Western blot analysis, we could trace the location of the epitopes on the HBsAg determinants. We also report the use of two specific methodology approaches based on molecular biology and biochemical techniques such as, respectively, cloning and expression of preS1 major neutralizing epitope of the HBsAg in Escherichia coli and ELISA accomplished to chemical reduction with dithiothreitol (DTT), which were able to complete the MAb anti-HBs characterization. Our results showed that the majority of the MAbs anti-HBs were directed to the HBV common determinant a. One MAb recognizes a discontinuous epitope present in all forms of the HBsAg when evaluated by Western blot.     

Prevalence of serologic markers of the hepatitis B virus in pregnant women of Bamako, Mali

A prospective study of the serological markers of hepatitis B virus (HBV) including hepatitis B virus surface antigen (HBsAg) and hepatitis B surface antibody (anti HBs) was conducted over 5 years in Bamako. The aims of this study were to assess the prevalence of HBsAg in pregnant women and to determine the risk of HBV infection for this population. The study involved 829 pregnant women for whom blood samples were collected after the first quarter of pregnancy. HBsAg and anti HBs were detected in all cases by radioimmunoassay. The prevalence of HBsAg and anti HBs in pregnant women was respectively 15.5% and 16.9%. This prevalence of HBsAg, higher than in the general population, points to the fact that pregnant women are a high risk group for hepatitis B infection. In addition, scarification and tattooing practices increase significantly the risk of infection by hepatitis B virus (OR = 2.03; 1.07 < OR < 3.82; chi 2 = 5.62; p: 1%). Thus, we can presumably conclude that infants and new borns in such conditions are largely exposed to hepatitis B virus infection, even though hepatitis B core antibody and hepatitis B e antigen were not investigated for technical reasons. In conclusion, the authors believe that infants and new borns must be systematically immunised against hepatitis B virus infection in Bamako.     

Detection of a premature stop codon in the surface gene of hepatitis B virus from an HBsAg and antiHBc negative blood donor.

BACKGROUND: In blood donors, HBV infection is detected by the presence of serum hepatitis B surface antigen (HBsAg). However, some mutations in the surface gene region may result in altered or truncated HBsAg that can escape from immunoassay-based diagnosis. Such diagnostic escape mutants pose a potential risk for blood transfusion services. RESULTS: In the present study, we report a blood donor seronegative for HBsAg and antiHBc, but positive for antiHBs who was HBV DNA positive by PCR. Sequencing of the HBsAg gene revealed presence of a point mutation (T-A) at 207th nucleotide of the HBsAg ORF, which resulted in a premature stop codon at position 69. This results in a truncated HBsAg gene lacking the entire 'a' determinant region. However, follow-up of the donor after 2 years revealed clearance of HBV DNA from the serum. CONCLUSION: The case illustrates an unusual mutation, which causes HBsAg negativity. The finding emphasizes the importance of molecular assays in reducing the possibility of HBV transmission through blood transfusion. However, developing more sensitive serological assays, capable of detecting HBV mutants, is an alternative to expensive and complex amplification-based assays for developing countries.     

Comparison of polyclonal and monoclonal antibodies in determination of glomerular deposits of hepatitis B virus antigens in hepatitis B virus-associated glomerulonephritides

The nature of hepatitis B virus (HBV) antigens in HBV-associated glomerulonephritides was investigated in 7 hepatitis B surface antigen (HBsAg) carriers with membranous nephropathy, 16 HBsAg carriers with mesangial IgA nephropathy, and 1 HBsAg carrier with a mixed picture of membranous and IgA nephropathies. Consecutive frozen sections of renal biopsy specimens were stained with polyclonal and monoclonal antibodies against HBV antigens. Glomerular capillary deposits of HBeAg and HBcAg were detected in 66% and 57% of renal biopsies from HBsAg carriers with membranous nephropathy by monoclonal and polyclonal antibodies, respectively. The discrepancy in the immunofluorescence findings resulted from the cross-reactivity of the polyclonal anti-HBcAg antiserum because it contains both anti-HBcAg and anti-HBeAg activities. Mesangial deposits of HBsAg were detected in 40% and 21% of renal biopsies from HBsAg carriers with mesangial IgA nephropathy by polyclonal and monoclonal antibodies, respectively. The authors' study confirms that HBeAg is the predominant HBV antigen deposited in HBV-associated membranous nephropathy, and glomerular HBsAg deposits are detected in some HBsAg carrier with mesangial IgA nephropathy. Careful testing and evaluation of each antibody are necessary to prevent misinterpretation.     

Infections by hepatitis B surface antigen gene mutants in Europe and North America

Hepatitis B virus (HBV) mutants have usually been studied in patients in Asia because of the wider use of HBV immunization there and the resultant emergence of viral mutants. Nevertheless, HBV surface antigen (S) gene mutants also are found in Europe and North America. In Europe and North America, HBV with mutations in the portion of the S gene coding the "a" determinant of the hepatitis B surface antigen (HBsAg) have been documented in small numbers of infants born to HBV-infected mothers following post-natal HBV vaccine and hepatitis B immune globulin (HBIG) prophylaxis and in many liver transplant recipients who develop HBV re-infection despite HBIG prophylaxis. In some cases, these mutations have included a glycine to arginine substitution at position 145 (G145R), which results in a conformational change and different reactivity to monoclonal antibody reagents than that of the wild-type virus. Mutations in the a determinant (but not G145R) also have been reported in European patients with chronic HBV infection who have not received HBV vaccine or HBIG. However, it appears that such mutations are only responsible for a small proportion of "occult" or "silent" HBV infections, which are characterized by the presence of HBV DNA in serum in the absence of detectable HBsAg. However, some of these mutant forms of HBV in cases of occult HBV may theoretically escape detection and could present a risk to blood safety.     

Two vaccinia virus recombinants expressing HBsAg with different concentration of A- and pre-S2 antigenic determinants.

Comparative studies of two vaccinia virus (VV) recombinants expressing the hepatitis B virus (HBV) surface antigen (HBsAg) including the pre-S2 region (M-protein) showed that the L-pre-S2/15 recombinant expressed 5-fold more HBsAg as determined by the content of a-determinant than the recombinant v137. However, both recombinants expressed comparable amounts of the pre-S2 antigenic determinant as assessed by enzyme immunoassay with monoclonal antibodies. According to our calculations, one HBsAg unit expressed by the recombinant v137 contained 7-9 times more pre-S2 antigen than did one HBsAg unit expressed by the L-pre-S2/15 recombinant. Binding of pre-S2 region to polymerized human serum albumin was shown not to be an efficient assay at low pre-S2 concentration. HBsAg expressed by the v137 recombinant was less extensively secreted from cells as compared to that expressed by L-pre-S2/15 recombinant. Both recombinants induced the production of antibodies to the pre-S2 antigenic determinant in rabbits. L-pre-S2/15 induced anti-HBsAg a-determinant antibody as well.     

Diagnosis of type B hepatitis

Since 1965, when Blumberg discovered the Australia antigen, the hepatitis B surface antigen (HBsAg), the research on viral hepatitis has rapidly progressed. The identification of specific hepatitis B associated antigens and antibodies in blood, and liver tissue, together with the improvement of detection systems, have enhanced our knowledge about the mechanism of liver injury and the natural history of hepatitis B virus (HBV) infection. Now it has been recognized that HBV has no direct cytopathic effect on hepatocytes and that hepatocyte necrosis is associated with the virus induced immunological reaction of the host. From the reaction, there are two types of HBV infection, i.e., transient (acute) and persistent (chronic) infection. In addition to the conventional measurements, such as HBsAg, anti-HBs, anti-HBc, HBeAg, anti-HBe and anti-IgM HBc, recently pre S1, pre S2 antigen/antibody systems and polymerized human albumin receptor and antibody have been developed. The significance of the detection of these antigen/antibody systems was discussed. On the other hand, to determine the presence of HBV, the state of HBV replication or the infectivity directly, HBV associated DNA polymerase and HBVDNA should have been detected. (Very recently, the polymerase chain reaction method has been introduced to detect very small amounts of HBVDNA). In this presentation, the change of these viral markers in various cases was shown, and especially emphasized was anti-IgM HBc in acute hepatitis and HBeAg/Ab status in chronic liver disease. Lastly, the present state of Interferon therapy for type B chronic hepatitis was mentioned.