Occurrence of pre-S1 antigen in viremic and nonviremic carriers of hepatitis B surface antigen

The proteins of viral envelope, encoded by the pre-S1 region of HBV-DNA, were measured quantitatively with enzyme immunoassay using monoclonal antibodies directed to pre-S1 epitope and correlated with the expression of pre-S2 region encoded epitope and other HBV markers. In acute HBV infection, both pre-S encoded proteins were detected in sera along with markers of viral replication and disappeared shortly before complete virus clearance while high HBsAg titers were still present. Pre-S1 antigen was present in most (95.5%) symptomatic and asymptomatic chronic HBsAg carriers. There was no correlation between the presence of pre-S1 and HBeAg or HBV-DNA in serum: 73% of sera with pre-S1 determinants were anti-HBe positive, and only 25.4% were positive for HBV-DNA. Most pre-S1 activity in sera of viremic carriers was detected in fractions of sucrose gradient containing subviral 22-nm particles, and much less in those containing infectious virions. In asymptomatic, nonviremic HBsAg carriers, pre-S1 was located only on subviral 22-nm forms. Pre-S1 positive particles had no accessible pre-S2 epitope, which is recognized specifically by monoclonal anti-pre-S2 (F124) antibody. These results show that the synthesis of the large protein of HBV envelope may occur also in the absence of active viral replication, and in these cases pre-S1 encoded sequences are on subviral particles of HBsAg. Therefore, pre-S1 is not a serologic marker of infectious virus. Disappearance of pre-S1 epitopes on HBsAg occurs only before complete clearance of the virus, and this may have potential prognostic relevance.     

Detection of antibodies to pre-S2 encoded epitopes of hepatitis B virus by monoclonal antibody-enzyme immunoassay

An inhibition enzyme immunoassay (IEIA) for the detection of anti-pre-S2 antibody has been developed and used to evaluate anti-pre-S2 responses in the sera of patients recovering from acute type B hepatitis and in the sera of healthy recipients of HBV vaccine. In the assay, we used two monoclonal antibodies recognizing the nonoverlapping epitopes (pre-S2a and pre-S2b) of the pre-S2 protein of HBV envelope which compete with human anti-pre-S2 for the limited antibody-binding sites on recombinant HBsAg particles (pre-S and S gene product). Two variants of the method were assayed employing the reference pre-S2 antigen on the solid (IEIA-sp) or in the liquid phase (IEIA-lp). Two McAbs were used to detect antibodies reacting specifically with pre-S2a and pre-S2b epitopes of the pre-S2 sequence. Both variants gave similar results and were successfully used for the determination of anti-pre-s2 in sera. We demonstrated that during HBV infection as well as after vaccination against HBV both pre-S2 epitopes generate specific immune responses. Anti-pre-S2 were detected in 45.3% patients recovering from HBV infection and in 43.7% of healthy recipients of the HBV vaccine licensed in France. Anti-pre-S2a and anti-pre-S2b were detected in sera in dilutions up to 10(-5). IEIA may provide a specific and highly sensitive screening test for monitoring serum anti-pre-S2 levels during HBV infection and after immunization with HBV vaccine.     

Dissociated antibody responses to the S and pre-S2 regions of the hepatitis B virus after vaccination in hemophiliacs

The membranes of hepatocytes and the pre-S2 envelope protein of the hepatitis B virus (HBV) contain binding sites for polymerized human albumin, which is thought to act as a link between HBV and hepatocytes. Hence, anti-pre-S2 antibodies should prevent HBV uptake by the liver, and there is indeed preliminary evidence that they protect chimpanzees from HBV infection. To evaluate whether a plasma-derived vaccine containing the pre-S2 sequence induced an anti-pre-S2 response in 105 vaccinated hemophiliacs, anti-pre-S2 was measured in parallel with antibody to hepatitis B surface antigen (anti-HBs). Eighty-five percent of the hemophiliacs had both anti-pre-S2 and anti-HBs when vaccination was completed, 13% had anti-HBs alone, and 2% (two cases) had anti-pre-S2 alone. Eighty-seven percent of anti-pre-S2-positive hemophiliacs compared with only 50% of anti-pre-S2-negative hemophiliacs (P less than 0.001) developed high anti-HBs titers (greater than or equal to 1,000 mlU/ml). This study demonstrates, therefore, that the antibody responses to the S and pre-S2 regions of HBV may be dissociated after vaccination in hemophiliacs and that higher anti-HBs titers are attained in anti-pre-S2-positive hemophiliacs.     

Evaluation of the pre-S (pre-S(1)Ag/pre-S(2)Ab) system in hepatitis B virus infection.

The diagnostic and prognostic value of pre-S(1)Ag and pre-S(2)Ab was investigated in 69 HBsAg surface antigen positive patients--14 with acute hepatitis B, 30 with chronic liver disease (six chronic persistent hepatitis, 14 chronic active hepatitis, 10 with cirrhosis) and in 25 asymptomatic carriers. Pre-S(1)Ag was found in all patients with chronic hepatitis B virus (HBV) infection regardless of viral replication. In contrast, pre-S(2)Ab was not detected in any patients. Acute hepatitis was studied sequentially with periodic controls at 20 day intervals. Pre-S(1)Ag cleared before HBsAg in six of 14 (43%) patients who progressed favourably, and the two antigens cleared simultaneously in eight of 14 (57%) cases. Patients with early clearance of pre-S(1)Ag progressed favourably, thus indicating the prognostic value of this test, which, however, is still of limited practical application given the small temporal difference between the moment of clearance of the two antigens. The first markers to clear, however, were HBeAg and DNA-HBV, which showed significant differences with respect to the clearance of HBsAg. Moreover, pre-S(2)Ab appeared before HBsAb in 57.1% of our patients and was found in some patients before pre-S(1)Ag and HBsAg had cleared (42.8%), thus allowing complete viral clearance and acute HBV infection to be predicted earlier.     

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.     

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.     

Sequential studies of pre-S2 antigenemia and anti-pre-S2 antibodies in relation to viral replication in acute hepatitis B followed from the early incubation phase

To determine the time sequence of expression of pre-S2 peptide (120-150) during the presymptomatic phase of acute hepatitis B, we used a monoclonal antibody radioimmunoassay in five subjects followed from 30 to 70 days before the onset of liver damage. Pre-S2 peptide was present in serum at low levels from the early incubation phase and started to increase immediately after the first detection of HBV-DNA in serum, in parallel with the increase in HBsAg levels. During the symptomatic phase, levels of pre-S2 peptide declined rapidly; it was no longer detectable after recovery. Anti-pre-S2 antibodies were detected, in four patients, only in the recovery phase. These results demonstrate that expression of pre-S2 peptide occurs very early in the incubation phase of acute HBV infection and is cleared in parallel with HBsAg. Anti-pre-S2 antibodies seem to play no role in viral clearance in these patients.     

A solid-phase enzyme immunoassay for the determination of IgM and IgG antibodies against translation products of pre-S1 and pre-S2 regions of hepatitis B virus

The envelope of hepatitis B virus is coded for by pre-S1, pre-S2 regions and the S gene. A method was developed to determine antibody to the product of pre-S1 region (anti-pre-S1) and antibody to the product of pre-S2 region (anti-pre-S2), either of IgM or IgG class, by a solid-phase enzyme immunoassay. For the determination of anti-pre-S1, tubular particles containing translation products of pre-S1, pre-S2 regions and the S gene were broken into constituent envelope polypeptides and immobilized on a solid support. Serums were absorbed with spherical particles containing translation products of pre-S2 region and the S gene, obtained from plasma positive for hepatitis B e antigen (HBeAg) and deprived of particles carrying pre-S1 product by an affinity column. They were then tested for the binding with tubular polypeptides fixed on a solid support, and the bound antibody representing anti-pre-S1 was detected by monoclonal antibody to human IgM/mu or IgG/gamma labeled with horseradish peroxidase. For the determination of anti-pre-S2, test serums were absorbed with spherical particles containing the product of the S gene, obtained from plasma positive for antibody to HBeAg and deprived of particles bearing pre-S2 product by an affinity column. They were then tested for the binding with polypeptides, fixed on a solid support, composed of products of pre-S2 region and the S gene. The assay was applied to the determination of anti-pre-S1 and anti-pre-S2 of IgM or IgG class in asymptomatic carriers and in persons who had recovered from infection with hepatitis B virus.     

A monoclonal antibody enzyme immunoassay for the detection of epitopes encoded by the pre-S2 region of the hepatitis B virus genome

Pre-S2-coded sequences of the hepatitis B virus (HBV) represent important serological markers of HBV infection and elicit the antibodies essential for recovery from type B hepatitis. Monoclonal antibodies (McAbs) directed against two non-overlapping epitopes (pre-S2a and pre-S2b) of the pre-S2 protein of HBV were used to develop an enzyme immunosorbent assay (EIA). The assay was based on the solid-phase sandwich principle in which two different epitope-specific antibodies were used as immunadsorbents and as enzyme-labelled probes. The assay sensitivity was in the pg range and permitted precise quantitation of the pre-S2 sequences in sera. Using the 'site-specific' monoclonal assay we demonstrated that pre-S2a and pre-S2b epitopes are expressed on HBsAg particles of both ay and ad subtypes. The assay is the most sensitive currently available method for the detection of pre-S2 epitopes and may be used for routine immunodiagnosis of hepatitis B.     

Immunochemical structure of the hepatitis B surface antigen vaccine--II. Analysis of antibody responses in human sera against the envelope proteins

Antibody responses to the three envelope (env) proteins of hepatitis B viral particles (HB-VP): the S-encoded P25 polypeptide; the pre-S(2)- and S-encoded GP33/GP36 polypeptide; and the large entire env gene (pre-S + S) product, P39/GP42, were investigated using a Western immunoblotting assay (WIBA). HB-VP proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose by electroblotting were used as antigenic probes to determine the polypeptide specificity of these antibodies present in immune individuals. Antisera from human subjects either after a natural HBV infection or after active immunization with the hepatitis B vaccine licensed in France were selected on the basis of a positive serological RIA test for antibodies against hepatitis B surface antigen (HBsAg). In all studied cases, the lack of reactivity of the anti-HBs/P25 antibodies in blots from reduced SDS gels confirms that the S-related-determinants have a conformation sensitive to denaturing agents. In contrast, the anti-pre-S(2)/GP33-GP36 antibodies and the anti-pre-S(1)/P39-GP42 antibodies can be easily detected in WIBA, providing these antibodies recognize the disulfide-bond independent pre-S determinants on the denatured env proteins. However, antisera raised in guinea-pigs against individual HBsAg polypeptides contain antibodies reacting with denatured S-proteins, suggesting that the sequential S-determinants are lost during HBV morphogenesis. Antibody responses in HBV convalescing patients or vaccinated healthy donors are shown to be characterized by: an early transient polypeptide specific-antibody response to pre-S(2)-sequences (detected in WIBA); a persistent antibody response to conformation-dependent S-determinants (detected in RIA). This implies that effective long-term protection against HBV infection requires antibodies directed to native env proteins.     

Detection of hepatitis B virus pre-S1 and pre-S2 determinants in paraffin wax embedded liver tissue: importance of reagents used.

The presence of pre-S polypeptides in paraffin wax embedded liver sections from the biopsy specimens of 15 hepatitis B surface antigen (HBsAg) seropositive patients (five with chronic persistent hepatitis (CPH), four with chronic active hepatitis (CAH), four with cirrhosis and two "healthy" HBsAg carriers) was investigated using monoclonal antibodies directed to distinct epitopes on pre-S1 (18/7 and TO 606) and pre-S2 (5535 and Q 19/10). Pre-S1 was found in 13 cases when MA 18/7 was used but in only one specimen when TO 606 was used. Pre-S2 was detected in all the biopsy specimens with 5535 and in eight samples with Q 19/10. Mild enzymatic digestion annulled the staining of all monoclonal antibodies but Q 19/10. No association was observed between pre-S polypeptide expression and hepatitis B virus (HBV) replication or disease severity. Pre-S polypeptides can be detected readily in paraffin wax embedded material but the results obtained largely depend on the monoclonal antibodies used.     

Characterization of neutralizing anti-pre-S1 and anti-pre-S2 (HBV) monoclonal antibodies and their fragments.

Single-chain Fv fragments (scFv) were generated from two murine monoclonal antibodies directed to the neutralizing epitopes of the pre-S1 and pre-S2 region of hepatitis B virus, respectively, using different assembly cloning strategies. The scFv fragments were solubly expressed in E. coli. Dissociation constants were in the nanomolar range for all forms (whole IgG antibodies, Fab fragment and scFv fragments). The epitopes of both antibodies were mapped using solid phase peptide synthesis on continuous cellulose membranes and turned out to be linear determinants. The minimal epitope for the anti-pre-S2 antibody 1F6 was identified to be DPRVRGLYF (amino acid 133-141 of the pre-S region). For the anti-pre-S1 antibody MA 18/7 the minimal epitope proved to be the hexamer LDPAFR (amino acid 30-35 of the pre-S region). Complete substitutional analyses as well as truncation experiments revealed key residues for these antibody-antigen interactions. On the basis of those results we used computer-assisted modeling techniques to suggest models for both antibody-peptide interactions providing insight into the structural basis of these molecular recognitions.     

In vitro immune responses to hepatitis B surface antigen (pre-S2 and S) following remote infection by hepatitis B virus in humans

In this report we evaluate the human immune response to hepatitis B surface antigen (HBsAg) following remote infection with hepatitis B virus (HBV). HBsAg-reactive lymphocytes can be readily demonstrated in the peripheral blood of individuals with established immunity following infection with HBV.In vitro stimulation with small doses of plasma-derived HBsAg, yeast-derived HBsAg (S region) or pre-S2 peptide will induce specific IgG to HBsAg (anti-HBs) in the absence of a polyclonal increase in total IgG. The pre-S2 peptide will stimulate, in a T cell-dependent fashion, thein vitro production of anti-HBs with specificity for the S domain. This anti-HBs production is mediated by pre-S2-stimulated soluble T-cell factors. Peripheral blood mononuclear cells from individuals with established immunity proliferate to the yeast-derived HBsAg but not to the plasma-derived HBsAg or pre-S2 peptide. The chronic HBsAg carriers do not produce anti-HBs following stimulation with HBsAg regardless of the source or component of antigen used. Different study protocols failed to demonstrate HBsAg-specific responses in the peripheral blood mononuclear cells of chronic carriers.     

Characterization of monoclonal antibodies specific for the pre-S2 region of the hepatitis B virus envelope protein

Monoclonal antibodies (McAb) specific for the pre-S region of the hepatitis B virus (HBV) envelope protein were prepared using HBV particles of hepatitis B surface antigens (HBsAg) as immunogens. The antibodies reacted in Western blot analyses and in ELISA with pre-S2 sequences of the HBV envelope protein. Pepsin or protease V8 treatment of the antigen abolished reactivity. The fine specificity of one of the McAb (F376) was established by immunoassays using synthetic peptides and a pre-S2-beta-galactosidase fusion protein expressed in E. coli. The shortest peptide recognized by F376 is demarcated by residues pre-S(132) at the N-terminal and pre-S(140)-pre-S(145) at the C-terminal. The corresponding amino acid sequence (for HBV subtype adw2) is: QDPRVRGLY(LPAGG). Additional amino acid residues at the N-terminal, and possibly at the C-terminal ends contribute to the binding of McAb, probably due to conformational influences. The McAb was applied to immunoassays of pre-S2 sequences in purified HBsAg and in human sera containing HBsAg.     

Expression of pre-S2 region of hepatitis B surface antigen in Escherichia coli

We constructed a recombinant plasmid that can express the entire pre-S2 sequence of hepatitis B surface antigen (HBsAg) as a fusion protein in E. coli. The hybrid protein, which comprises the bacterial TrpLE protein and the pre-S2 sequence, was the prominent protein that was found in cell extracts. As determined by immune blot analysis, this protein reacted with human HBV convalescent sera, as well as with sera from animals immunized with either purified HBsAg or isolated polypeptides containing pre-S2. It bound specifically to 125I-polymerized human albumin cross-linked with glutaraldehyde but not to 125I-monomeric human albumin. A novel adjuvant formulation was used in place of Freund's adjuvant to immunize guinea pigs with the recombinant product. The antisera obtained from serial bleedings were found to react with HBsAg of both d and y subtypes. These antisera were also shown to react solely with HBsAg polypeptides which contain of HBsAg to solid-phase polymerized the binding of HBsAg to solid-phase polymerized human albumin.     

Immunity to hepatitis B: analysis of antibody and cellular responses in recipients of a plasma-derived vaccine using synthetic peptides mimicking S and pre-S regions.

The potential of a panel of synthetic HBsAg peptides as components of a synthetic hepatitis B vaccine was assessed. Each was used in turn as probes to analyse human immune responses to a licensed plasma-derived HBV vaccine. Both humoral and cellular responses were analysed with synthetic peptides representing residues 124-147 of the surface antigen of the virus (HBsAg) and residues 126-140 of the pre-S2 region. Antibody levels and affinities were assessed in radioimmunoassays with synthetic linear and cyclical forms of surface antigen peptides 124-137 and 139-147, with the gp30p25 polypeptide complex of HBsAg and with the linear pre-S2 peptide 126-140. Levels and affinities of antibodies to the antigens increased with time during immunization. However, antibodies binding the cyclical peptide representing amino acids 139 to 147 (C139) were present at higher levels and had higher affinities than were antibodies binding the other peptides, indicating that C139 more closely approximates a domain on the native antigen than do the other peptides. No humoral responses were measured with the pre-S2 peptide. Cellular responses were assessed by in vitro stimulation of peripheral blood lymphocytes by HBsAg and by the synthetic peptides. All vaccine recipients had demonstrable lymphocyte responsiveness to HBsAg after both second and third doses of the vaccine. Of the S and pre-S peptides used, only L124 failed to induce lymphocyte stimulation in all recipients. However, there were individual variations in both the time of initial responsiveness to peptides and in the level and time of maximal stimulation. Stimulation by native HBsAg particles, which corresponded to the appearance of anti-HBs antibody, preceded that observed using synthetic peptides. In all recipients, maximum stimulation indices with HBsAg were significantly higher than those observed with the peptides. In contrast to the absence of pre-S2 antibody, the lymphocytes from all recipients showed positive stimulation in response to the peptide representing residues 126-140 of the pre-S2 region. None of these individuals had antibodies to pre-S or an HB core peptide sequence nor did their lymphocytes respond to a synthetic peptide representing an HB core sequence.     

Enzyme-linked immunoassay of pre-S gene-coded sequences in hepatitis B vaccines

Pre-S gene coded domains of the hepatitis B virus (HBV) envelope protein are highly immunogenic in experimental animals and humans. Their presence in HBV and hepatitis B surface antigen (HBsAg) particles leads to production of anti-pre-S-specific antibodies during the course of HBV infection. Since antibodies specific for pre-S domains are capable of preventing the attachment of HBV to hepatocytes and are virus neutralizing, it would seem desirable to produce HBV vaccines with a standardized level of pre-S determinants to ensure their potential for eliciting the same repertoire of protective antibodies as found after recovery from natural infection. However, a test with appropriate sensitivity for detecting pre-S determinants to ensure their potential for eliciting the same repertoire of protective antibodies as found after (ELISA) for detecting pre-S determinants in vaccines containing less than or equal to 20 micrograms of HBsAg. The components of this assay are (1) antibodies to a synthetic peptide pre-S (120-145) adsorbed to polystyrene beads, and (2) beta-lactamase-labelled antibodies purified from anti-HBV serum on the basis of their affinity for a pre-S (120-174) beta-galactosidase fusion protein produced in Escherichia coli. Results of an evaluation of the pre-S content of HBV vaccines from two different commercial sources are discussed.     

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.     

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)     

New assays for quantitative determination of viral markers in management of chronic hepatitis B virus infection.

We performed a quantitative study of serum hepatitis B virus (HBV) markers, including new parameters such as pre-S1 antigen (Ag), pre-S2 Ag, and anti-HBx, in 88 chronic hepatitis B surface antigen (HBsAg) carriers. New IMx assays for HBsAg and immunoglobulin M (IgM) anti-HBc detection were also used. The population studied was composed of 65 chronic hepatitis cases (40 positive for hepatitis B antigen [HBeAg] and 25 positive for anti-HBe) and 23 anti-HBe-positive, asymptomatic HBsAg carriers. Serum HBsAg levels detected by IMx were higher in HBeAg-positive than in anti-HBe-positive HBsAg carriers (all patient subgroups included) and correlated with the serum HBV DNA level (P = 0.0001). Both pre-S1 and pre-S2 Ags were detected by enzyme immunoassays in almost all HBsAg carriers. Both pre-S1 and pre-S2 Ag titers correlated positively with the serum HBsAg concentration (P = 0.0001), but only the pre-S1 Ag titer correlated with the level of serum HBV DNA (P = 0.02). The detection of low levels of IgM anti-hepatitis B core (anti-HBc) antibodies by IMx was associated with the presence of liver disease (P = 0.05) but not with the level of viral replication. The prevalence of anti-HBx antibodies detected by the enzyme immunoassay was slightly, although not significantly, higher in patients with high levels of HBV DNA (greater than 100 pg/ml) than in patients without detectable HBV DNA (P = 0.16). In anti-HBe-positive chronic HBsAg carriers, the quantitative detection of serum HBV DNA, pre-S Ag titers, and IgM anti HBc allowed us to predict which patients suffered from chronic liver disease and/or supported viral replication (P < 0.05). In a follow-up study of eight patients undergoing antiviral therapy, the clearance of both pre-S1 Ag and HBV DNA was associated with a subsequent clearance of HBV. Therefore, the quantitative determination of HBV DNA, pre-S Ags, IgM anti-HBc may prove useful for the decision to use and the monitoring of antiviral therapy, especially in anti-HBe-positive HBsAg carriers.