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.

Immunological cross-reactivity between preS2 sequences of the hepatitis B virus envelope proteins corresponding to serological subtypes adw2 and ayw.

An immune response to epitopes localized on the preS region of the hepatitis B virus (HBV) envelope (env) is elicited during recovery from HBV infection and appears to play a role in virus clearance. Anti-preS antibodies (Ab) are expected to be protective against HBV infection as indicated by the virus-neutralizing capacity of Ab to a preS2-specific synthetic peptide preS(120-145). However, there is considerable amino acid variability between preS regions corresponding to distinct serological subtypes of HBV, raising the question whether the preS sequences are sufficiently related immunologically to have the potential of inducing cross-protective immunity. To answer this question concerning the preS2 region, antisera to synthetic peptides preS(120-153) and preS(128-153) corresponding to subtypes adw2 and ayw, as well as to the native env [ayw] protein were raised. Using the resulting polyclonal Ab, an immunological relatedness between preS2 sequences of subtypes adw2 and ayw was demonstrated. On the other hand, Ab selected by affinity chromatography or by cloning hybridoma cells may recognize with strong preference subtype-specific determinants within the preS2 region when the compared antigens are in solution rather than on the solid phase. These findings have implication for the design of: (1) preS2-specific immunogens and (2) immunoassays for quantitation of preS2 sequences in HBV env proteins.

Antibodies to synthetic peptides from the pre-S1 and pre-S2 regions of one subtype of the hepatitis B virus (HBV) envelope protein recognize all HBV subtypes.

Immunodominant B and T cell epitopes have been demonstrated recently on the preS1 and PreS2 regions of the hepatitis B virus (HBV) envelope protein. Synthetic peptide analogs corresponding to the preS2 region elicit virus-neutralizing antibodies and protect chimpanzees against HBV infection. Antibodies raised by immunization with peptides derived from the preS1 sequence block the site involved in HBV attachment to cell receptors, and are expected to be virus-neutralizing. Results presented here show that antisera raised against synthetic peptide analogs carrying the immunodominant epitope of the preS1 and preS2 sequence, respectively, and corresponding to two HBV subtypes, adw2 and ayw, each recognized preS1 and preS2 specific epitopes on all serological subtypes of the HBV envelope protein. Thus, the sequence variability within the preS1 and preS2 regions does not represent an impediment to the development of synthetic peptide or genetically engineered hepatitis B preS immunogens for worldwide immunization.

Hepatitis B vaccination in diabetic patients. Randomized trial comparing recombinant vaccines containing and not containing pre-S2 antigen.

OBJECTIVE: To investigate the immunogenicity of two recombinant hepatitis B vaccines containing S antigen alone (Engerix B) or both S and pre-S2 antigens (GenHevac B) in diabetic patients. RESEARCH DESIGN AND METHODS: Of the adult diabetic patients, 71 (26 IDDM, 45 NIDDM) were randomized to receive Engerix B or GenHevac B at 0, 1, 2, and 12 months in a single-blind clinical trial; if the antibody to hepatitis B surface antigen (anti-HBs) titers were < 10 i.u./l at month 4, a fourth injection of vaccine was given. A positive response was defined by anti-HBs titer > or = 10 IU/l at month 13. RESULTS: The anti-HBs response rate and the titers of anti-HBs did not differ significantly between the two types of vaccine. Overall, > 90% of the patients responded at month 13. In patients vaccinated with GenHevac B, anti-pre-S2 antibodies appeared earlier than anti-HBs. The anti-HBs response tended to decrease with age (P = 0.07) and tended to be higher in IDDM patients than in NIDDM patients (P = 0.06). Metabolic control, as assessed by HbA1c level, did not influence the response rate. The presence of the HLA DQ2 allele was associated with a low response. CONCLUSIONS: A large majority of diabetic patients can be efficiently vaccinated against the hepatitis B virus using a booster dose at month 4. The choice of the vaccine (with or without pre-S2 antigen) appears to have little influence, if any, on the response rate.

Structural and functional characterization of a monoclonal antibody specific for the preS1 region of hepatitis B virus.

The monoclonal antibody 5a19, raised against the ay serotype of hepatitis B virus, binds to the segment of the preS1 region comprising residues 37-43, which is implicated in attachment of the virus to hepatocytes. The dissociation constant, derived from kinetic studies using surface plasmon resonance techniques, is in the low nanomolar range. The nucleotide sequence of the variable domains has been determined and the corresponding germ-line genes have been identified. The three-dimensional structure of the Fab fragment has been determined by X-ray crystallography to 2.6 A resolution.

Enzyme immunoassay for the detection of hepatitis B virus core antigen.

A solid-phase enzyme-linked immunosorbent assay (ELISA) was developed for the detection of hepatitis B core antigen (HBcAg) in preparations of core particles isolated from hepatocyte nuclei and obtained from serum Dane particles. Comparison with counterelectrophoresis (CEP) showed that the sensitivity of ELISA is about 80-100 times greater than that of CEP. The enzyme immunoassay was applied to studies on conversion of HBcAg to HBeAg and it was shown that treatment of core particles with SDS and 2-ME leading to exposure of HBeAg determinants resulted in a complete loss of antigenic activity of HBcAg.

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.

Interest of immunomodulation as a mean to improve the preparation of polyclonal and monoclonal antibody reagents.

Immunomodulation by monoclonal antibodies (mAbs) was investigated in mice in order to improve the preparation of antibody reagents. Three different types of representative immunogens were chosen: a human soluble protein (secretory immunoglobulin A, SIgA), a bacterial polysaccharide from E. coli K1 and an envelope protein from the hepatitis B virus. These Ag are all of importance for diagnosis and exhibit different levels of immunogenicity. Antibody-mediated enhancement was observed against restricted and defined regions of each immunogen i.e.: the Fab epitopes of SIgA, the preS1 domain of the HBV envelope and associated cell wall components of the capsular PS. The epitopes which were enhanced appeared to be different from those recognized by the modulating mAb. Negative modulations were also observed. Moreover, new epitopes seemed to be generated. In both cases the level and direction of the modulation were irrespective of isotypy and affinity of the mAbs. Interestingly the positive modulatory effect was found to be correlated with an in vitro assay based on the binding of immune complex to antigen-presenting cells.

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.

PCR analysis of HBV infected sera: relationship between expression of pre-S antigens and viral replication.

In order to determine the biological significance of the pre-S antigens in HBV infection, HBsAg sera were tested for the presence of pre-S1 and pre-S2. HBV DNA was detected by spot-hybridization and PCR. The data show a complete correlation between pre-S antigenemia and HBV DNA replication in anti-HBe positive cases. PCR but not spot-hybridization was adequately sensitive to also detect HBV DNA in roughly half of the preS negative sera as well. Thus PCR appears to be a valuable technique for detection of potentially infectious anti-HBe carriers.

Immunochemical and morphological studies of hepatitis B core antigen isolated from the nuclei of hepatocytes.

Immunochemical and morphological properties of hepatitis B core antigen (HBc Ag) were studied in intranuclear particles isolated from human liver. Immunochemical integrity of the purified particles was indicated in the production by guinea pigs of antibody to HBc Ag (anti-HBc) that was immunochemically identical to human anti-HBc. The HBc Ag particles were 27-30 nm in diameter, displayed apparent icosahedral symmetry, and consisted of distinct subunits. The susceptibility of HBc Ag particles to proteolytic and glycolytic enzymes indicated the presence of proteins and glycoproteins. The structural integrity of core particles depended on disulfide, hydrophobic, and hydrogen bonds, and immunological activity relied on intact sulfhydryl groups. Agents active against lipids did not affect immunological reactivity or core structure, as seen by electron microscopy.

Hepatitis B virus pre-S gene-encoded antigenic specificity and anti-pre-S antibody: relationship between anti-pre-S response and recovery.

A solid-phase radioimmunoassay involving specific antibody was developed for determination of the pre-S gene-encoded epitopes of hepatitis B virus and anti-pre-S antibody in sera of hepatitis B patients. The reaction for pre-S determinants associated with HBsAg was quantitatively inhibited by soluble, polymerized human serum albumin, and the lower limit of the assay was about 1.6 ng of HBsAg per ml. Continuous expression of pre-S-coded antigenic sites on HBsAg particles in chronic hepatitis B patients seropositive for HBeAg or anti-HBe shows that these determinants may be considered as a marker of chronicity during hepatitis B virus infection. The anti-pre-S antibody was determined by inhibition of the reaction for pre-S determinants. This antibody, different from anti-HBs, was detected during HBsAg antigenemia in patients recovering from acute type B hepatitis, before anti-HBs response. Kinetics of synthesis of anti-pre-S antibody in the course of acute type B hepatitis, followed by elimination of HBsAg and recovery, suggest the possible role of this antibody in the immunological clearance of infective hepatitis B virus particles.

HLA linked immune response to S and pre-S2 gene products in hepatitis B vaccination.

In order to detect a possible HLA linked genetic control of human immune responses to hepatitis B virus, forty healthy adult persons of the same age typed for HLA-A, -B and -DR antigens, were vaccinated against virus hepatitis B and sequentially tested for anti-HBs and anti-pre-S2 antibodies. They received three injections of Hevac-B Pasteur vaccine, the second 1 month and the third 3 months after the first. Following the third immunization, 38 individuals (95%) had a protective level of anti-HBs antibodies and 17 (42.3%) had a positive level of anti-pre-S2 antibodies. HLA-A11 antigen was significantly more frequent (pc = 0.007) among anti-HBs high responders than low responders. In addition, anti-HBs high responders were more frequently HLA-DR1, and less frequently HLA-DR4 and DR7 positive; corrected values, however, were not significant. Anti-pre-S2 high responders showed an apparent increase of HLA-B7, B14 or DR3 antigens, when compared to low responders (pc not significant).

Pre-S1 and Pre-S2 gene-encoded proteins in liver and serum in chronic hepatitis delta infection.

Frozen cryostat sections and sera from 30 patients with chronic delta infection were examined for pre-S1 and pre-S2 gene-encoded proteins, and the results were compared to markers in liver and serum HBV and HDV replication. Pre-S1 and pre-S2 were detected by indirect immunofluorescence (IF) in the liver in all 26 patients with histochemically demonstrable HBsAg. Pre-S peptides were found by double IF to have a predominantly cytoplasmic expression and to be located in the same hepatocytes expressing HBsAg. Liver cells expressing hepatitis delta antigen (HDAg) were frequently negative or very weakly positive for HBsAg and pre-S peptides, but occasional HDAg positive hepatocytes were also strongly positive for HBsAg and for pre-S peptides, particularly pre-S2. Circulating pre-S1 was detected in 24 patients (80%) and pre-S2 in 27 (90%). Detection of pre-S peptides in liver and serum was independent of HBV and HDV replication and of the HBV-DNA integration state. There was no correlation between the amount of circulating pre-S peptides and serum HBV-DNA and HDV-RNA. These results indicate that in chronic HDV infection, formation and secretion of pre-S peptides and of HBsAg occur independently of HBV and HDV replication and secretion. They further indicate that in the acquisition by replicating HDV of an HBV-derived envelope in the liver, both HBsAg and pre-S peptides are concomitantly available but circulating HDV-RNA is not invariably associated with the presence of these peptides in serum.

Immunochemistry and polypeptide composition of hepatitis B core antigen (HBc Ag).

Core particles were isolated from a nuclear extract of a hepatitis B-infected liver labeled with 125I by using chloramine-T and further purified by rate zonal sedimentation on sucrose gradients. Iodinated HBcAg was used as a ligand in a sensitive double-antibody radioimmunoprecipitation (RIP) assay for antibody to HBcAg. The specificity of the RIP reaction was evaluated using defined anti-HBc sera and paired sera from six well-documented cases of hepatitis B infection. The polypeptide composition of the iodinated antigen was examined by SDS-polyacrylamide gel electrophoresis of solubilized complexes of 125I-HBcAg and anti-HBc. Two major polypeptides with apparent m.w. of 17,000 and 35,000 daltons were observed and designated as cP-1 and cP-2, respectively.

Isolation and characterization of liver-derived hepatitis B e antigen.

Two subpopulations of hepatitis B e antigen (HBeAg) were isolated from a human liver infected with hepatitis B virus. HBeAg extracted from liver homogenate subsequent to treatment with buffered 3 M NaSCN or 0.5 M MgCl2 banded at the density of 1.13 g/cm3 in CsCl and was polydispersed on gel filtration. In contrast, HBeAg released with phosphate-buffered saline (PBS) was detected mainly at a density of 1.20 g/cm3 in a CsCl gradient and consisted of low molecular weight species on gel chromatography. Polypeptides of 40,000 and 45,000 daltons were found in NaSCN and PBS-released HBeAg preparations, respectively. The results are interpreted as suggestive that liver HBeAg is a dimer of the major core particle polypeptide in different physicochemical forms.

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.