A monoclonal antibody specific for the hepatocyte receptor binding site on hepatitis B virus

doi:10.1016/0161-5890(89)90004-7

Molecular Immunology

Volume 26, Issue 6, June 1989, Pages 531-537

https://doi.org/10.1016/0161-5890(89)90004-7 Get rights and content

Abstract

The sequence of the preS1 region of the hepatitis B virus (HBV) envelope (env) proteins contains a dominant binding site for hepatocytes between residues preS21 and preS47. Purified HBV particles (subtype ad) were used as the immunogen to produce specific monoclonal antibodies (McAbs) against three antigenic regions (S, preS2 and preS1) of the HBV env protein. One McAb, F35.25, was found to be specific for the region 32–53 of the preS1 sequence of HBV, which largely overlapped the hepatocyte receptor binding site. The preSl-specific McAb F35.25 reacted with both HBV subtypes, ad and ay, in radioimmunoassays (RIA) and with the large surface proteins, P39 and GP42, as well as with tryptic fragments preS(1–99/103) and preS(1–113) in Western blotting experiments. This McAb F35.25 preferentially recognized, however, the homologous (ad) preS1 sequence in RIA. The ad/ay amino acid substitution within the hepatocyte receptor binding site at position 35 (Gly-Arg) may explain the relative subtype-specificity of F35.25. Finally, the F35.25 epitope was detected in all HBV particles purified from HBeAg-positive human sera, confirming that this preS1 region 32–53 is exposed at the surface of complete virions. Thus, we developed a RIA system allowing us to assess the infectivity of HBV particles by the detection ofpreS1 sequences associated with the viral hepatocyte receptor. Moreover, it is expected that F35.25 may be a virus-neutralizing antibody by blockage of the attachment of HBV to liver cells.

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Cited by (34)

A monoclonal antibody specific to the non-epitope region of hepatitis B virus preS1 contributes to more effective HBV detection
2013, Clinical Biochemistry
Citation Excerpt :
The N-terminal region of preS1 is thought to mediate the attachment of the virus to cells and to possess neutralizing epitopes [4]. The aa 21–47 region of preS1 has been recognized as the epitope region based on the following observations: 1) the majority of the established preS1-specific mAbs were confirmed to bind to this region [18]; 2) several mAbs based on this region were proven to neutralize viral activity and block the entrance of virus into host cells [6–9]; and 3) all of the commercially available mAbs for clinical HBV LHBS detection are specific to this region [12,19]. The presence of an antibody specific to aa 21–47 of preS1 is also considered to be a marker of improved health in hepatitis B patients [20].
The hepatitis B virus (HBV) preS1 protein is divided into an epitope region and a non-epitope region based on the respective antigenicities of these regions. Most of the antibodies that are currently used to detect the large surface protein of HBV (HBV LHB) are specific to the epitope region of preS1, which may contribute to the false negative results of HBV LHB detection assays. Here, we established a mouse monoclonal antibody (mAb) that could improve the efficiency of HBV LHB detection.
The HBV preS1 protein was expressed in E. coli strain BL21 and used to screen hybridoma clones. HBV preS1-specific mAb was produced by immunizing mice with a chemically synthesized peptide antigen derived from the non-epitope region of HBV preS1. The mAb was characterized by ELISA, Western blot, and immunocytochemistry and was subsequently used in serum sample tests.
Based on in silico B cell epitope predictions, the HBV preS1 aa 91–117 peptide was synthesized as an antigen. Recombinant HBV preS1 was expressed in E. coli and identified by SDS-PAGE. The mAb D8 (IgG2b) recognized the recombinant preS1 protein in both ELISA and Western blot assays and also recognized the preS1 protein expressed in plasmid-transfected HepG2.2.15 cells by immunocytochemistry. Furthermore, the D8 mAb, which is specific for the non-epitope region of preS1, contributed to the improved sensitivity and specificity of HBV detection.
We established an mAb that is specific to the non-epitope region of HBV preS1 and improved the detection of HBV LHB in an ELISA assay. This mAb could help increase the accuracy of the clinical measurement of preS1.
Broadly neutralizing anti-HBV antibody binds to non-epitope regions of preS1
2009, FEBS Letters
Broadly neutralizing anti-hepatitis B virus (HBV) antibody HzKR127 undergoes a fairly large conformational change of CDR H3 loop upon binding to HBV preS1 epitope peptide. In this study, we identified low-affinity antibody-binding sites in the largely unstructured preS1 region by nuclear magnetic resonance and biochemical studies, indicating that the antibody binds to the preS1 region outside the major immune epitope with low affinity. Surface plasma resonance experiments showed that the full-length preS1 has approximately three fold higher affinity for HzKR127 Fab than the preS1 epitope peptide, suggesting that the presence of low-affinity sites in the preS1 region increases the antibody-binding affinity. Therefore, the low-affinity binding of the antibody to non-epitope regions of preS1 may contribute to effective neutralization.
Hepatocellular Carcinoma Is Associated With an Increased Risk of Hepatitis B Virus Recurrence After Liver Transplantation
2008, Gastroenterology
Citation Excerpt :
Immunostaining was performed on sections of fixed liver tumor and nontumor samples using a commercial 3-step streptavidin-biotin technique, according to the manufacturer's instructions (Ventana Medical Systems, Strasbourg, France). The primary monoclonal antibodies employed were anti-HBsAg, anti-HBcAg (Dako, Copenhagen, Denmark), and anti-pre-S1 (kindly provided by Dr M. A. Petit).31 S gene sequences were obtained from sera and liver tissue using the following forward and reverse primers: 5′-TGGYTATCGCTGGATGTGTC-3′ and 5′-CCCAAAAGACCCACAATTC-3′.
Background & Aims: Hepatitis B virus (HBV) recurrence after orthotopic liver transplantation (OLT) is significantly reduced by prophylaxis with hyperimmune antibody to hepatitis B surface antigen (anti-HBs) globulins (HBIG) and antiviral drugs. The role of hepatocellular carcinoma (HCC) in HBV recurrence remains unclear. We investigated the association between HCC pre-OLT and HBV recurrence post-OLT. Methods: We studied 99 hepatitis B surface antigen-positive patients who underwent OLT for cirrhosis. The median follow-up period was 43 months. All patients received HBIG, and 51 also received lamivudine and/or adefovir. Of these 99 patients, 31 had HCC before OLT. Total HBV DNA and covalently closed circular (ccc)-DNA were measured in tumor and nontumor tissues from the explanted livers of 16 of these 31 HCC patients and, also, in a context of tumor recurrence, in 3 patients who developed HBV/HCC recurrence. Results: Fourteen patients (14.1%) developed HBV recurrence within a median period of 15 months post-OLT. HCC at OLT, a pre-OLT HBV DNA viral load ≥100,000 copies/mL, and HBIG monoprophylaxis were independently associated with HBV recurrence post-OLT. Eleven out of the 31 patients with HCC at OLT presented with HBV recurrence and 3 out of the 68 patients without HCC had HBV recurrence (P < .0001). HBV recurrence was more frequent in patients who developed HCC recurrence (7/8 patients, 87.5%) than in those who did not (4/23 patients, 17.4%) (P < .0001). In the 16 explanted livers, cccDNA was detectable in HCC cells in 11 and in nontumor cells in 12. cccDNA was detected in a context of HCC recurrence in 2 of the 3 patients tested who developed HBV/HCC recurrence. Conclusions: The associations of HCC pre-OLT, and HCC recurrence with HBV recurrence post-OLT, and the detection of HBV DNA and cccDNA in HCC suggest that HBV replication in tumor cells may contribute to HBV recurrence post-OLT.
Enveloped particles in the serum of chronic hepatitis C patients
2005, Virology
HCV particles were isolated from the plasma of chronically infected patients. The virus was analysed by sucrose density gradient centrifugation. The fractions were tested for viral RNA, core antigen and envelope proteins by using a monoclonal antibody directed against the natural E1E2 complex (D32.10). Two populations of particles containing RNA plus core antigen were separated: the first with a density of 1.06–1.08 g/ml did not contain the envelope proteins; the second with a density between 1.17 and 1.21 g/ml expressed both E1 and E2 glycoproteins. Electron microscopy of the enveloped population after immunoprecipitation with D32.10 showed spherical particles with a rather featureless surface and with a diameter around 40 nm. Immuno-gold staining gave evidence that the E1E2 complex was indeed positioned at the surface of these particles.
Mapping of a Conformational Epitope Shared between E1 and E2 on the Serum-derived Human Hepatitis C Virus Envelope
2003, Journal of Biological Chemistry
Citation Excerpt :
The results were considered as positive when superior to the cutoff, corresponding to the mean of negative controls multiplied by 2.1. Western Immunoblot Experiments—The untreated HCV-enriched pellet (HCV-L) was used as an antigenic probe (12, 13) at concentrations from 0.1 to 1 mg/ml. The antigen was subjected to SDS-PAGE on 12.5% gels under reducing or nonreducing conditions (2% SDS ± 5% β-mercaptoethanol).
Monoclonal antibody D32.10 produced by immunizing mice with a hepatitis C virus (HCV)-enriched pellet obtained from plasmapheresis of a chronically HCV1b-infected patient binds HCV particles derived from serum of different HCV1a- and HCV1b-infected patients. Moreover, this monoclonal has been shown to recognize both HCV envelope proteins E1 and E2. In an attempt to provide novel insight into the membrane topology of HCV envelope glycoproteins E1 and E2, we localized the epitope recognized by D32.10 on the E1 and/or E2 sequence using Ph.D.-12™ phage display peptide library technology. Mimotopes selected from the phage display dodecapeptide library by D32.10 shared partial similarities with ²⁹⁷RHWTTQGCNC³⁰⁶ of the HCV E1 glycoprotein and with both ⁶¹³YRLWHYPCT⁶²¹ and ⁴⁸⁰PDQRPYCWHYPPKPC⁴⁹⁴ of the HCV E2 glycoprotein. Immunoreactivity of D32.10 with overlapping peptides corresponding to these three HCV regions confirmed these localizations and suggested that the three regions identified are likely closely juxtaposed on the surface of serum-derived particles as predicted by the secondary model structure of HCV E2 derived from the tick-borne encephalitis virus E protein. This assertion was supported by the detection of specific antibodies directed against these three E1E2 regions in sera from HCV-infected patients.
Fine mapping of virus-neutralizing epitopes on hepatitis B virus preS1
2000, Virology
We identified the epitopes on the preS1 which induce antibodies that neutralize both ad and ay subtypes of hepatitis B virus (HBV). Previously we generated murine monoclonal antibodies KR359 and KR127 that bind specifically to the preS1 of HBV. In this study we have performed fine mappings of the epitopes of the antibodies by examining their reactivity with GST fusion proteins, which contain a series of deletion mutants of the preS1. KR359 and KR127 specifically recognize aa 19–26 and 37–45 of the preS1, respectively. The antibodies neutralized both adr and ayw subtypes of the virus in an in vitro neutralization assay using in vitro infection of adult human hepatocyte primary culture by HBV. The epitopes showed little sequence divergence and the antibodies bound to the preS1 of all the HBV subtypes and variants tested.

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^☆: This work was supported by research grants from INSERM.

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A monoclonal antibody specific for the hepatocyte receptor binding site on hepatitis B virus☆

Abstract

Cell

Molec. Immun.

Molec. Immun.

Production of hybrid lines secreting monoclonal anti-idiotype antibodies by cell fusion on membrane filters

Curr. Top. Microbiol. Immun.

Hepatitis B surface antigen: an unusual secreted protein initially synthesized as a transmembrane polypeptide

Molec. Cell. Biol.

The molecular biology of the hepatitis B viruses

A. Rev. Biochem.