A ‘first loop’ linear epitope accessible on native hepatitis B surface antigen that persists in the face of ‘second loop’ immune escape

Samreen Ijaz, R. Bridget Ferns and Richard S. Tedder

Department of Virology, Royal Free and University College Medical School, Windeyer Building, 46 Cleveland Street, London W1T 4JF, UK

Correspondence
Samreen Ijaz (at Central Public Health Laboratory)
SIjaz{at}PHLS.org.uk


   ABSTRACT
Top
ABSTRACT
Introduction
Methods
Results
Discussion
REFERENCES
 
Murine monoclonal antibodies (mAbs) were raised following immunization with native mutant hepatitis B surface antigen (HBsAg) purified from human sera. A set of antibodies binding to a linear epitope carried between residues 121 and 129 of the s region was demonstrated. These antibodies were shown by cross-competition assays to bind to a single epitope whose antigenicity was influenced by the TTP motif lying between residues 125 and 127. This first loop epitope remained accessible on the surface of HBsAg in spite of major second loop mutations abrogating the normal a conformational epitopes. The mAb and its binding region in the first loop are important diagnostically and may represent an importance immunological target, one that is stable in the face of immunologically driven escape.

Present address: Sexually Transmitted and Bloodborne Virus Laboratory, Central Public Health Laboratory, 61 Colindale Avenue, London NW9 5HT, UK.


   Introduction
Top
ABSTRACT
Introduction
Methods
Results
Discussion
REFERENCES
 
The a determinant of the hepatitis B surface antigen (HBsAg) is the major domain for both the diagnosis and immunoprophylaxis of hepatitis B virus (HBV). While much work has focused on the a determinant, its fine structure and spatial arrangement brought about by the three-dimensional folding of the amino acid backbone remain uncertain. From denaturation and alkylation experiments (Imai et al., 1974), and work using linear and cyclical peptides (Brown et al., 1984), it is clear that the a determinant is a conformationally dependant epitope. Based on the model of HBsAg proposed by Stirk et al. (1992), the a determinant is located on the extra-membranous loop that spans amino acid residues 101–159. This region contains eight cysteine residues and it is thought that disulphide bridges between the cysteines play a role in maintaining the correct conformation of the a determinant. Though most antibody directed against HBsAg (anti-HBs) found in sera binds to the epitopes carried between amino acids 124 and 147 (Howard et al., 1984), the use of phage display libraries (Chen et al., 1996) and structural predictions (Prange & Streeck, 1995) suggest that, rather than a single determinant, there are several epitopes located on different regions of the HBsAg particle.

Evidence has accumulated that HBsAg mutants selected in the face of immunological pressure pose a number of problems. Vaccine failure is of particular concern in an increasingly immunized world where HBsAg mutants may spread or become established within communities (Carman et al., 1990; Harrison et al., 1991; Karthigesu et al., 1994). So also is the possibility of reinfection, reactivation or breakthrough infections; these are particularly important in immunoprophylaxis against, and treatment of, HBV infection in liver transplant recipients, where infection is often associated with changes in HBsAg sequence (McMahon et al., 1992; Carman et al., 1996; Hawkins et al., 1996; Ghany et al., 1998; Protzer-Knolle et al., 1998; Terrault et al., 1998). Finally there is the loss of diagnostic accuracy as a result of failure of some monoclonal antibodies (mAbs) to detect the mutants (Yamamoto et al., 1994; Carman et al., 1995; Hou et al., 1995).

Arising out of a programme to develop mAbs to conserved and mutant-specific epitopes, antibodies were identified reacting with a linear epitope located in the putative ‘first loop’ of the a determinant. The antibodies reacted strongly in Western blot and were able to bind to wild-type and a range of ‘second loop’ mutant HBsAg, indicating that the epitope is displayed on the surface of the 22 nm form and remains preserved in escape mutants. Here we describe the raising of the mAbs and the characterization of their binding sites.


   Methods
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ABSTRACT
Introduction
Methods
Results
Discussion
REFERENCES
 
Identification of patients infected with wild-type and mutant HBsAg.
Mice were immunized with mutant HBsAg prepared from the sera of two renal transplant patients (M and P). Neither of the patients had been immunized previously and both were identified as a result of their inconsistent HBV serological markers. Patient P underwent renal transplantation in 1985. In 1990, the patient's serum was HBsAg-negative but positive for hepatitis B c antigen antibodies (anti-HBc). Haemodialysis was started in 1993 and later that year HBsAg was detected transiently by polyclonal-based assays only. Patient M, identified in 1986, was an HBV carrier whose serum contained anti-HBc and hepatitis B e antigen. This patient's serum was HBsAg-positive by polyclonal-based assays but unreactive in a monoclonal-based enzyme immunoassay. Sequence analysis of the a determinant in both patients revealed multiple point mutations (Table 1).


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Table 1. Amino acid substitutions in patients P and M

 
In addition, 50 diverse sera from patients known to be infected with HBV were available for testing further. PCR amplification and direct sequencing of the a determinant of HBsAg was carried out on these sera, as described previously (Hawkins et al., 1996).

Immunization and fusion.
HBsAg was purified from the sera of patients M and P and a wild-type carrier (wt) using gel filtration and isopycnic centrifugation methods, as described previously (Cameron et al., 1980). Female BALB/c mice were immunized separately with purified HBsAg from each carrier, in an equal volume of TiterMax (Sigma). After two boosts approximately 2 months apart and a final tail vein injection, the spleens from the mice were removed and fused with JKAg myeloma cells using 50 % polyethylene glycol, as described previously (Tedder et al., 1982).

Screening for anti-HBs.
Hybridomas were screened for the production of anti-HBs by a reverse-capture radioimmunoassay (RIA). Briefly, round-bottom, Maxisorb wells (Nunc, Gibco BRL) were coated overnight with anti-mouse IgG (Dako), washed with Tween/saline (0·1 M NaCl and 0·5 % Tween 20) and blocked with 0·5 % BSA in Tris buffer (Tris/BSA). The wells were sealed and stored moist at 4 °C.

Before use, the wells were aspirated to dryness. A 100 µl volume of a 1 : 10 dilution of hybridoma culture supernatant fluid in 0·02 M Tris, pH 7·6, containing 0·1 % sodium azide and 0·5 % BSA (TBSA) was added to the coated wells and incubated at 37 °C for 1 h. After washing, 100 µl of purified 125I-labelled HBsAg [45 nCi diluted in Tris buffer containing 2 % BSA and 20 % normal human serum (NHS), which was negative for all serological markers] was added and left at room temperature overnight. Bound label was measured in a 16-channel gamma counter (NE 1600, Nuclear Enterprises). Supernatant fluids from all cultures were tested in this assay using separate preparations of HBsAg from either a wt carrier, M, P or purified recombinant protein (rHBsAg) expressing the wt s region (adw2, SmithKline Beecham), each labelled with 125I.

Hybridomas reactive with one or more labels were cloned and propagated as ascitic tumours in female BALB/c mice primed previously with an intraperitoneal dose of 0·5 ml Pristane (Aldrich).

Purification and radioiodination.
The immunoglobulin fraction of ascites fluid was purified and labelled with 125I (Amersham), as described previously (Tedder et al., 1982).

Cross-competition assays.
A 100 µl volume of purified HBsAg containing 2 µg of wt, M or P antigen ml-1 was added to wells coated with polyclonal goat anti-HBs (Abbott–Murex) and left overnight at room temperature. The wells were washed and unlabelled purified immunoglobulin (5 µg in 50 µl TBSA) from monoclonal hybridomas was added separately to the wells, together with 50 µl of 125I-labelled, purified monoclonal IgG (50 nCi in 50 µl of Tris buffer containing 2 % BSA and 20 % NHS). Each unlabelled mAb was competed separately either with its own or each of the other labelled antibodies. The wells were incubated for 4 h at room temperature, washed and bound reactivity measured and expressed as a percentage of the maximum label-binding occurring when no competing anti-HBs-specific, unlabelled antibody had been added. Cross-competition assays were performed separately with wt, M or P HBsAg.

Western blot.
A total of 5 µg wt HBsAg per lane was boiled for 5 min in sample buffer (1·43 M 2-mercaptoethanol, 125 mM Tris/HCl, pH 6·8, 20 % glycerol, 6 % SDS and 0·004 % bromophenol blue) and then resolved on a 12 % SDS-polyacrylamide gel. The protein was electro-transferred onto a nitrocellulose membrane (Amersham) in Tris/glycine buffer (25 mM Tris and 192 mM glycine, pH 8·3) and 20 % methanol. The membrane was then blocked overnight in PBS containing 5 % milk powder. Following three 10 min washes in TBSTT (20 mM Tris/HCl, 500 mM NaCl, 0·2 % Tween 20 and 0·3 % Triton X-100), the membrane was cut into strips and incubated separately with 5 µg of each of the mAbs for 3 h at room temperature. After three further washes, a 1 : 1000 dilution of a horseradish peroxidase (HRP)-labelled, anti-mouse IgG was added to the membrane and incubated for 1 h. The membrane was then washed four times. Chemiluminescence detection was carried out using the ECL Plus kit (Amersham). Hyperfilm ECL film sheets (Amersham) were developed using an automated developer.

Culture of rHBsAg-expressing cells.
HepG2 cell lines expressing eight HBsAg mutants with mutations ranging from codon 126 to codon 145 were supplied kindly by T. Harrison (Royal Free and University College Medical School, London). Details of the production of the expression vectors used are described in Ren et al. (1995). HepG2 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10 % foetal calf serum, 2 mM glutamine and 300 U hygromycin B (Calbiochem) ml-1. Culture medium was harvested after 4 days and tested in the conserved and wt HBsAg detection assays described below.

Detection of HBsAg.
Wells coated with either mAb H3F5 (Tedder et al., 1983) or polyclonal anti-HBs (Abbott Laboratories; Murex Diagnostics) were used to capture HBsAg from serum or culture medium. HBsAg bound by mAb H3F5 was detected with 125I-labelled mAb D2H5 (50 nCi diluted in 0·02 M Tris buffer containing 2 % BSA and 20 % NHS) (Tedder et al., 1983). This assay was termed ‘the wt assay’. HBsAg bound by the polyclonal antibody was detected using 125I-labelled mAb P2D3 (raised in this study). This assay was termed ‘the conserved assay’. mAbs D2H5 and H3F5 used in the wt assay were known through cross-competition studies to bind to separate epitopes on wt HBsAg (Tedder et al., 1983). The binding of both mAbs is sensitive to conformational changes brought about by mutations in HBsAg (personal observation).

Oligopeptide studies.
Amino-terminal biotinylated oligopeptides (Genosys Biotechnologies; Abbott–Murex; Table 2) were captured at 37 °C for 1 h onto streptavidin-coated, round-bottom plates. After blocking with TBSA, the binding of mAbs to the oligopeptides was investigated by adding 100 µl of a dilution series of the mAb to the well for 4 h at 37 °C. After washing in Tween/saline, binding of IgG was visualized by HRP-conjugated anti-mouse IgG (Dako) using tetramethyl benzidine. The reactions were stopped after 20 min with 50 µl sulphuric acid. Absorbance values were measured at 450 nm.


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Table 2. Oligopeptides used in the characterization of mAbs

The positions of the peptides are relative to the s region. Amino acids in bold reflect described genetic variability at these residues.

 

   Results
Top
ABSTRACT
Introduction
Methods
Results
Discussion
REFERENCES
 
Identification and characterization of hybridomas
Reactivity against the P, M and wt HBsAg readily identified hybridomas secreting antibodies to their homologous surface antigen immunogen and those that were producing antibodies reactive with both homologous and heterologous antigens. Hybridomas could be categorized by reactivity as follows: (a) homologous, reactive only with the antigen against which the response was raised: (i) M-specific and (ii) P-specific; (b) heterologous, reactive against two antigens: (i) M/P cross and (ii) P/wt cross; (c) conserved, reactive against wt and both mutant HBsAg preparations: (i) M/P/wt cross. Of 12 hybridoma cultures, nine chosen for further investigation were cloned successfully by limiting dilution (Table 3).


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Table 3. Reactivity of nine mAbs tested in a reverse-capture RIA using three different HBsAg preparations

Binding of 125I is expressed as a binding ratio: 125I binding with test mAb/125I binding with negative control mAb.

 
Purified mAbs from each clone were tested at approximately a 1000-fold molar excess for the ability to inhibit the binding of labelled monoclonal immunoglobulin onto the wt, M and P antigens. Unlabelled mAbs effectively inhibited the binding of label made from their own immunoglobulin. The pattern of inhibition in these cross-competition studies divided the mAbs into the same five groups described previously. The data confirmed that, within each group, cross competition occurred between all members of the group and ranged from 80 to 98 % (data not shown). Furthermore, cross competition between the groups indicated that the display of the epitopes on HBsAg was discrete and separate. Table 4 shows cross-competition data from one member of each of the five groups.


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Table 4. Percentage inhibition values of labelled mAbs (at a 1000 molar excess) with unlabelled mAbs taken from each of the five groups

Percentages of cross inhibition of 125I-labelled mAbs, carried out on one or more of three HBsAg solid phases (M, P and wt), are shown. Less than 50 % inhibition is indicated as ‘–’.

 
Further characterization of the conserved mAbs was undertaken. Their binding to preS1 and preS2 was excluded by reaction with rHBsAg, which only expressed the s gene, with binding ratios of 378·0, 347·0 and 330·0 for mAbs P2D3, M3A10 and M4F5, respectively. No reactivity of this protein was observed with either the homologous or the heterologous groups of mAbs (binding ratios <1·5). Only the three mAbs in the conserved groups reacted with HBsAg on Western blotting, suggesting the possibility of binding to a linear antigen (data not shown).

Use of mAbs as diagnostic reagents
As part of a preliminary study to investigate the potential usefulness of the conserved antibodies as diagnostic reagents, a panel of 50 diverse samples, known to be HBsAg-positive by reverse-passive haemagglutination assays (Hepatest, Murex Diagnostics), was tested for HBsAg in both the conserved and wt RIAs. Comparison of the reactivity in the two assays demonstrated five sera that were reactive in the conserved but not the wt assay. Subsequent sequence analysis revealed point mutations resulting in single amino acid substitutions of G145->R (found in two patients), G145->A and D144->G. Two point mutations in the fifth patient resulted in a double amino acid change of D144->E and G145->R. All but five of the remaining 45 sera were reactive in both assays. Five samples identified to be unreactive on the conserved assay, whilst reacting strongly in the wt assay, were investigated further. Sequencing was carried out across the s region of these five samples and on 10 control samples reactive on both assays. The sequence data generated on these five sera and the 10 control sera were unremarkable, with changes associated only with described genetic variability noted. Alignment of these sequences against those of the controls identified two amino acid changes present only in the samples unreactive on the conserved assay. Substitutions of T->M and P->T at codons 125 and 127, respectively, were associated with the loss of reactivity to mAb P2D3. Comparison against published alignments of HBV genotypes indicated that these changes are exclusive to genotype D subtype ayw3 (Norder et al., 1993) and then only to certain strains within this genotype. These changes appeared to be sufficient to abolish the binding of mAb P2D3. As cross-competition data indicate that the mAbs in the conserved group bind to the same epitope, it would be expected that these changes would also perturb the binding of mAbs M4F5 and M3A10.

In order to assess the ability of mAb P2D3 to detect a wide range of mutants, a panel of rHBsAg mutants was tested in parallel in the conserved and wt assays. Results of the assays are illustrated in Fig. 1. Considering results with binding ratios above 2·0 to be positive, all rHBsAg particles tested were detected in the conserved assay using mAb P2D3. In comparison, only T126->S was found to be positive in the wt assay. The presence of the mutations in rHBsAg was confirmed by sequence analysis (data not shown).



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Fig. 1. Reactivity of a panel of rHBsAg, expressed in HepG2 cells, in the conserved and wt assays. Binding of 125I expressed as binding ratios (125I binding with test HBsAg/125I binding with negative control). Dotted line represents a binding ratio of 2·0.

 
Oligopeptide binding studies
Confirmation of the binding site came from studies with the synthetic oligopeptides. Peptides LT1 and LT2 (Table 2), synthesized to reflect the a determinant sequence between codons 121 and 134 of subtypes ayw2 and ayw3, respectively, differed by only two amino acids at positions 125 and 127. The P2D3 antibody reacted with the LT1 peptide (ayw2) but not with the LT2 peptide (ayw3). Peptide A2 (second loop of the a determinant from amino acid 138–149) and PS2 (preS2) were unreactive (Fig. 2). These findings confirmed that the binding site was likely to be between residues 121 and 134.



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Fig. 2. Binding of mAb P2D3 to a series of solid-phase oligopeptides. Different concentrations of mAb were added to wells coated with synthetic oligopeptides (see Table 2) and the binding visualized with rabbit anti-mouse IgG coupled to HRP.

 
To investigate further the effect of known natural genetic variation at codons 125 and 127 on the reactivity with mAb P2D3, 10 12-mer peptides (MBL series) were synthesized reflecting known subtype (codon 122) and genotype (codons 125 and/or 127) amino acids (Table 2). No reactivity of mAb P2D3 was observed with peptide MBL322, where T and P residues at codons 125 and 127 were replaced with M and T residues, respectively. A reduced reactivity was observed against peptide MBL320, where an L residue replaced the P residue at codon 127 (Fig. 3). Reduced reactivity was also seen on testing nine serum samples containing HBsAg of genotype E subtype ayw4, where an L residue is found at position 127 (data not shown).



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Fig. 3. Binding of mAb P2D3 to a series of solid-phase oligopeptides reflecting known subtype and genotype variation found between amino acids 118 and 129 of HBsAg.

 

   Discussion
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ABSTRACT
Introduction
Methods
Results
Discussion
REFERENCES
 
To identify conserved epitopes on immune escape mutant HBsAg, mAbs were raised against native mutant HBsAg isolated from the serum of patients identified previously. A hybridoma screening strategy was designed, employing both wt and mutant HBsAg in order to identify hybridomas producing antibodies against mutant-specific, mutant-independent and conserved epitopes.

This identified mAb P2D3 and its related hybridomas (M4F5 and M3A10), which displayed conserved reactivity against the wt HBsAg and against both of the mutant P and M HBsAg molecules (Table 3). Western blot and rHBsAg reactivity indicated that mAb P2D3 binding was likely to be to a linear epitope in the s gene.

Loss of mAb P2D3 reactivity was observed in five HBsAg-positive samples despite their wt phenotype. Sequence analysis identified the amino acid changes associated with naturally occurring genetic variation described in some strains of genotype D subtype ayw3. Amino acid substitutions at codons 125 and 127 of T->M and P->T residues, respectively, abrogated mAb P2D3 binding.

Oligopeptide studies confirmed that the binding site lay between residues 121 and 129 and was dependent upon the sequence present in residues 125–127. The common motif is TTP, flanked by conserved residues TGPC*TC***AQ. Variation is recorded at residue 122 as well as in the TTP motif. The ad->ay serotype change (K122->R) has little effect upon mAb P2D3 binding. Interestingly, TTT (the amino acid sequence found in some strains of genotype A), TTL (genotype E) and TIP (genotype C) sequences remain reactive, although a reduced avidity was seen with the TTL motif (Fig. 3).

Access to both native and rHBsAg mutants allowed the conservation of the P2D3 epitope to be more fully assessed. While the conserved assay detected all mutants ranging from codon 133 to 145, including a double point mutation at codon 144 and 145, poorer reactivity was observed with the 126 and 129 mutants. However, oligopeptide studies have shown that mutations in these residues may perturb the P2D3 epitope and result in reduced reactivity.

The use of phage display libraries to map epitopes recognized by a panel of mAbs (Chen et al., 1996) has provided an interesting model of HBsAg, indicating a complex structure made up of several epitopes located on different regions of HBsAg. Further evidence for this has been demonstrated by the use of peptide analysis to identify various mAb-binding sites (Qiu et al., 1996; Paulij et al., 1999). Our data indicate that the P2D3 antibody binds to an epitope carried between residues 121 and 129.

However, when using mAbs to identify important epitopes, it is essential to ensure that the immunogen is in a native conformation in order to reflect the properties of individual proteins, especially when considering complex conformational epitopes such as the a determinant. The use of native HBsAg in this study ensured that the protein was in its natural state and that associated post-translational modifications were correct. From the ability of mAb P2D3 to detect serum HBsAg, we conclude that the P2D3 epitope domain is accessible on the surface of the 22 nm particle. This region is exposed and could be immunologically important as a target.

Most naturally occurring anti-HBs is found to bind to the a determinant between codons 124 and 147. This region is essentially conserved. However, sequence alignments demonstrate a greater degree of genetic variation lying between codons 124 and 137 when compared to the region between codons 138 and 147. This variation seen among the genotypes may be functionally significant as the surface and polymerase genes do overlap and one would expect significant genetic constraint in this part of the gene.

It is well documented that amino acid substitutions and insertions in the a determinant result in antigenic and immunogenic changes in HBsAg (Carman et al., 1990, 1995; Harrison et al., 1991; Waters et al., 1992; Karthigesu et al., 1994; Yamamoto et al., 1994; Hou et al., 1995). Furthermore, site-directed mutagenesis and amino acid replacement studies (Ashton-Rickardt & Murray, 1989; Bruce & Murray, 1995; Steward et al., 1993) have identified important HBsAg residues by studying their effect to HBsAg antigenicity. For example, the major vaccine and immunological escape variant G145->R abrogates the binding of many (Waters et al., 1992) mAbs directed against the second loop and a conformational determinants. It is not known how the radical amino acid change brings this about, whether by altering the folding of the polypeptide loops or by removing part of the backbone from the accessible structure. Our studies indicate that no matter how this change occurs, the peptide sequence lying between residues 121 and 129 remains externalized in HBsAg. This observation confirms that the first loop forms a suitable diagnostic and perhaps immunological target in the face of major alteration in the second loop mutants.

The production of mAbs that display reactivity to various mutant HBsAg epitopes is beneficial in developing an understanding of the structure of HBsAg. Epitope mapping studies using mAbs in conjunction with oligopeptides can show which regions of HBsAg are accessible on the surface of the 22 nm particle. This in turn may give an indication of how the epitopes cluster on the three-dimensional structure and how peptide backbones are folded. Furthermore, it is likely that using mutant-specific mAbs, such as those that have yet to be characterized, will inform on a molecular level of how single point mutations, as in the case of G145->R, cause perturbations in the a determinant.


   ACKNOWLEDGEMENTS
 
We would like to thank Dr Julian Duncan and Mr Clive Dobson for their support and helpful discussion. We are also grateful to Dr T. Harrison and Dr R. Ling for providing the rHBsAg mutants and for their help. This work was funded by Murex Diagnostics Ltd.


   REFERENCES
Top
ABSTRACT
Introduction
Methods
Results
Discussion
REFERENCES
 
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Received 26 June 2002; accepted 10 October 2002.



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