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
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ABSTRACT |
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Present address: Sexually Transmitted and Bloodborne Virus Laboratory, Central Public Health Laboratory, 61 Colindale Avenue, London NW9 5HT, UK.
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Introduction |
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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.
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Methods |
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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 (AbbottMurex) 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; AbbottMurex; 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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Results |
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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 G145R (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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Discussion |
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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 TM 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 125127. 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 aday 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 G145R, cause perturbations in the a determinant.
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ACKNOWLEDGEMENTS |
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Received 26 June 2002;
accepted 10 October 2002.
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