Localization of a unique hepatitis B virus epitope sheds new light on the structure of hepatitis B virus surface antigen

W. P. Paulij1, P. L. M. de Wit1, C. M. G. Sünnen1, M. H. van Roosmalen1, A. Petersen-van Ettekoven1, M. P. Cooreman2 and R. A. Heijtink3

Organon Teknika, Boxtel, The Netherlands1
Departments of Gastroenterology and Hepatology, Academic Medical Center, University of Amsterdam, The Netherlands2
Erasmus Medical Centre Rotterdam, PO Box 1738, 3000 DR Rotterdam, The Netherlands3

Author for correspondence: R. A. Heijtink.Fax +31 10 4089485. e-mail heijtink{at}viro.fgg.eur.nl


   Abstract
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Abstract
Introduction
Methods
Results
Discussion
References
 
In a search for monoclonal antibodies (MAbs) that can bind hepatitis B virus surface antigen (HBsAg) with amino acid substitutions in the immune dominant `a' region (escape mutants) we investigated the epitope recognition site of the human MAb 4-7B. Pepscan analysis and experiments with alanine substitution as well as substitutions known from nature pointed to residues 178–186 in the small S protein with the amino acid sequence PFVQWFVGL (key amino acids in bold) as the minimal epitope. Single amino acid substitutions at positions 122(R/K)(d/y), 134(Y/F), 145(G/R), 148(T/A) and 160(K/R)(w/r), representing `a' region variants in recombinant HBsAg COS-I cells, did not influence binding of MAb 4-7B. Synthetic peptides (residues 175–189) including the 4-7B epitope sequence were able to evoke an anti-HBs response in rabbits. According to established polypeptide models, the 4-7B epitope region is located in the lipid layer of 20 nm HBsAg particles. The present results, however, suggest that residues 178–186 are exposed on the surface of the 20 nm particle. This may change our view of the structure of HBsAg.


   Introduction
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Abstract
Introduction
Methods
Results
Discussion
References
 
The hepatitis B virus (HBV) surface antigen (HBsAg) is the translation product of a large open reading frame that is divided into three domains. Each of these domains starts with an ATG codon functioning as a translation initiation site, thus defining three polypeptides referred to as the major protein (S protein), middle protein (M protein) and large protein (L protein). These three HBsAg polypeptides share an immune dominant and immune protective determinant located in the S region. This so-called `a' determinant is considered to be the most important target for diagnosis and immune prophylaxis.

Native and recombinant forms of HBsAg exposing the `a' determinant are commonly used as the basis for hepatitis B vaccines. Such vaccines induce complete protection against HBV infection after pre-exposure HBV immunization and post-exposure maternal–infant transmission after passive–active immunization. However, Carman et al. (1990) first described a vaccine-induced escape mutant of HBV in a child from Southern Italy who received passive–active post-exposure immunization. This escape mutant had a point mutation from guanosine to adenosine at nucleotide 587 (codon 145) in the `a' determinant. Subsequently, escape mutants induced by active or passive immunization with amino acid changes resulting in the loss of the `a' determinant were reported from other parts of the world (Carman, 1997 ).

The first HBV marker of choice in the diagnosis of hepatitis B disease is HBsAg. Since the `a' determinant is thought to be the most immunogenic, it is to be expected that assays for HBsAg are mainly based on antibodies with `a' region preference. As a consequence even third generation assays may not detect all cases of infection with variant viruses, due to the changes in the `a' region (Carman et al., 1995 , 1997 ; Grethe et al., 1998 ).

To overcome this deficit we started screening a large number of monoclonal antibodies (MAbs) for their ability to detect variant HBsAg and analysed the corresponding epitopes of the most promising ones. The study described in this paper deals with the finding of a unique human HBsAg epitope located outside the `a' determinant. Surprisingly, this epitope was located within the third passage of the HBsAg polypeptide through the lipid membrane, as described in classical models (Stirk et al., 1992 ; Berting et al., 1995 ; Gerlich et al., 1993 ).


   Methods
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Abstract
Introduction
Methods
Results
Discussion
References
 
{blacksquare} Biological material.
Murine MAbs (HBs.OT16, HBs.OT17, HBs.OT24 and HBs.OT40) were developed at Organon Teknika (Boxtel, The Netherlands) according to standard methods. These MAbs are directed against native HBsAg obtained from sera of infected patients. The human MAb 4-7B-producing cell line was originally developed at the Central Laboratory of The Netherlands Blood Transfusion Services, Amsterdam. A purified preparation of MAb 4-7B was kindly provided by D. Rohm (Biotest Pharma, Dreieich, Germany).

{blacksquare} Epitope mapping.
Based on the sequence described for HBsAg subtype adr published by Fujiyama et al. (1983) 12-mer peptides were prepared covering the complete sequence by shifting one amino acid compared to each previous peptide. The reactivity of each peptide was determined by using standard pepscan procedures (Geysen et al., 1986 ). The minimal epitope of MAb 4-7B (shortest reactive peptide) was determined by shortening the most immune reactive 12-mer peptide at the N or C terminus and determining the reactivity of the fragments by pepscan. The importance of each individual amino acid was determined by including an alanine substitution study, in which each amino acid was replaced by alanine (A). If alanine naturally appeared in the basic sequence it was replaced by serine (S). Again the reactivity of each peptide was determined using standard pepscan technology and the most reactive 12-mer peptide was used as the basis for further study.

As a control for pepscan analysis, the reactivity of the four reactive sequences and the minimal epitope was also determined by using Ata-coupled peptides directly coated on the solid phase of a microtitre plate according to the method of Loomans et al. (1997) .

The synthesis of the peptides was carried out on a Perkin Elmer/Applied Biosystems 433A peptide synthesizer, using standard FastMoc 0·25 mmol procedures with UV-monitoring and feedback option. The peptides were synthesized on a TentaGel S RAM Fmoc resin via Fmoc/tBu chemistry. The linker is of a Rink-amide type, which automatically yields a C-terminally amidated peptide. During solid phase peptide synthesis the amino side chains were protected with acid-labile protecting groups: the {epsilon}-amino group of lysine with Boc, the {delta}-guanidino group of arginine with Pbf, the {gamma}-carboxyl group of glutamic acid and the ß-carboxyl group of aspartic acid with OtBu, the {gamma}-amide group of glutamine and the ß-amide group of asparagine with Trt, histidine and cysteine with Trt, the ß-hydroxyl group of serine and threonine with tBu, and tyrosine with tBu. All reactants were dissolved in DMF. The cleavage of the Fmoc group was carried out with 20% (v/v) piperidine in NMP during at least two consecutive cycles of 1·5 min. Coupling of the Fmoc amino acid derivatives (Fmoc-Aaa-OH, 4 eq. 1 mmol) was performed by in situ activation with HBTU, HOBt (4 eq. 1 mmol) and DIPEA. After coupling of each amino acid derivative (at least 20 min), no check for completion of the acylation reaction was carried out. The acylation was followed by a capping step with acetic anhydride in NMP. Finally, the Ata group was introduced via an active ester coupling with SAMA-OPfp. The fully protected peptides were cleaved from the resin during a 2 h reaction with 5% (v/v) thioanisole, 3% (v/v) ethane dithiol, 2·5% (v/v) water and 2% (v/v) anisole in trifluoracetic acid followed by precipitation in diethyl ether. The crude peptides were washed twice in diethyl ether, dried in air, dissolved in water–acetonitrile (3:1) and lyophilized.

Abbreviations for protecting groups are according to the recommendations of the IUPAC–IUB Joint Commission on Biochemical Nomenclature as given in European Journal of Biochemistry vol. 138, pp. 9–37 (1984). (Other abbreviations: Ata, acetylthioacetyl; NMP, N-methylpyrrolidone; SAMA-OPfp, S-acetylthioglycolic acid pentafluorophenyl ester; Pbf, 2,2,4,6,7-pentamethyldihydrobenzofuran-5 sulfonyl).

{blacksquare} Reactivity of peptides with naturally appearing amino acid substitutions.
Based on the sequence of the most reactive 12-mer peptide various peptides were synthesized including all known naturally appearing amino acid substitutions as described in the literature (Norder et al., 1993 ; Grethe et al., 1998 ; Loncarevic et al., 1990 ; Mbayed et al., 1998 ). The reactivity of each peptide was measured according to standard pepscan procedures.

{blacksquare} Reactivity of recombinant HBsAg including (escape) mutants.
A DNA fragment of 900 bp including the complete preS2 and S gene (subtype ayw3) was used for the construction of several point-mutated DNA sequences with primer site-directed mutagenesis. Mutant constructs in plasmid pSP70 were digested with SmaI. The HBsAg gene fragments were isolated from agarose gel with the Gene Clean II kit (BIO 101) and ligated into the blunt-ended mammalian expression vector pSG8puro at the EcoRI site under the control of the simian virus 40 promotor. The correct orientation was verified with PCR using a vector and an insert primer. We obtained single point-mutated HBsAg proteins with the following amino acids replacements: 122 R/K, 134 Y/F, 145 G/R, 148 T/A, 160 K/R (122R/K, Peterson et al., 1984 ; 134Y/F, Ashton-Rickardt & Murray, 1989 ; 145G/R, Waters et al., 1992 ; 148T/A, Ohno et al., 1993 ; 160 K/R, Okamoto et al., 1989 ). Wild-type recombinant HBsAg was used as positive control. COS-I cells were chosen because of the high expression level, secretion into the medium and glycosylation of the translated products; 1·5x106 cells were transiently transfected with 10 µg plasmid DNA in 0·2 ml PBS in a 4 mm cuvette at 300 V and 125 µF using a Biorad electroporator. Cells were cultured for 3 days in Dulbecco's modified Eagle's medium containing 10% foetal calf serum, 2 mM glutamine, 50 µg/ml gentamycin and non-essential amino acids. Expression was controlled by Western blotting using murine MAb HBs.OT43 (Organon Teknika), directed against a linear sequence in the preS2 domain of the HBsAg product. Culture medium was harvested after 3 days and clarified by low speed centrifugation. Samples were diluted in normal human serum. Variant HBsAg was quantified in an experimental assay with preS2 MAbs recognizing linear epitopes that are not influenced by mutations in the `a' domain.

Human MAb 4-7B (5 µg/ml) was coated directly on the solid phase of 96-well microtitre plates according to standard methods. Murine anti-HBs MAbs (HBs.OT16, HBs.OT17, HBs.OT24, HBs.OT40) were only available in low concentration. To achieve high solid phase concentrations of these antibodies, microtitre plates were first coated with sheep anti-mouse antibodies. Thereafter, murine anti-HBs MAbs were added. After incubation for 1 h at 37 °C extensive washing with PBS–Tween was done. Subsequently, each recombinant HBsAg was diluted to a pre-set concentration (based on the preS2 reactivity) at which the wild-type HBsAg gave an absorption signal of 1·000 at 450 nm with each individual MAb. After incubation for 1 h at 37 °C and extensive washing sheep anti-HBs horseradish peroxidase (HRP) conjugate was added followed by incubation for 1 h at 37 °C. Tetramethylbenzidine peroxidase substrate was then added and followed by incubation for 30 min at ambient temperature. For each MAb the reaction was stopped using 1 M sulfuric acid and reactivity was measured by spectrophotometry at 450 nm. Culture medium of non-transfected COS-I cells was included as negative control.

{blacksquare} Reactivity of rabbit polyclonal anti-HBs antibodies.
In order to generate antibodies against the 4-7B epitope including all possible (see above) variations, a peptide mixture of all known sequences was made. In Table 1 an overview of the mixture is shown. All peptides were based on 15 amino acids and included the 4-7B epitope. Before immunization in two rabbits, peptides were coupled to keyhole limpet haemocyanin and tetanus toxoid and a pre-treatment sample was taken from each rabbit. Each rabbit was immunized with 100 µg peptide (50 µg intra-muscularly and 50 µg subcutaneously, both in adjuvant). Four weeks after the first immunization a similar second immunization was carried out and 19 days later the first blood samples were taken. The reactivity of all blood samples was tested in a standard enzyme immunoassay. Plasma-derived HBsAg (both HBsAg ad and ay, 50% each) was pre-treated with pepsin to remove adhering proteins. Pepsin-treated HBsAg and a control peptide based on the 4-7B epitope (sequence 175-LLVPFVQWFVGLSPT-189) were coated on the solid phase while using swine anti-rabbit HRP as conjugate.


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Table 1. Mixture of peptides based on the 4-7B epitope including all known appearing variations used for immunization of rabbits

 

   Results
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Introduction
Methods
Results
Discussion
References
 
Epitope characterization
Pepscan analysis of the complete sequence of HBsAg subtype adr (Fujiyama et al., 1983 ) by 12-mer peptides revealed reactivity of MAb 4-7B with only four peptides, covering the sequence 175-LLVPFVQWFVGLSPT-189 (Fig. 1). By shortening the 12-mer peptides the minimal reactive sequence was found to be 178-PFVQWFVGL-186 (Fig. 2). Alanine substitution experiments (Fig. 3) suggest that amino acids F(179), Q(181), W(182), G(185) and L(186) are essential for reactivity of the epitope. Therefore the epitope can be determined as xxxFxQWxxGL where x can be replaced by an amino acid such as alanine.



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Fig. 1. Pepscan analysis of MAb 4-7B on overlapping 12-mer peptides from residues 170–183.

Fig. 2. Pepscan analysis to elucidate the minimal epitope for MAb 4-7B by shortening the immune reactive 12-mer peptide (residue 175–186) at the N or C terminus.

Fig. 3. Alanine substitution in a 12-mer peptide reactive with MAb 4-7B.

 
These results were confirmed in an enzyme immunoassay using Ata-coupled peptides directly coated to the solid phase of a microtitre plate.

Naturally occurring variation in amino acids 175–189
Table 2 illustrates the reactivity of MAb 4-7B with various peptides with naturally observed amino acid substitutions. The amino acids L(175), F(179), V(180), Q(181) and L(186) have been found to be conserved in nature (Norder et al., 1994 ). Alanine substitution of L(176), V(177) and V(184) had no influence on the reactivity of these peptides for MAb 4-7B. In contrast, substitution of P(178) by Q or G and V(184) by D but not by M significantly influenced their reaction. These changes in reactivity were not observed in alanine substitution experiments. It is suggested that the type of (polar) amino acid at these positions is essential.


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Table 2. Reactivity of MAb 4-7B with aa 175–186 peptides including various naturally observed amino acid substitutions

 
Substitution of P(178) by Q and F(183) by C are characteristic for HBsAg strain adw4 (Norder, 1994 ). The decrease in reactivity upon substitution of W(182) by S or C and G(185) by E confirmed that W and G are key amino acids as observed in the alanine substitution study.

MAb 4-7B reactivity with `a' region variant HBsAg
Recombinant HBsAg from COS-I cells (HBsAg/ayw3) and some of the `a' region variants were used to compare the reactivity of MAb 4-7B antibodies with that of four different mouse MAbs with binding sites in the 120–160 region. Fig. 4 illustrates the impact of single amino acid substitutions in the `a' region on the reactivity of the mouse MAbs. For instance, the reactivity of antibody HBs.OT16 was strongly reduced if residue 145 (G) was replaced by 145 (R) or 148 (T) by 148 (A). Similar observations were made for HBs.OT40 (145G/R) although the effect of the 148T/A substitution was less distinct. Both antibodies probably recognize a conformational epitope located inside the `a' determinant. The opposite result was observed for HBs.OT17, which showed an increased reactivity with the 145 (R) variant. The `y' sub-determinant specificity of this antibody was clearly demonstrated by its sensitivity for a 122 R/K substitution. HBs.OT24 could recognize all `a' region variants although its reactivity was decreased for the residue 145/148 variants. In contrast to the four mouse MAbs, MAb 4-7B reactivity was very similar for all the `a' region substitutions in these experiments. These observations are in agreement with the finding that the 4-7B epitope is located outside the `a' region. Amino acid substitutions in the 120–160 region apparently do not affect the binding of MAb 4-7B to its corresponding epitope.



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Fig. 4. Reactivity of variant HBsAg expressed in COS-I cells with MAb 4-7B () and four mouse MAbs (HBs.OT16, ; HBs.OT17, ; HBs.OT24, ; HBs.OT40, {square}) that react with `a' region epitopes. HBsAg types are designated by the residue number and substitution in the HBsAg/ayw3 amino acid sequence. WT, Wild-type.

 
Rabbit anti-HBs induced by peptide 175–186
Two rabbits were immunized with a mixture of peptides representing all known variants in the amino acid sequence of a 15-mer based on the 4-7B epitope. As shown in Table 3 these two rabbits produced antibodies that reacted with pepsin-treated plasma-derived HBsAg directly coated on the solid phase as well as with the peptide representing the 4-7B epitope in the basic sequence only (Table 1).


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Table 3. Absorbance at 450 nm of rabbit serum for antibodies reactive with plasma-derived HBsAg and a peptide (residues 175–189) including the 4-7B epitope (residues 178–186)

 

   Discussion
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Abstract
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Methods
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Discussion
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The cell line producing the human MAb 4-7B was first described by Stricker et al. (1985) . Used for detection of HBsAg in an experimental assay MAb 4-7B could efficiently detect HBsAg while missing only one sample in 1000. Further characterization of MAb 4-7B as a suspected candidate for prophylactic use in HBsAg-positive liver transplant recipients revealed its reactivity for all strains of the Couroucé panel of subtypes except for HBsAg/adw4 (Heijtink et al., 1995 ).

In the present study peptide analysis located the MAb 4-7B-corresponding epitope between amino acids 175 and 186 in the small S protein. Further refinement restricted the minimal reactive sequence (epitope) to 9 amino acids, 178-PFVQWFVGL-186 (key amino acids in bold). F(179), V(180), Q(181) and L(186) never change in nature. P(178) and F(183) play a crucial role in the decreased affinity of MAb 4-7B for strain HBsAg/adw4. According to Norder (1994 ), a change from P(178) to Q and F(183) to C discriminates genotype F (HBsAg/adw4) from all other genotypes. This confirms our earlier observations (Heijtink et al., 1995 ). W(182) and G(185) are essential amino acids as observed in substitutions experiments with alanine but also among naturally observed substitutions.

The location of the MAb 4-7B epitope outside the `a' region in the classical models (Stirk et al., 1992 ; Berting et al., 1995 ; Gerlich et al., 1993 ) and the continuous character of the epitope suggest that MAb 4-7B binding is not disturbed by conformation changes in the `a' region. Indeed, the present study confirmed that MAb 4-7B binds easily to HBsAg variants with substitutions that have a great impact on the antigenic properties of this region (Heijtink et al., 1995 ).

Immunization of rabbits with a mixture of the 4-7B epitope (like) peptide(s) revealed an anti-HBs response with the 4-7B epitope and pepsin-treated HBsAg(ad/ay). Studies on the prevalence of 4-7B antibodies in convalescent sera and sera from vaccinees are in progress.

The proposed models for the structure of the 20 nm spherical HBsAg-containing particles carefully combined information on hydrophobicity, hydrophobic moments, flexibility, secondary structure prediction and antigenicity (Berting et al., 1995 ; Guerrero et al., 1988 ; Howard et al., 1988 ; Mangold et al., 1997 ; Stirk et al., 1992 ).

The Stirk model for the small S protein predicts four transmembrane helices that are located between amino acid residues 8–28, 79–100, 160–184 and 189–210. Similar predictions were made by Berting et al. (1995) and Mangold et al. (1997) . The model of Guerrero et al. (1988) is different from the previous models in that the peptide spans the lipid membrane six times. In this model the 4-7B epitope region would be located completely internally. According to Stirk et al. (1992) the helix C(160–184), including the 4-7B epitope region, is predicted with a low probability level. By biopanning from a filamentous phage peptide library Chen et al. (1996) obtained the amino acid sequences corresponding to four different mouse MAbs raised by plasma-derived HBsAg (Peterson et al., 1984 ). One of the mouse MAbs, H35, reacts with residues between 166 and 175. Another mouse MAb, H53, corresponds to residues 187–207. Although these two MAbs recognize discontinuous epitopes with participation of amino acid residues surrounding residue 120, these examples suggest the existence of a rather extended immune reactive area between residues 160–207 including residues 176–184 as detected by human 4-7B antibodies.

As mentioned above, the 4-7B epitope could be detected in all subtypes, however, with the restriction of HBsAg/adw4. Furthermore, MAb 4-7B could inhibit the binding of HBsAg as well as Dane particles to solid phase anti-HBs in Ausria II (HBsAg assay, Abbott Laboratories). MAb 4-7B was also successfully used to bind the arginine residue 145 variant from cell culture (this paper) as well from human plasma (Heijtink et al., 1995 ). MAb 4-7B could even bind formaldehyde-treated plasma-derived vaccine HBsAg and yeast recombinant hepatitis B vaccine HBsAg (results not shown). Therefore, we conclude that the 4-7B epitope region is readily accessible in any HBsAg S polypeptide. Moreover, including the antigenic areas from MAbs H35 and H53 (Chen et al., 1996 ) we suggest that in 20 nm HBsAg particles the region 160–207 does not cross the lipid membrane twice but is rather projected in its full-length over the surface of these particles. In this way proline residue 188 can easily interrupt the predicted {alpha}-helix. Due to its hydrophobic character this area may be a candidate for (non-specific) binding of HBsAg particles to human hepatocytes (Leenders et al., 1990 ; Gerlich et al., 1993 ; Hertogs et al., 1993 , 1994 ).


   Acknowledgments
 
The authors are grateful to W. Puijk, E. van Dijk and D. Parohi from the Institute for Animal Science and Health (ID-DLO, Lelystad, The Netherlands) and to R. Holleman (Organon Teknika) for excellent technical assistance. We thank Dr J. A. Hellings for helpful comments and review of the manuscript.


   References
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Abstract
Introduction
Methods
Results
Discussion
References
 
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Received 8 February 1999; accepted 21 April 1999.