journal of Virological method, 35 (1991) 199-206 0 1991 Elsevier Science Publishers B.V. All rights reserved. / 0166-0934/91/$03.50

199

VIRMET 01247

Epitope mapping of HBsAg using a panel of human anti-HBs antinomies A. Sa’adu’, D. Bidwell*, M. Locniskar’, C. Howard’, K.P.W.J. McAdam’ and A. Voller’T2 ‘Department of Clinical Sciences, London School of Hygiene and Tropicai Medicine and 2The Institute of Zoology, The Zooiogical Society of London, London, U.K. (Accepted 8 July 1991)

Summary

Mapping of B cell epitopes on HBsAg was performed using a panel of human anti-HBs antibodies. Synthetic peptides representing different regions of HBsAg failed to inhibit the binding of two antibodies which recognized noncon~~ational IIBsAg determinants in dot-blot ELISA and HBsAg polypeptide bands in immunoblot analysis. Cross-inhibition studies using five of the antibodies conjugated to horseradish peroxidase suggested that at least three different epitopes are recognised by the panel of antibodies, two of which are within the ‘a’ group determinant. Epitope mapping; HBsAg; Anti-HBs antibodies

Introduction

Although epitope mapping using mouse anti-HBs monoclonal antibodies has demonstrated epitope diversity within the ‘a’ group determinant (Kennedy et al., 1983; Wands et al., 1984; Waters, 1987), no comparative epitope mapping has been carried out using human anti-HBs monoclonal antibodies. Having developed and characterized a panel of 10 human anti-HBs antibodies (Sa’adu et al., 1991a,b), we proceeded to perform epitope mapping experiments. We have previously shown that five of our panel of 10 human anti-HBs Correspondence to: A. Sa’adu, Dept. of Clinical Sciences, London School of Hygiene and Tropical Medicine, Keppel Street, London WCIE 7HT, U.K.

antibodies recognized the common a group determinant in seven HBsAg serotypes from the World Health Organization (Sa’adu et al., 1991a). Waters (1987) has shown that the majority of mouse anti-HBs monoclonal antibodies binding to the a group determinant of HBsAg recognize conformational (discontinuous) rather than linear (continuous) epitopes. She found, therefore, that the majority of her panel of mouse monoclonal antibodies did not recognize HBsAg polypeptide bands using immunoblot techniques. This report describes studies employing immunoblot analysis with our panel of human anti-HBs antibodies to demonstrate linear B cell epitopes on HBsAg. Furthermore, we attempted to determine these epitopes using a number of synthetic peptides in inhibition assays. Cross-inhibition studies were employed to demonstrate groups of antibodies that recognized similar epitopes.

Materials and Methods Immunoblot

analysis

Anti-HBs dot-blot ELISA. Recombinant yeast-derived HBsAg (Merck Sharpe and Dohme Ltd., Hoddesdon, Herts., U.K.) was either diluted in phosphate-buffered saline containing 0.02% sodium azide (PBS-A) to serve as ‘native’ antigen or boiled in buffer (0.125 M Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, 10% 2- mercaptoethanol) to act as ‘denatured’ antigen. Nitrocellulose strips were dotted with both antigens, blocked in 2% milk powder (Marvel, Cadburys, Bournville, U.K.) and incubated with the panel of human anti-HBs antibody (Table 1). After three washes with phosphate-buffered saline TABLE 1 Derivation

of EBV-transformed

cell lines and clones secreting human anti-HB antibody

Donors

Lines

EB

EBA-B6”

JR

JRA-B5” JRB-Alb JRD-A2b

WS

WSC-C3b

PT

PTA-B6b

SCR

SCRC-B2C

Clones

’ Producing an IgGi, kappa anti-HBs antibody; b producing an IgGi, lambda anti-HBs antibody; ’ producing an IgG4, lambda anti-HBs antibody; d EBV-hybridoma produced by fusing the A2(N) EBV-transformed clone with the P3-X63-Ag8.653 mouse myeloma cell line.

201 TABLE 2 Amino acid sequences of synthetic peptides Name of peptide

Amino acid sequence

Sl-10 s35-50 S50-65 s139-147 S217-226 C75-84

met-glu-asn-ile-thr-ser-gly-phe-leu-gly trp-trp-thr-ser-leu-asn-phe-leu-gly-gly-ser-pro-val-cys-leu-gly gly-gin-asn-ser-gin-ser-pro-thr-ser-asn-his-~r-pro-thr-ser-cys-pro cys-thr-lys-pro-thr-asp-gly-asn-cys pro-ile-phe-phe-cys-leu-trp-val-tyr-ile asn-leu-glu-asp-pro-ala-ser-arg-asp-leu

containing 0.1% Tween 20 (PBS/T20), the strips were incubated in goat antihuman IgG peroxidase conjugate (Tago Inc., Burlingame, CA, U.S.A.). After washing again, the strips were soaked in the peroxidase substrate solution (51 mixture of 3,3’-diaminobenzidine tetrahydrochloride and 4-chlorol-1-napthol) to visualize antibody-antigen binding. SDS-PAGE analysis. The proteins of the recombinant yeast-derived HBsAg were separated by SDS-PAGE (Laemmli, 1970) and blotted on to nitrocellulose (Towbin et al., 1979). Nitrocellulose strips were incubated with one of the human anti-HBs antibodies and binding visualized as above. Inhibition ELISA assays Synthetic peptide inhibition ELISA. The JRB-Al and JRD-A2 antibodies were with each of the six synthetic peptides (Table 2). The reaction mixtures were transferred to Immulon 1 pIates (Dynatech Laboratories Inc.) coated with recombinant yeast-derived HBsAg and incubated at 37°C for 60 min. After incubating with goat anti-human IgG peroxidase conjugate (Tago) the substrate, ortho-phenylenediamine hydrochloride (OPD tablets; Sigma Co. Ltd., Poole, Dorset, U.K.) was added, the enzyme reactions stopped and absorbances (A) determined. Competitive inhibition ELISA. Immulon 1 plates coated with recombinant yeast-derived HBsAg were incubated with the human anti-HBs antibodies (Table 1). After incubating with one of the five peroxidase-labelled human antiHBs antibodies, the substrate reaction was determined as above. Results

Anti-HBs antibody dot-blot ELISA Fig. 1 depicts the nitrocellulose strips incubated with the panel of antibodies. Only the anti-HBs-positive human serum and the JRB-Al and JRD-A2 anti-

202

Fig. 1. Binding of antibodies to nature and denatured HBaAg. All the anti-HBs antibodies recognize ‘native’ HBsAg dots. However, only the positive control serum, the JRA-Bl and JRA-B2 anti-HBs antibodies recognize ‘denatured’ HBsAg dots.

HBs antibodies recognized denatured HBsAg, while all anti-HBs antibodies recognized the native HBsAg. The anti-HBs-negative serum did not react with either antigen. These results suggest that the JRA-Bl and JRA-B2 antibodies recognize continuous (non-conformational) epitopes whilst the other antibodies recognize discontinuous (conformational) epitopes.

Antigen KD 971--,

mdwt. mukl---

HB8Ag

Amido Bbck

Amido Bbck

HB8Ag

HB~AQ

HB8Ag

HB8Ag

67‘ 43‘ 30‘

Bt8ln8

AntbHB8 AntbHB8 Po8itive Negetive Serum Sen8n

JRS-Al

JRD-A2

Fig. 2. Western blot analysis of HBsAg, separated by 12% polyacrylamide gel electrophoresis. Lane 1 shows separation of molecular weight markers. Lane 2 shows HBsAg polypeptide bands stained with amido black. Lane 3 shows that the anti-HBs positive control serum recognizes the 24-, 44-, 66- and 87 kDa bands. Anti-HBs-negative control serum does not recognize any of the HBsAg bands (lane 4). The JRB-AI (lane 5) and JRD-A2 (lane 6) human anti-HBs antibodies bind to HBsAg polypeptide bands.

203

Immunoblot

analysis

The reactivity of the antibodies to HBsAg polypeptide bands was determined (Fig. 2). Anti-HBsAg-positive control serum recognized a 24-kDa band as well as three higher molecular weight bands (44, 66 and 87 kDa) which represent aggregates of the 24-kDa polypeptides (lane 3). Anti-HBs-negative control serum did not recognize any band; neither did 8 of the 10 antibodies (data not shown). Only the JRB-Al and JRD-A2 antibodies recognized HBsAg polypeptide bands (24, 44, 66 and 87 kDa). Synthetic peptide inhibition ELBA

Mixing the JRB-Al and JRD-A2 antibodies with the panel of HBsAg synthetic peptides did not inhibit their subsequent binding to wells coated with recombinant yeast-derived HBsAg (data not shown). This suggests that no synthetic peptide represents the epitope recognized by the JRB-Al and JRDA2 human anti-HBs antibodies. Competitive

inhibition ELBA

Fig. 3 shows the inhibition curves of five of the human anti-HBs antibodies against the WSC-C3 conjugate binding to recombinant yeast-derived HBsAg. The homologous WSC-C3 monoclonal antibody inhibited the WSC-C3 conjugate, as did the EBA-B6, Al(N), A2(N) and A2(N).X63 antibodies. The other antibodies (PTA-B6, JRA-BS, JRB-Al, JRD-A2) failed to show any ,oo Percentage Inhibition I’O0

Antibody Titre Fig. 3. Inhibition of the binding of the WSC-C3 peroxidase conjugate recombinant yeast-derived HBsAg by the panel of human anti-HBs antibodies. Inhibition is seen with the WSC-C3 (-+-), Al(N) ++-), A2(N).X63 (-_t), A2(N) (a) and EBA-B6 (-*-) antibodies. The other antibodies, JRA-B5, JRB-AI, JRD-AZ, PTA-B6 and SCRC-B2, (+) demonstrate no inhibition.

204

,oo rntage

ILX

1,oo

Inhibition

2

4

6

16

32

64

None

Antibody Titre Fig. 4. Inhibition of the binding of the SCRC-B2 peroxidase conjugate to recombinant yeast-derived HBsAg by the panel of human anti-HBs antibodies. Only the SCRC-B2 antibody (+) demonstrated any inhibition. The other antibodies, EBA-B6, Al(N), A2(N), A2(N).X63, JRA-B5, JRB-Al, JRD-AZ, WSCC3 and PTA-B6, (+-)) did not.

inhibition. Experiments performed using the A2(N), A2(N).X63 and the EBAB6 conjugates confirmed that the Al(N), A2(N), A2(N).X63, EBA-B6 and WSC-C3 human anti-HBs antibodies all inhibit each other (data not shown). The binding of the SCRCB2 conjugate is only inhibited by the SCRCB2 antibody (Fig. 4); none of the other antibodies demonstrate any inhibition. These data suggest that the panel antibodies form at least three groups in terms of their epitope reactivity: group 1, the Al(N), A2(N), A2(N).X63, EBA-B6, WSC-C3 antibodies; group 2, the SCRCB2 antibody; and group 3, the PTAB6, JRA-B5, JRB-Al, JRD-A2 antibodies.

Discussion There are at least three epitopes recognized by the human anti-HBs antibodies evaluated. It is likely that the EBA-B6, Al(N), A2(N) and A2(N).X63 antibodies, derived from the EBV transformation of PBMC of a single donor, secrete the same human antibody. They are all IgGr, kappa antibodies and recognize the same epitope (group 1). The cell lines and clones producing these antibodies may represent different clones of the same parent EBV-transformed B cell. Having previously demonstrated that the A2(N) and A2(N).X63 monoclonal antibodies recognize the a group determinant of HBsAg (Sa’adu et al., 1991a), we can now conclude that this epitope is a conformational one because none of them recognized ‘denatured’ or HBsAg polypeptide bands. The WSC-C3 antibody, derived from a different donor, is a distinct IgGi, lambda antibody which belongs in this group.

205

The majority (eight out of ten) of our panel of human anti-HBs antibodies recognize discontinuous epitopes. Similar results were obtained with a panel of mouse anti-HBs monoclonal antibodies produced by Waters (1987). Thus, both the mouse and human data support the classical immunological teaching that most B cell epitopes are continuous because they react with free antigens whose conformations are complementary to the Ig molecules on their surface. The synthetic peptides failed to inhibit the binding of the two antibodies which recognized continuous epitopes, suggesting that none of their sequences, not even the S139-147 peptide which is thought to represent a partial analogue of the a group determinants (Vyas, 1981; Brown et al., 1984a,b), represents these unique epitopes. It seems that neither antibody recognizes this a group determinant epitope. The SCRCB2 antibody recognizes a panel of HBsAg serotypes (Sa’adu et al., 1991a), but recognizes a different epitope to that recognized by the group 1 antibodies. It seems to be directed at a second discontinuous epitope within the a group determinants. Antigenic diversity within the a group determinant was first suggested by Imai et al. (1974) and confirmed serologically by Kennedy et al. (1983) and Wands et al. (1984) using mouse monoclonal antibodies. In fact, Waters (1987) found four different epitopes within the a group determinant. It is not known whether all monoclonal antibodies directed against the a group determinant are protective. Failure to grow HBV in vitro has prevented the development of a tissue culture neutralization assay. The cost and logistic problems of neutralization experiments in the chimpanzee model has prevented the routine assessment of anti-HBs monoclonal antibodies as passive vaccine candidates. Indeed, it is only the RFHBs 1 mouse anti-HBs monoclonal antibody that has been assessed in this way (Iwarson et al., 1985). However, if therapeutic benefit is to be reaped from the labour necessary to produce human anti-HBs monoclonal antibodies, they must be assessed for protective efficacy in the chimpanzee model. The report by Harada et al. (1989) suggests that a cocktail ‘of human anti-HBs monoclonal antibodies is not only effective in neutralizing the in vivo infection of chimpanzees by HBV but that it shows higher antibody titres in enzyme-immunoassays than commercially available human polyclonal antibody preparations. We have successfully demonstrated epitope diversity within the a group determinant of HBsAg with the use of our panel of human anti-HBs antibodies. It remains to be seen whether such antibodies can confer passive protection and be useful as immunoprophylactic agents. Acknowledgements

Dr. Sa’adu was supported by grants from the Williams Medical Research Fellowship and the Overseas Students’ Research Fund. The laboratories acknowledge support from the Lawson Tate Trust, the Overseas Development

206

Administration, the Rockefeller Foundation grateful to Mrs Amy Davey for preparation

and the Wellcome of the manuscript.

Trust. We are

References Brown, SE., Howard, C.R., Zuckerman, A.J. and Steward, M.W. (1984a) The determination of the affinity of antibodies to hepatitis B surface antigen. J. Immunol. Methods 72, 4148. Brown, S.E., Howard, C.R., Zuckerman, A.J. and Steward, M.W. (1984b) Affinity of antibody responses in man to hepatitis B vaccine determined with synthetic peptides. Lancet ii, 184187. Harada, K., Ichimori, Y., Sasano, K., Sasai, S., Kitano, K., Iwasa, S., Tsukamoto, K. and Sugino, Y. (1989) Human-human hybridomas secreting hepatitis B virus-neutralizing antibodies. Biotechnology 7, 375-377. Imai, M., Gotch, A., Nishioka, K., Kurashina, S., Miyakawa, Y. and Mayumi, M. (1974) Antigenicity of reduced and alkylated Australia antigen. J. Immunol. 112, 416449. Iwarson, S., Tabor, E., Thomas, H.C., Goodall, A., Waters, J., Snoy, P., Shih, J.W.K. and Gerety, R.J. (1985) Neutralisation of hepatitis B virus infection by a murine monoclonal antibody: an experimental study in the chimpanzee. J. Med. Virol. 16, 89-96. Kennedy, R.C., Ionescu-Matiu, I., Adler-Storthz, K., Henkel, R.D., Sanchez, Y. and Dressman, G.R. (1983) Characterisation of anti-hepatitis B surface antigen monoclonal antibodies. Intervirology 9, 176-l 80. Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 277, 680-685. Sa’adu, A., Locniskar, M.F., Bidwell, D., Howard, C., Voller, A. and McAdam, K.P.W.J. (1991a) Development and characterisation of human anti-HBs antibodies. Submitted for publication. Sa’adu, A., Locniskar, M.F., Bidwell, D., Howard, C., Voller, A. and McAdam, K.P.W.J. (1991 b) Comparison of an EBV transformed cell line and an EBV hybridoma cell line producing the same human anti-HBs monoclonal antibody. Submitted for publication. Towbin, H., Staehelin, T. and Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76, 435&4354. Vyas, G.N. (1981) Molecular immunology of the hepatitis B surface antigen (HBsAg). In: P. Maupas and P. Guestry (Eds), Hepatitis B Vaccine INSERM Symposium No. 18, Elsevier/ North Holland, Biomedical Press, Amsterdam, pp. 227-237. Wands, J.R., Ben-Porath, E. and Wang, E.A. (1984) Monoclonal antibodies and hepatitis B: a new perspective using highly sensitive and specific radioimmunoassays. In: G.N. Vyas, J.L. Dienstag and J.H. Hoofnagle (Eds), Viral Hepatitis and Liver Disease, Grune and Stratton, New York, pp. 543-559. Waters, J.A. (1987) In: The Use of Monoclonal Antibodies to Analyze the Immune Response During HBV infection (Ph.D. Thesis), Royal Free Hospital School of Medicine (University of London), pp. 38-133.

Epitope mapping of HBsAg using a panel of human anti-HBs antibodies.

Mapping of B cell epitopes on HBsAg was performed using a panel of human anti-HBs antibodies. Synthetic peptides representing different regions of HBs...
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