Skip to main content
NCBI home page
Journal of Virology logoLink to Journal of Virology
. 1990 Mar;64(3):1290–1297. doi: 10.1128/jvi.64.3.1290-1297.1990

Virus-neutralizing monoclonal antibody to a conserved epitope on the duck hepatitis B virus pre-S protein.

V Lambert 1, D Fernholz 1, R Sprengel 1, I Fourel 1, G Deléage 1, G Wildner 1, C Peyret 1, C Trépo 1, L Cova 1, H Will 1
PMCID: PMC249246  PMID: 1689393

Abstract

In this study we used duck hepatitis B virus (DHBV)-infected Pekin ducks and heron hepatitis B virus (HHBV)-infected heron tissue to search for epitopes responsible for virus neutralization on pre-S proteins. Monoclonal antibodies were produced by immunizing mice with purified DHBV particles. Of 10 anti-DHBV specific hybridomas obtained, 1 was selected for this study. This monoclonal antibody recognized in both DHBV-infected livers and viremic sera a major (36-kilodalton) protein and several minor pre-S proteins in all seven virus strains used. In contrast, pre-S proteins of HHBV-infected tissue or viremic sera did not react. Thus, the monoclonal antibody recognizes a highly conserved DHBV pre-S epitope. For mapping of the epitope, polypeptides from different regions of the DHBV pre-S/S gene were expressed in Escherichia coli and used as the substrate for immunoblotting. The epitope was delimited to a sequence of approximately 23 amino acids within the pre-S region, which is highly conserved in four cloned DHBV isolates and coincides with the main antigenic domain as predicted by computer algorithms. In in vitro neutralization assays performed with primary duck hepatocyte cultures, the antibody reduced DHBV infectivity by approximately 75%. These data demonstrate a conserved epitope of the DHBV pre-S protein which is located on the surface of the viral envelope and is recognized by virus-neutralizing antibodies.

Full text

PDF
1292

Images in this article

1292Image
on p.1292
1293Image
on p.1293
1295Image
on p.1295

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Budkowska A., Riottot M. M., Dubreuil P., Lazizi Y., Brechot C., Sobczak E., Petit M. A., Pillot J. Monoclonal antibody recognizing pre-S(2) epitope of hepatitis B virus: characterization of pre-S(2) epitope and anti-pre-S(2) antibody. J Med Virol. 1986 Oct;20(2):111–125. doi: 10.1002/jmv.1890200204. [DOI] [PubMed] [Google Scholar]
  2. Büscher M., Reiser W., Will H., Schaller H. Transcripts and the putative RNA pregenome of duck hepatitis B virus: implications for reverse transcription. Cell. 1985 Mar;40(3):717–724. doi: 10.1016/0092-8674(85)90220-x. [DOI] [PubMed] [Google Scholar]
  3. Cheung R. C., Robinson W. S., Marion P. L., Greenberg H. B. Epitope mapping of neutralizing monoclonal antibodies against duck hepatitis B virus. J Virol. 1989 Jun;63(6):2445–2451. doi: 10.1128/jvi.63.6.2445-2451.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chou P. Y., Fasman G. D. Prediction of the secondary structure of proteins from their amino acid sequence. Adv Enzymol Relat Areas Mol Biol. 1978;47:45–148. doi: 10.1002/9780470122921.ch2. [DOI] [PubMed] [Google Scholar]
  5. Cova L., Hantz O., Arliaud-Gassin M., Chevalier A., Berthillon P., Boulay J., Jacquet C., Chomel B., Vitvitski L., Trepo C. Comparative study of DHBV DNA levels and endogenous DNA polymerase activity in naturally infected ducklings in France. J Virol Methods. 1985 Mar;10(3):251–260. doi: 10.1016/0166-0934(85)90065-5. [DOI] [PubMed] [Google Scholar]
  6. Cova L., Lambert V., Chevallier A., Hantz O., Fourel I., Jacquet C., Pichoud C., Boulay J., Chomel B., Vitvitski L. Evidence for the presence of duck hepatitis B virus in wild migrating ducks. J Gen Virol. 1986 Mar;67(Pt 3):537–547. doi: 10.1099/0022-1317-67-3-537. [DOI] [PubMed] [Google Scholar]
  7. Deléage G., Clerc F. F., Roux B., Gautheron D. C. ANTHEPROT: a package for protein sequence analysis using a microcomputer. Comput Appl Biosci. 1988 Aug;4(3):351–356. doi: 10.1093/bioinformatics/4.3.351. [DOI] [PubMed] [Google Scholar]
  8. Deléage G., Roux B. An algorithm for protein secondary structure prediction based on class prediction. Protein Eng. 1987 Aug-Sep;1(4):289–294. doi: 10.1093/protein/1.4.289. [DOI] [PubMed] [Google Scholar]
  9. Dimmock N. J. Mechanisms of neutralization of animal viruses. J Gen Virol. 1984 Jun;65(Pt 6):1015–1022. doi: 10.1099/0022-1317-65-6-1015. [DOI] [PubMed] [Google Scholar]
  10. Dreesman G. R., Sanchez Y., Ionescu-Matiu I., Sparrow J. T., Six H. R., Peterson D. L., Hollinger F. B., Melnick J. L. Antibody to hepatitis B surface antigen after a single inoculation of uncoupled synthetic HBsAg peptides. Nature. 1982 Jan 14;295(5845):158–160. doi: 10.1038/295158a0. [DOI] [PubMed] [Google Scholar]
  11. Eble B. E., Lingappa V. R., Ganem D. Hepatitis B surface antigen: an unusual secreted protein initially synthesized as a transmembrane polypeptide. Mol Cell Biol. 1986 May;6(5):1454–1463. doi: 10.1128/mcb.6.5.1454. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Eble B. E., MacRae D. R., Lingappa V. R., Ganem D. Multiple topogenic sequences determine the transmembrane orientation of the hepatitis B surface antigen. Mol Cell Biol. 1987 Oct;7(10):3591–3601. doi: 10.1128/mcb.7.10.3591. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Emini E. A., Hughes J. V., Perlow D. S., Boger J. Induction of hepatitis A virus-neutralizing antibody by a virus-specific synthetic peptide. J Virol. 1985 Sep;55(3):836–839. doi: 10.1128/jvi.55.3.836-839.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Emini E. A., Kao S. Y., Lewis A. J., Crainic R., Wimmer E. Functional basis of poliovirus neutralization determined with monospecific neutralizing antibodies. J Virol. 1983 May;46(2):466–474. doi: 10.1128/jvi.46.2.466-474.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Emini E. A., Larson V., Eichberg J., Conard P., Garsky V. M., Lee D. R., Ellis R. W., Miller W. J., Anderson C. A., Gerety R. J. Protective effect of a synthetic peptide comprising the complete preS2 region of the hepatitis B virus surface protein. J Med Virol. 1989 May;28(1):7–12. doi: 10.1002/jmv.1890280103. [DOI] [PubMed] [Google Scholar]
  16. Engvall E., Perlmann P. Enzyme-linked immunosorbent assay, Elisa. 3. Quantitation of specific antibodies by enzyme-labeled anti-immunoglobulin in antigen-coated tubes. J Immunol. 1972 Jul;109(1):129–135. [PubMed] [Google Scholar]
  17. Feitelson M. A., Marion P. L., Robinson W. S. The nature of polypeptides larger in size than the major surface antigen components of hepatitis b and like viruses in ground squirrels, woodchucks, and ducks. Virology. 1983 Oct 15;130(1):76–90. doi: 10.1016/0042-6822(83)90119-8. [DOI] [PubMed] [Google Scholar]
  18. Fourel I., Gripon P., Hantz O., Cova L., Lambert V., Jacquet C., Watanabe K., Fox J., Guillouzo C., Trepo C. Prolonged duck hepatitis B virus replication in duck hepatocytes cocultivated with rat epithelial cells: a useful system for antiviral testing. Hepatology. 1989 Aug;10(2):186–191. doi: 10.1002/hep.1840100211. [DOI] [PubMed] [Google Scholar]
  19. Fourel I., Hantz O., Cova L., Allaudeen H. S., Trepo C. Main properties of duck hepatitis B virus DNA polymerase: comparison with the human and woodchuck hepatitis B virus DNA polymerases. Antiviral Res. 1987 Nov;8(4):189–199. doi: 10.1016/0166-3542(87)90073-8. [DOI] [PubMed] [Google Scholar]
  20. Garnier J., Osguthorpe D. J., Robson B. Analysis of the accuracy and implications of simple methods for predicting the secondary structure of globular proteins. J Mol Biol. 1978 Mar 25;120(1):97–120. doi: 10.1016/0022-2836(78)90297-8. [DOI] [PubMed] [Google Scholar]
  21. Gerin J. L., Alexander H., Shih J. W., Purcell R. H., Dapolito G., Engle R., Green N., Sutcliffe J. G., Shinnick T. M., Lerner R. A. Chemically synthesized peptides of hepatitis B surface antigen duplicate the d/y specificities and induce subtype-specific antibodies in chimpanzees. Proc Natl Acad Sci U S A. 1983 Apr;80(8):2365–2369. doi: 10.1073/pnas.80.8.2365. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Guldner H. H., Netter H. J., Szostecki C., Lakomek H. J., Will H. Epitope mapping with a recombinant human 68-kDa (U1) ribonucleoprotein antigen reveals heterogeneous autoantibody profiles in human autoimmune sera. J Immunol. 1988 Jul 15;141(2):469–475. [PubMed] [Google Scholar]
  23. Gust I. D., Burrell C. J., Coulepis A. G., Robinson W. S., Zuckerman A. J. Taxonomic classification of human hepatitis B virus. Intervirology. 1986;25(1):14–29. doi: 10.1159/000149651. [DOI] [PubMed] [Google Scholar]
  24. Heermann K. H., Kruse F., Seifer M., Gerlich W. H. Immunogenicity of the gene S and Pre-S domains in hepatitis B virions and HBsAg filaments. Intervirology. 1987;28(1):14–25. doi: 10.1159/000149993. [DOI] [PubMed] [Google Scholar]
  25. Hopp T. P., Woods K. R. Prediction of protein antigenic determinants from amino acid sequences. Proc Natl Acad Sci U S A. 1981 Jun;78(6):3824–3828. doi: 10.1073/pnas.78.6.3824. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Iorio R. M., Bratt M. A. Neutralization of Newcastle disease virus by monoclonal antibodies to the hemagglutinin-neuraminidase glycoprotein: requirement for antibodies to four sites for complete neutralization. J Virol. 1984 Aug;51(2):445–451. doi: 10.1128/jvi.51.2.445-451.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Itoh Y., Takai E., Ohnuma H., Kitajima K., Tsuda F., Machida A., Mishiro S., Nakamura T., Miyakawa Y., Mayumi M. A synthetic peptide vaccine involving the product of the pre-S(2) region of hepatitis B virus DNA: protective efficacy in chimpanzees. Proc Natl Acad Sci U S A. 1986 Dec;83(23):9174–9178. doi: 10.1073/pnas.83.23.9174. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Klinkert M. Q., Theilmann L., Pfaff E., Schaller H. Pre-S1 antigens and antibodies early in the course of acute hepatitis B virus infection. J Virol. 1986 May;58(2):522–525. doi: 10.1128/jvi.58.2.522-525.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Kyhse-Andersen J. Electroblotting of multiple gels: a simple apparatus without buffer tank for rapid transfer of proteins from polyacrylamide to nitrocellulose. J Biochem Biophys Methods. 1984 Dec;10(3-4):203–209. doi: 10.1016/0165-022x(84)90040-x. [DOI] [PubMed] [Google Scholar]
  30. Köhler G., Milstein C. Derivation of specific antibody-producing tissue culture and tumor lines by cell fusion. Eur J Immunol. 1976 Jul;6(7):511–519. doi: 10.1002/eji.1830060713. [DOI] [PubMed] [Google Scholar]
  31. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  32. Levin J. M., Robson B., Garnier J. An algorithm for secondary structure determination in proteins based on sequence similarity. FEBS Lett. 1986 Sep 15;205(2):303–308. doi: 10.1016/0014-5793(86)80917-6. [DOI] [PubMed] [Google Scholar]
  33. Li J. S., Cova L., Buckland R., Lambert V., Deléage G., Trépo C. Duck hepatitis B virus can tolerate insertion, deletion, and partial frameshift mutation in the distal pre-S region. J Virol. 1989 Nov;63(11):4965–4968. doi: 10.1128/jvi.63.11.4965-4968.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Machida A., Kishimoto S., Ohnuma H., Baba K., Ito Y., Miyamoto H., Funatsu G., Oda K., Usuda S., Togami S. A polypeptide containing 55 amino acid residues coded by the pre-S region of hepatitis B virus deoxyribonucleic acid bears the receptor for polymerized human as well as chimpanzee albumins. Gastroenterology. 1984 May;86(5 Pt 1):910–918. [PubMed] [Google Scholar]
  35. Mandart E., Kay A., Galibert F. Nucleotide sequence of a cloned duck hepatitis B virus genome: comparison with woodchuck and human hepatitis B virus sequences. J Virol. 1984 Mar;49(3):782–792. doi: 10.1128/jvi.49.3.782-792.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Marion P. L., Knight S. S., Feitelson M. A., Oshiro L. S., Robinson W. S. Major polypeptide of duck hepatitis B surface antigen particles. J Virol. 1983 Nov;48(2):534–541. doi: 10.1128/jvi.48.2.534-541.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Marion P. L., Oshiro L. S., Regnery D. C., Scullard G. H., Robinson W. S. A virus in Beechey ground squirrels that is related to hepatitis B virus of humans. Proc Natl Acad Sci U S A. 1980 May;77(5):2941–2945. doi: 10.1073/pnas.77.5.2941. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Mason W. S., Seal G., Summers J. Virus of Pekin ducks with structural and biological relatedness to human hepatitis B virus. J Virol. 1980 Dec;36(3):829–836. doi: 10.1128/jvi.36.3.829-836.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Milich D. R. Genetic and molecular basis for T- and B-cell recognition of hepatitis B viral antigens. Immunol Rev. 1987 Oct;99:71–103. doi: 10.1111/j.1600-065x.1987.tb01173.x. [DOI] [PubMed] [Google Scholar]
  40. Neurath A. R., Kent S. B., Parker K., Prince A. M., Strick N., Brotman B., Sproul P. Antibodies to a synthetic peptide from the preS 120-145 region of the hepatitis B virus envelope are virus neutralizing. Vaccine. 1986 Mar;4(1):35–37. doi: 10.1016/s0264-410x(86)80001-9. [DOI] [PubMed] [Google Scholar]
  41. Neurath A. R., Kent S. B., Strick N., Parker K. Identification and chemical synthesis of a host cell receptor binding site on hepatitis B virus. Cell. 1986 Aug 1;46(3):429–436. doi: 10.1016/0092-8674(86)90663-x. [DOI] [PubMed] [Google Scholar]
  42. Neurath A. R., Kent S. B. The pre-S region of hepadnavirus envelope proteins. Adv Virus Res. 1988;34:65–142. doi: 10.1016/s0065-3527(08)60516-3. [DOI] [PubMed] [Google Scholar]
  43. Parker J. M., Guo D., Hodges R. S. New hydrophilicity scale derived from high-performance liquid chromatography peptide retention data: correlation of predicted surface residues with antigenicity and X-ray-derived accessible sites. Biochemistry. 1986 Sep 23;25(19):5425–5432. doi: 10.1021/bi00367a013. [DOI] [PubMed] [Google Scholar]
  44. Persing D. H., Varmus H. E., Ganem D. The preS1 protein of hepatitis B virus is acylated at its amino terminus with myristic acid. J Virol. 1987 May;61(5):1672–1677. doi: 10.1128/jvi.61.5.1672-1677.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Pontisso P., Petit M. A., Bankowski M. J., Peeples M. E. Human liver plasma membranes contain receptors for the hepatitis B virus pre-S1 region and, via polymerized human serum albumin, for the pre-S2 region. J Virol. 1989 May;63(5):1981–1988. doi: 10.1128/jvi.63.5.1981-1988.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Pugh J. C., Sninsky J. J., Summers J. W., Schaeffer E. Characterization of a pre-S polypeptide on the surfaces of infectious avian hepadnavirus particles. J Virol. 1987 May;61(5):1384–1390. doi: 10.1128/jvi.61.5.1384-1390.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Purcell R. H., Gerin J. L. Prospects for second and third generation hepatitis B vaccines. Hepatology. 1985 Jan-Feb;5(1):159–163. doi: 10.1002/hep.1840050130. [DOI] [PubMed] [Google Scholar]
  48. Rigby P. W., Dieckmann M., Rhodes C., Berg P. Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J Mol Biol. 1977 Jun 15;113(1):237–251. doi: 10.1016/0022-2836(77)90052-3. [DOI] [PubMed] [Google Scholar]
  49. Schaeffer E., Sninsky J. J. Predicted secondary structure similarity in the absence of primary amino acid sequence homology: hepatitis B virus open reading frames. Proc Natl Acad Sci U S A. 1984 May;81(9):2902–2906. doi: 10.1073/pnas.81.9.2902. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Schlicht H. J., Kuhn C., Guhr B., Mattaliano R. J., Schaller H. Biochemical and immunological characterization of the duck hepatitis B virus envelope proteins. J Virol. 1987 Jul;61(7):2280–2285. doi: 10.1128/jvi.61.7.2280-2285.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Shulman M., Wilde C. D., Köhler G. A better cell line for making hybridomas secreting specific antibodies. Nature. 1978 Nov 16;276(5685):269–270. doi: 10.1038/276269a0. [DOI] [PubMed] [Google Scholar]
  52. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  53. Sprengel R., Kaleta E. F., Will H. Isolation and characterization of a hepatitis B virus endemic in herons. J Virol. 1988 Oct;62(10):3832–3839. doi: 10.1128/jvi.62.10.3832-3839.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Sprengel R., Kuhn C., Manso C., Will H. Cloned duck hepatitis B virus DNA is infectious in Pekin ducks. J Virol. 1984 Dec;52(3):932–937. doi: 10.1128/jvi.52.3.932-937.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Sprengel R., Kuhn C., Will H., Schaller H. Comparative sequence analysis of duck and human hepatitis B virus genomes. J Med Virol. 1985 Apr;15(4):323–333. doi: 10.1002/jmv.1890150402. [DOI] [PubMed] [Google Scholar]
  56. Strebel K., Beck E., Strohmaier K., Schaller H. Characterization of foot-and-mouth disease virus gene products with antisera against bacterially synthesized fusion proteins. J Virol. 1986 Mar;57(3):983–991. doi: 10.1128/jvi.57.3.983-991.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Summers J., Smolec J. M., Snyder R. A virus similar to human hepatitis B virus associated with hepatitis and hepatoma in woodchucks. Proc Natl Acad Sci U S A. 1978 Sep;75(9):4533–4537. doi: 10.1073/pnas.75.9.4533. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Tuttleman J. S., Pugh J. C., Summers J. W. In vitro experimental infection of primary duck hepatocyte cultures with duck hepatitis B virus. J Virol. 1986 Apr;58(1):17–25. doi: 10.1128/jvi.58.1.17-25.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Wahl G. M., Stern M., Stark G. R. Efficient transfer of large DNA fragments from agarose gels to diazobenzyloxymethyl-paper and rapid hybridization by using dextran sulfate. Proc Natl Acad Sci U S A. 1979 Aug;76(8):3683–3687. doi: 10.1073/pnas.76.8.3683. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Yokosuka O., Omata M., Ito Y. Expression of pre-S1, pre-S2, and C proteins in duck hepatitis B virus infection. Virology. 1988 Nov;167(1):82–86. doi: 10.1016/0042-6822(88)90056-6. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Virology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES