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Expression in Yeast of Amino-Terminal Peptide Fusions to Hepatitis B Core Antigen and Their Immunological Properties

Abstract

Hepatitis B core protein (HBcAg) is a potent antigen that gives both a T-cell dependent and a T-cell-independent antibody response. It has been shown that a foreign epitope can be fused to the amino terminus of HBcAg without affecting partide integrity, and that the resulting chimaeric cores retain the immunogenicity of the foreign epitope. Here we describe the efficient expression in yeast of two different chimaeric cores, carrying epitopes of Foot and Mouth Disease Virus (FMDV) or human chorionic gonadotrophin (hCG), which are candidates for FMD and contraceptive vaccines, respectively. These cores could not be produced in E. coli in soluble form but were expressed to high levels in yeast. We constructed a yeast expression vector that allows rapid production of different chimaeric cores by cloning in cassettes encoding foreign epitopes. Both FMDV and hCG-cores were shown to present the epitopes at the sur face of the particles. The FMDV-cores produced in yeast were efficient inducers of neutralising antibodies in guinea-pigs after one low dose.

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References

  1. Stahl, S., Mackay, P., Agazin, M., Bruce, S.A. and Murray, K. 1982. Hepatitis B virus core antigen: synthesis in Escherichia coli and application in diagnosis. Proc. Natl. Acad. Sci. USA. 79: 1606–1610.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Miyanohara, A., Imamura, T., Araki, M., Sugawara, K., Ohtomo, N. and Matsubara, K. 1986. Expression of hepatitis B virus core antigen gene in Saccharomyces cerevisiae: synthesis of two polypeptides translated from different initiation codons. J. Virol. 59: 176–180.

    CAS  PubMed  PubMed Central  Google Scholar 

  3. Jean-Jean, O., Levrero, M., Will, H., Pericaudet, M. and Rossignol, J.M. 1989. Expression mechanism of the Hepatitis B virus (HBV) C gene and biosynthesis of HBe antigen. Virology 170: 99–106.

    Article  CAS  PubMed  Google Scholar 

  4. Milich, D.R. and McLachlan, A. 1986. The nucleocapsid of the hepatitis B virus is both a T-cell independent and a T-cell dependent antigen. Science 234: 1398–1401.

    Article  CAS  PubMed  Google Scholar 

  5. Valenzuela, P., Coit, D., Medina-Selby, M.A., Kuo, C.H., Van Nest, G., Burke, R.L., Bull, P., Urdea, M.S. and Graves, P.V. 1985. Antigen engineering in yeast synthesis and assembly of hybrid hepatitis B surface antigen herpes simplex 1g D particles. Bio/Technology. 3: 323–326.

    CAS  Google Scholar 

  6. Adams, S.E., Dawson, K.M., Gull, K., Kingsman, S.M. and Kingsman, A.J. 1987. The expression of hybrid HIV:Ty virus like particles in yeast. NAture. 329: 68–70.

    Article  CAS  PubMed  Google Scholar 

  7. Clarke, B.E., Newton, S.E., Carroll, A.R., Francis, M.J., Appleyard, G., Syred, A.D., Highfield, P.E., Rowlands, D.J. and Brown, F. 1987. Improved immunogenicity of a peptide epitope after fusion to hepatitis B core protein. Nature 330: 381–384.

    Article  CAS  PubMed  Google Scholar 

  8. Clarke, B.E., Brown, A.L., Grace, K.G., Hastings, G.Z., Brown, F., Rowlands, D.J. and Francis, M.J. 1990. Presentation and immunogenicity of viral epitopes on the surface of hybrid hepatitis B virus core particles produced on bacteria. J. Gen. Virol. In press.

    Google Scholar 

  9. Stevens, V.C. 1986. A synthetic peptide vaccine against human chorionic gonadotrophin. Vaccines. 86. Cold Spring Harbor Laboratory, N.Y.

    Google Scholar 

  10. Beggs, J.D. 1978. Transformation of yeast by a replicating hybrid plasmid. Nature 275: 104–109.

    Article  CAS  PubMed  Google Scholar 

  11. Vieira, J. and Messing, J. . 1982. The pUC plasmids, an M13mp7, derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene 19: 259–268.

    Article  CAS  PubMed  Google Scholar 

  12. Tajima, M., Nogi, Y. and Fukasawa, T. . 1985. Primary structure of the Saccharomyces cerevisiae GAL7 gene. Yeast 1: 67–77.

    Article  CAS  PubMed  Google Scholar 

  13. Sutton, A. and Broach, J.R. 1985. Signals for transcription initiation and termination in the Saccharomyces cerevisiae plasmid 2μm circle. Mol. Cell. Biol. 5: 2770–2780.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Strohmaier, K., Franze, R. and Adam, K.-H. 1982. Location and characterisation of the antigenic portion of the FMDV immunising protein. J. Gen. Virol. 59: 295–306.

    Article  CAS  PubMed  Google Scholar 

  15. Broekhuijsen, M.P., Van Rijn, J.M.M., Blom, A.J.M., Pouwels, P.H., Enger-Volk, B.E., Brown, F. and Francis, M.J. . 1987. Fusion proteins with multiple copies of the major determinant of foot-and-mouth disease virus protect both the natural host and laboratory animals. J. Gen. Virol. 68: 3137–3143.

    Article  PubMed  Google Scholar 

  16. Parry, N.R., Ouldridge, E.J., Burnett, P.V., Clarke, B.E., Francis, M.J., Fox, J.D., Rowlands, D.J. and Brown, F. . 1989. Serological prospects for peptide vaccines against food-and-mouth disease virus. J. Gen. Virol. 70:In press.

    Google Scholar 

  17. Francis, M.J., Fry, C.M., Rowlands, D.J. and Brown, F. 1988. Qualitative and quantitative differences in the immune response to foot-and-mouth disease virus antigens and synthetic peptides. J. Gen. Virol. 69: 2483–2491.

    Article  CAS  PubMed  Google Scholar 

  18. Imamura, T., Sugahara, K., Adachi, S., Miyatsu, Y., Mizokami, H. and Matsusaka, T. . 1988. Purification and characterisation of the hepatitis B virus core antigen produced in the yeast Saccharomyces cerevisiae . J. Biotechnol. 8: 149–162.

    Article  CAS  Google Scholar 

  19. Kniskern, P.J., Hagopian, A., Montgomery, D.L., Burke, P., Dunn, N.R., Hofmann, K.J., Miller, W.J. and Ellis, R.W. 1986. Unusually high-level expression of a foreign gene (hepatitis B core antigen) in Saccharomyces cerevisiae . Gene 46: 135–141.

    Article  CAS  PubMed  Google Scholar 

  20. Baker, S.M., Johnston, S.A., Hopper, J.E. and Jaehning, J.A. 1987. Transcription of multiple copies of the yeast GAL7 gene is limited by specific factors in addition to GAL4 . Mol. Gen. Genet. 208: 127–134.

    Article  CAS  PubMed  Google Scholar 

  21. Hallewell, R.A., Mills, R., Tekamp-Olson, P., Blacher, R., Rosenberg, S., Otting, F., Masairz, F.R. and Scandella, C.S. 1987. Amino terminal acetylation of authentic human Cu, Zn superoxide dismutase. Bio/Technology 5: 363–366.

    CAS  Google Scholar 

  22. Cousens, L.S., Shuster, J.R., Gallegos, C., Ku, L., Stampien, M.M., Urdea, M.S., Sanchez-Pescador, R., Taylor, A., Tekamp-Olson, P. 1987. High level expression of proinsulin in the yeast, Saccharomyces cerevisiae . Gene 61: 265–275.

    Article  CAS  PubMed  Google Scholar 

  23. Braddock, M., Chambers, A., Wilson, W., Esnouf, M.P., Adams, S.E., Kingsman, A.J. and Kingsman, S.M. 1989. HIV-1 TAT activates presynthesised RNA in the nucleus. Cell 58: 269–279.

    Article  CAS  PubMed  Google Scholar 

  24. Pereira, H.G. 1978. Antigenic variation in relation to epidemiology and control of foot-and-mouth disease. Brit. Vet. J. 134: 58–62.

    Article  Google Scholar 

  25. Francis, M.J., Hastings, G.Z., Clarke, B.E., Brown, A.L., Beddell, C.R., Rowlands, D.J. and Brown, F. 1990. Neutralising antibodies to all seven serotypes of foor and mouth disease virus elicited by synthetic peptides. Immunology 69: 171–176.

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Francis, M.J., Clarke, B.E., Hastings, G.Z., Brown, A.L., Rowlands, D.J. and Brown, F. 1990. Immune response to peptide/hepatitis B core antigen fusion proteins. Vaccines '90, Cold Spring Harbor Laboratory, N.Y. In press.

    Google Scholar 

  27. Zanetti, M., Sercarz, E. and Salk, J. 1987. The immunology of new generation vaccines. Immunology Today 8: 18–25.

    Article  CAS  PubMed  Google Scholar 

  28. Argos, P. and Fuller, S.D. 1988. A model for hepatitis B virus core protein: prediction of antigenic sites and relationship to RNA virus capsid proteins. EMBO J 7: 819–824.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Stahl, S.J. and Murray, K. 1989. Immunogenicity of peptide fusions to Hepatitis B virus core antigen. Proc. Nat. Acad. Sci. 86: 6283–6287.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Milich, D.R., McLachlan, A., Stahl, S., Wingfield, P., Thornton, G.B., Hughes, J.L. and Jones, J.E. 1988. Comparative immunogenicity of hepatitis B virus core and e antigens. J. Immunol. 141: 3617–3624.

    CAS  PubMed  Google Scholar 

  31. Casadaban, M. and Cohen, S. 1980. Analysis of gene control signals by DNA fusion and cloning in Escherichia coli . J. Mol. Biol. 138: 179–207.

    Article  CAS  PubMed  Google Scholar 

  32. Sanger, F., Coulson, A.R., Barrell, B.G., Smith, A.J.H. and Roe, B.A. 1980. Cloning in single-standed bacteriophage as an aid to rapid DNA sequencing. J. Mol. Biol. 143: 161–178.

    Article  CAS  PubMed  Google Scholar 

  33. McLeod, M., Volkert, F. and Broach, J. 1984. Components of the site specific recombination system encoded by the yeast plasmid 2-micron circle. Cold Spring Harbor. Quant. Symp. Biol. 49: 779–787.

    Article  CAS  Google Scholar 

  34. Ito, H., Fukuda, Y., Murata, K. and Kimura, A. 1983. Transformation of intact yeast cell treated with alkali cations. J. Bact. 153: 163–168.

    CAS  PubMed  PubMed Central  Google Scholar 

  35. Sherman, F., Fink, G.R. and Lawrence, C.W. 1983. Methods in Yeast Genetics. Cold Spring Harbor, N.Y.

    Google Scholar 

  36. Laemmli, U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685.

    Article  CAS  PubMed  Google Scholar 

  37. Voller, A., Bartlett, A., Blunell, D.E., Clark, M.F. and Adams, A.N. 1976. The detection of virus by enzyme-linked immunosorbent assay (ELISA). J. Gen. Virol. 33: 165–167.

    Article  CAS  PubMed  Google Scholar 

  38. Francis, M.J. and Black, L. 1983. Antibody response in pig nasal fluid and serum followin foot-and-mouth disease infection or vaccination. J. Hygiene 91: 329–334.

    Article  CAS  Google Scholar 

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Beesley, K., Francis, M., Clarke, B. et al. Expression in Yeast of Amino-Terminal Peptide Fusions to Hepatitis B Core Antigen and Their Immunological Properties. Nat Biotechnol 8, 644–649 (1990). https://doi.org/10.1038/nbt0790-644

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