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Vol. 145, No. 3, 2008
Issue release date: February 2008
Int Arch Allergy Immunol 2008;145:193–206
(DOI:10.1159/000109288)

Characterization of a Hypoallergenic Recombinant Bet v 1 Variant as a Candidate for Allergen-Specific Immunotherapy

Kahlert H. · Suck R. · Weber B. · Nandy A. · Wald M. · Keller W. · Cromwell O. · Fiebig H.
aAllergopharma Joachim Ganzer KG, Reinbek, Germany; bInstitute of Chemistry, Structural Biology, Graz, Austria

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Abstract

Background: Recombinant allergens and especially their hypoallergenic variants are promising candidates for a more effective and safer specific immunotherapy. Methods: Physicochemical and immunological characteristics of a folding variant of recombinant Bet v 1 (rBet v 1-FV) were investigated in comparison to natural Bet v 1 (nBet v 1) and the correctly folded recombinant Bet v 1 (rBet v 1-WT) by SDS-PAGE, size exclusion chromatography, multi-angle light scattering, circular dichroism, immunoblotting and enzyme allergosorbent test inhibition assay for detection of IgE reactivity and ELISA with Bet v 1-specific monoclonal antibodies. The functional IgE reactivity of the different Bet v 1 proteins was investigated using basophil activation in terms of CD203c expression and histamine release. T cell reactivity was investigated using T cell lines raised from birch pollen-allergic subjects against nBet v 1. Immunogenicity was investigated in mice. Results: Physicochemical characterization revealed purity, homogeneity and monomeric properties of rBet v 1-FV. Unlike nBet v 1 and rBet v 1-WT, rBet v 1-FV showed almost no IgE binding in immunoblots. The reduction of allergenicity was further proved by IgE-binding inhibition assays, basophil activation and histamine release. T cell reactivity was completely conserved, as demonstrated by proliferation of Bet v 1-specific T cell lines with multiple epitope specificities. rBet v 1-FV showed strong immunogenicity in mice. Conclusions: Due to its reduced IgE reactivity and decreased capacity to activate basophils, but retained T cell reactivity and strong immunogenicity, rBet v 1-FV proved to be a very promising candidate for specific immunotherapy in birch pollen-allergic subjects.



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References

  1. D’Amato G, Spieksma F Th M, Liccardi G, Jäger S, Russo M, Kontou-Fili K, Nikkels H, Wüthrich B, Bonini S: Pollen-related allergy in Europe. Allergy 1998;53:567–578.
  2. Bousquet J, Lockey RF, Malling HJ: Allergen immunotherapy: therapeutic vaccines for allergic diseases. A WHO position paper. J Allergy Clin Immunol 1998;102:558–562
  3. Akdis CA, Blesken T, Akdis M, Wüthrich B, Blaser K: Role of interleukin 10 in specific immunotherapy. J Clin Invest 1998;102:98–106.
  4. Akdis M, Verhagen J, Taylor A, Karamloo F, Karagiannidis C, Crameri R, Thunberg S, Deniz G, Valenta R, Fiebig H, Kegel C, Disch R, Schmidt-Weber CB, Blaser K, Akdis CA: Immune responses in healthy and allergic individuals are characterized by a fine balance between allergen-specific T regulatory 1 and T helper 2 cells. J Exp Med 2004;199:1567–1575.
  5. Jutel M, Akdis M, Budak F, Aebischer-Casaulta C, Wrzyszcz M, Blaser K, Akdis CA: IL-10 and TGF-β cooperate in regulatory T cell response to mucosal allergens in normal immunity and specific immunotherapy. Eur J Immunol 2003;33:1205–1214.
  6. Francis JN, Till SJ, Durham SR: Induction of IL-10+CD4+CD25+ T cells by grass pollen Immunotherapy. J Allergy Clin Immunol 2003;111:1255–1261.
  7. Bellinghausen I, Metz G, Enk AH, Christmann S, Knop J, Saloga J: Insect venom immunotherapy induces interleukin-10 production and a Th2-to-Th1 shift, and changes surface marker expression in venom-allergic subjects. Eur J Immunol 1997;27:1131–1139.
  8. Secrist H, Chelen CJ, Wen Y, Marshall JD, Umetsu DT: Allergen immunotherapy decreases interleukin 4 production in CD4+ T cells from allergic individuals. J Exp Med 1993;178:2123–2130.
  9. Jutel M, Pichler WJ, Skrbic D, Urwyler A, Dahinden C, Müller UR: Bee venom immunotherapy results in decrease of IL-4 and IL-5 and increase of IFN-γ secretion in specific allergen-stimulated T cell cultures. J Immunol 1995;154:4187–4194.
  10. Klimek L, Dormann D, Jarman ER, Cromwell O, Riechelmann H, Reske-Kunz AB: Short-term preseasonal birch pollen allergoid immunotherapy influences symptoms, specific nasal provocation and cytokine levels in nasal secretions, but not peripheral T-cell responses, in patients with allergic rhinitis. Clin Exp Allergy 1999;29:1326–1335.
  11. Durham SR, Ying S, Varney VA, Jacobson MR, Sudderick RM, Mackay IS, Kay AB, Hamid OA: Grass pollen immunotherapy inhibits allergen-induced infiltration of CD4+ T lymphocytes and eosinophils in the nasal mucosa and increases the number of cells expressing messenger RNA for interferon-γ. J Allergy Clin Immunol 1996;97:1356–1365.
  12. Puggioni F, Durham SR, Francis JN: Monophosphoryl lipid A (MPL®) promotes allergen-induced immune deviation in favour of Th1 responses. Allergy 2005;60:678–684.
  13. Wachholz PA, Soni NK, Till SJ, Durham SR: Inhibition of allergen-IgE binding to B cells by IgG antibodies after grass pollen immunotherapy. J Allergy Clin Immunol 2003;112:915–922.
  14. Flicker S, Valenta R: Renaissance of the blocking antibody concept in type I allergy. Int Arch Allergy Immunol 2003;132:13–24.
  15. Van Neerven RJ, Wikborg T, Lund G, Jacobsen B, Brinch-Nielsen A, Arnved J, Ipsen H: Blocking antibodies induced by specific allergy vaccination prevent the activation of CD4+ T cells by inhibiting serum-IgE-facilitated allergen presentation. J Immunol 1999;163:2944–2952.
  16. Valenta R, Breiteneder H, Pettenburger K, Breitenbach M, Rumpold H, Kraft D, Scheiner O: Homology of the major birch-pollen allergen, Bet v 1, with the major pollen allergens of alder, hazel, and hornbeam at the nucleic acid level as determined by cross-hybridization. J Allergy Clin Immunol 1991;87:677–682.
  17. Jarolim E, Rumpold H, Endler AT, Ebner H, Breitenbach M, Scheiner O, Kraft D: IgE and IgG antibodies of patients with allergy to birch as tools to define the allergen profile of Betula verrucosa. Allergy 1989;44:385–395.
  18. Batard T, Didierlaurent A, Chabre H, Mothes N, Bussières L, Bohle B, Couret MN, Ball T, Lemoine P, Tejkl MF, Chenal A, Clément G, Dupont F, Valent P, Krauth M-T, André C, Valenta R, Moingeon P: Characterization of wild-type recombinant Bet v 1a as a candidate vaccine against birch pollen allergy. Int Arch Allergy Immunol 2005;136:239–249.
  19. Breiteneder H, Pettenburger K, Bito A, Valenta R, Kraft D, Rumpold H, Scheiner O, Breitenbach M: The gene coding for the major birch pollen allergen Bet v 1 is highly homologous to a pea disease resistance response gene. EMBO J 1989;8:1935–1938.
  20. Ferreira F, Hoffmann-Sommergruber K, Breiteneder H, Pettenburger K, Ebner C, Sommergruber W, Steiner R, Bohle B, Sperr ER, Valent P, Kungl AJ, Breitenbach M, Kraft D, Scheiner O: Purification and characterization of recombinant Bet v 1, the major birch pollen allergen. J Biol Chem 1993;268:19574–19580.
  21. Vrtala S, Hirtenlehner K, Vangelista L, Pastore A, Eichler H-G, Sperr WR, Valent P, Ebner C, Kraft D, Valenta R: Conversion of the major birch pollen allergen, Bet v 1, into two nonanaphylactic T cell epitope-containing fragments. J Clin Invest 1997;99:1673–1681.
  22. Vrtala S, Hirtenlehner K, Susani M, Hufnagl P, Binder BR, Vangelista L, Pastore A, Sperr WR, Valent P, Ebner C, Kraft D, Valenta R: Genetic engineering of recombinant hypoallergenic oligomers of the major birch pollen allergen, Bet v 1: candidates for specific immunotheray. Int Arch Allergy Immunol 1999;118:218–219.
  23. Ferreira F, Ebner C, Kramer B, Casari G, Briza P, Kungl AJ, Grimm R, Jahn-Schmid B, Breiteneder H, Kraft D, Breitenbach M, Rheinberger H-J, Scheiner O: Modulation of IgE reactivity of allergens by site-directed mutagenesis: potential use of hypoallergenic variants for immunotherapy. FASEB J 1998;12:231–242.
  24. Spangfort MD, Mirza O, Ipsen H, van Neerven RJ, Gajhede M, Larsen JN: Dominating IgE-binding epitope of Bet v 1, the major allergen of birch pollen, characterized by X-ray crystallography and site-directed mutagenesis. J Immunol 2003;171:3084–3090.
  25. Holm J, Gajhede M, Ferreras M, Henriksen M, Henriksen A, Ipsen H, Larsen JN, Lund L, Jacobi H, Millner A, Würtzen PA, Spangfort MD: Allergy vaccine engineering: epitope modulation of recombinant Bet v 1 reduces IgE binding but retains protein folding pattern for induction of protective blocking-antibody responses. J Immunol 2004;173:5258–5267.
  26. Schramm G, Kahlert H, Suck R, Weber B, Stüwe H-T, Müller W-D, Bufe A, Becker W-M, Schlaak MW, Jäger L, Cromwell O, Fiebig H: ‘Allergen engineering’: variants of the timothy grass pollen allergen Phl p 5b with reduced IgE-binding capacity but conserved T-cell reactivity. J Immunol 1999;162:2406–2414.
  27. Ebner C, Szépfalusi Z, Ferreira F, Jilek A, Valenta R, Parronchi P, Maggi E, Romagnani S, Scheiner O, Kraft D: Identification of multiple T cell epitopes on Bet v 1, the major birch pollen allergen, using specific T cell clones and overlapping peptides. J Immunol 1993;150:1047–1054.
  28. Ebner C, Schenk S, Szépfalusi Z, Hoffmann K, Ferreira F, Willheim M, Scheiner O, Kraft D: Multiple T cell specificities for Bet v 1, the major birch pollen allergen, within single individuals. Studies using specific T cell clones and overlapping peptides. Eur J Immunol 1993;23:1523–1527.
  29. Dormann D, Montermann E, Klimek L, Weber B, Ebner C, Valenta R, Kraft D, Reske-Kunz AB: Heterogeneity in the polyclonal T cell response to birch pollen allergens. Int Arch Allergy Immunol 1997;114:272–277.
  30. Van Neerven RJJ, Sparholt SH, Schou C, Larsen JN: Preserved epitope-specific T cell activation by recombinant Bet v 1-MBP fusion proteins. Clin Exp Allergy 1998;28:423–433.
  31. Jahn-Schmid B, Radakovics A, Lüttkopf D, Scheurer S, Vieths S, Ebner C, Bohle B: Bet v 1 142–156 is the dominant T-cell epitope of the major birch pollen allergen and important for cross-reactivity with Bet v 1-related food allergens. J Allergy Clin Immunol 2005;116:213–219.
  32. Swoboda I, Jilek A, Ferreira F, Engel E, Hoffmann-Sommergruber K, Scheiner O, Kraft D, Breiteneder H, Pittenauer E, Schmid E, Vicente O, Heberle-Bors E, Ahorn H, Breitenbach M: Isoforms of Bet v 1, the major birch pollen allergen, analyzed by liquid chromatography, mass spectrometry, and cDNA cloning. J Biol Chem 1995;270:2607–2613
  33. Ferreira F, Hirtenlehner K, Jilek A, Godnik-Cvar J, Breiteneder H, Grimm R, Hoffmann-Sommergruber K, Scheiner O, Kraft D, Breitenbach M, Rheinberger HJ, Ebner C: Dissection of immunoglobulin E and T lymphocyte reactivity of isoforms of the major birch pollen allergen Bet v 1: potential use of hypoallergenic isoforms for immunotherapy. J Exp Med 1996;183:599–609.
  34. Arquint O, Helbing A, Crameri R, Ferreira F, Breitenbach M, Pichler J: Reduced in vivo allergenicity of Bet v 1d isoform, a natural component of birch pollen. J Allergy Clin Immunol 1999;104:1239–1243.
  35. Weber B, Slamal H, Suck R: Size exclusion chromatography as a tool for quality control of recombinant allergens and hypoallergenic variants. J Biochem Biophys Methods 2003;56:219–232.
  36. Breiteneder H, Pettenburger K, Bito A, Valenta R, Kraft D, Rumpold H, Scheiner O, Breitenbach M: The gene coding for the major birch pollen allergen Bet v 1, is highly homologous to a pea disease resistance response gene. EMBO J 1989;8:1935–1938.
  37. Deleage G, Geourjon C: An interactive graphic program for calculating the secondary structure content of proteins from circular dichroism spectrum. Comput Appl Biosci 1993;2:197–199.
  38. Greenfield D, Fasmann GD: Computed circular dichroism spectra for the calculation of protein conformation. Biochemistry 1969;8:4108–4116.
  39. Wahlund KG, Giddings JC: Properties of an asymmetrical flow field-flow fractionation channel having one permeable wall. Anal Chem 1987;59:1332–1339.
  40. Litzén A: Separation speed, retention, and dispersion in asymmetrical-flow field-flow fractionation as functions of channel dimensions and flow rate. Anal Chem 1993;65:461–470.
  41. Kahlert H, Weber B, Teppke M, Wahl R, Cromwell O, Fiebig H: Characterization of major allergens of Parietaria officinales. Int Arch Allergy Immunol 1996;109:141–149.
  42. Weber B, Fiebig H, Cromwell O: Quantification of major allergens as part of the quality control of commercially produced allergen extracts. Allergologie 1998;21:116–124.

    External Resources

  43. Suck R, Weber B, Kahlert H, Hagen S, Cromwell O, Fiebig H: Purification and immunobiochemical characterization of folding variants of the recombinant major wasp allergen Ver v 5 (antigen 5). Int Arch Allergy Immunol 2000;121:284–291.
  44. Suck R, Nandy A, Weber B, Stock M, Fiebig H, Cromwell O: Rapid method for arrayed investigation of IgE-reactivity profiles using natural and recombinant allergens. Allergy 2002;57:821–824
  45. Kahlert H, Cromwell O, Fiebig H: Measurement of basophil-activating capacity of grass pollen allergens, allergoids and hypoallergenic recombinant derivatives by flow cytometry using anti-CD203c. Clin Exp Allergy 2003;33:1266–1272.
  46. Kahlert H, Weber B, Cromwell O, Fiebig H: Evaluation of the allergenicity of hypoallergenic recombinant derivatives of Bet v 1 using basophil activation by CD203c expression measurement; in Marone G (ed): Clinical Immunology and Allergy in Medicine. Naples, JGC Editions, 2003, pp 735–740.
  47. Kahlert H, Stüwe H-T, Cromwell O, Fiebig H: Reactivity of T cells with grass pollen allergen extract and allergoid. Int Arch Allergy Immunol 1999;120:146–157.
  48. Wilkins DK, Grimshaw SB, Receveur V, Dobson CM, Jones JA, Smith LJ: Hydrodynamic radii of native and denatured proteins measured by pulse field gradient NMR techniques. Biochemistry 1999;38:16424–16431.
  49. Johansen P, Senti G, Martínez Gómez JM, Wüthrich B, Bot A, Kündig TM: Heat denaturation, a simple method to improve the immunotherapeutic potential of allergens. Eur J Immunol 2005;35:3591–3598.
  50. Bohle B, Zwölfer B, Heratizadeh A, Jahn-Schmid B, Antonia YD, Alter M, Keller W, Zuidmeer L, van Ree R, Werfel T, Ebner C: Cooking birch pollen-related food: Divergent consequences for IgE- and T-cell-mediated reactivity in vitro and in vivo. J Allery Clin Immunol 2006;118:242–249.
  51. Kahlert H, Suck R, Hagen S, Vrtala S, Valenta R, Weber B, Cromwell O, Fiebig H: T cell reactivity of recombinant hypoallergenic Bet v 1 derivatives. Allergy 2001;56(suppl 68):82.


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