Glutaraldehyde-Modified Recombinant Fel d 1: A Hypoallergen With Negligible Biological Activity but Retained Immunogenicity

World Allergy Organization Journal

Ronald van Ree⁴  Fatima Ferreira³  Esther C de Jong¹  Stef J Koppelman⁵  R van den Hourt⁵  Toni M van Capel¹  Martin Himly³  Ingrid Bulder⁶  Serge A Versteeg² 

[1] Department of Cell Biology and Histology, Academic Medical Center, Amsterdam, The Netherlands;Department of Experimental Immunology, Academic Medical Center, Amsterdam, The Netherlands;Department of Molecular Biology, University of Salzburg, Salzburg, Austria;Department of Otorhinolaryngology, Academic Medical Center, Amsterdam, The Netherlands;R&D Department, HAL Allergy, Leiden, The Netherlands;Department of Immunopathology, Sanquin Research, Amsterdam, The Netherlands

关键词: rFel d 1;    immunotherapy;    hypoallergen;    Felis domesticus;    allergoid;   

Others  :  1145594
DOI  :  10.1097/WOX.0b013e3182228a39

【 摘 要 】

Background

Recombinant allergens are under investigation for replacing allergen extracts in immunotherapy. Site-directed mutagenesis has been suggested as a strategy to develop hypoallergenic molecules that will reduce the risk of side effects. For decades, chemically modified allergen extracts have been used for the same reason.

Aim

To evaluate whether glutaraldehyde modification is a good strategy to produce hypoallergenic recombinant allergens with retained immunogenicity.

Methods

Fel d 1 was cloned as a single construct linking both chains of the molecule and expressed in Escherichia coli and Pichia pastoris. After physicochemical purification, recombinant (r) Fel d 1 was chemically modified using glutaraldehyde. The effect of modification on immune reactivity was evaluated using radioallergosorbent test, CAP inhibition, competitive radioimmunoassay, enzyme-linked immunosorbent assay, basophil histamine release, and T-cell proliferation assays. Both natural and recombinant unmodified Fel d 1 were used as controls.

Results

rFel d 1 demonstrated similar IgE binding and biologic activity as its natural counterpart. Upon modification, IgE-binding potency decreased >1000-fold, translating into a >10⁶-fold reduction in biologic activity assessed by basophil histamine release. In contrast, the modified recombinant did not show a decreased but even a moderately increased capacity (1.5-fold) to stimulate proliferation of T cells (P < 0.01). Finally, it induced specific IgG antibodies in rabbits that recognized the unmodified allergen.

Conclusions

Chemical modification is a practical and highly effective approach for achieving hypoallergenicity of recombinant allergens with retained immunogenicity.

【 授权许可】

   
2011 World Allergy Organization; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

Figure 4.

Figure 5.

Figure 6.

【 参考文献 】

• [1]van Ree R, van Leeuwen WA, Bulder I, Bond J, Aalberse RC: Purified natural and recombinant Fel d 1 and cat albumin in in vitro diagnostics for cat allergy. J Allergy Clin Immunol 1999, 104:1223-1230.
• [2]Kleine-Tebbe J, Kleine-Tebbe A, Jeep S, Schou C, Lowenstein H, Kunkel G: Role of the major allergen (Fel d I) in patients sensitized to cat allergens. Int Arch Allergy Immunol 1993, 100:256-262.
• [3]Ohman JL Jr, Lowell FC, Bloch KJ: Allergens of mammalian origin. III. Properties of a major feline allergen. J Immunol 1974, 113:1668-1677.
• [4]Lowenstein H, Lind P, Weeke B: Identification and clinical significance of allergenic molecules of cat origin. Part of the DAS 76 Study. Allergy 1985, 40:430-441.
• [5]Duffort O, Carreira J, Lombardero M: Monoclonal antibodies against Fel d I and other clinically relevant cat allergens. Immunol Lett 1988, 17:71-77.
• [6]de Groot H, van Swieten P, van Leeuwen J, Lind P, Aalberse RC: Monoclonal antibodies to the major feline allergen Fel d I. I. Serologic and biologic activity of affinity-purified Fel d I and of Fel d I-depleted extract. J Allergy Clin Immunol 1988, 82:778-786.
• [7]Ewbank PA, Murray J, Sanders K, Curran-Everett D, Dreskin S, Nelson HS: A double-blind, placebo-controlled immunotherapy dose-response study with standardized cat extract. J Allergy Clin Immunol 2003, 111:155-161.
• [8]Varney VA, Edwards J, Tabbah K, Brewster H, Mavroleon G, Frew AJ: Clinical efficacy of specific immunotherapy to cat dander: a double-blind placebo-controlled trial. Clin Exp Allergy 1997, 27:860-867.
• [9]Kristensen AK, Schou C, Roepstorff P: Determination of isoforms, N-linked glycan structure and disulfide bond linkages of the major cat allergen Fel d1 by a mass spectrometric approach. Biol Chem 1997, 378:899-908.
• [10]Morgenstern JP, Griffith IJ, Brauer AW, Rogers BL, Bond JF, Chapman MD, Kuo MC: Amino acid sequence of Fel dI, the major allergen of the domestic cat: protein sequence analysis and cDNA cloning. Proc Natl Acad Sci USA 1991, 88:9690-9694.
• [11]Griffith IJ, Craig S, Pollock J, Yu XB, Morgenstern JP, Rogers BL: Expression and genomic structure of the genes encoding FdI, the major allergen from the domestic cat. Gene 1992, 113:263-268.
• [12]Kaiser L, Gronlund H, Sandalova T, Ljunggren HG, van Hage-Hamsten M, Achour A, Schneider G: The crystal structure of the major cat allergen Fel d 1, a member of the secretoglobin family. J Biol Chem 2003, 278:37730-37735.
• [13]Rogers BL, Morgenstern JP, Garman RD, Bond JF, Kuo MC: Recombinant Fel d.I: Expression, purification, IgE binding and reaction with cat-allergic human T cells. Mol Immunol 1993, 30:559-568.
• [14]Slunt JB, Rogers BL, Chapman MD: IgE antibodies to recombinant forms of Fel d I: dichotomy between fluid-phase and solid-phase binding studies. J Allergy Clin Immunol 1995, 95:1221-1228.
• [15]Keating KM, Segal DB, Craig SJ, Nault AK, Semensi V, et al.: Enhanced immunoreactivity and preferential heterodimer formation of reassociated Fel d I recombinant chains. Mol Immunol 1995, 32:287-293.
• [16]Ichikawa K, Wen W, Wu Z, Vailes LD, Guyre P, Chapman MD: Fully immunoreactive recombinant cat allergen, Fel d 1, expressed in baculovirus [abstract]. J Allergy Clin Immunol 1999, 103:S184.
• [17]Vailes LD, Sun AW, Ichikawa K, Wu Z, Sulahian TH, Chapman MD, Guyre PM: High-level expression of immunoreactive recombinant cat allergen (fel d 1): Targeting to antigen-presenting cells. J Allergy Clin Immunol 2002, 110:757-762.
• [18]Kaiser L, Gronlund H, Sandalova T, Ljunggren HG, Schneider G, van Hage-Hamsten M, Achour A: Production, crystallization and preliminary crystallographic study of the major cat allergen Fel d 1. Acta Crystallogr D Biol Crystallogr 2003, 59:1103-1105.
• [19]Ferreira F, Ebner C, Kramer B, Casari G, Briza P, et al.: Modulation of IgE reactivity of allergens by site-directed mutagenesis: potential use of hypoallergenic variants for immunotherapy. FASEB J 1998, 12:231-242.
• [20]Markovic-Housley Z, Degano M, Lamba D, von Roepenack-Lahaye E, Clemens S, et al.: Crystal structure of a hypoallergenic isoform of the major birch pollen allergen Bet v 1 and its likely biological function as a plant steroid carrier. J Mol Biol 2003, 325:123-133.
• [21]Rabjohn P, West CM, Connaughton C, Sampson HA, Helm RM, Burks AW, Bannon GA: Modification of peanut allergen Ara h 3: effects on IgE binding and T cell stimulation. Int Arch Allergy Immunol 2002, 128:15-23.
• [22]Bannon GA, Cockrell G, Connaughton C, West CM, Helm R, et al.: Engineering, characterization and in vitro efficacy of the major peanut allergens for use in immunotherapy. Int Arch Allergy Immunol 2001, 124:70-72.
• [23]Akdis CA, Blaser K: Regulation of specific immune responses by chemical and structural modifications of allergens. Int Arch Allergy Immunol 2000, 121:261-269.
• [24]Norman PS, Lichtenstein LM, Marsh DG: Studies on allergoids from naturally occurring allergens. IV. Efficacy and safety of long-term allergoid treatment of ragweed hay fever. J Allergy Clin Immunol 1981, 68:460-470.
• [25]Norman PS, Lichtenstein LM, Kagey-Sobotka A, Marsh DG: Controlled evaluation of allergoid in the immunotherapy of ragweed hay fever. J Allergy Clin Immunol 1982, 70:248-260.
• [26]De Jonge J, Brissinck J, Heirman C, Demanet C, Leo O, Moser M, Thielemans K: Production and characterization of bispecific single-chain antibody fragments. Mol Immunol 1995, 32:1405-1412.
• [27]Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, et al.: Measurement of protein using bicinchoninic acid. Anal Biochem 1985, 150:76-85.
• [28]van Milligen FJ, van Swieten P, Aalberse RC: Structure of the major cat allergen Fel d I in different allergen sources: an immunoblotting analysis with monoclonal antibodies against denatured Fel d I and human IgE. Int Arch Allergy Immunol 1992, 99:63-73.
• [29]Bolhaar ST, Ree R, Bruijnzeel-Koomen CA, Knulst AC, Zuidmeer L: Allergy to jackfruit: a novel example of Bet v 1-related food allergy. Allergy 2004, 59:1187-1192.
• [30]Carnes J, Himly M, Gallego M, Iraola V, Robinson DS, Fernández-Caldas E, Briza P: Detection of allergen composition and in vivo immunogenicity of depigmented allergoids of Betula alba. Clin Exp Allergy 2009, 39:426-434.
• [31]Aalberse RC, Koshte V, Clemens JG: Immunoglobulin E antibodies that crossreact with vegetable foods, pollen, and Hymenoptera venom. J Allergy Clin Immunol 1981, 68:356-364.
• [32]van Ree R, van Leeuwen WA, van den BM, Weller HH, Aalberse RC: IgE and IgG cross-reactivity among Lol p I and Lol p II/III. Identification of the C-termini of Lol p I, II, and III as cross-reactive structures. Allergy 1994, 49:254-261.
• [33]Chapman MD, Aalberse RC, Brown MJ, Platts-Mills TA: Monoclonal antibodies to the major feline allergen Fel d I. II. Single step affinity purification of Fel d I, N-terminal sequence analysis, and development of a sensitive two-site immunoassay to assess Fel d I exposure. J Immunol 1988, 140:812-818.
• [34]Kleine Budde I, de Heer PG, van der Zee JS, Aalberse RC: The stripped basophil histamine release bioassay as a tool for the detection of allergen-specific IgE in serum. Int Arch Allergy Immunol 2001, 126:277-285.
• [35]Knol EF, Kuijpers TW, Mul FP, Roos D: Stimulation of human basophils results in homotypic aggregation. A response independent of degranulation. J Immunol 1993, 151:4926-4933.
• [36]Siraganian RP: Refinements in the automated fluorometric histamine analysis system. J Immunol Methods 1975, 7:283-290.
• [37]Akdis CA, Akdis M: Mechanisms and treatment of allergic disease in the big picture of regulatory T cells. J Allergy Clin Immunol 2009, 123:735-746.