Background: Phl p 5 is a major allergen of Timothy grass (Phleum pratense). A recombinant native Phl p 5 has already been used in clinical trials of allergen-specific immunotherapy as a component of a cocktail of allergens. Recombinant hypoallergenic allergens should further improve the treatment by reducing the risk of anaphylactic reactions at an increased therapeutic dosage. Native Phl p 5 is formed by α-helical regions separated by regions containing prolines. In order to generate hypoallergenic mutants, we studied the effect of proline mutations in single and multiple regions. Methods: All mutants were analyzed by IgE inhibition assays and size exclusion chromatography with on-line mass determination. Selected mutants were additionally analyzed by field-flow fractionation, dynamic light scattering, circular dichroism spectroscopy, basophil activation and T-cell proliferation assays. Results: Variants lacking prolines in a single region were obtained as soluble monomers. Six of eight molecules showed a slightly reduced IgE-binding capacity. Mutants carrying proline deletions in multiple regions formed monomers, dimers or insoluble aggregates. The mutant MPV.7 with five proline deletions and a substitution of proline 211 to leucine is monomeric, shows a strongly diminished IgE binding and maintains T-cell reactivity. The hydrodynamic radius and the content of the α-helical structure of MPV.7 are well comparable with the wild-type allergen. Conclusions: The hypoallergenic Phl p 5 variant MPV.7 combines multiple proline deletions with a substitution of proline 211 to leucine and meets basic demands for a pharmaceutical application. MPV.7 is a promising candidate for grass pollen immunotherapy with a cocktail of recombinant hypoallergens.

1.
Andersson K, Lidholm J: Characteristics and immunobiology of grass pollen allergens. Int Arch Allergy Immunol 2003;130:87–107.
2.
Lorenz AR, Lüttkopf D, May S, Scheurer S, Vieths S: The principle of homologous groups in regulatory affairs of allergen products – a proposal. Int Arch Immunol 2009;148:1–17.
3.
Hrabina M, Peltre G, Van Ree R, Moingeon P: Grass pollen allergens. Clin Exp Allergy Rev 2008;8:7–11.
4.
Rossi RE, Monasterolo G, Operti D, Lucchese S, Operti R: Distribution of specific serum IgE to recombinant pollen allergens (rPhlp1, rPhlp2, rPhlp5 and rBetv2) and their relationship to each other and to their natural counterparts in patients allergic to grass pollen. Allergy Int 2000;49:93–97.
5.
Vrtala S, Sperr WR, Reimitzer I, van Ree R, Laffer S, Müller WD, et al: cDNA cloning of a major allergen from timothy grass (Phleum pratense) pollen; characterization of the recombinant Phl pV allergen. J Immunol 1993;151:4773–81.
6.
Bufe A, Schramm G, Keown MB, Schlaak M, Becker WM: Major allergen Phl p Vb in timothy grass is a novel pollen RNase. FEBS Lett 1995;363:6–12.
7.
Van Ree R, Chapman MD, Ferreira F, Vieths S, Bryan D, Cromwell O, et al: The CREATE project: development of certified reference materials for allergenic products and validation of methods for their quantification. Allergy 2008;63:310–326.
8.
Chapman MD, Ferreira F, Villalba M, Cromwell O, Bryan D, Becker WM, Fernández-Rivas M, Durham S, Vieths S, van Ree R: The European Union CREATE project: a model for international standardization of allergy diagnostics and vaccines. J Allergy Clin Immunol 2008;122:882–889.
9.
Jutel M, Jaeger L, Suck R, Meyer H, Fiebig H, Cromwell O: Allergen-specific immunotherapy with recombinant grass pollen allergens. J Allergy Clin Immunol 2005;116:608–613.
10.
Kaul S, Englert L, May S, Vieths S: Regulatory aspects of specific immunotherapy in Europe. Curr Opin Allergy Clin Immunol 2010;10:594–602.
11.
Valenta R, Linhart B, Swoboda I, Niederberger V: Recombinant allergens for allergen-specific immunotherapy: 10 years anniversary of immunotherapy with recombinant allergens. Allergy 2011;66:775–783.
12.
Valenta R, Niederberger V: Recombinant allergens for immunotherapy. J Allergy Clin Immunol 2007;119:826–830.
13.
Akdis M, Akdis CA: Mechanisms of allergen-specific immunotherapy. J Allergy Clin Immunol 2007;119:780–789.
14.
James LK, Durham SR: Update on mechanisms of allergen injection immunotherapy. Clin Exp Allergy 2008;38:1074–1088.
15.
Akdis CA, Akdis MA: Mechanisms and treatment of allergic disease in the big picture of regulatory T cells. J Allergy Clin Immunol 2009;123:735–746.
16.
Mutschlechner S, Deifl S, Bohle B: Genetic allergen modification in the development of novel approaches to specific immunotherapy. Clin Exp Allergy 2009;39:1635–1642.
17.
Cromwell O, Suck R, Kahlert H, Nandy A, Weber B, Fiebig H: Transition of recombinant allergens from bench to clinical application. Methods 2004;32:300–312.
18.
Cromwell O, Häfner D, Nandy A: Recombinant allergens for specific immunotherapy. J Allergy Clin Immunol 2011;127:865–872.
19.
Schramm G, Kahlert H, Suck R, Weber B, Stüwe HT, Müller WD, Bufe A, Becker WM, Schlaak MW, Jäger L, Cromwell O, Fiebig H: ‘Allergen engeneering’: 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.
20.
Thalhamer T, Dobias H, Stepanoska T, Pröll M, Stutz H, Dissertori O, Lackner P, Ferreira F, Wallner M, Thalhamer J, Hartl A: Designing hypoallergenic derivatives for allergy treatment by means of in silico mutation and screening. J Allergy Clin Immunol 2010;125:926–934.
21.
Gehlhar K, Rajashankar KR, Hofmann E, Betzel C, Weber W, Werner S, Bufe A: Lysine as a critical amino acid for IgE binding in Phl p 5b C terminus. Int Arch Immunol 2006;140:285–294.
22.
Wald M, Kahlert H, Weber B, Jankovic M, Keller W, Cromwell O, Fiebig H: Generation of a low IgE-binding mutant of the timothy grass pollen major allergen Phl p 5a. Clin Exp Allergy 2007;37:441–450.
23.
Chou PY, Fasman GD: Conformational parameters for amino acids in helical, beta-sheet, and random coil regions calculated from proteins. Biochemistry 1974;13:211–222.
24.
Chou PY, Fasman GD: Empirical predictions of protein conformation. Annu Rev Biochem 1978;47:251–257.
25.
Richardson JS, Richardson DC: Amino acid preferences for specific locations at the ends of alpha helices. Science 1988;240:1648–1652.
26.
Kim MK, Kang YK: Positional preference of proline in alpha-helices. Protein Sci 1999;8:1492–1499.
27.
Li SC, Goto NK, Williams KA, Deber CM: Alpha-helical, but not beta-sheet, propensity of proline is determined by peptide environment. Proc Natl Acad Sci USA 1996;93:6676–6681.
28.
Hantusch B, Krieger S, Untersmayr E, Schöll I, Knittelfelder R, Flicker S, Spitzauer S, Valenta R, Boltz-Nitulescu G, Scheiner O, Jensen-Jarolim E: Mapping of conformational IgE epitopes on Phl p 5a by using mimotopes from a phage display library. J Allergy Clin Immunol 2004;114:1294–1300.
29.
Schramm G, Bufe A, Petersen A, Haas H, Merget R, Schlaak M, Becker WM: Discontinuous IgE-binding epitopes contain multiple continuous epitope regions: results of an epitope mapping on recombinant Hol l 5, a major allergen from velvet grass pollen. Clin Exp Allergy 2001;1:331–341.
30.
Flicker S, Schirmer T, Madritsch C, Randow S, Reese G, Vieths S, Lupinek C, Ebner C, Valenta R, Markovic-Housley Z: High-affinity IgE recognition of a conformational epitope of the major respiratory allergen Phl p 2 as revealed by X-ray crystallography. J Immunol 2009;182:2141–2151.
31.
Flicker S, Steinberger P, Ball T, Krauth MT, Verdino P, Valent P, Almo S, Valenta R: Spatial clustering of the IgE epitopes on the major timothy grass pollen allergen Phl p 1: importance for allergenic activity. J Allergy Clin Immunol 2006;117:1336–1343.
32.
Ge L, Rudolph P: Simultaneous introduction of multiple mutations using overlap extension PCR. Biotechniques 1997;22:28–30.
33.
Wyatt PJ: Light scattering and the absolute characterization of macromolecules. Anal Chim Acta 1993;272:l-40.
34.
Zimm B: The scattering of light and the radial distribution function of high-polymer solutions. J Chem Phys 1948;16:1093–1099.
35.
Folta-Stogniew E, Williams KR: Determination of molecular masses of proteins in solution: implementation of an HPLC size exclusion chromatography and laser light scattering service in a core laboratory. J Biomol Tech 1999;10:51–63.
36.
Wahlund KG, Giddings JC: Properties of an asymmetrical flow field-flow fractionation channel having one permeable wall. Anal Chem 1987;59:1332–1339.
37.
Kahlert H, Cromwell O, Fiebig H: Measurement of basophil-activating capacity of grass pollen allergens, allergoids and hypoallergenic recombinant derivates by flow cytometry using anti-CD203c. Clin Exp Allergy 2003;33:1266–1272.
38.
Kahlert H: Production of T-cell lines; in Jones MG, Lympany P (eds): Methods in Molecular Medicine: Allergy Methods and Protocols. Totowa, Humana Press, 2008.
39.
Sreerama N, Woody RW: Estimation of protein secondary structure from circular dichroism spectra: comparison of CONTIN, SELCON, and CDSSTR methods with an expanded reference set. Anal Biochem 2000;287:252–260.
40.
Whitmore L, Wallace BA: Protein secondary structure analyses from circular dichroism spectroscopy: methods and reference databases. Biopolymers 2008;89:392–400.
41.
Pauli G, Larsen TH, Rak S, Horak F, Pastorello E, Valenta R, et al: Efficacy of recombinant birch pollen vaccine for the treatment of birch-allergic rhinoconjunctivitis. J Allergy Clin Immunol 2008;122:951–960.
42.
Bonura A, Colombo P: Genetic engineering of allergens for immunotherapy. Inflamm Allergy Drug Targets 2009;8:104–109.
43.
Kahlert H, Suck R, Weber B, Nandy A, Wald M, Keller W, et al: Characterisation of a hypoallergenic recombinant Bet v 1 variant as a candidate for allergen-specific immunotherapy. Int Arch Allergy Immunol 2008;145:193–206.
44.
Rajashankar H, Bufe A, Weber W, Eschenburg S, Lindner B, Betzel C: Structure of the functional domain of the major grass-pollen allergen Phl p 5b. Acta Crystallogr D Biol Christallogr 2002;58:1175–1181.
45.
Maglio O, Saldanha JW, Vrtala S, Spitzauer S, Valenta R, Pastore A: A major IgE epitope-containing grass pollen allergen domain from Phl p 5 folds as a four-helix bundle. Protein Eng 2002;15:635–642.
46.
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.
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