Hypoallergenic mutants of the Timothy grass pollen allergen Phl p 5 generated by proline mutations

Int Arch Allergy Immunol. 2012;159(2):130-42. doi: 10.1159/000336651. Epub 2012 May 30.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adult
  • Aged
  • Allergens / genetics*
  • Allergens / immunology*
  • Amino Acid Substitution
  • Basophils / immunology
  • Desensitization, Immunologic / methods
  • Female
  • Humans
  • Immunoglobulin E / metabolism
  • In Vitro Techniques
  • Male
  • Middle Aged
  • Models, Molecular
  • Mutagenesis, Site-Directed
  • Mutant Proteins / chemistry
  • Mutant Proteins / genetics
  • Mutant Proteins / immunology
  • Phleum / genetics
  • Phleum / immunology
  • Plant Proteins / chemistry
  • Plant Proteins / genetics*
  • Plant Proteins / immunology*
  • Pollen / genetics*
  • Pollen / immunology*
  • Proline / genetics
  • Protein Multimerization
  • Protein Structure, Secondary
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / immunology
  • Rhinitis, Allergic, Seasonal / immunology
  • Rhinitis, Allergic, Seasonal / therapy
  • Sequence Deletion
  • Solubility
  • T-Lymphocytes / immunology


  • Allergens
  • Mutant Proteins
  • Phl p V protein, Phleum pratense
  • Plant Proteins
  • Recombinant Proteins
  • Immunoglobulin E
  • Proline