title: Hypoallergenic mutants of Ole e 1, the major olive pollen allergen, as candidates for allergy vaccines.
abstract: BACKGROUND
  The C-terminal region of Ole e 1, a major allergen from olive pollen, is a dominant IgE-reactive site and offers a target for site-directed mutagenesis to produce variants with reduced IgE-binding capability.
  
  
  OBJECTIVE
  To evaluate in vitro and in vivo the immunogenic properties of three engineered derivatives of Ole e 1.
  
  
  METHODS
  One point (Y141A) and two deletion (135Delta10 and 140Delta5) mutants were generated by site-directed mutagenesis of Ole e 1-specific cDNA and produced in Pichia pastoris. Ole e 1 mutants were analysed for IgE reactivity by ELISA using sera from olive pollen-allergic patients. Their allergenicity was also investigated in both a mouse model of allergic sensitization and in basophil activation assays. IgG1 response was assayed by immunoblotting and competitive ELISA. T cell reactivity was evaluated by proliferation assays and cytokine production in splenocyte cultures.
  
  
  RESULTS
  The 135Delta10 mutant showed the strongest reduction in the IgE-binding capability of sera from olive pollen-allergic patients. Rat basophil leukaemia assays identified the deletion mutant 135Delta10 as the variant with the lowest beta-hexosaminidase-releasing capacity. Furthermore, the same 135Delta10 mutant induced the lowest IgE levels in a BALB/c mouse model of sensitization. All Ole e 1 mutants retained their allergen-specific T cell reactivity. Immunization of mice with the mutants induced IgG1 antibodies, which cross-reacted with Ole e 1 and Ole e 1-like allergens from ash, lilac and privet pollens. The ability of the human IgE to block the binding of anti-Ole e 1 mutant-specific mouse IgG1 antibodies to natural Ole e 1 demonstrated that Ole e 1 mutants are able to induce in vivo antibodies reactive to the natural allergen.
  
  
  CONCLUSIONS
  The 135Delta10 mutant with reduced allergenicity, intact T cell reactivity and capacity to induce blocking antibodies could provide a suitable candidate vaccine for efficient and safer therapy of olive pollen allergy.
authors.affiliation: Departamento de Bioquímica y Biología Molecular, Facultad de Química, Universidad Complutense, Madrid, Spain.
authors.forename: E G
authors.initials: EG
authors.lastname: Marazuela
authors.forename: R
authors.initials: R
authors.lastname: Rodríguez
authors.forename: D
authors.initials: D
authors.lastname: Barber
authors.forename: M
authors.initials: M
authors.lastname: Villalba
authors.forename: E
authors.initials: E
authors.lastname: Batanero
mesh_terms: D000485:Allergens / Q000276:immunology*; D000818:Animals; D000937:Antigen-Antibody Reactions / Q000276:immunology*; D052179:Antigens, Plant; D006801:Humans; D051379:Mice; D023421:Models, Animal; D029592:Oleaceae / Q000276:immunology*; D010940:Plant Proteins / Q000276:immunology*; D006255:Rhinitis, Allergic, Seasonal / Q000276:immunology*; D014614:Vaccines, Synthetic / Q000096:biosynthesis / Q000276:immunology*
chemical_list: D000485:Allergens; D052179:Antigens, Plant; C081946:Ole e I protein, Olea europaea; D010940:Plant Proteins; D014614:Vaccines, Synthetic