Abstract
The cross-linking of effector cell-bound IgE antibodies by allergens induces the release of inflammatory mediators which are responsible for the symptoms of allergy. We demonstrate that a recombinant hybrid molecule consisting of the major birch (Bet v 1) and grass (Phl p 5) pollen allergen exhibited reduced allergenic activity as compared to equimolar mixes of the isolated allergens in basophil activation experiments. The reduced allergenic activity of the hybrid was not due to reduced IgE reactivity as demonstrated by IgE binding experiments using sera from allergic patients. Physicochemical characterization of the hybrid by size exclusion chromatography, dynamic light scattering, negative-stain electron microscopy and circular dichroism showed that the hybrid occurred as folded aggregate whereas the isolated allergens were folded monomeric proteins. IgG antibodies raised in rabbits against epitopes of Bet v 1 and Phl p 5 showed reduced reactivity with the hybrid compared to the monomeric allergens. Our results thus demonstrate that aggregation can induce changes in the conformation of allergens and lead to the reduction of allergenic activity. This is a new mechanism for reducing the allergenic activity of allergens which may be important for modifying allergens to exhibit reduced side effects when used for allergen-specific immunotherapy.
Highlights
Bet v 1 and Phl p 5 have been produced as recombinant reference allergens for the standardization of allergen extracts[11]
1 hybrid molecule consisting of the complete mature Phl p 5a sequence fused to the Bet v 1a sequence without linker was expressed as C-terminally hexahistidine-tagged protein in BL21 E. coli (Fig. 1a)
It is of great interest to develop molecularly defined forms of allergen-specific immunotherapy (AIT) which are based on recombinant hypoallergenic molecules which exhibit reduced allergenic activity
Summary
Bet v 1 and Phl p 5 have been produced as recombinant reference allergens for the standardization of allergen extracts[11]. Almost all recombinant Bet v 1 or Phl p 5 hypoallergenic derivatives are characterized by a reduction of the IgE binding capacity compared to the corresponding wild-type allergens[18,19]. These recombinant hypoallergens are similar to denatured allergen extracts obtained by chemical treatment (i.e., allergoids) which represent high molecular mass aggregates with reduced IgE reactivity[20]. Much to our surprise the Phl p 5-Bet v 1 hybrid formed high molecular aggregates similar to the Bet v 1 trimer, that showed increased IgE reactivity but reduced allergenic activity. The biochemical, biophysical and immunological characterization of the Phl p 5-Bet v 1 hybrid is reported in this study
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