Polcalcins are important respiratory panallergens, whose IgE-binding capacity depends on the presence of calcium. Since specific immunotherapy is not yet available for the treatment of polcalcin-sensitized patients, we aimed to develop a molecule for efficient and safe immunotherapy. We generated a hypoallergenic variant of the grass pollen polcalcin Phl p 7 by introducing specific point mutations into the allergen’s calcium-binding regions. We thereby followed a mutation strategy that had previously resulted in a hypoallergenic mutant of a calcium-binding food allergen, the major fish allergen parvalbumin. Dot blot assays performed with sera from Phl p 7-sensitized patients showed a drastically reduced IgE reactivity of the Phl p 7 mutant in comparison to wildtype Phl p 7, and basophil activation assays indicated a significantly reduced allergenic activity. Rabbit IgG directed against mutant rPhl p 7 blocked patients’ IgE binding to wildtype Phl p 7, indicating the mutant’s potential applicability for immunotherapy. Mass spectrometry and circular dichroism experiments showed that the mutant had lost the calcium-binding capacity, but still represented a folded protein. In silico analyses revealed that the hypoallergenicity might be due to fewer negative charges on the molecule’s surface and an increased molecular flexibility. We thus generated a hypoallergenic Phl p 7 variant that could be used for immunotherapy of polcalcin-sensitized individuals.

Background: Delayed food anaphylaxis upon consumption of red meat is attributed to specific IgE-antibodies directed to galactose-α-1,3-galactose (α-Gal). Anaphylactic reactions may occur after ingestion of meat from different mammals, mainly beef and pork, but reactions to lamb, rabbit or horse have also been reported. In particular, pork kidney has been shown to trigger symptoms that were more severe and occurred within a shorter delay. The objective of the present study was the identification and characterization of pork kidney proteins carrying α-Gal carbohydrates and mediating delayed allergic reactions through specific IgE to α-Gal. Materials and methods: A cohort of 59 patients with specific IgE to α-Gal was screened by immunoblot for IgE-reactive proteins in pork kidney extract. Proteins were purified by affinity chromatography and identified by Edman sequencing and peptide mass fingerprinting. Isolated proteins were used in immunoassays using patient sera and α-Gal specific antibodies. Allergenicity was assayed in basophil activation and skin prick test. Results: Multiple IgE-binding proteins were detected in protein extracts of pork kidney by immunoblot using patient sera and an anti-α-Gal antibody. Reactive bands were located in the high molecular weight range of 100 to ≥200 kDa. Two major IgE-binding proteins were identified as porcine angiotensin I converting enzyme (ACE I) and aminopeptidase N (AP-N). IgE-binding to both proteins was lost by periodate treatment, resulting in oxidation of carbohydrates. Addition of α-Gal inhibited IgE-reactivity to both peptidases. Allergenicity was confirmed by activation of patient basophils and positive skin prick tests. Conclusions: Two IgE-reactive cell membrane peptidases carrying α-Gal epitopes were identified in pork kidney, a tissue which is known as potent inducer of red meat-induced anaphylaxis. Allergenicity and clinical relevance of these proteins were confirmed in patients with delayed anaphylaxis to red meat by skin prick test and basophil activation.