Fusarium culmorum causes strong degradation of pollen allergens in extract mixtures

Abstract

Background: Therapeutic allergen extracts are frequently stored as mixtures to provide preparations used for specific immunotherapy. Substantial protease activity, found in certain mold extracts, has been suspected to cause a loss of allergenic activity as a result of self-degradation, as well as by means of degradation of allergens from pollen and other allergenic sources. Objective: This study was performed to address possible deterioration of individual pollen allergens during storage of extract mixtures, with a mold extract as the source of proteolytic activity. Methods: Aqueous birch and timothy pollen extracts were stored for 60 days at 6°C with and without addition of an extract derived from the mold Fusarium culmorum. The stability of the pollen allergens Bet v 1, Bet v 6, Phl p 1, and Phl p 5, as well as 2 to-date-undefined F culmorum allergens was examined by using immunoblotting analysis with sera from allergic patients and allergen-specific mAbs. Furthermore, the residual allergenic activity of the pollen extracts was monitored by using the rat basophilic leukemia cell-mediator release assay. Proteolytic activity of extracts was determined by using a commercial protease assay and gelatinase zymography. Results: Pollen extracts were very stable, corresponding to the low proteolytic activity of these extracts. In contrast, high proteolytic activity was found for the F culmorum extract, resulting in self-degradation of mold proteins and deactivation of allergens. Similarly, the mixtures showed a strong decrease of allergenic potency in the mediator release assay. Bet v 1 and Phl p 1 were relatively stable, whereas Bet v 6 and Phl p 5 were almost entirely degraded within 1 day. Conclusions: Proteases of the mold F culmorum clearly affected the overall allergenic activity of pollen extracts within a short time period. Apart from general objections against the use of mixtures of non-cross-reacting allergens, mixing of pollen extracts with extracts derived from molds for immunotherapy is not recommended unless they are applied directly after preparation of the mixture.