Bases moleculares de la reactividad cruzada en alérgenos de origen vegetal

Abstract

Allergies have been becoming considerably more widespread since the middle of the last century, but the underlying causes of this increase are still unknown. The knowledge of the mechanism by which a protein that is, in principle, harmless to the human body becomes capable of inducing an allergic response provides us with a basis to prevent and treat these diseases. So far, the only treatment applied in food allergy is the avoidance of consumption of the allergenic source. However, the existence of cross-reactivity among allergens and specific sensitization profiles makes it difficult to predict which foods are related and, therefore, should be avoided by the patient. For the treatment of some allergies, such as bee venom, immunotherapy is yielding good results. Nevertheless, in food allergy, its use is not widespread because of the potential side effects that it may induce, such as anaphylactic shock, arising from the use of non-standardized extracts. To develop safe and effective immunotherapy, it is necessary to characterize the allergens involved at the molecular and immunological levels. LTPs and profilins are two important panallergen families, especially in southern Europe. They are responsible for cross-reactivity among different foods and/or pollen. Within these families, the peach LTP, Pru p 3, and the melon profilin, Cuc m 2, have been characterized as major allergens. The objective of this thesis was to study the molecular basis of the allergic reaction to these proteins in order to identify possible mechanisms that mediate sensitization and cross-reactivity, and to develop safe molecules for generating new strategies in immunotherapy. To this end, the relationship between Pru p 3 and Ole e 7 and Par j 1, LTPs and pollen allergens have been studied to determine whether they are involved in the `LTP syndrome¿. On the other hand, Pru p 3-T and B-cell epitopes, and others involved in cross-reactivity between this allergen and Tri a 14, the wheat LTP, have been identified. Based on these results, hypoallergenic mutants of Pru p 3 have been designed and produced. Their allergenic capacity has been characterized in vitro and in vivo, as has their immunogenic capacity to stimulate specific T-cell lines. Preliminary studies have been performed to study the interaction of Pru p 3 with intestinal epithelium and identify the mechanism of sensitization to LTPs. The expression of Pru p 3 gene in the different tissues of peach flower has been determined by quantitative PCR to clarify its role in the plant, and the connection with its allergenic capacity. On the other hand, conformational B-cell epitopes of Cuc m 2, which are possibly involved in cross-reactivity among profilins, have been identified, and hypoallergenic mutants of this allergen have been developed. These mutants were characterized in vitro by ELISA assays, ex vivo by basophil activation tests, and in vivo by skin prick tests. The findings presented here provide us with a better understanding of the molecular basis of cross-reactivity in plants, and the mechanism by which allergens can trigger an allergic response.