Peptide-based strategies for the prevention and treatment of egg allergy
- Autores: Daniel Lozano
- Directores de la Tesis: Rosina López Fandiño (dir. tes.), Elena Molina Hernández (codir. tes.)
- Lectura: En la Universidad Autónoma de Madrid ( España ) en 2017
- Idioma: español
- Tribunal Calificador de la Tesis: Isidra Recio (presid.), Juan Miguel Rodríguez Gómez (secret.), Naima Gisela Cortés Pérez (voc.), Maria Dolores Ibañez Sandín (voc.), J. J. Córdoba (voc.)
- Programa de doctorado: Programa de Doctorado en Química Agrícola por la Universidad Autónoma de Madrid
- Materias:
- Enlaces
- Tesis en acceso abierto en: Biblos-e Archivo
- Resumen
Egg allergy is among the most common food allergies in European children below the age of three, with a prevalence ranging between 0.2 and 2%. The management of this allergic disease is limited to strict avoidance of the offending food, although total evasion is difficult from a practical standpoint since egg and egg-derived products are ubiquitous ingredients. In this regard, oral immunotherapy (OIT) using whole egg white (EW) appears as an effective treatment option for egg allergy, with a success rate between 50 and 90%. However, a major drawback of OIT using intact egg allergens is the high risk of severe side effects. This obstacle has prompted the investigation of strategies aimed at reducing the allergenicity of the egg-derived preparations used for immunotherapy. Among the most promising approaches currently studied, stands the possibility of using allergen-derived immunomodulating peptides to induce oral tolerance towards offending food proteins or even to prevent allergic sensitisation. In this respect, hydrolysis of egg white proteins appears as an attractive and safe alternative for a reproducible and standardized production of immune-active peptides with low associated costs.
Given this scenario, the aim of this thesis was the development of enzymatic hydrolysates of egg white proteins with hypoallergenic and immunomodulating properties that could be used in the prevention and treatment of egg allergy. With this purpose, enzymatic hydrolysates of ovalbumin (OVA), lysozyme, ovomucoid and EW were prepared using pepsin, Neutrase and alcalase. These hydrolysates were fractionated, characterised by chromatographic and mass spectrometric techniques. All the hydrolysates produced with alcalase, and that of OVA with pepsin, presented a reduced binding capacity to IgE from egg allergic patients. The hypoallergenicity of these hydrolysates was confirmed by the evaluation of the local allergic responses induced after their administration to mice passively sensitised to egg white proteins.
The immunomodulating effect of the hydrolysates was first evaluated in murine spleen and mesenteric lymph node cells stimulated with T or B cell mitogens. The results suggested that the hydrolysates produced with alcalase decreased the production of Th2-biased cytokines. In addition, these hydrolysates, together with that of OVA with pepsin, inhibited IgG1 class switching, which is also part of the IL-4 mediated Th2 response, and reduced the release of reactive oxygen species (ROS). We next assessed their ability to hinder cytokine and IgE production by Th2-skewed human peripheral blood mononuclear cells (PBMCs), as well as the release of pro-inflammatory factors and generation of ROS from Th1-stimulated peripheral blood leukocytes (PBLs). Data showed that the hydrolysates with alcalase and that of OVA with pepsin helped to re-establish the Th1/Th2 balance in Th2-biased PBMCs, while they also inhibited the release of pro-inflammatory mediators and reduced oxidative stress in PBLs treated with inflammatory stimuli. Together, these cellular models allowed us to select hydrolysates with the ability to down-modulate Th2 cytokine and IgE secretion and to attenuate inflammatory responses.
Based on these previous screening tests, we explored the possibility that the selected hypoallergenic and immunostimulating hydrolysates could modulate T cell cytokine responses to egg allergens ex vivo, using splenocytes from BALB/c mice sensitised to individual egg proteins or to their mixtures in different proportions. The obtained results revealed that OVA hydrolysed with alcalase and pepsin could be regarded as two good candidates for peptide-based immunotherapy on the grounds of their ability to reduce Th2 cytokines induced by egg allergens in the spleen cell cultures. In addition, the hydrolysate of OVA with pepsin also enhanced Th1-related responses.
In the last part of this thesis, we evaluated the sensitising and eliciting capacities of the selected hydrolysates, as well as their preventive and therapeutic ability, using a mouse model of egg allergy. Results revealed that the hydrolysate of OVA with pepsin was weakly immunogenic and lacked sensitising and eliciting capacity. For its part, in vivo testing of OVA hydrolysed with alcalase revealed immunogenic and antigenic capacities not previously detected by in vitro methods. OVA hydrolysed with pepsin offered preventive and therapeutic protection against allergy to EW, exerting a marked reduction of Th2 and Th1 systemic responses, through the induction of regulatory T cells and the up-regulation of TGF-β, IL-10 and IL-17 in intestinal tissues. This hydrolysate was more effective than intact OVA in desensitising mice by virtue of its lower allergenicity and its immunomodulating capacity.
In general terms, the screening strategy followed in this work proved useful to select a hydrolysate that combines tolerance induction with the absence of undesired effects of IgE-crosslinking and inflammatory cell activation. The results presented in this thesis show that OVA hydrolysed with pepsin is hypoallergenic and contains immunomodulating peptides which lead to the prevention of egg allergy development and the promotion of long term desensitisation to egg white proteins, as well as to linked-epitope suppression.
