Cow’s milk is one of the most important and basic nutrients introduced early in life in our diet but can induce IgE-associated allergy. IgE-associated allergy to cow’s milk can cause severe allergic manifestations in the gut, skin and even in the respiratory tract and may lead to life-threatening anaphylactic shock due to the stability of certain cow’s milk allergens. Here, we provide an overview about the allergen molecules in cow’s milk and the advantages of the molecular diagnosis of IgE sensitization to cow’s milk by serology. In addition, we review current strategies for prevention and treatment of cow’s milk allergy and discuss how they could be improved in the future by innovative molecular approaches that are based on defined recombinant allergens, recombinant hypoallergenic allergen derivatives and synthetic peptides.

The FAST project (Food Allergy Specific Immunotherapy) aims at the development of safe and effective treatment of food allergies, targeting prevalent, persistent and severe allergy to fish and peach. Classical allergen‐specific immunotherapy (SIT), using subcutaneous injections with aqueous food extracts may be effective but has proven to be accompanied by too many anaphylactic side‐effects. FAST aims to develop a safe alternative by replacing food extracts with hypoallergenic recombinant major allergens as the active ingredients of SIT. Both severe fish and peach allergy are caused by a single major allergen, parvalbumin (Cyp c 1) and lipid transfer protein (Pru p 3), respectively. Two approaches are being evaluated for achieving hypoallergenicity, i.e. site‐directed mutagenesis and chemical modification. The most promising hypoallergens will be produced under GMP conditions. After pre‐clinical testing (toxicology testing and efficacy in mouse models), SCIT with alum‐absorbed hypoallergens will be evaluated in phase I/II^a and II^b randomized double‐blind placebo‐controlled (DBPC) clinical trials, with the DBPC food challenge as primary read‐out. To understand the underlying immune mechanisms in depth serological and cellular immune analyses will be performed, allowing identification of novel biomarkers for monitoring treatment efficacy. FAST aims at improving the quality of life of food allergic patients by providing a safe and effective treatment that will significantly lower their threshold for fish or peach intake, thereby decreasing their anxiety and dependence on rescue medication.

Fish is a frequent elicitor of severe IgE-mediated allergic reactions. Beside avoidance, there is currently no allergen-specific therapy available. Hypoallergenic variants of the major fish allergen, parvalbumin, for specific immunotherapy based on mutation of the 2 calcium-binding sites have been developed. This study sought to establish a mouse model of fish allergy resembling human disease and to investigate whether mouse and rabbit IgG antibodies induced by immunization with a hypoallergenic mutant of the major carp allergen protect against allergic symptoms in sensitized mice. C3H/HeJ mice were sensitized with recombinant wildtype Cyp c 1 or carp extract by intragastric gavage. Antibody, cellular immune responses, and epitope specificity in sensitized mice were investigated by ELISA, rat basophil leukemia assay, T-cell proliferation experiments using recombinant wildtype Cyp c 1, and overlapping peptides spanning the Cyp c 1 sequence. Anti-hypoallergenic Cyp c 1 mutant mouse and rabbit sera were tested for their ability to inhibit IgE recognition of Cyp c 1, Cyp c 1-specific basophil degranulation, and Cyp c 1-induced allergic symptoms in the mouse model. A mouse model of fish allergy mimicking human disease regarding IgE epitope recognition and symptoms as close as possible was established. Administration of antisera generated in mice and rabbits by immunization with a hypoallergenic Cyp c 1 mutant inhibited IgE binding to Cyp c 1, Cyp c 1-induced basophil degranulation, and allergic symptoms caused by allergen challenge in sensitized mice. Antibodies induced by immunization with a hypoallergenic Cyp c 1 mutant protect against allergic reactions in a murine model of fish allergy.

Summary: Background: Vaccines consisting of allergen‐derived peptides lacking IgE reactivity and allergen‐specific T cell epitopes bound to allergen‐unrelated carrier molecules have been suggested as candidates for allergen‐specific immunotherapy.Objective: To study whether prophylactic and therapeutic vaccination with carrier‐bound peptides from the major birch pollen allergen Bet v 1 lacking allergen‐specific T cell epitopes has influence on Bet v 1‐specific T cell responses.Methods: Three Bet v 1‐derived peptides, devoid of Bet v 1‐specific T cell epitopes, were coupled to KLH and adsorbed to aluminium hydroxide to obtain a Bet v 1‐specific allergy vaccine. Groups of BALB/c mice were immunized with the peptide vaccine before or after sensitization to Bet v 1. Bet v 1‐ and peptide‐specific antibody responses were analysed by ELISA. T cell and cytokine responses to Bet v 1, KLH, and the peptides were studied in proliferation assays. The effects of peptide‐specific and allergen‐specific antibodies on T cell responses and allergic lung inflammation were studied using specific antibodies.Results: Prophylactic and therapeutic vaccination with carrier‐bound Bet v 1 peptides induced a Bet v 1‐specific IgG antibody response without priming/boosting of Bet v 1‐specific T cells. Prophylactic and therapeutic vaccination of mice with the peptide vaccine induced Bet v 1‐specific antibodies which suppressed Bet v 1‐specific T cell responses and allergic lung inflammation.Conclusion and Clinical Relevance: Vaccination with carrier‐bound allergen‐derived peptides lacking allergen‐specific T cell epitopes induces allergen‐specific IgG antibodies which suppress allergen‐specific T cell responses and allergic lung inflammation.

Allergen-specific immunotherapy is currently based on the administration of allergen extracts containing natural allergens. However, its broad application is limited by the poor quality of these extracts. Based on recombinant allergens, well-defined allergy vaccines for allergen-specific immunotherapy can be produced. Furthermore, they can be modified to reduce their allergenic activity and to avoid IgE-mediated side effects. Here, we demonstrate that the immunogenicity of two grass pollen-derived hypoallergenic allergen derivatives could be increased by engineering them as a single hybrid molecule. We used a hypoallergenic Phl p 2 mosaic, generated by fragmentation of the Phl p 2 sequence and reassembly of the resulting peptides in an altered order, and a truncated Phl p 6 allergen, to produce a hybrid protein. The hybrid retained the reduction of IgE reactivity and allergenic activity of its components as shown by ELISA and basophil activation assays. Immunization with the hybrid molecule demonstrated the increased immunogenicity of this molecule, leading to higher levels of allergen-specific IgG antibodies compared to the single components. These antibodies could inhibit patients' IgE binding to the wild-type allergens. Thus, the described strategy allows the development of safer and more efficacious vaccines for the treatment of grass pollen allergy.