Abstract
Allergy and asthma to cat (Felis domesticus) affects about 10% of the population in affluent countries. Immediate allergic symptoms are primarily mediated via IgE antibodies binding to B cell epitopes, whereas late phase inflammatory reactions are mediated via activated T cell recognition of allergen-specific T cell epitopes. Allergen-specific immunotherapy relieves symptoms and is the only treatment inducing a long-lasting protection by induction of protective immune responses. The aim of this study was to produce an allergy vaccine designed with the combined features of attenuated T cell activation, reduced anaphylactic properties, retained molecular integrity and induction of efficient IgE blocking IgG antibodies for safer and efficacious treatment of patients with allergy and asthma to cat. The template gene coding for rFel d 1 was used to introduce random mutations, which was subsequently expressed in large phage libraries. Despite accumulated mutations by up to 7 rounds of iterative error-prone PCR and biopanning, surface topology and structure was essentially maintained using IgE-antibodies from cat allergic patients for phage enrichment. Four candidates were isolated, displaying similar or lower IgE binding, reduced anaphylactic activity as measured by their capacity to induce basophil degranulation and, importantly, a significantly lower T cell reactivity in lymphoproliferative assays compared to the original rFel d 1. In addition, all mutants showed ability to induce blocking antibodies in immunized mice.The approach presented here provides a straightforward procedure to generate a novel type of allergy vaccines for safer and efficacious treatment of allergic patients.
Highlights
The domestic cat (Felis domesticus) is one of the most frequent pets and approximately 10% of the general population in industrialized countries is sensitized to cat allergens [1]
Further analyses of the primary structure revealed a substitution of a cysteine to a tyrosine (6.7.1, 6.7.3) or arginine (11.5.2) at residue 73, resulting in loss of a disulfide bond with Cys 95 present in rFel d 1
Treatments utilizing high dose hypoallergens, such as attenuated derivatives of wild type proteins or short peptides, covering the T cell repertoires of allergens have been tested in clinical trials
Summary
The domestic cat (Felis domesticus) is one of the most frequent pets and approximately 10% of the general population in industrialized countries is sensitized to cat allergens [1]. The symptoms deriving from cat allergy manifest mainly as rhinoconjunctivitis with a strong tendency to progress to asthma, especially in children [2]. Proteins of the saliva is left to dry and spread as small airborne particles [3] where it can be detected in a variety of indoor environments [4,5]. The major allergen of the cat, formally termed Fel d 1, is a hetero-dimer member of the secretoglobin protein family [6,7] found in skin, lachrymal glands and in particular in the saliva. More than 95% of cat allergic patients show elevated serum IgE levels to Fel d 1 which is, the primary target for the development of immunotherapeutic vaccines for the treatment of cat allergy [8,9]
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