Allergy is a disease state occuring as an effect of unsuitable regulated immune response. It is connected with dysfunction of immune tolerance where harmless antigens become allergens. Allergens are responsible for eliciting type 2 T cell immune response. Allergic immune response is harmful to our organism and can trigger life-threatening systemic reactions. Specific immunotherapy is the only way to cure allergic disease. The use of natural and recombinant allergens in specific immunotherapy induces immune tolerance to allergens but represents a danger for triggering of anaphylactic shock. Hence, investigations in the last decades have been directed to finding substances with lower or completely removed allergenic activity. Research is underway on altered allergens, parts of allergens, peptides derived from allergens, and mimotopes of allergens. The first part of doctoral thesis includes review of allergen mimotopes discovered by phage display technology. Review mostly refers to usefulness of phage display peptide libraries. With the help of these libraries we can discover mimotopes with potential to develop immunotherapy. The libraries help us to identify dominant B-cell epitopes and to determine IgE profiles of individual patients. Epitope discovery and individual IgE profiles serve as help to develop diagnostics and immunotherapy for common use as for individual treatment. Phage display peptide libraries also help us in studying of cross-reactivity among allergens. All these different contributions of phage display peptide libraries are indispensable components for understanding allergic pathogenesis and for development of new immunotherapies. Beside function of bacteriophages in peptide libraries, they are increasingly used and tested as immunogenic carriers of different peptides. Bacteriophages combine mimotope discovery and immunization into one complete platform. In the second part of doctoral thesis I focused on cat allergy which often leads to asthma development in allergic individual. At present used preparations for treatment of cat allergy contain whole allergens which can lead to triggering anaphylaxis as effect of an overdose. Epitope and mimotope identification of cat allergens represents important discovery for development of effective and safe specific immunotherapy. We identified five conformational mimotopes of the major cat allergen Fel d 1 by using phage display technology. Identified mimotopes were mapped to the surface of the allergen and potential epitope spot was defined. Binding of specific IgE from sera of cat-allergic patients confirmed IgE reactivity of mimotopes. Mimotopes were displayed in multiple copies on the surface of filamentous bacteriophages which were chosen as immunogenic carriers able to elicit suitable immune response. We demonstrated that mimotopes alone are not allergenic because they did not activate basophils. This shows their potential for development of hypoallergenic therapeutical products. Prepared construct elicited strong type 1 directed T cell immune response. Mimotopes alone contributed to type 1 T cell response by increasing production of interleukin 2. Identified mimotopes and selected carriers can contribute to progress in development of hypoallergenic preparations for treatment of cat allergy. Subject of the third part of doctoral thesis was peanut allergy which represents high risk of triggering anaphylaxis in allergic individuals. Anaphylaxis, a systemic respons in allergic reaction, is a reason researches are directed into discovery of safe and effective products for oral immunotherapy. This was the reason we decided to investigate mimotopes which have lower allergenic activity in comparison with whole allergens. We successfully identified mimotopes of Ara h 2, the major peanut allergen, by using phage display technology. Discovered mimotopes were mapped to the dominant surface epitope of Ara h 2. Fused with anchoring domain we displayed mimotopes on the surface of Lactococcus lactis used as immunogenic carriers. Prepared constructs bound IgE antibodies from sera of peanut-allergic patients. Mimotopes had lower allergenic activity except for mimotope L7-N40, which partially preserved allergenic activity. Mentioned mimotope showed the highest similarity with dominant epitope. Our constructs, especially construct with mimotope L7-N40, were able to elicit T cell immune response. In comparison with allergen they had significantly higher production of IFN-γ demonstrating type 1 directed T cell response. Our constructs with mimotopes present interesting delivery system with immunomodulatory characteristics to target mucosal surfaces in gastrointestinal tract. Doctoral thesis is helping to understand and develop safe and effective specific immunotherapy which will contribute to treatment of cat and peanut allergy by using hypoallergenic therapeutical preparations.
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