Identifikacija in ovrednotenje mimotopov izbranih alergenov Fel d 1 in Ara h 2 : doktorska disertacija

Luzar, Jernej (author), Lunder, Mojca (mentor), Korošec, Peter (comentor)

Alergija je bolezensko stanje, ki se pojavi kot posledica neustrezno uravnavanega imunskega odziva. Povezana je z motnjami imunske tolerance, kjer neškodljivi antigeni postanejo alergeni. Alergeni so odgovorni za sprožitev T celičnega imunskega odziva tipa 2. Alergijski imunski odziv je škodljiv za naš organizem in lahko sproži življenjsko ogrožajoče sistemske reakcije. Specifična imunoterapija je edini pristop zdravljenja vzroka alergijske bolezni in ne le njenih simptomov. Z uporabo naravnih ali rekombinatnih alergenov v specifični imunoterapiji induciramo imunsko toleranco na alergene. Toda uporaba celotnih alergenov lahko predstavlja nevarnost sprožitve anafilaktičnega šoka. Zaradi tega so v zadnjih desetletjih raziskave usmerjene v iskanje molekul z zmanjšano ali popolnoma odstranjeno alergogenostjo. Raziskave potekajo na spremenjenih alergenih, delih alergenov, peptidnih fragmentih alergenov in mimotopih alergenov. Prvi del doktorske disertacije obsega pregled alergenskih mimotopov, ki so bili odkriti z uporabo tehnologije bakteriofagnega prikaza. Pregled se nanaša predvsem na uporabnost bakteriofagnih peptidnih knjižnic. S pomočjo teh knjižnic lahko odkrijemo mimotope s potencialom za razvoj imunoterapije. Knjižnice nam pomagajo pri identifikaciji prevladujočih B celičnih epitopov in določanju IgE profilov posameznih pacientov. Odkritje epitopov in individualnih IgE profilov nam služi kot pomoč pri razvoju diagnostike in imunoterapije tako za splošno uporabo kot tudi za individualizirano zdravljenje. Z bakteriofagnimi peptidnimi knjižnicami si pomagamo tudi pri preučevanju navzkrižne reaktivnosti med alergeni. Vsi omenjeni prispevki bakteriofagnih peptidnih knjižnic so nepogrešljivi sestavni deli za razumevanje alergijske patogeneze ter za razvoj novih imunoterapij. Poleg uporabe v bakteriofagnih peptidnih knjižnicah se bakteriofagi vedno bolj uporabljajo in testirajo kot imunogeni nosilci različnih peptidov. Bakteriofagi tako združujejo odkrivanje mimotopov in imunizacijo v eno skupno platformo. V drugem delu doktorske disertacije sem se osredotočil na alergijo za mačke, ki pri alergičnemu posamezniku pogosto vodi v razvoj astme. Za zdravljenje alergije za mačke se trenutno uporabljajo le pripravki s celotnimi alergeni, kar lahko pri prevelikem odmerku vodi do sprožitve anafilaksije. Zaradi tega predstavlja identifikacija epitopov in mimotopov mačjih alergenov pomembno odkritje za razvoj učinkovite in varne specifične imunoterapije. Z uporabo tehnologije bakteriofagnega prikaza smo identificirali pet strukturnih mimotopov glavnega mačjega alergena Fel d 1. Identificirane mimotope smo računalniško prilegali na površino alergena in definirali možno epitopno mesto na samem alergenu. Z vezavo specifičnih protiteles razreda IgE iz serumov pacientov alergičnih za mačke smo potrdili IgE reaktivnost mimotopov. Mimotope smo v večjem številu izrazili na površini nitastih bakteriofagov. Bakteriofage smo izbrali kot imunogeni nosilec, ki je zmožen spodbuditi ustrezen imunski odziv. Dokazali smo, da sami mimotopi niso alergogeni, saj niso aktivirali bazofilcev, kar kaže na njihov potencial za razvoj hipoalergenih terapevtskih produktov. Bakteriofagi z izraženimi mimotopi so spodbudili močan T celični imunski odziv usmerjen v tip 1. Sami mimotopi so prispevali k tipu 1 T celičnega odziva, saj so povečali produkcijo interlevkina 2. Identificirani mimotopi in izbrani nosilec lahko pripomorejo k razumevanju in načrtovanju razvoja hipoalergenih pripravkov za zdravljenje alergije za mačke. Tema tretjega dela doktorske disertacije je bila alergija za arašide, ki predstavlja veliko nevarnost za sprožitev anafilaksije. Anafilaksija je pri alergijski reakciji posledica sistemskih odzivov in predstavlja enega glavnih razlogov, da so raziskave usmerjenje v iskanje varnih in učinkovitih produktov za peroralno imunoterapijo. S tem razlogom smo se odločili identificirati mimotope z znižano alergogenostjo v primerjavi s celotnimi alergeni. Z uporabo tehnologije bakteriofagnega prikaza smo uspešno identificirali mimotope Ara h 2, ki je glavni alergen v arašidih. Odkriti mimotopi so se prilegali na površino strukture Ara h 2, kjer se nahaja tudi dominantni epitop. Združene s sidrno domeno smo mimotope izrazili na površini Lactoccoccus lactis, katere smo uporabili kot imunogene nosilce. Tako pripravljeni konstrukti so vezali protitelesa razreda IgE iz serumov pacientov alergičnih za arašide. Identificirani mimotopi so imeli znižano alergogenost z izjemo mimotopa L7-N40, ki je ohranil delno alergogenost. Omenjeni mimotop je izkazal največjo podobnost z dominantnim epitopom. Naši konstrukti, izrazito konstrukt z mimotopom L7-N40, so bili zmožni sprožiti T celični imunski odziv. V primerjavi s celotnim alergenom so signifikantno povišali produkcijo IFN-γ, kar kaže na usmeritev imunskega odziva v tip 1 T celični odziv. Naši konstrukti z mimotopi predstavljajo zanimiv dostavni sistem z imunomodulatornimi lastnostmi za ciljanje mukoznih površin v gastrointestinalnem traktu. Doktorska disertacija je pomoč k razumevanju in razvoju varne in učinkovite specifične imunoterapije, ki bo pripomogla k zdravljenju alergije za mačke in arašide z uporabo hipoalergenih terapevtskih pripravkov.

• Language: Slovenian
• Keywords: alergeni, imunska toleranca, molekularna alergologija, mimotopi, disertacije
• Work type: Doctoral dissertation
• Typology: 2.08 - Doctoral Dissertation
• Organization: FFA - Faculty of Pharmacy
• Place of publishing: Ljubljana
• Publisher: [J. Luzar]
• Year: 2016
• Number of pages: 139 str.
• PID: 20.500.12556/RUL-143782
• UDC: 615.37:616-056.43 (043.3)
• COBISS.SI-ID: 4264049
• Publication date in RUL: 12.01.2023

LUZAR, Jernej, 2016, Identifikacija in ovrednotenje mimotopov izbranih alergenov Fel d 1 in Ara h 2 : doktorska disertacija [online]. Doctoral dissertation. Ljubljana : J. Luzar. [Accessed 26 April 2025]. Retrieved from: https://repozitorij.uni-lj.si/IzpisGradiva.php?lang=eng&id=143782

Secondary language

• Language: English
• Title: Identification and characterization of allergens Fel d 1 and Ara h 2 mimotopes

Abstract

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.