Identifikacija in ovrednotenje mimotopov glavnega alergena čebeljega strupa Api m 1 za razvoj specifične imunoterpaije : doktorska disertacija

Zahirović, Abida (author), Lunder, Mojca (mentor), Korošec, Peter (co-mentor)

Alergija po piku ĉebele je nevarno bolezensko stanje, saj lahko povzroĉi hude, v nekaterih primerih ţivljenjsko ogroţajoĉe anafilaktiĉne reakcije, obenem pa je specifiĉna imunoterapija s ĉebeljim strupom dolgotrajna, naporna in povzroĉa neţelene stranske uĉinke, zato obstaja potreba po novih uĉinkovinah in alternativnih strategijah zdravljenja. Eno izmed perspektivnejših skupin novih imunoterapevtikov predstavljajo peptidni mimetiki epitopov alergenov (mimotopi). Po principu molekularne mimikrije so mimotopi sposobni sproţiti zelo podoben ali celo enak imunski odziv, kot epitopi, katere posnemajo. Poslediĉno lahko v imunoterapiji predstavljajo nadomestne ligande za alergene. V primerjavi z alergeni je njihova prednost v tem, da, kot kratki peptidi, nimajo vezavnih mest potrebnih za premreţenje IgE na površini efektorskih celic. Zaradi potencialne terapevtske uporabnosti smo v okviru doktorskega dela poiskali mimotope poglavitnega alergena ĉebeljega strupa, Api m 1. Z rešetanjem bakteriofagnih knjiţnic kratkih nakljuĉnih peptidov na tarĉnih protitelesih IgG, usmerjenih proti Api m 1, smo odkrili kolekcijo 46 edinstvenih peptidov. V testih vrednotenja je 36 peptidov izkazovalo moĉno in specifiĉno vezavo do tarĉnih protiteles ter kompeticijo z Api m 1 za iste paratope (ali vezišĉa v neposredni bliţini). Med peptidi smo odkrili dva izrazita aminokislinska motiva GP[S/N/D]ELG[R/K/H] in D[K/R/H]SKP(H). Vpliv prispevka posameznih aminokislinskih ostankov znotraj motiva za vezavo na tarĉna protitelesa smo ocenili s testom alaninskega rešetanja. Pri linearnem mimotopu GRMGPSELGPVIG sta se glutaminska kislina in lizin izkazali kot kljuĉni za vezavo, medtem ko alaninske razliĉice cikliĉnega mimotopa GCWDSLGRAC niso obdrţale vezavnih lastnosti izvornega peptida. Aminokislinska zaporedja pridobljenih peptidov smo in silico prilegali na Api m 1 in doloĉili epitopa, ki gradita površinsko izpostavljeni zanki znotraj regije 17-24 na N-koncu Api m 1 in znotraj regije 119-124 na C-koncu. Za namen aktivne imunizacije smo na podlagi analize rezultatov našega raziskovalnega dela in podatkov iz literature izbrali majhni plašĉni protein pIII iz bakteriofaga M13 kot samostojni nosilec mimotopov. Štiri predstavnike identificiranih epitopov smo sintetizirali in jih hkrati pripravili kot fuzijske proteine z nosilcem pIII, tako da smo jih izolirali iz periplazme E. coli. Tako pripravljenim konstruktom in sinteznim peptidom smo ovrednotili njihov imunoterapevtski potencial. Ker smo v procesu selekcije kot tarĉna protitelesa uporabili IgG iz zajĉjega seruma, smo preverili ali mimotopi ohranijo sposobnost vezave na protitelesa IgE iz seruma pacientov, alergiĉnih za strup ĉebele. S testom toĉkovnega nanosa (ang. dot blot) smo potrdili, da se IgE iz seruma 12 pacientov, reaktivnih na Api m 1, specifiĉno veţejo na štiri izbrane mimotope, spojene z nosilcem pIII, kar potrjuje, da so identificirani epitopi relevantni za oba izotipa protiteles. S pomoĉjo testa aktivacije bazofilcev (ang. basophil activation test, BAT) smo ugotovili, da mimotopi, spojeni s pIII, ne aktivirajo bazofilcev, torej niso alergogeni. Po stimulaciji s sinteznimi peptidi prav tako nismo zaznali aktivacije bazofilcev, s ĉimer smo izkljuĉili moţnost, da peptidni mimotopi aktivirajo bazofilce preko drugih od IgE neodvisnih mehanizmov. Dodatno smo preverili, ali lahko peptidni mimotopi, z blokado vezavnih mest na IgE, prepreĉijo degranulacijo bazofilcev, ki jo sproţi alergen Api m 1. Preliminarni rezultati kaţejo, da ni razlik med aktivacijo bazofilcev, ki jo je povzroĉi Api m 1, in aktivacijo, ki jo je povzroĉil Api m 1 v prisotnosti mimotopov. Vseeno opisan koncept predstavlja osnovo za nadaljnji razvoj tega novega pristopa k zdravljenju alergij. Vpliv mimotopov na T celiĉni odziv smo ovrednotili z doloĉanjem citokinov v kulturi mononuklearnih celic iz periferne krvi alergiĉnih pacientov. Ĉebelji strup in Api m 1 sta spodbudila preteţno citokine Th2, medtem ko je mimotop, spojen s pIII, poveĉal izloĉanje interferona gama (IFN-γ), kar nakazuje premik citokinskega profila iz Th2 v Th1. Izsledki raziskave tako opredeljujejo mimotope Api m 1 kot obetavne uĉinkovine za nadaljni razvoj hipoalergenih uĉinkovin za varnejšo imunoterapijo alergije po piku ĉebele. V drugem delu doktorske disertacije smo tehnologijo rešetanja bakteriofagnih knjiţnic uporabili za doloĉitev epitopov poglavitnega alergena ambrozije, Amb a 1, pri katerem je dodaten izziv predstavljalo dejstvo, da tridimenzionalna struktura proteina ni znana. Odkrili smo 6 peptidov, ki so specifiĉno vezali tako na tarĉna protitelesa IgG, usmerjena proti Amb a 1 kot tudi na IgE iz zmesi serumov alergiĉnih pacientov. Izselekcionirani peptidi so se le šibko ujemali s primarnim zaporedjem Amb a 1, kar je nakazovalo, da so epitopi konformacijski. Za doloĉitev konformacijskih epitopov smo peptide prilegali na homologni tridimenzionalni model Amb a 1, ki smo ga izdelali v raĉunalniškem programu za molekulsko modeliranje Phyre2. Po prileganju smo z EpiSearch analizo definirali lokacijo moţnih epitopov na površini molekule okoli aminokislinskih ostankov K104, S110, H214 in W312. V zadnjem delu smo se lotili razvoja nove metode za soĉasno analizo veĉ alergenov v testu aktivacije bazofilcev (multipleks BAT) s pomoĉjo fluorescenĉnega oznaĉevanja alergenov z nanokristali Qdot. Poglavitna alergena ĉebeljega strupa, Api m 1 in Api m 2, smo konjugirali z nanokristali Qdot dveh razliĉnih velikosti ter primerjalno ovrednotili njihovo primernost za analizo veĉ alergenov hkrati. Uporabili smo nanokristale karboksil-Qdot, pri katerih vezava na alergen poteka preko karboksilne skupine in amino-Qdot, pri katerih vezava poteka preko aminske skupine. Potrdili smo, da oznaĉevanje alergenov tako z nanokristali karboksil- kot tudi z amino-(PEG)-Qdot ni vplivalo na njihovo IgE reaktivnost. Nasprotno pa se je alergogena aktivnost oz. sposobnost prekriţanja IgE na površju bazofilcev ohranila le pri alergenih, oznaĉenih z nanokristali amino-(PEG)-Qdot. Slednje smo nato uporabili v multipleks testu BAT in rezultat obiĉajne analize BAT primerjali z rezultatom multipleks BAT. Ugotovili smo ujemanje pri 15 od 17 bolnikov in 6 od 6 kontrolnih osebah s primerljivimi krivuljami odziva na alergen, na podlagi ĉesar sklepamo, da so alergeni, oznaĉeni z nanokristali amino-(PEG)-Qdot, uporabni za soĉasno analizo aktivacije bazofilcev z veĉ alergeni. Multipleks BAT je pomembna nadgradnja trenutnega testa, ki bo pocenil in olajšal diagnostiĉno obravnavo dvojno pozitivnih in polisenzibiliziranih bolnikov, obenem pa odpira nove moţnosti za raziskave, kot je na primer preuĉevanje razporeditev površinskih IgE na efektorskih celicah.

• Language: Slovenian
• Keywords: specifična imunoterapija, alergeni čebeljega strupa, alergijski odziv, mimotop Api m 1, alergeni ambrozije, analiza alergenov
• Work type: Dissertation
• Typology: 2.08 - Doctoral Dissertation
• Organization: FFA - Faculty of Pharmacy
• Place of publishing: Ljubljana
• Publisher: [A. Zahirović]
• Year: 2020
• Number of pages: XVI, 199 str.
• PID: 20.500.12556/RUL-125355
• UDC: 615.37:616-056.43:595.799(043.3)
• COBISS.SI-ID: 25939715
• Publication date in RUL: 12.03.2021

Secondary language

• Language: English
• Title: Identification and characterization of major bee venom allergen Api m 1 mimotopes for development of specific immunotherpay

Abstract

Bee sting allergy is a dangerous disease condition which can cause severe, in some cases life-threatening anaphylactic reactions. Due to the fact that bee venom immunotherapy is time-consuming and laborious, frequently accompanied by side effects, new therapeutic agents and alternative treatment strategies are needed. One of the promising groups of new immunotherapeutics is peptide mimetics of allergen epitopes (mimotopes). On the basis of molecular mimicry, mimotopes can elicit a very similar or even the same immune response as epitopes which they mimic. They may therefore represent substituting ligands for allergens in immunotherapy. As short peptides mimotopes do not have the binding sites required for cross-linking of IgE on the surface of effector cells which represents their advantage over allergens. Due to the potential therapeutic utility, we aimed to identify mimotopes of the major bee venom allergen, Api m 1. By screening bacteriophage library of short random peptides against target anti-Api m 1 IgG antibodies we discovered a collection of 46 unique peptides. In evaluation assays, 36 peptides showed strong and specific binding to target antibodies and effectively competed with Api m 1 for the same paratopes (or sites in the close proximity). The peptides contained two distinct amino acid motifs either GP[S/N/D]ELG[R/K/H] or D[K/R/H]SKP(H). The contribution of individual amino acid residues within the motif to the target antibody binding was assessed by alanine screening assay. In the GRMGPSELGPVIG linear mimotope, glutamic acid and leucine proved to be critical for binding, whereas alanine variants of the GCWDSLGRAC cyclic mimotope did not retain the binding properties of the parental peptide. We then mapped amino acid sequences of the obtained peptides to Api m 1 in silico and determined the epitopes on the surface-exposed loops within the 17-24 region at the N-terminus of Api m 1 and within the 119-124 region at the C-terminus. Based on our research results and literature data we selected small coat protein pIII from M13 bacteriophage as a carrier of mimotopes for active immunization. Four representatives of the identified epitopes were synthesized and isolated as pIII-fused proteins from the periplasm of E. coli. Immunotherapeutic potential of pIII-fused constructs and synthetic peptides were evaluated. Given that rabbit IgG were used as target antibodies in the selection process, we checked whether the mimotopes retained the ability to bind to IgE from patients´ serum. The dot blot assay confirmed that IgE from the serum of 12 patients, reactive to Api m 1, specifically bind to four selected pIII-fused mimotopes, thus verifying that the identified epitopes are relevant for both antibody isotypes. Using a basophil activation assay (BAT), we confirmed that pIII-fused mimotopes did not activate basophils i.e they are non-allergenic. Also, no basophil degranulation was detected after stimulation with synthetic peptides, thus excluding the possibility that peptide mimotopes would activate basophils via other IgE-independent mechanisms. We further examined whether peptide mimotopes, by blocking binding sites to IgE, can prevent basophil degranulation triggered by Api m 1 allergen. Preliminary results indicate that there is no difference between basophil activation induced by Api m 1 and activation induced by Api m 1 in the presence of mimotopes. Nevertheless, the described concept represents the basic platform for further development of this new approach to treating allergies. The effect of mimotopes on the T cell response was evaluated by measuring the cytokines in the cultures of peripheral blood mononuclear cells from allergic patients. Bee venom and Api m 1 stimulated predominantly Th2 cytokines, whereas the pIII-fused mimotope increased interferon-gamma (IFN-γ) secretion, suggesting a shift in the cytokine profile from Th2 to Th1. The results of the research thus identify mimotopes Api m 1 as promising candidates for the further development of hypoallergenic therapeutic agents for safer immunotherapy of bee sting allergy. In the second part of the doctoral dissertation, we performed a screening of bacteriophage short peptide libraries to determine the epitopes of the main ragweed allergen Amb a 1 where an additional challenge was the fact that the three-dimensional structure of the protein is unknown. We detected 6 peptides that specifically bound both target IgG antibodies directed against Amb a 1 as well as IgE from a mixture of sera from allergic patients. The selected peptides only weakly aligned with the Amb a 1 primary sequence, thus suggesting that the epitopes are conformational. To determine the conformational epitopes, the peptides were mapped onto the homologous three-dimensional model of Amb a 1 generated with a program for molecular modelling Phyre2. By using EpiSearch analysis after mapping, we defined the location of possible epitopes on the surface of the molecule around the amino acid residues K104, S110, H214 and W312. In the last part, we developed a new method for the simultaneous analysis of several allergens in the basophil activation test (BAT multiplex) using fluorescence labelling of allergens with Qdot nanocrystals. The main allergens of bee venom, Api m 1 and Api m 2, were conjugated with Qdots of two different sizes and their suitability for the analysis of several allergens simultaneously was evaluated. Binding of carboxyl-Qdot to allergen is attained via carboxyl group, whereas amino-Qdot binds to allergen via an amine group. We confirmed that the labelling of allergens with both carboxyl- and amino-(PEG)-Qdot did not affect their IgE reactivity. In contrast, allergenic activity i.e. the ability to cross-link IgE on the surface of basophils was maintained only in allergens labelled with amino-(PEG)-Qdot nanocrystals. The latter were then used in the multiplex BAT and the result of the BAT analysis was compared with the result of the multiplex BAT. Matching results were found in 15 of 17 patients and 6 of 6 controls with comparable allergen response curves, suggesting that allergens labelled with amino-(PEG)-Qdot nanocrystals are suitable for concomitant analysis of basophil activation with multiple allergens. Multiplex BAT represents an important improvement of the current test, which will reduce the cost and facilitate the diagnostic workup of double-positive and polysensitized patients, while opening up new possibilities for research, such as studying the distribution of surface IgE on effector cells.

Projects

• Funder: ARRS - Slovenian Research Agency
• Project number: P4-0127
• Name: Farmacevtska biotehnologija: znanost za zdravje