Identifikacija in ovrednotenje mimotopov glavnega alergena čebeljega strupa Api m 1 za razvoj specifične imunoterapije

Zahirović, Abida (author), Lunder, Mojca (mentor), Korošec, Peter (comentor)

Alergija po piku čebele je nevarno bolezensko stanje, saj lahko povzroči hude, v nekaterih primerih ţivljenjsko ogroţajoče anafilaktične reakcije, obenem pa je specifična imunoterapija s čebeljim strupom dolgotrajna, naporna in povzroča neţelene stranske učinke, zato obstaja potreba po novih učinkovinah 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. Posledično 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 čebeljega strupa, Api m 1. Z rešetanjem bakteriofagnih knjiţnic kratkih naključnih peptidov na tarčnih protitelesih IgG, usmerjenih proti Api m 1, smo odkrili kolekcijo 46 edinstvenih peptidov. V testih vrednotenja je 36 peptidov izkazovalo močno in specifično vezavo do tarčnih protiteles ter kompeticijo z Api m 1 za iste paratope (ali vezišča 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 tarčna protitelesa smo ocenili s testom alaninskega rešetanja. Pri linearnem mimotopu GRMGPSELGPVIG sta se glutaminska kislina in lizin izkazali kot ključni za vezavo, medtem ko alaninske različice cikličnega mimotopa GCWDSLGRAC niso obdrţale vezavnih lastnosti izvornega peptida. Aminokislinska zaporedja pridobljenih peptidov smo in silico prilegali na Api m 1 in določili 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 plaščni 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 tarčna protitelesa uporabili IgG iz zajčjega seruma, smo preverili ali mimotopi ohranijo sposobnost vezave na protitelesa IgE iz seruma pacientov, alergičnih za strup čebele. S testom točkovnega nanosa (ang. dot blot) smo potrdili, da se IgE iz seruma 12 pacientov, reaktivnih na Api m 1, specifično veţejo na štiri izbrane mimotope, spojene z nosilcem pIII, kar potrjuje, da so identificirani epitopi relevantni za oba izotipa protiteles. S pomočjo 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 čimer smo izključili 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, preprečijo degranulacijo bazofilcev, ki jo sproţi alergen Api m 1. Preliminarni rezultati kaţejo, da ni razlik med aktivacijo bazofilcev, ki jo je povzroči Api m 1, in aktivacijo, ki jo je povzročil Api m 1 v prisotnosti mimotopov. Vseeno opisan koncept predstavlja osnovo za nadaljnji razvoj tega novega pristopa k zdravljenju alergij. Vpliv mimotopov na T celični odziv smo ovrednotili z določanjem citokinov v kulturi mononuklearnih celic iz periferne krvi alergičnih pacientov. Čebelji strup in Api m 1 sta spodbudila preteţno citokine Th2, medtem ko je mimotop, spojen s pIII, povečal izločanje interferona gama (IFN-?), kar nakazuje premik citokinskega profila iz Th2 v Th1. Izsledki raziskave tako opredeljujejo mimotope Api m 1 kot obetavne učinkovine za nadaljni razvoj hipoalergenih učinkovin za varnejšo imunoterapijo alergije po piku čebele. V drugem delu doktorske disertacije smo tehnologijo rešetanja bakteriofagnih knjiţnic uporabili za določitev 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 specifično vezali tako na tarčna protitelesa IgG, usmerjena proti Amb a 1 kot tudi na IgE iz zmesi serumov alergičnih pacientov. Izselekcionirani peptidi so se le šibko ujemali s primarnim zaporedjem Amb a 1, kar je nakazovalo, da so epitopi konformacijski. Za določitev konformacijskih epitopov smo peptide prilegali na homologni tridimenzionalni model Amb a 1, ki smo ga izdelali v računalniškem 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 sočasno analizo več alergenov v testu aktivacije bazofilcev (multipleks BAT) s pomočjo fluorescenčnega označevanja alergenov z nanokristali Qdot. Poglavitna alergena čebeljega strupa, Api m 1 in Api m 2, smo konjugirali z nanokristali Qdot dveh različnih velikosti ter primerjalno ovrednotili njihovo primernost za analizo več 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 označevanje 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, označenih z nanokristali amino-(PEG)-Qdot. Slednje smo nato uporabili v multipleks testu BAT in rezultat običajne 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 česar sklepamo, da so alergeni, označeni z nanokristali amino-(PEG)-Qdot, uporabni za sočasno analizo aktivacije bazofilcev z več alergeni. Multipleks BAT je pomembna nadgradnja trenutnega testa, ki bo pocenil in olajšal diagnostično obravnavo dvojno pozitivnih in polisenzibiliziranih bolnikov, obenem pa odpira nove moţnosti za raziskave, kot je na primer preučevanje 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: Doctoral dissertation
• Organization: FFA - Faculty of Pharmacy
• Year: 2020
• PID: 20.500.12556/RUL-125335
• Publication date in RUL: 11.03.2021

Secondary language

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

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.

• Keywords: specific immunotherapy, bee venom allergens, allergic response, mimotope Api m 1, ragweed allergens, allergen analysis