Prevention of Birch Pollen-Related Food Allergy by Mucosal Treatment with Multi-Allergen-Chimers in Mice

Among birch pollen allergic patients up to 70% develop allergic reactions to Bet v 1-homologue food allergens such as Api g 1 (celery) or Dau c 1 (carrot), termed as birch pollen-related food allergy. In most cases, specific immunotherapy with birch pollen extracts does not reduce allergic symptoms to the homologue food allergens. We therefore genetically engineered a multi-allergen chimer and tested if mucosal treatment with this construct could represent a novel approach for prevention of birch pollen-related food allergy.BALB/c mice were poly-sensitized with a mixture of Bet v 1, Api g 1 and Dau c 1 followed by a sublingual challenge with carrot, celery and birch pollen extracts. For prevention of allergy sensitization an allergen chimer composed of immunodominant T cell epitopes of Api g 1 and Dau c 1 linked to the whole Bet v 1 allergen, was intranasally applied prior to sensitization.Intranasal pretreatment with the allergen chimer led to significantly decreased antigen-specific IgE-dependent β-hexosaminidase release, but enhanced allergen-specific IgG2a and IgA antibodies. Accordingly, IL-4 levels in spleen cell cultures and IL-5 levels in restimulated spleen and cervical lymph node cell cultures were markedly reduced, while IFN-γ levels were increased. Immunomodulation was associated with increased IL-10, TGF-β and Foxp3 mRNA levels in NALT and Foxp3 in oral mucosal tissues. Treatment with anti-TGF-β, anti-IL10R or anti-CD25 antibodies abrogated the suppression of allergic responses induced by the chimer.Our results indicate that mucosal application of the allergen chimer led to decreased Th2 immune responses against Bet v 1 and its homologue food allergens Api g 1 and Dau c 1 by regulatory and Th1-biased immune responses. These data suggest that mucosal treatment with a multi-allergen vaccine could be a promising treatment strategy to prevent birch pollen-related food allergy

Mucosal tolerance induction for treatment of type I allergy

In den letzten Jahrzehnten wurde ein ständiger Anstieg der Prävalenz von Type I-Allergien beobachtet, so dass mittlerweile mehr als 25% der Bevölkerung in westlichen Ländern an Allergien leiden. Häufig werden Allergiker mit zunehmendem Alter gegen mehrere Allergene co-sensibiliisert, wobei diese Patienten schwierig zu behandeln werden. Goldstandard der Allergiebehandlung ist die Allergen-spezifische Immuntherapie. Jedoch birgt diese Therapieform auch einige Nachteile, wie zum Beispiel regelmäßige Injektionen über einen Zeitraum von 2-3 Jahre, Gefahr von anaphylaktischen Nebenwirkungen sowie eine geringe Erfolgsquote bei der Behandlung von Mehrfachallergien. Um die Compliance der Patienten zu erhöhen, aber auch um die Effizienz der Therapie zu verbessern, ist in den letzten Jahren an folgenden Ansätzen gearbeitet worden: Verwendung von rekombinanten Allergenen, die dem Sensibilisierungsprofil der Patienten entsprechen, Applikation effektiver Adjuvantien, und der Wechsel zu einer mukosalen (z.B. oral, nasal) Route der Applikation zur Umgehung der häufigen Injektionen. Entsprechend wurden im Zuge dieser Doktorarbeit neue Behandlungsansätze entwickelt und neue Mausmodele für Einfach- und Mehrfachsensibilisierung etabliert, um Effektivität und Wirkungsmechanismen dieser neuen Ansätze in vivo und in vitro testen zu können. (i) Eine vielversprechende Strategie zur Behandlung von Allergien sind rekombinant hergestellte Allergene. Die rekombinante Produktion garantiert eine genaue strukturelle wie auch immunologische Definition des Allergens. Die Identifizierung von Allergenen, wie auch die Herstellung als rekombinantes Protein sind die Grundsteine bei der Entwicklung von neuen Formen von Immunotherapeutika wie hypoallergene Varianten von Allergenen, Fusionsmoleküle oder Multiallergenkonstrukte. Wir beschreiben die Charakterisierung und rekombinante Herstellung eines neuen Allergens der Dörrobstmotte, das Thioredoxin Plo i 2. In in vivo Studien in Mäusen konnten wir das allerogene Potential dieses Allergens, im Vergleich mit einem anderen Mottenallergen (Plo i 1), untersuchen. Mittels Immunoblot-Experimenten mit humanen Sera wurde die IgE-Reaktivität des Thioredoxins Plo i 2 analysiert, und so Plo i 2 als neue relevante Allergiequelle für mehrfachsensibilisierte Haus- und Meeresfrüchteallergiker, definiert. (ii) Pollen-allergische Patienten sind häufig co-sensibilisiert gegen homologe Lebensmittelallergene; dies wird als sogenannte Pollen-assoziierte-Lebensmittelallergie bezeichnet und manifestiert sich klinisch als „orales Allergiesyndrom“ (OAS). Mehr als 70% der Birkenpollenallergiker leiden unter einer Pollen-assoziierten-Lebensmittelallergie, somit zählt diese Form der Birkenpollen-assoziierten-Lebensmittelallergie zu einer der häufigsten Varianten der Mehrfachallergien. Da das OAS im Rahmen der Behandlung gegen Birkenpollenallergie meist nur ungenügend beeinflusst werden kann, haben wir ein Multiallergenkonstrukt entwickelt, das eine gleichzeitige Behandlung der Birkenpollen- und Nahrungsmittelallergien ermöglichen soll. Dieses Multiallergenkonstrukt wurde gentechnologische hergestellt,, bestehend aus dem gesamten Hauptbirkenpollenallergen Bet v 1 verlinkt mit den immundominanten T-Zell-Epitopen der Birkenpollen-assoziierten- Lebensmittelallergene Dau c 1 (Hauptallergen der Karotte) und Api g 1 (Hauptallergen des Selleries). Um das immunmodulatorische Potential dieses Konstrukts auszutesten, wurde ein Mausmodel für Mehrfachsensibilisierung mit oralem Allergiesyndrom etabliert. Wir konnten zeigen, dass nach intranasaler Applikation dieses Konstrukts die allergische Immunantwort in Richtung Th1 Antwort moduliert wurde und eine Suppression der allergischen Immunantworten durch Induktion von regulatorischen Immunmechansimen erreicht werden konnte. (iii) Um die Effektivität von Toleranzinduktion mittels rekombinanter Allergene oder Allergenkonstrukte zu erhöhen, können mukosale Adjuvantien als sogenannte „mukosale Transportsysteme“ verwende werden. Eines der mukosalen Adjuvantien mit tolerogenem Potenzial ist Choleratoxin (CT) bzw. die B-Untereinheit von Choleratoxin (CTB). In der vorliegenden Arbeit beschreiben wir die gentechnologische Konstruktion eines Fusionsmoleküls von CTB und Bet v 1. Dieses Fusionsmolekül wurde intranasal in einem Mausmodel für Birkenpollenallergie appliziert um dessen immunmodulatorische Kapazität zu untersuchen. Die Allergie-reduzierende Wirkung des neuen Konstrukts beruhte auf der gleichzeitigen Modulierung der Th2 Immunantwort Richtung Th1 und der Induktion von lokalen, protektiven IgA Antikörpern. Zusammenfassend, wir haben drei verschiedene Strategien (rekombinante Allergene, Multiallergenkonstrukte für mukosale Applikation und Verwendung neuer mukosaler Adjuvantien) zur Verbesserung der Prävention (und möglicherweise auch Therapie) von Typ I-Allergie entwickelt, deren Nutzen durch eine erfolgreiche Suppression der allergischen Immunantwort in Einfach- und Mehrfachsensibilisierungsmodele demonstriert wurde. Derartige experimentelle Studien sollen dazu beitragen, bestehende Behandlungsansätze zu verbessern, sowie auch neue Behandlungsstrategien für allergische Erkrankungen zu entwickeln.The prevalence of type I allergy is constantly increasing in industrialized countries: more than 25% of the population suffer from allergic disorders. With increasing age allergic individuals become co-sensitized to additional allergens, and become difficult to treat due to the multi-sensitization. Allergen-specific immunotherapy is state of the art treatment of allergic diseases. However, due to a low compliance of patients to frequent injections, and the reduced efficacy in multi-sensitized individuals, a change to a less invasive route of application via the mucosa offers a promising approach. The aim of this thesis was to develop new treatment strategies for primary and secondary prevention of type I allergy, focusing on strategies being applied via the mucosal surfaces. Therefore, three novel approaches were developed and tested that might lead to improvement of the current treatment strategies: (i) Use of recombinant allergens, (ii) development of multi-allergen constructs for mucosal tolerance induction, and (iii) use of mucosal adjuvants systems for improved mucosal treatment strategies. Furthermore, murine models of mono- and poly-sensitization were established, to investigate the immunological mechanisms of mucosal tolerance induction as well. Ad i) Recombinant forms of allergens are promising tools for treatment of allergy, because of their defined structural and immunological properties as well as the standardized production procedures. Furthermore, identification and production of allergens as recombinant proteins are the essential steps to develop advanced forms of immunotherapeutica such as hypoallergenic variants, fusion molecules, or multi-allergen constructs. A novel allergen of Indianmeal moth, the thioredoxin Plo i 2 was identified and recombinantly produced. The allergenic potential of Plo i 2 was investigated by in vivo studies in mice, in comparison to the moth allergen Plo i 1. IgE-reactivity tested by blotting experiments with human sera, defined thioredoxin Plo i 2 as new relevant source in multi-sensitized indoor and seafood allergic patients. Ad ii) In pollen allergic patients co-sensitization to homologue food allergens often occurs, termed as pollen-related food allergy and clinically manifested as “oral allergy syndrome” (OAS). With about 70% of birch pollen allergic patients, birch pollen-related food allergy is the most common form of this multi-sensitivity. For a new experimental approach to treat birch pollen-related food allergy, a multi-allergen construct composed of the whole major birch pollen allergen Bet v 1 linked to the immunodominant T cell epitopes of the Bet v 1-related food allergens Dau c 1 (major allergen of carrot) and Api g 1 (major allergen of celery), were genetically engineered. In order to investigate immunomodulatory potential of this chimer, a murine model of poly-sensitization and OAS was established. Intranasal application of the chimer modulated allergic immune responses towards Th1 immune responses via regulatory mechanisms. Ad iii) In order to enhance the effectiveness of mucosal tolerance induction with recombinant allergens or allergen constructs, mucosal adjuvants were used as mucosal delivery systems. The strong tolerogenic nontoxic B subunit of cholera toxin (CTB) served as mucosal adjuvant which was genetically conjugated to the birch pollen allergen Bet v 1. For improvement of mucosal tolerance induction the CTB-Bet v 1 fusion molecule was intranasally applied in a murine model of birch pollen allergy. The immunomodulatory properties of the fusion molecule were demonstrated by a modulation of Th2 immune responses towards Th1, accompanied by induction of protective IgA antibodies. In summary, we have presented three different strategies - (i) production of recombinant allergens or allergen constructs, (ii) use of mucosal route of application, (iii) use of mucosal adjuvants - to improve prevention of type I allergy. The benefit of mucosal application of recombinant allergens conjugated to adjuvants as fusion protein or to other allergens as multi-allergen construct was demonstrated by successful suppression of allergic immune responses in mono- and poly-sensitized models. Such experimental studies will contribute to the improvement of new treatment approaches with increased effectiveness against the constantly increasing number of allergic diseases

Thioredoxin from the Indianmeal moth Plodia interpunctella: cloning and test of the allergenic potential in mice.

BACKGROUND/OBJECTIVE: The Indianmeal moth Plodia interpunctella is a highly prevalent food pest in human dwellings, and has been shown to contain a number of allergens. So far, only one of these, the arginine kinase (Plo i 1) has been identified. OBJECTIVE: The aim of this study was to identify further allergens and characterise these in comparison to Plo i 1. METHOD: A cDNA library from whole adult P. interpunctella was screened with the serum of a patient with indoor allergy and IgE to moths, and thioredoxin was identified as an IgE-binding protein. Recombinant thioredoxin was generated in E. coli, and tested together with Plo i 1 and whole moth extracts in IgE immunoblots against a large panel of indoor allergic patients' sera. BALB/c mice were immunised with recombinant thioredoxin and Plo i 1, and antibody production, mediator release from RBL cells, T-cell proliferation and cytokine production were measured. RESULT: For the first time a thioredoxin from an animal species was identified as allergen. About 8% of the sera from patients with IgE against moth extracts reacted with recombinant P. interpunctella thioredoxin, compared to 25% reacting with recombinant Plo i 1. In immunised BALB/c mice, the recombinant allergens both induced classical Th2-biased immune responses such as induction IgE and IgG1 antibodies, upregulation of IL-5 and IL-4 and basophil degranulation. CONCLUSION: Thioredoxin from moths like Plo i 1 acts like a classical Type I allergen as do the thioredoxins from wheat or corn. This clearly supports the pan-allergen nature of thioredoxin. The designation Plo i 2 is suggested for the new P. interpunctella allergen

Oesophagostomum dentatum extract modulates T cell-dependent immune responses to bystander antigens and prevents the development of allergy in mice.

One third of the human population is currently infected by one or more species of parasitic helminths. Certain helminths establish long-term chronic infections resulting in a modulation of the host's immune system with attenuated responsiveness to "bystander" antigens such as allergens or vaccines. In this study we investigated whether parasite-derived products suppress the development of allergic inflammation in a mouse model. We show that extract derived from adult male Oesophagostomum dentatum (eMOD) induced Th2 and regulatory responses in BALB/c mice. Stimulation of bone marrow-derived dendritic cells induced production of regulatory cytokines IL-10 and TGF-beta. In a mouse model of birch pollen allergy, co-administration of eMOD with sensitizing allergen Bet v 1 markedly reduced the production of allergen-specific antibodies in serum as well as IgE-dependent basophil degranulation. Furthermore, eMOD prevented the development of airway inflammation, as demonstrated by attenuation of bronchoalveolar lavages eosinophil influx, peribronchial inflammatory infiltrate, and mucus secretion in lungs and IL-4 and IL-5 levels in lung cell cultures. Reduced secretion of Th2-related cytokines by birch pollen-re-stimulated splenocytes and mesenteric lymph node cells was observed in eMOD-treated/sensitized and challenged mice in comparison to sensitized and challenged controls. The suppressive effects of eMOD were heat-stable. Immunization with model antigens in the presence of eMOD reduced production of antibodies to thymus-dependent but not to thymus-independent antigen, suggesting that suppression of the immune responses by eMOD was mediated by interference with antigen presenting cell or T helper cell function but did not directly suppress B cell function. In conclusion, we have shown that eMOD possesses immunomodulatory properties and that heat-stable factors in eMOD are responsible for the dramatic suppression of allergic responses in a mouse model of type I allergy. The identification and characterization of parasite-derived immune-modulating molecules might have potential for designing novel prophylactic/therapeutic strategies for immune-mediated diseases

IgE immunoblots with patients' sera.

<p>Top panels: extracts from whole mediterranean flour moths (<i>E. kuehniella</i>) were separated by polyacrylamide electrophoresis, blotted onto nitrocellulose, and probed with the sera from various groups of patients with indoor allergy or seafood allergy as well as from control individuals. The indoor allergic patient groups were: M, housedust mite allergic patients (n = 69), M+D, patients allergic to mites and animal dander (n = 43), D−M, patients with allergy to animal dander but not to mites (n = 20). Patients SF, seafood allergic patients (n = 22), NC, nonallergic control individuals (n = 2), BC, buffer control without serum, only secondary anti-IgE antibody. Molecular weights [kDa] are indicated at the left side. Bottom panels, the recombinant <i>P. interpunctella</i> 40 kDa arginine kinase (AK) and 12 kDa thioredoxin (TRX) were probed with those patients' sera containing IgE to <i>E. kuehniella</i>, and only the parts of the strips containing the two recombinant allergens are shown.</p

In vitro splenocyte stimulation.

<p>Stimulation index [SI] of thioredoxin or arginine kinase restimulated spleen cell cultures of mice, treated with 1 µg (black bars), 5 µg (grey bars) or 25 µg (striped bars) antigen, compared to sham-treated controls (white bars). **p<0.01.</p

Immunoblot inhibition experiment.

<p>Extracts from <i>E. kuehniella</i> were separated by polyacrylamide electrophoresis, blotted onto nitrocellulose, and strips were probed with serum samples preincubated with or without 10 µg of recombinant allergen overnight. Lane 1, thioredoxin (Plo i 2) positive serum; lane 2, same serum preincubated with recombinant <i>P. interpunctella</i> thioredoxin; lane 3, arginine kinase (Plo i 1) positive serum; lane 4, same serum preincubated with recombinant Plo i 1; lane 5, nonallergic control serum, lane 6, same serum preincubated with Plo i 2; lane 7, same serum preincubated with Plo i 1; lane 8, buffer control. Molecular weights [kDa] are indicated at the left side, the positions of thioredoxin (TRX) and arginine kinase (AK) at the right side.</p

Cellular immune responses.

<p>IL-5, IL-4 and IFN-γ levels in supernatants of restimulated spleen cell cultures of mice, treated with 1 µg (black bars), 5 µg (grey bars) or 25 µg (striped bars) of (A), thioredoxin or (B), arginine kinase, or sham-treated (white bars). Groups were compared to sham-treated controls. *p<0.05, **p<0.01.</p

Construction and characterization of the pollen-food-chimer.

<p>(<b>A</b>) Design of the <i>pHis-parallel 2</i>-chimer composed of Bet v 1 protein, flanked by peptides from Api g 1 and Dau c 1. (<b>B</b>) Immunoblot: IgE binding to the chimer of sera from chimer-pretreated/poly-sensitized mice (lane 1) and of BP allergic patients with BPRFA (lane 5, 6, 7), and of Bet v 1 monoclonal antibody (lane 3). Negative controls sera from untreated mouse and non-allergic patient, or buffer control were run in parallel (lane 2, 4, 8. 9).</p