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Division of Immunopathology
Department of Pathophysiology and Allergy Research
Center of Pathophysiology, Infectiology & Immunology

Medical University of Vienna

Vienna General Hospital, AKH, 3Q
Waehringer Guertel 18-20
A-1090 Vienna, Austria
 

Allergen mimotopes for prophylaxis, therapy and diagnosis of type I allergy [Jensen-Jarolim]

Project summary

It is planned to continue the strategies started in the first and second project period, where mimotopes were generated and successfully applied for epitope analysis and immunizations. Now, it is aimed to go one step further and use mimotopes in therapeutic settings in murine models of respiratory and food allergy, and for diagnosis of human allergy. In detail, we plan the following experiments: A) Mimotopes and anti-idiotypic Fab "mimotopes" for prophylaxis and therapy: Anti-idiotypic Fab fragments with mimicry potential to grass pollen allergen Phl p 1 have been selected and are presently sequenced. Much further advanced are our Phl p 5-specific mimotope reagents. Therefore, right after the onset of a new project period the prophylactic and therapeutic potencies of these anti-idiotypic Fab fragments and mimotope peptides can be compared side by side in an established murine asthma model. The readouts will focus on the humoral and cytokine patterns to comprehend the immune modulation resulting from mimotope or anti-Id versus allergen treatments. In a later stage, the experiments will be repeated using the generated Phl p 1 anti-idiotypes, followed by a combined Phl p 1/Phl p5 mimotope treatment to optimize the effects. To elaborate the advantage of mimotopes over T-cell epitope peptides, special emphasis will be given on the analysis of late phase reactions in mucosa and skin within mimotope vaccinations. Like in all described murine models, tissue analyses of the respective target organs will be performed where inflammatory cells, T-lymphocytes and cytokines will be stained and semiquantitatively evaluated by Tissue FACS technology. Additionally, splenocytes of the treated mice will be harvested and analysed in ELISpot assay. In parallel, affected tissues as well as cells of interest, e.g. splenocytes, will be snap-frozen in liquid nitrogen followed by RNA isolation for subsequent gene expression profiling of immunity/allergy-related markers assayed by quantitative real-time PCR. Furthermore, in cooperation with Salzburg, Phl p 5 mimotopes and anti- idiotypic Fabs, translated into DNA vaccines will be tested in murine models. Among them DNA (peptide) mimotopes were already tested in preliminary immunization experiments, indicating the induction of blocking antibodies. The anti-idiotypic Fab inserts are presently cloned into the vectors.
Similarly, the Der p 1 and Der p 2 mimotopes of house dust mite (HDM) are scheduled for prophylactic and therapeutic applications. Here, a murine mite allergy model has to be established first. Since the dermal route has been shown to be effective in allergy induction and especially relevant for HDM, we aim to establish novel animal models in our lab. 1.) HDM asthma model: BALB/c mice will be sensitized intraperitoneally with HDM and consecutive aerosol challenges to achieve an asthma phenotype. 2.) HDM atopic dermatitis model: Dermatitis-prone inbred mice NC/Nga will be treated by repetitive epicutaneous applications of house dust mite allergens and staphylococcal enterotoxin B. These mice develop additionally asthma when aerosol challenged thereafter (1). Both models will serve to monitor the changes of disease through mimotope vaccination in prophylactic and therapeutic settings. When successful, the method will be expanded to grass pollen allergens, where cutaneous sensitization may also play a role and atopic eczema aggravates through contact with aeroallergens (2).
The generated parvalbumin mimotopes will be applied in immunization and therapy of food-allergic mice in our antiacid model, where digestion of fed protein is reduced by pharmacologic stomach acid suppression and neutralization. The resulting phenotype resembles human food allergy and we expect to be able to modulate it by the mimotope vaccines. Again, major focus of readouts will be on tissue morphology. Additionally, much focus will be given on the association of food allergy with asthma and skin symptoms, like atopic dermatitis. Therefore, a murine “skin – food – asthma model “ will be developed, where sensitization is supported by percutaneous allergen applications.
B) To the best of our knowledge mimotopes are today not yet applied for diagnosis. However, since the immune reactions to allergens are biologically divergent depending on the epitope specificity of the induced antibodies (3-5), this approach might be valuable. Therefore, we aim to couple mimotope peptides onto microchips for testing of large cohorts of healthy persons, allergic patients and patients before, during and after allergen immunotherapy. The study will be done in collaboration with Prof. Zsolt Szepfalusi, University Clinics of Pediatrics, Dr. Adriano Mari, IDI-IRCCS, Rome, Italy, and Dr. Harwanegg, VBC Genomics, Vienna. Moreover, we are planning to post mimotope informations of allergens on a web-based information tool (“www.mimotope.atâ€) to create an archive of structural epitopes. Taken together, this project aims to further develop and improve mimotopes for their application in prophylaxis, therapy and diagnosis.