Novel allergen preparations for use in allergen-specific immunotherapy

Allergy is a common disease in the industrialized countries, affecting approximately 25% of the population. Therefore, there is a need to find new treatment strategies to improve the quality of life for allergic individuals. Today the only treatment that gives long-lasting reduction of allergic symptoms is allergen-specific immunotherapy (SIT). However, despite successful clinical outcome, the method as it is performed today has some drawbacks such as therapy associated side-effects. There is thus an urge to optimize the treatment strategy. This can be approached in different ways, e.g. to use alternative adjuvants instead of the commonly used aluminum hydroxide (alum), which has for instance been linked to granuloma formation, or create hypoallergens with reduced IgE binding in order to minimize the risk of side-effects upon injection. The aim with this thesis was to evaluate carbohydrate-based particles (CBPs) as a novel adjuvant and allergen carrier for use in SIT. Furthermore, we wanted to employ a novel strategy, DNA shuffling, to generate hypoallergens of mite group 2 allergens from the mite species Lepidoglyphus destructor and Glycyphagus domesticus. The CBPs were first evaluated in an in vitro system with immature human monocyte-derived dendritic cells (iMDDCs). Here we showed that the major recombinant cat allergen Fel d 1 (rFel d 1) covalently coupled to the particles was readily ingested by the iMDDCs. Upon uptake of the CBP coupled rFel d 1, the cells upregulated cell surface expression of the co-stimulatory molecule CD86. Moreover, the cells produced increased levels of TNFand IL-8. Taken together, the MDDCs showed a semi-mature phenotype, and in the context of SIT, this might be beneficial. Instead of initiating a strong Th1 response, these dendritic cells might induce a mixed Th1/Th2 response or perhaps a regulatory response. To further investigate this, a mouse model for cat allergy was established. In this model, mice were sensitized with rFel d 1 followed by intranasal challenge in order to establish an allergic inflammation in the airways, characterized by an infiltration of eosinophils and an enhanced airway hyperreactivity (AHR). By utilizing this model we could show that by treating mice with rFel d 1 coupled to CBPs, they responded with a reduced AHR after allergen challenge compared to mice treated with empty CBPs. This was also reflected in the bronchoalveolar lavage (BAL) fluid with reduced levels of infiltrating eosinophils. Another possible application for the CBPs could be in prophylactic settings and therefore we moreover administered the CBP coupled rFel d 1 before the mice were sensitized, to investigate whether we could prevent the onset of allergy. This was indeed demonstrated, with mice pretreated responding with lower AHR and lower levels of infiltrating eosinophils in the BAL than sham pre-treated controls. To further investigate the in vivo faith of the CBPs, the particles were either labelled with FITC or coupled to radioactive [Se]-labelled rFel d 1. By performing immunizations with these preparations we could show that the particles are taken up by a CD11cF4/80 cell population and transported both to the draining lymph nodes and later also the spleen. Interestingly, the majority of the injected particles remained at the site of injection five days later and radioactivity originating from rFel d 1 could be detected at the site one week after injection. This supports the role of CBPs as an allergen carrier which prolongs the antigen exposure and thereby can induce tolerance to the allergen. This thesis also supports the use of DNA shuffling as a method to create hypoallergens for use in SIT. We successfully created two hypoallergen candidates from the major mite allergens Lep d 2 and Gly d 2 which demonstrated reduced IgE binding but retained T-cell epitopes. When applied in vivo, antibodies with blocking capacity were produced which could reduce the degranulation of rat basophil leukaemia cells sensitized with serum from mite allergic patients. In summary, this method has been proven useful to create hypoallergens without using prior knowledge either of Band T-cell epitopes or structure of the parental proteins. In conclusion, the work presented here contributes to the aim of creating tools for novel treatment strategies in order to make SIT safer and more efficient, which ultimately will be beneficial to patients suffering from allergic disease. LIST OF PUBLICATIONS I. Andersson TN, Ekman GJ, Gronlund H, Buentke E, Eriksson TL, Scheynius A, Van Hage-Hamsten M, Gafvelin G. A novel adjuvant-allergen complex, CBP-rFel d 1, induces up-regulation of CD86 expression and enhances cytokine release by human dendritic cells in vitro. Immunology, 2004, Oct 113(2):253-9 II. Neimert-Andersson T, Thunberg S*, Swedin L*, Wiedermann U, JacobssonEkman G, Dahlen S-E, Scheynius A, Gronlund H, van Hage M, Gafvelin G. Carbohydrate-based particles reduce allergic inflammation in a mouse model for cat allergy. Allergy, 2008, In press * Shared second authorship ¤ These authors contributed equally III. Thunberg S*, Neimert-Andersson T*, Cheng Q, Wermeling F, Bergstrom U, Swedin L, Dahlen S, Arner E, Scheynius A, Karlsson M, Gafvelin G, van Hage M, Gronlund H. Prolonged antigen-exposure with carbohydrate particle based vaccination prevents allergic immune responses. Submitted * Shared first authorship ¤ These authors contributed equally IV. Gafvelin G*, Parmley S*, Neimert-Andersson T, Blank U, Eriksson TL, van Hage M, Punnonen J. Hypoallergens for allergen-specific immunotherapy by directed molecular evolution of mite group 2 allergens. J Biol Chem, 2007, Feb 9;282(6):3778-87 *These authors contributed equally Publication not included in the thesis: 1. Cirkovic Velickovic T, Thunberg S, Polovic N, Neimert-Andersson T, Gronlund H, van Hage M and Gafvelin G. Low levels of endotoxin enhance allergen-stimulated proliferation and reduce the threshold for activation in human peripheral blood cells. Int Arch Allergy Immunol, 2007, Dec 14;146(1):1-10