CD4 + inkt Cells ; New Target for Treatment of Asthma in Human

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1 Award 2005

2 Award CD4 + inkt Cells ; New Target for Treatment of Asthma in Human Omid Akbari, Ph.D. Division of Immunology Children s Hospital Boston Department of Pediatrics Harvard Medical School, Karp Research Laboratories One Blackfan Circle, 10 th floor Boston, MA USA Tel: omid.akbari@childrens.harvard.edu

3 Introduction Asthma is an inflammatory disease of the respiratory tract that has increased in prevalence over the past two decades, reaching epidemic proportions in industrialized countries 1,2. Asthma is characterized by airway inflammation dominated by eosinophils and CD4 + T lymphocytes 3,4. The lung CD4 + cells in asthmatic subjects produce predominantly Th2 cytokines IL-4, IL-5 and IL-13, which play essential roles in asthma by enhancing the growth, differentiation, and recruitment of eosinophils, basophils, mast cells, and IgE producing B cells 5, and by directly inducing airway hyperreactivity (AHR) 6,7, a cardinal feature of asthma. Thus, conventional MHC class II restricted CD4 + Th2 cells, which have been observed in the airways of virtually all patients with asthma 8, are thought to play an obligatory role in the pathogenesis of bronchial asthma 9. The CD4 cell surface marker is expressed not only by conventional CD4 + T cells, but also by NKT cells, which comprise a unique subset of lymphocytes that express features of both classical T cells and natural killer (NK) cells. In humans, NKT cells express CD4, or CD8 (a small subset), or neither (CD4/CD8 double negative). Many NKT cells express a highly restricted T cell receptor (TCR) repertoire consisting of Vα14-Jα18 (in mice) or Vα24-Jα18 (in humans), and are called invariant TCR + NKT cells (inkt) 10. Through this invariant TCR, inkt cells recognize glycolipid antigens presented by the nonpolymorphic MHC class I protein, CD1d and rapidly produce large quantities of cytokines including IL-4 and IFN-γ, which enhance the function of dendritic cells, NK cells, B cells, as well as conventional CD4 + and CD8 + T cells 11. This rapid production of cytokines by inkt cells is a manifestation of innate-like immunity, and endows inkt cells with the capacity to critically amplify and regulate adaptive immune responses, and thus link innate and adaptive immunity Moreover, inkt cells have been shown in animal models to regulate the development of autoimmune, as well as antimicrobial, anti-tumor and anti-transplant immune responses Furthermore, we and others recently showed that allergen-induced AHR in mice failed to occur in the absence of inkt cells producing IL-4 and IL-13 18,19, demonstrating an essential role for inkt cells in the development of murine allergic asthma. To investigate whether inkt cells exhibit a similar important role in human asthma, we studied 13 patients with bronchial asthma, as defined by the American Thoracic Society 20, as well as six normal healthy subjects and five patients with biopsy proven sarcoidosis, a respiratory inflammatory disease in which large numbers of CD4 + Th1 cells are present in the lungs 21,22. Nine of the 13 asthmatic subjects received inhaled corticosteroids to control their symptoms, and all had airflow obstruction documented by computerized spirometry 4 (Table 1). Subject Characteristics Asthma Sarcoidosis Healthy n=13 n=5 Controls n = 6 Male gender (%) 8 (62%) 4 (80%) 2 (33%) Age + SD Mean total serum IgE (IU/ ml) * not done FEV 1 + SD (% predicted) FEV 1 / FVC + SD Table 1: Subject characteristics and lung function of patients with asthma, sarcoidosis and healthy control subjects. * p < 0.05 by Mann-Whitney t test (comparing the means of the asthma and control groups). p < 0.01 by one-way ANOVA, (post test Tukey-Kramer multiple comparisons test demonstrated a significant difference between the Asthma group and the Sarcoidosis group). Award winner 2005 Omar Akbari

4 Ten of the 13 asthmatic patients had the most common form of asthma, allergic asthma, with elevated total IgE (191 IU/ml compared to 21 IU/ml in control subjects), allergen specific IgE to at least one of a panel of common allergens, and a history of bronchospasm after allergen exposure. We examined bronchoalveolar lavage (BAL) fluid and airway endobronchial biopsies from these subjects (obtained by flexible fiberoptic bronchoscopy) for the presence of CD4 +, CD8 + and inkt cells. As expected, there was an increase in the proportion of lymphocytes in BAL fluid from asthmatic and sarcoidosis patients compared with healthy subjects. In both groups the majority of lymphocytes were CD4 + (Table 2). Bronchoalveolar Fluid Cell Composition Mean + SD (range) Asthma n = 13 Sarcoidosis n = 5 Healthy Controls n = 6 Cell count (total cells in BAL) (range) % Macrophage (range) % Neutrophils (range) % Eosinophils (range) % Lymphocytes (range) % CD3 (range) % CD4 (range) % CD8 (range) 5.2 x 10 6 ( x 10 6 ) (72-92) (0-5) (0-16) (4 -v 26) (87 95) (36-85) (9-51) 8.2 x 10 6 ( x 10 6 ) (48-94) (0-2) 0 (0-0) (4-40) (87 94) (63-83) (16-35) 2.0 x 10 6 ** ( x 10 6 ) (83-94) (0-7) (0-1) (2-15) (87 97) (16-60) (31-77) Table 2: BAL fluid cell profiles of patients with asthma and sarcoidosis, and healthy controls. Nine of the 13 patients with asthma were being treated with inhaled corticosteroids. Four patients had not received corticosteroids for more than three months. Data are presented as Mean + SEM (with range). ** p < 0.01 by ANOVA. post test Tukey-Kramer multiple comparisons test demonstrated a significant difference between the Asthma group and the Sarcoidosis group (p < 0.05). post test Tukey-Kramer multiple comparisons test demonstrated a significant difference between the Sarcoidosis group and the control group (p < 0.01). Award winner 2005 Omar Akbari

5 Fig 1: Significant numbers of inkt cells are present in the BAL fluid of patients with asthma but not of healthy control subjects. Cells from the BAL fluid of patients with asthma were stained with both FITC conjugated monoclonal antibody against inkt TCR (6B11) and PE conjugated α Gal Cer loaded CD1d tetramer and analyzed for double positive cells. Three representative dot plots from patients with asthma (Patients A, B, C) and three representative dot plots from control subjects (Subjects E, F and G) are shown. The great variability in double positive cells in the patients with asthma (13.7% to 71%) is due to the presence of red blood cells in the BAL fluid of Patients. The cursor placement is determined by results with negative controls, and is different for each sample. An example of a representative negative control (staining with isotype control mab and empty CD1d tetramer) of cells from Patient C is shown. Numbers in each quadrant represent the percentage of positive cells. Significant numbers of inkt cells are present in the BAL fluid of patients with asthma We also examined the BAL fluid for the presence of inkt cells using α-galactocyl-ceramide (α-galcer) loaded CD1d tetramers, which specifically bind to the invariant TCR of inkt cells 23, and with a monoclonal antibody, 6B11, that specifically recognizes the CDR3 region of the Vα24Jα18 TCR of human inkt cells 24. Figure 1 shows that both reagents stained a large number of cells in the BAL fluid from patients with asthma, indicating that inkt cells were present in the lungs of patients with asthma. By contrast, virtually no inkt cells were present in the BAL fluid from healthy subjects. The majority of CD4+ cells in BAL fluid of patients with asthma are inkt cells Because inkt cells can express the CD4 cell surface marker, and because large numbers of CD4 + cells are known to be present in the lungs of patients with asthma, we asked, what fraction of the CD4 + T cells in BAL fluid of the asthmatic individuals were inkt cells Surprisingly, we Award winner 2005 Omar Akbari

6 found that the majority of the CD4 + T cells in the BAL fluid of patients with asthma were inkt cells. In the 13 asthmatic subjects, 60-85% of the CD4 cells expressed the invariant TCR Vα24 (Figure 2a), and essentially no CD8 cells expressed the invariant TCR Vα24 (data not shown). In contrast, normal individuals had essentially no CD1d tetramer staining cells in their BAL fluid (data not shown). FIGURE 2: The majority of CD4 + cells in BAL fluid of patients with asthma are inkt cells. Fig 2: Analysis of BAL inkt cells for expression of CD4 and CD8. Cells from the BAL fluid were stained with anti- CD3 mab, α-galcer loaded CD1d tetramers and anti-cd4 mab. We gated on CD3 + cells to exclude red blood cells, which contaminated some BAL fluid specimens, and to focus the analysis on pulmonary T cells. Numbers in each quadrant represent the percentage of positive cells. Similar results were obtained using immuno-fluorescence and confocal laser scanning microscopy of biopsy specimens from patients with asthma. Figure 3a shows a photomicrograph of one biopsy, which demonstrates the typical features of bronchial asthma, with basement membrane (laminareticularis) thickening, epithelial disruption, and the presence of a monocnuclear cell infiltrate (including inkt cells) in the submucosa/lamina propria. Figure 3b demonstrates using confocal laser microscopy that nearly all of the lymphocytes in the lamina propria express both CD4 and the invariant TCR Vα24. In contrast, Figure 3c shows that in sarcoidosis the lymphocytes express CD4, but not Vα24, and therefore are not NKT cells. FIGURE 3: The CD4 + cells in the airway of patients with asthma, but not patients with sarcoidosis, are inkt cells. Fig 3A: (Left) In this photomicrograph of an endobronchial biopsy from a patient with asthma, cells immediately beneath the lamina reticularis demonstrate intense staining with FITC conjugated antibody (6B11) (directed against NKT cells). The background tissue morphology demonstrates features typical of asthma, including basement Award winner 2005 Omar Akbari

7 membrane (lamina reticularis) thickening (triangle), epithelial disruption, and the presence of monocnuclear cell infiltrate (including inkt cells) (arrow) in the submucosa/lamina propria. (Right) Section from the same endobronchial biopsy sample stained with H&E showing the typical features of chronic asthma. Fig 3B and C: These double immunofluorescence staining and laser confocal images of bronchial biopsies from a patient with asthma (Fig 3B) and a patient with sarcoidosis (Fig 3C) demonstrate that the CD4 + cells in bronchial asthma are inkt cells, but those in sarcoidosis are not. Lung biopsies were obtained in an identical manner in both patients and were stained with PE conjugated CD4 (red), and FITC conjugated 6B11 mab (blue) and analyzed with confocal microscopy. The overlay results (pink) indicated that while almost none of the CD4 + lymphocytes in the lungs of a patient with sarcoidosis expressed the invariant TCR Vα24 (Fig 3C), nearly all of the CD4 + infiltrating lymphocytes in asthma co-express the invariant TCR Vα24 (Fig 3B). High percentage of CD3 positive cells are inkt cells in BAL fluid of patients with asthma Analysis of the BAL fluid of patients with asthma, indicated that 55-85% of the CD3 + cells were inkt cells, whereas in patients with sarcoidosis, <3% were inkt cells (Figure 3d). The presence of inkt cells in the lungs of patients with asthma did not appear to be due to inhaled corticosteroid therapy, because four of the 13 patients had not been treated with corticosteroids for >3 months prior to bronchoscopy, and all four subjects had large number of pulmonary inkt cells (Patients A, E, F and G, Figure 3d). These studies indicate that CD4 + inkt cells are essentially absent from the lungs of healthy subjects and patients with sarcoidosis, but are specificall present in high numbers in asthma, whether or not patients receive corticosteroids. Fig 3D: Percentage of CD3 positive cells that are inkt cells in BAL fluid obtained from patients with asthma or sarcoidosis. BAL was performed on patients with asthma (n=5) or patients with sarcoid (n=7). Asthma patients A, E, F and G did not receive corticosteroids for >3 months prior to bronchoscopy. Cells were harvested and stained with anti- CD3 mab and -GalCer loaded CD1d tetramers. BAL fluid inkt cells from patients with asthma produce IL-4 and IL-13, but not IFN-γ. Like inkt cells in mouse models of asthma, the inkt in the lungs of patients with asthma produced both IL-4 and IL-13, but very little IFN-γ (Figure 4a). In contrast, inkt cells in the peripheral blood of all of our subjects (asthmatic, sarcoidosis and normal individuals) produced all three cytokines (Figure 4b), suggesting the compartmentalization of one subset of inkt cells, (i.e. those producing Th2 cytokines) in the lungs of patients with bronchial asthma. Thus, the inkt cell population in the lungs of patients with asthma appears to be unique and distinct, consistent with the fact that in the lungs of patients with asthma, >95% of the inkt cells are CD4 + (Figure 4c) (and inkt cells that are CD8 + or CD4/CD8 double negative are rare), while in the peripheral blood of asthmatic patients, normal individuals and sarcoidosis patients only about 44% of the inkt cells are CD4 + (53% are CD4/CD8 double negative and 3% are CD8 + ) (Figure 4d). Award winner 2005 Omar Akbari

8 FIGURE 4: BAL fluid inkt cells produce primarily IL-4 and IL-13, but not IFN-γ. Fig 4A & B: Both BAL fluid cells and PBMCs from patients with asthma were isolated and stimulated with PMA/ ionomycin, double stained for intracellular cytokines, and examined by flow cytometry, gating on CD1d tetramer positive cells. Fig 4C: The vast majority of inkt cell in BAL fluid from patients with asthma are CD4 +. Cells from BAL fluid of 6 patients with asthma were purified and stained with α GalCer loaded CD1d tetramers and anti-cd4 and CD8 mabs. The inkt cells were isolated by MACs and analyzed for expression of CD4 or CD8 by flow cytometry. The bars demonstrate the percentages of CD4, CD8 or double negative inkt cells in each individual. Fig 4D: Comparison of inkt cell subsets in peripheral blood of patients with asthma, sarcoidosis and from healthy subjects. inkt cells were purified from peripheral blood and stained with α-galcer loaded CD1d tetramers, anti- CD4 and CD8 mabs. The inkt cells were isolated by MACs and analyzed for expression of CD4 or CD8 by flow cytometry. Award winner 2005 Omar Akbari

9 Future direction and implications for drug development: Our studies, showing that CD4 + inkt cells are specifically present in the lungs of patients with asthma and not in the lungs of normal healthy subjects or patients with sarcoidosis, strongly suggest that inkt cells play a central role in the pathogenesis of human asthma. The number of inkt cells in the lungs of patients with asthma is striking and unexpected, especially given the fact that in peripheral blood inkt cells constitute <0.1% of mononuclear cells and <1% of CD4 + T cells 25. In addition, the fact that >90% of the inkt cells in the lungs of patients with asthma are CD4 +, while only about 50% of inkt cells in peripheral blood are CD4 +, suggests that in asthma the CD4 + subset of inkt cells is recruited preferentially into the lungs, and enriched at least 100-fold over the frequency found in peripheral blood. The preferential recruitment of such inkt cells may be related to differential expression of chemokine receptors on the CD4+ inkt cell subset, which is thought to preferentially produce IL-4 and IL Accordingly, our studies indicate that the immunology of asthma must be studied not by examination of peripheral blood, but rather by evaluation of cells from within the target organ (e.g., the lung in asthma). This same principle may hold true for other diseases in which inkt cells have been suggested to play an important role. The CD4 + inkt cells that drive the development of asthma express an invariant TCR that recognizes glycolipid antigens 12 that are just now being defined. These antigens appear to be highly conserved among mice and humans, and include the synthetic glycolipd, α-galcer, the self glycolipid isoglobotrihexosylceramide (igb3) 29,30 and bacterial glycosphingolipid 31,32. However, because many different protein antigens/allergens induce the development of asthma, we believe that conventional antigen-specific CD4 + Th2 cells respond on allergen challenge in the airways, altering self-glycolipid antigens within the lung. We propose that these modified self-glycolipid antigens, uncovered following allergen challenge or pulmonary injury, activate inkt cells that then induce the symptoms of asthma. While conventional CD4 + T cells may be important in the initial response to exogenous allergens, our studies suggest that inkt cells play the major pathogenic role once allergen sensitization is established. Finally, because inkt cells play such a prominent role in the pathogenesis of asthma, our studies suggest that future therapies for asthma that target pulmonary inkt cells may be highly effective. Award winner 2005 Omar Akbari

10 References 1. Holgate, S. T. The epidemic of allergy and asthma. Nature 402, b2-4 (1999). 2. Umetsu, D., McIntire, J., Macaubas, C., Akbari, O. & DeKruyff, R. Asthma: an epidemic of dysregulated immunity. Nature Immunology 3, (2002). 3. Busse, W. W. & Lemanske, R. F., Jr. Asthma. N Engl J Med 344, (2001). 4. Faul, J., Demers, E., Burke, C. & Poulter, L. The reproducibility of repeat measures of airway inflammation in stable atopic asthma. Am J Respir Crit Care Med 160, (1999). 5. Holt, P. G., Macaubas, C., Stumbles, P. A. & Sly, P. D. The role of allergy in the development of asthma. Nature 402, B12-17 (1999). 6. Grunig, G. et al. Requirement for IL-13 independently of IL-4 in experimental asthma. Science 282, (1998). 7. Wills-Karp, M. et al. Interleukin-13: central mediator of allergic asthma. Science 282, (1998). 8. Robinson, D. S. et al. Predominant Th2-like bronchoalveolar T-lymphocyte population in atopic asthma. N. Engl. J. Med. 326, (1992). 9. Cohn, L., Elias, J. A. & Chupp, G. L. Asthma: mechanisms of disease persistence and progression. Annu Rev Immunol 22, (2004). 10. Taniguchi, M., Harada, M., Kojo, S., Nakayama, T. & Wakao, H. The regulatory role of Valpha14 NKT cells in innate and acquired immune response. Annu Rev Immunol 21, (2003). 11. Kronenberg, M. & Gapin, L. The unconventional lifestyle of NKT cells. Nat Rev Immunol 2, (2002). 12. Brigl, M. & Brenner, M. B. CD1: antigen presentation and T cell function. Annu Rev Immunol 22, (2004). 13. Heller, F., Fuss, I., Nieuwenhuis, E., Blumberg, R. & Strober, W. Oxazolone colitis, a Th2 colitis model resembling ulcerative colitis, is mediated by IL-13-producing NK-T cells. Immunity 17, (2002). 14. Nieuwenhuis, E. E. et al. CD1d-dependent macrophage-mediated clearance of Pseudomonas aeruginosa from lung. Nat Med 8, (2002). 15. Terabe, M. et al. NKT cell-mediated repression of tumor immunosurveillance by IL-13 and the IL-4R-STAT6 pathway. Nat Immunol 1, (2000). 16. Cui, J. et al. Requirement for Valpha14 NKT cells in IL-12-mediated rejection of tumors. Science 278, (1997). 17. Wang, B., Geng, Y. & Wang, C. CD1-restricted NK T cells protect nonobese diabetic mice from developing diabetes. J Exp Med 194, (2001). 18. Akbari, O. et al. Essential role of NKT cells producing IL-4 and IL-13 in the development of allergen-induced airway hyperreactivity. Nature Medicine 9, (2003). 19. Lisbonne, M. et al. Cutting edge: invariant V alpha 14 NKT cells are required for allergen-induced airway inflammation and hyperreactivity in an experimental asthma model. J Immunol 171, (2003). Award winner 2005 Omar Akbari

11 20. Standards for the diagnosis and care of patients with chronic obstructive pulmonary disease (COPD) and asthma. Am Rev Respir Dis 136, (1987). 21. Wahlstrom, J. et al. Analysis of intracellular cytokines in CD4+ and CD8+ lung and blood T cells in sarcoidosis. Am J Respir Crit Care Med 163, (2001). 22. Agostini, C., Meneghin, A. & Semenzato, G. T-lymphocytes and cytokines in sarcoidosis. Curr Opin Pulm Med 8, (2002). 23. Sidobre, S. & Kronenberg, M. CD1 tetramers: a powerful tool for the analysis of glycolipidreactive T cells. J Immunol Methods 268, (2002). 24. Tahir, S. M. et al. Loss of IFN-gamma production by invariant NK T cells in advanced cancer. J Immunol 167, (2001). 25. Lee, P. et al. Testing the NKT cell hypothesis of human IDDM pathogenesis. J Clin Invest 110, (2002). 26. Gumperz, J. E., Miyake, S., Yamamura, T. & Brenner, M. B. Functionally distinct subsets of CD1d-restricted natural killer T cells revealed by CD1d tetramer staining. J Exp Med 195, (2002). 27. Lee, P. T., Benlagha, K., Teyton, L. & Bendelac, A. Distinct functional lineages of human V(alpha)24 natural killer T cells. J Exp Med 195, (2002). 28. Kim, C., Johnston, B. & Butcher, E. Trafficking machinery of NKT cells: shared and differential chemokine receptor expression among V alpha 24(+)V beta 11(+) NKT cell subsets with distinct cytokine-producing capacity. Blood 100, 11-6 (2002). 29. Naidenko, O. V. et al. Binding and antigen presentation of ceramide-containing glycolipids by soluble mouse and human CD1d molecules. J Exp Med 190, (1999). 30. Zhou, D. et al. Lysosomal Glycosphingolipid Recognition by NKT cells. Science in press, on line, PMID: (2004). 31. Kinjo, Y. et al. Recognition of bacterial glycosphingolipids by natural killer T cells. Nature (in press). 32. Mattner, J. et al. Both exogenous and endogenous glycolipid antigens activate NKT cells during microbial infections. Nature (in press). 33. Brossay, L. et al. CD1d-mediated recognition of an alpha-galactosylceramide by natural killer T cells is highly conserved through mammalian evolution. J Exp Med 188, (1998). Award winner 2005 Omar Akbari

12 Honorable Mention The transition of recombinant allergens from the bench into clinical applications Dr Verena Niederberger Medical University of Vienna Department of Otorhinolaryngology Waehringer Guertel A-1090 Vienna Austria Tel:

13 Introduction For nearly 100 years, diagnosis and immunotherapy of allergy was performed with extracts from allergen sources (e.g., pollen, mites, animal dander) (1). These extracts consist of complex mixtures of various allergens and non-allergenic components (2). I entered the research field of allergy when several research groups had made progress in the molecular characterization of allergens. Several important allergens from different allergen sources had been cloned and produced as recombinant proteins (reviewed in 3). Immunological studies had revealed that many of these recombinant allergens were similar to their natural counterparts. Cross-inhibition studies had shown that the allergen repertoire of important allergen sources was less complex than originally anticipated, and there was hope that it may be possible to eventually replace all important IgE epitopes from different allergen sources with recombinant allergens (reviewed in 3). The application of genetic engineering had resulted in the production of first examples of allergen derivatives with reduced IgE reactivity (reviewed in 4). Progress in the field of molecular allergen characterization thus promised to positively influence the development of diagnosis and therapy of allergic disease in the future. However, several important questions were open: It had yet to be shown that recombinant allergens carry all the relevant IgE epitopes contained in extracts from different allergen sources. The panel of recombinant allergens had (and, in some cases, still has) to be further completed by cloning and characterization of important allergens. It was necessary to perform experiments showing that recombinant allergens can be used for improved allergy diagnosis, and that this can result in improved indication for immunotherapy. Using recombinant allergens as new tools for immunological studies it was possible to investigate the mechanisms underlying the beginning and maintenance of allergic diseases as well as to get new insights into the immunological effects of immunotherapy. Recombinant allergen derivatives had to be carefully evaluated concerning their safety and usefulness for the treatment of allergic patients. Finally, it was necessary to show that recombinant allergen derivatives can successfully be used for vaccination of allergic patients and to study the immunological mechanisms underlying such a treatment. This review summarizes work done to advance recombinant birch and grass pollen allergens from the bench into clinical applications. Recombinant pollen allergens resemble the epitope spectrum of natural allergen extracts Grass and birch pollen allergic patients usually exhibit allergic symptoms upon contact with a wide variety of pollens from different grasses or birch-related trees (5, 6). Immunotherapy against grass or birch pollen allergy should thus ideally target symptoms caused by many different pollen species. We investigated the exact level of IgE-epitopes shared between crude pollen extracts from different grass or tree species and panels of grass and birch pollen allergens (7, 8). These experiments revealed that recombinant allergens derived from just one grass species and from birch alone would be sufficient for diagnosis and immunotherapy of grass and birch pollen allergy for patients from different geographic and climatic regions. Among trees of the order Fagales (birch, alder, hazel, hornbeam and oak), birch is regarded as the most relevant for allergic patients, because it sheds the usually primarily sensitizing pollen species (6, 9). Bet v 1, the major birch pollen allergen, is recognized by IgE antibodies of more than 95% of birch allergic patients (10, 11) and 60% are sensitized exclusively to Bet v 1 (12). Bet v 2, birch profilin, represents a target for IgE antibodies from 10-20% of these patients (13). Using sera from 102 patients from different parts of Europe we demonstrated that pollen from birch, alder, hornbeam, hazel, and oak contain allergens that share IgE epitopes with recombinant birch pollen allergens Bet v 1 and Bet v 2 (7). These two allergens accounted for an Honorable Mention 2005 Verena Niederberger

14 average level of 82% of Fagales pollen-specific IgE. The two molecules were thus shown to be excellent candidate molecules for the development of a vaccine that can be used to treat allergy caused by pollen from all Fagales trees. Given the fact that only 10-20% of patients have IgE antibodies to profilin (13), it appeared possible to develop a vaccine for birch pollen sensitization using only rbet v 1 as a basis. A similar approach was taken to evaluate allergens for clinical use in grass pollen allergic patients (8). Four different pollen allergens (Phl p 1, Phl p 2, Phl p 5, profilin) had been shown to be sufficient to diagnose grass pollen allergy in almost all grass allergic patients and were thus regarded as candidate molecules for the development of a grass pollen vaccine (14). A thorough characterization of pollen extracts from nine different grass species revealed that extracts from taxonomically less closely related species differed somewhat in their allergen content (8). Nevertheless, IgE inhibition experiments using 193 sera from grass pollen-allergic patients of different origin (Europe, America, and Asia) demonstrated that the four recombinant allergens accounted for the majority of IgE epitopes present in the extracts from the 9 grass species. The high average level (59%) of grass pollen-specific IgE directed against the mixture of the four recombinant allergens confirmed that these molecules were good candidates for the development of a vaccine, but also revealed that the panel of Timothy grass pollen allergens may require further completion. Molecular characterization of the cross-reactive allergen Phl p 7 from timothy grass pollen Besides Phl p 11 (15) and Aln g 4 (16), I have worked on Phl p 7, a highly cross-reactive Timothy grass pollen allergen (17). Phl p 7, an 8.6 kilo Dalton protein, was expressed in E. coli and purified to homogeneity. In-vivo tests in allergic patients demonstrated that although this allergen is recognized by IgE antibodies from only 10% of allergic patients the allergen is biologically highly active; small Phl p 7 doses are sufficient to induce strong allergic responses. Sequence analysis revealed that this allergen belongs to a family of pollen-specific calcium-binding proteins (16-18). Interestingly, IgE antibodies from allergic patients reacted much stronger with the calcium-bound form than with the calcium-depleted apo-rphl p 7, which we interpreted as a footprint of a preferential sensitization to the calcium-bound form (17). Because Phl p 7-homologous allergens are highly conserved and cross-reactive between pollen from different unrelated plants (18), patients who are sensitized against one allergen of this family often suffer from allergic symptoms throughout the whole flowering period of trees, grasses and weeds (19). Indication for immunotherapy in these patients needs careful consideration, because they usually exhibit positive test results with tree, grass and weed pollen, even though they are often genuinely sensitized against only against one of these allergen sources (2, 19). Figure 2 shows the skin test results of a patient who could easily be mistaken to be sensitized against birch pollen if no component-resolved diagnosis is performed. This patient shows clinically relevant sensitization to birch, grass and weed pollen although genuine sensitization to birch can be excluded due to lack of reactivity to the major birch pollen allergen, Bet v 1. To avoid immunotherapy with an extract from a source against which the patient is not genuinely sensitized, it is particularly important to differentiate between co-sensitization (i.e. genuine sensitization to each of the sources) and cross-sensitization (cross-reactivity to allergens contained in each of the sources) (2, 19-21). The importance of Phl p 7 thus lies in its use as a marker allergen for broad pollen sensitization, and recombinant Phl p 7 is now being used together with major source-specific recombinant allergens for the gate keeping test, a routine diagnostic test which allows the improved selection of patients for immunotherapy (22). Honorable Mention 2005 Verena Niederberger

15 Recombinant allergens to study immune responses Crude extracts of allergen sources are unsuitable for exact measurements of allergen-specific IgE, IgG and IgM antibody levels, because extracts are composed of varying amounts of allergens and other proteins, and a standardized measurement of antibodies to extract components is difficult to achieve, if not impossible. The measurement of IgG and IgM antibody levels is further complicated by the fact that allergic as well as non-allergic individuals physiologically mount IgG and IgM antibodies against various non-allergenic pollen proteins (reviewed in 23, Niederberger et al., unpublished results). The use of recombinant allergens for the measurement of allergen-specific antibody levels has enabled us to overcome the above mentioned problems and allowed us to study the development of allergen-specific antibody production during sensitization in childhood, during the boosting of the IgE response caused by seasonal allergen exposure and nasal provocation with allergens, and during immunotherapy with a grass pollen extract. Furthermore, we used recombinant allergens to study the effect of various factors and cytokines of importance in allergy on the permeability of the respiratory epithelium for allergens. The development of type I allergy is characterized by the formation of allergen-specific IgE antibodies. Whether isotype switching from IgM to IgE in allergic patients occurs directly or sequentially is a controversial issue that has been indirectly approached by in vitro studies and via experimental animal models (24, 25). By analysing the evolution of IgE, IgG 1-4 and IgM responses to different recombinant birch and grass pollen allergens in the sera from children who developed birch pollen and/or grass pollen allergy during the observation period, we aimed to determine whether there is a pattern in the antibody development to allergens that gives away information about class switch to IgE (26). The development of IgG 1-4 and IgE antibodies to the allergens was only partly synchronized and dissociated, and a rise of IgG antibodies was not preceded by an elevation of IgM antibody levels. These findings can best be explained by a partly sequential class switch involving few switch stations, or more likely, by direct class switching. Further experiments in adult allergic patients revealed that airway pollen exposure during the pollen season and during nasal provocation leads to a boost of allergen-specific IgE antibody levels and to a mild rise of IgG 4 antibodies, without inducing IgM antibody responses or novel sensitizations (Niederberger et al., unpublished data). IgE levels and systemic sensitivity increased after nasal but not after skin exposure, demonstrating the importance of the route of allergen contact. Kinetics and courses of allergen-specific antibody responses (IgM, IgG 1-4, IgE) during sensitization and later allergen exposure suggest that sensitization to a pattern of allergens is developed at one time (often during early childhood), while allergen-specific IgE responses in established allergy are driven by antigen contact rather than by cytokines controlling class switch to IgE (26). These findings are of importance for the development of treatment strategies, because they show that established allergy may be difficult to target via blockage of class-switch to IgE. Immunotherapy is the only causal form of treatment for type I allergic disease (27). Whether an induction of blocking IgG antibodies is relevant for the success of this treatment has remained controversial (reviewed in 28). We hypothesized that these conflicting results were caused by the impossibility to perform exact measurements of allergen-specific IgG antibody levels with allergen extracts. When we applied our assays to measure IgE, IgG 1-4 and IgM antibody levels to different timothy grass pollen allergens induced by immunotherapy with a monophosphoryl lipid A-adjuvanted vaccine, we found a strong induction of allergen-specific IgG 1 and IgG 4 antibody levels in actively treated patients, which were associated with clinical improvement (29). However, there was a great variation of IgG responses against the individual grass pollen Honorable Mention 2005 Verena Niederberger

16 allergens, presumably caused by their under-representation and varying immunogenicity in the extract-based vaccine. Furthermore, some patients mounted IgG antibodies primarily to non-allergenic extract components. This study thus demonstrated that it is impossible to correlate IgG antibody levels and clinical improvement using crude allergen extracts, and that on a component-resolved level there is an association between the levels of immunotherapy-induced IgG antibodies and the success of treatment. Using 125 I-labeled recombinant allergens we also studied the influence of various factors of allergic inflammation on the integrity and barrier function of respiratory epithelium for allergens (30). In a model using a respiratory epithelial cell line as a surrogate for respiratory epithelium, interferon gamma was identified as a potent factor capable of reducing epithelial barrier properties and enhancing trans-epithelial allergen penetration. The biological significance of this effect was demonstrated by experiments which yielded a more than sevenfold increase of histamine release from sensitized basophils due to the increase of submucosal allergen concentrations caused by interferon gamma. These findings indicate a hitherto unknown link between chronic and acute allergic reactions, and indicate that therapeutic anti-allergic strategies should aim to control both, the acute and chronic facets of allergic inflammation. Recombinant allergens for in-vivo allergy testing For the selection of allergens for the preparation of a vaccine it is crucial to know their ability to induce allergic reactions, i.e., their biological activity. In a series of experiments we compared different diagnostic systems for the evaluation of the biological activity of respiratory allergens (31). Recombinant pollen allergens were used for a comparison of the levels of allergen-specific serum IgE and IgG levels, the weal and flare reaction in skin prick tests, and the allergic reaction to allergen exposure in nasal provocation tests, a test system that closely simulates natural respiratory allergen exposure. All IgE-reactive allergens induced immediate skin and nasal reactions, and there was a significant correlation between results from skin prick tests and nasal provocation tests, demonstrating on a molecular level that skin testing provides a good reflection of immediate type respiratory sensitivity. Unexpectedly, some less frequently detected allergens with low IgE-binding capacity (i.e. rphl p 2, rbet v 2) induced rather strong allergic reactions, comparable to major allergens with high IgE binding capacity. Standardization of allergen extracts is usually performed exclusively regarding the content of major allergens (32), which may be a disadvantage for patients who biologically react strongly with a minor allergen. Development and characterization of hypoallergenic allergen molecules for therapy of allergy Based on the knowledge of the cdna and structure of common and relevant allergens it has become possible to develop a variety of molecular immunotherapy strategies. Wildtype-like allergens, chemically modified allergens, T-cell and B-cell peptides, mimotopes, and genetically engineered whole recombinant allergen derivatives with reduced allergenic activity have been proposed for treatment (reviewed in 1). The advantage of the latter approach is that is possible in most cases to preserve the repertoire of T-cell epitopes, which are necessary to induce T-cell tolerance, and the immunogenic structures that are essential to inducing blocking antibody responses, while at the same time the ability of the molecules to induce type I allergic reactions can be reduced (reviewed in 4). One example of whole allergen derivatives as vaccine candidates was the production of mutants of the major ryegrass allergen, Lol p 5 (33, 34). Allergen derivatives were produced by site-directed mutagenesis, based on B- and T-cell mapping studies. The resulting molecules exhibited reduced allergenic activity as determined by basophil histamine release and skin prick Honorable Mention 2005 Verena Niederberger

17 test studies, but retained the ability to induce proliferation of group 5 allergen-specific T cell lines and clones (33, 34). A different approach was used to produce hypoallergenic derivatives of the major birch pollen allergen, Bet v 1. The division of the molecule into two large fragments (F1: aa1-74, F2: aa75-160) led to the nearly complete loss of the IgE binding capacity, which is dependent on the presence of conformational epitopes on the intact and folded allergen (35). The production of a Bet v 1 trimer demonstrated that it is not necessary to completely destroy IgE reactivity to achieve a strong reduction of allergenic activity (36). This allergen derivative exhibited a greatly reduced allergenic activity, presumably based on a re-orientation of IgE epitopes on the trimeric molecule. T cell studies revealed that this molecule has interesting immunological properties, because it has a greatly enhanced ability to induce a Th1-like immune response as compared to the monomeric Bet v 1. As a prerequisite for the use of these Bet v 1-derivatives as a birch pollen vaccine we performed a systematic safety evaluation by provocation testing in a large number of birch pollen allergic patients and control individuals (37-40). Skin reactions to the genetically engineered allergen derivatives were compared with reactions induced by commercial birch pollen extract in skin prick and intracutaneous tests. These experiments showed that the ability of both hypoallergenic formulations to induce type I allergic reactions was more than 100fold reduced. It was therefore assumed that vaccination with Bet v 1 fragments and Bet v 1 trimer would have a greatly reduced risk of inducing immediate type anaphylactic side effects, and that higher doses of allergens could be used for treatment of patients. Immunotherapy with genetically modified allergens Recently we performed the first immunotherapy trial with genetically engineered allergen derivatives of the major birch pollen allergen, Bet v 1 (41). In a double-blind, placebo-controlled study performed in 3 treatment centres (Vienna, Stockholm, Strasbourg), 124 birch pollen-allergic patients received subcutaneous injections containing either one of the two Bet v 1 derivatives, or aluminium hydroxide alone, which was used as adjuvant for the subcutaneous injections. Treatment was performed with increasing doses (1-80µg) in one- to two-weekly intervals as one pre-seasonal treatment course. Because of the strongly reduced allergenic activity of the recombinant allergen derivatives, maximal doses of 80 µg of the active preparations per injection were tolerated by most of the patients. A reduction of cutaneous reactivity and improvement of symptoms was observed in actively treated but not in placebo-treated patients. We performed an in-depth analysis of the immunological mechanisms underlying this treatment in the 72 patients in whose treatment I was involved in Vienna. Vaccination with the hypoallergenic derivatives induced strong de-novo IgG responses to the wildtype allergen, which were of the IgG 1, IgG 2 and IgG 4 isotype (41). While extract-based injection immunotherapy is known to mainly induce Th2-like immune responses (i.e., induction of IgG 4 antibodies), treatment with the Bet v 1 derivatives led to changes of the allergic immune response towards a mixed Th1/Th2 phenotype, characterized by allergen-specific IgG 2 antibodies. As patients were treated exclusively with Bet v 1-derivatives, we observed highly specific immune responses to Bet v 1 and Bet v 1-cross-reactive allergens. An induction of new reactivities to other birch pollen components, which have been shown for extract-based treatment (42), was not observed. The newly induced IgG antibodies were able to inhibit basophil degranulation induced by Bet v 1 as well as by cross-reactive allergens from plant-derived food and other Fagales pollen. A protective effect to cross-reactive allergens was reflected by a clinical improvement in actively-treated patients with oral allergy syndrome. Our immunological analysis indicated that treatment with Honorable Mention 2005 Verena Niederberger

18 the Bet v 1-derivatives has vaccination character because of two reasons: Active treatment led to a subtle rise of Bet v 1-specific IgA and IgM antibodies and actively but not placebo-treated patients developed IgG antibody responses against new epitopes (i.e., Bet v 1 fragments). Moreover, we found that seasonally induced boosts of IgE production were significantly reduced in actively treated patients with high levels of allergen-specific IgG antibodies (Figure 3), suggesting that therapy-induced IgG antibodies also prevent the activation of allergen-specific IgE memory responses. We detected allergen-specific IgG antibodies not only in serum but also in nasal secretions of actively treated patients (43). By objectively evaluating nasal symptoms by nasal provocation experiments and rhinomanometry, we showed that these mucosal antibodies were significantly associated with a reduction of allergen sensitivity in the nose. Presumably, these mucosal antibodies exhibit a protective function by neutralizing intruding allergens and preventing them from inducing memory IgE production and allergic inflammation (30). An analysis of treatment-induced allergen-specific cytokine responses revealed that trimertreatment led to a reduction in the Th2-response to Bet v 1 (reduced number of IL-4, significantly reduced number of IL-5 and IL-13 producing cells) and to a Bet v 1-dependent increase in the number of cells producing the Th1-promoting cytokine IL-12 (44). Our data suggest that the application of high doses of genetically engineered allergen derivatives induces a healthy (i.e., mixed Th2/Th1) allergen-specific immune response, thus representing a new form of causative treatment for type I allergy. Directions for future research, Implications Since we have conducted the first immunotherapy study with genetically modified birch pollen allergen, Bet v 1, several other immunotherapy studies with recombinant allergens or allergen derivatives have been and are currently being conducted. It is hoped that recombinant allergens will substantially contribute to the improvement of allergen-specific immunotherapy and replace traditional extract-based treatment in the near future. Furthermore, it may be possible to develop even prophylactic allergy vaccines based on recombinant allergen technology. Regarding diagnostic testing, we see already first recombinant allergen-based tests in routine clinical use and expect that a wealth of new additional information will be obtained by allergen molecule/ epitope-based testing. My plans are to further foster the use of recombinant allergens in clinical applications, to conduct clinical research studies investigating the immunological mechanisms of allergy directly in humans and to develop better treatments for allergy Conclusion Since I have started to work in allergy research, recombinant allergens have made the transition from representing pure laboratory tools to being used for the benefit of allergic patients. Diagnostic tests with recombinant allergens are already being used in clinical routine. We have demonstrated that vaccination with genetically engineered hypoallergenic allergen derivatives has the potential to reduce the pathological IgE response underlying allergic disease and, hence, may prevent the progression of disease. Furthermore, we have provided an explanation for the basic mechanisms underlying this form of treatment. These results have the potential to lead to the development of more effective vaccines for the treatment of the most common forms of allergy and even for prophylactic vaccination, and may profoundly change the clinical management of type I allergy in the near future. Honorable Mention 2005 Verena Niederberger

19 Acknowledgements The research in my laboratory is supported by grant SFB F1818-B13 of the Austrian Science Fund. I am grateful to Susanne Spitzauer and Rudolf Valenta, who were my teachers and then encouraged and supported me when I set up a research group at the Department of Otorhinolaryngology. Many thanks also to my students and collaborators. References 1. Valenta R, Ball T, Focke M, Linhart B, Mothes N, Niederberger V, et al. Immunotherapy of allergic disease. Adv Immunol. 2004; 82: Valenta R, Lidholm J, Niederberger V, Hayek B, Kraft D, Grönlund H. The recombinant allergen-based concept of component-resolved diagnosis and immunotherapy (CRD & CRIT). Clin Exp Allergy 1999: 29: Valenta R, Kraft D. Recombinant allergens for diagnosis and therapy of allergic diseases. Curr Opin Immunol 1995; 7: Valenta R. The future of antigen-specific immunotherapy of allergy. Nature Rev Immunol 2002; 2: Pauli G. Evolution in the understanding of cross-reactivities of respiratory allergens: the role of recombinant allergens. Int Arch Allergy Immunol 2000; 123: Valenta R, Niederberger V, Fischer S, Jäger S, Spitzauer S, Kraft D. Tree pollen allergens. Allergens and Allergen Immunotherapy, 2nd edition, Eds.: R.F.Lockey, E. Fernandez-Caldas, S.C. Bukantz; Chapman &Hall 1998, chapter 5, p Niederberger V, Pauli G, Grönlund H, Fröschl R, Rumpold H, Kraft D, et al. Recombinant birch pollen allergens (rbet v1, rbet v 2) contain most of the IgE epitopes present in birch, alder, hornbeam, hazel and oak pollen. A quantitative IgE inhibition study using sera from different populations. J Allergy Clin Immunol 1998; 102: Niederberger V, Laffer S, Fröschl R, Kraft D, Rumpold H, Kapiotis S, et al. IgE antibodies to recombinant allergens (Phl p 1, Phl p 2, Phl p 5, Bet v 2) account for a high percentage of grass pollen-specific IgE. J Allergy Clin Immunol 1998; 101: Allergy. Which allergens? Pharmacia: Västra Aros, Västeras; Ipsen H, Loewenstein H. Isolation and immunochemical characterization of the major allergen of birch pollen (Betula verrucosa). J Allergy Clin Immunol 1983; 72: Ipsen H, Hansen OC. Structural similarities among major allergens of tree pollens. In: Sehon AH, Kraft D, Kunkel G, editors. Epitopes of atopic allergens. Brussels: The UCB Institute of Allergy; pp Jarolim E, Rumpold H, Endler AT, Ebner H, Breitenbach M, Scheiner O, et al. IgE and IgG antibodies of patients with allergy to birch pollen as tools to define the allergen profile of Betula verrucosa. Allergy 1989;44: Valenta R, Duchene M, Pettenburger K, Sillaber C, Valent P, Bettelheim P, et al. Identification of profilin as a novel pollen allergen: IgE autoreactivity in sensitized individuals. Science 1991; 253: Laffer S, Spitzauer S, Susani M, Pairleitner H, Schweiger C, Grönlund H, et al. Comparison of recombinant timothy grass pollen allergens with natural extract for diagnosis of grass pollen allergy in different populations. J Allergy Clin Immunol 1996; 98: Honorable Mention 2005 Verena Niederberger

20 15. Marknell De Witt A, Niederberger V, Lehtonen P, Spitzauer S, Sperr WR, Valent P, et al. Molecular and immunological characterization of a novel timothy grass (Phleum pratense) pollen allergen, Phl p 11. Clin Exp Allergy 2002; 32: Hayek B, Vangelista L, Pastore A, Sperr WR, Valent P, Vrtala S, et al. Molecular and immunologic characterization of a highly cross-reactive two EF-hand calcium-binding alder pollen allergen, Aln g 4. Molecular basis for calcium-modulated IgE recognition. J Immunol 1998; 161: Niederberger V, Hayek B, Vrtala S, Laffer S, Vangelista L, Sperr WR, et al. Calcium-dependent immunoglobulin E binding to the apo-and calcium-bound form of a cross-reactive two EF-hand timothy grass pollen allergen, Phl p 7. FASEB-J 1999; 13: Valenta R, Hayek B, Seiberler S, Bugajska-Schretter A, Niederberger V, Twardosz A, et al. Calcium binding allergens: from plants to man. Int Arch Allergy Immunol 1998; 117: Kazemi-Shirazi L, Niederberger V, Kraft D, Valenta R. Cross-reactive Recombinant Allergen Components: The Concept of the Marker Allergen for Diagnosis and Immunotherapy of Type I Allergy. Int Arch Allergy Immunol 2002; 127: Niederberger V., Valenta R. Genetically modified allergens. Immunol Allergy Clin North Am. 2004; 24: Niederberger V., Valenta R. Recombinant allergens for immunotherapy. Where do we stand? Curr Opin Allergy Clin Immunol. 2004; 4: Hiller R, Laffer S, Harwanegg C, Huber M, Schmidt WM, Twardosz A, et al. Microarrayed allergen-molecules: diagnostic gatekeepers for allergy treatment. FASEB J 2002;16: Flicker S, Valenta R. Renaissance of the blocking antibody concept in type I allergy. Int Arch Allergy Immunol 2003;132: Mandler R, Rinkelman RD, Leveine AD, Snapper CM. IL-4 induction of IgE class switching by lipopolysaccharide-activated muring B cells occurs predominantly though sequential switching. J. Immunol. 1993;150: Jung S, Siebenkotten G, Radbruch A. Frequency of immunoglobulin E class switching is autonomously determined and independent of prior switching to other classes. J Exp Med 1994;179: Niederberger V, Niggemann B, Kraft D, Spitzauer S, Valenta R. Evolution of IgM, IgE and IgG 1-4 antibody responses in early childhood monitored with recombinant allergen components: implications for class switch mechanisms. Eur J Immunol 2002 ; 32: Bousquet J, Lockey RF, Malling HJ. Allergen immunotherapy: therapeutic vaccines for allergic diseases: a WHO position paper. J Allergy Clin Immunol 1998;102: Till SJ, Francis JN, Nouri-Aria K, Durham SR. Mechanisms of immunotherapy. J Allergy Clin Immunol 2004;113: Mothes N, Heinzkill M, Drachenberg KJ, Sperr WR, Krauth MT, Majlesi Y, et al. Allergenspecific immunotherapy with a monophosphoryl lipid A-adjuvanted vaccine: reduced seasonally boosted immunoglobulin E production and inhibition of basophil histamine release by therapy-induced blocking antibodies. Clin Exp Allergy 2003; 33: Honorable Mention 2005 Verena Niederberger

21 30. Reisinger J., Triendl A., Kuechler E., Bohle B., Krauth M.-T., Valent P., et al. IFN- -enhanced allergen penetration across respiratory epithelium augments allergic inflammation. J Allergy Clin Immunol (in press). 31. Niederberger V, Stübner P, Spitzauer S, Kraft D, Valenta R, Ehrenberger K, et al. Skin test results but not serology reflect immediate type respiratory sensitivity: a study performed with recombinant allergen molecules. J Invest Dermatol 2001;117: Lowenstein H, Larsen JN. Recombinant allergens/allergen standardization. Curr Allergy Asthma Rep 2001;1: Swoboda I, de Weerd N, Bhalla LP, Niederberger V, Sperr WR, Valent P, et al. Hypoallergenic Forms of the Ryegrass Pollen Allergen Lol p 5 as Candidates for Immunotherapy. Int Arch Allergy Immunol 2001; 124: Swoboda I, De Weerd N, Bahlla PL, Niederberger V, Sperr WR, Valent P, et al. Mutants of the Major Ryegrass Pollen Allergen, Lol p 5, with Reduced IgE Binding Capacity: candidates for Grass Pollen-Specific Immunotherapy. Eur J Immunol 2002: 32: Vrtala S, Hirtenlehner K, Vangelista L, Pastore A, Eichler HG, Sperr WR, et al. Conversion of the major birch pollen allergen, Bet v 1, into two nonanaphylactic T cell epitope-containing fragments: candidates for a novel form of specific immunotherapy. J Clin Invest 1997; 99: Vrtala S, Hirtenlehner K, Susani M, Akdis M, Kussebi F, Akdis CA, et al. Genetic engineering of a hypoallergenic trimer of the major birch pollen allergen Bet v 1. FASEB J 2001; 11: Pauli G, Purohit A, Oster J-P, de Blay F, Vrtala S, Niederberger V, et al. Clinical evaluation of genetically engineered hypoallergenic rbet v 1 derivatives. Int Arch Allergy Immunol 1999; 118: van Hage-Hamsten M, Kronqvist M, Zetterstrom O, Johansson E, Niederberger V, Vrtala S, et al. Skin test evaluation of genetically engineered hypoallergenic derivates of the major birch pollen allergen, Bet v 1. Results obtained with a mix of two recombinant Bet v 1 fragments and recombinant Bet v 1 trimer in a Swedish population before the birch pollen season. J Allergy Clin Immunol 1999; 104: Pauli G, Purohit A, Oster J-P, de Blay R, Vrtala S, Niederberger V, et al. Skin testing with wild-type recombinant birch pollen allergens and hypoallergenic modified molecules. Arb Paul Ehrlich Inst Bundesamt Sera Impfstoffe Frankf A M 1999; 93: Pauli G, Purohit A, Oster J-P, de Blay F, Vrtala S, Niederberger V, et al. Comparison of genetically engineered hypoallergenic rbet v 1 derivatives with Bet v 1 wild-type by skin prick and intradermal testing: results obtained in a French population. Clin Exp Allergy 2000; 30: Niederberger V., Horak F., Vrtala S., Spitzauer S., Krauth M.-T., Valent P., et al. Vaccination with genetically engineered allergens prevents progression of allergic disease. Proc Natl Acad Sci U S A. 2004; 101: [Epub 2004 Aug 13] 42. Movérare R, Elfman L, Vesterinen E, Metso T, Haahtela T. Development of new IgE specifities to allergenic components in birch pollen extract during specific immunotherapy studied with immunoblotting and Pharmacia CAP system. Allergy 2002; 57: Reisinger J., Horak F., Pauli G., van Hage-Hamsten M., Cromwell O., König F., et al. Al- Honorable Mention 2005 Verena Niederberger

22 lergen-specific IgG antibodies in nasal secretions induced by vaccination with genetically modified allergens are associated with reduced nasal allergen sensitivity. J Allergy Clin Immunol (in press). 44. Gafvelin G, Thunberg S, Kronqvist M, Grönlund H, Grönneberg R, Troye-Blomberg M, et al. Cytokine and antibody responses in birch pollen allergic patients treated with genetically modified derivatives of the major birch pollen allergen Bet v 1. Int Arch Allergy Immunol 2005 (in press). Figures Figure 1 Steps on the way of recombinant allergens from the bench into clinical applications. After identification of important allergen sources (1), extraction of RNA (2) and transcription into cdna (3), and after insertion of cdna into expression systems (4), recombinant allergens were produced (5). The comparison of the epitope spectrum of panels of recombinant allergens with natural allergen sources (6) was a prerequisite for the use recombinant allergens for component-resolved diagnosis and disease monitoring (7). This has resulted in the development of diagnostic systems for improved indication for immunotherapy (8). Different strategies have been applied to convert recombinant allergens into hypoallergenic molecules by genetic engineering (9: red: division into two fragments, green: modification of IgE binding sites by site-directed mutagenesis, blue: polymerization). The safety of recombinant allergens and their derivatives had to be evaluated in clinical studies (10) before these molecules could be used for vaccination of allergic patients (11). Honorable Mention 2005 Verena Niederberger

23 Figure 2 Skin prick test from a 27-year-old male patient with allergic symptoms during the flowering period of trees, grasses and weeds (spring to autumn) as an example for a clinically relevant sensitization to cross-reactive calcium-binding allergens. Immunoblot results (data not shown) had demonstrated that reactivity to birch pollen and ragweed pollen were mainly due to a two-ef-hand allergen from birch and ragweed which cross-reacts with the Phl p 7 allergen from timothy grass pollen. This patient lacked IgE antibodies and skin reactivity to Bet v 1 and Bet v 2; birch pollen is therefore unlikely as a primary sensitizer. IgE reactivity to Phl p 1, Phl p 2 and Phl p 5 demonstrated sensitization to grass pollen and in addition there were IgE antibodies to the calcium-binding allergen Phl p 7. The immediate type skin reaction to birch pollen extract was apparently caused by IgE cross-reactivity between Phl p 7 and the homologous protein in birch pollen, Bet v 4. Figure 3 Vaccination with rbet v 1 fragments and rbet v 1 trimer reduces the saisonal boost of IgE levels. Bet v 1-specific IgE levels (percentage alteration compared to the baseline) before treatment (November/December 2000), after treatment (February 2001), after the birch pollen season (May 2001), and in October 2001 in the three patient groups (placebo; fragments; trimer). Honorable Mention 2005 Verena Niederberger