1. Recombinant Weed Allergens Nicole Wopfner Christian Doppler Laboratory for Allergy Diagnosis and Therapy Department of Molecular Biology University of Salzburg EAACI Allergy School 2007 Recombinant Allergens: From Fundamental Aspects to Clinical Applications

2. Mugwort pollen allergens Art v 1 PR-12 protein, defensin-like 70-90% Art v 2 PR-1 protein 21% Art v 3 nsLTP 70% Art v 4 profilin 36% Art v 5 polcalcin 10-15% Art v 6 pectate lyase 20-26% Wopfner et al. Int Arch Allergy Immunol 138:337-346, 2005

3. Ragweed pollen allergens Amb a 1 pectate lyase > 90% Amb a 2 Amb a 1 isoallergen 70% Amb a 5 10-20% Amb a 6 nsLTP 20-35% Amb a 9 2 EF-hand CBP 10-15% Amb a 8 profilin 20-36% Amb a 10 3 EF-hand CBP 10-15% Amb a 3 30-50% Wopfner et al. Int Arch Allergy Immunol 138:337-346, 2005

4. IgE-reactivity to ragweed and mugwort pollen allergens Austrian patients 1999 Austrian patients 2003 Italian patients Canadian patients Sensitization to Amb a 1 increased in Austrian patients, whereas sensitization to Art v 1 slightly decreased Amb a 1 represents the most important allergen for ragweed allergic individuals Austrian and Canadian patients react with ragweed and mugwort profilin Wopfner et al. (unpublished) 0 10 20 30 40 50 60 70 80 90 100 0 10 20 30 40 50 60 70 80 90 100 nAmb a 1 rAmb a 1rAmb a 5rAmb a 6rAmb a 8rAmb a 9 rAmb a 10 nArt v 1 rArt v 1 rArt v 4 rArt v 5 rArt v 6

5. Art v 1 – major mugwort pollen allergen Himly et al. FASEB J. 17:106, 2003 Purified nArt v 1 N-terminal hydrophobic signal sequence post-translational modifications: Cleavage of signal peptide Disulfide bond formation O-glycosylation of hydroxy-proline mature sequence: 108 amino acids 2 domains N-terminal “head”: cysteine-rich region C-terminal “tail”: extended (hydroxy)proline-rich domain IgE-reactivity up to 90% in mugwort sensitized individuals

6. Influence of post-translational modifications on immune recognition of Art v 1 RAST PROLIFERATION OF PBMC Himly et al., FASEB J. 17:106-108, 2003 Jahn-Schmidt et al., J. Immunol. 169:6005-11, 2002  In a sub-group of patients, post-translational modifications are important for IgE reactivity  Post-translational modifications play no role in T cell recognition

7. Different patients Peptides spanning the allergen sequence Art v 1 contains one single immunodominant T cell epitope AGSKLCEKTSKTYSGKCDNKKCDKKCIEWEKAQHGACHKREAGKESCFCYFDCSK SPPGATPAPPGAAPPPAAGGSPSPPADGGSPPPPADGGSPPVDGGSPPPPSTH 2536 1 108 Jahn-Schmid et al, JI (2002), Jahn-Schmid et al, JACI (2005) HLA-DRB1*01 main restriction element for presentation of immunodominant T cell epitope

8. Amb a 1 (formerly antigen E)  6% of total protein in an aqueous ragweed extract  Acidic single chain 40 kDa protein  4 isoforms: Amb a 1.1, 1.2, 1.3, 1.4  Family of pectate lyases  Homology to Jun a 1 and Cry j 1  95% of ragweed allergic patients display IgE antibodies to Amb a 1 Amb a 1, the major ragweed pollen allergen Natural Amb a 1  nAmb a 1 is a very instable protein  non-glycosylated  cleaved in two chains,  - and  -chain  both chains are reactive with IgE Amb a 1  -chain  -chain

9. purified rAmb a 1 Recombinant Amb a 1 Different patients Amb a 1 (AgE): multiple T cell epitopes Peptides spanning the allergen sequence