Immunological basis of ragweed Amb 1 a allergenicity

The pectate lyase Amb a 1 represents the major allergen from ragweed pollen and plays an important role in allergic disease. In fact ragweed allergy is rapidly increasing and a growing health concern in Europe. Amb a 1 comprises about 6% of the total protein in aqueous extracts of ragweed pollen and is a 397 residue 38 kDa protein with a beta-helix fold stabilized by three disulfide bridges. Even though the cDNA coding for Amb a 1 was isolated from ragweed pollen in 1991 by Rafnar et al., a complete immunological characterization of Amb a 1 has been hampered by the failure to express and purify large amounts of recombinant Amb a 1 that shows an immuno- reactivity comparable to natural Amb a 1. The major goal of this study is the engineering of Amb a 1 to obtain a recombinant molecule with comparable IgE- binding capacity to the natural counterpart. This goal will be achieved with the following three aims: (i) Generation of an immuno-reactive recombinant Amb a 1; (ii) Generation of a hypoallergenic Amb a 1; (iii) and Immunological characterization and structural basis of Amb a 1 allergenicity. These aims will be achieved using different approaches. Amb a 1 isoforms will be evaluated in terms of IgE reactivity and better expression and purification characteristics. Additionally an immuno-reactive engineered Amb a 1 and hypoallergenic variants will be produced using random mutagenesis. In parallel alternative expression systems like yeast cells and tobacco plants will be evaluated. A future goal is the characterization of B cell and T cell epitopes. These engineered forms of recombinant Amb a 1 will be a critical contribution towards its use in diagnosis and therapy of ragweed pollen allergy.

Ragweed is one of the primary causes of seasonal allergies in the United States and in many parts of Europe. Amb a 1, a pectate lyase protein, represents the major allergen from ragweed pollen and plays an important role in allergic disease. It was one of the first allergens to be cloned and sequenced over two decades ago. However, while other important allergens have been produced recombinant and used to develop new vaccines, little work has been carried out on the expression of recombinant Amb a 1 or on the immunologic reactivity of the recombinant protein. Therefore the major goal of this study was the engineering of Amb a 1 to obtain a recombinant molecule with comparable IgE-binding capacity to the natural counterpart and immunological characterization and structural basis of Amb a 1 allergenicity. First we tried to produce Amb a 1 recombinant in tobacco plants and yeast cells. The expression level in yeast was very low and in the tobacco plants we have not been able to detect any protein expression at all. So we focused on the random mutagenesis approach for the generation of an immunoactive Amb a 1. Amb a 1 was randomly mutated using PCR and expressed in E. coli using a small scale test expressions. Additionally a GFP (green fluorescent protein) tag was added to visualize the solubility of Amb a 1. All promising clones were further analyzed but in the end we have not been able to produce a full-length immune-reactive Amb a 1 in an E.coli based expression system. So we decided to focus on the construction of shorter fragments of the protein and screen these variants for solubility. With the help of the protein production platform (P-CUBE) in Grenoble we have been able to screen thousands of different protein fragments and tested the solubility and the behavior of these clones. We mainly focused on all soluble expressing clones since the solubility of Amb a 1 is the most critical and challenging part in the recombinant production of the protein. Therefore only these important soluble expressing fragments have been sequenced. Sequence information was further used to evaluate protein behavior using bioinformatics tools. Results of these sequence analysis demonstrated the complex structure of Amb a 1, conserved throughout species lines, and stabilized by several crucial disulfide bonds. Our results show that it is therefore, prone to weak expression and latent problems with folding. Although the expression analysis in this project obtained mostly negative data it is a valuable contribution towards the quest for a recombinant Amb a 1 in therapy of ragweed pollen allergy