Marie-Noëlle Couret

Production and Proteomic Characterization of Pharmaceutical-Grade Dermatophagoides pteronyssinus and Dermatophagoides farinae Extracts for Allergy Vaccines

Background: House dust mites (HDM) such as Dermatophagoides pteronyssinus and Dermatophagoides farinae represent a major cause of type 1 allergies worldwide. Hence large quantities of well-characterized HDM extracts are needed to prepare pharmaceutical-grade allergy vaccines. To this aim, the present study was undertaken to define optimal conditions for large-scale cultures. Methods:D. pteronyssinus and D. farinae were grown on different media combining various proportions of wheat germ, yeast and synthetic amino acids (the latter resembling the composition of the human stratum corneum). Extracts thus obtained were analyzed for their total allergenic activity, as well as major allergen and protein contents, using immunosorbent assays, HPLC, immunoblotting, two-dimensional electrophoresis and peptide mass fingerprinting. Results: An optimal culture medium (Stalmite APF®) based on wheat germ, yeast and amino acids in defined proportion (42, 42 and 15% w/w, respectively) was selected to grow various HDM species with high yields. A detailed proteomic analysis revealed that D. pteronyssinus extracts generated under such conditions did not contain allergens originating from culture medium components and that major prevalent HDM allergens (i.e. groups 1, 2, 7, 10, 13 and 20) are found among the most abundant proteins in the D. pteronyssinus extract. Semiquantitative dot-blot assays confirmed the presence of Der p 3–10 as well as Der p 13 and 14 allergens within the extracts. Conclusions: We developed a well-defined medium allowing to grow various HDM species at an industrial scale in a highly reproducible manner. Extracts from mites produced under such pharmaceutical conditions contain all the relevant allergens for desensitization purposes and in vivodiagnosis.

Characterization of Wild-Type Recombinant Bet v 1a as a Candidate Vaccine against Birch Pollen Allergy

Background: We describe the production in Escherichia coli as a recombinant protein of clinical grade wild-type Bet v 1a (rBet v 1a), to be used as a candidate vaccine against birch pollen allergy. Methods: This recombinant protein was purified by hydrophobic interaction and ion exchange chromatography and characterized by SDS-PAGE, immunoprint and circular dichroism in parallel with natural Bet v 1 (nBet v 1) purified from a birch pollen extract. We also compared rBet v 1 and nBet v 1 for their capacity to induce histamine release from basophils and to stimulate T lymphocyte proliferation. Results: rBet v 1a appears in SDS-PAGE as an 18-kDa monomeric protein, whereas purified nBet v 1 comprises a mixture of isoforms (resolving as three distinct bands and six spots after 1-dimensional and 2-dimensional electrophoresis, respectively). Both recombinant and natural purified Bet v 1 molecules are recognized by IgE from birch pollen-allergic patients as well as anti-Bet v 1 murine monoclonal antibodies, suggesting that the recombinant protein is correctly folded in a native configuration. Circular dichroism analysis confirmed that the two Bet v 1 molecules exhibit similar 3-dimensional structures, even if rBet v 1a appears more compact and stable in thermodenaturation/renaturation experiments. Both rBet v 1 and nBet v 1 induce the degranulation of sensitized basophils and proliferation of Bet v 1-specific T lymphocytes in a similar manner. Conclusions: On the basis of these structural and biological properties, rBet v 1a is a valid candidate vaccine against birch pollen allergy, currently evaluated in humans.

Identification of the cysteine protease Amb a 11 as a novel major allergen from short ragweed

BACKGROUND Allergy to pollen from short ragweed (Ambrosia artemisiifolia) is a serious and expanding health problem in the United States and in Europe. OBJECTIVE We sought to investigate the presence of undescribed allergens in ragweed pollen. METHODS Ragweed pollen proteins were submitted to high-resolution gel electrophoresis and tested for IgE reactivity by using sera from 92 American or European donors with ragweed allergy. Pollen transcriptome sequencing, mass spectrometry (MS), and recombinant DNA technologies were applied to characterize new IgE-binding proteins. RESULTS High-resolution IgE immunoblotting experiments revealed that 50 (54%) of 92 patients with ragweed allergy were sensitized to a 37-kDa allergen distinct from Amb a 1. The full-length cDNA sequence for this molecule was obtained by means of PCR cloning after MS sequencing of the protein combined with ragweed pollen RNA sequencing. The purified allergen, termed Amb a 11, was fully characterized by MS and confirmed to react with IgEs from 66% of patients. This molecule is a 262-amino-acid thiol protease of the papain family expressed as a combination of isoforms and glycoforms after proteolytic removal of N- and C-terminal propeptides from a proform. Three-dimensional modeling revealed a high structural homology with known cysteine proteases, including the mite Der p 1 allergen. The protease activity of Amb a 11, as well as its capacity to activate basophils from patients with ragweed allergy, were confirmed. The production of a nonglycosylated recombinant form of Amb a 11 in Escherichia coli established that glycosylation is not required for IgE binding. CONCLUSION We identified the cysteine protease Amb a 11 as a new major allergen from ragweed pollen. Given the similar physicochemical properties shared by the 2 major allergens, we hypothesize that part of the allergenic activity previously ascribed to Amb a 1 is rather borne by Amb a 11.