Pierrick Lemoine

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.

Patterns of IgE sensitization in house dust mite-allergic patients: implications for allergen immunotherapy

Understanding patterns of IgE sensitization in Dermatophagoides‐allergic patients living in various geographical areas is necessary to design a product suitable for worldwide allergen immunotherapy (AIT).

Development and evaluation of a sublingual tablet based on recombinant Bet v 1 in birch pollen-allergic patients

Sublingual immunotherapy (SLIT) applied to type I respiratory allergies is commonly performed with natural allergen extracts. Herein, we developed a sublingual tablet made of pharmaceutical‐grade recombinant Bet v 1.0101 (rBet v 1) and investigated its clinical safety and efficacy in birch pollen (BP)‐allergic patients.

Expression and characterization of natural-like recombinant Der p 2 for sublingual immunotherapy.

Background: Recombinant allergens with a native conformation represent an alternative to natural extracts for immunotherapy and diagnostic purposes. Methods: We produced the Der p 2 mite allergen in Pichia pastoris and Escherichia coli. After purification by cation exchange chromatography, recombinant molecules were compared to their natural counterpart based upon structural (disulfide bonds, secondary structure, thermal stability) and immunological properties (antibody reactivity, basophil and T cell activation, tolerance induction in a murine sublingual immunotherapy model). Results: The Der p 2.0101 isoform was confirmed to be prevalent in Dermatophagoides pteronyssinus extracts. It was then produced as a secreted molecule in P. pastoris or refolded from E. coli inclusion bodies. The yeast-expressed rDer p 2 molecule exhibits a natural-like disulfide bridge distribution and secondary structure, whereas the E. coli-derived rDer p 2 presents some heterogeneity in cysteine bonds and a lower stability following thermal stress. The two recombinant as well as natural Der p 2 molecules exhibit comparable IgE recognition and activate basophil and CD4+ T cells. Sublingual immunotherapy of nDer p 2- sensitized mice using either one of the rDer p 2 molecules efficiently decreases airway hyperresponsiveness as well as Th2 responses. Conclusions: Natural and recombinant Der p 2 molecules produced in P. pastoris and E. coli exhibit comparable immunological properties despite distinct structural features. Natural-like cysteine pairing is a critical parameter to identify stable, well-folded and homogenous proteins appropriate for immunotherapy and diagnostic purposes.

Recombinant Fusion Proteins Assembling Der p 1 and Der p 2 Allergens from Dermatophagoides pteronyssinus

Background: Fusion proteins assembling multiple allergens can be engineered by recombinant DNA technologies in order to produce tools for diagnostic and immunotherapeutic purposes. Herein, we developed and characterized chimeras assembling Der p 1 and Der p 2 allergens as potential candidate vaccines against house dust mite allergy. Methods: Fusion proteins encompassing Der p 2 with either mature or proDer p 1 were expressed in Escherichia coli or Pichia pastoris. Forms with mutation in Der p 1 catalytic site were also engineered. Purified chimeras were characterized by immunoblotting, circular dichroism, disulfide bond mapping, basophil and T lymphocyte stimulation assays. Results: Four fusion proteins were expressed in E. coli as inclusion bodies, whereas only chimeras comprising proDer p 1 were obtained in yeast. All such hybrids formed polymers and aggregates, and yeast-expressed chimeras were unstable. Circular dichroism analysis performed after refolding of bacteria expressed chimeras encompassing mature Der p 1 confirmed partial folding, consistent with the occurrence of both correct and inappropriate intramolecular disulfide bonds. All fusion molecules were recognized by Der p 1- and Der p 2-specific human IgEs, monoclonal and polyclonal antibodies. Fusion proteins activate basophils from mite-allergic patients and trigger the proliferation of specific CD4+ T cells, albeit to a lower level when compared to individual allergens. Conclusions: Production of multiple Der p 1-Der p 2 fusion proteins exhibiting partial folding and proper antigenic properties has been achieved. Nonetheless, significant solubility and stability issues currently limit the application of such chimeras for immunotherapy or diagnostic.