Expression and Characterization of Natural-Like Recombinant Der p 2 for Sublingual Immunotherapy

Abstract

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.

A Recombinant Sal k 1 Isoform as an Alternative to the Polymorphic Allergen from <b><i>Salsola kali</i></b> Pollen for Allergy Diagnosis

“…As previously reported, nonglycosylated PMEs like that of Diospyros kaki conserved their 3-dimensional folding and its enzymatic activity when comparing with other glycosylated PMEs [34]. Thus, we expressed Sal k 1-specific cDNA in bacteria because glycosylation should not be critical for the 3-dimensional folding of PMEs [34], and it has also been successfully used to express other glycosylated allergens, which maintained intact structural and immunological properties [35,36]. …”

Consideration of methods for identifying mite allergens

“…Olsson et al [ 49 ] reported that recombinant Lep d 2 expressed in E. coli or in S. frugiperda had IgE- binding and basophil activation activities similar to the native protein. Bordas-Le Floch et al [ 50 ] found that recombinant Der p 2 expressed in E. coli did have structural differences detected by circular dichroism but had Ig-E binding and basophil stimulation activities comparable to those of the native or recombinant Der p 2 expressed in P. pastoris . In contrast, Der p 1 or Der f 1 expressed in E coli had significantly less IgE-binding activity when compared to the native form [ 51 ].…”

“…Additionally, for purification of allergen homologues, cross-reactive antibodies identified in one species can be applied to a new species [ 18 ]. In the absence of specific antibodies, alternative chromatography techniques, including ion exchange, gel filtration and hydrophobic exchange, have been used to fractionate whole mite or feces extracts [ 6 , 27 , 30 , 50 ]. Fractions were then tested for IgE-binding activity and/or desired enzymatic activity [ 6 ] and assayed for homogeneity.…”

“…Animal models have been developed which recapitulate aspects of respiratory allergies in humans. Bordas-Le Floch et al [ 50 ] examined T cells isolated by bronchial lavage from Der p 2-sensitized mice demonstrating that recombinant Der p 2 could stimulate cytokine release. They also used an in vivo mouse model of asthma to test the effectiveness of Der p 2 protein immunotherapy.…”

Dermatophagoides farinae allergen Der f 9: Cloning, expression, purification, characterization and IgE‐binding in children with atopic asthma

“…Because of this high reactivity, recombinant groups 1 and 2 allergens have been generated from Dermatophagoides pteronyssinus . However, the recombinant Der p 1 is only weakly reactive, while the recombinant Der p 2 exhibits IgE reactivity comparable to the native allergen . Although the groups 1 and 2 allergens are the major allergens from Dermatophagoides spp., other allergens from these species contribute to allergic disease.…”