Production and Proteomic Characterization of Pharmaceutical-Grade <i>Dermatophagoides pteronyssinus </i>and <i>Dermatophagoides farinae </i>Extracts for Allergy Vaccines

Abstract

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.

Recombinant Fusion Proteins Assembling Der p 1 and Der p 2 Allergens from <i>Dermatophagoides pteronyssinus</i>

“…Natural Der p 1 and Der p 2 allergens were purified from a D. pteronyssinus extract [1] by two chromatographic steps. The two allergens were first separated onto a phenyl sepharose column (GE Healthcare) using an ammonium sulfate gradient.…”

“…Prevalent mite species in Europe and North America include Dermatophagoides pteronyssinus and D. farinae which share antigenic specificities. Current therapeutic vaccines against HDM allergy are based on complex biological extracts made from either one or both species [1]. Given significant amino acid sequence homologies between D. pteronyssinus and D. farinae allergens and resulting IgE cross-reactivities [2,3], a vaccine based solely on D. pteronyssinus allergens could likely be useful to treat allergies to either one of those two mite species.…”

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

“…Natural Der p 2 was purified from D. pteronyssinus mites cultured at our facilities [36]. Allergen purification was first performed from mite extracts by hydrophobic interaction chromatography using a phenyl sepharose column (GE Healthcare, Saclay, France) and 20 m M sodium phosphate, 1.8 M ammonium sulphate pH 6.2, as well as 20 m M sodium phosphate, 5% isopropanol pH 7.5 as binding and elution buffers, respectively.…”

Allergen-Specific Immunotherapy Follow-Up by Measuring Allergen-Specific IgG as an Objective Parameter

“…The intensity of the immune response to allergens is crucial to develop an allergic condition mediated by IgE antibody, or a healthy condition depending on the individual gene susceptibility, environmental pollutants, features of allergens, among others [22][23][24]. Other antibody classes have been analyzed because of this variation of response between allergic and healthy subjects, such as IgA and IgG subclasses [23][24][25].…”

“…Other antibody classes have been analyzed because of this variation of response between allergic and healthy subjects, such as IgA and IgG subclasses [23][24][25]. In healthy individuals, B cell response to house dust mite allergens ranges from no response to predominantly production of IgG antibodies specific to allergens, particularly IgG1 or IgG4 subclass, in the absence or low concentration of IgE.…”