title: Production and proteomic characterization of pharmaceutical-grade Dermatophagoides pteronyssinus and Dermatophagoides farinae 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 vivo diagnosis.
authors.affiliation: Recherche et Développement, Stallergènes, Antony, France.
authors.forename: Thierry
authors.initials: T
authors.lastname: Batard
authors.forename: Alexandre
authors.initials: A
authors.lastname: Hrabina
authors.forename: Xue Zhi
authors.initials: XZ
authors.lastname: Bi
authors.forename: Henri
authors.initials: H
authors.lastname: Chabre
authors.forename: Pierrick
authors.initials: P
authors.lastname: Lemoine
authors.forename: Marie-Noëlle
authors.initials: MN
authors.lastname: Couret
authors.forename: Dominique
authors.initials: D
authors.lastname: Faccenda
authors.forename: Bertrand
authors.initials: B
authors.lastname: Villet
authors.forename: Philippe
authors.initials: P
authors.lastname: Harzic
authors.forename: Françoise
authors.initials: F
authors.lastname: André
authors.forename: Sock Yong
authors.initials: SY
authors.lastname: Goh
authors.forename: Claude
authors.initials: C
authors.lastname: André
authors.forename: Fook Tim
authors.initials: FT
authors.lastname: Chew
authors.forename: Philippe
authors.initials: P
authors.lastname: Moingeon
mesh_terms: D000818:Animals; D039741:Antigens, Dermatophagoides / Q000032:analysis / Q000302:isolation & purification*; D060829:Arthropod Proteins; D002851:Chromatography, High Pressure Liquid; D003546:Cysteine Endopeptidases; D040001:Dermatophagoides farinae / Q000737:chemistry*; D040002:Dermatophagoides pteronyssinus / Q000737:chemistry*; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D006967:Hypersensitivity / Q000188:drug therapy* / Q000276:immunology; D040901:Proteomics / Q000379:methods; D011852:Radioallergosorbent Test; D014908:Triticum / Q000276:immunology; D014612:Vaccines / Q000276:immunology / Q000302:isolation & purification* / Q000627:therapeutic use; D015003:Yeasts / Q000276:immunology
chemical_list: D039741:Antigens, Dermatophagoides; D060829:Arthropod Proteins; C451163:Dermatophagoides pteronyssinus antigen p 2; D014612:Vaccines; D003546:Cysteine Endopeptidases; C451162:Dermatophagoides pteronyssinus antigen p 1