Allergy diagnosis based on purified allergen molecules provides detailed information regarding the individual sensitization profile of allergic patients, allows monitoring of the development of allergic disease and of the effect of therapies on the immune response to individual allergen molecules. Allergen microarrays contain a large variety of allergen molecules and thus allow the simultaneous detection of allergic patients' antibody reactivity profiles towards each of the MeDALL is a European research program in which allergen microarray technology is used for the monitoring of the development of allergic disease in childhood, to draw a geographic map of the recognition of clinically relevant allergens in different populations and to establish reactivity profiles which are associated with and predict certain disease manifestations. We describe technical advances of the MeDALL allergen-chip regarding specificity, sensitivity and its ability to deliver test results which are close to in vivo reactivity. In addition, the usefulness and numerous advantages of allergen microarrays for allergy research, refined allergy diagnosis, monitoring of disease, of the effects of therapies, for improving the prescription of specific immunotherapy and for prevention are discussed. Europe PMC Funders Group

House dust mites (HDMs) belong to the most potent indoor allergen sources worldwide and are associated with allergic manifestations in the respiratory tract and the skin. Here we studied the importance of the high-molecular-weight group 11 allergen from Dermatophagoides pteronyssinus (Der p 11) in HDM allergy. Sequence analysis showed that Der p 11 has high homology to paramyosins from mites, ticks, and other invertebrates. A synthetic gene coding for Der p 11 was expressed in Escherichia coli and rDer p 11 purified to homogeneity as folded, alpha-helical protein as determined by circular dichroism spectroscopy. Using antibodies raised against rDer p 11 and immunogold electron microscopy, the allergen was localized in the muscle beneath the skin of mite bodies but not in feces. IgE reactivity of rDer p 11 was tested with sera from HDM-allergic patients from Europe and Africa in radioallergosorbent test-based dot-blot assays. Interestingly, we found that Der p 11 is a major allergen for patients suffering from atopic dermatitis (AD), whereas it is only a minor allergen for patients suffering from respiratory forms of HDM allergy. Thus, rDer p 11 might be a useful serological marker allergen for the identification of a subgroup of HDM-allergic patients suffering from HDM-associated AD.

Der p 23, a new, major house dust mite (HDM) allergen that is recognized by >70% of HDM-allergic patients, has high allergenic activity and, therefore, must be considered an important component for HDM-specific immunotherapy. We constructed and characterized a hypoallergenic Der p 23 vaccine for HDM immunotherapy. Three nonallergenic peptides from the C-terminal IgE epitope-containing part of Der p 23 (P4, P5) and P6, a mutant peptide containing serines instead of cysteines, were identified. Peptides were fused to the hepatitis B virus–derived PreS domain as recombinant fusion proteins (i.e., PreS-2XP4P5 and PreS-4XP6) that were expressed in Escherichia coli and purified to homogeneity. Compared with Der p 23, PreS-2XP4P5 and PreS-4XP6 showed no relevant IgE reactivity and exhibited considerably reduced allergenic activity in basophil activation tests using blood from HDM-allergic patients. Upon immunization of rabbits, only PreS-2XP4P5 induced high levels of Der p 23–specific IgG Abs that inhibited binding of patients’ IgE to Der p 23, comparable to IgG Abs induced with Der p 23, whereas Abs induced with PreS-4XP6 had only low blocking capacity. Additionally, IgG Abs induced with PreS-2XP4P5 inhibited Der p 23–induced basophil activation comparable to IgG Abs induced with Der p 23. Compared with Der p 23, PreS-2XP4P5 induced lower T cell proliferation but higher levels of the tolerogenic cytokine IL-10 and the Th1 cytokine IFN-γ in PBMCs from HDM-allergic patients, indicating an immunomodulatory capacity of the fusion protein. Therefore, PreS-2XP4P5 represents a promising candidate for immunotherapy of HDM-allergic patients.

Background: Inthehousedustmite(HDM)Dermatophagoides pteronyssinus,Derp 1,2,5,7,21,and23havebeenidentifiedasthemostimportantallergens.Theaimof thisstudywastodefinehypoallergenicpeptidesderivedfromthesequencesofthe sixallergensandtousethepeptidesandthecompleteallergenstostudyantibody, Tcell,andcytokineresponsesinsensitizedandnonsensitizedsubjects. Methods: IgE reactivity of HDM-allergic and non-HDM-sensitized individuals to 15 HDM allergens was established using ImmunoCAP ISAC technology. Thirtythree peptides covering the sequences of the six HDM allergens were synthesized. Allergens and peptides were tested for IgE and IgG reactivity by ELISA and ImmunoCAP,respectively.Allergenicactivitywasdeterminedbybasophilactivation. CD4+TcellandcytokineresponsesweredeterminedinPBMCculturesbyCFSEdilu-tionandLuminextechnology,respectively. Results: HousedustmiteallergicsshowedIgEreactivityonlytocompleteallergens, whereas 31 of the 33 peptides lacked relevant IgE reactivity and allergenic activity.IgGantibodiesofHDM-allergicandnonsensitizedsubjectsweredirectedagainst peptideepitopesandhigherallergen-specificIgGlevelswerefoundinHDMallergics. PBMCfromHDM-allergicsproducedhigherlevelsofIL-5whereasnon-HDM-sensitized individuals mounted higher levels of IFN-gamma, IL-17, pro-inflammatory cytokines,andIL-10. Conclusion: IgG antibodies in HDM-allergic patients recognize peptide epitopes whicharedifferentfromtheepitopesrecognizedbyIgE.Thismayexplainwhynaturallyoccurringallergen-specificIgGantibodiesdonotprotectagainstIgE-mediated allergicinflammation.AmixofhypoallergenicpeptidescontainingTcellepitopesof themostimportantHDMallergenswasidentified. K E Y W O R D Sallergen,housedustmiteallergy,recombinantallergen,syntheticpeptide,Tcellepitope ThisisanopenaccessarticleunderthetermsoftheCreativeCommonsAttribution-NonCommercialLicense,whichpermitsuse,distributionandreproduction inanymedium,providedtheoriginalworkisproperlycitedandisnotusedforcommercialpurposes.

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