Cloning, production and characterization of antigen 5 like proteins from Simulium vittatum and Culicoides nubeculosus, the first cross-reactive allergen associated with equine insect bite hypersensitivity.
Insect bite hypersensitivity (IBH) is an IgE-mediated seasonal dermatitis of the horses associated with bites of Simulium (black fly) and Culicoides (midge) species. Although cross-reactivity between Simulium and Culicoides salivary gland extracts has been demonstrated, the molecular nature of the allergens responsible for the observed cross-reactivity remains to be elucidated. In this report we demonstrate for the first time in veterinary medicine that a homologous allergen, present in the salivary glands of both insects, shows extended IgE cross-reactivity in vitro and in vivo. The cDNA sequences coding for both antigen 5 like allergens termed Sim v 1 and Cul n 1 were amplified by PCR, subcloned in high level expression vectors, and produced as [His](6)-tagged proteins in Escherichia coli. The highly pure recombinant proteins were used to investigate the prevalence of sensitization in IBH-affected horses by ELISA and their cross-reactive nature by Western blot analyses, inhibition ELISA and intradermal skin tests (IDT). The prevalence of sensitization to Sim v 1 and Cul n 1 among 48 IBH-affected horses was 37% and 35%, respectively. In contrast, serum IgE levels to both allergens in 24 unaffected horses did not show any value above background. Both proteins strongly bound serum IgE from IBH-affected horses in Western blot analyses, demonstrating the allergenic nature of the recombinant proteins. Extended inhibition ELISA experiments clearly showed that Sim v 1 in fluid phase is able to strongly inhibit binding of serum IgE to solid phase coated Cul n 1 in a concentration dependent manner and vice versa. This crucial experiment shows that the allergens share common IgE-binding epitopes. IDT with Sim v 1 and Cul n 1 showed clear immediate and late phase reactions to the allergen challenges IBH-affected horses, whereas unaffected control horses do not develop relevant immediate hypersensitivity reactions. In some horses, however, mild late phase reactions were observed 4h post-challenge, a phenomenon reported to occur also in challenge experiments with Simulium and Culicoides crude extracts probably related to lipopolysaccaride contaminations which are also present in E. coli-expressed recombinant proteins. In conclusion our data demonstrate that IgE-mediated cross-reactivity to homologous allergens, a well-known clinically relevant phenomenon in human allergy, also occurs in veterinary allergy.
Swiss Institute of Allergy and Asthma Research (SIAF), University of Zürich, Obere Strasse 22, CH-7270 Davos, Switzerland.
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Schaffartzik
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Marti
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Crameri
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Rhyner
D000485:Allergens / Q000235:genetics / Q000276:immunology*; D000818:Animals; D002519:Ceratopogonidae / Q000276:immunology*; D003001:Cloning, Molecular; D003429:Cross Reactions; D004797:Enzyme-Linked Immunosorbent Assay; D006734:Horse Diseases / Q000209:etiology*; D006736:Horses; D006967:Hypersensitivity / Q000209:etiology / Q000662:veterinary*; D007073:Immunoglobulin E / Q000276:immunology; D007299:Insect Bites and Stings / Q000662:veterinary*; D011994:Recombinant Proteins / Q000276:immunology; D012843:Simuliidae / Q000276:immunology*; D012882:Skin Tests; D014862:Wasp Venoms / Q000276:immunology*
D000485:Allergens; D011994:Recombinant Proteins; D014862:Wasp Venoms; C040583:white-faced hornet venom antigen 5; D007073:Immunoglobulin E