Recombinant Culicoides obsoletus complex allergens stimulate antigen-specific T cells of insect bite hypersensitive Shetland ponies in vitro.

BACKGROUND: Ponies may suffer from Insect bite hypersensitivity (IBH), an allergic IgE-mediated pruritic skin disorder, induced by allergens from biting midges of the Culicoides spp. HYPOTHESIS/OBJECTIVES: To determine whether recombinant Culicoides obsoletus allergens are able to activate T cells of ponies exposed to C. obsoletus and whether these allergen-specific responses differ between IBH-affected and healthy ponies. ANIMALS: Ten IBH-affected Shetland ponies and 10 age-matched healthy controls taken from the same stables, to ensure similar exposure to midges. METHOD: Peripheral blood mononuclear cells (PBMC) were cultured with two different pools of recombinant C. obsoletus complex allergens to expand the allergen-specific T cells. These PBMC cultures were subsequently co-cultured with mature dendritic cells (DCs) loaded with the same antigens. Induction of Th1, Th2 and regulatory T (Treg) cells in these DC/PBMC co-cultures was assessed by analysis of IFN-γ, IL-4, IL-10 and FoxP3 expression levels using quantitative RT-PCR and phenotyping by flow cytometry. RESULTS: Recombinant C. obsoletus allergens increased IFN-γ mRNA expression levels, percentages of IFN-γ expressing (Th1) cells and CD25(high) FoxP3(+) IL-10(+) Tregs compared to unstimulated DC/PBMC co-cultures. Stimulation of IL-4 expressing Th2 cells by the recombinant allergens was far less pronounced. The DC/PBMC co-cultures did not reveal significant differences between healthy and IBH-affected ponies for any of the analysed parameters, except for higher IL-4 mRNA levels in IBH affected ponies after stimulation with one of the two allergen pools. CONCLUSION AND CLINICAL IMPORTANCE: The recombinant C. obsoletus complex allergens can stimulate antigen-specific Th1 and IL10 producing Treg cells and are therefore promising candidates for the immunotherapy of IBH.

Allergen-Specific Cytokine Polarization Protects Shetland Ponies against Culicoides obsoletus-Induced Insect Bite Hypersensitivity.

The immunological mechanisms explaining development of an allergy in some individuals and not in others remain incompletely understood. Insect bite hypersensitivity (IBH) is a common, seasonal, IgE-mediated, pruritic skin disorder that affects considerable proportions of horses of different breeds, which is caused by bites of the insect Culicoides obsoletus (C. obsoletus). We investigated the allergen-specific immune status of individual horses that had either been diagnosed to be healthy or to suffer of IBH. Following intradermal allergen injection, skin biopsies were taken of IBH-affected and healthy ponies and cytokine expression was determined by RT-PCR. In addition, allergen-specific antibody titers were measured and cytokine expression of in vitro stimulated, allergen-specific CD4 T-cells was determined. 24 hrs after allergen injection, a significant increase in mRNA expression of the type-2 cytokine IL-4 was observed in the skin of IBH-affected Shetland ponies. In the skin of healthy ponies, however, an increase in IFNγ mRNA expression was found. Analysis of allergen-specific antibody titers revealed that all animals produced allergen-specific antibodies, and allergen-specific stimulation of CD4 T-cells revealed a significant higher percentage of IFNγ-expressing CD4 T-cells in healthy ponies compared to IBH-affected ponies. These data indicate that horses not affected by IBH, in contrast to the so far established dogma, are not immunologically ignorant but have a Th1-skewed allergen-specific immune response that appears to protect against IBH-associated symptoms. To our knowledge this is the first demonstration of a natural situation, in which an allergen-specific immune skewing is protective in an allergic disorder.

Factors associated with Culicoides Obsoletus complex spp.-specific IgE reactivity in Icelandic horses and Shetland ponies.

Insect bite hypersensitivity (IBH) is a common allergic skin disease in horses, caused by biting insects of the Culicoides spp. In The Netherlands, Culicoides spp. of the Obsoletus complex are the most important midges involved in IBH. The aim of the present study was to identify and quantify associations between several endogenous (host) and exogenous (environmental) factors and immunoglobulin E (IgE) reactivity against Obsoletus complex-derived whole body extract or seven recombinant allergens, measured by ELISA. Data from 143 Icelandic horses and 177 Shetland ponies were analysed using multivariable models. In addition, the relationship between IgE reactivity and severity of clinical signs in IBH-affected horses was examined. Positive correlations were found between Obsoletus complex-specific IgE and severity of clinical signs. Disease status (IBH affected or control), breed and the interaction between IBH status and breed were significantly associated with IgE reactivity against several Obsoletus complex allergens. Significantly greater IgE reactivity was seen in IBH-affected horses compared to controls. The differences in IgE values between cases and controls were most pronounced in Icelandic horses. Shetland pony controls had significantly greater IgE reactivity compared to Icelandic horse controls, while differences in IgE values comparing Shetland pony cases and Icelandic horse cases were not significant. Severity of clinical signs and IgE reactivity in IBH-affected horses against several Obsoletus complex allergens appeared to be related. Consideration of the factors associated with Obsoletus complex-specific IgE in horses might further improve interpretation and accuracy of IgE ELISA test results within these breeds, although further research is required.

Evaluation of a diagnostic ELISA for insect bite hypersensitivity in horses using recombinant Obsoletus complex allergens.

Culicoides spp. of the Obsoletus complex belong to the most important species of midge, involved in causing insect bite hypersensitivity (IBH) in horses in The Netherlands. The aim of the current study was to evaluate seven different Obsoletus complex-derived recombinant allergens (Cul o 1-Cul o 7) and to compare these with Obsoletus complex whole body extract (WBE) in an IgE ELISA, using sera of 194 clinically-confirmed cases of IBH and 175 unaffected horses. The highest test accuracy was obtained with WBE, followed by Cul o 2, 3 and 5. Two ELISAs with a combination of recombinant allergens, Combi-1 (Cul o 3, 5 and 7) and Combi-2 (Cul o 1, 2, 5 and 7) were additionally performed and both resulted in high test accuracies close to that obtained with WBE. Combi-1 resulted in the best sensitivity and specificity, both 89%. Both Combi-1 and Combi-2 performed less well with samples collected in winter, but over 70% of the IBH-affected horses could still be identified. In conclusion, a combination of three Obsoletus complex recombinant allergens (Cul o 3, 5 and 7) could potentially replace Obsoletus complex WBE in an IgE ELISA for diagnosis of IBH in horses.

Cloning and expression of candidate allergens from Culicoides obsoletus for diagnosis of insect bite hypersensitivity in horses.

Insect bite hypersensitivity (IBH) is an IgE-mediated (Type I) hypersensitivity reaction induced by allergens from biting midges of the Culicoides spp. The aim of the present study was to identify, clone and express recombinant allergens from C. obsoletus, the main species found feeding on horses in the Netherlands, by sequence homology searches on the C. obsoletus specific RNA database, with previously described allergens from C. nubeculosus and C. sonorensis. BLAST searches with these described allergens resulted in similarity hits with 7 genes coding for C. obsoletus allergens. These allergens were expressed as hexahistidine tagged recombinant proteins in E. coli. Allergens were termed Cul o 1-Cul o 7. A maltase (Cul o 1) plus Cul s 1 (maltase of C. sonorensis) were additionally expressed in insect cells using the baculovirus expression system to compare homologous allergens from different species produced with different expression systems in diagnostic in vitro and in vivo tests. We demonstrate that IBH affected horses in the Netherlands show higher IgE levels to Cul o 1 than to Cul s 1, as determined by an IgE ELISA. Furthermore, we show that Cul o 1 produced in E. coli is at least as suitable for in vitro diagnosis of IBH affected horses as Cul o 1 produced in the baculovirus/insect cell expression system. The resulting proteins were evaluated for their ability to discriminate IBH affected and healthy horses by ELISA and intradermal testing. The frequency of positive test results by ELISA within IBH affected horses ranged from 38% to 67% for the different allergens. When results of IgE-binding to Cul o 1-Cul o 7 were combined the test had a sensitivity of 92% and specificity of 85%. The capability of the allergens to induce Type I hypersensitivity reaction in IBH affected horses was demonstrated by an intradermal test. The results show that E. coli expressed recombinant allergens from C. obsoletus are valuable tools to determine the allergen specific sensitisation profile (component resolved diagnosis) in horses with IBH in countries were C. obsoletus is the most abundant species and may facilitate in the development of future immunotherapy.

Culicoides obsoletus extract relevant for diagnostics of insect bite hypersensitivity in horses.

Insect bite hypersensitivity (IBH) is an allergic dermatitis in horses caused by the bites of Culicoides species. The aim of the present study was to evaluate the applicability of whole body extracts of C. obsoletus (the main species found feeding on horses in the Netherlands), C. nubeculosus (rarely found in The Netherlands) and C. sonorensis (typical for North America) for diagnosis of IBH in horses in The Netherlands. Blood and serum samples of 10 clinically confirmed IBH affected and 10 healthy control horses were used to evaluate the IgE titers (ELISA) against the Culicoides whole body extracts of the three Culicoides species. Basophil degranulation was assessed by histamine release test (HRT) after stimulation with these extracts at 5, 0.5 and 0.05 µg/ml. IBH affected horses had significantly higher IgE titers against C. obsoletus than against C. nubeculosus and C. sonorensis. Furthermore, C. obsoletus induced significantly higher histamine release in whole blood of IBH affected horses compared to the other extracts at 0.5 µg/ml. Western blot data revealed IgE binding to many proteins in C. obsoletus extract. This interaction was absent or weak in C. nubeculosus and C. sonorensis extracts for IBH affected horses. Results on individual level indicate that the HRT is more sensitive than ELISA in diagnosing IBH. However, ELISA is more practical as a routine test, therefore the ELISA was further evaluated using C. obsoletus extract on 103 IBH affected and 100 healthy horses, which resulted in a test sensitivity and specificity of 93.2% and 90.0%, respectively. The IgE ELISA readings enabled the analysis of the predicted probability of being IBH affected. From an optical density 450nm value of 0.33 onwards, the probability of IBH affected was more than 0.9. The results presented in this paper show that the use of native Culicoides spp. that feed on horse, is important for improved diagnosis and that the described ELISA based on C. obsoletus can be used routinely to diagnose IBH in countries where this species is the main Culicoides feeding on horses.