Sublingual Omp16-driven redirection of the allergic intestinal response in a pre-clinical model of food allergy.

BACKGROUND: IgE-mediated food allergy remains a significant and growing worldwide problem. Sublingual immunotherapy (SLIT) shows an excellent safety profile for food allergy, but the clinical efficacy needs to be improved. This study assessed the effects of the Toll-like receptor 4 agonist outer membrane protein (Omp) 16 from Brucella abortus combined with cow´s milk proteins (CMP) through the sublingual route to modulate cow's milk allergy in an experimental model. METHODS: Mice sensitized with cholera toxin and CMP were orally challenged with the allergen to elicit hypersensitivity reactions. Then, mice were treated with a very low amount of CMP along with Omp16 as a mucosal adjuvant, and finally, animals were re-exposed to CMP. Systemic and mucosal immune parameters were assessed in vivo and in vitro. RESULTS: We found that the sublingual administration of Omp16 + CMP induced a buccal Th1 immune response that modulated the intestinal allergic response with the suppression of symptoms, reduction of IgE and IL-5, and up-regulation of IgG2a and IFN-γ. The adoptive transfer of submandibular IFN-γ-producing α4ß7+ CD4+ and CD8+ cells conferred protection against allergic sensitization. The use of Omp16 + CMP promoted enhanced protection compared to CMP alone. CONCLUSION: In conclusion, Omp16 represents a promising mucosal adjuvant that can be used to improve the clinical and immune efficacy of SLIT for food allergy.

Avances en las inmunoterapias para alergias alimentarias / New insights in immunotherapies for food allergies / Novas perspectivas em imunoterapias para alergias alimentares

Las enfermedades alérgicas son las inmunopatologías que con mayor prevalencia se presentan en el mundo. Pueden o no estar mediadas por anticuerpos IgE, sin embargo estas últimas son las que más intensamente se han estudiado por el riesgo que presentan para la vida del paciente. Si bien el único tratamiento que logra revertir estos mecanismos es la no exposición al alergeno, esto no siempre es posible. Por esta razón, y a partir del mayor conocimiento alcanzado del sistema inmune de mucosas junto al desarrollo de modelos animales de alergia, existe un marcado interés en la especialidad para el desarrollo de inmunoterapias que controlen y reviertan el estado de alergia. A partir de los ensayos pre-clínicos en animales y la aplicación de protocolos terapéuticos en ensayos clínicos, se han desarrollado terapias mucosales que logran inducir mecanismos de tolerancia específicos del alergeno, los cuales son capaces de revertir la sensibilización alérgica. Dado que el principal escollo siguen siendo las reacciones adversas inducidas durante el tratamiento, se requiere profundizar los estudios para desarrollar protocolos terapéuticos más seguros. En este punto la medicina traslacional encuentra un campo próspero para fortalecer las interacciones entre la ciencia básica, la aplicada y la clínica.

Allergic diseases are the most prevalent immunopathologies worldwide. Although different mechanisms -IgE-independent or IgE-dependent- can be involved in the immunopathogenesis, the latter are the most studied reactions since they can be life-threatening. Nowadays, allergen avoidance is the unique effective treatment for allergic patients. However, this is rather difficult to implement. For this reason, and based on the new insights into the mucosal immune system and the development of animal models of allergy, there is an increasing interest in developing novel therapies to control or reverse allergic disorders. Pre-clinical studies and clinical trials have been successful to prove that immunotherapies may accomplish mucosal mechanisms of allergen-specific tolerance, which are able to revoke the allergic sensitization. Since the main obstacle in these therapies still has adverse reactions induced during treatment, further studies are required to explore safe and effective therapeutic protocols. At this point, translational medicine is a flourishing field in the areas of basic science, applied science, and clinical research.

As doenças alérgicas são as imunopatologias mais prevalentes em todo o mundo. Embora possam estar mediadas ou não por anticorpos IgE, estas últimas são as reacções mais intensamente estudadas, devido ao risco que apresentam para a vida do paciente. Ainda que o único tratamento eficaz para reverter este mecanismos seja a não exposição dos pacientes ao alergeno, isto nem sempre é possível. Por este motivo, e com base nas novas perspectivas sobre o sistema imune de mucosas, junto com o desenvolvimento de modelos e para o animais de alergia, existe um interesse crescente na especialidade para o desenvolvimento de imunoterapias que controlem e revertam o estado de alergia. A partir de estudos pré-clínicos em animais e a aplicação de protocolos terapêuticos em ensaios clínico, foram desenvolvidas terapias mucosas que conseguem induzir mecanismos de tolerância específicos do alergeno, que são capazes de reverter a sensibilização alérgica. Devido a que o principal obstáculo nestas terapias continuam sendo as reações adversas induzidas durante o tratamento, é necessário realizar mais estudos para desenvolver protocolos terapêuticos mais seguros. Neste ponto, medicina translacional é um campo próspero para fortalecer as interações entre a ciência básica, a aplicada e a clínica.

The Major Soybean Allergen Gly m Bd 28K Induces Hypersensitivity Reactions in Mice Sensitized to Cow's Milk Proteins.

Reactions to soy have been reported in a proportion of patients with IgE-mediated cow's milk allergy (CMA). In this work, we analyzed if Gly m Bd 28K/P28, one of the major soybean allergens, is a cross-reactive allergen with cow milk proteins (CMP). We showed that P28 was recognized by IgE sera from CMA patients and activated human peripheral basophils degranulation. Moreover, IgE sera of mice exclusively sensitized to CMP recognized P28. Splenocytes from sensitized animals secreted IL-5 and IL-13 when incubated with CMP or soy proteins, but only IL-13 when treated with P28. In addition, a skin test was strongly positive for CMP and weakly positive for P28. Remarkably, milk-sensitized mice showed hypersensitivity symptoms following sublingual challenge with P28 or CMP. With the use of bioinformatics' tools seven putative cross-reactive epitopes were identified. In conclusion, using in vitro and in vivo tests we demonstrated that P28 is a novel cross-reactive allergen with CMP.

Orally-Induced Intestinal CD4+ CD25+ FoxP3+ Treg Controlled Undesired Responses towards Oral Antigens and Effectively Dampened Food Allergic Reactions.

The induction of peripheral tolerance may constitute a disease-modifying treatment for allergic patients. We studied how oral immunotherapy (OIT) with milk proteins controlled allergy in sensitized mice (cholera toxin plus milk proteins) upon exposure to the allergen. Symptoms were alleviated, skin test was negativized, serum specific IgE and IgG1 were abrogated, a substantial reduction in the secretion of IL-5 and IL-13 by antigen-stimulated spleen cells was observed, while IL-13 gene expression in jejunum was down-regulated, and IL-10 and TGF-ß were increased. In addition, we observed an induction of CD4+CD25+FoxP3+ cells and IL-10- and TGF-ß-producing regulatory T cells in the lamina propria. Finally, transfer experiments confirmed the central role of these cells in tolerance induction. We demonstrated that the oral administration of milk proteins pre- or post-sensitization controlled the Th2-immune response through the elicitation of mucosal IL-10- and TGF-ß-producing Tregs that inhibited hypersensitivity symptoms and the allergic response.

Cross-reactivity between the soybean protein p34 and bovine caseins.

PURPOSE: Soy-based formulas are widely used as dairy substitutes to treat milk allergy patients. However, reactions to soy have been reported in a small proportion of patients with IgE-mediated milk allergies. The aim of this work was to explore whether P34, a mayor soybean allergen, is involved in this cross-reactivity. METHODS: In vitro recognition of P34 was evaluated by immunoblotting, competitive ELISA and basophil activation tests (BAT) using sera from allergic patients. In vivo cross-reactivity was examined using an IgE-mediated milk allergy mouse model. RESULTS: P34 was recognized by IgE antibodies from the sera of milk allergic patients, casein-specific monoclonal antibodies, and sera from milk-allergic mice. Spleen cells from sensitized mice incubated with milk, soy or P34 secreted IL-5 and IL-13, while IFN-γ remained unchanged. In addition, the cutaneous test was positive with cow's milk proteins (CMP) and P34 in the milk allergy mouse model. Moreover, milk-sensitized mice developed immediate symptoms following sublingual exposure to P34. CONCLUSIONS: Our results demonstrate that P34 shares epitopes with bovine casein, which is responsible for inducing hypersensitivity symptoms in milk allergic mice. This is the first report of the in vivo cross-allergenicity of P34.

Oral delivery of Brucella spp. recombinant protein U-Omp16 abrogates the IgE-mediated milk allergy.

Food allergies are increasingly common disorders and no therapeutic strategies are yet approved. The unlipidated Omp16 (U-Omp16) is the outer membrane protein of 16 kDa from B. abortus and possesses a mucosal adjuvant property. In this study, we aimed to examine the U-Omp16 capacity to abrogate an allergen-specific Th2 immune response when it is administered as an oral adjuvant in a mouse model of food allergy.   Balb/c mice were sensitized with cholera toxin and cow's milk proteins (CMP) by gavage and simultaneously treated with U-Omp16 and CMP. Oral challenge with CMP was performed to evaluate the allergic status of mice. Symptoms, local (small bowel cytokine and transcription factor gene expression) and systemic (specific isotypes and spleen cell-secreted cytokines) parameters, and skin tests were done to evaluate the immune response. We found that the oral administration of U-Omp16 with CMP during sensitization dampened the allergic symptoms, with negativization of immediate skin test and increased skin DTH response. Serum specific IgE and IL-5 were inhibited and a Th1 response was promoted (specific IgG2a antibodies and CMP-induced IFN-γ secretion). We found at the mucosal site an inhibition of the gene expression corresponding to IL-13 and Gata-3, with an induction of IFN-γ and T-bet. These results indicated that the oral administration of U-Omp16 significantly controlled the allergic response in sensitized mice with a shift of the balance of Th1- and Th2-T cells toward Th1 predominance. These findings suggest that U-Omp16 may be useful as a Th1-directing adjuvant in an oral vaccine.