RESUMO
Allergic reactions to Hymenoptera venom, which could lead to systemic and even fatal symptoms, is characterized by hypersensitivity reactions mediated by specific IgE (sIgE) driven to venom allergens. Patients multisensitized to sIgE usually recognize more than one allergen in different Hymenoptera species. However, the presence of sIgE directed against Cross-Reactive Carbohydrate Determinant (CCD), which occurs in some allergens from Hymenoptera venom, hampers the identification of the culprit insects. CCD is also present in plants, pollen, fruits, but not in mammals. Bromelain (Brl) extracted from pineapples is a glycoprotein commonly used for reference to sIgE-CCD detection and analysis. In sera of fifty-one Hymenoptera allergic patients with specific IgE ≥ 1.0 KU/L, we assessed by immunoblotting the reactivity of sIgE to the major allergens of Apis mellifera, Polybia paulista and Solenopsis invicta venoms. We also distinguished, using sera adsorption procedures, the cases of CCD cross-reaction using Brl as a marker and inhibitor of CCD epitopes. The presence of reactivity for bromelain (24-28 kDa) was obtained in 43% of the patients, in which 64% presented reactivity for more than one Hymenoptera venom in radioallergosorbent (RAST) tests, and 90% showed reactivity in immunoblot analysis to the major allergens of Apis mellifera, Polybia paulista and Solenopsis invicta venoms. Sera adsorption procedures with Brl lead to a significant reduction in patients' sera reactivity to the Hymenoptera allergens. Immunoblotting assay using pre- and post-Brl adsorption sera from wasp-allergic patients blotted with non-glycosylated recombinant antigens (rPoly p1, rPoly p5) from Polybia paulista wasp venom showed no change in reactivity pattern of sIgE that recognize allergen peptide epitopes. Our results, using Brl as a marker and CCD inhibitor to test sIgE reactivity, suggest that it could complement diagnostic methods and help to differentiate specific reactivity to allergens' peptide epitopes from cross-reactivity caused by CCD, which is extremely useful in clinical practice.
Assuntos
Alérgenos/imunologia , Venenos de Formiga/imunologia , Venenos de Abelha/imunologia , Carboidratos/imunologia , Hipersensibilidade/imunologia , Imunoglobulina E/imunologia , Mordeduras e Picadas de Insetos/imunologia , Venenos de Vespas/imunologia , Adolescente , Adulto , Especificidade de Anticorpos , Bromelaínas/imunologia , Criança , Pré-Escolar , Reações Cruzadas , Epitopos , Feminino , Humanos , Hipersensibilidade/sangue , Hipersensibilidade/diagnóstico , Imunoglobulina E/sangue , Testes Imunológicos , Mordeduras e Picadas de Insetos/sangue , Mordeduras e Picadas de Insetos/diagnóstico , Masculino , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Adulto JovemRESUMO
Insect venom can cause systemic allergic reactions, including anaphylaxis. Improvements in diagnosis and venom immunotherapy (VIT) are based on a better understanding of an immunological response triggered by venom allergens. Previously, we demonstrated that the recombinant phospholipase A1 (rPoly p 1) from Polybia paulista wasp venom induces specific IgE and IgG antibodies in sensitized mice, which recognized the native allergen. Here, we addressed the T cell immune response of rPoly p 1-sensitized BALB/c mice. Cultures of splenocytes were stimulated with Polybia paulista venom extract and the proliferation of CD8+ and CD4+ T cells and the frequency of T regulatory cells (Tregs) populations were assessed by flow cytometry. Cytokines were quantified in cell culture supernatants in ELISA assays. The in vitro stimulation of T cells from sensitized mice induces a significant proliferation of CD4+ T cells, but not of CD8+ T cells. The cytokine pattern showed a high concentration of IFN-γ and IL-6, and no significant differences to IL-4, IL-1ß and TGF-ß1 production. In addition, the rPoly p 1 group showed a pronounced expansion of CD4+CD25+FoxP3+ and CD4+CD25-FoxP3+ Tregs. rPoly p 1 sensitization induces a Th1/Treg profile in CD4+ T cell subset, suggesting its potential use in wasp venom immunotherapy.
Assuntos
Alérgenos/farmacologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Dessensibilização Imunológica , Proteínas de Insetos/farmacologia , Fosfolipases A1/farmacologia , Venenos de Vespas/farmacologia , Alérgenos/imunologia , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Citocinas/metabolismo , Feminino , Hipersensibilidade/imunologia , Hipersensibilidade/metabolismo , Hipersensibilidade/terapia , Mordeduras e Picadas de Insetos/imunologia , Mordeduras e Picadas de Insetos/metabolismo , Mordeduras e Picadas de Insetos/terapia , Proteínas de Insetos/imunologia , Ativação Linfocitária/efeitos dos fármacos , Camundongos Endogâmicos BALB C , Fosfolipases A1/imunologia , Venenos de Vespas/imunologiaRESUMO
Phospholipase A1 (PLA1) is one of the three major allergens identified in the venom of P. paulista (Hymenoptera: Vespidae), a clinically relevant wasp from southeastern Brazil. The recombinant form of this allergen (rPoly p 1) could be used for the development of molecular diagnostic of venom allergy. Early attempts to produce rPoly p 1 using Escherichia coli BL21 (DE3) cells rendered high yields of the insoluble rPoly p 1 but with low levels of solubilized protein recovery (12%). Here, we aimed to improve the production of rPoly p 1 in E. coli by testing different conditions of expression, solubilization of the inclusion bodies and protein purification. The results showed that the expression at 16 °C and 0.1 mM of IPTG increased the production of rPoly p 1, still in the insoluble form, but with high solubilized protein yields after incubation with citrate-phosphate buffer with 0.15 M NaCl, 6 M urea, pH 2.6 at 25 ºC for 2 h. The venom allergen was also cloned in pPICZαA vector for soluble expression as a secreted protein in Pichia pastoris X-33 cells, rendering almost undetectable levels (nanograms) in the culture supernatant. In contrast, a sevenfold increase of the solubilized and purified rPoly p 1 yields (1.5 g/L of fermentation broth) was obtained after improved production in E. coli. The identity of the protein was confirmed with an anti-His antibody and MS spectra. Allergen-specific IgE (sIgE)-mediated recognition was evaluated in immunoblotting with sera of allergic patients (n = 40). Moreover, rPoly p 1 showed high levels of diagnostic sensitivity (95%). The optimized strategy for rPoly p 1 production described here, will provide the amounts of allergen necessary for the subsequent protein refolding, immunological characterization steps, and ultimately, to the development of molecular diagnostic for P. paulista venom allergy.
RESUMO
Molecular cross-reactivity caused by allergen homology or cross-reactive carbohydrate determinants (CCDs) is a major challenge for diagnosis and immunotherapy of insect venom allergy. Venom phospholipases A1 (PLA1s) are classical, mostly non-glycosylated wasp and ant allergens that provide diagnostic benefit for differentiation of genuine sensitizations from cross-reactivity. As CCD-free molecules, venom PLA1s are not causative for CCD-based cross-reactivity. Little is known however about the protein-based cross-reactivity of PLA1 within vespid species. Here, we address PLA1-based cross-reactivity among ten clinically relevant Hymenoptera venoms from Neotropical and temperate regions including Polybia paulista (paulistinha) venom and Vespula vulgaris (yellow jacket) venom. In order to evaluate cross-reactivity, sera of mice sensitized with recombinant PLA1 (rPoly p 1) from P. paulista wasp venom were used. Pronounced IgE and IgG based cross-reactivity was detected for wasp venoms regardless the geographical region of origin. The cross-reactivity correlated well with the identity of the primary sequence and 3-D models of PLA1 proteins. In contrast, these mice sera showed no reaction with honeybee (HBV) and fire ant venom. Furthermore, sera from patients monosensitized to HBV and fire ants did not recognize the rPoly p 1 in immunoblotting. Our findings reveal the presence of conserved epitopes in the PLA1s from several clinically relevant wasps as major cause of PLA1-based in vitro cross-reactivity. These findings emphasize the limitations but also the potential of PLA1-based HVA diagnostics.