Murilo Luiz Bazon

The social wasp Polybia paulista (Hymenoptera, Vespidae) is highly aggressive, being responsible for many medical occurrences. One of the most allergenic components of this venom is Antigen 5 (Poly p 5). The possible modulation of the in vitro immune response induced by antigen 5 from P. paulista venom, expressed recombinantly (rPoly p 5), on BALB/c mice peritoneal macrophages, activated or not with LPS, was assessed. Here, we analyzed cell viability changes, expression of the phosphorylated form of p65 NF-κB subunit, nitric oxide (NO), proinflammatory cytokines production, and co-stimulatory molecules (CD80, CD86). The results suggest that rPoly p 5 does not affect NO production nor the expression of co-stimulatory molecules in mouse peritoneal macrophages. On the other hand, rPoly p 5 induced an increase in IL-1β production in non-activated macrophages and a reduction in the production of TNF-α and MCP-1 cytokines in activated macrophages. rPoly p 5 decreased the in vitro production of the phosphorylated p65 NF-κB subunit in non-activated macrophages. These findings suggest an essential role of this allergen in the polarization of functional M2 macrophage phenotypes, when analyzed in previously activated macrophages. Further investigations, mainly in in vivo studies, should be conducted to elucidate Polybia paulista Ag5 biological role in the macrophage functional profile modulation.

Purpose of Review: Stings of Hymenoptera of the superfamily Vespoidea such as yellow jackets, paper wasps or stinging ants are common triggers for severe and even fatal allergic reactions. Antigen 5 allergens are potent allergens in the majority of these venoms with major importance for diagnosis and therapy. Reviewed here are the characteristics of antigen 5 allergens, their role in component-resolved diagnostics as well as current limitations of the available diagnostics for proper therapeutic decisions. Recent Findings: Antigens 5 are proteins of unknown function in Hymenoptera venoms with high allergenic potency. They represent key elements in component-resolved diagnosis to discriminate between honeybee and vespid venom allergy. However, due to their pronounced cross-reactivity, there are remaining diagnostic and therapeutic challenges that have to be addressed. Summary: Antigens 5 are highly relevant venom allergens of the Vespoidea superfamily. Although their use in component-resolved diagnosis facilitates dissection of cross-reactivity and primary allergy in double sensitization to honeybee and vespid venom, new diagnostic concepts are needed to discriminate between allergies to different vespid species.

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-1β and TGF-β1 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.

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.

Although systemic reactions caused by allergenic proteins present in venoms affect a small part of the world population, Hymenoptera stings are among the main causes of immediate hypersensitivity responses, with risk of anaphylactic shock. In the attempt to obtain therapeutic treatments and prophylaxis to hypersensitivity responses, interest in the molecular characterization of these allergens has grown in the scientific community due to the promising results obtained in immunological and clinical studies. The present review provides an update on the knowledge regarding the immune response and the therapeutic potential of Antigen 5 derived from Hymenoptera venom. The results confirm that the identification and topology of epitopes, associated with molecular regions that interact with antibodies, are crucial to the improvement of hypersensitivity diagnostic methods.

Polybia paulista (Hymenoptera: Vespidae) is responsible for a high number of sting accidents and anaphylaxis events in Southeast Brazil, Argentina and Paraguay. The specific detection of allergy to the venom of this wasp is often hampered by the lack of recombinant allergens currently available for molecular diagnosis. Antigen 5 (~23 kDa) from P. paulista venom (Poly p 5) is a highly abundant and glycosylated allergenic protein that could be used for development of component-resolved diagnosis (CRD). Here, we describe the cloning and heterologous expression of the antigen 5 (rPoly p 5) from P. paulista venom using the eukaryotic system Pichia pastoris. The expression as a secreted protein yielded high levels of soluble rPoly p 5. The recombinant allergen was further purified to homogeneity (99%) using a two-step chromatographic procedure. Simultaneously, the native form of the allergen (nPoly p 5) was purified from the wasp venom by Ion exchange chromatography. The rPoly p 5 and nPoly p 5 were then submitted to a comparative analysis of IgE-mediated immunodetection using sera from patients previously diagnosed with sensitization to wasp venoms. Both rPoly p 5 and nPoly p 5 were recognized by specific IgE (sIgE) in the sera of the allergic individuals. The high levels of identity found between nPoly p 5 and rPoly p 5 by the alignment of its primary sequences as well as by 3-D models support the results obtained in the immunoblot. Overall, we showed that P. pastoris is a suitable system for production of soluble rPoly p 5 and that the recombinant allergen represents a potential candidate for molecular diagnosis of P.paulista venom allergy.