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The fibroin-based silk fibers of weaver ants are an alternative biomaterial to be investigated and explored for potential biomedical applications. In this context, the silk fibers from the nest of the weaver ant Camponotus textor was solubilized and fractionated by gel permeation. The different fractions were collected, pooled and submitted to analysis with a series of biochemical methods, nuclear magnetic resonance (NMR) spectroscopy, analytical proteomic strategies, and data treatment with bioinformatic tools to perform the structural characterization of the fibroin-based silk fibers produced by the ant. Our data demonstrated the identification of one fibroin proteoform in the ant silk fibers. The protein chracterized as a glycoprotein with MW around 40 kDa and presenting 66% (w/w) of total sugars attached to it through O-linked carbohydrates. The 3D of protein was modeled, revealing a structure predominantly constituted of coiled-coil secondary units in the whole model, featuring at least four superhelices (arrangement with multiple α-helices). The scientific outcomes reported herein may be relevant for the development of novel approaches for the synthetic or recombinant production of novel silk-based polymers for biomedical applications. BIOLOGICAL SIGNIFICANCE: The present investigation significantly expanded knowledge regarding to the fibroin-based silk fibers from weaver ants, contributing to improvements in our understanding of the properties and characteristics of these silk fibers. For example, as reported here, carbohydrates were detected in the ants' silk for the first time presenting the fibroin as a glycoprotein. Moreover, the 3D structure provided new insights into the secondary structures considering the whole model of the protein.

Insect venom phospholipases have been identified in nearly all clinically relevant social Hymenoptera, including bees, wasps and ants. Among other biological roles, during the envenoming process these enzymes cause the disruption of cellular membranes and induce hypersensitive reactions, including life threatening anaphylaxis. While phospholipase A2 (PLA2) is a predominant component of bee venoms, phospholipase A1 (PLA1) is highly abundant in wasps and ants. The pronounced prevalence of IgE-mediated reactivity to these allergens in sensitized patients emphasizes their important role as major elicitors of Hymenoptera venom allergy (HVA). PLA1 and -A2 represent valuable marker allergens for differentiation of genuine sensitizations to bee and/or wasp venoms from cross-reactivity. Moreover, in massive attacks, insect venom phospholipases often cause several pathologies that can lead to fatalities. This review summarizes the available data related to structure, model of enzymatic activity and pathophysiological roles during envenoming process of insect venom phospholipases A1 and -A2.

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 &ge, 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&ndash, 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&rsquo, 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&rsquo, peptide epitopes from cross-reactivity caused by CCD, which is extremely useful in clinical practice.

Made available in DSpace on 2020-12-12T02:04:03Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-05-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) 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. Center for the Study of Social Insects Department of General and Applied Biology University of Sao Paulo State (UNESP/RC) Center for Genetic Engineering and Biotechnology. Biomedical Research Division System Biology Department, Ave. 31, e/ 158 and 190, Cubanacan, Playa, P.O. Box 6162 Laboratory of Translational Immunology School of Medical Sciences University of Campinas (UNICAMP), Campinas Arthropod Molecular Biology Laboratory-LBMA-IB University of Sao Paulo State (UNESP/RC), Ave. 24-A, 1515- Bela Vista Venoms and Venomous Animal Studies Center-CEVAP University of São Paulo State (UNESP) Lageado Experimental Farm, José Barbosa de Barros Street, 1780 Center for the Study of Social Insects Department of General and Applied Biology University of Sao Paulo State (UNESP/RC) Arthropod Molecular Biology Laboratory-LBMA-IB University of Sao Paulo State (UNESP/RC), Ave. 24-A, 1515- Bela Vista Venoms and Venomous Animal Studies Center-CEVAP University of São Paulo State (UNESP) Lageado Experimental Farm, José Barbosa de Barros Street, 1780 FAPESP: # 2014/13936-7

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. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s13205-020-02202-8) contains supplementary material, which is available to authorized users.

Purpose of Review In Hymenoptera venom allergy, the research focus has moved from whole venoms to individual allergenic molecules. Api m 10 (icarapin) has been described as a major allergen of honeybee venom (HBV) with potentially high relevance for diagnostics and therapy of venom allergy. Here, we review recent studies on Api m 10 characteristics as well as its role in component-resolved diagnostics and potential implications for venom-specific immunotherapy (VIT). Recent Findings Api m 10 is a major allergen of low abundance in HBV. It is an obviously unstable protein of unknown function that exhibits homologs in other insect species. Despite its low abundance in HBV, 35 to 72% of HBV-allergic patients show relevant sensitization to this allergen. Api m 10 is a marker allergen for HBV sensitization, which in many cases can help to identify primary sensitization to HBV and, hence, to discriminate between genuine sensitization and cross-reactivity. Moreover, Api m 10 might support personalized risk stratification in VIT, as dominant sensitization to Api m 10 has been identified as risk factor for treatment failure. This might be of particular importance since Api m 10 is strongly underrepresented in some therapeutic preparations commonly used for VIT. Summary Although the role of Api m 10 in HBV allergy and tolerance induction during VIT is not fully understood, it certainly is a useful tool to unravel primary sensitization and individual sensitization profiles in component-resolved diagnostics (CRD). Moreover, a potential of Api m 10 to contribute to personalized treatment strategies in HBV allergy is emerging.

Polybia paulista (Hymenoptera: Vespidae) is a neotropical social wasp from southeast Brazil. As most social Hymenoptera, venom from P. paulista comprises a complex mixture of bioactive toxins ranging from low molecular weight compounds to peptides and proteins. Several efforts have been made to elucidate the molecular composition of the P. paulista venom. Data derived from proteomic, peptidomic and allergomic analyses has enhanced our understanding of the whole envenoming process caused by the insect sting. The combined use of bioinformatics, -omics- and molecular biology tools have allowed the identification, characterization, in vitro synthesis and recombinant expression of several wasp venom toxins. Some of these P. paulista - derived bioactive compounds have been evaluated for the rational design of antivenoms and the improvement of allergy specific diagnosis and immunotherapy. Molecular characterization of crude venom extract has enabled the description and isolation of novel toxins with potential biotechnological applications. Here, we review the different approaches that have been used to unravel the venom composition of P. paulista. We also describe the main groups of P. paulista - venom toxins currently identified and analyze their potential in the development of component-resolved diagnosis of allergy, and in the rational design of antivenoms and novel bioactive drugs.

Made available in DSpace on 2019-10-04T12:37:47Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-05-30 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) The partial proteome of Polybia paulista wasp venom was previously reported elsewhere using a gel-dependent approach and resulted in the identification of a limited number of venom toxins. Here, we reinvestigated the P. paulista venom using a gel-free shotgun proteomic approach; the highly dynamic range of this approach facilitated the detection and identification of 1673 proteins, of which 23 venom proteins presented N-linked glycosylation as a posttranslational modification. Three different molecular forms of PLAT were identified as allergenic proteins, and two of these forms were modified by N-linked glycosylation. This study reveals an extensive repertoire of hitherto undescribed proteins that were classified into the following six different functional groups: (i) typical venom proteins; (ii) proteins related to the folding/conformation and PTMs of toxins; (iii) proteins that protect toxins from oxidative stress; (iv) proteins involved in chemical communication; (v) housekeeping proteins; and (vi) uncharacterized proteins. It was possible to identify venom toxin-like proteins that are commonly reported in other animal venoms, including arthropods such as spiders and scorpions. Thus, the findings reported here may contribute to improving our understanding of the composition of P. paulista venom, its envenoming mechanism and the pathologies experienced by the victim after the wasp stinging accident. Biological significance: The present study significantly expanded the number of proteins identified in P. paulista venom, contributing to improvements in our understanding of the envenoming mechanism produced by sting accidents caused by this wasp. For example, novel wasp venom neurotoxins have been identified, but no studies have assessed the presence of this type of toxin in social wasp venoms. In addition, 23 N-linked glycosylated venom proteins were identified in the P. paulista venom proteome, and some of these proteins might be relevant allergens that are immunoreactive to human IgE. Sao Paulo State Univ, Inst Biosci Rio Claro, Dept Biol, Ctr Study Social Insects, BR-13500 Rio Claro, SP, Brazil Univ Campinas UNICAMP, Fac Med, Lab Translat Immunol, Cidade Univ Zeferino Vaz, BR-13083887 Campinas, SP, Brazil Sao Paulo State Univ, Inst Biosci Rio Claro, Dept Biol, Ctr Study Social Insects, BR-13500 Rio Claro, SP, Brazil FAPESP: 2013/26451-9 FAPESP: 2016/16212-5 FAPESP: 2017/04680-7 FAPESP: 2017/10373-0 FAPESP: 2017/22405-3 CNPq: 301656/2013-4 CNPq: 150699/2017-4

Made available in DSpace on 2020-12-12T02:10:46Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-06-01 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- and IL-6, and no significant differences to IL-4, IL-1 and TGF- 1 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. Laboratory of Translational Immunology Medicine School FCM University of Campinas Center of the Study of Social Insects Department of General and Applied Biology Institute of Biosciences of Rio Claro Institute of Biosciences of Rio Claro S o Paulo State University Laboratory of Arthropods Molecular Biology IBRC UNESP S o Paulo State University Venoms and Venomous Animal Studies Center CEVAP S o Paulo State University Laboratory of Arthropods Molecular Biology IBRC UNESP S o Paulo State University

Made available in DSpace on 2018-12-11T16:52:01Z (GMT). No. of bitstreams: 0 Previous issue date: 2018-05-01 Institute of Biosciences University of Sao Paulo State (UNESP) Immunological Biotechnology Department of Engineering Aarhus University Euroimmun AG Laboratory of Translational Immunology Faculty of Medicine University of Campinas (UNICAMP) Department of Dermatology and Allergology University Medical Center Giessen and Marburg Justus-Liebig University Giessen Institute of Biosciences University of Sao Paulo State (UNESP)

Made available in DSpace on 2018-11-26T17:52:01Z (GMT). No. of bitstreams: 0 Previous issue date: 2018-06-15 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Among venomous animals, Hymenoptera have been suggested as a rich source of natural toxins. Due to their broad ecological diversity, venom from Hymenoptera insects (bees, wasps and ants) have evolved differentially thus widening the types and biological functions of their components. To date, insect toxinology analysis have scarcely uncovered the complex composition of bee, wasp and ant venoms which include low molecular weight compounds, highly abundant peptides and proteins, including several allergens. In Hymenoptera, these complex mixtures of toxins represent a potent arsenal of biological weapons that are used for self-defense, to repel intruders and to capture prey. Consequently, Hymenoptera venom components have a broad range of pharmacological targets and have been extensively studied, as promising sources of new drugs and biopesticides. In addition, the identification and molecular characterization of Hymenoptera venom allergens have allowed for the rational design of component-resolved diagnosis of allergy, finally improving the outcome of venom immunotherapy (VIT). Until recently, a limited number of Hymenoptera venoms had been unveiled due to the technical limitations of the approaches used to date. Nevertheless, the application of novel techniques with high dynamic range has significantly increased the number of identified peptidic and proteinaceous toxins. Considering this, the present review summarizes the current knowledge about the most representative Hymenoptera venom peptides and proteins which are under study for a better understanding of the insect-caused envenoming process and the development of new drugs and biopesticides. (C) 2018 Elsevier Ltd. All rights reserved. Sao Paulo State Univ, Biosci Inst Rio Claro, Biol Dept, Social Insect Study Ctr, BR-13500 Rio Claro, SP, Brazil Ctr Genet Engn & Biotechnol, Biomed Res Div, Syst Biol Dept, Ave 31,E-158 & 190,POB 6162, Havana 10600, Cuba Sao Paulo State Univ, Biosci Inst Rio Claro, Biol Dept, Social Insect Study Ctr, BR-13500 Rio Claro, SP, Brazil FAPESP: 2016/16212-5 CNPq: 301656/2013-4 FAPESP: 2017/22405-3 CNPq: 150699/2017-4

Made available in DSpace on 2018-12-11T17:16:09Z (GMT). No. of bitstreams: 0 Previous issue date: 2018-01-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) 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. Laboratório de Biologia Molecular de Artrópodes-LBMA-IBRC-UNESP (Univ Estadual Paulista), Av. 24-A, n_1515, Bela Vista Laboratório de Imunologia Translacional Faculdade de Ciências Médicas FCM Universidade Estadual de Campinas-UNICAMP, Rua Vital Brasil, n_ 300, Cidade Universitária “Zeferino Vaz” Center for Genetic Engineering and Biotechnology Biomedical Research Division System Biology Department, Ave. 31, e/158 and 190, P.O. Box 6162, Cubanacan, Playa Centro de Estudos de Insetos Sociais-CEIS-IBRC-UNESP (Univ Estadual Paulista), Av. 24-A, n° 1515, Bela Vista Immunological Engineering Department of Engineering Aarhus University, Gustav Wieds Vej 10 Euroimmun AG, Seekamp 21 Centro de Estudos de Venenos e Animais Peçonhentos-CEVAP (Univ Estadual Paulista), Rua José Barbosa de Barros, 1780, Fazenda Experimental Lageado Laboratório de Biologia Molecular de Artrópodes-LBMA-IBRC-UNESP (Univ Estadual Paulista), Av. 24-A, n_1515, Bela Vista Centro de Estudos de Insetos Sociais-CEIS-IBRC-UNESP (Univ Estadual Paulista), Av. 24-A, n° 1515, Bela Vista Centro de Estudos de Venenos e Animais Peçonhentos-CEVAP (Univ Estadual Paulista), Rua José Barbosa de Barros, 1780, Fazenda Experimental Lageado FAPESP: 2013/26451-9

Made available in DSpace on 2018-12-11T17:07:45Z (GMT). No. of bitstreams: 0 Previous issue date: 2016-12-15 Polybia paulista (Hymenoptera: Vespidae) is a clinically relevant social wasp that frequently causes stinging accidents in southeast Brazil. To date, diagnosis and specific immunotherapy (SIT) of allergy are based on the use of crude venom extracts. Production of recombinant forms of major allergens from P. paulista venom will improve diagnosis and SIT of allergic patients by reducing the incidence of cross-reactivity and non-specific sensitization. Here, we describe the molecular cloning, heterologous expression, purification and IgE-mediated immunodetection of phospholipase A1 (Poly p 1), a major allergen from P. paulista venom. The cDNA of Poly p 1 was extracted from venom glands and then cloned, and further expression of the recombinant allergen (rPoly p 1) was achieved in Escherichia coli BL21 (DE3) cells. Purification of rPoly p 1 was performed using immobilized Ni2+ metal affinity chromatography. Also, a single-step chromatographic method allowed the purification of native Poly p 1 (nPoly p 1) from the wasp's venom glands. We used western blotting to evaluate IgE-reactivity of the sera from 10 P. paulista venom-allergic patients to rPoly p 1 and nPoly p 1. High levels of insoluble rPoly p 1 were obtained during heterologous expression. After solubilization of inclusion bodies and purification of the recombinant protein, a unique band of ∼34 kDa was detected in SDS-PAGE analysis. Allergen-specific IgE (sIgE) from allergic patients' sera recognized rPoly p 1, nPoly p 1 and crude venom extract to a similar extent. Our results showed that rPoly p 1 could be used for development of component-resolved diagnosis (CRD) and molecular-defined SIT of P. paulista venom allergy. Laboratório de Biologia Molecular de Artrópodes-LBMA-IBRC-UNESP (Univ Estadual Paulista), Av. 24-A, nº 1515, Bela Vista, CEP Laboratório de Mutagênese Ambiental, Avenida 24-A, nº 1515, Bela Vista, CEP Center for Genetic Engineering and Biotechnology Biomedical Research Division System Biology Department, Ave. 31, e/ 158 and 190, P.O. Box 6162, Cubanacan Laboratório de Imunologia Translacional Faculdade de Ciências Médicas FCM Universidade Estadual de Campinas-UNICAMP, Rua Vital Brasil, nº 300, Cidade Universitária “Zeferino Vaz”, CEP Centro de Estudos de Insetos Sociais-CEIS-IBRC-UNESP (Univ Estadual Paulista), Av. 24-A, nº 1515, Bela Vista, CEP Instituto de Pesquisa em Bioenergia (IPBEN) (Univ Estadual Paulista), Av. 24-A, nº1515, Bela Vista, CEP Centro de Estudos de Venenos e Animais Peçonhentos-CEVAP (Univ Estadual Paulista), Rua José Barbosa de Barros, 1780, Fazenda Experimental Lageado Laboratório de Biologia Molecular de Artrópodes-LBMA-IBRC-UNESP (Univ Estadual Paulista), Av. 24-A, nº 1515, Bela Vista, CEP Centro de Estudos de Insetos Sociais-CEIS-IBRC-UNESP (Univ Estadual Paulista), Av. 24-A, nº 1515, Bela Vista, CEP Instituto de Pesquisa em Bioenergia (IPBEN) (Univ Estadual Paulista), Av. 24-A, nº1515, Bela Vista, CEP Centro de Estudos de Venenos e Animais Peçonhentos-CEVAP (Univ Estadual Paulista), Rua José Barbosa de Barros, 1780, Fazenda Experimental Lageado

Made available in DSpace on 2015-12-07T15:33:07Z (GMT). No. of bitstreams: 0 Previous issue date: 2015. Added 1 bitstream(s) on 2015-12-07T15:52:35Z : No. of bitstreams: 1 PMC4516928.pdf: 648864 bytes, checksum: 75ea738e8072856dd8981382ff890978 (MD5) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Fundação para o Desenvolvimento da UNESP (FUNDUNESP) Coordenadação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Along with food and drug allergic reactions, a Hymenoptera insect Sting (Apoidea, Vespidae, Formicidae) is one of the most common causes of anaphylaxis worldwide. Diagnoses of Hymenoptera venom allergy (HVA) and specific immunotherapy (SIT) have been based on the use of crude venom extracts. However, the incidence of cross-reactivity and low levels of sensibility during diagnosis, as well as the occurrence of nonspecific sensitization and undesired side effects during SIT, encourage the search for novel allergenic materials. Recombinant allergens are an interesting approach to improve allergy diagnosis and SIT because they circumvent major problems associated with the use of crude venom. Production of recombinant allergens depends on the profound molecular characterization of the natural counterpart by combining some omics approaches with high-throughput screening techniques and the selection of an appropriate system for heterologous expression. To date, several clinically relevant allergens and novel venom toxins have been identified, cloned and characterized, enabling a better understanding of the whole allergenic and envenoming processes. Here, we review recent findings on identification, molecular characterization and recombinant expression of Hymenoptera venom allergens and on the evaluation of these heterologous proteins as valuable tools for tackling remaining pitfalls on HVA diagnosis and immunotherapy. Universidade Estadual de Campinas, Departamento de Clínica Médica, Faculdade de Ciências Médicas Universidade Estadual Paulista, Departamento de Biologia, Instituto de Biociências de Rio Claro Universidade Estadual Paulista, Centro de Estudos de Venenos e Animais Peçonhentos de Botucatu FAPESP: 2014/13936-7 FUNDUNESP: 0119710 FAPESP: 2009/51539-1

Made available in DSpace on 2018-12-11T17:14:26Z (GMT). No. of bitstreams: 0 Previous issue date: 2017-09-01 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) 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. Laboratório de Biologia Molecular de Artrópodes-LBMA-IB-RC-UNESP Univ Estadual Paulista, Av. 24-A, n◦ 1515, Bela Vista Centro de Estudos de Insetos Sociais-CEIS-IBRC-UNESP Univ Estadual Paulista, Av. 24-A, n◦ 1515, Bela Vista Laboratório de Imunologia Translacional-LIT Departamento de Clínica Médica Faculdade de Ciências Médicas FCM Universidade Estadual de Campinas-UNICAMP, Rua Tessália Vieira de Camargo n◦ 126, Cidade Universitária “Zeferino Vaz” System Biology Department Biomedical Research Division Center for Genetic Engineering and Biotechnology, Ave. 31, e/158 and 190, P.O. Box 6162, Cubanacan Centro de Estudos de Venenos e Animais Peçonhentos-CEVAP Univ Estadual Paulista, Rua José Barbosa de Barros, 1780, Fazenda Experimental Lageado Laboratório de Biologia Molecular de Artrópodes-LBMA-IB-RC-UNESP Univ Estadual Paulista, Av. 24-A, n◦ 1515, Bela Vista Centro de Estudos de Insetos Sociais-CEIS-IBRC-UNESP Univ Estadual Paulista, Av. 24-A, n◦ 1515, Bela Vista Centro de Estudos de Venenos e Animais Peçonhentos-CEVAP Univ Estadual Paulista, Rua José Barbosa de Barros, 1780, Fazenda Experimental Lageado FAPESP: 2013/26451-9 FAPESP: 2014/13936-7 CNPq: 455422/2014-1