EPI-peptide designer: a tool for designing peptide ligand libraries based on epitope–paratope interactions

International audienc

Anti-loxoscelic horse serum produced against a recombinant dermonecrotic protein of Brazilian Loxosceles intermedia spider neutralize lethal effects of Loxosceles laeta venom from Peru

AbstractIn this work, an anti-loxoscelic serum was produced by immunizing horses with a recombinant dermonecrotic protein from Loxosceles intermedia (rLiD1). Anti-rLiD1 antibodies were able to recognize different species of Loxosceles venoms by Western Blot and ELISA. The efficacy of anti-rLiD1 serum against the toxic effects of Loxosceles laeta (Peru) venom was tested, showing that anti-rLiD1 serum can neutralize those effects. This study confirms that recombinant proteins can be good candidates to replace crude venoms for antivenom production

L-amino acid oxidase from Bothrops atrox snake venom triggers autophagy, apoptosis and necrosis in normal human keratinocytes

Snake venom L-amino acid oxidases (LAAOs) are flavoproteins, which perform diverse biological activities in the victim such as edema, myotoxicity and cytotoxicity, contributing to the development of clinical symptoms of envenomation. LAAO cytotoxicity has been described, but the temporal cascade of events leading to cell death has not been explored so far. This study evaluates the involvement of LAAO in dermonecrosis in mice and its cytotoxic effects in normal human keratinocytes, the major cell type in the epidermis, a tissue that undergoes extensive necrosis at the snakebite site. Pharmacological inhibition by the antioxidant NAC (N-acetyl cysteine) prevented B. atrox venom-induced necrosis. Consistent with the potential role of oxidative stress in wounding, treatment with purified LAAO decreased keratinocyte viability with an Effective Concentration (EC50) of 5.1 μg/mL. Cytotoxicity caused by LAAO was mediated by H2O2 and treated cells underwent autophagy, followed by apoptosis and necrosis. LAAO induced morphological alterations that precede cell death. Our results show the chronological events leading to cell death and the temporal resolution from autophagy, apoptosis and necrosis as distinct mechanisms triggered by LAAO. Fluorescently-labelled LAAO was efficiently and rapidly internalized by keratinocytes, suggesting that catalysis of intracellular substrates may contribute to LAAO toxicity. A better understanding of LAAO cytotoxicity and its mechanism of action will help to identify potential therapeutic strategies to ameliorate localized snake envenomation symptoms

Induction of neutralizing antibodies against mutalysin-II from Lachesis muta muta Snake Venom Elicited by a conformational B-cell epitope predicted by Blue Star Sting data base.

Mutalysin-II, from Lachesis muta muta snake venom, is an endopeptidase with hemorrhagic activity. To identify a conformational epitope we used Blue Star Sting, the latest version of the web based Sting Millenium Suite, as alternative computational analysis tool to select and design peptides. Pre-selected cut-off values of the accessibility and hydrophilicity parameters were used to select amino acid residues as potential conformational epitopes. A peptide (P117-Y116-C115-Q194-C195-L197-N198-K199-P200-Y5-L48) was manually drawn on Swiss-PDB-Viewer package and synthesized by Fmoc-synthesis. Immunization of rabbits with this peptide induced antibodies that recognized Mutalysin-II and protected against the hemorrhagic factors present in Lachesis venom. The Sting Millennium Suite was able to predict conformational epitopes in this class of proteins. Three amino acids (K199, Y5 and L48) were identified as essential in the interaction between the peptide and the neutralizing antibodies

Biochemical, biological and molecular characterization of an L-Amino acid oxidase (LAAO) purified from Bothrops pictus Peruvian snake venom

We thank Prof. J.A. Eble, from the Institute for Physiological Chemistry and Pathobiochemistry, University of Münster, Germany for critical reading the manuscript. This work was supported by Convenio de Cooperación Bilateral CONCYTEC (Perú) - CNPq (Brazil), Grant 490269/2013-3 , Fundação de Amparo a Pesquisa do Estado de Minas Gerais (FAPEMIG, Brazil) and Programa Nacional de Innovación para la Competitividad y Productividad - Innóvate Perú (Contrato N° 131-FINCyT-IB-2013 ). This report is part of a dissertation for Doctoral Degree of Fanny Lazo to Post Graduate School in Biological Sciences, UNMSM.Consejo Nacional de Ciencia, Tecnología e Innovación Tecnológica - Concyte

Recombinant Protein Containing B-Cell Epitopes of Different Loxosceles Spider Toxins Generates Neutralizing Antibodies in Immunized Rabbits

Loxoscelism is the most important form of araneism in South America. The treatment of these accidents uses heterologous antivenoms obtained from immunization of production animals with crude loxoscelic venom. Due to the scarcity of this immunogen, new alternatives for its substitution in antivenom production are of medical interest. In the present work, three linear epitopes for Loxosceles astacin-like protease 1 (LALP-1) (SLGRGCTDFGTILHE, ENNTRTIGPFDYDSIMLYGAY, and KLYKCPPVNPYPGGIRPYVNV) and two for hyaluronidase (LiHYAL) (NGGIPQLGDLKAHLEKSAVDI and ILDKSATGLRIIDWEAWR) from Loxosceles intermedia spider venom were identified by SPOT-synthesis technique. One formerly characterized linear epitope (DFSGPYLPSLPTLDA) of sphingomyelinase D (SMase D) SMase-I from Loxosceles laeta was also chosen to constitute a new recombinant multiepitopic protein. These epitopes were combined with a previously produced chimeric multiepitopic protein (rCpLi) composed by linear and conformational B-cell epitopes from SMase D from L. intermedia venom, generating a new recombinant multiepitopic protein derived from loxoscelic toxins (rMEPLox). We demonstrated that rMEPLox is non-toxic and antibodies elicited in rabbits against this antigen present reactivity in ELISA and immunoblot assays with Brazilian L. intermedia, L. laeta, L. gaucho, and L. similis spider venoms. In vivo and in vitro neutralization assays showed that anti-rMEPLox antibodies can efficiently neutralize the sphingomyelinase, hyaluronidase, and metalloproteinase activity of L. intermedia venom. This study suggests that this multiepitopic protein can be a suitable candidate for experimental vaccination approaches or for antivenom production against Loxosceles spp. venoms

Mesenchymal Stromal Cell-Based Therapies as Promising Treatments for Muscle Regeneration After Snakebite Envenoming

Snakebite envenoming is a global neglected disease with an incidence of up to 2.7 million new cases every year. Although antivenoms are so-far the most effective treatment to reverse the acute systemic effects induced by snakebite envenoming, they have a limited therapeutic potential, being unable to completely neutralize the local venom effects. Local damage, such as dermonecrosis and myonecrosis, can lead to permanent sequelae with physical, social, and psychological implications. The strong inflammatory process induced by snake venoms is associated with poor tissue regeneration, in particular the lack of or reduced skeletal muscle regeneration. Mesenchymal stromal cells (MSCs)-based therapies have shown both anti-inflammatory and pro-regenerative properties. We postulate that using allogeneic MSCs or their cell-free products can induce skeletal muscle regeneration in snakebite victims, improving all the three steps of the skeletal muscle regeneration process, mainly by anti-inflammatory activity, paracrine effects, neovascularization induction, and inhibition of tissue damage, instrumental for microenvironment remodeling and regeneration. Since snakebite envenoming occurs mainly in areas with poor healthcare, we enlist the principles and potential of MSCs-based therapies and discuss regulatory issues, good manufacturing practices, transportation, storage, and related-procedures that could allow the administration of these therapies, looking forward to a safe and cost-effective treatment for a so far unsolved and neglected health problem.The authors are supported by the University Pablo de Olavide (Sevilla), the University Miguel Hernández (Elche, Alicante), National University Toribio Rodriguez de Mendoza (Chachapoyas, Peru) Grants: Contrato N° 09-2019-FONDECYT-BM-INC.INV to JRT, JDRF 2-SRA-2019-837-S-B and AVI-GVA COVID-19-68 to BS, Fundación Andaluza de I+D and Al-Andalus Biopharma Project (FAID-2018-1). The authors CC-O, CG-D, and TCSA were supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico, Brazil (CNPq) (Process: 406163/2018-9), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, Brazil - CAPES (Program COFECUB Process: 88881.191812/2018-01) and by Fundação de Amparo à Pesquisa do Estado de Minas Gerais, Brazil (FAPEMIG).Ye