Use of a Synthetic Biosensor for Neutralizing Activity-Biased Selection of Monoclonal Antibodies against Atroxlysin-I, an Hemorrhagic Metalloproteinase from Bothrops atrox Snake Venom

Francisco Santos Schneider,Claude Granier,Eladio Flores Sanchez,Christophe Nguyen,Dung Le Nguyen,Nivia De Assis Ferreira,Sandra Cobo,Karen Larissa Castro,Ricardo Andrez Machado De Avila,Pascale Galéa,Franck Molina,Carlos Chávez-Olortegui,José María Gutiérrez

doi:10.1371/journal.pntd.0002826

Abstract

BackgroundThe snake Bothrops atrox is responsible for the majority of envenomings in the northern region of South America. Severe local effects, including hemorrhage, which are mainly caused by snake venom metalloproteinases (SVMPs), are not fully neutralized by conventional serum therapy. Little is known about the immunochemistry of the P-I SVMPs since few monoclonal antibodies (mAbs) against these molecules have been obtained. In addition, producing toxin-neutralizing mAbs remains very challenging.Methodology/Principal FindingsHere, we report on the set-up of a functional screening based on a synthetic peptide used as a biosensor to select neutralizing mAbs against SVMPs and the successful production of neutralizing mAbs against Atroxlysin-I (Atr-I), a P-I SVMP from B. atrox. Hybridomas producing supernatants with inhibitory effect against the proteolytic activity of Atr-I towards the FRET peptide Abz-LVEALYQ-EDDnp were selected. Six IgG1 Mabs were obtained (named mAbatr1 to mAbatr6) and also two IgM. mAbatrs1, 2, 3 and 6 were purified. All showed a high specific reactivity, recognizing only Atr-I and B. atrox venom in ELISA and a high affinity, showing equilibrium constants in the nM range for Atr-I. These mAbatrs were not able to bind to Atr-I overlapping peptides, suggesting that they recognize conformational epitopes.Conclusions/SignificanceFor the first time a functional screening based on a synthetic biosensor was successfully used for the selection of neutralizing mAbs against SVMPs.

Highlights

Snakebites cause up to 1,800 000 envenomations per year, mainly in tropical areas [1,2,3,4]
We propose a new screening strategy to produce monoclonal antibodies against Atr-I, a P-I class snake venom metalloproteinases (SVMPs) from Bothrops atrox, which is the snake responsible for the majority of the accidents in South America
SVMPs are the main toxic factors in Bothrops venom causing systemic and local hemorrhage, which may evolve to inflammation and/or necrosis

Summary

Introduction

Snakebites cause up to 1,800 000 envenomations per year, mainly in tropical areas [1,2,3,4]. In the Brazilian Amazon region, 9,000 snakebites occur per year with an incidence fourfold higher than that found in the rest of Brazil. Bothrops atrox is found in tropical lowlands and rainforests in the north of South America and is responsible for the majority of envenomations in this area, causing approximately 80% of snake bites [8,9,10]. The snake Bothrops atrox is responsible for the majority of envenomings in the northern region of South America. Severe local effects, including hemorrhage, which are mainly caused by snake venom metalloproteinases (SVMPs), are not fully neutralized by conventional serum therapy.

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Results

Discussion

Conclusion