Inhibition of the Myotoxicity Induced by Bothrops jararacussu Venom and Isolated Phospholipases A2 by Specific Camelid Single-Domain Antibody Fragments.

Nidiane D R Prado,Rodrigo G Stabeli,Leandro S Moreira-Dill,Soraya S Pereira,Andreimar M Soares,Fernando B Zanchi,Maribel E F Huacca,Anderson M Kayano,Cleberson F Fernandes,Carla F C Fernandes,Michelle S S Morais,Michele P Da Silva,Luiz H Pereira Da Silva,André L Fuly,Juliana P Zuliani,Leonardo A Calderon,Marcos B Luiz,Bruno Lomonte

doi:10.1371/journal.pone.0151363

Abstract

Antivenoms, produced using animal hyperimmune plasma, remains the standard therapy for snakebites. Although effective against systemic damages, conventional antivenoms have limited efficacy against local tissue damage. Additionally, the hypersensitivity reactions, often elicited by antivenoms, the high costs for animal maintenance, the difficulty of producing homogeneous lots, and the instability of biological products instigate the search for innovative products for antivenom therapy. In this study, camelid antibody fragments (VHH) with specificity to Bothropstoxin I and II (BthTX-I and BthTX-II), two myotoxic phospholipases from Bothrops jararacussu venom, were selected from an immune VHH phage display library. After biopanning, 28 and 6 clones recognized BthTX-I and BthTX-II by ELISA, respectively. Complementarity determining regions (CDRs) and immunoglobulin frameworks (FRs) of 13 VHH-deduced amino acid sequences were identified, as well as the camelid hallmark amino acid substitutions in FR2. Three VHH clones (KF498607, KF498608, and KC329718) were capable of recognizing BthTX-I by Western blot and showed affinity constants in the nanomolar range against both toxins. VHHs inhibited the BthTX-II phospholipase A2 activity, and when tested for cross-reactivity, presented specificity to the Bothrops genus in ELISA. Furthermore, two clones (KC329718 and KF498607) neutralized the myotoxic effects induced by B. jararacussu venom, BthTX-I, BthTX-II, and by a myotoxin from Bothrops brazili venom (MTX-I) in mice. Molecular docking revealed that VHH CDRs are expected to bind the C-terminal of both toxins, essential for myotoxic activity, and to epitopes in the BthTX-II enzymatic cleft. Identified VHHs could be a biotechnological tool to improve the treatment for snake envenomation, an important and neglected world public health problem.

Highlights

Snakebites represent a relevant public health problem, especially in subtropical and tropical countries
Evaluating the features of VHH, the characteristics of bothropstoxins, and the necessity for developing new approaches to support the treatment or even to diagnose snakebites, this study aimed to select VHHs capable of recognizing and neutralizing myotoxic effects triggered by BthTX-I, BthTX-II, and B. jararacussu venom
The co-infection of VHH-pHEN-1-6xHis transfected TG1 E. coli strain with the M13KO7 helper phage was performed in the phage rescuing strategy, a total of 8 x 106 phage particles were obtained

Summary

Introduction

Snakebites represent a relevant public health problem, especially in subtropical and tropical countries. Affecting mainly the rural population, about 5 million snakebites occur each year worldwide, causing approximately 100,000 deaths [1,2,3]. A large number of the victims experience permanent physical sequelaes due to inflammatory, hemorrhagic, coagulant, neurotoxic and myotoxic effects occasioned by the venom components. These signs are often aggravated by the difficulty of accessing health services in less developed regions [2,4]. It has been described that, worldwide, there are four families of venomous snakes, i.e. Viperidae, Elapidae, Atractaspididae, and Colubridae [5]. Viperidae and Elapidae families cause the most venomous bites. Beloging to the Viperidae family, the Bothrops genus is responsible for the majority of snake envenoming in Central and South America, occasioning both high morbidity and mortality [6]

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