In Vivo Neutralization of α-Cobratoxin with High-Affinity Llama Single-Domain Antibodies (VHHs) and a VHH-Fc Antibody

Gabrielle Richard,Ashley J Meyers,Roger Mackenzie,Michael D Mclean,Mehdi Arbabi-Ghahroudi,J Christopher Hall,Paulo Lee Ho

doi:10.1371/journal.pone.0069495

Gabrielle Richard, Ashley J Meyers + Show 5 more

Open Access

https://doi.org/10.1371/journal.pone.0069495

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Journal: PloS one	Publication Date: Jul 22, 2013
Citations: 132	License type: CC BY 4.0

Affiliation: National Research Council Canada, University of Guelph

Abstract

Small recombinant antibody fragments (e.g. scFvs and VHHs), which are highly tissue permeable, are being investigated for antivenom production as conventional antivenoms consisting of IgG or F(ab’)2 antibody fragments do not effectively neutralize venom toxins located in deep tissues. However, antivenoms composed entirely of small antibody fragments may have poor therapeutic efficacy due to their short serum half-lives. To increase serum persistence and maintain tissue penetration, we prepared low and high molecular mass antivenom antibodies. Four llama VHHs were isolated from an immune VHH-displayed phage library and were shown to have high affinity, in the low nM range, for α-cobratoxin (α–Cbtx), the most lethal component of Naja kaouthia venom. Subsequently, our highest affinity VHH (C2) was fused to a human Fc fragment to create a VHH2-Fc antibody that would offer prolonged serum persistence. After in planta (Nicotiana benthamiana) expression and purification, we show that our VHH2-Fc antibody retained high affinity binding to α–Cbtx. Mouse α–Cbtx challenge studies showed that our highest affinity VHHs (C2 and C20) and the VHH2-Fc antibody effectively neutralized lethality induced by α–Cbtx at an antibody:toxin molar ratio as low as ca. 0.75×:1. Further research towards the development of an antivenom therapeutic involving these anti-α-Cbtx VHHs and VHH2-Fc antibody molecules should involve testing them as a combination, to determine whether they maintain tissue penetration capability and low immunogenicity, and whether they exhibit improved serum persistence and therapeutic efficacy.

Highlights

Snake bite envenomation is a serious global public health problem especially in tropical and subtropical countries where poisonous snakes are abundant and agriculture activities are high [1]
We report the isolation of high affinity VHHs that offer full protection (100% mice survival) against a–Cbtx
After in planta expression and purification, we show that our VHH2-Fc antibody retained high affinity binding to a–Cbtx and has potent in vivo neutralizing capacity against a–Cbtx

Summary

Introduction

Snake bite envenomation is a serious global public health problem especially in tropical and subtropical countries where poisonous snakes are abundant and agriculture activities are high [1]. Since most snakebite victims are young male agricultural workers with families, their disability has serious social and economical impacts, especially in developing countries [1,3]. Since their first development and use in the late 1800s by Calmette, conventional antivenoms remain the only specific treatments for envenomation. Intravenous administration of antivenom is generally efficacious in treating systemic envenomation [3]; because of the rapid development of localized pathologies and the inability of antivenom antibodies to penetrate affected tissues, conventional antivenoms are generally ineffective in treating local effects on tissues near the snake bite, often resulting in permanent physical disability [3,4,5]. Conventional antivenoms often elicit life-threatening adverse reactions such as anaphylaxis or serum sickness in patients [4]

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