Diabody Mixture Providing Full Protection against Experimental Scorpion Envenoming with Crude Androctonus australis Venom

Marie-France Martin-Eauclaire,Isabelle Dimier-Poisson,Matthieu O. Juste,Philippe Billiald,Anne di Tommaso,Nicolas Aubrey

doi:10.1074/jbc.m112.348912

Abstract

Androctonus australis is primarily involved in envenomations in North Africa, notably in Tunisia and Algeria, and constitutes a significant public health problem in this region. The toxicity of the venom is mainly due to various neurotoxins that belong to two distinct structural and immunological groups, group I (the AahI and AahIII toxins) and group II (AahII). Here, we report the use of a diabody mixture in which the molar ratio matches the characteristics of toxins and polymorphism of the venom. The mixture consists of the Db9C2 diabody (anti-group I) and the Db4C1op diabody (anti-AahII), the latter being modified to facilitate in vitro production and purification. The effectiveness of the antivenom was tested in vivo under conditions simulating scorpion envenomation. The intraperitoneal injection of 30 μg of the diabody mixture protected almost all the mice exposed to 3 LD(50) s.c. of venom. We also show that the presence of both diabodies is necessary for the animals to survive. Our results are the first demonstration of the strong protective power of small quantities of antivenom used in the context of severe envenomation with crude venom.

Highlights

The only known treatment for scorpion envenomation is serotherapy with a heterologous polyclonal antibody displaying low specificity
The recombinant clones carrying the plasmid containing the insert fused in-frame with the PelB sequence were selected by PCR screening, and DNA sequencing was performed to verify that no mutation appeared (Fig. 1A). pSW1Db4C1op encodes diabody 4C1op, free of tag, in which the VH C-terminal Ser-128 is joined to the VL N-terminal D1 according to IMGT௡ by the short rigid (Gly4-Ser) intramolecular linker
Many antibody fragments have been described and have been shown to be of real interest as a potential antivenom therapy instead of the conventional, albeit controversial, immunotherapy based on the use of equine F(abЈ)2

Summary

Background

The only known treatment for scorpion envenomation is serotherapy with a heterologous polyclonal antibody displaying low specificity. Results: The injection of a diabody mixture allowed mice to survive in a test that mimics severe envenomation with the crude venom. Conclusion: The diabody mixture displayed better protective power than any other known remedy. Significance: This could herald the generation of antivenoms. We report the use of a diabody mixture in which the molar ratio matches the characteristics of toxins and polymorphism of the venom. The intraperitoneal injection of 30 ␮g of the diabody mixture protected almost all the mice exposed to 3 LD50 s.c. of venom. Our results are the first demonstration of the strong protective power of small quantities of antivenom used in the context of severe envenomation with crude venom. Scorpion envenomation is widespread in several countries, where it still constitutes a significant public health problem. The incidence varies from five scorpion stings per

To whom correspondence should be addressed

The abbreviations used are

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION