Generation of antibodies against disintegrin and cysteine-rich domains by DNA immunization: An approach to neutralize snake venom-induced haemorrhage

Objective To explore whether a DNA immunization approach targeting the major haemorrhage molecule of a prothrombin activator-like metalloproteinase from Echis ocellatus (E. ocellatus) venom could be conceived to inspire antibodies with more prominent specificity and equal adequacy to current conventional antivenoms systems. Methods The isolated DNA EoMP-6 was used as the template for PCR amplification using the EoDC-2-specific forward and reverse primers. A PCR product of approximately 700 bp was obtained and cloned into pSecTag-B expression vector where anti-EoDC-2 antibodies were generated and analysed for their efficacy to neutralise local haemorrhage in vitro and in vivo. Results Our results suggest that the generated anti-EoDC-2 showed a remarkable efficacy by (a) interfering with the interaction of the recombinant disintegrin “EoDC-2” isolated from the E. ocellatus as well as other viper species to the α₂β₁-integrins on platelets; (b) complete inhibition of the catalytic site of the metalloproteinase molecules in vitro using an adaptation antibody zymography assay. Furthermore, it has a polyspecific potential and constitutively expressed significant inhibition by cross-reaction and neutralised venom-induced local haemorrhage exerted by different viper species in vivo. The potential characteristic of EoDC-2 against one part (the non-catalytic domain) as opposed to the whole molecule to neutralise its haemorrhagic activity is of crucial importance as it represents a novel approach with greater immunological specificity and fewer hazards, if any, than conventional systems of antivenom production, by exposure large animals that usually being used for the current antivenom production to a less injurious than expression of the whole molecule containing the catalytic metalloprotease domain. Hence, we report for the first time that our preliminary results hold a promising future for antivenom development. Conclusions Antibodies generated against the E. ocellatus venom prothrombin activator-like metalloprotease and disintegrin-cysteine-rich domains modulated and inhibited the catalytic activity both in vitro and in vivo of venom metalloproteinase disintegrin cysteine rich molecules. Thus, generating of venom specific-toxin antibodies by DNA immunization offer a more rational treatment of snake envenoming than conventional antivenom.