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

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Abstract

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.

Original languageEnglish
Pages (from-to)198-207
Number of pages10
JournalAsian Pacific Journal of Tropical Biomedicine
Volume7
Issue number3
DOIs
Publication statusPublished - Mar 1 2017

Fingerprint

Immunization
Antivenins
Disintegrins
Snake Venoms
Venoms
Metalloproteases
Cysteine
Hemorrhage
Molecules
Antibodies
DNA
Prothrombin
Catalytic Domain
Polymerase Chain Reaction
Snakes
Cross Reactions
Platelets
Integrins
Amplification
Assays

Keywords

  • Antibody zymography
  • Antivenoms
  • DNA-immunization
  • Echis ocellatus
  • GeneGun
  • Neutralization
  • Snake

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology (miscellaneous)

Cite this

@article{0894e5aca3b142169b20ebac9af5ca21,
title = "Generation of antibodies against disintegrin and cysteine-rich domains by DNA immunization: An approach to neutralize snake venom-induced haemorrhage",
abstract = "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.",
keywords = "Antibody zymography, Antivenoms, DNA-immunization, Echis ocellatus, GeneGun, Neutralization, Snake",
author = "Hasson, {Sidgi Syed Anwer Abdo}",
year = "2017",
month = "3",
day = "1",
doi = "10.1016/j.apjtb.2016.12.015",
language = "English",
volume = "7",
pages = "198--207",
journal = "Asian Pacific Journal of Tropical Biomedicine",
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publisher = "Elsevier BV",
number = "3",

}

TY - JOUR

T1 - Generation of antibodies against disintegrin and cysteine-rich domains by DNA immunization

T2 - An approach to neutralize snake venom-induced haemorrhage

AU - Hasson, Sidgi Syed Anwer Abdo

PY - 2017/3/1

Y1 - 2017/3/1

N2 - 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.

AB - 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.

KW - Antibody zymography

KW - Antivenoms

KW - DNA-immunization

KW - Echis ocellatus

KW - GeneGun

KW - Neutralization

KW - Snake

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