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

Prado ND, Pereira SS, da Silva MP, Morais MS, Kayano AM, Moreira-Dill LS, Luiz MB, Zanchi FB, Fuly AL, Huacca ME, Fernandes CF, Calderon LA, Zuliani JP, Pereira da Silva LH, Soares AM, Stabeli RG, Fernandes CF - PLoS ONE (2016)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Fundação Oswaldo Cruz, Fiocruz Rondônia, Porto Velho-RO, Brazil.

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.

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Cross-reactivity of anti-BthTX-I and BthTX-II VHHs.In vitro reactivity showing different levels of interaction of selected VHHs (A) KF498607; (B) KF498608; (C) KF329715; (D) KC329718 with a variety of snake venoms (Bothrops alternatus, Bothrops atrox, Bothrops bilineata, Bothrops brazili, Bothrops diporus, Bothrops insularis, Bothrops jararaca, Bothrops leucurus, Bothrops marajoensis, Bothrops matogrossensis, Bothrops moojeni, Bothrops pauloensis, Bothrops pirajai, Bothrops urutu, Calloselasma rhodostoma, Crotalus atrox, Crotalus durissus cascavella, Crotalus durissus collilineatus, Crotalus durissus cumanensis, Crotalus durissus terrificus, Micrurus spixii), and isolated toxins (PLA2 from Bothrops atrox, and PLA2-I, convulxin, crotamin crotapotin, crotoxin, and giroxin from Crotalus durissus terrificus). After being coated on the wells, venoms and toxins were probed with selected VHHs. Samples were incubated with mouse anti-His antibody and the reactive signals were detected after incubation with HRP-conjugated anti-mouse IgG produced in goat and TMB. All clones that showed an absorbance value (OD 450nm) higher than the stipulated cut-off point (2 mean OD from negative samples plus 2 standard deviations) were considered positive. The dashed lines represent the cut off. All measurements were performed in triplicate. For the negative control (NC), wells were not coated with venoms or toxins. Error bars represent standard deviation.
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pone.0151363.g006: Cross-reactivity of anti-BthTX-I and BthTX-II VHHs.In vitro reactivity showing different levels of interaction of selected VHHs (A) KF498607; (B) KF498608; (C) KF329715; (D) KC329718 with a variety of snake venoms (Bothrops alternatus, Bothrops atrox, Bothrops bilineata, Bothrops brazili, Bothrops diporus, Bothrops insularis, Bothrops jararaca, Bothrops leucurus, Bothrops marajoensis, Bothrops matogrossensis, Bothrops moojeni, Bothrops pauloensis, Bothrops pirajai, Bothrops urutu, Calloselasma rhodostoma, Crotalus atrox, Crotalus durissus cascavella, Crotalus durissus collilineatus, Crotalus durissus cumanensis, Crotalus durissus terrificus, Micrurus spixii), and isolated toxins (PLA2 from Bothrops atrox, and PLA2-I, convulxin, crotamin crotapotin, crotoxin, and giroxin from Crotalus durissus terrificus). After being coated on the wells, venoms and toxins were probed with selected VHHs. Samples were incubated with mouse anti-His antibody and the reactive signals were detected after incubation with HRP-conjugated anti-mouse IgG produced in goat and TMB. All clones that showed an absorbance value (OD 450nm) higher than the stipulated cut-off point (2 mean OD from negative samples plus 2 standard deviations) were considered positive. The dashed lines represent the cut off. All measurements were performed in triplicate. For the negative control (NC), wells were not coated with venoms or toxins. Error bars represent standard deviation.

Mentions: KF498607, KF498608, KC329715 and KC329718 selected for cross reacivity showed interactions, at different levels, with the majority of venoms and PLA2s from different species of the Bothrops genus. On the other hand, the clones presented no reaction with venoms or toxins isolated from other members of the Viperidae or Elapidae families, indicating genus specificity (Fig 6). Myotoxicity induced by B. jararacussu venom, BthTX-I and BthTX-II was determined by CK activity. In a proportion of 1:5 (w/w), 10 μg BthTX-I or venom to 50 μg VHH, and 15 μg BthTX-II to 75 μg VHH, KF498607 was capable of inhibiting about 75% of the myotoxicity induced by BthTX-I, BthTX-II and venom. In the same proportion, KC329718 inhibited about 48% and 56% of BthTX-I and BthTX-II-induced myotoxicity, respectively, but showed no significant venom inhibition. However, when the assay was performed increasing the proportion to 1:10 (w/w) of venom:VHH, KC329718 presented about 73% of myotoxicity inhibition. Furthermore, when the capacity of both clones to inhibit MTX-I was analyzed, VHHs were able to neutralize around 50–55% of toxin activity (Fig 7).


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

Prado ND, Pereira SS, da Silva MP, Morais MS, Kayano AM, Moreira-Dill LS, Luiz MB, Zanchi FB, Fuly AL, Huacca ME, Fernandes CF, Calderon LA, Zuliani JP, Pereira da Silva LH, Soares AM, Stabeli RG, Fernandes CF - PLoS ONE (2016)

Cross-reactivity of anti-BthTX-I and BthTX-II VHHs.In vitro reactivity showing different levels of interaction of selected VHHs (A) KF498607; (B) KF498608; (C) KF329715; (D) KC329718 with a variety of snake venoms (Bothrops alternatus, Bothrops atrox, Bothrops bilineata, Bothrops brazili, Bothrops diporus, Bothrops insularis, Bothrops jararaca, Bothrops leucurus, Bothrops marajoensis, Bothrops matogrossensis, Bothrops moojeni, Bothrops pauloensis, Bothrops pirajai, Bothrops urutu, Calloselasma rhodostoma, Crotalus atrox, Crotalus durissus cascavella, Crotalus durissus collilineatus, Crotalus durissus cumanensis, Crotalus durissus terrificus, Micrurus spixii), and isolated toxins (PLA2 from Bothrops atrox, and PLA2-I, convulxin, crotamin crotapotin, crotoxin, and giroxin from Crotalus durissus terrificus). After being coated on the wells, venoms and toxins were probed with selected VHHs. Samples were incubated with mouse anti-His antibody and the reactive signals were detected after incubation with HRP-conjugated anti-mouse IgG produced in goat and TMB. All clones that showed an absorbance value (OD 450nm) higher than the stipulated cut-off point (2 mean OD from negative samples plus 2 standard deviations) were considered positive. The dashed lines represent the cut off. All measurements were performed in triplicate. For the negative control (NC), wells were not coated with venoms or toxins. Error bars represent standard deviation.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4814101&req=5

pone.0151363.g006: Cross-reactivity of anti-BthTX-I and BthTX-II VHHs.In vitro reactivity showing different levels of interaction of selected VHHs (A) KF498607; (B) KF498608; (C) KF329715; (D) KC329718 with a variety of snake venoms (Bothrops alternatus, Bothrops atrox, Bothrops bilineata, Bothrops brazili, Bothrops diporus, Bothrops insularis, Bothrops jararaca, Bothrops leucurus, Bothrops marajoensis, Bothrops matogrossensis, Bothrops moojeni, Bothrops pauloensis, Bothrops pirajai, Bothrops urutu, Calloselasma rhodostoma, Crotalus atrox, Crotalus durissus cascavella, Crotalus durissus collilineatus, Crotalus durissus cumanensis, Crotalus durissus terrificus, Micrurus spixii), and isolated toxins (PLA2 from Bothrops atrox, and PLA2-I, convulxin, crotamin crotapotin, crotoxin, and giroxin from Crotalus durissus terrificus). After being coated on the wells, venoms and toxins were probed with selected VHHs. Samples were incubated with mouse anti-His antibody and the reactive signals were detected after incubation with HRP-conjugated anti-mouse IgG produced in goat and TMB. All clones that showed an absorbance value (OD 450nm) higher than the stipulated cut-off point (2 mean OD from negative samples plus 2 standard deviations) were considered positive. The dashed lines represent the cut off. All measurements were performed in triplicate. For the negative control (NC), wells were not coated with venoms or toxins. Error bars represent standard deviation.
Mentions: KF498607, KF498608, KC329715 and KC329718 selected for cross reacivity showed interactions, at different levels, with the majority of venoms and PLA2s from different species of the Bothrops genus. On the other hand, the clones presented no reaction with venoms or toxins isolated from other members of the Viperidae or Elapidae families, indicating genus specificity (Fig 6). Myotoxicity induced by B. jararacussu venom, BthTX-I and BthTX-II was determined by CK activity. In a proportion of 1:5 (w/w), 10 μg BthTX-I or venom to 50 μg VHH, and 15 μg BthTX-II to 75 μg VHH, KF498607 was capable of inhibiting about 75% of the myotoxicity induced by BthTX-I, BthTX-II and venom. In the same proportion, KC329718 inhibited about 48% and 56% of BthTX-I and BthTX-II-induced myotoxicity, respectively, but showed no significant venom inhibition. However, when the assay was performed increasing the proportion to 1:10 (w/w) of venom:VHH, KC329718 presented about 73% of myotoxicity inhibition. Furthermore, when the capacity of both clones to inhibit MTX-I was analyzed, VHHs were able to neutralize around 50–55% of toxin activity (Fig 7).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Fundação Oswaldo Cruz, Fiocruz Rondônia, Porto Velho-RO, Brazil.

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.

Show MeSH
Related in: MedlinePlus