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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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Western blot of purified VHHs against BthTX-I.15% SDS-PAGE was carried out to resolve BthTX-I and BthTX-II under reducing conditions. After electrophoresis, BthTX-I and BthTX-II were electrotransferred to a nitrocellulose membrane and probed with selected VHHs (KF498607, KF498608, KC329715, and KC329718). Samples were incubated with mouse anti-His antibody followed by HRP-conjugated anti-mouse IgG produced in goat. Reactive signals were detected by DAB staining in the presence of hydrogen peroxide. Lama glama pre-immune serum was used as negative control (-), and Lama glama immune serum, as positive control (+).
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pone.0151363.g003: Western blot of purified VHHs against BthTX-I.15% SDS-PAGE was carried out to resolve BthTX-I and BthTX-II under reducing conditions. After electrophoresis, BthTX-I and BthTX-II were electrotransferred to a nitrocellulose membrane and probed with selected VHHs (KF498607, KF498608, KC329715, and KC329718). Samples were incubated with mouse anti-His antibody followed by HRP-conjugated anti-mouse IgG produced in goat. Reactive signals were detected by DAB staining in the presence of hydrogen peroxide. Lama glama pre-immune serum was used as negative control (-), and Lama glama immune serum, as positive control (+).

Mentions: Clones KF498607, KF498608, and KC329718 were able to bind to BthTX-I and BthTX-II, used for VHH library construction. On the other hand, KC329715 showed no or weak interaction with the toxins. Pre-immune serum was not capable of recognizing the toxins in WB (Fig 3).


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)

Western blot of purified VHHs against BthTX-I.15% SDS-PAGE was carried out to resolve BthTX-I and BthTX-II under reducing conditions. After electrophoresis, BthTX-I and BthTX-II were electrotransferred to a nitrocellulose membrane and probed with selected VHHs (KF498607, KF498608, KC329715, and KC329718). Samples were incubated with mouse anti-His antibody followed by HRP-conjugated anti-mouse IgG produced in goat. Reactive signals were detected by DAB staining in the presence of hydrogen peroxide. Lama glama pre-immune serum was used as negative control (-), and Lama glama immune serum, as positive control (+).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0151363.g003: Western blot of purified VHHs against BthTX-I.15% SDS-PAGE was carried out to resolve BthTX-I and BthTX-II under reducing conditions. After electrophoresis, BthTX-I and BthTX-II were electrotransferred to a nitrocellulose membrane and probed with selected VHHs (KF498607, KF498608, KC329715, and KC329718). Samples were incubated with mouse anti-His antibody followed by HRP-conjugated anti-mouse IgG produced in goat. Reactive signals were detected by DAB staining in the presence of hydrogen peroxide. Lama glama pre-immune serum was used as negative control (-), and Lama glama immune serum, as positive control (+).
Mentions: Clones KF498607, KF498608, and KC329718 were able to bind to BthTX-I and BthTX-II, used for VHH library construction. On the other hand, KC329715 showed no or weak interaction with the toxins. Pre-immune serum was not capable of recognizing the toxins in WB (Fig 3).

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