Inhibition of the Myotoxicity Induced by Bothrops jararacussu Venom and Isolated Phospholipases A2 by Specific Camelid Single-Domain Antibody Fragments |
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| Authors: | Nidiane D R Prado Soraya S Pereira Michele P da Silva Michelle S S Morais Anderson M Kayano Leandro S Moreira-Dill Marcos B Luiz Fernando B Zanchi André L Fuly Maribel E F Huacca Cleberson F Fernandes Leonardo A Calderon Juliana P Zuliani Luiz H Pereira da Silva Andreimar M Soares Rodrigo G Stabeli Carla F C Fernandes |
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| Institution: | 1. Fundação Oswaldo Cruz, Fiocruz Rondônia, Porto Velho-RO, Brazil;2. Universidade Federal Fluminense, UFF, Rio de Janeiro-RJ, Brazil;3. Universidade Federal de Rondônia, UNIR, Porto Velho-RO, Brazil;4. Empresa Brasileira de Pesquisa Agropecuária, Embrapa, Porto Velho-RO, Brazil;5. Centro de Pesquisa em Medicina Tropical, CEPEM, Porto Velho-RO, Brazil;Universidad de Costa Rica, COSTA RICA |
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| 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 ({"type":"entrez-nucleotide","attrs":{"text":"KF498607","term_id":"558064687"}}KF498607, {"type":"entrez-nucleotide","attrs":{"text":"KF498608","term_id":"558064693"}}KF498608, and {"type":"entrez-nucleotide","attrs":{"text":"KC329718","term_id":"468362338"}}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 ({"type":"entrez-nucleotide","attrs":{"text":"KC329718","term_id":"468362338"}}KC329718 and {"type":"entrez-nucleotide","attrs":{"text":"KF498607","term_id":"558064687"}}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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