title: Inhibition of the Myotoxicity Induced by Bothrops jararacussu Venom and Isolated Phospholipases A2 by Specific Camelid Single-Domain Antibody Fragments.
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.
authors.affiliation: Fundação Oswaldo Cruz, Fiocruz Rondônia, Porto Velho-RO, Brazil.
authors.forename: Nidiane D R
authors.initials: ND
authors.lastname: Prado
authors.affiliation: Fundação Oswaldo Cruz, Fiocruz Rondônia, Porto Velho-RO, Brazil.
authors.forename: Soraya S
authors.initials: SS
authors.lastname: Pereira
authors.affiliation: Fundação Oswaldo Cruz, Fiocruz Rondônia, Porto Velho-RO, Brazil.
authors.forename: Michele P
authors.initials: MP
authors.lastname: da Silva
authors.affiliation: Fundação Oswaldo Cruz, Fiocruz Rondônia, Porto Velho-RO, Brazil.
authors.forename: Michelle S S
authors.initials: MS
authors.lastname: Morais
authors.affiliation: Fundação Oswaldo Cruz, Fiocruz Rondônia, Porto Velho-RO, Brazil.
authors.forename: Anderson M
authors.initials: AM
authors.lastname: Kayano
authors.affiliation: Fundação Oswaldo Cruz, Fiocruz Rondônia, Porto Velho-RO, Brazil.
authors.forename: Leandro S
authors.initials: LS
authors.lastname: Moreira-Dill
authors.affiliation: Fundação Oswaldo Cruz, Fiocruz Rondônia, Porto Velho-RO, Brazil.
authors.forename: Marcos B
authors.initials: MB
authors.lastname: Luiz
authors.affiliation: Fundação Oswaldo Cruz, Fiocruz Rondônia, Porto Velho-RO, Brazil.
authors.forename: Fernando B
authors.initials: FB
authors.lastname: Zanchi
authors.affiliation: Universidade Federal Fluminense, UFF, Rio de Janeiro-RJ, Brazil.
authors.forename: André L
authors.initials: AL
authors.lastname: Fuly
authors.affiliation: Universidade Federal de Rondônia, UNIR, Porto Velho-RO, Brazil.
authors.forename: Maribel E F
authors.initials: ME
authors.lastname: Huacca
authors.affiliation: Empresa Brasileira de Pesquisa Agropecuária, Embrapa, Porto Velho-RO, Brazil.
authors.forename: Cleberson F
authors.initials: CF
authors.lastname: Fernandes
authors.affiliation: Fundação Oswaldo Cruz, Fiocruz Rondônia, Porto Velho-RO, Brazil.|Universidade Federal de Rondônia, UNIR, Porto Velho-RO, Brazil.
authors.forename: Leonardo A
authors.initials: LA
authors.lastname: Calderon
authors.affiliation: Fundação Oswaldo Cruz, Fiocruz Rondônia, Porto Velho-RO, Brazil.|Universidade Federal de Rondônia, UNIR, Porto Velho-RO, Brazil.
authors.forename: Juliana P
authors.initials: JP
authors.lastname: Zuliani
authors.affiliation: Fundação Oswaldo Cruz, Fiocruz Rondônia, Porto Velho-RO, Brazil.
authors.forename: Luiz H
authors.initials: LH
authors.lastname: Pereira da Silva
authors.affiliation: Fundação Oswaldo Cruz, Fiocruz Rondônia, Porto Velho-RO, Brazil.
authors.forename: Andreimar M
authors.initials: AM
authors.lastname: Soares
authors.affiliation: Fundação Oswaldo Cruz, Fiocruz Rondônia, Porto Velho-RO, Brazil.|Universidade Federal de Rondônia, UNIR, Porto Velho-RO, Brazil.
authors.forename: Rodrigo G
authors.initials: RG
authors.lastname: Stabeli
authors.affiliation: Fundação Oswaldo Cruz, Fiocruz Rondônia, Porto Velho-RO, Brazil.|Centro de Pesquisa em Medicina Tropical, CEPEM, Porto Velho-RO, Brazil.
authors.forename: Carla F C
authors.initials: CF
authors.lastname: Fernandes
mesh_terms: D000818:Animals; D000997:Antivenins / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D017837:Bothrops; D002161:Camelids, New World / Q000235:genetics / Q000276:immunology; D003435:Crotalid Venoms / Q000737:chemistry / Q000276:immunology / Q000633:toxicity; D054501:Group II Phospholipases A2 / Q000737:chemistry / Q000276:immunology / Q000633:toxicity; D008297:Male; D051379:Mice; D062105:Molecular Docking Simulation; D057127:Single-Chain Antibodies / Q000737:chemistry / Q000235:genetics / Q000276:immunology
chemical_list: D000997:Antivenins; D003435:Crotalid Venoms; D057127:Single-Chain Antibodies; C078097:bothropstoxin; D054501:Group II Phospholipases A2; C072031:bothropstoxin II