Camelid Single-Domain Antibodies (VHHs) against Crotoxin: A Basis for Developing Modular Building Blocks for the Enhancement of Treatment or Diagnosis of Crotalic Envenoming
Marcos B. Luiz,
Soraya S. Pereira,
Nidiane D. R. Prado,
Naan R. Gonçalves,
Anderson M. Kayano,
Leandro S. Moreira-Dill,
Juliana C. Sobrinho,
Fernando B. Zanchi,
André L. Fuly,
Cleberson F. Fernandes,
Juliana P. Zuliani,
Andreimar M. Soares,
Rodrigo G. Stabeli,
Carla F. C. Fernandes
Affiliations
Marcos B. Luiz
Fundação Oswaldo Cruz, Fiocruz Rondônia, Porto Velho, 76812-245 Rondônia, Brazil
Soraya S. Pereira
Fundação Oswaldo Cruz, Fiocruz Rondônia, Porto Velho, 76812-245 Rondônia, Brazil
Nidiane D. R. Prado
Fundação Oswaldo Cruz, Fiocruz Rondônia, Porto Velho, 76812-245 Rondônia, Brazil
Naan R. Gonçalves
Fundação Oswaldo Cruz, Fiocruz Rondônia, Porto Velho, 76812-245 Rondônia, Brazil
Anderson M. Kayano
Fundação Oswaldo Cruz, Fiocruz Rondônia, Porto Velho, 76812-245 Rondônia, Brazil
Leandro S. Moreira-Dill
Fundação Oswaldo Cruz, Fiocruz Rondônia, Porto Velho, 76812-245 Rondônia, Brazil
Juliana C. Sobrinho
Fundação Oswaldo Cruz, Fiocruz Rondônia, Porto Velho, 76812-245 Rondônia, Brazil
Fernando B. Zanchi
Fundação Oswaldo Cruz, Fiocruz Rondônia, Porto Velho, 76812-245 Rondônia, Brazil
André L. Fuly
Universidade Federal Fluminense, UFF, Rio de Janeiro, 24220-900 Rio de Janeiro, Brazil
Cleberson F. Fernandes
Embrapa Agroindústria Tropical, Fortaleza, 60020-181 Ceará, Brazil
Juliana P. Zuliani
Fundação Oswaldo Cruz, Fiocruz Rondônia, Porto Velho, 76812-245 Rondônia, Brazil
Andreimar M. Soares
Fundação Oswaldo Cruz, Fiocruz Rondônia, Porto Velho, 76812-245 Rondônia, Brazil
Rodrigo G. Stabeli
Plataforma Bi-institucional de Pesquisa em Medicina Translacional, Fiocruz-USP, Ribeirão Preto, 14049-900 São Paulo, Brazil
Carla F. C. Fernandes
Fundação Oswaldo Cruz, Fiocruz Rondônia, Porto Velho, 76812-245 Rondônia, Brazil
Toxic effects triggered by crotalic envenoming are mainly related to crotoxin (CTX), composed of a phospholipase A2 (CB) and a subunit with no toxic activity (CA). Camelids produce immunoglobulins G devoid of light chains, in which the antigen recognition domain is called VHH. Given their unique characteristics, VHHs were selected using Phage Display against CTX from Crotalus durissus terrificus. After three rounds of biopanning, four sequence profiles for CB (KF498602, KF498603, KF498604, and KF498605) and one for CA (KF498606) were revealed. All clones presented the VHH hallmark in FR2 and a long CDR3, with the exception of KF498606. After expressing pET22b-VHHs in E. coli, approximately 2 to 6 mg of protein per liter of culture were obtained. When tested for cross-reactivity, VHHs presented specificity for the Crotalus genus and were capable of recognizing CB through Western blot. KF498602 and KF498604 showed thermostability, and displayed affinity constants for CTX in the micro or nanomolar range. They inhibited in vitro CTX PLA2 activity, and CB cytotoxicity. Furthermore, KF498604 inhibited the CTX-induced myotoxicity in mice by 78.8%. Molecular docking revealed that KF498604 interacts with the CA–CB interface of CTX, seeming to block substrate access. Selected VHHs may be alternatives for the crotalic envenoming treatment.
