RESUMEN
BaP1 is a P-I class snake venom metalloproteinase (SVMP) relevant in the local tissue damage associated with envenomings by Bothrops asper, a medically important snake species in Central America and parts of South and North America. The main treatment for these accidents is the passive immunotherapy using antibodies raised in horses. In order to obtain more specific and batch-to-batch consistent antivenons, recombinant antibodies are considered a good option compared to animal immunization. We constructed a recombinant single chain variable fragment (scFv) from a monoclonal antibody against BaP1 (MABaP1) formerly secreted by a hybridoma clone. This recombinant antibody was cloned into pMST3 vector in fusion with SUMO protein and contains VH and VL domains linked by a flexible (G4S)3 polypeptide (scFvBaP1). The aim of this work was to produce scFvBaP1 and to evaluate its potential concerning the neutralization of biologically important activities of BaP1. The cytoplasmic expression of this construct was successfully achieved in C43 (DE3) bacteria. Our results showed that scFvBaP1-SUMO fusion protein presented an electrophoretic band of around 43 kDa from which SUMO alone corresponded to 13.6 kDa, and only the scFv was able to recognize BaP1 as well as the whole venom by ELISA. In contrast, neither an irrelevant scFv anti-LDL nor its MoAb partner recognized it. BaP1-induced fibrinolysis was significantly neutralized by scFvBaP1, but not by SUMO, in a concentration-dependent manner. In addition, scFvBaP1, as well as MaBaP1, completely neutralized in vivo hemorrhage, muscle necrosis, and inflammation induced by the toxin. Docking analyses revealed possible modes of interaction of the recombinant antibody with BaP1. Our data showed that scFv recognized BaP1 and whole B. asper venom, and neutralized biological effects of this SVMP. This scFv antibody can be used for understanding the molecular mechanisms of neutralization of SVMPs, and for exploring the potential of recombinant antibody fragments for improving the neutralization of local tissue damage in snakebite envenoming.
Asunto(s)
Antivenenos/farmacología , Bothrops/metabolismo , Hemorragia/inducido químicamente , Metaloproteasas/antagonistas & inhibidores , Metaloproteasas/toxicidad , Venenos de Serpiente/antagonistas & inhibidores , Proteínas Supresoras de Tumor/antagonistas & inhibidores , Ubiquitina Tiolesterasa/antagonistas & inhibidores , Secuencia de Aminoácidos , Animales , Especificidad de Anticuerpos , Antivenenos/química , Escherichia coli/metabolismo , Femenino , Fragmentos de Inmunoglobulinas/farmacología , Inflamación/inducido químicamente , Inflamación/prevención & control , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Pruebas de Neutralización , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacología , Proteínas Supresoras de Tumor/química , Proteínas Supresoras de Tumor/inmunología , Ubiquitina Tiolesterasa/química , Ubiquitina Tiolesterasa/inmunologíaRESUMEN
BaP1 is a P-I class of Snake Venom Metalloproteinase (SVMP) relevant in the local tissue damage associated with envenomations by Bothrops asper, a medically-important species in Central America and parts of South America. Six monoclonal antibodies (MoAb) against BaP1 (MABaP1) were produced and characterized regarding their isotype, dissociation constant (K(d)), specificity and ability to neutralize BaP1-induced hemorrhagic and proteolytic activity. Two MABaP1 are IgM, three are IgG1 and one is IgG2b. The K(d)s of IgG MoAbs were in the nM range. All IgG MoAbs recognized conformational epitopes of BaP1 and B. asper venom components but failed to recognize venoms from 27 species of Viperidae, Colubridae and Elapidae families. Clone 7 cross-reacted with three P-I SVMPs tested (moojeni protease, insularinase and neuwiedase). BaP1-induced hemorrhage was totally neutralized by clones 3, 6 and 8 but not by clone 7. Inhibition of BaP1 enzymatic activity on a synthetic substrate by MABaP1 was totally achieved by clones 3 and 6, and partially by clone 8, but not by clone 7. In conclusion, these neutralizing MoAbs against BaP1 may become important tools to understand structure-function relationships of BaP1 and the role of P-I class SVMP in snakebite envenomation.
Asunto(s)
Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/inmunología , Bothrops/fisiología , Venenos de Crotálidos/enzimología , Metaloendopeptidasas/inmunología , Animales , Anticuerpos Monoclonales/aislamiento & purificación , Reacciones Cruzadas , Venenos de Crotálidos/antagonistas & inhibidores , Venenos de Crotálidos/toxicidad , Edema/inducido químicamente , Edema/prevención & control , Ensayo de Inmunoadsorción Enzimática , Mapeo Epitopo , Hemorragia/inducido químicamente , Hemorragia/prevención & control , Immunoblotting , Inmunoglobulinas , Inyecciones Intraperitoneales , Metaloendopeptidasas/antagonistas & inhibidores , Metaloendopeptidasas/toxicidad , Ratones , Ratones Endogámicos BALB C , Pruebas de NeutralizaciónRESUMEN
BaP1 is a P-I class of Snake Venom Metalloproteinase (SVMP) relevant in the local tissue damage associated with envenomations by Bothrops asper, a medically-important species in Central America and parts of South America. Six monoclonal antibodies (MoAb) against BaP1 (MABaP1) were produced and characterized regarding their isotype, dissociation constant (Kd), specificity and ability to neutralize BaP1-induced hemorrhagic and proteolytic activity. Two MABaP1 are IgM, three are IgG1 and one is IgG2b. The Kds of IgG MoAbs were in the nM range. All IgG MoAbs recognized conformational epitopes of BaP1 and B. asper venom components but failed to recognize venoms from 27 species of Viperidae, Colubridae and Elapidae families. Clone 7 cross-reacted with three P-I SVMPs tested (moojeni protease, insularinase and neuwiedase). BaP1-induced hemorrhage was totally neutralized by clones 3, 6 and 8 but not by clone 7. Inhibition of BaP1 enzymatic activity on a synthetic substrate by MABaP1 was totally achieved by clones 3 and 6, and partially by clone 8, but not by clone 7. In conclusion, these neutralizing MoAbs against BaP1 may become important tools to understand structurefunction relationships of BaP1 and the role of P-I class SVMP in snakebite envenomation.