ABSTRACT
Bushmasters (Lachesis spp) and lancehead vipers (Bothrops spp) are two of the most dangerous snakes found in Latin America. Victims of envenoming by these snakes require urgent administration of antivenom. Here, we report the identification of a small set of broadly neutralizing human monoclonal single-chain variable fragment (scFv) antibodies targeting key phospholipases A2 from Lachesis and Bothrops spp using phage display technology and demonstrate their in vitro efficacy using a hemolysis assay.
Subject(s)
Crotalid Venoms , Single-Chain Antibodies/immunology , Viperidae , Animals , Antivenins , Bothrops/immunology , Humans , Snake BitesABSTRACT
Accidents involving venomous snakes are a public health problem worldwide, causing a large number of deaths per year. In Brazil, the majority of accidents are caused by the Bothrops and Crotalus genera, which are responsible for approximately 80% of severe envenoming cases. The cross-neutralization of snake venoms by antibodies is an important issue for development of more effective treatments. Our group has previously reported the construction of human monoclonal antibody fragments towards Bothrops jararacussu and Crotalus durissus terrificus' venoms. This study aimed to select human single-chain variable fragments (scFvs) that recognize both bothropic and crotalic crude venoms following venoms neutralizing capacity in vitro and in vivo. The cross-reactivity of Cro-Bothrumabs were demonstrated by ELISA and in vitro and in vivo experiments showed that a combination of scFvs neutralizes in vitro toxic activities (e.g. indirect hemolysis and plasma-clotting) of crotalic and bothropic venoms as well as prolonged survival time of envenomed animals. Our results may contribute to the development of the first human polyvalent antivenom against Bothrops jararacussu and Crotalus durissus terrificus venoms, overcoming some undesirable effects caused by conventional serotherapy.
Subject(s)
Antivenins/pharmacology , Bothrops , Crotalid Venoms/immunology , Crotalus , Single-Chain Antibodies/pharmacology , Animals , Antibodies, Monoclonal , Antivenins/immunology , Brazil , Cross Reactions , Enzyme-Linked Immunosorbent Assay , Humans , Single-Chain Antibodies/immunologyABSTRACT
Africanized Apis mellifera bees, also known as killer bees, have an exceptional defensive instinct, characterized by mass attacks that may cause envenomation or death. From the years 2000-2013, 77,066 bee accidents occurred in Brazil. Bee venom comprises several substances, including melittin and phospholipase A2 (PLA2). Due to the lack of antivenom for bee envenomation, this study aimed to produce human monoclonal antibody fragments (single chain fragment variable; scFv), by using phage display technology. These fragments targeted melittin and PLA2, the two major components of bee venom, to minimize their toxic effects in cases of mass envenomation. Two phage antibody selections were performed using purified melittin. As the commercial melittin is contaminated with PLA2, phages specific to PLA2 were also obtained during one of the selections. Specific clones for melittin and PLA2 were selected for the production of soluble scFvs, named here Afribumabs: prefix: afrib- (from Africanized bee); stem/suffix: -umab (fully human antibody). Afribumabs 1 and 2 were tested in in vitro and in vivo assays to assess their ability to inhibit the toxic actions of purified melittin, PLA2, and crude bee venom. Afribumabs reduced hemolysis caused by purified melittin and PLA2 and by crude venom in vitro and reduced edema formation in the paws of mice and prolonged the survival of venom-injected animals in vivo. These results demonstrate that Afribumabs may contribute to the production of the first non-heterologous antivenom treatment against bee envenomation. Such a treatment may overcome some of the difficulties associated with conventional immunotherapy techniques.
Subject(s)
Antivenins/therapeutic use , Bee Venoms/antagonists & inhibitors , Drug Design , Insect Bites and Stings/drug therapy , Insect Proteins/antagonists & inhibitors , Melitten/antagonists & inhibitors , Single-Chain Antibodies/therapeutic use , Animals , Antivenins/genetics , Antivenins/metabolism , Antivenins/pharmacology , Bee Venoms/chemistry , Bee Venoms/enzymology , Bee Venoms/toxicity , Cell Surface Display Techniques , Clone Cells , Drug Therapy, Combination , Edema/etiology , Edema/prevention & control , Hemolysis/drug effects , Humans , Insect Bites and Stings/physiopathology , Insect Proteins/analysis , Insect Proteins/toxicity , Male , Melitten/analysis , Melitten/toxicity , Mice , Phospholipase A2 Inhibitors/pharmacology , Phospholipase A2 Inhibitors/therapeutic use , Phospholipases A2, Secretory/antagonists & inhibitors , Phospholipases A2, Secretory/toxicity , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Recombinant Proteins/pharmacology , Recombinant Proteins/therapeutic use , Single-Chain Antibodies/genetics , Single-Chain Antibodies/metabolism , Single-Chain Antibodies/pharmacology , Subcutaneous Tissue/drug effects , Survival AnalysisABSTRACT
In various types of snake venom, the major toxic components are proteinases and members of the phospholipase A2 family, although other enzymes also contribute to the toxicity. In this study, we evaluated the proteolytic, phospholipase, and L-Amino acid oxidase activities in the venom of five Bothrops species-Bothrops jararaca, Bothrops jararacussu, Bothrops moojeni, Bothrops neuwiedi, and Bothrops alternatus-all of which are used in the production of commercial antivenom, prepared in horses. The enzymatic activities of each species' venom were classified as high, moderate, or low. B. moojeni venom demonstrated the highest enzymatic activity profile, followed by the venom of B. neuwiedi, B. jararacussu, B. jararaca, and B. alternatus. To our knowledge, this is the first study to compare all of these enzymes from multiple species, which is significant in view of the activity of L-amino acid oxidase across Bothrops species.
Subject(s)
Bothrops , Crotalid Venoms/enzymology , Animals , Brazil , Cattle , Crotalid Venoms/chemistry , L-Amino Acid Oxidase/chemistry , Peptide Hydrolases/chemistry , Phospholipases/chemistry , Proteolysis , Sheep , Species SpecificityABSTRACT
The hybrid created from the crossbreeding of European and African bees, known as the Africanised bee, has provided numerous advantages for current beekeeping. However, this new species exhibits undesirable behaviours, such as colony defence instinct and a propensity to attack en masse, which can result in serious accidents. To date, there is no effective treatment for cases of Africanised bee envenomation. One promising technique for developing an efficient antivenom is the use of phage display technology, which enables the production of human antibodies, thus avoiding the complications of serum therapy, such as anaphylaxis and serum sickness. The aim of this study was to produce human monoclonal single-chain Fv (scFv) antibody fragments capable of inhibiting the toxic effects of Africanised bee venom. We conducted four rounds of selection of antibodies against the venom and three rounds of selection of antibodies against purified melittin. Three clones were selected and tested by enzyme-linked immunosorbent assay to verify their specificity for melittin and phospholipase A2. Two clones (C5 and C12) were specific for melittin, and one (A7) was specific for phospholipase A2. In a kinetic haemolytic assay, these clones were evaluated individually and in pairs. The A7-C12 combination had the best synergistic effect and was chosen to be used in the assays of myotoxicity inhibition and lethality. The A7-C12 combination inhibited the in vivo myotoxic effect of the venom and increased the survival of treated animals.
