title: Human scFv antibodies (Afribumabs) against Africanized bee venom: Advances in melittin recognition.
abstract: 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.
authors.affiliation: Department of Biochemistry and Immunology, School of Medicine of Ribeirão Preto, University of São Paulo, Ribeirão Preto, Av. Bandeirantes, 3900, 14049-900, Ribeirão Preto, SP, Brazil. Electronic address: gapessenda@usp.br.
authors.forename: Gabriela
authors.initials: G
authors.lastname: Pessenda
authors.affiliation: Department of Biochemistry and Immunology, School of Medicine of Ribeirão Preto, University of São Paulo, Ribeirão Preto, Av. Bandeirantes, 3900, 14049-900, Ribeirão Preto, SP, Brazil. Electronic address: luciano.kosta@usp.br.
authors.forename: Luciano C
authors.initials: LC
authors.lastname: Silva
authors.affiliation: Department of Biochemistry and Immunology, School of Medicine of Ribeirão Preto, University of São Paulo, Ribeirão Preto, Av. Bandeirantes, 3900, 14049-900, Ribeirão Preto, SP, Brazil. Electronic address: luc_bqi@yahoo.com.br.
authors.forename: Lucas B
authors.initials: LB
authors.lastname: Campos
authors.affiliation: Department of Biochemistry and Immunology, School of Medicine of Ribeirão Preto, University of São Paulo, Ribeirão Preto, Av. Bandeirantes, 3900, 14049-900, Ribeirão Preto, SP, Brazil. Electronic address: pacelloele@yahoo.com.br.
authors.forename: Elenice M
authors.initials: EM
authors.lastname: Pacello
authors.affiliation: Department of Physics and Chemistry, School of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Av. do Café, s/n, 14040-903, Ribeirão Preto, SP, Brazil. Electronic address: manupucca@usp.br.
authors.forename: Manuela B
authors.initials: MB
authors.lastname: Pucca
authors.affiliation: Department of Social Medicine, School of Medicine of Ribeirão Preto, University of São Paulo, Ribeirão Preto, Av. Bandeirantes, 3900, 14049-900, Ribeirão Preto, SP, Brazil. Electronic address: edson@fmrp.usp.br.
authors.forename: Edson Z
authors.initials: EZ
authors.lastname: Martinez
authors.affiliation: Department of Biochemistry and Immunology, School of Medicine of Ribeirão Preto, University of São Paulo, Ribeirão Preto, Av. Bandeirantes, 3900, 14049-900, Ribeirão Preto, SP, Brazil. Electronic address: jebarbos@fmrp.usp.br.
authors.forename: José E
authors.initials: JE
authors.lastname: Barbosa
mesh_terms: D000818:Animals; D000997:Antivenins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology / Q000627:therapeutic use*; D001514:Bee Venoms / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000201:enzymology / Q000633:toxicity; D061505:Cell Surface Display Techniques; D002999:Clone Cells; D015195:Drug Design; D004359:Drug Therapy, Combination; D004487:Edema / Q000209:etiology / Q000517:prevention & control; D006461:Hemolysis / Q000187:drug effects; D006801:Humans; D007299:Insect Bites and Stings / Q000188:drug therapy* / Q000503:physiopathology; D019476:Insect Proteins / Q000032:analysis / Q000037:antagonists & inhibitors* / Q000633:toxicity; D008297:Male; D008555:Melitten / Q000032:analysis / Q000037:antagonists & inhibitors* / Q000633:toxicity; D051379:Mice; D064801:Phospholipase A2 Inhibitors / Q000494:pharmacology / Q000627:therapeutic use; D054497:Phospholipases A2, Secretory / Q000037:antagonists & inhibitors / Q000633:toxicity; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology / Q000627:therapeutic use; D057127:Single-Chain Antibodies / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology / Q000627:therapeutic use*; D040521:Subcutaneous Tissue / Q000187:drug effects; D016019:Survival Analysis
chemical_list: C000612201:Afribumab 1; C000612202:Afribumab 2; D000997:Antivenins; D001514:Bee Venoms; D019476:Insect Proteins; D064801:Phospholipase A2 Inhibitors; D011994:Recombinant Proteins; D057127:Single-Chain Antibodies; D008555:Melitten; D054497:Phospholipases A2, Secretory
keywords: Africanized bees; Antivenom; Apis mellifera; Melittin; PLA(2); ScFv