Expression of an scFv antibody fragment in Nicotiana benthamiana and in vitro assessment of its neutralizing potential against the snake venom metalloproteinase BaP1 from Bothrops asper

Abstract

Human accidents with venomous snakes represent an overwhelming public health problem, mainly in ruralpopulations of underdeveloped countries. Their high incidence and the severity of the accidents result in 81,000to 138,000 deaths per year. The treatment is based on the administration of purified antibodies, produced byhyper immunization of animals to generate immunoglobulins (Igs), and then obtained by fractionating hyperimmune plasma. The use of recombinant antibodies is an alternative to conventional treatment of snakebiteenvenoming, particularly the Fv fragment, named the single-chain variable fragment (scFv). We have producedrecombinant single chain variable fragment scFv against the venom of the pit viperBothrops asperat high levelsexpressed transiently and stably in transgenic plants andin vitrocultures that is reactive to BaP1 (a metallo-proteinase fromB. aspervenom). The yield from stably transformed plants was significantly (p > 0.05) higherthan the results in from transient expression. In addition, scFvBaP1 yields from systems derived from stabletransformation were: transgenic callus 62µg/g ( ± 2); biomass from cell suspension cultures 83µg/g ( ± 0.2);culture medium from suspensions 71.75 mg/L ( ± 6.18). The activity of scFvBaP1 was confirmed by binding andneutralization of thefibrin degradation induced by BnP1 toxins fromB. neuwiediand by Atroxlysin Ia fromB.atroxvenoms. In the present work, we demonstrated the potential use of plant cells to produce scFvBaP1 to beused in the future as a biotechnological alternative to horse immunization protocols to produce anti-venoms tobe used in human therapy against snakebites.