Production and characterization of murine and chimeric monoclonal antibodies against Bothrops jararaca venom

Introduction: Snakebite accidents are classified in Category A of Neglected Tropical Diseases by the World Health Organization (WHO) and represent a public health concern, particularly in low and middle-income tropical and subtropical countries.In Brazil, snakes of the genus Bothrops, including B. jararaca, are responsible for most incidents, accounting for 80-90% of the cases.Antivenom therapy is the only specific treatment recommended by the WHO.Although it is a highly effective intervention, it has some limitations, such as the number of animals required for sera production, the low concentration of neutralizing antibodies, the variation in the potency and composition among batches, and the possibility of allergic reactions due to its heterologous nature. Objectives:In this context, our team has directed efforts towards the production of monoclonal antibodies (mAbs) against the main toxins of Bothrops jararaca venom (Bjv).Besides that, this work also sought the production and characterization of a chimeric antibody (AcQ) from the murine mAb produced.This approach aims to increase the specificity and therapeutic efficacy of the treatment, meets the society's demands for more ethical and sustainable practices, and may minimize potential adverse reactions.Methodology: Monoclonal antibodies were produced by the hybridoma technique and the mAb which showed the highest reactivity (mAb4E6B5) against Bjv was further immunocharacterized by Western Blot (WB) and Enzyme-linked immunosorbent assay (ELISA), followed by fibrinogenolytic and coagulation neutralizing assays.In order to reduce the mAb4E6B5 immunogenicity, a chimeric antibody was produced by Gibson Assembly technique, in which the murine Fc region was replaced by a human Fc region.Results: WB results demonstrated that the mAb binds to bands of 50 kDa and 25 kDa of Bjv, suggesting the recognition of a conserved domain across different toxins.The mAb neutralized the degradation of fibrinogen chain and affected the coagulation induced by Bjv.The production of AcQ in mammalian cells was successful, maintaining the binding specificity to Bjv and the ability to neutralize the fibrinogenolytic activity, in a similar way to the pro-coagulant effect observed with the original murine mAb.ELISAs using venom fractions or purified proteins indicate that this antibody likely targets toxins of the metalloprotease family. Conclusion:In conclusion, this study demonstrated the feasibility of producing an effective AcQ from the original murine mAb, while retaining binding, recognition, and neutralization properties.