Improving the bothropic antivenom through affinity chromatography and proteomics

Every year, more than 100,000 deaths occur worldwide as a consequence of snakebite envenoming, and around 400,000 people suffer lasting injuries or disfigurements. The World Health Organization added snakebite envenoming to the list of Neglected Tropical Diseases in 2017 due to the severity of the disease and a lack of antivenoms in certain regions of the world, such as Sub-Saharan Africa and Asian populations. Antivenoms have been the only effective therapy for snakebite envenoming since the end of the XIX century. Despite their relevance, antivenoms have limitations, such as a high rate of adverse responses when injected into the human body. Although there has been significant progress in the study of snake venom composition and antivenom effectiveness, little is known about antivenoms at the molecular level. By affinity chromatography purification of specific antibodies against the venom toxins of Bothrops jararaca, we started with the bothropic antivenom (BAv) and produced an improved bothropic antivenom (iBAv). Then, BAv and iBAv were characterized by quantitative proteomic analysis, and the affinities of both antivenoms were evaluated by surface plasmon resonance (SPR) on chips covered with venom toxins. In addition, in vivo neutralization assays were performed in mice model. A total of 105 proteins were identified by proteomics and 55 were quantified. The results showed that serum proteins were significantly depleted in the iBAv, resulting in an antivenom enriched in heavy and light chains of immunoglobulins. iBAV was 2.8 times more effective to neutralize B. jararaca venom lethal activity. In vitro SPR assays revealed that iBAv generated 2.9 higher binding responses to B. jararaca venom toxins in comparison to BAv. The results of this study may aid in the characterization of existing antivenoms and contribute to the development of a new generation of enhanced antivenoms.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)88887.336072/2019-002017/20106-988887.336072/2019­002017/20106­