Snake envenoming is a globally neglected public health problem. Antivenoms produced using animal hyperimmune plasma remain the standard therapy for snakebites. Although effective against systemic effects, conventional antivenoms have limited efficacy against local tissue damage. In addition, potential hypersensitivity reactions, high costs for animal maintenance, and difficulties in obtaining batch-to-batch homogeneity are some of the factors that have motivated the search for innovative and improved therapeutic products against such envenoming. In this study, we have developed a set of nanobodies (recombinant single-domain antigen-binding fragments from camelid heavy chain-only antibodies) against Bothrops atrox snake venom hemorrhagic and myotoxic components. An immune library was constructed after immunizing a Lama glama with whole venom of B. atrox, from which nanobodies were selected by phage display using partially purified hemorrhagic and myotoxic proteins. Biopanning selections retrieved 18 and eight different nanobodies against the hemorrhagic and the myotoxic proteins, respectively. In vivo assays in mice showed that five nanobodies inhibited the hemorrhagic activity of the proteins; three neutralized the hemorrhagic activity of whole B. atrox venom, while four nanobodies inhibited the myotoxic protein. A mixture of the anti-hemorrhagic and anti-myotoxic nanobodies neutralized the local tissue hemorrhage and myonecrosis induced by the whole venom, although the nanobody mixture failed to prevent the venom lethality. Nevertheless, our results demonstrate the efficacy and usefulness of these nanobodies to neutralize important pathologies of the venom, highlighting their potential as innovative therapeutic agents against envenoming by B. atrox, a viperid species causing many casualties in South America.
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We acknowledge Centro Nacional de Productos Biol gicos (CNPB-INS) for providing the experimental animals, as well as facilities to support this research. We thank Carlos Chavez (Universidade Federal de Minas Gerais), Armando Yarlequ (UNMSM), Elizabeth Sanchez, Silvia Seraylan, Rosio Inga, Juana Choque, Karin Flores (INS) for helping in the snake venom analysis. We also thank Siever Morales (Universidad Cient fica del Sur), Alejandro Arenas, Yesler Pariona, and Elvis Arana (INS) for supporting the llama management. Funding. This work was financially supported by the Peruvian National Council of Science and Technology (CONCYTEC-FONDECYT), grant 188-2015-FONDECYT. This research was partly supported by the master thesis in Molecular Biology of VY at the UNMSM.
This work was financially supported by the Peruvian National Council of Science and Technology (CONCYTEC-FONDECYT), grant 188-2015-FONDECYT. This research was partly supported by the master thesis in Molecular Biology of VY at the UNMSM.
© Copyright © 2020 Bailon Calderon, Yaniro Coronel, Cáceres Rey, Colque Alave, Leiva Duran, Padilla Rojas, Montejo Arevalo, García Neyra, Galarza Pérez, Bonilla, Tintaya, Ricciardi, Smiejkowska, Romão, Vincke, Lévano, Celys, Lomonte and Muyldermans.
- Bothrops atrox