Abstract
The bacterium Clostridium botulinum is the causative agent of botulism—a severe intoxication caused by botulinum neurotoxin (BoNT) and characterized by damage to the nervous system. In an effort to develop novel C. botulinum immunotherapeutics, camelid single-domain antibodies (sdAbs, VHHs, or nanobodies) could be used due to their unique structure and characteristics. In this study, VHHs were produced using phage display technology. A total of 15 different monoclonal VHHs were selected based on their comlementarity-determining region 3 (CDR3) sequences. Different toxin lethal dose (LD50) challenges with each selected phage clone were conducted in vivo to check their neutralizing potency. We demonstrated that modification of neutralizing VHHs with a human immunoglobulin G (IgG)1 Fc (fragment crystallizable) fragment (fusionbody, VHH-Fc) significantly increased the circulation time in the blood (up to 14 days). At the same time, VHH-Fc showed the protective activity 1000 times higher than monomeric form when challenged with 5 LD50. Moreover, VHH-Fcs remained protective even 14 days after antibody administration. These results indicate that this VHH-Fc could be used as an effective long term antitoxin protection against botulinum type A.
Highlights
IntroductionBotulinum neurotoxin (BoNT) is the strongest organic poison for humans and animals
Botulinum neurotoxin (BoNT) is the strongest organic poison for humans and animals.It is produced by the anaerobic, Gram-positive, spore-forming, rod-shaped bacterium Clostridium botulinum [1,2]
After 24 and 49 days post-immunization, blood was collected, and the botulinum neurotoxin (BoNT)/A specific antibodies titer was measured by enzyme-linked immunosorbent assay (ELISA)
Summary
Botulinum neurotoxin (BoNT) is the strongest organic poison for humans and animals. It is produced by the anaerobic, Gram-positive, spore-forming, rod-shaped bacterium Clostridium botulinum [1,2]. The estimated human lethal dose is about 10 nanograms per kilogram of bodyweight if the toxin is inhaled and one microgram if it is taken orally [3,4]. The most common forms of natural botulism are food-borne, wound, and infant [2]. Food-borne botulism occurs through contaminated food ingestion, and the case fatality rate is about 5–10% in developed countries (as opposed to 60–70% before 1950). Wound botulism occurs when an open wound is exposed to C. botulinum spores, and the case fatality is approximately 10–15% of patients even with aggressive treatment
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