Development of an Equine Antitoxin by Immunizing the Halla Horse with the Receptor-Binding Domain of Botulinum Neurotoxin Type A1

Botulinum neurotoxins (BoNTs), produced by Clostridium botulinum, are the most toxic substances known. However, the number of currently approved medical countermeasures for these toxins is very limited. Therefore, studies on therapeutic antitoxins are essential to prepare for toxin-related emergencies. Currently, more than 10,000 Halla horses, a crossbreed between the native Jeju and Thoroughbred horses, are being raised in Jeju Island of Korea. They can be used for equine antitoxin experiments and production of hyperimmune serum against BoNT/A1. Instead of the inactivated BoNT/A1 toxoid, Halla horse was immunized with the receptor-binding domain present in the C-terminus of heavy chain of BoNT/A1 (BoNT/A1-HCR) expressed in Escherichia coli. The anti-BoNT/A1-HCR antibody titer increased rapidly by week 4, and this level was maintained for several weeks after boosting immunization. Notably, $20{\mu}l$ of the week-24 BoNT/A1-HCR(-immunized) equine serum showed an in vitro neutralizing activity of over 8 international units (IU) of a reference equine antitoxin. Furthermore, $20{\mu}l$ of equine serum and $100{\mu}g$ of purified equine $F(ab^{\prime})_2$ showed 100% neutralization of 10,000 $LD_{50}$ in vivo. The results of this study shall contribute towards optimizing antitoxin production for BoNT/A1, which is essential for emergency preparedness and response.