ABSTRACT
Botulinum neurotoxin (BoNT) is one of the most dangerous bacterial toxins and a potential biological weapon component. BoNT mechanism of pathological action is based on inhibiting the release of neurotransmitters from nerve endings. To date, anti-BoNT therapy is reduced to the use of horse hyperimmune serum, which causes many side effects, as well as FDA-approved drug BabyBig which consists of human-derived anti-BoNT antibodies (IgG) for infant botulinum treatment. Therapeutics for botulism treatment based on safer monoclonal antibodies are undergoing clinical trials. In addition, agents have been developed for the specific prevention of botulism, but their effectiveness has not been proved. In this work, we have obtained a recombinant adeno-associated virus (rAAV-B11-Fc) expressing a single-domain antibody fused to the human IgG Fc-fragment (B11-Fc) and specific to botulinum toxin type A (BoNT/A). We have demonstrated that B11-Fc antibody, expressed via rAAV-B11-Fc treatment, can protect animals from lethal doses of botulinum toxin type A, starting from day 3 and at least 120 days after administration. Thus, our results showed that rAAV-B11-Fc can provide long-term expression of B11-Fc-neutralizing antibody in vivo and provide long-term protection against BoNT/A intoxication. Consequently, our study demonstrates the applicability of rAAV expressing protective antibodies for the prevention of intoxication caused by botulinum toxins.
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.