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.

Single-domain antibodies (sdAbs, VHHs, or nanobodies) are a promising tool for the treatment of both infectious and somatic diseases. Their small size greatly simplifies any genetic engineering manipulations. Such antibodies have the ability to bind hard-to-reach antigenic epitopes through long parts of the variable chains, the third complementarity-determining regions (CDR3s). VHH fusion with the canonical immunoglobulin Fc fragment allows the Fc-fusion single-domain antibodies (VHH-Fc) to significantly increase their neutralizing activity and serum half-life. Previously we have developed and characterized VHH-Fc specific to botulinum neurotoxin A (BoNT/A), that showed a 1000-fold higher protective activity than monomeric form when challenged with five times the lethal dose (5 LD50) of BoNT/A. During the COVID-19 pandemic, mRNA vaccines based on lipid nanoparticles (LNP) as a delivery system have become an important translational technology that has significantly accelerated the clinical introduction of mRNA platforms. We have developed an mRNA platform that provides long-term expression after both intramuscular and intravenous application. The platform has been extensively characterized using firefly luciferase (Fluc) as a reporter. An intramuscular administration of LNP-mRNA encoding VHH-Fc antibody made it possible to achieve its rapid expression in mice and resulted in 100% protection when challenged with up to 100 LD50 of BoNT/A. The presented approach for the delivery of sdAbs using mRNA technology greatly simplifies drug development for antibody therapy and can be used for emergency prophylaxis.

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.

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Staphylococcal enterotoxins cause food poisoning of various degrees of severity. For milk and meat products, there is a high probability of contamination with staphylococcal enterotoxin H (SEH). In this regard specific and sensitive methods are required to be developed for its detection and monitoring. In this work, the gene seh was expressed and a preparation of recombinant toxin was obtained. Using hybridoma technology, a panel of high-affinity monoclonal antibodies (mAbs) to SEH was produced. The antibodies were characterized and shown to have no cross-reactivity towards the main staphylococcal enterotoxins (A, B, C1, D, E, G and I). Based on these mAbs, a method for specific and quantitative detection of SEH was developed in the format of sandwich enzyme immunoassay (linear range, 0.2-3 ng/ml). All the mAbs produced revealed SEH by immunoblotting. Immunochemical analysis of the culture fluids of staphylococcal isolates obtained from the milk of mastitis-infected cows by immunoblotting and sandwich enzyme immunoassay demonstrated the conformity of these methods. Using the developed method, the toxin was revealed in blood serum and liquid food products practically to 100%. From non-liquid foods, it was shown to be extracted to a maximum with a buffer of pH 4.0-4.5.