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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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Derkaev, Ryabova, Esmagambetov, Shcheblyakov, Godakova, Vinogradova, Noskov, Logunov, Naroditsky and Gintsburg.)

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 (LD 50 ) 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 LD 50 . 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., Competing Interests: The authors declare no conflict of interest.

Mouse monoclonal antibodies against the most acutely toxic substances, botulinum neurotoxins (BoNTs) of types A, B, E, and F, was generated and characterized, that recognize their respective toxins in natural toxin complex. Based on these antibodies, we developed sandwich-ELISA for quantitative detection of these toxins. For each respective toxin the detection limit of the assay was: BoNT/A - 0.4 ng/ml, BoNT/B - 0.5 ng/ml; BoNT/E - 0.1 ng/ml; and for BoNT/F - 2.4 ng/ml. The developed assays permitted quantitative identification of the BoNTs in canned meat and vegetables. The BNTA-4.1 and BNTA-9.1 antibodies possessed neutralizing activity against natural complex of the botulinium toxin type A in vivo, both individually and in mixture, the mixture of the antibodies neutralized the higher dose of the toxin. The BNTA-4.1 antibody binds specifically the light chain (the chain with protease activity) of the toxin, whereas BNTA-9.1 interacts with the heavy chain. We believe that the BNTA-4.1 and BNTA-9.1 monoclonal antibodies are prospective candidates for development of humanized therapeutic antibodies for treatment of BoNT/A-caused botulism.

Test systems for indirect hemagglutination (IHA) test for detection of S. aureus and S. epidermidis teichoic acids and S. aureus alpha-toxin in patients' sera have been developed on the basis of immunoglobulins isolated from monospecific sera. Test system for IHA test for detection of antitoxin in donor and patients' sera has been created on the basis of highly purified alpha-toxin. Thirty donor sera and 61 sera from patients with pneumonia were analyzed. Low antibody levels in the patients may be due to the fact that the sera were collected during the first days of disease. Group of patients with high content of staphylococcal antigens and antitoxin in the blood was particularly interesting. These patients developed severe pneumonia, among whose etiological agents were S. aureus and S. epidermidis. Diagnostic analysis of patients' sera by IHA test for detection of staphylococcal antigens was more effective, accurate, and rapid in comparison with the bacteriological method; moreover, it confirmed the significance of staphylococci in the pathogenesis of pneumonia.

Kinetic and concentration-mediated dependences of urea effects on toxicity and upon values of negative extremes (279, 287, 292 nm), shifts of lambda max fluorescence from 332 nm to the red region, decrease of fluorescence intensity, changes in accessibility of SH-groups were found. These data point to direct correlation between conformational changes in neurotoxin in urea solutions and decrease of toxicity. An investigation was carried out on renaturation of conformational changed neurotoxin in 8 M urea solution. In no cases was the toxicity restored, though there was a shift of lambda max fluorescence in protein from 340 to 333 nm, that was the evidence for reverse transition of the neurotoxin molecule from a disordered structure to the globule shape. The data lead to the conclusion that the toxicity region is probably a conformationally depending determinant.

A procedure for isolation of highly purified hemagglutinin from a toxic complex of culture filtrates of Cl. botulinum type A is described. This procedure includes precipitation with (NH4)2SO4, chromatography on Sephadex G-100, G-200 and DEAE-cellulose, specific adsorption on human erythrocytes and affinity chromatography. Using polyacrylamide gel electrophoresis, it was shown that hemagglutinin is a heteropolymeric protein consisting of a monomer (Mr 53 000) and a trimer (Mr 160 000). The monomer is made up of two subunits with Mr 13 000 and one subunit with Mr 27 000 covalently linked by SS-crosslinks. The number and nature of the SS-crosslinks and SH-groups in the protein molecule were determined and a hypothetical structural model of hemagglutinin was proposed. Using immunochemical analysis, it was shown that some (but not all) serological properties of the highly purified protein from Cl. botulinum type A and of its partially purified counterpart are similar to those of hemagglutinin from Cl. botulinum type B.

A comparative amino acid analysis of botulinum neurotoxin type A and its subunits has been carried out. The heavy and light chains of neurotoxin have the same ratios of polar and non-polar amino acids (1.3:1), the amount of tryptophan residues in the heavy chain is 4 times as much as that in the light chain, and the number of SH-groups exceeds that in the light chains 2-fold. In neurotoxin, two N-terminal amino acid residues--alanine and leucine--were identified. Alanine was found to be the N-terminus of the heavy chain. The fluorescence spectra of neurotoxin subunits indicate differences in the conformational state of the polypeptide chains. The antigenic non-identity of botulinum neurotoxin A subunits suggests the presence in the neurotoxin molecule of at least two antigenic determinants, corresponding to the heavy and light chains.

A procedure for preparation of electrophoretically and serologically homogeneous neurotoxin and a highly purified hemagglutinin from the culture fluid of Cl. botulinum A, strain 501 is described. The yield of neurotoxin with specific activity of 80-100 X 10(6) DLM/mg of protein is 5-20%. Neurotoxin has a molecular weight of 150,000, sedimentation coefficient of 7.1S, pI of 6.2-6.3; the maximum of its fluorescence corresponds to 332 nm. The toxin molecule contains 4 SH-groups. Neurotoxin consists of two subunits with molecular weights of 98,000 and 56,000. The storage of neurotoxin at -20 degrees C causes inactivation and electrophoretical heterogeneity of the protein. The inactivation leads to an alteration of the toxin molecule charge, a shift of the lambda max of fluorescence and a loss of the SH-group reactivity without affecting the molecular weight or serological properties of the protein. The data obtained suggest that the conformational state of toxin molecules is essential for its biological activity.