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Botulinum neurotoxins (BoNTs) cause botulism and are the deadliest naturally-occurring substances known to humans. BoNTs have been classified as one of the category A agents by the Centers for Disease Control and Prevention, indicating their potential use as bioweapons. To counter bio-threat and naturally-occurring botulism cases, well-tolerated antibodies by humans that neutralize BoNTs are relevant. In our previous work, we showed the neutralizing potential of macaque (Macaca fascicularis)-derived scFv-Fc (scFv-Fc ELC18) by in vitro endopeptidase immunoassay and ex vivo mouse phrenic nerve-hemidiaphragm assay by targeting the light chain of the botulinum neurotoxin type E (BoNT/E). In the present study, we germline-humanized scFv-Fc ELC18 into a full IgG hu8ELC18 to increase its immunotolerance by humans. We demonstrated the protection and prophylaxis capacity of hu8ELC18 against BoNT/E in a mouse model. A concentration of 2.5 ng/mouse of hu8ELC18 protected against 5 mouse lethal dose (MLD) in a mouse protection assay and complete neutralization of 1 LD50 of pure BoNT/E toxin was achieved with 8 ng of hu8ELC18 in mouse paralysis assay. Furthermore, hu8ELC18 protected mice from 5 MLD if injected up to 14 days prior to intraperitoneal BoNT/E administration. This newly-developed humanized IgG is expected to have high tolerance in humans.

AbstractBotulinum neurotoxins (BoNTs), the causative agents of botulism, are proteins consisting of an N-terminal catalytic Light Chain (LC) and a C-terminal Heavy Chain (HC), which comprises a LC-translocation domain (HC N) and a receptor binding domain (HC C). There are seven BoNT serotypes (A-G) and multiple subtypes. Currently, there is no approved vaccine against botulism available for human use. Several studies have investigated BoNT domains, multi-domains, or mutated holotoxins as candidate BoNT vaccines. However, residual toxicity has been observed in mutated full-length BoNTs and multi-domain (LCHC N) vaccines. In this study, six-individual point substitutions (6M) were introduced into the substrate binding and cleavage sites, zinc binding motif, and LC translocation region of LCHC N/A1 and LCHC N/B1, which possessed no detectable toxicity in mice at 200 μg, being >40-million-fold less toxic than full-length BoNT. IP immunization of mice with 6MLCHC N/A1 or 6MLCHC N/B1 elicited a strong IgG response that protected against high dose challenge with BoNT/A1 or BoNT/B1, respectively. 4MBoNT/B1 and 7MBoNT/B1 were also engineered and found to elicit strong IgG responses that protected against high dose challenge with BoNT/B1. Thus, multi-domain inactivation may yield potent and safe vaccines against other BoNT-serotypes and BoNT-like proteins. The strategy of systematic inactivation of multiple functional domains by targeted single amino acid substitutions to decrease toxicity, combined with investigations of the most immunogenic and protective domains, provides a robust platform for protein toxin vaccines for humans and animals.

Background. Only Clostridium botulinum strain IBCA10-7060 produces the recently described novel botulinum neurotoxin type H (BoNT/H). BoNT/H (N-terminal two-thirds most homologous to BoNT/F and C-terminal one-third most homologous to BoNT/A) requires antitoxin to toxin ratios ≥1190:1 for neutralization by existing antitoxins. Hence, more potent and safer antitoxins against BoNT/H are needed. Methods. We therefore evaluated our existing monoclonal antibodies (mAbs) to BoNT/A and BoNT/F for BoNT/H binding, created yeast-displayed mutants to select for higher-affinity-binding mAbs by using flow cytometry, and evaluated the mAbs' ability to neutralize BoNT/H in the standard mouse bioassay. Results. Anti-BoNT/A HCC-binding mAbs RAZ1 and CR2 bound BoNT/H with high affinity. However, only 1 of 6 BoNT/F mAbs (4E17.2A) bound BoNT/H but with an affinity >800-fold lower (equilibrium dissociation binding constant [KD] = 7.56 × 10−8 M) than its BoNT/F affinity (KD = 9.1 × 10−11 M), indicating that the N-terminal two-thirds of BoNT/H is immunologically unique. The affinity of 4E17.2A for BoNT/H was increased >500-fold to KD = 1.48 × 10−10 M (mAb 4E17.2D). A combination of mAbs RAZ1, CR2, and 4E17.2D completely protected mice challenged with 280 mouse median lethal doses of BoNT/H at a mAb dose as low as 5 μg of total antibody. Conclusions. This 3-mAb combination potently neutralized BoNT/H and represents a potential human antitoxin that could be developed for the prevention and treatment of type H botulism.

Botulinum neurotoxin (BoNT) produced by is one of the most potent known toxins. Moreover, BoNT is classified as one of the most important biological warfare agents that threatens the biosafety of the world. Currently, the approved treatment for botulism in humans is the use of polyvalent horse serum antitoxins. However, they are greatly limited because of insufficient supply and adverse reactions. Thus, treatment of human botulism requires the development of effective toxin-neutralizing antibodies. Considering their advantages, neutralizing nanobodies will play an increasing role as BoNTs therapeutics. Herein, neutralizing nanobodies binding to the heavy chain (Hc) domain of BoNT/B (BHc) were screened from a phage display library. Then, BoNT/B-specific clones were identified and fused with the human Fc fragment (hFc) to form chimeric heavy chain antibodies. Finally, the affinity, specificity, and neutralizing activity of antibodies against BoNT/B were evaluated. The B5-hFc, B9-hFc and B12-hFc antibodies demonstrated high affinity for BHc in the nanomolar range. The three antibodies were proven to have potent neutralizing activity against BoNT/B . The results demonstrate that inhibiting toxin binding to the host receptor is an efficient strategy and the three antibodies could be used as candidates for the further development of drugs to prevent and treat botulism.

Botulinum neurotoxin (BoNT), produced by Clostridium botulinum , is the most toxic protein known, capable of causing severe paralysis and posing a significant bioterrorism threat due to its extreme lethality even in minute quantities. Despite this, there are currently no FDA-approved vaccines for widespread public use. To address this urgent need, we have developed an innovative vaccine platform by fusing the neuronal binding domain of BoNT/E (Hc/E) with core-streptavidin (CS), resulting in a stable CS-Hc/E vaccine. Mice vaccinated with CS-Hc/E exhibited superior antibody titers compared to those receiving Hc/E alone. To develop a trivalent vaccine against BoNT/A, BoNT/B, and BoNT/E— key contributors to the vast majority of human botulism—we conjugated CS-Hc/E with a biotinylated atoxic chimeric protein incorporating neutralizing epitopes from BoNT/A and BoNT/B. This chimeric protein includes the binding domain of BoNT/A, along with the protease-inactive light chain and translocation domains of BoNT/B. The interaction between CS and biotin formed a stable tetrameric antigen, EBA. Vaccination with EBA in mice elicited robust antibody responses and provided complete protection against lethal doses of BoNT/A, BoNT/B, and BoNT/E. Our findings highlight EBA’s potential as a stable and effective broad-spectrum vaccine against BoNT. Moreover, our technology offers a versatile platform for developing multivalent, stable vaccines targeting various biological threats by substituting the BoNT domain(s) with neutralizing epitopes from other life-threatening pathogens, thereby enhancing public health preparedness and biodefense strategies.

•In a mouse model of botulism, M-BoNT/A1 was not toxic at >106-fold greater amounts than native BoNT/A.•M-BoNT/A1(W1266A) (M-BoNT/A1W) was created to prevent neuronal cell binding.•M-BoNT/A1 vaccination protected against challenge by 106 LD50 of native BoNT/A1.•LCHCN elicited a higher neutralizing antibody titer than HCC, showing neutralizing epitopes within LCHCN.•Engineered BoNT with defects in catalysis and receptor binding is a novel vaccine strategy against botulism. Botulinum neurotoxins (BoNT) are the most toxic proteins for humans. BoNTs are single chain proteins with an N-terminal light chain (LC) and a C-terminal heavy chain (HC). HC comprises a translocation domain (HCN) and a receptor binding domain (HCC). Currently, there are no approved vaccines against botulism. This study tests a recombinant, full-length BoNT/A1 versus LCHCN/A1 and HCC/A1 as vaccine candidates against botulism. Recombinant, full-length BoNT/A1 was detoxified by engineering 3-amino acid mutations (E224A/R363A/Y366F) (M-BoNT/A1) into the LC to eliminate catalytic activity, which reduced toxicity in a mouse model of botulism by >106-fold relative to native BoNT/A1. As a second step to improve vaccine safety, an additional mutation (W1266A) was engineered in the ganglioside binding pocket, resulting in reduced receptor binding, to produce M-BoNT/A1W. M-BoNT/A1W vaccination protected against challenge by 106 LD50 Units of native BoNT/A1, while M-BoNT/A1 or M-BoNT/A1W vaccination equally protected against challenge by native BoNT/A2, a BoNT subtype. Mice vaccinated with M-BoNT/A1W surviving BoNT challenge had dominant antibody responses to the LCHCN domain, but varied antibody responses to HCC. Sera from mice vaccinated with M-BoNT/A1W also neutralized BoNT/A1 action on cultured neuronal cells. The cell- and mouse-based assays measured different BoNT-neutralizing antibodies, where M-BoNT/A1W elicited a strong neutralizing response in both assays. Overall, M-BoNT/A1W, with defects in multiple toxin functions, elicits a potent immune response to BoNT/A challenge as a vaccine strategy against botulism and other toxin-mediated diseases.

Botulinum neurotoxin serotype A (BoNT/A) is the most potent known neurotoxin. While its light chain (LC) catalytic domain is a prime target for next-generation vaccines and therapeutics, the functional differences among BoNT/A subtype LCs (A1, A2, A3) remain to be definitively characterized, despite notable sequence variation. This work aimed to systematically compare the proteolytic activity and immunoprotective efficacy of recombinant BoNT/A1-LC, A2-LC, and A3-LC. Recombinant A1-LC-His, A2-LC-His, A3-LC-His, and A3-LC-Twin-Strep proteins were expressed in Escherichia coli (E. coli) and purified with affinity chromatography. Their proteolytic activity was assessed via in vitro SNAP-25 cleavage assays. The protective potency of these antigens was evaluated in a mouse model. In vitro cleavage assays revealed a substrate cleavage efficiency order of A2-LC > A1-LC > A3-LC. In vivo, both A1-LC and A2-LC immunization conferred robust, broad protection against high-dose challenges with all three toxin subtypes. In stark contrast, A3-LC provided only minimal protection against its homologous toxin and none against heterologous subtypes. Crucially, the functional deficit of A3-LC was confirmed to be an intrinsic property, as the A3-LC-TS variant, designed to exclude tag-specific interference, exhibited comparable low efficacy. According to structural research, A3-LC’s compromised function may be caused by a four-amino-acid loss. The inferior performance of A3-LC is inherent to its primary structure. This work identified A1-LC or A2-LC as the potential proteolytic activity molecule and vaccine antigen by demonstrating functional differences among BoNT/A subtype LCs. These findings provide crucial insights for developing subtype-specific countermeasures against botulism.

Botulinum toxin (BoNT) from Clostridium botulinum is the most toxic biotoxin known and is also an important bioterrorism agent. After poisoning, the only effective treatment is injection of antitoxin. However, neutralizing nanoantibodies are safer and more effective, representing a promising therapeutic approach. Therefore, it is important to obtain effective neutralizing nanoantibodies. Hence, the present study aimed to construct a phage antibody library by immunizing a camel and screening specific clones that bind to the L-HN domain of BoNT/F and constructing chimeric heavy-chain antibodies by fusing them with a human Fc fragment. The antibodies’ affinity and in vivo neutralizing activities were evaluated. The results showed that 2 µg of F20 antibody could completely neutralize 20 × the median lethal dose (LD50) of BoNT/F in vitro. Injection of 5 mg/kg F20 at 1 h, 2 h, 3 h, and 4 h into mice after BoNT/F challenge resulted in complete survival in vivo. Overall, the antibody might be a candidate for the development of new drugs to treat botulism.

Botulinum neurotoxins (BoNTs) are the most potent toxins on Earth and are classified as Category A biological agents. BoNTs lead to paralysis in humans and cause botulism. Antibody therapeutics can effectively treat toxin-mediated infectious diseases. In this study, we generated a pharmaceutical humanized monoclonal antibody (HZ45 mAb) to prevent or treat botulism. HZ45 binds to the heavy chain receptor (HCR) domain of the toxin, preventing the toxin from entering the cell. The mAb was produced using hybridoma technology and phage display. We evaluated HZ45 mAb for the neutralization of BoNT serotype A (BoNT/A) in mice and rabbits. The survival results showed that pretreatment with HZ45 mAb provided 100% protection at a dose of 0.1 mg per mouse against a maximum of 100 LD50 of BoNT/A. To assess the therapeutic efficacy of HZ45 mAb in New Zealand white rabbits (NZWs), a 5 mg dose was administered 4 or 8 h after challenge with 10 LD50. The results indicated that 5 mg of HZ45 could treat the NZWs within 8 h after exposure to 10 LD50 botulinum. Consequently, in an in vivo context, including mice and rabbits, HZ45 mAb could protect against botulinum type A intoxication.

Botulism is one of the most serious food intoxications, manifesting as prolonged paralytic conditions. This disease is usually the result of the consumption of poor quality canned or smoked foods, so the inhabitants of many countries of the world are exposed to the risk of this kind of poisoning every year. In view of the severity of poisonings caused by botulinum neurotoxins, monoclonal antibodies (mAbs) show great promise because of their targeting action, lack of allergic reactions and serum sickness. The use of a cocktail of mAbs increases the “functional specificity” of their mixture, allowing them to bind to the active domains of different toxin chains and block their action. In this work, we obtained 14 murine mAbs to the catalytic and receptor-binding domain of botulinum toxin type A. The Sp2/0-Ag14 murine myeloma cell line and splenocytes from immunized mice of the BALB/c line were used as fusion partners. We have shown that the selected cocktail of three antibodies neutralizes native toxin more effectively than antibodies separately—complete neutralization is achieved at a toxin dose of 3LD50 and partial neutralization at 5LD50. We presume that this cocktail may be promising as a prototype for the creation of a therapeutic drug capable of neutralizing the toxin in the blood of patients.