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De novo protein design holds promise for creating small stable proteins with shapes customized to bind therapeutic targets. We describe a massively parallel approach for designing, manufacturing and screening mini-protein binders, integrating large-scale computational design, oligonucleotide synthesis, yeast display screening and next-generation sequencing. We designed and tested 22,660 mini-proteins of 37–43 residues that target influenza haemagglutinin and botulinum neurotoxin B, along with 6,286 control sequences to probe contributions to folding and binding, and identified 2,618 high-affinity binders. Comparison of the binding and non-binding design sets, which are two orders of magnitude larger than any previously investigated, enabled the evaluation and improvement of the computational model. Biophysical characterization of a subset of the binder designs showed that they are extremely stable and, unlike antibodies, do not lose activity after exposure to high temperatures. The designs elicit little or no immune response and provide potent prophylactic and therapeutic protection against influenza, even after extensive repeated dosing. A massively parallel computational and experimental approach for de novo designing and screening small hyperstable proteins targeting influenza haemagglutinin and botulinum neurotoxin B identifies new therapeutic candidates more robust than traditional antibody therapies. Designer proteins De novo protein design is a powerful tool for preparing small proteins with desired folds and functions. In this work, David Baker and colleagues report a combined computational and experimental approach to designing and screening folded mini-proteins, consisting of around 40 residues, to bind and target influenza haemagglutinin, a protein on the surface of the flu virus, and botulinum neurotoxin B, a cause of botulism. This high-throughput method produces binding proteins that are more stable and much smaller than traditional antibody therapies, that can be readily modulated and that elicit very little immune response. The optimal haemagglutinin binders show protection against influenza infection in vivo , illustrating the potential of this method for antiviral and other therapeutic applications.

Botulinum toxins (BoNTs) produced by Clostridium botulinum are the most potent known bacterial toxins. The BoNT complex from serotype B-Okra (LPTC/B Okra ) exerts at least 80-fold higher oral toxicity in mice compared with that from serotype A1 (L-PTC/A 62A ). Here, we show that L-PTC/B Okra is predominantly absorbed through enterocytes, whereas LPTC/A 62A targets intestinal microfold cells. Furthermore, α1,2-fucosylation of intestinal mucin determines the oral toxicity of L-PTCs as well as their entry routes, due to differential carbohydrate-binding spectrum of one of the L-PTC components, the hemagglutinin (HA) complex. Fucosylation-deficient mice display reduced intestinal mucin penetration of L-PTC/B Okra via HA, and lower susceptibility to oral intoxication with this toxin. Thus, our results shed light on the molecular mechanisms by which the oral toxicity of BoNTs is increased after crossing intestinal mucus layers Botulinum toxins vary in oral toxicity, but the reasons are unclear. Here, the authors show that the differences can be due to variations in one of the toxin’s components, the hemagglutinin complex, which influence mucin binding and mucus layer penetration

Botulinum neurotoxin (BoNTs; serotypes A, B, E, and F) cause botulism disease in humans, which could be effectively treated using antitoxins. Herein, we established a novel receptor-binding domain (RBD)-based antitoxin using recombinant C terminal heavy chain (Hc) domains of BoNTs as immunogens. Immunization of horses with these recombinant Hc domains allowed the purification and digestion of IgGs from hyper-immune sera to produce high-quality and high-efficiency monovalent botulism antitoxin F(ab′)2 against each BoNT (M-BATs). However, these M-BATs could not bind or neutralize other serotypes of BoNTs, and that there were no cross-protective effects among these M-BATs. This suggested the need to prepare tetravalent antitoxins to neutralize the four BoNTs simultaneously. Thus, these M-BATs were formulated into a novel tetravalent botulism antitoxin (T-BAT), in which a 10-ml volume contained 10000 IU of BoNT/A and 5000 IU of BoNT/B, BoNT/E, and BoNT/F antitoxins. The novel antitoxin preparation could prevent and treat the four mixed botulinum neurotoxins simultaneously in vivo, representing strong efficacy in an animal poisoning model. Moreover, these antibodies in T-BAT could bind the RBD, whereas conventional antitoxins based on inactivated toxins mainly bind the light chain or heavy chain translocation domain (HN) and weakly bind the important RBD in current experimental conditions. The high levels of RBD-specific novel antitoxins can efficiently bind the RBD and neutralize natural or recombinant toxins containing this RBD. The findings of the present study experimentally support the use of RBD-specific antitoxins to treat BoNT serotype A, B, E, and F-mediated botulism. This study demonstrated the concept of developing potent novel multivalent antitoxins against all BoNTs or other toxins, using the RBD of these toxins as an alternative antigen to inactivated toxins.Key points• Antitoxins based on the receptor-binding domains of botulinum neurotoxins were made.• Novel antitoxin binds RBD; traditional antitoxin mainly binds light chain or HN domain.• A tetravalent antitoxin could prevent and treat the four mixed neurotoxins in vivo.

Botulinum neurotoxin type B (BoNT/B) recognizes nerve terminals by binding to 2 receptor components: a polysialoganglioside, predominantly GT1b, and synaptotagmin 1/2. It is widely thought that BoNT/B initially binds to GT1b then diffuses in the plane of the membrane to interact with synaptotagmin. We have addressed the hypothesis that a GT1b–synaptotagmin cis complex forms the BoNT/B receptor. We identified a consensus glycosphingolipid-binding motif in the extracellular juxtamembrane domain of synaptotagmins 1/2 and confirmed by Langmuir monolayer, surface plasmon resonance, and circular dichroism that GT1b interacts with synaptotagmin peptides containing this sequence, inducing α-helical structure. Molecular modeling and tryptophan fluorescence spectroscopy were consistent with the intertwining of GT1b and synaptotagmin, involving cis interactions between the oligosaccharide and ceramide moieties of GT1b and the juxtamembrane and transmembrane domains of synaptotagmin, respectively. Furthermore, a point mutation on synaptotagmin, located outside of the BoNT/B-binding segment, inhibited GT1b binding and blocked GT1b-induced potentiation of BoNT/B binding to synaptotagmin-expressing cells. Our findings are consistent with amodel in which a preassembled GT1b–synaptotagmin complex constitutes the high-affinity BoNT/B receptor.

Botulinum toxin (BoNT) causes flaccid paralysis by blocking the release of neurotransmitters. BoNTs associate with neurotoxin-associated proteins to form medium and large progenitor toxin complexes. The large progenitor toxin complex serotype A-62A (L-PTC/A-62A) specifically targets intestinal M cells for invasion, whereas large progenitor toxin complex serotype B-Okra (L-PTC/B-Okra) is mainly taken up by enterocytes and exhibits higher toxicity via the oral route. Hemagglutinin (HA) is a neurotoxin-associated protein that promotes BoNT absorption from the intestine and has carbohydrate-binding and barrier-disrupting activities. In this study, we established an in vitro reconstitution and purification system for recombinant L-PTC/B-Okra and created a recombinant L-PTC/B-Okra mutant rL-PTC/B-KA with carbohydrate-binding activity but not barrier-disrupting activity. rL-PTC/B-KA showed significantly reduced oral toxicity. Our results demonstrate that the B-Okra toxin disrupts the epithelial barrier of enterocytes and exerts oral toxicity.

Botulinum neurotoxins (BoNTs) are valuable tools to unveil molecular mechanisms of exocytosis in neuronal and non-neuronal cells due to their peptidase activity on exocytic isoforms of SNARE proteins. They are produced by Clostridia as single-chain polypeptides that are proteolytically cleaved into light, catalytic domains covalently linked via disulfide bonds to heavy, targeting domains. This format of two subunits linked by disulfide bonds is required for the full neurotoxicity of BoNTs. We have generated a recombinant version of BoNT/B that consists of the light chain of the toxin fused to the protein transduction domain of the human immunodeficiency virus-1 (TAT peptide) and a hexahistidine tag. His6-TAT-BoNT/B-LC, expressed in Escherichia coli and purified by affinity chromatography, penetrated membranes and exhibited strong enzymatic activity, as evidenced by cleavage of the SNARE synaptobrevin from rat brain synaptosomes and human sperm cells. Proteolytic attack of synaptobrevin hindered exocytosis triggered by a calcium ionophore in the latter. The novel tool reported herein disrupts the function of a SNARE protein within minutes in cells that may or may not express the receptors for the BoNT/B heavy chain, and without the need for transient transfection or permeabilization.

A lumbar sympathetic ganglion block (LSB) is a therapeutic method for complex regional pain syndrome (CRPS) affecting the lower limbs. Recently, LSB with botulinum toxin type A and B was introduced as a novel method to achieve longer duration of analgesia. In this study, we compared the botulinum toxin type A (BTA) with botulinum toxin type B (BTB) in performing LSB on patients with CRPS. LSB was performed with either BTA or BTB on patients with CRPS in their lower extremities. The length of time taken for patients to return to the pre-LSB pain score and the adverse effect of LSB with BTA/BTB were investigated. The median length of time taken for the patients to return to the pre-LSB pain score was 15 days for the BTA group and 69 days for the BTB group (P = 0.002). Scores on a visual analogue scale decreased in the patients of both groups, and no significant adverse effects were experienced. In conclusion, the administration of either BTA or BTB for LSB is a safe method to prolong the sympathetic blocking effect in patients with CRPS. BTB is more effective than BTA to prolong the sympathetic blocking effect in CRPS patients.

Botulinum toxin type B (BoNT-B), known as Myobloc® in the United States and as Neurobloc® in Europe, is a new therapeutically available serotype among the botulinum toxin family. During the last years several data have been reported in literature investigating its efficacy and safety, as well as defining the dosing and application regiments of BoNT-B in the treatment of hyperhidrosis. Moreover, recent studies have been examining its safety profile, which may be different from those known about BoNT-A. The aim of this review is to provide information about what is currently known about BoNT-B in regards to the treatment of focal hyperhidrosis.

Botulinum neurotoxin (BoNT) inhibits neurotransmitter release in motor nerve endings, causing botulism, a condition often resulting from ingestion of the toxin or toxin-producing bacteria. BoNTs are always produced as large protein complexes by associating with a non-toxic protein, non-toxic non-hemagglutinin (NTNH), and some toxin complexes contain another non-toxic protein, hemagglutinin (HA), in addition to NTNH. These accessory proteins are known to increase the oral toxicity of the toxin dramatically. NTNH has a protective role against the harsh conditions in the digestive tract, while HA is considered to facilitate intestinal absorption of the toxin by intestinal binding and disruption of the epithelial barrier. Two specific activities of HA, carbohydrate and E-cadherin binding, appear to be involved in these processes; however, the exact roles of these activities in the pathogenesis of botulism remain unclear. The toxin is conventionally divided into seven serotypes, designated A through G. In this study, we identified the amino acid residues critical for carbohydrate and E-cadherin binding in serotype B HA. We constructed mutants defective in each of these two activities and examined the relationship of these activities using an in vitro intestinal cell culture model. Our results show that the carbohydrate and E-cadherin binding activities are functionally and structurally independent. Carbohydrate binding potentiates the epithelial barrier-disrupting activity by enhancing cell surface binding, while E-cadherin binding is essential for the barrier disruption.

Botulinum neurotoxins (BoNT) are some of the most toxic proteins known and can induce respiratory failure requiring long-term intensive care. Treatment of botulism includes the administration of antitoxins. Monoclonal antibodies (mAbs) hold considerable promise as BoNT therapeutics and prophylactics, due to their potency and safety. A three-mAb combination has been developed that specifically neutralizes BoNT serotype A (BoNT/A), and a separate three mAb combination has been developed that specifically neutralizes BoNT serotype B (BoNT/B). A six mAb cocktail, designated G03-52-01, has been developed that combines the anti-BoNT/A and anti-BoNT/B mAbs. The pharmacokinetics and neutralizing antibody concentration (NAC) of G03-52-01 has been determined in guinea pigs, and these parameters were correlated with protection against an inhalation challenge of BoNT/A1 or BoNT/B1. Previously, it was shown that each antibody demonstrated a dose-dependent mAb serum concentration and reached maximum circulating concentrations within 48 h after intramuscular (IM) or intraperitoneal (IP) injection and that a single IM injection of G03-52-01 administered 48 h pre-exposure protected guinea pigs against an inhalation challenge of up to 93 LD50s of BoNT/A1 and 116 LD50s of BoNT/B1. The data presented here advance our understanding of the relationship of the neutralizing NAC to the measured circulating antibody concentration and provide additional support that a single IM or intravenous (IV) administration of G03-52-01 will provide pre-exposure prophylaxis against botulism from an aerosol exposure of BoNT/A and BoNT/B.