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1.  Extraction and Inhibition of Enzymatic Activity of Botulinum Neurotoxins/A1, /A2, and /A3 by a Panel of Monoclonal Anti-BoNT/A Antibodies 
PLoS ONE  2009;4(4):e5355.
Botulinum neurotoxins (BoNTs) are extremely potent toxins that are capable of causing death or respiratory failure leading to long-term intensive care. Treatment includes serotype-specific antitoxins, which must be administered early in the course of the intoxication. Rapidly determining human exposure to BoNT is an important public health goal. In previous work, our laboratory focused on developing Endopep-MS, a mass spectrometry-based endopeptidase method for detecting and differentiating BoNT/A–G serotypes in buffer and BoNT/A, /B, /E, and /F in clinical samples. We have previously reported the effectiveness of antibody-capture to purify and concentrate BoNTs from complex matrices, such as clinical samples. Because some antibodies inhibit or neutralize the activity of BoNT, the choice of antibody with which to extract the toxin is critical. In this work, we evaluated a panel of 16 anti-BoNT/A monoclonal antibodies (mAbs) for their ability to inhibit the in vitro activity of BoNT/A1, /A2, and /A3 complex as well as the recombinant LC of A1. We also evaluated the same antibody panel for the ability to extract BoNT/A1, /A2, and /A3. Among the mAbs, there were significant differences in extraction efficiency, ability to extract BoNT/A subtypes, and inhibitory effect on BoNT catalytic activity. The mAbs binding the C-terminal portion of the BoNT/A heavy chain had optimal properties for use in the Endopep-MS assay.
doi:10.1371/journal.pone.0005355
PMCID: PMC2670495  PMID: 19399171
2.  Extraction of BoNT/A, /B, /E, and /F with a Single, High Affinity Monoclonal Antibody for Detection of Botulinum Neurotoxin by Endopep-MS 
PLoS ONE  2010;5(8):e12237.
Botulinum neurotoxins (BoNTs) are extremely potent toxins that are capable of causing respiratory failure leading to long-term intensive care or death. The best treatment for botulism includes serotype-specific antitoxins, which are most effective when administered early in the course of the intoxication. Early confirmation of human exposure to any serotype of BoNT is an important public health goal. In previous work, we focused on developing Endopep-MS, a mass spectrometry-based endopeptidase method for detecting and differentiating the seven serotypes (BoNT/A-G) in buffer and BoNT/A, /B, /E, and /F (the four serotypes that commonly affect humans) in clinical samples. We have previously reported the success of antibody-capture to purify and concentrate BoNTs from complex matrices, such as clinical samples. However, to check for any one of the four serotypes of BoNT/A, /B, /E, or /F, each sample is split into 4 aliquots, and tested for the specific serotypes separately. The discovery of a unique monoclonal antibody that recognizes all four serotypes of BoNT/A, /B, /E and /F allows us to perform simultaneous detection of all of them. When applied in conjunction with the Endopep-MS assay, the detection limit for each serotype of BoNT with this multi-specific monoclonal antibody is similar to that obtained when using other serotype-specific antibodies.
doi:10.1371/journal.pone.0012237
PMCID: PMC2923190  PMID: 20808925
3.  Selection and Characterization of a Human Monoclonal Neutralizing Antibody for Clostridium Botulinum Neurotoxin Serotype B 
Botulinum neurotoxins (BoNTs) are causative agents for botulism and are identified as a category A bioterror agents by the Centers for Disease Control and Prevention (CDC). Current antitoxins against BoNTs intoxication have some limitations including side effects or limited supply. As an alternative, neutralizing monoclonal antibodies will play an increasing role as BoNTs therapeutics. To date, no human anti-BoNT/B neutralizing monoclonal antibodies have yet to be reported. Herein, we describe an improved selection approach and characterization of a human monoclonal antibody, F2, which is capable of binding BoNT/B with high specificity and displays neutralizing activity in an in vitro cell-based assay. Through surface plasmon resonance studies, we have determined its association and dissociation rate constants. In sum, our data demonstrate that monoclonal antibody F2 is a promising BoNT/B therapeutic lead for further development.
doi:10.1016/j.bmcl.2008.12.055
PMCID: PMC2645068  PMID: 19112017
Phage display; Botulinum neurotoxin; monoclonal antibody; neutralizing antibody
4.  Botulinum Neurotoxins and Botulism: A Novel Therapeutic Approach  
Toxins  2011;3(5):469-488.
Specific treatment is not available for human botulism. Current remedial mainstay is the passive administration of polyclonal antibody to botulinum neurotoxin (BoNT) derived from heterologous species (immunized animal or mouse hybridoma) together with supportive and symptomatic management. The antibody works extracellularly, probably by blocking the binding of receptor binding (R) domain to the neuronal receptors; thus inhibiting cellular entry of the holo-BoNT. The antibody cannot neutralize the intracellular toxin. Moreover, a conventional antibody with relatively large molecular size (150 kDa) is not accessible to the enzymatic groove and, thus, cannot directly inhibit the BoNT zinc metalloprotease activity. Recently, a 15–20 kDa single domain antibody (VHH) that binds specifically to light chain of BoNT serotype A was produced from a humanized-camel VH/VHH phage display library. The VHH has high sequence homology (>80%) to the human VH and could block the enzymatic activity of the BoNT. Molecular docking revealed not only the interface binding between the VHH and the toxin but also an insertion of the VHH CDR3 into the toxin enzymatic pocket. It is envisaged that, by molecular linking the VHH to a cell penetrating peptide (CPP), the CPP-VHH fusion protein would be able to traverse the hydrophobic cell membrane into the cytoplasm and inhibit the intracellular BoNT. This presents a novel and safe immunotherapeutic strategy for botulism by using a cell penetrating, humanized-single domain antibody that inhibits the BoNT by means of a direct blockade of the groove of the menace enzyme.
doi:10.3390/toxins3050469
PMCID: PMC3202833  PMID: 22069720
botulinum neurotoxin; botulism; zinc metalloprotease; immunotherapy; serum therapy; therapeutic antibody; chimeric antibody; humanized antibody; single chain antibody variable fragment (ScFv); heavy chain antibody (HCAb); single domain antibody (sdAb); VH; VL; VHH; humanized-camel phage display library; nanobody; transbody; cell penetrating peptide (CPP); phage display
5.  A Single Domain Llama Antibody Potently Inhibits the Enzymatic Activity of Botulinum Neurotoxin by Binding to the Non-Catalytic Alpha-Exosite Binding Region 
Journal of molecular biology  2010;397(4):1106-1118.
Ingestion or inhalation of botulinum neurotoxin (BoNT) results in botulism, a severe and frequently fatal disease. Current treatments rely on antitoxins, which while effective cannot reverse symptoms once BoNT has entered the neuron. For treatments that can reverse intoxication, interest has focused on developing inhibitors of the enzymatic BoNT light chain (BoNT Lc). Such inhibitors typically mimic substrate and bind in or around the substrate cleavage pocket. To explore the full range of binding sites for serotype A light chain (BoNT/A Lc) inhibitors, we created a library of non-immune llama single domain VHH antibodies displayed on the surface of the yeast Saccharomyces cerevisiae. Library selection on BoNT/A Lc yielded 15 yeast displayed VHH with equilibrium dissociation constants (KD) from 230 to 0.03 nM measured by flow cytometry. Eight of 15 VHH inhibited the cleavage of substrate SNAP25 by BoNT/A Lc. The most potent VHH (Aa1) had a solution KD for BoNT/A Lc of 1.47 × 10-10 M, an IC50 of 4.7 × 10-10 M, and was resistant to heat denaturation and reducing conditions. To understand the mechanism by which Aa1 inhibited catalysis, the X-ray crystal structure of the BoNT/A Lc - Aa1 VHH complex was solved at 2.6 Å resolution. The structure reveals that the Aa1 VHH binds in the alpha-exosite of the BoNT/A Lc, far from the active site for catalysis. The study validates the utility of non-immune llama VHH libraries as a source of enzyme inhibitors and identifies the BoNT/A Lc alpha-exosite as a target for inhibitor development.
doi:10.1016/j.jmb.2010.01.070
PMCID: PMC2903050  PMID: 20138889
botulinum neurotoxin type A; llama single VHH; single domain antibody; alpha-exosite; Naïve yeast-displayed library
6.  Botulinum Neurotoxin Detection and Differentiation by Mass Spectrometry 
Emerging Infectious Diseases  2005;11(10):1578-1583.
A new rapid, mass spectrometry-based method to detect and differentiate botulinal neurotoxins is described.
Botulinum neurotoxins (BoNTs) are proteases that cleave specific cellular proteins essential for neurotransmitter release. Seven BoNT serotypes (A–G) exist; 4 usually cause human botulism (A, B, E, and F). We developed a rapid, mass spectrometry–based method (Endopep-MS) to detect and differentiate active BoNTs A, B, E, and F. This method uses the highly specific protease activity of the toxins with target peptides specific for each toxin serotype. The product peptides derived from the endopeptidase activities of BoNTs are detected by matrix-assisted laser-desorption ionization time-of-flight mass spectrometry. In buffer, this method can detect toxin equivalents of as little as 0.01 mouse lethal dose (MLD)50 and concentrations as low as 0.62 MLD50/mL. A high-performance liquid chromatography–tandem mass spectrometry method for quantifying active toxin, where the amount of toxin can be correlated to the amount of product peptides, is also described.
doi:10.3201/eid1110.041279
PMCID: PMC3366733  PMID: 16318699
bioterrorism; botulism; mass spectrometry; botulinum neurotoxin; research
7.  Sequence Variation within Botulinum Neurotoxin Serotypes Impacts Antibody Binding and Neutralization  
Infection and Immunity  2005;73(9):5450-5457.
The botulinum neurotoxins (BoNTs) are category A biothreat agents which have been the focus of intensive efforts to develop vaccines and antibody-based prophylaxis and treatment. Such approaches must take into account the extensive BoNT sequence variability; the seven BoNT serotypes differ by up to 70% at the amino acid level. Here, we have analyzed 49 complete published sequences of BoNTs and show that all toxins also exhibit variability within serotypes ranging between 2.6 and 31.6%. To determine the impact of such sequence differences on immune recognition, we studied the binding and neutralization capacity of six BoNT serotype A (BoNT/A) monoclonal antibodies (MAbs) to BoNT/A1 and BoNT/A2, which differ by 10% at the amino acid level. While all six MAbs bound BoNT/A1 with high affinity, three of the six MAbs showed a marked reduction in binding affinity of 500- to more than 1,000-fold to BoNT/A2 toxin. Binding results predicted in vivo toxin neutralization; MAbs or MAb combinations that potently neutralized A1 toxin but did not bind A2 toxin had minimal neutralizing capacity for A2 toxin. This was most striking for a combination of three binding domain MAbs which together neutralized >40,000 mouse 50% lethal doses (LD50s) of A1 toxin but less than 500 LD50s of A2 toxin. Combining three MAbs which bound both A1 and A2 toxins potently neutralized both toxins. We conclude that sequence variability exists within all toxin serotypes, and this impacts monoclonal antibody binding and neutralization. Such subtype sequence variability must be accounted for when generating and evaluating diagnostic and therapeutic antibodies.
doi:10.1128/IAI.73.9.5450-5457.2005
PMCID: PMC1231122  PMID: 16113261
8.  Botulinum neurotoxin subtype A2 enters neuronal cells faster than subtype A1 
FEBS letters  2010;585(1):199-206.
Botulinum neurotoxins (BoNTs), the causative agent of human botulism, are the most potent naturally occurring toxins known. BoNT/A1, the most studied BoNT, is also used as an important biopharmaceutical. In this study, the biological activity of BoNT/A1 is compared to that of BoNT/A2 using neuronal cell models. The data obtained indicate faster and increased intoxication of neuronal cells by BoNT/A2 than BoNT/A1, and that the mechanism underlying this increased toxicity is faster and more efficient cell entry that is independent of ganglioside binding. These results have important implications for the development of new BoNT based therapeutics and BoNT countermeasures.
doi:10.1016/j.febslet.2010.11.045
PMCID: PMC3041152  PMID: 21126520
botulinum neurotoxin; BoNT/A1; BoNT/A2; cell based assay; cell entry; neuron
9.  Unique Biological Activity of Botulinum D/C Mosaic Neurotoxin in Murine Species 
Infection and Immunity  2012;80(8):2886-2893.
Clostridium botulinum types C and D cause animal botulism by the production of serotype-specific or mosaic botulinum neurotoxin (BoNT). The D/C mosaic BoNT (BoNT/DC), which is produced by the isolate from bovine botulism in Japan, exhibits the highest toxicity to mice among all BoNTs. In contrast, rats appeared to be very resistant to BoNT/DC in type C and D BoNTs and their mosaic BoNTs. We attempted to characterize the enzymatic and receptor-binding activities of BoNT/DC by comparison with those of type C and D BoNTs (BoNT/C and BoNT/D). BoNT/DC and D showed similar toxic effects on cerebellar granule cells (CGCs) derived from the mouse, but the former showed less toxicity to rat CGCs. In recombinant murine-derived vesicle-associated membrane protein (VAMP), the enzymatic activities of both BoNTs to rat isoform 1 VAMP (VAMP1) were lower than those to the other VAMP homologues. We then examined the physiological significance of gangliosides as the binding components for types C and D, and mosaic BoNTs. BoNT/DC and C were found to cleave an intracellular substrate of PC12 cells upon the exogenous addition of GM1a and GT1b gangliosides, respectively, suggesting that each BoNT recognizes a different ganglioside moiety. The effect of BoNT/DC on glutamate release from CGCs was prevented by cholera toxin B-subunit (CTB) but not by a site-directed mutant of CTB that did not bind to GM1a. Bovine adrenal chromaffin cells appeared to be more sensitive to BoNT/DC than to BoNT/C and D. These results suggest that a unique mechanism of receptor binding of BoNT/DC may differentially regulate its biological activities in animals.
doi:10.1128/IAI.00302-12
PMCID: PMC3434576  PMID: 22665374
10.  Production and Characterisation of a Neutralising Chimeric Antibody against Botulinum Neurotoxin A 
PLoS ONE  2010;5(10):e13245.
Botulinum neurotoxins, produced by Clostridium botulinum bacteria, are the causative agent of botulism. This disease only affects a few hundred people each year, thus ranking it among the orphan diseases. However, botulinum toxin type A (BoNT/A) is the most potent toxin known to man. Due to their potency and ease of production, these toxins were classified by the Centers for Disease Control and Prevention (CDC) as Category A biothreat agents. For several biothreat agents, like BoNT/A, passive immunotherapy remains the only possible effective treatment allowing in vivo neutralization, despite possible major side effects. Recently, several mouse monoclonal antibodies directed against a recombinant fragment of BoNT/A were produced in our laboratory and most efficiently neutralised the neurotoxin. In the present work, the most powerful one, TA12, was selected for chimerisation. The variable regions of this antibody were thus cloned and fused with the constant counterparts of human IgG1 (kappa light and gamma 1 heavy chains). Chimeric antibody production was evaluated in mammalian myeloma cells (SP2/0-Ag14) and insect cells (Sf9). After purifying the recombinant antibody by affinity chromatography, the biochemical properties of chimeric and mouse antibody were compared. Both have the same very low affinity constant (close to 10 pM) and the chimeric antibody exhibited a similar capacity to its parent counterpart in neutralising the toxin in vivo. Its strong affinity and high neutralising potency make this chimeric antibody interesting for immunotherapy treatment in humans in cases of poisoning, particularly as there is a probable limitation of the immunological side effects observed with classical polyclonal antisera from heterologous species.
doi:10.1371/journal.pone.0013245
PMCID: PMC2953832  PMID: 20967241
11.  Potent Neutralization of Botulinum Neurotoxin/B by Synergistic Action of Antibodies Recognizing Protein and Ganglioside Receptor Binding Domain 
PLoS ONE  2012;7(8):e43845.
Background
Botulinum neurotoxins (BoNTs), the causative agents for life-threatening human disease botulism, have been recognized as biological warfare agents. Monoclonal antibody (mAb) therapeutics hold considerable promise as BoNT therapeutics, but the potencies of mAbs against BoNTs are usually less than that of polyclonal antibodies (or oligoclonal antibodies). The confirmation of key epitopes with development of effective mAb is urgently needed.
Methods and Findings
We selected 3 neutralizing mAbs which recognize different non-overlapping epitopes of BoNT/B from a panel of neutralizing antibodies against BoNT/B. By comparing the neutralizing effects among different combination groups, we found that 8E10, response to ganglioside receptor binding site, could synergy with 5G10 and 2F4, recognizing non-overlapping epitopes within Syt II binding sites. However, the combination of 5G10 with 2F4 blocking protein receptor binding sites did not achieve synergistical effects. Moreover, we found that the binding epitope of 8E10 was conserved among BoNT A, B, E, and F, which might cross-protect the challenge of different serotypes of BoNTs in vivo.
Conclusions
The combination of two mAbs recognizing different receptors' binding domain in BoNTs has a synergistic effect. 8E10 is a potential universal partner for the synergistical combination with other mAb against protein receptor binding domain in BoNTs of other serotypes.
doi:10.1371/journal.pone.0043845
PMCID: PMC3430616  PMID: 22952786
12.  Purification and Characterization of a Novel Subtype A3 Botulinum Neurotoxin 
Botulinum neurotoxins (BoNTs) produced by Clostridium botulinum are of considerable importance due to their being the cause of human and animal botulism, their potential as bioterrorism agents, and their utility as important pharmaceuticals. Type A is prominent due to its high toxicity and long duration of action. Five subtypes of type A BoNT are currently recognized; BoNT/A1, -/A2, and -/A5 have been purified, and their properties have been studied. BoNT/A3 is intriguing because it is not effectively neutralized by polyclonal anti-BoNT/A1 antibodies, and thus, it may potentially replace BoNT/A1 for patients who have become refractive to treatment with BoNT/A1 due to antibody formation or other modes of resistance. Purification of BoNT/A3 has been challenging because of its low levels of production in culture and the need for innovative purification procedures. In this study, modified Mueller-Miller medium was used in place of traditional toxin production medium (TPM) to culture C. botulinum A3 (CDC strain) and boost toxin production. BoNT/A3 titers were at least 10-fold higher than those produced in TPM. A purification method was developed to obtain greater than 95% pure BoNT/A3. The specific toxicity of BoNT/A3 as determined by mouse bioassay was 5.8 × 107 50% lethal doses (LD50)/mg. Neutralization of BoNT/A3 toxicity by a polyclonal anti-BoNT/A1 antibody was approximately 10-fold less than the neutralization of BoNT/A1 toxicity. In addition, differences in symptoms were observed between mice that were injected with BoNT/A3 and those that were injected with BoNT/A1. These results indicate that BoNT/A3 has novel biochemical and pharmacological properties compared to those of other subtype A toxins.
doi:10.1128/AEM.07967-11
PMCID: PMC3346436  PMID: 22367089
13.  Recombinant botulinum neurotoxin A heavy chain-based delivery vehicles for neuronal cell targeting 
The long half-life of botulinum neurotoxin serotype A (BoNT/A) in cells poses a challenge in developing post-exposure therapeutics complementary to existing antitoxin strategies. Delivery vehicles consisting of the toxin heavy chain (HC), including the receptor-binding domain and translocation domain, connected to an inhibitory cargo offer a possible solution for rescuing intoxicated neurons in victims paralyzed from botulism. Here, we report the expression and purification of soluble recombinant prototype green fluorescent protein (GFP) cargo proteins fused to the entire BoNT/A-HC (residues 544–1295) in Escherichia coli with up to a 40 amino acid linker inserted between the cargo and BoNT/A-HC vehicle. We show that these GFP-HC fusion proteins are functionally active and readily taken up by cultured neuronal cells as well as by neuronal cells in mouse motor nerve endings.
doi:10.1093/protein/gzq093
PMCID: PMC3038457  PMID: 21051321
delivery vehicle; cargo; neurotoxin; antitoxin
14.  Pharmacophore Refinement Guides the Rational Design of Nanomolar-Range Inhibitors of the Botulinum Neurotoxin Serotype A Metalloprotease 
ACS medicinal chemistry letters  2010;1(7):301-305.
Botulinum neurotoxins (BoNTs) are the deadliest of microbial toxins. The enzymes’ Zinc(II) metalloprotease, referred to as the light chain (LC) component, inhibits acetylcholine release into neuromuscular junctions, resulting in the disease botulism. Currently, no therapies counter BoNT poisoning post-neuronal intoxication; however, it is hypothesized that small molecules may be used to inhibit BoNT LC activity in the neuronal cytosol. Herein, we describe the pharmacophore-based design and chemical synthesis of potent (non-Zinc(II) chelating) small molecule (non-peptidic) inhibitors (SMNPIs) of the BoNT serotype A LC (the most toxic of the BoNT serotype LCs). Specifically, the three-dimensional superimpositions of 2-[4-(4-amidinephenoxy)-phenyl]-indole-6-amidine-based SMNPI regioisomers (Ki = 0.600 μM (± 0.100 μM)), with a novel lead bis-[3-amide-5-(imidazolino)-phenyl]-terephthalamide (BAIPT)-based SMNPI (Ki = 8.52 μM (± 0.53 μM)), resulted in a refined 4-zone pharmacophore. The refined model guided the design of BAIPT-based SMNPIs possessing Ki values = 0.572 μM (± 0.041 μM) and 0.900 μM (± 0.078 μM).
doi:10.1021/ml100056v
PMCID: PMC2992458  PMID: 21116458
botulinum neurotoxin; small molecule inhibitor; gas-phase pharmacophore; rational design; biothreat agent
15.  Fine and domain-level epitope mapping of botulinum neurotoxin type A neutralizing antibodies by yeast surface display 
Journal of molecular biology  2006;365(1):196-210.
Botulinum neurotoxin (BoNT), the most poisonous substance known, causes naturally occurring human disease (botulism) and is one of the top six biothreat agents. Botulism is treated with polyclonal antibodies produced in horses which are associated with a high incidence of systemic reactions. Human monoclonal antibodies (mAbs) are under development as a safer therapy. Identifying neutralizing epitopes on BoNTs is an important step in generating neutralizing mAbs, and also has implications for vaccine development. Here we show that the three domains of BoNT serotype A (BoNT/A) can be displayed on the surface of yeast and used to epitope map six mAbs to the toxin domains they bind. The use of yeast obviates the need to express and purify each domain, and it should prove possible to display domains of other BoNT subtypes and serotypes for epitope mapping. Using a library of yeast displayed BoNT/A binding domain (HC) mutants and selecting for loss of binding, the fine epitopes of three neutralizing BoNT/A mAbs were identified. Two mAbs bind the C-terminal subdomain of HC, with one binding near the toxin sialoganglioside binding site. The most potently neutralizing mAb binds the N-terminal subdomain of HC, in an area not previously thought to be functionally important. Modeling the epitopes shows how all three mAbs could bind BoNT/A simultaneously and may partially explain the dramatic synergy observed on in vivo toxin neutralization when these antibodies are combined. The results demonstrate how yeast display can be used for domain-level and fine mapping of conformational BoNT antibody epitopes and the mapping results identify three neutralizing BoNT/A epitopes.
doi:10.1016/j.jmb.2006.09.084
PMCID: PMC1994578  PMID: 17059824
epitope mapping; yeast surface display; botulinum neurotoxin; affinity; alanine scanning mutagenesis
16.  Simultaneous and sensitive detection of six serotypes of botulinum neurotoxin using enzyme-linked immunosorbent assay-based protein antibody microarrays 
Analytical biochemistry  2012;430(2):185-192.
Botulinum neurotoxins (BoNTs), produced by Clostridium botulinum, are a group of seven (A–G) immunologically distinct proteins and cause the paralytic disease botulism. These toxins are the most poisonous substances known to humans and are potential bioweapon agents. Therefore, it is necessary to develop highly sensitive assays for the detection of BoNTs in both clinical and environmental samples. In the current study, we have developed an enzyme-linked immunosorbent assay (ELISA)-based protein antibody microarray for the sensitive and simultaneous detection of BoNT serotypes A, B, C, D, E, and F. With engineered high-affinity antibodies, the BoNT assays have sensitivities in buffer ranging from 1.3 fM (0.2 pg/ml) to 14.7 fM (2.2 pg/ml). Using clinical and food matrices (serum and milk), the microarray is capable of detecting BoNT serotypes A to F to similar levels as in standard buffer. Cross-reactivity between assays for individual serotype was also analyzed. These simultaneous, rapid, and sensitive assays have the potential to measure botulinum toxins in a high-throughput manner in complex clinical, food, and environmental samples.
doi:10.1016/j.ab.2012.08.021
PMCID: PMC3589981  PMID: 22935296
Botulinum neurotoxin; Biodefense; ELISA; Protein microarray; Antibody; High-throughput assays
17.  Recombinant Holotoxoid Vaccine against Botulism▿  
Infection and Immunity  2007;76(1):437-442.
The botulinum neurotoxins (BoNT) are the most toxic proteins for humans and designated “Category A Select Agents.” The current vaccine against botulism is in limited supply, and there is a need to develop new vaccine strategies. A recombinant BoNT/A toxoid was produced in Clostridium botulinum that contained a double amino acid substitution, R363A Y365F (termed BoNT/ARYM). BoNT/ARYM was noncatalytic for SNAP25 and nontoxic for mice. Immunization with BoNT/ARYM protected mice from challenge at levels that were similar to chemically inactivated BoNT/A toxoid. BoNT/ARYM elicited an immune response against the light-chain and heavy-chain components of the toxin. Neutralizing anti-BoNT/ARYM sera blocked BoNT toxicity in primary cortical neurons and blocked ganglioside binding by the heavy chain. BoNT/ARYM represents a viable vaccine candidate for a holotoxoid against botulism.
doi:10.1128/IAI.00843-07
PMCID: PMC2223665  PMID: 17967862
18.  Efficient Serum Clearance of Botulinum Neurotoxin Achieved Using a Pool of Small Antitoxin Binding Agents▿  
Infection and Immunity  2009;78(2):756-763.
Antitoxins for botulinum neurotoxins (BoNTs) and other toxins are needed that can be produced economically with improved safety and shelf-life properties compared to conventional therapeutics with large-animal antisera. Here we show that protection from BoNT lethality and rapid BoNT clearance through the liver can be elicited in mice by administration of a pool of epitope-tagged small protein binding agents together with a single anti-tag monoclonal antibody (MAb). The protein binding agents used in this study were single-chain Fv domains (scFvs) with high affinity for BoNT serotype A (BoNT/A). The addition of increasing numbers of differently tagged scFvs synergistically increased the level of protection against BoNT/A. It was not necessary that any of the BoNT/A binding agents possess toxin-neutralizing activity. Mice were protected from a dose equivalent to 1,000 to 10,000 50% lethal doses (LD50) of BoNT/A when given three or four different anti-BoNT scFvs, each fused to an E-tag peptide, and an anti-E-tag IgG1 MAb. Toxin protection was enhanced when an scFv contained two copies of the E tag. Pharmacokinetic studies demonstrated that BoNT/A was rapidly cleared from the sera of mice given a pool of anti-BoNT/A scFvs and an anti-tag MAb but not from the sera of mice given scFvs alone or anti-tag MAb alone. The scFv pool and anti-tag MAb protected mice from lethality when administered up to 2 h following exposure of mice to a dose equivalent to 10 LD50 of BoNT/A. These results suggest that it will be possible to rapidly and economically develop and produce therapeutic antitoxins consisting of pools of tagged binding agents that are administered with a single, stockpiled anti-tag MAb.
doi:10.1128/IAI.01084-09
PMCID: PMC2812214  PMID: 19917718
19.  MOLECULAR BASIS OF ACTIVATION OF ENDOPEPTIDASE ACTIVITY OF BOTULINUM NEUROTOXIN TYPE E 
Biochemistry  2010;49(11):2510-2519.
Botulinum neurotoxins (BoNTs) are a group of large proteins that are responsible for the clinical syndrome of botulism. The seven immunologically distinct serotypes of BoNTs (A-G), each produced by various strains of Clostridium botulinum, act on the neuromuscular junction by blocking the release of the neurotransmitter acetylcholine thereby resulting in flaccid muscle paralysis. BoNTs are synthesized as single inactive polypeptide chains that are cleaved by endogenous or exogenous proteases to generate the active di-chain form of the toxin. Nicking of the single chain BoNT/E to the di-chain form is associated with 100-fold increase in toxicity. Here we investigated the activation mechanism of botulinum neurotoxin type E upon nicking and subsequent reduction of disulfide bond. It was observed that nicking of BoNT/E significantly enhances its endopeptidase activity and that at the physiological temperature of 37 °C, the reduced form of nicked BoNT/E adopts a dynamically flexible conformation resulting from the exposure of hydrophobic segments and facilitating optimal cleavage of its substrate SNAP-25. Such reduction induced increase in the flexibility of the polypeptide folding provides a rationale for the mechanism of BoNT/E endopeptidase against its intracellular substrate, SNAP-25, and complements current understanding of the mechanistics of interaction between the substrate and BoNT endopeptidase.
doi:10.1021/bi902096r
PMCID: PMC3720690  PMID: 20178376
botulinum neurotoxin; botulism; endopeptidase; SNAP-25; light chain
20.  The botulinum toxin complex meets E-cadherin on the way to its destination 
Cell Adhesion & Migration  2011;5(1):34-36.
Botulinum neurotoxin (BoNT) causes the disease botulism, which is characterized by flaccid paralysis in humans and animals. The metalloprotease activity of BoNT inhibits neurotransmitter release at neuro-muscular junctions. In most cases, poisoning occurs when BoNT is ingested. Therefore, BoNT must pass through the epithelial barrier of the gastrointestinal tract to enter the systemic circulation and reach the target site. BoNT forms large protein complexes by associating with non-toxic components referred to as non-toxic non-hemagglutinin (NTNH) and hemagglutinin (HA). These proteins protect BoNT from the low pH and proteases in the digestive tract. We recently determined that HA has an unexpected function of disrupting the intercellular epithelial barrier by directly binding to E-cadherin. HA binds to E-cadherin and disrupts its function in a species-specific manner, and this interaction is essential to disrupt tight junctions. This activity is thought to facilitate the absorption of BoNT through the paracellular route of the intestinal epithelium in susceptible species.
doi:10.4161/cam.5.1.13574
PMCID: PMC3038094  PMID: 20935473
botulinum toxin; Clostridium botulinum; hemagglutinin; food-borne botulism; E-cadherin; epithelial barrier; tight junction
21.  Detection of Botulinum Neurotoxin Serotype B at Sub Mouse LD50 Levels by a Sandwich Immunoassay and Its Application to Toxin Detection in Milk 
PLoS ONE  2010;5(6):e11047.
Background
Botulinum neurotoxin (BoNT), the causative agent of botulism, a serious neuroparylatic disease, is produced by the anaerobic bacterium Clostridium botulinum and consists of a family of seven serotypes (A-H). We previously reported production of high-affinity monoclonal antibodies to BoNT serotype A.
Methods and Findings
Recombinant peptide fragments of the light chain, the transmembrane and receptor-binding domains of the heavy chain of botulinum neurotoxin type B (BoNT/B) were expressed in Escherichia coli as GST-fusion proteins and purified. These proteins were used to immunize BALB/cJ mice for the generation of monoclonal antibodies (mAbs). Antibody-producing hybridomas were detected using either a direct binding ELISA binding to plate-immobilized BoNT/B, or with a capture-capture ELISA whereby the capacity of the antibody to capture BoNT/B from solution was tested. A total of five mAbs were selected, two of which bound the toxin light chain and three bound the receptor-binding domain of BoNT/B heavy chain. MAb MCS6-27 was identified via capture-capture ELISA and was the only mAb able to bind BoNT/B in solution under physiological conditions. MAbs F24-1, F26-16, F27-33 and F29-40 were identified via direct binding ELISA, and were able to capture BoNT/B in solution only in the presence of 0.5–0.9 mM sodium dodecyl sulphate (SDS). MAb MCS6-27 and an anti-BoNT/B polyclonal antibody were incorporated into a sandwich ELISA that did not require SDS.
Conclusions
We report here the generation of monoclonal antibodies to serotype B and the subsequent development of a sensitive sandwich immunoassay. This immunoassay has a detection limit of 100 fg BoNT/B, fifty times more sensitive than the mouse bioassay detection limit of 5 pg BoNT/B. Additionally, this assay detected as little as 39 pg/mL of toxin in skim, 2% and whole milk.
doi:10.1371/journal.pone.0011047
PMCID: PMC2883556  PMID: 20548779
22.  Association of Botulinum neurotoxins with synaptic vesicle protein complexes 
Botulinum neurotoxins (BoNTs) elicit flaccid paralysis by cleaving SNARE proteins within peripheral neurons. BoNTs are classified into seven serotypes, termed A-G, based upon antibody cross neutralization. Clostridia produce BoNTs are single chain toxins that are cleaved into a di-chain protein that comprises an N-terminal zinc metalloprotease domain that is linked by a disulfide bond to the C-terminal translocation/receptor binding domain. BoNT/A and BoNT/B utilize synaptic vesicle protein 2 (SV2) and synaptotagmin, respectively, as receptors for entry into neurons. Using affinity chromatography, BoNT/A and BoNT/B were found to bind a synaptic vesicle protein complex in CHAPS extracts of synaptic vesicles. Mass spectroscopy identified synaptic vesicle protein 2, synaptotagmin I, synaptophysin, vesicle-associated membrane protein 2, and the vacuolar ATPase-proton pump as components of the BoNT - synaptic vesicle protein complex. BoNT/A and BoNT/B possessed unique density gradient profiles when bound to synaptic vesicle protein complexes. The identification of BoNT/A and BoNT/B bound to synaptic vesicle protein complexes provides insight into the interactions of BoNT and neuronal receptors.
doi:10.1016/j.toxicon.2009.01.040
PMCID: PMC2730980  PMID: 19362106
Botulinum neurotoxins; SV2; synaptotagmin; VAMP-2
23.  Interaction of Botulinum Toxin with the Epithelial Barrier 
Botulinum neurotoxin (BoNT) is a protein toxin (~150 kDa), which possesses a metalloprotease activity. Food-borne botulism is manifested when BoNT is absorbed from the digestive tract to the blood stream and enters the peripheral nerves, where the toxin cleaves core proteins of the neuroexocytosis apparatus and elicits the inhibition of neurotransmitter release. The initial obstacle to orally ingested BoNT entering the body is the epithelial barrier of the digestive tract. Recent cell biology and molecular biology studies are beginning to elucidate the mechanism by which this large protein toxin crosses the epithelial barrier. In this review, we provide an overview of the structural features of botulinum toxins (BoNT and BoNT complex) and the interaction of these toxins with the epithelial barrier.
doi:10.1155/2010/974943
PMCID: PMC2822237  PMID: 20169001
24.  Protective immunity against botulism provided by a single dose vaccination with an adenovirus-vectored vaccine 
Vaccine  2007;25(43):7540-7548.
Botulinum neurotoxins cause botulism, a neuroparalytic disease in humans and animals. We constructed a replication-incompetent adenovirus encoding a synthesized codon-optimized gene for expression of the heavy chain C-fragment (HC50) of botulinum neurotoxin type C (BoNT/C). This recombinant human serotype 5 adenoviral vector (Ad5) was evaluated as a genetic vaccine candidate against botulism caused by BoNT/C in a mouse model. A one-time intramuscular injection with 105 to 2 × 107 pfu of adenoviral vectors elicited robust serum antibody responses against HC50 of BoNT/C as assessed by ELISA. Immune sera showed high potency in neutralizing the active BoNT/C in vitro. After a single dose of 2 × 107 pfu adenoviral vectors, the animals were completely protected against intraperitoneal challenge with 100 × MLD50 of active BoNT/C. The protective immunity appeared to be vaccine dose-dependent. The anti-toxin protective immunity could last for at least 7 months without a booster injection. In addition, we observed that pre-existing immunity to the wild type Ad5 in the host had no significant influence on the protective efficacy of vaccination. The data suggest that an adenovirus-vectored genetic vaccine is a highly efficient prophylaxis candidate against botulism.
doi:10.1016/j.vaccine.2007.08.035
PMCID: PMC2077857  PMID: 17897756
Botulism Vaccine; Protective immunity; Replication-incompetent adenovirus
25.  Identification of a Natural Product Antagonist against the Botulinum Neurotoxin Light Chain Protease 
ACS medicinal chemistry letters  2010;1(6):268-272.
Botulinum neurotoxins (BoNTs) are the etiological agents responsible for botulism, a disease characterized by peripheral neuromuscular blockade and a characteristic flaccid paralysis of humans. BoNTs are the most lethal known poisons affecting humans and has been recognized as a potential bioterrorist threat. Current treatments for botulinum poisoning are predominately prophylactic in nature relying on passive immunization with antitoxins. Inhibition of the BoNT light chain metalloprotease (LC) has emerged as a new therapeutic strategy for the treatment of botulism that may provide an effective post-exposure remedy. A high-throughput screening effort against the light chain of BoNT serotype A (LC/A) was conducted with the John Hopkins Clinical Compound Library comprised of over 1,500 existing drugs. Lomofungin, a natural product first isolated in the late 1960’s, was identified as an inhibitor of LC/A, displaying classical noncompetitive inhibition kinetics with a Ki of 6.7 ± 0.7 µM. Inhibitor combination studies reveal that lomofungin binding is nonmutually exclusive (synergistic). The inhibition profile of lomofungin has been delineated by the use of both an active site inhibitor, 2,4-dichlorocinnamic hydroxamate, and a noncompetitive inhibitor d-chicoric acid; the mechanistic implications of these observations are discussed. Lastly, cellular efficacy was investigated using a rat primary cell model which demonstrated that lomofungin can protect against SNAP-25 cleavage, the intracellular protein target of LC/A.
doi:10.1021/ml100074s
PMCID: PMC2955888  PMID: 20959871
Botulinum neurotoxin; zinc-dependent metalloprotease; high-throughput screening; small molecule inhibitor; natural product

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