The Light Chain Defines the Duration of Action of Botulinum Toxin Serotype A Subtypes.

Botulinum neurotoxin (BoNT) is the causative agent of botulism and a widely used pharmaceutical to treat a variety of neurological diseases. BoNTs are 150-kDa protein toxins organized into heavy chain (HC) and light chain (LC) domains linked by a disulfide bond. The HC selectively binds to neurons and aids cell entry of the enzymatically active LC. There are seven immunological BoNT serotypes (A to G); each serotype includes genetic variants, termed subtypes. Only two subtypes, BoNT/A1 and BoNT/B1, are currently used as therapeutics. BoNT serotype A (BoNT/A) subtypes A2 to A8 show distinct potency, duration of action, and pathology relative to BoNT/A1. Specifically, BoNT/A3 possesses shorter duration of action and elicits distinct symptoms in mice at high toxin doses. In this report, we analyzed the roles of LC and HC of BoNT/A3 for duration of action, neuronal cell entry, and mouse pathology by using clostridium-derived recombinant hybrid BoNTs consisting of reciprocal LC and HC (BoNTA1/A3 and BoNTA3/A1). Hybrid toxins were processed in their expression host to a dichain BoNT consisting of LC and HC linked via a disulfide bond. The LC and HC defined BoNT potency in mice and BoNT toxicity for cultured neuronal cells, while the LC defined the duration of BoNT action in cell and mouse models. Protein alignment identified a previously unrecognized region within the LC subtype A3 (LC/A3) relative to the other LC serotype A (LC/A) subtypes (low primary acid homology [LPH]) that correlated to intracellular LC localization. This study shows the utility of recombinant hybrid BoNTs with new therapeutic potential, while remaining sensitive to antitoxins and therapies to native BoNT.IMPORTANCE Botulinum neurotoxins are the most potent protein toxins for humans and potential bioterrorism threats, but they are also widely used as pharmaceuticals. Within the large family of BoNTs, only two subtypes are currently used as pharmaceuticals, with a large number of BoNT subtypes remaining as untapped potential sources for unique pharmaceuticals. Here, two recombinant hybrid toxins were engineered, consisting of domains from two BoNT subtypes that possess distinct duration of action and activity in human neurons and mice. We define the functional domains responsible for BoNT action and demonstrate creation of functional hybrid BoNTs with new therapeutic potential, while remaining sensitive to antitoxins and therapies to native BoNT.

Purification and Characterization of Botulinum Neurotoxin FA from a Genetically Modified Clostridium botulinum Strain.

Botulinum neurotoxins (BoNTs), produced by neurotoxigenic clostridial species, are the cause of the severe disease botulism in humans and animals. Early research on BoNTs has led to their classification into seven serotypes (serotypes A to G) based upon the selective neutralization of their toxicity in mice by homologous antibodies. Recently, a report of a potential eighth serotype of BoNT, designated "type H," has been controversial. This novel BoNT was produced together with BoNT/B2 in a dual-toxin-producing Clostridium botulinum strain. The data used to designate this novel toxin as a new serotype were derived from culture supernatant containing both BoNT/B2 and novel toxin and from sequence information, although data from two independent laboratories indicated neutralization by antibodies raised against BoNT/A1, and classification as BoNT/FA was proposed. The sequence data indicate a chimeric structure consisting of a BoNT/A1 receptor binding domain, a BoNT/F5 light-chain domain, and a novel translocation domain most closely related to BoNT/F1. Here, we describe characterization of this toxin purified from the native strain in which expression of the second BoNT (BoNT/B) has been eliminated. Mass spectrometry analysis indicated that the toxin preparation contained only BoNT/FA and confirmed catalytic activity analogous to that of BoNT/F5. The in vivo mouse bioassay indicated a specific activity of this toxin of 3.8 × 10(7) mouse 50% lethal dose (mLD50) units/mg, whereas activity in cultured human neurons was very high (50% effective concentration [EC50] = 0.02 mLD50/well). Neutralization assays in cells and mice both indicated full neutralization by various antibodies raised against BoNT/A1, although at 16- to 20-fold-lower efficiency than for BoNT/A1. IMPORTANCE Botulinum neurotoxins (BoNTs), produced by anaerobic bacteria, are the cause of the potentially deadly, neuroparalytic disease botulism. BoNTs have been classified into seven serotypes, serotypes A to G, based upon their selective neutralization by homologous antiserum, which is relevant for clinical and diagnostic purposes. Even though supportive care dramatically reduces the death rate of botulism, the only pharmaceutical intervention to reduce symptom severity and recovery time is early administration of antitoxin (antiserum raised against BoNTs). A recent report of a novel BoNT serotype, serotype H, raised concern of a "treatment-resistant" and highly potent toxin. However, the toxin's chimeric structure and characteristics indicate a chimeric BoNT/FA. Here we describe the first characterization of this novel toxin in purified form. BoNT/FA was neutralized by available antitoxins, supporting classification as BoNT/FA. BoNT/FA required proteolytic activation to achieve full toxicity and had relatively low potency in mice compared to BoNT/A1 but surprisingly high activity in cultured neurons.

A Novel Botulinum Neurotoxin, Previously Reported as Serotype H, Has a Hybrid-Like Structure With Regions of Similarity to the Structures of Serotypes A and F and Is Neutralized With Serotype A Antitoxin.

Botulism is a potentially fatal paralytic disease caused by the action of botulinum neurotoxin (BoNT) on nerve cells. There are 7 known serotypes (A-G) of BoNT and up to 40 genetic variants. Clostridium botulinum strain IBCA10-7060 was recently reported to produce BoNT serotype B (BoNT/B) and a novel BoNT, designated as BoNT/H. The BoNT gene (bont) sequence of BoNT/H was compared to known bont sequences. Genetic analysis suggested that BoNT/H has a hybrid-like structure containing regions of similarity to the structures of BoNT/A1 and BoNT/F5. This novel BoNT was serologically characterized by the mouse neutralization assay and a neuronal cell-based assay. The toxic effects of this hybrid-like BoNT were completely eliminated by existing serotype A antitoxins, including those contained in multivalent therapeutic antitoxin products that are the mainstay of human botulism treatment.

Human Induced Pluripotent Stem Cell Derived Neuronal Cells Cultured on Chemically-Defined Hydrogels for Sensitive In Vitro Detection of Botulinum Neurotoxin.

Botulinum neurotoxin (BoNT) detection provides a useful model for validating cell-based neurotoxicity screening approaches, as sensitivity is dependent on functionally competent neurons and clear quantitative endpoints are available for correlating results to approved animal testing protocols. Here, human induced pluripotent stem cell (iPSC)-derived neuronal cells were cultured on chemically-defined poly(ethylene glycol) (PEG) hydrogels formed by "thiol-ene" photopolymerization and tested as a cell-based neurotoxicity assay by determining sensitivity to active BoNT/A1. BoNT/A1 sensitivity was comparable to the approved in vivo mouse bioassay for human iPSC-derived neurons and neural stem cells (iPSC-NSCs) cultured on PEG hydrogels or treated tissue culture polystyrene (TCP) surfaces. However, maximum sensitivity for BoNT detection was achieved two weeks earlier for iPSC-NSCs that were differentiated and matured on PEG hydrogels compared to TCP. Therefore, chemically-defined synthetic hydrogels offer benefits over standard platforms when optimizing culture conditions for cell-based screening and achieve sensitivities comparable to an approved animal testing protocol.

Activity of botulinum neurotoxin type D (strain 1873) in human neurons.

Botulinum Neurotoxin type D (BoNT/D) causes periodic outbreaks of botulism in cattle and horses, but is rarely associated with human botulism. Previous studies have shown that humans responded poorly to peripheral injection of up to 10U of BoNT/D. Isolated human pyramidalis muscle preparations were resistant to BoNT/D, whereas isolated human intercostal muscle preparations responded to BoNT/D similarly as to other BoNT serotypes. In vitro data indicate that BoNT/D does not cleave human VAMP1 efficiently, and differential expression of the VAMP 1 and 2 isoforms may be responsible for the above observations. Here we examined sensitivity of cultured human neurons derived from human induced pluripotent stem cells to BoNT/D. Our data indicate that BoNT/D can enter and cleave VAMP 2 in human neurons, but at significantly lower efficiency than other BoNT serotypes. In addition, BoNT/D had a short duration of action in the cultured neurons, similar to that of BoNT/E. In vivo analyses indicated a slower time to death in mice, as well as a later onset and shorter duration of action than BoNT/A1. Finally, examination of BoNT/D activity in various rodent and human cell models resulted in dramatic differences in sensitivity, indicating a unique cell entry mechanism of BoNT/D.

Prevalence of toxin-producing Clostridium botulinum associated with the macroalga Cladophora in three Great Lakes: growth and management.

The reemergence of avian botulism caused by Clostridium botulinum type E has been observed across the Great Lakes in recent years. Evidence suggests an association between the nuisance algae, Cladophora spp., and C. botulinum in nearshore areas of the Great Lakes. However, the nature of the association between Cladophora and C. botulinum is not fully understood due, in part, to the complex food web interactions in this disease etiology. In this study, we extensively evaluated their association by quantitatively examining population size and serotypes of C. botulinum in algal mats collected from wide geographic areas in lakes Michigan, Ontario, and Erie in 2011-2012 and comparing them with frequencies in other matrices such as sand and water. A high prevalence (96%) of C. botulinum type E was observed in Cladophora mats collected from shorelines of the Great Lakes in 2012. Among the algae samples containing detectable C. botulinum, the population size of C. Botulinum type E was 10(0)-10(4) MPN/g dried algae, which was much greater (up to 10(3) fold) than that found in sand or the water column, indicating that Cladophora mats are sources of this pathogen. Mouse toxinantitoxin bioassays confirmed that the putative C. botulinum belonged to the type E serotype. Steam treatment was effective in reducing or eliminating C. botulinum type E viable cells in Cladophora mats, thereby breaking the potential transmission route of toxin up to the food chain. Consequently, our data suggest that steam treatment incorporated with a beach cleaning machine may be an effective treatment of Cladophora-borne C. botulinum and may reduce bird mortality and human health risks.

Characterization of botulinum neurotoxin A subtypes 1 through 5 by investigation of activities in mice, in neuronal cell cultures, and in vitro.

Botulinum neurotoxins (BoNTs) are synthesized by Clostridium botulinum and exist as seven immunologically distinct serotypes designated A through G. For most serotypes, several subtypes have now been described based on nominal differences in the amino acid sequences. BoNT/A1 is the most well-characterized subtype of the BoNT/A serotype, and many of its properties, including its potency, its prevalence as a food poison, and its utility as a pharmaceutical, have been thoroughly studied. In contrast, much remains unknown of the other BoNT/A subtypes. In this study, BoNT/A subtype 1 (BoNT/A1) to BoNT/A5 were characterized utilizing a mouse bioassay, an in vitro cleavage assay, and several neuronal cell-based assays. The data indicate that BoNT/A1 to -5 have distinct in vitro and in vivo toxicological properties and that, unlike those for BoNT/A1, the neuronal and mouse results for BoNT/A2 to -5 do not correlate with their enzymatic activity. These results indicate that BoNT/A1 to -5 have distinct characteristics, which are of importance for a greater understanding of botulism and for pharmaceutical applications.

Model for studying Clostridium botulinum neurotoxin using differentiated motor neuron-like NG108-15 cells.

Cancerous cell lines have traditionally shown low sensitivity to laboratory or pharmaceutical preparations of botulinum neurotoxin. The work presented here demonstrates that the mouse neuroblastoma/rat glioma hybrid cell line NG108-15 is capable of more sensitively detecting BoNT/A1 than any cell line previously described. This cell line has previously been described to have motor neuron like characteristics, therefore making it a good model to study BoNTs. Differentiation of NG108-15 cells in serum-free medium containing retinoic acid and purmorphamine dramatically increased sensitivity of the neurons to BoNT/A (EC(50) = ~16 LD(50) U). Additional pre-treatment with triasialoganglioside GT1B prior to toxin exposure reduced the EC(50) further to ~11 LD(50) U. Co-culture of the neurons with C2C12 myotubes also significantly increased BoNT/A sensitivity of NG108-15 cells (EC(50) = 26 U) in the absence of differentiation factors.

Sensitive and quantitative detection of botulinum neurotoxin in neurons derived from mouse embryonic stem cells.

Botulinum neurotoxins (BoNTs), the most poisonous protein toxins known, represent a serious bioterrorism threat but are also used as a unique and important bio-pharmaceutical to treat an increasing myriad of neurological disorders. The only currently accepted detection method by the United States Food and Drug Administration for biological activity of BoNTs and for potency determination of pharmaceutical preparations is the mouse bioassay (MBA). Recent advances have indicated that cell-based assays using primary neuronal cells can provide an equally sensitive and robust detection platform as the MBA to reliably and quantitatively detect biologically active BoNTs. This study reports for the first time a BoNT detection assay using mouse embryonic stem cells to produce a neuronal cell culture. The data presented indicate that this assay can reliably detect BoNT/A with a similar sensitivity as the MBA.

Botulinum neurotoxin subtype A2 enters neuronal cells faster than subtype A1.

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.

Expression of the Clostridium botulinum A2 neurotoxin gene cluster proteins and characterization of the A2 complex.

Clostridium botulinum subtype A2 possesses a botulinum neurotoxin type A (BoNT/A) gene cluster consisting of an orfX cluster containing open reading frames (ORFs) of unknown functions. To better understand the association between the BoNT/A2 complex proteins, first, the orfX cluster proteins (ORFX1, ORFX3, P47, and the middle part of NTNH) from C. botulinum A2 strain Kyoto F and NTNH of A1 strain ATCC 3502 were expressed by using either an Escherichia coli or a C. botulinum expression system. Polyclonal antibodies against individual orfX cluster proteins were prepared by immunizing a rabbit and mice against the expressed proteins. Antibodies were then utilized as probes to determine which of the A2 orfX cluster genes were expressed in the native A2 culture. N-terminal protein sequencing was also employed to specifically detect ORFX2. Results showed that all of the neurotoxin cluster proteins, except ORFX1, were expressed in the A2 culture. A BoNT/A2 toxin complex (TC) was purified which showed that C. botulinum A2 formed a medium-size (300-kDa) TC composed of BoNT/A2 and NTNH without any of the other OrfX cluster proteins. NTNH subtype-specific immunoreactivity was also discovered, allowing for the differentiation of subtypes based on cluster proteins associated with BoNT.

Catalytic properties of botulinum neurotoxin subtypes A3 and A4.

Botulinum toxins (BoNT) are zinc proteases (serotypes A-G) which cause flaccid paralysis through the cleavage of SNARE proteins within motor neurons. BoNT/A was originally organized into two subtypes, BoNT/A1 and BoNT/A2, which are approximately 95% homologous and possess similar catalytic activities. Subsequently, two additional subtypes were identified, BoNT/A3 (Loch Maree) and BoNT/A4 (657Ba), which are 81 and 88% homologous with BoNT/A1, respectively. Alignment studies predicted that BoNT/A3 and BoNT/A4 were sufficiently different from BoNT/A1 to affect SNAP25 binding and cleavage. Recombinant light chain (LC) of BoNT/A3 (LC/A3) and BoNT/A4 (LC/A4) were subjected to biochemical analysis. LC/A3 cleaved SNAP25 at 50% of the rate of LC/A1 but cleaved SNAPtide at a faster rate than LC/A1, while LC/A4 cleaved SNAP25 and SNAPtide at slower rates than LC/A1. LC/A3 and LC/A4 had similar K(m) values for SNAP25 relative to LC/A1, while the k(cat) for LC/A4 was 10-fold slower than that for LC/A1, suggesting a defect in substrate cleavage. Neither LC/A3 nor LC/A4 possessed autocatalytic activity, a property of LC/A1 and LC/A2. Thus, the four subtypes of BoNT/A bind SNAP25 with similar affinity but have different catalytic capacities for SNAP25 cleavage, SNAPtide cleavage, and autocatalysis. The catalytic properties identified among the subtypes of LC/A may influence strategies for the development of small molecule or peptide inhibitors as therapies against botulism.

Subunit vaccine against the seven serotypes of botulism.

Botulinum neurotoxins (BoNTs) are the most toxic proteins for humans and are classified as category A toxins. There are seven serotypes of BoNTs defined by the lack of cross-serotype toxin neutralization. Thus, an effective vaccine must neutralize each BoNT serotype. BoNTs are organized as dichain A-B toxins, where the N-terminal domain (light chain) is a zinc metalloprotease targeting soluble NSF attachment receptor proteins that is linked to the C-terminal domain (heavy chain [HC]) by a disulfide bond. The HC comprises a translocation domain and a C-terminal receptor binding domain (HCR). HCRs of the seven serotypes of BoNTs (hepta-HCR) were engineered for expression in Escherichia coli, and each HCR was purified from E. coli lysates. Immunization of mice with the E. coli-derived hepta-serotype HCR vaccine elicited an antibody response to each of the seven BoNT HCRs and neutralized challenge by 10,000 50% lethal doses of each of the seven BoNT serotypes. A solid-phase assay showed that the anti-hepta-serotype HCR sera inhibited the binding of HCR serotypes A and B to the ganglioside GT1b, the first step in BoNT intoxication of neurons. This is the first E. coli-derived vaccine that effectively neutralizes each of the seven BoNT serotypes.

Recombinant holotoxoid vaccine against botulism.

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/A(RYM)). BoNT/A(RYM) was noncatalytic for SNAP25 and nontoxic for mice. Immunization with BoNT/A(RYM) protected mice from challenge at levels that were similar to chemically inactivated BoNT/A toxoid. BoNT/A(RYM) elicited an immune response against the light-chain and heavy-chain components of the toxin. Neutralizing anti-BoNT/A(RYM) sera blocked BoNT toxicity in primary cortical neurons and blocked ganglioside binding by the heavy chain. BoNT/A(RYM) represents a viable vaccine candidate for a holotoxoid against botulism.

Characterization of the antibody response to the receptor binding domain of botulinum neurotoxin serotypes A and E.

Clostridium botulinum neurotoxins (BoNTs) are the most toxic proteins for humans. The current clostridial-derived vaccines against BoNT intoxication have limitations including production and accessibility. Conditions were established to express the soluble receptor binding domain (heavy-chain receptor [HCR]) of BoNT serotypes A and E in Escherichia coli. Sera isolated from mice and rabbits immunized with recombinant HCR/A1 (rHCR/A1) from the classical type A-Hall strain (ATCC 3502) (BoNT/A1) and rHCR/E from BoNT serotype E Beluga (BoNT/E(B)) neutralized the homologous serotype of BoNT but displayed differences in cross-recognition and cross-protection. Enzyme-linked immunosorbent assay and Western blotting showed that alpha-rHCR/A1 recognized epitopes within the C terminus of the HCR/A and HCR/E, while alpha-rHCR/E recognized epitopes within the N terminus or interface between the N and C termini of the HCR proteins. alpha-rHCR/E(B) sera possessed detectable neutralizing capacity for BoNT/A1, while alpha-rHCR/A1 did not neutralize BoNT/E. rHCR/A was an effective immunogen against BoNT/A1 and the Kyoto F infant strain (BoNT/A2), but not BoNT serotype E Alaska (BoNT/E(A)), while rHCR/E(B) neutralized BoNT/E(A), and under hyperimmunization conditions protected against BoNT/A1 and BoNT/A2. The protection elicited by rHCR/A1 to BoNT/A1 and BoNT/A2 and by rHCR/E(B) to BoNT/E(A) indicate that immunization with receptor binding domains elicit protection within sub-serotypes of BoNT. The protection elicited by hyperimmunization with rHCR/E against BoNT/A suggests the presence of common neutralizing epitopes between the serotypes E and A. These results show that a receptor binding domain subunit vaccine protects against serotype variants of BoNTs.