Purification, Potency, and Efficacy of the Botulinum Neurotoxin Type A Binding Domain from Pichia pastoris as a Recombinant Vaccine Candidate

Recombinant botulinum neurotoxin serotype A binding domain [BoNT/A(H_c)], expressed in Pichia pastoris, was developed as a vaccine candidate for preventing botulinum neurotoxin type A (BoNT/A) intoxication. After fermentation and cell disruption, BoNT/A(H_c) was purified by using a three-step chromatographic process consisting of expanded-bed chromatography, Mono S cation-exchange chromatography, and hydrophobic interaction chromatography. Two pools of immunogenic product were separated on the Mono S column and processed individually. Both products were more than 95% pure and indistinguishable by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blot analysis, and enzyme-linked immunosorbent assay (ELISA). Each protein was assayed for potency in mice at immunogen doses ranging from 2.4 ng to 10 μg, followed by challenge with 1,000 mouse intraperitoneal 50% lethal doses (i.p. LD₅₀) of BoNT/A. The calculated 50% effective dose for both peaks was approximately 0.1 μg/mouse. Peak 1 was evaluated further in a mouse efficacy assay. Mice were injected either once, twice, or three times at five different doses and subsequently challenged with 100,000 mouse i.p. LD₅₀ of BoNT/A. In general, multiple injections protected better than one, with complete or nearly complete protection realized at doses of ≥0.5 μg/mouse. Serum neutralization and ELISA titers were also determined. Tellingly, 82 of 83 mice with antibody titers of ≥1,600, as measured by ELISA, survived, but only 6 of 42 mice with titers of ≤100 survived. This work shows that the purified BoNT/A(H_c) produced was a highly effective immunogen, able to protect against a high challenge dose of neurotoxin.     

Evaluation of a recombinant Hc of Clostridium botulinum neurotoxin serotype F as an effective subunit vaccine

A new gene encoding the Hc domain of Clostridium botulinum neurotoxin serotype F (FHc) was designed and completely synthesized with oligonucleotides. A soluble recombinant Hc of C. botulinum neurotoxin serotype F was highly expressed in Escherichia coli with this synthetic FHc gene. Subsequently, the purified FHc was used to vaccinate mice and evaluate their survival against challenge with active botulinum neurotoxin serotype F (BoNT/F). After the administration of FHc protein mixed with Freund adjuvant via the subcutaneous route, a strong protective immune response was elicited in the vaccinated mice. Mice that were given two or three vaccinations with a dosage of 1 or 10 μg of FHc were completely protected against an intraperitoneal administration of 20,000 50% lethal doses (LD₅₀) of BoNT/F. The BoNT/F neutralization assay showed that the sera from these vaccinated mice contained high titers of protective antibodies. Furthermore, mice were vaccinated once, twice, or three times at four different dosages of FHc using Alhydrogel (Sigma) adjuvant via the intramuscular route and subsequently challenged with 20,000 LD₅₀ of neurotoxin serotype F. A dose response was observed in both the antibody titer and the protective efficacy with increasing dosage of FHc and number of vaccinations. Mice that received one injection of 5 μg or two injections of ≥0.04 μg of FHc were completely protected. These findings suggest that the recombinant FHc expressed in E. coli is efficacious in protecting mice against challenge with BoNT/F and that the recombinant FHc subunit vaccine may be useful in humans.     

The C-Terminal Heavy-Chain Domain of Botulinum Neurotoxin A Is Not the Only Site That Binds Neurons,as the N-Terminal Heavy-Chain Domain Also Plays a Very Active Role in Toxin-Cell Binding and Interactions

Botulinum neurotoxins (BoNTs) possess unique specificity for nerve terminals. They bind to the presynaptic membrane and then translocate intracellularly, where the light-chain endopeptidase cleaves the SNARE complex proteins, subverting the synaptic exocytosis responsible for acetylcholine release to the synaptic cleft. This inhibits acetylcholine binding to its receptor, causing paralysis. Binding, an obligate event for cell intoxication, is believed to occur through the heavy-chain C-terminal (H_C) domain. It is followed by toxin translocation and entry into the cell cytoplasm, which is thought to be mediated by the heavy-chain N-terminal (H_N) domain. Submolecular mapping analysis by using synthetic peptides spanning BoNT serotype A (BoNT/A) and mouse brain synaptosomes (SNPs) and protective antibodies against toxin from mice and cervical dystonia patients undergoing BoNT/A treatment revealed that not only regions of the H_C domain but also regions of the H_N domain are involved in the toxin binding process. Based on these findings, we expressed a peptide corresponding to the BoNT/A region comprising H_N domain residues 729 to 845 (H_N729–845). H_N729–845 bound directly to mouse brain SNPs and substantially inhibited BoNT/A binding to SNPs. The binding involved gangliosides GT1b and GD1a and a few membrane lipids. The peptide bound to human or mouse neuroblastoma cells within 1 min. Peptide H_N729–845 protected mice completely against a lethal BoNT/A dose (1.05 times the 100% lethal dose). This protective activity was obtained at a dose comparable to that of the peptide from positions 967 to 1296 in the H_C domain. These findings strongly indicate that H_N729–845 and, by extension, the H_N domain are fully programmed and equipped to bind to neuronal cells and in the free state can even inhibit the binding of the toxin.     

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.     

Bivalent Recombinant Vaccine for Botulinum Neurotoxin Types A and B Based on a Polypeptide Comprising Their Effector and Translocation Domains That Is Protective against the Predominant A and B Subtypes

The botulinum neurotoxins (BoNTs) are a large family of extremely potent, neuroparalytic, dichain proteins which act at the peripheral nervous system. The wide genetic diversity observed with this neurotoxin family poses a significant challenge for the development of an effective botulinum vaccine. The present study describes a vaccine development platform based on protein fragments representing the N-terminal two-thirds of each toxin molecule. These fragments, designated LH_N, comprise the light chain and translocation domains of each neurotoxin and are devoid of any neuron-binding activity. Using codon-optimized genes, LH_N fragments derived from BoNT serotypes A and B were expressed in Escherichia coli in high yield with >1 g of purified, soluble fragment recoverable from 4.5 liter-scale fermentations. The protective efficacy of LH_N/A was significantly enhanced by treatment with formaldehyde, which induced intramolecular cross-linking but virtually no aggregation of the fragment. A single immunization of the modified fragment protected mice from challenge with a 10³ 50% lethal dose (LD₅₀) of BoNT/A₁ with an 50% effective dose (ED₅₀) of 50 ng of the vaccine. In similar experiments, the LH_N/A vaccine was shown to protect mice against challenge with BoNT/A subtypes A₁, A₂, and A₃, which is the first demonstration of single-dose protection by a vaccine against the principal toxin subtypes of BoNT/A. The LH_N/B vaccine was also highly efficacious, giving an ED₅₀ of ∼140 ng to a challenge of 10³ LD₅₀ of BoNT/B₁. In addition, LH_N/B provided single-dose protection in mice against BoNT/B₄ (nonproteolytic toxin subtype).The clostridial neurotoxins include tetanus toxin and the seven antigenically different botulinum neurotoxins (BoNTs), all of which exert their action by blocking the calcium-mediated release of neurotransmitters (24). The BoNTs act principally on the peripheral nervous system, where they inhibit the release of acetylcholine at the neuromuscular junction, an action that results in a widespread descending flaccid paralysis and ultimately the syndrome botulism. Because of the high potencies of the BoNTs, they are considered potential reagents for bioterrorist use and are currently designated by the Centers for Disease Control and Prevention as category A biothreat agents (1).In their most active forms, the BoNTs consist of two subunits: a light chain (∼50 kDa) linked by a disulfide bond to a heavy chain (∼100 kDa). Structurally, these subunits are arranged into three distinct domains (17, 30): a 50-kDa H_C domain that consists of two subdomains (of which the C-terminal subdomain is involved in neuronal acceptor binding), a translocation domain represented by the N-terminal half of the heavy chain (H_N domain), and a light-chain, effector domain (LC). Collectively, these domains enable the BoNTs to bind and translocate to within the presynaptic nerve terminal (6), where they act, via highly specific, zinc-dependent protease actions within the LC domain, to disable the process of calcium-mediated transmitter release (24).While architecturally and mechanistically similar, the various serotypes of the BoNTs differ significantly in their primary structures (19) with the result that antibodies raised against one BoNT serotype offer no, or very little, protection against the biological action of another. Separate antigens are therefore required for each serotype to provide complete protection against the full spectrum of BoNTs. Vaccine development is further complicated by the occurrence of subtypes within most of the BoNT serotypes (13). For BoNT serotype A, for example, four subtypes have thus far been identified (designated BoNT/A₁ to BoNT/A₄) which display between 7 and 16% heterology in their primary nucleic acid sequences (2). These sequence variations occur primarily within surface-exposed regions on the molecule, thus maximizing their impact on antibody binding and neutralization and hence vaccine efficacy. Providing adequate cross-protection against the principal subtypes of each BoNT serotype must therefore be an important consideration in design of both vaccines and antibody-based therapeutics for the BoNTs.Current vaccines for the BoNTs consist of formaldehyde-inactivated toxin complexes which were first developed in the 1950s. Although these vaccines are effective, they require specialized high containment manufacturing facilities and are difficult and expensive to manufacture in large quantities (9). The initial design of recombinant vaccines was undertaken with the rationale of inhibiting a key facet of the biological activity of the BoNTs, such as receptor binding. Thus, first-generation recombinant vaccines under development are based on the receptor-binding domains (H_C fragments) of each BoNT. These fragments, produced in Pichia pastoris, have been shown to provide a protective immune response in mice and have recently entered clinical trials (3, 4, 27). The H_C fragments derived from the various BoNTs, however, differ markedly in their isoelectric points (pIs 5.7 to 9.1), which make formulation of a multivalent vaccine difficult. More recent studies indicate that antibodies directed against the light chain and the H_N region of the BoNT molecule can also provide a neutralizing immune response (5, 6).The LH_N fragment of the BoNTs is a polypeptide of ∼100 kDa consisting of the light-chain domain in close association with the translocation domain (Fig. ​(Fig.1).1). A polypeptide belt from the latter surrounds the light chain under nonreducing conditions. In initial studies, the LH_N fragment of BoNT/A was produced by prolonged trypsin digestion of the neurotoxin and shown to be a soluble, immunoreactive fragment (26). Subsequently, LH_N fragments from several BoNT serotypes have been produced by recombinant DNA technology and demonstrated to be useful as the core of a range of potential novel therapeutics (10, 29). In the present study, LH_N fragment-based vaccines for BoNT/A and BoNT/B are described. A derivative of the LH_N/A vaccine is shown to have exceptional efficacy in animal studies providing single-dose protection against BoNT/A subtypes A₁, A₂, and A₃. The LH_N/B vaccine is shown to provide protection against BoNT/B subtypes B₁ and B₄ (nonproteolytic).Open in a separate windowFIG. 1.Structure and function of the BoNTs. A diagram of the structure of BoNT/A shows the organization of the domains and the composition of the LH_N fragment. The H_C (binding) domain binds to neuronal receptors, after which the H_N (translocation) domain mediates the entry of the light chain (effector) into the nerve cell.     

Efficient Serum Clearance of Botulinum Neurotoxin Achieved Using a Pool of Small Antitoxin Binding Agents

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.Microbial toxins are the cause of many serious human diseases, and several of these toxins are listed among the NIAID category A and B priority pathogens. Specifically, botulinum neurotoxin (BoNT) is a category A threat, and ricin, epsilon toxin, Staphylococcus enterotoxin B, and Shiga toxins are category B threat agents. Other microbial toxins, such as those produced by Clostridium difficile, Clostridium tetani, Staphylococcus, Bordetella pertussis, and Corynebacterium diphtheriae, also cause serious human diseases. Toxins produced by some animals and plants, such as insects, spiders, snails, snakes, and jellyfish, also can cause human disease. Currently, most toxin-induced diseases are treated with antitoxins, usually polyclonal antisera produced in animals. These antitoxins are generally produced by immunizing large animals with a chemically inactivated form of the toxin or a nontoxic portion of the toxin that elicits polyclonal antisera which bind the holotoxin and prevent its uptake into cells and/or accelerate its clearance. While effective, antitoxins are often expensive to manufacture and problematic for quality control, and they have a limited shelf life. Furthermore, products derived from serum can cause serum sickness and may have pathogen contamination. There is thus a clear need for improved agents to prevent or treat intoxication.Botulism is a flaccid paralysis resulting from exposure to BoNT, usually by an oral route (reviewed in reference 24). This toxin is considered among the most dangerous biodefense threats because of its extreme potency, wide availability, relative ease of production, stability, and lack of specific treatment modalities (5). Seven different BoNT serotypes (A to G) are known to exist, and further subtype variants are found within some serotypes. Each BoNT serotype contains a 100-kDa heavy chain, responsible for transcytosis across mucosal membranes and for neuron receptor binding and internalization, and a 50-kDa light chain protease that cleaves one or more SNARE proteins to inactivate neurotransmitter exocytosis. Because antidote therapies do not exist, significant intoxication by BoNT will result in death due to paralysis of the muscles associated with respiration unless constant, intensive, and prolonged supportive care is provided.Botulism symptoms can be reduced or prevented if proper antitoxins are administered within about a day following exposure to moderate doses. While polyclonal antitoxin products are available to treat human botulism, it is widely accepted that improved antitoxin agents are badly needed (27). One approach showing promise is to replace polyclonal antisera with monoclonal antibodies (MAbs) (1, 2, 4, 9, 17, 20). This approach has been applied successfully in mice, using anti-BoNT MAbs (17). Although a single MAb was weakly protective, pools of two and three different MAbs led to synergistic improvements in efficacy. The requirement for perhaps 21 different MAbs to protect against all seven known BoNT serotypes and problems relating to high MAb development and production costs and limited shelf life are likely to hinder widespread stockpiling of such antitoxin agents. Here we provide evidence that a pool of small tagged antitoxin binding agents administered with a single anti-tag MAb protects mice with the same efficacy as that of polyclonal antitoxin sera. Therapeutic agents of this type should be more rapid to develop and more economical to produce and stockpile than conventional antitoxins.     

Inhalational Botulism in Rhesus Macaques Exposed to Botulinum Neurotoxin Complex Serotypes A1 and B1

A recombinant botulinum vaccine (rBV A/B) is being developed for protection against inhalational intoxication with botulinum neurotoxin (BoNT) complex serotype A, subtype A1 (BoNT/A1), and BoNT serotype B, subtype B1 (BoNT/B1). A critical component for evaluating rBV A/B efficacy will be the use of animal models in which the pathophysiology and dose-response relationships following aerosol exposure to well-characterized BoNT are thoroughly understood and documented. This study was designed to estimate inhaled 50% lethal doses (LD₅₀) and to estimate 50% lethal exposure concentrations relative to time (LCt₅₀) in rhesus macaques exposed to well-characterized BoNT/A1 and BoNT/B1. During the course of this study, clinical observations, body weights, clinical hematology results, clinical chemistry results, circulating neurotoxin levels, and telemetric parameters were documented to aid in the understanding of disease progression. The inhaled LD₅₀ and LCt₅₀ for BoNT/A1 and BoNT/B1 in rhesus macaques were determined using well-characterized challenge material. Clinical observations were consistent with the recognized pattern of botulism disease progression. A dose response was demonstrated with regard to the onset of these clinical signs for both BoNT/A1 and BoNT/B1. Dose-related changes in physiologic parameters measured by telemetry were also observed. In contrast, notable changes in body weight, hematology, and clinical chemistry parameters were not observed. Circulating levels of BoNT/B1 were detected in animals exposed to the highest levels of BoNT/B1; however, BoNT/A1 was not detected in the circulation at any aerosol exposure level. The rhesus macaque aerosol challenge model will be used for future evaluations of rBV A/B efficacy against inhalational BoNT/A1 and BoNT/B1 intoxication.Botulinum neurotoxins (BoNTs) produced by the bacterium Clostridium botulinum and related clostridial species are the causative agents of the human disease botulism (7). Approximately 145 cases of naturally occurring botulism are reported in the United States each year. Infant botulism accounts for more than 50% of the cases, with food-borne and wound botulism making up the balance (9). The Centers for Disease Control and Prevention (CDC) consider BoNTs to be a serious potential threat and have listed them as a category A threat, a highest-risk threat agent for bioterrorism (8). Use of these BoNTs offensively could potentially result in catastrophic consequences to the nation. Aerosol dissemination represents the most likely method for the use of BoNT as a biological weapon (2).Treatment of botulism is largely supportive, and any medical care must be initiated quickly to be effective. Current therapies include supportive intensive care (with ventilation) and treatment with antitoxin (2). A licensed vaccine is not currently available to provide protection against botulism. An investigational pentavalent vaccine (pentavalent botulinum toxoid vaccine [PBT]) that induces neutralizing antibodies against BoNT serotypes A, B, C, D, and E has been available for use in “at-risk” laboratory workers for 30 years (21).A new-generation recombinant botulinum vaccine (rBV A/B) is being developed to prevent fatal botulism following exposure to aerosolized botulinum neurotoxin complex serotype A, subtype A1 (BoNT/A1) and serotype B, subtype B1 (BoNT/B1). The component antigens of rBV A/B, developed by the U.S. Army Medical Research Institute for Infectious Diseases (5), are identified as antigen A and antigen B and were derived from the nontoxic 50-kDa C-terminal domains of BoNT/A1 and BoNT/B1, respectively. The efficacy of rBV A/B cannot be determined directly in humans because the incidence of botulism in the general population is extremely low and direct-challenge studies in humans are unethical. Therefore, rBV A/B efficacy will be evaluated according to the animal rule of U.S. law (9a).Evaluation of rBV A/B efficacy according to the animal rule requires the development and use of relevant animal models. Draft guidelines describing essential elements of animal models required to address efficacy under the animal rule were made available by the FDA in 2009 (6). Animal studies under the animal rule require a thorough understanding of the pathophysiological mechanism of the pathogenic agent and demonstration of similarities to known aspects of human disease. The challenge agent used for animal studies should be identical to the etiologic agent that causes the human disease, and the purity of the challenge preparation should be documented to the extent possible. Evaluation of efficacy in animal models will use a route of exposure to the etiological agent that is the same as the anticipated human exposure route. Reliable quantification and reproducibility of the challenge dose should be demonstrated. Acceptance of the animal model for evaluation of rBV A/B efficacy involves the performance of well-controlled, well-documented studies which incorporate these requirements.The rhesus macaque was selected based upon its historic use for evaluation of investigational vaccines and antitoxin therapeutics (4, 13, 14, 18, 20). Available nonclinical data suggest that the pathophysiological response to aerosol exposure in rhesus macaques is relevant to the human disease. Clinical signs in rhesus macaques exposed to aerosolized BoNT/A or BoNT/B include mild muscular weakness, intermittent ptosis, severe weakness of postural muscles of the neck, occasional mouth breathing, serous nasal discharge, salivation, dysphagia, rales, anorexia, severe weakness, and lateral recumbency (4, 13).The use of well-characterized challenge material is important for maintaining consistency among studies. The diversity of neurotoxin-producing C. botulinum strains has become well recognized, and the quality of BoNT preparations produced by different production facilities varies. Therefore, stocks of well-characterized BoNT/A1 and BoNT/B1 were established for use in the rhesus macaque model development study and all future rBV A/B efficacy studies.The following study was designed to establish the rhesus macaque BoNT/A1 and BoNT/B1 aerosol challenge models for use in future rBV A/B efficacy studies. The lethality of well-characterized BoNT/A1 and BoNT/B1 challenge material administered by the inhalational route of exposure was evaluated in rhesus macaques. In addition, clinical signs of disease were assessed through clinical observations, body weight measurements, clinical hematology, clinical chemistry, and telemetric monitoring to aid in the understanding of botulism disease progression. The relationships of the estimated total inhaled BoNT/A1 and BoNT/B1 dosages and circulating BoNT/A1 and BoNT/B1 levels to disease progression were also evaluated.     

Mapping of the regions on the heavy chain of botulinum neurotoxin A (BoNT/A) recognized by antibodies of cervical dystonia patients with immunoresistance to BoNT/A

The purpose of this work was to map the entire recognition profile of the H chain of botulinum neurotoxin A (BoNT/A) by Abs in sera that have protective anti-BoNT/A Abs by the mouse protection assay (MPA) from cervical dystonia (CD) patients who had been treated with botulinum neurotoxin, serotype A (BOTOX). In previous studies we found that human anti-tetanus neurotoxin (TeNT) Abs cross-react with BoNT/A and BoNT/B. In the present work we devised an assay procedure for measuring specific anti-BoNT/A Abs in human sera by absorbing out or inhibiting the anti-TeNT Abs with TeNT before analyzing the sera for the anti-BoNT/A Abs. The sera were obtained from 28 CD patients who had become unresponsive to treatment with BoNT/A and the sera were found to protect mice against a lethal dose of BoNT/A. For localization of the Ab-binding regions on the H chain we employed a set of sixty, 19-residue synthetic peptides (except for peptide C31 which was 22 residues) that encompassed the entire H chain sequence 449-1296 and overlapped consecutively by five residues. The pattern of Ab recognition varied from patient to patient, but a very limited set of peptides were recognized by most of the patients. These were, in decreasing amounts of Ab binding, peptide N25 (H chain residues 785-803), C9/C10 (967-985/981-999), C31 (1275-1296), C15 (1051-1069), C20 (1121-1139), N16 (659-677), N22 (743-761), and N4 (491-509). But not every serum recognized all these peptides. The finding that the binding profile was not the same for all the patients is consistent with previous observations that immune responses to protein antigens are under genetic control and that the response to each epitope within a protein is under separate genetic control. Except for the region within C9/C10, the other regions either coincided (N16 and C31), or overlapped (N4, N22, N25, C15 and C20), with the recently mapped synaptosomes (snps)-binding regions on the H chain. The molecular and clinical implications of these findings are discussed.     

Critical Roles of Clostridium difficile Toxin B Enzymatic Activities in Pathogenesis

TcdB is one of the key virulence factors of Clostridium difficile that is responsible for causing serious and potentially fatal colitis. The toxin contains at least two enzymatic domains: an effector glucosyltransferase domain for inactivating host Rho GTPases and a cysteine protease domain for the delivery of the effector domain into host cytosol. Here, we describe a novel intrabody approach to examine the role of these enzymes of TcdB in cellular intoxication. By screening a single-domain heavy chain (V_HH) library raised against TcdB, we identified two V_HH antibodies, 7F and E3, that specifically inhibit TcdB cysteine protease and glucosyltransferase activities, respectively. Cytoplasmic expression of 7F intrabody in Vero cells inhibited TcdB autoprocessing and delayed cellular intoxication, whereas E3 intrabody completely blocked the cytopathic effects of TcdB holotoxin. These data also demonstrate for the first time that toxin autoprocessing occurs after cysteine protease and glucosyltransferase domains translocate into the cytosol of target cells. We further determined the role of the enzymatic activities of TcdB in in vivo toxicity using a sensitive systemic challenge model in mice. Consistent with these in vitro results, a cysteine protease noncleavable mutant, TcdB-L543A, delayed toxicity in mice, whereas glycosyltransferase-deficient TcdB demonstrated no toxicity up to 500-fold of the 50% lethal dose (LD₅₀) when it was injected systemically. Thus, glucosyltransferase but not cysteine protease activity is critical for TcdB-mediated cytopathic effects and TcdB systemic toxicity, highlighting the importance of targeting toxin glucosyltransferase activity for future therapy.     

抗肉毒神经毒素A(BoNT/A)单克隆抗体的制备与鉴定

目的 :制备抗肉毒毒素A(BoNT/A)的单克隆抗体 (mAb)。方法 :用纯化的重组BoNT/A Hc片段免疫BALB/c小鼠 ,取其脾细胞与骨髓瘤Sp2 /0融合 ,经间接ELISA筛选和克隆化制备杂交瘤细胞系 ,及Western免疫印迹分析等方法对mAb进行特异性鉴定。结果 :获得 3株杂交瘤细胞株 :命名为4A8、2F7和 4F2 ,IgG亚类鉴定均为IgG1,腹水mAb的效价在 1× 10 -4～ 1× 10 -6之间。其中 ,4A8和 4F2能稳定分泌抗BoNT AmAb,并可特异性地识别重组BoNT/A和天然BoNT/A ,特别是 4A8可保护小鼠抵抗 10LD50 BoNT/A的攻击。结论 :成功地制备 3株特异性抗BoNT/AmAb ,并有 1株属于中和性mAb ,为BoNT/A的检测和肉毒中毒的临床治疗奠定了基础     

The Regions on the Light Chain of Botulinum Neurotoxin Type A Recognized by T Cells from Toxin-Treated Cervical Dystonia Patients. The Complete Human T-Cell Recognition Map of the Toxin Molecule

We have recently mapped the in vitro proliferative responses of T cells from botulinum neurotoxin type A (BoNT/A)-treated cervical dystonia (CD) patients with overlapping peptides encompassing BoNT/A heavy chain (residues 449–1296). In the present study, we determined the recognition profiles, by peripheral blood lymphocytes (PBL) from the same set of patients, of BoNT/A light (L) chain (residues 1–453) by using 32 synthetic overlapping peptides that encompassed the entire L chain. Profiles of the T-cell responses (expressed in stimulation index, SI; Z score based on transformed SI) to the peptides varied among the patients. Samples from 14 patients treated solely with BoNT/A recognized 3–13 (average 7.2) peptides/sample at Z > 3.0 level. Two peptide regions representing residues 113–131 and 225–243 were recognized by around 40% of these patients. Regarding treatment parameters, treatment history with current BOTOX^® only group produced significantly lower average T-cell responses to the 32 L-chain peptides compared to treatments with mix of type A including original and current BOTOX^®. Influence of other treatment parameters on T-cell recognition of the L-chain peptides was also observed. Results of the submolecular T-cell recognition of the L chain are compared to those of the H chain and the T-cell recognition profile of the entire BoNT/A molecule is discussed.

Abbreviations used: BoNT/A, botulinum neurotoxin type A; BoNT/A_i, inactivated BoNT/A; BoNT/B, botulinum neurotoxin type B; CD, cervical dystonia; L chain, the light chain (residues 1–448) of BoNT/A; LNC, lymph node cells; H chain, the heavy chain (residues 449–1296) of BoNT/A; H_C, C-terminal domain (residues 855–1296) of H chain; H_N, N-terminal domain (residues 449–859) of H chain; MPA, mouse protection assay; SI, stimulation index (SI = cpm of ³H-thymidine incorporated by antigen-stimulated T cells/cpm incorporated by unstimulated cells); TeNT, tetanus neurotoxin; TeNT_i, inactivated TeNT.     

Enhanced Toxicity for Mice of Combinations of Antibiotics with Escherichia coli Cells or Salmonella typhosa Endotoxin

Enhanced lethality for BALB/c mice has been observed after the administration of Salmonella typhosa endotoxin with either actinomycin D, cycloheximide, or nogalamycin. The dose of actinomycin D required to kill half of the mice (LD₅₀) was 0.8 mg/kg in normal animals, 0.35 mg/kg in mice administered 0.08 mg of endotoxin per kg, and 0.28 mg/kg in mice administered 0.2 mg of endotoxin per kg. The LD₅₀ of endotoxin in normal mice was 12 mg/kg and in mice given 0.4 mg of actinomycin D per kg was 0.067 mg/kg. The LD₅₀ of actinomycin D in mice administered 1.8 × 10⁸ live Escherichia coli cells per kg or 1.8 × 10⁹ heat-killed E. coli cells per kg was reduced to 0.4 mg/kg. The LD₅₀ of cycloheximide was 181 mg/kg in normal animals and 28 mg/kg in mice administered 4 mg of endotoxin per kg. The LD₅₀ of endotoxin in mice given 120 mg of cycloheximide per kg was 0.02 mg/kg. Enhanced lethality due to various combinations of cycloheximide and endotoxin was abolished by pretreatment of mice with endotoxin. The LD₅₀ of nogalamycin was 21 mg/kg in normal mice and 13 mg/kg in mice receiving 1 mg of endotoxin per kg.     

High-affinity, protective antibodies to the binding domain of botulinum neurotoxin type A

Monoclonal antibodies (MAbs) were prepared against the putative binding domain of botulinum neurotoxin A (BoNT/A), a nontoxic 50-kDa fragment. Initially, all fusion products were screened against the holotoxin BoNT/A and against the binding fragment, BoNT/A H(C). Eleven neutralizing hybridomas were cloned, and their specific binding to BoNT/A H(C) was demonstrated by surface plasmon resonance, with dissociation constants ranging from 0.9 to <0.06 nM. Epitope mapping by real-time surface plasmon resonance showed that the antibodies bound to at least two distinct regions of the BoNT/A H(C) fragment. These MAbs will be useful tools for studying BoNT/A interactions with its receptor, and they have potential diagnostic and therapeutic applications.     

Protection with a recombinant Hc of Clostridium Botulinum neurotoxin serotype A from Escherichia coli as an effective subunit vaccine

A recombinant Hc of Clostridium botulinum neurotoxin serotype A (AHc) was successfully expressed in Escherichia coli for use as an antigen, and the purified AHc was used to vaccinate mice and evaluate their survival against challenge with active botulinum neurotoxin serotype A. The mice, given twice or third subcutaneous vaccinations with a dosage of 1 μg AHc mixed with Freund adjuvant, were completely protected against an intraperitoneal administration of 1,000,000 50% lethal doses (LD₅₀) of neurotoxin serotype A. Following the administration of AHc using alhydrogel adjuvant via the intramuscular route, a strong protective immune response was also elicited in the vaccinated mice. A dose-response was observed in protective efficacy with increasing AHc dosage and number of vaccinations. Mice that received two injections of ≥ 0.2 μg and three injections of ≥ 0.04 μg were completely protected when challenged with 100,000 LD₅₀ of neurotoxin serotype A. These results clearly suggest that the recombinant AHc highly expressed in Escherichia coli is very efficacious in protecting against challenge with active botulinum neurotoxin serotype A in mouse model and a good subunit candidate vaccine against botulinum neurotoxin serotype A for human use.     

Botulinum neurotoxin type A counteracts neuropathic pain and facilitates functional recovery after peripheral nerve injury in animal models

A growing interest was recently focused on the use of Botulinum neurotoxin serotype A (BoNT/A) for fighting pain. The aim of this study was to investigate the effects of BoNT/A on neuropathic pain. It was observed that BoNT/A is able to counteract neuropathic pain induced by chronic constriction injury (CCI) to the sciatic nerve both in mice and in rats. This effect is already present after a single intraplantar (i.pl.) or intrathecal (i.t.) neurotoxin administration that significantly reduces the sciatic nerve ligation-induced mechanical allodynia in mice and rats and thermal hyperalgesia in rats. This effect was evident starting 24 h after the administration of BoNT/A and it was long-lasting, being present 81 or 25 days after i.pl. injection of the higher dose in mice (15 pg/paw) and rats (75 pg/paw), respectively, and 35 days after i.t. injection in rats (75 pg/rat). Moreover, BoNT/A-injected mice showed a quicker recovery of the walking pattern and weight bearing compared to control groups. The behavioral improvement was accompanied by structural modifications, as revealed by the expression of cell division cycle 2 (Cdc2) and growth associated protein 43 (GAP-43) regeneration associated proteins, investigated by immunofluorescence and Western blotting in the sciatic nerve, and by the immunofluorescence expression of S100β and glial fibrillary acidic protein (GFAP) Schwann cells proteins. In conclusion, the present research demonstrate long-lasting anti-allodynic and anti-hyperalgesic effects of BoNT/A in animal models of neuropathic pain together with an acceleration of regenerative processes in the injured nerve, as evidenced by both behavioral and immunohistochemistry/blotting analysis. These results may have important implications in the therapy of neuropathic pain.     

Mapping of the Antibody and T Cell Recognition Profiles of the HN Domain (Residues 449–859) of the Heavy Chain of Botulinum Neurotoxin A in Two High-Responder Mouse Strains

Using a set of synthetic overlapping peptides, encompassing the entire N-terminal domain (H_N,) of the heavy (H) chain of botulinum neurotoxin serotype A (BoNT/A), we have mapped on H_N, the regions recognized by Abs (B cells) and by T cells in two inbred mouse strains. After one BoNT/A toxoid injection, BALB/c T cells mounted a weak in vitro response to a region within overlap 687–705/701–719. The remaining peptides stimulated no detectable responses. After 3 injections, BALB/c T cells gave stronger responses to an expanded region within the overlap 687–705/701–719/715–733, peaking at 701–719. BoNT/A-primed BALB/c T cells showed substantial cross-reaction with BoNT/B but did not respond to TeNT. Unlike BALB/c T cells, BoNT/A-primed T cells of SJL cross-reacted well with both BoNT/B and with TeNT. They also recognized a lager epitope profile than the corresponding BALB/c T cells. After one injection with BoNT/A toxoid, SJL T cells responded in vitro to a number of the H_N peptides. Regionswithin peptides 617–635 and 561–579 stimulated strong in vitro responses. Several peptides (463–481, 589–607, 659–677, 729–747, 827–845, and 841–859 revoked weak-to-medium proliferative activities. Four other peptides stimulated very low but reproducible responses (SI between 2.0 and 3.0). After 3 BoNT/A injections, SJL T cells responded in vitro strongly to peptides 463–481, 561–579, 617–635, 743–761, and 841–859. There were medium or weak responses to at least 10 other peptides. The cells also responded well to the l-chain peptide 218–231. Antisera of BALB/c and SJL, obtained after 3 injections with BoNT/A toxoid, protected at very high dilutions recipient mice against LD₁₀₅ of BoNT/A. BALB/c Abs showed medium-to-high binding to peptides 533–551/547–565, 785–803, and 813–831/827–845. Four other peptides showed very low binding. The corresponding SJL Abs had high binding to the overlap 533–551/547–565/561–579, and peptides 743–761, 785–803, and 813–831. Three other peptides bound low amounts of Abs. The results indicate that the responses to each Ab or T cell epitope is under separate genetic control and that, in a given strain, the Ab and T cell recognition regions may coincide but, in addition, H_N contains regions that are recognized only by Abs or only by T cells.     

Development and evaluation of candidate vaccine and antitoxin against botulinum neurotoxin serotype F

To produce a vaccine suitable for human use, a recombinant non His-tagged isoform of the Hc domain of botulinum neurotoxin serotype F (rFHc) was expressed in Escherichia coli and purified by sequential chromatography. The rFHc was evaluated as a subunit vaccine candidate in mouse model of botulism. A dose-response was observed in both antibody titer and protective efficacy with increasing dosage of rFHc and number of vaccinations. These findings suggest that the rFHc is an effective botulism vaccine candidate. Further, we developed a new antitoxin against botulinum neurotoxin serotype F (BoNT/F) by purifying F(ab′)₂ fragments from pepsin digested serum IgGs of horses inoculated with rFHc. The protective effect of the F(ab′)₂ antitoxin against BoNT/F was determined both in vitro and in vivo. The results showed that the F(ab′)₂ antitoxin could prevent botulism in mice challenged with BoNT/F and effectively delayed progression of paralysis from botulism in the therapeutic setting. Thus, our results provide valuable experimental data for this new antitoxin as a potential candidate for treatment of botulism caused by BoNT/F.     

Genetic Characterization of Clostridium botulinum Associated with Type B Infant Botulism in Japan

The 15 proteolytic Clostridium botulinum type B strains, including 3 isolates associated with infant botulism in Japan, were genetically characterized by phylogenetic analysis of boNT/B gene sequences, genotyping, and determination of the boNT/B gene location by using pulsed-field gel electrophoresis (PFGE) for molecular epidemiological analysis of infant botulism in Japan. Strain Osaka05, isolated from a case in 2005, showed a unique boNT/B gene sequence and was considered to be a new BoNT/B subtype by phylogenetic analysis. Strain Osaka06, isolated from a case in 2006, was classified as the B2 subtype, the same as strain 111, isolated from a case in 1995. The five isolates associated with infant botulism in the United States were classified into the B1 subtype. Isolates from food samples in Japan were divided into the B1 and the B2 subtypes, although no relation with infant botulism was shown by PFGE genotyping. The results of PFGE and Southern blot hybridization with undigested DNA suggested that the boNT/B gene is located on large plasmids (approximately 150 kbp, 260 kbp, 275 kbp, or 280 kbp) in five strains belonging to three BoNT/B subtypes from various sources. The botulinum neurotoxin (BoNT) of Osaka05 was suggested to have an antigenicity different from the antigenicities of BoNT/B1 and BoNT/B2 by a sandwich enzyme-linked immunosorbent assay with the recombinant BoNT/B-C-terminal domain. We established a multiplex PCR assay for BoNT/B subtyping which will be useful for epidemiological studies of type B strains and the infectious diseases that they cause.Infant botulism is neuromuscular paralysis caused by the botulinum neurotoxin (BoNT) produced in the intestines after the germination and outgrowth of ingested spores of Clostridium botulinum, which is an anaerobic spore-forming bacterium (7, 9). On the basis of the antigenic specificity of BoNT, C. botulinum strains are divided into seven serotypes (serotypes A to G), and the species has been separated into four groups (groups I to IV) by cultural characteristics (24). BoNT is encoded by an approximately 3.8-kb gene, which is preceded by several nontoxic component genes (17, 30). BoNT is released from the bacteria as a single polypeptide chain of 150 kDa and is cleaved by endogenous or exogenous proteases into a 50-kDa light chain and a 100-kDa heavy chain. The heavy chain contains two functional domains, the N-terminal domain (H_N) and the C-terminal domain (H_C). H_C can be further divided into two distinct subdomains: the N-terminal domain (H_CN) and the C-terminal domain (H_CC) (5, 38).Recently, the subtype classification was confirmed by the diversity of the amino acid sequences within each serotype (13, 37). BoNT serotype A (BoNT/A) has been divided into four subtypes (subtypes A1, A2, A3, and A4) (2, 10). BoNT/B has been divided into three subtypes from type B group I (subtypes B1, B2, and B3), one subtype from group I bivalent strains that express another BoNT type, in addition to BoNT/B (bivalent), and one subtype from type B group II (nonproteolytic) (13). BoNT/E has been divided into four subtypes from C. botulinum type E (subtypes E1, E2, E3, and E6) and two subtypes from BoNT/E-producing C. butyricum (subtypes E4 and E5) (6).Since infant botulism was first recognized in the United States in 1976 (27, 31), it is now the most common disease caused by C. botulinum. This disease affects children up to 6 months old, but with rare exceptions it affects individuals of other ages. The symptoms are characterized by constipation, generalized weakness, and various neurological disorders (9). Cases represent a spectrum of disease, ranging from subclinical infection to the most fulminant form of the disease, which is unexpected sudden death (3). Almost all cases of infant botulism have been caused by proteolytic C. botulinum type A and B strains. Since the first occurrence of infant botulism in Japan caused by C. botulinum type A in 1986 (29), there have been 24 cases; 16 were caused by type A strains, 3 were caused by type B strains, 1 was caused by a type C strain, and 1 was caused by a C. butyricum strain producing BoNT/E. The types of toxin in the other three cases were not described (16). We previously indicated that the original BoNT/B2 produced by strain 111, which was isolated from the first case of type B infant botulism in Japan in 1995, showed antigenic and biological properties different from those of the authentic BoNT/B (B1) produced by strain Okra (15, 20, 22). Two additional cases of type B infant botulism with typical symptoms occurred in Osaka Prefecture in 2005 and 2006. We eventually isolated two proteolytic C. botulinum type B strains, designated Osaka05 and Osaka06, respectively.In the study described here, to better understand the background of type B infant botulism, we determined the genetic characteristics of proteolytic C. botulinum type B isolates by comparison of the nucleotide sequences of boNT/B and nontoxic component genes, the pulsed-field gel electrophoresis (PFGE) genotypes, the boNT/B gene location by PFGE and Southern blot hybridization, and the antigenicity of the new BoNT/B subtype. We developed multiplex PCR assays for the detection and identification of the BoNT/B subtypes.     

Enhanced expression of soluble recombinant tetanus neurotoxin Hc in Escherichia coli as a tetanus vaccine candidate

The expression of the carboxyl fragment of the heavy chain of tetanus neurotoxin (TeNT-Hc) in Escherichia coli has been hampered by the unusually high AT content and the presence of rarely used codons by Clostridium. The gene encoding TeNT-Hc was optimized for E. coli by replacing rare codons and decreasing the AT pairs from 72.57% to 52.47%. The reconstructed gene was expressed in E. coli BL21(DE3) and resulted in a soluble product which was about 46% of the total bacterial protein. TeNT-Hc produced in a 42 L fermentor was purified to >95% at 87 g/kg of cell paste (approximately 333 mg/L). BALB/c mice vaccinated with three bi-weekly doses of TeNT-Hc with Freund's adjuvant were fully protected against an intraperitoneally challenge of 2 × 10³ 50% lethal doses (LD₅₀s) of tetanus neurotoxin. NIH mice vaccinated with TeNT-Hc adsorbed to aluminum hydroxide gel adjuvant demonstrated a potency of 168 IU/mL, which was 2 times higher than the national standard for tetanus vaccines. These results suggest that TeNT-Hc may be a promising second-generation vaccine candidate for clinical use against tetanus neurotoxin.     

Vaccination of Rabbits with an Alkylated Toxoid Rapidly Elicits Potent Neutralizing Antibodies against Botulinum Neurotoxin Serotype B

New Zealand White (NZW) rabbits were immunized with several different nontoxic botulinum neurotoxin serotype B (BoNT/B) preparations in an effort to optimize the production of a rapid and highly potent, effective neutralizing antibody response. The immunogens included a recombinant heavy chain (rHc) protein produced in Escherichia coli, a commercially available formaldehyde-inactivated toxoid, and an alkylated toxoid produced by urea-iodoacetamide inactivation of the purified active toxin. All three immunogens elicited an antibody response to BoNT/B, detected by enzyme-linked immunosorbent assay (ELISA) and by toxin neutralization assay, by the use of two distinct mouse toxin challenge models. The induction period and the ultimate potency of the observed immune response varied for each immunogen, and the ELISA titer was not reliably predictive of the potency of toxin neutralization. The kinetics of the BoNT/B-specific binding immune response were nearly identical for the formaldehyde toxoid and alkylated toxoid immunogens, but immunization with the alkylated toxoid generated an approximately 10-fold higher neutralization potency that endured throughout the study, and after just 49 days, each milliliter of serum was capable of neutralizing 10⁷ 50% lethal doses of the toxin. Overall, the immunization of rabbits with alkylated BoNT/B toxoid appears to have induced a neutralizing immune response more rapid and more potent than the responses generated by vaccination with formaldehyde toxoid or rHc preparations.Botulinum neurotoxin (BoNT), the causative agent of botulism, is the most potent of all the known toxins (7). BoNT is a secreted protein produced by the anaerobic soil organisms Clostridium botulinum, Clostridium baratii, and Clostridium butyricum in seven distinct serotypes (serotypes A to G) (9, 23, 28). The BoNT serotypes are all synthesized as single-chain polypeptides with molecular masses of approximately 150 kDa. Posttranslational cleavage of the original polypeptide monomer results in the formation of a disulfide-linked dichain product composed of light chain (LC) and heavy chain (HC) domains. The HC is divided into two distinct functional domains; the first mediates toxin binding and uptake by peripheral neuronal cells, and the second mediates translocation of the LC subunit into the target cell cytosol. Once it is in the cytosol, the zinc metalloprotease of the LC specifically cleaves the soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptors (SNAREs) responsible for synaptic vesicle docking and neurotransmitter release at the neuromuscular synapse.Human botulism typically results from the ingestion of contaminated foods (often improperly prepared canned goods), although BoNT intoxication can also result from wound colonization by one or more species of Clostridium. Similarly, infant botulism results from exposure to actively secreted toxin following the germination of ingested Clostridium spores, which proliferate in the immature gastrointestinal tract. Regardless of the route of exposure, BoNT intoxication occurs by the same mechanism, once the toxin enters the circulation. Although there is no cure for botulism after the onset of symptoms, an effective circulating antibody response can completely neutralize an otherwise intoxicating dose of BoNT. Widespread immunization against the toxin is precluded by the growing number of clinical applications of BoNT for the treatment of various neuromuscular spasticity disorders, yet BoNT vaccine development continues for the purposes of immunizing at-risk populations, such as laboratory workers, first responders, and military personnel (26).A number of BoNT immunogens and a variety of vaccination strategies have successfully been used to elicit neutralizing antibody responses against individual BoNT serotypes (3, 19, 20, 29, 32). The immune responses to BoNT vary according to the animal species, the toxin serotype, and the antigen preparation. Additionally, the development of a potent neutralizing antibody response to BoNT serotype B (BoNT/B) has proven problematic, prompting a demand for alternative toxin-derived immunogens (25, 27).In the present study, we tested three BoNT/B immunogens in New Zealand White (NZW) rabbits using a rapid vaccination scheme to develop a potent toxin-neutralizing immune response in a short time period (12). Rabbits were immunized with BoNT/B recombinant heavy chain (rHc) or toxoid preparations derived from formaldehyde inactivation or urea- iodoacetamide alkylation of active toxin (15). All three immunogens elicited toxin-neutralizing antibody responses by the end of the study; however, vaccination with the alkylated toxoid preparation induced a more rapid and more potent BoNT/B-neutralizing response than the other immunogens.