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.     

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.     

Antibody Protection against Botulinum Neurotoxin Intoxication in Mice

Adulteration of food or feed with any of the seven serotypes of botulinum neurotoxin (BoNT) is a potential bioterrorism concern. Currently, there is strong interest in the development of detection reagents, vaccines, therapeutics, and other countermeasures. A sensitive immunoassay for detecting BoNT serotype A (BoNT/A), based on monoclonal antibodies (MAbs) F1-2 and F1-40, has been developed and used in complex matrices. The epitope for F1-2 has been mapped to the heavy chain of BoNT/A, and the epitope of F1-40 has been mapped to the light chain. The ability of these MAbs to provide therapeutic protection against BoNT/A intoxication in mouse intravenous and oral intoxication models was tested. High dosages of individual MAbs protected mice well both pre- and postexposure to BoNT/A holotoxin. A combination therapy consisting of antibodies against both the light and heavy chains of the toxin, however, significantly increased protection, even at a lower MAb dosage. An in vitro peptide assay for measuring toxin activity showed that pretreatment of toxin with these MAbs did not block catalytic activity but instead blocked toxin entry into primary and cultured neuronal cells. The timing of antibody rescue in the mouse intoxication models revealed windows of opportunity for antibody therapeutic treatment that correlated well with the biologic half-life of the toxin in the serum. Knowledge of BoNT intoxication and antibody clearance in these mouse models and understanding of the pharmacokinetics of BoNT are invaluable for future development of antibodies and therapeutics against intoxication by BoNT.Botulinum neurotoxins (BoNTs) are considered some of the most potent toxins known and are potential bioterrorist threat agents. Yet, BoNT serotype A (BoNT/A) and BoNT/B are also used therapeutically in a wide array of medical conditions, such as dystonia and eye disorders like strabismus and blepharospasms, for pain management, and more (3, 25). Thus, there is a need to protect humans and animals against toxin exposure from contaminated food or feed and yet preserve the medical benefits of BoNT. A better understanding of the biology of the toxin, such as toxin distribution and mechanisms of toxin neutralization following intoxication, is needed to aid further development of improved therapies, as well as bona fide use of toxin to treat serious medical conditions.BoNTs are 150-kDa endopeptidase toxins that are produced by Clostridium botulinum, C. butyricum, and C. baratii (20, 26, 27). The toxin polypeptide is cleaved upon secretion from the cell by bacterial proteases or proteases in the animal host into a disulfide bond-linked dipeptide consisting of a 100-kDa heavy chain (Hc) and a 50-kDa light chain (Lc). The 50-kDa Lc contains the active site or catalytic domain that targets the soluble N-ethylmaleimide-sensitive factor attachment protein receptor. Specifically, the synaptosome-associated 25-kDa protein (SNAP25) is the target for BoNT/A. Cleavage of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor results in the inhibition of neurotransmitter vesicle docking and subsequent vesicle fusion and release of the neurotransmitter acetylcholine. The toxin Hc contains the cell receptor binding and translocation domains necessary for the uptake of the 150-kDa holotoxin by neurons (18, 24).Currently, the therapies available for botulism patients are supportive intensive care, such as mechanical ventilation, and antibody treatment postintoxication with equine antitoxins or a botulinum immunoglobulin (BabyBIG) human antibody (3, 6, 9). Drawbacks of those therapies are serious side effects such as serum sickness and anaphylaxis for the use of equine antitoxins and the limited availability of the human antibody product. Neither treatment reverses paralysis completely or immediately, since they act by neutralizing toxin in the circulation. Thus, both must be administered relatively early before toxin uptake by neurons in order to be effective. Currently, there is intense effort to develop better vaccines and antitoxins against BoNTs. Antibodies and other molecular targets, such as small peptides and receptor mimics, and various functional domains of the toxin, such as the catalytic domain and the receptor binding or translocation domain, have previously been developed as therapeutic targets (7, 8, 15, 25, 26).Studies identifying the antibody binding sites and their toxin neutralization potential have helped identify targets for the design of antibody therapeutics (11). However, to facilitate vaccine approval and development of additional therapeutics, animal model data are needed. Information gleaned from animal models will lead to a better understanding of the efficacy of treatment, as well as define the window of opportunity for optimal treatment. Such knowledge will result in the design of better therapeutics and treatment regiments. Thus far, the mouse model of botulism has been used extensively for toxin detection, for toxin concentration standardization, and in the development of therapeutics (9, 12).We previously described the development of high-affinity monoclonal antibodies (MAbs) F1-2 (against the Hc) and F1-40 (against the Lc) (28). These MAbs were used successfully in detection assays for BoNT/A in complex food matrices. The binding site for MAb F1-40 has been mapped to the exposed loop between the β4 and β5 regions of the Lc. While this site is located on the Lc, it is distal from the catalytic domain. The binding site for MAb F1-2 is conformational and has been mapped to the region between R564 and K595 within the transmembrane domain of the toxin (22, 23).By using animal studies, one can measure the pharmacokinetics of toxins, the physiological distribution and stability of toxins, and how they are cleared from the system by MAbs. By using ¹²⁵I-labeled BoNT/A and BoNT/B, the biological half-lives (t_1/2s) of such toxins have been determined in blood (2, 21). Furthermore, BoNTs were shown not to go through biotransformation during epithelial membrane translocation or by interaction with blood components. Following antibody treatment, toxin was found sequestered mainly in the liver of the animal; however, the mechanism of antibody clearance has not yet been determined.Here we report the in vivo neutralization of BoNT/A by MAbs in systemic and oral models of botulism. The effects of antibody dosage and timing of administration were tested, and a window of opportunity for MAb rescue following intoxication was defined. To better understand MAb neutralization, the toxin levels in mouse serum over time were determined and compared with the timing of MAb rescue postintoxication. A direct correlation of toxin pharmacokinetics with antibody rescue was found. The individual MAbs also were tested in vitro for enzymatic activity inhibition and blockade of SNAP25 cleavage. These latter studies were designed to mimic conditions found in human intoxication. Increased knowledge of the pharmacokinetics of the toxin and how antibodies clear the toxin will aid in the design of more effective treatments for botulism.     

Comparison of extracellular and intracellular potency of botulinum neurotoxins

Levels of botulinum neurotoxin (BoNT) proteolytic activity were compared using a cell-free assay and living neurons to measure extracellular and intracellular enzymatic activity. Within the cell-free reaction model, BoNT serotypes A and E (BoNT/A and BoNT/E, respectively) were reversibly inhibited by chelating Zn2+ with N,N,N',N'-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN). BoNT/E required relatively long incubation with TPEN to achieve total inhibition, whereas BoNT/A was inhibited immediately upon mixing. When na?ve Zn2+-containing BoNTs were applied to cultured neurons, the cellular action of each BoNT was rapidly inhibited by subsequent addition of TPEN, which is membrane permeable. Excess Zn2+ added to the culture medium several hours after poisoning fully restored intracellular toxin activity. Unlike TPEN, EDTA irreversibly inhibited both BoNT/A and -E within the cell-free in vitro reaction. Excess Zn2+ did not reactivate the EDTA-treated toxins. However, application of EDTA-treated BoNT/A or -E to cultured neurons demonstrated normal toxin action in terms of both blocking neurotransmission and SNAP-25 proteolysis. Different concentrations of EDTA produced toxin preparations with incrementally reduced in vitro proteolytic activities, which, when applied to living neurons showed undiminished cellular potency. This suggests that EDTA renders the BoNT proteolytic domain conformationally inactive when tested with the cell-free reaction, but this change is corrected during entry into neurons. The effect of EDTA is unrelated to Zn2+ because TPEN could be applied to living cells before or after poisoning to produce rapid and reversible inhibition of both BoNTs. Therefore, bound Zn2+ is not required for toxin entry into neurons, and removal of Zn2+ from cytosolic BoNTs does not irreversibly alter toxin structure or function. We conclude that EDTA directly alters both BoNTs in a manner that is independent of Zn2+.     

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.     

Antigenic comparison of different foot-and-mouth disease virus types using monoclonal antibodies defining multiple neutralizing epitopes on FMDV A5 subtypes

Thirteen monoclonal antibodies (MAbs) were elicited with A5 Spain-86 virus, the cause of the most recent foot-and-mouth disease virus (FMDV) outbreak in Spain. The MAbs were tested for ability to bind 140S virions and 12S protein subunits by liquid-phase radioimmunoassay (RIA), and to bind VP1 capsid protein by Western immunoblot assay. One of the thirteen MAb was virion (140S) specific, seven recognized 140S and 12S subunits, one bound to 140S, 12S and VP1 and four were 12S specific. These MAbs presented varying binding patterns when tested against different FMDV subtypes and serotypes, indicating the presence of conserved and non-conserved epitopes among FMDV serotypes and subtypes. Neutralization assays, in vivo and in vitro, showed that none of the 140S specific MAbs or 12S specific MAbs were neutralizing, but notably several of the 12S specific MAbs bound to all the different FMDV serotypes and can be useful diagnostic reagent for the detection of FMDV. The remaining MAbs showed varying behavior with the different types tested: not all types to which the MAbs bound were neutralized, demonstrating that the presence of an epitope and subsequent neutralization of infectivity are not necessarily correlated. Five type A12 neutralizing MAbs, previously characterized, have been used in this work. Four bound to A5 Spain-86 virus, but only one neutralized viral infectivity. On the basis of differential reactivity and neutralization among various FMDV subtypes and serotypes, and cross-inhibition binding assays between these MAbs, seven neutralization related epitopes have been defined on A5 Spain-86 virus.     

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.     

Tetanus toxin and botulinum toxin a utilize unique mechanisms to enter neurons of the central nervous system

Botulinum neurotoxins (BoNTs) and tetanus neurotoxin (TeNT) are the most toxic proteins for humans. While BoNTs cause flaccid paralysis, TeNT causes spastic paralysis. Characterized BoNT serotypes enter neurons upon binding dual receptors, a ganglioside and a neuron-specific protein, either synaptic vesicle protein 2 (SV2) or synaptotagmin, while TeNT enters upon binding gangliosides as dual receptors. Recently, TeNT was reported to enter central nervous system (CNS) neurons upon synaptic vesicle cycling that was mediated by the direct binding to SV2, implying that TeNT and BoNT utilize common mechanisms to enter CNS neurons. This prompted an assessment of TeNT entry into CNS neurons, using the prototypic BoNT serotype A as a reference for SV2-mediated entry into synaptic vesicles, analyzing the heavy-chain receptor binding domain (HCR) of each toxin. Synaptic vesicle cycling stimulated the entry of HCR/A into neurons, while HCR/T entered neurons with similar levels of efficiency in depolarized and nondepolarized neurons. ImageJ analysis identified two populations of cell-associated HCR/T in synaptic vesicle cycling neurons, a major population which segregated from HCR/A and a minor population which colocalized with HCR/A. HCR/T did not inhibit HCR/A entry into neurons in competition experiments and did not bind SV2, the protein receptor for BoNT/A. Intoxication experiments showed that TeNT efficiently cleaved VAMP2 in depolarized neurons and neurons blocked for synaptic vesicle cycling. These experiments demonstrate that TeNT enters neurons by two pathways, one independent of stimulated synaptic vesicle cycling and one by synaptic vesicles independent of SV2, showing that TeNT and BoNT/A enter neurons by unique mechanisms.     

Unique biological activity of botulinum D/C mosaic neurotoxin in murine species

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.     

Letter from the Editor

The present studies were carried out in order to investigate the cross-reaction of botulinum neurotoxins (BoNTs) with human and mouse antibodies against tetanus neurotoxin (TeNT) and determine whether injection of BoNT into a host that has been primed with TeNT would result in boosting of the response to the injected BoNT. Human antisera against TeNT obtained from 9 individuals were found to exhibit substantial cross-reaction with BoNTs A and B. We prepared antibodies (Abs) against inactivated tetanus neurotoxin (TeNT) in outbred mice and determined the binding of these Abs to active TeNT and active botulinum neurotoxins (BoNTs) A and B. Blood samples were collected before immunization (day 0) and on days 42, 82 and 125 after the first injection. The reactions of these sera with the immunizing antigen (inactivated TeNT), active TeNT, active BoNT/A and active BoNT/B were determined. At a fixed dilution (1:62.5 v/v), the sera contained high levels of Abs that reacted with TeNT and also with BoNTs A and B. Throughout the test period (up through day 125) and at different dilutions the cross-reactions of the antisera with BoNT/B were almost twice those with BoNT/A. The reactions of the antisera with the immunizing antigen (inactive TeNT) or with active TeNT were essentially equal throughout the dilution range tested (1:16–1:500 v/v). To determine whether injection of BoNT/A or B into a host that had been primed with TeNT resulted in boosting of the response to the priming antigen (TeNT) as well as BoNT/A or B, mice were primed with TeNT and boosted 21 days later with TeNT, BoNT/A or BoNT/B. Appropriate controls were also employed. Blood samples were collected prior to TeNT priming (day -1) and on days 21, 32, 46 and 67 after priming. In TeNT-primed mice, BoNTs A or B boosted the anti-TeNT Ab responses slightly but had no significant boosting effect on the Ab populations that bind to BoNTs A or B. It is concluded that while Abs against TeNT cross react with BoNTs and the cross reaction with BoNT/B is almost double that of BoNT/A, injection of BoNTs A or B in the presence of a prior active immunity against TeNT is not very likely to make the host mount an Ab response against the injected BoNT.     

Cross reaction of tetanus and botulinum neurotoxins A and B and the boosting effect of botulinum neurotoxins A and B on a primary anti-tetanus antibody response

The present studies were carried out in order to investigate the cross-reaction of botulinum neurotoxins (BoNTs) with human and mouse antibodies against tetanus neurotoxin (TeNT) and determine whether injection of BoNT into a host that has been primed with TeNT would result in boosting of the response to the injected BoNT. Human antisera against TeNT obtained from 9 individuals were found to exhibit substantial cross-reaction with BoNTs A and B. We prepared antibodies (Abs) against inactivated tetanus neurotoxin (TeNT) in outbred mice and determined the binding of these Abs to active TeNT and active botulinum neurotoxins (BoNTs) A and B. Blood samples were collected before immunization (day 0) and on days 42, 82 and 125 after the first injection. The reactions of these sera with the immunizing antigen (inactivated TeNT), active TeNT, active BoNT/A and active BoNT/B were determined. At a fixed dilution (1:62.5 v/v), the sera contained high levels of Abs that reacted with TeNT and also with BoNTs A and B. Throughout the test period (up through day 125) and at different dilutions the cross-reactions of the antisera with BoNT/B were almost twice those with BoNT/A. The reactions of the antisera with the immunizing antigen (inactive TeNT) or with active TeNT were essentially equal throughout the dilution range tested (1:16-1:500 v/v). To determine whether injection of BoNT/A or B into a host that had been primed with TeNT resulted in boosting of the response to the priming antigen (TeNT) as well as BoNT/A or B, mice were primed with TeNT and boosted 21 days later with TeNT, BoNT/A or BoNT/B. Appropriate controls were also employed. Blood samples were collected prior to TeNT priming (day -1) and on days 21, 32, 46 and 67 after priming. In TeNT-primed mice, BoNTs A or B boosted the anti-TeNT Ab responses slightly but had no significant boosting effect on the Ab populations that bind to BoNTs A or B. It is concluded that while Abs against TeNT cross react with BoNTs and the cross reaction with BoNT/B is almost double that of BoNT/A, injection of BoNTs A or B in the presence of a prior active immunity against TeNT is not very likely to make the host mount an Ab response against the injected BoNT.     

CROSS REACTION OF TETANUS AND BOTULINUM NEUROTOXINS A AND B AND THE BOOSTING EFFECT OF BOTULINUM NEUROTOXINS A AND B ON A PRIMARY ANTI-TETANUS ANTIBODY RESPONSE

The present studies were carried out in order to investigate the cross-reaction of botulinum neurotoxins (BoNTs) with human and mouse antibodies against tetanus neurotoxin (TeNT) and determine whether injection of BoNT into a host that has been primed with TeNT would result in boosting of the response to the injected BoNT. Human antisera against TeNT obtained from 9 individuals were found to exhibit substantial cross-reaction with BoNTs A and B. We prepared antibodies (Abs) against inactivated tetanus neurotoxin (TeNT) in outbred mice and determined the binding of these Abs to active TeNT and active botulinum neurotoxins (BoNTs) A and B. Blood samples were collected before immunization (day 0) and on days 42, 82 and 125 after the first injection. The reactions of these sera with the immunizing antigen (inactivated TeNT), active TeNT, active BoNT/A and active BoNT/B were determined. At a fixed dilution (1:62.5 v/v), the sera contained high levels of Abs that reacted with TeNT and also with BoNTs A and B. Throughout the test period (up through day 125) and at different dilutions the cross-reactions of the antisera with BoNT/B were almost twice those with BoNT/A. The reactions of the antisera with the immunizing antigen (inactive TeNT) or with active TeNT were essentially equal throughout the dilution range tested (1:16-1:500 v/v). To determine whether injection of BoNT/A or B into a host that had been primed with TeNT resulted in boosting of the response to the priming antigen (TeNT) as well as BoNT/A or B, mice were primed with TeNT and boosted 21 days later with TeNT, BoNT/A or BoNT/B. Appropriate controls were also employed. Blood samples were collected prior to TeNT priming (day -1) and on days 21, 32, 46 and 67 after priming. In TeNT-primed mice, BoNTs A or B boosted the anti-TeNT Ab responses slightly but had no significant boosting effect on the Ab populations that bind to BoNTs A or B. It is concluded that while Abs against TeNT cross react with BoNTs and the cross reaction with BoNT/B is almost double that of BoNT/A, injection of BoNTs A or B in the presence of a prior active immunity against TeNT is not very likely to make the host mount an Ab response against the injected BoNT.     

Purification of Shiga toxin and Shiga-like toxins I and II by receptor analog affinity chromatography with immobilized P1 glycoprotein and production of cross-reactive monoclonal antibodies.

Shiga toxin from Shigella dysenteriae 60R was purified to homogeneity by a novel one-step receptor analog affinity chromatography method. The method was based on the binding affinity of Shiga toxin for a specific disaccharide, Gal alpha 1----4Gal, which was also present in glycoproteins with P1 blood group seroreactivity produced in hydatid cysts from sheep infected with Echinococcus granulosus. Having shown that cyst fluid P1 glycoprotein bound Shiga toxin on a solid phase, a P1 glycoprotein affinity column was made by coupling P1-active substance to Sepharose 4B. Shiga toxin was purified by this method in large quantities (5 to 10 mg/20-liter batch) with a consistently good yield (greater than 80% of starting toxin). Shiga-like toxins I and II (SLT-I and -II, respectively) from Escherichia coli were also purified by the same method. A preparation containing SLT-II and SLT-I purified by receptor analog affinity chromatography was used to raise four monoclonal antibodies (MAbs) that were reactive with SLT-II by enzyme-linked immunosorbent assay. Three of these antibodies also reacted with Shiga toxin, which was the first clear demonstration of cross-reactivity between these toxins. One MAb, 4D1, which was specific for the B subunit of SLT-II and Shiga toxin, neutralized both toxins in a HeLa cell cytotoxicity assay. Two MAbs recognized the A subunit of both SLT-II and Shiga toxin by Western blot (immunoblot) analysis but were unable to neutralize either toxin. In addition, one B-subunit-specific MAb neutralized SLT-II alone, and a previously described Shiga toxin B-subunit-specific MAb was shown to be specific for Shiga toxin but not SLT-II.     

Generation of High-Titer Neutralizing Antibodies against Botulinum Toxins A,B, and E by DNA Electrotransfer

Botulinum neurotoxins are known to be among the most toxic known substances. They produce severe paralysis by preventing the release of acetylcholine at the neuromuscular junction. Thus, new strategies for efficient production of safe and effective anti-botulinum neurotoxin antisera have been a high priority. Here we describe the use of DNA electrotransfer into the skeletal muscle to enhance antiserum titers against botulinum toxin serotypes A, B, and E in mice. We treated animals with codon-optimized plasmid DNA encoding the nontoxic but highly immunogenic C-terminal heavy chain fragment of the toxin. By employing both codon optimization and the electrotransfer procedure, the immune response and corresponding neutralizing antiserum titers were markedly increased. The cellular localization of the antigen and the immunization regimens were also shown to increase neutralizing titers to >100 IU/ml. This study demonstrates that DNA electrotransfer is an effective procedure for raising neutralizing antiserum titers to remarkably high levels.Botulinum neurotoxins (BoNTs) are among the most toxic known substances and have been characterized as the most potent substances known. They have accounted for several food poisoning cases in humans and animals (1, 24). Among the seven serologically distinct types of BoNTs (types A to G), BoNT types A, B, E, and F are commonly linked to human disease. BoNT/A is the deadliest of the seven toxins, with a very high potency; the theoretical lethal dose is estimated to be on the order of 1 nanogram per kilogram of body weight (1, 31).BoNT consists of a poorly active single polypeptide chain of 150 kDa, which is proteolytically cleaved to an active double chain comprised of a light subunit (about 50 kDa) and a heavy subunit (about 100 kDa) linked by a disulfide bridge. The toxin is composed of three functional domains (50). The C-terminal half of the heavy chain (fragment C [Fc]) mediates binding to the target neurons, which triggers the internalization of the whole toxin into endocytic vesicles. The N-terminal half of the heavy chain mediates the translocation of the light chain, which is the intracellular active domain, into the cytoplasm of the neuron. In motor nerve endings and autonomic cholinergic junctions, BoNTs cleave one of three SNARE (soluble NSF attachment protein receptor) proteins, synaptobrevin, SNAP-25, and syntaxin, which constitute the synaptic fusion complex and have a determinant role in neuroexocytosis. Thus, BoNTs block the release of acetylcholine, leading to flaccid paralysis (36).Botulism is naturally a relatively rare disease in humans. However, based on their high toxicity, BoNTs are considered potential biological weapons via aerosols, which could raise the necessity to develop a vaccine against these toxins. However, on the other hand, BoNTs are currently used as FDA-approved therapeutic agents for the treatment of numerous diseases, such as dystonias and strabismus, or for cosmetic surgery (8); multiple novel applications (not FDA approved) are currently being used for the treatment of various disorders in a variety of medical fields (26). Because of these implications, the use of toxoid vaccine may not be suitable, and thus, better strategies to neutralize BoNTs, including the production of safe and effective anti-BoNT antisera, are needed.Current therapies for botulism consist mainly of supportive care, active vaccination, and passive immunization with anti-BoNT antibodies. Although these antibodies will not reverse existing paralysis, they prevent additional nerve intoxication if given before all circulating toxins bind to the neuromuscular junction. Antitoxin antibodies used in adults are of equine origin, including the bivalent equine botulinum antitoxin for serotypes A and B and equine botulinum antitoxin type E. The U.S. Army has developed an investigational heptavalent botulinum antitoxin (serotypes A to G). However, its efficacy in humans is not yet known (1).Genetic immunization by intramuscular DNA electrotransfer is a cost-effective and widely used technique involving the application of electrical pulses after intramuscular injection of plasmid DNA encoding antigens to enhance immunogenicity and vaccine efficiency (3, 35, 48). This technique requires only plasmid DNA, which can easily be produced under good manufacturing production conditions. Furthermore, intramuscular electrotransfer leads to sustained production in muscles for more than several months, with secretion into the blood circulation (5). Thus, long-lasting antibody production is expected in treated animals.In this study, we investigated the possibility of antiserum production using in vivo intramuscular DNA electrotransfer. We focused on the production of antisera against BoNT/A, BoNT/B, and BoNT/E, which are the most potent forms of BoNT identified so far (38). We treated animals with plasmid DNA encoding the nontoxic C-terminal heavy chain fragment of each toxin. This fragment is responsible for the interaction of BoNTs with the extracellular membrane and has been described as the best minimal part of the protein to elicit efficient production of neutralizing antibodies (20, 47).     

Comparison of toxins of Clostridium butyricum and Clostridium botulinum type E.

The toxin of Clostridium butyricum strains isolated from two infants with botulism is neutralized by antitoxin for type E botulinum toxin. This toxin and that of a C. botulinum type E strain were purified by the same protocol. Both toxins were Mr 145,000 proteins which, when activated with trypsin, were composed of an H subunit of Mr 105,000 and an L subunit of Mr 50,000. The activated specific toxicity of purified butyricum toxin based on an intravenous assay was 2 X 10(8) mouse 50% lethal doses (LD50s)/mg of protein, but that based on an intraperitoneal assay was 7 X 10(7) LD50s/mg, compared with 6 X 10(7) LD50s/mg for type E toxin as determined by both methods. Immunodiffusion tests with antitoxin raised with type E toxin indicated that the two toxins were serologically very similar except for a spur formed by type E toxin. The close similarities of the two toxins suggest that toxigenic C. butyricum could arise when a wild-type strain, which is normally nontoxigenic, acquires the toxin gene of a C. botulinum type E strain.     

Functionally distinct monoclonal antibodies reactive with enzymatically active and binding domains of Pseudomonas aeruginosa toxin A.

Monoclonal antibodies (MAbs) are described which react with two discrete structural domains of Pseudomonas aeruginosa toxin A and which have two distinct functional profiles. The MAbs designated T3-1C7 and T4-1F2 reacted with a 46,000-dalton peptide similar to the putative B or binding fragment of toxin A. These antibodies neutralized the cytotoxic and lethal properties of toxin but had no effect on its ADP-ribosyl transferase activity. T4-1F2 interfered with the binding of toxin A to membrane receptors on mouse fibroblasts (L cells), although the epitope for the antibody appears to be distinct from the actual receptor binding site. The MAb designated T2-1H2 reacted with intact toxin A and with a cloned, enzymatically active carboxy-terminal polypeptide similar to the toxin A fragment. This MAb neutralized the ADP-ribosyl transferase activity of activated holotoxin and of the cloned peptide, but inhibited neither binding of toxin to membrane receptors nor its cytotoxic and lethal actions. The complementary specificity and function of these MAbs confirm the functional specialization of discrete structural domains within the toxin A molecule. Our findings suggest the greater antitoxic potential of antibodies that block binding, compared with those which inhibit the enzymatic activity of toxin A.     

Enhancing the Protective Immune Response against Botulism

The need for a vaccine against botulism has increased since the discontinuation of the pentavalent (ABCDE) botulinum toxoid vaccine by the Centers for Disease Control and Prevention. The botulinum toxins (BoNTs) are the primary virulence factors and vaccine components against botulism. BoNTs comprise three domains which are involved in catalysis (LC), translocation (HCT), and host receptor binding (HCR). Recombinant HCR subunits have been used to develop the next generation of BoNT vaccines. Using structural studies and the known entry properties of BoNT/A, an HCR subunit vaccine against BoNT/A that contained the point mutation W1266A within the ganglioside binding pocket was designed. HCR/A(W1266A) did not enter primary neurons, and the crystal structure of HCR/A(W1266A) was virtually identical to that of wild-type HCR/A. Using a mouse model, experiments were performed using a high-dose vaccine and a low-dose vaccine. At a high vaccine dose, HCR/A and HCR/A(W1266A) elicited a protective immune response to BoNT/A challenge. At the low-dose vaccination, HCR/A(W1266A) was a more protective vaccine than HCR/A. α-HCR IgG titers correlated with protection from BoNT challenge, although titers to block HCR/A entry were greater in serum in HCR/A-vaccinated mice than in HCR/A(W1266A)-vaccinated mice. This study shows that removal of receptor binding capacity enhances potency of the subunit HCR vaccine. Vaccines that lack receptor binding capacity have the added property of limited off-target toxicity.     

Human monoclonal antibodies against anthrax lethal factor and protective antigen act independently to protect against Bacillus anthracis infection and enhance endogenous immunity to anthrax

The unpredictable nature of bioterrorism and the absence of real-time detection systems have highlighted the need for an efficient postexposure therapy for Bacillus anthracis infection. One approach is passive immunization through the administration of antibodies that mitigate the biological action of anthrax toxin. We isolated and characterized two protective fully human monoclonal antibodies with specificity for protective antigen (PA) and lethal factor (LF). These antibodies, designated IQNPA (anti-PA) and IQNLF (anti-LF), were developed as hybridomas from individuals immunized with licensed anthrax vaccine. The effective concentration of IQNPA that neutralized 50% of the toxin in anthrax toxin neutralization assays was 0.3 nM, while 0.1 nM IQNLF neutralized the same amount of toxin. When combined, the antibodies had additive neutralization efficacy. IQNPA binds to domain IV of PA containing the host cell receptor binding site, while IQNLF recognizes domain I containing the PA binding region in LF. A single 180-mug dose of either antibody given to A/J mice 2.5 h before challenge conferred 100% protection against a lethal intraperitoneal spore challenge with 24 50% lethal doses [LD50s] of B. anthracis Sterne and against rechallenge on day 20 with a more aggressive challenge dose of 41 LD50s. Mice treated with either antibody and infected with B. anthracis Sterne developed detectable murine anti-PA and anti-LF immunoglobulin G antibody responses by day 17 that were dependent on which antibody the mice had received. Based on these results, IQNPA and IQNLF act independently during prophylactic anthrax treatment and do not interfere with the establishment of endogenous immunity.     

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.     

Characterization of monoclonal antibodies against feline infectious peritonitis virus type II and antigenic relationship between feline,porcine, and canine coronaviruses

Summary Seven monoclonal antibodies (MAbs) with neutralizing activity against feline infectious peritonitis virus (FIPV) strain 79-1149 (type II) were prepared. When the polypeptide specificity recognized by these monoclonal antibodies (MAbs) was investigated by Western immunoblotting, all of the MAbs reacted with peplomer glycoprotein (S) of the virus. By competitive binding assay these MAbs were found to recognize at least 3 different epitopes. The reactivity of these MAbs with 6 viruses classified as FIPV type I (UCD-1, UCD-2, UCD-3, UCD-4, NW-1, and Black), feline enteric coronavirus (FECV) type II strain 79-1683, canine coronavirus (CCV) strain 1-71, and transmissible gastroenteritis virus (TGEV) strains TO-163 and SH was examined by neutralization tests. All MAbs neutralized FECV strain 79-1683, CCV strain 1-71, and TGEV strains TO-163 and SH, while they did not neutralize the 6 FIPV type I viruses. Moreover, the MAb against TGEV strain TO-163, which has strong neutralizing activity against 7 TGEV viruses, neutralized CCV strain 1-71, FECV strain 79-1683, and FIPV strain 79-1146, but did not neutralize the 6 FIPV type I viruses.These results demonstrated that there are at least 3 epitopes involved in the neutralization of FIPV type II strain 79-1146, and that these epitopes are not present in FIPV type I viruses but are present in FECV strain 79-1683 which does not induce feline infectious peritonitis, TGEV strains TO-163 and SH, and CCV strain 1-71. These results suggest the presence of 2 serotypes of FIPV which can be clearly distinguished by the neutralization test using MAbs.