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.     

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.     

Sequence variation within botulinum neurotoxin serotypes impacts antibody binding and neutralization

The botulinum neurotoxins (BoNTs) are category A biothreat agents which have been the focus of intensive efforts to develop vaccines and antibody-based prophylaxis and treatment. Such approaches must take into account the extensive BoNT sequence variability; the seven BoNT serotypes differ by up to 70% at the amino acid level. Here, we have analyzed 49 complete published sequences of BoNTs and show that all toxins also exhibit variability within serotypes ranging between 2.6 and 31.6%. To determine the impact of such sequence differences on immune recognition, we studied the binding and neutralization capacity of six BoNT serotype A (BoNT/A) monoclonal antibodies (MAbs) to BoNT/A1 and BoNT/A2, which differ by 10% at the amino acid level. While all six MAbs bound BoNT/A1 with high affinity, three of the six MAbs showed a marked reduction in binding affinity of 500- to more than 1,000-fold to BoNT/A2 toxin. Binding results predicted in vivo toxin neutralization; MAbs or MAb combinations that potently neutralized A1 toxin but did not bind A2 toxin had minimal neutralizing capacity for A2 toxin. This was most striking for a combination of three binding domain MAbs which together neutralized >40,000 mouse 50% lethal doses (LD(50)s) of A1 toxin but less than 500 LD(50)s of A2 toxin. Combining three MAbs which bound both A1 and A2 toxins potently neutralized both toxins. We conclude that sequence variability exists within all toxin serotypes, and this impacts monoclonal antibody binding and neutralization. Such subtype sequence variability must be accounted for when generating and evaluating diagnostic and therapeutic antibodies.     

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.     

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.     

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.     

Molecular characterization of murine humoral immune response to botulinum neurotoxin type A binding domain as assessed by using phage antibody libraries.

To produce antibodies capable of neutralizing botulinum neurotoxin type A (BoNT/A), the murine humoral immune response to BoNT/A binding domain (H(C)) was characterized at the molecular level by using phage antibody libraries. Mice were immunized with BoNT/A H(C), the spleens were harvested, and single-chain Fv (scFv) phage antibody libraries were constructed from the immunoglobulin heavy and light chain variable region genes. Phage expressing BoNT/A binding scFv were isolated by selection on immobilized BoNT/A and BoNT/A H(C). Twenty-eight unique BoNT/A H(C) binding scFv were identified by enzyme-linked immunosorbent assay and DNA sequencing. Epitope mapping using surface plasmon resonance in a BIAcore revealed that the 28 scFv bound to only 4 nonoverlapping epitopes with equilibrium constants (Kd) ranging from 7.3 x 10(-8) to 1.1 x 10(-9) M. In a mouse hemidiaphragm assay, scFv binding epitopes 1 and 2 significantly prolonged the time to neuroparalysis, 1.5- and 2.7-fold, respectively, compared to toxin control. scFv binding to epitopes 3 and 4 showed no protection against neuroparalysis. A combination of scFv binding epitopes 1 and 2 had an additive effect on time to neuroparalysis, which increased to 3.9-fold compared to the control. The results suggest that there are two "productive" receptor binding sites on H(C) which lead to toxin internalization and toxicity. Blockade of these two epitopes with monoclonal antibodies may provide effective immunoprophylaxis or therapy against BoNT/A intoxication.     

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.     

Reactivity of VP4-specific monoclonal antibodies to a serotype 4 porcine rotavirus with distinct serotypes of human (symptomatic and asymptomatic) and animal rotaviruses.

Thirteen hybridomas secreting VP4-specific monoclonal antibodies against the Gottfried strain of porcine rotavirus (serotype 4) were produced and characterized. Nine of the hybridomas secreted neutralizing monoclonal antibodies (N-MAbs) against Gottfried rotavirus. These N-MAbs were divided into five distinct groups (groups I to V) according to their patterns of reactivity with different serotypes of human and animal rotaviruses. Group I N-MAbs (n = 3) were cross-reactive with five different serotypes of human rotavirus examined by a plaque reduction virus neutralization test. Group II N-MAbs (n = 3) neutralized all symptomatic human rotavirus serotypes tested and asymptomatic human rotavirus serotype 4 to a low titer. The single group III N-MAb neutralized mainly symptomatic human rotavirus serotypes 2 and 9 and none of the asymptomatic human rotavirus serotypes. The one N-MAb in group IV reacted at low titers with only asymptomatic human rotavirus serotypes 1 through 4. A group V N-MAb recognized serotype 4 porcine rotaviruses (Gottfried and SB-2) but no other human or animal rotaviruses examined. None of the N-MAbs recognized any animal rotaviruses tested (SA-11, RRV, OSU, NCDV, and B223), except for the Gottfried and SB-2 rotaviruses. The failure of N-MAbs (groups I to IV) to react with any animal rotaviruses tested but their ability to react variably with all human rotaviruses tested suggest that neutralizing epitopes on the VP4 protein are highly conserved between the Gottfried porcine and human rotaviruses. The Gottfried rotavirus may possibly represent a naturally occurring reassortant between pig and human rotaviruses or a rotavirus which is human in origin but pathogenic for swine.     

Serologic analysis of human rotavirus serotypes P1A and P2 by using monoclonal antibodies.

Three human rotavirus (HRV) VP4 serotypes and one subtype have been described on the basis of a fourfold or an eightfold-or-greater difference in neutralization titer when tested with hyperimmune antisera to recombinant VP4 or VP8* (serotypes P1A, P1B, P2, and P3). To start to analyze the antigenic basis underlying serotype specificity, we produced a library of 13 VP4-specific neutralizing monoclonal antibodies (NMAbs) to two HRVs, the serotype P1A strain Wa and the serotype P2 strain ST3, and characterized the reactivity of these NMAbs with a panel of serotypically diverse HRV strains by neutralization assay and enzyme-linked immunosorbent assay (ELISA). We characterized the serotypic specificity of the NMAbs by using a fourfold or an eightfold-or-greater difference in titer against the homologous (i.e., immunogen) and heterologous strains as a criterion for serotype. Some ST3-derived NMAbs reacted specifically with serotype P2 HRVs by ELISA and/or neutralization assay, while some Wa-derived NMAbs reacted specifically by ELISA and/or neutralization assay with some or all serotype P1A HRVs. Other Wa- and ST3-derived NMAbs reacted with some or all serotype P1A and P2 HRV strains by neutralization assay and ELISA. Most NMAbs did not react with serotype P1B or P3 strains. In previous studies, three distinct operationally defined epitopes have been identified on VP4 by examining the reactivity patterns of selected antigenic variants of HRV strain KU. At least one of the NMAbs described here recognizes an epitope unrelated to these previously identified epitopes, since it neutralized both KU and its variants.     

The Receptor Binding Domain of Botulinum Neurotoxin Serotype A (BoNT/A) Inhibits BoNT/A and BoNT/E Intoxications In Vivo

The receptor binding domain of botulinum neurotoxin (BoNT), also designated the C terminus of the heavy chain (H_C), is a promising vaccine candidate against botulism. In this study, a highly efficient expression system for the protein was developed in Escherichia coli, which provided yields that were 1 order of magnitude higher than those reported to date (350 mg H_C per liter). The product was highly immunogenic, protecting mice from a challenge with 10⁵ 50% lethal dose (LD₅₀) after a single vaccination and generating a neutralizing titer of 49.98 IU/ml after three immunizations. In addition, a single boost with H_C increased neutralizing titers by up to 1 order of magnitude in rabbits hyperimmunized against toxoid. Moreover, we demonstrate here for the first time in vivo inhibition of BoNT/A intoxication by H_C/A, presumably due to a blockade of the neurotoxin protein receptor SV2. Administration of H_C/A delayed the time to death from 10.4 to 27.3 h in mice exposed to a lethal dose of BoNT/A (P = 0.0005). Since BoNT/A and BoNT/E partially share SV2 isoforms as their protein receptors, the ability of H_C/A to cross-inhibit BoNT/E intoxication was evaluated. The administration of H_C/A together with BoNT/E led to 50% survival and significantly delayed the time to death for the nonsurviving mice (P = 0.003). Furthermore, a combination of H_C/A and a subprotective dose of antitoxin E fully protected mice against 850 mouse LD₅₀ of BoNT/E, suggesting complementary mechanisms of protection consisting of toxin neutralization by antibodies and receptor blocking by H_C/A.     

Neutralizing antibodies to heterologous animal rotavirus serotypes 5, 6, 7, and 10 in sera from Ecuadorian children.

Serum samples from 870 Ecuadorian children who underwent natural rotavirus exposure were tested for neutralizing serum antibody to heterologous animal rotavirus (RV) serotypes. Six percent of the sera neutralized porcine RV OSU (serotype 5), 10% neutralized bovine RV NCDV (serotype 6), 4% neutralized avian RV Ch-2 (serotype 7), and 8% neutralized bovine RV V1005 (serotype 10). Neutralization was defined as a 90% reduction in infectious virus at a 1:100 serum dilution. The prevalence of antibody to all four heterotypic viruses increased with the age of the children and the number of human RV serotypes neutralized, but prevalences did not differ significantly between children from rural and urban areas of Ecuador. No serum sample that specifically neutralized bovine RV NCDV was identified. We inferred from the seroepidemiological analysis that human RVs contain immunorecessive neutralization epitopes that can stimulate cross-neutralizing antibody to heterotypic animal RVs. This occurs increasingly with age and with the number of human serotypes recognized by a child's neutralizing antibody. Thus, it appears that a broadened immune response to the heterotypic strains occurs with repetitive RV infections.     

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.     

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+.     

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).     

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.     

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.     

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.     

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.