DNA vaccination using the fragment C of botulinum neurotoxin type A provided protective immunity in mice

Botulinum neurotoxin (BoNT) is one of the most toxic substances known to produce severe neuromuscular paralysis. The currently used vaccine is prepared mainly from biohazardous toxins. Thus, we studied an alternative method and demonstrated that DNA immunization provided sufficient protection against botulism in a murine model. A plasmid of pBoNT/A-Hc, which encodes the fragment C gene of type A botulinum neurotoxin, was constructed and fused with an Igkappa leader sequence under the control of a human cytomegalovirus promoter. After 10 cycles of DNA inoculation with this plasmid, mice survived lethal doses of type A botulinum neurotoxin challenges. Immunized mice also elicited cross-protection to the challenges of type E botulinum neurotoxin. This is the first study demonstrating the potential use of DNA vaccination for botulinum neurotoxins.     

Isolation and molecular size of Clostridium botulinum type C toxin.

A procedure is described for the purification of hemagglutinin-free Clostridium botulinum type C toxin. The toxin was purified approximately 1,000-fold from the original culture supernatant in an overall yield of 60% to a final specific toxicity of 4.4 x 10(7) minimal lethal doses/mg of protein. The toxin had a molecular weight of 141,000 and consisted of a heavy and a light chain. The molecular weights of the subunits were approximately 98,000 and 53,000. When comparing the molecular size and composition of type C toxin to that of botulinum toxins of different types, some common features may be suggested; i.e., the toxin has a molecular weight between 141,000 to 160,000 and is comprised of a heavy and a light chain linked by disulfide bonds (or bond).     

Isolation and molecular size of Clostridium botulinum type C toxin.

A procedure is described for the purification of hemagglutinin-free Clostridium botulinum type C toxin. The toxin was purified approximately 1,000-fold from the original culture supernatant in an overall yield of 60% to a final specific toxicity of 4.4 x 10(7) minimal lethal doses/mg of protein. The toxin had a molecular weight of 141,000 and consisted of a heavy and a light chain. The molecular weights of the subunits were approximately 98,000 and 53,000. When comparing the molecular size and composition of type C toxin to that of botulinum toxins of different types, some common features may be suggested; i.e., the toxin has a molecular weight between 141,000 to 160,000 and is comprised of a heavy and a light chain linked by disulfide bonds (or bond).     

Botulinum type F neurotoxin. Large-scale purification and characterization of its binding to rat cerebrocortical synaptosomes.

1. A large-scale purification procedure has been developed for Clostridium botulinum type F neurotoxin. Commencing with 160 litres of bacterial culture, 101 mg of purified type F neurotoxin with a specific toxicity of 2 x 10(7) mouse LD50 (median lethal dose).mg-1 were obtained. 2. Purified type F neurotoxin was labelled to high specific radioactivity (900-1360 Ci/mmol) without loss of biological activity using a chloramine-T procedure. Of the two neurotoxin subunits, the heavy chain was preferentially radiolabelled. 3. Radiolabelled type F neurotoxin displayed specific saturable binding to rat synaptosomes. At least two pools of acceptors were evident: a low content of high-affinity acceptors sites [KD approximately 0.15 nM; Bmax (maximal binding) 20 fmol/mg] and a larger pool of lower-affinity sites (KD greater than 20 nM; Bmax greater than 700 fmol/mg). Both pools of acceptors were sensitive to trypsin and neuraminidase treatment, which suggests that protein and sialic acid residues are components of the synaptosomal acceptors. 4. Experiments investigating competition among botulinum neurotoxin types A, B, E and F for acceptors on rat brain synaptosomes showed that type F neurotoxin binds to acceptor molecules which are completely distinct from those of the other three neurotoxins.     

A peptide-based immunoassay for antibodies against botulinum neurotoxin A

Cervical dystonia (CD) is due to neck-muscle spasms that cause pain and involuntary contractions resulting in abnormal neck movements and posture. Symptoms can be relieved by injecting the affected muscle with a botulinum neurotoxin (BoNT, usually type A or type B). The therapeutic benefits are impermanent and toxin injections need to be repeated every 3-6 months. In a very small percentage of patients (less with BoNT/A than with BoNT/B) the treatment elicits blocking anti-toxin antibodies (Abs), which reduce or terminate the patient's responsiveness to further treatment. We have recently mapped (Dolimbek et al., 2006) the CD sera Ab-binding profile using a panel of 60, 19-residue peptides that encompassed the entire H chain sequence 449-1296 and overlapped consecutively by 5 residues. Abs in CD sera bound to one or more of the peptides N25, C10, C15, C20, and C31. This suggested the possibility that binding to these peptides could be used for assay of Abs in CD sera. Data analysis reported here found that Ab binding to these regions showed very significant deviations from the control responses. Of these four peptides, C10 showed the most significant level of separation between patient and control groups (p = 5 x 10(-7)) and the theoretical resolution (i.e., ability to distinguish CD patients from control, see full definition under 'Statistical analysis' in Methods), 84%, was about 4% higher than the least resolved response, C31 (p = 6 x 10(-6), resolution 80%). Since the amounts of Abs bound to a given peptide varied with the patient and not all the patients necessarily recognized all four peptides, there was the possibility that binding to combinations of two or more peptides might give a better discriminatory capability. Using two peptides, C10 plus C31, the resolution improved to 87% (p = 4 x 10(-8)). These two peptides appeared to compliment each other and negate the lower resolution of C31. Combination of three peptides gave resolutions that ranged from 85 (N25 + C15 + C31; p = 2 x 10(-7)) to 88% (C10 + C15 + C31; p = 1 x 10(-8)). Finally, using the data of all four peptides, N25 + C10 + C15 + C31, gave a resolution of 86% (p = 1 x 10(-7)). Although these levels of resolution are somewhat lower than that obtained with whole BoNT/A (resolution 97%; p = 6 x 10(-12)), it may be concluded that the two-peptide combination C10 + C31, or the three-peptide combination C10 + C15 + C31 (affording resolutions of 87 and 88%, respectively) provide a good diagnostic, toxin-free procedure for assay of total specific anti-toxin Abs in BoNT/A-treated CD patients.     

Detection of six serotypes of botulinum neurotoxin using fluorogenic reporters

Methods that do not require animal sacrifice to detect botulinum neurotoxins (BoNTs) are critical for BoNT antagonist discovery and the advancement of quantitative assays for biodefense and pharmaceutical applications. Here we describe the development and optimization of fluorogenic reporters that detect the proteolytic activity of BoNT/A, B, D, E, F, and G serotypes in real time with femtomolar to picomolar sensitivity. Notably, the reporters can detect femtomolar concentrations of BoNT/A in 4 h and BoNT/E in 20 h, sensitivity that equals that of animal-based methods. The reporters can be used to determine the specific activity of BoNT preparations with intra- and inter-assay coefficients of variation of approximately 10%. Finally, we find that the greater sensitivity of our reporters compared with those used in other commercially available assays makes the former attractive candidates for high-throughput screening of BoNT antagonists.     

C terminal half fragment (50 kDa) of heavy chain components of Clostridium botulinum type C and D neurotoxins can be used as an effective vaccine

Recombinant whole heavy chains (H, 100 kDa) and their N-terminal (Hn, 50 kDa) and C-terminal (Hc, 50 kDa) half fragments of Clostridium botulinum type C and D neurotoxins were expressed as glutathione S-transferase (GST) fusion proteins in Escherichia coli. GST eliminated-preparations of H (10 microg), Hn (5 microg), Hc (5 microg), or a mixture of Hn (5 microg) and Hc (5 microg) of types C and D were mixed with an equal volume of adjuvant, and then were twice injected into mice subcutaneously. After immunization, the mice were challenged with up to 10(6) the minimum lethal doses (MLD)/0.5 ml of C or D toxin, the type of which was same as that of the immunogens. All of the mice immunized with antigens except for Hn survived against 10(5) to 10(6) MLD/0.5 ml of the toxins, but the mice immunized with Hn were killed by 100 MLD/0.5 ml. The mice immunized with a mixture of C-Hc and D-Hc, each 5 microg, also showed a high level of resistance against both C and D toxins. Antibody levels immunized with GST fused-or GST eliminatedpreparation were quite similar. These results indicate that recombinant GST-fused Hc can be used as a safe and effective vaccine for type C and D botulism in animals. It also became clear that one time inoculation with a large amount of C-Hc or D-Hc, 100 microg, is useful for vaccine trials in mice.     

Sensitive detection of botulinum neurotoxin types C and D with an immunoaffinity chromatographic column test

A sensitive and specific immunoassay for the simultaneous detection of Clostridium botulinum type C (BoNT/C) and type D neurotoxin was developed. Goat anti-mouse immunoglobulin G was bound to polyethylene disks in a small disposable column used for this assay. The sample was preincubated together with monoclonal antibodies specific for the heavy chain of BoNT/C and D and affinity-purified, biotinylated polyclonal antibodies against these neurotoxins. This complex was captured on the assay disk. Streptavidin-poly-horseradish peroxidase was used as a conjugate, and a precipitating substrate allowed the direct semiquantitative readout of the assay, if necessary. For a more accurate quantitative detection, the substrate can be eluted and measured in a photometer. Depending on the preincubation time, a sensitivity of 1 mouse lethal dose ml(-1) was achieved in culture supernatants.     

Properties and use of botulinum toxin and other microbial neurotoxins in medicine.

Crystalline botulinum toxin type A was licensed in December 1989 by the Food and Drug Administration for treatment of certain spasmodic muscle disorders following 10 or more years of experimental treatment on human volunteers. Botulinum toxin exerts its action on a muscle indirectly by blocking the release of the neurotransmitter acetylcholine at the nerve ending, resulting in reduced muscle activity or paralysis. The injection of only nanogram quantities (1 ng = 30 mouse 50% lethal doses [U]) of the toxin into a spastic muscle is required to bring about the desired muscle control. The type A toxin produced in anaerobic culture and purified in crystalline form has a specific toxicity in mice of 3 x 10(7) U/mg. The crystalline toxin is a high-molecular-weight protein of 900,000 Mr and is composed of two molecules of neurotoxin (ca. 150,000 Mr) noncovalently bound to nontoxic proteins that play an important role in the stability of the toxic unit and its effective toxicity. Because the toxin is administered by injection directly into neuromuscular tissue, the methods of culturing and purification are vital. Its chemical, physical, and biological properties as applied to its use in medicine are described. Dilution and drying of the toxin for dispensing causes some detoxification, and the mouse assay is the only means of evaluation for human treatment. Other microbial neurotoxins may have uses in medicine; these include serotypes of botulinum toxins and tetanus toxin. Certain neurotoxins produced by dinoflagellates, including saxitoxin and tetrodotoxin, cause muscle paralysis through their effect on the action potential at the voltage-gated sodium channel. Saxitoxin used with anaesthetics lengthens the effect of the anaesthetic and may enhance the effectiveness of other medical drugs. Combining toxins with drugs could increase their effectiveness in treatment of human disease.