Safety and Pharmacokinetics of a Four Monoclonal Antibody Combination Against Botulinum C and D Neurotoxins.

Botulism is caused by botulinum neurotoxin (BoNT), the most poisonous substance known. BoNTs are also classified as Tier 1 biothreat agents due to their high potency and lethality. The existence of seven BoNT serotypes (A-G), which differ between 35% to 68% in amino acid sequence, necessitates the development of serotype specific countermeasures. We present results of a Phase 1 clinical study of an anti-toxin to BoNT serotypes C and D, NTM-1634, which consists of an equimolar mixture of four fully human IgG1 monoclonal antibodies (mAbs), each binding to non-overlapping epitopes on BoNT serotypes C and D resulting in potent toxin neutralization in rodents. This first-in-human study evaluated the safety and pharmacokinetics of escalating doses of NTM-1634 administered intravenously to healthy adults (NCT03046550). Three cohorts of eight healthy subjects received a single intravenous dose of NTM-1634 or placebo at 0.33 mg/kg, 0.66 mg/kg or 1 mg/kg. Follow-up examinations and pharmacokinetic evaluations were continued up to 121 days post-infusion. Subjects were monitored using physical examinations, hematology and chemistry blood tests, and electrocardiograms. Pharmacokinetic parameters were estimated using noncompartmental methods. The results demonstrated that the materials were safe and well-tolerated with the expected half-lives for human mAbs and with minimal anti-drug antibodies detected over the dose ranges and duration of the study

Botulinum Neurotoxin Detection and Differentiation by Mass Spectrometry

A new rapid, mass spectrometry-based method to detect and differentiate botulinal neurotoxins is described

Snake and spider toxins induce a rapid recovery of function of botulinum neurotoxin paralysed neuromuscular junction

Botulinum neurotoxins (BoNTs) and some animal neurotoxins (-Bungarotoxin, -Btx, from elapid snakes and -Latrotoxin, -Ltx, from black widow spiders) are pre-synaptic neurotoxins that paralyse motor axon terminals with similar clinical outcomes in patients. However, their mechanism of action is different, leading to a largely-different duration of neuromuscular junction (NMJ) blockade. BoNTs induce a long-lasting paralysis without nerve terminal degeneration acting via proteolytic cleavage of SNARE proteins, whereas animal neurotoxins cause an acute and complete degeneration of motor axon terminals, followed by a rapid recovery. In this study, the injection of animal neurotoxins in mice muscles previously paralyzed by BoNT/A or /B accelerates the recovery of neurotransmission, as assessed by electrophysiology and morphological analysis. This result provides a proof of principle that, by causing the complete degeneration, reabsorption, and regeneration of a paralysed nerve terminal, one could favour the recovery of function of a biochemically- or genetically-altered motor axon terminal. These observations might be relevant to dying-back neuropathies, where pathological changes first occur at the neuromuscular junction and then progress proximally toward the cell body

Botulinum neurotoxins are zinc proteins.

The available amino acid sequences of 150-kDa botulinum and tetanus neurotoxins show the presence of a closely homologous segment in the middle of the light chain (NH2-terminal 50 kDa), which is the intracellularly active portion of the toxin. This segment contains the zinc binding motif of metalloendopeptidases, HEXXH. Atomic adsorption analysis of botulinum neurotoxins (serotypes A, B, and E) made on the basis of this observation demonstrated the presence of one zinc atom/molecule of 150-kDa neurotoxin. Conditions were found for the removal of the zinc ion with chelating agents and for the restoration of the normal metal content. The conserved segment, which includes the zinc binding motif, was synthesized and shown to bind [65Zn]2+. Chemical modification experiments indicated that two histidines and no cysteines are involved in Zn2+ coordination in agreement with a probable catalytic role for the zinc ion. The present findings suggest the possibility that botulinum neurotoxins are zinc proteases

Current Methods for Detecting the Presence of Botulinum Neurotoxins in Food and Other Biological Samples

https://scholarlycommons.pacific.edu/cop-facbooks/1205/thumbnail.jp

Immunological Characterization and Neutralizing Ability of Monoclonal Antibodies Directed Against Botulinum Neurotoxin Type H.

BackgroundOnly Clostridium botulinum strain IBCA10-7060 produces the recently described novel botulinum neurotoxin type H (BoNT/H). BoNT/H (N-terminal two-thirds most homologous to BoNT/F and C-terminal one-third most homologous to BoNT/A) requires antitoxin to toxin ratios ≥1190:1 for neutralization by existing antitoxins. Hence, more potent and safer antitoxins against BoNT/H are needed.MethodsWe therefore evaluated our existing monoclonal antibodies (mAbs) to BoNT/A and BoNT/F for BoNT/H binding, created yeast-displayed mutants to select for higher-affinity-binding mAbs by using flow cytometry, and evaluated the mAbs' ability to neutralize BoNT/H in the standard mouse bioassay.ResultsAnti-BoNT/A HCC-binding mAbs RAZ1 and CR2 bound BoNT/H with high affinity. However, only 1 of 6 BoNT/F mAbs (4E17.2A) bound BoNT/H but with an affinity >800-fold lower (equilibrium dissociation binding constant [KD] = 7.56 × 10(-8)M) than its BoNT/F affinity (KD= 9.1 × 10(-11)M), indicating that the N-terminal two-thirds of BoNT/H is immunologically unique. The affinity of 4E17.2A for BoNT/H was increased >500-fold to KD= 1.48 × 10(-10)M (mAb 4E17.2D). A combination of mAbs RAZ1, CR2, and 4E17.2D completely protected mice challenged with 280 mouse median lethal doses of BoNT/H at a mAb dose as low as 5 µg of total antibody.ConclusionsThis 3-mAb combination potently neutralized BoNT/H and represents a potential human antitoxin that could be developed for the prevention and treatment of type H botulism

A historical and proteomic analysis of botulinum neurotoxin type/G

<p>Abstract</p> <p>Background</p> <p><it>Clostridium botulinum </it>is the taxonomic designation for at least six diverse species that produce botulinum neurotoxins (BoNTs). There are seven known serotypes of BoNTs (/A through/G), all of which are potent toxins classified as category A bioterrorism agents. BoNT/G is the least studied of the seven serotypes. In an effort to further characterize the holotoxin and neurotoxin-associated proteins (NAPs), we conducted an <it>in silico </it>and proteomic analysis of commercial BoNT/G complex. We describe the relative quantification of the proteins present in the/G complex and confirm our ability to detect the toxin activity <it>in vitro</it>. In addition, we review previous literature to provide a complete description of the BoNT/G complex.</p> <p>Results</p> <p>An in-depth comparison of protein sequences indicated that BoNT/G shares the most sequence similarity with the/B serotype. A temperature-modified Endopep-MS activity assay was successful in the detection of BoNT/G activity. Gel electrophoresis and in gel digestions, followed by MS/MS analysis of/G complex, revealed the presence of four proteins in the complexes: neurotoxin (BoNT) and three NAPs--nontoxic-nonhemagglutinin (NTNH) and two hemagglutinins (HA70 and HA17). Rapid high-temperature in-solution tryptic digestions, coupled with MS/MS analysis, generated higher than previously reported sequence coverages for all proteins associated with the complex: BoNT 66%, NTNH 57%, HA70 91%, and HA17 99%. Label-free relative quantification determined that the complex contains 30% BoNT, 38% NTNH, 28% HA70, and 4% HA17 by weight comparison and 17% BoNT, 23% NTNH, 42% HA70, and 17% HA17 by molecular comparison.</p> <p>Conclusions</p> <p>The <it>in silico </it>protein sequence comparisons established that the/G complex is phenetically related to the other six serotypes of <it>C. botulinum</it>. Proteomic analyses and Endopep-MS confirmed the presence of BoNT and NAPs, along with the activity of the commercial/G complex. The use of data-independent MS^Edata analysis, coupled to label-free quantification software, suggested that the weight ratio BoNT:NAPs is 1:3, whereas the molar ratio of BoNT:NTNH:HA70:HA17 is 1:1:2:1, within the BoNT/G progenitor toxin.</p

Accelerated Neuronal Cell Recovery from Botulinum Neurotoxin Intoxication by Targeted Ubiquitination

Botulinum neurotoxin (BoNT), a Category A biodefense agent, delivers a protease to motor neuron cytosol that cleaves one or more soluble NSF attachment protein receptors (SNARE) proteins involved in neurotransmission to cause a flaccid paralysis. No antidotes exist to reverse symptoms of BoNT intoxication so severely affected patients require artificial respiration with prolonged intensive care. Time to recovery depends on toxin serotype because the intraneuronal persistence of the seven known BoNT serotypes varies widely from days to many months. Our therapeutic antidote strategy is to develop ‘targeted F-box’ (TFB) agents that target the different intraneuronal BoNT proteases for accelerated degradation by the ubiquitin proteasome system (UPS), thus promoting rapid recovery from all serotypes. These agents consist of a camelid heavy chain-only VH (VHH) domain specific for a BoNT protease fused to an F-box domain recognized by an intraneuronal E3-ligase. A fusion protein containing the 14 kDa anti-BoNT/A protease VHH, ALcB8, joined to a 15 kDa F-box domain region of TrCP (D5) was sufficient to cause increased ubiquitination and accelerate turnover of the targeted BoNT/A protease within neurons. Neuronal cells expressing this TFB, called D5-B8, were also substantially resistant to BoNT/A intoxication and recovered from intoxication at least 2.5 fold quicker than control neurons. Fusion of D5 to a VHH specific for BoNT/B protease (BLcB10) led to accelerated turnover of the targeted protease within neurons, thus demonstrating the modular nature of these therapeutic agents and suggesting that development of similar therapeutic agents specific to all botulinum serotypes should be readily achievable