Tetanus and tetanus neurotoxin: From peripheral uptake to central nervous tissue targets.

Tetanus is a deadly but preventable disease caused by a protein neurotoxin produced by Clostridium tetani. Spores of C. tetani may contaminate a necrotic wound and germinate into a vegetative bacterium that releases a toxin, termed tetanus neurotoxin (TeNT). TeNT enters the general circulation, binds to peripheral motor neurons and sensory neurons, and is transported retroaxonally to the spinal cord. It then enters inhibitory interneurons and blocks the release of glycine or GABA causing a spastic paralysis. This review attempts to correlate the metalloprotease activity of TeNT and its trafficking and localization into the vertebrate body to the nature and sequence of appearance of the symptoms of tetanus.

Exceptionally potent human monoclonal antibodies are effective for prophylaxis and treatment of tetanus in mice.

We used human monoclonal antibodies (humAbs) to study the mechanism of neuron intoxication by tetanus neurotoxin and to evaluate these antibodies as a safe preventive and therapeutic substitute for hyperimmune sera to treat tetanus in mice. By screening memory B cells from immune donors, we selected 2 tetanus neurotoxin-specific mAbs with exceptionally high neutralizing activities and extensively characterized them both structurally and functionally. We found that these antibodies interfered with the binding and translocation of the neurotoxin into neurons by interacting with 2 epitopes, whose identification pinpoints crucial events in the cellular pathogenesis of tetanus. Our observations explain the neutralization ability of these antibodies, which we found to be exceptionally potent in preventing experimental tetanus when injected into mice long before the toxin. Moreover, their Fab derivatives neutralized tetanus neurotoxin in post-exposure experiments, suggesting their potential for therapeutic use via intrathecal injection. As such, we believe these humAbs, as well as their Fab derivatives, meet the requirements to be considered for prophylactic and therapeutic use in human tetanus and are ready for clinical trials.

Transglutaminase participates in the blockade of neurotransmitter release by tetanus toxin: evidence for a novel biological function.

Inhibition of neuroexocytosis by tetanus neurotoxin (TeNT) involves VAMP-2/synaptobrevin-2 cleavage. However, deletion of the TeNT activity does not completely abolish its inhibitory action. TeNT is a potent activator of the cross-linking enzyme transglutaminase 2 (TGase 2) in vitro. The role of the latter mechanism in TeNT poisoning was investigated in isolated nerve terminals and intact neurons. TeNT-induced inhibition of glutamate release from rat cortical synaptosomes was associated with a simultaneous activation of neuronal transglutaminase (TGase) activity. The TeNT-induced blockade of neuroexocytosis was strongly attenuated by pretreatment of either live Aplysia neurons or isolated nerve terminals with specific TGase inhibitors or neutralizing antibodies. The same treatments completely abolished the residual blockade of neuroexocytosis of a non-proteolytic mutant of TeNT light chain. Electrophysiological studies indicated that TGase activation occurs at an early step of TeNT poisoning and contributes to the inhibition of transmitter release. Bioinformatics and biochemical analyses identified synapsin I and SNAP-25 as potential presynaptic TGase substrates in isolated nerve terminals, which are potentially involved in the inhibitory action of TeNT. The results suggest that neuronal TGase activity plays an important role in the regulation of neuroexocytosis and is one of the intracellular targets of TeNT in neurons.

Contribution of Fc fragment of monoclonal antibodies to tetanus toxin neutralization.

BACKGROUND: Monoclonal antibodies (MAbs) against neurotoxin of Clostridium tetani are considered as a novel source of immunoglobulins for passive immunotherapy of tetanus. Toxin neutralization is classically attributed to the Fab and F(ab')2 fragments of antibodies. Herein, we generated Fab and F(ab')2 fragments of three toxin neutralizing mouse MAbs and compared their neutralizing activities to those of their intact molecules. METHODS: Fab and F (ab')2 fragments of the antibodies were generated by papain and pepsin digestions, respectively, and their toxin neutralizing activities were compared with those of the intact antibodies in an in vivo toxin neutralization assay. RESULTS: While low doses of the intact MAbs were able to fully protect the mice against tetanus toxin, none of the mice which received Fab or F(ab')2 fragments survived until day 14, even at the highest administered dose. All mice receiving human polyclonal anti-tetanus immunoglobulin or their fragments were fully protected. CONCLUSION: Reduction in toxin neutralization activities of Fab and F(ab')2 fragments of our MAbs seems to be influenced by their Fc regions. Steric hindrance of the Fc region on the receptor-binding site of the toxin may explain the stronger neutralization of the toxin by the intact MAbs in comparison to their fragments.

Transcutaneous delivery of tetanus toxin Hc fragment induces superior tetanus toxin neutralizing antibody response compared to tetanus toxoid.

Transcutaneous immunization is a promising vaccination delivery strategy which targets potent immune cells residing in the outer layer of the skin. In this study, the immunogenicity and neutralizing potency of the non-toxic Hc fragment of tetanus toxin (HcWT) and a mutant of Hc lacking ganglioside binding activity were compared with that of tetanus toxoid (TTxd) following transcutaneous immunization (TCI) of mice. Mice immunized with HcWT in the absence of an adjuvant induced highest anti-toxoid and anti-Hc antibody titres, with a significant increase in the toxin neutralizing antibody response compared with TTxd. These results are in contrast to previous studies employing subcutaneous delivery, where TTxd was found to be a more potent immunogen than the Hc fragment of the toxin. We conclude that the HcWT protein is more immunogenic than TTxd when given via the transcutaneous route. Our results suggest that TCI may provide an opportunity for effective delivery of toxin-like antigens which harbor protective epitopes and that traditional toxoid proteins may not be optimal antigens for skin immunization.

Activity-dependent changes in partial VAMP complexes during neurotransmitter release.

The temporal sequence of SNARE protein interactions that cause exocytosis is unknown. Blockade of synaptic neurotransmitter release through cleavage of VAMP/synaptobrevin by tetanus toxin light chain (TeNT-LC) was accelerated by nerve stimulation. Botulinum/B neurotoxin light chain (BoNT/B-LC), which cleaves VAMP at the same site as TeNT-LC, did not require stimulation. Because TeNT-LC requires the N-terminal coil domain of VAMP for binding but BoNT/B-LC requires the C-terminal coil domain, it seems that, before nerve activity, the N-terminal domain is shielded in a protein complex, but the C-terminal domain is exposed. This N-terminal complex lasts until nerve activity occurs and may serve to cock synaptic vesicles for immediate exocytosis upon Ca2+ entry.

Cooperative binding of anti-tetanus toxin monoclonal antibodies: Implications for designing an efficient biclonal preparation to prevent tetanus toxin intoxication.

Oligoclonal combinations of several monoclonal antibodies (MAbs) are being considered for the treatment of various infectious pathologies. These combinations are less sensitive to antigen structural changes than individual MAbs; at the same time, their characteristics can be more efficiently controlled than those of polyclonal antibodies. The main goal of this study was to evaluate the binding characteristics of six biclonal equimolar preparations (BEP) of tetanus toxin (TeNT)-specific MAbs and to investigate how the MAb combination influences the BEPs' protective capacity. We show that a combination of TeNT-specific MAbs, which not only bind TeNT but also exert positive cooperative effects, results in a BEP with superior binding characteristics and protective capacity, when compared with the individual component MAbs. Furthermore, we show that a MAb with only partial protective capacity but positive effects on the binding of the other BEP component can be used as a valuable constituent of the BEP.

Key protection factors against tetanus: Anti-tetanus toxin antibody affinity and its ability to prevent tetanus toxin - ganglioside interaction.

Antibodies capable to neutralize tetanus toxin (TeNT) are key factors in protection against tetanus disease. Although antibody-based therapeutics for treatment of tetanus exist on the market its production is tedious. Hence, the tetanus-specific antibodies preparation that could be easily produced in large scale in vitro would be beneficial. Monoclonal antibodies (MAbs) are considered for a long time as a reagent of choice, but the core drawback is how to select a MAb that would be safe in providing efficacious protection. In this study we have investigated the parameters crucial for a single MAb to be assigned as protective. Eight murine MAbs were characterized in vitro for their reactivity toward TeNT and assessed in vivo for protectiveness against TeNT intoxication. Correlation of in vitro and in vivo data has revealed that in vitro selection of MAb that is protective in vivo could be performed by a combination of two assays: the measurement of MAb affinity toward TeNT taking Ka 1 × 10(8) M(-1) as a threshold level, and the evaluation of its capability to prevent TeNT-ganglioside interaction. Single MAb could be taken into consideration as a potential therapeutic only if it has a capacity to completely inhibits TeNT-ganglioside complex formation.

Design and construction of immune phage antibody library against Tetanus neurotoxin: Production of single chain antibody fragments.

BACKGROUND: Tetanus neurotoxin (TeNT) is composed of a light (LC) and heavy chain (HC) polypeptides, released by anaerobic bacterium Clostridium tetani and can cause fatal life-threatening infectious disease. Toxin HC and LC modules represents receptor binding and zinc metalloprotease activity, respectively. The passive administration of animal-derived antibodies against tetanus toxin has been considered as the mainstay therapy for years. However, this treatment is associated with several adverse effects due to the presence of anti-isotype antibodies. OBJECTIVE: In the present study, we have produced the fully human single chain antibody fragments (HuScFv) from two human antibody phage display libraries. MATERIAL AND METHODS: Twenty-four different HuscFvs were isolated from two anti TeNT immune libraries. Our produced human ScFv (HuScFv) were converted to IgG platform and analyzed regarding their specific reactivity to TeNT. RESULTS: All of the selected scFvs have the same VL but different VH. Three HuscFvs from the first library (TTX15, 51, 75) and two HuscFvs from the second library (TTX16, 20) were chosen to convert to IgG1 using pOptiVEC and pcDNA3.3 systems. Production of IgG1 from transfected DG44 and binding capacity of them to tetanus toxin and toxoid were measured by ELISA. ELISA results showed no detectable production of TTX16 and TTX20 IgG1. Although, TTX51 and TTX75 were converted and produced as IgG1, no reactivity to tetanus toxin and toxoid was observed. However, TTX15 was successfully produced as whole IgG1 platform with reactivity to both tetanus toxin and toxoid. The latter would be an appropriate replacement for conventional polyclonal antibodies if would meet the further characterization including specificity determination, affinity measurement and toxin neutralizing assays. CONCLUSION: Our results demonstrated production of functional IgG1 derived from TTX15 scFv and might be an appropriate replacement for polyclonal Tetabulin but it needs further characterization.

Effects of tetanus toxin after intracerebral microinjection are antagonized by drugs enhancing GABAergic transmission in adult fowls.

In adult fowls (Gallus domesticus), the behavioural effects of small doses of tetanus toxin, after unilateral microinjection into the nucleus mesencephalicus profundus (NMP), homologous to the mammalian substantia nigra, were studied. A rich pattern of stereotyped movements, vocalization, ipsilateral head-neck rotation, wing abduction, occasional ipsilateral circling and escape responses from the box were observed; the intensity of such symptomatology was dose-related and fully reversible within approx. 4 hr. Pretreatment with sodium valproate (100 and 200 mg/kg, i.m.) or with ethanolamine-sulphate (EOS, 1.6 mumol into the NMP) completely prevented the effects of tetanus toxin. The present findings show that fowls can represent an ideal species for studying acute central effects of tetanus toxin and more interestingly that drugs enhancing GABAergic mechanisms are able to prevent the effects of tetanus toxin.

Metallopeptidase inhibitors of tetanus toxin: A combinatorial approach.

The bacterial protein tetanus toxin (TeNt), which belongs to the family of zinc endopeptidases, cleaves synaptobrevin, an essential synaptic protein component of the neurotransmitter exocytosis apparatus, at a single peptide bond (Gln76-Phe77). This protease activity is a particularly attractive target for designing potent and selective synthetic inhibitors as a possible drug therapy for tetanus. beta-Aminothiols mimicking Gln76 of synaptobrevin have been previously shown to inhibit the tetanus neurotoxin enzymatic activity in the 35-250 microM range. These compounds have now been modified to interact with S' subsites of the TeNt active site, with the aim of increasing their inhibitory potencies. Combinatorial libraries of pseudotripeptides, containing an ethylene sulfonamide or an m-sulfonamidophenyl moiety as the P1 side chain and natural amino acids in P1' and P2' positions, were synthesized. The best inhibitory activity was observed with Tyr and His as P1' and P2' components, respectively. This led to new inhibitors of TeNt with Ki values in the 3-4 microM range. These molecules are the most potent inhibitors of TeNt described so far.

Beta-amino-thiols inhibit the zinc metallopeptidase activity of tetanus toxin light chain.

Tetanus neurotoxin is a 150-kDa protein produced by Clostridium tetani, which causes the lethal spastic paralytic syndromes of tetanus by blocking inhibitory neurotransmitter release at central synapses. The toxin light chain (50 kDa) has a zinc endopeptidase activity specific for synaptobrevin, an essential component of the neuroexocytosis apparatus. Previous unsuccessful attempts to block the proteolytic activity of this neurotoxin with well-known inhibitors of other zinc proteases led us to study the design of specific inhibitors as a possible drug therapy to prevent the progressive evolution of tetanus following infection. Starting from the synaptobrevin sequence at the level of the cleavage site by tetanus neurotoxin (Gln76-Phe77), a thiol analogue of glutamine demonstrated inhibitory activities in the millimolar range. A structure-activity relationship performed with this compound led us to determine the requirement for the correct positioning of the thiol group, the primary amino group, and a carboxamide or sulfonamide group on the side chain. This resulted in the design of a beta-amino-(4-sulfamoylphenyl)glycine-thiol, the first significantly efficient inhibitor of tetanus neurotoxin with a Ki value of 35 +/- 5 microM.

Exploring the functional domain and the target of the tetanus toxin light chain in neurohypophysial terminals.

The tetanus toxin light chain blocks calcium induced vasopressin release from neurohypophysial nerve terminals. Here we show that histidine residue 233 within the putative zinc binding motif of the tetanus toxin light chain is essential for the inhibition of exocytosis, in the rat. The zinc chelating agent dipicolinic acid as well as captopril, an inhibitor of zinc-dependent peptidases, counteract the effect of the neurotoxin. Synthetic peptides, the sequences of which correspond to motifs present in the cytoplasmic domain of the synaptic vesicle membrane protein synaptobrevin 1 and 2, prevent the effect of the tetanus toxin light chain. Our results indicate that zinc bound to the zinc binding motif constitutes the active site of the tetanus toxin light chain. Moreover they suggest that cleavage of synaptobrevin by the neurotoxin causes the inhibition of exocytotic release of vasopressin from secretory granules.

Prevention by the NMDA receptor antagonist, MK801 of neuronal loss produced by tetanus toxin in the rat hippocampus.

1. The behavioural and neuropathological effects of tetanus toxin, microinjected directly into the hippocampus, were studied in rats. 2. A single dose (1000 minimum lethal doses, MLDs) of tetanus toxin, injected unilaterally into the hippocampus produced a time-dependent neuronal loss in the CA1 pyramidal cell layer. In comparison with the contralateral, untreated side these effects became statistically significant (P less than 0.05) 7 days (22.0 +/- 1.1% reduction) and 10 days (29.2 +/- 1.7% reduction) after the injection. No significant changes were observed 7 days after treatment with 500 MLDs whereas a reduction of 37.5 +/- 3.1% in the CA1 area cell number was produced 4 days after the injection of 2000 MLDs. 3. Behavioral stimulatory effects were also induced by tetanus toxin (1000 MLDs) within 48 h of the injection and these culminated in generalized convulsions 5-7 days later. Convulsions were observed after a shorter period of latency in rats receiving 2000 MLDs tetanus toxin whereas 500 MLDs were ineffective. 4. No behavioural and neuropathological effects were observed in rats treated with neutralized tetanus toxin (1000 MLDs), bovine serum albumin or phosphate buffer. 5. Pretreatment with MK801 (0.3 mg kg-1, i.p., given 1 h before and after the injection with tetanus toxin and then once daily for 4 or 7 days) prevented the behavioural and neuropathological effects induced by tetanus toxin (1000-2000 MLDs). In addition, such treatment fully protected the animals from the lethal effects induced by 1000 MLDs tetanus toxin. In addition, such treatment fully protected the animals from the lethal effects induced by 1000MLDs tetanus toxin. By contrast, pretreatment with diazepam (3.Omgkg-1, i.p.) using the same schedule as for MK801 did not antagonize the effects of tetanus toxin (1000-2000 MLDs). 6. In conclusion, the present experiments have demonstrated that the intrahippocampal injection of tetanus toxin produces in rats a dose- and time-dependent behavioural stimulation and neuronal loss in the CAl pyramidal cell layer which can be prevented by the non-competitive NMDA antagonist, MK801.

Black tea extract, thearubigin fraction, counteracts the effect of tetanus toxin in mice.

The aim of this study was to find an inactivating substance for tetanus toxin in natural foodstuff. Tetanus toxin (4 micrograms/ml) abolished indirect twitches in In vitro mouse phrenic nerve-diaphragm preparations within 2.5 hr. Hot water infusion of black tea mixed with tetanus toxin blocked the inhibitory effect of the toxin. Mixing the toxin with thearubigin fraction extracted from black tea infusion produced an identical result. Furthermore, thearubigin fraction mixed with the toxin protected against the in vivo paralytic effect of the toxin. Thearubigin fraction had no protective effect on other toxins, such as tetrodotoxin and saxitoxin. The specific binding of [125I]tetanus toxin to rat cerebrocortical synaptosomes was inhibited by mixing iodinated toxin with thearubigin fraction. These results imply that thearubigin fraction counteracts the effect of tetanus toxin by binding with toxin, and also suggest that this fraction may be able to apply for prophylaxis of tetanus.

New trends in the management of tetanus.

The prevalence of tetanus reflects a failure of immunization. Prompt diagnosis and prediction of severity are crucial for the prevention of early life threatening complications and the institution of appropriate management. The current symptomatic treatment of heavy sedation, paralysis and artificial ventilation for 3-5 weeks for moderate and severe tetanus, is, even in the best centers, still associated with unacceptably high mortality, due to the disease and complications of the therapy itself. It is especially inappropriate for the developing world where intensive care resources are minimal. New options reported to avoid artificial ventilation and sedation are dantrolene (Dantrium, Procter and Gamble Pharmaceuticals), baclofen (Lioresal, Novartis) and magnesium. Magnesium therapy has the advantages of controlling spasms and sympathetic over activity without sedation. This simplifies nursing care and minimizes the need for ventilatory support except in the very severe disease and the elderly. Magnesium is recommended as the first-line therapy in tetanus.

A mechanism of the thearubigin fraction of black tea (Camellia sinensis) extract protecting against the effect of tetanus toxin.

The aim of the present study was to elucidate the mechanism of the protective effect of black tea extract's thearubigin fraction against the action of tetanus toxin. The effects of thearubigin fraction extracted from a black tea infusion were examined for neuromuscular blocking action on tetanus toxin in mouse phrenic nerve-diaphragm preparations and on the binding of this toxin to the synaptosomal membrane preparations of rat cerebral cortices. The interaction between tetanus toxin and thearubigin fraction was also investigated. Tetanus toxin (4 micrograms/ml) abolished indirect twitches in mouse phrenic nerve-diaphragm preparations within 150 min. Thearubigin fraction mixed with tetanus toxin blocked the inhibitory effect of the toxin. Mixing iodinated toxin with thearubigin fraction inhibited the specific binding of [125I]tetanus toxin to the synaptosomal membrane preparation. The effects of thearubigin fraction were dose-dependent. The elution profile of [125I]tetanus toxin on Sephadex G-50 column chromatography was different from that of toxin mixed with thearubigin fraction. These findings indicate that thearubigin fraction protects against the action of tetanus toxin by binding with the toxin.

Comparative in vitro and in vivo assessment of toxin neutralization by anti-tetanus toxin monoclonal antibodies.

Tetanus is caused by the tetanus neurotoxin (TeNT), a 150 kDa single polypeptide molecule which is cleaved into an active two-chain molecule composed of a 50 kDa N-terminal light (L) and a 100 kDa C-terminal heavy (H) chains. Recently, extensive effort has focused on characterization of TeNT binding receptors and toxin neutralization by monoclonal antibodies (mAbs). Toxin binding inhibition and neutralization is routinely assessed either in vitro by the ganglioside GT1b binding inhibition assay or in vivo using an animal model. These two assay systems have never been compared. In the present study, we report characterization of eleven mAbs against different parts of TeNT. The toxin inhibitory and neutralization activity of the mAbs was assessed in vitro and in vivo respectively. Our data demonstrated that seven mAbs bind to fragment C of the heavy chain, two mAbs react with the light chain, one mAb recognizes both chains and one mAb reacts with neither light chain nor fragment C. Six fragment C specific mAbs were able to inhibit TeNT binding to GT1b ganglioside in vitro but three failed to neutralize the toxin in vivo. One in vitro inhibitory mAb (1F3E3) was found to synergize with the in vivo neutralizing mAbs to reduce toxin lethal activity in vivo. Sequencing of the immunoglobulin heavy and light chain variable region genes revealed that the three in vivo neutralizing mAbs were derived from a common origin. Altogether, our data suggests that fragment C specific mAbs contribute to toxin neutralization in both systems, though some of the GT1b binding inhibitory mAbs may not be able to neutralize TeNT in vivo.

Clostridial neurotoxins: mode of substrate recognition and novel therapy development.

The clostridial neurotoxins (CNTs) are among the most potent protein toxins known to humans. CNTs include seven serotypes (A~G) of botulinum toxins (BoNTs), which cause botulism, a flaccid paralysis, and tetanus toxin (TeNT), which causes spastic paralysis. BoNTs are classified as category A agent and may be used as potential bioterrorism weapons. On the other hand, the ability of an extremely low dosage of BoNTs (less than 1 ng) to cause reversible partial paralysis upon injection into muscle has turned BoNTs, in particular serotypes A and B, from deadly agents to novel therapeutic agents for treatment of a wide range of clinical conditions associated with involuntary muscle spasm and contractions. In addition to clinical use, they may also be used in cosmetics. Further indications for BoNTs will continue to be developed, although current BoNT therapies have encountered some limitations due to the pharmacological properties of BoNTs, such as their ability to elicit immunoresistance in patients upon periodical injections. This review summarizes the present knowledge of the mechanisms of action of CNTs, with particular focus on the mode of substrate recognition by CNT catalytic domains and knowledge regarding substrate recognition can be utilized to develop novel BoNT products to extend its usefulness in therapeutic interventions and overcome the immunoresistance problems.

Construction of scFv library of human antibodies against tetanus toxin.

BACKGROUND: Over the last three decades, monoclonal antibodies have become powerful therapeutics and diagnostics tools. Progress in the antibody engineering, and the appearance of great selection technology such as phage display has made human antibodies production possible against antigens with high affinities. OBJECTIVE: The purpose of this study was to construct an immune antibody library from a vaccinated donor against tetanus toxin. METHODS: A blood sample was drawn from the donor who was vaccinated with tetanus toxoid. PBMC were isolated by using ficoll. After RNA extraction and cDNA synthesis. In order to amplify VH and VL regions, two uniplex PCRs were performed and put considering NcoI & HindIII, MluI & NotI restriction sites respectively for their cloning. These amplicons were cloned to pSEX81 vector and transformed to E. coli XL1blue strain. Eventually, recombinant plasmids were extracted and sequenced. RESULTS: The result showed acceptable similarity between antibody gene library nucleotide sequences and the antibody genes were deposited in this database. CONCLUSION: In this study, the VH and VL genes human antibody library was constructed and confirmed by using DNA sequencing and alignment of sequences with blast database.