Localization of the sites and characterization of the mechanisms by which anti-light chain antibodies neutralize the actions of the botulinum holotoxin

Takahashi, Tsuyoshi; Joshi, Suresh G.; Al-Saleem, Fetweh H.; Ancharski, Denise M.; Singh, Ajay K.; Nasser, Zidoon; Simpson, Lance L.

doi:10.1016/j.vaccine.2009.02.051

Cited by 28 publications

(23 citation statements)

References 20 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…A decline in PBT potency was first observed in the year 2001, which raised the demand for the renewal of botulinum countermeasures for prophylactic treatment. Extensive study was devoted in the past decade to develop second generation botulinum vaccines that are based on recombinant nontoxic fragments of the neurotoxin (47,52). The 50-kDa carboxy terminal of the neurotoxin heavy chain (Hc) was found to carry most of the neutralizing epitopes (58) and is the leading candidate for recombinant botulinum vac- (5).…”

Section: Discussionmentioning

confidence: 99%

Efficacy of a Potential Trivalent Vaccine Based on Hc Fragments of Botulinum Toxins A, B, and E Produced in a Cell-Free Expression System

Zichel

Mimran

Keren

et al. 2010

Clin Vaccine Immunol

View full text Add to dashboard Cite

Botulinum toxins produced by the anaerobic bacterium Clostridium botulinum are the most potent biological toxins in nature. Traditionally, people at risk are immunized with a formaldehyde-inactivated toxin complex. Second generation vaccines are based on the recombinant carboxy-terminal heavy-chain (Hc) fragment of the neurotoxin. However, the materialization of this approach is challenging, mainly due to the high AT content of clostridial genes. Herein, we present an alternative strategy in which the native genes encoding Hc proteins of botulinum toxins A, B, and E were used to express the recombinant Hc fragments in a cell-free expression system. We used the unique property of this open system to introduce different combinations of chaperone systems, protein disulfide isomerase (PDI), and reducing/oxidizing environments directly to the expression reaction. Optimized expression conditions led to increased production of soluble Hc protein, which was successfully scaled up using a continuous exchange (CE) cell-free system. Hc proteins were produced at a concentration of more than 1 mg/ml and purified by one-step Ni ؉ affinity chromatography. Mice immunized with three injections containing 5 g of any of the in vitro-expressed, alum-absorbed, Hc vaccines generated a serum enzyme-linked immunosorbent assay (ELISA) titer of 10 5 against the native toxin complex, which enabled protection against a high-dose toxin challenge (10 3 to 10 6 mouse 50% lethal dose [MsLD 50 ]). Finally, immunization with a trivalent HcA, HcB, and HcE vaccine protected mice against the corresponding trivalent 10 5 MsLD 50 toxin challenge. Our results together with the latest developments in scalability of the in vitro protein expression systems offer alternative routes for the preparation of botulinum vaccine.

show abstract

Section: Discussionmentioning

confidence: 99%

Efficacy of a Potential Trivalent Vaccine Based on Hc Fragments of Botulinum Toxins A, B, and E Produced in a Cell-Free Expression System

Zichel

Mimran

Keren

et al. 2010

Clin Vaccine Immunol

View full text Add to dashboard Cite

show abstract

“…The gene segment encoding the light chain of botulinum neurotoxin type A was cloned into 6x His vector pET30 a+ (Novagen; Gibstown, NJ) to construct the expression plasmid for LC/A (18). The Escherichia coli [BL21-codon plus (DE3)-RIL (Stratagene: La Jolla, CA) was used as host strain for expression of recombinant polypeptide.…”

Section: Methodsmentioning

confidence: 99%

Anti-nociceptive effect of a conjugate of substance P and light chain of botulinum neurotoxin type A

Mustafa

Anderson

Bokrand-Donatelli

et al. 2013

Pain

View full text Add to dashboard Cite

Neuropathic pain is a debilitating condition resulting from damage to sensory transmission pathways in the peripheral and central nervous system. A potential new way of treating chronic neuropathic pain is to target specific pain processing neurons based on their expression of particular receptor molecules. We hypothesized that a toxin-neuropeptide conjugate would alter pain by first being taken up by specific receptors for the neuropeptide expressed on the neuronal cells. Then, once inside the cell the toxin would inhibit the neurons’ activity without killing the neurons, thereby providing pain relief without lesioning the nervous system. In an effort to inactivate the nociceptive neurons in the trigeminal nucleus caudalis in mice we targeted the NK1 receptor (NK1R) using substance P (SP). The catalytically active light chain of botulinum neurotoxin type A (LC/A) was conjugated with SP. Our results indicate that the conjugate BoNT/A-LC:SP is internalized in cultured NK1R expressing neurons and also cleaves the target of botulinum toxin, a component docking motif necessary for release of neurotransmitters called SNAP-25. The conjugate was next tested in a mouse model of Taxol induced neuropathic pain. An intracisternal injection of BoNT/A-LC:SP decreased thermal hyperalgesia as measured by the operant orofacial nociception assay. These findings indicate that conjugates of the light chain of botulinum toxin are extremely promising agents for use in suppressing neuronal activity for extended time periods and that BoNT/A-LC:SP may be a useful agent for treating chronic pain.

show abstract

“…Rabbit antiserum was titrated for antibody using a standard protocol (Takahashi et al, 2009). Flat-bottom 96-well microplates were coated with recombinant antigen (200 ng; 100 l/well) overnight at 4°C, followed by three washes with PBS containing 0.05% Tween 20, pH 7.4.…”

Section: Methodsmentioning

confidence: 99%

Identification of the Factors That Govern the Ability of Therapeutic Antibodies to Provide Postchallenge Protection Against Botulinum Toxin: A Model for Assessing Postchallenge Efficacy of Medical Countermeasures against Agents of Bioterrorism and Biological Warfare

Al-Saleem¹,

Nasser²,

Olson³

et al. 2011

J Pharmacol Exp Ther

Self Cite

View full text Add to dashboard Cite

Therapeutic antibodies are one of the major classes of medical countermeasures that can provide protection against potential bioweapons such as botulinum toxin. Although a broad array of antibodies are being evaluated for their ability to neutralize the toxin, there is little information that defines the circumstances under which these antibodies can be used. In the present study, an effort was made to quantify the temporal factors that govern therapeutic antibody use in a postchallenge scenario. Experiments were done involving inhalation administration of toxin to mice, intravenous administration to mice, and direct application to murine phrenic nerve-hemidiaphragm preparations. As part of this study, several pharmacokinetic characteristics of botulinum toxin and neutralizing antibodies were measured. The core observation that emerged from the work was that the window of opportunity within which postchallenge administration of antibodies exerted a beneficial effect increased as the challenge dose of toxin decreased. The critical factor in establishing the window of opportunity was the amount of time needed for fractional redistribution of a neuroparalytic quantum of toxin from the extraneuronal space to the intraneuronal space. This redistribution event was a dose-dependent phenomenon. It is likely that the approach used to identify the factors that govern postchallenge efficacy of antibodies against botulinum toxin can be used to assess the factors that govern postchallenge efficacy of medical countermeasures against any agent of bioterrorism or biological warfare.

show abstract

Localization of the sites and characterization of the mechanisms by which anti-light chain antibodies neutralize the actions of the botulinum holotoxin

Cited by 28 publications

References 20 publications

Efficacy of a Potential Trivalent Vaccine Based on Hc Fragments of Botulinum Toxins A, B, and E Produced in a Cell-Free Expression System

Efficacy of a Potential Trivalent Vaccine Based on Hc Fragments of Botulinum Toxins A, B, and E Produced in a Cell-Free Expression System

Anti-nociceptive effect of a conjugate of substance P and light chain of botulinum neurotoxin type A

Identification of the Factors That Govern the Ability of Therapeutic Antibodies to Provide Postchallenge Protection Against Botulinum Toxin: A Model for Assessing Postchallenge Efficacy of Medical Countermeasures against Agents of Bioterrorism and Biological Warfare

Contact Info

Product

Resources

About