July 17, 2017

By: douano@listlabs.com

By: Mary N. Wessling, Ph.D. ELS

Tetanus Toxin’s Use as a Protein Carrier and Antigen

Tetanus toxin (TT) is the major virulence factor of the Gram-positive bacterium Clostridium tetani. Infection with this bacterium in unvaccinated persons produces muscle spasms by binding to nerve endings and moving throughout the nervous system in a specific way. Eventually, almost total paralysis results. The deactivated toxin is the basis for a vaccine, which can even be administered to pregnant women, usually as part of a combination vaccine also aimed at preventing neonatal pertussis.¹ Worldwide, the mortality of infection among unvaccinated persons reaches 10%.² It is the binding specificity of this dangerous toxin that makes it valuable as a research material. List Biological Laboratories (List Labs) offers inactivated tetanus toxin and six related products, used in intricate and fascinating ways in research as a model antigen and protein carrier.

 

List Labs’ Tetanus Toxoid in Immunosupression Research

Existing antibody treatments for rheumatic arthritis (RA), for example doses of rituximab every six months, suppress autoreactive B cells by killing them. The effects of the treatment fade over the 6-month period; this increases the inherent risk of infection and progressive multifocal leukoencephalopathy. Chu et al studied a treatment that characterized B-cell immunosuppression by an engineered antibody; List Labs’ tetanus toxoid (TTd)

was used in an elegant series of explorations of the mechanism of action of an engineered antibody (XmAb581) that enhanced the action of a B-cell antigen receptor complex currently under clinical development for treatment of RA, and enhanced both its safety and efficacy.³

 

Tetanus Toxoid in B Cell-Driven Autoimmune Disease Research

In a study with a broader purpose, Klose et al, expanding a previous murine study, developed a protein engineering strategy to selectively target and eradicate human memory B cells. These authors built a fusion protein that combined a model antigen TTd fragment C with a truncated version of exotoxin A derived from Pseudomonas aeruginosa. A fluorescein isocyanate-labelled TT fragment C produced by List Laboratories, used as a control in the binding analysis, played an important supporting role. This research offers a promising approach for the specific depletion of autoreactive B-lymphocytes in B cell-driven autoimmune diseases.⁴

 Prompted by the lower prevalence of these diseases in children who lived near farm animals and in unhygienic environments, Iwasaki et al studied the key role of intestinal infection in development of allergic respiratory disease in children. Their study used List Labs’ TTd antigen

to compare total antibody binding between asthmatic and non-asthmatic children. The authors found an association between lower antibody titers in asthmatic children to echovirus, and in a previous study, a heightened response to rhinovirus. These findings support a key role for intestinal infection in the development of allergic respiratory disease.⁵

 

Further Innovative Applications of List Labs’ Tetanus Toxin and Tetanus Toxoid

List Labs’ tetanus toxin (TT)

was used in a study that challenged an accepted mechanism for cell death in injured human retinal ganglion cells; Li Y et al showed that dysregulation of mobile zinc is to blame. In anesthetized animals, they used TTd to cleave the synaptic vesicle protein and then injected the zinc formulation. Fluorescent images showed that there was a rapid accumulation of Zn²⁺ in amacrine cell processes after optic nerve injury.⁶ Investigating yet another problem that affects injured persons, i.e., the necessity to keep an injured limb immobile, which results in muscle atrophy, Matthews et al reported that inactivity can result in 20% to 30% atrophy despite the use of exercise-based or neuromuscular electronic stimulation. They injected either saline or a very dilute solution of List Labs’ TT in physiological saline in the tibialis anterior of rats and compared the health of muscle fibers after immobilization. They found that the TT prevented loss of size in all 3 myofiber types, and therefore was protective against muscle loss during immobility.⁷

Finally, animal studies are used to evaluate the efficacy of pharmaceutical and other products for human use; although there are stringent conditions that assure ethical treatment of animals, the process is often time-inefficient, inaccurate, and costly. Temann et al explored using precision-cut lung slices (PCLS) from lungs of donors that were not suitable for use in transplantation as an alternative to animal studies. They evaluated a culture system using PCLS stimulated by List Labs’ TTd

and found that these slices could be held in culture for up to 14 days to study cytotoxic, inflammatory, and immune responses.⁸

The studies we cite here are only a small sample of what can be accomplished using List Labs’ TT and related products. We invite you to visit our citations page to explore how TT and our other products can augment your experimental design.

 

1. Chu HY, Englund JA. Maternal immunization. Birth Defects Research. 2017;109(5):379-386. PMID: 28398678
2. da Silva Antunes R, Paul S, Sidney J, et al. Definition of Human Epitopes Recognized in Tetanus Toxoid and Development of an Assay Strategy to Detect Ex Vivo Tetanus CD4+ T Cell Responses. PloS One. 2017;12(1):e0169086. PMID: 28081174
3. Chu SY, Yeter K, Kotha R, et al. Suppression of rheumatoid arthritis B cells by XmAb5871, an anti-CD19 antibody that coengages B cell antigen receptor complex and Fcgamma receptor IIb inhibitory receptor. Arthritis & Rheumatology (Hoboken, NJ). 2014;66(5):1153-1164. PMID: 24782179
4. Klose D, Saunders U, Barth S, Fischer R, Jacobi AM, Nachreiner T. Novel fusion proteins for the antigen-specific staining and elimination of B cell receptor-positive cell populations demonstrated by a tetanus toxoid fragment C (TTC) model antigen. BMC Biotechnology. 2016;16:18. PMCID: PMC4756516
5. Iwasaki J, Chai LY, Khoo SK, et al. Lower anti-echovirus antibody responses in children presenting to hospital with asthma exacerbations. Clinical and Experimental Allergy : Journal of the British Society for Allergy and Clinical Immunology. 2015;45(10):1523-1530. PMID: 25640320
6. Li Y, Andereggen L, Yuki K, et al. Mobile zinc increases rapidly in the retina after optic nerve injury and regulates ganglion cell survival and optic nerve regeneration. Proceedings of the National Academy of Sciences of the United States of America. 2017;114(2):E209-e218. PMCID: PMC5240690
7. Matthews CC, Lovering RM, Bowen TG, Fishman PS. Tetanus toxin preserves skeletal muscle contractile force and size during limb immobilization. Muscle & Nerve. 2014;50(5):759-766. PMID: 24590678
8. Temann A, Golovina T, Neuhaus V, et al. Evaluation of inflammatory and immune responses in long-term cultured human precision-cut lung slices. Human Vaccines & Immunotherapeutics. 2017;13(2):351-358. PMID: 27929748