Animal trials
All animal procedures were performed in accordance with protocols approved by the Animal Ethics committee of the Royal Melbourne Institute of Technology (RMIT) University, Melbourne. All protocols adhere to the Victorian Prevention of Cruelty to Animals Act 1986, the associated Regulations (2008), and the Australian Code for the Care and Use of Animals for Scientific Purposes (2013) (the ‘Animal Code’) and the Australian Code for the Responsible Conduct of Research 2018.
Mice were randomised, after being deemed to have generated antibody to tetanus toxin. Blinded observations were not deemed necessary- a progressive scheme for the progression of tetany was followed (see below).
The local effects of tetanus toxin and decoy solutions were determined by injecting toxin solutions directly into the gastrocnemius muscle of one hind limb of female, C57BL6 mice. In brief, the TeNT and/or decoy proteins were diluted to the desired concentration in PBS in a total volume of 15–25 µL. Mice were anaesthetised individually by isofluorane inhalation and injected with the solution using a 27-gauge tuberculin syringe. The mice were monitored consistently at 1–4 h intervals for symptoms of localised tetany based on a modified motor behavior scale reported by Webster and Laurence, 1963 (Laurence and Webster, 1963). Stage 1 = limb tetanus that involved only part of limb, stage 2 = limb tetanus consistently evoked on attempted limb movement and usually involving the entire limb, stage 3 = intermittent spontaneous limb tetanus, and stage 4 = sustained localized limb tetanus. The trial endpoint was considered when the mice reached either stage 4, or 48 hours post-injection, whichever occurred first. The group size was n = 6 per dose for vaccinated animals and n = 3 per dose for non-vaccinated animals. Group sizes were determined based on pilot studies to analyse variations based on individual susceptibility to TeNT and variance in titres of anti-TeNT antibodies in vaccinated animals.
To induce anti-tetanus immunity, 4-week-old mice were vaccinated three times with human divalent diphtheria – tetanus vaccine (ADT, Sequiris) at a dose of 2 IU of tetanus toxoid at 3-week intervals