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