Induction of tetany in vaccinated mice
The minimum dose of TeNT where tetany was detected in vaccinated mice was 250 ng, with one third of mice observed with stage one tetany within 48 hours (Figure 4A and B). At a 500 ng dose, half of the mice exhibited stage one tetany symptoms within 48 hours. Half of the mice reached stage 4 tetany by 48 hours with 1 µg and 2 µg TeNT doses, and all mice reached stage 4 tetany in 24 hours with a dose of 4 µg (Figures 4A and B), with symptoms developing more rapidly at greater doses. These results broadly concur with previous studies of native TeNT administered to vaccinated mice (Fishman et al. , 2009). When administered with 100 µg of decoy, a TeNT dose of 500 ng induced stage 4 tetany in all mice by 24 hours (Figures 4A and C). This suggests an activity similar to 4 µg of TeNT alone, an 8-fold increase in activity in vaccinated mice. The lowest dose tested, 0.2 ng TeNT with 100 µg of equimolar decoy solution, induced stage 1 tetany in two thirds of mice, greater than the effect shown when 250 ng of TeNT was administered alone (Figures 4A, B and C). A direct comparison of the time course of tetany development in mice administered 500 ng and 250 ng of TeNT, with and without 100 µg of equimolar decoy solution, demonstrated a significant increase in activity of the decoy-accompanied treatment, with greater tetany stages exhibited in a shorter time (Figures 4 D and E). Together, these data demonstrated that the decoy effectively improved the activity of TeNT and that the effect is more pronounced at lower toxin concentrations. Furthermore, the increase between a minimal observable effect dose and the dose required to induce stage four tetany in under 24 hours was considerably greater with the decoy present.