Transcutaneous immunisation (TCI) is a novel needle-free method of vaccine delivery,
which involves the application of soluble antigens onto the surface of intact skin. In this
thesis, the immunogenicity and neutralising potency of tetanus neurotoxin (TeNT)
fragments were compared to that of tetanus toxoid (TTxd) following TCI. In addition, to
understand the mechanisms of induction of immune response by TCI, in vitro and in vivo
interaction of tetanus proteins with skin immune cells was also investigated. The 50kDa
recombinant carboxyl-terminal fragment of tetanus toxin (HCWT) and a 50kDa HC mutant
(HCM115) which is devoid of neuronal binding properties, were expressed and purified by
affinity chromatography, and excess endotoxin removed by size exclusion
chromatography. Mice immunized with HCWT, in the absence of adjuvant, induced the
highest anti-toxoid and anti-HCWT antibody titers, with significant increases in the toxin
neutralising antibody response when compared with TTxd. In vitro studies demonstrated
that both HC fragments and TTxd were capable of up regulating the surface expression of
activation marker ICAM-1 on murine bone-marrow derived dendritic cells (DC), but not
on the human keratinocyte cell line HaCaT. Real-time reverse transcriptase-PCR (Realtime
RT-PCR) analysis showed that TNF-α expression was up regulated in vivo as early as
10 minutes following TCI, and this was caused by shaving rather than by tetanus proteins
themselves. Immunohistochemistry staining to monitor the translocation of HCWT
fragment and TTxd through the skin following TCI showed that HCWT protein could be
detected in both the epidermis and dermis within four hours post-application. However,
TTxd translocation appeared to be much slower in comparison. Collectively, the results
presented in this thesis suggest that TCI may provide an opportunity for effective delivery
of toxin-like antigens, which harbor protective epitopes and that traditional toxoid proteins
may not be optimal antigens for skin immunisation.