Transcutaneous immunisation with antigens derived from tetanus toxin

Abstract

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.