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The effectiveness of vaccination programs would be enhanced greatly through the availability of vaccines that can be administered simply and, preferably, painlessly without the need for timed booster injections. Tetanus is a prime example of a disease that is readily preventable by vaccination but remains a major threat to public health due to the problems associated with administration of the present vaccine. Here we show that a protective immune response against live Clostridium tetani infection in mice can be elicited by an adenovirus vector encoding the tetanus toxin C fragment when administered as a nasal or epicutaneous vaccine. The results suggest that these vaccination modalities would be effective needle-free alternatives. This is the first demonstration that absorption of a small number of vectored vaccines into the skin following topical application of a patch can provide protection against live bacteria in a disease setting.Tetanus continues to be a threat to public health with more than half a million fatalities each year being associated with infection with Clostridium tetani (23). Due to the ubiquitous distribution of the causal agent, vaccination is the most effective medical intervention for protection of the public against this deadly disease. The effectiveness of the vaccine is due, at least in part, to the fact that the sequences of the neurotoxin molecules are conserved among different strains of C. tetani, which permits elicitation of a protective immune response against all C. tetani strains through the use of a single vaccine (23). The effectiveness of the vaccine is limited, however, by the needle-based delivery method currently in use. Effective protection requires injection of three consecutive doses of the tetanus toxoid vaccine (16). Moreover, booster injections must be administered periodically during adulthood to compensate for the age-related decline in antitoxin levels (16). In developing countries, vaccine coverage against this disease is generally low due to failure to follow up as well as a lack of the trained medical personnel and facilities required for administration of the vaccine. In developed countries, although vaccine coverage in childhood is high, there is a general lack of compliance of adults with recommended schedules for booster injections (16). These factors have led to the realization that tetanus vaccination programs would be improved significantly worldwide through the development of low-cost, needle-free vaccines.Needle-free vaccination requires the development of novel vaccines that can be administered safely and effectively. Several routes of administration are being considered. Both nasal and oral immunizations have been shown to be effective in eliciting an immune response against a number of pathogens. An alternative new modality is noninvasive vaccination onto the skin (NIVS) by topical application of epicutaneous vaccines (8,10,11,17,18,22), which would offer a greater safety margin and eliminate the discomfort associated with injections. Prior to our ...

Mammalian skin is regularly exposed to different environmental stresses, each of which results in specific compensatory changes in protein expression that can be assessed by proteomic analysis. We have established a reference proteome map of BALB/c murine skin allowing the resolution of greater than 500 protein spots in a single two-dimensional polyacrylamide gel. Forty-four protein spots, corresponding to 28 different cutaneous proteins, were identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and the Mascot online database searching algorithm. Twenty-five proteins were expressed at higher levels in the epidermis, whereas only nine were found predominantly in the subepidermal tissues. A subset of protein spots exhibited strain-specific expression. Proteins of diverse function were identified, including those involved in stress response, apoptosis, growth inhibition, the maintenance of structural integrity, translational control, energy metabolism, calcium binding, cholesterol transport, and the scavenging of free radicals. Prohibitin expression was detected cutaneously, with more abundant protein and mRNA levels in the epidermis. Five molecular chaperones including protein di-sulfide isomerase, 78 kDa glucose-regulated protein precursor, heat shock protein 60 (HSP60), HSP70, and HSP27 were also identified. Of these, HSP27 expression was confined mainly to the epidermis, and expression of protein disulfide isomerase was found primarily in the subepidermal tissues. Proteomic analysis of skin following heat or cold shock resulted in increased levels of HSP27, HSP60, and HSP70 suggesting involvement of these chaperones in the cutaneous response mechanism to temperature stress. These data establish numerous reference markers within the proteome map of murine skin and provide an important framework for future efforts aimed at characterization of the epidermal and subepidermal responses to environmental changes.

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