Administration of recombinant adenovirus-based vaccines to the mucosa has also conferred sufficient protection against challenge with a variety of pathogens in the presence of preexisting immunity to the vaccine carrier in mice and other preclinical models of disease [5, 19, 32, 51, 52] . This served as a basis for the present study. A single intranasal dose of a recombinant Ad5 vaccine expressing the Zaire Ebola glycoprotein conferred 100% protection in both naïve mice and those with pre-existing immunity despite the fact that the strength of the immune response generated by this route of administration was quantitatively lower than that seen in animals vaccinated by intramuscular injection. It is also important to note that pre-existing immunity induced by intramuscular injection did not severely compromise the T cell-mediated response at either the systemic or mucosal levels in these animals. The level of anti-Ebola GP antibodies in the circulation and in the lung was also not significantly compromised by pre-existing immunity to adenovirus. While these results suggest that intranasal vaccination with an Ad-based vaccine is indeed a promising strategy to overcome pre-existing immunity, one might wonder how accurately our results translate to natural exposure to the wild type virus via the respiratory tract. This is somewhat difficult to establish in the mouse model since the ability of the wild-type adenovirus to replicate is limited [53] . In addition, the amount of neutralizing antibody present in the nasal cavity of those in the general population with pre-existing immunity to adenovirus 5 has not been assessed to the degree that serum neutralizing antibodies have, making it difficult to set a relative parameter for one to target in pre-clinical animal models. Lack of this data may be due to the invasive nature of the technique for acquiring samples and/or the fact that antibody levels in the mucosa of individuals with established pre-existing immunity to adenovirus type 5 are quite low and transient in contrast to systemic levels of anti-adenovirus neutralizing antibodies which are quite robust and persist over time (unreported observations). Thus, for these studies, we decided to induce pre-existing immunity against adenovirus by intramuscular injection at a dose that has been shown to induce production of systemic neutralizing antibodies at 1:320, a level mostly below what was reported in humans with documented preexisting immunity [8, 11, 12] . Additional studies designed to assess the amount of neutralizing antibody to AdHu5 in the lung over time after intranasal administration of varying doses of AdHu5 are currently underway in an effort to further define stringent conditions under which pre-existing immunity can be established for experimental testing.