We have previously found that a single dose of a recombinant adenovirus expressing Ebola Zaire GP given by either the oral or the nasal route is capable of affording protection against lethal challenge in naïve mice and that mucosal immunization can stimulate a broad, prolonged T cell-mediated immune response in both the systemic and mucosal compartments [37] . The primary objective of this study was to test the hypothesis that administration of an Ad5-based Ebola vaccine by either the nasal or oral route can circumvent pre-existing immunity and confer full protection upon challenge. Systemic and mucosal T and B cell responses to Ebola GP were assessed in both naïve mice and those with pre-existing immunity. The influence of PEGylation of the vaccine carrier on the immune response after oral immunization is also described.