Data obtained from animals vaccinated by the oral route provided several insights about the immunological requirements for protection against Ebola in a mouse model. Immunization by this route induced quantitatively lower T and B cell mediated immune responses against Ebola glycoprotein with respect to that achieved by either intramuscular or intranasal immunization. Despite this, every naïve animal immunized with a single oral dose of Ad-ZGP survived challenge, giving better precision on the minimal threshold of immunity required to achieve protection. As seen with intranasal immunization, the T cell-mediated response was not compromised by pre-existing immunity 10 days after oral vaccination. The number of IFN-c secreting T cells in both mucosal and systemic compartments of these animals at day 45, however, was significantly reduced by pre-existing immunity. It is possible that the peak T cell response at day 10 does not reflect the extent by which pre-existing immunity decreases the Ad5-ZGP-induced T cell response. Alternatively, it is also possible that the ELISPOT assay could detect subtle variations that flow cytometery could not monitor accurately due to differences in the sensitivity of each assay. Neutralizing antibody was not detected in the serum of animals with pre-existing immunity given a single oral dose of the vaccine. More importantly, none of these animals survived challenge with mouse adapted Ebola.