In an effort to correlate markers of immunity with protection against Ebola infection after mucosal immunization, T and B cell specific immune responses against Ebola glycoprotein were analyzed in mice in the presence or absence of pre-existing immunity (PEI) to adenovirus 10 days after vaccination with a first generation adenovirus serotype 5 vector expressing the Zaire Ebola glycoprotein (Ad5-ZGP). Intracellular staining and flow cytometry (FACS) revealed that Ebola glycoprotein peptide-specific activation of CD8+ T cells, as measured by production of IFN-c, occurred in naïve animals immunized by the nasal route at a frequency of 3.961% (I.N., Figure 1A ). Pre-existing immunity was induced by intramuscular administration of recombinant Ad5 expressing a nonrelevant antigen, beta-galactosidase (AdlacZ), 30 days prior to vaccination with Ad5-ZGP. At the time of vaccination, mice had an average anti-adenovirus circulating NAB titer of 1:320. Pre-existing immunity did not significantly alter activation of CD8+ T cells when the vaccine was given intranasally (I.N.+PEI, 3.661%, p = 0.07). Oral immunization lowered the response against Ebola glycoprotein (P.O., 260.5%). Samples obtained from animals with pre-existing immunity and vaccinated in the same manner were barely above the positive threshold (three times the average background level obtained from animals treated with the irrelevant virus, AdlacZ). Samples obtained from naïve animals immunized by the intramuscular route (I.M.) contained the largest population of IFN-c positive CD8+ T cells (1062%). Animals treated with the AdlacZ vector alone (AdlacZ-control) served as negative controls and produced 0.5% CD8+, IFN-c+ T cells in response to the Ebola glycoproteinspecific peptide.