Pre-existing immunity to adenovirus serotype 5 was established by injecting 5610 10 particles of adenovirus expressing betagalactosidase (AdlacZ) by intramuscular injection in the right hindlimb 30 days prior to vaccination with Ad5-ZGP. This protocol has been documented to activate T and B cells against virus capsid proteins and elicit humoral immunity [8, 11] . At the time of vaccination, mice had an average anti-adenovirus circulating NAB titer of 1:320, which falls within lower range of average values reported in humans after natural infection [11] . Mice were challenged by intraperitoneal injection of 2006LD 50 of mouse-adapted Ebola virus, Zaire strain (MA-ZEBOV) in 200 ml sterile saline [40] . After challenge, the animals were weighed daily for 13 days and monitored for clinical signs of Ebola infection using an approved scoring sheet. All procedures and the scoring method were approved by the Institutional Animal Care Committee at the National Microbiology Laboratory (NML) of the Public Health Agency of Canada (PHAC) according to the guidelines of the Canadian Council on Animal Care. All infectious work was performed in the 'Biosafety Level 4' (BSL4) facility at NML, PHAC.