B10.BR mice were immunized with 1×1010 particles of recombinant virus per mouse either by intramuscular injection (50 µl) in the right hindlimb, or by oral gavage (100 µl) using oral feeding needles (18G, 2.25 mm dia., Popper & Sons, Inc, New Hyde Park, NY). For nasal immunization, mice were anesthetized with isoflurane. Once anesthesia was achieved, 1×1010 particles of virus slowly delivered as a bolus into the nostrils using a standard micropipette (Gilson, Middleton, WI) as previously described [39] (link).
Pre-existing immunity to adenovirus serotype 5 was established by injecting 5×1010 particles of adenovirus expressing beta-galactosidase (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] (link), [11] (link). 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] (link). Mice were challenged by intraperitoneal injection of 200× LD50 of mouse-adapted Ebola virus, Zaire strain (MA-ZEBOV) in 200 µl sterile saline [40] (link). 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.
Pre-existing immunity to adenovirus serotype 5 was established by injecting 5×1010 particles of adenovirus expressing beta-galactosidase (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] (link), [11] (link). 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] (link). Mice were challenged by intraperitoneal injection of 200× LD50 of mouse-adapted Ebola virus, Zaire strain (MA-ZEBOV) in 200 µl sterile saline [40] (link). 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.
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