Mucosal delivery of recombinant adenovirus serotype 5 (rAd5)-based vaccine preparations are appealing for vaccine development in terms of lowering toxicity induced by high viral loads and substantial liver accumulation following systemic injection of the vaccine. However, this mode of delivery is currently under-developed due to the relatively low T-cell mediated immune responses generated against the encoded transgene. The first study described in this thesis demonstrated that sublingual immunization induces rapid migration of MHCII+, CD11C+ antigen presenting cells to the delivery site and elicit antigen-specific T and B cell-mediated immune responses in naïve mice and those with pre-existing immunity (PEI) to Ad5 at a level higher than that achieved after oral immunization. More importantly, this strategy improved protection of animals with PEI to Ad in contrast to poor protection after IM injection. The second study was designed to establish a method for inducing PEI that most accurately reflects natural infection in rodents and identifies the immunologic parameters elicited by rAd5-based Ebola vaccine necessary for protection against lethal infection. When immunization occurred by the same route in which PEI was induced, the antigen-specific multifunctional CD8+ T cell and antibody responses were significantly reduced. This correlated with poor survival after challenge with a lethal dose of Ebola Zaire in rodents. The data suggests that 1) establishment of PEI by the same route used for immunization is the most stringent test for a novel formulation designed to be effective in those with PEI to Ad5, and 2) for a formulation to be effective in those with PEI, it must be capable of restoring antigen-specific multifunctional CD8+ T cell and antibody responses, compromised by PEI. The third study screened novel formulations for their ability to improve in vitro transduction efficiency and immunogenicity and efficacy in vivo in the presence of anti-Ad5 neutralizing antibodies. Formulations consisting of pharmaceutically acceptable, non-immunogenic excipients that can prime the arms of immune response compromised by PEI improved survival after lethal challenge with Ebola Zaire challenge for rAd5-based Ebola vaccine in rodents with PEI. Taken together, these studies provide insight on how to reconstitute necessary immune responses in vaccine protocols by establishing a reliable PEI model in rodents, testing routes of administration, and formulations of the rAd5-based Ebola vaccine. / text