This study demonstrates that a single-dose of codon-optimized adenovirus-based H1N1 vaccine expressing HA derived from influenza virus A/California/04/2009 (H1N1)pdm efficiently induces HA-specific antibodies and T-cells in mice that correlates with protection when measured as the elimination of detectable virus titers in lungs and nasal turbinates 3 days after challenge. The intensity of the immune response was greater in the group of mice immunized with AdHA.cod, indicating a benefit associated with optimizing the genetic code of antigen-expressing genes in recombinant vaccines as observed in other studies 
. Though the correlation of induction of HA-specific antibodies to vaccine doses was clearly demonstrated, we did not measure T-cell frequencies following immunization with different vaccine doses. Thus, future studies to determine the impact of T cell frequency and the correlation with HA antigen expression levels and protection would be of great interest.
These findings can be extended to other genetically engineered H1N1 influenza vaccines using reverse genetic techniques 
or using other recombinant viral vectors 
, and warrant the use of a codon-optimized version of the gene encoding HA. Enhancing the HA expression in the reassortant influenza vaccine strain could result in higher yields of the HA protein purified from the allantoic fluid of virus infected chicken eggs and consequently more vaccine doses per batch.
The use of recombinant adenoviral vectors to immunize against infectious diseases and cancer has been proven safe in more than 100 phase I, II, and III clinical trials. The effective use of adenoviral-based influenza HA vaccines was demonstrated in previous studies in mice, chickens and humans 
showing promising results indicating the adenoviral platform as a safe and practicable alternative to propagating vaccines with conventional methods in embryonated chicken eggs. Moreover, the ability to produce the adenoviral seed stock generation only 2–3 weeks after the appearance of the newly emerged influenza strain is one of the most valuable properties of this vaccine technology. However, natural pre-existing immunity against adenoviral vectors 
could potentially reduce vaccine efficacy. While the impact of natural neutralizing antibodies on the induction of effective immune responses is still controversial, the effective delivery of an adenovirus-based influenza HA vaccine via the mucosal route has been demonstrated, by van Kampen et al and our own results (data not shown), effective despite the presence of anti-adenoviral antibodies, suggesting that vector-specific immunity may be overcome 
. Extensive in vivo
work comparing different routes of administration as a way to overcome pre-existing adenoviral neutralizing immunity is underway in our laboratory and will be the subject of future manuscripts.
Alternatively, a wide range of different human and animal adenovirus serotypes, as well as structural and nonstructural modifications of adenoviral vectors, have been investigated to circumvent pre-existing adenoviral immunity 
In conclusion, this study further emphasizes the vaccine potential of an adenoviral-based platform with the advantages of speed for the seed stock generation and high yields of vaccine virus in a large-scale production. Furthermore, this vaccine elicits robust cellular and humoral immunity after a single dose immunization. These properties are invaluable when rapid vaccine development and production are required to control the virus spread and mitigate the public health impact of pandemic influenza. Although still experimental, these results mandate further testing of adenovirus-based influenza vaccine technology in human clinical trials.