A Porcine Adenovirus Vaccine Vector against H5N1 Figure 3. T-cell responses detected by ELISPOT-IFNc and flow cytometry. Groups of 4 BALB/c mice were vaccinated with 1010 vp/mouse of either PAV3-HA ( ) or AdHu5-HA ( ). Mouse spleens were harvested 8, 10, 14, and 21 days post-immunization and positive T-cell responses following peptide restimulation were determined through IFNc secretion, as indicated by spot formation on the membrane. (A) Pooled T-cell responses. Cells were restimulated with a peptide library of overlapping 15mers spanning the entire H05-HA protein. Bars represent the total number of spot-forming cells/million mononuclear cells for all peptide pools combined. The data represent average values and standard deviations from 4 experiments performed with 3 independent vector preparations of each vaccine (* represents p,0.005). (B) T-cell responses following stimulation with immunodominant peptide. Splenocytes were restimulated with individual 9mer or 8mer peptides representing the immunodominant epitope of H05-HA (IYS) or control peptides (PR8-NP TYQ (), ZGP). Positive responses for IFNc secretion were detected by ELISPOT. Bars represent the total number of spot-forming cells/106 mononuclear cells. The data represent average values and standard deviations from 4 experiments performed with 3 independent vector preparations of each vaccine (** represents p,0.05). C) Flow cytometric analysis representing the percentage of CD8+IFNc secreting T-lymphocytes day ten post-immunization. The data represent average values and standard deviations from 3 experiments performed with one vector preparation of each vaccine. doi:10.1371/journal.pone.0015301.g003 such as Ebola and avian influenza (H5N1) viruses [4,26,27] Complete protection and long-term memory responses have also been reported in different animal models [7,26,28]; however, the final development of AdHu5-based vector to approved human vaccines has been hampered by the presence of natural pre- existing immunity to AdHu5 which is present in a large fraction of the human population. Neutralizing antibody to porcine adeno- virus 3 (PAV3) was not detected from pooled immune globulin representing 10 to 60 thousand human sera suggesting that pre- existing immunity to PAV3-based vaccines is not likely to be of concern for human applications. In addition, the documented compatibility of porcine and human tissues could translate into PAV3-based vaccines being efficacious while of low toxicity in humans [23]. The further development of a new adenovirus serotype also increases the spectrum of possible applications such as sequential vaccination with other adenovirus vectors (e.g. prime/boost or a second Ad-based vaccination against a different agent) [20,29]. The present study compares in parallel the immune responses and protection generated by a PAV3-based vector and a similar AdHu5-based vector using an avian influenza H5N1 disease model of infection. PAV3-based vector expressing the H5N1 HA antigen was able to generate quick and robust immune responses against H5N1. This finding was supported by the rapid induction of improved immediate protection by the PAV3-HA vaccine compared to AdHu5-HA. Interestingly, both vaccines shared similar in vivo expression, suggesting that the increase in vaccine efficacy observed with PAV3-HA may be due to specific PLoS ONE | www.plosone.org 5 December 2010 | Volume 5 | Issue 12 | e15301