the PAV3-HA and AdHu5-HA (p = 0.57). Based on clinical data, a reference HI antibody titre of 40 is recommended for influenza vaccine candidates as the minimum level to confer fifty percent protection in humans [24,25]. Both vaccines met this requirement at all doses prior to challenge. Serum samples had an average NAB titre of 65620, 75614, or 106646 reciprocal dilution at 108, 109 or 1010 PAV3-HA vp/mouse respectively (Figure 2d). The AdHu5-HA vaccine generated NAB titres of 65620, 55620 or 133646 at 108, 109 or 1010 vp/mouse, respectively. Cellular immune responses over time Although a strong antibody response is important for immediate and long-term protection against influenza viruses, the induction of early cellular immune responses following vaccination may enhance clearance of virus-infected cells following H5N1 influenza virus infection. T-cell responses were assayed using an enzyme- linked immunosorbent spot (ELISPOT) assay to detect secretion of interferon gamma (IFNc) by activated lymphocytes. Splenocytes were obtained from mice vaccinated at days 8, 10, 14, and 21 with 1010 vp/mouse of PAV3-HA or AdHu5-HA and the cells were restimulated with pools of overlapping peptides corresponding to the entire H05-HA protein. The peptide corresponding to the immunodominant H5N1-H05 HA (IYSTVASSL, conserved influenza A virus) epitope was evaluated alongside an unrelated control peptide (TYQRTRALV, A/PuertoRico/8/34 H1N1 nucleoprotein). In mice vaccinated with PAV3-HA, 13,59862066, 14,4426 2541, 69546392, or 20566633 spot-forming cells (sfc) per million splenocytes were detected at days 8, 10, 14, or 21 respectively (Figure 3a). Immunization with the AdHu5-HA vaccine generated 1,9656341, 12,85861749, 7332693, and 19026372 sfc/million, respectively. The number of sfc/million splenocytes detected at day 8 was statistically significant between PAV3-HA and AdHu5- HA vaccines (p,0.005). The H05-HA immunodominant epitope also stimulated the strongest T-cell responses on day 8 for the PAV3-HA vaccine compared to AdHu5-HA. PAV3-HA vacci- nated mice generated an average of 8126138 sfc/million at day 8 whereas 2206133 sfc/million were detected from AdHu5-HA mice at the same time point (Figure 3b, p = 0.048). At day 10, average responses following stimulation with the H05-HA immunodominant epitope were 1,2166178 or 1,364636 sfc/ million from PAV3-HA or AdHu5-HA immunized mice respec- tively (p = 0.258). T-cell responses of 770674 and 453684 were detected from PAV3-HA or 791658 and 590684 from AdHu5- HA at days 14 and 21 post-immunization, respectively. Re- stimulation of splenocytes from PAV3-HA or AdHu5-HA immunized mice by the NP control peptide repeatedly generated less than 65 sfc/million. Cellular responses following vaccination with PAV3-HA were further characterized by detecting the relative frequency of peptide-specific CD8+ T cells expressing IFN-c by flow cytometry. Splenocytes were harvested ten days post-immunization and re- stimulated in vitro with the H05-HA immunodominant peptide and unrelated NP or ZGP (TELRTFSI, Zaire Ebolavirus glycoprotein) control peptides. Cells were stained with anti-mouse CD8-FITC (fluorescein isothiocyanate) and an anti-mouse IFNc-PE (phyco- erythrin). The frequency of IFNc positive CD8+ T-cells was 3.460.3 and 2.860.3 percent for PAV3-HA or AdHu5-HA, respectively (Figure 3c). Cells stimulated with control peptide NP or ZGP showed frequencies of CD8+ IFNc positive T-cells equal to 0.360.05 or 0.660.05 percent, respectively. These results suggest that the PAV3-based vaccine can stimulate a robust immune response in mice faster than AdHu5HA and comparable in strength. A Porcine Adenovirus Vaccine Vector against H5N1 Protective efficacy of PAV3-HA following lethal challenge The early detection of the T-cell response at day 8 post- vaccination with the PAV3-HA vaccine suggest that it may be a good candidate for immediate administration just prior or following a suspected exposure to H5N1 virus. The success of rapid vaccination regimen was evaluated in two parts: first, overall survival and second, total viral load in lung tissue, as determined by TCID50 assay. BALB/c mice were challenged with a lethal dose of H5N1-H05 virus at 5, 8 and 10 days post-vaccination and lungs were harvested at day 3 post-infection. Although mice challenged 5 days post-vaccination did not survive lethal challenge (Figure 4a), animals challenged 8 days post-vaccination experienced significant- ly greater survival with the PAV3-HA (66% survival) vaccine than for AdHu5-HA (11% survival) and had less signs of disease and weight loss (Figure 4b, p = 0.038). Both vaccines were fully protective with minimal signs of disease when administered 10 days prior to challenge (Figure 4c). Lung viral titres were significantly lower at all time points with both vaccines compared to unvaccinated control animals (Figure 4d). Following vaccina- tion with PAV3-HA, virus titers were 3.57610666.266106, 1.40610664.876106, and 4.82610666.576106 TCID50/g, for days 5, 8, and 10 respectively and 1.64610768.606106, 1.03610766.96106, and 3.52610665.766106 logTCID50/g for AdHu5-HA. The PAV3-HA vaccine was associated with signifi- cantly lower viral load in animals vaccinated at both days 5 and 8 compared to AdHu5-HA (p = 0.032 and p = 0.048, respectively). Both vaccines were similar on day 10 (p = 0.72). Survival was also assessed in groups of 10 BALB/c mice vaccinated with 108, 109, or 1010 virus particles of recombinant adenovirus PAV3-HA or AdHu5-HA by intramuscular (I.M.) administration, challenged 28 days later with lethal homologous H5N1-H05 virus. Protection against clinical signs of disease with minimal weight loss and full survival was observed at the 1010 vp/ mouse dose for both Ad-based vaccines (Figure 5). Complete survival was also observed for both vaccines at a dose of 109 vp/ mouse, with the PAV3-HA vaccinated mice either asymptomatic or having milder signs of disease (14% weight loss) compared to AdHu5-HA (21% weight loss). Another desirable characteristic that influenza vaccines should provide is long-term protection. Therefore, long term immunity was evaluated in mice challenged 12 months after vaccination in order to determine whether protective immune responses could be maintained. PAV3-HA afforded full protection in mice challenged with 100 LD50 of H5N1-H05 virus 12 months post-immunization whereas 50% of mice vaccinated with AdHu5-HA succumbed (Figure 6a). Higher HI antibody titers for PAV3-HA compared to AdHu5-HA (186665 and 60623, respectively (p = 0.006)) may have translated directly to the improved survival observed with the PAV3-HA vaccine. NAB titers were 23615 and 10611 for AdHu5-HA (Figure 6b). An ELISA assay was also performed to detect total IgG antibody titres against the H5N1-HA antigen. Serum was obtained from mice 25 days and 1 year post-vaccination, and unvaccinated control mice (Figure 6c). Total antibody titers were significantly lower for both vaccines after 1 year. On average, higher levels of IgG antibodies were detected for the PAV3-HA vaccine, however the difference was not statistically significant compared to AdHu5-HA (p = 0.241). Discussion Currently, replication-deficient human adenovirus serotype 5 (AdHu5) vaccines are being evaluated against several pathogens. Several candidates have been shown to induce protective immune responses against emerging or re-emerging infectious pathogens PLoS ONE | www.plosone.org 4 December 2010 | Volume 5 | Issue 12 | e15301