A Porcine Adenovirus Vaccine Vector against H5N1 The presence of neutralizing antibodies to inhibit active H5N1- H05 infection was also evaluated as a measure of the humoral response. Serial dilutions of treated serum were incubated with 100 pfu/well of H5N1-H05 virus and cells were monitored for the presence or absence of cytopathic effects (CPE) using a light microscope. The highest serum dilution which did not exhibit CPE was scored as positive for neutralizing antibody and titres were reported as the reciprocal of the dilution. NAB titres of 060, 7612, 7612 and 90650 or 060, 7612, 10610 and 4060 reciprocal dilutions were observed with the PAV3-HA or AdHu5- HA vaccines at 8, 10, 14, and 21 days, respectively (Figure 2b). The increased neutralizing antibody response following PAV3-HA vaccination was statistically significant at day 21 (p = 0.045) relative to AdHu5-HA. In order to assess antibody titres immediately before challenge, mice were vaccinated with different doses of each vaccine and serum was obtained 25-days post-immunization. HI reciprocal titres of 9866326, 10936293, and 1280 for the PAV3-HA vaccine or 10666330, 10666330, and 11736261 with AdHu5- HA were detected for 108, 109 or 1010 vp/mouse doses respectively (Figure 2c). Both vaccines had similar HI antibody titres at all doses, with no significant differences detected between Figure 2. Hemagglutination inhibition (HI) and neutralizing antibody (NAB) assays for PAV3-HA and AdHu5-HA. Serum was obtained from groups of PAV3-HA ( ) or AdHu5-HA ( ) vaccinated BALB/c mice and treated overnight at receptor-destroying enzyme (RDE), followed by complement inactivation the following day. (A) Kinetics of the HI antibody response. Serum was obtained from groups of 4 BALB/c mice on days 8, 10, 14, and 21 post-vaccination with 1010 vp/mouse of PAV3-HA or AdHu5-HA. Four agglutinating doses of H5N1-H05 virus were added to serial dilutions of serum and samples were incubated with horse red blood cells. HI antibody titre was determined as the reciprocal of the highest serum dilution which did not cause red blood cell agglutination. (B) Kinetics of the NAB response. Serum from obtained on 8, 10, 14, and 21 days post- vaccination with 1010 vp/mouse of PAV3-HA or AdHu5-HA. Serial dilutions of was also incubated with 100 pfu of H5N1-H05 virus, added to MDCK cells and monitored for the presence of cytopathic effects (CPE) over 48 hours. The NAB titre was scored as the highest serum dilution with the absence of CPE. (C) HI titre against H5N1-H05. Serum was obtained 25 days post-immunization from groups of 5–10 BALB/c mice were vaccinated with 108, 109, and 1010 vp/mouse of AdHu5-HA or PAV3-HA and the HI titre was assayed. (D) NAB titres against H5N1-H05. Serum from 25 days post- immunization was also monitored for NAB titre. The data represent average values and standard deviations from 3 to 5 experiments performed with 2 independent vector preparations of each vaccine (* represents p,0.05). doi:10.1371/journal.pone.0015301.g002 PLoS ONE | www.plosone.org 3 December 2010 | Volume 5 | Issue 12 | e15301