A Porcine Adenovirus Vaccine Vector against H5N1 Figure 6. Long-term protection in BALB/c mice. Long-term protection was evaluated in groups of six BALB/c mice vaccinated with PAV3-HA 109 (,) vp/mouse, AdHu5-HA 109 (.) vp/mouse, and PBS Control (N). (A) Mice were challenged 12 months following vaccination with 100LD50 of H5N1- H05 virus and monitored for (i) survival and (ii) weight loss and clinical signs of disease. (B) Long-term antibody responses. Serum was collected 12 months post-vaccination from mice immunized with PAV3-HA ( ) or AdHu5-HA ( ). HI and NAB responses were evaluated. The data represent average values and standard deviations from one experiment performed with one vector preparation of each vaccine (* represents p = 0.006). (C) ELISA detecting total IgG antibody titres against the H5 HA antigen. Endpoint titers were assessed for PAV3-HA ( ) or AdHu5-HA ( ) 25 days and 1 year post-vaccination. doi:10.1371/journal.pone.0015301.g006 suggesting that a replication-competent PAV3 vector which would be easy to produce at high titres could also be a promising vaccine candidate. Nevertheless, performance of this new vaccine vector will need to be addressed in other animal species, including nonhuman primates, before its real utility as a human vaccine can be predicted more accurately. Overall, this study supports a complementary role for cellular immunity during early H5N1 infection and the further development of porcine adenoviruses as human vaccine candidates. Materials and Methods Ethics Statement All animal procedures and scoring sheets were first approved by the Animal Care Committee (Animal Use Document ID# H-08- 010) at the Canadian Science Centre for Human and Animal Health, according to the guidelines set by the Canadian Council on Animal Care. Cells and viruses Human embryonic kidney (HEK) 293 cells, Madin-Darby canine kidney cells (MDCK), and mouse AB12 cells were maintained in Dulbecco’s modified eagle’s medium (DMEM), supplemented with 10% fetal bovine serum (FBS), L-glutamate, sodium pyruvate (NaPyr), and antibiotics. Fetal porcine retina cells (VR1BL E1), expressing human adenovirus 5 (AdHu5) E1a and porcine adenovirus 3 (PAV3) E1b large genes, were maintained in minimum essential medium (MEM) alpha, supplemented with 10% FBS, L-glutamate, NaPyr, non-essential amino acids, HEPES buffered saline, penicillin/streptomycin, and 50 mg/ml Hygro- PLoS ONE | www.plosone.org 8 December 2010 | Volume 5 | Issue 12 | e15301