A Porcine Adenovirus Vaccine Vector against H5N1 Figure 4. Protection following vaccination with PAV3-HA or AdHu5-HA vaccine. (A–C) Groups of 9 BALB/c mice were vaccinated with 1010 vp/mouse of PAV3-HA (n), AdHu5-HA (m), or PBS Control (N) and challenged with 100LD50 of H5N1-H05 on days 5, 8, and 10 post-vaccination. (A) Protection afforded 5-days post-vaccination. (i) survival and (ii) weight loss. (B) Protection afforded 8-days post-vaccination. (i) survival and (ii) weight loss. (C) Protection afforded 10-days post-vaccination. (i) survival and (ii) weight loss. The data represent average values and standard deviations from 2 experiments performed with 2 independent vector preparations of each vaccine (D) Lung viral titres 3 days after lethal challenge. Groups of 6 BALB/c were vaccinated with 1010 vp/mouse of PAV3-HA () or AdHu5-HA (), or no vaccine (Control, ()) and challenged with 100LD50 of H5N1-H05 on days 5, 8, and 10 post-vaccination. Lungs were harvested from the mice on day 3 post-challenge. Virus titre was determined by TCID50 assay using serial dilution of lung homogenates on MDCK cells and monitoring for the presence of CPE over 48 hours. The TCID50 titre was calculated by the Reed & Muench method [39] and normalized/gram of lung tissue. Data is presented as log10 TCID50/g of lung tissue. The data represent average values and standard deviations from one experiment performed with one vector preparation of each vaccine (* and ** represents p,0.05). doi:10.1371/journal.pone.0015301.g004 interactions with the immune system rather than enhanced expression of the antigen. A recent study suggests that PAV3- HA may activate different innate immune pathways to AdHu5- HA [30]. Additionally, the PAV3-HA offered improved long-term protection against lethal H5N1 virus challenge with higher levels of detectable antibodies by HI assay. Although full short-term (28 day) survival was observed following vaccination with PAV3-HA or AdHu5-HA at the 109 vp/mouse dose, greater weight loss was observed with the AdHu5-HA vaccine. Comparing HI and NAB antibody titres with observed clinical outcome, this suggests that there may have been incomplete neutralization of the virus following vaccination with AdHu5-HA, perhaps explaining why there were differences in the levels of protection observed following challenge 1-year post-vaccination. It has been shown that levels of neutralizing antibodies in serum decrease over time following H5N1 infection [31]. Following vaccination, the exact levels of neutralizing antibodies correlating with full protection from infection with homologous virus is still uncertain. In comparison with total IgG antibody levels, neutralizing antibody titres correlated better with early and long-term protection, similar to previously described reports [19,32,33]. Together the data suggests that PAV3 vector is an additional option to AdHu5 vector and could have several important applications including rapid or post-exposure protection against emerging influenza viruses or other infectious agents. A similar study described a BAV3-HA vaccine sharing comparable protective efficacy to the AdHu5-HA against an H5N1 virus. Two doses of the BAV3 vector generated similar humoral and cell- mediated immune responses and was able to evade pre-existing neutralizing antibodies against AdHu5 [12]. Overall protective efficacy offered by PAV3-HA was similar to AdHu5-HA one month post-immunization and the immune response kinetics was also generally comparable at later time points. Although not explored in the current study, previous studies have evaluated the impact of pre-existing immunity to PAV3 and the potential reuse of PAV3-based vectors against different pathogens. Groups of outbred pigs with high PAV3 neutralizing antibody titres were vaccinated with a PAV3-based vaccine and vector re-administra- tion did not result in hepatotoxicity or reduced transgene expression [14]. Doses of 1013 particles/kg AdHu5 vector can also bypass pre-existing immunity, however, several pathologies including liver damage indicated by elevated transaminase, low platelets count, and lymphocytopenia were observed in nonhuman primates administered similar doses [9,10,34,35]. Even though conventional vaccines have been relatively successful against influenza A infection, the ability of adenoviral vectors to rapidly generate a strong immune response may be useful to specific applications such as rapid immunization of health care workers in anticipation of a probable exposure to an emerging virulent pandemic virus such as H1N1-1918. In addition to strong antibody responses against the H5N1-H05 virus, both survival and cellular responses suggest that induction of an earlier T-cell response by the PAV3-may complement the developing antibody response to improve protection against H5N1 challenge and contribute directly towards lower viral load. Although the influenza glycoproteins have high antigenic variation, the generation of faster T-cell responses by a PAV3 vector against well-conserved influenza antigens may supplement a mismatched antibody response and may improve protection against a wider range of influenza viruses. Previous studies have shown that PAV3 vectors can transduce several human cell lines resulting in full transgene expression and adenoviral coat proteins [20,21]. An additional safety feature is that wild-type PAV3 does not replicate in human cells [21], Figure 5. Groups of 10 BALB/c mice were vaccinated with PAV3-HA at a dose of 108 (e), 109 (,), and 1010 (n) vp/mouse, AdHu5-HA 108 (¤), 109 (.), and 1010 (m), or PBS Control (N). Twenty-eight days post-vaccination, the mice were challenged with 100LD50 of H5N1-H05 virus and monitored for (i) survival and (ii) weight loss and clinical signs of disease. doi:10.1371/journal.pone.0015301.g005 PLoS ONE | www.plosone.org 7 December 2010 | Volume 5 | Issue 12 | e15301