adenovirus 3 (PAV3) vector expressing the HA gene from the A/ Hanoi/30408/2005 H5N1 (H5N1-H05) influenza A isolate (PAV3-HA). The immunogenicity of HA and the success of previous AdHu5 H5N1-HA vaccines [1,19] suggested that avian influenza H5N1 may be a good comparative model to evaluate the efficacy of a similar PAV3 vector. Results Seroprevalence of PAV3 in pooled human Ig Previous studies showed that PAV3 does not exhibit cross- reactivity with AdHu5 or BAV3 neutralizing antibodies [20,21]. Additionally, PAV3 was not neutralized by the lowest dilution of 1:4 from 50 randomly selected human sera [20] In order to further address neutralization of PAV3 by an extended number of human sera, human Ig made of pooled sera from 10,000–60,000 individuals was evaluated. AdHu5, used as a control, was neutralized at the highest dilution of 1:160 (6.2561023 mg/ml human Ig). In contrast, neutralization of PAV3 was not detected at 1:20 (5.061022 mg/ml), the lowest dilution tested. Previous studies showed little cross-reactivity between cell-mediated im- mune responses against AdHu5 and PAV3 [22]. Vector construction and humoral immune responses over time An optimized expression cassette containing the codon- optimized H5N1-HA was inserted by homologous recombination A Porcine Adenovirus Vaccine Vector against H5N1 into a replication-incompetent PAV3 vector containing deletions in the E1/E3 genes (described in [23]). An AdHu5-HA vaccine was also developed in parallel as a control to compare levels of protection and immune responses. Expression of the PAV3-HA and AdHu5-HA vaccines was evaluated in VRIBL E1, HEK 293, and mouse AB12 cells (Figure 1a). BALB/c mice were also vaccinated with 1010 virus particles (vp) and in vivo expression of PAV3-HA or AdHu5-HA in muscle tissue was detected 4 days post-immunization (Figure 1b). A robust antibody response is important for the prevention of influenza virus infection. The ability of the PAV3-HA vaccine to generate humoral immune responses following immunization was assayed through detection of HA-specific antibodies by hemagglutination inhibition (HI) or neutralizing antibody (NAB) titres. BALB/c mice were vaccinated with 1010 vp/mouse of PAV3-HA or AdHu5-HA and the development of HI and NAB antibody responses was monitored from serum collected at days 8, 10, 14, and 21 post-immunization. All samples were treated with receptor-destroying enzyme (RDE), followed by complement inactivation the next day. An HI assay was performed using serial dilutions of the treated serum combined with H5N1-H05 virus and horse red blood cells to detect inhibition of cell agglutination by serum antibody. The reciprocal of the highest dilution which did not agglutinate red blood cells was scored as the HI antibody titre. HI titres of 2060, 120669, 213692, 5336184 were detected for PAV3-HA and 27611, 67623, 213692, 4266184 for AdHu5-HA, respectively, with no statistical difference between the two vaccines (Figure 2a). Figure 1. Comparative detection of H5N1-H05 HA protein expressed by AdHu5-HA and PAV3-HA. Transgene expression was detected by Western Blot using polyclonal anti-H5N1 mouse sera and a goat anti-mouse-HRP secondary antibody. (A) M = Marker, Lane 1 = expression of AdHu5-HA in HEK 293 cells, Lane 2 = expression of PAV3-HA in VRIBLE1 cells, Lanes 3 and 4 expression of AdHu5-HA and PAV3-HA in mouse syngenic AB12 cells, (2) negative control, untransfected cell lysate. (B) In vivo expression in mouse muscle tissues 4 days following vaccination. 75 mg of muscle tissue was loaded in each lane to compare protein expression. M = Marker, Lanes 1–6 = PAV3-HA, Lanes 8–13 = AdHu5-HA, (2) = unvaccinated muscle tissue. doi:10.1371/journal.pone.0015301.g001 PLoS ONE | www.plosone.org 2 December 2010 | Volume 5 | Issue 12 | e15301