Detection was performed on a STORM 820 phosphoimager (MOLECULAR DYNAMICS) after a standard chemiluminescence reaction (ECL plus detection system; GE HEALTHCARE). To determine the 50% of lethal dose (LD50) of vNA and FLU-SAG2, female BALB/c mice were anesthetized with 15 mg/kg of ketamine and 0.6 mg/kg of xylazine and inoculated intranasally with 103 to 105 pfu of either virus in 25 μl of PBS. Survival of inoculated animals was followed for 30 days and LD50 endpoint was calculated

according to Reed and Muench’s method [43]. To evaluate influenza multiplication in mouse lungs, female BALB/c mice were anesthetized and infected as described above. Five days later, the animals were sacrificed and lung homogenates were prepared in 3 ml of PBS. Viral loads in lungs were assessed by standard titration under agarose overlay on MDCK cells. Viral RNA was extracted from 250 μl of lung homogenates with Trizol reagent see more (INVITROGEN) and analyzed by RT-PCR as described above. Heterologous prime-boost immunizations were performed as follows: Mice were anesthetized EGFR tumor as described above and received, by intranasal (IN) route, a dose of 103 pfu of vNA or FLU-SAG2 in 25 μl of PBS. Four weeks later, the animals received, by IN or subcutaneous (SC) routes, a boost dose of 108 pfu of Ad-Ctrl

or Ad-SAG2 diluted in 100 μl of PBS. Other groups were prime-immunized by IN route with 103 pfu of vNA and boosted 4 weeks later with a SC dose of 108 pfu of Ad-SAG2 or received a single SC immunization

with 108 pfu of Ad-SAG2. Homologous prime-boost protocols were performed as follows: animals were immunized twice within an 8-week interval by SC route with 108 pfu of Ad-Ctrl or Ad-SAG2 diluted in 100 μl of PBS. Serum and bronchoalveolar lavage (BAL) samples were obtained from vaccinated mice 2 weeks after the prime (serum) and boost immunization (serum and BAL), as previously described [39] and [44]. Specific Antibodies (total IgG, IgG2a or IgG1) against SAG2 protein were detected by enzyme-linked STK38 immunosorbent assay (ELISA) as previously described [40]. Briefly, 96-well plates (Maxisorp®, NUNC) were coated overnight with a T. gondii tachyzoite membrane extract enriched for GPI-anchored proteins (F3 fraction), as previously described [40], diluted to 1 μg/ml in 0.2 M sodium carbonate buffer pH = 9.6, at 4 °C. Plates were blocked with PBS supplemented with 2% skimmed milk (block buffer) for 2 h at 37 °C. Undiluted BAL or serum samples diluted 1:50 in block buffer were incubated for 2 h at 37 °C. Secondary antibody consisted of peroxidase-conjugated goat anti-mouse IgG (SIGMA) and it was incubated for 1 h at 37 °C. Reactions were detected with 3,3′,5,5′-tetramethylbenzidine (TMB) reagent (SIGMA), stopped with 2N sulfuric acid and read at 450 nm.