Analysis of IFN-γ and IL-4 by ELISPOT assays.

<p>On day 14 following the second immunization, mice were sacrificed and single-cell suspensions were prepared from the spleen, cultured for 48 h, and stimulated with 5 μg/mL purified Ah01/AA viral antigen. IFN-γ (A) and IL-4 (B) secretion by splenocytes was determined by ELISPOT in triplicate wells. Values and bars represent means ± SD. ** <i>p</i><0.001 compared to the PBS group.</p

Live attenuated monovalent influenza A (H7N9) vaccines induce antibody responses in mice.

<p>Groups of mice were i.n. immunized at weeks 0 and 2 with various doses of 10⁴ CCID₅₀, 10⁵ CCID₅₀, and 10⁶ CCID₅₀ of the Ah01/AA ca vaccine or mock-infected with PBS. Levels of HI (A) and NT (B) antibodies against wt Influenza H7N9 virus in sera 2 weeks after prime and boost. Error bars indicate SDs (n = 8).</p

The reassortant influenza H7N9 ca virus is <i>ca</i> and <i>ts</i> in CEF cells.

<p>^a<i>ca</i> = difference between the mean CCID₅₀ at 33°C and 25°C ≥100-fold.</p><p>^<i>b</i><i>ts</i> = difference between the mean CCID₅₀ at 33°C and 39°C ≥100-fold.</p><p>The reassortant influenza H7N9 ca virus is <i>ca</i> and <i>ts</i> in CEF cells.</p

Production of an influenza Ah01/AA ca vaccine candidate using reverse genetics.

<p>The characteristics of Ah01/AA ca transfectant viruses were confirmed by visualizing the shape and size distribution of the virus particles. (A) Recombinant Ah01/AA ca virus particles were visualized using electron microscopy. (B) Ah01/AA ca virus particles were measured; 82% (out of 200 particles measured) ranged in size between 80 and 120 nm.</p

Lung viral titers and histopathological changes of vaccinated mice following challenge with live influenza Ah01/H7N9 virus.

<p>Two weeks after the boost, vaccinated mice were i.n. infected with Ah01/H7N9 (50LD₅₀) and lung tissues were collected 3 days later. (A) Viral titers in the lung of infected mice. The data were determined in triplicate by CCID₅₀ assay in MDCK cells 3 days post infection and expressed as log₁₀ CCID₅₀/g tissue. Data are means ± SD. *p<0.01; ** <i>p</i><0.001 (B) Lung histopathological changes following virus challenge. Representative histopathological images of lung damage by H&E staining from five mice per group.</p

Mucosal antibody response in BALB/c.

<p>Secretion of anti-HA IgA antibodies against VN/1203, ID/05, and AH/01 H5N1 inactivated antigen in nasal and lung lavage (dilution 1∶5) from mice immunized i.n. with single H5N1 influenza split vaccines (3 µg HA per dose) and a trivalent vaccine containing 1 µg HA per dose of each single vaccine in combination with adjuvant. The values are means ± SEM from six mice. * p<0.05 and ** p<0.01. The dashed horizontal line indicates the lower limit of detection.</p

HAI titers of mice immunized with single or trivalent H5N1 influenza split vaccines.

<p>Mice were immunized i.n. with single H5N1 influenza split vaccines (3 µg HA per dose) and a trivalent vaccine that contained 1 µg HA per dose of each single vaccine in combination with adjuvant on day 0 and 14, and bled on day 28. Four HA units of VN/1203 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030252#pone-0030252-g001" target="_blank">Fig. 1A</a>), ID/05 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030252#pone-0030252-g001" target="_blank">Fig. 1B</a>), AH/01 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030252#pone-0030252-g001" target="_blank">Fig. 1C</a>), and China097 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030252#pone-0030252-g001" target="_blank">Fig. 1D</a>) viral antigen were used. Results are the geometric mean titers of positive sera (HI titer >10). The values are means ± SEM from six mice. * p<0.05 and ** p<0.01. The dashed horizontal line indicates the lower limit of detection.</p

Antibody responses to the H7N9/PR8 split vaccine in mice.

<p>Groups of 20 BALB/c mice were immunized intramuscularly at weeks 0 and 2 with 7.5, 15, 30, or 45 µg (HA levels) of the H7N9/PR8 split vaccine. (A) HI antibody responses to the (wt) AnHui virus after vaccination as described above. Serum samples were collected on day 0; 2 weeks after priming; and 2, 4, and 8 weeks after boosting. (B) Serum IgG titers against the (wt) AnHui virus measured 2 weeks after both the first and second doses of vaccine. (C) The ratios of serum IgG1 to IgG2a titers against the (wt) AnHui virus, calculated 2 weeks after the second dose of vaccine. The data are presented as means ± SDs of the data from three experiments, each performed in duplicate. HI, hemagglutination inhibition; PBS, phosphate-buffered saline. 2WPD1: 2 weeks post-vaccination with dose 1. 2WPD2: 2 weeks post-vaccination with dose 2; 4WPD2: 4 weeks post-vaccination with dose 2; 8WPD2: 8 weeks post-vaccination with dose 2.</p