Protective effect of multiple-clade influenza vaccine in mice challenged with A/OT/SZ/097/03.

<p>Groups of mice (<i>n</i> = 10/group) 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 and challenged i.n. with 50LD₅₀ OT/SZ/097/03 virus suspension. Mice were monitored for weight change (A) and survival rates (B) throughout a 14-day observation period.</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

Production of the H7N9/PR8 influenza vaccine candidate using reverse genetics, and the size distribution and shapes of the virus particles.

<p>(A) Preparation of the H7N9 vaccine seed strain using eight-plasmid reverse genetics. (B) Electron micrograph of recombinant H7N9/PR8 virus particles. (C) Most (73%) of 200 H7N9/PR8 virus particles were 80–120 nm in diameter.</p

Characterization of the 2009 Pandemic A/Beijing/501/2009 H1N1 Influenza Strain in Human Airway Epithelial Cells and Ferrets

<div><h3>Background</h3><p>A novel 2009 swine-origin influenza A H1N1 virus (S-OIV H1N1) has been transmitted among humans worldwide. However, the pathogenesis of this virus in human airway epithelial cells and mammals is not well understood.</p> <h3>Methodology/Principal Finding</h3><p>In this study, we showed that a 2009 A (H1N1) influenza virus strain, A/Beijing/501/2009, isolated from a human patient, caused typical influenza-like symptoms including weight loss, fluctuations in body temperature, and pulmonary pathological changes in ferrets. We demonstrated that the human lung adenocarcinoma epithelial cell line A549 was susceptible to infection and that the infected cells underwent apoptosis at 24 h post-infection. In contrast to the seasonal H1N1 influenza virus, the 2009 A (H1N1) influenza virus strain A/Beijing/501/2009 induced more cell death involving caspase-3-dependent apoptosis in A549 cells. Additionally, ferrets infected with the A/Beijing/501/2009 H1N1 virus strain exhibited increased body temperature, greater weight loss, and higher viral titers in the lungs. Therefore, the A/Beijing/501/2009 H1N1 isolate successfully infected the lungs of ferrets and caused more pathological lesions than the seasonal influenza virus. Our findings demonstrate that the difference in virulence of the 2009 pandemic H1N1 influenza virus and the seasonal H1N1 influenza virus <em>in vitro</em> and <em>in vivo</em> may have been mediated by different mechanisms.</p> <h3>Conclusion/Significance</h3><p>Our understanding of the pathogenesis of the 2009 A (H1N1) influenza virus infection in both humans and animals is broadened by our findings that apoptotic cell death is involved in the cytopathic effect observed <em>in vitro</em> and that the pathological alterations in the lungs of S-OIV H1N1-infected ferrets are much more severe.</p> </div

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

Antibody responses of ferrets immunized with H7N9/PR8 virus.

<p>HI antibody responses to the (wt) AnHui virus after intramuscular vaccination with 7.5, 15, 30, or 45 µg (HA levels) of H7N9/PR8 split vaccine, in 200-µl volumes. Serum samples were collected on day 0; 2 weeks after priming; and 2, 4, and 8 weeks after boosting. 2WPD1: 2 weeks post-vaccination dose 1. 2WPD2: 2 weeks post-vaccination dose 2; 4WPD2: 4 weeks post-vaccination dose 2; 8WPD2: 8 weeks post-vaccination dose 2. The lower limit of detection is indicated by the dashed horizontal line.</p

A/Beijing/501 induces apoptosis in the A549 cells.

<p>(A) Western blot analysis of mock-infected and H1N1-infected A549 cell lysate with anti-caspase 3, anti-PARP and anti-β-actin antibodies after a 24 h infection. (B–C) Cells were infected with seasonal H1N1, A/Beijing/501 H1N1 and A/CA/07 H1N1 influenza viruses. In situ apoptosis was detected using a FITC-dUTP labeled TUNEL assay and statistical analysis of relative proportion of TUNEL positive cells. For quantification, >1000 cells were scored in three independent experiments. (D–E) Knockdown of caspase 3 inhibits A/Beijing/501 H1N1 virus replication. A549 cells were transfected with the control, siRNA-1 and siRNA-2 against caspase 3 (50 nm). 24 hours later, Western blot analysis (D) and MTT assay (E) of A/Beijing/501 H1N1-infected A549 cells. **p<0.001.</p

Pathogenicity and replication of a reassortant H7N9/PR8 virus in BALB/c mice^a.

a<p>BALB/c mice were inoculated intranasally with 10⁶ EID₅₀ of the indicated viruses and euthanized 3 days later. Virus titers in lung, brain, spleen, and liver tissue were determined by titration in MDCK cells. Geometric mean titers ± SDs are shown.</p>b<p>p<0.01 compared with the tiers in the corresponding organs of the AnHui 01 or PR8-inoculated mice.</p>c<p>The lower limit of detection was 10 TCID₅₀/g of tissue.</p

Immunogenicity of a H7N9 split vaccine prepared from the H7N9/PR8 transfectant virus.

a<p>Groups of mice and ferrets received two doses of 7.5, 15, 30 or 45 µg monovalent influenza A (H7N9) split vaccine or PBS i.m in 14 days intervals.</p>b<p>Serum samples were collected and assayed at 2 weeks after boost.</p