Protection following vaccination with PAV3-HA or AdHu5-HA vaccine.

<p>(A–C) Groups of 9 BALB/c mice were vaccinated with 10¹⁰ vp/mouse of PAV3-HA (▵), AdHu5-HA (▴), or PBS Control (•) 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015301#pone.0015301-Reed1" target="_blank">[39]</a> 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).</p

Comparative detection of H5N1-H05 HA protein expressed by AdHu5-HA and PAV3-HA.

<p>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, (−) negative control, untransfected cell lysate. (B) <i>In vivo</i> expression in mouse muscle tissues 4 days following vaccination. 75 µg of muscle tissue was loaded in each lane to compare protein expression. M = Marker, Lanes 1–6 = PAV3-HA, Lanes 8–13 = AdHu5-HA, (−) = unvaccinated muscle tissue.</p

Long-term protection in BALB/c mice.

<p>Long-term protection was evaluated in groups of six BALB/c mice vaccinated with PAV3-HA 10⁹ (▿) vp/mouse, AdHu5-HA 10⁹ (▾) vp/mouse, and PBS Control (•). (A) Mice were challenged 12 months following vaccination with 100LD50 of H5N1-H05 virus and monitored for (i) survival and (ii) weight loss and clinical signs of disease. (B) Long-term antibody responses. Serum was collected 12 months post-vaccination from mice immunized with PAV3-HA () or AdHu5-HA (). HI and NAB responses were evaluated. The data represent average values and standard deviations from one experiment performed with one vector preparation of each vaccine (* represents p = 0.006). (C) ELISA detecting total IgG antibody titres against the H5 HA antigen. Endpoint titers were assessed for PAV3-HA () or AdHu5-HA () 25 days and 1 year post-vaccination.</p

T-cell responses detected by ELISPOT-IFNγ and flow cytometry.

<p>Groups of 4 BALB/c mice were vaccinated with 10¹⁰ vp/mouse of either PAV3-HA () or AdHu5-HA (). Mouse spleens were harvested 8, 10, 14, and 21 days post-immunization and positive T-cell responses following peptide restimulation were determined through IFNγ secretion, as indicated by spot formation on the membrane. (A) Pooled T-cell responses. Cells were restimulated with a peptide library of overlapping 15mers spanning the entire H05-HA protein. Bars represent the total number of spot-forming cells/million mononuclear cells for all peptide pools combined. The data represent average values and standard deviations from 4 experiments performed with 3 independent vector preparations of each vaccine (* represents p<0.005). (B) T-cell responses following stimulation with immunodominant peptide. Splenocytes were restimulated with individual 9mer or 8mer peptides representing the immunodominant epitope of H05-HA (IYS) or control peptides (PR8-NP TYQ (), ZGP). Positive responses for IFNγ secretion were detected by ELISPOT. Bars represent the total number of spot-forming cells/10⁶ mononuclear cells. The data represent average values and standard deviations from 4 experiments performed with 3 independent vector preparations of each vaccine (** represents p<0.05). C) Flow cytometric analysis representing the percentage of CD8⁺IFNγ secreting T-lymphocytes day ten post-immunization. The data represent average values and standard deviations from 3 experiments performed with one vector preparation of each vaccine.</p

Hemagglutination inhibition (HI) and neutralizing antibody (NAB) assays for PAV3-HA and AdHu5-HA.

<p>Serum was obtained from groups of PAV3-HA () or AdHu5-HA () vaccinated BALB/c mice and treated overnight at receptor-destroying enzyme (RDE), followed by complement inactivation the following day. (A) Kinetics of the HI antibody response. Serum was obtained from groups of 4 BALB/c mice on days 8, 10, 14, and 21 post-vaccination with 10¹⁰ vp/mouse of PAV3-HA or AdHu5-HA. Four agglutinating doses of H5N1-H05 virus were added to serial dilutions of serum and samples were incubated with horse red blood cells. HI antibody titre was determined as the reciprocal of the highest serum dilution which did not cause red blood cell agglutination. (B) Kinetics of the NAB response. Serum from obtained on 8, 10, 14, and 21 days post-vaccination with 10¹⁰ vp/mouse of PAV3-HA or AdHu5-HA. Serial dilutions of was also incubated with 100 pfu of H5N1-H05 virus, added to MDCK cells and monitored for the presence of cytopathic effects (CPE) over 48 hours. The NAB titre was scored as the highest serum dilution with the absence of CPE. (C) HI titre against H5N1-H05. Serum was obtained 25 days post-immunization from groups of 5–10 BALB/c mice were vaccinated with 10⁸, 10⁹, and 10¹⁰ vp/mouse of AdHu5-HA or PAV3-HA and the HI titre was assayed. (D) NAB titres against H5N1-H05. Serum from 25 days post-immunization was also monitored for NAB titre. The data represent average values and standard deviations from 3 to 5 experiments performed with 2 independent vector preparations of each vaccine (* represents p<0.05).</p

Induction of proapoptotic molecules in lungs from pigs infected with <i>Zaire ebolavirus.</i>

<p>Data was obtained by microarray analysis of RNA from lungs of pigs infected with <i>Zaire ebolavirus</i> in experiment 1. Numbers represent fold change in transcription relative to tissues from uninfected controls. DPI, days post infection. N = 2 pigs for controls, DPI 3, 5 and 7.</p

Antibodies from H1N1 and H1N2 infected chickens reduce H5N1 plaque size.

<p>MDCK cells were infected with H5N1 in the presence of sera from H1N1-infected (open histograms), H1N2-infected (grey histograms) or naïve (black histograms) chicken and plaque size (mm) determined after 3 days. Data represents mean and error bars are standard deviation for sera from at least 5 chickens per group.</p

Model for pulmonary disease pathogenesis in <i>Zaire ebolavirus</i> (ZEBOV)-infected pigs based on microarray data.

<p>Arrows show the proposed sequence of events that take place from the time of ZEBOV infection to the development of lung inflammation and respiratory distress. The grey shaded blocks represent the anti-viral/anti-inflammatory machinery. The scale suggests that the series of events favour the proinflammatory pathway.</p

Up-regulation of pattern recognition receptors, transcription factors and interferon stimulated genes in lungs from pigs infected with <i>Zaire ebolavirus.</i>

<p>Data was obtained by microarray analysis of RNA from lungs of pigs infected with <i>Zaire ebolavirus</i> in experiment 1. Numbers represent fold change in transcription relative to tissues from uninfected controls. DPI, days post infection. N = 2 pigs for controls, DPI 3, 5 and 7.</p

Virus detection in tissues from pigs infected with <i>Zaire ebolavirus</i> (ZEBOV).

<p>Virus detection for pigs infected with <i>Zaire ebolavirus</i> in experiment 2 was done by quantitative real time RT-PCR targeting the L gene of ZEBOV and L gene copy numbers estimated based on a standard curve. Data represents log₁₀ copies/mL of tissue suspension (numbers in brackets represent standard deviations of 3 replicates for each sample). SLN, submandibular lymph node; BLN, bronchial lymph node; MLN, mesenteric lymph nodes, U, virus was either absent or below levels detectable by the current assay.</p