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

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

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

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

Renal pathology scores in TgFVB and HIV-1 transgenic congenic and sub-congenic strains.

<p>Renal pathology scores in TgFVB and HIV-1 transgenic congenic and sub-congenic strains.</p

Albuminuria in TgFVB and the HIV-1 transgenic sub-congenic strains.

<p>Albuminuria is expressed as μg albumin/mg creatinine ratio. Statistically significant increase in albuminuria was observed in Sub-III and Sub-IV groups, but not in the Sub-II group when compared to TgFVB mice. (nonparametric P-value: *-p<0.05, **-p<0.01).</p

Map of the <i>HIVAN1</i> locus, congenic and sub-congenic regions.

<p>The rectangles depict the congenic and sub- congenic segments. The top line shows the position of the limiting markers in Mb (genome build 38p.3/mm10). The limiting markers with FVB genotypes are shown in blue, and those with CAST genotypes in red. The segments carrying CAST alleles are shown in grey. (Con = congenic strain)</p

Pathway analysis of differentially expressed genes encoded outside the <i>HIVAN1</i> locus

<p>Pathway analysis of differentially expressed genes encoded outside the <i>HIVAN1</i> locus</p

Oral Vaccination with PEGylated Adenovirus Improves the B-cell Mediated but Not the T-cell Mediated Immune Response Against Ebola Glycoprotein.

<p>Naïve mice and those with pre-existing immunity were vaccinated with 1×10¹⁰ particles of either unmodified (Vaccine) or PEGylated (PEG-Vaccine) Ad5-ZGP by oral gavage. Pre-existing immunity (PEI) was induced by I.M. injection with 5×10¹⁰ particles of adenovirus expressing beta-galactosidase (AdlacZ). (A) Frequency analysis of IFN-γ secreting CD8+ T cells harvested from splenocytes 10 days post-immunization (n = 4/group). (B) Neutralizing antibody (NAB) levels against ZEBOV-EGFP were evaluated 25 days post-vaccination (n = 10/group). (C) Profile of anti-Ebola-specific IgG antibodies. (D) Profile of anti-Ebola-specific IgA antibodies. In all panels, error bars represent the standard deviation of the data.</p