Effect of H3N2 (X-31) influenza A virus infection on viral titer and body weight in wild type (WT) and Nox2^−/y mice.

<p>Mice were treated with 1×10⁴ PFU of the low virulence H3N2 strain of influenza A virus and (A) viral titer determined 3 days post infection and (B) body weight recorded for up to 7 days post infection. Data are shown as mean ± SD for 6–8 mice per group. *<i>P</i>&lt;0.05 vs WT mice (Students' unpaired <i>t</i> test).</p

Superoxide production from BALF cells using L-O12-enhanced chemiluminescence.

<p>BALF cells obtained from H3N2 (X-31) influenza A virus-infected WT and Nox2^−/y mice 3 days post infection. Data are shown as mean ± SD relative light units per second (RLU/s) for 6–8 mice per treatment group. *<i>P</i>&lt;0.05 vs WT no virus mice, #<i>P</i>&lt;0.05 vs respective WT mice (ANOVA and Dunnett's <i>post hoc</i> test).</p

Effect of H3N2 (X-31) influenza A virus infection on BALF cellularity in wild type (WT) and Nox2^−/y mice.

<p>Mice were treated with 1×10⁴ PFU of the low virulence H3N2 strain of influenza A virus and the number of (A) total cells, (B) macrophages and (C) neutrophils counted in the BALF 3 and 7 days post infection. Data are shown as mean ± SD for 6–8 mice per group. *<i>P</i>&lt;0.05 vs respective no virus group, #<i>P</i>&lt;0.05 vs D3 X-31-treated WT mice mice, ∧<i>P</i>&lt;0.05 vs D7 X-31-treated WT mice (ANOVA and Dunnett's <i>post hoc</i> test).</p

Peroxynitrite production in mouse lung infected with H3N2 (X-31) influenza A virus using 3-nitrotyrosine (3-NT) immunofluorescence.

<p>Representative sections of lung tissue obtained from WT (A) and Nox2^−/y mice (B) infected with X-31 were incubated with mouse monoclonal anti-3-nitrotyrosine antibody (1∶50) followed by biotinylated anti-mouse IgG reagent. (C and D) The same sections showing corresponding light microscope images. WT mice lung sections displayed strong immunofluorescence for 3-NT in the inflammatory cells that infiltrated the airways and in the alveolar tissue. In contrast, Nox2^−/y mice displayed markedly less immunofluorescence for 3-NT.</p

Cleaved caspase 3 immunofluorescence to assess lung alveolar epithelial apoptosis.

<p>Representative sections of lung tissue obtained from WT (A) and Nox2^−/y mice (B) infected with influenza A virus (H3N2; X-31) were incubated with rabbit polyclonal anti-cleaved caspase 3 antibody (1∶250) followed by goat anti-rabbit Alexa fluor 488 (Invitrogen; 1∶500) secondary antibody. (C and D) The same sections showing corresponding light microscope images. WT mice lung sections displayed strong immunofluorescence for cleaved caspase 3 in the alveolar tissue. In contrast, Nox2^−/y mice displayed markedly less immunofluorescence for cleaved caspase 3.</p

Number of the CD8+ T cell response.

<p>Enriched CD8+ T cells from BALF (A–C) or spleen (D–F) were obtained from influenza A virus (X-31)-infected WT and Nox2^−/y mice 7 days post infection. These were then stained with fluorescently labelled tetramer complexes specific for the two immunodominant CD8+ T cell epitopes (D^bNP_366–372 and D^bPA_224–232) and data analysed by flow cytometry. Data are shown as mean ± SD of 4 individual mice.</p

Restriction of RA9 CD8 T cell epitope to <i>Mane-A*10</i>.

<p>(<b>A</b>) CD8 T cell response to influenza RA9 peptide in influenza vaccinated animal 26359 (<b>B</b>) Expansion of RA9 specific CD8 T cells. RA9-specific CD8 T cell response in fresh blood is shown in comparison to a 2 week <i>in vitro</i> expansion as described in methods. (<b>C</b>) <i>Mane-A*10</i> restriction of RA9 response. Transfected (<i>Mane-A*10 or Mane-B*02</i>) and untransfected C1R cells were pulsed with either DMSO or RA9 peptide. These C1R cells were incubated separately with <i>in vitro</i> cultured RA9-specific CD8 T cells and IFN-γ and TNF-α expression measured.</p

Exhaustion marker expression on influenza and SIV-specific CD8 T-cells.

<p>(<b>A</b>) Comparison of PD-1 expression on influenza RA9 tetramer-positive CD8 T-cells (top) and SIV KP9 tetramer-positive CD8 T-cells (bottom). RA9-tetramer staining was performed on frozen PBMC samples from animals either influenza vaccinated (influenza vaccinated after SIV infection or influenza vaccinated then SIV infected. Samples were typically tested at around 2–3 weeks post final vaccination. Tetramer staining of KP9-positive CD8 T-cells was performed on frozen PBMC samples from animals either, SIV vaccinated, SIV infected then vaccinated or SIV infected. Samples were compared using a One-way ANOVA with overall p = 0.956 for the influenza-RA9 comparison and p<0.001 for SIV-KP9 comparison (<b>B</b>) Magnitude of the tetramer responses in the same groups as for (B). Samples were compared as above using a One-way ANOVA overall p>0.05 for both influenza-RA9 comparison and SIV-KP9 comparison. (<b>C</b>) Frozen PBMC samples from two animals inoculated with recombinant influenza-SIV viruses (animals 1335 and 2374) were stained for CD28 expression on RA9 or KP9 tetramer positive cells prior to and following challenge with SIVmac251.</p

Mane-A*10+ pigtail macaques studied for Influenza- and SIV-specific CD8 T cell responses.

*<p>All SIV CTL epitopes were minimal epitopes expressed from within the stalk of influenza neuraminidase protein.</p>**<p>The DNA and fowlpoxvirus vaccines expressed full length SIV Gag and Pol proteins from standard promoters.</p

Comparison of polyfunctional influenza and SIV-specific CD8 T cell responses following recombinant influenza-SIV vaccination.

<p>Polyfunctional ICS assay was performed on whole blood from the animal 26359 at day 133 after initial influenza vaccination stimulating with either RA9 or KVA10 peptides. (<b>A</b>) Gating strategy on SEB stimulated sample (<b>B</b>) Summary of functional profile; the proportions of the number of effector molecules produced by cells when stimulated with either RA9 or KVA10 peptide. (<b>C</b>) The frequency of total effector molecules produced by RA9 (black bars) and KVA10 (white bars) specific CD8 T cells for animals 26359 and B0526.</p