Protection in immunized young adult mice is associated with improved cellular immunity and reduced viral titers.

<p>Young adult mice (6–8 weeks) vaccinated twice, 2 weeks apart, with Fluzone (FZ x2), Fluzone+CpG (FZ+CpG) and unvaccinated controls were challenged with 1x10⁴ p.f.u of A/California/09 H1N1. Five mice per group were sacrificed at day 7 post-infection. (A) The frequency of neutrophils (CD3-/Ly6C+) and (B) CD3+/CD8+ T-cells was measured by flow cytometry in homogenates of the right lobe of the lung. Dots represent individual mice and horizontal lines represent average +/-SEM where *p<0.05. (C) Lung virus titers from pooled mice (n = 5 per group) were determined day 7 post challenge using homogenates from the left lobe of the lung. Equal volumes of lung homogenate were pooled from 5 mice in each group and the horizontal bar represents average of duplicate wells. Data shown is representative of 2 independent experiments.</p

Single Fluzone vaccination with or without adjuvants is non-protective in aged mice.

<p>Young adult (6–8 weeks) and aged (18–24 months) C57/BL6 mice (n = 5–8 mice per group) were vaccinated with Fluzone (FZ), Fluzone+CpG (FZ+CpG) or left unvaccinated as controls. Approximately 4 weeks later, all groups were challenged with 1x10⁴ p.f.u of A/California/09 H1N1 virus. Weight loss (A) and (C) and survival (B) and (D) were monitored daily in young adult and aged mice respectively. Individual data points represent average weight loss within the group of surviving mice +/-SEM.</p

Antigen presenting cells from aged mice do not upregulate MHC class II expression in response to vaccine components <i>in vitro</i>.

<p>Bone marrow derived macrophages (BMM) or dendritic cells (BMDC) from young adult (6–8 weeks, dark bars) and aged (18–24 months, light bars) were stimulated <i>in vitro</i> with CpG, Fluzone (FZ) or both (FZ+CpG) overnight. Up regulation of MHC class II relative to unstimulated cells (media) was measured using flow cytometry on (A) BMM (F4/80+/CD11b+) and (C) BMDC (CD11c+/CD11b^med /F4/80-). TNF-α production was measured by ELISA in supernatants from (B) BMM and (D) BMDC. Readings were taken as an average of triplicate wells. Results shown are mean values + S.D and are representative of 2 independent experiments.</p

Heme agglutination inhibition and Influenza-specific IgG titers in sera of immunized mice.

<p>Heme agglutination inhibition and Influenza-specific IgG titers in sera of immunized mice.</p

Immune responses following infection by the tail scarification and i.p. routes.

<p>Mice were infected with 1×10⁶ PFU VACV-WR or vGK5 by the i.p. and t.s. routes. (A) Lung lymphocytes and splenocytes obtained from mice (n = 4 mice/group except for infection with VACV-WR by the t.s. route where splenocytes and lung lymphocytes from 2 mice were pooled together) infected 7 days prior were stained with B8R_20–27 tetramer. The data shown represent frequencies of cells that were tetramer positive within the CD3+CD8+ gate. Each symbol represents the frequency of tetramer+ T cells obtained in target organs of individual mice; median values are denoted by horizontal lines. (B) Seven days post infection, splenocytes were isolated and CTL assays were carried out using RMA cells infected with VACV-WR (moi = 5), vGK5 (moi = 5) at different (E/T) ratios. Data shown are representative of 2–3 experiments performed for each condition for the i.p. route. (C) PRNT50 antibody titers were measured in sera of mice immunized 3 months prior with 10⁶ PFU of VACV-WR (n = 3) or vGK5 (n = 4). (D) VACV titers were determined in organs 5 days post infection by the i.p. route and expressed as log₁₀ PFU per gram of lung and spleen tissue and PFU/ovary. – represents median values of titers in respective organs. N.S. = Not significant. P values were determined by Student's <i>t</i> test.</p

Cytokine responses and cytolytic activity in target organs.

<p>(A) IFN-γ responses of splenocytes from intranasally infected mice were measured in response to 3 VACV-specific CD8 T cell peptides (1 µg/ml) in an Elispot assay. Assays were performed using triplicate wells for each condition and individual mice/group. * = no responses detected. Seven days post infection, (B) splenocytes and (C) lung lymphocytes were isolated and CTL assays were carried out using RMA cells infected with VACV-WR (moi = 5), vGK5 (moi = 5) at different effector to target (E/T) ratios. Data shown are 1of 2 experiments performed. (D) PRNT50 antibody titers were measured in the sera of mice immunized 5 months prior with varying doses of vGK5 or 10^3.5 PFU of VACV-WR (n = 5/group).</p

Viral titers following i.n. infections.

<p>(A) Spleens and (B) lungs (n = 4–8 per group) were harvested on day 7 from mice infected with VACV-WR and vGK5 by the i.n. route. VACV titers were determined and expressed as log₁₀ PFU per gram of lung and spleen tissue. – represents median values of titers in respective organs. N.S. = not significant. Each symbol represents the titer obtained in target organs of individual mice; median values are denoted by horizontal lines. P values were determined by Student's <i>t</i> test.</p

Weight loss curves of individual mice and splenocyte counts following i.n. VACV infection of C57/BL6 mice.

<p>(A) Groups of female C57/BL6 mice (n = 5–8) were infected with 10⁵, 10⁶ and 10⁷ PFU of vGK5 by the i.n. route. The percentage of weight relative to the initial body weight (100%) was plotted and the data are presented as percent change in body weight following infection. † depicts days that individual mice were last alive. (B) Average spleen counts±standard deviation of mice were assessed by trypan blue exclusion at days 3, 5 and 7 post infection with 10⁶ VACV-WR and vGK5 by the i.n. route. Data shown are representative of 2 experiments performed and demonstrate that high dose VACV-WR i.n. infections result in significantly (p<0.05) lower lymphocytes in the spleens during acute infection.</p

Mice immunized with vGK5 are protected from a lethal challenge with VACV-WR.

<p>Mice were infected with 10^4.5, 10⁶ PFU of vGK5 (n = 3/group) by the intranasal route. 1 month later immunized mice and age matched naïve controls were challenged with a lethal dose of VACV-WR (10⁶ PFU) by the i.n. route. (A) Weights of mice were monitored over 5 days. Viral titers were measured in the (B) lungs and (C) ovaries and expressed as viral titers/gm lung tissue or ovaries. Each symbol represents the titer obtained in target organs of individual mice.</p