Morbidity, survival, and lung viral titers after influenza virus challenge of young and middle-aged mice.

<p>Groups of 14 BALB/cAnNCr mice of age 2 months (A, C, and E) or 11 months (B, D, and F) were immunized as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153195#pone.0153195.g001" target="_blank">Fig 1</a>. In addition, 6 mice per age group were left unvaccinated (naïve, Fig 4E and 4F). One month after rAd, animals were challenged with 10⁴ TCID₅₀ A/FM-ma. Panels A and B show the mean % starting body weight + SD of 8 mice per group. Panels C and D show survival until weight loss endpoint; *p≤0.001 by the log-rank (Mantel-Cox) for all groups compared to B/NP control. Lung virus titers (E and F) were determined by TCID₅₀ assay on the lungs of 3 animals per group harvested on day 3 (open bars) and day 5 (solid bars) after challenge, along with lungs from naïve mice of the same age infected at the same time. Shown are the mean ± SEM of 3 mice per group with the limit of detection indicated by a horizontal line. ** p≤0.01 was determined by ANOVA vs. the B/NP and naïve control groups. The difference was statistically significant (**p<0.01) on day 5 for two-month-old animals. The 2- and 11-month-old groups were not performed simultaneously, so we cannot conclude that there was superior control of viral replication in one age group.</p

Characterization of lung T cells from young and middle-aged mice.

<p>BALB/cAnNCr mice were immunized at 2 months (A, C, and E) or 11 months old (B, D, and F) as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153195#pone.0153195.g001" target="_blank">Fig 1</a> or left unvaccinated (naïve). Lung T cells were prepared and analyzed by flow cytometry. (A, B) Total numbers of K^d-NP_147–155-tetramer⁺ CD8⁺ T cells recovered from the lungs 12–13 weeks after vaccination were determined by multicolor flow cytometry. Bars show mean ± SEM of 3–4 animals per group for the two-month-old mice (A). For 11- month-old mice, cell yield after Ficoll was low and lung cells had to be pooled to permit analysis (B). (C, D) Lung CD8⁺ T cell phenotypic analysis: Proportions of activated effector memory (CD62L^lo, CD127^lo), effector memory (CD62L^lo, CD127^hi) and central memory (CD62L^hi) among tetramer⁺ CD8⁺ T cells from A/NP+M2 vaccinated animals are displayed as pie charts. (E, F) Activation status of pooled lung CD8⁺ T cells characterized by positive staining for both tetramer and CD69 marker. Fluorescence minus one (FMO) controls were used to identify gating boundaries and adjusted based on internal negative controls for each treatment group. Percent of total CD8⁺ T cells are displayed per quadrant.</p

Morbidity and survival following rAd given twice and the impact of prior vaccination.

<p>(A-B) Groups of 4 (A/NP) or 9 (A/M2) or 4 (B/NP) BALB/cAnNCr mice were available from groups given 1×10¹⁰ particles of the respective rAds at two and three months of age. They were then challenged with 10³ TCID₅₀ of A/FM-ma at 20 months of age. (A) Weight loss after challenge, shown as the mean + SD. (B) Survival after challenge until weight loss endpoint, p = 0.016 for A/M2 vs B/NP control group (*). The A/NP group was too small to reach significance for protection versus the B/NP control group. (C-F) Groups of BALB/cAnNCr mice (N = 4 to 7) were given 1×10¹⁰ particles of individual rAd, or 5×10⁹ of each rAd in the mixture of A/NP and M2 at two and three months of age (Fig 7C-D) or 20 and 21 months (Fig 7E and 7F), then challenged with 10³ TCID₅₀ of A/FM-ma a month after the boost. (C, E) Weight loss after challenge shown as the mean + SD. (D, F) Survival after challenge until weight loss endpoint.</p

Morbidity and survival following challenge in young and elderly mice.

<p>(A-D) Groups of BALB/cAnNCr mice or (E-H) BALB/cJ mice were immunized to A/NP+M2 (N = 6 to 8) or B/NP (N = 6 to 7) by DNA priming followed by rAd boosting. They were challenged with 200 TCID₅₀ of A/FM-ma one month after rAd. (A, C, E, and G) Weight loss after challenge shown as the mean + SD. (B, D, F, and H) Survival until weight loss endpoint after challenge. Animals were euthanized if weight decreased by more than 30% of the individual starting weight (a pre-determined endpoint for this experiment so that even weak protection would have been seen in 20-month-old mice, if present). Not all groups were vaccinated and challenged simultaneously, but each old group was vaccinated and challenged simultaneously with a young control group of the same BALB/c subline. Young animals in different groups showed similar results.</p

Cytotoxic activity.

<p>BALB/cBy mice of age 2 months were immunized as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153195#pone.0153195.g001" target="_blank">Fig 1</a>, and lung cells analyzed about 3 weeks after the last vaccination. Lung cells from mice infected 6 days earlier with A/Mex were used as a positive control for cytotoxicity. (A) Total number of CD3⁺CD8⁺CD107⁺Tetramer⁺ cells in lung cells pooled from 4 animals was determined by multicolor flow cytometry after stimulation with peptides NP_147–155+NP_55–69+M2_e2–24 (NP/M2e; solid bars), or SARS M peptide control (open bars). (B) Splenocytes were labeled with CFSE or CMTMR, and pulsed with peptides as indicated. 200,000 of these splenocytes were then incubated with 2x10⁶ lung cells of A/NP+M2- or B/NP-immune or A/Mex-infected mice for 5.5 hours. Staining with CFSE is displayed on the horizontal axis, staining with CMTMR on the vertical axis. Dead cells were defined as Vivid⁺ and values shown in the top right corner for each treatment. Top panel: Both CFSE- and CMTMR-labeled cells were control-pulsed (SARS M peptides). Bottom panel: CFSE-labeled target cells were pulsed with NP/M2e peptides, CMTMR-labeled target cells were control-pulsed. (C) Percent specific cell death calculated from the flow cytometry data as described in Materials and Methods. All results shown are representative of two experiments. We were not able to obtain enough elderly mice for comparison of CTL activity to that in young mice.</p

Immune responses in young and elderly mice.

<p>BALB/cBy mice of age 2 months (A, C, D, G, and H) or 20 months (B, E, F, I, and J) were immunized as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153195#pone.0153195.g001" target="_blank">Fig 1</a>. <b>T cell responses:</b> Three weeks after the rAd immunization, T cell responses in lungs were determined by IFN-γ ELISPOT (A, B). IFN-γ responses of lung T cells are shown following stimulation with peptides NP_147–155, NP_55–69, and M2_e2–24. SARS M stimulated cells were used as controls. Bars show mean total lung IFN-γ secreting cell number per organ ± SEM for 3 or 4 mice per group. ***p≤0.05 comparison of A/NP+M2 versus B/NP by Mann-Whitney rank sum test determined for each peptide, performed only for those samples significantly above SARS M. <b>Antibody responses:</b> (C-J) Samples were collected three weeks after rAd from A/NP+M2 DNA prime-rAd boost (solid circles) and B/NP DNA prime-rAd boost (solid triangles) mice. Sera (C-F) or BAL (G-J) were analyzed by ELISA. Results show the mean of absorbance ± SEM of sera from 3–12 mice or BAL samples from 3–4 mice per group. Background from secondary antibody alone was subtracted.</p

Vaccination to Conserved Influenza Antigens in Mice Using a Novel Simian Adenovirus Vector, PanAd3, Derived from the Bonobo <em>Pan paniscus</em>

<div><p>Among approximately 1000 adenoviruses from chimpanzees and bonobos studied recently, the Pan Adenovirus type 3 (PanAd3, isolated from a bonobo, <i>Pan paniscus</i>) has one of the best profiles for a vaccine vector, combining potent transgene immunogenicity with minimal pre-existing immunity in the human population. In this study, we inserted into a replication defective PanAd3 a transgene expressing a fusion protein of conserved influenza antigens nucleoprotein (NP) and matrix 1 (M1). We then studied antibody and T cell responses as well as protection from challenge infection in a mouse model. A single intranasal administration of PanAd3-NPM1 vaccine induced strong antibody and T cell responses, and protected against high dose lethal influenza virus challenge. Thus PanAd3 is a promising candidate vector for vaccines, including universal influenza vaccines.</p> </div

NPM1 fusion protein insert.

<p>a) Design of the insert showing CMV promoter, NPM1 transgene, and BGH-polyadenylation cassettes. b) Complete amino acid sequence of the consensus NPM1 fusion protein. NP is indicated in red, linker sequence is shown in black, and M1 is green. The deletion of the nuclear localization signal by mutation of TKR to AAA in NP is indicated in bold text.</p

T cell responses to different doses of PanAd3-NPM1.

<p>Mice were immunized as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055435#pone-0055435-g003" target="_blank"><b>Figure 3</b></a>. Four weeks post-immunization, T -cells of three mice per group were assayed by IFN-γ ELISPOT. Results shown are for lung T cells, and are reported as number of IFN-γ secreting cells per 10⁶ cells. Bars show mean ± SEM of results for lungs of individual mice.</p

Protection against lethal influenza challenge by PanAd3-NPM1.

<p>Mice were immunized as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055435#pone-0055435-g003" target="_blank"><b>Figure 3</b></a>. Approximately 9 weeks post-immunization, mice were challenged with influenza virus A/FM at a dose of 10⁴ TCID₅₀ (100 LD₅₀) per mouse, and monitored for body weight and mortality. Survival of the PanAd3-NPM1 group at the dose of 10⁹ vp differs significantly (p<0.05) from the PanAd3-RSV control group. Error bars indicate mean ± SEM.</p