Figure 1.
Single-dose i.n. rAd immunization protects from lethal influenza virus challenge.
Groups of ten 12-week old BALB/cAnNCr mice were immunized with 1×1010 particles of A/NP-rAd or M2-rAd, or with 1×1010 particles each of A/NP and M2-rAd, via the i.m. or i.n. routes. 4 weeks after immunization, animals were challenged i.n. with 104 TCID50 (100 LD50) A/FM/1/47-ma (H1N1) and monitored for survival (A) and weight loss (B).
Figure 2.
Immune responses after single-dose rAd immunization.
BALB/cAnNCr mice were immunized with 5×109 particles each of A/NP-rAd and M2-rAd, or 1×1010 particles of B/NP-rAd i.n. or i.m., or were unimmunized (naïve). Analyses were performed at one month (A, C, E, G) or 10 months (B, D, F, H) post-immunization. (A, B) M2e-specific IgG (left panels) and IgA (right panels) responses in serum and BAL were measured by ELISA as described. Bars show mean ± SEM of 3 mice per group. The dashed line indicates limit of detection. (C, D) Pro-inflammatory cytokine levels in BAL were measured as described. Bars show mean ± SEM of 4 mice per group at 1 month or 3 mice per group at 10 months. T-cell responses in spleen (E, F) and lung (G, H) were measured by IFN-γ ELISPOT of triplicate wells after stimulation with NP147–155, Hex486–494, Dbp413–421, M2e2–24, NP55–69 peptides. Unstimulated cells (no peptide) were used as controls. Bars show mean total IFN-γ secreting cell number per organ ± SEM of 4 mice per group at month or 3 mice per group at 10 months. Statistically significant differences are indicated as follows: * P<0.05 compared to all other groups; ‡ P<0.05 compared to i.m. and naïve groups; † P<0.05 compared to i.n. and naïve groups; ** P<0.05 compared to B/NP-rAd and naïve groups; § P<0.05 compared to A/NP+M2-rAd i.n. and naïve groups; # P<0.05 compared to all other groups except A/NP+M2-rAd i.m.
Figure 3.
BALB/cAnNCr mice were immunized with 5×109 particles each of A/NP-rAd and M2-rAd, or 1×1010 particles of B/NP-rAd i.n. or i.m., or were unimmunized (naïve). Lung T cells were isolated and analyzed by flow cytometry. (A) Total number of Kd-NP147–155-tetramer positive CD8+ T cells recovered from the lungs of mice, as determined by multicolor flow cytometry. Bars show mean ± SEM of 3 animals per group. (B) Phenotypic analysis of lung CD8+ T cells. Pie charts show relative proportions of central memory (CD62Lhi), effector memory (CD62Llo, CD127hi) and activated effector memory (CD62Llo, CD127lo) among tetramer positive (upper pies) and tetramer negative (lower pies) CD8+ T-cells. (C) Activation status of lung CD8+ T cells, as determined by staining for tetramer vs. CD69. Each plot shows one representative mouse per group of 3 mice assessed. Numbers in plots indicate % of CD8+ T cells per quadrant. * Indicates a statistically significant difference (P<0.05) compared to all other groups.
Figure 4.
Morbidity and mortality after H5N1 challenge following single-dose rAd immunization.
Groups of 10 (at one month) or 8 (at 10 months) BALB/cAnNCr mice were challenged with 102.84 EID50 (∼10 LD50) of A/VN1203 one month (A, B, C) or 10 months (D, E, F) after immunization. (A, D) Survival after challenge. (B, E) Weight loss after challenge. When challenged one month post-boosting, statistically significant differences in weight loss (P<0.05) were observed between A/NP+M2-rAd i.n. and all other groups at days 4–12. When challenged at 10 months, weight loss in A/NP+M2-rAd i.n. immunized mice was significantly (P<0.05) different from all other groups from days 4–16. (C, F) Virus titers in the lungs at days 3 and 5 after challenge as determined by plaque assay. Bars show log10 geometric mean titer ± SEM of 4 mice per group. The dashed line shows limit of detection. * indicates a statistically significant difference (P<0.05) compared to all other groups at the same time point; ** indicates a significant difference from B/NP-rAd and naïve groups.
Figure 5.
IgA is not required for protection after i.n. rAd immunization.
Groups of 6 IgA−/−-BALB/c mice were immunized with 5×109 particles each of A/NP-rAd and M2-rAd i.n. or i.m., or 1×1010 particles of B/NP-rAd i.n. (A) IgG responses against M2e were measured by ELISA using serum obtained 2 weeks after immunization. The dashed line indicates limit of detection. (B) Antigen-specific T-cell responses at 3 weeks post-immunization were determined in peripheral blood pooled from these animals by IFN-γ ELISPOT using NP147–155, NP55–69 or M2e2-24 peptides as stimulus. Unstimulated cells (no peptide) were used as a control. Bars show mean ± SEM of triplicate wells for each group per stimulus. At one month post-immunization, animals were challenged with 104 TCID50 (100 LD50) of A/FM and monitored for survival (C) and weight loss (D). Error bars in weight loss graph indicate mean ± SEM. Statistically significant differences in weight loss (P<0.05) were observed between A/NP+M2-rAd i.n. and i.m. at days 2–15, between A/NP+M2-rAd i.n. and B/NP-rAd at days 3–13, and between A/NP+M2-rAd i.n. and B/NP-rAd at days 2, 3, 9 and 13.
Figure 6.
Kinetics of protection after single-dose rAd immunization.
BALB/cAnNCr mice were immunized with 5×109 particles each of A/NP-rAd and M2-rAd, or 1×1010 particles of B/NP-rAd i.n. or i.m. and challenged with 104 TCID50 (∼100 LD50) of A/FM at 1, 2, 3, or 4 weeks or 6 months later. Left panels show survival and right panels show weight loss after challenge. Groups consisted of 10 mice per immunization per challenge time. Error bars in weight loss graphs indicate mean ± SEM. Statistically significant (P<0.05) differences in survival were as follows: A/NP+M2-rAd i.n. was significantly different from A/NP+M2-rAd i.m. at weeks 2, 3, and 4, and from B/NP-rAd i.n. or i.m. at all times; A/NP+M2-rAd i.m. was significantly different from B/NP-rAd i.m. at 2 weeks and 6 months, but not different from B/NP-rAd i.n. at any time. In terms of weight loss, no statistically significant differences were seen between groups at week 1. At week 2 A/NP+M2-rAd i.n. differed (P<0.05) from all other groups on days 2–7, and also from B/NP-rAd i.n. on day 1; A/NP+M2-rAd i.m. was significantly different from B/NP-rAd i.n. on days 2–3 and from B/NP-rAd i.m. on day 1. At week 3 A/NP+M2-rAd differed from all other groups on days 2–8; B/NP-rAd i.n. differed from all other groups on day 3. At week 4 A/NP+M2-rAd i.n. was significantly different from all other groups on days 2–15; A/NP+M2-rAd i.m. differed from B/NP-rAd i.m. on day 11. At 6 months A/NP+M2-rAd i.n. was significantly different from all other groups on days 2–10, and from A/NP+M2-rAd i.m. on days 11–15.
Figure 7.
Kinetics of virus clearance following H1N1 challenge.
BALB/cAnNCr mice were immunized with 5×109 particles each of A/NP-rAd and M2-rAd, or 1×1010 particles of B/NP-rAd i.n. or i.m. and challenged with 104 TCID50 (∼100 LD50) of A/FM one month later. Lung virus titers were measured by TCID50 at the indicated time points, and are expressed as geometric mean titer ± SEM of 3 mice per group per time point, except for day 7 in the B/NP-rAd i.m. group and day 10 in the B/NP-rAd i.n. group where only 1 mouse remained alive (indicated by †). The dashed line shows limit of detection. * indicates a statistically significant difference (P<0.001) compared to all other groups at the same time point.