Mice were infected with varying doses of vGK5 by the i.n. route. (A) We used a gating strategy to identify live CD3⁺CD8⁺ T lymphocytes (live dead aqua negative and forward scatter positive; CD3+CD8+). Frequencies of B8R_20–27 tetramer+ T cells were assessed in the (B) splenocytes and (C) lung lymphocytes 7 days after i.n. infection. (D) The absolute numbers of B8R_20–27 tetramer+ T cells in the lung following i.n. infection. Each symbol represents the frequency of tetramer+ T cells obtained in target organs of individual mice; median values are denoted by horizontal lines.

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 t test.

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.

(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.

(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).

(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 t test.