Fig. 6: Cell proliferation and IFNγ production in mice immunized with rAd5-YFV vaccine during long-term study.

Mice (n = 10/group) were immunized (i.n.) twice 21 days apart with rAd5-YFV vaccine (a). Forty-two days after the second vaccination (day 63 of the study), spleens were harvested from mice (n = 5) and also on day 3 post-challenge with Y. pestis CO92-lux (b). For cell proliferation study after the second vaccination, mouse splenocytes were stimulated with rF1-V and treated with BrdU for the post-vaccination timepoint (n = 5) as described in panel b. For the post-challenge timepoint, mice (n = 5) was i.n. challenged on day 85 after immunization (day 105 of the study) with Y. pestis CO92-lux, and i.p. injected with BrdU (b). On day 3 p.i., spleens were harvested and splenocytes were then stained for T- and B-cell surface markers (CD3 and CD19) as well as for incorporated BrdU, and analyzed by flow cytometry. The percent of BrdU incorporation in CD3 (c) or CD19 (d) positive cells was plotted. For IFNγ studies (e, f), splenocytes were stimulated with PMA, Ionomycin, and Brefeldin A. Cells were then stained with T-cell surface markers CD3, CD4, and CD8 followed by intracellular IFNγ staining. Percentages of CD4⁺ IFNγ⁺ (e) and CD8⁺ IFNγ⁺ cells (f) were shown. Student’s t-test was used to determine statistical significance between T-cell population from control and rAd5-YFV vaccinated groups. Asterisks above columns represent comparison to the control group. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Two biological replicates were performed, and data plotted.