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. 2018 May 3;8:7009. doi: 10.1038/s41598-018-24581-y

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© The Author(s) 2018

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Two immunizations by homologous priming-boosting with rLVS ΔcapB/iglABC induces strong protection against respiratory challenge with the virulent F. tularensis Schu S4 strain. (a) Schedule - Experiment VI. Mice (n = 8/group) were immunized once with PBS (Sham), 10⁴ CFU LVS, 10⁶ CFU LVS ΔcapB vector, or 10⁶ CFU rLVS ΔcapB/iglABC; or twice with 10⁶ CFU rLVS ΔcapB/iglABC; challenged i.n. with F. tularensis Schu S4 (10 CFU, ~10 LD₅₀) at Week 10; and monitored for signs of illness, weight change, and death for 3 weeks, as indicated. (b) Survival after vaccination and challenge – Experiment VI. The survival curve of each vaccinated group is compared with that of the Sham (Group A) or LVS ΔcapB vector (Group C) group by the log-rank test (Mantel-cox); P values for vaccine groups that are significantly different from the Sham or LVS ΔcapB vector control group are marked with asterisk(s) and multiple “§”, respectively, color-coded to the color of the vaccine symbol. *P < 0.05; ***P < 0.001; and ****P < 0.0001 vs. Group A (Sham); ^§§P < 0.01; ^§§§P < 0.001 vs. Group C (vector control). (c) Schedule - Experiment VII. Mice (n = 8/group) were immunized once with PBS (Sham), 10⁴ CFU LVS, or 10⁶ CFU rLVS ΔcapB/iglABC three times at Weeks 0, 4, and 8, or twice at Weeks 4 and 8, bled at Week 13, and challenged i.n. with 2 CFU (2 LD₅₀) or 6 CFU (6 LD₅₀) F. tularensis Schu S4 at Week 14, and monitored for 3 weeks, as indicated. (d) Survival after vaccination and challenge and serum antibody pre-challenge – Experiment VII. Upper panels – Survival. The survival curve after challenge with 2 LD₅₀ (left panel) or 6 LD₆₀ (right panel) of each vaccinated group is compared with that of either the sham- or LVS-immunized group by the log-rank test (Mantel-cox); P values that are significantly different from the control group are marked with asterisk(s) color-coded to the color of the vaccine symbol. ***P < 0.001 vs. Group A (Sham); *P < 0.05 vs. Group B (LVS). Lower panel- Serum antibody after vaccination. Sera were assayed for IgG and subtypes IgG1 and IgG2a specific to heat-inactivated LVS. Data are mean + SEM of serum antibody endpoint titer for n = 8 per group. Differences among individual groups were compared by two-way ANOVA with Tukey’s correction. Values that are significantly different between two groups are marked with asterisk(s) over an open horizontal line crossing above the two groups. As indicated by the asterisks above the Sham group, its titers were significantly different from all other groups. *P < 0.05; **P < 0.01; and ****P < 0.0001.