Skip to main content
. 2020 Nov 6;11:585359. doi: 10.3389/fimmu.2020.585359

Figure 3.

Figure 3

H83, but not H89, mediate long-term protection in chronically infected mice. (A) Percentage of of Rv1198-specific CD4 T cells was assessed in Mtb9.9-vaccinated CB6F1 mice by ICS. Splenocytes were restimulated Rv1198 pepmix or media two weeks post 3rd vaccination (n = 4). Paired t-test, two-tailed, t-values (3.75, df = 3). (B) The bacterial burden were determined in the lungs of saline, Rv1198 and ESAT-6-vaccinated mice 4 weeks post Mtb infection (n = 8) as shown by CFUs relative to mice immunized with saline. An Ordinary One-way ANOVA with Tukey’s multiple comparison test, F-value (5, 41) = 41.59, was performed and the exact p-values indicated. (C) H89 was constructed by replacing ESAT-6 with an ESAT-6 like antigen, Rv1198, in a vaccine consisting of the same highly immunogenic antigens as in H83. (D) Percentage of antigen-specific CD4 T cells in H89-vaccinated mice. Splenocytes were restimulated with H89, MPT70, Rv1198, Rv3020c, Rv3019c, or media two weeks after 3rd vaccination (n = 4). Brown-Forsythe and Welch ANOVA tests with Dunnett’s multiple comparison test, F-value (3.000, 3.045) = 8.660, W-value (3.000, 5.882) = 7.711. (E) Schematic overview of the standard preventive model. Mice were vaccinated s.c. three times with the CAF01 adjuvant only or 0.5 µg H83 or H89 at the base of the tail with two-week interval (black arrows) and challenged with 50–100 CFU Mtb Erdman 6 weeks after the 3rd vaccination (red arrow). CFUs were determined 4 and 24 weeks after Mtb challenge. (F) Early (week 4) and late chronic (week 24) vaccine protection of H83 and H89 in a preventive setting (n = 8). A Two-Way ANOVA with Tukey’s multiple comparison test comparing cell means regardless of rows and columns, Finteraction (3, 56) = 3.134, Frow (1, 56) = 6.819, Fcolumn (3, 56) = 12.77. Post infection (p.i.) (G) Schematic overview of the re-infection model (post-exposure vaccination). CB6F1 mice were infected by the aerosol route with 25-50 CFU Mtb Erdman (1st infection, red arrow). One group of mice were left uninfected. Four weeks into the infection mice were administered antibiotics in their drinking water for 12 weeks to clear the primary infection. At week 14, 16, and 18, mice were immunized with 0.5 µg H83 or H89 s.c. at the base of the tail or left non-vaccinated (black arrows). All mice were then challenged with 50–100 CFU Mtb Erdman (2nd infection, red arrow) 6 weeks post 3rd vaccination and level of lung infection was quantified 4 and 8 weeks into 2nd Mtb infection. (H) In the reinfection model: Total lung CFU in non-vaccinated mice (2nd infection only), Mtb memory mice (1st & 2nd infection), H83 and H89-vaccinated mice 4 and 8 weeks after the 2nd Mtb infection (n = 8). Post 2nd infection = (p.2nd.i). A Two-Way ANOVA with Tukey’s multiple comparison test comparing cell means regardless of rows and columns, Finteraction (3, 55) = 5.503, Frow (1, 55) = 11.48, Fcolumn (3, 55) = 85.10. Exact p-values are indicated. Data shows box plots with whiskers indicating minimum and maximum values.