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1.
Figure 4

Figure 4. From: A recombinant Mycobacterium smegmatis induces potent bactericidal immunity against Mycobacterium tuberculosis.

Improvement of histopathology in IKEPLUS-immunized mice during resolution of Mtb infection. (a) Representative images are shown for lungs (left) and livers (right) of C57BL/6 mice immunized with IKEPLUS and challenged with Mtb as described in Figure 3. Top and middle, H&E staining; bottom, AFB staining. Scale bars correspond to 650 nm, 40 nm and 10 nm in top, middle and bottom rows, respectively. The asterisk indicates an area of residual dense granulomatous infiltrate. Filled arrowheads indicate occasional macrophages in open alveoli in the lungs of mice with resolving inflammation at day 202. Open arrowheads mark granulomatous foci in the liver, which were also substantially resolved by day 202. AFBs were visualized in macrophages in lung and liver sections at both time points. (b) Quantitative scoring of histopathology confirming that all three groups showed similar pathology in the lungs and liver at day 100 (P > 0.05, two-way ANOVA). However, at day 202 the survivors from the IKEPLUS-immunized group showed significantly reduced pathology scores (P < 0.05 compared to all other groups at day 100; one-way ANOVA with Tukey post-test).

Kari A Sweeney, et al. Nat Med. ;17(10):1261-1268.
2.
Figure 2

Figure 2. From: A recombinant Mycobacterium smegmatis induces potent bactericidal immunity against Mycobacterium tuberculosis.

Characterization of innate immune responses against the Msmeg IKEPLUS strain. (a) Serum concentrations of IL-12 p40, IL-12p70 and IFN-γ in C57BL/6 mice infected with IKE or IKEPLUS (*P < 0.01, ANOVA with Bonferroni post-test). (b) Growth of bacteria (CFU) in the lungs (left) and kidneys (right) of Rag1−/− mice after intravenous inoculation of Msmeg parental, IKE or IKEPLUS strains. Differences in CFU were statistically significant (P < 0.001, two-way ANOVA) for Msmeg versus either IKE or IKEPLUS at day 3. # at the bases of y axes indicate that no colonies were obtained at day 35, consistent with complete clearance of IKE and IKEPLUS. (c) Average time to death of mice after intravenous inoculation of IKEPLUS in C57BL/6 (n = 10), Rag1−/− (n = 5) and Myd88−/− (n = 5) mice. Survival of wild-type C57BL/6 and Rag1−/− strains was significantly different than that of Myd88−/− mice (P < 0.05, log-rank test). All inoculations were done at a high intravenous dose (5 × 107 CFU per mouse). Data from a and b are mean ± s.e.m. of three mice per group. Data for all panels are representative of four independent experiments.

Kari A Sweeney, et al. Nat Med. ;17(10):1261-1268.
3.
Figure 1

Figure 1. From: A recombinant Mycobacterium smegmatis induces potent bactericidal immunity against Mycobacterium tuberculosis.

Role of the esx-3 region in evasion of innate immunity by M. smegmatis in a high-dose infection model. (a) Serum concentrations of IL-12 p40, IL-6 and IFN-γ in mice (C57BL/6) infected intravenously with 5 × 107 CFU per mouse of parental Msmeg (strain mc2155), IKE (Δesx-3) or Δesx-1 strains. (b) Survival after intravenous inoculation of 5 × 107 CFU parental Msmeg or IKE in C57BL/6 (n = 10), Rag1−/− (n = 5) or Myd88−/− (n = 5) mice. Survival was significantly different for IKE versus parental Msmeg in C57BL/6 and Rag1−/− mice (P < 0.001, log-rank test), but not in Myd88−/− mice. (c) CFU in lungs and kidneys of C57BL/6 mice after intravenous inoculation of high doses (5 × 107 CFU per mouse) of various Msmeg strains. # at the bases of y axes indicate that no colonies were obtained at day 35, consistent with complete clearance of IKE infection. (d) CFU in lungs and kidneys of Myd88−/− mice after intravenous inoculation of high doses (5 × 107 CFU per mouse) of various Msmeg strains. For a, c and d, data are mean ± s.e.m. of three mice per group. † indicates death or killing owing to extreme morbidity of all mice in a group; *P < 0.01 (analysis of variance (ANOVA)). Data for all panels are representative of six independent experiments.

Kari A Sweeney, et al. Nat Med. ;17(10):1261-1268.
4.
Figure 5

Figure 5. From: A recombinant Mycobacterium smegmatis induces potent bactericidal immunity against Mycobacterium tuberculosis.

Protection from Mtb challenge by subcutaneous immunization with IKEPLUS. (a) Survival (left) of C57BL/6 mice (n = 10–14 per group) immunized subcutaneously with PBS, IKEPLUS (1 × 108 CFU per mouse), or BCG (1 × 106 CFU per mouse) and challenged 5 weeks later with a high intravenous dose (1 × 108 CFU per mouse) of Mtb H37Rv. CFU (right) data from the same experiment show means of three mice per group. The survival curve for IKEPLUS-immunized mice was significantly different from that of the BCG-immunized mice (P < 0.001, log-rank test). The CFU levels in lungs of IKEPLUS-immunized mice were significantly different from those of surviving saline-immunized mice at days 14 and 20 (P < 0.01, two-way ANOVA) and compared to BCG-immunized mice at day 20 (P < 0.01, two-way ANOVA). (b) Survival (left) of mice immunized subcutaneously with PBS (n = 10), 1 × 106 CFU of BCG (n = 10) or 1 × 108 CFU of IKEPLUS (n = 9) and challenged 1 month later with ~100 CFU of aerosolized Mtb H37Rv. The survival curves for IKEPLUS- and BCG-immunized mice were significantly different from that of PBS-immunized mice (P < 0.05, log-rank test). The survival curve for IKEPLUS was not significantly different from that of BCG-immunized mice (P = 0.0898, log-rank test). Lung CFU (right) from three mice per group from the same experiment showed that the IKEPLUS- and BCG-vaccinated groups had significantly reduced CFU compared to saline-immunized mice at days 21, 56 and 112 (P < 0.05, two-way ANOVA). IKEPLUS-immunized mice also showed lower CFU versus saline- or BCG-immunized mice at day 175 (*P < 0.05, two-way ANOVA). Results are representative of three independent experiments.

Kari A Sweeney, et al. Nat Med. ;17(10):1261-1268.
5.
Figure 3

Figure 3. From: A recombinant Mycobacterium smegmatis induces potent bactericidal immunity against Mycobacterium tuberculosis.

Bactericidal immunity against Mtb in mice vaccinated with IKEPLUS. (a) Survival of C57BL/6 mice that were sham immunized (intravenous (i.v.) PBS injection; n = 21) or immunized by intravenous infection with IKEPLUS (5 × 107 CFU per mouse; n = 20) or by subcutaneous (s.c.) infection with BCG (1 × 107 CFU per mouse; n = 18) and subsequently challenged 8 weeks later with a high intravenous dose (1 × 107 CFU per mouse) of Mtb H37Rv. Differences in survival were significant for PBS versus BCG (P = 0.0389, log-rank test), PBS versus IKEPLUS (P < 0.0001, log-rank test) or BCG versus IKEPLUS (P < 0.0001, log-rank test). (b) Measurement of CFU from the lungs, spleens and livers of C57BL/6 mice in a separate experiment in which mice were immunized and challenged as described in a. Each symbol represents one mouse. # at base of the y axis for the liver CFU plot indicates that no colonies were obtained from IKEPLUS-vaccinated mice at day 202, consistent with clearance of Mtb infection in that tissue (entire organs were plated to give a limit of detection of 1 CFU). The CFU at day 100 was not significantly different between BCG- or IKEPLUS-immunized mice in any organ (P > 0.05, ANOVA). Data shown are pooled from two independent experiments. (c) Survival and lung CFU of C57BL/6 mice that were sham immunized (i.v. PBS; n = 6) or immunized by intravenous infection with IKE (5 × 107 CFU per mouse; n = 6) or IKEPLUS (5 × 107 CFU per mouse; n = 6) and subsequently challenged 6 weeks later with a high intravenous dose (5 × 107 CFU per mouse) of Mtb H37Rv. Differences in survival curves were significant for PBS versus IKEPLUS (P = 0.0007, log-rank test), PBS versus IKE (P = 0.0049, log-rank test) and IKEPLUS versus IKE (P = 0.0246, log-rank test). For lung CFU, asterisks indicate significant differences (P < 0.05, two-way ANOVA) compared to the PBS control group. Results shown are representative of four independent experiments.

Kari A Sweeney, et al. Nat Med. ;17(10):1261-1268.
6.
Figure 6

Figure 6. From: A recombinant Mycobacterium smegmatis induces potent bactericidal immunity against Mycobacterium tuberculosis.

Role of CD4+ T cells in IKEPLUS-induced protective immunity. (a) Contributions of CD4+ and CD8+ subsets, as assessed by adoptive transfer of T cells from IKEPLUS-immunized mice. Naive recipient mice (n = 5 per group) were challenged 1 d after T cell transfer with M. tuberculosis (H37Rv, 1 × 107 CFU i.v. per mouse). Left, lung CFU of Mtb for mice killed 3 weeks after Mtb challenge. The dashed line indicates mean CFU in the lungs of five mice that were directly immunized with IKEPLUS (5 × 107 CFU per mouse i.v.) at 3 weeks before challenge. A negative control group of five sham-immunized mice that did not receive T cell transfer before challenge had CFU levels that were not significantly different from those of recipients of T cells from saline-immunized donors (not shown). *P < 0.05 compared to negative control group (ANOVA). This experiment was performed three times with similar results. Right, survival curves from similarly treated groups (n = 5) of mice from one experiment. IKEPLUS CD4+, adoptive transfer of CD4+ T cells from IKEPLUS-immunized donors; BCG CD4+, transfer of CD4+ T cells from BCG-immunized donors. Mice immunized intravenously with IKEPLUS directly or injected with PBS only were included as controls. The IKEPLUS CD4+ group was significantly different from the PBS group (P = 0.035, log-rank test) but not significantly different from the directly IKEPLUS-immunized group (P = 0.3177). (b) Cytokine production by CD4+ T cells in the lungs of mice immunized intravenously with PBS (sham) or IKEPLUS (5 × 107 CFU per mouse) or subcutaneously with BCG (1 × 106 CFU per mouse) and challenged 8 weeks later with Mtb H37Rv (1 × 107 CFU i.v.) (n = 5 mice per time point). The graphs indicate the absolute numbers of CD4+ T cells staining positively for three, two or one of the cytokines analyzed (IFN-γ, TNF and IL-2) after re-stimulation in vitro with a peptide representing the immunodominant epitope of TB9.8. Significant differences between the IKEPLUS- and BCG-vaccinated groups are indicated (*P < 0.05, **P < 0.01, ***P < 0.001, two-way ANOVA). Results shown are representative of two similar experiments. (c) Same as in b except that the cells were re-stimulated with plate-bound monoclonal antibodies to CD3 and CD28 before analysis. (d) Pie charts showing the relative fractions of CD4+ T cells at 14 d after Mtb challenge producing three, two or one of the cytokines with either specific antigen re-stimulation in vitro (TB9.8 peptide) or polyclonal activation (anti-CD3).

Kari A Sweeney, et al. Nat Med. ;17(10):1261-1268.

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