Journal: PLOS Pathogens

Article Title: Mucosal immunization with the lung Lactobacillus -derived amphiphilic exopolysaccharide adjuvanted recombinant vaccine improved protection against P . aeruginosa infection

doi: 10.1371/journal.ppat.1012696

Figure Lengend Snippet: Mice (n = 3-5/group) were immunized (i.n.) twice 14 days apart with rPcrV, CTB+ rPcrV or EPS301@rPcrV, with animals receiving PBS served as controls. Mice were inoculated with 40 μl bacterial slurry (lower dose, 1×10 7 CFUs of P . aeruginosa PAO1) as above. Vaccinated mice were sacrificed at 12 hours post-challenge on day 7 or day 112 after the second vaccination lung tissue were prepared. Number of IFN-γ + CD4 + T cells, IL-17A + CD4 + T cells (A), IFN-γ + γδ + T cells, IL-17A + γδ + T cells (B) in lung at 12 hours post-challenge on day 7 after the second vaccination were estimated by intracellular cytokine. Number of IFN-γ + CD4 + T cells, IL-17A + CD4 + T cells (C), IFN-γ + γδ + T cells, IL-17A + γδ + T cells (D) in lung at 12 hours post-challenge on day 112 after the second vaccination were estimated by intracellular cytokine. IFN-γ and IL-17 levels, determined by ELISA in a supernatant of lung tissue homogenate were analyzed. The IFN-γ levels and IL-17A levels (E) in lung at 12 hours post-challenge on day 7 and day 112 after the second vaccination were determined by ELISA. Data are presented as means ± SEM. Significant differences were calculated with One-way ANOVA followed by Tukey’s multiple comparisons test. *p < 0.05; **p < 0.01; ***p < 0.001 for comparison with EPS301@rPcrV immunized mice.

Article Snippet: To define the IL-17A and IFN-γ levels in lung and spleen tissue homogenate, the murine bioplex ELISA kits (R&D SYSTEMS Mouse IL-17A/F Heterodimer DuoSet ELISA DY5390 and R&D SYSTEMS Mouse IFN-gamma DuoSet ELISA DY485) were used according to the manufacturer’s recommendations.

Techniques: Enzyme-linked Immunosorbent Assay, Comparison

Journal: PLOS Pathogens

Article Title: Mucosal immunization with the lung Lactobacillus -derived amphiphilic exopolysaccharide adjuvanted recombinant vaccine improved protection against P . aeruginosa infection

doi: 10.1371/journal.ppat.1012696

Figure Lengend Snippet: (A) Comparative analysis of opsonophagocytosis against PAO1 using antisera from differently immunized mice. (B) Timeline of passive transfer and P . aeruginosa -induced pneumonia model. (C) Passive transfer of immunized and non-immunized mice was evaluated by i.v. injection of pooled serum (100 μl) to naïve C57BL/6 mice (n = 10/group). 24 hours after serum transfer, mice were inoculated with 40 μl bacterial slurry (1×10 9 CFUs of P . aeruginosa PAO1) via intranasal route (i.n.). Survival of mice from PBS, rPcrV, rPcrV+CTB and EPS301@rPcrV groups were observed for 48 h. The data for survival test were analyzed by Wilcoxon log-rank survival test (ns, not significant). (D) A lower dose of bacterial slurry (1×10 7 CFUs of P . aeruginosa PAO1) was inoculated to recipient mice via intranasal route (i.n.). Bacterial loads in lung and BALF were detected at 12 h post infection. (E) 12 hours post-challenge, lung tissue was prepared. IL-17A and IFN-γ levels determined by ELISA in a supernatant of lung tissue homogenate were analyzed. (F) Timeline of adoptive transfer and P . aeruginosa -induced pneumonia model. Lung and splenic CD3 + CD4 + T cells from EPS301@rPcrV–vaccinated congenic mice were purified on day 7 after second vaccination. CD3 + CD4 + T cells (5×10 4 ) were intravenously transferred into naïve C57BL/6 mice. (G) 24 hours after adoptive transfer, mice were inoculated with 40 μl bacterial slurry (1×10 9 CFUs of P . aeruginosa PAO1) via intranasal route (i.n.). Survival of mice were observed for 48 h. Survival after P . aeruginosa challenge (data were pooled from two independent experiments (n = 8–10). The data for survival test were analyzed by Wilcoxon log-rank survival test (***p < 0.001). (H) A lower dose of bacterial slurry (1×10 7 CFUs of P . aeruginosa PAO1) was inoculated to recipient mice via intranasal route (i.n.). Bacterial loads in lung and BALF were detected at 12 h post infection. (I) 12 hours post-challenge, lung tissue was prepared. IL-17A and IFN-γ levels determined by ELISA in a supernatant of lung tissue homogenate were analyzed. (J) Coexpression of CD44 and CD69 on non-stimulated lung CD4 + or γδ + T cells (gated in CD45 + CD4 + T cells). (K) FTY720 was initially dissolved in DMSO to create a 50 mg/mL stock solution. This stock solution was subsequently diluted with saline for intraperitoneal administration at a dosage of 30 μg per mice, and with distilled water for administration via stomach intubation at a dosage of 50 μg per mice. A lower dose of bacterial slurry (1×10 7 CFUs of P . aeruginosa PAO1) was inoculated to FTY720 treated and FTY720 non-treated mice via intranasal route (i.n.). Bacterial loads in lung were detected at 12 h post P . aeruginosa infection. Data are presented as means ± SEM. Significant differences were calculated with One-way ANOVA followed by Tukey’s multiple comparisons test (D, E, H and L), Mann-Whitney U test (J) or unpaired t test (K). *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: To define the IL-17A and IFN-γ levels in lung and spleen tissue homogenate, the murine bioplex ELISA kits (R&D SYSTEMS Mouse IL-17A/F Heterodimer DuoSet ELISA DY5390 and R&D SYSTEMS Mouse IFN-gamma DuoSet ELISA DY485) were used according to the manufacturer’s recommendations.

Techniques: Injection, Infection, Enzyme-linked Immunosorbent Assay, Adoptive Transfer Assay, Purification, Saline, MANN-WHITNEY

Journal: PLOS Pathogens

Article Title: Mucosal immunization with the lung Lactobacillus -derived amphiphilic exopolysaccharide adjuvanted recombinant vaccine improved protection against P . aeruginosa infection

doi: 10.1371/journal.ppat.1012696

Figure Lengend Snippet: TCR δ-deficient mice were vaccinated with EPS301@rPcrV and survival, bacterial loads and the lung pathology were detected on day 7 post second vaccination. Mice were inoculated with 40 μl bacterial slurry (1×10 9 CFUs of P . aeruginosa PAO1) via intranasal route (i.n.). (A) The number and percentage of Th 17 cells in WT and TCR δ-deficient mice before immunization, after immunization and post infection. (B) Survival of mice were observed for 48 h. Mice were inoculated with 40 μl bacterial slurry (1×10 7 CFUs of P . aeruginosa PAO1) via intranasal route (i.n.). The data for survival test were analyzed by Wilcoxon log-rank survival test (***p < 0.001). (C) Bacterial burdens of mice were counted at 12 h post infection. (D) Histological evaluation of lung sections by light microscopy. Lung specimens were fixed, sectioned, and stained with H&E (n = 3–5). (E) Timeline of adoptive transfer and P . aeruginosa -induced pneumonia model. Lung and splenic CD3 + γδ T cells from EPS301@rPcrV–vaccinated congenic mice were purified on day 7 after second vaccination. CD3 + γδ T cells (1 × 10 4 ) were intravenously transferred into naïve C57BL/6 mice. (F) 24 hours after adoptive transfer, mice were inoculated with 40 μl bacterial slurry (1×10 9 CFUs of P . aeruginosa PAO1) via intranasal route (i.n.). Survival of mice were observed for 48 h. Survival after P . aeruginosa challenge (data were pooled from two independent experiments (n = 8–10). The data for survival test were analyzed by Wilcoxon log-rank survival test (**p < 0.01, ***p < 0.001). A lower dose of bacterial slurry (1×10 7 CFUs of P . aeruginosa PAO1) was inoculated to recipient mice via i.n.. (G) Bacterial loads in lung and BALF were detected at 12 h post infection. (H) 12 hours post-challenge, lung tissue was prepared. IL-17A and IFN-γ levels determined by ELISA in a supernatant of lung tissue homogenate were analyzed. Data are presented as means ± SEM. Significant differences were calculated with unpaired t test (A) and One-way ANOVA followed by Tukey’s multiple comparisons test (C, G and H). ns, not significant, *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: To define the IL-17A and IFN-γ levels in lung and spleen tissue homogenate, the murine bioplex ELISA kits (R&D SYSTEMS Mouse IL-17A/F Heterodimer DuoSet ELISA DY5390 and R&D SYSTEMS Mouse IFN-gamma DuoSet ELISA DY485) were used according to the manufacturer’s recommendations.

Techniques: Infection, Light Microscopy, Staining, Adoptive Transfer Assay, Purification, Enzyme-linked Immunosorbent Assay

Journal: PLOS Pathogens

Article Title: Mucosal immunization with the lung Lactobacillus -derived amphiphilic exopolysaccharide adjuvanted recombinant vaccine improved protection against P . aeruginosa infection

doi: 10.1371/journal.ppat.1012696

Figure Lengend Snippet: (A) Experimental design and timeline of prime and booster regimen to IL-17A-deficient, IFN-γ-deficient and WT mice of EPS301-adjuvanted vaccination (i.n.) and challenge. (B) 7 days post booster vaccination, level of IgG in serum was detected by ELISA. (C) 7 days post booster vaccination, level of IgA in lung was detected by ELISA. (D) IL-17A-deficient, IFN-γ-deficient and WT mice were inoculated with 40 μl bacterial slurry (1×10 9 CFUs of P . aeruginosa PAO1) via left nostril on day 7 post booster vaccination and held upright for 1 min. Representative survival rates from two independent experiments are shown (n = 8–10). The data for survival test were analyzed by Wilcoxon log-rank survival test (**p < 0.01, ***p < 0.001). (E) Mice were inoculated with 40 μl bacterial slurry (lower dose: 1×10 7 CFUs of P . aeruginosa PAO1) as above. At 12 h, the numbers of bacteria in lung and BALF were counted (n = 5–8). (F) Mice were inoculated with the lower dose of PAO1 as above. At 12 h post challenge, histological evaluation of lung sections by light microscopy. Lung specimens were fixed, sectioned, and stained with H&E (n = 3–5). Data are presented as means ± SEM. Significant differences were calculated with One-way ANOVA followed by Tukey’s multiple comparisons test. ns, not significant, *p < 0.05, **p < 0.01, ***p < 0.001).

Article Snippet: To define the IL-17A and IFN-γ levels in lung and spleen tissue homogenate, the murine bioplex ELISA kits (R&D SYSTEMS Mouse IL-17A/F Heterodimer DuoSet ELISA DY5390 and R&D SYSTEMS Mouse IFN-gamma DuoSet ELISA DY485) were used according to the manufacturer’s recommendations.

Techniques: Enzyme-linked Immunosorbent Assay, Bacteria, Light Microscopy, Staining