Serum cytokine/chemokine responses in mice immunized with the subunit vaccine and infected with FT LVS via the respiratory route.

<p>Levels of various cytokines/chemokines in sera of GPI control mice or of mice immunized with DnaK+GPI, Tul4+GPI, or DnaK+Tul4+GPI and then challenged with FT LVS (1.5×10⁶ CFU) via the i.n. route were assessed using a mouse multiplex kit. Serum samples were collected on days 0, 2 and 3, and then every other day until the mice succumbed to infection or until day 15 from surviving mice. There were 5 mice per group and the values are expressed as the mean.</p

Induction of equivalent DnaK- and Tul4-specific mucosal and serum antibody responses following i.n. immunization with DnaK+Tul4+GPI.

<p>Mice were immunized via the i.n. route with DnaK+GPI, Tul4+GPI or DnaK+Tul4+GPI on days 0, 14 and 28. (A) Individual saliva, vaginal wash and serum samples were collected 2 weeks following the last immunization. The levels of DnaK- and Tul4-specific IgA in the saliva and vaginal wash samples and IgG in the serum were determined by ELISA. (B) Individual bronchoalveolar lung lavage samples were collected 2 weeks following the last immunization and the levels of DnaK- and Tul4-specific IgA and IgG were determined by ELISA. There were 4 mice per experimental group and values are expressed as the mean ± SEM. None detected (ND), not significant (<i>ns</i>).</p

Cytokine production by spleen and purified CD4⁺ T cells from mice immunized with DnaK+Tul4+GPI.

<p>Mice were immunized via the i.n. route with DnaK+Tul4+GPI on days 0, 14 and 28. Seven days after the last immunization (A) total splenocytes or (B) purified splenic CD4⁺ T cells co-cultured with irradiated splenocytes/APC at different ratios were stimulated with DnaK or Tul4 at various concentrations, as indicated in figures. Culture supernatants were harvested on day 4 and assessed for the levels of IFN-γ, IL-10 and IL-17 by ELISA. Values are expressed as the mean ± SEM and are representative of 2 separate experiments. Values are significantly different at <i>P</i><0.001 (*), <i>P</i><0.01 (**) and <i>P</i><0.05 (***).</p

Immunization with subunit vaccine results in reduced bacterial burden.

<p>GPI and DnaK+Tul4+GPI immunized mice were challenged with 1.5×10⁶ CFU of FT LVS via the i.n. route. The results show the relative levels of FT LVS-specific 16S rDNA present in the spleen, liver and lungs of infected mice five days after infection. There were 4 mice per group and the values are expressed as the mean ± SEM. Significant differences were seen at <i>P</i><0.01 (*) and <i>P<</i>0.05 (**) compared to mice immunized with GPI only.</p

DnaK and Tul4 are recognized by CD4⁺ and CD8⁺ T cells during respiratory FT LVS infection.

<p>Spleen cells from FT LVS infected mice were cultured for 18–24 h with DnaK, Tul4, DnaK+Tul4 or with FT LVS extract (FT extract), which served as a positive control. Negative control wells were cultured with an irrelevant protein, SBR, or media alone. Protein transport inhibitor, brefeldin A, was added to cultures during the last 4 h of incubation, and then cells were stained with fluorochrome-labeled antibodies followed by FACS analysis. Representative FACS dot plots for intracellular IFN-γ positive CD4⁺ and CD8⁺ T cells are shown.</p

Immune reactivity to DnaK and Tul4 confers protection against a lethal respiratory challenge with FT LVS.

<p>Mice were immunized on day 0, 14 and 28 with DnaK+GPI, Tul4+GPI or DnaK+Tul4+GPI. Control groups of mice received PBS or GPI. Two weeks after the last immunization, mice were challenged with either (A) 1.5×10⁶ CFU or (B) 8×10⁶ CFU of FT LVS via the i.n. route and survival was monitored for 30 days. The results show pooled data of two independent experiments (a total of 14 mice per group). Significant differences were seen at <i>P</i><0.001 (*) compared to mice immunized with GPI only.</p

Tul4-specific mucosal and serum antibody responses following i.n. immunization with Tul4+GPI.

<p>Mice were immunized via the i.n. route with Tul4 (1 or 10 µg) with or without GPI (100 µg) on days 0, 14 and 28. Individual saliva, vaginal wash and serum samples were collected prior to and at approximately 2-week intervals following immunization. Levels of Tul4-specific IgA in the saliva and vaginal wash samples and IgG in the vaginal wash samples (A), and of IgG, IgG1 and IgG2c in the serum (B), were determined by ELISA. There were 6 mice per experimental group and values are expressed as the mean ± SEM. Significant differences were seen at <i>P</i><0.001 (*) compared with mice immunized with equivalent doses of Tul4 only.</p

DnaK-specific mucosal and serum antibody responses following i.n. immunization with DnaK+GPI.

<p>Mice were immunized via the i.n. route with DnaK (10 or 20 µg)+GPI (100 µg) on days 0 and 14 or on days 0, 14 and 28. Individual saliva, vaginal wash and serum samples were collected prior to and at approximately 2-week intervals following immunization. Levels of DnaK-specific IgA in the saliva and vaginal wash samples and IgG in the vaginal wash samples (A), and of IgG, IgG1 and IgG2c in the serum (B), were determined by ELISA. There were 6 mice per experimental group and values are expressed as the mean ± SEM. Significant differences were seen at <i>P</i><0.001 (*) and <i>P</i><0.01 (**) compared with mice receiving only two immunizations with equivalent doses of DnaK and GPI.</p

Core supra-gingival microbiome in root caries and health.

<p>Red field represents health-associated core species with significantly higher prevalence and relative abundance in Healthy_controls than in Patient_cases; green field represents root caries-associated core species with significantly higher prevalence and relative abundance in Patient_cases than in Healthy_controls; brown field represents core species in both health and root caries with no significantly different prevalence and relative abundance between the two groups. In each field, the prevalences of species were at least 1/2. Inner circles labeled 1 contain the species with high prevalence (prevalence ≥ 2/3) and high abundance (average relative abundance ≥ 2%); circles labeled 2 contain species with high prevalence (prevalence ≥ 2/3) but low abundance (average relative abundance < 2%); circles labeled 3 contain species with moderate prevalence (1/2 ≤ prevalence < 2/3) but high abundance (average relative abundance ≥ 2%); circles labeled 4 contain species with moderate prevalence (1/2 ≤ prevalence < 2/3) and low abundance (average relative abundance < 2%).</p

Maximum likelihood phylogenetic tree at the species level.

<p>Inner loop displays species with color ranges for the class level (see color key at the bottom left); middle loop indicates mean relative abundance in each group (see color key at the top left); outer loop indicates significantly different species detected between groups (see color key at the top left). An online tool of iTOL was used to construct this tree.</p