Article Figures & Data
Figures
- Fig. 1.
Vaccine internalization across the NALT FAE layer was accelerated and sustained by TLR4 activation. Mice were IN inoculated with the following: rSEBv and adjuvant (rSEBv-Adj) (A to F), rSEBv only (rSEBv) (G to L), rSEBv with adjuvant and anti-TLR4 monoclonal antibody (rSEBv-Adj/a-TLR4) (M to R), or PBS only (UNT) (S and T). The mice were euthanized at the specified times and paraffin cross sections of the nasal cavities were prepared. Vaccine was localized (brown staining) across the FAE layer of the NALT using SEB-specific antibodies, and tissue was counterstained with hematoxylin. These images represent one of two similar experiments. Original magnification, ×40.
- Fig. 2.
Germinal center formation in the NALT did not require adjuvant, whereas the generation of IgA+ and IgG+ cells was enhanced by a TLR4 agonist. Mice were IN inoculated three times (2 weeks apart) with vaccine and a TLR4 agonist (IN rSEBv-Adj) (C, G, and K) or with vaccine only (IN rSEBv) (B, F, and J). Control mice were inoculated either IN with PBS (UNT) (A, E, and I) or IP with vaccine and a TLR4 agonist (IP rSEBv-Adj) (D, H, and L). Two weeks after treatments, paraffin cross sections of NALT were probed with PNA (A to D) to detect B-cell germinal centers or probed with isotype-specific antibodies to detect B cells that produced IgA (E to H) and IgG (I to L). Sample tissues were counterstained with hematoxylin. Each panel contains two opposite cross sections of the NALT of each mouse. This figure represents one of two similar experiments. Original magnification, ×40.
- Fig. 3.
Secretion of antibody specific for SEB into serum and saliva resulting from IN vaccination. Groups of mice (n = 10) were vaccinated IN or IP with three doses of rSEBv and a TLR4 agonist (rSEBv-Adj) in 2-week intervals. Controls received only IN PBS. Two weeks after the last inoculation, saliva (A) and serum (B) samples were collected and analyzed for rSEBv-specific IgA and IgG. The vertical axis displays the average OD in each group ± the standard error of the mean (SEM). Statistical significance (P ≤ 0.05) between indicated groups is shown (‡). This figure shows results from one of two similar experiments.
- Fig. 4.
Levels of SEB-specific antibody secreted by NALT cell cultures were similar to those detected in serum and saliva of vaccinated mice. Groups of mice (n = 6 each) were vaccinated IN or IP with either rSEBv and a TLR4 agonist (rSEBv-Adj) or IN with rSEBv alone. A control group was inoculated IN with PBS. The mice received three doses 2 weeks apart. The mice were euthanized 2 weeks after the last dose, and the NALT was removed for placement in separate cultures. Every day one-third of the spent medium was removed for analysis and replaced with fresh medium. Anti-rSEBv IgA (top) and IgG (bottom) content in the spent medium collected on the third day was measured by ELISA. The vertical axis displays the average OD in each group ± SEM. Statistical significance (P ≤ 0.01) between indicated groups is shown (+). The figure shows the collective data from two similar experiments.
- Fig. 5.
NALT disruption renders mice unresponsive to IN vaccination. NALT were surgically removed (No NALT) and the mice were vaccinated IN with rSEBv and TLR4 agonist (rSEBv-Adj). Three doses were applied in 2-week intervals. Non-manipulated or sham surgery control groups were similarly vaccinated or IN inoculated with PBS. Two weeks after the last treatment, mice were sedated and samples collected. The levels of SEB-specific IgA and IgG in the saliva (A), nasal wash (B), and serum (C) were measured by ELISA. The vertical axis displays the average OD in each group ± SEM. Statistical significances between indicated groups is shown (+, P ≤ 0.01; ‡, P ≤ 0.05).
- Fig. 6.
NALT is required for intranasal vaccination to convey protection against toxic shock. Mouse NALTs were surgically removed (No NALT), and the mice were vaccinated IN with rSEBv and TLR4 agonist (No NALT, IN rSEBv-Adj; n = 11) or with PBS (No NALT, IN PBS; n = 4). Sham surgery control mice were also IN inoculated with rSEBv and a TLR4 agonist (Sham, IN rSEBv-Adj; n = 25) or with PBS (Sham, IN PBS; n = 29). A control group of normal mice was IN inoculated with PBS alone (IN PBS; n = 10). Two weeks after the last inoculation, all mice were IN challenged with wt SEB. The graph shows the percent survival (y axis) versus time postchallenge (x axis; h).
Additional Files
Supplemental material
Files in this Data Supplement:
- Supplemental file 1
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Fig. S1. H&E staining of histological cross-sections, corresponding to experiments with results shown in Fig. 5.
Zipped MS PowerPoint file, 218K. - Supplemental file 2
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Fig. S2. H&E staining of histological cross-sections, corresponding to experiments with results shown in Fig. 6.
Zipped MS PowerPoint file, 411K.
- Supplemental file 1
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Fig. S1. H&E staining of histological cross-sections, corresponding to experiments with results shown in Fig. 5.
