Article Figures & Data
Figures
- FIG 1
S1 and S2 show pH-dependent thermal stability. Empirical phase diagrams for S1 (A) and S2 (B) are shown. The three-index empirical phase diagrams illustrate the thermal stability for each fusion protein as a function of pH 3 to 8. The red, green, and blue panels define individual component indices for secondary structure (CD), tertiary structure (fluorescence peak position), and aggregation behavior (SLS), respectively.
- FIG 2
Immunization elicits an antigen-specific serum IgG response. Mice (n = 15) were immunized intramuscularly (i.m.) on days 0, 14, and 28 with adjuvanted S1, S2, or S1S2. Two groups were vaccinated orally on day 28, with one group receiving S. Typhimurium ΔSPI-1/2 and one group receiving S. Enteritidis ΔSPI-1/2. Blood and fecal samples were collected at days −1, 13, 27, 41, and 55. Serum IgG antibodies specific for SipD (A), SipB (B), SseB (C), and SseC (D) were analyzed by enzyme-linked immunosorbent assay (ELISA). The individual titers for serum IgG are represented as endotoxin units (EU) ml−1. Titers under 100 EU ml−1 were adjusted to baseline titer. Data are represented as mean ± SEM for 10 mice per group. The serum IgG response was tested for levels of significance using an unpaired t test (**, P < 0.001 [S1 versus S1S2], #, P < 0.05 [S2 versus S1S2] using a two-way ANOVA). No antigen-specific IgA was detected in any of the fecal samples.
- FIG 3
Immunization triggers production of antibody-secreting cells. Antibody-secreting cells (ASCs) were quantified in bone marrow (A) and spleen (B). Mice (n = 5 per group concomitant with mice for Fig. 2) were vaccinated on days 0, 14, and 28 and euthanized on day 55 to collect spleen and bone marrow for assessment of antibody-secreting cells. Single-cell suspensions obtained from spleen and bone marrow were incubated in SipD-, SipB-, SseB-, or SseC-precoated plates. IgG-secreting cells were detected by ELISpot assay, counted, and plotted as specific ASCs/106 cells ± standard deviation (SD). *, P < 0.05 (t test). No IgA-secreting cells were detected.
- FIG 4
Protective efficacy of the vaccine formulations. Mice (n = 10 per group used for Fig. 2) were vaccinated on days 0, 14, and 28. On day 56, 2 × 108 CFU of wild-type S. Typhimurium SL1344 (A) and 5 × 107 CFU of wild-type S. Enteritidis 125109 (B) were administered by gavage. Survival was monitored for 20 days postchallenge. *, P < 0.05 compared to survival of mice vaccinated with PBS using the log rank test; n.s., not significant when survival of the S1S2-immunized group was compared with those of the S. Typhimurium ΔSPI-1/2- and S. Enteritidis ΔSPI-1/2-vaccinated groups (P > 0.05 using log rank test).
- FIG 5
Cecal inflammation during lethal challenges with S. Typhimurium. Immunized mice (n = 5; as described in Materials and Methods) were treated on day 55 with streptomycin to clear gut flora. On day 56, all mice were challenged with 200 CFU of wild-type S. Typhimurium SL1344. The cecum of each mouse was collected at day 4 postchallenge (day 60) and fixed in OCT. Thin cryosections (4 μm) of cecum were stained with hematoxylin and eosin. Sections were visualized and photographed at a magnification of ×10 (A to E), whereas the pathoscore was evaluated at a magnification of ×400 (F). The stained sections were evaluated independently on the basis of pathological changes that included submucosal edema (score, 0 to 3), PMN infiltration (score, 0 to 4), loss of goblet cells (score, 0 to 3), and epithelial integrity (score, 0 to 3), with a final pathoscore of 0 to 12, reflecting the overall degree of inflammation. The pathoscores were determined by averaging the scores. The combined scores ranged from 0 to 12 arbitrary units covering the inflammation levels as follows: intact intestine (pathoscore, 0); minimal inflammation (pathoscore, 1 or 2), which is commonly found in the ceca of mice; slight inflammation (pathoscore, 3 or 4); moderate inflammation (pathoscore, 5 to 8); and significant inflammation (pathoscore, 9 to 12) (11). Pathoscores are represented graphically as mean ± SEM. All the sections were imaged at a magnification of ×10 with a Nikon Eclipse 80i microscope, and the best representative image from each group is presented. Representative sections are presented to show differences in the cecal inflammation with respect to the individual vaccine formulation. SE, submucosal edema; Lu, cecal lumen; LP, lamina propria. Scale bar, 100 μm. *, P < 0.05 using ANOVA; **, P < 0.01; n.s. not significant.
Additional Files
Supplemental material
- Supplemental file 1 -
Fig. S1. Far-UV circular dichroism analysis of S1 and S2. Fig. S2. Intrinsic Trp fluorescence peak position and static light scattering of S1 and S2. Fig. S3. SDS-PAGE of purified S1 and S2 with extinction coefficient and molecular weight.
PDF, 621K
- Supplemental file 1 -
