Article Figures & Data
Figures
- FIG 1
Generation of a prototype biofilm matrix protein vaccine. (A) Schematic demonstrating the use of RbmA to anchor the B subunit of cholera toxin (CTB), a secreted protein, to the surface of the cell by fusion to the C terminus of RbmA. (B) Genotype of strains expressing CTB from the tac promoter on a multicopy plasmid either alone (pCTB) or genetically coupled to the 3′ end of rbmA (pR-CTB). MO10(pR-CTB) is a prototype antigen-boosted whole-cell vaccine. (C) Western blot analysis of cells harboring an empty expression plasmid (EV) or the same plasmid expressing either CTB or the RbmA-CTB fusion protein (R-CTB). A polyclonal primary antibody against CTB was used. The molecular masses of purified, monomeric CTB and the RbmA-CTB fusion protein are 11.6 kDa and 42.7 kDa, respectively (arrows). Supt, supernatant. (D) Quantification of R-CTB per cell in a Vc(pR-CTB) prototype whole-cell vaccine or a Dukoral (WC-rBS) equivalent. (E) Western blot analysis showing that R-CTB is released into the supernatant by a V. cholerae ΔvpsA mutant. Data for the pellets and supernatants from three independent cultures representing biological triplicates are shown for each condition. (F) Quantification of integrated band intensities in panel E by densitometry. Mean measurements are shown; error bars represent standard deviations. One-way ANOVA was used to calculate statistical significance. **, P < 0.01. pel, pellet.
- FIG 2
A prototype inactivated vaccine administered via the orogastric route does not elicit CTB-specific antibody responses. (A) Western blot analysis of CTB and R-CTB cell association after formalin treatment of a prototype vaccine. R-CTB (arrow), but not native CTB, remains in the cellular fraction after formalin treatment. Formalin treatment results in the cross-linking of R-CTB. (B) Vaccination and sample collection time line. Arrowheads indicate vaccination. Arrows indicate blood and stool collection. (C) Fold changes in CTB-specific IgA and IgG levels in serum and CTB-specific IgA levels in stool. Antibody levels were measured 4 weeks after the second vaccine booster (D56, day 56). The fold change was calculated by using the antibody response of PBS-immunized mice as the denominator. Each vaccination group included 10 mice. Horizontal bars mark the means. Asterisks indicate significant differences (**, P ≤ 0.01) using one-way ANOVA followed by Tukey's test; ns, not significant.
- FIG 3
A sublingually delivered live-attenuated whole-cell vaccine expressing R-CTB elicits LPS- and CTB-specific antibodies in serum and stool and is protective in an infant mouse model of cholera. (A) Genotype of the live-attenuated V. cholerae strain harboring pR-CTB that was used for vaccination. (B) Vaccination and sample collection time line. Arrowheads indicate vaccination. Arrows indicate blood and stool collection. (C) Concentrations of live V. cholerae bacteria recovered from stool pellets after sublingual immunization. Bacterial shedding ceased after 24 h. The limit of detection is 440 CFU/g stool and is indicated by the dotted line. Data for groups receiving the vaccine strain alone or the vaccine strain expressing R-CTB are shown. Each group included 10 mice. vacc., vaccination. (D) Fold changes in levels of LPS-specific IgG and IgA in the serum and LPS-specific IgA in the stool specimens of mice immunized with the sublingual live-attenuated vaccine. Each vaccination group included 10 mice. (E) Comparison of serum vibriocidal titers at day 42 after immunization with the sublingual live-attenuated (Live) vaccine or the orogastrically administered formalin-inactivated vaccine (Inact). (F) Colonization of the small and large intestines (sm. int. and lg. int., respectively) of suckling mice challenged with wild-type MO10. Pups were born to unvaccinated dams (Ctrl) or to dams that received the live-attenuated sublingual vaccine (Vacc). Litters from vaccinated and unvaccinated dams included 16 and 11 pups, respectively. (G and H) Representative intestinal fluid accumulation in the large intestine and cecum (triangles) (G) and skin turgor of pups (H) in vaccinated or control litters. Bars, 1 cm. (I) Fold changes in levels of CTB-specific IgG and IgA in the serum and CTB-specific IgA in the stool specimens of mice immunized with a control vaccine strain or a vaccine strain expressing R-CTB. Each vaccination group included 10 mice. Horizontal bars mark the medians in panels C, E, and F and the means in panels D and I. Asterisks indicate significant differences (*, P ≤ 0.5; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001) using one-way ANOVA followed by Tukey's test in panels D and I; ns, not significant. A complete set of statistical comparisons is given in Table S2 in the supplemental material. A two-tailed, unpaired Mann-Whitney U test was used for panels C, E, and F.
Additional Files
Supplemental material
- Supplemental file 1 -
Fig. S1 (Total protein loaded for Western blot analysis of R-CTB) and Tables S1 (Strains and plasmids) and S2 (Statistical analysis of fold change in antigen-specific antibody production)
PDF, 290K
- Supplemental file 1 -
