BAL of orally and i.p. vaccinated mice. For lung lavages of orally (A) and i.p. (B) inoculated mice, animals were anesthetized and the tracheas were exposed by standard procedures and cannulated. A total of 2 ml of PBS was instilled into the lungs and carefully extracted. BAL fluid was analyzed by an ELISA on plates coated with PA103 whole organisms and probed for bound IgG and IgA. BAL fluid dilutions are plotted as log₁₀ units as a function of reactivity at OD₄₀₅. Error bars indicate standard deviations.

Reactivity of serum from orally vaccinated mice. (A) Western immunoblot analysis of whole-cell lysates from five heterologous serogroup O11 strains, a serogroup O6 strain, and an O-antigen-deficient mutant of PA103 (galU) (13) with pooled sera from orally vaccinated animals. (B) Competition ELISA with diluted pooled sera from orally vaccinated mice (1:5,000) and increasing amounts of free serogroup O11 LPS on ELISA plates coated with PA103 whole organisms. Data were analyzed by nonlinear regression and are represented as percent inhibition by antisera from vaccine-inoculated mice. Error bars indicate standard deviations.

Survival rates for orally and i.p. vaccinated animals after intranasal challenge with P. aeruginosa strain 9882-80. Animals immunized by oral (A and B) and i.p. (C) inoculations were challenged intranasally with P. aeruginosa strain 9882-80 (serogroup O11) and carefully monitored for survival. PBS- and vector-immunized mice served as controls. Infectious doses were calculated to be 1.12 × 10⁸ CFU (A), 2.34 × 10⁸ CFU (B), and 1.13 × 10⁸ CFU (C). Results are represented as Kaplan-Meier survival curves, and differences in survival were calculated by the log-rank test. (A) In the first oral vaccination study, increased survival was observed only for mice that received the vaccine strain (single asterisk) (P values determined by log-rank analysis: for PBS versus vaccine, 0.0128; for vector versus vaccine, 0.0084; and for PBS versus vector, 0.2813). Median survival times were as follows: with PBS, 54 h; with vector, 36 h; and with vaccine, 78 h. (B) In the second oral vaccination study, increased survival was again seen for mice that received the vaccine strain (double asterisks) (P value for vaccine versus vector: 0.0002). Median survival times were as follows: with vector, 22 h; and with vaccine, 42.5 h. (C) No difference in survival was observed for mice that received the vaccine strain i.p. versus those that received control treatments (P values: for PBS versus vaccine, 0.135; for vector versus vaccine, 0.1178; and for PBS versus vector, 0.2341). Median survival times were as follows: with PBS, 42 h; with vector, 88.5 h; and with vaccine, 92.3 h. Values in parentheses indicate the numbers of animals used.

Complete antibody titers for PBS- and SL3261-vaccinated mice after oral and i.p. vaccinations. (A) Serum P. aeruginosa PA103-specific (O11) total IgG and IgA from orally vaccinated BALB/c mice. (B) PA103-specific total IgG from i.p. vaccinated animals. (C) Total IgG to heterologous serogroup O11 strains. (D) IgG subtype responses in orally and i.p. inoculated animals, as determined by an ELISA on plates coated with Pseudomonas whole organisms. (A and B) Box and whisker plots show endpoint titers in log₁₀ units as determined by linear regression. The box is marked by the median and depicts the first and third quartiles, while the whiskers depict the range. Significant differences in the titers of Pseudomonas-specific antibodies were observed for orally vaccinated mice in panel A (single asterisk) (P value determined by Kruskal-Wallis analysis for IgG and IgA: <0.0001; P values determined by pairwise Mann-Whitney analysis for IgG and IgA: for PBS versus vaccine, <0.0001, and for vector versus vaccine, <0.0001) and for i.p. vaccinated mice in panel B (single asterisk) (P value determined by Kruskal-Wallis analysis: <0.0001; P values determined by pairwise Mann-Whitney analysis: for PBS versus vaccine, <0.0001, and for vector versus vaccine, <0.0001). i.p. vaccination with the vaccine strain induced significantly higher levels of serum total IgG than did the orally delivered vaccine strain (double asterisks) (P value determined by Mann-Whitney analysis: 0.0034). (C) Endpoint titers were determined by linear regression for pooled sera derived from animals receiving oral or i.p. inoculations. Error bars indicate 95% confidence intervals. (D) Level of each IgG subtype calculated from standard curves that were generated for secondary antibody reactivity to each IgG subtype. Error bars indicate standard deviations.

Killing of P. aeruginosa organisms in opsonophagocytosis assays and clearance from mouse lungs at 6 and 12 h after infection with P. aeruginosa 9882-80. (A) Opsonophagocytosis assay of P. aeruginosa serogroup O11 strains and a heterologous serogroup O6 strain, 6294, with dilutions of pooled sera raised to the vaccine strain by oral vaccination of BALB/c mice. Shaded bars represent the mean percent killing, and error bars represent the standard deviation. Results were determined from two independent experiments performed in duplicate and were calculated relative to results obtained with a preimmune serum control. Biological significance was obtained at 50% killing. (B) Orally vaccinated mice received a nonlethal dose of P. aeruginosa 9882-80 (1.2 × 10⁷ CFU), and lungs were removed, homogenized, and plated for identification of bacterial CFU at the indicated hours. Box and whisker plots show bacterial CFU in log₁₀ units. The box is marked by the median and depicts the first and third quartiles, while the whiskers depict the range. Differences in viable CFU were determined by one-way analysis of variance. No difference in viable CFU was seen among PBS, vector, and vaccine groups at 6 h postinfection (P = 0.9613). However, a significant decrease in CFU was observed at 12 h postinfection for vaccine-inoculated mice compared to PBS- and vector-treated mice (single asterisk) (P values: overall, for all data sets, 0.0424; for vector versus vaccine, 0.022; and for PBS versus vaccine, 0.043). Double asterisks indicate biologically significant data, i.e., 50% reduction of bacteria by vaccine versus controls.