Protection against oral <i>B. abortus</i> challenge in BALB/c mice immunized i.g. with <i>E. coli</i>- or <i>N. benthamiana</i>- derived purified U-Omp19 without adjuvants.

a<p>The content of bacteria in spleens is represented as the mean log₁₀ CFU ± SD per group.</p>b<p>Significantly different from PBS-immunized mice <i>P</i><0.01 estimated by Dunnett's test.</p>c<p>Significantly different from <i>B. abortus</i> RB51 immunized mice <i>P</i><0.01 estimated by Dunnett's test.</p

<i>In vivo</i> activation of DCs after U-Omp19 injection.

<p>U-Omp19 either untreated or digested with proteinase K, <i>E. coli</i> LPS or PBS alone was injected i.v. to BALB/c mice. Splenic CD11c⁺ DCs were analyzed for their activation status 20 h after injection by assessing the surface expression of (<b>A</b>) CD40, (<b>B</b>) CD80 and (<b>C</b>) CD86 molecules by flow cytometry. This experiment was conducted three times with similar results. Histograms display results from one representative experiment.</p

Cellular immune responses elicited after i.g. or i.p. administration of adjuvant-free Omp19.

<p>Mice were immunized with U-Omp19 or PBS i.g. (<b>A–B</b>) or i.p. (<b>C–D</b>) as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016203#s4" target="_blank">materials and methods</a> and in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016203#pone-0016203-g001" target="_blank">Figure 1</a>. Specific production of IFN-γ (<b>A and C</b>) and IL-17 (<b>B and D</b>) by splenocytes of i.g. or i.p. U-Omp19 immunized mice was evaluated 30 days after the last immunization. Spleen cells (4×10⁶ cells/ml) were cultured with HS (20 µg/ml) or complete medium alone (RPMI) for 72 h. Each sample was assayed in duplicate wells. Cytokines in culture supernatants were measured by sandwich ELISA. Data represent the mean ± SEM from each group of five mice. (★) Significantly different from the cytokine level induced by the same stimulus in PBS immunized mice (<i>P</i><0.05). These experiments were conducted three times with similar results.</p

Oral administration of U-Omp19 expressing crude leaf material without adjuvant induces protection against oral <i>B. abortus</i> challenge in BALB/c mice.

a<p>The content of bacteria in spleens is represented as the mean log₁₀ CFU ± SD per group.</p>b<p>Significantly different from PBS-immunized mice <i>P</i><0.01 estimated by Dunnett's test.</p>c<p>Significantly different from U-Omp19 immunized mice <i>P</i><0.01 estimated by Dunnett's test.</p

Parenteral L-Omp19 or U-Omp19 inoculation without adjuvant induce protection against <i>B. abortus</i> infection in BALB/c mice.

a<p>The content of bacteria in spleens is represented as the mean log₁₀ CFU ± SD per group.</p>b<p>Significantly different from PBS-immunized mice <i>P</i><0.01 estimated by Dunnett's test.</p>c<p>Significantly different from <i>B. abortus</i> S19 immunized mice <i>P</i><0.01 estimated by Dunnett's test.</p>d<p>Significantly different from <i>B. abortus</i> S19 immunized mice <i>P</i><0.05 estimated by Dunnett's test.</p

Specific DTH response elicited after i.g. administration of adjuvant-free Omp19.

<p>Three weeks after the last i.g. immunization mice were intradermally challenged with U-Omp19 in their left footpad and an equal volume of saline into their right footpad. DTH response was measured at 72 h following injection of antigen. Each bar represents the mean increase in the footpad thickness ± SEM from 5 mice per group. (★) Significantly different from the mean increase thickness measured in PBS immunized mice (<i>P</i><0.05).These experiments were conducted three times with similar results.</p

Immunization and experimental design scheme.

<p>(<b>A</b>) In order to analyze the immune responses and the elicited protective responses induced after oral immunization, mice were immunized i.g. on days 0, 7 and 14. For the protection experiments mice were challenged one month after the last immunization with virulent bacteria and 30 days later were sacrificed to analyze the bacterial burden in the spleens. For the analysis of the DTH response immunized mice were challenged with U-Omp19 3 weeks after the last immunization. Cytokine production by splenocytes was determined one month after the last immunization. (<b>B</b>) In order to analyze the immune responses and the elicited protective responses after parenteral immunization, mice were immunized i.p. on days 0 and 15. For the protection experiments mice were challenged one month after the last immunization with virulent bacteria and 30 days later were sacrificed to analyze the bacterial burden in the spleens. For the analysis of the cytokine production the spleens of immunized mice were obtained one month after the last immunization.</p

Protection against oral <i>B. abortus</i> infection in BALB/c mice vaccinated orally with L-Omp19 or U-Omp19 without adjuvants or with U-Omp19+CT.

a<p>The content of bacteria in spleens is represented as the mean log₁₀ CFU ± SD per group.</p>b<p>Significantly different from PBS-immunized mice <i>P</i><0.01 estimated by Dunnett's test.</p>c<p>Significantly different from PBS-immunized mice <i>P</i><0.05 estimated by Dunnett's test.</p>d<p>Significantly different from <i>B. abortus</i> RB51 immunized mice <i>P</i><0.01 estimated by Dunnett's test.</p

Kinetic of the specific humoral responses elicited after administration of adjuvant-free Omp19.

<p>BALB/c mice were immunized i.g. (<b>A</b>) or i.p. (<b>B</b>) with L-Omp19 (•) or U-Omp19 (<b>○</b>) without adjuvants as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016203#s4" target="_blank">materials and methods</a> and the kinetic of the Omp19 specific humoral response elicited after immunizations was determined. Serum samples were obtained at the indicated time after the first immunization. Omp19-specific IgG Ab titers were determined by ELISA. Each point represents the mean ± S.E.M of the Ab titer from 5 mice per group. (★) and (★ ★) significantly different from the U-Omp19 immunized group, (<i>P</i><0.05) and (<i>P</i><0.01), respectively. This experiment was conducted three times with similar results.</p