Colonic Immune Stimulation by Targeted Oral Vaccine

<div><h3>Background</h3><p>Currently, sufficient data exist to support the use of lactobacilli as candidates for the development of new oral targeted vaccines. To this end, we have previously shown that <em>Lactobacillus gasseri</em> expressing the protective antigen (PA) component of anthrax toxin genetically fused to a dendritic cell (DC)-binding peptide (DCpep) induced efficacious humoral and T cell-mediated immune responses against <em>Bacillus anthracis</em> Sterne challenge.</p> <h3>Methodology/Principal Finding</h3><p>In the present study, we investigated the effects of a dose dependent treatment of mice with <em>L. gasseri</em> expressing the PA-DCpep fusion protein on intestinal and systemic immune responses and confirmed its safety. Treatment of mice with different doses of <em>L. gasseri</em> expressing PA-DCpep stimulated colonic immune responses, resulting in the activation of innate immune cells, including dendritic cells, which induced robust Th1, Th17, CD4⁺Foxp3⁺ and CD8⁺Foxp3⁺ T cell immune responses. Notably, high doses of <em>L. gasseri</em> expressing PA-DCpep (10¹² CFU) were not toxic to the mice. Treatment of mice with <em>L. gasseri</em> expressing PA-DCpep triggered phenotypic maturation and the release of proinflammatory cytokines by dendritic cells and macrophages. Moreover, treatment of mice with <em>L. gasseri</em> expressing PA-DCpep enhanced antibody immune responses, including IgA, IgG₁, IgG_2b, IgG_2c and IgG₃. <em>L. gasseri</em> expressing PA-DCpep also increased the gene expression of numerous pattern recognition receptors, including Toll-like receptors, C-type lectin receptors and NOD-like receptors.</p> <h3>Conclusion/Significance</h3><p>These findings suggest that <em>L. gasseri</em> expressing PA-DCpep has substantial immunopotentiating properties, as it can induce humoral and T cell-mediated immune responses upon oral administration and may be used as a safe oral vaccine against anthrax challenge.</p> </div

<i>L. gasseri</i> expressing PA-DCpep promotes Th17 responses.

<p>C57BL/6 mice were orally gavaged with increasing doses of <i>L. gasseri</i> expressing PA-DCpep (10⁷, 10⁹ and 10¹² CFU) or PBS; LPLs were harvested by collagenase digestion after days 1, 3, 7 and 14, and stained with antibodies against CD4, CD8, RORγT, IL-17 and IL-22 (A&B) before analysis by flow cytometry. Data are representative of two independent experiments. Error bars represent ±SEM. *P<0.05 and **P<0.01 compared with PBS. (C) C57BL/6 mice were orally gavaged with increasing doses of <i>L. gasseri</i> expressing PA-DCpep (10⁷, 10⁹ and 10¹² CFU) or PBS; colonic sections were stained with RORγT (red) and CD4 (green) antibodies, and visualized using confocal microscopy.</p

Activation of colonic DCs by <i>L. gasseri</i> expressing PA-DCpep.

<p>Groups of C57BL/6 mice (n = 3) were fed with different doses (10⁷, 10⁹ and 10¹² CFU) of <i>L. gasseri</i> expressing PA-DCpep and sacrificed on days 1, 3, 7 and 14 post-inoculation. The activation of colonic DCs was evaluated by the surface expression of CD40, CD86 and B7H1, and analyzed by flow cytometry (A). Lamina propria lymphocytes (LPLs) were also stained with antibodies against CD11c, CD11b, IL-10 and IL-12, and analyzed by flow cytometry (B). Data are representative of two independent experiments. Error bars represent ±SEM. *P<0.05 and **P<0.01 compared with PBS. (C) C57BL/6 mice were orally gavaged with increasing doses of <i>L. gasseri</i> expressing PA-DCpep (10⁷, 10⁹ and 10¹² CFU) or PBS and colonic sections were stained with CD11c (magenta), CD11b (red) and either TNFα, IL-12 or IL-10 (green) antibodies, and visualized using confocal microscopy.</p

<i>L. gasseri</i> expressing PA-DCpep is safe for mice at high doses.

<p>C57BL/6 mice were orally gavaged with 10⁹ CFU of <i>L. gasseri</i> expressing PA-DCpep once and the CFU per gram of feces was determined (A) in MRS plates with erythromycin (5 µg/mL). C57BL/6 mice were orally gavaged with increasing doses of <i>L. gasseri</i> expressing PA-DCpep (10⁷, 10⁹ and 10¹² CFU) or PBS, serum was collected after days 1, 3 and 7, and the enzyme activities of ALT/SGPT (B) and AST/SGOT (C) were analyzed by ELISA. CCl₄ was used as a positive control for toxicity (B & C). Tissues from C57BL/6 mice orally gavaged with increasing doses of <i>L. gasseri</i> expressing PA-DCpep (10⁷, 10⁹ and 10¹² CFU) or PBS were collected and sections stained with H&E at days 1, 3 and 7 of treatment. (D) Photomicrographs of H&E sections of liver A–D, kidney E–H, spleen I–L, colon M–P, and cecum Q–T. Insets of a representative glomerulus and surrounding proximal tubules are included in each of the kidney photomicrographs. Insets of high magnification of the lymphoid tissue are included in each of the spleen photomicrographs. For each of the organs, the PBS control is pictured in the left column A, E, I, M and Q, respectively. The three photomicrographs on the right are from increasing concentrations of bacteria from 10⁷, 10⁹ and 10¹² from left to right.</p

<i>L. gasseri</i> expressing PA-DCpep enhances the induction of regulatory T cells.

<p>C57BL/6 mice were orally gavaged with increasing doses of <i>L. gasseri</i> expressing PA-DCpep (10⁷, 10⁹ and 10¹² CFU) or PBS; LPLs were harvested by collagenase digestion after days 1, 3, 7 and 14, stained with antibodies against CD4, CD8, IFNγ (A&B), TGFβ and FoxP3 (C&D), and analyzed by flow cytometry. Data are representative of two independent experiments. Error bars represent ±SEM. *P<0.05 and **P<0.01 compared with PBS.</p

Activation of PRR-genes by <i>L. gasseri</i> expressing PA-DCpep.

<p>C57BL/6 mice were orally gavaged with increasing doses of <i>L. gasseri</i> expressing PA-DCpep (10⁷, 10⁹ and 10¹² CFU), or PBS; RNA was isolated from colon tissue after days 1, 3 and 7 of treatment. Quantitative real-time PCR was performed to measure changes in gene expression. Hierarchical matrix cluster analysis (A) and comparative gene analysis presented as a heat map (B). Data are representative of two independent experiments with duplicates.</p