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

<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

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

<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 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

Colonic Immune Suppression, Barrier Dysfunction, and Dysbiosis by Gastrointestinal <i>Bacillus anthracis</i> Infection

<div><p>Gastrointestinal (GI) anthrax results from the ingestion of <i>Bacillus anthracis</i>. Herein, we investigated the pathogenesis of GI anthrax in animals orally infected with toxigenic non-encapsulated <i>B. anthracis</i> Sterne strain (pXO1⁺ pXO2⁻) spores that resulted in rapid animal death. <i>B. anthracis</i> Sterne induced significant breakdown of intestinal barrier function and led to gut dysbiosis, resulting in systemic dissemination of not only <i>B. anthracis</i>, but also of commensals. Disease progression significantly correlated with the deterioration of innate and T cell functions. Our studies provide critical immunologic and physiologic insights into the pathogenesis of GI anthrax infection, whereupon cleavage of mitogen-activated protein kinases (MAPKs) in immune cells may play a central role in promoting dysfunctional immune responses against this deadly pathogen.</p></div

T Cell Responses in Sterne-Infected A/J Mice.

<p>A/J mice were orally gavaged with 10⁹ spores of the Sterne strain of <i>B. anthracis</i> and adaptive immune responses in the colon analyzed at various time points by flow cytometry. <b>A</b>. Gating strategy for the analysis of colonic T cells. <b>B</b>. Th1, Th17, and regulatory T cells responses were tested by flow cytometry. Representative plots indicate cytokine production of uninfected and day 5-infected mice. <b>C</b>. Surface expression of PD1 in colonic T cells. <b>D</b>. Gene expression profile of the distal colon of Sterne-infected A/J mice. Data represent observations from four independent experiments and are shown as mean +/− SEM. *P<0.05, **P<0.01, ***P<0.001 compared with PBS.</p

Cleavage of MAPKs in DCs of Sterne-Infected A/J Mice.

<p>A/J mice were orally gavaged with 10⁹ spores of the Sterne strain of <i>B. anthracis</i> and DCs analyzed at various time points. <b>A</b>. Gating strategy for the analysis of CD45⁺MHCII^hiCD11c⁺F4/80⁻CD11b⁺ colonic DCs. <b>B</b>. Cell surface expression of CD86, CD80, and B7-H1 by colonic DCs was analyzed by flow cytometry. Gray tinted line = isotype control; black line = PBS group; green line = day 1 Sterne-infected A/J mice; magenta line = day 3 Sterne-infected A/J mice; blue line = day 5 Sterne-infected mice. <b>C</b>. Activity of MAPKs in colonic DCs of infected versus uninfected mice was analyzed by flow cytometry. Gray tinted line = isotype control; black line = PBS group; blue line = day 5 Sterne-infected mice. <b>D</b>. Colonic LP cells were isolated from uninfected A/J mice and incubated with 1 MOI of <i>B. anthracis</i> spores for 1, 3, or 6 hours. Activity of p38 and Erk1/2 was subsequently analyzed in colonic DCs. Gray tinted line = isotype control; black line = PBS group; green line = 1 hour treatment; magenta line = 3 hour treatment; blue line = 6 hour treatment. Data represent observations from three independent experiments and are shown as mean +/− SEM. *P<0.05, **P<0.01, ***P<0.001 compared with PBS.</p

Lethality and Systemic <i>B. anthracis</i> Sterne Dissemination in A/J Mice.

<p>A. A/J mice were orally gavaged with 10⁵, 10⁷, or 10⁹ spores of the Sterne strain of <i>B. anthracis</i>. Lethal infection was established within 3 days in A/J mice receiving 10⁹ spores; n = 10 mice/group (10⁵ and 10⁷), n = 20 mice/group (10⁹). Experiments were performed a minimum of three times. Statistical significance was calculated using the log-rank test. After 3 days of infection, A/J mice were sacrificed; both spores and vegetative bacilli (marked with *) were observed in the colon (<b>B</b>), MLNs (<b>C</b>), spleen (<b>D</b>, <b>E</b>), liver (<b>F</b>), kidneys (<b>G</b>), lungs (<b>H</b>), and in bronchoalveolar lavage (BAL) fluid (<b>I</b>). Bar = .</p