Evaluation of intestinal and serum immune responses from vaccinated mice groups.

<p><b>A:</b> Assessment of antibody response (serum IgG and intestinal IgA) by bacterial FACS. Gut-wash/serum samples from the mice immunized with PBS (n = 4), MT12 (n = 4) or MT13 (n = 4) were serially diluted and analyzed by FACS. Representative FACS graphs for IgG response from diluted (1∶120) serum sample and intestinal IgA response from diluted (1∶9) sample from gut-wash are shown. Each plotted line in a panel (PBS/MT12/MT13) indicates data from individual mice of a group. <b>B:</b> Representative western blots of mucosal secretory IgA response and <b>C:</b> serum IgG response as observed on final treatment with α-mouse IgA (for gut wash) and α-mouse IgG (for serum).</p

Bacterial strains, plasmids and primers used in study.

<p>Bacterial strains, plasmids and primers used in study.</p

Colonisation of vaccine candidate and its systemic access.

<p>Vaccination-challenge experiment to analyze the immunogenic potential of <i>S.</i> Typhimurium M12 and MT13 strain. Groups of C57BL/6 mice were vaccinated with 5×10⁷ cfu of <i>Salmonella</i> Typhimurium MT12 (<i>ssaV</i> mutant; closed grey circles; n = 8) and MT13 (<i>ssaV</i> and <i>fur</i> double mutant; closed black circles; n = 8) by gavage. One group of mice was treated with PBS (open circles; n = 8) to serve as control. Mice groups were kept under observation for next 30 days. <b>A:</b> Fecal shedding of <i>Salmonella</i> strains as analyzed by plating at various time-points in days. Four mice from each vaccinated group were sacrificed at day 30 post-vaccination (p.v.) to assess the bacterial population at different host sites. <b>B:</b> Enumeration of bacterial loads at various host sites. Broken lines in the graphs shows minimum detection limit. n.s.: not significant, *: statistically significant (P<0.05, t-test).</p

Attenuation status of MT12 and MT13 <i>S</i>. Typhimurium strains in immunocompromised mice.

<p>To identify the attenuation properties of developed <i>Salmonella</i> strains, groups of various immunocompromised mice (<i>iNos^−/−, IL10^−/−, CD40L^−/−</i> in C57BL/6 background along with wild-type mice as control group) were infected with 5×10⁷ cfu of different strains of <i>Salmonella</i> Typhimurium (wild-type: SB300; <i>ssaV</i> mutant: MT12; <i>ssaV</i> and <i>fur</i> double mutant: MT13) by gavage. Mice were sacrificed at 4 days p.i. and bacterial colonization was assessed. Graph represents colonization of <i>S.</i> Typhimurium strains at different host sites; <b>A:</b> cecum, <b>B:</b> mesenteric lymph nodes, <b>C:</b> spleen and <b>D:</b> liver. Broken lines in the graphs shows minimum detection limit. n.s.: not significant, *: statistically significant (P<0.05, t-test).</p

Evaluation of T-cell immune responses from vaccinated mice groups.

<p>Tissue homogenates of mesenteric lymph nodes (mLN), collected from mice groups vaccinated with MT12, MT13 and PBS at day 30 p.v., were FACS analyzed for T-cell response. Representative FACS graphs depicting CD4⁺ (pacific blue labelled) and CD8⁺ (FITC labelled) cell populations (second quadrant in each graph) in various immunized mice groups. Serum and mucosal antibody response from immunized mice was analyzed; serum and gut wash were collected at day 30 p.v.; bacterial lysate of overnight cultures of SB300 (wild-type <i>S.</i>Typhimurium), M1525 (wild-type <i>S.</i> Enteritidis P125109), MT11 (S.Tm <i>fur</i> mutant), MT12 (S.Tm <i>ssaV</i> mutant) and MT13 (S.Tm <i>ssaV</i> and <i>fur</i> double mutant) were developed with serum or gut wash from immunized mice.</p

<i>S.</i> Typhimurium challenge experiment: an assessment of vaccination efficacy.

<p>Vaccinated C57BL/6 mice groups (PBS: n = 4, MT12: n = 4 and MT13: n = 4) were ampicillin-treated (25 mg by gavage), challenged with wild-type SB300 (amp^r, sm^r) and euthanized at day 3 post-challenge (p.c.) i.e. day 34 post-vaccination. <b>A:</b> Colonization at various host sites after SB300 challenge. <b>B:</b> Enumeration of cecal pathology in terms of 13 point pathoscore scale was determined as described above (see material and methods). Broken lines in the graphs shows minimum detection limit. n.s.: not significant, *: statistically significant (P<0.05, t-test). <b>C:</b> Representative HE-stained cecum sections obtained from SB300-challenged mice groups immunized with PBS, MT12, MT13. Bar, 200 µm. S, submucosal edema; Lp, lamina propria; L, lumen.</p

Live Attenuated <em>S</em>. Typhimurium Vaccine with Improved Safety in Immuno-Compromised Mice

<div><p>Live attenuated vaccines are of great value for preventing infectious diseases. They represent a delicate compromise between sufficient colonization-mediated adaptive immunity and minimizing the risk for infection by the vaccine strain itself. Immune defects can predispose to vaccine strain infections. It has remained unclear whether vaccine safety could be improved via mutations attenuating a vaccine in immune-deficient individuals without compromising the vaccine's performance in the normal host. We have addressed this hypothesis using a mouse model for <em>Salmonella</em> diarrhea and a live attenuated <em>Salmonella</em> Typhimurium strain (<em>ssaV</em>). Vaccination with this strain elicited protective immunity in wild type mice, but a fatal systemic infection in immune-deficient <em>cybb</em>^−/−<em>nos2</em>^−/− animals lacking NADPH oxidase and inducible NO synthase. In <em>cybb</em>^−/−<em>nos2</em>^−/− mice, we analyzed the attenuation of 35 <em>ssaV</em> strains carrying one additional mutation each. One strain, Z234 (<em>ssaV SL1344_3093</em>), was >1000-fold attenuated in <em>cybb</em>^−/−<em>nos2</em>^−/− mice and ≈100 fold attenuated in <em>tnfr1</em>^−/− animals. However, in wt mice, Z234 was as efficient as <em>ssaV</em> with respect to host colonization and the elicitation of a protective, O-antigen specific mucosal secretory IgA (sIgA) response. These data suggest that it is possible to engineer live attenuated vaccines which are specifically attenuated in immuno-compromised hosts. This might help to improve vaccine safety.</p> </div

Verification of the attenuation of Z234.

<p>Streptomycin treated <i>cybb</i>^−/−<i>nos2</i>^−/− (2A) or C57BL/6 (2B) mice were infected either with <i>ssaV</i> (open), Z234 (grey) or Z234 p_compl. (black; 5×10⁷ cfu by gavage; n = 5–10 mice). Bacterial loads in the cecum contents, the MLN and the spleens were quantified by plating at day 4 post infection; *, statistically significant (p <.05; Mann-Whitney U-test).</p

Vaccination-challenge analysis of the immunogenic potential of Z234.

<p>C57BL/6 mice were vaccinated with PBS (n = 4; open symbols), <i>ssaV</i> (5×10⁷ cfu; n = 9; grey) or Z234 (5×10⁷ cfu; n = 13; black). 40 days post immunization (PBS (n = 4); M2735 (n = 5); Z234 (n = 7)), mice were ampicillin-treated (20 mg by gavage) and challenged with wt SB300 (amp^r, sm^r) for two days.. Colonization of cecum (A), MLN (C), Spleen (D) and cecal pathology (B) after wt <i>S</i>. Typhimurium, day 2 post challenge was shown accordingly. Disease parameters (vaccination control data) were determined as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045433#pone.0045433.s003" target="_blank">Figure S3B</a>. Striped line: detection limit.</p

Screening experiment identifying Z234.

<p>Streptomycin pre-treated <i>cybb</i>^−/−<i>nos2</i>^−/− mice (n = 3) were co-infected with a 1∶1∶1∶1∶1∶1∶1 mixture of 6 different double mutants including Z234 (WITS tag 21) and the isogenic parental strain M2735 (<i>ssaV::cat;</i> WITS 17) and; <i>ssaV STM2616</i> (WITS 1), <i>ssaV STM2998</i> (WITS 2), <i>ssaV STM2011</i> (WITS11), <i>ssaV STM2231</i> (WITS13), <i>ssaV STM2570</i> (WITS19). Real-time PCR analysis of the sequence tags established the relative abundance of Z234 (black symbols) as compared to the parental strain and the five other double-mutants analyzed in these mice (open symbols).</p