Evaluation of efficacy of a new live Salmonella Typhimurium vaccine candidate in a murine model

Comparative Immunology, Microbiology and Infectious Diseases 34 (2011) 171–177

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Evaluation of efficacy of a new live Salmonella Typhimurium vaccine candidate in a murine model Jin Hur, Mi Young Kim, John Hwa Lee ∗ College of Veterinary Medicine and Bio-Safety Research Institute, Chonbuk National University, Jeonju 561-756, South Korea

a r t i c l e

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Article history: Received 26 August 2010 Accepted 29 October 2010 Keywords: Salmonella Typhimurium Live attenuated vaccine Immune response Cytokine Protection

a b s t r a c t Efficacy of a new live Salmonella Typhimurium vaccine candidate attenuated by two virulence gene deletions was evaluated in this study. Live form of the vaccine was orally administered and formalin-inactivated form was intramuscularly (IM) administered. 105 female BALB/c mice were used and divided into 5 groups, A to E, containing 21 mice per group. Serum IgG and secretory IgA titers and levels of serum IFN-␥, IL-4, TNF-␣, IL-12 of the immunized groups, especially group C (oral prime-oral booster) significantly increased. In addition, all animals in groups C showed no clinical symptoms and survived the virulent challenges, whereas all or some of mice from the other groups died of the challenge. These indicate that the vaccine candidate can be an effective tool for prevention of Salmonella infections by inducing robustly protective immune responses and leading to the production of inflammatory cytokines. Booster immunization, especially via oral administration, is necessary to optimize its protection. © 2010 Elsevier Ltd. All rights reserved.

1. Introduction Salmonella enterica serovar Typhimurium (S. Typhimurium) causes gastroenteritis in humans and animals, and is a major food borne pathogen [1,2]. In mouse infection models S. Typhimurium can induce enteric fever with symptoms similar to those observed in humans after infection with S. Typhi [2]. Vaccination against salmonellosis is an effective means to prevent this disease [1,3,4]. It is generally accepted that the cellular immune response is crucial for effective protection against Salmonella [5,6], and antibodies including serum immunoglobulin (Ig) G and secretory IgA (sIgA) are also known to contribute to the clearing of Salmonella infection under some circumstances [3,5]. Vaccination with either attenuated or killed Salmonella induces protection against infection with an otherwise lethal dose of virulent bacteria,

∗ Corresponding author. Tel.: +82 63 270 2553; fax: +82 63 270 3780. E-mail address: [email protected] (J.H. Lee). 0147-9571/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.cimid.2010.11.001

and this protection can be passively transferred to offspring through the serum [6–9]. An ideal vaccine giving long lasting, protective immunity is needed for the prevention of salmonellosis. Many attenuated vaccine strains of Salmonella have been generated by mutating metabolic genes or virulence-associated pathogenicity islands in an otherwise wild type background [4,10–12]. Many important virulence factors are encoded within different Salmonella pathogenicity islands (SPIs), including SPI-1 and SPI-2. SPI-1 is required for membrane ruffling, invasion of epithelial cells, and intestinal pathogenesis [13,14], whereas SPI-2 is required for survival and replication within the host cells, and is necessary for systemic spread [14–16]. The expression of SPI-1 and SPI-2 genes appears to be regulated at several levels by many regulators [17–19]. It has been reported that the ATP dependent Lon protease is a negative regulator of epithelial cell invasion and the expression of invasion genes carried on SPI-1 [2,19]. Despite this, Lon is important for successful systemic infection [20]. In addition to Lon, the CpxRA two-component regulatory system senses a variety of diverse signals, including pH, misfolded pili

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subunits, overproduction of outer membrane lipoproteins, copper ions, and surface contact [21–23]. Furthermore, the Cpx pathway is required for the invasion of host cells by diverse pathogenic bacteria including Escherichia coli (enterohemorrhagic E. coli and uropathogenic E. coli), S. Typhimurium, S. Typhi, Shigella sonnei, Yersinia enterocolitica, and Legionella pneumophila [21,22,24,25]. Serum cytokine levels after vaccination can be used as indicators of specific immune responses. Tumor necrosis factor-alpha (TNF-␣), a proinflammatory cytokine, is produced mainly by macrophages [26] and induces the inflammatory response in order to control Salmonella [27–29] infection. Interleukin (IL)-12 induces interferongamma (IFN-␥) production by T cells and has the potential to drive T helper (Th) 1 cell differentiation [30,31]. It has been suggested that IL-12 may augment the protective immune response to Salmonella [32,33]. IFN- is secreted by Th1 cells, dendritic cells and natural killer cells [25]. The induction of the IFN-␥ response during infection with phagosome- or endosome-bound intracellular bacterial pathogens has also been considered pivotal for the resolution of infection [31]. Finally, Th2-type cytokines such as IL-4 and IL-5, have been known to induce mucosal IgA and serum IgG antibodies [27,34] in response to infection. The objective of this study was to examine a new live Salmonella Typhimurium (S. Typhimurium loncpxR) vaccine candidate and optimize an immunization strategy that provides maximum protection against salmonellosis in a murine model. We investigated both the immune responses and the induction of cytokines including TNF␣, IL-12, IFN-␥ and IL-4. In addition, we performed histopathological and bacteriological analysis of the vaccinated mice. 2. Materials and methods 2.1. Bacterial strains and plasmids The attenuated S. Typhimurium strain, JOL911, was constructed by deletion of the lon and cpxR genes from a wild type S. Typhimurium isolate, using the suicide plasmid pMEG375 and gene specific primers, as previously described [35], and used as vaccine candidate. A wild type virulent S. Typhimurium isolate, JOL398, was used as a virulent challenge strain. 2.2. Preparation of live and formalin-inactivated vaccines In order to prepare live vaccine, JOL911 (S. Typhimurium loncpxR strain) was grown in LB broth (Becton, Dickinson and Company, Sparks, MD, USA) at 37 ◦ C to an optical density (OD)600 of 0.8. Cells were harvested by centrifugation at 4000 rpm for 20 min and were resuspended in sterile phosphate-buffered saline (PBS) containing 20% sucrose (PBS-sucrose) to 2 × 1011 colony-forming units (CFU)/ml. Mice were then orally immunized with the live vaccine on the same day of preparation. For formalin-inactivated vaccine preparation, the strain was grown in LB broth overnight at 37 ◦ C. Cells were washed and diluted in sterile PBS to a density of 1 × 109 CFU/ml. Formalin was added

to a final concentration of 1% (v/v), and the suspension was held at 4 ◦ C for 24 h. Cells were washed with sterile PBS. The remaining pellets were diluted to 2 × 1010 cells in 10 mg/ml of aluminum hydroxide (Sigma–Aldrich, St. Louis, MO, USA), which has been used as a parenteral adjuvant. The formalin-inactivated vaccine was stored at 4 ◦ C until use [36]. 2.3. Immunization and sample collection A total of 105 5-week-old female BALB/c mice were divided into 5 groups, containing 21 mice per group. All groups of mice were primed at 6 weeks of age, and all groups except group A were boosted at 9 weeks. In group A, mice were orally administrated with 20 ␮l of live vaccine. In group B, mice were orally primed with 20 ␮l of live vaccine, then intramuscularly boosted with 100 ␮l of formalin-inactivated vaccine. In group C, mice were both orally primed and boosted with 20 ␮l of live vaccine. In group D, mice were intramuscularly primed with 100 ␮l of formalin-inactivated vaccine and then orally boosted with 20 ␮l of live vaccine. In control group E, mice were both orally primed and boosted with 20 ␮l of sterile PBS-sucrose. Food and water were withdrawn 4 h prior to immunization and supplied 30 min after immunization. In order to evaluate serum IgG and IgA titers, blood samples were obtained by retro-orbital puncture with a Pasteur pipette at 0, 3, and 6 weeks post prime immunization (PPI). To determine the induction of TNF-␣, IL-12, IFN-␥ and IL-4 in serum, blood samples were obtained on days 3 and 7 PPI, and days 3, 7, and 14 post booster immunization (PBI). Sera were obtained from the whole blood by centrifugation at 4000 × g for 5 min. Vaginal secretions were collected by washing the vaginal tracts with 100 ␮l of sterile PBS. All samples were stored at −80 ◦ C until use. The animal experiments described in this study were conducted under ethics approval from the Chonbuk National University Animal Ethics Committee in accordance with the guidelines of the Korean Council on Animal Care. 2.4. Immune responses and cytokines measurement by ELISA Salmonella Typhimurium LPS was prepared from the wild type S. Typhimurium with an LPS extraction kit (iNtRON Biotechnology, Seongnam, South Korea) according to the manufacturer’s instructions and stored at −70 ◦ C until use. A standard enzyme-linked immunosorbent assay (ELISA) was used to determine the Salmonella-specific IgG, IgG1, IgG2a and IgA antibodies in serum, and sIgA in vaginal exudates, as previously described with slight modification [37]. Briefly, ELISA plates (Greiner Bio-One, Frickenhausen, Germany) were coated with 0.5 ␮g of S. Typhimurium LPS per well at 4 ◦ C overnight. Sera were diluted to 1:400 for examination of IgG, IgG1 and IgG2a titers, and sera and vaginal secretions were diluted to 1:16 for examination of IgA and sIgA titers, respectively. The plates were treated with horseradish peroxidase-conjugated goat antimouse antibodies (Southern Biotechnology Associates Inc., Birmingham, AL, USA). Enzymatic reactions were developed with o-phenylenediamine (Sigma–Aldrich, St. Louis,

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MO, USA) and measured with an automated ELISA spectrophotometer (TECAN, Salzburg, Austria) at 492 nm. A standard curve was generated describing the relationship between the concentration of standards and their absorbance, and the concentration of antibody for each sample was expressed as nanogram per milliliter (ng/ml). ELISA was also used to measure the concentration of cytokines in mouse serum, including TNF-␣, IL-12, IFN-␥ and IL-4, in using the mouse cytokine ELISA Ready-SET-GO reagent set according to the manufacturer’s instructions (eBioscience Inc., San Diego, CA, USA). Enzymatic reactions were developed with 3,3 5,5 -tetramethylbenzidine. 2.5. Fecal shedding of vaccine strain In order to investigate shedding of the vaccine strain in feces, five mice of groups A and B were tested daily for 3 weeks PPI of oral inoculation, while five mice of groups C and D were tested daily for 3 weeks PBI of oral inoculation. The fecal samples were initially pre-enriched in buffered peptone water for 18 h at 37 ◦ C. Pre-enriched broth (100 ␮l) was transferred to Rappaport-Vassiliadis R10 broth (10 ml, Becton, Dickinson and Company, Sparks, MD, USA) and incubated under aerobic conditions for 24 h at 42 ◦ C. The enrichment medium (100 ␮l) was then streaked on Brilliant Green agar (BGA) (Becton, Dickinson and Company, Sparks, MD, USA) and identified using the API 20E system (bioMérieuxsa, Marcy, I Étoile, France). Identities of the vaccine and challenge strains were confirmed by PCR using Salmonella specific primers, OMPC (OMPCF: 5 -ATCGCTGACTTATGCAAT CG; OMPCR: CGGGTTGCGTTATAGGTCTG-3 ) for Salmonella spp., TYPH (TYPHF: 5 -TTGTTCACTTTTTACCCCTGAA-3 ; TYPHR: 5 CCCTGACAGCCGTTAGATATT-3 ) for S. Typhimurium. CpxR (cpxR-F: 5 -GATAATTTACCGTTAACGAC-3 ; cpxR-R: 5 -CATCATCTGCGGGTTGCAGC-3 ) and lon (lon-F: 5 -CAGGAGTTCTTACAGGTAGA-3 ; lon-R: 5 CCACACTCCGCTGTAGGTGA-3 ) primers were used to identify the vaccine candidate [38]. 2.6. Challenge experiment The virulent challenge strain, JOL398, was grown in LB broth overnight at 37 ◦ C, diluted 1:20 in LB broth, and then grown at 37 ◦ C to an OD600 of 0.8. Cells were harvested by centrifugation at 4,000 rpm for 20 min. Cells were diluted to approximately 2 × 108 CFU in 20 ␮l of sterile PBS-sucrose after washing twice with sterile PBS, and all mice were orally challenged with 20 ␮l of the diluted solution at week 6 PPI. All mice were subsequently monitored for mortality for 14 days after challenge. 2.7. Bacteriological and histological examinations of infected organs Five mice from each group were sacrificed on day 8 after challenge and their livers and spleens were harvested. Approximately half of the liver and spleen were homogenized, then liver and spleen homogenates were diluted 1:10 and 1:3 in PBS (w/v), respectively. Serially diluted liver and spleen homogenates were plated on BGA.

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Colonies were counted after incubation at 37 ◦ C for 24 h. The other half of each organ was fixed in 10% buffered formaldehyde and embedded in paraffin for histological examinations. Sections (5 ␮m thick) were cut, stained by hematoxylin and eosin, and examined under the light microscope. 2.8. Statistical analysis Independent-samples t-test was used to determine significant differences in antibody titers between immunized and control groups, and significant differences in the antibody titers of IgG2a and IgG1 in immunized groups. Statistical results were considered significant when P-values were <0.05. All statistical analyses were carried out with the SPSS 16.0 program (SPSS Inc., Chicago, IL, USA). 3. Results 3.1. Systemic and mucosal immune responses induced by vaccination The antibody responses to Salmonella antigen in the sera and the vaginal secretions of immunized mice are presented in Fig. 1. At 3 weeks PPI, the serum IgG titers of groups A (single administered orally), B (orally primed and boosted intramuscularly), C (both primed and boosted by the oral route) and D (intramuscularly primed and boosted orally) were 721.7, 721.5, 727.5 and 706.6 ng/ml, respectively, while the titer of control group E (administered with PBS) was approximately 358.6 ng/ml (P < 0.01). At 6 weeks PPI levels for groups A–D were increased to 1074.0, 1006.6, 1149.4 and 1087.1 ng/ml, respectively, while the level in control group was 381.5 ng/ml (P < 0.01) (Fig. 1A). At 6 weeks PPI the serum IgA titers of groups A, B, C and D were 114.5, 114.8, 226.5 and 93.0 ng/ml, respectively, while the titer in control group was 67.3 ng/ml (Fig. 1B). At 3 weeks PPI the sIgA titers of the vaginal secretions of groups A, B, C and D were 3.9, 3.6, 4.9, and 3.3 ng/ml, respectively, while the titer in control group was 1.8 ng/ml (P < 0.05), At 6 weeks PPI the titers were increased to 78.8, 58.5, 137.9 and 131.9 ng/ml, respectively, while the titer in control group was 2.0 ng/ml (P < 0.01) (Fig. 1C). 3.2. IgG isotype analyses The nature of the immune response to Salmonellaspecific antigen was further examined by measuring the levels of IgG isotype subclasses IgG2a and IgG1 at 6 weeks PPI. As shown in Fig. 1D, the IgG2a isotype was the dominant response in the sera of all immunized groups in response to Salmonella antigen. The IgG2a/IgG1 ratios in groups A, B, C and D were approximately 4.0, 1.9, 1.5 and 4.4, respectively (P < 0.05). 3.3. Cytokine analysis Serum TNF-␣ of all immunized groups started to increase on day 3 PPI and their significantly high levels remained until day 14 PBI (P < 0.01) (Fig. 2A). Serum IL-4, IL-12 and IFN-␥ also started to increase from day 3 PPI, and

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Fig. 1. Immune response to Salmonella-specific antigen in mice inoculated with the Salmonella vaccine candidate with/without adjuvant via oral and/or intramuscular routes. (A) Serum Salmonella-specific IgG. (B) Serum Salmonella-specific IgA. (C) Vaginal Salmonella-specific sIgA. (D) Serum Salmonellaspecific IgG1 and IgG2a. Group A mice were single administered orally, group B mice were orally primed and boosted intramuscularly, group C mice were both primed and boosted by the oral route group D mice were intramuscularly primed and boosted orally, and group E mice were administered with PBSsucrose as a control. Data are the mean of all mice in each group; error bars show standard deviation. Asterisks indicate a significant difference between the values of groups A, B, C and D mice (*P < .05, **P < .01), and those of the control group mice.

especially, the mice of groups C and D maintained significant high levels of IL-12 and IFN-␥ until day 14 PBI (P < 0.01) (Fig. 2B and C), while group C mice induced significantly high level of IL-4 until the end of experiment (P < 0.05) (Fig. 2D).

3.4. Fecal shedding of the vaccine strain Shedding of the vaccine strain in feces was investigated for 3 weeks following oral inoculation. Five mice of each group were tested daily. Among all daily fecal samples, only

Fig. 2. Cytokine titers in serum of mice immunized with Salmonella vaccine candidate with/without adjuvant via oral and/or intramuscular routes. (A) Serum mouse TNF-␣. (B) Serum mouse IL-12. (C) Serum mouse IFN-. (D) Serum mouse IL-4. Group A, (); group B, ( ); group C, (); group D, ( ); group E, ( ). Data are the mean of all mice in each group; error bars show SD. Asterisks indicate a significant difference between the values of groups A, B, C and D mice (* P < .05, ** P < .01), and those of the control group mice.

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Fig. 3. Survival rate of mice challenged with wild type S. Typhimurium 6 weeks post prime immunization. Group A, (); group B, (); groups C and D, (♦) and group E, ().

a sample from one mouse, taken on the 5th day contained the strain. 3.5. Protection after challenge All mice in each group were orally challenged with 2 × 108 CFU of the virulent challenge strain at 6 weeks PPI. As shown in Fig. 3, all mice that were orally primed and orally boosted (group C), or intramuscularly primed and orally boosted (group D), did not show any clinical signs and survived from the challenge through the end of this study. However, mice in the control group began to show mortality at day 6 after challenge and all mice died by day 11. Three mice from group A and 4 mice from group B died between days 8 and 10 after challenge. 3.6. Bacteriological and pathological examinations Isolation of the virulent challenge strain from the liver and spleen of mice was performed on day 8 after challenge. As shown in Table 1, the virulent challenge strain was isolated from the livers of all mice in groups A, B and E, and an average of 2 × 103 , 4 × 105 and 8 × 106 CFU/g of bacteria were found, respectively. The bacterium was also found in the spleens of all mice from groups A, B and E, with an average of 8 × 103 , 3 × 103 and 2 × 107 CFU/g, respectively. Strikingly, the organs of only one mouse in group D con-

tained bacteria, and the numbers of bacteria found in its liver and spleen were 2 × 101 and 1 × 102 CFU/g, respectively. No bacteria were detected in the organs of group C mice. In order to examine pathological changes in the infected organs of these mice, gross lesions and histological sections of liver and spleen were investigated from each group. As shown in Table 1, gross lesions of the liver and spleen were rarely observed in group C, whereas gross lesions, including paleness, hypertrophy and white coloration, and red and/or yellow spots, were observed in the liver and spleen from other groups. The examination of liver tissue sections from group C mice found no necrosis or degeneration of hepatocytes, or any neutrophil infiltration, whereas liver sections from the challenged control group mice showed an increased prevalence of necrotic tissue, degeneration of cells, and neutrophil infiltration. Spleen tissue sections from group C mice were normal, whereas spleen sections from control group mice exhibited notable necrosis and the degeneration of splenocytes. 4. Discussion Previous studies have suggested that Lon regulates epithelial cell invasion and is required for intestinal pathogenesis [2,19,20]. It also potentially regulates SPI-2, effects macrophage survival and is required for systemic infection

Table 1 Bacteriological and pathological examinations of the mouse organs after challenge. Groupa

Organ

No. of infected organb

No. of isolated bacteria (CFU/g)

Gross lesion

A

Liver Spleen

5/5 5/5

2 × 103 ± 9.3 × 102 8 × 103 ± 1.1 × 103

Pale, hypertrophy, many white spots Pale, hypertrophy, many white spots

B

Liver Spleen

5/5 5/5

4 × 105 ± 1.5 × 103 3 × 103 ± 1.5 × 103

Pale, hypertrophy, many white spots Pale, hypertrophy, white and yellow spots

C

Liver Spleen

0/5 0/5

NDc ND

Normal Normal

D

Liver Spleen

1/5 1/5

2 × 101 1 × 102

Slightly pale and hypertrophy Slight hypertrophy

E

Liver Spleen

5/5 5/5

8 × 106 ± 1.8 × 106 2 × 107 ± 5.2 × 106

Pale, hypertrophy, many white and yellow spots Pale, hypertrophy, many white and yellow spots

a Group A mice were single administered orally; group B mice were orally primed and boosted intramuscularly; group C mice were both primed and boosted by the oral route; group D mice were intramuscularly primed and boosted orally, and group E mice were administered with PBS-sucrose as a control. b The number of infected organs/the number of tested organs. c Not detected.

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in mice [2,19,20]. Its deletion renders cells sensitive to hydrogen peroxide (which contributes to the bactericidal capacity of phagocytes) [2,20]. The maintenance of the cellular envelope is critically important for the survival of Gram-negative bacteria [21,22]. CpxR is a central regulator of the maintenance of the bacterial envelope, and integrates many input signals to coordinate gene expression and mediate bacterial virulence [21,23,25]. Based on this, we hypothesized that S. Typhimurium lon and cpxR gene deletion would result in bacterial attenuation, and despite leading to increased epithelial cell invasion, would not cause systemic infection or enterocolitis. Salmonella is a facultative intracellular pathogen, and a phagocyte associated Th1 response begins a few hours post-infection, eventually resulting in the elimination of Salmonella [3,14,39]. Macrophages are critical to the control of Salmonella infection. However, bacterial uptake is not directed by the macrophage alone, and is enhanced after opsonization of Salmonella with antibodies including serum IgG and secretory IgA [5,6,14,40]. In the intestinal lumen, these antibodies block a secondary infection of Salmonella by inhibiting bacterial adhesion and invasion of epithelial cells and M cells [3,6,14,31,32]. In this study, in order to evaluate the vaccine potential of a double mutant Salmonella strain (loncpxR), a total of 105 female BALB/c mice were immunized by multiple administration routes with live and/or killed bacteria. We investigated protective immunity, as measured by levels of serum IgG, IgA and mucosal sIgA, and by the induction of cytokines such as TNF-␣, IL-12, INF-␥ and IL-4. IgG levels against Salmonella antigen were significantly increased in all groups from 3 weeks till 6 weeks PPI compared to control group (administered with PBS) (P < 0.01). The sIgA levels from the vaginal secretions of all groups were significantly increased compared to control mice from 3 weeks PPI (P < 0.05) until the end of the study (P < 0.01). This suggests that our vaccine candidate can effectively induce both systemic and mucosal immune responses regardless of the administration route. In addition, the data also shows that serum IgG2a titers were higher than that of IgG1 for all immunized groups (P < 0.05), suggesting that this vaccine candidate induces a stronger Th1 type cellular immune response. The concerted actions of several cytokines, including TNF-␣, IL-12 and IFN-␥, are necessary for the suppression of bacterial growth in the plateau phase of Salmonella infection [14,27,33]. TNF-␣, a proinflammatory cytokine, is generally an indicator of the early onset of disease and stimulates IL-12 secretion during late infection [27]. IL-12, another proinflammatory cytokine, is believed to induce IFN-␥ secretion by T cells and contributes to the resolution of Salmonella infections [14,27]. Further, IL-12 has been shown to play a crucial role in the development of Th1 type immunity [1]. IFN-␥ stimulates activation of macrophages and promotes the elimination of Salmonella in macrophages [14]. Although Salmonella vaccines need to generate a Th1 type cellular response, the production of IFN-␥ [4,26,27,34], and IL-4 is also necessary for the secretion of mucosal IgA and protection against secondary Salmonella infections [34]. In other words, to control Salmonella infection, the host immune system needs to

release not only TNF-␣ and IL-12, but also INF-␥ and IL-4 [1,27,29]. Our results showed that serum IL-12 and IFN␥ levels of groups C and D were significantly higher in general throughout the experiment period, compared to that of control group. In addition, serum IL-4 level of group C remained significantly high until the end of experiment compared to that of control group. These results showed that immunization with oral booster more effectively induces the cytokines, which are related to cellular and humoral immune responses. Furthermore, serum TNF␣ of all immunized mice were significantly higher. These results indicate that the secretions of proinflammatory cytokines, which are necessary to eliminate Salmonella infection, were also significantly increased by our vaccine candidate. Strikingly, all mice from group C were fully protected against salmonellosis after challenge with a virulent wild type strain, while all control mice, 14.3% of mice from group A and 19% of mice from group B died after challenge. The virulent strain was isolated from the livers and spleens of all mice except group C after challenge. In addition, gross lesions including hypertrophy, necrosis and degeneration of hepatocytes and splenocytes were observed in the livers and spleens from all mice except group C. These results demonstrate that for the strain described here immunization using both oral primer and oral booster is the most efficient vaccination strategy. In order to examine fecal shedding of the vaccine strain in mice, we attempted to isolate the bacteria daily from fecal samples of 5 randomly selected mice from each immunized group for 3 weeks after vaccination. No vaccine strain was found from any fecal samples, with the exception of one vaccinated mouse, which indicates that the vaccine strain is rarely excreted into feces and would not contaminate the environment. No adverse reactions such as diarrhea or weight loss were observed in the vaccinated mice during the experimental period. This indicates that the locpxR strain can be a very safe vaccine for both the host and the environment. In conclusion, our results in the present study indicate that the vaccine candidate can be a safe and effective tool for prevention of Salmonella infections. It can induce a robustly protective cellular/humoral immune responses, and lead to the production of inflammatory cytokines. Booster immunization, especially via oral administration, is necessary to optimize its protection against salmonellosis. In addition, S. Typhimurium infection in mice can induce enteric fever with symptoms similar to those of typhoid fever by S. Typhi infection in humans. Therefore, our results may provide basic information on development of novel vaccine for prevention of typhoid fever in humans. Conflict of interest The authors declare no conflict of interest. Acknowledgments This work was supported by Mid-career Researcher Program through NRF grant funded by the MEST (no. 20100000325), and the Brain Korea 21 Project in Republic Korea.

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