Mice orally vaccinated with Edwardsiella tarda ghosts are significantly protected against infection

Vaccine 27 (2009) 1571–1578

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Mice orally vaccinated with Edwardsiella tarda ghosts are significantly protected against infection Xuepeng Wang a,b , Chengping Lu a,∗ a b

Key Lab Animal Disease Diagnostic & Immunology, Ministry of Agriculture, Nanjing Agricultural University, Nanjing 210095, PR China College of Animal Science and Technology, Shandong Agricultural University, Taian 271018, PR China

a r t i c l e

i n f o

Article history: Received 25 November 2008 Received in revised form 1 January 2009 Accepted 3 January 2009 Available online 21 January 2009 Keywords: Edwardsiella tarda Bacterial ghosts Oral vaccination Cellular immunity Humoral immunity

a b s t r a c t Bacterial ghosts may be generated by the controlled expression of the phiX174 lysis gene E in Gramnegative bacteria and they are intriguing vaccine candidates since ghosts retain functional antigenic cellular determinants often lost during traditional inactivation procedures. The Edwardsiella tarda ghost (ETG) vaccine was prepared using this technology and tested in vaccination trials. Control groups included mice immunized with formalin-killed E. tarda (FKC) or mice treated with phosphate-buffered saline (PBS), respectively. The results showed that serum IgA and IgG antibody titers were significantly higher in the ETG-vaccinated group compared to the other groups. In addition, CD8+ T cell counts in peripheral blood were elevated in the ETG groups. Most important, ETG-immunized mice were significantly protected against E. tarda challenge (86.7% survival) compared to 73.3 and 33.3% survival in the FKC-immunized and PBS-treated control, respectively, suggesting that an ETG oral vaccine could confer protection against infection in a mouse model of disease. © 2009 Elsevier Ltd. All rights reserved.

1. Introduction Edwardsiella tarda (E. tarda) is an intracellular, rod-shaped Gramnegative, non-capsulated, motile, facultative anaerobic bacteria that was first isolated from a pond-cultured eel by Hoshina [1]. E. tarda is widely distributed in aquatic environments [2,3] and is infectious to variety of animals including humans [4–7], fish [8–13], amphibians [14,15], reptiles [14,16,17] and birds [18,19]. This organism’s versatility with respect to the broad-range of hosts highlight the importance of developing strategies for the protection of both animals and humans from E. tarda infections. In recent years, chemotherapy has been used effectively in controling fish infections [20], however, there is significant concern regarding food safety following chemotherapeutic interventions in addition to the danger of selecting for antibiotic-resistant E. tarda isolates which have been reported worldwide [21–23]. These concerns have prompted the development of novel vaccination strategies for the control of E. tarda infections. Over the last decade, vaccination has become an important prevention strategy against numerous infectious agents affecting humans and farm animals [24,25]. Although, the development of E. tarda vaccines has been attempted, their efficacy against challenge has been inconsistent [26–29]. The commercial vac-

∗ Corresponding author at: College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, PR China. Tel.: +86 25 8439 6517; fax: +86 25 8439 6517. E-mail addresses: [email protected] (X. Wang), [email protected] (C. Lu). 0264-410X/$ – see front matter © 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2009.01.002

cines presently available consist of heat- or formalin-inactivated E. tarda formulations, however, these strategies can affect the physiochemical/structural properties of surface antigens thereby negatively affecting the development of protective immunity [30,31]. Bacterial ghosts are empty cell envelopes that are produced, for example, by the controlled expression of the phiX174 lysis gene E in Gram-negative bacteria. Expression of lysis gene E leads to the formation of trans-membrane tunnels which consequently lead to the loss of cytoplasmic contents [31,32]. The resulting bacterial ghosts have been demonstrated to retain functional and antigenic determinants of the envelope [33]. Even highly sensitive and fragile structures such as pili are well protected following ghost formation as demonstrated during the generation of Vibrio cholerae ghosts expressing the toxin-coregulated pilus [34]. These data suggested that ghosts could be used in place of the traditional live-attenuated vaccine preparations to elicit immunity [25,35–37]. During the development of conventional, nonviable whole-cell vaccines formulations antigenic epitopes can be potentially altered as a result of the inactivation process that is absent in the generation of bacterial-ghosts [30,31]. Furthermore, inactivated whole-cell vaccine preparations and subunit vaccines are often less immunogenic, necessitating the use of adjuvants which may have significant negative side effects on the host [25,30,31]. In contrast, bacterial ghost formulations possess inherent adjuvant-like properties thereby negating the need of additional immunostimulatory formulations [25]. Because economic losses due to E. tarda infections of fish are significant and human infections can result in gastroenteritis, vari-

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ous vaccine candidates have been developed and tested in fish and mouse models of disease [38–40]. Although, E. tarda ghosts vaccine have been reported to confer protection in Oreochromis mosambicus (tilapia) and the Paralichthys olivaceus (olive flounder) infection models [32,41], the fish immune system is not conducive to testing cellular immunity. In this study we demonstrate that ETG vaccination stimulated both cellular and humoral immunity and conferred significant protection against E. tarda infection in a mouse model of disease.

at 72 ◦ C for 7 min. The PCR product was visualized on 1.5% agarose gels stained with ethidium bromide, purified with a gel extraction kit (Takara, Dlian, China) and cloned into the PMD19 T easy vector (Takara) and designated PMD19-E. Following BamHI (Takara) and PstI (Takara) digestion of PMD19-E and PBV220, gene E was inserted into the PBV220 plasmid and designated pLysis E (Fig. 1B). The pLysis E plasmid was first propagated in the E. coli strain DH5␣ (Invitrogen, CA, USA) at 28 ◦ C, and then extracted using a plasmid extraction kit (Takara).

2. Materials and methods

2.2. Bacterial strains and culture conditions

2.1. The pLysis E plasmid

E. tarda strain CD (16S ribosomal RNA gene GenBank accession number EF467289) was isolated in 1991 from a moribund child during an Edwardsiellosis outbreak on a farm in China. E. tarda Strain CD cells were grown in tryptic soy broth (TSB) (Oxoid Ltd., Basingsoke, Hampshire, England) at 28 ◦ C and E. coli DH5␣ were grown in Luria Broth (LB) (Oxoid Ltd.) at 37 ◦ C. Transformed E. tarda cells were grown in TSB containing 100 ␮g/ml ampicillin (Sigma, MO, USA) and transformed E. coli DH5␣ strain cells were grown in LB containing 100 ␮g/ml ampicillin at 28 ◦ C. Incubation temperatures for repression and expression of the lysis gene in transformants were 28 ◦ C and 42 ◦ C, respectively. Growth and lysis of bacterial cultures was monitored by measuring the optical density at 600 nm (OD600 ), and colony-forming units (CFUs) were determined by plating serial dilutions onto tryptic soy broth agar (TSA) and counting colonies.

The plasmid pElysis [42] carrying the E lysis gene and the PBV220 (Fig. 1A) plasmid carrying the PR/CI857 regulatory system were used in the generation of the E. tarda ghosts. For the construction of the lysis plasmid, the E gene was amplified by PCR from the pElysis plasmid with oligonucleotides E-Forward (5 -GGATCCATGGTACGCTGGACTTTG-3 ) and E-Reverse (5 -CTGCAGTCACTCCTTCTGCACGTA-3 ) containing the BamHI and PstI restriction enzyme sites, respectively (italicized). PCR amplification steps consisted of one 3 min cycle at 95 ◦ C, 30 cycles at 94 ◦ C for 30 s, 58 ◦ C for 30 s and 72 ◦ C for 45 s with a final extension step

2.3. Transformation of bacteria E. tarda were inoculated into 100 ml TSB and grown at 28 ◦ C to an OD600 of 0.45 with vigorous agitation (200 rpm). Competent E. tarda cells were transformed with pLysis E and mixtures of E. tarda and pLysis E were incubated on ice for 30 min and heat-shocked at 42 ◦ C using a circulating water bath for 90 s [43]. After cooling, 1 ml of TSB medium was added to the cells and incubated for 1 h at 28 ◦ C. After incubation, transformed E. tarda were plated onto TSA ampicillin (100 ␮g/ml) plates. 2.4. Production of E. tarda ghosts and generation of formalin-killed E. tarda

Fig. 1. Structure of the PBV220 and the pLysis E plasmids used for the production of ETG. The plasmid harbors an E lysis cassette, consisting of a lysis gene E (gene E), the leftward Lambda promoter (PR), the rightward Lambda promoter (PL), a temperature sensitive repressor CI857 (CIts857), and an ampicillin resistance gene (Ampr).

E. tarda harboring pLysis E were inoculated into 300 ml TSB broth containing 100 ␮g/ml ampicillin and incubated at 28 ◦ C. When the cultures reached an OD600 of 0.3–0.4 E gene expression was induced by a temperature shift from 28 to 42 ◦ C and at different time points after expression the optical density was measured until no further decreases were detected. After lysis was completed, ghosts were collected by centrifugation and washed three times with PBS (pH 7.2) at 1/3, 1/6, and 1/12 of the starting culture volume. The final cell pellet was resuspended in 20 ml distilled water and freeze-dried for 24 h. Ten milligrams of the lyophilized ghost preparations were inoculated in TSB, incubated for 1 week at 28 ◦ C and analyzed for living-cells by plating on TSA plates. The pLysis E plasmid was extracted from the final product using a plasmid extraction kit (Takara), and the Amp gene was amplified by PCR from the final product with oligonucleotides Amp-Forward (5 -TTTCCGTGTCGCCCTTAT-3 ) and Amp-Reverse (5 GCAACTTTATCCGCCTCC-3 ). PCR amplification steps consisted of one 3 min cycle at 95 ◦ C, 30 cycles at 94 ◦ C for 40 s, 53 ◦ C for 40 s and 72 ◦ C for 50 s with a final extension step at 72 ◦ C for 7 min. The PCR product was visualized on 1.5% agarose gels stained with ethidium bromide. Morphological features of E. tarda and ETG were examined by scanning electron microscopy (Hitachi S-2400) and transmission electron microscopy (7650; Hitachi) as previously described in Refs. [43–45].

X. Wang, C. Lu / Vaccine 27 (2009) 1571–1578

For FKC preparation, formalin was added to 24 h cultures of the bacterium to a final concentration of 0.5%. After a 24-h incubation, cells were washed three times with PBS and resuspended in 10 ml PBS and the suspensions plated on TSA to confirm inactivation. FKC was stored at 4 ◦ C until use. 2.5. Immunization protocol and challenge infection Inbred mice (ICR, National Rodent Laboratory Animal Resources, Shanghai Branch, China) were housed with access to food and water ad libitum. Mice were divided into three immunization groups (A–C, n = 30 for each group) and mice from all groups were fasted for 24 h prior to oral immunization. Ten minutes before immunization, 30 ␮l of 10% sodium hydrocarbonate was administered orally [25]. Mice from groups A and B mice were immunized on d0 and d21 with either ETG or FKC. Group C mice were immunized with PBS (negative control) at similar times. For group A immunizations, the oral dose was 1 mg of freeze-dried ETG (corresponding to 5 × 109 dead bacterial ghosts) in 30 ␮l of PBS applied through a polyethylene catheter and group B mice were immunized with FKC (5 × 109 CFU dead bacterial) in a similar fashion. On d35 post-primary immunization mice from all groups divided into six subgroups (1–6, n = 5 for each subgroup) were intragastrically challenged with 2 × 108 E. tarda/mouse (100 ␮l). Dead mice were necropsied and liver samples homogenized and plated onto Salmonella shigella agar (SSA, Luqiao, Beijing, China) and E. tarda infection confirmed by quantifying colonies with black pigmentation. Mice were lightly anesthetized with methoxyflurane and serum collected from the retro-orbital sinus at d7, d14, d21, d28, d35, d42 and d49 for the analysis of anti-E. tarda antibody responses. 2.6. Antibody response assessment The presence of specific immunoglobulin A (IgA) and immunoglobulin G (IgG) antibodies against E. tarda following immunization was determined by an enzyme-linked immunosorbent assay (ELISA). Briefly, 100 ␮l carbonate–bicarbonate buffer (pH 9.6) containing 1 × 105 FKC were added to respective microtiter plate wells and incubated for 20 h at 37 ◦ C. The plates were washed three times with PBS containing 0.05% Tween 20 (PBS-T) and blocked for 24 h at 4 ◦ C with blocking buffer (0.5% BSA in PBS-T). After the plate was washed three times, sera from immunized and control mice, at a starting dilution of 1:40 in PBS-T, were titrated through a twofold dilution series. Plates were incubated at 37 ◦ C for 1 h and washed four times with PBS-T then probed with 50 ␮l horseradish peroxidase-conjugated goat anti-mouse IgG (1:10,000) or IgA (1:5000) (Sigma) for 1 h at 37 ◦ C. Plates were washed four times with PBS-T and binding visualized by adding TMB (Tiangen, Beijing, China) according to the manufacturer’s instructions (100 ␮l/well). The plates were incubated at room temperature for 20 min and the reaction stopped with 100 ␮l of 2 M H2 SO4 and the absorbance read at 450 nm. The results were plotted as the mean OD ± S.D. of triplicate wells versus dilution. Titers at halfmaximal OD were determined by linear interpolation between the measured points neighboring the half maximal OD. Linear interpolation was calculated using the logarithm of the titer values as previously described by Fritz et al. [46] and Van et al. [47].

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were incubated with appropriately diluted fluorescein isothiocyanate (FITC)-labeled anti-CD3 (Invitrogen) and phycoerythrin (PE)-labeled anti-CD8a (Invitrogen) or FITC-labeled anti-CD3 and PE-labeled anti-CD4 (Invitrogen) for 30 min at room temperature. The samples were then incubated with 2 ml flow cytometry (FCM) lysing solution (Invitrogen) for 10 min at room temperature. After incubation, all samples were washed by centrifugation two times with PBS and resuspended in 0.5 ml of PBS. Analysis was performed using a BD FACScan equipped with Cell Quest software on the same day. A total of 10,000 events per sample were recorded and the absolute number of positive cells for the respective markers per was determined. 2.8. Statistical analysis Statistical significance was determined by ANOVA analysis. Differences were considered significant at P < 0.01 or P < 0.05. 3. Results 3.1. Production and characterization of bacterial ghosts Lysis plasmid pLysis E (Fig. 1B) was successfully constructed and cotransformed into E. tarda Strain CD and ghosts produced by protein E-mediated lysis of bacterial cells. Production of ghosts in E. tarda transformants was carried out using temperature increments to activate the pLysis E gene (up to 42 ◦ C). The OD of the transformed E. tarda cultures decreased during the first hour after induction of gene E expression as a function of cell lysis and remained constant for the next 8 h until the bacterial ghosts were harvested (Fig. 2). The number of CFU decreased after expression of gene E until the time of ghost harvest (Fig. 2). Approximately 9 h after lysis induction the viability of the culture decreased by 107 orders of magnitude. After washing and freeze-drying, plating of the material revealed no growth, confirming E. tarda inactivation. Although, the pLysis E plasmid was not found in the final product, the Amp gene was found by PCR (Fig. 3). Electron microscopic analysis of protein E-lysed E. tarda cells revealed no gross alterations in cellular morphology compared to unlysed cells (Fig. 4B and D) except for the lysis pore (arrow, Fig. 4A). The morphology of the cell, including all cell surface structures, were unaffected by lysis. Pores ranging from 200 to 500 nm in diameter were observed in E. tarda ghosts by scanning electron microscopy (Fig. 4A). The loss of cytoplasmic material and structural integrity were observed in E. tarda ghosts by transmission electron microscopy (Fig. 4A and C).

2.7. Flow cytometric analysis T cell markers were examined by collecting peripheral blood lymphocytes (PBL) from 100 ␮l blood taken from the retro-orbital sinuses of methoxyflurane-anesthetized mice from each group at various time points postvaccination, mixed with 20 ␮l heparin (140 IU/ml) and divided into two tubes. The blood cells

Fig. 2. Growth and lysis curves of naive E. tarda strains CD and E following induction. The CD strain harbors the plysis E plasmid with expression of gene E being temperature induced. Induction occurred at time zero, when the cultures were shifted from 28 to 42 ◦ C. Both OD600 (left y-axis) and CFU (right y-axis) values are shown in the graph.

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X. Wang, C. Lu / Vaccine 27 (2009) 1571–1578 Table 1 Survival rates following vaccination and E. tarda challenge.a . Group

Ac Bf Cg a b c d e f g

Fig. 3. The presence of the Amp gene and the pLysis E plasmid in the final ETG vaccine. (1) The pLysis E plasmid, (2) Amp gene, (3) Marker.

3.2. ETG and FKC immunizations and E. tarda infections After the first and the second oral immunizations with ETG, FKC or PBS, the mice behaved normally and did not exhibit any signs of

Subgroups (n = 5)b

Survival rate (%)

1

2

3

4

5

6

0 1 2

2 1 3

0 2 4

0 2 4

1 0 3

1 2 4

86.7d , e 73.3d 33.3

Mice were infected intragastrically. Number of dead mice/subgroup postchallenge. Mice immunized with ETG twice (d0 and d21). Difference between group A or B and group C, p < 0.01. Difference between group A and B, p < 0.05. Mice immunized with FKC twice (d0 and d21). Mice immunized with PBS twice (control for group A and B).

illness. Following a challenge infection with E. tarda the mice were monitored daily for 2 weeks postchallenge. Disease manifestations appeared between days 1 and 5 postchallenge and included reduced activity, lethargy, anorexia, convulsions and death. All mice were euthanized 14 days postchallenge. Survival differences (p < 0.01–0.05) were observed between the vaccination groups (Table 1). ETG-immunized mice showed the highest survival rates (26/30, 86.7% survival) and FKC-immunized mice (22/30, 73.3% survival) were better protected than PBS-treated

Fig. 4. Evaluation of E. tarda and E. tarda ghosts by SEM (A and B) and TEM (C and D). (A) E. tarda ghosts. Arrow shows the trans-membrane lysis tunnel. (B) Naive E. tarda examined by SEM. (C) Loss of cytoplasmic material of E. tarda ghosts. (D) Naive E. tarda examined by TEM.

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nized with FKC or PBS (the highest titers at half maximal OD were 7502, 4532 and 2702 in E. tarda-immunized mice). FKC-immunized mice did not show significant differences (p > 0.05) in IgG antibody titers when compared PBS-immunized mice in the whole observed period. 3.4. Flow cytometry

Fig. 5. Anti-E. tarda titers in mice following oral immunization with either ETG, FKC or PBS and intragastrically challenge with E. tarda. Bars represent the half-maximal titer ± S.D. of four mice analyzed in three separate experiments. Serum IgA (A) and IgG (B) titers to E. tarda over time. Mice from all groups were challenged intragastrically with 2 × 108 CFU E. tarda on d35. * p < 0.05 for groups A or B compared to group C; ** p < 0.01 between groups A or B compared to group C; † p < 0.05 between groups A and B; ‡ p < 0.01 for between groups A and B.

controls (10/30, 33.3% survival). E. tarda infection was confirmed in all dead mice by finding black pigments on SSA. 3.3. Antibody response analysis The IgA- and IgG-specific E. tarda antibody responses were examined by ELISA (Fig. 5). Specific anti-E. tarda IgA and IgG antibodies were detected in the serum of mice immunized with either ETG or FKC. No antibody reactivity could be detected in the serum collected from control mice. Animals immunized twice with either ETG or FKC on days 0 and 21 (groups A and B) showed the highest IgA and IgG titers in serum samples collected on d35 postvaccination. Mice immunized with ETG showed significantly higher IgA titers than mice immunized with either FKC or treated with PBS (p < 0.01) and the highest titers at half maximal OD were 763, 426 and 10. ETG-immunized mice also had elevated IgG titers compared to mice immunized with either FKC (p < 0.05) or PBS (p < 0.01) and the highest IgG titers at half maximal OD were 2545, 1122 and 127. All antibody determinations showed a statistical IgA or IgG (p < 0.01) increase and mice immunized with ETG developed significantly (p < 0.05) higher IgA antibody titers than mice immunized with FKC or PBS (the highest titers at half maximal OD were 1317, 712 and 142 for E. tarda-vaccinated mice). ETG-immunized mice had significantly (p < 0.05) higher IgG antibody titers than mice immu-

The CD3+, CD4+ and CD8+ T-cell subpopulations in PBL from the respective immunization groups were determined by flow cytometry. Different CD3+, CD4+, CD8+ percentages were observed in PBL from ETG, FKC immunized and PBS treated mice in the whole period (Table 2). Animals immunized twice with ETG on days 0 and 21 (group A) had the highest percentages of CD3+ (62.09%) and CD3+ CD4+ CD8− (44.20%) lymphocytes in peripheral blood samples on d35 compared to animals immunized twice with FKC which had the highest percentages on day 28 for the same cell populations (57.71 and 43.57%, respectively). The percentage of CD3+ CD4−CD8+ lymphocytes in peripheral blood reached its highest peaks on d7 in ETG-immunized mice compared to FKCimmunized on d14 and PBS-treated mice which had their highest CD3+ CD4−CD8+ counts on d35 (18.72, 15.01 and 11.80%, respectively). Analysis of the T cell populations in the respective vaccination groups revealed significant changes in the profile of the T cell populations in ETG-vaccinated mice compared to FKCimmunized and PBS-treated mice (Table 2). First, ETG-immunized mice developed statistically (p < 0.01) higher frequencies of CD3+, CD3+ CD4+ CD8− and CD3+ CD4−CD8+ PBL than control mice, whereas FKC-immunized mice developed significantly (p < 0.05) higher frequencies of CD3+ and CD3+ CD4+ CD8− in PBL than control mice (no differences in the CD3+ CD4−CD8+ population were observed between FKC-immunized mice and controls). Second, ETG-immunized mice developed (p < 0.05) higher frequencies of CD3+ cells than FKC-immunized mice. Third, significant increases in the CD3+, CD3+ CD4+ CD8− and CD3+ CD4−CD8+ populations in PBL were observed in ETG-immunized mice on d7, i.e., increases of 75, 105 and 97% over d0 readings. In contrast, the percent increase in FKC-immunized mice for the same cell populations was 8, 45, 0%. Fourth, a drop in the percentages of CD3+, CD4+ and CD8+ cells was observed in group A mice following infection 1 week later on d42, i.e., decreases of 48, 54 and 35% compared to values recorded on d35. In contrast changes in CD3+, CD4+ and CD8+ cell populations in mice from groups B and C were not evident. Finally, an evident increase in the percentages of CD3+/CD4+/CD8+ cells was observed in group A mice on d49 (2 weeks postinfections), i.e., increases of 111, 123 and 93% over the percentages recorded on d42 whereas the percentages of CD3+/CD4+/CD8+ cells in group B mice increased by only 22, 10 and 41% and in group C mice they increased by 17, 2.5 and 27%, respectively. 4. Discussion Although E. tarda ghost vaccines have been reported to protect tilapia (Oreochromis mosambicus) and the olive flounder (Paralichthys olivaceus) against E. tarda infections [32,41], determining the capacity of this vaccination strategy to elicit T cell-mediated immunity cannot be carried out in fish models. We therefore developed an infection model in mice that allowed us to examine both humoral and cellular response to E. tarda and this study is the first to determine immune response in mouse following oral vaccination using E. tarda ghosts. Various techniques are available for bacterial inactivation, including physical or chemical processing treatments such as heating or formalin fixation used in the production of nonviable, whole-cell vaccine formulations. However, these vaccines have

47.95 ± 15.91 30.12 ± 11.91 13.25 ± 3.76

h

f

g

e

c

d

a

b

Ch

Values represent the ratios (the mean ± standard deviation) of T cell subsets from three mice per time point. Mice were immunized on d0, challenged on d21 and infected on d35. ETG-vaccinated mice. Difference between group A or B and group C, p < 0.01. Difference between group A and B, p < 0.05. Difference between group A and B, p < 0.01. Difference between group A or B and group C, p < 0.05. FKC-vaccinated mice. PBS-treated mice.

40.93 ± 2.00 29.38 ± 0.43 10.42 ± 1.89 46.26 ± 9.7 33.31 ± 9.45 11.8 ± 2.08 45.71 ± 10.52 32.96 ± 9.95 11.53 ± 2.42 40.64 ± 11.26 30.42 ± 11.16 11.22 ± 2.56 33.31 ± 6.59 20.27 ± 1.33 10.81 ± 2.28 31.98 ± 5.13 19.51 ± 0.19 9.50 ± 0.18 CD3+ CD4+ CD8+

Bg

38.79 ± 8.36 26.7 ± 4.75 10.2 ± 0.87

52.96 ± 17.96 30.24 ± 14.45 20.66 ± 2.90 43.3 ± 3.99 27.33 ± 1.24 14.62 ± 4.38f 44.10 ± 8.15 33.44 ± 4.67 11.57 ± 5.27 57.71 ± 7.77f 43.57 ± 5.45 14.01 ± 3.73 47.39 ± 6.72 37.3 ± 3.69 10.05 ± 1.95 34.63 ± 14.11 28.27 ± 9.04c 8.05 ± 4.07 31.98 ± 5.13 19.51 ± 0.19 9.50 ± 0.18 CD3+f CD4+f CD8+

Ab

48.98 ± 13.97 34.47 ± 12.50 15.01 ± 1.75f

d42

32.05 ± 3.73c , e 20.21 ± 1.89c , e 11.25 ± 1.78 62.09 ± 5.14c , e 44.20 ± 0.94 17.25 ± 1.49

d35 d28

50.57 ± 11.2 39.72 ± 9.15 13.09 ± 2.56 56.23 ± 6.65c , d 42.37 ± 3.94f 13.44 ± 3.43

d21

56.00 ± 4.94c , e 39.92 ± 0.73c , d 18.72 ± 3.23f , d 31.98 ± 5.13 19.51 ± 0.19 9.50 ± 0.18

52.06 ± 4.97f 34.98 ± 4.13 16.82 ± 3.49c

d14 d7 d0

CD3+c , d CD4+c CD8+c

Percentage of total T cell subsets (mean ± S.D.)a T cell subsets Group

Table 2 T cell subset profile following vaccination.

67.75 ± 8.58f 45.09 ± 6.82 21.69 ± 5.16f

X. Wang, C. Lu / Vaccine 27 (2009) 1571–1578

d49

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been shown to be less immunogenic when delivered orally since inactivation procedures often alter the antigenicity of potentially protective epitopes making then non-viable in an immunologic context [25]. Vaccination efficacy against enteric pathogens has been improved by combining different vaccines with adjuvants whose potential negative side effects are well defined [25]. One advantage of using bacterial ghosts is the preservation of surface antigens in their natural conformations [33], e.g., even highly sensitive and fragile structures like pili are well preserved [34]. Furthermore, conventional adjuvants need not be utilized in combination with bacterial ghost formulations since these preparations possess adjuvant-like properties conferred by molecules such as LPS, lipids, and peptidoglycans [25]. The data presented in this report demonstrated that formation of E. tarda ghosts as a result of lysis gene E expression followed by freeze-drying resulted in effective E. tarda inactivation. Although the intact antibiotic resistance gene was found in the ETG, as earlier reported it can be completely degraded by nucleotidase [25]. In addition the freeze-dried ghosts were well tolerated by mice and have the potential to be used as an oral vaccine in other animals or humans. Mohanty and Sahoo [48] reported that E. tarda infections in humans were primarily associated with gastroenteritis and wound infections and E. tarda-infected fish presented with excessive mucus secretion in a few cases, suggesting that humoral mucosal immunity to E. tarda in these different systems was likely to be needed for protection. As noted earlier, immunity to enteropathogens is mediated by mucosal immune mechanisms [49]. In the infection model presented in this report we found that ETG orally vaccinated mice had higher serum IgA antibody titers specific to E. tarda and elevated CD4+ counts in PBL than FKC-immunized or PBS-treated controls. Furthermore, mice immunized with ETG showed statistically higher IgG antibody titers than mice immunized with FKC over the course of the study. In contrast FKC-immunized mice did not develop significantly different IgG antibody titers compared to PBS-treated controls. All these results suggested that ETG significantly stimulated antibody production and based on previous observations likely stimulated mucosal immunity in the present study [25]. Most important, E. tarda ghosts conferred immunity against a lethal challenge given on d35 (2 weeks post-secondary boost). ETG-immunized mice were significantly protected compared to FKC-immunized and PBS-treated controls suggesting that ETG effectively elicited protective immune responses. The overall percentage of CD3+, CD4+ and CD8+ T-cell subpopulations increased markedly in response to immunization with ETG, especially the percentage of CD8+ T-cell subpopulations. These results confirmed that oral ETG vaccination significantly modified the T cell population profile, especially the CD8+ T-cell subpopulations. Recently, it was shown that immunization with E. coli ghosts elevated interferon (IFN)-␥ production by spleen cells and that recombinant V. cholerae ghosts expressing Chlamydia trachomatis antigens induced a Chlamydia-specific Th1 response defined by splenic T cell production of IFN-␥ [25,50]. In the model study presented here, we found that the percentages of CD4+ and CD8+ T cells in vaccinated mice increased, which may have enhanced IFN␥ production or the INF-␥ production increased the respective T cell populations observed. Previous reports demonstrated that elevated CD4+ and CD8+ T cells in vaccinated mice were associated with IFN-␥ secretion that could result in the up-regulation of MHC I surface expression. Increased MHC class I expression could in turn enhance the CD8+ T cell responses to respective immunogens [51–53]. In vitro it was shown that bacterial ghosts could be efficiently processed by dendritic cells and macrophages [54,55]. In all, these results suggested that ETG elicited cellular immune response in mice.

X. Wang, C. Lu / Vaccine 27 (2009) 1571–1578

Bacterial ghosts are viable vaccine candidates that can function alone (described here) or as carriers of drugs, nucleic acids, viral and/or bacterial antigens [56,57] comprising multivalent vaccines designed to protect against animal and human diseases. Ghost formulations represent a new, improved nonliving bacterial vaccine delivery strategy that is safe and highly immunogenic. The data presented in this report suggest that oral immunization with ETG provided efficacious immune protection against infection, suggesting that it is possible to develop an E. tarda-based bacterial-ghost vaccine for use in animals or humans. Acknowledgements We thank Y.Y. Wang and R.B. Cao for providing the E. tarda strain CD, and the plasmid PBV220, respectively.

[26]

[27] [28] [29]

[30]

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