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Original Paper	Pdf file on Synergy omments
Acta Biochim Biophys Sin 2006, 38: 844-856
doi:10.1111/j.1745-7270.2006.00232.x

Removal of Antibiotic Resistance of Live Vaccine Strain Escherichia coli MM-3 and Evaluation of the Immunogenicity of the New Strain

Sheng-Ling YUAN, Peng WANG, Hao-Xia TAO, Xiang-Xin LIU, Yan-Chun WANG, De-Wen ZHAN, Chun-Jie LIU, and Zhao-Shan ZHANG*

Beijing Institute of Biotechnology, Beijing 100071, China

Received: May 18, 2006

Accepted: September 8, 2006

*Corresponding author: Tel, 86-10-63834140; Fax, 86-10-63833521; E-mail, zhangzs@nic.bmi.ac.cn

Abstract MM-3 was a live vaccine strain candidate for protecting neonatal piglets from diarrhea. Designed in the 1980s, a high degree of protection from colibacillosis was afforded to piglets in a challenge study and field trials, however MM-3 had a drawback of carrying the antibiotic resistance gene (chloramphenicol acetyltransferase gene, cat). The introduction of a host-plasmid balanced lethal system into the vaccine was a good idea to solve the problem. The l-Red recombination system was adopted in this study to realize the replacement of cat by aspartate-semialdehyde dehydrogenase gene (asd) in the plasmid pMM085. The new plasmid named pMMASD was introduced into an Escherichia coli strain c6097 and Salmonella typhimurium c4072 where the asd gene had been knocked out in their chromosomes. Cultured in an Erlenmeyer flask, expression levels of two antigens K88ac fimbriae and heat-labile enterotoxin B subunit (LTB) in cell lysate were similar among MM-3, c4072(pMMASD) and c6097(pMMASD). However, c4072(pMMASD) possessed the more effective secretion mechanism to transport LTB enterotoxin into culture liquid. The relatively higher stability of pMMASD in Salmonella typhimurium c4072 than that of pMM085 in MM-3 was determined both in vitro in the absence of selective pressure, and in vivo following oral inoculation. Oral immunization of BALB/c mice with c4072(pMMASD) or c6097(pMMASD) was sufficient to elicit IgA responses in mucosal tissues as well as systemic IgG antibody responses to the K88 fimbriae, while MM-3 failed to elicit specific antibody responses to K88 fimbriae in mucosal tissues. Among three live strains, only c4072(pMMASD) could develop strong humoral responses against LTB enterotoxin. The results suggest that c4072(pMMASD) is expected to be a promising live vaccine strain.

Key words l-Red recombination system; host-plasmid balanced lethal system; K88ac fimbriae; LTB enterotoxin; ETEC; live vaccine strain; Salmonella

Diarrheal disease caused by enterotoxigenic strains of Escherichia coli (ETEC) expressing the K88 (F4) fimbrial adhesin was a significant source of mortality and morbidity among newborn and weaned piglets. Recent vaccination strategies include the oral immunization of pregnant sows with a vaccine containing purified K88, K99 and 987P fimbriae, and B subunit of LT enterotoxin (LTB) [1], the utilization of killed recombinant strain expressing enterotoxigenic E. coli K88ac-ST1-LTB fusion protein [2], using Salmonella typhimurium as a vector to express the ETEC ST1-LTB and having a non-antibiotic selection marker (asd+) [3], or oral immunization of new born piglets with live avirulent E. coli vaccine strains expressing K88ac fimbriae and LTB subunit [4].

The use of live attenuated bacteria to deliver recombinant protein vaccine antigens was a promising approach to overcoming the difficulties and costs of vaccine antigen purification, and it enhanced presentation to the immune system [5]. Bacterial vectors may mimic natural infection and therefore interact with the mucosal, humoral and cellular compartments of the immune system [6]. Live bacteria vaccine might be superior to an orally administered bacterin if the attenuated strain colonized the intestine, thus stimulating the immune system over a longer period of time than that would be possible with killed bacteria [4]. When this approach was used, genes encoding vaccine antigens were expressed from multicopy plasmids in the live attenuated bacterial vectors.

LTB was heat-labile toxin B subunit and was able to bind to the ganglioside GM1 and other related receptors presented on enterocyte [7]. K88 fimbrial adhesins were filamentous surface appendages whose lectin (carbohydrate binding) activity allowed K88 ETEC to attach to specific glycoconjugates (receptors) on porcine intestinal epithelial cells, which was an essential step in the colonization of the small intestine by ETEC [8]. Live bacterial vector MM-3 was designed by Chen and colleagues [9] and played a function of protecting neonatal piglets from diarrhea. The strain MM-3 carried pMM085, which possessed ETEC immunogens of both LTB and fimbriae antigen K88ac. In a challenge study and field trials, a high degree of protection from colibacillosis was afforded to piglets [9,10].

MM-3 always lost pMM085 in vivo because the plasmid brought the chloramphenicol resistance gene (cat). Antibiotic pressure worked well for plasmid maintenance in vitro, however, it was not presented in vivo. As a result, under the circumstance of lacking selective pressure, plasmid would be quickly lost during cell division. Furthermore, the antibiotic resistance gene was no longer recommended in live bacterial vaccines [11]. Removal of cat gene in MM-3 seemed necessary. b-galactosidase as a tracking marker was also attempted in the work of Chen et al. [9]. Although this tracking marker was safer than the antibiotic resistance gene, the common problem of instability remained.

Stability of protective antigen expression in vivo was essential for recombinant vaccine efficacy. The use of a balanced-lethal host-vector system wherein the plasmid possessed a gene complementing to a chromosomal deletion mutation of a vital gene, such as a gene for cell wall synthesis or DNA stability and replication, ensured that the plasmid was maintained [5]. The other approaches to ensure retention of the plasmid included the "Hok-Sok" strategy [12] and "Operator-Repressor Titration" [13] which also resulted in bacterial cell death if the plasmid was lost.

A classic example of the balanced lethal host-vector system was the asd system. A deletion of the chromosomal aspartate-semialdehyde dehydrogenase gene (asd) was made in the bacterial vector, resulting in an absolute requirement for diaminopimelic acid, an essential component of the peptidoglycan of the cell wall of gram-negative bacteria that was not found in mammalian hosts. The asd gene was supplied on a plasmid and complemented the mutation. In the original work describing this system in serovar typhimurium, the plasmid was stable in the absence of any exogenous selective pressure in vitro or in vivo [14]. In this study, the host-plasmid balanced lethal system was adopted and the work included the replacement of the cat gene by the asd gene and the transformation of the new plasmid into the strain which the asd gene was knocked out.

Considering that the pMM085 was constructed in 1980s and it was difficult to find its accurate physical map, the l-Red recombination system was introduced to mediate the cat gene removal and the asd gene substitution. Recent advances in homologous recombination-based DNA engineering technology, termed "recombinogenic engineering" or "recombineering", have led to the development of highly efficient procedures with which chromosomal or plasmid DNA could be modified in E. coli by the introduction of mutations, such as single base substitutions, deletions and insertions. These methods were based on the rac-encoded RecET system or the bacteriophage l-Red recombination system, eliminating the need for in vitro manipulations using restriction enzymes or DNA ligases. The three proteins of the Red system, Exo, Beta and Gam, mediated recombination between a linear double-stranded DNA donor and its homologous target sequence by promoting double-strand break repair [15-17]. The results of this study also indicated that the l-Red recombination system was convenient and efficient to re-construct plasmid.

Recombinant attenuated Salmonella strains were receiving much attention regarding their potential as an antigen delivery system for mucosal immunization. They were particularly useful as oral vaccines since these organisms colonized a major mucosal inductive site, i.e., the gut-associated lymphoid tissue (GALT). Specifically, the Salmonella were taken-up by the specialized M cells interspersed throughout the luminal epithelium of the Peyer's patches in the GALT and transported to underlying lymphoid cells [18,19]. S. typhimurium DcyaDcrpDasd mutants, which lacking adenylate cyclase and the cyclic AMP (cAMP) receptor protein had been shown to be stable and safe for in vivo use [11,20].

In this study, the new plasmid possessing asd gene was introduced into attenuated DcyaDcrpDasd S. typhimurium c4072. BALB/c mice orally immunized with the S. typhimurium c4072(pMMASD), which expressed heat-labile enterotoxin LTB and fimbriae antigen K88ac, produced stronger humoral and mucosal antibody responses against the two antigens than the former live vaccine strain MM-3.

Materials and Methods

Bacterial strains, media, plasmids and antisera

Escherichia coli MM-3, containing pMM085, expressing K88ac fimbriae and LTA-B+, with cmR antibiotic resistance gene was kindly provided by Prof. T. M. CHEN (Beijing Institute of Biotechnology, Beijing, China) [9]. E. coli c6097, a Dasd mutant strain, and S. typhimurium c4072, a DcyaDcrpDasd mutant strain, were kindly provided by Curtiss et al. [11,20]. S. typhimurium c3730, a DgalEDhsdDasd mutant strain which was a restriction-negative, modification-positive strain used as an intermediate for the transfer of plasmids from E. coli to Salmonella, was also provided by Roy Curtiss III. The plasmid pKD46 was kindly provided by Dr. B. L. WANNER [16], expressing Red proteins-Exo, Beta and Gam when induced by arabinose and containing ampR gene. The plasmid pZL21, containing asd gene was preserved in our lab. MM-3 and E. coli containing pKD46 were cultured in LB media with corresponding antibiotics. E. coli c6097, S. typhimurium c3730 and S. typhimurium c4072 were grown in LB media supplemented with diaminopimelic acid (50 mg/ml). All strains and plasmids used in this study are provided in Table 1. The mouse anti-K88ac fimbriae antiserum and mouse anti-CTB enterotoxin antiserum were prepared in our laboratory.

Preparation of DNA fragments for recombineering

Polymerase chain reaction (PCR) was used for the amplification of DNA fragments with Thermocycler (Biometra, Gottingen, UK). The high-fidelity Pfu DNA polymerase (Tiangen, Beijing, China) was used in all of the reactions, prepared as described by the supplier. DNA fragments were generated by touchdown PCR amplification and the appropriate primers are shown in Table 2. Primers Forcatasd and Revcatasd were used to amplify a 1.7-kb DNA fragment containing the full-length asd gene using plasmid pZL21 as a template. The PCR conditions included an initial denaturation at 94 ºC for 5 min, followed by touchdown procedure of 16 cycles of denaturation at 94 ºC for 40 s, annealing at 58 ºC for 40 s (-0.5 ºC increment of every cycle) and a final extension at 72 ºC for 2 min, followed by general conditioning including 20 cycles of denaturation at 94 ºC for 40 s, annealing at 50 ºC for 40 s and extension at 72 ºC for 2 min. PCR products were used as templates in the second round of PCR with the same primers and the same reaction conditions to decrease the concentration of pZL21 [21]. The final PCR products were gel-purified and suspended in elution buffer (10 mM Tris, pH 8.0). Primers Forcat and Revcat were used to identify the correct mutants.

Genetic manipulation of Escherichia coli c6097

The plasmid pMM085 and pKD46 were transformed into E. coli c6097 and the transformants were selected from an LB agar plate supplemented with 100 mg/ml ampicillin, 75 mg/ml chloramphenicol and 50 mg/ml diaminopimelic acid (DAP). Transformants containing a Red helper plasmid were grown in 50 ml LB cultures with appropriate selective pressure and L-arabinose at 30 ºC to allow the expression of Exo, Beta and Gam when A600 reached 0.5, then made electrocompetent by concentrating 100 folds and washing three times with ice-cold 10% glycerol [17]. A total of 25 ml of competent cells were mixed with 20-25 ng of the PCR amplified DNA in a pre-cooled 0.1 cm Gene Pulser cuvette (Bio-Rad, Hercules, USA) and electroporated using the Bio-Rad Gene Pulser with conditions as 1.8 kV, 25 mF and 200 W. Following electroporation, the cells were grown in 1 ml of LB at 37 ºC for 1 h, plated 200 ml of cells on every LB agar plate without supplementation, and incubated at 37 ºC for 20 h. The correct mutants were identified by whole cell PCR using Primers Forcat and Revcat.

Expression of K88ac and LTA-B+ (LTB) in different live vaccine strains

The verified pMMASD obtained from E. coli c6097 was transformed into S. typhimurium c3730, which had a characteristic of DgalEDhsdDasd. Plasmid pMMASD was then extracted from c3730 and transformed into S. typhimurium c4072 by the CaCl2 transformation method. The strains were cultured at 37 ºC in 75 ml of LB medium (250 ml Erlenmeyer flask) for 12 h, and when A600 reached 45, 30 ml of culture was removed for centrifugation. The precipitants were resuspended in 10 ml of 1´phosphate-buffered saline (PBS) buffer and lysed by ultrasonication, centrifuged at 12,000 g for 20 min at 4 ºC. The lysate supernatant and culture supernatant were preserved at -20 ºC for checking the expression level of both K88ac fimbriae and LTB enterotoxin as described as follows in the enzyme-linked immunosorbent assay (ELISA).

In vitro plasmid maintenance

A single colony of S. typhimurium c4072(pMMASD) was inoculated into 5 ml of nonselective medium (LB supplemented with 50 mg/ml DAP), grown at 37 ºC in an orbital shaker at 220 rpm for 12 h, then successively subcultured with 1:100 into flesh nonselective media every 12 h until the total time was 48 h. The culture of the first 12 h and the fourth 12 h was diluted 1:160,000 and 40 ml was plated on the nonselective agar plates respectively. After incubation at 37 ºC for 20 h, 100 colonies on the plate were selected randomly and inoculated on a selective medium (LB agar plate), and viable colonies were enumerated. The cells grew approximately three generations per hour, so there were 36 generations and 144 generations after 12 h growth and 48 h growth respectively.

The procedure of determining in vitro plasmid maintenance of E. coli MM-3 was almost the same as that of S. typhimurium c4072 (pMMASD), except that the nonselective and selective media were changed respectively to LB media and LB media plus chloramphenicol.

In vivo plasmid maintenance

Bacteria were cultured in an Erlenmeyer flask for 12 h, and centrifuged at 4000 g for 10 min. The density of the bacteria was adjusted by optical density measurement at 600 nm and confirmed by serial dilutions on LB agar plates. Female BALB/c mice were obtained from the Central Animal House of Beijing Institute of Biotechnology (Beijing, China) and were 8 weeks old at the commencement of experiments. Mice were fasted for 4 h before feeding the bacteria. After treatment with 300 ml of 50% saturated sodium bicarbonate solution, each mouse received a single oral dose of 1.0109 colony forming units (200 ml) of S. typhimurium c4072(pMMASD) vaccine or 1.0´109 CFU of E. coli MM-3 vaccine and fasted for an additional 2 h.

On day 10 postinoculation, 10 mice were killed by cervical dislocation, and the spleen and 6-7 Peyer's patches of each mouse were removed. The spleen or the Peyer's patches were homogenized in 1.8 ml of sterile PBS using 50 mm pore size cell strainers, and 50 ml of the mixture was plated onto suitable agar for enumeration. Specifically, the samples were plated onto LB agar and LB agar containing chloramphenicol respectively for checking E. coli MM-3, and were plated onto Hektoen enteric (HE) agar and HE agar containing diaminopimelic acid for checking S. typhimurium c4072(pMMASD).

Plasmid maintenance in the alimentary canal was also detected. The fecal pellets of each group were collected every day and resuspended in 60 ml of enrichment medium. Enrichment medium of selenite broth was employed for detection of S. typhimurium c4072(pMMASD) in fecal specimens since the vaccine strains represented only a small percentage of the intestinal flora [22]. After incubation at 37 ºC for 10-12 h, 50 ml of broth was plated on HE agar plates, and the suspicious colonies on HE agar plates were green or transparent without a black center (lactose negative, no production of H2S), and were further identified by whole cell PCR using primers Forcat and Revcat. For detection of MM-3 in fecal specimens, LB plus chloramphenicol was used as an enrichment medium and selective plate medium. The suspicious colonies were further identified by whole cell PCR.

Enzyme-linked immunosorbent assay

For detecting the expression level of K88ac fimbriae, the lysate supernatants were diluted to 1:10 and the culture supernatants were diluted to 1:3. The diluted antigens were then used to coat 96-well microtiter plates (Greiner Bio-One, Frickenhausen, Germany) at 100 ml per well. The plates were incubated overnight at 4 ºC, washed with PBST (PBS containing 0.05% Tween 20) three times, then blocked by 0.1% bovine serum albumin (BSA), and followed by incubation with mouse anti-K88ac fimbriae antiserum (1:2000 dilution). The bound Ag-Ab complex was detected using goat anti-mouse IgG conjugated to horseradish peroxidase (1:10,000 dilution) and color was developed using o-phenylene dimine (OPD) as a chromogen and H2O2 as substrate and the absorbance was read at 492 nm. For detecting the expression level of LTB, ganglioside GM1 at 2 mg/ml was used to coat 96-well microtiter plates, blocked by 0.1% BSA, and incubated with diluted lysate supernatant dilution or culture supernatant dilution. After being washed with PBST three times, mouse anti-CTB enterotoxin antiserum (1:2000) was added and the following steps were the same as those used when detecting the expression level of K88ac.

Oral immunization of mice

The preparation of bacteria was in the same way as in the method of "in vivo plasmid maintenance". Before immunization, mice were fasted for 4 h, and then pretreated with 300 ml of 50% saturated sodium bicarbonate solution. Finally, the mice were fed with feeding needles for intragastrical delivery of 200 ml of bacterial suspensions (2.0109 CFU for E. coli strains or 1.4108 CFU for Salmonella strains) and fasted for an additional 30 min. The mice were immunized at day 0, 7 and 21.

Assessment of immune responses

Mice of each group were killed on day 35. The blood was collected and centrifuged at 10,000 g for 5 min and the serum was stored at 20 ºC. Whole small intestines, from the duodenum to the ileocecal junction, were excised, snipped into pieces, and resuspended with 800 ml of PBS with 50 mM EDTA (pH 8.0). Recovered intestinal contents were vortexed vigorously for 5 min. After centrifugation at 10,000 g for 10 min at 4 ºC, supernatants were collected and stored at -20 ºC. For each immunized group of mice, fecal pellets were collected on day 34. Approximately 100 mg of feces were added to tubes containing 400 ml of PBS with 50 mM EDTA (pH 8.0) and incubated ice for 15 min. And then, tubes were agitated vigorously for 10 min on a vortex mixer at maximum speed. The suspensions were centrifuged at 10,000 g for 10 min at 4 ºC, and the supernatants were stored at 20 ºC.

Purification of K88ac fimbriae was as follows: S. typhimurium c4072(pMMASD) was cultured in 150 ml of LB medium for 12 h at 37 ºC and shaken at 200 rpm. The cell suspensions were collected by centrifugation at 4000 g for 10 min, and the pellets were resuspended in 30 ml of 50 mM Tris buffer (pH 7.45) containing 1.0 M NaCl. The bacteria were subjected to heat shock at 63 ºC for 20 min to obtain K88ac fimbriae. The bacteria were pelleted at 4000 g for 10 min, and the K88ac fimbriae were precipitated from the supernatant by adding ammonium sulfate (30% saturation) and stirring for 1 h at 4 ºC. The precipitate was collected by centrifugation at 10,000 g for 10 min, resuspended in 2 ml of 50 mM Tris buffer (pH 7.45), and dialyzed against the same buffer for 16 h at 4 ºC. The purity of the isolated K88ac fimbriae was evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The protein concentration was determined by the Lowry method. The purified K88ac fimbriae were used to coat the 96-well ELISA plates.

Antibody responses were assayed by ELISA. Individual mouse serum, intestinal washes and fecal pellet extract were tested for IgG and IgA antibodies against K88ac fimbriae by ELISA. The 96-well plates were coated with purified K88ac fimbriae (0. 5 mg in 100 ml of 0.1 M carbonate buffer, pH 9.6, per well) overnight at 4 ºC. The plates were blocked with 0.1% BSA in PBST at 37 ºC for 2 h, washed three times with PBST, and incubated respectively with dilutions of serum, intestinal washes and fecal pellet extract samples in PBST-0.1% BSA for 1 h at 37 ºC. For detecting IgG and IgA antibodies against enterotoxin LTB, ganglioside GM1 (0.2 mg in 100 ml of 0.1 M carbonate buffer, pH 9.6, per well) was used to coat 96-well microtiter plates, blocked by 0.1% BSA, and followed by incubation with diluted cell lysate supernatants as described in "Materials and Methods". After being washed with PBST three times, diluted serum, intestinal washes, or fecal pellet extract samples in PBST-0.1% BSA were added, respectively. The bound Ag-Ab complexes were detected using goat anti-mouse IgG conjugated to horseradish peroxidase (1:10,000 dilution). The color was developed using OPD as a chromogen and H2O2 as substrate. The absorbance was read at 492 nm.

Statistical analysis

The absorbance at 492 nm (A492) in ELISA was plotted using GraphPad Prism. Probability values were determined by the two-tailed, unpaired t test with Welch's correction (95% confidence interval) for comparisons of two sets of data.

Results

Construction of plasmid pMMASD

Using the l-Red recombination system presented in pKD46, the cat gene was knocked out and the asd gene was knocked in through homologous recombination between pMM085 and a PCR-amplified asd gene fragment. The homologous recombination was between a plasmid pMM085 containing the cat gene sequence and the PCR-generated DNA fragment containing asd sequence flanked by 38 to 39 bp of cat sequence, corresponding to the 5' and 3' regions of the intended site of mutagenesis, respectively. When the l-Red recombination system was activated, the flanking sequences of homology to normal cat sequence were sufficient to allow homologous recombination in E. coli c6097 to promote the incorporation of the asd gene into the cat sequence, with an efficiency of 2.2102 recombinants/mg DNA when growth was selected in conditional medium lacking DAP. Among the 21 colonies selected, 20 colonies showed a varying mixture of parentally and correctly recombined plasmids by PCR amplification of the region corresponding to the site of asd gene integration (Fig. 1). Colonies containing only the recombinant plasmid pMMASD were obtained by retransformation in E. coli c6097 and selected for asd function. The correct recombination event was also confirmed by restriction enzyme and sequence analysis.

Expression of LTB and K88ac in Salmonella typhimurium c4072 and in Escherichia coli c6097

Salmonella typhimurium c3730 in which the asd gene was knocked out was the DgalE mutant strain of S. typhimurium LT2-Z and lost the effect of restriction but had the effect of modification. Therefore, the plasmid transformed into S. typhimurium c3730 could obtain the methylated model of S. typhimurium and thus existed in this strain stably. The plasmid with the methylated model extracted from c3730 was then transformed into the final host strain S. typhimurium c4072 without any restriction. Five vaccine strains, E. coli MM-3, E. coli 6097, E. coli 6097(pMMASD), S. typhimurium 4072 and S. typhimurium 4072(pMMASD), were cultured in LB medium at 37 ºC for 12 h. When A600 reached 4.0 to 5.0, 30 ml of mixture was obtained for analyzing the expression levels of K88ac fimbriae and LTB enterotoxin, respectively by ELISA.

Salmonella typhimurium c4072(pMMASD) and E. coli c6097(pMMASD) were found to stably express K88ac fimbriae and LTB enterotoxin without antibiotics. As shown in Fig. 2, E. coli MM-3, E. coli c6097(pMMASD) and S. typhimurium c4072(pMMASD) expressed similar levels of LTB in the cell lysate. However, the secretion level of LTB enterotoxin in c4072(pMMASD) was highest, indicating that S. typhimurium had an advantage of secreting antigen over E. coli. Meanwhile, the negative contrasts c6097 and c4072 had a very low background, suggesting that the "GM1 sandwich" method was suitable for checking the LTB enterotoxin. As to the expression of K88ac fimbriae, although the background of negative contrasts c6097 and c4072 were relatively high in culture supernatant, the conclusion that K88ac fimbriae were displayed on the surface of the bacteria and dropped into culture fluid was easy to make. In the cell lysate of MM-3, c6097(pMMASD) as well as c4072(pMMASD), the expression levels of K88ac fimbriae were almost the same as others, with c6097(pMMASD) possessing the relatively highest ability to display the K88ac fimbriae.

The asd balanced-lethal system was used to stabilize plasmid maintenance for in vivo experiments. Data showed in this study that the changes in the reconstructed plasmid pMMASD had no negative effect on the expression of K88ac fimbriae and LTB enterotoxin; on the other hand, the new host cell S. typhimurium c4072 possessed the more effective secretion mechanism to transport LTB enterotoxin into culture liquid, indicating that S. typhimurium c4072(pMMASD) might be the better vaccine strain to elicit the humoral and mucosal antibody responses against the LTB enterotoxin.

In vitro plasmid maintenance

To demonstrate whether pMMASD would be stably maintained in a host-plasmid balanced lethal system, in vitro plasmid maintenance of S. typhimurium c4072(pMMASD) was investigated compared with that of E. coli MM-3. Serial subcultures in nonselective broth showed that more than 70% of equivalent colony numbers were obtained on selective agar plates for total viable cells after the 144 generations. In contrast, there were less than a quarter of equivalent colony numbers obtained in MM-3, which maintained its plasmid with antibiotic selective pressure, indicating that the plasmids were more stable in the host-plasmid balanced lethal system than in the antibiotic selective system (Fig. 3).

In vivo plasmid maintenance

Maintenance of plasmid pMMASD in S. typhimurium c4072, as well as pMM085 in MM-3 in vivo was evaluated. In vivo, recombinant bacteria could be detected neither in the spleens and in Peyer's patches of BALB/c mice orally inoculated with 1.0´109 of E. coli MM-3, nor in the spleens of BALB/c mice orally inoculated with 1.0´109 of c4072(pMMASD). Only three of ten mice inoculated with c4072(pMMASD) were detected having the invasion of the vaccine strain in Peyer's patches (Table 3).

S. typhimurium c4072 was a DcyaDcrp mutant strain in which the adenylate cyclase and the cAMP receptor protein genes were deleted. These proteins were necessary for the transcription of many genes and operons concerning with the transportation and breakdown of catabolites [23]. It was said that the DcyaDcrp mutant abolishes the synthesis of at least two adhesins that S. typhimurium uses for attachment to, and thus invasion of enterocytes [24]. Curtiss et al. also proved that the DcyaDcrp mutations do not significantly impair the ability of S. typhimurium to attach to, invade and persist in Peyer's patches, but do impair the ability to reach or survive in the spleen [25]. Although the invasion ability of S. typhimurium c4072 in spleens and Peyer's patches was relatively poor, the conclusion that the plasmid in the vaccine strain was stably maintained could be made according to the percentages of retaining plasmid in Peyer's patches that were from 60% to 115% in three of ten susceptible mice. Furthermore, when the total of 63 colonies on the nonselective agar plates (HE plus DAP) were selected and inoculated on a selective medium, 61 colonies grew well, indicating the stability of plasmid pMMASD in vivo.

Maintenance of plasmid in vaccine strains in the alimentary canal was also evaluated (Table 4). If plasmid pMMASD or pMM085 were lost in the host cell, there was no vaccine strain in fecal pellets. The results suggested that the pMM085 had a greater chance of being lost from the host strains comparing with pMMASD, and the reason was perhaps that the environment in vivo did not provide an antibiotic pressure for the stable maintenance of pMM085. On the contrary, this environment provided a selective pressure (the lack of DAP) for the survival of pMMASD in the host-plasmid balanced lethal system. Perhaps the other reason for the relatively longer persistence in alimentary canal for c4072(pMMASD) was the ability of invasion of intestinal enterocytes.

Immune responses of BALB/c mice after oral immunization with the live vaccine strains

The immunogenicity of the K88ac fimbriae and LTB enterotoxin expressed in S. typhimurium c4072(pMMASD), E. coli c6097(pMMASD) and MM-3 were tested by oral administration of the strains to its corresponding group of BALB/c mice. Thirty mice were divided into six groups with five mice in each group. After three immunizations, each mouse was killed and the blood, small intestines and fecal pellets were collected separately for analyzing the effectiveness of different live vaccine strains delivering K88ac fimbriae and LTB enterotoxin to the immune system.

S. typhimurium c4072(pMMASD), E. coli c6097(pMMASD) and E. coli MM-3 developed significantly high levels of K88ac fimbriae-specific serum IgG [Fig. 4(D)]. However, K88ac fimbriae-specific mucosal secretory IgA levels determined in intestinal washes and fecal pellet washes for the mice immunized with c4072(pMMASD) and c6097(pMMASD) were significantly higher than secretory IgA levels for the mice immunized with E. coli MM-3 [Fig. 4(E,F)]. Meanwhile, serum IgG against the LTB enterotoxin were only developed in the mice immunized with S. typhimurium c4072(pMMASD), as visualized in absorbance of A492 by ELISA [Fig. 4(A)]. The results indicated that the more effective secretion system of S. typhimurium enhanced the immune response to the LTB enterotoxin. Comparison of the immune response with S. typhimurium c4072(pMMASD) versus E. coli MM-3 resulted in significant differences in both K88ac fimbriae-specific mucosal secretory IgA and LTB enterotoxin-specific serum IgG, suggesting that the use of S. typhimurium as a delivery system improved the immunogenicity of both K88ac fimbriae and LTB enterotoxin.

Although S. typhimurium c4072(pMMASD) elicited mucosal secretory IgA specific for LTB enterotoxin, the antibody levels were generally low [Fig. 4(B,C)]. There were no statistically significant differences in LTB-specific mucosal secretory IgA levels between the mice immunized with c4072 and the ones immunized with c4072(pMMASD), or between the control mice and ones immunized with c4072(pMMASD).

Discussion

Live bacterial vector MM-3 played a function of protecting neonatal piglets from diarrhea. In a challenge study and field trials, a high degree of protection from colibacillosis was afforded to piglets when their dams were immunized orally or parenterally [9,10]. However, since the plasmid of MM-3 contained the cat gene, it had a large chance of being lost in the environment where antibiotic selective pressure was not presented. b-galactosidase as a tracking marker was also attempted in the work of Chen et al. [9], but the same problem remained. The target of this study was the introduction of the asd gene to replace the cat gene to result in a stable maintenance of recombinant plasmid in the asd balanced-lethal system. Moreover, finding a more suitable host cell to deliver the K88ac fimbriae and LTB enterotoxin was also expected.

Recently, the development of recombineering using homologous recombination in bacteria has allowed DNA manipulation without the need for restriction enzymes. The target gene could be introduced anywhere in chromosomes, bacterial artificial chromosomes or a plasmid DNA sequence specifically by homologous recombination. The replacement of the cat gene by the asd gene in pMM085 was achieved by a l-Red recombination system. pMM085 was a multicopy plasmid, and this made the high frequency of recombination events in vivo for the greater chances of linear double-stranded DNA donor encountered its homologous target sequence. The recombined efficiency was only 2.2102 recombinants/mg DNA (in the work of Wong et al. [15], the recombined efficiency was 1.32104 recombinants/mg DNA), because the data were not gathered under the optimal conditions. Besides, this system showed a selection efficiency of more than 95% (20/21), requiring the screening of fewer colonies to ensure a successful outcome.

Recombinant plasmid pMMASD was transformed into two host cells: S. typhimurium c4072 and E. coli c6097. Cultured in LB medium, expression levels of K88ac fimbriae and LTB enterotoxin in cell lysate were similar among MM-3, c4072(pMMASD) and c6097(pMMASD), and c6097(pMMASD) had a slightly higher expression level of K88ac fimbriae. It seemed that c6097(pMMASD) was the candidate for taking the place of MM-3. However, analysis of the expression level of LTB enterotoxin in culture supernatant revealed that c4072(pMMASD) possessed the more effective secretion mechanism to transport LTB enterotoxin into culture liquid. Work in mice with serovar typhimurium expressing the recombinant Streptococcus pneumoniae antigen PspA compared expression in the cytoplasm with secretion into the periplasm and culture supernatant [26]. There was a 104 increase in IgG titer in animals that received the strain bearing a secreted antigen. The same results in this study were reached when oral immunization of BALB/c mice with different live vaccine strains, only c4072(pMMASD) developed a strong humoral response against LTB enterotoxin, while E. coli MM-3 and c6097(pMMASD) did not elicit the corresponding response.

Although c4072(pMMASD) failed to elicit strong IgA responses to LTB enterotoxin in mucosal tissues, expressed LTB might play an adjuvant effect on response to the K88 fimbriae. Consistent with this interpretation, only live E. coli strains carrying both K88ac and LTB antigens would afford litter pigs effective protection from the challenge inoculation, while strains carrying the K88ac antigen would afford pigs part protection to the challenge inoculation and the strain carrying LTB antigen afforded no protection to the pigs [4].

Enteroadhesive fimbriae played a critical role in the pathogenesis of ETEC. The binding of fimbriae to intestinal receptors ensures optimal mucosal colonization by the bacteria and efficient enterotoxin delivery to the enterocytes. Fimbriae can serve as an effective vaccine to induce an immune response against ETEC infections. The results of this study clearly demonstrate that oral immunization of BALB/c mice with the Salmonella-K88 construct c4072(pMMASD), as well as Escherichia-K88 construct c6097(pMMASD) were sufficient to elicit elevated IgA responses in mucosal tissues and increase the systemic IgG antibody responses to the K88 fimbriae. Although MM-3 could express ETEC K88 fimbriae and elicit good IgG responses, the ability of stimulating the mucosal inductive tissues was weaker than the other of two strains. Considering IgA was a major maternal antibody that passed to piglets through colostrum and milk and provided protection against infection, therefore, the higher level of IgA vaccine strains c4072(pMMASD) and c6097(pMMASD) elicited, the higher protection effects might be gained.

S. typhimurium naturally infects Peyer's patches, a major IgA inductive site, and subsequently disseminates to systemic tissues. During the past several years, numerous studies have evaluated recombinant Salmonella vaccine strains for their ability to induce mucosal and systemic immune responses to heterologous protein antigens [27-29]. Although S. typhimurium DcyaDcrp mutant c4072 had a weaker ability of invading and persisting in Peyer's patches and spleen than the DaroA mutants (defect in the aromatic amino acid synthesis pathway) such as SL3261 which invaded both Peyer抯 patches and spleen [13,30], the fact that S. typhimurium c4072(pMMASD) developed higher levels of specific IgA and IgG to K88ac fimbriae than that of E. coli c6097(pMMASD) proved that Salmonella strains c4072 as a living vector could enhance the mucosal and humoral immune response to the antigen.

Development of immunity to Salmonella infections relies on the cellular, humoral, and mucosal arms of the immune system. Although the role of humoral immunity has been long appreciated, recent work has shown the cellular responses stimulated by the Salmonella [31-33]. Furthermore, displaying of antigens or antigenic fragments by the autotransporter pathway on the surface of Salmonella vaccine strains would result in a pronounced T-cell response in mice [34,35]. The reason might be the efficient antigen presentation via the MHC class II pathway stimulating cellular immune response. Therefore, it would be speculated that the Salmonella strain c4072 plus K88 fimbriae displaying on the surface of this cell might be involved in the cellular immune responses.

In conclusion, S. typhimurium c4072(pMMASD) was identified in this work to have many advantages over the former live vaccine strain E. coli MM-3, not only in plasmid stability in vitro and in vivo, but also in developing immune response to both K88ac fimbriae and LTB enterotoxin, and was expected to be a safe and stable live vaccine strain. Therefore, this study gave an example of re-construction of a live vaccine strain carrying an antibiotic resistance gene.

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