- Research article
- Open Access
Recombinant porcine rotavirus VP4 and VP4-LTB expressed in Lactobacillus casei induced mucosal and systemic antibody responses in mice
© Qiao et al; licensee BioMed Central Ltd. 2009
Received: 25 November 2008
Accepted: 4 December 2009
Published: 4 December 2009
Porcine rotavirus infection is a significant cause of morbidity and mortality in the swine industry necessitating the development of effective vaccines for the prevention of infection. Immune responses associated with protection are primarily mucosal in nature and induction of mucosal immunity is important for preventing porcine rotavirus infection.
Lactobacillus casei expressing the major protective antigen VP4 of porcine rotavirus (pPG612.1-VP4) or VP4-LTB (heat-labile toxin B subunit from Echerichia coli) (pPG612.1-VP4-LTB) fusion protein was used to immunize mice orally. The expression of recombinant pPG612.1-VP4 and pPG612.1-VP4-LTB was confirmed by SDS-PAGE and Western blot analysis and surface-displayed expression on L. casei was verified by immunofluorescence. Mice orally immunized with recombinant protein-expressing L. casei produced high levels of serum immunoglobulin G (IgG) and mucosal IgA. The IgA titters from mice immunized with pPG612.1-VP4-LTB were higher than titters from pPG612.1-VP4-immunized mice. The induced antibodies demonstrated neutralizing effects on RV infection.
These results demonstrated that VP4 administered in the context of an L. casei expression system is an effective method for stimulating mucosal immunity and that LTB served to further stimulate mucosal immunity suggesting that this strategy can be adapted for use in pigs.
Rotaviruses are members of the family Reoviridae. Rotaviruses affecting pigs are classified as group A, B or C based on their respective inner capsid protein sequences. The rotavirus double-stranded RNA genome is composed of 11 segments enclosed by a nonenveloped, triple-layered icosahedral capsid . The outer capsid VP4 protein can induce neutralizing antibodies resulting in protecting herd from porcine rotavirus infection.
Porcine rotaviruses are the major cause of acute diarrhea in the piglets [3, 4] and can cause mild-severe diarrhea associated with potentially high morbidity and mortality. Group A rotaviruses cause diarrhea in pigs both before and after weaning  and can account for 53 and 44% pre- and post-weaning rotavirus-associated diarrhea in swine, respectively . A recent report attributed 89% of all rotavirus-associated diarrhea in commercial pig farms to group A rotavirus infections . Since rotaviruses can survive in the environment for long period of time and are transmitted via the fecal-oral route outbreaks are difficult to control. Virion replication occurs at the tips of epithelial cell in intestinal villi and destroy enterocytes primarily in the jejunum and ileum resulting in villous atrophy [8, 9]. Furthermore, nutrients cannot be digested or absorbed in the affected regions resulting in severe malabsorption . A better understanding of rotavirus epidemiology will contribute to the optimization of current vaccines and prevention programs for the control of rotavirus infection. Currently available vaccines (mostly killed) can not offer efficient immunity. To stimulate efficient immunity, a large vaccine dose and repeated administration are usually required. This often results in undesirable clinical signs. To overcome these shortcomings, the potential development of lactic acid bacteria (LAB) to deliver heterologous antigen to the mucosal immune system has been proposed.
Since rotaviruses are enteric pathogens, mucosal immunity is likely to play an important role in protective immunity. Innate immune responses in gut provide the first line of defense against pathogenic microorganisms and also initiate acquired immune responses. Furthermore, immune responses resulting from oral immunization are the only suitable method of stimulating gut immunity  since this route facilitates stimulation of gut-associated lymphoid tissue (GALT) enhancing the production of anti-viral IgA .
Compared to recombinant antigens or heat-killed formulations, 'live' vaccines elicit the most effective protective responses since they stimulate both systemic and mucosal immunity [13–17]. However, oralvaccination presents a challenge since the gut milieu often denatures and/or inactivates potential vaccinogens therefore large vaccination doses and repeated vaccinations are required[18, 19]. This often results in fecal shedding of the live vaccine in addition to causing fever and diarrhea [16, 18, 19]. These challenges can be overcome by using lactic acid bacteria (LAB) as antigen delivery system for the stimulation of mucosal immunity [20–25] owing to its safety. LAB are used in industrial food fermentation, preservation and have beneficial effects on the health of both humans and animals and 'generally regarded as safe, (GRAS'micro-organisms). In addition, many strains of LAB are able to survive and colonize the intestinal tract [26, 27] inducing a non-specific immunoadjuvant effect  which prompted studies aimed at determining the oral vaccine potential of LAB-derived vaccines.
Since genetically engineered vaccines composed of a single recombinant antigen are poorly immunogenic, it is important to increase their immunogenicity by combining with appropriate adjuvants. The E. coli heat-labile toxin B subunit (LTB) has been shown to be a potent mucosal adjuvant [29–33] with low potential of eliciting allergic responses [34, 35].
In this study, we tested the efficacy of the L. casei ATCC 393 expressing the heterologous VP4 porcine rotavirus protein and its ability acting as an antigen delivery system for oral vaccinations. We constructed recombinant strains expressing porcine rotavirus VP4 and VP4-LTB. The immunogenic potential of the two recombinant strains was analyzed after oral administration of live bacteria to mice. This is the first report describing the cloning and expression of porcine rotavirus genes in Lactobacillus. The data reported indicate that oral administration of two recombinant strains pPG612.1-VP4 or pPG612.1-VP4-LTB could induce specific anti-rotavirus mucosal and systemic immune responses. The potency of the immune responses measured was greater in animals immunized with L. casei-expressing the VP4-LTB fusion (compared to mice immunized with L. casei expressing VP4 only) demonstrating the efficacy of LTB as a mucosal adjuvant.
Expression of VP4 and VP4-LTB in L. casei
The sequences of the respective L. casei 393 transformants are confirmed by plasmid DNA sequencing and the result shows that there is no mutation in the transformants (data not shown).
Antibody responses following oral immunizations
Neutralization ability of the induced antibodies analysis
Porcine rotaviruses are the major cause of acute diarrhea in the piglets and can cause mild to severe diarrhea with potentially high morbidity and mortality rates. Infection with porcine rotavirus has been an economic concern to worldwide pig breeders. Vaccination is the main prophylatic method for the prevention of porcine rotavirus infections. Mucosal immunization offer a number of advantages over other routes of antigen delivery, including ease of administration, cost effectiveness and the capacity of inducing both local and systemic immune responses [36–41].
To assess mucosal immune responses, specific IgA anti-VP4 protein levels were examined from various mucosal surfaces. Oral administration of recombinant VP4 or VP4-LTB-expressing L. casei induced both systemic (IgG) and mucosal (IgA) immune responses. Specifically, IgA specific for VP4 could be isolated from the gastrointestinal tract, vagina and eye secretions compared to no detectable IgA anti-VP4 responses in control animals. These experiments suggested that L. casei expressing recombinant VP4 could be used in the vaccination of pigs, potentially protecting them from porcine rotavirus infections since this vector successfully elicited a significant and specific anti-VP4 IgA response.
The titers of anti-VP4 IgG in the serum from mice immunized with the L. casei pPG612.1-VP4 or pPG612.1-VP4-LTB were similar but higher than the control group. rLc393:pPG612.1-VP4-LTB induced even higher IgA specific for VP4 compared to mice immunized with the pPG612.1-VP4 as a result of the LTB mucosal adjuvant. It demonstrated the specific mucosal adjuvanticity of LTB, highlighting its potential use as a safe and effective mucosal adjuvant that can be used in conjunction with VP4 for the elicitation of specific anti-porcine rotavirus immunity.
Furthermore, in order to confirm the efficacy of the induced antibodies in inhibiting the virus, we tested whether sera collected from immunized mice could inhibit the infection of RV in MA104 cells by neutralization ability assay. The results showed that serum collected from mice immunized with recombinant strains demonstrated statistically significant inhibition. The neutralization by sera antibodies obtained from mice immunized with pPG612.1-VP4-LTB was more effective than that of mice immuned with the pPG612.1-VP4.
In this report, we described the methods for constructing two L. casei recombinant expression vectors expressing the porcine rotavirus VP4 antigen or VP4-LTB fusion protein. L. casei is an excellent delivery vector since it can withstand the rigors of the intestinal environment in addition to being able to colonize different mucosal sites (gastrointestinal and genital tracts) and guaranteeing proper (intact) presentation of the respective antigens to the immune system. In addition to the versatility of L. casei, it possesses probiotic properties making it an even more attractive vaccine delivery system i.e., immunization with L. casei expressing VP4-LTB elicited potent anti-VP4 IgA responses. Testing the efficacy in a porcine vaccination and infection model is a next step in testing the efficacy of this vaccine formulation.
Strains and culture conditions
L. casei ATCC 393 (a kind gift of Jos Seegers, NIZO, The Netherlands) was grown anaerobically in MRS broth (Sigma, St, Louis, MO) at 37°C without shaking. To analyze protein expression, transformed L. casei were grown in basal MRS medium (10 g peptone, 8 g beef extract, 4 g yeast extract, 2 g potassium phosphate, 5 g sodium acetate, 1 ml Tween 80, 2 g diammonium citrate, 0.2 g magnesium sulfate, and 0.05 g manganese sulfate per liter) supplemented with 2% xylose. L. casei was plated on MRS medium with 1.5% agar. The antibiotic concentration used for the selection of lactobacilli transformants was 10 μg/ml of chloromycetin (Cm; Sigma). Porcine rotavirus JL94 (belonging to P) was conserved in the laboratory.
Balb/c mice (female) weighing 25-30 g (7 weeks of age) were obtained from the inbred colony maintained at the Harbin Veterinary Research Institute. Each experimental and control group consisted of 10 mice. The animals were fed balanced rodent food and water ad libitum. The mice were handled and maintained under strict ethical conditions according to the international recommendations for animal welfare and the Ethical Committee for animals sciences of HeiLongJiang province (032/2006).
Mouse anti-VP4 antibodies
The mouse anti-VP4 antibodies used in Western-blot and immunofluorescence analysis had been prepared and stored in our laboratory. The recombinant plasmid VP4-pGEX-6P-1 was constructed and transformed into E. coli BL21(Yan Song). The recombinant strain was induced with IPTG. The serum was obtained from the Balb/c mice immunized with the purified VP4 protein. Western-blot test and neutralization test circumstantiate the expressed protein has biological activity(data not shown).
Expression plasmid construction
Electroporation of L. casei was carried out as previously described . Briefly, plasmid DNA (10 μl) was added to 150 μl of L. casei 393, gently mixed at 4°C for 5 min and subjected to a single electric pulse (25 μF of 2.5 kV/cm). The mix was then incubated in MRS medium without Cm at 37°C anaerobically for 2 h. Recombinant strains were selected on MRS-agar medium containing 10 μg/ml of Cm. The sequences of the respective L. casei 393 transformants were confirmed by plasmid DNA sequencing.
Protein expression and Western-blot analysis
To analyze the expression of the VP4 and VP4-LTB fusion protein following xylose induction of rLc393:pPG612.1-VP4 and pPG612.1-VP4-LTB, respectively, overnight cultures grown in basal MRS broth supplemented with xylose (or glucose as a negative induction control) and pellets collected by centrifugation at 12,000 × g for 10 min. The pellets were washed twice with sterile 50 mM Tris-Cl, pH 8.0 and treated with 10 mg/ml lysozyme at 37°C for 60 min. The lysates were centrifuged at 12000 × g for 10 min and subjected to 10% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and either stained with Coomassie blue or electrotransferred onto nitrocellulose membranes. The immunoblots were blocked with PBS containing 5% skimmed milk for 2 hr at 37°C. Blots were washed three times between all steps for ten minutes. Blots were incubated with 1:800 dilution(100 μL) of mouse anti-VP4 antibodies in phosphate-buffered saline (PBS), washed and then probed with a horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (Sigma) diluted at 1:2500(100 μL) in PBS. The blots were washed and incubated with the Chemiluminescent Substrate reagent (Pierce, Rockford, IL) according to the manufacturer's instruction. Control blots incubated with secondary antibody only did not result in visible protein band reactivity.
Immunofluorescence was used to analyze VP4 and VP4-LTB protein surface expression by either rLc393:pPG612.1-VP4 or pPG612.1-VP4-LTB as described previously . Briefly, 2 ml induced cultures were harvested to an OD600 = 0.5-0.6 and then resuspended in 1 ml sterile PBS 3% bovine serum albumin (BSA) containing anti-VP4 antibodies and then incubated overnight at 37°C. The cells were then pelleted, washed 3 times with sterile PBS 0.05% Tween 20. The cell-antibody complexes were then incubated for 6 h at 37°C in the dark with fluoreoscein isothiocyanate (FITC)-conjugated goat anti-mouse IgG (Sigma) containing 1% Evans blue. Cells were washed 3 times with PBS 0.05%, Tween 20 and then air-dried on a glass slide. Analysis was performed using a confocal microscope. Non-induced or glucose-induced recombinant strains were used as negative controls.
rLc393:pPG612.1-VP4 and rLc393:pPG612.1-VP4-LTB were cultured and centrifuged as described above. Cell pellets were washed once with sterile PBS and resuspended in PBS (pH 7.4). Mice were orally vaccinated with 0.2 ml 109 colony-forming units (c.f.u.)/ml of the recombinant strains, respectively. A control group of 10 mice received L. casei ATCC 393 containing the empty plasmid was also included. Mice in all groups were immunized on days 0, 1 and 2 and boosted on days 14, 15 and 16 and again on days 28, 29 and 30.
Enzyme-linked immunosorbent assay (ELISA)
Mouse serum was collected on days 7,14,21 and examined for specific anti-VP4 antibodies by ELISA. Feces was collected at 1, 2 and 7 days after every immunization as described previously . Ophthalmic washes were obtained by washing the eyes with 50 μl PBS 7 days after every immunization. Vaginal washes were collected by washing the vagina with 200 μl PBS 7 days after every immunization. All samples were stored at -20°C until assayed by ELISA.
Polystyrene microtitre plates were coated overnight at 4°C with either porcine rotavirus propagated on MA104 cells or with supernatants harvested from MA104 cells cultured without rotavirus as negative control. ELISA plates were washed 3 times with PBS 1%Tween 20 and then blocked with PBS 5% skim milk at 37°C for 2 h. Serum or mucosal wash samples were serially diluted in PBS 1% BSA and incubated at 37°C for 1 h, washed 3 times and then incubated with a 1:2000 dilution(100 μL) of an HRP-conjugated goat anti-mouse IgA (Sigma) or IgG (Sigma), washed and visualized following the addition of 100 μl of o-phenylene diamine dihydrochloride substrate(Sigma). The absorbance was measured at 490 nm. Differences in the samples between treatments were examined for the level of significance by ANOVA.
Neutralization ability of the induced antibodies
Serum samples from mice immunized with recombinant strains expressing VP4 or VP4-LTB were evaluated  to determine the neutralization ability of the induced antibodies. In brief, sera from mice fed with non-expressor strains was used as negative control. Fifty microliters of samples in serial dilutions (from 1:2 to 1:512) was prepared in a 96-cell plate. RV adjusted to 200 TCID50 in 50 μL of virus diluent (10% concentrated Hanks balanced salt solution, pH 7.4) was added to the cell plate containing serially diluted serum. The mixture of antibody and virus was mixed and incubated at 37°C for 1 h. Then 100 μL of MA104 cells (used for virus infection) was added to the antibody-virus mixture and incubated in a 5% CO2 incubator at 37°C for 5 days. The overlay medium was then discarded, after which the wells were washed three times with sterile PBS, pH 7.4, and stained with 1% crystal violet solution. Differences in the number of plaques formed between treatments were examined for the level of significance by ANOVA.
Statistical significance was determined using ANOVA, with a P value < 0.05 considered as significant.
This work was supported by grants from the National Science and Technology Foundation of China (No. 2006BAD06A07) and the Program for Innovative Research Team of NEAU (No. CXZ008). The authors wish to thank Jos Seegers for providing plasmid pPG611.1 and bacterial strain L. casei ATCC 393.
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