ABSTRACT
The aim of this study was to assess the cumulative immunogenicity properties of rZot and rAce combination and their potential ability to increase the clearance rate of pathogenic standard Vibrio cholerae strain in challenge experiments in mice model. The recombinant Zot and Ace proteins were produced and used to raise polyclonal antibodies of anti-Zot and anti-Ace recombinant proteins in rabbit. Six-week female BALB/c mice were immunized with different antigens via oral route. Blood samples were collected, and the total amount of IgG and IgA antibodies against rZot and rAce were measured in blood and stool samples of each immunized mouse. Challenge experiments were done with toxigenic V. cholerae strain. The anti-Zot and anti-Ace IgG titers were significantly higher in immunized mice in comparison with control group. The IgG and IgA titers were higher in the sera of mice immunized by recombinant Ace than in group immunized by rZot, indicating the higher immunogenicity of rAce than rZot. The use of rAce and rZot mixture led to synergistic activities in increasing the level of IgG and IgA in comparison with the use of each protein separately. The clearance rate was significantly higher in different challenge groups than in the control group, and the coherence between rZot and rAce reduced the bacterial shedding significantly. In conclusion, the use of recombinant Zot and Ace mixture can produce the proper amount of IgA and IgG against to toxigenic V. cholerae, reduce bacterial shedding in immunized mice significantly, and be used as a potent candidate in cholera vaccine research.
KEYWORDS: Ace, zot, vaccine, Vibrio cholerae
Introduction
Vibrio cholerae is the famous causative agent of cholera which is characterized as a major secretory diarrheal disease. Cholera is the second common cause of death in infants and young children, causing annual morbidities and mortalities in countries encountering cholera epidemics.1-3 Cholera has the ability to make explosive outbreaks which has remained as a critical health problem all over the world.4 The seven cholera pandemics were occurred during 19th and 20th centuries,5 the majority of which took place in Asia, especially in India.6 Only the strains of V. cholerae Serogroup O1 have the ability to make cholera epidemics7; however, in 1992, the O139 serogroup with epidemic potential emerged and caused another cholera epidemic.8,9 The pathogenicity of V. cholerae is related to the 3 main toxins including cholera toxin (CT), with some variations between the two biotypes,10 zonula occludens toxin (Zot), and accessory cholera enterotoxin (Ace), among which cholera toxin has attracted many studies to investigate its immunological properties and its potential use for inclusion in cholera vaccine. Regarding the binding properties of B subunit of CT, which is responsible for binding ability of bacteria to the epithelial cells, and its adjuvant properties, this subunit has been applied in several commercial vaccines and investigational vaccine candidates,11,12 alone or in combinations. The Ace toxin which is an approximately 11kDa protein can increase the transcellular ion exchange and cause fluid accumulation in intestinal cells.13 Some studies demonstrated that Ace protein can form a pore into eukaryotic cell membrane due to its ability in producing multimers.13,14 The Zot toxin has synergy with CTX to cause dehydrating diarrhea. The function of Zot is associated with the interaction of this toxin with epithelial cell receptor, activating the intracellular signaling, loosening the intercellular tight junctions, and increasing the permeability of the small intestine.15,16 Zot also has the property of cell specificity due to interaction with specific receptors on the surface of the host cells.17,18 Our previous studies proved significant ability of rZot and rAce to induce the immune responses in animal model, suggesting their possible role for inclusion in future cholera vaccines.19,20 Consequently, the aim of this study was to assess the cumulative immunogenicity properties of rZot and rAce combination and their potential ability to increase the clearance rate of pathogenic standard strain of V. cholerae in challenge experiments in mouse model.
Results
Anti-rZot and anti-rAce IgG titer in immunized mice
Six-week female BALB/c mice were immunized with different antigens via oral route (Table 1) and the total amount of IgG antibodies against rZot and rAce were measured in blood samples of each immunized mouse. According to the results of indirect ELISA in detecting the total amount of anti-Ace IgG in sera, a significant difference was detected in mice receiving rAce+CpG, rAce+rZot+CpG, and rZot+rAce in comparison with the control groups (P<.001). There was no significant difference in the anti-Ace IgG titer between rAce and rAce+CpG. Moreover, the titer of anti-Ace IgG was also similar in rZot+rAce andrAce+rZot+CpG groups. The total amount of anti-Ace IgG in mice receiving rZot+rAce with or without CpG was significantly higher than the group receiving rAce or rZot alone (P<.001). A significant disparity was also detected in rAce and rAce+CpG in comparison with vaccine and vaccine+CpG groups (P<.001) while there was no statistical difference in IgG titration among rAce, rZot, and rAce+rZot+CpG in contrast to vaccine and vaccine+CpG groups (Fig. 1).
Table 1.
Antigens administered to each group of mice.
| Group No. |
Antigen received |
| 1 | 100 µl sterile PBS containing 25 µgrAce |
| 2 | 100 µl sterile PBS containing 25 µg rAce and 10 µgCpG |
| 3 | 100 µl sterile PBS containing 25 µg rZot |
| 4 | 100 µl sterile PBS containing 25 µg rZot and 10 µg CpG |
| 5 | 100 µl sterile PBS containing 25 µg rAce and 25 µg rZot |
| 6 | 100 µl sterile PBS containing 25 µg rAce, 25 µg rZot and 10 µg CpG |
| 7 | 100 µl of 105 cfu.ml−1 of Dukoral |
| 8 | 100 µl of 105 cfu.ml−1 of Dukoral containing 10 µg CpG |
| 9 | 100 µl sterile PBS containing 10 µg CpG |
| 10 | 100 µl sterile sodium bicarbonate |
Figure 1.
The Mean Log of serum anti-rAce IgG in different groups of mice.The horizontal axis is related to different mice groups, and the vertical axis indicates the log10 of serum anti-rAce IgG in mice groups.
The total amount of anti-Zot IgG produced in rZot, rZot+CpG and rZot+rAce was significantly higher than the control (PBS) and CpG groups (P<.05 and P<.001, respectively) but still less than the vaccine and vaccine+CpG groups (P<.001 and P<.003, respectively). The mixture of rZot and rAce had no more immunogenicity than rZot alone. Addition of CpG not significantly increased the level of anti-Zot IgG in the rZot+rAce+CpG group in comparison with rZot+rAce group (Fig. 2).
Figure 2.
The Mean Log of serum anti-rZotIgG in different groups of mice.The horizontal axis is related to different mice groups, and the vertical axis indicates the log10 of serum anti-rZot IgG in mice groups.
Anti-Zot and anti-Ace IgA titer in immunized mice
The total amount of IgA antibodies against rZot and rAce were measured in fecal samples of each immunized mouse (Fig. 3 and Fig. 4). The highest anti-rAce IgA titer was related to the following groups: vaccine, vaccine+CpG, and rAce+rZot+CpG. There was a significant disparity in the level of anti-rAce IgA among rAce and rAc+CpG compared with control groups (PBS and CpG). The production of anti-rAce IgA rose up when a combination of rAce and rZot was used compared with the use of rAce alone (P<.001). There was no significant difference in the level of IgA in rAce+rZot and rAce groups with or without CpG (Fig. 3).
Figure 3.
The Mean Log of fecal anti-rAce IgA in different groups of mice.The horizontal axis is related to different mice groups, and the vertical axis indicates the log10 of fecal anti-rAce IgA in mice groups.
Figure 4.
The Mean Log of fecal anti-rZot IgA in different groups of mice.The horizontal axis is related to different mice groups, and the vertical axis indicates the log10 of fecal anti-rZot IgA in mice groups.
The Anti-rZot IgA level was statistically identical in rZot and rAce+rZot groups with or without CpG while these two groups produced higher amount of anti-rZot IgA than the control groups (P<.001). The highest titration was related to vaccine and vaccine+CpG groups, and the former showed significantly higher level of antibody than rZot, rZot+CpG, and rZot+rAce groups (P<.001).
Clearance rate of toxigenic Vibrio cholera in immunized mice
Fecal specimens were collected and cultured from mice after oral administration of 105 cfu.ml−1 of toxigenic V.cholerae ATCC14035 to immunized mice. The clearance rate of toxigenic V. cholerae in different mice groups was determined by culturing fecal samples. All collected data were subjected to statistical analysis. The results showed the same trend on Days 1, 2, and 7 after inoculation of toxigenic strains in immunized mice (Fig. 5). The highest clearance rate was related to the vaccine groups, reaching to 100% on Day 7 compared to control group (P<.001) with the consistency between rZot and rAce which significantly reduced the bacterial shedding (P<.001) The group receiving rAce+rZot+CpG was located in the second rank with the clearance rate of 41%. The descending order of clearance rate in other test groups was as follows: rAce+rZot (38%), rAce+CpG (29%), rZot+CpG (25%), rAce (22%), and rZot (12%).
Figure 5.
Mean Log of colony counts after 24h, 48h and 1 week.The horizontal axis is related to different mice groups, and the vertical axis indicatesthe log of colony count.
Discussion
Cholera has still remained as a critical health threat in developing countries. Several studies have been performed on producing and using different V. cholerae antigens or mixtures as vaccine candidates, some of which revealed acceptable results for using inactivated whole cell of V. cholerae strains in the mixture of cholera toxin B subunit.11,21,22
Epidemiological studies showed the simultaneous occurrence of V. cholerae virulence genes including ctxA, ctxB, zot, and ace in strains responsible for cholera outbreaks.13,15,16 Previously, recombinant CTB was produced, and its immunological activity was investigated in rabbit model.11 In the present study, we followed the hypothesis of using recombinant Zot and Ace toxins, alone or in combination, with or without CpG as adjuvant in comparison with commercial cholera vaccine with killed whole cell V. cholerae+ CTB formulation (Fig. 6). According to the studies conducted on different animal models, infant mice has been revealed as a proper model with parallel findings in human volunteers.23,24
Figure 6.
Percentage of clearance rate of immunized mice after 48h and 1 week.
According to the results of this study, anti-Zot and anti-Ace IgG titers were significantly higher in immunized mice in comparison with control group. The IgG and IgA titers were higher in the sera of mice immunized by recombinant Ace than the group immunized by rZot, indicating the higher immunogenicity of rAce than rZot. In addition, the use of rAce and rZot mixture leaded to the increased level of IgG and IgA in comparison with the groups immunized by the two recombinant proteins, separately; this finding signifies the i) the synergistic activities of the two recombinant proteins in provoking mice immune responses and/or, ii) the adjuvant activity of rZot which could raise the antibody titers against the rAce. Although CpG is known to have a mucosal adjuvant activity, it seems that rZot is more potent to induce the immune responses against rAce in mouse model as mice receiving rAce+rZot raised antibody titers much higher than the group receiving rAce+CpG. Marinaro and colleagues (1998) reported the same results for adjuvant activity of Zot, which could induce immunological responses 40 times(folds) higher in the mice receiving antigen with zot compared with the control group.25 No significant disparity was observed in anti-rAce IgG and IgA levels between rAce+rZot+CpG and rAce+rZot groups, showing the null role of CpG in immunological responses against Ace.
In different groups under study, the IgA level was higher than the IgG level. It was evidenced that oral immunization can efficiently increase the mucosal IgA secretion, which is supposed to have an important effect in preventing from bacterial colonization.
The clearance rate of toxigenic V. cholerae from immunized mice in different challenge groups was significantly higher than the control group. The coherence between rZot and rAce reduced the bacterial shedding significantly. Similar results were reported by Zhang and colleagues (2011) regarding adjuvant activity of Zot in nasal immunization of mice against Escherichia coli O157:H712. In other studies, it was proved that Zot has the ability to increase the penetration of anti-cancer drugs into endothelial cells, suggesting it as a potent candidate in drug delivery.26 Moreover, in our previous study, the antibacterial activity of Ace against standard strains of E.coli, Pseudomonas aeruginosa, and Staphylococcus aureus was proved.19
Previously, the construction and application of Δace/Δzot/ΔctxA derivatives of El Tor strains in CVD110 vaccine candidate was discussed.27 It was shown that although powerful immunogen but this vaccine candidate was insufficiently attenuated despite the absence of known potential enterotoxins of V. cholerae. Therefore, some other unrecognized toxin or colonization factors may be responsible for diarrhea caused by vaccine candidate in volunteers. This would increase the need for highly potent immunogen candidates to circumvent the necessity of inclusion of not fully characterized intra and extracellular compartments in vaccine development studies. Zot and Zce were used in this study to assess the rate of seroconversions in the challenge populations, providing a cost-effective preview of the vaccine candidate before undertaking further experiments and allowing for the rejection of vaccine candidates if proved poor immunogenicity. As expected, the two proteins were evidenced to be cumulatively potent immunogens whose adverse effect can be tolerated in mouse model. Considering their nature as extracellular toxins, which can abundantly provoke human immune system, their beneficial role in vaccine studies should not be ignored. Our mission in achieving a safe and immune vaccine candidate is to perform some nucleotide exchanges in the zot and ace gene sequences in order to retain the immunogenicity and to collapse the full reactogenicity of candidate proteins in challenge experiments. Their combination with commercially available vaccines would be of significant interest in the vaccine development in the future.
In conclusion, the use of recombinant Zot and Ace mixture can produce the proper amount of IgA and IgG against toxigenic V. cholerae and reduce bacterial shedding in immunized mice significantly. We have all reasons to claim that produced rZot and rAce in this study have a proper biological activity and can be used as a potent candidate in cholera vaccine research. However, some limitations exist including i) the toxic nature of ace and zot proteins which perimeter their production in different vector-host combinations and ii) their toxicity for mammalian cells which limits their high-dose use in different invivo experiments.
Materials and methods
Study ethics approval
The study was reviewed and approved by Medical Ethics Committee of Tarbiat Modares University of Medical Science (Code: IR.MODARES.REC) before the study began.
Preparation of polyclonal antibodies of anti-Zot and anti-Ace recombinant proteins
The recombinant Zot and Ace proteins were produced in (pET-28a/zot and pET-28a/ace)/ Escherichia coliTuner and BL21 expression system, respectively, as described previously.19,20 The proteins were purified by Ni-NTA affinity chromatography columns, concentrated with dialysis method, and subjected to Bradford assay to determine the protein concentration. Polyclonal antibodies were produced using two white New Zealand rabbits weighing about 2 kg, which were classified in 2 groups. The Group 1 received 500 µg of the recombinant Zot and Ace proteins mixture with complete Freund adjuvant, and Group 2 received 1 mL of 108cfu.mL−1 inactivated V. cholerae ATCC14035 with complete Freund adjuvant, subcutaneously. In the days of 14 and 28, after the first injection, the antigens were boosted for each group with the same dose with incomplete Freund adjuvant. In order to produce hyper immune serum in the day of 35 (2 weeks after last boost), the rabbit in Group 1 received 250 µg antigens, and the rabbit in Group 2 received 250 µL of inactivated V. cholerae ATCC14035, intravenously. Blood samples were collected in 72 hours after the last injection, and related serums were separated and stored at -20°C until used.
Mice immunization
The immunogenicity of recombinant Zot and Ace proteins was investigated using mouse as an animal model. Six-week female BALB/c mice were divided into 10 different groups, each with 5 members. Each group was immunized with different antigens via oral route. Naive group received sterile sodiumbicarbonate, and the test groups received antigens according to the Table 1. The commercial Dukoral vaccine (SBL Vaccine AB, Sweden), alone or in combination with CpG, was used as control. The final volume of suspension for immunization was 100 µL for each mouse, containing 25µg of recombinant proteins or 10 µg of CpG adjuvant or 105 cfu.ml−1 of Dukoral depending on different groups (Table 1). For better absorption of antigens, mice were fasted 5–6 hours before immunization, and the acidity of stomach was neutralized by sodium bicarbonate 5% few minutes before oral immunization. In the days of 14 and 28, after the first immunization, all the dosages were boosted for each group.
Blood and stool sample collection
Blood samples were collected in the days 0 (before immunization), 14, 28, and 42 from orbital sinus of each mouse. All blood samples were incubated at 37 °C for 30 min and centrifuged at 9000 rpm for 5 min. Serums were separated and stored at -70°C until used. Stool samples were collected on days 0 (before immunization), 14, 28, and 42 and mixed with Phosphate Buffered Saline with tween 20 and 0.05% sodium azide. About 1 gr of each stool was vortexed with10 mL of mentioned buffer and incubated at room temperature for 15 min and centrifuged at 10000 rpm for 20 min. The supernatant was collected and mixed with 1% phenylmethylsulfonyl fluoride (PMSF) and stored at -70°C until used.
Investigation of antibody responses in immunized mice
Total amount of IgG and IgA antibodies against rZot and rAce were measured in blood and stool samples of each immunized mouse. The indirect Enzyme Linked Immuno Assay (ELISA) was performed to determine the antibody titers. Checkerboard titration was done in order to optimize the concentration of recombinant proteins used in ELISA for both serum and stool samples. The optimal concentrations of rZot and rAce proteins were 3 and 1 µg per well, respectively, which together with 100 µL PBS were used in two different series of 96 microplates. All plates were covered with parafilm and incubated at 37°C overnight. Plates were washed three times with PBS-T (PBS + 005% v/w Tween 20) and blocked with 300 µL of blocking buffer containing 5% v/w skim milk in PBS and incubated for 90 min at 37°C. Wells were washed 5 times with PBS-T at the end of the final washing step; microplates were dried with taping several times on a paper tissue. All wells were poured with 100 µL of diluted sera of different mice groups and incubated at 37°C for 1 hour. Washing steps were applied 5 times with PBS-T, and then 100 µL of 1:5000 dilution of HRP-conjugated rabbit anti-mice IgG (Sigma) was applied and incubated at 37°C for 90 min. HRP-conjugated antibodies were visualized by the addition of 100 µL per well of 3,3',5,5'-Tetramethylbenzidine (TMB) as a chromogenic substrate. After 15min incubation at room temperature, the reaction was stopped by 100µL per well of 1 mol.l-1sulfuric acid, and the absorbance was measured at 450 nm by ELISA reader. The same procedure was done for titration of IgA in stool samples except for HRP-conjugated rabbit anti-goat IgA which was used as the secondary antibody.
Challenge experiments with toxigenic Vibrio choleraein immunized mice
The immunized mice were challenged with toxigenic V. cholerae ATCC 14035 to investigate the efficiency of immunization. Oral administration of 105 cfu.ml−1 of toxigenic V.cholerae ATCC14035 was performed to all groups of mice under study. Fecal specimens were collected from mice on days 1, 2, and 7 after inoculation and cultured. Colony count was applied to all fecal samples, and the numbers of V. cholerae colonies were counted. The disparity in colony counts in different groups was an indicative of clearance rate and the ability of immunized mice to clear the V. cholerae strains from intestine.
Statistical analysis
All the qualitative and quantitative data were subjected to statistical analysis (SPSS, ver2, IBM Corp., Armonk, NY, USA). Quantitative data with normal distribution were analyzed with One-Way ANOVA and Games-Howell tests considering 95% confidence interval (α = 0.05).
Supplementary Material
Disclosure of potential conflicts of interest
No potential conflicts of interest were disclosed.
Acknowledgments
We thank the research council of Tarbiat Modares University for supporting the project.
References
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