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Vaccine 25 (2007) 4595–4601
Oral administration of BCG encapsulated in alginate microspheres induces strong Th1 response in BALB/c mice Soheila Ajdary a,∗ , Faramarz Dobakhti b , Mohammad Taghikhani c , Farhad Riazi-Rad a , Shahnaz Rafiei d , Morteza Rafiee-Tehrani b a
Immunology Department, Pasteur Institute of Iran, Pasteur Ave., Tehran IR, Iran b School of Pharmacy, Zanjan University of Medical Sciences, Zanjan, Iran c Faculty of Medical Sciences, Tarbiat Modarres University, Tehran, Iran d Faculty of Medicine, Tehran University of Medical Sciences, Tehran, Iran
Received 26 August 2006; received in revised form 4 March 2007; accepted 21 March 2007 Available online 11 April 2007
Abstract Bacille Calmette-Guerin (BCG) is one of the first vaccines administered to the newborns in developing countries. As an alternative to parenteral administration of vaccines, oral vaccines offer significant logistical advantages. Successful oral immunization, however, requires that vaccine antigens be protected from gastric secretions. In the present study, BALB/c mice were vaccinated orally with BCG encapsulated in alginate microspheres and the immune responses and protective effect were compared with those of mice vaccinated with free BCG by subcutaneous and oral routes. Proliferative and delayed-type hypersensitivity (DTH) responses and IFN-␥ production were significantly higher in mice immunized orally with encapsulated BCG in comparison with results of mice immunized orally with free BCG. Following systemic infection with BCG, mice vaccinated with encapsulated BCG had lower mean bacterial count compared to those vaccinated orally with free BCG. The immune responses induced by oral administration of encapsulated BCG were equal to or better than the responses induced by standard BCG vaccination. © 2007 Elsevier Ltd. All rights reserved. Keywords: BCG; Oral vaccination; Alginate microspheres
1. Introduction Mycobacterium bovis (M. bovis) strain BCG is the only available vaccine for prevention of tuberculosis (TB). BCG has exhibited protection against sever and fatal tuberculosis in children but affords variable protection against the pulmonary form of disease in adults [1–3]. BCG is currently administered parenterally, which primarily stimulates systemic immune responses. Mucosal administration of vaccine offers the ability to trigger both mucosal and systemic immune responses, improved safety and compliance and reduces discomfort associated with administration of vaccines via needle and syringe [4]. Ease of vaccine delivery, especially in new∗ Corresponding author. Tel.: +98 21 66 96 88 57; fax: +98 21 66 96 88 57. E-mail address: [email protected] (S. Ajdary).
0264-410X/$ – see front matter © 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2007.03.039
borns is another advantage of mucosal delivery of vaccine. The best method of inducing mucosal immunity is to administer a vaccine directly to the site where a pathogen invades the host; however, this is not always the most practical one. BCG is given routinely to all infants at birth as a part of the national childhood immunization program. Nasal administration of BCG has been reported to induce protective response in experimental models [5,6]. However, it has been shown that the organogenesis of nasal-associated lymphoid tissue (NALT) is initiated after birth [7]. The effectiveness of vaccine administration via nasal route in neonates remains to be elucidated. The oral route used by Calmette for initial BCG vaccination gave protective immunity but showed deleterious effects such as suppurative cervical adenitis and weak or no response to the skin test [8]. Cervical lymphadenitis is due to translocation of bacteria across the oropharyngeal area after oral ingestion [9]. Furthermore, following oral deliv-
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ery bacilli are exposed to degradation by digestive enzymes. Microencapsulation of the bacteria can be used to overcome these drawbacks [10,11]. Encapsulation of BCG prevents its exposure to the bactericidal effects of gastric secretions that may also lead to a reduction in the doses of BCG required to obtain efficient vaccination, and minimizes the incidence of cervical adenitis [9,12,13]. Sodium alginate is a naturally occurring polysaccharide which can be easily cross-linked into a solid matrix by addition of divalent cations [14]. Cross linking in a water-in-oil emulsion results in the formation of microspheres. Alginate microspheres are biodegradable and safe to use in animals. They have been used to encapsulate proteins, live viruses and bacteria, and plasmid DNA [15–19]. In the present study, we have compared immune responses in BALB/c mice immunized with BCG encapsulated in alginate microspheres by oral route with those of immunized orally and parenterally with free BCG.
2.3. Cell proliferation assay
2. Materials and methods
Spleen cell suspensions were prepared as described above for cell proliferation assay. Cell culture supernatants from triplicate wells were pooled after 72 h incubation and stored at −70 ◦ C for cytokine assay. Cytokines were assessed by using commercial IFN-␥ and IL-4 capture enzyme-linked immunosorbent assays (Bendermed System, Austria) as recommended by the manufacturer.
2.1. Encapsulation of M. bovis BCG Fresh frozen M. bovis BCG Pasteur 1173-P2 produced in BCG unit of Pasteur’s Production Complex of Iran, and stored at −30 ◦ C until use. Bacteria were re-suspended to 2 × 109 CFU/ml, prior to use. The number of colony forming units was determined retrospectively by plating on Lowenstein medium. BCG was encapsulated in alginate microspheres as described elsewhere [20]. Briefly, equal volume of BCG was added 10 ml to sodium alginate 2% (w/v) (provided by FMC biopolymer, Norway) and mixed thoroughly. Twenty milliliters of this mixture was added to 30 ml of olive oil and stirred at 7000 rpm for 5 min to form a water-in-oil emulsion. Subsequently, 0.5 g of calcium chloride (Merck, Germany) was added and stirred to form jelling microspheres. The microspheres were pelleted and the oil residuals were carefully removed. Microspheres were tested for viability of BCG by culturing on Lowenstein medium. The size of microspheres was determined by Malvern Master Seizer particle size analyzer (Malvern, UK). 2.2. Immunizations of mice Female BALB/c mice 6–8 weeks old were used. Mice were immunized subcutaneously at the base of the tail with 100 l of 107 CFU of BCG. For oral immunization, two groups of mice immunized by intra-gastric administration of 1 ml of alginate microspheres, either loaded with 108 CFU of BCG or unloaded. Microspheres were introduced into the stomach with a blunt needle plugged onto a 1 ml syringe. A third group of mice received 108 CFU of non-formulated BCG orally. A group of non-immunized mice was used as control.
Spleen cells were removed and a single-cell suspension was prepared and cultured in RPMI 1640 medium (Sigma, Germany) supplemented with 10% FCS (Sigma), 2 mM lglutamine, 100 U/ml penicillin and 100 g/ml streptomycin. Cells were added to 96-well plates (Nunc, Denmark) in triplicate at a density of 4 × 105 cells per well and cultured with 10 g/ml of Mycobacterium tuberculosis-derived purified protein derivative (PPD, Razi Serum and Vaccine Research Institute, Iran) or with medium alone. The cultures were incubated for 4 days at 37 ◦ C under 5% CO2 and pulsed with 0.5 Ci of [3 H]-thymidine (Amersham, UK) per well for the last 18 h of incubation. Stimulation index (SI) was obtained by dividing the mean counts per minute (cpm) for the triplicate stimulated with PPD by the mean cpm for cells cultured with medium only. 2.4. Cytokine measurements
2.5. DTH Five weeks after immunization the mice were tested for delayed-type hypersensitivity (DTH) as an index of cellmediated immunity. Fifty microliter of PPD (80 g/ml) was injected into one footpad and 50 l of PBS was injected into the contralateral footpad. The DTH reaction was quantified 24 h later by using a dial caliper (sensitivity, 0.05 mm) to measure the difference between the thicknesses of footpads. 2.6. Evaluation of antigen-specific antibody levels Mice were bled retro-orbitally for serum preparation at 12 weeks post immunization. Total IgG, IgG2a and IgG1 antiBCG antibody responses were analyzed in different groups of mice. ELISA plates (Grinere, Germany) were coated with sonicated BCG (10 g/ml) overnight at 4 ◦ C. Non-specific binding sites were blocked by 1% bovine serum albuminPBS (BSA–PBS). Sera diluted in BSA–PBS were added and the plates were incubated for 2 h at 37 ◦ C. The plates were then incubated for 1 h at 37 ◦ C with peroxidase conjugated goat anti-mouse IgG, rabbit anti-mouse IgG1, or rabbit antimouse IgG2a (Sigma, Germany). Plates were developed with TMB (Sigma, Germany). 2.7. Challenge with BCG Control and immunized mice which received free or encapsulated BCG via oral route or free BCG via subcuta-
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neous (sc) route were challenged intravenously with 108 CFU of BCG 8 weeks after immunization. Five weeks later the spleen from each mouse was processed individually. Spleens were homogenized and appropriate dilutions were plated on Lowenstein media. The Petri dishes were kept in sealed plastic bags at 37 ◦ C for 4 weeks before counting. 2.8. Statistical analysis Data were subjected to ANOVA and Student’s t-test for statistical analysis, and a P value of <0.05 was considered to be significant.
3. Results 3.1. Vaccine formulation The mean size of 50% of the microspheres was 11.5 m. BCG formulation in alginate microspheres has no detrimental effect on bacterial viability, all bacilli in alginate microspheres survived at 4 ◦ C until 2 weeks. Moreover, we have previously shown that microencapsulation protects against simulated gastric fluid compared with free BCG [20]. 3.2. Proliferation assay The results of proliferative response showed that PPD induced proliferative response in splenocytes from all immunized mice (Fig. 1). Comparison of stimulation indices 5 weeks after immunization showed the proliferative response of mice orally immunized with encapsulated BCG was significantly higher than the response of mice received free BCG by oral route (P < 0.001). However, the difference between the groups immunized with encapsulated BCG by oral route and free BCG by sc route was not significant. The proliferative responses of all immunized groups were significantly higher than those of controls (P < 0.01) (Fig. 1a). After 12 weeks the proliferative response increased in all immunized groups except the group receiving free BCG orally (Fig. 1b); however, the differences between responses at 5 and 12 weeks were not significant.
Fig. 2. Mean IFN-␥ production produced by spleen cells ± S.D. (pg/ml) of six mice per group from two independent experiments. Mice immunized orally with encapsulated BCG produce significantly higher levels of IFN␥ than mice that received free BCG subcutaneously (P < 0.01), and orally (P < 0.001).
3.3. Cytokine assay Splenocytes were recovered and analyzed for IFN-␥ and IL-4 production 5 and 8 weeks after immunization. Splenocytes from all groups groups released little or no IFN-␥ and IL-4 in the absence of antigen stimulation. However, upon PPD-stimulation IFN-␥ production increased significantly (P < 0.001) (Fig. 2). As shown in Fig. 2, the highest level was found in mice immunized with encapsulated BCG followed by those immunized subcutaneously with free BCG and the difference observed between these two groups was significant (P < 0.01). Both groups produced significantly higher levels of IFN-␥ than mice immunized orally with free BCG (P < 0.001). Eight weeks after immunization, the production of IFN-␥ remained unchanged (data not shown). Low levels of IL-4 were produced 5 weeks after immunization. The mean levels of IL-4 (±S.D.) in mice immunized orally with encapsulated BCG (13.5 ± 3.4 pg/ml) and mice immunized subcutaneously with free BCG (10.8 ± 2.2 pg/ml) were significantly higher compared to mice immunized orally with free BCG (5.1 ± 2 pg/ml) (P < 0.05). No significant difference in IL-4 level was observed after PPD stimulation of lymphocytes from mice immunized subcutaneously with free BCG and mice immunized orally with encapsulated BCG. Control groups produced no detectable IL-4. Eight weeks
Fig. 1. Lymphoproliferative response to PPD 5 (a) and 12 weeks after vaccination (b). Results are mean stimulation indices ± S.D. of six mice per group from two independent experiments. Asterisks indicate values significantly greater than the corresponding value in group that received free BCG orally (P < 0.001).
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Fig. 4. Spleen bacterial load of immunized mice 8 weeks after BCG challenge. Bars represent the mean and standard deviation (error bar) of four mice per group. Asterisks indicate values significantly lower than the group that received free BCG orally (P < 0.05).
Fig. 3. Skin test responses 5 weeks after immunization. Bars represent the mean increase in footpad thickness and standard deviation (error bar) of four mice per group. Asterisks indicate values significantly greater than the group that received free BCG orally (P < 0.05).
IgG2a level was significantly higher in the group immunized with formulated BCG than the groups immunized with free BCG orally and subcutaneously (P < 0.001), however, immunization with free BCG either orally or subcutaneously produce similar level of IgG2a. Mice immunized with formulated BCG showed significantly higher level of IgG1 than those immunized with free BCG, whatever the route of immunization (P < 0.05). The IgG2a/IgG1 ratio could be an indicator whether a Th1 or a Th2 response dominates. In all immunized groups, the antibody responses were predominantly of IgG2a subclass. Encapsulated BCG produced the highest ratios (3.6) compared to free BCG administered by sc and oral routes (2.1 and 1.4, respectively).
after immunization IL-4 was not detectable in any of the groups (data not shown). 3.4. DTH responses Since a positive DTH response is an indicator of cellmediated immunity the mice were tested for DTH response 5 weeks after immunization. Mice immunized with encapsulated BCG and those immunized subcutaneously with free BCG developed high and comparable DTH responses (Fig. 3). Both groups showed significant DTH response compared to control groups (P < 0.005) and the group immunized orally with free BCG (P < 0.05). The DTH response of orally immunized mice with free BCG was also significantly higher than that of controls (P < 0.001).
3.6. Protection against BCG challenge
3.5. Antibody response
The same strain of BCG was used for vaccination and challenge. BCG was completely eliminated in spleens of orally vaccinated mice during the 8-week period. There were very few bacteria in the spleens of subcutaneously vaccinated mice. Five weeks after challenge the number of BCG in spleen was significantly lower in all immunized mice compared to control animals. The protective level in mice immunized with encapsulated BCG was significantly higher than mice that received free BCG orally (P < 0.05) (Fig. 4). Similar level of protection was observed for the groups immunized orally with encapsulated BCG and subcutaneously with free BCG.
Specific serum IgG, IgG1 and IgG2a responses were assayed by ELISA 12 weeks after immunization (Table 1). The IgG level in mice immunized with encapsulated BCG was significantly higher than mice that received free BCG orally (P < 0.001). There was no significant difference between the groups immunized with encapsulated BCG orally and the group immunized subcutaneously. Likewise, the differences among mice orally immunized with free BCG and control groups were not significant.
Table 1 Anti-BCG specific IgG, IgG1, IgG2a in sera from six mice were determined by ELISA, 12 weeks after immunization Study group
IgG
IgG1
IgG2a
IgG2a/IgG1
Encapsulated BCG Oral free BCG Subcutaneous BCG Free microspheres Non-vaccinated
1.58 ± 0.2 0.673 ± 0.1 1.333 ± 0.2 0.406 ± 0.08 0.304 ± 0.08
0.325 ± 0.07 0.074 ± 0.02 0.263 ± 0.05 N.D. N.D.
1.035 ± 0.1 0.10 ± 0.05 0.550 ± 0.05 N.D. N.D.
3.18 1.35 2.09 – –
N.D.: not detectable.
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4. Discussion In tuberculosis, induction of concurrent mucosal and systemic immunity protective against both pulmonary infection and systemic disease progression is desired [21]. Parenteral route of administration is effective in inducing systemic immunity; however, the generated immunity does not generally extend to the mucosal surfaces. In order to achieve mucosal immunity, a vaccine should be delivered to the mucosal associated lymphoid tissue located at mucosal surfaces. Mucosal delivery of BCG vaccine via nasal, oro-gastric and rectal routes has been proven to be efficient means of inducing protective responses [5,6,9,22]. Of mucosal routes of immunization, oral vaccination is the preferred route since it facilitates vaccine administration, uses a physiological delivery process and offers better compliance [4]. Nasal administration of vaccines to newborns and children may not be the ideal one because of the delay in organogenesis of NALT [7] and recurrent upper respiratory tract infections with rhinorrhea and rapid clearance of vaccine [23]. However, vaccine administration via oral route results in degradation in the gastrointestinal tract. The use of oral antigen delivery systems, made of biodegradable microparticles has allowed protecting orally administered antigens. Calcium alginate microspheres are extensively used in industry as stabilizer and thickening agent. Alginate microspheres have also been used successfully for oral immunization against a variety of antigens. Oral vaccination with this antigen delivery system elicits mucosal as well as systemic immune responses. [15,16,18]. Bowersock et al. have shown that oral administration of ovalbumin encapsulated in alginate microspheres results in a mucosal immune responses in the respiratory tract [24]. In this study, we have used alginate microspheres to encapsulate BCG and showed that microencapsulation enhances vaccine immunogenicity and protectivity following oral delivery. Mice were vaccinated orally with BCG encapsulated in alginate microspheres and PPD-induced lymphocyte proliferative response, cytokine production, mycobacteriumspecific DTH response, and antibody response were determined. All responses were significantly higher in group immunized orally with encapsulated BCG compared with the group vaccinated orally with free BCG. Immune responses of the mice immunized orally with encapsulated BCG were greater than or comparable to those obtained after sc immunization with free BCG. High IFN-␥ production and strong DTH response after oral immunization with encapsulated BCG suggest the development of Th1 response which is important in mycobacterial immunity [25–28]. Low levels of IL-4 were detected after BCG vaccination in all vaccinated groups. Although, significantly higher levels of IL-4 were detected in mice immunized orally with encapsulated BCG and subcutaneously with free BCG than in mice vaccinated with free BCG by oral route, the IFN-␥/IL-4 ratio in the two latter groups was higher than the group vaccinated with free BCG by oral route suggesting development of stronger Th1
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type response in those two groups. In keeping with our results, no detectable IL-4 production after oral, intra-gastric, intratracheal and sc administration of BCG have been reported [9,29,30]. It has been shown that IgG1 and IgG2a subclasses of IgG associate with Th2 and Th1 type response, respectively. In further support of Th1 response induction after oral vaccination with BCG encapsulated in alginate microspheres IgG1 and IgG2a levels were determined. Assessment of anti-BCG IgG isotypes (IgG2a/IgG1) showed a preferential Th1 type response in all immunized mice. Encapsulated BCG produced the highest IgG2a/IgG1 ratio followed by the ratios produced by free BCG administered subcutaneously and orally. The higher ratio is indicative of a stronger Th1 response in those receiving encapsulated BCG orally and free BCG subcutaneously. The capacity of BCG encapsulated in alginate but not free BCG to stimulate strong immune responses suggests that the alginate matrix protects BCG against degradation in gastrointestinal tract. It has been shown that alginate microspheres shrink at acidic pH and erode at alkaline pH; thus, it can effectively deliver vaccines and peptides without degradation or alteration into the intestine [16,31–33]. We have shown that alginate microspheres efficiently protect BCG against simulated gastric fluid [20]. These findings suggest alginate encapsulation of BCG is suitable for oral delivery of BCG vaccine without considerable loss of viability in stomach. Lipids have also been used to protect BCG against degradation in the stomach. In agreement with our data, lipidformulated BCG has been shown to be more effective in inducing immune responses than non-formulated BCG [13]. Although, it has been suggested that BCG vaccination may provide less protection against virulent strains [34], M. tuberculosis, M. bovis and BCG strains have been widely used to analyze the protective efficacy of vaccination with BCG [9,13,29,35,36]. Aldwell et al. have shown oral vaccination with lipid-encapsulated BCG provide significant protection against both virulent tubercle bacilli and less virulent strain (M. bovis) [13,37]. Considering safety issues we used BCG strain to determine the protective efficacy of alginate encapsulated BCG. The results showed that oral administration of BCG encapsulated in alginate microspheres confers significant protection and reduces bacterial burdens to levels comparable to those observed in subcutaneously vaccinated mice. Our study extends earlier studies which used lipidencapsulated BCG for oral vaccination of mice, cattle and possums against TB and showed it promotes protectivity against M. bovis and M. tuberculosis [13,37–39]. Aldwell et al., have shown that lipid-microencapsulation can prolong the in vivo survival of bacilli and results in greater recovery of viable bacilli from the mesenteric lymph nodes compared to non-formulated BCG [12,40]. Indeed, we found more acid fast bacilli in Peyer’s patches of mice immunized with encapsulated BCG compared to mice immunized orally with free BCG 4 days after immunization (data not shown). Alginate is mucoadhesive and is likely to adhere to intestinal mucosa for
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a long period of time [41]; which could lead to a more efficient uptake of the bacilli through the gastrointestinal mucosa facilitating bacterial access to lymphoid organs. Our data indicate that oral administration of BCG in alginate microspheres results in strong systemic protective immune responses; however, mucosal immunity and protection against pulmonary infection after oral vaccination with alginate-encapsulated BCG remains to be determined. Alginate microspheres have been used for microencapsulation of BCG [42,43]; however, to our knowledge, this is the first report on evaluating the immunogenicity and protective efficacy of alginate encapsulated BCG.
Acknowledgment We gratefully thank Dr. Anis Jafari for her valuable suggestions in revising the manuscript.
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Oral administration of BCG encapsulated in alginate microspheres induces strong Th1 response in BALB/c mice Soheila Ajdary a,∗ , Faramarz Dobakhti b , Mohammad Taghikhani c , Farhad Riazi-Rad a , Shahnaz Rafiei d , Morteza Rafiee-Tehrani b a
Immunology Department, Pasteur Institute of Iran, Pasteur Ave., Tehran IR, Iran b School of Pharmacy, Zanjan University of Medical Sciences, Zanjan, Iran c Faculty of Medical Sciences, Tarbiat Modarres University, Tehran, Iran d Faculty of Medicine, Tehran University of Medical Sciences, Tehran, Iran
Received 26 August 2006; received in revised form 4 March 2007; accepted 21 March 2007 Available online 11 April 2007
Abstract Bacille Calmette-Guerin (BCG) is one of the first vaccines administered to the newborns in developing countries. As an alternative to parenteral administration of vaccines, oral vaccines offer significant logistical advantages. Successful oral immunization, however, requires that vaccine antigens be protected from gastric secretions. In the present study, BALB/c mice were vaccinated orally with BCG encapsulated in alginate microspheres and the immune responses and protective effect were compared with those of mice vaccinated with free BCG by subcutaneous and oral routes. Proliferative and delayed-type hypersensitivity (DTH) responses and IFN-␥ production were significantly higher in mice immunized orally with encapsulated BCG in comparison with results of mice immunized orally with free BCG. Following systemic infection with BCG, mice vaccinated with encapsulated BCG had lower mean bacterial count compared to those vaccinated orally with free BCG. The immune responses induced by oral administration of encapsulated BCG were equal to or better than the responses induced by standard BCG vaccination. © 2007 Elsevier Ltd. All rights reserved. Keywords: BCG; Oral vaccination; Alginate microspheres
1. Introduction Mycobacterium bovis (M. bovis) strain BCG is the only available vaccine for prevention of tuberculosis (TB). BCG has exhibited protection against sever and fatal tuberculosis in children but affords variable protection against the pulmonary form of disease in adults [1–3]. BCG is currently administered parenterally, which primarily stimulates systemic immune responses. Mucosal administration of vaccine offers the ability to trigger both mucosal and systemic immune responses, improved safety and compliance and reduces discomfort associated with administration of vaccines via needle and syringe [4]. Ease of vaccine delivery, especially in new∗ Corresponding author. Tel.: +98 21 66 96 88 57; fax: +98 21 66 96 88 57. E-mail address: [email protected] (S. Ajdary).
0264-410X/$ – see front matter © 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2007.03.039
borns is another advantage of mucosal delivery of vaccine. The best method of inducing mucosal immunity is to administer a vaccine directly to the site where a pathogen invades the host; however, this is not always the most practical one. BCG is given routinely to all infants at birth as a part of the national childhood immunization program. Nasal administration of BCG has been reported to induce protective response in experimental models [5,6]. However, it has been shown that the organogenesis of nasal-associated lymphoid tissue (NALT) is initiated after birth [7]. The effectiveness of vaccine administration via nasal route in neonates remains to be elucidated. The oral route used by Calmette for initial BCG vaccination gave protective immunity but showed deleterious effects such as suppurative cervical adenitis and weak or no response to the skin test [8]. Cervical lymphadenitis is due to translocation of bacteria across the oropharyngeal area after oral ingestion [9]. Furthermore, following oral deliv-
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ery bacilli are exposed to degradation by digestive enzymes. Microencapsulation of the bacteria can be used to overcome these drawbacks [10,11]. Encapsulation of BCG prevents its exposure to the bactericidal effects of gastric secretions that may also lead to a reduction in the doses of BCG required to obtain efficient vaccination, and minimizes the incidence of cervical adenitis [9,12,13]. Sodium alginate is a naturally occurring polysaccharide which can be easily cross-linked into a solid matrix by addition of divalent cations [14]. Cross linking in a water-in-oil emulsion results in the formation of microspheres. Alginate microspheres are biodegradable and safe to use in animals. They have been used to encapsulate proteins, live viruses and bacteria, and plasmid DNA [15–19]. In the present study, we have compared immune responses in BALB/c mice immunized with BCG encapsulated in alginate microspheres by oral route with those of immunized orally and parenterally with free BCG.
2.3. Cell proliferation assay
2. Materials and methods
Spleen cell suspensions were prepared as described above for cell proliferation assay. Cell culture supernatants from triplicate wells were pooled after 72 h incubation and stored at −70 ◦ C for cytokine assay. Cytokines were assessed by using commercial IFN-␥ and IL-4 capture enzyme-linked immunosorbent assays (Bendermed System, Austria) as recommended by the manufacturer.
2.1. Encapsulation of M. bovis BCG Fresh frozen M. bovis BCG Pasteur 1173-P2 produced in BCG unit of Pasteur’s Production Complex of Iran, and stored at −30 ◦ C until use. Bacteria were re-suspended to 2 × 109 CFU/ml, prior to use. The number of colony forming units was determined retrospectively by plating on Lowenstein medium. BCG was encapsulated in alginate microspheres as described elsewhere [20]. Briefly, equal volume of BCG was added 10 ml to sodium alginate 2% (w/v) (provided by FMC biopolymer, Norway) and mixed thoroughly. Twenty milliliters of this mixture was added to 30 ml of olive oil and stirred at 7000 rpm for 5 min to form a water-in-oil emulsion. Subsequently, 0.5 g of calcium chloride (Merck, Germany) was added and stirred to form jelling microspheres. The microspheres were pelleted and the oil residuals were carefully removed. Microspheres were tested for viability of BCG by culturing on Lowenstein medium. The size of microspheres was determined by Malvern Master Seizer particle size analyzer (Malvern, UK). 2.2. Immunizations of mice Female BALB/c mice 6–8 weeks old were used. Mice were immunized subcutaneously at the base of the tail with 100 l of 107 CFU of BCG. For oral immunization, two groups of mice immunized by intra-gastric administration of 1 ml of alginate microspheres, either loaded with 108 CFU of BCG or unloaded. Microspheres were introduced into the stomach with a blunt needle plugged onto a 1 ml syringe. A third group of mice received 108 CFU of non-formulated BCG orally. A group of non-immunized mice was used as control.
Spleen cells were removed and a single-cell suspension was prepared and cultured in RPMI 1640 medium (Sigma, Germany) supplemented with 10% FCS (Sigma), 2 mM lglutamine, 100 U/ml penicillin and 100 g/ml streptomycin. Cells were added to 96-well plates (Nunc, Denmark) in triplicate at a density of 4 × 105 cells per well and cultured with 10 g/ml of Mycobacterium tuberculosis-derived purified protein derivative (PPD, Razi Serum and Vaccine Research Institute, Iran) or with medium alone. The cultures were incubated for 4 days at 37 ◦ C under 5% CO2 and pulsed with 0.5 Ci of [3 H]-thymidine (Amersham, UK) per well for the last 18 h of incubation. Stimulation index (SI) was obtained by dividing the mean counts per minute (cpm) for the triplicate stimulated with PPD by the mean cpm for cells cultured with medium only. 2.4. Cytokine measurements
2.5. DTH Five weeks after immunization the mice were tested for delayed-type hypersensitivity (DTH) as an index of cellmediated immunity. Fifty microliter of PPD (80 g/ml) was injected into one footpad and 50 l of PBS was injected into the contralateral footpad. The DTH reaction was quantified 24 h later by using a dial caliper (sensitivity, 0.05 mm) to measure the difference between the thicknesses of footpads. 2.6. Evaluation of antigen-specific antibody levels Mice were bled retro-orbitally for serum preparation at 12 weeks post immunization. Total IgG, IgG2a and IgG1 antiBCG antibody responses were analyzed in different groups of mice. ELISA plates (Grinere, Germany) were coated with sonicated BCG (10 g/ml) overnight at 4 ◦ C. Non-specific binding sites were blocked by 1% bovine serum albuminPBS (BSA–PBS). Sera diluted in BSA–PBS were added and the plates were incubated for 2 h at 37 ◦ C. The plates were then incubated for 1 h at 37 ◦ C with peroxidase conjugated goat anti-mouse IgG, rabbit anti-mouse IgG1, or rabbit antimouse IgG2a (Sigma, Germany). Plates were developed with TMB (Sigma, Germany). 2.7. Challenge with BCG Control and immunized mice which received free or encapsulated BCG via oral route or free BCG via subcuta-
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neous (sc) route were challenged intravenously with 108 CFU of BCG 8 weeks after immunization. Five weeks later the spleen from each mouse was processed individually. Spleens were homogenized and appropriate dilutions were plated on Lowenstein media. The Petri dishes were kept in sealed plastic bags at 37 ◦ C for 4 weeks before counting. 2.8. Statistical analysis Data were subjected to ANOVA and Student’s t-test for statistical analysis, and a P value of <0.05 was considered to be significant.
3. Results 3.1. Vaccine formulation The mean size of 50% of the microspheres was 11.5 m. BCG formulation in alginate microspheres has no detrimental effect on bacterial viability, all bacilli in alginate microspheres survived at 4 ◦ C until 2 weeks. Moreover, we have previously shown that microencapsulation protects against simulated gastric fluid compared with free BCG [20]. 3.2. Proliferation assay The results of proliferative response showed that PPD induced proliferative response in splenocytes from all immunized mice (Fig. 1). Comparison of stimulation indices 5 weeks after immunization showed the proliferative response of mice orally immunized with encapsulated BCG was significantly higher than the response of mice received free BCG by oral route (P < 0.001). However, the difference between the groups immunized with encapsulated BCG by oral route and free BCG by sc route was not significant. The proliferative responses of all immunized groups were significantly higher than those of controls (P < 0.01) (Fig. 1a). After 12 weeks the proliferative response increased in all immunized groups except the group receiving free BCG orally (Fig. 1b); however, the differences between responses at 5 and 12 weeks were not significant.
Fig. 2. Mean IFN-␥ production produced by spleen cells ± S.D. (pg/ml) of six mice per group from two independent experiments. Mice immunized orally with encapsulated BCG produce significantly higher levels of IFN␥ than mice that received free BCG subcutaneously (P < 0.01), and orally (P < 0.001).
3.3. Cytokine assay Splenocytes were recovered and analyzed for IFN-␥ and IL-4 production 5 and 8 weeks after immunization. Splenocytes from all groups groups released little or no IFN-␥ and IL-4 in the absence of antigen stimulation. However, upon PPD-stimulation IFN-␥ production increased significantly (P < 0.001) (Fig. 2). As shown in Fig. 2, the highest level was found in mice immunized with encapsulated BCG followed by those immunized subcutaneously with free BCG and the difference observed between these two groups was significant (P < 0.01). Both groups produced significantly higher levels of IFN-␥ than mice immunized orally with free BCG (P < 0.001). Eight weeks after immunization, the production of IFN-␥ remained unchanged (data not shown). Low levels of IL-4 were produced 5 weeks after immunization. The mean levels of IL-4 (±S.D.) in mice immunized orally with encapsulated BCG (13.5 ± 3.4 pg/ml) and mice immunized subcutaneously with free BCG (10.8 ± 2.2 pg/ml) were significantly higher compared to mice immunized orally with free BCG (5.1 ± 2 pg/ml) (P < 0.05). No significant difference in IL-4 level was observed after PPD stimulation of lymphocytes from mice immunized subcutaneously with free BCG and mice immunized orally with encapsulated BCG. Control groups produced no detectable IL-4. Eight weeks
Fig. 1. Lymphoproliferative response to PPD 5 (a) and 12 weeks after vaccination (b). Results are mean stimulation indices ± S.D. of six mice per group from two independent experiments. Asterisks indicate values significantly greater than the corresponding value in group that received free BCG orally (P < 0.001).
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Fig. 4. Spleen bacterial load of immunized mice 8 weeks after BCG challenge. Bars represent the mean and standard deviation (error bar) of four mice per group. Asterisks indicate values significantly lower than the group that received free BCG orally (P < 0.05).
Fig. 3. Skin test responses 5 weeks after immunization. Bars represent the mean increase in footpad thickness and standard deviation (error bar) of four mice per group. Asterisks indicate values significantly greater than the group that received free BCG orally (P < 0.05).
IgG2a level was significantly higher in the group immunized with formulated BCG than the groups immunized with free BCG orally and subcutaneously (P < 0.001), however, immunization with free BCG either orally or subcutaneously produce similar level of IgG2a. Mice immunized with formulated BCG showed significantly higher level of IgG1 than those immunized with free BCG, whatever the route of immunization (P < 0.05). The IgG2a/IgG1 ratio could be an indicator whether a Th1 or a Th2 response dominates. In all immunized groups, the antibody responses were predominantly of IgG2a subclass. Encapsulated BCG produced the highest ratios (3.6) compared to free BCG administered by sc and oral routes (2.1 and 1.4, respectively).
after immunization IL-4 was not detectable in any of the groups (data not shown). 3.4. DTH responses Since a positive DTH response is an indicator of cellmediated immunity the mice were tested for DTH response 5 weeks after immunization. Mice immunized with encapsulated BCG and those immunized subcutaneously with free BCG developed high and comparable DTH responses (Fig. 3). Both groups showed significant DTH response compared to control groups (P < 0.005) and the group immunized orally with free BCG (P < 0.05). The DTH response of orally immunized mice with free BCG was also significantly higher than that of controls (P < 0.001).
3.6. Protection against BCG challenge
3.5. Antibody response
The same strain of BCG was used for vaccination and challenge. BCG was completely eliminated in spleens of orally vaccinated mice during the 8-week period. There were very few bacteria in the spleens of subcutaneously vaccinated mice. Five weeks after challenge the number of BCG in spleen was significantly lower in all immunized mice compared to control animals. The protective level in mice immunized with encapsulated BCG was significantly higher than mice that received free BCG orally (P < 0.05) (Fig. 4). Similar level of protection was observed for the groups immunized orally with encapsulated BCG and subcutaneously with free BCG.
Specific serum IgG, IgG1 and IgG2a responses were assayed by ELISA 12 weeks after immunization (Table 1). The IgG level in mice immunized with encapsulated BCG was significantly higher than mice that received free BCG orally (P < 0.001). There was no significant difference between the groups immunized with encapsulated BCG orally and the group immunized subcutaneously. Likewise, the differences among mice orally immunized with free BCG and control groups were not significant.
Table 1 Anti-BCG specific IgG, IgG1, IgG2a in sera from six mice were determined by ELISA, 12 weeks after immunization Study group
IgG
IgG1
IgG2a
IgG2a/IgG1
Encapsulated BCG Oral free BCG Subcutaneous BCG Free microspheres Non-vaccinated
1.58 ± 0.2 0.673 ± 0.1 1.333 ± 0.2 0.406 ± 0.08 0.304 ± 0.08
0.325 ± 0.07 0.074 ± 0.02 0.263 ± 0.05 N.D. N.D.
1.035 ± 0.1 0.10 ± 0.05 0.550 ± 0.05 N.D. N.D.
3.18 1.35 2.09 – –
N.D.: not detectable.
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4. Discussion In tuberculosis, induction of concurrent mucosal and systemic immunity protective against both pulmonary infection and systemic disease progression is desired [21]. Parenteral route of administration is effective in inducing systemic immunity; however, the generated immunity does not generally extend to the mucosal surfaces. In order to achieve mucosal immunity, a vaccine should be delivered to the mucosal associated lymphoid tissue located at mucosal surfaces. Mucosal delivery of BCG vaccine via nasal, oro-gastric and rectal routes has been proven to be efficient means of inducing protective responses [5,6,9,22]. Of mucosal routes of immunization, oral vaccination is the preferred route since it facilitates vaccine administration, uses a physiological delivery process and offers better compliance [4]. Nasal administration of vaccines to newborns and children may not be the ideal one because of the delay in organogenesis of NALT [7] and recurrent upper respiratory tract infections with rhinorrhea and rapid clearance of vaccine [23]. However, vaccine administration via oral route results in degradation in the gastrointestinal tract. The use of oral antigen delivery systems, made of biodegradable microparticles has allowed protecting orally administered antigens. Calcium alginate microspheres are extensively used in industry as stabilizer and thickening agent. Alginate microspheres have also been used successfully for oral immunization against a variety of antigens. Oral vaccination with this antigen delivery system elicits mucosal as well as systemic immune responses. [15,16,18]. Bowersock et al. have shown that oral administration of ovalbumin encapsulated in alginate microspheres results in a mucosal immune responses in the respiratory tract [24]. In this study, we have used alginate microspheres to encapsulate BCG and showed that microencapsulation enhances vaccine immunogenicity and protectivity following oral delivery. Mice were vaccinated orally with BCG encapsulated in alginate microspheres and PPD-induced lymphocyte proliferative response, cytokine production, mycobacteriumspecific DTH response, and antibody response were determined. All responses were significantly higher in group immunized orally with encapsulated BCG compared with the group vaccinated orally with free BCG. Immune responses of the mice immunized orally with encapsulated BCG were greater than or comparable to those obtained after sc immunization with free BCG. High IFN-␥ production and strong DTH response after oral immunization with encapsulated BCG suggest the development of Th1 response which is important in mycobacterial immunity [25–28]. Low levels of IL-4 were detected after BCG vaccination in all vaccinated groups. Although, significantly higher levels of IL-4 were detected in mice immunized orally with encapsulated BCG and subcutaneously with free BCG than in mice vaccinated with free BCG by oral route, the IFN-␥/IL-4 ratio in the two latter groups was higher than the group vaccinated with free BCG by oral route suggesting development of stronger Th1
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type response in those two groups. In keeping with our results, no detectable IL-4 production after oral, intra-gastric, intratracheal and sc administration of BCG have been reported [9,29,30]. It has been shown that IgG1 and IgG2a subclasses of IgG associate with Th2 and Th1 type response, respectively. In further support of Th1 response induction after oral vaccination with BCG encapsulated in alginate microspheres IgG1 and IgG2a levels were determined. Assessment of anti-BCG IgG isotypes (IgG2a/IgG1) showed a preferential Th1 type response in all immunized mice. Encapsulated BCG produced the highest IgG2a/IgG1 ratio followed by the ratios produced by free BCG administered subcutaneously and orally. The higher ratio is indicative of a stronger Th1 response in those receiving encapsulated BCG orally and free BCG subcutaneously. The capacity of BCG encapsulated in alginate but not free BCG to stimulate strong immune responses suggests that the alginate matrix protects BCG against degradation in gastrointestinal tract. It has been shown that alginate microspheres shrink at acidic pH and erode at alkaline pH; thus, it can effectively deliver vaccines and peptides without degradation or alteration into the intestine [16,31–33]. We have shown that alginate microspheres efficiently protect BCG against simulated gastric fluid [20]. These findings suggest alginate encapsulation of BCG is suitable for oral delivery of BCG vaccine without considerable loss of viability in stomach. Lipids have also been used to protect BCG against degradation in the stomach. In agreement with our data, lipidformulated BCG has been shown to be more effective in inducing immune responses than non-formulated BCG [13]. Although, it has been suggested that BCG vaccination may provide less protection against virulent strains [34], M. tuberculosis, M. bovis and BCG strains have been widely used to analyze the protective efficacy of vaccination with BCG [9,13,29,35,36]. Aldwell et al. have shown oral vaccination with lipid-encapsulated BCG provide significant protection against both virulent tubercle bacilli and less virulent strain (M. bovis) [13,37]. Considering safety issues we used BCG strain to determine the protective efficacy of alginate encapsulated BCG. The results showed that oral administration of BCG encapsulated in alginate microspheres confers significant protection and reduces bacterial burdens to levels comparable to those observed in subcutaneously vaccinated mice. Our study extends earlier studies which used lipidencapsulated BCG for oral vaccination of mice, cattle and possums against TB and showed it promotes protectivity against M. bovis and M. tuberculosis [13,37–39]. Aldwell et al., have shown that lipid-microencapsulation can prolong the in vivo survival of bacilli and results in greater recovery of viable bacilli from the mesenteric lymph nodes compared to non-formulated BCG [12,40]. Indeed, we found more acid fast bacilli in Peyer’s patches of mice immunized with encapsulated BCG compared to mice immunized orally with free BCG 4 days after immunization (data not shown). Alginate is mucoadhesive and is likely to adhere to intestinal mucosa for
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a long period of time [41]; which could lead to a more efficient uptake of the bacilli through the gastrointestinal mucosa facilitating bacterial access to lymphoid organs. Our data indicate that oral administration of BCG in alginate microspheres results in strong systemic protective immune responses; however, mucosal immunity and protection against pulmonary infection after oral vaccination with alginate-encapsulated BCG remains to be determined. Alginate microspheres have been used for microencapsulation of BCG [42,43]; however, to our knowledge, this is the first report on evaluating the immunogenicity and protective efficacy of alginate encapsulated BCG.
Acknowledgment We gratefully thank Dr. Anis Jafari for her valuable suggestions in revising the manuscript.
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