IL-2-secreting recombinant bacillus Calmette Guerin can overcome a Type 2 immune response and corticosteroid-induced immunosuppression to elicit a Type 1 immune response
Sarah L. Young1,
Michael A. ODonnell2 and
Glenn S. Buchan1
1 Department of Microbiology, University of Otago, 700 Cumberland Street, PO Box 56, Dunedin, New Zealand 2 Department of Urology, Iowa University, Iowa City, IA 52242, USA
Correspondence to: G. Buchan; E-mail: glen.buchan{at}stonebow.otago.ac.nz
Transmitting editor: K. Shortman
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Abstract
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The efficacy of bacillus Calmette Guerin (BCG) as a vaccine against tuberculosis is adversely affected by both genetic and environmental factors on the immune system. In this study we have demonstrated that a recombinant BCG (rBCG) secreting biologically active IL-2 has the ability to induce a Th1 profile in both immunocompromised and in IL-4 transgenic (Tg) mice. Dexamethasone (DXM) was administered orally to mice prior to vaccination with either rBCG or normal BCG (nBCG). Six weeks post-vaccination with rBCG, splenocytes from DXM-treated mice exhibited a strong antigen-specific proliferative response, while also secreting large amounts of IFN-
and low levels of IgG1. The opposite profile occurred when DXM-treated mice were vaccinated with nBCG. Splenocytes from these mice showed no significant proliferation and produced a cytokine profile associated with a Th2 immune response, in addition to exhibiting high levels of serum IgG1. In the IL-4 Tg model, mice vaccinated with rBCG again produced a strong Th1 immune response, exhibiting a high antigen-specific IFN-
:IL-4 ratio and a concomitantly high IgG2a:IgG1 ratio. IL-4 Tg mice vaccinated with nBCG produced the opposite profile. These findings suggest that BCG can be made more robust by incorporating immunopotentiating cytokines into the vaccine.
Keywords: bacillus Calmette Guerin, cytokine, immunotherapy, Th1/Th2, tuberculosis, vaccination
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Introduction
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Tuberculosis (TB), the disease associated with Myco bacterium tuberculosis infection, is responsible for >3 million deaths annually (1). Cell-mediated (Type 1), but not humoral (Type 2) immune responses protect humans and animals against chronic mycobacterial diseases (24). Effective vaccination against TB must produce an immunological imprint so that a protective cell-mediated immune response is induced in all individuals.
The standard anti-TB vaccine is bacillus Calmette Guerin (BCG), a live attenuated form of Mycobacterium bovis. While some studies have shown BCG provides up to 80% protection, others have shown this vaccine confers no protection at all (5,6). Reports of varying efficacy have therefore resulted in the search for new TB vaccines.
However, the fact that BCG has already undergone the most extensive vaccine safety trial in the world, allied with its proven adjuvant properties, suggests it should not be discarded prematurely. The advent of recombinant technology has enabled BCG to be modified genetically. In 1994, ODonnell et al. produced a recombinant BCG that constitutively secreted IL-2 in a biologically active form (7). IL-2 is an important regulator of anti-mycobacterial immune responses as it drives the proliferation and activation of CD4 T cells (4). In addition, this cytokine activates and is a growth factor for NK (8) cells and 
T cells (9), which are known to be an early source of IFN-
(10,11). IFN-
is an important immunoregulatory cytokine that activates macrophages and promotes the development of a Type 1 response (12). The importance of this cytokine in TB has been demonstrated in IFN-
knockout mice, which are very susceptible to infection with virulent mycobacteria (13,14). Experiments have also shown that vaccination with recombinant IL-2-secreting BCG elicits high IFN-
production (7,15,16).
Despite extensive efforts to find an alternative vaccine against TB, experimental trials have been unable to identify a candidate that offers superior protection to BCG. A limiting factor in these trials has been that BCG usually performs well under controlled experimental conditions (17,18). In these situations it is difficult to show any statistically significant improvement in vaccine efficacy between BCG and potential alternatives. It has been proposed that environmental or genetic factors, which compromise the hosts immune response to BCG, may help explain why the efficacy of BCG appears to be unpredictable under field conditions (17,19,20,21).
In TB, the expression of a Type 2 immune profile is known to be associated with the disease state (1,13,14,22). Any genetic or environmental factor that predisposes an organism towards a Type 2-biased immune response is likely to weaken the protective effects of vaccination against TB (19,23,24). To test this hypothesis, we have compared an IL-2-secreting, BCG (rBCG) vaccine with normal BCG (nBCG) in two models designed to favor Type 2 responses. The first model exploits the observation that corticosteroids affect T cell activation in a number of ways, one of which is to enhance Th2 cytokine synthesis (25). A similar immunological state can be induced in individuals exposed to natural stresses such as malnutrition (26). The second model is based on the knowledge that IL-4 is the key cytokine involved in inducing differentiation and activation of Type 2 lymphocytes. Studies have shown that patients with TB have decreased production of antigen-specific IL-2 and IFN-
(27), while showing increased concentrations of serum IgG1 and IL-4, compared with healthy controls. Research on animal models has also shown that increased transcription of IL-4 is associated with a non-protective immune response to M. bovis and M. tuberculosis (4,28). We have used a transgenic (Tg) mouse model in which IL-4 is expressed under the control of the H-2K class 1 promoter, leading to constitutive expression in most organs (29). Consequently, this mouse strain exhibits a Type 2 immune profile in response to BCG vaccination.
Using these models we have compared the ability of nBCG and rBCG to elicit a protective Type 1 immune response in the presence of a transitory suppression of Type 1 responses [dexamethasone (DXM) model] or a pre-existing Type 2 immune response (IL-4 Tg model).
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Methods
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Animals
Healthy C57BL/6 and BALB/c mice were obtained from the Department of Animal Laboratory Sciences, University of Otago, Dunedin, New Zealand. Male mice were used at 68 weeks of age. C3HeJ wild-type and IL-4 Tg mice (29) were obtained from the Malaghan Institute for Medical Research (Wellington, New Zealand). Mice used were aged 68 weeks. All animals were housed in cages containing five animals, and kept on a daily 12-h cycle of light and dark. The mice were fed ad libitum on rat pellets (R-94).
Ethical approval for these experiments was granted by the Otago University Animal Ethics Committee (grant no. 39/98).
Immunizations
DXM study
DXM sodium phosphate (DXM) (100 µg/day) was administered orally in water for 7 days prior to vaccination. Water alone was used for control animals. Groups of five mice were immunized s.c. with 106 rBCG or nBCG. BCG was cultured in 7H9 broth (Difco, Detroit, MI) and organisms were allowed to grow to mid-log phase before vaccination. Mice were subsequently left for 6 weeks before sacrifice. Each experiment was repeated 3 times.
IL-4 Tg study
Groups of five mice were immunized s.c. with 106 rBCG or nBCG as above. Mice were subsequently left for 16 weeks before sacrifice. Each experiment was repeated 3 times.
Cell proliferation studies
Splenocytes were washed in medium 3 times and cultured in DMEM (Gibco/BRL, Life Technologies, Gaithersburg, MD) containing 5% FBS (Gibco/BRL), 25mM HEPES (Sigma, St Louis, MO) and 40 µg/ml gentamicin sulfate (David Bull, Melbourne, Australia). Cells (3 x 105) were plated along with either medium alone or medium containing 50 µg/ml purified protein derivative (PPD)-B (CSL, Parkville, Australia) or 12 µg/ml concanavalin A (Con A; Sigma) in 96-well round-bottom plates (Nunc, Roskilde, Denmark). The final volume was 150 µl in each well. Each sample was plated in triplicate and cultured for a period of 3 days in a humidified atmosphere of CO2 in air. Wells were pulsed with 0.5 µCi of [3H]thymidine (Amersham Life Science, Little Chalfont, UK) and cultured for a further 18 h before harvest onto microfiber filters (Whatman International, Maidstone, UK). Proliferation was determined by measuring [3H]thymidine incorporation using a Microbeta counter (Wallac, Turku, Finland), and was expressed as c.p.m. Background levels of proliferation for each experiment did not exceed 1000 c.p.m. and these values were subtracted from antigen-stimulated proliferation values.
Cytokine analysis
Lymphocytes were isolated, washed and 2 x 106cells were plated in 24-well plates (Nunc). Cells were cultured along with either DMEM, 50 µg/ml PPD-B or 12 µg/ml Con A (Sigma) for a period of 72 h. Supernatants were removed and analyzed for the presence of IL-4, IL-10 and IFN-
using a sandwich ELISA (PharMingen, San Diego, CA). Assays were performed according to the manufacturers instructions, using 2 µg/ml of primary anti-cytokine capture antibody and 1 µg/m of l anti-cytokine-detecting mAb. The OD was determined at 450nm, using a spectrophotometer (BioRad, Hercules, CA). The amount of each cytokine in the supernatant was extrapolated from the standard curve. The standards were recombinant cytokines (PharMingen) serially diluted in 1:2 dilutions from 1000 to 50 pg/ml for IL-4, 1000 to 15 pg/ml for IL-10 and 25,000 to 50 pg/ml for IFN-
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RT-PCR
Total cellular RNA was isolated from activated cells by acid guanidium isothiocyanatephenolchloroform extraction as previously described (30). Cytokine mRNA were assessed by a semiquantitative RT-PCR. RNA was reverse transcribed, using reverse transcriptase and oligo(dT) primers (Boehringer Mannheim, Mannheim, Germany) according to the manufacturers protocol. The cDNA was amplified using primers to murine IL-2, IL-4, IFN-
and ß-actin. PCR was performed in a reaction mixture containing 2 µl cDNA, 25 mM dNTP, 0.4 µmol/l of each primer and 2.5 U of Taq DNA polymerase (Boehringer Mannheim). Samples were amplified with a thermocycler (Hybaid, Teddington, UK) using the following protocol: initial denaturing of 95°C for 1 min, followed by cycles of 95°C for 30 s, 54°C for 45 s and 72°C for 45 s (32 cycles for each cytokine) with a final extension at 72°C for 5 min. Samples were separated by electrophoresis in 2% agarose gel, and the results were determined by OD readings and arbitrarily expressed as mRNA units. All data was normalized to ß-actin.
Flow cytometry
Splenocytes were cultured at a concentration of 3 x 106/ml in DMEM + 5% FCS with PPD-B (50 µg/ml) for 72 h in 24-well plates (Nunc), as above. Cells were then washed and incubated with 2 µg of either FITC-conjugated anti-CD4 or FITC-conjugated anti-CD8a (Sigma) and 2 µg of phycoerythrin (PE)-conjugated anti-CD25 (Sigma). The cell samples were incubated at 4°C for 30 min, washed and resuspended in 300 µl FACS buffer (PBS + 1% FBS + 0.1% sodium azide), Flow cytometric analysis was carried out on 10,000 events using a FACSCalibur (Becton Dickinson, Mountain View, CA). Fluorescence data was collected with logarithmic amplification and was analyzed using CellQuest software.
Serum antibody detection
Mice were heart-bed and the whole blood was allowed to clot. The serum was pooled from the various groups of mice. ELISA plates were coated overnight with 50 µg/ml of PPD-B, washed, blocked with PBS + 10% FBS, and serial dilutions of serum samples and standards were added. Plates were incubated overnight at 4°C. Anti-IgG1 and IgG2a antibodies (Sigma) were added at dilutions recommended by the manufacturer, and the plates were incubated for 1 h at room temperature. Peroxidase-conjugated goat anti-mouse mAb was added to each well and the plates were incubated for a further 45 min, before the addition of TMB substrate (BioRad). The reaction was stopped by addition of 1 N H2SO4 and the OD was read at 450 nm.
Statistical analysis
Data for each experiment were expressed as a mean value (± SEM) and the results were analyzed for statistical significance (unpaired Students t-test) using the software package InStat (2.01). The level of significance was set at P < 0.05.
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Results
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rBCG overcomes the immunosuppressive effects of DXM, and elicits greater numbers of T cells and enhanced antigen-specific proliferation
Table 1 shows that both C57BL/6 and BALB/c mice vaccinated with rBCG, after DXM treatment, generated significantly more antigen-specific CD4 T cells (34 and 38% respectively) than those vaccinated with nBCG (25 and 22% respectively) (P < 0.001). The presence of CD25 on these CD4 T cells indicated the proportion of activated splenocytes. Cells from the two strains vaccinated with rBCG contained more activated CD4 T cells (41 and 44%) than mice vaccinated with normal BCG (34 and 27%) (P = 0.001). Similar patterns were seen when we examined the levels of antigen-specific CD8 T cells elicited by the two vaccines after DXM administration. Mice vaccinated with rBCG produced a much larger population of CD8 T cells (19.1 and 20%) than mice vaccinated with the normal BCG (13.6 and 17%) (P < 0.001).
We then analyzed the antigen-specific proliferative response to PPD by the two strains of mice. In the absence of DXM immunosuppression, both nBCG and rBCG were able to sensitize T cells as shown by the ability of splenocytes to be re-stimulated in vitro by specific antigen. This response was significantly greater (P < 0.05) than that to rBCG vaccine was (Fig. 1). Prior administration of DXM reversed this trend. Splenocytes from both BALB/c and C57BL/6 mice vaccinated with standard BCG showed greatly reduced antigen responses after corticosteroid treatment. By contrast, cells from DXM-treated mice which had been vaccinated with rBCG continued to proliferate strongly to PPD. Their responses were significantly greater than those of animals vaccinated with nBCG (P < 0.005). Interestingly, the splenocytes from C57BL/6 mice pretreated with DXM and vaccinated with rBCG showed significantly enhanced proliferative responses, as compared to those not treated with DXM (P < 0.01). This trend was also apparent in BALB/c mice although it did not reach statistical significance.

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Fig. 1. Antigen-specific proliferation of DXM-treated mice. Spleno cyte proliferation in response to PPD stimulation was determined 6 weeks after vaccination with rBCG or nBCG. Splenocyte responses from mice treated with the corticosteroid DXM for 7 days prior to vaccination were compared to those of mice given no DXM before vaccination.
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DXM does not affect the ability of mice vaccinated with rBCG to produce a strong Type 1 immune response
Figure 2(a) compares the ability of normal or immunosuppressed mice to respond to vaccination with either rBCG or nBCG, as measured by antigen-specific IFN-
production. DXM treatment had no effect on the ability of either C57BL/6 or BALB/c mice, vaccinated with rBCG, to produce high levels of IFN-
. By contrast, mice vaccinated with nBCG were impaired in their ability to produce this cytokine after treatment with DXM, with both strains of mice exhibiting a 100-fold decrease in the amount of IFN-
produced following DXM administration (P < 0.001).
To confirm these findings, RT-PCR was used to detect cytokine transcription. Figure 2(b) shows the antigen-specific cytokine profiles elicited by the two different vaccines after DXM treatment. The levels of IFN-
mRNA transcribed by animals vaccinated with rBCG were significantly higher than those vaccinated with nBCG (P < 0.001). By contrast, the levels of IL-4 mRNA transcription were not significantly altered by pretreatment with DXM and rBCG did not appear to influence its production. The data show that there is an inverse relationship in the ratios of IFN-
:IL-4 mRNA transcribed in response to the two vaccines. In mice vaccinated with rBCG, there was a higher level of IFN-
produced compared to IL-4. The reverse situation occurred in mice vaccinated with nBCG. This pattern could also be detected at 4 weeks post-vaccination (data not shown). In addition to the IFN-
:IL-4 ratio, we also compared IL-2 and IL-10 mRNA production. In mice vaccinated with rBCG, there was much higher levels of IL-2 produced compared to IL-10. The reverse situation was observed for mice vaccinated with nBCG after DXM administration (Fig. 2c). Analysis of the differences in the production of IL-2 and IL-10 mRNA between the two vaccines showed them to be highly significant (P < 0.001).
As a further indicator of the immune phenotype produced by the two vaccines, we examined the antibody profile produced. BALB/c mice vaccinated with rBCG after DXM treatment produced high titers of IgG2a and exhibited a high IgG2a: IgG1 ratio (Fig. 3). This pattern is reversed in BALB/c mice vaccinated with nBCG after DXM administration. C57BL/6 mice vaccinated with rBCG do not appear to produce any antigen-specific IgG1 after DXM treatment while abundant PPD-specific IgG1 was detected in the same strain after vaccination with nBCG (data not shown). As C57BL/6 mice did not appear to produce the IgG2a isotype, no IgG2a:IgG1 ratio could be calculated.

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Fig. 3. Antibody isotype determination. The antigen-specific serum IgG isotype produced by BALB/c mice 6 weeks after vaccination with rBCG or nBCG were assayed by ELISA. Both groups of mice had been treated with the corticosteroid DXM for 7 days prior to vaccination.
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rBCG enhances the levels of CD4 and CD8 T cells, and evokes a high degree of antigen-specific proliferation in both the wild-type and the IL-4 Tg mice
Splenocytes from both IL-4 Tg and wild-type mice, vaccinated with either rBCG or nBCG, were cultured for 72 h with PPD-B to measure the antigen-specific proliferative response after vaccination (Fig. 4). It was evident that both vaccines maintain levels of proliferation which are 10-fold higher than background, for up to 4 months post-vaccination. rBCG evoked a high degree of proliferation in both the IL-4 Tg and wild-type strains of mice. Statistical analysis indicates that there is no significant difference in the levels of antigen-specific proliferation between the two strains in response to rBCG (P = 0.05). However, the proliferation induced by rBCG in the wild-type group was significantly higher than that induced by nBCG (P <0.001).

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Fig. 4. Antigen-specific proliferation by IL-4 Tg mice. Splenocytes were cultured with PPD-B for 72 h and antigen-specific proliferation was determined by [3H]thymidine incorporation. The responses elicited in IL-4 Tg and wild-type (C3HeJ) mice 16 weeks post-vaccination with either live rBCG or live nBCG are shown.
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Splenocytes from both strains of mice vaccinated with either rBCG or nBCG were analyzed for phenotype. Table 2 shows that animals vaccinated with rBCG generated significantly more antigen-specific CD4 T cells than those vaccinated with nBCG (P < 0.01). Both the IL-4 Tg and wild-type mice vaccinated with rBCG exhibited a significantly higher level of antigen-specific CD4 and CD8 T cells when compared to those mice vaccinated with nBCG. Cells from the groups vaccinated with rBCG contained more activated T cells than mice vaccinated with nBCG, as judged by CD25 expression (P = 0.001).
rBCG is able to evoke high levels of Type 1 cytokines in both the IL-4 Tg and wild-type mice, whereas nBCG cannot
Figure 5(a) compares the ability of both the IL-4 Tg and wild-type mice to respond to vaccination with either rBCG or nBCG, as measured by antigen-specific cytokine production. Three cytokines were initially assayed (IL-2, IFN-
and IL-10). However, as there was a low level of IL-2 and IL-10 produced by both vaccines (<1 ng/ml), they have been omitted from Fig. 5(a). The rBCG was able to evoke high levels of IFN-
in both the IL-4 Tg and wild-type mice, whereas nBCG did not. The difference between the two vaccines is highly significant (P < 0.001).
Because cytokines such as IL-4 are sometimes difficult to detect by ELISA, RT-PCR was used to confirm the findings. Background levels of mRNA were subtracted from the test samples, in order to see the cytokine profile obtained in response to antigen re-stimulation. The measured levels of mRNA showed a similar cytokine profile to the ELISA results. Figure 5(b) shows there was a high IFN-
:IL-4 ratio (Type 1) in mice vaccinated with rBCG, whereas mice vaccinated with nBCG exhibit the opposite profile (Type 2).
Mice vaccinated with rBCG also produced a higher IL-2:IL-10 ratio than mice vaccinated with nBCG (Fig. 5c). The overall analysis of the cytokine production results confirmed that rBCG preferentially elicited a Type 1 immune response, in both strains of mice, whereas nBCG produced a profile associated with a Type 2 response.
Both the IL-4 Tg and wild-type strains of mice produced high levels of IgG2a and exhibited a high IgG2a:IgG1 ratio after vaccination with rBCG (Fig. 6). This antibody profile is associated with a Type 1 immune response. Conversely, mice vaccinated with nBCG produced an antibody isotype pattern consistent with a Type 2 immune profile, with a high IgG1:IgG2a ratio.

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Fig. 6. Antibody isotype analysis. The antigen-specific, serum IgG isotype was determined by ELISA. Antibody titers produced by both IL-4 Tg and IL-4 wild-type mice 16 weeks post-vaccination with either rBCG or nBCG is shown.
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Discussion
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We postulate that the failure of BCG in the field may be at least partially explained by poor or inappropriate (Type 2) host responses rather than the poor immunogenicity of BCG. Rather than discard the vaccine, we suggest it may be more advantageous to alter the BCG, so that it is consistently able to induce protective, Type 1, immune responses, even under adverse conditions. Dai and McMurray (26) have shown that protein malnourishment induces an immunocompromised state which impairs responsiveness to vaccines against TB, supposedly by altering the cytokine profiles to favor the development of a Type 2 immune response. We have used DXM to mimic the immunosuppressive activities of cortisol, which is produced naturally during stress. In addition, we have also used an IL-4 Tg mouse model. Both of these models produce a Type 2 cytokine profile in response to mycobacterial antigen stimulation.
BCG strains secreting cytokines, such as granulocyte macrophage colony stimulating factor, IL-2 and IFN-
, have been shown to modify and potentiate the immune responses to BCG antigens (7,16,31). The inclusion of IL-2 with BCG has been shown to elicit a long-lasting Type 1 phenotype associated with cell-mediated immunity (7,16). This study takes the observation further to assess whether an IL-2-secreting rBCG is able to induce a protective Type 1 immune response in a host which defaults to a Type 2 response. Here we report that rBCG was able to evoke a strong Type 1 immune response in the immunocompromised host, whereas nBCG was not. The overall tendency of rBCG to elicit a Type 1 immune response and nBCG to evoke a Type 2 immune response after immunosuppression was observed in all three strains of mice studied.
It is interesting to note that in C57BL/6 mice and, to a lesser extent, BALB/c mice, the response to rBCG was actually higher after DXM treatment than when rBCG alone was administered. This phenomenon may be explained by the observation that DXM is capable of suppressing the expression of a broad spectrum of immunosuppressive cytokines such as transforming growth factor-ß and IL-10 (32,33,34).
The binding of corticosteroids to their receptor on the surface of leukocytes inhibits the production of most interleukins, with the postulated exception of IL-4. Previous studies have shown that IL-2 and IL-4 reduce glucocorticoid receptor binding affinity in T cells (35), causing lymphocytes to become unresponsive to the suppressive effects of DXM. This is a possible mechanism by which rBCG overcomes the suppressive effect of DXM. Alternatively, rBCG may overcome immunosuppression by replacing the missing IL-2 and thereby initiating normal production of other down-stream cytokines. The later hypothesis is supported by data showing that IFN-
levels increase after vaccination with IL-2-secreting rBCG. Due to the pleiotropic effects of IL-2, it is likely that it is acting at a number of levels to overcome the immunosuppressive effects of DXM.
Genetic or environmental factors which predispose an individual to produce a humoral, Type 2, immune response upon vaccination have also been postulated as a reason why the efficacy of BCG varies (20,21,36). A striking example is the failure of the vaccine to produce significant levels of immune protection in Southern India. In this BCG trial it was suggested that the presence of environmental, or saprophytic, mycobacteria in the region had already predisposed the population to produce a Th2 immune profile to mycobacterial antigens (37). A question which we have attempted to address, using IL-4 Tg mice, is whether established inappropriate immune responses, such as those that might develop in individuals whose previous antigenic experiences ensure that their immune responses to BCG defaults to a Type 2 response, can be rectified by co-administration of BCG and appropriate cytokines.
Tg mice were vaccinated with both rBCG and nBCG, and subsequently left for 16 weeks to compare the quality and longevity of the immune response to the vaccines. From the results obtained it is evident that nBCG was unable to alter the established immune pattern in mice, which constitutively express IL-4, and the Type 2 immune response continued to be observed post-vaccination.
Conversely, the inclusion of IL-2 in the BCG vaccine seemed capable of overcoming the natural Type 2 immune response and elicited a Type 1 immune profile. rBCG was also able to evoke a greater number of activated splenic CD4 and CD8 T cells than nBCG.
It is unclear how rBCG evoked this Type 1 immune response. Our results cannot be explained by an increase in the persistence of rBCG, as previous studies have shown that rBCG does not persist any longer than BCG, with 99.9% being eliminated within 14 weeks (15). Kong and Kunimoto (38) found that the bacterial numbers in the spleens of mice were significantly less than those in the spleens of mice infected with control BCG strains 6 weeks after infection. In our study, no detectable BCG or rBCG could be found in the spleens of vaccinated animals at 16 weeks post-vaccination.
It is possible that the production of relatively high concentrations of IL-2 in the microenvironment of the lymphoid organ where the antigen is presented to lymphocytes may overcome the more general diffuse and systemic expression of IL-4. IL-2 may be eliciting a different cell population early in the immune response to the vaccine. Gajewski et al. (39) have shown that if the microenvironment into which cells are migrating contains IFN-
, then a Type 1 profile will develop. Conversely, if the microenvironment contains Type 2 cytokines, then a Type 2 profile will ensue. It is well documented that IL-2 is a growth factor for cells that produce IFN-
, such as NK and 
T cells (8,9). These cell populations may be elicited early in the immune response to the vaccine and produce enough IFN-
to overcome the constitutive expression of IL-4 in this model.
In our experiments, high levels of IL-10 were produced in mice vaccinated with rBCG. IL-10 is usually associated with a Type 2 immune response and IL-10 may act as a regulator of the high levels of IFN-
. IL-4 could not be detected by ELISA in this experiment. This was unexpected, especially in the Tg mice where IL-4 is expressed in every cell. However, is has been shown that when IL-4 is produced there is an immediate up-regulation of its receptor, thereby rapidly removing free cytokine (40).
In conclusion, this study suggests that BCG can be made more robust by including immunopotentiating cytokines, such as IL-2, in the vaccine. The answer to improving protective immunity against TB may therefore lie in boosting the response of the host, rather than searching for new immunogens.
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Acknowledgements
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This work was funded by the Foundation for Research and Technology (New Zealand). We are grateful to Drs Franca Ronchese and Graham LeGros (Malaghan Institute, Wellington, New Zealand) for the gift of the IL-4 Tg mice.
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Abbreviations
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BCGbacillus Calmette Guerin
Con Aconcanavalin A
DXMdexamethasone
nBCGnormal bacillus Calmette Guerin
PEphycoerythrin
PPDpurified protein derivative
rBCGrecombinant bacillus Calmette Guerin
TBtuberculosis
Tgtransgenic
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