Gamma Interferon and Monophosphoryl Lipid A-Trehalose Dicorynomycolate Are Efficient Adjuvants for Mycobacterium tuberculosis Multivalent Ac
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感染与免疫杂志 2005年第1期
Department of Clinical Microbiology, Faculty of Medicine, The Hebrew University, Jerusalem, Israel
ABSTRACT
In this study, we examined the immunogenicity and protective efficacy of six immunodominant Mycobacterium tuberculosis recombinant antigens (85B, 38kDa, ESAT-6, CFP21, Mtb8.4, and 16kDa) in a multivalent vaccine preparation (6Ag). Gamma interferon (IFN-) and monophosphoryl lipid A-trehalose dicorynomycolate (Ribi) adjuvant systems were used separately or in combination for immunization with the recombinant antigens. Our results demonstrate that immunization of mice with Ribi emulsified antigens in the presence of IFN- (Ribi+6Ag+IFN-) resulted after challenge with a virulent M. tuberculosis strain in a significant reduction in the CFU counts that was comparable to that achieved with the BCG vaccine (0.9-log protection). Antigen-specific immunoglobulin G (IgG) titers in the Ribi+6Ag+IFN--immunized mice were lower than in mice immunized with Ribi+6Ag and were oriented toward a Th1-type response, as confirmed by elevated IgG2a levels. In addition, splenocyte proliferation, IFN- secretion, and NO production were significantly higher in splenocytes derived from Ribi+6Ag+IFN--immunized mice, whereas IL-10 secretion was decreased. These findings confirm the induction of a strong cellular immunity in the vaccinated mice that correlates well with their enhanced resistance to M. tuberculosis. The adjuvant effect of IFN- was dose dependent. A dose of 5 μg of IFN- per mouse per immunization gave optimal protection, whereas lower or higher amounts (0.5 or 50 μg/ mouse) of IFN- failed to enhance protection.
INTRODUCTION
Tuberculosis (TB) remains an urgent public health problem worldwide. Because of the rising incidence of TB caused by Mycobacterium tuberculosis infection, coupled with the oncurrent human immunodeficiency virus epidemic, the development of improved TB therapies and a more effective preventative vaccine is needed (8, 10). The appearance of multidrug-resistant M. tuberculosis strains aggravates this problem (39). The only TB vaccine currently available is the attenuated M. bovis strain bacillus Calmette-Guerin (BCG), which has been reported to have variable protective efficacies ranging from 0 to 85% in different controlled studies (9). Several attempts to improve the efficacy of BCG vaccine were performed by developing BCG strains expressing different cytokines and mycobacterial antigens (41, 49). Other studies used attenuated M. tuberculosis strains (42) or live recombinant vaccine vectors such as Salmonella and Listeria spp. to protect the mice against M. tuberculosis (35, 36). However, these live vaccines cannot be used in immunosuppressed individuals such as human immunodeficiency virus patients, emphasizing the need for an acellular vaccine.
Various recombinant mycobacterial antigens were tested for their immunogenicity and protective efficacy against M. tuberculosis challenge. Since M. tuberculosis is an intracellular pathogen, the relevant antigens are those that preferentially induce a Th1 immune response thought to be crucial for protection (13, 14). Thus far, the antigens that appeared most promising as protective antigens were 85B, 38kDa, ESAT-6, and Mtb8.4 (5, 11, 44, 48). The protection afforded by these antigens is variable, and the maximal protection levels ever reported were lower or similar to that achieved by the BCG vaccine. The efficacy of any vaccine depends not only on the selection of relevant antigens but also on the choice of adjuvants. An adjuvant modulates the immune response against the antigen enhancing its antigenicity and immunogenicity (21). Various adjuvant systems were tested for M. tuberculosis vaccination such as dimethyl dioctadecyl ammonium bromide, monophosphoryl lipid A (MPL), incomplete Freund adjuvant, and trehalose dicorynomycolate (TDM), a synthetic analogue of the mycobacterial trehalose dimycolate (24, 31, 50).
Cytokines such as interleukin-12 (IL-12), IL-18, and IL-15 were also tested for their immunomodulatory effect in M. tuberculosis vaccines (3, 46). Gamma interferon (IFN-) is a key cytokine in controlling mycobacterial infection; however, little is known about its effect when it is added to subunit or DNA vaccines against M. tuberculosis. Moreover, IFN- has been successfully used as an adjuvant in vaccines against other pathogens (2, 16, 47) and found to be safe in immunocompromised mice (23). We therefore analyzed here the effect of IFN- as an adjuvant in subunit vaccines against M. tuberculosis.
In the present study we immunized mice with a mixture of six immunodominant M. tuberculosis recombinant antigens, the secreted antigens 38kDa (Rv0934), ESAT-6 (Rv3875), CFP21 (Rv1984c), and Mtb8.4 (Rv1174c) and the cell wall proteins 85B (Rv1886c) and 16kDa (Rv2031c). All antigens were selected for their reported ability to be recognized by the immune system or to induce Th1-type immunity (5, 7, 11, 18, 44, 52). MPL and TDM that enhance Th1-type immunity (24, 45) were used as adjuvants in the presence or absence of IFN-. The immunogenicity and protective efficacy of the different vaccine preparations were analyzed in C57BL/6 mice.
MATERIALS AND METHODS
Mice. Specific-pathogen-free female C57BL/6 mice 5 to 6 weeks old were purchased from Harlan (Israel). Animals were maintained under specific-pathogen-free conditions throughout the experiments.
Cloning and purification of the recombinant proteins. M. tuberculosis DNA was prepared as previously described (4). Cloning and purification of the proteins were performed as described previously (26). Briefly, the recombinant mycobacterial antigens were amplified by PCR from M. tuberculosis DNA by using specific primers (Table 1) and then cloned in pQE60 or pQE70 vectors (Qiagen, Hilden, Germany) that add a His6 tag to the C terminus of the expressed proteins. The histidine tag of Mtb8.4 antigen was added to the N terminus of the protein. Sequences of the recombinant clones were confirmed by automated sequencing (ABI, Perkin-Elmer, Applied Biosystems). Resulting clones were introduced by transformation to Escherichia coli SG13009. Induction of the proteins was performed with 1 mM IPTG (isopropyl--D-thiogalactopyranoside; Ornat, Rehovot, Israel) for 2 to 3 h, and then bacteria were harvested and lysed. The proteins were purified in denaturing conditions on Ni-nitrilotriacetic acid agarose column (Qiagen, Hilden, Germany) and dialyzed against saline. Proteins sequences were confirmed by Edman degradation.
Immunization protocol. Groups of 10 mice were immunized subcutaneously twice, at 2-week intervals, with an antigen mixture consisting of 85B, CFP21, MTB8.4, 38kDa, 16kDa, and ESAT-6 antigens (5 μg of each antigen per mouse), emulsified or not in MPL+TDM (Ribi) adjuvant system (Sigma Chemical Co.). IFN- (specific biological activity > 107 U/mg; Peprotech, Rocky Hill, N.J.) was added to the vaccine preparation in the amount of 5 μg of per mouse. The amounts of Ribi (one vial per group of 10 mice) or IFN- (5 μg per mice) used for immunization were identical in each of the relevant vaccine preparations. To analyze the dose response of IFN- on protection, different amounts of IFN- (0.5, 5, or 50 μg per mouse) were added to the Ribi emulsified antigens. As a positive control, mice were vaccinated subcutaneously with 2 x 105 CFU of BCG Pasteur 1173 P2 (kindly provided by Gilles Marchal, Pasteur Institute, Paris, France). Mice immunized with saline, Ribi, or Ribi+IFN- constituted the negative control groups.
Serum analysis. Three weeks after the last immunization, blood was drawn from the mice, and the sera were kept at –70°C until used. For each mouse, antigen-specific immunoglobulin G (IgG), IgG1, and IgG2a antibodies were measured by enzyme-linked immunosorbent assay (ELISA). Ninety-six-well plates were coated overnight at 4°C with 1 μg/well of the recombinant antigen in phosphate-buffered saline (PBS; pH 7.4) solution. Plates were washed twice with PBS-0.005% Tween 20 and blocked with PBS-10% fetal calf serum for 2 h at room temperature. After two washes, mouse serum samples serially diluted in PBS were added to the plates for 3 h at room temperature. This was followed by four washes and the addition of anti-mouse IgG (Sigma), anti-mouse IgG1, and anti-mouse IgG2a (Southern Biotechnology). After incubation for 2 h at room temperature, plates were washed six times and p-nitrophenylphosphate solution (1 mg/ml) was added for 5 min (Kirkegaard & Perry Laboratories). Absorption was read at 405 nm by using an ELISA reader (ELX-800UV; Bio-Tec instruments).
Proliferation assay. Immunized mice were sacrificed and splenocytes were aseptically harvested for the proliferation assay. Red blood cells were lysed with ACK medium (0.15 M NH4Cl, 1.0 mM KHOC3, and 0.1 mM Na2EDTA) and splenocytes were pooled and grown in 96-well plates (Nunc, Roskilde, Denmark) at a concentration of 4 x 105 cells/well. RPMI 1640 medium supplemented with 10% fetal calf serum, 1 mM glutamine, 25 mM HEPES, penicillin (100 U/ml), streptomycin (100 μg/ml), and nystatin (12.5 U/ml; all purchased from Biological Industries, Beit Haemek, Israel) was used for in vitro cultures. Recombinant protein (5 μg/ml), concanavalin A (2.5 μg/ml), and purified protein derivate (PPD; 1 μg/ml) (Statens Serum Institute, Copenhagen, Denmark) were added to the splenocyte cultures. After 96 h, [methyl-3H]thymidine (0.5 μCi/well) was added for 16 h, and the cells were then harvested and lysed by a cell harvester. The amount of [methyl-3H]thymidine incorporation was measured with a -counter. We defined the stimulation index as the count-per-minute values obtained from the cells stimulated by the antigen divided by the count-per-minute values obtained from the cells without antigen stimulation. This experiment was repeated several times with the same results.
Cytokine assays. Secretion of IFN- and IL-10 was monitored in supernatants collected from splenocytes stimulated as described above after 96 h of incubation. Cytokine levels were determined by ELISA with commercial pairs of antibodies and recombinant cytokines (Pharmingen International) according to the manufacturer's instructions. Absorption was read by using an ELISA reader. The amount of cytokine in the samples was extrapolated from a standard curve established with the relevant cytokine.
Nitric oxide (NO) measurement. NO levels were measured by Griess assay (19). A 100-μl sample of 96-h-old splenocyte culture supernatant was added to a 96-well plate, followed by 100 μl of Griess reagent (Sigma). After a 4-min incubation at room temperature, absorption was read at 550 nm with an ELISA reader. Units of NO were determined by comparison with a standard curve with sodium nitrate (Sigma).
M. tuberculosis challenge. Four weeks after the last immunization, five mice from each group were infected intravenously with 5 x 105 CFU of M. tuberculosis H37Rv strain (kindly provided by Gilles Marchal). Five weeks after the challenge mice were sacrificed, spleens and lungs were homogenized in saline and serially diluted, and samples were plated on Middlebrook 7H9 agar (Difco) supplemented with 10% oleic acid-albumin-dextrose-catalase (Becton Dickinson). CFU numbers were determined after a 3-week period of incubation at 37°C.
Ethical considerations. All experiments were performed in accordance with the regulations of the animal experimentation ethic committee of The Hebrew University.
Statistics analysis. Data were analyzed by using the Student t test; P values of <0.05 were considered significant. The results shown are the means ± the standard deviations (SD).
RESULTS
Antigen recognition during experimental BCG infection. The six antigens used in our study were chosen based on their immune properties, which have been described elsewhere (5, 7, 11, 18, 44, 52). To compare their levels of recognition by the immune system during experimental mycobacterial infection, we measured IFN- and IL-10 secretion by splenocytes of BCG vaccinated mice after being stimulated with each antigen. High IFN- levels were elicited by 85B-, 38kDa-, Mtb8.4-, and CFP21-stimulated splenocytes (Table 2), whereas IL-10 levels were low, except for 38kDa-stimulated splenocytes that secrete high IL-10 levels. Stimulation of BCG-derived splenocytes with ESAT-6 or 16kDa antigen resulted in a weak IFN- and IL-10 secretion. The addition of PPD to splenocytes of BCG-infected mice induced the highest IFN- levels, whereas IL-10 levels were very low. These results indicate that the recombinant antigens in our multivalent vaccine are recognized by the immune system during BCG infection and therefore might be relevant candidates for a vaccine against M. tuberculosis.
Serum analysis of the immunized mice groups. Sera were collected from the mice 3 weeks after the last immunization, and antibody titers were tested individually for each antigen. Figure 1A shows that immunization of mice with the antigen mixture alone, resulted in high IgG titers for all antigens except for the ESAT-6 antigen. Increased IgG titers were found for 85B, CFP21, and Mtb8.5 antigens when the antigen mixture was emulsified in Ribi (P < 0.05). The addition of IFN- abolished this increase. Antibody response against antigen 38kDa was not affected by the presence of Ribi and IFN-, and actually the 38kDa-specific IgG titers were consistently high in all of the immunization procedures. The antigen 16kDa consistently induced weak IgG levels and ESAT-6 failed to induce a significant antibody production in any immunization protocol (Fig. 1A). Immunization of mice with the antigen mixture in the presence of IFN- but without Ribi did not improve IgG production (data not shown). Since IgG1 is associated with the Th2-type immune response and IgG2a is associated with the Th1 immune response in mice (43), we examined the ratio between the IgG1 and IgG2a titers. In the presence of Ribi, the vaccine preparations induced significant increase in the IgG2a titers, indicating a shift toward a Th1 immune response (Fig. 1B). The addition of IFN- to the emulsified antigens resulted in an additional increase of IgG2a production especially for the 85B and CFP21 antigens (P < 0.05). Addition of IFN- to the antigens without emulsification in Ribi adjuvant failed to affect the IgG2a titers (data not shown). As expected, immunization of mice with the control groups Ribi, IFN-, or Ribi+IFN- alone showed no antibody production (data not shown).
IFN- and Ribi enhance Th1-type immunity in the mice. Three weeks after the last immunization, splenocytes were prepared from the different immunized groups and stimulated with each antigen separately. No proliferation was found in splenocytes of 6Ag (Fig. 2) - or 6Ag/IFN--immunized mice (data not shown). Only splenocytes derived from mice immunized with Ribi+6Ag+IFN- were able to proliferate in a significant manner compared to the splenocytes of Ribi+6Ag- or 6Ag-immunized mice (P < 0.05) (Fig. 2). As in the case of the antibody response, the antigens 85B, 38kDa, Mtb8.4, and CFP21 were the most efficient immunogens, whereas the ESAT-6 and 16kDa antigens induced only a weak proliferation. These findings emphasized the synergism of Ribi and IFN- when used simultaneously as an adjuvant. In order to characterize the nature of the immune response in the immunized mice, we analyzed the cytokine secretion pattern in the stimulated splenocytes. The data presented in Fig. 3 show cytokine levels in the immunized groups after subtraction of the amount of cytokines secreted by the naive splenocyte control. IFN- secretion correlates well with the pattern of splenocyte proliferation. The highest level of IFN- secretion was found in splenocytes of Ribi+6Ag+IFN--immunized mice (Fig. 3A). All of the antigens except for antigen 16kDa induced high IFN- levels in this group, and the levels were notably higher from those secreted by Ribi+6Ag-derived splenocytes (P < 0.05). Splenocytes of Ribi+6Ag-immunized mice secreted slightly more IFN- than splenocytes of 6Ag-immunized mice. In contrast to the IFN- secretion, IL-10 levels in splenocytes of Ribi+6Ag+IFN-- and Ribi+6Ag-immunized mice were in general significantly lower than in 6Ag-derived splenocytes (Fig. 3B) (P < 0.05). The secretion patterns of IFN- and IL-10 by splenocytes of 6Ag+IFN--immunized mice were similar to those of Ribi+6Ag-immunized mice (data not shown). Our results indicate that administration of the antigens in the presence of Ribi or IFN- alone induced a limited Th1-type immune response that is necessary for protection against M. tuberculosis. However, the Th1-type immune response was remarkably enhanced when Ribi and IFN- were used together in the immunization protocol. An additional important factor in protective immunity against M. tuberculosis is NO production by macrophages (33). NO production was at its highest level in Ribi+6Ag+IFN--derived splenocytes. The addition of Ribi alone to the antigens increased NO levels only moderately (Fig. 3C). Thus, the high NO production, the enhanced proliferation, and the cytokine secretion pattern demonstrate the development of a strong cellular immunity in Ribi+6Ag+IFN--immunized mice. Indeed, in vitro stimulation of splenocytes with PPD supports the development of a Th1 immune response in these mice (Table 3). Splenocytes of Ribi+6Ag+IFN--immunized mice produce higher IFN- levels but lower IL-10 levels after PPD stimulation compared to the other immunized groups. A similar cytokine pattern was observed also in splenocytes of mice vaccinated with BCG, which is known to induce a strong Th1-type immunity. No proliferation or significant cytokine secretions were measured in splenocytes from the control groups Ribi, IFN-, and Ribi+IFN- (data not shown).
Ribi+6Ag+IFN- immunization conferred high protection against M. tuberculosis challenge. The immunological tests showed that the addition of IFN- to the Ribi emulsified antigens induced a strong Th1-type immune response. Therefore, we investigated whether this immune response correlated with protection against M. tuberculosis challenge. Figure 4A and B presents the results of the splenic CFU counts from two independent experiments. In both experiments, immunization of mice with Ribi+6Ag+IFN- resulted in high protection levels (0.85 to 0.95 log, P < 0.0005) that were similar or better than the protection achieved by the BCG vaccine. Immunizations of mice with Ribi emulsified antigens alone induced only a partial protection (0.5 log, P < 0.0005) (Fig. 4B). Partial protection was also obtained when the antigens were injected only with IFN-. CFU counts in the lungs showed results similar to those seen in the spleen (Fig. 4C). The highest reduction of lung CFU levels was found after immunization with Ribi+6Ag+IFN- (0.9 log, P < 0.0001).
The above experiments were done in vaccination protocol where 5 μg of IFN- was injected per vaccine dose and per mouse. We next examined the effect of different amounts of exogenous IFN- on the protection afforded by the Ribi emulsified antigens. Our results demonstrated that a low dose (0.5 μg/mouse) or a high dose (50 μg/mouse) of IFN- failed to confer higher protection than Ribi+6Ag-immunized mice (Fig. 5). In fact, the addition of 50 μg of IFN- per mouse resulted in a deleterious effect since the protection achieved by Ribi+6Ag immunization was completely abolished (Fig. 5). Only the addition of 5 μg of IFN- per mouse to Ribi emulsified antigens succeeded in a better protection than Ribi+6Ag vaccine preparation. No protection was found in the control groups immunized with Ribi (Fig. 4A), IFN- (Fig. 4A), or Ribi+IFN- (Fig. 4B and C).
DISCUSSION
In this study we examined the contribution of an IFN- and Ribi adjuvant system to the immunogenicity and protective efficacy of a multivalent acellular vaccine against M. tuberculosis. We showed that the selected recombinant antigens were recognized by the immune system during experimental mycobacterial infection, emphasizing their high potential for immunization. Significant IgG titers were measured in 6Ag- or Ribi+6Ag-immunized mice; however, the addition of IFN- to the Ribi+6Ag preparation resulted in a significant decrease in the IgG titers of 85B, CFP21, and Mtb8.4. The ability of IFN- to inhibit antibody production was also reported in Borrelia burgdorferi infection both in vitro and in vivo (37, 38) and is possibly due to its ability to prevent the expression of IL-4 functions, a well-known B-lymphocyte stimulator, thus impairs the generation of a strong humoral response (40). In contrast to 85B, CFP21, and Mtb8.4 antigens, the specific IgG titers against antigen 38kDa were consistently high in all immunization protocols. This is consistent with the presence of an acylation moiety of this antigen, since acylation has been reported to have an adjuvant-like activity (15, 25). In our study ESAT-6 antigen failed to induce antibody production in any formulation system. ESAT-6 is inherently a poor immunogen that induces protective immunity only in a selected adjuvant system (5, 24), and it is possible that our vaccine formulations were not effective for the EAST-6 antigen. Emulsifying the antigen mixture in Ribi or in Ribi with IFN- dramatically elevated the IgG2a titers against the 85B, 38kDa, Mtb8.4, and CFP21 antigens, indicating a shift toward a Th1 immune response. This is consistent with previous report showing that IFN- is involved in differential isotype secretion, since it enhances IgG2a while suppressing IgG1 production (27). In addition, coadministration of antigens and IL-12 to mice induced a Th1-type immune response through elevation of IFN- levels and consequently increasing Ag-specific IgG2a titers (17, 54). The high IFN- levels found in Ribi+6Ag+IFN--immunized mice are therefore responsible for the antibody isotype switching toward the Th1-type IgG2a isotype.
The concurrent addition of Ribi and IFN- to the antigen mixture directed antibody production toward a Th1-type immunity. Similarly, only splenocytes derived from Ribi+6Ag+IFN--immunized mice were able to proliferate significantly. IFN- is able to enhance antigen processing and presentation in antigen-presenting cells (34) and consequently to induce higher lymphocyte proliferation. It also mediates specific changes in the proteosome structure, resulting in the production and presentation of Th1-type specific epitopes by antigen-presenting cells (reviewed in reference 20). These functions of IFN- may thus explain the high splenocyte proliferation level in mice immunized with Ribi+6Ag+IFN- (Fig. 3A). Cytokine secretion pattern by stimulated splenocytes of Ribi+6Ag+IFN--immunized mice also confirmed the development of a strong Th1 immune response. In most cases (except for antigen 16kDa) IFN- levels were highest in Ribi+6Ag+IFN--derived splenocytes after stimulation with the antigens, whereas IL-10 secretion decreased significantly in comparison to 6Ag-derived splenocytes. These findings are supported by former studies showing that administration of exogenous IFN- stimulates secretion of IFN- but decreases IL-10 levels (22) and that this effect of IFN- can be enhanced by emulsifying the antigens in an adjuvant such as liposome (16). In our system, IFN- as a sole adjuvant induced only weak Th1 immunity; however, in mice immunized with Ribi+6Ag+IFN, IFN- enhanced the immunoadjuvant properties of Ribi. This enhancement could be the result of a slow release of IFN- in the presence of Ribi or, alternatively, the enhancement could result from an increased amount of immunostimulant compounds due to an additive effect of both adjuvants. In addition to IFN-, NO is an important mediator in controlling M. tuberculosis infection (33). In our experiments, NO production by splenocytes followed the pattern of IFN- secretion, as the presence of Ribi and even more Ribi with IFN- increased its production levels. The high NO levels detected in splenocytes of Ribi+6Ag+IFN--immunized mice may therefore enhance mouse resistance to M. tuberculosis infection.
The development of a Th1 immune response is a prerequisite for mounting efficient protection against M. tuberculosis challenge. There is a large dispute in the literature about the relevance of IFN- secretion as a marker of a protective immune response (1). Our results clearly demonstrate that the protection level correlates well with the level of IFN- secretion induced by the different immunization protocols. The best protection found in our study was achieved in mice immunized with Ribi+6Ag+IFN- (0.9 log), and the protection levels were similar or better than that of BCG vaccine. Mice immunized with Ribi+6Ag or 6Ag+IFN- that induce moderate cellular immune responses were only partially protected (0.3 to 0.5 log). These results indicated that the addition of IFN- to the Ribi emulsified antigens augmented the protective immunity in the immunized mice. However, the amount of IFN- used for immunization should be carefully determined, as we demonstrated that lower or higher amounts of IFN- (0.5 and 50 μg, respectively) failed to increase protection. The failure of 0.5 μg of IFN- to improve protection might be due to an inability of low IFN- amounts to trigger the immune system efficiently. The addition of the high IFN- amount not only failed to increase protection but even eliminated the protection achieved by the Ribi emulsified antigens alone (Fig. 5). This might be a result of suppression of the immune system, since a high IFN- level impairs T-cell activity by downregulating the subunit of the T-cell receptor complex (6). In the context of vaccine research and development, the choice of cytokines as immunomodulators and particularly IFN- should be carefully examined. Several studies tested IFN- as an adjuvant by administering it as a plasmid DNA (22, 30, 32) or by expressing it in live vaccination vehicles (29, 49, 53). In these studies, it is difficult to determine the exact amount of IFN- expressed in the animal, which, as we showed, has a critical role in protective immunity. Our study shows that administration of an optimal IFN- dose enhances Th1-type immunity induced by an adjuvant, resulting in an improved protection against M. tuberculosis. IFN- is one of the cytokines approved for human use. It is used for therapy in TB patients (12), and there are reports of its use in immunocompromised individuals (28). IFN- is therefore an excellent candidate for use as an adjuvant in TB vaccines.
In order to improve the protection level of our multivalent acellular vaccine the relative role of each antigen in protection should be examined. As determined by immunological analyses, it is clear that 85B, 38kDa, Mtb8.4, and CFP21 were the dominant immunogens, whereas ESAT-6 and 16kDa were relatively weak antigens. These results correlate well with the ability of the six antigens to stimulate splenocytes of BCG vaccinated mice in vitro (Table 2). Protective immunity against the ESAT-6 antigen was enhanced by fusing it to the 85B antigen, resulting in greater protection than that obtained when mice were immunized with each antigen alone (51). Such fusion antigens should be tested to improve the protective efficacy of a multivalent vaccine. Optimizing the dose of each antigen in a multivalent vaccine preparation might also augment the protection afforded by the vaccine. Finally, the level of protection achieved by the recombinant protein based vaccine that we have developed brings some hope in achieving the development of an efficient protective vaccine against TB that would be safe and welcome for a large population of immunosuppressed patients that are at high risk for TB.
ACKNOWLEDGMENTS
This study was supported by a grant from the Center for the Study of Emerging Diseases. The study was performed in the Peter A. Krueger P3 laboratory with the generous financial support of Nancy and Lawrence E. Glick.
We thank Itai R. Eyal for helpful comments.
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ABSTRACT
In this study, we examined the immunogenicity and protective efficacy of six immunodominant Mycobacterium tuberculosis recombinant antigens (85B, 38kDa, ESAT-6, CFP21, Mtb8.4, and 16kDa) in a multivalent vaccine preparation (6Ag). Gamma interferon (IFN-) and monophosphoryl lipid A-trehalose dicorynomycolate (Ribi) adjuvant systems were used separately or in combination for immunization with the recombinant antigens. Our results demonstrate that immunization of mice with Ribi emulsified antigens in the presence of IFN- (Ribi+6Ag+IFN-) resulted after challenge with a virulent M. tuberculosis strain in a significant reduction in the CFU counts that was comparable to that achieved with the BCG vaccine (0.9-log protection). Antigen-specific immunoglobulin G (IgG) titers in the Ribi+6Ag+IFN--immunized mice were lower than in mice immunized with Ribi+6Ag and were oriented toward a Th1-type response, as confirmed by elevated IgG2a levels. In addition, splenocyte proliferation, IFN- secretion, and NO production were significantly higher in splenocytes derived from Ribi+6Ag+IFN--immunized mice, whereas IL-10 secretion was decreased. These findings confirm the induction of a strong cellular immunity in the vaccinated mice that correlates well with their enhanced resistance to M. tuberculosis. The adjuvant effect of IFN- was dose dependent. A dose of 5 μg of IFN- per mouse per immunization gave optimal protection, whereas lower or higher amounts (0.5 or 50 μg/ mouse) of IFN- failed to enhance protection.
INTRODUCTION
Tuberculosis (TB) remains an urgent public health problem worldwide. Because of the rising incidence of TB caused by Mycobacterium tuberculosis infection, coupled with the oncurrent human immunodeficiency virus epidemic, the development of improved TB therapies and a more effective preventative vaccine is needed (8, 10). The appearance of multidrug-resistant M. tuberculosis strains aggravates this problem (39). The only TB vaccine currently available is the attenuated M. bovis strain bacillus Calmette-Guerin (BCG), which has been reported to have variable protective efficacies ranging from 0 to 85% in different controlled studies (9). Several attempts to improve the efficacy of BCG vaccine were performed by developing BCG strains expressing different cytokines and mycobacterial antigens (41, 49). Other studies used attenuated M. tuberculosis strains (42) or live recombinant vaccine vectors such as Salmonella and Listeria spp. to protect the mice against M. tuberculosis (35, 36). However, these live vaccines cannot be used in immunosuppressed individuals such as human immunodeficiency virus patients, emphasizing the need for an acellular vaccine.
Various recombinant mycobacterial antigens were tested for their immunogenicity and protective efficacy against M. tuberculosis challenge. Since M. tuberculosis is an intracellular pathogen, the relevant antigens are those that preferentially induce a Th1 immune response thought to be crucial for protection (13, 14). Thus far, the antigens that appeared most promising as protective antigens were 85B, 38kDa, ESAT-6, and Mtb8.4 (5, 11, 44, 48). The protection afforded by these antigens is variable, and the maximal protection levels ever reported were lower or similar to that achieved by the BCG vaccine. The efficacy of any vaccine depends not only on the selection of relevant antigens but also on the choice of adjuvants. An adjuvant modulates the immune response against the antigen enhancing its antigenicity and immunogenicity (21). Various adjuvant systems were tested for M. tuberculosis vaccination such as dimethyl dioctadecyl ammonium bromide, monophosphoryl lipid A (MPL), incomplete Freund adjuvant, and trehalose dicorynomycolate (TDM), a synthetic analogue of the mycobacterial trehalose dimycolate (24, 31, 50).
Cytokines such as interleukin-12 (IL-12), IL-18, and IL-15 were also tested for their immunomodulatory effect in M. tuberculosis vaccines (3, 46). Gamma interferon (IFN-) is a key cytokine in controlling mycobacterial infection; however, little is known about its effect when it is added to subunit or DNA vaccines against M. tuberculosis. Moreover, IFN- has been successfully used as an adjuvant in vaccines against other pathogens (2, 16, 47) and found to be safe in immunocompromised mice (23). We therefore analyzed here the effect of IFN- as an adjuvant in subunit vaccines against M. tuberculosis.
In the present study we immunized mice with a mixture of six immunodominant M. tuberculosis recombinant antigens, the secreted antigens 38kDa (Rv0934), ESAT-6 (Rv3875), CFP21 (Rv1984c), and Mtb8.4 (Rv1174c) and the cell wall proteins 85B (Rv1886c) and 16kDa (Rv2031c). All antigens were selected for their reported ability to be recognized by the immune system or to induce Th1-type immunity (5, 7, 11, 18, 44, 52). MPL and TDM that enhance Th1-type immunity (24, 45) were used as adjuvants in the presence or absence of IFN-. The immunogenicity and protective efficacy of the different vaccine preparations were analyzed in C57BL/6 mice.
MATERIALS AND METHODS
Mice. Specific-pathogen-free female C57BL/6 mice 5 to 6 weeks old were purchased from Harlan (Israel). Animals were maintained under specific-pathogen-free conditions throughout the experiments.
Cloning and purification of the recombinant proteins. M. tuberculosis DNA was prepared as previously described (4). Cloning and purification of the proteins were performed as described previously (26). Briefly, the recombinant mycobacterial antigens were amplified by PCR from M. tuberculosis DNA by using specific primers (Table 1) and then cloned in pQE60 or pQE70 vectors (Qiagen, Hilden, Germany) that add a His6 tag to the C terminus of the expressed proteins. The histidine tag of Mtb8.4 antigen was added to the N terminus of the protein. Sequences of the recombinant clones were confirmed by automated sequencing (ABI, Perkin-Elmer, Applied Biosystems). Resulting clones were introduced by transformation to Escherichia coli SG13009. Induction of the proteins was performed with 1 mM IPTG (isopropyl--D-thiogalactopyranoside; Ornat, Rehovot, Israel) for 2 to 3 h, and then bacteria were harvested and lysed. The proteins were purified in denaturing conditions on Ni-nitrilotriacetic acid agarose column (Qiagen, Hilden, Germany) and dialyzed against saline. Proteins sequences were confirmed by Edman degradation.
Immunization protocol. Groups of 10 mice were immunized subcutaneously twice, at 2-week intervals, with an antigen mixture consisting of 85B, CFP21, MTB8.4, 38kDa, 16kDa, and ESAT-6 antigens (5 μg of each antigen per mouse), emulsified or not in MPL+TDM (Ribi) adjuvant system (Sigma Chemical Co.). IFN- (specific biological activity > 107 U/mg; Peprotech, Rocky Hill, N.J.) was added to the vaccine preparation in the amount of 5 μg of per mouse. The amounts of Ribi (one vial per group of 10 mice) or IFN- (5 μg per mice) used for immunization were identical in each of the relevant vaccine preparations. To analyze the dose response of IFN- on protection, different amounts of IFN- (0.5, 5, or 50 μg per mouse) were added to the Ribi emulsified antigens. As a positive control, mice were vaccinated subcutaneously with 2 x 105 CFU of BCG Pasteur 1173 P2 (kindly provided by Gilles Marchal, Pasteur Institute, Paris, France). Mice immunized with saline, Ribi, or Ribi+IFN- constituted the negative control groups.
Serum analysis. Three weeks after the last immunization, blood was drawn from the mice, and the sera were kept at –70°C until used. For each mouse, antigen-specific immunoglobulin G (IgG), IgG1, and IgG2a antibodies were measured by enzyme-linked immunosorbent assay (ELISA). Ninety-six-well plates were coated overnight at 4°C with 1 μg/well of the recombinant antigen in phosphate-buffered saline (PBS; pH 7.4) solution. Plates were washed twice with PBS-0.005% Tween 20 and blocked with PBS-10% fetal calf serum for 2 h at room temperature. After two washes, mouse serum samples serially diluted in PBS were added to the plates for 3 h at room temperature. This was followed by four washes and the addition of anti-mouse IgG (Sigma), anti-mouse IgG1, and anti-mouse IgG2a (Southern Biotechnology). After incubation for 2 h at room temperature, plates were washed six times and p-nitrophenylphosphate solution (1 mg/ml) was added for 5 min (Kirkegaard & Perry Laboratories). Absorption was read at 405 nm by using an ELISA reader (ELX-800UV; Bio-Tec instruments).
Proliferation assay. Immunized mice were sacrificed and splenocytes were aseptically harvested for the proliferation assay. Red blood cells were lysed with ACK medium (0.15 M NH4Cl, 1.0 mM KHOC3, and 0.1 mM Na2EDTA) and splenocytes were pooled and grown in 96-well plates (Nunc, Roskilde, Denmark) at a concentration of 4 x 105 cells/well. RPMI 1640 medium supplemented with 10% fetal calf serum, 1 mM glutamine, 25 mM HEPES, penicillin (100 U/ml), streptomycin (100 μg/ml), and nystatin (12.5 U/ml; all purchased from Biological Industries, Beit Haemek, Israel) was used for in vitro cultures. Recombinant protein (5 μg/ml), concanavalin A (2.5 μg/ml), and purified protein derivate (PPD; 1 μg/ml) (Statens Serum Institute, Copenhagen, Denmark) were added to the splenocyte cultures. After 96 h, [methyl-3H]thymidine (0.5 μCi/well) was added for 16 h, and the cells were then harvested and lysed by a cell harvester. The amount of [methyl-3H]thymidine incorporation was measured with a -counter. We defined the stimulation index as the count-per-minute values obtained from the cells stimulated by the antigen divided by the count-per-minute values obtained from the cells without antigen stimulation. This experiment was repeated several times with the same results.
Cytokine assays. Secretion of IFN- and IL-10 was monitored in supernatants collected from splenocytes stimulated as described above after 96 h of incubation. Cytokine levels were determined by ELISA with commercial pairs of antibodies and recombinant cytokines (Pharmingen International) according to the manufacturer's instructions. Absorption was read by using an ELISA reader. The amount of cytokine in the samples was extrapolated from a standard curve established with the relevant cytokine.
Nitric oxide (NO) measurement. NO levels were measured by Griess assay (19). A 100-μl sample of 96-h-old splenocyte culture supernatant was added to a 96-well plate, followed by 100 μl of Griess reagent (Sigma). After a 4-min incubation at room temperature, absorption was read at 550 nm with an ELISA reader. Units of NO were determined by comparison with a standard curve with sodium nitrate (Sigma).
M. tuberculosis challenge. Four weeks after the last immunization, five mice from each group were infected intravenously with 5 x 105 CFU of M. tuberculosis H37Rv strain (kindly provided by Gilles Marchal). Five weeks after the challenge mice were sacrificed, spleens and lungs were homogenized in saline and serially diluted, and samples were plated on Middlebrook 7H9 agar (Difco) supplemented with 10% oleic acid-albumin-dextrose-catalase (Becton Dickinson). CFU numbers were determined after a 3-week period of incubation at 37°C.
Ethical considerations. All experiments were performed in accordance with the regulations of the animal experimentation ethic committee of The Hebrew University.
Statistics analysis. Data were analyzed by using the Student t test; P values of <0.05 were considered significant. The results shown are the means ± the standard deviations (SD).
RESULTS
Antigen recognition during experimental BCG infection. The six antigens used in our study were chosen based on their immune properties, which have been described elsewhere (5, 7, 11, 18, 44, 52). To compare their levels of recognition by the immune system during experimental mycobacterial infection, we measured IFN- and IL-10 secretion by splenocytes of BCG vaccinated mice after being stimulated with each antigen. High IFN- levels were elicited by 85B-, 38kDa-, Mtb8.4-, and CFP21-stimulated splenocytes (Table 2), whereas IL-10 levels were low, except for 38kDa-stimulated splenocytes that secrete high IL-10 levels. Stimulation of BCG-derived splenocytes with ESAT-6 or 16kDa antigen resulted in a weak IFN- and IL-10 secretion. The addition of PPD to splenocytes of BCG-infected mice induced the highest IFN- levels, whereas IL-10 levels were very low. These results indicate that the recombinant antigens in our multivalent vaccine are recognized by the immune system during BCG infection and therefore might be relevant candidates for a vaccine against M. tuberculosis.
Serum analysis of the immunized mice groups. Sera were collected from the mice 3 weeks after the last immunization, and antibody titers were tested individually for each antigen. Figure 1A shows that immunization of mice with the antigen mixture alone, resulted in high IgG titers for all antigens except for the ESAT-6 antigen. Increased IgG titers were found for 85B, CFP21, and Mtb8.5 antigens when the antigen mixture was emulsified in Ribi (P < 0.05). The addition of IFN- abolished this increase. Antibody response against antigen 38kDa was not affected by the presence of Ribi and IFN-, and actually the 38kDa-specific IgG titers were consistently high in all of the immunization procedures. The antigen 16kDa consistently induced weak IgG levels and ESAT-6 failed to induce a significant antibody production in any immunization protocol (Fig. 1A). Immunization of mice with the antigen mixture in the presence of IFN- but without Ribi did not improve IgG production (data not shown). Since IgG1 is associated with the Th2-type immune response and IgG2a is associated with the Th1 immune response in mice (43), we examined the ratio between the IgG1 and IgG2a titers. In the presence of Ribi, the vaccine preparations induced significant increase in the IgG2a titers, indicating a shift toward a Th1 immune response (Fig. 1B). The addition of IFN- to the emulsified antigens resulted in an additional increase of IgG2a production especially for the 85B and CFP21 antigens (P < 0.05). Addition of IFN- to the antigens without emulsification in Ribi adjuvant failed to affect the IgG2a titers (data not shown). As expected, immunization of mice with the control groups Ribi, IFN-, or Ribi+IFN- alone showed no antibody production (data not shown).
IFN- and Ribi enhance Th1-type immunity in the mice. Three weeks after the last immunization, splenocytes were prepared from the different immunized groups and stimulated with each antigen separately. No proliferation was found in splenocytes of 6Ag (Fig. 2) - or 6Ag/IFN--immunized mice (data not shown). Only splenocytes derived from mice immunized with Ribi+6Ag+IFN- were able to proliferate in a significant manner compared to the splenocytes of Ribi+6Ag- or 6Ag-immunized mice (P < 0.05) (Fig. 2). As in the case of the antibody response, the antigens 85B, 38kDa, Mtb8.4, and CFP21 were the most efficient immunogens, whereas the ESAT-6 and 16kDa antigens induced only a weak proliferation. These findings emphasized the synergism of Ribi and IFN- when used simultaneously as an adjuvant. In order to characterize the nature of the immune response in the immunized mice, we analyzed the cytokine secretion pattern in the stimulated splenocytes. The data presented in Fig. 3 show cytokine levels in the immunized groups after subtraction of the amount of cytokines secreted by the naive splenocyte control. IFN- secretion correlates well with the pattern of splenocyte proliferation. The highest level of IFN- secretion was found in splenocytes of Ribi+6Ag+IFN--immunized mice (Fig. 3A). All of the antigens except for antigen 16kDa induced high IFN- levels in this group, and the levels were notably higher from those secreted by Ribi+6Ag-derived splenocytes (P < 0.05). Splenocytes of Ribi+6Ag-immunized mice secreted slightly more IFN- than splenocytes of 6Ag-immunized mice. In contrast to the IFN- secretion, IL-10 levels in splenocytes of Ribi+6Ag+IFN-- and Ribi+6Ag-immunized mice were in general significantly lower than in 6Ag-derived splenocytes (Fig. 3B) (P < 0.05). The secretion patterns of IFN- and IL-10 by splenocytes of 6Ag+IFN--immunized mice were similar to those of Ribi+6Ag-immunized mice (data not shown). Our results indicate that administration of the antigens in the presence of Ribi or IFN- alone induced a limited Th1-type immune response that is necessary for protection against M. tuberculosis. However, the Th1-type immune response was remarkably enhanced when Ribi and IFN- were used together in the immunization protocol. An additional important factor in protective immunity against M. tuberculosis is NO production by macrophages (33). NO production was at its highest level in Ribi+6Ag+IFN--derived splenocytes. The addition of Ribi alone to the antigens increased NO levels only moderately (Fig. 3C). Thus, the high NO production, the enhanced proliferation, and the cytokine secretion pattern demonstrate the development of a strong cellular immunity in Ribi+6Ag+IFN--immunized mice. Indeed, in vitro stimulation of splenocytes with PPD supports the development of a Th1 immune response in these mice (Table 3). Splenocytes of Ribi+6Ag+IFN--immunized mice produce higher IFN- levels but lower IL-10 levels after PPD stimulation compared to the other immunized groups. A similar cytokine pattern was observed also in splenocytes of mice vaccinated with BCG, which is known to induce a strong Th1-type immunity. No proliferation or significant cytokine secretions were measured in splenocytes from the control groups Ribi, IFN-, and Ribi+IFN- (data not shown).
Ribi+6Ag+IFN- immunization conferred high protection against M. tuberculosis challenge. The immunological tests showed that the addition of IFN- to the Ribi emulsified antigens induced a strong Th1-type immune response. Therefore, we investigated whether this immune response correlated with protection against M. tuberculosis challenge. Figure 4A and B presents the results of the splenic CFU counts from two independent experiments. In both experiments, immunization of mice with Ribi+6Ag+IFN- resulted in high protection levels (0.85 to 0.95 log, P < 0.0005) that were similar or better than the protection achieved by the BCG vaccine. Immunizations of mice with Ribi emulsified antigens alone induced only a partial protection (0.5 log, P < 0.0005) (Fig. 4B). Partial protection was also obtained when the antigens were injected only with IFN-. CFU counts in the lungs showed results similar to those seen in the spleen (Fig. 4C). The highest reduction of lung CFU levels was found after immunization with Ribi+6Ag+IFN- (0.9 log, P < 0.0001).
The above experiments were done in vaccination protocol where 5 μg of IFN- was injected per vaccine dose and per mouse. We next examined the effect of different amounts of exogenous IFN- on the protection afforded by the Ribi emulsified antigens. Our results demonstrated that a low dose (0.5 μg/mouse) or a high dose (50 μg/mouse) of IFN- failed to confer higher protection than Ribi+6Ag-immunized mice (Fig. 5). In fact, the addition of 50 μg of IFN- per mouse resulted in a deleterious effect since the protection achieved by Ribi+6Ag immunization was completely abolished (Fig. 5). Only the addition of 5 μg of IFN- per mouse to Ribi emulsified antigens succeeded in a better protection than Ribi+6Ag vaccine preparation. No protection was found in the control groups immunized with Ribi (Fig. 4A), IFN- (Fig. 4A), or Ribi+IFN- (Fig. 4B and C).
DISCUSSION
In this study we examined the contribution of an IFN- and Ribi adjuvant system to the immunogenicity and protective efficacy of a multivalent acellular vaccine against M. tuberculosis. We showed that the selected recombinant antigens were recognized by the immune system during experimental mycobacterial infection, emphasizing their high potential for immunization. Significant IgG titers were measured in 6Ag- or Ribi+6Ag-immunized mice; however, the addition of IFN- to the Ribi+6Ag preparation resulted in a significant decrease in the IgG titers of 85B, CFP21, and Mtb8.4. The ability of IFN- to inhibit antibody production was also reported in Borrelia burgdorferi infection both in vitro and in vivo (37, 38) and is possibly due to its ability to prevent the expression of IL-4 functions, a well-known B-lymphocyte stimulator, thus impairs the generation of a strong humoral response (40). In contrast to 85B, CFP21, and Mtb8.4 antigens, the specific IgG titers against antigen 38kDa were consistently high in all immunization protocols. This is consistent with the presence of an acylation moiety of this antigen, since acylation has been reported to have an adjuvant-like activity (15, 25). In our study ESAT-6 antigen failed to induce antibody production in any formulation system. ESAT-6 is inherently a poor immunogen that induces protective immunity only in a selected adjuvant system (5, 24), and it is possible that our vaccine formulations were not effective for the EAST-6 antigen. Emulsifying the antigen mixture in Ribi or in Ribi with IFN- dramatically elevated the IgG2a titers against the 85B, 38kDa, Mtb8.4, and CFP21 antigens, indicating a shift toward a Th1 immune response. This is consistent with previous report showing that IFN- is involved in differential isotype secretion, since it enhances IgG2a while suppressing IgG1 production (27). In addition, coadministration of antigens and IL-12 to mice induced a Th1-type immune response through elevation of IFN- levels and consequently increasing Ag-specific IgG2a titers (17, 54). The high IFN- levels found in Ribi+6Ag+IFN--immunized mice are therefore responsible for the antibody isotype switching toward the Th1-type IgG2a isotype.
The concurrent addition of Ribi and IFN- to the antigen mixture directed antibody production toward a Th1-type immunity. Similarly, only splenocytes derived from Ribi+6Ag+IFN--immunized mice were able to proliferate significantly. IFN- is able to enhance antigen processing and presentation in antigen-presenting cells (34) and consequently to induce higher lymphocyte proliferation. It also mediates specific changes in the proteosome structure, resulting in the production and presentation of Th1-type specific epitopes by antigen-presenting cells (reviewed in reference 20). These functions of IFN- may thus explain the high splenocyte proliferation level in mice immunized with Ribi+6Ag+IFN- (Fig. 3A). Cytokine secretion pattern by stimulated splenocytes of Ribi+6Ag+IFN--immunized mice also confirmed the development of a strong Th1 immune response. In most cases (except for antigen 16kDa) IFN- levels were highest in Ribi+6Ag+IFN--derived splenocytes after stimulation with the antigens, whereas IL-10 secretion decreased significantly in comparison to 6Ag-derived splenocytes. These findings are supported by former studies showing that administration of exogenous IFN- stimulates secretion of IFN- but decreases IL-10 levels (22) and that this effect of IFN- can be enhanced by emulsifying the antigens in an adjuvant such as liposome (16). In our system, IFN- as a sole adjuvant induced only weak Th1 immunity; however, in mice immunized with Ribi+6Ag+IFN, IFN- enhanced the immunoadjuvant properties of Ribi. This enhancement could be the result of a slow release of IFN- in the presence of Ribi or, alternatively, the enhancement could result from an increased amount of immunostimulant compounds due to an additive effect of both adjuvants. In addition to IFN-, NO is an important mediator in controlling M. tuberculosis infection (33). In our experiments, NO production by splenocytes followed the pattern of IFN- secretion, as the presence of Ribi and even more Ribi with IFN- increased its production levels. The high NO levels detected in splenocytes of Ribi+6Ag+IFN--immunized mice may therefore enhance mouse resistance to M. tuberculosis infection.
The development of a Th1 immune response is a prerequisite for mounting efficient protection against M. tuberculosis challenge. There is a large dispute in the literature about the relevance of IFN- secretion as a marker of a protective immune response (1). Our results clearly demonstrate that the protection level correlates well with the level of IFN- secretion induced by the different immunization protocols. The best protection found in our study was achieved in mice immunized with Ribi+6Ag+IFN- (0.9 log), and the protection levels were similar or better than that of BCG vaccine. Mice immunized with Ribi+6Ag or 6Ag+IFN- that induce moderate cellular immune responses were only partially protected (0.3 to 0.5 log). These results indicated that the addition of IFN- to the Ribi emulsified antigens augmented the protective immunity in the immunized mice. However, the amount of IFN- used for immunization should be carefully determined, as we demonstrated that lower or higher amounts of IFN- (0.5 and 50 μg, respectively) failed to increase protection. The failure of 0.5 μg of IFN- to improve protection might be due to an inability of low IFN- amounts to trigger the immune system efficiently. The addition of the high IFN- amount not only failed to increase protection but even eliminated the protection achieved by the Ribi emulsified antigens alone (Fig. 5). This might be a result of suppression of the immune system, since a high IFN- level impairs T-cell activity by downregulating the subunit of the T-cell receptor complex (6). In the context of vaccine research and development, the choice of cytokines as immunomodulators and particularly IFN- should be carefully examined. Several studies tested IFN- as an adjuvant by administering it as a plasmid DNA (22, 30, 32) or by expressing it in live vaccination vehicles (29, 49, 53). In these studies, it is difficult to determine the exact amount of IFN- expressed in the animal, which, as we showed, has a critical role in protective immunity. Our study shows that administration of an optimal IFN- dose enhances Th1-type immunity induced by an adjuvant, resulting in an improved protection against M. tuberculosis. IFN- is one of the cytokines approved for human use. It is used for therapy in TB patients (12), and there are reports of its use in immunocompromised individuals (28). IFN- is therefore an excellent candidate for use as an adjuvant in TB vaccines.
In order to improve the protection level of our multivalent acellular vaccine the relative role of each antigen in protection should be examined. As determined by immunological analyses, it is clear that 85B, 38kDa, Mtb8.4, and CFP21 were the dominant immunogens, whereas ESAT-6 and 16kDa were relatively weak antigens. These results correlate well with the ability of the six antigens to stimulate splenocytes of BCG vaccinated mice in vitro (Table 2). Protective immunity against the ESAT-6 antigen was enhanced by fusing it to the 85B antigen, resulting in greater protection than that obtained when mice were immunized with each antigen alone (51). Such fusion antigens should be tested to improve the protective efficacy of a multivalent vaccine. Optimizing the dose of each antigen in a multivalent vaccine preparation might also augment the protection afforded by the vaccine. Finally, the level of protection achieved by the recombinant protein based vaccine that we have developed brings some hope in achieving the development of an efficient protective vaccine against TB that would be safe and welcome for a large population of immunosuppressed patients that are at high risk for TB.
ACKNOWLEDGMENTS
This study was supported by a grant from the Center for the Study of Emerging Diseases. The study was performed in the Peter A. Krueger P3 laboratory with the generous financial support of Nancy and Lawrence E. Glick.
We thank Itai R. Eyal for helpful comments.
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