Differential Effects of Prior Exposure to Environmental Mycobacteria on Vaccination with Mycobacterium bovis BCG or a Recombinant BCG Strain
http://www.100md.com
感染与免疫杂志 2005年第4期
Unite de Genetique Moleculaire Bacterienne, Institut Pasteur, Paris, France
Department of Infectious Disease Immunology, Statens Serum Institut, Copenhagen, Denmark
ABSTRACT
In silico analysis reveals that most protective antigens expressed by the antituberculous vaccine Mycobacterium bovis BCG (BCG) are conserved in M. avium, supporting the hypothesis that exposure to environmental mycobacteria generates cross-reactive immune responses blocking BCG activity. We investigated the impact of sensitization with M. avium, M. scrofulaceum, or M. vaccae on the protective efficacy of a recombinant BCG strain expressing RD1 antigens (BCG::RD1), using a mouse model of experimental tuberculosis (TB). No evidence that the RD1-encoded antigens ESAT-6, CFP-10, and PPE68 were expressed by these environmental strains could be demonstrated by Western blot analysis. Mice sensitized with each of these strains did not prime cellular immune responses cross-reacting with the immunodominant ESAT-6. Importantly, clearance of BCG::RD1 from the lungs and spleens of mice exposed to each of the environmental strains before vaccination was minimal compared to that of BCG. In mice sensitized with M. avium, increased persistence of BCG::RD1 correlated with stronger antimycobacterial gamma interferon responses and enhanced protection against aerosol infection with M. tuberculosis, compared to BCG. In contrast, animals exposed to M. scrofulaceum or M. vaccae prior to vaccination with BCG or BCG::RD1 were better protected against TB than were the unsensitized controls. Our results suggest that the inhibitory effect of environmental mycobacteria on the protective efficacy of BCG depends critically on the extent of cross-recognition of antigens shared with the vaccine. In hosts sensitized with M. avium, potent immunogenicity of ESAT-6 and increased persistence of BCG::RD1 may allow this recombinant vaccine to overcome preexisting antimycobacterial responses.
INTRODUCTION
Tuberculosis (TB) remains the single most important mycobacterial infection worldwide, with a third of the world's population being infected with Mycobacterium tuberculosis and 10 million new cases of clinical TB being reported to the World Health Organization each year. Meta-analysis of trials with the only currently available vaccine, M. bovis Bacille Calmette-Guerin (BCG), has concluded that BCG confers 50 and 80% protective efficacy against pulmonary and disseminated TB, respectively (7). Several lines of evidence suggest that exposure to environmental mycobacteria may reduce the protective efficacy of BCG against TB. In particular, epidemiological observations have shown that BCG is highly efficient at preventing TB in neonates vaccinated before exposure to environmental mycobacteria occurs (7) or in vaccination trials excluding strictly tuberculin skin test-positive individuals (13). In contrast, in areas like southern India, where isolates from the Mycobacterium avium-intracellulare-scrofulaceum complex are commonly found in water and dust (16), BCG is particularly ineffective and fails to protect adults against pulmonary TB (11, 12).
Recently, animal studies have provided additional evidence that sensitization with environmental mycobacteria may have a direct antagonistic effect on BCG vaccination. Mice presensitized with M. avium or with cocktails of M. avium, M. vaccae, and M. scrofulaceum developed antimycobacterial responses that control the multiplication of BCG, thereby reducing its protective efficacy against TB (2). Sensitization with M. avium or M. fortuitum before vaccination with BCG also showed a modulatory effect on the protective efficacy of BCG against experimental TB in guinea pigs (15). These results strongly suggest that prior exposure to live environmental mycobacteria primes the host immune system against mycobacterial antigens shared with BCG and that recall of this immune response on vaccination results in accelerated clearance of BCG and hence decreased protection against TB.
The persistence of BCG in vivo can be markedly augmented by stable insertion of RD1, a region of difference between attenuated mycobacterial strains (M. bovis BCG and M. microti) and pathogenic species such as M. bovis and M. tuberculosis (5, 8, 17, 22). When mice and guinea pigs were vaccinated with BCG::RD1 knock-in constructions (BCG::RD1), progression of TB was not significantly modified in the lungs but was markedly reduced in the spleen compared to that in BCG-immunized animals. Enhanced protection was associated with the expression of antigens encoded by RD1 (9, 23). Among them is ESAT-6 (6-kDa early secreted antigenic target), a potent Th1 T-cell inducer that protects mice against experimental infection with M. tuberculosis (3). To a lesser extent, CFP-10 (10-kDa culture filtrate protein) and PPE68 have also been identified as immunogenic components of RD1, and both proteins induce Th1-oriented T-cell responses in animal models (9, 20). Expression of CFP-10 and ESAT-6 has been detected in pathogenic species of the M. tuberculosis complex, M. leprae, M. kansasii, M. marinum, and M. smegmatis, although the amino acid sequences of the corresponding proteins vary among these species. As a member of the mycobacterial PPE protein family, PPE68 may show sequence homology to proteins from environmental strains. In fact, nonspecific reactivity of sera from healthy human donors with the PPE68 protein has been reported (6), and PPE68 stimulated high levels of gamma interferon (IFN-) secretion in peripheral blood mononuclear cells isolated from TB patients, as well as from a significant proportion of BCG-vaccinated donors (21). However, no equivalent of CFP-10 and ESAT-6 is detectable in filtrates from BCG or from most environmental species by Western blot analysis techniques (24; J. S. Rothel, personal communication). Importantly, nontuberculous donors do not show cellular responses to ESAT-6 or CFP-10 antigens (1), suggesting that no immune responses cross-reacting with these antigens are primed in hosts exposed to environmental mycobacteria.
In the present study, we postulated that the increased persistence and the antigenic specificity of BCG::RD1 may enable the recombinant vaccine to override immunity imparted by prior contact with environmental mycobacteria. We show that mice immunized with M. avium, M. vaccae, or M. scrofulaceum do not develop immune responses cross-reacting with ESAT-6. Moreover, our data demonstrate that BCG::RD1 persists significantly longer than BCG in animals exposed to each of these strains and that the protective efficacy of BCG::RD1 against TB is not reduced in animals sensitized with these environmental mycobacteria. These results suggest that BCG::RD1 may be a better alternative to BCG in areas where exposure to environmental mycobacteria is common.
MATERIALS AND METHODS
Mycobacteria. BCG::RD1 and BCG::pYUB412 vector control knock-in constructions (23), M. tuberculosis (H37Rv), M. avium (ATCC 15769), M. scrofulaceum (ATCC 19275), and M. vaccae (ATCC 15483) were grown in Middlebrook 7H9-ADC medium supplemented with 0.05% Tween 80 until the mid-log phase of bacterial growth. Mycobacteria were harvested by centrifugation and washed once with 50 mM sodium phosphate buffer (pH 7.0) before resuspension in the same buffer. The bacteria were then sonicated briefly and allowed to stand for 2 h to allow residual aggregates to settle. Bacterial suspensions were aliquoted and frozen at –80°C. For immunization studies, frozen aliquots were thawed and sonicated briefly before injection, and the viability of each inoculum was verified by titer determination on 7H11-OADC agar plates.
Animals and sensitization with environmental mycobacteria. Six-week-old female C57BL/6 mice (Charles Rivers, L'arbresle, France) were housed in specific-pathogen-free animal facilities of the Institut Pasteur. Vaccine persistence and protective efficacy studies with mice exposed to environmental mycobacteria were performed essentially as previously described (2). Briefly, animals were immunized by three consecutive subcutaneous (s.c.) injections of 2 x 106 CFU of either M. avium, M. scrofulaceum, or M. vaccae performed at 2-week intervals. Four weeks after the last injection, the mice were treated with a cocktail of rifampin (Sigma; 100 mg/liter), ethambutol (Sigma; 200 mg/liter), and clarithromycin (Abbott; 200 mg/liter) added to drinking water for 1 month. This treatment resulted in efficient elimination of environmental mycobacteria from the lungs and spleens of the mice (reference 2 and data not shown).
Immunization and infection procedures. To study the immune responses elicited by environmental mycobacteria, mice were immunized by two consecutive s.c. injections of 106 CFU of M. avium, M. scrofulaceum, or M. vaccae or a mixture of the three strains (5 x 105 CFU of each) performed at 2-week intervals. To compare the multiplication of BCG and BCG::RD1 in sensitized mice, the animals were immunized by intravenous injection of 106 CFU 4 weeks after the end of the antibiotic treatment. For protection studies, mice were vaccinated by s.c. injection of 105 CFU of BCG or BCG::RD1 at the base of the tail 4 weeks after the end of the antibiotic treatment. Animals were infected with approximately 100 CFU of M. tuberculosis per lung via the aerosol route 6 weeks postvaccination. The mice were sacrificed 4 weeks postinfection. Lungs and spleens were homogenized using an MM300 apparatus (Qiagen, Hilden, Germany) and 2.5-mm-diameter glass beads. Serial fivefold dilutions in phosphate-buffered saline were plated onto 7H11 agar plates, and CFU were determined after 3 weeks of growth at 37°C.
Mycobacterial antigen preparation and Western blot analysis. Mycobacterial cell lysates were prepared as follows. Culture samples were centrifuged, and bacterial pellets were washed twice with 20 mM Tris buffer (pH 7.5) before being resuspended in 500 μl of the same buffer with complete EDTA-free protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany). The cells were then lysed by shaking on an MM300 apparatus for 10 min at maximum speed with 500 μl of acid-washed 106-μm-diameter glass beads (Sigma, St. Louis, Mo.). Beads and unbroken cells were removed by centrifugation at 5,000 x g for 30 min, and the resulting supernatants were centrifuged at 15,000 x g for 30 min to yield bacterial cell lysates. Mycobacterial culture filtrates were prepared by concentrating culture supernatants on a Millipore filter with a 3-kDa cutoff (YM3). Both cell lysates and culture filtrates were aliquoted and stored at –20°C after protein quantification using a Bio-Rad protein assay. Immunoblot detections were performed as previously described (22), using rabbit polyclonal antibodies raised against the recombinant proteins Rv3873 (PPE68), Rv3874 (CFP-10), Rv1886c (Ag85B), and an anti-ESAT-6 monoclonal antibody (Hyb076-08; Statens Serum Institut, Denmark).
IFN- response to mycobacterial antigens. Single-cell suspensions of splenocytes were prepared by sieving through 200-μm mesh. After red blood cell lysis, cells were resuspended in synthetic HL-1 medium (Cambrex) supplemented with 2 mM L-glutamine, 100 IU of penicillin/ml, and 100 μg of streptomycin/ml. The cells were plated in 96-well plates at 5 x 105 cells per well in presence of antigen preparations, medium alone, PPD (Veterinary Laboratories Agency, Weybridge, United Kingdom), or concanavalin A (Sigma). T-cell responses to ESAT-6 were measured after in vitro stimulation with pESAT-6, a peptide corresponding to the first 20 amino acid residues of ESAT-6 (Neosystem, Strasbourg, France), which has been identified as an immunodominant T-cell epitope on ESAT-6 in the context of H-2b (4). The presence of IFN- in culture supernatants was then assessed after 2 days of culture at 37°C, as previously described (23).
Bioinformatics. Sequence alignments were performed with the Basic Local Alignment Search Tool (BLAST). M. tuberculosis H37Rv genes were analyzed with the TubercuList database (http://genolist.pasteur.fr/TubercuList). Homologues of M. tuberculosis genes in M. avium and BCG were identified by use of the unfinished genomic database of M. avium 104 (TIGR; http://www.tigr.org/tdb/) and our in-house BCG Pasteur database, the sequences of which are complete and annotation is in progress.
RESULTS
Most M. tuberculosis immunogens expressed by the vaccine strain BCG Pasteur are conserved in M. avium. Table 1 summarizes the results of alignments between the amino acid sequences of selected M. tuberculosis antigens, for which immunogenicity has been demonstrated, and their homologues in BCG Pasteur and M. avium 104. Not surprisingly, considering that BCG is a member of the M. tuberculosis complex, most of the M. tuberculosis antigens were strictly conserved in BCG Pasteur. With the exception of Mpt70 and the related Mpt83, all selected antigens were identified in M. avium 104 with significant sequence homology. Heat shock proteins Hsp70 and Hsp65, Ag85 complex members A-B, were identified with sequence identity greater than 80%. Alpha-crystallin and the phosphate transport proteins PstS1 and PstS3 were also identified, but with a much lower degree of sequence identity. In contrast, the RD1-encoded antigens ESAT-6, CFP-10, and PPE68 were absent from BCG and M. avium.
Immune responses elicited by environmental mycobacteria cross-react with the BCG 30-kDa antigen, but not with RD1 antigenic products. To examine further the antigenic specificity of RD1-encoded products, cellular extracts of M. avium, M. vaccae, or M. scrofulaceum were incubated with antibodies raised against the potent M. tuberculosis immunogen Ag85B (18) or the RD1 antigens CFP-10, ESAT-6, and PPE68. The Ag85B antiserum detected a 30-kDa product in the M. avium and M. scrofulaceum extracts, although with lower reactivity than with Ag85B from BCG and BCG::RD1 (Fig. 1). No antigen was detected with this antiserum in the M. vaccae preparation. This result was consistent with the observation that M. tuberculosis Ag85B is strictly conserved in BCG whereas M. scrofulaceum and M. avium Ag85B amino acid sequences have 84 and 85% identical residues, respectively, within a 27-kDa segment of this antigen and no significant homology is found with M. vaccae Ag85B.
C57BL/6 mice infected with M. avium, M. scrofulaceum, M. vaccae, or a cocktail of the three strains were then compared for the generation of cellular responses to Ag85B. For this purpose, animals were injected twice with 106 CFU a 2-week interval and sacrificed 3 weeks later. Figure 2 shows that animals exposed to M. avium, M. scrofulaceum, or the mix of environmental strains gave significant IFN- responses to Ag85B compared to unsensitized controls. In contrast, splenocytes from M. vaccae-infected mice produced IFN- in response to M. vaccae culture filtrate but not to Ag85B. In none of the sensitized groups could an IFN- response to PPD or BCG culture filtrate preparation be detected (data not shown).
We used the same approach to determine if immune responses generated by environmental mycobacteria cross-react with the antigenic products encoded by RD1, namely, ESAT-6, CFP-10, and PPE-68. A monoclonal antibody binding an epitope located at positions 3 to 15 in the 95-amino-acid sequence of ESAT-6 showed strong reactivity with the BCG::RD1 cell lysate, but no signal was observed with environmental strains or control BCG (Fig. 1). Similarly, polyclonal sera raised against CFP-10 and PPE-68 recognized their targets in the BCG::RD1 preparation but showed no reactivity with lysates from environmental strains or BCG. When sensitized mice were tested for IFN- production in response to ESAT-6 by use of a synthetic peptide corresponding to an immunodominant T-cell epitope in the context of H-2b (pESAT-6), no response was detected (Fig. 2). Taken together, these results suggested that exposure to M. avium or M. scrofulaceum may generate significant cellular immune responses cross-reacting with BCG antigens such as Ag85B, but not with the immunodominant RD1 antigen ESAT-6.
BCG::RD1 persists significantly longer than BCG in mice presensitized with environmental mycobacteria. BCG growth is severely hampered in mice presensitized with a cocktail of M. avium, M. scrofulaceum, and M. vaccae or with M. avium alone (2). Since persistence of BCG is significantly increased in host mice by stable expression of the RD1 region (22), we examined whether BCG and BCG::RD1 are differentially cleared in animals exposed to environmental mycobacteria. The rationale of this experiment was adapted from the procedure described by Brandt et al. (2) and is outlined in Fig. 3A. Briefly, C57BL/6 mice were immunized by repeated injections of either M. avium, M. scrofulaceum, or M. vaccae and treated for 1 month with a cocktail of rifampin, ethambutol, and clarithromycin added to drinking water in order to clear the remaining mycobacteria. Then, the animals were inoculated with BCG or BCG::RD1 intravenously and vaccine growth was monitored over time in the lungs and spleens. Figure 3B shows that BCG was significantly eliminated from the lungs of mice exposed to each of the environmental mycobacteria 3 weeks following vaccination, compared to naive mice. BCG was also cleared from the spleens of animals immunized with M. avium. In contrast, BCG::RD1 was still present in significant numbers in the lungs and spleens of mice exposed to each of the environmental strains. Therefore, the immune responses mounted by the mouse host immune system to environmental mycobacteria were more effective at clearing BCG than BCG::RD1.
Antimycobacterial cellular responses induced by BCG::RD1 vaccination are not altered by sensitization with environmental mycobacteria. To determine if exposure to environmental mycobacteria differentially impacts on the protective efficacy of BCG and BCG::RD1, we compared splenocytes from sensitized and naive mice for the generation of antimycobacterial IFN- responses following vaccination. In BCG-immunized mice, IFN- production in response to Ag85B or to a BCG culture filtrate was similar in animals preexposed to M. vaccae or M. scrofulaceum and in unsensitized mice (Fig. 4). However, in the group exposed to M. avium prior to vaccination, IFN- responses to BCG antigens were reduced, probably reflecting the clearance of mycobacteria from the spleen. In contrast, potent IFN- responses were generated against both ESAT-6, Ag85B, or BCG culture filtrate in mice vaccinated with BCG::RD1, irrespective of sensitization to environmental mycobacteria.
Protective efficacy of BCG::RD1 against M. tuberculosis infection is not diminished by presensitization with environmental mycobacteria. We then compared BCG and BCG::RD1 for protective efficacy against TB in mice exposed to M. avium, M. scrofulaceum, or M. vaccae prior to vaccination. For this purpose, C57BL/6 mice immunized by repeated injections of each of the environmental mycobacteria were treated with antibiotics, vaccinated, and subsequently challenged by aerosol infection with M. tuberculosis (Fig. 5A) Four weeks later, animals were sacrificed and the lung and spleen bacterial loads were measured. Figure 5B shows that exposure to M. vaccae or M. scrofulaceum did not modify M. tuberculosis growth in the lungs and spleens of vaccinated animals, irrespective of the vaccine strain used. In contrast, the lungs of animals sensitized with M. avium were significantly better protected by BCG::RD1 than by BCG. Interestingly, progression of TB was significantly reduced in the lungs of mice sensitized with each of the environmental mycobacteria before vaccination with BCG::RD1 compared to that in the lungs of unsensitized mice vaccinated with BCG::RD1. This effect was also observed in mice exposed to M. vaccae or M. scrofulaceum before being vaccinated with BCG.
DISCUSSION
Multiplication of BCG in the host is essential for the induction of protective antituberculous immunity. Administration of killed preparations of BCG or treatment with antibiotics after BCG vaccination significantly abrogates the protective efficacy of the vaccine (10). Similarly, clearance of BCG by antimycobacterial immune responses, elicited by sensitization to a cocktail of environmental mycobacteria containing M. avium, was associated with a failure of BCG to confer any protection against TB in CBA/J mice (2). Although we reproduced the observation that BCG is dramatically eliminated in animals sensitized with M. avium, loss of protection was not significant in our model. Two reasons may account for this difference. First, we used a mouse strain more resistant to TB (19). Second, the generation of cross-reactive immune responses blocking BCG growth was probably more important in the work of Brandt et al., since in their experiments sensitization was done with a combination of M. scrofulaceum, M. vaccae, and M. avium (2), whereas environmental mycobacteria were administered separately in our model. To investigate further the importance of prior exposure to M. avium, the vaccination efficacy of BCG and BCG::RD1 is currently being examined in the guinea pig model.
The outcome of prior exposure to M. scrofulaceum or M. vaccae on BCG multiplication was different from that of M. avium in our mouse model. Sensitization with M. avium resulted in total clearance of BCG from the lungs and spleens 21 days after inoculation of 106 live bacilli by the intravenous route, whereas the BCG count was maintained at the initial level in the spleen and decreased by only 10-fold in the lungs of unsensitized control (Fig. 3). In contrast, sensitization with M. scrofulaceum or M. vaccae resulted in the elimination of BCG from mouse lungs within 3 weeks, but the persistence of the vaccine in the spleens of vaccinated animals was not modified. These observations suggest that the inhibitory effect of environmental mycobacteria on BCG growth depends critically on the extent of cross-recognition of antigens shared with the vaccine. The fast-growing M. vaccae is phylogenetically more distant from BCG than the slow-growing M. scrofulaceum and M. avium, as exemplified by immune responses cross-reacting with M. tuberculosis Ag85B in mice exposed to these environmental mycobacteria. Ag85B is strictly conserved in BCG, to a large extent in M. avium or M. scrofulaceum, and not significantly in M. vaccae (Table 1 and data not shown). Accordingly, mice sensitized with M. avium or M. scrofulaceum, but not with M. vaccae, generated IFN--producing cells specific for Ag85B. Our in silico analysis of M. tuberculosis antigen specificity showed that the major T-cell antigens expressed by BCG are conserved in M. avium with a significant degree of sequence identity (Table 1), suggesting that the risk of cross-reactive immune responses is particularly high with this environmental strain.
Protective efficacy of BCG::RD1 against infection with M. tuberculosis was increased in all sensitized groups compared to unsensitized controls. The same effect was observed in BCG-vaccinated mice exposed to M. scrofulaceum or M. vaccae. We can postulate that the antigenic stimulation provided by the BCG bacilli colonizing the spleens of M. scrofulaceum- and M. vaccae-sensitized mice (Fig. 3) was sufficient to boost the protective immune responses primed by the environmental strains. Analysis of antimycobacterial cellular responses 21 days after vaccination with BCG supports this hypothesis, since IFN- production in response to BCG antigens was more important in the groups sensitized with M. scrofulaceum or M. vaccae than in the group sensitized with M. avium or the unsensitized controls (Fig. 4). These results confirm that minimal persistence of the vaccine in the host is required to induce protective immunity. Further, they suggest that prior contact with environmental mycobacteria may efficiently prime protective immune responses to M. tuberculosis infection and boost the efficacy of BCG when the vaccine is able to override the immunological control imparted by sensitization with these strains.
Immune responses elicited by exposure to environmental strains, particularly M. avium, were highly efficient at clearing BCG but not BCG::RD1 from host mice (Fig. 3). Two factors may account for this increased persistence. First, stable reintroduction of the RD1 locus into BCG significantly enhances the capacity of the vaccine to persist in immunocompetent mice (22). The mechanism by which RD1 confers increased virulence to BCG is not clearly defined yet. Since ESAT-6 mutants of M. tuberculosis failed to cause cytolysis of pneumocytes in vitro and showed reduced tissue invasiveness in vivo, ESAT-6 secretion was proposed to mediate virulence by promoting cell-to-cell bacterial spread (14, 25). Another reason why BCG::RD1 persistence is not influenced by sensitization with environmental mycobacteria may that immune responses generated by vaccination with BCG::RD1 are strongly oriented toward ESAT-6. Therefore, the recall of IFN- responses cross-reacting with BCG antigens may be less efficient after vaccination with BCG::RD1 than after vaccination with BCG. Taken together, our results suggest that the potent immunogenicity of ESAT-6 and increased persistence of BCG::RD1 render this recombinant vaccine more able to overcome antimycobacterial responses generated by exposure to some environmental mycobacteria. This vaccine thus represents an attractive alternative to BCG for use in areas where environmental mycobacteria are prevalent.
ACKNOWLEDGMENTS
This work was funded by the Institut Pasteur (PTR 110, Direction de la Valorisation et des Partenariats Industrials) and the European Community (QLRT-2001-02018 and LSHP-CT-2003-503367).
We gratefully acknowledge Colorado State University (under NIAID contract NO1 AI-75320 "Tuberculosis Research materials and Vaccine testing") for providing the Ag85 used in this study and the TIGR Center (Rockville, Md. USA) for giving us access to their M. avium 104 sequence data.
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Department of Infectious Disease Immunology, Statens Serum Institut, Copenhagen, Denmark
ABSTRACT
In silico analysis reveals that most protective antigens expressed by the antituberculous vaccine Mycobacterium bovis BCG (BCG) are conserved in M. avium, supporting the hypothesis that exposure to environmental mycobacteria generates cross-reactive immune responses blocking BCG activity. We investigated the impact of sensitization with M. avium, M. scrofulaceum, or M. vaccae on the protective efficacy of a recombinant BCG strain expressing RD1 antigens (BCG::RD1), using a mouse model of experimental tuberculosis (TB). No evidence that the RD1-encoded antigens ESAT-6, CFP-10, and PPE68 were expressed by these environmental strains could be demonstrated by Western blot analysis. Mice sensitized with each of these strains did not prime cellular immune responses cross-reacting with the immunodominant ESAT-6. Importantly, clearance of BCG::RD1 from the lungs and spleens of mice exposed to each of the environmental strains before vaccination was minimal compared to that of BCG. In mice sensitized with M. avium, increased persistence of BCG::RD1 correlated with stronger antimycobacterial gamma interferon responses and enhanced protection against aerosol infection with M. tuberculosis, compared to BCG. In contrast, animals exposed to M. scrofulaceum or M. vaccae prior to vaccination with BCG or BCG::RD1 were better protected against TB than were the unsensitized controls. Our results suggest that the inhibitory effect of environmental mycobacteria on the protective efficacy of BCG depends critically on the extent of cross-recognition of antigens shared with the vaccine. In hosts sensitized with M. avium, potent immunogenicity of ESAT-6 and increased persistence of BCG::RD1 may allow this recombinant vaccine to overcome preexisting antimycobacterial responses.
INTRODUCTION
Tuberculosis (TB) remains the single most important mycobacterial infection worldwide, with a third of the world's population being infected with Mycobacterium tuberculosis and 10 million new cases of clinical TB being reported to the World Health Organization each year. Meta-analysis of trials with the only currently available vaccine, M. bovis Bacille Calmette-Guerin (BCG), has concluded that BCG confers 50 and 80% protective efficacy against pulmonary and disseminated TB, respectively (7). Several lines of evidence suggest that exposure to environmental mycobacteria may reduce the protective efficacy of BCG against TB. In particular, epidemiological observations have shown that BCG is highly efficient at preventing TB in neonates vaccinated before exposure to environmental mycobacteria occurs (7) or in vaccination trials excluding strictly tuberculin skin test-positive individuals (13). In contrast, in areas like southern India, where isolates from the Mycobacterium avium-intracellulare-scrofulaceum complex are commonly found in water and dust (16), BCG is particularly ineffective and fails to protect adults against pulmonary TB (11, 12).
Recently, animal studies have provided additional evidence that sensitization with environmental mycobacteria may have a direct antagonistic effect on BCG vaccination. Mice presensitized with M. avium or with cocktails of M. avium, M. vaccae, and M. scrofulaceum developed antimycobacterial responses that control the multiplication of BCG, thereby reducing its protective efficacy against TB (2). Sensitization with M. avium or M. fortuitum before vaccination with BCG also showed a modulatory effect on the protective efficacy of BCG against experimental TB in guinea pigs (15). These results strongly suggest that prior exposure to live environmental mycobacteria primes the host immune system against mycobacterial antigens shared with BCG and that recall of this immune response on vaccination results in accelerated clearance of BCG and hence decreased protection against TB.
The persistence of BCG in vivo can be markedly augmented by stable insertion of RD1, a region of difference between attenuated mycobacterial strains (M. bovis BCG and M. microti) and pathogenic species such as M. bovis and M. tuberculosis (5, 8, 17, 22). When mice and guinea pigs were vaccinated with BCG::RD1 knock-in constructions (BCG::RD1), progression of TB was not significantly modified in the lungs but was markedly reduced in the spleen compared to that in BCG-immunized animals. Enhanced protection was associated with the expression of antigens encoded by RD1 (9, 23). Among them is ESAT-6 (6-kDa early secreted antigenic target), a potent Th1 T-cell inducer that protects mice against experimental infection with M. tuberculosis (3). To a lesser extent, CFP-10 (10-kDa culture filtrate protein) and PPE68 have also been identified as immunogenic components of RD1, and both proteins induce Th1-oriented T-cell responses in animal models (9, 20). Expression of CFP-10 and ESAT-6 has been detected in pathogenic species of the M. tuberculosis complex, M. leprae, M. kansasii, M. marinum, and M. smegmatis, although the amino acid sequences of the corresponding proteins vary among these species. As a member of the mycobacterial PPE protein family, PPE68 may show sequence homology to proteins from environmental strains. In fact, nonspecific reactivity of sera from healthy human donors with the PPE68 protein has been reported (6), and PPE68 stimulated high levels of gamma interferon (IFN-) secretion in peripheral blood mononuclear cells isolated from TB patients, as well as from a significant proportion of BCG-vaccinated donors (21). However, no equivalent of CFP-10 and ESAT-6 is detectable in filtrates from BCG or from most environmental species by Western blot analysis techniques (24; J. S. Rothel, personal communication). Importantly, nontuberculous donors do not show cellular responses to ESAT-6 or CFP-10 antigens (1), suggesting that no immune responses cross-reacting with these antigens are primed in hosts exposed to environmental mycobacteria.
In the present study, we postulated that the increased persistence and the antigenic specificity of BCG::RD1 may enable the recombinant vaccine to override immunity imparted by prior contact with environmental mycobacteria. We show that mice immunized with M. avium, M. vaccae, or M. scrofulaceum do not develop immune responses cross-reacting with ESAT-6. Moreover, our data demonstrate that BCG::RD1 persists significantly longer than BCG in animals exposed to each of these strains and that the protective efficacy of BCG::RD1 against TB is not reduced in animals sensitized with these environmental mycobacteria. These results suggest that BCG::RD1 may be a better alternative to BCG in areas where exposure to environmental mycobacteria is common.
MATERIALS AND METHODS
Mycobacteria. BCG::RD1 and BCG::pYUB412 vector control knock-in constructions (23), M. tuberculosis (H37Rv), M. avium (ATCC 15769), M. scrofulaceum (ATCC 19275), and M. vaccae (ATCC 15483) were grown in Middlebrook 7H9-ADC medium supplemented with 0.05% Tween 80 until the mid-log phase of bacterial growth. Mycobacteria were harvested by centrifugation and washed once with 50 mM sodium phosphate buffer (pH 7.0) before resuspension in the same buffer. The bacteria were then sonicated briefly and allowed to stand for 2 h to allow residual aggregates to settle. Bacterial suspensions were aliquoted and frozen at –80°C. For immunization studies, frozen aliquots were thawed and sonicated briefly before injection, and the viability of each inoculum was verified by titer determination on 7H11-OADC agar plates.
Animals and sensitization with environmental mycobacteria. Six-week-old female C57BL/6 mice (Charles Rivers, L'arbresle, France) were housed in specific-pathogen-free animal facilities of the Institut Pasteur. Vaccine persistence and protective efficacy studies with mice exposed to environmental mycobacteria were performed essentially as previously described (2). Briefly, animals were immunized by three consecutive subcutaneous (s.c.) injections of 2 x 106 CFU of either M. avium, M. scrofulaceum, or M. vaccae performed at 2-week intervals. Four weeks after the last injection, the mice were treated with a cocktail of rifampin (Sigma; 100 mg/liter), ethambutol (Sigma; 200 mg/liter), and clarithromycin (Abbott; 200 mg/liter) added to drinking water for 1 month. This treatment resulted in efficient elimination of environmental mycobacteria from the lungs and spleens of the mice (reference 2 and data not shown).
Immunization and infection procedures. To study the immune responses elicited by environmental mycobacteria, mice were immunized by two consecutive s.c. injections of 106 CFU of M. avium, M. scrofulaceum, or M. vaccae or a mixture of the three strains (5 x 105 CFU of each) performed at 2-week intervals. To compare the multiplication of BCG and BCG::RD1 in sensitized mice, the animals were immunized by intravenous injection of 106 CFU 4 weeks after the end of the antibiotic treatment. For protection studies, mice were vaccinated by s.c. injection of 105 CFU of BCG or BCG::RD1 at the base of the tail 4 weeks after the end of the antibiotic treatment. Animals were infected with approximately 100 CFU of M. tuberculosis per lung via the aerosol route 6 weeks postvaccination. The mice were sacrificed 4 weeks postinfection. Lungs and spleens were homogenized using an MM300 apparatus (Qiagen, Hilden, Germany) and 2.5-mm-diameter glass beads. Serial fivefold dilutions in phosphate-buffered saline were plated onto 7H11 agar plates, and CFU were determined after 3 weeks of growth at 37°C.
Mycobacterial antigen preparation and Western blot analysis. Mycobacterial cell lysates were prepared as follows. Culture samples were centrifuged, and bacterial pellets were washed twice with 20 mM Tris buffer (pH 7.5) before being resuspended in 500 μl of the same buffer with complete EDTA-free protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany). The cells were then lysed by shaking on an MM300 apparatus for 10 min at maximum speed with 500 μl of acid-washed 106-μm-diameter glass beads (Sigma, St. Louis, Mo.). Beads and unbroken cells were removed by centrifugation at 5,000 x g for 30 min, and the resulting supernatants were centrifuged at 15,000 x g for 30 min to yield bacterial cell lysates. Mycobacterial culture filtrates were prepared by concentrating culture supernatants on a Millipore filter with a 3-kDa cutoff (YM3). Both cell lysates and culture filtrates were aliquoted and stored at –20°C after protein quantification using a Bio-Rad protein assay. Immunoblot detections were performed as previously described (22), using rabbit polyclonal antibodies raised against the recombinant proteins Rv3873 (PPE68), Rv3874 (CFP-10), Rv1886c (Ag85B), and an anti-ESAT-6 monoclonal antibody (Hyb076-08; Statens Serum Institut, Denmark).
IFN- response to mycobacterial antigens. Single-cell suspensions of splenocytes were prepared by sieving through 200-μm mesh. After red blood cell lysis, cells were resuspended in synthetic HL-1 medium (Cambrex) supplemented with 2 mM L-glutamine, 100 IU of penicillin/ml, and 100 μg of streptomycin/ml. The cells were plated in 96-well plates at 5 x 105 cells per well in presence of antigen preparations, medium alone, PPD (Veterinary Laboratories Agency, Weybridge, United Kingdom), or concanavalin A (Sigma). T-cell responses to ESAT-6 were measured after in vitro stimulation with pESAT-6, a peptide corresponding to the first 20 amino acid residues of ESAT-6 (Neosystem, Strasbourg, France), which has been identified as an immunodominant T-cell epitope on ESAT-6 in the context of H-2b (4). The presence of IFN- in culture supernatants was then assessed after 2 days of culture at 37°C, as previously described (23).
Bioinformatics. Sequence alignments were performed with the Basic Local Alignment Search Tool (BLAST). M. tuberculosis H37Rv genes were analyzed with the TubercuList database (http://genolist.pasteur.fr/TubercuList). Homologues of M. tuberculosis genes in M. avium and BCG were identified by use of the unfinished genomic database of M. avium 104 (TIGR; http://www.tigr.org/tdb/) and our in-house BCG Pasteur database, the sequences of which are complete and annotation is in progress.
RESULTS
Most M. tuberculosis immunogens expressed by the vaccine strain BCG Pasteur are conserved in M. avium. Table 1 summarizes the results of alignments between the amino acid sequences of selected M. tuberculosis antigens, for which immunogenicity has been demonstrated, and their homologues in BCG Pasteur and M. avium 104. Not surprisingly, considering that BCG is a member of the M. tuberculosis complex, most of the M. tuberculosis antigens were strictly conserved in BCG Pasteur. With the exception of Mpt70 and the related Mpt83, all selected antigens were identified in M. avium 104 with significant sequence homology. Heat shock proteins Hsp70 and Hsp65, Ag85 complex members A-B, were identified with sequence identity greater than 80%. Alpha-crystallin and the phosphate transport proteins PstS1 and PstS3 were also identified, but with a much lower degree of sequence identity. In contrast, the RD1-encoded antigens ESAT-6, CFP-10, and PPE68 were absent from BCG and M. avium.
Immune responses elicited by environmental mycobacteria cross-react with the BCG 30-kDa antigen, but not with RD1 antigenic products. To examine further the antigenic specificity of RD1-encoded products, cellular extracts of M. avium, M. vaccae, or M. scrofulaceum were incubated with antibodies raised against the potent M. tuberculosis immunogen Ag85B (18) or the RD1 antigens CFP-10, ESAT-6, and PPE68. The Ag85B antiserum detected a 30-kDa product in the M. avium and M. scrofulaceum extracts, although with lower reactivity than with Ag85B from BCG and BCG::RD1 (Fig. 1). No antigen was detected with this antiserum in the M. vaccae preparation. This result was consistent with the observation that M. tuberculosis Ag85B is strictly conserved in BCG whereas M. scrofulaceum and M. avium Ag85B amino acid sequences have 84 and 85% identical residues, respectively, within a 27-kDa segment of this antigen and no significant homology is found with M. vaccae Ag85B.
C57BL/6 mice infected with M. avium, M. scrofulaceum, M. vaccae, or a cocktail of the three strains were then compared for the generation of cellular responses to Ag85B. For this purpose, animals were injected twice with 106 CFU a 2-week interval and sacrificed 3 weeks later. Figure 2 shows that animals exposed to M. avium, M. scrofulaceum, or the mix of environmental strains gave significant IFN- responses to Ag85B compared to unsensitized controls. In contrast, splenocytes from M. vaccae-infected mice produced IFN- in response to M. vaccae culture filtrate but not to Ag85B. In none of the sensitized groups could an IFN- response to PPD or BCG culture filtrate preparation be detected (data not shown).
We used the same approach to determine if immune responses generated by environmental mycobacteria cross-react with the antigenic products encoded by RD1, namely, ESAT-6, CFP-10, and PPE-68. A monoclonal antibody binding an epitope located at positions 3 to 15 in the 95-amino-acid sequence of ESAT-6 showed strong reactivity with the BCG::RD1 cell lysate, but no signal was observed with environmental strains or control BCG (Fig. 1). Similarly, polyclonal sera raised against CFP-10 and PPE-68 recognized their targets in the BCG::RD1 preparation but showed no reactivity with lysates from environmental strains or BCG. When sensitized mice were tested for IFN- production in response to ESAT-6 by use of a synthetic peptide corresponding to an immunodominant T-cell epitope in the context of H-2b (pESAT-6), no response was detected (Fig. 2). Taken together, these results suggested that exposure to M. avium or M. scrofulaceum may generate significant cellular immune responses cross-reacting with BCG antigens such as Ag85B, but not with the immunodominant RD1 antigen ESAT-6.
BCG::RD1 persists significantly longer than BCG in mice presensitized with environmental mycobacteria. BCG growth is severely hampered in mice presensitized with a cocktail of M. avium, M. scrofulaceum, and M. vaccae or with M. avium alone (2). Since persistence of BCG is significantly increased in host mice by stable expression of the RD1 region (22), we examined whether BCG and BCG::RD1 are differentially cleared in animals exposed to environmental mycobacteria. The rationale of this experiment was adapted from the procedure described by Brandt et al. (2) and is outlined in Fig. 3A. Briefly, C57BL/6 mice were immunized by repeated injections of either M. avium, M. scrofulaceum, or M. vaccae and treated for 1 month with a cocktail of rifampin, ethambutol, and clarithromycin added to drinking water in order to clear the remaining mycobacteria. Then, the animals were inoculated with BCG or BCG::RD1 intravenously and vaccine growth was monitored over time in the lungs and spleens. Figure 3B shows that BCG was significantly eliminated from the lungs of mice exposed to each of the environmental mycobacteria 3 weeks following vaccination, compared to naive mice. BCG was also cleared from the spleens of animals immunized with M. avium. In contrast, BCG::RD1 was still present in significant numbers in the lungs and spleens of mice exposed to each of the environmental strains. Therefore, the immune responses mounted by the mouse host immune system to environmental mycobacteria were more effective at clearing BCG than BCG::RD1.
Antimycobacterial cellular responses induced by BCG::RD1 vaccination are not altered by sensitization with environmental mycobacteria. To determine if exposure to environmental mycobacteria differentially impacts on the protective efficacy of BCG and BCG::RD1, we compared splenocytes from sensitized and naive mice for the generation of antimycobacterial IFN- responses following vaccination. In BCG-immunized mice, IFN- production in response to Ag85B or to a BCG culture filtrate was similar in animals preexposed to M. vaccae or M. scrofulaceum and in unsensitized mice (Fig. 4). However, in the group exposed to M. avium prior to vaccination, IFN- responses to BCG antigens were reduced, probably reflecting the clearance of mycobacteria from the spleen. In contrast, potent IFN- responses were generated against both ESAT-6, Ag85B, or BCG culture filtrate in mice vaccinated with BCG::RD1, irrespective of sensitization to environmental mycobacteria.
Protective efficacy of BCG::RD1 against M. tuberculosis infection is not diminished by presensitization with environmental mycobacteria. We then compared BCG and BCG::RD1 for protective efficacy against TB in mice exposed to M. avium, M. scrofulaceum, or M. vaccae prior to vaccination. For this purpose, C57BL/6 mice immunized by repeated injections of each of the environmental mycobacteria were treated with antibiotics, vaccinated, and subsequently challenged by aerosol infection with M. tuberculosis (Fig. 5A) Four weeks later, animals were sacrificed and the lung and spleen bacterial loads were measured. Figure 5B shows that exposure to M. vaccae or M. scrofulaceum did not modify M. tuberculosis growth in the lungs and spleens of vaccinated animals, irrespective of the vaccine strain used. In contrast, the lungs of animals sensitized with M. avium were significantly better protected by BCG::RD1 than by BCG. Interestingly, progression of TB was significantly reduced in the lungs of mice sensitized with each of the environmental mycobacteria before vaccination with BCG::RD1 compared to that in the lungs of unsensitized mice vaccinated with BCG::RD1. This effect was also observed in mice exposed to M. vaccae or M. scrofulaceum before being vaccinated with BCG.
DISCUSSION
Multiplication of BCG in the host is essential for the induction of protective antituberculous immunity. Administration of killed preparations of BCG or treatment with antibiotics after BCG vaccination significantly abrogates the protective efficacy of the vaccine (10). Similarly, clearance of BCG by antimycobacterial immune responses, elicited by sensitization to a cocktail of environmental mycobacteria containing M. avium, was associated with a failure of BCG to confer any protection against TB in CBA/J mice (2). Although we reproduced the observation that BCG is dramatically eliminated in animals sensitized with M. avium, loss of protection was not significant in our model. Two reasons may account for this difference. First, we used a mouse strain more resistant to TB (19). Second, the generation of cross-reactive immune responses blocking BCG growth was probably more important in the work of Brandt et al., since in their experiments sensitization was done with a combination of M. scrofulaceum, M. vaccae, and M. avium (2), whereas environmental mycobacteria were administered separately in our model. To investigate further the importance of prior exposure to M. avium, the vaccination efficacy of BCG and BCG::RD1 is currently being examined in the guinea pig model.
The outcome of prior exposure to M. scrofulaceum or M. vaccae on BCG multiplication was different from that of M. avium in our mouse model. Sensitization with M. avium resulted in total clearance of BCG from the lungs and spleens 21 days after inoculation of 106 live bacilli by the intravenous route, whereas the BCG count was maintained at the initial level in the spleen and decreased by only 10-fold in the lungs of unsensitized control (Fig. 3). In contrast, sensitization with M. scrofulaceum or M. vaccae resulted in the elimination of BCG from mouse lungs within 3 weeks, but the persistence of the vaccine in the spleens of vaccinated animals was not modified. These observations suggest that the inhibitory effect of environmental mycobacteria on BCG growth depends critically on the extent of cross-recognition of antigens shared with the vaccine. The fast-growing M. vaccae is phylogenetically more distant from BCG than the slow-growing M. scrofulaceum and M. avium, as exemplified by immune responses cross-reacting with M. tuberculosis Ag85B in mice exposed to these environmental mycobacteria. Ag85B is strictly conserved in BCG, to a large extent in M. avium or M. scrofulaceum, and not significantly in M. vaccae (Table 1 and data not shown). Accordingly, mice sensitized with M. avium or M. scrofulaceum, but not with M. vaccae, generated IFN--producing cells specific for Ag85B. Our in silico analysis of M. tuberculosis antigen specificity showed that the major T-cell antigens expressed by BCG are conserved in M. avium with a significant degree of sequence identity (Table 1), suggesting that the risk of cross-reactive immune responses is particularly high with this environmental strain.
Protective efficacy of BCG::RD1 against infection with M. tuberculosis was increased in all sensitized groups compared to unsensitized controls. The same effect was observed in BCG-vaccinated mice exposed to M. scrofulaceum or M. vaccae. We can postulate that the antigenic stimulation provided by the BCG bacilli colonizing the spleens of M. scrofulaceum- and M. vaccae-sensitized mice (Fig. 3) was sufficient to boost the protective immune responses primed by the environmental strains. Analysis of antimycobacterial cellular responses 21 days after vaccination with BCG supports this hypothesis, since IFN- production in response to BCG antigens was more important in the groups sensitized with M. scrofulaceum or M. vaccae than in the group sensitized with M. avium or the unsensitized controls (Fig. 4). These results confirm that minimal persistence of the vaccine in the host is required to induce protective immunity. Further, they suggest that prior contact with environmental mycobacteria may efficiently prime protective immune responses to M. tuberculosis infection and boost the efficacy of BCG when the vaccine is able to override the immunological control imparted by sensitization with these strains.
Immune responses elicited by exposure to environmental strains, particularly M. avium, were highly efficient at clearing BCG but not BCG::RD1 from host mice (Fig. 3). Two factors may account for this increased persistence. First, stable reintroduction of the RD1 locus into BCG significantly enhances the capacity of the vaccine to persist in immunocompetent mice (22). The mechanism by which RD1 confers increased virulence to BCG is not clearly defined yet. Since ESAT-6 mutants of M. tuberculosis failed to cause cytolysis of pneumocytes in vitro and showed reduced tissue invasiveness in vivo, ESAT-6 secretion was proposed to mediate virulence by promoting cell-to-cell bacterial spread (14, 25). Another reason why BCG::RD1 persistence is not influenced by sensitization with environmental mycobacteria may that immune responses generated by vaccination with BCG::RD1 are strongly oriented toward ESAT-6. Therefore, the recall of IFN- responses cross-reacting with BCG antigens may be less efficient after vaccination with BCG::RD1 than after vaccination with BCG. Taken together, our results suggest that the potent immunogenicity of ESAT-6 and increased persistence of BCG::RD1 render this recombinant vaccine more able to overcome antimycobacterial responses generated by exposure to some environmental mycobacteria. This vaccine thus represents an attractive alternative to BCG for use in areas where environmental mycobacteria are prevalent.
ACKNOWLEDGMENTS
This work was funded by the Institut Pasteur (PTR 110, Direction de la Valorisation et des Partenariats Industrials) and the European Community (QLRT-2001-02018 and LSHP-CT-2003-503367).
We gratefully acknowledge Colorado State University (under NIAID contract NO1 AI-75320 "Tuberculosis Research materials and Vaccine testing") for providing the Ag85 used in this study and the TIGR Center (Rockville, Md. USA) for giving us access to their M. avium 104 sequence data.
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