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Department of Medicine, School of Medicine, University of California, Los Angeles, Los Angeles, California 90095
Received 2 October 2002/ Returned for modification 14 November 2002/ Accepted 2 January 2003
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ABSTRACT |
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INTRODUCTION |
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In a previous study (20), two recombinant BCG vaccines (rBCG30) overexpressing the major secretory protein of M. tuberculosis, a 30-kDa mycolyl transferase (2, 27), were described. This protein is not only the major secretory protein of M. tuberculosis in broth culture (19) but it is also among the major proteins of all M. tuberculosis proteins expressed in human macrophages (15, 21). Derived from the commercially available Connaught (Conn) and Tice strains of BCG, the recombinant vaccines were tested in the demanding guinea pig model of pulmonary tuberculosis. In contrast to other small animals used for models of tuberculosis, guinea pigs are much more susceptible to tuberculosis than humans, yet they develop disease that closely mimics human disease clinically, immunologically, and pathologically. Guinea pigs immunized with the recombinant vaccines and then challenged by aerosol with a high dose of the highly virulent Erdman strain of M. tuberculosis had 0.5 log fewer M. tuberculosis bacteria in their lungs and 1 log fewer bacteria in their spleens 10 weeks after challenge than guinea pigs immunized with the parental BCG vaccines, differences that statistically were highly significant. Moreover, the rBCG30-immunized animals had significantly less lung pathology and fewer tubercles in their lungs, livers, and spleens than the animals immunized with the parental BCG vaccines. However, aside from these laboratory and pathological findings, a superior effect of the recombinant vaccines on the well-being of the animals was not demonstrated.
In the present study, we examine the impact of recombinant vaccines on the survival of guinea pigs challenged with M. tuberculosis. We demonstrate that animals immunized with the recombinant rBCG30 vaccines survive significantly longer than animals immunized with the parental conventional BCG vaccines after aerosol challenge with virulent M. tuberculosis. We also show that the recombinant and parental vaccines initially multiply in guinea pig lungs, spleens, and regional lymph nodes and are subsequently cleared from these tissues at the same rate, although, interestingly, low levels of organisms persist for at least a half of a year after immunization.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The following strains were used for bacterial mating experiments: Escherichiacoli strains DH5
[recA1 endA1 gyrA96 thi-1 hsdR17(rK-mK+) supE44 relA1 lacZ+], Y1090 {[D(lac)U169 
lon araD139 strA supF mcrA trpC22::Tn10(Tetr)]}(pMC9AmprTetr), and XL1-Blue {recA1 endA1 gyrA96 thi-1 hsdR17 supE44 relA1 lac[F' proAB lacIqZM15 Tn10(Tetr)]}, all obtained from Stratagene; E. coli strains S17(
pir+ recA thi pro hsdR-M+)(pLC28StrpsCamr) and SM10 (pir+ Strpr Cams), both gifts from Olaf Schneewind, University of Chicago, Chicago, Ill. (7); M. bovis BCG Tice, purchased from Organon; Mycobacterium smegmatis 1-2c [sodA::Tn5(Kanr)], generated by the chromosomal insertion of a resistance marker (M. Tullius, G. Harth, and M. A. Horwitz, unpublished data) into wild-type M. smegmatis 1-2c, originally obtained from Peadar O'Gaora, Imperial College, London, United Kingdom (17); and M. tuberculosis Erdman [glnA1::Tn5(Kanr)], generated from the wild type (see strains listed above) in the same way as M. smegmatis 1-2c [sodA::Tn5(Kanr)].
Plasmids.
The following plasmids were studied in bacterial mating experiments: pLC28, a conjugative R6K derivative containing the mob region of plasmid RP4 and a mutant oriR6K that requires a functional 
protein in trans for replication (provided by E. coli S17's pir lysogen); pGB9, a derivative of the cryptic Mycobacterium fortuitum plasmid pMF1 conferring kanamycin resistance that was obtained from K. G. Papavinasasundaram, National Institute for Medical Research, London, United Kingdom (1); pMTB30 (Fig. 1); and pVK173T, an E. coli-mycobacterium shuttle plasmid conferring resistance to ampicillin, apramycin, and hygromycin, that was obtained from Julian Davies, University of British Columbia, Vancouver, Canada (24).
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Cutaneous delayed-type hypersensitivity (DTH). Guinea pigs were shaved over the back and injected intradermally with 10 µg of purified recombinant M. tuberculosis 30-kDa major secretory protein (r30) in 100 µl of phosphate-buffered saline. r30 was purified as described previously (15, 19). The diameter of hard induration was measured using a blunt instrument. (A separate group of sham-immunized animals from the one used in the challenge studies was used for skin testing. Sham-immunized animals used in challenge studies were not skin tested, to eliminate the possibility that the skin test itself might influence the outcome).
Guinea pig challenge.
Guinea pigs were challenged with an aerosol generated from a 10-ml single-cell suspension containing a total of 5 x 104 CFU of M. tuberculosis Erdman strain, a dose that delivers 
20 live bacteria to the lungs of each animal, as described previously (19). Afterwards, guinea pigs were individually housed in stainless steel cages contained within a laminar flow biohazard safety enclosure and allowed free access to standard laboratory food and water.
Bacterial mating experiments. Donor and recipient bacteria were mixed on an Amicon YM3-43 filter at ratios of 1 x 108:1 x 108 or 1 x 108:1 x 109 and incubated either for 4 or 16 h at 37°C. Bacteria were washed with 1 to 2 ml of medium (Luria-Bertani or 7H9), and exconjugants were assayed by plating on selective medium and incubation for 24 h at 37°C (E. coli), 3 to 5 days at 37°C in 5% CO2-95% air (M. smegmatis), or 2 weeks at 37°C in 5% CO2-95% air (BCG and M. tuberculosis).
Assay for pMTB30 expression in a nonmycobacterium.
The recombinant plasmid pMTB30 was transformed into E. coli DH5, and 2 clones were analyzed in detail for the expression of r30. The clones were grown for 16 h at 37°C in 10 ml of Luria-Bertani medium containing 250 µg of hygromycin/ml to a density of 1 x 109 to 2 x 109 bacteria/ml. The cultures were centrifuged at 3,500 x g for 15 min to separate bacteria from the culture medium. The bacteria were resuspended in 250 µl of polyacrylamide gel sample buffer, boiled for 5 min, and centrifuged at 10,000 x g for 3 min to pellet the insoluble material. The culture supernate was filtered and concentrated with a Centricon 10 unit to 250 µl. Both the pellet and supernate fractions were adjusted so as to reflect the equivalent of approximately 5 x 109 cells in 50 µl of soluble material and then electrophoresed on a 12.5% denaturing polyacrylamide gel. The proteins were transferred electrophoretically to a nitrocellulase membrane and incubated with polyvalent rabbit mycolyl transferase-specific antibodies (raised to the purified M. tuberculosis 30-kDa protein) at a dilution of 1:2,500 for 24 h at 4°C. After incubation of the membrane with secondary goat anti-rabbit, alkaline phosphatase-conjugated antibodies at a dilution of 1:2,500 for 24 h at 4°C, the immunoblot was developed with the alkaline phosphatase-specific substrates 5-bromo-4-chloro-3-indolyl-phosphate-p-toluidine and nitroblue tetrazolium for 30 min at room temperature. As positive controls, culture supernates obtained from the equivalent of 5 x 109 rBCG30 Tice (harboring the plasmid pMTB30) and 1 µg of purified 30-kDa protein (r30) were electrophoresed on the same gel, immunoblotted, and probed with the antibodies on the same membrane.
Antibiotic susceptibility. The two parental BCG strains (BCG Tice and BCG Conn) and the two recombinant strains (rBCG30 Tice and rBCG30 Conn) were tested by the UCLA Clinical Microbiology Laboratory (Microbiology Reference Laboratory, Cypress, Calif.) for sensitivity to the following antibiotics (concentrations are given in parentheses): ethambutol (2.5 µg/ml), isoniazid (0.1 and 0.4 µg/ml), pyrazinamide (100 µg/ml), rifampin (2.0 µg/ml), streptomycin (2.0 µg/ml), ethionamide (5.0 µg/ml), capreomycin (5.0 µg/ml), ciprofloxacin (2.0 µg/ml), amikacin (5.0 µg/ml), and p-aminosalicylic acid (4.0 or 5.0 µg/ml).
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RESULTS |
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(i) Assay for self-transmissibility of plasmid pMTB30.
We first investigated whether the plasmid pMTB30 was capable of self-transmissibility from one bacterium to another and especially from the vaccine strain to other bacteria. We tested the self-transmissibility of plasmid pMTB30 (Hygr) from (i) the recombinant E. coli strain DH5 to E. coli XL1-Blue, (ii) the recombinant M. bovis rBCG30 Tice strain to the E. coli strains DH5 and Y1090 (Tetr), and (iii) the recombinant M. bovis rBCG30 Tice strain to the kanamycin-resistant mycobacteria M. smegmatis 1-2c [sodA::Tn5(Kanr)] and M. tuberculosis Erdman [glnA1::Tn5(Kanr)]. We observed no hygromycin (250 µg/ml)- or hygromycin (250 µg/ml)- and tetracycline (12.5 µg/ml)-resistant E. coli colonies, no hygromycin (50 µg/ml)- and kanamycin (20 µg/ml)-resistant M. smegmatis, and no hygromycin (50 µg/ml)- and kanamycin (20 µg/ml)-resistant M. tuberculosis (M. tuberculosis plates were also supplemented with 20 mM L-glutamine to compensate for the glutamine synthetase deficiency). In a parallel series of experiments, we tested the self-transmissibility of the E. coli-mycobacterium shuttle plasmid pVK173T. We replicated the mating experiments described for pMTB30, and again we observed no apramycin (50 µg/ml)- or apramycin (50 µg/ml)- and tetracycline (12.5 µg/ml)-resistant E. coli colonies and no apramycin (30 µg/ml)- and kanamycin (20 µg/ml)-resistant mycobacterial colonies.
In a set of control experiments, we assayed the transfer of a conjugative plasmid (pLC28StrpsCamr) between E. coli strains under the same conditions. We mixed E. coli S17 (harboring the plasmid) with E. coli SM10 (Strpr Cams) and screened for streptomycin- and chloramphenicol (both at 30 µg/ml)-resistant exconjugants. Exconjugates were obtained at a frequency of 9.5 x 10-7.
These studies demonstrated that plasmid pMTB30 is not self-transmissible to E. coli or mycobacteria.
(ii) Assay for mobilization of plasmid pMTB30. Although the above studies showed that plasmid pMTB30 was not self-transmissible, it remained possible that the plasmid could be mobilizable by a helper plasmid and thus transferred to another bacterial species. We tested this possibility by first introducing pMTB30 into E. coli S17 harboring the conjugative plasmid pLC28StrpsCamr and then mating this strain with E. coli SM10 (Strpr Cams). No streptomycin (30 µg/ml)- and hygromycin (250 µg/ml)-resistant exconjugants were obtained. More importantly, when this same E. coli S17 strain was mated with the mycobacterial strains M. smegmatis 1-2c [sodA::Tn5(Kanr)] and M. tuberculosis Erdman [glnA1::Tn5(Kanr)], no hygromycin (50 µg/ml)- and kanamycin (20 µg/ml)-resistant mycobacterial colonies were obtained (M. tuberculosis Erdman plates were again supplemented with 20 mM L-glutamine). As a positive control, we introduced the mobilizable E. coli-mycobacterium shuttle plasmid pVK173T into E. coli S17 (containing pLC28StrpsCamr) and then mated this strain with the mycobacterial strains M. smegmatis 1-2c [sodA::Tn5(Kanr)] and M. tuberculosis Erdman [glnA1::Tn5(Kanr)]. These matings resulted in hygromycin (50 µg/ml)-, apramycin (30 µg/ml)-, and kanamycin (20 µg/ml)-resistant M. smegmatis colonies at a frequency of 7.7 x 10-7 and M. tuberculosis colonies with the same resistance markers at a frequency of 1.3 x 10-7, demonstrating that this plasmid can be mobilized to other mycobacteria.
Although these studies showed that pMTB30 was not mobilizable with an E. coli-based helper plasmid, it remained possible that the plasmid was mobilizable to other mycobacteria with a mycobacterial helper plasmid. We investigated this possibility by introducing plasmid pGB9(Kanr) into the wild-type strains M. smegmatis 1-2c and M. tuberculosis Erdman and then mating these strains with the recombinant rBCG30 Tice[pMTB30(Hygr)]. In both cases, hygromycin (50 µg/ml)- and kanamycin (20 µg/ml)-resistant exconjugant BCG Tice colonies were obtained at frequencies of 8.0 x 10-7. However, when recombinant BCG Tice[pMTB30(Hygr), pGB9(Kanr)] bacteria were mixed with M. smegmatis 1-2c or M. tuberculosis Erdman wild-type bacteria, no hygromycin (50 µg/ml)- and kanamycin (20 µg/ml)-resistant colonies were obtained, except, of course, for the donor bacteria. (M. smegmatis is differentiated from BCG and M. tuberculosis by its much faster growth [visible colonies after an incubation period of 3 to 4 days versus 10 to 12 days for BCG and M. tuberculosis], and BCG is differentiated from M. tuberculosis by replating the bacteria on plates containing 50 µg of 2-thiophene carboxylic acid hydrazide/ml, which allows growth of only M. tuberculosis.) Thus, pGB9 is self-transmissible but cannot mobilize pMTB30 into mycobacteria.
These studies showed that pMTB30 cannot be mobilized with the help of a conjugative plasmid, a not-unexpected result, since the sequence of pMTB30 lacks both a mob locus and an oriT element.
(iii) Assay for pMTB30 expression in a nonmycobacterium.
To determine if the plasmid pMTB30 could be expressed in a nonmycobacterium, we transformed the plasmid by electroporation into E. coli DH5, cultured the transformed organism in the presence of hygromycin to maintain the plasmid, and assayed expression of the 30-kDa protein in both the bacterial pellet and the culture medium by immunoblot analysis by using polyvalent mycolyl transferase-specific antibodies. The antibodies did not react with either of the E. coli-derived protein fractions, demonstrating that E. coli cannot express the 30-kDa protein gene as contained in the plasmid pMTB30. In contrast, the antibodies reacted strongly with both the recombinant protein expressed in rBCG30 Tice and the purified 30-kDa protein (r30). This analysis showed that even when pMTB30 is forced into E. coli by electroporation, it does not express the M. tuberculosis 30-kDa major secretory protein, a result consistent with nonrecognition of the mycobacterial promoter by E. coli.
(iv) Antibiotic susceptibility. Both parent BCG strains and recombinant BCG strains were resistant to pyrazinamide, as is typical of M. bovis. All were sensitive to the other nine antibiotics tested. However, the recombinant rBCG30 Tice strain was slightly less sensitive to isoniazid (resistant to a concentration of 0.1 µg/ml but sensitive to a concentration of 0.4 µg/ml) than the parental BCG Tice, the recombinant rBCG30 Conn, and the parental BCG Conn (all sensitive to concentrations of both 0.1 and 0.4 µg/ml). Possibly, the rBCG30 Tice strain's much greater expression of the 30-kDa mycolyl transferase, a protein involved in the synthesis of the cell wall component trehalose dimycolate, rendered this strain slightly more resistant to isoniazid, an inhibitor of mycolic acid synthesis.
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The clear superiority of the higher-producing rBCG30 Tice strain compared with the lower-producing rBCG30 Conn strain in this study was not demonstrated in the predecessor study in which the two recombinant vaccines induced comparable reductions in bacterial load in the lungs and spleen (20). Hence, the assessment of survival but not bacterial load after immunization and challenge allowed for differentiation between the vaccines in potency. However, even in the predecessor study, the rBCG30 Tice-immunized animals had less organ pathology than the rBCG30 Conn-immunized animals.
Many different strains of BCG have been used as vaccines throughout the world, reflecting a lack of evidence that any one of them is superior to any other. Colditz et al. (9), in their meta-analysis of the published literature on BCG trials, investigated the impact of BCG pedigree on between-study variability and found no impact. They additionally concluded that trials with Mycobacterium microti (vole bacillus), a strain less-closely related to M. tuberculosis than M. bovis BCG, gave protection equal to that of BCG. Recently, the Tice and Conn BCG strains were compared for immunogenicity and reactogenicity in a human clinical trial, and no difference was detected between them (18). Similarly, in this and our previous study, no significant difference in efficacy was observed between the parental BCG Conn and Tice strains. These and four other BCG strains secreted comparably low amounts of the 30-kDa protein.
This study lends further support to the extracellular protein hypothesis for vaccines against intracellular pathogens, which holds that proteins secreted or otherwise released into the intracellular compartment of such pathogens are potentially potent immunoprotective molecules (3-6, 16, 19, 20, 25). Such proteins are available for processing and presentation to the immune system as major histocompatibility complex-peptide complexes on host cells. These complexes serve to alert the host immune system to the presence of a pathogen sequestered within the host cell, allowing it to mount an appropriate antimicrobial response. As in previous studies (3-5), in which immunization with either of two major extracellular proteins of Legionella pneumophila, the agent of Legionnaires' disease, was demonstrated to enhance the survival of guinea pigs after challenge by aerosol with L. pneumophila, this study shows that immunization with a major extracellular protein of M. tuberculosis, albeit via a live vector rather than as a protein in an adjuvant, enhances survival after challenge by aerosol with M. tuberculosis in a highly susceptible animal model.
Prior to this study, an alternative explanation for the enhanced efficacy of the recombinant BCG vaccine compared with that of the parental BCG vaccine was that the recombinant vaccine was more virulent in the guinea pig model and, for this reason, it induced a stronger immune response. However, the parental and recombinant BCG vaccines were found to be equally avirulent for guinea pigs, ruling out this possibility.
Of the small-animal models of tuberculosis, the guinea pig model most closely resembles human disease. Like humans, but unlike mice and rats, guinea pigs are susceptible to low doses of aerosolized M. tuberculosis, exhibit a high sensitivity to tuberculin, develop a cutaneous DTH response characterized by a dense mononuclear cell infiltrate, and exhibit Langhans giant cells and caseation in their lung lesions. A major difference between humans and guinea pigs, however, is the susceptibility to disease after infection with M. tuberculosis. Whereas only about 10% of humans develop active disease after infection, some soon after and some after a period of latency, 100% of guinea pigs develop active rapidly progressive disease to which they quickly succumb. The high susceptibility of guinea pigs to the development of active rapidly fatal disease renders this model especially suitable for assessing the capacity of a vaccine against tuberculosis to enhance survival after M. tuberculosis challenge.
Assessing survival in guinea pigs after respiratory challenge with M. tuberculosis is a time-consuming and expensive undertaking. While not often done, our study is not the first to do so. In a study conducted over three decades ago, Wiegeshaus et al. (26), using experimental conditions very different from ours (lower challenge dose, less-virulent challenge strain, shorter immunization-challenge interval, and different housing conditions, etc.), found that guinea pigs immunized with the BCG vaccine survived significantly longer than sham-immunized animals after aerosol challenge with M. tuberculosis, which was similar to our results with the parental BCG vaccines. Coincidentally, the BCG vaccine studied by Wiegeshaus et al. originated at Tice Laboratory at the University of Illinois, as did one of our parental vaccines.
Thus far, our studies of the rBCG30 Tice vaccine have demonstrated that, in the highly susceptible guinea pig model of pulmonary tuberculosis, immunization with the vaccine results in a marked reduction in bacterial burden, number of tubercles, and pathology in the lungs and other organs, and in enhanced survival—all to an extent greater than that achieved by immunization with parental BCG Tice. To what extent this effect of the rBCG30 vaccine will translate to humans and whether the vaccine will have an impact on reactivation tuberculosis remains to be determined in forthcoming clinical studies. However, given the relatively high innate resistance of even unvaccinated humans to M. tuberculosis, it seems reasonable to postulate that an immunologic intervention such as the rBCG30 Tice vaccine that is likely to shift the balance of power between the host and pathogen further toward the host will have a beneficial impact on human disease incidence. Even a modest reduction in disease incidence would translate into hundreds of thousands of lives saved.
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ACKNOWLEDGMENTS |
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a Maslea-Gali
, and Chalermchai Chaloyphian for technical assistance, Jeffrey Gornbein and Lena Ting for assistance with statistical analyses, David Bruckner for assistance with antibiotic susceptibility testing, and the Sequella Global Tuberculosis Foundation for encouragement of these studies. This work was supported by National Institutes of Health grant AI31338.
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FOOTNOTES |
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REFERENCES |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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| 1. | Bachrach, G., M. J. Colston, H. Bercovier, D. Bar-Nir, C. Anderson, and K. G. Papavinasasundaram. 2000. A new single-copy mycobacterial plasmid, pMF1, from Mycobacterium fortuitum which is compatible with the pAL5000 replicon. Microbiology 146:297-303. |
| 2. | Belisle, J. T., V. D. Vissa, T. Sievert, K. Takayama, P. J. Brennan, and G. S. Besra. 1997. Role of the major antigen of Mycobacterium tuberculosis in cell wall biogenesis. Science 276:1420-1422. |
| 3. | Blander, S. J., and M. A. Horwitz. 1989. Vaccination with the major secretory protein of Legionella pneumophila induces cell-mediated and protective immunity in a guinea pig model of Legionnaires' disease. J. Exp. Med. 169:691-705. |
| 4. | Blander, S. J., and M. A. Horwitz. 1991. Vaccination with the major secretory protein of Legionella induces humoral and cell-mediated immune responses and protective immunity across different serogroups of Legionella pneumophila and different species of Legionella. J. Immunol. 147:285-291.[Abstract] |
| 5. | Blander, S. J., and M. A. Horwitz. 1993. The major cytoplasmic membrane protein of Legionella pneumophila, a genus common antigen and member of the hsp 60 family of heat shock proteins, induces protective immunity in a guinea pig model of Legionnaires' disease. J. Clin. Investig. 91:717-723. |
| 6. | Breiman, R. F., and M. A. Horwitz. 1987. The major secretory protein of Legionella pneumophila stimulates proliferation of splenic lymphocytes from immunized guinea pigs. Clin. Res. 35:469A. |
| 7. | Cheng, L. W., D. M. Anderson, and O. Schneewind. 1997. Two independent type III secretion mechanisms for YopE in Yersinia enterocolitica. Mol. Microbiol. 24:757-765.[CrossRef][Medline] |
| 8. | Cohn, D. L., F. Bustreo, and M. C. Raviglione. 1997. Drug-resistant tuberculosis: review of the worldwide situation and the W. H. O./IUATLD Global Surveillance Project. Clin. Infect. Dis. 24:S121-S130. |
| 9. | Colditz, G. A., T. F. Brewer, C. S. Berkey, M. E. Wilson, E. Burdick, H. V. Fineberg, and F. Mosteller. 1994. Efficacy of BCG vaccine in the prevention of tuberculosis. Meta-analysis of the published literature. JAMA 271:698-702.[Abstract] |
| 10. | Dye, C., S. Scheele, P. Dolin, V. Pathania, and M. C. Raviglione. 1999. Consensus statement. Global burden of tuberculosis: estimated incidence, prevalence, and mortality by country. W. H. O. Global Surveillance and Monitoring Project. JAMA 282:677-686. |
| 11. | Ensernik, M. 2001. Driving a stake into resurgent TB. Science 293:234-235. |
| 12. | Fine, P. E. M. 1989. The BCG story: lessons from the past and implications for the future. Rev. Infect. Dis. 11:(Suppl. 2):S353-S359. |
| 13. | Garbe, T. R., J. Barathi, S. Barnini, Y. Zhang, C. Abou-Zeid, D. Tang, R. Mukherjee, and D. B. Young. 1994. Transformation of mycobacterial species using hygromycin resistance as a selectable marker. Microbiology 140:133-138.[Abstract] |
| 14. | Haas, D. W., and R. M. Des Prez. 1995. Mycobacterium tuberculosis, p. 2213-2243. In G. L. Mandell, J. E. Bennett, and R. Dolin (ed.), Principles and practice of infectious diseases. Churchill Livingstone, Inc., New York, N.Y. |
| 15. | Harth, G., B.-Y.Lee, J. Wang, D. L. Clemens, and M. A. Horwitz. 1996. Novel insights into the genetics, biochemistry, and immunocytochemistry of the 30-kilodalton major extracellular protein of Mycobacterium tuberculosis. Infect. Immun. 64:3038-3047.[Abstract] |
| 16. | Harth, G., B-Y. Lee, and M. A. Horwitz. 1997. High-level heterologous expression and secretion in rapidly growing nonpathogenic mycobacteria of four major Mycobacterium tuberculosis extracellular proteins considered to be leading vaccine candidates and drug targets. Infect. Immun. 65:2321-2328.[Abstract] |
| 17. | Herrmann, J. L., P. O'Gaora, A. Gallagher, J. E. R. Thole, and D. B. Young.1996. Bacterial glycoproteins: a link between glycosylation and proteolytic cleavage of a 19 kDa antigen from Mycobacterium tuberculosis. EMBO J. 15:3547-3554.[Medline] |
| 18. | Hoft, D. F., E. B. Kemp, M. Marinaro, O. Cruz, H. Kiyono, J. R. McGhee, J. T. Belisle, T. W. Milligan, J. P. Miller, and R. B. Belshe. 1999. A double-blind, placebo-controlled study of Mycobacterium-specific human immune responses induced by intradermal bacille Calmette-Guerin vaccination. J. Lab. Clin. Med. 134:244-252.[CrossRef][Medline] |
| 19. | Horwitz, M. A., B.-W. E. Lee, B. J. Dillon, and G. Harth. 1995. Protective immunity against tuberculosis induced by vaccination with major extracellular proteins of Mycobacterium tuberculosis. Proc. Natl. Acad. Sci. USA 92:1530-1534. |
| 20. | Horwitz, M. A., G. Harth, B. J. Dillon, and S. Malesa-Galic. 2000. Recombinant BCG vaccines expressing the Mycobacterium tuberculosis 30 kDa major secretory protein induce greater protective immunity against tuberculosis than conventional BCG vaccines in a highly susceptible animal model. Proc. Natl. Acad. Sci. USA 97:13853-13858. |
| 21. | Lee, B,-Y., and M. A. Horwitz. 1995. Identification of macrophage and stress induced proteins of Mycobacterium tuberculosis. J. Clin. Investig. 96:245-249. |
| 22. | Murray, C. J. L., K. Styblo, and A. Rouillon. 1990. Tuberculosis in developing countries: burden, intervention, and cost. Bull. Int. Union Tuberc. Lung Dis. 65:6-24.[Medline] |
| 23. | Orme, I. M., D. N. McMurray, and J. T. Belisle. 2001. Tuberculosis vaccine development: recent progress. Trends Microbiol. 9:115-118.[CrossRef][Medline] |
| 24. | Paget, E., and J. Davies. 1996. Apramycin resistance as a selective marker for gene transfer in mycobacteria. J. Bacteriol. 178:6357-6360. |
| 25. | Pal, P. G., and M. A. Horwitz. 1992. Immunization with extracellular proteins of Mycobacterium tuberculosis induces cell-mediated immune responses and substantial protective immunity in a guinea pig model of pulmonary tuberculosis. Infect. Immun. 60:4781-4792. |
| 26. | Wiegeshaus, E. H., D. N. McMurray, A. A. Grover, G. E. Harding, and D. W. Smith. 1970. Host-parasite relationships in experimental airborne tuberculosis. III. Relevance of microbial enumeration to acquired resistance in guinea pigs. Am. Rev. Respir. Dis. 102:422-429.[Medline] |
| 27. | Wiker, H. G., and M. Harboe. 1992. The antigen 85 complex: a major secretion product of Mycobacterium tuberculosis. Microbiol. Rev. 56:648-661. |
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