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. 2006 Sep;13(9):1014-21.
doi: 10.1128/CVI.00120-06.

Unique model of dormant infection for tuberculosis vaccine development

Suely S Kashino  1 , Pamela Ovendale, Angelo Izzo, Antonio Campos-Neto
Affiliations

Unique model of dormant infection for tuberculosis vaccine development

Suely S Kashino et al. Clin Vaccine Immunol. 2006 Sep.

Abstract

Most individuals exposed to Mycobacterium tuberculosis become infected but hinder the infectious process in dormant foci, known as latent tuberculosis. This limited infection usually stimulates strong T-cell responses, which provide lifelong resistance to tuberculosis. However, latent tuberculosis is still poorly understood, particularly because of the lack of a reliable animal model of dormant infection. Here we show that inoculation of mice with a unique streptomycin-auxotrophic mutant of Mycobacterium tuberculosis recapitulates dormant infection. The mutant grows unimpaired in the presence of streptomycin and no longer grows but remains viable for long periods of time after substrate removal, shifting from the log growth phase to the latent stage, as indicated by augmented production of alpha-crystallin. Mice challenged with the mutant and inoculated with streptomycin for approximately 3 weeks developed a limited infection characterized by a low bacteriological burden and the presence of typical granulomas. After substrate withdrawal, the infection was hindered but few microorganisms remained viable (dormant) in the animals' tissues for at least 6 months. In addition, the animals developed both potent T-cell responses to M. tuberculosis antigens, such as early culture filtrate, Ag85B, and ESAT-6, and resistance to reinfection with virulent M. tuberculosis. Therefore, infection of mice or other animals (e.g., guinea pigs) with M. tuberculosis strain 18b constitutes a simple and attractive animal model for evaluation of antituberculosis vaccines in the context of an M. tuberculosis-presensitized host, a prevailing condition among humans in need of a vaccine.

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Figures

FIG. 1.
FIG. 1.
Production of α-crystallin by Mycobacterium tuberculosis strain 18b. Approximately 108 CFU of the mycobacterium was incubated for 10 days in Middlebrook 7H9 medium either in the presence of 50 μg/ml of streptomycin (A) or in the absence of the antibiotic (B). Bacterial samples were harvested daily, lysed, normalized for the same protein concentration, and analyzed for the presence of α-crystallin by Western blotting with the specific MAb 04.CS49. Lane R, purified recombinant α-crystallin; lanes 1 to 10, days of Mycobacterium growth in culture. Note the steady augmentation of α-crystallin in the samples obtained from the Mycobacterium incubated in streptomycin-free medium.
FIG. 2.
FIG. 2.
Growth of M. tuberculosis strain 18b in tissues of infected mice. BALB/c mice were infected via the lateral tail vein with 106 CFU of M. tuberculosis strain 18b. One group of mice (filled symbols) received daily s.c. injections of streptomycin sulfate (2 mg/mouse) for 4 weeks. A second group of mice (open symbols) was injected with saline only. The animals were euthanized on days 7, 14, 21, and 28 (A) or at 6 months (B) after the challenge; and their spleens and lungs were removed, homogenized, and plated on Middlebrook 7H10 Bacto agar plates containing 50 μg/ml of streptomycin sulfate. The numbers of CFU were determined after incubation for 3 to 4 weeks at 37°C. Bars represent the standard errors of the means of the results for five mice per group. This figure shows the results of one representative experiment of three experiments performed, with essentially the same results obtained in each experiment.
FIG. 3.
FIG. 3.
Histopathology of liver, lung, and spleen tissue from BALB/c mice infected with M. tuberculosis strain 18b. The mice were infected as described in the legend to Fig. 2, followed by daily injections (s.c.) of 2 mg of streptomycin sulfate for 2 weeks. The mice were euthanized, and the lungs, spleen, and liver were removed. Tissue sections were stained with hematoxylin-eosin (A and B) or by the Ziehl-Neelsen method (C and D) for acid-fast bacilli. Typical epithelioid granulomas are seen in the liver and spleen sections (A, C, and D). Perivascular infiltration of mononuclear cells is clearly seen in the lung tissue (B). Arrows point to several acid-fast M. tuberculosis organisms inside the spleen granulomas (C and D). Magnifications, ×200 (A and B) and ×1,000 (C and D).
FIG. 4.
FIG. 4.
Immunohistochemical staining of CD11b+ cells and CD4+ and CD8+ lymphocytes in frozen sections of lung tissue from BALB/c mice infected with M. tuberculosis strain 18b. The mice were infected as described in the legend to Fig. 2, followed by daily injections (s.c.) of 2 mg of streptomycin sulfate for 3 weeks. The mice were euthanized, and the lungs were removed and processed as described in Material and Methods. Note the characteristic granuloma organization with the central accumulation of CD11b+ cells (macrophages) surrounded by a dense mantle of CD4+ lymphocytes and the sparse distribution of CD8+ lymphocytes. These are serial sections (magnification, ×200) 5 μm thick and are representative of the findings obtained in three experiments.
FIG. 5.
FIG. 5.
Cytokine production by spleen cells of M. tuberculosis-infected mice. BALB/c mice were infected i.v. with M. tuberculosis strain 18b and supplemented daily (s.c. injections) with streptomycin (2 mg/mouse) for 2 weeks. The animals were euthanized 4 weeks after the withdrawal of streptomycin. Mononuclear spleen cells were obtained and stimulated for 3 days with CF antigens of M. tuberculosis H37Rv, Ag85 complex, and ESAT-6. Supernatants were harvested and assayed for the presence of IFN-γ by ELISA. Bars are the standard deviations of the means obtained from triplicate cultures. This figure shows the results of one representative experiment of three experiments performed, with essentially the same results obtained in each experiment.
FIG. 6.
FIG. 6.
Growth of M. tuberculosis H37Rv in the tissues of mice with established latent infection with M. tuberculosis strain 18b (Mtb 18b). Dormant infection was induced by infecting five mice i.v. with 106 CFU of M. tuberculosis strain 18b, followed by daily s.c. injections of streptomycin (2 mg/mouse) for 2 weeks (preinfection was with M. tuberculosis strain 18b). Control groups of mice were either treated with saline only (saline) or vaccinated with BCG. Ten weeks after infection with the auxotrophic mutant, the animals were challenged by the respiratory route with 200 aerosolized CFU of M. tuberculosis H37Rv. The animals were euthanized 4 weeks later, and the CFU in the spleens and lungs were enumerated on 7H10 agar plates. Bars represent the standard errors of the means of the results for five mice. This figure shows the results of one representative experiment of two experiments performed, with essentially the same results obtained in each experiment.
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References

    1. Andersen, P. 1994. Effective vaccination of mice against Mycobacterium tuberculosis infection with a soluble mixture of secreted mycobacterial proteins. Infect. Immun. 62:2536-2544. - PMC - PubMed
    1. Brock, I., M. E. Munk, A. Kok-Jensen, and P. Andersen. 2001. Performance of whole blood IFN-gamma test for tuberculosis diagnosis based on PPD or the specific antigens ESAT-6 and CFP-10. Int. J. Tuberc. Lung Dis. 5:462-467. - PubMed
    1. Cardoso, F. L., P. R. Antas, A. S. Milagres, A. Geluk, K. L. Franken, E. B. Oliveira, H. C. Teixeira, S. A. Nogueira, E. N. Sarno, P. Klatser, T. H. Ottenhoff, and E. P. Sampaio. 2002. T-cell responses to the Mycobacterium tuberculosis-specific antigen ESAT-6 in Brazilian tuberculosis patients. Infect. Immun. 70:6707-6714. - PMC - PubMed
    1. Collins, F. M., and V. Montalbine. 1976. Distribution of mycobacteria grown in vivo in the organs of intravenously infected mice. Am. Rev. Respir. Dis. 113:281-286. - PubMed
    1. Cunningham, A. F., and C. L. Spreadbury. 1998. Mycobacterial stationary phase induced by low oxygen tension: cell wall thickening and localization of the 16-kilodalton alpha-crystallin homolog. J. Bacteriol. 180:801-808. - PMC - PubMed

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