Ryhor Harbacheuski

Virulence of Selected Mycobacterium tuberculosis Clinical Isolates in the Rabbit Model of Meningitis Is Dependent on Phenolic Glycolipid Produced by the Bacilli

Infection with Mycobacterium tuberculosis in humans results in active disease in approximately 10% of immune-competent individuals, with the most-severe clinical manifestations observed when the bacilli infect the central nervous system (CNS). Here, we use a rabbit model of tuberculous meningitis to evaluate the severity of disease caused by the M. tuberculosis clinical isolates CDC1551, a highly immunogenic strain, and HN878 or W4, 2 members of the W/Beijing family of strains. Compared with infection with CDC1551, CNS infection with HN878 or W4 resulted in higher bacillary loads in the cerebrospinal fluid and brain, increased dissemination of bacilli to other organs, persistent levels of tumor necrosis factor-alpha , higher leukocytosis, and more-severe clinical manifestations. This pathogenic process is associated with the production by HN878 of a polyketide synthase-derived phenolic glycolipid (PGL), as demonstrated by reduced virulence in rabbits infected with an HN878 mutant disrupted in the pks1-15 gene, which is required for PGL synthesis.

Bacillus Calmette-Guérin Vaccination of Human Newborns Induces T Cells with Complex Cytokine and Phenotypic Profiles

The immune response to vaccination with bacillus Calmette-Guérin (BCG), the only tuberculosis vaccine available, has not been fully characterized. We used multiparameter flow cytometry to examine specific T cell cytokine production and phenotypic profiles in blood from 10-wk-old infants routinely vaccinated with BCG at birth. Ex vivo stimulation of whole blood with BCG for 12 h induced expression of predominantly IFN-γ, IL-2, and TNF-α in CD4+ T cells in seven distinct cytokine combinations. IL-4 and IL-10 expression was detected in CD4+ T cells at low frequencies and only in cells that did not coexpress type 1 cytokines. Specific CD8+ T cells were less frequent than CD4+ T cells and produced mainly IFN-γ and/or IL-2 and less TNF-α, IL-4, and IL-10. Importantly, many mycobacteria-specific CD4+ and CD8+ T cells did not produce IFN-γ. The predominant phenotype of BCG-specific type 1 T cells was that of effector cells, i.e., CD45RA−CCR7−CD27+, which may reflect persistence of Mycobacterium bovis BCG in infants until 10 wk of age. Among five phenotypic patterns of CD4+ T cells, central memory cells were more likely to be IL-2+ and effector cells were more likely to be IFN-γ+. We concluded that neonatal vaccination with BCG induces T cells with a complex pattern of cytokine expression and phenotypes. Measuring IFN-γ production alone underestimates the magnitude and complexity of the host cytokine response to BCG vaccination and may not be an optimal readout in studies of BCG and novel tuberculosis vaccination.

The Mycobacterium tuberculosis iniA gene is essential for activity of an efflux pump that confers drug tolerance to both isoniazid and ethambutol

Little is known about the intracellular events that occur following the initial inhibition of Mycobacterium tuberculosis by the first‐line antituberculosis drugs isoniazid (INH) and ethambutol (EMB). Understanding these pathways should provide significant insights into the adaptive strategies M. tuberculosis undertakes to survive antibiotics. We have discovered that the M. tuberculosis iniA gene (Rv 0342) participates in the development of tolerance to both INH and EMB. This gene is strongly induced along with iniB and iniC (Rv 0341 and Rv 0343) by treatment of Mycobacterium bovis BCG or M. tuberculosis with INH or EMB. BCG strains overexpressing M. tuberculosis iniA grew and survived longer than control strains upon exposure to inhibitory concentrations of either INH or EMB. An M. tuberculosis strain containing an iniA deletion showed increased susceptibility to INH. Additional studies showed that overexpression of M. tuberculosis iniA in BCG conferred resistance to ethidium bromide, and the deletion of iniA in M. tuberculosis resulted in increased accumulation of intracellular ethidium bromide. The pump inhibitor reserpine reversed both tolerance to INH and resistance to ethidium bromide in BCG. These results suggest that iniA functions through an MDR‐pump like mechanism, although IniA does not appear to directly transport INH from the cell. Analysis of two‐dimensional crystals of the IniA protein revealed that this predicted transmembrane protein forms multimeric structures containing a central pore, providing further evidence that iniA is a pump component. Our studies elucidate a potentially unique adaptive pathway in mycobacteria. Drugs designed to inhibit the iniA gene product may shorten the time required to treat tuberculosis and may help prevent the clinical emergence of drug resistance.

Bacillus Calmette Guerin Vaccination of Human Newborns Induces a Specific, Functional CD8+ T Cell Response

Mounting evidence points to CD8+ T cells playing an important role in protective immunity against Mycobacterium tuberculosis. The only available vaccine against tuberculosis, bacillus Calmette Guérin (BCG), has traditionally been viewed not to induce these cells optimally. In this study, we show that vaccination of human newborns with BCG does indeed induce a specific CD8+ T cell response. These cells degranulated or secreted IFN-γ, but not both, when infant blood was incubated with BCG. This stimulation also resulted in proliferation and up-regulation of cytotoxic molecules. Overall, the specific CD8+ T cell response was quantitatively smaller than the BCG-induced CD4+ T cell response. Incubation of whole blood with M. tuberculosis also caused CD8+ T cell IFN-γ expression. We conclude that BCG induces a robust CD8+ T cell response, which may contribute to vaccination-induced protection against tuberculosis.

Evaluation of the Mtb72F Polyprotein Vaccine in a Rabbit Model of Tuberculous Meningitis

ABSTRACT Using a rabbit model of tuberculous meningitis, we evaluated the protective efficacy of vaccination with the recombinant polyprotein Mtb72F, which is formulated in two alternative adjuvants, AS02A and AS01B, and compared this to vaccination with Mycobacterium bovis bacillus Calmette-Guérin (BCG) alone or as a BCG prime/Mtb72F-boost regimen. Vaccination with Mtb72F formulated in AS02A (Mtb72F+AS02A) or Mtb72F formulated in AS01B (Mtb72F+AS01B) was protective against central nervous system (CNS) challenge with Mycobacterium tuberculosis H37Rv to an extent comparable to that of vaccination with BCG. Similar accelerated clearances of bacilli from the cerebrospinal fluid, reduced leukocytosis, and less pathology of the brain and lungs were noted. Weight loss of infected rabbits was less extensive for Mtb72F+AS02A-vaccinated rabbits. In addition, protection against M. tuberculosis H37Rv CNS infection afforded by BCG/Mtb72F in a prime-boost strategy was similar to that by BCG alone. Interestingly, Mtb72F+AS01B induced better protection against leukocytosis and weight loss, suggesting that the polyprotein in this adjuvant may boost immunity without exacerbating inflammation in previously BCG-vaccinated individuals.

Aerosol Exposure System for Rabbits: Application to M. Tuberculosis Infection

The development and testing of new antitubercular vaccines and drugs require the use of experimental animal models that resemble pulmonary tuberculosis in humans. We evaluated the effectiveness of an aerosol exposure system for studying experimental pulmonary tuberculosis in rabbits. The device is a snout-only exposure system with 6-animal capacity that uses an individual facemask for each animal. The system is operated from a single jet Bio Aerosol Nebulizing Generator (BANG). The device is placed inside a laminar flow biosafety enclosure, specifically constructed to contain the exposure system. The entire aerosol unit is located within the Animal BSL-3 facility at the Public Health Research Institute (PHRI). After establishing and validating the technical parameters of operation, rabbits were infected via the respiratory route with the avirulent M. bovis strain Bacille Calmette-Guerin (BCG). A suspension of 5–10×106 CFU/ml resulted in 3.6 log10 organisms deposited in the lungs; these were almost fully cleared by 14 days. Next, rabbits were infected with the same infectious dose of the virulent clinical isolate of M. tuberculosis HN878. The bacillary load in the lung at 14 days reached 5.3 log10 CFU and increased progressively. Large granulomatous lesions, distributed evenly in the lung parenchyma developed. We conclude that the aerosol exposure unit is easy to operate, yields a consistent implantation of mycobacteria in the lungs, is safe for the investigators, and induces disease similar to that observed in humans infected with M. tuberculosis.