Despite issues relating to variable efficacy in the past, the Mycobacterium bovis BCG vaccine remains the basis for new-generation recombinant vaccines currently in clinical trials. To date, vaccines have been tested mostly against laboratory strains and not against the newly emerging clinical strains. In this study, we evaluated the ability of BCG Pasteur to protect mice from aerosol infections with two highly virulent W-Beijing clinical strains, HN878 and SA161. In a conventional 30-day protection assay, BCG was highly protective against both strains, but by day 60 of the assay, this protection was diminished. Histological examination of the lungs of vaccinated animals showed reduced lung consolidation and smaller and more-organized granulomas in the vaccinated mice after 30 days, but in both cases, these tissues demonstrated worsening pathology over time. Effector T cell responses were increased in the vaccinated mice infected with HN878, but these diminished in number after day 30 of the infections concomitant with increased CD4 ؉ Foxp3 ؉ T cells in the lungs, draining lymph nodes, and the spleen. Given the concomitant decrease in effector immunity and continued expansion of regulatory Foxp3؉ cells observed here, it is reasonable to hypothesize that downregulation of effector immunity by these cells may be a serious impediment to the efficacy of BCG-based vaccines.The global epidemic of disease caused by Mycobacterium tuberculosis continues unabated, with recent figures indicating approximately 8 million new cases of tuberculosis (TB) each year, with about 2 million deaths (5,8,9,13,31,40). Among these infections, clinical isolates typed as belonging to the W-Beijing family of strains are becoming increasingly prevalent; in fact, it is believed that the Beijing genotype family is responsible for approximately 50% of TB cases in East Asia and now accounts for at least 13% of all isolates worldwide (4,17,29,31,33,37,38). The reason for the success of this family is unclear and is compounded by the suggestion that the current vaccine for tuberculosis, Mycobacterium bovis BCG, has actually selected for the emergence of this family (1).Despite recent recommendations (10), we know little about the actual basic biology of the newly emerging clinical strains of M. tuberculosis. Only one W-Beijing strain, HN878, has been extensively studied with animal models to date (19)(20)(21)(22)36); the data obtained shows that this isolate is extremely virulent, at least in comparison with the modestly virulent laboratory strains H37Rv and Erdman. A major difference, as recently demonstrated (22), is that while all the above strains potently induce effector immunity 20 to 30 days after low-dose aerosol infection of mice, this is soon replaced in HN878-infected mice by the emergence of CD4 cells expressing Foxp3, with many of these staining in addition for the immunosuppressive cytokine interleukin-10 (IL-10).Because vaccines for tuberculosis are usually tested against the laboratory strains, there is very limited information as...

The purpose of this study was 2-fold. First, we evaluated standard chemotherapy in the guinea pig model of tuberculosis to determine if this animal species could productively be used for this purpose. Second, given the similarities of the pathology of disease in guinea pigs and humans, we wished to evaluate additional parameters, including magnetic resonance imaging, microscopy, and cytokine expression and lymphocyte phenotypes, in response to an infection treated with drug therapy. This study shows that conventional rifampin-isoniazidpyrazinamide chemotherapy significantly decreased the numbers of the highly virulent Erdman K01 strain of Mycobacterium tuberculosis, with most of the bacilli being eliminated in a month. Despite this result, bacteria could still be detected in the lungs and other tissues for at least another 3 to 4 months. Resolution of the nonnecrotic granulomas in the lungs and lymph nodes could be clearly visualized by magnetic resonance imaging at the macroscopic level. Microscopically, the majority of the pulmonary and extrapulmonary inflammation resolved spontaneously, leaving residual lesions composed of dystrophic calcification and fibrosis marking the site of necrosis of the primary lesion. Residual calcified lesions, which were also associated with pulmonary lymphangitis, contained acid-fast bacilli even following aggressive chemotherapy. The presence of intact extracellular bacilli within these lesions suggests that these could serve as the primary sites of disease reactivation. The chemotherapy reduced the level of T-cell influx into infected tissues and was accompanied by a large and sustained increase in TH1 cytokine expression. Chemotherapy also prevented the emergence in lung tissues of high levels of interleukin-10 and Foxp3-positive cells, known markers of regulatory T cells.

SUMMARY There is increasing evidence that clinical isolates of Mycobacterium tuberculosis that belong to the W-Beijing genotype of newly emerging strains are often of very high virulence when tested in small animal models, including the mouse and guinea pig. In this report we provide further evidence to support this contention, and show that two W-Beijing strains are of very high virulence when introduced by low dose aerosol into out-bred guinea pigs. In addition to severe lung pathology, each of these infections was associated with large influxes of activated CD4 and CD8 T cells into the lungs. Large influxes of macrophages were also observed, but the fraction of these showing evidence of activation by Class-II expression was relatively low. A progressive increase in neutrophils was also seen, with highest levels accumulating in the lungs of the W-Beijing infected animals. In the case of these two infections mRNA levels for TH1 cytokines was elevated early, but these then declined, and were replaced by increasing levels of message encoding for Foxp3, IL-10, and TGFβ. These observations support the hypothesis that W-Beijing strains are potent inducers of regulatory T cells, and that this event may enhance survival and transmission of these bacilli.

Metronidazole, which is used for the treatment of infections caused by anaerobic organisms, was evaluated in Mycobacterium tuberculosis-infected guinea pigs. M. tuberculosis can adapt to hypoxia, which is present in the primary lesions of infected guinea pigs. Metronidazole treatment (for 6 weeks at 100 mg/kg of body weight) resulted in no reduction in the bacillary burden and significantly worsened lesion inflammation.The excellent distribution of metronidazole (MET) in all organs and its good bactericidal activity, including its activity against quiescent bacteria, make it the compound of choice for the treatment of many infections caused by anaerobic organisms. Mycobacterium tuberculosis also has the ability to adapt to gradual oxygen depletion or stationary growth. This phenomenon has been studied extensively by Wayne and Hayes and has been described as a sequential progression through two stages, defined as nonreplicating phase 1 (NRP-1) and NRP-2 (15). MET has no effect on exponentially growing bacilli or on NRP-1 bacilli, but it is bactericidal for bacilli in stationary phase and NRP-2 (6, 16).Nonprogressive M. tuberculosis lesions in the lungs often have limited vascularization (due to necrosis and dystrophic mineralization), causing a limited O 2 supply. Bacterial persistence in these hypoxic lesions has been thought to be accompanied by susceptibility to MET. In mouse models of tuberculosis (TB), MET has failed to show consistent activity (5, 6, 10). This is not surprising, as hypoxia was found to be completely absent in the M. tuberculosis lesions of infected mice (1, 11), and the progression of disease rarely reaches the stages of extensive necrosis (2,3,8,12). Lung lesions in guinea pigs infected with M. tuberculosis, on the other hand, show caseous necrosis, mineralization, and hypoxia, which are also seen in natural infections in humans (13,14). Guinea pigs develop necrotic primary lesions that differ in their morphology compared to those of the secondary lesions that result after the activation of adaptive immunity (8). In an earlier paper, we described that the persisting, acid-fast bacilli are primarily found extracellularly in a hypoxic microenvironment of primary lesion necrosis and that only few a bacilli are localized within the secondary granulomas (8). The same study also showed evidence of hypoxia in these primary lesions of guinea pigs when pimonidazole was used (8), and therefore, we chose to explore the potential bactericidal activity of MET in the guinea pig model.To establish appropriate drug doses in guinea pigs for the anti-M. tuberculosis drugs used in this study, pharmacokinetic analysis was performed with a single dose each of isoniazid (INH), rifampin (RIF), pyrazinamide (PZA), and MET. All drugs were obtained from Sigma Chemical Co. (St. Louis, MO). INH, PZA, and MET were dissolved in distilled water, while RIF was dissolved in dimethyl sulfoxide (final concentration, 0.5%) with sucrose (40%, wt/vol) to increase its palatability. The pharmacokinetic data obtained by validated...

The experimental compound TMC207 is showing promise against infections caused by Mycobacterium tuberculosis both in a variety of animal studies and in the field. In this study, we used the guinea pig model, a species that shows several similarities to human tuberculosis, including the hallmark of primary granuloma necrosis, to determine the efficacy of a combination regimen combining TMC207 with rifampin and pyrazinamide. This drug regimen rapidly reduced the bacterial load in the lungs to undetectable levels by 8 weeks of treatment. This reduction was associated with a substantial improvement in lung pathology, but despite this effect areas of residual necrosis still remained. In the draining lymph nodes, however, tissue damage was rapid and not significantly reversed by the drug treatment. Approximately 10 to 11 months after the treatment had ended, the animals began to trigger a Karnovsky scale indicating bacterial regrowth and potential relapse, an event confirmed by the new development of both pulmonary and extrapulmonary granulomatous lesions. Interestingly, a similar rate of relapse was also seen in animals receiving 24 weeks of rifampin, pyrazinamide, and isoniazid standard chemotherapy. These data indicate that TMC207 could be a useful addition to current treatment regimens for tuberculosis.The increasing incidence of drug-resistant forms of Mycobacterium tuberculosis, now thought to exceed half a million new cases a year (8,10,11,26), further complicates the critical need to discover new antituberculosis drugs. Equally troubling, newly emerging evidence suggests that a significant percentage of new clinical isolates of M. tuberculosis are of extremely high virulence (8). Within the current drug discovery pipeline (12), a particularly attractive new candidate is the diarylquinolone TMC207 (2). This investigational drug potently inhibits the mycobacterial enzyme complex ATP synthase, thus interfering with energy production and homeostasis. As a consequence, TMC207 is highly active against both drug-sensitive and drugresistant isolates of M. tuberculosis.In animal studies, the majority of data has been obtained from the mouse model, where TMC207 has activity equivalent to, and synergizes with, standard rifampin, isoniazid, and pyrazinamide therapy (RHZ) (3,13,14,17,18,30). However, although the mouse is an excellent and cost-effective screening tool (15), it lacks elements of the disease pathogenesis in humans, including granulomatous lymphadenitis, lymphangitis, and the development of both pulmonary and extrapulmonary lesions exhibiting caseous necrosis with dystrophic calcification (15). In contrast, the guinea pig model of tuberculosis mimics multiple elements of the pathogenesis of the disease in humans (4, 6, 7).Virtually all studies evaluating drug efficacy in animal models utilize the laboratory strains M. tuberculosis H37Rv or Erdman. However, it is becoming increasingly apparent that newly emerging isolates are of very high virulence, including the W-Beijing family of M. tuberculosis, which is no...