Randall J. Basaraba

Enhanced priming of adaptive immunity by a proapoptotic mutant of Mycobacterium tuberculosis

The inhibition of apoptosis of infected host cells is a well-known but poorly understood function of pathogenic mycobacteria. We show that inactivation of the secA2 gene in Mycobacterium tuberculosis, which encodes a component of a virulence-associated protein secretion system, enhanced the apoptosis of infected macrophages by diminishing secretion of mycobacterial superoxide dismutase. Deletion of secA2 markedly increased priming of antigen-specific CD8(+) T cells in vivo, and vaccination of mice and guinea pigs with a secA2 mutant significantly increased resistance to M. tuberculosis challenge compared with standard M. bovis bacille Calmette-Guérin vaccination. Our results define a mechanism for a key immune evasion strategy of M. tuberculosis and provide what we believe to be a novel approach for improving mycobacterial vaccines.

Location of Persisting Mycobacteria in a Guinea Pig Model of Tuberculosis Revealed by R207910

ABSTRACT The lengthy chemotherapy of tuberculosis reflects the ability of a small subpopulation of Mycobacterium tuberculosis bacteria to persist in infected individuals. To date, the exact location of these persisting bacteria is not known. Lung lesions in guinea pigs infected with M. tuberculosis have striking similarities, such as necrosis, mineralization, and hypoxia, to natural infections in humans. Guinea pigs develop necrotic primary lesions after aerosol infection that differ in their morphology compared to secondary lesions resulting from hematogenous dissemination. In infected guinea pigs conventional therapy for tuberculosis during 6 weeks reduced the bacterial load by 1.7 logs in the lungs and, although this completely reversed lung inflammation associated with secondary lesions, the primary granulomas remained largely unaffected. Treatment of animals with the experimental drug R207910 (TMC207) for 6 weeks was highly effective with almost complete eradication of the bacteria throughout both the primary and the secondary lesions. Most importantly, the few remnants of acid-fast bacilli remaining after R207910 treatment were to be found extracellular, in a microenvironment of residual primary lesion necrosis with incomplete dystrophic calcification. This zone of the primary granuloma is hypoxic and is morphologically similar to what has been described for human lung lesions. These results show that this acellular rim may, therefore, be a primary location of persisting bacilli withstanding drug treatment.

The Hypervirulent Mycobacterium tuberculosis Strain HN878 Induces a Potent TH1 Response followed by Rapid Down-Regulation

The HN878 strain of Mycobacterium tuberculosis is regarded as “hypervirulent” due to its rapid growth and reduced survival of infected mice when compared with other clinical isolates. This property has been ascribed due to an early increase in type I IFNs and a failure to generate TH1-mediated immunity, induced by a response to an unusual cell wall phenolic glycolipid expressed by the HN878 isolate. We show, however, that although type I IFN does play an inhibitory role, this response was most apparent during the chronic disease stage and was common to all M. tuberculosis strains tested. In addition, we further demonstrate that the HN878 infection was associated with a potent TH1 response, characterized by the emergence of both CD4 and CD8 T cell subsets secreting IFN-γ. However, where HN878 differed to the other strains tested was a subsequent reduction in TH1 immunity, which was temporally associated with the rapid emergence of a CD4+CD25+FoxP3+CD223+IL-10+ regulatory T cell population. This association may explain the paradoxical initial emergence of a TH1 response in these mice but their relatively short time of survival.

Azithromycin blocks autophagy and may predispose cystic fibrosis patients to mycobacterial infection

Azithromycin is a potent macrolide antibiotic with poorly understood antiinflammatory properties. Long-term use of azithromycin in patients with chronic inflammatory lung diseases, such as cystic fibrosis (CF), results in improved outcomes. Paradoxically, a recent study reported that azithromycin use in patients with CF is associated with increased infection with nontuberculous mycobacteria (NTM). Here, we confirm that long-term azithromycin use by adults with CF is associated with the development of infection with NTM, particularly the multi-drug-resistant species Mycobacterium abscessus, and identify an underlying mechanism. We found that in primary human macrophages, concentrations of azithromycin achieved during therapeutic dosing blocked autophagosome clearance by preventing lysosomal acidification, thereby impairing autophagic and phagosomal degradation. As a consequence, azithromycin treatment inhibited intracellular killing of mycobacteria within macrophages and resulted in chronic infection with NTM in mice. Our findings emphasize the essential role for autophagy in the host response to infection with NTM, reveal why chronic use of azithromycin may predispose to mycobacterial disease, and highlight the dangers of inadvertent pharmacological blockade of autophagy in patients at risk of infection with drug-resistant pathogens.

Copper resistance is essential for virulence of Mycobacterium tuberculosis

Copper (Cu) is essential for many biological processes, but is toxic when present in excessive amounts. In this study, we provide evidence that Cu plays a crucial role in controlling tuberculosis. A Mycobacterium tuberculosis (Mtb) mutant lacking the outer membrane channel protein Rv1698 accumulated 100-fold more Cu and was more susceptible to Cu toxicity than WT Mtb. Similar phenotypes were observed for a M. smegmatis mutant lacking the homolog Ms3747, demonstrating that these mycobacterial copper transport proteins B (MctB) are essential for Cu resistance and maintenance of low intracellular Cu levels. Guinea pigs responded to infection with Mtb by increasing the Cu concentration in lung lesions. Loss of MctB resulted in a 1,000- and 100-fold reduced bacterial burden in lungs and lymph nodes, respectively, in guinea pigs infected with Mtb. In mice, the persistence defect of the Mtb mctB mutant was exacerbated by the addition of Cu to the diet. These experiments provide evidence that Cu is used by the mammalian host to control Mtb infection and that Cu resistance mechanisms are crucial for Mtb virulence. Importantly, Mtb is much more susceptible to Cu than other bacteria and is killed in vitro by Cu concentrations lower than those found in phagosomes of macrophages. Hence, this study reveals an Achilles heel of Mtb that might be a promising target for tuberculosis chemotherapy.

The Protective Effect of the Mycobacterium bovis BCG Vaccine Is Increased by Coadministration with the Mycobacterium tuberculosis 72-Kilodalton Fusion Polyprotein Mtb72F in M. tuberculosis-Infected Guinea Pigs

ABSTRACT A tuberculosis vaccine candidate consisting of a 72-kDa polyprotein or fusion protein based upon the Mtb32 and Mtb39 antigens of Mycobacterium tuberculosis and designated Mtb72F was tested for its protective capacity as a potential adjunct to the Mycobacterium bovis BCG vaccine in the mouse and guinea pig models of this disease. Formulation of recombinant Mtb72F (rMtb72F) in an AS02A adjuvant enhanced the Th1 response to BCG in mice but did not further reduce the bacterial load in the lungs after aerosol challenge infection. In the more stringent guinea pig disease model, rMtb72F delivered by coadministration with BCG vaccination significantly improved the survival of these animals compared to BCG alone, with some animals still alive and healthy in their appearance at >100 weeks post-aerosol challenge. A similar trend was observed with guinea pigs in which BCG vaccination was boosted by DNA vaccination, although this increase was not statistically significant due to excellent protection conferred by BCG alone. Histological examination of the lungs of test animals indicated that while BCG controls eventually died from overwhelming lung consolidation, the majority of guinea pigs receiving BCG mixed with rMtb72F or boosted twice with Mtb72F DNA had mostly clear lungs with minimal granulomatous lesions. Lesions were still prominent in guinea pigs receiving BCG and the Mtb72F DNA boost, but there was considerable evidence of lesion healing and airway remodeling and reestablishment. These data support the hypothesis that the coadministration or boosting of BCG vaccination with Mtb72F may limit the lung consolidation seen with BCG alone and may promote lesion resolution and healing. Collectively, these data suggest that enhancing BCG is a valid vaccination strategy for tuberculosis that is worthy of clinical evaluation.

Immunopathogenesis of Pulmonary Granulomas in the Guinea Pig after Infection with Mycobacterium tuberculosis

ABSTRACT Pulmonary tuberculosis in guinea pigs is similar to the disease in humans and is accordingly widely used as a model to test tuberculosis vaccines. The primary site of expression of acquired immunity and the hallmark of tuberculosis is the granuloma. Granuloma morphology is well described, but there is limited information regarding T-cell subset influx. We monitored the course of pulmonary tuberculosis in guinea pigs and observed four distinct immunohistopathological stages. In all stages there were similar numbers and arrangement of CD4 and CD8 T cells. There were only small numbers of apoptotic lymphocytes, scattered around and within the necrotic core, and acid-fast bacilli were visible both within macrophages and free within airway debris. A key finding of the study was the observation that the development of the necrotic core was an early event and almost certainly preceded the emergence of the acquired immune response. This in turn suggests that innate mechanisms are the basis of the early lesions and that subsequent acquired responses are unable to moderate them. This hypothesis differs from the current dogma that excessive activity of T cells mediates delayed-type hypersensitivity and that cellular cytolysis is the root cause of the necrosis.

Role for Matrix Metalloproteinase 9 in Granuloma Formation during Pulmonary Mycobacterium tuberculosis Infection

ABSTRACT Recent studies have shown that matrix metalloproteinases (MMPs) are induced by Mycobacterium tuberculosis during pulmonary infection. Here, expression of MMP-9 during pulmonary M. tuberculosis infection was characterized to determine whether its production correlated with disease resistance in vivo and to determine what role, if any, MMP-9 might have in granuloma formation. Following aerosol infection with M. tuberculosis, dissemination of bacilli occurred earlier in the C57BL/6 resistant mouse strain than in the susceptible CBA/J strain, as was evident from an increased number of bacteria in the blood, spleen, and liver at day 14 after infection. In addition, early dissemination of the bacilli was associated with early induction of protective immunity as assessed from gamma interferon levels. Nonspecific blocking of MMPs in C57BL/6 mice early during infection reduced hematogenous spread of the bacilli, suggesting that MMPs indeed play a role in facilitating dissemination, likely via extracellular matrix degradation. The concentration of active MMP-9, specifically, was greater in the lungs of C57BL/6 mice than in those of the CBA/J mice at day 28, thereby suggesting that MMP-9 is not one of the MMPs directly involved in promoting early dissemination of M. tuberculosis. Instead, however, histological lung sections and flow cytometric analysis of lung cells from MMP-9-knockout mice showed that MMP-9 is involved in macrophage recruitment and granuloma development. These combined data support the idea that early MMP activity is an essential component of resistance to pulmonary mycobacterial infection and that MMP-9, specifically, is required for recruitment of macrophages and tissue remodeling to allow for the formation of tight, well-organized granulomas.

Defined tuberculosis vaccine, Mtb72F/AS02A, evidence of protection in cynomolgus monkeys

The development of a vaccine for tuberculosis requires a combination of antigens and adjuvants capable of inducing appropriate and long-lasting T cell immunity. We evaluated Mtb72F formulated in AS02A in the cynomolgus monkey model. The vaccine was immunogenic and caused no adverse reactions. When monkeys were immunized with bacillus Calmette–Guérin (BCG) and then boosted with Mtb72F in AS02A, protection superior to that afforded by using BCG alone was achieved, as measured by clinical parameters, pathology, and survival. We observed long-term survival and evidence of reversal of disease progression in monkeys immunized with the prime-boost regimen. Antigen-specific responses from protected monkeys receiving BCG and Mtb72F/AS02A had a distinctive cytokine profile characterized by an increased ratio between 3 Th1 cytokines, IFN-γ, TNF, and IL-2 and an innate cytokine, IL-6. To our knowledge, this is an initial report of a vaccine capable of inducing long-term protection against tuberculosis in a nonhuman primate model, as determined by protection against severe disease and death, and by other clinical and histopathological parameters.

Pulmonary Necrosis Resulting from DNA Vaccination against Tuberculosis

ABSTRACT The use of DNA constructs encoding mycobacterial proteins is a promising new approach to vaccination against tuberculosis. A DNA vaccine encoding the hsp60 molecule of Mycobacterium leprae has previously been shown to protect against intravenous infection of mice with Mycobacterium tuberculosis in both the prophylactic and immunotherapeutic modes. It is shown here, however, that this vaccine was not effective in a more realistic aerosol infection model or in a model of latent tuberculosis in the lungs. Moreover, when given in an immunotherapeutic model the immunized mice developed classical Koch reactions characterized by multifocal discrete regions of cellular necrosis throughout the lung granulomas. Similar and equally severe reactions were seen in mice given a vaccine with DNA coding for the Ag85 antigen of M. tuberculosis. This previously unanticipated safety problem indicates that DNA vaccines should be used with caution in individuals who may have already been exposed to tuberculosis.