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.

Background Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), is a facultative intracellular pathogen that can persist within the host. The bacteria are thought to be in a state of reduced replication and metabolism as part of the chronic lung infection. Many in vitro studies have dissected the hypothesized environment within the infected lung, defining the bacterial response to pH, starvation and hypoxia. While these experiments have afforded great insight, the picture remains incomplete. The only way to study the combined effects of these environmental factors and the mycobacterial response is to study the bacterial response in vivo.Methodology/Principal FindingsWe used the guinea pig model of tuberculosis to examine the bacterial proteome during the early and chronic stages of disease. Lungs were harvested thirty and ninety days after aerosol challenge with Mtb, and analyzed by liquid chromatography-mass spectrometry. To date, in vivo proteomics of the tubercle bacillus has not been described and this work has generated the first large-scale shotgun proteomic data set, comprising over 500 unique protein identifications. Cell wall and cell wall processes, and intermediary metabolism and respiration were the two major functional classes of proteins represented in the infected lung. These classes of proteins displayed the greatest heterogeneity indicating important biological processes for establishment of a productive bacterial infection and its persistence. Proteins necessary for adaptation throughout infection, such as nitrate/nitrite reduction were found at both time points. The PE-PPE protein class, while not well characterized, represented the third most abundant category and showed the most consistent expression during the infection.Conclusions/SignificanceCumulatively, the results of this work may provide the basis for rational drug design – identifying numerous Mtb proteins, from essential kinases to products involved in metal regulation and cell wall remodeling, all present throughout the course of infection.

A crucial and distinctive feature of tuberculosis infection is that Mycobacterium tuberculosis (Mtb) resides in granulomatous lesion at various stages of disease development and necrosis, an aspect that is little understood. We used a novel approach, applying high resolution magic angle spinning nuclear magnetic resonance spectroscopy (HRMAS NMR) directly to infected tissues, allowing us to study the development of tuberculosis granulomas in guinea pigs in an untargeted manner. Significant up-regulation of lactate, alanine, acetate, glutamate, oxidized and the reduced form of glutathione, aspartate, creatine, phosphocholine, glycerophosphocholine, betaine, trimethylamine N-oxide, myo-inositol, scyllo-inositol, and dihydroxyacetone was clearly visualized and was identified as the infection progressed. Concomitantly, phosphatidylcholine was down-regulated. Principal component analysis of NMR data revealed clear group separation between infected and uninfected tissues. These metabolites are suggestive of utilization of alternate energy sources by the infiltrating cells that generate much of the metabolites in the increasingly necrotic and hypoxic developing granuloma through the glycolytic, pentose phosphate, and tricarboxylic acid pathways. The most relevant changes seen are, surprisingly, very similar to metabolic changes seen in cancer during tumor development.

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.

Chronic lung disease as a result of Mycobacterium abscessus is an emerging infection in the United States. We characterized the lung immune responses in mice and guinea pigs infected with M. abscessus. C57BL/6 and leptin-deficient ob/ob mice challenged with a low-dose aerosol (LDA) of M. abscessus did not develop an infection. However, when challenged with a high-dose aerosol (HDA), C57BL/6 and ob/ob mice developed an established infection and a pulmonary immune response consisting of an early influx of IFN-gamma+ CD4+ T cells; this immune response preceded the successful clearance of M. abscessus in both strains of mice, although mycobacterial elimination was delayed in the ob/ob mice. Infected guinea pigs showed an increased influx of lymphocytes into the lungs with bacterial clearance by Day 60. In contrast to the C57BL/6 and ob/ob mice and guinea pigs, IFN-gamma knockout (GKO) mice challenged with a LDA or HDA of M. abscessus showed a progressive lung infection despite a robust influx of T cells, macrophages, and dendritic cells, culminating in extensive lung consolidation. Furthermore, with HDA challenge of the GKO mice, emergence of IL-4- and IL-10-producing CD4+ and CD8+ T cells was seen in the lungs. In conclusion, IFN-gamma is critically important in the host defense against M. abscessus. As the number of effective drugs against M. abscessus is limited, the GKO mice provide a model for in vivo testing of novel drugs.

BackgroundPreviously we have shown that Ag85B-TB10.4 is a highly efficient vaccine against tuberculosis when delivered in a Th1 inducing adjuvant based on cationic liposomes. Another Th1 inducing adjuvant, which has shown a very promising profile in both preclinical and clinical trials, is IC31®. In this study, we examined the potential of Ag85B-TB10.4 delivered in the adjuvant IC31® for the ability to induce protection against infection with Mycobacterium tuberculosis. In addition, we examined if the antigen dose could influence the phenotype of the induced T cells.Methods and FindingsWe found that vaccination with the combination of Ag85B-TB10.4 and IC31® resulted in high numbers of polyfunctional CD4 T cells co-expressing IL-2, IFN-γ and TNF-α. This correlated with protection against subsequent challenge with M.tb in the mouse TB model. Importantly, our results also showed that both the vaccine induced T cell response, and the protective efficacy, was highly dependent on the antigen dose. Thus, whereas antigen doses of 5 and 15 µg did not induce significant protection against M.tb, reducing the dose to 0.5 µg selectively increased the number of polyfunctional T cells and induced a strong protection against infection with M.tb. The influence of antigen dose was also observed in the guinea pig model of aerosol infection with M.tb. In this model a 2.5 fold increase in the antigen dose reduced the protection against infection with M.tb to the level observed in non-vaccinated animals.Conclusions/SignificanceSmall changes in the antigen dose can greatly influence the induction of specific T cell subpopulations and the dose is therefore a crucial factor when testing new vaccines. However, the adjuvant IC31® can, with the optimal dose of Ag85B-TB10.4, induce strong protection against Mycobacterium tuberculosis. This vaccine has now entered clinical trials.

Mycobacterium tuberculosis (M. tuberculosis), the causative agent of human tuberculosis (TB), is estimated to be harbored by up to 2 billion people in a latent TB infection (LTBI) state. The only TB vaccine approved for use in humans, BCG, does not confer protection against establishment of or reactivation from LTBI, so new vaccine candidates are needed to specifically address this need. Following the hypothesis that mycobacterial biofilms resemble aspects of LTBI, we modified BCG by deleting the BCG1419c gene to create the BCGΔBCG1419c vaccine strain. In this study, we compared cytokine profiles, bacterial burden, and lung lesions after immunization with BCG or BCGΔBCG1419c before and after 6 months of aerosol infection with M. tuberculosis H37Rv in the resistant C57BL/6 mouse model. Our results show that in infected mice, BCGΔBCG1419c significantly reduced lung lesions and IL-6 in comparison to the unmodified BCG strain, and was the only vaccine that decreased production of TNF-α and IL-10 compared to non-vaccinated mice, while vaccination with BCG or BCGΔBCG1419c significantly reduced IFN-γ production. Moreover, transcriptome profiling of BCGΔBCG1419c suggests that compared to BCG, it has decreased expression of genes involved in mycolic acids (MAs) metabolism, and antigenic chaperones, which might be involved in reduced pathology compared to BCG-vaccinated mice.

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