Low-dose Aerosol Infection Research Articles

B cells in perivascular and peribronchiolar granuloma-associated lymphoid tissue and B-cell signatures identify asymptomatic Mycobacterium tuberculosis lung infection in Diversity Outbred mice.

Because most humans resist Mycobacterium tuberculosis infection, there is a paucity of lung samples to study. To address this gap, we infected Diversity Outbred mice with M. tuberculosis and studied the lungs of mice in different disease states. After a low-dose aerosol infection, progressors succumbed to acute, inflammatory lung disease within 60 days, while controllers maintained asymptomatic infection for at least 60 days, and then developed chronic pulmonary tuberculosis (TB) lasting months to more than 1 year. Here, we identified features of asymptomatic M. tuberculosis infection by applying computational and statistical approaches to multimodal data sets. Cytokines and anti-M. tuberculosis cell wall antibodies discriminated progressors vs controllers with chronic pulmonary TB but could not classify mice with asymptomatic infection. However, a novel deep-learning neural network trained on lung granuloma images was able to accurately classify asymptomatically infected lungs vs acute pulmonary TB in progressors vs chronic pulmonary TB in controllers, and discrimination was based on perivascular and peribronchiolar lymphocytes. Because the discriminatory lesion was rich in lymphocytes and CD4 T cell-mediated immunity is required for resistance, we expected CD4 T-cell genes would be elevated in asymptomatic infection. However, the significantly different, highly expressed genes were from B-cell pathways (e.g., Bank1, Cd19, Cd79, Fcmr, Ms4a1, Pax5, and H2-Ob), and CD20+ B cells were enriched in the perivascular and peribronchiolar regions of mice with asymptomatic M. tuberculosis infection. Together, these results indicate that genetically controlled B-cell responses are important for establishing asymptomatic M. tuberculosis lung infection.

Low-dose M.tb infection but not BCG or MTBVAC vaccination enhances heterologous antibody titres in non-human primates.

Mycobacteria are known to exert a range of heterologous effects on the immune system. The mycobacteria-based Freund's Complete Adjuvant is a potent non-specific stimulator of the immune response used in immunization protocols promoting antibody production, and Mycobacterium bovis Bacille Calmette Guérin (BCG) vaccination has been linked with decreased morbidity and mortality beyond the specific protection it provides against tuberculosis (TB) in some populations and age groups. The role of heterologous antibodies in this phenomenon, if any, remains unclear and under-studied. We set out to evaluate antibody responses to a range of unrelated pathogens following infection with Mycobacterium tuberculosis (M.tb) and vaccination with BCG or a candidate TB vaccine, MTBVAC, in non-human primates. We demonstrate a significant increase in the titer of antibodies against SARS-CoV-2, cytomegalovirus, Epstein-Barr virus, tetanus toxoid, and respiratory syncytial virus antigens following low-dose aerosol infection with M.tb. The magnitude of some of these responses correlated with TB disease severity. However, vaccination with BCG administered by the intradermal, intravenous or aerosol routes, or intradermal delivery of MTBVAC, did not increase antibody responses against unrelated pathogens. Our findings suggest that it is unlikely that heterologous antibodies contribute to the non-specific effects of these vaccines. The apparent dysregulation of B cell responses associated with TB disease warrants further investigation, with potential implications for risk of B cell cancers and novel therapeutic strategies.

Pulmonary Mycobacterium tuberculosisinfection alters the hepatic immunometabolic landscape and compromises insulin responsiveness early after infection

Abstract Immunity against Mycobacterium tuberculosis(Mtb) is associated with both regional and systemic inflammation as well as with metabolic disruption in tuberculosis (TB) patients. The metabolic and immunological responses are intertwined but the specific impact of localized disease on systemic metabolism is not well defined. We investigated the impact of low dose aerosol infection of the lung with Mtbon the systemic metabolic regulatory organ – the liver. Using an unbiased approach we defined the mRNA and protein dysregulation occurring in the early stage of infection. We found that infection leads to immune pathway activation and reduced metabolic activity in liver. Comparison of the gene expression patterns between of the lung, liver and blood revealed organ specific metabolic gene dysregulation in the context of non-organ specific activation of interferon response pathways. We have also found that infection leads to suppression of carbohydrate and lipid metabolism, without alteration of fasting or random blood glucose levels. Infected mice show reduced sensitivity to exogenous insulin but the glucose tolerance remains intact indicating insulin resistance. Analysis of metabolomics data from public repository reveals a metabolic signature of insulin resistance correlates with TB progression. Taken together, our finding shows that aerosol Mtbinfection influences hepatic metabolism very soon after infection and may contribute to early metabolic disease. Defining the early interplay between localised infection, inflammation and metabolic regulation is key to understanding the impact of latent and subclinical TB on the large proportion of the World’s population who do not exhibit TB symptoms but who are infected with Mtb. Supported by Grants from Royal Society Wolfson Merit Award (AMC) Newton Fellowship (MKD), Wellcome Trust Institutional Support Fund Fellowship (MKD), MRC award MR/P011136/1 (AMC).

Metformin potentiates BCG efficacy against Mtb infection

Abstract Tuberculosis (TB) is a chronic inflammatory disease that is caused by the bacterium Mycobacterium tuberculosis (Mtb) and is currently the leading cause of death by an infectious disease worldwide. Presently, TB therapeutic strategies include prolonged treatments with antimicrobial drugs, which often produce toxic side-effects, and vaccination with Bacille Calmette-Guérin (BCG), the only available and only partially protective TB vaccine. Considering the critical need for better prevention strategies, there have been multiple attempts to enhance the efficacy of BCG, yet none have significantly outperformed BCG alone. Previously, we have demonstrated that the anti-diabetic drug, metformin, has immunomodulatory effects and is associated with increased host resistance against TB. Based on our findings we hypothesized that following BCG vaccination, treatment with metformin would also enhance the vaccine response to Mtb. To test our hypothesis, we vaccinated guinea pigs with BCG Pasteur, dosed them with metformin or mock treatment, and performed low dose aerosol infection with Mtb H37Rv. At 90 days following infection, histological evaluation revealed that guinea pigs that received metformin- BCG had significantly reduced lung lesion burden compared to BCG alone. In addition, we evaluated the effects of metformin on T cell differentiation using an in vitro model of anti-CD3/anti-CD28 activation and found that metformin enhances memory T cell marker expression. Moreover, we show that mitochondrial hyperpolarization, a critical component of T cell activation, is dampened by metformin, which may ultimately alter T cell differentiation towards a memory-like phenotype and help explain metformin’s protective efficacy.

The Efficacy of the BCG Vaccine against Newly Emerging Clinical Strains of Mycobacterium tuberculosis.

To date, most new vaccines against Mycobacterium tuberculosis, including new recombinant versions of the current BCG vaccine, have usually been screened against the laboratory strains H37Rv or Erdman. In this study we took advantage of our recent work in characterizing an increasingly large panel of newly emerging clinical isolates [from the United States or from the Western Cape region of South Africa], to determine to what extent vaccines would protect against these [mostly high virulence] strains. We show here that both BCG Pasteur and recombinant BCG Aeras-422 [used here as a good example of the new generation BCG vaccines] protected well in both mouse and guinea pig low dose aerosol infection models against the majority of clinical isolates tested. However, Aeras-422 was not effective in a long term survival assay compared to BCG Pasteur. Protection was very strongly expressed against all of the Western Cape strains tested, reinforcing our viewpoint that any attempt at boosting BCG would be very difficult to achieve statistically. This observation is discussed in the context of the growing argument made by others that the failure of a recent vaccine trial disqualifies the further use of animal models to predict vaccine efficacy. This viewpoint is in our opinion completely erroneous, and that it is the fitness of prevalent strains in the trial site area that is the centrally important factor, an issue that is not being addressed by the field.

Evaluation of moxifloxacin-containing regimens in pathologically distinct murine tuberculosis models.

In the recently concluded REMox-TB trial, two 4-month moxifloxacin-containing regimens did not meet the criteria for noninferiority compared to the current 6-month first-line regimen to treat tuberculosis (TB). Despite the disappointing result, this phase 3 clinical trial provides a rare opportunity to gauge the predictive accuracy of the nonclinical models used to support regimen development. In parallel with the REMox-TB trial, we compared the efficacy of the same three regimens against chronic TB infection in the commonly used BALB/c mouse strain and in C3HeB/FeJ mice, which have attracted recent interest as a nonclinical efficacy model because they develop caseous lung lesions which may better resemble human TB. In long-term treatment experiments at two institutions, using low-dose aerosol infection models with 6- to 8-week incubation periods in both mouse strains, control mice received rifampin, isoniazid, pyrazinamide, and ethambutol (RHZE), and test mice received the same regimen with moxifloxacin replacing isoniazid (RMZE) or ethambutol (RHZM). Outcome measures were lung CFU counts during treatment and relapse after various durations of treatment. At both institutions and in both mouse strains, RMZE and RHZM reduced by approximately 1 month and 0 to 1 month, respectively, the treatment duration needed to produce the same relapse rate as RHZE. These results demonstrating generally similar treatment-shortening effects of the moxifloxacin-containing regimens in each mouse strain, with effect sizes consistent with the REMox-TB trial results, reinforce the predictive value of murine models for TB regimen development.

In vitro and in vivo activities of the nitroimidazole TBA-354 against Mycobacterium tuberculosis.

Nitroimidazoles are a promising new class of antitubercular agents. The nitroimidazo-oxazole delamanid (OPC-67683, Deltyba) is in phase III trials for the treatment of multidrug-resistant tuberculosis, while the nitroimidazo-oxazine PA-824 is entering phase III for drug-sensitive and drug-resistant tuberculosis. TBA-354 (SN31354[(S)-2-nitro-6-((6-(4-trifluoromethoxy)phenyl)pyridine-3-yl)methoxy)-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazine]) is a pyridine-containing biaryl compound with exceptional efficacy against chronic murine tuberculosis and favorable bioavailability in preliminary rodent studies. It was selected as a potential next-generation antituberculosis nitroimidazole following an extensive medicinal chemistry effort. Here, we further evaluate the pharmacokinetic properties and activity of TBA-354 against Mycobacterium tuberculosis. TBA-354 is narrow spectrum and bactericidal in vitro against replicating and nonreplicating Mycobacterium tuberculosis, with potency similar to that of delamanid and greater than that of PA-824. The addition of serum protein or albumin does not significantly alter this activity. TBA-354 maintains activity against Mycobacterium tuberculosis H37Rv isogenic monoresistant strains and clinical drug-sensitive and drug-resistant isolates. Spontaneous resistant mutants appear at a frequency of 3 × 10(-7). In vitro studies and in vivo studies in mice confirm that TBA-354 has high bioavailability and a long elimination half-life. In vitro studies suggest a low risk of drug-drug interactions. Low-dose aerosol infection models of acute and chronic murine tuberculosis reveal time- and dose-dependent in vivo bactericidal activity that is at least as potent as that of delamanid and more potent than that of PA-824. Its superior potency and pharmacokinetic profile that predicts suitability for once-daily oral dosing suggest that TBA-354 be studied further for its potential as a next-generation nitroimidazole.

Host Defense and Recruitment of Foxp3+ T Regulatory Cells to the Lungs in Chronic Mycobacterium tuberculosis Infection Requires Toll-like Receptor 2

Acute resistance to low dose M. tuberculosis (Mtb) infection is not dependent on Toll-like receptor (TLR) 2. However, whether TLR2 contributes to resistance in chronic Mtb infection has remained uncertain. Here we report that, following low dose aerosol infection with Mtb, mice lacking TLR2 (TLR2KO), in comparison with wild type (WT) mice, exhibit enhanced cellular infiltration and inflammation in the lungs, and fail to stably control bacterial burden during chronic infection. IFNγ and IL-17 was expressed at equivalent levels in the two groups; however, the characteristic accumulation of Foxp3+ T regulatory cells (Tregs) in pulmonary granulomas was significantly reduced in TLR2KO mice. Nonetheless, this reduction in Tregs was independent of whether Tregs expressed TLR2 or not. To directly link the reduced number of Tregs to the increased inflammation present in the TLR2KO mice, we used a macrophage adoptive transfer model. At seven weeks post-Mtb infection, TLR2KO mice, which were adoptively transferred with WT macrophages, displayed enhanced accumulation of Tregs in the lungs and a concomitant reduction in inflammation in contrast with control mice that received TLR2KO macrophages. However, the pulmonary bacterial burden between the two groups remained similar indicating that TLR2's role in modulating immunopathology is functionally distinct from its role in restricting Mtb growth in chronic infection. Together, these findings unequivocally demonstrate that TLR2 contributes to host resistance against chronic Mtb infection and reveal a novel role for TLR2 in mediating the recruitment of Foxp3+ Tregs to the lungs to control inflammation.

Heme Catabolism by Heme Oxygenase-1 Confers Host Resistance to Mycobacterium Infection

Heme oxygenases (HO) catalyze the rate-limiting step of heme degradation. The cytoprotective action of the inducible HO-1 isoform, encoded by the Hmox1 gene, is required for host protection against systemic infections. Here we report that upregulation of HO-1 expression in macrophages (M) is strictly required for protection against mycobacterial infection in mice. HO-1-deficient (Hmox1(-/-)) mice are more susceptible to intravenous Mycobacterium avium infection, failing to mount a protective granulomatous response and developing higher pathogen loads, than infected wild-type (Hmox1(+/+)) controls. Furthermore, Hmox1(-/-) mice also develop higher pathogen loads and ultimately succumb when challenged with a low-dose aerosol infection with Mycobacterium tuberculosis. The protective effect of HO-1 acts independently of adaptive immunity, as revealed in M. avium-infected Hmox1(-/-) versus Hmox1(+/+) SCID mice lacking mature B and T cells. In the absence of HO-1, heme accumulation acts as a cytotoxic pro-oxidant in infected M, an effect mimicked by exogenous heme administration to M. avium-infected wild-type M in vitro or to mice in vivo. In conclusion, HO-1 prevents the cytotoxic effect of heme in M, contributing critically to host resistance to Mycobacterium infection.

Lipoarabinomannan mannose caps do not affect mycobacterial virulence or the induction of protective immunity in experimental animal models of infection and have minimal impact onin vitroinflammatory responses

Mannose-capped lipoarabinomannan (ManLAM) is considered an important virulence factor of Mycobacterium tuberculosis. However, while mannose caps have been reported to be responsible for various immunosuppressive activities of ManLAM observed in vitro, there is conflicting evidence about their contribution to mycobacterial virulence in vivo. Therefore, we used Mycobacterium bovis BCG and M. tuberculosis mutants that lack the mannose cap of LAM to assess the role of ManLAM in the interaction of mycobacteria with the host cells, to evaluate vaccine-induced protection and to determine its importance in M. tuberculosis virulence. Deletion of the mannose cap did not affect BCG survival and replication in macrophages, although the capless mutant induced a somewhat higher production of TNF. In dendritic cells, the capless mutant was able to induce the upregulation of co-stimulatory molecules and the only difference we detected was the secretion of slightly higher amounts of IL-10 as compared to the wild type strain. In mice, capless BCG survived equally well and induced an immune response similar to the parental strain. Furthermore, the efficacy of vaccination against a M. tuberculosis challenge in low-dose aerosol infection models in mice and guinea pigs was not affected by the absence of the mannose caps in the BCG. Finally, the lack of the mannose cap in M. tuberculosis did not affect its virulence in mice nor its interaction with macrophages in vitro. Thus, these results do not support a major role for the mannose caps of LAM in determining mycobacterial virulence and immunogenicity in vivo in experimental animal models of infection, possibly because of redundancy of function.

Francisella DnaK Inhibits Tissue-nonspecific Alkaline Phosphatase

Following pulmonary infection with Francisella tularensis, we observed an unexpected but significant reduction of alkaline phosphatase, an enzyme normally up-regulated following inflammation. However, no reduction was observed in mice infected with a closely related gram-negative pneumonic organism (Klebsiella pneumoniae) suggesting the inhibition may be Francisella-specific. In similar fashion to in vivo observations, addition of Francisella lysate to exogenous alkaline phosphatase (tissue-nonspecific isozyme) was inhibitory. Partial purification and subsequent proteomic analysis indicated the inhibitory factor to be the heat shock protein DnaK. Incubation with increasing amounts of anti-DnaK antibody reduced the inhibitory effect in a dose-dependent manner. Furthermore, DnaK contains an adenosine triphosphate binding domain at its N terminus, and addition of adenosine triphosphate enhances dissociation of DnaK with its target protein, e.g. alkaline phosphatase. Addition of adenosine triphosphate resulted in decreased DnaK co-immunoprecipitated with alkaline phosphatase as well as reduction of Francisella-mediated alkaline phosphatase inhibition further supporting the binding of Francisella DnaK to alkaline phosphatase. Release of DnaK via secretion and/or bacterial cell lysis into the extracellular milieu and inhibition of plasma alkaline phosphatase could promote an orchestrated, inflammatory response advantageous to Francisella.

Use of mouse models to study the variability in virulence associated with specific genotypic lineages of Mycobacterium tuberculosis

The host response against Mycobacterium tuberculosis show a wide spectrum of clinical manifestations in those patients who fail to control the infection. The course of the infection and its epidemiological consequences depend upon a complex interplay of host, environmental and bacterial factors. Experimental animal models have helped to define the influence of bacterial genetic diversity on virulence and on the immune response that is induced. For this purpose, experimental animals such as mice, guinea pigs and rabbits have been infected with selected clinical isolates obtained from outbreaks or from clinical epidemiology settings. Here we review the contribution of mouse models to defining the variability in virulence and immune response in relation to mycobacterial genetic diversity. Low dose aerosol infection in C57Bl mice or high dose intratracheal infection in BALB/c mice have demonstrated wide variability in virulence and immune responses induced by different bacterial genotypes, and each genotype has different phenotypes, with high and low virulence variants. In general, these studies have shown that high prevalent strains from big clusters are more virulent than low prevalent sporadic clinical isolates, and highly virulent strains induce non-protective immune responses with some correlation with clinical–epidemiological data. In the future selected strains from these types of studies should be analyzed with molecular technologies. These kinds of study will contribute to the identification of mycobacterial genes associated with virulence and immunogenicity.

Molecular cloning and expression of the IL-10 gene from guinea pigs

The Guinea pig (Cavia porcellus) is one of the most relevant small animals for modeling human tuberculosis (TB) in terms of susceptibility to low dose aerosol infection, the organization of granulomas, extrapulmonary dissemination and vaccine-induced protection. It is also considered to be a gold standard for a number of other infectious and non-infectious diseases; however, this animal model has a major disadvantage due to the lack of readily available immunological reagents. In the present study, we successfully cloned a cDNA for the critical Th2 cytokine, interleukin-10 (IL-10), from inbred Strain 2 guinea pigs using the DNA sequence information provided by the genome project. The complete open reading frame (ORF) consists of 537 base pairs which encodes a protein of 179 amino acids. This cDNA sequence exhibited 87% homology with human IL-10. Surprisingly, it showed only 84% homology with the previously published IL-10 sequence from the C4-deficient (C4D) guinea pig, leading us to clone IL-10 cDNA from the Hartley strain of guinea pig. The IL-10 gene from the Hartley strain showed 100% homology with the IL-10 sequence of Strain 2 guinea pigs. In order to validate the only published IL-10 sequence existing in Genbank reported from C4D guinea pigs, genomic DNA was isolated from tissues of C4D guinea pigs. Amplification with various sets of primers showed that the IL-10 sequence reported from C4D guinea pigs contained numerous errors. Hence the IL-10 sequence that is being reported by us replaces the earlier sequence making our IL-10 sequence to be the first one accurate from guinea pig. Recombinant guinea pig IL-10 proteins were subsequently expressed in both prokaryotic and eukaryotic cells, purified and were confirmed by N-terminal sequencing. Polyclonal anti-IL-10 antibodies were generated in rabbits using the recombinant IL-10 protein expressed in this study. Taken together, our results indicate that the DNA sequence information provided by the genome project is useful to directly clone much needed cDNAs necessary to study TB in the guinea pig. The newly cloned guinea pig IL-10 cDNA and recombinant proteins will serve as valuable resources for immunological studies in the guinea pig model of TB and other diseases.

Recombinant Adenovirus Delivery of Calreticulin–ESAT‐6 Produces an Antigen‐Specific Immune Response but no Protection Against a Mycobacterium Tuberculosis Challenge

Bacillus Calmette-Guerin (BCG) has failed to efficaciously control the worldwide spread of the disease. New vaccine development targets virulence antigens of Mycobacterium tuberculosis that are deleted in Mycobacterium bovis BCG. Immunization with ESAT-6 and CFP10 provides protection against M.tuberculosis in a murine infection model. Further, previous studies have shown that calreticulin increases the cell-mediated immune responses to antigens. Therefore, to test whether calreticulin enhances the immune response against M.tuberculosis antigens, we fused ESAT-6 to calreticulin and constructed a recombinant replication-deficient adenovirus to express the resulting fusion protein (AdCRT-ESAT-6). The adjuvant effect of calreticulin was assayed by measuring cytokine responses specific to ESAT-6. Recombinant adenovirus expressing the fusion protein produced higher levels of interferon-γ and tumour necrosis factor-α in response to ESAT-6. This immune response was not improved by the addition of CFP-10 to the CRT-ESAT-6 fusion protein (AdCRT-ESAT-6-CFP10). Mice immunized with these recombinant adenoviruses did not decrease the mycobacterial burden after low-dose aerosol infection with M.tuberculosis. We conclude that calreticulin can be used as an adjuvant to enhance the immune response against mycobacterial antigens, but it is not enough to protect against tuberculosis.

Immune Responses of HIV-1 Tat Transgenic Mice to Mycobacterium Tuberculosis W-Beijing SA161

Background:Mycobacterium tuberculosis remains among the leading causes of death from an infectious agent in the world and exacerbates disease caused by the human immunodeficiency virus (HIV). HIV infected individuals are prone to lung infections by a variety of microbial pathogens, including M. tuberculosis. While the destruction of the adaptive immune response by HIV is well understood, the actual pathogenesis of tuberculosis in co-infected individuals remains unclear. Tat is an HIV protein essential for efficient viral gene transcription, is secreted from infected cells, and is known to influence a variety of host inflammatory responses. We hypothesize Tat contributes to pathophysiological changes in the lung microenvironment, resulting in impaired host immune responses to infection by M. tuberculosis.Results:Herein, we show transgenic mice that express Tat by lung alveolar cells are more susceptible than non-transgenic control littermates to a low-dose aerosol infection of M. tuberculosis W-Beijing SA161. Survival assays demonstrate accelerated mortality rates of the Tat transgenic mice compared to non-transgenics. Tat transgenic mice also showed poorly organized lung granulomata-like lesions. Analysis of the host immune response using quantitative RT-PCR, flow cytometry for surface markers, and intracellular cytokine staining showed increased expression of pro-inflammatory cytokines in the lungs, increased numbers of cells expressing ICAM1, increased numbers of CD4+CD25+Foxp3+ T regulatory cells, and IL-4 producing CD4+ T cells in the Tat transgenics compared to infected non-tg mice.Conclusions:Our data show quantitative differences in the inflammatory response to the SA161 clinical isolate of M. tuberculosis W-Beijing between Tat transgenic and non-transgenic mice, suggesting Tat contributes to the pathogenesis of tuberculosis.

Reactivation of latent tuberculosis in rhesus macaques by coinfection with simian immunodeficiency virus

Tuberculosis (TB) and AIDS together present a devastating public health challenge. Over 3 million deaths every year are attributed to these twin epidemics. Annually, ∼11 million people are coinfected with HIV and Mycobacterium tuberculosis (Mtb). AIDS is thought to alter the spontaneous rate of latent TB reactivation. Macaques are excellent models of both TB and AIDS. Therefore, it is conceivable that they can also be used to model coinfection. Using clinical, pathological, and microbiological data, we addressed whether latent TB infection in rhesus macaques can be reactivated by infection with simian immunodeficiency virus (SIV). A low-dose aerosol infection of rhesus macaques with Mtb caused latent, asymptomatic TB infection. Infection of macaques exhibiting latent TB with a rhesus-specific strain of SIV significantly reactivated TB. Rhesus macaques are excellent model of TB/AIDS coinfection and can be used to study the phenomena of TB latency and reactivation.

Short-Course Chemotherapy with TMC207 and Rifapentine in a Murine Model of Latent Tuberculosis Infection

Multidrug-resistant and extensively drug-resistant tuberculosis (MDR/XDR-TB) is an emerging global health threat. Proper management of close contacts of infectious patients is increasingly important. However, no evidence-based recommendations for treating latent TB infection (LTBI) after MDR/XDR-TB exposure (DR-LTBI) exist. An ultrashort regimen for LTBI caused by drug-susceptible strains (DS-LTBI) is also desirable. TMC207 has bactericidal and sterilizing activity in animal models of TB and improves the activity of current MDR-TB therapy in patients. The objective of this study was to determine whether TMC207 might enable short-course treatment of DR-LTBI and ultrashort treatment of DS-LTBI. Using an established experimental model of LTBI chemotherapy in which mice are aerosol-immunized with a recombinant bacillus Calmette-Guérin vaccine before low-dose aerosol infection with Mycobacterium tuberculosis, the efficacy of TMC207 alone and in combination with rifapentine was compared with currently recommended control regimens as well as once-weekly rifapentine + isoniazid and daily rifapentine ± isoniazid. Outcomes included monthly lung colony-forming unit counts and relapse rates. Lung colony-forming unit counts were stable at about 3.75 log(10) for up to 7.5 months postinfection in untreated mice. Rifamycin-containing regimens were superior to isoniazid monotherapy. TMC207 exhibited sterilizing activity at least as strong as that of rifampin alone and similar to that of rifampin + isoniazid, but daily rifapentine +/- isoniazid was superior to TMC207. Addition of TMC207 to rifapentine did not improve the sterilizing activity of rifapentine in this model. TMC207 has substantial sterilizing activity and may enable treatment of DR-LTBI in 3-4 months.

Vaccination of guinea pigs using mce operon mutants of Mycobacterium tuberculosis

The limited efficacy of the BCG vaccine for tuberculosis, coupled with emerging information suggesting that it is poorly protective against newly emerging strains of Mycobacterium tuberculosis such as the W-Beijing isolates, makes it paramount to search for more potent alternatives. One such class of candidates is attenuated mutants derived from M. tuberculosis itself. We demonstrate here, in an initial short term assay, that mutants derived from disruption of the mce genes of the bacillus were highly protective in guinea pigs exposed by low dose aerosol infection with the virulent W-Beijing isolate SA161. This protection was demonstrated by a significant reduction in the numbers of bacilli harvested from the lungs, and dramatic improvements in lung histopathology.

Toll-like receptor 2 signaling regulates the tubercle granuloma (58.6)

Abstract The granuloma developed in the host in response to Mycobacterium tuberculosis (Mtb) is a protective structure that curtails the spread of the pathogen and controls reactivation of latent/persistent Mtb. Despite this knowledge, the molecular pathways that regulate the structural reorganization of immune cells in the lung remains poorly understood. We have found that TLR2KO mice can resist acute infection following low dose aerosol infection with Mtb, but are unable to maintain the granulomatous response and control chronic infection. Consequently, the goal of this study was to identify molecular pathways downstream of TLR2 signaling that may regulate granuloma maintenance in the chronic phase. We therefore examined the transcriptional signature of WT and TLR2KO lungs obtained at several time points following Mtb infection using Affymetrix GeneChip Mouse Genome 430A 2.0 Array. Gene set enrichment analysis (GSEA) indicated that enriched upregulated gene sets in the TLR2KO mice corresponded to Cell cycle, cell proliferation and cell activation related gene sets, while enriched down-regulated gene sets in the TLR2KO mice corresponded to extracellular matrix related gene sets, specifically matrix metalloproteinases (MMPs). Together with earlier reports that MMPs regulate the developing tubercle granuloma, our finding of reduced MMP expression in the lungs of TLR2KO mice indicates a novel role for TLR2 in host defense against Mtb through the control of MMP secretion.

Quinoxaline 1,4-di-N-Oxide and the Potential for Treating Tuberculosis

New drugs active against drug-resistant tuberculosis are urgently needed to extend the range of TB treatment options to cover drug resistant infections. Quinoxaline derivatives show very interesting biological properties (antibacterial, antiviral, anticancer, antifungal, antihelmintic, insecticidal) and evaluation of their medicinal chemistry is still in progress. In this review we report the properties and the recent developments of quinoxaline 1,4-di-N-oxide derivatives as potential anti-tuberculosis agents. Specific agents are reviewed that have excellent antitubercular drug properties, are active on drug resistant strains and non-replicating mycobacteria. The properties of select analogs that have in vivo activity in the low dose aerosol infection model in mice will be reviewed.