In Vitro Evaluation of Inhalable Verapamil-Rifapentine Particles for Tuberculosis Therapy.

Recent studies have demonstrated that efflux pumps of Mycobacterium tuberculosis (M. tb) provide a crucial mechanism in the development of drug resistant to antimycobacterial drugs. Drugs that inhibit these efflux pumps, such as verapamil, have shown the potential in enhancing the treatment success. We therefore hypothesized that the combined inhaled administration of verapamil and a first-line rifamycin antibiotic will further improve the treatment efficacy. An inhalable dry powder consisting of amorphous verapamil and crystalline rifapentine with l-leucine as an excipient was produced by spray drying. The in vitro aerosol characteristic of the powder, its microbiological activity and stability were assessed. When the powder was dispersed by an Osmohaler, the total fine particle fraction (FPFtotal, wt % of particles in aerosol <5 µm) of verapamil and rifapentine was 77.4 ± 1.1% and 71.5 ± 2.0%, respectively. The combination drug formulation showed a minimum inhibitory concentration (MIC90) similar to that of rifapentine alone when tested against both M. tb H37Ra and M. tb H37Rv strains. Importantly, the combination resulted in increased killing of M. tb H37Ra within the infected macrophage cells compared to either verapamil or rifapentine alone. In assessing cellular toxicity, the combination exhibited an acceptable half maximal inhibitory concentration (IC50) values (62.5 µg/mL) on both human monocytic (THP-1) and lung alveolar basal epithelial (A549) cell lines. Finally, the powder was stable after 3 months storage in 0% relative humidity at 20 ± 3 °C.

Inactivation of the Mycobacterium tuberculosis fadB4 gene results in increased virulence in host cell and mice.

The genome of Mycobacterium tuberculosis encodes many proteins involved in fatty acid metabolism, a subset of which are required for virulence. The Mycobacterium tuberculosis fadB4 gene, which shares strong similarity with oxidoreductases and fatty acid synthases, is up-regulated during infection of macrophages and is predicted to protect the bacterium from the hostile environment of the host cell. In order to determine if fadB4 plays a role in the virulence of M. tuberculosis, we constructed a M. tuberculosis mutant in which the fadB4 had been disrupted (DeltafadB4). Surprisingly, DeltafadB4, grew more rapidly in host cells compared to wild-type M. tuberculosis or the DeltafadB4 or the gene-disrupted strain complemented with fadB4. In addition, macrophages infected with DeltafadB4 displayed reduced secretion of the cytokine TNF-alpha, suggesting a role for the FadB4 protein in influencing the pro-inflammatory host response to M. tuberculosis. After infection of mice, DeltafadB4 demonstrated an increased replication at early time-points post-infection compared to the growth of wild-type M. tuberculosis. This increased capacity of DeltafadB4 to replicate in vivo was reflected in the decreased time to death of immuno-deficient RAG-1(-/-) mice infected with M. tuberculosis lacking the fadB4 gene. Therefore fadB4 is part of the family of genes whose expression serves to regulate the virulence of M. tuberculosis within the host.

Sequential pulmonary immunization with heterologous recombinant influenza A virus tuberculosis vaccines protects against murine M. tuberculosis infection.

Tuberculosis (TB) infection affects a quarter of the global population resulting in a large burden of TB disease and mortality. The long-term control of TB requires vaccines with greater efficacy and durability than the current Mycobacterium bovis Bacille Calmette-Guérin (BCG). Pulmonary immunization may increase and prolong immunity at the site of Mycobacterium tuberculosis infection. We have investigated recombinant influenza A viruses (rIAVs) expressing the p25 CD4+ T cell epitope of M. tuberculosis Ag85B240-254 for single and sequential immunization against M. tuberculosis infection. Intranasal immunization with single dose of rIAV X31 (H3N2 strain) expressing the p25 epitope (X31-p25), induced p25-specific CD4+ T cells and conferred protection against aerosol delivery of M. tuberculosis infection in the lungs. To enhance this effect, prime-boost immunization with hetero-subtypic rIAVs was examined. Sequential immunization with X31-p25 and a second rIAV, PR8 (H1N1 strain) expressing the same epitope (PR8-p25), increased the frequency of p25-specific IFN-γ T cell responses and polyfunctional CD4+ T cells producing IFN-γ, IL-2, and TNF, compared to immunization with each rIAV alone. This combination resulted in protection against M. tuberculosis in both the lungs and spleen. Therefore, our study revealed that rIAV is not only an efficient vector to induce protective immunity in the lungs, but also has a potential use for sequential immunization with heterologous rIAV to boost the immunogenicity and improve the protection against M. tuberculosis.

Effect of phthiocerol dimycocerosate deficiency on the transcriptional response of human macrophages to Mycobacterium tuberculosis.

The control of mycobacterial infections is dependent on the finely tuned synergism between the innate and adaptive immune responses. The macrophage is the major host cell for Mycobacterium tuberculosis and the degree of virulence of mycobacteria may influence the initial macrophage response to infection. The cell wall molecule, phthiocerol dimycocerosate (DIM), is an important virulence factor that influences the early growth of M. tuberculosis in the lungs. To explore the basis for this effect we have compared the early gene response of human THP-1 macrophages to infection with virulent M. tuberculosis and the DIM-deficient DeltafadD26 M. tuberculosis strain using microarrays. Detailed analysis revealed a common core of macrophage genes, which were rapidly induced following infection with both strains, and deficiency of DIM had no significant effect on this initial macrophage transcriptional responses. In addition to chemokines and pro-inflammatory cytokines, the early response genes included components of the Toll-like receptor signalling, antigen presentation and apoptotic pathways, interferon response genes, cell surface receptors and their ligands, including TNF-related apoptosis inducing ligand (TRAIL) and CD40, and other novel genes. Therefore, although fadD26 deficiency is responsible for the early attenuation of the growth of M. tuberculosis in vivo, this effect is not associated with differences in the initial macrophage transcriptional response.

In vitro evaluation of novel inhalable dry powders consisting of thioridazine and rifapentine for rapid tuberculosis treatment.

Thioridazine is an orally administered antipsychotic drug with potential for treatment of drug-resistant tuberculosis (TB). However, drug-induced adverse cardiac effects have been reported when thioridazine was used at an efficacious oral dose of 200mg/day to treat TB. Pulmonary delivery of thioridazine could be a rational approach to reduce dose-related side effects while enabling high drug concentrations at the primary site of infection. The present study compares in vitro aerosol performance, storage stability, and in vitro antimicrobial activity and cytotoxicity of two inhalable powders composed of thioridazine and a first-line anti-TB drug, rifapentine. Formulation 1 is a combination of amorphous thioridazine and crystalline rifapentine, while Formulation 2 consisted of both drugs as amorphous forms. Both thioridazine-rifapentine formulations were found suitable for inhalation with a total fine particle fraction (<5µm) of 68-76%. The two powders had similar MIC90 to rifapentine alone, being 0.000625µg/mL and 0.005µg/ml against Mycobacterium tuberculosis H37Ra and M. tuberculosis H37Rv, respectively. In contrast, thioridazine alone had a MIC90 of 12.5µg/mL and 500µg/mL, against M. tuberculosis H37Ra and M. tuberculosis H37Rv, respectively, demonstrating no synergistic anti-TB activity. However, thioridazine and rifapentine in a ratio of 1:3 enhanced the killing of M. tuberculosis H37Ra within the human monocyte-derived macrophages (THP-1) compared to the single drug treatments. Both powders showed an acceptable half maximal inhibitory concentration (IC50) of 31.25µg/mL on both THP-1 and human lung epithelial (A549) cells. However, Formulation 1 showed greater chemical stability than Formulation 2 after three months of storage under low humidity (vacuum) at 20±3°C. In conclusion, we have demonstrated a novel inhalable powder consisted of amorphous thioridazine and crystalline rifapentine (Formulation 1) with a good aerosol performance, potent anti-TB activity and storage stability, which deserves further in vivo investigations.

BCG vaccination against tuberculosis: past disappointments and future hopes.

Three billion doses of the bacille Calmette-Guérin (BCG) vaccine have been given over the past 60 years. BCG has a widely variable ability to protect humans against tuberculosis. This article examines some of the reasons for this variability, and discusses ways in which BCG vaccination might be improved.

A novel levansucrase-levanase gene cluster in Bacillus stearothermophilus ATCC12980.

A 3.1 kb fragment of Bacillus stearothermophilus ATCC12980 DNA permitted growth of Escherichia coli on sucrose. The fragment encoded genes for a levansucrase (surB) and also a levanase (surC). SurB and SurC shared 97% and 43% amino acid identity with the corresponding SacB and SacC proteins of Bacillus subtilis, whose sacB and sacC genes are organised very differently.

Analysis of stress- and host cell-induced expression of the Mycobacterium tuberculosis inorganic pyrophosphatase.

BACKGROUND: The gene encoding the inorganic pyrophosphatase (PPase) of the intracellular pathogen Legionella pneumophila is induced during intracellular infection, but is constitutively expressed in Escherichia coli. The causative agent of tuberculosis, Mycobacterium tuberculosis, contains a well conserved copy of PPase. We sought to determine if expression of the M. tuberculosis PPase is regulated by the intracellular environment. RESULTS: A strain of Mycobacterium bovis bacille Calmette-Guérin (BCG) was constructed in which the Aequoria victoria green fluorescent protein (GFP) is controlled by the promoter of the M. tuberculosis ppa gene. After prolonged exposure of the recombinant BCG strain within murine bone-marrow-derived macrophages, there was no observed increased activity of the ppa promoter. Furthermore, there was no change in promoter activity after exposure to various stress stimuli such as reduced pH, osmotic shock, nutrient limitation or oxidative stress. CONCLUSIONS: These results suggest that macrophage induction of ppa is not a general phenomenon among intracellular pathogens.

An inducible expression system permitting the efficient purification of a recombinant antigen from Mycobacterium smegmatis.

A novel expression vector utilising the highly inducible acetamidase promoter of Mycobacterium smegmatis was constructed. High-level induction of a model antigen, the Mycobacterium leprae 35 kDa protein, was demonstrated in recombinant M. smegmatis grown in the presence of the acetamidase inducer acetamide. The recombinant protein could be simply and efficiently purified from the bacterial sonicate by virtue of a C-terminal 6-histidine tag, demonstrating that this purification strategy can be used for the mycobacteria. The histidine tag had no apparent effect on the protein conformation or immunogenicity, suggesting that the vector described may prove useful for the purification of native-like recombinant mycobacterial proteins from fast-growing mycobacterial hosts.

Life on the inside: probing mycobacterium tuberculosis gene expression during infection.

The identification of Mycobacterium tuberculosis genes specifically expressed during infection is a key step in understanding mycobacterial pathogenesis. Such genes most likely encode products required for survival within the host and for progressive infection. Recent advances in mycobacterial genetics have permitted the development of new techniques and the adaptation of existing methods to analyse mycobacterial in vivo gene expression and virulence. This has revealed a subset of M. tuberculosis genes that are differentially expressed during infection and has demonstrated that a number of components contribute to the virulence of the organism. This information is expected to provide new strategies to prevent tuberculosis infection, new targets for antimicrobial therapy and new insights into the infectious process.

Specific serological diagnosis of leprosy with a recombinant Mycobacterium leprae protein purified from a rapidly growing mycobacterial host.

In this report we demonstrate the utility of an monoclonal antibody inhibition enzyme-linked immunosorbent assay based on the Mycobacterium leprae 35-kDa protein, purified from the rapidly growing host Mycobacterium smegmatis, for the serodiagnosis of multibacillary leprosy. The assay proved highly specific (97.5%) and sensitive (90%) and compared favorably with two other established methods routinely utilized for leprosy serodiagnosis.

Protection against virulent Mycobacterium avium infection following DNA vaccination with the 35-kilodalton antigen is accompanied by induction of gamma interferon-secreting CD4(+) T cells.

Mycobacterium avium is an opportunistic pathogen that primarily infects immunocompromised individuals, although the frequency of M. avium infection is also increasing in the immunocompetent population. The antigen repertoire of M. avium varies from that of Mycobacterium tuberculosis, with the immunodominant 35-kDa protein being present in M. avium and Mycobacterium leprae but not in members of the M. tuberculosis complex. Here we show that a DNA vector encoding this M. avium 35-kDa antigen (DNA-35) induces protective immunity against virulent M. avium infection, and this protective effect persists over 14 weeks of infection. In C57BL/6 mice, DNA vaccines expressing the 35-kDa protein as a cytoplasmic or secreted protein, both induced strong T-cell gamma interferon (IFN-gamma) and humoral immune responses. Furthermore, the antibody response was to conformational determinants, confirming that the vector-encoded protein had adopted the native conformation. DNA-35 immunization resulted in an increased activated/memory CD4(+) T-cell response, with an accumulation of CD4(+) CD44(hi) CD45RB(lo) T cells and an increase in antigen-specific IFN-gamma production. The protective effect of the DNA-35 vectors against M. avium infection was comparable to that of vaccination with Mycobacterium bovis BCG and significantly greater than that for previous treated infection with M. avium. These results illustrate the importance of the 35-kDa protein in the protective response to M. avium infection and indicate that DNA vaccination successfully promotes a sustained level of protection during chronic M. avium infection.

Expression of Mycobacterium tuberculosis MPT64 in recombinant Myco. smegmatis: purification, immunogenicity and application to skin tests for tuberculosis.

Proteins secreted across the cell wall of mycobacteria are important antigens recognized early in the host response to mycobacterial infection. MPT64 is a 23-kD secreted protein restricted to members of the Mycobacterium tuberculosis complex which elicits T cell responses and cutaneous DTH reactions in Myco. tuberculosis-infected animals. Patients with tuberculosis and their tuberculin-positive contacts respond to the protein, but recipients of bacille Calmette-Guérin (BCG) vaccine strains lacking the mpt64 gene do not. In the present study, we describe the development of a unique recombinant mycobacterial vector which secretes the encoded Myco. tuberculosis protein MPT64 at high levels into the culture filtrate, from which the protein is isolated by a single-step affinity chromatographic step. The purified protein was recognized by both polyclonal and monoclonal anti-MPT64 antibodies. The T cell reactivity of the protein was confirmed by its ability to stimulate human anti-rMPB64 T cell lines. The Myco. smegmatis recombinant MPT64 protein was superior to the Escherichia coli rMPB64 protein, which has identical amino acid sequence, in eliciting cutaneous DTH reactions in guinea pigs sensitized with Myco. tuberculosis. Animals sensitized with BCG strains lacking the mpb64 gene failed to respond to MPT64. Similarly, interferon-gamma (IFN-gamma) responses in tuberculosis patients and their contacts were higher to the Myco. smegmatis form of the protein. The potential of this form of the Myco. tuberculosis MPT64 protein as a skin test reagent for tuberculosis is discussed.

Use of fluorescence induction and sucrose counterselection to identify Mycobacterium tuberculosis genes expressed within host cells.

The identification of Mycobacterium tuberculosis genes expressed within host cells would contribute greatly to the development of new strategies to combat tuberculosis. By combining the natural fluorescence of the Aequoria victoria green fluorescent protein (GFP) with the counterselectable property of the Bacillus subtilis SacB protein, M. tuberculosis promoters displaying enhanced in vivo activity have been isolated. Macrophages were infected with recombinant Mycobacterium bovis bacille Calmette-Guérin containing a library of M. tuberculosis promoters controlling gfp and sacB expression, and fluorescent bacteria recovered by fluorescence-activated cell sorting. The expression of sacB was used to eliminate clones with strong promoter activity outside the macrophage, resulting in the isolation of seven clones containing M. tuberculosis promoters with greater activity intracellularly. The gene products identified displayed similarity to proteins from other organisms whose functions include nutrient utilization, protection from oxidative stress and defence against xenobiotics. These proposed functions are consistent with conditions encountered within the host cell and thus suggest that the augmented activity of the isolated promoters/genes may represent strategies employed by M. tuberculosis to enhance intracellular survival and promote infection.

DNA encoding a single mycobacterial antigen protects against leprosy infection.

The continuing incidence of leprosy infection around the world and the inability of Mycobacterium bovis bacille Calmette-Guérin (BCG) to protect certain populations clearly indicates that an improved vaccine against leprosy is needed. The immuno dominant 35 kDa protein, shared by Mycobacterium leprae and Mycobacterium avium, but not Mycobacterium tuberculosis or BCG, is recognised by >90% of leprosy patients, making it an ideal candidate antigen for a subunit vaccine. Immunization of outbred Swiss Albino mice with a DNA-35 vaccine stimulated specific T cell activation and IFN-gamma production. DNA-35 immunization induced significant levels of protection against M. leprae footpad infection, comparable to that produced by BCG. Therefore, DNA immunization with the 35 kDa antigen is effective against M. leprae infection and genetic immunization with a combination of antigens holds the potential for an improved vaccine against leprosy.

Comparative protective effects of recombinant DNA and Mycobacterium bovis bacille Calmette-Guérin vaccines against M. avium infection.

A range of strategies are being explored to develop more effective vaccines against mycobacterial infection, including immunization with DNA plasmids encoding single mycobacterial bacterial genes and the use of recombinant live vectors based on the current vaccine, Mycobacterium bovis bacille Calmette-Guérin (BCG). We have compared these two approaches using a model of virulent M. avium infection, and the gene for the immunodominant 35 kDa protein which is shared by M. avium and M. leprae, but absent from BCG. Recombinant BCG over-expressing the M. avium 35 kDa protein (BCG-35) induced strong antigen-specific proliferative and interferon-gamma (IFN-gamma)-secreting T cell responses. These were comparable to those induced by a single immunization with a plasmid expressing the same antigen (DNA-35); however, repeat DNA-35 immunization evoked the strongest IFN-gamma release. Immunization with BCG-35 significantly reduced the growth of virulent M. avium, although this effect was similar to that induced by wild-type BCG. Immunization with DNA-35 resulted in significantly greater (2 x log(10)) reduction in the growth of M. avium. Prime-boost strategies combining DNA-35 and BCG-35 increased the protective effect above that achieved by BCG-35, but they were not more protective than DNA-35 alone. Therefore, recombinant BCG-35 and BCG induced similar levels of protection in this model, and maximal protection against M. avium infection was attained by immunization with DNA encoding the 35 kDa protein.

Differential T cell responses to mycobacteria-secreted proteins distinguish vaccination with bacille Calmette-Guérin from infection with Mycobacterium tuberculosis.

The immune responses of healthy recipients of Mycobacterium bovis bacille Calmette-Guérin (BCG) vaccine, tuberculosis (TB) patients, and contacts of TB patients were examined to three major secretory proteins of Mycobacterium tuberculosis, MPB59, MPB64, and MPB70. MPB59 evoked a T cell response in 78% of BCG vaccines, 62% of TB patients, and 60% of contacts. MPB64 and MPB70 were recognized by < 15% of BCG vaccinees, half of TB patients, and three-quarters of contacts. TB and leprosy patients had antibody responses to MPB59, but few had antibodies to MPB64 or MPB70. Hybridization of mycobacterial DNA with specific gene probes demonstrated the absence of a gene for MBP64 in the vaccine strain of BCG, but the MPB70 gene was found in all virulent and vaccine BCG strains tested. Since MPB64 and MPB70 can induce delayed-type hypersensitivity reactions in infected animals, either of these proteins may have potential as skin test reagents for detecting infection with M. tuberculosis.

A 35-kilodalton protein is a major target of the human immune response to Mycobacterium leprae.

The control of leprosy will be facilitated by the identification of major Mycobacterium leprae-specific antigens which mirror the immune response to the organism across the leprosy spectrum. We have investigated the host response to a 35-kDa protein of M. leprae. Recombinant 35-kDa protein purified from Mycobacterium smegmatis resembled the native antigen in the formation of multimeric complexes and binding by monoclonal antibodies and sera from leprosy patients. These properties were not shared by two forms of 35-kDa protein purified from Escherichia coli. The M. smegmatis-derived 35-kDa protein stimulated a gamma interferon-secreting T-cell proliferative response in the majority of paucibacillary leprosy patients and healthy contacts of leprosy patients tested. Cellular responses to the protein in patients with multibacillary leprosy were weak or absent, consistent with hyporesponsiveness to M. leprae characteristic of this form of the disease. Almost all leprosy patients and contacts recognized the 35-kDa protein by either a T-cell proliferative or an immunoglobulin G antibody response, whereas few tuberculosis patients recognized the antigen. This specificity was confirmed in guinea pigs, with the 35-kDa protein eliciting strong delayed-type hypersensitivity in M. leprae-sensitized animals but not in those sensitized with Mycobacterium tuberculosis or Mycobacterium bovis BCG. Therefore, the M. leprae 35-kDa protein appears to be a major and relatively specific target of the human immune response to M. leprae and is a potential component of a diagnostic test to detect exposure to leprosy or a vaccine to combat the disease.

Molecular and immunological analyses of the Mycobacterium avium homolog of the immunodominant Mycobacterium leprae 35-kilodalton protein.

The analysis of host immunity to mycobacteria and the development of discriminatory diagnostic reagents relies on the characterization of conserved and species-specific mycobacterial antigens. In this report, we have characterized the Mycobacterium avium homolog of the highly immunogenic M. leprae 35-kDa protein. The genes encoding these two proteins were well conserved, having 82% DNA identity and 90% identity at the amino acid level. Moreover both proteins, purified from the fast-growing host M. smegmatis, formed multimeric complexes of around 1000 kDa in size and were antigenically related as assessed through their recognition by antibodies and T cells from M. leprae-infected individuals. The 35-kDa protein exhibited significant sequence identity with proteins from Streptomyces griseus and the cyanobacterium Synechoccocus sp. strain PCC 7942 that are up-regulated under conditions of nutrient deprivation. The 67% amino acid identity between the M. avium 35-kDa protein and SrpI of Synechoccocus was spread across the sequences of both proteins, while the homologous regions of the 35-kDa protein and the P3 sporulation protein of S. griseus were interrupted in the P3 protein by a divergent central region. Assessment by PCR demonstrated that the gene encoding the M. avium 35-kDa protein was present in all 30 M. avium clinical isolates tested but absent from M. intracellulare, M. tuberculosis, or M. bovis BCG. Mice infected with M. avium, but not M. bovis BCG, developed specific immunoglobulin G antibodies to the 35-kDa protein, consistent with the observation that tuberculosis patients do not recognize the antigen. Strong delayed-type hypersensitivity was elicited by the protein in guinea pigs sensitized with M. avium.