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N-acetylcysteine (NAC) is most commonly used for the treatment of acetaminophen overdose and acetaminophen-induced liver injury. In patients infected with Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), NAC is given to treat hepatotoxicity induced by TB drugs. We had previously shown that cysteine, a derivative of NAC, potentiated the activity of isoniazid, a first-line TB drug, by preventing the emergence of INH resistance and persistence in M. tuberculosis in vitro. Herein, we demonstrate that in vitro, NAC has the same boosting activity with various combinations of first- and second-line TB drugs against drug-susceptible and multidrug-resistant M. tuberculosis strains. Similar to cysteine, NAC increased M. tuberculosis respiration. However, in M. tuberculosis-infected mice, the addition of NAC did not augment the activity of first- or second-line TB drugs. A comparison of the activity of NAC combined with TB drugs in murine and human macrophage cell lines revealed that studies in mice might not be recapitulated during host infection in vivo.

The bacterial pathogen Mycobacterium tuberculosis can enter into a persistent state in which M. tuberculosis can evade host immunity, thereby reducing the effectiveness of current tuberculosis vaccines. Understanding the factors that contribute to persistence would enable the rational design of vaccines effective against persisters., Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, can enter into a persistent state that confers resistance to antibacterial agents. Many observations suggest that persistent M. tuberculosis cells also evade the antimycobacterial immune mechanisms, thereby reducing the effectiveness of the current tuberculosis vaccine. Understanding the factors that contribute to persistence may enable the rational design of vaccines that stimulate effective immune killing mechanisms against persister cells. Independent mutations targeting the methionine and arginine biosynthetic pathways are bactericidal for M. tuberculosis in mice. However, in this study, we discovered that the addition of leucine and pantothenate auxotrophy altered the bactericidality of methionine auxotrophy. Whereas the leucine/pantothenate/methionine auxotrophic M. tuberculosis strain H37Rv ΔleuCD ΔpanCD ΔmetA was eliminated in immunocompetent mice, this strain persisted in multiple organs of immunodeficient Rag1−/− mice for at least a year. In contrast, the leucine/pantothenate/arginine auxotroph H37Rv ΔleuCD ΔpanCD ΔargB was eliminated in both immunocompetent and immunodeficient Rag1−/− mice. Our results showed that leucine and pantothenate starvation metabolically blocked the sterilization mechanisms of methionine starvation but not those of arginine starvation. These triple-auxotrophic strains should be invaluable tools for unravelling the bacterial and host factors that enable persistence and for vaccine development studies to assess the efficacy of vaccines that boost immune recognition of M. tuberculosis in the persistent state. The sterilization of the ΔleuCD ΔpanCD ΔmetA auxotroph in immunocompetent mice, but not in mice lacking an adaptive immune response, could provide a new system for studying the antimycobacterial killing mechanisms of adaptive immunity.

Mycobacterium tuberculosis, the causative agent of tuberculosis disease, remains a public health crisis on a global scale, and development of new interventions and identification of drug resistance are pillars in the World Health Organization End TB Strategy. Leveraging the tractability of the TM4 mycobacteriophage and the sensitivity of the nanoluciferase reporter enzyme, the present work describes an evolution of phage-mediated detection and drug susceptibility testing of M. tuberculosis, adding a valuable tool in drug discovery and basic biology research. With additional validation, this system may play a role as a quantitative phenotypic reference method and complement to genotypic methods for diagnosis and antibiotic susceptibility testing., Phenotypic testing for drug susceptibility of Mycobacterium tuberculosis is critical to basic research and managing the evolving problem of antimicrobial resistance in tuberculosis management, but it remains a specialized technique to which access is severely limited. Here, we report on the development and validation of an improved phage-mediated detection system for M. tuberculosis. We incorporated a nanoluciferase (Nluc) reporter gene cassette into the TM4 mycobacteriophage genome to create phage TM4-nluc. We assessed the performance of this reporter phage in the context of cellular limit of detection and drug susceptibility testing using multiple biosafety level 2 drug-sensitive and -resistant auxotrophs as well as virulent M. tuberculosis strains. For both limit of detection and drug susceptibility testing, we developed a standardized method consisting of a 96-hour cell preculture followed by a 72-hour experimental window for M. tuberculosis detection with or without antibiotic exposure. The cellular limit of detection of M. tuberculosis in a 96-well plate batch culture was ≤102 CFU. Consistent with other phenotypic methods for drug susceptibility testing, we found TM4-nluc to be compatible with antibiotics representing multiple classes and mechanisms of action, including inhibition of core central dogma functions, cell wall homeostasis, metabolic inhibitors, compounds currently in clinical trials (SQ109 and Q203), and susceptibility testing for bedaquiline, pretomanid, and linezolid (components of the BPaL regimen for the treatment of multi- and extensively drug-resistant tuberculosis). Using the same method, we accurately identified rifampin-resistant and multidrug-resistant M. tuberculosis strains. IMPORTANCE Mycobacterium tuberculosis, the causative agent of tuberculosis disease, remains a public health crisis on a global scale, and development of new interventions and identification of drug resistance are pillars in the World Health Organization End TB Strategy. Leveraging the tractability of the TM4 mycobacteriophage and the sensitivity of the nanoluciferase reporter enzyme, the present work describes an evolution of phage-mediated detection and drug susceptibility testing of M. tuberculosis, adding a valuable tool in drug discovery and basic biology research. With additional validation, this system may play a role as a quantitative phenotypic reference method and complement to genotypic methods for diagnosis and antibiotic susceptibility testing.

Large genomic deletions (LGDs) (6 to 63 kbp) were observed in isoniazid-resistant Mycobacterium tuberculosis mutants derived from four M. tuberculosis strains. These LGDs had no growth defect in vitro but could be defective in intracellular growth and showed various sensitivities toward oxidative stress despite lacking katG . The LGD regions comprise 74 genes, mostly of unknown function, that may be important for M. tuberculosis intracellular growth and protection against oxidative stress.

Herpes simplex virus 1 (HSV-1) is a leading cause of infectious blindness, highlighting the need for effective vaccines. A single-cycle HSV-2 strain with the deletion of glycoprotein D, ΔgD-2, completely protected mice from HSV-1 and HSV-2 skin or vaginal disease and prevented latency following active or passive immunization in preclinical studies. The antibodies functioned primarily by activating Fc receptors to mediate antibody-dependent cellular cytotoxicity (ADCC). The ability of ADCC to protect the immune-privileged eye, however, may differ from skin or vaginal infections. Thus, the current studies were designed to compare active and passive immunization with ΔgD-2 versus an adjuvanted gD subunit vaccine (rgD-2) in a primary lethal ocular murine model. ΔgD-2 provided significantly greater protection than rgD-2 following a two-dose vaccine regimen, although both vaccines were protective compared to an uninfected cell lysate. However, only immune serum from ΔgD-2-vaccinated, but not rgD-2-vaccinated, mice provided significant protection against lethality in passive transfer studies. The significantly greater passive protection afforded by ΔgD-2 persisted after controlling for the total amount of HSV-specific IgG in the transferred serum. The antibodies elicited by rgD-2 had significantly higher neutralizing titers, whereas those elicited by ΔgD-2 had significantly more C1q binding and Fc gamma receptor activation, a surrogate for ADCC function. Together, the findings suggest ADCC is protective in the eye and that nonneutralizing antibodies elicited by ΔgD-2 provide greater protection than neutralizing antibodies elicited by rgD-2 against primary ocular HSV disease. The findings support advancement of vaccines, including ΔgD-2, that elicit polyfunctional antibody responses. IMPORTANCE Herpes simplex virus 1 is the leading cause of infectious corneal blindness in the United States and Europe. Developing vaccines to prevent ocular disease is challenging because the eye is a relatively immune-privileged site. In this study, we compared a single-cycle viral vaccine candidate, which is unique in that it elicits predominantly nonneutralizing antibodies that activate Fc receptors and bind complement, and a glycoprotein D subunit vaccine that elicits neutralizing but not Fc receptor-activating or complement-binding responses. Only the single-cycle vaccine provided both active and passive protection against a lethal ocular challenge. These findings greatly expand our understanding of the types of immune responses needed to protect the eye and will inform future prophylactic and therapeutic strategies.

The emergence of drug resistance in Helicobacter pylori has resulted in a greater need for susceptibility-guided treatment. While the alleles associated with resistance to clarithromycin and levofloxacin have been defined, there are limited data regarding the molecular mechanisms underlying resistance to other antimicrobials. Using H. pylori isolates from 42 clinical specimens, we compared phenotypic and whole-genome sequencing (WGS)-based detection of resistance. Phenotypic resistance correlated with the presence of alleles of 23S rRNA (A2142G/A2143G) for clarithromycin (kappa coefficient, 0.84; 95% confidence interval [CI], 0.67 to 1.0) and gyrA (N87I/N87K/D91Y/D91N/D91G/D99N) for levofloxacin (kappa coefficient, 0.90; 95% CI, 0.77 to 1.0). Phenotypic resistance to amoxicillin in three isolates correlated with mutations in pbp1, pbp2, and/or pbp3 within coding regions near known amoxicillin binding motifs. All isolates were phenotypically susceptible to tetracycline, although four bore a mutation in 16S rRNA (A926G). For metronidazole, nonsense mutations and R16H substitutions in rdxA correlated with phenotypic resistance (kappa coefficient, 0.76; 95% CI, 0.56 to 0.96). Previously identified mutations in the rpoB rifampin resistance-determining region (RRDR) were not present, but 14 novel mutations outside the RRDR were found in rifampin-resistant isolates. WGS also allowed for strain lineage determination, which may be important for future studies in associating precise MICs with specific resistance alleles. In summary, WGS allows for broad analyses of H. pylori isolates, and our findings support the use of WGS for the detection of clarithromycin and levofloxacin resistance. Additional studies are warranted to better define mutations conferring resistance to amoxicillin, tetracycline, and rifampin, but combinatorial analyses for rdxA gene truncations and R16H mutations have utility for determining metronidazole resistance.

A critical gap in tuberculosis (TB) treatment is detection of emergent drug resistance. We hypothesized that advanced phenotyping with whole-genome sequencing (WGS) will detect low-frequency Mycobacterium tuberculosis drug resistance. We assessed a reporter mycobacteriophage (Φ(2)GFP10) in vitro to detect drug-resistant subpopulations and predict M. tuberculosis bactericidal activity in this pilot study. Subsequently, we prospectively studied 20 TB patients with serial Φ(2)GFP10, Xpert MTB/RIF, and M. tuberculosis culture through end of treatment. WGS was performed, and single nucleotide polymorphisms (SNPs) were examined to detect mixed infection in selected M. tuberculosis isolates. Resistant M. tuberculosis isolates were detected at 1:100,000, and changes in cytometry-gated events were predictive of in vitro M. tuberculosis bactericidal activity using the Φ(2)GFP10 assay. Emergent drug resistance was detected in one patient by Φ(2)GFP10 at 3 weeks but not by conventional testing (M. tuberculosis culture and GeneXpert). WGS revealed a phylogeographically distinct extensively drug-resistant tuberculosis (XDR-TB) genome, identical to an XDR-TB isolate from the patient’s spouse. Variant lineage-specific SNPs were present early, suggesting mixed infection as the etiology of emergent resistance with temporal trends providing evidence for selection during treatment. Φ(2)GFP10 can detect low-frequency drug-resistant M. tuberculosis and with WGS characterize emergent M. tuberculosis resistance. In areas of high TB transmission and drug resistance, rapid screening for heteroresistance should be considered.

A critical gap in tuberculosis (TB) treatment is detection of emergent drug resistance. We hypothesized that advanced phenotyping with whole-genome sequencing (WGS) will detect low-frequency Mycobacterium tuberculosis drug resistance. We assessed a reporter mycobacteriophage (Φ2GFP10) in vitro to detect drug-resistant subpopulations and predict M. tuberculosis bactericidal activity in this pilot study. Subsequently, we prospectively studied 20 TB patients with serial Φ2GFP10, Xpert MTB/RIF, and M. tuberculosis culture through end of treatment. WGS was performed, and single nucleotide polymorphisms (SNPs) were examined to detect mixed infection in selected M. tuberculosis isolates. Resistant M. tuberculosis isolates were detected at 1:100,000, and changes in cytometry-gated events were predictive of in vitro M. tuberculosis bactericidal activity using the Φ2GFP10 assay. Emergent drug resistance was detected in one patient by Φ2GFP10 at 3 weeks but not by conventional testing (M. tuberculosis culture and GeneXpert). WGS revealed a phylogeographically distinct extensively drug-resistant tuberculosis (XDR-TB) genome, identical to an XDR-TB isolate from the patient's spouse. Variant lineage-specific SNPs were present early, suggesting mixed infection as the etiology of emergent resistance with temporal trends providing evidence for selection during treatment. Φ2GFP10 can detect low-frequency drug-resistant M. tuberculosis and with WGS characterize emergent M. tuberculosis resistance. In areas of high TB transmission and drug resistance, rapid screening for heteroresistance should be considered.

Mycobacterium tuberculosis remains a threat to global health, and a more efficacious vaccine is needed to prevent disease caused by M. tuberculosis We previously reported that the mycobacterial ribosome is a major target of CD4+ T cells in mice immunized with a genetically modified Mycobacterium smegmatis strain (IKEPLUS) but not in mice immunized with Mycobacterium bovis BCG. Two specific ribosomal proteins, RplJ and RpsA, were identified as cross-reactive targets of M. tuberculosis, but the breadth of the CD4+ T cell response to M. tuberculosis ribosomes was not determined. In the present study, a library of M. tuberculosis ribosomal proteins and in silico-predicted peptide libraries were used to screen CD4+ T cell responses in IKEPLUS-immunized mice. This identified 24 out of 57 M. tuberculosis ribosomal proteins distributed over both large and small ribosome subunits as specific CD4+ T cell targets. Although BCG did not induce detectable responses against ribosomal proteins or peptide epitopes, the M. tuberculosis ribosomal protein RplJ produced a robust and multifunctional Th1-like CD4+ T cell population when administered as a booster vaccine to previously BCG-primed mice. Boosting of BCG-primed immunity with the M. tuberculosis RplJ protein led to significantly reduced lung pathology compared to that in BCG-immunized animals and reductions in the bacterial burdens in the mediastinal lymph node compared to those in naive and standard BCG-vaccinated mice. These results identify the mycobacterial ribosome as a potential source of cryptic or subdominant antigenic targets of protective CD4+ T cell responses and suggest that supplementing BCG with ribosomal antigens may enhance protective vaccination against M. tuberculosis.

Multidrug-resistant (MDR) tuberculosis, defined as tuberculosis resistant to the two first-line drugs isoniazid and rifampin, poses a serious problem for global tuberculosis control strategies. Lack of a safe and convenient model organism hampers progress in combating the spread of MDR strains of Mycobacterium tuberculosis. We reasoned that auxotrophic MDR mutants of M. tuberculosis would provide a safe means for studying MDR M. tuberculosis without the need for a biosafety level 3 (BSL3) laboratory. Two different sets of triple auxotrophic mutants of M. tuberculosis were generated, which were auxotrophic for the nutrients leucine, pantothenate, and arginine or for leucine, pantothenate, and methionine. These triple auxotrophic strains retained their acid-fastness, their ability to generate both a drug persistence phenotype and drug-resistant mutants, and their susceptibility to plaque-forming mycobacterial phages. MDR triple auxotrophic mutants were obtained in a two-step fashion, selecting first for solely isoniazid-resistant or rifampin-resistant mutants. Interestingly, selection for isoniazid-resistant mutants of the methionine auxotroph generated isolates with single point mutations in katG, which encodes an isoniazid-activating enzyme, whereas similar selection using the arginine auxotroph yielded isoniazid-resistant mutants with large deletions in the chromosomal region containing katG. These M. tuberculosis MDR strains were readily sterilized by second-line tuberculosis drugs and failed to kill immunocompromised mice. These strains provide attractive candidates for M. tuberculosis biology studies and drug screening outside the BSL3 facility., IMPORTANCE Elimination of Mycobacterium tuberculosis, the bacterium causing tuberculosis, requires enhanced understanding of its biology in order to identify new drugs against drug-susceptible and drug-resistant M. tuberculosis as well as uncovering novel pathways that lead to M. tuberculosis death. To circumvent the need for a biosafety level 3 (BSL3) laboratory when conducting research on M. tuberculosis, we have generated drug-susceptible and drug-resistant triple auxotrophic strains of M. tuberculosis suitable for use in a BSL2 laboratory. These strains originate from a double auxotrophic M. tuberculosis strain, H37Rv ΔpanCD ΔleuCD, which was reclassified as a BSL2 strain based on its lack of lethality in immunocompromised and immunocompetent mice. A third auxotrophy (methionine or arginine) was introduced via deletion of metA or argB, respectively, since M. tuberculosis ΔmetA and M. tuberculosis ΔargB are unable to survive amino acid auxotrophy and infect their host. The resulting triple auxotrophic M. tuberculosis strains retained characteristics of M. tuberculosis relevant for most types of investigations.

The treatment of drug-susceptible tuberculosis (TB) is long and cumbersome. Mismanagement of TB treatment can lead to the emergence of drug resistance in patients, so shortening the treatment duration could significantly improve TB chemotherapy and prevent the development of drug resistance. We previously discovered that high concentrations of vitamin C sterilize cultures of drug-susceptible and drug-resistant Mycobacterium tuberculosis . Here, we tested subinhibitory concentration of vitamin C in combination with TB drugs against M. tuberculosis in vitro and in a mouse model of M. tuberculosis infection. In vivo , we showed that the vitamin C level in mouse serum can be increased by intraperitoneal injection of vitamin C to reach vitamin C levels close to the concentrations required for activity in vitro . Although vitamin C had no activity by itself in M. tuberculosis -infected mice, the combination of vitamin C with the first-line TB drugs isoniazid and rifampin reduced the bacterial burden in the lungs of M. tuberculosis -infected mice faster than isoniazid and rifampin combined in two independent experiments. These experiments suggest that the addition of vitamin C to first-line TB drugs could shorten TB treatment. Vitamin C, an inexpensive and nontoxic compound, could easily be added to the TB pharmacopeia to substantially improve chemotherapy outcome, which would have a significant impact on the worldwide TB community.

Improved diagnostics and drug susceptibility testing for Mycobacterium tuberculosis are urgently needed. We developed a more powerful mycobacteriophage (Φ 2 GFP10) with a fluorescent reporter. Fluorescence-activated cell sorting (FACS) allows for rapid enumeration of metabolically active bacilli after phage infection. We compared the reporter phage assay to GeneXpert MTB/RIF for detection of M. tuberculosis and rifampin (RIF) resistance in sputum. Patients suspected to have tuberculosis were prospectively enrolled in Durban, South Africa. Sputum was incubated with Φ 2 GFP10, in the presence and absence of RIF, and bacilli were enumerated using FACS. Sensitivity and specificity were compared to those of GeneXpert MTB/RIF with an M. tuberculosis culture as the reference standard. A total of 158 patients were prospectively enrolled. Overall sensitivity for M. tuberculosis was 95.90% (95% confidence interval (CI), 90.69% to 98.64%), and specificity was 83.33% (95% CI, 67.18% to 93.59%). In acid-fast bacillus (AFB)-negative sputum, sensitivity was 88.89% (95% CI, 73.92% to 96.82%), and specificity was 83.33% (95% CI, 67.18% to 93.59%). Sensitivity for RIF-resistant M. tuberculosis in AFB-negative sputum was 90.00% (95% CI, 55.46% to 98.34%), and specificity was 91.94% (95% CI, 82.16% to 97.30%). Compared to GeneXpert, the reporter phage was more sensitive in AFB smear-negative sputum, but specificity was lower. The Φ 2 GFP10 reporter phage showed high sensitivity for detection of M. tuberculosis and RIF resistance, including in AFB-negative sputum, and has the potential to improve phenotypic testing for complex drug resistance, paucibacillary sputum, response to treatment, and detection of mixed infection in clinical specimens.

The well-established safety profile of the tuberculosis vaccine strain,Mycobacterium bovisbacille Calmette-Guérin (BCG), makes it an attractive vehicle for heterologous expression of antigens from clinically relevant pathogens. However, successful generation of recombinant BCG strains possessing consistent insert expression has encountered challenges in stability. Here, we describe a method for the development of large recombinant BCG accession lots which stably express the lentiviral antigens, human immunodeficiency virus (HIV) gp120 and simian immunodeficiency virus (SIV) Gag, using selectable leucine auxotrophic complementation. Successful establishment of vaccine stability stems from stringent quality control criteria which not only screen for highly stable complemented BCG ΔleuCDtransformants but also thoroughly characterize postproduction quality. These parameters include consistent production of correctly sized antigen, retention of sequence-pure plasmid DNA, freeze-thaw recovery, enumeration of CFU, and assessment of cellular aggregates. Importantly, these quality assurance procedures were indicative of overall vaccine stability, were predictive for successful antigen expression in subsequent passaging bothin vitroandin vivo, and correlated with induction of immune responses in murine models. This study has yielded a quality-controlled BCG ΔleuCDvaccine expressing HIV gp120 that retained stable full-length expression after 1024-fold amplificationin vitroand following 60 days of growth in mice. A second vaccine lot expressed full-length SIV Gag for >1068-fold amplificationin vitroand induced potent antigen-specific T cell populations in vaccinated mice. Production of large, well-defined recombinant BCG ΔleuCDlots can allow confidence that vaccine materials for immunogenicity and protection studies are not negatively affected by instability or differences between freshly grown production batches.

Herpes simplex virus (HSV) entry requires multiple interactions at the cell surface and activation of a complex calcium signaling cascade. Previous studies demonstrated that integrins participate in this process, but their precise role has not been determined. These studies were designed to test the hypothesis that integrin αvβ3 signaling promotes the release of intracellular calcium (Ca 2+ ) stores and contributes to viral entry and cell-to-cell spread. Transfection of cells with small interfering RNA (siRNA) targeting integrin αvβ3, but not other integrin subunits, or treatment with cilengitide, an Arg-Gly-Asp (RGD) mimetic, impaired HSV-induced Ca 2+ release, viral entry, plaque formation, and cell-to-cell spread of HSV-1 and HSV-2 in human cervical and primary genital tract epithelial cells. Coimmunoprecipitation studies and proximity ligation assays indicated that integrin αvβ3 interacts with glycoprotein H (gH). An HSV-2 gH-null virus was engineered to further assess the role of gH in the virus-induced signaling cascade. The gH-2-null virus bound to cells and activated Akt to induce a small Ca 2+ response at the plasma membrane, but it failed to trigger the release of cytoplasmic Ca 2+ stores and was impaired for entry and cell-to-cell spread. Silencing of integrin αvβ3 and deletion of gH prevented phosphorylation of focal adhesion kinase (FAK) and the transport of viral capsids to the nuclear pore. Together, these findings demonstrate that integrin signaling is activated downstream of virus-induced Akt signaling and facilitates viral entry through interactions with gH by activating the release of intracellular Ca 2+ and FAK phosphorylation. These findings suggest a new target for HSV treatment and suppression. IMPORTANCE Herpes simplex viruses are the leading cause of genital disease worldwide, the most common infection associated with neonatal encephalitis, and a major cofactor for HIV acquisition and transmission. There is no effective vaccine. These epidemiological findings underscore the urgency to develop novel HSV treatment or prevention strategies. This study addresses this gap by further defining the signaling pathways the virus usurps to enter human genital tract epithelial cells. Specifically, the study defines the role played by integrins and by the viral envelope glycoprotein H in entry and cell-to-cell spread. This knowledge will facilitate the identification of new targets for the development of treatment and prevention.

Mycobacterium tuberculosis releases membrane vesicles packed with molecules that can modulate the immune response. Because environmental conditions often influence the production and content of bacterial vesicles, this study examined M. tuberculosis microvesicles released under iron limitation, a common condition faced by pathogens inside the host. The findings indicate that M. tuberculosis increases microvesicle production in response to iron restriction and that these microvesicles contain mycobactin, which can serve as an iron donor and supports replication of iron-starved mycobacteria. Consequently, the results revealed a role of microvesicles in iron acquisition in M. tuberculosis , which can be critical for survival in the host.

We previously reported that a recombinant pantothenate auxotroph of Mycobacterium bovis BCG expressing human immunodeficiency virus type 1 (HIV-1) subtype C Gag (rBCGpan-Gag) efficiently primes the mouse immune system for a boost with a recombinant modified vaccinia virus Ankara (rMVA) vaccine. In this study, we further evaluated the immunogenicity of rBCGpan-Gag in a nonhuman primate model. Two groups of chacma baboons were primed or mock primed twice with either rBCGpan-Gag or a control BCG. Both groups were boosted with HIV-1 Pr55 gag virus-like particles (Gag VLPs). The magnitude and breadth of HIV-specific cellular responses were measured using a gamma interferon (IFN-γ) enzyme-linked immunosorbent spot (ELISPOT) assay, and the cytokine profiles and memory phenotypes of T cells were evaluated by polychromatic flow cytometry. Gag-specific responses were detected in all animals after the second inoculation with rBCGpan-Gag. Boosting with Gag VLPs significantly increased the magnitude and breadth of the responses in the baboons that were primed with rBCGpan-Gag. These responses targeted an average of 12 Gag peptides per animal, compared to an average of 3 peptides per animal for the mock-primed controls. Robust responses of Gag-specific polyfunctional T cells capable of simultaneously producing IFN-γ, tumor necrosis alpha (TNF-α), and interleukin-2 (IL-2) were detected in the rBCGpan-Gag-primed animals. Gag-specific memory T cells were skewed toward a central memory phenotype in both CD4 + and CD8 + T cell populations. These data show that the rBCGpan-Gag prime and Gag VLP boost vaccine regimen is highly immunogenic, inducing a broad and polyfunctional central memory T cell response. This report further indicates the feasibility of developing a BCG-based HIV vaccine that is safe for childhood HIV immunization.

Our goal is to develop a pediatric combination vaccine to protect the vulnerable infant population against human immunodeficiency virus type 1 (HIV-1) and tuberculosis (TB) infections. The vaccine consists of an auxotroph Mycobacterium tuberculosis strain that coexpresses HIV antigens. Utilizing an infant rhesus macaque model, we have previously shown that this attenuated M. tuberculosis (A Mtb )-simian immunodeficiency virus (SIV) vaccine is immunogenic, and although the vaccine did not prevent oral SIV infection, a subset of vaccinated animals was able to partially control virus replication. However, unexpectedly, vaccinated infants required fewer SIV exposures to become infected compared to naive controls. Considering that the current TB vaccine, Mycobacterium bovis bacillus Calmette-Guérin (BCG), can induce potent innate immune responses and confer pathogen-unspecific trained immunity, we hypothesized that an imbalance between enhanced myeloid cell function and immune activation might have influenced the outcome of oral SIV challenge in A Mtb -SIV-vaccinated infants. To address this question, we used archived samples from unchallenged animals from our previous A Mtb -SIV vaccine studies and vaccinated additional infant macaques with BCG or A Mtb only. Our results show that vaccinated infants, regardless of vaccine strain or regimen, had enhanced myeloid cell responses. However, CD4 + T cells were concurrently activated, and the persistence of these activated target cells in oral and/or gastrointestinal tissues may have facilitated oral SIV infection. Immune activation was more pronounced in BCG-vaccinated infant macaques than in A Mtb -vaccinated infant macaques, indicating a role for vaccine attenuation. These findings underline the importance of understanding the interplay of vaccine-induced immunity and immune activation and its effect on HIV acquisition risk and outcome in infants.

Mycobacteriophage DS6A is unique among the more than 8,000 isolated mycobacteriophages due to its ability to form plaques exclusively on mycobacteria belonging to the Mycobacterium tuberculosis complex (MTBC). Speculation surrounding this specificity has led to unsupported assertions in published studies and patents that nontuberculous mycobacteria (NTM) are wholly resistant to DS6A infection. In this study, we identified two independent nonessential regions in the DS6A genome and replaced them with an mVenus-expressing plasmid to generate fluorescent reporter phages Φ 2 GFP12 and Φ 2 GFP13. We show that even though DS6A is able to form plaques only on MTBC bacteria, infection of various NTM results in mVenus expression in transduced cells. The efficiency of DS6A in delivering DNA varied between NTM species. Additionally, we saw a striking difference in the efficiency of DNA delivery between the closely related members of the Mycobacterium abscessus complex, M. abscessus and Mycobacterium massiliense . We also demonstrated that TM4 and DS6A, two phages that do not form plaques on M. massiliense , differ in their ability to deliver DNA, suggesting that there is a phage-specific restriction between mycobacterial species. Phylogenetic analysis reveals that the DS6A genome has a characteristically mosaic structure but provided few insights into the basis for the specificity for MTBC hosts. This study demonstrates that the inability of the MTBC-specific phage DS6A to form plaques on NTM is more complex than previously thought. Moreover, the DS6A-derived fluorophages provide important new tools for the study of mycobacterial biology. IMPORTANCE The coevolution of bacteria and their infecting phages involves a constant arms race for bacteria to prevent phage infection and phage to overcome these preventions. Although a diverse array of phage defense systems is well characterized in bacteria, very few phage restriction systems are known in mycobacteria. The DS6A mycobacteriophage is unique in the mycobacterial world in that it forms plaques only on members of the Mycobacterium tuberculosis complex. However, the novel DS6A reporter phages developed in this work demonstrate that DS6A can infect nontuberculous mycobacteria at various efficiencies. By comparing the abilities of DS6A and another phage, TM4, to infect and form plaques on various mycobacterial species, we can begin to discern new phage restriction systems employed within the genus.

Despite significant progress in reducing peripartum mother-to-child transmission (MTCT) of human immunodeficiency virus (HIV) with antiretroviral therapy (ART), continued access to ART throughout the breastfeeding period is still a limiting factor, and breast milk exposure to HIV accounts for up to 44% of MTCT. As abstinence from breastfeeding is not recommended, alternative means are needed to prevent MTCT of HIV. We have previously shown that oral vaccination at birth with live attenuated Mycobacterium tuberculosis strains expressing simian immunodeficiency virus (SIV) genes safely induces persistent SIV-specific cellular and humoral immune responses both systemically and at the oral and intestinal mucosa. Here, we tested the ability of oral M. tuberculosis vaccine strains expressing SIV Env and Gag proteins, followed by systemic heterologous (MVA-SIV Env/Gag/Pol) boosting, to protect neonatal macaques against oral SIV challenge. While vaccination did not protect infant macaques against oral SIV acquisition, a subset of immunized animals had significantly lower peak viremia which inversely correlated with prechallenge SIV Env-specific salivary and intestinal IgA responses and higher-avidity SIV Env-specific IgG in plasma. These controller animals also maintained CD4 + T cell populations better and showed reduced tissue pathology compared to noncontroller animals. We show that infants vaccinated at birth can develop vaccine-induced SIV-specific IgA and IgG antibodies and cellular immune responses within weeks of life. Our data further suggest that affinity maturation of vaccine-induced plasma antibodies and induction of mucosal IgA responses at potential SIV entry sites are associated with better control of viral replication, thereby likely reducing SIV morbidity. IMPORTANCE Despite significant progress in reducing peripartum MTCT of HIV with ART, continued access to ART throughout the breastfeeding period is still a limiting factor. Breast milk exposure to HIV accounts for up to 44% of MTCT. Alternative measures, in addition to ART, are needed to achieve the goal of an AIDS-free generation. Pediatric HIV vaccines constitute a core component of such efforts. The results of our pediatric vaccine study highlight the potential importance of vaccine-elicited mucosal Env-specific IgA responses in combination with high-avidity systemic Env-specific IgG in protection against oral SIV transmission and control of viral replication in infant macaques. The induction of potent mucosal IgA antibodies by our vaccine is remarkable considering the age-dependent development of mucosal IgA responses postbirth. A deeper understanding of postnatal immune development may inform the design of improved vaccine strategies to enhance systemic and mucosal SIV/HIV antibody responses.

Tumor necrosis factor alpha (TNF) plays a critical role in the control of Mycobacterium tuberculosis, in part by augmenting T cell responses through promoting macrophage phagolysosomal fusion (thereby optimizing CD4+ T cell immunity by enhancing antigen presentation) and apoptosis (a process that can lead to cross-priming of CD8+ T cells). M. tuberculosis can evade antituberculosis (anti-TB) immunity by inhibiting host cell TNF production via expression of specific mycobacterial components. We hypothesized that M. tuberculosis mutants with an increased capacity to induce host cell TNF production (TNF-enhancing mutants) and thus with enhanced immunogenicity can be useful for vaccine development. To identify mycobacterial genes that regulate host cell TNF production, we used a TNF reporter macrophage clone to screen an H37Rv M. tuberculosis cosmid library constructed in M. smegmatis. The screen has identified a set of TNF-downregulating mycobacterial genes that, when deleted in H37Rv, generate TNF-enhancing mutants. Analysis of mutants disrupted for a subset of TNF-downregulating genes, annotated to code for triacylglycerol synthases and fatty acyl-coenzyme A (acyl-CoA) synthetase, enzymes that concern lipid biosynthesis and metabolism, has revealed that these strains can promote macrophage phagolysosomal fusion and apoptosis better than wild-type (WT) bacilli. Immunization of mice with the TNF-enhancing M. tuberculosis mutants elicits CD4+ and CD8+ T cell responses that are superior to those engendered by WT H37Rv. The results suggest that TNF-upregulating M. tuberculosis genes can be targeted to enhance the immunogenicity of mycobacterial strains that can serve as the substrates for the development of novel anti-TB vaccines., IMPORTANCE One way to control tuberculosis (TB), which remains a major global public health burden, is by immunization with an effective vaccine. The efficacy of Mycobacterium bovis BCG, the only currently approved TB vaccine, is inconsistent. Tumor necrosis factor alpha (TNF) is a cytokine that plays an important role in controlling TB. M. tuberculosis, the causative agent of TB, can counter this TNF-based defense by decreasing host cell TNF production. This study identified M. tuberculosis genes that can mediate inhibition of TNF production by macrophage (an immune cell critical to the control of TB). We have knocked out a number of these genes to generate M. tuberculosis mutants that can enhance macrophage TNF production. Immunization with these mutants in mice triggered a T cell response stronger than that elicited by the parental bacillus. Since T cell immunity is pivotal in controlling M. tuberculosis, the TNF-enhancing mutants can be used to develop novel TB vaccines.

The challenges of developing new drugs to treat tuberculosis (TB) are indicated by the relatively small number of candidates entering clinical trials in the past decade. To overcome these issues, we reexamined two FDA-approved antibacterial drugs, sulfamethoxazole (SMX) and trimethoprim (TMP), for use in TB treatment. SMX and TMP inhibit folic acid biosynthesis and are used in combination to treat infections of the respiratory, urinary, and gastrointestinal tracts. The MICs of SMX and TMP, alone and in combination, were determined for drug-susceptible, multidrug-resistant (MDR), and extensively drug-resistant Mycobacterium tuberculosis strains. While TMP alone was not effective against M. tuberculosis , the combination of TMP and SMX was bacteriostatic against M. tuberculosis . Surprisingly, the combination of SMX and TMP was also active against a subset of MDR M. tuberculosis strains. Treatment of M. tuberculosis with TMP-SMX and a first-line anti-TB drug, either isoniazid or rifampin, was bactericidal, demonstrating that the combination of TMP and SMX with isoniazid or rifampin was not antagonistic. Moreover, the addition of SMX-TMP in combination with either isoniazid or rifampin also prevented the emergence of drug resistance in vitro . In conclusion, this study further illustrates the opportunity to reevaluate the activity of TMP-SMX in vivo to prevent the emergence of drug-resistant M. tuberculosis .

The difficulty of diagnosing active tuberculosis (TB) and lack of rapid drug susceptibility testing (DST) at the point of care remain critical obstacles to TB control. This report describes a high-intensity mycobacterium-specific-fluorophage (ϕ 2 GFP10) that for the first time allows direct visualization of Mycobacterium tuberculosis in clinical sputum samples. Engineered features distinguishing ϕ 2 GFP10 from previous reporter phages include an improved vector backbone with increased cloning capacity and superior expression of fluorescent reporter genes through use of an efficient phage promoter. ϕ 2 GFP10 produces a 100-fold increase in fluorescence per cell compared to existing reporter phages. DST for isoniazid and oxofloxacin, carried out in cultured samples, was complete within 36 h. Use of ϕ 2 GFP10 detected M. tuberculosis in clinical sputum samples collected from TB patients. DST for rifampin and kanamycin from sputum samples yielded results after 12 h of incubation with ϕ 2 GFP10. Fluorophage ϕ 2 GFP10 has potential for clinical development as a rapid, sensitive, and inexpensive point-of-care diagnostic tool for M. tuberculosis infection and for rapid DST.

We tested a new method for detecting drug-resistant strains of Mycobacterium tuberculosis that uses a TM4 mycobacteriophage phAE87::hsp60-EGFP (EGFP-phage) engineered to contain the gene encoding enhanced green fluorescent protein (EGFP). After promising results in preliminary studies, the EGFP-phage was used to detect isoniazid (INH), rifampin (RIF), and streptomycin (STR) resistance in 155 strains of M. tuberculosis, and the results were compared to the resazurin microplate technique, with the proportion method serving as the reference standard. The resazurin technique yielded sensitivities of 94% for INH and RIF and 98% for STR and specificities of 97% for INH, 95% for RIF, and 98% for STR. The sensitivity of EGFP-phage was 94% for all three antibiotics, with specificities of 90% for INH, 93% for RIF, and 95% for STR. The EGFP-phage results were available in 2 days for RIF and STR and in 3 days for INH, with an estimated cost of ∼2$ to test the three antibiotics. Using a more stringent criterion for resistance improved the specificity of the EGFP-phage for INH and RIF without affecting the sensitivity. In preliminary studies, the EGFP-phage could also effectively detect resistance to the fluoroquinolones. The EGFP-phage method has the potential to be a valuable rapid and economic screen for detecting drug-resistant tuberculosis if the procedure can be simplified, if it can be adapted to clinical material, and if its sensitivity can be improved. Fil: Rondon Salazar, Liliana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina. Instituto Venezolano de Investigaciones Científicas; Venezuela Fil: Piuri, Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentina. Instituto Venezolano de Investigaciones Científicas; Venezuela Fil: Jacobs Jr., William R.. Instituto Venezolano de Investigaciones Científicas; Venezuela Fil: De Waard, Jacobus. Hospital Vargas; Venezuela Fil: Hatfull, Graham F.. University of Pittsburgh; Estados Unidos Fil: Takiff, Howard Eugene. Instituto Venezolano de Investigaciones Científicas; Venezuela

The pyrazinamide (PZA) analog 5-chloropyrazinamide (5-Cl PZA) is active against mycobacterial species, including PZA-resistant strains of Mycobacterium tuberculosis . In M. smegmatis , overexpression of the type 1 fatty acid synthase (FAS I) confers resistance to 5-Cl PZA, a potent FAS I inhibitor. Since M. tuberculosis and M. bovis cannot tolerate FAS I overexpression, 5-Cl PZA resistance mutations have yet to be described for tubercle bacilli. In an attempt to identify other factors that govern the activity of 5-Cl PZA, we selected for 5-Cl PZA-resistant isolates from a library of transposon-mutagenized M. smegmatis isolates. Here, we report that increased expression of the M. smegmatis pyrazinamidase PzaA confers resistance to 5-Cl PZA and susceptibility to PZA in M. smegmatis , M. tuberculosis , and M. bovis . In contrast, while ectopic overexpression of the M. tuberculosis pyrazinamidase PncA increases PZA susceptibility, this amidase does not mediate resistance to 5-Cl PZA. We conclude that PncA-independent turnover of 5-Cl PZA represents a potential mechanism of resistance to this compound for M. tuberculosis , which will likely translate into enhanced PZA susceptibility. Thus, countersusceptibility can be manipulated as a resistance-proofing strategy for PZA-based compounds when these agents are used simultaneously.

The publication of the complete genome sequence for Mycobacterium tuberculosis H37Rv in 1998 has had a great impact on the research community. Nonetheless, it is suspected that genetic differences have arisen in stocks of H37Rv that are maintained in different laboratories. In order to assess the consistency of the genome sequences among H37Rv strains in use and the extent to which they have diverged from the original strain sequenced, we carried out whole-genome sequencing on six strains of H37Rv from different laboratories. Polymorphisms at 73 sites were observed, which were shared among the lab strains, though 72 of these were also shared with H37Ra and are likely to be due to sequencing errors in the original H37Rv reference sequence. An updated H37Rv genome sequence should be valuable to the tuberculosis research community as well as the broader microbial research community. In addition, several polymorphisms unique to individual strains and several shared polymorphisms were identified and shown to be consistent with the known provenance of these strains. Aside from nucleotide substitutions and insertion/deletions, multiple IS 6110 transposition events were observed, supporting the theory that they play a significant role in plasticity of the M. tuberculosis genome. This genome-wide catalog of genetic differences can help explain any phenotypic differences that might be found, including a frameshift mutation in the mycocerosic acid synthase gene which causes two of the strains to be deficient in biosynthesis of the surface glycolipid phthiocerol dimycocerosate (PDIM). The resequencing of these six lab strains represents a fortuitous “ in vitro evolution” experiment that demonstrates how the M. tuberculosis genome continues to evolve even in a controlled environment.

Persistence ofMycobacterium tuberculosisin humans represents a major roadblock to elimination of tuberculosis. We describe identification of a locus inM. tuberculosis, mel2, that displays similarity to bacterial bioluminescent loci and plays an important role during persistence in mice. We constructed a deletion of themel2locus and found that the mutant displays increased susceptibility to reactive oxygen species (ROS). Upon infection of mice by aerosol the mutant grows normally until the persistent stage, where it does not persist as well as wild type. Histopathological analyses show that infection with themel2mutant results in reduced pathology and both CFU and histopathology indicate that dissemination of themel2mutant to the spleen is delayed. These data along with growth in activated macrophages and infection of Phox−/−and iNOS−/−mice and bone marrow-derived macrophages suggest that the primary mechanism by whichmel2affects pathogenesis is through its ability to confer resistance to ROS. These studies provide the first insight into the mechanism of action for this novel class of genes that are related to bioluminescence genes. The role ofmel2in resistance to ROS is important for persistence and dissemination ofM. tuberculosisand suggests that homologues in other bacterial species are likely to play a role in pathogenesis.

While mycobacteria have been proposed as vaccine vectors because of their persistence and safety, little has been done systematically to optimize their immunogenicity in nonhuman primates. We successfully generated recombinant Mycobacterium bovis BCG (rBCG) expressing simian immunodeficiency virus (SIV) Gag and Pol as multigenic, nonintegrating vectors, but rBCG-expressing SIV Env was unstable. A dose and route determination study in rhesus monkeys revealed that intramuscular administration of rBCG was associated with local reactogenicity, whereas intravenous and intradermal administration of 10 6 to 10 8 CFU of rBCG was well tolerated. After single or repeat rBCG inoculations, monkeys developed high-frequency gamma interferon enzyme-linked immunospot responses against BCG purified protein derivative. However, the same animals developed only modest SIV-specific CD8 + T-cell responses. Nevertheless, high-frequency SIV-specific cellular responses were observed in the rBCG-primed monkeys after boosting with recombinant adenovirus 5 (rAd5) expressing the SIV antigens. These cellular responses were of greater magnitude and more persistent than those generated after vaccination with rAd5 alone. The vaccine-elicited cellular responses were predominantly polyfunctional CD8 + T cells. These findings support the further exploration of mycobacteria as priming vaccine vectors.

Little is known about the immune distribution and localization of antigen-specific T cells in mucosal interfaces of tissues/organs during infection of humans. In this study, we made use of a macaque model ofMycobacterium tuberculosisinfection to assess phosphoantigen-specific Vγ2Vδ2 T cells regarding their tissue distribution, anatomical localization, and correlation with the presence or absence of tuberculosis (TB) lesions in lymphoid and nonlymphoid organs/tissues in the progression of severe pulmonary TB. Progression of pulmonaryM. tuberculosisinfection generated diverse distribution patterns of Vγ2Vδ2 T cells, with remarkable accumulation of these cells in lungs, bronchial lymph nodes, spleens, and remote nonlymphoid organs but not in blood. Increased numbers of Vγ2Vδ2 T cells in tissues were associated withM. tuberculosisinfection but were independent of the severity of TB lesions. In lungs with apparent TB lesions, Vγ2Vδ2 T cells were present within TB granulomas. In extrathoracic organs, Vγ2Vδ2 T cells were localized in the interstitial compartment of nonlymphoid tissues, and the interstitial localization was present despite the absence of detectable TB lesions. Finally, Vγ2Vδ2 T cells accumulated in tissues appeared to possess cytokine production function, since granzyme B was detectable in the γδ T cells present within granulomas. Thus, clonally expanded Vγ2Vδ2 T cells appeared to undergotrans-endothelial migration, interstitial localization, and granuloma infiltration as immune responses toM. tuberculosisinfection.

T helper 17 (Th17)-associated cytokines are integral to the immune responses to tuberculosis, initiating both protective and harmful inflammatory responses. The aim of the present study was to evaluate applied aspects of interleukin-17 (IL-17) biology in the context of Mycobacterium bovis infection of cattle. Using transcriptome sequencing (RNA-Seq), numerous Th17-associated cytokine genes (including IL-17A, IL-17F, IL-22, IL-19, and IL-27) were upregulated >9-fold in response to purified protein derivative stimulation of peripheral blood mononuclear cells from experimentally M. bovis -infected cattle. Protective vaccines elicited IL-17A, IL-17F, IL-22, and IL-27 responses. Reduced IL-17A responses by vaccine recipients, compared to nonvaccinated animals, at 2.5 weeks after M. bovis challenge correlated with reduced disease burdens. Additionally, IL-17A and interferon gamma (IFN-γ) responses were highly correlated and exhibited similar diagnostic capacities. The present findings support the use of Th17-associated cytokines as biomarkers of infection and protection in the immune responses to bovine tuberculosis.

Mycobacteria target and persist within phagocytic monocytes and are strong adjuvants, making them attractive candidate vectors for DNA vaccines. We characterized the ability of mycobacteria to deliver transgenes to mammalian cells and the effects of various bacterial chromosomal mutations on the efficiency of transfer in vivo and in vitro. First, we observed green fluorescent protein expression via microscopy and fluorescence-activated cell sorting analysis after infection of phagocytic and nonphagocytic cell lines byMycobacterium smegmatisorM. bovisBCG harboring a plasmid encoding the fluorescence gene under the control of a eukaryotic promoter. Next, we compared the efficiencies of gene transfer usingM. smegmatisor BCG containing chromosomal insertions or deletions that cause early lysis, hyperconjugation, or an increased plasmid copy number. We observed a significant—albeit only 1.7-fold—increase in the level of plasmid transfer to eukaryotic cells infected withM. smegmatishyperconjugation mutants.M. smegmatisstrains that overexpressed replication proteins (Rep) of pAL5000, a plasmid whose replicon is incorporated in many mycobacterial constructs, generated a 10-fold increase in plasmid copy number and 3.5-fold and 3-fold increases in gene transfer efficiency to HeLa cells and J774 cells, respectively. Although BCG strains overexpressing Rep could not be recovered, BCG harboring a plasmid with a copy-up mutation inoriMresulted in a threefold increase in gene transfer to J774 cells. Moreover,M. smegmatisstrains overexpressing Rep enhanced gene transfer in vivo compared with a wild-type control. Immunization of mice with mycobacteria harboring a plasmid (pgp120hE) encoding human immunodeficiency virus gp120 elicited gp120-specific CD8 T-cell responses among splenocytes and peripheral blood mononuclear cells that were up to twofold (P< 0.05) and threefold (P< 0.001) higher, respectively, in strains supporting higher copy numbers. The magnitude of these responses was approximately one-half of that observed after intramuscular immunization with pgp120hE.M. smegmatisand other nonpathogenic mycobacteria are promising candidate vectors for DNA vaccine delivery.

Phosphate import is required for the growth of mycobacteria and is regulated by environmental inorganic phosphate (P i ) concentrations, although the mechanism of this regulation has not been characterized. The expression of genes involved in P i acquisition is frequently regulated by two-component regulatory systems (2CRs) consisting of a sensor histidine kinase and a DNA-binding response regulator. In this work, we have identified the senX3-regX3 2CR as a P i -dependent regulator of genes involved in phosphate acquisition in Mycobacterium smegmatis . Characterization of senX3 mutants with different PhoA phenotypes suggests a dual role for SenX3 as a phosphatase or a phosphodonor for the response regulator RegX3, depending upon P i availability. Expression of PhoA activity required phosphorylation of RegX3, consistent with a role for phosphorylated RegX3 (RegX3∼P) as a transcriptional activator of phoA . Furthermore, purified RegX3∼P bound to promoter sequences from phoA , senX3 , and the high-affinity phosphate transporter component pstS , demonstrating direct transcriptional control of all three genes. DNase I footprinting and primer extension analyses have further defined the DNA-binding region and transcriptional start site within the phoA promoter. A DNA motif consisting of an inverted repeat was identified in each of the promoters bound by RegX3∼P. Based upon our findings, we propose a model for P i -regulated gene expression mediated by SenX3-RegX3 in mycobacteria.

A successful vaccine vector for human immunodeficiency virus type 1 (HIV-1) should induce anti-HIV-1 T-cell immune responses at mucosal sites. We have constructed recombinant Mycobacterium bovis bacillus Calmette-Guérin (rBCG) expressing an HIV-1 group M consensus envelope (Env) either as a surface, intracellular, or secreted protein as an immunogen. rBCG containing HIV-1 env plasmids engineered for secretion induced optimal Env-specific T-cell gamma interferon enzyme-linked immunospot responses in murine spleen, female reproductive tract, and lungs. While rBCG-induced T-cell responses to HIV-1 envelope in spleen were lower than those induced by adenovirus prime/recombinant vaccinia virus (rAd-rVV) boost, rBCG induced comparable responses to rAd-rVV immunization in the female reproductive tract and lungs. T-cell responses induced by rBCG were primarily CD4 + , although rBCG alone did not induce anti-HIV-1 antibody. However, rBCG could prime for a protein boost by HIV-1 envelope protein. Thus, rBCG can serve as a vector for induction of anti-HIV-1 consensus Env cellular responses at mucosal sites.

A successful vaccine vector for human immunodeficiency virus type 1 (HIV-1) should induce anti-HIV-1 immune responses at mucosal sites. We have generated recombinant Mycobacterium smegmatis vectors that express the HIV-1 group M consensus envelope protein (Env) as a surface, intracellular, or secreted protein and have tested them in animals for induction of both anti-HIV-1 T-cell and antibody responses. Recombinant M. smegmatis engineered for expression of secreted protein induced optimal T-cell gamma interferon enzyme-linked immunospot assay responses to HIV-1 envelope in the spleen, female reproductive tract, and lungs. Unlike with the induction of T-cell responses, priming and boosting with recombinant M. smegmatis did not induce anti-HIV-1 envelope antibody responses, due primarily to insufficient protein expression of the insert. However, immunization with recombinant M. smegmatis expressing HIV-1 Env was able to prime for an HIV-1 Env protein boost for the induction of anti-HIV-1 antibody responses.

We generated four individual glutamine synthetase (GS) mutants (Δ glnA1 , Δ glnA2 , Δ glnA3 , and Δ glnA4 ) and one triple mutant (Δ glnA1EA2 ) of Mycobacterium tuberculosis to investigate the roles of GS enzymes. Subcutaneous immunization with the Δ glnA1EA2 and Δ glnA1 glutamine auxotrophic mutants conferred protection on C57BL/6 mice against an aerosol challenge with virulent M. tuberculosis , which was comparable to that provided by Mycobacterium bovis BCG vaccination.

Approximately one-third of the human population is latently infected withMycobacterium tuberculosis, comprising a critical reservoir for disease reactivation. Despite the importance of latency in maintainingM. tuberculosisin the human population, little is known about the mycobacterial factors that regulate persistence and reactivation. Previous in vitro studies have implicated a family of five relatedM. tuberculosisproteins, called resuscitation promoting factors (Rpfs), in regulating mycobacterial growth. We studied the in vivo role ofM. tuberculosis rpfgenes in an established mouse model ofM. tuberculosispersistence and reactivation. After an aerosol infection with theM. tuberculosisErdman wild type (Erdman) or single-deletionrpfmutants to establish chronic infections in mice, reactivation was induced by administration of the nitric oxide (NO) synthase inhibitor aminoguanidine. Of the fiverpfdeletion mutants tested, one (ΔRv1009) exhibited a delayed reactivation phenotype, manifested by delayed postreactivation growth kinetics and prolonged median survival times among infected animals. Immunophenotypic analysis suggested differences in pulmonary B-cell responses between Erdman- and ΔRv1009-infected mice at advanced stages of reactivation. Analysis ofrpfgene expression in the lungs of Erdman-infected mice revealed that relative expression of four of the fiverpf-like genes was diminished at late times following reactivation, when bacterial numbers had increased substantially, suggesting thatrpfgene expression may be regulated in a growth phase-dependent manner. To our knowledge, ΔRv1009 is the firstM. tuberculosismutant to have a specific defect in reactivation without accompanying growth defects in vitro or during acute infection in vivo.

Arabinomannan (AM) is a polysaccharide of the mycobacterial capsule. The capsular polysaccharides of various microorganisms are diverse, and this diversity is important for classification of organisms into serotypes and vaccine development. In the present study we examined the prevalence and diversity of AM amongMycobacterium tuberculosisstrains using four AM-binding monoclonal antibodies (MAbs). One of these MAbs, MAb 9d8, is known to bind to AM specifically. By whole-cell enzyme-linked immunosorbent assay (ELISA), the AM recognized by MAb 9d8 was detected on the surfaces of 9 of 11 strains, while 2 strains showed no reactivity with MAb 9d8. However, the AM recognized by MAb 9d8 was found in the culture supernatants of all 11M. tuberculosisstrains tested, as demonstrated by capture ELISA. Other AM-binding MAbs reacted both with the surfaces and with the culture supernatants of all 11 strains. Mice immunized with an experimental AM-recombinantPseudomonas aeruginosaexoprotein A (rEPA) conjugate vaccine had an increased antibody response to AM and a moderate reduction in the numbers of CFU in their organs 7 days after challenge. Our results indicate that AM was detected in allM. tuberculosisstrains tested, with differences in epitope distributions of certain strains. In addition, our results suggest that an experimental AM-rEPA vaccine has a moderate effect on the numbers of CFU in organs early after infection.

We developed a live, fully attenuated Mycobacterium tuberculosis vaccine candidate strain with two independent attenuating auxotrophic mutations in leucine and pantothenate biosynthesis. The Δ leuD Δ panCD double auxotroph is fully attenuated in the SCID mouse model and highly immunogenic and protective in the extremely sensitive guinea pig tuberculosis model, reducing both bacterial burden and disease pathology.

Mycobacterium tuberculosispossesses five genes with significant homology to the resuscitation-promoting factor (Rpf) ofMicrococcus luteus. TheM. luteusRpf is a secreted ∼16-kDa protein which restores active growth to cultures ofM. luteusrendered dormant by prolonged incubation in stationary phase. More recently, the Rpf-like proteins ofM. tuberculosishave been shown to stimulate the growth of extended-stationary-phase cultures ofMycobacterium bovisBCG. These data suggest that the Rpf proteins can influence the growth of mycobacteria; however, the studies do not demonstrate specific functions for the various members of this protein family, nor do they assess the function ofM. tuberculosisRpf homologues in vivo. To address these questions, we have disrupted each of the fiverpf-like genes inM. tuberculosisErdman, and analyzed the mutants for their growth in vitro and in vivo. In contrast toM. luteus, for whichrpfis an essential gene, we find that all of theM. tuberculosis rpfdeletion mutant strains are viable; in addition, all show growth kinetics similar to Erdman wild type both in vitro and in mouse organs following aerosol infection. Analysis ofrpfexpression inM. tuberculosiscultures from early log phase through late stationary phase indicates that expression of therpf-like genes is growth phase-dependent, and that the expression patterns of the fiveM. tuberculosis rpfgenes, while overlapping to various degrees, are not uniform. We also provide evidence that mycobacterialrpfgenes are expressed in vivo in the lungs of mice acutely infected with virulentM. tuberculosis.

Mycobacterium tuberculosis and Mycobacterium avium are pathogenic slow-growing mycobacteria that cause distinct human diseases. In contrast to recent advances in M. tuberculosis genetics and pathogenesis investigation, M. avium has remained genetically intractable and, consequently, its pathogenic strategies remain poorly understood. Here we report the successful development of efficient allelic exchange and transposon mutagenesis in an opaque clinical strain of M. avium by specialized transduction. Efforts to disrupt the leuD gene of M. avium by specialized transduction were successful but were complicated by inefficient isolation of recombinants secondary to high spontaneous antibiotic resistance. However, by using this leucine auxotroph as a genetic host and the Streptomyces coelicolor leuD gene as a selectable marker, we achieved efficient allelic exchange at the M. avium pcaA locus. A leuD -marked transposon delivered by specialized transduction mutagenized M. avium with efficiencies similar to M. tuberculosis . These results establish a system for random and directed mutagenesis of M. avium . In combination with the forthcoming M. avium genome sequence, these tools will allow the distinct physiologic and pathogenic properties of M. avium to be dissected in molecular detail.

Pathogenic and nonpathogenic species of bacteria and fungi release membrane vesicles (MV), containing proteins, polysaccharides, and lipids, into the extracellular milieu. Previously, we demonstrated that several mycobacterial species, including bacillus Calmette-Guerin (BCG) and Mycobacterium tuberculosis, release MV containing lipids and proteins that subvert host immune response in a Toll-like receptor 2 (TLR2)-dependent manner (R. Prados-Rosales et al., J. Clin. Invest. 121:1471–1483, 2011, doi:10.1172/JCI44261). In this work, we analyzed the vaccine potential of MV in a mouse model and compared the effects of immunization with MV to those of standard BCG vaccination. Immunization with MV from BCG or M. tuberculosis elicited a mixed humoral and cellular response directed to both membrane and cell wall components, such as lipoproteins. However, only vaccination with M. tuberculosis MV was able to protect as well as live BCG immunization. M. tuberculosis MV boosted BCG vaccine efficacy. In summary, MV are highly immunogenic without adjuvants and elicit immune responses comparable to those achieved with BCG in protection against M. tuberculosis., IMPORTANCE This work offers a new vaccine approach against tuberculosis using mycobacterial MV. Mycobacterium MV are a naturally released product combining immunogenic antigens in the context of a lipid structure. The fact that MV do not need adjuvants and elicit protection comparable to that elicited by the BCG vaccine encourages vaccine approaches that combine protein antigens and lipids. Consequently, mycobacterium MV establish a new type of vaccine formulation.

Live attenuated nonpathogenic Mycobacterium bovis bacillus Calmette-Guérin (BCG) mediates long-lasting immune responses, has been safely administered as a tuberculosis vaccine to billions of humans, and is affordable to produce as a vaccine vector. These characteristics make it very attractive as a human immunodeficiency virus (HIV) vaccine vector candidate. Here, we assessed the immunogenicity of recombinant BCG (rBCG) constructs with different simian immunodeficiency virus (SIV) gag expression cassettes as priming agents followed by a recombinant replication-incompetent New York vaccinia virus (NYVAC) boost in rhesus macaques. Unmutated rBCG constructs were used in comparison to mutants with gene deletions identified in an in vitro screen for augmented immunogenicity. We demonstrated that BCG-SIV gag is able to elicit robust transgene-specific priming responses, resulting in strong SIV epitope-specific cellular immune responses. While enhanced immunogenicity was sustained at moderate levels for >1 year following the heterologous boost vaccination, we were unable to demonstrate a protective effect after repeated rectal mucosal challenges with pathogenic SIVmac251. Our findings highlight the potential for rBCG vaccines to stimulate effective cross-priming and enhanced major histocompatibility complex class I presentation, suggesting that combining this approach with other immunogens may contribute to the development of effective vaccine regimens against HIV.

The new medicinal compound bedaquiline (BDQ) kills Mycobacterium tuberculosis by inhibiting F1Fo-ATP synthase. BDQ is bacteriostatic for 4 to 7 days and kills relatively slowly compared to other frontline tuberculosis (TB) drugs. Here we show that killing with BDQ can be improved significantly by inhibiting cytochrome bd oxidase, a non-proton-pumping terminal oxidase. BDQ was instantly bactericidal against a cytochrome bd oxidase null mutant of M. tuberculosis, and the rate of killing was increased by more than 50%. We propose that this exclusively bacterial enzyme should be a high-priority target for new drug discovery., IMPORTANCE A major drawback of current TB chemotherapy is its long duration. New drug regimens with rapid killing kinetics are desperately needed. Our study demonstrates that inhibition of a nonessential bacterial enzyme greatly improves the efficacy of the latest TB drug bedaquiline and emphasizes that screening for compounds with synergistic killing mechanisms is a promising strategy.

Genetic engineering has contributed greatly to our understanding of Mycobacterium tuberculosis biology and has facilitated antimycobacterial and vaccine development. However, methods to generate M. tuberculosis deletion mutants remain labor-intensive and relatively inefficient. Here, methods are described that significantly enhance the efficiency (greater than 100-fold) of recovering deletion mutants by the expression of mycobacteriophage recombineering functions during the course of infection with specialized transducing phages delivering allelic exchange substrates. This system has been successfully applied to the CDC1551 strain of M. tuberculosis, as well as to a ΔrecD mutant generated in the CDC1551 parental strain. The latter studies were undertaken as there were precedents in both the Escherichia coli literature and mycobacterial literature for enhancement of homologous recombination in strains lacking RecD. In combination, these measures yielded a dramatic increase in the recovery of deletion mutants and are expected to facilitate construction of a comprehensive library of mutants with every nonessential gene of M. tuberculosis deleted. The findings also open up the potential for sophisticated genetic screens, such as synthetic lethal analyses, which have so far not been feasible for the slow-growing mycobacteria., IMPORTANCE Genetic manipulation of M. tuberculosis is hampered by laborious and relatively inefficient methods for generating deletion mutant strains. The combined use of phage-based transduction and recombineering methods greatly enhances the efficiency by which knockout strains can be generated. The additional elimination of recD further enhances this efficiency. The methods described herein will facilitate the construction of comprehensive gene knockout libraries and expedite the isolation of previously difficult to recover mutants, promoting antimicrobial and vaccine development.

Infectious diseases have plagued humankind throughout history and have posed serious public health problems. Yet vaccines have eradicated smallpox and antibiotics have drastically decreased the mortality rate of many infectious agents. These remarkable successes in the control of infections came from knowing the causative agents of the diseases, followed by serendipitous discoveries of attenuated viruses and antibiotics. The discovery of DNA as genetic material and the understanding of how this information translates into specific phenotypes have changed the paradigm for developing new vaccines, drugs, and diagnostic tests. Knowledge of the mechanisms of immunity and mechanisms of action of drugs has led to new vaccines and new antimicrobial agents. The key to the acquisition of the knowledge of these mechanisms has been identifying the elemental causes (i.e., genes and their products) that mediate immunity and drug resistance. The identification of these genes is made possible by being able to transfer the genes or mutated forms of the genes into causative agents or surrogate hosts. Such an approach was limited in Mycobacterium tuberculosis by the difficulty of transferring genes or alleles into M. tuberculosis or a suitable surrogate mycobacterial host. The construction of shuttle phasmids—chimeric molecules that replicate in Escherichia coli as plasmids and in mycobacteria as mycobacteriophages—was instrumental in developing gene transfer systems for M. tuberculosis. This review will discuss M. tuberculosis genetic systems and their impact on tuberculosis research.

The utility of luciferase reporter mycobacteriophages (LRPs) for detection, identification, and antibiotic susceptibility testing of Mycobacterium tuberculosis was prospectively evaluated in a clinical microbiology laboratory in Mexico City, Mexico. Five hundred twenty-three consecutive sputum samples submitted to the laboratory during a 5-month period were included in this study. These specimens were cultivated in Middlebrook 7H9 (MADC), MGIT, and Löwenstein-Jensen (LJ) media. Of the 71 mycobacterial isolates recovered with any of the three media, 76% were detected with the LRPs, 97% were detected with the MGIT 960 method, and 90% were detected with LJ medium. When contaminated specimens were excluded from the analysis, the LRPs detected 92% (54 of 59) of the cultures. The median time to detection of bacteria was 7 days with both the LRPs and the MGIT 960 method. LRP detection of growth in the presence of p -nitro-α-acetylamino-β-hydroxypropiophenone (NAP) was used for selective identification of M. tuberculosis complex (MTC) and compared to identification with BACTEC 460. Using the LRP NAP test, 47 (94%) out of 50 isolates were correctly identified as tuberculosis complex. The accuracy and speed of LRP antibiotic susceptibility testing with rifampin, streptomycin, isoniazid, and ethambutol were compared to those of the BACTEC 460 method, and discrepant results were checked by the conventional proportion method. In total, 50 MTC isolates were tested. The overall agreement between the LRP and BACTEC 460 results was 98.5%. The median LRP-based susceptibility turnaround time was 2 days (range, 2 to 4 days) compared to 10.5 days (range, 7 to 16 days) by the BACTEC 460 method. Phage resistance was not detected in any of the 243 MTC isolates tested. Mycobacteriophage-based approaches to tuberculosis diagnostics can be implemented in clinical laboratories with sensitivity, specificity, and rapidity that compare favorably with those of the MGIT 960 and BACTEC 460 methods. The phages currently provide the fastest phenotypic assay for susceptibility testing.

Mycobacterium tuberculosisis a specialized intracellular pathogen that must regulate gene expression to overcome stresses produced by host defenses during infection. SigH is an alternative sigma factor that we have previously shown plays a role in the response to stress of the saprophyteMycobacterium smegmatis. In this work we investigated the role ofsigHin theM.tuberculosisresponse to heat and oxidative stress. We determined that aM.tuberculosis sigHmutant is more susceptible to oxidative stresses and that the inducible expression of the thioredoxin reductase/thioredoxin genestrxB2/trxCand a gene of unknown function, Rv2466c, is regulated bysigHvia expression from promoters directly recognized by SigH. We also determined that thesigHmutant is more susceptible to heat stress and that inducible expression of the heat shock genesdnaKandclpBis positively regulated bysigH. The induction of these heat shock gene promoters but not of other SigH-dependent promoters was markedly greater in response to heat versus oxidative stress, consistent with their additional regulation by a heat-labile repressor. To further understand the role ofsigHin theM.tuberculosisstress response, we investigated the regulation of the stress-responsive sigma factor genessigEandsigB. We determined that inducible expression ofsigEis regulated bysigHand that basal and inducible expression ofsigBis dependent onsigEandsigH. These data indicate thatsigHplays a central role in a network that regulates heat and oxidative-stress responses that are likely to be important inM.tuberculosispathogenesis.

Tuberculosis remains one of the most significant diseases of humans and animals. The only currently available vaccine against this disease is a live, attenuated vaccine, bacillus Calmette-Guérin (BCG), which was originally derived from Mycobacterium bovis and despite its variable efficacy is the most widely administered vaccine in the world. With the advent of the human immunodeficiency virus-AIDS pandemic concern has been raised over the safety of BCG. Moreover, since BCG sensitizes vaccinated individuals to the tuberculin test, vaccination with BCG prevents diagnosis of infection in vaccinated individuals. Recently, auxotrophic strains of BCG have been generated by insertional mutagenesis which have been shown to be safer than the parent BCG strain following administration to mice with severe combined immunodeficiency disease. These strains have also been shown to give comparable protection against intravenous and intratracheal challenge of BALB/c mice with M. tuberculosis relative to conventional BCG. Here we report that one of these mutants, a leucine auxotroph of BCG, conferred significant protection of the lungs and spleens of guinea pigs infected with M. bovis and protection of the spleens of guinea pigs infected with M. tuberculosis in the absence of a cutaneous hypersensitivity reaction to tuberculin. Therefore, protective immunity to tuberculosis may, at least in part, be achieved without sensitization to the tuberculin skin test. These results indicate that it may be possible to develop a new generation of vaccines based on BCG that are protective, are safe for use in the immunocompromised, and do not preclude the use of the tuberculin skin test in both humans and animals.

Mycobacterium tuberculosis continues to pose a serious health threat to people throughout the world. With an estimated 2.9 million people having died from tuberculosis in 1997, M. tuberculosis remains the infectious agent responsible for the most deaths worldwide (64). In order to develop new drugs, treatments, and vaccines for tuberculosis, a better understanding of M. tuberculosis pathogenesis and M. tuberculosis antigens that elicit a protective immune response is required. Secreted and cell envelope-associated proteins are likely to play a critical role in M. tuberculosis disease. Research on several bacterial pathogens has revealed that the majority of virulence factors are secreted (22), and it was recently shown that the secreted ERP protein contributes to the virulence of M. tuberculosis (6). A key component of M. tuberculosis infection is the ability of the bacillus to survive within phagocytic cells. Since secreted and surface proteins are ideally positioned to interact with the host, they could facilitate this survival by influencing phagosome maturation, by enabling access to the cytoplasm, or by countering the antimicrobial attacks of the phagocyte. Secreted proteins of M. tuberculosis also play an important role in the generation of a protective immune response. The most striking demonstration of this property comes from experiments in which mice or guinea pigs were immunized with extracellular proteins and significant protective immunity ensued (2, 32, 33, 45, 51). It has been reported that M. tuberculosis secretes an extensive number of proteins, and there are even more proteins that must be cell surface associated (3, 50, 56). Many of these proteins have not yet been identified and even fewer have been tested for a role in virulence and protective immunity. PhoA (Escherichia coli alkaline phosphatase) protein fusions have been used in many different organisms to identify exported proteins, including ones that are important to bacterial virulence (7, 17, 26, 36, 49). Importantly, PhoA has been shown to function as a reporter for secreted proteins in M. smegmatis (60). Moreover, when a multicopy plasmid library of phoA fusions to M. tuberculosis genomic DNA was screened in M. smegmatis, four active PhoA fusions were identified, one of which involved the ERP protein (see above) (40). Typically, phoA fusions are made either by using in vivo transposition with transposons such as TnphoA (a derivative of Tn5) or by cloning genomic DNA upstream of a truncated ′phoA gene (43). The ′phoA gene in both of these cases lacks signals for expression and export. Since PhoA is active only when it is located outside of the cytosol, enzymatically active PhoA fusions identify proteins that have export signals. A simple and efficient in vitro transposition system for Tn552, a transposon first identified in Staphylococcus aureus (52), has recently been developed (25, 39). An extensive analysis of transposon insertion sites produced by this in vitro system demonstrated that the transposon inserts randomly within different target DNAs, including mycobacterial DNA (25). Thus, it appears to be ideal for constructing large complex libraries of gene fusions. We set out to use this system to create a random library of phoA fusions in cosmids containing M. tuberculosis genomic DNA to identify genes encoding exported proteins. We constructed Tn552′phoA transposons and inserted them into cosmids containing M. tuberculosis DNA by using in vitro transposition reactions. The resulting population of transposon-containing cosmids was integrated into the M. smegmatis genome in single copy, and colonies were screened for active phoA fusions. To our knowledge, this is the first report of an in vitro transposition system for producing phoA fusions. It provides a simple alternative to constructing libraries by the standard methods. Initial screening of our M. tuberculosis DNA-′phoA fusion libraries identified 31 secreted and membrane proteins. An additional feature of this system is that cosmids containing a transposon insertion can later be used as a substrate for allele exchange in M. tuberculosis. This should enable a relatively rapid transition from the identification of an exported protein to the construction of the corresponding M. tuberculosis mutant and evaluation of its role in pathogenesis and protective immunity.

Attenuated mutants ofMycobacterium tuberculosisrepresent potential vaccine candidates for the prevention of tuberculosis. It is known that auxotrophs of a variety of bacteria are attenuated in vivo and yet provide protection against challenge with wild-type organisms. A leucine auxotroph ofM. tuberculosiswas created by allelic exchange, replacing wild-typeleuD(Rv2987c), encoding isopropyl malate isomerase, with a mutant copy of the gene in which 359 bp had been deleted, creating a strain requiring exogenous leucine supplementation for growth in vitro. The frequency of reversion to prototrophy was −11. In contrast to wild-typeM. tuberculosis, the ΔleuDmutant was unable to replicate in macrophages in vitro. Its attenuation in vivo and safety as a vaccine were established by the fact that it caused no deaths in immunodeficient SCID mice. Complementation of the mutant with wild-typeleuDabolished the requirement for leucine supplementation and restored the ability of the strain to grow both in macrophages and in SCID mice, thus confirming that the attenuated phenotype was due to the ΔleuDmutation. As a test of the vaccine potential of the leucine auxotroph, immunocompetent BALB/c mice, susceptible to fatal infection with wild-typeM. tuberculosis, were immunized with the ΔleuDmutant and subsequently challenged with virulentM. tuberculosisby both the intravenous and aerosol routes. A comparison group of mice was immunized with conventionalMycobacterium bovisBCG vaccine. Whereas all unvaccinated mice succumbed to intravenous infection within 15 weeks, mice immunized with either BCG or the ΔleuDmutant ofM. tuberculosisexhibited enhanced and statistically equivalent survival curves. However, theleuDauxotroph was less effective than live BCG in reducing organ burdens and tissue pathology of mice challenged by either route. We conclude that attenuation and protection againstM. tuberculosischallenge can be achieved with a leucine auxotroph and suggest that to induce optimal protection, attenuated strains ofM. tuberculosisshould persist long enough and be sufficiently metabolically active to synthesize relevant antigens for an extended period of time.