Safety and Immunogenicity of the ID93 + GLA-SE Tuberculosis Vaccine in BCG-Vaccinated Healthy Adults: A Randomized, Double-Blind, Placebo-Controlled Phase 2 Trial.

INTRODUCTION: This randomized, double-blind, placebo-controlled, phase 2a trial was conducted to evaluate the safety and immunogenicity of the ID93 + glucopyranosyl lipid adjuvant (GLA)-stable emulsion (SE) vaccine in human immunodeficiency virus (HIV)-negative, previously Bacillus Calmette-Guérin (BCG)-vaccinated, and QuantiFERON-TB-negative healthy adults in South Korea. METHODS: Adults (n = 107) with no signs or symptoms of tuberculosis were randomly assigned to receive three intramuscular injections of 2 µg ID93 + 5 µg GLA-SE, 10 µg ID93 + 5 µg GLA-SE, or 0.9% normal saline placebo on days 0, 28, and 56. For safety assessment, data on solicited adverse events (AEs), unsolicited AEs, serious AEs (SAEs), and special interest AEs were collected. Antigen-specific antibody responses were measured using serum enzyme-linked immunosorbent assay. T-cell immune responses were measured using enzyme-linked immunospot and intracellular cytokine staining. RESULTS: No SAEs, deaths, or AEs leading to treatment discontinuation were found. The solicited local and systemic AEs observed were consistent with those previously reported. Compared with adults administered with the placebo, those administered with three intramuscular vaccine injections exhibited significantly higher antigen-specific antibody levels and Type 1 T-helper cellular immune responses. CONCLUSION: The ID93 + GLA-SE vaccine induced antigen-specific cellular and humoral immune responses, with an acceptable safety profile in previously healthy, BCG-vaccinated, Mycobacterium tuberculosis-uninfected adult healthcare workers. TRIAL REGISTRATION: This clinical trial was retrospectively registered on 16 January 2019 at Clinicaltrials.gov (NCT03806686).

A Rapid Assessing Method of Drug Susceptibility Using Flow Cytometry for Mycobacterium tuberculosis Isolates Resistant to Isoniazid, Rifampin, and Ethambutol.

BACKGROUND: The current conventional drug susceptibility test (DST) for Mycobacterium tuberculosis (Mtb) takes several weeks of incubation to obtain results. As a rapid method, molecular DST requires only a few days to get the results but does not fully cover the phenotypic resistance. A new rapid method based on the ability of viable Mtb bacilli to hydrolyze fluorescein diacetate to free fluorescein with detection of fluorescent mycobacteria by flow cytometric analysis, was recently developed. METHODS: To evaluate this cytometric method, we tested 39 clinical isolates which were susceptible or resistant to isoniazid (INH) or rifampin (RIF), or ethambutol (EMB) by phenotypic or molecular DST methods and compared the results. RESULTS: The susceptibility was determined by measuring the viability rate of Mtb and all the isolates which were tested with INH, RIF, and EMB showed susceptibility results concordant with those by the phenotypic solid and liquid media methods. The isolates having no mutations in the molecular DST but resistance in the conventional phenotypic DST were also resistant in this cytometric method. These results suggest that the flow cytometric DST method is faster than conventional agar phenotypic DST and may complement the results of molecular DST. CONCLUSION: In conclusion, the cytometric method could provide quick and more accurate information that would help clinicians to choose more effective drugs.

Identification of MicroRNAs as Potential Blood-Based Biomarkers for Diagnosis and Therapeutic Monitoring of Active Tuberculosis.

Early diagnosis increases the treatment success rate for active tuberculosis (ATB) and decreases mortality. MicroRNAs (miRNAs) have been studied as blood-based markers of several infectious diseases. We performed miRNA profiling to identify differentially expressed (DE) miRNAs using whole blood samples from 10 healthy controls (HCs), 15 subjects with latent tuberculosis infection (LTBI), and 12 patients with ATB, and investigated the expression of the top six miRNAs at diagnosis and over the treatment period in addition to performing miRNA-target gene network and gene ontology analyses. miRNA profiling identified 84 DE miRNAs in patients with ATB, including 80 upregulated and four downregulated miRNAs. Receiver operating characteristic curves of the top six miRNAs exhibited excellent distinguishing efficiency with an area under curve (AUC) value > 0.85. Among them, miR-199a-3p and miR-6886-3p can differentiate between ATB and LTBI. Anti-TB treatment restored the levels of miR-199b-3p, miR-199a-3p, miR-16-5p, and miR-374c-5p to HC levels. Furthermore, 108 predicted target genes were related to the regulation of cellular amide metabolism, intrinsic apoptotic signaling, translation, transforming growth factor beta receptor signaling, and cysteine-type endopeptidase activity. The DE miRNAs identified herein are potential biomarkers for diagnosis and therapeutic monitoring in ATB.

Development of a bivalent conjugate vaccine candidate against rotaviral diarrhea and tuberculosis using polysaccharide from Mycobacterium tuberculosis conjugated to ΔVP8* protein from rotavirus.

Conjugation of carbohydrate antigens with a carrier protein is a clinically proven strategy to overcome the poor immunogenicity of bacterial polysaccharide. In addition to its primary role, which is to help generate a T cell-mediate long-lasting immune response directed against the carbohydrate antigen, the carrier protein in a glycoconjugate vaccine can also play an important role as a protective antigen. Among carrier proteins currently used in licensed conjugate vaccines, non-typeable Haemophilus influenzae protein D has been used as an antigenically active carrier protein. Our previous studies also indicate that some carrier proteins provide B cell epitopes, along with T cell helper epitopes. Herein we investigated the dual role of truncated rotavirus spike protein ΔVP8* as a carrier and a protective antigen. Capsular polysaccharide lipoarabinomannan (LAM), purified from Mycobacterium tuberculosis (M.tb), was chemically conjugated with ΔVP8*. Mouse immunization experiments showed that the resultant conjugates elicited strong and specific immune responses against the polysaccharide antigen, and the responses were comparable to those induced by Diphtheria toxoid (DT)-based conjugates. The conjugate vaccine induced enhanced antibody titers and functional antibodies against ΔVP8* when compared to immunization with the unconjugated ΔVP8*. Thus, these results indicate that ΔVP8* can be a relevant carrier protein for glycoconjugate vaccine and the glycoconjugates consisting of ΔVP8* with LAM are effective bivalent vaccine candidates against rotavirus and tuberculosis.

Characterization of Drug-Resistant Lipid-Dependent Differentially Detectable Mycobacterium tuberculosis.

An estimated 15-20% of patients who are treated for pulmonary tuberculosis (TB) are culture-negative at the time of diagnosis. Recent work has focused on the existence of differentially detectable Mycobacterium tuberculosis (Mtb) bacilli that do not grow under routine solid culture conditions without the addition of supplementary stimuli. We identified a cohort of TB patients in Lima, Peru, in whom acid-fast bacilli could be detected by sputum smear microscopy, but from whom Mtb could not be grown in standard solid culture media. When we attempted to re-grow Mtb from the frozen sputum samples of these patients, we found that 10 out of 15 could be grown in a glycerol-poor/lipid-rich medium. These fell into the following two groups: a subset that could be regrown in glycerol after "lipid-resuscitation", and a group that displayed a heritable glycerol-sensitive phenotype that were unable to grow in the presence of this carbon source. Notably, all of the glycerol-sensitive strains were found to be multidrug resistant. Although whole-genome sequencing of the lipid-resuscitated strains identified 20 unique mutations compared to closely related strains, no single genetic lesion could be associated with this phenotype. In summary, we found that lipid-based media effectively fostered the growth of Mtb from a series of sputum smear-positive samples that were not culturable in glycerol-based Lowenstein-Jensen or 7H9 media, which is consistent with Mtb's known preference for non-glycolytic sources during infection. Analysis of the recovered strains demonstrated that both genetic and non-genetic mechanisms contribute to the observed differential capturability, and suggested that this phenotype may be associated with drug resistance.

Screening for Mycobacterium tuberculosis Infection Using Beijing/K Strain-Specific Peptides in a School Outbreak Cohort.

Background: The Beijing strain of Mycobacterium tuberculosis (M. tb) has been most frequently isolated from TB patients in South Korea, and the hyper-virulent Beijing/K genotype is associated with TB outbreaks. To examine the diagnostic potential of Beijing/K-specific peptides, we performed IFN-γ release assays (IGRA) using a MTBK antigen tube containing Beijing/K MTBK_24800, ESAT-6, and CFP-10 peptides in a cohort studied during a school TB outbreak. Methods: A total of 758 contacts were investigated for M. tb infection, and 43 contacts with latent TB infection (LTBI) and 25 active TB patients were enrolled based on serial screening with QuantiFERON-TB Gold In-Tube tests followed by clinical examinations. Blood collected in MTBK antigen tubes was utilized for IGRA and multiplex cytokine bead arrays. Immune responses were retested in 24 patients after TB treatment, and disease progression was investigated in subjects with LTBI. Results: Total proportions of active disease and LTBI during the outbreak were 3.7% (28/758) and 9.2% (70/758), respectively. All clinical isolates had a Beijing/K M. tb genotype. IFN-γ responses to the MTBK antigen identified M. tb infection and distinguished between active disease and LTBI. After anti-TB treatment, IFN-γ responses to the MTBK antigen were significantly reduced, and strong TNF-α responses at diagnosis were dramatically decreased. Conclusions: MTBK antigen-specific IFN-γ has diagnostic potential for differentiating M. tb infection from healthy controls, and between active TB and LTBI as well. In addition, TNF-α is a promising marker for monitoring therapeutic responses. These data provide informative readouts for TB diagnostics and vaccine studies in regions where the Beijing/K strain is endemic.

Identification of serum biomarkers for active pulmonary tuberculosis using a targeted metabolomics approach.

Although tuberculosis (TB) is a severe health problem worldwide, the current diagnostic methods are far from optimal. Metabolomics is increasingly being used in the study of infectious diseases. We performed metabolome profiling to identify potential biomarkers in patients with active TB. Serum samples from 21 patients with active pulmonary TB, 20 subjects with latent TB infection (LTBI), and 28 healthy controls were analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS) followed by multivariate and univariate analyses. Metabolic profiles indicated higher serum levels of glutamate, sulfoxy methionine, and aspartate and lower serum levels of glutamine, methionine, and asparagine in active TB patients than in LTBI subjects or healthy controls. The ratios between metabolically related partners (glutamate/glutamine, sulfoxy methionine/methionine, and aspartate/asparagine) were also elevated in the active TB group. There was no significant difference in the serum concentration of these metabolites according to the disease extent or risk of relapse in active TB patients. Novel serum biomarkers such as glutamate, sulfoxy methionine, aspartate, glutamine, methionine, and asparagine are potentially useful for adjunctive, rapid, and noninvasive pulmonary TB diagnosis.

Does the Effectiveness and Mechanical Strength of Kanamycin-Loaded Bone Cement in Musculoskeletal Tuberculosis Compare to Vancomycin-Loaded Bone Cement.

BACKGROUND: Antibiotic-loaded bone cement (ALBC) is used to deliver antimycobacterial agents into the focal lesion of musculoskeletal tuberculosis. Although kanamycin is currently used as an antimycobacterial agent for the treatment of multidrug-resistant tuberculosis, there is no information about its suitability in ALBC. METHODS: An in vitro experiment was conducted with cylindrical shape of 40 g of bone cement with 1, 2, and 3 g of kanamycin. Eluate (1 mL) was extracted from each specimen to measure the level of elution and antimycobacterial activity on days 1, 4, 7, 14, and 30. The quantity of kanamycin in eluates was evaluated by a liquid chromatography-mass spectrometry system, and the antimycobacterial activity of eluates against Mycobacterium tuberculosis H37Rv was calculated by comparing the minimal inhibitory concentration. The ultimate compression strength was conducted using a material testing system machine (Instron 3366; Instron, Norwood, MA) before and after elution. RESULTS: Eluates from ALBC containing 2 and 3 g of kanamycin had effective antimycobacterial activity for 30 days, whereas eluates from ALBC containing 1 g of kanamycin were partially active until day 30. The pre-eluted compression strength of kanamycin-loaded cement and vancomycin-loaded cement was weaker as they contained a larger amount of antibiotics. There was no statistical difference between the strength of all kanamycin regimens and 1 g of vancomycin in the ultimate compression test. After 30 days of elution, the strength of all kanamycin-loaded cement and vancomycin-loaded cement cylinders was significantly lower than that of initial specimens (P < .05). CONCLUSION: The antimycobacterial activity of ALBC containing more than 2 g of kanamycin was effective during a 30-day period. The ultimate compression strength of bone cement loaded with 1-3 g of kanamycin was comparable with 1 g of vancomycin while maintaining effective elution until day 30.

Long-term protective efficacy with a BCG-prime ID93/GLA-SE boost regimen against the hyper-virulent Mycobacterium tuberculosis strain K in a mouse model.

Since ID93/GLA-SE was developed as a targeted BCG-prime booster vaccine, in the present study, we evaluated the protective efficacy of ID93/GLA-SE as a boost to a BCG-prime against the hypervirulent Mycobacterium tuberculosis (Mtb) K challenge to provide further information on the development and application of this vaccine candidate. Boosting BCG with the ID93/GLA-SE vaccine significantly reduced bacterial burden at 16 weeks post-challenge while the BCG vaccine alone did not confer significant protection against Mtb K. The pathological analysis of the lung from the challenged mice also showed the remarkably protective boosting effect of ID93/GLA-SE on BCG-immunised animals. Moreover, qualitative and quantitative analysis of the immune responses following ID93/GLA-SE-immunisation demonstrated that ID93/GLA-SE was able to elicit robust and sustained Th1-biased antigen-specific multifunctional CD4+ T-cell responses up to 16 weeks post-challenge as well as a high magnitude of an antigen-specific IgG response. Our findings demonstrate that the ID93/GLA-SE vaccine candidate given as a BCG-prime boost regimen confers a high level of long-term protection against the hypervirulent Mtb Beijing infection. These findings will provide further and more feasible validation for the potential utility of this vaccine candidate particularly in East-Asian countries, with the predominance of the Beijing genotype, after BCG vaccination.

Construction and Characterization of the Mycobacterium tuberculosis sigE fadD26 Unmarked Double Mutant as a Vaccine Candidate.

Despite the great increase in the understanding of the biology and pathogenesis of Mycobacterium tuberculosis achieved by the scientific community in recent decades, tuberculosis (TB) still represents one of the major threats to global human health. The only available vaccine (Mycobacterium bovis BCG) protects children from disseminated forms of TB but does not effectively protect adults from the respiratory form of the disease, making the development of new and more-efficacious vaccines against the pulmonary forms of TB a major goal for the improvement of global health. Among the different strategies being developed to reach this goal is the construction of attenuated strains more efficacious and safer than BCG. We recently showed that a sigE mutant of M. tuberculosis was more attenuated and more efficacious than BCG in a mouse model of infection. In this paper, we describe the construction and characterization of an M. tuberculosissigE fadD26 unmarked double mutant fulfilling the criteria of the Geneva Consensus for entering human clinical trials. The data presented suggest that this mutant is even more attenuated and slightly more efficacious than the previous sigE mutant in different mouse models of infection and is equivalent to BCG in a guinea pig model of infection.

Common Variants in the Glycerol Kinase Gene Reduce Tuberculosis Drug Efficacy.

Despite the administration of multiple drugs that are highly effective in vitro, tuberculosis (TB) treatment requires prolonged drug administration and is confounded by the emergence of drug-resistant strains. To understand the mechanisms that limit antibiotic efficacy, we performed a comprehensive genetic study to identify Mycobacterium tuberculosis genes that alter the rate of bacterial clearance in drug-treated mice. Several functionally distinct bacterial genes were found to alter bacterial clearance, and prominent among these was the glpK gene that encodes the glycerol-3-kinase enzyme that is necessary for glycerol catabolism. Growth on glycerol generally increased the sensitivity of M. tuberculosis to antibiotics in vitro, and glpK-deficient bacteria persisted during antibiotic treatment in vivo, particularly during exposure to pyrazinamide-containing regimens. Frameshift mutations in a hypervariable homopolymeric region of the glpK gene were found to be a specific marker of multidrug resistance in clinical M. tuberculosis isolates, and these loss-of-function alleles were also enriched in extensively drug-resistant clones. These data indicate that frequently observed variation in the glpK coding sequence produces a drug-tolerant phenotype that can reduce antibiotic efficacy and may contribute to the evolution of resistance.IMPORTANCE TB control is limited in part by the length of antibiotic treatment needed to prevent recurrent disease. To probe mechanisms underlying survival under antibiotic pressure, we performed a genetic screen for M. tuberculosis mutants with altered susceptibility to treatment using the mouse model of TB. We identified multiple genes involved in a range of functions which alter sensitivity to antibiotics. In particular, we found glycerol catabolism mutants were less susceptible to treatment and that common variation in a homopolymeric region in the glpK gene was associated with drug resistance in clinical isolates. These studies indicate that reversible high-frequency variation in carbon metabolic pathways can produce phenotypically drug-tolerant clones and have a role in the development of resistance.

PPE39 of the Mycobacterium tuberculosis strain Beijing/K induces Th1-cell polarization through dendritic cell maturation.

In a previous study, we have identified MTBK_24820, the complete protein form of PPE39 in the hypervirulent Mycobacterium tuberculosis (Mtb) strain Beijing/K by using comparative genomic analysis. PPE39 exhibited vaccine potential against Mtb challenge in a murine model. Thus, in this present study, we characterize PPE39-induced immunological features by investigating the interaction of PPE39 with dendritic cells (DCs). PPE39-treated DCs display reduced dextran uptake and enhanced MHC-I, MHC-II, CD80 and CD86 expression, indicating that this PPE protein induces phenotypic DC maturation. In addition, PPE39-treated DCs produce TNF-α, IL-6 and IL-12p70 to a similar and/or greater extent than lipopolysaccharide-treated DCs in a dose-dependent manner. The activating effect of PPE39 on DCs was mediated by TLR4 through downstream MAPK and NF-κB signaling pathways. Moreover, PPE39-treated DCs promoted naïve CD4+ T-cell proliferation accompanied by remarkable increases of IFN-γ and IL-2 secretion levels, and an increase in the Th1-related transcription factor T-bet but not in Th2-associated expression of GATA-3, suggesting that PPE39 induces Th1-type T-cell responses through DC activation. Collectively, the results indicate that the complete form of PPE39 is a so-far-unknown TLR4 agonist that induces Th1-cell biased immune responses by interacting with DCs.This article has an associated First Person interview with the first author of the paper.

Transient drug-tolerance and permanent drug-resistance rely on the trehalose-catalytic shift in Mycobacterium tuberculosis.

Stochastic formation of Mycobacterium tuberculosis (Mtb) persisters achieves a high level of antibiotic-tolerance and serves as a source of multidrug-resistant (MDR) mutations. As conventional treatment is not effective against infections by persisters and MDR-Mtb, novel therapeutics are needed. Several approaches were proposed to kill persisters by altering their metabolism, obviating the need to target active processes. Here, we adapted a biofilm culture to model Mtb persister-like bacilli (PLB) and demonstrated that PLB underwent trehalose metabolism remodeling. PLB use trehalose as an internal carbon to biosynthesize central carbon metabolism intermediates instead of cell surface glycolipids, thus maintaining levels of ATP and antioxidants. Similar changes were identified in Mtb following antibiotic-treatment, and MDR-Mtb as mechanisms to circumvent antibiotic effects. This suggests that trehalose metabolism is associated not only with transient drug-tolerance but also permanent drug-resistance, and serves as a source of adjunctive therapeutic options, potentiating antibiotic efficacy by interfering with adaptive strategies.

In vitro Mycobacterial Growth Inhibition in South Korean Adults With Latent TB Infection.

Background: It is important to understand the ability to inhibit mycobacterial growth in healthy adults who would have been Bacillus Calmette-Guérin (BCG) vaccinated in childhood as this group will be the potential target population for novel booster TB vaccine trials. In this study we investigated not only the long-term immunity induced by childhood BCG vaccination but also protective immunity in terms of the ability to inhibit mycobacterial growth in those who were BCG vaccinated in childhood, with evidence of recent or remote TB infection. Methods: We measured the baseline immune response using a functional mycobacterial growth inhibition assay (MGIA) as a novel approach and an intracellular cytokine staining (ICS) assay as a reference approach in healthy adults, with different status of Mycobacterium tuberculosis (Mtb) infection. Results: Based on MGIA responses in historically BCG-vaccinated healthy adults, demographical characteristics including age, and gender did not affect mycobacterial growth inhibition in PBMC. However, the uninfected healthy control (HC) group showed a greater ability to inhibit mycobacterial growth compared with the latent TB infection (LTBI) group (P = 0.0005). In terms of the M. tuberculosis antigen-specific T-cell immune response in diluted whole blood quantitated using an ICS assay, the LTBI group had a higher frequency of polyfunctional CD 4+ T cells compared with the HC group (P = 0.0002), although there was no correlation between ICS and the MGIA assay. Conclusion: The Mtb infection status had a significant impact on mycobacterial growth inhibition in PBMC from healthy adults in South Korea, a country with an intermediate burden of tuberculosis, with healthy controls showing the greatest mycobacterial growth inhibition.

Immunogenicity and Vaccine Potential of InsB, an ESAT-6-Like Antigen Identified in the Highly Virulent Mycobacterium tuberculosis Beijing K Strain.

Our group recently identified InsB, an ESAT-6-like antigen belonging to the Mtb9.9 subfamily within the Esx family, in the Mycobacterium tuberculosis Korean Beijing strain (Mtb K) via a comparative genomic analysis with that of the reference Mtb H37Rv and characterized its immunogenicity and its induced immune response in patients with tuberculosis (TB). However, the vaccine potential of InsB has not been fully elucidated. In the present study, InsB was evaluated as a subunit vaccine in comparison with the most well-known ESAT-6 against the hypervirulent Mtb K. Mice immunized with InsB/MPL-DDA exhibited an antigen-specific IFN-γ response along with antigen-specific effector/memory T cell expansion in the lungs and spleen upon antigen restimulation. In addition, InsB immunization markedly induced multifunctional Th1-type CD4+ T cells coexpressing TNF-α, IL-2, and IFN-γ in the lungs following Mtb K challenge. Finally, we found that InsB immunization conferred long-term protection against Mtb K comparable to that conferred by ESAT-6 immunization, as evidenced by a similar level of CFU reduction in the lung and spleen and reduced lung inflammation. These results suggest that InsB may be an excellent vaccine antigen component for developing a multiantigenic Mtb subunit vaccine by generating Th1-biased memory T cells with a multifunctional capacity and may confer durable protection against the highly virulent Mtb K.

Comparison of QFT-Plus and QFT-GIT tests for diagnosis of M. tuberculosis infection in immunocompetent Korean subjects.

BACKGROUND: QFT-Plus is a recently developed next-generation QuantiFERON-TB Gold In-Tube (QFT-GIT) test. Unlike the QFT-GIT test, it includes a TB2 antigen tube with peptides that may stimulate CD8+ T cells. This study evaluated the diagnostic performance of QFT-Plus and compared it with that of QFT-GIT. METHODS: QFT-Plus and QFT-GIT tests were performed in 33 patients with active tuberculosis (TB) and 57 healthy controls including subjects with latent TB infection (LTBI). Positivity and negativity of IFN-γ responses were compared between tests, and total concordance of the outcome was analyzed. RESULTS: Positive and negative outcomes of QFT-Plus and QFT-GIT tests showed substantial agreement (91.1%, kappa=0.8). The sensitivity and the specificity of QFT-Plus (93.9% sensitivity, 92.6% specificity) were similar with those of QFT-GIT (93.9% sensitivity, 100% specificity). Of eight discordant results, five (5.56%) and three (3.3%) were positive in QFT-GIT alone and QFT-plus alone, respectively. Reactivity in the TB2 tube contributes to the difference between QFT-GIT and QFT-Plus. Median IFN-γ production in TB2 (10.0 IU/mL in TB, 3.850 IU/mL in LTBI, P=0.001) is significantly higher in the TB group than the LTBI group. The QFT-Plus did not clearly discriminate between active TB and latent TB, although it showed significantly lower IFN-γ concentrations compared with the QFT-GIT in individuals with LTBI (3.850 vs. 7.205 IU/mL). CONCLUSIONS: Similar accuracy of detecting Mycobacterium tuberculosis infection was observed for QFT-Plus and QFT-GIT tests in immunocompetent patients and healthy controls, including those with LTBI. Improved efficacy for identifying M. tuberculosis infection was not found with the QFT-Plus, but further studies in a larger population may confirm the clinical significance of positive response in the TB2 tube of QFT-Plus.

Validation and comparison of ELISA kits to measure interferon gamma responses in QuantiFERON cultural supernatants for diagnosis of tuberculosis.

Much effort has been made to reduce the cost of LTBI diagnosis with equivalent efficacy and efficiency of interferon-γ release assay (IGRA). This study showed that repeatability, intermediate precision, and accuracy of the Bionote-ELISA were comparable to QFT-ELISA. The Bionote-ELISA could provide the alternative method.

Urine IP-10 as a biomarker of therapeutic response in patients with active pulmonary tuberculosis.

BACKGROUND: Prior to clinical trials of new TB drugs or therapeutic vaccines, it is necessary to develop monitoring tools to predict treatment outcomes in TB patients. Urine interferon gamma inducible protein 10 (IP-10) is a potential biomarker of treatment response in chronic hepatitis C virus infection and lung diseases, including tuberculosis. In this study, we assessed IP-10 levels in urine samples from patients with active TB at diagnosis, during treatment, and at completion, and compared these with levels in serum samples collected in parallel from matched patients to determine whether urine IP-10 can be used to monitor treatment response in patients with active TB. METHODS: IP-10 was measured using enzyme-linked immunosorbent assays in urine and serum samples collected concomitantly from 23 patients with active TB and 21 healthy adults (44 total individuals). The Mann-Whitney U test and Wilcoxon matched-pairs signed rank test were used for comparisons among healthy controls and patients at three time points, and LOESS regression was used for longitudinal data. RESULTS: The levels of IP-10 in urine increased significantly after 2 months of treatment (P = 0.0163), but decreased by the completion of treatment (P = 0.0035). Serum IP-10 levels exhibited a similar trend, but did not increase significantly after 2 months of treatment in patients with active TB. CONCLUSIONS: Unstimulated IP-10 in urine can be used as a biomarker to monitor treatment response in patients with active pulmonary TB.

Investigation of bovine tuberculosis outbreaks by using a trace-back system and molecular typing in Korean Hanwoo beef cattle.

Bovine tuberculosis is a chronic contagious disease responsible for major agricultural economic losses. Abattoir monitoring and trace-back systems are an appropriate method to control bovine tuberculosis, particularly in beef cattle. In the present study, a trace-back system was applied to bovine tuberculosis cases in Korean native Hanwoo beef cattle. Bovine tuberculosis was detected in three index beef cattle during abattoir monitoring in Jeonbuk Province, Korea, and the original herds were traced back from each index cow. All cattle in each original herd were subjected to tuberculin skin test. The positive rates in the tuberculin skin test were 64.6% (62 of 96), 4.8% (2 of 42), and 8.1% (3 of 37) at farms A, B, and C, respectively. On post-mortem examination of 56 tuberculin-positive cattle, 62% had granulomatous lesions, and Mycobacterium bovis was cultured from 40 (71.4%) of the cattle. Molecular typing by spoligotyping and the mycobacterial interspersed repetitive unit-variable-number tandem repeat assay revealed the genotype of the M. bovis strains from the index cattle were same as the M. bovis genotype in each original herd. The results suggest that tracing back from index cattle to the original herd is an effective method to control bovine tuberculosis in beef cattle.

Protective Vaccine Efficacy of the Complete Form of PPE39 Protein from Mycobacterium tuberculosis Beijing/K Strain in Mice.

The aim of this study was to evaluate the protective efficacy of MTBK_24820, a complete form of PPE39 protein derived from a predominant Beijing/K strain of Mycobacterium tuberculosis in South Korea. Mice were immunized with MTKB_24820, M. bovis Bacilli Calmette-Guérin (BCG), or adjuvant prior to a high-dosed Beijing/K strain aerosol infection. After 4 and 9 weeks, bacterial loads were determined and histopathologic and immunologic features in the lungs and spleens of the M. tuberculosis-infected mice were analyzed. Putative immunogenic T-cell epitopes were examined using synthetic overlapping peptides. Successful immunization of MTBK_24820 in mice was confirmed by increased IgG responses (P < 0.05) and recalled gamma interferon (IFN-γ), interleukin-2 (IL-2), IL-6, and IL-17 responses (P < 0.05 or P < 0.01) to MTBK_24820. After challenge with the Beijing/K strain, an approximately 0.5 to 1.0 log10 reduction in CFU in lungs and fewer lung inflammation lesions were observed in MTBK_24820-immunized mice compared to those for control mice. Moreover, MTBK_24820 immunization elicited significantly higher numbers of CD4+ T cells producing protective cytokines, such as IFN-γ and IL-17, in lungs and spleens (P < 0.01) and CD4+ multifunctional T cells producing IFN-γ, tumor necrosis factor alpha (TNF-α), and/or IL-17 (P < 0.01) than in control mice, suggesting protection comparable to that of BCG against the hypervirulent Beijing/K strain. The dominant immunogenic T-cell epitopes that induced IFN-γ production were at the N terminus (amino acids 85 to 102 and 217 to 234). Its vaccine potential, along with protective immune responses in vivo, may be informative for vaccine development, particularly in regions where the M. tuberculosis Beijing/K-strain is frequently isolated from TB patients.