B Cell-Based Vaccine Transduced With ESAT6-Expressing Vaccinia Virus and Presenting ?-Galactosylceramide Is a Novel Vaccine Candidate Against ESAT6-Expressing Mycobacterial Diseases.
ABSTRACT: Early secretory antigenic target-6 (ESAT6) is a potent immunogenic antigen expressed in Mycobacterium tuberculosis as well as in some non-tuberculous mycobacteria (NTM), such as M. kansasii. M. kansasii is one of the most clinically relevant species of NTM that causes mycobacterial lung disease, which is clinically indistinguishable from tuberculosis. In the current study, we designed a novel cell-based vaccine using B cells that were transduced with vaccinia virus expressing ESAT6 (vacESAT6), and presenting ?-galactosylceramide (?GC), a ligand of invariant NKT cells. We found that B cells loaded with ?GC had increased levels of CD80 and CD86 after in vitro stimulation with NKT cells. Immunization of mice with B/?GC/vacESAT6 induced CD4+ T cells producing TNF-? and IFN-? in response to heat-killed M. tuberculosis. Immunization of mice with B/?GC/vacESAT6 ameliorated severe lung inflammation caused by M. kansasii infection. We also confirmed that immunization with B/?GC/vacESAT6 reduced M. kansasii bacterial burden in the lungs. In addition, therapeutic administration of B/?GC/vacESAT6 increased IFN-?+ CD4+ T cells and inhibited the progression of lung pathology caused by M. kansasii infection. Thus, B/?GC/vacESAT6 could be a potent vaccine candidate for the prevention and treatment of ESAT6-expressing mycobacterial infection caused by M. kansasii.
Project description:ESAT6 has recently been demonstrated to cause haemolysis and macrophage lysis. Our studies demonstrate that ESAT6 causes cytolysis of type 1 and type 2 pneumocytes. Both types of pneumocytes express membrane laminin, and ESAT6 exhibits dose-dependent binding to both cell types and to purified human laminin. While minimal ESAT6 was detected on the surface of Mycobacterium tuberculosis grown in vitro, exogenously provided ESAT6 specifically associated with the bacterial cell surface, and the bacterium-associated ESAT6 retained its cytolytic ability. esat6 transcripts were upregulated approximately 4- to approximately 13-fold in bacteria replicating in type 1 cells, and approximately 3- to approximately 5 fold in type 2 cells. In vivo, laminin is primarily concentrated at the basolateral surface of pneumocytes where they rest on the basement membrane, which is composed primarily of laminin and collagen. The upregulation of esat6 transcripts in bacteria replicating in pneumocytes, the specific association of ESAT6 with the bacterial surface, the binding of ESAT6 to laminin and the lysis of pneumocytes by free and bacterium-associated ESAT6 together suggest a scenario wherein Mycobacterium tuberculosis replicating in pneumocytes may utilize surface ESAT6 to anchor onto the basolateral laminin-expressing surface of the pneumocytes, and damage the cells and the basement membrane to directly disseminate through the alveolar wall.
Project description:The prevalence of tuberculosis continues to be high, and nontuberculous mycobacterial (NTM) infection has also emerged worldwide. Moreover, differential and accurate identification of mycobacteria to the species or subspecies level is an unmet clinical need. Here, we developed a one-step multiplex PCR assay using whole-genome analysis and bioinformatics to identify novel molecular targets. The aims of this assay were to (i) discriminate between the Mycobacterium tuberculosis complex (MTBC) and NTM using rv0577 or RD750, (ii) differentiate M. tuberculosis (M. tuberculosis) from MTBC using RD9, (iii) selectively identify the widespread M. tuberculosis Beijing genotype by targeting mtbk_20680, and (iv) simultaneously detect five clinically important NTM (M. avium, M. intracellulare, M. abscessus, M. massiliense, and M. kansasii) by targeting IS1311, DT1, mass_3210, and mkan_rs12360 An initial evaluation of the multiplex PCR assay using reference strains demonstrated 100% specificity for the targeted Mycobacterium species. Analytical sensitivity ranged from 1 to 10 pg for extracted DNA and was 103 and 104 CFU for pure cultures and nonhomogenized artificial sputum cultures, respectively, of the targeted species. The accuracy of the multiplex PCR assay was further evaluated using 55 reference strains and 94 mycobacterial clinical isolates. Spoligotyping, multilocus sequence analysis, and a commercial real-time PCR assay were employed as standard assays to evaluate the multiplex PCR assay with clinical M. tuberculosis and NTM isolates. The PCR assay displayed 100% identification agreement with the standard assays. Our multiplex PCR assay is a simple, convenient, and reliable technique for differential identification of MTBC, M. tuberculosis, M. tuberculosis Beijing genotype, and major NTM species.
Project description:OBJECTIVE:Nontuberculous mycobacteria (NTM) often cause disease that is clinically indistinguishable from tuberculosis. Specific identification is important as treatment varies according to Mycobacterium species causing the infection. This study used multiplex PCR (mPCR) assay for rapid differentiation of mycobacterial growth indicator tube 960 system (MGIT) cultures as Mycobacterium tuberculosis (MTB) or NTM together with INNO LiPA Mycobacteria v2 assay (LiPA) and/or PCR sequencing of rDNA for species-specific identification of selected MTB and all NTM isolates in Kuwait. MATERIALS AND METHODS:DNA was extracted from MGIT cultures (n = 1,033) grown from 664 pulmonary and 369 extrapulmonary specimens from 1,033 suspected tuberculosis patients. mPCR was performed to differentiate MTB from NTM. LiPA was performed and results were interpreted according to kit instructions. rDNA was amplified and sequenced by using panmycobacterial primers. RESULTS:mPCR identified 979 isolates as MTB, 53 as NTM and 1 isolate as mixed culture. LiPA and/or PCR sequencing confirmed 112 of 979 selected isolates as MTB. Mixed culture contained M. tuberculosis and M. fortuitum. LiPA yielded 12 patterns and identified 10 species/species complexes among 47 NTM, M. kansasii + M. scrofulaceum in one culture and 5 isolates only at genus level. PCR sequencing yielded more specific identification for 22 isolates at the species/subspecies level. CONCLUSIONS:mPCR rapidly differentiated MTB from NTM. LiPA identified 44 of 52 NTM isolates at the species/species complex level and 2 mixed cultures. PCR sequencing yielded more specific identification at the species/subspecies level. Rapid differentiation as MTB or NTM by mPCR, followed by species-specific NTM identification by LiPA/PCR sequencing is suitable for the proper management of mycobacterial infections in Kuwait.
Project description:Mycobacterium kansasii is a nontuberculous mycobacterial (NTM) pathogen, frequently isolated from clinical samples and responsible for a large part of NTM infections in the human population. Here, we report the draft genome sequences of 12 M. kansasii strains isolated from clinical and host-associated sources from the Netherlands, Germany, and Poland.
Project description:BACKGROUND:The burden of non-tuberculous mycobacterial (NTM) disease is increasing worldwide but still its diagnosis is delayed and it is mistaken as multidrug-resistant tuberculosis (MDR-TB).The present study was performed to develop a multiplex PCR assay for detection and identification of clinically most common NTM to the species level from pulmonary samples. RESULTS:Out of 50 isolates, 26 were identified as Mycobacterium kansasii (MK), 20 were identified as Mycobacterium abscessus (MA) and 4 were identified as Mycobacterium avium complex (MAC) through multiplex PCR and further confirmed by sequencing. CONCLUSION:Our study showed that multiplex PCR assay is a simple, convenient, and reliable technique for detection and differential identification of major NTM species.
Project description:Infections caused by non-tuberculous mycobacteria (NTM) is increasing wordwide. Due to the difference in treatment of NTM infections and tuberculosis, rapid species identification of mycobacterial clinical isolates is necessary for the effective management of mycobacterial diseases treatment and their control strategy. In this study, a cost-effective technique, real-time PCR coupled with high-resolution melting (HRM) analysis, was developed for the differentiation of Mycobacterial species using a novel rpoBC sequence. A total of 107 mycobacterial isolates (nine references and 98 clinical isolates) were subjected to differentiation using rpoBC locus sequence in a real-time PCR-HRM assay scheme. From 98 Mycobacterium clinical isolates, 88 species (89.7%), were identified at the species level by rpoBC locus sequence analysis as a gold standard method. M. simiae was the most frequently encountered species (41 isolates), followed by M. fortuitum (20 isolates), M. tuberculosis (15 isolates), M. kansassi (10 isolates), M. abscessus group (5 isolates), M. avium (5 isolates), and M. chelonae and M. intracellulare one isolate each. The HRM analysis generated six unique specific groups representing M. tuberculosis complex, M. kansasii, M. simiae, M. fortuitum, M. abscessus-M. chelonae group, and M. avium complex. In conclusion, this study showed that the rpoBC-based real-time PCR followed by HRM analysis could differentiate the majority of mycobacterial species that are commonly encountered in clinical specimens.
Project description:Mycobacteria cause a variety of illnesses that differ in severity and public health implications. The differentiation of Mycobacterium tuberculosis from nontuberculous mycobacteria (NTM) is of primary importance for infection control and choice of antimicrobial therapy. Despite advances in molecular diagnostics, the ability to rapidly diagnose M. tuberculosis infections by PCR is still inadequate, largely because of the possibility of false-negative reactions. We designed and validated a real-time PCR for mycobacteria by using the LightCycler system with 18 reference strains and 168 clinical mycobacterial isolates. All clinically significant mycobacteria were detected; the mean melting temperatures (with 99.9% confidence intervals [99.9% CI] in parentheses) for the different mycobacteria were as follows: M. tuberculosis, 64.35 degrees C (63.27 to 65.42 degrees C); M. kansasii, 59.20 degrees C (58.07 to 60.33 degrees C); M. avium, 57.82 degrees C (57.05 to 58.60 degrees C); M. intracellulare, 54.46 degrees C (53.69 to 55.23 degrees C); M. marinum, 58.91 degrees C (58.28 to 59.55 degrees C); rapidly growing mycobacteria, 53.09 degrees C (50.97 to 55.20 degrees C) or 43.19 degrees C (42.19 to 44.49 degrees C). This real-time PCR assay with melting curve analysis consistently accurately detected and differentiated M. tuberculosis from NTM. Detection of an NTM helps ensure that the negative result for M. tuberculosis is a true negative. The specific melting temperature also provides a suggestion of the identity of the NTM present, when the most commonly encountered mycobacterial species are considered. In a parallel comparison, both the LightCycler assay and the COBAS Amplicor M. tuberculosis assay correctly categorized 48 of 50 specimens that were proven by culture to contain M. tuberculosis, and the LightCycler assay correctly characterized 3 of 3 specimens that contained NTM.
Project description:RATIONALE:Nontuberculous mycobacteria (NTM) are a diverse group of environmental organisms that infrequently cause human disease. Understanding of the epidemiologic and clinical characteristics associated with NTM disease is needed to refine diagnostic and treatment strategies, particularly among the less commonly isolated species. OBJECTIVES:To improve knowledge of geographic variance of NTM species, to correlate detailed clinical information with isolation of specific NTM, and to examine the decision to treat and outcomes for specific NTM. METHODS:Mycobacterial cultures submitted to the University of Washington mycobacterial laboratory from 1998 to 2011 were examined. We report isolation frequency and demographic information from all samples with clinical variables. We also examined treatment decisions and outcomes in a subset of patients with Mycobacterium abscessus complex, Mycobacterium chelonae, Mycobacterium fortuitum, Mycobacterium gordonae, Mycobacterium kansasii, Mycobacterium lentiflavum, Mycobacterium porcinum, and Mycobacterium xenopi. RESULTS:Cultures of NTM were available from 3,470 patients, 937 of whom had clinical data available. When we compared patients born within or outside Washington State, we found that the mycobacterial species frequency varied. Among 168 patients with one of the studied environmental mycobacteria, 72% had major comorbid conditions. Bronchiectasis was common among patients with pulmonary isolation of any NTM, including those with nonpathogenic M. gordonae. Although mortality was high (37%), few deaths were directly attributable to mycobacterial infection. Among 56 patients who met American Thoracic Society criteria for NTM lung disease, 22 were treated, and 19 of those had M. abscessus complex and M. kansasii. The treatment regimens used tended to follow published guidelines. CONCLUSIONS:Isolation of NTM varied by geographic region of origin and location within Washington State. Several clinical risk factors were specific to individual species. Comorbid conditions were common in patients with and without mycobacterial disease. Among patients with one of the studied organisms, there was a high mortality rate more frequently related to comorbid conditions than to mycobacterial disease.
Project description:BackgroundTo investigate the species distribution of non-tuberculous mycobacteria (NTM) among tuberculosis (TB) specimens collected from January 2013 to December 2018 at Peking Union Medical Hospital (Beijing), China. NTM species identification was carried out by DNA microarray chip.ResultsMycobacterial species were detected in 1514 specimens from 1508 patients, among which NTM accounted for 37.3% (565/1514), increasing from a proportion of 15.6% in 2013 to 46.1% in 2018 (P?<?0.001). Among the 565 NTM positive specimens, the majority (55.2%) were from female patients. Furthermore, patients aged 45–65?years accounted for 49.6% of the total patients tested. Among 223 NTM positive specimens characterized further, the majority (86.2%) were from respiratory tract, whilst 3.6 and 3.1% were from lymph nodes and pus, respectively. Mycobacterium intracellulare (31.8%) and Mycobacterium chelonae / Mycobacterium abscessus (21.5%) were the most frequently detected species, followed by M. avium (13.5%), M. gordonae (11.7%), M. kansasii (7.6%), and others.ConclusionThe proportion of NTM among mycobacterial species detected in a tertiary hospital in Beijing, China, increased rapidly from year 2013 to 2018. Middle-aged patients are more likely to be infected with NTM, especially females. Mycobacterium intracellulare and Mycobacterium chelonae/ Mycobacterium abscessus were the most frequently detected NTM pathogens. Accurate and timely identification of NTM is important for diagnosis and treatment.
Project description:The most commonly used tools for tuberculosis testing in cattle, the tuberculin skin test and the interferon-? release assay, detect immune reactivity to various antigens of Mycobacterium bovis, including ESAT-6 and CFP-10. However, some non-tuberculous mycobacteria (NTM) can also harbor the cfp-10 and/or esat-6 genes, which can lead to false-positive results. We tested 77 NTM isolates belonging to 22 different species from lymph nodes of healthy slaughtered cattle for the occurrence of cfp-10 and esat-6. Most isolates did not harbor cfp-10 and esat-6. However, M. gordonae, 'M. lymphaticum', M. kansasii, and M. persicum were cfp-10 positive. The esat-6 gene was found in M. kansasii and M. persicum. Protein expression of cfp-10 and esat-6 could be detected for M. kansasii and M. persicum. An effective tuberculosis control program based on interferon-? release assays and tuberculin skin testing is dependent on further monitoring and characterization of NTM in a cattle population.