Corrigendum: Activation of M1 Macrophages in Response to Recombinant TB Vaccines With Enhanced Antimycobacterial Activity.

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Activation of M1 Macrophages in Response to Recombinant TB Vaccines With Enhanced Antimycobacterial Activity.

Pulmonary tuberculosis (TB) is a difficult-to-eliminate disease. Although the Bacille Calmette-Guérin (BCG) vaccine against Mycobacterium tuberculosis (MTB) has been available for decades, its efficacy is variable and has lessened over time. Furthermore, the BCG vaccine no longer protects against newly emerged Beijing strains which are responsible for many current infections in adults. Development of a novel vaccine is urgently needed. In this study, we first tested the efficacy of our recombinant BCG vaccines rBCG1 and rBCG2, compared to parental BCG, against MTB strain H37Ra in mice. Both the bacterial load and the level of lymphocyte infiltration decreased dramatically in the three groups treated with vaccine, especially rBCG1 and rBCG2. Furthermore, the Th1 and Th17 responses increased and macrophage numbers rose in the vaccination groups. Th1-mediated production of cytokines TNF-α, IFN-γ, and MCP-1 as well as M1-polarized cells all increased in lung tissue of the rBCG1 and rBCG2 groups. Clodronate-induced depletion of macrophages reduced the level of protection. Based on these results, we conclude that rBCG vaccines induce a significant increase in the number of M1 macrophages, which augments their potential as TB vaccine candidates.

Simultaneous detections of genetic fragment and single nucleotide mutation with a three-tiered output for tuberculosis diagnosis.

Tuberculosis (TB) remains one of the major infectious diseases worldwide. The pathogenic bacterium, Mycobacterium tuberculosis (M.tb), continuously evolves strains carrying drug-resistance genes, thus posing a growing challenge to TB prevention and treatment. We report a diagnostic system that uses a molecular beacon probe and an assistant strand as the core to simultaneously interact with an M.tb-specific fragment (in IS6110) and a single nucleotide substitution (SNS)-encoded segment (in rpoB) associated with drug resistance. A single fluorescent output in three-tiered levels was produced for combinatorial interpretations based on formation of a four-way DNA junction (4WJ). The SNS caused the 4WJ to partially dissociate, thus resulting in medium-level fluorescence. By contrast, high- and low-level fluorescence, represented the complete complementary complex and absence of either targeted fragments, respectively. Manipulating the length of the analyte-binding arm realized the medium output. The thermodynamics and kinetics of 4WJ construction were investigated to maximize the tiered-output performance. Biocatalytic amplification driven by the Klenow Fragment and Nt.AlwI was incorporated into the method to enhance the signal 64-fold and ensure long-term stability of the three-tiered output. The detection accuracy of the sensing system was verified using unpurified amplicons with templates of extracted DNA and boiled bacterial solutions. The tiered-output mechanism was usable at bacterial loads ranging from 4 × 100 to 4 × 103 CFU per reaction. The interference caused by nontuberculous mycobacteria was minimal. The results demonstrated the integrity of the sensing method as an alternative strategy for rapid screening of M.tb and detecting rifampin-resistance.

Combining molecular typing and spatial pattern analysis to identify areas of high tuberculosis transmission in a moderate-incidence county in Taiwan.

In total, 303 randomly selected clinical Mycobacterium tuberculosis (MTB) isolates from 303 patients (collected January to December 2012) in central Taiwan were examined. The major lineages found were Beijing (N = 114, 37.62%), Haarlem (N = 76, 25.08%) and East African-Indian (EAI) (N = 42, 13.86%). Notably, younger persons (≤30 years old) were 6.58 times more likely to be infected with a Beijing genotype compared to older persons (>70 years) (p < 0.05). Combining molecular typing methods and geographical information system (GIS) analysis, we uncovered a twofold higher incidence of Beijing strains in a hotspot area (33%) compared to non-hotspot areas (17%). By 24 MIRU-VNTR typing, persons in clustered groups were 1.96 times more likely to be infected with a Beijing strain compared with non-clustered persons, suggesting recent spread and emergence of MTB. Finally, we observed a trend in which TB incidence increased as the density/concentration of analyzed environmental factors increased, suggesting that environmental factors are associated with TB transmission; however, only population density was found to be significantly associated with increased risk of TB (p < 0.05). Molecular typing methods combined with spatial analysis suggest possible TB transmission. Early intervention to interrupt transmission may be most effective if targeted to hot zones of TB.

Lineage-specific SNPs for genotyping of Mycobacterium tuberculosis clinical isolates.

Tuberculosis (TB) is a severe infectious disease worldwide. Genetic variation of the causative agent, Mycobacterium tuberculosis (MTB), determines the outcomes of infection and anti-TB treatment. Until recently, there has been no effective and convenient way for classifying clinical isolates based on the DNA sequences of the divergent lineages of MTB infecting human populations. Here, we identified single nucleotide polymorphisms (SNPs) of six representative strains from Taiwan by whole-genome sequencing and comparing the results to the sequence of the H37Rv reference strain. One hundred and ten SNPs, each unique to one of the six strains, were used to genotype 150 additional isolates by applying DNA mass spectrometry. Lineage-specific SNPs were identified that could distinguish the major lineages of the clinical isolates. A subset including 32 SNPs was found to be sufficient to type four major groups of MTB isolates in Taiwan (ancient Beijing, modern Beijing, East African-Indian, and Latin-American Mediterranean). However, there was high genetic homozygosity within the Euro-American lineage, which included spoligotype-classified Haarlem and T strains. By whole-genome sequencing of 12 representative Euro-American isolates, we identified multiple subtype-specific SNPs which allowed us to distinguish two major branches within the Euro-American lineage.

Recombinant bacille Calmette-Guerin coexpressing Ag85b, CFP10, and interleukin-12 elicits effective protection against Mycobacterium tuberculosis.

BACKGROUND: The tuberculosis (TB) pandemic remains a leading cause of human morbidity and mortality, despite widespread use of the only licensed anti-TB vaccine, bacille Calmette-Guerin (BCG). The protective efficacy of BCG in preventing pulmonary TB is highly variable; therefore, an effective new vaccine is urgently required. METHODS: In the present study, we assessed the ability of novel recombinant BCG vaccine (rBCG) against Mycobacterium tuberculosis by using modern immunological methods. RESULTS: Enzyme-linked immunospot assays demonstrated that the rBCG vaccine, which coexpresses two mycobacterial antigens (Ag85B and CFP10) and human interleukin (IL)-12 (rBCG2) elicits greater interferon-γ (IFN-γ) release in the mouse lung and spleen, compared to the parental BCG. In addition, rBCG2 triggers a Th1-polarized response. Our results also showed that rBCG2 vaccination significantly limits M. tuberculosis H37Rv multiplication in macrophages. The rBCG2 vaccine surprisingly induces significantly higher tumor necrosis factor-α (TNF-α) production by peripheral blood mononuclear cells that were exposed to a nonmycobacterial stimulus, compared to the parental BCG. CONCLUSION: In this study, we demonstrated that the novel rBCG2 vaccine may be a promising candidate vaccine against M. tuberculosis infection.

Genetic diversity of the Mycobacterium tuberculosis East African-Indian family in three tropical Asian countries.

BACKGROUND: The Beijing lineage of Mycobacterium tuberculosis (MTB) is the most predominant MTB strain in Asian countries and is spreading worldwide, however, the East African-Indian (EAI) lineage is also particularly prevalent in many tropical Asian countries. The evolutionary relationships among MTB EAI isolates from Taiwan and those of tropical Asian countries remain unknown. METHODS: The EAI strains collected from patients in Taiwan were analyzed using spacer oligonucleotide typing and mycobacterial interspersed repetitive unit-variable number of tandem repeats (MIRU-VNTR) typing, and compared with published profiles from Cambodia and Singapore to investigate potential epidemiological linkages. RESULTS: Among the three countries, the EAI lineage was most prevalent in Cambodia (60%; Singapore, 25.62%; and Taiwan, 21.85%), having also the highest rates of multidrug resistance and lowest rates of clustering of MTB isolates. We describe a convenient method using seven selected MIRU-VNTR loci for first-line typing to discriminate Beijing and EAI lineages. A potential epidemiological linkage in these tropical Asian countries is also discussed based on a minimum-spanning tree constructed using 24 MIRU-VNTR loci of MTB EAI strains. CONCLUSION: This study identified evolutionary relationships among MTB EAI isolates from Taiwan and those of two other tropical Asian countries, Cambodia and Singapore.

Genomics Study of Mycobacterium tuberculosis Strains from Different Ethnic Populations in Taiwan.

To better understand the transmission and evolution of Mycobacterium tuberculosis (MTB) in Taiwan, six different MTB isolates (representatives of the Beijing ancient sublineage, Beijing modern sublineage, Haarlem, East-African Indian, T1, and Latin-American Mediterranean (LAM)) were characterized and their genomes were sequenced. Discriminating among large sequence polymorphisms (LSPs) that occur once versus those that occur repeatedly in a genomic region may help to elucidate the biological roles of LSPs and to identify the useful phylogenetic relationships. In contrast to our previous LSP-based phylogeny, the sequencing data allowed us to determine actual genetic distances and to define precisely the phylogenetic relationships between the main lineages of the MTB complex. Comparative genomics analyses revealed more nonsynonymous substitutions than synonymous changes in the coding sequences. Furthermore, MTB isolate M7, a LAM-3 clinical strain isolated from a patient of Taiwanese aboriginal origin, is closely related to F11 (LAM), an epidemic tuberculosis strain isolated in the Western Cape of South Africa. The PE/PPE protein family showed a higher dn/ds ratio compared to that for all protein-coding genes. Finally, we found Haarlem-3 and LAM-3 isolates to be circulating in the aboriginal community in Taiwan, suggesting that they may have originated with post-Columbus Europeans. Taken together, our results revealed an interesting association with historical migrations of different ethnic populations, thus providing a good model to explore the global evolution and spread of MTB.

Draft Genome Sequence of Mycobacterium tuberculosis Clinical Strain W06, a Prevalent Beijing Genotype Isolated in Taiwan.

Mycobacterium tuberculosis strain W06, analyzed by molecular methods, was classified as a modern Beijing M. tuberculosis strain, the most predominant strain in Taiwan. To our knowledge, this is the first draft genome announcement of a Beijing M. tuberculosis strain in Taiwan.

Molecular epidemiology of tuberculosis in Kaohsiung City located at southern Taiwan, 2000-2008.

BACKGROUND: We present the first comprehensive analysis of Mycobacterium tuberculosis (MTB) isolates circulating in southern Taiwan. In this 9-year population-based study, the TB situation in the Kaohsiung region was characterized by genotypic analysis of 421 MTB isolates. METHODS: All 421 isolates of MTB were analyzed by spoligotyping and MIRU-VNTR typing. Drug-resistance patterns were also analyzed. RESULTS: The percentage of EAI (East African-Indian) strains increased across sampling years (2000-2008) in southern Taiwan, whereas the proportion of Beijing lineages remained unchanged. Clustering was more frequent with EAI genotype infections (odds ratio = 3.6, p<0.0001) when compared to Beijing genotypes. Notably, MTB resistance to streptomycin (STR) had significantly increased over time, but resistance to other antibiotics, including multidrug resistance, had not. Three major genes (gidB, rpsL and rrs) implicated in STR resistance were sequenced and specific mutations identified. CONCLUSIONS: This study revealed that EAI strains were highly transmissible and that STR resistance has increased between 2000 and 2008 in Kaohsiung, Taiwan.

Distinct modes of transmission of tuberculosis in aboriginal and non-aboriginal populations in Taiwan.

Tuberculosis incidence among aborigines is significantly higher than for Han Chinese in Taiwan, but the extent to which Mycobacterium tuberculosis (MTB) strain characteristics contribute to this difference is not well understood. MTB isolates from aborigines and Han Chinese living in eastern and southern Taiwan, the major regions of aborigines, were analyzed by spoligotyping and 24-loci MIRU-VNTR. In eastern Taiwan, 60% of aboriginal patients were ≤20 years old, significantly younger than the non-aboriginal patients there; aborigines were more likely to have clustered MTB isolates than Han Chinese (odds ratio (OR) = 5.98, p<0.0001). MTB lineages with high clustering were EAI (54.9%) among southern people, and Beijing (62.5%) and Haarlem (52.9%) among eastern aborigines. Resistance to first-line drugs and multidrug resistance (MDR) were significantly higher among eastern aborigines (≥15%) than in any other geographic and ethnic group (p<0.05); MDR was detected in 5 of 28 eastern aboriginal patients ≤20 years old. Among patients from the eastern region, clustered strains (p = 0.01) and aboriginal ethnicity (p = 0.04) were independent risk factors for MDR. The lifestyles of aborigines in eastern Taiwan may explain why the percentage of infected aborigines is much higher than for their Han Chinese counterparts. The significantly higher percentage of the MDR-MTB strains in the aboriginal population warrants close attention to control policy and vaccination strategy.

Draft Genome Sequences of the Mycobacterium tuberculosis Clinical Strains A2 and A4, Isolated from a Relapse Patient in Taiwan.

The recurrence rate of Mycobacterium tuberculosis in Taiwan is 3%. Here, we present the draft genome sequences of M. tuberculosis strains A2 and A4 from a relapse patient. The draft genome sequences comprise 4,443,031 bp and 4,487,096 bp, revealing 4,220 and 4,143 coding sequences for A2 and A4, respectively, as well as 49 tRNA genes for the both isolates.

Draft Genome Sequence of the Mycobacterium tuberculosis Clinical Isolate C2, Belonging to the Latin American-Mediterranean Family.

Tuberculosis remains a major infectious disease in Taiwan. Here we present the draft genome sequence of the Mycobacterium tuberculosis C2 strain, belonging to the Latin American-Mediterranean lineage. The draft genome sequence comprises 4,453,307 bp with a G+C content of 65.6%, revealing 4,390 coding genes and 45 tRNA genes.

The pattern of cytokine production in vitro induced by ancient and modern Beijing Mycobacterium tuberculosis strains.

It is unclear to what extent the host-responses elicited by Beijing versus non-Beijing strains of Mycobacterium tuberculosis (MTB) contribute to the predominance of modern Beijing strains in Taiwan and some other Asian countries. The purpose of this study was to compare the expression profiles of virulence-related genes in human monocyte-derived macrophages infected in vitro with Beijing (ancient and modern strains) and non-Beijing strains (EAI strains) of MTB that are epidemic in Taiwan. We found that modern Beijing strains induced lower levels of pro-inflammatory cytokines, whereas EAI strains induced higher levels. Notably, the most prevalent modern Beijing sub-lineage, possessing intact RD150 and RD142 chromosomal regions, induced very low levels of pro-inflammatory cytokines, especially interleukin-1ß. Moreover, in an intracellular growth assay, the survival of the same modern Beijing strain in human monocyte-derived macrophages was significantly higher than that of an ancient Beijing strain and an EAI strain. Taken together, these results may explain why modern Beijing strains of MTB predominate in Taiwan.

Molecular epidemiology of Mycobacterium tuberculosis in aboriginal peoples of Taiwan, 2006-2011.

Previous research revealed a 6-fold higher incidence of tuberculosis (TB) amongst aborigines compared to Han Chinese in Taiwan. To investigate the reasons for this disparity, we genotyped Mycobacterium tuberculosis (MTB) strains obtained from members of different aboriginal tribes in different geographical regions of Taiwan by using molecular methods. In total, 177 isolates of MTB collected from patients at four hospitals in Taiwan from January 2006 to December 2011 were analysed by spoligotyping, mycobacterial interspersed repetitive unit-variable number tandem-repeat (MIRU-VNTR) typing. The most prevalent strains in the eastern and central regions of Taiwan were Beijing (45.7% in eastern) and Haarlem (39.1% in eastern, 37.1% in central) lineages, whereas in southern regions the most prevalent strains were EAI (47.7%) and Haarlem (20.5%) lineages. The high prevalence of EAI in southern Taiwan aborigines may be closely associated with Austronesian culture. This study provides a first overview of the M. tuberculosis strains circulating in aboriginal populations in Taiwan. The high prevalences of certain MTB lineages within aboriginal sub-populations suggest that transmission of MTB may have been restricted to close contacts.

Clonal expansion of both modern and ancient genotypes of Mycobacterium tuberculosis in southern Taiwan.

We present the first comprehensive analysis of Mycobacterium tuberculosis isolates circulating in the Kaohsiung region of southern Taiwan. The major spoligotypes found in the 224 isolates studied were Beijing lineages (n = 97; 43.3%), EAI lineages (n = 72; 32.1%) and Haarlem lineages (n = 18; 8.0%). By 24 MIRU-VNTR typing, 174 patterns were identified, including 24 clusters of 74 isolates and 150 unique patterns. The combination of spoligotyping and 12-MIRU-VNTR revealed that 129 (57.6%) of the 224 isolates were clustered in 18 genotypes. Moreover, 63.6% (7/11) of infected persons younger than 30 years had a Beijing strain, which could suggest recent spread among younger persons by this family of TB strains in Kaohsiung. Among the 94 Beijing family (SIT1, SIT250 and SIT1674) isolates further analyzed for SNPs by mass spectrometry, the most frequent strain found was ST10 (n = 49; 52%), followed by ST22 (n = 17; 18%) and ST19 (n = 11; 12%). Among the EAI-Manila family isolates analyzed by region deletion-based subtyping, the most frequent strain found was RD type 1 (n = 63; 87.5%), followed by RD type 2 (n = 9; 12.5%). In our previous study, the proportion of modern Beijing strains (52.5%) in northern Taiwan was significantly higher than the proportion of EAI strains (11%). In contrast, in the present study, EAI strains comprised up to 32% of Beijing strains in southern Taiwan. In conclusion, both 'modern' (Beijing) and 'ancient' (EAI) M. tuberculosis strains are prevalent in the Kaohsiung region, perhaps suggesting that both strains are somehow more adapted to southern Taiwan. It will be interesting to investigate the dynamics of the lineage composition by different selection pressures.

Genetic diversity of the Mycobacterium tuberculosis Beijing family based on SNP and VNTR typing profiles in Asian countries.

The Mycobacterium tuberculosis (MTB) Beijing strain is highly virulent, drug resistant, and endemic over Asia. To explore the genetic diversity of this family in several different regions of eastern Asia, 338 Beijing strains collected in Taiwan (Republic of China) were analyzed by mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) typing and compared with published MIRU-VNTR profiles and by the Hunter-Gaston diversity index (HGDI) of Beijing strains from Japan and South Korea. The results revealed that VNTR2163b (HGDI>0.6) and five other loci (VNTR424, VNTR4052, VNTR1955, VNTR4156 and VNTR 2996; HGDI>0.3) could be used to discriminate the Beijing strains in a given geographic region. Analysis based on the number of VNTR repeats showed three VNTRs (VNTR424, 3192, and 1955) to be phylogenetically informative loci. In addition, to determine the geographic variation of sequence types in MTB populations, we also compared sequence type (ST) data of our strains with published ST profiles of Beijing strains from Japan and Thailand. ST10, ST22, and ST19 were found to be prevalent in Taiwan (82%) and Thailand (92%). Furthermore, classification of Beijing sublineages as ancient or modern in Taiwan was found to depend on the repeat number of VNTR424. Finally, phylogenetic relationships of MTB isolates in Taiwan, South Korea, and Japan were revealed by a minimum spanning tree based on MIRU-VNTR genotyping. In this topology, the MIRU-VNTR genotypes of the respective clusters were tightly correlated to other genotypic characters. These results are consistent with the hypothesis that clonal evolution of these MTB lineages has occurred.

Recombinant BCG coexpressing Ag85B, CFP10, and interleukin-12 induces multifunctional Th1 and memory T cells in mice.

Mycobacterium tuberculosis (MTB) continues to be a leading cause of human deaths due to an infectious agent. Current efforts are focused on making better TB vaccines. We describe the generation and immunological characterization of recombinant BCG (rBCG). This rBCG was generated by incorporating an expression plasmid encoding two mycobacterial antigens (Ag85B and CFP10) and human interleukin (IL)-12 into a BCG strain. Immunogenicity studies in mice showed that rBCG coexpressing Ag85B, CFP10, and IL-12 (rBCG::Ag85B-CFP10-IL-12) induces a robust immune response in mice. The rBCG vaccine promotes a T-cell response against MTB that is characterized by a high proportion of polyfunctional and memory T cells in spleen and lung. Our results showed strong immunogenicity and mycobacterial growth inhibition of rBCG::Ag85B-CFP10 plus IL-12 than that of BCG vaccine.

Utility and evaluation of new variable-number tandem-repeat systems for genotyping mycobacterial tuberculosis isolates.

We compared mycobacterial interspersed repetitive unit (MIRU)-variable number tandem repeat (VNTR) typing to traditional spoligotyping for discriminating Mycobacterium tuberculosis (MTB) strains. Our 17-loci MIRU-VNTR typing method was found to be superior to spoligotyping for non-Beijing family strains. To extend the method we also established PCR-based rapid genotyping protocols for Beijing, East-African-Indian and U lineages.

Molecular epidemiology and evolutionary genetics of Mycobacterium tuberculosis in Taipei.

BACKGROUND: The control of tuberculosis in densely populated cities is complicated by close human-to-human contacts and potential transmission of pathogens from multiple sources. We conducted a molecular epidemiologic analysis of 356 Mycobacterium tuberculosis (MTB) isolates from patients presenting pulmonary tuberculosis in metropolitan Taipei. Classical antibiogram studies and genetic characterization, using mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) typing and spoligotyping, were applied after culture. METHODS: A total of 356 isolates were genotyped by standard spoligotyping and the strains were compared with in the international spoligotyping database (SpolDB4). All isolates were also categorized using the 15 loci MIRU-VNTR typing method and combin with NTF locus and RD deletion analyses. RESULTS: Of 356 isolates spoligotyped, 290 (81.4%) displayed known spoligotypes and 66 were not identified in the database. Major spoligotypes found were Beijing lineages (52.5%), followed by Haarlem lineages (13.5%) and EAI plus EAI-like lineages (11%). When MIRU-VNTR was employed, 140 patterns were identified, including 36 clusters by 252 isolates and 104 unique patterns, and the largest cluster comprised 95 isolates from the Beijing family. The combination of spoligotyping and MIRU-VNTR revealed that 236 (67%) of the 356 isolates were clustered in 43 genotypes. Strains of the Beijing family was more likely to be of modern strain and a higher percentage of multiple drug resistance than other families combined (P = 0.08). Patients infected with Beijing strains were younger than those with other strains (mean 58.7 vs. 64.2, p = 0.02). Moreover, 85.3% of infected persons younger than 25 years had Beijing modern strain, suggesting a possible recent spread in the young population by this family of TB strain in Taipei. CONCLUSION: Our data on MTB genotype in Taipei suggest that MTB infection has not been optimally controlled. Control efforts should be reinforced in view of the high prevalence of the Beijing strain in young population and association with drug resistance.