Different Contributions of embB and ubiA Mutations to Variable Level of Ethambutol Resistance in Mycobacterium tuberculosis Isolates.

Objective: To explore the association between the variant mutations within embB and ubiA, and the degree of ethambutol (EMB) resistance of Mycobacterium tuberculosis (M. tuberculosis) isolates. Methods: A total of 146 M. tuberculosis isolates were used to determine the minimum inhibitory concentrations (MICs) of EMB with a 96-well microplate-based assay. The mutations within embB and ubiA among these isolates were identified with DNA sequencing. Moreover, a multivariate regression model and a computer model were established to assess the effects of mutations on EMB resistance. Results: Our data showed that overall 100 isolates exhibited 28 mutated patterns within the sequenced embB and ubiA. Statistical analysis indicated that embB mutations Met306Val, Met306Ile, Gly406Ala, and Gln497Arg, were strongly associated with EMB resistance. Of these mutations, Met306Val and Gln497Arg were significantly associated with high-level EMB resistance. Almost all multiple mutations occurred in high-level EMB-resistant isolates. Although the mutation within ubiA accompanied with embB mutation presented exclusively in EMB-resistant isolates, four single ubiA mutations (Ala39Glu, Ser173Ala, Trp175Cys, and Val283Leu) leading to protein instability were observed in EMB-susceptible isolates. Conclusion: This study highlighted the complexity of EMB resistance. Some individual mutations and multiple mutations within embB and ubiA contributed to the different levels of EMB resistance.

Candidate Genes and Favorable Haplotypes Associated with Iron Toxicity Tolerance in Rice.

Iron (Fe) toxicity is a major issue adversely affecting rice production worldwide. Unfortunately, the physiological and genetic mechanisms underlying Fe toxicity tolerance in rice remain relatively unknown. In this study, we conducted a genome-wide association study using a diverse panel consisting of 551 rice accessions to identify genetic mechanisms and candidate genes associated with Fe toxicity tolerance. Of the 29 quantitative trait loci (QTL) for Fe toxicity tolerance detected on chromosomes 1, 2, 5, and 12, five (qSH_Fe5, qSFW_Fe2.3, qRRL5.1, qRSFW1.1, and qRSFW12) were selected to identify candidate genes according to haplotype and bioinformatics analyses. The following five genes were revealed as promising candidates: LOC_Os05g40160, LOC_Os05g40180, LOC_Os12g36890, LOC_Os12g36900, and LOC_Os12g36940. The physiological characteristics of rice accessions with contrasting Fe toxicity tolerance reflected the importance of reactive oxygen species-scavenging antioxidant enzymes and Fe homeostasis for mitigating the negative effects of Fe toxicity on rice. Our findings have clarified the genetic and physiological mechanisms underlying Fe toxicity tolerance in rice. Furthermore, we identified valuable genetic resources for future functional analyses and the development of Fe toxicity-tolerant rice varieties via marker-assisted selection.

Analysis on epidemiological and drug resistance characteristics of lymph node tuberculosis from Hunan province, China.

Objectives: To investigate the clinical epidemiological and drug resistance (DR) characteristics of lymph node tuberculosis (LNTB) in Hunan Province which locates in South-central China, and to provide scientific clues for effective prevention and treatment of LNTB. Methods: We retrospectively collected LNTB patients with Mycobacterium tuberculosis culture positive at Hunan Chest Hospital, the biggest TB reference hospital in South-central China, from January 2013 to December 2021. The multiple demographic, clinical and drug susceptibility data of patients were collected from the hospital's electronic patient records. Descriptive statistical methods, Chi-square test and logistic regression analysis were employed as statistical methods. Results: Of the 577 LNTB cases, 373 (64.64%) were males, 352 (61.01%) were farmers; majority (161, 33.10%) aged at 20-29 years old; 147 (25.48%) had simple LNTB, 350 (60.66%) had LNTB combined with pulmonary TB (PTB) (defined as LNTB-PTB), and 80 (13.86%) had LNTB combined with other extrapulmonary TB (EPTB) (defined as LNTB-EPTB). A total of 345 (59.79%, 345/577) LNTB patients had cervical node infection, and the simple LNTB patients (81.63%, 120/147) had higher proportion of this infection than LNTB-PTB (51.71%, 181/350) and LNTB-EPTB (55.00%, 44/80) (both p values <0.017), respectively. LNTB-EPTB was more inclined to have abdominal tuberculous LNs (20%, 16/80) and at least four tuberculous lesions (22.50%, 18/80) than simple LNTB and LNTB-PTB. Seventy-seven (13.34%) and 119 (20.62%) were resistant to rifampicin (RIF) and isoniazid (INH), respectively; 72 (12.48%) were multi-drug resistant (MDR), and a total of 150 (26.00%) were DR (resistant to at least one of RIF, INH, ethambutol and streptomycin). LNTB patients aged 30-34 and 50-54 years old (compared to those aged <30 years) were independent predictors of RIF resistance (RR) (ORs were 3.47 and 2.83, respectively; 95% CIs were 1.64-7.35 and 1.08-7.46, respectively). Conclusion: Our study disclosed the epidemiological and DR characteristics of LNTB in Hunan Province, China. High LNTB prevalence was found in younger people while high RR LNTB prevalence was found in older ones, suggesting that we should conduct further studies to clarify the occurrence of RR in LNTB, meanwhile, strengthen the diagnoses and treatments of LNTB to prevent the emergence of RR.

A novel pathogenic species of genus Stenotrophomonas: Stenotrophomonas pigmentata sp. nov.

Introduction: Stenotrophomonas is a prominent genus owing to its dual nature. Species of this genus have many applications in industry and agriculture as plant growth-promoting rhizobacteria and microbial biological control agents, whereas species such as Stenotrophomonas maltophilia are considered one of the leading gram-negative multi-drug-resistant bacterial pathogens because of their high contribution to the increase in crude mortality and significant clinical challenge. Pathogenic Stenotrophomonas species and most clinical isolates belong to the Stenotrophomonas maltophilia complex (SMc). However, a strain highly homologous to S. terrae was isolated from a patient with pulmonary tuberculosis (TB), which aroused our interest, as S. terrae belongs to a relatively distant clade from SMc and there have been no human association reports. Methods: The pathogenicity, immunological and biochemical characteristics of 610A2T were systematically evaluated. Results: 610A2T is a new species of genus Stenotrophomonas, which is named as Stenotrophomonas pigmentata sp. nov. for its obvious brown water-soluble pigment. 610A2T is pathogenic and caused significant weight loss, pulmonary congestion, and blood transmission in mice because it has multiple virulence factors, haemolysis, and strong biofilm formation abilities. In addition, the cytokine response induced by this strain was similar to that observed in patients with TB, and the strain was resistant to half of the anti-TB drugs. Conclusions: The pathogenicity of 610A2T may not be weaker than that of S. maltophilia. Its isolation extended the opportunistic pathogenic species to all 3 major clades of the genus Stenotrophomonas, indicating that the clinical importance of species of Stenotrophomonas other than S. maltophilia and potential risks to biological safety associated with the use of Stenotrophomonas require more attention.

Identification of Key Genes and Pathways for Anaerobic Germination Tolerance in Rice Using Weighted Gene Co-Expression Network Analysis (WGCNA) in Association with Quantitative Trait Locus (QTL) Mapping.

BACKGROUND: Rice is one of the most important food crops in the world, and with the development of direct seeding methods for rice, exposure to anaerobic stress has become a major factor limiting its growth. RESULTS: In this experiment, we tested the tolerance to anaerobic germination of rice varieties NIP and HD84, and they were used as parents to construct a DH (doubled-haploid) population. The transcriptomes of NIP (highly tolerant) and HD86 (intolerant), and their progeny HR (highly tolerant) and NHR (intolerant) were sequenced from normal and anaerobic environments. The differentially-expressed genes (DEGs) were subjected to GO (Gene ontology), KEGG (Kyoto Encyclopedia of Genes and Genomes), and WGCNA analyses. QTL mapping of the DH population identified tolerance to anaerobic germination-related chromosomal segments. The transcriptome results from 24 samples were combined with the anaerobic stress QTL results for 159 DH population lines to construct a metabolic network to identify key pathways and a gene interaction network to study the key genes. Essential genes were initially subjected to rigorous functional validation, followed by a comprehensive analysis aimed at elucidating their potential utility in domestication and breeding practices, particularly focusing on the exploitation of dominant haplotypes. CONCLUSION: The results show that pyruvate decarboxylase (PDC) and alcohol dehydrogenase (ADH) are the starting signals of energy metabolism for coleoptile length growth, the auxin transporter EXPA is the determining signal for coleoptile length growth. The pivotal genes Os05g0498700 and Os01g0866100 exert a negative regulatory influence on coleoptile length, ultimately enhancing tolerance to anaerobic germination in rice. Analyses of breeding potential underscore the additional value of Os05g0498700-hyp2 and Os01g0866100-hyp2, highlighting their potential utility in further improving rice through breeding programs. The results of our study will provide a theoretical basis for breeding anaerobic-tolerant rice varieties.

Identification of positively selected genes in Mycobacterium tuberculosis from southern Xinjiang Uygur autonomous region of China.

Background: Tuberculosis (TB), mainly caused by Mycobacterium tuberculosis (Mtb), remains a serious public health problem. Increasing evidence supports that selective evolution is an important force affecting genomic determinants of Mtb phenotypes. It is necessary to further understand the Mtb selective evolution and identify the positively selected genes that probably drive the phenotype of Mtb. Methods: This study mainly focused on the positive selection of 807 Mtb strains from Southern Xinjiang of China using whole genome sequencing (WGS). PAML software was used for identifying the genes and sites under positive selection in 807 Mtb strains. Results: Lineage 2 (62.70%) strains were the dominant strains in this area, followed by lineage 3 (19.45%) and lineage 4 (17.84%) strains. There were 239 codons in 47 genes under positive selection, and the genes were majorly associated with the functions of transcription, defense mechanisms, and cell wall/membrane/envelope biogenesis. There were 28 codons (43 mutations) in eight genes (gyrA, rpoB, rpoC, katG, pncA, embB, gid, and cut1) under positive selection in multi-drug resistance (MDR) strains but not in drug-susceptible (DS) strains, in which 27 mutations were drug-resistant loci, 9 mutations were non-drug-resistant loci but were in drug-resistant genes, 2 mutations were compensatory mutations, and 5 mutations were in unknown drug-resistant gene of cut1. There was a codon in Rv0336 under positive selection in L3 strains but not in L2 and L4 strains. The epitopes of T and B cells were both hyper-conserved, particularly in the T-cell epitopes. Conclusion: This study revealed the ongoing selective evolution of Mtb. We found some special genes and sites under positive selection which may contribute to the advantage of MDR and L3 strains. It is necessary to further study these mutations to understand their impact on phenotypes for providing more useful information to develop new TB interventions.

Pedigree genome data of an early-matured Geng/japonica glutinous rice mega variety Longgeng 57.

By using PacBio HiFi technology, we produced over 700 Gb of long-read sequencing (LRS) raw data; and by using Illumina paired-end whole-genome shotgun (WGS) sequencing technology, we generated more than 70 Gb of short-read sequencing (SRS) data. With LRS data, we assembled one genome and then generate a set of annotation data for an early-matured Geng/japonica glutinous rice mega variety genome, Longgeng 57 (LG57), which carries multiple elite traits including good grain quality and wide adaptability. Together with the SRS data from three parents of LG57, pedigree genome variations were called for three representative types of genes. These data sets can be used for deep variation mining, aid in the discovery of new insights into genome structure, function, and evolution, and help to provide essential support to biological research in general.

Exploring the immunogenicity of Rv2201-519: A T-cell epitope-based antigen derived from Mycobacterium tuberculosis AsnB with implications for tuberculosis infection detection and vaccine development.

Research dedicated to diagnostic reagents and vaccine development for tuberculosis (TB) is challenging due to the paucity of immunodominant antigens that can predict disease risk and exhibit protective potential. Therefore, it is crucial to identify T-cell epitope-based Mycobacterium tuberculosis (MTB) antigens characterized by specific and prominent recognition by the immune system. In this study, we constructed a T-cell epitope-rich tripeptide-splicing fragment (nucleotide positions 131-194, 334-377, and 579-643) of Rv2201 (also known as the 72 kDa AsnB)from the MTB genome, ultimately yielding the recombinant protein Rv2201-519 in Escherichia coli BL21 (DE3). Subsequently, we gauged the recombinant protein's ability to detect tuberculosis infection through ELISpot and assessed its immunostimulatory effect on mouse models using flow cytometry and ELISA. Our results indicated that Rv2201-519 possessed promising sensitivity; however, the sensitivity was lower than that of a commercial diagnostic kit containing ESAT-6, CFP-10, and Rv3615c (80.56 % vs. 94.44 %). The Rv2201-519 group exhibited a propensity for a CD4+ Th1 cell immune response in inoculated BALB/c mice that manifested as higher levels of antigen-specific IgG production (IgG2a/IgG1 > 1). In comparison to Ag85B, Rv2201-519 induced a more robust Th1-type cellular immune response as evidenced by a notable rise in the ratio of IFN-γ/IL-4 and IL-12 cytokine production and increased CD4+ T cell activation with a higher percentage of CD4+IFN-γ+ T cells. Rv2201-519 also induced a higher level of IL-6 compared with Ag85B, a higher percentage of CD8+ T cells specific for Rv2201-519, and a lower percentage of CD8+IL-4+ T cells. Collectively, the current evidence suggests that Rv2201-519 could potentially serve as an immunodominant protein for tuberculosis infection screening, laying the groundwork for further evaluation in recombinant Bacillus Calmette-Guérin (BCG) and subunit vaccines against MTB challenges in future studies.

Comparison of the Immunogenicity and Efficacy of rBCG-EPCP009, BCG Prime-EPCP009 Booster, and EPCP009 Protein Regimens as Tuberculosis Vaccine Candidates.

Bacillus Calmette-Guérin (BCG) is the only widely used prophylactic tuberculosis (TB) vaccine that can prevent severe TB in infants. However, it provides poor protection in adults, and therefore, there is ongoing research into new TB vaccines and immunization strategies with more durable immune effects. The recombinant BCG and BCG prime-protein booster are two important vaccine strategies that have recently been developed based on BCG and could improve immune responses. In this study, three immune strategies based on four protective antigens, namely, ESAT-6, CFP-10, nPPE18, and nPstS1, were applied to construct recombinant rBCG-EPCP009, EPCP009 subunit protein, and BCG prime-EPCP009 booster vaccine candidates. The short- and long-term immune effects after vaccination in Balb/c mice were evaluated based on humoral immunity, cellular immunity, and the ability of spleen cells to inhibit in vitro mycobacterial growth. At 8 and 12 weeks after the initial immunization, splenocytes from mice inoculated with the BCG prime-EPCP009 protein booster secreted higher levels of PPD- and EPCP009-specific IFN-γ, IL-2, TNF-α, IL-17, GM-CSF, and IL-12 and had a higher IFN-γ+CD4+ TEM:IL-2+CD8+ TCM cell ratio than splenocytes from mice inoculated with the rBCG-EPCP009 and EPCP009 proteins. In addition, the EPCPE009-specific IgG2a/IgG1 ratio was slightly higher in the BCG prime-EPCP009 protein booster group than in the other two groups. The in vitro mycobacterial inhibition assay showed that the splenocytes of mice from the BCG prime-EPCP009 protein booster group exhibited stronger inhibition of Mycobacterium tuberculosis (M. tuberculosis) growth than the splenocytes of mice from the other two groups. These results indicate that the BCG prime-EPCP009 protein booster exhibited superior immunogenicity and M. tuberculosis growth inhibition to the parental BCG, rBCG-EPCP009, and EPCP009 proteins under in vitro conditions. Thus, the BCG prime-EPCP009 protein booster may be important for the development of a more effective adult TB vaccine.

A novel multistage antigens ERA005f confer protection against Mycobacterium tuberculosis by driving Th-1 and Th-17 type T cell immune responses.

Introduction: Tuberculosis (TB) is a major threat to human health. In 2021, TB was the second leading cause of death after COVID-19 among infectious diseases. The Bacillus Calmette-Guérin vaccine (BCG), the only licensed TB vaccine, is ineffective against adult TB. Therefore, there is an urgent need to develop new effective vaccines. Methods: In this study, we developed a novel multistage subunit vaccine (ERA005f) comprising various proteins expressed in metabolic states, based on three immunodominant antigens (ESAT-6, Rv2628, and Ag85B). We utilized the E. coli prokaryotic expression system to express ERA005f and subsequently purified the protein using nickel affinity chromatography and anion exchange. Immunogenicity and protective efficacy of ERA005f and ERA005m were evaluated in BALB/c mice. Results: ERA005f was consistently expressed as an inclusion body in a prokaryotic expression system, and a highly pure form of the protein was successfully obtained. Both ERA005f and ERA005m significantly improved IgG titers in the serum. In addition, mice immunized with ERA005f and ERA005m generated higher titers of antigen-specific IgG2a than the other groups. Elispot results showed that, compared with other groups, ERA005f increased the numbers of IFN-γ-secreting and IL-4-secreting T cells, especially the number of IFN-γ-secreting T cells. Meanwhile, ERA005f induced a higher number of IFN-γ+ T lymphocytes than ERA005m did. In addition, ERA005f improved the expression of cytokines, including IFN-γ, IL-12p70, TNF-α, IL-17, and GM-CSF and so on. Importantly, both ERA005f and ERA005m significantly inhibited the growth of Mtb. Conclusion: The novel multistage antigen ERA005f elicited a strong antigen-specific humoral response and Th-1 and Th-17 cell-mediated immunity in mice. Meanwhile, it can effectively inhibit H37Rv growth in vitro, and represents a correlate of protection in vivo, indicating that ERA005f may exhibit excellent protective efficacy against Mycobacterium tuberculosis H37Rv infection. Our study suggests that ERA005f has the potential to be a promising multistage tuberculosis vaccine candidate.

A case report of Andersen-Tawil syndrome misdiagnosed with myodystrophy.

Andersen-Tawil syndrome (ATS) is a rare periodic paralysis caused by the KCNJ2 gene mutation. Here, we report on an ATS patient misdiagnosed with myodystrophy. A 66-year-old man presented with a 60-year history of episodic weakness in the proximal muscles of the upper and lower limbs. The man has been diagnosed with muscle pathology and has undergone genetic examinations in many hospitals since childhood. We conducted a correct diagnosis in combination with the patient's history, electrical physiology, and genetic analysis and identified a heterozygous KCNJ2 gene variant (c.220A > G; p.T74A). Patients with ATS can develop permanent myasthenia characterized by chronic progressive myopathy. ATS patients should also pay special attention to the risks of anesthesia in surgery, including malignant hyperthermia (MH), muscle spasms affecting tracheal intubation or ventilation, and ventilator weakness. Early diagnosis and therapy could help delay the onset of myasthenia and prevent risks associated with anesthesia accidents.

Rapid detection of fluoroquinolone resistance in Mycobacterium tuberculosis using a novel multienzyme isothermal rapid assay.

Simple, rapid, and accurate detection of Fluoroquinolone (FQ) resistance is essential for early initiation of appropriate anti-tuberculosis treatment regimen among rifampicin-resistant tuberculosis (RR-TB). In this study, we developed a new assay, which combines multienzyme isothermal rapid amplification and a lateral flow strip (MIRA-LF), to identify the mutations on codons 90 and 94 of gyrA for detecting levofloxacin (LFX) resistance. Compared to conventional phenotypic drug susceptibility testing, the new assay detected fluoroquinolone resistance with a sensitivity, specificity, and accuracy of 92.4%, 98.5%, and 96.5%, respectively. Thus, these characteristics of the newly developed MIRA-LF assay make it particularly useful and accurate for detecting FQ resistance in Mycobacterium tuberculosis in resource-limited condition.

Association of Single-Nucleotide Polymorphisms in the VDR Gene with Tuberculosis and Infection of Beijing Genotype Mycobacterium tuberculosis.

Background: The aim of the present study was to investigate the association between vitamin D receptor (VDR) gene polymorphism and tuberculosis susceptibility, as well as the potential interaction of host genetic factors with the heterogeneity of Mycobacterium tuberculosis in the population from Xinjiang, China. Methods: From January 2019 to January 2020, we enrolled 221 tuberculosis patients as the case group and 363 staff with no clinical symptoms as the control group from four designated tuberculosis hospitals in southern Xinjiang, China. The polymorphisms of Fok I, Taq I, Apa I, Bsm I, rs3847987 and rs739837 in the VDR were detected by sequencing. M. tuberculosis isolates were collected from the case group and identified as Beijing or non-Beijing lineage by multiplex PCR. Propensity score (PS), univariate analysis and multivariable logistic regression models were used to perform the analysis. Results: Our results showed that the allele and genotype frequencies of Fok I, Taq I, Apa I, Bsm I, rs3847987 and rs739837 in VDR were not correlated with tuberculosis susceptibility or lineages of M. tuberculosis. Two out of six loci of the VDR gene formed one haplotype block, and none of the haplotypes was found to correlate with tuberculosis susceptibility or lineages of M. tuberculosis infected. Conclusion: Polymorphisms in the VDR gene may not indicate susceptibility to tuberculosis. There was also no evidence on the interaction between the VDR gene of host and the lineages of M. tuberculosis in the population from Xinjiang, China. Further studies are nonetheless required to prove our conclusions.

Genome-wide association and epistasis studies reveal the genetic basis of saline-alkali tolerance at the germination stage in rice.

Introduction: Saline-alkali stress is one of the main abiotic factors limiting rice production worldwide. With the widespread use of rice direct seeding technology, it has become increasingly important to improve rice saline-alkali tolerance at the germination stage. Methods: To understand the genetic basis of saline-alkali tolerance and facilitate breeding efforts for developing saline-alkali tolerant rice varieties, the genetic basis of rice saline-alkali tolerance was dissected by phenotyping seven germination-related traits of 736 diverse rice accessions under the saline-alkali stress and control conditions using genome-wide association and epistasis analysis (GWAES). Results: Totally, 165 main-effect quantitative trait nucleotides (QTNs) and 124 additional epistatic QTNs were identified as significantly associated with saline-alkali tolerance, which explained a significant portion of the total phenotypic variation of the saline-alkali tolerance traits in the 736 rice accessions. Most of these QTNs were located in genomic regions either harboring saline-alkali tolerance QTNs or known genes for saline-alkali tolerance reported previously. Epistasis as an important genetic basis of rice saline-alkali tolerance was validated by genomic best linear unbiased prediction in which inclusion of both main-effect and epistatic QTNs showed a consistently better prediction accuracy than either main-effect or epistatic QTNs alone. Candidate genes for two pairs of important epistatic QTNs were suggested based on combined evidence from the high-resolution mapping plus their reported molecular functions. The first pair included a glycosyltransferase gene LOC_Os02g51900 (UGT85E1) and an E3 ligase gene LOC_Os04g01490 (OsSIRP4), while the second pair comprised an ethylene-responsive transcriptional factor, AP59 (LOC_Os02g43790), and a Bcl-2-associated athanogene gene, OsBAG1 (LOC_Os09g35630) for salt tolerance. Detailed haplotype analyses at both gene promoter and CDS regions of these candidate genes for important QTNs identified favorable haplotype combinations with large effects on saline-alkali tolerance, which can be used to improve rice saline-alkali tolerance by selective introgression. Discussion: Our findings provided saline-alkali tolerant germplasm resources and valuable genetic information to be used in future functional genomic and breeding efforts of rice saline-alkali tolerance at the germination stage.

A novel multi-component protein vaccine ECP001 containing a protein polypeptide antigen nPstS1 riching in T-cell epitopes showed good immunogenicity and protection in mice.

Tuberculosis (TB) is an infectious disease that seriously affects human health. Until now, the only anti-TB vaccine approved for use is the live attenuated Mycobacterium bovis (M. bovis) vaccine - BCG vaccine, but its protective efficacy is relatively low and does not provide satisfactory protection against TB in adults. Therefore, there is an urgent need for more effective vaccines to reduce the global TB epidemic. In this study, ESAT-6, CFP-10, two antigens full-length and the T-cell epitope polypeptide antigen of PstS1, named nPstS1, were selected to form one multi-component protein antigens, named ECP001, which include two types, one is a mixed protein antigen named ECP001m, the other is a fusion expression protein antigen named ECP001f, as candidates for protein subunit vaccines. were prepared by constructing one novel subunit vaccine by mixing or fusing the three proteins and combining them with aluminum hydroxide adjuvant, and the immunogenicity and protective properties of the vaccine was evaluated in mice. The results showed that ECP001 stimulated mice to produce high titre levels of IgG, IgG1 and IgG2a antibodies; meanwhile, high levels of IFN-γ and a broad range of specific cytokines were secreted by mouse splenocytes; in addition, ECP001 inhibited the proliferation of Mycobacterium tuberculosis in vitro with a capacity comparable to that of BCG. It can be concluded that ECP001 is a novel effective multicomponent subunit vaccine candidate with potential as BCG Initial Immunisation-ECP001 Booster Immunisation or therapeutic vaccine for M. tuberculosis infection.

Genetic Characterization Conferred Co-Resistance to Isoniazid and Ethionamide in Mycobacterium tuberculosis Isolates from Southern Xinjiang, China.

Background: Ethionamide (ETH), a structural analogue of isoniazid (INH), is used for treating multidrug-resistant tuberculosis (MDR-TB). Due to the common target InhA, INH and ETH showed cross-resistance in M. tuberculosis. This study aimed to explore the INH and ETH resistant profiles and genetic mutations conferring independent INH- or ETH-resistance and INH-ETH cross-resistance in M. tuberculosis circulating in south of Xinjiang, China. Methods: From Sep 2017 to Dec 2018, 312 isolates were included using drug susceptibility testing (DST), spoligotyping, and whole genome sequencing (WGS) to analyze the resistance characteristics for INH and/or ETH. Results: Among the 312 isolates, 185 (58.3%) and 127 (40.7%) belonged to the Beijing family and non-Beijing family, respectively; 90 (28.9%) were INH-resistant (INHR) with mutation rates of 74.4% in katG, 13.3% in inhA and its promoter, 11.1% in ahpC and its upstream region, 2.2% in ndh, 0.0% in mshA, whilst 34 (10.9%) were ETH-resistant (ETHR) with mutation rates of 38.2% in ethA, 26.2% in inhA and its promoter, and 5.9% in ndh, 0.0% in ethR or mshA; and 25 (8.0%) were INH-ETH co-resistant (INHRETHR) with mutation rates of 40.0% in inhA and its promoter, and 8% in ndh. katG mutants tended to display high-level resistant to INH; and more inhA and its promoter mutants showed low-level of INH and ETH resistance. The optimal gene combinations by WGS for the prediction of INHR, ETHR, and INHRETHR were, respectively, katG+inhA and its promoter (sensitivity: 81.11%, specificity: 90.54%), ethA+inhA and its promoter+ndh (sensitivity: 61.76%, specificity: 76.62%), and inhA and its promoter+ndh (sensitivity: 48.00%, specificity: 97.65%). Conclusion: This study revealed the high diversity of genetic mutations conferring INH and/or ETH resistance among M. tuberculosis isolates, which would facilitate the study on INHR and/or ETHR mechanisms and provide clues for choosing ETH for MDR treatment and molecular DST methods in south of Xinjiang, China.

Immunogenicity and efficacy analyses of EPC002, ECA006, and EPCP009 protein subunit combinations as tuberculosis vaccine candidates.

Tuberculosis (TB) is the leading cause of death from infectious diseases worldwide, and developing a new TB vaccine is a priority for TB control. Combining multiple immunodominant antigens to form a novel multicomponent vaccine with broad-spectrum antigens to induce protective immune responses is a trend in TB vaccine development. In this study, we used T-cell epitope-rich protein subunits to construct three antigenic combinations: EPC002, ECA006, and EPCP009. Fusion expression of purified protein EPC002f (CFP-10-linker-ESAT-6-linker-nPPE18), ECA006f (CFP-10-linker-ESAT-6-linker-Ag85B), and EPCP009f (CFP-10-linker-ESAT-6-linker-nPPE18-linker-nPstS1) and recombinant purified protein mixtures EPC002m (mix of CFP-10, ESAT-6, and nPPE18), ECA006m (mix of CFP-10, ESAT-6, and Ag85B), and EPCP009m (mix of CFP-10, ESAT-6, nPPE18, and nPstS1) were used as antigens, formulated with alum adjuvant, and the immunogenicity and efficacy were analyzed using immunity experiments with BALB/c mice. All protein-immunized groups elicited higher levels of humoral immunity, including IgG and IgG1. The IgG2a/IgG1 ratio of the EPCP009m-immunized group was the highest, followed by that of the EPCP009f-immunized group, which was significantly higher than the ratios of the other four groups. The multiplex microsphere-based cytokine immunoassay revealed that EPCP009f and EPCP009m induced the production of a wider range of cytokines than EPC002f, EPC002m, ECA006f, and ECA006m, which included Th1-type (IL-2, IFN-γ, TNF-α), Th2-type (IL-4, IL-6, IL-10), Th17-type (IL-17), and other proinflammatory cytokines (GM-CSF, IL-12). The enzyme-linked immunospot assays demonstrated that the EPCP009f- and EPCP009m-immunized groups had significantly higher amounts of IFN-γ than the other four groups. The in vitro mycobacterial growth inhibition assay demonstrated that EPCP009m inhibited Mycobacterium tuberculosis (Mtb) growth most strongly, followed by EPCP009f, which was significantly better than that of the other four vaccine candidates. These results indicated that EPCP009m containing four immunodominant antigens exhibited better immunogenicity and Mtb growth inhibition in vitro and may be a promising candidate vaccine for the control of TB.

The Evolution and Transmission Dynamics of Multidrug-Resistant Tuberculosis in an Isolated High-Plateau Population of Tibet, China.

On the Tibetan Plateau, most tuberculosis is caused by indigenous Mycobacterium tuberculosis strains with a monophyletic structure and high-level drug resistance. This study investigated the emergence, evolution, and transmission dynamics of multidrug-resistant tuberculosis (MDR-TB) in Tibet. The whole-genome sequences of 576 clinical strains from Tibet were analyzed with the TB-profiler tool to identify drug-resistance mutations. The evolution of the drug resistance was then inferred based on maximum-likelihood phylogeny and dated trees that traced the serial acquisition of mutations conferring resistance to different drugs. Among the 576 clinical M. tuberculosis strains, 346 (60.1%) carried at least 1 resistance-conferring mutation and 231 (40.1%) were MDR-TB. Using a pairwise distance of 50 single nucleotide polymorphisms (SNPs), most strains (89.9%, 518/576) were phylogenetically separated into 50 long-term transmission clusters. Eleven large drug-resistant clusters contained 76.1% (176/231) of the local multidrug-resistant strains. A total of 85.2% of the isoniazid-resistant strains were highly transmitted with an average of 6.6 cases per cluster, of which most shared the mutation KatG Ser315Thr. A lower proportion (71.6%) of multidrug-resistant strains were transmitted, with an average cluster size of 2.9 cases. The isoniazid-resistant clusters appear to have undergone substantial bacterial population growth in the 1970s to 1990s and then subsequently accumulated multiple rifampicin-resistance mutations and caused the current local MDR-TB burden. These findings highlight the importance of detecting and curing isoniazid-resistant strains to prevent the emergence of endemic MDR-TB. IMPORTANCE Emerging isoniazid resistance in the 1970s allowed M. tuberculosis strains to spread and form into large multidrug-resistant tuberculosis clusters in the isolated plateau of Tibet, China. The epidemic was driven by the high risk of transmission as well as the potential of acquiring further drug resistance from isoniazid-resistant strains. Eleven large drug-resistant clusters consisted of the majority of local multidrug-resistant cases. Among the clusters, isoniazid resistance overwhelmingly evolved before all the other resistance types. A large bacterial population growth of isoniazid-resistant clusters occurred between 1970s and 1990s, which subsequently accumulated rifampicin-resistance-conferring mutations in parallel and accounted for the local multidrug-resistant tuberculosis burden. The results of our study indicate that it may be possible to restrict MDR-TB evolution and dissemination by prioritizing screening for isoniazid (INH)-resistant TB strains before they become MDR-TB and by adopting measures that can limit their transmission.

Molecular Epidemiology of Clinical Mycobacterium tuberculosis Isolates from Southern Xinjiang, China Using Spoligotyping and 15-Locus MIRU-VNTR Typing.

Background: In the last decades, the molecular epidemiological investigation of Mycobacterium tuberculosis has significantly increased our understanding of tuberculosis epidemiology. However, few such studies have been done in southern Xinjiang, China. We aimed to clarify the molecular epidemic characteristics and their association with drug resistance in the M. tuberculosis isolates circulating in this area. Methods: A total of 347 isolates obtained from southern Xinjiang, China between Sep, 2017 and Sep, 2019 were included to characterize using a 15-locus MIRU-VNTR (VNTR-15China) typing and spoligotyping, and test for drug susceptibility profiles. Then the lineages and clustering of the isolates were analyzed, as well as their association with drug resistance. Results: Spoligotyping results showed that 60 spoligotype international types (SITs) containing 35 predefined SITs and 25 Orphan or New patterns, and 12 definite genotypes were found, and the top three prevalent genotypes were Beijing genotype (207, 59.7%), followed by CAS1-Delhi (46, 13.6%), and Ural-2 (30, 8.6%). The prevalence of Beijing genotype infection in the younger age group (≤30) was more frequent than the two older groups (30~59 and ≥60 years old, both P values <0.05). The Beijing genotype showed significantly higher prevalence of resistance to isoniazid, rifampicin, ethambutol, multi-drug or at least one drug than the non-Beijing genotype (All P values ≤0.05). The estimated proportion of tuberculosis cases due to transmission was 18.4% according to the cluster rate acquired by VNTR-15China typing, and the Beijing genotype was the risk factor for the clustering (OR 9.15, 95% CI: 4.18-20.05). Conclusion: Our data demonstrated that the Beijing genotype is the dominant lineage, associated with drug resistance, and was more likely to infect young people and contributed to tuberculosis transmission in southern Xinjiang, China. These findings will contribute to a better understanding of tuberculosis epidemiology in this area.

A Multistage Antigen Complex Epera013 Promotes Efficient and Comprehensive Immune Responses in BALB/c Mice.

Tuberculosis (TB) remains a serious global health problem. Despite the widespread use of the Mycobacterium bovis bacillus Calmette-Guerin (BCG) vaccine, the primary factor for the TB pandemic and deaths is adult TB, which mainly result from endogenous reactivation of latent Mycobacterium tuberculosis (MTB) infection. Improved new TB vaccines with eligible safety and long-lasting protective efficacy remains a crucial step toward the prevention and control of TB. In this study, five immunodominant antigens, including three early secreted antigens and two latency associated antigens, were used to construct a single recombinant fusion protein (Epera013f) and a protein mixture (Epera013m). When formulated with aluminum adjuvant, the two subunit vaccines Epera013m and Epera013f were administered to BALB/c mice. The humoral immune responses, cellular responses and MTB growth inhibiting capacity elicited after Epera013m and Epera013f immunization were analyzed. In the present study, we demonstrated that both the Epera013f and Epera013m were capable of inducing a considerable immune response and protective efficacy against H37Rv infection compared with BCG groups. In addition, Epera013f generated a more comprehensive and balanced immune status, including Th1, Th2 and innate immune response, over Epera013f and BCG. The multistage antigen complex Epera013f possesses considerable immunogenicity and protective efficacy against MTB infection ex vivo indicating its potential and promising applications in further TB vaccine development.