BACKGROUND:The global tuberculosis (TB) epidemic remains severe. We aimed to develop a therapeutic DNA vaccine as an adjunct to TB treatment to improve efficacy. METHODS:The W545 DNA vaccine was constructed using the M. tuberculosis (MTB) antigens Ag85A and Rv1419, integrated with epitopes from the Ag85B, Rv3407, and Rv2628. Bioinformatics tools were used to predict and analyze the physicochemical properties, structure modelling and molecular docking, epitopes (HTL, CTL, and B-cell), safety, population coverage, and simulated immunization of the W545 vaccine protein. Animal studies were then performed to evaluate the vaccine's immunogenicity by measuring Th1-type immune responses (IFN-γ, IL-2) and IgG antibody levels, as well as its therapeutic efficacy in reducing lung inflammation and pathological damage in a murine TB model. RESULTS:The vaccine protein is a 70 kDa hydrophilic protein with a half-life of 30 h, an instability index of 43.33, and strong affinity to Toll-like receptor (TLR) 2 and TLR4. It contains 397 helper T cell (HTL) epitopes, 248 cytotoxic T cell (CTL) epitopes, and 27 B cell epitopes, with broad population coverage (global: 99.7 %, Chinese: 97.6 %). The W545 vaccine significantly induced a Th1-type immune response, producing high levels of IFN-γ (5.38 pg/ml ± 0.89 pg/ml) and IgG antibodies (OD450: 0.13 ± 0.06). It also reduced the lung weight index, tissue lesions, and severity in the murine TB model. CONCLUSION:The W545 DNA vaccine effectively induces a Th1-type immune response, alleviates pathological damage, and demonstrates potential as an immunotherapeutic agent. Bioinformatics analysis provides valuable guidance for vaccine design and optimization.

BACKGROUND:Mycobacterium tuberculosis (MTB) Ag85A has become a component of multiple new tuberculosis vaccines. It is necessary to evaluate the immunogenicity, biological distribution, and safety of ag85a plasmid DNA (pDNA) to lay the foundation for the design of new vaccines. METHOD:Chronic toxicity test: cynomolgus monkeys were injected intramuscularly with different doses of ag85a pDNA, and the vaccine absorption kinetics, tissue distribution, and toxicity were observed. Their immune function was evaluated. Acute toxicity test: Mice were injected intramuscularly 0.5 ml saline, and injected intramuscularly and intravenously 0.5 mg/0.5 ml ag85a pDNAs, respectively. The toxicity and death of the mice were observed continuously for 14 days. Allergic test: Guinea pigs were intraperitoneally injected with different doses of ag85a pDNA. After stimulation, the allergic reaction and its severity were observed. RESULTS:Chronic and acute toxicity tests demonstrated that ag85a pDNA injections caused no clinical symptoms or tissue damage. Repeated intramuscular injections in cynomolgus monkeys enhanced specific Th1 immune responses, with pDNA rapidly entering the bloodstream and its concentration positively correlating with dosage. After 8 weeks, ag85a gene was detected only in muscles, myocardium, iliac lymph nodes, and blood. Guinea pig allergy tests showed no weight changes or allergic reactions, even after multiple sensitizations. CONCLUSIONS:The ag85a pDNA showed good safety in cynomolgus monkeys, mice, and guinea pigs, and induced high levels of antibodies and T-cell responses, making it a candidate antigen for the construction of a new tuberculosis vaccine.

Rifampin (RIF) resistance in Mycobacterium tuberculosis (M.tb) is primarily caused by mutations in the rpoB gene. Rapid and accurate detection of RIF resistance is critical for effective tuberculosis (TB) control. Nucleotide matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is an emerging technology used to detect RIF resistance-associated rpoB mutations in 210 M.tb clinical isolates, including 107 RIF-sensitive and 103 RIF-resistant strains, as determined by phenotypic drug susceptibility testing (DST). DNA sequencing was used as the reference method to validate nucleotide MALDI-TOF MS results. Nucleotide MALDI-TOF MS demonstrated a sensitivity of 93.2%, specificity of 98.1%, and an overall accuracy of 95.7% compared to phenotypic DST. The Kappa value between nucleotide MALDI-TOF MS and phenotypic DST was 0.91, indicating excellent agreement. DNA sequencing confirmed that nucleotide MALDI-TOF MS successfully identified RIF resistance-associated mutations, particularly in codons 450, 445, and 435 of the rpoB gene. Among the 61 isolates analyzed by DNA sequencing, nucleotide MALDI-TOF MS and sequencing results were consistent for 52 of 56 RIF-resistant strains and all five RIF-sensitive strains, with an overall concordance of 93.4%. Importantly, nucleotide MALDI-TOF MS accurately detected heteroresistance in eight isolates (14.3%), confirmed by sequencing. These results support that nucleotide MALDI-TOF MS is a rapid, accurate, and reliable method for detecting rpoB mutations associated with RIF resistance in M.tb. Its high concordance with DNA sequencing, excellent diagnostic performance, and ability to identify heteroresistance highlight its potential as a valuable tool for early TB diagnostics and improve the precision of chemotherapy regimen development.IMPORTANCEThe emergence of multidrug-resistant tuberculosis (MDR-TB) and rifampin-resistant tuberculosis (RR-TB) poses a significant challenge to global tuberculosis (TB) control efforts. Rifampin (RIF) resistance is a critical marker for MDR-TB, which requires more complex, prolonged, and costly treatment regimens. Early and accurate detection of RIF resistance is crucial for effective TB control. This study evaluates the performance of nucleotide MALDI-TOF MS, an innovative technology, for detecting RIF resistance-associated mutations in the rpoB gene. The method demonstrates high sensitivity (93.2%) and specificity (98.1%), with the added advantage of identifying heteroresistance, capabilities that are lacking in conventional methods. These capabilities are crucial for early diagnosis, guiding personalized treatment regimens, and curbing the transmission of drug-resistant TB. The findings demonstrate that nucleotide MALDI-TOF MS provides a rapid, high-throughput, and cost-effective alternative for detecting rpoB gene mutations associated with RIF resistance.

Our previous research developed a novel tuberculosis (TB) DNA vaccine ag85a/b showed a significant therapeutic effect on the mouse tuberculosis model by intramuscular injection (IM) and electroporation (EP). However, the action mechanisms between these two vaccine immunization methods remain unclear. In a previous study, 96 M. tuberculosis (MTB) H37Rv-infected BALB/c mice were treated with PBS, 10μg, 50μg, 100μg, and 200μg ag85a/b DNA vaccine delivered by IM and EP three times at two-week intervals, respectively. In this study, peripheral blood mononuclear cells (PBMCs) from 3 mice in each group were isolated to extract total RNA. The gene expression profiles were analyzed using gene microarray technology to obtain differentially expressed (DE) genes. Finally, DE genes were validated by real-time reverse transcription-quantitive PCR (RT-qPCR) and the GEO database. After MTB infection, most of the up-regulated DE genes were related to the digestion and absorption of nutrients or neuroendocrine, for example, Iapp, Scg2, Chga, Amy2a5, etc, and most of the down-regulated DE genes were related to cellular structural and functional proteins, especially the structure and function proteins of alveolar epithelial cell, for example, Sftpc, Sftpd, Pdpn, etc. Most of the abnormally up-regulated or down-regulated DE genes in the TB model group were recovered in the 100μg and 200μg ag85a/b DNA IM groups and four DNA EP groups. The pancreatic secretion pathway down-regulated and Rap1 signal pathway up-regulated had particularly significant changes during the immunotherapy of the ag85a/b DNA vaccine on the mouse TB model. The action target and mechanism of IM and EP are highly consistent. Tuberculosis infection caused rapid catabolism and slow anabolism in mice. For the first time, we found that the effective dose of the ag85a/b DNA vaccine immunized whether by IM or EP could significantly up-regulate immune-related pathways and recover the metabolic disorder and the injury caused by MTB.

ABSTRACTBackgroundVaccination is one of the effective measures to prevent latent tuberculosis infection (LTBI) from developing into active tuberculosis (TB). Applying bioinformatics methods to pre‐evaluate the biological characteristics and immunogenicity of vaccines can improve the efficiency of vaccine development.ObjectivesTo evaluate the immunogenicity of TB vaccine W541 and to explore the application of bioinformatics technology in TB vaccine research.MethodsThis study concatenated the immunodominant sequences of Ag85A, Ag85B, Rv3407, and Rv1733c to construct the W541 DNA vaccine. Then, bioinformatics methods were used to analyze the physicochemical properties, antigenicity, allergenicity, toxicity, and population coverage of the vaccine, to identify its epitopes, and to perform molecular docking with MHC alleles and Toll‐like receptor 4 (TLR4) of the host. Finally, the immunogenicity of the vaccine was evaluated in animal experiments.ResultsThe W541 vaccine protein is a soluble cytoplasmic protein with a half‐life of 1.1 h in vivo and an instability index of 45.37. It has good antigenicity and wide population coverage without allergenicity and toxicity. It contains 138 HTL epitopes, 73 CTL epitopes, 8 linear and 14 discontinuous B cell epitopes, and has a strong affinity for TLR4. Immune simulations have shown that it can effectively stimulate innate and adaptive immune responses. Animal experiments confirmed that the W541 DNA vaccine could effectively activate Th1‐ and Th17‐type immune responses, producing high levels of IFN‐γ and IL‐17A, but could not significantly increase antibody levels.ConclusionThe W541 DNA vaccine can induce strong cellular immune responses. However, further optimization of the vaccine design is needed to make the expressed protein more stable in vivo. Bioinformatics analysis could reveal the physicochemical and immunological information of vaccines, which is critical for guiding vaccine design and development.

Our previous research developed a novel tuberculosis (TB) DNA vaccine ag85a/b that showed a significant therapeutic effect on the mouse tuberculosis model by intramuscular injection (IM) and electroporation (EP). However, the action mechanisms between these two vaccine immunization methods remain unclear. In a previous study, 96 Mycobacterium tuberculosis (MTB) H37 Rv-infected BALB/c mice were treated with phosphate-buffered saline, 10, 50, 100, and 200 μg ag85a/b DNA vaccine delivered by IM and EP three times at 2-week intervals, respectively. In this study, peripheral blood mononuclear cells (PBMCs) from three mice in each group were isolated to extract total RNA. The gene expression profiles were analyzed using gene microarray technology to obtain differentially expressed (DE) genes. Finally, DE genes were validated by real-time reverse transcription-quantitive polymerase chain reaction and the GEO database. After MTB infection, most of the upregulated DE genes were related to the digestion and absorption of nutrients or neuroendocrine (such as Iapp, Scg2, Chga, Amy2a5), and most of the downregulated DE genes were related to cellular structural and functional proteins, especially the structure and function proteins of the alveolar epithelial cell (such as Sftpc, Sftpd, Pdpn). Most of the abnormally upregulated or downregulated DE genes in the TB model group were recovered in the 100 and 200 μg ag85a/b DNA IM groups and four DNA EP groups. The pancreatic secretion pathway downregulated and the Rap1 signal pathway upregulated had particularly significant changes during the immunotherapy of the ag85a/b DNA vaccine on the mouse TB model. The action targets and mechanisms of IM and EP are highly consistent. Tuberculosis infection causes rapid catabolism and slow anabolism in mice. For the first time, we found that the effective dose of the ag85a/b DNA vaccine immunized whether by IM or EP could significantly up-regulate immune-related pathways and recover the metabolic disorder and the injury caused by MTB.

BackgroundAbout a quarter of the world’s population with latent tuberculosis infection (LTBI) are the main source of active tuberculosis. Bacillus Calmette Guerin (BCG) cannot effectively control LTBI individuals from developing diseases. Latency-related antigens can induce T lymphocytes of LTBI individuals to produce higher IFN-γ levels than tuberculosis patients and normal subjects. Herein, we firstly compared the effects of M. tuberculosis (MTB) ag85ab and 7 latent DNA vaccines on clearing latent MTB and preventing its activation in the mouse LTBI model.MethodsA mouse LTBI model was established, and then immunized respectively with PBS, pVAX1 vector, Vaccae vaccine, ag85ab DNA and 7 kinds of latent DNAs (including rv1733c, rv2660c, rv1813c, rv2029c, rv2628, rv2659c and rv3407) for three times. The mice with LTBI were injected with hydroprednisone to activate the latent MTB. Then, the mice were sacrificed for the bacterial count, histopathological examination, and immunological evaluation.ResultsUsing chemotherapy made the MTB latent in the infected mice, and then using hormone treatment reactivated the latent MTB, indicating that the mouse LTBI model was successfully established. After the mouse LTBI model was immunized with the vaccines, the lung colony-forming units (CFUs) and lesion degree of mice in all vaccines group were significantly decreased than those in the PBS group and vector group (P<0.0001, P<0.05). These vaccines could induce antigen-specific cellular immune responses. The number of IFN-γ effector T cells spots secreted by spleen lymphocytes in the ag85ab DNA group was significantly increased than those in the control groups (P<0.05). In the splenocyte culture supernatant, IFN-γ and IL-2 levels in the ag85ab, rv2029c, and rv2659c DNA groups significantly increased (P<0.05), and IL-17A levels in ag85ab and rv2659c DNA groups also significantly increased (P<0.05). Compared with the PBS and vector groups, the proportion of CD4+CD25+FOXP3+ regulatory T cells in spleen lymphocytes of ag85ab, rv2660c, rv2029c, and rv3407 DNA groups were significantly reduced (P<0.05).ConclusionsMTB ag85ab and 7 kinds of latent DNA vaccines showed immune preventive efficacies on a mouse model of LTBI, especially the rv2659c, and rv1733c DNA. Our findings will provide candidates for the development of new multi-stage vaccines against TB.

Background: Verapamil enhances the sensitivity of Mycobacterium tuberculosis to anti-tuberculosis (TB) drugs, promotes the macrophage anti-TB ability, and reduces drug resistance, but its mechanism is unclear. Herein, we have investigated the effect of verapamil on cytokine expression in mouse peritoneal macrophages. Methods: Macrophages from mice infected with M. tuberculosis or S. aureus were cultured with verapamil, the cytokines were detected by enzyme-linked immunosorbent assay, and the RNA was measured with quantitative real-time polymerase chain reaction and agarose gel electrophoresis. The intracellular calcium signaling was measured by confocal microscopy. Results: Significantly higher levels of NF-κB, IL-12, TNF-α, and IL-1β were observed after TB infection. The levels of NF-κB and IL-12 increased when verapamil concentration was less than 50 μg/ml, but decreased when verapamil concentration was greater than 50μg/ml. With the increase in verapamil concentration, TNF-α and IL-1β expressed by macrophages decreased. The L-type calcium channel transcription significantly increased in M. tuberculosis rather than S. aureus-infected macrophages. Furthermore, during bacillus Calmette-Guerin (BCG) infection, verapamil stimulated a sharp peak in calcium concentration in macrophages, while calcium concentration increased mildly and decreased smoothly over time in the absence of verapamil. Conclusions: Verapamil enhanced macrophage immunity via the NF-κB pathway, and its effects on cytokine expression may be achieved by its regulation of intracellular calcium signaling.

Background Tuberculosis (TB) is a major global public health problem. New treatment methods on TB are urgently demanded. Methods Ninety-six female BALB/c mice were challenged with 2×10 4 colony-forming units (CFUs) of MTB H 37 Rv through tail vein injection, then was treated with 10μg, 50μg, 100μg, and 200μg of Mycobacterium tuberculosis (MTB) ag85a/b chimeric DNA vaccine delivered by intramuscular injection (IM) and electroporation (EP), respectively. The immunotherapeutic effects were evaluated immunologically, bacteriologically, and pathologically. Results Compared with the phosphate-buffered saline (PBS) group, the CD4 + IFN-γ + T cells% in whole blood from 200 µg DNA IM group and four DNA EP groups increased significantly ( P <0.05), CD8 + IFN-γ + T cells% (in 200 μg DNA EP group), CD4 + IL-4 + T cells% (50 μg DNA IM group) and CD8 + IL-4 + T cells% (50 μg and 100 μg DNA IM group, 100 μg and 200 μg DNA EP group) increased significantly only in a few DNA groups ( P < 0.05). The CD4 + CD25 + Treg cells% decreased significantly in all DNA vaccine groups ( P <0.01). Except for the 10 μg DNA IM group, the lung and spleen colony-forming units (CFUs) of the other seven DNA immunization groups decreased significantly ( P <0.001, P <0.01), especially the 100 μg DNA IM group and 50 μg DNA EP group significantly reduced the pulmonary bacterial loads and lung lesions than the other DNA groups. Conclusions An MTB ag85a/b chimeric DNA vaccine could induce Th1-type cellular immune reactions. DNA immunization by EP could improve the immunogenicity of the low-dose DNA vaccine, reduce DNA dose, and produce good immunotherapeutic effects on the mouse TB model, to provide the basis for the future human clinical trial of MTB ag85a/b chimeric DNA vaccine.

Background. The Traditional Chinese Medicine NiuBeiXiaoHe (NBXH) is a valid antituberculosis (TB) prescription from the experience of clinical practice. However, the mechanism of NBXH extracts’ immunotherapy has been poorly understood. Herein, the immunotherapeutic efficacy and the differentially expressed (DE) genes of NBXH extracts were evaluated and identified in BALB/c mice. Methods. The total RNA was extracted from peripheral blood mononuclear cells, and the DE genes were identified by gene chip. The enrichment and signaling pathway analyses were performed using Gene Ontology (GO) and KEGG database. Results. It was shown that the treatment of NBXH extracts (high dose) significantly reduced mycobacteria loads and histopathological lesions in mice infected by Mycobacterium tuberculosis and resulted in 3,454 DE upregulated genes and 3,594 downregulated DE genes. Furthermore, NBXH extracts killed mycobacteria by inhibiting the supply of necessary ingredients for their growth and proliferation. They restored the disordered immune microenvironments by up- or downregulating immune and inflammation-related pathways. Conclusions. Taken together, NBXH extracts not only efficiently decreased the mycobacteria loads but also balanced the immune disorders in mice. These new findings provide a fresh perspective for elucidating the immunotherapeutic mechanism of NBXH extracts and pointed out the direction for improving the treatment efficacy of NBXH extracts.

Background: Immune- and inflammation-related genes （IIRGs） play an important role in the pathogenesis of tuberculosis （TB）. However, the relationship between IIRG polymorphisms and TB risk remains unknown. In this study, the gene polymorphisms and their association with tuberculosis were determined in a Chinese population.Methods: We performed a case-control study involving 1016 patients with TB and 507 healthy controls of Han Chinese origin. Sixty-four single-nucleotide polymorphisms （SNPs） belonging to 18 IIRGs were genotyped by the PCR-MassArray assay, and the obtained data was analyzed with χ2-test, Bonferroni correction, and unconditional logistic regression analysis.Results: We observed significant differences in the allele frequency ofLTA rs2229094*C （P = 0.015）,MBL2 rs2099902*C （P = 0.001）,MBL2 rs930507*G （P = 0.004）,MBL2 rs10824793*G （P = 0.004）, andIL12RB1 rs2305740*G （P = 0.040） between the TB and healthy groups. Increased TB risk was identified in the rs930507 G/G genotype （Padjusted = 0.027） under a codominant genetic model as well as in the rs2099902 （C/T + C/C） vs T/T genotype （Padjusted = 0.020）, rs930507 （C/G + G/G） vs C/C genotype （Padjusted = 0.027）, and rs10824793 （G/A + G/G） vs A/A genotype （Padjusted = 0.017） under a dominant genetic model after Bonferroni correction in the analysis of the overall TB group rather than the TB subgroups. Furthermore, the rs10824793 r s7916582*GT and rs10824793 r s7916582*GC haplotypes were significantly associated with increased TB risk （P = 0.001, odds ratio [OR] = 1.421, 95% confidence interval [CI]: 1.152-1.753; andP = 0.018,OR = 1.364, 95%CI: 1.055-1.765, respectively）. Moreover, the rs10824793 r s7916582*AT/AT or rs10824793 r s7916582*GT/GT diplotype showed a protective （P = 0.003,OR = 0.530, 95%CI: 0.349-0.805） or harmful （P = 0.009,OR = 1.396, 95%CI: 1.087-1.793） effect against the development of TB.Conclusions: This study indicated thatMBL2 polymorphisms, haplotypes, and diplotypes were associated with TB susceptibility in the Han Chinese population. Additionally, larger sample size studies are needed to further confirm these findings in the future.

Background: Tuberculosis is a leading cause of death worldwide. BCG is an effective vaccine, but not widely used in many parts of the world due to a variety of issues. Mycobacterium vaccae（M. vaccae） is another vaccine used in human subjects to prevent tuberculosis. In the current study, we investigated the potential mechanisms of M. vaccae vaccination by determining differentially expressed genes in mice infected with M. tuberculosis before and after M. vaccae vaccination.Methods: Three days after exposure to M. tuberculosis H37 Rv strain（5×10~5 CFU）, adult BALB/c mice randomly received either M. vaccae vaccine（22.5 μg） or vehicle via intramuscular injection（n=8）. Booster immunization was conducted 14 and 28 days after the primary immunization. Differentially expressed genes were identified by microarray followed by standard bioinformatics analysis.Results: M. vaccae vaccination provided protection against M. tuberculosis infection（most prominent in the lungs）. We identified 2,326 upregulated and 2,221 downregulated genes in vaccinated mice. These changes could be mapped to a total of 123 signaling pathways（68 upregulated and 55 downregulated）. Further analysis pinpointed to the MyD88-dependent TLR signaling pathway and PI3 K-Akt signaling pathway as most likely to be functional.Conclusions: M. vaccae vaccine provided good protection in mice against M. tuberculosis infection, via a highly complex set of molecular changes. Our findings may provide clue to guide development of more effective vaccine against tuberculosis.

Additional file 2: Table S1. Raw and log2 value of the normalized intensity of each sample in the control group and the M. vaccae group.

The currently purified protein derivative (PPD) skin test and 2 interferon (IFN)-γ release assays (IGRAs) were usually used to detect Mycobacterium tuberculosis infection. We try to evaluate the performance of these methods to detect latent tuberculosis infection (LTBI) in this study. Each subject of the 876 recruits (19.05 ± 1.55, 17-24) underwent the PPD test, enzyme-linked immunospot (ELISPOT) assay, and chemiluminescent enzyme immunoassay (CLEIA). The prevalence of LTBI among the participants, as estimated by PPD, ELISPOT, and CLEIA, was 49.89% (437/876), 25.34% (222/876), and 28.77% (252/876), respectively. Of the participants, positive results were noted in 12.79% (112/876) for both ELISPOT and PPD, 19.52% (171/876) for both CLEIA and PPD; 9.82% (86/876) for 2 IGRAs; and 6.62% (58/876) for all 3 methods. Overall, the consistency among the 3 tests was 36.99% (324/876). ELISPOT-positive rate (41.38%) in the recruits with a PPD result ≥20 mm was higher than PPD <20 mm (24.76%; P < 0.05). Increased PPD skin reactions were associated with significantly increased CLEIA-positive rates and IFN-γ levels. Of 307 recruits without the bacillus Calmette-Guérin (BCG) vaccination, 2 IGRA (42.19%)-positive rates in the PPD-positive group were significantly higher than those in the PPD-negative group (28.40% and 23.05%; P < 0.05 and P < 0.01, respectively).There was low correlation and poor consistency among 2 IGRAs and PPD in healthy recruits, but IGRAs may be more accurate screening methods for TB infection in the countries with BCG vaccination.

In this study, the Mycobacterium tuberculosis (MTB) latency-associated antigens Rv2660c, Rv1733c, Rv1813c, Rv2628, Rv2029c, and Rv2659c were compared regarding their immunogenicity and potential therapeutic effects in an MTB reactivation mouse model. Normal mice or MTB reactivation mice were immunized intramuscularly three times at 2-week intervals with saline, plasmid vector pVAX1, Mycobacterium vaccae vaccine (a commercial inactivated vaccine), rv1813c DNA, rv2628 DNA, rv2029c DNA, rv2659c DNA, rv1733c DNA, or rv2660c DNA. The normal mice immunized with rv2628 DNA or rv2659c DNA had low numbers of Th1 cells and a lower ratio of Th1:Th2 immune cells in whole blood (p < 0.05). Compared to the saline group, Tc1 cells in the rv2029c DNA group and Tc1:Tc2 cell ratio in the rv1813c DNA, rv2628 DNA, and rv2029c DNA groups were significantly decreased (p < 0.05). The proportion of Foxp3+CD4+ T cells in the rv2628 DNA and rv2659c DNA groups and the proportion of CD4+CD25+ T cells in the rv2029c DNA group were significantly increased (p < 0.05). The level of anti-Rv1813c-immunoglobulin G (IgG) in the rv1813c DNA group was significantly increased (p < 0.01). The levels of specific IgG, IgG1, and IgG2a in the rv2628 DNA, rv2029c DNA, and rv2659c DNA groups were significantly increased (p < 0.05). Lung colony-forming units in M. vaccae and the six DNA groups decreased to different degrees in the MTB reactivation mouse model, but only the lung colony-forming units in the rv2628 DNA group (4.38 ± 0.70 log10) significantly decreased compared to the vector group (5.90 ± 0.42 log10; p < 0.05). The MTB rv1813c DNA, rv2628 DNA, rv2029c DNA, and rv2659c DNA could elicit a strong humoral immune response and a higher proportion of CD4+CD25+or CD4+Foxp3+ T cells but could not increase the proportions of Th1 and Tc1 cells. These results suggest that latency-associated DNA vaccines, especially rv2628 DNA, had some therapeutic effect on the endogenous resurgence mouse tuberculosis model.

Relapse of pulmonary tuberculosis (PTB) is associated with a failure of the host immune system to control the invading Mycobacterium tuberculosis. Severe immunodeficiency or immune disorders may be the main reason for TB recurrence. This study aimed to quantify serum inflammatory cytokine and soluble adhesion molecule levels in Re-treated smear-positive PTB patients before and after re-anti-TB drug therapy. Serum samples were collected from 30 healthy controls and 215 Treated active PTB patients at baseline and 2, 4, and 6 months post-re treatment. Levels of 18 serum cytokines and soluble adhesion molecules were measured by a high-throughput Cytometric Bead Array. At baseline, IL-1, IL-2, IL-12P70, and soluble CD62E levels were significantly higher in PTB patients than those in the healthy controls (p < 0.05); IL-4, IL-5, IL-7, IL-8, IL-10, IL-17, IL-21, soluble CD54, MIG, and TGF-beta levels in PTB patients were significantly lower than those in the healthy controls (p < 0.05), of which TGF-beta, IL-7, IL-8, IL-10, soluble CD54, and MIG were most notably (p < 0.0005). After re treatment, IFN-gamma, IL-2, IL-7, and soluble CD54 levels and IL-2/IL-10 and IFN-gamma/IL-10 ratios showed an upward trend during the re-treatment period. They were more sensitive than other cytokines and adhesion molecules and could be effective as serum indicators for re-treatment response. The immune response was imbalance in treated smear-positive PTB patients: Th1 response was elevated, but Th2 and Th17 responses were reduced. Systematic and comprehensive understanding of the cytokine and soluble adhesion molecule profiles provides a theoretical basis for immuno-diagnosis, immunotherapy, and immuno-monitoring of Re-treated PTB patients.

耐药结核病(TB)尤其是耐多药结核病(MDR-TB)、广泛耐药结核病(XDR-TB)的快速诊断和有效治疗是TB防控中亟需解决的难题.结核分枝杆菌(Mtb)耐药的主要机制是由于药物作用靶标或药物代谢酶编码基因突变所致.临床上常用的Mtb药物敏感性试验方法主要包括表型药敏方法和分子药敏方法,本文还简要地概述了通过药敏试验所揭示的一些值得临床医师关注的临床意义.

Objective To study the clinical value of Mycobacterium tuberculosis IgG and IgM antibody detection reagents.Methods The clinical value was detected in two study periods.In the first study period,the patients who visited the 309th Hospital of Chinese PLA in May 2014 were included.By retrospectively analyzing the medical records,there were 92 patients with pulmonary tuberculosis (TB) and 99 patients without TB.Ninety-four healthy volunteers in the same period were selected as controls.In the second study period,118 patients who visited the 309th Hospital of Chinese PLA between December 2016 and March 2017 were collected,including 62 cases with pulmonary TB and 56 patients without TB.The samples of all subjects were collected for smear detection and evaluation using Mycobacterium tuberculosis IgG and IgM antibody detection kit (i.e.Immune colloidal gold tech nique).Results In the first study,the sensitivity,specificity,positive predictive value,negative predictive value and consistency rate of IgG and IgM antibody detection were 64.1％ (59/92),89.1 ％ (172/193),73.8％ (59/80),83.9％ (172/205) and 81.1％ (231/285),respectively.Of the 92 pulmonary TB cases,the positive rate of IgG antibody detection was 64.1％.The positive rate was 73.8％ (31/42) in the smear-positive pulmonary TB cases and 56.0％ (28/50) in the smear-negative pulmonary TB cases;the difference was not statistical significant (x2 =3.15,P=0.076).All pulmonary TB cases were detected as negative by IgM antibody detection.In the second study,the sensitivity,specificity,positive predictive value,negative predictive value and consistency rate of IgG and IgM antibody detection for the TB diagnosis were 45.2％ (28/62),78.6％ (44/56),70.7％ (29/41),57.1％ (44/77)and 61.9％ (73/118),respectively.Among the 62 pulmonary TB cases,the positive rate of IgG antibody detection was 45.2％ (28/62).The positive rate was 56.3％ (18/32) in the smear-positive pulmonary TB cases,which was significantly higher than that of 29.2％ (7/24) in the smear-negative pulmonary TB cases (x2 =4.07,P=0.044).Whereas there was only one case detected as positive by IgM antibody detection,yielding a positive rate of 1.6％(1/62).Conclusion The IgG in Mycobacterium tuberculosis IgG and IgM antibody tests had a better auxiliary diagnostic value for active TB,while the clinical value of IgM test requires to be further evaluated.

Tuberculosis (TB) is a major global public health problem. Latent TB infection (LTBI) is a major source of active TB. New vaccines to treat LTBI are urgently demanded. In this study, the gene encoding latency-associated antigen Rv3407 of Mycobacterium tuberculosis (MTB) rv3407 DNA vaccine was used to prepare and the immunogenicity and therapeutic effects were evaluated. Normal mice were immunized intramuscularly three times at two-week intervals with sterile water for injection, plasmid vector pVAX1, M. vaccae vaccine, ag85a DNA or rv3407 DNA. TB-infected mice were immunized intramuscularly three times at two-week intervals with phosphate-buffered saline (PBS) and rv3407 DNA. The normal mice immunized with rv3407 DNA or ag85a DNA showed higher levels of interferon-gamma (IFN-gamma) in stimulated spleen lymphocyte culture supernatants, and had more Th1 cells and an elevated ratio of Th1/Th2 immune cells in whole blood, indicating that a Th1-type immune response was predominant. The levels of anti-Ag85A or anti-Rv3407 IgG antibody were significantly increased in the ag85a DNA and rv3407 DNA groups compared to the sterile water for injection, vector, and M. vaccae groups (p < .0001). Compared with the PBS group, the rv3407 DNA group had pulmonary bacterial loads that were lower by 0.56 log(10) (p < .01)(.) The mice vaccinated with rv3407 DNA developed antigen-specific cellular and humoral responses. The rv3407 DNA is a potential DNA vaccine candidate against TB.