A lack of effective antitumor T-cell immunity often drives immune evasion and immunotherapy resistance. Here, we demonstrated that tumor-intrinsic ARHGEF3 reprogrammed the tumor microenvironment into a T-cell-inflamed state, resulting in potent antitumor effects. Mechanistically, ARHGEF3 functioned as a guanine nucleotide exchange factor that activated the RHOA-ROCK-PTEN cascade and inhibited AKT signaling. This inhibition upregulated IRF1-dependent chemokines CXCL10 and CXCL11 to drive T-cell infiltration, while suppressing FASN-mediated fatty acid synthesis to limit myeloid cell-mediated immunosuppression. The dual effects elicited robust T-cell immunity and overcame tumor resistance to immunotherapy. In human tumors, ARHGEF3 expression correlated positively with T-cell-inflamed signatures, improved clinical outcomes, and responsiveness to immunotherapy. Collectively, these findings identify ARHGEF3 as a key modulator linking chemokine signaling with lipid availability to shape T-cell immunity, offering a promising therapeutic strategy to overcome immunotherapy resistance.

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.

Objective: The interferon-γ (IFN-γ) release assays still fail to meet the diagnostic needs of etiology negative tuberculosis (TB) patients. The purpose of this paper is to explore the value of the IL-2-enzyme-linked immunospot (ELISPOT) assay in the auxiliary diagnosis of TB. Methods: We recruited 127 TB patients, 49 non-TB patients with other diseases (NTB), and 198 healthy people, whose peripheral blood mononuclear cells were isolated and stimulated by Mtb-specific antigen CFP-10/ESAT-6 fusion protein to study the changes of T cells secreting IFN-γ and Interleukin-2 (IL-2) by ELISPOT method. Results: The combination of IFN-γ and IL-2 had an area under the curve (AUC) of 0.867 (IFN-γ: 0.835, IL-2: 0.866) in the diagnosis of TB and could increase the sensitivity of IFN-γ detection alone from 79.5% to 86.6%. The cut-off value for IL-2 and IFN-γ was 16. IL-2 could increase the positive diagnostic rates of IFN-γ-ELISPOT (-) in the etiologically positive and negative ATB patients by 9.4% and 7.1%. In the ATB group, the positive rates and the numbers of spot-forming cells (SFC) of IFN-γ and IL-2 showed significant differences compared to the normal control subjects and NTB group (P<0.05). And SFCs of IFN-γ-ELISPOT were significantly higher than that of IL-2-ELISPOT in ATB patients. Before the treatment, the SFC values of the etiologically positive TB patients were higher than those of the etiologically negative patients. However, this situation was totally reversed after anti-TB treatment. After effective treatment, the positive rates and SFC values of IFN-γ and IL-2 in TB patients decreased and exhibited a decreasing and increasing trend in etiologically positive and negative TB patients, respectively. Conclusions: The combination of IL-2 and IFN-γ could improve the diagnostic sensitivity of single-factor detection in TB patients and these two cytokines could be used as effective biomarkers for the treatment monitoring in TB patients.

ObjectiveTo investigate the influencing factors on the quality of life of patients with pulmonary tuberculosis under initial treatment.MethodsUsing convenience sampling,a total of 288 initial treatment pulmonary tuberculosis patients admitted to a tertiary grade A hospital in Tianjin city from June to December 2023 were selected.All patients were investigated using the general information questionnaire,Hospital Anxiety and Depression Scale(HADS),General Self⁃Efficacy Scale(GSES),Perceived Social Support Scale(PSSS),Mishel Uncertainty in Illness Scale⁃adult(MUIS⁃A),and Quality of Life Instruments for Chronic Diseases⁃Pulmonary Tuberculosis(QLICD⁃PT).ResultsThe QLICD⁃PT score of initial treatment pulmonary tuberculosis patients was (136.14±24.12) points.Pearson correlation analysis showed that the total score of QLICD⁃PT in patients was negatively correlated with the HADS and MUIS⁃A scores(P<0.001),and positively correlated with the PSSS and GSES scores(P<0.001).Structural equation modeling analysis showed that patients' self⁃care ability,perceived social support,and self⁃efficacy directly or indirectly had positive effects on the quality of life.However,anti⁃tuberculosis drug⁃induced liver injury,anxiety and depression,and uncertainty in illness had negative effects on the quality of life.ConclusionsClinical medical staff should pay more attention to the prevention and treatment of liver injury caused by anti⁃tuberculosis drugs.They can provide targeted psychological care based on the characteristics of patients,increase social support,help alleviate anxiety,depression,and uncertainty in illness,enhance self⁃efficacy,and thereby improve patients' quality of life.

Background: Tuberculosis (TB) is a respiratory infectious disease, and the current TB vaccine has low local lung protection. We aim to optimize immune pathways to improve the immunogenicity of vaccines. Methods: In the immunogenicity study, 50 BALB/c mice were randomly divided into the following: (1) phosphate buffered saline (PBS)+intramuscular injection combined with electroporation (EP) group (100 μL), (2) pVAX1+EP group (50 μg/100 μL), (3) ag85ab+EP group (50 μg/100 μL), (4) pVAX1+pulmonary delivery (PD) group (50 μg/50 μL), and (5) ag85ab+PD group (50 μg/50 μL). Immunization was given once every 2 weeks for a total of three times. The number of IFN-γ-secreting lung and spleen lymphocytes was determined by enzyme-linked immunospot assay (ELISPOT). The levels of Th1, Th2, and Th17 cytokines in the culture supernatants of lung and spleen lymphocytes were detected with the Luminex method. The proportion of FoxP3 regulatory T cells in splenocytes was determined by flow cytometry. The levels of IgG-, IgG1-, and IgG2a-specific antibodies in plasma and IgA antibody in bronchoalveolar lavage fluid (BALF) were determined by enzyme-linked immunosorbent assay (ELISA). Results: The PD and EP routes of Mycobacterium tuberculosis (M. tb) ag85ab DNA vaccine can effectively induce the responses of IFN-γ-secreting lung and spleen lymphocytes, and induce dominant Th1 and Th17 cell immune responses. The PD route can induce earlier, greater numbers and stronger responses of pulmonary effector T cells, with higher levels of the specific antibody IgA detected in BALF. High levels of the specific antibodies IgG, IgG1, and IgG2α were detected in the plasma of mice immunized by the EP route. Conclusions: The PD route of DNA vaccines can more effectively stimulate the body to produce strong cellular and mucosal immunity than the EP route, especially local cellular immunity in the lungs, which can provide early protection for the lungs. It can significantly improve the immunogenicity of the ag85ab DNA vaccine, suggesting a feasible and effective approach to DNA immunization.

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.

Objective: This study was aimed at screening differentially expressed genes (DEGs) and exploring the potential immune mechanism induced by the Bacillus Calmette-Guerin (BCG) vaccine in a humanized mouse model. Methods: Candidate DEGs between mice vaccinated with BCG or injected with PBS were identified through transcriptomics, and their biological functions, signaling pathways, and protein interaction networks were analyzed through bioinformatics. Results: A total of 1035 DEGs were identified by transcriptomics: 398 up-regulated and 637 down-regulated. GO analysis indicated that these DEGs were significantly enriched in cell adhesion, oxygen transport, receptor complex, carbohydrate binding, serine-type endopeptidase activity, and peroxidase activity terms. KEGG analysis indicated that these DEGs were involved in the Rap1 signaling pathway, axon guidance, PI3K-Akt signaling pathway, natural killer cell mediated cytotoxicity, and cytokine-cytokine receptor interaction. Protein interaction network analysis demonstrated that the Myc, Vegfa, and Itgb3 proteins had the highest aggregation degree, aggregation coefficient, and connectivity. Conclusion: The BCG vaccine induced 1035 DEGs in humanized mice. Among them, the differentially expressed down-regulated genes myc and itgb3 involved in the PI3K-Akt signaling pathway may play essential roles in the immune mechanism of the BCG vaccine.

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.

目的:对抗结核中药牛贝消核提取物进行血清药物化学初步研究,通过分析入血成分,探讨牛贝消核发挥药效的物质基础.方法:2018年4月至2019年6月在中国人民解放军总医院第八医学中心全军结核病研究所制备桔梗、白及、鱼腥草和牛蒡子标准品溶液及牛贝消核供试品;55只小鼠随机分为11组,每组5只:1～5组为牛贝消核提取物组,每只小鼠每天给予1.25 mg/g牛贝消核提取物灌胃;6～10组为牛蒡苷组,每只小鼠每天给予0.3mg/g牛蒡苷灌胃;11组为空白对照组,给予蒸馏水灌胃;连续灌胃7 d.于末次给药后0.5、1、2、4、6 h小鼠眼眶后静脉丛取血,制备血清供试品溶液.采用高效液相色谱法(high-performance liquid chromatography,HPLC)分析各中药标准品、牛贝消核提取物供试品及各组小鼠给药后血清供试品溶液,分析色谱图中各物质保留时间;然后参照对照品初步鉴定牛贝消核提取物的入血成分.结果:通过牛贝消核提取物和各药材标准品重叠色谱图中可以看出,牛贝消核提取物保留时间为7.1 min处的色谱峰所对应的物质来源于桔梗,保留时间为13.5 min、15.5 min、21.2 min处的色谱峰所对应的物质来源于白及,保留时间为16.3 min的色谱峰所对应的物质来源于牛蒡子,并进一步鉴定为牛蒡苷化合物.牛贝消核提取物和牛蒡苷给药后血清样本与空白血清比较,均在17.5～22.5 min发现有吸收峰.结论:HPLC分析可作为牛贝消核中桔梗、白及、鱼腥草和牛蒡子提取的质量控制方法,检出的牛贝消核提取物血中移行成分可能是牛蒡苷代谢产物,其余药效成分入血微量未检出.

BACKGROUND:Tuberculosis (TB) is a global infectious disease, but there is no ideal vaccine against TB except the Bacille Calmette-Guérin (BCG) vaccine.METHODS:Herein, 25 candidate peptides were predicted from four antigens of Mycobacterium tuberculosis based on their high-affinity binding capacity for the human leukocyte antigen (HLA) DRB1∗0101. Three T-helper 1 (Th1) immunodominant peptides (Ag85B12-26, CFP2112-26, and PPE18149-163) were identified by ELISPOT assays in the humanized C57BL/6 mice. They resulted in a novel Th1 peptide-based vaccine ACP named by the first letter of the three peptides. In addition, the protective efficacy was evaluated in humanized or wild-type C57BL/6 mice and the humoral and cellular immune responses were confirmed in vitro.RESULTS:Compared with the PBS group, the ACP vaccinated mice showed slight decreases in colony-forming units (CFUs) and pathological lesions. However, when using it as a booster, the ACP vaccine did not significantly enhance the protective efficacy of BCG in humanized or wild-type mice. Interestingly, we found that ACP vaccination significantly increased the number of interferon-γ positive (IFN-γ+) T lymphocytes and the levels of IFN-γ cytokines as well as antibodies. Furthermore, the IL-2 level was significantly higher in humanized mice prime-boosted with BCG and ACP.CONCLUSIONS:Our results suggested that ACP vaccination could stimulate higher levels of cytokines and antibodies but failed to improve the protective efficacy of BCG in mice, indicating that the secretion level of IFN-γ may not be positively correlated with the protection efficiency of the vaccine. These findings provided important information on the feasibility of a peptide vaccine as a booster for enhancing the protective efficacy of BCG.

Abstract Background The aim of the present study is to investigate the clinical value and characteristics of peripheral blood lymphocyte subsets in patients with pulmonary tuberculosis (PTB) using flow cytometry. Methods The absolute counts of T, CD4+T, CD8+T, natural killer (NK), NKT and B lymphocytes in 217 cases of PTB were detected, and the variations in lymphocyte subset counts between different ages and genders and between aetiological detection results and chest radiography results were analysed. Results In 75.3% of the patients with PTB, six subset counts were lower than the normal reference range, and 44% showed lower‐than‐normal CD4+T lymphocyte levels. The counts of T, CD4+T, CD8+T and B lymphocytes were significantly lower in patients aged >60 years, and the NKT cell counts were significantly lower in female patients than in male patients. Among the patients with positive aetiological results, 40.8% had reduced CD8+T counts; these were significantly lower than those in patients with negative aetiological results (P = 0.0295). The cell counts of T, CD4+T, CD8+T and B lymphocytes reduced as lesion lobe numbers increased. The counts of T, CD4+T and CD8+T lymphocytes were significantly higher in the group with lesions affecting one lobe than in the groups with two to three lobes or four to five lobes, and the counts of B lymphocytes were significantly higher in the group with one lobe and the group with two to three lobes than in the group with four to five lobes. The counts of CD4+T and CD8+T lymphocytes were highest in the no cavity group and showed a downward trend with the increase in cavities; the T lymphocyte count was significantly higher in the no cavity group than in the group with five or more cavities (P = 0.014), and the CD8+T lymphocyte count was significantly higher in the no cavity group than in the group with one to two cavities and the group with five or more cavities (P = 0.001 and 0.01, respectively). Conclusions In most patients with tuberculosis, immune function is impaired. The absolute counts of peripheral blood lymphocyte subsets are closely related to the aetiological results and lesion severity in patients with PTB; this could be used as evidence for immune intervention and monitoring curative effects.

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.

Latent tuberculosis infection (LTBI) is the primary source of tuberculosis (TB) but there is no suitable detection method to distinguish LTBI from active tuberculosis (ATB). In this study, five antigens of Mycobacterium tuberculosis belonging to LTBI and regions of difference (RDs) were selected to predict Th1 and cytotoxic T lymphocyte (CTL) epitopes. The immunodominant Th1 and CTL peptides were identified in mouse models, and their performance in distinguishing LTBI from ATB was determined in mice and humans. Ten Th1 and ten CTL immunodominant peptides were predicted and synthesized in vitro. The enzyme-linked immunosorbent spot assay results showed that the combination of five Th1 peptides (area under the curve [AUC] = 1, P, 0.0001; sensitivity = 100% and specificity = 93.33%), the combination of seven CTL peptides (AUC = 1, P, 0.0001; 100 and 95.24%), and the combination of four peptide pools (AUC = 1, P, 0.0001; sensitivity = 100% and specificity = 91.67%) could significantly discriminate mice with LTBI from mice with ATB or uninfected controls (UCs). The combined peptides or peptide pools induced significantly different cytokine levels between the three groups, improving their ability to differentiate ATB from LTBI. Furthermore, it was found that pool 2 could distinguish patients with ATB from UCs (AUC = 0.6728, P = 0.0041; sensitivity = 72.58% and specificity = 59.46%). The combination of Th1 and CTL immunodominant peptides derived from LTBI-RD antigens might be a promising strategy for diagnosing ATB and LTBI in mice and patients with ATB and uninfected controls. IMPORTANCE Latent tuberculosis infection (LTBI) is a challenging problem in preventing, diagnosing, and treating tuberculosis (TB). The innate and adaptive immune responses are essential for eliminating or killing the mycobacteria. Antigen-presenting cells (APCs) present and display mycobacterium peptides on their surfaces, and recognition between T cells and APCs is based on some essential peptides rather than the full-length protein. Therefore, the selection of candidate antigens and the prediction and screening of potential immunodominant peptides have become a key to designing a new generation of TB diagnostic biomarkers. This study is the first to report that the combination of Th1 and CTL immunodominant peptides derived from LTBI-RD antigens can distinguish LTBI from active TB (ATB) in animals and ATB patients from uninfected individuals. These findings provide a novel insight for discovering potential biomarkers for the differential diagnosis of ATB and LTBI in the future.

BackgroundTuberculosis (TB) is still a global infectious disease that seriously threatens human beings. The only licensed TB vaccine Bacille Calmette-Guérin (BCG)’s protective efficacy varies significantly among populations and regions. It is very urgent to develop more effective vaccines.MethodsIn this study, eleven candidate proteins of Mycobacterium tuberculosis were selected to predict peptides with high-affinity binding capacity for the HLA-DRB1*01:01 molecule. The immunodominant peptides were identified with the enzyme-linked immunospot assay (ELISPOT) and linked in silico to result in a novel polypeptide vaccine in Escherichia coli cells. The vaccine’s protective efficacy was evaluated in humanized and wild-type C57BL/6 mice. The potential immune protective mechanisms were explored with Enzyme-linked Immunosorbent Assay (ELISA), flow cytometry, and ELISPOT.ResultsSix immunodominant peptides screened from 50 predicted peptides were used to construct a new polypeptide vaccine named MP3RT. After challenge with M. tuberculosis, the colony-forming units (CFUs), lung lesion area, and the number of inflammatory cells in humanized mice rather than wild-type mice vaccinated with MP3RT were significantly lower than these in mice immunized with PBS. The humanized mice vaccinated with MP3RT revealed significant increases in IFN-γ cytokine production, IFN-γ+ T lymphocytes, CD3+IFN-γ+ T lymphocytes, and the MP3RT-specific IgG antibody.ConclusionsTaken together, MP3RT is a promising peptides-based TB vaccine characterized by inducing high levels of IFN-γ and CD3+IFN-γ+ T lymphocytes in humanized mice. These new findings will lay a foundation for the development of peptides-based vaccines against TB.

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 The traditional Chinese medicine NiuBeiXiaoHe (NBXH) extract and Chinese medicine preparation JieHeWan (JHW) exhibit anti-tuberculosis effects. The anti- tuberculosis effect of NBXH was compared with that of JHW to elucidate the mechanism of action of NBXH. Methods BALB/c mice aged 6-8 weeks were randomly divided into a normal control group, Tuberculosis (TB) model group, JHW treatment group, and NBXH treatment group. After 3 and 13 weeks of treatment, the therapeutic effect in each group was evaluated by comparing lung histopathology, lung and liver colony counts, the number of spots representing effector T cells secreting IFN-γ in an ELISPOT, and the levels of Th1, Th2, and Th17 cytokines, which were measured by a cytometric bead array (CBA). Mouse RNA samples were subjected to transcriptome sequencing. Results After 13 weeks of treatment, the mean histopathological lesion area of the NBXH group was significantly smaller than that of the TB model group ( P < 0.05). Compared with those in the TB model group, the lung colony counts in the JHW and NBXH groups were significantly decreased ( P < 0.05), and the IL-2 and IL-4 levels in the NBXH group were significantly increased ( P < 0.05). NBXH partly restored significant changes in gene expression caused by Mycobacterium tuberculosis ( M. tuberculosis ) infection. According to GO and KEGG analyses, the changes in biological process (BP), cell composition (CC) and molecular function (MF) terms and in signaling pathways caused by NBXH and JHW treatment were not completely consistent, but they were mainly related to the immune response and inflammatory response in the mouse TB model. Conclusions NBXH had therapeutic effects similar to those of JHW in improving lung histopathology, reducing lung colony counts, and regulating the levels of cytokines. NBXH restored significant changes in gene expression and repaired cell damage caused by M. tuberculosis infection by regulating immune-related pathways, which clarified the mechanism of action of NBXH.

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.