Stages Of Mycobacterium Tuberculosis Infection Research Articles

A comprehensive approach to developing a multi-epitope vaccine against Mycobacterium tuberculosis: from in silico design to in vitro immunization evaluation.

The Bacillus Calmette-Guérin (BCG) vaccine, currently used against tuberculosis (TB), exhibits inconsistent efficacy, highlighting the need for more potent TB vaccines. In this study, we employed reverse vaccinology techniques to develop a promising multi-epitope vaccine (MEV) candidate, called PP13138R, for TB prevention. PP13138R comprises 34 epitopes, including B-cell, cytotoxic T lymphocyte, and helper T lymphocyte epitopes. Using bioinformatics and immunoinformatics tools, we assessed the physicochemical properties, structural features, and immunological characteristics of PP13138R. The vaccine candidate demonstrated excellent antigenicity, immunogenicity, and solubility without any signs of toxicity or sensitization. In silico analyses revealed that PP13138R interacts strongly with Toll-like receptor 2 and 4, stimulating innate and adaptive immune cells to produce abundant antigen-specific antibodies and cytokines. In vitro experiments further supported the efficacy of PP13138R by significantly increasing the population of IFN-γ+ T lymphocytes and the production of IFN-γ, TNF-α, IL-6, and IL-10 cytokines in active tuberculosis patients, latent tuberculosis infection individuals, and healthy controls, revealing the immunological characteristics and compare the immune responses elicited by the PP13138R vaccine across different stages of Mycobacterium tuberculosis infection. These findings highlight the potential of PP13138R as a promising MEV candidate, characterized by favorable antigenicity, immunogenicity, and solubility, without any toxicity or sensitization.

Alterations in the intestinal microbiota associated with active tuberculosis and latent tuberculosis infection

ObjectivesTo study the characteristics of intestinal microbiota at different stages of Mycobacterium tuberculosis infection. MethodsFecal samples of 19 active tuberculosis (ATB) patients, 21 latent tuberculosis infection (LTBI) individuals, and 20 healthy controls (HC) were collected. Gut microbiota of all the participants were analyzed by 16S rDNA sequencing. Clinical information of ATB patients was also collected and analyzed. ResultsBoth ATB and LTBI groups showed significant decreases in microbial diversity and decline of Clostridia. For ATB patients, bacteria within phylum Proteobacteria increased. While for LTBI individuals, genera Prevotella and Rosburia enriched. The abundance of Faecalibacterium, Clostridia and Gammaproteobacteria has the potential to diagnose ATB, with the area under the curve (AUC) of 0.808, 0.784 and 0.717. And Prevotella and Rosburia has the potential to diagnose LTBI, with the AUC of 0.689 and 0.689. Notably, in ATB patients, the relative abundance of Blautia was negatively correlated with the proportions of peripheral T cells and CD8+T cells. And serum direct bilirubin was positively correlated with Bacteroidales, while negatively correlated with Clostridiales in ATB patients. ConclusionsThe specifically changed bacteria are promising markers for ATB and LTBI diagnosis. Some gut bacteria contribute to anti-MTB immunity through interactions with T cells and bilirubin.

Accuracy of QuantiFERON-TB Gold Plus Test for Diagnosis of Mycobacterium tuberculosis Infection in Children.

Compared to its predecessor QuantiFERON-TB Gold In Tube (QFT-IT), QuantiFERON-TB Gold Plus (QFT-Plus) contains an additional antigen tube (TB2), stimulating both CD4+ and CD8+ T cells. The ability to discriminate CD4+ and CD8+ responses is suggested to be useful in differentiating stages of Mycobacterium tuberculosis infection. While QFT-Plus has already been evaluated in adults, there are not enough data in children evaluated for suspected active tuberculosis (TB) or latent TB infection (LTBI). A prospective cross-sectional study was conducted among children aged 0 to 17 years who were evaluated for suspected active TB or screened for LTBI. All children underwent QFT-Plus and further clinical, radiological, and/or microbiological analyses according to clinical scenario. Of the 198 children enrolled, 43 (21.7%) were tested because of suspicion of active TB. A total of 12/43 (27.9%) were diagnosed with active TB, and among these, 10/12 (83.3%) had a positive QFT-Plus assay. Of the 155 children screened for LTBI, 18 (11.6%) had a positive QFT-Plus, and 5 (2.5%) had an indeterminate result. TB1 and TB2 quantitative responses were not able to discriminate active disease from latent infection. The percent agreement between TB1 and TB2 was 100%. QFT-Plus assay showed good sensitivity for active TB and was particularly useful for the evaluation of children with suspected LTBI, giving a low rate of indeterminate results in this group. More studies are needed to properly evaluate QFT-Plus ability in discriminating active disease from latent infection.

Construction and Evaluation of a Tuberculosis Multistage Vaccine Candidate Based on Recombinant Listeria ivanovii

To construct a recombinant Listeria ivanovii (LI) strain that expressed Mycobacterium tuberculosis (MTB) specific antigen protein as a novel multistage tuberculosis (TB) vaccine candidate, and evaluate the biosafety and immunogenicity in mouse model. T cell epitopes of four genes related to different stages of MTB infection were fused in series to form an antigen gene, i.e. the multistage antigen gene (named msv). Then msv was inserted into the targeting plasmid that contained LI homologous sequences. Recombinant LI strain was obtained by transfecting LI with targeting plasmid and screening the recombinant LI strain that carried msv in the genome after series of homologous gene recombination processes. The growth rate of the recombinant LI strain in vitro was observed and the expression of target protein was verified by Western blot. The 50% lethal dose (LD 50) of the recombinant strain to C57BL/6 mice was measured. Mice were intravenously inoculated with vaccine candidate in dose of 0.1×LD 50.The serum alanine aminotransferase (ALT) levels, bacterial load in organs, and organ pathological sections before and 1, 2, 3, 5, 7, 14 d after vaccination were used to evaluate the safety of vaccine candidate strain. To analyze the immunogenicity of vaccine candidate strain, mice were intravenously inoculated with LI- msv, LI, and NS respectively. Nine days post immunization, the spleens were isolated under sterile conditions and splenocytes were collected and stimulated. Lyphocytes which secret specific cytokines, interferon (IFN)-γ, tumor necrosis factor (TNF)-α and interleukin (IL)-2 were analyzed by flow cytometry. A recombinant strain named LI- msv which was capable of expressing the multistage TB antigen protein was successfully constructed. The LD 50 value of LI- msv for C57BL/6 mice (i.v.) was 3.3×10 8 CFU. After intravenously immunized the mice, this strain mainly multiplied in the liver and spleen, and was cleared at 7 d post innoculation. Such infection process caused transient pathological damages of the liver and spleen. Results of flow cytometry showed specific IFN-γ + CD4 + and IFN-γ + CD8 +T lymphocytes were successfully induced in LI -msv immunized mice spleen lymphocytes. The frequency of IFN-γ positive CD4 + and CD8 +T cells was significantly higher than those of vector control group and NS control group ( P<0.005). Additionally, the frequency of specific TNF-α + CD4 + T cell in LI -msv immunized group was significantly higher than that of vector control ( P<0.01) and NS control group ( P<0.005), and TNF-α + CD8 + T cell frequency obviously increased than NS control group ( P<0.005). A novel multistage TB vaccine candidate expressing TB multistage antigen based on LI was successfully constructed. This vaccine candidate is safe and can induce specific cellular immune response to some extent. It is promising to be further studied as a candidate vaccine against tuberculosis.

Differed IL-1 Beta Response between Active TB and LTBI Cases by Ex Vivo Stimulation of Human Monocyte-Derived Macrophage with TB-Specific Antigen.

Background The difference of macrophage-specific interleukin-1 beta (IL-1b) response between latent tuberculosis infection (LTBI) and active tuberculosis (TB) remains less studied. Method We performed this prospective study and recruited active TB patients, contacts with LTBI, and uninfected contacts. The gene and protein expression of human monocyte-derived macrophage (hMDM) after ex vivo stimulation by early secretory antigenic target-6KD (ESAT-6) and tuberculin purified protein derivatives (PPD) was studied by real-time PCR and flow cytometry. The effect of caspase-1 inhibitor was also studied. Result The IL-1b gene expression after 6 hr ESAT-6 1 μg/ml stimulation was different among active TB patients (n = 12), LTBI cases (n = 12), and uninfected contacts (n = 23) (log fold change: 0.98 ± 1.26 vs. 2.20 ± 0.96 vs. 2.20 ± 0.96, P = 0.013). The IL-1b gene expression at 24 hours was higher than that at 6 hours in LTBI cases (n = 4) and uninfected contacts (n = 6). After 24 hr ESAT-6 1 μg/ml stimulation, the percentage of IL-1b-expressed hMDM was borderline lower in the active TB patients (n = 9) than in the LTBI cases (n = 10) (14.0 ± 11.2% vs. 31.6 ± 22.5%, P = 0.065). Compared with ESAT-6 1 μg/ml stimulation but without the addition of caspase-1 inhibitor (CasI) (55.6 ± 16.3%), the percentage of IL-1b-positive hMDMs decreased after addition of CasI (50 μg/ml CasI: 49.8 ± 18.2%, P = 0.078; 100 μg/ml CasI: 46.6 ± 20.8%, P = 0.030; 150 μg/ml CasI: 33.7 ± 15.5%, P = 0.016). Conclusions This study revealed that macrophage-specific IL-1b response differed among different stages of Mycobacterium tuberculosis infection. The role of IL-1b and inflammasome in the process of LTBI progressing to active TB warrants further investigation.

Diagnostic performance of plasma cytokine biosignature combination and MCP-1 as individual biomarkers for differentiating stages Mycobacterium tuberculosis infection

We aimed to identify plasma cytokine biomarkers that differentiate the infection stages of Mycobacterium tuberculosis (MTB). This study included a total of 227 subjects consisting of active tuberculosis (ATB) patients, latent tuberculosis infection (LTBI) individuals, and healthy controls (HC). We analyzed the expressions of 38 plasma cytokines in the discovery cohort to identify the biosignatures for differentiating MTB infection states, area under the curve (AUC) were used to evaluate the diagnostic efficiency. The AUC of unique plasma biomarker was confirmed in the validation cohort. In the discovery cohort, the AUC of the 8-marker biosignature (eotaxin, MIP-1α, MDC, IP-10, MCP-1, IL-1α, IL-10, and TNF-α) in diagnosing ATB was 1.0. The sensitivity and specificity of the 5-marker biosignature (IP-10, MCP-1, IL-1α, IL-10, and TNF-α) in diagnosing LTBI were 94% and 81.25%, respectively. The AUC of the 3-signature biosignature (eotaxin, MDC, MCP-1) in differentiating ATB from LTBI was 0.94, with the sensitivity and specificity of 87.76% and 91.84%, respectively. Moreover, among all the single cytokine biomarkers, MCP-1 exhibited the highest AUC in diagnosing ATB (0.98) and differentiating ATB from LTBI (0.91). In the subsequent validation cohort analysis, we identified that the AUC of MCP-1 in diagnosing ATB and differentiating ATB from LTBI were 0.97 and 0.89, respectively, which was generally consistent with the results of the discovery cohort. Cytokine levels in plasma can be used as biosignatures to diagnose ATB. The cytokine concentrations vary during the different stages of MTB infection, which might serve as biomarkers in differentiating ATB from LTBI. Future studies with a larger population and data from multiple institutions are needed to validate our findings.

Cathelicidin LL37: Defense Roles in the Early Stages of Mycobacterium Tuberculosis Infection

Cathelicidin LL37: Defense Roles in the Early Stages of Mycobacterium Tuberculosis Infection

IgG subclasses' response to a set of mycobacterial antigens in different stages of Mycobacterium tuberculosis infection

Despite the reported high heterogeneity of the human immune response to tuberculosis (TB), new studies may contribute to the understanding of Mycobacterium tuberculosis immunopathogenesis. To investigate the patterns of humoral response during latent (LTBI) and active TB, we evaluated specific IgG subclasses' response, by ELISA, to a set of mycobacterial antigens (Rv2029c, Rv2031c, Rv2034, Rv2628, Rv3353c ESAT6:CFP10, and the new chimeric PstS1(285-374):CFP10) in plasma samples from exposed uninfected controls (ExC, n = 24), LTBI (n = 61), and TB (n = 15) donors. In general, the TB group showed statistically higher levels of IgG1, and lower levels of IgG3. Keeping specificities ≥90%, the highest sensitivity for TB detection was observed for IgG1-ESAT6:CFP10 (93.3%), followed by IgG2-Rv3353 (86.7%), IgG1-Rv3353 (69.2%) and IgG1-PstS1(285-374):CFP10 (53.3%). The combinatory of high IgG1-ESAT6:CFP10, followed by low IgG2-Rv3353c titers increased the specificity for TB detection to 100%. Only IgG3-ESAT6:CFP10 showed statistical differences between ExC and LTBI, detecting 50% of the LTBI donors. For the first time, higher levels of IgG2-PstS1(285-374):CFP10 and IgG2-Rv3353 were observed in LTBI and ExC, as compared with a lower or absent immunoreactivity among TB. This study demonstrates differential modulation of subclasses' profiles for the stages of infection, which may contribute to the further development of new diagnostic tools.

Transcriptomic Biomarkers for Tuberculosis: Evaluation of DOCK9. EPHA4, and NPC2 mRNA Expression in Peripheral Blood.

Lately, much effort has been made to find mRNA biomarkers for tuberculosis (TB) disease/infection with microarray-based approaches. In a pilot investigation, through RNA sequencing technology, we observed a prominent modulation of DOCK9, EPHA4, and NPC2 mRNA abundance in the blood of TB patients. To corroborate these findings, independent validations were performed in cohorts from different areas. Gene expression levels in blood were evaluated by quantitative real-time PCR (Brazil, n = 129) or reanalysis of public microarray data (UK: n = 96; South Africa: n = 51; Germany: n = 26; and UK/France: n = 63). In the Brazilian cohort, significant modulation of all target-genes was observed comparing TB vs. healthy recent close TB contacts (rCt). With a 92% specificity, NPC2 mRNA high expression (NPC2high) showed the highest sensitivity (85%, 95% CI 65%–96%; area under the ROC curve [AUROC] = 0.88), followed by EPHA4 (53%, 95% CI 33%–73%, AUROC = 0.73) and DOCK9 (19%, 95% CI 7%–40%; AUROC = 0.66). All the other reanalyzed cohorts corroborated the potential of NPC2high as a biomarker for TB (sensitivity: 82–100%; specificity: 94–97%). An NPC2high profile was also observed in 60% (29/48) of the tuberculin skin test positive rCt, and additional follow-up evaluation revealed changes in the expression levels of NPC2 during the different stages of Mycobacterium tuberculosis infection, suggesting that further studies are needed to evaluate modulation of this gene during latent TB and/or progression to active disease. Considering its high specificity, our data indicate, for the first time, that NPC2high might serve as an accurate single-gene biomarker for TB.

To achieve an earlier IFN-γ response is not sufficient to control Mycobacterium tuberculosis infection in mice.

The temporo-spatial relationship between the three organs (lung, spleen and lymph node) involved during the initial stages of Mycobacterium tuberculosis infection has been poorly studied. As such, we performed an experimental study to evaluate the bacillary load in each organ after aerosol or intravenous infection and developed a mathematical approach using the data obtained in order to extract conclusions. The results showed that higher bacillary doses result in an earlier IFN-γ response, that a certain bacillary load (BL) needs to be reached to trigger the IFN-γ response, and that control of the BL is not immediate after onset of the IFN-γ response, which might be a consequence of the spatial dimension. This study may have an important impact when it comes to designing new vaccine candidates as it suggests that triggering an earlier IFN-γ response might not guarantee good infection control, and therefore that additional properties should be considered for these candidates.