Latent Mycobacterium Tuberculosis Infection Research Articles

Assay for interferon gamma release as a novel marker in pediatric patients with systemic lupus erythematosus

BackgroundThe interferon-gamma (IFN-γ) release assay (IGRA) is an important laboratory diagnosis for latent Mycobacterium tuberculosis (TB) infection. The TB-IGRA measures the release of IFN-γ from peripheral blood cells, who are exposed to TB antigen (Ag), mitogen (MT), or negative/nil control (NL) in vitro. While, an exceptional higher TB Ag-NL level will reflect an elevation of peripheral lymphocytes released IFN-γ in a same condition. Therefore, we found that the elevated levels of TB Ag-NL could become a new biomarker for the diagnosis and treatment of pediatric systemic lupus erythematosus (SLE) patients.MethodsWe have analyzed the clinical data of 776 children who are underwent TB-IGRA testing in the Department of Allergy and Rheumatology of Guangzhou Women and Children’s Medical Center from 2018 to 2020. To investigate the association between TB Ag-NL and SLE, we have analyzed the clinical data of 47 SLE patients and TB Ag-NL testing results, and then evaluated the association between TB Ag-NL and SLE disease activity.ResultsThe TB Ag-NL levels were significantly higher in patients with active SLE than those in inactive SLE (p = 0.0002). The TB Ag-NL levels were positively correlated with the SLE disease activity index (SLEDAI) and laboratory diagnosis parameters. The mean value of TB Ag-NL in SLE patients (0.04191 ± 0.07955, IU/mL) were significantly higher than those in patients with juvenile dermatomyositis (JDM) (0.0158 ± 0.0337, IU/mL, p = 0.036), juvenile idiopathic arthritis (JIA) (0.0162 ± 0.0388, IU/mL, p = 0.001), and healthy controls (HC) (0.0001 ± 0.0027, IU/mL, p = 0.0003). Therefore, the elevated TB Ag-NL levels could serve as a potential diagnostic biomarker of SLE, especially for the active SLE.ConclusionThe detection of IFN-γ release levels by the TB-IGRA may be useful to assess SLE disease activity in pediatric patients with active SLE.

Specific CD4+ T cell phenotypes associate with bacterial control in people who ‘resist’ infection with Mycobacterium tuberculosis

A subset of individuals exposed to Mycobacterium tuberculosis (Mtb) that we refer to as ‘resisters’ (RSTR) show evidence of IFN-γ− T cell responses to Mtb-specific antigens despite serially negative results on clinical testing. Here we found that Mtb-specific T cells in RSTR were clonally expanded, confirming the priming of adaptive immune responses following Mtb exposure. RSTR CD4+ T cells showed enrichment of TH17 and regulatory T cell-like functional programs compared to Mtb-specific T cells from individuals with latent Mtb infection. Using public datasets, we showed that these TH17 cell-like functional programs were associated with lack of progression to active tuberculosis among South African adolescents with latent Mtb infection and with bacterial control in nonhuman primates. Our findings suggested that RSTR may successfully control Mtb following exposure and immune priming and established a set of T cell biomarkers to facilitate further study of this clinical phenotype.

Exosomal Small RNA Sequencing Profiles in Plasma from Subjects with Latent Mycobacterium tuberculosis Infection.

Despite huge efforts, tuberculosis (TB) is still a major public health threat worldwide, with approximately 23% of the human population harboring a latent TB infection (LTBI). LTBI can reactivate and progress to active and transmissible TB disease, contributing to its spread within the population. The challenges in diagnosing and treating LTBI patients have been major factors contributing to this phenomenon. Exosomes offer a novel avenue for investigating the process of TB infection. In this study, we conducted small RNA sequencing to investigate the small RNA profiles of plasma exosomes derived from individuals with LTBI and healthy controls. Our findings revealed distinct miRNA profiles in the exosomes between the two groups. We identified 12 differentially expressed miRNAs through this analysis, which were further validated via qRT-PCR using the same exosomes. Notably, six miRNAs (hsa-miR-7850-5p, hsa-miR-1306-5p, hsa-miR-363-5p, hsa-miR-374a-5p, hsa-miR-4654, has-miR-6529-5p, and hsa-miR-140-5p) exhibited specifically elevated expression in individuals with LTBI. Gene ontology and KEGG pathway analyses revealed that the targets of these miRNAs were enriched in functions associated with ferroptosis and fatty acid metabolism, underscoring the critical role of these miRNAs in regulating the intracellular survival of Mycobacterium tuberculosis (Mtb). Furthermore, our results indicated that the overexpression of miR-7850-5p downregulated the expression of the SLC11A1 protein in both Mtb-infected and Mtb-uninfected THP1 cells. Additionally, we observed that miR-7850-5p promoted the intracellular survival of Mtb by suppressing the expression of the SLC11A1 protein. Overall, our findings provide valuable insights into the role of miRNAs and repetitive region-derived small RNAs in exosomes during the infectious process of Mtb and contribute to the identification of potential molecular targets for the detection and diagnosis of latent tuberculosis.

Active Tuberculosis Is Associated with Depletion of HIV-Specific CD4 and CD8 T Cells in People with HIV.

Infection with Mycobacterium tuberculosis (Mtb) in people with HIV (PWH) is associated with depletion of Mtb-specific CD4 T cell responses, increased risk of progression to active tuberculosis (TB) disease, and increased immune activation. Although higher HIV viral loads have been reported in Mtb/HIV co-infection, the extent to which Mtb infection and TB disease impact the frequency and phenotype of HIV-specific T cell responses has not been well described. We enrolled a cohort of PWH in Kenya across a spectrum of Mtb infection states, including those with no evidence of Mtb infection, latent Mtb infection (LTBI), and active pulmonary TB disease, and evaluated the frequency, immune activation, and cytotoxicity phenotype of HIV-specific CD4 and CD8 T cell responses in peripheral blood by flow cytometry. We found evidence of depletion of HIV-specific CD4 and CD8 T cells in people with TB, but not with LTBI. Expression of the immune activation markers human leukocyte antigen-DR isotype (HLA-DR) and Ki67 and of the cytotoxic molecules granzyme B and perforin were increased in total CD4 and CD8 T cell populations in individuals with TB, although expression of these markers by HIV-specific CD4 and CD8 T cells did not differ by Mtb infection status. These data suggest that TB is associated with overall increased T cell activation and cytotoxicity and with depletion of HIV-specific CD4 and CD8 T cells, which may contribute to further impairment of T cell-mediated immune control of HIV replication in the setting of TB.

Integrated Analysis of Single-Cell and Bulk RNA Sequencing Data Reveals Memory-like NK Cell Subset Associated with Mycobacterium tuberculosis Latency.

Natural killer (NK) cells are innate-like lymphocytes that belong to the family of type-1 innate lymphoid cells and rapidly respond to virus-infected and tumor cells. In this study, we have combined scRNA-seq data and bulk RNA-seq data to define the phenotypic and molecular characteristics of peripheral blood NK cells. While the role of NK cells in immune surveillance against virus infections and tumors has been well established, their contribution to protective responses to other intracellular microorganisms, such as Mycobacterium tuberculosis (Mtb), is still poorly understood. In this study, we have combined scRNA-seq data and bulk RNA-seq data to illuminate the molecular characteristics of circulating NK cells in patients with active tuberculosis (TB) disease and subjects with latent Mtb infection (LTBI) and compared these characteristics with those of healthy donors (HDs) and patients with non-TB other pulmonary infectious diseases (ODs). We show here that the NK cell cluster was significantly increased in LTBI subjects, as compared to patients with active TB or other non-TB pulmonary diseases and HD, and this was mostly attributable to the expansion of an NK cell population expressing KLRC2, CD52, CCL5 and HLA-DRB1, which most likely corresponds to memory-like NK2.1 cells. These data were validated by flow cytometry analysis in a small cohort of samples, showing that LTBI subjects have a significant expansion of NK cells characterized by the prevalence of memory-like CD52+ NKG2C+ NK cells. Altogether, our results provide some new information on the role of NK cells in protective immune responses to Mtb.

Individualized lipid profile in urine-derived extracellular vesicles from clinical patients with Mycobacterium tuberculosis infections.

Lipids are a key nutrient source for the growth and reproduction of Mycobacterium tuberculosis (Mtb). Urine-derived extracellular vesicles (EVs), because of its non-invasive sampling, lipid enrichment, and specific sorting character, have been recognized as a promising research target for biomarker discovery and pathogenesis elucidation in tuberculosis (TB). We aim to profile lipidome of Mtb-infected individuals, offer novel lipid signatures for the development of urine-based TB testing, and provide new insights into the lipid metabolism after Mtb infection. Urine-derived extracellular vesicles from 41 participants (including healthy, pulmonary tuberculosis, latent tuberculosis patients, and other lung disease groups) were isolated and individually detected using targeted lipidomics and proteomics technology platforms. Biomarkers were screened by multivariate and univariate statistical analysis and evaluated by SPSS software. Correlation analyses were performed on lipids and proteins using the R Hmisc package. Overall, we identified 226 lipids belonging to 14 classes. Of these, 7 potential lipid biomarkers for TB and 6 for latent TB infection (LTBI) were identified, all of which were classified into diacylglycerol (DAG), monoacylglycerol (MAG), free fatty acid (FFA), and cholesteryl ester (CE). Among them, FFA (20:1) was the most promising biomarker target in diagnosing TB/LTBI from other compared groups and also have great diagnostic performance in distinguishing TB from LTBI with AUC of 0.952. In addition, enhanced lipolysis happened as early as individuals got latent Mtb infection, and ratio of raft lipids was gradually elevated along TB progression. This study demonstrated individualized lipid profile of urinary EVs in patients with Mtb infection, revealed novel potential lipid biomarkers for TB/LTBI diagnosis, and explored mechanisms by which EV lipid raft-dependent bio-processes might affect pathogenesis. It lays a solid foundation for the subsequent diagnosis and therapeutic intervention of TB.

Evaluation of a Lateral Flow Assay for Rapid and Simple Detection of IFN-γ for the Diagnosis of Latent Mycobacterium tuberculosis Infection

Evaluation of a Lateral Flow Assay for Rapid and Simple Detection of IFN-γ for the Diagnosis of Latent Mycobacterium tuberculosis Infection

Mucosal and systemic antigen-specific antibody responses correlate with protection against active tuberculosis in nonhuman primates

Increasing evidence supports that antibodies can protect against active tuberculosis (TB) but knowledge of potentially protective antigens, especially in the airways, is limited. The main objective of this study was to identify antigen-specific airway and systemic immunoglobulin isotype responses associated with the outcome of controlled latent Mycobacterium tuberculosis (Mtb) infection (LTBI) versus uncontrolled infection (TB) in nonhuman primates. In a case-control design, using non-parametric group comparisons with false discovery rate adjustments, we assessed antibodies in 57 cynomolgus macaques which, following low-dose airway Mtb infection, developed either LTBI or TB. We investigated airway and systemic IgG, IgA, and IgM responses in paired bronchoalveolar lavage and plasma samples prior to, two-, and 5-6-months post Mtb infection using an antigen-unbiased approach with Mtb glycan and proteome-wide microarrays. Macaques that developed LTBI (n=36) had significantly increased airway and plasma IgA reactivities to specific arabinomannan (AM) motifs prior to Mtb infection compared to those that developed TB (n=21; p<0.01, q<0.05). Furthermore, LTBI macaques had higher plasma IgG reactivity to protein MTB32A (Rv0125) early post Mtb infection (p<0.05) and increasing airway IgG responses to some proteins over time. Our results support a protective role of pre-existing mucosal (lung) and systemic IgA to specific Mtb glycan motifs, suggesting that prior exposure to nontuberculous mycobacteria could be protective against TB. They further suggest that IgG to Mtb proteins early post infection could provide an additional protective mechanism. These findings could inform TB vaccine development strategies. NIH/NIAID AI117927, AI146329, and AI127173 to JMA.

Nursing professionals’ leading role in expanding the treatment for latent Mycobacterium tuberculosis infection in Brazil

Nursing professionals’ leading role in expanding the treatment for latent Mycobacterium tuberculosis infection in Brazil

Assessment of the Cepheid 3-gene Host Response Fingerstick Blood Test (MTB-HR) on rapid diagnosis of tuberculosis

ABSTRACT The World Health Organization has identified high-priority target product profiles for new TB diagnostics which include rapid biomarker-based, non-sputum-based diagnostic testing, using an easily accessible sample. The Cepheid 3-gene Host Response Fingerstick Blood Prototype Test (MTB-HR) quantifies relative mRNA levels of a 3-gene signature (GBP5, DUSP3, and KLF2) from a whole-blood sample on the GeneXpert platform. The objective of the present study was to evaluate the performance of the MTB-HR to distinguish between active tuberculosis (ATB), latent Mycobacterium tuberculosis infection (LTBI), other pulmonary diseases, and healthy volunteers at a tertiary care centre. Among 653 participants enrolled in this study, 192 were diagnosed as having ATB, and the remaining 461 were classified as non-ATB, including 137 cases of LTBI, 224 cases of other pulmonary diseases, and 100 healthy volunteers. The corresponding AUCs of the MTB-HR in distinguishing untreated ATB from non-ATB, LTBI, other pulmonary diseases, and healthy volunteers were 0.814 (95% CI, 0.760-0.868, sensitivity 76.1%, specificity 71.6%), 0.739 (95% CI, 0.667-0.812, sensitivity 59.7%, specificity 78.1%), 0.825 (95% CI, 0.770-0.880, sensitivity 82.1%, specificity 65.6%), 0.892 (95% CI, 0.839-0.945, sensitivity 76.1%, specificity 88.0%), respectively. When only samples with TAT of less than 1 h were included, the AUC of the MTB-HR in distinguishing untreated ATB from non-ATB was largest, 0.920 (95% CI, 0.822-1.000, sensitivity 81.3%, specificity 87.7%). In conclusion, the MTB-HR assay shows potential as a rapid, blood-based screening and triage test for ATB, especially for untreated ATB, with the advantage of increased diagnostic yield since blood is more readily available.

P-472 Evaluation of the usefulness of the QuantiFERON-TB gold in-tube test for the detection of latent mycobacterium tuberculosis infections in patients underwent autologous stem cell transplantation

P-472 Evaluation of the usefulness of the QuantiFERON-TB gold in-tube test for the detection of latent mycobacterium tuberculosis infections in patients underwent autologous stem cell transplantation

Mycobacterium tuberculosis impairs human memory CD4+ T cell recognition of M2 but not M1-like macrophages

Direct recognition of Mycobacterium tuberculosis (Mtb)-infected cells is required for protection by CD4+ Tcells. While impaired Tcell recognition of Mtb-infected macrophages was demonstrated in mice, data are lacking for humans. Using Tcells and monocyte-derived macrophages (MDMs) from individuals with latent Mtb infection (LTBI), we quantified the frequency of memory CD4+ Tcell activation in response to autologous MDMs infected with virulent Mtb. We observed robust Tcell activation in response to Mtb infection of M1-like macrophages differentiated using GM-CSF, while M2-like macrophages differentiated using M-CSF were poorly recognized. However, non-infected GM-CSF and M-CSF MDMs loaded with exogenous antigens elicited similar CD4+ Tcell activation. IL-10 was preferentially secreted by infected M-CSF MDMs, and neutralization improved Tcell activation. These results suggest that preferential infection of macrophages with an M2-like phenotype limits Tcell-mediated protection against Mtb. Vaccine development should focus on Tcell recognition of Mtb-infected macrophages.

The interaction of macrophages and CD8 T cells in bronchoalveolar lavage fluid is associated with latent tuberculosis infection

ABSTRACT Mycobacterium tuberculosis (Mtb) infection, including active tuberculosis (TB) and latent Mtb infection (LTBI), leads to diverse outcomes owing to different host immune responses. However, the immune mechanisms that govern the progression from LTBI to TB remain poorly defined in humans. Here, we profiled the lung immune cell populations within the bronchoalveolar lavage fluid (BALF) from patients with LTBI or TB using single-cell RNA sequencing (scRNA-seq). We found that Mtb infection substantially changed the immune cell compartments in the BALF, especially for the three subsets of macrophages, monocyte macrophage (MM)-CCL23, MM-FCN1, and MM-SPP1, which were found to be associated with the disease status of TB infection. Notably, MM-CCL23 cells derived from monocytes after stimulation with Mtb were characterized by high levels of chemokine (CCL23 and CXCL5) production and might serve as a marker for Mtb infection. The MM-CCL23 population mainly recruited CD8-CCR6 T cells through CCL20/CCR6, which was a prominent feature associated with protection immunity in LTBI. This study improves our understanding of the lung immune landscape during Mtb infection, which may inform future vaccine design for protective immunity.

Reactivation of latent Mycobacterium tuberculosis infection with different outcomes after in vitro fertilization and embryo transfer: two case reports

Reactivation of latent Mycobacterium tuberculosis infection with different outcomes after in vitro fertilization and embryo transfer: two case reports

Prevalence of latent Mycobacterium tuberculosis infection in hematopoietic stem cell transplantation comparing tuberculin skin test and interferon-gamma release assay.

The aim of this study was to evaluate the prevalence of latent Mycobacterium tuberculosis infection in hematopoietic stem cell transplantation candidates, using tuberculin skin test and QuantiFERON-TB Gold-Plus, in a high-burden tuberculosis country. Adult candidates for hematopoietic stem cell transplantation performed both tests before and those submitted to transplantation were followed up for 12 months. The prevalence of latent Mycobacterium tuberculosis infection was 17.1% and a moderate agreement between QuantiFERON-TB Gold-Plus and tuberculin skin test was observed in this population. Previous tuberculosis exposure was a risk factor for latent Mycobacterium tuberculosis infection. No cases of tuberculosis were diagnosed during follow-up period.

Inhibition of Mycobacterium tuberculosis resuscitation-promoting factor B (RpfB) by microbially derived natural compounds: a computational study

The majority of the world population (around 25%) has latent Mycobacterium tuberculosis (Mtb) infection, among which only 5–10% of individuals develop active tuberculosis (TB), and 90–95% continue to have latent tuberculosis infection. This makes it the biggest global health concern. It has been reported that the resuscitation-promoting factor B (RpfB) is an exciting potential target for tuberculosis drug discovery due to its significant role in the reactivation of latent TB infection to an active infection. Several attempts have been made to investigate potential inhibitors against RpfB utilizing in-silico approaches. The present study also utilized a computational approach to investigate microbially derived natural compounds against the Mtb RpfB protein which is a very cost-effective This evaluation used structure-based virtual screening (SBVS), drug-likeness profiling, molecular docking, molecular dynamics simulation, and free-binding energy calculations. Six potential natural compounds, viz. Cyclizidine I, Boremexin C, Xenocoumacin 2, PM-94128, Cutinostatin B, and (+)1-O-demethylvariecolorquinone A were selected, which displayed a potential binding affinity between −52.39 and −60.87 Kcal/mol MMGBSA score and docking energy between −7.307 Kcal/mol to −6.972 Kcal/mol. All the complexes showed acceptable stability (<2.7 Å RMSD) during 100 ns MD simulation time except the RpfB protein-xenocoumacin 2 complex. This result exhibited that the selected compounds have high efficiency in inhibiting the Mtb RpfB and can be taken into account for additional in vitro and in vivo experimental validation. Communicated by Ramaswamy H. Sarma

Combined Detection of IFN-γ and Lymphocyte Subsets with Activation Indicators in the Clinical Application of Mycobacterium Tuberculosis Infection at Different Times

The immune status of mycobacterium tuberculosis (MTB) infection is essential for the diagnosis and treatment of this disease. In this work, we aim to evaluate the clinical significance of the combination of serum IFN-γ, IGRAs (Interferon-Gamma Release Assay), lymphocyte subset with activation indicators detection in active and latent tuberculosis infection patients. For this study, anticoagulant whole blood were collected from 45 active tuberculosis (AT group), 44 latent tuberculosis (LT group) and 32 healthy controls (HCs group). The serum IFN-γ and IGRAs detected by chemiluminescence, and the percentage of lymphocyte subsets and activated lymphocytes detected by flow cytometry. The results showed combined IGRAs, serum IFN-γ and NKT cells not only has good diagnostic efficiency for the AT, but also provides a laboratory diagnostic method to distinguish AT from LT. Activation indicator of CD3+HLA-DR+T and CD4+HLA-DR+T can effectively distinguish LT from HCs. While combined CD3+T, CD4+T, CD8+CD28+T, Treg and CD16+CD56+CD69+ cells can distinguish AT from HCs. This study showed combined direct detection of serum IFN-γ and IGRAs as well as lymphocyte subsets with activation indicators which may provide laboratory basis for the diagnosis and differential diagnosis of active and latent MTB infection.

A pro-oxidant property of vitamin C to overcome the burden of latent Mycobacterium tuberculosis infection: A cross-talk review with Fenton reaction.

Tuberculosis (TB), caused by the bacillus M. tuberculosis, is one of the deadliest infectious illnesses of our day, along with HIV and malaria.Chemotherapy, the cornerstone of TB control efforts, is jeopardized by the advent of M. tuberculosis strains resistant to many, if not all, of the existing medications.Isoniazid (INH), rifampicin (RIF), pyrazinamide, and ethambutol are used to treat drug-susceptible TB for two months, followed by four months of INH and RIF, but chemotherapy with potentially harmful side effects is sometimes needed to treat multidrug-resistant (MDR) TB for up to two years. Chemotherapy might be greatly shortened by drugs that kill M. tuberculosis more quickly while simultaneously limiting the emergence of drug resistance.Regardless of their intended target, bactericidal medicines commonly kill pathogenic bacteria (gram-negative and gram-positive) by producing hydroxyl radicals via the Fenton reaction.Researchers have concentrated on vitamins with bactericidal properties to address the rising cases globally and have discovered that these vitamins are effective when given along with first-line drugs. The presence of elevated iron content, reactive oxygen species (ROS) generation, and DNA damage all contributed to VC's sterilizing action on M. tb in vitro. Moreover, it has a pleiotropic effect on a variety of biological processes such as detoxification, protein folding - chaperons, cell wall processes, information pathways, regulatory, virulence, metabolism etc.In this review report, the authors extensively discussed the effects of VC on M. tb., such as the generation of free radicals and bactericidal mechanisms with existing treatments, and their further drug development based on ROS production.

Differentially expressed transcript isoforms associate with resistance to tuberculin skin test and interferon gamma release assay conversion.

A mechanistic understanding of uncommon immune outcomes such as resistance to infection has led to the development of novel therapies. Using gene level analytic methods, we previously found distinct monocyte transcriptional responses associated with resistance to Mycobacterium tuberculosis (Mtb) infection defined as persistently negative tuberculin skin test (TST) and interferon gamma release assay (IGRA) reactivity among highly exposed contacts (RSTR phenotype). Using transcript isoform analyses, we aimed to identify novel RSTR-associated genes hypothesizing that previous gene-level differential expression analysis obscures isoform-specific differences that contribute to phenotype. Monocytes from 49 RSTR versus 52 subjects with latent Mtb infection (LTBI) were infected with M. tuberculosis (H37Rv) or left unstimulated (media) prior to RNA isolation and sequencing. RSTR-associated gene expression was then identified using differential transcript isoform analysis. We identified 81 differentially expressed transcripts (DETs) in 70 genes (FDR <0.05) comparing RSTR and LTBI phenotypes with the majority (n = 79 DETs) identified under Mtb-stimulated conditions. Seventeen of these genes were previously identified with gene-level bulk RNAseq analyses including genes in the IFNγ response that had increased expression among LTBI subjects, findings consistent with a clinical phenotype based on IGRA reactivity. Among the subset of 23 genes with positive differential expression among Mtb-infected RSTR monocytes, 13 were not previously identified. These novel DET genes included PDE4A and ZEB2, which each had multiple DETs with higher expression among RSTR subjects, and ACSL4 and GAPDH that each had a single transcript isoform associated with RSTR. Transcript isoform-specific analyses identify transcriptional associations, such as those associated with resistance to TST/IGRA conversion, that are obscured when using gene-level approaches. These findings should be validated with additional RSTR cohorts and whether the newly identified candidate resistance genes directly influence the monocyte Mtb response requires functional study.

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.