Antigens ESAT-6 Research Articles

Immune profiles of MCP-1 with M tb antigens and recombinant cytokines stimulation in tuberculosis

Tuberculosis is caused by Mycobacterium tuberculosis (M tb), which is recognized by macrophages and produces inflammatory cytokines, and chemokines at the site of infection. The present study was proposed to understand the interaction of M tb antigens, cytokines, and chemokines. We have evaluated the chemokine MCP-1 levels and its expression in PBMCs stimulated with M tb antigens Ag85A, ESAT6 and recombinant cytokines rhTNF-α, rhIFN-γ, rhTGF-β, and rhIL-10 in active pulmonary TB (APTB) patients, household contacts (HHC) at 0 months, 6 months and healthy controls (HC). We have observed low levels of MCP-1 with Ag85A, ESAT6, and rhTNF-α stimulations in APTB 0M compared to HHC and HC (p < 0.0067, p < 0.0001, p < 0.01, p < 0.005, p < 0.0065, p < 0.0001) and significantly increased after treatment with rhTNF-α. The MCP-1 levels with rhIFN-γ were high in APTB, HHC at 0 M and significant between APTB 0 M vs. 6 M, HHC vs. HC, and HHC 0M vs. 6M (p < 0.0352, p < 0.0252, p < 0.00062). The rhTGF-β, rhIL-10 induced high MCP-1 levels in APTB, HHC compared to HC (p < 0.0414, p < 0.0312, p < 0.004, p < 0.0001) and significantly decreased after treatment with rhIL-10 (p < 0.0001). The MCP-1 expression was low with all the stimulations in APTB 0M when compared to HC and after treatment. Whereas, HHC shown low MCP-1 expression with rhTNF-α, rhIFN-γ and Ag85A and high with rhTGF-β, rhIL-10 and ESAT6. In conclusion, the study determined the differential expression and production of MCP-1 with M tb antigens and recombinant cytokines. Further, cohort studies are required to study these interaction to identify the high risk individuals, which might help for TB control.

Diagnostic value of tuberculosis-specific antigens ESAT-6 and CFP10 in lymph node tuberculosis

ObjectiveTo assess the diagnostic value of immunohistochemical (IHC) staining for detecting the tuberculosis-secreted antigens ESAT-6 and CFP10 in lymph node tuberculosis. MethodsArchived, paraffin-embedded lymph node specimens from 72 patients diagnosed with lymph node tuberculosis and 68 patients with lymphoma were retrospectively collected from the Department of Pathology at the Affiliated Hospital of North Sichuan Medical College, Nanchong, Sichuan Province, China between January 2016 and March 2023. These specimens were subjected to acid-fast and immunohistochemical staining to compare the effectiveness of these methods, with their sensitivity and specificity evaluated against a comprehensive reference standard. ResultsAcid-fast staining demonstrated a sensitivity of 12.3% and a specificity of 100%. IHC staining for ESAT-6 showed a sensitivity of 87.5% and a specificity of 85.3%, whereas IHC staining for CFP10 exhibited a sensitivity of 75.0% and a specificity of 89.7%. ConclusionThe study indicates that IHC detection of ESAT-6 and CFP10 in paraffin-embedded lymph node tuberculosis tissues has a markedly higher sensitivity compared to acid-fast staining. Thus, IHC staining may serve as a supplementary diagnostic tool for the pathological evaluation of lymph node tuberculosis.

Intradermal Tuberculin Test in Water Buffalo (Bubalus bubalis): Experimental use of Mycobacterial Antigens for the Diagnosis of Bovine Tuberculosis

The study aims to evaluate the potential use of mycobacterial ESAT6 and CFP10 antigens, Early Secretory Proteins (ESP) in the Skin Test used for bovine tuberculosis (TB) diagnosis in Water Buffalo.&#x0D; A pilot study was performed on 21 buffaloes from a TB outbreak and 11 buffaloes from a TB-free herd. Three concentrations of ESAT6-CFP10 (10, 20, and 30 mg) and two of ESP (50 and 100 µg) were inoculated in the Skin Test, along with PPDB, PPDA, and PBS as a negative control. Skin thickness was measured with calipers before the test and every 24 hours for 4 days. Then, to evaluate the specificity of the antigens, a field study was conducted, and 100 buffaloes from a TB-free herd were inoculated using the best antigens concentration derived from the pilot study.&#x0D; In the positive buffaloes, the strongest skin response was to PPDB at 24h, with some subjects becoming inconclusive at 72 and 96 h. A peak response to PPDA at 48 hours was detected, followed by a slight decrease. The response to ESP-100 µg remained high at 24 and 48 h, then decreased, remaining positive at 72 h. In the 100 TB-free buffaloes, the best specificity was observed using ESAT6-CFP10 and ESP.&#x0D; ESP yielded the best results, showing higher reactivity in infected animals and no reactivity in the healthy ones at 72 h. Therefore, ESP could be an excellent candidate for further extensive studies in the buffalo species to improve Skin Test performance.

Recombinant BCG vaccine expressing multistage antigens of Mycobacterium tuberculosis provides long-term immunity against tuberculosis in BALB/c mice

ABSTRACT Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) persistently kills nearly 1.5 million lives per year in the world, whereas the only licensed TB vaccine BCG exhibits unsatisfactory efficacy in adults. Taking BCG as a vehicle to express Mtb antigens is a promising way to enhance its efficacy against Mtb infection. In this study, the immune efficacy of recombination BCG (rBCG-ECD003) expressing specific antigens ESAT-6, CFP-10, and nDnaK was evaluated at different time points after immunizing BALB/c mice. The results revealed that rBCG-ECD003 induced multiple Th1 cytokine secretion including IFN-γ, TNF-α, IL-2, and IL-12 when compared to the parental BCG. Under the action of PPD or ECD003, rBCG-ECD003 immunization resulted in a significant increase in the proportion of IL-2+ and IFN-γ+IL-2+ CD4+T cells. Importantly, rBCG-ECD003 induced a stronger long-term humoral immune response without compromising the safety of the parental BCG vaccine. By means of the protective efficacy assay in vitro, rBCG-ECD003 showed a greater capacity to inhibit Mtb growth in the long term. Collectively, these features of rBCG-ECD003 indicate long-term protection and the promising effect of controlling Mtb infection.

PPE59 antibodies in tuberculous patients and potential use for diagnosis when assayed with other rapid biomarkers.

PPE 59, which is absent from bacillus Calmette Guérin (BCG) strains, seems to induce a humoral immune response in patients with tuberculosis (TB). Additional studies are needed to better evaluate this protein in immune response to tuberculosis. To evaluate the response of antibodies to PPE59 in TB individuals, its combination with IgG response to other, previously tested mycobacterial antigens (Ag) and with sputum smear microbiology (SM) results. We have cloned and expressed the rv3429 gene that encodes PPE59, then IgG, IgM, and IgA against PPE59 antigens measured by enzyme-linked immunosorbent assay (ELISA) in 212 sera samples obtained from the following subject cohorts: TB residents from Italy (79) and in Brazil (52); and an all-Brazilian cohort of 55 patients with other respiratory disorders; 10 patients infected with non-tuberculous mycobacteria, and 16 asymptomatic subjects. Drawing on results from a previous study(17) of serum samples from Brazilian subjects tested for IgG by ELISA against mycobacterial antigens ESAT-6, 16kDa, MT10.3, MPT-64 and 38kDa, the results were analysed in combination with those of the PPE59 and SM tests. Keeping the specificity rate at 97%, the overall PPE59 IgA sensitivity was 42.7%, while IgG and IgM showed lower performance (p < 0.0001). Combining PPE59 IgA/16kDa IgG results increased sensitivity to 71%, and even higher rates when the results were combined with SM results (86.5%, p = 0.001), at 88.9% specificity. Positive IgA was associated with pulmonary image alterations of high TB probability (p < 0.05). Tests with TB patients found a moderate frequency of positivity for PPE59 IgA. However, the higher level of sensitivity attained in combination with PPE59 IgA/16kDa IgG/SM results unheard of before, although imperfect, suggests that this may be a potential additional tool for rapid detection of TB in low-resource areas.

Fluorescent nanodiamond immunosensors for clinical diagnostics of tuberculosis.

Fluorescent nanodiamonds (FNDs) are carbon nanoparticles containing a dense ensemble of nitrogen-vacancy defects as color centers. These centers have exceptional photostability and unique quantum properties, making them useful for ultrasensitive biosensing applications. This work employed FNDs conjugated with antibodies as magneto-optical immunosensors for tuberculosis (TB) diagnostics using competitive spin-enhanced lateral flow immunoassay (SELFIA). ESAT6 (6-kDa early secretory antigenic target) of Mycobacterium tuberculosis is a clinical marker of TB. We evaluated the assay's performance using the recombinant ESAT6 antigen and its antibodies noncovalently coated on FNDs. A detection limit of ∼0.02 ng mL-1 was achieved with the lateral flow membrane strip pre-structured with a narrow channel of 1 mm width. Adopting a cut-off value of 24.0 ng mm-1 for 100-nm FNDs on the strips, the method detected 49 out of 50 clinical samples with Mycobacterium tuberculosis complexes. In contrast, none of the assays for 10 clinical samples with non-tuberculous mycobacteria (NTM) isolates exhibited the presence of ESAT6. These results suggest that the SELFIA platform is applicable for TB detection and can differentiate TB from NTM infections, which also affect the human respiratory system. The FND-enabled immunosensing techniques are versatile and promising for early detection of TB and other diseases, opening a new avenue for biomedical applications of carbon-based nanomaterials.

Development of a Multiplex HIV/TB Diagnostic Assay Based on the Microarray Technology.

Currently there are diagnostic tests available for human immunodeficiency virus (HIV) and tuberculosis (TB); however, they are still diagnosed separately, which can delay treatment in cases of co-infection. Here we report on a multiplex microarray technology for the detection of HIV and TB antibodies using p24 as well as TB CFP10, ESAT6 and pstS1 antigens on epoxy-silane slides. To test this technology for antigen-antibody interactions, immobilized antigens were exposed to human sera spiked with physiological concentrations of primary antibodies, followed by secondary antibodies conjugated to a fluorescent reporter. HIV and TB antibodies were captured with no cross-reactivity observed. The sensitivity of the slides was compared to that of high-binding plates. We found that the slides were more sensitive, with the detection limit being 0.000954 µg/mL compared to 4.637 µg/mL for the plates. Furthermore, stability studies revealed that the immobilized antigens could be stored dry for at least 90 days and remained stable across all pH and temperatures assessed, with pH 7.4 and 25 °C being optimal. The data collectively suggested that the HIV/TB multiplex detection technology we developed has the potential for use to diagnose HIV and TB co-infection, and thus can be developed further for the purpose.

Quantitation of Circulating Mycobacterium tuberculosis Antigens by Nanopore Biosensing in Children Evaluated for Pulmonary Tuberculosis in South Africa.

Nanopore sensing of proteomic biomarkers lacks accuracy due to the ultralow abundance of targets, a wide variety of interferents in clinical samples, and the mismatch between pore and analyte sizes. By converting antigens to DNA probes via click chemistry and quantifying their characteristic signals, we show a nanopore assay with several amplification mechanisms to achieve an attomolar level limit of detection that enables quantitation of the circulating Mycobacterium tuberculosis (Mtb) antigen ESAT-6/CFP-10 complex in human serum. The assay's nonsputum-based feature and low-volume sample requirements make it particularly well-suited for detecting pediatric tuberculosis (TB) disease, where establishing an accurate diagnosis is greatly complicated by the paucibacillary nature of respiratory secretions, nonspecific symptoms, and challenges with sample collection. In the clinical assessment, the assay was applied to analyze ESAT-6/CFP-10 levels in serum samples collected during baseline investigation for TB in 75 children, aged 0-12 years, enrolled in a diagnostic study conducted in Cape Town, South Africa. This nanopore assay showed superior sensitivity in children with confirmed TB (94.4%) compared to clinical "gold standard" diagnostic technologies (Xpert MTB/RIF 44.4% and Mtb culture 72.2%) and filled the diagnostic gap for children with unconfirmed TB, where these traditional technologies fell short. We envision that, in combination with automated sample processing and portable nanopore devices, this methodology will offer a powerful tool to support the diagnosis of pulmonary TB in children.

The Immunogenicity and Safety of Mycobacterium tuberculosis-mosR-Based Double Deletion Strain in Mice.

Mycobacterium tuberculosis (M. tuberculosis) remains a significant global health threat, accounting for ~1.7 million deaths annually. The efficacy of the current vaccine, M. bovis BCG, ranges from 0 to 80% in children and does not prevent adulthood tuberculosis. We explored the immune profile and safety of a live-attenuated M. tuberculosis construct with double deletions of the mosR and echA7 genes, where previously, single mutations were protective against an M. tuberculosis aerosol challenge. Over 32 weeks post-vaccination (WPV), immunized mice with M. tuberculosisΔmosRΔechA7 (double mutant) were sacrificed to evaluate the vaccine persistence, histopathology, and immune responses. Interestingly, despite similar tissue colonization between the vaccine double mutant and wild-type M. tuberculosis, the vaccine construct showed a greater reaction to the ESAT-6, TB.10, and Ag85B antigens with peptide stimulation. Additionally, there was a greater number of antigen-specific CD4 T cells in the vaccine group, accompanied by significant polyfunctional T-cell responses not observed in the other groups. Histologically, mild but widely distributed inflammatory responses were recorded in the livers and lungs of the immunized animals at early timepoints, which turned into organized inflammatory foci via 32WPV, a pathology not observed in BCG-immunized mice. A lower double-mutant dose resulted in significantly less tissue colonization and less tissue inflammation. Overall, the double-mutant vaccine elicited robust immune responses dominated by antigen-specific CD4 T cells, but also triggered tissue damage and vaccine persistence. The findings highlight key features associated with the immunogenicity and safety of the examined vaccine construct that can benefit the future evaluation of other live vaccines.

Safety and Protective Efficacy of a Candidate Vector-Based Vaccine for Bovine Tuberculosis.

This study presents the results of a survey of the safety and protective efficacy of a candidate vector-based vaccine for bovine tuberculosis, using an influenza vector with the NS1 mutation and expressing M. bovis protective antigens ESAT-6 and TB10.4. We vaccinated Balb/c outbred mice two times at 21 days apart. Our experimental design includes mice immunised with the candidate vaccine with or without adjuvant 15% Montanide Gel. The candidate vaccine's safety was determined by biometric analysis, and protective efficacy was assessed by bacteriological and histological experiments following a virulent M. bovis-8 strain challenge. Our data indicated that the adjuvant-free version of the vaccine ensured complete protection from the M. bovis-8 infection in mice.

IFN-γ independent markers of Mycobacterium tuberculosis exposure among male South African gold miners.

The prevalence of tuberculosis among men who work in the gold mines of South Africa is among the highest in the world, but a fraction of miners demonstrate consistently negative results upon tuberculin skin test (TST) and IFN-γ release assay (IGRA). We hypothesized that these "resisters" (RSTRs) may display unconventional immune signatures of exposure to M.tuberculosis (M.tb). In a cohort of RSTRs and matched controls with latent TB infection (LTBI), we profiled the functional breadth of M.tb antigen-specific T cell and antibody responses using multi-parameter flow cytometry and systems serology, respectively. RSTRs and LTBI controls both exhibited IFN-γ independent T-cell and IgG antibody responses to M.tb-specific antigens ESAT-6 and CFP-10. Antigen-specific antibody Fc galactosylation and sialylation were higher among RSTRs. In a combined T-cell and antibody analysis, M.tb lysate-stimulated TNF secretion by T cells correlated positively with levels of purified protein derivative-specific IgG. A multivariate model of the combined data was able to differentiate RSTR and LTBI subjects. IFN-γ independent immune signatures of exposure to M.tb, which are not detected by approved clinical diagnostics, are readily detectable in an occupational cohort uniquely characterized by intense and long-term infection pressure. Further, TNF may mediate a coordinated response between M.tb-specific T-cells and B-cells. This work was supported by the US National Institutes of Health (R01-AI124348 to Boom, Stein, and Hawn; R01-AI125189 and R01-AI146072 to Seshadri; and 75N93019C00071 to Fortune, Alter, Seshadri, and Boom), the Doris Duke Charitable Foundation (Davies), the Bill & Melinda Gates Foundation (OPP1151836 and OPP1109001 to Hawn; and OPP1151840 to Alter), Mass Life Science Foundation (Fortune), and Good Ventures Fund (Fortune).

In Silico Evaluation of Expressed TB Fusion CFP10-ESAT-6 Reveals Promising Diagnostic and Therapeutic Potential

Tuberculosis (TB) an opportunistic disease still remains a major cause of death globally after COVID-19 pandemic and HIV. Enhanced microbial resistance to therapeutic drugs has paved way to introducing new vaccines for prophylactic control of the disease. Timely diagnosis of TB is also highly needed for effective treatment. Therefore, a fast reliable diagnostic method with high sensitivity is need of the day. In our study, CFP10 and ESAT-6 antigens were cloned, expressed, and purified individually and as a chimeric construct in Escherichia coli BL21. Epitopic analysis revealed that the CFP10-ESAT-6 (CEP) chimeric construct may be more sensitive than the individual sensitivities of CFP10 and ESAT-6. Moreover, molecular analysis of secondary structure and 3-D modelling of each construct also validated that CEP may prove a better immunodiagnostic tool in addition to previously reported EC skin test.

Comparative Evaluation of Specific Antibody against Mycobacterium tuberculosis ESAT-6 Recombinant Antigen in Healthy Subject with Positive and Negative Skin Test.

Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (M. tuberculosis). The laboratory diagnosis of the disease includes various bacteriologic and immunologic methods. Despite the effectiveness of many of these methods in diagnosing active TB, their high cost and time-consuming nature have led researchers to adopt more accurate and rapid screening methods based on specific antigens for M. tuberculosis. The present study aimed to measure specific antibody serum levels against the early secretory antigenic target 6 kDa (ESAT-6) recombinant protein in healthy people and compare it to TB patients. The target population included 27 TB patients and 87 healthy individuals with no clinical TB symptoms. The healthy population was divided into two groups, including positive purified protein derivative (PPD) and negative PPD (35 and 52 people, respectively), using the Tuberculin skin test. The specific antibody level against the ESAT-6 recombinant antigen and the PPD protein was measured using an indirect Enzyme-Linked Immunosorbent Assay (ELISA) test. The results of the study showed that the majority of the healthy population with no symptoms of clinical TB and having negative skin test results did not have antibodies against the recombinant ESAT-6 (98%) and PPD (96%) antigens. On the other hand, there was a high level of the specific antibody of the ESAT-6 recombinant and PPD antigens in TB patients (77%). It is notable that in people with positive skin test results, the level of the antibody against the ESAT-6 recombinant antigen and PPD antigen was 94%. The results demonstrated that the ELISA method based on the measurement of antibodies against the ESAT-6 recombinant antigen can be a proper diagnostic method for rapid and accurate screening of healthy from infected people.

Conductive nano-gold self-assembled MXene@hemin with high catalytic activity achieved by strong metal-support interactions: A powerful nanozyme for development of electrochemical aptasensor in tuberculosis diagnosis

Despite multiple advances in tuberculosis (TB), there is still a lack of effective methods available for its diagnosis. To this end, we developed a novel nanozyme-mediated electrochemical aptasensor for TB diagnosis. We showed that hemin decorated with multi-layered MXene enhanced the metal-support interaction with nano-gold via charge transfer, resulting in excellent peroxidase-like activity aside from the highly conductive feature. TheKmvalue of hemin functionalized MXene decorated with nano-gold (MXene@hemin-Au) was 0.37 mM and 0.23 mM for H2O2 and TMB, respectively, which was smaller to that of MXene@hemin (4.01 mM and 0.39 mM) or horseradish peroxidase (3.7 mM and 0.434 mM), indicating its high affinity to both substrates, showing improved stability in harsh conditions and excellent biocompatibility. Given these advantages, an ultrasensitive detection method was established based on a typical sandwich format using a combination of specific Mycobacterium tuberculosis (MTB) ESAT-6 antigen aptamers with MXene@hemin-Au as probes for signal amplification and gold nanoparticles supported nitrogen-doped carbon nanotube (N-CNTs-Au) as a sensing platform. It exhibited a wide detection linear for ESAT-6 antigen from 100 fg mL−1to 50 ng mL−1, with limit of detection and limit of quantification of 2.36 fg mL−1 and 7.87 fg mL−1, respectively. For its diagnostic application, of note, the proposed method outperformed diagnostic performance for TB in contract to a commercial ELISA method in terms of specificity (100% vs. 20.59%) and area under curve (0.987 vs. 0.596), verifying its great potential in clinical practice.

Preclinical Evaluation of TB/FLU-04L-An Intranasal Influenza Vector-Based Boost Vaccine against Tuberculosis.

Tuberculosis is a major global threat to human health. Since the widely used BCG vaccine is poorly effective in adults, there is a demand for the development of a new type of boost tuberculosis vaccine. We designed a novel intranasal tuberculosis vaccine candidate, TB/FLU-04L, which is based on an attenuated influenza A virus vector encoding two mycobacterium antigens, Ag85A and ESAT-6. As tuberculosis is an airborne disease, the ability to induce mucosal immunity is one of the potential advantages of influenza vectors. Sequences of ESAT-6 and Ag85A antigens were inserted into the NS1 open reading frame of the influenza A virus to replace the deleted carboxyl part of the NS1 protein. The vector expressing chimeric NS1 protein appeared to be genetically stable and replication-deficient in mice and non-human primates. Intranasal immunization of C57BL/6 mice or cynomolgus macaques with the TB/FLU-04L vaccine candidate induced Mtb-specific Th1 immune response. Single TB/FLU-04L immunization in mice showed commensurate levels of protection in comparison to BCG and significantly increased the protective effect of BCG when applied in a "prime-boost" scheme. Our findings show that intranasal immunization with the TB/FLU-04L vaccine, which carries two mycobacterium antigens, is safe, and induces a protective immune response against virulent M. tuberculosis.

TB skin test recombinant proteins as vaccine candidates

Incomplete protection of BCG vaccines, high variability of tuberculosis strains, together with the growing antibiotic resistance of mycobacterium tuberculosis, actualize the need to develop new anti-tuberculosis vaccines. Several novel experimental candidate vaccines based on recombinant proteins, such as those based on the M. tuberculosis ESAT-6 and CFP-10 antigens, are currently being studied in clinical trials. The genome region coding for ESAT-6 and CFP-10 antigens is deleted in BCG strains, so the BCG-immunized individuals cannot develop an immune response against the recombinant ESAT-6/CFP-10 antigen. Therefore, a positive immune reaction to these antigens in TB tests indicates the tested individual has earlier been exposed to M. tuberculosis. The ESAT-6/CFP-10 fusion recombinant antigen was, thus, selected as an immunogen to be evaluated on its potential to induce protective immunity against tuberculosis in a mice model when combined with a birch bark betulin-based vaccine adjuvant. The effect of use was assessed based on the results of histological evaluation of the infected lung tissue in mice and the Mtb lung content. The results herein reported eventually demonstrated that the use of corpuscular adjuvant-based (betulin) ESAT-6/CFP-10 vaccine preparation can induce the immune response commensurate to that of when immunized with the BCG vaccine.

Effect of ESAT-6 on Phagocytosis Activity, ROS, NO, IFN-γ, and IL-10 in Peripheral Blood Mononuclear Cells of Pulmonary Tuberculosis Patients

Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (MTB) that lives intracellularly. MTB can inhibit lysosomal enzymes and phagolysosomal fusion, which is challenging to eliminate. These are due to ESAT-6/CFP-10 originating from the RD1 region genome that expresses the Esx-1 type VII secretion system. Esx-1 encodes Esx-A (ESAT-6) and Esx-B (CFP-10), potential vaccine candidates still under research and development. ESAT-6/CFP-10 is predicted to affect macrophage phagocytic activity, IFN-γ, ROS/NO, and IL-10 levels. Several studies have begun to focus on the ESAT-6 antigen due to the high levels of ESAT-6 antibody found in pleural effusion and granuloma fluid. They can last up to 1 year compared to CFP-10 in experimental animals. This study aimed to analyze the effect of ESAT-6 on the phagocytic activity of macrophages, ROS/NO, IFN-γ, and IL-10 in peripheral blood mononuclear cells (PBMCs) cultures of pulmonary TB patients. It is experimental laboratory research with a post-test-only control group design with PBMCs from October 2019 to December 2020 in the Aretha Laboratory Bandung. There were two groups: the negative group (without ESAT-6) and the positive group (with ESAT-6). Six subjects were sampled at the Pindad Hospital in Bandung, and the research was carried out at the Aretha Laboratory in Bandung. Statistical analysis using paired t test. There was a significant difference between the negative and positive groups (p&lt;0.05). ESAT-6 can decrease macrophage phagocytic activity, intracellular ROS/NO, and IFN-γ but increase IL-10 levels.

Evaluating the Performance of PPE44, HSPX, ESAT-6 and CFP-10 Factors in Tuberculosis Subunit Vaccines

Mycobacterium tuberculosis (M. tuberculosis) is an intracellular pathogen causing long-term infection in humans that mainly attacks macrophages and can escape from the immune system with the various mechanisms. The only FDA-approved vaccine against M. tuberculosis (MTB) is Mycobacterium bovis bacillus Calmette-Guérin (BCG). The protection of this vaccine typically lasts 10–15 years. Due to the increasing number of people becoming ill with MTB each year worldwide, the need to develop a new effective treatment against the disease has been increased. During the past two decades, the research budget for TB vaccine has quadrupled to over half a billion dollars. Most of these research projects were based on amplifying and stimulating the response of T-cells and developing the subunit vaccines. Additionally, these studies have demonstrated that secretory and immunogenic proteins of MTB play a key role in the pathogenesis of the bacteria. Therefore, these proteins were used to develop the new subunit vaccines. In this review, based on the use of these proteins in the successful new subunit vaccines, the PPE44, HSPX, CFP-10 and ESAT-6 antigens were selected and the role of these antigens in designing and developing new subunit vaccines against TB and for the prevention of TB were investigated.

Test performance data demonstrates utility of a cattle DIVA skin test reagent (DST-F) compatible with BCG vaccination

Bacillus Calmette-Guérin (BCG), an attenuated strain of Mycobacterium bovis (M. bovis), is the lead candidate vaccine for control of bovine tuberculosis (TB) in cattle. However, BCG vaccination sensitises cattle to bovine tuberculin, thus compromising the use of the current bovine TB surveillance tests. To address this, we have developed a diagnostic skin test that is not compromised by BCG vaccination and is able to detect BCG vaccinated animals that subsequently develop bovine TB following exposure to M. bovis. Building on previous work using ‘in house’ formulated protein cocktail reagents, we herein present test performance data for a single fusion protein (DST-F) containing the mycobacterial antigens ESAT-6, CFP-10 and Rv3615c formulated as a ‘ready to use’ reagent by a commercial manufacturer. Our results demonstrate that, unlike tuberculin reagents, a diagnostic skin test using DST-F maintained high specificity in BCG vaccinated animals. Furthermore, the DST-F skin test demonstrated a high relative sensitivity in identifying M. bovis infected animals, including those where BCG vaccination failed to prevent bovine TB pathology following experimental exposure to M. bovis. The DST-F is currently undergoing field trials in Great Britain to support its licensure and commercialisation.

ESAT-6 and CFP-10 antigens as a biotechnology molecule substrate. Applications in medicine

Recombinant technologies have been long widely used in medicine. This article presents a review on the application of medical technologies based on ESAT-6 and CFP-10 proteins in diagnostics and prevention of tuberculosis. ESAT-6 and CFP-10 are specific proteins whose genes are encoded in the RD-1 zone (region of difference) of M. tuberculosis. M. bovis BCG and in most nontuberculous mycobacteria lack the RD-1 genome fragment. The discovery of ESAT-6 and CFP-10 antigens allowed to make the first and so far, the only breakthrough in improving the diagnostics of latent tuberculosis infection after the first tuberculin skin test (TST) was implemented. The article describes the principle of action and the experience with diagnostic tools based on ESAT-6 and CFP-10 such as in vitro interferon-gamma release assays (IGRA) and in vivo recombinant tuberculosis allergen (RTA, Diaskintest). RTA is inoculated intradermally similar to TST followed by developing delayed-type immune reaction detected in the area closest to M. tuberculosis. Combined use of ESAT-6 and CFP-10 for early detection of tuberculosis infection allowed to ameliorate for many drawbacks related to TST. High sensitivity and specificity was confirmed for ESAT-6- and CFP-10-based tests, so that former BCG vaccination had no more effect on test results and lowered frequency of false positive results due to reaction to non-tuberculous mycobacteria. The results of a large-scale meta-analysis on studies with patients at high risk demonstrated that the risk of developing tuberculosis in subjects with positive vs. negative IGRA was increased by 9.35-fold (95% confidence interval (CI [6.4813.49]), whereas for TST by 4.24-fold (95% CI [3.35.46]). 95.1%, (95% CI [95.0695.1]). Analyzing available publications demonstrated sufficient evidence base regarding efficacy of using ESAT-6CFP-10-based tests in tuberculosis diagnostics. Finally, there are also reviewed the diagnostic tests and vaccines based on using such proteins currently being under development.