Mycobacterial Antigen ESAT-6 Research Articles

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.

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.

A New Intranasal Influenza Vector-Based Vaccine TB/FLU-04L Against Tuberculosis: Preclinical Safety Studies.

A recombinant vector vaccine TB/FLU-04L for the prevention of tuberculosis was developed in RIBSP CS MES RK and SRII. The vaccine is based on the attenuated influenza strain Flu NS106/ESAT-6_Ag85A expressing mycobacterial antigens Esat-6 and Ag85A. This research aimed to conduct pre-clinical safety studies of the vaccine as one of the basic and mandatory stages in the development and introduction of immunobiological preparations. The studies were performed at the research centers of the Republic of Kazakhstan and the Russian Federation.The experiment was conducted on ferrets, monkeys, and rabbits. The TB/FLU-04L vaccine was administered intranasally (7.5 lg TCID50/animal). The clinical signs, body weight, temperature, hematological parameters, and local irritant effects were monitored throughout the study. The results of the study demonstrated the safety of the TB/FLU-04L intranasal vector vaccine against tuberculosis since its administration in laboratory animals led to no adverse effects in any of the monitored parameters. No influenza A virus particles were isolated from samples of nasal washes.

CXCL10 is overexpressed in active tuberculosis patients compared to M. tuberculosis-exposed household contacts

IntroductionVariability in clinical outcome of tuberculosis infection is dependent, among other factors, on variation in host immunological response to the infection, which is modulated, in part by genetic variations present in the host. We undertook a study to identify host factors associated with such clinical variability. Study design and methodsA comparative study between groups of active TB patients vs. clinically normal household contacts, family members living under the same roof with the patients for a long period of time, was carried out. We screened 22 candidate cytokines and chemokines in the plasma of 119 pairs (“discovery set”) of TB patients and their asymptomatic household contacts. Identified associations were validated in an independent cohort of 78 patient-household contact pairs (“validation set”). Validated associations were further cross-validated by gene expression assays using RT-PCR and in-vitro whole blood stimulation by mycobacterial antigens ESAT6 and Rv2031c, two well-characterized antigens that are expressed in active and latent phases of disease, respectively. In a concomitant SNP association study, we have sequenced the validated gene in these patients and household contacts. ResultCXCL10 was found to be the most significantly (p = 0.0002) elevated chemokine – discovered and validated -- in patients' plasma compared to their household contacts. We found that CXCL10 was overexpressed by 5-fold at the RNA level in patients compared to asymptomatic household contacts (p = 0.004). On stimulation of whole blood collected from normal healthy volunteers with mycobacterial antigens ESAT6 and Rv2031c, we found that production of CXCL10 by ESAT6 was significantly higher (p = 2.8 × 10−12) than Rv2031c. The production of CXCL10 was 20-fold more than IFN-γ, the most widely validated cytokine, by ESAT6 stimulation (p = 4.6 × 10−8). One of the polymorphisms in promoter of CXCL10, rs4508917 (−1447 A > G), was identified as a proteinQTL (pQTL). Reduced expression of CXCL10 was observed among individuals with GG genotype, but the reduction was statistically significant only among controls, but not among patients. Among patients, the expression level was very high compared to the controls irrespective of the genotypes at this locus. ConclusionPlasma level of CXCL10 is predictive of the active phase of TB infection.

Rv2204c, Rv0753c and Rv0009 antigens specific T cell responses in latent and active TB – a flow cytometry-based analysis

High global prevalence of latent TB infection (LTBI) is a key challenge in distinguishing patients with active pulmonary TB (PTB) from those with LTBI. The functional profile of CD4+ and CD8+ T cell cytokines produced as a response to Mycobacterium tuberculosis antigens vary during the course of tuberculosis (TB) infection. We evaluated antigen-specific CD4+ and CD8+ T cell cytokine response after overnight in vitro stimulation of peripheral blood with mycobacterial antigens ESAT-6, CFP-10, Rv2204c, Rv0753c and Rv0009 by flow cytometry. A significantly higher frequency of antigen-specific CD4+ or CD8+ IFN-γ+ T cells were found in LTBI than in PTB. Among all the antigens used, Rv2204c-specific CD8+ IFN-γ+ displayed the positivity of 72% and 24% in LTBI and PTB respectively. In contrast to IFN-γ, the frequencies of CD4+ or CD8+ secreting TNF-α+ cells were significantly high in PTB compared to LTBI. CD8+TNF-α+ analysis showed 60% positivity in PTB and 13.6% positivity in LTBI against Rv0753c antigen stimulation. We also predicted Rv2204c specific CD8+ T cells secreting IL-10 or IL-4 showed maximum differentiation between LTBI and PTB. In conclusion, altered expression of Rv2204c-specific CD4+IFN-γ+ and CD8+IL-4+ T cells in LTBI and PTB might be a useful biomarker to differentially diagnose LTBI and active TB.

ESAT-6 Targeting to DEC205+ Antigen Presenting Cells Induces Specific-T Cell Responses against ESAT-6 and Reduces Pulmonary Infection with Virulent Mycobacterium tuberculosis.

Airways infection with Mycobacterium tuberculosis (Mtb) is contained mostly by T cell responses, however, Mtb has developed evasion mechanisms which affect antigen presenting cell (APC) maturation/recruitment delaying the onset of Ag-specific T cell responses. Hypothetically, bypassing the natural infection routes by delivering antigens directly to APCs may overcome the pathogen’s naturally evolved evasion mechanisms, thus facilitating the induction of protective immune responses. We generated a murine monoclonal fusion antibody (α-DEC-ESAT) to deliver Early Secretory Antigen Target (ESAT)-6 directly to DEC205+ APCs and to assess its in vivo effects on protection associated responses (IFN-γ production, in vivo CTL killing, and pulmonary mycobacterial load). Treatment with α-DEC-ESAT alone induced ESAT-6-specific IFN-γ producing CD4+ T cells and prime-boost immunization prior to Mtb infection resulted in early influx (d14 post-infection) and increased IFN-γ+ production by specific T cells in the lungs, compared to scarce IFN-γ production in control mice. In vivo CTL killing was quantified in relevant tissues upon transferring target cells loaded with mycobacterial antigens. During infection, α-DEC-ESAT-treated mice showed increased target cell killing in the lungs, where histology revealed cellular infiltrate and considerably reduced bacterial burden. Targeting the mycobacterial antigen ESAT-6 to DEC205+ APCs before infection expands specific T cell clones responsible for early T cell responses (IFN-γ production and CTL activity) and substantially reduces lung bacterial burden. Delivering mycobacterial antigens directly to APCs provides a unique approach to study in vivo the role of APCs and specific T cell responses to assess their potential anti-mycobacterial functions.

Regulatory T-cell subsets in response to specific Mycobacterium tuberculosis antigens in vitro distinguish among individuals with different QTF and TST reactivity

Regulatory T cells (Tregs), a subset of CD4+ T cells related with immune regulation, have been associated with active and latent tuberculosis infection (LTBI). Treg frequencies were evaluated by multicolor flow cytometry (FC) in peripheral blood mononuclear cells (PBMCs) stimulated with mycobacterial antigens ESAT-6, CFP-10, and TB7.7 to assess their capacity to distinguish subjects with different reactivity to the QuantiFERON-TB® Gold In-Tube (QFT-IT) test and the tuberculin skin test (TST). Increased frequencies of CD4+CD25highCD39+ cells were found for the [TST+, QTF+] compared with the [TST+, QTF−] group. Also, higher frequencies were observed for the [TST+, QTF+] compared with the [TST+, QTF−] and [TST−, QTF−] groups in CD4+CD25highFoxp3+ and CD4+CD25highCD39+Foxp3+ populations. Receiver operating characteristics (ROC curve) analysis confirmed these discriminating results. QFT–IT and TST quantitative values correlated with several Treg population frequencies.

Exposure to a Mycobacterial Antigen, ESAT-6, Exacerbates Granulomatous and Fibrotic Changes in a Multiwall Carbon Nanotube Model of Chronic Pulmonary Disease.

Recent studies suggest additive effects of environmental pollutants and microbial antigens on respiratory disease. We established a granuloma model in which instilled multiwall carbon nanotubes (MWCNT) elicit granulomatous pathology. We hypothesized that mycobacterial antigen ESAT-6, a T cell activator associated with tuberculosis and sarcoidosis, might alter pathology. Wild-type C57Bl/6 mice received MWCNT with or without ESAT-6 peptide. Controls received vehicle (surfactant-PBS) or ESAT-6 alone. Mice were evaluated 60 days later for granulomas, fibrosis, and bronchoalveolar lavage (BAL) cell expression of inflammatory mediators (CCL2, MMP-12, and Osteopontin). Results indicated increased granulomas, fibrosis, and inflammatory mediators in mice receiving the combination of MWCNT+ESAT-6 compared to MWCNT or vehicle alone. ESAT-6 alone showed no significant effect on these pathological endpoints. However, CD3 (+) lymphocyte infiltration of lung tissue increased with MWCNT+ESAT-6 versus MWCNT alone. Findings suggest that concurrent exposure to microbial antigen and MWCNT exacerbates chronic pulmonary disease.

Prime-boost BCG vaccination with DNA vaccines based in β-defensin-2 and mycobacterial antigens ESAT6 or Ag85B improve protection in a tuberculosis experimental model

The World Health Organization (WHO) has estimated that there are about 8 million new cases annually of active Tuberculosis (TB). Despite its irregular effectiveness (0–89%), the Bacillus Calmette-Guérin) BCG is the only vaccine available worldwide for prevention of TB; thus, the design is important of novel and more efficient vaccination strategies. Considering that β-defensin-2 is an antimicrobial peptide that induces dendritic cell maturation through the TLR-4 receptor and that both ESAT-6 and Ag85B are immunodominant mycobacterial antigens and efficient activators of the protective immune response, we constructed two DNA vaccines by the fusion of the gene encoding β-defensin-2 and antigens ESAT6 (pDE) and 85B (pDA). After confirming efficient local antigen expression that induced high and stable Interferon gamma (IFN-γ) production in intramuscular (i.m.) vaccinated Balb/c mice, groups of mice were vaccinated with DNA vaccines in a prime-boost regimen with BCG and with BCG alone, and 2 months later were challenged with the mild virulence reference strain H37Rv and the highly virulent clinical isolate LAM 5186. The level of protection was evaluated by survival, lung bacilli burdens, and extension of tissue damage (pneumonia). Vaccination with both DNA vaccines showed similar protection to that of BCG. After the challenge with the highly virulent Mycobacterium tuberculosis strain, animals that were prime-boosted with BCG and then boosted with both DNA vaccines showed significant higher survival and less tissue damage than mice vaccinated only with BCG. These results suggest that improvement of BCG vaccination, such as the prime-boost DNA vaccine, represents a more efficient vaccination scheme against TB.

Improved Skin Test for Differential Diagnosis of Bovine Tuberculosis by the Addition of Rv3020c-Derived Peptides

A peptide cocktail derived from the mycobacterial antigens ESAT-6, CFP-10, and Rv3615c allowed differentiation between Mycobacterium bovis-infected and M. bovis bacillus Calmette-Guérin (BCG)-vaccinated cattle when used as a skin test reagent for a "DIVA" test (i.e., a test capable of differentiating infected and uninfected vaccinated animals). Addition of the antigen Rv3020c improves the diagnostic sensitivity without compromising specificity in the face of BCG or Johne's disease vaccination.

Endoplasmic reticulum stress response is involved in Mycobacterium tuberculosis protein ESAT-6-mediated apoptosis

Mycobacterium tuberculosis (Mtb) infection leads to the induction of the apoptotic response, which is associated with bacilli killing. The early secreted mycobacterial antigen ESAT-6 of Mtb has been shown to induce apoptosis in human macrophages and epithelial cells. In the present study, we demonstrate that the stimulation of human epithelial A549 cells by ESAT-6 induces the endoplasmic reticulum (ER) stress response. We observed that ESAT-6 treatment increases intracellular Ca2+ concentration, which results in ROS accumulation, and therefore induces the onset of ER stress-induced apoptosis. Our results uncover a novel apoptotic mechanism of ESAT-6 through ER stress responses in pathologic conditions such as tuberculosis.

Screening of Highly Expressed Mycobacterial Genes Identifies Rv3615c as a Useful Differential Diagnostic Antigen for theMycobacterium tuberculosisComplex

Tuberculous infections caused by mycobacteria, especially tuberculosis of humans and cattle, are important both clinically and economically. Human populations can be vaccinated with Mycobacterium bovis bacille Calmette-Guérin (BCG), and control measures for cattle involving vaccination are now being actively considered. However, diagnostic tests based on tuberculin cannot distinguish between genuine infection and vaccination with BCG. Therefore, identification of differential diagnostic antigens capable of making this distinction is required, and until now sequence-based approaches have been predominant. Here we explored the link between antigenicity and mRNA expression level, as well as the possibility that we may be able to detect differential antigens by analyzing quantified global transcriptional profiles. We generated a list of 14 candidate antigens that are highly expressed in Mycobacterium tuberculosis and M. bovis under a variety of growth conditions. These candidates were screened in M. bovis-infected and naïve cattle for the ability to stimulate a gamma interferon (IFN-gamma) response. We identified one antigen, Rv3615c, which stimulated IFN-gamma responses in a significant proportion of M. bovis-infected cattle (11 of 30 cattle [37%] [P < 0.01]) but not in naïve or BCG-vaccinated animals. Importantly, the same antigen stimulated IFN-gamma responses in a significant proportion of infected cattle that did not respond to the well-characterized mycobacterial antigens ESAT-6 and CFP-10. Therefore, use of the Rv3615c epitope in combination with previously described differential tests based on ESAT-6 and CFP-10 has the potential to significantly increase diagnostic sensitivity without reducing specificity in BCG-vaccinated populations.

Elicitation of efficient, protective immune responses by using DNA vaccines against tuberculosis

DNA vaccination is an effective method for elicitation of strong humoral as well as cellular immune responses. DNA vaccines expressing mycobacterial antigens ESAT-6 (Rv3875), α-crystallin (Rv2031c) and superoxide dismutase A (Rv3846) were evaluated for their immune responses in Balb/c mice and protective efficacy in guinea pigs. Immunization of mice with the DNA vaccines expressing superoxide dismutase A and α-crystallin resulted in markedly higher levels of IFN-γ as compared to the levels of IL-10. The DNA vaccine expressing ESAT-6 elicited a mixed Th1/Th2 response. Immunization of guinea pigs with these DNA vaccines and subsequent challenge of animals with Mycobacterium tuberculosis H 37Rv, showed that DNA vaccine expressing superoxide dismutase imparted the maximum protection as observed by a 50 and 10 folds reduction in bacillary load in spleens and lungs, respectively, in comparison to immunization with vector control.

Toward the development of diagnostic assays to discriminate between Mycobacterium bovis infection and bacille Calmette-Guérin vaccination in cattle.

A scientific review of the recent sharp increase in bovine tuberculosis in Great Britain has concluded that the development of a cattle vaccine holds the best prospect for long-term disease control. It is important to develop a diagnostic test that differentiates between vaccinated and Mycobacterium bovis-infected animals, to ensure that test-and-slaughter control strategies can continue alongside vaccination. The mycobacterial antigens ESAT-6, MPB64, and MPB83 are expressed at high levels in M. bovis but are expressed at low levels or not at all in bacille Calmette-Guérin (BCG) Pasteur. Promiscuous bovine T cell epitopes of these antigens were identified and formulated into a peptide cocktail. This cocktail and a cocktail composed of recombinant forms of the 3 antigens was able to distinguish cattle infected with virulent M. bovis from those vaccinated with BCG and from those sensitized to avian tuberculin in lymphocyte transformation and interferon-gamma assays.