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Fusion Molecules of Heat Shock Protein HSPX with Other Antigens of Mycobacterium tuberculosis Show High Potential in Serodiagnosis of Tuberculosis.

ABSTRACT: Variable individual response against the antigens of Mycobacterium tuberculosis necessitates detection of multiple antibodies for enhancing reliability of serodiagnosis of tuberculosis. Fusion molecules consisting of two or more antigens showing high sensitivity would be helpful in achieving this objective. Antigens of M. tuberculosis HSPX and PE35 were expressed in a soluble form whereas tnPstS1 and FbpC1 were expressed as inclusion bodies at 37°C. Heat shock protein HSPX when attached to the N-termini of the antigens PE35, tnPstS1 and FbpC1, all the fusion molecules were expressed at high levels in E. coli in a soluble form. ELISA analysis of the plasma samples of TB patients against HSPX-tnPstS1 showed 57.7% sensitivity which is nearly the same as the expected combined value obtained after deducting the number of plasma samples (32) containing the antibodies against both the individual antigens. Likewise, the 54.4% sensitivity of HSPX-PE35 was nearly the same as that expected from the combined values of the contributing antigens. Structural analysis of all the fusion molecules by CD spectroscopy showed that ?-helical and ?-sheet contents were found close to those obtained through molecular modeling. Molecular modeling studies of HSPX-tnPstS1 and HSPX-PE35 support the analytical results as most of the epitopes of the contributing antigens were found to be available for binding to the corresponding antibodies. Using these fusion molecules in combination with other antigenic molecules should reduce the number of antigenic proteins required for a more reliable and economical serodiagnosis of tuberculosis. Also, HSPX seems to have potential application in soluble expression of heterologous proteins in E. coli.

SUBMITTER: Khalid R

PROVIDER: S-EPMC5031420 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Fusion Molecules of Heat Shock Protein HSPX with Other Antigens of Mycobacterium tuberculosis Show High Potential in Serodiagnosis of Tuberculosis.

Khalid Ruqyya R Afzal Madeeha M Khurshid Sana S Paracha Rehan Zafar RZ Khan Imran H IH Akhtar Muhammad Waheed MW

PloS one 20160921 9

Variable individual response against the antigens of Mycobacterium tuberculosis necessitates detection of multiple antibodies for enhancing reliability of serodiagnosis of tuberculosis. Fusion molecules consisting of two or more antigens showing high sensitivity would be helpful in achieving this objective. Antigens of M. tuberculosis HSPX and PE35 were expressed in a soluble form whereas tnPstS1 and FbpC1 were expressed as inclusion bodies at 37°C. Heat shock protein HSPX when attached to the N ...[more]

PMID: 27654048

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Project description:Background: In individuals living with HIV infection the development of tuberculosis (TB) is associated with rapid progression from asymptomatic TB infection to active TB disease. Sputum-based diagnostic tests for TB have low sensitivity in minimal and subclinical TB precluding early diagnosis. The immune response to novel Mycobacterium tuberculosis in-vivo expressed and latency associated antigens may help to measure the early stages of infection and disease progression and thereby improve early diagnosis of active TB disease. Methods: Serial prospectively sampled cryopreserved lymphocytes from patients of the Swiss HIV Cohort Study developing TB disease ("cases") and matched patients with no TB disease ("controls") were stimulated with 10 novel Mycobacterium tuberculosis antigens. Cytokine concentrations were measured in cases and controls at four time points prior to diagnosis of TB: T1-T4 with T4 being the closest time point to diagnosis. Results: 50 samples from nine cases and nine controls were included. Median CD4 cell count at T4 was 289/ul for the TB-group and 456/ul for the control group. Viral loads were suppressed in both groups. At T4 Rv2431c-induced and Rv3614/15c-induced interferon gamma-induced protein (IP)-10 responses and Rv2031c-induced and Rv2346/Rv2347c-induced tumor necrosis factor (TNF)-α responses were significantly higher in cases compared to controls (p < 0.004). At T3 - being up to 2 years prior to TB diagnosis - Rv2031c-induced TNF-α was significantly higher in cases compared to controls (p < 0.004). Area under the receiver operating characteristics (AUROC) curves resulted in an AUC > 0.92 for all four antigen-cytokine pairs. Conclusion: The in vitro Mycobacterium tuberculosis-specific immune response in HIV-infected individuals that progress toward developing TB disease is different from those in HIV-infected individuals that do not progress to developing TB. These differences precede the clinical diagnosis of active TB up to 2 years, paving the way for the development of immune based diagnostics to predict TB disease at an early stage.

Project description:Serological antibody detection tests for tuberculosis may offer the potential to improve diagnosis. Recent meta-analyses have shown that commercially available tests have variable accuracies and a limited clinical role. We reviewed the immunodiagnostic potential of antigens evaluated in research laboratories (in-house) for the serodiagnosis of pulmonary tuberculosis and conducted a meta-analysis to evaluate the performance of comparable antigens. Selection criteria included the participation of at least 25 pulmonary tuberculosis patients and the use of purified antigens. Studies evaluating 38 kDa, MPT51, malate synthase, culture filtrate protein 10, TbF6, antigen 85B, alpha-crystallin, 2,3-diacyltrehalose, 2,3,6-triacyltrehalose, 2,3,6,6'-tetraacyltrehalose 2'-sulfate, cord factor, and TbF6 plus DPEP (multiple antigen) were included in the meta-analysis. The results demonstrated that (i) in sputum smear-positive patients, sensitivities significantly >or=50% were provided for recombinant malate synthase (73%; 95% confidence interval [CI], 58 to 85) and TbF6 plus DPEP (75%; 95% CI, 50 to 91); (ii) protein antigens achieved high specificities; (iii) among the lipid antigens, cord factor had the best overall performance (sensitivity, 69% [95% CI, 28 to 94]; specificity, 91% [95% CI, 78 to 97]); (iv) compared with the sensitivities achieved with single antigens (median sensitivity, 53%; range, 2% to 100%), multiple antigens yielded higher sensitivities (median sensitivity, 76%; range, 16% to 96%); (v) in human immunodeficiency virus (HIV)-infected patients who are sputum smear positive, antibodies to several single and multiple antigens were detected; and (vi) data on seroreactivity to antigens in sputum smear-negative or pediatric patients were insufficient. Potential candidate antigens for an antibody detection test for pulmonary tuberculosis in HIV-infected and -uninfected patients have been identified, although no antigen achieves sufficient sensitivity to replace sputum smear microscopy. Combinations of select antigens provide higher sensitivities than single antigens. The use of a case-control design with healthy controls for the majority of studies was a limitation of the review. Efforts are needed to improve the methodological quality of tuberculosis diagnostic studies.

Dataset Information

Fusion Molecules of Heat Shock Protein HSPX with Other Antigens of Mycobacterium tuberculosis Show High Potential in Serodiagnosis of Tuberculosis.

Publications

Fusion Molecules of Heat Shock Protein HSPX with Other Antigens of Mycobacterium tuberculosis Show High Potential in Serodiagnosis of Tuberculosis.

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