A potent subset of Mycobacterium tuberculosis glycoproteins as relevant candidates for vaccine and therapeutic target.

Tuberculosis (TB) remains one of the most afflictive bacterial infections globally. In high burden TB countries, surveillance, diagnosis and treatment of drug resistant TB (RR and X/MDRTB) display a crucial public health challenge. Therefore, we need new TB vaccines; diagnostic and therapeutic strategies to briskly prevent disease promotion; reduce drug-resistant TB and protect everyone from disease. The study identified various potent membrane and cell wall M. tuberculosis glycolipoproteins that are relevant for diagnostics, drug and vaccine discovery. A M. tuberculosis Proskauer and Beck broth culture was extracted for total proteins by ammonium sulfate method. After ConA-Affinity Chromatography reputed glycoproteins were collected followed by 2DE gel electrophoresis and LC Mass spectrometry. A total of 293 glycoproteins were identified using GlycoPP and IEDB database. Probable conserved trans-membrane protein (Rv0954), LpqN (Rv0583), PPE68 (Rv3873), Phosphate-binding protein (Rv0932c), PPE61 (Rv3532) and LprA (Rv1270c), had the highest glycosylation percentage value with 13.86%, 11.84%, 11.68%, 11.1%, 10.59% and10.2%, respectively. Our study discloses several dominant glycoproteins that play roles in M. tuberculosis survival, and immunogenicity. These include glycoproteins involved in antigenicity, transport and biosynthesis of M. tuberculosis cell envelope, pathogen-host interaction and drug efflux pumps, which are considered as a feasible drug targets or TB new vaccine candidates.

Recognition of specific immunogenic antigens with potential diagnostic value in multi-drug resistant Mycobacterium tuberculosis inducing humoral immunity in MDR-TB patients.

Tuberculosis (TB) as a public health crisis is caused by the intracellular bacterium Mycobacterium tuberculosis. Detection of immunogenic proteins in TB is valuable for the development of diagnostic tests, vaccine formulations and monitoring treatment outcome. In this study, we differentiated the immune-reactivity of proteins in multidrug-resistant tuberculosis (MDRTB) and drug-susceptible strains using purified anti-MDRTB antibodies isolated from inpatients. Our data showed that the anti- MDRTB antibody was well able to detect the MDR strain in the patient's sputum. The immunogenic proteins of MDRTB were purified by affinity chromatography and subjected to matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. Analysis of the data revealed that seven MDRTB immunogenic proteins, including Rv2986c (HupB), Rv3699, Rv1133c (MetE), Rv0440 (GroEL), Rv3057c, Rv2558 and Rv2971 are involved in DNA stability, metabolism, cellular processes and some unknown functions. Similarities in the electrophoresis protein profiles were evident between the extracts of MDR and sensitive TB strains. However, the protein expression patterns of MDRTB isolates were distinguishable from that formed by susceptible TB strains.

Comparing mRNA expression and protein abundance in MDR Mycobacterium tuberculosis: Novel protein candidates, Rv0443, Rv0379 and Rv0147 as TB potential diagnostic or therapeutic targets.

Tuberculosis (TB) is a sizable public health threat in the world. This study was conducted to determine the differential protein composition between susceptible and MDRTB strains. Tuberculosis proteins were extracted by Triton™ X-114 and ammonium sulfate. Two-dimensional gel electrophoresis protein spots were selected for identification by mass spectrometry and mRNA expression levels were measured by real- time PCR. 2DE-Western blot and T cell epitope prediction for identified proteins were made by the IEDB server. The result shows at least six protein spots (Rv0147, Rv3597c, Rv0379, Rv3699, Rv1392 and Rv0443) were differentially expressed in MDRTB isolates. However, difference in mRNA gene expression was not found in the six mRNA genes. 2DE-Western blot procedures indicated strong reaction against MDRTB proteins corresponds to 13, 16 and 55 kDa areas that might be used as new diagnostic tools. In conclusion, these MDRTB proteins identified in this study could be reliable TB diagnostic candidates or therapeutic targets.

Cellular immune response in MDR-TB patients to different protein expression of MDR and susceptible Mycobacterium tuberculosis: Rv0147, a novel MDR-TB biomarker.

Tuberculosis (TB) is a crucial public health problem with prevalence of multidrug resistant (MDR) rising. An accurate TB biomarker is urgently needed to monitor the response to treatment in patients with MDR tuberculosis. To analyze interaction between selected MDR-TB purified protein and immune cells, dendritic cells from MDR-TB patients and healthy subjects were stimulated by 55KDa protein fractions (Rv0147). The purified proteins identified by proteomic techniques (two-dimensional gel electrophoresis, mass spectrometry) and peptide sequences are known to bind a MHC class I alleles which are extracted from the Immune Epitope Database and Analysis Resource database ( www.iedb.org ). T cells were isolated from PBMC by negative selection and cells were cultured in RPMI-1640 at 37 °C and 5% CO2. Cell culture was assayed for cytokine IL-10 and INF-γ by ELISA. We found that INF-γ production was significantly (335 ± 35.5 pg/ml, P ˂ 0.05) upregulated after protein candidate (Rv0147) stimulation by dendritic cells from MDR-TB patients, whereas IL-10 production was greatly reduced compared with production in healthy subjects (212 ± 9.94 pg/ml, P ˂ 0.05). In fact, the purified protein, Rv0147, stimulated dendritic cells from MDR-TB patients, failed to produce IL-10 and directly stimulates INF-γ production by T cells. These results suggest that the purified protein, Rv0147, may stimulate Th1 type protective cytokine response in MDR-TB patients but not in normal subjects. The production of INF-γ but not IL-10 in the presence of purified protein, Rv0147, may be shifted to Th1 responses in MDR-TB patients and supports its potential as protein vaccine candidates against TB.

Development of high-affinity monoclonal antibody using CD44 overexpressed cells as a candidate for targeted immunotherapy and diagnosis of acute myeloid leukemia.

AIM: CD44s antigens have been suggested as an efficient biomarker for cancer stem cells. Current study aimed to develop a hybridoma that producing a high affinity murine anti-human CD44 monoclonal antibody for early diagnostic laboratory tests of some cancer. MATERIALS AND METHODS: To make hybridoma against CD44, mice were immunized with MDA-MB-468 cells. Resulted hybridomas using three culture media were screened by indirect ELISA, then cloned by limiting dilution, and isotype was determined after obtaining ascitic fluid and antibody purification. RESULTS: We obtained a stable secreting clone, capable of secreting a high-affinity monoclonal antibody against CD44 protein, IgG2a kappa, with the affinity of 5.4 × 10-8 M without cross-reactivity. CONCLUSION: We could establish a hybridoma in a native form. This stable and high-affinity anti-CD44 mAb has a potential for diagnostic procedures and laboratory research. Thus, it could be exploited as a suitable tool for target-specific diagnosis and even treatment in several cancers.

A comparative study of various methods for detection of IL28B rs12979860 in chronic hepatitis C.

Interleukin-28B (IL28B) single-nucleotide polymorphisms (SNPs) constitute important host-related factors influencing the response rate to Hepatitis C virus (HCV) standard antiviral therapy. In the last few years, several new technologies for SNP detection have been developed. However, the sensitivity and specificity of various methods are different and needs evaluation. Five different methods (resolution melting curve [RMC], polymerase chain reaction-restriction fragment length polymorphism [PCR-RFLP], PCR-sequencing analysis, amplification refractory mutation system [ARMS], and zip nucleic acid probe-based real-time PCR [ZNA]) were developed for genotyping rs12979860 associated with IL28B. In this study, limit of detection (LD), costs and turnaround time of these methods were compared in 350 subjects. As for IL28B rs12979860 polymorphisms, 348/350 (99.4%) samples were consistent among the five methods, while results for 2/350 (0.57%) samples were concordant by ZNAs and PCR-sequencing, and discordant by other methods. Without considering the cost of DNA extraction, the price of each reaction for ARMS-PCR, RMC, PCR-RFLP, ZNA and PCR-sequencing were respectively: US$3.10, US$5.0, US$5.50, US$8.50 and US$17.0. RMC was the fastest method, while the ZNA method was easy to use, reliable and effective. Lower LD was determined to be 50-60 copies/µL for the PCR-RFLP, RMC and ARMS-PCR assays; whilst ZNA assay was able to detect 2-3 copies/µL. In conclusion, in the current study, all four methods are suitable for IL28B rs12979860 genotyping, but the ZNA assay can be a reliable tool. Due to its lower LD for SNP identification, this method is better than others for detecting this type of polymorphism.

Proteomic analysis of drug-resistant Mycobacterium tuberculosis by one-dimensional gel electrophoresis and charge chromatography.

Multidrug-resistant tuberculosis (MDR-TB) is a form of TB caused by Mycobacterium tuberculosis (M. tuberculosis) that do not respond to, at least, isoniazid and rifampicin, the two most powerful, first-line (or standard) anti-TB drugs. Novel intervention strategies for eliminating this disease were based on finding proteins that can be used for designing new drugs or new and reliable kits for diagnosis. The aim of this study was to compare the protein profiles of MDR-TB with sensitive isolates. Proteomic analysis of M. tuberculosis MDR-TB and sensitive isolates was obtained with ion exchange chromatography coupled with MALDI-TOF-TOF (matrix-assisted laser desorption/ionization) in order to identify individual proteins that have different expression in MDR-TB to be used as a drug target or diagnostic marker for designing valuable TB vaccines or TB rapid tests. We identified eight proteins in MDR-TB isolates, and analyses showed that these proteins are absent in M. tuberculosis-sensitive isolates: (Rv2140c, Rv0009, Rv1932, Rv0251c, Rv2558, Rv1284, Rv3699 and MMP major membrane proteins). These data will provide valuable clues in further investigation for suitable TB rapid tests or drug targets against drug-resistant and sensitive M. tuberculosis isolates.

Haarlem 3 is the predominant genotype family in multidrug-resistant and extensively drug-resistant Mycobacterium tuberculosis in the capital of Iran: A 5-year survey.

The objective of this study was to further understand the genetic diversity of multidrug-resistant (MDR) and extensively drug-resistant (XDR) Mycobacterium tuberculosis isolates prevalent in Tehran, the capital city of Iran. From January 2010 to March 2015, a total of 723 M. tuberculosis strains were isolated from patients with pulmonary tuberculosis (TB). A total of 23 MDR, pre-XDR and XDR M. tuberculosis isolates were genotyped by spoligotyping and 24-loci mycobacterial interspersed repetitive unit-variable-number tandem repeat (MIRU-VNTR) typing. The results showed that the MDR, pre-XDR and XDR M. tuberculosis strains mainly belonged to the Haarlem 3 genotype (11/23; 47.8%), followed by the Beijing family (9/23; 39.1%). In addition, the 23 strains were clustered into 21 genotypes using a 24-loci MIRU-VNTR. In conclusion, Haarlem 3 genotype was the predominant genotype among the isolates from MDR-TB cases in this study, which could be of special concern.

High prevalence of Mycobacterium tuberculosis mixed infection in the capital of moderate tuberculosis incidence country.

OBJECTIVE: Recent studies using molecular epidemiological techniques have demonstrated mixed infection with multiple strains of Mycobacterium tuberculosis especially in countries with high tuberculosis (TB) burden. We aimed to determine the prevalence of mixed infection among patients with TB in the capital of Iran as a country with moderate incidence rate. METHODS: Samples were collected randomly from January 2011 to December 2013 in Tehran, capital of Iran. A total of 75 M. tuberculosis isolates were genotyped by 24 loci mycobacterial interspersed repetitive unit-variable number tandem repeat typing (MIRU-VNTR) for screening the mixed infection. RESULTS: Twenty patients (20/75) were identified with mixed infection, and the estimated rate of mixed infection was 26.6%. Thirteen out of the 24 loci were able to detect the mixed infection in our study. CONCLUSIONS: Mixed infections occur at high prevalence among studied Iranian TB patients. Further research is inevitable to evaluate the association of mixed infection and disease progression and treatment.

Proteome-scale MDR-TB-antibody responses for identification of putative biomarkers for the diagnosis of drug-resistant Mycobacterium tuberculosis.

OBJECTIVE: Multidrug-resistant tuberculosis (MDR-TB) is caused by Mycobacterium tuberculosis strains that do not respond to isoniazid and rifampicin, the two most effective first-line anti-TB drugs. Here, we designed and produced antibodies based on biomarkers that exist only in MDR-TB. METHODS: Bacilli were cultured for 4weeks at 37°C, and protein extraction was performed by sequential extraction. Bacterial cells were sonicated, centrifuged at 5000rpm for 45min, and the supernatant was collected and subjected to multiple rounds of treatment to prior to protein isolation. Protein concentration was determined using the Bradford method, and extracted proteins (50µg) from each strain (drug-sensitive- and MDR-TB isolates) were visualized on polyacrylamide gels (5-15%) with Coomassie Brilliant Blue R-250 staining. Three extracts were mixed and dialyzed against 0.1M ammonium bicarbonate (pH 8.0), followed by mass spectrometry. Specific polyclonal antibodies against purified MDR-TB proteins were purified by affinity chromatography and prepared in rabbits using three booster injections. The ELIZA test was performed for evaluation the antibody production. The antibody was treated with normal oral flora to remove any non specificity and cross reactivity. Analyses of different protein patterns (drug-sensitive- and MDR-TB) were performed by western blot. RESULTS: Our revealed that the MDR-TB strains contained specific antigens, and that the protein profiles of drug-sensitive TB strains differed from those of the MDR-TB isolates. Five bands from the MDR-TB fractions were detected as diagnostic antigens and were not observed in drug-sensitive-TB fractions. Western blot results showed that the MDR-TB antigenic fractions showed immunogenic bands at 50.0kDa and 70.0kDa, with the five antigenic MDR-TB-specific bands were identified as Rv3248c, Rv0350, Rv0440, Rv0475, and Rv3588c. CONCLUSION: Western blot data revealed dynamic properties of antibody responses that led to actionable findings for further research. Moreover, specific anti-mycobacterial antibodies, such as MDR-TB antibodies, can be essential tools in the identification of species-restricted antigens, such as drug-resistant TB antigens. The MDR-TB antibodies described here might promote identification of mycobacterial antigens during the course of infection, which could be helpful for the development of newer TB-vaccine candidates or therapeutic agents for improved TB treatment or diagnosis.

T cell cytokine responses in peripheral blood mononuclear cells from patients with multidrug-resistant tuberculosis following stimulation with proteins purified from Mycobacterium tuberculosis MDR clinical isolates.

OBJECTIVE: Tuberculosis (TB) is a devastating disease that remains a major health threat worldwide. The appearance of Mycobacterium tuberculosis strains resistance to current antibiotics is a growing problem, both in the third world and in developed countries. Completion of genomic sequencing of M. tuberculosis provides a strong foundation for subsequent identification of proteins to aid the understanding of protein function and the discovery of new drug targets or a TB vaccine. This study employed a proteomics approach to identify proteins from antibiotic resistant M. tuberculosis isolates and compare them to drug-sensitive isolates to determine the role of T cells in multidrug-resistant (MDR)-TB patients against M. tuberculosis-purified proteins (Rv0147) as compared with healthy subjects. METHODS: Proteins were extracted by Triton X-114 detergent-phase separation and precipitated by adding saturated ammonium sulfate to the supernatant. Following isoelectric focusing, proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Mass spectrometry was performed, and protein sequences were determined. Peripheral bloom mononuclear cells (PBMCs) were cultured, and autologous T cells were isolated from PBMCs by negative selection. Cells were subsequently cultured at 37°C in 5% CO2, followed by stimulation with 10µg/mL of the protein candidate (Rv0147) for 72h. Culture supernatants were assayed for interleukin (IL)-10 and interferon (IFN)-γ by enzyme-linked immunosorbent assay. RESULTS: The identified proteins included Rv3057c, Rv0009, Rv3161c, Rv3614c, Rv0685, Rv2986c, Rv0443, Rv2114, Rv3311, Rv0831, Rv3804, and Rv3614c, and our results showed that the majority of upregulated or overexpressed proteins belonged to pathways associated with cellular metabolism, cell wall integrity, respiration, or cell membrane construction. Additionally, Rv1876 from MDR-TB isolates was predicted to be involved in the expression of bacterioferritin exclusively in MDR-TB-related resistance to first-line TB drugs. Furthermore, Rv2031c (HspX) was induced under oxygen-deficient conditions, and hypothetical protein (Rv2744c) and two membrane- and cell-wall-fraction proteins (Rv0379 and Rv1886c) were also identified. Analysis revealed increased percentages of INF-γ and decreased IL-10 levels in MDR-TB patients as compared with those observed in normal subjects. CONCLUSION: Four identified membrane or membrane-associated proteins, including bacterioferritin, GroEs, HspX, and Ef-Tu, may be potential targets for the development of novel prophylactic diagnostics and therapeutic strategies against TB. Our results suggested that T cells stimulated by the protein candidate Rv0147 may be shifted to T helper 1 status in MDR-TB patients.

Modified TB rapid test by proteinase K for rapid diagnosis of pleural tuberculosis.

The diagnosis of pleural tuberculosis continues to be a challenge due to the low sensitivity of traditional diagnostic methods. Better and more rapid tests are needed for diagnosis of pleural TB. In this study, pleural fluids were tested with rapid test to determine Mycobacterium tuberculosis (MTB antigen). Affinity chromatography was used to purify specific polyclonal antibodies against MTB antigen. Pleural samples after decontamination were treated with proteinase K. Rapid test for pleural fluids was prepared by specific antibody. Rapid test was performed on 85 pleural fluid patients. The patients had a mean age of 46.55 ± 15.96 years and 38 were men. The performance of rapid test, using proteinase K, was found to be the most impressive: sensitivity 93%, specificity 94%, PPV 90%, and NPV 96% compared with adenosine deaminase test (ADA), PCR, smear, and culture. The present study did demonstrate that modified TB rapid test can substantially improve the diagnosis of extrapulmonary TB.

MDR-TB Antibody Response (Western Blot) to Fractions of Isoniazid and Rifampicin Resistant Antigens of Mycobacterium tuberculosis.

Drug-resistant TB poses a major threat to control of TB worldwide. Despite progress in the detection of Multidrug-resistant TB (MDR-TB) cases, a major diagnostic gap remains: 55% of reported TB patients estimated to have MDR-TB were not detected in 2013. MDR-TB antigens were conjugated to CNBr-activated Sepharose 4B. Specific polyclonal antibodies against MDR-TB Ags were prepared in rabbits using two boosted injections of the MDR-TB antigen. The antibodies were purified and treated with susceptible TB to remove any non-specific and cross-reactive antibodies. In the present study, comparative analysis of electrophoretic pattern of different antigens of INH/RIF-resistant TB were studied for identifying protein profiles. A RIF-resistant TB antigen was shown here to have different protein profiles from INH-resistant TB isolate. The results of Western blotting analysis showed that in the RIF- and INH-resistant antigenic fractions some bands of 14.4 and 45 kDa as immunogenic were common. Moreover, four bands of RIF-resistant TB antigen fractions (16, 19, 21, and 45 KDa) and one band of INH-resistant TB (about 26 KDa) were detected as diagnostic antigens. This study suggests that the Western blot is an accurate test to survey INH- and RIF-resistant TB antigens of M. tuberculosis infection. These findings indicate that MDR-TB diagnosis (based on Ag detection) could be useful in the identification of disease stages that precede symptomatic and microbiologically positive TB, such as subclinical and incipient TB.

RETRACTED: High level Rifampin Resistance Correlates with Multiple Mutations in the rpoB Gene of Pulmonary Tuberculosis Isolates from the Afghanistan Border of Iran.

This article has been retracted.