Enhancement of the immunogenicity of a Mycobacterium tuberculosis fusion protein using ISCOMATRIX and PLUSCOM nano-adjuvants as prophylactic vaccine after nasal administration in mice.

Objectives: Tuberculosis (TB), a contagious disease caused by Mycobacterium tuberculosis (M. tuberculosis), remains a health problem worldwide and this infection has the highest mortality rate among bacterial infections. Current studies suggest that intranasal administration of new TB vaccines could enhance the immunogenicity of M. tuberculosis antigens. Hence, we aim to evaluate the protective efficacy and immunogenicity of HspX/EsxS fusion protein of M. tuberculosis along with ISCOMATRIX and PLUSCOM nano-adjuvants and MPLA through intranasal administration in a mice model. Materials and Methods: In the present study, the recombinant fusion protein was expressed in Escherichia coli and purified and used to prepare different nanoparticle formulations in combination with ISCOMATRIX and PLUSCOM nano-adjuvants and MPLA. Mice were intranasally vaccinated with each formulation three times at an interval of 2 weeks. Three weeks after the final vaccination, IFN-γ, IL-4. IL-17, and TGF-ß concentrations in the supernatant of cultured splenocytes of vaccinated mice as well as serum titers of IgG1 and IgG2a and sIgA titers in nasal lavage were determined. Results: According to obtained results, intranasally vaccinated mice with formulations containing ISCOMATRIX and PLUSCOM nano-adjuvants and MPLA could effectively induce IFN-γ and sIgA responses. Moreover, both HspX/EsxS/ISCOMATRIX/MPLA and HspX/EsxS/PLUSCOM/MPLA and their BCG booster formulation could strongly stimulate the immune system and enhance the immunogenicity of M. tuberculosis antigens. Conclusion: The results demonstrate the potential of HspX/EsxS-fused protein in combination with ISCOMATRIX, PLUSCOM, and MPLA after nasal administration in enhancing the immune response against M. tuberculosis antigens. Both nanoparticles were good adjuvants in order to promote the immunogenicity of TB-fused antigens. So, nasal immunization with these formulations, could induce immune responses and be considered a new TB vaccine or a BCG booster.

Characterization of carbapenem-resistant Escherichia coli and Klebsiella: a role for AmpC-producing isolates.

Aim: This study aimed to investigate the role of AmpC enzymes in carbapenem resistance among AmpC/extended-spectrum ß-lactamase (ESBL)-producing clinical isolates of Escherichia coli and Klebsiella spp. Methods: Fifty-six bacterial strains that were AmpC producers were examined. The antibiotic susceptibility test was performed by the disk diffusion and E-test. The prevalence of the plasmid carbapenemase was determined using PCR. Results: The resistance to meropenem in the AmpC+/ESBL+ group was 64%, higher than that reported for the AmpC-/ESBL+ group. Ten isolates of the carbapenem-resistant AmpC producers were negative for carbapenemase-encoding genes. Conclusion: Carbapenem resistance among AmpC-producing isolates with negative results for carbapenemase-encoding genes potentially demonstrates the role of AmpC enzymes among these isolates.

A Comparative Study on Visual Detection of Mycobacterium tuberculosis by Closed Tube Loop-Mediated Isothermal Amplification: Shedding Light on the Use of Eriochrome Black T.

Loop-mediated isothermal amplification is a promising candidate for the rapid detection of Mycobacterium tuberculosis. However, the high potential for carry-over contamination is the main obstacle to its routine use. Here, a closed tube LAMP was intended for the visual detection of Mtb to compare turbidimetric and two more favorable colorimetric methods using calcein and hydroxy naphthol blue (HNB). Additionally, a less studied dye (i.e., eriochrome black T (EBT)) was optimized in detail in the reaction for the first time. Mtb purified DNA and 30 clinical specimens were used to respectively determine the analytical and diagnostic sensitivities of each method. The turbidimetric method resulted in the best analytical sensitivity (100 fg DNA/reaction), diagnostic sensitivity and specificity (100%), and time-to-positivity of the test (15 min). However, this method is highly prone to subjective error in reading the results. Moreover, HNB-, calcein-, and EBT-LAMP could respectively detect 100 fg, 1 pg, and 1 pg DNA/reaction (the analytical sensitivities) in 30, 15, and 30 min, while the diagnostic sensitivity and specificity were respectively 93.3% and 100% for them all. Interestingly, EBT-LAMP showed the lowest potential for subjective error in reading the results. This report helps judiciously choose the most appropriate visual method, taking a step forward toward the field applicability of LAMP for the detection of Mtb, particularly in resource-limited settings.

Development of detection methods for the diagnosis and analysis of highly toxic metal phosphides: A comprehensive and critical review.

Metal phosphides, especially aluminum phosphide, and phosphine (PH3 ) are widely used as insecticides and rodenticides for protection of grains during process of storage and transportation. The main reason of poisoning with this compound is related to the conscious ingestion of salts or accidental inhalation of PH3 . So the early and accurate diagnosis of poisoning can significantly help to the effective clinical treatment or recognition of death cause. PH3 is somewhat unstable due to reaction with oxygen or hemoglobin leading to formation of oxy-acids phosphorous. Here, we critically reviewed the literature introducing the quantitative and qualitative methods for the detection of metal phosphides, PH3 , and its products. This study obviously demonstrates that during past years, different diagnosis methods have been remarkably progressed. Head-space gas chromatography and confirmatory colorimetric methods have been as the most popular techniques. Also, the gas sensors are a promising method that must be more progressed.

The first diagnostic test for specific detection of Mycobacterium simiae using an electrochemical label-free DNA nanobiosensor.

Mycobacterium simiae has been reported to be the most prevalent species of Nontuberculous mycobacteria (NTM) in many countries. As both phenotypic and molecular detection of M. simiae and other NTMs have limitations, finding an accurate, fast, and low-cost diagnostic method is critical for the management of infections. Here, we report the development of a new type of label-free electrochemical biosensor using a gold electrode decorated with l-cysteine/PAMAM dendrimer for specific targeting of M. simiae ITS sequence. DNA hybridization was monitored by measuring changes in the free guanine electrical signal with changing ssDNA target concentrations by differential pulse voltammetry (DPV) method. Response surface methodology (RSM) was applied for the optimization of variables affecting biosensor response. Under optimal conditions, the biosensor revealed a wide linear range from 10-14 M to 10-6 M and a detection limit of 1.40 fM. The fabricated biosensor showed an excellent selectivity to M. simiae in the presence of other similar pathogenic bacteria. Moreover, experimental results confirmed that this biosensor exhibited great precision and high reproducibility, hence provides a low-cost, label-free, and faster detection analysis, representing a novel strategy in detecting other NTMs.

Mycobacterium szulgai pulmonary infection in a vitamin D-deficient patient: A case report.

Closer attention should be paid to vitamin D status in patients with mycobacterial diseases.

Response surface methodology optimized electrochemical DNA biosensor based on HAPNPTs/PPY/MWCNTs nanocomposite for detecting Mycobacterium tuberculosis.

An important issue in the prognosis of tuberculosis (TB) is a short period between correct diagnosis and start the suitable antibiotic therapy. So, a rapid and valid method for detection of Mycobacterium tuberculosis (M. tb) complex is considered as a necessity. Herein, a rapid, low-cost, and PCR-free DNA biosensor was developed based on multi-walled carbon nanotubes (MWCNTs), polypyrrole (PPy), and hydroxyapatite nanoparticles (HAPNPs) for highly sensitive and specific recognition of M.tb. The biosensor consisted of M.tb ssDNA probe covalently attached to the HANPs/PPy/MWCNTs/GCE surface that hybridized to a complementary target sequence to form a duplex DNA. The M.tb target recognition was based on the oxidation signal of the electroactive Methylene Blue (MB) on the surface of the modified GCE using differential pulse voltammetry (DPV) method. It is worth to mention that for the first time Plackett-Burman (PB) screening design and response surface method (RSM) based on central composite design (CCD) was applied as a powerful and an efficient approach to find optimal conditions for maximum M.tb biosensor performance leading to simplicity and rapidity of operation. The proposed DNA biosensor exhibits a wide detection range from 0.25 to 200.0 nM with a low detection limit of 0.141 nM. The performance of designed biosensor for clinical diagnosis and practical applications was revealed through hybridization between DNA probe-modified GCE and extracted DNA from sputum clinical samples.

A case of multidrug-resistant Mycobacterium simiae in an elderly woman.

Mycobacterium simiae is an emerging and spreading pathogen in Iran and little data about its drug susceptibility test (DST) and no standard treatment regimen are available. We report a case of multidrug-resistant M. simiae respiratory infection in a 65-year-old woman with a history of previous Mycobacterium tuberculosis infection. The patient was treated with clarithromycin, levofloxacin, and cotrimoxazole for one year and eventually died while still suffering from respiratory problems. For DST, broth microdilution method was used according to the Clinical and Laboratory Standards Institute guidelines as well as molecular DST in clinical isolate. Mycobacterium simiae was resistant to streptomycin, moxifloxacin, clarithromycin, and cotrimoxazole antibiotics and was sensitive to clofazimine and amikacin antibiotics. Inappropriate use of antibiotics without determining the pattern of antibiotic resistance increases the likelihood of resistance and, for resistant specimens, the need to review the treatment protocol and replace antibiotics. Effectiveness based on antibiotic resistance pattern is essential.

Mycobacterium simiae pulmonary infection: a case series and literature review.

Incidence of Mycobacterium simiae pulmonary infection is increasing and diagnosis and treatment are challenging. We surveyed the clinical features, risk factors, diagnosis, and management in 20 patients from northeastern Iran diagnosed by line probe assay and confirmed by sequencing the ITS (16S-23S) rRNA region and carried out a literature review using the keywords "pulmonary infection" and "Mycobacterium simiae." The mean age of patients was 55.1 years, with 80% female and 90% diagnosed by sputum. Clinical symptoms included severe cough (90%), sputum production (70%), haemoptysis (50%), and chest pain (35%). Comorbidities included a history of tuberculosis (60%), smoking (40%), or chronic obstructive pulmonary disease (20%). Patients were treated with levofloxacin, clarithromycin, and co-trimoxazole. Except for two patients, the clinical symptoms improved. Mycobacterium simiae pulmonary infection is increasing in people with underlying diseases. Although choosing the most appropriate treatment remains a challenge, combining successful treatments could be useful in treating these patients.

Nanotechnology-driven advances in the treatment of diabetic wounds.

Diabetic foot ulcers (DFUs) are chronic severe complications of diabetes disease and remain a worldwide clinical challenge with social and economic consequences. Diabetic wounds can cause infection, amputation of lower extremities, and even death. Several factors including impaired angiogenesis, vascular insufficiency, and bacterial infections result in a delayed process of wound healing in diabetic patients. Treatment of wound infections using traditional antibiotics has become a critical status. Thus, finding new therapeutic strategies to manage diabetic wounds is urgently needed. Nanotechnology has emerged as an efficient approach for this purpose. This review aimed to summarize recent advances using nanotechnology for the treatment of diabetic wounds.

The overview and perspectives of biosensors and Mycobacterium tuberculosis: A systematic review.

Tuberculosis (TB) is referred to as a "consumption" or phthisis, which has been a fatal human disease for thousands of years. Mycobacterium tuberculosis (M. tb) might have been responsible for the death of more humans than any other bacterial pathogens. Therefore, the rapid diagnosis of this bacterial infection plays a pivotal role in the timely and appropriate treatment of the patients, as well as the prevention of disease spread. More than 98% of TB cases are reported in developing countries, and due to the lack of well-equipped and specialized diagnostic laboratories, development of effective diagnostic methods based on biosensors is essential for this bacterium. In this review, original articles published in English were retrieved from multiple databases, such as PubMed, Scopus, Google Scholar, Science Direct, and Cochrane Library during January 2010-October 2019. In addition, the reference lists of the articles were also searched. Among 109 electronically searched citations, 42 articles met the inclusion criteria. The highest potential and wide usage of biosensors for the diagnosis of M. tb and its drug resistance belonged to DNA electrochemical biosensors (isoniazid and rifampin strains). Use of biosensors is expanding for the detection of resistant strains of anti-TB antibiotics with high sensitivity and accuracy, while the speed of these sensory methods is considered essential as well. Furthermore, the lowest limit of detection (0.9 fg/ml) from an electrochemical DNA biosensor was based on graphene-modified iron-oxide chitosan hybrid deposited on fluorine tin oxide for the MPT64 antigen target. According to the results, the most common methods used for M. tb detection include acid-fast staining, cultivation, and polymerase chain reaction (PCR). Although molecular techniques (e.g., PCR and real-time PCR) are rapid and sensitive, they require sophisticated laboratory and apparatuses, as well as skilled personnel and expertise in the commentary of the results. Biosensors are fast, valid, and cost-efficient diagnostic method, and the improvement of their quality is of paramount importance in resource-constrained settings.

Bayesian Spatial Survival Analysis of Duration to Cure among New Smear-Positive Pulmonary Tuberculosis (PTB) Patients in Iran, during 2011-2018.

Mycobacterium tuberculosis is the causative agent of tuberculosis (TB), and pulmonary TB is the most prevalent form of the disease worldwide. One of the most concrete actions to ensure an effective TB control program is monitoring TB treatment outcomes, particularly duration to cure; but, there is no strong evidence in this respect. Thus, the primary aim of this study was to examine the possible spatial variations of duration to cure and its associated factors in Iran using the Bayesian spatial survival model. All new smear-positive PTB patients have diagnosed from March 2011 to March 2018 were included in the study. Out of 34,744 patients, 27,752 (79.90%) patients cured and 6992 (20.10%) cases were censored. For inferential purposes, the Markov chain Monte Carlo algorithms are applied in a Bayesian framework. According to the Bayesian estimates of the regression parameters in the proposed model, a Bayesian spatial log-logistic model, the variables gender (male vs. female, TR = 1.09), altitude (>750 m vs. ≤750 m, TR = 1.05), bacilli density in initial smear (3+ and 2+ vs. 1-9 Basil & 1+, TR = 1.09 and TR = 1.02, respectively), delayed diagnosis (>3 months vs. <1 month, TR = 1.02), nationality (Iranian vs. other, TR = 1.02), and location (urban vs. rural, TR = 1.02) had a significant influence on prolonging the duration to cure. Indeed, pretreatment weight (TR = 0.99) was substantially associated with shorter duration to cure. In summary, the spatial log-logistic model with convolution prior represented a better performance to analyze the duration to cure of PTB patients. Also, our results provide valuable information on critical determinants of duration to cure. Prolonged duration to cure was observed in provinces with low TB incidence and high average altitude as well. Accordingly, it is essential to pay a special attention to such provinces and monitor them carefully to reduce the duration to cure while maintaining a focus on high-risk provinces in terms of TB prevalence.

Antimicrobial Resistance among Community-Acquired Uropathogens in Mashhad, Iran.

Background: Antimicrobial resistance among community-acquired uropathogens is an emerging concern over the past decades that warrants a continuing reevaluation of the appropriateness of recommended empiric antimicrobial regimens for treatment of urinary tract infections (UTIs). Aims: To describe the microbial spectrum and resistance profile of community-acquired uropathogens and predictors of isolation of resistant strains. Methods: Between October 2017 and June 2019, individuals who visited the outpatient clinics for diagnosis of UTIs or screening of asymptomatic bacteriuria were included in the study if they were tested for urine culture in one of the three main medical diagnostic laboratories of Mashhad, Iran. The standard disk diffusion antimicrobial susceptibility testing was used, with the Clinical and Laboratory Standards Institute (CLSI) threshold cutoffs for susceptibility of isolated uropathogens. Results: Three hundred thirty cases were included with a median age of 47 years. Two hundred seventy-six (83.6%) were female. The most common isolated uropathogens were Escherichia coli in 201 (60.9%) cases and Klebsiella species in 46 (13.9%) cases. E. coli isolates showed the highest rates of susceptibility to nitrofurantoin (89.3%), cefixime (75%), and gentamicin (72.4%). Exposure to antibiotics in the past 3 months was a predictor of resistance to ciprofloxacin (OR: 2.8, 95% CI: 1.33-6.28), and older age was a predictor of resistance to TMP-SMX (OR: 2.1, 95% CI: 1.07-3.97) among E. coli isolates. Conclusion. E. coli and Klebsiella species accounted for about two-thirds of community-acquired uropathogens. In regard to the high susceptibility rates, nitrofurantoin was identified as the first-choice agent for empiric treatment of community-acquired cystitis, while cefixime and gentamicin might be the second-choice alternatives. Ciprofloxacin and TMP-SMX, on the other hand, cannot be considered appropriate agents for empiric therapy of community-acquired UTIs, particularly in those who had exposure to antibiotics in the past 3 months and the elderly.

Development of a Cost-Effective Line Probe Assay for Rapid Detection and Differentiation of Mycobacterium Species: A Pilot Study.

BACKGROUND: The line probe assay (LPA) is one of the most accurate diagnostic tools for detection of different Mycobacterium species. Several commercial kits based on the LPA for detection of Mycobacterium species are currently available. Because of their high cost, especially for underdeveloped and developing countries, and the discrepancy of non-tuberculous mycobacteria (NTM) prevalence across geographic regions, it would be reasonable to consider the development of an in-house LPA. The aim of this study was to develop an LPA to detect and differentiate mycobacterial species and to evaluate the usefulness of PCR-LPA for direct application on clinical samples. METHODS: One pair of biotinylated primers and 15 designed DNA oligonucleotide probes were used based on multiple aligned internal transcribed spacer (ITS) sequences. Specific binding of the PCR-amplified products to the probes immobilized on nitrocellulose membrane strips was evaluated by the hybridization method. Experiments were performed three times on separate days to evaluate the assay's repeatability. The PCR-LPA was evaluated directly on nine clinical samples and their cultivated isolates. RESULTS: All 15 probes used in this study hybridized specifically to ITS sequences of the corresponding standard species. Results were reproducible for all the strains on different days. Mycobacterium species of the nine clinical specimens and their cultivated isolates were correctly identified by PCR-LPA and confirmed by sequencing. CONCLUSION: In this study, we describe a PCR-LPA that is readily applicable in the clinical laboratory. The assay is fast, cost-effective, highly specific, and requires no radioactive materials.

Design and Construction of a Eukaryotic Cloning Vector Encoding the mpt51 Gene of Mycobacterium tuberculosis.

BACKGROUND: Tuberculosis (TB) is the leading cause of death by infectious diseases worldwide, and especially prevalent in developing countries. Several vaccines against TB have been developed, recently. The aim of the present study was to design and construct a cloning vector encoding Mycobacterium tuberculosis (MTB) mpt51 gene. METHODS: DNA was extracted from MTB H37Rv strain. Gene-specific primers were designed using Gene Runner software and the mpt51 gene was amplified by PCR. The amplified fragment and pcDNA3.1(+) cloning vector were both digested with restriction enzymes, the mpt51 fragment was ligated into the vector, and the Escherichia coli (E. coli) TOP10 strain were transformed by the recombinant plasmid. Positive clones were identified by colony PCR, restriction enzyme digestion, and DNA sequencing. RESULTS: The mpt51 gene was successfully cloned into pcDNA3.1(+). A 6400 bp band for the pcDNA3.1(+)/mpt51 recombinant plasmid and a 926 bp band for mpt51 were observed by colony PCR, and restriction enzyme digestion on agarose gels. The DNA sequence was 100% homologous with the mpt51 fragment of H37Rv in GenBank. CONCLUSION: In the current study, the mpt51 gene of MTB was correctly cloned into pcDNA3.1(+). The expression of this recombinant vector can be studied in eukaryotic cells. Moreover, it is possible to determine the efficacy of this vector as a DNA vaccine candidate, and to test its protective function compared to BCG in animal models in future.

Autophagy induction plays time-dependent role in viral load of HCV infected Huh7.5 cell line.

Autophagy provides an initial membranous platform for incoming hepatitis C virus (HCV) RNA translation and immune evasion. Once HCV replication is established, this infrastructure will be unnecessary for translation of HCV RNA progeny. So, the autophagy plays key role in the replication and immune pathogenesis of HCV virus. The aim of this study was to study the effect of autophagy induction in Huh7.5 cell on virus titer. The Huh7.5 cell was transfected with recombinant pcDNA-Beclin1. The autophagy induction was evaluated via microtubule associated protein 1 light chain 3 staining as autophagy formation marker using flow cytometry. The HCV (JFH1) was inoculated 12-h post-transfection. Next, to evaluate the viral load, viral RNA was extracted after 24 and 48 h and virus titer was calculated using real-time PCR. The result of the current study shows that the induction of autophagy before virus infection was able to enhance virus yield from 4 × 103 copies/mL to 1 × 104 copies/mL at 24-h post-infection, but reduced viral load after 48 h up to 6 × 103 copies/mL. The study of cross-talk between autophagy and HCV may bring new hope for human intervention and treatment of HCV. Also, it opens new avenue to improve virus cultivation in cell culture and understanding HCV and host cell responses. © 2018 IUBMB Life, 71(1):41-44, 2019.

Designing and Construction of a Cloning Vector Containing mpt64 Gene of Mycobacterium tuberculosis.

BACKGROUND: Tuberculosis caused by Mycobacterium tuberculosis (M. tuberculosis), remains as one of the leading causes of deaths worldwide, with nearly two million death cases annually. BCG (Bacille Calmette-Guerin) continues to be the most widely used vaccine in the world, but the protective immunity differs in different parts of the world. Accordingly, new strategies including DNA vaccines are essentially needed. This study was aimed to design and construct a cloning vector containing mpt64 gene of M. tuberculosis. MATERIALS AND METHODS: M. tuberculosis H37Rv was cultured on Lowenstein Jensen medium, and genomic DNA was extracted. The mpt64 gene was amplified by PCR using designed specific primers. After the digestion of mpt64 and pcDNA3.1 (+) by BamHI and EcoRI restriction enzymes, the mpt64 fragment was ligated into the digested vector using T4 DNA ligase enzyme. Then, the recombinant vector was transformed into competent Escherichia coli (E. coli) TOP10 strain. To confirm the colonies of transformed bacteria, antibiotic resistance, colony-PCR, restriction enzyme digestion and DNA sequencing were used. RESULTS: To confirm the clones, colony-PCR using mpt64 specific primers was performed and the fragment of 718 bp was observed by gel electrophoresis. Clones were also verified by restriction enzyme digestion using BamHI and EcoRI restriction enzymes and the 718 bp fragment was observed. Furthermore, results of DNA sequencing showed 100% homology with the mpt64 fragment of H37Rv in GenBank. CONCLUSION: In this study, the mpt64 fragment was successfully cloned in pcDNA3.1 (+) vector. This construct can be used in future studies as a DNA vaccine in animal models to induce immune system responses.

Designing and Construction of a Cloning Vector Encoding mtb32C and mpt51 Fragments of Mycobacterium tuberculosis as a DNA Vaccine Candidate.

BACKGROUND & OBJECTIVE: Tuberculosis (TB) remains a major cause of death around the world. Bacillus Calmette Guérin (BCG) is the only vaccine used in TB prevention that has a protective effect in children, but its effectiveness declines in adults. Design and development of new vaccines is the most effective way against TB.The aim of this study was to design and construct a DNA vaccine encoding mtb32C and mpt51 fusion genes of Mycobacterium tuberculosis. METHODS: First, mpt51 fragment was amplified by PCR method. The pcDNA3.1+/mtb32C plasmid was transformed into E. coli JM109 and then extracted. The mpt51 gene and pcDNA3.1+/mtb32C plasmid were both digested with EcoRI and BamHI restriction enzymes followed by ligation of mpt51 fragment into the digested vector. The recombinant plasmid containing mtb32C and mpt51 was subsequently transformed into competent E. coli TOP10 strain. The clones were confirmed by colony-PCR, restriction enzyme digestion and sequencing. RESULTS: Using agarose gel electrophoresis, a 926 bp fragment corresponded to mpt51 was observed. Digestion of the vector pcDNa3.1+/mtb32C and mpt51 gene was confirmed by electrophoresis. Then, the pcDNA3.1+/mtb32C plasmid was extracted. Sequencing results confirmed the accuracy of the desired plasmid. CONCLUSION: In this study, we constructed a cloning vector encoding mtb32C/mpt51 gene of M. tuberculosis. The eukaryotic expression of this vector can be confirmed in future studies. It can be considered as a DNA vaccine in animal models later. Successful cloning provides a basis for the development of new DNA vaccines against TB.

Mycobacterium tuberculosis HspX/EsxS Fusion Protein: Gene Cloning, Protein Expression, and Purification in Escherichia coli.

BACKGROUND: The purpose of this study was to clone, express, and purify a novel multidomain fusion protein of Micobacterium tuberculosis (Mtb) in a prokaryotic system. METHODS: An hspX/esxS gene construct was synthesized and ligated into a pGH plasmid, E. coli TOP10 cells were transformed, and the vector was purified. The vector containing the construct and pET-21b (+) plasmid were digested with the same enzymes and the construct was ligated into pET-21b (+). The accuracy of cloning was confirmed by colony PCR and sequencing. E. coli BL21 cells were transformed with the pET-21b (+)/hspX/esxS expression vector and protein expression was evaluated. Finally, the expressed fusion protein was purified on a Ni-IDA column and verified by SDS-PAGE and western blotting. RESULTS: The hspX/esxS gene construct was inserted into pET-21b (+) and recombinant protein expression was induced with IPTG in E. coli BL21 cells. Various concentrations of IPTG were tested to determine the optimum concentration for expression induction. The recombinant protein was expressed in insoluble inclusion bodies. Three molar guanidine HCl was used to solubilize the insoluble protein. CONCLUSION: An HspX/EsxS Mtb fusion protein was expressed in E. coli and the recombinant protein was purified. After immunological analysis, the HspX/EsxS fusion protein might be an anti-tuberculosis vaccine candidate in future clinical trial studies.

Fc-based delivery system enhances immunogenicity of a tuberculosis subunit vaccine candidate consisting of the ESAT-6:CFP-10 complex.

Tuberculosis (TB) remains a major global health threat despite chemotherapy and Bacilli Calmette-Guérin (BCG) vaccination. Therefore, a safer and more effective vaccine against TB is urgently needed. This study evaluated the immunogenicity of a recombinant fusion protein consisting of early secreted antigenic target protein 6 kDa (ESAT-6), culture filtrate protein 10 kDa (CFP-10) and the Fc-domain of mouse IgG2a as a novel subunit vaccine. The recombinant expression vectors (pPICZαA-ESAT-6:CFP-10:Fcγ2a and pPICZαA-ESAT-6:CFP-10:His) were transferred into Pichia pastoris. After SDS-PAGE and immunoblotting, the immunogenicity of the recombinant proteins was evaluated in mice. When both recombinant proteins (ESAT-6:CFP-10:Fcγ2a and ESAT-6:CFP-10:His) were used for vaccination, Th1-type cellular responses were induced producing high levels of IFN-γ and IL-12. However, the Fc-tagged recombinant protein induced more effective Th1-type cellular responses with a small increase in IL-4 as compared to the BCG and ESAT-6:CFP-10:His groups. Moreover, mice primed with BCG and then supplemented with ESAT-6:CFP-10:Fcγ2a produced the highest levels of IFN-γ and IL-12 in immunized groups. The findings indicate that when Fcγ2a is fused to the ESAT-6:CFP-10 complex, as a delivery vehicle, there could be an increase in the immunogenicity of this type of subunit vaccine. Therefore, additional investigations are necessary for the development of appropriate Fc-based tuberculosis vaccines.