Mycobacterium Tuberculosis Protein Research Articles

MycobacteriumtuberculosisProtein PE6 (Rv0335c), a Novel TLR4 Agonist, Evokes an Inflammatory Response and Modulates the Cell Death Pathways in Macrophages to Enhance Intracellular Survival.

Mycobacterium tuberculosis (M. tb) is an intracellular pathogen that exploits moonlighting functions of its proteins to interfere with host cell functions. PE/PPE proteins utilize host inflammatory signaling and cell death pathways to promote pathogenesis. We report that M. tb PE6 protein (Rv0335c) is a secretory protein effector that interacts with innate immune toll-like receptor TLR4 on the macrophage cell surface and promotes activation of the canonical NFĸB signaling pathway to stimulate secretion of proinflammatory cytokines TNF-α, IL-12, and IL-6. Using mouse macrophage TLRs knockout cell lines, we demonstrate that PE6 induced secretion of proinflammatory cytokines dependent on TLR4 and adaptor Myd88. PE6 possesses nuclear and mitochondrial targeting sequences and displayed time-dependent differential localization into nucleus/nucleolus and mitochondria, and exhibited strong Nucleolin activation. PE6 strongly induces apoptosis via increased production of pro-apoptotic molecules Bax, Cytochrome C, and pcMyc. Mechanistic details revealed that PE6 activates Caspases 3 and 9 and induces endoplasmic reticulum-associated unfolded protein response pathways to induce apoptosis through increased production of ATF6, Chop, BIP, eIF2α, IRE1α, and Calnexin. Despite being a potent inducer of apoptosis, PE6 suppresses innate immune defense strategy autophagy by inducing inhibitory phosphorylation of autophagy initiating kinase ULK1. Inversely, PE6 induces activatory phosphorylation of autophagy master regulator MtorC1, which is reflected by lower conversion of autophagy markers LC3BI to LC3BII and increased accumulation of autophagy substrate p62 which is also dependent on innate immune receptor TLR4. The use of pharmacological agents, rapamycin and bafilomycin A1, confirms the inhibitory effect of PE6 on autophagy, evidenced by the reduced conversion of LC3BI to LC3BII and increased accumulation of p62 in the presence of rapamycin and bafilomycin A1. We also observed that PE6 binds DNA, which could have significant implications in virulence. Furthermore, our analyses reveal that PE6 efficiently binds iron to likely aid in intracellular survival. Recombinant Mycobacterium smegmatis (M. smegmatis) containing pe6 displayed robust growth in iron chelated media compared to vector alone transformed cells, which suggests a role of PE6 in iron acquisition. These findings unravel novel mechanisms exploited by PE6 protein to subdue host immunity, thereby providing insights relevant to a better understanding of host–pathogen interaction during M. tb infection.

Computational identification and characterization of antigenic properties of Rv3899c of Mycobacterium tuberculosis and its interaction with human leukocyte antigen (HLA).

A rise in drug-resistant tuberculosis (TB) cases demands continued efforts towards the discovery and development of drugs and vaccines. Secretory proteins of Mycobacterium tuberculosis (H37Rv) are frequently studied for their antigenicity and their scope as protein subunit vaccines requiresfurther analysis. In this study, Rv3899c of H37Rv emerges as a potential vaccine candidate on its evaluation by several bioinformatics tools. It is a non-toxic, secretory protein with an 'immunoglobulin-like' fold which does not show similarity with a human protein. Through BlastP and MEME suite analysis, we found Rv3899c homologs in several mycobacterial species and its antigenic score (0.54) to compare well with the known immunogens such as ESAT-6 (0.56) and Rv1860 (0.52). Structural examination of Rv3899c predicted ten antigenic peptides, an accessibility profile of the antigenic determinants constituting B cell epitope-rich regions and a low abundance of antigenic regions (AAR) value. Significantly, STRING analysis showed ESX-2 secretion system proteins and antigenic PE/PPE proteins of H37Rv as the interacting partners of Rv3899c. Further, molecular docking predicted Rv3899c to interact with human leukocyte antigen HLA-DRB1*04:01 through its antigenically conserved motif (RAAEQQRLQRIVDAVARQEPRISWAAGLRDDGTT). Interestingly, the binding affinity was observed to increase on citrullination of its Arg1 residue. Taken together, the computational characterization and predictive information suggest Rv3899c to be a promising TB vaccine candidate, which should be validated experimentally.

Exploration of potential inhibitors for tuberculosis via structure-based drug design, molecular docking, and molecular dynamics simulation studies.

Drug resistance in tuberculosis is major threat to human population. In the present investigation, we aimed to identify novel and potent benzimidazole molecules to overcome the resistance management. A series of 20 benzimidazole derivatives were examined for its activity as selective antitubercular agents. Initially, AutodockVina algorithm was performed to assess the efficacy of the molecules. The results are further enriched by redocking by means of Glide algorithm. The binding free energies of the compounds were then calculated by MM-generalized-born surface area method. Molecular docking studies elucidated that benzimidazole derivatives has revealed formation of hydrogen bond and strong binding affinity in the active site of Mycobacterium tuberculosis protein. Note that ARG308, GLY189, VAL312, LEU403, and LEU190 amino acid residues of Mycobacterium tuberculosis protein PrpR are involved in binding with ligands of benzimidazoles. Interestingly, the ligands exhibited same binding potential to the active site of protein complex PrpR in both the docking programs. In essence, the result portrays that benzimidazole derivatives such as 1p, 1q, and 1t could be potent and selective antitubercular agents than the standard drug isoniazid. These compounds were then subjected to molecular dynamics simulation to validate the dynamics activity of the compounds against PrpR. Finally, the inhibitory behavior of compounds was predicted using a machine learning algorithm trained on a data collection of 15,000 compounds utilizing graph-based signatures. Overall, the study concludes that designed benzimidazoles can be employed as antitubercular agents. Indeed, the results are helpful for the experimental biologists to develop safe and non-toxic drugs against tuberculosis.

EmPAI-assisted strategy enhances screening and assessment of Mycobacteriumtuberculosis infection serological markers.

SummaryDiscovering new serological markers of Mycobacterium tuberculosis (MTB) infection and establishing a rapid and efficient detection technology is of great significance for the prevention and control of tuberculosis. In this study, we established an exponentially modified protein abundance index (emPAI) value‐assisted strategy to investigate and improve the screening efficiency of serological biomarkers of tuberculosis. First, we used LC‐MS/MS to analyse MTB culture filtrate proteins (MTB‐CFPs), and 632 MTB proteins were identified. Then, the characteristic values of MTB‐CFPs – including emPAI value, molecular weight (Mw), isoelectric point (pI), grand average of hydropathy (GRAVY), transmembrane domain (TMD) and functional groups were calculated. Next, we successfully prepared 10 MTB proteins with emPAI value > 1.0 and recombinantly expressed these proteins in Escherichia coli. At the same time, 3 MTB proteins with emPAI between 0.1 and 0.5 were randomly selected as the control groups, and the immunogenicity of the recombinant MTB proteins was detected using ELISA. The sensitivity and receiver operating characteristic (ROC) curves were calculated for each recombinant MTB protein. The results showed that the areas under the curve (AUC) value of Rv2031c, Rv0577, Rv0831c, Rv0934 and Rv3248c were all higher than those of Rv3875 (AUC, 0.6643). Further analysis of the relationship between emPAI value and antibody sensitivity, AUC value and antibody affinity in mice immunized with recombinant MTB protein showed that emPAI values were positively correlated with them, and R‐squared value ranged from 0.64 to 0.79. The only exception was ESAT‐6 (encoded by the Rv3875 gene), which AUC value was relatively low owing to its strong immunosuppressive properties. This study provides a rationale for the serological marker screening of emPAI‐assisted tuberculosis clinical test. The results also provide new technical support for the screening of candidate serological markers of infectious diseases in the future.

Structural insights into the functional divergence of WhiB-like proteins in Mycobacterium tuberculosis

WhiB7 represents a distinct subclass of transcription factors in the WhiB-Like (Wbl) family, a unique group of iron-sulfur (4Fe-4S] cluster-containing proteins exclusive to the phylum of Actinobacteria. In Mycobacterium tuberculosis (Mtb), WhiB7 interacts with domain 4 of the primary sigma factor (σA4) in the RNA polymerase holoenzyme and activates genes involved in multiple drug resistance and redox homeostasis. Here, we report crystal structures of the WhiB7:σA4 complex alone and bound to its target promoter DNA at 1.55-Å and 2.6-Å resolution, respectively. These structures show how WhiB7 regulates gene expression by interacting with both σA4 and the AT-rich sequence upstream of the -35 promoter DNA via its C-terminal DNA-binding motif, the AT-hook. By combining comparative structural analysis of the two high-resolution σA4-bound Wbl structures with molecular and biochemical approaches, we identify the structural basis of the functional divergence between the two distinct subclasses of Wbl proteins in Mtb.

Mycobacterium tuberculosis protein PPE2 binds to DNA region containing promoter activity

ppe2 gene is predicted to be present in operon with non pe/ppe genes, cobq1 and cobu as ppe2-cobq1-cobu. Thus, we were interested to investigate the role of ppe2 in operonic organization. We performed microscale thermophoresis (MST) experiment which revealed that PPE2 protein could bind to upstream DNA segments of ppe2-cobq1-cobu operon. Upstream region of ppe2 had shown promoter activity in β-gal assay. In this study, for the first time, a physical interaction between PPE2 protein and DNA fragment was reported, suggesting that PPE2 protein plays a role in the regulation of the putative ppe2-cobq1-cobu operon, via unknown mechanism.

Morten Harboe (1929‐2021), one of the founders of Scandinavian Journal of Immunology

Morten Harboe (1929‐2021), one of the founders of Scandinavian Journal of Immunology

In-vivo expressed Mycobacterium tuberculosis antigens recognised in three mouse strains after infection and BCG vaccination

Novel tuberculosis (TB)-vaccines preferably should (i) boost host immune responses induced by previous BCG vaccination and (ii) be directed against Mycobacterium tuberculosis (Mtb) proteins expressed throughout the Mtb infection-cycle. Human Mtb antigen-discovery screens identified antigens encoded by Mtb-genes highly expressed during in vivo murine infection (IVE-TB antigens). To translate these findings towards animal models, we determined which IVE-TB-antigens are recognised by T-cells following Mtb challenge or BCG vaccination in three different mouse strains. Eleven Mtb-antigens were recognised across TB-resistant and susceptible mice. Confirming previous human data, several Mtb-antigens induced cytokines other than IFN-γ. Pulmonary cells from susceptible C3HeB/FeJ mice produced less TNF-α, agreeing with the TB-susceptibility phenotype. In addition, responses to several antigens were induced by BCG in C3HeB/FeJ mice, offering potential for boosting. Thus, recognition of promising Mtb-antigens identified in humans validates across multiple mouse TB-infection models with widely differing TB-susceptibilities. This offers translational tools to evaluate IVE-TB-antigens as diagnostic and vaccine antigens.

In silico identification of novel chemical compounds with antituberculosis activity for the inhibition of InhA and EthR proteins from Mycobacterium tuberculosis

Tuberculosis (TB) continuously poses a major public health concern around the globe, with a mounting death toll of approximately 1.4 million in 2019. Reduced bioavailability, elevated toxicity, increasedside effects, and resistance of multiple first-line and second-line TB medications, including isoniazid, ethionamide necessitate studies of new drugs. The method of computational biology and bioinformatics approach allows virtual screening of a large number of drugs, reduces growing side effects of medications, and predicts potential drug resistance over time. In this study, we have analyzed fifty small molecules with antituberculosis properties using in silico approach including molecular docking, drug-likeness assessment, ADMET (absorption, distribution, metabolism, excretion, toxicity) profile evaluation, P450 site of metabolism prediction, and molecular dynamics simulation. Among those fifty compounds, 3-[3-(4-Fluorophenyl)-1,2,4-oxadiazol-5-yl]-N-(2-methylphenyl) piperidine-1-carboxamide (C22) and 5-(4-Ethyl-phenyl)-2-(1H-tetrazol-5-ylmethyl)-2H-tetrazole (C29) were found to pass the two-step molecular docking, P450 site of metabolism prediction and pharmacokinetics analysis successfully. Their binding stability for target proteins has been evaluated through root mean square deviation and root mean square fluctuation, Radius of gyration analysis from 10 ns Molecular Dynamics Simulation (MDS). Our identified drugs (C22 and C29) performed better than the control drugs (Isoniazid, Ethionamide) regarding binding affinity and molecular stability with the regulatory proteins (InhA, EthR) of Mycobacterium tuberculosis. The study proposed these compounds as effective therapeutic agents for Tuberculosis drug discovery, but further in vitro and in vivo testing are needed to substantiate their potential as novel drugs and modes of action.

Anti-TB evaluation of novel 2,3-dihydroquinazolin-4(1H)-ones and in silico studies of the active compounds

In vitro anti-tubercular activity of a series of 15 novel 2,3-dihydroquinazolin-4(1H)-one analogues were evaluated against Mycobacterium tuberculosis H37Ra (ATCC 25177 strain). Among the series, seven compounds showed moderate to good anti-TB activity with minimum inhibitory concentration (MIC) values ranging from 25.0–12.5 μg/mL. Further, in silico experiments were carried out to identify the probable ligand-protein interaction. Molecular docking of the target compounds into the active site of enzymes 1DQY Antigen 85C from Mycobacterium Tuberculosis and 2NSD Enoyl Acyl Carrier Protein Reductase reveals notable information on the possible binding interactions.

Label-Free Comparative Proteomics of Differentially Expressed Mycobacterium tuberculosis Protein in Rifampicin-Related Drug-Resistant Strains.

Rifampicin (RIF) is one of the most important first-line anti-tuberculosis (TB) drugs, and more than 90% of RIF-resistant (RR) Mycobacterium tuberculosis clinical isolates belong to multidrug-resistant (MDR) and extensively drug-resistant (XDR) TB. In order to identify specific candidate target proteins as diagnostic markers or drug targets, differential protein expression between drug-sensitive (DS) and drug-resistant (DR) strains remains to be investigated. In the present study, a label-free, quantitative proteomics technique was performed to compare the proteome of DS, RR, MDR, and XDR clinical strains. We found iniC, Rv2141c, folB, and Rv2561 were up-regulated in both RR and MDR strains, while fadE9, espB, espL, esxK, and Rv3175 were down-regulated in the three DR strains when compared to the DS strain. In addition, lprF, mce2R, mce2B, and Rv2627c were specifically expressed in the three DR strains, and 41 proteins were not detected in the DS strain. Functional category showed that these differentially expressed proteins were mainly involved in the cell wall and cell processes. When compared to the RR strain, Rv2272, smtB, lpqB, icd1, and folK were up-regulated, while esxK, PPE19, Rv1534, rpmI, ureA, tpx, mpt64, frr, Rv3678c, esxB, esxA, and espL were down-regulated in both MDR and XDR strains. Additionally, nrp, PPE3, mntH, Rv1188, Rv1473, nadB, PPE36, and sseA were specifically expressed in both MDR and XDR strains, whereas 292 proteins were not identified when compared to the RR strain. When compared between MDR and XDR strains, 52 proteins were up-regulated, while 45 proteins were down-regulated in the XDR strain. 316 proteins were especially expressed in the XDR strain, while 92 proteins were especially detected in the MDR strain. Protein interaction networks further revealed the mechanism of their involvement in virulence and drug resistance. Therefore, these differentially expressed proteins are of great significance for exploring effective control strategies of DR-TB.

Impact of the E57D and N189L mutations on catalysis of M. tuberculosis IGPS

Indole-3-glycerol phosphate synthesis (IGPS) is an enzyme that catalyzes the ring closure in 1-(o-carbocyphenylamino)-1-deoxyribulose 5-phosphate (CdRP). Multiple steps are expected to be involved in formation of the pyrrole ring including dehydration, decarboxylation, cyclization, and condensation. IGPS is an essential protein in the pathogen Mycobacterium tuberculosis and a potential target in the treatment of drug-resistant tuberculosis. In order to better understand the function of Mycobacterium tuberculosis IGPS (MtIGPS), we introduced single-point mutations into active site residues. I expressed and purified MtIGPS mutants N189L and E57D. The catalytic activities of the mutants were lower than that of the wild type MtIGPS, indicating that these residues play important roles in catalysis. The Michaelis constant, pH-dependence, and temperature dependence of the mutants were also determined. Wildtype MtIGPS was found to have a Km of 3.0 ± 0.6uM and a kcat value of 1.8 ± 0.13s-1 whereas E57D kinetics revealed a Km of 11.6 ± 2.1 uM and a kcat value of 0.047 ± 0.0009s-1. N189L indicated no measurable activity and could not be further characterized. The data on E57D show an altered pH profile, possibly resulting from the lower pKa of D compared to E, and support the hypothesis that E57 serves as the catalytic base in MtIGPS mechanism. Together these data contribute to an improved understanding of the MtIGPS catalysis and ligand binding.

Peptides-Based Vaccine MP3RT Induced Protective Immunity Against Mycobacterium Tuberculosis Infection in a Humanized Mouse Model.

BackgroundTuberculosis (TB) is still a global infectious disease that seriously threatens human beings. The only licensed TB vaccine Bacille Calmette-Guérin (BCG)’s protective efficacy varies significantly among populations and regions. It is very urgent to develop more effective vaccines.MethodsIn this study, eleven candidate proteins of Mycobacterium tuberculosis were selected to predict peptides with high-affinity binding capacity for the HLA-DRB1*01:01 molecule. The immunodominant peptides were identified with the enzyme-linked immunospot assay (ELISPOT) and linked in silico to result in a novel polypeptide vaccine in Escherichia coli cells. The vaccine’s protective efficacy was evaluated in humanized and wild-type C57BL/6 mice. The potential immune protective mechanisms were explored with Enzyme-linked Immunosorbent Assay (ELISA), flow cytometry, and ELISPOT.ResultsSix immunodominant peptides screened from 50 predicted peptides were used to construct a new polypeptide vaccine named MP3RT. After challenge with M. tuberculosis, the colony-forming units (CFUs), lung lesion area, and the number of inflammatory cells in humanized mice rather than wild-type mice vaccinated with MP3RT were significantly lower than these in mice immunized with PBS. The humanized mice vaccinated with MP3RT revealed significant increases in IFN-γ cytokine production, IFN-γ+ T lymphocytes, CD3+IFN-γ+ T lymphocytes, and the MP3RT-specific IgG antibody.ConclusionsTaken together, MP3RT is a promising peptides-based TB vaccine characterized by inducing high levels of IFN-γ and CD3+IFN-γ+ T lymphocytes in humanized mice. These new findings will lay a foundation for the development of peptides-based vaccines against TB.

Improvement of the immunogenicity of ESAT-6 via fusion with the dodecameric protein dodecin of Mycobacterium tuberculosis

Tuberculosis (TB) is a chronic infectious disease that creates a heavy medical burden worldwide. The only approved vaccine, Bacillus Calmette-Guérin (BCG), cannot fully protect adolescents and adults from TB. Therefore, there is an urgent need to develop an effective new vaccine. Previous studies have found that dodecin, a flavin-binding protein of Mycobacterium tuberculosis (Mtb), can form stable dodecamers and has the potential to improve the immunogenicity of Mtb antigens. In this study, we constructed the fusion protein dodecin-ESAT-6 and evaluated the immunogenicity of dodecin, ESAT-6, and dodecin-ESAT-6 separately. Our results showed that dodecin-ESAT-6 is a dodecameric protein that can withstand heat at 95 °C and under SDS-PAGE conditions. Dodecin-ESAT-6 increased the expression of the costimulatory molecules CD80, CD86, and major histocompatibility complex class II (MHC-II) on the surface of RAW264.7 macrophages. Mice immunized with dodecin-ESAT-6 exhibited higher percentages of antigen-specific CD4+ and CD8+ T lymphocytes, higher levels of spleen lymphocyte proliferation and IFN-γ and IL-2 secretion, and a lower level of IL-4 secretion than those immunized with ESAT-6. The IgG, IgG1, and IgG2a titers of the dodecin-ESAT-6 group were significantly higher than those of the ESAT-6 group. Dodecin-ESAT-6 elicited a high IgG2a/IgG1 ratio and tended to produce a predominantly Th1-like response. These results support the conclusion that the dodecin-ESAT-6 dodecameric protein induced strong Th1 immune responses and improved the immunogenicity of ESAT-6, which provides a new strategy for TB vaccine development.

Recombinant antigens and synthetic peptides to characterize Mycobacterium tuberculosis proteins for immunological reactivity

The control and eventual eradication of tuberculosis (TB) requires an effective vaccine and reagents for specific diagnosis. The only available vaccine against TB is the bacillus Calmette Guerin (BCG), but the protection imparted by BCG against pulmonary TB in adults varies between nil to 80%. Moreover, the use of BCG vaccination faces two additional problems: i. BCG vaccination induces a delayed type hypersensitivity response that cannot be distinguished from exposure to M. tuberculosis, and therefore it compromises the use of purified protein derivative (PPD) of M. tuberculosis for diagnostic purposes. ii. BCG being a live vaccine is contraindicated in HIV infected individuals for fear of causing disease by itself.

Comparison of simple and rapid extracting methods of free-tags Mycobacterium tuberculosis protein 64 Recombinant Protein from polyacrylamide gel: Electroelution and the optimized passive elution.

ABSTRACTIn this study, the Mycobacterium tuberculosis protein 64 (MPT64) protein was constructed without any tags to facilitate the purification using column affinity chromatography, but the MPT64 must be obtained as a pure protein. This study was purpose to ensure the efficient extracting method to purify protein MPT64 directly from the polyacrylamide gel. The crude extract of extracellular protein containing MPT64 protein was separated into single protein band and the targeted protein which is located in the size of 24 kDa was excised. Each of the six bands was collected in a sterile microtube to be eluted using electroelution and the optimized of the passive-elution method. Both the elution methods demonstrated the purity level of the MPT64 protein by detecting a solely band on the gel at the 24 kDa. Among the variety of passive-elution time, the highest MPT64 protein concentration was 0.549 mg/ml after elution for 72 h. However, the electroelution result provided higher MPT64 protein concentration, i.e., 0.683 mg/mL. However, based on the recognition of the purified MPT64 protein on commercial detection kit of MPT64 protein, it showed that the positive result was only showed by the passive-elution extracting protein. Therefore, for purifying the protein MPT64 from the sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels, the efficient method was passive elution.

PE_PGRS3 ensures provision of the vital phospholipids cardiolipin and phosphatidylinositols by promoting the interaction between M. tuberculosis and host cells

ABSTRACT PE_PGRS proteins of Mycobacterium tuberculosis (Mtb) constitute a large family of complex modular proteins whose role is still unclear. Among those, we have previously shown, using the heterologous expression in Mycobacterium smegmatis, that PE_PGRS3 containing a unique arginine-rich C-terminal domain, promotes adhesion to host cells. In this study, we investigate the role of PE_PGRS3 and its C-terminal domain directly in Mtb using functional deletion mutants. The results obtained here show that PE_PGRS3 is localized on the mycobacterial cell wall and its arginine-rich C-terminal region protrudes from the mycobacterial membrane and mediates Mtb entry into epithelial cells. Most importantly, this positively charged helical domain specifically binds phosphorylated phosphatidylinositols and cardiolipin, whereas it is unable to bind other phospholipids. Interestingly, administration of cardiolipin and phosphatidylinositol but no other phospholipids was able to turn-off expression of pe_pgrs3 activated by phosphate starvation conditions. These findings suggest that PE_PGRS3 has the key role to serve as a bridge between mycobacteria and host cells by interacting with specific host phospholipids and extracting them from host cells, for their direct integration or as a source of phosphate, during phases of TB pathogenesis when Mtb is short of phosphate supply.

Immunogenicity of HspX/EsxS fusion protein of Mycobacterium tuberculosis along with ISCOMATRIX and PLUSCOM nano-adjuvants after subcutaneous administration in animal model

BackgroundTuberculosis (TB), caused by Mycobacterium tuberculosis (M. tuberculosis), is one of the most common and dangerous infectious diseases in the world. Despite vaccination with BCG, it is still considered as a major health problem. Therefore, design and production of an effective novel vaccine against TB is necessary. Our aim was to evaluate immunogenicity of HspX/EsxS fusion protein of M. tuberculosis along with ISCOMATRIX, PLUSCOM nano-adjuvants and MPLA through the subcutaneous route in mice model. MethodsHspX/EsxS fused protein of M. tuberculosis was cloned, expressed and purified in the prokaryotic system. ISCOMATRIX and PLUSCOM nano-adjuvants were prepared by film hydration method. Subcutaneous immunization of BALB/c mice was performed by different formulations. IFN-γ, IL-4, IL-17 and TGF-β cytokines levels as well as serum IgG1, IgG2a. ResultsOur results showed that subcutaneous administration of mice with HspX/EsxS along with three adjuvants, ISCOMATRIX, PLUSCOM and MPLA increased immunogenicity of multi-stage fusion protein of M. tuberculosis. Additionally, HspX/EsxS protein + ISCOMATRIX or + PLUSCOM nano-adjuvants induced stronger Th1, IgG2a and IgG1 immune responses compared to MPLA adjuvant. Totally, HspX/EsxS/ISCOMATRIX/MPLA, HspX/EsxS/PLUSCOM/MPLA and two BCG booster groups could significantly induce higher Th1 and IgG2a immune responses. ConclusionWith regard to ability of ISCOMATRIX, PLUSCOM and MPLA adjuvants to increase immunogenicity of HspX/EsxS protein through induction of IFN-γ and IgG2a immune responses, it seems that these adjuvants and especially ISCOMATRIX and PLUSCOM, could also improve BCG efficacy as a BCG booster.

Immune Responses Following BCG Immunization of Infants in Uganda and United Kingdom Are Similar for Purified Protein Derivative but Differ for Secretory Proteins of Mycobacterium tuberculosis.

Introduction: The immunogenicity of BCG vaccination in infants differs between populations. We hypothesized that prenatal exposure to mycobacterial antigens might explain the differences in immune responses to BCG seen in other studies of infants in Africa and the United Kingdom (UK) and we explored this in birth cohorts in Uganda and the UK.Materials and Methods: Blood samples were obtained from BCG-immunized infants of mothers with (n = 110) and without (n = 121) latent Mycobacterium tuberculosis infection (LTBI) in Uganda and BCG-immunized infants of mothers without LTBI (n = 25) in the UK at 10 and 52 weeks after birth. Cytokine and chemokine responses to PPD were measured to assess responses to BCG immunization, and to ESAT6/CFP10 to assess exposure to or infection with M. tuberculosis or non-tuberculous mycobacteria (NTM) in 6-day whole blood culture supernatants by a 17-plex Luminex assay. Median responses were compared between Ugandan infants (together, and separated by maternal LTBI status) and UK infants.Results: The IFN-γ response to BCG vaccination was similar between Ugandan and UK infants at 10 and 52 weeks. At week 52, TNF production was marginally higher in Ugandan infants, but after adjusting for multiple comparisons this difference was not significant. At weeks 10 and 52, stimulation of blood with ESAT6/CFP10 produced significantly higher IFN-γ, TNF, IL-12p40, IL-1α, IL-1β, IL-1Ra, IP-10, MIP-1α, MIP-1β, and GM-CSF in Ugandan compared to UK infants. Stimulation of blood with ESAT6/CFP10 produced significantly higher amounts of IL-8 (p = 0.0001), IL-10 (p = 0.0022), and IL-13 (p = 0.0020) in the UK than in Ugandan infants of mothers without LTBI at week 10, but not at week 52.Conclusions: Immune responses to mycobacterial antigens following BCG immunization are similar for PPD, but differ for ESAT6/CFP10, between infants in Uganda and the UK. Neither maternal LTBI nor infant exposure to or infection with mycobacteria impacts the response to BCG. The observed global differences in immune response to BCG immunization are likely to be due to other causes.

Engineered Mycobacterium tuberculosis antigen assembly into core-shell nanobeads for diagnosis of tuberculosis

Despite recent advances in diagnosis, tuberculosis (TB) remains one of the ten leading causes of death worldwide. Here, we engineered Mycobacterium tuberculosis (Mtb) proteins (ESAT6, CFP10, and MTB7.7) to self-assemble into core-shell nanobeads for enhanced TB diagnosis. Respective purified Mtb antigen-coated polyester beads were characterized and their functionality in TB diagnosis was tested in whole blood cytokine release assays. Sensitivity and specificity were studied in 11 pulmonary TB patients (PTB) and 26 healthy individuals composed of 14 Tuberculin Skin Test negative (TSTn) and 12 TST positive (TSTp). The production of 6 cytokines was determined (IFNγ, IP10, IL2, TNFα, CCL3, and CCL11). To differentiate PTB from healthy individuals (TSTp + TSTn), the best individual cytokines were IL2 and CCL11 (>80% sensitivity and specificity) and the best combination was IP10 + IL2 (>90% sensitivity and specificity). We describe an innovative approach using full-length antigens attached to biopolyester nanobeads enabling sensitive and specific detection of human TB.