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A single ssDNA aptamer binding to ManLAM of BCG enhances immunoprotective effects against Tuberculosis
Abstract
Because of the Mycobacterium bovis, termed Bacillus Calmette–Guérin (BCG), the only available used Tuberculosis (TB) vaccine, retaining immunomodulatory properties that limit its protective immunogenicity, there are continuous efforts to identify the immunosuppression mechanism as well as new strategies for improving immunogenicity of BCG. Here, an ssDNA aptamer “antibody” BM2 that specifically bound to Mannose-capped lipoarabinomannan (ManLAM) of BCG was selected. BM2 significantly blocked ManLAM-Mannose Receptor (MR) binding and triggered ManLAM-CD44 signaling, and enhanced M1 macrophage and Th1 activation via cellular surface CD44 in vitro and in vivo. BM2 enhanced immunoprotective effects of BCG against virulent Mycobacterium tuberculosis (M. tb) H37Rv infection in mice and monkeys models. Thus, we report a new mechanism of the interaction between ManLAM and CD44 on macrophages and CD4+ T cells, and reveal that ManLAM-binding membrane molecule CD44 is a novel target for the enhancement of BCG immunogenicity and BM2 has strong potential as an immune enhancer for BCG.
... The MPI anchors of ManLAM are recognized by CD1 (including CD1b and CD1d), Toll like receptor 2 (TLR2), dendritic cell immunoactivating receptor (DCAR), mannose-binding protein (MBP), and lactosylceramide enriched lipid rafts in the plasma membrane ( Figure 1) [10][11][12][13][14][15][16]. Other receptors and molecules that bind to ManLAM include pulmonary surfactant protein A (PS-A), Dectin 2 and CD44 [17][18][19][20][21], however, the ManLAM binding domains that interact with these molecules remain unknown. ...
... On the one hand, macrophages can develop into M1-polarized cells that engulf and eliminate the invading M. tb [30], while on the other hand, macrophages provide a critical intracellular niche that is required for M. tb to establish infection [31]. Early studies observed increased tumour necrosis factor (TNF) production by both human and murine macrophages following ManLAM stimulation [32], while later studies demonstrated that ManLAM inhibits various interferon γ (IFN-γ)-mediated microbicidal and tumoricidal activities, and triggers macrophages to produce anti-inflammatory cytokine IL-10 [19,26,33,34]. Previously, we reported that ManLAM recognized by CD44 causes the upregulation of IL-1β, IL-12 and iNOS (inducible nitric oxide synthase) expression in macrophages, indicating that ManLAM-CD44 signalling enhances M1 macrophage polarization [19,20]. ...
... Early studies observed increased tumour necrosis factor (TNF) production by both human and murine macrophages following ManLAM stimulation [32], while later studies demonstrated that ManLAM inhibits various interferon γ (IFN-γ)-mediated microbicidal and tumoricidal activities, and triggers macrophages to produce anti-inflammatory cytokine IL-10 [19,26,33,34]. Previously, we reported that ManLAM recognized by CD44 causes the upregulation of IL-1β, IL-12 and iNOS (inducible nitric oxide synthase) expression in macrophages, indicating that ManLAM-CD44 signalling enhances M1 macrophage polarization [19,20]. However, the binding of ManLAM to MR might lead to IL-10 production and impair M1 polarization [19]. ...
Mannose-capped lipoarabinomannan (ManLAM) is a high molecular mass amphipathic lipoglycan identified in pathogenic Mycobacterium tuberculosis (M. tb) and M. bovis Bacillus Calmette-Guérin (BCG). ManLAM, serves as both an immunogen and a modulator of the host immune system, and its critical role in mycobacterial survival during infection has been well-characterized. ManLAM can be recognized by various types of receptors on both innate and adaptive immune cells, including macrophages, dendritic cells (DCs), neutrophils, natural killer T (NKT) cells, T cells and B cells. MamLAM has been shown to affect phagocytosis, cytokine production, antigen presentation, T cell activation and polarization, as well as antibody production. Exploring the mechanisms underlying the roles of ManLAM during mycobacterial infection will aid in improving tuberculosis (TB) prevention, diagnosis and treatment interventions. In this review, we highlight the interaction between ManLAM and receptors, intracellular signalling pathways triggered by ManLAM and its roles in both innate and adaptive immune responses.
... Our group has selected several aptamers specifically binding to M. tb whole cells 18 and surface lipoglycan. [19][20][21] We first implemented whole-cell SELEX and generated ssDNA aptamer NK2, which binds to virulent M. tb H37Rv with high affinity and specificity. 18 We then selected ssDNA aptamer ZXL1, which specifically binds to ManLAM from virulent M. tb strain H37Rv, 19 , 21 and significantly reduces the progression of M. tb H37Rv infections as well as bacterial loads in mice and rhesus monkeys. ...
... 18 We then selected ssDNA aptamer ZXL1, which specifically binds to ManLAM from virulent M. tb strain H37Rv, 19 , 21 and significantly reduces the progression of M. tb H37Rv infections as well as bacterial loads in mice and rhesus monkeys. 19 Based on the different structures of ManLAM among different Mycobacteria , we next selected ssDNA aptamer BM2 that specifically binds to ManLAM from BCG. 20 Aptamer BM2 acts as an adjuvant enhancing immunoprotective effects of BCG against virulent M. tb infection in murine and monkey models. 20 To our knowledge, this was the first report regarding an aptamer serving as an adjuvant. ...
... 19 Based on the different structures of ManLAM among different Mycobacteria , we next selected ssDNA aptamer BM2 that specifically binds to ManLAM from BCG. 20 Aptamer BM2 acts as an adjuvant enhancing immunoprotective effects of BCG against virulent M. tb infection in murine and monkey models. 20 To our knowledge, this was the first report regarding an aptamer serving as an adjuvant. Other groups have also reported aptamers potential for TB therapy. ...
The entire human population is at risk of infectious diseases worldwide. Thus far, the diagnosis and treatment of human infectious diseases at the molecular and nanoscale levels have been extremely challenging tasks because of the lack of effective probes to identify and recognize biomarkers of pathogens. Oligonucleotide aptamers are a class of small nucleic acid ligands that are composed of single-stranded DNA (ssDNA) or RNA and act as affinity probes or molecular recognition elements for a variety of targets. These aptamers have an exciting potential for diagnose and/or treatment of specific diseases. In this review, we highlight areas where aptamers have been developed as diagnostic and therapeutic agents for both bacterial and viral infectious diseases as well as aptamer-based detection.
... To test the performance of ddPCR as a tool to evaluate the protective efficacy in vaccine studies performed in nonhuman primates, we used banked blood specimens from a previously reported monkey study 14 . Because of the expense, a total of eight rhesus monkeys (RM) were used in three groups (PBS (n = 2), BCG (n = 3), and BCG + BM2 (ssDNA aptamer "antibody" BM2 specifically bound to the mannose-capped lipoarabinomannan (ManLAM) of BCG) (n = 3) groups) 14 . ...
... To test the performance of ddPCR as a tool to evaluate the protective efficacy in vaccine studies performed in nonhuman primates, we used banked blood specimens from a previously reported monkey study 14 . Because of the expense, a total of eight rhesus monkeys (RM) were used in three groups (PBS (n = 2), BCG (n = 3), and BCG + BM2 (ssDNA aptamer "antibody" BM2 specifically bound to the mannose-capped lipoarabinomannan (ManLAM) of BCG) (n = 3) groups) 14 . Briefly, at week -5, the RMs were vaccinated with BCG or BCG + BM2. ...
... At week 0, all RMs were infected with 100 CFUs of M. tb H37Rv. Then blood samples were collected and stored at 3, 5, 10, 16, 19 weeks after H37Rv infection 14 . The bacterial CFU burden in the lung has been detected and histopathological analysis has been performed to confirm that BCG/BCG + BM2 vaccine groups elicited protective efficiency, compared with the PBS group in our previous report 14 . ...
Mycobacterium tuberculosis (M. tb) is emerging as a more serious pathogen due to the increased multidrug-resistant TB and co-infection of human immunodeficiency virus (HIV). The development of an effective and sensitive detection method is urgently needed for bacterial load evaluation in vaccine development, early TB diagnosis, and TB treatment. Droplet digital polymerase chain reaction (ddPCR) is a newly developed sensitive PCR method for the absolute quantification of nucleic acid concentrations. Here, we used ddPCR to quantify the circulating virulent M. tb-specific CFP10 (10-kDa culture filtrate protein, Rv3874) and Rv1768 DNA copy numbers in the blood samples from Bacille Calmette-Guerin (BCG)-vaccinated and/or virulent M. tb H37Rv-challenged rhesus monkeys. We found that ddPCR was more sensitive compared to real-time fluorescence quantitative PCR (qPCR), as the detection limits of CFP10 were 1.2 copies/μl for ddPCR, but 15.8 copies/μl for qPCR. We demonstrated that ddPCR could detect CFP10 and Rv1768 DNA after 3 weeks of infection and at least two weeks earlier than qPCR in M.tb H37Rv-challenged rhesus monkey models. DdPCR could also successfully quantify CFP10 and Rv1768 DNA copy numbers in clinical TB patients’ blood samples (active pulmonary TB, extrapulmonary TB (EPTB), and infant TB). To our knowledge, this study is the first to demonstrate that ddPCR is an effective and sensitive method of measuring the circulating CFP10 and Rv1768 DNA for vaccine development, bacterial load evaluation in vivo, and early TB (including EPTB and infant TB) diagnosis as well.
... Because of their high affinity and specificity, aptamers have been used as therapeutic or diagnostic tools in numerous investigations (19,20). In our previous studies, we generated the ssDNA aptamer ZXL1 specifically against ManLAM from virulent Mtb H37Rv and the ssDNA aptamer BM2, which specifically binds to ManLAM from Mycobacterium bovis (termed BCG), based on the different structures of ManLAMs in different Mycobacteria (21,22). We reported that ZXL1 competes with the MR for binding to ManLAM and inhibits ManLAM-induced immunosuppression of DCs and that BM2 promotes M1 macrophage polarization (21,22). ...
... In our previous studies, we generated the ssDNA aptamer ZXL1 specifically against ManLAM from virulent Mtb H37Rv and the ssDNA aptamer BM2, which specifically binds to ManLAM from Mycobacterium bovis (termed BCG), based on the different structures of ManLAMs in different Mycobacteria (21,22). We reported that ZXL1 competes with the MR for binding to ManLAM and inhibits ManLAM-induced immunosuppression of DCs and that BM2 promotes M1 macrophage polarization (21,22). However, we do not know the effects of ZXL1 on macrophages or the exact nature of the cellular signaling pathway triggered by anti-ManLAM aptamers. ...
... Enzyme-linked oligonucleotide assays were performed according to our previous report (21,22). ELISA plates were coated with ManLAM (1 mg/mL) overnight at 4°C. ...
Mannose-capped lipoarabinomannan (ManLAM) is an immunomodulatory epitope of Mycobacterium tuberculosis (Mtb). We previously generated an aptamer (ZXL1) that specifically binds to ManLAM from the virulent Mtb H37Rv strain and reported that ZXL1 functioned as an antagonist, inhibiting the ManLAM-induced immunosuppression of dendritic cells (DCs). In the present study, we found that ZXL1 inhibited Mtb entry into murine macrophages. ZXL1 enhanced IL-1β and IL-12 mRNA expression and cytokine production in ManLAM-treated macrophages but decreased IL-10 production. Inducible nitric oxide synthase (iNOS) expression in the macrophages was upregulated in the presence of ZXL1 after stimulation with ManLAM. ZXL1 also inhibited the expression of the lipid-sensing nuclear receptor peroxisome proliferator-activated receptor γ (PPARγ). These results suggest that ZXL1 promotes anti-tuberculosis activity through the downregulation of PPAR γ expression, which may contribute to M1 macrophage polarization and Mtb killing by macrophages.
... Its precursors, including LM, phosphatidyl-myoinositol mannosides (PIMs), have been shown to have potential regulatory effects in vitro experiments on immune cells (Dao et al. 2004;Gilleron et al. 2003). Mannose-capped lipoarabinomannan (ManLAM) is a major cell wall lipoglycan and an important immunomodulatory component of mycobacteria (Kang et al. 2005;Sun et al. 2016;Tang et al. 2017;Gringhuis et al. 2009;Yonekawa et al. 2014;Yuan et al. 2019;Torrelles and Schlesinger 2010) (Table 11.3). Bacterial ManLAM can also be secreted and recognized by macrophages and DCs via pattern recognition receptors, including MR, Toll-like receptor 2 (TLR2), DC-SIGN, CD1d, sphingosine-1phosphate receptor 1 (S1P1), Dectin-2, and CD44, and triggers several cell signaling pathways (Sun et al. 2016;Yonekawa et al. 2014;Pan et al. 2014;Osanya et al. 2011;Zajonc et al. 2006). ...
... Mannose-capped lipoarabinomannan (ManLAM) is a major cell wall lipoglycan and an important immunomodulatory component of mycobacteria (Kang et al. 2005;Sun et al. 2016;Tang et al. 2017;Gringhuis et al. 2009;Yonekawa et al. 2014;Yuan et al. 2019;Torrelles and Schlesinger 2010) (Table 11.3). Bacterial ManLAM can also be secreted and recognized by macrophages and DCs via pattern recognition receptors, including MR, Toll-like receptor 2 (TLR2), DC-SIGN, CD1d, sphingosine-1phosphate receptor 1 (S1P1), Dectin-2, and CD44, and triggers several cell signaling pathways (Sun et al. 2016;Yonekawa et al. 2014;Pan et al. 2014;Osanya et al. 2011;Zajonc et al. 2006). It is well known that ManLAM inhibits phagosome maturation of macrophages, DC maturation, and CD4 + T cell activation (Osanya et al. 2011;Fratti et al. 2003;Mahon et al. 2012). ...
Glycosylation plays an important role in infectious diseases. Many important interactions between pathogens and hosts involve their carbohydrate structures (glycans). Glycan interactions can mediate adhesion, recognition, invasion, and immune evasion of pathogens. To date, changes in many protein N/O-linked glycosylation have been identified as biomarkers for the development of infectious diseases and cancers. In this review, we will discuss the principal findings and the roles of glycosylation of both pathogens and host cells in the context of human important infectious diseases. Understanding the role and mechanism of glycan-lectin interaction between pathogens and hosts may create a new paradigm for discovering novel glycan-based therapies that can lead to eradication or functional cure of pathogens infection.
... ManLAM was prepared from M tb H37Rv (ATCC strain 93009) or BCG (ATCC strain 35734) as previously described. 16,18 ManLAM was extracted from delipidated bacteria, purified by high-performance liquid chromatography (HPLC) and identified as our previous reports. 16,18 Briefly, the bacteria were maintained on L-J (Lowenstein-Jensen) medium and were harvested while in log phase growth. ...
... 16,18 ManLAM was extracted from delipidated bacteria, purified by high-performance liquid chromatography (HPLC) and identified as our previous reports. 16,18 Briefly, the bacteria were maintained on L-J (Lowenstein-Jensen) medium and were harvested while in log phase growth. The bacterial cells were delipidated using CHCl 3 : CH 3 OH (2:1, v/v) at 37°C for 12 hours. ...
IBD (inflammatory bowel disease) is a chronic, non‐specific, inflammatory gastrointestinal disease that mainly consists of Crohn's disease and ulcerative colitis. However, the etiology and pathogenesis of IBD are still unclear. B10 (IL‐10 producing regulatory B) cells, a subset of regulatory B cells, are known to contribute to intestinal homeostasis and the aberrant frequency of B10 cells is associated with IBD. We have recently reported that B10 cells can be induced by ManLAM (mannose‐capped lipoarabinomannan), a major cell‐wall lipoglycan of M.tb (Mycobacterium tuberculosis). In the current study, the ManLAM‐induced B10 cells were adoptively transferred into IL(interleukin)‐10‐/‐ mice and the roles of ManLAM‐induced B10 cells were investigated in DSS (dextran sodium sulfate)‐induced IBD model. ManLAM‐induced B10 cells decrease colitis severity in the mice. The B10 cells downregulate Th1 polarization in spleen and MLNs (mesenteric lymph nodes) of DSS‐treated mice. These results suggest that IL‐10 production by ManLAM‐treated B cells contributes to keeping the balance between CD4+ T cell subsets and protect mice from DSS‐induced IBD.
... Each C57BL/6 mouse (male, ages between 8 and 10 weeks) was infected with H37Rv, H37RvEST12, BCG, and BCG-EST12 (5 × 10 4 CFUs of M.tb/50 l diluted with PBS) by intranasal administration on day 0. On day 30, the mice were euthanized. The bacterial loads in the lungs of infected mice were assessed as previously described (73). The serum cytokine production was assessed by ELISA. ...
Pyroptosis, an inflammatory form of programmed cell death, has been implicated in eliminating pathogenic infections. However, macrophage pyroptosis-related proteins from Mycobacterium tuberculosis (M.tb) have largely gone unexplored. Here, we identified a cell pyroptosis-inducing protein, Rv1579c, named EST12, secreted from the M.tb H37Rv region of difference 3. EST12 binds to the receptor for activated C kinase 1 (RACK1) in macrophages, and the EST12-RACK1 complex recruits the deubiquitinase UCHL5 to promote the K48-linked deubiquitination of NLRP3, subsequently leading to an NLRP3 inflammasome caspase-1/11-pyroptosis gasdermin D-interleukin-1 immune process. Analysis of the crystal structure of EST12 reveals that the amino acid Y80 acts as a critical binding site for RACK1. An EST12-deficient strain (H37RvEST12) displayed higher susceptibility to M.tb infection in vitro and in vivo. These results provide the first proof that RACK1 acts as an endogenous host sensor for pathogens and that EST12-RACK1-induced pyroptosis plays a pivotal role in M.tb-induced immunity.
... The aptamers 16 are a small segment of oligonucleotide sequences that have been screened in vitro and can bind to the corresponding ligand with high affinity and strong specificity. The appearance of aptamers has provided a new efficient and rapid identification research platform for the chemical, 17 biological 18,19 and biomedical fields. ...
Electrochemistry will bring revolutionary detection methods for bisphenol A (BPA) which is a endocrine disrupter. Electrochemical aptasensor appears to be a promising approach to this end because of its brilliant specificity, high affinity, low cost and rapid response time. However, electrochemical aptasensor suffers from reducing signal when anti-BPA aptamers are modified on electrode surface. In response, we report an electrochemical aptasensor based on gold nanoparticles (AuNPs) and nitrogen, sulfur, and phosphorus co-doped carbon dots (N,S,P-CDs) modified glassy carbon electrode(GCE) for amplified signal detection of BPA. The thiol-gold (S-Au) bonds between the AuNPs and the thiol termini on anti-BPA aptamers anchor the anti-BPA aptamers to the GCE. N,S,P-CDs as electrode modifications are applied to expand the active area and enhance electron-transport ability. In this work, the presences of N,S,P-CDs and the AuNPs were assessed via Fourier transform infrared (FTIR), field emission scanning electron microscopy (FESEM), transmission electron microscopy (TEM), X-ray diffractometer (XRD), etc. The sensing scheme and electrochemical response of the proposed sensor were investigated by chronocoulometry (CC), cyclic voltammetry (CV), electrochemical impedance spectra (EIS) and differential pulse voltammetry (DPV). Meanwhile, we focused on discussing the kinetic parameters of [Fe(CN)6]3−/4− on the composite electrode that had reacted with BPA, such as active surface areas (A), surface concentration (Г⁰), the standard rate constant (k) and electron transfer coefficient (α). Aptasensor revealed a high sensitivity and had a linear electrochemical response to BPA in the concentration range of 0.01 μM–120 μM, with a low detection limit of 5.273 × 10−10 M. Moreover, the proposed aptasensor could be applied for the detection of BPA in real samples with great precision, high sensitivity, excellent selectivity, brilliant reproducibility and satisfactory results. Therefore, this convenient and sensitive aptasensor is proved to a promising and reliable tool for detecting BPA in food and environmental pollution.
... M. tb H37Rv (strain American Type Culture Collection (ATCC) 93 009) and Mycobacterium bovis BCG (ATCC strain 35 734) were purchased from the Beijing Biological Product Institute (Beijing, China) (Sun et al. 2016;Tang et al. 2017). The mycobacterial strain was grown in Middlebrook 7H9 broth (BD Biosciences, NJ, USA) supplemented with 10% oleic acid-albumin-dextrose-catalase (OADC, BD Biosciences, NJ, USA) and 0.05% Tween-80. ...
Macrophages are the primary host target cells of Mycobacterium tuberculosis (M. tb). As a subunit of immunoregulatory cytokines IL-27 and IL-35, Epstein–Barr virus-induced gene 3 (EBI3) has typically been explored as the secreted form and assessed in terms of its effects triggered by extracellular EBI3. However, little is known about intracellular EBI3 function. In the current study, we report that EBI3 production by macrophages is elevated in TB patients. We further demonstrate that increased EBI3 accumulates in virulent M. tb-treated murine macrophages. Eukaryotic translation elongation factor 1-alpha 1 (eEF1A1) binds to intracellular EBI3 to reduce Lys48 (K48)-linked ubiquitination of EBI3, leading to EBI3 accumulation. Moreover, the intracellular EBI3 inhibits caspase-3-mediated apoptosis in M. tb-treated macrophages. Herein, we propose a novel mechanism for accumulating intracellular EBI3 and its regulation of macrophage apoptosis in response to virulent M. tb.
This study presented a nanoparticle-enhanced aptamer-recognizing homogeneous detection system combined with a portable instrument (NASPI) to quantify lipoarabinomannan (LAM). This system leveraged the high binding affinity of aptamers, the high...
Tuberculosis (TB) is a chronic infectious disease mainly caused by Mycobacterium tuberculosis (M. tuberculosis). The structures of polysaccharides and glycolipids at M. tuberculosis cell wall vary among different strains, which affect the physiology and pathogenesis of mycobacteria by activating or inhibiting innate and acquired immunity. Among them, some components such as lipomannan (LM) and lipoarabinomannan (LAM) activate innate immunity by recognizing some kinds of pattern recognition receptors (PRRs) like Toll‐like receptors, while other components like mannose‐capped lipoarabinomannan (ManLAM) could prevent innate immune responses by inhibiting the secretion of pro‐inflammatory cytokines and maturation of phagosomes. In addition, many glycolipids can activate natural killer T (NKT) cells and CD1‐restricted T cells to produce interferon‐γ (IFN‐γ). Furthermore, humoral immunity against cell wall components, such as antibodies against LAM, plays a role in immunity against M. tuberculosis infection. Cell wall polysaccharides and glycolipids of M. tuberculosis have potential applications as antigens and adjuvants for novel TB subunit vaccines.
Tuberculosis, caused by Mycobacterium tuberculosis, claims ∼1.5 million lives annually. Effective chemotherapy is essential to control TB, however the emergence of drug-resistant strains of TB have seriously threatened global attempts to control and eradicate this deadly pathogen. Trehalose recycling via the LpqY-SugABC importer is essential for the virulence and survival of Mtb and inhibiting or hijacking this transport system is an attractive approach for the development of novel anti-tubercular and diagnostic agents. Therefore, we interrogated the drug-like compounds in the open-source Medicines for Malaria Pathogen Box and successfully identified seven compounds from the TB, kinetoplastids and reference compound disease sets that recognise LpqY. The molecules have diverse chemical scaffolds, are not specific trehalose analogues, and may be used as novel templates to facilitate the development of therapeutics that kill Mtb with a novel mechanism of action via the mycobacterial trehalose LpqY-SugABC transport system.
Glycans are carbohydrates that are made by all organisms and covalently conjugated to other biomolecules. Glycans cover the surface of both human cells and pathogens and are fundamental to defining the identity of a cell or an organism, thereby contributing to discriminating self from non‐self. As such, glycans are a class of “Self‐Associated Molecular Patterns” that can fine‐tune host inflammatory processes. In fact, glycans can be sensed and recognized by a variety of glycan‐binding proteins (GBP) expressed by immune cells, such as galectins, siglecs and C‐type lectins, which recognize changes in the cellular glycosylation, instructing both pro‐inflammatory or anti‐inflammatory responses. In this review, we introduce glycans as cell‐identification structures, discussing how glycans modulate host‐pathogen interactions and how they can fine‐tune inflammatory processes associated with infection, inflammation and autoimmunity. Finally, from the clinical standpoint, we discuss how glycoscience research can benefit life sciences and clinical medicine by providing a source of valuable biomarkers and therapeutic targets for immunity. Glycans have an enormous power to discriminate self/non‐self as they constitute a fundamental molecular ID of both human cells and pathogens. We discuss the concept of glycan mimicry associated with the breach of immune tolerance and the potential application of glycans as promising diagnostic/prognostic biomarkers or as novel immunomodulatory therapies against infections and autoimmune diseases.
Purpose
The rapid emergence of multidrug-resistant Mycobacterium tuberculosis (MTB) poses a significant challenge to the treatment of tuberculosis (TB). Sonodynamic antibacterial chemotherapy (SACT) combined with sonosensitizer-loaded nanoparticles with targeted therapeutic function is highly expected to eliminate bacteria without fear of drug resistance. This study aimed to investigate the antibacterial effect and underlying mechanism of levofloxacin-loaded nanosonosensitizer with targeted therapeutic function against Bacillus Calmette-Guérin bacteria (BCG, an MTB model).
Methods
This study developed levofloxacin-loaded PLGA-PEG (poly lactide-co-glycolide-polyethylene glycol) nanoparticles with BM2 aptamer conjugation on its surface using the crosslinking agents EDC and NHS (BM2-LVFX-NPs). The average diameter, zeta potential, morphology, drug-loading properties, and drug release efficiency of the BM2-LVFX-NPs were investigated. In addition, the targeting and toxicity of BM2-LVFX-NPs in the subcutaneous BCG infection model were evaluated. The biosafety, reactive oxygen species (ROS) production, cellular phagocytic effect, and antibacterial effect of BM2-LVFX-NPs in the presence of ultrasound stimulations (42 kHz, 0.67 W/cm², 5 min) were also systematically evaluated.
Results
BM2-LVFX-NPs not only specifically recognized BCG bacteria in vitro but also gathered accurately in the lesion tissues. Drugs loaded in BM2-LVFX-NPs with the ultrasound-responsive feature were effectively released compared to the natural state. In addition, BM2-LVFX-NPs exhibited significant SACT efficiency with higher ROS production levels than others, resulting in the effective elimination of bacteria in vitro. Meanwhile, in vivo experiments, compared with other options, BM2-LVFX-NPs also exhibited an excellent therapeutic effect in a rat model with BCG infection after exposure to ultrasound.
Conclusion
Our work demonstrated that a nanosonosensitizer formulation with LVFX could efficiently translocate therapeutic drugs into the cell and improve the bactericidal effects under ultrasound, which could be a promising strategy for targeted therapy for MTB infections with high biosafety.
During an epidemic or pandemic, the primary task is to rapidly develop precise diagnostic approaches and effective therapeutics. Oligonucleotide aptamer-based pathogen detection assays and control therapeutics are promising, as aptamers that specifically recognize and block pathogens can be quickly developed and produced through simple chemical synthesis. This work reviews common aptamer-based diagnostic techniques for communicable diseases and summarizes currently available aptamers that target various pathogens, including the SARS-CoV-2 virus. Moreover, this review discusses how oligonucleotide aptamers might be leveraged to control pathogen propagation and improve host immune system responses. This review offers a comprehensive data source to the further develop aptamer-based diagnostics and therapeutics specific for infectious diseases.
As a key virulence factor for persistent colonization, urease B subunit (UreB) is considered to be an ideal vaccine antigen against Helicobacter pylori infection. However, the role and molecular mechanisms of UreB involved in immune microenvironment dysregulation still remain largely unknown. In the present study, we evaluated the effects of UreB on macrophage activation and found that UreB induced PD-L1 accumulation on bone marrow-derived macrophages (BMDMs). Co-culture assays further revealed that UreB-induced PD-L1 expression on BMDMs significantly decreased the proliferation and secretion of cytolytic molecules (granzyme B and perforin) of splenic CD8+ T cells isolated from inactivated H. pylori-immunized mice. More importantly, using liquid chromatography–tandem mass spectrometry (LC-MS/MS) and co-immunoprecipitation techniques, it has been confirmed that myosin heavy chain 9 (Myh9) is a direct membrane receptor for UreB and is required for PD-L1 up-regulation on BMDMs. Molecular studies further demonstrated that the interaction between UreB and Myh9 decreased GCN2 autophosphorylation and enhanced the intracellular pool of amino acids, leading to the up-regulation of S6K phosphorylation, a commonly used marker for monitoring activation of mTORC1 signaling activity. Furthermore, blocking mTORC1 activation with its inhibitor Temsirolimus reversed the UreB-induced PD-L1 up-regulation and the subsequent inhibitory effects of BMDMs on activation of cytotoxic CD8+ T-cell responses. Overall, our data unveil a novel immunosuppressive mechanism of UreB during H. pylori infection, which may provide valuable clues for the optimization of H. pylori vaccine.
Tuberculosis (TB) has been plaguing human civilization for centuries, and currently around one-third of the global population is affected with TB. Development of novel intervention tools for early diagnosis and therapeutics against Mycobacterium tuberculosis (M.tb) is the main thrust area in today’s scenario. In this direction global efforts were made to use aptamers, the chemical antibodies as tool for TB diagnostics and therapeutics. This review describes the various aptamers introduced for targeting M.tb and highlights the need for development of novel aptamers to selectively target virulent proteins of M.tb for vaccine and anti-TB drugs. The objective of this review is to highlight the diagnostic and therapeutic application of aptamers used for tuberculosis. The discovery of aptamers, SELEX technology, different types of SELEX development processes, DNA and RNA aptamers reported for diseases and pathogenic agents as well have also been described in detail. But the emphasis of this review is on the development of aptamers which can block the function of virulent mycobacterial components for developing newer TB vaccine candidates and/or drug targets. Aptamers designed to target M.tb cell wall proteins, virulent factors, secretory proteins, or combination could orchestrate advanced diagnosis and therapeutic measures for tuberculosis.
Mycobacterial arabinogalactan (AG) is an essential cell wall component of mycobacteria and a frequent structural and bio-synthetical target for anti-tuberculosis (TB) drug development. Here, we report that mycobacterial AG is recognized by galectin-9 and exacerbates mycobacterial infection. Administration of AG-specific aptamers inhibits cellular infiltration caused by Mycobacterium tuberculosis (Mtb) or Mycobacterium bovis BCG, and moderately increases survival of Mtb-infected mice or Mycobacterium marinum-infected zebrafish. AG interacts with carbohydrate recognition domain (CRD) 2 of galectin-9 with high affinity, and galectin-9 associates with transforming growth factor β-activated kinase 1 (TAK1) via CRD2 to trigger subsequent activation of extracellular signal-regulated kinase (ERK) as well as induction of the expression of matrix metalloproteinases (MMPs). Moreover, deletion of galectin-9 or inhibition of MMPs blocks AG-induced pathological impairments in the lung, and the AG-galectin-9 axis aggravates the process of Mtb infection in mice. These results demonstrate that AG is an important virulence factor of mycobacteria and galectin-9 is a novel receptor for Mtb and other mycobacteria, paving the way for the development of novel effective TB immune modulators.
Aptamers have gained an increasing role as the molecular recognition element (MRE) in diagnostic assay development, since their first conception thirty years ago. The process to screen for nucleic acid-based binding elements (aptamers) was first described in 1990 by the Gold Laboratory. In the last three decades, many aptamers have been identified for a wide array of targets. In particular, the number of reports on investigating single-stranded DNA (ssDNA) aptamer applications in biosensing and diagnostic platforms have increased significantly in recent years. This review article summarizes the recent (2015 to 2020) progress of ssDNA aptamer research on bacteria, proteins, and lipids of bacterial origins that have implications for human infections. The basic process of aptamer selection, the principles of aptamer-based biosensors, and future perspectives will also be discussed.
The development of a tumor-targeted immunotherapy is highly required. The most advanced application is the use of CD19 chimeric antigen receptor (CAR) T (CAR-T) cells to B-cell malignancies, but there are still side effects including potential carcinogenicity of lentiviral or retroviral insertion into the host cell genome. Here we developed a non-viral aptamer-T-cell targeted strategy for tumor therapy. Tumor cells surface-specific ssDNA aptamers were conjugated to CD3+T cells (aptamer-T cells) using N-azidomannosamine (ManNAz) sugar metabolic cell labeling and click chemistry. We found that the aptamer-T cells could specifically target and bind to tumor cells (such as SGC-7901 gastric cancer cell and CT26 colon carcinoma cell) in vitro and in mice after adoptively transfer in. Aptamer-T cells led to significant regression in tumor volume due to being enriched at tumor microenvironment and producing strong cytotoxicity activities of CD3+T cells with enhanced perforin, granzyme B, CD107a, CD69 and FasL expression. Moreover, aptamer-T displayed even stronger anti-tumor effects than an anti-PD1 immune-checkpoint monoclonal antibody (mAb) treatment in mice and combination with anti-PD1 yielded synergic anti-tumor effects. This study uncovers the strong potential of the adoptive non-viral aptamer-T cell strategy as a feasible and efficacious approach for tumor-targeted immunotherapy application.
Nanomedicine uses nanotechnology-based strategies for precision tumor therapy, including passive and ligand-mediated active tumor targeting by nanocarriers. However, the possible biotoxicity of chemosynthetic nanovectors limits their clinical applications. A novel natural egg yolk lipid nanovector (EYLNs) was developed for effective loading and delivery of therapeutic agents. Lipids were extracted from egg yolks and reassembled into nano-sized particles. EYLNs stability, cellular uptake, toxicity, and delivery capacity for therapeutic agents were evaluated in vitro. Systemic toxicity and biodistribution of EYLNs were analyzed in normal mice, and the therapeutic effects of doxorubicin (Dox)-loaded EYLNs were evaluated in mouse breast cancer and hepatoma models. EYLNs had a particle size of ~40 nm and a surface zeta potential of −45 mV and were effectively internalized by tumor cells, without showing toxicity and side effects in vitro and in vivo. Importantly, their excellent permeability and retention effect significantly enhanced the distribution of EYLNs to tumor sites, and EYLN-Dox effectively inhibited the tumor growth in both mouse models. Targeted modification with folic acid further promoted vector-mediated drug distribution in tumors. This study demonstrates that lipids with specific proportions in the egg yolk can be used to construct natural drug vectors, providing a new strategy for nano-oncology research.
Worldwide the infectious diseases are a major threat to the human population. However, the most important parameter is the prompt and sensitive diagnosis of pathogens. Therefore, tremendous efforts have been made for the development of rapid and portable detection techniques. The identification and treatment of infectious diseases at the nano and molecular levels is a hard task to achieve because of the scarcity of effective probes for characterization and recognition of biomarkers of these pathogens. Nonetheless, if it made possible simultaneous diagnosis and treatment at the specific spot, i.e., theranostics can be beneficial for treating the disease at a cellular level and can be helpful to understand the disease system. However, for theranostics a sensing system should be able to detect and measure biomarkers quickly. In this regard, aptamers (oligonucleotide polymers consist of single-stranded DNA (ssDNA) or RNA) have displayed the ability to be used as probes for the recognition of various targets at molecular level. DNA and RNA have the capacity of doing much lot than just keeping genetic information and therefore are also known as functional nucleic acids. Aptamer-based biosensors would be an attractive format because they can be developed for various molecules using the same sensing format. Therefore, the aptasensors utilizing aptamers for various bacterial infections have stimulating theranostic potential as well. In this chapter, the potential of aptamers as theranostic agent for bacterial infections has been discussed. The advantages and limitations of aptamer-based theranostics for the development of personalized medicine are also discussed.
As the first line defensive mediators against Mycobacterium tuberculosis (M.tb) infection, macrophages can be modulated by M.tb to influence innate and adaptive immunity. Recently, we have identified several potential immunodominant T-cell antigens from the region of deletion (RD) of M.tb H37Rv, including Rv1768 from RD14. In this study, we further determined that Rv1768 was highly conserved among virulent M.tb strains and mainly distributed as a secreted protein. Exposure to recombinant purified Rv1768 (rRv1768) induced apoptosis of bone marrow derived macrophages (BMDMs) but showed no dose-dependent manner. Regarding macrophage activation, significant higher levels of iNOS and pro-inflammatory cytokines (like IL-6 and TNF-α) were detected in rRv1768-challenged BMDMs, whereas arginase 1 (Arg1) expression was markedly decreased. Meanwhile, MHC-II expression and antigen presentation activity of BMDMs were also enhanced by rRv1768 stimulation, leading to significantly increased IFN-γ expression of CD4⁺ T cells isolated from H37Rv-infected mice. It is worthy to note that Rv1768-induced IFN-γ production of peripheral blood mononuclear cells (PBMCs) and Rv1768-specific immunoglobulins was specifically observed in H37Rv-infected mice, but not BCG-infected or normal mice. Analysis of clinical blood samples further revealed that Rv1768 had a higher sensitivity and specificity (91.38 and 96.83%) for tuberculosis diagnosis than the results obtained from clinical CFP10 and ESAT6 peptides (CE)-based enzyme-linked immunospot (ELISPOT) assay. The area under ROC curve of Rv1768 was 0.9618 (95% CI: 0.919–1.000) when cutoff value set as 7 spots. In addition, Rv1768-specific IgG and IgM also exhibited moderate diagnostic performance for tuberculosis compared with CE specific antibodies. Our data suggest that Rv1768 is an antigen that strongly activates macrophages and has potential to serve as a novel ELISPOT-based TB diagnostic agent.
Aptamers are in vitro selected oligonucleotides (DNA, RNA, oligos with modified nucleotides) that can have high affinity and specificity for a broad range of potential targets with high affinity and specificity. Here we focus on their applications as biosensors in the diagnostic field, although they can also be used as therapeutic agents. A small number of peptide aptamers have also been identified. In analytical settings, aptamers have the potential to extend the limit of current techniques as they offer many advantages over antibodies and can be used for real-time biomarker detection, cancer clinical testing, and detection of infectious microorganisms and viruses. Once optimized and validated, aptasensor technologies are expected to be highly beneficial to clinicians by providing a larger range and more rapid output of diagnostic readings than current technologies and support personalized medicine and faster implementation of optimal treatments.
Mycobacterium tuberculosis (Mtb) has plagued humanity for tens of thousands of years, yet still remains a threat to human health. Its pathology is largely associated with pulmonary tuberculosis with symptoms including fever, hemoptysis, and chest pain. Mtb, however, also manifests in other extrapulmonary organs, such as the pleura, bones, gastrointestinal tract, central nervous system, and lymph nodes. Compared to the knowledge of pulmonary tuberculosis, extrapulmonary pathologies of Mtb are quite understudied. Lymph node tuberculosis is one of the most common extrapulmonary manifestations of tuberculosis, and presents significant challenges in its diagnosis, management, and treatment due to its elusive etiologies and pathologies. The objective of this review is to overview the current understanding of the tropism and pathogenesis of Mtb in endothelial cells of the extrapulmonary tissues, particularly, in lymph nodes. Lymphatic endothelial cells (LECs) are derived from blood vascular endothelial cells (BECs) during development, and these two types of endothelial cells demonstrate substantial molecular, cellular and genetic similarities. Therefore, systemic comparison of the differential and common responses of BECs vs. LECs to Mtb invasion could provide new insights into its pathogenesis, and may promote new investigations into this deadly disease.
There is a need to develop protective vaccines against tuberculosis (TB). Recently, we identified an immunodominant T-cell antigen, Rv2645, from the region of deletion 13 (RD13) of M. tuberculosis (M. tb) H37Rv, which is absent in Bacille Calmette-Guérin (BCG). Here, a recombinant BCG expressing Rv2645, namely, BCG::Rv2645, was constructed. Compared to BCG, we found that BCG::Rv2645 improved the antigen presentation capacity of dendritic cells (DCs) and elicited much stronger Th1 and Th17 responses, higher CD44highCD62low effector memory CD4+ T cells (TEM), and fewer T regulated cells (Treg) and regulatory B10 in mice. Importantly, BCG::Rv2645 exhibited enhanced protective efficacy against virulent M. tb H37Rv challenge in both mice and rhesus monkeys, showing less severe pathology and reduced pathogens. Further, transcriptomic analysis and reverse transcription-quantitative real time PCR revealed that the mRNA levels of ISGylation (Isg)-related genes such as interferon-stimulated gene 15 (Isg15), and Th1- and Th17-related genes such as interferon-γ (IFN-γ) and interleukin-17A (IL-17A) were significantly up-regulated in splenocytes and macrophages after stimulation with Rv2645. This study shows that BCG::Rv2645 is a promising TB vaccine candidate with enhanced protective immunity. The enhanced Th1/Th17 immune responses and up-regulation of ISGylation-related genes induced by Rv2645 may be major factors contributing to the protective immunity of BCG::Rv2645.
Polypeptides play a vital role in physiological processes of life. The pharmacological and medical value of polypeptides has attracted the attention of researchers in recent years. Aptamers are short, single stranded DNA or RNA developed by an in vitro process called systematic evolution of ligands by exponential enrichment (SELEX). Owing to the high affinity and specificity to the target, aptamer is also called “chemical antibody” or “chemist's antibody”. To date, there are two main application aspects for polypeptides-targeted aptamers. First, aptamer can be used as specific binding elements based on their ability of recognition, which would be applied to polypeptides sensing or imaging. The other one is that aptamer can also be used as antagonists based on their ability of inhibiting, which can restrict the activity of polypeptides and block the downstream signaling pathways in vivo, thus can be used to treat the disease associated with polypeptides. In this review, we summarize a number of polypeptides-targeted aptamers and the related applications in vitro and in vivo. Current issues and development trends throughout the screening, characterizing and applying of polypeptides-targeted aptamers are also discussed. © 2017 Changchun Institute of Applied Chemistry, Chinese Academy of Sciences
It is well established for a broad range of disease states, including cancer and Mycobacterium tuberculosis infection, that pathogenesis is bolstered by polarisation of macrophages towards an anti-inflammatory phenotype, known as M2. As these innate immune cells are relatively long-lived, their re-polarisation to pro-inflammatory, phagocytic and bactericidal "classically activated" M1 macrophages is an attractive therapeutic approach. On the other hand, there are scenarios where the resolving inflammation, wound healing and tissue remodelling properties of M2 macrophages are beneficial - for example the successful introduction of biomedical implants. Although there are numerous endogenous and exogenous factors that have an impact on the macrophage polarisation spectrum, this review will focus specifically on prominent macrophage-modulating carbohydrate motifs with a view towards highlighting structure-function relationships and therapeutic potential.
Tuberculosis is the leading infectious cause of mortality worldwide. The global epidemic, caused by Mycobacterium tuberculosis, has prompted renewed interest in the development of novel vaccines for disease prevention and control. The cell envelope of M. tuberculosis is decorated with an assortment of glycan structures, including glycolipids, that are involved in disease pathogenesis. Phenolic glycolipids and the structurally related para-hydroxybenzoic acid derivatives, display potent immunomodulatory activities and have particular relevance for both understanding the interaction of the bacterium with the host immune system and also in the design of new vaccine and therapeutic candidates. Interest in glycobiology has grown exponentially over the last decade, with advancements paving the way for effective carbohydrate based vaccines. This review highlights recent advances in our understanding of phenolic glycans, including their biosynthesis and role as virulence factors in M. Tuberculosis. Recent chemical synthesis approaches and biochemical analysis of synthetic glycans and their conjugates have led to fundamental insights into their roles in host-pathogen interactions. The applications of these synthetic glycans as potential vaccine candidates are discussed.
Macrophages are the primary host target cells of Mycobacterium tuberculosis (M.tb). However, little is known about the changes of membrane glycopatterns of macrophages in response to M. tb infection. Using lectin microarrays we compared the differential expression of glycopatterns of macrophages upon stimulation with the heat-inactivated virulent M.tb H37Rv or attenuate M.tb H37Ra. We found that widespread alteration of macrophage membrane glycopatterns were induced by the heat-inactivated virulent M. tb H37Rv, as shown by the significantly changed binding abilities of 11 lectins (sugar binding proteins) among 40 lectins tested. The binding ability of the lectin ABA to macrophages showed the greatest increase after virulent M. tb H37Rv treatment, which suggests that the expression of N-acetyl-d-lactosamine (ABA binding ligand Galβ1-3GalNAc, O-link glycan) is mainly increased on macrophages during virulent M.tb infection. Addition of ABA blocked the attachment/engulfment of M. tb H37Rv, but not H37Ra, to macrophages. Further, increased glycosylated CD44, one of ABA-binding glycoproteins on macrophages, was identified by pull-down assays with ABA-agarose, followed by mass spectrometry and western blotting. ABA directly binds with Galβ1-3GalNAc-glycosylated CD44 on macrophage, and inhibits M. tb mannose-capped lipoarabinomannan (ManLAM) binding to glycosylated CD44. Moreover, ABA increases IL-6, but reduces IL-10 production of ManLAM-treated macrophages and inhibits M. tb H37Rv-induced necrosis in macrophages. Our study will help to reveal the mechanism of pathogenicity and virulence of M. tb from a new perspective and provide a potential new diagnostic and therapeutic strategy for tuberculosis based on glycopatterns, ABA and its ligand Galβ1-3GalNAc-glycosylated CD44 target molecule on macrophage.
Human ficolin-2 (FCN-2) and mouse ficolin-A (FCN-A, a ficolin-2-like molecule in mouse) are activators of the lectin complement pathway, present in normal plasma and usually associated with infectious diseases, but little is known about the role of FCN-A/2 in inflammatory bowel disease (IBD). In our present study, we found that IBD patients exhibited much higher serum FCN-2 levels than healthy controls. In the dextran sulfate sodium (DSS)-induced acute colitis mouse model, FCN-A knockout mice showed much milder disease symptoms with less histological damage, lower expression levels of pro-inflammatory cytokines (interleukin (IL)-6, IL-1β and tumor necrosis factor alpha (TNF-α)), chemokines (CXCL1/2/10 and CCL4) and higher levels of anti-inflammatory cytokine IL-10 compared to WT mice. We demonstrated that FCN-A/2 exacerbated the inflammatory pathogenesis of IBD by stimulating M1 polarization through the TLR4/MyD88/MAPK/NF-κB signaling pathway in macrophages. Thus, our data suggest FCN-A/2 may be used as a novel therapeutic target for IBD. This article is protected by copyright. All rights reserved.
Regardless of the fact that potent drug-regimen is currently available, tuberculosis continues to kill 1.5 million people annually. Tuberculosis patients are not only inflicted by the trauma of disease but they also suffer from the harmful side-effects, immune suppression and drug resistance instigated by prolonged therapy. It is an exigency to introduce radical changes in the existing drug-regime and discover safer and better therapeutic measures. Hence, we designed a novel therapeutic strategy by reinforcing the efficacy of drugs to kill Mtb by concurrently boosting host immunity by L91. L91 is chimera of promiscuous epitope of Acr1 antigen of Mtb and TLR-2 agonist Pam2Cys. The adjunct therapy using drugs and L91 (D-L91) significantly declined the bacterial load in Mtb infected animals. The mechanism involved was through enhancement of IFN-γ+TNF-α+ polyfunctional Th1 cells and IL-17A+IFN-γ+ Th17 cells, enduring memory CD4 T cells and downregulation of PD-1. The down-regulation of PD-1 prevents CD4 T cells from undergoing exhaustion and improves their function against Mtb. Importantly, the immune response observed in animals could be replicated using T cells of tuberculosis patients on drug therapy. In future, D-L91 therapy can invigorate drugs potency to treat tuberculosis patients and reduce the dose and duration of drug-regime.
TB is an underappreciated public health threat in developed nations. In 2014, an estimated 9.6 million TB cases and 1.5 million
deaths occurred worldwide; 3.3% of these cases resulted from multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant
tuberculosis (XDR-TB) strains. These figures underestimate the economic burden associated with MDR-TB and XDR-TB, as the cost
of treating disease caused by these strains can be 9–25 times higher than treating drug-susceptible TB. Developing new drugs,
improved diagnostics and new TB vaccines are critical components of a strategy to combat TB in general, and drug-resistant
TB in particular. Because Mycobacterium tuberculosis (MTB) has demonstrated a capacity to develop resistance to drugs developed to combat it, it is unlikely that drug-resistant
MTB would be ‘resistant’ to vaccines capable of preventing disease or established infection with drug-sensitive MTB strains.
Accordingly, the development of TB vaccines represents an important long-term investment in preventing the spread of drug-resistant
TB and achieving WHO's goal of ending the global TB epidemic by 2035. Our current understanding of the epidemiology of drug-resistant
TB and the interventions needed to limit its spread, reviewed in this article, illustrates the need for increased financial
support for developing new TB drugs, diagnostics and vaccines to meet the WHO goal of TB elimination by 2035.
Lipoarabinomannan is a major immunomodulatory lipoglycan found in the cell envelope of Mycobacterium tuberculosis and related human pathogens. It reproduces several salient properties of M. tuberculosis in phagocytic cells, including inhibition of pro-inflammatory cytokine production, inhibition of phagolysosome biogenesis, and inhibition of apoptosis as well as autophagy. In this review, we present our current knowledge on lipoarabinomannan structure and ability to manipulate the endocytic pathway as well as phagocyte functions. A special focus is put on the molecular mechanisms employed and the signaling pathways hijacked. Available information is discussed in the context of M. tuberculosis pathogenesis.
Complement is a critical system of enzymes, regulatory proteins, and receptors that regulates both innate and adaptive immune responses. Natural mutations in complement molecules highlight their requirement in regulation of a variety of human conditions including infectious disease and autoimmunity. As sentinels of the immune system, macrophages are specialized to respond to infectious microbes, as well as normal and altered self, and dictate appropriate immune responses. Complement components such as anaphylatoxins (C3a and C5a) and opsonins [C3b, C1q, mannan binding lectin (MBL)] influence macrophage responses. While anaphylatoxins C3a and C5a trigger inflammasome activation, opsonins such as C1q and related molecules (MBL and adiponectin) downregulate inflammasome activation and inflammation, and upregulate engulfment of apoptotic cells consistent with a pro-resolving or M2 macrophage phenotype. This review summarizes our current understanding of the influence of the complement system on macrophage polarization with an emphasis on C1q and related molecules.
Tuberculosis remains second only to HIV/AIDS as the leading cause of mortality worldwide due to a single infectious agent. Despite chemotherapy, the global tuberculosis epidemic has intensified because of HIV co-infection, the lack of an effective vaccine and the emergence of multi-drug-resistant bacteria. Alternative host-directed strategies could be exploited to improve treatment efficacy and outcome, contain drug-resistant strains and reduce disease severity and mortality. The innate inflammatory response elicited by Mycobacterium tuberculosis (Mtb) represents a logical host target. Here we demonstrate that interleukin-1 (IL-1) confers host resistance through the induction of eicosanoids that limit excessive type I interferon (IFN) production and foster bacterial containment. We further show that, in infected mice and patients, reduced IL-1 responses and/or excessive type I IFN induction are linked to an eicosanoid imbalance associated with disease exacerbation. Host-directed immunotherapy with clinically approved drugs that augment prostaglandin E2 levels in these settings prevented acute mortality of Mtb-infected mice. Thus, IL-1 and type I IFNs represent two major counter-regulatory classes of inflammatory cytokines that control the outcome of Mtb infection and are functionally linked via eicosanoids. Our findings establish proof of concept for host-directed treatment strategies that manipulate the host eicosanoid network and represent feasible alternatives to conventional chemotherapy.
The major surface lipoglycan of Mycobacterium tuberculosis (M. tb), mannose-capped lipoarabinomannan (ManLAM) is an immunosuppressive epitope of M. tb. We used Systematic Evolution of Ligands by EXponential enrichment (SELEX) to generate an aptamer (ZXL1) that specifically bound to ManLAM from the virulent M. tb strain H37Rv. Aptamer ZXL1 had the highest binding affinity with equilibrium dissociation constant-Kd of 436.3±37.84 nM and competed with the mannose receptor for binding to ManLAM and M. tb H37Rv. ZXL1 significantly inhibited the ManLAM-induced immunosuppression of CD11c(+) dendritic cells (DCs) and enhanced the M. tb-antigen-presenting activity of DCs for naïve CD4(+) Th1 cell activation. More importantly, we demonstrated that injection of aptamer ZXL1 significantly reduced the progression of M. tb H37Rv infections and bacterial loads in lungs in mice and rhesus monkeys. These results suggest that aptamer ZXL1 is a new potential antimycobacterial agent and TB vaccine immune-adjuvant.Molecular Therapy (2014); doi:10.1038/mt.2014.31.
Macrophages in granulomas are both antimycobacterial effector and host cell for Mycobacterium tuberculosis, yet basic aspects of macrophage diversity and function within the complex structures of granulomas remain poorly understood. To address this, we examined myeloid cell phenotypes and expression of enzymes correlated with host defense in macaque and human granulomas. Macaque granulomas had upregulated inducible and endothelial NO synthase (iNOS and eNOS) and arginase (Arg1 and Arg2) expression and enzyme activity compared with nongranulomatous tissue. Immunohistochemical analysis indicated macrophages adjacent to uninvolved normal tissue were more likely to express CD163, whereas epithelioid macrophages in regions where bacteria reside strongly expressed CD11c, CD68, and HAM56. Calprotectin-positive neutrophils were abundant in regions adjacent to caseum. iNOS, eNOS, Arg1, and Arg2 proteins were identified in macrophages and localized similarly in granulomas across species, with greater eNOS expression and ratio of iNOS/Arg1 expression in epithelioid macrophages as compared with cells in the lymphocyte cuff. iNOS, Arg1, and Arg2 expression in neutrophils was also identified. The combination of phenotypic and functional markers support that macrophages with anti-inflammatory phenotypes localized to outer regions of granulomas, whereas the inner regions were more likely to contain macrophages with proinflammatory, presumably bactericidal, phenotypes. Together, these data support the concept that granulomas have organized microenvironments that balance antimicrobial anti-inflammatory responses to limit pathology in the lungs.
Objective.
—To quantify the efficacy of BCG vaccine against tuberculosis (TB).
Chemokine receptor cross-desensitization provides an important mechanism to regulate immune cell recruitment at sites of inflammation. We previously reported that the mycobacterial cell wall glycophospholipid mannose-capped lipoarabinomannan (ManLAM) could induce human peripheral blood T cell chemotaxis. Therefore, we examined the ability of ManLAM to desensitize T cells to other chemoattractants as a potential mechanism for impaired T cell homing and delayed lung recruitment during mycobacterial infection. We found that ManLAM pretreatment inhibited in vitro migration of naive human or mouse T cells to the lymph node egress signal sphingosine-1-phosphate (S1P). Intratracheal administration of ManLAM in mice resulted in significant increases in T cells, primarily CCR5(+) (Th1) cells, in lung-draining lymph nodes. To investigate the selective CCR5 effect, mouse T cells were differentiated into Th1 or Th2 populations in vitro, and their ability to migrate to S1P with or without ManLAM pretreatment was analyzed. ManLAM pretreatment of Th1 populations inhibited S1P-induced migration but had no effect on Th2 cell S1P-directed migration, suggesting a differential effect by S1P on the two subsets. The PI3K/AKT inhibitor Ly294002 inhibited S1P-directed migration by Th1 cells, whereas the ERK inhibitor U0126 inhibited Th2 cell S1P-directed migration. These observations demonstrate that S1P-induced migratory responses in Th1 and Th2 lymphocytes occurs via different signaling pathways and suggests further that the production of ManLAM during Mycobacterium tuberculosis infection may function to sequester Th1 cells in lung-draining lymph nodes, thereby delaying their recruitment to the lung.
Mycobacterium tuberculosis (Mtb) is able to evade the immune defenses and may persist for years, decades and even lifelong in the infected host. Mtb cell wall components may contribute to such persistence by modulating several pivotal types of immune cells. Dendritic cells (DCs) are the most potent antigen-presenting cells and hence play a crucial role in the initial immune response to infections by connecting the innate with the adaptive immune system.
We investigated the effects of two of the major mycobacterial cell wall-associated types of glycolipids, mannose-capped lipoarabinomannan (ManLAM) and phosphatidylinositol mannosides (PIMs) purified from the Mtb strains H37Rv and Mycobacterium bovis, on the maturation and cytokine profiles of immature human monocyte-derived DCs. ManLAM from Mtb H37Rv stimulated the release of pro-inflammatory cytokines TNF, IL-12, and IL-6 and expression of co-stimulatory (CD80, CD86) and antigen-presenting molecules (MHC class II). ManLAM from M. bovis also induced TNF, IL-12 and IL-6 but at significantly lower levels. Importantly, while ManLAM was found to augment LPS-induced DC maturation and pro-inflammatory cytokine production, addition of PIMs from both Mtb H37Rv and M. bovis strongly reduced this stimulatory effect.
These results indicate that the mycobacterial cell wall contains macromolecules of glycolipid nature which are able to induce strong and divergent effects on human DCs; i.e while ManLAM is immune-stimulatory, PIMs act as powerful inhibitors of DC cytokine responses. Thus PIMs may be important Mtb-associated virulence factors contributing to the pathogenesis of tuberculosis disease. These findings may also aid in the understanding of some earlier conflicting reports on the immunomodulatory effects exerted by different ManLAM preparations.
Brucella abortus is a Gram-negative intracellular bacterium that induces MyD88-dependent IL-12 production in dentritic cells (DCs) and a subsequent protective Th1 immune response. Previous studies have shown that the Toll-like receptor 2 (TLR2) is required for tumor-necrosis factor (TNF) production, whereas TLR9 is responsible for IL-12 induction in DCs after exposure to heat-killed Brucella abortus (HKBA). TLR2 is located on the cell surface and is required for optimal microorganism-induced phagocytosis by innate immune cells; thus, phagocytosis is an indispensable preliminary step for bacterial genomic DNA recognition by TLR9 in late-endosomal compartments. Here, we hypothesized that TLR2-triggered signals after HKBA stimulation might cross-regulate TLR9 signaling through the indirect modulation of the phagocytic function of DCs or the direct modulation of cytokine gene expression. Our results indicate that HKBA phagocytosis was TLR2-dependent and an essential step for IL-12p40 induction. In addition, HKBA exposure triggered the TLR2-mediated activation of both p38 and extracellular signal-regulated kinase 1/2 (ERK1/2). Interestingly, although p38 was required for HKBA phagocytosis and phagosome maturation, ERK1/2 did not affect these processes but negatively regulated IL-12 production. Although p38 inhibitors tempered both TNF and IL-12 responses to HKBA, pre-treatment with an ERK1/2 inhibitor significantly increased IL-12p40 and abrogated TNF production in HKBA-stimulated DCs. Further experiments showed that the signaling events that mediated ERK1/2 activation after TLR2 triggering also required HKBA-induced Ras activation. Furthermore, Ras-guanine nucleotide-releasing protein 1 (RasGRP1) mediated the TLR2-induced ERK1/2 activation and inhibition of IL-12p40 production. Taken together, our results demonstrated that HKBA-mediated TLR2-triggering activates both the p38 and ERK1/2 signaling subpathways, which divergently regulate TLR9 activation at several levels to induce an appropriate protective IL-12 response.
Immune evasion is required for Mycobacterium tuberculosis to survive in the face of robust CD4(+) T cell responses. We have shown previously that M. tuberculosis cell wall glycolipids, including mannose capped lipoarabinomannan (ManLAM), directly inhibit polyclonal murine CD4(+) T cell activation by blocking ZAP-70 phosphorylation. We extended these studies to antigen-specific murine CD4(+) T cells and primary human T cells and found that ManLAM inhibited them as well. Lck and LAT phosphorylation also were inhibited by ManLAM without affecting their localization to lipid rafts. Inhibition of proximal TCR signaling was temperature sensitive, suggesting that ManLAM insertion into T cell membranes was required. Thus, M. tuberculosis ManLAM inhibits antigen-specific CD4(+) T cell activation by interfering with very early events in TCR signaling through ManLAM's insertion in T cell membranes.
Pathogen glycolipids, including Leishmania spp. lipophosphoglycan (LPG) and Mycobacterium tuberculosis mannosylated lipoarabinomannan (ManLAM), modulate essential interactions with host phagocytic cells. Polysaccharide and lipid components promote immunomodulation. Owing to the stereochemistry required to synthesize oligosaccharides, the roles for oligosaccharides in the pathogenesis of infectious diseases have remained largely unknown. Recent advances in carbohydrate chemistry allowed us to synthesize pathogen surface oligosaccharides to discern their immune response-altering activities. Trimannose cap carbohydrates from ManLAM and LPG altered the production of proinflammatory cytokines via a toll-like receptor (TLR2)-mediated mechanism in vitro and in vivo. In vivo treatment with trimannose led to increased Th1-polarizing, IL-12p40-producing cells from the draining lymph nodes of treated Leishmania major-infected mice compared with cells from untreated infected mice. Trimannose treatment increased the production of other Th1 proinflammatory cytokines (ie, interferon-γ, IL-6, and tumor necrosis factor-α) critical for a productive immune response to either pathogen. This significant difference in cytokine production between trimannose cap sugar-treated and control groups was not observed in draining lymph node cells from TLR2(-/-) mice. Type of inflammation and rate of bead entry into macrophages and dendritic cells were different for trimannose-coated beads compared with control oligosaccharide-coated beads, indicating selective lectin receptor/oligosaccharide interactions mediating cell entry and cytokine production. These novel findings may prompt the development of targeted oligosaccharide adjuvants against chronic infections.
The continued spread of the HIV epidemic underscores the need to interrupt transmission. One attractive strategy is a topical vaginal microbicide. Sexual transmission of herpes simplex virus type 2 (HSV-2) in mice can be inhibited by intravaginal siRNA application. To overcome the challenges of knocking down gene expression in immune cells susceptible to HIV infection, we used chimeric RNAs composed of an aptamer fused to an siRNA for targeted gene knockdown in cells bearing an aptamer-binding receptor. Here, we showed that CD4 aptamer-siRNA chimeras (CD4-AsiCs) specifically suppress gene expression in CD4⁺ T cells and macrophages in vitro, in polarized cervicovaginal tissue explants, and in the female genital tract of humanized mice. CD4-AsiCs do not activate lymphocytes or stimulate innate immunity. CD4-AsiCs that knock down HIV genes and/or CCR5 inhibited HIV infection in vitro and in tissue explants. When applied intravaginally to humanized mice, CD4-AsiCs protected against HIV vaginal transmission. Thus, CD4-AsiCs could be used as the active ingredient of a microbicide to prevent HIV sexual transmission.
Mycobacterium bovis bacille Calmette-Guerin (BCG) provides only limited protection against pulmonary tuberculosis. We tested the hypothesis that BCG might have retained immunomodulatory properties from its pathogenic parent that limit its protective immunogenicity. Mutation of the molecules involved in immunomodulation might then improve its vaccine potential. We studied the vaccine potential of BCG mutants deficient in the secreted acid phosphatase, SapM, or in the capping of the immunomodulatory ManLAM cell wall component with α-1,2-oligomannoside. Both systemic and intratracheal challenge of mice with Mycobacterium tuberculosis following vaccination showed that the SapM mutant, compared to the parental BCG vaccine, provided better protection: it led to longer-term survival. Persistence of the SapM-mutated BCG in vivo resembled that of the parental BCG indicating that this mutation will likely not compromise the safety of the BCG vaccine. The SapM mutant BCG vaccine was more effective than the parental vaccine in inducing recruitment and activation of CD11c(+) MHC-II(int) CD40(int) dendritic cells (DCs) to the draining lymph nodes. Thus, SapM acts by inhibiting recruitment of DCs and their activation at the site of vaccination.
Mycobacterium tuberculosis enhances its survival in macrophages by suppressing immune responses in part through its complex cell wall structures. Peroxisome proliferator-activated receptor gamma (PPARgamma), a nuclear receptor superfamily member, is a transcriptional factor that regulates inflammation and has high expression in alternatively activated alveolar macrophages and macrophage-derived foam cells, both cell types relevant to tuberculosis pathogenesis. In this study, we show that virulent M. tuberculosis and its cell wall mannose-capped lipoarabinomannan induce PPARgamma expression through a macrophage mannose receptor-dependent pathway. When activated, PPARgamma promotes IL-8 and cyclooxygenase 2 expression, a process modulated by a PPARgamma agonist or antagonist. Upstream, MAPK-p38 mediates cytosolic phospholipase A(2) activation, which is required for PPARgamma ligand production. The induced IL-8 response mediated by mannose-capped lipoarabinomannan and the mannose receptor is independent of TLR2 and NF-kappaB activation. In contrast, the attenuated Mycobacterium bovis bacillus Calmette-Guérin induces less PPARgamma and preferentially uses the NF-kappaB-mediated pathway to induce IL-8 production. Finally, PPARgamma knockdown in human macrophages enhances TNF production and controls the intracellular growth of M. tuberculosis. These data identify a new molecular pathway that links engagement of the mannose receptor, an important pattern recognition receptor for M. tuberculosis, with PPARgamma activation, which regulates the macrophage inflammatory response, thereby playing a role in tuberculosis pathogenesis.
According to WHO, about one third of the world's population is infected with bacteria of the Mycobacterium tuberculosis complex. Currently there is globally 9.15 million recorded cases of overt tuberculosis (TB) annually and due to lack of adequate diagnostics presumably a large but unknown number of non-recorded cases. TB is estimated to cause 1.65 million deaths per annum which accounts for one-fifth of all deaths by infectious diseases of adults in low-income countries. During recent years a rapid spread of multi-drug resistant bacteria causing about 0.5 million TB cases per year has worsened the problem. The live attenuated Bacillus Calmette-Guérin (BCG) vaccine which is the only currently available TB vaccine does not confer any significant protection against the most common and contagious form of TB-adult pulmonary TB.
Mycobacterium tuberculosis modulates host immune responses through proteins and complex glycolipids. Here, we report that the glycosylphosphatidylinositol
anchor phosphatidyl-myo-inositol hexamannosides PIM6 or PIM2 exert potent anti-inflammatory activities. PIM strongly inhibited the Toll-like receptor (TLR4) and myeloid differentiation
protein 88 (MyD88)-mediated release of NO, cytokines, and chemokines, including tumor necrosis factor (TNF), interleukin 12
(IL-12) p40, IL-6, keratinocyte-derived chemokine, and also IL-10 by lipopolysaccharide (LPS)-activated macrophages. This
effect was independent of the presence of TLR2. PIM also reduced the LPS-induced MyD88-independent, TIR domain-containing
adaptor protein inducing interferon β (TRIF)-mediated expression of co-stimulatory receptors. PIM inhibited LPS/TLR4-induced
NFκB translocation. Synthetic PIM1 and a PIM2 mimetic recapitulated these in vitro activities and inhibited endotoxin-induced airway inflammation, TNF and keratinocyte-derived chemokine secretion, and neutrophil
recruitment in vivo. Mannosyl, two acyl chains, and phosphatidyl residues are essential for PIM anti-inflammatory activity, whereas the inosityl
moiety is dispensable. Therefore, PIM exert potent antiinflammatory effects both in vitro and in vivo that may contribute to the strategy developed by mycobacteria for repressing the host innate immunity, and synthetic PIM
analogs represent powerful anti-inflammatory leads.
Converging studies have shown that M1 and M2 macrophages are functionally polarized in response to microorganisms and host mediators. Gene expression profiling of macrophages reveals that various Gram-negative and Gram-positive bacteria induce the transcriptional activity of a "common host response," which includes genes belonging to the M1 program. However, excessive or prolonged M1 polarization can lead to tissue injury and contribute to pathogenesis. The so-called M2 macrophages play a critical role in the resolution of inflammation by producing anti-inflammatory mediators. These M2 cells cover a continuum of cells with different phenotypic and functional properties. In addition, some bacterial pathogens induce specific M2 programs in macrophages. In this review, we discuss the relevance of macrophage polarization in three domains of infectious diseases: resistance to infection, infectious pathogenesis, and chronic evolution of infectious diseases.
Subpopulations of RNA molecules that bind specifically to a variety of organic dyes have been isolated from a population of random sequence RNA molecules. Roughly one in 10(10) random sequence RNA molecules folds in such a way as to create a specific binding site for small ligands.
Mutagenesis of the host immune system has helped identify response pathways necessary to combat tuberculosis. Several such pathways may function as activators of a common protective gene: inducible nitric oxide synthase (NOS2). Here we provide direct evidence for this gene controlling primary Mycobacterium tuberculosis infection using mice homozygous for a disrupted NOS2 allele. NOS2(-/-) mice proved highly susceptible, resembling wild-type littermates immunosuppressed by high-dose glucocorticoids, and allowed Mycobacterium tuberculosis to replicate faster in the lungs than reported for other gene-deficient hosts. Susceptibility appeared to be independent of the only known naturally inherited antimicrobial locus, NRAMP1. Progression of chronic tuberculosis in wild-type mice was accelerated by specifically inhibiting NOS2 via administration of N6-(1-iminoethyl)-L-lysine. Together these findings identify NOS2 as a critical host gene for tuberculostasis.
Mycobacterium bovis bacillus Calmette-Guérin (BCG) is the only vaccine approved for prevention of tuberculosis. It has been postulated that serial passage of BCG over the years may have resulted in attenuation of its effectiveness. Because interleukin-12 (IL-12) and oligodeoxynucleotides (ODN) containing cytidine phosphate guanosine (CpG) motifs have been shown to enhance Th1 responses in vivo, they were chosen as adjuvants to increase the effectiveness of BCG vaccination. In this report, mice were vaccinated with BCG with or without IL-12 or CpG ODN and then challenged 6 weeks later via the aerosol route with the Erdman strain of M. tuberculosis. Mice vaccinated with BCG alone showed a 1- to 2-log reduction in bacterial load compared with control mice that did not receive any vaccination prior to M. tuberculosis challenge. Moreover, the bacterial loads of mice vaccinated with BCG plus IL-12 or CpG ODN were a further two- to fivefold lower than those of mice vaccinated with BCG alone. As an immune correlate, the antigen-specific production IFN-gamma and mRNA expression in spleen cells prior to challenge were evaluated. Mice vaccinated with BCG plus IL-12 or CpG ODN showed enhanced production of IFN-gamma compared with mice vaccinated with BCG alone. Finally, granulomas in BCG-vaccinated mice were smaller and more lymphocyte rich than those in unvaccinated mice; however, the addition of IL-12 or CpG ODN to BCG vaccination did not alter granuloma formation or result in added pulmonary damage. These observations support a role for immune adjuvants given with BCG vaccination to enhance its biologic efficacy.
IL-12 is a key cytokine in directing the development of type 1 Th cells, which are critical to eradicate intracellular pathogens such as Mycobacterium tuberculosis. Here, we report that mannose-capped lipoarabinomannans (ManLAMs) from Mycobacterium bovis bacillus Calmette-Guérin and Mycobacterium tuberculosis inhibited, in a dose-dependent manner, the LPS-induced IL-12 production by human dendritic cells. The inhibitory activity was abolished by the loss of the mannose caps or the GPI acyl residues. Mannan, which is a ligand for the mannose receptor (MR) as well as an mAb specific for the MR, also inhibited the LPS-induced IL-12 production by dendritic cells. Our results indicate that ManLAMs may act as virulence factors that contribute to the persistence of M. bovis bacillus Calmette-Guérin and M. tuberculosis within phagocytic cells by suppressing IL-12 responses. Our data also suggest that engagement of the MR by ManLAMs delivers a negative signal that interferes with the LPS-induced positive signals delivered by the Toll-like receptors.
Murine macrophages effect potent antimycobacterial function via the production of nitric oxide by the inducible isoform of
the enzyme nitric oxide synthase (NOS2). The protective role of reactive nitrogen intermediates (RNI) against Mycobacterium tuberculosisinfection has been well established in various murine experimental tuberculosis models using laboratory strains of the tubercle
bacillus to establish infection by the intravenous route. However, important questions remain about the in vivo importance
of RNI in host defense against M. tuberculosis. There is some evidence that RNI play a lesser role following aerogenic, rather than intravenous,M. tuberculosis infection of mice. Furthermore, in vitro studies have demonstrated that different strains of M. tuberculosis, including clinical isolates, vary widely in their susceptibility to the antimycobacterial effects of RNI. Thus, we sought
to test rigorously the protective role of RNI against infection with recent clinical isolates of M. tuberculosis following both aerogenic and intravenous challenges. Three recently isolated and unique M. tuberculosis strains were used to infect both wild-type (wt) C57BL/6 and NOS2 gene-disrupted mice. Regardless of the route of infection, NOS2−/− mice were much more susceptible than wt mice to any of the clinical isolates or to either the Erdman or H37Rv laboratory
strain of M. tuberculosis. Mycobacteria replicated to much higher levels in the organs of NOS2−/− mice than in those of wt mice. Although the clinical isolates all exhibited enhanced virulence in NOS2−/− mice, they displayed distinct growth rates in vivo. The present study has provided results indicating that RNI are required
for the control of murine tuberculous infection caused by both laboratory and clinical strains of M. tuberculosis. This protective role of RNI is essential for the control of infection established by either intravenous or aerogenic challenge.
Interactions between dendritic cells (DCs) and Mycobacterium tuberculosis, the etiological agent of tuberculosis, most likely play a key role in anti-mycobacterial immunity. We have recently shown that M. tuberculosis binds to and infects DCs through ligation of the DC-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) and that M. tuberculosis mannose-capped lipoarabinomannan (ManLAM) inhibits binding of the bacilli to the lectin, suggesting that ManLAM might be a key DC-SIGN ligand. In the present study, we investigated the molecular basis of DC-SIGN ligation by LAM. Contrary to what was found for slow growing mycobacteria, such as M. tuberculosis and the vaccine strain Mycobacterium bovis bacillus Calmette-Guérin, our data demonstrate that the fast growing saprophytic species Mycobacterium smegmatis hardly binds to DC-SIGN. Consistent with the former finding, we show that M. smegmatis-derived lipoarabinomannan, which is capped by phosphoinositide residues (PILAM), exhibits a limited ability to inhibit M. tuberculosis binding to DC-SIGN. Moreover, using enzymatically demannosylated and chemically deacylated ManLAM molecules, we demonstrate that both the acyl chains on the ManLAM mannosylphosphatidylinositol anchor and the mannooligosaccharide caps play a critical role in DC-SIGN-ManLAM interaction. Finally, we report that DC-SIGN binds poorly to the PILAM and uncapped AraLAM-containing species Mycobacterium fortuitum and Mycobacterium chelonae, respectively. Interestingly, smooth colony-forming Mycobacterium avium, in which ManLAM is capped with single mannose residues, was also poorly recognized by the lectin. Altogether, our results provide molecular insight into the mechanisms of mycobacteria-DC-SIGN interaction, and suggest that DC-SIGN may act as a pattern recognition receptor and discriminate between Mycobacterium species through selective recognition of the mannose caps on LAM molecules.
Mycobacterium tuberculosis represents a world-wide health risk and immunosuppression is a particular problem in M. tuberculosis infections. Although macrophages are primarily infected, dendritic cells (DCs) are important in inducing cellular immune responses against M. tuberculosis. We hypothesized that DCs represent a target for M. tuberculosis and that the observed immuno-suppression results from modulation of DC functions. We demonstrate that the DC-specific C-type lectin DC-SIGN is an important receptor on DCs that captures and internalizes intact Mycobacterium bovis bacillus Calmette-Guérin (BCG) through the mycobacterial cell wall component ManLAM. Antibodies against DC-SIGN block M. bovis BCG infection of DCs. ManLAM is also secreted by M. tuberculosis-infected macrophages and has been implicated as a virulence factor. Strikingly, ManLAM binding to DC-SIGN prevents mycobacteria- or LPS-induced DC maturation. Both mycobacteria and LPS induce DC maturation through Toll-like receptor (TLR) signaling, suggesting that DC-SIGN, upon binding of ManLAM, interferes with TLR-mediated signals. Blocking antibodies against DC-SIGN reverse the ManLAM-mediated immunosuppressive effects. Our results suggest that M. tuberculosis targets DC-SIGN both to infect DCs and to down-regulate DC-mediated immune responses. Moreover, we demonstrate that DC-SIGN has a broader pathogen recognition profile than previously shown, suggesting that DC-SIGN may represent a molecular target for clinical intervention in infections other than HIV-1.
The tubercle bacillus parasitizes macrophages by inhibiting phagosome maturation into the phagolysosome. This phenomenon underlies the tuberculosis pandemic involving 2 billion people. We report here how Mycobacterium tuberculosis causes phagosome maturation arrest. A glycosylated M. tuberculosis phosphatidylinositol [mannose-capped lipoarabinomannan (ManLAM)] interfered with the phagosomal acquisition of the lysosomal cargo and syntaxin 6 from the trans-Golgi network. ManLAM specifically inhibited the pathway dependent on phosphatidylinositol 3-kinase activity and phosphatidylinositol 3-phosphate-binding effectors. These findings identify ManLAM as the M. tuberculosis product responsible for the inhibition of phagosomal maturation.
The cell wall component lipoarabinomannan (ManLAM) from Mycobacterium tuberculosis is involved in the inhibition of phagosome maturation, apoptosis and interferon (IFN)-gamma signalling in macrophages and interleukin (IL)-12 cytokine secretion of dendritic cells (DC). All these processes are important for the host to mount an efficient immune response. Conversely, LAM isolated from non-pathogenic mycobacteria (PILAM) have the opposite effect, by inducing a potent proinflammatory response in macrophages and DCs. LAMs from diverse mycobacterial species differ in the modification of their terminal arabinose residues. The strong proinflammatory response induced by PILAM correlates with the presence of phospho-myo-inositol on the terminal arabinose. Interestingly, recent work indicates that the biosynthetic precursor of LAM, lipomannan (LM), which is also present in the cell wall, displays strong proinflammatory effects, independently of which mycobacterial species it is isolated from. Results from in vitro assays and knock-out mice suggest that LM, like PILAM, mediates its biological activity via Toll-like receptor 2. We hypothesize that the LAM/LM ratio might be a crucial factor in determining the virulence of a mycobacterial species and the outcome of the infection. Recent progress in the identification of genes involved in the biosynthesis of LAM is discussed, in particular with respect to the fact that enzymes controlling the LAM/LM balance might represent targets for new antitubercular drugs. In addition, inactivation of these genes may lead to attenuated strains of M. tuberculosis for the development of new vaccine candidates.
The Mycobacterium tuberculosis (M.tb) envelope is highly mannosylated with phosphatidyl-myo-inositol mannosides (PIMs), lipomannan, and mannose-capped lipoarabinomannan (ManLAM). Little is known regarding the interaction between specific PIM types and host cell C-type lectin pattern recognition receptors. The macrophage mannose receptor (MR) and dendritic cell-specific ICAM-3-grabbing nonintegrin on dendritic cells engage ManLAM mannose caps and regulate several host responses. In this study, we analyzed the association of purified PIM families (f, separated by carbohydrate number) and individual PIM species (further separated by fatty acid number) from M.tb H(37)R(v) with human monocyte-derived macrophages (MDMs) and lectin-expressing cell lines using an established bead model. Higher-order PIMs preferentially associated with the MR as demonstrated by their reduced association with MDMs upon MR blockade and increased binding to COS-1-MR. In contrast, the lower-order PIM(2)f associated poorly with MDMs and did not bind to COS-1-MR. Triacylated PIM species were recognized by MDM lectins better than tetra-acylated species and the degree of acylation influenced higher-order PIM association with the MR. Moreover, only higher-order PIMs that bind the MR showed a significant increase in phagosome-lysosome fusion upon MR blockade. In contrast with the MR, the PIM(2)f and lipomannan were recognized by DC-SIGN comparable to higher-order PIMs and ManLAM, and the association was independent of their degree of acylation. Thus, recognition of M.tb PIMs by host cell C-type lectins is dependent on both the nature of the terminal carbohydrates and degree of acylation. Subtle structural differences among the PIMs impact host cell recognition and response and are predicted to influence the intracellular fate of M.tb.
Mycobacterial phosphatidylinositol tetramannosides (PIM4) are agonists for a distinct population of invariant human (Valpha24) and mouse (Valpha14) NKT cells, when presented by CD1d. We determined the crystal structure at 2.6-A resolution of mouse CD1d bound to a synthetic dipalmitoyl-PIM2. Natural PIM2, which differs in its fatty acid composition is a biosynthetic precursor of PIM4, PIM6, lipomannan, and lipoarabinomannan. The PIM2 headgroup (inositol-dimannoside) is the most complex to date among all the crystallized CD1d ligands and is remarkably ordered in the CD1d binding groove. A specific hydrogen-bonding network between PIM2 and CD1d orients the headgroup in the center of the binding groove and above the A' pocket. A central cluster of hydrophilic CD1d residues (Asp(153), Thr(156), Ser(76), Arg(79)) interacts with the phosphate, inositol, and alpha1-alpha6-linked mannose of the headgroup, whereas additional specificity for the alpha1- and alpha2-linked mannose is conferred by Thr(159). The additional two mannoses in PIM4, relative to PIM2, are located at the distal 6' carbon of the alpha1-alpha6-linked mannose and would project away from the CD1d binding groove for interaction with the TCR. Compared with other CD1d-sphingolipid structures, PIM2 has an increased number of polar interactions between its headgroup and CD1, but reduced specificity for the diacylglycerol backbone. Thus, novel NKT cell agonists can be designed that focus on substitutions of the headgroup rather than on reducing lipid chain length, as in OCH and PBS-25, two potent variants of the highly stimulatory invariant NKT cell agonist alpha-galactosylceramide.
We evaluated the role of regulatory T cells (CD4(+) CD25(+) Foxp3(+) cells, Tregs) in human Mycobacterium tuberculosis infection. Tregs were expanded in response to M. tuberculosis in healthy tuberculin reactors, but not in tuberculin-negative individuals. The M. tuberculosis mannose-capped lipoarabinomannan (ManLAM) resulted in regulatory T cell expansion, whereas the M. tuberculosis 19-kDa protein and heat shock protein 65 had no effect. Anti-IL-10 and anti-TGF-beta alone or in combination, did not reduce expansion of Tregs. In contrast, the cyclooxygenase enzyme-2 inhibitor NS398 significantly inhibited expansion of Tregs, indicating that prostaglandin E2 (PGE2) contributes to Treg expansion. Monocytes produced PGE2 upon culturing with heat-killed M. tuberculosis or ManLAM, and T cells from healthy tuberculin reactors enhanced PGE2 production by monocytes. Expanded Tregs produced significant amounts of TGF-beta and IL-10 and depletion of Tregs from PBMC of these individuals increased the frequency of M. tuberculosis-responsive CD4(+) IFN-gamma cells. Culturing M. tuberculosis-expanded Tregs with autologous CD8(+) cells decreased the frequency of IFN-gamma(+)cells. Freshly isolated PBMC from tuberculosis patients had increased percentages of Tregs, compared to healthy tuberculin reactors. These findings demonstrate that Tregs expand in response to M. tuberculosis through mechanisms that depend on ManLAM and PGE2.
Lipoarabinomannan (LAM) is one of the key virulence factors for Mycobacterium tuberculosis, the etiological agent of tuberculosis. During uptake of mycobacteria, LAM interacts with the cell membrane of the host macrophage
and can be detected throughout the cell upon infection. LAM can inhibit phagosomal maturation as well as induce a proinflammatory
response in bystander cells. The aim of this study was to investigate how LAM exerts its action on human macrophages. We show
that LAM is incorporated into membrane rafts of the macrophage cell membrane via its glycosylphosphatidylinositol anchor and
that incorporation of mannose-capped LAM from M. tuberculosis results in reduced phagosomal maturation. This is dependent on successful insertion of the glycosylphosphatidylinositol anchor.
LAM does not, however, induce the phagosomal maturation block through activation of p38 mitogen-activated protein kinase,
contradicting some previous suggestions.
Throughout development, hematopoietic stem cells migrate to specific microenvironments, where their fate is in part, extrinsically controlled. CD44 standard as a member of the cell adhesion molecule family, is extensively expressed within adult bone marrow and has been previously reported to play important roles in adult hematopoietic regulation via CD44 standard-ligand interactions. In this manuscript, CD44 expression and function are further assessed and characterized on both fetal and adult hematopoietic stem cells. Using a CD44-/- mouse model, conserved functional roles of CD44 are revealed throughout development: CD44 is critical in the maintenance of hematopoietic stem and progenitor pools, as well as in hematopoietic stem cell migration. CD44 expression on hematopoietic stem cells as well as other hematopoietic cells within the bone marrow microenvironment is important in the homing and lodgment of adult hematopoietic stem cells isolated from the bone/bone marrow interface. CD44 is also involved in fetal hematopoietic stem cell migration out of the liver, via a process involving stromal cell-derived factor-1α. The absence of CD44 in neonatal bone marrow has no impact on the size of the long-term reconstituting hematopoietic stem cell pool, but results in an enhanced long-term engraftment potential of hematopoietic stem cells.
Mycobacteria possess various immunomodulatory molecules on the cell wall. Mannose-capped lipoarabinomannan (Man-LAM), a major lipoglycan of Mycobacterium tuberculosis, has long been known to have both inhibitory and stimulatory effects on host immunity. However, the direct Man-LAM receptor that explains its pleiotropic activities has not been clearly identified. Here, we report that a C-type lectin receptor Dectin-2 (gene symbol Clec4n) is a direct receptor for Man-LAM. Man-LAM activated bone-marrow-derived dendritic cells (BMDCs) to produce pro- and anti-inflammatory cytokines, whereas it was completely abrogated in Clec4n–/– BMDCs. Man-LAM promoted antigen-specific T cell responses through Dectin-2 on DCs. Furthermore, Man-LAM induced experimental autoimmune encephalitis (EAE) as an adjuvant in mice, whereas Clec4n–/– mice were resistant. Upon mycobacterial infection, Clec4n–/– mice showed augmented lung pathology. These results demonstrate that Dectin-2 contributes to host immunity against mycobacterial infection through the recognition of Man-LAM.
Bacillus Calmette-Guérin (BCG) has been used to treat non-muscle-invasive bladder cancer for more than 30 years. It is one of the most successful biotherapies for cancer in use. Despite long clinical experience with BCG, the mechanism of its therapeutic effect is still under investigation. Available evidence suggests that urothelial cells (including bladder cancer cells themselves) and cells of the immune system both have crucial roles in the therapeutic antitumour effect of BCG. The possible involvement of bladder cancer cells includes attachment and internalization of BCG, secretion of cytokines and chemokines, and presentation of BCG and/or cancer cell antigens to cells of the immune system. Immune system cell subsets that have potential roles in BCG therapy include CD4(+) and CD8(+) lymphocytes, natural killer cells, granulocytes, macrophages, and dendritic cells. Bladder cancer cells are killed through direct cytotoxicity by these cells, by secretion of soluble factors such as TRAIL (tumour necrosis factor-related apoptosis-inducing ligand), and, to some degree, by the direct action of BCG. Several gaps still exist in our knowledge that should be addressed in future efforts to understand this biotherapy of cancer.
Bacille Calmette-Guerin (BCG) is an attenuated strain of Mycobacterium bovis that is used widely as a vaccine for tuberculosis and is used as an effective treatment for superficial bladder carcinoma. Despite being the most successful cancer biotherapy, its mechanism of action and response determinants remain obscure. Here, we establish a model system to analyze BCG interaction with bladder cancer cells, using it to show that these cells vary dramatically in their susceptibility to BCG infection. Unexpectedly, the uptake of BCG by bladder cancer cells occurs by macropinocytosis rather than phagocytosis. BCG entry into bladder cancer cells relied upon Rac1, Cdc42, and their effector kinase Pak1. The difference in susceptibility between BCG-permissive and -resistant bladder cancer cells was due to oncogenic activation of signaling pathways that activate macropinocytosis, with phosphoinositide 3-kinase inhibitor activation stimulating BCG uptake independently of Akt. Similarly, activated Ras strongly activated Pak1-dependent uptake of BCG. These results reveal that oncogenic activation of macropinocytosis determines BCG uptake by bladder cancer cells, implying that tumor responsiveness to BCG may be governed by the specific mutations present in the treated cancer cell. Cancer Res; 73(3); 1156-67. ©2013 AACR.
RNA aptamers can fold into complex structures and bind with high affinity and selectivity to various macromolecules, viruses, and cells. They are isolated from a large pool of nucleic acids by a conceptually straightforward iterative selection process called SELEX. Aptamers have enormous potential as therapeutics due to their ability to bind to proteins and specifically inhibit their functions with minimal or no harmful side-effects. The first aptamer therapeutic was FDA approved in 2005 and a number of novel aptamer-based therapeutics are currently undergoing clinical trials for treating diseases such as macular degeneration, choroidal neovascularization, intravascular thrombus, acute coronary syndrome, von Willebrand factor related disorders, von Hippel-Lindau syndrome (VHL), angiomas, acute myeloid leukemia, renal cell carcinoma, non-small cell lung cancer, thrombotic thrombocytopenic purpura, and several others. In this review, we present aptamers in on-going, completed, and terminated clinical studies highlighting their mechanism of action as well as the inherent challenges of aptamer production and use.
Mannose-capped lipoarabinomannan (ManLAM) is considered an important virulence factor of Mycobacterium tuberculosis. However, while mannose caps have been reported to be responsible for various immunosuppressive activities of ManLAM observed in vitro, there is conflicting evidence about their contribution to mycobacterial virulence in vivo. Therefore, we used M. bovis BCG and M. tuberculosis mutants that lack the mannose cap of LAM to assess the role of ManLAM in the interaction of mycobacteria with the host cells, to evaluate vaccine-induced protection and to determine its importance in M. tuberculosis virulence. Deletion of the mannose cap did not affect BCG survival and replication in macrophages, although the capless mutant induced a somewhat higher production of TNF. In dendritic cells, the capless mutant was able to induce the up-regulation of co-stimulatory molecules and the only difference we detected was the secretion of slightly higher amounts of IL-10 as compared to the wild type strain. In mice, capless BCG survived equally well and induced an immune response similar to the parental strain. Furthermore, the efficacy of vaccination against a M. tuberculosis challenge in low-dose aerosol infection models in mice and guinea pigs was not affected by the absence of the mannose caps in the BCG. Finally, the lack of the mannose cap in M. tuberculosis did not affect its virulence in mice nor its interaction with macrophages in vitro. Thus, these results do not support a major role for the mannose caps of LAM in determining mycobacterial virulence and immunogenicity in vivo in experimental animal models of infection, possibly due to redundancy of function.
The World Health Organization recently revised its recommendations for tuberculosis (TB) diagnosis in people with HIV. Most studies cited to support these policies involved HIV-uninfected patients and only evaluated sputum specimens.
To evaluate the performance of acid-fast bacilli smear and mycobacterial culture on sputum and nonsputum specimens for TB diagnosis in a cross-sectional survey of HIV-infected patients.
In Thailand and Vietnam, we enrolled people with HIV regardless of signs or symptoms. Enrolled patients provided three sputum, one urine, one stool, one blood, and, for patients with palpable peripheral adenopathy, one lymph node aspirate specimen for acid-fast bacilli microscopy and mycobacterial culture on solid and broth-based media. We classified any patient with at least one specimen culture positive for Mycobacterium tuberculosis as having TB.
Of 1,060 patients enrolled, 147 (14%) had TB. Of 126 with pulmonary TB, the incremental yield of performing a third sputum smear over two smears was 2% (95% confidence interval, 0-6), 90 (71%) patients were detected on broth-based culture of the first sputum specimen, and an additional 21 (17%) and 12 (10%) patients were diagnosed with the second and third specimens cultured. Of 82 lymph nodes cultured, 34 (42%) grew M. tuberculosis. In patients with two negative sputum smears, broth-based culture of three sputum specimens had the highest yield of any testing strategy.
In people with HIV living in settings where mycobacterial culture is not routinely available to all patients, a third sputum smear adds little to the diagnosis of TB. Broth-based culture of three sputum specimens diagnoses most TB cases, and lymph node aspiration provides the highest incremental yield of any nonpulmonary specimen test for TB.
Taking a strand: Aptamers are small single-stranded oligonucleotides that fold into a well-defined 3D structure and interact with high affinity and specificity with their target molecules, thereby inhibiting their biological functions. Aptamers can be synthesized by either chemical and/or enzymatic procedures and can thus be considered as both chemical and biological substances. The current status and new developments in this area are described.
Aptamers are small single-stranded nucleic acids that fold into a well-defined three-dimensional structure. They show a high affinity and specificity for their target molecules and inhibit their biological functions. Aptamers belong to the nucleic acids family and can be synthesized by chemical or enzymatic procedures, or a combination of the two. They can, therefore, be considered as both chemical and biological substances. This Review summarizes the most convenient approaches to their preparation and new developments in the field of aptamers. The application of aptamers in chemical biology is also discussed.
High-affinity nucleic acid ligands for a protein were isolated by a procedure that depends on alternate cycles of ligand selection from pools of variant sequences and amplification of the bound species. Multiple rounds exponentially enrich the population for the highest affinity species that can be clonally isolated and characterized. In particular one eight-base region of an RNA that interacts with the T4 DNA polymerase was chosen and randomized. Two different sequences were selected by this procedure from the calculated pool of 65,536 species. One is the wild-type sequence found in the bacteriophage mRNA; one is varied from wild type at four positions. The binding constants of these two RNA's to T4 DNA polymerase are equivalent. These protocols with minimal modification can yield high-affinity ligands for any protein that binds nucleic acids as part of its function; high-affinity ligands could conceivably be developed for any target molecule.
The principal efferent role of the macrophage in acquired resistance to intracellular pathogens depends on activation by T-cell lymphokines, primarily gamma interferon (IFN-gamma). However, mouse macrophages that are heavily burdened with Mycobacterium leprae are refractory to activation by IFN-gamma and are thus severely compromised in their capacity for both enhanced microbicidal and tumoricidal activities. We report here that lipoarabinomannan (LAM), a highly immunogenic lipopolysaccharide that is a prominent component of the cell walls of M. leprae and M. tuberculosis, was a potent inhibitor of IFN-gamma-mediated activation of mouse macrophages in vitro. Inhibition of macrophage activation by LAM required preincubation for approximately 24 h, resulting in uptake of LAM into cytoplasmic vacuoles of macrophages. Intact LAM was necessary to inhibit IFN-gamma-mediated activation, as this property was lost when the acyl side chains were removed from LAM by mild alkaline hydrolysis. In addition, LAM was an abundant constituent of macrophages isolated from lepromatous granulomas of M. leprae-infected nude mice and likely contributed to the defective activation of granuloma macrophages by IFN-gamma.
Lipoarabinomannan (LAM) is a major structural surface component of all mycobacteria, and has been reported to have a wide range of biological effects. Immunogenic LAM specific oligosaccharide protein conjugates were synthesized and immunologically characterized. Oligosaccharides were derived from LAM purified from Mycobacterium tuberculosis H37 Rv and covalently conjugated to tetanus toxoid and cross reactive mutant (CRM197) diphtheria toxoid. Both types of LAM oligosaccharide protein conjugates proved to be highly immunogenic, inducing a boosterable T helper cell dependent IgG response. These conjugates are currently evaluated as components in a subcellular experimental TB vaccine.
The molecular bases of Mycobacterium tuberculosis pathogenicity remain unclear. We report here how M. tuberculosis mannosylated lipoarabinomannans contribute to the survival of bacilli in the human reservoir by (i) inhibiting IL-12 production by macrophages and dendritic cells and (ii) modulating M. tuberculosis-induced macrophage apoptosis.
Mycobacterium tuberculosis, the causative agent of tuberculosis, is one of the most effective human pathogens and the molecular basis of its virulence remains poorly understood. Here, we review our current knowledge about the structure and biosynthesis of the mycobacterial cell-wall lipoglycans, lipoarabinomannans (LAM). LAM are ubiquitous of mycobacteria and appear as the most potent non-peptidic molecules to modulate the host immune response. Nevertheless, LAM structure differs according to the mycobacterial species and three types of LAM have been described: mannose-capped LAM (ManLAM), phospho-myo-inositol-capped LAM (PILAM) and non-capped LAM (AraLAM). The type of capping is a major structural feature determining the ability of LAM to modulate the immune response. ManLAM, found in slow-growing mycobacteria, such as M. tuberculosis, have been demonstrated to be powerful anti-inflammatory molecules and emerge as key virulence factors that may be relevant drug targets. LAM-like molecules are not only confined to mycobacteria but are also present in actinomycetes (including the genera Rhodococcus, Corynebacterium or Gordonia). This offers the possibility of comparative studies that should help in deciphering the structure-function relationships and biosynthesis of these complex molecules in the future.
Dendritic cells (DCs) are crucial in the defence against pathogens. Invading pathogens are recognized by Toll-like receptors (TLRs) and receptors such as C-type lectins expressed on the surface of DCs. However, it is becoming evident that some pathogens, including viruses, such as HIV-1, and non-viral pathogens, such as Mycobacterium tuberculosis, subvert DC functions to escape immune surveillance by targeting the C-type lectin DC-SIGN (DC-specific intercellular adhesion molecule-grabbing nonintegrin). Notably, these pathogens misuse DC-SIGN by distinct mechanisms that either circumvent antigen processing or alter TLR-mediated signalling, skewing T-cell responses. This implies that adaptation of pathogens to target DC-SIGN might support pathogen survival.
The hyperbolic parabola is commonly used to summarize kinetics for enzyme reactions and receptor binding kinetics. Depending on the experimental conditions, certain assumptions are valid but others might be violated so that the parameters used to fit this hyperbolic function, the maximum asymptote and the equilibrium constant, require different interpretations. The first topic of this review compares enzyme-induced transformations and receptor binding in terms of the appropriate assumptions. The second topic considers the complication of adding a competitive inhibitor as an enzyme substrate or a receptor ligand and the subtleties of inferring the equilibrium dissociation constant from the concentration of inhibitor (for example unlabeled drug) that leads to the midpoint, IC(50,) of an inhibition curve. Receptor binding may be measured directly by a concentration assay or as a pharmacodynamic response variable.
Lipoarabinomannan (LAM) is a major structural carbohydrate antigen of the outer surface of Mycobacterium tuberculosis. High antibody titres against LAM are often seen in active tuberculosis (TB). The role of such LAM-specific antibodies in the immune response against TB is unknown. Here we have investigated a monoclonal antibody (MoAb) SMITB14 of IgG1 subclass and its corresponding F(ab')(2) fragment directed against LAM from M. tuberculosis strain H37Rv. MoAb SMITB14 was shown by immunofluorescence to bind to whole cells of the clinical isolate M. tuberculosis strain Harlingen as well as to M. tuberculosis H37Rv. The binding of MoAb SMITB14 to LAM was inhibited by arabinomannan (AM) and oligosaccharides (5.2 kDa) derived from LAM, showing that the MoAb binds specifically to the AM carbohydrate portion of LAM. In passive protection experiments BALB/c mice were infected intravenously with M. tuberculosis Harlingen. MoAb SMITB14 was added intravenously either prior to, or together with, the bacteria. The antibody proved to be protective against the M. tuberculosis infection in terms of a dose-dependent reduction in bacterial load in spleens and lungs, reduced weight loss and, most importantly, increased long-term survival.
We have developed a novel tuberculosis (TB) vaccine; a combination of the DNA vaccines expressing mycobacterial heat shock protein 65 (HSP65) and interleukin 12 (IL-12) delivered by the hemagglutinating virus of Japan (HVJ)-liposome (HSP65+IL-12/HVJ). This vaccine provided remarkable protective efficacy in mouse and guinea pig models compared to the BCG vaccine, on the basis of an induction of the CTL activity and improvement of the histopathological tuberculosis lesions, respectively. Furthermore, we extended our studies to a cynomolgus monkey model, which is currently the best animal model of human tuberculosis. This novel vaccine provided a higher level of the protective efficacy than BCG based upon the assessment of mortality, the ESR, body weight, chest X-ray findings and immune responses. Furthermore, the combination of HSP65+IL-12/HVJ and BCG by the priming-booster method showed a synergistic effect in the TB-infected cynomolgus monkey (100% survival). These data indicate that our novel DNA vaccine might be useful against Mycobacterium tuberculosis for human clinical trials.