Cell wall skeleton of Mycobacterium bovis BCG enhances the vaccine potential of antigen 85B against tuberculosis by inducing Th1 and Th17 responses.

A safe and effective adjuvant is necessary to induce reliable protective efficacy of the protein-based vaccines against tuberculosis (TB). Mycobacterial components, such as synthetic cord factor and arabinogalactan, have been used as one of the adjuvant components. Mycobacterium bovis bacillus Calmette- Guérin cell-wall skeleton (BCG-CWS) has been used as an effective immune-stimulator. However, it is not proven whether BCG-CWS can be an effective adjuvant for the subunit protein vaccine of TB. In this study, we demonstrated that the BCG-CWS effectively coupled with Ag85B and enhanced the conjugated Ag85B activity on the maturation of dendritic cells (DCs). Ag85B-BCG-CWS-matured DCs induced significant Th1 and Th17 responses when compared to BCG-CWS or Ag85B alone. In addition, significant Ag85B-specific Th1 and Th17 responses were induced in Ag85B-BCG-CWS-immunized mice before infection with M. tuberculosis and maintained after infection. Moreover, Ag85B-BCG-CWS showed significant protective effect comparable to live BCG at 6 weeks after infection and maintained its protective efficacy at 32 weeks post-challenge, whereas live BCG did not. These results suggest that the BCG-CWS may be an effective adjuvant candidate for a protein-based vaccine against TB.

Mycobacterial cell-wall skeleton as a universal vaccine vehicle for antigen conjugation.

Mycobacterial cell-wall skeleton (CWS) is an immunoactive and biodegradable particulate adjuvant and has been used for immunotherapy in patients with cancer. The CWS of Mycobacterium bovis bacillus Calmette-Guérin (BCG-CWS) was studied as a universal vaccine vehicle for antigen conjugation, to develop potentially effective and safe vaccines. Here, we describe experiments in which protein antigens, such as keyhole limpet haemocyanin (KLH), ovalbumin (OVA) and bovine serum albumin (BSA) were highly efficiently coupled to 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide (EDC/NHS)-activated carboxyl groups of BCG-CWS, and tested the immunogenicity of OVA-conjugated BCG-CWS vaccine. We found that a strong immune response was induced in mice immunised with OVA-conjugated BCG-CWS, which was similar to the enhancement of the immune responses in mice immunised with OVA and complete Freund's adjuvant. Covalent conjugation of OVA to BCG-CWS was essential for Th1-skewed immune responses, with prominent expression of IFN-γ. Furthermore, antigen-conjugated BCG-CWS vaccine is simple to manufacture, safe, and easy to use. Our results suggest that mycobacterial CWS as a universal vaccine vehicle for conjugation of a wide variety of antigens constitutes a breakthrough for development of the most promising vaccines for infections, allergic diseases, and cancer.

Bacillus calmette-guerin cell wall cytoskeleton enhances colon cancer radiosensitivity through autophagy.

The cell wall skeleton of Mycobacterium bovis Bacillus Calmette-Guerin (BCG/CWS) is an effective antitumor immunotherapy agent. Here, we demonstrate that BCG/CWS has a radiosensitizing effect on colon cancer cells through the induction of autophagic cell death. Exposure of HCT116 colon cancer cells to BCG/CWS before ionizing radiation (IR) resulted in increased cell death in a caspase-independent manner. Treatment with BCG/CWS plus IR resulted in the induction of autophagy in colon cancer cells. Either the autophagy inhibitor 3-methyladenine or knockdown of beclin 1 or Atg7 significantly reduced tumor cell death induced by BCG/CWS plus IR, whereas the caspase inhibitor z-VAD-fmk failed to do so. BCG/CWS plus IR-mediated autophagy and cell death was mediated predominantly by the generation of reactive oxygen species (ROS). The c-Jun NH(2)-terminal kinase pathway functioned upstream of ROS generation in the induction of autophagy and cell death in HCT116 cells after co-treatment with BCG/CWS and IR. Furthermore, toll-like receptor (TLR) 2, and in part, TLR4, were responsible for BCG/CWS-induced radiosensitization. In vivo studies revealed that BCG/CWS-mediated radiosensitization of HCT116 xenograft growth is accompanied predominantly by autophagy. Our data suggest that BCG/CWS in combination with IR is a promising therapeutic strategy for enhancing radiation therapy in colon cancer cells through the induction of autophagy.

Roles of reactive oxygen species in CXCL8 and CCL2 expression in response to the 30-kDa antigen of Mycobacterium tuberculosis.

BACKGROUND: The 30-kDa antigen (Ag) of Mycobacterium tuberculosis (M. tuberculosis) is a strong inducer of innate and adaptive immune responses in human tuberculosis. The generation of reactive oxygen species (ROS) plays an important role in inflammatory signaling as well as antimicrobial defense. MATERIALS AND METHODS: In this study, we investigated the role of ROS in the activation of mitogen-activated protein kinases (MAPKs) and secretion of the CXC chemokine ligand 8 (CXCL8) and CC chemokine ligand 2 (CCL2) by human monocytes stimulated with the 30-kDa Ag of M. tuberculosis H37Rv. RESULTS: Treatment of human monocytes with the 30-kDa Ag activated rapid superoxide generation. In addition, the 30-kDa Ag activated mRNA and protein expression of CXCL8 and CCL2 in human primary monocytes through nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-dependent ROS generation. Analysis of MAPK activation (extracellular signal-regulated kinase (ERK) 1/2 and p38) showed rapid phosphorylation of both subfamilies in response to the 30-kDa Ag. In addition, 30-kDa-induced MAPK activation was inhibited in a dose-dependent manner by pretreatment with ROS scavengers. Toll-like receptor (TLR) 2 was required for ROS generation, chemokine production, and MAPK activation following stimulation with the 30-kDa Ag. Using highly specific signaling pathway inhibitors, we found that both p38 and ERK1/2 activation are essential for 30-kDa Ag-induced CCL2 but not CXCL8 production in human monocytes. CONCLUSION: These results indicate that TLR2-ROS signaling plays a crucial role in the 30-kDa Ag-mediated expression of CXCL8 and CCL2 in human monocytes. In addition, both p38 and ERK1/2 activation are essential for 30-kDa Ag-stimulated CCL2 production by monocytes.

Protein kinase C zeta plays an essential role for Mycobacterium tuberculosis-induced extracellular signal-regulated kinase 1/2 activation in monocytes/macrophages via Toll-like receptor 2.

This study characterized the upstream signalling molecules involved in extracellular signal-regulated kinase (ERK) 1/2 activation and determined their effects on differential tumour necrosis factor (TNF)-alpha expression by monocytes/macrophages infected with virulent or avirulent mycobacteria. The avirulent Mycobacterium tuberculosis (MTB) strain H37Ra (MTBRa) induced higher levels of activation of ERK 1/2 and the upstream MAPK kinase (MEK)1 and, subsequently, higher levels of TNF-alpha expression in human primary monocytes and monocyte-derived macrophages, as compared with MTB strain H37Rv (MTBRv). The MTB-induced activation of ERK 1/2 was not dependent on Ras or Raf. However, inhibition of the activity of atypical protein kinase C (PKC) zeta decreased the in vitro phosphorylation of MEK, ERK 1/2 activation and subsequent TNF-alpha induction caused by MTBRv or MTBRa. Toll-like receptor (TLR) 2 was found to play a major role in MTB-induced TNF-alpha expression and PKCzeta phosphorylation. Co-immunoprecipitation experiments showed that PKCzeta interacts physically with TLR2 after MTB stimulation. Moreover, PKCzeta phosphorylation was increased more in macrophages following MTBRa, versus MTBRv, infection. This is the first demonstration that PKCzeta interacts with TLR2 to play an essential role in MTB-induced ERK 1/2 activation and subsequent TNF-alpha expression in monocytes/macrophages.

Mycobacterium tuberculosis HBHA protein reacts strongly with the serum immunoglobulin M of tuberculosis patients.

Identification and characterization of serologically active mycobacterial antigens are prerequisites for the development of diagnostic reagents. We examined the humoral immune responses of active tuberculosis (TB) patients against Triton-soluble proteins extracted from Mycobacterium tuberculosis by immunoblotting. A 29-kDa protein reacted with immunoglobulin M (IgM) in the pooled sera of the patients, and its N-terminal amino acid sequence matched that of the heparin-binding hemagglutinin (HBHA). Recombinant full-length HBHA was expressed in Escherichia coli (rEC-HBHA) and M. smegmatis (rMS-HBHA). In immunoblot analysis, the IgM antibodies of the TB patients reacted strongly with rMS-HBHA but not with rEC-HBHA, whereas the IgG antibodies of these patients reacted weakly with both recombinant HBHA proteins. In enzyme-linked immunosorbent assay analysis using rMS-HBHA and 85B as antigens, the mean levels and sensitivities of the anti-HBHA IgM antibodies of the TB patients were significantly higher than those of the anti-antigen 85B IgM antibodies, while the IgG antibodies showed the opposite results. Of interest in this respect, the pooled sera from the TB patients that contained anti-HBHA IgM antibodies neutralized the entry of M. tuberculosis into epithelial cells. These findings suggest that IgM antibody to HBHA may play a role in protection against extrapulmonary dissemination.

Intracellular network of phosphatidylinositol 3-kinase, mammalian target of the rapamycin/70 kDa ribosomal S6 kinase 1, and mitogen-activated protein kinases pathways for regulating mycobacteria-induced IL-23 expression in human macrophages.

We previously demonstrated that Mycobacterium tuberculosis (M. tbc)-induced interleukin (IL)-12 expression is negatively regulated by the phosphatidylinositol 3-kinase (PI3K) and extracellular signal-regulated kinase (ERK) 1/2 pathways in human monocyte-derived macrophages (MDMs). To extend these studies, we examined the nature of the involvement of toll-like receptors (TLRs) and intracellular signalling pathways downstream from PI3K in M. tbc-induced IL-23 expression in human MDMs. M. tbc-induced Akt activation and IL-23 expression were essentially dependent on TLR2. Blockade of the mammalian targets of rapamycin (mTOR)/70 kDa ribosomal S6 kinase 1 (S6K1) pathway by the specific inhibitor rapamycin greatly enhanced M. tbc-induced IL-12/IL-23 p40 (p40) and IL-23 p19 (p19) mRNA and IL-23 protein expression. In sharp contrast, p38 mitogen-activated protein kinase (MAPK) inhibition abrogated the p40 and p19 mRNA and IL-23 protein expression induced by M. tbc. Furthermore, the inhibition of PI3K-Akt, but not ERK 1/2 pathway, attenuated M. tbc-induced S6K1 phosphorylation, whereas PI3K inhibition enhanced p38 phosphorylation and apoptosis signal-regulating kinase 1 activity during exposure to M. tbc. Although the negative or positive regulation of IL-23 was not reversed by neutralization of IL-10, it was significantly modulated by blocking TLR2. Collectively, these findings provide new insight into the homeostatic mechanism controlling type 1 immune responses during mycobacterial infection involving the intracellular network of PI3K, S6K1, ERK 1/2 and p38 MAPK pathways in a TLR2-dependent manner.

The mycobacterial 38-kilodalton glycolipoprotein antigen activates the mitogen-activated protein kinase pathway and release of proinflammatory cytokines through Toll-like receptors 2 and 4 in human monocytes.

Although the 38-kDa glycolipoprotein of Mycobacterium tuberculosis H37Rv is known to evoke prominent cellular and humoral immune responses in human tuberculosis (TB), little information is known about intracellular regulatory mechanisms involved in 38-kDa antigen (Ag)-induced host responses. In this study, we found that purified 38-kDa glycolipoprotein activates mitogen-activated protein kinases (MAPKs; extracellular signal-regulated kinase 1/2 [ERK1/2] and p38) and induces tumor necrosis factor alpha (TNF-alpha) and interleukin 6 (IL-6) in human monocytes. When the 38-kDa Ag was applied to monocytes from TB patients and healthy controls, the activation of ERK1/2 and p38 MAPK and the subsequent cytokine secretion were greater in the monocytes from the active pulmonary TB patients than in monocytes from the healthy controls. Additionally, neutralizing antibodies for Toll-like receptor 2 (TLR2) or TLR4 significantly reduced the ERK1/2 and p38 activation induced by the 38-kDa protein when the antibodies were applied to HEK293 cells overexpressing TLR2 or TLR4 as well as human primary monocytes. Furthermore, the inhibition of TLR2 significantly, and that of TLR4 partially, decreased the 38-kDa Ag-induced secretion of TNF-alpha and IL-6 in human monocytes. The intact protein moieties of the 38-kDa protein were responsible for biologic activities by this Ag. These data collectively demonstrate that the 38-kDa glycolipoprotein, acting through both TLR2 and TLR4, induces the activation of the ERK1/2 and p38 MAPK pathways, which in turn play an essential role in TNF-alpha and IL-6 expression during mycobacterial infection.

Role of the phosphatidylinositol 3-kinase and mitogen-activated protein kinase pathways in the secretion of tumor necrosis factor-alpha and interleukin-10 by the PPD antigen of Mycobacterium tuberculosis.

Here we investigated the role of the phosphatidylinositol 3-kinase (PI 3-K) and mitogen-activated protein kinase (MAPK) pathways in the secretion of tumor necrosis factor (TNF)-alpha and interleukin (IL)-10 in human primary monocytes after stimulation with the PPD antigen of Mycobacterium tuberculosis. MAPK [extracellular signal-regulated kinase (ERK) 1/2 and p38] and Akt are rapidly phosphorylated in human monocytes stimulated with PPD. We found that the PI 3-K-Akt pathway stimulated by PPD is essential for both IL-10 and TNF-alpha production, although the inhibition of IL-10 production was more pronounced. The analysis of cytokine production using specific inhibitors of the MAPK pathway revealed that both p38 and ERK activation are essential for PPD-induced TNF-alpha production, whereas p38, but not ERK, activation is essential for IL-10 secretion. The inhibition of PI 3-K did not significantly activate p38 MAPK or ERK 1/2 in PPD-stimulated human monocytes. Further, the Src inhibitor PP2 inhibited the release of TNF-alpha but enhanced IL-10 release, suggesting the differential regulation of Src kinase in upstream signaling. Collectively, these data suggest that the PI 3-K and MAPK pathways play a central role in the regulation of both pro- and anti-inflammatory cytokines by the PPD antigen of M. tuberculosis.

Identification of the new T-cell-stimulating antigens from Mycobacterium tuberculosis culture filtrate.

The proteins secreted by Mycobacterium tuberculosis are an important target for vaccine development. To identify the antigens from M. tuberculosis culture filtrate (CF) that strongly stimulate T-cells, the CF was fractionated by ion-exchange chromatography and then non-reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis with mini-whole gel elution. Each fraction was screened for its ability to induce interferon-gamma (IFN-gamma) production in peripheral blood mononuclear cells isolated from healthy tuberculin reactors. The protein bands that strongly induced IFN-gamma production were subjected to N-terminal sequencing. Two new proteins, a 17-kDa protein (Rv0164, MTSP17) and an 11-kDa (Rv3204, MTSP11) protein, were identified. The recombinant MTSP17 (rMTSP17) and rMTSP11 induced significant production of IFN-gamma and interleukin (IL)-12p40 in peripheral blood mononuclear cells from healthy tuberculin reactors. Interestingly, IL-12p40 production in response to rMTSP11 was significantly higher than that in response to rMTSP17 or the three components of the antigen 85 complex. These results suggest that MTSP11 antigen should be further evaluated as a component of a subunit vaccine.

Interleukin-8 is differentially expressed by human-derived monocytic cell line U937 infected with Mycobacterium tuberculosis H37Rv and Mycobacterium marinum.

Although Mycobacterium marinum is closely related to Mycobacterium tuberculosis H37Rv genomically, the clinical outcome in humans is quite different for M. marinum and M. tuberculosis infections. We investigated possible factors in the host macrophages for determining differential pathological responses to M. tuberculosis and M. marinum using an in vitro model of mycobacterial infection. Using suppression-subtractive hybridization, we identified 12 differentially expressed genes in the human monocytic cell line U937 infected with M. tuberculosis and M. marinum. Of those genes, the most frequently recovered transcript encoded interleukin-8 (IL-8). Northern hybridization revealed that IL-8 mRNA was highly upregulated in M. tuberculosis-infected U937 cells compared with M. marinum-infected cells. In addition, enzyme-linked immunosorbent assay showed that IL-8 protein secretion was significantly elevated in M. tuberculosis-infected U937 cells, human primary monocytes, and monocyte-derived macrophages compared with that in M. marinum-infected cells. The depressed IL-8 expression was unique in M. marinum-infected cells compared with cells infected with other strains of mycobacteria, including M. tuberculosis H37Ra, Mycobacterium bovis BCG, or Mycobacterium smegmatis. When the expression of NF-kappaB was assessed in mycobacterium-infected U937 cells, IkappaBalpha proteins were significantly degraded in M. tuberculosis-infected cells compared with M. marinum-infected cells. Collectively, these results suggest that differential IL-8 expression in human macrophages infected with M. tuberculosis and M. marinum may be critically associated with distinct host responses in tuberculosis. Additionally, our data indicate that differential signal transduction pathways may underlie the distinct patterns of IL-8 secretion in cells infected by the two mycobacteria.

Role of mitogen-activated protein kinase pathways in the production of tumor necrosis factor-alpha, interleukin-10, and monocyte chemotactic protein-1 by Mycobacterium tuberculosis H37Rv-infected human monocytes.

The role of mitogen-activated protein kinase (MAPK) pathways in the secretion of tumor necrosis factor (TNF)-alpha, interleukin (IL)-10, IL-8, and monocyte chemotactic protein-1 (MCP-1) was investigated in human monocytes that were infected with Mycobacterium tuberculosis H37Rv. Analysis of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and p38 kinase showed rapid phosphorylation of both subfamilies in response to M. tuberculosis H37Rv. Using highly specific inhibitors of p38 (SB203580) and of MAPK kinase-1 (U0126 and PD98059), we found that both p38 and ERK were essential for M. tuberculosis H37Rv-induced TNF-alpha production, whereas activation of the p38 pathway, but not that of ERK, was essential for M. tuberculosis H37Rv-induced IL-10 production. Interestingly, the ERK pathway, but not that of p38, was critical for MCP-1 secretion from human monocytes that were infected with M. tuberculosis H37Rv. However, IL-8 secretion was not regulated by ERK1/2 or p38 MAPK. Collectively, these results suggest that induction of the MAPK pathway is required for the expression of TNF-alpha, IL-10, and MCP-1 by human monocytes during M. tuberculosis H37Rv infection.

Depressed interleukin-12 production by peripheral blood mononuclear cells after in vitro stimulation with the 30-kDa antigen in recurrent pulmonary tuberculosis patients.

Some patients develop recurrent tuberculosis (R-TB), even after successfully completing initial anti-tubercular treatment. Although R-TB may be caused by relapse or exogenous reinfection, little is known about the underlying host responses associated with R-TB. This study investigated the profile of cytokines [interferon (IFN)-gamma, interleukin (IL)-12, tumor necrosis factor (TNF)-alpha, IL-6, and IL-10] present in peripheral blood mononuclear cells (PBMCs) of 17 R-TB patients after stimulation with the 30-kDa antigen (Ag) or purified protein derivative (PPD) Ag of Mycobacterium tuberculosis. These data were compared with data obtained from 15 patients with newly diagnosed pulmonary TB (N-TB), 22 patients with treatment failure (TF-TB), and 19 healthy tuberculin reactors (HTR). N-TB and R-TB patients were enrolled in this study within 1 month of beginning anti-tubercular chemotherapy. ELISA results showed that IFN-gamma production following stimulation with the 30-kDa Ag was significantly lower in each group of TB patients than in the HTR controls. In addition, patients with R-TB showed the most significant IL-12 depression among the subject groups after in vitro stimulation with either Ag. Furthermore, a significant decrease in TNF-alpha and IL-10 levels was observed in R-TB patients relative to N-TB patients. However, there was no statistical difference in TNF-alpha and IL-10 production between R-TB patients, TF-TB patients, and HTR controls. Our findings suggest that the underlying mechanisms of cytokine regulation might differ between N-TB and R-TB patients, and that decreased IL-12 production in response to the 30-kDa or PPD Ag might be involved in the immunopathogenesis of human R-TB.

Isolation and partial characterisation of the Triton X-100 solubilised protein antigen from Mycobacterium tuberculosis.

This report describes extraction of a new native antigen fraction from Mycobacterium tuberculosis without massive degradation of proteins by Triton X-100. The Triton X-100 solubilised protein (TSP) antigen showed a characteristic antigen profile and reproducible extraction pattern. To characterise the nature of their composition, the TSP antigen was fractionated by Triton X-114 phase partitioning. The TSP antigen contained a variety of lipids and glycoconjugates as well as diverse proteins. Most proteins were partitioned into the aqueous phase during phase fractionation, whereas non-protein molecules and lipoproteins were recovered in the detergent phase. The lymphoproliferative responses to the TSP aqueous fraction in healthy tuberculin reactors were significantly higher than those to the purified protein derivative (PPD) and unfractionated TSP. In contrast, the antibody responses to TSP aqueous fraction in tuberculosis patients showed weak reactivity. This study suggests that the TSP aqueous fraction can be used as a T-cell antigen associated with protective immunity against tuberculosis.