Cell wall skeleton of Mycobacterium bovis BCG enhances the vaccine potential of antigen 85B against tuberculosis by inducing Th1 and Th17 responses.
ABSTRACT: 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.
Project description:Tuberculosis (TB) still remains a major public health issue despite the current available vaccine for TB, Bacille Calmette Guerin (BCG). An effective vaccine against TB remains a top priority in the fight against this pandemic bacterial infection. Adequate protection against TB is associated with the development of TH1-type and CD8(+) T cell responses. One alarmin cytokine, interleukin 33 (IL-33), has now been implicated in the development of both CD4(+) TH1 and CD8(+) T cell immunity. In this study, we determined whether the administration of IL-33 as an adjuvant, encoded in a DNA plasmid, could enhance the immunogenicity of a TB DNA vaccine. We report that the co-immunization of IL-33 with a DNA vaccine expressing the Mycobacterium Tuberculosis (Mtb) antigen 85B (Ag85B) induced robust Ag85B-specific IFNγ responses by ELISpot compared to Ag85B alone. Furthermore, these enhanced responses were characterized by higher frequencies of Ag85B-specific, multifunctional CD4(+) and CD8(+) T cells. Vaccination with IL-33 also increased the ability of the Ag85B-specific CD8(+) T cells to undergo degranulation and to secrete IFNγ and TNFα cytokines. These finding highlights IL-33 as a promising adjuvant to significantly improve the immunogenicity of TB DNA vaccines and support further study of this effective vaccine strategy against TB.
Project description:Liposomes have been long considered as a vaccine delivery system but this technology remains to be fully utilized. Here, we describe a novel liposome-based subunit vaccine formulation for tuberculosis (TB) based on phosphatidylserine encapsulating two prominent TB antigens, Ag85B, and ESAT-6. We show that the resulting liposomes (Lipo-AE) are stable upon storage and can be readily taken up by antigen presenting cells and that their antigenic cargo is delivered and processed within endosomal cell compartments. The Lipo-AE vaccine formulation combined with the PolyIC adjuvant induced a mixed Th1/Th17-Th2 immune response to Ag85B but only a weak response to ESAT-6. An immunization regimen based on systemic delivery followed by mucosal boost with Lipo-AE resulted in the accumulation of resident memory T cells in the lungs. Most importantly though, when Lipo-AE vaccine candidate was administered to BCG-immunized mice subsequently challenged with low dose aerosol <i>Mycobacterium tuberculosis</i>, we observed a significant reduction of the bacterial load in the lungs and spleen compared to BCG alone. We therefore conclude that the immunization with mycobacterial antigens delivered by phosphatidylserine based liposomes in combination with Poly:IC adjuvant may represent a novel BCG boosting vaccination strategy.
Project description:Tuberculosis (TB) is one of the deadliest diseases, causing ?2 million deaths annually worldwide. Mycobacterium bovis bacillus Calmette-Guérin (BCG), the only TB vaccine in common use, is effective against disseminated and meningeal TB in young children but is not effective against adult pulmonary TB. T helper 1 (Th1) cells producing interferon gamma (IFN-?) and Th17 cells producing interleukin-17 (IL-17) play key roles in host protection against TB, whereas Th2 cells producing IL-4 and regulatory T cells (Tregs) facilitate TB disease progression by inhibiting protective Th1 and Th17 responses. Furthermore, the longevity of vaccine efficacy critically depends on the magnitude of long-lasting central memory T (TCM) cell responses. Hence, immunomodulators that promote TCM responses of the Th1 and Th17 cell lineages may improve BCG vaccine efficacy. Here, we show that curcumin nanoparticles enhance various antigen-presenting cell (APC) functions, including autophagy, costimulatory activity, and the production of inflammatory cytokines and other mediators. We further show that curcumin nanoparticles enhance the capacity of BCG to induce TCM cells of the Th1 and Th17 lineages, which augments host protection against TB infection. Thus, curcumin nanoparticles hold promise for enhancing the efficacy of TB vaccines.
Project description:Despite its relatively poor efficacy, Bacillus Calmette-Guérin (BCG) has been used as a tuberculosis (TB) vaccine since its development in 1921. BCG induces robust T helper 1 (Th1) immune responses but, for many individuals, this is not sufficient for host resistance against Mycobacterium tuberculosis (M. tb) infection. Here we provide evidence that early secreted antigenic target protein 6 (ESAT-6), expressed by the virulent M. tb strain H37Rv but not by BCG, promotes vaccine-enhancing Th17 cell responses. These activities of ESAT-6 were dependent on TLR-2/MyD88 signalling and involved IL-6 and TGF-? production by dendritic cells. Thus, animals that were previously infected with H37Rv or recombinant BCG containing the RD1 region (BCG::RD1) exhibited improved protection upon re-challenge with virulent H37Rv compared with mice previously infected with BCG or RD1-deficient H37Rv (H37Rv?RD1). However, TLR-2 knockout (TLR-2?/?) animals neither showed Th17 responses nor exhibited improved protection in response to immunization with H37Rv. Furthermore, H37Rv and BCG::RD1 infection had little effect on the expression of the anti-inflammatory microRNA-146a (miR146a) in dendritic cells (DCs), whereas BCG and H37Rv?RD1 profoundly induced its expression in DCs. Consistent with these findings, ESAT-6 had no effect on miR146a expression in uninfected DCs, but dramatically inhibited its upregulation in BCG-infected or LPS-treated DCs. Collectively, our findings indicate that, in addition to Th1 immunity induced by BCG, RD1/ESAT-6-induced Th17 immune responses are essential for optimal vaccine efficacy.
Project description:Protein subunit vaccines present a compelling new area of research for control of tuberculosis (TB). Based on the interaction between Mycobacterium tuberculosis and its host, five stage-specific antigens of M. tuberculosis that participate in TB pathogenesis--Rv1813, Rv2660c, Ag85B, Rv2623, and HspX--were selected. These antigens were verified to be recognized by T cells from a total of 42 whole blood samples obtained from active TB patients, patients with latent TB infections (LTBIs), and healthy control donors. The multistage polyprotein A1D4 was developed using the selected five antigens as a potentially more effective novel subunit vaccine. The immunogenicity and protective efficacy of A1D4 emulsified in the adjuvant MTO [monophosphoryl lipid A (MPL), trehalose-6,6'-dibehenate (TDB), components of MF59] was compared with Bacillus Calmette-Guerin (BCG) in C57BL/6 mice. Our results demonstrated that A1D4/MTO could provide more significant protection against M. tuberculosis infection than the PBS control or MTO adjuvant alone judging from the A1D4-specific Th1-type immune response; however, its efficacy was inferior to BCG as demonstrated by the bacterial load in the lung and spleen, and by the pathological changes in the lung. Antigen-specific single IL-2-secreting cells and different combinations with IL-2-secreting CD4+ T cells were beneficial and correlated with BCG vaccine-induced protection against TB. Antigen-specific IFN-?+ IL-2+ CD4+ T cells were the only effective biomarker significantly induced by A1D4/MTO. Among all groups, A1D4/MTO immunization also conferred the highest number of antigen-specific single IFN-?+ and IFN-?+ TNF-?+ CD4+ T cells, which might be related to the antigen load in vivo, and single IFN-?+ CD8+ T cells by mimicking the immune patterns of LTBIs or curable TB patients. Our strategy seems promising for the development of a TB vaccine based on multistage antigens, and subunit antigen A1D4 suspended in MTO adjuvant warrants preclinical evaluation in animal models of latent infection and may boost BCG vaccination.
Project description:Novel adjuvants are in demand for improving the efficacy of human vaccines. The immunomodulatory properties of Mycobacterium tuberculosis cell wall components have been highlighted in the formulation of complete Freund's adjuvant (CFA). We have explored the adjuvant potential of poly-α-l-glutamine (PLG), a lesser-known constituent of the pathogenic mycobacterial cell wall. Immune parameters indicated that the adjuvant potency of PLG was statistically comparable to that of CFA and better than that of alum in the context of H1 antigen (Ag85B and ESAT-6 fusion). At 1 mg/dose, PLG augmented the immune response of Ag85B, BP26, and protective antigen (PA) by increasing serum antibodies and cytokines in the culture supernatant of antigen-stimulated splenocytes. PLG modulated the humoral response of vaccine candidate ESAT-6, eliciting significantly higher levels of total IgG and isotypes (IgG1, IgG2a, and IgG2b). Additionally, the splenocytes from PLG-adjuvanted mice displayed a robust increase in the Th1-specific gamma interferon, tumor necrosis factor alpha, interleukin-2 (IL-2), Th2-specific IL-6 and IL-10, and Th17-specific IL-17A cytokines upon antigenic stimulation. PLG improved the protective efficacy of ESAT-6 by reducing bacillary load in the lung and spleen as well as granuloma formation, and it helped in maintaining vital health parameters of mice challenged with M. tuberculosis The median survival time of PLG-adjuvanted mice was 205 days, compared to 146 days for dimethyl-dioctadecyl ammonium bromide-monophosphoryl lipid A (DDA-MPL)-vaccinated groups and 224 days for Mycobacterium bovis BCG-vaccinated groups. PLG enhanced the efficiency of the ESAT-6 vaccine to the level of BCG and better than that of DDA-MPL (P < 0.05), with no ill effect in C57BL/6J mice. Our results propose that PLG is a promising adjuvant candidate for advanced experimentation.
Project description:The attenuated vaccine Mycobacterium bovis BCG (Bacille Calmette Guerin) has limited protective efficacy against TB. The development of more effective TB vaccines has focused on the mycobacterial antigens that cause strong T helper 1 (Th1) responses. Mtb protein Rv3841 (bacterioferritin B; BfrB) is known to play a crucial role in the growth of Mtb. Nonetheless, it is unclear whether Rv3841 can induce protective immunity against Mtb. Here, we studied the action of Rv3841 in maturation of dendritic cells (DCs) and its engagement in the development of T-cell immunity. We found that Rv3841 functionally activated DCs by upregulating costimulatory molecules and increased secretion of proinflammatory cytokines. Activation of DCs by Rv3841 was mediated by Toll-like receptor 4 (TLR4), followed by triggering of mitogen-activated protein kinase and nuclear factor-?B signaling pathways. In addition, Rv3841-matured DCs effectively proliferated and polarized Th1 immune response of naïve CD4+ and CD8+ T-cells. Moreover, Rv3841 specifically caused the expansion of CD4+CD44highCD62Llow T-cells from Mtb-infected mice; besides, the T-cells activated by Rv3841-matured DCs inhibited intracellular mycobacterial growth. Our data suggest that Rv3841 induces DC maturation and protective immune responses, a finding that may provide candidate of effective TB vaccines.
Project description:One third of the world is infected with Mycobacterium tuberculosis (Mtb) with eight million new cases of active tuberculosis (TB) each year. Development of a new vaccine to augment or replace the only approved TB vaccine, BCG, is needed to control this disease. Mtb infection is primarily controlled by TH1 cells through the production of IFN-? and TNF which activate infected macrophages to kill the bacterium. Here we examine an array of adjuvant formulations containing the TLR4 agonist GLA to identify candidate adjuvants to pair with ID93, a lead TB vaccine antigen, to elicit protective TH1 responses. We evaluate a variety of adjuvant formulations including alum, liposomes, and oil-in-water emulsions to determine how changes in formulation composition alter adjuvant activity. We find that alum and an aqueous nanosuspension of GLA synergize to enhance generation of ID93-specific TH1 responses, whereas neither on their own are effective adjuvants for generation of ID93-specific TH1 responses. For GLA containing oil-in-water emulsions, the selection of the oil component is critical for adjuvant activity, whereas a variety of lipid components may be used in liposomal formulations of GLA. The composition of the liposome formulation of ID93/GLA does alter the magnitude of the TH1 response. These results demonstrate that there are multiple solutions for an effective formulation of a novel TB vaccine candidate that enhances both TH1 generation and protective efficacy.
Project description:BACKGROUND: The current vaccine against tuberculosis (TB), BCG, has failed to control TB worldwide and the protective efficacy is moreover limited to 10-15 years. A vaccine that could efficiently boost a BCG-induced immune response and thus prolong protective immunity would therefore have a significant impact on the global TB-burden. METHODS/FINDINGS: In the present study we show that the fusion protein HyVac4 (H4), consisting of the mycobacterial antigens Ag85B and TB10.4, given in the adjuvant IC31® or DDA/MPL effectively boosted and prolonged immunity induced by BCG, leading to improved protection against infection with virulent M. tuberculosis (M.tb). Increased protection correlated with an increased percentage of TB10.4 specific IFN?/TNF?/IL-2 or TNF?/IL-2 producing CD4 T cells at the site of infection. Moreover, this vaccine strategy did not compromise the use of ESAT-6 as an accurate correlate of disease development/vaccine efficacy. Indeed both CD4 and CD8 ESAT-6 specific T cells showed significant correlation with bacterial levels. CONCLUSIONS/SIGNIFICANCE: H4-IC31® can efficiently boost BCG-primed immunity leading to an increased protective anti-M.tb immune response dominated by IFN?/TNF?/IL-2 or TNF?/IL2 producing CD4 T cells. H4 in the CD4 T cell inducing adjuvant IC31® is presently in clinical trials.
Project description:Tuberculosis (TB) is one of the deadliest diseases, claiming ~2 million deaths annually worldwide. The majority of people in TB endemic regions are vaccinated with Bacillus Calmette Guerin (BCG), which is the only usable vaccine available. BCG is efficacious against meningeal and disseminated TB in children, but protective responses are relatively short-lived and fail to protect against adult pulmonary TB. The longevity of vaccine efficacy critically depends on the magnitude of long-lasting central memory T (TCM) cells, a major source of which is stem cell-like memory T (TSM) cells. These TSM cells exhibit enhanced self-renewal capacity as well as to rapidly respond to antigen and generate protective poly-functional T cells producing IFN-?, TNF-?, IL-2 and IL-17. It is now evident that T helper Th 1 and Th17 cells are essential for host protection against TB. Recent reports have indicated that Th17 cells preserve the molecular signature for TSM cells, which eventually differentiate into IFN-?-producing effector cells. BCG is ineffective in inducing Th17 cell responses, which might explain its inadequate vaccine efficacy. Here, we show that revaccination with BCG along with clofazimine treatment promotes TSM differentiation, which continuously restores TCM and T effector memory (TEM) cells and drastically increases vaccine efficacy in BCG-primed animals. Analyses of these TSM cells revealed that they are predominantly precursors to host protective Th1 and Th17 cells. Taken together, these findings revealed that clofazimine treatment at the time of BCG revaccination provides superior host protection against TB by increasing long-lasting TSM cells.