Delta inulin-based adjuvants promote the generation of polyfunctional CD4+ T cell responses and protection against Mycobacterium tuberculosis infection

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.There is an urgent need for the rational design of safe and effective vaccines to protect against chronic bacterial pathogens such as Mycobacterium tuberculosis. Advax™ is a novel adjuvant based on delta inulin microparticles that enhances immunity with a minimal inflammatory profile and has entered human trials to protect against viral pathogens. In this report we determined if Advax displays broad applicability against important human pathogens by assessing protective immunity against infection with M. tuberculosis. The fusion protein CysVac2, comprising the M. tuberculosis antigens Ag85B (Rv1886c) and CysD (Rv1285) formulated with Advax provided significant protection in the lungs of M. tuberculosis-infected mice. Protection was associated with the generation of CysVac2-specific multifunctional CD4+ T cells (IFN-γ+TNF+IL-2+). Addition to Advax of the TLR9 agonist, CpG oligonucleotide (AdvaxCpG), improved both the immunogenicity and protective efficacy of CysVac2. Immunisation with CysVac2/AdvaxCpG resulted in heightened release of the chemoattractants, CXCL1, CCL3, and TNF, and rapid influx of monocytes and neutrophils to the site of vaccination, with pronounced early priming of CysVac2-specific CD4+ T cells. As delta inulin adjuvants have shown an excellent safety and tolerability profile in humans, CysVac2/AdvaxCpG is a strong candidate for further preclinical evaluation for progression to human trials

Non-toxic metal-cyclam complexes, a new class of compounds with potency against drug-resistant Mycobacterium tuberculosis

Tuberculosis (TB) accounted for 1.5 million deaths in 2014 and new classes of anti-TB drugs are required. We re-port a class of functionalized 1,8-disubstituted cyclam derivatives that display low micromolar activity against pathogenic myco-bacteria. These compounds inhibit intracellular growth of Mycobacterium tuberculosis, are non-toxic to human cell lines and are active against multidrug-resistant M. tuberculosis strains, indicating a distinct mode of action. These compounds warrant further appraisal as novel agents to control TB in humans.This work was supported by the National Health and Medical Research Council (NHMRC) Project APP1084266, the NHMRC Center of Research Excellence in Tuberculosis Control (APP1043225) and the University of Sydney Sydnovate Fund. We acknowledge the Centre for Drug Candidate Optimization, Monash University for the ADME studie

The effect of the Roquin mutation on immunity to mycobacterium tuberculosis infection

Mycobacterium tuberculosis is a major cause of mortality and morbidity worldwide. IFN-gamma (IFN-γ) is essential for the control of M. tuberculosis infection and increased expression of the costimulatory molecule, Inducible T cell Costimulator (ICOS) on CD4+ T cells has been found to enhance mycobacteria-specific IFN-γ production in TB patients. ICOS is a marker of T cell activation and is also expressed by T follicular helper (TFH) cells, the role of which in immunity to M. tuberculosis is not well understood. Sanroque (SAN) mice carry a point mutation in the E3 ligase gene, roquin, and display increased ICOS expression on their CD4+ T cells, resulting in enhanced T cell activation and increased numbers of TFH cells. Further, SAN mice also progressively develop autoimmune disease. In this thesis, young SAN mice were utilised to examine whether their increased ICOS expression and higher numbers of TFH cells would improve control of M. tuberculosis infection. SAN mice displayed enhanced control of early M. tuberculosis infection, resulting in delayed bacterial dissemination from the lung. By 8 weeks post infection however, despite the enhanced T cell activation and increased mycobacteria-specific IFN-γ production, the SAN mice were unable to maintain the control of chronic M. tuberculosis infection, with significantly higher bacterial load in the lungs and dysregulated granuloma formation compared with wild type (WT) mice. The mechanisms leading to this increased susceptibility were then investigated. Further findings indicated that the expression of the roquin mutation itself in both T cells and myeloid cells, and potentially non-haematopoietic cells is the cause of increased susceptibility to M. tuberculosis infection and this is independent of the numbers of TFH cells seen in the SAN mice. The role of TFH cells in immunity to intracellular pathogens is not clear. M. tuberculosis infection increases the number of TFH cells in C57Bl/6 mice. To dissect the function of TFH cells in response to M. tuberculosis, we differentiated naïve M. tuberculosis-specific CD4+ T cells in vitro into TFH-like cells producing high IL-21 and low levels of IFN-γ. When these cells were transferred into RAG1-/- mice, which were then infected with M. tuberculosis, they upregulated IFN-γ production, like TH1 cells and controlled M. tuberculosis infection more effectively than the transferred TH0 cells. These data suggest that TFH-like cells retain a degree of plasticity and can modulate their response to infection depending upon the stimuli received. In summary, these data demonstrate that both roquin and TFH cells play important roles in protective immunity against M. tuberculosis infection

New tuberculosis drug leads from naturally occurring compounds

Tuberculosis (TB) continues to be a significant cause of mortality and morbidity worldwide. An estimated 2 billion individuals are infected with Mycobacterium tuberculosis and annually there are approximately 10 million new cases of clinical TB and 1.5 million deaths. Currently available drugs and vaccines have had no significant impact on TB control. In addition, the emergence of drug resistant TB is considered a public health crisis, with some strains now resistant to all available drugs. Unfortunately, the growing burden of antibiotic resistance is coupled with decreased effort in the development of new antibiotics. Natural sources are attractive starting points in the search for anti-tubercular drugs because they are extremely rich in chemical diversity and have privileged antimicrobial activity. This review will discuss recent advances in the development of TB drug leads from natural products, with a particular focus on anti-mycobacterial compounds in late-stage preclinical and clinical development

Modulation of roquin function in myeloid cells reduces mycobacterium tuberculosis-induced inflammation

Damaging inflammation is a hallmark of Mycobacterium tuberculosis infection, and understanding how this is regulated is important for the development of new therapies to limit excessive inflammation. The E3 ubiquitin ligase, Roquin, is involved in immune regulation; however, its role in immunity to M. tuberculosis is unknown. To address this, we infected mice with a point mutation in Roquin1/Rc3h1 (sanroque). Aerosol-infected sanroque mice showed enhanced control of M. tuberculosis infection associated with delayed bacterial dissemination and upregulated TNF production in the lungs after 2 wk. However, this early control of infection was not maintained, and by 8 wk postinfection sanroque mice demonstrated an increased bacterial burden and dysregulated inflammation in the lungs. As the inflammation in the lungs of the sanroque mice could have been influenced by emerging autoimmune conditions that are characteristic of the mice aging, the function of Roquin was examined in immune cell subsets in the absence of autoimmune complications. M. bovis bacillus Calmette–Guérin-primed sanroque T cells transferred into Rag1−/− mice provided equivalent protection in the spleen and liver. Interestingly, the transfer of mycobacteria-specific (P25 CD4+ TCR transgenic) wild-type spleen cells into sanroque.Rag1−/− mice actually led to enhanced protection with reduced bacterial load, decreased chemokine expression, and reduced inflammation in the lungs compared with transfers into Rag1−/− mice expressing intact Roquin. These studies suggest that modulation of Roquin in myeloid cells may reduce both inflammation and bacterial growth during the chronic phase of M. tuberculosis infection

Rapid Antibacterial Activity of Cannabichromenic Acid against Methicillin-Resistant Staphylococcus aureus

Methicillin-resistant Staphylococcus aureus (MRSA) has proven to be an imminent threat to public health, intensifying the need for novel therapeutics. Previous evidence suggests that cannabinoids harbour potent antibacterial activity. In this study, a group of previously inaccessible phytocannabinoids and synthetic analogues were examined for potential antibacterial activity. The minimum inhibitory concentrations and dynamics of bacterial inhibition, determined through resazurin reduction and time-kill assays, revealed the potent antibacterial activity of the phytocannabinoids against gram-positive antibiotic-resistant bacterial species, including MRSA. One phytocannabinoid, cannabichromenic acid (CBCA), demonstrated faster and more potent bactericidal activity than vancomycin, the currently recommended antibiotic for the treatment of MRSA infections. Such bactericidal activity was sustained against low-and high-dose inoculums as well as exponential- and stationary-phase MRSA cells. Further, mammalian cell viability was maintained in the presence of CBCA. Finally, microscopic evaluation suggests that CBCA may function through the degradation of the bacterial lipid membrane and alteration of the bacterial nucleoid. The results of the current study provide encouraging evidence that cannabinoids may serve as a previously unrecognised resource for the generation of novel antibiotics active against MRSA

Inhalation of Respirable Crystalline Rifapentine Particles Induces Pulmonary Inflammation

Rifapentine is an anti-tuberculosis (anti-TB) drug with a prolonged half-life, but oral delivery results in low concentrations in the lungs because of its high binding (98%) to plasma proteins. We have shown that inhalation of crystalline rifapentine overcomes the limitations of oral delivery by significantly enhancing and prolonging the drug concentration in the lungs. The delivery of crystalline particles to the lungs may promote inflammation. This <i>in vivo</i> study characterizes the inflammatory response caused by pulmonary deposition of the rifapentine particles. The rifapentine powder was delivered to BALB/c mice by intratracheal insufflation at a dose of 20 mg/kg. The inflammatory response in the lungs and bronchoalveolar lavage (BAL) was examined at 12 h, 24 h, and 7 days post-treatment by flow cytometry and histopathology. At 12 and 24 h post-treatment, there was a significant influx of neutrophils into the lungs, and this returned to normal by day 7. A significant recruitment of macrophages occurred in the BAL at 24 h. Consistent with these findings, histopathological analysis demonstrated pulmonary vascular congestion and significant macrophage recruitment at 12 and 24 h post-treatment. In conclusion, the pulmonary delivery of crystalline rifapentine caused a transient neutrophil-associated inflammatory response in the lungs that resolved over 7 days. This observation may limit pulmonary delivery of rifapentine to once a week at a dose of 20 mg/kg or less. The effectiveness of weekly dosing with inhalable rifapentine will be assessed in murine <i>Mycobacterium tuberculosis</i> infection