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Global control of COVID-19 requires broadly accessible vaccines that are effective against SARS-CoV-2 variants. In this report, we exploit the immunostimulatory properties of bacille Calmette-Guérin (BCG), the existing tuberculosis vaccine, to deliver a vaccination regimen with potent SARS-CoV-2-specific protective immunity. Combination of BCG with a stabilised, trimeric form of SARS-CoV-2 spike antigen promoted rapid development of virus-specific IgG antibodies in the blood of vaccinated mice, that was further augmented by the addition of alum. This vaccine formulation, BCG:CoVac, induced high-titre SARS-CoV-2 neutralising antibodies (NAbs) and Th1-biased cytokine release by vaccine-specific T cells, which correlated with the early emergence of T follicular helper cells in local lymph nodes and heightened levels of antigen-specific plasma B cells after vaccination. Vaccination of K18-hACE2 mice with a single dose of BCG:CoVac almost completely abrogated disease after SARS-CoV-2 challenge, with minimal inflammation and no detectable virus in the lungs of infected animals. Boosting BCG:CoVac-primed mice with a heterologous vaccine further increased SARS-CoV-2-specific antibody responses, which effectively neutralised B.1.1.7 and B.1.351 SARS-CoV-2 variants of concern. These findings demonstrate the potential for BCG-based vaccination to protect against major SARS-CoV-2 variants circulating globally.

There is an urgent need for novel vaccination strategies to combat respiratory pathogens. Mucosal vaccine delivery is an attractive option as it directly targets the site of infection; however, preclinical development has been hindered by a lack of suitable mucosal adjuvants and a limited understanding of their immune effects in the lung environment. Herein, we define the early immune events following the intrapulmonary delivery of a vaccine incorporating the adjuvant delta-inulin. Analysis of the early inflammatory response showed vaccine-induced innate cell recruitment to lungs and local lymph nodes (LN) was transient and non-polarised, correlating with an increase in pulmonary chemotactic factors. Use of fluorescently labelled adjuvant revealed widespread tissue dissemination of adjuvant particles, coupled with broad cellular uptake and transit to the lung-draining LN by a range of innate immune cells. Mass cytometric analysis revealed extensive phenotypic changes in innate and adaptive cell subsets induced by vaccination; this included identification of unconventional lymphocytes such as γδ-T cells and MAIT cells that increased following vaccination and displayed an activated phenotype. This study details a comprehensive view of the immune response to intrapulmonary adjuvant administration and provides pre-clinical evidence to support delta-inulin as a suitable adjuvant for pulmonary vaccines.

Mycobacterial granuloma formation involves significant stromal remodeling including the growth of leaky, granuloma-associated vasculature. These permeable blood vessels aid mycobacterial growth, as antiangiogenic or vascular normalizing therapies are beneficial host-directed therapies in preclinical models of tuberculosis across host-mycobacterial pairings. Using the zebrafish–Mycobacterium marinum infection model, we demonstrate that vascular normalization by inhibition of vascular endothelial protein tyrosine phosphatase (VE-PTP) decreases granuloma hypoxia, the opposite effect of hypoxia-inducing antiangiogenic therapy. Inhibition of VE-PTP decreased neutrophil recruitment to granulomas in adult and larval zebrafish, and decreased the proportion of neutrophils that extravasated distal to granulomas. Furthermore, VE-PTP inhibition increased the accumulation of T cells at M. marinum granulomas. Our study provides evidence that, similar to the effect in solid tumors, vascular normalization during mycobacterial infection increases the T cell:neutrophil ratio in lesions which may be correlates of protective immunity.

Pathogenic mycobacteria inhibit inflammasome activation to establish infection. Although it is known that potassium efflux is a trigger for inflammasome activation, the interaction between mycobacterial infection, potassium efflux, and inflammasome activation has not been investigated. Here, we use Mycobacterium marinum infection of zebrafish embryos and Mycobacterium tuberculosis infection of THP-1 cells to demonstrate that pathogenic mycobacteria up-regulate the host WNK signalling pathway kinases SPAK and OXSR1 which control intracellular potassium balance. We show that genetic depletion or inhibition of OXSR1 decreases bacterial burden and intracellular potassium levels. The protective effects of OXSR1 depletion are at least partially mediated by NLRP3 inflammasome activation, caspase-mediated release of IL-1β, and downstream activation of protective TNF-α. The elucidation of this druggable pathway to potentiate inflammasome activation provides a new avenue for the development of host-directed therapies against intracellular infections.

biBackground:/i/bIvermectin has received worldwide attention as a potential COVID-19 treatment after showing antiviral activity against SARS-CoV-2iin vitro./iHowever, the pharmacokinetic limitations associated with oral administration have been postulated as limiting factors to its bioavailability and efficacy. These limitations can be overcome by targeted delivery to the lungs. In this study, inhalable dry powders of ivermectin and lactose crystals were prepared and characterized for the potential treatment of COVID-19.biMethods:/i/bIvermectin was co-spray dried with lactose monohydrate crystals and conditioned by storage at two different relative humidity points (43% and 58% RH) for a week. Theiin vitro/idispersion performance of the stored powders was examined using a medium-high resistance Osmohaler connecting to a next-generation impactor at 60 L/min flow rate. The solid-state characteristics including particle size distribution and morphology, crystallinity, and moisture sorption profiles of raw and spray-dried ivermectin samples were assessed by laser diffraction, scanning electron microscopy, Raman spectroscopy, X-ray powder diffraction, thermogravimetric analysis, differential scanning calorimetry, and dynamic vapor sorption.biResults:/i/bAll the freshly spray-dried formulation (T0) and the conditioned samples could achieve the anticipated therapeutic dose with fine particle dose of 300 μg, FPFsubrecovered/subof 70%, and FPFsubemitted/subof 83%. In addition, the formulations showed a similar volume median diameter of 4.3 μm and span of 1.9. The spray-dried formulations were stable even after conditioning and exposing to different RH points as ivermectin remained amorphous with predominantly crystalline lactose.biConclusion:/i/bAn inhalable and stable dry powder of ivermectin and lactose crystals was successfully formulated. This powder inhaler ivermectin candidate therapy appears to be able to deliver doses that could be safe and effective to treat the SARS-COV-2 infection. Further development of this therapy is warranted.

Uropathogenic Escherichia coli (UPEC) causes urinary tract infections that can result in sepsis. The haemostatic system is protective in the pyelonephritis stage of ascending UPEC infection, but the role of the haemostatic system has not been investigated during sepsis. Here we utilize a zebrafish-UPEC systemic infection model to visualize infection-induced coagulation and examine the effects of commonly prescribed anti-haemostatic medications on the infection severity. Treatment of systemically infected zebrafish with warfarin, aspirin, or ticagrelor reduced host survival, while stabilization of clots with aminocaproic acid increased host survival. Anti-haemostatic drug treatment increased UPEC burden. Our findings provide evidence that commonly prescribed anti-haemostatic medications may worsen the outcome of severe UPEC infection.

Viral mutations are an emerging concern in reducing SARS-CoV-2 vaccination efficacy. Second generation vaccines will need to elicit neutralizing antibodies against sites that are evolutionarily conserved across the sarbecovirus subgenus. Here, we immunized mice containing a human antibody repertoire with diverse sarbecovirus receptor binding domains (RBDs) to identify antibodies targeting conserved sites of vulnerability. Antibodies with broad reactivity against diverse clade B RBDs targeting the conserved class 4 epitope, with recurring IGHV/IGKV pairs, were readily elicited but were non-neutralizing. However, rare class 4 antibodies binding this conserved RBD supersite showed potent neutralization of SARS-CoV-2 and all variants of concern. Structural analysis revealed that neutralizing ability of cross-reactive antibodies was reserved only for those with an elongated CDRH3 that extends the antiparallel beta-sheet RBD core and orients the antibody light chain to obstruct ACE2-RBD interactions. These results identify a structurally defined pathway for vaccine strategies eliciting escape-resistant SARS-CoV-2 neutralizing antibodies., Graphical Abstract, Viral mutations are an emerging concern in reducing SARS-CoV-2 vaccination efficacy. Burnett et al. immunized humanized mice with different diverse sarbecovirus RBDs to elicit antibodies targeting conserved sites. Non-neutralizing cross-reactive antibodies targeting the conserved class 4 epitope were readily elicited. Neutralizing ability was reserved only for antibodies binding this conserved supersite through an elongated CDRH3 that obstructed ACE2-RBD interactions.

Hyperglycaemia damages the microvasculature in part through the reduced recruitment of immune cells and interference with platelet signalling, leading to poor wound healing and accelerated lipid deposition in mammals. We investigated the utility of zebrafish larvae to model the effect of exogenous glucose on neutrophil and macrophage recruitment to a tail wound, wound-induced haemostasis, and chicken egg yolk feed challenge-induced hyperlipidaemia by supplementing larvae with exogenous glucose by immersion or injection. Neither method of glucose supplementation affected the recruitment of neutrophils and macrophages following tail transection. Glucose injection reduced thrombocyte retention and fibrin plug formation while only thrombocyte retention was reduced by glucose immersion following tail transection. We observed accelerated lipid accumulation in glucose-injected larvae challenged with high fat chicken egg yolk feeding. Our study identifies conserved and divergent effects of high glucose on inflammation, haemostasis, and hyperlipidaemia in zebrafish larvae compared to mammals.

Cystic fibrosis (CF) is a genetic disorder causing dysfunctional ion transport resulting in accumulation of viscous mucus that fosters chronic bacterial biofilm-associated infection in the airways. Achromobacter xylosoxidans and Stenotrophomonas maltophilia are increasingly prevalent CF pathogens and while Burkholderia cencocepacia is slowly decreasing, all are complicated by multidrug resistance that is enhanced by biofilm formation. This study investigates potential synergy between the antibiotics ciprofloxacin (0.5–128 µg/mL), colistin (0.5–128 µg/mL) and tobramycin (0.5–128 µg/mL) when combined with the neutral pH form of N-Acetylcysteine (NACneutral) (0.5–16.3 mg/mL) against 11 cystic fibrosis strains of Burkholderia, Stenotrophomonas and Achromobacter sp. in planktonic and biofilm cultures. We screened for potential synergism using checkerboard assays from which fraction inhibitory concentration indices (FICI) were calculated. Synergistic (FICI ≤ 0.5) and additive (0.5 &gt, FICI ≥ 1) combinations were tested on irreversibly attached bacteria and 48 h mature biofilms via time-course and colony forming units (CFU/mL) assays. This study suggests that planktonic FICI analysis does not necessarily translate to reduction in bacterial loads in a biofilm model. Future directions include refining synergy testing and determining further mechanisms of action of NAC to understand how it may interact with antibiotics to better predict synergy.

Currently available vaccines fail to provide consistent protection against tuberculosis (TB). New, improved vaccines are urgently needed for controlling the disease. The mycobacterial antigen fusions H4 (Ag85B-TB10.4) and H28 (Ag85B-TB10.4-Rv2660c) have been shown to be very immunogenic and have been considered as potential candidates for TB vaccine development. However, soluble protein vaccines are often poorly immunogenic, but augmented immune responses can be induced when selected antigens are delivered in particulate form. This study investigated whether the mycobacterial antigen fusions H4 and H28 can induce protective immunity when assembled into particulate vaccines (polyester nanoparticle-H4, polyester nanoparticle-H28, H4 nanoparticles and H28 nanoparticles). The particulate mycobacterial vaccines were assembled inside an engineered endotoxin-free production strain of Escherichia coli at high yield. Vaccine nanoparticles were purified and induced long-lasting antigen-specific T cell responses and protective immunity in mice challenged by aerosol with virulent Mycobacterium tuberculosis. A significant reduction of M. tuberculosis CFU, up to 0.7-log10 protection, occurred in the lungs of mice immunized with particulate vaccines in comparison to placebo-vaccinated mice (p &lt, 0.0001). Polyester nanoparticles displaying the mycobacterial antigen fusion H4 induced a similar level of protective immunity in the lung when compared to M. bovis bacillus Calmette-Guérin (BCG), the currently approved TB vaccine. The safe and immunogenic polyester nanoparticle-H4 vaccine is a promising subunit vaccine candidate, as it can be cost-effectively manufactured and efficiently induces protection against TB.

Pathogenic mycobacteria actively dysregulate protective host immune signalling pathways during infection to drive the formation of permissive granuloma microenvironments. Dynamic regulation of host microRNA (miRNA) expression is a conserved feature of mycobacterial infections across host-pathogen pairings. Here we examine the role of miR-206 in the zebrafish model of Mycobacterium marinum infection, which allows investigation of the early stages of granuloma formation. We find miR-206 is upregulated following infection by pathogenic M. marinum and that antagomir-mediated knockdown of miR-206 is protective against infection. We observed striking upregulation of cxcl12a and cxcr4b in infected miR-206 knockdown zebrafish embryos and live imaging revealed enhanced recruitment of neutrophils to sites of infection. We used CRISPR/Cas9-mediated knockdown of cxcl12a and cxcr4b expression and AMD3100 inhibition of Cxcr4 to show that the enhanced neutrophil response and reduced bacterial burden caused by miR-206 knockdown was dependent on the Cxcl12/Cxcr4 signalling axis. Together, our data illustrate a pathway through which pathogenic mycobacteria induce host miR-206 expression to suppress Cxcl12/Cxcr4 signalling and prevent protective neutrophil recruitment to granulomas., Author summary Mycobacterial infections cause significant disease burden to humans and animals, the most widely known example being tuberculosis which has killed more humans than any other infectious disease throughout history. Infectious mycobacteria are highly evolved to hijack host processes, including the very immune cells tasked with destroying them. MicroRNAs are host molecules that control wide-ranging programs of host gene expression and are important in the immune response to infections. Here we use the zebrafish model of mycobacterial infection to determine the role of the infection-induced microRNA miR-206 in the host response to infection. We found pathogenic mycobacteria trigger the host to produce more miR-206 in order to suppress the otherwise protective recruitment of neutrophils to sites of infection via the host Cxcl12/Cxcr4 signalling pathway. Our study provides new insight into the role of mycobacterial infection-induced miR-206 function in the context of a whole host.

The current Mycobacterium bovis BCG vaccine provides inconsistent protection against pulmonary infection with Mycobacterium tuberculosis. Immunity induced by subcutaneous immunization with BCG wanes and does not promote early recruitment of T cell to the lungs after M. tuberculosis infection. Delivery of Tuberculosis (TB) vaccines to the lungs may increase and prolong immunity at the primary site of M. tuberculosis infection. Pulmonary immunization with recombinant influenza A viruses (rIAVs) expressing an immune-dominant M. tuberculosis CD4+ T cell epitope (PR8-p25 and X31-p25) stimulates protective immunity against lung TB infection. Here, we investigated the potential use of rIAVs to improve the efficacy of BCG using simultaneous immunization (SIM) and prime-boost strategies. SIM with parenteral BCG and intranasal PR8-p25 resulted in equivalent protection to BCG alone against early, acute and chronic M. tuberculosis infection. Boosting BCG with rIAVs increased the frequency of IFN-γ-secreting specific T cells (p+ T cells (pagainst M. tuberculosis compared to BCG alone. Therefore, sequential pulmonary immunization with these rIAVs after BCG increased M. tuberculosis-specific memory T cell responses in the lung, but not protection against M. tuberculosis infection.

Objectives Non‐sputum‐based tests to accurately identify active tuberculosis (TB) disease and monitor response to therapy are urgently needed. This study examined the biomarker capacity of a panel of plasma proteins alone, and in conjunction with a previously identified miRNA signature, to identify active TB disease. Methods The expression of nine proteins (IP‐10, MCP‐1, sTNFR1, RANTES, VEGF, IL‐6, IL‐10, TNF and Eotaxin) was measured in the plasma of 100 control subjects and 100 TB patients, at diagnosis (treatment naïve) and over the course of treatment (1‐, 2‐ and 6‐month intervals). The diagnostic performance of the nine proteins alone, and with the miRNA, was assessed. Results Six proteins were significantly up‐regulated in the plasma of TB patients at diagnosis compared to controls. Receiver operator characteristic curve analysis demonstrated that IP‐10 with an AUC = 0.874, sensitivity of 75% and specificity of 87% was the best single biomarker candidate to distinguish TB patients from controls. IP‐10 and IL‐6 levels fell significantly within one month of commencing treatment and may have potential as indicators of a positive response to therapy. The combined protein and miRNA panel gave an AUC of 1.00. A smaller panel of only five analytes (IP‐10, miR‐29a, miR‐146a, miR‐99b and miR‐221) showed an AUC = 0.995, sensitivity of 96% and specificity of 97%. Conclusions A novel combination of miRNA and proteins significantly improves the sensitivity and specificity as a biosignature over single biomarker candidates and may be useful for the development of a non‐sputum test to aid the diagnosis of active TB disease., The biomarker potential of plasma proteins to identify tuberculosis (TB) patients was examined. Six proteins were significantly elevated in TB patients with IP‐10 and IL‐6 declining with treatment. In conjunction with miRNA previously detected, a five analyte biosignature could identify TB patients with an AUC = 0.995, sensitivity of 96% and specificity of 97%. IP‐10 was the best single biomarker candidate, showing utility as a triage tool, to quickly and easily identify potential TB patients and monitor their response to therapy.

Short-chain fatty acids (SCFAs) are produced by microbial fermentation of dietary fiber in the gut. Butyrate is a particularly important SCFA with anti-inflammatory properties and is generally present at lower levels in inflammatory diseases associated with gut microbiota dysbiosis in mammals. We aimed to determine if SCFAs are produced by the zebrafish microbiome and if SCFAs exert conserved effects on zebrafish immunity as an example of the non-mammalian vertebrate immune system. We demonstrate that bacterial communities from adult zebrafish intestines synthesize all three main SCFA in vitro, although SCFA were below our detectable limits in zebrafish intestines in vivo. Immersion in butyrate, but not acetate or propionate, reduced the recruitment of neutrophils and M1-type pro-inflammatory macrophages to wounds. We found conservation of butyrate sensing by neutrophils via orthologs of the hydroxycarboxylic acid receptor 1 (hcar1) gene. Neutrophils from Hcar1-depleted embryos were no longer responsive to the anti-inflammatory effects of butyrate, while macrophage sensitivity to butyrate was independent of Hcar1. Our data demonstrate conservation of anti-inflammatory butyrate effects and identify the presence of a conserved molecular receptor in fish.

The development of effective vaccines against bacterial lung infections requires the induction of protective, pathogen-specific immune responses without deleterious inflammation within the pulmonary environment. Here, we made use of a polysaccharide-adjuvanted vaccine approach to elicit resident pulmonary T cells to protect against aerosol Mycobacterium tuberculosis infection. Intratracheal administration of the multistage fusion protein CysVac2 and the delta-inulin adjuvant Advax™ (formulated with a TLR9 agonist) provided superior protection against aerosol M. tuberculosis infection in mice, compared to parenteral delivery. Surprisingly, removal of the TLR9 agonist did not impact vaccine protection despite a reduction in cytokine-secreting T cell subsets, particularly CD4+IFN-γ+IL-2+TNF+ multifunctional T cells. CysVac2/Advax-mediated protection was associated with the induction of lung-resident, antigen-specific memory CD4+ T cells that expressed IL-17 and RORγT, the master transcriptional regulator of Th17 differentiation. IL-17 was identified as a key mediator of vaccine efficacy, with blocking of IL-17 during M. tuberculosis challenge reducing phagocyte influx, suppressing priming of pathogen-specific CD4+ T cells in local lymph nodes and ablating vaccine-induced protection. These findings suggest that tuberculosis vaccines such as CysVac2/Advax that are capable of eliciting Th17 lung-resident memory T cells are promising candidates for progression to human trials.

This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic.; International audience; COVID-19 is causing a major once-in-a-century global pandemic. The scientific and clinical community is in a race to define and develop effective preventions and treatments. The major features of disease are described but clinical trials have been hampered by competing interests, small scale, lack of defined patient cohorts and defined readouts. What is needed now is head-to-head comparison of existing drugs, testing of safety including in the background of predisposing chronic diseases, and the development of new and targeted preventions and treatments. This is most efficiently achieved using representative animal models of primary infection including in the background of chronic disease with validation of findings in primary human cells and tissues. We explore and discuss the diverse animal, cell and tissue models that are being used and developed and collectively recapitulate many critical aspects of disease manifestation in humans to develop and test new preventions and treatments.

BackgroundHuman Papillomavirus (HPV) associated oropharyngeal squamous cell carcinoma (OPSCC) is one of the fastest growing cancers in the Western world. When compared to OPSCCs induced by smoking or alcohol, patients with HPV+ OPSCC, have better survival and the mechanisms remain unclear.MethodsThe Cancer Genome Atlas (TCGA) database was examined for genes associated with tissue-resident CD8+ T cells. Multiplex immunohistochemistry (IHC) staining was performed on tumor specimen taken from 35 HPV+ and 27 HPV- OPSCC patients.ResultsTCGA database revealed that the expression of genes encoding CD103 and CD69 were significantly higher in HPV+ head and neck SCCs (HNSCC) than in HPV- HNSCC. Higher expression levels of these two genes were also associated with better overall survival. IHC staining showed that the proportion of CD103+ tumor-resident CD8+ T cells were significantly higher in HPV+ OPSCCs when compared to HPV- OPSCC. This higher level was also associated with both lower risk of loco-regional failure, and better overall survival. Importantly, patients with HPV- OPSCC who had comparable levels of CD103+ tumor-resident CD8+ T cells to those with HPV+ OPSCC demonstrated similar survival as those with HPV+OPSCC.ConclusionOur results show that CD103+ tumor-resident CD8+ T cells are critical for protective immunity in both types of OPSCCs. Our data further suggest that the enhanced local protective immunity provided by tumor-resident T cell responses is the underlying factor driving favorable clinical outcomes in HPV+ OPSCCs over HPV- OPSCCs.

Type I interferons (IFN), best known for their anti-viral functions, have been shown to impair host resistance to intracellular bacteria in mice. However, the precise role of type I IFN signaling in bacterial infection in humans is unclear. Here, we show that genetic variation in the human IFNAR1 gene is associated with decreased susceptibility to tuberculosis and an increased risk of viral hepatitis in Chinese populations. Receptor mutagenesis and cell signaling studies establish that the IFNAR1 mutation corresponding to a proline deletion in the hinge region of the membrane-proximal domain of IFNAR1 decreases the binding affinity of IFNAR1 to IFN-β, impeding type I IFN signaling. Our findings suggest that IFNAR1 signaling underlies an increased risk of tuberculosis in humans and reveals a function for the IFNAR1 inter-domain region in cytokine–cytokine receptor interaction and signal transduction., The role of type I interferons in bacterial infection is less clear than it is in viral infection. Here, the authors show that genetic variation of the human IFNAR1 gene is associated with decreased susceptibility to tuberculosis and identify a role for the IFNAR1 inter-domain region in the cytokine response.

This study aimed to assess the robustness of using a spray drying approach and formulation design in producing inhalable phage powders. Two types of Pseudomonas phages, PEV2 (Podovirus) and PEV40 (Myovirus) in two formulations containing different amounts of trehalose (70% and 60%) and leucine (30% and 40%) were studied. Most of the surface of the produced powders was found to be covered in crystalline leucine. The powders were stored at 4 °C and 20 °C under vacuum. The phage stability and in vitro aerosol performance of the phage powders were examined on the day of production and after 1, 3 and 12 months of storage. A minor titer loss during production was observed for both phages (0.2–0.8 log10 pfu/ml). The storage stability of the produced phage powders was found to be phage and formulation dependent. No further reduction in titer occurred for PEV2 powders stored at 4 °C across the study. The formulation containing 30% leucine maintained the viability of PEV2 at 20 °C, while the formulation containing 40% leucine gradually lost titer over time with a storage reduction of ∼0.9 log10 pfu/ml measured after 12 months. In comparison, the PEV40 phage powders generally had a ∼ 0.5 log10 pfu/ml loss upon storage regardless of temperature. When aerosolized, the total in vitro lung doses of PEV2 were of the order of 107 pfu, except the formulation containing 40% leucine stored at 20 °C which had a lower lung dose. The PEV40 powders also had lung doses of 106–107 pfu. The results demonstrate that spray dried Myoviridae and Podoviridae phage in a simple formulation of leucine and trehalose can be successfully stored for one year at 4 °C and 20 °C with vacuum packaging. The University of Sydney; Australian Research Council; National Institute of Allergy and Infectious Diseases of the National Institutes of Health; National Health and Medical Research Council Centre of Research Excellence in Tuberculosis Control

© 2015 The Authors. Tuberculosis (TB) remains a major public health issue. New tests to aid diagnoses and monitor the response to therapy are urgently required. There is growing interest in the use of microRNA (miRNA) profiles as diagnostic, prognostic or predictive markers in a range of clinical and infectious diseases, including Mycobacterium tuberculosis infection, however, challenges exist to accurately normalise miRNA levels in cohorts. This study examined the appropriateness of 12 miRs and RNU6B to normalise circulating plasma miRNA levels in individuals with active TB from 2 different geographical and ethnic regions. Twelve miRs (let-7, miR-16, miR-22, miR-26, miR-93, miR-103, miR-191, miR-192, miR-221, miR-423, miR-425 and miR-451) and RNU6B were selected based on their reported production by lung cells, expression in blood and previous use as a reference miRNA. Expression levels were analysed in the plasma of newly diagnosed TB patients from Australia and China compared with individuals with latent TB infection and healthy volunteers. Analysis with both geNorm and NormFinder software identified miR-93 as the most suitable reference miR in both cohorts, either when analysed separately or collectively. Interestingly, there were large variations in the expression levels of some miRs, in particular miR-192 and let-7, between the two cohorts, independent of disease status. These data identify miR-93 is a suitable reference miR for normalizing miRNA levels in TB patients, and highlight how environmental, and possibly ethnic, factors influence miRNA expression levels, demonstrating the necessity of assessing the suitability of reference miRs within the study population.

The total syntheses of the marine-derived lipopeptide natural product fellutamide B and deoxy-fellutamides B, C, and D are reported. These compounds were accessed through a novel solid-phase synthetic strategy using Weinreb amide-derived resin. As part of the synthesis, a new enantioselective route to (3R)-hydroxy lauric acid was developed utilizing a Brown allylation reaction followed by an oxidative cleavage-oxidation sequence as the key steps. The activity of these natural products, and natural product analogues was also assessed against Mycobacterium tuberculosis in vitro.

Disruption of T cell memory during severe immune suppression results in reactivation of chronic viral infections, such as Epstein Barr virus (EBV) and Cytomegalovirus (CMV). How different subsets of memory T cells contribute to the protective immunity against these viruses remains poorly defined. In this study we examined the compartmentalization of virus-specific, tissue resident memory CD8+ T cells in human lymphoid organs. This revealed two distinct populations of memory CD8+ T cells, that were CD69+CD103+ and CD69+CD103—, and were retained within the spleen and tonsils in the absence of recent T cell stimulation. These two types of memory cells were distinct not only in their phenotype and transcriptional profile, but also in their anatomical localization within tonsils and spleen. The EBV-specific, but not CMV-specific, CD8+ memory T cells preferentially accumulated in the tonsils and acquired a phenotype that ensured their retention at the epithelial sites where EBV replicates. In vitro studies revealed that the cytokine IL-15 can potentiate the retention of circulating effector memory CD8+ T cells by down-regulating the expression of sphingosine-1-phosphate receptor, required for T cell exit from tissues, and its transcriptional activator, Kruppel-like factor 2 (KLF2). Within the tonsils the expression of IL-15 was detected in regions where CD8+ T cells localized, further supporting a role for this cytokine in T cell retention. Together this study provides evidence for the compartmentalization of distinct types of resident memory T cells that could contribute to the long-term protection against persisting viral infections., Author Summary Some viruses have the capacity to establish chronic infections in humans. How different T cell populations effectively control these infections has not been clear. Continuous circulation of memory T cells was thought to be crucial for effective immune surveillance against such infections. Recent studies in mice however, have shown that non-circulating tissue resident memory populations can also contribute to protective immunity. In this study we have examined the distribution, localization and characteristics of Epstein-Barr virus and Cytomegalovirus-specific T cells in different human tissues. This showed that virus-specific T cells were differentially distributed in different tissues and there was preferential accumulation of EBV-specific resident memory T cells at sites where EBV reactivates. In vitro studies showed that IL-15 and TGF-β could cooperate to extinguish tissue exit signals in T cells and therefore potentiate their retention within tissues. IL-15 expression was also detected in areas where T cells aggregated within the tissue. Together our study provides insight into how distinct memory T cells are compartmentalized in tissues to maintain long-term protection against persisting viral infections.

BackgroundMore than 120 million doses of BCG vaccine are administered worldwide each year. Most infants are given BCG at birth in accordance with WHO recommendations. However, the effect of the maturing neonatal immune system on the immune response and protection conferred by BCG remains uncertain. Previous studies investigating the influence of age at immunisation on the immune response induced by BCG have reported conflicting results. This study compared BCG given at birth and at two months of age in infants in Australia.MethodsInfants born in Melbourne were randomly allocated to immunisation with BCG-Denmark at birth or two months of age. Ten weeks after immunisation, anti-mycobacterial immune responses were measured in a whole blood assay using intracellular cytokine assays and xMAP multiplex cytokine analysis.ResultsResult from 98 BCG-immunised infants were included in the final analysis. BCG immunisation at birth (n = 54) and at 2 months of age (n = 44) induced comparable proportions of mycobacteria-specific cytokine-producing CD4 and CD8 T cells, as well as comparable proportions of polyfunctional (TNF+ IL-2+ IFN-?+) CD4 T cells. Concentrations of cytokines in supernatants were also similar in both groups.ConclusionsCellular immunity measured 10 weeks after BCG immunisation was similar in infants given BCG at birth and in those given BCG at 2 months of age. Although definitive correlates of protection against TB remain uncertain, these results suggest that delaying BCG immunisation does not confer any immunological advantage in cellular immunity.

Host control of influenza A virus (IAV) is associated with exuberant pulmonary inflammation characterized by the influx of myeloid cells and production of proinflammatory cytokines including interferons (IFNs). It is unclear, however, how the immune system clears the virus without causing lethal immunopathology. Here, we demonstrate that in addition to its known anti-viral activity, STAT1 signaling coordinates host inflammation during IAV infection in mice. This regulatory mechanism is dependent on both type I IFN and IFN-γ receptor signaling and, importantly, requires the functional interplay between the two pathways. The protective function of type I IFNs is associated with not only the recruitment of classical inflammatory Ly6Chi monocytes into IAV-infected lungs, but also the prevention of excessive monocyte activation by IFN-γ. Unexpectedly, type I IFNs preferentially regulate IFN-γ signaling in Ly6Clo rather than inflammatory Ly6Chi mononuclear cell populations. In the absence of type I IFN signaling, Ly6Clo monocytes/macrophages, become phenotypically and functionally more proinflammatory than Ly6Chi cells, revealing an unanticipated function of the Ly6Clo mononuclear cell subset in tissue inflammation. In addition, we show that type I IFNs employ distinct mechanisms to regulate monocyte and neutrophil trafficking. Type I IFN signaling is necessary, but not sufficient, for preventing neutrophil recruitment into the lungs of IAV-infected mice. Instead, the cooperation of type I IFNs and lymphocyte-produced IFN-γ is required to regulate the tissue neutrophilic response to IAV. Our study demonstrates that IFN interplay links innate and adaptive anti-viral immunity to orchestrate tissue inflammation and reveals an additional level of complexity for IFN-dependent regulatory mechanisms that function to prevent excessive immunopathology while preserving anti-microbial functions., Author Summary Influenza A virus (IAV) is a leading cause of respiratory infection and induces a strong acute inflammation manifested by the recruitment of monocytes and neutrophils as well as the production of proinflammatory cytokines in infected lungs. The interferons (IFNs) are strongly induced by IAV and are known to mediate host resistance to the infection. However, in contrast to their well-studied inhibitory effect on viral replication, the effects of IFNs on host inflammatory responses are less well understood. In this manuscript, we demonstrate that anti-viral IFN signaling is also required for the orchestration of a tissue response associated with the protection against IAV infection in mice. Importantly, we identify that type I IFNs cross-regulate and cooperate with IFN-γ to inhibit monocyte activation and neutrophil infiltration, respectively. This study also demonstrates that Ly6Clo monocytes/macrophages can potentially mediate influenza virus-induced inflammation, suggesting that IFNs dictate the homeostasis versus inflammatory function of mononuclear phagocytes in viral infection. Our study reveals a novel IFN-dependent regulatory mechanism designed to prevent the excessive immunopathology while preserving its anti-microbial functions. Moreover, these observations have particular relevance for understanding the mechanisms underlying the strong inflammatory response associated with lethal IAV strains and have implications for the development of new immunotherapies to treat influenza.