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Introduction: Tuberculosis (TB) remains a major health threat and it is now clear that the current vaccine, BCG, is unable to arrest the global TB epidemic. A new vaccine is needed to either replace or boost BCG so that a better level of protection could be achieved. The route of entry of Mycobacterium tuberculosis, the causative organism, is via inhalation making TB primarily a respiratory disease. There is therefore good reason to hypothesize that a mucosally delivered vaccine against TB could be more effective than one delivered via the systemic route. Areas covered: This review summarizes the progress that has been made in the area of TB mucosal vaccines in the last few years. It highlights some of the strengths and shortcomings of the published evidence and aims to discuss immunological and practical considerations in the development of mucosal vaccines. Expert opinion: There is a growing body of evidence that the mucosal approach to vaccination against TB is feasible and should be pursued. However, further key studies are necessary to both improve our understanding of the protective immune mechanisms operating in the mucosa and the technical aspects of aerosolized delivery, before such a vaccine could become a feasible, deployable strategy.

Summary Classical activation of macrophages (M(LPS+IFNγ)) elicits the expression of inducible nitric oxide synthase (iNOS), generating large amounts of NO and inhibiting mitochondrial respiration. Upregulation of glycolysis and a disrupted tricarboxylic acid (TCA) cycle underpin this switch to a pro-inflammatory phenotype. We show that the NOS cofactor tetrahydrobiopterin (BH4) modulates IL-1β production and key aspects of metabolic remodeling in activated murine macrophages via NO production. Using two complementary genetic models, we reveal that NO modulates levels of the essential TCA cycle metabolites citrate and succinate, as well as the inflammatory mediator itaconate. Furthermore, NO regulates macrophage respiratory function via changes in the abundance of critical N-module subunits in Complex I. However, NO-deficient cells can still upregulate glycolysis despite changes in the abundance of glycolytic intermediates and proteins involved in glucose metabolism. Our findings reveal a fundamental role for iNOS-derived NO in regulating metabolic remodeling and cytokine production in the pro-inflammatory macrophage., Graphical Abstract, Highlights • NO orchestrates metabolic remodeling in macrophages responding to LPS+IFNγ • NO regulates itaconate metabolism in two models of infection and inflammation • NO determines Complex I subunit abundance in inflammatory macrophages • Glycolysis is increased in activated NO-deficient cells despite metabolic changes, Metabolic remodeling underpins inflammatory macrophage activation, but the modulatory mechanisms are still being elucidated. Bailey et al. show that NO regulates specific changes in the abundance of TCA cycle metabolites, itaconate, and catalytic subunits of Complex I in the respiratory chain in inflammatory murine macrophages both in vitro and in vivo.

Several thermal indices have been proposed for quantification of various aspects of the thermal environment. Their applicability however is a methodological conundrum. The aim of the present study (part of the Biometeorological Aspect of Thermal environment and Health (BeAT Heat) project) was to assess the outdoor thermal environment using data on thermal sensation. In particular, the study tried to identify the thermal index that best simulates thermal sensation in the temperate and dry Mediterranean climate of Cyprus. Field surveys were conducted in summer 2019 in public urban spaces of five districts in Cyprus. These surveys involved monitoring of micrometeorological conditions and conducting questionnaire-based interviews with pedestrians in order to collect data on actual thermal sensation (ATS), demographics, and physiological and psychological variables. The study participants, using a predefined thermal sensation scale, reported their ATS. Variations of thermal sensation across the participants were studied in relation to the outdoor thermal environment and to physiological factors. ATS was compared with simulated thermal sensation (STS) produced by operationally used thermal indices under matching weather conditions, i.e., Heat Index, Humidex, and Wet-Bulb Globe Temperature. Physiologically Equivalent Temperature was additionally considered. All indices showed low predictability of ATS. The thermal index that performed best to the temperate and dry Mediterranean climate of Cyprus was the Heat Index using an assessment scale modified for the Mediterranean climate. © 2020, Springer-Verlag GmbH Austria, part of Springer Nature.

Immune activation is associated with increased risk of tuberculosis (TB) disease in infants. We performed a case control analysis to identify drivers of immune activation and disease risk. Among 49 infants who developed TB disease over the first two years of life, and 129 matched controls who remained healthy, we found the cytomegalovirus (CMV) stimulated IFNγ response at age 4-6 months to be associated with CD8+ T cell activation (Spearmans rho, P = 6 x 10-8). A CMV specific IFNγ response was also associated with increased risk of developing TB disease (Conditional Logistic Regression, P = 0.043, OR 2.2, 95% CI 1.02-4.83), and shorter time to TB diagnosis (Log Rank Mantel-Cox P = 0.037). CMV positive infants who developed TB disease had lower expression of natural killer cell associated gene signatures and a lower frequency of CD3-CD4-CD8- lymphocytes. We identified transcriptional signatures predictive of risk of TB disease among CMV ELISpot positive (AUROC 0.98, accuracy 92.57%) and negative (AUROC 0.9, accuracy 79.3%) infants; the CMV negative signature validated in an independent infant study (AUROC 0.71, accuracy 63.9%). Understanding and controlling the microbial drivers of T cell activation, such as CMV, could guide new strategies for prevention of TB disease in infants.

BCG, the only vaccine licensed against tuberculosis, demonstrates variable efficacy in humans. Recent preclinical studies highlight the potential for mucosal BCG vaccination to improve protection. Lung tissue-resident memory T cells reside within the parenchyma, potentially playing an important role in protective immunity to tuberculosis. We hypothesised that mucosal BCG vaccination may enhance generation of lung tissue-resident T cells, affording improved protection against Mycobacterium tuberculosis. In a mouse model, mucosal intranasal (IN) BCG vaccination conferred superior protection in the lungs compared to the systemic intradermal (ID) route. Intravascular staining allowed discrimination of lung tissue-resident CD4+ T cells from those in the lung vasculature, revealing that mucosal vaccination resulted in an increased frequency of antigen-specific tissue-resident CD4+ T cells compared to systemic vaccination. Tissue-resident CD4+ T cells induced by mucosal BCG displayed enhanced proliferative capacity compared to lung vascular and splenic CD4+ T cells. Only mucosal BCG induced antigen-specific tissue-resident T cells expressing a PD-1+ KLRG1− cell-surface phenotype. These cells constitute a BCG-induced population which may be responsible for the enhanced protection observed with IN vaccination. We demonstrate that mucosal BCG vaccination significantly improves protection over systemic BCG and this correlates with a novel population of BCG-induced lung tissue-resident CD4+ T cells.

© 2018, The Author(s). A major contribution to the burden of Tuberculosis (TB) comes from latent Mycobacterium tuberculosis infections (LTBI) becoming clinically active. TB and LTBI probably exist as a spectrum and currently there are no correlates available to identify individuals with LTBI most at risk of developing active disease. We set out to identify immune parameters associated with ex vivo mycobacterial growth control among individuals with active TB disease or LTBI to define the spectrum of TB infection. We used a whole blood mycobacterial growth inhibition assay to generate a functional profile of growth control among individuals with TB, LTBI or uninfected controls. We subsequently used a multi-platform approach to identify an immune signature associated with this profile. We show, for the first time, that patients with active disease had the greatest control of mycobacterial growth, whilst there was a continuum of responses among latently infected patients, likely related to the degree of immune activation in response to bacillary load. Control correlated with multiple factors including inflammatory monocytes, activated and atypical memory B cells, IgG1 responses to TB-specific antigens and serum cytokines/chemokines. Our findings offer a method to stratify subclinical TB infections and the future potential to identify individuals most at risk of progressing to active disease and benefit from chemoprophylaxis. Wellcome Trust (grant number 103420/Z/13/Z to M.K.O.) and the British Lung Foundation (grant number BHPG13-2 to M.K.O.)

Tuberculosis (TB) is the biggest cause of human mortality from an infectious disease. The only vaccine currently available, bacille Calmette-Guérin (BCG), demonstrates some protection against disseminated disease in childhood but very variable efficacy against pulmonary disease in adults. A greater understanding of protective host immune responses is required in order to aid the development of improved vaccines. Tissue-resident memory T cells (TRM) are a recently-identified subset of T cells which may represent an important component of protective immunity to TB. Here, we demonstrate that intradermal BCG vaccination induces a population of antigen-specific CD4+ T cells within the lung parenchyma which persist for >12 months post-vaccination. Comprehensive flow cytometric analysis reveals this population is phenotypically and functionally heterogeneous, and shares characteristics with lung vascular and splenic CD4+ T cells. This underlines the importance of utilising the intravascular staining technique for definitive identification of tissue-resident T cells, and also suggests that these anatomically distinct cellular subsets are not necessarily permanently resident within a particular tissue compartment but can migrate between compartments. This lung parenchymal population merits further investigation as a critical component of a protective immune response against Mycobacterium tuberculosis (M. tb).

A replication-deficient chimpanzee adenovirus expressing Ag85A (ChAdOx1.85A) was assessed, both alone and in combination with modified vaccinia Ankara also expressing Ag85A (MVA85A), for its immunogenicity and protective efficacy against a Mycobacterium tuberculosis (M.tb) challenge in mice. Naïve and BCG-primed mice were vaccinated or boosted with ChAdOx1.85A and MVA85A in different combinations. Although intranasally administered ChAdOx1.85A induced strong immune responses in the lungs, it failed to consistently protect against aerosol M.tb challenge. In contrast, ChAdOx1.85A followed by MVA85A administered either mucosally or systemically, induced strong immune responses and was able to improve the protective efficacy of BCG. This vaccination regime has consistently shown superior protection over BCG alone and should be evaluated further.

Vaccines to protect against tuberculosis (TB) are urgently needed. We performed a case–control analysis to identify immune correlates of TB disease risk in Bacille Calmette–Guerin (BCG) immunized infants from the MVA85A efficacy trial. Among 53 TB case infants and 205 matched controls, the frequency of activated HLA-DR+ CD4+ T cells associates with increased TB disease risk (OR=1.828, 95% CI=1.25–2.68, P=0.002, FDR=0.04, conditional logistic regression). In an independent study of Mycobacterium tuberculosis-infected adolescents, activated HLA-DR+ CD4+ T cells also associate with increased TB disease risk (OR=1.387, 95% CI=1.068–1.801, P=0.014, conditional logistic regression). In infants, BCG-specific T cells secreting IFN-γ associate with reduced risk of TB (OR=0.502, 95% CI=0.29–0.86, P=0.013, FDR=0.14). The causes and impact of T-cell activation on disease risk should be considered when designing and testing TB vaccine candidates for these populations., BCG vaccine confers only partial protection against tuberculosis. Here the authors show that the risk of tuberculosis infection and progression to disease in BCG-immunized children positively correlates with the frequency of activated HLA-DR+CD4+ T cells.

The efficacy of Bacillus Calmette-Guerin (BCG) vaccination in protection against pulmonary tuberculosis (TB) is highly variable between populations. One possible explanation for this variability is increased exposure of certain populations to non-tuberculous mycobacteria (NTM). This study used a murine model to determine the effect that exposure to NTM after BCG vaccination had on the efficacy of BCG against aerosol Mycobacterium tuberculosis challenge. The effects of administering live Mycobacterium avium (MA) by an oral route and killed MA by a systemic route on BCG-induced protection were evaluated. CD4+ and CD8+ T cell responses were profiled to define the immunological mechanisms underlying any effect on BCG efficacy. BCG efficacy was enhanced by exposure to killed MA administered by a systemic route; T helper 1 and T helper 17 responses were associated with increased protection. BCG efficacy was reduced by exposure to live MA administered by the oral route; T helper 2 cells were associated with reduced protection. These findings demonstrate that exposure to NTM can induce opposite effects on BCG efficacy depending on route of exposure and viability of NTM. A reproducible model of NTM exposure would be valuable in the evaluation of novel TB vaccine candidates. © 2014 Elsevier Ltd. All rights reserved.

The regulation of the inhibitor of nuclear factor kappa B (I kappa B) by interleukin 1 (IL1) was investigated in HeLa cells. Two forms of I kappa B were resolved by ion-exchange chromatography. The major form (75%) was identified as MAD3 by specific antisera. IL1 generated rapidly (6 min) an electrophoretically retarded form of MAD3 that was stable in acid and was converted into the unmodified form by phosphatase 2A. It thus corresponded to a phosphorylation of the protein on serine or threonine. IL1 also caused the disappearance of MAD3 from the cells, which was complete 15 min after stimulation and coincided with a 46% reduction of cellular I kappa B activity. Newly-synthesized MAD3 accumulated to pre-stimulation levels between 60 and 90 min after stimulation and this coincided with the down-regulation of the phosphorylating activity. The serine proteinase inhibitors 3,4-dichloroisocoumarin (DCI) and tosylphenylalanyl chloromethylketone (TPCK) prevented phosphorylation and disappearance of MAD3. At the same concentrations (10-100 microM), they also increased basal phosphorylation of the small heat shock protein (hsp27) and prevented the IL1- and phorbol 12-myristate 13-acetate-induced increases of its phosphorylation. The inhibitors were thus interfering with protein kinases when blocking degradation of MAD3. Recombinant MAD3 phosphorylated in vitro by protein kinase C was not electrophoretically retarded, suggesting that MAD3 was phosphorylated by another kinase in IL1-stimulated cells. Our results suggest that the IL1-induced phosphorylation of MAD3 on serine or threonine leads to its degradation. DCI and TPCK blocked phosphorylation mechanisms and it could not be concluded that serine proteinases were involved in the breakdown of MAD3.

The development of a vaccine against tuberculosis (TB), a disease caused by Mycobacterium tuberculosis, is urgently needed. The only currently available vaccine, M. bovis BCG, has variable efficacy. One approach in the global vaccine development effort is focused on boosting BCG using subunit vaccines. The identification of novel antigens for inclusion in subunit vaccines is a critical step in the TB vaccine development pathway. We selected four novel mycobacterial antigens recognized during the course of human infection. A replication-deficient chimpanzee adenovirus (ChAdOx1) was constructed to express each antigen individually, and these vectors were evaluated for protective efficacy in murine M. tuberculosis challenge experiments. One antigen, PPE15 (Rv1039c), conferred significant and reproducible protection when administered alone and as a boost to BCG vaccination. We identified immunodominant epitopes to define the protective immune responses using tetramers and intravascular staining. Lung parenchymal CD4+ and CD8+ CXCR3+ KLRG1− T cells, previously associated with protection against M. tuberculosis, were enriched in the vaccinated groups compared to the control groups. Further work to evaluate the protective efficacy of PPE15 in more stringent preclinical animal models, together with the identification of further novel protective antigens using this selection strategy, is now merited.

This study describes experiments that compare the proteoglycans (PGs) extracted from the dialysate from patients receiving continuous peritoneal ambulatory dialysis (CAPD) with those secreted by metabolically labeled human peritoneal mesothelial cells in vitro. The PGs isolated from both sources were predominantly small chondroitin sulfate/dermatan sulfate PGs. Western blot of the core proteins obtained after chondroitin ABC lyase treatment with specific antibodies identified decorin and biglycan. With [35S]sulfate and [35S]methionine as labeling precursors it was shown that dermatan sulfate rather than chondroitin sulfate were the major glycosaminoglycan chains and that decorin was the predominant species. These data provide the first evidence that human peritoneal mesothelial cells may be the principal source of PGs in the peritoneum. Given the proposed functions of decorin and biglycan, the results suggest that these PGs may be involved in the control of transforming growth factor-beta activity and collagen fibril formation in the peritoneum.

A vaccine against tuberculosis (TB), a disease resulting after infection with Mycobacterium tuberculosis (M.tb), is urgently needed to prevent more than a million deaths per year. Bacillus Calmette–Guérin (BCG) is the only available vaccine against TB but its efficacy is variable throughout the world. A promising vaccination regimen composed of BCG prime, followed by vaccination with a chimpanzee adenoviral vector (ChAdOx1) and a modified vaccinia Ankara virus (MVA), both expressing Ag85A, was identified in our laboratory. This regimen significantly improved BCG efficacy in animal studies. Antibody, effector and memory T cell responses induced by BCG-ChAdOx1.85A-MVA85A (B-C-M), were evaluated, to identify immune correlates of protection. This regimen induced Ag85A-specific IgG and IgA responses as well as cytokine responses in CD4+ and CD8+ T cells, both in the systemic and pulmonary system. To discriminate lymphocytes in the vasculature from lymphocytes in the lung parenchyma, a fluorochrome-conjugated antibody was injected intravascularly before harvesting the lungs. Lung parenchymal Ag85A-specific memory CD4+ T cells that were CXCR3+ KLRG1-, significantly increased in B-C-M immunised mice compared to BCG alone at 4, 8 and 20 weeks post vaccination but the cell number decreased at the latter time point. These memory cells associated with protection against M.tb challenge, which was observed at 4 and 8 weeks but not when mice were challenged 20 weeks post vaccination. These putative T cells may have an important role against M.tb infection and could be a target for designing a protective vaccine. In parallel, six novel M.tb antigens were selected and expressed individually in a ChAdOx1 vector. The immunogenicity and protective efficacy of these vaccines were evaluated. Three antigens were not immunogenic when expressed in viral vectors and DNA. The immunogenic antigens induced cytokine production but failed to confer protection when delivered alone or with B-C-M regimen.