Identification and Evaluation of Novel Protective Antigens for the Development of a Candidate Tuberculosis Subunit Vaccine.

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.

Enhancing the Biological Relevance of Machine Learning Classifiers for Reverse Vaccinology.

Reverse vaccinology (RV) is a bioinformatics approach that can predict antigens with protective potential from the protein coding genomes of bacterial pathogens for subunit vaccine design. RV has become firmly established following the development of the BEXSERO® vaccine against Neisseria meningitidis serogroup B. RV studies have begun to incorporate machine learning (ML) techniques to distinguish bacterial protective antigens (BPAs) from non-BPAs. This research contributes significantly to the RV field by using permutation analysis to demonstrate that a signal for protective antigens can be curated from published data. Furthermore, the effects of the following on an ML approach to RV were also assessed: nested cross-validation, balancing selection of non-BPAs for subcellular localization, increasing the training data, and incorporating greater numbers of protein annotation tools for feature generation. These enhancements yielded a support vector machine (SVM) classifier that could discriminate BPAs (n = 200) from non-BPAs (n = 200) with an area under the curve (AUC) of 0.787. In addition, hierarchical clustering of BPAs revealed that intracellular BPAs clustered separately from extracellular BPAs. However, no immediate benefit was derived when training SVM classifiers on data sets exclusively containing intra- or extracellular BPAs. In conclusion, this work demonstrates that ML classifiers have great utility in RV approaches and will lead to new subunit vaccines in the future.

Human CD68 promoter GFP transgenic mice allow analysis of monocyte to macrophage differentiation in vivo.

The recruitment of monocytes and their differentiation into macrophages at sites of inflammation are key events in determining the outcome of the inflammatory response and initiating the return to tissue homeostasis. To study monocyte trafficking and macrophage differentiation in vivo, we have generated a novel transgenic reporter mouse expressing a green fluorescent protein (GFP) under the control of the human CD68 promoter. CD68-GFP mice express high levels of GFP in both monocyte and embryo-derived tissue resident macrophages in adult animals. The human CD68 promoter drives GFP expression in all CD115(+) monocytes of adult blood, spleen, and bone marrow; we took advantage of this to directly compare the trafficking of bone marrow-derived CD68-GFP monocytes to that of CX3CR1(GFP) monocytes in vivo using a sterile zymosan peritonitis model. Unlike CX3CR1(GFP) monocytes, which downregulate GFP expression on differentiation into macrophages in this model, CD68-GFP monocytes retain high-level GFP expression for 72 hours after differentiation into macrophages, allowing continued cell tracking during resolution of inflammation. In summary, this novel CD68-GFP transgenic reporter mouse line represents a powerful resource for analyzing monocyte mobilization and monocyte trafficking as well as studying the fate of recruited monocytes in models of acute and chronic inflammation.

A new tool for tuberculosis vaccine screening: Ex vivo Mycobacterial Growth Inhibition Assay indicates BCG-mediated protection in a murine model of tuberculosis.

BACKGROUND: In the absence of a validated animal model and/or an immune correlate which predict vaccine-mediated protection, large-scale clinical trials are currently the only option to prove efficacy of new tuberculosis candidate vaccines. Tools to facilitate testing of new tuberculosis (TB) vaccines are therefore urgently needed. METHODS: We present here an optimized ex vivo mycobacterial growth inhibition assay (MGIA) using a murine Mycobacterium tuberculosis infection model. This assay assesses the combined ability of host immune cells to inhibit mycobacterial growth in response to vaccination. C57BL/6 mice were immunized with Bacillus Calmette-Guérin (BCG) and growth inhibition of mycobacteria by splenocytes was assessed. Mice were also challenged with Mycobacterium tuberculosis Erdman, and bacterial burden was assessed in lungs and spleen. RESULTS: Using the growth inhibition assay, we find a reduction in BCG CFU of 0.3-0.8 log10 after co-culture with murine splenocytes from BCG vaccinated versus naïve C57BL/6 mice. BCG vaccination in our hands led to a reduction in bacterial burden after challenge with Mycobacterium tuberculosis of approx. 0.7 log10 CFU in lung and approx. 1 log10 CFU in spleen. This effect was also seen when using Mycobacterium smegmatis as the target of growth inhibition. An increase in mycobacterial numbers was found when splenocytes from interferon gamma-deficient mice were used, compared to wild type controls, indicating that immune mechanisms may also be investigated using this assay. CONCLUSIONS: We believe that the ex vivo mycobacterial growth inhibition assay could be a useful tool to help assess vaccine efficacy in future, alongside other established methods. It could also be a valuable tool for determination of underlying immune mechanisms.

Mucosal delivery of antigen-coated nanoparticles to lungs confers protective immunity against tuberculosis infection in mice.

Mucosal boosting of BCG-immunised individuals with a subunit tuberculosis (TB) vaccine would be highly desirable, considering that the lungs are the principal port of entry for Mycobacterium tuberculosis (MTB) and the site of the primary infection and reactivation. However, the main roadblock for subunit TB vaccine development is the lack of suitable adjuvants that could induce robust local and systemic immune responses. Here, we describe a novel vaccine delivery system that was designed to mimic, in part, the MTB pathogen itself. The surface of yellow carnauba wax nanoparticles was coated with the highly immunogenic Ag85B Ag of MTB and they were directed to the alveolar epithelial surfaces by the incorporation of the heparin-binding hemagglutinin adhesion (HBHA) protein. Our results showed that the i.n. immunisation of BCG-primed BALB/c mice with nanoparticles adsorbed with Ag85B-HBHA (Nano-AH vaccine) induced robust humoral and cellular immune responses and IFN-γ production, and multifunctional CD4⁺ T cells expressing IFN-γ, IL-2 and TNF-α. Mice challenged with H37Rv MTB had a significantly reduced bacterial load in their lungs when compared with controls immunised with BCG alone. We therefore conclude that this immunisation approach is an effective means of boosting the BCG-induced anti-TB immunity.

Plant-derived recombinant immune complexes as self-adjuvanting TB immunogens for mucosal boosting of BCG.

Progress with protein-based tuberculosis (TB) vaccines has been limited by poor availability of adjuvants suitable for human application. Here, we developed and tested a novel approach to molecular engineering of adjuvanticity that circumvents the need for exogenous adjuvants. Thus, we generated and expressed in transgenic tobacco plants the recombinant immune complexes (RICs) incorporating the early secreted Ag85B and the latency-associated Acr antigen of Mycobacterium tuberculosis, genetically fused as a single polypeptide to the heavy chain of a monoclonal antibody to Acr. The RICs were formed by virtue of the antibody binding to Acr from adjacent molecules, thus allowing self-polymerization of the complexes. TB-RICs were purified from the plant extracts and shown to be biologically active by demonstrating that they could bind to C1q component of the complement and also to the surface of antigen-presenting cells. Mice immunized with BCG and then boosted with two intranasal immunizations with TB-RICs developed antigen-specific serum IgG antibody responses with mean end-point titres of 1 : 8100 (Acr) and 1 : 24 300 (Ag85B) and their splenocytes responded to in vitro stimulation by producing interferon gamma. 25% of CD4+ proliferating cells simultaneously produced IFN-γ, IL-2 and TNF-α, a phenotype that has been linked with protective immune responses in TB. Importantly, mucosal boosting of BCG-immunized mice with TB-RICs led to a reduced M. tuberculosis infection in their lungs from log10 mean = 5.69 ± 0.1 to 5.04 ± 0.2, which was statistically significant. We therefore propose that the plant-expressed TB-RICs represent a novel molecular platform for developing self-adjuvanting mucosal vaccines.

Inhaled aerosol viral-vectored vaccines against tuberculosis.

Bacille Calmette-Guérin (BCG) remains the sole licensed vaccine against tuberculosis (TB), despite its variable efficacy in protecting against pulmonary TB. The development of effective TB vaccines faces significant challenges, marked by the absence of validated correlates of protection and predictive animal models. Strategic approaches to enhance TB vaccines and augment BCG efficacy include utilising prime-boost strategies with viral-vectored vaccines and exploring innovative delivery techniques, such as mucosal vaccine administration. Viral vectors offer numerous advantages, including the capacity to accommodate genes encoding extensive antigenic fragments and the induction of robust immune responses. Aerosol delivery aligns with the route of Mycobacterium tuberculosis infection and holds the potential to enhance protective mucosal immunity. Aerosolised viral-vectored vaccines overcome anti-vector immunity, facilitating repeated aerosol deliveries.

Safety of a controlled human infection model of tuberculosis with aerosolised, live-attenuated Mycobacterium bovis BCG versus intradermal BCG in BCG-naive adults in the UK: a dose-escalation, randomised, controlled, phase 1 trial.

BACKGROUND: Mycobacterium tuberculosis is the main causative agent of tuberculosis. BCG, the only licensed vaccine, provides inadequate protection against pulmonary tuberculosis. Controlled human infection models are useful tools for vaccine development. We aimed to determine a safe dose of aerosol-inhaled live-attenuated Mycobacterium bovis BCG as a surrogate for M tuberculosis infection, then compare the safety and tolerability of infection models established using aerosol-inhaled and intradermally administered BCG. METHODS: This phase 1 controlled human infection trial was conducted at two clinical research facilities in the UK. Healthy, immunocompetent adults aged 18-50 years, who were both M tuberculosis-naive and BCG-naive and had no history of asthma or other respiratory diseases, were eligible for the trial. Participants were initially enrolled into group 1 (receiving the BCG Danish strain); the trial was subsequently paused because of a worldwide shortage of BCG Danish and, after protocol amendment, was restarted using the BCG Bulgaria strain (group 2). After a dose-escalation study, during which participants were sequentially allocated to receive either 1 × 103, 1 × 104, 1 × 105, 1 × 106, or 1 × 107 colony-forming units (CFU) of aerosol BCG, the maximum tolerated dose was selected for the randomised controlled trial. Participants in this trial were randomly assigned (9:12), by variable block randomisation and using sequentially numbered sealed envelopes, to receive aerosol BCG (1 × 107 CFU) and intradermal saline or intradermal BCG (1 × 106 CFU) and aerosol saline. Participants were masked to treatment allocation until day 14. The primary outcome was to compare the safety of a controlled human infection model based on aerosol-inhaled BCG versus one based on intradermally administered BCG, and the secondary outcome was to evaluate BCG recovery in the airways of participants who received aerosol BCG or skin biopsies of participants who received intradermal BCG. BCG was detected by culture and by PCR. The trial is registered at ClinicalTrials.gov, NCT02709278, and is complete. FINDINGS: Participants were assessed for eligibility between April 7, 2016, and Sept 29, 2018. For group 1, 15 participants were screened, of whom 13 were enrolled and ten completed the study; for group 2, 60 were screened and 33 enrolled, all of whom completed the study. Doses up to 1 × 107 CFU aerosol-inhaled BCG were sufficiently well tolerated. No significant difference was observed in the frequency of adverse events between aerosol and intradermal groups (median percentage of solicited adverse events per participant, post-aerosol vs post-intradermal BCG: systemic 7% [IQR 2-11] vs 4% [1-13], p=0·62; respiratory 7% [1-19] vs 4% [1-9], p=0·56). More severe systemic adverse events occurred in the 2 weeks after aerosol BCG (15 [12%] of 122 reported systemic adverse events) than after intradermal BCG (one [1%] of 94; difference 11% [95% CI 5-17]; p=0·0013), but no difference was observed in the severity of respiratory adverse events (two [1%] of 144 vs zero [0%] of 97; 1% [-1 to 3]; p=0·52). All adverse events after aerosol BCG resolved spontaneously. One serious adverse event was reported-a participant in group 2 was admitted to hospital to receive analgesia for a pre-existing ovarian cyst, which was deemed unrelated to BCG infection. On day 14, BCG was cultured from bronchoalveolar lavage samples after aerosol infection and from skin biopsy samples after intradermal infection. INTERPRETATION: This first-in-human aerosol BCG controlled human infection model was sufficiently well tolerated. Further work will evaluate the utility of this model in assessing vaccine efficacy and identifying potential correlates of protection. FUNDING: Bill & Melinda Gates Foundation, Wellcome Trust, National Institute for Health Research Oxford Biomedical Research Centre, Thames Valley Clinical Research Network, and TBVAC2020.

Safety, tolerability, viral kinetics, and immune correlates of protection in healthy, seropositive UK adults inoculated with SARS-CoV-2: a single-centre, open-label, phase 1 controlled human infection study.

BACKGROUND: A SARS-CoV-2 controlled human infection model (CHIM) has been successfully established in seronegative individuals using a dose of 1×101 50% tissue culture infectious dose (TCID50) pre-alpha SARS-CoV-2 virus. Given the increasing prevalence of seropositivity to SARS-CoV-2, a CHIM that could be used for vaccine development will need to induce infection in those with pre-existing immunity. Our aim was to find a dose of pre-alpha SARS-CoV-2 virus that induced infection in previously infected individuals. METHODS: Healthy, UK volunteers aged 18-30 years, with proven (quantitative RT-PCR or lateral flow antigen test) previous SARS-CoV-2 infection (with or without vaccination) were inoculated intranasally in a stepwise dose escalation CHIM with either 1×101, 1×102, 1×10³, 1×104, or 1×105 TCID50 SARS-CoV-2/human/GBR/484861/2020, the same virus used in the seronegative CHIM. Post-inoculation, volunteers were quarantined in functionally negative pressure rooms (Oxford, UK) for 14 days and until 12-hourly combined oropharyngeal-nasal swabs were negative for viable virus by focus-forming assay. Outpatient follow-up continued for 12 months post-enrolment, with additional visits for those who developed community-acquired SARS-CoV-2 infection. The primary objective was to identify a safe, well tolerated dose that induced infection (defined as two consecutive SARS-CoV-2 positive PCRs starting 24 h after inoculation) in 50% of seropositive volunteers. This study is registered with ClinicalTrials.gov (NCT04864548); enrolment and follow-up to 12 months post-enrolment are complete. FINDINGS: Recruitment commenced on May 6, 2021, with the last volunteer enrolled into the dose escalation cohort on Nov 24, 2022. 36 volunteers were enrolled, with four to eight volunteers inoculated in each dosing group from 1×101 to 1×105 TCID50 SARS-CoV-2. All volunteers have completed quarantine, with follow-up to 12 months complete. Despite dose escalation to 1×105 TCID50, we were unable to induce sustained infection in any volunteers. Five (14%) of 36 volunteers were considered to have transient infection, based on the kinetic of their PCR-positive swabs. Transiently infected volunteers had significantly lower baseline mucosal and systemic SARS-CoV-2-specific antibody titres and significantly lower peripheral IFNγ responses against a CD8+ T-cell SARS-CoV-2 peptide pool than uninfected volunteers. 14 (39%) of 36 volunteers subsequently developed breakthrough infection with the omicron variant after discharge from quarantine. Most adverse events reported by volunteers in quarantine were mild, with fatigue (16 [44%]) and stuffy nose (16 [44%]) being the most common. There were no serious adverse events. INTERPRETATION: Our study demonstrates potent protective immunity induced by homologous vaccination and homologous or heterologous previous SARS-CoV-2 infection. The community breakthrough infections seen with the omicron variant supports the use of newer variants to establish a model with sufficient rate of infection for use in vaccine and therapeutic development. FUNDING: Wellcome Trust and Department for Health and Social Care.

A five-antigen Esx-5a fusion delivered as a prime-boost regimen protects against M.tb challenge.

The development of tuberculosis (TB) vaccines has been hindered by the complex nature of Mycobacterium tuberculosis (M.tb) and the absence of clearly defined immune markers of protection. While Bacillus Calmette-Guerin (BCG) is currently the only licensed TB vaccine, its effectiveness diminishes in adulthood. In our previous research, we identified that boosting BCG with an intranasally administered chimpanzee adenovirus expressing the PPE15 antigen of M.tb (ChAdOx1.PPE15) improved its protection. To enhance the vaccine's efficacy, we combined PPE15 with the other three members of the Esx-5a secretion system and Ag85A into a multi-antigen construct (5Ag). Leveraging the mucosal administration safety of ChAdOx1, we targeted the site of M.tb infection to induce localized mucosal responses, while employing modified vaccinia virus (MVA) to boost systemic immune responses. The combination of these antigens resulted in enhanced BCG protection in both the lungs and spleens of vaccinated mice. These findings provide support for advancing ChAdOx1.5Ag and MVA.5Ag to the next stages of vaccine development.

Episodic memory effects of gamma frequency precuneus transcranial magnetic stimulation in Alzheimer's disease: A randomized multiple baseline study.

Episodic memory decline is the prominent neuropsychological feature of typical Alzheimer's Disease (AD), for which current treatments have a limited clinical response. Recently, gamma entrainment therapy has been used as a non-invasive treatment in AD, providing evidence that it may have the potential to alleviate brain pathology and improve cognitive function in AD patients. At the same time, the precuneus (PC) has been recognized as a key area involved in AD related memory deficits and as a key node of the Default Mode Network. This study aimed to investigate the effectiveness of a 40 Hz Transcranial Magnetic Stimulation (TMS) intervention, delivered bilaterally to the precuneus for 10 days, in improving the patients' episodic memory performance. Secondary outcome variables investigated included general cognitive function, semantic and spatial memory, as well as attention and executive function. A concurrent multiple baseline design across five cases was employed. Four patients completed the study. Visual analysis combined with effect size indices were used to evaluate changes across phases. An increase in the average level of immediate recalled words was observed in three out of four patients. Effect size indices indicated significant improvement of attention skills in two patients. No treatment effect was observed for semantic and visual memory, or for executive function. An immediate treatment effect was observed in all patients' general cognitive function as assessed with the Alzheimer's Disease Assessment Scale (mean reduction of 5 points), which was maintained and improved further three months post-treatment. The neuropsychological evaluations indicated improved performance three months post-treatment in immediate and delayed recall, attention, phonological verbal fluency, anxiety, and neuropsychiatric symptoms. This study provides preliminary evidence for the efficacy of a novel non-pharmacological treatment using gamma-band TMS in addressing cognitive dysfunction in AD.

Molecular Pharming: future targets and aspirations.

Molecular Pharming represents an unprecedented opportunity to manufacture affordable modern medicines and make these available at a global scale. The area of greatest potential is in the prevention of infectious diseases, particular in underdeveloped countries where access to medicines and vaccines has historically been limited. This is why, at St. George's, we focus on diseases such as HIV, TB and rabies, and aim to develop production strategies that are simple and potentially easy to transfer to developing countries.

Using an effective TB vaccination regimen to identify immune responses associated with protection in the murine model.

A vaccine against tuberculosis (TB), a disease resulting from 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 varies throughout the world. Subunit vaccine candidates, based on recombinant viral vectors expressing mycobacterial antigens, are one of the strategies being developed to boost BCG-primed host immune responses and efficacy. A promising vaccination regimen composed of intradermal (i.d.) BCG prime, followed by intranasally (i.n.) administered chimpanzee adenoviral vector (ChAdOx1) and i.n. or i.d. modified vaccinia Ankara virus (MVA), both expressing Ag85A, has been previously reported to significantly improve BCG efficacy in mice. Effector and memory immune responses induced by BCG-ChAdOx1.85A-MVA85A (B-C-M), were evaluated to identify immune correlates of protection in mice. This protective regime induced strong Ag85A-specific cytokine responses in CD4+ and CD8+ T cells, both in the systemic and pulmonary compartments. Lung parenchymal CXCR3+ KLRG1- Ag85A-specific memory CD4+ T cells were significantly increased in B-C-M compared to BCG immunised mice at 4, 8 and 20 weeks post vaccination, but the number of these cells decreased at the latter time point. This cell population was associated with the protective efficacy of this regime and may have an important protective role against M.tb infection.

Cytokine interactions that determine the outcome of Mycobacterial infection of macrophages.

Macrophages are the target cells for mycobacterial infections. They are also largely responsible for intracellular killing of mycobacteria, which is dependent on the cytokine environment. Interferon-gamma (IFN-gamma) is chiefly responsible for macrophage activation and bactericidal capacity while Th2 cytokines have a contrasting effect. However, cytokines rarely act in isolation during an infection. Instead, multiple cytokines, both activating and inhibitory, are present and their concentration levels and mutual interactions are likely to determine the ultimate outcome of an infection. Here, we used an in vitro infection model of mouse macrophages to study the effect of cytokine interactions on the infection with Mycobacterium bovis strain BCG. We measured nitric oxide (NO) production and bacterial survival in cells following stimulation with various combinations of cytokines. The surprising finding was that high concentrations of IL-10 (i.e., above 16.5 ng/ml), which is generally considered to be a macrophage-suppressive cytokine, enhanced IFN-gamma-induced NO production. Furthermore, the simultaneous addition of either of the two Th2 cytokines IL-4 or IL-13, strongly inhibited IFN-gamma-mediated NO production and bacterial killing even at a low concentration of 0.62 ng/ml, but could not reverse the synergistic action of IFN-gamma and TNF-alpha, even when the Th2 cytokines were present at high concentrations (i.e., 50 ng/ml). Therefore, macrophage activity is heavily dependent on the cytokine micro-environment where the final outcome is determined in equal measures by the nature of cytokines present, the timing of their accumulation and their concentration levels.

Identification of antigens presented by MHC for vaccines against tuberculosis.

Mycobacterium tuberculosis (M.tb) is responsible for more deaths globally than any other pathogen. The only available vaccine, bacillus Calmette-Guérin (BCG), has variable efficacy throughout the world. A more effective vaccine is urgently needed. The immune response against tuberculosis relies, at least in part, on CD4+ T cells. Protective vaccines require the induction of antigen-specific CD4+ T cells via mycobacterial peptides presented by MHC class-II in infected macrophages. In order to identify mycobacterial antigens bound to MHC, we have immunoprecipitated MHC class-I and class-II complexes from THP-1 macrophages infected with BCG, purified MHC class-I and MHC class-II peptides and analysed them by liquid chromatography tandem mass spectrometry. We have successfully identified 94 mycobacterial peptides presented by MHC-II and 43 presented by MHC-I, from 76 and 41 antigens, respectively. These antigens were found to be highly expressed in infected macrophages. Gene ontology analysis suggests most of these antigens are associated with membranes and involved in lipid biosynthesis and transport. The sequences of selected peptides were confirmed by spectral match validation and immunogenicity evaluated by IFN-gamma ELISpot against peripheral blood mononuclear cell from volunteers vaccinated with BCG, M.tb latently infected subjects or patients with tuberculosis disease. Three antigens were expressed in viral vectors, and evaluated as vaccine candidates alone or in combination in a murine aerosol M.tb challenge model. When delivered in combination, the three candidate vaccines conferred significant protection in the lungs and spleen compared with BCG alone, demonstrating proof-of-concept for this unbiased approach to identifying new candidate antigens.

Mucosal delivery of tuberculosis vaccines: a review of current approaches and challenges.

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.

Author Correction: The influence of haemoglobin and iron on in vitro mycobacterial growth inhibition assays.

A correction has been published and is appended to both the HTML and PDF versions of this paper. The error has not been fixed in the paper.

Nitric Oxide Modulates Metabolic Remodeling in Inflammatory Macrophages through TCA Cycle Regulation and Itaconate Accumulation.

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-1ß 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.

Cytomegalovirus infection is a risk factor for tuberculosis disease in infants.

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 2 years of life, and 129 healthy matched controls, we found the cytomegalovirus-stimulated (CMV-stimulated) IFN-γ response to be associated with CD8+ T cell activation (Spearman's rho, P = 6 × 10-8). A CMV-specific IFN-γ 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+ infants who developed TB disease had lower expression of NK cell-associated gene signatures and a lower frequency of CD3-CD4-CD8- lymphocytes. We identified transcriptional signatures predictive of TB disease risk among CMV ELISpot-positive (area under the receiver operating characteristic [AUROC], 0.98, accuracy, 92.57%) and -negative (AUROC, 0.9; accuracy, 79.3%) infants; the CMV- signature was validated in an independent infant study (AUROC, 0.71; accuracy, 63.9%). A 16-gene signature that previously identified adolescents at risk of developing TB disease did not accurately classify case and control infants in this study. Understanding the microbial drivers of T cell activation, such as CMV, could guide new strategies for prevention of TB disease in infants.

Induction and maintenance of a phenotypically heterogeneous lung tissue-resident CD4+ T cell population following BCG immunisation.

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).