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Herein, we report the design and synthesis of inhibitors of Mycobacterium tuberculosis (Mtb) phospho-MurNAc-pentapeptide translocase I (MurX), the first membrane-associated step of peptidoglycan synthesis, leveraging the privileged structure of the sansanmycin family of uridylpeptide natural products. A number of analogues bearing hydrophobic amide modifications to the pseudo-peptidic end of the natural product scaffold were generated that exhibited nanomolar inhibitory activity against Mtb MurX and potent activity against Mtb in vitro. We show that a lead analogue bearing an appended neopentylamide moiety possesses rapid antimycobacterial effects with a profile similar to the frontline tuberculosis drug isoniazid. This molecule was also capable of inhibiting Mtb growth in macrophages where mycobacteria reside in vivo and reduced mycobacterial burden in an in vivo zebrafish model of tuberculosis.

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.

Macrophages display marked plasticity with functions in both inflammation and tissue repair. Evidence demonstrates that this spectrum of macrophage phenotypes is influenced by their local microenvironment and tissue origin. However, in vitro macrophage experiments often do not or cannot readily use macrophages from the most relevant tissue of origin. This study investigated if the origin of two C57BL/6 mouse macrophage cell lines of alveolar (AMJ2-C11) and peritoneal (IC-21) origin may influence their response to mycobacterial infection. Both cell lines equally controlled the growth of Mycobacterium bovis BCG and Mycobacterium tuberculosis, although the expression of all proinflammatory cytokines and chemokines measured (TNF, IL-6, MCP-1, MIP-1α, MIP-1β, and RANTES) was significantly higher in AMJ2-C11 cells than in IC-21 cells. During M. tuberculosis infection, IL-6, MCP-1, and RANTES expression increased 5-fold, and MIP-1β expression increased 30-fold. Additionally, AMJ2-C11 cells exhibited significantly higher inducible nitric oxide synthase activity than IC-21 cells, indicative of a more polarized M1 response. The expression of multiple surface markers was also assessed by flow cytometry. CD80 and CD86 were significantly upregulated in AMJ2-C11 cells and downregulated in IC-21 cells during M. tuberculosis infection. The results support the notion that the origin of tissue-resident macrophages influences their phenotype and antimicrobial response and demonstrate hereto unrecognized potential for these cell lines in in vitro studies.

Regulatory T cells (Tregs) play a critical role in immune regulation and peripheral tolerance. While different types of Tregs have been identified in both mice and humans, much of our understanding about how these cells maintain immune homeostasis is derived from animal models. In this study, we examined two distinct human lymphoid organs to understand how repeated exposure to infections at the mucosal surface influences the phenotype and tissue localization of Tregs. We show that while Tregs in both tonsils and spleen express a tissue-resident phenotype, they accumulate in greater numbers in tonsils. Tonsillar-resident Tregs exhibit a highly suppressive phenotype with significantly increased expression of CD39, ICOS and CTLA-4 compared with their counterparts in circulation or in the spleen. Functionally, resident Tregs are able effectively to suppress T cell proliferation. We further demonstrate that tonsillar-resident Tregs share key features of T follicular helper cells. Spatial analysis reveals that the vast majority of resident Tregs are localized at the border of the T-zone and B cell follicle, as well as within the lymphocyte pockets enriched with resident memory T cells. Together our findings suggest that resident Tregs are strategically co-localized to maintain immune homeostasis at sites of recurrent inflammation.

The development of safe and effective adjuvants is a critical goal of vaccine development programs. In this report, we defined the immunostimulatory profile and protective effect against aerosol Mycobacterium tuberculosis infection of vaccine formulations incorporating the semi-crystalline adjuvant δ-inulin (Advax). Advax formulated with CpG oligonucleotide and the QS-21 saponin (AdvaxCpQS) was the most effective combination, demonstrated by the capacity of CysVac2/AdvaxCpQS to significantly reduce the bacterial burden in the lungs of M. tuberculosis-infected mice. CysVac2/AdvaxCpQS protection was associated with rapid influx of neutrophils, macrophages and monocytes to the site of vaccination and the induction of antigen-specific IFN-γ+/IL-2+/TNF+ polyfunctional CD4+ T cells in the lung. When compared to the highly potent adjuvant combination of monophosphoryl lipid A and dimethyldioctadecylammonium bromide (MPL/DDA), AdvaxCpQS imparted a similar level of protective efficacy yet without the profound stimulation of inflammatory cytokines and vaccination site ulceration observed with MPL/DDA. Addition of DDA to CysVac2/AdvaxCpQS further improved the protective effect of the vaccine, which correlated with increased polyfunctional CD4+ T cells in the lung but with no increase in vaccine reactogenicity. The data demonstrate that Advax formulations can decouple protective tuberculosis immunity from reactogenicity, making them ideal candidates for human application.

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.

Combination treatment using bacteriophage and antibiotics is potentially an advanced approach to combatting antimicrobial-resistant bacterial infections. We have recently developed an inhalable powder by co-spray drying Pseudomonas phage PEV20 with ciprofloxacin. The purpose of this study was to assess the in vivo effect of the powder using a neutropenic mouse model of acute lung infection. The synergistic activity of PEV20 and ciprofloxacin was investigated by infecting mice with P. aeruginosa, then administering freshly spray-dried single PEV20 (10(6) PFU/mg), single ciprofloxacin (0.33 mg/mg) or combined PEV20-ciprofloxacin treatment using a dry powder insufflator. Lung tissues were then harvested for colony counting and flow cytometry analysis at 24 h post-treatment. PEV20 and ciprofloxacin combination powder significantly reduced the bacterial load of clinical P. aeruginosa strain in mouse lungs by 5.9 log(10) (p < 0.005). No obvious reduction in the bacterial load was observed when the animals were treated only with PEV20 or ciprofloxacin. Assessment of immunological responses in the lungs showed reduced inflammation associating with the bactericidal effect of the PEV20-ciprofloxacin powder. In conclusion, this study has demonstrated the synergistic potential of using the combination PEV20-ciprofloxacin powder for P. aeruginosa respiratory infections.

Mycobacterium abscessus infections are of increasing global prevalence and are often difficult to treat due to complex antibiotic resistance profiles. While there are similarities between the pathogenesis of M. abscessus and tuberculous mycobacteria, including granuloma formation and stromal remodeling, there are distinct molecular differences at the host-pathogen interface. Here we have used a zebrafish-M. abscessus model and host-directed therapies that were previously identified in the zebrafish-M. marinum model to identify potential host-directed therapies against M. abscessus infection. We find efficacy of anti-angiogenic and vascular normalizing therapies against rough M. abscessus infection, but no effect of anti-platelet drugs.

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.

Regulation of host microRNA (miRNA) expression is a contested node that controls the host immune response to mycobacterial infection. The host must overcome concerted subversive efforts of pathogenic mycobacteria to launch and maintain a protective immune response. Here we examine the role of miR-126 in the zebrafish model of Mycobacterium marinum infection and identify a protective role for this infection-induced miRNA through multiple effector pathways. Specifically, we analyse the impact of the miR-126 knockdown-induced tsc1a and cxcl12a/ccl2/ccr2 signalling axes during early host-M. marinum interactions. We find a strong detrimental effect of tsc1a upregulation that renders zebrafish embryos susceptible to higher bacterial burden and increased cell death despite dramatically higher recruitment of macrophages to the site of infection. We demonstrate that infection-induced miR-126 suppresses tsc1 and cxcl12a expression thus improving macrophage function early in infection, partially through activation of mTOR signalling and strongly through preventing the recruitment of Ccr2+ permissive macrophages, resulting in the recruitment of protective tnfa-expressing macrophages. Together our results demonstrate an important role for infection-induced miR-126 in shaping an effective immune response to M. marinum infection in zebrafish embryos.

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.

Gut dysbiosis is an important modifier of pathologies including cardiovascular disease but our understanding of the role of individual microbes is limited. Here, we have used transplantation of mouse microbiota into microbiota-deficient zebrafish larvae to study the interaction between members of a mammalian high fat diet-associated gut microbiota with a lipid rich diet challenge in a tractable model species. We find zebrafish larvae are more susceptible to hyperlipidaemia when exposed to the mouse high fat-diet-associated microbiota and that this effect can be driven by two individual bacterial species fractionated from the mouse high fat-diet-associated microbiota. We find Stenotrophomonas maltophilia increases the hyperlipidaemic potential of chicken egg yolk to zebrafish larvae independent of direct interaction between S. maltophilia and the zebrafish host. Colonisation by live, or exposure to heat-killed, Enterococcus faecalis accelerates hyperlipidaemia via host Myd88 signalling. The hyperlipidaemic effect is replicated by exposure to the Gram positive Toll-like receptor agonists peptidoglycan and lipoteichoic acid in a MyD88-dependent manner. In this work, we demonstrate the applicability of zebrafish as a tractable host for the identification of gut microbes that can induce conditional host phenotypes via microbiota transplantation and subsequent challenge with a high fat diet.

Cigarette smoke (CS)-induced inflammation leads to a range of diseases including chronic obstructive pulmonary disease and cancer. The gut microbiota is a major modifying environmental factor that determine the severity of cigarette smoke-induced pathology. Microbiomes and metabolites from CS-exposed mice exacerbate lung inflammation via the gut-lung axis of shared mucosal immunity in mice but these systems are expensive to establish and analyse. Zebrafish embryos and larvae have been used to model the effects of cigarette smoking on a range of physiological processes and offer an amenable platform for screening modifiers of cigarette smoke-induced pathologies with key features of low cost and rapid visual readouts. Here we exposed zebrafish larvae to cigarette smoke extract (CSE) and characterised a CSE-induced leukocytic inflammatory phenotype with increased neutrophilic and macrophage inflammation in the gut. The CSE-induced phenotype was exacerbated by co-exposure to microbiota from the faeces of CS-exposed mice, but not control mice. Microbiota could be recovered from the gut of zebrafish and studied in isolation in a screening setting. This demonstrates the utility of the zebrafish-CSE exposure platform for identifying environmental modifiers of cigarette smoking-associated pathology and demonstrates that the CS-exposed mouse gut microbiota potentiates the inflammatory effects of CSE across host species.

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.

Iron homeostasis is essential for both sides of the host-pathogen interface. Restricting access of iron slows bacterial growth while iron is also a necessary co-factor for host immunity. Heme oxygenase 1 (HMOX1) is a critical regulator of iron homeostasis that catalyses the liberation of iron during degradation of heme. It is also a stress-responsive protein that can be rapidly upregulated and confers protection to the host. Although a protective role of HMOX1 has been demonstrated in a variety of diseases, the role of HMOX1 in Mycobacterium tuberculosis infection is equivocal across experiments with different host-pathogen combinations. Here, we use the natural host-pathogen pairing of the zebrafish-Mycobacterium marinum infection platform to study the role of zebrafish heme oxygenase in mycobacterial infection. We identify zebrafish Hmox1a as the relevant functional paralog of mammalian HMOX1 and demonstrate a conserved role for Hmox1a in protecting the host from M. marinum infection. Using genetic and chemical tools, we show zebrafish Hmox1a protects the host against M. marinum infection by reducing infection-induced iron accumulation and ferrostatin-sensitive cell death.

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.

International audience; Tuberculosis remains a leading cause of mortality among infectious diseases worldwide, prompting the need to discover new drugs to fight this disease. We report here the design, synthesis, and antimycobacterial activity of isatin-mono/bis-isoniazid hybrids. Most of the compounds exhibited very high activity against Mycobacterium tuberculosis, with MICs in the range of 0.195 to 0.39 μg/ml, and exerted a more potent bactericidal effect than the standard antitubercular drug isoniazid (INH). Importantly, these compounds were found to be well tolerated at high doses (>200 μg/ml) on Vero kidney cells, leading to high selectivity indices. Two of the most promising hybrids were evaluated for activity in THP-1 macrophages infected with M. tuberculosis, among which compound 11e was found to be slightly more effective than INH. Overexpression of InhA along with cross-resistance determination of the most potent compounds, selection of resistant mutants, and biochemical analysis, allowed us to decipher their mode of action. These compounds effectively inhibited mycolic acid biosynthesis and required KatG to exert their biological effects. Collectively, this suggests that the synthesized isatin-INH hybrids are promising antitubercular molecules for further evaluation in preclinical settings.

Iron homeostasis is essential for both sides of the host-pathogen interface. Restricting access of iron slows bacterial growth while iron is also a necessary co-factor for host immunity. Heme oxygenase 1 (HMOX1) is a critical regulator of iron homeostasis that catalyses the liberation of iron during degradation of heme. It is also a stress-responsive protein that can be rapidly upregulated and confers protection to the host. Although a protective role of HMOX1 has been demonstrated in a variety of diseases, the role of HMOX1 in Mycobacterium tuberculosis infection is equivocal across experiments with different host-pathogen combinations. Here we use the natural host-pathogen pairing of the zebrafish-Mycobacterium marinum infection platform to study the role of zebrafish heme oxygenase in mycobacterial infection. We identify zebrafish Hmox1a as the relevant functional paralog of mammalian HMOX1 and demonstrate a conserved role for Hmox1a in protecting the host from mycobacterial infection. Using genetic and chemical tools, we show zebrafish Hmox1a protects the host against mycobacterial infection by reducing infection-induced iron accumulation and ferroptosis.

Summary Bacterial nutrition is an essential aspect of host–pathogen interaction. For the intracellular pathogen Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis in humans, fatty acids derived from lipid droplets are considered the major carbon source. However, many other soluble nutrients are available inside host cells and may be used as alternative carbon sources. Lactate and pyruvate are abundant in human cells and fluids, particularly during inflammation. In this work, we study Mtb metabolism of lactate and pyruvate combining classic microbial physiology with a ‘multi‐omics’ approach consisting of transposon‐directed insertion site sequencing (TraDIS), RNA‐seq transcriptomics, proteomics and stable isotopic labelling coupled with mass spectrometry‐based metabolomics. We discovered that Mtb is well adapted to use both lactate and pyruvate and that their metabolism requires gluconeogenesis, valine metabolism, the Krebs cycle, the GABA shunt, the glyoxylate shunt and the methylcitrate cycle. The last two pathways are traditionally associated with fatty acid metabolism and, unexpectedly, we found that in Mtb the methylcitrate cycle operates in reverse, to allow optimal metabolism of lactate and pyruvate. Our findings reveal a novel function for the methylcitrate cycle as a direct route for the biosynthesis of propionyl‐CoA, the essential precursor for the biosynthesis of the odd‐chain fatty acids., Mycobacterium tuberculosis assimilates lactate and pyruvate utilising glyoxylate shunt and methylcitrate cycle, two pathways traditionally associated with fatty acid metabolism in bacteria. Surprisingly, we found that M. tuberculosis synthesises propionyl‐CoA by a reverse methylcitrate cycle and another partially characterised (in this study) pathway. Our results point to a re‐interpretation of the mechanistic role and utilisation of these pathways by Mtb during infection, i.e., their potential reversibility and importance during lactate and pyruvate metabolism in vivo.

Tuberculosis (TB) remains a staggering burden on global public health. Novel preventative tools are desperately needed to reach the targets of the WHO post-2015 End-TB Strategy. Peptide or protein-based subunit vaccines offer potential as safe and effective generators of protection, and enhancement of local pulmonary immunity may be achieved by mucosal delivery. We describe the synthesis of a novel subunit vaccine via native chemical ligation. Two immunogenic epitopes, ESAT61−20 and TB10.43−11 from Mycobacterium tuberculosis (Mtb), were covalently conjugated to the TLR2-ligand Pam2Cys to generate a self-adjuvanting lipopeptide vaccine. When administered mucosally to mice, the vaccine enhanced pulmonary immunogenicity, inducing strong Th17 responses in the lungs and multifunctional peripheral T-lymphocytes. Mucosal, but not peripheral vaccination, provided substantial protection against Mtb infection, emphasizing the importance of delivery route for optimal efficacy. NHMRC

Tuberculosis is a chronic inflammatory disease caused by persistent infection with Mycobacterium tuberculosis. The rise of antibiotic resistant strains necessitates the design of novel treatments. Recent evidence shows that not only is M. tuberculosis highly resistant to oxidative killing, it also co-opts host oxidant production to induce phagocyte death facilitating bacterial dissemination. We have targeted this redox environment with the cyclic nitroxide derivative 4-methoxy-TEMPO (MetT) in the zebrafish-M. marinum infection model. MetT inhibited the production of mitochondrial ROS and decreased infection-induced cell death to aid containment of infection. We identify a second mechanism of action whereby stress conditions, including hypoxia, found in the infection microenvironment appear to sensitise M. marinum to killing by MetT both in vitro and in vivo. Together, our study demonstrates MetT inhibited the growth and dissemination of M. marinum through host and bacterial targets. This work was supported by the Australian National Health and Medical Research Council grants APP1099912 and APP1053407; The University of Sydney, Australia Fellowship G197581; NSW Ministry of Health, Australia under the NSW Health Early-Mid Career Fellowships Scheme H18/31086; and the Kenyon Family Inflammation Award (S.H.O.).

The lung is the primary site of infection with the major human pathogen, Mycobacterium tuberculosis. Effective vaccines against M. tuberculosis must stimulate memory T cells to provide early protection in the lung. Recently, tissue-resident memory T cells (TRM) were found to be phenotypically and transcriptional distinct from circulating memory T cells. Here, we identified M. tuberculosis-specific CD4+ T cells induced by recombinant influenza A viruses (rIAV) vaccines expressing M. tuberculosis peptides that persisted in the lung parenchyma with the phenotypic and transcriptional characteristics of TRMs. To determine if these rIAV-induced CD4+ TRM were protective independent of circulating memory T cells, mice previously immunized with the rIAV vaccine were treated with the sphingosine-1-phosphate receptor modulator, FTY720, prior to and during the first 17 days of M. tuberculosis challenge. This markedly reduced circulating T cells, but had no effect on the frequency of M. tuberculosis-specific CD4+ TRMs in the lung parenchyma or their cytokine response to infection. Importantly, mice immunized with the rIAV vaccine were protected against M. tuberculosis infection even when circulating T cells were profoundly depleted by the treatment. Therefore, pulmonary immunization with the rIAV vaccine stimulates lung-resident CD4+ memory T cells that are associated with early protection against tuberculosis infection.

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 global incidence of tuberculosis remains unacceptably high, with new preventative strategies needed to reduce the burden of disease. We describe here a method for the generation of synthetic self-adjuvanted protein vaccines and demonstrate application in vaccination against Mycobacterium tuberculosis. Two vaccine constructs were designed, consisting of full-length ESAT6 protein fused to the TLR2-targeting adjuvants Pam(2)Cys-SK(4) or Pam(3)Cys-SK(4). These were produced by chemical synthesis using a peptide ligation strategy. The synthetic self-adjuvanting vaccines generated powerful local CD4(+) T cell responses against ESAT6 and provided significant protection in the lungs from virulent M. tuberculosis aerosol challenge when administered to the pulmonary mucosa of mice. The flexible synthetic platform we describe, which allows incorporation of adjuvants to multiantigenic vaccines, represents a general approach that can be applied to rapidly assess vaccination strategies in preclinical models for a range of diseases, including against novel pandemic pathogens such as SARS-CoV-2.

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.

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 stabilized, 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 neutralizing 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 neutralized 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.

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.

The quality of T cell responses depends on the lymphocytes’ ability to undergo clonal expansion, acquire effector functions and traffic to the site of infection. Although TCR signal strength is thought to dominantly shape the T cell response, by using TCR transgenic CD4+ T cells with different pMHC binding affinity, we reveal that TCR affinity does not control Th1 effector function acquisition nor the functional output of individual effectors following mycobacterial infection. Rather, TCR affinity calibrates the rate of cell division to synchronize the distinct processes of T cell proliferation, differentiation and trafficking. By timing cell division-dependent IL-12R expression, TCR affinity controls when T cells become receptive to Th1-imprinting IL-12 signals, determining the emergence and magnitude of the Th1 effector pool. These findings reveal a distinct yet cooperative role for IL-12 and TCR signalling in Th1 differentiation and suggests that the temporal activation of clones with different TCR affinity is a major strategy to coordinate immune surveillance against persistent pathogens.

The ohmyungsamycin and ecumicin natural product families are structurally related cyclic depsipeptides that display potent antimycobacterial activity. Herein the total syntheses of ohmyungsamycin A, deoxyecumicin, and ecumicin are reported, together with the direct biological comparison of members of these natural product families against Mycobacterium tuberculosis (Mtb), the etiological agent of tuberculosis (TB). The synthesis of each of the natural products employed a solid-phase strategy to assemble the linear peptide precursor, involving a key on-resin esterification and an optional on-resin dimethylation step, before a final solution-phase macrolactamization between the non-proteinogenic N-methyl-4-methoxy-l-tryptophan amino acid and a bulky N-methyl-l-valine residue. The synthetic natural products possessed potent antimycobacterial activity against Mtb with MIC90 's ranging from 110-360 nm and retained activity against Mtb in Mtb-infected macrophages. Deoxyecumicin also exhibited rapid bactericidal killing against Mtb, sterilizing cultures after 21 days.

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 SCFAin vitro, although SCFA were below our detectable limits in zebrafish intestinesin 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 thehydroxycarboxylic acid receptor 1(hcar1) gene. Neutrophils from Hcar1-depeleted 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.

Infections caused by Mycobacterium abscessus are increasing in prevalence within patient groups with respiratory comorbidities. Initial colonisation by the smooth colony M. abscessus (S) can be followed by an irreversible genetic switch into a highly inflammatory rough colony M. abscessus (R), often associated with a decline in pulmonary function. Our understanding of the role of adaptive immunity in M. abscessus pathogenesis is largely unknown. Here, we have used intraperitoneal infection of adult zebrafish to model M. abscessus pathogenesis in the context of fully functioning host immunity. We find infection with the R variant penetrates host organs causing an inflammatory immune response leading to necrotic granuloma formation within 2 weeks. The R bacilli are targeted by T cell-mediated immunity and burden is constrained. Strikingly, the S variant colonises host internal surfaces at high loads and is met with a robust innate immune response but little T cell-mediated immunity. Invasive granuloma formation is delayed in S variant infection compared to R variant infection upon which T cell-mediated immunity is required to control infection. In mixed infections, the S variant outcompetes the R variant. We also find the R variant activates host immunity to the detriment of S variant M. abscessus in mixed infections. These findings demonstrate the applicability of the adult zebrafish to model persistent M. abscessus infection and provide insight into the immunopathogenesis of chronic M. abscessus infection.

The World Health Organization has set ambitious targets for the global elimination of tuberculosis. However, these targets will not be achieved at the current rate of progress.We performed a cluster-randomized, controlled trial in Ca Mau Province, Vietnam, to evaluate the effectiveness of active community-wide screening, as compared with standard passive case detection alone, for reducing the prevalence of tuberculosis. Persons 15 years of age or older who resided in 60 intervention clusters (subcommunes) were screened for pulmonary tuberculosis, regardless of symptoms, annually for 3 years, beginning in 2014, by means of rapid nucleic acid amplification testing of spontaneously expectorated sputum samples. Active screening was not performed in the 60 control clusters in the first 3 years. The primary outcome, measured in the fourth year, was the prevalence of microbiologically confirmed pulmonary tuberculosis among persons 15 years of age or older. The secondary outcome was the prevalence of tuberculosis infection, as assessed by an interferon gamma release assay in the fourth year, among children born in 2012.In the fourth-year prevalence survey, we tested 42,150 participants in the intervention group and 41,680 participants in the control group. A total of 53 participants in the intervention group (126 per 100,000 population) and 94 participants in the control group (226 per 100,000) had pulmonary tuberculosis, as confirmed by a positive nucleic acid amplification test forThree years of community-wide screening in persons 15 years of age or older who resided in Ca Mau Province, Vietnam, resulted in a lower prevalence of pulmonary tuberculosis in the fourth year than standard passive case detection alone. (Funded by the Australian National Health and Medical Research Council; ACT3 Australian New Zealand Clinical Trials Registry number, ACTRN12614000372684.).

Tuberculosis (TB) infection affects a quarter of the global population resulting in a large burden of TB disease and mortality. The long-term control of TB requires vaccines with greater efficacy and durability than the current Mycobacterium bovis Bacille Calmette-Guerin (BCG). Pulmonary immunization may increase and prolong immunity at the site of Mycobacterium tuberculosis infection. We have investigated recombinant influenza A viruses (rIAVs) expressing the p25 CD4+ T cell epitope of M. tuberculosis Ag85B240-254 for single and sequential immunization against M. tuberculosis infection. Intranasal immunization with single dose of rIAV X31 (H3N2 strain) expressing the p25 epitope (X31-p25), induced p25-specific CD4+ T cells and conferred protection against aerosol delivery of M. tuberculosis infection in the lungs. To enhance this effect, prime-boost immunization with hetero-subtypic rIAVs was examined. Sequential immunization with X31-p25 and a second rIAV, PR8 (H1N1 strain) expressing the same epitope (PR8-p25), increased the frequency of p25-specific IFN-γ T cell responses and polyfunctional CD4+ T cells producing IFN-γ, IL-2, and TNF, compared to immunization with each rIAV alone. This combination resulted in protection against M. tuberculosis in both the lungs and spleen. Therefore, our study revealed that rIAV is not only an efficient vector to induce protective immunity in the lungs, but also has a potential use for sequential immunization with heterologous rIAV to boost the immunogenicity and improve the protection against M. tuberculosis.

Background Active case finding is a top priority for the global control of tuberculosis, but robust evidence for its effectiveness in high-prevalence settings is lacking. We sought to evaluate the effectiveness of household-contact investigation, as compared with standard, passive measures alone, in Vietnam. Methods We performed a cluster-randomized, controlled trial at clinics in 70 districts (local government areas with an average population of approximately 500,000 in urban areas and 100,000 in rural areas) in eight provinces of Vietnam. Health workers at each district clinic or hospital were assigned to perform either household-contact intervention plus standard passive case finding (intervention group) or passive case finding alone (control group). In the intervention districts, household contacts of patients with positive results for tuberculosis on sputum smear microscopy (smear-positive tuberculosis) were invited for clinical assessment and chest radiography at baseline and at 6, 12, and ...

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.

The potential of bacteriophage therapy for the treatment of pulmonary infections caused by antibiotic-resistant bacteria has been well recognised. The purpose of this study was to investigate the effect of excipients on stabilisation and aerosolisation of spray dried powders of morphologically different phages – PEV podovirus and PEV myovirus. Seven anti-pseudomonal phages were screened against 90 clinical strains of bacterial hosts and three of the phages were selected for formulation study based on the host range. Design of experiments was utilised to assess the effect of different excipients on the stabilisation and aerosolisation of spray dried phages. Both podovirus and myovirus phages were stable in spray dried formulations containing trehalose or lactose and leucine as excipients with less than 1-log10 titre reduction during spray drying, with lactose providing superior phage protection over trehalose. Furthermore, the spray dried phage formulations dispersed in an Osmohaler at 85 L/min produced a high fine particle fraction of over 50 %. The results showed that the phages in this study can form respirable dry powder phage formulations using the same excipient composition. Spray dried various types of lytic phages hold significant potential for the treatment of pulmonary infections.

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

To compare titer reduction and delivery rate of active anti-tuberculosis bacteriophage (phage) D29 with three inhalation devices. Phage D29 lysate was amplified to a titer of 11.8 ± 0.3 log10(pfu/mL) and diluted 1:100 in isotonic saline. Filters captured the aerosolized saline D29 preparation emitted from three types of inhalation devices: 1) vibrating mesh nebulizer; 2) jet nebulizer; 3) soft mist inhaler. Full-plate plaque assays, performed in triplicate at multiple dilution levels with the surrogate host Mycobacterium smegmatis, were used to quantify phage titer. Respective titer reductions for the vibrating mesh nebulizer, jet nebulizer, and soft mist inhaler were 0.4 ± 0.1, 3.7 ± 0.1, and 0.6 ± 0.3 log10(pfu/mL). Active phage delivery rate was significantly greater (p

Tuberculosis (TB) remains a prevalent and lethal infectious disease. The glycobiology associated with Mycobacterium tuberculosis infection of frontline alveolar macrophages is still unresolved. Herein, we investigated the regulation of protein N-glycosylation in human macrophages and their secreted microparticles (MPs) used for intercellular communication upon M. tb infection. LC-MS/MS-based proteomics and glycomics were performed to monitor the regulation of glycosylation enzymes and receptors and the N-glycome in in vitro-differentiated macrophages and in isolated MPs upon M. tb infection. Infection promoted a dramatic regulation of the macrophage proteome. Most notably, significant infection-dependent down-regulation (4-26 fold) of 11 lysosomal exoglycosidases, e.g., β-galactosidase, β-hexosaminidases and α-/β-mannosidases, was observed. Relative weak infection-driven transcriptional regulation of these exoglycosidases and a stronger augmentation of the extracellular hexosaminidase activity demonstrated that the lysosome-centric changes may originate predominantly from infection-induced secretion of the lysosomal content. The macrophages showed heterogeneous N-glycan profiles and displayed significant up-regulation of complex-type glycosylation and concomitant down-regulation of paucimannosylation upon infection. Complementary intact N-glycopeptide analysis supported a subcellular-specific manipulation of the glycosylation machinery and altered glycosylation patterns of lysosomal N-glycoproteins within infected macrophages. Interestingly, the corresponding macrophage-derived MPs displayed unique N-glycome and proteome signatures supporting a preferential packaging from plasma membranes. The MPs were devoid of infection-dependent N-glycosylation signatures, but interestingly displayed increased levels of the glyco-initiating oligosaccharyltransferase complex and associated α-glucosidases that correlated with increased formation, N-glycan precursor levels and N-glycan density of infected MPs. In conclusion, this system-wide study provides new insight into the host- and pathogen-driven N-glycoproteome manipulation of macrophages in TB.

Thioridazine is an orally administered antipsychotic drug with potential for treatment of drug-resistant tuberculosis (TB). However, drug-induced adverse cardiac effects have been reported when thioridazine was used at an efficacious oral dose of 200mg/day to treat TB. Pulmonary delivery of thioridazine could be a rational approach to reduce dose-related side effects while enabling high drug concentrations at the primary site of infection. The present study compares in vitro aerosol performance, storage stability, and in vitro antimicrobial activity and cytotoxicity of two inhalable powders composed of thioridazine and a first-line anti-TB drug, rifapentine. Formulation 1 is a combination of amorphous thioridazine and crystalline rifapentine, while Formulation 2 consisted of both drugs as amorphous forms. Both thioridazine-rifapentine formulations were found suitable for inhalation with a total fine particle fraction (

T-lymphocytes are critical for protection against respiratory infections, such as Mycobacterium tuberculosis and influenza virus, with chemokine receptors playing an important role in directing these cells to the lungs. CXCR6 is expressed by activated T-lymphocytes and its ligand, CXCL16, is constitutively expressed by the bronchial epithelia, suggesting a role in T-lymphocyte recruitment and retention. However, it is unknown whether CXCR6 is required in responses to pulmonary infection, particularly on CD4+ T-lymphocytes. Analysis of CXCR6-reporter mice revealed that in naïve mice, lung leukocyte expression of CXCR6 was largely restricted to a small population of T-lymphocytes, but this population was highly upregulated after either infection. Nevertheless, pulmonary infection of CXCR6-deficient mice with M. tuberculosis or recombinant influenza A virus expressing P25 peptide (rIAV-P25), an I-Ab-restricted epitope from the immunodominant mycobacterial antigen, Ag85B, demonstrated that the receptor was redundant for recruitment of T-lymphocytes to the lungs. Interestingly, CXCR6-deficiency resulted in reduced bacterial burden in the lungs 6 weeks after M. tuberculosis infection, and reduced weight loss after rIAV-P25 infection compared to wild type controls. This was paradoxically associated with a decrease in Th1-cytokine responses in the lung parenchyma. Adoptive transfer of P25-specific CXCR6-deficient T-lymphocytes into WT mice revealed that this functional change in Th1-cytokine production was not due to a T-lymphocyte intrinsic mechanism. Moreover, there was no reduction in the number or function of CD4+ and CD8+ tissue resident memory cells in the lungs of CXCR6-deficient mice. Although CXCR6 was not required for T-lymphocyte recruitment or retention in the lungs, CXCR6 influenced the kinetics of the inflammatory response so that deficiency led to increased host control of M. tuberculosis and influenza virus.

Introduction The development of more effective tuberculosis (TB) vaccines is essential for the global control of TB. Recently, there have been major advances in the field, but an important hindrance remains the lack of correlates of protection against TB. This requires each vaccine candidate to undergo clinical efficacy trials based on data from animal protection studies, but the results from animal models do not necessarily predict efficacy in humans. Areas covered In this review we summarize our current knowledge of immune mechanisms that may contribute to protective immunity against TB following vaccination and relate these to protective efficacy in animal models and recent clinical trials. Although some initial trials did not reproduce protection against TB in humans, recent trials have demonstrated promising efficacy for three vaccine approaches. Expert opinion Although CD4+ T lymphocytes are essential for protection against TB, there is no clear correlation between conventional CD4+ or CD8+ T cell responses and protective efficacy of TB vaccines. Recent attention has focused on other immune responses, including donor unrestricted T cells, B lymphocytes, and antibodies. Prospective studies on samples from vaccinated individuals protected in recent trials will allow evaluation of these alternative immune mechanisms as potential correlates of protection.

Summary: Interferons (IFN) are pleiotropic cytokines essential for defense against infection, but the identity and tissue distribution of IFN-responsive cells in vivo are poorly defined. In this study, we generate a mouse strain capable of reporting IFN-signaling activated by all three types of IFNs and investigate the spatio-temporal dynamics and identity of IFN-responding cells following IFN injection and influenza virus infection. Despite ubiquitous expression of IFN receptors, cellular responses to IFNs are highly heterogenous in vivo and are determined by anatomical site, cell type, cellular preference to individual IFNs, and activation status. Unexpectedly, type I and II pneumocytes, the primary target of influenza infection, exhibit striking differences in the strength and temporal dynamics of IFN signaling associated with differential susceptibility to the viral infection. Our findings suggest that time- and cell-type-dependent integration of distinct IFN signals govern the specificity and magnitude of IFN responses in vivo. : Stifter et al. generated an Irgm1 reporter mouse sensitive to induction by all three types of IFNs. They show that cellular responses to IFNs are highly heterogenous in vivo. Furthermore, different types of IFNs act in a cell-type-dependent manner to convey synergistic, antagonistic, or non-redundant signaling during influenza infection. Keywords: interferons, interferon receptor signaling, viral infection, influenza, lung, epithelial cells, Irgm1

Endolysins are bacteriophage-derived protein molecules highly effective for bacterial killing. Cpl-1 and ClyJ-3 are native and chimeric endolysins, respectively, having antimicrobial activity against Streptococcus pneumoniae which causes lung infections. We conducted the first feasibility study on nebulisation of Cpl-1 and ClyJ-3, with a focus on the antimicrobial activity, structural changes of the proteins and aerosol performance. Bacterial colony counts, live cell imaging and Fourier-transform infrared (FTIR) spectroscopy were used to evaluate the proteins before and after jet or vibrating mesh nebulisation. These nebulised aerosols were inhalable with a volume median size of 3.8–4.2 µm (span 1.1–2.3) measured by laser diffraction. How­ever, neb­u­li­sa­tion caused al­most com­plete loss in bioac­tiv­ity of ClyJ-3, which were corroborated with the live cell imaging observation and protein structural damage with a large intensity reduction in the amide absorption bands between 1300 and 1700 cm−1. In contrast, the bactericidal activity of Cpl-1 showed no significant difference (p ≥ 0.05) before and after mesh nebulisation with 4.9 and 4.6-log10 bacterial count reduction, respectively. However, jet nebulisation reduced the bioactivity of Cpl-1 and the effect was time-dependent showing 1.7, 1.0-log10 bacterial count reduction at 7 and 14 min with complete loss of antimicrobial activity at 21 min after nebulisation, respectively. The results were consistent with time-dependent changes in live cell images and FTIR amide band changes at 1655, 1640, 1632 and 1548 cm−1. In conclusion, it is feasible to nebulise endolysins for inhalation delivery but it depends on both the protein and the nebuliser, with the mesh nebuliser being the preferred choice.

Tuberculosis (TB) is an intracellular infectious disease caused by the airborne bacterium, Mycobacterium tuberculosis. Despite considerable research efforts, the treatment of TB continues to be a great challenge in part due to the requirement of prolonged therapy with multiple high-dose drugs and associated side effects. The delivery of pharmacological agents directly to the respiratory system, following the natural route of infection, represents a logical therapeutic approach for treatment or vaccination against TB. Pulmonary delivery is non-invasive, avoids first-pass metabolism in the liver and enables targeting of therapeutic agents to the infection site. Inhaled delivery also potentially reduces the dose requirement and the accompanying side effects. Dry powder is a stable formulation of drug that can be stored without refrigeration compared to liquids and suspensions. The dry powder inhalers are easy to use and suitable for high-dose formulations. This review focuses on the current innovations of inhalable dry powder formulations of drug and vaccine delivery for TB, including the powder production method, preclinical and clinical evaluations of inhaled dry powder over the last decade. Finally, the risks associated with pulmonary therapy are addressed. A novel dry powder formulation with high percentages of respirable particles coupled with a cost effective inhaler device is an appealing platform for TB drug delivery.

The first total synthesis of the cyclic depsipeptide natural product teixobactin is described. Synthesis was achieved by solid-phase peptide synthesis, incorporating the unusual l-allo-enduracididine as a suitably protected synthetic cassette and employing a key on-resin esterification and solution-phase macrolactamization. The synthetic natural product was shown to possess potent antibacterial activity against a range of Gram-positive pathogenic bacteria, including a virulent strain of Mycobacterium tuberculosis and methicillin-resistant Staphylococcus aureus (MRSA).

Purpose The potential of aerosol phage therapy for treating lung infections has been demonstrated in animal models and clinical studies. This work compared the performance of two dry powder formation techniques, spray freeze drying (SFD) and spray drying (SD), in producing inhalable phage powders. Method A Pseudomonas podoviridae phage, PEV2, was incorporated into multi-component formulation systems consisting of trehalose, mannitol and L-leucine (F1 = 60:20:20 and F2 = 40:40:20). The phage titer loss after the SFD and SD processes and in vitro aerosol performance of the produced powders were assessed. Results A significant titer loss (~ 2 log) was noted for droplet generation using an ultrasonic nozzle employed in the SFD method, but the conventional two-fluid nozzle used in the SD method was less destructive for the phage (~0.75 log loss). The phage were more vulnerable during the evaporative drying process (~0.75 log further loss) compared with the freeze drying step, which caused negligible phage loss. In vitro aerosol performance showed that the SFD powders (~80% phage recovery) provided better phage protection than the SD powders (~20% phage recovery) during the aerosolization process. Despite this, higher total lung doses were obtained for the SD formulations (SD-F1 = 13.1 ± 1.7 ×104 pfu and SD-F2 = 11.0 ± 1.4 ×104 pfu) than from their counterpart SFD formulations (SFD-F1 = 8.3 ± 1.8 ×104 pfu and SFD-F2 = 2.1 ± 0.3 ×104 pfu). Conclusion Overall, the SD method caused less phage reduction during the powder formation process and the resulted powders achieved better aerosol performance for PEV2. This work was financially supported by the Australian Research Council (Discovery Project DP150103953). Authors are grateful to Tony Smithyman of Special Phage Services for his valuable discussion and advice. Sharon Leung is a research fellow supported by the University of Sydney. Thaigarajan Parumasivam is a recipient of the Malaysian Government Scholarship.