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The development of a universal influenza vaccine likely requires an understanding of previous exposure to influenza virus (through vaccination or infection) and how that shapes the antibody repertoire to vaccination, sometimes called Original Antigenic Sin or antigenic imprinting. Whilst animal models can have a much more defined exposure history, they lack a human B cell repertoire. Transgenic mice with the complete human immunoglobulin locus enable studies of controlled infection history leading to human-like antibody evolution. Here we evaluated responses to influenza in the Intelliselect Transgenic mouse (the Kymouse). We show the Kymouse is susceptible to disease following infection with either H1N1, H3N2 or B/Yam influenza viruses and that it induces a robust binding and neutralising antibody response to all three strains of influenza virus. This study demonstrates that human B cell repertoire mice can be used for influenza virus studies, providing a tool for further interrogation of the antibody response., (© The Author(s) 2024. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

The use of monoclonal antibodies for the control of drug resistant nosocomial bacteria may alleviate a reliance on broad spectrum antimicrobials for treatment of infection. We identify monoclonal antibodies that may prevent infection caused by carbapenem resistant Acinetobacter baumannii. We use human immune repertoire mice (Kymouse platform mice) as a surrogate for human B cell interrogation to establish an unbiased strategy to probe the antibody-accessible target landscape of clinically relevant A. baumannii. After immunisation of the Kymouse platform mice with A. baumannii derived outer membrane vesicles (OMV) we identify 297 antibodies and analyse 26 of these for functional potential. These antibodies target lipooligosaccharide (OCL1), the Oxa-23 protein, and the KL49 capsular polysaccharide. We identify a single monoclonal antibody (mAb1416) recognising KL49 capsular polysaccharide to demonstrate prophylactic in vivo protection against a carbapenem resistant A. baumannii lineage associated with neonatal sepsis mortality in Asia. Our end-to-end approach identifies functional monoclonal antibodies with prophylactic potential against major lineages of drug resistant bacteria accounting for phylogenetic diversity and clinical relevance without existing knowledge of a specific target antigen. Such an approach might be scaled for a additional clinically important bacterial pathogens in the post-antimicrobial era., (© 2024. The Author(s).)

Antimicrobial resistance (AMR) is a growing public health crisis that requires innovative solutions. Current susceptibility testing approaches limit our ability to rapidly distinguish between antimicrobial-susceptible and -resistant organisms. Salmonella Typhimurium (S. Typhimurium) is an enteric pathogen responsible for severe gastrointestinal illness and invasive disease. Despite widespread resistance, ciprofloxacin remains a common treatment for Salmonella infections, particularly in lower-resource settings, where the drug is given empirically. Here, we exploit high-content imaging to generate deep phenotyping of S. Typhimurium isolates longitudinally exposed to increasing concentrations of ciprofloxacin. We apply machine learning algorithms to the imaging data and demonstrate that individual isolates display distinct growth and morphological characteristics that cluster by time point and susceptibility to ciprofloxacin, which occur independently of ciprofloxacin exposure. Using a further set of S. Typhimurium clinical isolates, we find that machine learning classifiers can accurately predict ciprofloxacin susceptibility without exposure to it or any prior knowledge of resistance phenotype. These results demonstrate the principle of using high-content imaging with machine learning algorithms to predict drug susceptibility of clinical bacterial isolates. This technique may be an important tool in understanding the morphological impact of antimicrobials on the bacterial cell to identify drugs with new modes of action., (© 2024. The Author(s).)

The World Health Organization's aim to end the global tuberculosis (TB) epidemic by 2050 cannot be achieved without taking measures to identify people with asymptomatic Mycobacterium tuberculosis (Mtb) infection and offer them an intervention to reduce the risk of disease progression, such as preventive antimicrobial therapy. Implementation of this strategy is limited by the fact that existing tests for Mtb infection, which use immunosensitization to Mtb-specific antigens as a proxy for infection, have low positive predictive value for progression to TB. A blood test that detects Mtb deoxyribonucleic acid (DNA) could allow preventive therapy to be targeted at individuals with microbiological evidence of persistent infection. In this review, we summarize recent advances in the development of molecular microbial blood tests for Mtb infection and discuss potential explanations for discordance between their results and those of immunodiagnostic tests in adults with recent exposure to an infectious index case. We also present a roadmap for further development of molecular microbial blood tests for Mtb infection, and highlight the potential for research in this area to provide novel insights into the biology of Mtb infection and yield new tools to support efforts to control the global TB epidemic., Competing Interests: Declarations of competing interest STR has a pending patent that is relevant to the work (WO2017207825A1). All other authors have no competing interests to declare., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Objectives: The multidrug-resistant bacteria Acinetobacter baumannii is a major cause of hospital-associated infection; a vaccine could significantly reduce this burden. The aim was to develop a clinically relevant model of A. baumannii respiratory tract infection and to test the impact of different immunization routes on protective immunity provided by an outer membrane vesicle (OMV) vaccine., Methods: BALB/c mice were intranasally challenged with isolates of oxa23-positive global clone GC2 A. baumannii from the lungs of patients with ventilator-associated pneumonia. Mice were immunized with OMVs by the intramuscular, subcutaneous or intranasal routes; protection was determined by measuring local and systemic bacterial load., Results: Infection with A. baumannii clinical isolates led to a more disseminated infection than the prototype A. baumannii strain ATCC17978; with bacteria detectable in upper and lower airways and the spleen. Intramuscular immunization induced an antibody response but did not protect against bacterial infection. However, intranasal immunization significantly reduced airway colonization and prevented systemic bacterial dissemination., Conclusions: Use of clinically relevant isolates of A. baumannii provides stringent model for vaccine development. Intranasal immunization with OMVs was an effective route for providing protection, demonstrating that local immunity is important in preventing A. baumannii infection., Competing Interests: Conflict of interest SR is an employee of Kymab., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: The tuberculin skin test is commonly used to diagnose latent tuberculosis infection (LTBI) in resource-limited settings, but its specificity is limited by factors including cross-reactivity with BCG vaccine and environmental mycobacteria. Interferon-gamma release assays (IGRA) overcome this problem by detecting M. tuberculosis complex-specific responses, but studies to determine risk factors for IGRA-positivity in high TB burden settings are lacking., Methods: We conducted a cross-sectional study to determine factors associated with a positive IGRA by employing the QuantiFERON-TB® Gold-plus (QFT Plus) assay in a cohort of asymptomatic adult TB contacts in Kampala, Uganda. Multivariate logistic regression analysis with forward stepwise logit function was employed to identify independent correlates of QFT Plus-positivity., Results: Of the 202 participants enrolled, 129/202 (64%) were female, 173/202 (86%) had a BCG scar, and 67/202 (33%) were HIV-infected. Overall, 105/192 (54%, 95% CI 0.48-0.62) participants had a positive QFT Plus result. Increased risk of QFT-Plus positivity was independently associated with casual employment/unemployment vs. non-casual employment (adjusted odds ratio (aOR) 2.18, 95% CI 1.01-4.72), a family vs. non-family relation to the index patient (aOR 2.87, 95% CI 1.33-6.18), living in the same vs. a different house as the index (aOR 3.05, 95% CI 1.28-7.29), a higher body mass index (BMI) (aOR per additional kg/m2 1.09, 95% CI 1.00-1.18) and tobacco smoking vs. not (aOR 2.94, 95% CI 1.00-8.60). HIV infection was not associated with QFT-Plus positivity (aOR 0.91, 95% CI 0.42-1.96)., Conclusion: Interferon Gamma Release Assay positivity in this study population was lower than previously estimated. Tobacco smoking and BMI were determinants of IGRA positivity that were previously unappreciated., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Mayito et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Haematopoietic stem cells expressing the CD34 surface marker have been posited as a niche for Mycobacterium tuberculosis complex bacilli during latent tuberculosis infection. Our aim was to determine whether M tuberculosis complex DNA is detectable in CD34-positive peripheral blood mononuclear cells (PBMCs) isolated from asymptomatic adults living in a setting with a high tuberculosis burden., Methods: We did a cross-sectional study in Ethiopia between Nov 22, 2017, and Jan 10, 2019. Digital PCR (dPCR) was used to determine whether M tuberculosis complex DNA was detectable in PBMCs isolated from 100 mL blood taken from asymptomatic adults with HIV infection or a history of recent household or occupational exposure to an index case of human or bovine tuberculosis. Participants were recruited from HIV clinics, tuberculosis clinics, and cattle farms in and around Addis Ababa. A nested prospective study was done in a subset of HIV-infected individuals to evaluate whether administration of isoniazid preventive therapy was effective in clearing M tuberculosis complex DNA from PBMCs. Follow-up was done between July 20, 2018, and Feb 13, 2019. QuantiFERON-TB Gold assays were also done on all baseline and follow-up samples., Findings: Valid dPCR data (ie, droplet counts >10 000 per well) were available for paired CD34-positive and CD34-negative PBMC fractions from 197 (70%) of 284 participants who contributed data to cross-sectional analyses. M tuberculosis complex DNA was detected in PBMCs of 156 of 197 participants with valid dPCR data (79%, 95% CI 74-85). It was more commonly present in CD34-positive than in CD34-negative fractions (154 [73%] of 197 vs 46 [23%] of 197; p<0·0001). Prevalence of dPCR-detected M tuberculosis complex DNA did not differ between QuantiFERON-negative and QuantiFERON-positive participants (77 [78%] of 99 vs 79 [81%] of 98; p=0·73), but it was higher in HIV-infected than in HIV-uninfected participants (67 [89%] of 75 vs 89 [73%] of 122, p=0·0065). By contrast, the proportion of QuantiFERON-positive participants was lower in HIV-infected than in HIV-uninfected participants (25 [33%] of 75 vs 73 [60%] of 122; p<0·0001). Administration of isoniazid preventive therapy reduced the prevalence of dPCR-detected M tuberculosis complex DNA from 41 (95%) of 43 HIV-infected individuals at baseline to 23 (53%) of 43 after treatment (p<0·0001), but it did not affect the prevalence of QuantiFERON positivity (17 [40%] of 43 at baseline vs 13 [30%] of 43 after treatment; p=0·13)., Interpretation: We report a novel molecular microbiological biomarker of latent tuberculosis infection with properties that are distinct from those of a commercial interferon-γ release assay. Our findings implicate the bone marrow as a niche for M tuberculosis in latently infected individuals. Detection of M tuberculosis complex DNA in PBMCs has potential applications in the diagnosis of latent tuberculosis infection, in monitoring response to preventive therapy, and as an outcome measure in clinical trials of interventions to prevent or treat latent tuberculosis infection., Funding: UK Medical Research Council., Competing Interests: STR has a pending patent that is relevant to the work (WO2017207825A1). All other authors declare no competing interests. The views expressed are those of the authors and not necessarily those of the UK Medical Research Council or the UK Department of Health., (© 2021 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 license.)

The increase of antimicrobial resistance (AMR), and lack of new classes of licensed antimicrobials, have made alternative treatment options for AMR pathogens increasingly attractive. Recent studies have demonstrated anti-bacterial efficacy of a humanised monoclonal antibody (mAb) targeting the O25b O-antigen of Escherichia coli ST131. To evaluate the phenotypic effects of antibody binding to diverse clinical E. coli ST131 O25b bacterial isolates in high-throughput, we designed a novel mAb screening method using high-content imaging (HCI) and image-based morphological profiling to screen a mAb targeting the O25b O-antigen. Screening the antibody against a panel of 86 clinical E. coli ST131 O25:H4 isolates revealed 4 binding phenotypes: no binding (18.60%), weak binding (4.65%), strong binding (69.77%) and strong agglutinating binding (6.98%). Impaired antibody binding could be explained by the presence of insertion sequences or mutations in O-antigen or lipopolysaccharide core biosynthesis genes, affecting the amount, structure or chain length of the O-antigen. The agglutinating binding phenotype was linked with lower O-antigen density, enhanced antibody-mediated phagocytosis and increased serum susceptibly. This study highlights the need to screen candidate mAbs against large panels of clinically relevant isolates, and that HCI can be used to evaluate mAb binding affinity and potential functional efficacy against AMR bacteria.

Latent tuberculosis has been recognized for over a century, but discovery of new niches, where Mycobacterium tuberculosis resides, continues. We evaluated literature on M.tuberculosis locations during latency, highlighting that mesenchymal and hematopoietic stem cells harbor organisms in sensitized asymptomatic individuals., (© The Author(s) 2018. Published by Oxford University Press for the Infectious Diseases Society of America.)

Persistence of Mycobacterium tuberculosis within human bone marrow stem cells has been identified as a potential bacterial niche during latent tuberculosis. Using a murine model of tuberculosis, we show here that bone marrow stem and progenitor cells containing M. tuberculosis propagated tuberculosis when transferred to naive mice, given that both transferred cells and recipient mice were unable to express inducible nitric oxide synthase, which mediates killing of intracellular bacteria via nitric oxide. Our findings suggest that bone marrow stem and progenitor cells containing M. tuberculosis propagate hallmarks of disease if nitric oxide-mediated killing of bacteria is defective.

New vaccine platforms are needed to address the time gap between pathogen emergence and vaccine licensure. RNA-based vaccines are an attractive candidate for this role: they are safe, are produced cell free, and can be rapidly generated in response to pathogen emergence. Two RNA vaccine platforms are available: synthetic mRNA molecules encoding only the antigen of interest and self-amplifying RNA (sa-RNA). sa-RNA is virally derived and encodes both the antigen of interest and proteins enabling RNA vaccine replication. Both platforms have been shown to induce an immune response, but it is not clear which approach is optimal. In the current studies, we compared synthetic mRNA and sa-RNA expressing influenza virus hemagglutinin. Both platforms were protective, but equivalent levels of protection were achieved using 1.25 μg sa-RNA compared to 80 μg mRNA (64-fold less material). Having determined that sa-RNA was more effective than mRNA, we tested hemagglutinin from three strains of influenza H1N1, H3N2 (X31), and B (Massachusetts) as sa-RNA vaccines, and all protected against challenge infection. When sa-RNA was combined in a trivalent formulation, it protected against sequential H1N1 and H3N2 challenges. From this we conclude that sa-RNA is a promising platform for vaccines against viral diseases., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

During active TB in humans a spectrum of pulmonary granulomas with central necrosis and hypoxia exists. BALB/c mice, predominantly used in TB drug development, do not reproduce this complex pathology thereby inaccurately predicting clinical outcome. We found that Nos2 -/- mice incapable of NO-production in immune cells as microbial defence uniformly develop hypoxic necrotizing lung lesions, widely observed in human TB. To study the impact of hypoxic necrosis on the efficacy of antimycobacterials and drug candidates, we subjected Nos2 -/- mice with TB to monotherapy before or after establishment of human-like pathology. Isoniazid induced a drug-tolerant persister population only when necrotic lesions were present. Rifapentine was more potent than rifampin prior to development of human-like pathology and equally potent thereafter, in agreement with recent clinical trials. Pretomanid, delamanid and the pre-clinical candidate BTZ043 were bactericidal independent of pulmonary pathology. Linezolid was bacteriostatic in TB-infected Nos2 -/- mice but significantly improved lung pathology. Hypoxic necrotizing lesions rendered moxifloxacin less active. In conclusion, Nos2 -/- mice are a predictive TB drug development tool owing to their consistent development of human-like pathology.

An estimated third of the world's population is latently infected with Mycobacterium tuberculosis (Mtb), with no clinical signs of tuberculosis (TB), but lifelong risk of reactivation to active disease. The niches of persisting bacteria during latent TB infection remain unclear. We detect Mtb DNA in peripheral blood selectively in long-term repopulating pluripotent hematopoietic stem cells (LT-pHSCs) as well as in mesenchymal stem cells from latently infected human donors. In mice infected with low numbers of Mtb, that do not develop active disease we, again, find LT-pHSCs selectively infected with Mtb. In human and mouse LT-pHSCs Mtb are stressed or dormant, non-replicating bacteria. Intratracheal injection of Mtb-infected human and mouse LT-pHSCs into immune-deficient mice resuscitates Mtb to replicating bacteria within the lung, accompanied by signs of active infection. We conclude that LT-pHSCs, together with MSCs of Mtb-infected humans and mice serve as a hitherto unappreciated quiescent cellular depot for Mtb during latent TB infection., Competing Interests: The authors have declared that no competing interests exist.

The neonatal Fc receptor (FcRn) extends the systemic half-life of IgG antibodies by chaperoning bound Fc away from lysosomal degradation inside stromal and hematopoietic cells. FcRn also transports IgG across mucosal barriers into the lumen, and yet little is known about how FcRn modulates immunity in the lung during homeostasis or infection. We infected wild-type (WT) and FcRn-deficient (fcgrt(-/-)) mice with Pseudomonas aeruginosa or Mycobacterium tuberculosis to investigate whether recycling and transport of IgG via FcRn influences innate and adaptive immunity in the lung in response to bacterial infection. We found that FcRn expression maintains homeostatic IgG levels in lung and leads to preferential secretion of low-affinity IgG ligands into the lumen. Fcgrt(-/-) animals exhibited no evidence of developmental impairment of innate immunity in the lung and were able to efficiently recruit neutrophils in a model of acute bacterial pneumonia. Although local humoral immunity in lung increased independently of the presence of FcRn during tuberculosis, there was nonetheless a strong impact of FcRn deficiency on local adaptive immunity. We show that the quantity and quality of IgG in airways, as well as the abundance of dendritic cells in the lung, are maintained by FcRn. FcRn ablation transiently enhanced local T cell immunity and neutrophil recruitment during tuberculosis, leading to a lower bacterial burden in lung. This novel understanding of tissue-specific modulation of mucosal IgG isotypes in the lung by FcRn sheds light on the role of mucosal IgG in immune responses in the lung during homeostasis and bacterial disease., (Copyright © 2016 Vogelzang et al.)

Lung granulomas develop upon Mycobacterium tuberculosis (Mtb) infection as a hallmark of human tuberculosis (TB). They are structured aggregates consisting mainly of Mtb-infected and -uninfected macrophages and Mtb-specific T cells. The production of NO by granuloma macrophages expressing nitric oxide synthase-2 (NOS2) via l-arginine and oxygen is a key protective mechanism against mycobacteria. Despite this protection, TB granulomas are often hypoxic, and bacterial killing via NOS2 in these conditions is likely suboptimal. Arginase-1 (Arg1) also metabolizes l-arginine but does not require oxygen as a substrate and has been shown to regulate NOS2 via substrate competition. However, in other infectious diseases in which granulomas occur, such as leishmaniasis and schistosomiasis, Arg1 plays additional roles such as T-cell regulation and tissue repair that are independent of NOS2 suppression. To address whether Arg1 could perform similar functions in hypoxic regions of TB granulomas, we used a TB murine granuloma model in which NOS2 is absent. Abrogation of Arg1 expression in macrophages in this setting resulted in exacerbated lung granuloma pathology and bacterial burden. Arg1 expression in hypoxic granuloma regions correlated with decreased T-cell proliferation, suggesting that Arg1 regulation of T-cell immunity is involved in disease control. Our data argue that Arg1 plays a central role in the control of TB when NOS2 is rendered ineffective by hypoxia.

General interest in the biological functions of IFN type I in Mycobacterium tuberculosis (Mtb) infection increased after the recent identification of a distinct IFN gene expression signature in tuberculosis (TB) patients. Here, we demonstrate that TB-susceptible mice lacking the receptor for IFN I (IFNAR1) were protected from death upon aerogenic infection with Mtb. Using this experimental model to mimic primary progressive pulmonary TB, we dissected the immune processes affected by IFN I. IFNAR1 signaling did not affect T-cell responses, but markedly altered migration of inflammatory monocytes and neutrophils to the lung. This process was orchestrated by IFNAR1 expressed on both immune and tissue-resident radioresistant cells. IFNAR1-driven TB susceptibility was initiated by augmented Mtb replication and in situ death events, along with CXCL5/CXCL1-driven accumulation of neutrophils in alveoli, followed by the discrete compartmentalization of Mtb in lung phagocytes. Early depletion of neutrophils rescued TB-susceptible mice to levels observed in mice lacking IFNAR1. We conclude that IFN I alters early innate events at the site of Mtb invasion leading to fatal immunopathology. These data furnish a mechanistic explanation for the detrimental role of IFN I in pulmonary TB and form a basis for understanding the complex roles of IFN I in chronic inflammation., (© 2014 The Authors. European Journal of Immunology published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A protective malaria vaccine may induce both high levels of neutralising antibodies and strong T-cell responses. The Plasmodium falciparum circumsporozoite protein (CSp) is a leading pre-erythrocytic vaccine candidate. CSp is a week immunogen per se, but Mycobacterium bovis Bacille Calmette-Guérin (BCG) has excellent adjuvant activity and has been utilized as a vector to deliver heterologous vaccine candidate antigens. It is safe in immunocompetent individuals and inexpensive to produce. We assessed in vitro and in vivo a recombinant BCG-expressing CSp (BCG-CS) as malaria vaccine candidate. Immunisation of BALB/c mice with BCG-CS augmented numbers of dendritic cells (DCs) in draining lymph nodes and in the spleen. The activation markers MHC-class-II, CD40, CD80 and CD86 on DCs were significantly upregulated by BCG-CS as compared to wild-type BCG (wt-BCG). In vitro stimulation of bone marrow-derived DCs and macrophages with BCG-CS induced IL-12 and TNF-α production. BCG-CS induced higher phagocytic activity in macrophages as compared to wt-BCG. Immunogenicity studies show that BCG-CS induced CS-specific antibodies and IFN-γ-producing memory cells. In conclusion, BCG-CS is highly efficient in activating antigen-presenting cells (APCs) for priming of adaptive immunity. Implications for the rational design of novel vaccines against malaria and TB, the two major devastating poverty-related diseases, are discussed., (Copyright © 2012. Published by Elsevier Ltd.)

Background: Sustained antibody levels are a hallmark of immunity against many pathogens, and induction of long-term durable antibody titers is an essential feature of effective vaccines. Heterologous prime-boost approaches with vectors are optimal strategies to improve a broad and prolonged immunogenicity of malaria vaccines., Results: In this study, we demonstrate that the heterologous prime-boost regimen Ad35-CS/BCG-CS induces stronger immune responses by enhancing type 1 cellular producing-cells with high levels of CSp-specific IFN-γ and cytophilic IgG2a antibodies as compared to a homologous BCG-CS and a heterologous BCG-CS/CSp prime-boost regimen. Moreover, the heterologous prime-boost regimen elicits the highest level of LLPC-mediated immune responses., Conclusion: The increased IFN-γ-producing cell responses induced by the combination of Ad35-CS/BCG-CS and sustained type 1 antibody profile together with high levels of LLPCs may be essential for the development of long-term protective immunity against liver-stage parasites., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The granuloma in tuberculosis (TB), referred to as the tubercle, is a lesion containing multiple cell types and is the one definite hallmark of this disease. A number of tubercle phenotypes are seen during infection yet how these contribute to development of TB remains unclear. Here we highlight recent results using diverse models of tubercle development as well as recent findings from studies of human TB in an attempt to illustrate the plasticity of the tubercle and to place it between the poles of pathology and protection. Such insights could lead to future interventions to address TB as a global health issue., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

As a hallmark of tuberculosis (TB), Mycobacterium tuberculosis (MTB) induces granulomatous lung lesions and systemic inflammatory responses during active disease. Molecular regulation of inflammation is associated with inflammasome assembly. We determined the extent to which MTB triggers inflammasome activation and how this impacts on the severity of TB in a mouse model. MTB stimulated release of mature IL-1β in macrophages while attenuated M. bovis BCG failed to do so. Tubercle bacilli specifically activated the NLRP3 inflammasome and this propensity was strictly controlled by the virulence-associated RD1 locus of MTB. However, Nlrp3-deficient mice controlled pulmonary TB, a feature correlated with NLRP3-independent production of IL-1β in infected lungs. Our studies demonstrate that MTB activates the NLRP3 inflammasome in macrophages in an ESX-1-dependent manner. However, during TB, MTB promotes NLRP3- and caspase-1-independent IL-1β release in myeloid cells recruited to lung parenchyma and thus overcomes NLRP3 deficiency in vivo in experimental models., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Bacille Calmette-Guérin (BCG) is the vaccine against tuberculosis (TB), but has varied efficacy in different geographical locations. Recombinant strategies to genetically modify the organism to enhance the quality of the immune response have aimed at improving BCG efficacy. Here we describe such a strategy using rBCGΔureC∷hly expressing defined latency-associated antigens and test this construct for long-term protection against an isolate of the Mycobacterium tuberculosis (Mtb) Beijing/W lineage. Expression of the antigens Rv2659c, Rv3407 and Rv1733c by rBCGΔureC∷hly improved long-term efficacy in both lung and spleen at day 200 post-infection after intradermal vaccination of mice. Our data support expression of Mtb latency associated antigens by rBCG to improve protection against Mtb., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Tuberculosis (TB) is a complex disease, and the success of the bacterium as an intracellular pathogen is the outcome of its close and longstanding coevolution with the mammalian host. The dialogue between Mycobacterium tuberculosis and the host is becoming understandable at the molecular, cellular, and tissue level. This has led to the elucidation of the (i) interaction between pattern recognition receptors and pathogen-associated molecular patterns, (ii) cross-talk between immune cells, and (iii) mechanisms underlying granuloma development. Disease as an eventual but not a necessary consequence of infection results from a sensitive balance between protective immunity and destructive pathology. Early events, governed largely by conserved mechanisms of host recognition, impact not only on type and course of adaptive immunity but also on lung parenchymal function. New interpretations of how these responses shape the lung environment and direct granuloma development emphasize that the disease results from pathologic consequences of non-resolving inflammation. We review recent advances in TB research within the context of this ambitious view of TB., (© 2011 John Wiley & Sons A/S.)

A simple serodiagnostic test based on the Mycobacterium leprae-specific phenolic glycolipid I(PGL-I), for individuals with leprosy is nearly universally positive in leprosy patients with high bacillary loads but cannot be used as a stand-alone diagnostic test for the entire spectrum of the disease process. For patients with early infection with no detectable acid-fast bacilli in lesions or with low or no antibody titer to PGL-I, as in those at the tuberculoid end of the disease spectrum, this diagnostic approach has limited usefulness. To identify additional M. leprae antigens that might enhance the serological detection of these individuals, we have examined the reactivity patterns of patient sera to PGL-I, lipoarabinomannan (LAM), and six recombinant M. leprae proteins (ML1877, ML0841, ML2028, ML2038, ML0380, and ML0050) by Western blot analysis and enzyme-linked immunosorbent assay (ELISA). Overall, the responses to ML2028 (Ag85B) and ML2038 (bacterioferritin) were consistently high in both multibacillary and paucibacillary groups and weak or absent in endemic controls, while responses to other antigens showed considerable variability, from strongly positive to completely negative. This analysis has given a clearer understanding of some of the differences in the antibody responses between individuals at opposite ends of the disease spectrum, as well as illustrating the heterogeneity of antibody responses toward protein, carbohydrate, and glycolipid antigens within a clinical group. Correlating these response patterns with a particular disease state could allow for a more critical assessment of the form of disease within the leprosy spectrum and could lead to better patient management.

The hallmark of human Mycobacterium tuberculosis infection is the presence of lung granulomas. Lung granulomas can have different phenotypes, with caseous necrosis and hypoxia present within these structures during active tuberculosis. Production of NO by the inducible host enzyme NOS2 is a key antimycobacterial defense mechanism that requires oxygen as a substrate; it is therefore likely to perform inefficiently in hypoxic regions of granulomas in which M. tuberculosis persists. Here we have used Nos2-/- mice to investigate host-protective mechanisms within hypoxic granulomas and identified a role for host serine proteases in hypoxic granulomas in determining outcome of disease. Nos2-/- mice reproduced human-like granulomas in the lung when infected with M. tuberculosis in the ear dermis. The granulomas were hypoxic and contained large amounts of the serine protease cathepsin G and clade B serine protease inhibitors (serpins). Extrinsic inhibition of serine protease activity in vivo resulted in distorted granuloma structure, extensive hypoxia, and increased bacterial growth in this model. These data suggest that serine protease activity acts as a protective mechanism within hypoxic regions of lung granulomas and present a potential new strategy for the treatment of tuberculosis.

Tuberculosis causes 2 million deaths per year, yet in most cases the immune response successfully contains the infection and prevents disease outbreak. Induced lymphoid structures associated with pulmonary granuloma are observed during tuberculosis in both humans and mice and could orchestrate host defense. To investigate whether granuloma perform lymphoid functions, mice lacking secondary lymphoid organs (SLO) were infected with Mycobacterium tuberculosis (MTB). As in WT mice, granuloma developed, exponential growth of MTB was controlled, and antigen-specific T-cell responses including memory T cells were generated in the absence of SLO. Moreover, adoptively transferred T cells were primed locally in lungs in a granuloma-dependent manner. T-cell activation was delayed in the absence of SLO, but resulted in a normal development program including protective subsets and functional recall responses that protected mice against secondary MTB infection. Our data demonstrate that protective immune responses can be generated independently of SLO during MTB infection and implicate local pulmonary T-cell priming as a mechanism contributing to host defense.

Progression and outcome of tuberculosis is governed by extensive crosstalk between pathogen and host. Analyses of global changes in gene expression during immune response to infection with Mycobacterium tuberculosis (M.tb) can help identify molecular markers of disease state and progression. Global distribution of M.tb strains with different degrees of virulence and drug resistance, especially for the immunocompromised host, make closer analyses of host responses more pressing than ever. Here, we describe global transcriptional responses of inducible nitric oxide synthase-deficient (iNOS(-/-)) and WT mice infected with two related M.tb strains of markedly different virulence, namely the M.tb laboratory strains H37Rv and H37Ra. Both hosts exhibited highly similar resistance to infection with H37Ra. In contrast, iNOS(-/-) mice rapidly succumbed to H37Rv, whereas WT mice developed chronic course of disease. By differential analyses, virulence-specific changes in global host gene expression were analyzed to identify molecular markers characteristic for chronic versus acute infection. We identified several markers unique for different stages of disease progression and not previously associated with virulence-specific host responses in tuberculosis.

The detection of hundreds of thousands of new cases of leprosy every year suggests that transmission of Mycobacterium leprae infection still continues. Unfortunately, tools for identification of asymptomatic disease and/or early-stage M. leprae infection (likely sources of transmission) are lacking. The recent identification of M. leprae-unique genes has allowed the analysis of human T-cell responses to novel M. leprae antigens. Antigens with the most-promising diagnostic potential were tested for their ability to induce cytokine secretion by using peripheral blood mononuclear cells from leprosy patients and controls in five different areas where leprosy is endemic; 246 individuals from Brazil, Nepal, Bangladesh, Pakistan, and Ethiopia were analyzed for gamma interferon responses to five recombinant proteins (ML1989, ML1990, ML2283, ML2346, and ML2567) and 22 synthetic peptides. Of these, the M. leprae-unique protein ML1989 was the most frequently recognized and ML2283 the most specific for M. leprae infection/exposure, as only a limited number of tuberculosis patients responded to this antigen. However, all proteins were recognized by a significant number of controls in areas of endemicity. T-cell responses correlated with in vitro response to M. leprae, suggesting that healthy controls in areas where leprosy is endemic are exposed to M. leprae. Importantly, 50% of the healthy household contacts and 59% of the controls in areas of endemicity had no detectable immunoglobulin M antibodies to M. leprae-specific PGL-I but responded in T-cell assays to >or=1 M. leprae protein. T-cell responses specific for leprosy patients and healthy household contacts were observed for ML2283- and ML0126-derived peptides, indicating that M. leprae peptides hold potential as diagnostic tools. Future work should concentrate on the development of a sensitive and field-friendly assay and identification of additional peptides and proteins that can induce M. leprae-specific T-cell responses.

The identification of human T-cell antigens of Mycobacterium leprae could improve treatment and help to disrupt the transmission of leprosy by directing diagnosis and vaccine programs. This study screened a panel of M. leprae recombinant proteins for T-cell recall responses, measured by gamma interferon (IFN-gamma) production, among leprosy patients. After initial studies using peripheral blood mononuclear cells from leprosy patients, we transitioned our studies to simple whole-blood assays (WBA), which are more applicable in field or clinical settings. T-cell responses generated in WBA using blood from individuals in Goiânia, Brazil, demonstrated that several M. leprae antigens (ML0276, ML0840, ML1623, ML2044, and 46f) elicited >0.5 IU/ml IFN-gamma, and these proteins were classified as immunogenic and leprosy specific. Several of these individual antigens were recognized by cells from >60% of Brazilian paucibacillary (PB) leprosy patients, and ML0276, ML0840, ML1623, and 46f complemented each other such that 82% of PB patients had strong (>1.25 IU/ml IFN-gamma) responses to at least one of these proteins. These proteins were also recognized by cells from a significant proportion of the household contacts of multibacillary leprosy patients, but in contrast, few responses were observed in active tuberculosis patients or healthy control groups from areas of endemicity. Our results indicate several potential candidate antigens which may be useful for either leprosy diagnosis or vaccination and demonstrate the utility of leprosy WBA that can be applied broadly in clinical or field settings.

Tuberculosis represents a serious problem for public health worldwide, and effective vaccines are urgently required. This represents a significant challenge as the causative bacterial agent, Mycobacterium tuberculosis, has developed strategies to persist in infected hosts despite the presence of potent T-cell-mediated immune responses. New advances in basic immunology are giving us improved understanding of what constitutes a protective immune response and ways this response is manipulated by the bacillus. Such insights should inform us how to design more effective vaccination strategies against intracellular pathogens.

Leprosy is a chronic and debilitating human disease caused by infection with the Mycobacterium leprae bacillus. Despite the marked reduction in the number of registered worldwide leprosy cases as a result of the widespread use of multidrug therapy, the number of new cases detected each year remains relatively stable. This indicates that M. leprae is still being transmitted and that, without earlier diagnosis, M. leprae infection will continue to pose a health problem. Current diagnostic techniques, based on the appearance of clinical symptoms or of immunoglobulin M (IgM) antibodies that recognize the bacterial phenolic glycolipid I, are unable to reliably identify early-stage leprosy. In this study we examine the ability of IgG within leprosy patient sera to bind several M. leprae protein antigens. As expected, multibacillary leprosy patients provided stronger responses than paucibacillary leprosy patients. We demonstrate that the geographic locations of the patients can influence the antigens they recognize but that ML0405 and ML2331 are recognized by sera from diverse regions (the Philippines, coastal and central Brazil, and Japan). A fusion construct of these two proteins (designated leprosy IDRI diagnostic 1 [LID-1]) retained the diagnostic activity of the component antigens. Upon testing against a panel of prospective sera from individuals who developed leprosy, we determined that LID-1 was capable of diagnosing leprosy 6 to 8 months before the onset of clinical symptoms. A serological diagnostic test capable of identifying and allowing treatment of early-stage leprosy could reduce transmission, prevent functional disabilities and stigmatizing deformities, and facilitate leprosy eradication.

Leprosy is caused by infection with Mycobacterium leprae. The immune response of leprosy patients can be highly diverse, ranging from strong cellular responses accompanied by an apparent deficit of M. leprae-specific antibodies to strong humoral responses with a deficit of cell-mediated responses. Leprosy takes many years to manifest, and this has precluded analyses of disease and immune response development in infected humans. In an attempt to better define development of the immune response during leprosy we have developed an M. leprae ear infection model. Intradermal inoculation of M. leprae into the ear supported not only infection but also the development of a chronic inflammatory response. The inflammatory response was localized, comprising a T-cell infiltration into the ear and congestion of cells in the draining lymph nodes. The development of local chronic inflammation was prevented by rifampin treatment. Importantly, and in contrast to subcutaneous M. leprae footpad infection, systemic M. leprae-specific gamma interferon and antibody responses were detected following intradermal ear infection. These results indicate the utility of intradermal ear infection for both induction and understanding of the immune response during M. leprae infection and the identification or testing of new leprosy treatments.

Despite the success of multidrug therapy in reducing the number of registered leprosy cases worldwide, evidence suggests that Mycobacterium leprae continues to be transmitted. A serological diagnostic test capable of identifying and allowing treatment of early-stage disease could reduce transmission and prevent the onset of the disability, a common complication of the disease in later stages. Serological diagnosis based on antibody recognition of phenolic glycolipid I (PGL-I) cannot reliably identify individuals with lower bacterial indices (BI). One strategy that might improve this situation is the provision of highly specific serological antigens that may be combined with PGL-I to improve the sensitivity of diagnosis. Using serological expression cloning with a serum pool of untreated lepromatous leprosy (LL) patients, we identified 14 strongly reactive M. leprae proteins, 5 of which were previously unstudied. We present results suggesting that two of these proteins, ML0405 and ML2331, demonstrate the ability to specifically identify LL/borderline lepromatous (BL) patients on the basis of immunoglobulin G (IgG) reactivity. In a household contact study, LL index cases were identified on the basis of this reactivity, while household contacts of these patients demonstrated undetectable reactivity. At a serum dilution of 1:800, suitable to reduce background PGL-I IgM reactivity, two BL patients with a BI of <4 showed anti-human polyvalent immunoglobulin G, A, and M reactivity measured with a combination of ML0405, ML2331, and natural disaccharide O-linked human serum albumin (NDOHSA) (synthetic PGL-I) that was markedly higher than IgM reactivity to NDOHSA alone. We suggest that ML0405 and ML2331 may have utility in serological leprosy diagnosis.

Tuberculin shock due to inoculation of Mycobacterium tuberculosis antigens in patients with tuberculosis is a serious syndrome originally described over 100 years ago by Robert Koch. Here, we present experimental evidence that a single M. tuberculosis recombinant protein, CFP-10, triggers this syndrome. Intradermal inoculation of CFP-10 elicits in M. tuberculosis-infected mice high levels of serum tumor necrosis factor alpha and causes tuberculin shock in infected guinea pigs characterized by hypothermia and death within 6 to 48 h after the antigen inoculation. Autopsies of these animals revealed intense polycythemia and hemorrhagic patches in the lung parenchyma, a pathological observation consistent with tuberculin shock. These results point to the possible occurrence of tuberculin shock in sensitive individuals inoculated with highly purified M. tuberculosis recombinant proteins as vaccine candidates or skin test reagents.