Toward a molecular microbial blood test for tuberculosis infection.

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.

Determinants of QuantiFERON Plus-diagnosed tuberculosis infection in adult Ugandan TB contacts: A cross-sectional study.

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.

Intranasal immunization with outer membrane vesicles (OMV) protects against airway colonization and systemic infection with Acinetobacter baumannii.

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.

Detection of Mycobacterium tuberculosis complex DNA in CD34-positive peripheral blood mononuclear cells of asymptomatic tuberculosis contacts: an observational study.

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.

A novel therapeutic antibody screening method using bacterial high-content imaging reveals functional antibody binding phenotypes of Escherichia coli ST131.

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.

Anatomic and Cellular Niches for Mycobacterium tuberculosis in Latent Tuberculosis Infection.

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.

Mycobacterium tuberculosis-Infected Hematopoietic Stem and Progenitor Cells Unable to Express Inducible Nitric Oxide Synthase Propagate Tuberculosis in Mice.

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.

Self-Amplifying RNA Vaccines Give Equivalent Protection against Influenza to mRNA Vaccines but at Much Lower Doses.

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.

NOS2-deficient mice with hypoxic necrotizing lung lesions predict outcomes of tuberculosis chemotherapy in humans.

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.

Human and Mouse Hematopoietic Stem Cells Are a Depot for Dormant Mycobacterium tuberculosis.

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.

Neonatal Fc Receptor Regulation of Lung Immunoglobulin and CD103+ Dendritic Cells Confers Transient Susceptibility to Tuberculosis.

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.

Macrophage arginase-1 controls bacterial growth and pathology in hypoxic tuberculosis granulomas.

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.

Type I IFN signaling triggers immunopathology in tuberculosis-susceptible mice by modulating lung phagocyte dynamics.

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.

Heterologous prime-boost regimen adenovector 35-circumsporozoite protein vaccine/recombinant Bacillus Calmette-Guérin expressing the Plasmodium falciparum circumsporozoite induces enhanced long-term memory immunity in BALB/c mice.

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.

A recombinant Bacille Calmette-Guérin construct expressing the Plasmodium falciparum circumsporozoite protein enhances dendritic cell activation and primes for circumsporozoite-specific memory cells in BALB/c mice.

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.

Activation of the NLRP3 inflammasome by Mycobacterium tuberculosis is uncoupled from susceptibility to active tuberculosis.

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-1ß 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-1ß 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-1ß release in myeloid cells recruited to lung parenchyma and thus overcomes NLRP3 deficiency in vivo in experimental models.

Floating between the poles of pathology and protection: can we pin down the granuloma in tuberculosis?

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.

Improved long-term protection against Mycobacterium tuberculosis Beijing/W in mice after intra-dermal inoculation of recombinant BCG expressing latency associated antigens.

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.

For better or for worse: the immune response against Mycobacterium tuberculosis balances pathology and protection.

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.