Age and sex influence antibody profiles associated with tuberculosis progression.

Antibody features vary with tuberculosis (TB) disease state. Whether clinical variables, such as age or sex, influence associations between Mycobacterium tuberculosis-specific antibody responses and disease state is not well explored. Here we profiled Mycobacterium tuberculosis-specific antibody responses in 140 TB-exposed South African individuals from the Adolescent Cohort Study. We identified distinct response features in individuals progressing to active TB from non-progressing, matched controls. A multivariate antibody score differentially associated with progression (SeroScore) identified progressors up to 2 years before TB diagnosis, earlier than that achieved with the RISK6 transcriptional signature of progression. We validated these antibody response features in the Grand Challenges 6-74 cohort. Both the SeroScore and RISK6 correlated better with risk of TB progression in adolescents compared with adults, and in males compared with females. This suggests that age and sex are important, underappreciated modifiers of antibody responses associated with TB progression.

Cynomolgus macaques as a translational model of human immune responses to yellow fever 17D vaccination.

The non-human primate (NHP) model (specifically rhesus and cynomolgus macaques) has facilitated our understanding of the pathogenic mechanisms of yellow fever (YF) disease and allowed the evaluation of the safety and efficacy of YF-17D vaccines. However, the accuracy of this model in mimicking vaccine-induced immunity in humans remains to be fully determined. We used a systems biology approach to compare hematological, biochemical, transcriptomic, and innate and antibody-mediated immune responses in cynomolgus macaques and human participants following YF-17D vaccination. Immune response progression in cynomolgus macaques followed a similar course as in adult humans but with a slightly earlier onset. Yellow fever virus neutralizing antibody responses occurred earlier in cynomolgus macaques [by Day 7[(D7)], but titers > 10 were reached in both species by D14 post-vaccination and were not significantly different by D28 [plaque reduction neutralization assay (PRNT)50 titers 3.6 Log vs 3.5 Log in cynomolgus macaques and human participants, respectively; P = 0.821]. Changes in neutrophils, NK cells, monocytes, and T- and B-cell frequencies were higher in cynomolgus macaques and persisted for 4 weeks versus less than 2 weeks in humans. Low levels of systemic inflammatory cytokines (IL-1RA, IL-8, MIP-1α, IP-10, MCP-1, or VEGF) were detected in either or both species but with no or only slight changes versus baseline. Similar changes in gene expression profiles were elicited in both species. These included enriched and up-regulated type I IFN-associated viral sensing, antiviral innate response, and dendritic cell activation pathways D3-D7 post-vaccination in both species. Hematological and blood biochemical parameters remained relatively unchanged versus baseline in both species. Low-level YF-17D viremia (RNAemia) was transiently detected in some cynomolgus macaques [28% (5/18)] but generally absent in humans [except one participant (5%; 1/20)].IMPORTANCECynomolgus macaques were confirmed as a valid surrogate model for replicating YF-17D vaccine-induced responses in humans and suggest a key role for type I IFN.

Activin A levels are raised during human tuberculosis and blockade of the activin signaling axis influences murine responses to M. tuberculosis infection.

Activin A strongly influences immune responses; yet, few studies have examined its role in infectious diseases. We measured serum activin A levels in two independent tuberculosis (TB) patient cohorts and in patients with pneumonia and sarcoidosis. Serum activin A levels were increased in TB patients compared to healthy controls, including those with positive tuberculin skin tests, and paralleled severity of disease, assessed by X-ray scores. In pneumonia patients, serum activin A levels were also raised, but in sarcoidosis patients, levels were lower. To determine whether blockade of the activin A signaling axis could play a functional role in TB, we harnessed a soluble activin type IIB receptor fused to human IgG1 Fc, ActRIIB-Fc, as a ligand trap in a murine TB model. The administration of ActRIIB-Fc to Mycobacterium tuberculosis-infected mice resulted in decreased bacterial loads and increased numbers of CD4 effector T cells and tissue-resident memory T cells in the lung. Increased frequencies of tissue-resident memory T cells corresponded with downregulated T-bet expression in lung CD4 and CD8 T cells. Altogether, the results suggest a disease-exacerbating role of ActRIIB signaling pathways. Serum activin A may be useful as a biomarker for diagnostic triage of active TB or monitoring of anti-tuberculosis therapy. IMPORTANCE: Tuberculosis remains the leading cause of death by a bacterial pathogen. The etiologic agent of tuberculosis, Mycobacterium tuberculosis, can remain dormant in the infected host for years before causing disease. Significant effort has been made to identify biomarkers that can discriminate between latently infected and actively diseased individuals. We found that serum levels of the cytokine activin A were associated with increased lung pathology and could discriminate between active tuberculosis and tuberculin skin-test-positive healthy controls. Activin A signals through the ActRIIB receptor, which can be blocked by administration of the ligand trap ActRIIB-Fc, a soluble activin type IIB receptor fused to human IgG1 Fc. In a murine model of tuberculosis, we found that ActRIIB-Fc treatment reduced mycobacterial loads. Strikingly, ActRIIB-Fc treatment significantly increased the number of tissue-resident memory T cells. These results suggest a role for ActRIIB signaling pathways in host responses to Mycobacterium tuberculosis and activin A as a biomarker of ongoing disease.

Reversible Host-Guest Crosslinks in Supramolecular Hydrogels for On-Demand Mechanical Stimulation of Human Mesenchymal Stem Cells.

Stem cells are regulated not only by biochemical signals but also by biophysical properties of extracellular matrix (ECM). The ECM is constantly monitored and remodeled because the fate of stem cells can be misdirected when the mechanical interaction between cells and ECM is imbalanced. A well-defined ECM model for bone marrow-derived human mesenchymal stem cells (hMSCs) based on supramolecular hydrogels containing reversible host-guest crosslinks is fabricated. The stiffness (Young's modulus E) of the hydrogels can be switched reversibly by altering the concentration of non-cytotoxic, free guest molecules dissolved in the culture medium. Fine-adjustment of substrate stiffness enables the authors to determine the critical stiffness level E* at which hMSCs turn the mechano-sensory machinery on or off. Next, the substrate stiffness across E* is switched and the dynamic adaptation characteristics such as morphology, traction force, and YAP/TAZ signaling of hMSCs are monitored. These data demonstrate the instantaneous switching of traction force, which is followed by YAP/TAZ signaling and morphological adaptation. Periodical switching of the substrate stiffness across E* proves that frequent applications of mechanical stimuli drastically suppress hMSC proliferation. Mechanical stimulation across E* level using dynamic hydrogels is a promising strategy for the on-demand control of hMSC transcription and proliferation.

Ixodes scapularis nymph saliva protein blocks host inflammation and complement-mediated killing of Lyme disease agent, Borrelia burgdorferi.

Tick serine protease inhibitors (serpins) play crucial roles in tick feeding and pathogen transmission. We demonstrate that Ixodes scapularis (Ixs) nymph tick saliva serpin (S) 41 (IxsS41), secreted by Borrelia burgdorferi (Bb)-infected ticks at high abundance, is involved in regulating tick evasion of host innate immunity and promoting host colonization by Bb. Recombinant (r) proteins were expressed in Pichia pastoris, and substrate hydrolysis assays were used to determine. Ex vivo (complement and hemostasis function related) and in vivo (paw edema and effect on Bb colonization of C3H/HeN mice organs) assays were conducted to validate function. We demonstrate that rIxsS41 inhibits chymase and cathepsin G, pro-inflammatory proteases that are released by mast cells and neutrophils, the first immune cells at the tick feeding site. Importantly, stoichiometry of inhibition analysis revealed that 2.2 and 2.8 molecules of rIxsS41 are needed to 100% inhibit 1 molecule of chymase and cathepsin G, respectively, suggesting that findings here are likely events at the tick feeding site. Furthermore, chymase-mediated paw edema, induced by the mast cell degranulator, compound 48/80 (C48/80), was blocked by rIxsS41. Likewise, rIxsS41 reduced membrane attack complex (MAC) deposition via the alternative and lectin complement activation pathways and dose-dependently protected Bb from complement killing. Additionally, co-inoculating C3H/HeN mice with Bb together with rIxsS41 or with a mixture (rIxsS41 and C48/80). Findings in this study suggest that IxsS41 markedly contributes to tick feeding and host colonization by Bb. Therefore, we conclude that IxsS41 is a potential candidate for an anti-tick vaccine to prevent transmission of the Lyme disease agent.

Tuberculosis prevention: current strategies and future directions.

BACKGROUND: An estimated one fourth of the world's population is infected with Mycobacterium tuberculosis, and 5-10% of those infected develop tuberculosis in their lifetime. Preventing tuberculosis is one of the most underutilized but essential components of curtailing the tuberculosis epidemic. Moreover, current evidence illustrates that tuberculosis manifestations occur along a dynamic spectrum from infection to disease rather than a binary state as historically conceptualized. Elucidating determinants of transition between these states is crucial to decreasing the tuberculosis burden and reaching the END-TB Strategy goals as defined by the WHO. Vaccination, detection of infection, and provision of preventive treatment are key elements of tuberculosis prevention. OBJECTIVES: This review provides a comprehensive summary of recent evidence and state-of-the-art updates on advancements to prevent tuberculosis in various settings and high-risk populations. SOURCES: We identified relevant studies in the literature and synthesized the findings to provide an overview of the current state of tuberculosis prevention strategies and latest research developments. CONTENT: We present the current knowledge and recommendations regarding tuberculosis prevention, with a focus on M. bovis Bacille-Calmette-Guérin vaccination and novel vaccine candidates, tests for latent infection with M. tuberculosis, regimens available for tuberculosis preventive treatment and recommendations in low- and high-burden settings. IMPLICATIONS: Effective tuberculosis prevention worldwide requires a multipronged approach that addresses social determinants, and improves access to tuberculosis detection and to new short tuberculosis preventive treatment regimens. Robust collaboration and innovative research are needed to reduce the global burden of tuberculosis and develop new detection tools, vaccines, and preventive treatments that serve all populations and ages.

Vaccine development against tuberculosis before and after Covid-19.

Coronavirus disease (Covid-19) has not only shaped awareness of the impact of infectious diseases on global health. It has also provided instructive lessons for better prevention strategies against new and current infectious diseases of major importance. Tuberculosis (TB) is a major current health threat caused by Mycobacterium tuberculosis (Mtb) which has claimed more lives than any other pathogen over the last few centuries. Hence, better intervention measures, notably novel vaccines, are urgently needed to accomplish the goal of the World Health Organization to end TB by 2030. This article describes how the research and development of TB vaccines can benefit from recent developments in the Covid-19 vaccine pipeline from research to clinical development and outlines how the field of TB research can pursue its own approaches. It begins with a brief discussion of major vaccine platforms in general terms followed by a short description of the most widely applied Covid-19 vaccines. Next, different vaccination regimes and particular hurdles for TB vaccine research and development are described. This specifically considers the complex immune mechanisms underlying protection and pathology in TB which involve innate as well as acquired immune mechanisms and strongly depend on fine tuning the response. A brief description of the TB vaccine candidates that have entered clinical trials follows. Finally, it discusses how experiences from Covid-19 vaccine research, development, and rollout can and have been applied to the TB vaccine pipeline, emphasizing similarities and dissimilarities.

Human GBP1 Is Involved in the Repair of Damaged Phagosomes/Endolysosomes.

Mouse guanylate-binding proteins (mGBPs) are recruited to various invasive pathogens, thereby conferring cell-autonomous immunity against these pathogens. However, whether and how human GBPs (hGBPs) target M. tuberculosis (Mtb) and L. monocytogenes (Lm) remains unclear. Here, we describe hGBPs association with intracellular Mtb and Lm, which was dependent on the ability of bacteria to induce disruption of phagosomal membranes. hGBP1 formed puncta structures which were recruited to ruptured endolysosomes. Furthermore, both GTP-binding and isoprenylation of hGBP1 were required for its puncta formation. hGBP1 was required for the recovery of endolysosomal integrity. In vitro lipid-binding assays demonstrated direct binding of hGBP1 to PI4P. Upon endolysosomal damage, hGBP1 was targeted to PI4P and PI(3,4)P2-positive endolysosomes in cells. Finally, live-cell imaging demonstrated that hGBP1 was recruited to damaged endolysosomes, and consequently mediated endolysosomal repair. In summary, we uncover a novel interferon-inducible mechanism in which hGBP1 contributes to the repair of damaged phagosomes/endolysosomes.

Mycobacterium tuberculosis hijacks host TRIM21- and NCOA4-dependent ferritinophagy to enhance intracellular growth.

Ferritin, a key regulator of iron homeostasis in macrophages, has been reported to confer host defenses against Mycobacterium tuberculosis (Mtb) infection. Nuclear receptor coactivator 4 (NCOA4) was recently identified as a cargo receptor in ferritin degradation. Here, we show that Mtb infection enhanced NCOA4-mediated ferritin degradation in macrophages, which in turn increased the bioavailability of iron to intracellular Mtb and therefore promoted bacterial growth. Of clinical relevance, the upregulation of FTH1 in macrophages was associated with tuberculosis (TB) disease progression in humans. Mechanistically, Mtb infection enhanced NCOA4-mediated ferritin degradation through p38/AKT1- and TRIM21-mediated proteasomal degradation of HERC2, an E3 ligase of NCOA4. Finally, we confirmed that NCOA4 deficiency in myeloid cells expedites the clearance of Mtb infection in a murine model. Together, our findings revealed a strategy by which Mtb hijacks host ferritin metabolism for its own intracellular survival. Therefore, this represents a potential target for host-directed therapy against tuberculosis.

Wnt/ß-catenin signaling induces axial elasticity patterns of Hydra extracellular matrix.

The extracellular matrix (ECM) plays crucial roles in animal development and diseases. Here, we report that Wnt/ß-catenin signaling induces the ECM remodeling during Hydra axis formation. We determined the micro- and nanoscopic arrangement of fibrillar type I collagen along Hydra's body axis using high-resolution microscopy and X-ray scattering. Elasticity mapping of the ECM ex vivo revealed distinctive elasticity patterns along the body axis. A proteomic analysis of the ECM showed that these elasticity patterns correlate with a gradient-like distribution of metalloproteases along the body axis. Activation of the Wnt/ß-catenin pathway in wild-type and transgenic animals alters these patterns toward low ECM elasticity patterns. This suggests a mechanism whereby high protease activity under control of Wnt/ß-catenin signaling causes remodeling and softening of the ECM. This Wnt-dependent spatiotemporal coordination of biochemical and biomechanical cues in ECM formation was likely a central evolutionary innovation for animal tissue morphogenesis.

Safety and Immunogenicity of Recombinant Bacille Calmette-Guérin Strain VPM1002 and Its Derivatives in a Goat Model.

A more effective vaccine against tuberculosis than Bacille Calmette-Guérin (BCG) is urgently needed. BCG derived recombinant VPM1002 has been found to be more efficacious and safer than the parental strain in mice models. Newer candidates, such as VPM1002 Δpdx1 (PDX) and VPM1002 ΔnuoG (NUOG), were generated to further improve the safety profile or efficacy of the vaccine. Herein, we assessed the safety and immunogenicity of VPM1002 and its derivatives, PDX and NUOG, in juvenile goats. Vaccination did not affect the goats' health in regards to clinical/hematological features. However, all three tested vaccine candidates and BCG induced granulomas at the site of injection, with some of the nodules developing ulcerations approximately one month post-vaccination. Viable vaccine strains were cultured from the injection site wounds in a few NUOG- and PDX- vaccinated animals. At necropsy (127 days post-vaccination), BCG, VPM1002, and NUOG, but not PDX, still persisted at the injection granulomas. All strains, apart from NUOG, induced granuloma formation only in the lymph nodes draining the injection site. In one animal, the administered BCG strain was recovered from the mediastinal lymph nodes. Interferon gamma (IFN-γ) release assay showed that VPM1002 and NUOG induced a strong antigen-specific response comparable to that elicited by BCG, while the response to PDX was delayed. Flow cytometry analysis of IFN-γ production by CD4+, CD8+, and γδ T cells showed that CD4+ T cells of VPM1002- and NUOG-vaccinated goats produced more IFN-γ compared to BCG-vaccinated and mock-treated animals. In summary, the subcutaneous application of VPM1002 and NUOG induced anti-tuberculous immunity, while exhibiting a comparable safety profile to BCG in goats.

T cell receptor repertoires associated with control and disease progression following Mycobacterium tuberculosis infection.

Antigen-specific, MHC-restricted αß T cells are necessary for protective immunity against Mycobacterium tuberculosis, but the ability to broadly study these responses has been limited. In the present study, we used single-cell and bulk T cell receptor (TCR) sequencing and the GLIPH2 algorithm to analyze M. tuberculosis-specific sequences in two longitudinal cohorts, comprising 166 individuals with M. tuberculosis infection who progressed to either tuberculosis (n = 48) or controlled infection (n = 118). We found 24 T cell groups with similar TCR-ß sequences, predicted by GLIPH2 to have common TCR specificities, which were associated with control of infection (n = 17), and others that were associated with progression to disease (n = 7). Using a genome-wide M. tuberculosis antigen screen, we identified peptides targeted by T cell similarity groups enriched either in controllers or in progressors. We propose that antigens recognized by T cell similarity groups associated with control of infection can be considered as high-priority targets for future vaccine development.

VPM1002 as Prophylaxis Against Severe Respiratory Tract Infections Including Coronavirus Disease 2019 in the Elderly: A Phase 3 Randomized, Double-Blind, Placebo-Controlled, Multicenter Clinical Study.

BACKGROUND: Bacille Calmette-Guérin (BCG) vaccination can potentially reduce the rate of respiratory infections in vulnerable populations. This study evaluates the safety and efficacy of VPM1002 (a genetically modified BCG) as prophylaxis against severe respiratory tract infections including coronavirus disease 2019 (COVID-19) in an elderly population. METHODS: In this phase 3, randomized, double-blind, placebo-controlled, multicenter clinical trial, healthy elderly volunteers (N = 2064) were enrolled, randomized (1:1) to receive either VPM1002 or placebo, and followed up remotely for 240 days. The primary outcome was the mean number of days with severe respiratory infections at hospital and/or at home. Secondary endpoints included the incidence of self-reported fever, number of hospital and intensive care unit (ICU) admissions, and number of adverse events. RESULTS: A total of 31 participants in the VPM1002 group reported at least 1 day with severe respiratory disease and a mean number of days with severe respiratory disease of 9.39 ± 9.28 while in the placebo group; 38 participants reported a mean of 14.29 ± 16.25 days with severe respiratory disease. The incidence of self-reported fever was lower in the VPM1002 group (odds ratio, 0.46 [95% confidence interval, .28-.74]; P = .001), and consistent trends to fewer hospitalization and ICU admissions due to COVID-19 were observed after VPM1002 vaccination. Local reactions typical for BCG were observed in the VPM1002-vaccinated group, which were mostly of mild intensity. CONCLUSIONS: Vaccination with VPM1002 is well tolerated and seems to have a prophylactic effect against severe respiratory disease in the elderly. CLINICAL TRIALS REGISTRATION: NCT04435379.

Association Between Human Immunodeficiency Virus Viremia and Compromised Neutralization of Severe Acute Respiratory Syndrome Coronavirus 2 Beta Variant.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection may be associated with worse clinical outcomes in people with human immunodeficiency virus (HIV) (PWH). We report anti-SARS-CoV-2 antibody responses in patients hospitalized with coronavirus disease 2019 in Durban, South Africa, during the second SARS-CoV-2 infection wave dominated by the Beta (B.1.351) variant. METHODS: Thirty-four participants with confirmed SARS-CoV-2 infection were followed up with weekly blood sampling to examine antibody levels and neutralization potency against SARS-CoV-2 variants. Participants included 18 PWH, of whom 11 were HIV viremic. RESULTS: SARS-CoV-2-specific antibody concentrations were generally lower in viremic PWH than in virologically suppressed PWH and HIV-negative participants, and neutralization of the Beta variant was 4.9-fold lower in viremic PWH. Most HIV-negative participants and antiretroviral therapy-suppressed PWH also neutralized the Delta (B.1.617.2) variant, whereas the majority of viremic PWH did not. CD4 cell counts <500/µL were associated with lower frequencies of immunoglobulin G and A seroconversion. In addition, there was a high correlation between a surrogate virus neutralization test and live virus neutralization against ancestral SARS-CoV-2 virus in both PWH and HIV-negative individuals, but correlation decreased for the Beta variant neutralization in PWH. CONCLUSIONS: HIV viremia was associated with reduced Beta variant neutralization. This highlights the importance of HIV suppression in maintaining an effective SARS-CoV-2 neutralization response.

Neutrophil degranulation, NETosis and platelet degranulation pathway genes are co-induced in whole blood up to six months before tuberculosis diagnosis.

Mycobacterium tuberculosis (M.tb) causes tuberculosis (TB) and remains one of the leading causes of mortality due to an infectious pathogen. Host immune responses have been implicated in driving the progression from infection to severe lung disease. We analyzed longitudinal RNA sequencing (RNAseq) data from the whole blood of 74 TB progressors whose samples were grouped into four six-month intervals preceding diagnosis (the GC6-74 study). We additionally analyzed RNAseq data from an independent cohort of 90 TB patients with positron emission tomography-computed tomography (PET-CT) scan results which were used to categorize them into groups with high and low levels of lung damage (the Catalysis TB Biomarker study). These groups were compared to non-TB controls to obtain a complete whole blood transcriptional profile for individuals spanning from early stages of M.tb infection to TB diagnosis. The results revealed a steady increase in the number of genes that were differentially expressed in progressors at time points closer to diagnosis with 278 genes at 13-18 months, 742 at 7-12 months and 5,131 detected 1-6 months before diagnosis and 9,205 detected in TB patients. A total of 2,144 differentially expressed genes were detected when comparing TB patients with high and low levels of lung damage. There was a large overlap in the genes upregulated in progressors 1-6 months before diagnosis (86%) with those in TB patients. A comprehensive pathway analysis revealed a potent activation of neutrophil and platelet mediated defenses including neutrophil and platelet degranulation, and NET formation at both time points. These pathways were also enriched in TB patients with high levels of lung damage compared to those with low. These findings suggest that neutrophils and platelets play a critical role in TB pathogenesis, and provide details of the timing of specific effector mechanisms that may contribute to TB lung pathology.

Vaccine-Induced Subcutaneous Granulomas in Goats Reflect Differences in Host-Mycobacterium Interactions between BCG- and Recombinant BCG-Derivative Vaccines.

Tuberculous granulomas are highly dynamic structures reflecting the complex host-mycobacterium interactions. The objective of this study was to compare granuloma development at the site of vaccination with BCG and its recombinant derivatives in goats. To characterize the host response, epithelioid cells, multinucleated giant cells (MNGC), T cell subsets, B cells, plasma cells, dendritic cells and mycobacterial antigen were labelled by immunohistochemistry, and lipids and acid-fast bacteria (AFB) were labelled by specific staining. Granulomas with central caseous necrosis developed at the injection site of most goats though lesion size and extent of necrosis differed between vaccine strains. CD4+ T and B cells were more scarce and CD8+ cells were more numerous in granulomas induced by recombinant derivatives compared to their parental BCG strain. Further, the numbers of MNGCs and cells with lipid bodies were markedly lower in groups administered with recombinant BCG strains. Microscopic detection of AFB and mycobacterial antigen was rather frequent in the area of central necrosis, however, the isolation of bacteria in culture was rarely successful. In summary, BCG and its recombinant derivatives induced reproducibly subcutaneous caseous granulomas in goats that can be easily monitored and surgically removed for further studies. The granulomas reflected the genetic modifications of the recombinant BCG-derivatives and are therefore suitable models to compare reactions to different mycobacteria or TB vaccines.

100 Years of BCG Immunization: Past, Present, and Future.

The 100th anniversary of the introduction of Bacille-Calmette-Guérin (BCG) as a tuberculosis (TB) vaccine is an occasion warranting further investigation of the early attempts which culminated in the introduction of BCG as a TB vaccine, as well as of subsequent recognition of failures, new findings that broaden its applications, outstanding questions, and approaches towards the development of novel vaccine candidates [...].

Pathogenesis of tuberculosis: the 1930 Lübeck disaster revisited.

During the 1930 Lübeck Mycobacterium bovis bacille Calmette-Guérin (BCG) disaster, 251 neonates received three oral BCG doses accidentally contaminated by virulent Mycobacterium tuberculosis; 67 (26.7%) infants died of tuberculosis. BCG reversion to pathogenicity did not occur. Detailed post mortem examinations clarified contested aspects of tuberculosis pathogenesis. Gastrointestinal infection was seldom "silent" and did not cause typical primary pulmonary lesions. In 15 infants, primary pulmonary foci were found but these resulted from vaccine ingestion and aspiration and were not caused by gastrointestinal infection spreading to the lungs without trace of its journey, as claimed by prominent researchers such as Calmette and von Behring. Further, among 60 infants in whom post mortem evaluation was completed, a "silent" gastrointestinal infection without an intestinal primary focus was found in only one. Lymphohaematogenous-disseminated tuberculosis caused death in 24/67 (35.8%) infants and tuberculous meningitis in a further 17/67 (25.4%). Gastrointestinal tuberculosis complications caused death in 26/67 (38.8%) infants. Half of the tuberculosis-attributed deaths had occurred by 3 months, 93% by 6 months and 100% by 12 months; remarkably no further deaths or tuberculosis recurrences occurred within 5 years post-vaccination/infection. These findings provide graphic confirmation that the early introduction of chemoprophylaxis in recently M. tuberculosis-infected young children is critical and urgent.