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HSP90B1, also known as gp96, is a chaperone for multiple Toll-like receptors (TLRs) and is necessary for TLR-mediated inflammatory responses in murine myeloid cells. The molecule is also expressed in T-cells though its specific role is unknown. We hypothesized that human HSP90B1 regulates monocyte and T-cell responses to Mycobacterium tuberculosis (Mtb) and bacilli Calmette-Guerin (BCG) and that its variants are associated with susceptibility to TB disease. We screened 17 haplotype-tagging SNPs in the HSP90B1 gene region for association with BCG-induced T-cell cytokine responses using both an ex-vivo whole blood assay (N = 295) and an intracellular cytokine staining assay (N = 180) on samples collected 10 weeks after birth. Using a case-control study design, we evaluated the same SNPs for association with TB disease in a South African pediatric cohort (N = 217 cases, 604 controls). A subset of these SNPs was evaluated for association with HSP90B1 expression in human monocytes, monocyte-derived dendritic cells, and T-cells using RT-PCR. Lastly, we used CRISPR/Cas9 gene editing to knock down HSP90B1 expression in a human monocyte cell line (U937). Knockdown and control cell lines were tested for TLR surface expression and control of Mtb replication. We identified three SNPs, rs10507172, rs10507173 and rs1920413, that were associated with BCG-induced IL-2 secretion (p = 0.017 for rs10507172 and p = 0.03 for rs10507173 and rs1920413, Mann-Whitney, dominant model). SNPs rs10507172 and rs10507173 were associated with TB disease in an unadjusted analysis (p = 0.036 and 0.025, respectively, dominant model) that strengthened with sensitivity analysis of the definite TB cases, which included only those patients with microbiologically confirmed Mtb (p = 0.007 and 0.012, respectively). Knockdowns of HSP90B1 in monocyte cell lines with CRISPR did not alter TLR2 surface expression nor influence Mtb replication relative to controls. Among infants, an HSP90B1 gene-region variant is associated with BCG-induced IL-2 production and may be associated with protection from TB disease. HSP90B1 knockdown in human monocyte-like cell lines did not influence TLR2 surface localization nor Mtb replication. Together, these data suggest that HSP90B1 regulates T-cell, but not monocyte, responses to mycobacteria in humans.

Maintenance of long-lasting immunity is thought to depend on stem cell memory T cells (T ), which have superior self-renewing capacity, longevity and proliferative potential compared with central memory (T ) or effector (T ) T cells. Our knowledge of T derives primarily from studies of virus-specific CD8 T . We aimed to determine if infection with ( ), the etiological agent of tuberculosis, generates antigen-specific CD4 T and to characterize their functional ontology. We studied T cell responses to natural infection in a longitudinal adolescent cohort of recent QuantiFERON-TB Gold (QFT) converters and three cross-sectional QFT adult cohorts; and to bacillus Calmette-Guerin (BCG) vaccination in infants. and/or BCG-specific CD4 T cells were detected by flow cytometry using major histocompatibility complex class II tetramers bearing Ag85, CFP-10, or ESAT-6 peptides, or by intracellular cytokine staining. Transcriptomic analyses of -specific tetramer CD4 T (CD45RA CCR7 CD27 ) were performed by microfluidic qRT-PCR, and functional and phenotypic characteristics were confirmed by measuring expression of chemokine receptors, cytotoxic molecules and cytokines using flow cytometry. -specific T were not detected in QFT-negative persons. After QFT conversion frequencies of T increased to measurable levels and remained detectable thereafter, suggesting that primary infection induces T cells. Gene expression (GE) profiling of tetramer T showed that these cells were distinct from bulk CD4 naïve T cells (T ) and shared features of bulk T and -specific tetramer T and T cells. These T were predominantly CD95 and CXCR3 , markers typical of CD8 T . Tetramer T expressed significantly higher protein levels of CCR5, CCR6, CXCR3, granzyme A, granzyme K, and granulysin than bulk T and T cells. -specific T were also functional, producing IL-2, IFN-γ, and TNF-α upon antigen stimulation, and their frequencies correlated positively with long-term BCG-specific CD4 T cell proliferative potential after infant vaccination. Human infection with induced distinct, antigen-specific CD4 T cells endowed with effector functions, including expression of cytotoxic molecules and Th1 cytokines, and displayed chemokine receptor profiles consistent with memory Th1/17 cells. Induction of CD4 T should be considered for vaccination approaches that aim to generate long-lived memory T cells against .

Background Sensitive point-of-care screening tests are urgently needed to identify individuals at highest risk of tuberculosis. We prospectively tested performance of host-blood transcriptomic tuberculosis signatures. Methods Adults without suspicion of tuberculosis were recruited from five endemic South African communities. Eight parsimonious host-blood transcriptomic tuberculosis signatures were measured by microfluidic RT-qPCR at enrolment. Upper respiratory swab specimens were tested with a multiplex bacterial-viral RT-qPCR panel in a subset of participants. Diagnostic and prognostic performance for microbiologically confirmed prevalent and incident pulmonary tuberculosis was tested in all participants at baseline and during active surveillance through 15 months follow-up, respectively. Results Among 20,207 HIV-uninfected and 963 HIV-infected adults screened; 2923 and 861 were enroled. There were 61 HIV-uninfected (weighted prevalence 1.1%) and 10 HIV-infected (prevalence 1.2%) tuberculosis cases at baseline. Parsimonious signature diagnostic performance was superior among symptomatic (AUCs 0.85–0.98) as compared to asymptomatic (AUCs 0.61–0.78) HIV-uninfected participants. Thereafter, 24 HIV-uninfected and 9 HIV-infected participants progressed to incident tuberculosis (1.1 and 1.0 per 100 person-years, respectively). Among HIV-uninfected individuals, prognostic performance for incident tuberculosis occurring within 6–12 months was higher relative to 15 months. 1000 HIV-uninfected participants were tested for respiratory microorganisms and 413 HIV-infected for HIV plasma viral load; 7/8 signature scores were higher ( p  < 0.05) in participants with viral respiratory infections or detectable HIV viraemia than those without. Conclusions Several parsimonious tuberculosis transcriptomic signatures met triage test targets among symptomatic participants, and incipient test targets within 6 months. However, the signatures were upregulated with viral infection and offered poor specificity for diagnosing sub-clinical tuberculosis. Mendelsohn et al. evaluated in a prospective multicentre validation study whether host-blood transcriptomic tuberculosis (TB) signatures could be used to find active cases of TB. Whilst most of the signatures met WHO Target Product Profile criteria for a triage test to diagnose symptomatic TB; most signatures did not meet the criteria for asymptomatic TB. Plain Language Summary Delays in tuberculosis (TB) diagnosis result in increased disease severity, risk of death, and infection of further individuals. The presence of symptoms is typically used to find  people with TB. However, about half of individuals with TB are asymptomatic. We evaluated blood samples from individuals living in areas with high incidence of TB to see whether there were changes in components of the blood following infection with TB. Markers were identified that diagnosed TB in symptomatic adults, but were not as accurate to detect TB in those without symptoms. Most markers tested were able to accurately predict progression to TB within 6 months in HIV-uninfected individuals. These markers in the blood could enable the screening of symptomatic adults and predict TB risk, thus enabling targeting of therapy.

Myeloid-derived suppressor cells (MDSC) have been identified in the peripheral blood and granulomas of patients with active TB disease, but their phenotype-, function-, and immunosuppressive mechanism- spectrum remains unclear. Importantly, the frequency and signaling pathways of MDSC at the site of disease is unknown with no indication how this compares to MDSC identified in peripheral blood or to those of related myeloid counterparts such as alveolar macrophages and monocytes. Most phenotypic and functional markers have been described in oncological studies but have not yet been validated in TB. Using a panel of 43 genes selected from pathways previously shown to contribute to tumor-derived MDSC, we set out to evaluate if the expression of these additional functional markers and properties may also be relevant to TB-derived MDSC. Differential expression was investigated between MDSC, alveolar macrophages and monocytes enriched from bronchoalveolar lavage fluid and peripheral blood of patients with active TB, patients with other lung diseases (OLD). Results demonstrated that anatomical compartments may drive compartment-specific immunological responses and subsequent MDSC immunosuppressive functions, demonstrated by the observation that MDSC and/or monocytes from PB alone can discriminate, via hierarchical clustering, between patients with active TB disease and OLD. Our data show that the gene expression patterns of MDSC in peripheral blood and bronchoalveolar lavage fluid do not cluster according to disease states (TB vs OLD). This suggests that MDSC from TB patients may display similar gene expression profiles to those found for MDSC in cancer, but this needs to be validated in a larger cohort. These are important observations for TB research and may provide direction for future studies aimed at repurposing and validating cancer immunotherapies for use in TB.

Tuberculosis (TB) remains a major public health problem and we lack a comprehensive understanding of how Mycobacterium tuberculosis ( M. tb ) infection impacts host immune responses. We compared the induced immune response to TB antigen, BCG and IL-1β stimulation between latently M. tb infected individuals (LTBI) and active TB patients. This revealed distinct responses between TB/LTBI at transcriptomic, proteomic and metabolomic levels. At baseline, we identified a novel immune-metabolic association between pregnane steroids, the PPARγ pathway and elevated plasma IL-1ra in TB. We observed dysregulated IL-1 responses after BCG stimulation in TB patients, with elevated IL-1ra responses being explained by upstream TNF differences. Additionally, distinct secretion of IL-1α/IL-1β in LTBI/TB after BCG stimulation was associated with downstream differences in granzyme mediated cleavage. Finally, IL-1β driven signalling was dramatically perturbed in TB disease but was completely restored after successful treatment. This study improves our knowledge of how immune responses are altered during TB disease, and may support the design of improved preventive and therapeutic tools, including host-directed strategies.

•The TB vaccine development pipeline moves slowly and includes few candidates.•TB progression-associated candidate antigens have recently been identified.•Investigation to identify vaccine-induced correlates of protection against TB is underway.•The mRNA platform may accelerate screening and manufacturing of new TB vaccines.•Transparency and community engagement are critical for mRNA vaccine acceptability.•Capacity building in lesser resourced regions may improve equitable TB vaccine access. Tuberculosis (TB) is a global health emergency. Across the globe, approximately 2 billion people are currently infected with Mycobacterium tuberculosis (Mtb), and of those, 5–10% may progress to become ill and potentially transmit the bacterium. In 2021, nearly 10.6 million people developed TB disease and 1.6 million died. There is an urgent need for accelerated development of new TB-focused interventions, in particular, improved TB vaccines. However, progress in developing highly effective TB vaccines has been slow and is chronically under-resourced. The mRNA vaccine platform may offer an opportunity to accelerate development of new TB vaccines. In April 2023, the World Health Organization convened global experts to discuss the feasibility and potential value of mRNA-based vaccines for TB. Here we report on meeting deliberations related to the current TB vaccine pipeline and potential novel antigens, the status of efforts to identify correlates of protection, potential clinical development strategies and considerations for community acceptance of new TB vaccines based on this relatively new platform. The role of industry collaborations, ethics, social science, and responsibility to the global community regarding transparency and manufacturing capacity building were discussed through expert presentations and panel sessions. The overall conclusion of the meeting is that mRNA-based vaccines constitute a potentially powerful new tool for reducing the global burden of TB.

Individuals with a history of tuberculosis (TB) disease are at elevated risk of disease recurrence. The underlying cause is not known, but one explanation is that previous disease results in less-effective immunity against Mycobacterium tuberculosis (Mtb). We hypothesized that the repertoire of Mtb-derived epitopes recognized by T cells from individuals with latent Mtb infection differs as a function of previous diagnosis of active TB disease. T-cell responses to peptide pools in samples collected from an adult screening and an adolescent validation cohort were measured by IFN-γ enzyme-linked immunospot assay or intracellular cytokine staining. We identified a set of \"type 2\" T-cell epitopes that were recognized at 10-fold-lower levels in Mtb-infected individuals with a history of TB disease less than 6 years ago than in those without previous TB. By contrast, \"type 1\" epitopes were recognized equally well in individuals with or without previous TB. The differential epitope recognition was not due to differences in HLA class II binding, memory phenotypes, or gene expression in the responding T cells. Instead, \"TB disease history-sensitive\" type 2 epitopes were significantly (P < 0.0001) more homologous to sequences from bacteria found in the human microbiome than type 1 epitopes. Preferential loss of T-cell reactivity to Mtb epitopes that are homologous to bacteria in the microbiome in persons with previous TB disease may reflect long-term effects of antibiotic TB treatment on the microbiome.

The VALIDATE Network brings together scientists addressing vaccine development for neglected infectious diseases caused by intracellular pathogens. In September 2023, the first workshop on One Health approaches to research and capacity building was held in Paarl, South Africa, with a focus on tuberculosis and leishmaniasis. Thirty-two scientists from 15 countries presented and discussed broad topics pertinent to zoonotic diseases, cross-species disease transmission, disease mitigation strategies, inequitable access to medicines and health technologies, and health system challenges in One Health. In this report, we summarize the gaps, challenges, and opportunities identified during the 2023 VALIDATE One Health workshop. We anticipate that the experiences and dialogues will be informative for animal, human, and environmental health investigators to guide the development of research projects and vaccine development with a One Health vision.

The molecular mechanisms that regulate tuberculosis susceptibility and bacillus Calmette-Guérin (BCG)-induced immunity are mostly unknown. However, induction of the adaptive immune response is a critical step in host control of Mycobacterium tuberculosis. Toll-interacting protein (TOLLIP) is a ubiquitin-binding protein that regulates innate immune responses, including Toll-like receptor signaling, which initiate adaptive immunity. TOLLIP variation is associated with susceptibility to tuberculosis, but the mechanism by which it regulates tuberculosis immunity is poorly understood. To identify functional TOLLIP variants and evaluate the role of TOLLIP variation on innate and adaptive immune responses to mycobacteria and susceptibility to tuberculosis. We used human cellular immunology approaches to characterize the role of a functional TOLLIP variant on monocyte mRNA expression and M. tuberculosis-induced monocyte immune functions. We also examined the association of TOLLIP variation with BCG-induced T-cell responses and susceptibility to latent tuberculosis infection. We identified a functional TOLLIP promoter region single-nucleotide polymorphism, rs5743854, which was associated with decreased TOLLIP mRNA expression in infant monocytes. After M. tuberculosis infection, TOLLIP-deficient monocytes demonstrated increased IL-6, increased nitrite, and decreased bacterial replication. The TOLLIP-deficiency G/G genotype was associated with decreased BCG-specific IL-2 CD4 T-cell frequency and proliferation. This genotype was also associated with increased susceptibility to latent tuberculosis infection. TOLLIP deficiency is associated with decreased BCG-specific T-cell responses and increased susceptibility to tuberculosis. We hypothesize that the heightened antibacterial monocyte responses after vaccination of TOLLIP-deficient infants are responsible for decreased BCG-specific T-cell responses. Activating TOLLIP may provide a novel adjuvant strategy for BCG vaccination.

BackgroundNew safe and effective TB vaccine strategies to replace infant BCG vaccination are needed urgently. We evaluated the safety, reactogenicity and immunogenicity of three doses of the live-attenuated Mycobacterium tuberculosis (Mtb) vaccine candidate, MTBVAC, in comparison to BCG in South African newborns.MethodsHealthy infants, HIV-unexposed, BCG-naïve newborns without history of close TB contact were randomly allocated into three sequential cohorts to receive a single intradermal dose of BCG (SSI, 2.5×105 CFU) or MTBVAC (2.5×104; 2.5×105 CFU; or 2.5×106 CFU).Results228 pregnant women consented, and 99 newborns were enrolled. Seventy-eight infants across all 3 cohorts had local reactions, all rated mild, except one grade 2 erythema. Induration, swelling, and erythema was more common with increased MTBVAC dosage. Induration and swelling were more common in MTBVAC 2.5x106 than in BCG and reactogenicity in MTBVAC 2.5x105 was same as BCG. Twelve infants experienced 14 vaccine-unrelated SAEs including one death due to bronchopneumonia. Eight infants commenced TB treatment for unconfirmed pulmonary TB (BCG n=4 and MTBVAC 2.5x104 CFU n=4) and one for unconfirmed TB meningitis (BCG). MTBVAC was highly immunogenic at all 3 doses, inducing predominantly Th1-cytokine-expressing CD4 T-cells, which peaked at day 56 and waned thereafter. The 2.5×105 and 2.5×106 CFU MTBVAC doses were more immunogenic than BCG, inducing very similar response magnitudes and phenotypes. Vaccination with any MTBVAC dose resulted in QFT conversion in most infants at Day 56, but these responses waned and reverted to QFT-negative in more than half by the end of the 1-year follow-up period. ConclusionMTBVAC appeared safe and well tolerated and immunogenic at doses between 2.5×104 CFU and 2.5×106 CFU in South Africans newborns. The 2.5×105 CFU MTBVAC dose was selected for the ongoing phase 3 trial in a high TB prevalence setting.