Single-cell profiling of tuberculosis lung granulomas reveals functional lymphocyte signatures of bacterial control [preprint]

In humans and nonhuman primates, Mycobacterium tuberculosis lung infection yields a complex multicellular structure—the tuberculosis granuloma. All granulomas are not equivalent, however, even within the same host: in some, local immune activity promotes bacterial clearance, while in others, it allows persistence or outgrowth. Here, we used single-cell RNA-sequencing to define holistically cellular responses associated with control in cynomolgus macaques. Granulomas that facilitated bacterial killing contained significantly higher proportions of CD4+ and CD8+ T cells expressing hybrid Type1-Type17 immune responses or stem-like features and CD8-enriched T cells with specific cytotoxic functions; failure to control correlated with mast cell, plasma cell and fibroblast abundance. Co-registering these data with serial PET-CT imaging suggests that a degree of early immune control can be achieved through cytotoxic activity, but that more robust restriction only arises after the priming of specific adaptive immune responses, defining new targets for vaccination and treatment

Neutrophil Dynamics Affect Mycobacterium tuberculosis Granuloma Outcomes and Dissemination

Neutrophil infiltration into tuberculous granulomas is often associated with higher bacteria loads and severe disease but the basis for this relationship is not well understood. To better elucidate the connection between neutrophils and pathology in primate systems, we paired data from experimental studies with our next generation computational model GranSim to identify neutrophil-related factors, including neutrophil recruitment, lifespan, and intracellular bacteria numbers, that drive granuloma-level outcomes. We predict mechanisms underlying spatial organization of neutrophils within granulomas and identify how neutrophils contribute to granuloma dissemination. We also performed virtual deletion and depletion of neutrophils within granulomas and found that neutrophils play a nuanced role in determining granuloma outcome, promoting uncontrolled bacterial growth in some and working to contain bacterial growth in others. Here, we present three key results: We show that neutrophils can facilitate local dissemination of granulomas and thereby enable the spread of infection. We suggest that neutrophils influence CFU burden during both innate and adaptive immune responses, implying that they may be targets for therapeutic interventions during later stages of infection. Further, through the use of uncertainty and sensitivity analyses, we predict which neutrophil processes drive granuloma severity and structure

Population tailored modification of tuberculosis specific interferon-gamma release assay

Objectives: Blood-based Interferon-Gamma Release Assays (IGRA) identify Mycobacterium tuberculosis (MTB) sensitisation with increased specificity, but sensitivity remains impaired in human immunodeficiency virus (HIV) infected persons. The QuantiFERON-TB Gold In-Tube test contains peptide 38e55 of Rv2654c, based on data indicating differential recognition between tuberculosis patients and BCG vaccinated controls in Europe. We aimed to fine map the T cell response to Rv2654c with the view of improving sensitivity. Methods: Interferon-gamma ELISpot assay was used in HIV uninfected persons with latent and active tuberculosis to map peptide epitopes of Rv2654c. A modified IGRA was tested in two further groups of 55 HIV uninfected and 44 HIV infected persons, recruited in South Africa. Results: The most prominently recognised peptide was between amino acids 51e65. Using p51-65 to boost the QuantiFERON-TB Gold In-Tube assay, the quantitative performance of the modified IGRA increased from 1.83 IU/ml (IQR 0.30e7.35) to 2.83 (IQR 0.28e12.2; p Z 0.002) in the HIV uninfected group. In the HIV infected cohort the percentage of positive responders increased from 57% to 64% but only after 3 months of ART (p Z ns)

High prevalence of subclinical tuberculosis in HIV-1-infected persons without advanced immunodeficiency: implications for TB screening

Background The prevalence of asymptomatic tuberculosis (TB) in recently diagnosed HIV-1-infected persons attending pre-antiretroviral therapy (ART) clinics is not well described. In addition, it is unclear if the detection of Mycobacterium tuberculosis in these patients clearly represents an early asymptomatic phase leading to progressive disease or transient excretion of bacilli. Objective To describe the prevalence and outcome of subclinical TB disease in HIV-1-infected persons not eligible for ART. Methods The study was conducted in 274 asymptomatic ART-naive HIV-1-infected persons in Khayelitsha Day Hospital, Cape Town, South Africa. All participants were screened for TB using a symptom screen and spoligotyping was performed to determine genotypes. Results The prevalence of subclinical TB disease was 8.5% (95% CI 5.1% to 13.0%) (n = 18; median days to culture positivity 17 days), with 22% of patients being smear-positive. Spoligotyping showed a diverse variety of genotypes with all paired isolates being of the same spoligotype, effectively excluding cross-contamination. 56% of patients followed up developed symptoms 3 days to 2 months later. All were well and still in care 6-12 months after TB diagnosis; 60% were started on ART. A positive tuberculin skin test (OR 4.96, p = 0.064), low CD4 count (OR 0.996, p = 0.06) and number of years since HIV diagnosis (OR 1.006, p = 0.056) showed trends towards predicting TB disease. Conclusion This study found a high prevalence but good outcome (retained in care) of subclinical TB disease in HIV-1-infected persons. The results suggest that, in high HIV/TB endemic settings, a positive HIV-1 test should prompt TB screening by sputum culture irrespective of symptoms, particularly in those with a positive tuberculin skin test, longer history of HIV infection and low CD4 count. Operational difficulties in resource-constrained settings with respect to screening with TB culture highlight the need for rapid and affordable point-of-care tests to identify persons with clinical and subclinical TB disease.Immunogenetics and cellular immunology of bacterial infectious disease

Impairment of IFN-Gamma Response to Synthetic Peptides of Mycobacterium tuberculosis in a 7-Day Whole Blood Assay

Studies on Mycobacterium tuberculosis (MTB) antigens are of interest in order to improve vaccine efficacy and to define biomarkers for diagnosis and treatment monitoring. The methodologies used for these investigations differ greatly between laboratories and discordant results are common. The IFN-gamma response to two well characterized MTB antigens ESAT-6 and CFP-10, in the form of recombinant proteins and synthetic peptides, was evaluated in HIV-1 uninfected persons in both long-term (7 day) and 24 hour, commercially available QuantiFERON TB Gold in Tube (QFT-GIT), whole blood assays. Our findings showed differences in the IFN-gamma response between 24 hour and 7 day cultures, with recombinant proteins inducing a significantly higher response than the peptide pools in 7 day whole blood assays. The activity of peptides and recombinant proteins did not differ in 24 hour whole blood or peripheral blood mononuclear cell (PBMC) based assays, nor in the ELISpot assay. Further analysis by SELDI-TOF mass spectrometry showed that the peptides are degraded over the course of 7 days of incubation in whole blood whilst the recombinant proteins remain intact. This study therefore demonstrates that screening antigenic candidates as synthetic peptides in long-term whole blood assays may underestimate immunogenicity

International genome-wide meta-analysis identifies new primary biliary cirrhosis risk loci and targetable pathogenic pathways.

Primary biliary cirrhosis (PBC) is a classical autoimmune liver disease for which effective immunomodulatory therapy is lacking. Here we perform meta-analyses of discovery data sets from genome-wide association studies of European subjects (n=2,764 cases and 10,475 controls) followed by validation genotyping in an independent cohort (n=3,716 cases and 4,261 controls). We discover and validate six previously unknown risk loci for PBC (Pcombined<5 × 10(-8)) and used pathway analysis to identify JAK-STAT/IL12/IL27 signalling and cytokine-cytokine pathways, for which relevant therapies exist

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Global age-sex-specific mortality, life expectancy, and population estimates in 204 countries and territories and 811 subnational locations, 1950–2021, and the impact of the COVID-19 pandemic: a comprehensive demographic analysis for the Global Burden of Disease Study 2021

Background: Estimates of demographic metrics are crucial to assess levels and trends of population health outcomes. The profound impact of the COVID-19 pandemic on populations worldwide has underscored the need for timely estimates to understand this unprecedented event within the context of long-term population health trends. The Global Burden of Diseases, Injuries, and Risk Factors Study (GBD) 2021 provides new demographic estimates for 204 countries and territories and 811 additional subnational locations from 1950 to 2021, with a particular emphasis on changes in mortality and life expectancy that occurred during the 2020–21 COVID-19 pandemic period. Methods: 22 223 data sources from vital registration, sample registration, surveys, censuses, and other sources were used to estimate mortality, with a subset of these sources used exclusively to estimate excess mortality due to the COVID-19 pandemic. 2026 data sources were used for population estimation. Additional sources were used to estimate migration; the effects of the HIV epidemic; and demographic discontinuities due to conflicts, famines, natural disasters, and pandemics, which are used as inputs for estimating mortality and population. Spatiotemporal Gaussian process regression (ST-GPR) was used to generate under-5 mortality rates, which synthesised 30 763 location-years of vital registration and sample registration data, 1365 surveys and censuses, and 80 other sources. ST-GPR was also used to estimate adult mortality (between ages 15 and 59 years) based on information from 31 642 location-years of vital registration and sample registration data, 355 surveys and censuses, and 24 other sources. Estimates of child and adult mortality rates were then used to generate life tables with a relational model life table system. For countries with large HIV epidemics, life tables were adjusted using independent estimates of HIV-specific mortality generated via an epidemiological analysis of HIV prevalence surveys, antenatal clinic serosurveillance, and other data sources. Excess mortality due to the COVID-19 pandemic in 2020 and 2021 was determined by subtracting observed all-cause mortality (adjusted for late registration and mortality anomalies) from the mortality expected in the absence of the pandemic. Expected mortality was calculated based on historical trends using an ensemble of models. In location-years where all-cause mortality data were unavailable, we estimated excess mortality rates using a regression model with covariates pertaining to the pandemic. Population size was computed using a Bayesian hierarchical cohort component model. Life expectancy was calculated using age-specific mortality rates and standard demographic methods. Uncertainty intervals (UIs) were calculated for every metric using the 25th and 975th ordered values from a 1000-draw posterior distribution. Findings: Global all-cause mortality followed two distinct patterns over the study period: age-standardised mortality rates declined between 1950 and 2019 (a 62·8% [95% UI 60·5–65·1] decline), and increased during the COVID-19 pandemic period (2020–21; 5·1% [0·9–9·6] increase). In contrast with the overall reverse in mortality trends during the pandemic period, child mortality continued to decline, with 4·66 million (3·98–5·50) global deaths in children younger than 5 years in 2021 compared with 5·21 million (4·50–6·01) in 2019. An estimated 131 million (126–137) people died globally from all causes in 2020 and 2021 combined, of which 15·9 million (14·7–17·2) were due to the COVID-19 pandemic (measured by excess mortality, which includes deaths directly due to SARS-CoV-2 infection and those indirectly due to other social, economic, or behavioural changes associated with the pandemic). Excess mortality rates exceeded 150 deaths per 100 000 population during at least one year of the pandemic in 80 countries and territories, whereas 20 nations had a negative excess mortality rate in 2020 or 2021, indicating that all-cause mortality in these countries was lower during the pandemic than expected based on historical trends. Between 1950 and 2021, global life expectancy at birth increased by 22·7 years (20·8–24·8), from 49·0 years (46·7–51·3) to 71·7 years (70·9–72·5). Global life expectancy at birth declined by 1·6 years (1·0–2·2) between 2019 and 2021, reversing historical trends. An increase in life expectancy was only observed in 32 (15·7%) of 204 countries and territories between 2019 and 2021. The global population reached 7·89 billion (7·67–8·13) people in 2021, by which time 56 of 204 countries and territories had peaked and subsequently populations have declined. The largest proportion of population growth between 2020 and 2021 was in sub-Saharan Africa (39·5% [28·4–52·7]) and south Asia (26·3% [9·0–44·7]). From 2000 to 2021, the ratio of the population aged 65 years and older to the population aged younger than 15 years increased in 188 (92·2%) of 204 nations. Interpretation: Global adult mortality rates markedly increased during the COVID-19 pandemic in 2020 and 2021, reversing past decreasing trends, while child mortality rates continued to decline, albeit more slowly than in earlier years. Although COVID-19 had a substantial impact on many demographic indicators during the first 2 years of the pandemic, overall global health progress over the 72 years evaluated has been profound, with considerable improvements in mortality and life expectancy. Additionally, we observed a deceleration of global population growth since 2017, despite steady or increasing growth in lower-income countries, combined with a continued global shift of population age structures towards older ages. These demographic changes will likely present future challenges to health systems, economies, and societies. The comprehensive demographic estimates reported here will enable researchers, policy makers, health practitioners, and other key stakeholders to better understand and address the profound changes that have occurred in the global health landscape following the first 2 years of the COVID-19 pandemic, and longer-term trends beyond the pandemic