Michael Levin

Vascular injury and coagulopathy are key drivers of mortality in bacterial sepsis. In Neisseria meningitidis infection, endothelial adhesion and thrombosis cause the characteristic petechial rash and, in the most severe cases, purpura fulminans. Although antibiotics rapidly kill bacteria, inflammation and vascular injury often persist or worsen after bacterial clearance, suggesting ongoing toxicity from released bacterial components. Here we identify bacterial histone-like proteins (HLPs), small positively charged DNA-binding proteins conserved across bacterial species, as previously unrecognized mediators of vascular damage. In vitro HLPs are released following antibiotic exposure, disrupting endothelial integrity. In patients with severe sepsis, they are detectable in plasma and tissue, colocalising with areas of vascular leak and coagulopathy. Non-anticoagulant heparins and anti-HLP antibodies neutralize HLP-induced endothelial disruption and toxicity in vitro and in vivo . These findings reveal HLPs as antibiotic-released bacterial toxins and suggest new therapeutic strategies to prevent vascular injury in sepsis. One sentence summary: Antibiotic treatment of meningococcal and other sepsis causative bacteria triggers the release of histone-like proteins (HLPs) that damage the endothelium and drive coagulopathy, but these effects can be neutralized by non-anticoagulant heparins or anti-HLP antibodies.

Controlled human influenza infection studies can uniquely interrogate the early immune factors associated with clinical outcome. In this study, 27 healthy volunteers with low strain-specific serum neutralizing antibody levels were challenged with influenza A/H3N2 virus. Twenty-two became infected, with 18 developing mild-to-moderate symptoms and four remaining asymptomatic. Local and systemic immune profiling revealed innate pathways that engaged more rapidly and to a higher level in symptomatic participants. Earlier monocyte and dendritic cell activation correlated with higher symptom scores but also enhanced natural killer and CD8 ⁺ T cell activation thereafter. At baseline, peripheral blood mononuclear cells from symptomatic participants were more responsive to in vitro challenge, indicating a predisposition to divergent immunological outcomes at the time of virus exposure that was subsequently modulated by infection. These results show that human innate cell responsiveness is a predeterminant of both symptomatic disease and cellular immune responses known to promote viral clearance, suggesting potential targets for therapeutic intervention if decoupled.

Mycobacterium tuberculosis infection is a dynamic continuum. Clinical outcomes reflect complex host-pathogen interactions. Epidemiological and animal studies have suggested influenza coinfection as a risk factor for progression from contained infection to active disease, but human studies have been lacking. Using a whole blood luminescent mycobacterial growth inhibition assay within a human influenza challenge study, we show that influenza infection reduces immunological control of mycobacterial growth. Transcriptome-wide RNA sequencing, cytokine and cellular analyses of subjects’ blood before and after influenza infection reveal that innate immune pathways, including type 1 interferon signalling, are activated by influenza but their subsequent responsiveness to mycobacteria is reduced, with multiple genes’ responses to BCG lux infection repressed by influenza coinfection. Our data suggest that influenza infection impairs immune mechanisms that contain mycobacterial growth and may be a risk factor for tuberculosis (TB) disease. Influenza vaccination might offer high risk, high prevalence populations protection against TB disease.

Transcriptomic analyses reveal the status of cells, tissues, or organisms, across states of health and disease. RNA velocity adds a temporal dimension to single cell analyses, predicting future transcriptomic and phenotypic states, based on the current spliced and unspliced mRNA of each cell. We hypothesized that RNA velocity could be adapted to predict future clinical state of individuals with acute and chronic illnesses, using their whole-blood transcriptomes. We developed VeloCD, a method for quantitative prediction of transitions in clinical state from a single time-point RNA sample. This predicts transcriptomic trajectories and future infection status in influenza A and SARS-CoV-2 controlled human infection studies, which are consistent with trajectories in naturally acquired infections. In HIV-TB coinfected individuals, VeloCD predicts the onset of immune reconstitution inflammatory syndrome. In individuals receiving biological therapy for inflammatory bowel disease, whole blood RNA velocity after the first dose of treatment indicates whether remission will be achieved by the end of the treatment course. In a multinational observational study of acutely unwell febrile children, VeloCD predicts those with greatest medical care requirements. Our results demonstrate proof-of-concept for the use of RNA velocity to predict trajectories of human diseases.

Introduction: Young children and children living with HIV are at high risk of progressing to tuberculosis (TB) disease following Mycobacterium tuberculosis ( Mtb ) exposure and infection, and also of developing severe forms of disease and TB-related mortality. Identifying children who have very early (sub-clinical) TB disease, prior to progression to clinically apparent TB, would mean that TB preventive treatment (TPT) could be more efficiently targeted to this group. Identifying biomarker changes on drug therapy in children with Mtb infection or very early disease could pave the way for the development of tests that can identify which children have viable bacilli and are therefore at increased risk of disease progression. Methods and analysis: The INTREPID study will use already collected samples taken from well-phenotyped paediatric cohorts in three clinical studies conducted in South Africa in children Mtb exposure to disease and from children treated for Mtb infection and early TB disease, as well as targeted Mtb antibody analysis. Data on viral co-infections and relevant clinical and epidemiological parameters will be integrated and evaluated to identify the optimal biosignatures that can predict future progression to clinically overt disease in children below 5 years of age, including those living with HIV. Ethics and dissemination: The study protocol received ethical approval from the Stellenbosch University Health Research Ethics Committee (N23/03/025). The study findings will be disseminated through peer-reviewed publications, scientific conferences and formal presentations to healthcare professionals and to local communities, in collaboration with the Desmond Tutu TB Centre Community Advisory Board.

Background: Identifying the cause of infection is important for clinical management and public health decisions, including vaccination strategies. In low-resource settings, causes of fever are often not identified. In this study, molecular testing panels were used to identify the causes of pediatric fever in the Kathmandu Valley, Nepal. A dengue fever outbreak facilitated the investigation of dengue diagnostics. Methods: Children under 14 years of age were recruited to this prospective cohort study at Patan Hospital, Nepal. Clinical data and routine diagnostics were used to classify cases, including nonstructural protein 1 (NS1) antigen testing for dengue. Additional molecular diagnostics were performed on blood (12 viral, 26 bacterial and 6 fungal targets) and respiratory samples (17 viral and 3 bacterial targets). Results: From September 1, 2021, to April 19, 2023, 565 children were enrolled, median age 3 (interquartile-range 1–7) years. Pathogens identified included dengue virus (n = 101), respiratory syncytial virus (n = 30), influenza (n = 25), typhoidal Salmonella spp. (n = 7) and Neisseria meningitidis (n = 2). During the dengue outbreak, dengue polymerase chain reaction (PCR) and NS1 positivity rates were both high early in dengue disease, but if >3 days of symptoms, PCR positivity rates declined (10.3%) while NS1 positivity remained high into the second week of illness (80%). Conclusions: This prospective cohort study is the most comprehensive effort to date to describe the causes of pediatric fever in the Kathmandu Valley, Nepal. The United States Centers for Disease Control and Prevention recommends dengue PCR or NS1 antigen testing during the first 7 days of dengue fever. Our data indicate that PCR positivity declines after 3 days of symptoms, resulting in missed cases when relying solely on PCR.

Expression Atlas (https://www.ebi.ac.uk/gxa/home) is EMBL-EBI’s comprehensive knowledgebase for gene and protein expression across tissues, cell types, conditions, and multiple species. Since our last update, Expression Atlas has expanded substantially in both content and functionality, now comprising >4500 studies from 67 species, with increased proteomics coverage and updated Genotype-Tissue Expression (GTEx) tissue profiles. The resource also includes hundreds of single-cell RNA-seq experiments spanning 21 species, among them externally analysed community datasets such as Tabula Sapiens and GTEx single-nucleus profiles, allowing exploration of curated atlases while maintaining their original analytical framework. Key methodological advances include a new marker gene analysis module for bulk baseline experiments, alongside workflow updates that improve reproducibility. Expression Atlas data are integrated into EMBL-EBI resources such as Ensembl, UniProt, and Europe PMC and disseminated through collaboration with model organism communities such as FlyBase and Gramene. The resource also supports translational research through the European Diagnostic Transcriptomic Library and integration with the Open Targets platform. Future directions include modernizing analysis pipelines, enhancing programmatic access, and delivering AI-ready data formats, strengthening Expression Atlas as a findable, accessible, interoperable, and reusable (FAIR) community-driven resource for both fundamental and translational discovery.

Mycobacterium tuberculosis infection is a dynamic continuum. Clinical outcomes reflect complex host-pathogen interactions. Epidemiological and animal studies have suggested influenza coinfection as a risk factor for progression from contained infection to active disease, but human studies have been lacking. Using a whole blood luminescent mycobacterial growth inhibition assay within a human influenza challenge study, we show that influenza infection reduces immunological control of mycobacterial growth. Transcriptome-wide RNA sequencing, cytokine and cellular analyses of subjects’ blood before and after influenza infection showed that innate immune pathways, including type 1 interferon signalling, are activated by influenza but their subsequent responsiveness to mycobacteria is reduced, with multiple genes’ responses to BCG lux infection repressed by influenza coinfection. The data suggest that influenza infection impairs immune mechanisms that contain mycobacterial growth and may be a risk factor for tuberculosis (TB) disease. Influenza vaccination might offer high risk, high prevalence populations protection against TB disease.

Importance: The upsurge in invasive disease caused by Streptococcus pyogenes among children reported in several European countries during 2022-2023 has not been fully explained. Objective: To test the hypothesis that changes in circulation of common respiratory pathogens associated with the introduction of non-pharmaceutical interventions (NPIs) during the COVID-19 pandemic altered acquisition of immunity to S. pyogenes and common respiratory viruses. Design: Cross-sectional study recruiting before (September 2016 to March 2020) and after (April 2020 to July 2023) introduction of NPIs. Setting: European hospitals in 10 countries. Participants: Children with suspected infection and afebrile controls, aged 0-4 years. Main Outcomes: Molecular detection of bacterial and viral pathogens on throat swabs and age-stratified total serum IgG reactivity to S. pyogenes cell wall extract from two strains (measured by ELISA), and respiratory syncytial virus (RSV), five influenza viruses and four common cold coronaviruses and SARS-CoV-2 measured by Mesoscale immunoassay. Results: Throat swabs from 1942 children under 5-years of age were tested for respiratory pathogens (1449 recruited before introduction of NPIs; 493 recruited after). A decrease in detection of S. pyogenes, RSV, common cold coronaviruses, and influenza viruses was observed between March 2020 to July 2021, corresponding to the maximal period of NPIs. Antibodies to S. pyogenes were measured in 252 children recruited before NPIs and 200 thereafter. Antibodies to viral antigens were measured in 230 before NPIs and 92 thereafter. Total IgG to S. pyogenes and RSV was significantly lower in children aged 3-4 years recruited after NPI introduction compared with before ( S. pyogenes emm1 : after, n=67, median 0.13 vs before, n=87, 0.35 IVIG relative-units, p=0.007. RSV: after, n=30, median 49604 vs before, n=76, 141782 mesoscale-units pS. pyogenes and RSV in children aged 3-4 years after introduction of NPIs. Equivalent to approximately a year delay in acquisition of immunity, these data provide a biological basis for the 2022-2023 upsurge in severe S. pyogenes infections in this age group.

Transcriptomic analyses reveal the status of cells, tissues, or organisms, across states of health and disease. RNA velocity adds a temporal dimension to single cell analyses, predicting future transcriptomic and phenotypic states, based on current spliced and unspliced mRNA of each cell. We hypothesized that RNA velocity could be adapted to predict future clinical status of individuals with acute illness using their whole-blood transcriptome. We developed a method for quantitative prediction of transitions in clinical state from a single time-point sample, which we call VeloCD. This predicted transcriptomic trajectories and future infection status in influenza A and SARS-CoV-2 human challenge studies. In HIV-TB coinfected individuals, it predicted the onset of immune reconstitution inflammatory syndrome. In a multinational observational study of acutely unwell febrile children, VeloCD predicted those with greatest medical care requirements. Our results demonstrate a novel application of RNA velocity to predict the trajectory of acute illness.