Targeted metagenomics reveals association between severity and pathogen co-detection in infants with respiratory syncytial virus.

Respiratory syncytial virus (RSV) is the leading cause of hospitalisation for respiratory infection in young children. RSV disease severity is known to be age-dependent and highest in young infants, but other correlates of severity, particularly the presence of additional respiratory pathogens, are less well understood. In this study, nasopharyngeal swabs were collected from two cohorts of RSV-positive infants <12 months in Spain, the UK, and the Netherlands during 2017-20. We show, using targeted metagenomic sequencing of >100 pathogens, including all common respiratory viruses and bacteria, from samples collected from 433 infants, that burden of additional viruses is common (111/433, 26%) but only modestly correlates with RSV disease severity. In contrast, there is strong evidence in both cohorts and across age groups that presence of Haemophilus bacteria (194/433, 45%) is associated with higher severity, including much higher rates of hospitalisation (odds ratio 4.25, 95% CI 2.03-9.31). There is no evidence for association between higher severity and other detected bacteria, and no difference in severity between RSV genotypes. Our findings reveal the genomic diversity of additional pathogens during RSV infection in infants, and provide an evidence base for future causal investigations of the impact of co-infection on RSV disease severity.

RSV-related Community COPD Exacerbations and Novel Diagnostics: A Binational Prospective Cohort Study.

RATIONALE: Respiratory syncytial virus (RSV) is a common global respiratory virus increasingly recognized as a major pathogen in frail older adults and as a cause of chronic obstructive pulmonary disease (COPD) exacerbations. There is no single test for RSV in adults with acceptable diagnostic accuracy. Trials of RSV vaccines have recently shown excellent safety and efficacy against RSV in older adults; defining the frequency of RSV-related community infections and COPD exacerbations is important for vaccine deployment decisions. OBJECTIVES: This prospective study aimed to establish the frequency of outpatient-managed RSV-related exacerbations of COPD in two well-characterized patient cohorts using a combination of diagnostic methods. METHODS: Participants were recruited at specialist clinics in London, UK and Groningen, NL from 2017 and observed for three consecutive RSV seasons, during exacerbations and at least twice yearly. RSV infections were detected by reverse transcription-polymerase chain reaction (RT-PCR) and serologic testing. MEASUREMENTS AND MAIN RESULTS: 377 patients with COPD attended 1,999 clinic visits and reported 310 exacerbations. There were 27 RSV-related exacerbations (8·7% of total); of these, seven were detected only on PCR, 16 only on serology and 4 by both methods. Increases in RSV specific N-protein antibody were as sensitive as antibody to pre-F or post-F for serodiagnosis of RSV related exacerbations. CONCLUSIONS: RSV is associated with 8.7% of outpatient managed COPD exacerbations in this study. Antibodies to RSV-N protein may have diagnostic value, potentially important in a vaccinated population. The introduction of vaccines that prevent RSV is expected to benefit patients with COPD. This article is open access and distributed under the terms of the Creative Commons Attribution 4.0 International License (https://creativecommons.org/licenses/by/4.0/).

A Genome-Wide Association Study of Respiratory Syncytial Virus Infection Severity in Infants.

BACKGROUND: Respiratory syncytial virus (RSV) is a significant cause of infant morbidity and mortality worldwide. Most children experience at least one 1 RSV infection by the age of two 2 years, but not all develop severe disease. However, the understanding of genetic risk factors for severe RSV is incomplete. Consequently, we conducted a genome-wide association study of RSV severity. METHODS: Disease severity was assessed by the ReSVinet scale, in a cohort of 251 infants aged 1 week to 1 year. Genotyping data were collected from multiple European study sites as part of the RESCEU Consortium. Linear regression models were used to assess the impact of genotype on RSV severity and gene expression as measured by microarray. RESULTS: While no SNPs reached the genome-wide statistical significance threshold (P < 5 × 10-8), we identified 816 candidate SNPs with a P-value of <1 × 10-4. Functional annotation of candidate SNPs highlighted genes relevant to neutrophil trafficking and cytoskeletal functions, including LSP1 and RAB27A. Moreover, SNPs within the RAB27A locus significantly altered gene expression (false discovery rate, FDR P < .05). CONCLUSIONS: These findings may provide insights into genetic mechanisms driving severe RSV infection, offering biologically relevant information for future investigations.

External Validation of the Discriminative Validity of the ReSVinet Score and Development of Simplified ReSVinet Scores in Secondary Care.

BACKGROUND: There is no consensus on how to best quantify disease severity in infants with respiratory syncytial virus (RSV) and/or bronchiolitis; this lack of a sufficiently validated score complicates the provision of clinical care and, the evaluation of trials of therapeutics and vaccines. The ReSVinet score appears to be one of the most promising; however, it is too time consuming to be incorporated into routine clinical care. We aimed to develop and externally validate simplified versions of this score. METHODS: Data from a multinational (the Netherlands, Spain, and United Kingdom) multicenter case-control study of infants with RSV were used to develop simplified versions of the ReSVinet score by conducting a grid search to determine the best combination of equally weighted parameters to maximize for the discriminative ability (measured by area under the receiver operating characteristic curve [AUROC]) across a range of outcomes (hospitalization, intensive care unit admission, ventilation requirement). Subsequently discriminative validity of the score for a range of secondary care outcomes was externally validated by secondary analysis of datasets from Rwanda and Colombia. RESULTS: Three candidate simplified scores were identified using the development dataset; they were excellent (AUROC >0.9) at discriminating for a range of outcomes, and their performance was not significantly different from the original ReSVinet score despite having fewer parameters. In the external validation datasets, the simplified scores were moderate to excellent (AUROC, 0.7-1) across a range of outcomes. In all outcomes, except in a single dataset for predicting admission to the high-dependency unit, they performed at least as well as the original ReSVinet score. CONCLUSIONS: The candidate simplified scores developed require further external validation in larger datasets, ideally from resource-limited settings before any recommendation regarding their use.

Single-cell immune profiling reveals markers of emergency myelopoiesis that distinguish severe from mild respiratory syncytial virus disease in infants.

Whereas most infants infected with respiratory syncytial virus (RSV) show no or only mild symptoms, an estimated 3 million children under five are hospitalized annually due to RSV disease. This study aimed to investigate biological mechanisms and associated biomarkers underlying RSV disease heterogeneity in young infants, enabling the potential to objectively categorize RSV-infected infants according to their medical needs. Immunophenotypic and functional profiling demonstrated the emergence of immature and progenitor-like neutrophils, proliferative monocytes (HLA-DRLow , Ki67+), impaired antigen-presenting function, downregulation of T cell response and low abundance of HLA-DRLow B cells in severe RSV disease. HLA-DRLow monocytes were found as a hallmark of RSV-infected infants requiring hospitalization. Complementary transcriptomics identified genes associated with disease severity and pointed to the emergency myelopoiesis response. These results shed new light on mechanisms underlying the pathogenesis and development of severe RSV disease and identified potential new candidate biomarkers for patient stratification.

T cells, more than antibodies, may prevent symptoms developing from respiratory syncytial virus infections in older adults.

Introduction: The immune mechanisms supporting partial protection from reinfection and disease by the respiratory syncytial virus (RSV) have not been fully characterized. In older adults, symptoms are typically mild but can be serious in patients with comorbidities when the infection extends to the lower respiratory tract. Methods: This study formed part of the RESCEU older-adults prospective-cohort study in Northern Europe (2017-2019; NCT03621930) in which a thousand participants were followed over an RSV season. Peripheral-blood samples (taken pre-season, post-season, during illness and convalescence) were analyzed from participants who (i) had a symptomatic acute respiratory tract infection by RSV (RSV-ARTI; N=35) or (ii) asymptomatic RSV infection (RSV-Asymptomatic; N=16). These analyses included evaluations of antibody (Fc-mediated-) functional features and cell-mediated immunity, in which univariate and machine-learning (ML) models were used to explore differences between groups. Results: Pre-RSV-season peripheral-blood biomarkers were predictive of symptomatic RSV infection. T-cell data were more predictive than functional antibody data (area under receiver operating characteristic curve [AUROC] for the models were 99% and 76%, respectively). The pre-RSV season T-cell phenotypes which were selected by the ML modelling and which were more frequent in RSV-Asymptomatic group than in the RSV-ARTI group, coincided with prominent phenotypes identified during convalescence from RSV-ARTI (e.g., IFN-γ+, TNF-α+ and CD40L+ for CD4+, and IFN-γ+ and 4-1BB+ for CD8+). Conclusion: The evaluation and statistical modelling of numerous immunological parameters over the RSV season suggests a primary role of cellular immunity in preventing symptomatic RSV infections in older adults.

Enolase represents a metabolic checkpoint controlling the differential exhaustion programmes of hepatitis virus-specific CD8+ T cells.

OBJECTIVE: Exhausted T cells with limited effector function are enriched in chronic hepatitis B and C virus (HBV and HCV) infection. Metabolic regulation contributes to exhaustion, but it remains unclear how metabolism relates to different exhaustion states, is impacted by antiviral therapy, and if metabolic checkpoints regulate dysfunction. DESIGN: Metabolic state, exhaustion and transcriptome of virus-specific CD8+ T cells from chronic HBV-infected (n=31) and HCV-infected patients (n=52) were determined ex vivo and during direct-acting antiviral (DAA) therapy. Metabolic flux and metabolic checkpoints were tested in vitro. Intrahepatic virus-specific CD8+ T cells were analysed by scRNA-Seq in a HBV-replicating murine in vivo model of acute and chronic infection. RESULTS: HBV-specific (core18-27, polymerase455-463) and HCV-specific (NS31073-1081, NS31406-1415, NS5B2594-2602) CD8+ T cell responses exhibit heterogeneous metabolic profiles connected to their exhaustion states. The metabolic state was connected to the exhaustion profile rather than the aetiology of infection. Mitochondrial impairment despite intact glucose uptake was prominent in severely exhausted T cells linked to elevated liver inflammation in chronic HCV infection and in HBV polymerase455-463 -specific CD8+ T cell responses. In contrast, relative metabolic fitness was observed in HBeAg-negative HBV infection in HBV core18-27-specific responses. DAA therapy partially improved mitochondrial programmes in severely exhausted HCV-specific T cells and enriched metabolically fit precursors. We identified enolase as a metabolic checkpoint in exhausted T cells. Metabolic bypassing improved glycolysis and T cell effector function. Similarly, enolase deficiency was observed in intrahepatic HBV-specific CD8+ T cells in a murine model of chronic infection. CONCLUSION: Metabolism of HBV-specific and HCV-specific T cells is strongly connected to their exhaustion severity. Our results highlight enolase as metabolic regulator of severely exhausted T cells. They connect differential bioenergetic fitness with distinct exhaustion subtypes and varying liver disease, with implications for therapeutic strategies.

Single-cell RNA sequencing of liver fine-needle aspirates captures immune diversity in the blood and liver in chronic hepatitis B patients.

BACKGROUND AND AIMS: HBV infection is restricted to the liver, where it drives exhaustion of virus-specific T and B cells and pathogenesis through dysregulation of intrahepatic immunity. Our understanding of liver-specific events related to viral control and liver damage has relied almost solely on animal models, and we lack useable peripheral biomarkers to quantify intrahepatic immune activation beyond cytokine measurement. Our objective was to overcome the practical obstacles of liver sampling using fine-needle aspiration and develop an optimized workflow to comprehensively compare the blood and liver compartments within patients with chronic hepatitis B using single-cell RNA sequencing. APPROACH AND RESULTS: We developed a workflow that enabled multi-site international studies and centralized single-cell RNA sequencing. Blood and liver fine-needle aspirations were collected, and cellular and molecular captures were compared between the Seq-Well S 3 picowell-based and the 10× Chromium reverse-emulsion droplet-based single-cell RNA sequencing technologies. Both technologies captured the cellular diversity of the liver, but Seq-Well S 3 effectively captured neutrophils, which were absent in the 10× dataset. CD8 T cells and neutrophils displayed distinct transcriptional profiles between blood and liver. In addition, liver fine-needle aspirations captured a heterogeneous liver macrophage population. Comparison between untreated patients with chronic hepatitis B and patients treated with nucleoside analogs showed that myeloid cells were highly sensitive to environmental changes while lymphocytes displayed minimal differences. CONCLUSIONS: The ability to electively sample and intensively profile the immune landscape of the liver, and generate high-resolution data, will enable multi-site clinical studies to identify biomarkers for intrahepatic immune activity in HBV and beyond.

Clinical and Viral Factors Associated With Disease Severity and Subsequent Wheezing in Infants With Respiratory Syncytial Virus Infection.

Respiratory syncytial virus (RSV) causes substantial morbidity and mortality in infants and young children worldwide. Here we evaluated host demographic and viral factors associated with RSV disease severity in 325 RSV-infected infants under 1 year of age from 3 European countries during 2017-2020. Younger infants had a higher clinical severity (ReSViNET) score and were more likely to require hospitalization, intensive care, respiratory support, and/or mechanical ventilation than older infants (<3 months vs 3 to <6 months and 3 to <6 months vs ≥6 months). Older age (≥6 months vs <3 months), higher viral load, and RSV-A were associated with a greater probability of fever. RSV-A and RSV-B caused similar disease severity and had similar viral dynamics. Infants with a more severe RSV infection, demonstrated by having a higher ReSViNET score, fever, and requiring hospitalization and intensive care, were more likely to have developed subsequent wheezing at 1 year of age. CLINICAL TRIALS REGISTRATION: NCT03756766.

The merit of pressure dependent kinetic modelling in steam cracking.

Renewable cracking feedstocks from plastic waste and the need for novel reactor designs related to electrification of steam crackers drives the development of accurate and fundamental kinetic models for this process, despite its large scale implementation for more than half a century. Pressure dependent kinetics have mostly been omitted in fundamental steam cracking models, while they are crucial in combustion models. Therefore, we have assessed the importance of pressure dependent kinetics for steam cracking via in-depth modelling and experimental studies. In particular we have studied the influence of considering fall-off on the product yields for ethane and propane steam cracking. A high-pressure limit fundamental kinetic model is generated, based on quantum chemical data and group additive values, and supplemented with literature values for pressure dependent kinetic parameters for ß-scission reactions and homolytic bond scissions of C2 and C3 species. Model simulations with high-pressure limit rate coefficients and pressure dependent kinetics are compared to new experimental measurements. Steam cracking experiments for pure ethane and propane feeds are performed on a tubular bench-scale reactor at 0.17 MPa and temperatures ranging from 1058 to 1178 K. All important product species are identified using a comprehensive GC × GC-FID/q-MS. For homolytic bond scissions, the inclusion of pressure dependent kinetics has a significant effect on the conversion profile for ethane steam cracking. On the other hand, pressure dependence of C2 ß-scissions significantly influences conversion and product species profiles for both ethane and propane steam cracking. The C3 ß-scissions pressure dependence has a negligible effect in ethane steam cracking, while for propane steam cracking the effect is non-negligible on the product species profiles.

Differential gene expression, irrespective of circulating Hepatitis B Surface Antigen levels, between Inactive Carrier and Nucleos(t)ide Analogue-Treated Hepatitis B Virus patients.

Long-term viremia control in chronic HBV patients occurs either spontaneously in inactive carrier (IC) patients or therapy-induced by nucleos(t)ide analogues (NUC). To better understand the characteristics of viremia control, we evaluated gene expression in purified leukocyte subsets from IC versus NUC-treated patients, and evaluated the putative modulatory effects of hepatitis B surface antigen (HBsAg). We observed that gene expression in NUC-treated patients differed markedly from IC patients, especially in dendritic cells, monocytes, and CD8+ T cells, while serum HBsAg levels had little effect. Nevertheless, based on our findings it cannot be excluded that HBsAg may act locally in the infected liver or preferentially affects HBV-specific cells.

A Systematic Review and Meta-analysis of Animal Studies Investigating the Relationship Between Serum Antibody, T Lymphocytes, and Respiratory Syncytial Virus Disease.

BACKGROUND: Respiratory syncytial virus (RSV) infections occur in human populations around the globe, causing disease of variable severity, disproportionately affecting infants and older adults (>65 years of age). Immune responses can be protective but also contribute to disease. Experimental studies in animals enable detailed investigation of immune responses, provide insights into clinical questions, and accelerate the development of passive and active vaccination. We aimed to review the role of antibody and T-cell responses in relation to RSV disease severity in animals. METHODS: Systematic review and meta-analysis of animal studies examining the association between T-cell responses/phenotype or antibody titers and severity of RSV disease. The PubMed, Zoological Record, and Embase databases were screened from January 1980 to May 2018 to identify animal studies of RSV infection that assessed serum antibody titer or T lymphocytes with disease severity as an outcome. Sixty-three studies were included in the final review. RESULTS: RSV-specific antibody appears to protect from disease in mice, but such an effect was less evident in bovine RSV. Strong T-cell, Th1, Th2, Th17, CD4/CD8 responses, and weak Treg responses accompany severe disease in mice. CONCLUSIONS: Murine studies suggest that measures of T-lymphocyte activity (particularly CD4 and CD8 T cells) may be predictive biomarkers of severity. Further inquiry is merited to validate these results and assess relevance as biomarkers for human disease.

Distinct patterns of within-host virus populations between two subgroups of human respiratory syncytial virus.

Human respiratory syncytial virus (RSV) is a major cause of lower respiratory tract infection in young children globally, but little is known about within-host RSV diversity. Here, we characterised within-host RSV populations using deep-sequencing data from 319 nasopharyngeal swabs collected during 2017-2020. RSV-B had lower consensus diversity than RSV-A at the population level, while exhibiting greater within-host diversity. Two RSV-B consensus sequences had an amino acid alteration (K68N) in the fusion (F) protein, which has been associated with reduced susceptibility to nirsevimab (MEDI8897), a novel RSV monoclonal antibody under development. In addition, several minor variants were identified in the antigenic sites of the F protein, one of which may confer resistance to palivizumab, the only licensed RSV monoclonal antibody. The differences in within-host virus populations emphasise the importance of monitoring for vaccine efficacy and may help to explain the different prevalences of monoclonal antibody-escape mutants between the two subgroups.

Burden of respiratory syncytial virus infection in community-dwelling older adults in Europe (RESCEU): an international prospective cohort study.

BACKGROUND: Respiratory syncytial virus (RSV) infection in older adults is recognised as an important health issue. We aimed to assess the community burden of RSV in Europe in older adults aged ≥60 years. METHODS: This international, prospective, observational cohort study is part of work by the REspiratory Syncytial virus Consortium in EUrope (RESCEU). Participants were recruited through general practitioners' (GPs) offices before two independent RSV seasons. Participants reported weekly about symptoms of acute respiratory tract infection (ARTI) during one RSV season. ARTI patients were tested for RSV during home visits and completed a daily symptom diary. RSV illness included PCR-confirmed ARTI and those showing seroconversion over the season. RSV ARTI was based on PCR alone (ClinicalTrials.gov, NCT03621930). RESULTS: We recruited 1040 participants (527 in season 2017-2018 and 513 in season 2018-2019) with a median age of 75 years (range 60-100 years). Of these, 1023 (99%) lived independently at home at baseline. RSV illness incidence was 22 out of 527 (4.2%) and 37 out of 513 (7.2%) in the respective seasons. RSV illness did not affect frailty or cardiopulmonary status during the course of the study. No patients were hospitalised or died from RSV illness. In the 36 patients with PCR confirmed RSV ARTI, symptom duration averaged 19 days, while a doctor's visit took place in 11 out of 36 cases (31%). RSV ARTI could not be differentiated clinically from all other ARTIs based on symptoms. CONCLUSION: This European study showed that RSV is prevalent in community-dwelling older adults and rarely causes severe disease. This suggests that watchful waiting, using a continuity of care approach to identify those who do need more intensive care, is often justified when RSV is suspected in family practice.

Biomarkers for Disease Severity in Children Infected With Respiratory Syncytial Virus: A Systematic Literature Review.

BACKGROUND: Clinical manifestations of respiratory syncytial virus (RSV) infection vary widely from mild, self-limiting illness to severe life-threatening disease. There are gaps in knowledge of biomarkers to objectively define severe disease and predict clinical outcomes. METHODS: A systematic search was performed, 1945-March 2019 in databases Ovid Medline, Embase, Global health, Scopus, and Web of Science. Risk of bias was assessed using the Cochrane tool. RESULTS: A total of 25 132 abstracts were screened and studies were assessed for quality, risk of bias, and extracted data; 111 studies met the inclusion criteria. RSV severity was correlated with antibody titers, reduced T and B cells, dysregulated innate immunity, neutrophil mobilization to the lungs and blood, decreased Th1 response, and Th2 weighted shift. Microbial exposures in respiratory tract may contribute to neutrophil mobilization to the lungs of the infants with severe RSV compared with mild RSV disease. CONCLUSIONS: Although a wide range of biomarkers have been associated with RSV disease severity, robust validated biomarkers are lacking. This review illustrates the broad heterogeneity of study designs and high variability in the definition of severe RSV disease. Prospective studies are required to validate biomarkers. Additional research investigating epigenetics, metabolomics, and microbiome holds promise for novel biomarkers.

Presumed Risk Factors and Biomarkers for Severe Respiratory Syncytial Virus Disease and Related Sequelae: Protocol for an Observational Multicenter, Case-Control Study From the Respiratory Syncytial Virus Consortium in Europe (RESCEU).

Respiratory syncytial virus (RSV) is the leading viral pathogen associated with acute lower respiratory tract infection and hospitalization in children < 5 years of age worldwide. While there are known clinical risk factors for severe RSV infection, the majority of those hospitalized are previously healthy infants. There is consequently an unmet need to identify biomarkers that predict host response, disease severity, and sequelae. The primary objective is to identify biomarkers of severe RSV acute respiratory tract infection (ARTI) in infants. Secondary objectives include establishing biomarkers associated with respiratory sequelae following RSV infection and characterizing the viral load, RSV whole-genome sequencing, host immune response, and transcriptomic, proteomic, metabolomic and epigenetic signatures associated with RSV disease severity. Six hundred thirty infants will be recruited across 3 European countries: the Netherlands, Spain, and the United Kingdom. Participants will be recruited into 2 groups: (1) infants with confirmed RSV ARTI (includes upper and lower respiratory tract infections), 500 without and 50 with comorbidities; and (2) 80 healthy controls. At baseline, participants will have nasopharyngeal, blood, buccal, stool, and urine samples collected, plus complete a questionnaire and 14-day symptom diary. At convalescence (7 weeks ± 1 week post-ARTI), specimen collection will be repeated. Laboratory measures will be correlated with symptom severity scores to identify corresponding biomarkers of disease severity. CLINICAL TRIALS REGISTRATION: NCT03756766.

Simultaneous Viral Whole-Genome Sequencing and Differential Expression Profiling in Respiratory Syncytial Virus Infection of Infants.

Targeted metagenomics using strand-specific libraries with target enrichment is a sensitive, generalized approach to pathogen sequencing and transcriptome profiling. Using this method, we recovered 13 (76%) complete human respiratory syncytial virus (RSV) genomes from 17 clinical respiratory samples, reconstructed the phylogeny of the infecting viruses, and detected differential gene expression between 2 RSV subgroups, specifically, a lower expression of the P gene and a higher expression of the M2 gene in RSV-A than in RSV-B. This methodology can help to relate viral genetics to clinical phenotype and facilitate ongoing population-level RSV surveillance and vaccine development. Clinical Trials Registration. NCT03627572 and NCT03756766.

Commentary on ICH guideline on genomic sampling and data management-enabling opportunities in drug development and patient treatment.

The ability to benefit from knowledge of human genomic data in medicine has been anticipated since the sequencing of the human genome. That promise has experienced some degree of realization, particularly in oncology where biomarker-specific clinical trials and patient treatment specific to the genetics of their tumours now occur. With whole genome sequencing and related technologies becoming more affordable, and the generation and management of vast amounts of data and information, more capable, new opportunities to benefit from these developments lie ahead. Already emerging are many studies describing the association of genomic variation with molecular underpinnings of disease, association with patient response to drugs and informing the nomination of new drug targets. These developments are accompanied by some ethical, legal and regulatory challenges, which we discuss in this article.