A novel whole blood assay to quantify the release of T cell associated cytokines in response to Bordetella pertussis antigens.

BACKGROUND: Bordetella pertussis continues to cause whooping cough globally even in countries with high immunisation coverage. Booster vaccinations with acellular pertussis vaccines are thus used in children, adolescents, and adults. T cell immunity is crucial for orchestrating the immune response after vaccination. However, T cell assays can be expensive and difficult to implement in large clinical trials. In this study, a whole blood (WB) stimulation assay was developed to identify secreted T cell associated cytokines in different age groups after acellular pertussis booster vaccination. MATERIAL AND METHODS: Longitudinal WB samples were collected from a small set of subjects (n = 38) aged 7-70 years participating in a larger ongoing clinical trial. For assay development, samples were diluted and incubated with purified inactivated pertussis toxin (PT), filamentous haemagglutinin (FHA), inactivated B. pertussis lysate, and complete medium (M) as stimulating conditions, with anti-CD28 and anti-CD49d as co-stimulants. Different timepoints around the vaccination (D0, D7, D14, D28), WB dilution factor (1:2, 1:4) and incubation time (24 h, 48 h, 72 h) were compared. Responses to 15 cytokines were tested with Luminex/multiplex immunoassay. RESULTS: The optimized assay consisted of WB incubation with M, PT, and FHA (including the two co-stimulants). After 48 h incubation, supernatants were collected for measurement of seven selected T cell associated cytokines (IL-2, IL-5, IL-10, IL-13, IL-17 A, IL-17F, and IFN-y) from samples before and 28 days after vaccination. PT stimulation showed a trend for upregulation of IL-2, IL-13, and IL-17 A/F for adult subjects, whereas the responses of all cytokines were downregulated for the paediatric subjects. Furthermore, PT and FHA-stimulated WB showed diverse cytokine producing profiles. CONCLUSIONS: The developed WB-based cytokine assay was shown to be less costly, easy to perform, and functional in differently aged individuals. Further, it requires only a small amount of fresh blood, which is beneficial especially for studies including infants. Our results support the use of this assay for other immunological studies in the future.

Computational mining of B cell receptor repertoires reveals antigen-specific and convergent responses to Ebola vaccination.

Outbreaks of Ebolaviruses, such as Sudanvirus (SUDV) in Uganda in 2022, demonstrate that species other than the Zaire ebolavirus (EBOV), which is currently the sole virus represented in current licensed vaccines, remain a major threat to global health. There is a pressing need to develop effective pan-species vaccines and novel monoclonal antibody-based therapeutics for Ebolavirus disease. In response to recent outbreaks, the two dose, heterologous Ad26.ZEBOV/MVA-BN-Filo vaccine regimen was developed and was tested in a large phase II clinical trial (EBL2001) as part of the EBOVAC2 consortium. Here, we perform bulk sequencing of the variable heavy chain (VH) of B cell receptors (BCR) in forty participants from the EBL2001 trial in order to characterize the BCR repertoire in response to vaccination with Ad26.ZEBOV/MVA-BN-Filo. We develop a comprehensive database, EBOV-AbDab, of publicly available Ebolavirus-specific antibody sequences. We then use our database to predict the antigen-specific component of the vaccinee repertoires. Our results show striking convergence in VH germline gene usage across participants following the MVA-BN-Filo dose, and provide further evidence of the role of IGHV3-15 and IGHV3-13 antibodies in the B cell response to Ebolavirus glycoprotein. Furthermore, we found that previously described Ebola-specific mAb sequences present in EBOV-AbDab were sufficient to describe at least one of the ten most expanded BCR clonotypes in more than two thirds of our cohort of vaccinees following the boost, providing proof of principle for the utility of computational mining of immune repertoires.

Normalization of C1 Inhibitor in a Patient with Hereditary Angioedema.

Hereditary angioedema is a potentially life-threatening autosomal dominant condition, causing attacks of angioedema due to failure to regulate bradykinin. Nearly all cases of hereditary angioedema are caused by mutations in the gene encoding C1 inhibitor, SERPING1. C1 inhibitor is a multifunctional protein produced in the liver that regulates the kallikrein-kinin system at multiple points. An infant with genetically confirmed hereditary angioedema and low C1 inhibitor levels (but without previous episodes of angioedema) underwent liver transplantation for biliary atresia, an unrelated condition. Liver transplantation led to normalization of the C1 inhibitor level and function. To our knowledge, this represents the first patient to be potentially cured of hereditary angioedema.

Decline in pneumococcal vaccine serotype carriage, multiple-serotype carriage, and carriage density in Nepalese children after PCV10 introduction: A pre-post comparison study.

BACKGROUND: Carriage studies are an efficient means for assessing pneumococcal conjugate vaccine effect in settings where pneumococcal disease surveillance programmes are not well established. In this study the effect of 10-valent pneumococcal conjugate vaccine (PCV10) introduction on pneumococcal carriage and density among Nepalese children using a bacterial microarray and qPCR was examined. METHODS: PCV10 was introduced into the Nepalese infant immunisation schedule in August 2015. Nasopharyngeal swabs were collected from healthy Nepalese children in Kathmandu between April 2014 and December 2021. Samples were plated on blood agar, incubated overnight, and DNA extracted from plate sweeps. Pneumococcal serotyping was done using the Senti-SPv1.5 microarray (BUGS Bioscience, UK). DNA was extracted from swab media and qPCR performed for pneumococcal autolysin (lytA). RESULTS: A significant decline in prevalence of PCV10 serotypes was observed when comparing pre-PCV10 with post-PCV10 collection periods (36.5 %, 454/1244 vs 10.3 %, 243/2353, p < 0.0001). Multiple-serotype carriage was also observed to significantly decline when comparing pre-PCV10 with post-PCV10 periods (31.4 %, 390/1244 vs 22.2 %, 522/2353, p < 0.0001). Additionally, a significant decline in median pneumococcal density was observed when comparing pre-PCV10 with post-PCV10 periods (3.3 vs 3.25 log10 GE/ml, p = 0.0196). CONCLUSIONS: PCV10 introduction was associated with reduced, prevalence of all PCV10 serotypes, multiple serotype carriage, and pneumococcal carriage density.

Evaluation of Acute and Convalescent Antibody Concentration Against Pneumococcal Capsular Polysaccharides for the Diagnosis of Pneumococcal Infection in Children with Community-Acquired Pneumonia.

We evaluated whether the quantification of IgG to pneumococcal capsular polysaccharides is an accurate diagnostic test for pneumococcal infection in children with pneumonia in Nepal. Children with pneumococcal pneumonia did not have higher convalescent, or higher fold change, IgG to pneumococcal polysaccharides than children with other causes of pneumonia. Caution is needed in interpreting antibody responses in pneumococcal infections.

Antiviral responses induced by Tdap-IPV vaccination are associated with persistent humoral immunity to Bordetella pertussis.

Many countries continue to experience pertussis epidemics despite widespread vaccination. Waning protection after booster vaccination has highlighted the need for a better understanding of the immunological factors that promote durable protection. Here we apply systems vaccinology to investigate antibody responses in adolescents in the Netherlands (N = 14; NL) and the United Kingdom (N = 12; UK) receiving a tetanus-diphtheria-acellular pertussis-inactivated poliovirus (Tdap-IPV) vaccine. We report that early antiviral and interferon gene expression signatures in blood correlate to persistence of pertussis-specific antibody responses. Single-cell analyses of the innate response identified monocytes and myeloid dendritic cells (MoDC) as principal responders that upregulate antiviral gene expression and type-I interferon cytokine production. With public data, we show that Tdap vaccination stimulates significantly lower antiviral/type-I interferon responses than Tdap-IPV, suggesting that IPV may promote antiviral gene expression. Subsequent in vitro stimulation experiments demonstrate TLR-dependent, IPV-specific activation of the pro-inflammatory p38 MAP kinase pathway in MoDCs. Together, our data provide insights into the molecular host response to pertussis booster vaccination and demonstrate that IPV enhances innate immune activity associated with persistent, pertussis-specific antibody responses.

Oral Valganciclovir Initiated Beyond 1 Month of Age as Treatment of Sensorineural Hearing Loss Caused by Congenital Cytomegalovirus Infection: A Randomized Clinical Trial.

OBJECTIVE: The objective of this study was to determine if valganciclovir initiated after 1 month of age improves congenital cytomegalovirus-associated sensorineural hearing loss. STUDY DESIGN: We conducted a randomized, double-blind, placebo-controlled phase 2 trial of 6 weeks of oral valganciclovir at US (n = 12) and UK (n = 9) sites. Patients of ages 1 month through 3 years with baseline sensorineural hearing loss were enrolled. The primary outcome was change in total ear hearing between baseline and study month 6. Secondary outcome measures included change in best ear hearing and reduction in cytomegalovirus viral load in blood, saliva, and urine. RESULTS: Of 54 participants enrolled, 35 were documented to have congenital cytomegalovirus infection and were randomized (active group: 17; placebo group: 18). Mean age at enrollment was 17.8 ± 15.8 months (valganciclovir) vs 19.5 ± 13.1 months (placebo). Twenty (76.9%) of the 26 ears from subjects in the active treatment group did not have worsening of hearing, compared with 27 (96.4%) of 28 ears from subjects in the placebo group (P = .09). All other comparisons of total ear or best ear hearing outcomes were also not statistically significant. Saliva and urine viral loads decreased significantly in the valganciclovir group but did not correlate with change in hearing outcome. CONCLUSIONS: In this randomized controlled trial, initiation of antiviral therapy beyond the first month of age did not improve hearing outcomes in children with congenital cytomegalovirus-associated sensorineural hearing loss. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov identifier NCT01649869.

100 days of Adolescence: Elucidating Externalizing Behaviors Through the Daily Assessment of Inhibitory Control.

Inhibitory control is a transdiagnostic risk factor for externalizing behaviors, particularly during adolescence. Despite advances in understanding links between inhibitory control and externalizing behaviors across youth on average, significant questions remain about how these links play out in the day-to-day lives of individual adolescents. The goals of the current study were to: (1) validate a novel 100-occasion measure of inhibitory control; (2) assess links between day-to-day fluctuations in inhibitory control and individual differences in externalizing behaviors; and (3) illustrate the potential of intensive longitudinal studies for person-specific analyses of adolescent externalizing behaviors. Participants were 106 youth (57.5% female, Mage = 13.34 years; SDage = 1.92) who completed a virtual baseline session followed by 100 daily surveys, including an adapted Stroop Color Word task designed to assess inhibitory control. Results suggested that the novel task was generally reliable and valid, and that inhibitory control fluctuated across days in ways that were meaningfully associated with individual differences in baseline impulsive behaviors. Results of illustrative personalized analyses suggested that inhibitory control had more influence in the daily networks of adolescents who used substances during the 100 days than in a matched set of adolescents who did not. This work marks a path forward in intensive longitudinal research by validating a novel inhibitory control measure, revealing that daily fluctuations in inhibitory control may be a unique construct broadly relevant to adolescent externalizing problems, and at the same time, highlighting that links between daily inhibitory control and impulsive behaviors are adolescent-specific.

Structural and non-coding variants increase the diagnostic yield of clinical whole genome sequencing for rare diseases.

BACKGROUND: Whole genome sequencing is increasingly being used for the diagnosis of patients with rare diseases. However, the diagnostic yields of many studies, particularly those conducted in a healthcare setting, are often disappointingly low, at 25-30%. This is in part because although entire genomes are sequenced, analysis is often confined to in silico gene panels or coding regions of the genome. METHODS: We undertook WGS on a cohort of 122 unrelated rare disease patients and their relatives (300 genomes) who had been pre-screened by gene panels or arrays. Patients were recruited from a broad spectrum of clinical specialties. We applied a bioinformatics pipeline that would allow comprehensive analysis of all variant types. We combined established bioinformatics tools for phenotypic and genomic analysis with our novel algorithms (SVRare, ALTSPLICE and GREEN-DB) to detect and annotate structural, splice site and non-coding variants. RESULTS: Our diagnostic yield was 43/122 cases (35%), although 47/122 cases (39%) were considered solved when considering novel candidate genes with supporting functional data into account. Structural, splice site and deep intronic variants contributed to 20/47 (43%) of our solved cases. Five genes that are novel, or were novel at the time of discovery, were identified, whilst a further three genes are putative novel disease genes with evidence of causality. We identified variants of uncertain significance in a further fourteen candidate genes. The phenotypic spectrum associated with RMND1 was expanded to include polymicrogyria. Two patients with secondary findings in FBN1 and KCNQ1 were confirmed to have previously unidentified Marfan and long QT syndromes, respectively, and were referred for further clinical interventions. Clinical diagnoses were changed in six patients and treatment adjustments made for eight individuals, which for five patients was considered life-saving. CONCLUSIONS: Genome sequencing is increasingly being considered as a first-line genetic test in routine clinical settings and can make a substantial contribution to rapidly identifying a causal aetiology for many patients, shortening their diagnostic odyssey. We have demonstrated that structural, splice site and intronic variants make a significant contribution to diagnostic yield and that comprehensive analysis of the entire genome is essential to maximise the value of clinical genome sequencing.

Daily Gender and Cognition: A Person-Specific Behavioral Network Analysis.

Gender is person-specific, and it influences and is influenced by a breadth of multidimensional psychological factors, including cognition. Directionality is important for research on gender and cognition, as debate surrounds, for instance, whether masculine self-concepts precede spatial skills, or whether the reverse is true. In order to provide novel insights into the individualized nature of these relations, a person-specific network approach devised by Peter Molenaar and the first author - group iterative multiple model estimation for multiple solutions (GIMME-MS) - was applied to 75-day intensive longitudinal data on gender self-concept (i.e., femininity-masculinity, instrumentality, and expressivity) and cognition (i.e., mental rotations and verbal recall) from 103 young adults. GIMME-MS estimates individualized networks that contain same-day and next-day directed relations, prioritizing relations common across participants. It is ideal for analyzing behavioral time series with unclear directionality, as it generates multiple solutions from which an optimal one is selected. GIMME-MS revealed notable heterogeneity in the presence, direction, and nature of relations from gender self-concept to cognition (∼26% of participants) and vice versa (∼21% of participants). Findings are wholly novel in revealing the person-specific nature of gender and its cognitive dynamics, yet somehow, unsurprising given the revolutionary corpus of Peter Molenaar.

The daily association between affect and alcohol use: A meta-analysis of individual participant data.

Influential psychological theories hypothesize that people consume alcohol in response to the experience of both negative and positive emotions. Despite two decades of daily diary and ecological momentary assessment research, it remains unclear whether people consume more alcohol on days they experience higher negative and positive affect in everyday life. In this preregistered meta-analysis, we synthesized the evidence for these daily associations between affect and alcohol use. We included individual participant data from 69 studies (N = 12,394), which used daily and momentary surveys to assess affect and the number of alcoholic drinks consumed. Results indicate that people are not more likely to drink on days they experience high negative affect, but are more likely to drink and drink heavily on days high in positive affect. People self-reporting a motivational tendency to drink-to-cope and drink-to-enhance consumed more alcohol, but not on days they experienced higher negative and positive affect. Results were robust across different operationalizations of affect, study designs, study populations, and individual characteristics. These findings challenge the long-held belief that people drink more alcohol following increases in negative affect. Integrating these findings under different theoretical models and limitations of this field of research, we collectively propose an agenda for future research to explore open questions surrounding affect and alcohol use.

Intracranial Empyema in Children: A Single-center Retrospective Case Series.

We conducted a retrospective, observational study of 42 children with intracranial empyema admitted to a pediatric neurosurgical center over a 9-year period. Intracranial empyema is rare, but causes significant morbidity and mortality. Twenty-eight cases had neurosurgical source control, more commonly for subdural collections. Streptococcus anginosus group bacteria are important pathogens in subdural empyema, whose isolation predicts more complicated postoperative courses.

Molecular correlates of vaccine-induced protection against typhoid fever.

BACKGROUNDTyphoid fever is caused by the Gram-negative bacterium Salmonella enterica serovar Typhi and poses a substantial public health burden worldwide. Vaccines have been developed based on the surface Vi-capsular polysaccharide of S. Typhi; these include a plain-polysaccharide-based vaccine, ViPS, and a glycoconjugate vaccine, ViTT. To understand immune responses to these vaccines and their vaccine-induced immunological protection, molecular signatures were analyzed using bioinformatic approaches.METHODSBulk RNA-Seq data were generated from blood samples obtained from adult human volunteers enrolled in a vaccine trial, who were then challenged with S. Typhi in a controlled human infection model (CHIM). These data were used to conduct differential gene expression analyses, gene set and modular analyses, B cell repertoire analyses, and time-course analyses at various post-vaccination and post-challenge time points between participants receiving ViTT, ViPS, or a control meningococcal vaccine.RESULTSTranscriptomic responses revealed strong differential molecular signatures between the 2 typhoid vaccines, mostly driven by the upregulation in humoral immune signatures, including selective usage of immunoglobulin heavy chain variable region (IGHV) genes and more polarized clonal expansions. We describe several molecular correlates of protection against S. Typhi infection, including clusters of B cell receptor (BCR) clonotypes associated with protection, with known binders of Vi-polysaccharide among these.CONCLUSIONThe study reports a series of contemporary analyses that reveal the transcriptomic signatures after vaccination and infectious challenge, while identifying molecular correlates of protection that may inform future vaccine design and assessment.TRIAL REGISTRATIONClinicalTrials.gov NCT02324751.

Pulmonary Cystic Echinococcosis in a Child Presenting in the United Kingdom with Fever and Chest Pain: A Brief Report and Discussion on Management.

Cystic echinococcosis is a zoonosis caused by the larvae of Echinococcus granulosus . Pulmonary disease may be asymptomatic until the cyst ruptures or becomes secondarily infected. We report a case of pulmonary cystic echinococcosis presenting in the United Kingdom, with discussion on management: optimum antihelminthic agent, length of treatment and type of operative intervention. Treatment should be individualized to the clinical scenario.

Characterisation of the immune repertoire of a humanised transgenic mouse through immunophenotyping and high-throughput sequencing.

Immunoglobulin loci-transgenic animals are widely used in antibody discovery and increasingly in vaccine response modelling. In this study, we phenotypically characterised B-cell populations from the Intelliselect Transgenic mouse (Kymouse) demonstrating full B-cell development competence. Comparison of the naïve B-cell receptor (BCR) repertoires of Kymice BCRs, naïve human, and murine BCR repertoires revealed key differences in germline gene usage and junctional diversification. These differences result in Kymice having CDRH3 length and diversity intermediate between mice and humans. To compare the structural space explored by CDRH3s in each species' repertoire, we used computational structure prediction to show that Kymouse naïve BCR repertoires are more human-like than mouse-like in their predicted distribution of CDRH3 shape. Our combined sequence and structural analysis indicates that the naïve Kymouse BCR repertoire is diverse with key similarities to human repertoires, while immunophenotyping confirms that selected naïve B cells are able to go through complete development.

Management of neonatal central nervous system viral infections: Knowledge gaps and research priorities.

Congenital CMV, enteroviruses, human parechovirus and herpes simplex virus are all common causes of severe central nervous system (CNS) infection in neonates. The introduction of screening (i.e. newborn hearing screening programme), integration of molecular syndromic testing (i.e. multiplex polymerase chain reaction assays) and increase in sexually transmitted infections (i.e. anogenital herpes) have contributed to increases in each of these infections over the last decade. However, therapeutic options are highly limited in part due to the lack of epidemiological data informing trials. This review will describe our current understanding of the clinical burden and epidemiology of these severe neonatal CNS infections, outline the novel antiviral and vaccines in the pipeline and suggest future research studies which could help develop new therapeutics.

Mediating effect of pubertal stages on the family environment and neurodevelopment: An open-data replication and multiverse analysis of an ABCD Study®.

Increasing evidence demonstrates that environmental factors meaningfully impact the development of the brain (Hyde et al., 2020; McEwen and Akil, 2020). Recent work from the Adolescent Brain Cognitive Development (ABCD) Study® suggests that puberty may indirectly account for some association between the family environment and brain structure and function (Thijssen et al., 2020). However, a limited number of large studies have evaluated what, how, and why environmental factors impact neurodevelopment. When these topics are investigated, there is typically inconsistent operationalization of variables between studies which may be measuring different aspects of the environment and thus different associations in the analytic models. Multiverse analyses (Steegen et al., 2016) are an efficacious technique for investigating the effect of different operationalizations of the same construct on underlying interpretations. While one of the assets of Thijssen et al. (2020) was its large sample from the ABCD data, the authors used an early release that contained 38% of the full ABCD sample. Then, the analyses used several 'researcher degrees of freedom' (Gelman and Loken, 2014) to operationalize key independent, mediating and dependent variables, including but not limited to, the use of a latent factor of preadolescents' environment comprised of different subfactors, such as parental monitoring and child-reported family conflict. While latent factors can improve reliability of constructs, the nuances of each subfactor and measure that comprise the environment may be lost, making the latent factors difficult to interpret in the context of individual differences. This study extends the work of Thijssen et al. (2020) by evaluating the extent to which the analytic choices in their study affected their conclusions. In Aim 1, using the same variables and models, we replicate findings from the original study using the full sample in Release 3.0. Then, in Aim 2, using a multiverse analysis we extend findings by considering nine alternative operationalizations of family environment, three of puberty, and five of brain measures (total of 135 models) to evaluate the impact on conclusions from Aim 1. In these results, 90% of the directions of effects and 60% of the p-values (e.g. p > .05 and p < .05) across effects were comparable between the two studies. However, raters agreed that only 60% of the effects had replicated. Across the multiverse analyses, there was a degree of variability in beta estimates across the environmental variables, and lack of consensus between parent reported and child reported pubertal development for the indirect effects. This study demonstrates the challenge in defining which effects replicate, the nuance across environmental variables in the ABCD data, and the lack of consensus across parent and child reported puberty scales in youth.

Effect of the of 10-valent pneumococcal conjugate vaccine in Nepal 4 years after introduction: an observational cohort study.

BACKGROUND: In Nepal, Streptococcus pneumoniae (pneumococcus) is a common cause of bacterial pneumonia in children, and is a major health concern. There are few data on the effect of vaccination on the disease or colonisation with pneumococci in the nasopharynx of children in this setting. The 10-valent pneumococcal conjugate vaccine (PCV10) was introduced into the routine infant immunisation schedule in Nepal in 2015. We aimed to investigate the effect of the introduction of PCV10 on pneumococcal carriage and disease in children in Nepal. METHODS: We did an observational cohort study in children in Nepal. The hospital surveillance study took place in Patan Hospital, Kathmandu, and community studies in healthy children took place in Kathmandu and Okhaldhunga district. For the surveillance study, all children admitted to Patan Hospital between March 20, 2014, and Dec 31, 2019, aged between 2 months and 14 years with clinician-suspected pneumonia, were eligible for enrolment. For the community study, healthy children aged 0-8 weeks, 6-23 months, and 24-59 months were recruited from Kathmandu, and healthy children aged 6-23 months were recruited from Okhaldhunga. We assessed the programmatic effect of PCV10 introduction using surveillance for nasopharyngeal colonisation, pneumonia, and invasive bacterial disease from 1·5 years before vaccine introduction and 4·5 years after vaccine introduction. For the surveillance study, nasopharyngeal swabs, blood cultures, and chest radiographs were obtained from children admitted to Patan Hospital with suspected pneumonia or invasive bacterial disease. For the community study, nasopharyngeal swabs were obtained from healthy children in the urban and rural settings. Pneumonia outcomes were analysed using log-binomial models and adjusted prevalence ratios (aPR) comparing each calendar year after the introduction of the vaccine into the national programme with the pre-vaccine period (2014-15), adjusted for calendar month, age, and sex. FINDINGS: Between March 20, 2014, and Dec 31, 2019, we enrolled 2051 children with suspected pneumonia, and 11 354 healthy children (8483 children aged 6-23 months, 761 aged 24-59 months, and 2110 aged 0-8 weeks) to assess nasopharyngeal colonisation. Among clinical pneumonia cases younger than 2 years, vaccine serotype carriage declined 82% (aPR 0·18 [95% CI 0·07-0·50]) by 2019. There was no decrease in vaccine serotype carriage in cases among older unvaccinated age groups. Carriage of the additional serotypes in PCV13 was 2·2 times higher by 2019 (aPR 2·17 [95% CI 1·16-4·05]), due to increases in serotypes 19A and 3. Vaccine serotype carriage in healthy children declined by 75% in those aged 6-23 months (aPR 0·25 [95% CI 0·19-0·33]) but not in those aged 24-59 months (aPR 0·59 [0·29-1·19]). A decrease in overall vaccine serotype carriage of 61% by 2019 (aPR 0·39 [95% CI 0·18-0·85]) was also observed in children younger than 8 weeks who were not yet immunised. Carriage of the additional PCV13 serotypes in children aged 6-23 months increased after PCV10 introduction for serotype 3 and 19A, but not for serotype 6A. The proportion of clinical pneumonia cases with endpoint consolidation on chest radiographs declined from 41% in the pre-vaccine period to 25% by 2018, but rose again in 2019 to 36%. INTERPRETATION: The introduction of the PCV10 vaccine into the routine immunisation programme in Nepal has reduced vaccine serotype carriage in both healthy children and children younger than 2 years with pneumonia. Increases in serotypes 19A and 3 highlight the importance of continued surveillance to monitor the effect of vaccine programmes. This analysis demonstrates a robust approach to assessing vaccine effect in situations in which pneumococcal disease endpoint effectiveness studies are not possible. FUNDING: Gavi, the Vaccine Alliance and the World Health Organization.

Effect of childhood vaccination and antibiotic use on pneumococcal populations and genome-wide associations with disease among children in Nepal: an observational study.

BACKGROUND: Pneumococcal disease is a leading cause of bacterial pneumonia and invasive bacterial disease among children globally. The reason some strains of pneumococci are more likely to cause disease, and how interventions such as vaccines and antibiotics affect pneumococcal strains is poorly understood. We aimed to identify genetic regions under selective pressure and those associated with disease through the analysis of pneumococcal whole-genome sequences. METHODS: Whole-genome sequencing was performed on pneumococcal isolates collected between January, 2005, and May, 2018, in Kathmandu, Nepal, which included programmatic ten-valent pneumococcal conjugate vaccine (PCV10) introduction in 2015. Isolates were from three distinct cohorts: nasopharyngeal swabs of healthy community-based children, nasopharyngeal swabs of children admitted to hospital with pneumonia, and sterile-site cultures from children admitted to hospital. Across these cohorts we examined serotype distribution, antibiotic resistance, strain distribution, and regions of recombination to determine genes that were undergoing diversifying selection. Genome-wide association studies comparing pneumonia and sterile-site isolates with healthy carriage were used to determine novel variants associated with disease. FINDINGS: After programmatic introduction of PCV10, there was a decline in vaccine covered serotypes; however, strains that had expressed these serotypes continued to exist in the post-PCV population. We identified GPSC9 to be a strain of concern due to its high prevalence in disease, multidrug resistance, and ability to switch to an unencapsulated phenotype via insertion of virulence factor pspC into the cps locus. Antibiotic resistance loci to co-trimoxazole were found to be prevalent (pre-PCV10 78% vs post-PCV10 81%; p=0·27) and increasingly prevalent to penicillin (pre-PCV10 15% vs post-PCV10 32%; p<0·0001). Regions with multiple recombinations were identified spanning the surface protein virulence factors pspA and pspC and antibiotic targets pbpX, folA, folC, folE, and folP. Furthermore, we identified variants in lacE2 to be strongly associated with isolates from children with pneumonia and PRIP to be strongly associated with isolates from sterile sites. INTERPRETATION: Our work highlights the effect of pneumococcal conjugate vaccines, antibiotics, and host-pathogen interaction in pneumococcal variation, and the pathogen's capability of adapting to these factors at both population-wide and strain-specific levels. Ongoing surveillance of disease-associated strains and further investigation of lacE2 and PRIP as serotype-independent targets for therapeutic interventions is required. FUNDING: Gavi, The Vaccine Alliance; WHO; Bill & Melinda Gates Foundation; Wellcome Sanger Institute; and US Centers for Disease Control and Prevention.