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Abstract The immune checkpoint protein, Lymphocyte activation gene-3 (LAG3), binds Major Histocompatibility Complex Class II (MHC-II) and suppresses T cell activation. Despite the recent FDA approval of a LAG3 inhibitor for the treatment of melanoma, how LAG3 engages MHC-II on the cell surface remains poorly understood. Here, we determine the 3.84 Å-resolution structure of mouse LAG3 bound to the MHC-II molecule I-Ab, revealing that domain 1 (D1) of LAG3 binds a conserved, membrane-proximal region of MHC-II spanning both the α2 and β2 subdomains. LAG3 dimerization restricts the intermolecular spacing of MHC-II molecules, which may attenuate T cell activation by enforcing suboptimal signaling geometry. The LAG3-MHC-II interface overlaps with the MHC-II-binding site of the T cell coreceptor CD4, implicating disruption of CD4-MHC-II interactions as a mechanism for LAG3 immunosuppressive function. Lastly, antibody epitope analysis indicates that multiple LAG3 inhibitors do not recognize the MHC-II-binding interface of LAG3, suggesting a role for functionally distinct mechanisms of LAG3 antagonism in therapeutic development.

IntroductionSeagrass sediments are important ‘blue carbon’ reservoirs which store climatically significant quantities of organic carbon (Corg) at the global scale. Seagrass meadows that overly these sediments also provide a range of critical ecosystem services including shoreline stabilization, storm surge protection, and fisheries nursery grounds. However, the controls over accumulation and the sources of organic C to these sediments beds are highly variable and poorly understood with the relative importance of hydrodynamic setting, species composition and canopy density being unclear.MethodsHere we address these questions using the first observation-based estimates of Corg stocks and provenance on Turneffe Atoll, Belize, made via remotely-sensed habitat extent, local Corg data and isotopic data. Sedimentary Corg was highest in sediments underlying the most sheltered meadows and decreased with increasing exposure to wind and wave energy with the seagrass meadows in the central lagoon containing an extensive deposit of mangrove derived organic carbon, stabilized and protected by the overlying seagrass meadow.ResultsThe influence of species composition appeared weak with the ubiquitous species T. testudinum occurring across a wide range of hydrodynamic regimes ranging from the most sheltered to the most energetic and being associated with a wide range of sedimentary organic C concentrations. Importantly from the perspective of remote sensing, org C concentrations were unrelated to canopy density. We hypothesize that this decoupling of organic C concentration from seagrass canopy cover reflects a much longer timescale for carbon storage in the sediments than the lifespan of the seagrass plants themselves and/or a substantial non seagrass derived organic C burden in seagrass sediments. Overall, we conservatively estimate that the top 30cm of sediments underlying the seagrass meadows overlying carbonate sediments on the atoll exterior store 0.58 x 106 Mg Corg, most of which is seagrass-derived, whilst the sediments underlying the meadows within the central lagoon store an additional 1.28 x 106 Mg Corg. When the maximum possible extent of seagrass is considered, this estimate increases to 3.54 x 106 Mg Corg. Substantial Corg stocks extending >1m depth were observed across all sites, and so these inventories are considered conservative.DiscussionA preliminary ‘cost of loss’ for sedimentary Corg in the top 30 cm of Turneffe Atoll’s seagrass meadows, based on a carbon trading value of €60 tCO2 (eq), is estimated at €42 million for the outer atoll, increasing to €136 million when the mangrove-derived sediments of the central atoll are considered and €260 million when turbid areas are assumed to contain seagrass.

Abstract Streptococcus dysgalactiae subspecies equisimilis (SDSE) and Streptococcus pyogenes share skin and throat niches with extensive genomic homology and horizontal gene transfer (HGT) possibly underlying shared disease phenotypes. It is unknown if cross-species transmission interaction occurs. Here, we conduct a genomic analysis of a longitudinal household survey in remote Australian First Nations communities for patterns of cross-species transmission interaction and HGT. Collected from 4547 person-consultations, we analyse 294 SDSE and 315 S. pyogenes genomes. We find SDSE and S. pyogenes transmission intersects extensively among households and show that patterns of co-occurrence and transmission links are consistent with independent transmission without inter-species interference. We identify at least one of three near-identical cross-species mobile genetic elements (MGEs) carrying antimicrobial resistance or streptodornase virulence genes in 55 (19%) SDSE and 23 (7%) S. pyogenes isolates. These findings demonstrate co-circulation of both pathogens and HGT in communities with a high burden of streptococcal disease, supporting a need to integrate SDSE and S. pyogenes surveillance and control efforts.

Abstract Background Reducing antibiotic use in Australia, and the subsequent impact on antimicrobial resistance, requires multiple, sustained approaches with appropriate resources and support. Additional strategies to reduce antibiotic prescribing include effective vaccines, against pathogens such as Streptococcus pyogenes, the most common bacterial cause of sore throat. As part of efforts towards assessing the benefits of introducing new strategies to reduce antimicrobial prescribing, we aimed to determine the burden of antimicrobial prescribing for sore throat in general practice. Methods General practice activity data from 2013 – 2017 derived from the first 8 practices participating in the ‘Primary Care Audit, Teaching and Research Open Network’ (Patron) program were analysed according to reason for visit (upper respiratory tract infection, URTI, or sore throat) and antibiotic prescription. The main outcome measures were percentage of sore throat or URTI presentations with antibiotic prescription by age. Results A total of 722,339 visits to general practice were made by 65,449 patients; 5.7% of visits were for URTI with 0.8% meeting the more specific criteria for sore throat. 66.1% of sore throat visits and 36.2% of URTI visits resulted in antibiotic prescription. Penicillin, the recommended antibiotic for sore throat when indicated, was the antibiotic of choice in only 52.9% of sore throat cases prescribed antibiotics. Broader spectrum antibiotics were prescribed more frequently in older age groups. Conclusions Frequency of antibiotic prescribing for sore throat is high and broad, despite Australian Therapeutic guideline recommendations. Multiple, sustained interventions to reduce prescribing, including availability of effective S. pyogenes vaccines that could reduce the incidence of streptococcal pharyngitis, could obviate the need to prescribe antibiotics and support ongoing efforts to promote antimicrobial stewardship.

Pyrimidine nucleotide biosynthesis is a druggable metabolic dependency of cancer cells, and chemotherapy agents targeting pyrimidine metabolism are the backbone of treatment for many cancers. Dihydroorotate dehydrogenase (DHODH) is an essential enzyme in the de novo pyrimidine biosynthesis pathway that can be targeted by clinically approved inhibitors. However, despite robust preclinical anticancer efficacy, DHODH inhibitors have shown limited single-agent activity in phase 1 and 2 clinical trials. Therefore, novel combination therapy strategies are necessary to realize the potential of these drugs. To search for therapeutic vulnerabilities induced by DHODH inhibition, we examined gene expression changes in cancer cells treated with the potent and selective DHODH inhibitor brequinar (BQ). This revealed that BQ treatment causes upregulation of antigen presentation pathway genes and cell surface MHC class I expression. Mechanistic studies showed that this effect is (1) strictly dependent on pyrimidine nucleotide depletion, (2) independent of canonical antigen presentation pathway transcriptional regulators, and (3) mediated by RNA polymerase II elongation control by positive transcription elongation factor B (P-TEFb). Furthermore, BQ showed impressive single-agent efficacy in the immunocompetent B16F10 melanoma model, and combination treatment with BQ and dual immune checkpoint blockade (anti-CTLA-4 plus anti-PD-1) significantly prolonged mouse survival compared to either therapy alone. Our results have important implications for the clinical development of DHODH inhibitors and provide a rationale for combination therapy with BQ and immune checkpoint blockade.

Summary: Background: In Australia the incidence of HIV has declined steadily, yet sustained reduction of HIV transmission in this setting requires improved public health responses. As enhanced public health responses and prioritisation of resources may be guided by molecular epidemiological data, here we aimed to assess the applicability of these approaches in Victoria, Australia. Methods: A comprehensive collection of HIV-1 pol sequences from individuals diagnosed with HIV in Victoria, Australia, between January 1st 2000 and December 31st 2020 were deidentified and used as the basis of our assessment. These sequences were subtyped and surveillance drug resistance mutations (SDRMs) identified, before definition of transmission groups was performed using HIV-TRACE (0.4.4). Phylodynamic methods were applied using BEAST (2.6.6), assessing effective reproductive numbers for large groups, and additional demographic data were integrated to provide a high resolution view of HIV transmission in Victoria on a decadal time scale. Findings: Based on standard settings for HIV-TRACE, 70% (2438/3507) of analysed HIV-1 pol sequences were readily assigned to a transmission group. Individuals in transmission groups were more commonly males (aOR 1.50), those born in Australia (aOR 2.13), those with probable place of acquisition as Victoria (aOR 6.73), and/or those reporting injectable drug use (aOR 2.13). SDRMs were identified in 375 patients (10.7%), with sustained transmission of these limited to a subset of smaller groups. Informative patterns of epidemic growth, stabilisation, and decline were observed; many transmission groups showed effective reproductive numbers (Re) values reaching greater than 4.0, representing considerable epidemic growth, while others maintained low Re values. Interpretation: This study provides a high resolution view of HIV transmission in Victoria, Australia, and highlights the potential of molecular epidemiology to guide and enhance public health responses in this setting. This informs ongoing discussions with community groups on the acceptability and place of molecular epidemiological approaches in Australia. Funding: National Health and Medical Research Council, Australian Research Council.

Background: Increasing frequency of intermittent oral corticosteroid (OCS) prescription and cumulative OCS exposure increase the risk of OCS-related adverse outcomes. Objective: We sought to describe the evolution and trajectory of intermittent OCS prescription patterns in patients with asthma and investigate risk factors independently associated with transitioning to a frequent prescription pattern. Methods: This historical cohort study included patients with active asthma managed in UK primary care and included in the Optimum Patient Care Research Database (OPCRD; opcrd.co.uk). Intermittent OCS prescription patterns were categorized as sporadic, infrequent, moderately frequent, or frequent. Prescription pattern sequences were described for those who had a frequent sequence in their final year of prescribing. We examined associations between OCS prescription pattern and the hazard of transitioning into a frequent intermittent OCS prescription pattern using multivariable Cox regression with a 10-year look-back period. Results: Of 105,229 patients with intermittent OCS prescriptions, 57.1% (n = 60,083) had a frequent OCS prescription pattern at some point. Irrespective of baseline pattern, most patients transitioned to frequent prescription during the look back. The strongest risk factors were a more frequent prescription pattern at the start of look-back period, a lower percentage peak expiratory flow rate, and higher Global Initiative for Asthma treatment step. Older age, female sex, obesity, and active smoking were also associated with a higher risk of transitioning. Conclusion: Our findings help identify those most at risk of transitioning to frequent intermittent OCS receipt and encourage earlier intervention with OCS-sparing treatments.

BackgroundTo date, studies investigating the association between pre-biologic biomarker levels and post-biologic outcomes have been limited to single biomarkers and assessment of biologic efficacy from structured clinical trials.AimTo elucidate the associations of pre-biologic individual biomarker levels or their combinations with pre-to-post biologic changes in asthma outcomes in real-life.MethodsThis was a registry-based, cohort study using data from 23 countries, which shared data with the International Severe Asthma Registry (May 2017-February 2023). The investigated biomarkers (highest pre-biologic levels) were immunoglobulin E (IgE), blood eosinophil count (BEC) and fractional exhaled nitric oxide (FeNO). Pre- to approximately 12-month post-biologic change for each of three asthma outcome domains (i.e. exacerbation rate, symptom control and lung function), and the association of this change with pre-biologic biomarkers was investigated for individual and combined biomarkers.ResultsOverall, 3751 patients initiated biologics and were included in the analysis. No association was found between pre-biologic BEC and pre-to-post biologic change in exacerbation rate for any biologic class. However, higher pre-biologic BEC and FeNO were both associated with greater post-biologic improvement in FEV1 for both anti-IgE and anti-IL5/5R, with a trend for anti-IL4Rα. Mean FEV1 improved by 27-178 mL post-anti-IgE as pre-biologic BEC increased (250 to 1000 cells/µL), and by 43-216 mL and 129-250 mL post-anti-IL5/5R and -anti-IL4Rα, respectively along the same BEC gradient. Corresponding improvements along a FeNO gradient (25-100 ppb) were 41-274 mL, 69-207 mL and 148-224 mL for anti-IgE, anti-IL5/5R, and anti-IL4Rα, respectively. Higher baseline BEC was also associated with lower probability of uncontrolled asthma (OR 0.392; p=0.001) post-biologic for anti-IL5/5R. Pre-biologic IgE was a poor predictor of subsequent pre-to-post-biologic change for all outcomes assessed for all biologics. The combination of BEC + FeNO marginally improved the prediction of post-biologic FEV1 increase (adjusted R2: 0.751), compared to BEC (adjusted R2: 0.747) or FeNO alone (adjusted R2: 0.743) (p=0.005 and

Abstract The Staphylococcus aureus Network Adaptive Platform (SNAP) trial is a multifactorial Bayesian adaptive platform trial that aims to improve the way that S. aureus bloodstream infection, a globally common and severe infectious disease, is treated. In a world first, the SNAP trial will simultaneously investigate the effects of multiple intervention modalities within multiple groups of participants with different forms of S. aureus bloodstream infection. Here, we formalise the trial structure, modelling approach, and decision rules that will be used for the SNAP trial. By summarising the statistical principles governing the design, our hope is that the SNAP trial will serve as an adaptable template that can be used to improve comparative effectiveness research efficiency in other disease areas. Trial registration NCT05137119 . Registered on 30 November 2021.

Allergic rhinitis (AR) is a chronic respiratory condition that internationally continues to be burdensome and impacts quality of life. Despite availability of medicines and guidelines for healthcare providers for the optimal management of AR, optimisation of its management in the community continues to be elusive. The reasons for this are multi-faceted and include both environmental and healthcare related factors. One factor that we can no longer ignore is that AR management is no longer limited to the domain of healthcare provider and that people with AR make their own choices when choosing how to manage their condition, without seeking advice from a health care provider. We must build a bridge between healthcare provider knowledge and guidelines and patient decision-making. With this commentary, we propose that a shared decision-making approach between healthcare professionals and people with AR be developed and promoted, with a focus on patient health literacy. As custodians of AR knowledge, we have a responsibility to ensure it is accessible to those that matter most—the people with AR.

Background: Longitudinal predictors of persistent poor asthma control in severe asthma (SA) cohort remain scarce. The predictive value of the asthma severity scoring system (ASSESS) in the SA cohort outside the original study and in the Asian population is unknown. Objective: We sought to determine the 5-year longitudinal outcome of patients with SA and validate the use of ASSESS score in predicting future outcomes in SA. Methods: A prospective longitudinal observational study of patients with SA attending the multidisciplinary specialist SA clinic of the Singapore General Hospital from 2011 to 2021 was conducted. The number of exacerbations and asthma control test results were recorded yearly for 5 consecutive years. The ASSESS score was computed at baseline, and the area under the receiver-operating characteristic curve for predicting persistent poor asthma control was generated. Results: Of the 489 patients recruited into the study, 306 patients with 5-year follow-up data were analyzed. Seventy-three percent had type 2 inflammation with increased overall exacerbations over 5 years (rate ratio, 2.55; 95% CI, 1.31-4.96; P = .006) relative to non–type 2 SA. In the multivariate model, bronchiectasis, gastroesophageal reflux disease, and an asthma control test score of less than 20 were significantly associated with persistent poor asthma control over 5 years. ASSESS scores were good at predicting persistent poor asthma control with an area under the receiver-operating characteristic curve of 0.71 (95% CI, 0.57-0.84). Conclusions: Bronchiectasis and gastroesophageal reflux disease are predictors for persistent poor asthma control and targeted traits for precision medicine in SA. The ASSESS score has a good prediction for persistent poor asthma control over 5 years.

Women in malaria-endemic areas receive sulphadoxine-pyrimethamine (SP) as Intermittent Preventive Treatment in Pregnancy (IPTp) to reduce malaria. While dihydroartemisinin-piperaquine (DP) has superior antimalarial properties as IPTp, SP is associated with superior fetal growth. As maternal inflammation influences fetal growth, we investigated whether SP alters the relationship between inflammation and birth outcomes. We measured C-reactive protein (CRP) and alpha-1-acid glycoprotein (AGP) at enrollment (16-28 gestation weeks (gw)), visit 3 (24-36 gw) and delivery in 1319 Malawian women randomized to receive monthly SP, DP, or DP and single-dose azithromycin (AZ) in the IMPROVE trial (NCT03208179). Logistic regression was used to assess the relationship between adverse outcomes, inflammation, and treatment arm. Elevated AGP at enrollment was associated with adverse birth outcome (aRR 1.40, 95% CI: 1.15, 1.70), with similar associations observed across treatment arms, exceptions being that elevated AGP was associated with low maternal weight gain in SP recipients (aRR 1.94, 95% CI: 1.36, 2.76) and with small for gestational age in DP+AZ recepients (aRR 1.49, 95% CI 1.02, 2.17). At visit 3 there were few associations between inflammation andoutcomes. At delivery, women with elevated AGP receiving either DP or DP+AZ had an increased risk of adverse birth outcomes (aRR 1.60, 95% CI: 1.28, 2.00), including low birth weight, pre-term birth and foetal loss, this was not seen in women receiving SP (aRR 0.82, 95% CI: 0.54, 1.26). The risk of an association between elevated AGP and adverse birth outcome was higher in those receiving DP or DP+AZ compared to those receiving SP (aRR 1.95, 95% CI: 1.21, 3.13). No clear associations between CRP and adverse outcomes were observed. AGP identified women at risk of adverse pregnancy outcomes. SP modifies the relationship between inflammatory biomarkers and adverse outcomes. Our findings provide insights into potential mechanisms by which SP may improve pregnancy outcomes.

BackgroundMalaria transmission modelling has demonstrated the potential impact of semiquantitative glucose-6-phosphate dehydrogenase (G6PD) testing and treatment with single-dose tafenoquine for Plasmodium vivax radical cure but has not investigated the associated costs. This study evaluated the cost-effectiveness of P. vivax treatment with tafenoquine after G6PD testing using a transmission model.Methods and findingsWe explored the cost-effectiveness of using tafenoquine after G6PD screening as compared to usual practice (7-day low-dose primaquine (0.5 mg/kg/day) without G6PD screening) in Brazil using a 10-year time horizon with 5% discounting considering 4 scenarios: (1) tafenoquine for adults only assuming 66.7% primaquine treatment adherence; (2) tafenoquine for adults and children aged >2 years assuming 66.7% primaquine adherence; (3) tafenoquine for adults only assuming 90% primaquine adherence; and (4) tafenoquine for adults only assuming 30% primaquine adherence. The incremental cost-effectiveness ratios (ICERs) were estimated by dividing the incremental costs by the disability-adjusted life years (DALYs) averted. These were compared to a willingness to pay (WTP) threshold of US$7,800 for Brazil, and one-way and probabilistic sensitivity analyses were performed. All 4 scenarios were cost-effective in the base case analysis using this WTP threshold with ICERs ranging from US$154 to US$1,836. One-way sensitivity analyses showed that the results were most sensitive to severity and mortality due to vivax malaria, the lifetime and number of semiquantitative G6PD analysers needed, cost per malaria episode and per G6PD test strips, and life expectancy. All scenarios had a 100% likelihood of being cost-effective at the WTP threshold. The main limitations of this study are due to parameter uncertainty around our cost estimates for low transmission settings, the costs of G6PD screening, and the severity of vivax malaria.ConclusionsIn our modelling study that incorporated impact on transmission, tafenoquine prescribed after a semiquantitative G6PD testing was highly likely to be cost-effective in Brazil. These results demonstrate the potential health and economic importance of ensuring safe and effective radical cure.

BackgroundPlasmodium knowlesi is a zoonotic parasite that causes malaria in humans. The pathogen has a natural host reservoir in certain macaque species and is transmitted to humans via mosquitoes of the Anopheles Leucosphyrus Group. The risk of human P. knowlesi infection varies across Southeast Asia and is dependent upon environmental factors. Understanding this geographic variation in risk is important both for enabling appropriate diagnosis and treatment of the disease and for improving the planning and evaluation of malaria elimination. However, the data available on P. knowlesi occurrence are biased towards regions with greater surveillance and sampling effort. Predicting the spatial variation in risk of P. knowlesi malaria requires methods that can both incorporate environmental risk factors and account for spatial bias in detection.Methods & resultsWe extend and apply an environmental niche modelling framework as implemented by a previous mapping study of P. knowlesi transmission risk which included data up to 2015. We reviewed the literature from October 2015 through to March 2020 and identified 264 new records of P. knowlesi, with a total of 524 occurrences included in the current study following consolidation with the 2015 study. The modelling framework used in the 2015 study was extended, with changes including the addition of new covariates to capture the effect of deforestation and urbanisation on P. knowlesi transmission.DiscussionOur map of P. knowlesi relative transmission suitability estimates that the risk posed by the pathogen is highest in Malaysia and Indonesia, with localised areas of high risk also predicted in the Greater Mekong Subregion, The Philippines and Northeast India. These results highlight areas of priority for P. knowlesi surveillance and prospective sampling to address the challenge the disease poses to malaria elimination planning.

Abstract Drug regulatory institutions, infrastructures, and systems are becoming increasingly interconnected across national boundaries and increasingly global in outlook. This process is reflected in the broadening and deepening application of the principles and practice of Regulatory Reliance, and parallel initiatives to strengthen the capacities of regulatory institutions in low‐ and middle‐income countries (LMICs). Although these developments are important and constructive, they have tended to be framed in terms of the transfer of systems, knowledge, and skills from relatively “mature” regulatory agencies in high‐income countries (HICs) to less‐well‐resourced regulatory agencies in LMICs. This framing recognizes and foregrounds the considerable practical challenges that many LMIC regulatory agencies face, but in doing so, also backgrounds and underestimates the significance of the different contextual insights that LMIC health researchers and regulators can bring to the regulatory deliberations of their HIC counterparts. This position paper argues that the systematic pursuit, identification, and sharing of these different contextual insights—a dimension of regulatory science that we term “Regulatory Complementarity”—can augment the current practice and goals of Regulatory Reliance, and further invigorate the emerging global regulatory ecosystem.

Disease surveillance aims to collect data at different times or locations, to assist public health authorities to respond appropriately. Surveillance of the simian malaria parasite, Plasmodium knowlesi, is sparse in some endemic areas and the spatial extent of transmission is uncertain. Zoonotic transmission of Plasmodium knowlesi has been demonstrated throughout Southeast Asia and represents a major hurdle to regional malaria elimination efforts. Given an arbitrary spatial prediction of relative disease risk, we develop a flexible framework for surveillance site selection, drawing on principles from multi-criteria decision-making. To demonstrate the utility of our framework, we apply it to the case study of Plasmodium knowlesi malaria surveillance site selection in western Indonesia. We demonstrate how statistical predictions of relative disease risk can be quantitatively incorporated into public health decision-making, with specific application to active human surveillance of zoonotic malaria. This approach can be used in other contexts to extend the utility of modelling outputs.

Abstract As of January 2021, Australia had effectively controlled local transmission of COVID-19 despite a steady influx of imported cases and several local, but contained, outbreaks in 2020. Throughout 2020, state and territory public health responses were informed by weekly situational reports that included an ensemble forecast of daily COVID-19 cases for each jurisdiction. We present here an analysis of one forecasting model included in this ensemble across the variety of scenarios experienced by each jurisdiction from May to October 2020. We examine how successfully the forecasts characterised future case incidence, subject to variations in data timeliness and completeness, showcase how we adapted these forecasts to support decisions of public health priority in rapidly-evolving situations, evaluate the impact of key model features on forecast skill, and demonstrate how to assess forecast skill in real-time before the ground truth is known. Conditioning the model on the most recent, but incomplete, data improved the forecast skill, emphasising the importance of developing strong quantitative models of surveillance system characteristics, such as ascertainment delay distributions. Forecast skill was highest when there were at least 10 reported cases per day, the circumstances in which authorities were most in need of forecasts to aid in planning and response.

Abstract Despite intensive research since the emergence of SARS-CoV-2, it has remained unclear precisely which components of the early immune response protect against the development of severe COVID-19. Here, we perform a comprehensive immunogenetic and virologic analysis of nasopharyngeal and peripheral blood samples obtained during the acute phase of infection with SARS-CoV-2. We find that soluble and transcriptional markers of systemic inflammation peak during the first week after symptom onset and correlate directly with upper airways viral loads (UA-VLs), whereas the contemporaneous frequencies of circulating viral nucleocapsid (NC)-specific CD4+ and CD8+ T cells correlate inversely with various inflammatory markers and UA-VLs. In addition, we show that high frequencies of activated CD4+ and CD8+ T cells are present in acutely infected nasopharyngeal tissue, many of which express genes encoding various effector molecules, such as cytotoxic proteins and IFN-γ. The presence of IFNG mRNA-expressing CD4+ and CD8+ T cells in the infected epithelium is further linked with common patterns of gene expression among virus-susceptible target cells and better local control of SARS-CoV-2. Collectively, these results identify an immune correlate of protection against SARS-CoV-2, which could inform the development of more effective vaccines to combat the acute and chronic illnesses attributable to COVID-19.

Abstract Adoptive immune therapies based on the transfer of antigen-specific T cells have been used successfully to treat various cancers and viral infections, but improved techniques are needed to identify optimally protective human T cell receptors (TCRs). Here we present a high-throughput approach to the identification of natively paired human TCRα and TCRβ (TCRα:β) genes encoding heterodimeric TCRs that recognize specific peptide antigens bound to major histocompatibility complex molecules (pMHCs). We first captured and cloned TCRα:β genes from individual cells, ensuring fidelity using a suppression PCR. We then screened TCRα:β libraries expressed in an immortalized cell line using peptide-pulsed antigen-presenting cells and sequenced activated clones to identify the cognate TCRs. Our results validated an experimental pipeline that allows large-scale repertoire datasets to be annotated with functional specificity information, facilitating the discovery of therapeutically relevant TCRs.

Background Patients with chronic obstructive pulmonary disease (COPD) may be prescribed multiple inhalers that require different techniques for optimal performance. Mixing devices has been associated with poorer COPD outcomes suggesting that it leads to inappropriate inhaler technique. However, empirical evidence is lacking.Aims Compare the nature and frequency of dry powder inhaler (DPI) technique errors in patients with COPD using (1) a single DPI or (2) mixed-devices (a DPI and pressurised metered dose inhaler (pMDI)).Methods Data from the PIFotal study—a cross-sectional study on Peak Inspiratory Flow in patients with COPD using a DPI as maintenance therapy, capturing data from 1434 patients on demographic characteristics, COPD health status and inhaler technique—were used to select 291 patients using mixed-devices. Frequency matching based on country of residence and DPI device type was used to select 291 patients using a DPI-only for comparison. Predetermined checklists were used for the evaluation of DPI video recordings and complemented with additional errors that were observed in ≥10%. Error proportions were calculated for the (1) individual and total number of errors, (2) number of critical errors and (3) number of pMDI-related errors.Results The study sample contained 582 patients (mean (SD) age 69.6 (9.4) years, 47.1% female). DPI technique errors were common, but not significantly different between the groups. The majority of patients made at least one critical error (DPI-only: 90.7% vs mixed-devices: 92.8%). Proportions of total, ‘pMDI-related’ and critical errors did not significantly differ between the groups.Conclusion The nature and frequency of inhaler technique errors did not substantially differ between patients prescribed with a single DPI and mixed-devices. Currently, ‘pMDI-related errors’ in DPI use are not accounted for in existing checklists.Trial registration number ENCEPP/EUPAS48776.

Abstract Background Autism spectrum condition or ‘autism’ is associated with numerous genetic risk factors including the polygenic 16p11.2 microdeletion. The balance between excitatory and inhibitory neurons in the cerebral cortex is hypothesised to be critical for the aetiology of autism making improved understanding of how risk factors impact on the development of these cells an important area of research. In the current study we aim to combine bioinformatics analysis of human foetal cerebral cortex gene expression data with anatomical and electrophysiological analysis of a 16p11.2 +/- rat model to investigate how genetic risk factors impact on inhibitory neuron development. Methods We performed bioinformatics analysis of single cell transcriptomes from gestational week (GW) 8–26 human foetal prefrontal cortex and anatomical and electrophysiological analysis of 16p11.2 +/- rat cerebral cortex and hippocampus at post-natal day (P) 21. Results We identified a subset of human interneurons (INs) first appearing at GW23 with enriched expression of a large fraction of risk factor transcripts including those expressed from the 16p11.2 locus. This suggests the hypothesis that these foetal INs are vulnerable to mutations causing autism. We investigated this in a rat model of the 16p11.2 microdeletion. We found no change in the numbers or position of either excitatory or inhibitory neurons in the somatosensory cortex or CA1 of 16p11.2 +/- rats but found that CA1 Sst INs were hyperexcitable with an enlarged axon initial segment, which was not the case for CA1 pyramidal cells. Limitations The human foetal gene expression data was acquired from cerebral cortex between gestational week (GW) 8 to 26. We cannot draw inferences about potential vulnerabilities to genetic autism risk factors for cells not present in the developing cerebral cortex at these stages. The analysis 16p11.2 +/- rat phenotypes reported in the current study was restricted to 3-week old (P21) animals around the time of weaning and to a single interneuron cell-type while in human 16p11.2 microdeletion carriers symptoms likely involve multiple cell types and manifest in the first few years of life and on into adulthood. Conclusions We have identified developing interneurons in human foetal cerebral cortex as potentially vulnerable to monogenic autism risk factors and the 16p11.2 microdeletion and report interneuron phenotypes in post-natal 16p11.2 +/- rats.

Abstract Background The distribution of the duration that clinical cases of COVID-19 occupy hospital beds (the ‘length of stay’) is a key factor in determining how incident caseloads translate into health system burden. Robust estimation of length of stay in real-time requires the use of survival methods that can account for right-censoring induced by yet unobserved events in patient progression (e.g. discharge, death). In this study, we estimate in real-time the length of stay distributions of hospitalised COVID-19 cases in New South Wales, Australia, comparing estimates between a period where Delta was the dominant variant and a subsequent period where Omicron was dominant. Methods Using data on the hospital stays of 19,574 individuals who tested positive to COVID-19 prior to admission, we performed a competing-risk survival analysis of COVID-19 clinical progression. Results During the mixed Omicron-Delta epidemic, we found that the mean length of stay for individuals who were discharged directly from ward without an ICU stay was, for age groups 0–39, 40–69 and 70 +, respectively, 2.16 (95% CI: 2.12–2.21), 3.93 (95% CI: 3.78–4.07) and 7.61 days (95% CI: 7.31–8.01), compared to 3.60 (95% CI: 3.48–3.81), 5.78 (95% CI: 5.59–5.99) and 12.31 days (95% CI: 11.75–12.95) across the preceding Delta epidemic (1 July 2021–15 December 2021). We also considered data on the stays of individuals within the Hunter New England Local Health District, where it was reported that Omicron was the only circulating variant, and found mean ward-to-discharge length of stays of 2.05 (95% CI: 1.80–2.30), 2.92 (95% CI: 2.50–3.67) and 6.02 days (95% CI: 4.91–7.01) for the same age groups. Conclusions Hospital length of stay was substantially reduced across all clinical pathways during a mixed Omicron-Delta epidemic compared to a prior Delta epidemic, contributing to a lessened health system burden despite a greatly increased infection burden. Our results demonstrate the utility of survival analysis in producing real-time estimates of hospital length of stay for assisting in situational assessment and planning of the COVID-19 response.

Abstract Background Electronic consultation (eConsult) programs are crucial components of modern healthcare that facilitate communication between primary care providers (PCPs) and specialists. eConsults between PCPs and specialists. They also provide a unique opportunity to use real-world patient scenarios for reflective learning as part of professional development. However, tools that guide and document learning from eConsults are limited. The purpose of this study was to develop and pilot two eConsult reflective learning tools (RLTs), one for PCPs and one for specialists, for those participating in eConsults. Methods We performed a four-phase pragmatic mixed methods study recruiting PCPs and specialists from two public health systems located in two countries: eConsult BASE in Canada and San Francisco Health Network eConsult in the United States. In phase 1, subject matter experts developed preliminary RLTs for PCPs and specialists. During phase 2, a Delphi survey among 20 PCPs and 16 specialists led to consensus on items for each RLT. In phase 3, we conducted cognitive interviews with three PCPs and five specialists as they applied the RLTs on previously completed consults. In phase 4, we piloted the RLTs with eConsult users. Results The RLTs were perceived to elicit critical reflection among participants regarding their knowledge and practice habits and could be used for quality improvement and continuing professional development. Conclusion PCPs and specialists alike perceived that eConsult systems provided opportunities for self-directed learning wherein they were motivated to investigate topics further through the course of eConsult exchanges. We recommend the RLTs be subject to further evaluation through implementation studies at other sites.

In peripheral blood mononuclear cells from healthy human donors, the superantigens SpeC and TSST-1 are shown to specifically activate TRBV12-3/12-4+ memory T cells.

Brain-wide mapping of synapse molecular composition in Pax6 mutant mice shows remodelling and restoration of synaptome architecture during development, a possible means of conferring resilience to genetic disorders.

Introduction: Antiretroviral therapy for people living with HIV-1 must be taken lifelong due to the persistence of latent virus in long-lived and proliferating CD4+ T cells. Vitamin D3 is a steroidal gene transcription regulator which exerts diverse effects on immune and epithelial cells including reductions in CD4+ T cell proliferation and improvement in gut barrier integrity. We hypothesised that a high dose of vitamin D3 would reduce the size of the HIV-1 reservoir by reducing CD4+ T cell proliferation. Methods: We performed a randomised placebo-controlled trial evaluating the effect of 24 weeks of vitamin D3 (10,000 international units per day) on the HIV-1 reservoir and immunologic parameters in 30 adults on antiretroviral therapy; participants were followed for 12 weeks post-treatment. The primary endpoint was the effect on total HIV-1 DNA at week 24. Parameters were assessed using mixed-effects models. Results: We found no effect of vitamin D3 on the change in total HIV-1 DNA from week 0 to week 24 relative to placebo. There were also no changes in integrated HIV-1 DNA, 2-long-terminal repeat (2-LTR) circles or cell-associated HIV-1 RNA. Vitamin D3 induced a significant increase in the proportion of central memory CD4+ and CD8+ T cells, a reduction in the proportion of senescent CD8+ T cells and a reduction in the natural killer cell frequency at all time points including week 36, 12 weeks after the study drug cessation. At week 36, there was a significant reduction in total HIV-1 DNA relative to placebo and persistently elevated 25-hydroxyvitamin D levels. No significant safety issues were identified. Conclusions: Vitamin D3 administration had a significant impact on the T cell differentiation but overall effects on the HIV-1 reservoir were limited and a reduction in HIV-1 DNA was only seen following cessation of the study drug. Additional studies are required to determine whether the dose and duration of vitamin D3 can be optimised to promote a continued depletion of the HIV-1 reservoir over time. Trial registration: ClinicalTrials.gov NCT03426592.

The fundamental basis of T cell memory remains elusive. It is established that antigen stimulation drives clonal proliferation and differentiation, but the relationship between cellular phenotype, replicative history, and longevity, which is likely essential for durable memory, has proven difficult to elucidate. To address these issues, we used conventional markers of differentiation to identify and isolate various subsets of CD8+ memory T cells and measured telomere lengths in these phenotypically defined populations using the most sensitive technique developed to date, namely single telomere length analysis (STELA). Naive cells were excluded on the basis of dual expression of CCR7 and CD45RA. Memory subsets were sorted as CD27+CD45RA+, CD27intCD45RA+, CD27−CD45RA+, CD27+CD45RAint, CD27−CD45RAint, CD27+CD45RA−, and CD27−CD45RA− at >98% purity. The shortest median telomere lengths were detected among subsets that lacked expression of CD45RA, and the longest median telomere lengths were detected among subsets that expressed CD45RA. Longer median telomere lengths were also a feature of subsets that expressed CD27 in compartments defined by the absence or presence of CD45RA. Collectively, these data suggested a disconnect between replicative history and CD8+ memory T cell differentiation, which is classically thought to be a linear process that culminates with revertant expression of CD45RA.

Inhibitory CD4+ T cells have been linked with suboptimal immune responses against cancer and pathogen chronicity. However, the mechanisms that underpin the development of these regulatory cells, especially in the context of ongoing antigen exposure, have remained obscure. To address this knowledge gap, we undertook a comprehensive functional, phenotypic, and transcriptomic analysis of interleukin (IL)-10-producing CD4+ T cells induced by chronic infection with murine cytomegalovirus (MCMV). We identified these cells as clonally expanded and highly differentiated TH1-like cells that developed in a T-bet-dependent manner and coexpressed arginase-1 (Arg1), which promotes the catalytic breakdown of L-arginine. Mice lacking Arg1-expressing CD4+ T cells exhibited more robust antiviral immunity and were better able to control MCMV. Conditional deletion of T-bet in the CD4+ lineage suppressed the development of these inhibitory cells and also enhanced immune control of MCMV. Collectively, these data elucidated the ontogeny of IL-10-producing CD4+ T cells and revealed a previously unappreciated mechanism of immune regulation, whereby viral persistence was facilitated by the site-specific delivery of Arg1.

Background: Measurement of antibody titers directed against mosquito salivary antigens in blood samples has been proposed as an outcome measure to assess human exposure to vector bites. However, only a handful of antigens have been identified and the specificity and longitudinal dynamics of antibody responses are not well known. We report the protocol of a clinical trial of controlled exposure to mosquito bites that aims to identify and validate biomarkers of exposure to bites of mosquito vector species that transmit malaria and dengue in Southeast Asia and some other parts of the world. Methods: This study is an exploratory factorial randomized control trial of controlled exposure to mosquito bites with 10 arms corresponding to different species (Aedes aegypti, Ae. albopictus, Anopheles dirus, An. maculatus and An. minimus) and numbers of bites (35 or 305 bites in total over 6 weeks). Blood samples will be collected from study participants before, during and after mosquito biting challenges. Candidate peptides will be identified from published literature with antigen prediction algorithms using mosquito DNA sequence data and with immunoblotting assays carried out using protein extracts of dissected mosquito salivary glands and participants samples. Antibody titers against candidate peptides will be determined in participants samples with high-throughput cutting-edge immuno-assays. Quantification of the antibody response profile over time (including an estimate of the decay rate) and the effect of the number of bites on the antibody response will be determined using linear and logistic mixed-effects models for the continuous and the binary response, respectively. Conclusion: This research is expected to generate important knowledge for vector sero-surveillance and evaluation of vector-control interventions against malaria and dengue in the Greater Mekong Subregion. Registration: This study is registered with clinicaltrials.gov (NCT04478370) on July 20th, 2020.

Introduction: Sickle cell trait (SCT) has been associated with chronic kidney disease (CKD) in African Americans, although evidence for its impact in Africans and people with HIV is currently lacking. We conducted a cross-sectional study investigating the association between SCT and kidney disease in people of African ancestry with HIV in the UK. Methods: The primary outcome was estimated glomerular filtration rate (eGFR) 50 mg/mmol), and albuminuria (albumin-to-creatinine ratio >3 mg/mmol). Multivariable logistic regression was used to estimate the associations between SCT and kidney disease outcomes. Results: A total of 2895 participants (mean age 48.1 [SD 10.3], 57.2% female) were included, of whom 335 (11.6%) had SCT and 352 (12.2%) had eGFR

Summary: Background: CD8+ T cells equipped with a full arsenal of antiviral effector functions are critical for effective immune control of HIV-1. It has nonetheless remained unclear how best to elicit such potent cellular immune responses in the context of immunotherapy or vaccination. HIV-2 has been associated with milder disease manifestations and more commonly elicits functionally replete virus-specific CD8+ T cell responses compared with HIV-1. We aimed to learn from this immunological dichotomy and to develop informed strategies that could enhance the induction of robust CD8+ T cell responses against HIV-1. Methods: We developed an unbiased in vitro system to compare the de novo induction of antigen-specific CD8+ T cell responses after exposure to HIV-1 or HIV-2. The functional properties of primed CD8+ T cells were assessed using flow cytometry and molecular analyses of gene transcription. Findings: HIV-2 primed functionally optimal antigen-specific CD8+ T cells with enhanced survival properties more effectively than HIV-1. This superior induction process was dependent on type I interferons (IFNs) and could be mimicked via the adjuvant delivery of cyclic GMP-AMP (cGAMP), a known agonist of the stimulator of interferon genes (STING). CD8+ T cells elicited in the presence of cGAMP were polyfunctional and highly sensitive to antigen stimulation, even after priming from people living with HIV-1. Interpretation: HIV-2 primes CD8+ T cells with potent antiviral functionality by activating the cyclic GMP-AMP synthase (cGAS)/STING pathway, which results in the production of type I IFNs. This process may be amenable to therapeutic development via the use of cGAMP or other STING agonists to bolster CD8+ T cell-mediated immunity against HIV-1. Funding: This work was funded by INSERM, the Institut Curie, and the University of Bordeaux (Senior IdEx Chair) and by grants from Sidaction (17-1-AAE-11097, 17-1-FJC-11199, VIH2016126002, 20-2-AEQ-12822-2, and 22-2-AEQ-13411), the Agence Nationale de la Recherche sur le SIDA (ECTZ36691, ECTZ25472, ECTZ71745, and ECTZ118797), and the Fondation pour la Recherche Médicale (EQ U202103012774). D.A.P. was supported by a Wellcome Trust Senior Investigator Award (100326/Z/12/Z).

Here the authors digested chromatin with DNA fragmentation factor (DFF) prior to chromatin immunoprecipitation (DFF-ChIP) to depict transcription complex interactions with neighboring nucleosomes in cells. Applying this method to human cytomegalovirus (HMCV)-infected cells, they find that the viral genome is underchromatinized, leading to fewer transcription complex interactions with nucleosomes.

Abstract Background Malaria remains a major public health threat and tools sensitive to detect infections in low malaria transmission areas are needed to progress elimination efforts. Pregnant women are particularly vulnerable to malaria infections. Throughout pregnancy they access routine antenatal care, presenting a unique sentinel population to apply novel sero-surveillance tools to measure malaria transmission. The aim of this study was to quantify the dynamic antibody responses to multiple antigens during pregnancy so as to identify a single or multiple antibody response of exposure to malaria in pregnancy. Methods This study involved a secondary analysis of antibody responses to six parasite antigens [five commonly studied merozoite antigens and the variant surface antigen 2-chondroitin sulphate A (VAR2CSA), a pregnancy-specific erythrocytic antigen] measured by enzyme-linked immunosorbent assay (ELISA) over the gestation period until delivery (median of 7 measurements/woman) in 250 pregnant women who attended antenatal clinics located at the Thai-Myanmar border. A multivariate mixture linear mixed model was used to cluster the pregnant women into groups that have similar longitudinal antibody responses to all six antigens over the gestational period using a Bayesian approach. The variable-specific entropy was calculated to identify the antibody responses that have the highest influence on the classification of the women into clusters, and subsequent agreement with grouping of women based on exposure to malaria during pregnancy. Results Of the 250 pregnant women, 135 had a Plasmodium infection detected by light microscopy during pregnancy (39% Plasmodium falciparum only, 33% Plasmodium vivax only and 28% mixed/other species), defined as cases. The antibody responses to all six antigens accurately identified the women who did not have a malaria infection detected during pregnancy (93%, 107/115 controls). Antibody responses to P. falciparum merozoite surface protein 3 (PfMSP3) and P. vivax apical membrane antigen 1 (PvAMA1) were the least dynamic. Antibody responses to the antigens P. falciparum apical membrane antigen 1 (PfAMA1) and PfVAR2CSA were able to identify the majority of the cases more accurately (63%, 85/135). Conclusion These findings suggest that the combination of antibodies, PfAMA1 and PfVAR2CSA, may be useful for sero-surveillance of malaria infections in pregnant women, particularly in low malaria transmission settings. Further investigation of other antibody markers is warranted considering these antibodies combined only detected 63% of the malaria infections during pregnancy.

Introduction Severe asthma is associated with a disproportionally high disease burden, including the risk of severe exacerbations. Accurate prediction of the risk of severe exacerbations may enable clinicians to tailor treatment plans to an individual patient. This study aims to develop and validate a novel risk prediction model for severe exacerbations in patients with severe asthma, and to examine the potential clinical utility of this tool.Methods and analysis The target population is patients aged 18 years or older with severe asthma. Based on the data from the International Severe Asthma Registry (n=8925), a prediction model will be developed using a penalised, zero-inflated count model that predicts the rate or risk of exacerbation in the next 12 months. The risk prediction tool will be externally validated among patients with physician-assessed severe asthma in an international observational cohort, the NOVEL observational longiTudinal studY (n=1652). Validation will include examining model calibration (ie, the agreement between observed and predicted rates), model discrimination (ie, the extent to which the model can distinguish between high-risk and low-risk individuals) and the clinical utility at a range of risk thresholds.Ethics and dissemination This study has obtained ethics approval from the Institutional Review Board of National University of Singapore (NUS-IRB-2021-877), the Anonymised Data Ethics and Protocol Transparency Committee (ADEPT1924) and the University of British Columbia (H22-01737). Results will be published in an international peer-reviewed journal.Trial registration number European Union electronic Register of Post-Authorisation Studies, EU PAS Register (EUPAS46088).

Against a backdrop of widespread global transmission, a number of countries have successfully brought large outbreaks of COVID-19 under control and maintained near-elimination status. A key element of epidemic response is the tracking of disease transmissibility in near real-time. During major outbreaks, the effective reproduction number can be estimated from a time-series of case, hospitalisation or death counts. In low or zero incidence settings, knowing the potential for the virus to spread is a response priority. Absence of case data means that this potential cannot be estimated directly. We present a semi-mechanistic modelling framework that draws on time-series of both behavioural data and case data (when disease activity is present) to estimate the transmissibility of SARS-CoV-2 from periods of high to low – or zero – case incidence, with a coherent transition in interpretation across the changing epidemiological situations. Of note, during periods of epidemic activity, our analysis recovers the effective reproduction number, while during periods of low – or zero – case incidence, it provides an estimate of transmission risk. This enables tracking and planning of progress towards the control of large outbreaks, maintenance of virus suppression, and monitoring the risk posed by re-introduction of the virus. We demonstrate the value of our methods by reporting on their use throughout 2020 in Australia, where they have become a central component of the national COVID-19 response.

Summary: Background: HIV can infect multiple cells in the liver including hepatocytes, Kupffer cells and infiltrating T cells, but whether HIV can persist in the liver in people with HIV (PWH) on suppressive antiretroviral therapy (ART) remains unknown. Methods: In a prospective longitudinal cohort of PWH and hepatitis B virus (HBV) co-infection living in Bangkok, Thailand, we collected blood and liver biopsies from 18 participants prior to and following ART and quantified HIV and HBV persistence using quantitative (q)PCR and RNA/DNAscope. Antiretroviral (ARV) drug levels were quantified using mass spectroscopy. Findings: In liver biopsies taken prior to ART, HIV DNA and HIV RNA were detected by qPCR in 53% (9/17) and 47% (8/17) of participants respectively. Following a median ART duration of 3.4 years, HIV DNA was detected in liver in 61% (11/18) of participants by either qPCR, DNAscope or both, but only at very low and non-quantifiable levels. Using immunohistochemistry, HIV DNA was observed in both hepatocytes and liver infiltrating CD4+ T cells on ART. HIV RNA was not detected in liver biopsies collected on ART, by either qPCR or RNAscope. All ARVs were clearly detected in liver tissue. Interpretation: Persistence of HIV DNA in liver in PWH on ART represents an additional reservoir that warrants further investigation. Funding: National Health and Medical Research Council of Australia (Project Grant APP1101836, 1149990, and 1135851); This project has been funded in part with federal funds from the National Cancer Institute, National Institutes of Health, under Contract No. 75N91019D00024.

Abstract Background Convalescent plasma has been widely used to treat COVID-19 and is under investigation in numerous randomized clinical trials, but results are publicly available only for a small number of trials. The objective of this study was to assess the benefits of convalescent plasma treatment compared to placebo or no treatment and all-cause mortality in patients with COVID-19, using data from all available randomized clinical trials, including unpublished and ongoing trials (Open Science Framework, https://doi.org/10.17605/OSF.IO/GEHFX ). Methods In this collaborative systematic review and meta-analysis, clinical trial registries (ClinicalTrials.gov, WHO International Clinical Trials Registry Platform), the Cochrane COVID-19 register, the LOVE database, and PubMed were searched until April 8, 2021. Investigators of trials registered by March 1, 2021, without published results were contacted via email. Eligible were ongoing, discontinued and completed randomized clinical trials that compared convalescent plasma with placebo or no treatment in COVID-19 patients, regardless of setting or treatment schedule. Aggregated mortality data were extracted from publications or provided by investigators of unpublished trials and combined using the Hartung–Knapp–Sidik–Jonkman random effects model. We investigated the contribution of unpublished trials to the overall evidence. Results A total of 16,477 patients were included in 33 trials (20 unpublished with 3190 patients, 13 published with 13,287 patients). 32 trials enrolled only hospitalized patients (including 3 with only intensive care unit patients). Risk of bias was low for 29/33 trials. Of 8495 patients who received convalescent plasma, 1997 died (23%), and of 7982 control patients, 1952 died (24%). The combined risk ratio for all-cause mortality was 0.97 (95% confidence interval: 0.92; 1.02) with between-study heterogeneity not beyond chance (I2 = 0%). The RECOVERY trial had 69.8% and the unpublished evidence 25.3% of the weight in the meta-analysis. Conclusions Convalescent plasma treatment of patients with COVID-19 did not reduce all-cause mortality. These results provide strong evidence that convalescent plasma treatment for patients with COVID-19 should not be used outside of randomized trials. Evidence synthesis from collaborations among trial investigators can inform both evidence generation and evidence application in patient care.

Summary: Background: Whole genome sequencing (WGS) is increasingly used by tuberculosis (TB) programs to monitor Mycobacterium tuberculosis (Mtb) transmission. We aimed to characterise the molecular epidemiology of TB and Mtb transmission in the low-incidence setting of Victoria, Australia, and assess the utility of WGS. Methods: WGS was performed on all first Mtb isolates from TB cases from 2017 to 2020. Potential clusters (≤12 single nucleotide polymorphisms [SNPs]) were investigated for epidemiological links. Transmission events in highly-related (≤5 SNPs) clusters were classified as likely or possible, based on the presence or absence of an epidemiological link, respectively. Case characteristics and transmission settings (as defined by case relationship) were summarised. Poisson regression was used to examine associations with secondary case number. Findings: Of 1844 TB cases, 1276 (69.2%) had sequenced isolates, with 182 (14.2%) in 54 highly-related clusters, 2–40 cases in size. Following investigation, 140 cases (11.0% of sequenced) were classified as resulting from likely/possible local-transmission, including 82 (6.4%) for which transmission was likely. Common identified transmission settings were social/religious (26.4%), household (22.9%) and family living in different households (7.1%), but many were uncertain (41.4%). While household transmission featured in many clusters (n = 24), clusters were generally smaller (median = 3 cases) than the fewer that included transmission in social/religious settings (n = 12, median = 7.5 cases). Sputum-smear-positivity was associated with higher secondary case numbers. Interpretation: WGS results suggest Mtb transmission commonly occurs outside the household in our low-incidence setting. Further work is required to optimise the use of WGS in public health management of TB. Funding: The Victorian Tuberculosis Program receives block funding for activities including case management and contact tracing from the Victorian Department of Health. No specific funding for this report was received by manuscript authors or the Victorian Tuberculosis Program, and the funders had no role in the study design, data collection, data analysis, interpretation or report writing.

Summary: Background: First Few “X” (FFX) studies provide a platform to collect the required epidemiological, clinical and virological data to help address emerging information needs about the COVID-19 pandemic. Methods: We adapted the WHO FFX protocol for COVID-19 to understand severity and household transmission dynamics in the early stages of the pandemic in Australia. Implementation strategies were developed for participating sites; all household members were followed for 14 days from case identification. Household contacts completed symptom diaries and had multiple respiratory swabs taken irrespective of symptoms. We modelled the spread of COVID-19 within households using a susceptible-exposed-infectious-recovered-type model, and calculated the household secondary attack rate and key epidemiological parameters. Findings: 96 households with 101 cases and 286 household contacts were recruited into the study between April–October 2020. Forty household contacts tested positive for SARS-CoV-2 in the study follow-up period. Our model estimated the household secondary attack rate to be 15% (95% CI 8–25%), which scaled up with increasing household size. Our findings suggest children were less infectious than their adult counterparts but were also more susceptible to infection. Interpretation: Our study provides important baseline data characterising the transmission of early SARS-CoV-2 strains from children and adults in Australia, against which properties of variants of concern can be benchmarked. We encountered many challenges with respect to logistics, ethics, governance and data management. Continued efforts to invest in preparedness research will help to test, refine and further develop Australian FFX study protocols in advance of future outbreaks. Funding: Australian Government Department of Health.

Abstract Background Many primary care patients receive both medical and chiropractic care; however, interprofessional relations between physicians and chiropractors are often suboptimal which may adversely affect care of shared patients. We surveyed Canadian family physicians in 2010 to explore their attitudes towards chiropractic and re-administered the same survey a decade later to explore for changes in attitudes. Methods A 50-item survey administered to a random sample of Canadian family physicians in 2010, and again in 2019, that inquired about demographic variables, knowledge and use of chiropractic. Imbedded in our survey was a 20-item chiropractic attitude questionnaire (CAQ); scores could range from 0 to 80 with higher scores indicating more positive attitudes toward chiropractic. We constructed a multivariable regression model to explore factors associated with CAQ scores. Results Among eligible physicians, 251 of 685 in 2010 (37% response rate) and 162 of 2429 in 2019 (7% response rate) provided a completed survey. Approximately half of respondents (48%) endorsed a positive impression of chiropractic, 27% were uncertain, and 25% held negative views. Most respondents (72%) referred at least some patients for chiropractic care, mainly due to patient request or lack of response to medical care. Most physicians believed that chiropractors provide effective therapy for some musculoskeletal complaints (84%) and disagreed that chiropractic care was beneficial for non-musculoskeletal conditions (77%). The majority agreed that chiropractic care was a useful supplement to conventional care (65%) but most respondents (59%) also indicated that practice diversity among chiropractors presented a barrier to interprofessional collaboration. In our adjusted regression model, attitudes towards chiropractic showed trivial improvement from 2010 to 2019 (0.31 points on the 80-point CAQ; 95%CI 0.001 to 0.62). More negative attitudes were associated with older age (− 1.55 points for each 10-year increment from age 28; 95%CI − 2.67 to − 0.44), belief that adverse events are common with chiropractic care (− 1.41 points; 95% CI − 2.59 to − 0.23) and reported use of the research literature (− 6.04 points; 95% CI − 8.47 to − 3.61) or medical school (− 5.03 points; 95% CI − 7.89 to − 2.18) as sources of knowledge on chiropractic. More positive attitudes were associated with endorsing a relationship with a specific chiropractor (5.24 points; 95% CI 2.85 to 7.64), family and friends (4.06 points; 95% CI 1.53 to 6.60), or personal treatment experience (4.63 points; 95% CI 2.14 to 7.11) as sources of information regarding chiropractic. Conclusions Although generally positive, Canadian family physicians’ attitudes towards chiropractic are diverse, and most physicians felt that practice diversity among chiropractors was a barrier to interprofessional collaboration.

Abstract Background The 7SK small nuclear RNA (snRNA) found in most metazoans is a key regulator of P-TEFb which in turn regulates RNA polymerase II elongation. Although its primary sequence varies in protostomes, its secondary structure and function are conserved across evolutionary distant taxa. Results Here, we describe a novel ncRNA sharing many features characteristic of 7SK RNAs, in D. melanogaster. We examined the structure of the corresponding gene and determined the expression profiles of the encoded RNA, called snRNA:7SK:94F, during development. It is probably produced from the transcription of a lncRNA which is processed into a mature snRNA. We also addressed its biological function and we show that, like dm7SK, this alternative 7SK interacts in vivo with the different partners of the P-TEFb complex, i.e. HEXIM, LARP7 and Cyclin T. This novel RNA is widely expressed across tissues. Conclusion We propose that two distinct 7SK genes might contribute to the formation of the 7SK snRNP complex in D. melanogaster.

Abstract COVID-19-associated respiratory failure offers the unprecedented opportunity to evaluate the differential host response to a uniform pathogenic insult. Understanding whether there are distinct subphenotypes of severe COVID-19 may offer insight into its pathophysiology. Sequential Organ Failure Assessment (SOFA) score is an objective and comprehensive measurement that measures dysfunction severity of six organ systems, i.e., cardiovascular, central nervous system, coagulation, liver, renal, and respiration. Our aim was to identify and characterize distinct subphenotypes of COVID-19 critical illness defined by the post-intubation trajectory of SOFA score. Intubated COVID-19 patients at two hospitals in New York city were leveraged as development and validation cohorts. Patients were grouped into mild, intermediate, and severe strata by their baseline post-intubation SOFA. Hierarchical agglomerative clustering was performed within each stratum to detect subphenotypes based on similarities amongst SOFA score trajectories evaluated by Dynamic Time Warping. Distinct worsening and recovering subphenotypes were identified within each stratum, which had distinct 7-day post-intubation SOFA progression trends. Patients in the worsening suphenotypes had a higher mortality than those in the recovering subphenotypes within each stratum (mild stratum, 29.7% vs. 10.3%, p = 0.033; intermediate stratum, 29.3% vs. 8.0%, p = 0.002; severe stratum, 53.7% vs. 22.2%, p

The development of stable specialized cell types in multicellular organisms relies on mechanisms controlling inductive intercellular signals and the competence of cells to respond to such signals. In developing cerebral cortex, progenitors generate only glutamatergic excitatory neurons despite being exposed to signals with the potential to initiate the production of other neuronal types, suggesting that their competence is limited. Here, we tested the hypothesis that this limitation is due to their expression of transcription factor Pax6. We used bulk and single-cell RNAseq to show that conditional cortex-specific Pax6 deletion from the onset of cortical neurogenesis allowed some progenitors to generate abnormal lineages resembling those normally found outside the cortex. Analysis of selected gene expression showed that the changes occurred in specific spatiotemporal patterns. We then compared the responses of control and Pax6-deleted cortical cells to in vivo and in vitro manipulations of extracellular signals. We found that Pax6 loss increased cortical progenitors' competence to generate inappropriate lineages in response to extracellular factors normally present in developing cortex, including the morphogens Shh and Bmp4. Regional variation in the levels of these factors could explain spatiotemporal patterns of fate change following Pax6 deletion in vivo. We propose that Pax6's main role in developing cortical cells is to minimize the risk of their development being derailed by the potential side effects of morphogens engaged contemporaneously in other essential functions.

Abstract A balanced t(1;11) translocation that directly disrupts DISC1 is linked to schizophrenia and affective disorders. We previously showed that a mutant mouse, named Der1, recapitulates the effect of the translocation upon DISC1 expression. Here, RNAseq analysis of Der1 mouse brain tissue found enrichment for dysregulation of the same genes and molecular pathways as in neuron cultures generated previously from human t(1;11) translocation carriers via the induced pluripotent stem cell route. DISC1 disruption therefore apparently accounts for a substantial proportion of the effects of the t(1;11) translocation. RNAseq and pathway analysis of the mutant mouse predicts multiple Der1-induced alterations converging upon synapse function and plasticity. Synaptosome proteomics confirmed that the Der1 mutation impacts synapse composition, and electrophysiology found reduced AMPA:NMDA ratio in hippocampal neurons, indicating changed excitatory signalling. Moreover, hippocampal parvalbumin-positive interneuron density is increased, suggesting that the Der1 mutation affects inhibitory control of neuronal circuits. These phenotypes predict that neurotransmission is impacted at many levels by DISC1 disruption in human t(1;11) translocation carriers. Notably, genes implicated in schizophrenia, depression and bipolar disorder by large-scale genetic studies are enriched among the Der1-dysregulated genes, just as we previously observed for the t(1;11) translocation carrier-derived neurons. Furthermore, RNAseq analysis predicts that the Der1 mutation primarily targets a subset of cell types, pyramidal neurons and interneurons, previously shown to be vulnerable to the effects of common schizophrenia-associated genetic variants. In conclusion, DISC1 disruption by the t(1;11) translocation may contribute to the psychiatric disorders of translocation carriers through commonly affected pathways and processes in neurotransmission.

ABSTRACT Human cytomegalovirus (HCMV) immediate-early 2 (IE2) protein is a multifunctional transcription factor that is essential for lytic HCMV infection. IE2 functions as an activator of viral early genes, negatively regulates its own promoter, and is required for viral replication. The mechanisms by which IE2 executes these distinct functions are incompletely understood. Using PRO-Seq, which profiles nascent transcripts, and a recently developed DFF-chromatin immunoprecipitation (DFF-ChIP; employs chromatin digestion by the endonuclease DNA fragmentation factor prior to IP) approach that resolves occupancy and local chromatin environment, we show that IE2 controls viral gene transcription in three distinct capacities during late HCMV infection and reveal mechanisms that involve direct binding of IE2 to viral DNA. IE2 represses a subset of viral promoters by binding within their core promoter regions and blocking the assembly of preinitiation complexes (PICs). Remarkably, IE2 forms a repressive complex at the major immediate-early promoter region involving direct association of IE2 with nucleosomes and TBP. IE2 stimulates transcription by binding nearby, but not within, core promoter regions. In addition, IE2 functions as a direct roadblock to transcription elongation. At one locus, this function of IE2 appears to be important for the synthesis of a spliced viral RNA. Consistent with the minimal observed effects of IE2 depletion on host gene transcription, IE2 does not functionally engage the host genome. Our results reveal mechanisms of transcriptional control by IE2, uncover a previously unknown function of IE2 as a Pol II elongation modulator, and demonstrate that DFF-ChIP is a useful tool for probing transcription factor occupancy and interactions between transcription factors and nucleosomes at high resolution. IMPORTANCE HCMV infects more than half of the world population and persists lifelong in its hosts. Although generally asymptomatic, HCMV infection can lead to life-threating disease in immunosuppressed individuals. Moreover, HCMV is the leading infectious cause of birth defects in the United States. As there are no vaccines effective against HCMV and antiviral drugs exhibit toxicity and are undermined by resistant HCMV variants, other vulnerabilities in HCMV must be explored. Here, we characterize the mechanism by which IE2 controls transcription during late HCMV infection. We demonstrate that IE2 engages numerous consensus sites across the HCMV genome and functions as an activator, repressor, or elongation modulator depending on the context of IE2 binding sites in relation to Pol II initiation and elongation complexes. Our findings have important implications for the ongoing exploration of IE2 as an antiviral drug target.

The outcome of infection is dependent on the ability of viruses to manipulate the infected cell to evade immunity, and the ability of the immune response to overcome this evasion. Understanding this process is key to understanding pathogenesis, genetic risk factors, and both natural and vaccine-induced immunity. SARS-CoV-2 antagonises the innate interferon response, but whether it manipulates innate cellular immunity is unclear. An unbiased proteomic analysis determined how cell surface protein expression is altered on SARS-CoV-2-infected lung epithelial cells, showing downregulation of activating NK ligands B7-H6, MICA, ULBP2, and Nectin1, with minimal effects on MHC-I. This occurred at the level of protein synthesis, could be mediated by Nsp1 and Nsp14, and correlated with a reduction in NK cell activation. This identifies a novel mechanism by which SARS-CoV-2 host-shutoff antagonises innate immunity. Later in the disease process, strong antibody-dependent NK cell activation (ADNKA) developed. These responses were sustained for at least 6 months in most patients, and led to high levels of pro-inflammatory cytokine production. Depletion of spike-specific antibodies confirmed their dominant role in neutralisation, but these antibodies played only a minor role in ADNKA compared to antibodies to other proteins, including ORF3a, Membrane, and Nucleocapsid. In contrast, ADNKA induced following vaccination was focussed solely on spike, was weaker than ADNKA following natural infection, and was not boosted by the second dose. These insights have important implications for understanding disease progression, vaccine efficacy, and vaccine design.

Abstract Some autism spectrum disorders (ASD) likely arise as a result of abnormalities during early embryonic development of the brain. Studying human embryonic brain development directly is challenging, mainly due to ethical and practical constraints. However, the recent development of cerebral organoids provides a powerful tool for studying both normal human embryonic brain development and, potentially, the origins of neurodevelopmental disorders including ASD. Substantial evidence now indicates that cerebral organoids can mimic normal embryonic brain development and neural cells found in organoids closely resemble their in vivo counterparts. However, with prolonged culture, significant differences begin to arise. We suggest that cerebral organoids, in their current form, are most suitable to model earlier neurodevelopmental events and processes such as neurogenesis and cortical lamination. Processes implicated in ASDs which occur at later stages of development, such as synaptogenesis and neural circuit formation, may also be modeled using organoids. The accuracy of such models will benefit from continuous improvements to protocols for organoid differentiation.