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Blood and immune development in human fetal bone marrow and Down syndrome.
Haematopoiesis in the bone marrow (BM) maintains blood and immune cell production throughout postnatal life. Haematopoiesis first emerges in human BM at 11-12 weeks after conception1,2, yet almost nothing is known about how fetal BM (FBM) evolves to meet the highly specialized needs of the fetus and newborn. Here we detail the development of FBM, including stroma, using multi-omic assessment of mRNA and multiplexed protein epitope expression. We find that the full blood and immune cell repertoire is established in FBM in a short time window of 6-7 weeks early in the second trimester. FBM promotes rapid and extensive diversification of myeloid cells, with granulocytes, eosinophils and dendritic cell subsets emerging for the first time. The substantial expansion of B lymphocytes in FBM contrasts with fetal liver at the same gestational age. Haematopoietic progenitors from fetal liver, FBM and cord blood exhibit transcriptional and functional differences that contribute to tissue-specific identity and cellular diversification. Endothelial cell types form distinct vascular structures that we show are regionally compartmentalized within FBM. Finally, we reveal selective disruption of B lymphocyte, erythroid and myeloid development owing to a cell-intrinsic differentiation bias as well as extrinsic regulation through an altered microenvironment in Down syndrome (trisomy 21).
Evaluation of a novel malaria anti-sporozoite vaccine candidate, R21 in Matrix-M adjuvant, in the UK and Burkina Faso: two phase 1, first-in-human trials.
BACKGROUND: Malaria remains a substantial public health burden among young children in sub-Saharan Africa and a highly efficacious vaccine eliciting a durable immune response would be a useful tool for controlling malaria. R21 is a malaria vaccine comprising nanoparticles, formed from a circumsporozoite protein and hepatitis B surface antigen (HBsAg) fusion protein, without any unfused HBsAg, and is administered with the saponin-based Matrix-M adjuvant. This study aimed to assess the safety and immunogenicity of the malaria vaccine candidate, R21, administered with or without adjuvant Matrix-M in adults naïve to malaria infection and in healthy adults from malaria endemic areas. METHODS: In this Article we report two phase 1, first-in-human trials. The first trial was a phase 1a open-label study in the UK evaluating the safety and immunogenicity of R21 administered either alone, or with 50 μg of Matrix-M. The second trial was a phase 1b randomised controlled trial in Burkina Faso. Adults had to be aged 18-50 years for enrolment in the phase 1a trial, and 18-45 years in the phase 1b trial. The phase 1a trial doses were 2 μg, 10 μg, and 50 μg R21/Matrix-M, and 50 μg R21 only. The phase 1b trial doses were 10 μg R21/Matrix-M and saline placebo. Matrix-M was always dosed at 50 μg. Phase 1b implemented block randomisation by randomisation into study groups by an independent statistician based at the University of Oxford using a randomisation code list with allocation concealment using opaque sealed envelopes. The primary objective of the phase 1a trial was to assess the safety and tolerability of R21 with and without Matrix-M. The primary objective of the phase 1b trial was to assess the safety and tolerability of R21 with Matrix-M. Both trials are registered with ClinicalTrials.gov, NCT02572388 for phase 1a and NCT02925403 for phase 1b, and are completed. FINDINGS: Between Oct 1, 2015, and Jan 3, 2017, 31 individuals were enrolled in the phase 1a study. Six individuals were assigned to receive 2 μg R21/Matrix-M, 11 to 10 μg R21/Matrix-M, ten to 50 μg R21/Matrix-M, and four to 50 μg R21 only. Between Aug 26, 2016, and Sept 28, 2017, 13 individuals were enrolled in the phase 1b study. Eight individuals were assigned to receive 10 μg R21/Matrix-M, and five to placebo. Vaccinations were well tolerated, and most local and systemic adverse events were mild. There were no serious adverse events deemed related to vaccination. Two serious adverse events occurred. The first in the 10 μg R21/Matrix-M group was worsening of previously undisclosed or undiagnosed palindromic rheumatism and was deemed unlikely to be related to vaccination and the second in the 2 μg R21/Matrix-M was hospital admission for an unplanned excision of a pre-existing Bartholin's cyst, also unrelated to vaccination. In the phase 1a study, a total of 21 adverse events were recorded in the 2 μg R21/Matrix-M group, 103 in the 10 μg R21/Matrix-M group, 94 in the 50 μg R21/Matrix-M group, and 21 in the 50 μg R21 alone group. In the phase 1b study, twelve adverse events were recorded in the 10 μg R21/Matrix-M group and 0 in the placebo group. INTERPRETATION: R21 with Matrix-M adjuvant has an acceptable safety profile. These data have formed the basis for efficacy testing of this vaccine. FUNDING: The European Commission Framework 7 and The European & Developing Countries Clinical Trials Partnership.
Safety and immunogenicity of a bivalent Ebola virus and Sudan virus ChAdOx1 vectored vaccine in adults in the UK: an open-label, non-randomised, first-in-human, phase 1 clinical trial.
BACKGROUND: Four Orthoebolavirus species can cause Ebola disease, with Ebola virus (species Orthoebolavirus zairense) and Sudan virus (species Orthoebolavirus sudanense) responsible for the majority of outbreaks and cases. No vaccines have been approved against orthoebolaviruses other than Ebola virus. We aimed to evaluate the safety and immunogenicity of a non-replicating single-adenoviral vaccine (ChAdOx1 biEBOV) encoding both Ebola virus and Sudan virus glycoproteins. METHODS: In this open-label, non-randomised, first-in-human, phase 1, dose-escalation clinical trial of ChAdOx1 biEBOV, participants aged 18-55 years without clinically significant medical comorbidities or previous adenovirus vaccine exposure were recruited at a single site (Oxford, UK). Participants were non-randomly enrolled to a low-dose group (5 × 10⁹ viral particles [vp] of ChAdOx1 biEBOV), a medium-dose group (2·5 × 101⁰ vp), and a high-dose group (5 × 101⁰ vp). All doses were administered intramuscularly. After recruitment of all participants, the protocol was amended so that a subgroup from the high-dose group received a second high dose of vaccine 12 weeks after the first dose. Primary outcome measures were assessment of solicited adverse events for 7 days after vaccinations, unsolicited adverse events for 28 days after vaccinations, changes in clinical laboratory measures within 28 days after vaccination, and serious adverse events and adverse events of special interest for the study duration. Secondary outcomes were assessment of humoral and cellular immunity to Ebola virus and Sudan virus glycoprotein. This study is registered with ClinicalTrials.gov, NCT05079750. FINDINGS: Between Nov 11, 2021, and April 7, 2022, 40 individuals attended the trial screening visit, of whom 26 were enrolled (six in the low-dose group, six in the medium-dose group, and 14 in the high-dose group). Seven participants in the high-dose group received one vaccine dose and seven received two vaccine doses. Local solicited adverse events were reported by 17 (65%) of 26 participants after dose 1 and five (71%) of seven after dose 2. Systemic solicited adverse events were reported by 23 (88%) participants after dose 1 and five (71%) after dose 2. All solicited adverse events were mild or moderate, with no severe events reported. No serious adverse reactions were reported. Unsolicited adverse events related to vaccination were mostly mild or moderate and short-lived, such as joint pain or upper respiratory symptoms. One adverse event of special interest, thrombocytopenia, occurred transiently in one participant in the high-dose group. Rapidly resolving lymphopenia was common at the early post-vaccination timepoint. A single 5 × 101⁰ vp dose vaccination elicited seropositivity to Ebola virus in 14 (100%) participants in the high-dose group and elicited seropositivity to Sudan virus in 12 (86%) participants in the high-dose group; antibody titres were boosted in the two-dose group. INTERPRETATION: Our results suggest that the ChAdOx1 biEBOV vaccine was safe and well tolerated. Safety and tolerability data are consistent with other vaccines using the same vaccine backbone. A single 5 × 101⁰ vp dose of the vaccine was immunogenic, generating binding antibodies against both Ebola virus and Sudan virus glycoproteins, with antibody responses boosted in the subgroup receiving a second immunisation. Future research should focus on approaches to enhance antibody responses and to elicit neutralising antibodies to Sudan virus. FUNDING: UK Research and Innovation.
A feedback amplifier circuit with Notch and E2A orchestrates T-cell fate and suppresses the innate lymphoid cell lineages during thymic ontogeny.
External signals from the thymic microenvironment and the activities of lineage-specific transcription factors (TFs) instruct T-cell versus innate lymphoid cell (ILC) fates. However, mechanistic insights into how factors such as Notch1-Delta-like-4 (Dll4) signaling and E-protein TFs collaborate to establish T-cell identity remain rudimentary. Using multiple in vivo approaches and single-cell multiome analysis, we identified a feedback amplifier circuit that specifies fetal and adult T-cell fates. In early T progenitors (ETPs) in the fetal thymus, Notch signaling minimally lowered E-protein antagonist Id2 levels, and high Id2 abundance favored the differentiation of ETPs into ILCs. Conversely, in the adult thymus, Notch signaling markedly decreased Id2 abundance in ETPs, substantially elevating E-protein DNA binding and in turn promoting the activation of a T-cell lineage-specific gene expression program linked with V(D)J gene recombination and T-cell receptor signaling. Our findings indicate that, in the fetal versus the adult thymus, a simple feedback amplifier circuit dictated by Notch-mediated signals and Id2 abundance enforces T-cell identity and suppresses ILC development.
R21 in Matrix-M adjuvant in UK malaria-naive adult men and non-pregnant women aged 18-45 years: an open-label, partially blinded, phase 1-2a controlled human malaria infection study.
BACKGROUND: R21 is a novel malaria vaccine, composed of a fusion protein of the malaria circumsporozoite protein and hepatitis B surface antigen. Following favourable safety and immunogenicity in a phase 1 study, we aimed to assess the efficacy of R21 administered with Matrix-M (R21/MM) against clinical malaria in adults from the UK who were malaria naive in a controlled human malaria infection study. METHODS: In this open-label, partially blinded, phase 1-2A controlled human malaria infection study undertaken in Oxford, Southampton, and London, UK, we tested five novel vaccination regimens of R21/MM. A standard three-dose regimen (groups 1 and 6) was compared with a reduced (fractional) third dose (groups 2 and 5) of R21/MM, concomitant administration with viral vectors ChAd63-MVA expressing ME-TRAP (group 3), and a two-dose R21/MM regimen (group 7). Controlled Human Malaria Infection (CHMI) was delivered by mosquito bite at Imperial College London, London, UK, 3-4 weeks after final vaccination (or 18 months after final vaccination for group 6) alongside unvaccinated controls (groups 4A and 4B). The primary outcome measures were to assess safety of the vaccines in healthy malaria-naive volunteers and the efficacy (occurrence of blood-stage malaria infection) of the different vaccine regimens compared with non-vaccinated controls after CHMI. The trial was registered with ClinicalTrials.gov (NCT02905019). FINDINGS: 66 volunteers were enrolled with 59 undergoing subsequent CHMI. All vaccination schedules were well tolerated. The highest level of protection against CHMI was observed in participants receiving the standard three-dose regimen of R21/MM (group 1, nine of 11 volunteers protected) with protection maintained in three of five volunteers re-challenged by CHMI 7·5 months later. Protection against malaria was also seen in group 2, group 3, and group 5 compared with unvaccinated control participants. Total IgG antibody responses to the NANP repeat region of circumsporozoite protein peaked after the third dose of R21/MM in all volunteers and were well maintained to 90 days after challenge. Reducing the third dose did not affect protection or antibody concentrations. INTERPRETATION: Our study shows that R21/MM elicits high-level efficacy against clinical malaria in a controlled human infection model of malaria in adults who are malaria naive. These data supported the evaluation of R21/MM in field efficacy trials in the target population of young children in malaria-endemic areas. FUNDING: EU Horizon 2020, the UK Medical Research Council, the European Commission, the UK National Institute of Health Research, the Imperial NIHR Clinical Research Facility, the Oxford NIHR Biomedical Research Centre, and the Wellcome Trust.
Trajectories of interferon-gamma release assay results over two years in independent cohorts from China, South Africa, Tanzania, and the United States.
BACKGROUND: There is an ongoing debate about whether clearance of Mycobacterium tuberculosis infection occurs and at what magnitude. Recent studies quantifying 'uncertainty zones' of interferon-gamma release assays (IGRA) provide a more stringent estimate of reversion, potentially indicating clearance. RESEARCH QUESTION: When accounting for 'uncertainty zones' through stringent cutoffs, what are the trajectories of interferon-gamma release assays in cases of Mycobacterium tuberculosis infection? STUDY DESIGN AND METHODS: We followed five cohorts from South Africa, China, Tanzania, and the United States tested with an IGRA test three or more times for stringent conversion and reversion. The annual risk of IGRA reversion was assessed after an IGRA conversion and among those with baseline positivity. RESULTS: 26,596 IGRA measurements were taken over 13,593 years of follow-up (Nparticipants=7,683). Stringent reversion at year 2 after stringent conversion at year 1 varied between cohorts, occurring in 48% (43/90) for WANTAI, 37% (22/59) for QuantiFERON, and 17% (2/12) for T-SPOT.TB, respectively. In the U.S. cohorts, stringent reversion at year 1 after stringent conversion at 6 months was 58% (15/26) for QuantiFERON and 18% (12/60) for T-SPOT.TB. Stringent reversion at 1 year after baseline positivity occurred in 12% (47/404) for WANTAI, 21% (10/48) for QuantiFERON and 44% for T-SPOT.TB (45/102). In one cohort from (N=399; age range, 59 years [IQR, 48-67]), IGRA reversion was more common in younger participants (Adjusted Odds Ratio [aOR], 0.95; 95% CI, 0.93-0.97) and those without recent close tuberculosis exposure (aOR, 0.35; 95%CI, 0.11-1.03 in South Africa; 0.10; 95%CI, 0.01-0.61 in China). INTERPRETATION: These results suggest high annual rates of IGRA reversion, even with the use of 'uncertainty zones'; reversion rates decreased with time from exposure and at older ages.
Upadacitinib for Induction of Remission in Paediatric Crohn's Disease: An International Multicentre Retrospective Study.
BACKGROUND: There are scarce data available on upadacitinib in children with Crohn's disease (CD). AIM: To evaluate the effectiveness and safety of upadacitinib as an induction therapy in paediatric CD. METHODS: This was a multicentre retrospective study between 2022 and 2024 of children treated with upadacitinib for induction of remission of active CD conducted in 30 centres worldwide affiliated with the IBD Interest and Porto group of the ESPGHAN. We recorded demographic, clinical and laboratory data and adverse events (AEs) at week 8 post-induction. The analysis of the primary outcome was based upon the intention-to-treat (ITT) principle. RESULTS: We included 100 children (median age 15.8 [interquartile range 14.3-17.2]). All were previously treated with biologic therapies including 89 with ≥ 2 biologics. At the end of the 8-week induction period, we observed clinical response, clinical remission and corticosteroid- and exclusive enteral nutrition-free clinical remission (CFR) in 75%, 56% and 52%, respectively. By the end of induction, 68% had achieved normalisation of C-reactive protein, and 58% had faecal calprotectin (FC)
Pneumococcal Carriage and Disease in Adults in England, 2011-2019: The Importance of Adults as a Reservoir for Pneumococcus in Communities.
BACKGROUND: Pneumococcal carriage in healthy adults and its relationship to invasive pneumococcal disease (IPD) is not well understood. METHODS: Nasal wash samples from adults without close contact with young children (Liverpool, UK), 2011-2019, were cultured, and culture-negative samples tested by polymerase chain reaction (PCR). Pneumococcal carriage in adults 18-44 years was compared with carriage among pneumococcal conjugate vaccine-vaccinated children aged 13-48 months (nasopharyngeal swabs, Thames Valley, UK) and national IPD data, 2014-2019. Age group-specific serotype invasiveness was calculated and used with national IPD data to estimate carriage serotype distributions for ≥65 years. RESULTS: Overall, 98 isolates (97 carriers) were identified (3 solely by PCR) from 1631 ≥18 years adults (standardized carriage prevalence 6.4%). Despite different carriage and IPD serotype distributions between adults and children, serotype invasiveness was highly correlated (R = 0.9). Serotypes 3, 37, and 8 represented a higher proportion of adult carriage than expected. Predicted carriage serotype distributions for ≥65 years aligned closest with the young adult carriage serotype distribution. CONCLUSIONS: Nasal wash technique is highly sensitive. For some serotypes carried by adults aged ≥65 years, other adults may be an important reservoir for transmission. Age groups such as older children should also be considered.
Surface display of functional moieties on extracellular vesicles using lipid anchors.
Extracellular vesicles (EVs) are efficient natural vehicles for intercellular communication and are under extensive investigation for the delivery of diverse therapeutics including small molecule drugs, nucleic acids, and proteins. To understand the mechanisms behind the biological activities of EVs and develop EV therapeutics, it's fundamental to track EVs and engineer EVs in a customized manner. In this study, we identified, using single-vesicle flow cytometry and microscopy, the lipid DOPE (dioleoyl phosphatidyl ethanolamine) as an efficient anchor for isolated EVs. Notably, DOPE associated with EVs quickly, and the products remained stable under several challenging conditions. Moreover, conjugating fluorophores, receptor-targeting peptides or albumin-binding molecules with DOPE enabled tracking the cellular uptake, enhanceing the cellular uptake or extending the circulation time in mice of engineered EVs , respectively. Taken together, this study reports an efficient lipid anchor for exogenous engineering of EVs and further showcases its versatility for the functionalization of EVs.
Serological cross-reactivity between Crimean-Congo haemorrhagic fever virus and Nairobi sheep disease virus glycoprotein C
Introduction: Crimean-Congo haemorrhagic fever virus (CCHFV) and Nairobi sheep disease virus (NSDV) are orthonairoviruses of concern, able to cause haemorragic fever disease in humans and sheep, respectively. CCHFV and NSDV cocirculating in small ruminant populations across South Asia and East Africa. Cross-reactivity to viruses of the Orthonairovirus genus can potentially interfere with serological assays when employed for serosurveillance in regions where two or more genus members overlap in their distribution. Methods: In this study, sheep sera sampled from a region of confirmed CCHFV circulation and NSDV absence were utilized, thereby eliminating the possibility of co-exposure. Field sera were tested against in-house anti-NSDV ELISAs specific to the nucleoprotein (NSDV NP) and glycoprotein C (NSDV Gc) antigens as well as an in-house NSDV 80% plaque reduction neutralization test (PRNT80). We assessed whether there is a correlation between CCHFV- and NSDV-specific ELISAs. Furthermore, epitopes-derived from CCHFV antigens for sheep antibody that were available from the literature were analyzed. Results: When comparing NSDV antigen-specific antibody responses against previously tested CCHFV antigen-specific antibody responses, a strong positive correlation was observed between the Gc-specific responses, while a weak positive correlation was observed between the NP-specific responses. Consequently, NP-specific ELISAs have a higher assay specificity compared to Gc-specific ELISAs, making them more suitable for serosurveillance in regions where multiple orthonairoviruses co-circulate. Crucially, only one seropositive sample to NSDV Gc-specific out of a set of 224 (0.4%) showed a neutralizing capacity at the lowest serum dilution (1:8), suggesting these field sera have not been exposed to NSDV. Based on an analysis of known epitopes in NP targeted by antibodies in sheep serum, we propose that NP is less cross-reactive because dominant epitopes are highly dissimilar between CCHFV and NSDV. Discussion: Gc exhibited a strong cross-reaction while the NP was weakly cross-reactive due to dominant epitopes being highly dissimilar between CCHFV and NSDV. Our in-house PRNT80 assay can could be used as a confirmatory test in regions where CCHFV and NSDV circulate.
Effectiveness and Safety of Adalimumab in Patients With Very Early-Onset Inflammatory Bowel Disease: A Retrospective Study on Behalf of the Porto Inflammatory Bowel Disease Working Group of European Society for Pediatric Gastroenterology Hepatology and Nutrition.
BACKGROUND AND AIMS: Patients with very early-onset inflammatory bowel disease (VEO-IBD), with an age of onset
Short-chain fatty acids: linking diet, the microbiome and immunity.
The short-chain fatty acids (SCFAs) butyrate, propionate and acetate are microbial metabolites and their availability in the gut and other organs is determined by environmental factors, such as diet and use of antibiotics, that shape the diversity and metabolism of the microbiota. SCFAs regulate epithelial barrier function as well as mucosal and systemic immunity via evolutionary conserved processes that involve G protein-coupled receptor signalling or histone deacetylase activity. Indicatively, the anti-inflammatory role of butyrate is mediated through direct effects on the differentiation of intestinal epithelial cells, phagocytes, B cells and plasma cells, and regulatory and effector T cells. Intestinally derived SCFAs also directly and indirectly affect immunity at extra-intestinal sites, such as the liver, the lungs, the reproductive tract and the brain, and have been implicated in a range of disorders, including infections, intestinal inflammation, autoimmunity, food allergies, asthma and responses to cancer therapies. An ecological understanding of microbial communities and their interrelated metabolic states, as well as the engineering of butyrogenic bacteria may support SCFA-focused interventions for the prevention and treatment of immune-mediated diseases.
Past, present, and future of Phase 3 vaccine trial design: rethinking statistics for the 21st century.
Vaccines are crucial for protecting health globally; however, their widespread use relies on rigorous clinical development programmes. This includes Phase 3 randomized controlled trials (RCTs) to confirm their safety, immunogenicity, and efficacy. Traditionally, such trials used fixed designs with predetermined assumptions, lacking the flexibility to change during the trial or stop early due to overwhelming evidence of either efficacy or futility. Modern vaccine trials benefit from innovative approaches like adaptive designs, allowing for planned trial adaptations based on accumulating data. Here, we provide an overview of the evolution of Phase 3 vaccine trial design and statistical analysis methods from traditional to more innovative contemporary methods. This includes adaptive trial designs, which offer ethical advantages and enable early termination if indicated; Bayesian methods, which combine prior knowledge and observed trial data to increase efficiency and enhance result interpretation; modern statistical analysis methods, which enable more accurate and precise inferences; the estimand framework, which ensures the primary question of interest is addressed in a trial; novel approaches using machine learning methods to assess heterogeneity of treatment effects; and statistical advances in safety analysis to evaluate reactogenicity and clinical adverse events. We conclude with insights into the future direction of vaccine trials, aiming to inform clinicians and researchers about conventional and novel RCT design and analysis approaches to facilitate the conduct of efficient, timely trials.
An extracellular vesicle delivery platform based on the PTTG1IP protein.
Extracellular vesicles (EVs) are promising therapeutic delivery vehicles, although their potential is limited by a lack of efficient engineering strategies to enhance loading and functional cargo delivery. Using an in-house bioinformatics analysis, we identified N-glycosylation as a putative EV-sorting feature. PTTG1IP (a small, N-glycosylated, single-spanning transmembrane protein) was found to be a suitable scaffold for EV loading of therapeutic cargoes, with loading dependent on its N-glycosylation at two arginine residues. Chimeric proteins consisting of PTTG1IP fused with various cargo proteins, and separated by self-cleaving sequences (to promote cargo release), were shown to enable highly efficient functional delivery of Cre protein to recipient cell cultures and mouse xenograft tumors, and delivery of Cas9-sgRNA complexes to recipient reporter cells. The favorable membrane topology of PTTG1IP enabled facile engineering of further variants with improved properties, highlighting its versatility and potential as a platform for EV-based therapeutics.
Exploiting the biogenesis of extracellular vesicles for bioengineering and therapeutic cargo loading.
Extracellular vesicles (EVs) are gaining increasing attention for diagnostic and therapeutic applications in various diseases. These natural nanoparticles benefit from favorable safety profiles and unique biodistribution capabilities, rendering them attractive drug-delivery modalities over synthetic analogs. However, the widespread use of EVs is limited by technological shortcomings and biological knowledge gaps that fail to unravel their heterogeneity. An in-depth understanding of their biogenesis is crucial to unlocking their full therapeutic potential. Here, we explore how knowledge about EV biogenesis can be exploited for EV bioengineering to load therapeutic protein or nucleic acid cargos into or onto EVs. We summarize more than 75 articles and discuss their findings on the formation and composition of exosomes and microvesicles, revealing multiple pathways that may be stimulation and/or cargo dependent. Our analysis further identifies key regulators of natural EV cargo loading and we discuss how this knowledge is integrated to develop engineered EV biotherapeutics.