Biology subjects

Wright, D.

Publications and source records attributed to Wright, D..

At least 37 records · Page 2Linked to original sources

bioRxiv2021

A disease-linked lncRNA mutation in RNase MRP inhibits ribosome synthesis

RMRP encodes a non-coding RNA forming the core of the RNase MRP ribonucleoprotein complex. Mutations cause Cartilage Hair Hypoplasia (CHH), characterized by skeletal abnormalities and impaired T cell activation. Yeast RNase MRP cleaves a specific site in the pre-ribosomal RNA (pre-rRNA) during ribosome synthesis. CRISPR-mediated disruption of RMRP in human cells lines caused growth arrest, with pre-rRNA accumulation. Here, we analyzed disease-relevant primary cells, showing that mutations in RMRP impair mouse T cell activation and delay pre-rRNA processing. Patient-derived human fibroblasts with CHH-linked mutations showed similar pre-rRNA processing delay. Human cells engineered with the most common CHH mutation (70AG in RMRP) show specifically impaired pre-rRNA processing, resulting in reduced mature rRNA and a reduced ratio of cytosolic to mitochondrial ribosomes. Moreover, the 70AG mutation caused a reduction in intact RNase MRP complexes. Together, these results indicate that CHH is a ribosomopathy, and the first processing-specific human disorder to be described. HighlightsO_LIMutations in RMRP lncRNA impair pre-rRNA processing and T cell activation C_LIO_LIPatient derived fibroblasts show impaired pre-rRNA processing C_LIO_LICells with the most common disease-linked mutation have specific processing defects C_LIO_LICytoplasmic ribosomes and intact RNase MRP complexes are also reduced in these cells C_LI

cell biology↗

bioRxiv2021

Induction of specific antibodies, IgG-secreting plasmablasts and memory B cells following BCG vaccination

Many tuberculosis (TB) vaccine candidates are designed as a boost to BCG; an understanding of the BCG-induced immune response is therefore critical, and the opportunity to relate this to circumstances where BCG does protect may direct the design of more efficacious vaccines. While the T cell response to BCG vaccination has been well-characterised, little is known about the B cell and antibody response. We demonstrate BCG vaccine-mediated induction of specific antibodies in different human populations and macaque species which represent important preclinical models for TB vaccine development. We observe a strong correlation between antibody titres in serum versus plasma with modestly higher titres in serum. We also report for the first time the rapid and transient induction of antibody-secreting plasmablasts following BCG vaccination, together with a robust and durable memory B cell response in humans. Finally, we demonstrate a potential contribution of the antibody response to BCG vaccine-mediated control of mycobacterial growth in vitro. Taken together, our findings indicate that the humoral immune response in the context of BCG vaccination merits further attention to determine whether TB vaccine candidates could benefit from the induction of humoral as well as cellular immunity.

immunology↗

bioRxiv2021

Heterologous vaccination regimens with self-amplifying RNA and Adenoviral COVID vaccines induce superior immune responses than single dose vaccine regimens in mice

Several vaccines have demonstrated efficacy against SARS-CoV-2 mediated disease, yet there is limited data on the immune response induced by heterologous vaccination regimens using alternate vaccine modalities. Here, we present a detailed description of the immune response, in mice, following vaccination with a self-amplifying RNA (saRNA) vaccine and an adenoviral vectored vaccine (ChAdOx1 nCoV-19/AZD1222) against SARS-CoV-2. We demonstrate that antibody responses are higher in two dose heterologous vaccination regimens than single dose regimens. Neutralising titres after heterologous prime-boost were at least comparable or higher than the titres measured after homologous prime boost vaccination with viral vectors. Importantly, the cellular immune response after a heterologous regimen is dominated by cytotoxic T cells and Th1+ CD4 T cells which is superior to the response induced in homologous vaccination regimens in mice. These results underpin the need for clinical trials to investigate the immunogenicity of heterologous regimens with alternate vaccine technologies.

immunology↗

bioRxiv2021

Quantification of allosteric communications in matrix metalloprotease-1 on alpha-synuclein aggregates and substrate-dependent virtual screening

Alpha-synuclein (aSyn) has implications in pathological protein aggregations observed in neurodegenerative disorders, including Parkinsons and Alzheimers diseases. There are currently no approved prevention and cure for these diseases. In this context, matrix metalloproteases (MMPs) provide an opportunity because MMPs are broad-spectrum proteases and cleave aSyn. Previously, we showed that allosteric communications between the two domains of MMP1 on collagen fibril and fibrin depend on substrates, MMP1 activity, and ligands. However, allosteric communications in MMP1 on aSyn-induced aggregates have not been explored. Here we report quantification of allostery using single molecule measurements of MMP1 dynamics on aSyn-induced aggregates by calculating Forster Resonance Energy Transfer (FRET) between two dyes attached to the catalytic and hemopexin domains of MMP1. The two domains of MMP1 prefer open conformations, with the two domains well-separated. These open conformations are inhibited by a single point mutation E219Q of MMP1 and tetracycline, an MMP inhibitor. A two-state Poisson process describes the interdomain dynamics. The best-fit parameters for a Gaussian fit to the distributions of FRET values provide the two states. The ratio of the kinetic rates between the two states comes from the ratio of fitted areas around the two states. The decay rate of an exponential fit to the correlations between FRET values provides the sum of the kinetic rates. Since a crystal structure of aSyn-bound MMP1 is not available, we performed molecular docking of MMP1 with aSyn using ClusPro. We simulated MMP1 dynamics using different docking poses and matched the experimental and simulated interdomain dynamics to determine the most appropriate pose. We performed virtual screening against the potential ligand-binding sites on the appropriate aSyn-MMP1 binding pose and showed that lead molecules differ between free MMP1 and substrate-bound MMP1. In other words, virtual screening needs to take substrates into account for substrate-specific control of MMP1 activity. Molecular understanding of interactions between MMP1 and aSyn-induced aggregates may open up the possibility of degrading pathological aggregates in neurodegeneration by targeting MMPs. SignificanceWe have quantified MMP1 interdomain dynamics on aSyn-induced aggregates by a two-state Poisson process. Histograms and correlations of FRET values determine the kinetic rates of interconversion between the two states. We quantify the conformational dynamics of the whole MMP1 and allosteric communications by the two-dimensional matrix of correlations between every pair of amino acids from experimentally-validated all-atom simulations. The two-dimensional correlations lead to a Gray Level Co-occurrence Matrix and a measure of Shannon entropy describing the conformational fluctuations. As such, we address the quantification of allosteric communications, a leading challenge in defining allostery. We report that the potential ligand-binding sites and lead molecules change for MMP1 upon binding alpha-synuclein and depend on the binding pose selected. This suggests that one needs to take the substrate into account while targeting MMPs.

biophysics↗

bioRxiv2020

CRISPR-mediated deletion of the Protein tyrosine phosphatase, non-receptor type 22 (PTPN22) improves human T cell function for adoptive T cell therapy

Adoptive T cell transfer has improved the treatment of cancer patients. However, treatment of solid tumors is still challenging and new strategies that optimize T cell function and response duration in the tumor could be beneficial additions to cancer therapy. In this study, we deleted the intracellular phosphatase PTPN22 and the endogenous TCR chain from human PBMC-derived T cells using CRISPR/Cas9 and transduced them with TCRs specific for a defined antigen. Deletion of PTPN22 in human T cells increased the secretion of IFN{gamma} and GM-CSF in multiple donors. The cells retained a polyfunctional cytokine expression after re-stimulation and greater numbers of PTPN22KO T cells expressed inflammatory cytokines compared to unmutated control cells. PTPN22KO T cells seemed to be more polyfunctional at low antigen concentrations. Additionally, we were able to show that that PTPN22KO T cells were more effective in controlling tumor cell growth. This suggests that they might be more functional within the suppressive tumor microenvironment thereby overcoming the limitations of immunotherapy for solid tumors.

immunology↗

bioRxiv2020

A booster dose enhances immunogenicity of the COVID-19 vaccine candidate ChAdOx1 nCoV-19 in aged mice

The spread of SARS-CoV-2 has caused a global pandemic that has affected almost every aspect of human life. The development of an effective COVID-19 vaccine could limit the morbidity and mortality caused by infection, and may enable the relaxation of social distancing measures. Age is one of the most significant risk factors for poor health outcomes after SARS-CoV-2 infection, therefore it is desirable that any new vaccine candidates should elicit a robust immune response in older adults. Here, we test the immunogenicity of the adenoviral vectored vaccine ChAdOx1 nCoV-19 (AZD-1222) in aged mice. We find that a single dose of this vaccine induces cellular and humoral immunity in aged mice, but at a reduced magnitude than in younger adult mice. Furthermore, we report that a second dose enhances the immune response to this vaccine in aged mice, indicating that a primeboost strategy may be a rational approach to enhance immunogenicity in older persons.

immunology↗

bioRxiv2020

Open Targets Genetics: An open approach to systematically prioritize causal variants and genes at all published GWAS trait-associated loci

Genome-wide association studies (GWAS) have identified many variants robustly associated with complex traits but identifying the gene(s) mediating such associations is a major challenge. Here we present an open resource that provides systematic fine-mapping and protein-coding gene prioritization across 133,441 published human GWAS loci. We integrate diverse data sources, including genetics (from GWAS Catalog and UK Biobank) as well as transcriptomic, proteomic and epigenomic data across many tissues and cell types. We also provide systematic disease-disease and disease-molecular trait colocalization results across 92 cell types and tissues and identify 729 loci fine-mapped to a single coding causal variant and colocalized with a single gene. We trained a machine learning model using the fine mapped genetics and functional genomics data using 445 gold standard curated GWAS loci to distinguish causal genes from background genes at the same loci, outperforming a naive distance based model. Genes prioritized by our model are enriched for known approved drug targets (OR = 8.1, 95% CI: [5.7, 11.5]). These results will be regularly updated and are publicly available through a web portal, Open Targets Genetics (OTG, http://genetics.opentargets.org), enabling users to easily prioritize genes at disease-associated loci and assess their potential as drug targets.

genetics↗

bioRxiv2020

Evaluation of the immunogenicity of prime-boost vaccination with the replication-deficient viral vectored COVID-19 vaccine candidate ChAdOx1 nCoV-19

Clinical development of the COVID-19 vaccine candidate ChAdOx1 nCoV-19, a replication-deficient simian adenoviral vector expressing the full-length SARS-CoV-2 spike (S) protein was initiated in April 2020 following non-human primate studies using a single immunisation. Here, we compared the immunogenicity of one or two doses of ChAdOx1 nCoV-19 in both mice and pigs. Whilst a single dose induced antigen-specific antibody and T cells responses, a booster immunisation enhanced antibody responses, particularly in pigs, with a significant increase in SARS-CoV-2 neutralising titres.

immunology↗

bioRxiv2020

Reduced levels of membrane-bound alkaline phosphatase in Vip3Aa-resistant Heliothis virescens

The Vip3Aa insecticidal protein from Bacillus thuringiensis (Bt) is produced by specific transgenic corn and cotton varieties for efficient control of target lepidopteran pests. The main threat to this technology is the evolution of resistance in targeted insect pests, thus understanding the mechanistic basis of resistance is crucial to deploy the most appropriate strategies for resistance management. In this work, a laboratory-selected colony of Heliothis virescens (Vip-Sel) highly resistant to the Vip3Aa protein was used to test whether an alteration of membrane receptors in the insect midgut might explain the resistance phenotype. Binding of 125I-labeled Vip3Aa to brush border membrane vesicles (BBMV) from 3rd instar larvae from Vip-Sel was not significantly different from binding in the reference susceptible colony. Interestingly, BBMV from Vip-Sel larvae show dramatically reduced levels of alkaline phosphatase activity, which was further confirmed by a strong down-regulation of the membrane-bound alkaline phosphatase 1 (HvmALP1) gene. However, its involvement as a receptor for the Vip3Aa protein was not supported by ligand blotting and viability assays with insect cells expressing HvmALP1. These data support that reduced alkaline phosphatase, previously observed in insect colonies resistant to Cry proteins from Bt, may also serve as an indirect marker that is not mechanistically involved in resistance to Vip3Aa. IMPORTANCEThe Vip3Aa insecticidal protein remains the only lepidopteran-specific trait in transgenic Bt crops with no cases of field-evolved resistance. While laboratory-selected resistance to Vip3A has been reported elsewhere, the mechanism for resistance is unknown. Results in this work show lack of significant Vip3Aa binding alterations in resistant and reference colonies of H. virescens. These observations are in contrast to most cases of high levels of resistance to insecticidal Bt proteins for which decreased binding is commonly detected. In addition, this study provides the first evidence of down-regulation of membrane bound alkaline phosphatase (mALP) associated with Vip3Aa resistance, a phenomenon commonly associated with resistance to Cry proteins from Bt. Results from this work suggest that mALP down-regulation may be a useful biomarker yet reject its direct participation in resistance to Vip3Aa.

microbiology↗

bioRxiv2019

Allosteric communications between domains modulate activity of matrix metalloprotease-1

An understanding of the structure-dynamics relationship is essential for understanding how a protein works. Prior research has shown that the activity of a protein correlates with intra-domain dynamics occurring at picosecond to millisecond timescales. However, the correlation between inter-domain dynamics and the function of a protein is poorly understood. Here we show that communications between the catalytic and hemopexin domains of matrix metalloprotease-1 (MMP1) on type-1 collagen fibrils correlate with its activity. Using single-molecule FRET (smFRET), we identified functionally relevant open conformations where the two MMP1 domains are well-separated, which were significantly absent for catalytically inactive point mutant (E219Q) of MMP1 and could be modulated by an inhibitor or an enhancer of activity. The observed relevance of open conformations resolves the debate about the roles of open and closed MMP1 structures in function. A sum of two Gaussians fitted histograms, whereas an exponential fitted autocorrelations of smFRET values. We used a two-state Poisson process to describe the dynamics and used histograms and autocorrelations of conformations to calculate the kinetic rates between the two states. All-atom and coarse-grained simulations reproduced some of the experimental features and revealed substrate-dependent MMP1 dynamics. Our results suggest that an inter-domain separation facilitates opening up the catalytic pocket so that the collagen chains come closer to the MMP1 active site. Coordination of functional conformations at different parts of MMP1 occurs via allosteric communications that can take place via interactions mediated by collagen even if the linker between the domains is absent. Modeling dynamics as a Poisson process enables connecting the picosecond timescales of molecular dynamics simulations with the millisecond timescales of single molecule measurements. Water-soluble MMP1 interacting with water-insoluble collagen fibrils poses challenges for biochemical studies that the single molecule tracking can overcome for other insoluble substrates. Inter-domain communications are likely important for multidomain proteins. Statement of SignificanceIt is often challenging to distinguish functionally important dynamics because proteins are inherently flexible. MMP1 is a model enzyme because both the catalytic and hemopexin domains are necessary to degrade triple-helical type-1 collagen, the highly proteolysis-resistant structural component of the extracellular matrix. We report, for the first time, measurements of MMP1 inter-domain dynamics on type-1 collagen fibrils. We have identified functionally relevant MMP1 conformations where the two domains are far apart. Mutations and ligands can allosterically modulate the dynamics that correlate with activity. The dynamics follow a two-state Poisson process that connects the picosecond timescales of MD simulations with the millisecond timescales of experiments. The two domains can functionally communicate via collagen even when the physical linker is absent.

biophysics↗

bioRxiv2019

Intra-individual behavioural variability: a trait under genetic control

When individuals are measured more than once in the same context they do not behave in exactly the same way each time. The degree of predictability differs between individuals, with some individuals showing low levels of variation around their behavioural mean while others show high levels of variation. This intra-individual variation in behaviour has received much less attention than between-individual variance in behaviour and very little is known about the underlying mechanisms that affect this potentially large but understudied component of behavioural variation. In this study, we combine standardized behavioural tests to estimate intra-individual behavioural variance with a large-scale genetical genomics analysis to identify genes affecting intra-individual variability in an avian population. Our study shows that within-individual variance in behaviour has a direct genetic basis which is largely unique compared to the genetic architecture for the standard behavioural measures they are based on. We identify six candidate genes underlying variation in intra-individual behavioural variability many of which have previously been linked to behaviour and mental health. These findings demonstrate that within-individual variability in behavioural is a heritable trait in and of itself on which evolution can act.

evolutionary biology↗

bioRxiv2019

Targeting the Pregnane X Receptor Using Microbial Metabolite Mimicry

The human pregnane X receptor (PXR), a master regulator of drug metabolism, has important roles in intestinal homeostasis and abrogating inflammation. Existing PXR ligands have substantial off-target toxicity. Based on prior work that established microbial (indole) metabolites as PXR ligands, we proposed microbial metabolite mimicry as a novel strategy for drug discovery that allows to exploit previously unexplored parts of chemical space. Here we report functionalized indole-derivatives as first-in-class non-cytotoxic PXR agonists, as a proof-of-concept for microbial metabolite mimicry. The lead compound, FKK6, binds directly to PXR protein in solution, induces PXR specific target gene expression in, cells, human organoids, and mice. FKK6 significantly represses pro-inflammatory cytokine production cells and abrogates inflammation in mice expressing the human PXR gene. The development of FKK6 demonstrates for the first time that microbial metabolite mimicry is a viable strategy for drug discovery and opens the door to mine underexploited regions of chemical space.

biochemistry↗

bioRxiv2019

Bee visitation and fruit quality in berries under protected cropping vary along the length of polytunnels

Wild and managed bees provide effective crop pollination services worldwide. Protected cropping conditions are thought to alter the ambient environmental conditions in which pollinators forage for flowers, yet few studies have compared conditions at the edges and centre of growing tunnels. We measured environmental variables (temperature, relative humidity, wind speed, white light and UV light) and surveyed the activity of managed honeybees Apis mellifera, wild stingless bees Tetragonula carbonaria and sweat bees Homalictus urbanus along the length of 32 multiple open-ended polyethylene growing tunnels. These were spaced across 12 blocks at two commercial berry farms, in Coffs Harbour, New South Wales and Walkamin, North Queensland, Australia. Berry yield, fresh weight and other quality metrics were recorded at discrete increments along the length of the tunnels. We found a higher abundance and greater number of flower visits by stingless bees and honeybees at the end of tunnels, and less frequent visits to flowers toward the middle of tunnels. The centre of tunnels experienced higher temperatures and reduced wind speed. In raspberry, fruit shape was improved with greater pollinator abundance and was susceptible to higher temperatures. In blueberry, per plant yield and mean berry weight were positively associated with pollinator abundance and were lower at the centre of tunnels than at the edge. Fruit quality (crumbliness) in raspberries was improved with a greater number of visits by sweat bees, who were not as susceptible to climatic conditions within tunnels. Understanding bee foraging behaviour and changes to yield under protected cropping conditions is critical to inform the appropriate design of polytunnels and aid pollinator management within them.

ecology↗

bioRxiv2019

Targeting DNA damage response and Replication Stress in Pancreatic Cancer

Continuing recalcitrance to therapy cements pancreatic cancer (PC) as the most lethal malignancy, which is set to become the second leading cause of cancer death in our society. We interrogated the transcriptome, genome, proteome and functional characteristics of 61 novel PC patient-derived cell lines to define novel therapeutic strategies targeting the DNA damage response (DDR) and replication stress. We show that patient-derived cell lines faithfully recapitulate the epithelial component of pancreatic tumors including previously described molecular subtypes. Biomarkers of DDR deficiency, including a novel signature of homologous recombination deficiency, co-segregates with response to platinum and PARP inhibitor therapy in vitro and in vivo. We generated a novel signature of replication stress with potential clinical utility in predicting response to ATR and WEE1 inhibitor treatment. Replication stress and DDR deficiency are independent of each other, creating opportunities for therapy in DDR proficient PC, and post-platinum therapy.\n\n\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=145 SRC=\"FIGDIR/small/713545v1_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (47K):\norg.highwire.dtl.DTLVardef@106f2b8org.highwire.dtl.DTLVardef@1294d22org.highwire.dtl.DTLVardef@86f5d3org.highwire.dtl.DTLVardef@751e13_HPS_FORMAT_FIGEXP M_FIG C_FIG STATEMENT OF SIGNIFICANCEWe define therapeutic strategies that target subgroups of PC using novel signatures of DNA damage response deficiency and replication stress. This potentially offers patients with DNA repair defects therapeutic options outside standard of care platinum chemotherapy and is being tested in clinical trials on the Precision-Panc platform.

cancer biology↗

bioRxiv2019

Genomic selection for lentil breeding: empirical evidence

Genomic selection (GS) is a type of marker-based selection which was initially suggested for livestock breeding and is being encouraged for crop breeding. Several statistical models and approaches have been developed to implement GS; however, none of these methods have been tested for use in lentil breeding. This study was conducted to evaluate different GS models and prediction scenarios based on empirical data and to make recommendations for designing genomic selection strategies for lentil breeding. We evaluated nine single-trait models, two multiple-trait models, and models that account for population structure and genotype-by-environment interaction (GEI) using a lentil diversity panel and two recombinant inbred lines (RIL) populations that were genotyped using a custom exome capture assay. Within-population, across-population and across-environment predictions were made for five phenology traits. Prediction accuracy varied among the evaluated models, populations, prediction scenarios, traits, and statistical models. Single-trait models showed similar accuracy for each trait in the absence of large effect QTL but BayesB outperformed all models when there were QTL with relatively large effects. Models that accounted for GEI and multiple-trait (MT) models increased prediction accuracy for a low heritability trait by up to 66% and 14% but accuracy did not improve for traits of high heritability. Moderate to high accuracies were obtained for within-population and across-environment predictions but across-population prediction accuracy was very low. This suggests that GS can be implemented in lentil to make predictions within populations and across environments, but across-population prediction should not be considered when the population size is small.

genomics↗

bioRxiv2019

Genetic predisposition to mosaic Y chromosome loss in blood is associated with genomic instability in other tissues and susceptibility to non-haematological cancers

Mosaic loss of chromosome Y (LOY) in circulating white blood cells is the most common form of clonal mosaicism, yet our knowledge of the causes and consequences of this is limited. Using a newly developed approach, we estimate that 20% of the UK Biobank male population (N=205,011) has detectable LOY. We identify 156 autosomal genetic determinants of LOY, which we replicate in 757,114 men of European and Japanese ancestry. These loci highlight genes involved in cell-cycle regulation, cancer susceptibility, somatic drivers of tumour growth and cancer therapy targets. Genetic susceptibility to LOY is associated with non-haematological health outcomes in both men and women, supporting the hypothesis that clonal haematopoiesis is a biomarker of genome instability in other tissues. Single-cell RNA sequencing identifies dysregulated autosomal gene expression in leukocytes with LOY, providing insights into how LOY may confer cellular growth advantage. Collectively, these data highlight the utility of studying clonal mosaicism to uncover fundamental mechanisms underlying cancer and other ageing-related diseases.

genetics↗

bioRxiv2018

Structural and Biochemical Studies of Dihydrofolate Reductase from Streptococcus pyogenes as a Target for Antifolate Antibiotics

Streptococcus pyogenes, a beta-hemolytic bacterium, causes a wide spectrum of infections in human including pharyngitis, tonsillitis, scarlet fever, rheumatic fever, and necrotizing fasciitis. Streptococcal infections can also exist as co-infection with methicillin resistant Staphylococcus aureus (MRSA). Trimethoprim-sulfamethoxazole (TMP-SMX) combination has been used for treatment of S. pyogenes and MRSA co-infection. However, resistance to TMP, an inhibitor of dihydrofolate reductase enzyme (DHFR), has challenged the efficacy of TMP-SMX combination. We explored the activity of a series of novel DHFR inhibitors against S. pyogenes. This study identified potent inhibitors of DHFR enzyme from S. pyogenes with excellent inhibitory activity against the growth of the live bacteria. We determined, for the first time, the crystal structure of S. pyogenes DHFR which provides structural insights into design and development of antifolate agents against this global pathogen.

microbiology↗

bioRxiv2018

Characterisation of antigenic MHC Class I-restricted T cell epitopes in the glycoprotein of Ebolavirus

Ebolavirus is a pathogen capable of causing highly lethal haemorrhagic fever in humans. The envelope-displayed viral glycoprotein is the primary target of humoral immunity induced by both natural exposure and vaccination. The epitopes targeted by B cells have been thoroughly characterised by functional and structural analyses of the glycoprotein, GP, yet there is a paucity of information regarding the cellular immune response to Ebolavirus. To date, no T cell epitopes in the glycoprotein have been characterised in detail in humans.\n\nA recent Phase I clinical trial of a heterologous prime-boost vaccination regime with viral vectors encoding filovirus antigens elicited strong humoral and T cell responses in vaccinees. Using samples from this trial, the most frequently recognised peptide pools were studied in more detail to identify the minimal epitopes recognised by antigen-specific T cells and associated HLA-restrictions.\n\nUsing IFN{gamma} ELISPOT and flow cytometry, we characterised nine highly immunogenic T cell epitopes located on both the GP1 and GP2 subunits of the Ebolavirus GP. Epitope mapping revealed the location of these epitopes as presented on the mature virion. HLA-typing on all participants, combined with in silico epitope analysis, determined the likely MHC class I restriction elements. Thirteen HLA-A and -B alleles were predicted to present the identified epitopes, suggesting promiscuous recognition and induction of a broad immune response.\n\nThe glycoprotein of Ebolavirus is highly immunogenic, inducing both CD4+ and CD8+ T cell responses and we have shown here for the first time that these responses are associated with multiple HLA types. Delivery of this antigen using a heterologous prime-boost approach with ChAd3 and MVA is likely to be highly immunogenic in genetically diverse human populations, due to the induction of responses against multiple immunodominant epitopes.\n\nAuthor SummaryThe West African Ebolavirus epidemic in 2013-2016 claimed more than 11,000 lives, eclipsing all previously recorded outbreaks combined. Understanding the mechanisms of host-pathogen interactions informs rational vaccine design. B cell epitopes have been described extensively through the characterisation of monoclonal antibodies from survivors of Ebolavirus disease. Despite a clear role for cellular immunity in survival of Ebolavirus disease, data relating to minimal T cell epitopes in the glycoprotein has so far only been obtained in murine. This study describes the first identification of human T cell epitopes in the surface glycoprotein of the Ebolavirus. These epitopes have been identified using samples from a clinical trial of viral vectored vaccines against the Zaire Ebolavirus undertaken during the 2013-2016 West African outbreak. We also describe the recognition of these epitopes through multiple HLA alleles. As efforts to progress a vaccine against Ebola towards licensure proceed, detailed characterisation of immunity to the virus contribute to our understanding of the potential application of vaccines in diverse populations.

immunology↗