ORCID Profile
0000-0002-2849-9216
Current Organisation
Rigshospitalet
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Publisher: IEEE
Date: 07-2015
Publisher: Springer Science and Business Media LLC
Date: 24-11-2020
DOI: 10.1038/S41598-020-77565-2
Abstract: Cytotoxic CD8 + T cells are key for immune protection against viral infections. The breadth and cross-reactivity of these responses are important against rapidly mutating RNA viruses, such as dengue (DENV), yet how viral ersity affect T cell responses and their cross-reactivity against multiple variants of the virus remains poorly defined. In this study, an integrated analysis was performed to map experimentally validated CD8 + T cell epitopes onto the distribution of DENV genome sequences across the 4 serotypes worldwide. Despite the higher viral ersity observed within HLA-I restricted epitopes, mapping of 609 experimentally validated epitopes sequences on 3985 full-length viral genomes revealed 19 highly conserved epitopes across the four serotypes within the immunogenic regions of NS3, NS4B and NS5. These conserved epitopes were associated with a higher magnitude of IFN-γ response when compared to non-conserved epitopes and were restricted to 13 HLA class I genotypes, hence providing high coverage among human populations. Phylogeographic analyses showed that these epitopes are largely conserved in most of the endemic regions of the world, and with only some of these epitopes presenting distinct mutated variants circulating in South America and Asia.This study provides evidence for the existence of highly immunogenic and conserved epitopes across serotypes, which may impact design of new universal T-cell-inducing vaccine candidates that minimise detrimental effects of viral ersification and at the same time induce responses to a broad human population.
Publisher: Elsevier BV
Date: 07-2019
Publisher: Hindawi Limited
Date: 2014
DOI: 10.1155/2014/264519
Abstract: Rapidly mutating viruses, such as hepatitis C virus (HCV) and HIV, have adopted evolutionary strategies that allow escape from the host immune response via genomic mutations. Recent advances in high-throughput sequencing are reshaping the field of immuno-virology of viral infections, as these allow fast and cheap generation of genomic data. However, due to the large volumes of data generated, a thorough understanding of the biological and immunological significance of such information is often difficult. This paper proposes a pipeline that allows visualization and statistical analysis of viral mutations that are associated with immune escape. Taking next generation sequencing data from longitudinal analysis of HCV viral genomes during a single HCV infection, along with antigen specific T-cell responses detected from the same subject, we demonstrate the applicability of these tools in the context of primary HCV infection. We provide a statistical and visual explanation of the relationship between cooccurring mutations on the viral genome and the parallel adaptive immune response against HCV.
Publisher: Public Library of Science (PLoS)
Date: 13-03-2023
DOI: 10.1371/JOURNAL.PNTD.0010758
Abstract: At least a third of dengue patients develop plasma leakage with increased risk of life-threatening complications. Predicting plasma leakage using laboratory parameters obtained in early infection as means of triaging patients for hospital admission is important for resource-limited settings. A Sri Lankan cohort including 4,768 instances of clinical data from N = 877 patients (60.3% patients with confirmed dengue infection) recorded in the first 96 hours of fever was considered. After excluding incomplete instances, the dataset was randomly split into a development and a test set with 374 (70%) and 172 (30%) patients, respectively. From the development set, five most informative features were selected using the minimum description length (MDL) algorithm. Random forest and light gradient boosting machine (LightGBM) were used to develop a classification model using the development set based on nested cross validation. An ensemble of the learners via average stacking was used as the final model to predict plasma leakage. Lymphocyte count, haemoglobin, haematocrit, age, and aspartate aminotransferase were the most informative features to predict plasma leakage. The final model achieved the area under the receiver operating characteristics curve, AUC = 0.80 with positive predictive value, PPV = 76.9%, negative predictive value, NPV = 72.5%, specificity = 87.9%, and sensitivity = 54.8% on the test set. The early predictors of plasma leakage identified in this study are similar to those identified in several prior studies that used non-machine learning based methods. However, our observations strengthen the evidence base for these predictors by showing their relevance even when in idual data points, missing data and non-linear associations were considered. Testing the model on different populations using these low-cost observations would identify further strengths and limitations of the presented model.
Publisher: Springer Science and Business Media LLC
Date: 31-12-2016
DOI: 10.1038/S41598-019-49454-W
Abstract: Hepatitis C virus (HCV) is one of very few viruses that are either naturally cleared, or alternatively persist to cause chronic disease. Viral ersity and escape, as well as host adaptive immune factors, are believed to control the outcome. To date, there is limited understanding of the critical, early host-pathogen interactions. The asymptomatic nature of early HCV infection generally prevents identification of the transmitted/founder (T/F) virus, and thus the study of host responses directed against the autologous T/F strain. In this study, 14 rare subjects identified from very early in infection (4–45 days) with varied disease outcomes (n = 7 clearers) were examined in regard to the timing, breadth, and magnitude of the neutralizing antibody (nAb) response, as well as evolution of the T/F strain. Clearance was associated with earlier onset and more potent nAb responses appearing at a mean of 71 days post-infection (DPI), but these responses were narrowly directed against the autologous T/F virus or closely related variants. In contrast, a delayed onset of nAbs (mean 425 DPI) was observed in chronic progressors that appear to have targeted longitudinal variants rather than the T/F strain. The nAb responses in the chronic progressors mapped to known CD81 binding epitopes, and were associated with rapid emergence of new viral variants with reduced CD81 binding. We propose that the prolonged period of viremia in the absence of nAbs in these subjects was associated with an increase in viral ersity, affording the virus greater options to escape nAb pressure once it emerged. These findings indicate that timing of the nAb response is essential for clearance. Further investigation of the specificities of the early nAbs and the factors regulating early induction of protective nAbs is needed.
Publisher: Elsevier BV
Date: 07-2017
DOI: 10.1016/J.VIRUSRES.2016.10.014
Abstract: With the advent of affordable deep sequencing technologies, detection of low frequency variants within genetically erse viral populations can now be achieved with unprecedented depth and efficiency. The high-resolution data provided by next generation sequencing technologies is currently recognised as the gold standard in estimation of viral ersity. In the analysis of rapidly mutating viruses, longitudinal deep sequencing datasets from viral genomes during in idual infection episodes, as well as at the epidemiological level during outbreaks, now allow for more sophisticated analyses such as statistical estimates of the impact of complex mutation patterns on the evolution of the viral populations both within and between hosts. These analyses are revealing more accurate descriptions of the evolutionary dynamics that underpin the rapid adaptation of these viruses to the host response, and to drug therapies. This review assesses recent developments in methods and provide informative research ex les using deep sequencing data generated from rapidly mutating viruses infecting humans, particularly hepatitis C virus (HCV), human immunodeficiency virus (HIV), Ebola virus and influenza virus, to understand the evolution of viral genomes and to explore the relationship between viral mutations and the host adaptive immune response. Finally, we discuss limitations in current technologies, and future directions that take advantage of publically available large deep sequencing datasets.
Publisher: American Society for Microbiology
Date: 15-11-6349
DOI: 10.1128/JVI.03717-14
Abstract: The interaction between hepatitis C virus (HCV) and cellular immune responses during very early infection is critical for disease outcome. To date, the impact of antigen-specific cellular immune responses on the evolution of the viral population establishing infection and on potential escape has not been studied. Understanding these early host-virus dynamics is important for the development of a preventative vaccine. Three subjects who were followed longitudinally from the detection of viremia preseroconversion until disease outcome were analyzed. The evolution of transmitted/founder (T/F) viruses was undertaken using deep sequencing. CD8 + T cell responses were measured via enzyme-linked immunosorbent spot (ELISpot) assay using HLA class I-restricted T/F epitopes. T/F viruses were rapidly extinguished in all subjects associated with either viral clearance ( n = 1) or replacement with viral variants leading to establishment of chronic infection ( n = 2). CD8 + T cell responses against 11 T/F epitopes were detectable by 33 to 44 days postinfection, and 5 of these epitopes had not previously been reported. These responses declined rapidly in those who became chronically infected and were maintained in the subject who cleared infection. Higher-magnitude CD8 + T cell responses were associated with rapid development of immune escape variants at a rate of up to 0.1 per day. Rapid escape from CD8 + T cell responses has been quantified for the first time in the early phase of primary HCV infection. These rapid escape dynamics were associated with higher-magnitude CD8 + T cell responses. These findings raise questions regarding optimal selection of immunogens for HCV vaccine development and suggest that detailed analysis of in idual epitopes may be required. IMPORTANCE A major limitation in our detailed understanding of the role of immune response in HCV clearance has been the lack of data on very early primary infection when the transmitted viral variants successfully establish the acute infection. This study was made possible through the availability of specimens from a unique cohort of asymptomatic primary infection cases in whom the first available viremic s les were collected approximately 3 weeks postinfection and at regular intervals thereafter. The study included detailed examination of both the evolution of the viral population and the host cellular immune responses against the T/F viruses. The findings here provide the first evidence of host cellular responses targeting T/F variants and imposing a strong selective force toward viral escape. The results of this study provide useful insight on how virus escapes the host response and consequently on future analysis of vaccine-induced immunity.
Publisher: Springer Science and Business Media LLC
Date: 23-10-2020
DOI: 10.1038/S41598-020-75374-1
Abstract: Current methods for dengue virus (DENV) genome lification, lify parts of the genome in at least 5 overlapping segments and then combine the output to characterize a full genome. This process is laborious, costly and requires at least 10 primers per serotype, thus increasing the likelihood of PCR bias. We introduce an assay to lify near full-length dengue virus genomes as intact molecules, sequence these licons with third generation “nanopore” technology without fragmenting and use the sequence data to differentiate within-host viral variants with a bioinformatics tool (Nano-Q). The new assay successfully generated near full-length licons from DENV serotypes 1, 2 and 3 s les which were sequenced with nanopore technology. Consensus DENV sequences generated by nanopore sequencing had over 99.5% pairwise sequence similarity to Illumina generated counterparts provided the coverage was 100 with both platforms. Maximum likelihood phylogenetic trees generated from nanopore consensus sequences were able to reproduce the exact trees made from Illumina sequencing with a conservative 99% bootstrapping threshold (after 1000 replicates and 10% burn-in). Pairwise genetic distances of within host variants identified from the Nano-Q tool were less than that of between host variants, thus enabling the phylogenetic segregation of variants from the same host.
Publisher: Springer Science and Business Media LLC
Date: 02-03-2021
DOI: 10.1186/S12864-021-07460-1
Abstract: Hepatitis C (HCV) and many other RNA viruses exist as rapidly mutating quasi-species populations in a single infected host. High throughput characterization of full genome, within-host variants is still not possible despite advances in next generation sequencing. This limitation constrains viral genomic studies that depend on accurate identification of hemi-genome or whole genome, within-host variants, especially those occurring at low frequencies. With the advent of third generation long read sequencing technologies, including Oxford Nanopore Technology (ONT) and PacBio platforms, this problem is potentially surmountable. ONT is particularly attractive in this regard due to the portable nature of the MinION sequencer, which makes real-time sequencing in remote and resource-limited locations possible. However, this technology (termed here ‘nanopore sequencing’) has a comparatively high technical error rate. The present study aimed to assess the utility, accuracy and cost-effectiveness of nanopore sequencing for HCV genomes. We also introduce a new bioinformatics tool (Nano-Q) to differentiate within-host variants from nanopore sequencing. The Nanopore platform, when the coverage exceeded 300 reads, generated comparable consensus sequences to Illumina sequencing. Using HCV Envelope plasmids (~ 1800 nt) mixed in known proportions, the capacity of nanopore sequencing to reliably identify variants with an abundance as low as 0.1% was demonstrated, provided the autologous reference sequence was available to identify the matching reads. Successful pooling and nanopore sequencing of 52 s les from patients with HCV infection demonstrated its cost effectiveness (AUD$ 43 per s le with nanopore sequencing versus $100 with paired-end short read technology). The Nano-Q tool successfully separated between-host sequences, including those from the same subtype, by bulk sorting and phylogenetic clustering without an autologous reference sequence (using only a subtype-specific generic reference). The pipeline also identified within-host viral variants and their abundance when the parameters were appropriately adjusted. Cost effective HCV whole genome sequencing and within-host variant identification without haplotype reconstruction are potential advantages of nanopore sequencing.
Publisher: Wiley
Date: 22-01-2019
DOI: 10.1111/JVH.13051
Publisher: Springer Science and Business Media LLC
Date: 31-01-2017
DOI: 10.1038/SREP41719
Abstract: Resistance against new hepatitis C virus (HCV) antivirals is an area of increasing interest. Resistance-associated substitutions (RASs) have been identified in treatment-naïve in iduals, but pressures driving treatment-independent RAS emergence are poorly understood. We analysed the longitudinal evolution of RASs in twelve participants with early acute HCV infections. Full-genome deep sequences were analysed for changes in RAS frequency within NS3, NS5A and NS5B-coding regions over the course of the infection. Emergence of RASs relevant only to the polymerase non-nucleoside inhibitors (NNI) was detected, and these lay within CD8+ T-cell epitopes. Conversely, the loss of NNI RASs over time appeared likely to be driven by viral fitness constraints. These results highlight the importance of monitoring CD8+ T cell epitope-associated RASs in populations with dominant HLA types.
Publisher: American Society for Microbiology
Date: 10-2019
DOI: 10.1128/JVI.00202-19
Abstract: There are currently at least 71 million in iduals with chronic HCV worldwide and almost two million new infections annually. Although the advent of direct-acting antivirals (DAAs) offers highly effective therapy, considerable remaining challenges argue against reliance on DAAs for HCV elimination, including high drug cost, poorly developed health infrastructure, low screening rates, and significant reinfection rates. Accordingly, development of an effective vaccine is crucial to HCV elimination. An HCV vaccine that elicits T cell immunity in the liver will be highly protective for the following reasons: (i) T cell responses against nonstructural proteins of the virus are associated with clearance of primary infection, and (ii) long-lived liver-resident T cells alone can protect against malaria infection of hepatocytes. Thus, in this study we exploit promising vaccination platforms to highlight strategies that can be used to evoke highly functional and long-lived T cell responses in the liver for protection against HCV.
Publisher: Hindawi Limited
Date: 2014
DOI: 10.1155/2014/680249
Publisher: Elsevier BV
Date: 04-2017
Publisher: Springer Science and Business Media LLC
Date: 07-12-2022
DOI: 10.1038/S41467-022-35281-7
Abstract: T cell exhaustion is a hallmark of hepatitis C virus (HCV) infection and limits protective immunity in chronic viral infections and cancer. Limited knowledge exists of the initial viral and immune dynamics that characterise exhaustion in humans. We studied longitudinal blood s les from a unique cohort of in iduals with primary infection using single-cell multi-omics to identify the functions and phenotypes of HCV-specific CD8 + T cells. Early elevated IFN-γ response against the transmitted virus is associated with the rate of immune escape, larger clonal expansion, and early onset of exhaustion. Irrespective of disease outcome, we find heterogeneous subsets of progenitors of exhaustion, based on the level of PD-1 expression and loss of AP-1 transcription factors. Intra-clonal analysis shows distinct trajectories with multiple fates and evolutionary plasticity of precursor cells. These findings challenge the current paradigm on the contribution of CD8 + T cells to HCV disease outcome and provide data for future studies on T cell differentiation in human infections.
Publisher: Medknow
Date: 2016
Publisher: Springer Science and Business Media LLC
Date: 11-02-2019
DOI: 10.1038/S41598-018-38368-8
Abstract: Enteroviruses (EVs) are prime candidate environmental triggers of islet autoimmunity (IA), with potential as vaccine targets for type 1 diabetes prevention. However, the use of targeted virus detection methods and the selective focus on EVs by most studies increases the risk for substantial investigation bias and an overestimated association between EV and type 1 diabetes. Here we performed comprehensive virome-capture sequencing to examine all known vertebrate-infecting viruses without bias in 182 specimens (faeces and plasma) collected before or at seroconversion from 45 case children with IA and 48 matched controls. From .6 billion reads, 28 genera of viruses were detected and 62% of children (58/93) were positive for ≥1 vertebrate-infecting virus. We identified 129 viruses as differentially abundant between the gut of cases and controls, including 5 EV-A types significantly more abundant in the cases. Our findings further support EV’s hypothesised contribution to IA and corroborate the proposal that viral load may be an important parameter in disease pathogenesis. Furthermore, our data indicate a previously unrecognised association of IA with higher EV-A abundance in the gut of children and provide a catalog of viruses to be interrogated further to determine a causal link between virus infection and type 1 diabetes.
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 07-12-2022
No related grants have been discovered for Preston Leung.