ORCID Profile
0000-0002-6966-2294
Current Organisation
Murdoch University
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Publisher: Public Library of Science (PLoS)
Date: 10-12-2019
Publisher: Elsevier BV
Date: 02-2023
DOI: 10.1016/J.HUMIMM.2022.11.005
Abstract: Human cytomegalovirus (HCMV) is carried lifelong by ∼80 % of adults worldwide, generating distinct disease syndromes in transplant recipients, people with HIV (PWH) and neonates. Amino acids 15-23 encoded by the HCMV gene UL40 match positions 3-11 of HLA-A and HLA-C, and constitute a "signal peptide" able to stabilise cell surface HLA-E as a restriction element and a ligand of NKG2A and NKG2C. We present next generation sequencing of UL40 lified from 15 Australian renal transplant recipients (RTR), six healthy adults and four neonates, and 21 Indonesian PWH. We found no groupwise associations between the presence of multiple sequences and HCMV burden (highest in PWH) or HCMV-associated symptoms in neonates. Homology between UL40 and corresponding HLA-C and HLA-A peptides in 11 RTR revealed perfect matches with HLA-C in three in iduals, all carrying HCMV encoding only VMAPRTLIL - a peptide previously associated with viremia. However indices of the burden of HCMV did not segregate in our cohort.
Publisher: Cold Spring Harbor Laboratory
Date: 03-03-2021
DOI: 10.1101/2021.03.02.433156
Abstract: SARS-CoV-2 lineage B.1.1.7 viruses are more transmissible, may lead to greater clinical severity, and result in modest reductions in antibody neutralization. subgenomic RNA (sgRNA) is produced by discontinuous transcription of the SARS-CoV-2 genome and is a crucial step in the SARS-CoV-2 life cycle. Applying our tool (periscope) to ARTIC Network Oxford Nanopore genomic sequencing data from 4400 SARS-CoV-2 positive clinical s les, we show that normalised sgRNA expression profiles are significantly increased in B.1.1.7 infections (n=879). This increase is seen over the previous dominant circulating lineage in the UK, B.1.177 (n=943), which is independent of genomic reads, E gene cycle threshold and days since symptom onset at s ling. A noncanonical sgRNA which could represent ORF9b is found in 98.4% of B.1.1.7 SARS-CoV-2 infections compared with only 13.8% of other lineages, with a 16-fold increase in median expression. We hypothesise that this is a direct consequence of a triple nucleotide mutation in nucleocapsid (28280:GAT CAT, D3L) creating a transcription regulatory-like sequence complementary to a region 3’ of the genomic leader. These findings provide a unique insight into the biology of B.1.1.7 and support monitoring of sgRNA profiles in sequence data to evaluate emerging potential variants of concern. The recently emerged and more transmissible SARS-CoV-2 lineage B.1.1.7 shows greater subgenomic RNA expression in clinical infections and enhanced expression of a noncanonical subgenomic RNA near ORF9b.
Publisher: Cold Spring Harbor Laboratory
Date: 15-03-2021
Abstract: We have developed periscope, a tool for the detection and quantification of subgenomic RNA (sgRNA) in SARS-CoV-2 genomic sequence data. The translation of the SARS-CoV-2 RNA genome for most open reading frames (ORFs) occurs via RNA intermediates termed “subgenomic RNAs.” sgRNAs are produced through discontinuous transcription, which relies on homology between transcription regulatory sequences (TRS-B) upstream of the ORF start codons and that of the TRS-L, which is located in the 5′ UTR. TRS-L is immediately preceded by a leader sequence. This leader sequence is therefore found at the 5′ end of all sgRNA. We applied periscope to 1155 SARS-CoV-2 genomes from Sheffield, United Kingdom, and validated our findings using orthogonal data sets and in vitro cell systems. By using a simple local alignment to detect reads that contain the leader sequence, we were able to identify and quantify reads arising from canonical and noncanonical sgRNA. We were able to detect all canonical sgRNAs at the expected abundances, with the exception of ORF10. A number of recurrent noncanonical sgRNAs are detected. We show that the results are reproducible using technical replicates and determine the optimum number of reads for sgRNA analysis. In VeroE6 ACE2 +/− cell lines, periscope can detect the changes in the kinetics of sgRNA in orthogonal sequencing data sets. Finally, variants found in genomic RNA are transmitted to sgRNAs with high fidelity in most cases. This tool can be applied to all sequenced COVID-19 s les worldwide to provide comprehensive analysis of SARS-CoV-2 sgRNA.
Publisher: Elsevier BV
Date: 04-2017
Publisher: Cold Spring Harbor Laboratory
Date: 11-04-2020
DOI: 10.1101/2020.04.10.029454
Abstract: Genetic variations across the SARS-CoV-2 genome may influence transmissibility of the virus and the host’s anti-viral immune response, in turn affecting the frequency of variants over-time. In this study, we examined the adjacent amino acid polymorphisms in the nucleocapsid (R203K/G204R) of SARS-CoV-2 that arose on the background of the spike D614G change and describe how strains harboring these changes became dominant circulating strains globally. Deep sequencing data of SARS-CoV-2 from public databases and from clinical s les were analyzed to identify and map genetic variants and sub-genomic RNA transcripts across the genome. Sequence analysis suggests that the three adjacent nucleotide changes that result in the K203/R204 variant have arisen by homologous recombination from the core sequence (CS) of the leader transcription-regulating sequence (TRS) rather than by stepwise mutation. The resulting sequence changes generate a novel sub-genomic RNA transcript for the C-terminal dimerization domain of nucleocapsid. Deep sequencing data from 981 clinical s les confirmed the presence of the novel TRS-CS-dimerization domain RNA in in iduals with the K203/R204 variant. Quantification of sub-genomic RNA indicates that viruses with the K203/R204 variant may also have increased expression of sub-genomic RNA from other open reading frames. The finding that homologous recombination from the TRS may have occurred since the introduction of SARS-CoV-2 in humans resulting in both coding changes and novel sub-genomic RNA transcripts suggests this as a mechanism for ersification and adaptation within its new host.
Publisher: Frontiers Media SA
Date: 29-03-2023
DOI: 10.3389/FIMMU.2023.1133781
Abstract: A vaccine against influenza is available seasonally but is not 100% effective. A predictor of successful seroconversion in adults is an increase in activated circulating T follicular helper (cTfh) cells after vaccination. However, the impact of repeated annual vaccinations on long-term protection and seasonal vaccine efficacy remains unclear. In this study, we examined the T cell receptor (TCR) repertoire and transcriptional profile of vaccine-induced expanded cTfh cells in in iduals who received sequential seasonal influenza vaccines. We measured the magnitude of cTfh and plasmablast cell activation from day 0 (d0) to d7 post-vaccination as an indicator of a vaccine response. To assess TCR ersity and T cell expansion we sorted activated and resting cTfh cells at d0 and d7 post-vaccination and performed TCR sequencing. We also single cell sorted activated and resting cTfh cells for TCR analysis and transcriptome sequencing. The percent of activated cTfh cells significantly increased from d0 to d7 in each of the 2016-17 (p & 0.0001) and 2017-18 (p = 0.015) vaccine seasons with the magnitude of cTfh activation increase positively correlated with the frequency of circulating plasmablast cells in the 2016-17 (p = 0.0001) and 2017-18 (p = 0.003) seasons. At d7 post-vaccination, higher magnitudes of cTfh activation were associated with increased clonality of cTfh TCR repertoire. The TCRs from vaccine-expanded clonotypes were identified and tracked longitudinally with several TCRs found to be present in both years. The transcriptomic profile of these expanded cTfh cells at the single cell level demonstrated overrepresentation of transcripts of genes involved in the type-I interferon pathway, pathways involved in gene expression, and antigen presentation and recognition. These results identify the expansion and transcriptomic profile of vaccine-induced cTfh cells important for B cell help.
Publisher: Springer Science and Business Media LLC
Date: 16-02-2022
DOI: 10.1038/S42003-022-03058-9
Abstract: Pre-existing pathogen-specific memory T cell responses can contribute to multiple adverse outcomes including autoimmunity and drug hypersensitivity. How the specificity of the T cell receptor (TCR) is subverted or seconded in many of these diseases remains unclear. Here, we apply abacavir hypersensitivity (AHS) as a model to address this question because the disease is linked to memory T cell responses and the HLA risk allele, HLA-B*57:01, and the initiating insult, abacavir, are known. To investigate the role of pathogen-specific TCR specificity in mediating AHS we performed a genome-wide screen for HLA-B*57:01 restricted T cell responses to Epstein-Barr virus (EBV), one of the most prevalent human pathogens. T cell epitope mapping revealed HLA-B*57:01 restricted responses to 17 EBV open reading frames and identified an epitope encoded by EBNA3C. Using these data, we cloned the dominant TCR for EBNA3C and a previously defined epitope within EBNA3B. TCR specificity to each epitope was confirmed, however, cloned TCRs did not cross-react with abacavir plus self-peptide. Nevertheless, abacavir inhibited TCR interactions with their cognate ligands, demonstrating that TCR specificity may be subverted by a drug molecule. These results provide an experimental road map for future studies addressing the heterologous immune responses of TCRs including T cell mediated adverse drug reactions.
Publisher: Mary Ann Liebert Inc
Date: 09-2017
Abstract: Host hepatitis C virus (HCV)-specific T cell responses and the ability of the virus to escape this response are important correlates of infection outcome. Understanding this host-viral interplay has been difficult given the often asymptomatic nature of acute HCV infection. We studied a recent transmission case to determine whether adapted viral strains can be transmitted and influence the recipient's anti-HCV T cell response. The ersity of viral populations was examined using next-generation sequencing, and HCV-specific T cell interferon (IFN)-γ responses were assessed using a peptide panel representing the autologous viruses. HCV-specific T cell responses in the source were directed against peptides that did not match the dominant autologous virus but rather low-frequency variants, implying existing viral adaptation in the source strain. Most HCV T cell epitopes that elicited an IFN-γ response in the source did not in the recipient, despite the pair sharing human leukocyte antigen alleles that govern antigen presentation and similar autologous viruses. Intrahost HCV variation in the recipient fell within predicted T cell epitopes, suggesting alternative targets of the immune response. These data suggest that transmission of adapted viral species can direct the host's HCV-specific immune response profile during acute infection.
Publisher: Public Library of Science (PLoS)
Date: 05-02-0006
Publisher: Springer Science and Business Media LLC
Date: 05-07-0001
DOI: 10.1038/S42003-022-03565-9
Abstract: B.1.1.7 lineage SARS-CoV-2 is more transmissible, leads to greater clinical severity, and results in modest reductions in antibody neutralization. Subgenomic RNA (sgRNA) is produced by discontinuous transcription of the SARS-CoV-2 genome. Applying our tool (periscope) to ARTIC Network Oxford Nanopore Technologies genomic sequencing data from 4400 SARS-CoV-2 positive clinical s les, we show that normalised sgRNA is significantly increased in B.1.1.7 (alpha) infections ( n = 879). This increase is seen over the previous dominant lineage in the UK, B.1.177 ( n = 943), which is independent of genomic reads, E cycle threshold and days since symptom onset at s ling. A noncanonical sgRNA which could represent ORF9b is found in 98.4% of B.1.1.7 SARS-CoV-2 infections compared with only 13.8% of other lineages, with a 16-fold increase in median sgRNA abundance. We demonstrate that ORF9b protein levels are increased 6-fold in B.1.1.7 compared to a B lineage virus in vitro. We hypothesise that increased ORF9b in B.1.1.7 is a direct consequence of a triple nucleotide mutation in nucleocapsid (28280:GAT CAT, D3L) creating a transcription regulatory-like sequence complementary to a region 3’ of the genomic leader. These findings provide a unique insight into the biology of B.1.1.7 and support monitoring of sgRNA profiles to evaluate emerging potential variants of concern.
Publisher: MDPI AG
Date: 22-04-2022
DOI: 10.3390/IJMS23094644
Abstract: Human cytomegalovirus (HCMV) is a beta-herpesvirus carried by ~80% of adults worldwide. Acute infections are often asymptomatic in healthy in iduals but generate erse syndromes in neonates, renal transplant recipients (RTR), and people with HIV (PWH). The HCMV gene UL111a encodes a homolog of human interleukin-10 (IL-10) that interacts with the human IL-10 receptor. Deep sequencing technologies were used to sequence UL111a directly from 59 clinical s les from Indonesian PWH and Australian RTR, healthy adults, and neonates. Overall, 93% of s les contained more than one variant of HCMV, as defined by at least one nonsynonymous variation. Carriage of these variants differed between neonates and adults, Australians and Indonesians, and between saliva and blood leukocytes. The variant alleles of N41D and S71Y occurred together in Australian RTR and were associated with higher T-cell responses to HCMV pp65. The variant P122S was associated with lower levels of antibodies reactive with a lysate of HCMV-infected fibroblasts. L174F was associated with increased levels of antibodies reactive with HCMV lysate, immediate-early 1 (IE-1), and glycoprotein B (gB) in Australian RTR and Indonesians PWH, suggesting a higher viral burden. We conclude that variants of UL111a are common in all populations and may influence systemic responses to HCMV.
Publisher: Elsevier BV
Date: 02-0012
Publisher: PeerJ
Date: 18-05-2018
DOI: 10.7717/PEERJ.4803
Abstract: Helicobacter pylori requires genetic agility to infect new hosts and establish long-term colonization of changing gastric environments. In this study, we analyzed H. pylori genetic adaptation in the Mongolian gerbil model. This model is of particular interest because H. pylori -infected gerbils develop a high level of gastric inflammation and often develop gastric adenocarcinoma or gastric ulceration. We analyzed the whole genome sequences of H. pylori strains cultured from experimentally infected gerbils, in comparison to the genome sequence of the input strain. The mean annualized single nucleotide polymorphism (SNP) rate per site was 1.5e −5 , which is similar to the rates detected previously in H. pylori- infected humans. Many of the mutations occurred within or upstream of genes associated with iron-related functions ( fur , tonB1 , fecA2 , fecA3 , and frpB3 ) or encoding outer membrane proteins ( alpA, oipA, fecA2, fecA3, frpB3 and cagY ). Most of the SNPs within coding regions (86%) were non-synonymous mutations. Several deletion or insertion mutations led to disruption of open reading frames, suggesting that the corresponding gene products are not required or are deleterious during chronic H. pylori colonization of the gerbil stomach. Five variants (three SNPs and two deletions) were detected in isolates from multiple animals, which suggests that these mutations conferred a selective advantage. One of the mutations (FurR88H) detected in isolates from multiple animals was previously shown to confer increased resistance to oxidative stress, and we now show that this SNP also confers a survival advantage when H. pylori is co-cultured with neutrophils. Collectively, these analyses allow the identification of mutations that are positively selected during H. pylori colonization of the gerbil model.
Publisher: Elsevier BV
Date: 2018
Publisher: Elsevier BV
Date: 02-2021
Publisher: Elsevier BV
Date: 2019
DOI: 10.2139/SSRN.3489447
Publisher: Springer Science and Business Media LLC
Date: 13-01-2017
Publisher: Informa UK Limited
Date: 03-11-2020
Publisher: American Society for Microbiology
Date: 25-03-2021
DOI: 10.1128/JVI.02380-20
Abstract: Human T cells recognize portions of viral proteins bound to host molecules (human leukocyte antigens) on the surface of infected cells. T cells recognize these foreign proteins through their T cell receptors (TCRs), which are formed by the assortment of several available V, D, and J genes to create millions of combinations of unique TCRs.
Publisher: Cold Spring Harbor Laboratory
Date: 07-2020
DOI: 10.1101/2020.07.01.181867
Abstract: We have developed periscope, a tool for the detection and quantification of sub-genomic RNA (sgRNA) in SARS-CoV-2 genomic sequence data. The translation of the SARS-CoV-2 RNA genome for most open reading frames (ORFs) occurs via RNA intermediates termed “sub-genomic RNAs”. sgRNAs are produced through discontinuous transcription which relies on homology between transcription regulatory sequences (TRS-B) upstream of the ORF start codons and that of the TRS-L which is located in the 5’ UTR. TRS-L is immediately preceded by a leader sequence. This leader sequence is therefore found at the 5’ end of all sgRNA. We applied periscope to 1,155 SARS-CoV-2 genomes from Sheffield, UK and validated our findings using orthogonal datasets and in vitro cell systems. Using a simple local alignment to detect reads which contain the leader sequence we were able to identify and quantify reads arising from canonical and non-canonical sgRNA. We were able to detect all canonical sgRNAs at expected abundances, with the exception of ORF10. A number of recurrent non-canonical sgRNAs are detected. We show that the results are reproducible using technical replicates and determine the optimum number of reads for sgRNA analysis. In VeroE6 ACE2+/− cell lines, periscope can detect the changes in the kinetics of sgRNA in orthogonal sequencing datasets. Finally, variants found in genomic RNA are transmitted to sgRNAs with high fidelity in most cases. This tool can be applied to all sequenced COVID-19 s les worldwide to provide comprehensive analysis of SARS-CoV-2 sgRNA.
Publisher: Frontiers Media SA
Date: 26-10-2021
DOI: 10.3389/FIMMU.2021.746986
Abstract: Loss of T cell immunogenicity due to mutations in virally encoded epitopes is a well-described adaptation strategy to limit host anti-viral immunity. Another described, but less understood, adaptation strategy involves the selection of mutations within epitopes that retain immune recognition, suggesting a benefit for the virus despite continued immune pressure (termed non-classical adaptation). To understand this adaptation strategy, we utilized a single cell transcriptomic approach to identify features of the HIV-specific CD8 + T cell responses targeting non-adapted (NAE) and adapted (AE) forms of epitopes containing a non-classical adaptation. T cell receptor (TCR) repertoire and transcriptome were obtained from antigen-specific CD8 + T cells of chronic (n=7) and acute (n=4) HIV-infected subjects identified by either HLA class I tetramers or upregulation of activation markers following peptide stimulation. CD8 + T cells were predominantly dual tetramer + , confirming a large proportion of cross-reactive TCR clonotypes capable of recognizing the NAE and AE form. However, single-reactive CD8 + T cells were identified in acute HIV-infected subjects only, providing the potential for the selection of T cell clones over time. The transcriptomic profile of CD8 + T cells was dependent on the autologous virus: subjects whose virus encoded the NAE form of the epitope (and who transitioned to the AE form at a later timepoint) exhibited an ‘effective’ immune response, as indicated by expression of transcripts associated with polyfunctionality, cytotoxicity and apoptosis (largely driven by the genes GZMB, IFNɣ, CCL3, CCL4 and CCL5). These data suggest that viral adaptation at a single amino acid residue can provide an alternative strategy for viral survival by modulating the transcriptome of CD8 + T cells and potentially selecting for less effective T cell clones from the acute to chronic phase.
Publisher: MDPI AG
Date: 26-10-2022
Abstract: Around 80% of adults worldwide carry human cytomegaloviris (HCMV). The HCMV gene UL18 is a homolog of HLA class I genes and encodes a protein with high affinity for the NK and T-cell cytotoxicity inhibitor LIR-1. UL18 was deep sequenced from blood, saliva or urine from Indonesian people with HIV (PWH) (n = 28), Australian renal transplant recipients (RTR) (n = 21), healthy adults (n = 7) and neonates (n = 4). 95% of s les contained more than one variant of HCMV UL18, as defined by carriage of nonsynonymous variations. When aligned with immunological markers of the host’s burden of HCMV, the S318N variation associated with high levels of antibody reactive with HCMV lysate in PWH over 12 months on antiretroviral therapy. The A107T variation associated with HCMV antibody levels and inflammatory biomarkers in PWH at early timepoints. Variants D32G, D248N, V250A and E252D aligned with elevated HCMV antibody levels in RTR, while M191K, E196Q and F165L were associated with HCMV-reactive T-cells and proportions of Vδ2− γδ T-cells—populations linked with high burdens of HCMV. We conclude that UL18 is a highly variable gene, where variation may alter the persistent burden of HCMV and/or the host response to that burden.
Publisher: BMJ
Date: 18-09-2018
DOI: 10.1136/GUTJNL-2017-313863
Abstract: Helicobacter pylori is the strongest risk factor for gastric cancer however, the majority of infected in iduals do not develop disease. Pathological outcomes are mediated by complex interactions among bacterial, host and environmental constituents, and two dietary factors linked with gastric cancer risk are iron deficiency and high salt. We hypothesised that prolonged adaptation of H. pylori to in vivo carcinogenic microenvironments results in genetic modification important for disease. Whole genome sequencing of genetically related H. pylori strains that differ in virulence and targeted H. pylori sequencing following prolonged exposure of bacteria to in vitro carcinogenic conditions were performed. A total of 180 unique single nucleotide polymorphisms (SNPs) were identified among the collective genomes when compared with a reference H. pylori genome. Importantly, common SNPs were identified in isolates harvested from iron-depleted and high salt carcinogenic microenvironments, including an SNP within fur (FurR88H). To investigate the direct role of low iron and/or high salt, H. pylori was continuously cultured in vitro under low iron or high salt conditions to assess fur genetic variation. Exposure to low iron or high salt selected for the FurR88H variant after only 5 days. To extend these results, fur was sequenced in 339 clinical H. pylori strains. Among the isolates examined, 17% (40/232) of strains isolated from patients with premalignant lesions harboured the FurR88H variant, compared with only 6% (6/107) of strains from patients with non-atrophic gastritis alone (p=0.0034). These results indicate that specific genetic variation arises within H. pylori strains during in vivo adaptation to conditions conducive for gastric carcinogenesis.
Publisher: Elsevier BV
Date: 02-2018
Publisher: Public Library of Science (PLoS)
Date: 16-12-2022
DOI: 10.1371/JOURNAL.PPAT.1010965
Abstract: Adaptation to human leukocyte antigen (HLA)-associated immune pressure represents a major driver of human immunodeficiency virus (HIV) evolution at both the in idual and population level. To date, there has been limited exploration of the impact of the initial cellular immune response in driving viral adaptation, the dynamics of these changes during infection and their effect on circulating transmitting viruses at the population level. Capturing detailed virological and immunological data from acute and early HIV infection is challenging as this commonly precedes the diagnosis of HIV infection, potentially by many years. In addition, rapid initiation of antiretroviral treatment following a diagnosis is the standard of care, and central to global efforts towards HIV elimination. Yet, acute untreated infection is the critical period in which the ersity of proviral reservoirs is first established within in iduals, and associated with greater risk of onward transmissions in a population. Characterizing the viral adaptations evident in the earliest phases of infection, coinciding with the initial cellular immune responses is therefore relevant to understanding which changes are of greatest impact to HIV evolution at the population level. In this study, we utilized three separate cohorts to examine the initial CD8 + T cell immune response to HIV (cross-sectional acute infection cohort), track HIV evolution in response to CD8 + T cell-mediated immunity over time (longitudinal chronic infection cohort) and translate the impact of HLA-driven HIV evolution to the population level (cross-sectional HIV sequence data spanning 30 years). Using next generation viral sequencing and enzyme-linked immunospot interferon-gamma recall responses to peptides representing HLA class I-specific HIV T cell targets, we observed that CD8 + T cell responses can select viral adaptations prior to full antibody seroconversion. Using the longitudinal cohort, we uncover that viral adaptations have the propensity to be retained over time in a non-selective immune environment, which reflects the increasing proportion of pre-adapted HIV strains within the Western Australian population over an approximate 30-year period.
Publisher: American Society for Microbiology
Date: 31-10-2021
DOI: 10.1128/SPECTRUM.00020-21
Abstract: Human cytomegalovirus (HCMV) is a common viral pathogen of solid organ transplant recipients, neonates, and HIV-infected in iduals. HCMV encodes homologs of several host genes with the potential to influence viral persistence and/or pathogenesis.
Publisher: Springer Science and Business Media LLC
Date: 27-03-2017
Publisher: American Society for Microbiology
Date: 27-10-2020
DOI: 10.1128/JVI.01456-20
Abstract: Murine cytomegalovirus (MCMV) is widely used as a model of human CMV (HCMV) infection. However, this model relies on strains of MCMV that have been serially passaged in the laboratory for over four decades. These laboratory strains have been cloned as bacterial artificial chromosomes (BACs), which permits rapid and precise manipulation. Low-passage strains of MCMV add to the utility of the mouse model of HCMV infection but do not exist as cloned BACs. This study describes the first such low-passage MCMV BAC. This BAC-derived G4 was initially attenuated in vivo , with subsequent full genomic sequencing revealing a novel spliced transcript required for salivary gland tropism. These data suggest that MCMV, like HCMV, undergoes tissue culture adaptation that can limit in vivo growth and supports the use of BAC clones as a way of standardizing viral strains and minimizing interlaboratory strain variation.
Publisher: Frontiers Media SA
Date: 17-06-2021
DOI: 10.3389/FGENE.2021.642012
Abstract: Type B adverse drug reactions (ADRs) are iatrogenic immune-mediated syndromes with mechanistic etiologies that remain incompletely understood. Some of the most severe ADRs, including delayed drug hypersensitivity reactions, are T-cell mediated, restricted by specific human leukocyte antigen risk alleles and sometimes by public or oligoclonal T-cell receptors (TCRs), central to the immunopathogenesis of tissue-damaging response. However, the specific cellular signatures of effector, regulatory, and accessory immune populations that mediate disease, define reaction phenotype, and determine severity have not been defined. Recent development of single-cell platforms bringing together advances in genomics and immunology provides the tools to simultaneously examine the full transcriptome, TCRs, and surface protein markers of highly heterogeneous immune cell populations at the site of the pathological response at a single-cell level. However, the requirement for advanced bioinformatics expertise and computational hardware and software has often limited the ability of investigators with the understanding of diseases and biological models to exploit these new approaches. Here we describe the features and use of a state-of-the-art, fully integrated application for analysis and visualization of multiomic single-cell data called Visual Genomics Analysis Studio (VGAS). This unique user-friendly, Windows-based graphical user interface is specifically designed to enable investigators to interrogate their own data. While VGAS also includes tools for sequence alignment and identification of associations with host or organism genetic polymorphisms, in this review we focus on its application for analysis of single-cell TCR–RNA–Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE)-seq, enabling holistic cellular characterization by unbiased transcriptome and select surface proteome. Critically, VGAS does not require user-directed coding or access to high-performance computers, instead incorporating performance-optimized hidden code to provide application-based fast and intuitive tools for data analyses and production of high-resolution publication-ready graphics on standard specification laptops. Specifically, it allows analyses of comprehensive single-cell TCR sequencing (scTCR-seq) data, detailing (i) functional pairings of α–β heterodimer TCRs, (ii) one-click histograms to display entropy and gene rearrangements, and (iii) Circos and Sankey plots to visualize clonality and dominance. For unbiased single-cell RNA sequencing (scRNA-seq) analyses, users extract cell transcriptome signatures according to global structure via principal component analysis, t-distributed stochastic neighborhood embedding, or uniform manifold approximation and projection plots, with overlay of scTCR-seq enabling identification and selection of the immunodominant TCR-expressing populations. Further integration with similar sequence-based detection of surface protein markers using oligo-labeled antibodies (CITE-seq) provides comparative understanding of surface protein expression, with differential gene or protein analyses visualized using volcano plot or heatmap functions. These data can be compared to reference cell atlases or suitable controls to reveal discrete disease-specific subsets, from epithelial to tissue-resident memory T-cells, and activation status, from senescence through exhaustion, with more finite transcript expression displayed as violin and box plots. Importantly, guided tutorial videos are available, as are regular application updates based on the latest advances in bioinformatics and user feedback.
Publisher: Elsevier BV
Date: 06-2020
Publisher: Elsevier BV
Date: 12-2018
Publisher: Informa UK Limited
Date: 11-05-2015
Publisher: Wiley
Date: 07-2021
DOI: 10.1002/JIA2.25755
Publisher: Elsevier BV
Date: 02-2021
No related grants have been discovered for Shay Leary.