ORCID Profile
0000-0001-5008-9080
Current Organisation
Murdoch University
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Publisher: Springer Science and Business Media LLC
Date: 30-09-2023
Publisher: Springer Science and Business Media LLC
Date: 12-2018
Publisher: Elsevier BV
Date: 12-2022
Publisher: Springer Science and Business Media LLC
Date: 10-2009
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: 08-2019
Publisher: Elsevier BV
Date: 02-2018
Publisher: Springer Science and Business Media LLC
Date: 10-07-2022
DOI: 10.1007/S00018-022-04436-6
Abstract: Inherited retinal diseases (IRDs) are a heterogeneous group of blinding disorders, which result in dysfunction or death of the light-sensing cone and rod photoreceptors. Despite in idual IRDs (Inherited retinal disease) being rare, collectively, they affect up to 1:2000 people worldwide, causing a significant socioeconomic burden, especially when cone-mediated central vision is affected. This study uses the Pde6c cpfl1 mouse model of achromatopsia, a cone-specific vision loss IRD (Inherited retinal disease), to investigate the potential gene-independent therapeutic benefits of a histone demethylase inhibitor GSK-J4 on cone cell survival. We investigated the effects of GSK-J4 treatment on cone cell survival in vivo and ex vivo and changes in cone-specific gene expression via single-cell RNA sequencing. A single intravitreal GSK-J4 injection led to transcriptional changes in pathways involved in mitochondrial dysfunction, endoplasmic reticulum stress, among other key epigenetic pathways, highlighting the complex interplay between methylation and acetylation in healthy and diseased cones. Furthermore, continuous administration of GSK-J4 in retinal explants increased cone survival. Our results suggest that IRD (Inherited retinal disease)-affected cones respond positively to epigenetic modulation of histones, indicating the potential of this approach in developing a broad class of novel therapies to slow cone degeneration.
Publisher: Wiley
Date: 26-08-2022
DOI: 10.1111/TAN.14767
Abstract: HLA (HLA) alleles are risk factors for CD8+ T‐cell‐mediated drug hypersensitivity reactions. However, as most HLA associations are incompletely predictive and/or involve risk alleles at low frequency, costly sequence‐based typing can elude an economically productive cost: benefit ratio for clinical validation studies and diagnostic and/or preventative screening. Hence rapid and low‐cost detection assays are now required, both for single alleles but also across risk loci associated with broader multi‐disease risk exemplified by associations with erse alleles in HLA‐B*35, including HLA‐B * 35 : 01 and green tea‐ or co‐trimoxazole‐induced liver injury. Here, we developed a cost‐effective ( $10USD) qPCR assay for rapid ( .5 h) clinical detection of HLA‐B * 35 alleles. The assay was validated using 430 DNA s les with previous American society for histocompatibility and immunogenetics‐accredited sequence‐based high‐resolution HLA typing, positively detecting all HLA‐B * 35 allelic variants in our cohort, and as expected by primer design, the six s les that expressed low‐frequency B * 78 : 01 . The assay did not result in positive detection for any negative control allele. With expected detection of B * 35 and B * 78 , our assay sensitivity (95% CI, 95.07%–100.00%) and specificity (95% CI, 98.97%–100.00%) of 100% using as low as 10 ng of DNA provides a reliable HLA‐B * 35 screening tool for clinical validation and HLA–risk‐based prevention and diagnostics.
Publisher: European Respiratory Society
Date: 09-2016
Publisher: MDPI AG
Date: 22-04-2016
DOI: 10.3390/V8040118
Publisher: Public Library of Science (PLoS)
Date: 05-02-0006
Publisher: European Respiratory Society
Date: 09-2015
Publisher: Elsevier BV
Date: 11-2020
Publisher: Elsevier BV
Date: 12-2010
DOI: 10.1016/J.GENE.2010.08.009
Abstract: Measurements of population ersity are fundamental to the reconstruction of the evolutionary and epidemiological history of organisms. Commonly used protocols to measure population ersity using the polymerase chain reaction (PCR) are prone to the introduction of artificial chimeras. These are often difficult to detect and can confound the correct interpretation of results due to the false generation of recombinants when the underlying DNA s le contains multiple distinct templates. This study presents a standardised procedure to suppress the formation of artificial chimeras during PCR lification. The solution is based on the accurate determination of the efficiency and end point of the log-linear phase of a PCR. This procedure will facilitate the generation of data sets that more accurately reflect the underlying population ersity rather than artifacts introduced by the process itself.
Publisher: Elsevier BV
Date: 02-2021
Publisher: Springer Science and Business Media LLC
Date: 13-01-2017
Publisher: The American Association of Immunologists
Date: 15-04-2010
Abstract: Currently, 1.1 million in iduals in the United States are living with HIV-1 infection. Although this is a relatively small proportion of the global pandemic, the remarkable mix of ancestries in the United States, drawn together over the past two centuries of continuous population migrations, provides an important and unique perspective on adaptive interactions between HIV-1 and human genetic ersity. HIV-1 is a rapidly adaptable organism and mutates within or near immune epitopes that are determined by the HLA class I genotype of the infected host. We characterized HLA-associated polymorphisms across the full HIV-1 proteome in a large, ethnically erse national United States cohort of HIV-1–infected in iduals. We found a striking ergence in the immunoselection patterns associated with HLA variants that have very similar or identical peptide-binding specificities but are differentially distributed among racial/ethnic groups. Although their similarity in peptide binding functionally clusters these HLA variants into supertypes, their differences at sites within the peptide-binding groove contribute to race-specific selection effects on circulating HIV-1 viruses. This suggests that the interactions between the HLA/HIV peptide complex and the TCR vary significantly within HLA supertype groups, which, in turn, influences HIV-1 evolution.
Publisher: Wiley
Date: 31-10-2019
DOI: 10.1111/AJCO.13263
Publisher: Springer Science and Business Media LLC
Date: 12-1990
DOI: 10.1007/BF02927976
Publisher: Elsevier BV
Date: 06-2016
DOI: 10.1016/J.VIROL.2016.02.016
Abstract: The spatiotemporal dynamics of Hepatitis C Virus (HCV) RNA localisation are poorly understood. To address this we engineered HCV genomes harbouring MS2 bacteriophage RNA stem-loops within the 3'-untranslated region to allow tracking of HCV RNA via specific interaction with a MS2-Coat-mCherry fusion protein. Despite the impact of these insertions on viral fitness, live imaging revealed that replication of tagged-HCV genomes induced specific redistribution of the mCherry-tagged-MS2-Coat protein to motile and static foci. Further analysis showed that HCV RNA was associated with NS5A in both static and motile structures while a subset of motile NS5A structures was devoid of HCV RNA. Further investigation of viral RNA traffic with respect to lipid droplets (LDs) revealed HCV RNA-positive structures in close association with LDs. These studies provide new insights into the dynamics of HCV RNA traffic with NS5A and LDs and provide a platform for future investigations of HCV replication and assembly.
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: Ovid Technologies (Wolters Kluwer Health)
Date: 18-01-2011
DOI: 10.1002/HEP.24076
Publisher: Elsevier BV
Date: 09-2023
Publisher: SAGE Publications
Date: 2016
DOI: 10.3851/IMP3056
Publisher: Elsevier BV
Date: 06-2020
Publisher: Public Library of Science (PLoS)
Date: 09-2011
Publisher: Wiley
Date: 17-05-2012
DOI: 10.1038/ICB.2011.34
Publisher: Wiley
Date: 07-2021
DOI: 10.1002/JIA2.25755
Publisher: Springer Science and Business Media LLC
Date: 17-08-2017
DOI: 10.1038/S41598-017-08876-0
Abstract: Genes of the human leukocyte antigen (HLA) system encode cell-surface proteins involved in regulation of immune responses, and the way drugs interact with the HLA peptide binding groove is important in the immunopathogenesis of T-cell mediated drug hypersensitivity syndromes. Nevirapine (NVP), is an HIV-1 antiretroviral with treatment-limiting hypersensitivity reactions (HSRs) associated with multiple class I and II HLA alleles. Here we utilize a novel analytical approach to explore these multi-allelic associations by systematically examining HLA molecules for similarities in peptide binding specificities and binding pocket structure. We demonstrate that primary predisposition to cutaneous NVP HSR, seen across ancestral groups, can be attributed to a cluster of HLA-C alleles sharing a common binding groove F pocket with HLA-C*04:01 . An independent association with a group of class II alleles which share the HLA-DRB1-P4 pocket is also observed. In contrast, NVP HSR protection is afforded by a cluster of HLA-B alleles defined by a characteristic peptide binding groove B pocket. The results suggest drug-specific interactions within the antigen binding cleft can be shared across HLA molecules with similar binding pockets. We thereby provide an explanation for multiple HLA associations with cutaneous NVP HSR and advance insight into its pathogenic mechanisms.
Publisher: Elsevier BV
Date: 04-2017
Publisher: Elsevier BV
Date: 02-2022
Publisher: Public Library of Science (PLoS)
Date: 10-12-2019
Publisher: Springer Science and Business Media LLC
Date: 31-01-2023
DOI: 10.1038/S41598-023-28847-Y
Abstract: Natural killer cells (NK) have been associated with the pathophysiology of atopic dermatitis (AD). NK function is regulated by killer cell Ig-like receptor family (KIR) receptors that interact with HLA ligands. The study goal was to focus on allelic variation in genes KIR2DL5 , KIR2DS5 , and KIR2DS1 with respect to AD. This was a case–control study of in iduals with (n = 313) and without (n = 176) AD. Associations were estimated using logistic regression. The prevalence of KIR2DL5 was 52.5% (95% CI 48.0,57.0), KIR2DS5 was 33.0% (28.8,37.3), and KIR2DS1 was 33.6% (29.4,38.0). The presence of the KIR2DL5*001:01 increased the odds of having AD by about 86% (odds ratio (OR): 1.86(1.23,2.82) p = 0.003). The risk for in iduals homozygous for KIR2DL5*001:01 was even greater (OR: 2.16 (95% CI 1.31,3.53) p = 0.0023). The odds of having AD with KIR2DL5*001:01 was similar in Whites and Blacks. Allelic variation in KIR2DS5 and KIR2DS1 was not associated with AD. There is no known HLA binding ligand for KIR2DL5 . The effect of KIR2DL5*001:01 increased in the presence of HLA-B*-21TT leader sequence (2.46(1.37,4.41) p = 0.0025) and the HLA-C2 ligand (2.07 (1.37,4.41, p = 0.000002). Our study shows an independent association of the KIR2DL5*001:01 with AD and is the first study to associate AD with KIR allelic variation.
Publisher: BMJ
Date: 11-2020
Abstract: We aimed to assess the impact of genomic human leukocyte antigen (HLA)-I/II homozygosity on the survival benefit of patients with unresectable locally advanced, metastatic non-small lung cancer treated by single-agent programmed cell death protein-1 rogrammed death ligand 1 (PD1/PDL1) inhibitors. We collected blood from 170 patients with advanced lung cancer treated with immunotherapy at two major oncology centers in Western Australia. Genomic DNA was extracted from white blood cells and used for HLA-I/II high-resolution typing. HLA-I/II homozygosity was tested for association with survival outcomes. Univariable and multivariable Cox regression models were constructed to determine whether HLA homozygosity was an independent prognostic factor affecting Overall Survival (OS) and Progression Free Survival (PFS). We also investigated the association between in idual HLA-A and -B supertypes with OS. Homozygosity at HLA-I loci, but not HLA-II, was significantly associated with shorter OS (HR=2.17, 95% CI 1.13 to 4.17, p=0.02) in both univariable and multivariable analysis. The effect of HLA-I homozygosity in OS was particularly relevant for patients with tumors expressing PDL1 ≥50% (HR=3.93, 95% CI 1.30 to 11.85, p .001). The adverse effect of HLA-I homozygosity on PFS was only apparent after controlling for interactions between PDL1 status and HLA-I genotype (HR=2.21, 95% CI 1.04 to 4.70, p=0.038). The presence of HLA-A02 supertype was the only HLA-I supertype to be associated with improved OS (HR=0.56, 95% CI 0.34 to 0.93, p=0.023). Our results suggest that homozygosity at ≥1 HLA-I loci is associated with short OS and PFS in patients with advanced non-small cell lung cancer with PDL1 ≥50% treated with single-agent immunotherapy. Carriers of HLA-A02 supertype reported better survival outcomes in this cohort of patients.
Publisher: Cold Spring Harbor Laboratory
Date: 09-02-2022
DOI: 10.1101/2022.02.07.479069
Abstract: Inherited retinal diseases (IRDs) are a heterogeneous group of blinding disorders, which result in dysfunction or death of the light-sensing cone and rod photoreceptors. Despite in idual IRDs being rare, collectively, they affect up to 1:2000 people worldwide, causing a significant socioeconomic burden, especially when cone-mediated central vision is affected. This study uses the Pde6c cpfl1 mouse model of achromatopsia, a cone-specific vision loss IRD, to investigate the potential gene-independent therapeutic benefits of a histone demethylase inhibitor GSK-J4 on cone cell survival. We investigated the effects of GSK-J4 treatment on cone cell survival in vivo and ex vivo and changes in cone-specific gene expression via single-cell RNA sequencing. A single intravitreal GSK-J4 injection led to transcriptional changes in pathways involved in mitochondrial dysfunction, endoplasmic reticulum stress, among other key epigenetic pathways, highlighting the complex interplay between methylation and acetylation in healthy and diseased cones. Furthermore, continuous administration of GSK-J4 in retinal explants increased cone survival. Our results suggest that IRD-affected cones respond positively to epigenetic modulation of histones, indicating the potential of this approach in the development of a broad class of novel therapies to slow cone degeneration.
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: Elsevier BV
Date: 09-2019
DOI: 10.1111/AJT.15470
Abstract: Abacavir administration is associated with drug-induced hypersensitivity reactions in HIV+ in iduals expressing the HLA-B*57:01 allele. However, the immunological effects of abacavir administration in an HLA-B57 mismatched transplantation setting have not been studied. We hypothesized that abacavir exposure could induce de novo HLA-B57-specific allorecognition. HIV-specific CD8 T cell clones were generated from HIV+ in iduals, using single cell sorting based on HIV peptide/HLA tetramer staining. The T cell clones were assayed for alloreactivity against a panel of single HLA-expressing cell lines, in the presence or absence of abacavir. Cytokine assay, CD137 upregulation, and cytotoxicity were used as readout. Abacavir exposure can induce de novo HLA-B57 allorecognition by HIV-specific T cells. A HIV Gag RK9/HLA-A3-specific T cell did exhibit interferon-γ production, CD137 upregulation, and cytolytic effector function against allogeneic HLA-B57, but only in the presence of abacavir. Allorecognition was specific to the virus specificity, HLA restriction, and T cell receptor TRBV use of the T cell. We provide proof-of-principle evidence that administration of a drug could induce specific allorecognition of mismatched HLA molecules in the transplant setting. We suggest that HIV-seropositive recipients of an HLA-B57 mismatched graft should not receive abacavir until further studies are completed.
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: Elsevier BV
Date: 06-2017
DOI: 10.1016/J.CELLIMM.2017.03.004
Abstract: We hypothesized that HIV-specific CD8 T cell clonotypes can be stimulated by allogeneic HLA molecules. Multiple HIV-specific CD8 T cell clones were derived from 12 in iduals with chronic HIV infection, specific for 13 different HIV Gag antigens and restricted to 7 different HLA molecules. The generated T cell clones were assayed for alloreactivity against a panel of single HLA class I expressing cell lines (SALs). HIV-specific T cells recognising at least one allogeneic HLA molecule could be identified from 7 of 12 patients tested. Allorecognition was associated with IFNγ cytokine production, CD137 upregulation and cytotoxicity, suggesting high avidity allo-stimulation. Allo-HLA recognition by HIV-specific T cells was specific to the HIV target peptide/HLA restriction and TCR TRBV usage of the T cells. HIV-specific T cells do crossreact against allogeneic HLA molecules in an epitope and TRBV specific manner. Therefore allo-HLA stimulation could be exploited to induce or augment HIV-specific T cell responses.
Publisher: Elsevier BV
Date: 08-2010
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: American Society for Microbiology
Date: 04-2014
DOI: 10.1128/JVI.03136-13
Abstract: HIV undergoes high rates of mutation and recombination during reverse transcription, but it is not known whether these events occur independently or are linked mechanistically. Here we used a system of silent marker mutations in HIV and a single round of infection in primary T lymphocytes combined with a high-throughput sequencing and mathematical modeling approach to directly estimate the viral recombination and mutation rates. From million nucleotides (nt) of sequences from HIV infection, we observed 4,801 recombination events and 859 substitution mutations (≈1.51 and 0.12 events per 1,000 nt, respectively). We used experimental controls to account for PCR-induced and transfection-induced recombination and sequencing error. We found that the single-cycle virus-induced mutation rate is 4.6 × 10 −5 mutations per nt after correction. By sorting of our data into recombined and nonrecombined sequences, we found a significantly higher mutation rate in recombined regions ( P = 0.003 by Fisher's exact test). We used a permutation approach to eliminate a number of potential confounding factors and confirm that mutation occurs around the site of recombination and is not simply colocated in the genome. By comparing mutation rates in recombined and nonrecombined regions, we found that recombination-associated mutations account for 15 to 20% of all mutations occurring during reverse transcription.
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: Springer Science and Business Media LLC
Date: 09-04-2015
Publisher: Elsevier BV
Date: 02-2016
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: Elsevier BV
Date: 05-2009
Publisher: Frontiers Media SA
Date: 30-03-2023
DOI: 10.3389/FIMMU.2023.1153789
Abstract: Inclusion body myositis (IBM) is a progressive inflammatory myopathy characterised by skeletal muscle infiltration and myofibre invasion by CD8 + T lymphocytes. In some cases, IBM has been reported to be associated with a systemic lymphoproliferative disorder of CD8 + T cells exhibiting a highly differentiated effector phenotype known as T cell Large Granular Lymphocytic Leukemia (T-LGLL). We investigated the incidence of a CD8 + T-LGL lymphoproliferative disorder in 85 IBM patients and an aged-matched group of 56 Healthy Controls (HC). Further, we analysed the phenotypical characteristics of the expanded T-LGLs and investigated whether their occurrence was associated with any particular HLA alleles or clinical characteristics. Blood cell analysis by flow cytometry revealed expansion of T-LGLs in 34 of the 85 (40%) IBM patients. The T cell immunophenotype of T-LGL HIGH patients was characterised by increased expression of surface molecules including CD57 and KLRG1, and to a lesser extent of CD94 and CD56 predominantly in CD8 + T cells, although we also observed modest changes in CD4 + T cells and γδ T cells. Analysis of Ki67 in CD57 + KLRG1 + T cells revealed that only a small proportion of these cells was proliferating. Comparative analysis of CD8 + and CD4 + T cells isolated from matched blood and muscle s les donated by three patients indicated a consistent pattern of more pronounced alterations in muscles, although not significant due to small s le size. In the T-LGL HIGH patient group, we found increased frequencies of perforin-producing CD8 + and CD4 + T cells that were moderately correlated to combined CD57 and KLRG1 expression. Investigation of the HLA haplotypes of 75 IBM patients identified that carriage of the HLA-C*14:02:01 allele was significantly higher in T-LGL HIGH compared to T-LGL LOW in iduals. Expansion of T-LGL was not significantly associated with seropositivity patient status for anti-cytosolic 5'-nucleotidase 1A autoantibodies. Clinically, the age at disease onset and disease duration were similar in the T-LGL HIGH and T-LGL LOW patient groups. However, metadata analysis of functional alterations indicated that patients with expanded T-LGL more frequently relied on mobility aids than T-LGL LOW patients indicating greater disease severity. Altogether, these results suggest that T-LGL expansion occurring in IBM patients is correlated with exacerbated immune dysregulation and increased disease burden.
Publisher: Elsevier BV
Date: 02-0012
Publisher: Elsevier BV
Date: 05-2013
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: American Society for Microbiology
Date: 03-2014
DOI: 10.1128/JVI.03014-13
Abstract: HIV-1 infection is characterized by the rapid generation of genetic ersity that facilitates viral escape from immune selection and antiretroviral therapy. Despite recombination's crucial role in viral ersity and evolution, little is known about the genomic factors that influence recombination between highly similar genomes. In this study, we use a minimally modified full-length HIV-1 genome and high-throughput sequence analysis to study recombination in gag and pol in T cells. We find that recombination is favored at a number of recombination hot spots, where recombination occurs six times more frequently than at corresponding cold spots. Interestingly, these hot spots occur near important features of the HIV-1 genome but do not occur at sites immediately around protease inhibitor or reverse transcriptase inhibitor drug resistance mutations. We show that the recombination hot and cold spots are consistent across five blood donors and are independent of coreceptor-mediated entry. Finally, we check common experimental confounders and find that these are not driving the location of recombination hot spots. This is the first study to identify the location of recombination hot spots between two similar viral genomes with great statistical power and under conditions that closely reflect natural recombination events among HIV-1 quasispecies. IMPORTANCE The ability of HIV-1 to evade the immune system and antiretroviral therapy depends on genetic ersity within the viral quasispecies. Retroviral recombination is an important mechanism that helps to generate and maintain this genetic ersity, but little is known about how recombination rates vary within the HIV-1 genome. We measured recombination rates in gag and pol and identified recombination hot and cold spots, demonstrating that recombination is not random but depends on the underlying gene sequence. The strength and location of these recombination hot and cold spots can be used to improve models of viral dynamics and evolution, which will be useful for the design of robust antiretroviral therapies.
Publisher: Wiley
Date: 07-2019
DOI: 10.1002/JIA2.25327
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: SAGE Publications
Date: 10-2009
DOI: 10.3851/IMP1419
Abstract: All site-specific interactions between HIV type-1 (HIV-1) subtype, human leukocyte antigen (HLA)-associated immune selection and integrase inhibitor resistance are not completely understood. We examined naturally occurring polymorphisms in HIV-1-integrase sequences from 342 antiretroviral-naive in iduals from the Western Australian HIV Cohort Study and the Swiss HIV Cohort Study. Standard bulk sequencing and sequence-based typing were used to generate integrase sequences and high-resolution HLA genotypes, respectively. Viral residues were examined with respect to drug resistance mutations and CD8 + T-cell escape mutations. In both predominantly subtype B cohorts, 12 of 38 sites that mediate integrase inhibitor resistance mutations were absolutely conserved, and these included the primary resistance mutations. There were 18 codons with non-primary drug resistance-associated substitutions at rates of up to 58.8% and eight sites with alternative polymorphisms. Five viral residues were potentially subject to dual-drug and HLA-associated immune selection in which both selective pressures either drove the same amino acid substitution (codons 72, 157 and 163) or HLA alleles were associated with an alternative polymorphism that would alter the genetic barrier to resistance (codons 125 and 193). The common polymorphism T125A, which was characteristic of non-subtype B and was also associated with carriage of HLA-B*57/*5801, increased the mutational barrier to the resistance mutation T125K. Primary integrase inhibitor resistance mutations were not detected in the absence of drug exposure in keeping with sites of high constraint. Viral polymorphisms caused by immune selection and/or associated with non-subtype B might alter the genetic barrier to some non-primary resistance-associated mutations.
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 27-03-2013
Publisher: The American Association of Immunologists
Date: 09-2011
Abstract: Strong statistical associations between polymorphisms in HIV-1 population sequences and carriage of HLA class I alleles have been widely used to identify possible sites of CD8 T cell immune selection in vivo. However, there have been few attempts to prospectively and systematically test these genetic hypotheses arising from population-based studies at a cellular, functional level. We assayed CD8 T cell epitope-specific IFN-γ responses in 290 in iduals from the same cohort, which gave rise to 874 HLA–HIV associations in genetic analyses, taking into account autologous viral sequences and in idual HLA genotypes. We found immunological evidence for 58% of 374 associations tested as sites of primary immune selection and identified up to 50 novel HIV-1 epitopes using this reverse-genomics approach. Many HLA-adapted epitopes elicited equivalent or higher-magnitude IFN-γ responses than did the nonadapted epitopes, particularly in Nef. At a population level, inclusion of all of the immunoreactive variant CD8 T cell epitopes in Gag, Pol, Nef, and Env suggested that HIV adaptation leads to an inflation of Nef-directed immune responses relative to other proteins. We concluded that HLA–HIV associations mark viral epitopes subject to CD8 T cell selection. These results can be used to guide functional studies of specific epitopes and escape mutations, as well as to test, train, and evaluate analytical models of viral escape and fitness. The inflation of Nef and HLA-adapted variant responses may have negative effects on natural and vaccine immunity against HIV and, therefore, has implications for ersity coverage approaches in HIV vaccine design.
Publisher: American Society for Microbiology
Date: 05-2018
DOI: 10.1128/JVI.02066-17
Abstract: The hepatitis C virus (HCV) E2 glycoprotein is a major target of the neutralizing antibody (nAb) response, with multiple type-specific and broadly neutralizing antibody (bnAb) epitopes identified. The 412-to-423 region can generate bnAbs that block interaction with the cell surface receptor CD81, with activity toward multiple HCV genotypes. In this study, we reveal the structure of rodent monoclonal antibody 24 (MAb24) with an extensive contact area toward a peptide spanning the 412-to-423 region. The crystal structure of the MAb24–peptide 412-to-423 complex reveals the paratope bound to a peptide hairpin highly similar to that observed with human MAb HCV1 and rodent MAb AP33, but with a different angle of approach. In viral outgrowth experiments, we demonstrated three distinct genotype 2a viral populations that acquired resistance to MAb24 via N415D, N417S, and N415D/H386R mutations. Importantly, the MAb24-resistant viruses exhibited significant increases in sensitivity to the majority of bnAbs directed to epitopes within the 412-to-423 region and in additional antigenic determinants located within E2 and the E1E2 complex. This study suggests that modification of N415 causes a global change in glycoprotein structure that increases its vulnerability to neutralization by other antibodies. This finding suggests that in the context of an antibody response to viral infection, acquisition of escape mutations in the 412-to-423 region renders the virus more susceptible to neutralization by other specificities of nAbs, effectively reducing the immunological fitness of the virus. A vaccine for HCV that generates polyspecific humoral immunity with specificity for the 412-to-423 region and at least one other region of E2 is desirable. IMPORTANCE Understanding how antibodies neutralize hepatitis C virus (HCV) is essential for vaccine development. This study reveals for the first time that when HCV develops resistance to a major class of bnAbs targeting the 412-to-423 region of E2, this results in a concomitant increase in sensitivity to neutralization by a majority of other bnAb specificities. Vaccines for the prevention of HCV infection should therefore generate bnAbs directed toward the 412-to-423 region of E2 and additional bnAb epitopes within the viral glycoproteins.
Publisher: Frontiers Media SA
Date: 12-11-2020
Publisher: American Society for Microbiology
Date: 13-07-2022
DOI: 10.1128/JVI.00122-22
Abstract: The integration of human immunodeficiency virus type 1 (HIV-1) into chromosomal DNA establishes the long-term persistence of HIV-1 as proviruses despite effective antiretroviral therapy (ART). Characterizing proviruses is difficult because of their rarity in in iduals on long-term suppressive ART, their highly polymorphic sequences and genetic structures, and the need for efficient lification and sequencing of the provirus and its integration site.
Publisher: Public Library of Science (PLoS)
Date: 30-10-2017
DOI: 10.1371/JOURNAL.PPAT.1009214
Abstract: The precise role of CD4 T cell turnover in maintaining HIV persistence during antiretroviral therapy (ART) has not yet been well characterized. In resting CD4 T cell subpopulations from 24 HIV-infected ART-suppressed and 6 HIV-uninfected in iduals, we directly measured cellular turnover by heavy water labeling, HIV reservoir size by integrated HIV-DNA (intDNA) and cell-associated HIV-RNA (caRNA), and HIV reservoir clonality by proviral integration site sequencing. Compared to HIV-negatives, ART-suppressed in iduals had similar fractional replacement rates in all subpopulations, but lower absolute proliferation rates of all subpopulations other than effector memory (TEM) cells, and lower plasma IL-7 levels (p = 0.0004). Median CD4 T cell half-lives decreased with cell differentiation from naïve to TEM cells (3 years to 3 months, p .001). TEM had the fastest replacement rates, were most highly enriched for intDNA and caRNA, and contained the most clonal proviral expansion. Clonal proviruses detected in less mature subpopulations were more expanded in TEM, suggesting that they were maintained through cell differentiation. Earlier ART initiation was associated with lower levels of intDNA, caRNA and fractional replacement rates. In conclusion, circulating integrated HIV proviruses appear to be maintained both by slow turnover of immature CD4 subpopulations, and by clonal expansion as well as cell differentiation into effector cells with faster replacement rates.
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: Springer Science and Business Media LLC
Date: 08-1999
DOI: 10.1007/BF02924560
Publisher: MDPI AG
Date: 31-10-2023
DOI: 10.3390/V15112198
Publisher: The American Association of Immunologists
Date: 2023
DOI: 10.4049/IMMUNOHORIZONS.2200095
Abstract: Atopic dermatitis (AD) is a common chronic skin disease. Although generally thought to be a disease of T-cell dysregulation, recent studies have suggested that immune dysregulation of NK cells is also important. Killer cell Ig-like receptors (KIRs) are involved with NK cell regulation. The Pediatric Eczema Elective Registry is a U.S. nationwide longitudinal cohort with up to 10 y of follow-up in which 655 children had DNA available for full allelic KIR sequencing. Every 6 mo, AD activity was reported by Pediatric Eczema Elective Registry children. Using generalized estimating equations, we evaluated the association of KIR allelic variation in concert with known HLA binding ligands and whether the child reported AD in “remission” (no skin lesions and not using AD medication). KIR2DS4*001:01 (odds ratio 0.53, 95% CI [0.32, 0.88]) and KIR2DL4*001:02 (0.54, [0.33, 0.89]) in the presence of C*04:01 had the largest effect on decreasing the likelihood of AD remission. The haplotype KIR 2DL4*001:02 ∼ 2DS4*001:01 ∼ 3DL2*002:01 (0.77, [0.60, 0.99]) was also associated with a decreased likelihood of AD remission. Our findings add to the general body of evidence of a growing literature on the importance of NK cells with respect to the immunopathogenesis and natural history of AD.
Publisher: SAGE Publications
Date: 06-2014
DOI: 10.3851/IMP2821
Abstract: Direct-acting antivirals (DAAs) are predicted to transform hepatitis C therapy, yet little is known about the prevalence of naturally occurring resistance mutations in recently acquired HCV. This study aimed to determine the prevalence and frequency of drug resistance mutations in the viral quasispecies among HIV-positive and -negative in iduals with recent HCV. The NS3 protease, NS5A and NS5B polymerase genes were lified from 50 genotype 1a participants of the Australian Trial in Acute Hepatitis C. Amino acid variations at sites known to be associated with possible drug resistance were analysed by ultra-deep pyrosequencing. A total of 12% of in iduals harboured dominant resistance mutations, while 36% demonstrated non-dominant resistant variants below that detectable by bulk sequencing (that is, %) but above a threshold of 1%. Resistance variants ( %) were observed at most sites associated with DAA resistance from all classes, with the exception of sofosbuvir. Dominant resistant mutations were uncommonly observed in the setting of recent HCV. However, low-level mutations to all DAA classes were observed by deep sequencing at the majority of sites and in most in iduals. The significance of these variants and impact on future treatment options remains to be determined. Clinicaltrials.gov NCT00192569.
Publisher: American Society for Microbiology
Date: 21-07-2022
DOI: 10.1128/IAI.00004-22
Abstract: To evaluate potential effects of gastric inflammation on Helicobacter pylori ersification and evolution within the stomach, we experimentally infected Mongolian gerbils with an H. pylori strain in which Cag type IV secretion system (T4SS) activity is controlled by a TetR/ tetO system. Gerbils infected with H. pylori under conditions in which Cag T4SS activity was derepressed had significantly higher levels of gastric inflammation than gerbils infected under conditions with repressed Cag T4SS activity.
Publisher: Public Library of Science (PLoS)
Date: 24-04-2017
Publisher: American Society for Microbiology
Date: 12-2016
DOI: 10.1128/JVI.01701-16
Abstract: Rhinovirus (RV) species A and C are the most frequent cause of respiratory viral illness worldwide, and RV-C has been linked to more severe exacerbations of asthma in young children. Little is known about the immune responses to the different RV species, although studies comparing IgG1 antibody titers found impaired antibody responses to RV-C. Therefore, the aim of this study was to assess whether T-cell immunity to RV-C is similarly impaired. We measured T-cell proliferation to overlapping synthetic peptides covering the entire VP1 capsid protein of an RV-A and RV-C genotype for 20 healthy adult donors. Human leukocyte antigen (HLA) was typed in all the donors in order to investigate possible associations between the HLA type and RV peptide recognition. Total and specific IgG1 antibody titers to the VP1 proteins of both RV-A and RV-C were also measured to examine associations between the antibody and T-cell responses. We identified T-cell epitopes that are specific to and representative of each RV-A and RV-C species. These epitopes stimulated CD4 + -specific T-cell proliferation, with similar magnitudes of response for both RV species. All the donors, independent of their HLA-DR or -DQ type, were able to recognize the immunodominant RV-A and -C regions of VP1. Furthermore, the presence or absence of specific antibody titers was not related to changes in T-cell recognition. Our results indicate a dissociation between the antibody and T-cell responses to rhinoviruses. The species-representative T-cell epitopes identified in this study are valuable tools for future studies investigating T-cell responses to the different RV species. IMPORTANCE Rhinoviruses (RVs) are mostly associated with the common cold and asthma exacerbations, although their contributions to most upper and lower respiratory tract diseases have increasingly been reported. Species C (RV-C) has been associated with more frequent and severe asthma exacerbations in young children and, along with RV-A, is the most clinically relevant species. Little is known about how our immune system responds to rhinoviruses, and there are limited tools to study specific adaptive immunity against each rhinovirus species. In this study, we identified immunodominant T-cell epitopes of the VP1 proteins of RV-A and RV-C, which are representative of each species. The study found that T-cell responses to RV-A and RV-C were of similar magnitudes, in contrast with previous findings showing RV-C-specific antibody responses were low. These findings will provide the basis for future studies on the immune response to rhinoviruses and can help elucidate the mechanisms of severity of rhinovirus-induced infections.
Publisher: Wiley
Date: 08-2016
Publisher: Springer Science and Business Media LLC
Date: 27-03-2017
Publisher: Springer Science and Business Media LLC
Date: 27-07-2016
DOI: 10.1038/SREP30381
Abstract: We describe a method for determining the parental HLA haplotypes of a single in idual without recourse to conventional segregation genetics. Blood s les were cultured to identify and sort chromosome 6 by bivariate flow cytometry. Single chromosome 6 lification products were confirmed with a single nucleotide polymorphism (SNP) array and verified by deep sequencing to enable assignment of both alleles at the HLA loci, defining the two haplotypes. This study exemplifies a rapid and efficient method of haplotyping that can be applied to any chromosome pair, or indeed all chromosome pairs, using a single sorting operation. The method represents a cost-effective approach to complete phasing of SNPs, which will facilitate a deeper understanding of the links between SNPs, gene regulation and protein function.
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: The American Association of Immunologists
Date: 11-2017
Abstract: Select CMV epitopes drive life-long CD8+ T cell memory inflation, but the extent of CD4 memory inflation is poorly studied. CD4+ T cells specific for human CMV (HCMV) are elevated in HIV+ HCMV+ subjects. To determine whether HCMV epitope–specific CD4+ T cell memory inflation occurs during HIV infection, we used HLA-DR7 (DRB1*07:01) tetramers loaded with the glycoprotein B DYSNTHSTRYV (DYS) epitope to characterize circulating CD4+ T cells in coinfected HLA-DR7+ long-term nonprogressor HIV subjects with undetectable HCMV plasma viremia. DYS-specific CD4+ T cells were inflated among these HIV+ subjects compared with those from an HIV− HCMV+ HLA-DR7+ cohort or with HLA-DR7–restricted CD4+ T cells from the HIV-coinfected cohort that were specific for epitopes of HCMV phosphoprotein-65, tetanus toxoid precursor, EBV nuclear Ag 2, or HIV gag protein. Inflated DYS-specific CD4+ T cells consisted of effector memory or effector memory–RA+ subsets with restricted TCRβ usage and nearly monoclonal CDR3 containing novel conserved amino acids. Expression of this near-monoclonal TCR in a Jurkat cell–transfection system validated fine DYS specificity. Inflated cells were polyfunctional, not senescent, and displayed high ex vivo levels of granzyme B, CX3CR1, CD38, or HLA-DR but less often coexpressed CD38+ and HLA-DR+. The inflation mechanism did not involve apoptosis suppression, increased proliferation, or HIV gag cross-reactivity. Instead, the findings suggest that intermittent or chronic expression of epitopes, such as DYS, drive inflation of activated CD4+ T cells that home to endothelial cells and have the potential to mediate cytotoxicity and vascular disease.
Publisher: Elsevier BV
Date: 02-2021
Publisher: Elsevier BV
Date: 04-1997
Publisher: Oxford University Press (OUP)
Date: 28-09-2020
Abstract: Chronic norovirus infection in immunocompromised patients can be severe, and presently there is no effective treatment. Adoptive transfer of virus-specific T cells has proven to be safe and effective for the treatment of many viral infections, and this could represent a novel treatment approach for chronic norovirus infection. Hence, we sought to generate human norovirus-specific T cells (NSTs) that can recognize different viral sequences. Norovirus-specific T cells were generated from peripheral blood of healthy donors by stimulation with overlapping peptide libraries spanning the entire coding sequence of the norovirus genome. We successfully generated T cells targeting multiple norovirus antigens with a mean 4.2 ± 0.5-fold expansion after 10 days. Norovirus-specific T cells comprised both CD4+ and CD8+ T cells that expressed markers for central memory and effector memory phenotype with minimal expression of coinhibitory molecules, and they were polyfunctional based on cytokine production. We identified novel CD4- and CD8-restricted immunodominant epitopes within NS6 and VP1 antigens. Furthermore, NSTs showed a high degree of cross-reactivity to multiple variant epitopes from clinical isolates. Our findings identify immunodominant human norovirus T-cell epitopes and demonstrate that it is feasible to generate potent NSTs from third-party donors for use in antiviral immunotherapy.
Publisher: American Society for Microbiology
Date: 16-02-2023
DOI: 10.1128/IAI.00420-22
Abstract: Both Helicobacter pylori infection and a high-salt diet are risk factors for gastric cancer. We previously showed that a mutation in fur (encoding the ferric uptake regulator variant Fur-R88H) was positively selected in H. pylori strains isolated from experimentally infected Mongolian gerbils receiving a high-salt diet.
No related grants have been discovered for Abha Chopra.