Publications by authors named "Louise C Rowntree"

13 Publications

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SARS-CoV-2-specific CD8 T-cell responses and TCR signatures in the context of a prominent HLA-A*24:02 allomorph.

Immunol Cell Biol 2021 Jun 4. Epub 2021 Jun 4.

Department of Microbiology and Immunology, University of Melbourne, Peter Doherty Institute for Infection and Immunity, Melbourne, 3000.

In-depth understanding of human T cell-mediated immunity in COVID-19 is needed if we are to optimize vaccine strategies and immunotherapies. Identification of SARS-CoV-2 T cell epitopes and generation of peptide-HLA tetramers facilitates direct ex vivo analyses of SARS-CoV-2-specific T cells and their T cell receptor (TCR) repertoires. We utilized a combination of peptide prediction and in vitro peptide stimulation to validate novel SARS-CoV-2 epitopes restricted by HLA-A*24:02, one of the most prominent HLA class I alleles, especially in Indigenous and Asian populations. Of the peptides screened, three spike-derived peptides generated CD8 IFN-γ responses above background, S (QYIKWPWYI), S (NYNYLYRLF) and S (VFKNIDGYF), with S generating immunodominant CD8 IFN-γ responses. Using peptide-HLA-I tetramers, we performed direct ex vivo tetramer enrichment for HLA-A*24:02-restricted CD8 T cells in COVID-19 patients and pre-pandemic controls. The precursor frequencies for HLA-A*24:02-restricted epitopes were within the range previously observed for other SARS-CoV-2 epitopes for both COVID-19 patients and pre-pandemic individuals. Naïve A24/SARS-CoV-2-specific CD8 T cells increased ~7.5-fold above the average precursor frequency during COVID-19, gaining effector and memory phenotypes. Ex vivo single-cell analyses of TCRαβ repertoires found that the A24/S CD8 T cell TCRαβ repertoire was driven by a common TCRβ chain motif, while the A24/S CD8 TCRαβ repertoire was diverse across COVID-19 patients. Our study provides an in depth characterisation and important insights into SARS-CoV-2-specific CD8 T cell responses associated with a prominent HLA-A*24:02 allomorph. This contributes to our knowledge on adaptive immune responses during primary COVID-19 and could be exploited in vaccine or immunotherapeutic approaches.
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http://dx.doi.org/10.1111/imcb.12482DOI Listing
June 2021

CD8 T cells specific for an immunodominant SARS-CoV-2 nucleocapsid epitope display high naive precursor frequency and TCR promiscuity.

Immunity 2021 05 15;54(5):1066-1082.e5. Epub 2021 Apr 15.

Department of Infectious Diseases, Austin Hospital, Heidelberg, VIC 3084, Australia; Department of Medicine and Radiology, The University of Melbourne, Parkville, VIC 3000, Australia; Data Analytics Research and Evaluation (DARE) Centre, Austin Health and The University of Melbourne, Heidelberg, VIC 3084, Australia.

To better understand primary and recall T cell responses during coronavirus disease 2019 (COVID-19), it is important to examine unmanipulated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific T cells. By using peptide-human leukocyte antigen (HLA) tetramers for direct ex vivo analysis, we characterized CD8 T cells specific for SARS-CoV-2 epitopes in COVID-19 patients and unexposed individuals. Unlike CD8 T cells directed toward subdominant epitopes (B7/N, A2/S, and A24/S) CD8 T cells specific for the immunodominant B7/N epitope were detected at high frequencies in pre-pandemic samples and at increased frequencies during acute COVID-19 and convalescence. SARS-CoV-2-specific CD8 T cells in pre-pandemic samples from children, adults, and elderly individuals predominantly displayed a naive phenotype, indicating a lack of previous cross-reactive exposures. T cell receptor (TCR) analyses revealed diverse TCRαβ repertoires and promiscuous αβ-TCR pairing within B7/NCD8 T cells. Our study demonstrates high naive precursor frequency and TCRαβ diversity within immunodominant B7/N-specific CD8 T cells and provides insight into SARS-CoV-2-specific T cell origins and subsequent responses.
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http://dx.doi.org/10.1016/j.immuni.2021.04.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8049468PMC
May 2021

Systems serology detects functionally distinct coronavirus antibody features in children and elderly.

Nat Commun 2021 04 1;12(1):2037. Epub 2021 Apr 1.

Department of Microbiology and Immunology, Peter Doherty Institute for Infection and Immunity, University of Melbourne, Melbourne, VIC, Australia.

The hallmarks of COVID-19 are higher pathogenicity and mortality in the elderly compared to children. Examining baseline SARS-CoV-2 cross-reactive immunological responses, induced by circulating human coronaviruses (hCoVs), is needed to understand such divergent clinical outcomes. Here we show analysis of coronavirus antibody responses of pre-pandemic healthy children (n = 89), adults (n = 98), elderly (n = 57), and COVID-19 patients (n = 50) by systems serology. Moderate levels of cross-reactive, but non-neutralizing, SARS-CoV-2 antibodies are detected in pre-pandemic healthy individuals. SARS-CoV-2 antigen-specific Fcγ receptor binding accurately distinguishes COVID-19 patients from healthy individuals, suggesting that SARS-CoV-2 infection induces qualitative changes to antibody Fc, enhancing Fcγ receptor engagement. Higher cross-reactive SARS-CoV-2 IgA and IgG are observed in healthy elderly, while healthy children display elevated SARS-CoV-2 IgM, suggesting that children have fewer hCoV exposures, resulting in less-experienced but more polyreactive humoral immunity. Age-dependent analysis of COVID-19 patients, confirms elevated class-switched antibodies in elderly, while children have stronger Fc responses which we demonstrate are functionally different. These insights will inform COVID-19 vaccination strategies, improved serological diagnostics and therapeutics.
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http://dx.doi.org/10.1038/s41467-021-22236-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8016934PMC
April 2021

Robust correlations across six SARS-CoV-2 serology assays detecting distinct antibody features.

Clin Transl Immunology 2021 28;10(3):e1258. Epub 2021 Feb 28.

Department of Microbiology and Immunology University of Melbourne, at the Peter Doherty Institute for Infection and Immunity Melbourne VIC Australia.

Objectives: As the world transitions into a new era of the COVID-19 pandemic in which vaccines become available, there is an increasing demand for rapid reliable serological testing to identify individuals with levels of immunity considered protective by infection or vaccination.

Methods: We used 34 SARS-CoV-2 samples to perform a rapid surrogate virus neutralisation test (sVNT), applicable to many laboratories as it circumvents the need for biosafety level-3 containment. We correlated results from the sVNT with five additional commonly used SARS-CoV-2 serology techniques: the microneutralisation test (MNT), in-house ELISAs, commercial Euroimmun- and Wantai-based ELISAs (RBD, spike and nucleoprotein; IgG, IgA and IgM), antigen-binding avidity, and high-throughput multiplex analyses to profile isotype, subclass and Fc effector binding potential. We correlated antibody levels with antibody-secreting cell (ASC) and circulatory T follicular helper (cTfh) cell numbers.

Results: Antibody data obtained with commercial ELISAs closely reflected results using in-house ELISAs against RBD and spike. A correlation matrix across ten measured ELISA parameters revealed positive correlations for all factors. The frequency of inhibition by rapid sVNT strongly correlated with spike-specific IgG and IgA titres detected by both commercial and in-house ELISAs, and MNT titres. Multiplex analyses revealed strongest correlations between IgG, IgG1, FcR and C1q specific to spike and RBD. Acute cTfh-type 1 cell numbers correlated with spike and RBD-specific IgG antibodies measured by ELISAs and sVNT.

Conclusion: Our comprehensive analyses provide important insights into SARS-CoV-2 humoral immunity across distinct serology assays and their applicability for specific research and/or diagnostic questions to assess SARS-CoV-2-specific humoral responses.
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http://dx.doi.org/10.1002/cti2.1258DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7916820PMC
February 2021

Integrated immune dynamics define correlates of COVID-19 severity and antibody responses.

Cell Rep Med 2021 Mar 5;2(3):100208. Epub 2021 Feb 5.

Department of Medicine, Central Clinical School, Monash University, Melbourne, VIC, Australia.

SARS-CoV-2 causes a spectrum of COVID-19 disease, the immunological basis of which remains ill defined. We analyzed 85 SARS-CoV-2-infected individuals at acute and/or convalescent time points, up to 102 days after symptom onset, quantifying 184 immunological parameters. Acute COVID-19 presented with high levels of IL-6, IL-18, and IL-10 and broad activation marked by the upregulation of CD38 on innate and adaptive lymphocytes and myeloid cells. Importantly, activated CXCR3cT1 cells in acute COVID-19 significantly correlate with and predict antibody levels and their avidity at convalescence as well as acute neutralization activity. Strikingly, intensive care unit (ICU) patients with severe COVID-19 display higher levels of soluble IL-6, IL-6R, and IL-18, and hyperactivation of innate, adaptive, and myeloid compartments than patients with moderate disease. Our analyses provide a comprehensive map of longitudinal immunological responses in COVID-19 patients and integrate key cellular pathways of complex immune networks underpinning severe COVID-19, providing important insights into potential biomarkers and immunotherapies.
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http://dx.doi.org/10.1016/j.xcrm.2021.100208DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7862905PMC
March 2021

A Dual-Antigen Enzyme-Linked Immunosorbent Assay Allows the Assessment of Severe Acute Respiratory Syndrome Coronavirus 2 Antibody Seroprevalence in a Low-Transmission Setting.

J Infect Dis 2021 01;223(1):10-14

Department of Immunology and Infectious Disease, John Curtin School of Medical Research, The Australian National University, Canberra, Australia.

Estimates of seroprevalence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies have been hampered by inadequate assay sensitivity and specificity. Using an enzyme-linked immunosorbent assay-based approach that combines data about immunoglobulin G responses to both the nucleocapsid and spike receptor binding domain antigens, we show that excellent sensitivity and specificity can be achieved. We used this assay to assess the frequency of virus-specific antibodies in a cohort of elective surgery patients in Australia and estimated seroprevalence in Australia to be 0.28% (95% Confidence Interval, 0-1.15%). These data confirm the low level of transmission of SARS-CoV-2 in Australia before July 2020 and validate the specificity of our assay.
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http://dx.doi.org/10.1093/infdis/jiaa623DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7665523PMC
January 2021

Suboptimal SARS-CoV-2-specific CD8 T cell response associated with the prominent HLA-A*02:01 phenotype.

Proc Natl Acad Sci U S A 2020 09 10;117(39):24384-24391. Epub 2020 Sep 10.

Department of Microbiology and Immunology, Peter Doherty Institute for Infection and Immunity, University of Melbourne, Melbourne, VIC 3000, Australia;

An improved understanding of human T cell-mediated immunity in COVID-19 is important for optimizing therapeutic and vaccine strategies. Experience with influenza shows that infection primes CD8 T cell memory to peptides presented by common HLA types like HLA-A2, which enhances recovery and diminishes clinical severity upon reinfection. Stimulating peripheral blood mononuclear cells from COVID-19 convalescent patients with overlapping peptides from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) led to the clonal expansion of SARS-CoV-2-specific CD8 and CD4 T cells in vitro, with CD4 T cells being robust. We identified two HLA-A*02:01-restricted SARS-CoV-2-specfic CD8 T cell epitopes, A2/S and A2/Orf1ab Using peptide-HLA tetramer enrichment, direct ex vivo assessment of A2/SCD8 and A2/Orf1abCD8 populations indicated that A2/SCD8 T cells were detected at comparable frequencies (∼1.3 × 10) in acute and convalescent HLA-A*02:01 patients. These frequencies were higher than those found in uninfected HLA-A*02:01 donors (∼2.5 × 10), but low when compared to frequencies for influenza-specific (A2/M1) and Epstein-Barr virus (EBV)-specific (A2/BMLF) (∼1.38 × 10) populations. Phenotyping A2/SCD8 T cells from COVID-19 convalescents ex vivo showed that A2/SCD8 T cells were predominantly negative for CD38, HLA-DR, PD-1, and CD71 activation markers, although the majority of total CD8 T cells expressed granzymes and/or perforin. Furthermore, the bias toward naïve, stem cell memory and central memory A2/SCD8 T cells rather than effector memory populations suggests that SARS-CoV-2 infection may be compromising CD8 T cell activation. Priming with appropriate vaccines may thus be beneficial for optimizing CD8 T cell immunity in COVID-19.
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http://dx.doi.org/10.1073/pnas.2015486117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7533701PMC
September 2020

A Shared TCR Bias toward an Immunogenic EBV Epitope Dominates in HLA-B*07:02-Expressing Individuals.

J Immunol 2020 09 19;205(6):1524-1534. Epub 2020 Aug 19.

Department of Medicine, Monash University, Central Clinical School, The Alfred Hospital, Melbourne, Victoria 3004, Australia;

EBV is one of the most common viruses found in humans and is prototypic of a persistent viral infection characterized by periods of latency. Across many HLA class I molecules, the latent-specific CD8 T cell response is focused on epitopes derived from the EBNA-3 protein family. In the case of HLA-B*07:02 restriction, a highly frequent class I allele, the T cell response is dominated by an epitope spanning residues 379-387 of EBNA-3 (RPPIFIRRL [EBV]). However, little is known about either the TCR repertoire specific for this epitope or the molecular basis for this observed immunodominance. The EBV CD8 T cell response was common among both EBV-seropositive HLA-B*07:02 healthy and immunocompromised individuals. Similar TCRs were identified in EBV-specific CD8 T cell repertoires across multiple HLA-B7 individuals, indicating a shared Ag-driven bias in TCR usage. In particular, TRBV4-1 and TRAV38 usage was observed in five out of six individuals studied. In this study, we report the crystal structure of a TRBV4-1 TCR-HLA-B*07:02/EBV complex, which provides a molecular basis for the observed TRBV4-1 bias. These findings enhance our understanding of the CD8 T cell response toward a common EBV determinant in HLA-B*07:02 individuals.
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http://dx.doi.org/10.4049/jimmunol.2000249DOI Listing
September 2020

Preferential HLA-B27 Allorecognition Displayed by Multiple Cross-Reactive Antiviral CD8 T Cell Receptors.

Front Immunol 2020 19;11:248. Epub 2020 Feb 19.

Respiratory Medicine Laboratory, Department of Medicine, Central Clinical School, Monash University, Melbourne, VIC, Australia.

T cells provide essential immunosurveillance to combat and eliminate infection from pathogens, yet these cells can also induce unwanted immune responses via T cell receptor (TCR) cross-reactivity, also known as heterologous immunity. Indeed, pathogen-induced TCR cross-reactivity has shown to be a common, robust, and functionally potent mechanism that can trigger a spectrum of human immunopathologies associated with either transplant rejection, drug allergy, and autoimmunity. Here, we report that several virus-specific CD8 T cells directed against peptides derived from chronic viruses (EBV, CMV, and HIV-1) presented by high frequency HLA-A and -B allomorphs differentially cross-react toward HLA-B27 allotypes in a highly focused and hierarchical manner. Given the commonality of cross-reactive T cells and their potential contribution to adverse outcomes in allogeneic transplants, our study demonstrates that multiple antiviral T cells recognizing the same HLA allomorph could pose an extra layer of complexity for organ matching.
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http://dx.doi.org/10.3389/fimmu.2020.00248DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7042382PMC
March 2021

Inability To Detect Cross-Reactive Memory T Cells Challenges the Frequency of Heterologous Immunity among Common Viruses.

J Immunol 2018 06 7;200(12):3993-4003. Epub 2018 May 7.

Department of Medicine, Monash University, Central Clinical School, The Alfred Hospital, Melbourne, Victoria 3004, Australia;

Human memory T cells that cross-react with epitopes from unrelated viruses can potentially modulate immune responses to subsequent infections by a phenomenon termed heterologous immunity. However, it is unclear whether similarities in structure rather than sequence underpin heterologous T cell cross-reactivity. In this study, we aimed to explore the mechanism of heterologous immunity involving immunodominant epitopes derived from common viruses restricted to high-frequency HLA allotypes (HLA-A*02:01, -B*07:02, and -B*08:01). We examined EBV-specific memory T cells for their ability to cross-react with CMV or influenza A virus-derived epitopes. Following T cell immunoassays to determine phenotype and function, complemented with biophysical and structural investigations of peptide/HLA complexes, we did not detect cross-reactivity of EBV-specific memory T cells toward either CMV or influenza A virus epitopes presented by any of the selected HLA allomorphs. Thus, despite the ubiquitous nature of these human viruses and the dominant immune response directed toward the selected epitopes, heterologous virus-specific T cell cross-reactivity was not detected. This suggests that either heterologous immunity is not as common as previously reported, or that it requires a very specific biological context to develop and be clinically relevant.
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http://dx.doi.org/10.4049/jimmunol.1800010DOI Listing
June 2018

Deciphering the clinical relevance of allo-human leukocyte antigen cross-reactivity in mediating alloimmunity following transplantation.

Curr Opin Organ Transplant 2016 Feb;21(1):29-39

aDepartment of Allergy, Immunology and Respiratory Medicine, The Alfred Hospital, Commercial Road, Melbourne bInfection and Immunity Program, Monash Biomedicine Discovery Institute and Department of Biochemistry and Molecular Biology, School of Biomedical Sciences, Monash University, Clayton cDepartment of Microbiology and Immunology, The University of Melbourne, The Peter Doherty Institute for Infection and Immunity, Parkville dAustralian Research Council Centre of Excellence for Advanced Molecular Imaging eDepartment of Medicine, Central Clinical School, Monash University, Clayton, VIC, Australia.

Purpose Of Review: Despite a growing awareness regarding the potential of cross-reactive virus-specific memory T cells to mediate alloimmunity, there has been limited clinical evaluation on allograft immunopathology. This review will explore published models of human T-cell cross-reactivity and discuss criteria required to drive this mechanism as a contributing cause of allograft dysfunction in transplantation.

Recent Findings: Published models of human allogeneic (allo)-human leukocyte antigen (HLA) cross-reactivity have enabled dissection of the cross-reactive T cell receptor/peptide/major histocompatibility complex (TCR/peptide/MHC) interaction. In many of the models, the cross-reactive T cells express a unique TCR, although the relevance of a public cross-reactive TCR repertoire has yet to be determined. Equally, allopeptide identity, a vital component driving cross-recognition, remains unknown in the majority of models thereby prompting further characterization utilizing novel technologies. Although clinical studies examining the presence and impact of specific cross-reactive virus-specific T cells have been minimally explored, the existing data suggest that there may be a marginal set of requirements that need to be satisfied before the potentially damaging effects of allo-HLA cross-reactivity can be realized.

Summary: Our understanding of allo-HLA cross-reactivity continues to evolve as improved technology and novel strategies allow us to better question the contribution of allo-HLA cross-reactivity in clinically relevant allograft dysfunction.
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http://dx.doi.org/10.1097/MOT.0000000000000264DOI Listing
February 2016

Recognition of distinct cross-reactive virus-specific CD8+ T cells reveals a unique TCR signature in a clinical setting.

J Immunol 2014 Jun 28;192(11):5039-49. Epub 2014 Apr 28.

Department of Medicine, Monash University, Central Clinical School, The Alfred Centre, Melbourne, Victoria 3004, Australia; Department of Allergy, Immunology and Respiratory Medicine, The Alfred Hospital, Melbourne, Victoria 3004, Australia;

Human CMV still remains problematic in immunocompromised patients, particularly after solid organ transplantation. CMV primary disease and reactivation greatly increase the risks associated with incidences of chronic allograft rejection and decreased survival in transplant recipients. But whether this is due to direct viral effects, indirect viral effects including cross-reactive antiviral T cell immunopathology, or a combination of both remains undetermined. In this article, we report the novel TCR signature of cross-reactive HLA-A*02:01 (A2) CMV (NLVPMVATV [NLV])-specific CD8(+) T cells recognizing a specific array of HLA-B27 alleles using technical advancements that combine both IFN-γ secretion and multiplex nested RT-PCR for determining paired CDR3α/β sequences from a single cell. This study represents the first evidence, to our knowledge, of the same A2-restricted cross-reactive NLV-specific TCR-α/β signature (TRAV3TRAJ31_TRBV12-4TRBJ1-1) in two genetically distinct individuals. Longitudinal posttransplant monitoring of a lung transplant recipient (A2, CMV seropositive) who received a HLA-B27 bilateral lung allograft showed a dynamic expansion of the cross-reactive NLV-specific TCR repertoire before CMV reactivation. After resolution of the active viral infection, the frequency of cross-reactive NLV-specific CD8(+) T cells reduced to previremia levels, thereby demonstrating immune modulation of the T cell repertoire due to antigenic pressure. The dynamic changes in TCR repertoire, at a time when CMV reactivation was subclinical, illustrates that prospective monitoring in susceptible patients can reveal nuances in immune profiles that may be clinically relevant.
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http://dx.doi.org/10.4049/jimmunol.1303147DOI Listing
June 2014