Publications by authors named "Katherine Kedzierska"

168 Publications

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

Antibody mediated activation of natural killer cells in malaria exposed pregnant women.

Sci Rep 2021 Feb 18;11(1):4130. Epub 2021 Feb 18.

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

Immune effector responses against Plasmodium falciparum include antibody-mediated activation of innate immune cells, which can induce Fc effector functions, including antibody-dependent cellular cytotoxicity, and the secretion of cytokines and chemokines. These effector functions are regulated by the composition of immunoglobulin G (IgG) Fc N-linked glycans. However, a role for antibody-mediated natural killer (NK) cells activation or Fc N-linked glycans in pregnant women with malaria has not yet been established. Herein, we studied the capacity of IgG antibodies from pregnant women, with placental malaria or non-placental malaria, to induce NK cell activation in response to placental malaria-associated antigens DBL2 and DBL3. Antibody-mediated NK cell activation was observed in pregnant women with malaria, but no differences were associated with susceptibility to placental malaria. Elevated anti-inflammatory glycosylation patterns of IgG antibodies were observed in pregnant women with or without malaria infection, which were not seen in healthy non-pregnant controls. This suggests that pregnancy-associated anti-inflammatory Fc N-linked glycans may dampen the antibody-mediated activation of NK cells in pregnant women with malaria infection. Overall, although anti-inflammatory glycans and antibody-dependent NK cell activation were detected in pregnant women with malaria, a definitive role for these antibody features in protecting against placental malaria remains to be proven.
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http://dx.doi.org/10.1038/s41598-021-83093-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7893158PMC
February 2021

Atypical B cells are part of an alternative lineage of B cells that participates in responses to vaccination and infection in humans.

Cell Rep 2021 Feb;34(6):108684

Department of Immunology and Infectious Disease, John Curtin School of Medical Research, The Australian National University, Canberra, ACT 2601, Australia. Electronic address:

The diversity of circulating human B cells is unknown. We use single-cell RNA sequencing (RNA-seq) to examine the diversity of both antigen-specific and total B cells in healthy subjects and malaria-exposed individuals. This reveals two B cell lineages: a classical lineage of activated and resting memory B cells and an alternative lineage, which includes previously described atypical B cells. Although atypical B cells have previously been associated with disease states, the alternative lineage is common in healthy controls, as well as malaria-exposed individuals. We further track Plasmodium-specific B cells after malaria vaccination in naive volunteers. We find that alternative lineage cells are primed after the initial immunization and respond to booster doses. However, alternative lineage cells develop an atypical phenotype with repeated boosts. The data highlight that atypical cells are part of a wider alternative lineage of B cells that are a normal component of healthy immune responses.
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http://dx.doi.org/10.1016/j.celrep.2020.108684DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7873835PMC
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 Microbiology and Immunology, University of Melbourne at the Peter Doherty Institute for Infection and Immunity, 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

Genetic Bias, Diversity Indices, Physiochemical Properties and CDR3 Motifs Divide Auto-Reactive from Allo-Reactive T-Cell Repertoires.

Int J Mol Sci 2021 Feb 5;22(4). Epub 2021 Feb 5.

The Australian Institute of Tropical Health and Medicine (AITHM), James Cook University, Cairns, QLD 4811, Australia.

The distinct properties of allo-reactive T-cell repertoires are not well understood. To investigate whether auto-reactive and allo-reactive T-cell repertoires encoded distinct properties, we used dextramer enumeration, enrichment, single-cell T-cell receptor (TCR) sequencing and multiparameter analysis. We found auto-reactive and allo-reactive T-cells differed in mean ex vivo frequency which was antigen dependent. Allo-reactive T-cells showed clear differences in TCR architecture, with enriched usage of specific T-cell receptor variable () genes and broader use of T-cell receptor variable joining () genes. Auto-reactive T-cell repertoires exhibited complementary determining regions three (CDR3) lengths using a Gaussian distribution whereas allo-reactive T-cell repertoires exhibited distorted patterns in CDR3 length. CDR3 loops from allo-reactive T-cells showed distinct physical-chemical properties, tending to encode loops that were more acidic in charge. Allo-reactive T-cell repertoires differed in diversity metrics, tending to show increased overall diversity and increased homogeneity between repertoires. Motif analysis of CDR3 loops showed allo-reactive T-cell repertoires differed in motif preference which included broader motif use. Collectively, these data conclude that allo-reactive T-cell repertoires are indeed different to auto-reactive repertoires and provide tangible metrics for further investigations and validation. Given that the antigens studied here are overexpressed on multiple cancers and that allo-reactive TCRs often show increased ligand affinity, this new TCR bank also has translational potential for adoptive cell therapy, soluble TCR-based therapy and rational TCR design.
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http://dx.doi.org/10.3390/ijms22041625DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7915266PMC
February 2021

TLR2-mediated activation of innate responses in the upper airways confers antiviral protection of the lungs.

JCI Insight 2021 Mar 8;6(5). Epub 2021 Mar 8.

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

The impact of respiratory virus infections on global health is felt not just during a pandemic, but endemic seasonal infections pose an equal and ongoing risk of severe disease. Moreover, vaccines and antiviral drugs are not always effective or available for many respiratory viruses. We investigated how induction of effective and appropriate antigen-independent innate immunity in the upper airways can prevent the spread of respiratory virus infection to the vulnerable lower airways. Activation of TLR2, when restricted to the nasal turbinates, resulted in prompt induction of innate immune-driven antiviral responses through action of cytokines, chemokines, and cellular activity in the upper but not the lower airways. We have defined how nasal epithelial cells and recruitment of macrophages work in concert and play pivotal roles to limit progression of influenza virus to the lungs and sustain protection for up to 7 days. These results reveal underlying mechanisms of how control of viral infection in the upper airways can occur and support the implementation of strategies that can activate TLR2 in nasal passages to provide rapid protection, especially for at-risk populations, against severe respiratory infection when vaccines and antiviral drugs are not always effective or available.
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http://dx.doi.org/10.1172/jci.insight.140267DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8021123PMC
March 2021

Influenza, but not SARS-CoV-2, infection induces a rapid interferon response that wanes with age and diminished tissue-resident memory CD8 T cells.

Clin Transl Immunology 2021 26;10(1):e1242. Epub 2021 Jan 26.

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

Older individuals exhibit a diminished ability to respond to and clear respiratory pathogens and, as such, experience a higher rate of lung infections with a higher mortality rate. It is unclear why respiratory pathogens impact older people disproportionately. Using human lung tissue from donors aged 22-68 years, we assessed how the immune cell landscape in lungs changes throughout life and investigated how these immune cells respond following exposure to influenza virus and SARS-CoV-2, two clinically relevant respiratory viruses. While the frequency of most immune cell subsets profiled in the human lung remained stable with age, memory CD8 T cells declined, with the tissue-resident memory (Trm) CD8 T-cell subset being most susceptible to age-associated attrition. Infection of lung tissue with influenza virus resulted in an age-associated attenuation in the antiviral immune response, with aged donors producing less type I interferon (IFN), GM-CSF and IFNγ, the latter correlated with a reduction of IFNγ-producing memory CD8 T cells. In contrast, irrespective of donor age, exposure of human lung cells to SARS-CoV-2, a pathogen for which all donors were immunologically naïve, did not trigger activation of local immune cells and did not result in the induction of an early IFN response. Our findings show that the attrition of tissue-bound pathogen-specific Trm in the lung that occurs with advanced age, or their absence in immunologically naïve individuals, results in a diminished early antiviral immune response which creates a window of opportunity for respiratory pathogens to gain a greater foothold.
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http://dx.doi.org/10.1002/cti2.1242DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7837404PMC
January 2021

FOXO1 constrains activation and regulates senescence in CD8 T cells.

Cell Rep 2021 Jan;34(4):108674

Division of Biological Sciences, Molecular Biology Section, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0377, USA; Department of Cellular and Molecular Medicine, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0377, USA. Electronic address:

Naive and memory T cells are maintained in a quiescent state, yet capable of rapid response and differentiation to antigen challenge via molecular mechanisms that are not fully understood. In naive cells, the deletion of Foxo1 following thymic development results in the increased expression of multiple AP-1 family members, rendering T cells less able to respond to antigenic challenge. Similarly, in the absence of FOXO1, post-infection memory T cells exhibit the characteristics of extended activation and senescence. Age-based analysis of human peripheral T cells reveals that levels of FOXO1 and its downstream target, TCF7, are inversely related to host age, whereas the opposite is found for AP-1 factors. These characteristics of aging also correlate with the formation of T cells manifesting features of cellular senescence. Our work illustrates a role for FOXO1 in the active maintenance of stem-like properties in T cells at the timescales of acute infection and organismal life span.
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http://dx.doi.org/10.1016/j.celrep.2020.108674DOI Listing
January 2021

Immune responses to SARS-CoV-2 in three children of parents with symptomatic COVID-19.

Nat Commun 2020 11 11;11(1):5703. Epub 2020 Nov 11.

Department of Paediatrics, The University of Melbourne, Melbourne, Victoria, Australia.

Compared to adults, children with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have predominantly mild or asymptomatic infections, but the underlying immunological differences remain unclear. Here, we describe clinical features, virology, longitudinal cellular, and cytokine immune profile, SARS-CoV-2-specific serology and salivary antibody responses in a family of two parents with PCR-confirmed symptomatic SARS-CoV-2 infection and their three children, who tested repeatedly SARS-CoV-2 PCR negative. Cellular immune profiles and cytokine responses of all children are similar to their parents at all timepoints. All family members have salivary anti-SARS-CoV-2 antibodies detected, predominantly IgA, that coincide with symptom resolution in 3 of 4 symptomatic members. Plasma from both parents and one child have IgG antibody against the S1 protein and virus-neutralizing activity detected. Using a systems serology approach, we demonstrate higher levels of SARS-CoV-2-specific antibody features of these family members compared to healthy controls. These data indicate that children can mount an immune response to SARS-CoV-2 without virological confirmation of infection, raising the possibility that immunity in children can prevent the establishment of SARS-CoV-2 infection. Relying on routine virological and serological testing may not identify exposed children, with implications for epidemiological and clinical studies across the life-span.
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http://dx.doi.org/10.1038/s41467-020-19545-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7658256PMC
November 2020

The Dynamics of the Ferret Immune Response During H7N9 Influenza Virus Infection.

Front Immunol 2020 24;11:559113. Epub 2020 Sep 24.

Commonwealth Scientific and Industrial Research Organisation Health and Biosecurity, Australian Centre for Disease Prevention, East Geelong, VIC, Australia.

As the recent outbreak of SARS-CoV-2 has highlighted, the threat of a pandemic event from zoonotic viruses, such as the deadly influenza A/H7N9 virus subtype, continues to be a major global health concern. H7N9 virus strains appear to exhibit greater disease severity in mammalian hosts compared to natural avian hosts, though the exact mechanisms underlying this are somewhat unclear. Knowledge of the H7N9 host-pathogen interactions have mainly been constrained to natural sporadic human infections. To elucidate the cellular immune mechanisms associated with disease severity and progression, we used a ferret model to closely resemble disease outcomes in humans following influenza virus infection. Intriguingly, we observed variable disease outcomes when ferrets were inoculated with the A/Anhui/1/2013 (H7N9) strain. We observed relatively reduced antigen-presenting cell activation in lymphoid tissues which may be correlative with increased disease severity. Additionally, depletions in CD8 T cells were not apparent in sick animals. This study provides further insight into the ways that lymphocytes maturate and traffic in response to H7N9 infection in the ferret model.
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http://dx.doi.org/10.3389/fimmu.2020.559113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7541917PMC
October 2020

Unresponsiveness to inhaled antigen is governed by conventional dendritic cells and overridden during infection by monocytes.

Sci Immunol 2020 10;5(52)

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

The nasal-associated lymphoid tissues (NALTs) are mucosal-associated lymphoid organs embedded in the submucosa of the nasal passage. NALTs represent a known site for the deposition of inhaled antigens, but little is known of the mechanisms involved in the induction of immunity within this lymphoid tissue. We find that during the steady state, conventional dendritic cells (cDCs) within the NALTs suppress T cell responses. These cDCs, which are also prevalent within human NALTs (tonsils/adenoids), express a unique transcriptional profile and inhibit T cell proliferation via contact-independent mechanisms that can be diminished by blocking the actions of reactive oxygen species and prostaglandin E Although the prevention of unrestrained immune activation to inhaled antigens appears to be the default function of NALT cDCs, inflammation after localized virus infection recruited monocyte-derived DCs (moDCs) to this region, which diluted out the suppressive DC pool, and permitted local T cell priming. Accommodating for inflammation-induced temporal changes in NALT DC composition and function, we developed an intranasal vaccine delivery system that coupled the recruitment of moDCs with the sustained release of antigen into the NALTs, and we were able to substantially improve T cell responses after intranasal immunization. Thus, homeostasis and immunity to inhaled antigens is tuned by inflammatory signals that regulate the balance between conventional and moDC populations within the NALTs.
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http://dx.doi.org/10.1126/sciimmunol.abb5439DOI Listing
October 2020

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

Innate and adaptive immunity toward influenza B viruses.

Future Microbiol 2020 Jul 19;15:1045-1058. Epub 2020 Aug 19.

Department of Microbiology & Immunology, University of Melbourne, at the Peter Doherty Institute for Infection & Immunity, Parkville, Victoria 3010, Australia.

Despite annual vaccination, influenza B viruses (IBV) cause significant disease with substantial health and socio-economic impacts. Novel vaccination strategies inducing broadly protective and long-lasting immunity across IBV lineages are needed. However, as immune responses toward IBV are largely understudied, host-virus interactions and protective immune mechanisms need to be defined to rationally design such vaccines. Here, we summarize recent advances in our understanding of immunological mechanisms underpinning protection from IBV. We discuss how innate antiviral host factors inhibit IBV replication and the ways by which IBV escapes such restriction. We review the specificity of broadly cross-reactive antibodies and universal T cells, and the mechanisms by which they mediate protection. We highlight important knowledge gaps needing to be addressed to design improved IBV vaccines.
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http://dx.doi.org/10.2217/fmb-2019-0340DOI Listing
July 2020

HLA-B*27:05 alters immunodominance hierarchy of universal influenza-specific CD8+ T cells.

PLoS Pathog 2020 08 4;16(8):e1008714. Epub 2020 Aug 4.

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

Seasonal influenza virus infections cause 290,000-650,000 deaths annually and severe morbidity in 3-5 million people. CD8+ T-cell responses towards virus-derived peptide/human leukocyte antigen (HLA) complexes provide the broadest cross-reactive immunity against human influenza viruses. Several universally-conserved CD8+ T-cell specificities that elicit prominent responses against human influenza A viruses (IAVs) have been identified. These include HLA-A*02:01-M158-66 (A2/M158), HLA-A*03:01-NP265-273, HLA-B*08:01-NP225-233, HLA-B*18:01-NP219-226, HLA-B*27:05-NP383-391 and HLA-B*57:01-NP199-207. The immunodominance hierarchies across these universal CD8+ T-cell epitopes were however unknown. Here, we probed immunodominance status of influenza-specific universal CD8+ T-cells in HLA-I heterozygote individuals expressing two or more universal HLAs for IAV. We found that while CD8+ T-cell responses directed towards A2/M158 were generally immunodominant, A2/M158+CD8+ T-cells were markedly diminished (subdominant) in HLA-A*02:01/B*27:05-expressing donors following ex vivo and in vitro analyses. A2/M158+CD8+ T-cells in non-HLA-B*27:05 individuals were immunodominant, contained optimal public TRBV19/TRAV27 TCRαβ clonotypes and displayed highly polyfunctional and proliferative capacity, while A2/M158+CD8+ T cells in HLA-B*27:05-expressing donors were subdominant, with largely distinct TCRαβ clonotypes and consequently markedly reduced avidity, proliferative and polyfunctional efficacy. Our data illustrate altered immunodominance patterns and immunodomination within human influenza-specific CD8+ T-cells. Accordingly, our work highlights the importance of understanding immunodominance hierarchies within individual donors across a spectrum of prominent virus-specific CD8+ T-cell specificities prior to designing T cell-directed vaccines and immunotherapies, for influenza and other infectious diseases.
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http://dx.doi.org/10.1371/journal.ppat.1008714DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7428290PMC
August 2020

Immune profiling of influenza-specific B- and T-cell responses in macaques using flow cytometry-based assays.

Immunol Cell Biol 2021 Jan 7;99(1):97-106. Epub 2020 Sep 7.

Department of Microbiology and Immunology, University of Melbourne, Peter Doherty Institute for Infection and Immunity, Parkville, VIC, 3010, Australia.

Influenza remains a significant global public health burden, despite substantial annual vaccination efforts against circulating virus strains. As a result, novel vaccine approaches are needed to generate long-lasting and universal broadly cross-reactive immunity against distinct influenza virus strains and subtypes. Several new vaccine candidates are currently under development and/or in clinical trials. The successful development of new vaccines requires testing in animal models, other than mice, which capture the complexity of the human immune system. Importantly, following vaccination or challenge, the assessment of adaptive immunity at the antigen-specific level is particularly informative. In this study, using peripheral blood mononuclear cells (PBMCs) from cynomolgus macaques, we describe detection methods and in-depth analyses of influenza virus-specific B cells by recombinant hemagglutinin probes and flow cytometry, as well as the detection of influenza virus-specific CD8 and CD4 T cells by stimulation with live influenza A virus and intracellular cytokine staining. We highlight the potential of these assays to be used with PBMCs from other macaque species, including rhesus macaques, pigtail macaques and African green monkeys. We also demonstrate the use of a human cytometric bead array kit in detecting inflammatory cytokines and chemokines from cynomolgus macaques to assess cytokine/chemokine milieu. Overall, the detection of influenza virus-specific B and T cells, together with inflammatory responses, as described in our study, provides useful insights for evaluating novel influenza vaccines. Our data deciphering immune responses toward influenza viruses can be also adapted to understanding immunity to other infections or vaccination approaches in macaque models.
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http://dx.doi.org/10.1111/imcb.12383DOI Listing
January 2021

Publisher Correction: Metabolic characteristics of CD8 T cell subsets in young and aged individuals are not predictive of functionality.

Nat Commun 2020 07 9;11(1):3517. Epub 2020 Jul 9.

Department of Biochemistry and Molecular Biology, Monash Biomedicine Discovery Institute, Monash University, Clayton, VIC, 3800, Australia.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41467-020-17441-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7347533PMC
July 2020

Glycolipid-peptide vaccination induces liver-resident memory CD8 T cells that protect against rodent malaria.

Sci Immunol 2020 06;5(48)

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

Liver resident-memory CD8 T cells (T cells) can kill liver-stage -infected cells and prevent malaria, but simple vaccines for generating this important immune population are lacking. Here, we report the development of a fully synthetic self-adjuvanting glycolipid-peptide conjugate vaccine designed to efficiently induce liver T cells. Upon cleavage in vivo, the glycolipid-peptide conjugate vaccine releases an MHC I-restricted peptide epitope (to stimulate -specific CD8 T cells) and an adjuvant component, the NKT cell agonist α-galactosylceramide (α-GalCer). A single dose of this vaccine in mice induced substantial numbers of intrahepatic malaria-specific CD8 T cells expressing canonical markers of liver T cells (CD69, CXCR6, and CD101), and these cells could be further increased in number upon vaccine boosting. We show that modifications to the peptide, such as addition of proteasomal-cleavage sequences or epitope-flanking sequences, or the use of alternative conjugation methods to link the peptide to the glycolipid improved liver T cell generation and led to the development of a vaccine able to induce sterile protection in C57BL/6 mice against sporozoite challenge after a single dose. Furthermore, this vaccine induced endogenous liver T cells that were long-lived (half-life of ~425 days) and were able to maintain >90% sterile protection to day 200. Our findings describe an ideal synthetic vaccine platform for generating large numbers of liver T cells for effective control of liver-stage malaria and, potentially, a variety of other hepatotropic infections.
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http://dx.doi.org/10.1126/sciimmunol.aaz8035DOI Listing
June 2020

Viral burden, inflammatory milieu and CD8 T-cell responses to influenza virus in a second-generation thiazolide (RM-5061) and oseltamivir combination therapy study.

Influenza Other Respir Viruses 2020 11 25;14(6):678-687. Epub 2020 Jun 25.

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

Background: Influenza viruses cause significant morbidity and mortality, especially in young children, elderly, pregnant women and individuals with co-morbidities. Patients with severe influenza disease are typically treated with one neuraminidase inhibitor, oseltamivir or zanamivir. These antivirals need to be taken early to be most effective and often lead to the emergence of drug resistance and/or decreased drug susceptibility. Combining oseltamivir with another antiviral with an alternative mode of action has the potential to improve clinical effectiveness and reduce drug resistance.

Methods: In this study, we utilized a host-targeting molecule RM-5061, a second-generation thiazolide, in combination with oseltamivir to determine whether these compounds could reduce viral burden and understand their effects on the immune response to influenza virus infection in mice, compared with either monotherapy or placebo.

Results: The combination of RM-5061 and OST administered for 5 days after influenza infection reduced viral burden at day 5 post-infection, when compared to placebo and RM-5061 monotherapy, but was not significantly different from oseltamivir monotherapy. The inflammatory cytokine milieu was also reduced in animals which received a combination therapy when compared to RM-5061 and placebo-treated animals. Antiviral treatment in all groups led to a reduction in CD8 T-cell responses in the BAL when compared to placebo.

Conclusions: To our knowledge, this is the first time a combination of a host-targeting compound, RM-5061, and neuraminidase inhibitor, OST, has been tested in vivo. This antiviral combination was safe in mice and led to reduced inflammatory responses following viral infection when compared to untreated animals.
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http://dx.doi.org/10.1111/irv.12776DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7578329PMC
November 2020

A serological assay to detect SARS-CoV-2 seroconversion in humans.

medRxiv 2020 Apr 16. Epub 2020 Apr 16.

Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY, USA.

SARS-Cov-2 (severe acute respiratory disease coronavirus 2), which causes Coronavirus Disease 2019 (COVID19) was first detected in China in late 2019 and has since then caused a global pandemic. While molecular assays to directly detect the viral genetic material are available for the diagnosis of acute infection, we currently lack serological assays suitable to specifically detect SARS-CoV-2 antibodies. Here we describe serological enzyme-linked immunosorbent assays (ELISA) that we developed using recombinant antigens derived from the spike protein of SARS-CoV-2. Using negative control samples representing pre-COVID 19 background immunity in the general adult population as well as samples from COVID19 patients, we demonstrate that these assays are sensitive and specific, allowing for screening and identification of COVID19 seroconverters using human plasma/serum as early as two days post COVID19 symptoms onset. Importantly, these assays do not require handling of infectious virus, can be adjusted to detect different antibody types and are amendable to scaling. Such serological assays are of critical importance to determine seroprevalence in a given population, define previous exposure and identify highly reactive human donors for the generation of convalescent serum as therapeutic. Sensitive and specific identification of coronavirus SARS-Cov-2 antibody titers may, in the future, also support screening of health care workers to identify those who are already immune and can be deployed to care for infected patients minimizing the risk of viral spread to colleagues and other patients.
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http://dx.doi.org/10.1101/2020.03.17.20037713DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7239062PMC
April 2020

Metabolic characteristics of CD8 T cell subsets in young and aged individuals are not predictive of functionality.

Nat Commun 2020 06 5;11(1):2857. Epub 2020 Jun 5.

Department of Biochemistry and Molecular Biology, Monash Biomedicine Discovery Institute, Monash University, Clayton, VIC, 3800, Australia.

Virtual memory T (T) cells are antigen-naïve CD8 T cells that exist in a semi-differentiated state and exhibit marked proliferative dysfunction in advanced age. High spare respiratory capacity (SRC) has been proposed as a defining metabolic characteristic of antigen-experienced memory T (T) cells, facilitating rapid functionality and survival. Given the semi-differentiated state of T cells and their altered functionality with age, here we investigate T cell metabolism and its association with longevity and functionality. Elevated SRC is a feature of T, but not T, cells and it increases with age in both subsets. The elevated SRC observed in aged mouse T cells and human CD8 T cells from older individuals is associated with a heightened sensitivity to IL-15. We conclude that elevated SRC is a feature of T, but not T, cells, is driven by physiological levels of IL-15, and is not indicative of enhanced functionality in CD8 T cells.
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http://dx.doi.org/10.1038/s41467-020-16633-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7275080PMC
June 2020

Adoptive cellular therapy with T cells expressing the dendritic cell growth factor Flt3L drives epitope spreading and antitumor immunity.

Nat Immunol 2020 08 18;21(8):914-926. Epub 2020 May 18.

Cancer Immunology Program, Peter MacCallum Cancer Centre, Melbourne, Victoria, Australia.

Adoptive cell therapies using genetically engineered T cell receptor or chimeric antigen receptor T cells are emerging forms of immunotherapy that redirect T cells to specifically target cancer. However, tumor antigen heterogeneity remains a key challenge limiting their efficacy against solid cancers. Here, we engineered T cells to secrete the dendritic cell (DC) growth factor Fms-like tyrosine kinase 3 ligand (Flt3L). Flt3L-secreting T cells expanded intratumoral conventional type 1 DCs and substantially increased host DC and T cell activation when combined with immune agonists poly (I:C) and anti-4-1BB. Importantly, combination therapy led to enhanced inhibition of tumor growth and the induction of epitope spreading towards antigens beyond those recognized by adoptively transferred T cells in solid tumor models of T cell receptor and chimeric antigen receptor T cell therapy. Our data suggest that augmenting endogenous DCs is a promising strategy to overcome the clinical problem of antigen-negative tumor escape following adoptive cell therapy.
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http://dx.doi.org/10.1038/s41590-020-0676-7DOI Listing
August 2020

A serological assay to detect SARS-CoV-2 seroconversion in humans.

Nat Med 2020 07 12;26(7):1033-1036. Epub 2020 May 12.

Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY, USA.

Here, we describe a serological enzyme-linked immunosorbent assay for the screening and identification of human SARS-CoV-2 seroconverters. This assay does not require the handling of infectious virus, can be adjusted to detect different antibody types in serum and plasma and is amenable to scaling. Serological assays are of critical importance to help define previous exposure to SARS-CoV-2 in populations, identify highly reactive human donors for convalescent plasma therapy and investigate correlates of protection.
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http://dx.doi.org/10.1038/s41591-020-0913-5DOI Listing
July 2020

Monocyte apoptotic bodies are vehicles for influenza A virus propagation.

Commun Biol 2020 May 8;3(1):223. Epub 2020 May 8.

Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, VIC, 3086, Australia.

The disassembly of apoptotic cells into small membrane-bound vesicles termed apoptotic bodies (ApoBDs) is a hallmark of apoptosis; however, the functional significance of this process is not well defined. We recently discovered a new membrane protrusion (termed beaded apoptopodia) generated by apoptotic monocytes which fragments to release an abundance of ApoBDs. To investigate the function of apoptotic monocyte disassembly, we used influenza A virus (IAV) infection as a proof-of-concept model, as IAV commonly infects monocytes in physiological settings. We show that ApoBDs generated from IAV-infected monocytes contained IAV mRNA, protein and virions and consequently, could facilitate viral propagation in vitro and in vivo, and induce a robust antiviral immune response. We also identified an antipsychotic, Haloperidol, as an unexpected inhibitor of monocyte cell disassembly which could impair ApoBD-mediated viral propagation under in vitro conditions. Together, this study reveals a previously unrecognised function of apoptotic monocyte disassembly in the pathogenesis of IAV infections.
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http://dx.doi.org/10.1038/s42003-020-0955-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7210108PMC
May 2020

The ABC of Major Histocompatibility Complexes and T Cell Receptors in Health and Disease.

Viral Immunol 2020 04;33(3):160-178

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

A seminal discovery of major histocompatibility complex (MHC) restriction in T cell recognition by Peter Doherty and Rolf Zinkernagel has led to 45 years of exciting research on the mechanisms governing peptide MHC (pMHC) recognition by T cell receptors (TCRs) and their importance in health and disease. T cells provide a significant level of protection against viral, bacterial, and parasitic infections, as well as tumors, hence, the generation of protective T cell responses is a primary goal for cell-mediated vaccines and immunotherapies. Understanding the mechanisms underlying generation of optimal high-avidity effector T cell responses, memory development, maintenance, and recall is of major importance for the rational design of preventative and therapeutic vaccines/immunotherapies. In this review, we summarize the lessons learned over the last four decades and outline our current understanding of the basis and consequences of pMHC/TCR interactions on T cell development and function, and TCR diversity and composition, driving better clinical outcomes and prevention of viral escape. We also discuss the current models of T cell memory formation and determinants of immunodominant T cell responses in animal models and humans. As TCR composition and diversity can affect both the protective capacity of T cells and protection against viral escape, defining the spectrum of TCR selection has implications for improving the functional efficacy of effector T cell responsiveness and memory formation.
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http://dx.doi.org/10.1089/vim.2019.0184DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7185345PMC
April 2020

Multiplex Screening Assay for Identifying Cytotoxic CD8 T Cell Epitopes.

Front Immunol 2020 11;11:400. Epub 2020 Mar 11.

School of Public Health, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China.

The cytotoxicity of epitope-specific CD8 T cells is usually measured indirectly through IFNγ production. Existing assays that directly measure this activity are limited mainly to measurements of up to two specificities in a single reaction. Here, we develop a multiplex cytotoxicity assay that allows direct, simultaneous measurement of up to 23 different specificities of CD8 T cells in a single reaction. This can greatly reduce the amount of starting clinical materials for a systematic screening of CD8 T cell epitopes. In addition, this greatly enhanced capacity enables the incorporation of irrelevant epitopes for determining the non-specific killing activity of CD8 T cells, thereby allowing to measure the actual epitope-specific cytotoxicity activities. This technique is shown to be useful to study both human and mouse CD8 T cells. Besides, our results from human PBMCs and three independent infectious animal models (MERS, influenza and malaria) further reveal that IFNγ expression by epitope-specific CD8 T cells does not always correlate with their cell-killing potential, highlighting the need for using cytotoxicity assays in specific contexts (e.g., evaluating vaccine candidates). Overall, our approach opens up new possibilities for comprehensive analyses of CD8 T cell cytotoxicity in a practical manner.
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http://dx.doi.org/10.3389/fimmu.2020.00400DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7078160PMC
March 2021