Publications by authors named "Marios Koutsakos"

39 Publications

High expression of CD38 and MHC class II on CD8 T cells during severe influenza disease reflects bystander activation and trogocytosis.

Clin Transl Immunology 2021 8;10(9):e1336. Epub 2021 Sep 8.

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

Objectives: Although co-expression of CD38 and HLA-DR reflects T-cell activation during viral infections, high and prolonged CD38HLA-DR expression is associated with severe disease. To date, the mechanism underpinning expression of CD38HLA-DR is poorly understood.

Methods: We used mouse models of influenza A/H9N2, A/H7N9 and A/H3N2 infection to investigate mechanisms underpinning CD38MHC-II phenotype on CD8 T cells. To further understand MHC-II trogocytosis on murine CD8 T cells as well as the significance behind the scenario, we used adoptively transferred transgenic OT-I CD8 T cells and A/H3N2-SIINKEKL infection.

Results: Analysis of influenza-specific immunodominant DNP CD8 T-cell responses showed that CD38MHC-II co-expression was detected on both virus-specific and bystander CD8 T cells, with increased numbers of both CD38MHC-IICD8 T-cell populations observed in immune organs including the site of infection during severe viral challenge. OT-I cells adoptively transferred into MHC-II mice had no MHC-II after infection, suggesting that MHC-II was acquired via trogocytosis. The detection of CD19 on CD38MHC-II OT-I cells supports the proposition that MHC-II was acquired by trogocytosis sourced from B cells. Co-expression of CD38MHC-II on CD8 T cells was needed for optimal recall following secondary infection.

Conclusions: Overall, our study demonstrates that both virus-specific and bystander CD38MHC-II CD8 T cells are recruited to the site of infection during severe disease, and that MHC-II presence occurs via trogocytosis from antigen-presenting cells. Our findings highlight the importance of the CD38MHC-II phenotype for CD8 T-cell recall.
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http://dx.doi.org/10.1002/cti2.1336DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8426257PMC
September 2021

Potent priming by inactivated whole influenza virus particle vaccines is linked to viral RNA uptake into antigen presenting cells.

Vaccine 2021 06 2;39(29):3940-3951. Epub 2021 Jun 2.

Kino Consulting, Kumamoto, Japan.

Current detergent or ether-disrupted split vaccines (SVs) for influenza do not always induce adequate immune responses, especially in young children. This contrasts with the whole virus particle vaccines (WPVs) originally used against influenza that were immunogenic in both adults and children but were replaced by SV in the 1970s due to concerns with reactogenicity. In this study, we re-evaluated the immunogenicity of WPV and SV, prepared from the same batch of purified influenza virus, in cynomolgus macaques and confirmed that WPV is superior to SV in priming potency. In addition, we compared the ability of WPV and SV to induce innate immune responses, including the maturation of dendritic cells (DCs) in vitro. WPV stimulated greater production of inflammatory cytokines and type-I interferon in immune cells from mice and macaques compared to SV. Since these innate responses are likely triggered by the activation of pattern recognition receptors (PRRs) by viral RNA, the quantity and quality of viral RNA in each vaccine were assessed. Although the quantity of viral RNA was similar in the two vaccines, the amount of viral RNA of a length that can be recognized by PRRs was over 100-fold greater in WPV than in SV. More importantly, 1000-fold more viral RNA was delivered to DCs by WPV than by SV when exposed to preparations containing the same amount of HA protein. Furthermore, WPV induced up-regulation of the DC maturation marker CD86 on murine DCs, while SV did not. The present results suggest that the activation of antigen-presenting DCs, by PRR-recognizable viral RNA contained in WPV is responsible for the effective priming potency of WPV observed in naïve mice and macaques. WPV is thus recommended as an alternative option for seasonal influenza vaccines, especially for children.
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http://dx.doi.org/10.1016/j.vaccine.2021.05.065DOI Listing
June 2021

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 at the Peter Doherty Institute for Infection and Immunity, Melbourne, VIC 3000, Australia.

In-depth understanding of human T-cell-mediated immunity in coronavirus disease 2019 (COVID-19) is needed if we are to optimize vaccine strategies and immunotherapies. Identification of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) T-cell epitopes and generation of peptide-human leukocyte antigen (peptide-HLA) tetramers facilitate 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 IFNγ responses above background, S (QYIKWPWYI), S (NYNYLYRLF) and S (VFKNIDGYF), with S generating immunodominant CD8 IFNγ 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 prepandemic 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 prepandemic individuals. Naïve A24/SARS-CoV-2-specific CD8 T cells increased nearly 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, whereas the A24/S CD8 TCRαβ repertoire was diverse across COVID-19 patients. Our study provides an in depth characterization 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
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8242669PMC
June 2021

CD8 T cell landscape in Indigenous and non-Indigenous people restricted by influenza mortality-associated HLA-A*24:02 allomorph.

Nat Commun 2021 05 18;12(1):2931. Epub 2021 May 18.

Department of Biochemistry and Molecular Biology & Infection and Immunity Program, Biomedicine Discovery Institute, Monash University, Clayton, VIC, Australia.

Indigenous people worldwide are at high risk of developing severe influenza disease. HLA-A*24:02 allele, highly prevalent in Indigenous populations, is associated with influenza-induced mortality, although the basis for this association is unclear. Here, we define CD8 T-cell immune landscapes against influenza A (IAV) and B (IBV) viruses in HLA-A*24:02-expressing Indigenous and non-Indigenous individuals, human tissues, influenza-infected patients and HLA-A*24:02-transgenic mice. We identify immunodominant protective CD8 T-cell epitopes, one towards IAV and six towards IBV, with A24/PB2-specific CD8 T cells being cross-reactive between IAV and IBV. Memory CD8 T cells towards these specificities are present in blood (CD27CD45RA phenotype) and tissues (CD103CD69 phenotype) of healthy individuals, and effector CD27CD45RAPD-1CD38CD8 T cells in IAV/IBV patients. Our data show influenza-specific CD8 T-cell responses in Indigenous Australians, and advocate for T-cell-mediated vaccines that target and boost the breadth of IAV/IBV-specific CD8 T cells to protect high-risk HLA-A*24:02-expressing Indigenous and non-Indigenous populations from severe influenza disease.
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http://dx.doi.org/10.1038/s41467-021-23212-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8132304PMC
May 2021

Immune cellular networks underlying recovery from influenza virus infection in acute hospitalized patients.

Nat Commun 2021 05 11;12(1):2691. Epub 2021 May 11.

Department of Biochemistry and Genetics, La Trobe Institute For Molecular Science, La Trobe University, Bundoora, VIC, Australia.

How innate and adaptive immune responses work in concert to resolve influenza disease is yet to be fully investigated in one single study. Here, we utilize longitudinal samples from patients hospitalized with acute influenza to understand these immune responses. We report the dynamics of 18 important immune parameters, related to clinical, genetic and virological factors, in influenza patients across different severity levels. Influenza disease correlates with increases in IL-6/IL-8/MIP-1α/β cytokines and lower antibody responses. Robust activation of circulating T follicular helper cells correlates with peak antibody-secreting cells and influenza heamaglutinin-specific memory B-cell numbers, which phenotypically differs from vaccination-induced B-cell responses. Numbers of influenza-specific CD8 or CD4 T cells increase early in disease and retain an activated phenotype during patient recovery. We report the characterisation of immune cellular networks underlying recovery from influenza infection which are highly relevant to other infectious diseases.
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http://dx.doi.org/10.1038/s41467-021-23018-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8113517PMC
May 2021

Natural killer cell receptors regulate responses of HLA-E-restricted T cells.

Sci Immunol 2021 Apr;6(58)

Department of Microbiology and Immunology, University of Melbourne at the Peter Doherty Institute, Parkville, Victoria 3010, Australia.

Human cytomegalovirus (CMV) infection can stimulate robust human leukocyte antigen (HLA)-E-restricted CD8 T cell responses. These T cells recognize a peptide from UL40, which differs by as little as a single methyl group from self-peptides that also bind HLA-E, challenging their capacity to avoid self-reactivity. Unexpectedly, we showed that the UL40/HLA-E T cell receptor (TCR) repertoire included TCRs that had high affinities for HLA-E/self-peptide. However, paradoxically, lower cytokine responses were observed from UL40/HLA-E T cells bearing TCRs with high affinity for HLA-E. RNA sequencing and flow cytometric analysis revealed that these T cells were marked by the expression of inhibitory natural killer cell receptors (NKRs) KIR2DL1 and KIR2DL2/L3. On the other hand, UL40/HLA-E T cells bearing lower-affinity TCRs expressed the activating receptor NKG2C. Activation of T cells bearing higher-affinity TCRs was regulated by the interaction between KIR2D receptors and HLA-C. These findings identify a role for NKR signaling in regulating self/non-self discrimination by HLA-E-restricted T cells, allowing for antiviral responses while avoiding contemporaneous self-reactivity.
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http://dx.doi.org/10.1126/sciimmunol.abe9057DOI Listing
April 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

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 02;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 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

The rise and fall of bone marrow plasma cells after influenza vaccination.

Immunol Cell Biol 2021 Feb 9;99(2):130-132. Epub 2020 Nov 9.

Department of Pathology and Immunology, Washington University School of Medicine, St Louis, MO, USA.

A recent report by Davis et al. shows that following vaccination, B-cell activation results in the emergence of a population of antibody-secreting cells (plasmablasts - PBs) in blood and a population of plasma cells (PCs) in the bone marrow, which likely originate from the day 7 PBs. However, these newly arrived PCs do not become long-lived and their abundance decreases by 1 year post-vaccination.
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http://dx.doi.org/10.1111/imcb.12417DOI Listing
February 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/S CD8 and A2/Orf1ab CD8 populations indicated that A2/S CD8 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/S CD8 T cells from COVID-19 convalescents ex vivo showed that A2/S CD8 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/S CD8 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

Innate and adaptive immunity toward influenza B viruses.

Future Microbiol 2020 07 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

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

Monocyte apoptotic bodies are vehicles for influenza A virus propagation.

Commun Biol 2020 05 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

Study of MAIT Cell Activation in Viral Infections In Vivo.

Methods Mol Biol 2020 ;2098:261-281

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

MAIT cells are abundant, highly evolutionarily conserved innate-like lymphocytes expressing a semi-invariant T cell receptor (TCR), which recognizes microbially derived small intermediate molecules from the riboflavin biosynthetic pathway. However, in addition to their TCR-mediated functions they can also be activated in a TCR-independent manner via cytokines including IL-12, -15, -18, and type I interferon. Emerging data suggest that they are expanded and activated by a range of viral infections, and significantly that they can contribute to a protective anti-viral response. Here we describe methods used to investigate these anti-viral functions in vivo in murine models. To overcome the technical challenge that MAIT cells are rare in specific pathogen-free laboratory mice, we describe how pulmonary MAIT cells can be expanded using intranasal bacterial infection or a combination of synthetic MAIT cell antigen and TLR agonists. We also describe protocols for adoptive transfer of MAIT cells, methods for lung homogenization for plaque assays, and surface and intracellular cytokine staining to determine MAIT cell activation.
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http://dx.doi.org/10.1007/978-1-0716-0207-2_17DOI Listing
January 2021

Influenza A Virus-Infected Lung Epithelial Cell Co-Culture with Human Peripheral Blood Mononuclear Cells.

Methods Mol Biol 2020 ;2098:141-147

Respiratory Medicine Unit, Nuffield Department of Medicine Experimental Medicine, University of Oxford, Oxfordshire, UK.

Sensing of influenza A virus (IAV) infection by pattern recognition receptors can occur by either direct infection of lung epithelial cells or uptake of virus-infected cells by innate cells such as dendritic cells/monocytes. This triggers a series of downstream events including activation of the inflammasome, the production of cytokines, chemokines, and the upregulation of stress-induced ligands that can lead to the activation of innate cells. These cells include innate lymphocytes such as MAIT, NKT, NK, and γδ T cells. Here we describe a method used to allow activation of human innate lymphocytes in co-culture with an IAV-infected human lung epithelial cell line (A549) to measure ex vivo effector functions (TNF and IFNγ) in a mixed culture environment. We describe (1) infection of the human lung epithelial cell line, (2) co-culture with PBMC, and (3) measurement of activation using intracellular cytokine staining.
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http://dx.doi.org/10.1007/978-1-0716-0207-2_9DOI Listing
January 2021

Human γδ T-cell receptor repertoire is shaped by influenza viruses, age and tissue compartmentalisation.

Clin Transl Immunology 2019 23;8(9):e1079. Epub 2019 Sep 23.

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

Background: Although γδ T cells comprise up to 10% of human peripheral blood T cells, questions remain regarding their role in disease states and T-cell receptor (TCR) clonal expansions. We dissected anti-viral functions of human γδ T cells towards influenza viruses and defined influenza-reactive γδ TCRs in the context of γδ-TCRs across the human lifespan.

Methods: We performed Cr-killing assay and single-cell time-lapse live video microscopy to define mechanisms underlying γδ T-cell-mediated killing of influenza-infected targets. We assessed cytotoxic profiles of γδ T cells in influenza-infected patients and IFN-γ production towards influenza-infected lung epithelial cells. Using single-cell RT-PCR, we characterised paired TCRγδ clonotypes for influenza-reactive γδ T cells in comparison with TCRs from healthy neonates, adults, elderly donors and tissues.

Results: We provide the first visual evidence of γδ T-cell-mediated killing of influenza-infected targets and show distinct features to those reported for CD8 T cells. γδ T cells displayed poly-cytotoxic profiles in influenza-infected patients and produced IFN-γ towards influenza-infected cells. These IFN-γ-producing γδ T cells were skewed towards the γ9δ2 TCRs, particularly expressing the public GV9-TCRγ, capable of pairing with numerous TCR-δ chains, suggesting their significant role in γδ T-cell immunity. Neonatal γδ T cells displayed extensive non-overlapping TCRγδ repertoires, while adults had enriched γ9δ2-pairings with diverse CDR3γδ regions. Conversely, the elderly showed distinct γδ-pairings characterised by large clonal expansions, a profile also prominent in adult tissues.

Conclusion: Human TCRγδ repertoire is shaped by age, tissue compartmentalisation and the individual's history of infection, suggesting that these somewhat enigmatic γδ T cells indeed respond to antigen challenge.
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http://dx.doi.org/10.1002/cti2.1079DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6756999PMC
September 2019

Downregulation of MHC Class I Expression by Influenza A and B Viruses.

Front Immunol 2019 29;10:1158. Epub 2019 May 29.

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

Manipulation of the MHC-I presentation pathway, and thus limiting MHC-I cell surface expression, is used by many viruses to evade immune recognition. In particular, downregulation of MHC-I molecules at the cell surface can reduce the ability of CD8 T cells to recognize viral peptides presented by MHC-I molecules and thereby delay viral clearance by CD8 T cells. To date, MHC-I downregulation by influenza viruses has not been reported. Given that influenza virus infections are a global health concern and that CD8 T cells play an important role in promoting influenza virus clearance and recovery from influenza disease, we investigated whether influenza A and B viruses (IAV, IBV) downregulated MHC-I as a novel mechanism to evade cellular immunity. Here, we showed that infection of several cell types, including epithelial A549 cells, with a panel of IAV and IBV viruses downregulated the surface MHC-I expression on IAV/IBV-infected cells during the late stages of influenza virus infection . This observation was consistent across a panel of class I-reduced (C1R) cell lines expressing 14 different HLA-A or -B alleles and a panel of 721.221 cell lines expressing 11 HLA-C alleles. Interestingly, IBV infection caused more pronounced reduction in surface MHC-I expression compared to IAV. Importantly, the two viruses utilized two distinct mechanisms for MHC-I downregulation. Our data demonstrated that while IAV caused a global loss of MHC-I within influenza-infected cells, IBV infection resulted in the preferential loss of MHC-I molecules from the cell surface, consequent of delayed MHC-I trafficking to the cell surface, resulting from retaining MHC-I intracellularly during IBV infection. Overall, our study suggests that influenza viruses across both IAV and IBV subtypes have the potential to downregulate MHC-I surface expression levels. Our findings provide new insights into the host-pathogen interaction of influenza A and B viruses and inform the design of novel vaccine strategies against influenza viruses.
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http://dx.doi.org/10.3389/fimmu.2019.01158DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6548845PMC
July 2020

Human CD8 T cell cross-reactivity across influenza A, B and C viruses.

Nat Immunol 2019 05 18;20(5):613-625. Epub 2019 Feb 18.

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

Influenza A, B and C viruses (IAV, IBV and ICV, respectively) circulate globally and infect humans, with IAV and IBV causing the most severe disease. CD8 T cells confer cross-protection against IAV strains, however the responses of CD8 T cells to IBV and ICV are understudied. We investigated the breadth of CD8 T cell cross-recognition and provide evidence of CD8 T cell cross-reactivity across IAV, IBV and ICV. We identified immunodominant CD8 T cell epitopes from IBVs that were protective in mice and found memory CD8 T cells directed against universal and influenza-virus-type-specific epitopes in the blood and lungs of healthy humans. Lung-derived CD8 T cells displayed tissue-resident memory phenotypes. Notably, CD38Ki67CD8 effector T cells directed against novel epitopes were readily detected in IAV- or IBV-infected pediatric and adult subjects. Our study introduces a new paradigm whereby CD8 T cells confer unprecedented cross-reactivity across all influenza viruses, a key finding for the design of universal vaccines.
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http://dx.doi.org/10.1038/s41590-019-0320-6DOI Listing
May 2019

Cross-lineage protection by human antibodies binding the influenza B hemagglutinin.

Nat Commun 2019 01 18;10(1):324. Epub 2019 Jan 18.

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

Influenza B viruses (IBV) drive a significant proportion of influenza-related hospitalisations yet are understudied compared to influenza A. Current vaccines target the head of the viral hemagglutinin (HA) which undergoes rapid mutation, significantly reducing vaccine effectiveness. Improved vaccines to control IBV are needed. Here we developed novel IBV HA probes to interrogate humoral responses to IBV in humans. A significant proportion of IBV HA-specific B cells recognise both B/Victoria/2/87-like and B/Yamagata/16/88-like lineages in a distinct pattern of cross-reactivity. Monoclonal antibodies (mAbs) were reconstituted from IBV HA-specific B cells, including mAbs providing broad protection in murine models of lethal IBV infection. Protection was mediated by neutralising antibodies targeting the receptor binding domain, or via Fc-mediated functions of non-neutralising antibodies binding alternative epitopes including the IBV HA stem. This work defines antigenic cross-recognition between IBV lineages and provides guidance for the rational design of improved IBV vaccines for broad and durable protection.
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http://dx.doi.org/10.1038/s41467-018-08165-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6338745PMC
January 2019

Immune Responses to Avian Influenza Viruses.

J Immunol 2019 01;202(2):382-391

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

Avian influenza A viruses (IAVs) naturally infect different avian species, and aquatic birds are their natural reservoir. Sporadically, avian IAVs can be transmitted to humans, and some, such as H5N1 and H7N9 viruses, cause severe disease in humans. Antigenically novel avian influenza viruses that infect and cause disease in humans pose a potential pandemic threat if they are able to spread efficiently from person to person. The immune response of the host is crucial in determining disease pathogenesis and is the basis for the development of control strategies. In this review, we examine the innate and adaptive immune responses to avian influenza viruses and their role in disease and recovery. Furthermore, we discuss the progress in developing vaccines against avian IAVs and summarize obstacles in designing universal and pandemic influenza vaccines.
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http://dx.doi.org/10.4049/jimmunol.1801070DOI Listing
January 2019

With a Little Help from T Follicular Helper Friends: Humoral Immunity to Influenza Vaccination.

J Immunol 2019 01;202(2):360-367

Department of Microbiology and Immunology, University of Melbourne at The Peter Doherty Institute for Infection and Immunity, Parkville 3010, Victoria, Australia

Influenza remains a global and unpredictable threat. Annual vaccination against influenza A and B viruses promotes the induction of Abs and memory B cells, which can provide strain-specific protection against subsequent infections. The formation of effective memory B cell and Ab responses is highly dependent on the germinal center reaction, a well-orchestrated process involving B cells and a specialized CD4 T cell subset called T follicular helper (Tfh) cells. As Tfh cells predominantly reside within B cell follicles in secondary lymphoid organs, they are challenging to study in humans. Recent identification of a circulating counterpart of Tfh cells has allowed us to better understand the contribution of these circulating Tfh cells during human immune responses. In this article, we summarize the role of human Tfh cells during humoral immune responses and discuss the contribution of Tfh cells in promoting immunity to influenza viruses in healthy cohorts and high-risk groups.
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http://dx.doi.org/10.4049/jimmunol.1800986DOI Listing
January 2019

MAIT cells contribute to protection against lethal influenza infection in vivo.

Nat Commun 2018 11 9;9(1):4706. Epub 2018 Nov 9.

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

Mucosal associated invariant T (MAIT) cells are evolutionarily-conserved, innate-like lymphocytes which are abundant in human lungs and can contribute to protection against pulmonary bacterial infection. MAIT cells are also activated during human viral infections, yet it remains unknown whether MAIT cells play a significant protective or even detrimental role during viral infections in vivo. Using murine experimental challenge with two strains of influenza A virus, we show that MAIT cells accumulate and are activated early in infection, with upregulation of CD25, CD69 and Granzyme B, peaking at 5 days post-infection. Activation is modulated via cytokines independently of MR1. MAIT cell-deficient MR1 mice show enhanced weight loss and mortality to severe (H1N1) influenza. This is ameliorated by prior adoptive transfer of pulmonary MAIT cells in both immunocompetent and immunodeficient RAG2γC mice. Thus, MAIT cells contribute to protection during respiratory viral infections, and constitute a potential target for therapeutic manipulation.
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http://dx.doi.org/10.1038/s41467-018-07207-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6226485PMC
November 2018

Single-Cell Approach to Influenza-Specific CD8 T Cell Receptor Repertoires Across Different Age Groups, Tissues, and Following Influenza Virus Infection.

Front Immunol 2018 27;9:1453. Epub 2018 Jun 27.

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

CD8 T cells recognizing antigenic peptides derived from conserved internal viral proteins confer broad protection against distinct influenza viruses. As memory CD8 T cells change throughout the human lifetime and across tissue compartments, we investigated how T cell receptor (TCR) composition and diversity relate to memory CD8 T cells across anatomical sites and immunological phases of human life. We used peptide-HLA tetramer magnetic enrichment, single-cell multiplex RT-PCR for both the TCR-alpha (TCRα) and TCR-beta (TCRβ) chains, and new TCRdist and grouping of lymphocyte interactions by paratope hotspots (GLIPH) algorithms to compare TCRs directed against the most prominent human influenza epitope, HLA-A*02:01-M1 (A2M1). We dissected memory TCR repertoires directed toward A2M1 CD8 T cells within human tissues and compared them to human peripheral blood of young and elderly adults. Furthermore, we compared these memory CD8 T cell repertoires to A2M1 CD8 TCRs during acute influenza disease in patients hospitalized with avian A/H7N9 virus. Our study provides the first comparative analysis of paired antigen-specific TCR-α/β clonotypes across different tissues and peripheral blood across different age groups. We show that human A2M1 CD8 T cells can be readily detected in human lungs, spleens, and lymph nodes, and that tissue A2M1 TCRαβ repertoires reflect A2M1 TCRαβ clonotypes derived from peripheral blood in healthy adults and influenza-infected patients. A2M1 TCRαβ repertoires displayed distinct features only in elderly adults, with large private TCRαβ clonotypes replacing the prominent and public TRBV19/TRAV27 TCRs. Our study provides novel findings on influenza-specific TCRαβ repertoires within human tissues, raises the question of how we can prevent the loss of optimal TCRαβ signatures with aging, and provides important insights into the rational design of T cell-mediated vaccines and immunotherapies.
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http://dx.doi.org/10.3389/fimmu.2018.01453DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6030351PMC
June 2018
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