Publications by authors named "Effie Mouhtouris"

17 Publications

  • Page 1 of 1

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

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

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

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

The Role of Immunological and Clinical Biomarkers to Predict Clinical COVID-19 Severity and Response to Therapy-A Prospective Longitudinal Study.

Front Immunol 2021 17;12:646095. Epub 2021 Mar 17.

Centre for Antibiotic Allergy and Research, Department of Infectious Diseases, Austin Health, Heidelberg, VIC, Australia.

Background: The association of pro-inflammatory markers such as interleukin-6 (IL-6) and other biomarkers with severe coronavirus disease 2019 (COVID-19) is of increasing interest, however their kinetics, response to current COVID-related treatments, association with disease severity and comparison with other disease states associated with potential cytokine storm (CS) such as Staphylococcus aureus bacteraemia (SAB) are ill-defined.

Methods: A cohort of 55 hospitalized SARS-CoV-2 positive patients was prospectively recruited - blood sampling was performed at baseline, post-treatment and hospital discharge. Serum IL-6, C-reactive protein (CRP) and other laboratory investigations were compared between treatment groups and across timepoints. Acute serum IL-6 and CRP levels were then compared to those with suspected COVID-19 (SCOVID) and age and sex matched patients with SAB and patients hospitalized for any non-infectious condition (NIC).

Results: IL-6 was elevated at admission in the SARS-CoV-2 cohort but at lower levels compared to matched SAB patients. Median (IQR) IL-6 at admission was 73.89 pg/mL (30.9, 126.39) in SARS-CoV-2 compared to 92.76 pg/mL (21.75, 246.55) in SAB (p=0.017); 12.50 pg/mL (3.06, 35.77) in patients with NIC; and 95.51 pg/mL (52.17, 756.67) in SCOVID. Median IL-6 and CRP levels decreased between admission and discharge timepoints. This reduction was amplified in patients treated with remdesivir and/or dexamethasone. CRP and bedside vital signs were the strongest predictors of COVID-19 severity.

Conclusions: Knowledge of the kinetics of IL-6 did not offer enhanced predictive value for disease severity in COVID-19 over common investigations such as CRP and vital signs.
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http://dx.doi.org/10.3389/fimmu.2021.646095DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8009986PMC
April 2021

The Role of In Vivo and Ex Vivo Diagnostic Tools in Severe Delayed Immune-Mediated Adverse Antibiotic Drug Reactions.

J Allergy Clin Immunol Pract 2021 05 13;9(5):2010-2015.e4. Epub 2021 Jan 13.

Centre for Antibiotic Allergy and Research, Department of Infectious Diseases, Austin Health, Heidelberg, VIC, Australia; The National Centre for Infections in Cancer, Peter MacCallum Cancer Centre, Parkville, VIC, Australia; Department of Oncology, Peter MacCallum Cancer Centre, The University of Melbourne, Parkville, VIC, Australia; Department of Medicine (Austin Health), The University of Melbourne, Heidelberg, VIC, Australia.

Background: The use of in vivo and ex vivo diagnostic tools for delayed immune-mediated adverse drug reactions is currently ill defined.

Objective: To determine whether the combination of skin testing and/or IFN-γ enzyme-linked immunoSpot assay (ELISpot) can aid diagnosis of these allergy phenotypes.

Methods: Patients with antibiotic-associated severe delayed immune-mediated adverse drug reaction hypersensitivity, including Stevens-Johnson syndrome and toxic epidermal necrolysis, drug reaction with eosinophilia and systemic symptoms (DRESS), acute generalized exanthematous pustulosis, generalized bullous fixed drug eruption, and severe maculopapular exanthema, were prospectively recruited. In vivo testing was completed to the implicated drug(s), and ex vivo testing was performed with the patient's PBMCs stimulated with the relevant antibiotic concentrations for IFN-γ release ELISpot measurement.

Results: Eighty-one patients met the inclusion criteria, with DRESS (42; 51.9%) accounting for most cases. Among the 63 (78%) who had an ELISpot assay performed, 34 (54%) were positive to at least 1 implicated antibiotic (median spot-forming units/million cells, 99.5; interquartile range, 68-187), with glycopeptide being a strong predictor of positivity (adjusted odds ratio, 6.11; 95% CI, 1.74-21.42). In combination (in vivo and ex vivo), 51 (63%) of those tested were positive to an implicated antibiotic. For DRESS and severe maculopapular exanthema associated with penicillins and cephalosporins, this combination confirmed the culprit agent in 11 of the 12 cases and in 6 of 7 for DRESS associated with glycopeptides.

Conclusions: This study demonstrates that using in vivo in combination with ex vivo testing can enhance the diagnostic approach in these severe phenotypes by assisting with the identification of possible culprit antibiotics.
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http://dx.doi.org/10.1016/j.jaip.2020.12.052DOI Listing
May 2021

Cross-reactivity between vancomycin, teicoplanin, and telavancin in patients with HLA-A∗32:01-positive vancomycin-induced DRESS sharing an HLA class II haplotype.

J Allergy Clin Immunol 2021 Jan 19;147(1):403-405. Epub 2020 May 19.

Vanderbilt University Medical Center, Nashville, Tenn; Institute for Immunology and Infectious Diseases, Murdoch University, Perth, Australia. Electronic address:

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http://dx.doi.org/10.1016/j.jaci.2020.04.056DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7674263PMC
January 2021

Delayed hypersensitivity associated with amoxicillin-clavulanate.

Allergy 2020 10 26;75(10):2700-2702. Epub 2020 May 26.

Centre for Antibiotic Allergy and Research, Department of Infectious Diseases, Austin Health, Heidelberg, Vic, Australia.

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http://dx.doi.org/10.1111/all.14359DOI Listing
October 2020

Analysis of Skin-Resident Memory T Cells Following Drug Hypersensitivity Reactions.

J Invest Dermatol 2020 07 26;140(7):1442-1445.e4. Epub 2019 Dec 26.

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

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http://dx.doi.org/10.1016/j.jid.2019.11.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7369252PMC
July 2020

Safety of cephalosporins in penicillin class severe delayed hypersensitivity reactions.

J Allergy Clin Immunol Pract 2020 03 31;8(3):1142-1146.e4. Epub 2019 Oct 31.

Institute for Immunology and Infectious Diseases, Murdoch University, Murdoch, WA, Australia; Department of Infectious Diseases, Vanderbilt University, Nashville, Tenn. Electronic address:

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http://dx.doi.org/10.1016/j.jaip.2019.10.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7064395PMC
March 2020

Antigen-specific CD4 CD25 T cells induced by locally expressed ICOS-Ig: the role of Foxp3, Perforin, Granzyme B and IL-10 - an experimental study.

Transpl Int 2019 Nov 16;32(11):1203-1215. Epub 2019 Jul 16.

Department of Surgery, Austin Health, The University of Melbourne, Heidelberg, Vic., Australia.

We have previously reported that ICOS-Ig expressed locally by a PIEC xenograft induces a perigraft cellular accumulation of CD4 CD25 Foxp3 T cells and specific xenograft prolongation. In the present study we isolated and purified CD4 CD25 T cells from ICOS-Ig secreting PIEC grafts to examine their phenotype and mechanism of xenograft survival using knockout and mutant mice. CD4 CD25 T cells isolated from xenografts secreting ICOS-Ig were analysed by flow cytometry and gene expression by real-time PCR. Regulatory function was examined by suppression of xenogeneic or allogeneic primed CD4 T cells in vivo. Graft prolongation was shown to be dependent on a pre-existing Foxp3 Treg, IL-10, perforin and granzyme B. CD4 CD25 Foxp3 T cells isolated from xenografts secreting ICOS-Ig demonstrated a phenotype consistent with nTreg but with a higher expression of CD275 (ICOSL), expression of CD278 (ICOS) and MHC II and loss of CD73. Moreover, these cells were functional and specifically suppressed xenogeinic but not allogeneic primed T cells in vivo.
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http://dx.doi.org/10.1111/tri.13474DOI Listing
November 2019

An in vivo mouse model of intraosseous spinal cancer causing evolving paraplegia.

J Neurooncol 2013 Nov 18;115(2):189-96. Epub 2013 Aug 18.

Spinal Biology Research Laboratory, Department of Spinal Surgery, University of Melbourne Department of Surgery, Austin Health, 145 Studley Road, PO Box 5555, Heidelberg, VIC, 3084, Australia.

The spine is the commonest site of skeletal metastatic disease and uncontrolled growth of cancer in the spine will inevitably cause pain and neurologic compromise. Improved understanding of the pathobiology behind this devastating condition is urgently needed. For this reason, the aim of this study was to establish a clinically relevant, animal model of spinal cancer. A percutaneous orthotopic injection of human breast (MDA-MB-231) or human prostate (PC-3) cancer cells was administered into the upper lumbar spine of nude mice (n = 6). Animals were monitored twice daily for general welfare, gait asymmetry or disturbance, and hindlimb weakness. After sacrifice, plain radiographs, micro-CT imaging and histological analysis of the spines were performed on each mouse. All mice recovered fully from the inoculation procedure and displayed normal gait and behaviour patterns for at least 3 weeks post-inoculation. Subsequently, between 3 and 5 weeks post-inoculation, each mouse developed evolving paralysis in their hindlimbs over 48-72 h. All followed the same pattern of decline following onset of neurological dysfunction; from gait asymmetry and unilateral hindlimb weakness, to complete unilateral hindlimb paralysis and finally to complete bilateral hindlimb paralysis. Plain radiographs, micro-CT scanning and histological analysis confirmed local tumour growth and destruction of the spine in all six mice. An in vivo mouse model of human intraosseous spinal cancer has been established forming cancers that grow within the spine and cause epidural spinal cord compression, resulting in a reproducible, evolving neurological deficit and paralysis that closely resembles the human condition.
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http://dx.doi.org/10.1007/s11060-013-1226-zDOI Listing
November 2013

Studies on carbohydrate xenoantigens.

Methods Mol Biol 2012 ;885:47-56

Department of Surgery (Austin Health), University of Melbourne, Heidelberg, VIC, Australia.

Naturally occurring and elicited anti-carbohydrate antibodies play a major role in immune responses to xenografts. The original obstacles associated with the Gal antigen have been largely resolved by the generation of knockout pigs. In contrast, much less is known about the nature and role of non-Gal carbohydrate antigens and the antibodies recognizing these. These antibodies can be identified and characterized by enzyme-linked immunosorbent assay. Furthermore, the biological significance of the non-Gal antigen(s) can be determined by expression of the relevant glycosyltransferase(s) by transfection and analyzed by antibody and/or lectin binding.
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http://dx.doi.org/10.1007/978-1-61779-845-0_4DOI Listing
September 2012

Prolonged xenograft survival induced by inducible costimulator-Ig is associated with increased forkhead box P3(+) cells.

Transplantation 2011 May;91(10):1090-7

Department of Surgery, The University of Melbourne, Austin Health/Northern Health, Heidelberg, Victoria, Australia.

Background: Blockade of the inducible costimulator (ICOS) pathway has been shown to prolong allograft survival; however, its utility in xenotransplantation is unknown. We hypothesize that local expression of ICOS-Ig by the xenograft will suppress the T-cell response resulting in significant prolonged graft survival.

Methods: Pig iliac artery endothelial cells (PIEC) secreting ICOS-Ig were generated and examined for the following: (1) inhibition of allogeneic and xenogeneic proliferation of primed T cells in vitro and (2) prolongation of xenograft survival in vivo. Grafts were examined for Tregs by flow cytometry and cytokine levels determined by quantitative reverse-transcriptase polymerase chain reaction.

Results: Soluble ICOS-Ig markedly decreased allogeneic and xenogeneic primed T-cell proliferation in a dose-dependent manner. PIEC-ICOS-Ig grafts were significantly prolonged compared with wild-type grafts (median survival, 34 and 12 days, respectively) with 20% of PIEC-ICOS-Ig grafts surviving more than 170 days. Histological examination showed a perigraft cellular accumulation of Forkhead box P3 (Foxp3(+)) cells in the PIEC-ICOS-Ig grafts, these were also shown to be CD3(+)CD4(+)CD25(+). Survival of wild-type PIEC grafts in a recipient simultaneously transplanted with PIEC-ICOS-Ig were also prolonged, with a similar accumulation of Foxp3(+) cells at the periphery of the graft demonstrating ICOS-Ig induces systemic graft prolongation. However, this prolongation was specific for the priming xenograft. Intragraft cytokine analysis showed an increase in interleukin-10 levels, suggesting a potential role in induction/function of CD4(+)CD25(+)Foxp3(+) cells.

Conclusions: This study demonstrates prolonged xenograft survival by local expression of ICOS-Ig, we propose that the accumulation of CD4(+)CD25(+)Foxp3(+) cells at the periphery of the graft and secretion of interleukin-10 is responsible for this novel observation.
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http://dx.doi.org/10.1097/TP.0b013e31821774e0DOI Listing
May 2011

Antibody responses to glycolipid-borne carbohydrates require CD4+ T cells but not CD1 or NKT cells.

Immunol Cell Biol 2011 May 1;89(4):502-10. Epub 2011 Feb 1.

Department of Surgery, The University of Melbourne, Austin Health/Northern Health, Heidelberg, Victoria, Australia.

Naturally occurring anti-carbohydrate antibodies play a major role in both the innate and adaptive immune responses. To elicit an anti-carbohydrate immune response, glycoproteins can be processed to glycopeptides and presented by the classical antigen-presenting molecules, major histocompatibility complex (MHC) Class I and II. In contrast, much less is known about the mechanism(s) for anti-carbohydrate responses to glycolipids, although it is generally considered that the CD1 family of cell surface proteins presents glycolipids to T cells or natural killer T (NKT) cells. Using model carbohydrate systems (isogloboside 3 and B blood group antigen), we examined the anti-carbohydrate response on glycolipids using both antibody neutralisation and knockout mouse-based experiments. These studies showed that CD4(+) T cells were required to generate antibodies to the carbohydrates expressed on glycolipids, and unexpectedly, these antibody responses were CD1d and NKT cell independent. They also did not require peptide help. These data provide new insight into glycolipid antigen recognition by the immune system and indicate the existence of a previously unrecognised population of glycolipid antigen-specific, CD1-independent, CD4(+) T cells.
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http://dx.doi.org/10.1038/icb.2010.166DOI Listing
May 2011

Dendritic cells expressing soluble CTLA4Ig prolong antigen-specific skin graft survival.

Immunol Cell Biol 2010 Nov-Dec;88(8):846-50. Epub 2010 Apr 20.

Department of Nephrology, Austin Health, Heidelberg, Victoria, Australia.

Dendritic cells (DCs) and CTLA4Ig are important in regulating T-cell responses and therefore represent potential therapeutic tools in transplantation. In this study, CTLA4Ig was expressed in a C57BL/6 murine DC line (JAWS II) by lentiviral transduction and these cells were used to examine T-cell immunomodulatory effects in vitro and in vivo. A lower stimulation index to C57BL/6 was observed with splenocytes from BALB/c mice primed with JAWS II-CTLA4Ig compared with control JAWS II-green fluorescent protein (JAWS II-GFP). Mice primed with JAWS II-CTLA4Ig cells had significantly prolonged antigen-specific C57BL/6 skin graft survival compared with either JAWS II-GFP-primed or naïve mice (median 13, 11 and 11 days, respectively, P=0.0001). Furthermore, JAWS II-CTLA4Ig-primed mice that had been previously transplanted with skin grafts were re-transplanted with skin grafts 6 months later without immune manipulation. These mice demonstrated specific prolongation of second-set rejection responses, indicating systemic immune modulation induced by genetically modified DC. The mechanism was not due to expression of indoleamine 2,3-dioxygenase or induction of circulating regulatory T cells as assessed by flow cytometry of the peripheral blood lymphocytes. This potent effect demonstrated with skin grafts and second-set responses highlights the potential use of this strategy for transplantation more generally.
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http://dx.doi.org/10.1038/icb.2010.58DOI Listing
June 2011

Humans lack iGb3 due to the absence of functional iGb3-synthase: implications for NKT cell development and transplantation.

PLoS Biol 2008 Jul;6(7):e172

Department of Surgery, The University of Melbourne, Austin Health/Northern Health, Heidelberg, Victoria, Australia.

The glycosphingolipid isoglobotrihexosylceramide, or isogloboside 3 (iGb3), is believed to be critical for natural killer T (NKT) cell development and self-recognition in mice and humans. Furthermore, iGb3 may represent an important obstacle in xenotransplantation, in which this lipid represents the only other form of the major xenoepitope Galalpha(1,3)Gal. The role of iGb3 in NKT cell development is controversial, particularly with one study that suggested that NKT cell development is normal in mice that were rendered deficient for the enzyme iGb3 synthase (iGb3S). We demonstrate that spliced iGb3S mRNA was not detected after extensive analysis of human tissues, and furthermore, the iGb3S gene contains several mutations that render this product nonfunctional. We directly tested the potential functional activity of human iGb3S by expressing chimeric molecules containing the catalytic domain of human iGb3S. These hybrid molecules were unable to synthesize iGb3, due to at least one amino acid substitution. We also demonstrate that purified normal human anti-Gal immunoglobulin G can bind iGb3 lipid and mediate complement lysis of transfected human cells expressing iGb3. Collectively, our data suggest that iGb3S is not expressed in humans, and even if it were expressed, this enzyme would be inactive. Consequently, iGb3 is unlikely to represent a primary natural ligand for NKT cells in humans. Furthermore, the absence of iGb3 in humans implies that it is another source of foreign Galalpha(1,3)Gal xenoantigen, with obvious significance in the field of xenotransplantation.
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http://dx.doi.org/10.1371/journal.pbio.0060172DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2459210PMC
July 2008

Recognition of a carbohydrate xenoepitope by human NKRP1A (CD161).

Xenotransplantation 2006 Sep;13(5):440-6

Department of Surgery, The University of Melbourne, Austin Health/Northern Health, Heidelberg, Victoria, Australia.

Background: Many immunologically important interactions are mediated by leukocyte recognition of carbohydrates via cell surface receptors. Uncharacterized receptors on human natural killer (NK) cells interact with ligands containing the terminal Galalpha(1,3)Gal xenoepitope. The aim of this work was to isolate and characterize carbohydrate binding proteins from NK cells that bind alphaGal or other potential xenoepitopes, such as N-acetyllactosamine (NAcLac), created by the deletion of alpha1,3galactosyltransferase (GT) in animals.

Methods And Results: Initial analysis suggested the human C-type lectin NKRP1A bound to a pool of glycoconjugates, the majority of which contained the terminal Galalpha(1,3)Gal epitope. This was confirmed by high level binding of cells expressing NKRP1A to mouse laminin, which contains a large number of N-linked oligosaccharides with the Galalpha(1,3)Gal structure. The consequence of removing the terminal alphaGal was then investigated. Elevated NAcLac levels were observed on thymocytes from GT-/- mice. Exposing NAcLac on laminin, by alpha-galactosidase treatment, resulted in a significant increase in NKRP1A binding.

Conclusions: NKRPIA binds to the alphaGal epitope. Moreover, exposing NAcLac by removal of alphaGal resulted in an increase in binding. This may be relevant in the later phases of xenotransplant rejection if GT-/- pigs, like GT-/- mice, display increased NAcLac expression.
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http://dx.doi.org/10.1111/j.1399-3089.2006.00332.xDOI Listing
September 2006

Histidine 271 has a functional role in pig alpha-1,3galactosyltransferase enzyme activity.

Glycobiology 2002 Dec;12(12):793-802

John Connell Laboratory for Glycobiology, The Austin Research Institute, Austin and Repatriation Medical Centre, Studley Road, Heidelberg 3084, Australia.

Alpha(1,3)Galactosyltransferase (GT) is a Golgi-localized enzyme that catalyzes the transfer of a terminal galactose to N-acetyllactosamine to create Galalpha(1,3)Gal. This glycosyltransferase has been studied extensively because the Galalpha(1,3)Gal epitope is involved in hyperacute rejection of pig-to-human xenotransplants. The original crystal structure of bovine GT defines the amino acids forming the catalytic pocket; however, those directly involved in the interaction with the donor nucleotide sugars were not characterized. Comparison of amino acid sequences of GT from several species with the human A and B transferases suggest that His271 of pig GT may be critical for recognition of the donor substrate, UDP-Gal. Using pig GT as the representative member of the GT family, we show that replacement of His271 with Ala, Leu, or Gly caused complete loss of function, in contrast to replacement with Arg, another basic charged residue, which did not alter the ability of GT to produce Galalpha(1,3)Gal. Molecular modeling showed that His271 may interact directly with the Gal moiety of UDP-Gal, an interaction possibly retained by replacing His with Arg. However, replacing His271 with amino acids found in alpha(1,3)GalNAc transferases did not change the donor nucleotide specificity. Thus His271 is critical for enzymatic function of pig GT.
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http://dx.doi.org/10.1093/glycob/cwf092DOI Listing
December 2002