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1.
Organoids ; 1(1):2-27, 2022.
Article in English | MDPI | ID: covidwho-1715586

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19), which was classified as a pandemic in March 2020. As of 22 January 2022, globally more than 347 million cases of COVID-19 have been diagnosed, with 5.6 million deaths, making it the deadliest pandemic since the influenza pandemic in 1918. The clinical presentation of COVID-19-related illness spans from asymptomatic to mild respiratory symptoms akin to influenza infection to acute symptoms, including pneumonia necessitating hospitalisation and admission to intensive care units. COVID-19 starts in the upper respiratory tract and lungs but in severe cases can also involve the heart, blood vessels, brain, liver, kidneys and intestine. The increasing global health and economic burden of COVID-19 necessitates an urgent and global response. Understanding the functional characteristics and cellular tropism of SARS-CoV-2, and the pathogenesis that leads to multi-organ failure and death, has prompted an unprecedented adoption of organoid models. Successful drug discovery and vaccine development rely on pre-clinical models that faithfully recapitulate the viral life cycle and the host cell response to infection. Human stem cell-derived organoids fulfill these criteria. Here we highlight the role of organoids in the study of SARS-CoV-2 infection and modelling of COVID-19 pathogenesis.

2.
EuropePMC; 2020.
Preprint in English | EuropePMC | ID: ppcovidwho-317943

ABSTRACT

T-cell responses to SARS-CoV-2-derived peptide pools have been documented, however it remains largely unclear whether prominent SARS-CoV-2-specific T cell populations originate from naïve or pre-existing memory sets. As HLA-B*07:02-restricted N105-113 epitope (B7/N105) is the most dominant SARS-CoV-2 CD8+ T-cell specificity to date, we dissected CD8+ T-cell responses directed at this epitope by direct ex vivo analyses in peripheral blood mononuclear cells (PBMCs) from acute and convalescent COVID-19 patients, and pre-pandemic PBMCs, tonsils, lungs and spleens. Using peptide-HLA tetramers, immunodominant B7/N105+CD8+ T-cells were detected at a high frequency (∼2.18x10-4) in COVID-19 patients, comparable to the well-established influenza-specific A2/M158+CD8+ T-cell population. Remarkably, frequencies of B7/N105+CD8+ T-cells were also readily detectable in pre-pandemic PBMCs and tonsils (at 6.55x10-5 and 2.76x10-4, respectively), although they mainly displayed a naïve phenotype, indicating a lack of previous cross-reactive exposures. Ex vivo TCRαβ analyses revealed that a diverse TCRαβ repertoire and promiscuity in αβ TCR pairing underlie such high naïve precursor frequencies of B7/N105+CD8+ T-cells. Overall, our study demonstrates that high precursor frequency and plasticity of TCRα-TCRβ pairing underpin immunodominance of SARS-CoV-2-specific B7/N105+CD8+ T-cell responses and advocates for vaccine strategies which include the nucleocapsid protein to elicit immunodominant CD8+ T-cell responses in HLA-B*07:02 individuals.Funding: This work was supported by theClifford Craig Foundation to KLF and KK, NHMRC Leadership Investigator Grant to KK (1173871), NHMRC Program Grant to DLD (#1132975), Research Grants Council of theHong Kong Special Administrative Region, China (#T11-712/19-N) to KK, the Victorian Government (SJK, AKW), MRFF award (#2002073) to SJK and AKW, MRFFAward (#1202445) to KK, NHMRC program grant 1149990 (SJK) and NHMRC project grant 1162760 (AKW). AKW is supported by Emerging Leadership 1 Investigator Grant (#1173433), JAJ by an NHMRC Early Career Fellowship (ECF) (#1123673), KK by NHMRC SeniorResearch Fellowship (1102792), DLD by a NHMRC Principal Research Fellowship(#1137285) and SJK by NHMRC Senior Principal Research Fellowship (#1136322). CES has received funding from the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement (#792532). JR is supported by an ARC Laureate fellowship. JRH and WZ are supported by the Melbourne Research Scholarship from The University of Melbourne. LH is supported by the Melbourne International Research Scholarship (MIRS) and the Melbourne International Fee Remission Scholarship (MIFRS) from The University of Melbourne.Ethical Approval: Experiments conformed to the Declaration of Helsinki Principles and theAustralian National Health and Medical Research Council Code of Practice. Written informed consents were obtained from all blood donors prior to the study. Lung and spleen tissues were obtained from deceased organ donors after written informed consents from the next of kin.Written informed consents were obtained from participants’ parental or guardians for underage tonsil tissue donors. The study was approved by the Alfred Hospital (#280/14), Austin Health (HREC/63201/Austin-2020);the University of Melbourne (#2057366.1, #2056901.1,#2056689, #2056761, #1442952, #1955465, and #1443389), the Australian Red CrossLifeblood (ID 2015#8), the Tasmanian Health and Medical (ID H0017479) and the James Cook University (H7886) Human Research Ethics Committees.

3.
Int J Mol Sci ; 23(2)2022 Jan 13.
Article in English | MEDLINE | ID: covidwho-1625839

ABSTRACT

The global urgency to uncover medical countermeasures to combat the COVID-19 pandemic caused by the severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) has revealed an unmet need for robust tissue culture models that faithfully recapitulate key features of human tissues and disease. Infection of the nose is considered the dominant initial site for SARS-CoV-2 infection and models that replicate this entry portal offer the greatest potential for examining and demonstrating the effectiveness of countermeasures designed to prevent or manage this highly communicable disease. Here, we test an air-liquid-interface (ALI) differentiated human nasal epithelium (HNE) culture system as a model of authentic SARS-CoV-2 infection. Progenitor cells (basal cells) were isolated from nasal turbinate brushings, expanded under conditionally reprogrammed cell (CRC) culture conditions and differentiated at ALI. Differentiated cells were inoculated with different SARS-CoV-2 clinical isolates. Infectious virus release into apical washes was determined by TCID50, while infected cells were visualized by immunofluorescence and confocal microscopy. We demonstrate robust, reproducible SARS-CoV-2 infection of ALI-HNE established from different donors. Viral entry and release occurred from the apical surface, and infection was primarily observed in ciliated cells. In contrast to the ancestral clinical isolate, the Delta variant caused considerable cell damage. Successful establishment of ALI-HNE is donor dependent. ALI-HNE recapitulate key features of human SARS-CoV-2 infection of the nose and can serve as a pre-clinical model without the need for invasive collection of human respiratory tissue samples.


Subject(s)
COVID-19/virology , Nasal Mucosa/cytology , Nasal Mucosa/virology , Tissue Culture Techniques/methods , Adolescent , Adult , Angiotensin-Converting Enzyme 2/metabolism , Cell Culture Techniques , Cell Differentiation , Epithelial Cells/cytology , Epithelial Cells/virology , Female , Humans , Male , Middle Aged , Models, Biological , SARS-CoV-2 , Virus Internalization
4.
Immunity ; 54(5): 1066-1082.e5, 2021 05 11.
Article in English | MEDLINE | ID: covidwho-1216346

ABSTRACT

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/N257, A2/S269, and A24/S1,208) CD8+ T cells specific for the immunodominant B7/N105 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 TCRαß repertoires and promiscuous αß-TCR pairing within B7/N105+CD8+ T cells. Our study demonstrates high naive precursor frequency and TCRαß diversity within immunodominant B7/N105-specific CD8+ T cells and provides insight into SARS-CoV-2-specific T cell origins and subsequent responses.


Subject(s)
CD8-Positive T-Lymphocytes/immunology , COVID-19/immunology , Coronavirus Nucleocapsid Proteins/immunology , Immunodominant Epitopes/immunology , Receptors, Antigen, T-Cell/immunology , SARS-CoV-2/immunology , Adult , Aged , Amino Acid Motifs , CD4-Positive T-Lymphocytes , Child , Convalescence , Coronavirus Nucleocapsid Proteins/chemistry , Epitopes, T-Lymphocyte/chemistry , Epitopes, T-Lymphocyte/immunology , Female , Humans , Immunodominant Epitopes/chemistry , Male , Middle Aged , Phenotype , Phosphoproteins/chemistry , Phosphoproteins/immunology , Receptors, Antigen, T-Cell/chemistry , Receptors, Antigen, T-Cell/genetics , Receptors, Antigen, T-Cell, alpha-beta/chemistry , Receptors, Antigen, T-Cell, alpha-beta/genetics , Receptors, Antigen, T-Cell, alpha-beta/immunology , Spike Glycoprotein, Coronavirus/chemistry , Spike Glycoprotein, Coronavirus/immunology
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