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1.
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
2.
EBioMedicine ; 74: 103729, 2021 Dec.
Article in English | MEDLINE | ID: covidwho-1555409

ABSTRACT

BACKGROUND: As vaccines against SARS-CoV-2 are now being rolled out, a better understanding of immunity to the virus, whether from infection, or passive or active immunisation, and the durability of this protection is required. This will benefit from the ability to measure antibody-based protection to SARS-CoV-2, ideally with rapid turnaround and without the need for laboratory-based testing. METHODS: We have developed a lateral flow POC test that can measure levels of RBD-ACE2 neutralising antibody (NAb) from whole blood, with a result that can be determined by eye or quantitatively on a small instrument. We compared our lateral flow test with the gold-standard microneutralisation assay, using samples from convalescent and vaccinated donors, as well as immunised macaques. FINDINGS: We show a high correlation between our lateral flow test with conventional neutralisation and that this test is applicable with animal samples. We also show that this assay is readily adaptable to test for protection to newly emerging SARS-CoV-2 variants, including the beta variant which revealed a marked reduction in NAb activity. Lastly, using a cohort of vaccinated humans, we demonstrate that our whole-blood test correlates closely with microneutralisation assay data (specificity 100% and sensitivity 96% at a microneutralisation cutoff of 1:40) and that fingerprick whole blood samples are sufficient for this test. INTERPRETATION: Taken together, the COVID-19 NAb-testTM device described here provides a rapid readout of NAb based protection to SARS-CoV-2 at the point of care. FUNDING: Support was received from the Victorian Operational Infrastructure Support Program and the Australian Government Department of Health. This work was supported by grants from the Department of Health and Human Services of the Victorian State Government; the ARC (CE140100011, CE140100036), the NHMRC (1113293, 2002317 and 1116530), and Medical Research Future Fund Awards (2005544, 2002073, 2002132). Individual researchers were supported by an NHMRC Emerging Leadership Level 1 Investigator Grants (1194036), NHMRC APPRISE Research Fellowship (1116530), NHMRC Leadership Investigator Grant (1173871), NHMRC Principal Research Fellowship (1137285), NHMRC Investigator Grants (1177174 and 1174555) and NHMRC Senior Principal Research Fellowships (1117766 and 1136322). Grateful support was also received from the A2 Milk Company and the Jack Ma Foundation.


Subject(s)
Antibodies, Neutralizing/blood , Antibodies, Viral/blood , COVID-19 Serological Testing/methods , COVID-19/immunology , Point-of-Care Systems , SARS-CoV-2/immunology , Animals , Australia , COVID-19 Vaccines/immunology , Humans , Macaca/immunology , Neutralization Tests , Vaccination
3.
Angewandte Chemie ; 133(31):17239-17244, 2021.
Article in English | ProQuest Central | ID: covidwho-1315247

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) has resulted in an unprecedented need for diagnostic testing that is critical in controlling the spread of COVID‐19. We propose a portable infrared spectrometer with purpose‐built transflection accessory for rapid point‐of‐care detection of COVID‐19 markers in saliva. Initially, purified virion particles were characterized with Raman spectroscopy, synchrotron infrared (IR) and AFM‐IR. A data set comprising 171 transflection infrared spectra from 29 subjects testing positive for SARS‐CoV‐2 by RT‐qPCR and 28 testing negative, was modeled using Monte Carlo Double Cross Validation with 50 randomized test and model sets. The testing sensitivity was 93 % (27/29) with a specificity of 82 % (23/28) that included positive samples on the limit of detection for RT‐qPCR. Herein, we demonstrate a proof‐of‐concept high throughput infrared COVID‐19 test that is rapid, inexpensive, portable and utilizes sample self‐collection thus minimizing the risk to healthcare workers and ideally suited to mass screening.

4.
JCI Insight ; 6(16)2021 08 23.
Article in English | MEDLINE | ID: covidwho-1305530

ABSTRACT

The SARS-CoV-2 receptor binding domain (RBD) is both the principal target of neutralizing antibodies and one of the most rapidly evolving domains, which can result in the emergence of immune escape mutations, limiting the effectiveness of vaccines and antibody therapeutics. To facilitate surveillance, we developed a rapid, high-throughput, multiplex assay able to assess the inhibitory response of antibodies to 24 RBD natural variants simultaneously. We demonstrate how this assay can be implemented as a rapid surrogate assay for functional cell-based serological methods to measure the SARS-CoV-2 neutralizing capacity of antibodies at the angiotensin-converting enzyme 2-RBD (ACE2-RBD) interface. We describe the enhanced affinity of RBD variants N439K, S477N, Q493L, S494P, and N501Y to the ACE2 receptor and demonstrate the ability of this assay to bridge a major gap for SARS-CoV-2 research, informing selection of complementary monoclonal antibody candidates and the rapid identification of immune escape to emerging RBD variants following vaccination or natural infection.


Subject(s)
Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , SARS-CoV-2/immunology , Angiotensin-Converting Enzyme 2/metabolism , High-Throughput Screening Assays , Humans , Immune Evasion , Mutation
5.
Angew Chem Int Ed Engl ; 60(31): 17102-17107, 2021 07 26.
Article in English | MEDLINE | ID: covidwho-1245354

ABSTRACT

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in an unprecedented need for diagnostic testing that is critical in controlling the spread of COVID-19. We propose a portable infrared spectrometer with purpose-built transflection accessory for rapid point-of-care detection of COVID-19 markers in saliva. Initially, purified virion particles were characterized with Raman spectroscopy, synchrotron infrared (IR) and AFM-IR. A data set comprising 171 transflection infrared spectra from 29 subjects testing positive for SARS-CoV-2 by RT-qPCR and 28 testing negative, was modeled using Monte Carlo Double Cross Validation with 50 randomized test and model sets. The testing sensitivity was 93 % (27/29) with a specificity of 82 % (23/28) that included positive samples on the limit of detection for RT-qPCR. Herein, we demonstrate a proof-of-concept high throughput infrared COVID-19 test that is rapid, inexpensive, portable and utilizes sample self-collection thus minimizing the risk to healthcare workers and ideally suited to mass screening.


Subject(s)
COVID-19 Testing/methods , COVID-19/diagnosis , Saliva/chemistry , Animals , Chlorocebus aethiops , Cohort Studies , Discriminant Analysis , Humans , Least-Squares Analysis , Monte Carlo Method , Point-of-Care Testing , Proof of Concept Study , SARS-CoV-2 , Sensitivity and Specificity , Specimen Handling , Spectrophotometry, Infrared , Vero Cells
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