A comprehensive antigen production and characterisation study for easy-to-implement, specific and quantitative SARS-CoV-2 serotests

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Abstract

Background

Antibody tests are essential tools to investigate humoral immunity following SARS-CoV-2 infection or vaccination. While first-generation antibody tests have primarily provided qualitative results, accurate seroprevalence studies and tracking of antibody levels over time require highly specific, sensitive and quantitative test setups.

Methods

We have developed two quantitative, easy-to-implement SARS-CoV-2 antibody tests, based on the spike receptor binding domain and the nucleocapsid protein. Comprehensive evaluation of antigens from several biotechnological platforms enabled the identification of superior antigen designs for reliable serodiagnostic. Cut-off modelling based on unprecedented large and heterogeneous multicentric validation cohorts allowed us to define optimal thresholds for the tests’ broad applications in different aspects of clinical use, such as seroprevalence studies and convalescent plasma donor qualification.

Findings

Both developed serotests individually performed similarly-well as fully-automated CE-marked test systems. Our described sensitivity-improved orthogonal test approach assures highest specificity (99.8%); thereby enabling robust serodiagnosis in low-prevalence settings with simple test formats. The inclusion of a calibrator permits accurate quantitative monitoring of antibody concentrations in samples collected at different time points during the acute and convalescent phase of COVID-19 and disclosed antibody level thresholds that correlate well with robust neutralization of authentic SARS-CoV-2 virus.

Interpretation

We demonstrate that antigen source and purity strongly impact serotest performance. Comprehensive biotechnology-assisted selection of antigens and in-depth characterisation of the assays allowed us to overcome limitations of simple ELISA-based antibody test formats based on chromometric reporters, to yield comparable assay performance as fully-automated platforms.

Funding

WWTF, Project No. COV20–016; BOKU, LBI/LBG

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Most cited references 65

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SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor

Markus Hoffmann, Hannah Kleine-Weber, Simon Schroeder … (2020)

Summary The recent emergence of the novel, pathogenic SARS-coronavirus 2 (SARS-CoV-2) in China and its rapid national and international spread pose a global health emergency. Cell entry of coronaviruses depends on binding of the viral spike (S) proteins to cellular receptors and on S protein priming by host cell proteases. Unravelling which cellular factors are used by SARS-CoV-2 for entry might provide insights into viral transmission and reveal therapeutic targets. Here, we demonstrate that SARS-CoV-2 uses the SARS-CoV receptor ACE2 for entry and the serine protease TMPRSS2 for S protein priming. A TMPRSS2 inhibitor approved for clinical use blocked entry and might constitute a treatment option. Finally, we show that the sera from convalescent SARS patients cross-neutralized SARS-2-S-driven entry. Our results reveal important commonalities between SARS-CoV-2 and SARS-CoV infection and identify a potential target for antiviral intervention.

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A new coronavirus associated with human respiratory disease in China

Fan Wu, Su Zhao, Bin Yu … (2020)

Emerging infectious diseases, such as severe acute respiratory syndrome (SARS) and Zika virus disease, present a major threat to public health 1–3 . Despite intense research efforts, how, when and where new diseases appear are still a source of considerable uncertainty. A severe respiratory disease was recently reported in Wuhan, Hubei province, China. As of 25 January 2020, at least 1,975 cases had been reported since the first patient was hospitalized on 12 December 2019. Epidemiological investigations have suggested that the outbreak was associated with a seafood market in Wuhan. Here we study a single patient who was a worker at the market and who was admitted to the Central Hospital of Wuhan on 26 December 2019 while experiencing a severe respiratory syndrome that included fever, dizziness and a cough. Metagenomic RNA sequencing 4 of a sample of bronchoalveolar lavage fluid from the patient identified a new RNA virus strain from the family Coronaviridae, which is designated here ‘WH-Human 1’ coronavirus (and has also been referred to as ‘2019-nCoV’). Phylogenetic analysis of the complete viral genome (29,903 nucleotides) revealed that the virus was most closely related (89.1% nucleotide similarity) to a group of SARS-like coronaviruses (genus Betacoronavirus, subgenus Sarbecovirus) that had previously been found in bats in China 5 . This outbreak highlights the ongoing ability of viral spill-over from animals to cause severe disease in humans.

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Clinical and immunological assessment of asymptomatic SARS-CoV-2 infections

Quan-Xin Long, Xiao-Jun Tang, Qiu-Lin Shi … (2020)

The clinical features and immune responses of asymptomatic individuals infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have not been well described. We studied 37 asymptomatic individuals in the Wanzhou District who were diagnosed with RT-PCR-confirmed SARS-CoV-2 infections but without any relevant clinical symptoms in the preceding 14 d and during hospitalization. Asymptomatic individuals were admitted to the government-designated Wanzhou People's Hospital for centralized isolation in accordance with policy1. The median duration of viral shedding in the asymptomatic group was 19 d (interquartile range (IQR), 15-26 d). The asymptomatic group had a significantly longer duration of viral shedding than the symptomatic group (log-rank P = 0.028). The virus-specific IgG levels in the asymptomatic group (median S/CO, 3.4; IQR, 1.6-10.7) were significantly lower (P = 0.005) relative to the symptomatic group (median S/CO, 20.5; IQR, 5.8-38.2) in the acute phase. Of asymptomatic individuals, 93.3% (28/30) and 81.1% (30/37) had reduction in IgG and neutralizing antibody levels, respectively, during the early convalescent phase, as compared to 96.8% (30/31) and 62.2% (23/37) of symptomatic patients. Forty percent of asymptomatic individuals became seronegative and 12.9% of the symptomatic group became negative for IgG in the early convalescent phase. In addition, asymptomatic individuals exhibited lower levels of 18 pro- and anti-inflammatory cytokines. These data suggest that asymptomatic individuals had a weaker immune response to SARS-CoV-2 infection. The reduction in IgG and neutralizing antibody levels in the early convalescent phase might have implications for immunity strategy and serological surveys.

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Author and article information

Contributors

Florian Grebien

Christoph J. Binder

Reingard Grabherr

Journal

Journal ID (nlm-ta): EBioMedicine

Journal ID (iso-abbrev): EBioMedicine

Title: EBioMedicine

Publisher: Elsevier

ISSN (Electronic): 2352-3964

Publication date PMC-release: 25 April 2021

Publication date Collection: May 2021

Publication date (Electronic): 25 April 2021

Volume: 67

Electronic Location Identifier: 103348

Affiliations

[a ]Department of Biotechnology, University of Natural Resources and Life Sciences (BOKU) Vienna, Muthgasse 18, 1190 Vienna, Austria

[b ]Novasign GmbH Vienna, Austria

[c ]Department of Laboratory Medicine, Medical University of Vienna, Währinger Gürtel 18-20, 1090 Vienna, Austria

[d ]CD Laboratory for innovative Immunotherapeutics, Vienna, Austria

[e ]ACIB-Austrian Centre of Industrial Biotechnology, Graz, Austria

[f ]BOKU Core Facility Biomolecular & Cellular Analysis, University of Natural Resources and Life Sciences (BOKU),Vienna, Austria

[g ]Department of Chemical Engineering, Vrije Universiteit Brussel (VUB), Brussels, Belgium

[h ]Competence Unit Molecular Diagnostics, Center for Health and Bioresources, AIT Austrian Institute of Technology GmbH, Vienna, Austria

[i ]Austrian Agency for Health and Food Safety (AGES), Department for Animal Health, Moedling, Austria

[j ]Institute of Immunology, University of Veterinary Medicine, Vienna, Austria

[k ]Institute for Medical Biochemistry, University of Veterinary Medicine, Veterinärplatz 1, 1210 Vienna, Austria

[l ]Department of Laboratory Medicine, Konventhospital Barmherzige Brueder Linz and Ordensklinikum Linz Barmherzige Schwestern, Linz, Austria

[m ]BOKU Core Facility Mass Spectrometry, University of Natural Resources and Life Sciences (BOKU), Vienna, Austria

[n ]Department of Chemistry, University of Natural Resources and Life Sciences (BOKU) Vienna, Austria

[o ]enGenes Biotech GmbH, Vienna, Austria

[p ]Department of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences (BOKU) Vienna, Austria

[q ]Department of Medicine III, Division of Nephrology and Dialysis, Medical University of Vienna, Austria

[r ]Department of Respiratory and Critical Care Medicine and Ludwig Boltzmann Institute for Lung Health, Otto Wagner Hospital, Vienna, Austria

[s ]Division of Rheumatology, Department of Medicine III, Medical University of Vienna, Austria

[t ]Department of Blood Group Serology and Transfusion Medicine, Medical University of Vienna, Austria

[u ]Central Institute for Medical and Chemical Laboratory Diagnosis, Innsbruck University Hospital, Innsbruck, Austria

[v ]MLL Munich Leukemia Laboratory, Munich, Germany

[w ]Department of Internal Medicine II, Innsbruck Medical University, Innsbruck, Austria

[x ]Technoclone Herstellung von Diagnostika und Arzneimitteln GmbH, Vienna, Austria

[y ]Christian Doppler Laboratory for an Optimized Prediction of Vaccination Success in Pigs, University of Veterinary Medicine, Vienna, Austria

[z ]Present address: The Pirbright Institute, Pirbright, United Kingdom

Author notes

[* ]Corresponding authors. florian.grebien@ 123456vetmeduni.ac.at christoph.binder@ 123456meduniwien.ac.at reingard.grabherr@ 123456boku.ac.at

[1]

These authors contributed equally to this work.

[2]

On behalf of the CovILD study group.

Article

Publisher Item ID: S2352-3964(21)00141-9 Publisher ID: 103348

DOI: 10.1016/j.ebiom.2021.103348

PMC ID: 8099623

PubMed ID: 33906067

SO-VID: 0360fbb5-5f19-4c15-8017-75c388b0f60c

License:

This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

History

Date received : 9 February 2021

Date revision received : 15 March 2021

Date accepted : 2 April 2021

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A comprehensive antigen production and characterisation study for easy-to-implement, specific and quantitative SARS-CoV-2 serotests

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Abstract

Background

Methods

Findings

Interpretation

Funding

Related collections

Novel Coronavirus Disease COVID-19

Most cited references 65

SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor

A new coronavirus associated with human respiratory disease in China

Clinical and immunological assessment of asymptomatic SARS-CoV-2 infections

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Affiliations

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Article

History

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Comments

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Similar content 87

Cited by 25

Most referenced authors 2,230