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      Evaluating SARS-CoV-2 spike and nucleocapsid proteins as targets for antibody detection in severe and mild COVID-19 cases using a Luminex bead-based assay

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          Highlights

          • •

            We evaluated a bead-based Luminex assay for the detection of SARS-CoV-2 antibodies.

          • •

            We show that a combination of recombinant nucleocapsid protein and the receptor-binding domain results in a highly sensitive (96%) and specific (99%) bead-based assay that can detect positive cases at least five months after symptom onset.

          • •

            Because we find that IgG and neutralizing antibody levels can differ significantly between severe/mild and recent/old cases, we argue that serological tests should be validated on samples from all groups before use in serosurveillance studies.

          Abstract

          Large-scale serosurveillance of severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) will only be possible if serological tests are sufficiently reliable, rapid and affordable. Many assays are either labour-intensive and require specialised facilities (e.g. virus neutralization assays), or are expensive with suboptimal specificity (e.g. commercial ELISAs and RDTs). Bead-based assays offer a cost-effective alternative and allow for multiplexing to test for antibodies against multiple antigens and against other pathogens. Here, we compare the performance of spike (S) and nucleocapsid (NP) antigens for the detection of SARS-CoV-2 specific IgG, IgM and IgA antibodies in a panel of sera that includes recent (up to six weeks after symptom onset, severe n = 44; and mild cases n = 52) and old infections (five months after symptom onset, mild n = 104), using a Luminex-bead based assay and comparison to a virus neutralization test. While we show that neutralising antibody levels are significantly lower in mild than in severe cases, we demonstrate that a combination of the recombinant nucleocapsid protein (NP) and receptor-binding domain (RBD) results in highly specific (99%) IgG antibody detection five months after infection in 96% of cases. Although most severe Covid-19 cases developed a clear IgM and IgA response, titers fell below the detection threshold in more than 20% of mild cases in our bead-based assay. In conclusion, our data supports the use of RBD and NP for the development of SARS-CoV-2 serological IgG bead-based assays.

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          Structure, Function, and Antigenicity of the SARS-CoV-2 Spike Glycoprotein

          Alexandra C Walls, Young-Jun Park, M. Alejandra Tortorici … (2020)
          Summary The emergence of SARS-CoV-2 has resulted in >90,000 infections and >3,000 deaths. Coronavirus spike (S) glycoproteins promote entry into cells and are the main target of antibodies. We show that SARS-CoV-2 S uses ACE2 to enter cells and that the receptor-binding domains of SARS-CoV-2 S and SARS-CoV S bind with similar affinities to human ACE2, correlating with the efficient spread of SARS-CoV-2 among humans. We found that the SARS-CoV-2 S glycoprotein harbors a furin cleavage site at the boundary between the S1/S2 subunits, which is processed during biogenesis and sets this virus apart from SARS-CoV and SARS-related CoVs. We determined cryo-EM structures of the SARS-CoV-2 S ectodomain trimer, providing a blueprint for the design of vaccines and inhibitors of viral entry. Finally, we demonstrate that SARS-CoV S murine polyclonal antibodies potently inhibited SARS-CoV-2 S mediated entry into cells, indicating that cross-neutralizing antibodies targeting conserved S epitopes can be elicited upon vaccination.
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            Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR

            Victor M Corman, Olfert Landt, Marco Kaiser … (2020)
            Background The ongoing outbreak of the recently emerged novel coronavirus (2019-nCoV) poses a challenge for public health laboratories as virus isolates are unavailable while there is growing evidence that the outbreak is more widespread than initially thought, and international spread through travellers does already occur. Aim We aimed to develop and deploy robust diagnostic methodology for use in public health laboratory settings without having virus material available. Methods Here we present a validated diagnostic workflow for 2019-nCoV, its design relying on close genetic relatedness of 2019-nCoV with SARS coronavirus, making use of synthetic nucleic acid technology. Results The workflow reliably detects 2019-nCoV, and further discriminates 2019-nCoV from SARS-CoV. Through coordination between academic and public laboratories, we confirmed assay exclusivity based on 297 original clinical specimens containing a full spectrum of human respiratory viruses. Control material is made available through European Virus Archive – Global (EVAg), a European Union infrastructure project. Conclusion The present study demonstrates the enormous response capacity achieved through coordination of academic and public laboratories in national and European research networks.
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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

                Journal
                Journal ID (nlm-ta): J Virol Methods
                Journal ID (iso-abbrev): J Virol Methods
                Title: Journal of Virological Methods
                Publisher: The Author(s). Published by Elsevier B.V.
                ISSN (Print): 0166-0934
                ISSN (Electronic): 1879-0984
                Publication date PMC-release: 20 November 2020
                Publication date (Electronic): 20 November 2020
                Electronic Location Identifier: 114025
                Affiliations
                [a ]Outbreak Research Team, Institute of Tropical Medicine, Antwerp, Belgium
                [b ]Virology Unit, Department of Biomedical Sciences, Institute of Tropical Medicine, Antwerp, Belgium
                [c ]Department of Clinical Sciences, Institute of Tropical Medicine, Antwerp, Belgium
                [d ]Epidemiology and public health, Sciensano, Brussels, Belgium
                [e ]Immune response, Sciensano, Brussels, Belgium
                [f ]University Hospital Antwerp, Antwerp, Belgium
                [g ]University of Antwerp, Antwerp, Belgium
                Author notes
                [* ]Corresponding authors at: Virology Unit, Department of Biomedical Sciences, Institute of Tropical Medicine, Antwerp, Belgium.
                Article
                Publisher Item ID: S0166-0934(20)30277-9 Publisher ID: 114025
                DOI: 10.1016/j.jviromet.2020.114025
                PMC ID: 7678438
                PubMed ID: 33227340
                SO-VID: 276c2fdf-2cce-4551-87f4-6a06d7f8b55a
                Copyright © © 2020 The Author(s). Published by Elsevier B.V.
                License:

                Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active.

                History
                Date received : 4 August 2020
                Date revision received : 16 November 2020
                Date accepted : 18 November 2020
                Categories
                Subject: Article

                ScienceOpen disciplines: Microbiology & Virology
                Keywords: sars-cov-2,bead-based assay,serosurveillance,luminex antibody test,virus neutralization test
                Data availability:
                ScienceOpen disciplines: Microbiology & Virology
                Keywords: sars-cov-2, bead-based assay, serosurveillance, luminex antibody test, virus neutralization test

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