A serological assay to detect SARS-CoV-2 seroconversion in humans (original) (raw)

Data availability

The data shown in the manuscript are available upon request from the corresponding author. Nucleotide sequences of both constructs have been submitted to NCBI (GenBank IDs MT380724.1 and MT380725.1). Expression plasmids have been deposited to BEI Resources (https://www.beiresources.org/), and plasmids and sequences are also available from the corresponding author. A detailed protocol for expression and ELISA setup has been published (https://currentprotocols.onlinelibrary.wiley.com/doi/full/10.1002/cpmc.100).

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Acknowledgements

We thank Y.-Z. Zhang (Fudan University) and E. Holmes (University of Sydney) for sharing the sequence of the first SARS-CoV-2 isolate in a very timely manner. We thank J. Garlick and J. Roney (Alfred Hospital, Melbourne) for data and specimen collection, N. Aboelregal for making many different NHIG products (Mount Sinai) available and L. Martinez-Sobrido (Texas Biomedical Research Institute) for initially characterizing mAb 1C7. We are also thankful to Genewiz for speeding up gene synthesis for this project, and for being very accommodating to our needs. Furthermore, we thank D. Tidmore for help with ordering primers quickly, and finally S. Dong for commuting to New Jersey on several occasions to pick up reagents from Genewiz. We also thank the Mount Sinai Health System Translational Science Hub ‘ConduITS’ (NIH grant U54TR001433) for supporting sample collection. The work of the Personalized Virology Initiative is supported by institutional funds and philanthropic donations. This work was partially supported by the NIAID Centers of Excellence for Influenza Research and Surveillance (CEIRS; contract HHSN272201400008C to F.K. and A.G.-S.), the Collaborative Influenza Vaccine Innovation Centers (CIVIC; contract 75N93019C00051 to F.K. and A.G.-S.), Open Philanthropy, the Australian National Health and Medical Research Council (NHMRC Program Grant 1071916 and NHMRC Research Fellowship Level B (1102792) to K.K.), the Academy of Finland to O.V. and J.M.H., as well as Jane and Aatos Erkko Foundation and Helsinki University Hospital Funds to O.V. Furthermore, we thank our generous community for providing essential funds and support for our SARS-CoV-2 and COVID-19 research efforts. The following reagent was deposited by the Centers for Disease Control and Prevention and obtained through BEI Resources, NIAID, NIH: SARS-Related Coronavirus 2, Isolate USA-WA1/2020, NR-52281. Finally, we thank all of the study participants for their contribution to the research. We wish the patients with COVID-19 a speedy recovery.

Author information

Authors and Affiliations

  1. Department of Microbiology, Icahn School of Medicine at Mount Sinai, New York, NY, USA
    Fatima Amanat, Daniel Stadlbauer, Shirin Strohmeier, Veronika Chromikova, Meagan McMahon, Kaijun Jiang, Guha Asthagiri Arunkumar, Denise Jurczyszak, Jose Polanco, Maria Bermudez-Gonzalez, Giulio Kleiner, Teresa Aydillo, Lisa Miorin, Sean T. H. Liu, Thomas Moran, Adolfo García-Sastre, Viviana Simon & Florian Krammer
  2. Graduate School of Biomedical Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, USA
    Fatima Amanat, Guha Asthagiri Arunkumar & Denise Jurczyszak
  3. Department of Biotechnology, University of Natural Resources and Life Sciences, Vienna, Austria
    Shirin Strohmeier
  4. Department of Microbiology and Immunology, The Peter Doherty Institute for Infection and Immunity, University of Melbourne, Melbourne, Victoria, Australia
    Thi H. O. Nguyen & Katherine Kedzierska
  5. Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, USA
    Jose Polanco
  6. Global Health and Emerging Pathogens Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA
    Teresa Aydillo, Lisa Miorin, Adolfo García-Sastre & Viviana Simon
  7. Division of Infectious Diseases, Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, NY, USA
    Daniel S. Fierer, Luz Amarilis Lugo, Erna Milunka Kojic, Sean T. H. Liu, Adolfo García-Sastre & Viviana Simon
  8. Division of Pulmonary, Critical Care, and Sleep Medicine, Icahn School of Medicine at Mount Sinai, New York, NY, USA
    Jonathan Stoever
  9. Department of Medicine, Mount Sinai Queens, Astoria, NY, USA
    Sean T. H. Liu
  10. Division of Hospital Medicine, Mount Sinai Health System, New York, NY, USA
    Sean T. H. Liu
  11. Division of Clinical Immunology, Department of Medicine, Icahn School of Medicine at Mount Sinai, New York, NY, USA
    Charlotte Cunningham-Rundles
  12. Department of Pediatrics, the Icahn School of Medicine at Mount Sinai, NY, USA
    Charlotte Cunningham-Rundles
  13. Department of Physiology and Biophysics, University of California, Irvine, Irvine, CA, USA
    Philip L. Felgner
  14. Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA
    Adolfo García-Sastre
  15. Travel Medicine Program, Division of Infectious Diseases, Icahn School of Medicine at Mount Sinai, New York, NY, USA
    Daniel Caplivski
  16. School of Public Health and Preventive Medicine, Monash University and Infection Prevention and Healthcare Epidemiology Unit Alfred Health, Melbourne, Victoria, Australia
    Allen C. Cheng
  17. Department of Virology, Medicum, University of Helsinki, Helsinki, Finland
    Olli Vapalahti & Jussi M. Hepojoki
  18. Veterinary Biosciences, Veterinary Faculty, University of Helsinki, Helsinki, Finland
    Olli Vapalahti
  19. Department of Virology and Immunology, Helsinki University Hospital (HUSLAB), Helsinki, Finland
    Olli Vapalahti
  20. Institute of Veterinary Pathology, Vetsuisse Faculty, University of Zürich, Zürich, Switzerland
    Jussi M. Hepojoki

Authors

  1. Fatima Amanat
  2. Daniel Stadlbauer
  3. Shirin Strohmeier
  4. Thi H. O. Nguyen
  5. Veronika Chromikova
  6. Meagan McMahon
  7. Kaijun Jiang
  8. Guha Asthagiri Arunkumar
  9. Denise Jurczyszak
  10. Jose Polanco
  11. Maria Bermudez-Gonzalez
  12. Giulio Kleiner
  13. Teresa Aydillo
  14. Lisa Miorin
  15. Daniel S. Fierer
  16. Luz Amarilis Lugo
  17. Erna Milunka Kojic
  18. Jonathan Stoever
  19. Sean T. H. Liu
  20. Charlotte Cunningham-Rundles
  21. Philip L. Felgner
  22. Thomas Moran
  23. Adolfo García-Sastre
  24. Daniel Caplivski
  25. Allen C. Cheng
  26. Katherine Kedzierska
  27. Olli Vapalahti
  28. Jussi M. Hepojoki
  29. Viviana Simon
  30. Florian Krammer

Contributions

F.A., V.S. and F.K. conceived of and designed the study. F.A., D.S., S.S., T.H.O.N., V.C., M.M., K.J., G.A.A., D.J., J.P., M.B.-G., G.K., T.A., L.M., D.S.F., L.A.L., E.M.K., J.S., S.T.H.L., C.C.-R., P.L.F., A.G.-S., D.C., A.C.C., K.K., O.V. and J.M.H. collected data and contributed samples. F.A., V.S. and F.K. analyzed the data and wrote the manuscript.

Corresponding author

Correspondence toFlorian Krammer.

Ethics declarations

Competing interests

Mount Sinai is in the process of licensing out assays to commercial entities based on the assays described here and has filed for patent protection.

Additional information

Peer review information Alison Farrell is the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Constructs for recombinant protein expression.

a, Visualization of the trimeric spike protein of SARS-CoV-2 based on PBD # 6VXX using Pymol3. One monomer is colored in dark blue while the remaining two monomers are held in light blue. The receptor binding domain (RBD) of the dark blue trimer is highlighted in red. b, Schematic of the wild type full length spike protein with signal peptide, ectodomain, receptor binding domain, furin cleavage site, S1, S2, and transmembrane and endodomain domain indicated. c, Schematic of the soluble trimeric spike. The polybasic/furin cleavage site (RRAR) was replaced by a single A. The transmembrane and endodomain were replaced by a furin cleavage site, a T4 foldon tetramerization domain and a hexahistidine tag. Introduction of K986P and V987P has been shown to stabilize the trimer in the pre-fusion conformation. d, Schematic of the soluble receptor binding domain construct. All constructs are to scale. e Reducing SDS PAGE of insect cell and mammalian cell derived soluble trimerized spike protein (iSpike and mSpike). f Reducing SDS PAGE of insect cell derived and mammalian cell derived recombinant receptor binding domain (iRBD and mRBD). Experiments were performed six times with the same result.

Extended Data Fig. 2 Human normal immunoglobulin preparations and historic sera from HIV + patients do not react with the SAR-CoV-2 spike.

a, b, Reactivity of 21 different pools of human normal immunoglobulin (HNIG) preparations (27 different vials) to mRBD and mSpike of SARS-CoV-2. MAb CR3022 was used as positive control, three different irrelevant human mAbs were used as negative control. c, d shows reactivity of historic samples from 50 HIV + individuals to mRBD and mSpike of SARS-CoV-2. Both HNIG and serum samples from HIV + donors were collected before the SARS-CoV-2 pandemic. Experiments were performed once. MAb CR3022 was used as positive control at a starting concentration of 100 ug/ml. Of note, the experiments in A and C as well as B and D were done at the same time and their positive controls are shared and displayed in both panels. Experiments were performed once.

Extended Data Fig. 3 Isotypes and subtypes of antibodies from COVID19 patients to the soluble spike protein and microneutralization titers.

a, Mammalian cell derived spike protein was used to study isotype/subclass distribution of antibodies (n = 13 positive samples). Lines represent the geometric mean. b, Microneutralization assay (n = 12) performed with authentic SARS-CoV-2. Lines represent curves fitted using an inhibitor (log) versus response variable slope with four parameters function in Graphpad Prism. Experiments were performed once.

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Amanat, F., Stadlbauer, D., Strohmeier, S. et al. A serological assay to detect SARS-CoV-2 seroconversion in humans.Nat Med 26, 1033–1036 (2020). https://doi.org/10.1038/s41591-020-0913-5

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