A comparison of four serological assays for detecting anti-SARS-CoV-2 antibodies in human serum samples from different populations

Ludivine Grzelak, Sarah Temmam, Cyril Planchais, Caroline Demeret, Laura Tondeur, Christèle Huon, Florence Guivel-Benhassine, Isabelle Staropoli, Maxime Chazal, Jeremy Dufloo, Delphine Planas, Julian Buchrieser, Maaran Michael Rajah, Remy Robinot, Françoise Porrot, Mélanie Albert, Kuang-Yu Chen, Bernadette Crescenzo-Chaigne, Flora Donati, François Anna, Philippe Souque, Marion Gransagne, Jacques Bellalou, Mireille Nowakowski, Marija Backovic, Lila Bouadma, Lucie Le Fevre, Quentin Le Hingrat, Diane Descamps, Annabelle Pourbaix, Cédric Laouénan, Jade Ghosn, Yazdan Yazdanpanah, Camille Besombes, Nathalie Jolly, Sandrine Pellerin-Fernandes, Olivia Cheny, Marie-Noëlle Ungeheuer, Guillaume Mellon, Pascal Morel, Simon Rolland, Felix A Rey, Sylvie Behillil, Vincent Enouf, Audrey Lemaitre, Marie-Aude Créach, Stephane Petres, Nicolas Escriou, Pierre Charneau, Arnaud Fontanet, Bruno Hoen, Timothée Bruel, Marc Eloit, Hugo Mouquet, Olivier Schwartz, Sylvie van der Werf, Ludivine Grzelak, Sarah Temmam, Cyril Planchais, Caroline Demeret, Laura Tondeur, Christèle Huon, Florence Guivel-Benhassine, Isabelle Staropoli, Maxime Chazal, Jeremy Dufloo, Delphine Planas, Julian Buchrieser, Maaran Michael Rajah, Remy Robinot, Françoise Porrot, Mélanie Albert, Kuang-Yu Chen, Bernadette Crescenzo-Chaigne, Flora Donati, François Anna, Philippe Souque, Marion Gransagne, Jacques Bellalou, Mireille Nowakowski, Marija Backovic, Lila Bouadma, Lucie Le Fevre, Quentin Le Hingrat, Diane Descamps, Annabelle Pourbaix, Cédric Laouénan, Jade Ghosn, Yazdan Yazdanpanah, Camille Besombes, Nathalie Jolly, Sandrine Pellerin-Fernandes, Olivia Cheny, Marie-Noëlle Ungeheuer, Guillaume Mellon, Pascal Morel, Simon Rolland, Felix A Rey, Sylvie Behillil, Vincent Enouf, Audrey Lemaitre, Marie-Aude Créach, Stephane Petres, Nicolas Escriou, Pierre Charneau, Arnaud Fontanet, Bruno Hoen, Timothée Bruel, Marc Eloit, Hugo Mouquet, Olivier Schwartz, Sylvie van der Werf

Abstract

It is of paramount importance to evaluate the prevalence of both asymptomatic and symptomatic cases of SARS-CoV-2 infection and their differing antibody response profiles. Here, we performed a pilot study of four serological assays to assess the amounts of anti-SARS-CoV-2 antibodies in serum samples obtained from 491 healthy individuals before the SARS-CoV-2 pandemic, 51 individuals hospitalized with COVID-19, 209 suspected cases of COVID-19 with mild symptoms, and 200 healthy blood donors. We used two ELISA assays that recognized the full-length nucleoprotein (N) or trimeric spike (S) protein ectodomain of SARS-CoV-2. In addition, we developed the S-Flow assay that recognized the S protein expressed at the cell surface using flow cytometry, and the luciferase immunoprecipitation system (LIPS) assay that recognized diverse SARS-CoV-2 antigens including the S1 domain and the carboxyl-terminal domain of N by immunoprecipitation. We obtained similar results with the four serological assays. Differences in sensitivity were attributed to the technique and the antigen used. High anti-SARS-CoV-2 antibody titers were associated with neutralization activity, which was assessed using infectious SARS-CoV-2 or lentiviral-S pseudotype virus. In hospitalized patients with COVID-19, seroconversion and virus neutralization occurred between 5 and 14 days after symptom onset, confirming previous studies. Seropositivity was detected in 32% of mildly symptomatic individuals within 15 days of symptom onset and in 3% of healthy blood donors. The four antibody assays that we used enabled a broad evaluation of SARS-CoV-2 seroprevalence and antibody profiling in different subpopulations within one region.

Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Figures

Fig. 1. Serological survey of SARS-Cov-2 antibodies…
Fig. 1. Serological survey of SARS-Cov-2 antibodies in human serum samples.
Four serological assays were used to detect anti–SARS-Cov-2 antibodies in serum samples: (top row) individuals sampled between 2017 and 2019 (before pandemic), (second row) hospitalized cases with confirmed COVID-19, (third row) mildly symptomatic individuals from the Crépy-en-Vallois pandemic cluster with suspected COVID-19, and (bottom row) healthy blood donors. ELISA N and ELISA tri-S assays are conventional ELISAs using either N protein or the trimeric ectodomain of S protein as antigens. S-Flow is an assay detecting antibodies bound to cells expressing S protein by flow cytometry. The LIPS S1 and N assays detect either S1 or N protein fused to luciferase by immunoprecipitation. Pre-pandemic serum samples were used to determine the cutoff for each assay, which is indicated by a dotted line and a green area. The two ELISA assays were set to 95% specificity; the specificity of the S-Flow assay and LIPS assay was 99%. The number of positive samples is indicated. Each dot represents one sample. OD, optical density; StoN, signal-to-noise ratio.
Fig. 2. Antibody detection in serum samples…
Fig. 2. Antibody detection in serum samples from five hospitalized patients with COVID-19.
The kinetics of seroconversion in serum samples from five hospitalized patients with COVID-19 (B10 to B14) were measured by four different serological assays. At least five longitudinal serum samples were collected for each patient up to 20 days after symptom onset. All patients were admitted to the intensive care unit. Each line represents one patient. Dotted lines and green areas indicate cutoff for positivity in the seroprevalence assays.
Fig. 3. Comparison of positive serum samples.
Fig. 3. Comparison of positive serum samples.
The number of positive serum samples detected by each serological assay is shown for the three cohorts: hospitalized patients with COVID-19, mildly symptomatic individuals, and healthy blood donors. Correspondence of the positive results is shown among the four assays. For a given assay, each row indicates the number of positive samples that were also positive with the other three assays. Bold numbers indicate the number of positive samples for a given assay. The number of positive samples is color-coded: White corresponds to lower numbers, and green corresponds to higher numbers.
Fig. 4. Correlations among the four serological…
Fig. 4. Correlations among the four serological assays.
To compare the four serological assays, results from serum samples from mildly symptomatic individuals and hospitalized patients with COVID-19 (n = 329) were pooled. (A) Results obtained with one assay were correlated with those of the other three assays. Dotted lines indicate assay cutoff values for positivity. Values in pale green areas are positive in one assay, and values in darker green areas are positive in two assays. Each dot represents one study participant. (B) Pearson correlation coefficient (R2) of each comparison is shown. R2 values are color-coded, with white corresponding to the lowest value and dark blue corresponding to the highest value. All correlations are significant (P < 0.0001).
Fig. 5. Virus neutralizing activity in human…
Fig. 5. Virus neutralizing activity in human serum samples.
(A) Virus-neutralizing activity (dilution 1:100) of 12 serum samples from the mildly symptomatic cohort of individuals with suspected COVID-19 (C1 to C12) and 9 serum samples from hospitalized patients with COVID-19 (B1 to B9). Virus-neutralizing activity was determined by the pseudovirus neutralization assay and compared to serology data obtained with the four serological assays. Numbers in the top left quadrant indicate the Spearman correlation coefficient, r. All correlations are significant (P < 0.0001). (B) Neutralization activity of serum samples (B1 to B9) from hospitalized patients with COVID-19 was plotted against days after symptom onset. The black line corresponds to a nonlinear fit of the data.

References

    1. Wu F., Zhao S., Yu B., Chen Y.-M., Wang W., Song Z.-G., Hu Y., Tao Z.-W., Tian J.-H., Pei Y.-Y., Yuan M.-L., Zhang Y.-L., Dai F.-H., Liu Y., Wang Q.-M., Zheng J.-J., Xu L., Holmes E. C., Zhang Y.-Z., A new coronavirus associated with human respiratory disease in China. Nature 579, 265–269 (2020).
    1. Zhou P., Yang X.-L., Wang X.-G., Hu B., Zhang L., Zhang W., Si H.-R., Zhu Y., Li B., Huang C.-L., Chen H.-D., Chen J., Luo Y., Guo H., Jiang R.-D., Liu M.-Q., Chen Y., Shen X.-R., Wang X., Zheng X.-S., Zhao K., Chen Q.-J., Deng F., Liu L.-L., Yan B., Zhan F.-X., Wang Y.-Y., Xiao G.-F., Shi Z.-L., A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature 579, 270–273 (2020).
    1. Corman V. M., Landt O., Kaiser M., Molenkamp R., Meijer A., Chu D. K. W., Bleicker T., Brünink S., Schneider J., Schmidt M. L., Mulders D. G. J. C., Haagmans B. L., van der Veer B., van den Brink S., Wijsman L., Goderski G., Romette J.-L., Ellis J., Zambon M., Peiris M., Goossens H., Reusken C., Koopmans M. P. G., Drosten C., Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR. Euro Surveill. 25, 2000045 (2020).
    1. Chu D. K. W., Pan Y., Cheng S. M. S., Hui K. P. Y., Krishnan P., Liu Y., Ng D. Y. M., Wan C. K. C., Yang P., Wang Q., Peiris M., Poon L. L. M., Molecular diagnosis of a Novel Coronavirus (2019-nCoV) causing an outbreak of pneumonia. Clin. Chem. 66, 549–555 (2020).
    1. Pfefferle S., Reucher S., Nörz D., Lütgehetmann M., Evaluation of a quantitative RT-PCR assay for the detection of the emerging coronavirus SARS-CoV-2 using a high throughput system. Euro Surveill. 25, 2000152 (2020).
    1. Wölfel R., Corman V. M., Guggemos W., Seilmaier M., Zange S., Müller M. A., Niemeyer D., Jones T. C., Vollmar P., Rothe C., Hoelscher M., Bleicker T., Brünink S., Schneider J., Ehmann R., Zwirglmaier K., Drosten C., Wendtner C., Virological assessment of hospitalized patients with COVID-2019. Nature 581, 465–469 (2020).
    1. To K. K.-W., Tsang O. T.-Y., Leung W.-S., Tam A. R., Wu T.-C., Lung D. C., Yip C. C.-Y., Cai J.-P., Chan J. M.-C., Chik T. S.-H., Lau D. P.-L., Choi C. Y.-C., Chen L.-L., Chan W.-M., Chan K.-H., Ip J. D., Ng A. C.-K., Poon R. W.-S., Luo C.-T., Cheng V. C.-C., Chan J. F.-W., Hung I. F.-N., Chen Z., Chen H., Yuen K.-Y., Temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by SARS-CoV-2: An observational cohort study. Lancet Infect. Dis. 20, 565–574 (2020).
    1. Hoffmann M., Kleine-Weber H., Schroeder S., Krüger N., Herrler T., Erichsen S., Schiergens T. S., Herrler G., Wu N.-H., Nitsche A., Müller M. A., Drosten C., Pöhlmann S., SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2 and is blocked by a clinically proven protease inhibitor. Cell 181, 271–280.e8 (2020).
    1. Li F., Li W., Farzan M., Harrison S. C., Structure of SARS coronavirus spike receptor-binding domain complexed with receptor. Science 309, 1864–1868 (2005).
    1. Song W., Gui M., Wang X., Xiang Y., Cryo-EM structure of the SARS coronavirus spike glycoprotein in complex with its host cell receptor ACE2. PLOS Pathog. 14, e1007236 (2018).
    1. Yan R., Zhang Y., Li Y., Xia L., Guo Y., Zhou Q., Structural basis for the recognition of SARS-CoV-2 by full-length human ACE2. Science 367, 1444–1448 (2020).
    1. Walls A. C., Xiong X., Park Y.-J., Tortorici M. A., Snijder J., Quispe J., Cameroni E., Gopal R., Dai M., Lanzavecchia A., Zambon M., Rey F. A., Corti D., Veesler D., Unexpected receptor functional mimicry elucidates activation of coronavirus fusion. Cell 176, 1026–1039.e15 (2019).
    1. Amanat F., Nguyen T., Chromikova V., Strohmeier S., Stadlbauer D., Javier A., Jiang K., Asthagiri-Arunkumar G., Polanco J., Bermudez-Gonzalez M., Caplivski D., Cheng A., Kedzierska K., Vapalahti O., Hepojoki J., Simon V., Krammer F., A serological assay to detect SARS-CoV-2 seroconversion in humans. Nat. Med. 26, 1033–1036 (2020).
    1. Okba N. M. A., Müller M. A., Li W., Wang C., Geurts van Kessel C. H., Corman V. M., Lamers M. M., Sikkema R. S., de Bruin E., Chandler F. D., Yazdanpanah Y., Hingrat Q. L., Descamps D., Houhou-Fidouh N., Reusken C. B. E. M., Bosch B.-J., Drosten C., Koopmans M. P. G., Haagmans B. L., SARS-CoV-2 specific antibody responses in COVID-19 patients. Emerg. Infect. Dis. 26, 1478–1488 (2020).
    1. Zhao J., Yuan Q., Wang H., Liu W., Liao X., Su Y., Wang X., Yuan J., Li T., Li J., Qian S., Hong C., Wang F., Liu Y., Wang Z., He Q., Li Z., He B., Zhang T., Fu Y., Ge S., Liu L., Zhang J., Xia N., Zhang Z., Antibody responses to SARS-CoV-2 in patients of novel coronavirus disease 2019. Clin. Infect. Dis., ciaa344 (2020).
    1. Guo L., Ren L., Yang S., Xiao M., Chang D., Yang F., Dela Cruz C. S., Wang Y., Wu C., Xiao Y., Zhang L., Han L., Dang S., Xu Y., Yang Q.-W., Xu S.-Y., Zhu H.-D., Xu Y.-C., Jin Q., Sharma L., Wang L., Wang J., Profiling early humoral response to diagnose novel coronavirus disease (COVID-19). Clin. Infect. Dis. 71, 778–785 (2020).
    1. Jiang H., Li Y., Zhang H., Wang W., Yang X., Qi H., Li H., Men D., Zhou J., Tao S., SARS-CoV-2 proteome microarray for global profiling of COVID-19 specific IgG and IgM responses. Nat. Commun. 11, 3581 (2020).
    1. Müller M. A., Meyer B., Corman V. M., Al-Masri M., Turkestani A., Ritz D., Sieberg A., Aldabbagh S., Bosch B.-J., Lattwein E., Alhakeem R. F., Assiri A. M., Albarrak A. M., Al-Shangiti A. M., Al-Tawfiq J. A., Wikramaratna P., Alrabeeah A. A., Drosten C., Memish Z. A., Presence of Middle East respiratory syndrome coronavirus antibodies in Saudi Arabia: A nationwide, cross-sectional, serological study. Lancet Infect. Dis. 15, 629 (2015).
    1. Ou X., Liu Y., Lei X., Li P., Mi D., Ren L., Guo L., Guo R., Chen T., Hu J., Xiang Z., Mu Z., Chen X., Chen J., Hu K., Jin Q., Wang J., Qian Z., Characterization of spike glycoprotein of SARS-CoV-2 on virus entry and its immune cross-reactivity with SARS-CoV. Nat. Commun. 11, 1620 (2020).
    1. Wu F., Wang A., Liu M., Wang Q., Chen J., Xia S., Ling Y., Zhang Y., Xun J., Lu L., Jiang S., Lu H., Wen Y., Huang J., Neutralizing antibody responses to SARS-CoV-2 in a COVID-19 recovered patient cohort and their implications. medRxiv 2020.03.30.20047365, (2020).
    1. Poh C. M., Carissimo G., Wang B., Amrun S. N., Lee C. Y.-P., Chee R. S.-L., Fong S.-W., Yeo N. K.-W., Lee W.-H., Torres-Ruesta A., Leo Y.-S., Chen M. I.-C., Tan S.-Y., Chai L. Y. A., Kalimuddin S., Kheng S. S. G., Thien S.-Y., Young B. E., Lye D. C., Hanson B. J., Wang C.-I., Renia L., Ng L. F. P., Two linear epitopes on the SARS-CoV-2 spike protein that elicit neutralising antibodies in COVID-19 patients. Nat. Commun. 11, 2806 (2020).
    1. Ju B., Zhang Q., Ge J., Wang R., Sun J., Ge X., Yu J., Shan S., Zhou B., Song S., Tang X., Yu J., Lan J., Yuan J., Wang H., Zhao J., Zhang S., Wang Y., Shi X., Liu L., Zhao J., Wang X., Zhang Z., Zhang L., Human neutralizing antibodies elicited by SARS-CoV-2 infection. Nature 584, 115–119 (2020).
    1. Li R., Pei S., Chen B., Song Y., Zhang T., Yang W., Shaman J., Substantial undocumented infection facilitates the rapid dissemination of novel coronavirus (SARS-CoV2). Science 368, 489–493 (2020).
    1. Lescure F.-X., Bouadma L., Nguyen D., Parisey M., Wicky P.-H., Behillil S., Gaymard A., Bouscambert-Duchamp M., Donati F., Hingrat Q. L., Enouf V., Houhou-Fidouh N., Valette M., Mailles A., Lucet J.-C., Mentre F., Duval X., Descamps D., Malvy D., Timsit J.-F., Lina B., van-der-Werf S., Yazdanpanah Y., Clinical and virological data of the first cases of COVID-19 in Europe: A case series. Lancet Infect. Dis. 20, 697–706 (2020).
    1. Wajnberg A., Mansour M., Leven E., Bouvier N. M., Patel G., Firpo A., Mendu R., Jhang J., Arinsburg S., Gitman M., Houldsworth J., Baine I., Simon V., Aberg J., Krammer F., Reich D., Cordon-Cardo C., Humoral immune response and prolonged PCR positivity in a cohort of 1343 SARS-CoV 2 patients in the New York City region. medRxiv 2020.04.30.20085613, (2020).
    1. Fafi-Kremer S., Bruel T., Madec Y., Grant R., Tondeur L., Grzelak L., Staropoli I., Anna F., Souque P., Fernandes-Pellerin S., Jolly N., Renaudat C., Ungeheuer M.-N., Schmidt-Mutter C., Collongues N., Bolle A., Velay A., Lefebvre N., Mielcarek M., Meyer N., Rey D., Charneau P., Hoen B., De Seze J., Schwartz O., Fontanet A., Serologic responses to SARS-CoV-2 infection among hospital staff with mild disease in eastern France. EBioMedicine, 102915 (2020).
    1. Robbiani D. F., Gaebler C., Muecksch F., Lorenzi J. C. C., Wang Z., Cho A., Agudelo M., Barnes C. O., Gazumyan A., Finkin S., Hägglöf T., Oliveira T. Y., Viant C., Hurley A., Hoffmann H.-H., Millard K. G., Kost R. G., Cipolla M., Gordon K., Bianchini F., Chen S. T., Ramos V., Patel R., Dizon J., Shimeliovich I., Mendoza P., Hartweger H., Nogueira L., Pack M., Horowitz J., Schmidt F., Weisblum Y., Michailidis E., Ashbrook A. W., Waltari E., Pak J. E., Huey-Tubman K. E., Koranda N., Hoffman P. R., West A. P. Jr., Rice C. M., Hatziioannou T., Bjorkman P. J., Bieniasz P. D., Caskey M., Nussenzweig M. C., Convergent antibody responses to SARS-CoV-2 in convalescent individuals. Nature 10.1038/s41586-020-2456-9, (2020).
    1. Brochot E., Demey B., Touze A., Belouzard S., Dubuisson J., Schmit J.-L., Duverlie G., Francois C., Castelain S., Helle F., Anti-Spike, anti-Nucleocapsid and neutralizing antibodies in SARS-CoV-2 inpatients and asymptomatic carriers. medRxiv 2020.05.12.20098236, (2020).
    1. Long Q.-X., Tang X.-J., Shi Q.-L., Li Q., Deng H.-J., Yuan J., Hu J.-L., Xu W., Zhang Y., Lv F.-J., Su K., Zhang F., Gong J., Wu B., Liu X.-M., Li J.-J., Qiu J.-F., Chen J., Huang A.-L., Clinical and immunological assessment of asymptomatic SARS-CoV-2 infections. Nat. Med. 26, 1200–1204 (2020).
    1. Hohdatsu T., Yamada M., Tominaga R., Makino K., Kida K., Koyama H., Antibody-dependent enhancement of feline infectious peritonitis virus infection in feline alveolar macrophages and human monocyte cell line U937 by serum of cats experimentally or naturally infected with feline coronavirus. J. Vet. Med. Sci. 60, 49–55 (1998).
    1. Kam Y. W., Kien F., Roberts A., Cheung Y. C., Lamirande E. W., Vogel L., Chu S. L., Tse J., Guarner J., Zaki S. R., Subbarao K., Peiris M., Nal B., Altmeyer R., Antibodies against trimeric S glycoprotein protect hamsters against SARS-CoV challenge despite their capacity to mediate FcγRII-dependent entry into B cells in vitro. Vaccine 25, 729–740 (2007).
    1. Wan Y., Shang J., Sun S., Tai W., Chen J., Geng Q., He L., Chen Y., Wu J., Shi Z., Zhou Y., Du L., Li F., Molecular mechanism for antibody-dependent enhancement of coronavirus entry. J. Virol. 94, e02015–e02019 (2020).
    1. Wang S.-F., Tseng S.-P., Yen C.-H., Yang J.-Y., Tsao C.-H., Shen C.-W., Chen K.-H., Liu F.-T., Liu W.-T., Chen Y.-M. A., Huang J. C., Antibody-dependent SARS coronavirus infection is mediated by antibodies against spike proteins. Biochem. Biophys. Res. Commun. 451, 208–214 (2014).
    1. Esterre P., Ait-Saadi A., Arowas L., Chaouche S., Corre-Catelin N., Fanaud C., Laude H., Mellon V., Monceaux V., Morizot G., Najjar I., Ottone C., Perlaza B. L., Rimbault B., Sangari L., Ungeheuer M.-N., The ICAReB platform: A human biobank for the Institut Pasteur and beyond. Open J. Bioresour. 7, 1 (2020).
    1. Lorin V., Mouquet H., Efficient generation of human IgA monoclonal antibodies. J. Immunol. Methods 422, 102–110 (2015).
    1. Elbe S., Buckland-Merrett G., Data, disease and diplomacy: GISAID’s innovative contribution to global health. Glob. Chall. 1, 33–46 (2017).
    1. Temmam S., Chretien D., Bigot T., Dufour E., Petres S., Desquesnes M., Devillers E., Dumarest M., Yousfi L., Jittapalapong S., Karnchanabanthoeng A., Chaisiri K., Gagnieur L., Cosson J.-F., Vayssier-Taussat M., Morand S., Moutailler S., Eloit M., Monitoring silent spillovers before emergence: A pilot study at the tick/human interface in thailand. Front. Microbiol. 10, 2315 (2019).
    1. Iglesias M. C., Mollier K., Beignon A.-S., Souque P., Adotevi O., Lemonnier F., Charneau P., Lentiviral vectors encoding HIV-1 polyepitopes induce broad CTL responses in vivo. Mol. Ther. 15, 1203–1210 (2007).

Source: PubMed

Sponzoři a spolupracovníci

Zdravotní podmínky

Drogové intervence

3
Předplatit