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[Preprint]. 2021 Feb 12:2021.02.10.21251518.
doi: 10.1101/2021.02.10.21251518.

Antigen-based multiplex strategies to discriminate SARS-CoV-2 natural and vaccine induced immunity from seasonal human coronavirus humoral responses

Eric D Laing  1 , Spencer L Sterling  1   2 , Stephanie A Richard  2   3 , Nusrat J Epsi  2   3 , Si'Ana Coggins  1   2 , Emily C Samuels  1   2 , Shreshta Phogat  1   2 , Lianying Yan  1   2 , Nicole Moreno  2   3 , Christian L Coles  2   3 , Matthew Drew  4 , Jennifer Mehalko  4 , Caroline E English  2   3 , Scott Merritt  2   3   5 , Katrin Mende  2   3   5 , Vincent J Munster  6 , Emmie de Wit  6 , Kevin K Chung  7 , Eugene V Millar  2   3 , David R Tribble  3 , Mark P Simons  1   3 , Simon D Pollett  2   3 , Brian K Agan  2   3 , Dominic Esposito  4 , Charlotte Lanteri  2 , G Travis Clifton  5 , Edward Mitre  1 , Timothy H Burgess  2 , Christopher C Broder  1
Affiliations

Antigen-based multiplex strategies to discriminate SARS-CoV-2 natural and vaccine induced immunity from seasonal human coronavirus humoral responses

Eric D Laing et al. medRxiv. .

Abstract

Sensitive and specific SARS-CoV-2 antibody assays remain critical for community and hospital-based SARS-CoV-2 sero-surveillance. With the rollout of SARS-CoV-2 vaccines, such assays must be able to distinguish vaccine from natural immunity to SARS-CoV-2 and related human coronaviruses. Here, we developed and implemented multiplex microsphere-based immunoassay strategies for COVD-19 antibody studies that incorporates spike protein trimers of SARS-CoV-2 and the endemic seasonal human coronaviruses (HCoV), enabling high throughout measurement of pre-existing cross-reactive antibodies. We varied SARS-CoV-2 antigen compositions within the multiplex assay, allowing direct comparisons of the effects of spike protein, receptor-binding domain protein (RBD) and nucleocapsid protein (NP) based SARS-CoV-2 antibody detection. Multiplex immunoassay performance characteristics are antigen-dependent, and sensitivities and specificities range 92-99% and 94-100%, respectively, for human subject samples collected as early as 7-10 days from symptom onset. SARS-CoV-2 spike and RBD had a strong correlative relationship for the detection of IgG. Correlation between detectable IgG reactive with spike and NP also had strong relationship, however, several PCR-positive and spike IgG-positive serum samples were NP IgG-negative. This spike and NP multiplex immunoassay has the potential to be useful for differentiation between vaccination and natural infection induced antibody responses. We also assessed the induction of de novo SARS-CoV-2 IgG cross reactions with SARS-CoV and MERS-CoV spike proteins. Furthermore, multiplex immunoassays that incorporate spike proteins of SARS-CoV-2 and HCoVs will permit investigations into the influence of HCoV antibodies on COVID-19 clinical outcomes and SARS-CoV-2 antibody durability.

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Conflict of interest statement

CONFLICT OF INTEREST None of the authors have any conflicts of interest of relevance to disclose.

Figures

Figure 1.
Figure 1.. Pre-existing cross-reactivity with SARS-CoV-2 antigens informs threshold cutoffs for antibody positivity.
Acute and convalescent serum samples from SARS-CoV-2 naïve HCoV PCR-positive subjects were tested in β-CoV (A–B) and SARS-CoV-2 spike/NP (C) MMIA. SARS-CoV-2 antigens are indicated. Subjects are grouped together based on HCoV PCR confirmation, HCoV-OC43 (n= 16), HCoV-HKU1 (n= 6), HCoV-NL63 (n= 13) and HCoV-229E (n= 10). MFI, median fluorescence intensity; IgG MFI values represent the MEAN of independent experiments performed in technical duplicates.
Figure 2.
Figure 2.. Detection of SARS-CoV-2 reactive serum IgG with S glycoprotein and nucleoprotein antigens.
Serum samples were tested in β-CoV MMIA for reactivity with SARS-CoV-2 spike (A) and RBD (B). (C) Correlation analysis of IgG binding to SARS-CoV-2 spike and RBD when tested simultaneously in the β-CoV MMIA. Serum samples were tested in SARS-2 spike/NP MMIA for reactivity with SARS-CoV-2 spike (D) and NP (E). (F) Correlation analysis of IgG binding to SARS-CoV-2 spike and NP when tested simultaneously in the SARS-2 spike/NP MMIA.
Figure 3.
Figure 3.. SARS-2/HCoV MMIA detection of SARS-CoV-2 IgG in blood specimens.
(A) Serum samples were screened with a SARS-2/HCoV spike protein MMIA and reactivity to SARS-CoV-2 spike is shown; shaded grey bar indicates the threshold cutoff for IgG positivity. (B) Paired blood specimens (n= 22) collected from capillary blood as a dried blood spot (DBS) and serum collected by serum separator tubes (SST) were tested with the SARS-2/HCoV spike protein MMIA; SARS-CoV-2 spike reactive IgG MFI is indicated on x- and y-axes.
Figure 4.
Figure 4.. SARS-CoV and MERS-CoV IgG increases after SARS-CoV-2 infection.
(A) 99.7% probability distribution of ARIC HCoV PCR+ convalescent serum samples (n= 43) reactive with SARS-CoV (SARS-1) and MERS-CoV (MERS) spike; a dashed line and solid line indicates the SARS-CoV and MERS-CoV spike protein threshold cutoff values, respectively. (B) Sera from SARS-CoV-2 positive EPICC and JMS cohorts were tested in a β-CoV MMIA for IgG reactivity to SARS-CoV and MERS-CoV spike, a shaded grey line indicates the threshold cutoff for IgG positivity; error bars indicate the geometric mean and 95% confidence intervals; unpaired Mann-Whitney t-tests of EPICC and JMS compared to ARIC, **** P-values= < 0.0001.
Figure 5.
Figure 5.. SARS-CoV-2 infection is associated with rises in HCoV antibody levels.
(A) Serum samples collected 21 dpi from SARS-CoV-2 challenged non-human primates (n= 4) and tested in two independent experiments performed in technical duplicates with a SARS-2/HCoV spike protein MMIA; error bars represent mean±SD. IgG reactivity with seasonal HCoV, -HKU1 and -OC43 (β-CoVs) (B) and −229E and -NL63 (α-CoVs) (C), spike proteins were tested with a SARS-2/HCoV spike MMIA. Error bars indicate the geometric mean and 95% CI, IgG levels were compared by unpaired t-tests with Welch’s correction of EPICC and JMS compared to ARIC, **** P-values= < 0.0001.
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