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[Preprint]. 2020 Oct 16:2020.10.14.20207050.
doi: 10.1101/2020.10.14.20207050.

A betacoronavirus multiplex microsphere immunoassay detects early SARS-CoV-2 seroconversion and controls for pre-existing seasonal human coronavirus antibody cross-reactivity

Eric D Laing  1 , Spencer L Sterling  1   2 , Stephanie A Richard  3   2 , Shreshta Phogat  1   2 , Emily C Samuels  1   2 , Nusrat J Epsi  3   2 , Lianying Yan  1   2 , Nicole Moreno  3   2 , Christian Coles  3   2 , Jennifer Mehalko  4 , Matthew Drew  4 , Caroline English  3   2 , Kevin K Chung  5 , G Travis Clifton  6 , Vincent J Munster  7 , Emmie de Wit  7 , David Tribble  3 , Brian K Agan  3   2 , Dominic Esposito  4 , Charlotte Lanteri  3 , Edward Mitre  1 , Timothy H Burgess  3 , Christopher C Broder  1
Affiliations

A betacoronavirus multiplex microsphere immunoassay detects early SARS-CoV-2 seroconversion and controls for pre-existing seasonal human coronavirus antibody cross-reactivity

Eric D Laing et al. medRxiv. .

Abstract

With growing concern of persistent or multiple waves of SARS-CoV-2 in the United States, sensitive and specific SARS-CoV-2 antibody assays remain critical for community and hospital-based SARS-CoV-2 surveillance. Here, we describe the development and application of a multiplex microsphere-based immunoassay (MMIA) for COVD-19 antibody studies, utilizing serum samples from non-human primate SARS-CoV-2 infection models, an archived human sera bank and subjects enrolled at five U.S. military hospitals. The MMIA incorporates prefusion stabilized spike glycoprotein trimers of SARS-CoV-2, SARS-CoV-1, MERS-CoV, and the seasonal human coronaviruses HCoV-HKU1 and HCoV-OC43, into a multiplexing system that enables simultaneous measurement of off-target pre-existing cross-reactive antibodies. We report the sensitivity and specificity performances for this assay strategy at 98% sensitivity and 100% specificity for subject samples collected as early as 10 days after the onset of symptoms. In archival sera collected prior to 2019 and serum samples from subjects PCR negative for SARS-CoV-2, we detected seroprevalence of 72% and 98% for HCoV-HKU1 and HCoV-0C43, respectively. Requiring only 1.25 μL of sera, this approach permitted the simultaneous identification of SARS-CoV-2 seroconversion and polyclonal SARS-CoV-2 IgG antibody responses to SARS-CoV-1 and MERS-CoV, further demonstrating the presence of conserved epitopes in the spike glycoprotein of zoonotic betacoronaviruses. Application of this serology assay in observational studies with serum samples collected from subjects before and after SARS-CoV-2 infection will permit an investigation of the influences of HCoV-induced antibodies on COVID-19 clinical outcomes.

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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.. SARS-CoV-2 spike protein reactivity as a function of IgG concentration.
A sigmoidal curve was used to fit the MEAN±SEM of two independent experiments performed in technical triplicates. MFI, median fluorescence intensities.
Figure 2.
Figure 2.. MMIA displays enhanced sensitivity for SARS-CoV-2 IgG detection.
Serum samples from SARS-CoV-2 infected NHP collected 21 dpi were tested by SARS-CoV-2 spike protein (A) ELISA and (B) MMIA. (A) Dashed lines indicate 3-fold MFI above the NHP(−) serum sample(s) diluted 1:1000 and 1:2000. (B) A solid line indicates the lower limit of MFI linearity and a dashed line indicates 3-fold MFI above the NHP(−) serum sample diluted 1:4000. Positive samples are those above both the MFI level for curve linearity and 3-fold change in NHP(−) serum. MFI values represent MEAN±SD of two independent experiments performed in technical triplicates.
Figure 3.
Figure 3.. SARS-CoV-2 seroconversion in a non-human primate model.
Sera from SARS-CoV-2 infected NHP were screened for SARS-CoV-2 spike protein reactive (A) IgG and (B) IgM. Graphs represent the MEAN±SD of four SARS-CoV-2 challenged NHP screened in two independent experiments performed in technical duplicates. A solid line indicates a 4-fold rise in SARS-CoV-2 spike protein MFI from baseline (0 dpi) and was used as a threshold cutoff for SARS-CoV-2 IgG and IgM seroconversion; a dashed line indicates a 4-fold rise in SARS-CoV-2 RBD protein MFI from baseline.
Figure 4.
Figure 4.. Archival sera from subjects with seasonal HCoVs can display cross-reactivity with SARS-CoV-2 spike protein.
Acute and convalescent serum samples from HCoV PCR-positive subjects were tested in a β-CoV MMIA. Subjects are grouped together based on HCoV PCR confirmation, (A) OC43 (n= 16), (B) HKU1 (n= 6), (C) NL63 (n= 13) and (D) 229E (n= 10), A dashed line indicates the 3-fold change in the mean MFI of a mock antigen-coupled microsphere. MFI values represent the MEAN of two independent experiments performed in technical duplicates. MFI, median fluorescence intensity.
Figure 5.
Figure 5.. Archival sera generated threshold cutoffs for IgG and IgM antibodies confer specificity for SARS-CoV-2.
Convalescent serum samples (n= 43) from HCoV PCR-positive subjects were tested in the β-CoV MMIA with (A) IgG antibody and (B) IgM antibody. (C-D) Archival sera (n= 84), acute serum samples from HCoV PCR-positive subjects, acute/convalescent serum samples from rhinovirus PCR-positive subjects and acute/convalescent serum samples from ‘no pathogen detected’ subjects were tested for (C) IgG and (D) IgM antibody reactivity tested against the established threshold cutoffs. A solid line indicates the threshold cutoff for positivity with SARS-CoV-2 spike protein and a dashed line indicates the threshold cutoff for SARS-CoV-2 RBD. Colored dots in (C-D) indicate samples with MFI above the SARS-CoV-2 spike protein threshold cutoff for positive antibody. MFI is the average of sera diluted 1:400, adjusted with PBS controls and tested across technical duplicate plates. IgG data is a representation of three independent screenings.
Figure 6.
Figure 6.. A multiplex antibody test can detect SARS-CoV-2 specific and cross-reactive antibodies.
Serum samples from (A) SARS-CoV-2 PCR negative subjects (B) SARS-CoV-2 PCR-positive subjects collected ≥ 10 dpso were tested by β-CoV MMIA. Serum were diluted 1:400 and tested in duplicate plates. MFI, median fluorescence intensities, are the average of PBS-subtracted technical duplicates. A solid line indicates the IgG threshold cutoff and a dashed line indicates the IgM threshold cutoff. Colored dots indicate positive serum samples. SARS-2, SARS-COV-2; SARS-1, SARS-CoV-1; MERS, MERS-CoV; HKU1, HCoV-HKU1; OC43, HCoV-OC43.
Figure 7.
Figure 7.. Positive and negative results are reproducible over independent MMIA tests.
Selected positive(s) and negative serum samples were tested across independent experiments. CV, coefficient of variation, percentages are indicated on the graphs for each sample. A solid line indicates the threshold cutoff for positive IgG.
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