key: cord-0323743-xbf0x1he
authors: Brochot, E.; Souplet, V.; Follet, P.; Ponthieu, P.; Olivier, C.; Even, G.; Audebert, C.; Malbec, R.
title: A multiplex serological assay for the characterization of IgG immune response to SARS-CoV-2
date: 2021-09-24
journal: nan
DOI: 10.1101/2021.09.23.21262329
sha: 83a92804b0e5bad43ec8ddc853ff32aef5275d8b
doc_id: 323743
cord_uid: xbf0x1he

Background: In the fight against SARS-COV-2, the development of serological assays based on different antigenic domains represent a versatile tool to get a comprehensive picture of the immune response or differentiate infection from vaccination beyond simple diagnosis. Objectives: Here we use a combination of the Nucleoprotein (NP), the Spike 1 (S1) and Spike 2 (S2) subunits, and the receptor binding domain (RBD) and N-terminal domain (NTD) of the Spike antigens from the Syrius-CoViDiag multiplex IgG assay, to follow the immune response to SARS-CoV-2 infection over a long time period and depending on disease severity. Results: Using a panel of 209 sera collected from 61 patients up to eight months after infection, we observed that most patients develop an immune response against multiple viral epitope, but anti-S2 antibodies seemed to last longer. For all the tested IgGs, we have found higher titers for hospitalized patients than for non-hospitalized ones. Moreover the combination of the five different IgG titers increased the correlation to the neutralizing antibody titers than if considered individually. Conclusion: Multiplex immunoassays have the potential to improve diagnostic performances, especially for ancient infection or mild form of the disease presenting weaker antibody titers. Also the combined detection of anti-NP and anti-Spike-derived domains can be useful to differentiate vaccination from viral infection and accurately assess the antibody potential to neutralize the virus.

Background: In the fight against SARS-COV-2, the development of serological assays based on 24 different antigenic domains represent a versatile tool to get a comprehensive picture of the immune 25 response or differentiate infection from vaccination beyond simple diagnosis. 26

Objectives: Here we use a combination of the Nucleoprotein (NP), the Spike 1 (S1) and Spike 2 27 (S2) subunits, and the receptor binding domain (RBD) and N-terminal domain (NTD) of the Spike 28 antigens from the Syrius-CoViDiag ® multiplex IgG assay, to follow the immune response to 29 SARS-CoV-2 infection over a long time period and depending on disease severity. 30

Results: Using a panel of 209 sera collected from 61 patients up to eight months after infection, 31

we observed that most patients develop an immune response against multiple viral epitope, but 32 anti-S2 antibodies seemed to last longer. For all the tested IgGs, we have found higher titers for 33 hospitalized patients than for non-hospitalized ones. Moreover the combination of the five 34 different IgG titers increased the correlation to the neutralizing antibody titers than if considered 35 individually. 36

Conclusion: Multiplex immunoassays have the potential to improve diagnostic performances, 37 especially for ancient infection or mild form of the disease presenting weaker antibody titers. Also 38 the combined detection of anti-NP and anti-Spike-derived domains can be useful to differentiate 39 vaccination from viral infection and accurately assess the antibody potential to neutralize the virus. 40 41

Since its first detection in Wuhan (China) in December 2019, the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has rapidly spread to reach other countries worldwide as 48 the coronavirus 2019 disease (COVID-19) became pandemic (1) . 49

The virion has a nucleocapsid composed by genomic RNA and phosphorylated 50 Nucleocapsid (NP) protein, which is buried inside a phospholipid bilayer and covered by the Spike 51 proteins trimmers (S) that gives the CoVs their crown-like appearance on which their names are 52 based. The S protein has two subunits, the Spike 1 (S1) which contains the receptor-binding 53 domain (RBD) and N-terminal domain (NTD) and the Spike 2 (S2) (2). The choice of the antigenic 54 domain is important, as it must be specific to the SARS-CoV-2 for discrimination against other 55 hCoVs for example, and sensitive enough so infection would not be missed (Brochot et al., 2020) . 56

Also, anti-RBD antibodies are known to play a role in patients protection as this domain is used 57 by the virus to penetrate host cells (4). Most commercial serological assays have demonstrated 58 satisfying performances in terms of diagnostic sensitivity and specificity, based on one of those 59 main different antigenic domains (5,6). However, the combination of different immunogenic 60 antigens can give a more comprehensive picture of the humoral response strength and diversity 61 (7-9) while maintaining elevated diagnostic performances (10,11). In multiplex assays, positivity 62 thresholds can be adjusted to compensate for the use of antigenic domains more conserved between 63 coronaviruses (12). Moreover, as vaccines are based on the Spike protein, the additional detection 64 of anti-NP antibodies allows to differentiate viral infection from vaccination. 65

This study reports the design and use of the Syrius-CoViDiag ® multiplex IgG assay for the 66 characterization of the immune response against over time, depending on disease severity, and in 67 perspective of neutralizing antibody titers. 68 . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint The SirYus-CoViDiag® multiplex immunoassay targets IgG antibodies against five different 84 antigens of the virus: NP, S1, S2, RBD, and NTD ( Fig. 1 ). Note that the S1 and NP antigens have 85 been printed in dot replicates in the shape of an "S" and "N" letters, respectively. This design 86 allows for quick visual interpretation of seropositivity and vaccination status. The results have 87 been automatically delivered using the SciReader® plate reader (Scenion GmbH) and associated 88 analysis software, and an algorithm combining different cut-offs for the different antigens 89 according to the manufacturer instructions. The spot mean signal intensity (MSI) was calculated 90 as the average pixel value inside the spot perimeter minus the local background around the spot as 91 described in Malbec et al., 2020. When multiplexing, the positivity thresholds can be adjusted with 92 the number of different IgG antibodies detected. As NP and S2 antigens are more conserved 93 between coronaviruses, SARS-CoV-2 IgG positivity is declared when a single one of them gives 94 a signal over 40 MSI. However when NP and S2 antibodies are concomitantly present, the cut-off 95 is adjusted to 20 MSI. For S1, RBD, and NTD antibodies a cut-off of 10 MSI is applied for SARS-96

CoV-2 positivity. Based on this algorithm, the test has been accredited by the French National 97

Reference Center (CNR) in August 2020. On a reference cohort of 48 sera from patients positive 98

to Covid-19 and 48 sera from patients negative to Covid-19. 14 days post symptoms, the diagnostic 99 sensitivity was 90 %. The diagnostic specificity was 100 %. 100 . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. 

. CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint anti-S1 and anti-RBD titers is very similar, as RBD constitutes a domain of the Spike 1 protein. 194 For all the tested IgGs, we have found higher titers for hospitalized patients than for non-195 hospitalized ones. However, the differences were not statistically significant as a large number of 196 patients had no immune response detected for individual antigens, independently of the disease 197 severity. 198

It is noteworthy that most commercial assays performances have been established at the 199 beginning of the epidemic, when samples from hospitalized patients were the easier to collect. 200 . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. antibodies. We recommend using assays targeting IgGs for the evaluation of a long lasting 217 population protection and collective immunity. Furthermore, multiplexed assays have the 218 potential to slightly increase diagnostic performances, especially for ancient or weak infections 219 and be more representative of immune protection. For future epidemical studies, as the 220 vaccination based on the Spike protein progresses, multiplex serological assays may also help to 221 differentiate vaccination from viral infection. 222 223 . CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) preprint 

. CC-BY-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. 

A pneumonia outbreak 233 associated with a new coronavirus of probable bat origin

Coronavirus infections and immune 236 responses

Anti-Spike, 238 anti-Nucleocapsid and neutralizing antibodies in SARS-CoV-2 hospitalized patients and 239 asymptomatic carriers

The 242 receptor binding domain of the viral spike protein is an immunodominant and highly 243 specific target of antibodies in SARS-CoV-2 patients

Comparison of 246 different serological assays for SARS-CoV-2 in real life

Detection of SARS-CoV-2 antibodies using commercial assays and 249 seroconversion patterns in hospitalized patients

Comparison of 252 SARS-CoV-2 serological tests with different antigen targets

256 A multiplex chemiluminescent immunoassay for serological profiling of COVID-19-257 positive symptomatic and asymptomatic patients

Magnitude and kinetics of anti-SARS-CoV-2 antibody responses and their relationship to 260 disease severity

Analysis of 263 SARS-CoV-2 Antibodies in COVID-19 Convalescent Blood using a Coronavirus Antigen 264

Evaluating 267 SARS-CoV-2 spike and nucleocapsid proteins as targets for antibody detection in severe 268 and mild COVID-19 cases using a Luminex bead-based assay

A Sequence Homology 271 and Bioinformatic Approach Can Predict Candidate Targets for Immune Responses to 272 SARS-CoV-2

Longitudinal 274 Analysis and Comparison of Six Serological Assays up to Eight Months Post-COVID-19 275 Diagnosis

Agrodiag PorCoV: A multiplex immunoassay for the differential diagnosis of porcine 278 enteric coronaviruses