key: cord-0878424-vopfmuw0
authors: Carreño, Juan Manuel; Mendu, Damodara Rao; Simon, Viviana; Shariff, Masood A.; Singh, Gagandeep; Menon, Vidya; Krammer, Florian
title: Longitudinal analysis of SARS-CoV-2 seroprevalence using multiple serology platforms
date: 2021-07-31
journal: iScience
DOI: 10.1016/j.isci.2021.102937
sha: 599dda9a2989402fafeec98e22feac6d5a0dff9d
doc_id: 878424
cord_uid: vopfmuw0

Current SARS-CoV-2 serological tests are based on the full-length spike (S), the receptor binding domain (RBD), or the nucleoprotein (NP) as substrates. Here, we used samples from health care workers (HCWs) to perform a longitudinal analysis of the antibody responses using a research-grade RBD and spike based enzyme-linked immunosorbent assay (ELISA), a commercial RBD and spike based ELISA, and a commercial NP based chemiluminescent microparticle immunoassay. Seroprevalence ranged around 28% early during the pandemic and a good correlation was observed between RBD and spike based ELISAs. Modest correlations were observed between NP and both RBD and spike based assays. The antibody levels in HCWs declined over time, however the overall seroprevalence measured by RBD and spike based assays remained unchanged, while the seroprevalence of NP reactive antibodies significantly declined. Moreover, RBD and spike based assays effectively detected seroconversion in vaccinees. Overall, our results consolidate the strength of different serological assays to assess the magnitude and duration of antibodies to SARS-CoV-2.

In the advent of the current pandemic caused by the severe acute respiratory syndrome coronavirus 2 36 (SARS-CoV-2), pandemic, methods to detect the prevalence of recent and past infections are key to 37 determine public health and social countermeasures. Nucleic acid amplification tests (NAAT) provide an 38 accurate estimation of acute infections (Chu et al., 2020; Pan et al., 2020) , but they fail to inform about 39 past infections. Serological tests that detect antibodies directed against structural targets of the virus, not 40 only are useful to estimate the overall viral seroprevalence and rates of infection in the population (Angulo 41 for RBD-Spike reactive antibodies in phase I (May, 2020) using the Kantaro assay became undetectable 97 in phase II (Aug-Oct, 2020) (Fig. 2B, 2E) , whereas two individuals that tested negative for RBD-Spike 98 reactive antibodies in phase I (May, 2020) using our in-house assay seroconverted in phase II ( Fig. 2A) . 99

Of note, in these particular cases all the positive/negative values were close to the cutoff of the 100 corresponding assays. antibodies. We measured reactivity in serum of individuals who had received two doses of SARS-CoV-2 132 mRNA vaccines. The expectation was, that the RBD/spike based assays would detect a signal while the 133 NP based assay would not. Indeed, we measured high titers using the Spike based assay platforms (the 134 Mount Sinai and Kantaro assays) but the samples produced no signal in the NP-based assay (Fig. 5A-5C) . 135

Of note, the spike titers measured in the Mount Sinai and Kantaro assay correlated very well (Fig. 5D) . While antibody responses to acute SARS-CoV-2 infection are relatively well understood, less data is 139 available regarding antibody kinetics over longer time frames against different viral antigens. We 140 determined seroprevalence and antibody titers in SARS-CoV-2 infected individuals at two time points (1-141 2 months and 3-4 months post infection) using three different assays. One assay, the Mount Sinai ELISA, 142 is a laboratory-developed assay that uses an initial ELISA at a single serum dilution against the RBD 143 followed by a confirmation and titration against the full-length spike protein. The second assay tested, the 144 Kantaro SeroKlir assay, is based on the same principle, but commercially available. The third assay, the 145 Abbott Architect, targets the NP and is a CMIA. 146

There was high concordance among the three assays with respect to seroprevalence during phase 1. 147 However, the titers only correlated well for the two spike-based assays. During phase 2, the two spike- is short-lived (Grandjean et al., 2020) . Importantly, and as expected since no NP is included in the FDA 156 EUA approved vaccines used in the US, only the spike-based assays were able to detect antibodies induced 157 by SARS-CoV-2 mRNA vaccines (Krammer, 2020) . Our data highlight the need to understand assay 158 J o u r n a l P r e -p r o o f performance before a specific assay is used to study specific aspects of SARS-CoV-2 immunity. All three 159 assays are very valuable to assess seroconversion shortly after infection, but only the two spike-based 160 assays were reliable months after recovery. Similarly, only spike-based assays are fit for measuring 161 vaccine-induced antibodies, e.g. to determine if vaccination triggered immune responses. 162 163

We used samples from health care workers (HCWs) to perform a longitudinal analysis of the antibody 165 responses using three distinct serological assays. We detected good correlations between the spike-based 166 assays, but modest correlations between the NP-based assay and spike-based assays. The seroprevalence 167 as measured by the NP-based assay significantly declined, given that the Abbott test failed to detect a 

Further information and requests for resources should be directed to and will be fulfilled by the lead 268 contact, Florian Krammer (florian.krammer@mssm.edu). 269

Recombinant proteins generated in this study and plasmids encoding for recombinant proteins can be 271 requested to the Krammer laboratory (https://labs.icahn.mssm.edu/krammerlab/reagents/). 272

 All data reported in this paper and codes will be shared by the lead contact upon request 274  This paper does not report original code 275  Any additional information required to reanalyze the data reported in this paper is available upon 276 request to the lead contact 277

The samples used for the longitudinal study, were part of a cross sectional cohort of healthcare workers 279 three times with PBS-T and 50μl/well of anti-human IgG (Fab-specific) horseradish peroxidase antibody 310 (Sigma, A0293) diluted 1:3,000 in PBS-T 1% milk powder were added. Plates were incubated for 1 h at 311 room temperature, followed by three times washing with PBS-T and addition of developing solution 312 (100μl/well) sigmafast o-phenylenediamine dihydrochloride (Sigma). The reaction was led to proceed for 313 10 min, and stopped using 50μl/well of 3-molar hydrochloric acid (Thermo Fisher Scientific). Optical 314 density was measured at 490 nm using an automated plate reader (BioTek). Samples with an OD490nm 315 above 0.15 (cut-off value) were considered as presumptive positives and were further tested in the 316 confirmatory ELISA using the full-length recombinant spike protein. 317 318 Briefly, to perform the confirmatory ELISAs, plates were coated and blocked as described above, but 319 using full-length spike protein for coating. Presumptive positive plasma samples were serially diluted (1:3) 320 in 1%-milk prepared in PBS-T, starting at an initial dilution of 1:80. Serial dilutions (100μl/well) were 321 added to the plates, followed by 2-hour incubation at room temperature. The remaining steps were 322 performed as described above. Data was analyzed using GraphPad Prism 7. Samples with an OD490nm 323 CoV-2 antigen coated microparticles. The mixture is washed. Anti-human IgG acridinium-labeled 363 conjugate is added to create a reaction mixture and incubated. Following a wash cycle, Pre-Trigger and 364

Trigger Solutions are added. The resulting chemiluminescent reaction is measured as a relative light unit 365 (RLU). There is a direct relationship between the amount of IgG antibodies to SARS-CoV-2 in the sample 366 and the RLU detected by the system optics. This relationship is reflected in the calculated Index (S/C). 367

The presence or absence of IgG antibodies to SARS-CoV-2 in the sample is determined by comparing the 368 chemiluminescent RLU in the reaction to the calibrator RLU. 

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