key: cord-0793580-tfovpo7h
authors: Criscuolo, E.; Diotti, R. A.; Strollo, M.; Rolla, S.; Ambrosi, A.; Locatelli, M.; Burioni, R.; Mancini, N.; Clementi, M.; Clementi, N.
title: Poor correlation between antibody titers and neutralizing activity in sera from SARS-CoV-2 infected subjects
date: 2020-07-11
journal: nan
DOI: 10.1101/2020.07.10.20150375
sha: c48421cbad638e7aaaf388d5148f7e7c95f4eee3
doc_id: 793580
cord_uid: tfovpo7h

Plenty of serologic tests for SARS-CoV-2 have been developed so far, thus documenting the importance of evaluating the relevant features of the immune response to this viral agent. The performance of these assays is currently under investigation. Amongst them, LIAISON SARS-CoV-2 S1/S2 IgG by DiaSorin and Elecsys Anti-SARS-CoV-2 cobas by Roche are currently used by laboratory medicine hospital departments in Italy and many other countries. In the present study, we have firstly compared two serologic tests on serum samples collected at two different time points from forty-six laboratory-confirmed COVID-19 subjects. Secondly, eighty-five negative serum samples collected before the SARS-CoV-2 pandemic were analyzed. Thirdly, possible correlations between antibody levels and the resulting neutralizing activity against a clinical isolate of SARS-CoV-2 were evaluated. Results revealed that both tests are endowed with low sensitivity on the day of hospital admission, which increased to 97.8 and 100% for samples collected after 15 days for DiaSorin and Roche tests, respectively. The specificity of the two tests ranges from 96.5 to 100%, respectively. Importantly, a poor direct correlation between antibody titers and neutralizing activity levels was evidenced in the present study.

Tracking the seroprevalence of SARS-CoV-2 positive subjects certainly represents an urgent (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted July 11, 2020 . . https://doi.org/10.1101 3 from symptoms onset for and IgG within approximatively seven days (2-5). To date, a plethora 46 of serologic tests are invading the market, and some of them have been evaluated (6). However, 47 other assays deserve further analyses since there is no consensus so far on antigens used for the 48 antibody testing nor for the antibody isotype to be detected. These last aspects can be of pivotal 49 importance for evaluating antibody response to candidate vaccines, for selecting plasmas for 50 clinical trials, and to dissect unknown immunological aspects related to seroconversion.

Indeed, as of 8th July 2020, no correlations between seroconversion, neutralizing activity, and 52 immunity have been made (7). In the present study, we evaluated the performances of two 53 commercial serology tests, the LIAISON® SARS-CoV-2 S1/S2 IgG by DiaSorin and Elecsys the hospital (T0) and 15 days later (T15). Eighty-five "pre-pandemic" serum samples, spanning 66 from 2012 to 2018, were also tested for the presence of anti-SARS-CoV-2 antibodies. 67 68 All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted July 11, 2020. . https://doi.org/10. 1101 Immunoassays. Elecsys Anti-SARS-CoV-2 cobas® by Roche and LIAISON® SARS-CoV-2 69 S1/S2 IgG assay by DiaSorin were used for detecting anti-SARS-CoV-2 antibodies in all serum 70 samples. Analyses were performed according to manufacturer's instruction by using cobas® 71 and LIAISON® XL Analyzer platforms. In brief, Elecsys by Roche uses a recombinant SARS- (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted July 11, 2020. . https://doi.org/10.1101/2020.07.10.20150375 doi: medRxiv preprint 5 cells were infected with eight 10-fold dilutions of virus stock. After 1 h of adsorption at 37°C, 93 the cell-free virus was removed. Cells were then incubated for 48 h in DMEM containing 2% 94 FBS and 0.5% agarose. Cells were fixed and stained, and viral plaques were counted. In EDA, 95 Vero E6 cells were seeded into 96 wells plates and infected at 95% of confluency with base 10 96 dilutions of virus stock. After 1 h of adsorption at 37°C, the cell-free virus was removed, cells 97 were washed with PBS 1X and complete medium was added to cells. After 48h, cells were with the associated standard error (SE) and 95% confidence interval (CI). Differences between 115 sensitivities and specificities, respectively, were assessed by exact binomial test for paired 116 All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted July 11, 2020. . https://doi.org/10.1101/2020.07.10.20150375 doi: medRxiv preprint 6 study design (10). The overall performances were also measured, by means of the ROC curves 

Sensitivity and specificity of DiaSorin and Roche diagnostic tests. Pre-pandemic sera were 127 tested with DiaSorin and Roche diagnostic assays (Fig. 1A) . Three out of 85 samples tested 128 positive with DiaSorin, and one tested in the "grey-zone" (12.0-15.0 AU/mL) with the same 129 test. Thus, the diagnostic specificity observed on the tested samples for the DiaSorin assay was 130 96.5% (SE: 2%; 95% CI: 92.5-100%) (Fig. 1C) . Then, forty-six serum samples were randomly 131 collected from laboratory-confirmed symptomatic COVID-19 patients. Admission to the 132 hospital (T0) and 15 days later (T15) time points were evaluated for each patient, showing an 133 overall increase in serum IgG titers 15 days after hospital admission (Fig. 1B) . The diagnostic 134 sensitivity at T0 was 19.6% (SE: 5.8%; 95% CI: 8.1-31%), and at T15 was 100% (SE: 0%; 135 95% CI: 100-100%). On the other hand, the diagnostic specificity of the Roche test was 100% 136 (SE: 0%; 95% CI: 100-100%) and its diagnostic sensitivity at T0 was 45.7% (SE: 7.3%; 95% 137 CI: 31.2-60%), and at T15 was 100% (SE: 0%; 95% CI: 100-100%). Roc curves, calculated 138 for the two tests at the two different time points, show comparable performance at T15 (P = 139 All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted July 11, 2020. . https://doi.org/10.1101/2020.07.10.20150375 doi: medRxiv preprint 0.2961). Thus, Roche diagnostic test showed a statistically better performance than DiaSorin 140 at T0 on the tested samples (P < 0.001) (Fig. 1D) .

Neutralizing activity evaluation of a limited cohort of serum samples. Five patients were 143 randomly selected for the characterization of the neutralizing activity of their serum samples 144 against SARS-CoV-2, at both T0 and T15 (Fig. 2) . Results showed that samples collected at Neutralizing activity evaluation of all tested sera. As the previous analysis showed that no 156 neutralizing activity was detected at T0, all remaining sera were tested at T15. Moreover, all 157 T15 samples were tested at a 1:200 dilution, based on what observed in the five sera tested at 158 different dilutions. Sera neutralizing activity does not directly correlate with antibody titers 159 detected with both DiaSorin and Roche test, as highlighted by the graph (Fig. 3A) . Moreover, 160 no apparent relationship between ICU admission and both antibody titer and neutralizing 161 activity was observed. None of the five "pre-pandemic" sera checked for their neutralizing 162 activity, including those testing positive with the DiaSorin kit, neutralized the virus. Spearman 163 All rights reserved. No reuse allowed without permission.

(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted July 11, 2020. . https://doi.org/10.1101/2020.07.10.20150375 doi: medRxiv preprint 8 correlation analysis confirms the lack of correlation between anti-SARS-CoV-2 antibody 164 presence detected with both diagnostic serologic assays and the neutralizing activity (Fig. 3B) . (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted July 11, 2020 . . https://doi.org/10.1101 bind a recombinant form of nucleocapsid protein N of SARS-CoV-2. Whereas, DiaSorin assay 188 detects IgGs able to bind the S1 or the S2 recombinant portions of the virus spike glycoprotein.

The S protein mediates at least two crucial steps in the early phases of the viral productive (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this preprint this version posted July 11, 2020. . https://doi.org/10. 1101 investigations elucidating the clinical role of neutralizing antibodies and the possibility of 213 detecting them with binding assays will be of paramount importance for addressing the 214 development of effective vaccines.

SARS-CoV-2 infection serology: a useful tool to overcome lockdown?

Evaluation of Nucleocapsid and Spike Protein-Based 222

Enzyme-Linked Immunosorbent Assays for Detecting Antibodies against SARS-223

Temporal profiles of viral load in 228 posterior oropharyngeal saliva samples and serum antibody responses during 229 infection by SARS-CoV-2: an observational cohort study

Immune phenotyping based on neutrophil-to-lymphocyte ratio and IgG 233 predicts disease severity and outcome for patients with COVID-19

All rights reserved. No reuse allowed without permission.(which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint this version posted July 11, 2020. . https://doi.org/10.1101/2020.07.10.20150375 doi: medRxiv preprint 11 5.Long Q, Deng H, Chen J, Hu J, Liu B, Liao P, medRxiv YL, 2020 (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint this version posted July 11, 2020. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint this version posted July 11, 2020. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint this version posted July 11, 2020. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint this version posted July 11, 2020. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint this version posted July 11, 2020. (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint this version posted July 11, 2020. . https://doi.org/10. 1101