key: cord-0768814-6wf7saq8
authors: MENDRONE-JUNIOR, A.; DINARDO, C.; FERREIRA, S.; NISHIA, A.; SALLES, N.; ALMEIDA-NETO, C.; HAMASAKI, D.; FACINCANI, T.; ALVES, L.; MACHADO, R.; ARAUJO, D.; DURIGON, E.; ROCHA, V.; SABINO, E.
title: CORRELATION BETWEEN SARS-COV-2 ANTIBODY SCREENING BY IMMUNOASSAY AND NEUTRALIZING ANTIBODY TESTING
date: 2020-10-14
journal: nan
DOI: 10.1101/2020.10.11.20210005
sha: 1e07e3087b0f94f40918ade73cbf2bdfe9f460c2
doc_id: 768814
cord_uid: 6wf7saq8

Background: Passive antibody therapy with convalescent plasma (CP) represents a promising alternative for the treatment of SARS CoV 2 infection. The efficacy of CP therapy has been associated with high titers of neutralizing antibodies (nAbs) in the plasma of recovered patients, but the assays for quantifying nAbs are not widely available. Our goal was to develop a strategy to predict high titers of nAbs based on the results of anti-SARS CoV 2 immunoassays and the clinical characteristics of the CP potential donors. Methods: Two hundred and fourteen CP donors were enrolled and tested for the presence of anti-SARS CoV 2 antibodies using two commercial immunoassays (IA): Anti SARS CoV 2 ELISA IgG EUROIMMUN and Anti SARS CoV 2 Chemiluminescence IgG Abbott. In parallel, quantification of neutralizing antibodies (nAbs) was performed using the Cytopathic effect-based virus neutralization test (CPE VNT). Three criteria for identifying donors with high titers of nAbs (more than 160) were tested: Criterion1: Curve ROC Method; Criterion 2: Conditional decision tree considering only the results from the IA and Criterion 3: Conditional decision tree including both the IA results and the clinical variables. Results: The performance of Abbott and EUROIMMUN immunoassays was similar referring to both S/CO and predictive value for identifying nAbs titers more than 1:160. Regarding the three studied criteria for identifying CP donors with high nAbs titers (more than 1:160): 1) Criterion 1 showed 76.1% accuracy when the S/CO cut-off of 4.65 was used, 2) Criterion 2 presented 76.1% accuracy if the S/CO more than 4.57 was applied and 3) Criterion 3 had 71.6% accuracy if either S/CO more than 4.57 or S/CO between 2.68 and 4.57 and the last COVID-19-related symptoms occurred less than 19 days from donor recruiting were used. Conclusion: The results of SARS-CoV-2 immunoassays (S/CO) can be used to predict high nAbs titers of potential CP donors. This study has proposed three different criteria for identifying donors with more than 160 nAbs titer based on either solely S/CO results or S/CO together with clinical variables, all with high efficacy and accuracy.

The outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) represents an unprecedent challenge for the population, health workers and government all over the world, becoming a global public health emergency with growing impact on the global economy. On 11th March 2020, the World Health Organization (WHO) declared SARS-CoV-2 a pandemic. As of August 23 th , 2020, SARS-CoV-2 infection reached about 23 million confirmed cases worldwide in more than 213 countries and caused more than 800,000 deaths (https://covid19.who.int). To date, no specific treatment was proved to be effective for SARS-CoV-2 infection, beside supportive care.

Passive antibody therapy with convalescent plasma (CP), a classic adaptive immunotherapy, has been applied to the prevention and treatment of many infectious diseases over many decades, from A/H1N1 Spanish Flu in [1917] [1918] to SARS in 2012. 1 The efficacy of passive antibody therapy has been associated with the concentration of neutralizing antibodies (nAbs) in plasma of recovered patients. 2 CP from patients who have recovered from viral infection can be used to improve clinical conditions and survival rate of patients with acute viral infections, including SARS-CoV-2, without severe adverse effects. Preliminary data showed a reduction of viral load, shorter hospital stay and lower mortality in patients infected by SARS-CoV-2 treated with CP in comparison to those who were not. 3, [4] [5] [6] [7] [8] Possible mechanisms related to the efficacy of CP therapy in SARS-CoV-2 include the passive transfusion of neutralizing antibodies and an immunomodulatory effect via amelioration of severe inflammatory response. 9, 10 Patients infected with SARS-CoV-2 usually develop a primary immune response by days 10-14, which is followed by virus clearance. 11 Therefore, theoretically, it should be more effective to administer the CP at the early stage of disease. A recent matched study suggested that non-intubated patients may benefit more than those requiring mechanical ventilation. 12 However, other treatments might influence the relationship between CP and antibody level, including antiviral drugs, steroids, and intravenous immunoglobulin.

We are conducting a prospective randomized trial to evaluate the efficacy of CP for patients with moderate to severe SARS-CoV-2 disease. Convalescent donors have been recruited from the community. Pre-requisite for plasma donation include age (>18 years old); no previous pregnancy; time elapsed from the last day of symptoms (> 14 days); laboratorial evidence of prior infection by SARS-CoV-2; and screening negative for infectious diseases transmissible for blood (HIV 1+2, HTLV 1+2, hepatitis B and C, syphilis and Chagas Disease). Moreover, we also have evaluated the level of neutralizing antibodies (nAbs) and the absence of RNAemia in a blood samples before plasma collection. As nAbs play important roles on virus clearance and have been considered as a key immune product for treatment against viral diseases, in concordance with Food and Drug Administration (FDA), we stablished that CP unit for transfusion should contain nAbs with minimum titer of ≥1:160 (https://www.fda.gov/media/136798/download).

However, neutralization assays for SARS-CoV-2 are limited in availability and throughput, requiring biosafety level 3 facilities and skilled labor. Since such assay is often unavailable, one alternative is to perform the test later in a stored sample, or to perform another test to detect the presence of anti-SARS-CoV-2 antibody prior to issuing the plasma unit for transfusion.

The correlation between immunoassays antibodies titers and neutralizing antibodies has not been thoroughly investigated and the knowledge of this association can help to make better therapeutic decisions.

The aim of this study is to evaluate the performance of criteria based on the results of anti-SARS-CoV-2 immunoassays for the prediction of high nAbs titers in CP donors.

All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted October 14, 2020. . https://doi.org/10.1101/2020. 10.11.20210005 doi: medRxiv preprint Two hundred and sixty-three convalescent individuals were evaluated in April 2020 for plasma convalescent donation by apheresis. The SARS-Cov-2 infection was previously confirmed by Real Time Reverse Transcription-Polymerase Chain Reaction (RT-PCR) of material collected from the upper respiratory tract (nasopharynx or oropharynx). All candidates provided written informed consent and tested negative for SARS-CoV-2 by RT-PCR. Blood samples were collected from all participants for performing the SARS-CoV-2 IgG immunoassay and blood RT-PCR. Two hundred and fourteen were tested for neutralizing antibodies.

Two commercials immunoassays comprising the structural protein of SARS-CoV-2 (S1 domain) were tested in parallel with all collected samples: Anti-SARS-CoV-2 ELISA IgG EUROIMMUN (Lübeck, Germany) and Anti-SARS-CoV-2

Chemiluminescence IgG Abbott (Chicago, US). Tests were performed in accordance with the manufacturer's instructions.

Quantitative reverse-transcriptase polymerase chain reaction (RT-qPCR)

Blood samples with DO/CO ≥ 3 on the IgG immunoassay were subjected to SARS-CoV-2 RT-qPCR using TaqMan method. A quantitative in house real-time PCR assay amplifying the virus RdRp RNA-dependent RNA polymerase and envelope was applied to determine the copy number of SARS-CoV-2. 13 The test had sensitivity of approximately 100 copies/mL. In all amplification reactions, positive and negative controls and an exogenous internal control were used.

Two hundred and fourteen samples were tested for neutralizing antibodies (nAbs) were seeded 24 hours before the infection in a 96-well plate. Plasma samples were, initially, inactivated for 30 min at 56°C. We used 8 dilutions (two-fold) of each plasma (1:20 to 1:2560). Subsequently, plasma was mixed vol/vol with 10 3 TCID50/mL of SARS-CoV-2/human/BRA/SP02cc/2020 strain virus (GenBanK access number: MT350282.1) 16 and pre-incubated at 37°C for 1 hour to allow virus neutralization. Then, the plasma plus virus mixture was transferred onto the confluent cell monolayer and incubated for 3 days at 37°C, under 5% CO2. Virus neutralization titer referred to VNT100 is described as the highest dilution of serum that neutralized virus growth (absence of cytopathic effect). In each assay, a strong, assured internal positive control serum (RT-qPCR positive + PRNT90>640) 17 was used, as a negative pre-outbreak serum sample. All the procedures related to CPE-VNT were performed in a biosafety level 3 laboratory, in accordance with WHO recommendations 18 .

A descriptive analysis was carried out using frequencies, central tendency and position measures. Mann-Whitney and Kruskall-Wallis non-parametric tests were used to compare nAbs values in different groups and Bonferroni post-hoc method was applied to adjust results for multiple comparisons. The variables age and days since last symptom were tested according to groups from tertiles of the distribution values.

Simple linear regression models were used to assess the relationship between ELISA S/CO values and the concentration of nAbs titers. The predictive value of immunoassays tests (Abbott and Euroimmun) for the identification of nAbs ≥ 160 was assessed using ROC curve graphs. 19 To validate the proposed donation criteria, the total sample studied was divided into two parts (development sample and validation sample) according to the study All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted October 14, 2020. . were performed in the R environment using RStudio software. 19, 20 Results

There were 263 potential CP donors evaluated, of whom 49 were excluded from the analysis either because nAbs titers were lacking (n=35) or because the evaluation was performed less than 10 days after the symptom resolution (n=14)

Table1 shows the descriptive data of the analyzed sample. Most donors were male (57.9%) and young (median age was 35 years/IQR=15). The two most common clinical comorbidities were systemic arterial hypertension (8.7%) and pulmonary disease (6.5%).

The titers of nAbs of the studied sample varied widely Approximately 1 in each 5 donors (19.1%) had nAbs titers <1:80 (Figure2). Titers were significantly higher among: 1) men (difference of median=160 nAbs, p<0.001), 2) individuals in the upper tertile ofage (difference of median to lower tertile=160 nAbs, p=0.003) and

All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted October 14, 2020. . https://doi.org/10.1101/2020.10.11.20210005 doi: medRxiv preprint 3) individuals who needed hospitalization to treat SARS-CoV-2 (difference of median=1,120 nAbs, p<0.001). Donors with a shorter time between the end of symptoms and the enrollment had slightly higher nAbs titer, but without statistical significance (p=0.067) (Figure3). Validation of the S/CO cut-off as criteria for selecting donors with high nAbs titers All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted October 14, 2020. . https://doi.org/10.1101/2020. 10.11.20210005 doi: medRxiv preprint For validating the S/CO optimal cut-off, the initial sample of 214 donors was divided into tertiles from the date of the interview. The first two thirds (development sample) were used to develop the criteria, while the last third of the sample (validation sample) was used to validate the proposed criteria. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted October 14, 2020. . https://doi.org/10.1101/2020.10.11.20210005 doi: medRxiv preprint

The performance analysis of the three criteria tested in the validation sample showed similar results. In general, better results were observed for the prediction of nAbs titers ≥ 1:80 when compared to the results of the prediction of nAbs ≥ 160. Criterion 3 demonstrated a reduction in the rate of potential donors discarded (40.3%) and, above all, in the false negative rate. The overall accuracy of the prediction of nAbs ≥ 1:80 increased from 71.6% to 76.1% (Table4). On the other hand, the criterion 3 test in the validation sample also markedly increased the false positive rate for predicting nAbs ≥ 1:160 (Table5). perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted October 14, 2020. .

from two immunoassays analyzed, Abbott and Euroimmun (p<0.001). These data have already been observed in other manuscripts that used an immunoassay. [25] [26] [27] [28] However, this is the first report showing a positive correlation between nAbs tested by an ELISA and a Chemiluminescence assay. This correlation is very important since many services do not have access to measuring nAbs, and, in this scenario, an immunoassay can be used as a screening to detect the presence of antibodies anti SARS-CoV-2 in the samples of convalescent donors.

Also, we have tested three criteria for identifying convalescent plasma donors with high nAbs titers. According to Youden method, the best cut-off point for the This data reinforces previous information available in literature. 29 Conclusion All rights reserved. No reuse allowed without permission. perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted October 14, 2020. . https://doi.org/10.1101/2020. 10.11.20210005 doi: medRxiv preprint We evaluated the performance of Abbott and Euroimmun immunoassays for convalescent patient's IgG screening and the correlation between S/CO values with nAbs titers obtained by CPE-VNT. Our results show that S/CO cut-off value 4.57 can be applied to identify CP units with high nAbs titers. These findings support a CCP screening algorithm in which immunoassay for IgG testing could be first performed as a qualification testing and, in the presence of S/CO>4.57, CP units would be selected for transfusion. CP units with S/CO between 1.1 and 4.57 should be further tested using CPE-VNT to titrate nAbs and be issued for transfusion based on this result.

The authors declare no conflicts of interest. All rights reserved. No reuse allowed without permission.

perpetuity.

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

The copyright holder for this this version posted October 14, 2020. . https://doi.org/10.1101/2020.10.11.20210005 doi: medRxiv preprint perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted October 14, 2020. . https://doi.org/10.1101/2020.10.11.20210005 doi: medRxiv preprint perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted October 14, 2020. . https://doi.org/10.1101/2020.10.11.20210005 doi: medRxiv preprint perpetuity. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in The copyright holder for this this version posted October 14, 2020. . https://doi.org/10.1101/2020.10.11.20210005 doi: medRxiv preprint

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