key: cord-0771453-xdtv5rrs
authors: von Rhein, Christine; Scholz, Tatjana; Henss, Lisa; Kronstein-Wiedemann, Romy; Schwarz, Tatjana; Rodionov, Roman N; Corman, Victor M.; Tonn, Torsten; Schnierle, Barbara S.
title: Comparison of potency assays to assess SARS-CoV-2 neutralizing antibody capacity in COVID-19 convalescent plasma
date: 2020-12-01
journal: J Virol Methods
DOI: 10.1016/j.jviromet.2020.114031
sha: 4cf44f68c54468cf03b9e3b234b8949039f0c1b6
doc_id: 771453
cord_uid: xdtv5rrs

Convalescent plasma is plasma collected from individuals after resolution of an infection and the development of antibodies. Passive antibody administration by transfusion of convalescent plasma is currently in clinical evaluations to treat COVID-19 patients. The level of neutralizing antibodies vary among convalescent patients and fast and simple methods to identify suitable plasma donations are needed. We compared three methods to determine the SARS-CoV-2 neutralizing activity of human convalescent plasma: life virus neutralization by plaque reduction assay, a lentiviral vector based pseudotype neutralization assay and a competition ELISA-based surrogate virus neutralization assay (sVNT). Neutralization activity correlated among the different assays; however the sVNT assay was overvaluing the low neutralizing plasma. On the other hand, the sVNT assay required the lowest biosafety level, is fast and is sufficient to identify highly neutralizing plasma samples. Though weakly neutralizing samples were more reliable detected by the more challenging lentiviral vector based assays or virus neutralization assays. Spike receptor binding competition assays are suitable to identify highly neutralizing plasma samples under low biosafety requirements. Detailed analysis of in vitro neutralization activity requires more sophisticated methods that have to be performed under higher biosafety levels.

The ongoing worldwide pandemic of coronavirus disease 2019 , caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections, is a great threat to global public health.

Currently no specific treatment or vaccines are available. Passive immunization by plasma therapy however, has the potential to be used as an emergency treatment. Plasma was applied to humans already in 1917 for the treatment of patients with acute poliomyelitis (Amoss and Chesney, 1917) . Thereby, plasma collected from patients who have recovered from the disease, is transfused into acutely infected patients. This treatment has been successfully used as a safe and efficient therapy for infections with the coronaviruses SARS and MERS (Middle East respiratory coronavirus) and during the Ebola outbreak (Cheng et al., 2005) , (Ko et al., 2018) , (Winkler and Koepsell, 2015) .

Currently studies are ongoing and first successful treatments of COVID-19 patients are being published (Shen et al., 2020) , (Duan et al., 2020) , (Ye et al., 2020) , (Ahn et al., 2020) . Apart from finding the right clinical parameters, e.g. stage of disease or symptoms, for plasma transfusion, identifying plasma sample with therapeutic potential is the main challenge for this type of therapy.

Patients who have recovered from COVID-19 and have high neutralizing antibody titer are valuable donors. However, the amount of antibodies and the virus neutralizing activity in convalescent serum varies rigorously between patients (Robbiani et al., 2020) . The availability of high titer convalescent plasma is less abundant than expected. A recent study of 175 Chinese patients, who recovered from mild COVID-19, described that 6% of the patients did not produce detectable levels of neutralizing antibodies and 30% of them only very low neutralizing titers (Wu et al., 2020a) .

Here, we compared three different assay systems to determine in vitro SARS-CoV-2 neutralizing activity in convalescent plasma to identify a simple and reliable way to define samples with therapeutic potential.

HEK293T-hACE2 (Glowacka et al., 2010) , HEK293T (ATCC CRL-3216) and Vero E6 cells (ATCC CRL-1586) were cultured at 37°C under 5% CO2 and grown in Dulbecco's modified Eagle medium (DMEM;

Lonza, Verviers, Belgium) supplemented with 10% fetal bovine serum (PAA, Pasching, Austria) and 5% L-glutamine (200 mM; Lonza, Verviers, Belgium) and 1% penicillin/streptavidin (Fisher Scientific, Schwerte, Germany).

Human naïve and SARS-CoV-2 positive plasma was obtained from the German Red Cross from volunteer blood donors. Plasma samples were heat-inactivated at 56°C for 30 minutes.

Plaque reduction neutralization tests were done as described before (Wölfel et al., 2020) . In short, VeroE6 cells (4x10 5 cell/ml) were seeded in 24-well plates the day before. Prior to PRNT patient plasma were heat-inactivated at 56°C for 30 minutes and diluted 1:20 up to 1:640. For each dilution step PRNT testing was done in duplicates. For PRNT samples were diluted in OptiPro (Fisher scientific, Schwerte, Germany) and mixed 1:1 with virus solution containing 100 plaque forming units of SARS-CoV-2 (EPI ISL 406862) and incubated at 37°C for 1 hour. The solution was added onto two wells of a 24-well plate. After 1 hour at 37°C the supernatants were discarded, the cells were washed once with PBS and supplemented with 1.2% Avicel solution in DMEM (Merck, Darmstadt, Germany). After 3 days at 37°C, the supernatants were removed and the 24-well plates were fixed and inactivated using a 6% formaldehyde/PBS solution and stained with crystal violet as described (Herzog et al., 2008) .

Lentiviral vectors were prepared in HEK293T cells by co-transfection using Lipofectamine 2000 (Thermo Fisher, Darmstadt, Germany) as described previously (Henss et al., 2019) . Plasmids encoding HIV-1 Gag/pol, rev, the firefly luciferase encoding lentiviral vector genome and the SARS-Cov-2 fulllength spike gene (#MN908947) or VSV-G were transfected. Vectors were concentrated by ultracentrifugation and stored at -80 0 C. Pseudotyped vectors and serially diluted human plasma/serum (1:60 to 1:14,580) were incubated in triplicates for 30 min. at 37°C and used to transduce HEK293T-hACE2 cells. After 48 hours, luciferase substrate was added to measure luciferase activity. The reciprocal area under the curve (AUC) value calculated for each sample corresponds to the neutralization activity. AUC values were determined using the GraphPad Prism 7.04 software (La Jolla, CA, USA). Mean values and standard deviations were calculated in Excel.

The spike protein (S) receptor binding domain (RBD) is responsible for recognizing the cell surface receptor, angiotensin converting enzyme-2 (ACE2). The RBD of SARS-CoV-2 S protein strongly interacts with hACE2. The SARS-CoV-2 sVNT Kit (Genscript; Leiden, Netherlands) is a blocking ELISA, which mimics this virus receptor binding process. The protein-protein interaction between a horseradish peroxidase (HRP) conjugated recombinant SARS-CoV-2 RBD fragment (HRP-RBD) and hACE2 can be blocked by neutralizing antibodies against the SARS-CoV-2 RBD and residual HRP activity is measured as a surrogate for neutralization. The assay was performed with 1:50 diluted plasma following the manufacturer's instructions.

The SARS-Cov-2 neutralizing activity was determined by three different methods. First the gold standard method, virus neutralization assays was performed. Infectious SARS-Cov-2 was used to infect Vero E6 cells in the presence of decreasing amounts of convalescent plasma and the 50% plaque reduction titer was determined (Figure 1 A) . The assay was performed a second time with higher plasma dilutions because for some samples the endpoint titer could not be determined The different assay results were plotted against each other and showed linearity with a measure of certainty of r 2 = 0.79 for pseudotype against the virus neutralization assay (Figure 2A ). Slightly less linearity was observed for the sVNT and the virus neutralization with r 2 = 0.68 ( Figure 2B ). However, if the analysis was restricted to highly neutralizing plasma samples and the values of the virus neutralization assay with higher diluted plasma were used, a good correlation of pseudoneutralization and virus neutralization corresponding to a measure of certainty of r 2 = 0.84 was detectable ( Figure 2C ). This was less significant for the comparison of the sVNT versus the virus neutralization assay with r 2 = 0.60 ( Figure 2D ).

In general, the sVNT assay overestimated the samples with low neutralizing activities and was less reliable for these types of plasma samples ( Figure 2B , C and Figure 1C samples 7, 15, 25, 28, 29) .

However, highly neutralizing samples could be identified, when using a cut-off of 50% inhibition.

Transfusion of convalescent plasma might be a therapeutic option for the treatment of COVID-19, but the identification of suitable plasma donors is hampered by the substantially variable levels of neutralizing antibodies in convalescent patients. Therefore, we evaluated here, which assay would be suitable to identify donors. The three assays used are able to identify highly neutralizing plasma, but they require different biosafety levels to perform the assay. The virus neutralization has to be carried out under biosafety 3 level, the pseudotype neutralization assay requires biosafety level 2 and sVNT assay is a simple ELISA, which can be performed in any laboratory without special biosafety requirements. In addition, the pseudotype assay and the virus neutralization assay are time consuming and require 2 or 3 days respectively, until results are available. The sVNT assay can be performed in 2-3 hours. However, the sVNT assay overestimated the samples with low neutralizing activities, but increasing the cut-off to 50% inhibition enabled the identification of highly neutralizing samples. Therefore, the sVNT assay,

although not yet approved for diagnostic application, is superior for a clinical setting and allows the fast screening of convalescent plasma samples in low biosafety level laboratories. Also other ELISAbased sVNT assay systems have been developed recently (Bošnjak et al., 2020) , (Abe et al., 2020) , (Ding et al., 2020) and different kits are commercially available. Assay comparisons were performed by multiple groups and overall, the sVNT assays correlated well with the other neutralization assays (Abe et al., 2020) , (Meyer et al., 2020) , (Bond et al., 2020 ), (McGregor et al., 2020 ), (Bošnjak et al., 2020 , (Ding et al., 2020) . The sVNT assays are well suited for a rapid prescreening of patient plasma to identify donors with high neutralization activity. In addition, sVNT kits are useful to analyzed animal sera from preclinical vaccine studies, because no adaptation of the assay is required (Perera et al., 2020) . A general limitation of these assays is that they are only able to detect neutralizing antibodies that function by blocking the interaction between the RBD and ACE2, although most J o u r n a l P r e -p r o o f neutralizing antibodies fulfill this requirement, single antibodies have been described that use other mechanisms for neutralization , (Wu et al., 2020b) .

Detailed analysis of recipients of the plasma donation might require the more sophisticated assays.

For the clinical evaluation of this therapeutic option, also the recipients have to be tested because they might already have highly neutralizing antibodies and additional antibody treatment might not help (Arvind Gharbharan et al., 2020) . These more detailed analyses of SARS-CoV-2 neutralization activity require either, virus neutralization or pseudotype assays. In addition, basic research on the induction of SARS-CoV-2 immune responses should be performed with these assays. Both assays are comparable and a direct calibration of two assays will be possible when an international standard is available. 

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We are thankful to Heike Baumann, Marie Schmidt, Anja Richter and Felix Walper for technical assistant.J o u r n a l P r e -p r o o f