Evaluation of SARS‐CoV‐2 neutralizing antibodies using a CPE‐based colorimetric live virus micro‐neutralization assay in human serum samples

Abstract The micro‐neutralization assay is a fundamental test in virology, immunology, vaccine assessment, and epidemiology studies. Since the SARS‐CoV‐2 outbreak at the end of December 2019 in China, it has become extremely important to have well‐established and validated diagnostic and serological assays for this new emerging virus. Here, we present a micro‐neutralization assay with the use of SARS‐CoV‐2 wild type virus with two different methods of read‐out. We evaluated the performance of this assay using human serum samples taken from an Italian seroepidemiological study being performed at the University of Siena, along with the human monoclonal antibody CR3022 and some iper‐immune animal serum samples against Influenza and Adenovirus strains. The same panel of human samples have been previously tested in enzyme‐linked immunosorbent assay (ELISA) as a pre‐screening. Positive, borderline, and negative ELISA samples were evaluated in neutralization assay using two different methods of read‐out: subjective (by means of an inverted optical microscope) and objective (by means of a spectrophotometer). Our findings suggest that at least 50% of positive ELISA samples are positive in neutralization as well, and that method is able to quantify different antibody concentrations in a specific manner. Taken together, our results confirm that the colorimetric cytopathic effect‐based microneutralization assay could be used as a valid clinical test method for epidemiological and vaccine studies.

that a future pandemic may be caused by one of these agents, underlining the urgent need to prepare for such an eventuality, since no vaccines or approved therapies, are as yet available. 6 At the end of December 2019 in Wuhan, Hubei Province, China, a novel CoV strain, called SARS-CoV-2 by the International Committee on Taxonomy of Viruses (ICTV), caused 27 cases of pneumonia of unidentified etiology. 7 Due to the rapid and uncontrollable spread of the virus in almost every country in the world, the World Health Organization (WHO) officially declared the pandemic status in March 2020. The disease caused by SARS-CoV-2, named COVID-19, is considered a self-limiting infectious disease with five different possible outcomes: asymptomatic cases (1.2%), mild cases (80.9%), severe cases (13.8%), critical cases (4.7%), and deaths (2.3%). 7,8 However, some authors reported a higher percentage of asymptomatic infections in children under the age of 10 (15.8%). 9 Because of the lack of specific antiviral drugs or vaccines, several thousands of serious cases and deaths occur every day all over the world, and strict quarantine measures have been imposed either nationally or internationally. Since the antibody response of the serum, after a natural CoV infection remains detectable for a long time, 10 medical authorities in many countries are trying to calculate the percentage of the population that may be protected against the new circulating strain through the assessment of anti-SARS-CoV-2 Immunoglobulin G (IgG) and M (IgM) levels in serum samples. Principal serological tests used in these studies are ELISA-based assays. Most of these tests focus on different combinations of coatings on the viral spike (S) protein (S1; S1+S2; S1-S2 extracellular domain-ECD, receptor binding domain-RBD), due to the fact that the CoV's ability to attach and consequently enter the cell is mainly mediated by this protein. 11 Enzymelinked immunosorbent assays (ELISAs) certainly have advantages, such as high throughput, speed of testing, and the possibility of avoiding the requirement for a high containment laboratory, as BSL 3. However, most of these assays present some limitations, such as low specificity and sensitivity, and use of alternative purified proteins that can be produced in different hosts (human-derived cells vs insect cells). In addition, the mismatch between results obtained from the same samples, using different ELISA reagents and coatings (eg, source of antigen), may lead to confusion. 12 To date, the Micro-Neutralization assay (MN), currently considered the gold-standard is the most specific and sensitive serological assay capable of evaluating and detecting, functional neutralizing antibodies (nAbs). In this paper, a live virus-based MN assay is presented for the quantification of SARS-CoV-2-specific nAbs in human serum samples by two different methods of detection: a classical readout by checking the percentage of cytopathic effect (CPE) in the cell monolayer, and a colorimetric read-out by a spectrophotometer.

| Serum samples and human monoclonal antibody IgG1
A total of 83 human serum samples were collected as part of a seroepidemiological study that is being performed in the laboratory of    The SARS-CoV-2 virus was seeded and propagated in VERO, VERO E6, and Huh-7 cells by using EMEM (for VERO and Huh-7) and DMEM high glucose (for VERO E6) both supplemented with 2% FBS and 100 IU/mL penicillin-streptomycin.

| Virus and titration
Cells were seeded in T175 flasks at a density of 1 × 10 6 cells/mL. After 18 to 20 hours, the sub-confluent cell monolayer was washed twice with sterile Dulbeccos's phosphate buffered saline (DPBS).
After removal of the DPBS, the cells were infected with 3.5 mL of EMEM/DMEM 2% FBS containing the virus at a multiplicity of infection of 0.001 and 0.01. After 1 hour of incubation at 37°C in a humidified atmosphere with 5% CO 2 , 50 mL of EMEM/DMEM containing 2% FBS was added for VERO-Huh7/VERO E6. The flasks were daily observed and the virus was harvested when 80%-90% of the cells manifested CPE. The culture medium was centrifuged at +4°C 1600 rpm for 8 minutes, to remove the cell debris, then they aliquoted and stored at −80°C.

| Micro-neutralization assay
Serum samples were heat-inactivated for 30 minutes at 56°C; two-fold serial dilutions, starting from 1:10, were then mixed with an equal volume of viral solution containing 100 TCID50 of SARS-CoV-2.
The serum-virus mixture was incubated for 1 hour at 37°C in a humidified atmosphere with 5% CO 2 . After incubation, 100 µL of the mixture at each dilution was added in duplicate to a cell plate containing a semi-confluent VERO E6 monolayer. The plates were incubated for 4 days at 37°C in a humidified atmosphere with 5% CO 2 .

| CPE-read out
After 4 days of incubation, the plates were inspected by an inverted optical microscope. The highest serum dilution that protected more than the 50% of cells from CPE was taken as the neutralization titre. optical density (OD) value greater than the cut-off value, was considered as the neutralization titre. The cut-off value is calculated as the average of the OD values of the cell control wells divided by two.

| Enzyme-linked immunosorbent assay
Specific anti-SARS-CoV-2 IgG antibodies were detected through a commercial ELISA kit (Euroimmun, Lübeck, Germany). ELISA plates are coated with recombinant structural protein (S1 domain) of SARS-CoV-2.   To check the viral production in Huh-7 cells, we passed the supernatant in VERO E6 cells but no CPE was detected in this cell line. This confirms that Huh-7 cells are not able to support the viral replication of this CoV strain, as already showed by Harcourt et al. 16 The supernatants derived from VERO, VERO E6 and Huh-7 were titrated in 96-well plates, which were read after 72 hours; titres reached ranged from 10 6.2 to 10 7.8 TCID50/mL either for VERO and VERO E6-derived virus; no titre has been detected for Huh-7-derived virus (data not shown).  (Table 1). All animal samples tested against Influenza and Adenovirus type 4 proved completely negative, confirming the specificity of the MN assay in the detection of anti-SARS-CoV-2 nAbs.

| Absence of neutralizing activity for human IgG1 monoclonal antibody CR3022
As reported 17 that the CR3022 monoclonal antibody (mAb) has a high capability of neutralizing the SARS-CoV strain, we included this mAb (IgG1) within the human serum samples in our neutralization assay. The

| Neutralization assay read-out: subjective vs objective methods
The results obtained in the MN assay in all serum samples were evaluated through two methods of read-out: by inspecting the  (Table 1) suggest that a well-trained operator is able to read the CPE, thereby providing the same results as the spectrophotometer in terms of titre with no differences between the results provided by the two different operators and the spectrophotometric evaluation of the ODs.
One of the advantages of the colorimetric read-out is that, being a completely automated method, it offers a higher throughput, while

| DISCUSSION
The availability of a specific serological assay capable of providing the most reliable and accurate antibody response in a given sample is a crucial factor in all epidemiological studies. This is particularly important in an emergency situation, such as during a sudden epidemic or, even worse, a pandemic. Indeed, knowing which percentage of the  Sample ID ELISA   MN CPE  titre  analyst  1 100  TCID 50   MN CPE  titre  analyst  2 100  TCID 50   Colorimetric  MN 100  TCID 50   MN  CPE  titre 25  TCID 50   58  Positive  10  10  10  20   59  Positive  80  80  80  80   60  Positive  640  640  640  640   61  Positive  10  10  protein. 19

CONFLICT OF INTERESTS
The authors declare that there are no conflict of interests.