Cell based assays used to quantify neutralizing antibodies against SARS-CoV-2 in human samples

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 research-use-only assays for this new emerging virus. The microneutralization assay is a fundamentalserological test in virology, immunology, vaccine assessment and epidemiological studies, and represents one of the most used methods to evaluate the immune response induced by SARS-CoV-2 infection or vaccination. In this Phd project different microneutralization methods are presented which can be used to measure anti‐SARS‐CoV‐2 neutralizing antibodies (nAbs) in human serum samples. The aim of the first project of this thesis (Project I) was to compare a microneutralization assay (MN) with a read out based on the cytophatic effect (CPE) and a MN based on a colorimetric read out for the detection of nAbs against SARS-CoV-2 Wild type strain. In the first method the cell monolayers were microscopically inspected for inhibition of CPE at each serum dilution (subjective method), while in the MN based on a colorimetric read out the healthy cell monolayer was stained with neutral red solution, a vital dye. The plates were then read by a spectrophotometer at 540 nm (objective method). A panel of 83 human serum samples were previously tested in enzyme‐linked immunosorbent assay (ELISA) as a pre‐screening. All the samples found to be positive, borderline, and negative in this ELISA were then tested to determine the nAbs titers through the MN CPE and Colorimetric MN. The comparison between log2-trasformed MN titers obtained through these two methods showed comparable values, and the strong agreement in evaluating neutralizing antibodies against SARS-CoV-2 Wild type strain was also confirmed by Correlation (r 2=0,9955), Bland-Altman, and intra-class correlation (ICC) analysis (ICC value of 0.993, which is indicative of an excellent agreement). This suggests the suitability of performing the MN assay using an ‘objective’ colorimetric-based read out method. To better investigate if the classical MN CPE yielded similar results to those obtained with other MN methods, we compared this “classical” MN to a new MN platform: the Virospot MN assay (Project II). This method combines classic virus culture techniques with automated sensitive detection of immunostained virus infected cells. In the Virospot MN, a virus-specific immunostaining was used for plate reading and then the images of all wells were acquired by a CTL ImmunoSpot analyzer. The 80% (MN80) or 90% (MN90) neutralization titers are calculated according to the method described by Zielinska et al. 2005. This titer calculation is based on the serum dilutions above and below the reduction point, 80% or 90% neutralization. The MN CPE and Virospot methods were compared using a panel of 47 human serum samples against SARS-CoV-2 Wild type and Alpha variant. The results of this project showed that the these two different MN assays produce similar titer results against the Wild Type virus, with good correlation values (correlation MN80 r2=0,9091; correlation MN90 r2=0,8900). A lower agreement between the MN CPE and Virospot MN assay was observed when SARS-CoV-2 Alpha variant was used (correlation MN80 r2=0,7226; correlation MN90 r2=0,6673). Overall, these results showed a good agreement between the MN CPE assay and the two different MN methods, Colorimetric MN and Virospot MN assay, in detecting neutralizing antibodies against SARS-CoV-2 in human serum samples. Despite the need for further standardization and/or the differences noticed during the assessment of nAbs against SARS-COV-2 variants, the Colorimetric-based and Virospot MN demonstrate to have advantages over the classical MN CPE, both being completely automated methods, and hence offering a higher throughput, while inspection of each dilution well by means of the optical microscope slows down the process. However, to ensure that these correlation studies can provide meaningful results, further analysis with a bigger number of samples and with other SARS-CoV-2 variants would be an added value. Moreover, to make the data more comparable it would be necessary convert all the results to international standard unit (IU/mL) allowing the accurate calibration of assays to an arbitrary unit, thereby reducing inter-laboratory variation, and creating a common language for reporting data. The SARS-CoV-2 Virospot MN assay offers attractive advantages over the MN assay with a read out based on the cytophatic effect, including the relative insensitivity to variation in amount of infectious virus used in the test, independence from virus replication kinetics and suitability for high throughput analyses. Since many new SARS-CoV-2 variants occurred during the last two years, to make the Virospot more sensitive and robust in detecting neutralizing antibodies against these new variants, the third project (Project III) focused on the optimization study of this MN assay. Several new conditions were adapted to optimize the method and make it more sensitive for the analysis of samples against the Wild Type, Alpha, Beta, and Gamma SARS-CoV-2 variants. Carboxymethyl cellulose (CMC) overlay was introduced to make the spot count more accurate avoiding the viral spread after the first infection. Moreover, different sample matrices (serum and plasma), culture media with and without the CO2 supplementation, and different incubation time points and temperatures were assessed to evaluate and improve the assay performance and robustness. This optimization study has a planned follow-up, which can possibly include samples not only from infected/convalescent individuals but also from vaccinated donors (with two or more doses) or from people with hybrid immunity (such as breakthrough infections). Additionally, further analyses with additional SARS-CoV-2 variants to strengthen these finding will also be part of the next study. This project is worth to be conducted as the Virospot MN assay is likely to have importance for the pre-clinical evaluation and eventual licensing of the SARS-CoV-2 vaccines.