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.

Keywords: SARS coronavirus; epidemiology; humoral immunity; neutralization; pandemic.

Figure 1

Vero E6 cells at different stage of infection. A, Not infected VERO E6 cell monolayer after 72 hours, complete absence of CPE. B, SARS‐CoV‐2 infected VERO E6 cell monolayer after 36 hours postinfection, 20%‐30% of CPE recovered. C, SARS‐CoV‐2 infected VERO E6 after 52 hours postinfection, 80% of CPE recovered. CPE, cytopathic effect; SARS‐CoV‐2, Severe Acute Respiratory Syndrome‐Coronavirus‐2

Figure 2

Viral titres reached for VERO and VERO E6 in three different viral infection experiments in T‐175 flasks. A, Titres registered in triplicate (n = 3) for VERO cells after 36, 48 to 52 and 72 to 76 hours post infection. A significant increase in the viral titre has been registered after 48 to 52 hours according to Friedman test (P < .05), error bars indicate the standard deviation among the three independent measures. B, Titres registered (n = 3) for VERO E6 cells after 36, 48 to 52 and 72 to 76 hours post infection. A significant increase in the viral titre has been registered after 48 to 52 hours according to Friedman test (P < .05), error bars indicate the standard deviation among the three independent measures. C.1, Infection curves for VERO cells for three independent experiments of viral growth. C.2, Polynomial infection curve derived from the average of the three experimental curves for VERO cells. D.1, Infection curves for VERO E6 for three independent experiments of viral growth. D.2, Polynomial infection curve derived from the average of the three experimental curves for VERO E6 cells

Figure 3

Schematic overview of the colorimetric MN read‐out. A, SARS‐CoV‐2 virus titration. B, Titration of the working viral solution. C, Neutralization plate with a serum sample tested in quadruplicate. In each plate, the column highlighted in blue is the cell control (highest OD value), while the column highlighted in red is the virus control (no OD values). The cut‐off value is evaluated for each plate, and is equal to the average of the cell control ODs divided by two. Wells that show OD values lower than the cut‐off are considered virus‐positive, and hence infected. The viral titres in both the stock solution (A) and the working viral solution (B) are calculated by means of the Reed and Muench method. The titre of the serum sample (C) was calculated as the reciprocal of the highest dilution at which the OD value was higher than or equal to the cut‐off value. OD, optical density; ARS‐CoV‐2, Severe Acute Respiratory Syndrome‐Coronavirus‐2