Establishment and validation of a pseudovirus neutralization assay for SARS-CoV-2

Abstract

Pseudoviruses are useful virological tools because of their safety and versatility, especially for emerging and re-emerging viruses. Due to its high pathogenicity and infectivity and the lack of effective vaccines and therapeutics, live SARS-CoV-2 has to be handled under biosafety level 3 conditions, which has hindered the development of vaccines and therapeutics. Based on a VSV pseudovirus production system, a pseudovirus-based neutralization assay has been developed for evaluating neutralizing antibodies against SARS-CoV-2 in biosafety level 2 facilities. The key parameters for this assay were optimized, including cell types, cell numbers, virus inoculum. When tested against the SARS-CoV-2 pseudovirus, SARS-CoV-2 convalescent patient sera showed high neutralizing potency, which underscore its potential as therapeutics. The limit of detection for this assay was determined as 22.1 and 43.2 for human and mouse serum samples respectively using a panel of 120 negative samples. The cutoff values were set as 30 and 50 for human and mouse serum samples, respectively. This assay showed relatively low coefficient of variations with 15.9% and 16.2% for the intra- and inter-assay analyses respectively. Taken together, we established a robust pseudovirus-based neutralization assay for SARS-CoV-2 and are glad to share pseudoviruses and related protocols with the developers of vaccines or therapeutics to fight against this lethal virus.

Keywords: COVID-19; SARS-CoV-2; neutralization assay; neutralizing antibody; pseudovirus.

Figure 1.

Verification of the incorporaiton of SARS-CoV-2 spike protein in the pseudovirus. The surface proteins of the particle were investigated using western blotting (A), from left to right lane showing VSV pseudovirus, medium control and SARS-CoV-2 pseudovirus. The VSV pseudotyped virus and cell culture medium were prepared with the same procedure and used as negative control and pseudovirus negative control. The SARS-CoV-2 pseudovirus, together with G*ΔG-VSV, were tested against VSV G specific antibody (B).

Figure 2.

Selection of the cell line. Six types of cells were tested for VSV (A), and SARS-CoV-2 (B) pseudoviruses. The y-axis showed the absolute RLU value detected 24 h after pseudovirus infection. Cell backgrounds without pseudovirus infection were shown in figure C.

Figure 3.

Optimization of cell numbers for neutralization. (A) Effect of cells numbers on the titration of pseudovirus. The y-axis showed the RLU fold over the cell background. (B) Effect of cells numbers on the neutralization. Cell numbers and corresponding R² values were list in the tables. A serum sample from a healthy individual was tested as negative control shown in black lines and symbols.

Figure 4.

Optimization of virus dose for neutralization. The viral inocula with a dose range starting from 41 to 1300 TCID₅₀/well were tested for SARS-CoV-2 PBNA. A serum sample from a healthy individual was tested as negative control shown in black lines and symbols.

Figure 5.

Validation of the SARS-CoV-2 PBNA. (A) Specificity of the PBNA. A negative sample panel of seventy-four human and forty-six mouse serum samples were used to determine the specificity of this assay. (B) Inhibition curves for two SARS-CoV-2 convalescent patient serum samples and two negative serum samples derived from human and mouse. Typical four-parameter inhibition curves were observed for these two samples between log-transformed dilution and inhibition rate. The initial dilutions for positive and negative samples were 1:30 and 1:10 respectively, followed by 3-fold serial dilution. (C) Linear range for the PBNA. When the inhibition rate is in the range of 20%–80%, there is a linear relationship between the readout of the test sample and its dilution. (D) Reproducibility of the PBNA. A mixture of two SARS-CoV-2 coalescent patient serum samples was tested a total of 18 times on individual plates in three independent runs.