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

Cells and samples

HepG2 (American Type Culture Collection [ATCC], HB-8065), Huh-7 (Japanese Collection of Research Bioresources [JCRB], 0403), 293T (ATCC, CRL-3216), Vero (ATCC, CCL-81), CHO (ATCC, CCL-61), and MDCK (ATCC, CCL-34) cells were maintained in high glucose DMEM (GIBCO) supplemented with 10% FBS (GIBCO), penicillin (100 IU/ml), streptomycin (100 μg/ml), and HEPES (20 mM) in a 5% CO₂ environment at 37°C and passaged every 2–3 days.

VSV G specific antibody was generated by immunized Balb/c mouse with VSV G expressing plasmid at amount of 20 μg/mouse. Ten mice were immunized three times with two weeks interval. Two weeks after the final injection, serum samples from the ten mice were collected and pooled together as the VSV G specific antibody sample. The mice were housed and maintained in accordance with the relevant guidelines and regulations.

Two SARS-CoV-2 serum samples from convalescent patients, 2019-CS-1, 2019-CS-2 were generously provided by Mr. Qiang Gao from Sinovac Biotech Co., LTD. Written informed consents were obtained from all the volunteers.

Production and titration of pseudoviruses

For pseudoviruses construction, spike genes from strain Wuhan-Hu-1 (GenBank: MN908947) were codon-optimized for human cells and cloned into eukaryotic expression plasmid pcDNA3.1 to generate the envelope recombinant plasmids pcDNA3.1.S2.

The pseudoviruses were produced and titrated using methods similar to Rift valley fever pseudovirus, as described previously [19,20]. For this VSV pseudovirus system, the backbone was provided by VSV G pseudotyped virus (G*ΔG-VSV) that packages expression cassettes for firefly luciferase instead of VSV-G in the VSV genome. Briefly, 293T cells were transfected with pcDNA3.1.S2 (30 μg for a T75 flask) using Lipofectamine 3000 (Invitrogen, L3000015) following the manufacturer’s instruction. Twenty-four hours later, the transfected cells were infected with G*ΔG-VSV with a multiplicity of four. Two hours after infection, cells were washed with PBS three times, and then new complete culture medium was added. Twenty-four hours post infection, SARS-CoV-2 pseudoviruses containing culture supernatants were harvested, filtered (0.45-μm pore size, Millipore, SLHP033RB) and stored at −70°C in 2-ml aliquots until use. The 50% tissue culture infectious dose (TCID₅₀) of SARS-CoV-2 pseudovirus was determined using a single-use aliquot from the pseudovirus bank; all stocks were used only once to avoid inconsistencies that could have resulted from repeated freezing-thawing cycles. For titration of the SARS-CoV-2 pseudovirus, a 2-fold initial dilution was made in hexaplicate wells of 96-well culture plates followed by serial 3-fold dilutions (nine dilutions in total). The last column served as the cell control without the addition of pseudovirus. Then, the 96-well plates were seeded with trypsin-treated mammalian cells adjusted to a pre-defined concentration. After 24 h incubation in a 5% CO₂ environment at 37°C, the culture supernatant was aspirated gently to leave 100 μl in each well; then, 100 μl of luciferase substrate (Perkinelmer, 6066769) was added to each well. Two min after incubation at room temperature, 150 μl of lysate was transferred to white solid 96-well plates for the detection of luminescence using a microplate luminometer (PerkinElmer, Ensight). The positive well was determined as ten-fold relative luminescence unit (RLU) values higher than the cell background. The 50% tissue culture infectious dose (TCID₅₀) was calculated using the Reed–Muench method, as described previously [13,18,19].

Western blot analysis

Seven millilitres of SARS-CoV-2 pseudoviruses with a titre of 1.86 × 10⁵ TCID₅₀/ml were pelleted through a 25% sucrose cushion by ultra-centrifugation at 100,000× g for 3 h. The layers of supernatant and sucrose were removed, and the resulting viral pellets were re-suspended in 100 μl PBS. Sixty microlitre prepared pseudoviruses were mixed with 15 μl 6× SDS-sample buffer. The mixture was heated for 5 min at 100°C. Fifteen microlitre samples were subjected to SDS-PAGE and immunoblotting. The VSV pseudotyped virus was prepared with the same procedure and used as the pseudovirus negative control, cell culture medium as negative control. The incorporation of the spike protein on the pseudovirus surface was confirmed using Western bolt with SARS-CoV-2 convalescent serum sample as the detection antibody with a 500-fold dilution. Goat anti-human IgG (Jackson ImmunoResearch, 109-035-0030) was used with a 1:8000 dilution as the secondary antibody.

Pseudovirus based neutralization assay

Neutralization was measured by the reduction in luc gene expression, as described previously for the HIV pseudovirus neutralization assay [21]. The 50% inhibitory dilution (EC₅₀) was defined as the serum dilution at which the relative light units (RLUs) were reduced by 50% compared with the virus control wells (virus + cells) after subtraction of the background RLUs in the control groups with cells only. In brief, pseudovirus was incubated with serial dilutions of the test samples (six dilutions in a 3-fold step-wise manner) in duplicate for 1 h at 37°C, together with the virus control and cell control wells in hexaplicate. Then, freshly trypsinized cells were added to each well. Following 24 h of incubation in a 5% CO₂ environment at 37°C, the luminescence was measured as described in the section for pseudovirus titration. The EC₅₀ values were calculated with non-linear regression, i.e. log (inhibitor) vs. response (four parameters), using GraphPad Prism 8 (GraphPad Software, Inc., San Diego, CA, USA).

Optimization of SARS-CoV-2 PBNA

Having generated the SARS-CoV-2 S pseudotyped virus, we next set out to investigate the cell types for pseudovirus entry. The wide-type SARS-CoV-2 has been successfully isolated in both Vero (monkey kidney cell) and Huh7 (human hepatoma cell) [22]. Two similar cell lines, 293T (human kidney cell, similar to Vero) and Hep G2 (human hepatoma cell, similar to Huh7) were also included for the target cell investigation. In addition, another two mammal cell lines were also employed for target cell selection, including CHO (Chinese hamster ovary cell) and MDCK (dog kidney cell), which were widely employed in recombinant protein [23] or vaccine [24] production. Taken together, six types of cells were tested (Figure 2). As expected, all the cell lines showed high susceptibility to VSV pseudotyped virus. Huh7, 293T and Vero showed high susceptibility for SARS-CoV-2 pseudotypes, which was similar to SARS-CoV reported previously [25]. Huh7 cells were identified as the best cell substrate for SARS-CoV-2 pseudovirus infection, yielding the highest signals.

We next determined the optimal number of Huh7 cells for SARS-CoV-2 pseudovirus infection. Towards this end, we titrated the SARS-CoV-2 pseudotyped virus at a range of cell numbers (6.25 × 10³–2.00 × 10⁵/well). The highest titre was observed in a range of 1.25 × 10⁴–2.50 × 10⁴/well when equal amount of pseudovirus preparations was tested (Figure 3(A)). It is noted that the linear correlation coefficients (R²) obtained at cell inoculum from 1.25 × 10⁴ to 2.50 × 10⁴/well were found to be greater than 0.96, revealing an excellent linear curve fitting. Moreover, when neutralization assays were performed with different cell inocula, the EC₅₀ values were similar with cell numbers ranging from 6.25 × 10³ to 5.00 × 10⁴/well (Figure 3(B)). However, when the input cells numbers were more than 5.00 × 10⁴/well, the R² values for both titration and inhibition assay would decrease dramatically. Given these findings, we chose 5.00 × 10⁴/well for subsequent experiments. Although non-specific responses were observed in varied cell number conditions, the inhibition of pseudovirus have not reached 50% for normal human serum samples (Figure 3(B)).

We next tested the viral inocula with a dose range starting from 41 to 1300 TCID₅₀/well for SARS-CoV-2 PBNA. As expected, the absolute EC₅₀ values decreased gradually with the increasing amounts of the viral inocula (Figure 4). When the virus dose was lower than 325 TCID₅₀/well, the curve fit value would decrease significantly. Therefore, the viral inocula of 650 TCID₅₀/well was used as the optimal dose. Although non-specific responses were observed in varied viral inocula, the inhibition rate of pseudovirus have not reached 50% for normal human serum samples (Figure 4).

Establishment and validation of the SARS-CoV-2 PBNA

As the SARS-CoV-2 PBNA assay would be intended to be used for the analyses of both human and animal samples, a negative sample panel of seventy-four human and forty-six mouse sera were used to determine the specificity of this assay, with an initial dilution of 1:10 followed by 3-fold serial dilution. We first established the limit of detection (LOD) for SARS-CoV-2 PBNA assay. As shown in Figure 5(A), when assayed by PBNA, human negative serum samples showed relatively low background compared to the mouse negative samples. When the mean titre value of the negative samples +1.96 standard deviation (SD) was used to calculate the limit of detection (LOD), the LOD was 22.1 for human serum samples and 42.3 for mouse serum samples. The cutoff values were set as 30 and 50 for human and mouse serum samples, respectively. The non-specific background of mouse sera was higher than human serum samples, which was unexpected. The reason of the higher background for mouse sera would be investigated in future studies.

Linear range means that there is a direct correlation between the signal and the concentration of the substance within a specific range. To determine the linear range of the SARS-CoV-2 PBNA, two SARS-CoV-2 convalescent patient serum samples were tested against the pseudovirus. Typical four-parameter inhibition curves were observed for these two positive samples between log-transformed dilution and inhibition rate (Figure 5(B)). 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 for the positive samples, R²> 0.99 (Figure 5(C)). Non-specific inhibition was observed for both human and mouse negative serum samples in a lower dilution (Figure 2(B)).

In order to test the reproducibility of the PBNA, two SARS-CoV-2 coalescent patient serum samples were pooled together and used to test against the SARS-CoV-2 pseudovirus. This sample was tested a total of 18 times on individual plates in three independent runs (Figure 5(D)). The average coefficient of variation (CV) for the intra- and inter-assay was 15.9% and 16.2% respectively, which was acceptable for a cell-based assay.

Collectively, these data indicate that a robust PBNA assay has been established to test entry inhibition for SARS-CoV-2, which could be used for evaluation of candidate vaccine and therapeutics targeting the virus entry.