Cq1 confocal image cytometer

The pseudotyped virus displaying SARS-CoV-2 (Wuhan-1 reference strain and variant strains) S protein express GFP in infected cells. They were prepared as previously described [48 (link)]. Mice sera were 2-fold serially diluted and incubated with pseudotyped virus at 37°C for 1 h. Then the mixture was transferred to pre-plated VERO cell monolayers in 96-well plates. After incubation for 15 h, the transducing unit numbers were calculated on a CQ1 confocal image cytometer (Yokogawa). Neutralization titre was determined by fitting nonlinear regression curves using GraphPad Prism and calculating the reciprocal of the serum dilution required for 50% neutralization of infection. Neutralization titre below the limit of detection was determined as half the limit of detection. In this study, the GFP-encoding pesudotyped virus was only used in the comparison of neutralizing activity against Wuhan-1 reference strain and variant strains.

SARS-CoV-2 pseudotyped viruses were constructed using a GFP-encoding replication-deficient vesicular stomatitis virus (VSV) vector backbone (VSV-ΔG-GFP). HEK293T cells were transfected with 30 μg of the plasmid for spike protein expression. VSV-ΔG-GFP was added 24 h after transfection. The inoculum was removed after incubating for 1 h at 37 °C. The culture medium was then changed to DMEM supplemented with 10% FBS and 10 μg/mL of anti‐VSV‐G antibody (I1‐Hybridoma ATCC^® CRL2700) after washing cells with PBS. The pseudotyped viruses were harvested 20 h after inoculation, filtered, aliquoted and stored at -80°C.
Pseudovirus particles of SARS‐CoV‐2 were added to each well of a 96‐well plate containing HeLa‐bACE2-Ra cells. HeLa cells were used as controls. The plates were imaged at 15 h after transfection. Fluorescent cells were counted using a CQ1 confocal image cytometer (Yokogawa Electric, Tokyo, Japan). Each group contained three replicates.

Here, we employed a pseudovirus assay to evaluate NAbs titers in serum samples obtained from patients with BA.2 BTI and those who had been vaccinated twice with Convidecia against pseudoviruses of SARS-CoV, SARS-CoV-2 prototype, Delta and Omicron BA.1, BA.2, BA.2.12.1, BA.2.75, BA.4 and/or BA.5, BF.7, XBB, XBB.1.5, BQ.1, BQ.1.1 and CH.1.1. The pseudotyped virus neutralization assay was identical to that used in our previous work^{58 (link),59 (link)}. Briefly, pCAGGS was used as a vector to construct S protein expression plasmids for SARS-CoV and SARS-CoV-2 variants (Fig. 2a). Once the pseudoviruses were ready, the serum samples were heat-inactivated at 56 °C for 30 min and then diluted two-fold, starting at appropriate dilutions. An equivalent amount of pseudovirus (1000 transducing units, TU) was then incubated with sera at 37 °C for 1 h. The mixture was then added to pre-plated Vero cells (ATCC CCL81) in 96-well plates. After another 15 h of incubation, the TU numbers were measured using a CQ1 confocal image cytometer (Yokogawa, Japan). Each sample was tested in duplicate, and 50% pseudovirus neutralization titers (pVNT₅₀) were derived by non-linear regression using GraphPad Prism (8.4.3). The final results were illustrated using geometric mean titer with a 95% credibility interval). For more details, please refer to the references^{58 (link),59 (link)}.

Vesicular Stomatitis Virus (VSV)-backbone SARS-CoV-2 pseudotyped viruses of examined variants were purchase from Vazyme Biotech (SARS-CoV-2-Fluc WT, DD1502; SARS-CoV-2-Fluc Delta, DD1554; SARS-CoV-2-Fluc BA.1, DD1568; SARS-CoV-2 BA.2, DD1569; SARS-CoV-2 BA.2.12.1, DD1577; SARS-CoV-2 BA.2.75, DD1581; SARS-CoV-2 BA.3, DD1574; SARS-CoV-2 BA.5, DD1576; SARS-CoV-2 BF.7, DD1589). For neutralization assay, the heat-inactivated (56°C, 30 min) serum samples were subjected to a three-fold serial dilution started from 1:60. Each pseudotyped virus (650 TCID₅₀/well) was mixed with equal volume of serially diluted sera and incubated at 37°C for 1 h, and then 100 μl mixture was added onto pre-plated Vero cells in 96-well plate. After 15 h incubation, the transducing units (TU) numbers were calculated on a CQ1 confocal image cytometer (Yokogawa, Japan). Neutralization titers were calculated as EC50 titers by Reed-Muench method. Values below the limit of detection were determined as one third of the starting dilution factor.