[Development of a cell-based imaging assay for evaluating neutralizing antibodies against western equine encephalitis...

Objective To establish a rapid, high-throughput, and visualisation method for evaluating neutralizing antibodies against western equine encephalitis virus (WEEV) based on cell imaging technology. Methods Recombinant virus rVSV-WEEV-GFP, previously constructed by our research group, was propagated in baby hamster kidney cells (BHK-21) and subsequently titrated. The infection efficiency of rVSV-WEEV-GFP was assessed in three cell lines-BHK-21, African green monkey kidney cells (Vero), and mouse neuroblastoma cells (Neuro2a) via fluorescence intensity analysis based on a high-throughput microplate imaging system (Cytation5). A series of experiments were performed to optimize the key conditions for a neutralizing antibody assay including the mean fluorescence intensity in rVSV-WEEV-GFP-infected cells across different time points, MOIs, and cell seeding densities. Accordingly, a robust method based on cellular imaging was established. The consistency between the two methods was further evaluated by comparing the half-maximal inhibitory concentration (IC₅₀) of a broadly neutralizing antibody measured using both this novel imaging-based assay and the conventional plaque reduction neutralization test (PRNT). Finally, the neutralizing activity of previously prepared encephalitic alphavirus antibodies was analysed using the novel assay. Results Vero cells were regarded as the most efficient cell line for rVSV-WEEV-GFP infection. The optimal detection time and multiplicity of infection (MOI) were identified as 36 hours and 0.0625, respectively. And, the seeding optimal cell density was 10 000 cells per well. The cell imaging-based neutralization assay demonstrated high consistency with the conventional plaque reduction neutralization test (PRNT). Among six antibodies tested, three exhibited potent neutralizing activity against WEEV. Conclusion The cell imaging-based neutralization assay established in this study provides a reliable foundation for the screening of antibodies with high neutralizing activity against WEEV.