Development and characterization of viral pseudotypes as safe tools for investigating WHO priority pathogens

Recent outbreaks of highly pathogenic human RNA viruses have highlighted the importance of epidemic and pandemic research preparedness within society. Due to their biological risk, authentic viruses require handling by experienced personnel in high containment level laboratories; therefore, research efforts for the establishment of therapeutic and prevention strategies are hindered and slowed down. Pseudotyped viruses, which are surrogate, non- pathogenic recombinant viral particles bearing the envelope protein of the virus of interest, represent a safe alternative to circumvent the drawbacks highlighted by “live” viruses. Aims of this work are threefold: provide a library of viral pseudotypes for selected World Health Organization (WHO) priority pathogens, compare practical workflow and production yields across the three applied packaging platforms (lentiviral two/three-plasmids platforms and VSV based systems) and validate each construct through pseudotype based neutralization assays using appropriate controls in order to support evaluation of vaccines and antiviral agents in practice. Viral pseudotypes are produced in HEK293T/17 cells under standardized transfection and harvest conditions. Lentiviral systems used pNL4-3 (two-plasmid platform) or p8.91 with pCSFLW (three-plasmid platform) alongside pathogen-specific envelopes; VSV-ΔG-Luc particles were generated by transient glycoprotein expression followed by VSV-ΔG infection. Titration and neutralization assays are based on luciferase readout and employ target cells that match viral entry requirements. Three-plasmid lentiviral and VSV platforms generally out-performed the two-plasmid lentiviral system. Most pseudotypes achieved the titer threshold needed for their application in neutralization assays. In case of Nipah pseudotypes, where titers lagged, process refinements (freeze-thaw cycles and subsequent concentration) improved performance. Neutralization behaved as expected: positive controls neutralized pseudotypes (observed as a suppression of luciferase signal), whereas negative controls did not provide a protective titer. Overall, the work delivers reproducible routes to generate functional pseudotypes as a shareable resource that strengthens vaccine and antiviral testing for pandemic preparedness.