Transposon Vectors Enable Accelerated Development of Cellular Assay Platforms for the Identification and Characterization of Anti-Viral Small-Molecule Cell-Entry Inhibitors and Neutralizing Antibodies.

Background/Objectives: Screening for neutralizing antibodies (nAbs) or antiviral small-molecule inhibitors (SMIs) using live virus is laborious and often confined to higher biosafety levels, thus restricting accessibility. The use of murine leukemia virus (MLV)-derived pseudotype vector particles displaying spike or envelope proteins of the targeted human pathogen and susceptible target cells expressing the cognate virus receptor lowers biosafety requirements. Extended drug discovery campaigns demand larger quantities of pseudotype vector particles most favorably produced by stable high-titer suspension vector-producing cells (SVPCs). We aimed to demonstrate that this can be achieved by employing transposon vectors. Methods: Sleeping Beauty transposon vectors were used to rapidly establish MLV-derived SVPCs using two SARS-CoV-2 spike variants as well as highly susceptible target cells expressing the virus receptor ACE2 and the cellular protease TMPRSS2. Generated cell lines and viral vectors were characterized by employing syncytial-formation assay, Western blot analysis, vector titration and neutralization experiments using two SMIs and two nAbs. Results: The expression, fusogenicity and particle incorporation of two spike variants was confirmed and the production of high-titer MLV(SARS-CoV-2) particles was demonstrated. The components of this novel cellular assay platform consisting of stable susceptible target cells and high-yield SVPCs were successfully utilized to characterize nAbs directed against the spike protein and cell-entry-inhibiting SMIs targeting the cellular protease TMPRSS2. Conclusions: The proof-of-concept (POC) cellular assay platform components, namely, the two SVPCs and the highly susceptible target cell line, should prove useful in future small-molecule cell-entry inhibitor discovery campaigns, as well as the development of neutralizing antibodies and vaccine candidates directed against SARS-CoV-2. Moreover, the panel of transposon vectors should be easily adaptable to other non-cytotoxic viral pathogens provided these efficiently pseudotype MLV.