Engineered thermoswitch in the baculovirus expression vector system for production of virus-like particle vaccines with minimized baculovirus contaminants

The baculovirus expression vector system (BEVS) is commercially used for producing recombinant (glyco)proteins, gene therapy vectors, and virus-like particles (VLPs) in insect cells. A major challenge in VLP downstream processing (DSP) is the removal of similarly sized baculovirus particle contaminants. Herein we engineered a genetic ON/OFF thermoswitch in the baculovirus vector by mutating the essential gp41 gene. This temperature-sensitive (ts) baculovirus expression system (BacFree^ts) produces high baculovirus titers at 27°C, while contaminating baculovirus production switches off at elevated temperatures. We demonstrate scalable VLP production for chikungunya virus (CHIKV), West Nile virus (WNV), coxsackievirus A6, and foot-and-mouth disease virus (FMDV) at 33°C, concomitant with baculovirus particle reduction up to 99.97%. However, elevated temperature moderately affected the yield of two out of four VLP designs. BacFree^ts is compatible with commonly used insect cells and scalable infection strategies. BacFree^ts effectively reduces baculovirus contaminants in the upstream VLP production phase, which will facilitate DSP of human and veterinary VLP-based vaccines.