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.