Host-dependent optimization of rabies virus-like particle making in insect cells using a low-level control system

Rabies causes over 59,000 deaths annually, highlighting the need for scalable and cost-effective vaccines. Virus-like particles (VLP) are promising candidates, but their production depends on host physiology and process parameters. Here, rabies VLP were produced in Spodoptera frugiperda (Sf9) and High Five insect cells, employing a three-level full factorial design to optimize the impact of the expression system, time of infection (TOI), and harvest time (HT). The cell lines exhibited complementary expression profiles: Sf9 achieved the highest rabies virus glycoprotein (RVGP) expression, reaching 401.6 ng/mL (2.8-fold higher than High Five) at TOI 72 h and HT 96 hpi. In contrast, High Five maximized rabies membrane protein (RVM) expression, reaching 1294.5 ng/mL. Kinetic analyses further revealed distinct cellular behaviors: Sf9 supported efficient baculovirus propagation, while High Five maintained over 90% viability post-infection, consistent with a less effective infection profile. Transmission electron microscopy confirmed VLP integrity, with mean diameters of 119 nm in Sf9 and 144 nm in High Five, both suitable for immunogenicity. Statistical modeling identified Sf9 cells at TOI 72 h and HT 96 hpi as the optimal condition to maximize RVGP and RVM yields simultaneously. These results highlight the necessity of host-tailored upstream strategies, emphasizing RVGP, the primary immunogenic determinant, while ensuring sufficient RVM expression for particle assembly, thus supporting the rational design of robust processes for rabies VLP vaccines.