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

Area of Science:

• * Biotechnology
• * Virology
• * Vaccine Development

Background:

• * Rabies causes significant global mortality, necessitating scalable and cost-effective vaccines.
• * Virus-like particles (VLPs) are promising rabies vaccine candidates, but their production is sensitive to host cell physiology and process parameters.
• * Optimizing VLP production requires understanding the interplay between expression systems, infection timing, and harvest timing.

Purpose of the Study:

• * To optimize the production of rabies virus-like particles (VLPs) using different insect cell lines.
• * To investigate the impact of time of infection (TOI) and harvest time (HT) on VLP yield and integrity.
• * To identify optimal upstream strategies for maximizing both rabies virus glycoprotein (RVGP) and rabies membrane protein (RVM) expression for vaccine development.

Main Methods:

• * Production of rabies VLPs in Spodoptera frugiperda (Sf9) and High Five insect cells.
• * Application of a three-level full factorial design to optimize TOI and HT.
• * Kinetic analyses of cell behavior and baculovirus propagation.
• * Transmission electron microscopy (TEM) to assess VLP integrity and size.

Main Results:

• * Sf9 cells yielded significantly higher rabies virus glycoprotein (RVGP) (401.6 ng/mL) compared to High Five cells.
• * High Five cells achieved maximal rabies membrane protein (RVM) expression (1294.5 ng/mL).
• * Optimal conditions for maximizing both RVGP and RVM were identified as Sf9 cells at TOI 72 h and HT 96 h post-infection.
• * TEM confirmed VLP integrity with mean diameters of 119 nm (Sf9) and 144 nm (High Five), suitable for immunogenicity.

Conclusions:

• * Host cell choice significantly impacts rabies VLP component expression, with Sf9 excelling in RVGP and High Five in RVM.
• * Tailored upstream processing strategies, considering specific cell line kinetics and optimal TOI/HT, are crucial for efficient VLP vaccine production.
• * Maximizing RVGP, the key immunogen, while ensuring adequate RVM for assembly, supports the rational design of robust rabies VLP vaccine manufacturing processes.