Stability of recombinant baculoviruses in chemically defined medium

• Grant number: 24/17037-9
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: November 01, 2024
• End date: October 31, 2025
• Field of knowledge: Health Sciences - Pharmacy - Pharmaceutical Technology
• Principal Investigator: Eutimio Gustavo Fernández Núñez
• Grantee: Rodrigo Jorge Atanes Netto
• Host Institution: Escola de Artes, Ciências e Humanidades (EACH). Universidade de São Paulo (USP). São Paulo , SP, Brazil

Abstract

The production of viral vaccines, one of the most relevant classes of products within pharmaceutical biotechnology due to their positive impact on quality of life and the reduction of costs for national health systems, can be done through multiple platforms, such as through virus-like particles (VLP). Through this alternative, a protective immune response is generated with low doses. Consequently, the prevention against the viral pathogen is achieved, without risk of infection for the patient due to the absence of genetic material in the protein nanostructure which makes up the active pharmaceutical ingredient. In this context of VLP production, several technologies have been implemented, including expression systems using viral vectors based on recombinant baculoviruses and insect cells, an approach that takes advantage of the robustness and productivity of insect cells to manufacture complex heterologous proteins, as in the case of VLP, which often require post-translational modifications such as glycosylation and phosphorylation that a prokaryote would hardly be able to produce. Based on these favorable characteristics that VLP production using recombinant baculoviruses offers, several laboratories in Brazil and around the world have been proposing new methodologies for producing the most varied complex proteins, however with little published data on the stability of these recombinant baculoviruses throughout the storage period, as well as their ideal temperature or even whether the size of the inserted recombinant gene affects their infectivity during long-term storage in chemically defined media free of components of animal origin. Regarding these present issues, the present project aims to elucidate how these parameters influence the stability of recombinant baculoviruses in 1 year at 4 and -80 °C, with monthly sampling, using as a method for viral titration the determination of the 50% infective dose in culture of genetically modified insect cells Sf9 Easy Titre per unit volume (TCID_50/mL) then transformed into plaque-forming units per unit volume (TCID_50/mL). For this, batches of recombinant baculoviruses with genes of different sizes (kpb) will be generated in a chemically defined medium EX-CELL® CD (Sigma-Aldrich, MO, United States). With the collected data, mathematical models can be established to characterize the kinetics of loss of viral infectivity. The results of this proposal will help in the standardization of future commercial processes that use the recombinant baculovirus/insect cell system to produce recombinant proteins, especially VLP.