Zika virus-like particle production in a stirred tank bioreactor using a baculovirus/insect cell system

Abstract

Zika virus (ZIKV) was declared a public health emergency in 2016, yet effective vaccines are still needed. Among the immunization platforms under evaluation, virus-like particles (VLP) are promising candidates. Growth, metabolism, and respiration are among the cell host processes that are essential for optimizing and characterizing VLP upstream production stage. These cell functions can be influenced by factors such as culture medium composition and the multiplicity of infection (MOI) in viral vector-based expression systems. This study investigated the effects of three MOIs (2, 6, and 10) in a baculovirus/Sf9 insect cell system on ZIKV VLP production with and without medium supplemented with 0.028 mM cholesterol and 6 nM albumin. Medium supplementation during the growth phase increased the cell growth rate from 0.357 × 10⁴ to 0.565 × 10⁴ $\frac{\text{cells}}{\text{mL}·\text{h}}$ . In addition, cholesterol and albumin supplementation increased the expression of ZIKV structural proteins during infection. Higher MOIs led to increased substrate uptake and metabolite production, suggesting intensified cellular metabolism. Western blot analysis revealed that under nonsupplemented conditions, the highest MOI resulted in increased ZIKV envelope production, with a maximum protein concentration range of 1.049 $\frac{\text{mg}}{\text{L}}$ higher when comparing 6 to 2 $\frac{\text{PFU}}{\text{cell}}$ MOI via SDS‒PAGE densitometry. However, a lower MOI, 2 $\frac{\text{PFU}}{\text{cell}}$ , might be advantageous when a supplemented medium is used, which upper limit for ZIKV envelope protein concentration was 1.834 $\frac{\text{mg}}{\text{L}}$ higher than that from the nonsupplemented assay in semiquantitative analysis, which reached 23.504 $\frac{\text{mg}}{\text{L}}$ of ZIKV envelope protein. The resulting VLP had an average diameter of ~ 60 nm, making them suitable for vaccine applications.

Keywords: Baculovirus/insect cell system; Biochemical engineering; Medium supplementation; Multiplicity of infection; Virus-like particles; Zika virus.