Enhanced production of Chikungunya virus-like particles using a high-pH adapted spodoptera frugiperda insect cell line
Autor(es): Wagner James M, Pajerowski J David, Daniels Christopher L, McHugh Patrick M, Flynn Jessica A, Balliet John W, Casimiro Danilo R, Subramanian Shyamsundar
Resumo: Chikungunya virus-like particles (VLPs) have potential to be used as a prophylactic vaccine based on testing in multiple animal models - are currently being evaluated for human use in a Phase I clinical trial. The current method for producing these enveloped alphavirus VLPs by transient gene expression in mammalian cells presents challenges for scalable - robust industrial manufacturing, so the insect cell baculovirus expression vector system was evaluated as an alternative expression technology. Subsequent to recombinant baculovirus infection of Sf21 cells in st-ard culture media (pH 6.2-6.4), properly processed Chikungunya structural proteins were detected - assembled capsids were observed. However, an increase in culture pH to 6.6-6.8 was necessary to produce detectable concentrations of assembled VLPs. Since this elevated production pH exceeds the optimum for growth medium stability - Sf21 culture, medium modifications were made - a novel insect cell variant (SfBasic) was derived by exposure of Sf21 to elevated culture pH for a prolonged period of time. The high-pH adapted SfBasic insect cell line described herein is capable of maintaining normal cell growth into the typical mammalian cell culture pH range of 7.0-7.2 - produces 11-fold higher Chikungunya VLP yields relative to the parental Sf21 cell line. After scale-up into stirred tank bioreactors, SfBasic derived VLPs were chromatographically purified - shown to be similar in size - structure to a VLP st-ard derived from transient gene expression in HEK293 cells. Total serum anti-Chikungunya IgG - neutralizing titers from guinea pigs vaccinated with SfBasic derived VLPs or HEK293 derived VLPs were not significantly different with respect to production method, suggesting that this adapted insect cell line - production process could be useful for manufacturing Chikungunya VLPs for use as a vaccine. The adaptation of Sf21 to produce high levels of recombinant protein - VLPs in an elevated pH range may also have applications for other pH-sensitive protein or VLP targets.
Imprenta: PloS One, v. 9, n. 4, p. e94401, 2014
Identificador do objeto digital: 10.1371/journal.pone.0094401
Descritores: Chikungunya virus - Biosynthesis ; Chikungunya virus - Cell ; Chikungunya virus - Pathogenesis ; Chikungunya virus - Proteins ; Chikungunya Virus - Virus ; Chikungunya virus - Vaccine ; Chikungunya virus - Immunology
Data de publicação: 2014
