Enhanced Production of Chikungunya Virus-Like Particles Using a High-pH Adapted Spodoptera frugiperda Insect Cell Line

Abstract

Chikungunya virus-like particles (VLPs) have potential to be used as a prophylactic vaccine based on testing in multiple animal models and 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 and 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 standard culture media (pH 6.2–6.4), properly processed Chikungunya structural proteins were detected and 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 and Sf21 culture, medium modifications were made and 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 and 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 and shown to be similar in size and structure to a VLP standard derived from transient gene expression in HEK293 cells. Total serum anti-Chikungunya IgG and 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 and 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 and VLPs in an elevated pH range may also have applications for other pH-sensitive protein or VLP targets.

Alphavirus Nucleocapsid Packaging and Assembly

“…CHIKV virus-like particles(VLPs) have been generated from the heterologous expression of the structural proteins in both insect and mammalian cells [69][70][71]. These particles have been used in a Phase 1 clinical trial as a vaccine for CHIKV owing to the fact that the particles resemble wt particles and therefore illicit a relevant immune response [72].…”

Development and application of a reversed-phase high-performance liquid chromatographic method for quantitation and characterization of a Chikungunya virus-like particle vaccine

“…Instead of elongating the intermediate to produce complex glycans as in mammalian cells, insect cells trim the non-reducing GlcNAc to produce the major N-glycan Man 3 GlcNAc 2 Fuc (paucimannose) [30] [31]. To express CHIKV VLP in insect cells, Wagner et al developed a novel insect cell line - S f Basic by adapting Sf21 in elevated culture pH [19,32,33]. E2 from VLPs expressed in this cell substrate had different elution profile compared to HEK293 expressed VLP (Figure 5).…”

“…The expression and purification of CHIKV VLPs from the mammalian and the insect cell systems were described by Wagner et al . [19]. Briefly, HEK293 cells were transiently transfected with a plasmid DNA encoding the CHIKV structural genes.…”

Chikungunya virus vaccines: Current strategies and prospects for developing plant-made vaccines

“…This technology was adopted by the pharmaceutical company Merck that conducted studies consisting on the immunization of Guinea pigs with two doses (on days 0 and 14) that ranged 0.01-10 g along with the Adju-Phos aluminum adjuvant. The induction of specific IgG neutralizing antibodies in a dose-dependent manner was observed [58].…”