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.
Highlights
Chikungunya virus (CHIKV) is an arbovirus spread by mosquitoes and capable of causing debilitating, long-term joint pain and arthralgia similar to Dengue [1], [2]
To develop an alternative production platform, the cDNA sequence coding for CHIKV strain 37997 structural polyprotein was inserted into a recombinant baculovirus vector to generate AcMPNV-CHIKV37997
The same p62 band was detected in the HEK293 cell positive control lysate on the same gel, indicating that incomplete processing by furin in Sf21 cells is unlikely to preclude the production of CHIKV virus-like particles (VLPs)
Summary
Chikungunya virus (CHIKV) is an arbovirus (family Togaviridae, genus Alphavirus) spread by mosquitoes and capable of causing debilitating, long-term joint pain and arthralgia similar to Dengue [1], [2]. The recognized insect vector for CHIKV was historically Aedes aegypti, but a single point mutation in CHIKV has been associated with broadening of the geographical range of disease by increasing virus fitness for the alternative mosquito vector Aedes albopictus [8]. This potential for spread of the disease in new areas, including southern Europe, Australia, the Caribbean, and the Americas, has spurred increased research into methods for developing and producing an effective CHIKV vaccine [9], [10]. Many modalities have been evaluated or are currently under investigation, including an attenuated CHIKV vaccine [11], a formalin inactivated CHIKV vaccine [12], chimeric alphavirus vaccines [13], a consensus-based DNA vaccine [14], a Modified Vaccinia Ankara vector vaccine [15], an adenovirus vector vaccine [16], subunit vaccines [17], and virus-like particle (VLP) vaccines [18], [19]
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