Cargando…
Enhanced Production of Chikungunya Virus-Like Particles Using a High-pH Adapted Spodoptera frugiperda Insect Cell Line
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 expre...
Autores principales: | , , , , , , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2014
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3979839/ https://www.ncbi.nlm.nih.gov/pubmed/24713807 http://dx.doi.org/10.1371/journal.pone.0094401 |
_version_ | 1782310773347844096 |
---|---|
author | Wagner, James M. Pajerowski, J. David Daniels, Christopher L. McHugh, Patrick M. Flynn, Jessica A. Balliet, John W. Casimiro, Danilo R. Subramanian, Shyamsundar |
author_facet | Wagner, James M. Pajerowski, J. David Daniels, Christopher L. McHugh, Patrick M. Flynn, Jessica A. Balliet, John W. Casimiro, Danilo R. Subramanian, Shyamsundar |
author_sort | Wagner, James M. |
collection | PubMed |
description | 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. |
format | Online Article Text |
id | pubmed-3979839 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2014 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-39798392014-04-11 Enhanced Production of Chikungunya Virus-Like Particles Using a High-pH Adapted Spodoptera frugiperda Insect Cell Line Wagner, James M. Pajerowski, J. David Daniels, Christopher L. McHugh, Patrick M. Flynn, Jessica A. Balliet, John W. Casimiro, Danilo R. Subramanian, Shyamsundar PLoS One Research Article 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. Public Library of Science 2014-04-08 /pmc/articles/PMC3979839/ /pubmed/24713807 http://dx.doi.org/10.1371/journal.pone.0094401 Text en © 2014 Wagner et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited. |
spellingShingle | Research Article Wagner, James M. Pajerowski, J. David Daniels, Christopher L. McHugh, Patrick M. Flynn, Jessica A. Balliet, John W. Casimiro, Danilo R. Subramanian, Shyamsundar Enhanced Production of Chikungunya Virus-Like Particles Using a High-pH Adapted Spodoptera frugiperda Insect Cell Line |
title | Enhanced Production of Chikungunya Virus-Like Particles Using a High-pH Adapted Spodoptera frugiperda Insect Cell Line |
title_full | Enhanced Production of Chikungunya Virus-Like Particles Using a High-pH Adapted Spodoptera frugiperda Insect Cell Line |
title_fullStr | Enhanced Production of Chikungunya Virus-Like Particles Using a High-pH Adapted Spodoptera frugiperda Insect Cell Line |
title_full_unstemmed | Enhanced Production of Chikungunya Virus-Like Particles Using a High-pH Adapted Spodoptera frugiperda Insect Cell Line |
title_short | Enhanced Production of Chikungunya Virus-Like Particles Using a High-pH Adapted Spodoptera frugiperda Insect Cell Line |
title_sort | enhanced production of chikungunya virus-like particles using a high-ph adapted spodoptera frugiperda insect cell line |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3979839/ https://www.ncbi.nlm.nih.gov/pubmed/24713807 http://dx.doi.org/10.1371/journal.pone.0094401 |
work_keys_str_mv | AT wagnerjamesm enhancedproductionofchikungunyaviruslikeparticlesusingahighphadaptedspodopterafrugiperdainsectcellline AT pajerowskijdavid enhancedproductionofchikungunyaviruslikeparticlesusingahighphadaptedspodopterafrugiperdainsectcellline AT danielschristopherl enhancedproductionofchikungunyaviruslikeparticlesusingahighphadaptedspodopterafrugiperdainsectcellline AT mchughpatrickm enhancedproductionofchikungunyaviruslikeparticlesusingahighphadaptedspodopterafrugiperdainsectcellline AT flynnjessicaa enhancedproductionofchikungunyaviruslikeparticlesusingahighphadaptedspodopterafrugiperdainsectcellline AT ballietjohnw enhancedproductionofchikungunyaviruslikeparticlesusingahighphadaptedspodopterafrugiperdainsectcellline AT casimirodanilor enhancedproductionofchikungunyaviruslikeparticlesusingahighphadaptedspodopterafrugiperdainsectcellline AT subramanianshyamsundar enhancedproductionofchikungunyaviruslikeparticlesusingahighphadaptedspodopterafrugiperdainsectcellline |