Serum-Free Suspension Culture of the <i>Aedes albopictus</i> C6/36 Cell Line for Chimeric Orthoflavivirus Vaccine Production
Chimeric orthoflaviviruses derived from the insect-specific Binjari virus (BinJV) offer a promising basis for safe orthoflavivirus vaccines. However, these vaccines have so far only been produced using adherent C6/36 <i>Aedes albopictus</i> mosquito cell cultures grown in serum-supplemen...
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2025-02-01
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| Online Access: | https://www.mdpi.com/1999-4915/17/2/250 |
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| author | Joshua S. Dawurung Jessica J. Harrison Naphak Modhiran Roy A. Hall Jody Hobson-Peters Henry de Malmanche |
| author_facet | Joshua S. Dawurung Jessica J. Harrison Naphak Modhiran Roy A. Hall Jody Hobson-Peters Henry de Malmanche |
| author_sort | Joshua S. Dawurung |
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| description | Chimeric orthoflaviviruses derived from the insect-specific Binjari virus (BinJV) offer a promising basis for safe orthoflavivirus vaccines. However, these vaccines have so far only been produced using adherent C6/36 <i>Aedes albopictus</i> mosquito cell cultures grown in serum-supplemented media, limiting their scalable manufacture. To address this, we adapted C6/36 cells for serum-free suspension culture using Sf900-III medium, achieving high peak cell densities (up to 2.5 × 10<sup>7</sup> cells/mL). Higher agitation rates reduced cell aggregation, and cryopreservation and direct-to-suspension revival were successful, confirming the adapted line’s stability for research and industrial applications. Despite this, BinJV-based chimeric orthoflaviviruses, including BinJV/WNV<sub>KUN</sub>, a candidate vaccine for West Nile virus, and similar vaccines (BinJV/DENV2 and BinJV/JEV<sub>NSW22</sub>) for dengue 2 virus and Japanese encephalitis virus, respectively, exhibited substantially reduced titres in C6/36 cultures infected in Sf900-III, a phenomenon attributed to the medium’s acidic pH. Switching to the more alkaline, serum-free CD-FortiCHO medium enhanced the replication of these chimeric viruses to peak titres between 1.7 × 10<sup>7</sup> and 7.6 × 10<sup>9</sup> infectious units per mL whilst preserving viral integrity. These findings suggest that suspension-adapted C6/36 cultures in CD-FortiCHO medium can support high-yield vaccine production for various orthoflaviviruses and highlight the important role of cell culture media pH for orthoflavivirus bioprocessing. This scalable mosquito cell-based system could reduce production costs and improve vaccine accessibility, supporting efforts to combat arbovirus-related public health challenges. |
| format | Article |
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| institution | DOAJ |
| issn | 1999-4915 |
| language | English |
| publishDate | 2025-02-01 |
| publisher | MDPI AG |
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| series | Viruses |
| spelling | doaj-art-4c370b6743ed4f53bb84f6f7588ff5d22025-08-20T02:45:42ZengMDPI AGViruses1999-49152025-02-0117225010.3390/v17020250Serum-Free Suspension Culture of the <i>Aedes albopictus</i> C6/36 Cell Line for Chimeric Orthoflavivirus Vaccine ProductionJoshua S. Dawurung0Jessica J. Harrison1Naphak Modhiran2Roy A. Hall3Jody Hobson-Peters4Henry de Malmanche5School of Chemistry and Molecular Biosciences, The University of Queensland, St Lucia, QLD 4072, AustraliaSchool of Chemistry and Molecular Biosciences, The University of Queensland, St Lucia, QLD 4072, AustraliaSchool of Chemistry and Molecular Biosciences, The University of Queensland, St Lucia, QLD 4072, AustraliaSchool of Chemistry and Molecular Biosciences, The University of Queensland, St Lucia, QLD 4072, AustraliaSchool of Chemistry and Molecular Biosciences, The University of Queensland, St Lucia, QLD 4072, AustraliaSchool of Chemistry and Molecular Biosciences, The University of Queensland, St Lucia, QLD 4072, AustraliaChimeric orthoflaviviruses derived from the insect-specific Binjari virus (BinJV) offer a promising basis for safe orthoflavivirus vaccines. However, these vaccines have so far only been produced using adherent C6/36 <i>Aedes albopictus</i> mosquito cell cultures grown in serum-supplemented media, limiting their scalable manufacture. To address this, we adapted C6/36 cells for serum-free suspension culture using Sf900-III medium, achieving high peak cell densities (up to 2.5 × 10<sup>7</sup> cells/mL). Higher agitation rates reduced cell aggregation, and cryopreservation and direct-to-suspension revival were successful, confirming the adapted line’s stability for research and industrial applications. Despite this, BinJV-based chimeric orthoflaviviruses, including BinJV/WNV<sub>KUN</sub>, a candidate vaccine for West Nile virus, and similar vaccines (BinJV/DENV2 and BinJV/JEV<sub>NSW22</sub>) for dengue 2 virus and Japanese encephalitis virus, respectively, exhibited substantially reduced titres in C6/36 cultures infected in Sf900-III, a phenomenon attributed to the medium’s acidic pH. Switching to the more alkaline, serum-free CD-FortiCHO medium enhanced the replication of these chimeric viruses to peak titres between 1.7 × 10<sup>7</sup> and 7.6 × 10<sup>9</sup> infectious units per mL whilst preserving viral integrity. These findings suggest that suspension-adapted C6/36 cultures in CD-FortiCHO medium can support high-yield vaccine production for various orthoflaviviruses and highlight the important role of cell culture media pH for orthoflavivirus bioprocessing. This scalable mosquito cell-based system could reduce production costs and improve vaccine accessibility, supporting efforts to combat arbovirus-related public health challenges.https://www.mdpi.com/1999-4915/17/2/250suspension cultureC6/36vaccinesorthoflavivirus chimaerasvirus bioprocessing |
| spellingShingle | Joshua S. Dawurung Jessica J. Harrison Naphak Modhiran Roy A. Hall Jody Hobson-Peters Henry de Malmanche Serum-Free Suspension Culture of the <i>Aedes albopictus</i> C6/36 Cell Line for Chimeric Orthoflavivirus Vaccine Production Viruses suspension culture C6/36 vaccines orthoflavivirus chimaeras virus bioprocessing |
| title | Serum-Free Suspension Culture of the <i>Aedes albopictus</i> C6/36 Cell Line for Chimeric Orthoflavivirus Vaccine Production |
| title_full | Serum-Free Suspension Culture of the <i>Aedes albopictus</i> C6/36 Cell Line for Chimeric Orthoflavivirus Vaccine Production |
| title_fullStr | Serum-Free Suspension Culture of the <i>Aedes albopictus</i> C6/36 Cell Line for Chimeric Orthoflavivirus Vaccine Production |
| title_full_unstemmed | Serum-Free Suspension Culture of the <i>Aedes albopictus</i> C6/36 Cell Line for Chimeric Orthoflavivirus Vaccine Production |
| title_short | Serum-Free Suspension Culture of the <i>Aedes albopictus</i> C6/36 Cell Line for Chimeric Orthoflavivirus Vaccine Production |
| title_sort | serum free suspension culture of the i aedes albopictus i c6 36 cell line for chimeric orthoflavivirus vaccine production |
| topic | suspension culture C6/36 vaccines orthoflavivirus chimaeras virus bioprocessing |
| url | https://www.mdpi.com/1999-4915/17/2/250 |
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