Serum-Free Suspension Culture of the Aedes albopictus C6/36 Cell Line for Chimeric Orthoflavivirus Vaccine Production

Abstract

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 Aedes albopictus 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 × 10⁷ 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_KUN, a candidate vaccine for West Nile virus, and similar vaccines (BinJV/DENV2 and BinJV/JEV_NSW22) 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 × 10⁷ and 7.6 × 10⁹ 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.

Keywords: C6/36; orthoflavivirus chimaeras; suspension culture; vaccines; virus bioprocessing.

Figure 1

Growth kinetics and morphology of C6/36 cultures following adaptation to suspension. (A) Cell count and viability of 25 mL C6/36 suspension cultures following transition into shake flasks and continual passage via dilution into fresh media, tracking culture behaviour following cryopreservation and revival. Green areas represent live cells; red areas represent dead cells; and dotted line represents culture viability. (B) Culture morphology at low shake speed (120 rpm). (C) Culture morphology at high shake speed (250 rpm). (D) C6/36 suspension cultures were seeded in triplicate at two different cell densities and viable cell density was enumerated daily. Mean viable cell density is plotted with error bars representing +/− one standard deviation.

Figure 2

Impact of growth medium and pH on chimeric vaccine candidate BinJV/WNV_KUN infectivity and replication. (A) BinJV/WNV_KUN replication kinetics in adherent T-flask culture supernatant following infection in two different media at MOI of 0.1 as measured by TCID50 ELISA; mean titre of three biological replicates is plotted with error bars representing +/− 1 standard deviation. (B) Infectious titre of purified stock of BinJV/WNV_KUN in PBS following dilution and titration in two different media. Error bars indicate 95% confidence internal. (C) Infectious titre of purified stock of BinJV/WNV_KUN following incubation in PBS of various pH values. Error bars indicate 95% confidence internal. (D) Immunofluorescence of BinJV/WNV_KUN in infected C6/36 monolayers in different media types, based on DAPI (blue)/mAb 4G4 (green) staining of fixed cells at 3 days post-infection, were obtained for BinJV/WNV_KUN-infected C6/36 cell monolayers in various media types at three different MOIs.

Figure 3

Comparative infection dynamics of C6/36 suspension cultures inoculated with chimeric orthoflavivirus BinJV/WNV_KUN (MOI:0.1) in CD-FortiCHO and Sf900-III. (A) Suspension culture viable cell density enumerated daily following infection. (B) Replication kinetics of BinJV/WNV_KUN in suspension culture in two media as approximated by daily TCID50 ELISA on clarified culture supernatant. Each data point plotted is mean of three biological replicates, with error bars representing +/− one standard deviation.

Figure 4

Comparative infection dynamics of C6/36 suspension cultures infected with chimeric orthoflaviviruses BinJV/JEV_NSW22 or BinJV/DENV2 (MOI:0.1) in CD-FortiCHO and Sf900-III. (A) Replication kinetics of BinJV/JEV_NSW22 in suspension culture in either Sf900-III or CD-FortiCHO as quantified by TCID50 ELISA. (B) Replication kinetics of BinJV/DENV-2 in suspension culture in either Sf900-III or CD-FortiCHO as quantified by TCID50 ELISA. Each data point plotted is mean of three biological replicates, with error bars representing +/− one standard deviation.

Figure 5

Integrity and purity of virions grown in CD-FortiCHO in suspension culture. (A) Purified viruses were analysed using SDS-PAGE. Lane 1 (from left): molecular weight marker ladder (kDa); lane 2: BinJV/WNV_KUN; lane 3: BinJV/JEV_NSW22; lane 4: BinJV/DENV2. (B) Negative staining transmission electron micrograph of purified BinJV/WNV_KUN; (C) purified BinJV/JEV_NSW22; (D) purified BinJV/DENV2. TEM images are shown at 20,000× magnification and were stained with uranyl acetate.