Enhanced production of recombinant coxsackievirus A16 using a serum-free HEK293A suspension culture system for bivalent enterovirus vaccine development

Figures

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  Fig. 1. Construction of CVA16 infectious clone. (A) Structure of the CVA16ic plasmid. The pFL-CVA16-N5079 plasmid contains full-length CVA16 N5079 cDNA inserted into a modified pCMV-HA vector. (B) Agarose gel analysis of the CVA16ic plasmid digested by restriction enzymes XbaI (lane 2), XbaI/KpnI (lane 3), XhoI/EcoRV (lane 4), and ApaI/EcoRV (lane 5). A DNA ladder (M) was included in lane 1. (C) Cytopathic effects induced by CVA16ic P2 virus infection in RD cells at 3 DPI. RD cells were seeded at 5 × 105 cells per well in 6-well plates 24 h before virus infection. The medium was replaced with 1 mL of CVA16ic P1 virus-containing medium (directly from transfected cells) for 1 h, followed by replacement with 2 mL of fresh medium.

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  Fig. 2. Characterization of the CVA16ic virus. (A) Susceptibility of RD, Vero, and HEK293 cells to CVA16 and EV-A71 viruses. All cell lines were seeded at 2.5 × 105 cells per well in 6-well plates 24 h prior to viral infection. The cells were infected with CVA16wt, CVA16ic, and EV-A71 viruses at MOI=10-3. Infected cells were observed at 4 DPI. (B) Western blot analysis of samples collected from infected cells at 4 DPI. Ten μL of culture supernatant were loaded and detected with the VP1 (GTX132346) and the VP2 (mAb979) antibodies. The GAPDH antibody served as a loading proof control, but not for normalization due to different expression levels in these cell lines. (C) Plaque morphology of CVA16ic and CVA16wt. Vero cells were used to adsorb the two CVA16 viruses, and the overlay medium contained 1.2 % methylcellulose for plaque development. (D) Nucleotide differences in the capsid protein between CVA16ic and CVA16wt. Two silent mutations were engineered as molecular markers. (E) Sequencing results showing the retention of molecular markers in the virus working bank.

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  Fig. 3. Replication kinetics of two CVA16 viruses in Vero and HEK293A culture systems. Replication kinetics of CVA16ic and CVA16wt viruses were measured in Vero cells and HEK293A suspension cells. Vero cells were seeded in roller bottles with VP-SFM medium, and HEK293A cells were seeded at a density of 1 × 106 cells/mL in a 250 mL spinner with BalanCD293 medium. CVA16ic (A to C) or CVA16wt (D to F) infected both cell types at MOIs of (A and D) 10-3, (B and E) 10-4, and (C and F) 10-5. The TCID50 values were calculated using the Reed-Muench method, and results from triplicate experiments are presented with error bars indicating standard deviations.

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  Fig. 4. Purification and characterization of CVA16ic E- and F-particles. (A) Purification of CVA16ic by 10–60 % sucrose gradient zonal ultracentrifugation. M: protein ladder; C: sample loaded with CVA16ic from culture medium. The CVA16ic antigen was detected with the VP2 antibody. The numbers below the panels indicate the fractions collected during elution, and the numbers next to the protein ladder indicate protein sizes. CVA16ic antigen after ultracentrifugation was analyzed by western blotting. (B) Protein profiles of the purified CVA16ic E- and F-particles using SDS-PAGE 4–12 % gel. M: protein ladder; E: E-particle from fraction 6; and F: F-particle from fraction 13. (C) CVA16ic E-particles (left) and F-particles (right) analyzed by TEM. The scale bar represents 100 nm.

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  Fig. 5. Neutralizing antibody titers in mice. Sera from mice immunized with mock (PBS), CVA16 E-particle, CVA16 F-particle, and bivalent CVA16 F-particle and EV-A71 antigen were evaluated for neutralizing antibody titers against (A) CVA16 virus and (B) EV-A71 virus.

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  Fig. 6. Specific antibody titers in mice antisera. Sera from mice immunized with mock (PBS), CVA16 E-particle, CVA16 F-particle, and bivalent CVA16 F-particle and EV-A71 antigen were evaluated for specific antibody titers against (A) CVA16 virus and (B) EV-A71 virus.