Propagation and immunological characterization of coxsackievirus A10 in a serum-free HEK293A cell culture system

Abstract

Coxsackievirus A10 (CVA10) is one of enteroviral pathogens that cause the hand, foot, and mouth disease (HFMD). Since CVA10 was reported to be not easily propagated in the Vero cell culture, a feasible manufacture process for producing formalin-inactivated CVA10 vaccine is urgently needed. Several cell lines that commonly used for viral vaccine production was tested for CVA10 (M2014 strain) culture in this study, and our result showed that CVA10 could be easily propagated in the HEK293A cells. A serum-free HEK293A cell culture system was developed for CVA10 production and the yields have reached over 10⁸ TCID₅₀/mL. The biochemical and immunogenic properties of CVA10 particles obtained from this serum-free HEK293A culture were identical to our previous study. Two major particles of CVA10 were separated by ultracentrifugation, and only the infectious mature particles were capable of inducing CVA10 neutralizing antibody responses in the mouse immunogenicity studies. Additionally, we found that coxsackievirus A6 and enterovirus A71 could also be easily propagated using this serum-free HEK293A cell culture system. Our results provide a solution to overcome the obstacle in the propagation of CVA10 and facilitate the development of multivalent vaccines for prevention of HFMD.

Keywords: Coxsackievirus A10 (CVA10); HEK293A; Hand foot and mouth diseases; Inactivated whole virion vaccine; Serum-free cell culture.

Fig. 1

Propagation of CVA10 in RD, Vero, MDCK, and HEK293A cells. All tested cell lines were seeded with 1 × 10⁶ cells in T-25 flask. After 8 h of incubation, the medium was replaced with 10 mL fresh medium and the cells were infected with CVA10 at MOI = 10⁻⁴. (A) Cell morphologies of CVA10-infected and control cells. (B) The replication kinetics of CVA10 in different cell lines. The bar represents 100 µm.

Fig. 2

Propagation profiles of CVA10 in serum-free roller bottle culture. HEK293A cells were infected with CVA10 at an MOI of 10⁻⁴. (A) Morphology of serum-free HEK293A cells in roller bottle. (B) Morphology of HEK293A cells after CVA10 infection. (C) TCID₅₀ values of three roller bottles after counting CPE in infected RD cells. The TCID₅₀ values were calculated using the Reed-Muench method. The bar represents 100 µm.

Fig. 3

Propagation profiles of CVA10 in serum-free suspension culture. HEK293A cells were infected with CVA10 at an MOI of 10⁻⁴. (A) Suspension HEK293A cells in a spinner. (B) Suspension HEK293A cells after CVA10 infection. (C) TCID₅₀ values of four spinners after counting CPE in the infected RD cells. (D) Extracellular and intracellular TCID₅₀ values of serum-free bioreactor culture after counting CPE in infected RD cells. The TCID₅₀ values were calculated using the Reed-Muench method. The bar represents 100 µm.

Fig. 4

The purification profiles of CVA10 particles by two methods. (A) Purification of CVA10 by S-500 size exclusion chromatography. Total viral particles were eluted among fractions 18 to 22. (B) Purification of CVA10 by 10–60% sucrose gradient zonal ultracentrifugation. E-particles were eluted at fractions 6–7 and F-particles were eluted at fractions 12 to 16. M: protein ladder; C: concentrated CVA10 solution before purification. The numbers on the top of panels indicate the fraction collected during elution. The VP1 viral antigen was detected by western blot with a polyclonal antibody GTX132346.

Fig. 5

Viral antigen profile of CVA10 particles analyzed by SDS-PAGE and western blotting. (A) Samples of CVA10 particles analyzed on a NuPAGE 4–12% gel. (B) CVA10 VP1 protein detected by the GTX132346 antibody. Twenty µL of each sample was loaded for western blot analyses. M: Protein ladder; Conc.: concentrated CVA10 solution before purification; LC: viral fractions from size exclusion chromatography; E: E-particle fractions from sucrose gradient ultracentrifugation; and F: F-particle fractions from sucrose gradient ultracentrifugation.

Fig. 6

CVA10 particles analyzed by TEM. (A) CVA10 viral particles purified by size exclusion chromatography. (B) E-particle fractions of CVA10 purified from sucrose gradient ultracentrifugation. (C) F-particle fractions of CVA10 purified from sucrose gradient ultracentrifugation. Four µL of sample was loaded on carbon-vaporized copper grid (200-mesh) and stained with 2% uranyl acetate solution. The stained sample was examined by the Joel JEM-1400 TEM. The bar represents 100 nm.

Fig. 7

Antibody specificity assay of immunized mice to three enteroviruses (CVA6, CVA10 and EV-A71). Sera of mice immunized with CVA10 mixture particles (CVA10-LC), E-particles (CVA10-E), F-particles (CVA10-F), and mock group (PBS) were tested by ELISA. Anti-EV-A71 (mAb979) and anti-CVA6 VP1 (GTX132346) antibodies were included as controls.

Fig. 8

Propagation profiles of other HFMD-related viruses in serum-free HEK293A cell culture. HEK293A cells were infected with each virus (CVA6 and EV-A71) at MOI = 10⁻⁴. The culture and harvest conditions were identical to that described in the Method for CVA10 propagation. (A) Morphology of HEK293A cells after virus infection in roller bottle at 6 DPI. (B) Morphology of suspension HEK293A cells after virus infection in spinner at 4 DPI. (C) TCID₅₀ values of serum-free roller bottle cultures after counting CPE in infected RD cells. (D) TCID₅₀ values of serum-free spinner cultures after counting CPE in infected RD cells. The TCID₅₀ values were calculated using the Reed-Muench method. The bar represents 100 µm.