Abstract
Mammalian cell culture is an important technology for the production of viral vaccines. Microcarrier culture introduces the possibility the practical high yield culture of anchorage-dependent mammalian cells in suspension. A new trend of microcarrier technology is to utilize serum-free culture to overcome the drawbacks of serum-containing media. These drawbacks include high serum-protein content for complicating downstream purification process and the risk for potential contamination of prions due to the bovine resource. Enterovirus 71 (EV71) and dengue virus (DEN) are two important infectious diseases in Taiwan. Effective vaccines for enterovirus 71 and dengue virus are still been developed. This research has proposed the large-scale preparation by a scalable cell culture system and established a serum-free microcarrier-based cell culture for development of inactivated enterovirus 71 vaccine and live-attenuated dengue virus vaccine. In EV71 virus research, three strains (EV71-1207, EV71-075 and EV71-117) that contain two genotypes were propagated in a serum-free microcarrier culture. Vero cells were found to produce higher titers of EV71 than WI-38 and MRC-5 cells. Microcarrier Vero cell cultures were established using 5g/L Cytodex 1 microcarriers and found to promote the extracellular release of EV71s from infected Vero cells at 32oC. The large-scale preparation of EV71s can be achieved using serum-free microcarrier Vero cell culture in a 2-liter bioreactor. No significant differences were observed for the formalin-inactivation kinetics of the three EV71 strains in serum-free and serum-containing cultures. The immunogenicity of the inactivated EV71 virions produced in serum-free cultures elicited slight higher levels of neutralizing antibody response in immunized mice and exhibited a cross-neutralization ability to resist three EV71 virus strains. The inactivated virions of EV71-075 and EV71-117 strains elicited approximately 1-log increase in ID50 values than EV71-1207 strain. EV71-075 and EV71-117 has identical amino acid sequences in the VP1 protein, while EV71-1207 revealed 12 amino acid sequences differences. Nucleotide sequence analysis indicated that the VP1 protein of EV71-075 and EV71-117 belonging to the B4 subgenotype. In dengue virus research, four serotypes dengue virus strains (DEN-1 strain HAWAII, DEN-2 strain NGC, DEN-3 strain H-87 and DEN-4 strain H-241) and a DEN-4 infectious clone (strain 2A) were propagated in serum-free microcarrier cultures. Vero and MRC-5 cells were grown on microcarriers using 2g/L Cytodex 1 and propagated these dengue viruses in serum-free cultures. The virus titers of dengue virus produced in microcarrier Vero cell cultures were higher than that produced in microcarrier MRC-5 cell cultures for each of these dengue strains. The genetic quasispecises of DEN-4 infectious clone virus were observed that MRC-5 cells produced the lower sequence heterogeneity than Vero cells in microcarrier cultures. These results constitute valuable information on the development of a serum-free microcarrier cell culture process for producing inactivated EV71 vaccine and tetravalent live-attenuated dengue vaccines.