title: Pilot scale production of highly efficacious and stable enterovirus 71 vaccine candidates.
abstract: BACKGROUND
  Enterovirus 71 (EV71) has caused several epidemics of hand, foot and mouth diseases (HFMD) in Asia and now is being recognized as an important neurotropic virus. Effective medications and prophylactic vaccine against EV71 infection are urgently needed. Based on the success of inactivated poliovirus vaccine, a prototype chemically inactivated EV71 vaccine candidate has been developed and currently in human phase 1 clinical trial.
  
  
  RESULTS
  In this report, we present the development of a serum-free cell-based EV71 vaccine. The optimization at each step of the manufacturing process was investigated, characterized and quantified. In the up-stream process development, different commercially available cell culture media either containing serum or serum-free was screened for cell growth and virus yield using the roller-bottle technology. VP-SFM serum-free medium was selected based on the Vero cell growth profile and EV71 virus production. After the up-stream processes (virus harvest, diafiltration and concentration), a combination of gel-filtration liquid chromatography and/or sucrose-gradient ultracentrifugation down-stream purification processes were investigated at a pilot scale of 40 liters each. Although the combination of chromatography and sucrose-gradient ultracentrifugation produced extremely pure EV71 infectious virus particles, the overall yield of vaccine was 7-10% as determined by a VP2-based quantitative ELISA. Using chromatography as the downstream purification, the virus yield was 30-43%. To retain the integrity of virus neutralization epitopes and the stability of the vaccine product, the best virus inactivation was found to be 0.025% formalin-treatment at 37 °C for 3 to 6 days. Furthermore, the formalin-inactivated virion vaccine candidate was found to be stable for >18 months at 4 °C and a microgram of viral proteins formulated with alum adjuvant could induce strong virus-neutralizing antibody responses in mice, rats, rabbits, and non-human primates.
  
  
  CONCLUSIONS
  These results provide valuable information supporting the current cell-based serum-free EV71 vaccine candidate going into human Phase I clinical trials.
authors.affiliation: Vaccine R&D Center, National Institute of Infectious Diseases and Vaccinology, National Health Research Institutes, Zhunan, Taiwan.
authors.forename: Ai-Hsiang
authors.initials: AH
authors.lastname: Chou
authors.forename: Chia-Chyi
authors.initials: CC
authors.lastname: Liu
authors.forename: Cheng-Peng
authors.initials: CP
authors.lastname: Chang
authors.forename: Meng-Shin
authors.initials: MS
authors.lastname: Guo
authors.forename: Shih-Yang
authors.initials: SY
authors.lastname: Hsieh
authors.forename: Wen-Hsueh
authors.initials: WH
authors.lastname: Yang
authors.forename: Hsin-Ju
authors.initials: HJ
authors.lastname: Chao
authors.forename: Chien-Long
authors.initials: CL
authors.lastname: Wu
authors.forename: Ju-Lan
authors.initials: JL
authors.lastname: Huang
authors.forename: Min-Shi
authors.initials: MS
authors.lastname: Lee
authors.forename: Alan Yung-Chi
authors.initials: AY
authors.lastname: Hu
authors.forename: Sue-Chen
authors.initials: SC
authors.lastname: Lin
authors.forename: Yu-Yun
authors.initials: YY
authors.lastname: Huang
authors.forename: Mei-Hua
authors.initials: MH
authors.lastname: Hu
authors.forename: Yen-Hung
authors.initials: YH
authors.lastname: Chow
authors.forename: Jen-Ron
authors.initials: JR
authors.lastname: Chiang
authors.forename: Jui-Yuan
authors.initials: JY
authors.lastname: Chang
authors.forename: Pele
authors.initials: P
authors.lastname: Chong
mesh_terms: D017607:Aluminum Compounds; D000818:Animals; D061249:Batch Cell Culture Techniques; D019149:Bioreactors; D002522:Chlorocebus aethiops; D016895:Culture Media, Serum-Free; D029821:Enterovirus A, Human / Q000254:growth & development / Q000276:immunology*; D004769:Enterovirus Infections / Q000517:prevention & control*; D006801:Humans; D008251:Macaca; D010710:Phosphates; D015164:Vaccines, Inactivated / Q000276:immunology / Q000302:isolation & purification; D014709:Vero Cells; D014765:Viral Vaccines / Q000276:immunology / Q000302:isolation & purification; D038301:Virus Inactivation
chemical_list: D017607:Aluminum Compounds; D016895:Culture Media, Serum-Free; D010710:Phosphates; D015164:Vaccines, Inactivated; D014765:Viral Vaccines; C012714:aluminum phosphate