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.

Principal finding: 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.

Conclusion: These results provide valuable information supporting the current cell-based serum-free EV71 vaccine candidate going into human Phase I clinical trials.

Figure 1. The growth profiles of Vero cell grown in serum-contain medium (SC) and different serum-free media (SF).

Vero cells (2×10⁵ cell/ml) was cultured in 75T-flask and counted for viable cell number every 24 hours (0, 24, 48, 72, 96, 120 and 144 hours). The conditions for cell culture were described in the Materials and Methods section.

Figure 2. The optimization of the up-stream process for EV71 virus production.

(A) The T-flask was seeded with either 1.0∼1.5×10⁶ or 2.0∼2.5×10⁶ Vero cells, then after 2–3 days the cells were infected by the different ratio of E59/EV71 virus. The effects of different M.O.I were detected in the kinetic profile of virus produced from Vero cell grown in the VP-SFM medium. (B) The consistency of 3 Lots of EV71 virus production in the roller bottles. Virus titer was detected every day by TCID50 for 5 days.

Figure 3. The temperature effect in the kinetic of formalin-inactivation of EV71 virus.

Purified E59/EV71 was inactivated by 0.025% (v/v) formalin at different temperature and the residual of virus infectivity was detected by plaque assay performed triplicate at different time points.

Figure 4. The stability profiles of different Lots of EV71 vaccine products stored at 4°C for various time and analyzed by SDS-PAGE (Panel A) and Western blot (Panel B & C).

Lane M is MW Markers; lane 1 is EV71 vaccine product produced from serum-containing medium and stored at 4°C for 26 months; lanes 2 and 3 are EV71 vaccine products derived from Lot #1 and 2 and stored at 4°C for 13 and 4 months, respectively. Monoclonal antibody used in the Panels B and C are MAB979 specific for VP2 and N16 specific for VP1, respectively.