Inactivated Enterovirus 71 Vaccine Produced by 200-L Scale Serum-Free Microcarrier Bioreactor System Provides Cross-Protective Efficacy in Human SCARB2 Transgenic Mouse.

Chia-Ying Wu,Chi-Hsien Chan,Pei-Wen Hsiao,Chung-Cheng Liu,Yi-Wen Lin,Juine-Ruey Chen,Ching-Chuan Chang,Hsiu-Fen Tai,Yen-Hung Chow,Chia-Ho Kuo,Yung-Tsung Chen,Chien-Hung Pan,Wan-Hsin Liu,Ding Xiang Liu

doi:10.1371/journal.pone.0136420

Abstract

Epidemics and outbreaks caused by infections of several subgenotypes of EV71 and other serotypes of coxsackie A viruses have raised serious public health concerns in the Asia-Pacific region. These concerns highlight the urgent need to develop a scalable manufacturing platform for producing an effective and sufficient quantity of vaccines against deadly enteroviruses. In this report, we present a platform for the large-scale production of a vaccine based on the inactivated EV71(E59-B4) virus. The viruses were produced in Vero cells in a 200 L bioreactor with serum-free medium, and the viral titer reached 107 TCID50/mL 10 days after infection when using an MOI of 10−4. The EV71 virus particles were harvested and purified by sucrose density gradient centrifugation. Fractions containing viral particles were pooled based on ELISA and SDS-PAGE. TEM was used to characterize the morphologies of the viral particles. To evaluate the cross-protective efficacy of the EV71 vaccine, the pooled antigens were combined with squalene-based adjuvant (AddaVAX) or aluminum phosphate (AlPO4) and tested in human SCARB2 transgenic (Tg) mice. The Tg mice immunized with either the AddaVAX- or AlPO4-adjuvanted EV71 vaccine were fully protected from challenges by the subgenotype C2 and C4 viruses, and surviving animals did not show any degree of neurological paralysis symptoms or muscle damage. Vaccine treatments significantly reduced virus antigen presented in the central nervous system of Tg mice and alleviated the virus-associated inflammatory response. These results strongly suggest that this preparation results in an efficacious vaccine and that the microcarrier/bioreactor platform offers a superior alternative to the previously described roller-bottle system.

Highlights

Enterovirus 71 (EV71) is one of the major pathogens for hand-foot-and-mouth disease (HFMD), which is sometimes associated with severe neurological complications in young children leading to poliomyelitis-like paralysis, meningitis, brain stem encephalitis, and even death [1,2,3,4,5,6,7,8]
Most outbreaks in the Western Pacific region are caused by several subgenotypes of EV71 (B3, B4, B5, C1, C2, and C4) virus, which are frequently found to co-circulate with other serotype enteroviruses, such as the coxsackie A virus (CAV), another major causative agent of HFMD [9,10,11,12,13]
To test the efficacy of the EV71 vaccine produced from the bioreactor system, we investigated the humoral and cell-mediated immune responses elicited by various formulations in BALB/c mice, rabbits, and hSCARB2 transgenic (Tg) mice

Summary

Introduction

Enterovirus 71 (EV71) is one of the major pathogens for hand-foot-and-mouth disease (HFMD), which is sometimes associated with severe neurological complications in young children leading to poliomyelitis-like paralysis, meningitis, brain stem encephalitis, and even death [1,2,3,4,5,6,7,8]. Most outbreaks in the Western Pacific region are caused by several subgenotypes of EV71 (B3, B4, B5, C1, C2, and C4) virus, which are frequently found to co-circulate with other serotype enteroviruses, such as the coxsackie A virus (CAV), another major causative agent of HFMD [9,10,11,12,13]. This co-circulation increases the potential of genetic recombination among enteroviruses [10,12,14,15]. The development of an effective vaccine to control EV71 epidemics and prevent potential outbreaks is urgently needed

Methods

Results

Conclusion