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Enterovirus 71 (EV71) has emerged as a neurological virus causing life-threatening diseases in young children and infants. Although EV71 vaccines in development have presented promising results in several clinical trials, the identified key antigen for improving the broad protective efficacy of EV71 vaccines has not been well investigated. In this report, we show that different multiplicities of infection (MOIs) of the B4(E59) virus significantly affect EV71 vaccine production in a serum-free microcarrier bioreactor system. The antigens produced from high MOIs of 10 −1  and 10 −2  exhibited higher yield and more infectious full particle (FP) contents in the EV71 vaccines than those produced with low MOIs of 10 −4  and 10 −6 , leading to better cross-neutralizing efficacy. The C4(E36) neutralization results showed that only antisera raised from EV71 FPs provided substantial neutralizing titers against C4(E36), whereas empty particles (EPs) of EV71 conferred no efficacy. Competitive ELISA showed that anti-FP mainly binds to FPs and that 20% of antibodies bind to EPs, whereas most anti-EP binds EPs, with only 10% antibodies binding to FPs. VP1-adsorbed anti-FP lost most of the virus neutralization efficiency, suggesting that the VP1 subunit of FP is the major immunogenic antigen determining the ability of the EV71 vaccine to elicit cross-neutralizing antibodies against EV71 virus subtypes. These findings demonstrate that the high-MOI production approach is significantly correlated with FP productivity, thereby improving the cross-neutralization efficacy of an EV71 vaccine and providing the basis for a better vaccine design against widespread EV71 viruses.

The mature EV71 virion induced a broadly cross-neutralizing VP1 antibody against subtypes of the EV71 virus