Purification and Characterization of Enterovirus 71 Viral Particles Produced from Vero Cells Grown in a Serum-Free Microcarrier Bioreactor System

Abstract

BackgroundEnterovirus 71 (EV71) infections manifest most commonly as a childhood exanthema known as hand-foot-and-mouth disease (HFMD) and can cause neurological disease during acute infection.Principal FindingIn this study, we describe the production, purification and characterization of EV71 virus produced from Vero cells grown in a five-liter serum-free bioreactor system containing 5 g/L Cytodex 1 microcarrier. The viral titer was >106 TCID50/mL by 6 days post infection when a MOI of 10−5 was used at the initial infection. Two EV71 virus fractions were separated and detected when the harvested EV71 virus concentrate was purified by sucrose gradient zonal ultracentrifugation. The EV71 viral particles detected in the 24–28% sucrose fractions had an icosahedral structure 30–31 nm in diameter and had low viral infectivity and RNA content. Three major viral proteins (VP0, VP1 and VP3) were observed by SDS-PAGE. The EV71 viral particles detected in the fractions containing 35–38% sucrose were 33–35 nm in size, had high viral infectivity and RNA content, and were composed of four viral proteins (VP1, VP2, VP3 and VP4), as shown by SDS-PAGE analyses. The two virus fractions were formalin-inactivated and induced high virus neutralizing antibody responses in mouse immunogenicity studies. Both mouse antisera recognized the immunodominant linear neutralization epitope of VP1 (residues 211–225).ConclusionThese results provide important information for cell-based EV71 vaccine development, particularly for the preparation of working standards for viral antigen quantification.

Phage Display-Derived Cross-Reactive Neutralizing Antibody against Enterovirus 71 and Coxsackievirus A16

“…Two positive protein bands corresponding to the VP0 and VP2 proteins were detected with this system. This result is consistent with that of a previous report (31) and indicates that the collected-EV71 particles were a mixture of E-and F-particles. As expected, the VP0 precursor protein was visualized in collected EV71 particles by using the phage display-derived mAb under the applied electrophoresis conditions, and the results suggest that the isolated antibody is specific for the VP4 protein and has no cross-reactive response with other EV71 capsid proteins.…”

“…EV71 E-particles are immature viral particles with low infectivity in which the P1 and VP0 polyproteins are incompletely processed, whereas Fparticles are mature forms of EV71 (31). In our experiments, EV71 viral particles were harvested from the supernatant of cell cultures and loaded onto SDS-PAGE gel, and the commercial anti-VP2 antibody MAB979 was used to monitor the expression of VP2 and VP0 via western blot.…”

Tolerability and immunogenicity of an inactivated enterovirus 71 vaccine in Chinese healthy adults and children

“…13 The preclinical study showed that NT induced by inactivated EV71 vaccine can protect mice against lethal challenge of EV71 virus. 14,15 Two studies in Taiwan had demonstrated that passive immunization was able to protect against lethal EV71 challenge in new born mice. 1,16 Those results showed that humoral immunity played a key role in protecting against EV71-caused disease.…”

Chimeric Virus-Like Particle Vaccines Displaying Conserved Enterovirus 71 Epitopes Elicit Protective Neutralizing Antibodies in Mice through Divergent Mechanisms

“…Each peptide contains 15 amino acid residues. Cell culture-derived EV71 particles are present in two forms: the E-particle and the F-particle (20). E-particles are noninfectious, empty capsids consisting of VP1, VP3, and uncleaved VP0 proteins; F-particles are mature virions containing VP1, VP2, VP3, and VP4 proteins and an infectious viral genome (20).…”