Inactivated Enterovirus 71 Vaccine Produced by 200-L Scale Serum-Free Microcarrier Bioreactor System Provides Cross-Protective Efficacy in Human SCARB2 Transgenic Mouse.
Bottom Line: These concerns highlight the urgent need to develop a scalable manufacturing platform for producing an effective and sufficient quantity of vaccines against deadly enteroviruses.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.
Affiliation: Adimmune Corporation, Taichung, Taiwan.
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 10(7) 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.
No MeSH data available.
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Mentions: The harvested viruses were separated from cell debris and microcarriers and were concentrated by a PESU ultrafiltration cassette as described in the Materials and Methods. Subsequently, the concentrated viruses were purified using sucrose density gradient centrifugation. Sucrose density, total protein, ELISA, host cell protein (HCP) and protein profile by SDS-PAGE were performed to analyze the content of every fraction (Fig 2A and 2B). Based on an in-house sandwich ELISA assay, EV71 viral antigens appear to distribute among fractions 5–15 (Fig 2A). SDS-PAGE and silver staining analysis showed several viral antigens (e.g., VP0, VP1, VP2, and VP3) that co-migrated among fractions 5 to 14. Fractions 5–10 and fractions 11–14 from the sedimentation in the sucrose density gradient (Fig 2B) appear to be consistent with the infectious particles (F-particle) and non-infectious particles (E-particle) of EV71 described previously . Negative staining EM was performed to further observe the content of the different fractions. Fractions 5–10 and fractions 11–14 revealed that the physical appearances of viral particles approximately 30–35 nm in diameter were consistent with either full or empty morphologies as observed previously (Fig 2C) .
No MeSH data available.