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

Wu CY, Lin YW, Kuo CH, Liu WH, Tai HF, Pan CH, Chen YT, Hsiao PW, Chan CH, Chang CC, Liu CC, Chow YH, Chen JR - PLoS ONE (2015)

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.

View Article: PubMed Central - PubMed

Affiliation: Adimmune Corporation, Taichung, Taiwan.

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 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.


Related in: MedlinePlus

The purification and characterization of the EV71 virus.(A) Purification of EV71 by sucrose density gradient using zonal ultracentrifugation and fractionation analysis by sucrose percentage (Sucrose), contents of total protein (TP) and Vero cell-derived protein (HCP), and EV71-specific units (ELISA). (B) Definition of viral particles distributed in fractions using SDS-PAGE. (C) Electromicrographs of EV71 particles by transmission electron microscopy. (a) Fraction 10 presented a full-particle (F-particle) with infectious capability and a solid particle structure; (b) fraction 13 presented a non-infectious particle (E-particle) with defective particle structure; and (c) pooled viral particles for the vaccine bulk preparation. (D) Quality confirmation of EV71 pooled viral particles before and after formalin inactivation using SDS-PAGE and western blot analysis with a specific monoclonal antibody (Mab979). The molecular weight of VP0, 1, 2, and 3 are as indicated. The black triangle indicates the quantities of loading antigen from high to low.
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pone.0136420.g002: The purification and characterization of the EV71 virus.(A) Purification of EV71 by sucrose density gradient using zonal ultracentrifugation and fractionation analysis by sucrose percentage (Sucrose), contents of total protein (TP) and Vero cell-derived protein (HCP), and EV71-specific units (ELISA). (B) Definition of viral particles distributed in fractions using SDS-PAGE. (C) Electromicrographs of EV71 particles by transmission electron microscopy. (a) Fraction 10 presented a full-particle (F-particle) with infectious capability and a solid particle structure; (b) fraction 13 presented a non-infectious particle (E-particle) with defective particle structure; and (c) pooled viral particles for the vaccine bulk preparation. (D) Quality confirmation of EV71 pooled viral particles before and after formalin inactivation using SDS-PAGE and western blot analysis with a specific monoclonal antibody (Mab979). The molecular weight of VP0, 1, 2, and 3 are as indicated. The black triangle indicates the quantities of loading antigen from high to low.

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 [31]. 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) [31].


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

Wu CY, Lin YW, Kuo CH, Liu WH, Tai HF, Pan CH, Chen YT, Hsiao PW, Chan CH, Chang CC, Liu CC, Chow YH, Chen JR - PLoS ONE (2015)

The purification and characterization of the EV71 virus.(A) Purification of EV71 by sucrose density gradient using zonal ultracentrifugation and fractionation analysis by sucrose percentage (Sucrose), contents of total protein (TP) and Vero cell-derived protein (HCP), and EV71-specific units (ELISA). (B) Definition of viral particles distributed in fractions using SDS-PAGE. (C) Electromicrographs of EV71 particles by transmission electron microscopy. (a) Fraction 10 presented a full-particle (F-particle) with infectious capability and a solid particle structure; (b) fraction 13 presented a non-infectious particle (E-particle) with defective particle structure; and (c) pooled viral particles for the vaccine bulk preparation. (D) Quality confirmation of EV71 pooled viral particles before and after formalin inactivation using SDS-PAGE and western blot analysis with a specific monoclonal antibody (Mab979). The molecular weight of VP0, 1, 2, and 3 are as indicated. The black triangle indicates the quantities of loading antigen from high to low.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4543551&req=5

pone.0136420.g002: The purification and characterization of the EV71 virus.(A) Purification of EV71 by sucrose density gradient using zonal ultracentrifugation and fractionation analysis by sucrose percentage (Sucrose), contents of total protein (TP) and Vero cell-derived protein (HCP), and EV71-specific units (ELISA). (B) Definition of viral particles distributed in fractions using SDS-PAGE. (C) Electromicrographs of EV71 particles by transmission electron microscopy. (a) Fraction 10 presented a full-particle (F-particle) with infectious capability and a solid particle structure; (b) fraction 13 presented a non-infectious particle (E-particle) with defective particle structure; and (c) pooled viral particles for the vaccine bulk preparation. (D) Quality confirmation of EV71 pooled viral particles before and after formalin inactivation using SDS-PAGE and western blot analysis with a specific monoclonal antibody (Mab979). The molecular weight of VP0, 1, 2, and 3 are as indicated. The black triangle indicates the quantities of loading antigen from high to low.
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 [31]. 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) [31].

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.

View Article: PubMed Central - PubMed

Affiliation: Adimmune Corporation, Taichung, Taiwan.

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 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.


Related in: MedlinePlus