Limits...
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

Scale-up of Vero cell culture and EV71(E59-B4) virus production in a 200-L bioreactor.(A) Profile of Vero cell growth and EV71 amplification during a 200-L bioreactor process. (B) Metabolism profile by monitoring the contents of glucose, glutamine and lactate in a 200-L cell/EV71 virus culture. (C) Photomicrographs of Vero cells on microcarriers taken from the 200-L bioreactor immediately before infection with EV71 virus (a) and before harvest (b).
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4543551&req=5

pone.0136420.g001: Scale-up of Vero cell culture and EV71(E59-B4) virus production in a 200-L bioreactor.(A) Profile of Vero cell growth and EV71 amplification during a 200-L bioreactor process. (B) Metabolism profile by monitoring the contents of glucose, glutamine and lactate in a 200-L cell/EV71 virus culture. (C) Photomicrographs of Vero cells on microcarriers taken from the 200-L bioreactor immediately before infection with EV71 virus (a) and before harvest (b).

Mentions: To prepare Vero cells for EV71 virus infection in the bioreactor, the host Vero cells were serially amplified from a single vial to a final 200 L bioreactor using serum-free VP-SFM and TrypLE Select (Invitrogen, animal-component free). A typical profile of cell growth is shown in Fig 1A. The serial cell expansions were performed in T-flasks, roller bottles, spinner flasks, and a 50 L bioreactor. Then, the detached cells/microcarriers mixture from the 50 L bioreactor was transferred into a 200 L bioreactor at an initial cell density of 1 × 105 cells/mL. The cells grew to a cell density of approximately 1 × 106 cells/mL after 6 days (Fig 1A and 1C-a). The virus infection was performed at day 6 with an MOI of 10−4. The metabolism profile of cells (glucose, glutamine, and lactate concentration) was monitored daily, and more nutrients were added between days 6 and 8 when maintaining optimal conditions is necessary (Fig 1B). The virus production reached a maximal titer of 107 TCID50/mL, and the cells exhibited 95% cytopathic effect at day 10 post-infection (Fig 1A and 1C-b).


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)

Scale-up of Vero cell culture and EV71(E59-B4) virus production in a 200-L bioreactor.(A) Profile of Vero cell growth and EV71 amplification during a 200-L bioreactor process. (B) Metabolism profile by monitoring the contents of glucose, glutamine and lactate in a 200-L cell/EV71 virus culture. (C) Photomicrographs of Vero cells on microcarriers taken from the 200-L bioreactor immediately before infection with EV71 virus (a) and before harvest (b).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0136420.g001: Scale-up of Vero cell culture and EV71(E59-B4) virus production in a 200-L bioreactor.(A) Profile of Vero cell growth and EV71 amplification during a 200-L bioreactor process. (B) Metabolism profile by monitoring the contents of glucose, glutamine and lactate in a 200-L cell/EV71 virus culture. (C) Photomicrographs of Vero cells on microcarriers taken from the 200-L bioreactor immediately before infection with EV71 virus (a) and before harvest (b).
Mentions: To prepare Vero cells for EV71 virus infection in the bioreactor, the host Vero cells were serially amplified from a single vial to a final 200 L bioreactor using serum-free VP-SFM and TrypLE Select (Invitrogen, animal-component free). A typical profile of cell growth is shown in Fig 1A. The serial cell expansions were performed in T-flasks, roller bottles, spinner flasks, and a 50 L bioreactor. Then, the detached cells/microcarriers mixture from the 50 L bioreactor was transferred into a 200 L bioreactor at an initial cell density of 1 × 105 cells/mL. The cells grew to a cell density of approximately 1 × 106 cells/mL after 6 days (Fig 1A and 1C-a). The virus infection was performed at day 6 with an MOI of 10−4. The metabolism profile of cells (glucose, glutamine, and lactate concentration) was monitored daily, and more nutrients were added between days 6 and 8 when maintaining optimal conditions is necessary (Fig 1B). The virus production reached a maximal titer of 107 TCID50/mL, and the cells exhibited 95% cytopathic effect at day 10 post-infection (Fig 1A and 1C-b).

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