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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

Expression of pro-inflammatory cytokines/chemokines in the CNS compartments and muscle tissue of EV71 (C4)-infected mice.The vaccinated Tg mice were subcutaneously infected with 1×106 pfu of the EV71 3340 (C4) strain, and the RNAs extracted from the brain stem, spinal cord, and muscle at 6 days post-infection were subjected to quantitative RT-PCR analysis. Mock (M): uninfected mice group; Virus (V): EV71 3340 (C4) infected mice group. A schematic representation of the target gene expression and the statistical average from 5 mice per group is shown. The used vaccine formulations are as labeled. Significant difference between each group was shown as *: p ﹤ 0.05, **: p ﹤ 0.01, and ***: p ﹤ 0.001.
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pone.0136420.g010: Expression of pro-inflammatory cytokines/chemokines in the CNS compartments and muscle tissue of EV71 (C4)-infected mice.The vaccinated Tg mice were subcutaneously infected with 1×106 pfu of the EV71 3340 (C4) strain, and the RNAs extracted from the brain stem, spinal cord, and muscle at 6 days post-infection were subjected to quantitative RT-PCR analysis. Mock (M): uninfected mice group; Virus (V): EV71 3340 (C4) infected mice group. A schematic representation of the target gene expression and the statistical average from 5 mice per group is shown. The used vaccine formulations are as labeled. Significant difference between each group was shown as *: p ﹤ 0.05, **: p ﹤ 0.01, and ***: p ﹤ 0.001.

Mentions: Enterovirus infection can induce an extensive inflammatory response in the peripheral and central nervous system, which is responsible for the pathogenesis of EV71-associated disease [43,44,45]. A previous study demonstrated that the hypersecretion of CXCL10, CCL3, TNF-α, and IL-6 correlated to the severity of neurologic pathogenesis induced by EV71 challenge [36]. To confirm whether the vaccine prepared in our platform is able to prevent inflammation-related pathogenesis, the expression of pro-inflammatory indicators were measured after the virus infection in vaccinated Tg mice. As shown in Figs 9 and 10, the pro-inflammatory gene expression of CXCL10, TNF-α, and IFN-γ in the CNS and muscle tissues were up-regulated in the PBS group upon infection with EV71 5746 (C2) or 3340 (C4). In contrast, the expression level of CXCL10, TNF-α, and IFN-γ were significantly lower in the mice vaccinated with either AlPO4- or AddaVAX-EV71 vaccines upon infection with EV71 5746 (C2) or 3340 (C4). In these experiments, 2 μg of the AddaVAX-EV71 vaccine showed equivalent effectiveness as 6 μg of the AlPO4-EV71 vaccine, and it was also more potent than 2 μg of the AlPO4-EV71 vaccine in inhibiting the inflammatory responses caused by virus infection (Figs 9 and 10). These results suggest that the balance of the Th1/Th2 immune response induced by the AddaVAX-EV71 vaccine provides a role in alleviating the degree of EV71-induced neurologic pathogenesis (Figs 4, 8, 9 and 10).


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)

Expression of pro-inflammatory cytokines/chemokines in the CNS compartments and muscle tissue of EV71 (C4)-infected mice.The vaccinated Tg mice were subcutaneously infected with 1×106 pfu of the EV71 3340 (C4) strain, and the RNAs extracted from the brain stem, spinal cord, and muscle at 6 days post-infection were subjected to quantitative RT-PCR analysis. Mock (M): uninfected mice group; Virus (V): EV71 3340 (C4) infected mice group. A schematic representation of the target gene expression and the statistical average from 5 mice per group is shown. The used vaccine formulations are as labeled. Significant difference between each group was shown as *: p ﹤ 0.05, **: p ﹤ 0.01, and ***: p ﹤ 0.001.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0136420.g010: Expression of pro-inflammatory cytokines/chemokines in the CNS compartments and muscle tissue of EV71 (C4)-infected mice.The vaccinated Tg mice were subcutaneously infected with 1×106 pfu of the EV71 3340 (C4) strain, and the RNAs extracted from the brain stem, spinal cord, and muscle at 6 days post-infection were subjected to quantitative RT-PCR analysis. Mock (M): uninfected mice group; Virus (V): EV71 3340 (C4) infected mice group. A schematic representation of the target gene expression and the statistical average from 5 mice per group is shown. The used vaccine formulations are as labeled. Significant difference between each group was shown as *: p ﹤ 0.05, **: p ﹤ 0.01, and ***: p ﹤ 0.001.
Mentions: Enterovirus infection can induce an extensive inflammatory response in the peripheral and central nervous system, which is responsible for the pathogenesis of EV71-associated disease [43,44,45]. A previous study demonstrated that the hypersecretion of CXCL10, CCL3, TNF-α, and IL-6 correlated to the severity of neurologic pathogenesis induced by EV71 challenge [36]. To confirm whether the vaccine prepared in our platform is able to prevent inflammation-related pathogenesis, the expression of pro-inflammatory indicators were measured after the virus infection in vaccinated Tg mice. As shown in Figs 9 and 10, the pro-inflammatory gene expression of CXCL10, TNF-α, and IFN-γ in the CNS and muscle tissues were up-regulated in the PBS group upon infection with EV71 5746 (C2) or 3340 (C4). In contrast, the expression level of CXCL10, TNF-α, and IFN-γ were significantly lower in the mice vaccinated with either AlPO4- or AddaVAX-EV71 vaccines upon infection with EV71 5746 (C2) or 3340 (C4). In these experiments, 2 μg of the AddaVAX-EV71 vaccine showed equivalent effectiveness as 6 μg of the AlPO4-EV71 vaccine, and it was also more potent than 2 μg of the AlPO4-EV71 vaccine in inhibiting the inflammatory responses caused by virus infection (Figs 9 and 10). These results suggest that the balance of the Th1/Th2 immune response induced by the AddaVAX-EV71 vaccine provides a role in alleviating the degree of EV71-induced neurologic pathogenesis (Figs 4, 8, 9 and 10).

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