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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

Vaccine treatments protect Tg mice against a lethal-dose challenge of subgenotypes of the EV71 virus.The survival rate of immunized Tg mice challenged with 3×106 pfu of the C2 strain (A) or 1×106 pfu of the C4 strain (B). Mouse body weight change after EV71 C2 (C) or C4 (D) virus challenges. Scores of central nervous system (CNS)-like hind limb paralysis caused by EV71 C2 (E) or C4 (F) viral challenges. The severity of CNS symptoms was scored from 0 to 5 using the following criteria for scoring CNS diseases: 5 = severe front and rear limb paralysis (LP) and no movement, 4 = moderate two rear LP and hesitant movement, 3 = one rear LP with bending legs, 2 = mild rear limb bended, 1 = slightly rear limb bended, and 0 = normal movement. LP is defined as the rigidness of mouse legs that are resist movement. The PBS and vaccination groups are indicated as symbols. *: p < 0.05, **:p < 0.01, and ***: p < 0.001.
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pone.0136420.g005: Vaccine treatments protect Tg mice against a lethal-dose challenge of subgenotypes of the EV71 virus.The survival rate of immunized Tg mice challenged with 3×106 pfu of the C2 strain (A) or 1×106 pfu of the C4 strain (B). Mouse body weight change after EV71 C2 (C) or C4 (D) virus challenges. Scores of central nervous system (CNS)-like hind limb paralysis caused by EV71 C2 (E) or C4 (F) viral challenges. The severity of CNS symptoms was scored from 0 to 5 using the following criteria for scoring CNS diseases: 5 = severe front and rear limb paralysis (LP) and no movement, 4 = moderate two rear LP and hesitant movement, 3 = one rear LP with bending legs, 2 = mild rear limb bended, 1 = slightly rear limb bended, and 0 = normal movement. LP is defined as the rigidness of mouse legs that are resist movement. The PBS and vaccination groups are indicated as symbols. *: p < 0.05, **:p < 0.01, and ***: p < 0.001.

Mentions: To evaluate the cross-protective efficacy of EV71(E59-B4) antigen combined with adjuvants, the vaccinated Tg mice were challenged with the EV71 5746 (C2) or 3340 (C4) strains as previously described [36]. In contrast to the placebo group (PBS), the different groups of mice vaccinated with the EV71 vaccines were 100% protected against EV71 5746 (C2) and 3340 (C4) virus challenges (Fig 5A and 5B). All mice continually gained weight after different EV71 infections through the 15 days except those that received PBS (Fig 5C and 5D). In addition, the PBS-treated mice exhibited clear central nervous system (CNS) symptoms as early as 2–3 days post-infection (dpi) and peaked on 5–6 dpi depending on the virus strains of infection (Fig 5E and 5F). Representative photographs showed that the PBS-treated mice exhibited serious hind limb paralysis symptoms at 6 dpi, but no apparent symptoms appeared in vaccinated mice (Fig 6). To verify whether the vaccines can effectively prevent EV71-induced neurologic pathogenesis in Tg mice, the muscle and brain stem sections were examined by hematoxylin and eosin and immunohistochemical staining. The results showed clear and well-organized muscle fibers in uninfected mice (Fig 7A–7D, Mock) and vaccinated mice with infections at 6 dpi (Fig 7F–7H and 7J–7K). The inflammatory cells infiltrated into the muscle tissues and the destructed structures were only observed in the PBS group of EV71-infected mice (Fig 7E and 7I). The complete clearance of viral antigen in the brain stem was observed in mice vaccinated with 6 μg of the AlPO4-EV71 vaccine or 2 μg of the AddaVAX-EV71 vaccine following EV71 infections (Fig 8F–8Q and 8H–8L). However, numerous loci appeared in the brain stem of PBS-treated mice (Fig 8E–8P), and fewer loci were observed in the brain stem from the mice vaccinated with 2 μg of the AlPO4-EV71 vaccine after EV71 5746 (C2) infection (Fig 8G–8K).


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)

Vaccine treatments protect Tg mice against a lethal-dose challenge of subgenotypes of the EV71 virus.The survival rate of immunized Tg mice challenged with 3×106 pfu of the C2 strain (A) or 1×106 pfu of the C4 strain (B). Mouse body weight change after EV71 C2 (C) or C4 (D) virus challenges. Scores of central nervous system (CNS)-like hind limb paralysis caused by EV71 C2 (E) or C4 (F) viral challenges. The severity of CNS symptoms was scored from 0 to 5 using the following criteria for scoring CNS diseases: 5 = severe front and rear limb paralysis (LP) and no movement, 4 = moderate two rear LP and hesitant movement, 3 = one rear LP with bending legs, 2 = mild rear limb bended, 1 = slightly rear limb bended, and 0 = normal movement. LP is defined as the rigidness of mouse legs that are resist movement. The PBS and vaccination groups are indicated as symbols. *: p < 0.05, **:p < 0.01, and ***: p < 0.001.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4543551&req=5

pone.0136420.g005: Vaccine treatments protect Tg mice against a lethal-dose challenge of subgenotypes of the EV71 virus.The survival rate of immunized Tg mice challenged with 3×106 pfu of the C2 strain (A) or 1×106 pfu of the C4 strain (B). Mouse body weight change after EV71 C2 (C) or C4 (D) virus challenges. Scores of central nervous system (CNS)-like hind limb paralysis caused by EV71 C2 (E) or C4 (F) viral challenges. The severity of CNS symptoms was scored from 0 to 5 using the following criteria for scoring CNS diseases: 5 = severe front and rear limb paralysis (LP) and no movement, 4 = moderate two rear LP and hesitant movement, 3 = one rear LP with bending legs, 2 = mild rear limb bended, 1 = slightly rear limb bended, and 0 = normal movement. LP is defined as the rigidness of mouse legs that are resist movement. The PBS and vaccination groups are indicated as symbols. *: p < 0.05, **:p < 0.01, and ***: p < 0.001.
Mentions: To evaluate the cross-protective efficacy of EV71(E59-B4) antigen combined with adjuvants, the vaccinated Tg mice were challenged with the EV71 5746 (C2) or 3340 (C4) strains as previously described [36]. In contrast to the placebo group (PBS), the different groups of mice vaccinated with the EV71 vaccines were 100% protected against EV71 5746 (C2) and 3340 (C4) virus challenges (Fig 5A and 5B). All mice continually gained weight after different EV71 infections through the 15 days except those that received PBS (Fig 5C and 5D). In addition, the PBS-treated mice exhibited clear central nervous system (CNS) symptoms as early as 2–3 days post-infection (dpi) and peaked on 5–6 dpi depending on the virus strains of infection (Fig 5E and 5F). Representative photographs showed that the PBS-treated mice exhibited serious hind limb paralysis symptoms at 6 dpi, but no apparent symptoms appeared in vaccinated mice (Fig 6). To verify whether the vaccines can effectively prevent EV71-induced neurologic pathogenesis in Tg mice, the muscle and brain stem sections were examined by hematoxylin and eosin and immunohistochemical staining. The results showed clear and well-organized muscle fibers in uninfected mice (Fig 7A–7D, Mock) and vaccinated mice with infections at 6 dpi (Fig 7F–7H and 7J–7K). The inflammatory cells infiltrated into the muscle tissues and the destructed structures were only observed in the PBS group of EV71-infected mice (Fig 7E and 7I). The complete clearance of viral antigen in the brain stem was observed in mice vaccinated with 6 μg of the AlPO4-EV71 vaccine or 2 μg of the AddaVAX-EV71 vaccine following EV71 infections (Fig 8F–8Q and 8H–8L). However, numerous loci appeared in the brain stem of PBS-treated mice (Fig 8E–8P), and fewer loci were observed in the brain stem from the mice vaccinated with 2 μg of the AlPO4-EV71 vaccine after EV71 5746 (C2) infection (Fig 8G–8K).

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