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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

Antibody response to the EV71 vaccine in rabbits, hSCARB2 transgenic (Tg) mice and BALB/c mice.Measurement of vaccine elicited neutralizing antibody titers against EV71 viruses. The used vaccine formulations to immunize animals and the tested subgenotypes of EV71 virus are as labeled. “Prime” or “boost” indicate the detected viral neutralizing titers (NT) in antisera collected from animals that had been immunized once or twice.
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pone.0136420.g003: Antibody response to the EV71 vaccine in rabbits, hSCARB2 transgenic (Tg) mice and BALB/c mice.Measurement of vaccine elicited neutralizing antibody titers against EV71 viruses. The used vaccine formulations to immunize animals and the tested subgenotypes of EV71 virus are as labeled. “Prime” or “boost” indicate the detected viral neutralizing titers (NT) in antisera collected from animals that had been immunized once or twice.

Mentions: To test the efficacy of the EV71 vaccine produced from the bioreactor system, we investigated the humoral and cell-mediated immune responses elicited by various formulations in BALB/c mice, rabbits, and hSCARB2 transgenic (Tg) mice. Groups of BALB/c mice (6–8 weeks old, n = 5) were immunized twice with various formulations of 1 μg of vaccine antigen at day 0 and 14. The mice antisera were collected for the determination of viral-neutralization titers after 14 and 28 days, and the mice were sacrificed on day 28 post-vaccination to collect splenocytes to examine cell-mediated immune responses (S1 Text). As shown in Fig 3, the neutralizing titer with the sera collected 28 days after immunization was the highest from mice immunized with the AddaVAX-EV71 vaccine (1:16,384) against the EV71(E59-B4) strain compared with sera collected at the same time from mice immunized with the AlPO4-formulated vaccine (1:4,096) without adjuvants (1:1,024), and PBS (<1:8). The EV71 antigen without adjuvants exhibited no significant improvement in humoral immune response against the EV71 virus after booster vaccination (Fig 3, prime vs. boost). Both IL-4 and INF-γ were significantly higher in animal groups receiving either AddaVAX- or AlPO4-adjuvanted EV71 vaccines than the animal groups receiving antigen alone and PBS (Fig 4A and 4B). All of the above results suggested that the EV71(E59-B4) antigen formulated with adjuvants could achieve both higher humoral and cellular immune responses against homologous virus. To further examine whether the adjuvanted-EV71(E59-B4) vaccines can provide cross-protective immunity against heterotypic strains of EV71, rabbits and Tg mice were vaccinated (S1 Text). Antisera were collected and tested with a cross-strain viral-neutralization assay (Fig 3). The results revealed that the antisera collected from immunized rabbits or Tg mice achieved protective GMT titer ranging from 1:69 to 1:512 against the homotype B4 strain and exhibited significant cross-neutralizing activities against subgenotype 3340 (C4) virus (GMT titer: 1:45 to 1:327). In contrast, 80% of Tg mice vaccinated with the AlPO4-EV71 vaccine failed to induce neutralizing antibody to the subgenotype 5746 (C2) strain (GMT titer ≥ 1:8). In the AddaVAX-EV71 group, 60% of Tg mice developed weak cross-neutralizing antibody titers (GMT titer = 1:8) against the C2 strain (Fig 3). Similar results were obtained in rabbits vaccinated with AlPO4-formulation with levels of cross-reactive GMT titer (1:8 to 1:16) against the C2 strain (Fig 3).


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)

Antibody response to the EV71 vaccine in rabbits, hSCARB2 transgenic (Tg) mice and BALB/c mice.Measurement of vaccine elicited neutralizing antibody titers against EV71 viruses. The used vaccine formulations to immunize animals and the tested subgenotypes of EV71 virus are as labeled. “Prime” or “boost” indicate the detected viral neutralizing titers (NT) in antisera collected from animals that had been immunized once or twice.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0136420.g003: Antibody response to the EV71 vaccine in rabbits, hSCARB2 transgenic (Tg) mice and BALB/c mice.Measurement of vaccine elicited neutralizing antibody titers against EV71 viruses. The used vaccine formulations to immunize animals and the tested subgenotypes of EV71 virus are as labeled. “Prime” or “boost” indicate the detected viral neutralizing titers (NT) in antisera collected from animals that had been immunized once or twice.
Mentions: To test the efficacy of the EV71 vaccine produced from the bioreactor system, we investigated the humoral and cell-mediated immune responses elicited by various formulations in BALB/c mice, rabbits, and hSCARB2 transgenic (Tg) mice. Groups of BALB/c mice (6–8 weeks old, n = 5) were immunized twice with various formulations of 1 μg of vaccine antigen at day 0 and 14. The mice antisera were collected for the determination of viral-neutralization titers after 14 and 28 days, and the mice were sacrificed on day 28 post-vaccination to collect splenocytes to examine cell-mediated immune responses (S1 Text). As shown in Fig 3, the neutralizing titer with the sera collected 28 days after immunization was the highest from mice immunized with the AddaVAX-EV71 vaccine (1:16,384) against the EV71(E59-B4) strain compared with sera collected at the same time from mice immunized with the AlPO4-formulated vaccine (1:4,096) without adjuvants (1:1,024), and PBS (<1:8). The EV71 antigen without adjuvants exhibited no significant improvement in humoral immune response against the EV71 virus after booster vaccination (Fig 3, prime vs. boost). Both IL-4 and INF-γ were significantly higher in animal groups receiving either AddaVAX- or AlPO4-adjuvanted EV71 vaccines than the animal groups receiving antigen alone and PBS (Fig 4A and 4B). All of the above results suggested that the EV71(E59-B4) antigen formulated with adjuvants could achieve both higher humoral and cellular immune responses against homologous virus. To further examine whether the adjuvanted-EV71(E59-B4) vaccines can provide cross-protective immunity against heterotypic strains of EV71, rabbits and Tg mice were vaccinated (S1 Text). Antisera were collected and tested with a cross-strain viral-neutralization assay (Fig 3). The results revealed that the antisera collected from immunized rabbits or Tg mice achieved protective GMT titer ranging from 1:69 to 1:512 against the homotype B4 strain and exhibited significant cross-neutralizing activities against subgenotype 3340 (C4) virus (GMT titer: 1:45 to 1:327). In contrast, 80% of Tg mice vaccinated with the AlPO4-EV71 vaccine failed to induce neutralizing antibody to the subgenotype 5746 (C2) strain (GMT titer ≥ 1:8). In the AddaVAX-EV71 group, 60% of Tg mice developed weak cross-neutralizing antibody titers (GMT titer = 1:8) against the C2 strain (Fig 3). Similar results were obtained in rabbits vaccinated with AlPO4-formulation with levels of cross-reactive GMT titer (1:8 to 1:16) against the C2 strain (Fig 3).

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