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

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.

Squalene-adjuvanted H7N9 virus vaccine induces robust humoral immune response against H7N9 and H7N7 viruses.

Recent cases of avian influenza H7N9 have caused great concerns that virus may become transmittable between humans. It is imperative to develop an effective vaccine to fight against the pandemic potential of this H7N9 influenza virus to protect human from the disease. This study aims to investigate an optimized formulation for the development of H7N9 vaccines. Various doses of H7N9 inactivated whole or split-virus antigens (0.5, 1.5, or 3 µg based on hemagglutinin content) combined with squalene-based adjuvant (AddaVAX), aluminum hydroxide Al(OH)3 or without adjuvant were evaluated for the efficacy of H7N9 vaccine regiments in mice. With either H7N9 whole or split-virus based vaccines, AddaVAX-adjuvanted formulations were the most immunogenic in eliciting significant humoral immune response against H7N9 virus and exhibited strong cross-reactive response in hemagglutination inhibition (HAI) and viral-neutralization assays against H7N7 virus as well. In contrast, formulations with Al(OH)3 or without adjuvant were less immunogenic and elicited lower titers of HAI and microneutralization assays against both viruses. Dose-sparing experiments suggested that the formulation with as low as 0.004 µg of split or whole virus vaccine antigens together with 50% AddaVAX provided sufficient sero-protective HAI titers and achieved essential virus-neutralizing antibody titers against H7-subtype influenza viruses in mice. Protection experiments demonstrated that the formulation of 0.004 µg to 0.5 µg of split-virion vaccines with AddaVAX conferred full protection against viral challenge up to 100 LD50 of wild-type H7N9 virus, with 0% survival in placebo group. Taken together, our study demonstrates that squalene-based adjuvant can significantly enhance the protective efficacy of H7N9 virus vaccine and provides a useful strategy to confront the potential pandemic outbreaks of H7N9 virus.