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Propagation and immunological characterization of three enterovirus D68 strains using serum-free HEK293A suspension
Yu-Sheng Shen1, Chih-Yeu Fang1, Ting-Ting Chan2
1National Institute of Infectious Diseases and Vaccinology, National Health Research Institutes, Zhunan Town, Miaoli County, Taiwan.
Abstract:
Enterovirus D68 (EV-D68) is an emerging pathogen that typically causes respiratory illness but can also lead to acute flaccid myelitis, which has been reported in several outbreaks worldwide. As EV-D68 has been found to be difficult to infect and proliferate in the standard Vero cells, a viable manufacturing process for the development of inactivated EV-D68 vaccine is urgently needed. We performed cell line screening and found that the HEK293A is a suitable host for EV-D68 propagation. The EV-D68 TW2014 (B3) strain was used to develop the EV-D68 production using a serum-free HEK293A (sfHEK293A) suspension system. Two major EV-D68 particles, the empty and full viral particles, were separated by ultracentrifugation and were found to induce low and high neutralizing antibody responses, respectively, in animal studies. To facilitate the mass production of vaccine, a combination of size-exclusion and anion-exchange chromatography was developed to purify EV-D68 particles. High quality, pure EV-D68 particles could be obtained by this simple procedure for vaccine production. Additionally, we found that the EV-D68 MO (B1) and KY (A2) strains could also be propagated in this sfHEK293A cell system, but at a lower 33 °C condition. These three formalin-inactivated EV-D68s were shown to induce EV-D68-specific neutralizing responses and also neutralize a recently circulating strain, but failed to cross-neutralize enterovirus A71 (EV-A71) in sera assay. The combination of EV-D68 and EV-A71 in one vaccination induced the neutralizing responses against both viruses. Our results showed that EV-D68 particles produced from the sfHEK293A suspension culture could be used to prepare EV-D68 vaccine candidates. Purifying the full EV-D68 particles for vaccine production is recommended for inducing a high, specific immune response. Combination of EV-D68 and EV-A71 is beneficial for the development of bivalent enterovirus vaccine.
Insights
A new manufacturing process using HEK293A cells enables large-scale production of Enterovirus D68 (EV-D68) vaccine candidates. Purified full viral particles induce strong immune responses, suggesting potential for bivalent vaccines with Enterovirus A71.
Area of Science:
- Virology
- Vaccinology
- Biotechnology
Background:
- Enterovirus D68 (EV-D68) causes respiratory illness and acute flaccid myelitis.
- Current Vero cell culture is inefficient for EV-D68 vaccine development.
- A scalable and effective manufacturing process for EV-D68 vaccines is needed.
Purpose of the Study:
- To establish a robust cell culture system for EV-D68 propagation.
- To develop purification methods for EV-D68 vaccine production.
- To evaluate the immunogenicity of inactivated EV-D68 vaccine candidates.
Main Methods:
- Screening of cell lines for EV-D68 propagation, identifying HEK293A as suitable.
- Utilizing a serum-free HEK293A suspension system for EV-D68 production.
- Employing ultracentrifugation and chromatography for viral particle purification.
Main Results:
- HEK293A cells efficiently propagated EV-D68 strains.
- Purified full EV-D68 particles elicited high neutralizing antibody responses in animals.
- Formalin-inactivated EV-D68 vaccines induced specific neutralizing responses.
- Combined EV-D68 and EV-A71 vaccination generated responses against both viruses.
Conclusions:
- The sfHEK293A suspension culture system is viable for producing EV-D68 vaccine candidates.
- Purification of full EV-D68 particles is recommended for optimal immune response.
- A bivalent vaccine combining EV-D68 and EV-A71 shows promise.
