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Stable High-Producer Cell Clone Expressing Virus-Like Particles of the Japanese Encephalitis Virus E Protein for a Second-Generation Subunit Vaccine

Department of Pathology, National Institute of Infectious Diseases, Tokyo 162-8640,¹ Biological Science Laboratory, Zeon Corporation, Kanagawa 210-0863,² Institute of Glycotechnology, School of Engineering, Tokai University, Kanagawa 259-1292,³ Kanonji Institute, The Research Foundation for Microbial Diseases of Osaka University, Kagawa 768-0061,⁴ Department of Microbiology, Tokyo Metropolitan Institute for Neuroscience, Tokyo 183-0042, Japan⁵

Received 13 December 2002/ Accepted 27 May 2003

We produced and characterized a cell clone (J12#26 cells) that stably expresses Japanese encephalitis virus (JEV) cDNA, J12, which encodes the viral signal peptide, premembrane (prM), and envelope (E) proteins (amino acid positions 105 to 794). Rabbit kidney-derived RK13 cells were transfected with a J12 expression plasmid, selected by resistance to marker antibiotics, and cloned by two cycles of a limiting-dilution method in the presence of antibiotics, a procedure that prevents the successful generation of E-producing cell clones. J12#26 cells secreted virus-like particles containing the authentic E antigen (E-VLP) into the culture medium in a huge enzyme-linked immunosorbent assay-equivalent amount (2.5 µg per 10⁴ cells) to the internationally licensed JE vaccine JE-VAX. E-VLP production was stable after multiple cell passages and persisted over 1 year with 100% expressing cells without detectable cell fusion, apoptosis, or cell death, but was suspended when the cells grew to 100% confluency and contact inhibition occurred. Mice immunized with the purified J12#26 E-antigen without adjuvant developed high titers of neutralizing antibodies for at least 7 months and 100% protection against intraperitoneal challenge with 5 x 10⁶ PFU of JEV when examined according to the JE vaccine standardization protocol. These results suggest that the recombinant E-VLP antigen produced by the J12#26 cell clone is an effective, safe, and low-cost second-generation subunit JE vaccine.

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