title: Stable high-producer cell clone expressing virus-like particles of the Japanese encephalitis virus e protein for a second-generation subunit vaccine.
abstract: 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 micro g per 10(4) 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 10(6) 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.
authors.affiliation: Department of Pathology, National Institute of Infectious Diseases, Tokyo 162-8640, Japan. akojima@nih.go.jp
authors.forename: Asato
authors.initials: A
authors.lastname: Kojima
authors.forename: Atsushi
authors.initials: A
authors.lastname: Yasuda
authors.forename: Hideki
authors.initials: H
authors.lastname: Asanuma
authors.forename: Toyokazu
authors.initials: T
authors.lastname: Ishikawa
authors.forename: Akihisa
authors.initials: A
authors.lastname: Takamizawa
authors.forename: Kotaro
authors.initials: K
authors.lastname: Yasui
authors.forename: Takeshi
authors.initials: T
authors.lastname: Kurata
mesh_terms: D000818:Animals; D000956:Antigens, Viral / Q000276:immunology; D002460:Cell Line; D002999:Clone Cells; D005260:Female; D019084:Fluorescent Antibody Technique, Indirect; D008562:Membrane Glycoproteins / Q000235:genetics / Q000276:immunology*; D051379:Mice; D008807:Mice, Inbred BALB C; D008854:Microscopy, Electron; D014759:Viral Envelope Proteins / Q000235:genetics / Q000276:immunology*; D014765:Viral Vaccines / Q000276:immunology*
chemical_list: D000956:Antigens, Viral; D008562:Membrane Glycoproteins; D014759:Viral Envelope Proteins; D014765:Viral Vaccines; C080628:glycoprotein E, Japanese encephalitis virus