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 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 ؋ 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.Japanese encephalitis virus (JEV), a member of the flavivirus family, is the causative agent of Japanese encephalitis (JE), which is a pandemic infectious disease of major public health importance in Asia (9, 48). Vaccination is the only effective way to prevent flavivirus infection in humans and domestic animals. Inactivated JEV and tick-borne encephalitis virus vaccines and attenuated yellow fever virus vaccine are in widespread production and use, whereas other flavivirus vaccines are under development or in human trials (6,26,37). The only licensed JE vaccine, JE-VAX, which is distributed commercially and available internationally, is formalin-inactivated JEV prepared from a number of JEV-infected mouse brains. The brain-derived whole virion vaccine is costly to manufacture and carries potential risks of allergic reactions to brain basic proteins or contamination by mouse prion proteins, and there are biosafety issues of manufacturing an infectious pathogen. Thus, the development of second-generation JE vaccines that are not derived from the brain, do not involve infectious JEV, and are of low cost is a top priority.The 53-kDa envelope (E) glycoprotein of JEV has an important role in virus adhesion and entry into target cells through receptor binding (20,34) and, therefore, in inducing neutralizing antibodies that protect hosts against JEV infection (8,24,32,36). The protective epitopes on the E antigen are suggested to be formed in highly conformational structures of JEV virions (20, 34) based on antigenic analyses with panels of monoclonal ant...

Differential Modulation of prM Cleavage, Extracellular Particle Distribution, and Virus Infectivity by Conserved Residues at Nonfurin Consensus Positions of the Dengue Virus pr-M Junction

“…Changes in prM cleavage efficiency are known to affect the proportion of large and small flaviviral particles released from prMϩE-transfected cells (2,(23)(24)(25). Alterations in the proportion of virus particles were found in this study during the replication of several pr-M junction mutants.…”

“…The majority of particles released from dengue virus-infected Vero cells and C6/36 mosquito cells are virion-sized particles (24,34). These large particles predominate in Japanese encephalitis virus (JEV)-infected Vero cell cultures, whereas subviral particles are more abundant in cultures of infected C6/36 cells (20,23,25,29). Both types of particles are equally common following infection of COS-1 cells with tick-borne encephalitis virus (TBEV) (1).…”

N-glycosylation of the premembrane protein of Japanese encephalitis virus is critical for folding of the envelope protein and assembly of virus-like particles

“…Previous studies have shown that high level expression of VLPs could result in fusion, apoptosis, and death in some JEV susceptible cells (Kojima et al, 2003;Konishi et al, 2001). In this study, apoptosis induced by JEV VLPs was found in pCprME transfected 293FT cells, which suggested that the VLPs were also toxic for this cell line.…”

Evaluation of Extracellular Subviral Particles of Dengue Virus Type 2 and Japanese Encephalitis Virus Produced bySpodoptera frugiperdaCells for Use as Vaccine and Diagnostic Antigens

“…Cells expressing flavivirus prM and E proteins are known to secrete nucleocapsid-free subviral extracellular particles (EPs), which are similar to slowly sedimenting hemagglutinin (SHA) particles secreted from flavivirus-infected cells (47). EPs of JEV synthesized in mammalian expression systems have been evaluated for their immunogenicity and/or protective efficacy in mice (15,24,43). Two of these studies (24,43) demonstrated that the EPs induced neutralizing antibodies at levels comparable to those induced by an inactivated JE vaccine.…”