There is an urgent need for an effective, affordable and safer JE vaccine for humans and domestic animals. Biotechnically-engineered cell lines expressing the JEV-VLP antigen may meet this requirement. Here, we describe another alternative, a recombinant subunit vaccine candidate, based on a new mammalian cell line.
The BJ-ME cells described in this study produce extremely high levels of the JEV-VLP antigen—15–20 μg/ml—which is higher than that of any other reported cell lines such as J12#26 , F  and JE-4B . This value was determined under experimental conditions involving simple changes in the culture medium, and we believe that the titer could be further increased under optimal culture conditions. Moreover, time-course results suggest that 5 days of incubation is sufficient for antigen production.
BJ-ME cells had similar cell morphology and growth characteristics as the parental BHK-21 cells. In fact, cell clones with different morphogenetic characteristics found during the selection and cloning process were found to release the E antigen inefficiently. This phenomenon indicates that maintaining similar morphology and growth characteristics with the parental cells may be important in the engineering of cell lines with high efficiency and stable exogenous gene expression. Another factor that may have contributed to the high efficiency of JEV-VLP antigen expression in BJ-ME cells is the genetic codon optimization of the cDNA sequence encoding the prM and E proteins. Different species have codon usage bias, and optimizing the genetic codon according to the bias of the expressing host could increase the expression efficiency of the target gene [34–36]. The BJ-ME cells showed stable JEV-VLP production during cell culture and passage. Even after 20–25 passages, the JEV-VLP antigen-producing efficiency of BJ-ME cells was not significantly reduced.
A major obstacle to the stable expression of flavivirus membrane protein in mammalian cells is its cytotoxicity. In a previous report, attempts to establish cells with high expression of the prM-E protein from Vero cells were unsuccessful . This is probably because Vero cells are susceptible to JEV infection, which makes them sensitive to the cytotoxic effects of the JEV-VLP antigen. Although BHK-21 cells are susceptible to JEV infection, we did not observe any obvious cytotoxic effects of JEV VLP. It was possible to obtain dozens of G418-resistant cell colonies with a single transfection and selection cycle. The cell line BJ-ME constructed in this study has the same growth characteristics as the parent cell line BHK-21. So this cell line could easily be cultured in serum-free or low-serum medium. J12#26 cells derived from RK13, a rabbit kidney cell line, was the most stable and highest antigen-producing cell line in all previous reports . However, RK13 cells grow slowly and could not reach high cell density, so are not suitable for large-scale culture. Another important point is that RK13 cells contain bovine viral diarrhea virus , which make them unsuitable for vaccine antigen production.
To overcome the fusogenic cytotoxicity of JEV prM-E protein expression in mammalian cell lines, Konishi et al.  introduced a mutation at the prM/M cleavage site to prevent authentic processing and to reduce the toxic fusion activity of the expressed VLP, and established an F-cell line using CHO-k1 as the parent cell line. The elimination of cleavage of the prM protein resulted in the production of immature VLPs, which are quite different in their conformational structure from the mature VLPs produced by authentic cleavage of prM . These structural differences may eliminate virus conformational neutralizing epitopes. Moreover, reports have shown that most JEV-neutralizing epitopes are conformation dependent . The differences in the conformational structure will affect the immunogenicity of the F antigen. In fact, the protection rate in mice immunized with the F antigen with uncleaved prM protein did not exceed 50% . Recently Yamaji et al. further reported expressing the JEV prM protein mutated virus-like particles with baculovirus  and lepidopteran insect cell lines . Obviously the yield of E antigen were increased in both of two expressing systems, especially the E antigen yield reached about 30 μg/ml in lepidopteran insect cell line. However, these studies did not present immune protection result of these immature JEV virus-like particles with uncleaved prM protein. The differences in structural and immune protective effect between immature and mature JEV virus-like particles need further evaluation. Maybe DENV-related reports [42, 43] could provide some clues for future survey the relation of immune protective effect and structure of virus-like particles.
As expected be of particulate form, the BJ-ME antigen was immunogenic. With or without adjuvant, dose of 2 μg of BJ-ME induced high titers of neutralizing antibodies and provided complete protection against challenge. However we observed no enhancement effect of the adjuvant in this experiment which may because the antigen amount of 2 μg is overdosed for mice. Another experiment with different dose of antigen showed that 0.3 μg of BJ-ME antigen could induce effective protection neutralizing antibodies titer of 1:40 which is higher than surrogate maker for seroprotection of neutralizing antibody titer of 1:10.