Efficient production of Japanese encephalitis virus-like particles by recombinant lepidopteran insect cells

Abstract

The production of Japanese encephalitis (JE) virus-like particles (VLPs) in stably transformed lepidopteran insect cells was investigated. The DNA fragment encoding the JE virus (JEV) prM signal peptide, the precursor (prM) of the viral membrane protein (M), and the envelope glycoprotein (E) was cloned into the plasmid vector pIHAbla. The pIHAbla contained the Bombyx mori actin promoter downstream of the B. mori nucleopolyhedrovirus (BmNPV) IE-1 transactivator and the BmNPV HR3 enhancer for high-level expression, together with a blasticidin resistance gene for use as a selectable marker. DNA encoding a form of prM with a pr/M cleavage site mutation was used to suppress the cell-fusion activity of VLPs. After transfection with the resultant plasmid, Trichoplusia ni BTI-TN-5B1-4 (High Five) cells were incubated with blasticidin, and cells resistant to the antibiotic were obtained. Western blot analysis and enzyme-linked immunosorbent assay of a culture supernatant showed that transfected High Five cells secreted an E antigen equivalent to the authentic JEV E. Sucrose density-gradient sedimentation analysis of the culture supernatant from recombinant High Five cells indicated that secreted E antigen molecules were produced in a particulate form. VLPs recovered from the supernatant successfully induced neutralizing antibodies in mice, particularly when adsorbed to alum adjuvant. High yields (≈30 μg/ml) of E antigen were achieved in shake-flask cultures. These results indicate that recombinant insect cells may offer a novel approach for efficient VLP production.

Development of a Japanese encephalitis virus-like particle vaccine in silkworms using codon-optimised prM and envelope genes

“…Another type of JEV-VLP vaccine has also been produced in cloned BHK cells following genetic engineering, and the resulting cells showed increased JEV-E protein production [18]. Moreover, several JEV-VLP vaccines have been produced using the baculovirus system [19, 20, 21]. …”

Production of Japanese encephalitis virus-like particles in insect cells

“…In conclusion, recombinant insect cells may provide a breakthrough in the development and production of VLPs for use as 21 Bars represent the means ± SD obtained from three or four different determinations.…”

“…Recently, we investigated the production of JE VLPs in stably transformed lepidopteran insect cells. 21 The virion of flaviviruses, such as JE, dengue, West Nile, and yellow fever viruses, consists of a nucleocapsid structure surrounded by a lipid bilayer inserted with an envelope (E) glycoprotein and a membrane (M) protein. 5,6,[22][23][24][25] The E protein is the major surface protein with a role in cellular receptor binding and membrane fusion and induces neutralizing antibodies that protect hosts against disease.…”

“…This cleavage event results in changes in the oligomerization of these surface proteins from a prM/E heterodimer to an E homodimer, and, thereby, the formation of mature virions that can display cell-fusion activity. In our recent study, 21 Trichoplusia ni BTI-TN-5B1-4 (High Five) cells were stably transformed using a plasmid vector encoding the JE virus (JEV) prM signal sequence and the prM and E genes to produce a secreted form of JE VLPs (Fig. 1).…”

Generation and characterization of a new mammalian cell line continuously expressing virus-like particles of Japanese encephalitis virus for a subunit vaccine candidate

“…In fact, the protection rate in mice immunized with the F antigen with uncleaved prM protein did not exceed 50% [33]. Recently Yamaji et al further reported expressing the JEV prM protein mutated virus-like particles with baculovirus [40] and lepidopteran insect cell lines [41]. 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.…”