Baculovirus-insect cell expression, purification, and immunological studies of the full-length Japanese encephalitis virus envelope protein

Abstract

The E protein of Japanese encephalitis virus (JEV) is the major antigen to elicit neutralizing antibody response and protective immunity in hosts. In this study, we developed a baculovirus-based expression system of the Japanese encephalitis virus full-length E protein for production of recombinant vaccine. The expression of the full-length JEV E protein was not secreted but produced in the cytoplasm in spite of the use of prM-derived signal peptide. The full-length JEV E protein expressed in insect cells was extensively purified using immobilized metal affinity chromatography and emulsified with Freund’s adjuvant for immunological studies. Our studies demonstrated the mice immunized with the full-length JEV E protein successfully induced neutralizing antibody response and protective immunity toward a lethal challenge of the JEV. This finding provides valuable information for developing subunit vaccines for JEV antigenic complex viruses.

Introduction

Japanese encephalitis virus (JEV) (family Flaviviridae, genus Flavivirus) [1] is a flavivirus that is transmitted from amplifier animals such as pigs to humans through mosquito bites. The JEV infection occurs primarily in the Far East and Southeastern Asia and the northern Australia. In humans, JEV infection can cause severe central nerve system disease, including febrile headache syndrome, aseptic meningitis and encephalitis, which result in high mortality or developing permanent neurological sequelae in more than half of the survivors. Other flaviviruses like St. Louis encephalitis virus, Murray Valley encephalitis virus, West Nile virus, and Kunjin virus also belong to the JEV serocomplex [1].

For JEV, the inactivated mouse brain-derived vaccines have been used for many years in Japan, Korea, China, Taiwan, and Australia and demonstrated to be effective to reduce the disease rates in these areas [2], [3], [4], [5]. Nonetheless, the drawback of the inactivated JEV vaccines is that the mouse brain preparation is complicated in reactogenicity, requirement for multiple doses, cost, and dependence on a neurological tissue substrate [5]. The World Health Organization has been promoting the development of improved and/or new vaccines for JEV [5], [6].

The envelope (E) protein of flavivirus is associated with viral binding to cellular receptor(s) and membrane fusion, and the major antigen to elicit neutralizing antibodies and protection in hosts [1], [7]. The flavivirus E proteins consist of approximately 500 amino acids with six “conserved” disulfide bonds to maintain its three-dimensional structure which has been determined for the tick-borne encephalitis (TBE) virus using X-ray crystallography [8] and recently for the dengue virus using cryoelectron microscopy [9]. Expression of the recombinant E proteins has been achieved in Escherichia coli [10], yeasts [11], insect cells [12], [13], [14], [15], [16], [17], [18], and mammalian cells [19], [20], [21], [22]. Among these systems, the baculovirus-insect cell expression system mainly designed by using AcMNPV polyhedrin promoter provides the advantage of high-level expression of the recombinant proteins with proper folding and co-/post-translational processing [23].

We previously obtained the attenuated variant CH2195LA from a wild-type Taiwanese isolate CH2195 by plaque isolation in Vero cells [24], [25]. The complete nucleotide sequences of the attenuated variant CH2195LA was determined (accession number AF221499), showing its phylogenic nearness to JaOArS982 but distance to SA14-14-2, SA14-2-8, and RP-2ms of the attenuated JEV strains [26]. In this study, we constructed the recombinant baculovirus encoding a full-length JEV E protein with a N-terminal 24 amino-acid signal sequence derived from its adjacent prM, and a C-terminal six-histidine tag for immobilized metal affinity chromatography (IMAC) purification. The expressed protein was obtained from Sf9 cells infected with the recombinant baculovirus and purified by IMAC. The purified proteins were prepared at three different dose concentrations and emulsified with Freund’s adjuvant (FA) for immunological studies. Both the neutralizing antibody response and the protective immunity in mice were finally investigated.