Mosquito Cell-Derived Japanese Encephalitis Virus-Like Particles Induce Specific Humoral and Cellular Immune Responses in Mice

Abstract

The Japanese encephalitis virus (JEV) is the major cause of an acute encephalitis syndrome in many Asian countries, despite the fact that an effective vaccine has been developed. Virus-like particles (VLPs) are self-assembled multi-subunit protein structures which possess specific epitope antigenicities related to corresponding native viruses. These properties mean that VLPs are considered safe antigens that can be used in clinical applications. In this study, we developed a novel baculovirus/mosquito (BacMos) expression system which potentially enables the scalable production of JEV genotype III (GIII) VLPs (which are secreted from mosquito cells). The mosquito-cell-derived JEV VLPs comprised 30-nm spherical particles as well as precursor membrane protein (prM) and envelope (E) proteins with densities that ranged from 30% to 55% across a sucrose gradient. We used IgM antibody-capture enzyme-linked immunosorbent assays to assess the resemblance between VLPs and authentic virions and thereby characterized the epitope specific antigenicity of VLPs. VLP immunization was found to elicit a specific immune response toward a balanced IgG2a/IgG1 ratio. This response effectively neutralized both JEV GI and GIII and elicited a mixed Th1/Th2 response in mice. This study supports the development of mosquito cell-derived JEV VLPs to serve as candidate vaccines against JEV.

Keywords: Japanese encephalitis virus; baculovirus; mosquito; virus-like particles.

Figure 1

Schematic illustration of baculovirus/mosquito (BacMos) for Japanese encephalitis (JE) virus-like particles (VLPs) production. A recombinant baculovirus bearing a DNA casttest of PH-Mp-JEV precursor membrane protein and envelope (prME)-Rhir-EGFP generated by a Bac-N-Blue system are produced from Sf21 cell. Mosquito cells are efficiently transduced by baculoviruses (BacMos-JEV-prME), and JEV prME expression and VLP (hollow green circles) secretion were induced. Abbreviations: PH, polyhedrin promoter; Mp: Mosquito promoter (hr1pag1); JEV prME: an insect codon-optimized JEV prME gene (AAB66485, 105-794th amino acid); Rhir, RhPV 5′-UTR IRES; EGFP, enhanced green fluorescent protein gene.

Figure 2

Expression of JEV glycoproteins in BacMos-JEV prME-transduced mosquito cells. Immunofluorescence assays for the detection of JEV E (A) and prM (B) glycoproteins. Three mosquito cells, C6/36 (upper panels), CCL-125 (center panels), and AP-61 (lower panels), were transduced with BacMos-JEV-prME (left panels) or infected with JEV at an MOI of 5 (center panels). After 5 days (transduction) or 2 days (infection), BacMos-JEV-prME-transduced cells (left panels), JEV-infected cells (center panels), and mock cells (right panels) were fixed and stained using Mab specific to either JEV E (A) or prM (B) glycoproteins. Cells were examined using green (A) or red (B) filters. Scale bar = 100 μm. Western blot analysis results for the detection of JEV E glycoprotein in C6/36 cells transduced using BacMos-JEV-prME (C). Total cell lysates were harvested at indicated times (dpt) and subjected to Western blot analysis to detect JEV E glycoproteins (indicated by arrows on the left). Protein sizes (kDa) of markers are shown on the left.

Figure 3

Results of dot blot analysis for the detection of secreted JEV viral glycoproteins. Three mosquito cell lines (C6/36, CCL-125, and AP-61) were transduced with BacMos-JEV-prME at an MOI of 2. C6/36 cells were infected with JEV at an MOI of 0.1. Culture medium was harvested at the indicated dpi and subjected to dot blot analysis (A) and quantification (B).

Figure 4

VLPs characterization. (A) Sucrose gradient banding JE VLPs. Representative photograph (left panel) of the sucrose density gradient following ultracentrifugation to isolate JEV VLPs. Arrow on the left indicates the VLP band. Fractions on the right from 1 (top) to 8 (bottom) were subjected to dot blot analysis (right two panels) using the indicated antibodies. (B) Electron micrographs and size distribution of JEV VLPs. Purified JEV VLPs were examined using transmission electron microscopy (upper and lower right panels). The boxed area in the upper right panel contains the same tissue section as that shown in the bottom panel. Scale bars = 50 μm. The size distribution of JEV VLPs (lower left panel) was determined using DLS. (C) Identification of peptides from E proteins in VLPs. Partially purified VLPs from fraction 5 were resolved using SDS-PAGE, and the Coomassie blue-stained band corresponding to E proteins was analyzed using mass spectrometry. The amino acid sequences of JEV E glycoproteins are shown, with the matching peptides obtained from the LC–MS/MS analysis shown in red.

Figure 5

Epitope analysis of JEV VLPs. JEV VLPs (left) and virions (right) were subjected to MAC-ELISA for human sera, including six sera of JEV infection (JE sera): 58,912 (JE), 58,911 (JE), 58,833 (JE), 58,682 (JE), 58,655 (JE), and 58,556 (JE); as well as four normal sera (NC sera): S10700567 (NC), S10700568 (NC), S10700569 (NC), and S10700570 (NC).

Figure 6

Immunogenicity of mosquito cell-derived VLPs in mice. (A) JEV-specific IgG responses. BALB/c mice (5 mice/group) received 3 doses of either PBS, 1µg VLPs, 4 µg VLPs, or IMOJEV. The following JEV E-specific antibody responses were determined by ELISA: total IgG (left panel), IgG1 (center panel), and IgG2a (right panel). Error bars indicate the mean SEM of concentrations from specific treatment groups. We identified statistically significant differences (p < 0.005) between the group that received 1 µg VLPs and the group that received IMOJEV. (B) PRNT₅₀ titers against JEV GI and GIII. For both JEV GI and GIII, PRNT₅₀ titer values were calculated using 3 mice/group. Error bars indicate the mean SEM of concentrations from individual animals. (C) Virus-specific T cell responses. BALB/c mice received 2 doses of one of the following: PBS, 0.0625, 0.25, 1, 2 µg VLPs, or IMOJEV. Three months after the second immunization, splenocytes (from 1 mouse/group) were collected and stimulated with purified JEV VLP antigens. Splenocytes secreting either IFN-γ or IL-4 were quantified in triplicate using ELISpot assays. Spot-forming cells (SFCs) were counted and calculated. The data are shown as the means  ±  SEM, and the statistical significance between immunogen stimulations (PBS and VLP) was analyzed using Student’s t-test (* p < 0.05).