Figure 1.
CHIKV structural cassette expression and VLP production, using recombinant baculoviruses.
A) Schematic representation of the CHIKV structural cassette, as it was expressed in insect cells. Shaded areas represent transmembrane domain and ss signifies signal sequences. A, F and S indicate autocatalytic, furin and signalase cleavage sites, respectively. Asterisks indicate N-glycosylation sites and the molecular mass (in kDa) of the proteins is depicted. B) CHIKV E1 and E2 expression in the Sf21 cell (C) and medium (M) fraction was analysed by WB using α-E1 and α-E2 polyclonal antibodies. VLPs were precipitated using PEG-6000 (PEG) and subsequently purified using discontinuous sucrose gradient centrifugation (Suc). C) The glycosylation status of CHIKV E1 and E2 was analysed by WB after PNGase F treatment. Ac-GFP was used as a negative control.
Figure 2.
CHIKV-E1 and –E2 detection on the surface of Ac-S27 infected Sf21 cells.
Sf21 cells were infected with Ac-S27 and subjected to immunostaining using α-E1 and α-E2 antibodies. Cells were analysed by fluorescent microscopy where positive staining indicates E1 or E2 surface exposure.
Figure 3.
Syncytia formation assay on Ac-S27 infected insect cells.
Sf9 ET- cells were infected with Ac-S27 and treated 72 hpi with acidified culture medium of pH = 6.4, 5.8, 5.5 and 5.0 for 2 min. Cells were analysed 4 h post induction and syncytia are indicated with white arrows.
Figure 4.
CHIKV VLP production in Sf21 insect cells.
A) Sf21 cells were infected with Ac-S27 in duplo and VLP production was followed over time. Medium fraction samples were taken at the indicated time points and analysed on E2 content by sandwich ELISA. B) Electron micrographs of CHIKV-VLPs at a 12,000∶1 (top) and 40,000∶1 (middle and bottom) magnification. The medium fraction of infected Sf21 cells was analysed by transmission electron microscopy (TEM) for the presence of VLPs. C) The size of 200 VLPs were determined using TEM to determine the relative VLP size range.
Figure 5.
CHIKV neutralization and IgG-isotyping.
A) Serum of immunized mice were collected and tested for their neutralizing ability based on >95% protection against CHIKV induced CPE in Vero cells. BEI corresponds to the inactivated CHIKV positive control. B–C) Immunoglobulin-G1 and -2c isotypes were determined using ELISA upon serial dilution. Statistical analysis shows that all CHIKV antigen immunized groups are significantly different from the control groups.
Figure 6.
CHIKV VLP vaccination and CHIKV challenge.
A) At least 6 weeks old mice were vaccinated with 0.1 µg VLPs, 1 µg VLPs and 1 µg VLPs adjuvanted with Quil A (QA). PBS and GFP were used as a negative control and inactivated CHIKV virus as a positive control (n = 6 per group). Mice were challenged with the Rèunion Island isolate 5 w post infection. Viraemia levels were determined over 6 days. B) Foot size in mm2 (width×height) was determined over 16 days post challenge. Statistical analysis shows that mice vaccinated with inactivated CHIKV virus, 1 µg VLPs and 1 µg VLPs adjuvanted with Quil A display significant lack of viraemia or lack of footswelling at times of peak viraemia (1–3 dpi) and peak foot swelling (6–8 dpi) in the control groups, respectively.