Figure 1.
Genome organization of CHIKV and an expression vector.
A synthetic DNA fragment encoding human-preferred CHIKV structural protein genes, C-E3-E2-k6-E1, was subcloned into the plasmid pcDNA3.1/zeo+ to generate the expression vector. A DNA fragment encoding natural CHIKV structural protein genes was amplified and subcloned into the same vector.
Figure 2.
Western blot assay of structural proteins expressed in 293T cells.
We transfected 293T cells with an expression plasmid containing structural proteins derived from either natural or optimized codons. The supernatant was analyzed by western blot analysis using serum from a mouse immunized with formalin-inactivated CHIKV. Results for the following are shown: (1) the supernatants from 293T cells transfected with optimized structural protein genes, (2) natural structural protein genes, (3) plasmid vector, (4) no plasmid vector but lipofectamine, and (5) no transfection.
Figure 3.
TEM analysis of purified VLPs expressed with the optimized codons.
(A) Magnification at ×30,000, (B) ×150,000, including CHIKV virion (top, left corner).
Figure 4.
Time course of the formation of CHIKV VLPs.
293T cells were transfected with the expression plasmid, and the supernatant was collected for 10 consecutive days. The supernatant (20 µL) was subjected to the VLP concentration as described in the Materials and Methods, and analyzed by western blotting using anti-CHIKV mouse serum. (A) Time course of the expression by the plasmid with the natural codons. (B) Results for the same experiment but using the plasmid with optimized codons. Lanes 1 through 10 are days 1 to 10 p.t. (C) CsCl equilibrium density gradient centrifugation of the VLPs purified from 293F cells. The proteins in each fraction were analyzed by western blotting.
Figure 5.
The antigen-coated aggregation of the VLPs observed by immunoelectron microscopy.
The antigen-coated aggregation of the VLPs was observed by TEM. The VLPs incubated with (A) serum from a patient (32097), and (B) with serum from a healthy individual.
Figure 6.
Antigenicity and immunogenicity of VLPs.
(A) Anti-CHIKV IgG responses. Microplates were coated with purified VLPs (5 ng/well) and used to detect CHIKV-specific antibodies. IgG antibodies in the 1∶50 diluted serum from one rabbit (Rb1-post) and the sera from three mice (mouse #1, #2 and #3) immunized with inactivated CHIKV, and three sera from convalescent chikungunya patients (# 32095, 32097 and 32221) were examined. Preimmune (Rb1-pre) and PBS were included as controls. (B) Microneutralizaton assays to examine the neutralizing activity of anti-VLP sera. Serial twofold dilutions of the preimmune and postimmune sera were mixed with an equal volume of 100 TCID50 of the ROSS strain and used to inoculate 104 Vero cells. Cell viability was measured at 48 h after incubation by using a WST1 assay according to the manufacturer's instructions. The serum dilution at 50% cell viability indicated by a broken line was defined as the neutralizing antibody titer. pre Rb; rabbit preimmune serum, Rb 4w; rabbit serum from 4 wks postimmunization, Rb 5w; rabbit serum from 5 wks postimmunization, Gp 5w; guinea pigs serum from 5 wks postimmunization, vc; virus control, cc; cell control.
Figure 7.
Immunodetection of CHIKV antigens in the stable 293T cell line expressing VLPs.
The Zeocin-resistant cell lines were collected and immunostaining was performed with anti-CHIKV serum from a guinea pig. Untransfected 293F cells were used as a negative control. The cells were monitored under a fluorescence microscope.