Characterization of chikungunya virus-like particles.

Chikungunya virus (CHIKV) is becoming a global concern due to the increasing number of outbreaks throughout the world and the absence of any CHIKV-specific vaccine or treatment. Virus-like particles (VLPs) are multistructured proteins that mimic the organization and conformation of native viruses but lack the viral genome. They are noninfectious and potentially safer vaccine candidates. Recent studies demonstrated that the yield of CHIKV VLPs varies depending on the strains, despite the 95% amino acid similarity of the strains. This might be due to the codon usage, since protein expression is differently controlled by different organisms. We optimized the region encoding CHIKV structural proteins, C-E3-E2-6k-E1, inserted it into a mammalian expression vector, and used the resulting construct to transfect 293 cells. We detected 50-kDa proteins corresponding to E1 and/or E2 in the cell lysate and the supernatant. Transmission electron microscopy revealed spherical particles with a 50- to 60-nm diameter in the supernatant that resembled the native CHIKV virions. The buoyant density of the VLPs was 1.23 g/mL, and the yield was 20 µg purified VLPs per 108 cells. The VLPs aggregated when mixed with convalescent sera from chikungunya patients, indicating that their antigenicity is similar to that of native CHIKV. Antibodies elicited with the VLPs were capable of detecting native CHIKV, demonstrating that the VLPs retain immunogenicity similar to that of the native virion. These results indicated that CHIKV VLPs are morphologically, antigenically, and immunologically similar to the native CHIKV, suggesting that they have potential for use in chikungunya vaccines

Time course of the formation of CHIKV VLPs.

<p>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.</p

Antigenicity and immunogenicity of VLPs.

<p>(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.</p

TEM analysis of purified VLPs expressed with the optimized codons.

<p>(A) Magnification at ×30,000, (B) ×150,000, including CHIKV virion (top, left corner).</p

The antigen-coated aggregation of the VLPs observed by immunoelectron microscopy.

<p>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.</p

Western blot assay of structural proteins expressed in 293T cells.

<p>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.</p