Figure 1.
CHIKV structural polyprotein expression and processing in AcMNPV-CHIKV37997 infected Sf21 cells and pV1JNS-CHIKV37997 transfected HEK293 cells.
Cell lysate Western blots depicting (A) E2 expression and processing, detected by an E2 peptide-specific antibody. (B) E1 expression, detected by an E1 peptide-specific antibody. (C) E1/E2 (co-migrating) and capsid expression and processing, detected by an anti-CHIKV polyclonal antibody. AcMNPV-GFP infected Sf21 lysate was included as a negative control for insect cells and baculovirus vector.
Figure 2.
Transmission electron microscopy (TEM) images of thin-sections of AcMNPV-CHIKV37997 infected Sf21 cells and pV1JNS-CHIKV37997 transfected HEK293 cells.
(A) Putative CHIKV capsids formed in the cytoplasm of HEK293 cells. Capsid diameters are approximately 30–35 nm. (B) Baculovirus showing hallmark multiple nucleocapsids per envelope, with very electron dense nucleocapsids. Nucleocapsid diameter is approximately 40 nm. (C) Putative CHIKV capsids formed in the cytoplasm of Sf21 cells. Capsid diameters are approximately 30–35 nm. Scale bar is equivalent for all images and represents 500 nm.
Figure 3.
Effect of elevated culture pH on cell-surface localization of CHIKV glycoproteins and CHIKV VLP yield.
(A) Mean fluorescence intensity (MFI) from surface staining of AcMNPV-CHIKV37997 infected Sf21 and pV1JNS-CHIKV37997 transfected HEK293 cells with neutralizing antibody m242. AcMNPV-NC and a mock transfection were included as negative controls for Sf21 and HEK293, respectively. Error bars represent 95% confidence intervals (N = 3 independent infections/transfections). (B) E1/E2 Western blot and VLP concentration for supernatants from infected Sf21 and transfected HEK293. Error bars represent 95% confidence intervals (N = 4 assay replicates). <LOQ indicates a result less than the qELISA limit of quantitation, or <2 ng/mL.
Figure 4.
Characterization of the SfBasic cell line and comparison to the parental Sf21 cell line.
(A) Cell diameter (Ø) distribution histogram with a representative bright field image. (B) Fluorescence microscopy images of propidium iodide stained cells. Mean fluorescence intensity (MFI) of the G1-phase cell population from flow cytometry cell cycle analysis of the same sample is indicated below the corresponding image. (C) Cell line population doubling time (PDT) as a function of growth medium pH. The growth medium was Sf-900II-BES-MISS, adjusted to various pH levels by titration with 1 N NaOH.
Figure 5.
Volumetric CHIKV VLP yield enhancement resulting from adaptation of Sf21 to elevated culture pH.
(A) CHIKV VLP concentration of culture supernatants from Sf21 and SfBasic small-scale shake flask cultures. Error bars represent 95% confidence intervals (N = 10 independent infections). (B) CHIKV VLP concentration of culture supernatants from scale-up of SfBasic from shake flasks (SF) into a PID controlled 3-L stirred tank bioreactor (STBR).
Figure 6.
Biophysical characterization of CHIKV VLPs derived from SfBasic cells and comparison to a VLP standard derived from HEK293 cells.
(A) Western blot of density gradient ultracentrifugation fractions containing CHIKV VLPs, using E1, E2, and capsid peptide-specific antibodies. (B) Dynamic light scattering (DLS) distribution of purified CHIKV VLP diameters (Ø). (C) Raw/unprocessed and 2D class average transmission electron microscopy images of purified CHIKV VLPs.
Figure 7.
Immunogenicity assay titers from guinea pig sera after vaccination with adjuvanted SfBasic-derived and HEK293-derived CHIKV VLPs.
(A) Anti-CHIKV IgG geomean titer and CHIKV 181/25 geomean neutralizing titer (NT100) at study day 14 (14 days after first dose). (B) Anti-CHIKV IgG geomean titer and CHIKV 181/25 geomean neutralizing titer (NT100) at study day 21 (7 days after second dose). Geomean IgG ELISA background from pre-vaccination sera is indicated by a dashed line. Error bars represent 95% confidence intervals (N = 4 animals per group), and asterisks indicate a statistically significant increase in titer over background (p<0.05).