Supporting Information
Figure S1. 6pVLPs specifically bind to human ACE2 receptor
coated beads but not to recombinant IL-1β coated beads . Carboxyl
modified latex beads were coated with hACE2 or recombinant IL-1β and
then incubated with serially diluted CFSE labeled VLPs. Beads were
washed once and analyzed by flow cytometry for bead-bound VLP-CFSE
signal. Top dot plots show the gating strategy and the lower plots
represent bead-bound CFSE signal (y-axis) versus forward scatter
(x-axis). Histograms present VLP CFSE signal intensity (mean
fluorescence intensity, MFI) of serially diluted VLPs associated with
negative control IL-1β beads (top 4 histograms) or hACE2 coated beads
(lower 4 histograms).
Figure S2. Stability of SARS-CoV-2 VLPs under elevated
temperature (a) and following adsorption to alum and CpG adjuvantation
(b) . (a ) 6p VLPs were subjected to thermal stress for the
indicated time periods. Antigen identities were assessed by western blot
(anti-His Tag antibodies) and compared to a sample preserved at 5ºC for
one week. Positions of S, M and E proteins are indicated by arrows. (b)
Transmission electron microscopy image of formulated 6p VLPs demonstrate
that VLPs retain their morphology after adsorption to alum and CpG
adjuvantation.
Figure S3. VLPs elicit robust antibody responses in mice.BALB/c mice (n = 12 per group) were immunized on days 0 and 14 with 0.4
((a ) and (b ), low dose; LD) or 4 µg ((c ) and
(d ) high dose; HD) 6p VLP or 2p VLPs without or with Alum (5
µg/mouse), K3 CpG ODN (20 µg/mouse) or Alum+CpG ODN. Control BALB/c mice
were administered Alum or CpG ODN alone (black and gray). Sera were
collected 2 weeks post- 2 weeks post-boost and assessed for SARS-CoV-2
RBD- (a and c ) or N-specific (b andd ) IgG, IgG1 and IgG2a by enzyme-linked immunosorbent assay
(ELISA). Vaccinated groups were compared by one-way ANOVA with Dunnett’s
multiple comparisons test. *P < 0.05, **P < 0.01,
***P < 0.001, ****P < 0.0001. Data are presented as
GMT ± geometric SEM.
Figure S4. 6p VLP+Alum+CpG induce S-specific TH1 dominated
helper T cells. (a ) 1×106/250 µL splenocytes from naïve or
immunized BALB/c mice (n=6) were stimulated with recombinant
nucleocapsid (20 µg/ml) in the presence of 1 μg/mL anti-mouse CD28. T
helper cytokine levels were assessed from 48h culture supernatants using
the LEGENDplex™ MU Th Cytokine Panel (12-plex). Groups were compared by
one-way ANOVA with Dunnett’s multiple comparisons test. *P <
0.05, **P < 0.01, ***P < 0.001, ****P <
0.0001. Data are presented as mean cytokine levels ± SEM. (b )
Pie charts representing the proportions of individual secreted
N-specific T helper cytokines are presented.