Figure Legends
Figure 1. Development and characterization of the VLP vaccine.(A) Schematic representation of spike, membrane, envelope and
nucleocapsid protein designs. Included are a cleavable signal peptide
(CD33 SP), N-terminal domain (NTD), receptor binding domain (RBD), S1/S2
boundary (S1/S2), fusion peptide (FP), heptad repeat 1 (HR1), central
helix (CH), heptad repeat 2 (HR2), transmembrane domain (TM),
cytoplasmic tail (CT), tobacco etch virus protease cleavage site (TEV),
T4 fibritin trimerization domain (FD), thrombin cleavage site (THM) and
six histidine tag sequence (His). The native polybasic furin cleavage
site modifications and proline substitutions to generate the full-length
WT, prefusion stabilized 2p and 6p spike variants are also indicated.(B) Schematic representation of VLP production, purification
and formulation process. Representative transmission electron microscopy
image of VLP producing HEK293 cells (C) , scanning electron
microscopy and atomic force microscopy images of individual VLPs
((D) and (E) are shown. (F ) TRPS size
distribution measurement (nm) of WT, 2p and 6p spike variant
incorporating VLPs. Analysis of structural proteins assembled into
SARS-CoV-2 VLPs by Western blot using anti-His, anti-N (G) and
anti-S (H) antibodies.
Figure 2. VLPs elicit robust antibody and helper T cell
responses in mice. BALB/c mice (n = 12 per group) were immunized on
days 0 and 14 with 0.4 (low dose; LD) or 4 µg (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-prime(A) and 2 weeks post-boost (B) and assessed for
SARS-CoV-2 S-specific 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. (C) Spleens were
collected 2 weeks after booster (n=6). 1×106/250 µl
splenocytes from naïve or immunized mice were stimulated with
recombinant spike (5µg/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. (D)Pie charts representing the proportions of individual secreted
S-specific T helper cytokines are presented.
Figure 3. Immunogenicity of the VLP vaccine in mice, rats and
ferrets . (A) BALB/c mice (n=10/group) were subcutaneously
immunized with 6 different doses (24-0.75 µg) of 6p VLP/Alum/CpG on days
0 and 14. 2 weeks after the booster injection, IgG titers against the
whole inactivated virus was determined by ELISA. ED50 was determined by
non-linear regression curve fit in GraphPad Prism. (B) Sprague
Dawley rats (n=5) were immunized with 40 µg 6p VLP with Alum (600
µg/rat), K3 CpG ODN (300 µg/rat). Live virus neutralizing antibody
titers were evaluated 2 weeks after booster injection. (C)Ferrets (n=4/group) were vaccinated either with a 10 µg or a 40 µg dose
of the VLP with Alum (600 µg/ferret) and K3 CpG ODN (300 µg/ferret).
Live virus neutralizing antibody titers were determined 2 weeks after
priming and booster injection. (D) Analysis of spike protein expression in WT 6p or alpha variant 6p-S expressing VLPs by Western blot using anti-His (2.5 µg protein/well; 1:2 indicates two-fold diluted sample). (E-F) C57BL/6 mice (n=5-10/group) were subcutaneously immunized with 8 µg of either WT 6p-S VLP or alpha variant 6p-S VLP vaccine on days 0 and 14. Two-weeks after booster injection, (E) S-, inactivated virus- and N-specific IgG titers or (F) WT, alpha, beta or gamma variant RBD-specific IgG titers were determined by 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 4. Immunoprotective activity of the VLP vaccine in
K18-hACE2 transgenic mice. K18-hACE2 transgenic mice (n=10/group) were
subcutaneously immunized with 2 µg (Low dose; LD) or 8 µg (high dose;
HD) of the VLP vaccine on days 0 and 14. 2 weeks after booster
injection, (A) RBD-specific IgG, IgG1, IgG2c antibody titers
were determined by ELISA and (B) neutralizing antibody titers
against the authentic Wuhan strain and the B.1.1.7. Kent variant were
determined. 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. On day 21 after
booster, mice were challenged intranasally on 3 consecutive days with 50
µl of 1 × 105 pfu/mouse of SARS-CoV-2 (Wuhan strain).
Lungs were collected 7 days after last virus instillation. (C)Infectious virus loads in lung homogenates were assessed by qRT-PCR
against the nucleocapsid (NC1 and NC2). Bars represent the mean virus
load (n = 10/group) as 1/ct values. Comparisons were performed by
unpaired Student’s t-test; *P < 0.05, **P < 0.01,
***P < 0.001, ****P < 0.0001. (D)Histological micrographs showing healthy (first column), placebo (second
column), low dose vaccine (third column) and high dose vaccine (fourth
column) groups. A-D, Hematoxylin – Eosin (H&E), areas marked green
shows inflammed parts of the lungs; E-L, H&E, 20x; M-P, Gomori
Trichrome (GT), 40x. a, alveoli; b, bronchiole; v, blood vessel; blue
arrow, protein debris; red arrow, hyaline membrane. (E)Histomorphometric measurements. The descriptive statistics were
presented as median and interquartile range in all graphs except for
inflamed area percent (mean ±S.D). Statistical significance
(p<0.05): a, compared to healthy group; b, compared to placebo
group; c, compared to low dose vaccine group, d, compared to high dose
vaccine group. Nonparametric variables were compared between groups
using Kruskal-Wallis test. Pairwise comparisons were made with Dunn’s
test. Parametric variables were compared in multiple groups using
one-way analysis of variance. Pairwise comparisons were performed with
the Tukey test.