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.