Development and preclinical evaluation of virus-like particle vaccine against COVID-19 infection

Abstract

Background: Vaccines that incorporate multiple SARS-CoV-2 antigens can further broaden the breadth of virus-specific cellular and humoral immunity. This study describes the development and immunogenicity of SARS-CoV-2 VLP vaccine that incorporates the four structural proteins of SARS-CoV-2.

Methods: VLPs were generated in transiently transfected HEK293 cells, purified by multimodal chromatography, and characterized by tunable-resistive pulse sensing, AFM, SEM, and TEM. Immunoblotting studies verified the protein identities of VLPs. Cellular and humoral immune responses of immunized animals demonstrated the immune potency of the formulated VLP vaccine.

Results: Transiently transfected HEK293 cells reproducibly generated vesicular VLPs that were similar in size to and expressing all four structural proteins of SARS-CoV-2. Alum adsorbed, K3-CpG ODN-adjuvanted VLPs elicited high titer anti-S, anti-RBD, anti-N IgG, triggered multifunctional Th1-biased T-cell responses, reduced virus load, and prevented lung pathology upon live virus challenge in vaccinated animals.

Conclusion: These data suggest that VLPs expressing all four structural protein antigens of SARS-CoV-2 are immunogenic and can protect animals from developing COVID-19 infection following vaccination.

Keywords: COVID-19; CpG ODN Adjuvant; SARS-CoV-2; vaccine; virus-like particle.

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), 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/group) were immunized on days 0 and 14 with 0.4 µg (low dose; LD) or 4 µg (high dose; HD), 6p‐S VLP or 2p‐S VLPs without or with Alum (5 µg/mouse), without or with K3 CpG ODN (20 µg/mouse) or with 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 ELISA. Vaccinated groups were compared by one‐way ANOVA with Dunnett's multiple comparisons test. *P < .05, **P < .01, ***P < .001, ****P < .0001. Data are presented as GMT ± geometric SEM. (C) Spleens were collected 2 weeks after booster (n = 6). 1×10⁶/250 µl splenocytes from naive or immunized mice were stimulated with recombinant spike (5 µg/ml) in the presence of 1 μg/ml anti‐mouse CD28. T helper cell cytokine levels were assessed from 48 h 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 < .05, **P < .01, ***P < .001, ****P < .0001. Data are presented as mean cytokine levels ± SEM. (D) Pie charts representing the proportions of individual secreted S‐specific T helper cell 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 six different doses (24–0.75 µg) of 6p‐S 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/group) were immunized with 40 µg 6p‐S VLP with Alum (600 µg/rat), K3 CpG ODN (300 µg/rat). Live VNTs 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 VNTs 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 twofold diluted sample). (E–F) C57BL/6 mice (n = 5–10/group) were subcutaneously immunized with 8 µg of either WT (five mice/group) 6p‐S VLP (10mice/group) or alpha variant 6p‐S VLP (10mice/group) 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 < .05, **P < .01, ***P < .001, ****P < .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. Two 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. Alpha variant were determined. Groups were compared by one‐way ANOVA with Dunnett's multiple comparisons test. *P < .05, **P < .01, ***P < .001, ****P < .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 < .05, **P < .01, ***P < .001, ****P < .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 inflamed parts of the lungs; (E–L) H&E, 20×; M‐P, Gomori Trichrome (GT), 40×. 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 ± SD). Statistical significance (P < .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