Immunogenicity and protective potential of chimeric virus-like particles containing SARS-CoV-2 spike and H5N1 matrix 1 proteins

Abstract

Coronavirus Disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), has posed a constant threat to human beings and the world economy for more than two years. Vaccination is the first choice to control and prevent the pandemic. However, an effective SARS-CoV-2 vaccine against the virus infection is still needed. This study designed and prepared four kinds of virus-like particles (VLPs) using an insect expression system. Two constructs encoded wild-type SARS-CoV-2 spike (S) fused with or without H5N1 matrix 1 (M1) (S and SM). The other two constructs contained a codon-optimized spike gene and/or M1 gene (mS and mSM) based on protein expression, stability, and ADE avoidance. The results showed that the VLP-based vaccine could induce high SARS-CoV-2 specific antibodies in mice, including specific IgG, IgG1, and IgG2a. Moreover, the mSM group has the most robust ability to stimulate humoral immunity and cellular immunity than the other VLPs, suggesting the mSM is the best immunogen. Further studies showed that the mSM combined with Al/CpG adjuvant could stimulate animals to produce sustained high-level antibodies and establish an effective protective barrier to protect mice from challenges with mouse-adapted strain. The vaccine based on mSM and Al/CpG adjuvant is a promising candidate vaccine to prevent the COVID-19 pandemic.

Keywords: Coronavirus Disease 2019 (COVID-19); chimeric; severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2); spike; virus-like particle (VLP).

Figure 1

Generation of SARS-CoV-2 VLPs. (A) Schematic of SARS-CoV-2 S/M1 and its mutation. (B) Schematics of the four recombinant bacmid rFBD-SARS-CoV-2. (C, D) Expression of exogenous genes by recombinant baculoviruses identified by IFA (C) and Western blot (D). The scale bar corresponds to 150 μm. Convalescent serum of COVID-19 patient or Influenza A M1 as the primary antibody, and HRP-labeled Goat Anti-Mouse IgG (H+L) as the secondary antibody. Mock, wild baculoviruses infected cell. Unprocessed original images can be found in Supplemental Figure S6. (E) Transmission electron micrograph of negatively stained SARS-CoV-2 VLPs. The scale bar corresponds to 50 nm.

Figure 2

Evaluation of the immunogenicity of the VLP. Mice were primed with PBS or SARS-CoV-2 VLPs and boosted three weeks after the prime immunization using the same inocula used for priming. Blood samples were collected from the tail vein of mice at indicated times after the initial vaccination and used to analyze humoral immune responses using an antibody detection kit (The coating antigen used in the kit was RBD protein). Sera collected from the tail vein of pre-immune mice were used as a negative control. *, p< 0.05; **, p< 0.01; ***, p< 0.001; ****, p< 0.0001. The results are expressed as the mean ± standard deviation (SD). (A) Schematic diagram of immunization. (B) changes of total IgG during the immunization. (C) IgG titers at 4, 5, and 6 weeks post initial immunization. (D-F) RBD-specific IgG (D), IgG1 (E), and IgG2a (F) at six weeks post initial immunization. (G) Splenic lymphocyte subtypes.

Figure 3

Optimization of immunization strategy. (A) Adjuvant. (B, C) Immune interval duration. (D) RBD-specific IgG. (E) Proportion of positive serum of immunized mice in Figure3D (positive rate). Differences between vaccine groups are indicated by black asterisks. Differences between vaccine group and PBS are indicated by color asterisks.

Figure 4

Evaluation of the immune effect of the VLP in humanized mice. Humanized mice were primed with PBS or SARS-CoV-2 VLP and boosted three weeks after the prime immunization using the same inocula used for priming. Blood samples were collected from the tail vein of mice at indicated times after the initial vaccination and used to analyze humoral immune responses using an antibody detection kit. Sera collected from the tail vein of pre-immune mice were used as a negative control. *, P< 0.05; **, P< 0.01; ****, P< 0.0001. The results are expressed as the mean ± standard deviation (SD). (A) Schematic diagram of immunization. (B) RBD-specific IgG. (C) IgG titer. (D) Neutralizing antibody. (E) Bodyweight. (F) Virus loads detected by qRT-PCR.

Figure 5

The mSM-based vaccine is effective against virus challenges. Mice were primed with PBS or SARS-CoV-2 VLP and boosted three weeks after the prime immunization, followed by virus challenge at 31 days post-prime immunization. Blood samples were collected from the tail vein of mice at indicated times after the initial vaccination and used to analyze humoral immune responses using an antibody detection kit. Sera collected from the tail vein of pre-immune mice were used as a negative control. *, P< 0.05; **, P< 0.01; ***, P< 0.001; ****, P< 0.0001. The results are expressed as the mean ± standard deviation (SD). (A) Schematic diagram of immunization and infection. (B) RBD-specific IgG. (C) Bodyweight. (D) Animal survival. (E, F) Virus loads detected by qRT-PCR.