Influenza Virus-like Particle-Based Hybrid Vaccine Containing RBD Induces Immunity against Influenza and SARS-CoV-2 Viruses

Abstract

Several approaches have produced an effective vaccine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Since millions of people are exposed to influenza virus and SARS-CoV-2, it is of great interest to develop a two-in-one vaccine that will be able to protect against infection of both viruses. We have developed a hybrid vaccine for SARS-CoV-2 and influenza viruses using influenza virus-like particles (VLP) incorporated by protein transfer with glycosylphosphatidylinositol (GPI)-anchored SARS-CoV-2 RBD fused to GM-CSF as an adjuvant. GPI-RBD-GM-CSF fusion protein was expressed in CHO-S cells, purified and incorporated onto influenza VLPs to develop the hybrid vaccine. Our results show that the hybrid vaccine induced a strong antibody response and protected mice from both influenza virus and mouse-adapted SARS-CoV-2 challenges, with vaccinated mice having significantly lower lung viral titers compared to naive mice. These results suggest that a hybrid vaccine strategy is a promising approach for developing multivalent vaccines to prevent influenza A and SARS-CoV-2 infections.

Keywords: GM-CSF; IL-12; RBD; SARS-CoV-2; antibodies; influenza; mice; virus-like particles.

Figure 1

Design, expression and characterization of GPI-RBD-GM-CSF fusion protein. (A) Design of GPI-RBD-GM-CSF fusion protein gene, (B) GPI-RBD-GM-CSF fusion protein binds both anti-RBD mAb and anti-GM-CSF mAb on CHO-S cell transfectants, (C) PIPLC treatment of CHO-S cells expressing GPI-RBD-GM-CSF reduced the level of expression and (D) flow cytometry analysis showed binding of human ACE2 to GPI-RBD-GM-CSF fusion protein expressed in CHO-S cells.

Figure 2

Purified GPI-RBD-GM-CSF fusion protein retains functional activity. (A) Colloidal blue (lane 1) and Western blot (lanes 2–7) of the immunoaffinity column purified fusion protein from CHO-S cells probed with anti-RBD antibody (lanes 3 and 4) or anti-GM-CSF mAb (lanes 6 and 7). Lane 2 is control RBD probed with anti-RBD Ab, and lane 5 is control GM-CSF probed with anti-GM-CSF antibody. (B) ELISA for GPI-RBD-GM-CSF binding to antibodies in the human COVID-19 patients’ sera, and (C) ELISA of purified GPI-RBD-GM-CSF binding to ACE2 and RBD specific MM57 mAb. (D) FACS analysis of VLPs incorporated with GPI-IL-12 and GPI-RBD-GM-CSF fusion protein by protein transfer.

Figure 3

Hybrid vaccine induces antibody response against RBD in mice. ELISA plates were coated with GPI-RBD-GM-CSF, and serum samples from various groups of mice (n = 5) immunized with GPI-RBD-GM-CSF, VLP vaccine containing the GPI-RBD-GM-CSF or hybrid vaccine (VLP vaccine containing the GPI-RBD-GM-CSF + GPI-IL-12) were diluted and added to the wells after blocking the plates. (A) Total IgG levels 6 weeks after booster dose, and (B) IgG isotype in the sera 10 weeks after the booster dose. Anti-mouse IgG (A) or isotype-specific anti-mouse IgG-HRP conjugate (B) was used to detect the bound antibody.

Figure 4

Hybrid vaccine induces influenza A/PR8 virus-specific antibody response. (A–C) ELISA plates were coated with inactivated influenza A/PR8 H1N1. Serum samples from mice vaccinated with VLP, VLP with GPI-GM-CSF and GPI-IL-12 or hybrid vaccine were serially diluted (5-fold) and added to the wells after blocking the plates. (D) Hemagglutination inhibition (HAI) titer in the sera of mice. Isotype specific anti-mouse Ig-HRP conjugate was used to detect the isotype of the antibody bound. * p < 0.05.

Figure 5

Hybrid vaccine protects against SARS-CoV-2 challenge. BALB/c mice (n = 10; 2–3 months old) were administered with a hybrid vaccine, a hybrid vaccine without IL-12 or VLP in 50 ul volume via an intramuscular route. Control mice received either PBS or influenza VLP. A booster dose was given on d33 and blood was collected 2 weeks later (week 7). Mice were challenged with mouse-adapted SARS-CoV2 (n = 5) 16–18 weeks after the first dose. (A) Neutralizing antibody titers in the serum of BALB/c mice (n = 5–6 per group). Serum collected from BALB/c mice 2 weeks after the booster dose was serially diluted from 1:4 to 1:1024, and PRNT was conducted against SARS-CoV-2 (Wuhan virus). (B) BALB/c mice were inoculated intranasally with mouse-adapted SARS-CoV-2 (10⁵ plaque-forming units) 3 months after the booster dose. Percentage of daily body weight change in the animals. (C) The RNA levels of SARS-CoV-2 were determined in the lungs by qRT-PCR (n = 4–5 per group). Error bars represent SEM. The data are expressed as genome copies/μg of RNA. Each data point represents an individual mouse. Data are expressed as mean log₁₀ titer. For body weight changes, a two-way analysis of variance (ANOVA) with the post hoc Bonferroni test was used to calculate values of p. Mann–Whitney test was used to calculate the p values of the difference between viral titers. Differences of p < 0.05 were considered significant. * p < 0.05; ** p < 0.01 *** p < 0.001. Hybrid vaccine: VLP incorporated with GPI-RBD-GM-CSF and GPI-IL-12.

Figure 6

Hybrid vaccine protects against influenza A virus challenge. BALB/c mice were administered with a hybrid vaccine (10 μg/dose) or a hybrid vaccine without GPI-IL-12, as described in Figure 5. A booster dose was given after 33 days of the first dose. (A) HAI titer in the blood 2 weeks after the booster dose (day 48). Lung viral titer (B) and survival (C) of mice challenged with influenza A/PR8 H1N1 virus 3 months after the booster dose. The inoculation dose was 10 times LD₅₀. (C) All three groups (VLP, hybrid vaccine and hybrid vaccine without IL-12) survived from PR8 challenge; therefore, the lines are superimposed, and only the red symbol is visible. Hybrid vaccine: VLP incorporated with GPI-RBD-GM-CSF and GPI-IL-12. Statistical significance was calculated by one-way ANOVA and Dunnett’s post-multiple comparison tests. Error bars indicate the mean ± standard errors of the mean (SEM). ***; p < 0.001, ns; not significant.