Rational development of a combined mRNA vaccine against COVID-19 and influenza

Abstract

As the world continues to experience the COVID-19 pandemic, seasonal influenza remain a cause of severe morbidity and mortality globally. Worse yet, coinfection with SARS-CoV-2 and influenza A virus (IAV) leads to more severe clinical outcomes. The development of a combined vaccine against both COVID-19 and influenza is thus of high priority. Based on our established lipid nanoparticle (LNP)-encapsulated mRNA vaccine platform, we developed and characterized a novel mRNA vaccine encoding the HA antigen of influenza A (H1N1) virus, termed ARIAV. Then, ARIAV was combined with our COVID-19 mRNA vaccine ARCoV, which encodes the receptor-binding domain (RBD) of the SARS-CoV-2 S protein, to formulate the final combined vaccine, AR-CoV/IAV. Further characterization demonstrated that immunization with two doses of AR-CoV/IAV elicited robust protective antibodies as well as antigen-specific cellular immune responses against SARS-CoV-2 and IAV. More importantly, AR-CoV/IAV immunization protected mice from coinfection with IAV and the SARS-CoV-2 Alpha and Delta variants. Our results highlight the potential of the LNP-mRNA vaccine platform in preventing COVID-19 and influenza, as well as other respiratory diseases.

Fig. 1. Design and characterization of ARIAV mRNA-LNP encoding HA protein of influenza A (H1N1) virus as a vaccine candidate.

a Schematic diagram of ARIAV, encoding the full-length HA protein. b Indirect immunofluorescence assay of HA protein expression in HEK293T cells 48 h post-transfection. Scale bar, 20 μm. c HA expression in HEK293T cells was determined by immunoblotting. d HA-specific IgG antibody titers were determined by ELISA. e Hemagglutination inhibition (HAI) titers were determined 14 and 28 days post-initial immunization. Data are shown as the mean ± SEM (n = 8). Statistical differences were analyzed by using two-tailed unpaired t tests. *P < 0.05,**P < 0.01, ***P < 0.001.

Fig. 2. AR-CoV/IAV immunization elicits a humoral immune response in BALB/c mice.

a Schematic diagram of the AR-CoV/IAV formulation as a combination of ARCoV and ARIAV. b, c Representative intensity-size graphs of ARCoV and ARIAV. d HA and RBD protein expression in HEK293T cells was determined by immunoblotting. e Schematic diagram of the experimental design. Groups of mice were immunized with 30 µg of AR-CoV/IAV and boosted with the same dose after two weeks. Serum samples were collected 14 and 28 days post-initial immunization and subjected to antibody detection before an IAV or SARS-CoV-2 challenge. f, h IAV-HA- and SARS-CoV-2-RBD-specific IgG antibody titers were determined by ELISA. g HAI titers were determined 14 and 28 days post-initial immunization. i NT₅₀ titers against SARS-CoV-2 were determined by using VSV-based pseudovirus. Data are shown as the mean ± SEM (n = 8). Statistical differences were analyzed by using two-tailed unpaired t tests. ***P < 0.001.

Fig. 3. AR-CoV/IAV immunization elicits an antigen-specific T cell immune response in BALB/c mice.

a, b HA-specific CD4⁺ and CD8⁺ T cells producing IFN-γ, TNF-α and IL-2 were determined by flow cytometry. c, d RBD-specific CD4⁺ and CD8⁺ T cells producing IFN-γ, TNF-α and IL-2 were determined by flow cytometry. Data are shown as the mean ± SEM (n = 6). Statistical differences were analyzed by using unpaired t tests. **P < 0.01.

Fig. 4. AR-CoV/IAV protects mice from an IAV or SARS-CoV-2 challenge.

AR-CoV/IAV- or placebo-immunized mice were challenged with 1.5 × 10⁶ PFU of IAV (A/California/07/2009) (a–e) or 1.6 × 10⁴ PFU of SARS-CoV-2 (MASCp6) (f–h) at the indicated time points post-initial immunization. a Weight changes were recorded for 15 days and are shown as the mean ± SEM (AR-CoV/IAV, n = 7; placebo, n = 8). b Mortality was monitored for 21 days after inoculation. c Viral RNA copies in the lung tissues of IAV-infected mice (AR-CoV/IAV, n = 5; placebo, n = 4) were determined by qRT–PCR and are shown as the mean ± SEM. d Immunostaining results for HA protein in lung tissues. e H&E staining of lung tissues from IAV-infected mice. f Viral RNA copies in the lung tissues of SARS-CoV-2-infected mice (AR-CoV/IAV, n = 5; placebo, n = 6) were determined by qRT–PCR and are shown as the mean ± SEM. g ISH assay of lung tissues from SARS-CoV-2-infected mice. h H&E staining of lung tissues from SARS-CoV-2-infected mice. Scale bar, 100 μm. Statistical differences between groups were analyzed using two-tailed unpaired t tests. *P < 0.05, **P < 0.01.

Fig. 5. AR-CoV/IAV confers protection against coinfection with IAV and the SARS-CoV-2 Alpha variant in mice.

a Schematic diagram of experimental design. Groups of AR-CoV/IAV- or placebo-immunized mice were intranasally challenged with 2 × 10⁵ PFU of IAV (A/California/07/2009) 73 days after the initial immunization, followed by infection with 4 × 10³ PFU of the SARS-CoV-2 Alpha variant 2 days later. Mice were sacrificed 4 days after IAV infection for viral detection and histopathological analysis. b Weight changes in infected mice (n = 5) were recorded for 4 days post-infection and are shown as the mean ± SEM. c, d Viral RNA copies of IAV (c) and SARS-CoV-2 (d) in the lung tissues of infected mice (n = 5) were determined by qRT–PCR and are shown as the mean ± SEM. e Histopathologic analysis of lung sections from coinfected mice. Scale bar, 100 μm. f Serum cytokine and chemokine analyses were determined by Luminex and are presented as fold changes compared to samples collected before infection (AR-CoV/IAV, n = 5; placebo, n = 3). g–j Concentrations of cytokines and chemokines in serum samples collected post-infection. Data are shown as the mean ± SEM. Statistical differences were analyzed by using two-way ANOVA with multiple comparison tests or two-tailed unpaired t tests. *P < 0.05, **P < 0.01, ****P < 0.0001.

Fig. 6. AR-CoV/IAV elicits protection against coinfection with IAV and the SARS-CoV-2 Delta variant in mice.

a Schematic diagram of experimental design. Groups of AR-CoV/IAV- or placebo-immunized mice were intranasally challenged with 2 × 10⁵ PFU of IAV (A/California/07/2009) 73 days after the initial immunization, followed by infection with 1.2 × 10⁴ PFU of SARS-CoV-2 Delta variant 2 days later. Mice were sacrificed 4 days post-IAV infection for viral detection and histopathological analysis. b Weight changes of infected mice (n = 5) were recorded for 4 days post-infection and are shown as the mean ± SEM. c, d Viral RNA copies of IAV (c) and SARS-CoV-2 (d) in the lung tissues of infected mice (AR-CoV/IAV, n = 4; placebo, n = 5) were determined by qRT–PCR and are shown as the mean ± SEM. e Histopathologic analysis of lung sections from coinfected mice. Scale bar, 100 μm. Data are shown as the mean ± SEM. Statistical differences were analyzed by using two-way ANOVA with multiple comparison tests or two-tailed unpaired t tests. *P < 0.05, ****P < 0.0001.