A chimeric mRNA vaccine of S-RBD with HA conferring broad protection against influenza and COVID-19 variants

Abstract

Influenza and coronavirus disease 2019 (COVID-19) represent two respiratory diseases that have significantly impacted global health, resulting in substantial disease burden and mortality. An optimal solution would be a combined vaccine capable of addressing both diseases, thereby obviating the need for multiple vaccinations. Previously, we conceived a chimeric protein subunit vaccine targeting both influenza virus and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), utilizing the receptor binding domain of spike protein (S-RBD) and the stalk region of hemagglutinin protein (HA-stalk) components. By integrating the S-RBD from the SARS-CoV-2 Delta variant with the headless hemagglutinin (HA) from H1N1 influenza virus, we constructed stable trimeric structures that remain accessible to neutralizing antibodies. This vaccine has demonstrated its potential by conferring protection against a spectrum of strains in mouse models. In this study, we designed an mRNA vaccine candidate encoding the chimeric antigen. The resultant humoral and cellular immune responses were meticulously evaluated in mouse models. Furthermore, the protective efficacy of the vaccine was rigorously examined through challenges with either homologous or heterologous influenza viruses or SARS-CoV-2 strains. Our findings reveal that the mRNA vaccine exhibited robust immunogenicity, engendering high and sustained levels of neutralizing antibodies accompanied by robust and persistent cellular immunity. Notably, this vaccine effectively afforded complete protection to mice against H1N1 or heterosubtypic H5N8 subtypes, as well as the SARS-CoV-2 Delta and Omicron BA.2 variants. Additionally, our mRNA vaccine design can be easily adapted from Delta RBD to Omicron RBD antigens, providing protection against emerging variants. The development of two-in-one vaccine targeting both influenza and COVID-19, incorporating the mRNA platform, may provide a versatile approach to combating future pandemics.

Fig 1. H1Delta mRNA vaccine design and immunogenicity in mice.

(A) Schematic of the H1Delta mRNA vaccine design. The mRNA transcript includes a 5’ UTR, the H1Delta coding sequence [13], a 3’ UTR, and a poly-A tail, all synthesized from linear DNA templates. SP, signal peptide. For detailed descriptions of the mutation sites, please see the Materials and Methods section. (B-G) Groups of 6- to 8-week-old female BALB/c mice (n = 5) were vaccinated with two doses of H1Delta mRNA vaccine containing either 2 or 10 μg of immunogens at 3-week intervals. Serum samples were collected 19 and 35 days after the initial immunization for ELISA and neutralization assays. (B-D) H1 HA (B), H5 HA (C) and SARS-CoV-2 Delta RBD (D) specific IgG titers are determined by ELISA. (E-G) Neutralization assay results for sera from mice immunized with H1Delta mRNA. 50% neutralization titers of pseudovirus against Delta (E), BA.2 (F), and BA.4/5 (G) are shown. Limit of detections (LODs) were indicated by dashed lines.

Fig 2. H1Delta mRNA vaccine elicits robust cellular immune responses in mice.

Female BALB/c mice (n = 3) were vaccinated with 2 or 10 μg doses of H1Delta mRNA-LNP or empty LNP. Spleens were collected 120 days after the initial immunization. An ELISpot assay was performed to evaluate the capacity of splenocytes to secrete IFN-γ (A) and IL-4 (B) following re-stimulation with H1 stalk peptide pools. A flow cytometry with ICS assay was conducted to quantify the proportions of s CD8⁺ (C) and CD4⁺ (D) T cells secreting IFN-γ⁺, TNF-α⁺, IL-2⁺, IL-4⁺ or IL-5⁺ following re-stimulation with H1 stalk peptide pools. Another portion of the splenocytes was stimulated with SARS-CoV-2 RBD peptide pool to detect cytokine production. This was measured using ELISpot shown in (E) and (F), and ICS shown in (G) and (H).

Fig 3. Protection efficacy of H1Delta mRNA vaccine against homologous H1N1 and heterologous H5N8.

(A-D) Groups of 6- to 8-week-old female BALB/c mice (n = 8) were vaccinated with two doses of the H1Delta mRNA vaccine, containing either 2 or 10 μg immunogen, at 3-week intervals (A). Serum samples were collected 19 and 35 days after initial immunization. Mice were intranasally challenged with 20 × mLD₅₀ of A/Brisbane/02/2018 (H1N1) virus. Vaccine efficacy was assessed by measuring morbidity (weight loss) (B), mortality (survival) (C) in five mice. Lung tissues from the other 3 mice were harvested and split to test lung viral titers on 3 DPI (D), and histological pathology analyses (E). (F-J) Groups of 6- to 8-week-old female BALB/c mice (n = 8) were vaccinated with two doses of the H1Delta mRNA vaccine, containing either 2 or 10 μg immunogen, at 3-week intervals (F). Serum samples were collected 19 and 35 days after initial immunization. Mice were intranasally challenged with 10 × mLD₅₀ of reassortment A/Astrakhan/3212/2020(H5N8) virus. Vaccine efficacy was assessed by measuring morbidity (weight loss) (G) and mortality (survival) (H) in five mice. Lung tissues from the other 3 mice were harvested and split to test lung viral titers on 3 DPI (I), and histological pathology analyses (J). Representative lung sections were stained with H&E. Scale bars are labelled.

Fig 4. Protection efficacy of H1Delta mRNA vaccine against SARS-CoV-2.

(A-C) Groups of 6- to 8-week-old female BALB/c mice (n = 10) were vaccinated with two doses of the H1Delta mRNA vaccine, containing 10 μg immunogen or sham (control), at 3-week intervals. Five mice were randomly selected from each group and challenged with 5 × 10⁵ TCID₅₀ of Delta SARS-CoV-2 variant, and (D–F) the other five were challenged with 5 × 10⁵ TCID₅₀ of Omicron (BA.2) variant 140 days after the initial immunization. Mice challenged with Delta variant had previously received Ad5-hACE2 intranasally 5 days before the challenge. Mice were euthanized and necropsied on day 3 post-infection, with lung tissues collected for viral titration and pathological analysis. (A) Pulmonary Delta viral gRNA levels were detected by qRT-PCR. (B) Pulmonary Delta viral sgRNA levels were detected by qRT-PCR. (C) Plots show correlations and corresponding two-sided p values between pVNT₅₀ of Delta variant and Delta viral gRNA. (D) Pulmonary Omicron viral gRNA levels were detected by qRT-PCR. (E) Pulmonary Omicron viral sgRNA levels were detected by qRT-PCR. (F) Plots show correlations and corresponding two-sided p values between pVNT₅₀ of Omicron variant and Omicron viral gRNA. LODs were indicated by dashed lines. (G) Histopathological analyses of lung sections from mice challenged with Delta or Omicron. Shown are the representative lung sections by H&E staining. Scale bars are labelled.

Fig 5. Long-lasting protection of H1Delta mRNA vaccine.

(A) Time course of H1Delta vaccine immune antibody monitoring, viral challenge and measurement in mice. Groups of 6- to 8-week-old female BALB/c mice (n = 8) were vaccinated with two doses of the H1Delta mRNA vaccine, containing either 2 or 10 μg immunogen, at 3-week intervals. Specific IgG titers against H1 HA (B) and SARS-CoV-2 Delta RBD (C) are determined by ELISA at the indicated time points. Mice were intranasally challenged with 20 × mLD₅₀ of A/Brisbane/02/2018 (H1N1) virus. Vaccine efficacy was assessed by measuring morbidity (weight loss) (D) and mortality (survival) (E) in five mice over a 14-day period following virus infection. Lung tissues from the other three mice were harvested on 3 DPI and split to test lung viral titers (F) and for histological pathology analyses (G). Virus titers in the lungs were detected, with limits of detection (LODs) indicated by dashed lines. Representative lung sections are shown with H&E staining. Scale bars are labelled.

Fig 6. Development of H1BA.2 mRNA vaccine.

(A) Schematic representation of the H1BA.2 vaccine design. SP, signal peptide. Groups of 6- to 8-week-old female BALB/c mice (n = 5) were vaccinated with two doses of H1BA.2 mRNA vaccine containing either 2 or 10 μg of immunogens at 3-week intervals. Serum samples were collected 19 and 35 days after the first immunization for ELISA and neutralization assays. (B) ELISA shows the H1 specific IgG titers. Mice were intranasally challenged with 20 × mLD₅₀ of A/Brisbane/02/2018 (H1N1) virus at 63 days after the first immunization. Vaccine efficacy was assessed by measuring morbidity (weight loss) (C) and mortality (survival) (D) in five mice during the 14-day period. Lung tissues of the other three mice were harvested and split to test lung viral titers on 3 DPI (E). (F) ELISA shows the H5 specific IgG titers. Mice were intranasally challenged with 10 × mLD₅₀ of reassortment A/Astrakhan/3212/2020(H5N8) virus at 78 days after the first immunization. Vaccine efficacy was assessed by measuring morbidity (weight loss) and (G) mortality (survival) (H) in five mice. Lung tissues of the other three mice were harvested and split to test lung viral titers on 3 DPI (I). (J) ELISA depicting the SARS-CoV-2 Delta RBD specific IgG titers. (K) Determination of 50% neutralization titer for pseudotyped virus (Delta, BA.2, and BA.4/5) in serum. Five mice were randomly selected from each group and challenged with 5 × 10⁵ TCID₅₀ of Delta SARS-CoV-2 variant virus, and the other five were challenged with 5 × 10⁵ TCID₅₀ of Omicron (BA.2) variant virus140 days after the initial immunization. Mice challenged with Delta variant had received Ad5-hACE2 intranasally 5 days before. All tests were done after 3 days post either Delta or Omicron variant infection. (L) Quantification of pulmonary Delta viral gRNA levels via qRT-PCR. (M) Quantification of pulmonary Delta viral sgRNA levels via qRT-PCR. (N) Plots show correlations and corresponding two-sided p values between pVNT₅₀ of Delta variant and Delta viral gRNA. (O) Quantification of pulmonary Omicron viral gRNA levels via qRT-PCR. (P) Quantification of pulmonary Omicron viral sgRNA levels via qRT-PCR. (Q) Plots show correlations and corresponding two-sided p values between pVNT₅₀ of Omicron variant and Omicron viral gRNA. (R) Histological pathology analyses of lung sections of mice challenged with Delta or Omicron. Shown are the representative lung sections by H&E staining. Scale bars are labelled.