Chimeric hemagglutinin and M2 mRNA vaccine for broad influenza subtype protection

Abstract

Since multiple and unpredicted influenza viruses cause seasonal epidemics and even high-risk pandemics, developing a universal influenza vaccine is essential to provide broad protection against various influenza subtypes. Combined with the mRNA lipid nanoparticle-encapsulated (mRNA-LNP) vaccine platform and chimeric immunogen strategy, we developed a novel cocktail mRNA vaccine encoding chimeric HAs (cH5/1-BV, cH7/3) and intact M2 (termed Fluaxe), which confers broad protection against major circulating IAVs and IBVs, as well as highly pathogenic avian influenza. Two-dose intramuscular immunization of Fluaxe in mice elicited cross-reactive neutralizing antibodies, T cell responses, and long-lived immunity, resulting in robust protection against multiple lethal influenza virus infections and severe acute lung injuries. In particular, intramuscular administration stimulated systemic immunity together with a prominent lung tropism of memory cells. Moreover, Fluaxe immunization inhibited the inflammatory response induced by influenza infection. In summary, we conclude that Fluaxe can elicit broad cross-protection against numerous influenza subtypes.

Fig. 1. Immunogen design of Fluaxe.

A mRNA constructs of Fluaxe expressing the two chimeric HA and M2. Created in BioRender. Li (2025) https://BioRender.com/bithl81. B Monomeric structures of HA for four subtypes of IAV (H1N1, H5N1, H3N2, and H7N9) and IBV/Victoria. The disulfide bonds presented at the junction between the head and stem domains in all four IAV HA proteins are highlighted. For IBV/Victoria HA A56 and G302 residues are situated at the two equivalent structural positions instead of cysteines. C The predicted three-dimensional structure of Fluaxe chimeric sequences. The disulfide bond connecting the original head and stem fragments persists in the chimera. All structural figures were prepared using the PyMOL Molecular Graphics System, Version 3.0 (Schrödinger, LLC).

Fig. 2. Fluaxe-vaccination elicited broad humoral immunity.

A A schematic diagram of mice immunization and prime-boost vaccination was executed on Days 0 and 14. Female BALB/c mice were injected via i.m. with Fluaxe (5 μg for each mRNA) or empty LNP per mouse (n = 5) and boosted with an equivalent dose. Sera were collected on Days 7, 14, 21, and 28, respectively, for antibody analysis. Lungs and spleens were harvested on Day 28 to analyze cellular immunity. Splenocytes were collected on Day 104 for memory T or B cell detection. Created in BioRender. Li (2025) https://BioRender.com/bithl81. B Specific IgG antibodies against H1N1, H3N2, H5N1, H7N9, IBV/Victoria, and IBV/Yamagata were measured by ELISA. Data were shown as Mean ± SEM. Significance was determined by two-way ANOVA with Bonferroni’s test for multiple comparisons (**p < 0.01, ***p < 0.001). Neutralizing antibodies of sera collected on Day 28 against H1N1, H3N2, IBV/Victoria, and IBV/Yamagata influenza viruses were detected by C MN and D HAI assays, respectively. E Neutralizing responses against H5- and H7-containing viruses were measured by HAI assay. Each symbol represents one mouse, and sera from five mice were assessed. Multiple t-tests with correction for multiple comparisons by the Holm-Šídák method were used to calculate p values relative to the control LNP group (n.s. not significant; *p < 0.05, **p < 0.01, ***p < 0.001). F Pseudotype neutralizing assays were performed using influenza pseudovirus bearing multiple group 1 and group 2 HAs (HA1-16). Detection of 50% pseudovirus neutralization titer (PNT₅₀) of sera from immunized mice. The sera from five mice per group were pooled and tested in triplicate. Significance was calculated using two-tailed unpaired t-tests.

Fig. 3. Fluaxe vaccination elicited significant cellular immunity.

Lungs, spleens, and BALFs of vaccinated mice were harvested and homogenized on Day 28 post-immunization. A CD3⁺ T cell ratio and B CD8⁺/CD4⁺ within CD3⁺ population were determined, respectively. Lymphocytes were stimulated with a peptide cocktail containing cH5/1-BV, cH7/3, and M2 peptide pools. CD4⁺ or CD8⁺ cells from C splenocytes or D lungs were stained for intracellular IFN-γ, IL-2, and TNF-α followed by FACS analysis. Percentages of cytokines⁺ cells within CD8⁺ and CD4⁺ T cell populations were shown. E Ratios of CD8⁺ /CD4⁺ T cells within the CD3⁺cytokines⁺ population were exhibited. The red dotted line represents a ratio of 1. Each dot or square in the bar graphs represents one individual (n = 5). Error bars indicate SEM. p values were determined using multiple t-tests with correction for multiple comparisons by the Holm-Šídák’s method (n.s. not significant; *p < 0.05, **p < 0.01, ***p < 0.001).

Fig. 4. Fluaxe immunization induced significant memory T and B cells.

Spleens of prime-boost vaccinated mice were collected and homogenized on Day 104, lymphocytes were stimulated with a peptide cocktail containing cH5/1-BV, cH7/3, and M2 peptide pools. Memory cells were stained for intracellular IFN-γ. A Frequency of antigen-specific CD4⁺ and CD8⁺ Tcm (CD44⁺CD62L⁺IFN-γ⁺) in splenocytes. B Frequency of antigen-specific CD4⁺ and CD8⁺ Tem (CD44⁺CD62L⁻IFN-γ⁺) in splenocytes. C Frequency of antigen-specific CD4⁺ and CD8⁺ Trm (CD44⁺CD62L⁻CD69⁺CD103⁺IFN-γ⁺) in splenocytes. D Frequency of antigen-specific CD4⁺ and CD8⁺ CXCR3⁺Tm (CD44⁺CD62L⁻CD69⁻CXCR3⁺IFN-γ⁺) in splenocytes. E Frequency of H1N1 HA, H3N2 HA, H5N1 HA, H7N9 HA, IBV/Victoria HA, and IBV/Yamagata HA-specific MBC in splenocytes. Five mice per group, data are presented as Mean ± SEM. p values were determined using multiple t-tests followed by Holm-Šídák’s multiple comparisons.

Fig. 5. Fluaxe vaccination protected mice from multiple influenza virus infections.

A Schematic diagram of mice immunization and influenza virus challenge. Created in BioRender. Li (2025) https://BioRender.com/bithl81. Prime-boost vaccination was executed on Days 0 and 14. Four influenza subtypes (10LD₅₀) were used to challenge mice via intratracheal administration. Survival rate, weight loss, viral RNA in lung, and pathologic level were measured during 14 days post infection. The survival rate and weight loss of Fluaxe vaccinated mice were monitored post-challenge with B A/PR/8/1934 (H1N1) virus, D A/Aichi/2/1968 (H3N2) virus, F B/Brisbane/60/2008 (IBV/Victoria) virus, and H B/Phuket/3073/2013 (IBV/Yamagata) virus, respectively. Weight loss data are exhibited as Mean ± SD. The dotted lines in weight loss figures indicate the maximum body weight loss (25%) for the experiment. Kaplan–Meier survival curves of mice are shown, and significance testing was done by log-rank test (**p < 0.01). Viral RNA (vRNA) load in lungs of Fluaxe vaccinated mice were monitored post-challenge with C A/PR/8/1934 (H1N1) virus, E A/Aichi/2/1968 (H3N2) virus, G B/Brisbane/60/2008 (IBV/Victoria) virus, and I B/Phuket/3073/2013 (IBV/Yamagata) virus on Day 5 post-infection. Data of viral RNA are shown as Mean ± SEM (n = 5 per group) and significance was calculated using two-tailed unpaired t-tests (***p < 0.001), data are normalized to the LNP group, with the control value arbitrarily set to 1.

Fig. 6. Fluaxe immunization protected lung injury induced by influenza virus infection.

A Images of lungs dissected from mice (Fluaxe and LNP groups) challenged with A/PR/8/1934 (H1N1) on 7 dpi. B H&E staining of PR8-infected lung tissue pathology. Tissue lesions were indicated by narrows as the following: inflammatory cell infiltration predominantly composed of granulocytes (black arrow); congestion of capillaries and venous vessels (yellow arrow); epithelial exfoliation (brown arrow); perivascular edema (purple arrow); loose connective tissue accompanied by mild hemorrhage (green arrow); lymphocyte infiltration (red arrow). Representative images from 5 mice are shown. Scale bar = 100 μm. C Representative images of immunofluorescence staining of (upper panel, green) gap junction protein E-cadherin and (lower panel, green) tight junction protein ZO-1 expressed in lung tissues from 5 mice challenged with PR8. DNA was stained by DAPI in blue. Scale bars = 100 μm. Inflammation-associated cells in lungs were detected on Day 5 post-PR8 infection by FACS. D Frequencies of alveolar macrophages (CD64⁺, CD11c⁺, Siglec F⁺) and interstitial peribronchial macrophages (CD11b⁺, CD11c⁺, CD64⁺, Siglec F⁻, Ly6C⁻) in lungs after mock (PBS) or PR8 infection. Inflammatory macrophages were stained for intracellular NLRP3. E Frequencies classical monocytes (CD11b⁺, Ly6C⁺, CD64⁻) of monocytes-derived cells (CD11b⁺, Ly6C⁺, CD64⁺) and in lungs after mock or PR8 infection. Inflammatory monocytes were stained for intracellular NLRP3. n = 5, data were presented as Mean ± SEM. Statistics were calculated using multiple t-tests with Holm-Šídák’s multiple comparisons. F Influenza virus-induced cytokine expression in the BALFs. BALF cytokine concentrations were determined by a 23-plex assay kit. n = 3. Mean values were shown. p values were calculated by comparing the LNP group and Fluaxe-vaccinated group animals that all were infected with PR8. Significance was calculated using two-tailed unpaired t-tests.