Female C57BL/6 mice, aged 6 weeks, were procured from BEIJING HFK BIOSCIENCE Co., Ltd. The HEK-293T cells and PGEM-3Zf-n3 vector were maintained in our laboratory. HEK-293T cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS, Gibco) and 100 units/mL penicillin-streptomycin. Our laboratory possessed the following virus strains: A/Puerto/R/8/34(H1N1), A/Jilin/JYT-01/2018(H1N1), B/Massachusetts/2/2012(Yamagata), and B/Jilin/02/2022(Victoria). All experiments involving live viruses were conducted in biosafety level 2 facilities.

The HA2 domain, which contains epitopes targeted by broadly neutralizing antibodies, holds promise as a potential candidate for the development of vaccines with broad protective efficacy. In this study, we focused on screening HA2 epitopes that are validated and relatively conserved B-cell dominant epitopes (23). Additionally, M2e also is considered an important target for designing influenza vaccines due to its highly conserved nature across multiple strains of the virus. To obtain mRNA vaccine for subsequent in vivo utilization, the peptide sequences of M2e, HA2, and other relevant sequences were documented in Supplementary Table 1 and Figure 1A , along with their respective gene order. The aforementioned genes were subsequently cloned into the pGEM-3Zf-n3 vector utilizing Cla I and Pac I restriction enzymes (24). The resulting plasmids were subsequently linearized by Xho I cleavage in preparation for their use as DNA templates in an in vitro transcription reaction. The T7-FlashScribe Transcription Kit (CELLSCRIPT, USA) was used to generate mRNA from these DNA templates. To enhance in vivo stability, resistance to degradation, and translational efficiency, 1-Methylpseudouridine-5′-triphosphate (TriLink, USA) was employed as a substitute for uridine triphosphate (UTP). Subsequently, the mRNA molecules were purified using the MEGAclear Transcription Clean-Up Kit (Thermo Fisher Scientific, USA) and capped utilizing the ScriptCap Cap 1 Capping System (CELLSCRIPT, USA).

After seeding HEK293T cells in 12-well plates and allowing them to grow overnight until they reached approximately 80-90% confluence, the cells were transfected with mRNA using the Xfect RNA Transfection Reagent protocol (TAKARA, Japan). Cell lysates were collected at 24 hours post-transfection using RIPA lysis buffer for protein expression confirmation through western blotting with Influenza A Virus Hemagglutinin/HA Antibody, Rabbit Mab (Sino Biological, China), while mock-transfected cells served as controls. Indirect immunofluorescence technology was used to assess corresponding protein expression levels. The structure of MH-TF protein was observed by HT7800 transmission electron microscope.

A total of 36 female C57BL/6 mice, aged 6 weeks, were randomly divided into three groups for vaccine immunization and a negative control group, with each group consisting of 9 mice. Intramuscular injections were administered every two weeks for a total of three doses. Serum samples were collected at 7, 14, 21, 28, 35 and 42 days after the primary immunization and stored at −20°C for further analysis. On the 42^nd day following the primary immunization, mice were challenged with influenza virus (10×LD₅₀). Subsequently, body weight and survival of the mice were monitored daily for two weeks post-virus challenge.

The plates were coated with recombinant protein (5 μg/mL, 100 μL/well) at 4°C, followed by blocking with 3% bovine serum albumin for 2 hours. Subsequently, sera samples (a dilution of 1:100) were added and incubated at 37°C for 1.5 hours. Afterwards, the plates were washed and incubated with Goat Anti-Mouse IgG Human ads-HRP (SouthernBiotech, USA). The signal was developed using TMB as the substrate, and the reaction was terminated by adding 2M H₂SO₄. Finally, the optical density at 450 nm (OD₄₅₀) was measured using a microtiter plate reader.

Hemagglutination inhibition (HI) assay was measured as described earlier (25, 26). The immune serum samples were pretreated with receptor destroying enzyme II and diluted twofold before detecting HI in 96-well plates. Each well was incubated with 4 HA units of virus at room temperature for 30 min, followed by mixing with 1% chicken red blood cells and further incubation for another 30 min. The HI titer is defined as the reciprocal of the maximum serum dilution that inhibits erythrocyte viral hemagglutination.

The spleens of mice were harvested on the 42^nd day after the first vaccination using aseptic techniques. Single-cell suspensions were prepared by passing them through a 70 μm cell strainer and treating them with Red Blood Cell Lysis Buffer (Beyotime, China). The cells were then stimulated with the recombinant protein and treated with Brefeldin A Solution (1X) for 5 h. After stimulation, the cells were washed with PBS buffer and stained with FITC-conjugated anti-mouse CD3, APC-conjugated anti-mouse CD4, and PE-conjugated anti-mouse CD8a antibodies (BioLegend, USA). Following staining, the cells were fixed using Cytofix/Cytoperm™ Solution and labeled with PE-conjugated anti-mouse IFN-γ antibodies (BioLegend, USA). Finally, the stained cells were washed twice and analyzed using a flow cytometer (Beckman Coulter, USA) to detect immune responses mediated by antigen-specific CD4⁺ and CD8⁺ T lymphocytes.

On the 5^th day post-virus challenge, we collected nasal turbinates, trachea, and right lung from each experimental group to measure viral load. Total cellular RNA was extracted from homogenized supernatants using the QlAamp Viral RNA Mini Kit (Qiagen, Germany) according to the manufacturer’s instructions. The viral load and expression of cytokines (IL-1β, IL-6, TNF-α) were quantified using the HiScript//one-step qRT-PCR SYBR Green kit (Vazyme, China). Primer sequences used in this assay can be found in Supplementary Table 2.

On the 5^th day post-virus challenge, the mice were euthanized and their left lung lobes were fixed in 4% paraformaldehyde, embedded in paraffin, and subjected to H&E staining for histopathological examination to evaluate lung injury.

The data were presented as mean ± standard error of the mean (SEM). To determine statistically significant differences in group means, a one-way analysis of variance (ANOVA) with Tukey’s multiple comparison tests was performed using GraphPad Prism software. In the figures, significance levels were denoted as **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05, ns p > 0.05.

The immunogenicity of epitopes often results in comparatively weaker immune responses when compared to conventional vaccines, thereby limiting the efficacy of epitope-based vaccines. In this study, we developed three mRNA vaccines targeting influenza viruses: a monomeric structure (MH), a trimeric structure (MH-T), and an architecture based on ferritin (MH-TF) ( Figure 1A ). The successful expression of proteins encoded by the mRNA for MH, MH-T, and MH-TF immunogens was confirmed through Western blot analysis treated with reducing loading buffer ( Figure 1B ) and indirect immunofluorescence ( Figure 1C ) performed on the transfected cell lysates. Concurrently, non-reductive treatment was employed to examine the expression patterns of MH-T and MH-TF proteins ( Figure 1D ), TEM images analysis of MH-TF revealed nanoparticles with a diameter of nearly 50 nm ( Figure 1E ).

The humoral immune response is crucial for preventing virus entry and enhancing virus elimination. This study assessed the antibody levels produced by influenza virus mRNA vaccines in C57BL/6 mice. The humoral immune response of the mRNA vaccine was evaluated by administering three doses, each at a dose of 15 μg, to female C57BL/6 mice (n = 9), with a two-week interval between each vaccination( Figure 2A ). Enzyme-linked immunosorbent assay (ELISA) was employed to assess specific levels of immunoglobulin G (IgG) in serum samples collected on days 7, 14, 21, 28, 35, and 42 after the initial vaccination. All three vaccines induce specific antibodies within 14 days of the initial vaccination, with antibody titers peaking on day 42 in particular( Figure 2B ). The OD₄₅₀ values of MH, MH-T and MH-TF were significantly higher than those of the PBS group (p=0.007, p=0.0005, and p<0.0001). The evaluation of hemagglutination inhibition activity facilitates the identification of antibodies that specifically bind to the receptor binding domain of hemagglutinin. Generally, an HI titer equal to or greater than 40 is considered indicative of a protective effect. 42 days after the first immunization, compared to the antibody sera from the PBS group, significant HI activity was observed in the antibody sera from MH, MH-T, and MH-TF groups, with the highest HI titer detected in the MH-TF group ( Figures 2C–F ). However, no significant difference in HI titers targeting influenza B viruses was found between the MH group and PBS group ( Figures 2E–F ).

We further elucidated the cellular immune response elicited by mRNA-based vaccines. T lymphocytes isolated from the spleen of immunized and control mice (n=3) two weeks after the third immunization were assayed using ICS assay. All the three vaccine groups elicited antigen-specific CD4⁺ ( Figure 3A, E ) and CD8⁺ ( Figure 3B, F ) T cells. At the same time, the level of IFN-γ produced by CD3⁺ CD4⁺ T cells ( Figure 3C, G ) and CD3⁺ CD8⁺ T cells ( Figure 3D, H ) in three vaccine groups were found to be significantly higher compared to those in the control group. Notably, The level of IFN-γ produced by CD3⁺ CD8⁺ T cells in MH-T and MH-TF groups was significantly increased compared with that in MH group, and was 2.24 times and 2.06 times, respectively. These findings suggest that vaccination elicits augmented cellular immune response.

42 days after the first immunization, the mice were challenged intranasally with 10× LD₅₀ influenza virus. The weight and survival rate of the mice were monitored daily for two weeks( Figure 4 ). The results showed that mRNA vaccine-immunized mice exhibited protective effects against weight reduction and death, whereas control mice succumbed to the virus attack, and the protective efficiency of MH-T and MH-TF groups was 100%. On day 5 post-infection, viral load of the turbinate, trachea, and lungs exhibited a significantly diminished in the vaccinated group compared to the control group ( Figure 5 ).

The histological examination of lung tissue revealed a significant reduction in lung injury among vaccinated mice, accompanied by decreased inflammatory cell infiltration, preserved alveolar structure integrity, slightly thickened alveolar walls, and reduced epithelial cell necrosis ( Figure 6A ). To visually compare the variations in pathological damage across each group, we evaluated the outcomes of the pathological sections. A higher score indicates a more pronounced level of pathological damage ( Figure 6B ).

After influenza virus infection, the expression of inflammatory cytokines can exacerbate the infection. To investigate whether vaccines can mitigate the inflammatory response, we evaluated the expression of inflammation-associated cytokines, including IL-1β, IL-6, and TNF-α ( Figure 7 ). The relative real-time qPCR results revealed comparable IL-1β levels among all vaccine groups and the control group, without statistical significance. Notably, the expression of IL-6 and TNF-α genes in the vaccine group exhibited a significant reduction compared to the control group, except for IL-6 following B/Jilin/02/2022(Victoria) virus exposure.