Female BALB/c mice (6-8-week-old) were purchased from Beijing HFK Bioscience Co., Ltd. The Human embryonic kidney (HEK 293) cells were maintained in our laboratory. HEK 293 cells were cultivated in Dulbecco's Modified Eagle's medium (Gibco, USA) augmented with 10% fetal bovine serum (FBS; Gibco, USA) and a solution containing penicillin (100 U/mL) and -streptomycin (100 µg/mL). To harvest bone marrow, BALB/c mice were euthanized by cervical dislocation after being anesthetized with an intraperitoneal injection of tribromoethanol (2.01%, 20 μL/g body weight). Bone marrow was obtained from the femur and tibia of mice and washed with RPMI-1640 medium (Hyclone, USA). After filtration through a 70 μm filter, the cells were cultured in RPMI-1640 medium containing 100 U/mL penicillin and 100 μg/mL streptomycin. The culture medium was supplemented with 20 ng/mL recombinant murine granulocyte-macrophage colony-stimulating factor, 10 ng/mL recombinant IL-4, and 10% inactivated FBS at 37°C in an atmosphere of 5% CO₂. The medium was replenished half-way through the incubation period. This process yielded mouse bone marrow-derived DCs dendritic cells. B/Chicken embryos/2022 (Victoria) (GenBank: OR775570) and B/Massachusetts/2/2012 (Yamagata) (GenBank: MT056027) were obtained from our laboratory collection. All experiments involving infectious IBV were performed in a level-2 facility.

The HA gene of IBV Victoria lineage B/Austria/1359417/2021 (GISAID no. EPI1926631) was codon-optimized for human expression. The optimization process involved changes to the GC content, codon usage, distribution, and incorporation of restriction enzyme recognition sites. Approximately 1000 nucleotides underwent modification, yet the amino acid sequence remained unaltered. A Kozak sequence was incorporated after the upstream restriction site and Nde I and BamH I sites were introduced. Highly conserved epitopes consisting of the A α-helix of HA, the ectodomain of M2 and the HCA-2 of NA were connected into a continuous gene, and Not I and Xho I restriction sites were inserted 5' and 3' of the optimized gene (G3) sequence. The G3 epitopes ( Supplementary Table S2 ) were chosen based on IEDB conservation scores and HLA-binding predictions (HLA-DR4/DR7, common in humans). The IBV HA gene and epitope gene G3, which had incorporated homology arms, were ligated into the pVAX1-IRES vector, using seamless cloning method. The cytomegalovirus (CMV) promoter and internal ribosome entry site (IRES) sequence separately initiated two segments of the inserted gene.

Plasmid DNA was transfected into HEK293 or BMDC cells using the Advanced DNA/RNA Transfection Reagent (ZETA Life, USA). Briefly, 3×10⁵ cells were inoculated into 6-well plates. Following an overnight incubation, aliquots of a solution containing 2.5 μg of plasmid DNA and Advanced DNA/RNA Transfection Reagent were added to each well. The pVAX-1 vector was used as negative control. Following a 36-h incubation at 37°C, the cells were collected and transfected using Cell Lysis Buffer (Beyotime Biotechnology, China). The supernatant of the lysates was collected for western blot analysis as described next. Materials examined by an immunofluorescence assay as described below were washed and fixed with 4% paraformaldehyde in a 6-well plate.

The cell lysates were collected and the proteins resolved by 10% SDS-PAGE. The proteins were subsequently transferred to nitrocellulose membranes for immunoblotting. The membranes were blocked using 3% bovine serum albumin (BSA) for 1 h. Rabbit anti-IBV HA protein antibody (GeneTex, USA), rabbit anti-IAV M2 protein antibody (GeneTex, USA), and rabbit anti β-actin antibody (Cell Signaling Technology, USA) were each used at a 1:1000 dilution. Horseradish peroxidase (HRP)-labelled goat anti-rabbit IgG antibody (Beyotime Biotechnology, China) was used at a 1:25000 dilution. PBS as the MOCK control. The bands were visualized using SuperSignal West Femto (Thermo Fisher Scientific, USA).

The cells were permeabilized with 0.5% Triton X-100 after fixation, then blocked in 3% BSA for 2 h. The primary antibody used in this experiment was the same antibody used in western blotting, while fluorescein isothiocyanate (FITC)-labelled goat anti-rabbit IgG antibody (Beyotime Biotechnology, China) was used at a 1:1,000 dilution as the secondary antibody. Nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich, Germany), and images were obtained using an EVOS M5000 microscope (Invitrogen, USA).

Female, 6-8-week-old Balb/c mice were immunized by intramuscular injection of BHAG3 (100 μg) and introduced by intramuscular injection followed by electroporation (BHAG3 I.M. + EP) or an equivalent amount of pVAX1 (Mock) via intramuscular injection. The EP was applied to the injection site immediately following injection by six 10-ms pulses using a two-electrode array at a depth of 5 mm. Electric pulses were delivered by the Advaccine Biopharmaceutics (Suzhou) Co. LTD. (China). Intramuscular injection of BHAG3 in the absence of EP (BHAG3 I.M.) was used as a matched control ( Figure 1A ).

The mice were vaccinated via a sterile insulin syringe (0.3×8 mm, U-40/30G) into both hind legs. The first and second booster was given 21 and 42 days, respectively, after the initial vaccination.

To detect IBV HA-specific IgG and its subtypes, the ELISA was performed. The assay used 96-well polystyrene plates (Corning, USA) coated with IBV HA protein (Sino Biological, China) overnight at 4°C. After coating, the plates were washed three times with phosphate-buffered saline containing Tween (PBST). Five percent skim milk (BD, USA) was added to each well and incubated for 2 h at 37°C. After washing, plates were incubated with 2-fold serial dilutions of mouse sera starting from 1:100 and incubated at 37°C for 1 h. Following five washes in PBST, the plate was incubated for 45 min at 37°C with HRP-conjugated goat anti-mouse IgG (ZSGB, China), IgG1, or IgG2a antibody (Abcam, city, UK). Following five washes with PBST, 3,3,5,5′-tetramethylbenzidine (TMB; Sigma-Aldrich, Germany) was added to facilitate color development. The reaction was stopped by adding 2 mol/L H₂SO₄ at the appropriate time, and the optical density was measured at 450 nm using a 10M multimode microplate reader (Tecan, Switzerland).

Seven days after the final vaccination, the spleens of the mice were harvested. To harvest spleen, BALB/c mice were euthanized by cervical dislocation after being anesthetized with an intraperitoneal injection of tribromoethanol (2.01%, 20 μL/g body weight). A single-cell suspension was prepared and stimulated with inactivated influenza virus and treated with a solution of Brefeldin A for 6 h. Following stimulation, the cells were washed with PBS buffer and stained with CY5-coupled anti-mouse CD3, FITC-coupled anti-mouse CD4 and APC-coupled anti-mouse CD8a antibodies (BioLegend, USA). Following staining, cells were fixed using Cytofix/Cytoperm™ solution (BD, USA), divided into three portions and labelled using PE-coupled anti-mouse IFN-γ antibody (BioLegend, USA), PE-coupled anti-mouse IL-2 antibody (BioLegend, USA), or PE-coupled anti-mouse IL-4 antibody (BioLegend, USA), respectively. Subsequently, the stained cells were washed twice and analyzed using flow cytometry (Beckman Coulter, USA) to detect antigen-specific CD4⁺ and CD8⁺ T-lymphocyte-mediated immune responses.

The assay was performed as previously described (27, 28). Samples of sera were treated with receptor-destroying enzyme II (Denka Seiken, Japan) and diluted fivefold prior to the detection of HI in 96-well V-bottom microplates. The serum samples were adsorbed with 5% erythrocytes before testing. Each test sample was incubated for 30 min at room temperature with 4 HA units of virus (B/Chicken embryos/2022 or B/Massachusetts/2/2012), then mixed with 1% chicken red blood cells and further incubated for a further 30 min. The HI titer was defined as the reciprocal of the maximum serum dilution that inhibited viral hemagglutination.

Each group of BALB/c mice was divided into subgroups for challenge with either B/Chicken embryos/2022 (Victoria) (GenBank: OR775570) or B/Massachusetts/2/2012 (Yamagata) (GenBank: MT056027). The amino acid similarity between the Victoria lineage virus challenge variant used and the HA variant contained in the vaccine was 99.7%. Additionally, the amino acid similarity was 93% between the Yamagata lineage virus challenge variant used and the HA variant contained in the vaccine. The immunized mice were anesthetized with tribromoethanol and then challenged by via nasal installation of 50 µL of viral suspension, with an approximate dose of 5 mouse lethal dose 50 (5 × mLD₅₀ = 1 × 10^4.5 TCID₅₀). Body weight and mortality rates were monitored for 2 weeks. In another experiment, three mice were euthanized 5 days after challenge. Prior to euthanasia, mice were anesthetized via intraperitoneal injection with tribromoethanol (2.01%, 20 μL/g body weight) and then euthanized by cervical dislocation. The lung tissues were harvested for virus titration and histopathological analysis. In accordance with the ethical guidelines for animal experimentation, a humane endpoint of 25% weight loss was used in this study (21).

Five days following the viral challenge, the lungs of the mice were collected to measure the viral load. Viral RNA was extracted from the supernatant of the homogenate using the RaPure Viral RNA/DNA Kit (Magen, China) as described by the manufacturer. Viral RNA in lung tissue was quantified using the HiScript II U⁺ One Step qRT-PCR Probe Kit (Vazyme, China). The sequences of the primers and probe-specific used are provided as supplementary data ( Supplementary Table S1 ).

To determine whether the vaccine could attenuate the inflammatory response, Total protein was extracted from the homogenous lung tissue using the ProteinExt® Mammalian Total Protein Extraction Kit (TransGen, China) exactly as described by the manufacturer. The expressions of pro-inflammatory cytokines, including IL-1β, IL-6, IFN-γ, and TNF-α, were assessed by ELISA kits (R&D, USA) in lung tissue from mice infected with B/Victoria and B/Yamagata viruses.

Lungs were fixed in 10% formalin for approximately 12 h and subsequently embedded in paraffin blocks. The lung sections were stained with hematoxylin and eosin (H&E) prior to being scored for the presence of pulmonary hemorrhage, inflammatory cell infiltration, hyaline membrane formation, alveolar atrophy and collapse, alveolar wall thickening, pulmonary edema, and hemorrhage on a scale from 0–3. Immunohistochemistry was performed on the sections using rabbit anti-IBV NP antibody (GeneTex, USA). The percentage of positive cells was quantified.

The relationship between BHAG3-induced antibody production and early activation of the innate immune system was assessed. Bone marrow of 6-week-old mice was extracted and BMDCs isolated. DCs were stimulated with PBS, lipopolysaccharide (LPS), or BHAG3 at 37°C. The control was 4°C conditioned treatment. FITC-dextran treatment was added at the end of the stimulation. The percentage of DCs that phagocytosed FITC-dextran was determined by flow cytometry to indirectly assess the phagocytosis ability of DCs. The delivery capacity of DCs was evaluated using the mixed lymphocyte reaction. The lymphocyte stimulation index was calculated by adding CCK-8 after 36 h of co-culture and measuring the absorbance at 450 nm. The percentage of cells expressing CD40, CD80, CD86, and MHC-II surface markers (all from BioLegend, USA) was determined by flow cytometry following staining of antigen-stimulated DCs with antibodies against specific DC markers. The expression levels of inflammatory factors in culture supernatants of antigen-stimulated DCs were analyzed using ELISA kits (R&D, USA).

Statistical analyses were performed using the Prism 8.0.2 software (GraphPad, USA). One-way analysis of variance (ANOVA) was used for the comparison among multiple (>2) groups. Two-way ANOVA was used for comparison of antibody titers among different groups within the different phases. The antibody titers were compared by performing log-transformed values. P values <0.05, < 0.01, < 0.001, and < 0.0001 were considered statistically significant depending on the experiment.

To develop a safe and efficient IBV vaccine, seamless cloning was used to construct a recombinant DNA vector that carried the full-length IBV HA gene and the G3 genes encoding the HA, M2e, and NA antigenic epitopes ( Figure 2A ). The description of the various epitopes selected was in Supplementary Table S2 . The successful insertion of the genes was verified by PCR ( Figure 2B ) and double-enzymatic digestion analysis and sequencing ( Figure 2C ). The expression of the HA and M2 proteins in the constructs was detected using western blotting ( Figure 2D ). The expression of the HA protein was also detected using the immunofluorescence assay (IFA) ( Figure 2E ). The findings demonstrated the successful construction of a recombinant plasmid co-expressing the IBV HA gene and epitope gene G3 were successfully constructed in this study. The plasmid was designated BHAG3.

The immune responses elicited by BHAG3 in mice were assessed. Serum samples obtained at different and specific times were analyzed to evaluate the potency of specific antibodies. The findings indicated a pronounced IBV HA IgG specific response ( Figure 1B ). Analysis of IgG subtypes revealed that BHAG3 triggered a predominantly IgG2a immune response, whereas the IgG2a potency induced by BHAG3 I.M. + EP was 1.262 times higher than that induced by BHAG3 I.M. ( Figures 1C–E ). Moreover, the IgG2a/IgG1 ratio in both vaccine modalities exceeded 1.0, indicating that BHAG3 significantly increased IgG2a levels.

To further evaluate the antibody response to the vaccine, we examined HI antibodies in the collected sera. After the second immunization, the levels of vaccine-induced HI antibodies were significantly elevated, and EP further enhanced this antibody response. The peak geometric mean titer (GMT) induced by BHAG3 I.M. reached 1:105.6, while the GMT for BHAG3 I.M. + EP reached 1:320 at 56 days following the initial immunization ( Figure 1F ). Additionally, HI antibodies in the sera from the BHAG3-immunized group exhibited strain-specificity, with inhibition observed only against strains with matching HA gene profiles, but not against strains with mismatched HA gene profiles ( Supplementary Table S3 ).

To investigate the vaccine-induced cellular immune response, spleen lymphocytes were isolated from mice 7 days following the final vaccination. The proportion of activated T cells and cytokine expression were determined by flow cytometry. They demonstrated BHAG3-induced antigen-specific CD4⁺ T-cell responses ( Figure 3A ). No significant enhancement was observed in CD8⁺ T-cell responses. Furthermore, the cytokine results demonstrated that BHAG3 induced a notable elevation in the expressions of IL-2, IL-4, and IFN-γ expression ( Figures 3B–D ). In particular, BHAG3 I.M. + EP produced a 0.23-fold increase in IL-2 levels relative to BHAG3 I.M. These observations impllid that BHAG3 I.M. + EP may potentially elicit more robust cellular immune responses. Notably, the strategy also triggered cross-reactive T-cell immunity in response to stimuli from the B/Yamagata lineage of IBV, implying potential cross-protection against heterologous influenza attacks ( Figures 3E–H ). The data demonstrated that immunization with BHAG3 I.M. + EP elicited robust CD4⁺ T cell responses in BALB/c mice.

Although the B/Yamagata lineage has not been widely detected in recent years, it serves as a relevant model for assessing cross-lineage immunity. To evaluate the protective efficacy of BHAG3, the incidence of morbidity and mortality from weight loss was monitored over a 14-day period. All mice in the Mock group that were infected by the B/Victoria and B/Yamagata IBV lineages died of the viral infection within 5 days. The mice in the BHAG3 I.M. + EP group exhibited slight weight loss (up to 8% loss) on days 3−4 following infection by B/Victoria, which was followed by growth and weight gain. Mice infected by B/Yamagata also experienced transient weight loss (up to 9% loss) on days 1−3 days after infection, followed by growth and weight gain. All mice in the BHAG3 I.M. + EP group survived the infection by both B/Victoria and B/Yamagata IBV. Mice in the BHAG3 I.M. group exhibited significant and sustained weight loss (up to 20% loss) following infection by B/Victoria and B/Yamagata IBV ( Figure 4A ). However, these mice were protected with 100% survival ( Figure 4B ). This demonstrated that the vaccine could provide cross-protection against a heterologous lineage, even in the absence of high-titer HI antibodies against the challenge strain, underscoring the potential of the conserved epitope-based approach to broaden protection.

The expression of inflammatory cytokines exacerbates symptoms and inflammatory responses that develop upon infection with influenza virus. In the present study, mouse lung tissues of comparable dimensions were pulverized, and proteins were extracted to detect inflammatory cytokines. The four inflammation-associated factors IL-1β, IL-6, IFN-γ, and tumor necrosis factor-alpha (TNF-α), exhibited a similar trend ( Figure 4C ) and their levels were significantly lower in both BHAG3-immunized groups compared to the Mock group (P < 0.0001). Notably, the BHAG3 I.M. + EP group exhibited a lower expression of inflammatory factors compared to the BHAG3 I.M. group. Additionally, histological analysis revealed that mice in the Mock group exhibited significant lung tissue damage following infection with both B/Victoria and B/Yamagata IBV. This damage manifested as edema, hemorrhage, thickening of the alveolar wall, infiltration of inflammatory cells, and atrophy and collapse of the alveoli. In contrast, mice in the BHAG3 I.M. + EP group exhibited minimal abnormalities and a more defined alveolar wall structure. Lung structural integrity was maintained in mice in the BHAG3 I.M. group, although they displayed slight inflammatory cell infiltration and dilated and congested capillaries in the alveolar walls ( Figures 4D, E ). The lung tissues of the mice were weighed at the time of collection. The calculated lung index results were homologous with the histological scoring values ( Figure 4F ).

Immunohistochemical analysis of mouse lung tissues revealed that immunization by both BHAG3 I.M. + EP and BHAG3 I.M. resulted in a notable reduction in the deposition of IBV NP. Moreover, the percentage of NP-positive cells in the BHAG3 I.M. + EP group following immunization was < 1%, indicating a greater ability to inhibit IBV replication ( Figures 5A, B ). To further confirm the ability of vaccination to clear IBV, the viral load in lung tissues was assessed by RT-qPCR. Viral loads of B/Victoria and B/Yamagata in lung tissues were significantly reduced in the BHAG3 I.M. group. The BHAG3 I.M. + EP group exhibited a nearly 1000-fold reduction in viral RNA load in lung tissue compared to the Mock group ( Figure 5C ; P < 0.0001). The results indicated that BHAG3 I.M. + EP provided comprehensive protection against both lethal doses of B/Victoria and B/Yamagata, while also effectively reducing the viral load in lung tissues. BHAG3 I.M. only provided partial protection against lethal doses of B/Victoria and B/Yamagata.

The objective of the in vitro experiments was to investigate the uptake and delivery capacity of BHAG3 by DCs and to analyze the co-stimulatory factors that are upregulated on the surface of DCs and the pro-inflammatory cytokines that are secreted by DCs following the uptake of BHAG3. The results demonstrated the uptake of BHAG3 and delivered by DCs in substantial quantities ( Figures 6A, B ). In comparison to the PBS group, BHAG3 significantly stimulated the expression of DC surface co-stimulatory molecules CD80 and CD86, and T-cell co-stimulatory molecules CD40 and major histocompatibility complex II (MHC-II) ( Figure 6C ). BHAG3 significantly increased the secretion of the TNF-α, IFN-γ, IL-1β, IL-6, IL-10, and IL-12p70 cytokines ( Figure 6D ). These findings indicated that BHAG3 activated the maturation of DCs and induced the release of a substantial number of pro-inflammatory cytokines, which in turn promoted the proliferation and differentiation of other immune cells and had the capacity to rapidly activate the innate immune response.