Ferritin-Based HA DNA Vaccine Outperforms Conventional Designs in Inducing Protective Immunity Against Seasonal Influenza

Abstract

Background: Influenza remains a persistent public health challenge due to antigenic drift and shift, necessitating vaccines capable of eliciting broad and durable immunity. Hemagglutinin (HA) antigen serves as the critical target for eliciting protective immune responses against influenza. DNA vaccines offer distinct advantages over conventional platforms, including accelerated development and induction of both humoral and cellular immune responses. Methods: To optimize HA antigen presentation, we designed and systematically compared the immunogenicity and protective efficacy of HA antigen display strategies-bacteriophage T4 fibritin (HA-Foldon) and ferritin-based virus-like particles (HA-Ferritin)-versus monomeric HA DNA vaccines against seasonal influenza viruses. Results: HA-Ferritin showed superior structural stability. All vaccines induced similar HA-specific antibody levels, but HA-Ferritin elicited higher neutralizing antibodies and stronger T cell responses. Upon challenge, HA-Ferritin and HA-Foldon protected mice from weight loss and reduced lung virus loads by 3.27 and 0.76 times, respectively. Monomeric HA provided limited protection, with only 40% survival and minimal viral or pathological reduction. Conclusions: The HA-Ferritin DNA vaccine demonstrated enhanced immunogenicity and protection, supporting structured antigen display as a promising strategy for influenza DNA vaccine development.

Keywords: DNA vaccine; ferritin-based vaccine; hemagglutinin (HA) antigen; immunogenicity; influenza.

Figure 1

Vaccine designs and characterizations of influenza HA constructs in vitro. (A) Schematic illustration of HA, HA-Foldon, and HA-Ferritin vaccine designs and in vitro assembly. HA is the major surface antigenic protein of influenza virus. HA-Foldon was constructed by fusing HA with the bacteriophage T4 fibritin trimerization motif to stabilize its prefusion conformation. HA-Ferritin displays HA antigens on ferritin nanoparticles, which self-assemble into spherical structures (~20–50 nm in diameter) composed of 24 subunits, thereby enhancing immunogenicity. (B) Expression analysis of vaccine antigens by Western Blot. Plasmids encoding influenza HA antigens were transfected into Expi293F cells. At day 5 post transfection, cell supernatants were collected and mixed with SDS-containing loading buffer, followed by denaturation at 100 °C for 10 min. Protein expression was detected using an anti-His monoclonal antibody to confirm successful antigen expression and compare expression levels. (C) Oligomeric state analysis by Native PAGE under non-reducing conditions. Cell supernatants containing HA and HA-Foldon were purified via His-tag affinity chromatography, while HA-Ferritin was purified by SEC. The purified antigens were then analyzed by Native PAGE to verify the formation of higher-order oligomers. (D) TEM imaging of SEC-purified HA-Ferritin. The morphology and size of HA-Ferritin nanoparticles were directly visualized by transmission electron microscopy (TEM). Scale bar: 100 nm.

Figure 2

Humoral immune responses following vaccination. (A) Immunization schedule. BALB/c mice (n = 5 per group) were intramuscularly immunized with 25 μg of each antigen-encoding plasmid delivered via electroporation, followed by booster immunization on day 21. Blood samples were collected immediately before and two weeks after the booster immunization for serological analysis. Spleens were harvested for subsequent cellular immune response analysis. (B) HA-specific IgG antibody titers measured by an ELISA in serum samples collected immediately before and two weeks after the second immunization. Dotted lines indicate the seropositivity threshold (OD450 = 0.1), with values above this cutoff considered positive. (C) Hemagglutination inhibition (HI) titers against the homologous viral strain measured two weeks post boost immunization. Dotted lines represent the HI titer of 40, the threshold for protective immunity. Data points represent individual animals. Values are expressed as mean ± SEM. Statistical significance was determined by two-way ANOVA (B) or one-way ANOVA (C) with the following thresholds: ns, not significant; * p < 0.05; ** p < 0.01; *** p < 0.001; and **** p < 0.0001.

Figure 3

T cell immune responses following vaccination. Mice were immunized with monovalent seasonal influenza vaccines based on H1N1 and H3N2 strains. Two weeks after the booster immunization, spleens were collected and splenocytes (1 × 10⁶ cells per well) were stimulated in vitro with H1 or H3 antigenic peptides. IFN-γ-secreting cells were detected using an ELISPOT assay after incubation with anti-IFN-γ antibody. (A) Representative ELISPOT images showing spot-forming units (SFUs), with each image representing results from an individual mouse. (B) Quantification of IFN-γ-secreting lymphocytes in splenocytes. Data points represent the number of IFN-γ-secreting cell clones per 1 × 10⁶ splenocytes from individual animals (n = 5). Data are presented as mean ± SEM. Statistical significance was determined by one-way ANOVA: ns, not significant; * p < 0.05; ** p < 0.01; and *** p < 0.001.

Figure 4

Protective efficacy of vaccination against homologous viral challenge. (A) Experimental timeline of immunization and viral challenge. BALB/c mice (n = 10 per group) were intramuscularly immunized with 25 μg of monovalent DNA vaccine (based on the H1N1 PR8 strain) delivered via electroporation. Two weeks post boost, mice were intranasally challenged with 20 μL of 10 LD₅₀ homologous virus. Body weight and survival were monitored daily for 14 days post challenge (n = 5 for survival analysis). (B) Body weight changes (percentage of initial weight) and survival rates during the monitoring period. (C) Lung viral copy numbers quantified by absolute qPCR in lung tissues collected 5 days post challenge (n = 5). (D) Correlation of HI antibody titers with pulmonary viral loads was assessed through logarithmic transformation and linear regression modeling, with 95% confidence intervals for predicted values. (E) Representative H&E-stained lung sections from paraffin-embedded tissues (n = 3) and corresponding pathological scores. Scoring criteria: Grade 1, no lesions; Grade 2, <25% lobar involvement; Grade 3, 25–50%; Grade 4, 50–75%; and Grade 5, >75% involvement (perivascular/parenchymal infiltration). Higher scores indicate more severe pathology. All data points represent individual animals (mean ± SEM). Data are presented as mean ± SEM. Statistical significance was determined by one-way ANOVA: ns, not significant; * p < 0.05; ** p < 0.01; *** p < 0.001; and **** p < 0.0001.

Figure 5

Immunogenicity of trivalent seasonal influenza vaccines. Based on monovalent HA-Ferritin vaccine groups (25 μg per HA subtype), two trivalent formulations were evaluated: (1) equal-dose trivalent (25 μg per HA subtype) and (2) low-dose trivalent (8 μg per HA subtype). Following the immunization schedule in Figure 2, serum samples collected 2 weeks post boost were analyzed for homologous strain-specific IgG antibodies by an ELISA (A) and hemagglutination inhibition (HI) titers (B). Data points represent individual animal measurements (mean ± SEM). Statistical comparisons were performed using one-way ANOVA: ns, not significant; * p < 0.05; ** p < 0.01; *** p < 0.001.