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. 2026 Sep 16;9(10):2939–2949. doi: 10.1021/acsptsci.6c00419

Ferritin Complex Vaccine against Influenza A Using Screened Immunogenic Sequences from Fv-Antibody Library

Tae-Hun Kim †, Soonil Kwon †, Jaeyong Jung †, Jeong Soo Sung †, Dong-Ho Han †, Min-Jung Kang ‡, Joachim Jose §, Misu Lee ∥, Jae-Chul Pyun †,*
PMCID: PMC13659839  PMID: 42872118

Abstract

A ferritin-based influenza A vaccine was developed. The immunogenic sequences of the nucleoprotein (NP) were screened using an autodisplayed Fv-antibody library, and the paratopes with a high-affinity binding affinity were analyzed through docking simulations. The resulting antigenic sequences were engineered into single-domain constructs using a nonimmunogenic linker. These immunogenic sequences were then expressed in conjunction with ferritin heavy-chain proteins to form spherical nanoparticles through self-assembly. These NP-ferritin complex vaccines were subsequently injected into mice, and antibody responses, cytokine secretion, and T-cell activation profiles were subsequently analyzed to demonstrate that the ferritin-based NP vaccines activated innate as well as acquired immune responses.

Keywords: influenza A, vaccine, ferritin, Fv-antibody, library

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The influenza A virus (Inf-A) remains a persistent global health threat due to its high mutation rate, broad host range, and potential to cause seasonal epidemics as well as sporadic pandemics. − Despite widespread vaccination efforts, current influenza vaccines primarily target surface glycoproteins, such as hemagglutinin (HA) and neuraminidase (NA), which undergo rapid antigenic drift. , As a result, vaccine efficacy varies annually and often declines when emerging strains differ from vaccine-matched variants. , Consequently, the development of vaccines targeting highly conserved internal viral proteins has emerged as a promising strategy to induce broader and more durable immunity. As a major target of vaccine development against the influenza virus, the nucleoprotein (NP), which is the most abundant protein in the influenza virus, plays an essential role in viral replication, transcription, and ribonucleoprotein complex formation. Notably, NP contains multiple conserved immunogenic epitopes capable of activating both humoral and cellular immune responses, particularly cytotoxic T-lymphocyte (CTL) responses, which contribute to cross-protection across diverse influenza subtypes. − Therefore, NP-targeted vaccines offer the possibility of a universal or broadly protective influenza vaccination, addressing several limitations associated with strain-specific HA-based approaches.

Early influenza vaccine platforms relied on live-attenuated or inactivated viruses, which provide strong immune activation but display inherent drawbacks, including the risks of reversion to virulence, incomplete inactivation, and the requirement for large antigen quantities. These limitations parallel those historically observed in other viral vaccine systems, for which concerns regarding safety, stability, and production efficiency motivated the exploration of next-generation platforms, including subunit vaccines, virus-like particles, nanoparticle scaffolds, and nucleic acid technologies. , Among these modern platforms, protein-nanoparticle vaccines, particularly those based on ferritin, have demonstrated exceptional promise owing to their ability to self-assemble into uniform 24-mer nanocages, present antigens in multivalent forms, and enhance immunogenicity without an excessive adjuvant burden. ,

In parallel with advances in nanoparticle vaccines, the use of Fv-antibody libraries has provided a powerful method for identifying immunogenic peptide sequences in viral proteins. , The Fv-antibody represents the antigen-binding site of immunoglobulin G, which is composed of three complementarity-determining regions (CDRs) and four frame regions (FRs). The Fv-antibody library was prepared by site-directed mutagenesis of CDR3 with 11 amino acid residues , and expressed on the outer membrane of E. coli using autodisplay technology. − The prepared Fv-antibody library exhibits a high surface density of >105 Fv-antibodies/E. coli and a high diversity of >105 clones/library. ,, From these properties of the Fv-antibody library, Fv-antibodies with a high-affinity for target antigens can be screened without repeated spanning process. − Recently, the autodisplay-based Fv-antibody library platform has enabled the expression of a large number of diversified antigen-binding domains on the surface of Escherichia coli (E. coli), allowing high-throughput screening of peptides that bind with strong affinity to target viral proteins. − This strategy has been successfully applied in previous studies on viral vaccine development, including ferritin-based SARS-CoV-2 and porcine epidemic diarrhea virus (PEDV) vaccines, in which screened Fv-antibody binding sites were used to derive immunogenic sequences for nanoparticle display. , Building on this approach, the same methodology can be adapted for Inf-A by screening Fv-antibodies that specifically recognize conserved regions of NP and computationally map their binding interfaces to extract candidate immunogenic sequences.

In this study, we developed a ferritin-based Inf-A vaccine, in which immunogenic sequences derived from NP were identified using an autodisplayed Fv-antibody library and refined through docking simulations to map high-affinity binding residues, as shown in Scheme . The resulting antigenic sequences were engineered into single-domain constructs using nonimmunogenic linker modules and expressed in conjunction with ferritin heavy-chain proteins to form spherical nanoparticles through self-assembly. These NP-ferritin complexes were subsequently evaluated in mice to assess antibody responses, cytokine secretion, and T-cell activation profiles to demonstrate that ferritin-based NP vaccines activate innate as well as acquired immune responses.

1. Screening of Fv-Antibodies against Inf-A NP from the Fv-Antibody Library .

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a (A) Screening of target clones and docking simulation between NP and the Fv-antibody of the target clone. (B) Analysis of the antigenic sequence after a docking simulation. (C) Analysis of immune activity after the vaccination of ferritin complexes.

Materials and Methods

Materials

Custom-synthesized plasmids for Inf-A NP (residues: 184–498, Code: A/Hong Kong/H090-701-V10/2009) were supplied by Gyeongsang National University (Jinju, Korea). Mouse ferritin H (labeled with an immunogenic sequence) was purchased from Cosmo Genetech (Seoul, Korea). Fetal bovine serum (FBS) was purchased from Cytiva (Petaluma, CA, USA). Dulbecco’s Modified Eagle’s Medium (DMEM; 11995065), TMB reagent (#34021), 96-well microplates (Maxisorp), and penicillin-streptomycin (PS; 15140122) were purchased from Thermo Fisher Scientific (Waltham, MA, USA). IL-4 (ELM-IL4-1), IL-12 (ELM-Il12p40p70-1), IFN-γ (ELM-IFNg-1), and TNF-α (ELM-TNFa-1) mouse ELISA kits were purchased from RayBiotech (Norcross, GA, USA). Anti-CD4 monoclonal antibody (#11-0041-82, RRID:AB_464892) with FITC labeling and anti-CD8 monoclonal antibody (#12-0081-82, RRID:AB_465530) with PE labeling were purchased from eBioscience (San Diego, CA, USA). For the analysis of IgG subtypes, anti-mouse IgG1 (ab97240, RRID:AB_10695944), IgG2a (ab97245 RRID:AB_10680049), IgG2b (ab97250, RRID:AB_10695945), and IgG3 (ab97260, RRID:AB_10680425) antibodies labeled with HRP were obtained from Abcam (Cambridge, MA, USA). AutoDock Vina docking simulation software was purchased from Scripps Research (La Jolla, CA, USA). The Discovery Studio Visualizer from Dassault Systèmes BIOVIA (San Diego, CA, USA) and PyMOL software from DeLano Scientific LLC (San Francisco, CA, USA) were used for analysis.

Anti-NP Fv Screening

As documented in previous studies, an Fv-antibody library was constructed. ,,,,, The Fv-antibody library, which is autodisplayed on the outer membrane, was utilized to screen clones exhibiting binding affinity to Inf-A NP following the established protocol. First, the Fv-antibody library, incorporating random amino acid sequences of CDR3, was expressed on the outer membrane of E. coli. Inf-A NP (residues: 184–498, Code: A/Hong Kong/H090-701-V10/2009) was incubated with the Fv-antibody library under mild rotation. Following washing with PBST (PBS with 0.01% tween-20 addition), E. coli against Inf-A NP bound to His-tagged magnetic beads was spread onto agar plate (Figure A). Second, the binding specificity of the screened E. coli clones to the NP probe was evaluated by flow cytometry using a FACSCalibur system (Becton Dickinson, Franklin Lakes, NJ, USA). The cells were incubated with the NP probe (1 μM) for 1 h under gentle rotation, followed by washing with 0.1% PBST and PBS. After incubation with an anti-NP antibody and subsequent washing, the cells were labeled with an FITC-conjugated anti-mouse secondary antibody. Quantitative binding interactions were analyzed based on the fluorescence intensity (Figure B). Third, based on the DNA sequencing analysis, different amino acid sequences of the CDR3 region of the screened clones were selected. The oligonucleotide and amino acid sequences of the CDR3 region are shown in Figure C. Fourth, the plasmids for expression of the Fv-antibody were constructed through DNA cloning using the polymerase chain reaction (PCR). − Following purification of the Fv-antibodies, the binding affinity (K D) of the screened Fv-antibodies was determined using a surface plasmon resonance (SPR) biosensor. The Fv-antibodies exhibited binding affinities (K D) ranging from 1 to 500 nM. To evaluate the binding affinity using SPR, several colonies were used to evaluate the binding interactions between the NP probe and Fv-antibodies immobilized on gold-coated SPR gold chips (i-Cluebio, Seongnam, Korea). , Purified Fv-antibodies were incubated on SPR gold chips at 20 °C for 1 h. Following washing with PBS, bovine serum albumin (BSA) was used to block nonspecific binding sites. Following a further PBS washing, the association step involved flowing the NP probe (20–500 nM) over SPR gold chips at 10 μL/min for 1000 s, followed by a dissociation step using PBS under the same flow conditions for 1000 s. Following the dissociation phase, the SPR signals corresponding to the quantity of bound analytes were recorded. The SPR signals at different concentrations of the NP probe were evaluated using a dose–response curve fitted to the Hill’s equation model. −

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Screening of Fv-antibodies against Inf-A NP from the Fv-antibody library. (A) Screening of target clones using flow cytometry. (B) Selection of target clones using flow cytometry. (C) Oligonucleotide sequence (and amino acid sequences) of CDR3 of screened clones and binding constants (K D) to the NP probe from flow cytometry analysis.

Docking Simulation between Anti-NP Fv and Inf-A NP

The structure and amino acid sequence of Inf-A NP (UniProt ID: H9U5F3) were acquired from the UniProt database. To analyze the distribution of amino acid residues in the NP between CDR3 of the selected anti-NP Fv and Inf-A NP, an AutoDock Vina molecular docking simulation was conducted. ,− The docking region was analyzed and visualized using PyMOL software and the Discovery Studio Visualizer.

Purification of Ferritin H

A plasmid for NP fused ferritin H expression was inserted into BL21­(DE3) competent cells by electroporation for expression of mouse-derived ferritin H. Following plasmid insertion, E. coli was grown in 100 mL of LB medium supplemented with 100 μg/mL of carbenicillin and 1 mM of IPTG and cultured at 37 °C shaking at 150 rpm for 16 h. The E. coli cells were harvested by centrifugation and resuspended in 20 mL of 5 mM Tris-HCl buffer (pH 8.0) containing 6 M urea. E. coli cells were lysed using a VCX-130 sonicator (Sonics, Newtown, CT, USA) for 10 min using alternating cycles of 5 s on and 5 s off. After centrifugation at 25,000g for 10 min, the remaining cellular debris was discarded, and the supernatant was applied to a His-tag affinity column (Roche, Basel, Switzerland). The column was washed with 100 mL of 5 mM Tris-HCl buffer (pH 8.0) containing 6 M urea and 20 mM imidazole, and ferritin H was subsequently eluted with 10 mL of 5 mM Tris-HCl buffer (pH 8.0) containing 6 M urea and 500 mM imidazole. Urea and imidazole were removed from the eluted ferritin H sample for the generation of ferritin complexes using a dialysis membrane with mild rotation at 4 °C. During dialysis, the urea concentration was gradually reduced from 4 to 0 M, allowing ferritin H to self-assemble into ferritin complexes. − The spherical shape of the generated ferritin complexes was observed using scanning electron microscopy (SEM).

Mouse Immunization

Four groups of BALB/c mice at ages 6-weeks were obtained from Samtako (Seoul, Korea). For each mouse (five mice per group), intramuscular injections were administered three times at 14-day intervals into the gastrocnemius muscle. − The negative control group was administered only ferritin complexes along with of alum adjuvant (50 μL). The positive control group was administered ferritin complexes with NP fragment (aa 184–198, 100 μg) combined with alum adjuvant (50 μL). , Antisera were obtained 42 days after the initial intramuscular injections. All the experimental procedures were conducted in compliance with the guidelines for the “Care and Use of Laboratory Animals”. This study was approved by the Institutional Animal Care and Use Committee (IACUC, approval number: INU-ANIM-2023-25).

Evaluating the Subtypes of Antibody and Cytokine Levels in Immunized Mice

ELISA was performed to evaluate antibody subtypes. The experiments were performed with antisera after the injection of two types of ferritin complexes and two controls (negative control: only ferritin; positive control: ferritin complex with NP fragment). Initially, a 96-well Maxisorp microplate was coated with Inf-A NP at a concentration of 100 μg/mL and incubated for 16 h at 4 °C. BSA blocking was then performed to prevent nonspecific binding in each well. Subsequently, the gradient-diluted antisera were added. Each well was then incubated with anti-mouse antibodies labeled with horseradish peroxidase (HRP) and anti-mouse antibody subtypes (IgG1, IgG2a, IgG2b, and IgG3) labeled with HRP. After washing with PBST, the colorimetric reaction was performed with the addition of TMB substrate. An equal volume of sulfuric acid (2 M) was then added to quench the reaction, and the optical density (absorbance at 450 nm) was analyzed using a Microplate Reader. Each step was followed by PBS washing three times. The response level of cytokine (IL-4, IL-12, IFN-γ, and TNF-α) was measured using ELISA kits (ELM-IL4-1, ELM-Il12p40p70-1, ELM-IFNg-1, and ELM-TNFa-1) from RayBiotech (Norcross, GA, USA). The cytokine levels were analyzed according to the manufacturer’s instructions.

Competitive Binding Assay

To evaluate the specificity of antibodies in antisera against Inf-A NP, a competitive binding assay was performed. A 96-well microplate was coated with 100 μL of Inf-A NP antigen (100 μg/mL) and incubated at 37 °C for 2 h. The plate was then washed with 0.1% PBST and PBS. Subsequently, 100 μL of 1000-fold diluted mouse serum was added together with different concentrations of Fv-antibody and incubated for 1 h. Followed by washing PBST and PBS, 100 μL of HRP-conjugated anti-mouse antibody (100 ng/mL) was added and incubated for 1 h. The wells were washed again using the same washing procedure, followed by the addition of TMB substrate. After 5 min, the reaction was quenched with 2 M sulfuric acid, and the optical density was measured at 450 nm using a microplate reader.

Evaluating CD4+/CD8+ Cell Levels in the Immunized Mice

The edge of the tail of the immunized mice was clipped to collect blood samples, which were then resuspended in PBS containing 500 nM ethylenediaminetetraacetic acid. Following the addition of ammonium–chloride–potassium (ACK) lysing medium, the mixture was gently rotated for 2 min. Subsequently, the mixture was centrifuged at 4000g for 5 min, and the supernatant was removed. The pellet was resuspended in PBS (supplemented with 2% FBS and 1% PS) containing 300-fold diluted anti-mouse CD4+ monoclonal antibody with FITC labeled and 300-fold diluted anti-mouse CD8+ monoclonal antibody with PE labeled. The samples were then incubated in the dark for 30 min in a cold room. After incubation, the samples were rinsed with PBS by centrifugation at 300g for 5 min and resuspended in PBS. Finally, the CD4+/CD8+ cell levels in the samples were evaluated using flow cytometry.

Results and Discussion

Screening of Immunogenic Sequences

The immunogenic sequences (antigens) for vaccines against the nucleocapsid protein of influenza virus A were screened using an Fv-antibody library. The Fv-antibody represents the antigen-binding site of immunoglobulin G, which is composed of three CDRs and four FRs. The Fv-antibody library was prepared by site-directed mutagenesis of CDR3 with 11 amino acid residues , and expressed on the outer membrane of E. coli using autodisplay technology. − , The prepared Fv-antibody library exhibited a high surface density of >105 Fv-antibodies/E. coli and a high diversity of >105 clones/library. ,, Based on these properties, Fv-antibody libraries have previously been used to search for immunogenic sequences of various vaccines such as SARS-CoV-2 and porcine epidemic diarrhea virus (PEDV). , In this study, Fv-antibodies with a high-affinity for Inf-A NP were screened from an Fv-antibody library. The NP of Inf-A (code: A/Hong Kong/H090-701-V10/2009) was expressed as a screening probe (Figure S1). The identity and similarity of the amino acid sequence of the expressed Inf-A NP were compared with those of Inf-B NP (B/Lee/1940) and Inf-C NP (C/Lee/1978) (Figure S2A and S2B). The identity was estimated to be 37.2% for Inf-B NP and 22.0% for Inf-C NP, while the similarity was estimated to be 55.5% for Inf-B NP and 42.9% for Inf-C. These results showed that the amino acid sequences of Inf-A NP were highly conserved with Inf-B in comparison with Inf-C (Figure S2C). The expression of Inf-A NP with a molecular weight of 38 kDa was confirmed using SDS-PAGE. Fv-antibodies were screened from the Fv-antibody library using Inf-A NP. Two clones with high binding affinities were screened using flow cytometry. The screened Fv-antibodies were then expressed as soluble proteins, along with GFP. The expression of Fv-antibodies with a molecular weight of 40 kDa was confirmed by SDS-PAGE (Figure S3). The binding affinity of the Fv-antibodies was estimated after treatment with different concentrations of Inf-A NP. From the SPR analysis, the binding affinity (K D) was calculated to be 224 nM for Anti-NP-1 Fv and 123 nM for Anti-NP-2 Fv, and the sequences of screened Fv-antibodies are summarized in Figure C and Figure S4.

Preparation of a Ferritin-Based Inf-A Vaccine

The antigenic regions of the screened anti-NP Fv-antibodies were first analyzed using computer-aided docking simulations to rationally design immunogenic sequences suitable for ferritin-based nanoparticle displays. The docking results revealed that Anti-NP-1 Fv and Anti-NP-2 Fv recognize distinct surface regions of the Inf-A NP, exhibiting calculated Gibbs free energies (ΔG) of −9.2 and −7.4 kcal/mol, respectively (Figure A and C). These values indicate stable and specific interactions between the selected Fv-antibodies and NP, supporting their suitability as sources of immunogenic sequence information.

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Analysis of immunogenic sequences for vaccine development using docking simulations. (A) Distribution of highly interactive amino acids in NP with Anti-NP Fv1. (B) Ferritin complex #1 synthesis with immunogenic sequence. (C) Distribution of highly interactive amino acids in NP with Anti-NP Fv2. (D) Ferritin complex #2 synthesis with immunogenic sequence. The gaps between these highly interactive amino acids in the NP were filled with nonimmunogenic amino acid sequences (the repeating module of GGS).

Mapping of the corresponding antigenic sequences onto the three-dimensional structure of the NP showed that the identified binding regions were spatially proximal but discontinuous along the primary amino acid sequence. Because effective ferritin-based vaccines require immunogenic sequences to be expressed as a single-domain construct, direct linear expression of the native binding regions was not feasible. To address this structural constraint, the distributed antigenic segments were rationally connected using nonimmunogenic glycine–glycine–serine (GGS) linker modules. The linker lengths were determined based on the inter-residue distances derived from the NP three-dimensional structure, enabling preservation of the native spatial arrangement of the antigenic determinants while maintaining structural flexibility (Figure B and D).

The resulting engineered immunogenic sequences derived from Anti-NP-1 Fv and Anti-NP-2 Fv were subsequently genetically fused to the N-terminus of ferritin heavy-chain (H) molecules. To further enhance antigen density and multivalent presentation, each immunogenic sequence was repeated five times within a single ferritin-conjugated construct (Figure A). This design strategy was intended to maximize epitope exposure on the ferritin surface, a key factor known to promote efficient B-cell receptor cross-linking and immune activation in nanoparticle-based vaccines. Upon expression and purification, the ferritin conjugates formed nanoparticles with a predominantly spherical morphology. SEM images showed clearly distinguishable individual nanoparticles in both ferritin conjugates (Figure B). Consistent with the morphological observations, dynamic light scattering analysis indicated an average particle diameter of approximately 40–50 nm (Figure C), which is within the optimal size range for uptake by antigen-presenting cells. Collectively, these results demonstrate that the rationally designed immunogenic sequences can be successfully integrated into ferritin nanostructures without disrupting their self-assembly, thereby providing a structurally defined platform for subsequent immunological evaluation.

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Expression of the screened immunogenic sequences (antigens) with Ferritin H and analysis of antigen-conjugated ferritin complexes. (A) Schematic of ferritin complex expression. (B) SEM images of ferritin complexes. (C) Analysis of the size distribution of ferritin complexes using dynamic laser scattering.

Analysis of Antisera after Vaccination

Two ferritin vaccines (NP1 and NP2) were injected into the mice (n = 5), and two booster shots were injected every 2 weeks. As shown in Figure A, the mice were harvested and antisera were taken at the 42th day. The antibody titer was measured using 1000-fold diluted antisera in an Inf-A NP-immobilized microplate. As shown in Figure B, the titer of antibodies by ferritin vaccines (NP1 and NP2) was estimated to be higher than that by obtained the injection of ferritin-only as negative control and ferritin with NP fragment as positive control. The specificity of antibodies against NP1 and NP2 was estimated using a competition assay. As shown in Figure C, Inf-A NP was immobilized on the microplate, and the number of bound antibodies in the two types of antisera was measured by treatment with Fv-antibodies against Inf-A NP. As shown in Figure D, the binding of antibodies in the two types of antisera to the Inf-A NP decreased when the concentration of Fv-antibodies increased (7.9 nM for NP1 and 39.1 nM for NP2). These results indicate that the antibodies in the antisera specifically recognized Inf-A NP, and these antibodies competed with Fv-antibodies for Inf-A NP binding.

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Analysis of antibodies in the antisera from the immunization of mice using four ferritin complexes with immunogenic sequences. (A) Schematic of the immunization of mice with ferritin complexes. (B) Estimation of antibody activity in the antisera. (C) Schematic of the competitive assay between the antisera and Fv-antibody. (D) Competitive assay of the antisera for four kinds of ferritin complexes and Fv-antibody.

The influence of immunization with the two ferritin vaccines (NP1 and NP2) was analyzed by IgG subtyping. As shown in Figure A, IgG1, related to the activation of Th2, was increased in mice immunized with the two ferritin vaccines (NP1 and NP2) compared with the ferritin-only negative control (NC). Specifically, IgG1 levels increased by 444% and 183% in the NP1 and NP2 groups, respectively. IgG2 and IgG3, which are related to the activation of Th1, were also increased in mice immunized with the two ferritin vaccines (NP1 and NP2) compared with the ferritin-only negative control (NC). Compared with the NC group, IgG2a levels increased by 813% and 397%, IgG2b by 381% and 153%, and IgG3 by 153% and 36% in the NP1 and NP2 groups, respectively. Furthermore, the antibody subtype levels induced by NP1 and NP2 were comparable to those elicited by the positive control group immunized with ferritin complexes with Inf-A NP fragment. These results demonstrate that immunization with the NP1 and NP2 ferritin vaccines effectively induced a balanced Th1- and Th2-mediated immune response, resulting in robust production of multiple IgG subclasses.

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Evaluation of antibody, cytokine, and CD4+/CD8+ response level in the antisera from immunized mice with the injection of ferritin complexes. (A) Analysis of IgG subtypes (IgG1, IgG2a, IgG2b, and IgG3) in the antisera. (B) Analysis of cytokines (IL-4, IL12, IFN-γ, and TNF-α) in the antisera. (C) Analysis of CD4+ and CD8+ cells after immunization with ferritin complexes.

The influence of immunization with the two ferritin vaccines (NP1 and NP2) was also analyzed by measuring vaccine-related cytokines. As shown in Figure B, IL-1 and IFN-γ, related to the activation of Th1, were observed to be increased for two ferritin vaccines (NP1 and NP2) in comparison with the injection of ferritin with Inf-A NP fragment. IL-12 and TNF-α, related to the activation of Th2, were also increased for the two ferritin vaccines (NP1 and NP2) in comparison with the injection of ferritin with NP fragment. These results indicated that the immune response through Th1 and Th2 was evenly activated to produce vaccine-related cytokines by immunization with the two ferritin vaccines (NP1 and NP2).

The influence of immunization with the two ferritin vaccines (NP1 and NP2) was also analyzed by analyzing the CD4+ and CD8+ cells. As shown in Figure C, CD4+ cells, related to the production of helper T-cells, increased for the two ferritin vaccines (41.8% for NP1 and 38.6% for NP2) in comparison with the negative control level (15.2%). CD8+ cells, related to the production of killer T-cells, also increased for the two ferritin vaccines (10.1% for NP1 and 9.7% for NP2) in comparison with the negative control level (4.4%). These results indicate that the immune response through CD4+ and CD8+ cells was evenly activated to produce vaccine-related cytokines by immunization with the two ferritin vaccines (NP1 and NP2).

Conclusions

A ferritin-based Inf-A vaccine was developed. The immunogenic sequences of NP were screened using an autodisplayed Fv-antibody library, and paratopes with a high-affinity binding affinity were analyzed through docking simulations. The resulting antigenic sequences were engineered into single-domain constructs using a nonimmunogenic linker. These immunogenic sequences were expressed in conjunction with ferritin heavy-chain proteins to form spherical nanoparticles through self-assembly. These NP-ferritin complex vaccines were subsequently injected into mice, and antibody responses, cytokine secretion, and T-cell activation profiles were analyzed to demonstrate that the ferritin-based NP vaccines activated innate and acquired immune responses.

Supplementary Material

pt6c00419_si_001.pdf (479.1KB, pdf)

Acknowledgments

This work was supported by the National Research Foundation of Korea (NRF) [grant numbers RS-2026-25480724, RS-2020-NR049541, RS-2025-25460111, and RS-2024-00401422] and the Korea Health Industry Development Institute (KHIDI) [grant number RS-2025-02213534].

The data are provided in the Supporting Material, and additional data are available from the corresponding author upon reasonable request.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsptsci.6c00419.

  • Amino acids sequence of expressed NP (amino acid number of 184–498) of Inf-A nucleoprotein (Uniprot ID: H9U4F3) for screening against the Fv-antibody library (Figure S1); amino acids sequence of other influenza strains; amino acid sequences of (A) Inf-A NP and (B) Inf-B NP, (C) amino acid sequence identity and similarity of Inf-A NP between Inf-B NP and Inf-C NP (Figure S2); expression of Fv-antibodies (Anti-NP-1 Fv and Anti-NP-2 Fv) as a fusion protein by co-expression of GFP (Figure S3); estimation of the binding affinities of (A) Fv-anti-NP-1 and (B) Fv-anti-NP-2 to the NP probe (Figure S4); the binding properties of Anti-NP-1 Fv and Anti-NP-2 Fv against Inf-A NP, Inf-B NP, and SARS-CoV-2 NP (Figure S5) (PDF)

T-H.K.: investigation, formal analysis, and writing-original draft. S.K. investigation. J.J.: Investigation. J.S.S.: Investigation. D-H.H.: investigation. M-J.K.: investigation. J.Jo.: supervision. M.L.: supervision. J-C.P.: supervision, funding acquisition, writingoriginal draft, and review & editing.

The authors declare no competing financial interest.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

pt6c00419_si_001.pdf (479.1KB, pdf)

Data Availability Statement

The data are provided in the Supporting Material, and additional data are available from the corresponding author upon reasonable request.

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