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In the last decade, the interest in ferritin-based vaccines has been increasing due to their safety and immunogenicity. Candidates against a wide range of pathogens are now on Phase I clinical trials namely for influenza, Epstein-Barr, and SARS-CoV-2 viruses. Manufacturing challenges related to particle heterogeneity, improper folding of fused antigens, and antigen interference with intersubunit interactions still need to be overcome. In addition, protocols need to be standardized so that the production bioprocess becomes reproducible, allowing ferritin-based therapeutics to become readily available. In this review, the building blocks that enable the formulation of ferritin-based vaccines at an experimental stage, including design, production, and purification are presented. Novel bioengineering strategies of functionalizing ferritin nanoparticles based on modular assembly, allowing the challenges associated with genetic fusion to be circumvented, are discussed. Distinct up/down-stream approaches to produce ferritin-based vaccines and their impact on production yield and vaccine efficacy are compared. Finally, ferritin nanoparticles currently used in vaccine development and clinical trials are summarized.

Ferritin (Ft) nanoparticles are promising scaffolds for antigen display in vaccine design due to their stability, defined architecture, and biocompatibility. Enzymatic methods, such as tyrosinase catalysis, enable covalent antigen conjugation by oxidizing tyrosine residues into o -quinones that react with accessible cysteine thiols. Here, we engineered Pyrococcus furiosus ferritin (PfFt) by introducing single cysteines at defined positions (K8C, D33C, and E92C) to enable site-specific bioconjugation. All PfFt variants retained their quaternary nanoparticle structure, as confirmed by mass spectrometry, dynamic light scattering, HPLC, and mass photometry. Thiol accessibility was verified by Ellman’s assay. Using tyrosinase-mediated catalysis, we conjugated two tyrosine-tagged antigens, Rift Valley fever virus Gn and SARS-CoV-2 receptor-binding domain, to the engineered cysteines. Up to 13 antigens were displayed per 24-mer nanoparticle. Conjugation was highly specific to the engineered cysteines, and the resulting antigen-PfFt conjugates bound neutralizing antibodies with nanomolar affinities (2–7 nM), comparable to their soluble antigen counterparts. This work establishes a robust and modular strategy for precise antigen display on ferritin nanocages using tyrosinase-mediated cysteine conjugation. The platform shows strong potential for next-generation protein-based vaccines and other bioconjugate therapeutics.

Rift Valley fever (RVF) is a mosquito-borne zoonosis of major concern for human and animal health, yet no licensed human vaccine exists. Here, we engineered a self-assembling nanoparticle vaccine candidate by genetically fusing the RVF virus glycoprotein Gn to the N-terminus of a hybrid bacterial ferritin, generating nanoparticles that display 24 copies of Gn on their surface. Cryo-electron microscopy at 6 Å resolution confirmed ordered and symmetric presentation of the antigens, consistent with the structural models of ferritin and Gn. When incubated with human monocyte-derived dendritic cells, the Gn-ferritin nanoparticles were efficiently internalized and induced robust expression of maturation markers (e.g., CD54, CD83, CD86) and secretion of pro-inflammatory cytokines (e.g., IL-1β, IL-6, IL-12p40, TNF-α), in contrast to soluble Gn or ferritin controls. These findings demonstrate that ferritin nanoparticles provide a structurally defined and immunologically active platform for RVF virus antigen display, establishing a foundation for the development of safe and effective subunit vaccines against this emerging pathogen. Graphical Abstract

Self-assembling protein nanoparticles are used as a novel vaccine design platform to improve the stability and immunogenicity of safe subunit vaccines, while providing broader protection against viral infections. Infectious Hematopoietic Necrosis virus (IHNV) is the causative agent of the WOAH-listed IHN diseases for which there are currently no therapeutic treatments and no globally available commercial vaccine. In this study, by genetically fusing the virus glycoprotein to the H. pylori ferritin as a scaffold, we constructed a self-assembling IHNV nanovaccine (FerritVac). Despite the introduction of an exogenous fragment, the FerritVac NPs show excellent stability same as Ferritin NPs under different storage, pH, and temperature conditions, mimicking the harsh gastrointestinal condition of the virus main host (trout). MTT viability assays showed no cytotoxicity of FerritVac or Ferritin NPs in zebrafish cell culture (ZFL cells) incubated with different doses of up to 100 µg/mL for 14 hours. FerritVac NPs also upregulated expression of innate antiviral immunity, IHNV, and other fish rhabdovirus infection gene markers (mx, vig1, ifit5, and isg-15) in the macrophage cells of the host. In this study, we demonstrate the development of a soluble recombinant glycoprotein of IHNV in the E. coli system using the ferritin self-assembling nanoplatform, as a biocompatible, stable, and effective foundation to rescue and produce soluble protein and enable oral administration and antiviral induction for development of a complete IHNV vaccine. This self-assembling protein nanocages as novel vaccine approach offers significant commercial potential for non-mammalian and enveloped viruses.

Serious infections of (PA) in hospitals and the emergence and increase of multidrug resistance have raised an urgent need for effective vaccines. However, no vaccine has been approved to date. One possible reason for this is the limited immune response due to the lack of an efficient delivery system. Self-assembled ferritin nanoparticles are good carriers of heterogeneous antigens, which enhance the activation of immunological responses. In this study, two well-studied antigen candidates, PcrV and OprI, were selected and connected to the ferritin nanoparticle by the Spytag/SpyCatcher system to generate the nanovaccine rePO-FN. Compared to recombinant PcrV-OprI formulated with aluminum adjuvants, intramuscular immunization with adjuvant-free rePO-FN induced quick and efficient immunity and conferred protection against PA pneumonia in mice. In addition, intranasal immunization with adjuvant-free rePO-FN enhanced protective mucosal immunity. Moreover, rePO-FN exhibited good biocompatibility and safety. Our results suggest that rePO-FN is a promising vaccine candidate, as well as, provide additional evidence for the success of ferritin-based nanovaccines.

Self-assembling ferritin nanoparticle technology is a widely used vaccine development platform for enhancing the efficacy of subunit vaccines by displaying multiple antigens on nanocages. The dengue virus (DENV) envelope domain III (EDIII) protein, the most promising antigen for DENV, has been applied in vaccine development, and it is essential to evaluate the relative immunogenicity of the EDIII protein and EDIII-conjugated ferritin to show the efficiency of the ferritin delivery system compared with EDIII. In this study, we optimized the conditions for the expression of the EDIII protein in E. coli, protein purification, and refolding, and these optimization techniques were applied for the purification of EDIII ferritin nanoparticles. Thus, purified DENV2 EDIII and EDIII human ferritin heavy chain nanoparticles were immunized intramuscularly into BALB/c mice without an adjuvant, and the immunogenicity was analyzed using IgG ELISA and a serum-neutralizing assay. Purified, properly refolded, aggregate-free EDIII and EDIII ferritin proteins were obtained, and ferritin nanoparticles were identified using an electron microscope. By analyzing the immunogenicity of mouse serum, EDIII ferritin generated significantly higher IgG responses and neutralizing activity than EDIII-immunized mice. The IgG ELISA results confirmed that EDIII ferritin can induce a significantly higher IgG titer (O.D.:1.8) than EDIII (O.D.:0.05). Furthermore, EDIII ferritin produced a neutralizing titer of 1:68, whereas EDIII protein produced an average titer of 1:16, which is the serum dilution that inhibited 90% of the viruses. The longevity of the immune responses was analyzed using the serum obtained 2 months after the final immunization, and the results confirmed that EDIII ferritin induced constant immunity throughout the period.

The newly emerged Omicron subvariants demonstrate resistance to current therapeutic antibodies and an enhanced ability to evade the vaccine-induced immune responses. Among them, JN.1 sublineages are considered highly immune-evasive, underscoring the urgent need for broadly protective vaccines. Ferritin nanoparticles, with their unique hollow nanocage structure, provide an efficient antigen-display platform for next-generation vaccine development. Based on the previously constructed Delta-6P-S recombinant protein vaccine with broad-spectrum protective effects, this study optimized the S protein structure displayed on the surface of ferritin nanoparticles by comparing the immune responses induced in C57BL/6J mice. Delta-4S1158 nanoparticles, containing a truncated S-6P structure with four additional mutation sites, elicited robust S-specific immunoglobulin G (IgG), potent neutralizing antibodies, and a Th2-biased T-cell response in C57BL/6J mice, demonstrating favorable immunogenicity and safety. The JN.1-4S1158 nanoparticles, based on this structural design, induced a strong cross-neutralizing antibody response in C57BL/6J mice and conferred effective protection against Omicron BA.5, XBB, and JN.1 variants. Vaccinated mice exhibited significantly reduced viral genomic loads in trachea and lung tissues compared to controls, with no infectious virus detected. Lung tissue pathology was minimal in vaccinated mice. The JN.1-4S1158 nanoparticle vaccine demonstrates broad-spectrum protective effects against Omicron subvariants and shows potential for further development. It also provides a basis for the development of a universal SARS-CoV-2 vaccine.

AbstractFerritin, a natural iron-storage protein, has emerged as a versatile platform in nanotechnology and biomedicine due to its biocompatible 12 nm nanocage, intrinsic targeting via the transferrin receptor 1, and adaptability for diverse applications. This review integrates recent experimental and computational advances in ferritin-based nanoparticles, Ferritin is used for drug delivery, vaccine delivery, gene therapy, imaging and diagnostics, antioxidant therapy, and anti-inflammatory and neuroprotective therapies. Experimentally, ferritin nanocages achieve high-capacity loading (up to 400 molecules per cage) of therapeutics such as doxorubicin, siRNA, and CRISPR-Cas9 through pH-responsive disassembly, passive diffusion, and engineered self-assembly. Its natural TfR1 affinity enables precise tumor targeting and blood–brain barrier penetration, improving outcomes in cancers, infectious diseases, and neurological disorders. Computationally, molecular dynamics simulations predict stable antigen-ferritin interfaces. Density functional theory elucidates metal-oxide interactions in catalytic nanozymes. Machine learning classifiers leverage ferritin biomarkers for iron deficiency anemia detection, and bioinformatics tools like weighted gene co-expression network analysis and protein–protein interaction networks reveal ferritinophagy mechanisms in neurodegeneration and cancer. Docking-guided designs enhance vaccine epitope exposure and PROTAC degradation efficiency, fostering precision diagnostics and sustainable nanocarrier optimization. Despite promising preclinical results, challenges in scalability, long-term immunogenicity, and regulatory validation persist. This review highlights ferritin’s revolutionary potential in nanomedicine, proposing future directions for AI-assisted design, personalized therapies, and sustainable nanotechnology to overcome barriers for clinical use.Graphical abstract

Photodynamic therapy (PDT) using endoperoxides is to produce toxic singlet oxygen of reactive oxygen species through heat from the endoperoxides present in the medium. Compared with conventional PDT, PDT using endoperoxides has some advantages, for instance, in the presence of endoperoxide, singlet oxygen efficiency is not affected by hypoxic environment, because endoperoxides are the source of singlet oxygen. On the other hand, the kinetic stabilities of endoperoxides are important in the body temperature. Another advantage of endoperoxides is that they can produce singlet oxygen upon warming, so regardless of the wavelength of the emitted light, they can selectively produce singlet oxygen in the heated region. The aim of this study is to synthesize core-shell nanocages (10-Aft-CuS) with the synergistic effect of both photothermal therapy (PTT) and PDT by functionalizing the surface of the Apoferritin (Aft) nanocage platform with an endoperoxide derivative and forming ultra-small CuS nanoparticles in its inner cavity for the first time. So, first of all, Aft-CuS nanoparticles were obtained by the synthesis of ultra-small CuS nanoparticles in the inner cavity of Aft nanocages. Then, the surfaces of these nanocages have been functionalized with the Compound 10, an endoperoxide anthracene derivative. While the synthesized nanoparticles in this way have the synergistic effects of PTT and PDT therapies, achieving this with a natural nanoparticle has also been tested in cell cultures in vitro and in mice with progressed melanoma in vivo by increasing the effectiveness against cancer cells. 10-Aft-CuS nanocages showed high anti-tumor efficacy against malignant melanoma in vitro and in vivo.

Bone homeostasis plays a major role in supporting and protecting various organs as well as a body structure by maintaining the balance of activities of the osteoblasts and osteoclasts. Unbalanced differentiation and functions of these cells result in various skeletal diseases, such as osteoporosis, osteopetrosis, and Paget’s disease. Although various synthetic nanomaterials have been developed for bone imaging and therapy through the chemical conjugation, they are associated with serious drawbacks, including heterogeneity and random orientation, in turn resulting in low efficiency. Here, we report the synthesis of bone-targeting ferritin nanoparticles for bone imaging. Ferritin, which is a globular protein composed of 24 subunits, was employed as a carrier molecule. Bone-targeting peptides that have been reported to specifically bind to osteoblast and hydroxyapatite were genetically fused to the N-terminus of the heavy subunit of human ferritin in such a way that the peptides faced outwards. Ferritin nanoparticles with fused bone-targeting peptides were also conjugated with fluorescent dyes to assess their binding ability using osteoblast imaging and a hydroxyapatite binding assay; the results showed their specific binding with osteoblasts and hydroxyapatite. Using in vivo analysis, a specific fluorescent signal from the lower limb was observed, demonstrating a highly selective affinity of the modified nanoparticles for the bone tissue. These promising results indicate a specific binding ability of the nanoscale targeting system to the bone tissue, which might potentially be used for bone disease therapy in future clinical applications.