Chemistry and biology of ferritin.

Iron is an essential element required by cells and has been described as a key player in ferroptosis. Ferritin operates as a fundamental iron storage protein in cells forming multimeric assemblies with crystalline iron cores. We discuss the latest findings on ferritin structure and activity and its link to cell metabolism and ferroptosis. The chemistry of iron, including its oxidation states, is important for its biological functions, its reactivity, and the biology of ferritin. Ferritin can be localized in different cellular compartments and secreted by cells with a variety of functions depending on its spatial context. Here, we discuss how cellular ferritin localization is tightly linked to its function in a tissue-specific manner, and how impairment of iron homeostasis is implicated in diseases, including cancer and coronavirus disease 2019. Ferritin is a potential biomarker and we discuss latest research where it has been employed for imaging purposes and drug delivery.

Ferritin - a multifaceted protein scaffold for biotherapeutics.

The ferritin nanocage is an endogenous protein that exists in almost all mammals. Its hollow spherical structure that naturally stores iron ions has been diversely exploited by researchers in biotherapeutics. Ferritin has excellent biosafety profiles, and the nanosized particles exhibit rapid dispersion and controlled/sustained release pharmacokinetics. Moreover, the large surface-to-volume ratio and the disassembly/reassembly behavior of the 24 monomer subunits into a sphere allow diverse modifications by chemical and genetic methods on the surface and inner cage of ferritin. Here, we critically review ferritin and its applications. We (i) introduce the application of ferritin in drug delivery; (ii) present an overview of the use of ferritin in imaging and diagnosis for biomedical purposes; (iii) discuss ferritin-based vaccines; and (iv) review ferritin-based agents currently in clinical trials. Although there are no currently approved drugs based on ferritin, this multifunctional protein scaffold shows immense potential in drug development in diverse categories, and ferritin-based drugs have recently entered phase I clinical trials. This golden shortlist of recent developments will be of immediate benefit and interest to researchers studying ferritin and other protein-based biotherapeutics.

Ultradiluted SARS-CoV-2 Spike Protein mitigates hyperinflammation in lung via ferritin and MMP-9 regulation in BALB/c mice.

AIM: This study investigated the prophylactic and therapeutic role of ultradiluted preparation of the Delta variant of SARS-CoV-2 recombinant spike (S) protein during S antigen-induced inflammatory process of disease progression along with the probable mechanism of action. MAIN METHODS: Ultradiluted S protein (UDSP) was prepared and administered orally to adult BALB/c mice before and after administration of S antigen intranasally. After an observation period of 72 h, animals were sacrificed and expression level of ferritin was assayed through ELISA. The genetic expressions of cytokines, IL-6, IL-10, IL-1ß, TNFα, IL-17, MMP-9, TIMP-1, ferritin light and heavy chains, and mitochondrial ferritin from lung tissues were investigated through RT-PCR. Formalin-fixed lung tissue sections were stained with hematoxylin and eosin to observe the degree of pathological changes. The activity of MMP-9 in lung tissues was investigated through gelatin zymography and immunofluorescence of MMP-9 in lung tissue sections was performed to revalidate the finding from gelatin zymography. Systems biology approach was used to elucidate a probable pathway where UDSP attenuated the inflammation through the regulation of pro- and anti-inflammatory cytokines. KEY FINDINGS: UDSP attenuated the S antigen-induced hyperinflammation in the lung by regulating pro- and anti-inflammatory cytokines, calming cytokine storm, reducing ferritin level both in transcriptional and translational levels, and restoring critical ratio of MMP-9: TIMP-1. SIGNIFICANCE: Our findings suggest a probable pathway by which UDSP might have attenuated inflammation through the regulation of cytokines, receptors, and other molecules. This proclaims UDSP as a promising antiviral agent in the treatment of COVID-19-induced immunopathogenesis.

Shark nanobodies with potent SARS-CoV-2 neutralizing activity and broad sarbecovirus reactivity.

Despite rapid and ongoing vaccine and therapeutic development, SARS-CoV-2 continues to evolve and evade, presenting a need for next-generation diverse therapeutic modalities. Here we show that nurse sharks immunized with SARS-CoV-2 recombinant receptor binding domain (RBD), RBD-ferritin (RFN), or spike protein ferritin nanoparticle (SpFN) immunogens elicit a set of new antigen receptor antibody (IgNAR) molecules that target two non-overlapping conserved epitopes on the spike RBD. Representative shark antibody variable NAR-Fc chimeras (ShAbs) targeting either of the two epitopes mediate cell-effector functions, with high affinity to all SARS-CoV-2 viral variants of concern, including the divergent Omicron strains. The ShAbs potently cross-neutralize SARS-CoV-2 WA-1, Alpha, Beta, Delta, Omicron BA.1 and BA.5, and SARS-CoV-1 pseudoviruses, and confer protection against SARS-CoV-2 challenge in the K18-hACE2 transgenic mouse model. Structural definition of the RBD-ShAb01-ShAb02 complex enabled design and production of multi-specific nanobodies with enhanced neutralization capacity, and picomolar affinity to divergent sarbecovirus clade 1a, 1b and 2 RBD molecules. These shark nanobodies represent potent immunotherapeutics both for current use, and future sarbecovirus pandemic preparation.

A Multivalent Vaccine Based on Ferritin Nanocage Elicits Potent Protective Immune Responses against SARS-CoV-2 Mutations.

The SARS-CoV-2 pandemic has created a global public crisis and heavily affected personal lives, healthcare systems, and global economies. Virus variants are continuously emerging, and, thus, the pandemic has been ongoing for over two years. Vaccines were rapidly developed based on the original SARS-CoV-2 (Wuhan-Hu-1) to build immunity against the coronavirus disease. However, they had a very low effect on the virus' variants due to their low cross-reactivity. In this study, a multivalent SARS-CoV-2 vaccine was developed using ferritin nanocages, which display the spike protein from the Wuhan-Hu-1, B.1.351, or B.1.429 SARS-CoV-2 on their surfaces. We show that the mixture of three SARS-CoV-2 spike-protein-displaying nanocages elicits CD4+ and CD8+ T cells and B-cell immunity successfully in vivo. Furthermore, they generate a more consistent antibody response against the B.1.351 and B.1.429 variants than a monovalent vaccine. This leads us to believe that the proposed ferritin-nanocage-based multivalent vaccine platform will provide strong protection against emerging SARS-CoV-2 variants of concern (VOCs).

mRNA-encoded HIV-1 Env trimer ferritin nanoparticles induce monoclonal antibodies that neutralize heterologous HIV-1 isolates in mice.

The success of nucleoside-modified mRNAs in lipid nanoparticles (mRNA-LNP) as COVID-19 vaccines heralded a new era of vaccine development. For HIV-1, multivalent envelope (Env) trimer protein nanoparticles are superior immunogens compared with trimers alone for priming of broadly neutralizing antibody (bnAb) B cell lineages. The successful expression of complex multivalent nanoparticle immunogens with mRNAs has not been demonstrated. Here, we show that mRNAs can encode antigenic Env trimers on ferritin nanoparticles that initiate bnAb precursor B cell expansion and induce serum autologous tier 2 neutralizing activity in bnAb precursor VH + VL knock-in mice. Next-generation sequencing demonstrates acquisition of critical mutations, and monoclonal antibodies that neutralize heterologous HIV-1 isolates are isolated. Thus, mRNA-LNP can encode complex immunogens and may be of use in design of germline-targeting and sequential boosting immunogens for HIV-1 vaccine development.

A platform incorporating trimeric antigens into self-assembling nanoparticles reveals SARS-CoV-2-spike nanoparticles to elicit substantially higher neutralizing responses than spike alone.

Antigens displayed on self-assembling nanoparticles can stimulate strong immune responses and have been playing an increasingly prominent role in structure-based vaccines. However, the development of such immunogens is often complicated by inefficiencies in their production. To alleviate this issue, we developed a plug-and-play platform using the spontaneous isopeptide-bond formation of the SpyTag:SpyCatcher system to display trimeric antigens on self-assembling nanoparticles, including the 60-subunit Aquifex aeolicus lumazine synthase (LuS) and the 24-subunit Helicobacter pylori ferritin. LuS and ferritin coupled to SpyTag expressed well in a mammalian expression system when an N-linked glycan was added to the nanoparticle surface. The respiratory syncytial virus fusion (F) glycoprotein trimer-stabilized in the prefusion conformation and fused with SpyCatcher-could be efficiently conjugated to LuS-SpyTag or ferritin-SpyTag, enabling multivalent display of F trimers with prefusion antigenicity. Similarly, F-glycoprotein trimers from human parainfluenza virus-type 3 and spike-glycoprotein trimers from SARS-CoV-2 could be displayed on LuS nanoparticles with decent yield and antigenicity. Notably, murine vaccination with 0.08 µg of SARS-CoV-2 spike-LuS nanoparticle elicited similar neutralizing responses as 2.0 µg of spike, which was ~ 25-fold higher on a weight-per-weight basis. The versatile platform described here thus allows for multivalent plug-and-play presentation on self-assembling nanoparticles of trimeric viral antigens, with SARS-CoV-2 spike-LuS nanoparticles inducing particularly potent neutralizing responses.

An immune-based biomarker signature is associated with mortality in COVID-19 patients.

Immune and inflammatory responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) contribute to disease severity of coronavirus disease 2019 (COVID-19). However, the utility of specific immune-based biomarkers to predict clinical outcome remains elusive. Here, we analyzed levels of 66 soluble biomarkers in 175 Italian patients with COVID-19 ranging from mild/moderate to critical severity and assessed type I IFN-, type II IFN-, and NF-κB-dependent whole-blood transcriptional signatures. A broad inflammatory signature was observed, implicating activation of various immune and nonhematopoietic cell subsets. Discordance between IFN-α2a protein and IFNA2 transcript levels in blood suggests that type I IFNs during COVID-19 may be primarily produced by tissue-resident cells. Multivariable analysis of patients' first samples revealed 12 biomarkers (CCL2, IL-15, soluble ST2 [sST2], NGAL, sTNFRSF1A, ferritin, IL-6, S100A9, MMP-9, IL-2, sVEGFR1, IL-10) that when increased were independently associated with mortality. Multivariate analyses of longitudinal biomarker trajectories identified 8 of the aforementioned biomarkers (IL-15, IL-2, NGAL, CCL2, MMP-9, sTNFRSF1A, sST2, IL-10) and 2 additional biomarkers (lactoferrin, CXCL9) that were substantially associated with mortality when increased, while IL-1α was associated with mortality when decreased. Among these, sST2, sTNFRSF1A, IL-10, and IL-15 were consistently higher throughout the hospitalization in patients who died versus those who recovered, suggesting that these biomarkers may provide an early warning of eventual disease outcome.