Engineering-modified universal ferritin vector vaccine and application thereof

Abstract

The invention discloses a universal ferritin carrier vaccine subjected to engineering modification and application of the universal ferritin carrier vaccine. The invention discloses a ferritin adapter, a human ferritin heavy chain mutant, a universal ferritin carrier system, an antigen fusion protein and a ferritin carrier vaccine, nucleic acid for encoding the ferritin adapter, the human ferritin heavy chain mutant, the universal ferritin carrier system, the antigen fusion protein and the ferritin carrier vaccine, a recombinant expression vector and a transformant containing the ferritin adapter, the human ferritin heavy chain mutant and the universal ferritin carrier system. According to the invention, the fact that NCOA4 (residual 484-499) is a real binding epitope of FTH1 is determined for the first time. And the Peptide and FTH1 mutants with obviously improved affinity are designed. The rabies nanoparticle vaccine based on the general type ferritin carrier system is higher in stability and can induce wider, lasting and strong immune response. 100% of protection efficiency can be generated through two-dose immunization, and a better prevention effect is achieved compared with a commercially available standard inactivated vaccine BRP.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and specifically relates to an engineered universal ferritin carrier vaccine and its application. Background Art

[0002] Ferritin is ubiquitous in various organisms, including animals, plants, bacteria, and fungi. Its primary physiological function is to immobilize iron ions in an insoluble, nontoxic state, maintaining their intracellular bioavailability. This process is crucial for maintaining iron homeostasis. Over the past few decades, protein subunit nanoparticle vaccines have attracted considerable attention due to their ability to possess repeat antigen arrays similar to those on viral surfaces, resulting in high safety, rapid production, broad applicability, strong immunogenicity, excellent stability, high biocompatibility, and the ability to elicit sustained and robust immune responses at low doses. Virus-like particles (VLPs) that deliver protein antigens have been shown to further enhance antigen immunogenicity by promoting antigen drainage to lymph nodes, enhancing antigen-presenting cell uptake, and cross-linking of B cell receptors. Due to its high safety and biocompatibility, ferritin has become a promising antigen presentation platform. Its excellent antigen presentation properties have led to its widespread application in the development of subunit nanoparticle vaccines targeting various disease-associated antigens, including influenza, Epstein-Barr virus (EBV), HIV, SARS-CoV-2, and tumors. However, the preparation of ferritin-based nanoparticle vaccines remains challenging. For example, surface antigen cross-linking through chemical coupling faces heterogeneity in coupling sites and particle coverage. Uneven modification can lead to poor antigen display and inefficient B cell activation against specific neutralizing epitopes. While the method of using target antigens to express by fusion with self-assembling protein backbone genes circumvents the introduction of non-essential immunogens, it has disadvantages such as inability of the fusion construct to express correctly or inhibition of proper folding of nanoparticles, low yield, and difficulty in large-scale production. Using Tag / Catcher technology for antigen label coupling increases the antigen valence but also leads to immune responses to non-essential adapter proteins. Therefore, addressing these challenges is crucial to promoting the development of ferritin nanoparticle-based vaccines.

[0003] For ferritin, a well-known receptor is human transferrin receptor 1 (hTfR1, also known as CD71), which is a widely expressed and multifunctional cell entry carrier whose main function is to load transferrin and ensure the import of iron supply through endocytosis when bound to ferritin in response to changes in the intracellular concentration of this essential element. The complex structure shows that FTH1 binds to the CD71 receptor through four specific contact areas of the top structure, covering a total area of This extensive interaction makes CD71 an unsuitable candidate protein for FTH1 adaptor design. Interestingly, previous studies have shown that nuclear receptor coactivator 4 (NCOA4) is also a selective receptor for ferritin, highly enriched in autophagosomes, mediating the autophagic degradation of ferritin and maintaining the body's iron homeostasis. The direct interaction between FTH1 and NCOA4 is mediated by a key surface arginine (R23) in FTH1 and the C-terminal portion of NCOA4 (residues 383-522), but the molecular mechanism of this interaction remains unclear.

[0004] Rabies, a disease of historical significance, dating back to the earliest interactions between humans and dogs nearly 40,000 years ago, remains a neglected disease, resulting in approximately 60,000 deaths annually. Currently, no treatment is available for patients who present with clinical neurological symptoms following viral exposure. Rabies vaccines have proven to be one of the most effective means of preventing infection with this deadly animal pathogen, available for both prophylactic and post-exposure prophylaxis. Since Louis Pasteur's first use of a nerve tissue-inactivated rabies vaccine in 1885, significant advances have been made in the vaccine field, including cell-cultured vaccines, modified live attenuated virus vaccines, polymer-adjuvanted vaccines, gene-modified vaccines, recombinant vaccines, nucleic acid vaccines, and oral vaccines, some of which are currently in clinical use or under development. The global strategic plan introduced in 2018 aims to eliminate global human rabies deaths from dogs by 2030. Consequently, the demand for vaccines with enhanced safety, stability, and improved immunogenicity is driving the development of next-generation rabies vaccines, such as new recombinant vaccines. The rabies virus outer membrane glycoprotein (RABV-G) is a type III viral membrane fusion protein that has similar topological domains to the fusion proteins found in vesicular stomatitis virus, Chandipura virus, baculovirus, and herpes virus. RABV-G is the only exposed spike protein on the surface of rabies virus and is a key target for interaction with neutralizing antibodies. Previous studies have shown that the third domain (G DIII ), also known as the pluvitin homology domain (PHD domain), is highly conserved in the Rhabdoviridae family or the lyssavirus genus. Several well-characterized neutralizing antibodies have been identified that recognize this specific region, effectively blocking the conformational changes of the G protein induced by the acidic environment required for membrane fusion. Summary of the Invention

[0005] In order to address the lack of an effective and universal nanoparticle vaccine platform based on human ferritin heavy chain (FTH1) in the existing technology, a new universal nanoparticle delivery platform with an engineered design is developed based on the complex structure of FTH1 and NCOA4. The present invention provides an engineered universal ferritin carrier vaccine and its application.

[0006] In order to develop a novel, universal nanoparticle vaccine platform based on human ferritin heavy chain (FTH1) and mediate the presentation of antigenic proteins through a novel adapter tag, the present invention resolved the atomic resolution complex structure of the C-terminal region of FTH1 and NCOA4 by cryo-electron microscopy (Cryo-EM), providing a structural basis for monitoring the bioavailability of intracellular iron ions under the regulation of ferritin autophagy regulation mechanism. In addition, the present invention uses AI-assisted design to engineer the wild-type C-terminal domain into a short peptide containing 16 amino acid residues with significantly improved binding affinity to ferritin, and named it Fagy-tag. In order to evaluate the prospects of this multifunctional nanoparticle platform in practical applications, the present invention selected rabies virus as the starting point for application examples, while also taking into account the clinical needs associated with this serious infectious disease.

[0007] The present invention will G DIII A fusion protein containing a transfer linker tag was obtained by gene fusion expression with Fagy-tag, which was then co-incubated with an engineered FTH1-1 high-affinity mutant to prepare a G-binding protein based on the new Fagy-tag delivery platform. DIII -Ferritin candidate rabies nanoparticle vaccine. This G DIII Ferritin nanoparticle vaccine is more stable and can induce broader and more durable humoral immune responses in the test mice, as well as stronger CD4 Th1-biased cellular immune responses and cytotoxic CD8 + T lymphocyte immune response. And G DIII - Two doses of ferritin nanoparticle vaccine can produce 100% protection efficiency against mice challenged with rabies virus intracerebral poisoning, which shows a better preventive and protective effect compared with the standard inactivated vaccine on the market. In addition, the efficient cellular immune response and humoral immunity produce neutralizing antibodies in an efficient synergistic manner, effectively clearing cells invaded by rabies virus and preventing further spread of the virus, providing a better treatment opportunity for infected animals after exposure to rabies virus, and has significant immune advantages and research and development value. The research of this invention not only provides an effective molecular biological basis for the study of the autophagy iron homeostasis regulation mechanism of ferritin and the development of ferroptosis-mediated anti-tumor inducer drugs, but also promotes the expansion of the ferritin nanobioscaffold platform, providing a promising vaccination strategy for the development of rabies vaccine.

[0008] To solve the above technical problems, one of the technical solutions provided by the present invention is: a ferritin adaptor, wherein the amino acid sequence of the ferritin adaptor is shown in SEQ ID NOs: 5 to 22.

[0009] To solve the above technical problems, the present invention provides a second technical solution: a human ferritin heavy chain mutant, the amino acid sequence of the human ferritin heavy chain mutant is shown in SEQ ID NO: 4.

[0010] To solve the above technical problems, the third technical solution provided by the present invention is: a universal ferritin carrier system, which comprises: a ferritin adapter as described in one of the technical solutions, and a ferritin nanoscaffold containing human ferritin heavy chain.

[0011] In a specific embodiment of the present invention, the amino acid sequence of the human ferritin heavy chain is as shown in SEQ ID NO: 3 or 4; and / or, the ferritin nanoscaffold is in the form of 24-mers.

[0012] In a specific embodiment of the present invention, the N-terminus and / or C-terminus of the ferritin adaptor further comprises a protection zone.

[0013] In the present invention, the binding site for the human ferritin heavy chain involves only the ferritin adaptor (i.e., the NCOA4 (484-499) region). Peptides within the N-terminal and C-terminal protection zones of the ferritin adaptor do not participate in binding to the human ferritin heavy chain. Therefore, the polypeptides within the N-terminal and C-terminal protection zones are not essential components of the universal ferritin carrier system of the present invention; their function is to maintain the stability of the ferritin adaptor to a certain extent.

[0014] In a specific embodiment of the present invention, the amino acid sequence of the N-terminal protection zone of the ferritin adaptor is shown as SEQ ID NO: 32, and / or the amino acid sequence of the C-terminal protection zone of the ferritin adaptor is shown as SEQ ID NO: 33.

[0015] To solve the above technical problems, the fourth technical solution provided by the present invention is: an antigen fusion protein, the structure of the antigen fusion protein from N-terminus to C-terminus comprises: an antigen-connector peptide-ferritin adapter, and the ferritin adapter is as described in one of the technical solutions.

[0016] In a specific embodiment of the present invention, the structure of the antigen fusion protein from N-terminus to C-terminus is: antigen-connector peptide-ferritin adapter N-terminal protection zone-ferritin adapter-ferritin adapter C-terminal protection zone.

[0017] In the present invention, the ferritin adaptor N-terminal protection zone and the ferritin adaptor C-terminal protection zone are not essential structures of the antigen fusion protein of the present invention. Their function is to maintain the stability of the ferritin adaptor to a certain extent.

[0018] In a specific embodiment of the present invention, the amino acid sequence of the connecting peptide is shown in SEQ ID NO: 28; and / or the amino acid sequence of the N-terminal protection zone of the ferritin adaptor is shown in SEQ ID NO: 32 and / or the amino acid sequence of the C-terminal protection zone of the ferritin adaptor is shown in SEQ ID NO: 33.

[0019] In a specific embodiment of the present invention, the amino acid sequence of the connecting peptide is shown in SEQ ID NO: 28, the amino acid sequence of the N-terminal protection zone of the ferritin adaptor is shown in SEQ ID NO: 32, the amino acid sequence of the ferritin adaptor is shown in SEQ ID NO: 5, 15 or 22, and the amino acid sequence of the C-terminal protection zone of the ferritin adaptor is shown in SEQ ID NO: 33.

[0020] In a specific embodiment of the present invention, the antigen is a viral antigen, such as a rabies virus antigen.

[0021] In a specific embodiment of the present invention, the rabies virus antigen is the rabies virus outer membrane glycoprotein; preferably, the third domain of the rabies virus outer membrane glycoprotein.

[0022] In a specific embodiment of the present invention, the antigen comprises a serially connected antigen fragment 1 and an antigen fragment 2, wherein the antigen fragment 1 comprises amino acids 31 to 56 of the sequence shown in SEQ ID NO: 30, and the antigen fragment 2 comprises amino acids 182 to 262 of the sequence shown in SEQ ID NO: 30.

[0023] In a specific embodiment of the present invention, the amino acid sequence of the antigen is shown in SEQ ID NO: 1.

[0024] To solve the above technical problems, the fifth technical solution provided by the present invention is: a ferritin carrier vaccine, which comprises the antigen fusion protein as described in the fourth technical solution, and a ferritin nanoscaffold containing human ferritin heavy chain.

[0025] In a specific embodiment of the present invention, the amino acid sequence of the human ferritin heavy chain is as shown in SEQ ID NO: 3 or 4; and / or, the ferritin nanoscaffold is in the form of 24-mers.

[0026] To solve the above technical problems, the sixth technical solution provided by the present invention is: a rabies nanoparticle vaccine, which comprises a rabies virus antigen fusion protein and a ferritin nanoscaffold containing a human ferritin heavy chain; the structure of the rabies virus antigen fusion protein from the N-terminus to the C-terminus comprises: a rabies virus antigen-connecting peptide-ferritin adapter, and the ferritin adapter is as described in one of the technical solutions.

[0027] In a specific embodiment of the present invention, the amino acid sequence of the human ferritin heavy chain is as shown in SEQ ID NO: 3 or 4; and / or, the ferritin nanoscaffold is in the form of 24-mers.

[0028] In a specific embodiment of the present invention, the structure of the rabies virus antigen fusion protein from N-terminus to C-terminus is: rabies virus antigen-connector peptide-ferritin adapter N-terminal protection zone-ferritin adapter-ferritin adapter C-terminal protection zone.

[0029] In the present invention, the ferritin adaptor N-terminal protection zone and the ferritin adaptor C-terminal protection zone are not essential structures of the rabies virus antigen fusion protein of the present invention. Their function is to maintain the stability of the ferritin adaptor to a certain extent.

[0030] In a specific embodiment of the present invention, the amino acid sequence of the connecting peptide is shown in SEQ ID NO: 28; and / or the amino acid sequence of the N-terminal protection zone of the ferritin adaptor is shown in SEQ ID NO: 32 and / or the amino acid sequence of the C-terminal protection zone of the ferritin adaptor is shown in SEQ ID NO: 33.

[0031] In a specific embodiment of the present invention, the amino acid sequence of the connecting peptide is shown in SEQ ID NO: 28, the amino acid sequence of the N-terminal protection zone of the ferritin adaptor is shown in SEQ ID NO: 32, the amino acid sequence of the ferritin adaptor is shown in SEQ ID NO: 5, 15 or 22, and the amino acid sequence of the C-terminal protection zone of the ferritin adaptor is shown in SEQ ID NO: 33.

[0032] In a specific embodiment of the present invention, the rabies virus antigen is the rabies virus outer membrane glycoprotein; preferably, the third domain of the rabies virus outer membrane glycoprotein.

[0033] In a specific embodiment of the present invention, the rabies virus antigen comprises a tandem antigen fragment 1 and an antigen fragment 2, wherein the antigen fragment 1 comprises amino acids 31 to 56 of the sequence shown in SEQ ID NO: 30, and the antigen fragment 2 comprises amino acids 182 to 262 of the sequence shown in SEQ ID NO: 30.

[0034] In a specific embodiment of the present invention, the amino acid sequence of the rabies virus antigen is shown in SEQ ID NO: 1.

[0035] To solve the above technical problems, the present invention provides a seventh technical solution: an isolated nucleic acid, which encodes the ferritin adaptor as described in one of the technical solutions, the human ferritin heavy chain mutant as described in the second technical solution, the universal ferritin carrier system as described in the third technical solution, the antigen fusion protein as described in the fourth technical solution, the ferritin carrier vaccine as described in the fifth technical solution, or the rabies nanoparticle vaccine as described in the sixth technical solution.

[0036] To solve the above technical problems, the eighth technical solution provided by the present invention is: a recombinant expression vector, which contains the nucleic acid as described in the seventh technical solution.

[0037] In a specific embodiment of the present invention, the backbone of the recombinant expression vector is pGEX-6P-1.

[0038] To solve the above technical problems, the present invention provides a ninth technical solution: a transformant comprising the nucleic acid as described in the seventh technical solution or the recombinant expression vector as described in the eighth technical solution.

[0039] In a specific embodiment of the present invention, the starting bacteria of the transformant is Escherichia coli, preferably Escherichia coli BL21.

[0040] To solve the above technical problems, the present invention provides a tenth technical solution: a method for preparing the ferritin adapter as described in one of the technical solutions, the human ferritin heavy chain mutant as described in the second technical solution, the universal ferritin carrier system as described in the third technical solution, the antigen fusion protein as described in the fourth technical solution, the ferritin carrier vaccine as described in the fifth technical solution, or the rabies nanoparticle vaccine as described in the sixth technical solution, the method comprising culturing the transformant as described in the ninth technical solution and obtaining the target product from the culture.

[0041] To solve the above technical problems, the present invention provides an eleventh technical solution, which is: use of the ferritin adapter described in one of the technical solutions, the human ferritin heavy chain mutant described in the second technical solution, the universal ferritin carrier system described in the third technical solution, the antigen fusion protein described in the fourth technical solution, the ferritin carrier vaccine described in the fifth technical solution, the rabies nanoparticle vaccine described in the sixth technical solution, the nucleic acid described in the seventh technical solution, the recombinant expression vector described in the eighth technical solution, or the transformant described in the ninth technical solution in the preparation of a drug for preventing and / or treating a disease.

[0042] In a specific embodiment of the present invention, the disease is an infectious disease, an autoimmune disease and / or a tumor.

[0043] In a specific embodiment of the present invention, the infectious disease is rabies, influenza, EBV, HIV or SARS-CoV-2.

[0044] On the basis of conforming to the common sense in this field, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0045] The reagents and raw materials used in the present invention are commercially available.

[0046] The positive progress effect of the present invention is:

[0047] 1. The present invention first identified NCOA4 (residue 484-499, peptide WT) as the true binding epitope of FTH1; and designed peptide mutants with higher affinity for FTH1 than peptide WT.

[0048] 2. By engineering the binding interface of wild-type FTH1, a high-affinity FTH1-1 mutant was obtained.

[0049] 3. G of the present invention DIII The Ferritin nanoparticle vaccine exhibits enhanced stability, inducing a broader and more sustained humoral immune response and a stronger cellular immune response in the mice. Furthermore, a two-dose vaccination provided 100% protection against intracerebral rabies virus challenge in mice, demonstrating superior preventive efficacy compared to the standard inactivated vaccine available on the market. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 a: 24-mer cryo-electron microscopy density map of the NCOA4(484-499) / FTH1 complex. b: Interaction analysis of the NCOA4(484-499) / FTH1 complex.

[0051] Figure 2 a: Predominant sequence selection based on key ProteinMPNN prediction parameters. b: Accurate sequence screening based on ProteinMPNNscore and AlphaFold2 pLDDT confidence. c: Multiple sequence homology alignment between peptide WT and 17 designed peptides.

[0052] Figure 3a: Peptide expression and SDS-PAGE purity analysis before and after modification. Different lanes represent different peptide mutants, marked with dots of different colors. The dot colors correspond to the peptide numbers of the same color in panel b. b: ELISA affinity curves for 17 peptides with FTH1, and for peptide 10-1 with FTH1-1. c: Specific affinity values ​​for the corresponding peptides with ferritin in panel b.

[0053] Figure 4 a: Analysis of the interaction between peptide 10 and FTH1. b: Analysis of the interaction between peptide 10-1 and FTH1-1.

[0054] Figure 5 a: Accuracy assessment of the computational design model. Structural alignment of the initial peptide model and the high-affinity computational design model. The pairwise alignment shows the RMSD values ​​between the different peptide backbones. b: Effects of key amino acid mutations on affinity.

[0055] Figure 6 .a:G DIII - Assembly model of Ferritin nanoparticle vaccine. b: AUC determination of Ferritin and G DIII -The state of Ferritin nanoparticle vaccine in solution. c:G DIII -Negatively stained electron microscopy image of Ferritin nanoparticle vaccine. Original image (left), two-dimensional classification (right). d: G DIII -Ferritin nanoparticle vaccine negative staining electron microscopy single particle three-dimensional reconstruction electron density map. e: G DIII -Three-dimensional reconstruction map of Ferritin nanoparticle vaccine and RABV G DIII PDB structural model, fitting diagram of the Peptide 10-1 / FTH1-1 PDB structural model (PDB: 8WIE).

[0056] Figure 7 a: Hydration kinetics and particle size distribution of four different rabies subunit vaccines stored at 4°C for 15 days. b: SDS-PAGE analysis of protein stability of four different rabies subunit vaccines stored at 4°C and 16°C for 15 days. c: SDS-PAGE analysis of protein stability of four different rabies subunit vaccines after multiple freeze-thaw cycles.

[0057] Figure 8 . For four rabies subunit vaccines, scaffold ferritin and three G DIIISPR kinetic test of affinity between epitope-specific neutralizing antibodies (Docaravimab, Rafivirumab, RVC20).

[0058] Figure 9 a: Immunization flow chart of BALB / c mice. b: G of each vaccine group at 2, 5, 8 and 11 weeks after the initial immunization. DIII c: Peptide10-1 / FTH1-1 complex-specific antibody titer in serum of BALB / c mice 11 weeks after full-dose immunization. d: H. pylori ferritin-specific antibody titer in serum of BALB / c mice 11 weeks after full-dose immunization.

[0059] Figure 10 a: Titers of neutralizing antibodies in the serum of mice in each immunization group at 2, 5 and 11 weeks after the initial immunization. b: Antibody-secreting cells (CD138 + c: Statistical analysis of the frequency of IgG-specific memory B cells in the germinal centers of the spleen induced by vaccines in each immunization group.

[0060] Figure 11 .a: Statistical analysis of the frequency of T and B cells in spleen lymphocytes of mice in different immunization groups reflects the bias of humoral immunity and cellular immunity. + The statistical analysis of the frequency of different T cell subtypes in T cells reflects the activation status of various T cell subsets. b: IFN-γ + Th1, IL-4 + The frequency of Th2 cells was statistically analyzed to reflect the different functional CD4 + The activation status of T cell subsets. + IFN-γ + Statistical analysis of the frequency of T lymphocytes reflects the activation status of cytotoxic effector T cells. c: Quantitative analysis of characteristic cytokines secreted by T lymphocytes isolated from the spleen of experimental mice before and after activation.

[0061] Figure 12 .a: Schematic diagram of vaccination strategy and rabies virus challenge scheme for BALB / c mice in different experimental groups. Group 1 was used as negative control and received two subcutaneous immunizations; Group 2 was the standard vaccine (BRP) group and received two subcutaneous immunizations; Group 3 was G DIII - Ferritin nanoparticle vaccine group, received one subcutaneous immunization; Group 4 was G DIII-Ferritin nanoparticle vaccine group received two subcutaneous immunizations. All experimental mice were challenged with rabies virus intracranial injection on day 14 after the initial immunization. b: Body weight change of BALB / c mice after challenge with the CVS-11 rabies virus strain (n=5). c: Virus-neutralizing antibody titer in serum of mice that survived the RABV challenge on day 30. d: Survival rate of mice within 30 days after the CVS-11 challenge (n=5). e: Real-time quantitative PCR (qRT-PCR) analysis of RABV (CVS-11) genomic RNA content in mouse brains. f: Representative images of direct immunofluorescence analysis of virus localization in mouse brain tissue. Bright apple green fluorescent particles represent rabies virus-specific nucleoprotein. Brain tissue sections from deceased mice are underlined in red, and sections from surviving mice are underlined in green. DETAILED DESCRIPTION

[0062] In recent years, ferritin-based nanoparticle vaccines have attracted widespread attention due to their good safety and superior immunogenicity. It is worth noting that candidate ferritin nanoparticle vaccines for various pathogens such as influenza, EB, SARS-CoV-2, etc. have entered the Phase I clinical trial stage. However, this technical field is faced with related problems such as particle heterogeneity, improper assembly of antigen-ferritin fusion proteins, and additional immunogenicity caused by bulky protein adapters. In response to these challenges, the present invention designed a universal ferritin adapter tag platform based on the structure of the natural ferritin-autophagy complex of human ferritin heavy chain (FTH1) and nuclear receptor coactivator 4 (NCOA4). The engineered short peptide containing 16 amino acid residues derived from NCOA4 is called Fagy tag. Although the binding epitope of ferritin is shortened to half of the original length determined in previous studies, it still shows significant improvement in binding affinity to ferritin. Subsequently, the present invention displays the third structure of the glycoprotein of rabies virus - Fagy tag fusion protein (G DIII -Fagy tag) onto 24-mer ferritin nanoparticles, rationally designing a self-assembling rabies virus nanoparticle vaccine. This innovative recombinant rabies vaccine induced rapid, potent, and long-lasting humoral and cellular immune responses. Two doses completely protected mice challenged with rabies virus from acute infection, presenting a promising vaccine approach for the prevention of rabies. Importantly, this universal vaccine platform effectively presents antigens on ferritin, maintaining excellent homogeneity and immunogenicity. It has significant potential for the development of self-assembling vaccines for infectious diseases, autoimmune diseases, malignant tumors, and other diseases.

[0063] Unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, procedures in molecular genetics, nucleic acid chemistry, chemistry, molecular biology, biochemistry, cell culture, microbiology, cell biology, genomics, and recombinant DNA used herein are conventional procedures widely used in the relevant fields.

[0064] The present invention is further illustrated by way of examples below, but the present invention is not limited to the scope of the examples. Experimental methods in the following examples where specific conditions are not specified were performed according to conventional methods and conditions, or selected according to the product specifications.

[0065] Example 1: Analysis of the cryo-electron microscopy complex structure of FTH1 and NCOA4 to clarify the true binding epitope of FTH1

[0066] First, the wild-type NCOA4 (residue 475-511, SEQ ID NO: 2) gene and the wild-type FTH1 (SEQ ID NO: 3) gene were genetically fused to generate a construct (NCOA4 (475-511)-linker-FTH1, whose sequence is shown in SEQ ID NO: 24, wherein the amino acid sequence of the linker is shown in SEQ ID NO: 23). After prokaryotic codon optimization, the fusion gene was cloned into the pGEX-6P-1 expression vector (Ubao Bio) and expressed in BL21 (DE3) E. coli cells using the E. coli expression system. Protein expression was carried out for 20 hours under the induction of IPTG (isopropyl-β-D-thiogalactopyranoside) at a final concentration of 700 μM, and preliminary purification was performed using a GST-NTA column (GE Healthcare). After removal of the GST tag by HRV3C protease (1% wt / wt), the NCOA4(475-511)-linker-FTH1 fusion protein was further purified using a Superose 6 Increase 10 / 300GL column (GE Healthcare) and equilibration buffer (50 mM Tris, 200 mM NaCl, pH 8.0). The purity of the target protein was analyzed by SDS-PAGE, and the fractions corresponding to the single major peak were collected and concentrated. The concentration was determined using a Nanodrop and stored for future use.

[0067] 3.5 μL of purified NCOA4(475-511)-linker-FTH1 fusion protein was loaded onto glow-discharged holey carbon-film Quantifoil Cu grids, adsorbed with a layer of Ted Pella filter paper for 3 seconds at 4°C and 100% humidity, and then rapidly immersed in liquid nitrogen using a Vitrobot Mark IV (Thermo Fisher Scientific). Sample images were acquired automatically in super-resolution mode using EPU 3 software on a FEI Tecnai F30 300 kV transmission electron microscope (Thermo Fisher Scientific) equipped with a K3 detector. Drift correction, particle picking, 3D reconstruction, and model refinement were performed on the acquired data using RELION 3.1.1 and CryoSPARC (v.4.0.1). The atomic-resolution structure of the FTH1 / NCOA4 complex was ultimately solved.

[0068] Structural analysis revealed for the first time that a short peptide containing 16 amino acid residues (residues 484-499) in NCOA4 (SEQ ID NO:5) is the key epitope mediating ferritin transport and autophagic degradation. NCOA4 (484-499) is composed of two short, continuous α-helices that primarily interact with Helix A, Helix C, and the BC loop of FTH1. This binding interface is reinforced by hydrogen bonds, salt bridges, and two substantial hydrophobic cores. Hydrophobic core I comprises residues L27 and Y30 of FTH1 Helix A, residues L107 and V111 of Helix C, and residues F486 and I489 of NCOA4. Hydrophobic core II comprises residues A19 and A20 of FTH1 Helix A, residues I81 and F82 of the BC loop, and residues L494, W497, L498, and I499 of NCOA4. The interaction between FTH1 and NCOA4 is also strengthened by a salt bridge between E117 and K490, and hydrogen bonds between N22 and W497, between R23 and S492, and between D90 and S485 ( Figure 1 ).

[0069] Although NCOA4 (484-499) (Peptide WT) has good affinity with FTH1, it lacks stability and engineering flexibility. In order to obtain a peptide with higher affinity for FTH1, the present invention uses ProteinMPNN to calculate and generate a targeted high-throughput peptide adapter mutation sequence set based on the complex structure analyzed by Cryo-EM. In order to improve the efficiency of subsequent calculations and the accuracy of sequence modeling, the present invention statistically analyzed the three key parameter values ​​of ddg, contact_molecular_surface and score for each predicted sequence. Sequences with advantages in ddg and contact_molecular_surface parameter evaluation were selected as the original files for subsequent structural model prediction ( Figure 2 Subsequently, under the constraints of self-assembly parameters, the initially preferred sequences were input into AlphaFold2 to generate a structural model. Finally, based on a comprehensive evaluation of multiple key parameters, the present invention selected the top 16 sequences with the highest AlphaFold2pLDDT confidence median values ​​for further analysis and characterization ( Figure 2 This multi-step sequence design and screening approach laid a solid foundation for subsequent analysis and experimental characterization.

[0070] Subsequently, the present invention fused 16 selected peptides (Peptides 01-16, SEQ ID NOs: 6-21) to the C-terminus of GST tags for short peptide expression and display. After obtaining highly pure fusion peptides, they were screened for specific FTH1 binding by ELISA. Peptide 10 exhibited an affinity of 53.11 nM for FTH1, an approximately 5.5-fold increase compared to the WT peptide. Next, the present invention simultaneously performed constraint-based computational design on Peptide 10 and FTH1, selecting the combination with the highest confidence, Peptide 10-1 (SEQ ID NO: 22) / FTH1-1 (SEQ ID NO: 4), for in vitro recombinant expression and ELISA affinity testing. Analysis showed that compared with Peptide WT / FTH1, the affinity of the peptide10-1 / FTH1-1 elite combination increased by nearly an order of magnitude to 34.08nM, laying a solid foundation for future applications ( Figure 3 ).

[0071] To evaluate the intrinsic molecular mechanism of the enhanced affinity of Peptide 10 / FTH1 and Peptide 10-1 / FTH1-1, the present invention conducted in-depth structural studies on the two complexes. Both complexes were prepared in the form of fusion proteins (Peptide 10-FTH1, SEQ ID NO: 25; Peptide 10-1-FTH1-1, SEQ ID NO: 26), and the purity of the complexes was determined by gel filtration chromatography, the hydrodynamic particle size was measured by dynamic light scattering (DLS), and the particle dispersibility was verified by negative staining electron microscopy (NS-EM). Subsequently, the present invention successfully used cryo-EM single particle technology to The high-resolution structure of the Peptide 10 / FTH1 complex was solved at a resolution of 100 nm. Structural analysis revealed that the binding pattern of Peptide 10 / FTH1 is closely similar to that of Peptide WT / FTH1, consistent with the original design of the present invention, which maintains the primary binding site unchanged. Specifically, hydrophobic core I is composed of residues L27 and Y30 from Helix_A of FTH1, residues L107 and V111 from Helix_C of FTH1, and residues F486 and I489 from Peptide 10. Hydrophobic core II is composed of residues A19 and A20 from Helix_A of FTH1, residues I81 and F82 from the BC_loop of FTH1, and residues L494, F497, L498, and A499 from Peptide 10. Interactions within the hydrophobic core are further facilitated by salt bridges between E117 and R490 and a complex hydrogen bond network between R23 and Q492, R23 and R490, D90 and T485, and N22 and F497 ( Figure 4 At the same time, the present invention also solved the crystal structure of Peptide 10-1 / FTH1-1 with a resolution of In this structure, the binding mode of the complex remains essentially unchanged compared to that of peptide WT / FTH1 and peptide 10 / FTH1. Hydrophobic core I includes residues L27 and Y30 of Helix_A of FTH1-1, residues L107 and V111 of Helix_C, and residues L486, I489, and I490 of peptide 10-1. In addition, hydrophobic core II includes residues A19 and A20 of Helix_A of FTH1-1, residues I81 and F82 of BC_loop, and residues L494, F497, and L498 of peptide 10-1. The hydrogen bonds between K23 and L490, D90 and T485, and N22 and F497 play an important role in strengthening the binding force, while the salt bridge between E117 and R490 in peptide 10 / FTH1 no longer exists because the residue at position 490 is replaced by Ile ( Figure 4 b).

[0072] Structural comparisons showed that the folding structures of peptide WT, peptide 10, and peptide 10-1 were almost identical, with the root mean square deviation (RMSD) values ​​of all atoms being less than or equal to 0.5 ( Figure 5 A). Engineered peptides have enhanced affinity for interaction with FTH1 or FTH1-1 due to minor sequence changes. For peptide 10 / FTH1, the serine at position 485 is replaced with a threonine, whose side chain methyl group increases the size of the hydrophobic core II. At the same time, the arginine at position 490 forms a strong salt bridge with E117 at a distance of Significantly shorter than that in Peptide WT / FTH1 Furthermore, because phenylalanine is more hydrophobic than tryptophan, the substitution at position 497 increases hydrophobic interactions within hydrophobic core I. Simultaneously, this substitution results in a slight conformational change in R23, leading to a more complex hydrogen-bonding network with the carbonyl oxygens of R490 and Q492. Thus, the engineering design of the present invention does not alter the intrinsic interaction mode with FTH1, yet significantly increases binding affinity. Peptide 10-1 / FTH1-1 evolved from Peptide 10 / FTH1, retaining the substitutions at positions 485 and 497. The structure reveals that the mutations at positions 486 and 490 to leucine and isoleucine, respectively, eliminate the salt bridges seen in the Peptide WT / FTH1 and Peptide 10 / FTH1 complexes, but form a more stable and compact hydrophobic core II. Furthermore, the mutations S114E and N110E in FTH1-1 contribute to the formation of hydrophobic core II due to their longer side chain methylene groups. Furthermore, the methylene group at K494 provides more hydrophobic regions for hydrophobic core I, and the Q15R mutation on FTH1-1 creates another hydrophobic pocket centered around L498. Overall, the structural analysis validates the high effectiveness of the engineering design of peptides and FTH1 according to the present invention, which is highly consistent with the design concept of the present invention.

[0073] Example 2: Study on the practicality of the novel Fagy tag nanoparticle delivery platform in the preparation of rabies nanoparticle vaccines

[0074] Based on the solid foundation of structural biology and computational biology, the present invention finally obtained a high-affinity ferritin adapter peptide 10-1 containing 16 amino acid residues derived from NCOA4 and named it Fagy tag. Then, the present invention conducted in-depth research on the practicality of the novel nanoparticle delivery platform of Fagy tag in the preparation of rabies nanoparticle vaccine. In the nanoparticle vaccine composition system, it includes: multiple G DIII -Fagytag antigen fusion protein (sequence as shown in SEQ ID NO: 27; wherein, G DIII The sequence is shown in SEQ ID NO: 1, G DIII The connection method between Fagy tag is: G DIII The C-terminus of the ferritin is connected to the N-terminus of the Fagy tag, and the intermediate linker is G3S, SEQ ID NO: 28) and a 24-mer ferritin nanoscaffold (24-mer form of FTH1-1, i.e., an octahedral nanocage structure formed by the spontaneous assembly of 24 FTH1-1 ferritin monomers), G DIII -Fagy tag antigen fusion protein spontaneously assembled onto the surface of the nanoscaffold through Fagy tag to form a multivalent nanoparticle vaccine (G DIII -Ferritin, Figure 6 In order to further study the effect of antigen presentation efficiency and potential presentation mode on vaccine immunogenicity, the present invention prepared G DIII - Ferritin nanoparticle vaccine, the average molecular weight was assessed using analytical ultracentrifugation (AUC) and statistically calculated; the results showed that each nanoparticle displayed approximately 19 G DIII Antigen, achieving nearly 80% antigen presentation efficiency, laying the foundation for the high immunogenicity of the vaccine ( Figure 6 b). Subsequently, the present invention collects G DIII -Ferritin nanoparticle vaccine negative staining electron microscopy data set, using the obtained images for single particle selection and two-dimensional classification, comparative analysis found that there is a clear consistency between the antigen presentation efficiency and the AUC calculation results ( Figure 6 c). In order to clarify G DIII The present invention uses the symmetry of O to reconstruct the single particle set of nanoparticles in three dimensions. The reconstructed electron cloud density (map) shows that G DIII The distribution of G on ferritin nanoparticles revealed that the conformation of the antigen and ferritin monomer tended to be parallel. DIII 、The PDB structural model of Peptide10-1 / FTH1-1 and G DIII-Ferritin nanoparticles reconstructed map fit, the present invention found that both PDB structures can be reasonably matched to the corresponding regions of the map, further supporting the credibility of the antigen presentation mode ( Figure 6 d, e).

[0075] In order to verify G DIII -Ferritin nanoparticle vaccine has immunological advantages over different forms of rabies subunit vaccines. In addition, G DIII Monomeric protein (G DIII , SEQ ID NO: 1), G DIII Fusion protein of human antibody Fc domain in series (G DIII The immunogenicity of three rabies virus subunit vaccines, i.e., the full-length G extracellular domain monomer protein (G, SEQ ID NO: 30), was compared in parallel. When DLS was used to measure the hydrodynamic particle size changes of the four rabies vaccines under 4°C storage conditions for 20 days, the present invention found that compared with the particle aggregation or protein degradation of the other three vaccines, G DIII -Ferritin nanoparticle vaccine showed good stability ( Figure 7 At the same time, by monitoring the protein degradation of the four vaccines when they were stored at different temperatures, it was found that under different temperature conditions, G DIII -Ferritin nanoparticle vaccines can maintain their stability, and no obvious degradation or inactivation is observed. It is exciting that the assembly of nano vaccines significantly improves G DIII Structural stability of antigenic proteins ( Figure 7 b). In addition, the present invention also carried out repeated freeze-thaw treatment on 4 vaccines and found that G DIII -Ferritin nanoparticle vaccine can still maintain its stability after multiple freeze-thaw cycles, reflecting its good adaptability in coping with cold chain transportation and storage ( Figure 7 c).

[0076] The effective display of antigen neutralizing epitopes is crucial for achieving optimal vaccine efficacy. DIII Specific neutralizing antibodies (Docaravimab, Rafivirumab and RVC20), of which Docaravimab and Rafivirumab are currently undergoing phase II clinical studies, and RVC20 has also been tested for neutralizing activity in previous articles. These antibodies target G DIII Different neutralizing epitopes of antigens. SPR affinity analysis results showed that G DIII- Ferritin nanoparticles cleverly display neutralizing epitopes of the antigen, with the expected binding profile without the risk of epitope shielding, indicating potent cross-linking and activation of rabies virus G DIII The ability of G DIII The G protein showed significant differences in affinity for the three antibodies, which may be attributed to its highly glycosylated form or pH-sensitive conformation. This finding also provides valuable insights for the development of rabies vaccines. The comprehensive analysis of all in vitro experiments showed that G protein had a higher affinity for the three antibodies than other types of rabies subunit vaccines. DIII -Ferritin nanovaccine exhibits remarkable stability and antigenic efficacy ( Figure 8 ).

[0077] For further comparison, G DIII - Ferritin nanoparticle vaccine and other rabies subunit vaccines, the present invention evaluated the immune response in mice. Mice were subcutaneously injected with 50% (v / v) Freund's adjuvant and 30μg G DIII , 30μg G DIII -Ferritin, 30μg G DIII -Fc and 30μg G protein vaccine emulsion. Immunization was performed at 0, 3 and 6 weeks, blood samples were collected at 2, 5, 8 and 11 weeks, and serum samples were analyzed by ELISA to determine G DIII The titer of G-specific IgG binding antibody was measured, and the negative control group received parallel immunization ( Figure 9 a). After the initial immunization, G DIII Ferritin nanoparticle vaccine rapidly induced G-mediated DIII The combination of antibodies and G antigens, and this immune response showed an upward trend over time, and the binding antibody titer was higher than that of other subunit vaccine groups at all blood collection time points. This result strongly reflects the G DIII -Ferritin nanoparticle vaccine has high immunogenicity. The results of four serum binding antibody ELISA experiments clearly showed that the administration of equal mass of G DIII Monomers also significantly increased IgG binding antibody levels when this given dose was normalized to the molar amount of antigen, although G DIII -G in Ferritin Nanoparticle Vaccine DIIIThe molar amount of antigen was reduced by about 50%, but it still induced a higher level of binding antibodies, which also reflects the potential correlation between the valence of the antigen and the speed and strength of the induced humoral immune response. When comparing the IgG specific binding antibody titers in serum samples after immunization with the two types of nanoparticle vaccines, the present invention observed that G DIII -Ferritin nanoparticle vaccine induced specific serum binding antibody levels significantly higher than G DIII -Fc nanoparticle vaccine, embodies G DIII -Ferritin nanoparticle vaccine induces G DIII and G protein in terms of immune response. In addition, by comparing G DIII -Ferritin and G protein immunization groups serum antibody titers at each blood collection, G DIII -Ferritin nanoparticle vaccine can induce higher IgG binding antibody titers, showing its obvious advantages in epitope aggregation and providing new insights into the development of rabies vaccines ( Figure 9 b). To further demonstrate the low immunogenicity of the scaffold, the present invention simultaneously prepared Helicobacter pylori (H. pylori) ferritin (the amino acid sequence of its monomer is shown in SEQ ID NO: 31, also prepared in the form of 24-mers) and carried out simultaneous immunization. Eleven weeks after the initial immunization, the mouse serum was identified by ELISA for antibodies that specifically bind to the ferritin protein. The results showed that the new nanoparticle scaffold has a significant advantage in reducing the introduction of non-essential immunogens ( Figure 9 c, d).

[0078] In addition to the extensive binding antibody production intensity, the ability to induce neutralizing antibodies is another key factor affecting the quality of immunity. Therefore, in order to further evaluate the neutralizing antibody levels induced by different vaccines in animal models, the present invention carried out live virus neutralization activity tests at the cellular level on the serum of mice in each immunization group at the 2nd week, the 5th week and the 11th week respectively. In the neutralization test based on live rabies virus (CVS-11), the serum antibodies of all subunit vaccine groups of the present invention exceeded the virus neutralization titer (VNT) of the preventive rabies vaccination threshold specified by the World Health Organization in the second week after single-dose immunization, and maintained a high level at different subsequent time points. Even more exciting is that the serum neutralizing antibody titer at week 11 after the initial immunization reached the highest value of the assay and showed significant differences, G DIII -Ferritin nanoparticle vaccine immunization group G DIII group is 4.5 times higher than G DIII-Fc group was 6 times higher than that of G group, and 47 times higher than that of G group. This result further verified the significant immunogenicity of the new nanoparticle vaccine. DIII -The specific BCR cross-linking caused by the superior antigen valence on the surface of the Ferritin nanoparticle vaccine is more extensive, making it easier to activate the downstream signaling pathways of B cells, which plays a decisive role in the immune effect of the vaccine. At the same time, the present invention also notes that the fusion expression of antigens and antibody Fc domains may form larger oligomers with uneven antigen presentation due to the interaction of some amino acid side chains or proteins, thereby shielding some dominant antigen epitopes, reducing the efficiency of vaccine activation on B cells, and failing to achieve the expected effect. It also provides some valuable references for the current vaccine research and development ( Figure 10 a). It is clear that G DIII After studying the important role of Ferritin nanoparticle vaccine in humoral immunity, the present invention also conducted further analysis on the related B cell response, hoping to evaluate the possible protection time of the vaccine by studying the persistence of humoral immune response. At week 11, the mice were euthanized and lymphocytes were isolated from the spleen. The plasma cell phenotype with time-sensitive antibody production and secretion was analyzed by flow cytometry, and it was found that G DIII Ferritin nanoparticle vaccine-induced CD138 + The percentage of plasma cells in the total lymphocytes of the immunized group was significantly higher than that of the other immunized groups, and the results of plasma cell activation were also very consistent with the humoral immune response reflected by the serum binding antibody titer. In order to verify the persistence and specificity of activated B cells, the present invention analyzed the frequency of IgG-specific memory B cells in the germinal center of the spleen of the test animals in different immunized groups (PBS: 0.465%, GDIII: 1.455%, GDIII-Ferritin: 2.045%, GDIII-HFC: 1.1125%, G: 1.16%), and clarified the G DIII -The remarkable ability of ferritin nanoparticle vaccine in inducing specific memory B cells ( Figure 10 b, c). In summary, it is not difficult to see that G DIII -Ferritin nanoparticle vaccine's outstanding characteristics in inducing plasma cell differentiation, IgG-specific memory B cell storage, and providing long-lasting protective humoral immune response further demonstrate its great potential as a rabies vaccine candidate.

[0079] On this basis, the present invention used flow cytometry cell surface marker staining and intracellular cytokine staining (ICS) to perform statistical analysis of the frequency of representative cell populations and activation analysis of different T cell subsets on lymphocytes isolated from the spleen, further quantifying the overall situation of vaccine-induced cellular immune response. The present invention found that different vaccines have biased activation of T cells, G DIII -Ferritin nanoparticle vaccine showed obvious advantages in inducing T cell proliferation, especially in inducing the activation of cytotoxic T cells ( Figure 11 a). It is well known that CD4 + Th cells are very important for inducing optimal antibody responses and activating cellular immunity to clear viruses. In order to further evaluate the effect of vaccines on helper T cells and verify the activation of specific T cells presented by flow cytometry, the present invention uses G DIII The isolated T cells were stimulated with antigen and PMA at the same time, and the frequency percentage of active T cell subsets was determined by flow cytometry when Golgi blockers were added. DIII -Ferritin nanoparticle vaccine and G DIII Both induced higher CD4 + IFN-γ + Th1 cells respond, while G DIII -Ferritin nano-vaccine has a more significant induction bias. DIII -Ferritin and G DIII Vaccines against CD4 + IL-4 + There was no significant difference in the activation of Th2 cells compared with the PBS control group, while G DIII CD4 in the spleen of mice immunized with Fc and G vaccines + IL-4 + The frequency percentage of Th2 cells increased slightly, which may have the risk of inducing inflammatory response and needs further verification. DIII -Ferritin nanoparticle vaccine against CD8 + IFN-γ + The effect of effector T cells is more obvious, and IFN-γ + Cytotoxic T cells in CD8 + The mean percentage of T cells was higher than that of PBS control, G DIII , G DIII -Fc and G immunization groups were ∼4-fold, ∼1.8-fold, ∼3.4-fold, and ∼2.5-fold higher, respectively ( Figure 11b). At the same time, the present invention also detected the changes in the levels of three characteristic cytokines, IFN-γ, IL-4 and Granzyme B, in the cell supernatant culture fluid before and after T cell activation without adding Golgi blockers, and obtained experimental results consistent with those of flow cytometry quantitative detection, further proving that G DIII - Ferritin's unique advantage in inducing strong and safe T cell responses ( Figure 11 c).

[0080] In order to study G DIII -Ferritin nanoparticle vaccine induces higher neutralizing antibody titers, specific T cell immune responses and better antiviral protection. The present invention conducted experimental research on rabies virus intracerebral challenge in BALB / c mouse model. It is well known that the currently available rabies inactivated vaccine must adopt a three-dose immunization strategy (0d, 7d, 28d) to produce a good protective neutralizing antibody titer in animals. In order to explore the G DIII - Ferritin's novel rabies nanoparticle vaccine has the best immunization strategy and adjustable vaccination dose. Before the brain challenge of the test animals, the present invention set up 4 parallel immunization groups, firstly activated the immune response induced by vaccination. On the 14th day after the initial immunization, each test mouse was injected with 100×LD 50 CVS-11 strain, and then monitored the survival rate and weight changes of mice caused by infection within one month ( Figure 12 After intracerebral challenge, all mice showed a trend of weight loss, with the mock group experiencing a more pronounced rate of weight loss. By day 8 of challenge, the median weight had dropped to approximately 60% of the starting weight, and all mice succumbed to the acute infection. In the standard vaccine group (BRP, commercial information: EDQM-R0100000), mice developed flushing and abnormal behavior on days 8, 9, and 10 of challenge, respectively, and succumbed to the viral infection. By day 12, the mortality rate for this group reached 100%. DIII -3 mice in the Ferritin single-dose group died of infection on the 12th day after challenge, while the surviving mice began to regain weight after the 10th day and recovered from the infection in the following days. DIII -The survival rate of mice in the Ferritin 2-dose group reached 100%, and all mice began to quickly recover their weight 10 days after the challenge. All surviving mice in the experimental groups remained healthy one month later without any adverse reactions or behavioral abnormalities ( Figure 12 b, d). When the serum of surviving mice on day 30 after the virus challenge was tested for cell-level live virus neutralization activity, it was surprisingly found that all mice maintained ultra-high levels of virus neutralizing antibody titers ( Figure 12 c). It can be seen that the relatively mild rate of weight loss in the vaccine-immunized group of mice is significantly correlated with the titer of specific neutralizing antibodies, while there is a certain delay in the activation of the body's immune response by commercially available standard vaccines, which also explains the necessity of multiple-dose immunization. At the same time, the present invention also harvested brain tissue samples of infected mice when they died after the challenge, as well as brain tissue samples of mice that survived one month after the challenge, and measured the content of RABV (CVS-11) genomic RNA in brain tissue homogenate and direct immunofluorescence localization of rabies virus in brain tissue sections. It can be clearly seen that the two doses of G DIII -Ferritin nanoparticle vaccine completely inhibited viral replication in the brains of challenged mice, which was consistent with its 100% protection efficacy. Compared with the adjuvant control group, a single dose of G DIII -Ferritin nanoparticle vaccine also significantly reduced the rabies virus load in the brains of the mice that died. Combined with the 40% survival rate of the animals in the experimental group, it is not difficult to see that a single dose of G DIII Ferritin nanoparticle vaccine immunization also has a partial protective effect on experimental animals. Compared with the adjuvant control group, the viral load in the brain of animals in all standard vaccine (BRP) groups was only non-significantly reduced, which may be related to its delayed immune activation in the body, and is also the reason why the rabies disease in the test mice in this group was more severe ( Figure 12 In conclusion, these findings further reveal that G DIII The high internalization efficiency and B cell activation response of the Ferritin nanoparticle vaccine can induce the production of larger and more concentrated neutralizing antibodies, which in turn activate the antibody-dependent cytotoxicity and complement-dependent cytotoxicity mechanisms associated with viral clearance. Furthermore, the cytotoxic effector T cells activated by this efficient cellular immune response may synergize with the neutralizing antibodies produced by humoral immunity to eliminate rabies virus-invaded cells, preventing further spread of the virus and providing a better opportunity for treatment for those infected after rabies virus exposure.

[0081] The key amino acid sequences used in the present invention are shown in Table 1 below.

[0082] Table 1

[0083]

[0084]

[0085]

[0086] Among them, the flexible connections between fusion proteins are shown in bold and underlined, and the key amino acid mutation sites are shown in bold and italics.

Claims

1. A ferritin adaptor, characterized in that The amino acid sequence of the ferritin adaptor is shown in SEQ ID NOs: 5 to 22.

2. A human ferritin heavy chain mutant, characterized in that: The amino acid sequence of the human ferritin heavy chain mutant is shown in SEQ ID NO:

4.

3. A universal ferritin carrier system, characterized in that: The universal ferritin carrier system comprises: the ferritin adaptor according to claim 1, and a ferritin nanoscaffold containing a human ferritin heavy chain; Preferably, the amino acid sequence of the human ferritin heavy chain is as shown in SEQ ID NO: 3 or 4; and / or, the ferritin nanoscaffold is in the form of 24-mers.

4. An antigen fusion protein, characterized in that The structure of the antigen fusion protein from N-terminus to C-terminus comprises: antigen-connector peptide-ferritin adaptor, and the ferritin adaptor is as described in claim 1; Preferably, the structure of the antigen fusion protein from N-terminus to C-terminus is: antigen-connector peptide-ferritin adapter N-terminal protection zone-ferritin adapter-ferritin adapter C-terminal protection zone; More preferably, the amino acid sequence of the connecting peptide is shown as SEQ ID NO: 28; and / or the amino acid sequence of the N-terminal protection zone of the ferritin adaptor is shown as SEQ ID NO: 32 and / or the amino acid sequence of the C-terminal protection zone of the ferritin adaptor is shown as SEQ ID NO: 33; Further more preferably, the amino acid sequence of the connecting peptide is shown as SEQ ID NO: 28, the amino acid sequence of the N-terminal protection zone of the ferritin adaptor is shown as SEQ ID NO: 32, the amino acid sequence of the ferritin adaptor is shown as SEQ ID NO: 5, 15 or 22, and the amino acid sequence of the C-terminal protection zone of the ferritin adaptor is shown as SEQ ID NO:

33.

5. The antigen fusion protein according to claim 4, characterized in that The antigen is a viral antigen, such as a rabies virus antigen; Preferably, the rabies virus antigen is the rabies virus outer membrane glycoprotein; preferably, the third domain of the rabies virus outer membrane glycoprotein; More preferably, the antigen comprises a tandem antigen fragment 1 and an antigen fragment 2, wherein the antigen fragment 1 comprises amino acids 31 to 56 of the sequence shown in SEQ ID NO: 30, and the antigen fragment 2 comprises amino acids 182 to 262 of the sequence shown in SEQ ID NO: 30; More preferably, the amino acid sequence of the antigen is as shown in SEQ ID NO:

1.

6. A ferritin carrier vaccine, characterized in that The ferritin carrier vaccine comprises the antigen fusion protein according to claim 4 or 5, and a ferritin nanoscaffold containing a human ferritin heavy chain; Preferably, the amino acid sequence of the human ferritin heavy chain is as shown in SEQ ID NO: 3 or 4; and / or, the ferritin nanoscaffold is in the form of 24-mers.

7. An isolated nucleic acid, characterized in that The nucleic acid encodes the ferritin adaptor according to claim 1, the human ferritin heavy chain mutant according to claim 2, the universal ferritin carrier system according to claim 3, the antigen fusion protein according to claim 4 or 5, or the ferritin carrier vaccine according to claim 6.

8. A recombinant expression vector, characterized in that: The recombinant expression vector comprises the nucleic acid according to claim 7; Preferably, the backbone of the recombinant expression vector is pGEX-6P-1.

9. A transformant, characterized in that The transformant comprises the nucleic acid according to claim 7 or the recombinant expression vector according to claim 8; Preferably, the starting bacteria of the transformant is Escherichia coli, preferably Escherichia coli BL21.

10. A method for preparing the ferritin adaptor according to claim 1, the human ferritin heavy chain mutant according to claim 2, the universal ferritin carrier system according to claim 3, the antigen fusion protein according to claim 4 or 5, or the ferritin carrier vaccine according to claim 6, characterized in that: The method comprises culturing the transformant according to claim 9 and obtaining a target product from the culture.

11. Use of the ferritin adaptor according to claim 1, the human ferritin heavy chain mutant according to claim 2, the universal ferritin carrier system according to claim 3, the antigen fusion protein according to claim 4 or 5, the ferritin carrier vaccine according to claim 6, the nucleic acid according to claim 7, the recombinant expression vector according to claim 8, or the transformant according to claim 9 in the preparation of a medicament for preventing and / or treating a disease; Preferably, the disease is an infectious disease, an autoimmune disease and / or a tumor; More preferably, the infectious disease is rabies, influenza, EBV, HIV or SARS-CoV-2.

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