Molecular glue and nanoparticle vaccine compositions

Abstract

The present disclosure relates to a molecular glue based on the SpaA isopeptide bond forming pilus associated protein of B. finitimus. The present invention also relates to a nanoparticle vaccine composition formed by the molecular glue, covalent display of an immunogen on the surface of a nanoparticle by the molecular glue.

Description

Technical Field

[0001] This disclosure relates to a molecular gel, particularly a molecular gel based on the SpaA isopeptide bond formation of fimbriae-associated proteins in *Gastrodinium coeruleus*. The invention also relates to nanoparticle vaccine compositions formed via said molecular gel. Background Technology

[0002] Nanoparticle vaccines are an emerging vaccine technology that stimulates the immune system more efficiently by encapsulating antigens or nucleic acids on or inside nanoscale carriers (such as liposomes, polymers, or protein particles), mimicking the size and structure of viruses. Compared to traditional vaccines, nanoparticle vaccines offer several advantages: First, their tiny size and precise structure enhance antigen delivery efficiency and promote uptake by immune cells such as dendritic cells. Second, nanocarriers can protect antigens or nucleic acids from degradation, improving stability and immunogenicity. Furthermore, nanoparticles can be targeted and delivered via the lymphatic system, simultaneously activating humoral and cellular immunity, and even inducing mucosal immunity, thus providing more comprehensive protection. This technology also enables multivalent vaccine design, integrating different mutant strains of the same pathogen or heterologous antigens from different pathogens onto a single particle, reducing the frequency of vaccinations. Currently, nanoparticle vaccines have shown great potential in the development of vaccines for infectious diseases and cancer. Among them, bio-assembled protein nanoparticles such as Helicobacter pylori ferritin (HPF) tetratetram, Lumazine synthase (LS) hexadecimer, and fully synthetic nanoparticle I53-50 120-mer have been widely used in research on vaccines against the novel coronavirus (SARS-CoV-2) and human immunodeficiency virus type 1 (HIV-1), demonstrating great potential to induce neutralizing antibodies.

[0003] Commonly used methods for displaying antigens on nanoparticle carriers include direct fusion and indirect conjugation. Direct gene fusion presents significant challenges in expressing antigens and nanoparticle carriers within the same system. Indirect conjugation, on the other hand, involves constructing, expressing, and purifying the nanoparticle carrier and antigen separately. Both carriers carry specific tags from the conjugation system, i.e., a molecular gel. After mixing the nanoparticle carrier and antigen in vitro, the antigen specifically binds to the nanoparticles due to the tags, thus displaying the antigen on the nanoparticles. Currently, several molecular gel conjugation systems have been applied in vaccine design, including Spytag / Spycatcher, Sortase A, Streptavidin / Biotin, and Fc Tag / protein A Tag. Among these, the Spytag / Spycatcher molecular gel system has undergone several upgrades, with the latest version, Spytag003 / Spycatcher003, currently exhibiting the highest conjugation efficiency. However, designing nanoparticle vaccines that display multiple antigens presents the challenge of controlling the proportion of each antigen. Therefore, there is an urgent need to develop various cross-reactive molecular gels for use in nanoparticle vaccine design and even other fields. Summary of the Invention

[0004] To overcome the shortcomings of currently available isopeptide-based molecular glues, this disclosure provides novel molecular glues for displaying antigens on nanoparticles. This disclosure also provides a nanoparticle vaccine display system based on said molecular glue, in which an immunogen is covalently displayed on the surface of nanoparticles using the molecular glue system to prepare a nanoparticle vaccine.

[0005] According to one aspect of this disclosure, a peptide composition is provided, the peptide composition comprising a first peptide and a second peptide, the first peptide comprising amino acids corresponding to positions 88-103 of the amino acid sequence shown in SEQ ID NO: 1, or an amino acid sequence having one or more substituted, deleted, or inserted amino acid residues compared to the amino acid sequence shown therein, and the amino acid corresponding to position 97 of the amino acid sequence shown in SEQ ID NO: 1 being D; the second peptide comprising amino acids corresponding to positions 1-93 of the amino acid sequence shown in SEQ ID NO: 1, or an amino acid sequence having one or more substituted, deleted, or inserted amino acid residues compared to the amino acid sequence shown therein, and the amino acid corresponding to position 7 of the amino acid sequence shown in SEQ ID NO: 1 being K and / or the amino acid corresponding to position 54 being E.

[0006] In some embodiments, the first peptide and the second peptide can be covalently bonded to form an isopeptide bond.

[0007] In some embodiments, the first peptide comprises the amino acid sequence shown in SEQ ID NO: 2, or an amino acid sequence having one or more substituted, deleted, or inserted amino acid residues compared to SEQ ID NO: 2, wherein the 10th amino acid in the amino acid sequence of the first peptide is D.

[0008] In some embodiments, the second peptide comprises the amino acid sequence shown in SEQ ID NO: 3, or an amino acid sequence having one or more substituted, deleted, or inserted amino acid residues compared to SEQ ID NO: 3, wherein the 7th amino acid in the amino acid sequence of the second peptide is K and the 54th amino acid is E.

[0009] In some embodiments, the first peptide and the second peptide are derived from the SpaA isopeptide bond of Finegoldiamagna (FM) to form a fimbriae-associated protein.

[0010] In some embodiments, the 10th amino acid D of the first peptide and the 7th amino acid K of the second peptide form an isopeptide bond in the presence of the 54th amino acid E of the second peptide.

[0011] According to another aspect of this disclosure, a nucleic acid molecule is provided, comprising a nucleotide sequence encoding a first peptide and / or a second peptide in the peptide composition described herein.

[0012] According to another aspect of this disclosure, an expression vector is provided, comprising the nucleic acid molecules described herein.

[0013] According to another aspect of this disclosure, a host cell is provided, comprising the nucleic acid molecule or the expression vector described herein.

[0014] According to another aspect of this disclosure, a molecular glue system is provided, comprising a molecular glue formed from a first peptide and a second peptide in the peptide composition described in this disclosure.

[0015] According to another aspect of this disclosure, the application of the peptide composition, the nucleic acid molecule, the expression vector, the host cell, or the molecular glue system described herein is provided in the self-assembly of at least two molecules or components via isopeptide bonds.

[0016] In some embodiments, the two molecules or components are respectively linked to a first peptide and a second peptide in the peptide composition of this disclosure.

[0017] According to another aspect of this disclosure, a method for self-assembling two molecules or components via isopeptide bonds is provided, comprising the following steps: S1: Provide a first molecule or component and a second molecule or component comprising the first peptide and the second peptide in the peptide composition described herein; S2: Contact the first molecule or component with the second molecule or component, and self-assemble the first molecule or component with the second molecule or component through isopeptide bonds to form a complex.

[0018] According to another aspect of this disclosure, the use of the peptide composition, the nucleic acid molecule, the expression vector, the host cell, or the molecular gel system described herein in the preparation of nanoparticle vaccine compositions is provided.

[0019] According to another aspect of this disclosure, a nanoparticle vaccine composition is provided, comprising a nanoparticle core; and a peptide composition as described in this disclosure.

[0020] In some embodiments, the first peptide is linked to one of the nanoparticle core and the antigen portion to form a first fusion protein, and the second peptide is linked to the other of the nanoparticle core and the antigen portion to form a second fusion protein.

[0021] In some embodiments, the antigen portion is exposed on the outer surface of the nanoparticles.

[0022] In some embodiments, the first peptide is linked to the antigen portion.

[0023] In some embodiments, the second peptide is linked to the nanoparticle core.

[0024] In some embodiments, the nanoparticle core comprises one or more of the following: Helicobacter pylori ferritin (HPF) tetratriamer, Lumazine synthase (LS) hexadecimer, or I53-50 120-mer of fully synthetic nanoparticles.

[0025] In some embodiments, the nanoparticle vaccine includes infectious disease vaccines and / or tumor vaccines.

[0026] In some embodiments, the antigen portion comprises an active fragment of one or more of the following viruses: Middle East Respiratory Syndrome Coronavirus, novel coronavirus, influenza virus, respiratory syncytial virus, measles virus, mumps virus, rubella virus, adenovirus, varicella-zoster virus, human papillomavirus, rotavirus, hepatitis virus, or human immunodeficiency virus.

[0027] In some embodiments, the first fusion protein and the second fusion protein self-assemble to form nanoparticles.

[0028] In some embodiments, the first fusion protein includes the first peptide and the receptor-binding domain of the MERS-CoV virus Spike protein, and the second fusion protein includes the second peptide and Helicobacter pylori ferritin tetratrimester.

[0029] In some embodiments, the first fusion protein comprises the amino acid sequence shown in SEQ ID NO: 11, or an amino acid sequence having one or more substituted, deleted, or inserted amino acid residues compared to SEQ ID NO: 11.

[0030] In some embodiments, the second fusion protein comprises the amino acid sequence shown in SEQ ID NO: 6, or an amino acid sequence having one or more substituted, deleted, or inserted amino acid residues compared to SEQ ID NO: 6.

[0031] Previous reports on molecular glues have mostly been derived from the Spytag / Spycatcher system. This disclosure presents a novel molecular glue coupling system derived from SpaA isopeptide-forming pilin-related protein (GenBank: WP_172975141.1) on Finegoldia magna (FM), namely the FMtag / FMcatcher (FMt / FMc) molecular glue, which addresses the current lack of diversity in molecular glues. Through site-directed mutagenesis, it was determined that aspartic acid (D) at position 10 of FMT and lysine (K) at position 7 of FMC form an intermolecular isopeptide bond under the catalysis of glutamate (E) at position 54 of FMC. By tandemly expressing FMt with immunogens such as RBD (MERS) and FMc with nanoparticles such as HPF, the immunogen and nanoparticles of the coupled molecular glue were obtained. The two were then co-incubated to obtain a nanoparticle vaccine, such as an HPF nanoparticle vaccine that covalently presents twenty-four RBDs (MERS). The RBD(MERS)-FM-HPF nanoparticle vaccine induced RBD-specific antibody and neutralizing antibody titers that were superior to those induced by RBD(MERS) monomers, and were equivalent to the antibody titers induced by the known molecular gel nanovaccine RBD(MERS)-ST003-HPF. Attached Figure Description

[0032] Figure 1 The diagram shows the predicted structures of FM and its components FMC and FMT. FM is divided into two parts: the FMcatcher (FMc) protein and the FMtag (FMt) peptide.

[0033] Figure 2 The diagram shows the results of the binding verification of FMC and FMt. FMC was tandemly expressed at the N-terminus of GFP, and the proteins were purified separately as FMC and FMt-GFP. After incubation, the two proteins were subjected to SDS-PAGE electrophoresis and Coomassie Brilliant Blue staining. The results showed that the molecular weight of FMC was 11.390 kDa, and the molecular weight of FMt-GFP was 30.188 kDa. Furthermore, a protein band with a molecular weight approximately equal to the sum of the molecular weights of the two proteins appeared at the 41.578 kDa position in the co-incubation lane, indicating that FMC and FMt-GFP bound together and formed a covalent isopeptide bond.

[0034] Figure 3 The diagram shows the verification results of the key amino acid sites for the formation of heteropeptide bonds between FMC and FMT. FMcatcher (FMc) was tandemly expressed at the N-terminus of Ferritin (HPF) to obtain the FMC-HPF protein. Simultaneously, FMC(K7A)-HPF, FMC(E54A)-HPF, and FMT(D10A)-GFP-GST proteins were expressed and purified. FMtag (FMt) was tandemly expressed at the N-terminus of GFP-GST to obtain the FMT-GFP-GST protein. Subsequently, proteins tandemly expressed with FMT and proteins tandemly expressed with FMC were co-incubated pairwise and subjected to SDS-PAGE electrophoresis. The results in the left figure show that FMC and FMT do not bind when there is a K7A mutation in FMC or a D10A mutation in FMT. The results in the right figure show that when there is an E54A mutation in FMc, FMc and FMt will not bind, indicating that an isopeptide bond is formed between the K amino acid at position 7 of FMc and the D amino acid at position 10 of FMt, and that the E amino acid at position 54 of FMc affects the formation of the isopeptide bond.

[0035] Figure 4 A schematic diagram of the construction of the RBD(MERS)-FM-HPF nanoparticle vaccine, along with SEC and negative staining electron microscopy results, are shown. The RBD(MERS) nanoparticle vaccine, using HPF as the nanocore and FM as the molecular gel coupling system, was successfully prepared. During size exclusion chromatography (SEC) separation on a Superose 6 Increase 10 / 300 GL gel filtration column, the elution peak position was 11 mL, earlier than that of FMC-HPF and FMt-RBD(MERS), indicating the formation of uniform nanoparticles of approximately 1100 kDa. Negative staining electron microscopy results show that the nanoparticles formed a cage-like structure with uniform distribution and a particle size of approximately 50 nm.

[0036] Figure 5The results show the RBD-specific antibody titers induced by the RBD(MERS)-FM-HPF nanoparticle vaccine. Specifically, the RBD(MERS)-FM-HPF nanoparticle vaccine induced higher levels of RBD(MERS)-specific IgG antibody titers in mice, with levels superior to those induced by the RBD(MERS) monomer group. The antibody titers induced by this nanoparticle vaccine are equivalent to those induced by RBD(MERS)-ST003-HPF.

[0037] Figure 6 The neutralizing antibody titers induced by the RBD(MERS)-FM-HPF nanoparticle vaccine are shown. The RBD(MERS)-FM-HPF nanoparticle vaccine induced higher levels of neutralizing antibody titers against the MERS-CoV (Strain: HCoV-EMC / 2012) pseudovirus in C57BL / 6 mice, with antibody titer levels superior to those induced by the RBD(MERS) monomer group. The neutralizing antibody titers induced by this nanoparticle vaccine are equivalent to those induced by RBD(MERS)-ST003-HPF. Detailed Implementation

[0038] Isopeptide bonds, similar to peptide bonds, are covalent bonds. Specifically, they are amide bonds formed by the condensation of an amino or carboxyl group of at least one non-α-amino acid in a protein sequence. Examples include covalent bonds formed spontaneously by the combination of an amino group on the Lys side chain and a carboxamide group on the Asn side chain, or a carboxyl group on the Asp side chain. These include both intermolecular and intramolecular isopeptide bonds. Intermolecular isopeptide bonds are mainly formed by Lys-dominated acyl transfer reactions and play important roles in many physiological processes such as ubiquitination and glutamineting. The first reported protein to spontaneously form intramolecular isopeptide bonds was a fimbriae protein from *Streptococcus pyogenes*. The polypeptide sequence forming the isopeptide bond can be artificially split into two fragments. When expressed separately and then remixed, the two fragments can recognize each other and bind to form a functional protein. Based on this, scientists have conducted systematic research on isopeptide bonds and the stability of related protein molecules, and have developed molecular glue coupling systems for protein cross-linking, including isopeptide-N / pilin-N, isopeptide-C, Dogtag / Dogcatcher, and Spytag / Spycatcher. In addition, researchers have found that the Snooptag and Snoopcatcher peptides from the Streptococcus pneumoniae adhesive RrgA also carry Lys and Asn residues, respectively, indicating the potential for intramolecular isopeptide bond formation. However, the number of proteins currently reported that can form intramolecular isopeptide bonds is limited, restricting the development of molecular glues based on intramolecular isopeptide bonds and their application in vaccines.

[0039] The Spytag / Spycatcher conjugate protein exhibits stable isopeptide bond binding, short peptide chains, and rapid reaction, along with good thermal stability and resilience. Specifically, it originates from a region within the CnaB2 domain of the fibronectin-binding protein FbaB from Streptococcus pyogenes that spontaneously forms isopeptide bonds between Lys and Asp. Researchers have prepared peptides (Spytag) and proteins (Spycatcher) capable of forming amide bonds within minutes by dissecting this region. These peptides are then genetically encoded and fused to either end or within different proteins. When the two proteins carrying Spytag and Spycatcher are mixed, covalent fusion of the two proteins is achieved. This conjugate protein is suitable for various in vitro and in vivo applications, such as the assembly of various nonlinear protein structures, vaccine synthesis, nanobioreactors, and protein hydrogels.

[0040] However, my country currently lacks any isopeptide bond-based molecular glues with completely independent intellectual property rights, and only a few molecular glues based on isopeptide bond linkages have been reported abroad, with their cross-linking properties requiring further investigation. Therefore, there is an urgent need to develop novel molecular glues that utilize the binding specificity of different molecular glues to solve the technical challenge of simultaneously displaying equal amounts of different antigens on the same nanoparticle, thus promoting the development of nanoparticle vaccines. This would also significantly fill the intellectual property gap in this field in my country and increase the diversity of protein conjugation tools.

[0041] The technical problem this disclosure aims to solve is to overcome the shortcomings of currently available isopeptide-based molecular glues and develop more novel molecular glues for displaying antigens on nanoparticles. This disclosure extracts the intramolecular isopeptide-forming pilin-related protein (SpaA, GenBank: WP_172975141.1) from Finegoldiamagna (FM). Using AlphaFold3 prediction and structural biology analysis, the domain is split into two protein components capable of forming isopeptide bonds: the FMtag (FMt) peptide and the FMcatcher (FMc) protein. Antigens and nanoparticles carrying molecular glue tags are constructed by fusing FMt with the receptor-binding domain (RBD) of the Middle East respiratory syndrome coronavirus (MERS-CoV) Spike protein and by fusing FMc with the 24-twitch ferritin (HPF) ferrite from Helicobacter pylori. Subsequently, through in vitro spontaneous assembly, isopeptide bonds were formed to construct an RBD(MERS)-FM-HPF nanoparticle vaccine displaying twenty-four RBD(MERS) antigens. The key amino acid sites for isopeptide bond formation were confirmed using site-directed mutagenesis experiments. The antibody-inducing ability of the formed nanoparticle vaccine was verified through mouse immunization experiments.

[0042] This disclosure provides a novel molecular glue FM-based nanoparticle vaccine display system. The RBD (MERS) immunogen is covalently displayed on the surface of Helicobacter pylori ferritin (HPF) 24-twitch polymer using the FM molecular glue system to prepare a nanoparticle vaccine, and the effectiveness of the nanoparticle vaccine is fully verified.

[0043] The above-mentioned objectives of this disclosure are achieved through the following technical solutions: This disclosure first selects the SpaA isopeptide-forming pilin-related protein (GenBank: WP_172975141.1) from Finegoldia magna (FM), which contains a domain capable of forming intramolecular isopeptide bonds, as the source protein for molecular gel construction (SEQ ID NO:1). Using AlphaFold3, the domain was structurally predicted and analyzed, resulting in its separation into two molecular gel components: the FMtag (FMt) peptide and the FMcatcher (FMc) protein (SEQ ID NO:2 and SEQ ID NO:3).

[0044] Subsequently, this disclosure purified the FMc protein with a 6His tag (SEQ ID NO: 4) and constructed the FMt-GFP protein with a 6His tag by tandem expression of FMt and GFP (SEQ ID NO: 5). The ability of FMc and FMt-GFP proteins to form covalent isopeptide bonds was tested by co-incubating them.

[0045] Furthermore, this disclosure describes the tandem expression of FMc with Helicobacter pylori ferritin (HPF) nanoparticles with a 6His tag to obtain FMc-HPF protein (SEQ ID NO: 6), and the tandem expression of FMt with GFP-GST protein with a 6His tag to obtain FMt-GFP-GST protein (SEQ ID NO: 7). The 7th amino acid (lysine, K) on FMc, which is involved in the formation of isopeptide bonds, is mutated to alanine (A) to construct the 6His-tagged FMc(K7A)-HPF protein (SEQ ID NO: 8). The 54th amino acid (glutamic acid, E) on FMc, which catalyzes the formation of isopeptide bonds, is mutated to alanine (A) to obtain the 6His-tagged FMc(E54A)-HPF protein (SEQ ID NO: 9). The aspartic acid (D) at position 10 of FMt, which is involved in the formation of isopeptide bonds, was mutated to alanine (A), resulting in the FMt(D10A)-GFP-GST protein (SEQ ID NO: 10) with a 6His tag. Subsequently, HPF proteins containing FMc and its mutants were co-incubated with GFP-GST proteins containing FMt and its mutants to observe their ability to form isopeptide bonds, thereby determining the key amino acid positions for isopeptide bond formation and catalysis.

[0046] To investigate the ability of the FMt / FMc molecular gel system to present vaccine antigens, this disclosure constructs an antigen expressing the receptor-binding domain (RBD) of the Middle East respiratory syndrome coronavirus (MERS-CoV) Spike protein in tandem with the FMt peptide, namely the FMt-RBD(MERS) protein (SEQ ID NO: 11) with a 6His tag. FMt-RBD(MERS) was then co-incubated with FMC-HPF to obtain the nanoparticle vaccine RBD(MERS)-FM-HPF based on the FMt / FMc molecular gel system. The previously reported Spytag003(ST003) / Spycatcher003(SC003) molecular gel system was selected as a control. RBD(MERS) antigen conjugated with ST003 with a 6His tag (SEQ ID NO:12), HPF nanoparticles conjugated with SC003 with a 6His tag (SEQ ID NO:13), and RBD(MERS)-ST003-HPF nanoparticle vaccines were constructed.

[0047] Based on the constructed RBD (MERS) monomeric vaccine and HPF nanoparticle vaccine, C57BL / 6 mice were immunized twice with Prime / Boost. One week after the second immunization, the mice were euthanized, and serum samples were collected to determine the titers of RBD (MERS)-specific antibodies and neutralizing antibodies against the MERS-CoV (Strain: HCoV-EMC / 2012) pseudovirus. The antibody titers induced by the RBD (MERS) monomeric vaccine and the HPF nanoparticle vaccine were compared. Simultaneously, the antibody titers induced by nanoparticle vaccines based on the FMt / FMc molecular glue system and the ST003 / SC003 molecular glue system were compared to evaluate the effectiveness of the novel molecular glue system in inducing antibodies against nanoparticle vaccines.

[0048] The amino acid sequences involved in the above technical solutions are shown in Table 1 below.

[0049] Table 1

[0050] Through the above technical solutions, the present disclosure has achieved the following beneficial effects: (i) This disclosure develops a novel molecular glue FM system for vaccine antigen conjugation, which can covalently bind vaccine antigens to nanoparticle carriers by forming intermolecular isopeptide bonds, ultimately achieving high-density display of vaccine antigens.

[0051] (ii) The novel FM molecular gel system developed in this disclosure consists of two components: FMtag (FMt) peptide and FMcatcher (FMc) protein. By tandemly expressing FMt with the receptor-binding domain (RBD) of a vaccine antigen such as the coronavirus Spike protein, and tandemly expressing FMc with the nanoparticle core such as the 24-tetrameric ferritin (HPF) of Helicobacter pylori, and then co-incubating the two tandemly expressed proteins, it is possible to display antigens with the same number of valence states as the nanoparticles on the outer surface of the nanoparticles.

[0052] (III) This disclosure also identifies the key amino acids for forming and catalyzing isopeptide bonds in the novel FM molecular gel system. Aspartic acid (D) at position 10 of FMt and lysine (K) at position 7 of FMC form an intermolecular isopeptide bond under the catalysis of glutamic acid (E) at position 54 of FMC. Any mutation at these three sites prevents FMt from covalently binding with FMC.

[0053] (iv) This disclosure utilizes a novel FM molecular glue system to develop a nanoparticle vaccine against Middle East Respiratory Syndrome Coronavirus (MERS-CoV) based on the 24-tetrameric form of Helicobacter pylori ferritin (HPF). This disclosure involves fusing FMt with the RBD domain of the MERS-CoV Spike protein to obtain the FMt-RBD(MERS) antigen, and fusing FMc with HPF to obtain the FMc-HPF nanocore. Subsequently, the antigen and nanoparticles are co-incubated to obtain the RBD(MERS)-FM-HPF nanoparticle vaccine. The RBD(MERS) antigen displayed in this nanoparticle vaccine is covalently coupled to the outer surface of the nanoparticles through the action of isopeptide bonds in the FM molecular glue system. The number of RBD(MERS) antigens displayed on a single nanoparticle is equal to the valence state of the nanoparticle, both being 24.

[0054] (V) The RBD(MERS)-FM-HPF nanoparticle vaccine developed based on the novel FM molecular glue covalent coupling system disclosed in this paper has a molecular weight of 1100 kDa, a particle size of approximately 50 nm, and a uniform distribution. Through Prime / Boost double immunization in C57BL / 6 mice, it was found that the RBD(MERS)-specific antibody titer and the neutralizing antibody titer against the MERS-CoV (Strain: HCoV-EMC / 2012) pseudovirus induced by this nanoparticle vaccine were significantly higher than those in the RBD(MERS) monomeric vaccine group. Furthermore, the antibody titer induced by this novel FM molecular glue nanoparticle vaccine is equivalent to the antibody titer induced by nanoparticle vaccines developed based on the known molecular glue system Spytag003 (ST003) / Spycatcher003 (SC003).

[0055] Therefore, this disclosure develops a molecular glue FM system for displaying vaccine antigen conjugation, specifically comprising two components: an FMt peptide and an FMc protein. Simultaneously, an HPF nanoparticle vaccine targeting the RBD domain on the MERS-CoV Spike, based on the FM molecular glue system, was developed. This vaccine induced significantly higher antibody titers than the monoclonal vaccine. The novel molecular glue FM system developed in this disclosure can not only be used to display the RBD (MERS) antigen but also has reference value for vaccines against other infectious diseases and tumors. Furthermore, this FM molecular glue system can also be used for antigen display technologies mediated by other bio-self-assembled nanoparticle carriers.

[0056] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly used in the field to which this invention pertains. For the purposes of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural forms, and vice versa.

[0057] Unless the context clearly indicates otherwise, the terms “a” and “an” as used herein include plural references. For example, reference to “a cell” includes multiple such cells and equivalents known to those skilled in the art, etc.

[0058] As used herein, the term "about" indicates a range of ±20% of the following value. In some embodiments, the term "about" indicates a range of ±10% of the following value. In some embodiments, the term "about" indicates a range of ±5% of the following value.

[0059] As used in this article, the term "antigen" refers to a substance that can stimulate the body to produce a (specific) immune response and can bind in vitro with the immune response products, antibodies and sensitized lymphocytes, to produce an immune effect (specific reaction). Antigens have two basic characteristics: one is their ability to induce an immune response, i.e., immunogenicity; the other is their ability to react with the products of the immune response, i.e., antigenicity.

[0060] As used herein, the antigenic component also includes active fragments, derivatives, and analogs of the antigenic component. As used herein, the terms “fragment,” “derived,” “derived protein,” and “analyte” refer to proteins that substantially retain their activity in activating an immune response against meningococcal group B. Fragments, derivatives, or analogs of the proteins disclosed herein may be (i) polypeptides in which one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) are substituted, or (ii) polypeptides having substituents in one or more amino acid residues, or (iii) polypeptides formed by fusing the protein of the disclosed invention with another compound (e.g., a compound that prolongs the half-life of the polypeptide, such as polyethylene glycol), or (iv) polypeptides formed by fusing an additional amino acid sequence to this polypeptide sequence (derived proteins formed by fusing with a leader sequence, secretion sequence, or tag sequence such as 6His). In accordance with the teachings herein, these fragments, derivatives, and analogs may be wild-type or mutant, both of which are well known to those skilled in the art.

[0061] As used herein, the antigenic component also includes analogs of the antigenic component. These analogs may differ from the natural antigenic component of this disclosure in terms of amino acid sequence differences, or in the form of modifications that do not affect the sequence, or both. Analogs also include those having residues different from natural L-amino acids (such as D-amino acids), and those having non-naturally occurring or synthetic amino acids (such as β- or γ-amino acids). It should be understood that the polypeptides of this disclosure are not limited to the representative polypeptides exemplified above.

[0062] As used herein, the term "polynucleotide" refers to a polymer of nucleotides of any length, including ribonucleotides, deoxyribonucleotides, their analogues, or mixtures thereof. This term refers to the primary structure of the molecule. Therefore, the term includes triple-stranded, double-stranded, and single-stranded deoxyribonucleic acid ("DNA"), and triple-stranded, double-stranded, and single-stranded ribonucleic acid ("RNA"). It also includes modified (e.g., by alkylation and / or by capping) and unmodified forms of polynucleotides. More specifically, the term "polynucleotide" includes polydeoxyribonucleotides (containing 2-deoxy-D-ribose); polyribonucleotides (containing D-ribose), including tRNA, rRNA, hRNA, siRNA, and mRNA, whether spliced ​​or unspliced; any other type of polynucleotide that is an N- or C-glycoside of a purine or pyrimidine base; and other polymers containing a positive nucleotide backbone, such as polyamides (e.g., peptide nucleic acid "PNA") and polymorpholino polymers; and other synthetic sequence-specific nucleic acid polymers, provided that the polymer contains nucleosides in a configuration that allows base pairing and base stacking, as found in DNA and RNA.

[0063] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to amino acid polymers of any length. The polymers may include modified amino acids. The term also covers amino acid polymers that have been modified naturally or through intervention; for example, by disulfide bond formation, glycosylation, esterification, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeled component. The definition also includes, for example, polypeptides containing one or more amino acid analogs (including, for example, non-natural amino acids such as homocysteine, ornithine, p-acetylphenylalanine, D-amino acids, and creatine) and other modifications known in the art.

[0064] In this text, the amino acid sequence of the peptide represents: A for alanine (Ala), R for arginine (Arg), N for asparagine (Asn), D for aspartic acid (Asp), C for cysteine ​​(Cys), Q for glutamine (Gln), and E for glutamic acid (Glutamic acid). (Acid, Glu), G represents glycine (Gly), H represents histidine (His), I represents isoleucine (Ile), L represents leucine (Leu), K represents lysine (Lys), F represents phenylalanine (Phe), P represents proline (Pro), S represents serine (Ser), T represents threonine (Thr), W represents tryptophan (Trp), Y represents tyrosine (Tyr), and V represents valine (Val). Unless otherwise stated, amino acids (residues) mentioned herein may be D-type or L-type. Unless otherwise stated, the amino acid sequences mentioned herein are in N-terminus to C-terminus order from left to right.

[0065] The peptides of the present invention may be obtained by substituting one or more conserved amino acids into the peptide. In some embodiments, the substitution of a conserved amino acid may mean replacing an amino acid residue with a biologically similar residue. Particularly preferred substitutions are generally conserved in nature, i.e., those that occur within amino acid families. For example, amino acids are generally classified into four families: (1) acidic - aspartic acid (N) and glutamic acid (E); (2) basic - lysine (K), arginine (R), histidine (H); (3) nonpolar - alanine (A), valine (V), leucine (L), isoleucine (I), proline (P), phenylalanine (F), methionine (M), tryptophan (W); (4) uncharged polar - glycine (G), asparagine (D), glutamine (Q), cysteine ​​(C), serine (S), threonine (T), tyrosine (Y). Phenylalanine, tryptophan, and tyrosine are sometimes classified as aromatic amino acids. Examples of conserved changes include replacing one hydrophobic residue with another, such as isoleucine, valine, leucine, or methionine; or replacing one polar residue with another, such as replacing lysine with arginine, aspartic acid with glutamic acid, or asparagine with glutamine; or similar conserved substitutions of amino acids for structurally related amino acids that do not significantly affect biological activity. Therefore, proteins having a substantially identical amino acid sequence to the reference molecule but with a few amino acid substitutions that substantially do not affect protein activity fall within the definition of the reference polypeptide.

[0066] As used herein, the term "pharmaceuticalally acceptable carrier" refers to a component of a pharmaceutical composition that is non-toxic to the subject, other than the active ingredient. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives. In some embodiments, said pharmaceutically acceptable carriers include: polymers of acrylic acid or methacrylic acid, maleic anhydride, and alkenyl derivatives; immunostimulatory sequences (ISS), such as oligodeoxyribonucleotide sequences (CpG ODN) having one or more unmethylated CpG units; water-in-oil (W / O), oil-in-water (O / W), or water-in-oil-in-oil (W / O / W) adjuvants, such as Freund's adjuvant, SPT emulsion, MF59, ISA 206, ISA72, adjuvant-65, SAF, etc.; cationic lipids containing quaternary ammonium salts such as DDA; cytokines; aluminum hydroxide or aluminum phosphate; saponins (e.g., Quil A, QS-21, GPI-0100); or any combination or mixture thereof.

[0067] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention in any way. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concepts of this disclosure. Such structures and techniques have also been described in many publications.

[0068] Example 1. FM Structure Prediction and Decomposition into FMcatcher (FMc) and FMtag (FMt) Component Design In this embodiment, the intramolecular isopeptide-forming pilin-related protein (GenBank: WP_172975141.1) of *Finegoldia magna* (FM) was analyzed using AlphaFold3 structural prediction, which split the FM protein into two components: FMcatcher (FMc) protein and FMtag (FMt) peptide. Figure 1 The following experiment will verify whether FMC and FMT can form heteropeptide bonds.

[0069] Example 2. Verification of the binding of FMC and FMT and the formation of key amino acid sites for isopeptide bonds. In this embodiment, FMc protein (FMc-6His, SEQ ID NO: 4) and FMT-GFP protein (FMt-GFP-6His, SEQ ID NO: 5), which expresses FMt fused to the N-terminus of GFP, were purified and used to verify the binding ability of FMc and FMt. The purpose of fusing FMt with GFP is to reduce measurement bias caused by the small molecular weight of FMt and to separate it from FMc protein as much as possible during electrophoresis. The specific implementation steps are as follows: 1. Construction of expression cloning plasmids: The FMC expression gene and the FMT-GFP fusion gene were constructed into the pET28a plasmid vector using molecular cloning technology.

[0070] 2. Plasmid transformation into prokaryotic cells: The plasmid was transformed into competent E. coli BL21 cells and plated on LB agar plates (containing 0.05 mg / mL kanamycin). Single colonies were picked and transferred to LB medium containing 0.05 mg / mL kanamycin and cultured on a shaker at 220 rpm and 37°C. When the OD 600 nm absorbance of the bacterial culture reached 0.5, 1 mM of the protein expression inducer isopropyl β-D-thiogalactoside (IPTG) was added. The cells were then cultured on a shaker at 220 rpm and 16°C for 16 hours.

[0071] 3. Protein purification using affinity chromatography: Centrifuge the overnight culture at 9000 rpm for 3 minutes and discard the supernatant. Resuspend the bacteria in buffer (20 mM Tirs-HCl, 50 mM NaCl, pH=7.4) and autoclave the bacterial pellet. Centrifuge again at 9000 rpm, 4℃ for 30 minutes, collect the supernatant, and filter once through a 0.45 μm filter membrane. Since both FMC and FMt-GFP proteins carry a 6His tag, incubating the supernatant with cobalt affinity chromatography material will enrich the target protein. Proteins were eluted by adding buffers containing multiple concentrations of imidazole (10 mM, 20 mM, 50 mM, 100 mM, 200 mM, 300 mM) one by one to the cobalt packing material. Samples were collected in separate tubes, and each tube was sampled for SDS-PAGE electrophoresis and Coomassie brilliant blue staining. The protein eluent with better purity was selected and concentrated using an ultrafiltration tube to obtain FMC and FMt-GFP proteins.

[0072] 4. After incubating the two proteins, FMc and FMt-GFP, SDS-PAGE electrophoresis and Coomassie brilliant blue staining were performed. The results showed that the molecular weight of FMc protein was 11.390 kDa, and the molecular weight of FMt-GFP protein was 30.188 kDa. A protein band with a molecular weight approximately equal to the sum of the two proteins' molecular weights (41.578 kDa) was observed after incubation, indicating that FMc and FMt-GFP were covalently bound and formed heteropeptide bonds. Figure 2 ).

[0073] Furthermore, in this embodiment, the same experimental method described above was used to express and purify the FMC-HPF protein (FMc-HPF-6His, SEQ ID NO: 6) which fused to the N-terminus of Helicobacter pylori ferritin (HPF) and the FMC-GFP-GST protein (FMt-GFP-GST-6His, SEQ ID NO: 7) which fused to the N-terminus of GFP-GST protein. Based on the AlphaFold3 structure prediction results of the FM protein in Example 1, it was found that the 7th amino acid lysine (K) of FMC and the 10th amino acid aspartic acid (D) of FMC are most likely to form an isopeptide bond under the catalysis of the 54th amino acid glutamate (E) of FMC. Therefore, this embodiment also expresses and purifies mutant proteins with these sites mutated to alanine (A), including: FMc(K7A)-HPF (FMc(K7A)-HPF-6His, SEQ ID NO: 8), FMc(E54A)-HPF (FMc(E54A)-HPF-6His, SEQ ID NO: 9) and FMt(D10A)-GFP-GST (FMt(D10A)-GFP-GST-6His, SEQ ID NO: 10).

[0074] The proteins were incubated in pairs and then subjected to SDS-PAGE electrophoresis and Coomassie brilliant blue staining. The results showed that when FMC contained a K7A mutation or E54A mutation, or FMt contained a D10A mutation, the proteins expressing both FMC and FMt did not undergo covalent binding. Figure 3 These results indicate that an isopeptide bond is formed between the 7th K amino acid in FMc and the 10th D amino acid in FMt, and that the 54th E amino acid in FMc is responsible for catalyzing the formation of this isopeptide bond.

[0075] Example 3. Construction of RBD(MERS)-FM-HPF nanoparticle vaccine Using the experimental method described in Example 2, the FMc-HPF fusion gene was constructed into the pET28a plasmid vector and transformed into E. coli competent cells BL21, induced expression, and purified to obtain the FMc-HPF protein.

[0076] To verify the ability of FM molecular glue as a covalent coupling system to express vaccine antigens onto nanoparticles, this embodiment uses Middle East respiratory syndrome coronavirus (MERS-CoV) as the vaccine research object. The receptor-binding domain (RBD), a key structural domain on the MERS-CoV Spike protein that mediates the production of neutralizing antibodies, was selected as the test antigen. The FMt-RBD(MERS) protein (FMt-RBD(MERS)-6His, SEQ ID NO:11), in which the FMt peptide is fused to the N-terminus of the RBD(MERS) protein, was expressed and purified. Figure 4 The specific implementation steps are as follows: 1. Construction of expression cloning plasmid: A signal peptide (SP) sequence (MGILPSPGMPALLSLVSLLSVLLMGCVA, SEQ ID NO: 14) was added to the N-terminus of the FMt-RBD(MERS) fusion gene, and a 6His tag sequence was added to the C-terminus. This was then constructed into the pcDNA3.1 plasmid vector, transformed into *E. coli* DH5α competent cells, and plated on LB agar plates containing ampicillin (0.1 mg / mL). Single colonies were picked and cultured on LB medium (containing ampicillin 0.1 mg / mL) at 220 rpm and 37°C for 16 h. Afterwards, the cells were centrifuged at 9000 rpm for 3 min, the supernatant was discarded, and the plasmid was extracted using a plasmid extraction kit.

[0077] 2. Plasmid transfection into eukaryotic cells: The pcDNA3.1-SP-FMt-RBD(MERS)-6His plasmid was transfected into HEK293F eukaryotic cells at a ratio of 1.25 μg plasmid per 1 mL of cells. The amount of PEIMAX transfection reagent used was 4 times the mass of the plasmid, i.e., 5 μL. If transfecting 500 mL of cells, add 2.5 mL of PEIMAX to 20 mL of Opti-MEM medium, mix well, and let stand for 5 min; add 625 μg of plasmid to 20 mL of Opti-MEM medium and mix well; after 5 min, add diluted PEIMAX to the diluted plasmid tube, mix well, and let stand for 20 min; then add the mixture dropwise to 500 mL of cells and incubate in a 140 rpm, 8% CO2 cell shaker; 6 h after transfection, add 3.5 mL of sodium valproate (VPA) to inhibit cell growth and promote protein expression; add cell feed once on day 1 and once on day 3 after transfection.

[0078] 3. Protein purification using affinity chromatography: Seven days after transfection, the cell culture supernatant was collected by centrifugation at 9000 rpm for 5 min. The supernatant was then incubated with nickel affinity chromatography packing material. Proteins were eluted by adding different concentrations of imidazole buffer (10 mM, 20 mM, 50 mM, 100 mM, 200 mM, 500 mM) to the nickel packing material. Samples were collected in separate tubes, and each tube was subjected to SDS-PAGE electrophoresis and Coomassie brilliant blue staining. The protein eluent with the highest purity was concentrated using ultrafiltration to obtain FMt-RBD(MERS) protein. FMt-RBD(MERS) protein was stored in buffer (20 mM Tirs-HCl, 50 mM NaCl, pH=7.4).

[0079] In this embodiment, FMc-HPF and FMt-RBD (MERS) proteins were mixed and incubated at a 1:1 molar ratio to form RBD(MERS)-FM-HPF nanoparticle vaccines. These vaccines were then loaded onto a Superose 6 Increase 10 / 300GL gel filtration chromatography column for size exclusion chromatography (SEC). Protein solutions at the A280 peak were collected in 1 mL samples per tube, and each tube was sampled for SDS-PAGE electrophoresis and Coomassie Brilliant Blue staining. Based on the Coomassie Brilliant Blue staining results, the peak position of FMt-RBD (MERS) was 16 mL, the peak position of FMc-HPF was 13 mL, and the peak position of RBD(MERS)-FM-HPF was 11 mL. Figure 4 The above results indicate that FMt-RBD(MERS) was successfully covalently coupled to HPF nanoparticles.

[0080] In this embodiment, the RBD(MERS)-FM-HPF nanoparticle vaccine separated by a Superose 6 Increase 10 / 300 GL chromatography column was further characterized by negative staining electron microscopy. The specific implementation steps are as follows: 1. Glow discharge: The copper mesh required for the nanoparticle vaccine was subjected to glow discharge for 30 seconds using a glow discharge instrument.

[0081] 2. Prepare protein samples and staining solution: Centrifuge the RBD(MERS)-FM-HPF nanoparticle protein samples and staining solution (2% uranium acetate) at 12000 rpm for 3 min respectively, and transfer the supernatant to a new tube to remove insoluble precipitates.

[0082] 3. Loading the sample onto the copper mesh: Add 5 μL of RBD(MERS)-FM-HPF protein sample onto the sealing film. Hold the copper mesh with self-locking tweezers and bring it close to the sample on the sealing film. The sample will be adsorbed onto the copper mesh. Let the sample solution stand on the membrane for about 1 minute so that the sample particles can be adsorbed onto the membrane surface. Then, bring the copper mesh close to the filter paper to absorb the excess sample solution.

[0083] 4. Staining: Add 5 μL of staining solution (2% uranium acetate) to the sealing film and stain three times. Hold the copper mesh with self-locking tweezers and bring it close to the staining solution on the sealing film. The staining agent will be absorbed onto the copper mesh. Remove the staining solution with filter paper immediately after the first drop is applied; remove the staining solution with filter paper immediately after the second drop is applied; remove the excess staining solution with filter paper after 2 minutes of staining with the third drop and let the copper mesh dry.

[0084] 5. Electron Microscopy Observation: Nanoparticles on the copper mesh were observed using a 120 kV Talos L120C transmission electron microscope. The results showed that the RBD(MERS)-FM-HPF nanoparticles formed a cage-like structure with uniform distribution and a particle size of approximately 50 nm. Figure 4 ).

[0085] Example 4. Mouse Immunization Experiment of RBD(MERS)-FM-HPF Nanoparticle Vaccine To evaluate the antibody-inducing ability of the RBD(MERS)-FM-HPF nanoparticle vaccine, this embodiment mixed the RBD(MERS)-FM-HPF nanoparticle vaccine with an equal volume of aluminum adjuvant and subcutaneously immunized 6-8 week old C57BL / 6 mice, with each mouse receiving 5 μg in 100 μL. This embodiment also included a control group immunized with an equimolar amount of FMt-RBD(MERS) monomer, equivalent to 5 μg of RBD(MERS)-FM-HPF.

[0086] To compare the potential differences in antibody titers induced by the FM molecular glue nanoparticle vaccine with those of previously reported molecular glue nanoparticle vaccines, this embodiment constructed ST003-RBD(MERS) monomeric vaccine and RBD(MERS)-ST003-HPF nanoparticle vaccine using Spytag003(ST003) / Spycatcher003(SC003) as the molecular glue conjugation system. The amino acid sequence of ST003-RBD(MERS)-6His is shown in SEQ ID NO: 12, and the amino acid sequence of SC003-HPF-6His is shown in SEQ ID NO: 13. The immunization dose of the RBD(MERS)-ST003-HPF nanoparticle vaccine was equimolar with that of the RBD(MERS)-FM-HPF group, with a volume of 100 μL. The immunization dose of ST003-RBD(MERS) was also equimolar with that of RBD(MERS)-FM-HPF, with a volume of 100 μL.

[0087] In this embodiment, the mouse experiment employed a Prime / Boost double-dose immunization strategy: the first immunization was administered in week 0, followed by a second immunization four weeks later in week 4. One week after the second immunization, on week 5, the mice were euthanized, and peripheral blood was collected to separate serum samples for the determination of antigen-specific antibody titers and neutralizing antibody titers.

[0088] Example 5. Assay of antigen-specific antibodies induced by RBD(MERS)-FM-HPF nanoparticle vaccine This embodiment uses enzyme-linked immunosorbent assay (ELISA) to detect the level of MERS-CoV RBD-specific IgG antibodies in mouse serum samples obtained in the mouse experiment of Example 4. The specific implementation steps are as follows: 1. Coating antigen onto ELISA plate: Dilute RBD (MERS) protein to 1 μg / mL with coating buffer (pH=9.6, containing 3.03 g / L Na2CO3 and 6 g / L NaHCO3), add 50 μL to each well of a 96-well ELISA plate, and incubate overnight at 4°C to allow the protein to adsorb and coat the wells.

[0089] 2. Blocking the ELISA plate: On the second day, discard the liquid by swiping the plate. Add 200 μL of PBST solution (PBS containing 0.05% Tween-20) to each well and discard the liquid by swiping the plate again. Repeat the washing process twice. After patting the plate dry on absorbent paper, add 100 μL of 5% skim milk powder solution to each well and incubate at 37°C for 1 h to block the non-specific binding sites in the plate.

[0090] 3. Add serum to the plate: In a brand new 96-well U-plate, serially dilute mouse serum with PBS at seven dilutions (1:30, 1:300, 1:3000, 1:30000, 1:300000, 1:3000000, 1:30000000, 1:30000000). Reserve at least 110 μL for each well after dilution. After blocking for 1 h, discard the skim milk powder solution from the ELISA plate and wash each well with 200 μL of PBST solution. Repeat the washing process twice. After patting the plate dry on absorbent paper, transfer 100 μL of the diluted serum to the ELISA plate. Add 100 μL of PBS to each well in the last row as a blank control. Incubate at 37°C for 1 h.

[0091] 4. Add secondary antibody to plate: After 1 h, swirl the ELISA plate to discard the liquid, add 200 μL of PBST solution to each well for washing, repeat the washing 4 times, pat the plate dry on absorbent paper, add 100 μL of anti-mouse IgG secondary antibody diluted with PBS and conjugated with horseradish peroxidase (HRP) to each well, and incubate at 37°C for 1 h.

[0092] 5. Color development, termination, and assay: After 1 hour, discard the secondary antibody solution in the ELISA plate. Add 200 μL of PBST solution to each well for washing, repeating the washing process four times. Then, pat the plate dry on absorbent paper. Add 100 μL of TMB substrate chromogenic solution to each well and incubate at room temperature in the dark for 20 minutes. Finally, add 100 μL of 1 M sulfuric acid solution to each well to terminate the color development. Place the ELISA plate in a microplate reader to measure the absorbance at OD 450 nm for each well and export the data.

[0093] 6. Data Processing: Nonlinear regression analysis was used in GraphPad Prism software to generate data with a logarithmic structure. 10 The four-parameter Logistic curve is plotted with [serum dilution factor] as the x-axis and OD 450 nm as the y-axis. The x-value corresponding to each serum when y = the reference value is calculated, taking twice the average of the last row of OD 450 nm values ​​as the reference value. This is the endpoint antibody titer level of the serum.

[0094] The results showed that the RBD(MERS)-FM-HPF nanoparticle vaccine induced mice to produce high levels of RBD-specific antibodies, with an average titer of 5 × 10⁻⁶. 4 Furthermore, the antibody titer level was superior to that of the FMt-RBD(MERS) monomer group. Figure 5Furthermore, the antibody titer induced by this nanoparticle vaccine is equivalent to the antibody titer induced by RBD(MERS)-ST003-HPF.

[0095] Example 6. Assay of neutralizing antibodies induced by RBD(MERS)-FM-HPF nanoparticle vaccine This embodiment uses a MERS-CoV pseudovirus neutralization assay to detect the level of anti-MERS-CoV neutralizing antibodies in mouse serum. The specific implementation steps are as follows: 1. Pseudovirus Packaging and Titer Determination: To evaluate the neutralizing antibody titers induced by the nanovaccine, this embodiment constructed a pseudovirus based on the Spike protein of MERS-CoV (Strain: HCoV-EMC / 2012) to determine these antibody titers. HEK293T cells at 70-80% density were co-transfected with PEIMAX transfection reagent containing 12 μg of the MERS-CoV Spike expression plasmid, 6 μg of the packaging plasmid psPAX2, and 6 μg of the pHIV-luciferase plasmid. Six hours after transfection, the supernatant was replaced with fresh DMEM medium containing 10% FBS. The pseudovirus supernatant was collected 48 hours post-transfection and centrifuged at 3000 g for 5 min to remove cell debris. The obtained pseudovirus supernatant was serially diluted 10 times at a 1:2 ratio with DMEM medium (containing 10% FBS). 100 μL of each diluted virus solution was then co-cultured with HEK293T-hDPP4 cells. Cells were lysed 48 h after infection, and the expression level of luciferase was measured using a luciferin substrate. The relative expression level of luciferase also indirectly reflects the infection rate and titer of MERS-CoV pseudovirus.

[0096] 2. HEK293T-hDPP4 Cell Plating: The receptor for MERS-CoV virus is human dipeptidyl peptidase 4 (hDPP4). This example constructed HEK293T cells stably expressing the hDPP4 receptor, namely HEK293T-hDPP4. The night before the experiment, 50 μL of poly-L-lysine solution (10 μg / mL, molecular weight 150,000-300,000) was added to each well of a 96-well plate and incubated at 37°C for 20 min. Afterwards, the poly-L-lysine solution was discarded, and cells were plated at 5 × 10⁻⁶ cells per well. 4 Add 100 μL of cells per well to a 96-well plate and incubate overnight at 37°C in a 5% CO2 incubator.

[0097] 3. Serum Neutralization of Pseudovirus: On the second day after cell plating, serum was diluted in 96-well U-plates using Opti-MEM at six dilutions (1:5, 1:50, 1:500, 1:5000, 1:50000, 1:50000, 1:500000). Each well contained 55 μL of diluted serum. Based on the pre-determined MERS-CoV (Strain: HCoV-EMC / 2012) pseudovirus titer, the pseudovirus was diluted with DMEM medium (containing 10% FBS). 55 μL of the pseudovirus was then added to each well of the serum sample in the U-plate, gently mixed, and incubated at 37°C for 1 h. After 1 h of co-incubation with the pseudovirus, the supernatant from the HEK293T-hDPP4 cell plate was discarded, and the serum and pseudovirus mixture was transferred to each well in 100 μL. The first row is the cell control, with 100 μL of DMEM medium (containing 10% FBS) added to each well. The last row is the pseudovirus control, with 50 μL of Opti-MEM and 50 μL of pseudovirus added to each well. The cell culture plates co-incubated with serum / pseudovirus were then incubated at 37°C for 48 h.

[0098] 4. Cell lysis: After 48 h, discard the supernatant in the cell plate, add 100 μL of PBS to each well to wash the cells, then discard the washing buffer and add 50 μL of lysis buffer (50 mM Tris-HCl, 1 mM DTT, 0.1% Triton X-100, pH=7.5) to each well, place on a shaker and shake at 220 rpm for 30 min to lyse the cells.

[0099] 5. Color development and instrument detection: Take 20 μL of the lysed product from each well and add it to a 96-well white opaque plate. Under light-protected conditions, add 100 μL of luciferase substrate to each well of the white plate, gently tap the plate to mix it, and then use an instrument to detect the luciferase luminescence value and export the data.

[0100] 6. Data Processing: Using the fluorescein reading in the cell control group as the lower limit of detection and the pure pseudovirus control group as the upper limit of detection, the percentage of luminescence values ​​in each serum / pseudovirus treatment group within this range and the (1-percentage) value were calculated. Nonlinear regression analysis was used in GraphPad Prism software to generate a logarithmic graph. 10 A four-parameter logistic curve with [serum dilution factor] as the x-axis and (1-percentage) as the y-axis is used to calculate the reciprocal (IC50) of the serum dilution ratio that inhibits 50% pseudovirus infection. 50 This refers to the titer of neutralizing antibodies in the serum.

[0101] The results showed that the RBD(MERS)-FM-HPF nanoparticle vaccine induced mice to produce high levels of neutralizing antibody titers against the MERS-CoV (Strain: HCoV-EMC / 2012) pseudovirus, with an average titer of 5 × 10⁻⁶. 3 ( Figure 6 Furthermore, the neutralizing antibody titer level was superior to that induced by the RBD(MERS) monomer group, and equivalent to the neutralizing antibody titer induced by the known molecular gel nanovaccine RBD(MERS)-ST003-HPF.

[0102] In summary, this publication discloses the development of an isopeptide-forming pilin-related protein (GenBank: WP_172975141.1) from Finegoldia magna (FM), namely the FM molecular glue system. This FM molecular glue system consists of two components: the FMtag (FMt) peptide and the FMcatcher (FMc) protein. An isopeptide bond is formed between the aspartic acid (D) at position 10 of FMt and the lysine (K) at position 7 of FMc, catalyzed by the glutamate (E) at position 54 of FMc, achieving covalent bonding between FMt and FMc.

[0103] This disclosure discloses an RBD(MERS)-FM-HPF nanoparticle vaccine developed based on the FM molecular glue system, using RBD on a MERS-CoV spike as the antigen. This nanovaccine covalently displays an equal number of RBD(MERS) immunogens on the surface of the HPF 24-tetrator. The induced RBD(MERS) antigen-specific IgG antibody titers and neutralizing antibody titers against the MERS-CoV (Strain: HCoV-EMC / 2012) pseudovirus are significantly higher than those induced by RBD(MERS) monomeric vaccines, and are equivalent to the antibody titers induced by nanoparticle vaccines developed using the known molecular glue systems Spytag003 (ST003) / Spycatcher003 (SC003). The development of this FM molecular glue system overcomes the shortcomings of currently available molecular glue systems, helping to solve the technical challenge of displaying different antigens in equal proportions on nanoparticles using different molecular glues. Furthermore, the FM molecular glue system developed in this disclosure can also be used to display other infectious disease antigens or tumor antigens, and to present these antigens to different types of bio-self-assembled nanoparticle carriers.

[0104] The technical solutions of the present invention are not limited to the specific embodiments described above. Any technical modifications made in accordance with the technical solutions of the present invention fall within the protection scope of the present invention.

Claims

1. A molecular glue system comprising a molecular glue formed by a first peptide and a second peptide in a peptide composition; The amino acid sequence of the first peptide is shown in SEQ ID NO: 2; The amino acid sequence of the second peptide is shown in SEQ ID NO:

3.

2. The molecular glue system of claim 1, wherein, The first peptide and the second peptide can covalently bind to form an isopeptide bond.

3. The molecular glue system of claim 1, wherein, The first peptide and the second peptide are derived from the SpaA isopeptide bond of *Granifera macrophyte* to form fimbriae-associated proteins.

4. The molecular adhesive system according to claim 1, characterized in that, The 10th amino acid D of the first peptide and the 7th amino acid K of the second peptide form an isopeptide bond in the presence of the 54th amino acid E of the second peptide.

5. The use of the molecular glue system according to any one of claims 1 to 4 in the preparation of nanoparticle vaccine compositions.

6. A nanoparticle vaccine composition, comprising: Nanoparticle core; and peptide compositions; The peptide composition includes a first peptide and a second peptide. The amino acid sequence of the first peptide is shown in SEQ ID NO: 2; The amino acid sequence of the second peptide is shown in SEQ ID NO: 3; The first peptide is linked to one of the nanoparticle core and the antigen portion to form a first fusion protein, and the second peptide is linked to the other of the nanoparticle core and the antigen portion to form a second fusion protein.

7. The nanoparticle vaccine composition according to claim 6, characterized in that, The first peptide and the second peptide can covalently bind to form an isopeptide bond.

8. The nanoparticle vaccine composition according to claim 6, characterized in that, The first peptide and the second peptide are derived from the SpaA isopeptide bond of *Granifera macrophyte* to form fimbriae-associated proteins.

9. The nanoparticle vaccine composition according to claim 6, characterized in that, The 10th amino acid D of the first peptide and the 7th amino acid K of the second peptide form an isopeptide bond in the presence of the 54th amino acid E of the second peptide.

10. The nanoparticle vaccine composition according to claim 6, characterized in that, The antigen portion is displayed on the outer surface of the nanoparticles; and / or The first peptide is linked to the antigen moiety; and / or The second peptide connects to the nanoparticle core; and / or The nanoparticle core comprises one or more of the following: Helicobacter pylori ferritin tetratrichomer, Aerosol-producing bacteria 2,4-dioxetine synthase hexadecimer, or fully synthetic nanoparticle I53-50 120-mer; and / or The nanoparticle vaccines include infectious disease vaccines and / or cancer vaccines; and / or The antigen portion includes an active fragment of one or more viruses selected from Middle East Respiratory Syndrome Coronavirus, novel coronavirus, influenza virus, respiratory syncytial virus, measles virus, mumps virus, rubella virus, adenovirus, varicella-zoster virus, human papillomavirus, rotavirus, hepatitis virus, or human immunodeficiency virus.

11. The nanoparticle vaccine composition according to any one of claims 6 to 10, characterized in that, The first fusion protein and the second fusion protein self-assemble to form nanoparticles.

12. The nanoparticle vaccine composition according to claim 11, characterized in that, The first fusion protein includes the first peptide and the receptor-binding domain of the MERS-CoV virus Spike protein, and the second fusion protein includes the second peptide and Helicobacter pylori ferritin tetratrimester.

13. The nanoparticle vaccine composition according to claim 12, characterized in that, The amino acid sequence of the first fusion protein is shown in SEQ ID NO: 11; and / or The amino acid sequence of the second fusion protein is shown in SEQ ID NO: 6.

Images