Molecular glue and nanoparticle vaccine composition

Abstract

The invention relates to molecular glue for forming fimbriae related protein based on SpaA isopeptide bonds of Vinci bacteria. The invention also relates to a nanoparticle vaccine composition formed by the molecular glue, and the immunogen is covalently displayed on the surface of the nanoparticle by the molecular glue.

Description

TECHNICAL FIELD

[0001] The present disclosure relates to a molecular glue, in particular a molecular glue based on SpaA isopeptide bond forming pilus associated protein of B. finitimus. The present invention further relates to a nanoparticle vaccine composition formed by the molecular glue. BACKGROUND

[0002] Nanoparticle vaccine is an emerging vaccine technology that encapsulates antigens or nucleic acids on the surface or inside of nanoscale carriers such as liposomes, polymers or protein particles, mimicking the size and structure of viruses to stimulate the immune system more efficiently. Compared with traditional vaccines, nanoparticle vaccines have multiple advantages: first, their small size and precise structure can enhance antigen delivery efficiency and promote the uptake of immune cells such as dendritic cells. Second, nanocarriers can protect antigens or nucleic acids from degradation, improving stability and immunogenicity. In addition, nanoparticles can target delivery through the lymphatic system, while activating both humoral and cellular immunity, and even inducing mucosal immunity, thus providing more comprehensive protection. This technology also enables multivalent vaccine design, integrating different mutant strain antigens of the same pathogen or heterologous antigens of different pathogens into a single particle, reducing the frequency of vaccination. Currently, nanoparticle vaccines have shown great potential in the development of infectious disease and cancer vaccines. Among them, biological self-assembled protein nanoparticles such as Helicobacter pylori ferritin (Ferritin, HPF) twenty-fourmer, Sporosarcina mavi 2,4-dioxygen tetrahydropteridine synthase (Lumazine synthase, LS) sixtymer, and fully synthetic nanoparticle I53-50 one hundred and twentymer have been widely used in the research of vaccines for Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and Human immunodeficiency virus 1 (HIV-1), showing great potential in inducing neutralizing antibodies.

[0003] The two main ways to display antigen on nanoparticle carriers are direct fusion and indirect coupling. It is difficult to express the antigen and nanoparticle carrier in the same system by direct gene fusion. Indirect coupling is to construct, express and purify the nanoparticle carrier and antigen separately, and the two have specific tags of the molecular glue system. After mixing the nanoparticle carrier and antigen in vitro, the antigen and nanoparticle specifically bind due to the specific binding of the tag, so as to realize the display of the antigen on the nanoparticle. Currently, a variety of molecular glue coupling systems have been applied to vaccine design, including Spytag / Spycatcher, SortaseA, Streptavidin / Biotin and Fc Tag / protein A Tag, etc. Among them, the Spytag / Spycatcher molecular glue system has been upgraded several times, and the latest version of Spytag003 / Spycatcher003 has the highest coupling efficiency. However, when designing a nanoparticle vaccine displaying multiple antigens, there is a problem that the proportion of each antigen cannot be controlled, so it is urgent to develop multiple molecular glues without cross-reactivity for nanoparticle vaccine design and even other fields. SUMMARY

[0004] In order to overcome the deficiencies of the currently available heteropeptide bond-based molecular glue, the present disclosure provides a new molecular glue for displaying antigens on nanoparticles. The present disclosure also provides a nanoparticle vaccine display system based on the molecular glue, which covalently displays immunogens on the surface of nanoparticles through the molecular glue system to prepare a nanoparticle vaccine.

[0005] According to one aspect of the present disclosure, a peptide composition is provided, which includes a first peptide and a second peptide, the first peptide including an amino acid sequence corresponding to amino acids 88-103 of the amino acid sequence set forth in SEQ ID NO: 1, or an amino acid sequence having one or more substitutions, deletions, or insertions of amino acid residues compared thereto, and the amino acid corresponding to amino acid 97 of the amino acid sequence set forth in SEQ ID NO: 1 is D; and the second peptide including an amino acid sequence corresponding to amino acids 1-93 of the amino acid sequence set forth in SEQ ID NO: 1, or an amino acid sequence having one or more substitutions, deletions, or insertions of amino acid residues compared thereto, and the amino acid corresponding to amino acid 7 of the amino acid sequence set forth in SEQ ID NO: 1 is K and / or the amino acid corresponding to amino acid 54 of the amino acid sequence set forth in SEQ ID NO: 1 is E.

[0006] In some embodiments, the first peptide and the second peptide can covalently bind to form a heteropeptide bond.

[0007] In some embodiments, the first peptide comprises an amino acid sequence set forth in SEQ ID NO: 2, or an amino acid sequence having one or more substitutions, deletions, or insertions of 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 an amino acid sequence set forth in SEQ ID NO: 3, or an amino acid sequence having one or more substitutions, deletions, or insertions of amino acid residues compared to SEQ ID NO: 3, wherein the 7th amino acid and the 54th amino acid in the amino acid sequence of the second peptide are K and E, respectively.

[0009] In some embodiments, the first peptide and the second peptide are derived from a SpaA isopeptide bond-forming pilus-related protein of Finegoldia magna (FM).

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

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

[0012] According to yet another aspect of the present disclosure, there is provided an expression vector comprising the nucleic acid molecule of the present disclosure.

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

[0014] According to yet another aspect of the present disclosure, there is provided a molecular glue system comprising a molecular glue formed by the first peptide and the second peptide in the peptide composition of the present disclosure.

[0015] According to yet another aspect of the present disclosure, there is provided a use of the peptide composition, the nucleic acid molecule, the expression vector, the host cell, or the molecular glue system of the present disclosure in self-assembly of at least two molecules or components via isopeptide bond.

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

[0017] According to yet another aspect of the present disclosure, there is provided a method of self-assembly of two molecules or components via isopeptide bond, comprising the following steps: S1: providing a first molecule or component and a second molecule or component comprising a first peptide and a second peptide, respectively, in a peptide composition according to the present disclosure; S2: contacting the first molecule or component and the second molecule or component to self-assemble the first molecule or component and the second molecule or component through isopeptide bond to form a complex.

[0018] According to yet another aspect of the present disclosure, there is provided use of a peptide composition according to the present disclosure, a nucleic acid molecule according to the present disclosure, an expression vector according to the present disclosure, a host cell according to the present disclosure, or a molecular glue system according to the present disclosure in the preparation of a nanoparticle vaccine composition.

[0019] According to yet another aspect of the present disclosure, there is provided a nanoparticle vaccine composition comprising a nanoparticle inner core; and, a peptide composition according to the present disclosure.

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

[0021] In some embodiments, the antigenic moiety is displayed on an outer surface of the nanoparticle.

[0022] In some embodiments, the first peptide is linked to the antigenic moiety.

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

[0024] In some embodiments, the nanoparticle inner core comprises one or more of Helicobacter pylori ferritin (Ferritin, HPF) twenty-fourmer, Lumazine synthase (LS) sixtymer, or fully synthetic nanoparticle I53-50 one-hundred-twentymer.

[0025] In some embodiments, the nanoparticle vaccine comprises an infectious disease vaccine and / or a tumor vaccine.

[0026] In some embodiments, the antigenic moiety comprises an active fragment of one or more of 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 the nanoparticle.

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

[0029] In some embodiments, the first fusion protein comprises an amino acid sequence set forth in SEQ ID NO: 11, or an amino acid sequence having one or more substitutions, deletions, or insertions of amino acid residues compared to SEQ ID NO: 11.

[0030] In some embodiments, the second fusion protein comprises an amino acid sequence set forth in SEQ ID NO: 6, or an amino acid sequence having one or more substitutions, deletions, or insertions of amino acid residues compared to SEQ ID NO: 6.

[0031] The Spytag / Spycatcher system has been reported in the past. The present disclosure provides a new molecular glue coupling system derived from the SpaA isopeptide-forming pilin-related protein (GenBank: WP_172975141.1) of Finegoldia magna (FM), i.e., the FMtag / FMcatcher (FMt / FMc) molecular glue, which addresses the problem of insufficient diversity of current molecular glues. Through site-directed mutagenesis, it is determined that the 10th amino acid aspartic acid (D) of FMt and the 7th lysine (K) of FMc form an intermolecular isopeptide bond under the catalysis of the 54th glutamic acid (E) of FMc. By expressing FMt in tandem with an immunogen such as RBD(MERS) and expressing FMc in tandem with a nanoparticle such as HPF, an immunogen and a nanoparticle coupled with molecular glue are obtained. After co-incubation of the two, a nanoparticle vaccine is obtained, such as a HPF nanoparticle vaccine covalently presenting twenty-four RBD(MERS). The RBD-specific antibody and neutralizing antibody titers induced by the RBD(MERS)-FM-HPF nanoparticle vaccine are superior to the antibody titers induced by RBD(MERS) monomers, and are equivalent to the antibody titers induced by the known molecular glue nanoparticle RBD(MERS)-ST003-HPF. BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 1 The structure prediction of FM and its split into FMc and FMt is shown. FM is split into FMcatcher (FMc) protein and FMtag (FMt) peptide.

[0033] Figure 2 The figure shows the binding verification results of FMc and FMt. Among them, FMt is expressed in series to the N-terminal of GFP, and FMc and FMt-GFP proteins are respectively expressed and purified. After mixing and incubating the two proteins, SDS-PAGE electrophoresis and Coomassie brilliant blue staining are performed. The results show that the molecular weight of FMc is 11.390 kDa, and the molecular weight of FMt-GFP is 30.188 kDa. In addition, a protein band with a molecular weight of about the sum of the molecular weights of the two proteins appears at the position of 41.578 kDa after co-incubation, indicating that FMc and FMt-GFP bind and form a covalent isopeptide bond.

[0034] Figure 3 The figure shows the verification results of the key amino acid sites for forming isopeptide bonds between FMc and FMt. Among them, FMcatcher (FMc) is expressed in series to the N-terminal of Ferritin (HPF) to obtain FMc-HPF protein. FMc(K7A)-HPF protein, FMc(E54A)-HPF protein and FMt(D10A)-GFP-GST protein are purified by simultaneous expression. FMtag (FMt) is expressed in series to the N-terminal of GFP-GST to obtain FMt-GFP-GST protein. Then, the proteins expressed in series with FMt are co-incubated with the proteins expressed in series with FMc, and SDS-PAGE electrophoresis is performed. The left figure shows that when K7A mutation occurs in FMc or D10A mutation occurs in FMt, FMc and FMt do not bind. The right figure shows that when E54A mutation occurs in FMc, FMc and FMt do not bind, indicating that the 7th K amino acid in FMc and the 10th D amino acid in FMt form an isopeptide bond, and the 54th E amino acid in FMc affects the formation of the isopeptide bond.

[0035] Figure 4 The figure shows the construction schematic diagram, SEC and negative staining electron microscopy results of RBD(MERS)-FM-HPF nanoparticle vaccine. Among them, RBD(MERS) nanoparticle vaccine with HPF as the nanometer core and FM as the molecular glue coupling system is successfully prepared. Its peak position is 11 mL in the molecular sieve exclusion chromatography (Size exclusion chromatography, SEC) separation process in the gel filtration chromatography column Superose 6 Increase10 / 300 GL, and it is earlier than the peak of FMc-HPF and FMt-RBD(MERS), indicating that it forms a uniform nanoparticle of about 1100 kDa. The negative staining electron microscopy results show that the nanoparticle forms a cage-like structure and is uniformly distributed with a particle size of about 50 nm.

[0036] Figure 5RBD (MERS)-FM-HPF nanoparticle vaccine induced RBD-specific antibody titers are shown. The RBD (MERS)-FM-HPF nanoparticle vaccine induced high levels of RBD (MERS)-specific IgG antibody titers in mice, and the antibody titer level was superior to that of the RBD (MERS) monomer group. The antibody titer induced by the nanoparticle vaccine was equivalent to that induced by RBD (MERS)-ST003-HPF.

[0037] Figure 6 RBD (MERS)-FM-HPF nanoparticle vaccine induced neutralizing antibody titers are shown. The RBD (MERS)-FM-HPF nanoparticle vaccine induced C57BL / 6 mice to produce high levels of anti-MERS-CoV (Strain: HCoV-EMC / 2012) pseudovirus neutralizing antibody titers, and the antibody titer level was superior to that of the RBD (MERS) monomer group. The neutralizing antibody titer induced by the nanoparticle vaccine was equivalent to that induced by RBD (MERS)-ST003-HPF. DETAILED DESCRIPTION

[0038] Isopeptide bond is a covalent bond similar to peptide bond. Specifically, it refers to an amide bond formed by condensation of at least one amino group or carboxyl group of a non-alpha amino acid in a protein sequence, such as a covalent bond formed by spontaneous combination of a Lys side chain amino group and an Asn side chain carboxamide group or an Asp side chain carboxyl group, including intermolecular and intramolecular isopeptide bonds. Intermolecular isopeptide bonds are mainly formed by acyl transfer reaction dominated by Lys, and play an important role in many physiological processes such as ubiquitination and glutamylation. The first reported protein that spontaneously forms intramolecular isopeptide bonds is pilin of Streptococcus pyogenes. The polypeptide sequence forming the isopeptide bond can be artificially divided into two fragments. When expressed separately and mixed again, the two fragments can recognize and bind to each other to form a functional protein. Based on this, scientists have carried out systematic research on isopeptide bonds and related protein molecular stability, and developed a molecular glue system for protein cross-linking, including isopeptag-N / pilin-N, isopeptag / pilin-C, Dogtag / Dogcatcher and Spytag / Spycatcher. In addition, researchers have also found that Snooptag and Snoopcatcher peptide segments from S. pneumoniae adhesin RrgA carry Lys residues and Asn residues, respectively, indicating that intramolecular isopeptide bonds can be formed. However, there are few reports of proteins that can form intramolecular isopeptide bonds, which limits the development of intramolecular isopeptide bond-based molecular glue and vaccine applications.

[0039] The isopeptide bond formed by Spytag / Spycatcher coupling protein has stable bond, short peptide chain and rapid reaction, and has good thermal stability and resistance. It is a region between Lys and Asp in the CnaB2 domain of fibronectin binding protein FbaB from Streptococcus pyogenes that spontaneously forms an isopeptide bond. Researchers split this region to prepare a peptide (Spytag) and a protein (Spycatcher) that can form an amide bond in a few minutes, and then fuse them to either end or inside of different proteins through gene coding. When two proteins carrying Spytag and Spycatcher are mixed, the two proteins are covalently fused. This coupling protein is suitable for various in vitro and in vivo applications, such as assembly of various nonlinear protein structures, vaccine synthesis, nanobioreactor, and protein hydrogel.

[0040] However, there is no isopeptide bond molecular glue with complete independent intellectual property rights in China at present, and only a few molecular glues based on isopeptide bond connection have been reported abroad, and their cross-binding properties need further study. Therefore, it is urgent to develop new molecular glues to solve the technical problem of simultaneously displaying different antigens on the same nanoparticle, promote the development of nanoparticle vaccines, and also greatly fill the intellectual property gap in this field in China and increase the diversity of protein coupling tools.

[0041] The technical problem to be solved by the present disclosure is to overcome the shortcomings of the currently available isopeptide bond-based molecular glue, develop more new molecular glues for displaying antigens on nanoparticles. The present disclosure intercepts the domain that can form intramolecular isopeptide bond from SpaA isopeptide-forming pilin-related protein (GenBank: WP_172975141.1) of Finegoldia magna (FM). And through AlphaFold3 prediction and structural biology analysis, the domain is split into two protein components that can form isopeptide bond, namely FMtag (FMt) peptide and FMcatcher (FMc) protein. By fusing and expressing FMt with the receptor-binding domain (RBD) of the Middle East respiratory syndrome coronavirus (MERS-CoV) Spike protein, and fusing and expressing Mc with the Helicobacter pylori ferritin (Ferritin, HPF) tetradecamer, antigens and nanoparticles carrying molecular glue tags are constructed, respectively. Then through in vitro spontaneous assembly to form isopeptide bond, RBD (MERS)-FM-HPF nanoparticle vaccine displaying twenty-four RBD (MERS) antigens is constructed. The key amino acid sites for forming isopeptide bond are confirmed by molecular biology site-directed mutation experiment. The antibody production ability of the formed nanoparticle vaccine is verified by mouse immunization experiment.

[0042] The present disclosure provides a novel FM-based nanoparticle vaccine display system, which covalently displays RBD (MERS) immunogens on the surface of Helicobacter pylori ferritin (Ferritin, HPF) tetradecamer through FM molecular glue system, prepares nanoparticle vaccine, and fully verifies the effectiveness of the nanoparticle vaccine.

[0043] The above object of the present disclosure is achieved by the following technical solutions: The present disclosure first selected the domain containing intramolecular isopeptide bond forming structure on SpaA isopeptide-forming pilin-related protein (SpaA isopeptide-forming pilin-related protein, GenBank: WP_172975141.1) of Finegoldia magna (FM) as the source protein for molecular glue construction (SEQ ID NO: 1). The domain was split into FMtag (FMt) peptide and FMcatcher (FMc) protein two molecular glue components (SEQ ID NO: 2 and SEQ ID NO: 3) by AlphaFold3 structure prediction and analysis.

[0044] Subsequently, the present disclosure purified the 6His-tagged FMc protein (SEQ ID NO: 4), and constructed the 6His-tagged FMt-GFP protein (SEQ ID NO: 5) by tandem expression of FMt and GFP. The ability of FMc and FMt-GFP protein to form covalent isopeptide bond was tested by co-incubation of the two.

[0045] Further, the present disclosure obtained FMc-HPF protein (SEQ ID NO: 6) by tandem expression of FMc and 6His-tagged Helicobacter pylori ferritin (Ferritin, HPF) nanoparticles, and obtained FMt-GFP-GST protein (SEQ ID NO: 7) by tandem expression of FMt and 6His-tagged GFP-GST protein. The 7th amino acid lysine (Lysine, K) on FMc involved in isopeptide bond formation was mutated to alanine (Alanine, A) to construct 6His-tagged FMc(K7A)-HPF protein (SEQ ID NO: 8). The 54th glutamic acid (Glutamic acid, E) on FMc catalyzing isopeptide bond formation was mutated to alanine (Alanine, A) to obtain 6His-tagged FMc(E54A)-HPF protein (SEQ ID NO: 9). The 10th aspartic acid (Aspartic acid, D) on FMt involved in isopeptide bond formation was mutated to alanine (Alanine, A) to obtain 6His-tagged FMt(D10A)-GFP-GST protein (SEQ ID NO: 10). Subsequently, the HPF protein containing FMc and its mutants was co-incubated with the GFP-GST protein containing FMt and its mutants, and the ability to form isopeptide bond was observed to determine the key amino acid positions for isopeptide bond formation and catalysis of isopeptide bond formation.

[0046] To investigate the ability of FMt / FMc molecular glue system to present vaccine antigens, the present disclosure constructed an antigen of the receptor-binding domain (RBD) of the Middle East respiratory syndrome coronavirus (MERS-CoV) Spike protein expressing FMt peptide in tandem, i.e., a 6His-tagged FMt-RBD(MERS) protein (SEQ ID NO: 11). The FMt-RBD(MERS) was co-incubated with the FMc-HPF, and a nanoparticle vaccine RBD(MERS)-FM-HPF based on the FMt / FMc molecular glue system was obtained. At the same time, the present disclosure selected a reported Spytag003 (ST003) / Spycatcher003 (SC003) molecular glue system as a control. A 6His-tagged RBD(MERS) antigen coupled with ST003 (SEQ ID NO: 12), a 6His-tagged HPF nanoparticle coupled with SC003 (SEQ ID NO: 13), and a RBD(MERS)-ST003-HPF nanoparticle vaccine were constructed.

[0047] Based on the above-constructed RBD(MERS) monomer vaccine and HPF nanoparticle vaccine, C57BL / 6 mice were subjected to Prime / Boost double immunization, and the mice were euthanized one week after the second immunization, and serum was collected to determine the RBD(MERS)-specific antibody and neutralizing antibody titers against MERS-CoV (Strain: HCoV-EMC / 2012) pseudovirus. The antibody titers induced by the RBD(MERS) monomer vaccine and the HPF nanoparticle vaccine were compared, and the antibody titers induced by the nanoparticle vaccines based on the FMt / FMc molecular glue system and the ST003 / SC003 molecular glue system were compared, so as to evaluate the effect of the novel molecular glue system on the antibody induced by the nanoparticle vaccine.

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

[0049] Table 1

[0050] Through the above technical solutions, the present disclosure has the following beneficial effects: (I) The present disclosure develops a new molecular glue FM system for vaccine antigen coupling, which can covalently bind vaccine antigens to nanoparticle carriers in the form of intermolecular isopeptide bonds, and ultimately achieve high-density display of vaccine antigens.

[0051] (ii) The new FM molecular glue system developed by the present disclosure is composed of two components, i.e. FMtag (FMt) peptide and FMcatcher (FMc) protein. By tandem expression of FMt with vaccine antigens such as receptor-binding domain (RBD) of coronavirus Spike protein, and tandem expression of Mc with nanoparticle inner core such as Helicobacter pylori ferritin (Ferritin, HPF) 24-mer, and then co-incubation of the two tandem expressed proteins, it can be realized that the same number of antigens as the valence of the nanoparticles are displayed on the outer surface of the nanoparticles.

[0052] (iii) The present disclosure also identifies the key amino acids on the new FM molecular glue system that form isopeptide bond and catalyze the formation of isopeptide bond. The 10th aspartic acid (D) on FMt and the 7th lysine (K) on Mc form an intermolecular isopeptide bond under the catalysis of the 54th glutamic acid (E) on Mc. Any mutation of these three sites makes FMt unable to covalently bind to Mc.

[0053] (iv) The present disclosure develops a nanoparticle vaccine against Middle East respiratory syndrome coronavirus (MERS-CoV) based on Helicobacter pylori ferritin (Ferritin, HPF) 24-mer using the new FM molecular glue system. The present disclosure fuses and expresses FMt with the RBD domain of MERS-CoV Spike protein to obtain FMt-RBD(MERS) antigen, and fuses and expresses Mc with HPF to obtain Mc-HPF nanoparticle inner core. Then, the antigen and the nanoparticle are co-incubated to obtain RBD(MERS)-FM-HPF nanoparticle vaccine. The RBD(MERS) antigen displayed by the nanoparticle vaccine is covalently coupled to the outer surface of the nanoparticle under the action of the isopeptide bond of the FM molecular glue system. The number of RBD(MERS) antigens displayed by the vaccine on one nanoparticle is equal to the valence of the nanoparticle, both of which are twenty-four.

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

[0055] Therefore, the present disclosure develops a molecular glue FM system for vaccine antigen coupling display, which specifically includes two components of FMt peptide and FMc protein. At the same time, a HPF nanoparticle vaccine based on the FM molecular glue system for the RBD domain on MERS-CoV Spike is developed, and the antibody titer induced by the vaccine is significantly higher than that induced by the monomer vaccine. The new molecular glue FM system developed by the present disclosure can not only be used for display of RBD(MERS) antigen, but also has reference for other infectious disease vaccines and tumor vaccines. In addition, the FM molecular glue system can also be used for other biological self-assembly nanoparticle carrier mediated antigen display technology.

[0056] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The following definitions are applied to the descriptions and claims herein and, unless otherwise specified, take precedence over any contradictory definitions made elsewhere herein for purposes of interpreting this description.

[0057] Unless the context clearly indicates otherwise, as used herein, the construction "a" or "an" is taken to include plural referents unless the context clearly indicates otherwise. For example, reference to "a cell" includes a plurality of such cells as well as equivalents thereof known to those skilled in the art and so forth.

[0058] As used herein, the term "about" means ±20% of the number that it precedes. In some embodiments, the term "about" means ±10% of the number that it precedes. In some embodiments, the term "about" means ±5% of the number that it precedes.

[0059] As used herein, the term "antigen" refers to a substance that is capable of stimulating the body to produce a (specific) immune response and to bind to the products of the immune response, antibodies and sensitized lymphocytes, in vitro, and to effect immunological (specific) reactions. The essential properties of an antigen are two-fold, the ability to induce an immune response, i.e., immunogenicity, and the ability to react with the products of the immune response, i.e., antigenicity.

[0060] As used herein, the antigen component also includes active fragments, derivatives, and analogs of the antigen component. As used herein, the terms "fragment," "derivative," "derivative protein," and "analog" refer to a protein that substantially retains the activity of activating the body's immune response to meningococcal group B. Fragments, derivatives, or analogs of the proteins of the present disclosure can be (i) a polypeptide having one or several conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, or (ii) a polypeptide having a substituent group at one or more amino acid residues, or (iii) a polypeptide formed by fusion of the protein of the present disclosure with another compound (such as a compound that prolongs the half-life of the polypeptide, for example, polyethylene glycol), or (iv) a polypeptide formed by fusion of an additional amino acid sequence to the polypeptide sequence (a derivative protein formed by fusion with a leader sequence, a secretion sequence, or a 6His tag sequence, etc.). These fragments, derivatives, and analogs can be wild-type or mutant, according to the teachings herein, which are within the purview of one of skill in the art.

[0061] As used herein, the antigen component also includes analogs of the antigen component. These analogs can differ from the native antigen component of the present disclosure in the amino acid sequence, in the form of a modification that does not affect the sequence, or both. Analog also includes analogs having residues other than naturally occurring L-amino acids (e.g., D-amino acids), as well as analogs having non-naturally occurring or synthetic amino acids (e.g., β, γ-amino acids). It is understood that the polypeptides of the present disclosure are not limited to the representative polypeptides exemplified above.

[0062] The term "polynucleotide" as used herein refers to a polymer of nucleotides of any length, including ribonucleotides, deoxyribonucleotides, analogs thereof, or mixtures thereof. The term refers to the primary structure of the molecule. Thus, the term includes triple-stranded, double-stranded, and single- stranded deoxyribonucleic acids ("DNA"), as well as triple-stranded, double-stranded, and single-stranded ribonucleic acids ("RNA"). It also includes polynucleotides that have been modified (for example, by alkylations and / or by capping) and unmodified forms of the polynucleotide. More particularly, 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 which is a N- or C-glycoside of a purine or pyrimidine base; and other polymers which contain a positive nucleotide backbone, such as polyamide (e.g., peptide nucleic acid "PNA") and polyphosphoramide polymers; and other synthetic sequence-specific nucleic acid polymers, provided that the polymer contains nucleobases in a configuration which allows for 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 a polymer of amino acids of any length. The polymer can comprise modified amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids such as homoarginine, ornithine, p-acetylphenylalanine, D-amino acids, and creatine), and other modifications known in the art.

[0064] In the amino acid sequences of the peptides described herein, A represents Alanine (Ala), R represents Arginine (Arg), N represents Asparagine (Asn), D represents Aspartic acid (Asp), C represents Cysteine (Cys), Q represents Glutamine (Gln), E represents Glutamic 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 specified, the amino acids (residues) referred to herein can be of the D or L form. Unless otherwise specified, the amino acid sequences referred to herein are in the order N-terminal to C-terminal from left to right.

[0065] The peptides of the present application can include those obtained by substitution of one or more conservative amino acids of the peptide. In some embodiments, substitution of a conservative amino acid can mean the replacement of one amino acid residue with a biologically similar residue. Particularly preferred substitutions are generally conservative in nature, i.e., those substitutions of amino acids within the family. For example, amino acids are generally divided into four families: (1) acidic- aspartic acid (D) and glutamic acid (E); (2) basic- lysine (K), arginine (R), histidine (H); (3) non-polar- alanine (A), valine (V), leucine (L), isoleucine (I), proline (P), phenylalanine (F), methionine (M), tryptophan (W); (4) uncharged polar- glycine (G), asparagine (N), glutamine (Q), cysteine (C), serine (S), threonine (T), tyrosine (Y). Phenylalanine, tryptophan, and tyrosine are sometimes classified as aromatic amino acids. Examples of conservative changes include the substitution of one hydrophobic residue such as isoleucine, valine, leucine or methionine for another; or the substitution of one polar residue for another, such as aspartic acid for glutamic acid, or glutamine for asparagine, etc.; or like amino acid changes that result in a conservative alteration; or the substitution of a structural similar amino acid for an amino acid that will not have a significant effect on biological activity. Thus, proteins that have substantially the same amino acid sequence as a reference molecule but have a small number of amino acid substitutions that do not substantially affect the activity of the protein are within the definition of the reference polypeptide.

[0066] The term "pharmaceutically acceptable carrier" as used herein refers to a component of a pharmaceutical composition other than the active ingredient that is not toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives. In some embodiments, the pharmaceutically acceptable carrier includes: polymers of acrylic or methacrylic acid, maleic anhydride, and alkenyl derivative polymers; immunostimulatory sequences (ISS), such as oligodeoxynucleotide sequences with one or more non-methylated CpG units (CpG ODN); water-in-oil (W / O) adjuvants, oil-in-water (O / W) adjuvants, or water-in-oil-in-water (W / O / W) adjuvants, such as Freund's adjuvant, SPT emulsion, MF59, ISA 206, ISA 72, Adjuvant-65, SAF, etc.; cationic lipids containing quaternary ammonium salts, e.g., DDA; cytokines; aluminum hydroxide or aluminum phosphate; saponins (e.g., Quil A, QS-21, GPI-0100); or, any combination or mixture thereof.

[0067] In order to make the purpose, technical solutions and advantages of the present application clearer and more apparent, the present application will be further described in detail below in combination with the drawings and examples. The specific examples described herein are only used to explain the present application and do not constitute any limitation on the present application. In addition, in the following description, the description of well-known structures and technologies is omitted to avoid unnecessary confusion of the concept of the present disclosure. Such structures and technologies are also described in many publications.

[0068] Example 1. FM structure prediction and split into FMcatcher (FMc) and FMtag (FMt) component design This example performs AlphaFold3 structure prediction on the domain that can form an intramolecular isopeptide bond on the SpaA isopeptide-forming pilin-related protein (SpaA isopeptide-forming pilin-related protein, GenBank: WP_172975141.1) of Finegoldia magna (FM), and splits the FM protein into two components of FMcatcher (FMc) protein and FMtag (FMt) peptide (FMc and FMt) Figure 1 ), and the following experiments verify whether FMc and FMt can form isopeptide bonds.

[0069] Example 2. Verification of key amino acid sites for FMc and FMt binding and isopeptide bond formation This example purifies the FMc protein (FMc-6His, SEQ ID NO: 4) and the FMt-GFP protein (FMt-GFP-6His, SEQ ID NO: 5) fused with FMt at the N-terminus of GFP to verify the binding ability of FMc and FMt. The purpose of fusing FMt with GFP is to reduce the measurement deviation caused by the small molecular weight of FMt and to separate FMt from FMc protein as much as possible during electrophoresis. The specific implementation steps are as follows: 1. Construct expression clone plasmid: use molecular cloning technology to construct FMc expression gene and FMt-GFP fusion gene into pET28a plasmid vector, respectively.

[0070] 2. Plasmid transformation into prokaryotic cells: The plasmid was transformed into E. coli competent BL21, and plated on LB solid plates containing 0.05 mg / mL kanamycin. A single colony was picked and inoculated into LB medium containing 0.05 mg / mL kanamycin, and incubated in a 220 rpm, 37°C shaking incubator. When the OD600nm absorbance value reached 0.5, 1 mM of the protein expression inducer Isopropyl β-D-thiogalactoside (IPTG) was added. The culture was then incubated in a 220 rpm, 16°C shaking incubator for 16 hours.

[0071] 3. Purification of proteins using an affinity chromatography column: The overnight culture was centrifuged at 9000 rpm for 3 minutes, and the supernatant was discarded. The bacterial pellet was resuspended in buffer (20 mM Tris-HCl, 50 mM NaCl, pH=7.4) and high-pressure broken. The pellet was then centrifuged at 9000 rpm, 4°C for 30 minutes, and the supernatant was collected and filtered once through a 0.45 μm filter. Since both FMc and FMt-GFP proteins have a 6His tag, the target proteins can be enriched after incubation of the supernatant with an affinity chromatography cobalt filler. The proteins were eluted by adding buffer containing multiple concentrations of imidazole (10 mM, 20 mM, 50 mM, 100 mM, 200 mM, 300 mM) to the cobalt filler one by one, and each eluate was collected in a separate tube. Samples were taken from each tube for SDS-PAGE electrophoresis and Coomassie blue staining, and the protein eluate with the best purity was concentrated using an ultrafiltration tube to obtain FMc and FMt-GFP proteins.

[0072] 4. After incubation of FMc and FMt-GFP proteins, SDS-PAGE electrophoresis and Coomassie 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. After incubation of the two proteins, a protein band with a molecular weight of about 41.578 kDa, which was the sum of the molecular weights of the two proteins, was observed, indicating that FMc and FMt-GFP had covalently combined and formed an isopeptide bond Figure 2 ).

[0073] In addition, the same experimental method as described above was used in this embodiment to express and purify FMc-HPF protein (FMc-HPF-6His, SEQ ID NO: 6) fused to FMc at the N terminus of Helicobacter pylori ferritin (Ferritin, HPF) and FMt-GFP-GST protein (FMt-GFP-GST-6His, SEQ ID NO: 7) fused to FMt at the N terminus of GFP-GST protein. According to the AlphaFold3 structure prediction results of the FM protein in Example 1, it was found that the 7th amino acid lysine (Lysine, K) of FMc and the 10th aspartic acid (Aspartic acid, D) of FMt were most likely to form an isopeptide bond under the catalysis of the 54th glutamic acid (Glutamic acid, E) of FMc. Therefore, this embodiment also expressed and purified mutant proteins in which these sites were mutated to alanine (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] After incubation of the above-mentioned proteins in pairs, SDS-PAGE electrophoresis and Coomassie blue staining were performed. The results showed that when K7A mutation occurred in FMc or E54A mutation occurred in FMt, or D10A mutation occurred in FMt, the proteins fused to express FMc and FMt did not covalently combine 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 the 54th E amino acid in FMc is responsible for catalyzing the formation of the isopeptide bond.

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

[0076] To verify the ability of FM molecular glue to covalently tether vaccine antigens to nanoparticles, this example used Middle East respiratory syndrome coronavirus (MERS-CoV) as a vaccine research object, and selected the receptor-binding domain (RBD) of the MERS-CoV Spike protein as a test antigen, which mediates the production of neutralizing antibodies. The FMt-RBD(MERS) protein (FMt-RBD(MERS)-6His, SEQ ID NO: 11) was expressed and purified, in which the FMt peptide was fused to the N terminus of the RBD(MERS) protein. Figure 4 The specific implementation steps are as follows: 1. Construct the expression clone plasmid: add a signal peptide (SP) sequence (MGILPSPGMPALLSLVSLLSVLLMGCVA, SEQ ID NO: 14) to the N terminus of the FMt-RBD(MERS) fusion gene and a 6His tag sequence to the C terminus, then construct them into the pcDNA3.1 plasmid vector, and then transform them into E. coli DH5α competent cells and plate them on LB agar plates containing ampicillin (0.1 mg / mL). Pick a single colony into LB medium containing ampicillin (0.1 mg / mL) and then place it in a 220 rpm, 37°C shaking incubator for 16 h. Then centrifuge at 9000 rpm for 3 min and discard the supernatant, and use a plasmid extraction kit to extract the plasmid.

[0077] 2. Transfect the plasmid into eukaryotic cells: transfect the pcDNA3.1-SP-FMt-RBD(MERS)-6His plasmid into eukaryotic cells HEK293F, with a ratio of 1 mL of cells to 1.25 μg of plasmid, and the amount of PEIMAX transfection reagent is 4 times the volume of the plasmid mass, i.e. 5 μL. If 500 mL of cells are transfected, add 2.5 mL of PEIMAX to 20 mL of Opti-MEM medium and mix well, stand for 5 min; add 625 μg of plasmid to 20 mL of Opti-MEM medium and mix well; after 5 min, add the diluted PEIMAX to the diluted plasmid tube and mix well, and stand for 20 min; then add the mixture dropwise to the 500 mL of cells, and place it in a 140 rpm, 8% CO2 cell incubator; 6 h after transfection, add 3.5 mL of valproic acid sodium (VPA) reagent to inhibit cell growth and promote protein expression; add cell feed once after 1 day and 3 days of transfection.

[0078] 3. Purification of protein by affinity chromatography column: After 7 days of transfection, the cell culture supernatant was collected by centrifugation at 9000 rpm for 5 min, and the supernatant was incubated with the affinity chromatography nickel filler. The protein was eluted by adding a buffer containing multiple concentrations of imidazole (10 mM, 20 mM, 50 mM, 100 mM, 200 mM, and 500 mM) to the nickel filler one by one. The eluate was collected in separate tubes, and samples were taken from each tube for SDS-PAGE electrophoresis and Coomassie blue staining. The protein eluate with better purity was concentrated using an ultrafiltration tube to obtain the FMt-RBD(MERS) protein. The FMt-RBD(MERS) protein was stored in a buffer (20 mM Tirs-HCl, 50 mM NaCl, pH=7.4).

[0079] In this example, FMc-HPF and FMt-RBD(MERS) protein were mixed and incubated at a 1:1 molar ratio to form RBD(MERS)-FM-HPF nanoparticle vaccine, which was then loaded onto a gel filtration chromatography column Superose 6 Increase 10 / 300 GL for size exclusion chromatography (SEC) separation. The A280 wave peak protein liquid was collected at 1 mL per tube, and samples were taken from each tube for SDS-PAGE electrophoresis and Coomassie blue staining. According to the Coomassie blue staining results, it can be found that the peak position of FMt-RBD(MERS) is 16 mL, the peak position of FMc-HPF is 13 mL, and the peak position of RBD(MERS)-FM-HPF is 11 mL. Figure 4 ). The above results show that FMt-RBD(MERS) is successfully covalently coupled to the HPF nanoparticle.

[0080] In this example, the RBD(MERS)-FM-HPF nanoparticle vaccine separated by the 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. Preparation of protein sample and staining solution: The RBD(MERS)-FM-HPF nanoparticle protein sample and the staining solution (2% uranyl acetate) were centrifuged at 12000 rpm for 3 min, and the supernatant was transferred to a new tube to remove the insoluble precipitate.

[0082] 3. Loading onto copper mesh: 5 μL of RBD(MERS)-FM-HPF protein sample was added dropwise onto the sealing film, and the copper mesh was clamped with a self-locking forceps and then placed close to the sample on the sealing film. The sample was then adsorbed onto the copper mesh, and the sample solution was allowed to stand on the film for about 1 min to allow the sample particles to be adsorbed on the film surface. Then, the copper mesh was placed close to the filter paper to remove the excess sample solution.

[0083] 4. Staining: 5 μL of staining solution (2% uranyl acetate) was added dropwise onto the sealing film for three times of staining. The copper mesh was clamped with a self-locking forceps and then placed close to the staining solution on the sealing film. The staining agent was then adsorbed onto the copper mesh. The first drop of staining was removed immediately with filter paper; the second drop of staining was removed immediately with filter paper; and the third drop of staining was removed with filter paper after 2 min and the copper mesh was dried.

[0084] 5. Electron microscope observation: 120 kV Talos L120C transmission electron microscope was used to observe the nanoparticles on the copper mesh. The results showed that the RBD(MERS)-FM-HPF nanoparticles formed a cage-like structure, and the distribution was uniform, with a particle size of about 50 nm. Figure 4 ).

[0085] Example 4. Mouse immunization experiment of RBD(MERS)-FM-HPF nanoparticle vaccine To evaluate the antibody production ability of RBD(MERS)-FM-HPF nanoparticle vaccine, the RBD(MERS)-FM-HPF nanoparticle vaccine was mixed with an equal volume of aluminum adjuvant, and then 6-8 week-old C57BL / 6 mice were immunized subcutaneously, with 5 μg per mouse and a volume of 100 μL. This example also set up a FMt-RBD(MERS) monomer immunization group with an equal molar amount of 5 μg RBD(MERS)-FM-HPF as a control group.

[0086] To compare the antibody titers induced by FM molecular glue nanoparticle vaccine with the possible differences between the reported molecular glue nanoparticle vaccines in parallel, this embodiment uses Spytag003 (ST003) / Spycatcher003 (SC003) as a molecular glue coupling system to construct ST003-RBD (MERS) monomer vaccine and RBD (MERS) -ST003-HPF nanoparticle vaccine. Among them, 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 RBD (MERS) -ST003-HPF nanoparticle vaccine is the same molar number as that of RBD (MERS) -FM-HPF, and the volume is 100 μL. The immunization dose of ST003-RBD (MERS) is also the same molar number as that of RBD (MERS) -FM-HPF, and the volume is 100 μL.

[0087] The mouse experiment in this embodiment adopts a Prime / Boost double-needle immunization strategy, that is, the first needle is immunized at Week 0, and the second needle is immunized at Week 4 after four weeks of interval. One week after the second immunization, that is, at Week 5, the mice are euthanized, and peripheral blood is collected to separate serum samples for antigen-specific antibody titer and neutralizing antibody titer determination.

[0088] Example 5. Antigen-specific antibody determination induced by RBD (MERS) -FM-HPF nanoparticle vaccine This embodiment uses enzyme-linked immunosorbent assay (ELISA) to detect the IgG antibody level specific to MERS-CoV RBD in the serum of mice obtained from the mouse experiment in Example 4. The specific implementation steps are as follows: 1. Coating antigen on ELISA plate: dilute RBD (MERS) protein to 1 μg / mL with coating solution (pH = 9.6, containing 3.03 g / l Na2CO3 and 6 g / l NaHCO3), add 50 μL per well to a 96-well ELISA plate, and place it in a 4℃ incubator overnight to allow the protein to be adsorbed and coated in the plate well.

[0089] 2. Seal the ELISA plate: the next day, shake off the liquid, add 200 μL of PBST solution (containing 0.05% Tween-20 in PBS) per well and shake off the liquid, repeat the cleaning for 2 more times, then dry the well plate on the absorbent paper, add 100 μL of 5% skimmed milk powder solution per well, and incubate in a 37℃ incubator for 1 h to seal the non-specific binding sites in the well plate.

[0090] 3. Add serum to the plate: In a brand new 96-well U-bottom plate, dilute mouse serum with PBS at 7 dilution ratios (1:30, 1:300, 1:3000, 1:30000, 1:300000, 1:3000000, 1:30000000) and leave at least 110 μL per well after dilution. After blocking for 1 h, discard the defatted milk powder solution in the ELISA plate and wash each well with 200 μL of PBST solution. After repeating the washing for 2 more times, dry the plate on absorbent paper, transfer 100 μL of the diluted serum to the ELISA plate, and finally add 100 μL of PBS to the last row of each well as a blank control. Incubate in a 37°C incubator for 1 h.

[0091] 4. Add secondary antibody to the plate: After 1 h, shake the ELISA plate to discard the solution, wash each well with 200 μL of PBST solution, and repeat the washing for 4 more times. Dry the plate on absorbent paper, add 100 μL of anti-mouse IgG secondary antibody coupled with horseradish peroxidase (HRP) diluted with PBS to each well, and incubate in a 37°C incubator for 1 h.

[0092] 5. Color development, termination, and machine detection: After 1 h, discard the secondary antibody solution in the ELISA plate, wash each well with 200 μL of PBST solution, and repeat the washing for 4 more times. Dry the plate on absorbent paper, add 100 μL of TMB substrate color development solution to each well, and incubate at room temperature in the dark for 20 min. Add 100 μL of 1 M sulfuric acid solution to each well to terminate the color development. Place the ELISA plate in an enzyme label meter to detect the OD 450 nm absorbance value of each well and export the data.

[0093] 6. Data processing: In the GraphPad Prism software, use non-linear regression analysis to make a four-parameter Logistic curve with the serum dilution ratio as the x-axis and the OD 450 nm value as the y-axis. Take the average value of the OD 450 nm value of the last row as the reference value, calculate the x value corresponding to y = reference value for each serum, which is the endpoint antibody titer level of the serum. 10 [serum dilution ratio] as the x-axis and the OD 450 nm value as the y-axis, and take the average value of the OD 450 nm value of the last row as the reference value. Calculate the x value corresponding to y = reference value for each serum, which is the endpoint antibody titer level of the serum.

[0094] The results show that the RBD(MERS)-FM-HPF nanoparticle vaccine induced a high level of RBD-specific antibodies in mice, with an average titer of 5 x 10 4 , and the antibody titer level was better than that of the FMt-RBD(MERS) monomer group ( Figure 5). In addition, the antibody titers induced by the nanoparticle vaccine were equivalent to the antibody titers induced by RBD (MERS)-ST003-HPF.

[0095] Example 6. Neutralizing antibody assay induced by RBD (MERS)-FM-HPF nanoparticle vaccine This example used a MERS-CoV pseudovirus neutralization experiment method to detect the level of anti-MERS-CoV neutralizing antibodies in mouse serum. The specific implementation steps are as follows: 1. Packaging and titer determination of pseudovirus: In order to evaluate the neutralizing antibody titers induced by the nanoparticle vaccine, this example constructed a pseudovirus based on the Spike protein of MERS-CoV (Strain: HCoV-EMC / 2012) for determining these antibody titers. 12 μg of MERS-CoV Spike expression plasmid, 6 μg of packaging plasmid psPAX2 and 6 μg of pHIV-luciferase plasmid were co-transfected in 70~80% density HEK293T cells using transfection reagent PEI MAX. After 6 h of transfection, the supernatant was replaced with fresh DMEM medium containing 10% FBS, and the pseudovirus supernatant was collected 48 h after transfection, and the cell debris was removed by centrifugation at 3000 g for 5 min. The obtained pseudovirus supernatant was diluted by 10 dilutions with DMEM medium (containing 10% FBS) at a ratio of 1:2, and then 100 μL of diluted virus solution was taken from each dilution and co-cultured with HEK293T-hDPP4 cells. After 48 h of infection, the cells were lysed, and the expression amount of luciferase was determined using luciferin substrate, and the relative expression amount of luciferase also indirectly reflects the infection rate and titer of MERS-CoV pseudovirus.

[0096] 2. HEK293T-hDPP4 cell plating: The receptor of MERS-CoV virus is human dipeptidyl peptidase 4 (human dipeptidyl peptidase 4, hDPP4). In this example, HEK293T cells stably expressing hDPP4 receptor, i.e. HEK293T-hDPP4, were constructed. One night before the experiment, 50 μL of polylysine solution (10 μg / mL, molecular weight 150-300 kDa) was added to each well of a 96-well flat-bottom plate, and incubated in a 37°C incubator for 20 min. Then the polylysine solution was discarded, and 5x10 4 cells / 100 μL / well were added to the 96-well plate, and incubated in a 37°C, 5% CO2 incubator overnight.

[0097] 3. Serum neutralization pseudovirus: On the second day after cell plating, dilute the serum in a 96-well U-bottom plate, and dilute the mouse serum by 6 dilution ratios (1:5, 1:50, 1:500, 1:5000, 1:50000, 1:500000) using Opti-MEM, ensuring that 55 μL of diluted serum sample is left in each well after dilution. According to the previously determined MERS-CoV (Strain: HCoV-EMC / 2012) pseudovirus titer, dilute the pseudovirus in DMEM medium (containing 10% FBS), and then add 55 μL of the pseudovirus to the serum sample U-bottom plate, mix gently, and incubate in a 37°C incubator for 1 h. After 1 h of co-incubation of the serum and the pseudovirus, discard the supernatant in the HEK293T-hDPP4 cell plate, and transfer the serum and pseudovirus mixture to the cell plate, 100 μL per well. The first row is the cell control, and 100 μL of DMEM medium (containing 10% FBS) is added to each well. The last row is the pseudovirus control, and 50 μL of Opti-MEM and 50 μL of pseudovirus are added to each well. Then, place the serum / pseudovirus co-incubated cell culture plate in a 37°C incubator for 48 h.

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

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

[0100] 6. Data processing: Take the luciferin reading value of the cell control group as the lower limit of detection, and the pure pseudovirus control group as the upper limit of detection, calculate the proportion of the luminescence value in this interval in each serum / pseudovirus treatment group and the (1-proportion) value, and use non-linear regression analysis in GraphPad Prism software to make a four-parameter Logistic curve with the Log 10 [serum dilution ratio] value as the x-axis and the (1-proportion) value as the y-axis. The reciprocal of the serum dilution ratio corresponding to 50% inhibition of pseudovirus infection (IC 50 ) is the neutralizing antibody titer of the serum.

[0101] The results showed that RBD(MERS)-FM-HPF nanoparticle vaccine induced high levels of anti-MERS-CoV (Strain: HCoV-EMC / 2012) pseudovirus neutralizing antibody titers in mice, with an average titer of 5 x 10 3 Figure 6 . And the neutralizing antibody titer level is better than the antibody titer induced by RBD(MERS) monomer group, equivalent to the neutralizing antibody titer induced by the known molecular glue nanoparticle vaccine RBD(MERS)-ST003-HPF.

[0102] In summary, the present disclosure develops an isopeptide covalent bond coupling system of SpaA isopeptide-forming pilin-related protein (SpaA isopeptide-forming pilin-related protein, GenBank: WP_172975141.1) of Finegoldia magna (FM), namely FM molecular glue system. The FM molecular glue system is composed of two components of FMtag (FMt) peptide and FMcatcher (FMc) protein. The aspartic acid (D) at the 10th position on FMt and the lysine (K) at the 7th position on FMc can form an isopeptide bond under the catalysis of glutamic acid (E) at the 54th position on FMc, realizing the covalent binding of FMt and FMc.

[0103] Based on the FM molecular glue system, the present disclosure develops a RBD(MERS)-FM-HPF nanoparticle vaccine with RBD on MERS-CoV Spike as the antigen. The nanoparticle vaccine covalently displays an equal number of RBD(MERS) immunogens on the surface of HPF twenty-fourmer. The RBD(MERS) antigen-specific IgG antibody titer and the neutralizing antibody titer against MERS-CoV (Strain: HCoV-EMC / 2012) pseudovirus induced by the nanoparticle vaccine are significantly higher than the antibody titer induced by the RBD(MERS) monomer vaccine, and equivalent to the antibody titer induced by the nanoparticle vaccine developed by the known molecular glue system Spytag003 (ST003) / Spycatcher003 (SC003). The development of the FM molecular glue system makes up for the shortcomings of the currently available molecular glue system, and helps to solve the technical difficulties of using different molecular glues to display different antigens on nanoparticles in equal proportions. In addition, the FM molecular glue system developed by the present disclosure can also be used to display other infectious disease antigens or tumor antigens, and to present these antigens to different types of biological self-assembly nanoparticle carriers.

[0104] ​The technical solutions of the present application are not limited to the above specific embodiments, and any technical variations made according to the technical solutions of the present application fall within the protection scope of the present application.

Claims

1. A peptide composition comprising a first peptide and a second peptide, The first peptide comprises amino acids 88 to 103 corresponding to 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 it, and the amino acid 97 corresponding to the amino acid sequence shown in SEQ ID NO: 1 is D; The second peptide comprises amino acids 1 to 93 corresponding to 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 it, and the 7th amino acid corresponding to the amino acid sequence shown in SEQ ID NO: 1 is K and / or the 54th amino acid is E.

2. The peptide composition according to claim 1, characterized in that, The first peptide and the second peptide can covalently bind to form heteropeptide bonds; Preferably, 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; Preferably, 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; Preferably, the first peptide and the second peptide are derived from the SpaA isopeptide bond of *Granifera macrophyte* to form a fimbrial-associated protein. Preferably, 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.

3. A nucleic acid molecule comprising a nucleotide sequence encoding a first peptide and / or a second peptide in the peptide composition of claim 1 or 2.

4. An expression vector comprising the nucleic acid molecule of claim 3.

5. A host cell comprising the nucleic acid molecule of claim 3 or the expression vector of claim 4.

6. A molecular glue system comprising a molecular glue formed from a first peptide and a second peptide in the peptide composition of claim 1 or 2.

7. The application of the peptide composition of claim 1 or 2, the nucleic acid molecule of claim 3, the expression vector of claim 4, the host cell of claim 5, or the molecular glue system of claim 6 in the self-assembly of at least two molecules or components via isopeptide bonds; Preferably, the two molecules or components are respectively linked to the first peptide and the second peptide in the peptide composition of claim 1 or 2.

8. A method for self-assembling two molecules or components via isopeptide bonds, 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 of claim 1 or 2, respectively; 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.

9. The use of the peptide composition of claim 1 or 2, the nucleic acid molecule of claim 3, the expression vector of claim 4, the host cell of claim 5, or the molecular gel system of claim 6 in the preparation of nanoparticle vaccine compositions.

10. A nanoparticle vaccine composition, comprising: Nanoparticle core; and The peptide composition according to claim 1 or 2.

11. The nanoparticle vaccine composition according to claim 10, characterized in that, 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; Preferably, the antigen portion is exposed on the outer surface of the nanoparticles; Preferably, the first peptide is linked to the antigen moiety; Preferably, the second peptide is connected to the nanoparticle core; Preferably, the nanoparticle core comprises one or more of the following: Helicobacter pylori ferritin tetratriamer, Aerosol-producing bacteria 2,4-dioxane-tetrahydropteridine synthase hexamer, or fully synthetic nanoparticle I53-50 120-mer. Preferably, the nanoparticle vaccine includes infectious disease vaccines and / or tumor vaccines; Preferably, the antigen portion comprises 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.

12. The nanoparticle vaccine composition according to claim 10 or 11, characterized in that, The first fusion protein and the second fusion protein self-assemble to form nanoparticles. Preferably, the first fusion protein comprises the first peptide and the receptor-binding domain of the MERS-CoV virus Spike protein, and the second fusion protein comprises the second peptide and Helicobacter pylori ferritin tetratrimester; Preferably, 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; Preferably, 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.

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