A multimeric protein nanoparticle and its application in preparation of a novel coronavirus broad-spectrum vaccine

Abstract

The application discloses a kind of multimer protein nanoparticles and its application in preparation of novel coronavirus broad-spectrum vaccine, the multimer protein nanoparticles are: using the heptad repeat region HR1, HR2 on coronavirus S protein and fusion peptide FP, form pentamer complex FP-HR5 fusion protein, on this basis, with Ferritin assembly, prepare novel coronavirus antigen multimer complex FP-HR5-NP protein nanoparticle.It is prepared as immunogen to obtain the novel coronavirus general nanoparticle vaccine that can be inoculated by injection or respiratory system, and the immunoprotective effect is good.The immunoprotective effect of the novel coronavirus vaccine obtained by the application is good, can significantly activate mucosal immunity after respiratory system inoculation, and can be free from infection of different mutant strains of novel coronavirus after inoculation, and the preparation method is simple, protein expression purification technology route is mature, safety is high, and can be applied to clinical test faster.

Description

Technical Field

[0001] This invention relates to the field of immunology, and more specifically, to a multimeric protein nanoparticle and its application in the preparation of a broad-spectrum vaccine against the novel coronavirus. Background Technology

[0002] Coronaviruses are a large family of viruses known to cause more serious illnesses such as the common cold, Middle East Respiratory Syndrome (MERS), and Severe Acute Respiratory Syndrome (SARS).

[0003] The genome of coronaviruses is a single-sense positive-sense RNA, with a sequence length between 26.2 and 31.7 kb, making it the largest genome among all RNA viruses. Its genome is highly mutable during viral replication, allowing some mutant strains to evade the previously established immune barrier when facing pressure from the host's immune system.

[0004] The coronavirus genome has 6 to 10 open reading frames (ORFs), with a fixed sequence of structural protein genes at the 3' end: (HE)-SEMN. During viral replication, the genome is packaged into a helical complex by the nucleocapsid protein N expressed by the N gene, which is then encapsulated by a host-derived lipid bilayer to form the viral particle envelope. The envelope contains at least three viral proteins: spike protein (S), membrane protein (M), and envelope protein (E). Among these, the S protein is the most important protein mediating viral-host cell recognition and membrane fusion. It determines the virus's tissue and cell tropism, and antibodies that can block the binding of the S protein to its receptor have a strong neutralizing effect. Therefore, the S protein is also the most important antigenic source in coronavirus vaccine design.

[0005] The S protein sequence of coronaviruses can be divided into two parts, S1 and S2, based on the sites on which it is cleaved by host proteases. The S1 region is crucial for viral receptor recognition, and its receptor-binding domain (RBD) is considered the primary antigenic target region for inducing neutralizing antibodies, such as the RBD of SARS-CoV-2, which recognizes the host cell receptor ACE2. However, viral mutations often lead to the RBD remaining unrecognized by previously generated neutralizing antibodies while retaining its receptor-binding ability, resulting in decreased vaccine efficacy. Therefore, choosing the RBD as the core of vaccine antigen design has certain limitations. The S2 region is a key region mediating viral-host cell membrane fusion and exhibits highly conserved features, particularly high sequence homology among various mutant strains of SARS-CoV-2 and different coronaviruses within the genus Coronavirus. This includes the heptacap repeat domain, which mediates the dynamic folding of the S protein, and the fusion peptide sequence, which mediates fusion.

[0006] The novel coronavirus SARS-CoV-2 is highly transmissible, and its infection has a significant negative impact on people with underlying medical conditions. In severe cases, infection can lead to pneumonia, severe acute respiratory syndrome, kidney failure, and even death. There is no specific treatment for the disease caused by the novel coronavirus; furthermore, the immune barrier formed after vaccination can be easily evaded by novel mutant strains. Therefore, effectively mitigating the disease burden caused by the SARS-CoV-2 epidemic remains a major infectious disease challenge that global public health needs to address. Although several antiviral drugs have been developed and launched, the most critical and important measure to overcome the infectious disease crisis remains safe and effective vaccination, establishing a strong herd immunity barrier, and focusing on developing broad-spectrum vaccines to combat seasonal outbreaks of novel mutant strains.

[0007] However, vaccines currently designed based on the earliest original strains of SARS-CoV-2, or early mutant strains, have poor efficacy against subsequent emerging mutant strains. Therefore, developing a universal vaccine capable of combating potentially future circulating mutant strains is a pressing technical challenge.

[0008] In conclusion, there is a need to develop a universal vaccine that can induce potent and effective infections against various mutated coronaviruses to address the problem of immune escape.

[0009] In the native conformation of the SARS-CoV-2 virus, most of the S2 subunit is shielded by the S1 subunit. Therefore, the S2 subunit cannot induce antibodies or be recognized by antibodies in most cases, and the chance of mutation is relatively small. The HR1 (Heptad Repeat region 1, HR1) and HR2 (Heptad Repeat region 2, HR2) domains in the S2 subunit are only briefly exposed for a few minutes immediately after the RBD in S1 binds to the ACE2 receptor on the target cell. The HR1 and HR2 domains are the most conserved regions of the coronavirus S protein, mainly mediating the dynamic folding of the membrane protein after viral recognition of the viral receptor, and playing an important role in the membrane fusion of the virus and the host cell. Since the S protein exists in the form of a trimer on the viral envelope in the native state, and there are also intermolecular forces between HR1 and HR2, the HR region can fold to form an HR hexamer composed of tightly bound HR1 and HR2 during membrane fusion. In previous studies, nanoparticle vaccines using HR1 / HR2 as antigens have shown effective efficacy against the novel coronavirus SARS-CoV-2 (PMID:33275896), but the potency of HR1 / HR2 nanoparticle vaccines alone is still low. Summary of the Invention

[0010] The purpose of this invention is to overcome the shortcomings of the prior art and provide a multimeric protein nanoparticle and its application in the preparation of a broad-spectrum vaccine against the novel coronavirus.

[0011] The first objective of this invention is to provide a fusion protein.

[0012] A second objective of this invention is to provide a nucleic acid molecule.

[0013] A third objective of this invention is to provide a biomaterial.

[0014] A fourth object of the present invention is to provide a fusion protein composition.

[0015] The fifth objective of this invention is to provide a multimeric protein nanoparticle.

[0016] The sixth objective of this invention is to provide a method for preparing multimeric protein nanoparticles.

[0017] A seventh objective of this invention is to provide the application of the said multimeric protein nanoparticles in the preparation of a novel coronavirus vaccine.

[0018] The eighth object of the present invention is to provide a composition.

[0019] A ninth object of the present invention is to provide another composition.

[0020] The tenth object of the present invention is to provide the use of the composition in the preparation of a novel coronavirus vaccine.

[0021] To achieve the above objectives, the present invention is implemented through the following technical solution:

[0022] This invention utilizes three HR1 fragments and two HR2 fragments in tandem to design a viral antigen pentamer HR5, mimicking the HR multimer conformation during natural membrane fusion and reserving spatial epitopes for antibody recognition and binding with the HR fragments. This facilitates the induction of protective efficacy based on the HR folding process during membrane fusion. Furthermore, the FP-HR5 fusion protein was designed using FP and HR5 fusion, which facilitates the induction of antibody protection that can recognize and block the fusion peptide FP. Using the GvTagOpti / SdCatcher system (Gv / Sd system, see Chinese patent CN113621031A), the dual-region antigen FP-HR5 is covalently coupled to the surface of Ferritin to form 24-tetriameric nanoparticles FP-HR5-NP, which can effectively elicit a stronger immune response from the receptor. Furthermore, by utilizing the high conservation of FP and HR5 sequences in different coronaviruses, neutralizing antibodies that prevent coronavirus invasion of target cells are generated. The 24-tetrameric FP-HR5-NP protein nanoparticles of this invention overcome the shortcomings of insufficient broad-spectrum antigen RBD in traditional coronavirus vaccines, significantly improving the broad-spectrum and titer of neutralizing antibodies through multi-antigen display. Immunization of mice via subcutaneous and respiratory routes revealed that the prepared universal nanoparticle vaccine for the novel coronavirus induced high-titer antigen-specific antibody production, and respiratory administration protected mice from infection with different mutant strains of the novel coronavirus.

[0023] Therefore, the present invention claims a fusion protein having an amino acid sequence as shown in any one of SEQ ID NO:4, 5 or 7.

[0024] This invention relates to a polyantigen HR5 fusion protein formed by fusing three HR1 fragments and two HR2 fragments in series via a linker, in the form of HR1-HR2-HR1-HR2-HR1. The C-terminus of the HR1 fragment is fused with the N-terminus of the HR2 fragment using a GGSGG linker, and the C-terminus of the HR2 fragment is fused with the N-terminus of the HR1 fragment using a GSSGG linker. The amino acid sequence of the resulting HR5 is shown in SEQ ID NO:4.

[0025] The HR5 and FP are fused together via a linker to form the viral antigen polymeric complex FP-HR5 fusion protein. The linker is GSG, and its amino acid sequence is shown in SEQ ID NO:5.

[0026] The amino acid sequence of the fusion protein obtained by linking the C-terminus of the viral antigen polymeric complex FP-HR5 fusion protein with the Sd tag sequence shown in SEQ ID NO.6 via a GSG linker is shown in SEQ ID NO.7.

[0027] Further protection is sought for a nucleic acid molecule that encodes the gene for the fusion protein, or the reverse complementary sequence of the gene for the fusion protein.

[0028] And a biological material, which is any one of the following:

[0029] (1) An expression cassette containing the nucleic acid molecule;

[0030] (2) A recombinant vector containing the nucleic acid molecule described in (1) or the expression cassette described in (1);

[0031] (3) A recombinant microorganism containing the nucleic acid molecule described in (1), the expression cassette described in (1), or the recombinant vector described in (2);

[0032] (4) A cell line containing the nucleic acid molecule described in (1), the expression cassette described in (2), or the recombinant vector described in (3).

[0033] Further protection is claimed for a fusion protein composition comprising a Gv-Ferritin fusion protein with the amino acid sequence as shown in SEQ ID NO.10 and an FP-HR5-Sd fusion protein with the amino acid sequence as shown in SEQ ID NO.7.

[0034] Preferably, the molar ratio of the Gv-Ferritin fusion protein to the FP-HR5-Sd fusion protein is 4:5.

[0035] And a multimeric protein nanoparticle, which is obtained by self-assembly of Gv-Ferritin fusion protein with amino acid sequence as shown in SEQ ID NO.10 and FP-HR5-Sd fusion protein with amino acid sequence as shown in SEQ ID NO.7.

[0036] A method for preparing multimeric protein nanoparticles, wherein the Gv-Ferritin fusion protein with the amino acid sequence as shown in SEQ ID NO.10 and the FP-HR5-Sd fusion protein with the amino acid sequence as shown in SEQ ID NO.7 are incubated in a buffer solution.

[0037] Preferably, the buffer solution is 20 mM Tris-HCl, 50 mM NaCl, pH 7.5.

[0038] Preferably, the incubation period is no more than 15 hours.

[0039] It also claims protection for the use of the said multimeric protein nanoparticles in the preparation of a novel coronavirus vaccine.

[0040] Preferably, the application is for the preparation of vaccines against the novel coronavirus Omicron and / or Delta strains.

[0041] The following compositions also require protection:

[0042] A composition comprising the fusion protein composition and an adjuvant.

[0043] A composition comprising the said polymeric protein nanoparticles and an adjuvant.

[0044] Preferably, the adjuvant is cycloguanosine monophosphate or an aluminum adjuvant.

[0045] It also claims protection for the use of the composition in the preparation of a novel coronavirus vaccine.

[0046] Preferably, the novel coronavirus vaccine is a respiratory vaccine, and the adjuvant is cyclic diguanosine monophosphate (c-di-GMP) adjuvant.

[0047] More preferably, the mass ratio of the polymeric protein nanoparticles to the cyclic diguanylic acid adjuvant is 5:1.

[0048] Preferably, the novel coronavirus vaccine is an injectable vaccine, the adjuvant is an aluminum adjuvant, and the volume ratio of the novel coronavirus antigen polymeric complex to the aluminum adjuvant is 1:1.

[0049] Compared with the prior art, the present invention has the following beneficial effects:

[0050] This invention utilizes the heptapeptide repeat regions HR1 and HR2 and the fusion peptide FP on the coronavirus S protein to obtain a novel coronavirus antigen. A pentamer complex FP-HR5 fusion protein is formed by linking FP, HR1, and HR2 together via a linker. Based on this, a Gv-Ferritin fusion protein is self-assembled with the GvTagOpti / SdCatcher linker system to prepare novel coronavirus antigen polymeric complex FP-HR5-NP protein nanoparticles. These nanoparticles are used as immunogens to prepare a universal novel coronavirus nanoparticle vaccine that can be administered via injection or respiratory route and exhibits good immunoprotective efficacy. The novel coronavirus vaccine obtained by this invention has good immunoprotective efficacy, significantly activates mucosal immunity after respiratory route inoculation, and protects against infection by different mutant strains (Omicron and Delta) of the novel coronavirus. Furthermore, the preparation method is simple, the protein expression and purification technology is mature, the safety is high, and it can be rapidly applied to clinical trials. Attached Figure Description

[0051] Figure 1 This is a schematic diagram of the structural simulation of the novel coronavirus antigen FP-HR5-Sd fusion protein and Gv-Ferritin protein.

[0052] Figure 2 This is a structural simulation diagram of the novel coronavirus polymeric complex FP-HR5-NP formed by the covalent binding of the novel coronavirus antigen FP-HR5-Sd with the multimeric nanoparticle protein Gv-Ferritin.

[0053] Figure 3 This is a schematic diagram of the expression vector PET28a-FP-HR5-Sd for the FP-HR5-Sd fusion protein.

[0054] Figure 4 This is a schematic diagram of the structure of the expression vector PET28a-Gv-Ferritin for the Gv-Ferritin fusion protein.

[0055] Figure 5 The results of SDS-PAGE electrophoresis and Coomassie brilliant blue staining are shown for the expression and purification of the 24-tetrameric complex Gv-Ferritin protein nanoparticles, FP-HR5-Sd fusion protein, and 24-tetrameric complex FP-HR5-NP protein nanoparticles.

[0056] Figure 6 The image shows the peaks obtained after purification of the 24-tetrameric complex FP-HR5-NP protein nanoparticle sample using a gel chromatography column (Superose6Increase 10 / 300GL column, GE).

[0057] Figure 7 The results show the particle size of the 24-tetrameric complex FP-HR5-NP protein nanoparticles measured using a Baxter nanoparticle size potential analyzer (Zeta-potential distribution analysis).

[0058] Figure 8 This image shows the results of observation under a transmission electron microscope (TEM) of the novel coronavirus antigen polymer complex FP-HR5-NP sample.

[0059] Figure 9 An immunization strategy diagram for immunizing C57BL / 6 mice with the novel coronavirus FP-HR5-NP protein nanoparticle inhalation vaccine.

[0060] Figure 10Eight weeks after the first immunization, the antigen-specific IgG antibody in mouse serum was measured. The control group consisted of mice that had been inoculated with Gv-Ferritin via the respiratory system (HPF(in) group). * indicates statistically significant difference (p < 0.05), ** indicates p < 0.01, *** indicates p < 0.001, and **** indicates highly significant difference (p < 0.0001).

[0061] Figure 11 The results show the titers of IgA and IgG antibodies in bronchoalveolar lavage fluid of mice 8 weeks after the first immunization; the control group consisted of mice that were inoculated with Gv-Ferritin in their respiratory system (HPF(in) group). * indicates statistical difference, p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, and **** indicates extremely significant difference, p < 0.0001.

[0062] Figure 12 Eight weeks after the initial immunization, resident memory B cells (B cells) in the lung tissue of mice were observed. RM The proportion of the subgroups; the control group consisted of mice whose respiratory system was inoculated with Gv-Ferritin (HPF(in) group). * indicates statistical difference, p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, **** indicates extremely significant difference, p < 0.0001.

[0063] Figure 13 Eight weeks after the initial immunization, resident memory T cells (T cells) in the lung tissue of mice were observed. RM The proportion of subgroups (Figure D) is shown in the figure. The control group in the figure is mice whose respiratory system was inoculated with Gv-Ferritin (HPF(in) group). * indicates statistical difference, p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, and **** indicates extremely significant difference, p < 0.0001.

[0064] Figure 14 The results show the in vitro titer of neutralizing antibody against the Omicron strain of the novel coronavirus in mouse serum 8 weeks after the first immunization.

[0065] Figure 15 The results show the RNA copy number of the novel coronavirus in the lungs of mice 8 weeks after the initial immunization, following challenge with the Omicron and Delta mutant strains of the novel coronavirus.

[0066] Figure 16 The results are pathological sections of the lungs of mice 8 weeks after the first immunization, following challenge with the novel coronavirus Omicron and Delta mutant strains. Detailed Implementation

[0067] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; the materials and reagents used, unless otherwise specified, are commercially available.

[0068] The Omicron and Delta strains of the novel coronavirus used were both provided by the Guangdong Provincial Center for Disease Control and Prevention.

[0069] Example 1: Design of a universal viral antigen for the novel coronavirus

[0070] The structural diagram of the universal viral antigen of the novel coronavirus is shown below. Figure 1 As shown:

[0071] The sequences located in heptad repeat region 1 (HR1) and heptad repeat region 2 (HR2) were tandemly fused multiple times in the order of HR1-HR2-HR1-HR2-HR1 to form the HR5 pentamer complex antigen. The HR5 pentamer mimics the polymeric structure of HR in the viral natural protein multimer. The C-terminus of the HR1 fragment is fused with the N-terminus of HR2 using a GGSGG linker, and the C-terminus of HR2 is fused with the N-terminus of HR1 using a GSSGG linker.

[0072] Then, HR5 and the novel coronavirus fusion peptide (FP) are fused together using a linker (GSG) to form the composite antigen FP-HR5 fusion protein.

[0073] The amino acid sequence of SARS-CoV-2 HR1 is shown in SEQ ID NO:1, the amino acid sequence of HR2 is shown in SEQ ID NO:2, and the amino acid sequence of FP is shown in SEQ ID NO:3.

[0074] SEQ ID NO:1(HR1):

[0075] GVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQD;

[0076] SEQ ID NO:2(HR2):

[0077] DISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQ;

[0078] SEQ ID NO:3 (FP sequence):

[0079] SKPSKRSFIEDLLFNKVTLA;

[0080] SEQ ID NO:4(HR5):

[0081] GVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDGGSGGDISGINAS

[0082] VVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQGSSGGGVTQNVLYENQKLIA

[0083] NQFNSAIGKIQDSLSSTASALGKLQDGGSGGDISGINASVVNIQKEIDRLNEVA

[0084] KNLNESLIDLQELGKYEQGSSGGGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDGG;

[0085] SEQ ID NO:5(FP-HR5):

[0086] SKPSKRSFIEDLLFNKVTLAGSGGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDGGSGGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQGSSGGGVTQNVLYEN QKLIANQFNSAIGKIQDSLSSTASALGKLQDGGSGGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQGSSGGGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDGG.

[0087] The novel coronavirus antigen FP-HR5 fusion protein was linked to the multimeric complex Ferritin protein nanoparticles using the GvTagOpti / SdCatcher (Gv / Sd) isopeptide linking system to construct the novel coronavirus antigen multimeric complex FP-HR5-NP. Therefore, the Sd sequence shown in SEQ ID NO.6 was fused to the C-terminus of FP-HR5 via the GSG linker. The amino acid sequence of the resulting fusion protein FP-HR5-Sd is shown in SEQ ID NO.7.

[0088] SEQ ID NO.6(Sd):

[0089] SGETGQSGNTTIEEDSTTHVKFSKRDINGKELAGAMIELRNLSGQTIQSWVSDGTVKDFYLMPGTYQFVETAAPEGYELAAPITFTIDEKGQIWVDSTLIVGDDPI;

[0090] SEQ ID NO.7 (FP-HR5-Sd):

[0091] SKPSKRSFIEDLLFNKVTLAGSGGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDGGSGGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQGSSGGGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDGGSGGDISGINASVVNIQKEIDRL NEVAKNLNESLIDLQELGKYEQGSSGGGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDGGGSGSGETGQSGNTTIEEDSTTHVKFSKRDINGKELAGAMIELRNLSGQTIQSWVSDGTVKDFYLMPGTYQFVETAAPEGYELAAPITFTIDEKGQIWVDSTLIVGDDPI.

[0092] Meanwhile, the Gv sequence shown in SEQ ID NO. 9 was fused to the N-termin of Ferritin shown in SEQ ID NO. 8, and the amino acid sequence of the resulting fusion protein Gv-Ferritin is shown in SEQ ID NO. 10.

[0093] SEQ ID NO.8 (Ferritin):

[0094] DIIKLLNEQVNKEMQSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS;

[0095] SEQ ID NO.9(Gv):

[0096] KVGNTIVMVD KLKEVPTP;

[0097] SEQ ID NO.10 (Gv-Ferritin):

[0098] KVGNTIVMVDKLKEVPTPGSGDIIKLLNEQVNKEMQSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISAPEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYVKGIAKSRKS.

[0099] Example 2: Preparation of a universal nanoparticle antigen for the novel coronavirus

[0100] The novel coronavirus antigen FP-HR5 fusion protein was assembled onto the surface of ferritin-based nanoparticles, forming the novel coronavirus antigen-ticodrimer complex FP-HR5-NP protein nanoparticles, as shown in the schematic diagram. Figure 2 As shown.

[0101] I. Protein Expression

[0102] (I) Experimental Methods

[0103] 1. Expression of fusion proteins FP-HR5-Sd and Gv-Ferritin

[0104] (1) Preparation of FP-HR5-Sd fusion protein

[0105] The FP-HR5-Sd protein was expressed and purified in Escherichia coli (E. coli) using the following method:

[0106] (A) Construction of recombinant vectors

[0107] The encoding DNA sequence of the FP-HR5-Sd fusion protein shown in SEQ ID NO.7 was cloned into the prokaryotic expression vector PET28a to construct the expression vector PET28a-FP-HR5-Sd (as shown in SEQ ID NO.7). Figure 3 As shown in the figure, this allows the protein to be expressed naturally by fusing the six his tags originally located on the PET28a vector to the C-terminus of the FP-HR5-Sd protein.

[0108] (B) Expression of FP-HR5-Sd fusion protein

[0109] The expression vector PET28a-FP-HR5-Sd was transformed into BL21(Takara) cells; then, single clones were amplified by shaking culture in LB medium containing kanamycin at 37°C; isopropyl-β-D-thiogalactoside (IPTG) was added to the bacterial culture to induce protein expression; after 18 hours of induction, the bacteria expressing the protein were harvested and subjected to high-pressure disruption, and the supernatant was collected.

[0110] (C) Purification of FP-HR5-Sd fusion protein

[0111] The supernatant was initially purified using a 10K ultrafiltration tube (Millipore), followed by incubation on a Ni-NTA agarose column (GE) to enrich the His-labeled FP-HR5-Sd fusion protein. The obtained FP-HR5-Sd fusion protein was washed with Tris buffer containing 500 mM Imidazole (20 mM Tris-HCl and 50 mM NaCl, pH 7.5) to remove the protein. The purified protein was concentrated, and the buffer was replaced with a standard Imidazole-free Tris buffer (20 mM Tris-HCl and 50 mM NaCl, pH 7.5). Finally, the concentration of the FP-HR5-Sd fusion protein was determined using the BCA assay.

[0112] (2) Preparation of Gv-Ferritin fusion protein

[0113] The Gv-Ferritin protein was expressed and purified in Escherichia coli (E. coli) using the following method:

[0114] (A) Construction of recombinant vectors

[0115] The encoding DNA sequence of the Gv-Ferritin fusion protein shown in SEQ ID NO.10 was cloned into the prokaryotic expression vector PET28a to construct the expression vector PET28a-Gv-Ferritin (as shown in SEQ ID NO.10). Figure 4 As shown in the figure, this allows the protein to be expressed naturally by fusing the six his tags originally located on the PET28a vector to the C-terminus of the Gv-Ferritin protein.

[0116] (B) Expression of Gv-Ferritin fusion protein

[0117] Same as above.

[0118] (C) Purification of Gv-Ferritin fusion protein

[0119] Same as above.

[0120] (II) Experimental Results

[0121] The prepared Gv-Ferritin fusion protein, FP-HR5-Sd fusion protein, and FP-HR5-NP fusion protein were analyzed by SDS-PAGE. Results Figure 5 As shown. By Figure 5 It can be seen that the present invention successfully prepared Gv-Ferritin fusion protein, FP-HR5-Sd fusion protein and FP-HR5-NP fusion protein, and the size was consistent with the expectation.

[0122] II. Self-assembly of novel coronavirus universal viral nanoparticle antigens

[0123] (I) Experimental Methods

[0124] The FP-HR5-Sd fusion protein (SEQ ID NO.7) prepared at 5 μM and the Gv-Ferritin fusion protein (SEQ ID NO.10) prepared at 4 μM were placed in 800 μL of enzyme-free buffer (20 mM Tris-HCl, 50 mM NaCl, pH 7.5) and incubated at 25 °C for 15 hours. The FP-HR5-Sd fusion protein and the Gv-Ferritin fusion protein were covalently bound through the GvTagOpti / Sdcatcher system (Gv / Sd, see Chinese Patent CN113621031A) to prepare 24-tetrameric complex FP-HR5-NP protein nanoparticles.

[0125] Then, the incubated system was purified by size-exclusion chromatography (SEC) using a Superose 6Increase10 / 300GL column to obtain high-purity 24-tetrameric complex FP-HR5-NP protein nanoparticles. The SEC buffer and the storage solvent for the purified FP-HR5-NP protein were a buffer containing 20 mM Tris-HCl and 50 mM NaCl at pH 7.5.

[0126] (II) Experimental Results

[0127] The SEC purification peak diagram of the tetratetramic complex FP-HR5-NP protein nanoparticles is shown below. Figure 6 As shown, the purified product has a purity higher than 99%.

[0128] Example 3 Characterization of the tetratetramic complex FP-HR5-NP protein nanoparticles

[0129] I. Particle Size Detection

[0130] 1. Experimental Methods

[0131] The 24-tetrameric complex FP-HR5-NP protein nanoparticles prepared in Example 2 were separated and collected by SEC, then concentrated, and the particle size of the obtained 24-tetrameric complex FP-HR5-NP protein nanoparticles was measured using a Baxter nanoparticle size potential analyzer (Zeta-potential distribution analysis).

[0132] 2. Experimental Results

[0133] Particle size detection results are as follows Figure 7 As shown, the UV280 peak of the obtained tetratrimester complex FP-HR5-NP protein nanoparticles is single; the particle size is slightly larger than that of the Gv-Ferritin fusion protein without FP-HR5 fusion protein, around 20 nm, indicating that the present invention has obtained tetratrimester complex FP-HR5-NP protein nanoparticles with a purity higher than 99%.

[0134] II. Uniformity Test

[0135] 1. Experimental Methods

[0136] The FP-HR5-NP protein nanoparticles, which are 24-tetramic complexes prepared in Example 2, were separated and collected by SEC, then concentrated and observed by transmission electron microscopy (TEM).

[0137] 2. Experimental Results

[0138] Transmission electron microscopy (TEM) image of the tetratetramic complex FP-HR5-NP protein nanoparticles as shown below. Figure 8 As shown, after FP-HR5 is covalently bound to Ferritin nanoparticles, the 24-tetrameric complex FP-HR5-NP protein nanoparticles exhibit homogeneous hollow particles, with the antigen FP-HR5 protruding from the spherical core to form spike-like structures.

[0139] The results above demonstrate that FP-HR5 was successfully enriched on the surface of Ferritin nanoparticles, which can be used to construct nanoparticle vaccines.

[0140] Example 4: Immunization of the tetratetramic complex FP-HR5-NP protein nanoparticles

[0141] I. Experimental Methods

[0142] Mice were immunized by subcutaneous (sc) and respiratory (in) administration, respectively, to test the immunoprotective effect of the vaccine constructed from the 24-tetrameric complex FP-HR5-NP protein nanoparticles prepared in Example 2.

[0143] (1) The FP-HR5-NP protein nanoparticles, which are 24-tetramic complexes prepared in Example 2, are mixed with the adjuvant cyclic diguanosine monophosphate (c-di-GMP) at a mass ratio of 5:1 to obtain the FP-HR5-NP protein nanoparticle inhalation vaccine.

[0144] C57BL / 6 mice were immunized via respiratory inoculation (in) to detect the respiratory immunoprotective effect of the FP-HR5-NP protein nanoparticle inhaled vaccine.

[0145] (2) The FP-HR5-NP protein nanoparticles of the tetratriamer complex prepared in Example 2 were mixed with aluminum adjuvant at a mass ratio of 1:1 to obtain the FP-HR5-NP protein nanoparticle injection vaccine.

[0146] The subcutaneous immunoprotective effect of FP-HR5-NP protein nanoparticle injection vaccine was tested by immunizing C57BL / 6 mice via subcutaneous injection (sc).

[0147] (3) Immune strategies such as Figure 9 As shown, both subcutaneous (sc) and respiratory (in) immunization of mice involved administering 10 μg of the tetratetramic complex FP-HR5-NP protein nanoparticles prepared in Example 2 to each mouse, using a three-immunization strategy, namely, immunization at week 0, week 3, and week 6.

[0148] Samples were collected and tested two weeks after the third vaccination (8 weeks after the first vaccination):

[0149] Enzyme-linked immunosorbent assay (ELISA) was used to detect antigen-specific IgG antibodies in serum. The secondary antibody used was either horseradish peroxidase (HRP)-labeled goat anti-mouse IgG secondary antibody (abcam) or HRP-labeled goat anti-mouse IgA secondary antibody (abcam).

[0150] The titers of IgA and IgG antibodies in bronchoalveolar lavage fluid were detected using enzyme-linked immunosorbent assay (ELISA).

[0151] Resident memory B cells (B cells) in mouse lung tissue were analyzed using flow cytometry. RM ) and resident memory T cells (T RM ) to conduct testing;

[0152] The neutralizing protective ability of the serum of immunized mice was determined by neutralization titration experiment using the Omicron strain of the novel coronavirus. 500 FFU of live Omicron strain was mixed with diluted serum at a volume ratio of 1:1 for neutralization titration. The primary antibody used was anti-SARS-CoV-2 nucleocapsid (N) protein antibody (Sino-US), and the secondary antibody was HRP-labeled rabbit anti-IgG secondary antibody (Sino-US).

[0153] Transgenic C57BL / 6 mice (hACE2 mice, purchased from Jiangsu Jicui Pharmaceutical Co., Ltd., strain number T037659) expressing the human SARS-CoV-2 receptor ACE2 were infected with the respiratory system by intranasal inoculation with the Omicron and Delta strains of SARS-CoV-2 for 8 weeks. The specific infection method was as follows: after the mice were anesthetized with isoflurane, 2 × 10⁻⁶ styrosine dextrose intranasally. 4 FFU virus; on the third day after inoculation, lung tissue of mice was collected for virus content detection, and lung tissue of mice was collected for pathological examination and evaluation by HE section staining.

[0154] II. Experimental Results

[0155] See results Figures 10 to 16 The FP-HR5-NP protein nanoparticle inhalation vaccine and FP-HR5-NP protein nanoparticle injection vaccine constructed from the 24-tetrameric complex FP-HR5-NP protein nanoparticles prepared in Example 2 can induce a systemic immune response after inhalation (in) and subcutaneous injection (sc), respectively, achieving protection against Omicron and Delta mutant strains. In particular, the FP-HR5-NP protein nanoparticle inhalation vaccine after inhalation (in) showed a better pulmonary immune response.

[0156] Specifically, compared with the control group, both the FP-HR5-NP protein nanoparticle inhalation vaccine and the FP-HR5-NP protein nanoparticle injection vaccine induced higher titers of FP and HR5 antigen-specific IgG antibodies in peripheral blood after immunization. Figure 10 );

[0157] Compared with the control group and subcutaneous injection, inhaled FP-HR5-NP protein nanoparticle vaccine via respiratory administration induces higher titers of antigen-specific IgA and IgG antibodies in the alveolar environment after immunization, thereby exerting the respiratory immune barrier's resistance to the virus through IgA mucosal immunity and IgG immune protection. Figure 11 );

[0158] Compared with the control group, subcutaneous injection of the FP-HR5-NP protein nanoparticle inhaled vaccine induced high levels of antigen-specific IgG1 in lung tissue after immunization.+ CD38 + B RM and IgA + CD38 + B RM This induces a strong humoral immune response, thereby exerting the respiratory immune barrier's resistance against the virus within the lung tissue. Figure 12 );

[0159] Compared with the control group, subcutaneous injection and respiratory administration of the FP-HR5-NP protein nanoparticle inhaled vaccine induced high levels of CD4 in lung tissue after immunization. + CD44 + CD69 + CD103 + T RM and CD8 + CD44 + CD69 + CD103 + T RM Subgroups, thereby inducing a potent cellular immune response, exert a respiratory immune barrier effect against the virus within the lung tissue. Figure 13 ).

[0160] Compared with the control group, both the FP-HR5-NP protein nanoparticle inhalation vaccine and the FP-HR5-NP protein nanoparticle injection vaccine induced high levels of neutralizing antibodies in peripheral blood after immunization, and both showed neutralizing activity against the novel mutant strain Omicron. Figure 14 ).

[0161] Compared with the control group, the FP-HR5-NP protein nanoparticle inhalation vaccine immunized hACE2 mice exerted a good protective effect, resulting in viral RNA levels in the lungs of mice below the detection limit on day 3 post-exposure. Furthermore, it demonstrated broad-spectrum protective efficacy against both Omicron and Delta mutant strains. Figure 15 ).

[0162] Compared with the control group, the FP-HR5-NP protein nanoparticle inhalation vaccine immunized hACE2 mice exerted a good protective effect, maintaining the alveolar and bronchial structure under normal physiological conditions in the lung tissue of mice on the 3rd day after exposure, reducing inflammatory infiltration, and also exerting a protective effect against both Omicron and Delta mutant strains, demonstrating broad-spectrum protective efficacy. Figure 16 ).

Claims

1. A fusion protein, characterized in that, Its amino acid sequence is shown in either SEQ ID NO: 5 or 7.

2. A nucleic acid molecule, characterized in that, The encoding gene of the fusion protein of claim 1, or the reverse complementary sequence of the encoding gene.

3. A biomaterial, characterized in that, It is any one of the following: (1) an expression cassette containing the nucleic acid molecule of claim 2; (2) a recombinant vector containing the nucleic acid molecule of claim 2 or the expression cassette of (1); (3) a recombinant microorganism containing the nucleic acid molecule of claim 2, the expression cassette of (1) or the recombinant vector of (2); (4) a cell line containing the nucleic acid molecule of claim 2, the expression cassette of (1), the recombinant vector of (2) or the recombinant microorganism of (3).

4. A fusion protein composition, characterized in that, This includes the Gv-Ferritin fusion protein with the amino acid sequence shown in SEQ ID NO: 10 and the FP-HR5-Sd fusion protein with the amino acid sequence shown in SEQ ID NO:

7.

5. A polymeric protein nanoparticle, characterized in that, It was obtained by self-assembly using the Gv-Ferritin fusion protein with the amino acid sequence shown in SEQ ID NO: 10 and the FP-HR5-Sd fusion protein with the amino acid sequence shown in SEQ ID NO:

7.

6. A method for preparing polymeric protein nanoparticles, characterized in that, The Gv-Ferritin fusion protein with the amino acid sequence shown in SEQ ID NO: 10 and the FP-HR5-Sd fusion protein with the amino acid sequence shown in SEQ ID NO: 7 are obtained by incubating in a buffer solution.

7. The application of the polymeric protein nanoparticles according to claim 5 in the preparation of a novel coronavirus vaccine, characterized in that, The novel coronavirus is either SARS-CoV-2 Omicron or SARS-CoV-2 Delta mutant strain.

8. A composition, characterized in that, It contains the fusion protein composition of claim 4 and an adjuvant.

9. A composition, characterized in that, It contains the polymeric protein nanoparticles of claim 5 and an adjuvant.

10. The use of the composition according to claim 8 or 9 in the preparation of a novel coronavirus vaccine, characterized in that, The novel coronavirus is either SARS-CoV-2 Omicron or SARS-CoV-2 Delta mutant strain.

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