A novel coronavirus nano-vaccine with targeted synergistic enhancement

Abstract

The present invention discloses a novel coronavirus nano-vaccine with targeted synergistic effect, belonging to the field of vaccines. The active ingredient of the vaccine is a fusion protein, which includes the amino acid sequence of the RBD domain of the novel coronavirus. The N-terminus of the amino acid sequence of the RBD domain is connected to XCL1 through a flexible linker GSG, and the C-terminus of the RBD domain sequence is connected to the Ferritin protein through a flexible linker GSG. The purified XCL1-RBD-Ferritin can be assembled into nanoparticles of 24-mers, improving the efficiency of RBD antigen in activating DCs, especially DC1s, thereby efficiently initiating humoral and cellular immune responses.

Description

Technical Field

[0001] The present invention belongs to the field of vaccines, and particularly relates to a novel coronavirus nano-vaccine with targeted synergistic enhancement. Background Art

[0002] The pathogen of COVID-19 is the novel coronavirus (severe acute respiratory syndrome coronavirus 2, SARS-CoV-2). The extremely high mutation rate of the virus genes has brought great difficulties to the prevention and control of the disease.

[0003] Currently, the mutated novel coronaviruses mainly include six categories: Alpha, Beta, Gamma, Delta, Lambda, and Omicron. In terms of both the transmission speed and the fatality rate, Omicron is much more severe than influenza and other previous mutated strains of the new coronavirus.

[0004] Currently, the COVID-19 vaccines already on the market in China include: inactivated vaccines, adenovirus vector vaccines, and recombinant subunit vaccines; many enterprises have also actively carried out research on mRNA vaccines. Inactivated vaccines have the advantages of a short preparation cycle and high safety, but their immunogenicity is relatively low, and mainly induce humoral immunity. Adenovirus vector vaccines use harmless adenoviruses after modification as vectors, loaded with the S protein gene of the new coronavirus to stimulate the human body to produce antibodies. However, the fact that some people have been infected with adenoviruses may affect the immune effect of the vaccine. mRNA vaccines have good immunogenicity and can effectively stimulate the body to produce high levels of neutralizing antibodies. However, due to the relatively new technology, their safety still needs to be further clinically tracked and determined. The main component of recombinant subunit vaccines is to express a large amount of S protein or RBD antigen through biotechnology methods. This kind of vaccine has a single component, very good safety, and a relatively mature mass production process. However, due to relatively weak immunogenicity, it is necessary to optimize the vaccine components and construction methods.

[0005] The frequent mutation of SARS-CoV-2 has brought great challenges to the effectiveness of existing vaccines. Clinical follow-up studies have shown that the protective effects of mRNA vaccines (ChAdOx-1 and BNT162b2) against the Delta variant have decreased by an average of 8 times, and the protective efficiency against the Omicron variant has decreased by 41.1 times; the neutralizing ability of the serum neutralizing antibodies of recovered COVID-19 patients against the Delta variant has decreased by an average of 6 times, and decreased by 10.14 times against the Omicron variant. Existing studies have shown that cellular immune responses are not only correlated with the severity and development of COVID-19, but also the early CD8 +T cell immune responses are also very important for the body's defense and inhibition of the spread of SARS-CoV-2. Long-term studies on recovered patients from SARS in 2003 showed that cellular immune memory was maintained for a relatively long time, some even up to 17 years, while the humoral immune memory of recovered patients was relatively short. However, compared with live attenuated vaccines, other types of vaccines have limited ability to induce CD8 + T cell immune responses, and live attenuated vaccines themselves have a potential risk of spreading the virus, and are not suitable for COVID-19 prevention and control.

[0006] Therefore, new research strategies are needed to improve the ability of SARS-CoV-2 vaccines to induce CD8 + T cell immune responses and maintain immune memory, in order to cope with the possibility of repeated infections caused by the continuous mutation of the virus leading to severe patients. Summary of the Invention

[0007] The purpose of the present invention is to overcome at least one deficiency of the prior art and provide a novel coronavirus nano-vaccine (antigen) with targeted synergistic effect. After immunizing the body with this antigen, it is expected to stimulate high-level humoral immunity and CD8 + T cell immune responses in the body, and the immune memory is maintained for a longer time.

[0008] The technical solution adopted by the present invention is:

[0009] A fusion protein, comprising the amino acid sequence of the RBD domain of the novel coronavirus, the N-terminus of the amino acid sequence of the RBD domain is connected to XCL1 through a flexible linker GSG, and the C-terminus of the RBD domain sequence is connected to the Ferritin protein through a flexible linker GSG.

[0010] The Ferritin protein is Helicobacter pylori nonhemeferritin (hereinafter simply referred to as Ferritin protein)

[0011] In some embodiments, the amino acid sequence of the RBD domain is as shown in SEQ ID NO.1, the amino acid sequence of XCL1 is as shown in SEQ ID NO.2, and the amino acid sequence of the Ferritin protein is as shown in SEQ ID NO.3.

[0012] In some embodiments, the amino acid sequence of the fusion protein is as shown in SEQ ID NO.4.

[0013] A gene encoding the fusion protein described in any one of the above.

[0014] In some of these embodiments, the nucleotide sequence encoding the RBD domain of the novel coronavirus is as shown in SEQ ID NO.5, the nucleotide sequence encoding XCL1 is as shown in SEQ ID NO.6, and the nucleotide sequence encoding the Ferritin protein is as shown in SEQ ID NO.7. The nucleotide sequences of XCL1, RBD, and Ferritin of the present invention are obtained by optimizing according to the preference of the E.Coli expression system.

[0015] In some of these embodiments, the nucleotide sequence encoding the flexible linker GSG between XCL1 and RBD is GGTAGCGGT, and the nucleotide sequence encoding the flexible linker GSG between RBD and Ferritin is GGCAGTGGT.

[0016] The nucleotide sequences of the GSG linkers are inconsistent, which means that there are multiple combinations of the preference of E. coli for the GSG linker. Through optimization by the inventors, the best combination is achieved to effectively increase the protein expression level.

[0017] In some of these embodiments, the nucleotide sequence encoding the fusion protein is as shown in SEQ ID NO.9.

[0018] A vaccine, wherein the active ingredient of the vaccine is the above-mentioned fusion protein.

[0019] In some of these embodiments, the preparation form of the vaccine is an aqueous solution or a freeze-dried preparation.

[0020] In some of these embodiments, the vaccine contains an adjuvant.

[0021] In some of these embodiments, the adjuvant is any one of CpG, QS21, aluminum phosphate, a mixture of CpG and aluminum phosphate, or a mixture of QS21 and aluminum phosphate.

[0022] The fusion protein of the present application is designed such that antibodies against the RBD region of the S protein of the novel coronavirus can play a key neutralizing role. For people vaccinated with the S protein vaccine, more than 90% of the neutralizing activity is caused by antibodies in the RBD region; existing research shows that after removing the RBD antibodies, the titer of the remaining neutralizing antibodies directly drops from the original antibody titer >1000 to <25. Selecting RBD as the antigen also has several potential advantages: The neutralizing antigenic epitopes of the S protein are mainly concentrated in the RBD region, and the content of neutralizing epitopes in other regions is less and there are a certain number of non-neutralizing epitopes. Therefore, under the same vaccination quality, the ability of the RBD antigen to stimulate the body to produce neutralizing antibodies is stronger, and the possibility of inducing non-neutralizing antibodies is lower, so the safety is higher.

[0023] Subunit vaccines are highly safe and have mature production technologies. However, their immunogenicity is relatively low. Antigens need to be optimized or combined with adjuvants, and multiple immunizations are required to improve the humoral immune effect. However, after the above optimizations, their ability to induce CD8 + T cell immune responses is still extremely limited. To overcome the poor immunogenicity of the RBD monomer protein obtained from the Escherichia coli expression system, in this application, the amino acid sequence of the RBD of the novel coronavirus Omicron strain and the amino acid sequence of Helicobacter pylori non-heme iron Ferritin were first fused using the flexible Linker GSG, in order to obtain a multimerized RBD nanoparticle antigen using the self-assembly ability of Ferritin. After expressing it using the Escherichia coli expression system, the following problems were found in the preparation of the RBD-Ferritin antigen: ① A large amount of the target protein RBD-Ferritin was lost during the protein inclusion body harvesting process; ② During the concentration process after the purification of the target protein RBD-Ferritin, the protein was prone to aggregation and the loss was relatively large. In the immune effect evaluation, it was found that compared with the RBD monomer, the RBD-Ferritin nanoparticles could effectively improve the humoral immune response level, but the ability to enhance CD8 + T cell immune responses still needed to be further improved. Therefore, finding a soluble small protein molecule that can improve the solubility of the RBD-Ferritin recombinant protein and further enhance the ability of the RBD-Ferritin nanoparticles to induce adaptive immune responses after fusion expression is crucial for the development of a novel SARS-CoV-2 vaccine with a simple production preparation process and high immunogenicity. Dendritic cells (DCs) are the key antigen-presenting cells (APCs) that initiate adaptive immunity. The activation of DCs determines the ability of T cells and the development direction of immune responses. DCs can be divided into two subsets: classical dendritic cells (cDCs) and plasmacytoid dendritic cells (pDCs). Among them, cDCs can be further divided into classical type 1 DCs (cDC1s) and classical type 2 DCs (cDC2s). The cDC1s subset cells have the ability to cross-present antigens and induce cytotoxic T cell responses, and play a key role in antiviral immunity. Chemokine receptor 1 (XCR1) is specifically expressed on mouse and human cDC1s, and chemokine ligand 1 (XCL1) is the specific ligand of XCR1, with a size of approximately 13KDa. It is a small molecule secreted protein with strong solubility. Therefore, based on the targeting effect of XCL1, after fusion with the target antigen, it can improve the recognition and presentation efficiency of cDC1s to the target antigen, and promote CD8 +The occurrence of T cell immune response; due to its highly soluble and small molecular weight characteristics, it can also improve the solubility of the target antigen expressed by fusion expression during the expression process and have less influence on the formation of its natural spatial structure.

[0024] Ferritin is an endogenous protein present in various organisms. According to its structural characteristics, it can usually be divided into four subfamilies, namely classical ferritin, bacterial ferritin, DNA-binding protein of starved cells, and encapsulin. Helicobacter pylori ferritin (abbreviated as Ferritin) belongs to classical ferritin and is composed of 24 subunit units, forming a symmetric hollow spherical structure, which can efficiently display the target antigen on its surface, improve the recognition efficiency of the immune system for the target antigen; and has low autoimmunogenicity, high safety, and good stability.

[0025] Therefore, in this application, the amino acid sequence of human XCL1, the amino acid sequence of the RBD of the Omicron strain of the novel coronavirus, and the amino acid sequence of Helicobacter pylori non-heme iron Ferritin are fused using the flexible Linker GSG. The XCL1-RBD-Ferritin recombinant protein has a low loss during protein purification and concentration, and can be assembled into a 24-mer nanoparticle structure. This nanoparticle antigen can effectively activate cDC1s, efficiently induce humoral and CD8 + T cell immune responses and maintain more persistent immune memory.

[0026] A method for constructing a fusion protein expression vector, comprising the following steps:

[0027] (1) Connect the N-terminus of the nucleotide sequence of the antigen protein to be expressed to the nucleotide sequence encoding a flexible linker, and then connect it to XCL1;

[0028] (2) Connect the C-terminus of the nucleotide sequence of the antigen protein to be expressed to the nucleotide sequence encoding a flexible linker, and then connect it to the nucleotide sequence encoding the Ferritin protein;

[0029] (3) Transfer the sequence obtained above into an expression vector to obtain a fusion protein expression vector.

[0030] The antigen protein can be the full-length sequence of the structural protein of a certain pathogen or a partial fragment carrying the neutralizing epitope in the pathogen.

[0031] After the fusion protein expression vector is transferred into cells for expression, a fusion protein is obtained. The fusion protein is a target nanoparticle antigen that can self-assemble into a 24-mer nanostructure and target DC1s cells. It can be expected that the nanoparticle antigen can effectively activate cDC1s, efficiently induce humoral and CD8 + T cell immune responses and maintain more persistent immune memory.

[0032] The protein expression vector includes a prokaryotic expression vector and a eukaryotic expression vector.

[0033] In the fields of vaccine design and biotechnology, a "flexible linker" refers to a stretch of amino acid sequence that connects two different molecules or protein domains. The main function of this linker sequence is to provide sufficient flexibility so that the two connected parts can fold and move independently without interfering with each other. Those skilled in the art can select a suitable flexible linker according to their needs.

[0034] A fusion protein expression vector includes an expression vector, a nucleotide sequence encoding XCL1, a nucleotide sequence encoding Ferritin protein, and a nucleotide sequence encoding the target protein to be expressed. Among them, the N-terminus of the nucleotide sequence encoding the target protein to be expressed is connected to the nucleotide sequence encoding XCL1, and the C-terminus is connected to the nucleotide sequence encoding Ferritin protein. The resulting sequence is then connected to the expression vector.

[0035] The expression vector includes a prokaryotic expression vector and a eukaryotic expression vector, and those skilled in the art can make adjustments according to the actual situation.

[0036] A method for preventing a disease caused by the novel coronavirus, the method comprising administering an effective amount of a vaccine to a subject in need thereof.

[0037] The beneficial effects of the present application are:

[0038] 1. High efficiency in antigen expression, purification and concentration, and good immunogenicity

[0039] To overcome the problems of poor immunogenicity of the RBD protein monomer, low purification and concentration efficiency of RBD-Ferritin, and still limited level of CD8 + T cell immune response, the inventors fused the human XCL1 amino acid sequence, the RBD amino acid sequence of the novel coronavirus Omicron strain, and the Ferritin amino acid sequence of Helicobacter pylori subafameloglobin iron using the flexible Linker GSG. According to the codon bias of the Escherichia coli (E.coli) expression system, the XCL1-RBD-Ferritin nucleotide sequence was optimized and synthesized, and cloned into the pET28a(+) prokaryotic expression plasmid. The E.coli BL21(DE3) expression strain was used to express the XCL1-RBD-Ferritin fusion protein. The purified XCL1-RBD-Ferritin can assemble into 24-mer nanoparticles, improving the efficiency of RBD antigen activating DCs, especially DC1s, thereby efficiently initiating humoral and cellular immune responses.

[0040] 2. Simple preparation and suitable for large-scale production

[0041] In this application, the expression of the fusion protein using the Escherichia coli prokaryotic expression system is carried out through the prokaryotic expression system. The addition of the XCL1 molecule improves the expression, purification, and concentration efficiency of the XCL1-RBD-Ferritin protein, effectively increasing the harvest of XCL1-RBD-Ferritin nanoparticles, which is beneficial to the batch production of vaccine antigens.

[0042] 3. High level of humoral immune response and long-lasting immune memory

[0043] It can effectively activate TFH, induce a high level of humoral immune response, and the immune memory is more persistent, providing long-term and effective immune protection.

[0044] 4. Achieve the targeting of antigens to cDC1s cells and improve the level of antigen-induced CD8 + T cell immune response level.

[0045] The XCL1 molecule can target antigens to DC1s with cross-presentation function, achieve antigen cross-presentation, and thus induce a high-efficiency humoral and CD8 + T cell immune response. Brief Description of the Drawings

[0046] Figure 1 Results of identification of the target protein expression strain; among them, A is the identification result of the RBD expression strain; B is the identification result of the RBD-Ferritin expression strain; C is the identification result of the XCL1-RBD-Ferritin expression strain.

[0047] Figure 2 Results of purification and identification of the target protein; among them, A is the purification and renaturation result of the RBD protein; B is the purification and renaturation result of the RBD-Ferritin protein; C is the purification and renaturation result of the XCL1-RBD-Ferritin protein.

[0048] Figure 3 Results of analysis of nanoparticle assembly; among them, A is the simulated diagram of XCL1-RBD-Ferritin nanoparticle assembly; B is the transmission electron microscopy analysis result of Ferrtin / RBD-Ferritin / XCL1-RBD-Ferrtin nanoparticle assembly.

[0049] Figure 4 Analysis of the initiation of adaptive immune response by nanoparticles using flow cytometry technology, where A is the schematic diagram of the experimental principle; B is the DCs level; C is the DC1s level.

[0050] Figure 5Analysis results of the humoral immune response induced by the targeted nanoparticle antigen; among them, A is the analysis of the reactivity of sera from different antigen-immunized groups with the antigen by Western Blot; B is the level of RBD-specific IgG antibodies in different immunized groups 6 weeks after the first immunization; C is the level of Memory B cells in different immunized groups 6 weeks after the first immunization.

[0051] Figure 6 Analysis results of the cellular immune response induced by the targeted nanoparticle antigen; 6 weeks after the first immunization, A is the level of IFNγ + CD8 + T cells in different antigen-immunized groups; B is the level of TNFα + CD8 + T cells in different antigen-immunized groups; C is the level of Th1 cells; D is the level of Th2 cells; E is the level of CD8 + T cells; F is the analysis of the level of IFNγ + T cells by ELSpot technique.

[0052] Figure 7 Analysis results of the maintenance of immune memory; among them, A is the blood sample collection time; B is the dynamic analysis results of RBD-specific IgG antibodies in sera of different antigen-immunized groups; C is the level of Memory B cells in different immunized groups 18 weeks after the first immunization; D is the level of CD8 + TCM cells 18 weeks after the first immunization. Detailed implementation manners

[0053] For the experimental methods without specific conditions noted in the following examples of the present invention, they are generally carried out under conventional conditions, such as the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. All common chemical reagents used in the examples are commercially available products.

[0054] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those skilled in the technical field to which the present invention belongs. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0055] The terms "comprising" and "having" and any variations thereof in the present invention are intended to cover non-exclusive inclusion. For example, a process, method, device, product or equipment that includes a series of steps is not limited to the listed steps or modules, but optionally further includes steps not listed, or optionally further includes other steps inherent to these processes, methods, devices, products or equipment.

[0056] Example 1 Invention Design of XCL1-RBD-Ferritin

[0057] Currently, commercially available COVID-19 vaccines mainly include mRNA vaccines, inactivated vaccines, viral vector vaccines designed based on adenovirus (AdV), and subunit vaccines. Inactivated vaccines can be made by traditional methods. The antibodies induced by inactivated vaccines target all viral structural proteins, and most of these proteins are not related to the production of neutralizing antibodies. However, due to the damage of the natural spatial structure of the spike S protein or the receptor-binding domain (RBD) region caused by virus inactivation, its immunogenicity is reduced, and the ability to induce the body to produce neutralizing antibodies is limited. Research shows that except for live attenuated vaccines, the ability of other types of vaccines to induce CD8 + T cell immune responses is extremely limited. Therefore, new research strategies should be explored to improve the ability of vaccines to induce CD8 + T cell immune responses.

[0058] To improve the immunogenicity of RBD, this application pioneered the fusion of the DC1s-targeting factor XCL1, the SARS-CoV-2 RBD domain, and the Ferritin protein with self-assembly ability, and developed a SARS-CoV-2 nanoparticle vaccine with targeting ability. Using the self-assembly characteristics of Ferritin, the RBD protein is displayed on the surface of the nanoparticles, improving the efficiency of recognition of the neutralizing antigenic epitopes of the RBD protein by antigen-presenting cells. Furthermore, XCL1 can induce antigen-targeted activation of DC1s cells, and DC1s have cross-presentation function, which can more efficiently induce humoral and CD8 + T cell immune responses. At the same time, the addition of the XCL1 molecule further improves the expression, purification, and concentration efficiency of the target protein, which is more conducive to the large-scale production of vaccine antigens.

[0059] The experimental design flow chart is as Figure 1 shown.

[0060] Among them, the amino acid sequence of SARS-CoV-2 RBD is truncated from omicron spike gp delta [synthetic construct] GenBank: BDY33930.1. The specific sequence is as follows:

[0061] SFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFDEVFNATRFASVYAWNRKRISNCVADYSVLYNLAPFFTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKVSGNYNYLYRLFRKSNLKPFERDISTEIYQAGNKPCNGVAGFNCYFPLRSYSFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLKGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEIL(SEQ ID No.1).

[0062] The amino acid sequence of XCL1, selected from lymphotactin precursor [Homo sapiens] (NCBI Reference Sequence: NP_002986.1). The specific sequence is shown as follows:

[0063] MRLLILALLGICSLTAYIVEGVGSEVSDKRTCVSLTTQRLPVSRIKTYTITEGSLRAVIFITKRGLKVCADPQATWVRDVVRSMDRKSNTRNNMIQTKPTGTQQSTNTAVTLTG(SEQ ID No.2).

[0064] The amino acid sequence of Ferritin, selected from ferritin [Helicobacter pylori] (NCBI Reference Sequence: WP_021436151.1), the specific sequence is shown as follows:

[0065] MLSKEIIKLLNEQVNKEMNSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYDHAKKLIVFLNENNVPVQLNSISAPEHKFESLTQIFQKAYKHEQDISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYIKGIAKSKKS(SEQ ID No.3).

[0066] Determine the amino acid sequence of the target protein as follows:

[0067] XCL1-RBD-Ferritin amino acid sequence, flexible linker: GSG, His purification tag: HHHHHH

[0068] MRLLILALLGICSLTAYIVEGVGSEVSDKRTCVSLTTQRLPVSRIKTYTITEGSLRAVIFITKRGLKVCADPQATWVRDVVRSMDRKSNTRNNMIQTKPTGTQQSTNTAVTLTG GSG SFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFDEVFNATRFASVYAWNRKRISNCVADYSVLYNLAPFFTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKVSGNYNYLYRLFRKSNLKPFERDISTEIYQAGNKPCNGVAGFNCYFPLRSYSFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLKGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEIL GSG MLSKEIIKLLNEQVNKEMNSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYDHAKKLIVFLNENNVPVQLNSISAPEHKFESLTQIFQKAYKHEQDISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYIKGIAKSKKSHHHHHH(SEQ ID No.4).

[0069] Further optimize the XCL1, RBD, and Ferritin nucleotide sequences according to the codon bias of the E. coli expression system.

[0070] The optimized SARS-CoV-2 RBD nucleotide sequence is as follows:[[]]END]]

[0071] TCTTTCACCGTGGAAAAAGGTATTTATCAGACCAGCAACTTCCGCGTCCAACCGACCGAGAGCATCGTGCGCTTCCCGAATATCACCAATCTGTGTCCGTTCGACGAAGTATTTAACGCGACGCGTTTTGCTAGCGTCTATGCGTGGAACCGTAAGCGTATCTCCAACTGCGTCGCGGACTATTCCGTGCTGTACAATCTCGCGCCGTTTTTTACTTTTAAGTGCTACGGCGTGTCGCCGACTAAACTTAACGATCTGTGCTTTACCAATGTTTATGCCGATTCCTTCGTGATCCGTGGTGACGAGGTCCGCCAGATTGCTCCGGGCCAGACCGGGAACATTGCGGACTACAATTACAAGCTCCCAGATGATTTTACGGGCTGCGTGATTGCGTGGAATAGCAACAAACTGGATTCCAAGGTGAGCGGTAACTATAACTACCTGTATCGTCTCTTCCGCAAAAGCAATCTGAAACCGTTCGAGCGCGACATCTCCACCGAGATCTACCAAGCGGGTAATAAGCCGTGTAATGGCGTTGCTGGTTTTAACTGCTACTTCCCGCTGCGTAGCTATAGCTTTCGTCCGACCTACGGCGTTGGTCATCAGCCGTATCGCGTTGTTGTTCTCTCCTTCGAGTTGCTTCATGCGCCGGCAACGGTGTGCGGTCCGAAAAAGTCCACCAATTTGGTTAAAAACAAGTGCGTTAATTTTAATTTCAACGGCTTGAAGGGTACGGGCGTCTTGACCGAGAGCAACAAGAAATTCCTGCCGTTCCAACAATTTGGTCGTGACATCGCCGACACCACCGATGCAGTTCGTGACCCACAGACGCTGGAAATCCTG(SEQ ID No.5).

[0072] Optimized XCL1 nucleotide sequence, the specific sequence is as follows:

[0073] ATGAGGCTATTGATATTAGCTCTGCTTGGAATCTGCAGCTTGACGGCCTATATTGTTGAAGGCGTGGGCTCTGAGGTTAGCGATAAACGCACCTGCGTGTCTTTGACTACCCAGCGTCTGCCGGTTTCTCGTATTAAAACCTACACCATTACTGAGGGTTCTCTGCGCGCAGTGATCTTCATCACCAAGCGTGGCTTGAAGGTGTGTGCAGATCCGCAAGCTACTTGGGTCAGAGACGTGGTGCGTAGCATGGATCGTAAAAGCAACACCCGCAACAACATGATCCAGACGAAACCGACCGGTACACAGCAAAGCACCAACACTGCGGTGACCCTGACCGGT(SEQ ID No.6).

[0074] Optimized Ferritin nucleotide sequence is as follows:

[0075] ATGCTGAGCAAAGAAATTATTAAGCTTCTGAACGAGCAAGTTAATAAGGAGATGAATAGCAGCAACCTGTACATGTCTATGAGCAGCTGGTGTTACACCCATAGCCTGGACGGCGCAGGTCTATTCTTGTTTGACCACGCGGCGGAAGAATACGATCATGCTAAAAAACTGATCGTGTTCCTGAACGAAAACAACGTTCCGGTTCAACTGAACAGCATTTCGGCCCCAGAACATAAATTTGAGTCGTTAACCCAGATTTTCCAAAAGGCGTACAAGCACGAACAGGATATCTCTGAATCCATCAACAATATTGTAGATCACGCGATTAAGTCAAAGGACCACGCTACGTTCAACTTTCTGCAGTGGTATGTTGCGGAACAGCATGAAGAGGAGGTTCTGTTCAAAGACATTCTGGACAAGATCGAGCTGATAGGCAACGAGAACCACGGCCTGTACTTAGCCGATCAGTATATCAAGGGTATTGCGAAGAGCAAAAAATCA(SEQ ID No.7).

[0076] The nucleotide sequence of the flexible linker GSG amino acid fragment between XCL1 and RBD is GGTAGCGGT.

[0077] The nucleotide sequence of the flexible linker GSG amino acid fragment between RBD and Ferritin is GGCAGTGGT.

[0078] The nucleotide sequence of 6×His Tag: CATCACCACCACCACCAC (SEQ ID No.8).

[0079] The nucleotide sequence of XCL1-RBD-Ferritin optimized according to the codon bias of E.Coli expression system is:

[0080] ATGAGGCTATTGATATTAGCTCTGCTTGGAATCTGCAGCTTGACGGCCTATATTGTTGAAGGCGTGGG CTCTGAGGTTAGCGATAAACGCACCTGCGTGTCTTTGACTACCCAGCGTCTGCCGGTTTCTCGTATTAAAACCTAC ACCATTACTGAGGGTTCTCTGCGCGCAGTGATCTTCATCACCAAGCGTGGCTTGAAGGTGTGTGCAGATCCGCAAG CTACTTGGGTCAGAGACGTGGTGCGTAGCATGGATCGTAAAAGCAACACCCGCAACAACATGATCCAGACGAAACC GACCGGTACACAGCAAAGCACCAACACTGCGGTGACCCTGACCGGTGGTAGCGGTTCTTTCACCGTGGAAAAAGGTATTTATCAGACCAGCAACTTCCGCGTCCAACCGACCGAGAGCATCGTGCGCTTCCCGAATATCACCAATCTGTGTCCGTTCGACGAAGTATTTAACGCGACGCGTTTTGCTAGCGTCTATGCGTGGAACCGTAAGCGTATCTCCAACTGCGTCGCGGACTATTCCGTGCTGTACAATCTCGCGCCGTTTTTTACTTTTAAGTGCTACGGCGTGTCGCCGACTAAACTTAACGATCTGTGCTTTACCAATGTTTATGCCGATTCCTTCGTGATCCGTGGTGACGAGGTCCGCCAGATTGCTCCGGGCCAGACCGGGAACATTGCGGACTACAATTACAAGCTCCCAGATGATTTTACGGGCTGCGTGATTGCGTGGAATAGCAACAAACTGGATTCCAAGGTGAGCGGTAACTATAACTACCTGTATCGTCTCTTCCGCAAAAGCAATCTGAAACCGTTCGAGCGCGACATCTCCACCGAGATCTACCAAGCGGGTAATAAGCCGTGTAATGGCGTTGCTGGTTTTAACTGCTACTTCCCGCTGCGTAGCTATAGCTTTCGTCCGACCTACGGCGTTGGTCATCAGCCGTATCGCGTTGTTGTTCTCTCCTTCGAGTTGCTTCATGCGCCGGCAACGGTGTGCGGTCCGAAAAAGTCCACCAATTTGGTTAAAAACAAGTGCGTTAATTTTAATTTCAACGGCTTGAAGGGTACGGGCGTCTTGACCGAGAGCAACAAGAAATTCCTGCCGTTCCAACAATTTGGTCGTGACATCGCCGACACCACCGATGCAGTTCGTGACCCACAGACGCTGGAAATCCTGGGCAGTGGT ATGCT GAGCAAAGAAATTATTAAGCTTCTGAACGAGCAAGTTAATAAGGAGATGAATAGCAGCAACCTGTACATGTCTATG AGCAGCTGGTGTTACACCCATAGCCTGGACGGCGCAGGTCTATTCTTGTTTGACCACGCGGCGGAAGAATACGATC ATGCTAAAAAACTGATCGTGTTCCTGAACGAAAACAACGTTCCGGTTCAACTGAACAGCATTTCGGCCCCAGAACA TAAATTTGAGTCGTTAACCCAGATTTTCCAAAAGGCGTACAAGCACGAACAGGATATCTCTGAATCCATCAACAAT ATTGTAGATCACGCGATTAAGTCAAAGGACCACGCTACGTTCAACTTTCTGCAGTGGTATGTTGCGGAACAGCATG AAGAGGAGGTTCTGTTCAAAGACATTCTGGACAAGATCGAGCTGATAGGCAACGAGAACCACGGCCTGTACTTAGC CGATCAGTATATCAAGGGTATTGCGAAGAGCAAAAAATCA CATCACCACCACCACCACTGA(SEQ ID No.9).

[0081] Example 2 Construction of XCL1-RBD-Ferritin Recombinant Protein Expression Strain

[0082] The amino acid sequences of human XCL1, RBD, and Helicobacter pylori ferritin (Ferritin) were fused using the flexible linker GSG. According to the codon bias of the Escherichia coli (E. coli) expression system, the XCL1-RBD-Ferritin nucleotide sequence (as shown in SEQ ID NO.9) was designed and synthesized by GenScript Biotech Corporation.

[0083] It was further cloned into the pET28a(+) prokaryotic expression plasmid to obtain the pET28a-XCL1-RBD-Ferritin recombinant expression plasmid. The pET28a-XCL1-RBD-Ferritin recombinant plasmid was transformed into the E. coli BL21(DE3) expression strain. After picking colonies and culturing overnight for 12 h, the bacterial solution was inoculated into LB medium containing kanamycin resistance at a ratio of 1:100. When the OD value reached between 0.6 and 0.8, an IPTG inducer with a final concentration of 0.8 M was added, and the bacterial solution was harvested after culturing for 5 h. The expression of the XCL1-RBD-Ferritin recombinant protein was analyzed using SDS electrophoresis technology. The XCL1-RBD-Ferritin expression strain was mixed with 40% glycerol at a ratio of 1:1 and stored at -80 °C for later use.

[0084] Meanwhile, for the comparative experiment, the pET28a recombinant plasmids pET28a-RBD and pET28a-RBD-Ferritin carrying the RBD and RBD-Ferritin nucleotide sequences were transformed into the E. coli BL21(DE3) expression strain. After that, the bacterial solution was inoculated into LB medium containing kanamycin resistance at a ratio of 1:100. When the OD value reached between 0.6 and 0.8, an IPTG inducer with a final concentration of 0.8 M was added, and the bacterial solution was harvested after culturing for 5 h. The expression of the RBD and RBD-Ferritin recombinant proteins was analyzed using SDS electrophoresis technology. The RBD and RBD-Ferritin expression strains were respectively mixed with 40% glycerol at a ratio of 1:1 and stored at -80 °C for later use.

[0085] The expression identification of the RBD, RBD-Ferritin, and XCL1-RBD-Ferritin protein strains is as Figure 1 shown: RBD( Figure 1 A), RBD-Ferritin( Figure 1 B), XCL1-RBD-Ferritin( Figure 1C) After induction with IPTG, the expression strain was able to express recombinant proteins of RBD, RBD-Ferritin, and XCL1-RBD-Ferritin with sizes of 29KDa, 48KDa, and 61KDa.

[0086] Example 3 Preparation of Recombinant Protein

[0087] 1. Identification of Expression Strain

[0088] 1.1 Inoculate the XCL1-RBD-Ferritin expression strain into LB medium containing kanamycin resistance at a ratio of 1:1000, and culture it overnight at 37°C in a shaker at 200 rpm for 12 h;

[0089] 1.2 Inoculate the overnight-activated bacterial solution into 200 mL of LB medium containing kanamycin resistance at a ratio of 1:100, culture it at 37°C and 200 rpm for 2 hours and 40 minutes. When the OD value reaches 0.71, add IPTG inducer with a final concentration of 0.8 M, and continue to culture at 37°C and 200 rpm for 5 h. After the culture is completed, centrifuge to harvest all the bacterial cells at 6000 rmp, 4°C, for 10 min.

[0090] According to the steps of 1.1 and 1.2, expand the bacterial cells of RBD and RBD-Ferritin plasmids and induce them with IPTG to analyze the expression of RBD and RBE-Ferritin recombinant proteins for subsequent protein purification.

[0091] 2. Inclusion Body Treatment

[0092] 2.1 Resuspend all the bacterial cells with 6 mL of non-denaturing lysis buffer and transfer them to a 10 mL centrifuge tube. Place it in an ice-water mixture, and use an ultrasonic crusher (parameter settings: power: 30%, time: 60 min, ultrasound on for 3 s and off for 3 s). The ultrasonic probe should be placed 1 / 2 below the liquid level;

[0093] 2.2 After the ultrasound is completed, transfer the ultrasonicated bacterial solution to 3 2 mL centrifuge tubes, centrifuge at 4°C and 12000 rpm for 25 min, completely discard the supernatant. Add 1 mL of 0.1 M PBS buffer to each tube to resuspend and wash the precipitate, and then centrifuge at 4°C and 12000 rpm for 5 min again. Discard the supernatant and retain the precipitate;

[0094] 2.3 Add 3 mL of Binding / washing buffer containing 8 M urea to each tube of precipitate, resuspend and dissolve the precipitate; transfer the resuspended material to a 10 mL centrifuge tube, add a DTT solution with a final concentration of 1 mM, mix, and use an ultrasonic crusher to promote the dissolution of the precipitate (parameters set: power 10%, time 20 min, ultrasound on for 3 s and off for 3 s). The whole process should be completed in an ice-water mixture, and the ultrasonic probe should be kept at a position 1 / 2 below the liquid surface;

[0095] 2.4 Divide the ultrasonicated product equally and transfer it to 2 mL centrifuge tubes, centrifuge again at 4 °C and 12,000 rpm for 20 min, collect the supernatant into a 15 mL centrifuge tube, and discard the insoluble precipitate;

[0096] 2.5 Use Binding / washing buffer containing 8 M urea to make up the total volume of the supernatant to 10 mL for subsequent purification experiments.

[0097] 3. Purification process of recombinant protein

[0098] 3.1 Take out the Ni-TED protein purification column (Sangon) from the 4 °C refrigerator, drain the storage solution, add 10 ml of Binding / washing buffer (Sangon) containing 8 M urea to balance the purification column;

[0099] 3.2 Add the inclusion body lysate to the Ni-TED protein purification column, pass through it repeatedly 3 times, and collect the passing-through liquid into a 10 mL centrifuge tube;

[0100] 3.3 Wash the Ni-TED protein purification column with 10 mL of Binding / washing buffer containing 20 mM imidazole and 8 M urea, and collect the washing solution into a 10 mL centrifuge tube (this step is repeated 5 times);

[0101] 3.4 Wash the Ni-TED protein purification column with 10 mL of Binding / washing buffer containing 30 mM imidazole and 8 M urea, and collect the washing solution into a 10 mL centrifuge tube (this step is repeated 2 times);

[0102] 3.5 Use 5 ml of eluent (Sangon) containing 8 M urea to elute the target protein adsorbed on the Ni-TED protein purification column and collect it into a 10 ml centrifuge tube. This step is repeated 3 times to obtain a total of 30 mL of protein purification solution.

[0103] 4. Protein renaturation and concentration

[0104] 4.1 Place 30 mL of the protein purification solution (obtained in step 3.5) in a dialysis bag and place it in 1 L of a dialysis solution containing 4 M urea for 4 h at 4 °C;

[0105] 4.2 Transfer the dialysis bag containing the protein purification solution to 1 L of a dialysis solution containing 2 M urea for 4 h at 4 °C;

[0106] 4.3 Transfer the dialysis bag liquid to a 100 mL beaker, add reduced glutathione with a final concentration of 0.5 mM, and incubate overnight at 4 °C;

[0107] 4.4 Take out the renatured protein purification solution, centrifuge it at 12,000 rpm at 4 °C for 10 min in a low-temperature centrifuge, collect the supernatant, and remove the denatured and precipitated protein precipitate.

[0108] 4.5 Add the renatured protein to an ultrafiltration tube with an inner tube volume of 15 mL and centrifuge it at 4000 rmp at 4 °C for 7 min until the liquid level of the protein concentrate reaches below the 1.5 ml mark;

[0109] 4.6 Add PBS buffer with a volume 3 times that of the protein concentrate, mix well, and centrifuge it at 4000 rmp at 4 °C for 7 min until the liquid level reaches below the 1.5 mL mark; repeat this step 3 times to finally replace all of the protein dissolution solution with PBS buffer.

[0110] Analyze the protein purification and concentration using SDS-PAGE electrophoresis technology, and the results are as Figure 2 shown: Using the Ni-TED protein purification column, RBD ( Figure 2 A), RBD-Ferritin ( Figure 2 B), and XCL1-RBD-Ferritin ( Figure 2C) Recombinant protein. However, during the purification process, it was found that for the RBD-Ferritin expression strain, after the induction expression ended and the protein expressed in the form of inclusion bodies was collected, the recovery rate of inclusion bodies was low, and RBD-Ferritin showed problems of low solubility and easy denaturation and precipitation during the protein purification, renaturation and concentration processes. After fusing XCL1 with RBD-Ferritin for expression, the inclusion body recovery rate was high, and a large amount of XCL1-RBD-Ferritin recombinant protein with good solubility could be obtained after protein purification, renaturation and concentration. Using an ultra-micro nucleic acid and protein detector to detect the protein concentration, the results showed that when the cell concentration OD value of each 200 mL of activated RBD, RBD-Ferritin, and XCL1-RBD-Ferritin expression strains was 0.7, after induction with IPTG at a final concentration of 0.8 M, 131.2 μg of RBD protein, 56.3 μg of RBD-Ferritin protein, and 112.7 μg of XCL1-RBD-Ferritin protein were obtained respectively after purification, renaturation and concentration (the results are shown in Table 1).

[0111] Table 1. Protein harvest

[0112] Protein Name Induced Bacterial Quantity Protein Harvest Quantity RBD 200 mL 131.2 μg RBD-Ferritin 200 mL 56.3 μg XCL1-RBD-Ferritin 200 mL 112.7 μg

[0113] The above results also indicate that although the addition of the XCL1 molecule increases the molecular weight of the antigen to be expressed, it improves the solubility of the fusion protein.

[0114] Taking the Ferritin protein that can self-assemble into nanoparticles stored in the laboratory as a control, Ferritin, RBD-Ferritin, and XCL1-RBD-Ferritin were respectively dropped onto a 400-mesh copper grid with a carbon coating at a concentration of 0.3 mg / mL. After acting for 2 min, they were stained with 0.5% phosphotungstic acid for 30 s. All samples were imaged using a Hitachi H-7650 electron microscope at 80 kV. The microscope was equipped with an Olympus Cantega G2 bottom-mounted CCD TEM camera, and the TEM images were processed and analyzed using iTEM (Olympus). The results are shown in Figure 3 , where A is a simulated diagram of the assembly of XCL1-RBD-Ferritin nanoparticles; B is the transmission electron microscopy analysis result of the assembly of Ferrtin / RBD-Ferritin / XCL1-RBD-Ferrtin nanoparticles. As Figure 3 shown, both RBD-Ferritin and XCL1-RBD-Ferrtin can assemble into 80-nm multimerized nanoparticle structures similar to the structure of Ferritin nanoparticles.

[0115] Study on the Immune Initiation Ability of XCL1-RBD-Ferritin Nanoparticles in Example 4

[0116] Using 6-week-old female BALB / c mice as an animal model, they were divided into 5 groups (3 mice in each group). Freund's adjuvant was used as an immune adjuvant and mixed with the antigen and PBS at a ratio of 1:1. Ferritin nanoparticles, RBD monomer protein, RBD-Ferritin nanoparticles, and XCL1-RBD-Ferritin nanoparticles were immunized respectively, and the blank group was only inoculated with PBS buffer; Immunization was carried out on days 0 and 28, for a total of 2 times, and all were carried out by abdominal subcutaneous injection. Each time of immunization, the blank control group was injected with 50 μL of PBS buffer, the Ferritin group was injected with 6.45 μg of antigen, the RBD group was injected with 10 μg of antigen, the RBD-Ferritin group was injected with 16.6 μg of antigen, and the XCL1-RBD-Ferritin group was injected with 21 μg of antigen to ensure that the total amount of RBD antigen received by each experimental animal was the same (the immunization protocol is shown in Table 2).

[0117] Table 2. Immunization Protocol for Experimental Animals

[0118]

[0119] Flow cytometry was used to analyze the activation levels of DCs and cDC1s cells, including the following experiments:

[0120] 1. Preparation of single-cell suspension

[0121] The inguinal lymph nodes and spleens of mice were taken, and the lymph and spleen were ground separately in a cell sieve and placed in a 15 mL centrifuge tube. Centrifugation was carried out at 4°C and 1500 rmp for 7 min. The supernatant was discarded, and the lymph was resuspended with 1 mL of PBS buffer. The spleen was lysed on ice with 1 mL of erythrocyte lysate for 15 min, then centrifuged at 4°C and 1500 rmp for 7 min, the supernatant was discarded, and it was resuspended with 1 mL of PBS.

[0122] 2. Cell surface molecule staining

[0123] The single-cell suspension was dispensed into flow cytometry tubes, surface molecule staining antibodies were added, and after staining for 30 min, 1 mL of PBS was added to terminate the staining, then centrifuged at 4°C and 1500 rmp for 7 min. The supernatant was discarded, and it was resuspended with 200 μL of PBS. The staining protocol was as follows: MHC II-FITC, CD11c-BV421, XCR1-PE.

[0124] 3. Analysis of the activation levels of DCs and cDC1s cells by flow cytometry

[0125] The activation levels of DCs and cDC1s cells were analyzed by flow cytometry, that is, the analysis of the initiation of the adaptive immune response by nanoparticles. The results are shown in Figure 4 , where A is the schematic diagram of the experimental principle; B is the level of DCs; C is the level of DC1s. The specific data are shown in Table 3.

[0126] At 4 h after immunization, the average activation level of DCs in the Control group was 0.37%, in the Ferritin group was 0.41%, in the RBD group was 0.52%, in the RBD-Ferritin group was 0.68%, and in the XCL1-RBD-Ferritin group was 0.81%. The XCL1-RBD-Ferritin group was significantly higher than the RBD and RBD-Ferritin immunized groups (*p < 0.05, **p < 0.01, ***p < 0.001), and each immunized group was significantly higher than the Control group and the Ferritin group (*p < 0.05, **p < 0.01, ***p < 0.001) (see Figure 4 B). The average proportion of cDC1s in total DCs in the Control group was 29.3%, in the Ferritin group was 33.3%, in the RBD group was 36.8%, in the RBD-Ferritin group was 37.8%, and in the XCL1-RBD-Ferritin group was 48.0%. The XCL1-RBD-Ferritin group was higher than the RBD and RBD-Ferritin immunized groups (**p < 0.01) (see Figure 4 C).

[0127] The above results indicate that compared with the non-targeted RBD-Ferritin nanoparticle antigen and the common monomer antigen RBD, the XCL1-RBD-Ferritin nanoparticle antigen with targeting ability can more effectively activate DCs, especially cDC1s, and further initiate the RBD-specific immune response.

[0128] Table 3. Average activation levels of DCs and cDC1s cells

[0129] Animal Grouping DCs (%) cDC1s (%) PBS Control group 0.37 29.3 Ferritin group 0.41 33.3 RBD group 0.52 36.8 RBD-Ferritin group 0.68 37.8 XCL1-RBD-Ferritin group 0.81 48.0

[0130] Example 5 Analysis of the ability of XCL1-RBD-Ferritin nanoparticles to induce humoral immune responses

[0131] Six-week-old female BALB / c mice were used as animal models and divided into 5 groups (5 mice in each group). They were immunized with Ferritin nanoparticles, RBD monomer protein, RBD-Ferritin nanoparticles, and XCL1-RBD-Ferritin nanoparticles respectively. The blank group was only inoculated with PBS buffer. Immunization was carried out on days 0 and 28, for a total of 2 times. The abdominal subcutaneous immunization method was used for each immunization. In each immunization, the blank control group was injected with 50 μL of PBS buffer, the Ferritin group was injected with 6.45 μg of antigen, the RBD group was injected with 10 μg of antigen, the RBD-Ferritin group was injected with 16.6 μg of antigen, and the XCL1-RBD-Ferritin group was injected with 21 μg of antigen to ensure that the total amount of RBD antigen received by each experimental animal was the same (see Table 4). Six weeks after the first immunization, Western Blot technology, ELISA experiments, and flow cytometry were used to analyze the ability of XCL1-RBD-Ferritin nanoparticles to induce humoral immune responses.

[0132] Table 4. Immunization procedures and sampling times for experimental animals

[0133]

[0134] 1. Western Blot experiment

[0135] SDS-PAGE electrophoresis: SDS-PAGE separation electrophoresis was performed on the purified products of Ferritin, RBD, RBD-Ferritin, and XCL1-RBD-Ferrtin proteins. The following steps were included:

[0136] (1) Transfer membrane: Cut a nitrocellulose membrane (NC membrane) of appropriate size, moisten the NC membrane with 1× transfer buffer, place the protein gel and NC membrane in the transfer device in the corresponding order, and set the constant current mode to transfer the membrane for 90 min at 180 mA under ice bath conditions.

[0137] (2) Blocking: Transfer the transferred NC membrane to an antibody hybridization box, add 10 mL of 5% skim milk, and block it at room temperature under the condition of 60 rpm for 1 h.

[0138] (3) Incubate with primary antibody: Recover the blocking solution, and add 10 mL of mouse serum immunized with RBD / RBD-Ferritin / XCL1-RBD-Ferrtin (three proteins) diluted 1:1000, and incubate it overnight at 4°C on a horizontal shaker under the condition of 60 rpm.

[0139] (4) Membrane washing: Recover the primary antibody, wash the NC membrane with 1×TBST at room temperature at a speed of 90 rpm for 10 min, and wash the membrane 3 times.

[0140] (5) Incubate the secondary antibody: Add 10 mL of goat anti-mouse-HRP IgG antibody diluted 1:2000 times, and incubate it on a horizontal shaker at room temperature at 60 rpm for 1 h.

[0141] (6) Membrane washing: Recover the secondary antibody, wash the NC membrane with 1×TBST at room temperature at a speed of 90 rpm for 10 min, and wash the membrane 3 times.

[0142] (7) Development: According to the instructions of the ECL kit, mix solution A and solution B in a ratio of 1:1 to prepare the developing working solution. Use a pipette to evenly drop it on the NC membrane, and perform development and photography with a chemiluminescence instrument.

[0143] 2. Determine the titer of RBD protein antibody in mouse serum by ELISA detection method

[0144] Six weeks after the first immunization, collect blood from the tail vein of the mouse and collect the serum. Use the established ELISA detection method in the laboratory to determine the level of RBD-specific antibody in the mouse serum. The specific experimental operation steps are as follows:

[0145] (1) Take the tail vein blood of the immunized BALB / c mouse, let it stand for 1 hour, centrifuge at 2500 rmp at 4℃ for 10 min, and take the supernatant; (2) Coating 100 μl of 4 μg / mL RBD protein per well on the antigen adsorption plate, place it at 4℃ for 12 h; (3) After discarding the liquid in the well, add 150 μl / well of blocking solution, and block it at 37℃ for 90 min; (4) Add 300 μL / well of PBST and wash 3 times; (5) Add 100 μL of the appropriately diluted serum to be tested to each well and react, incubate at 37℃ for 1 h; (6) Add 300 μL / well of PBST and wash 3 times; (7) Add 100 μL of HRP-labeled goat anti-mouse secondary antibody (diluted 1:5000) to each well, incubate at 37℃ for 1 h; (8) Add 300 μL / well of PBST and wash 3 times; (9) Add 200 μL of TMB substrate solution to each well, react at 37℃ in the dark for 10 min; terminate the reaction with 50 μL of 1M H2SO4 per well; detect the OD value at 450 nm absorbance with an enzyme-linked immunosorbent assay instrument. Perform statistical analysis on the results.

[0146] 3. Analyze the level of Memory B cells related to humoral immune memory by flow cytometry

[0147] Single-cell suspension preparation: Take the inguinal lymph nodes and spleens of mice 6 weeks after the first immunization. Grind the lymph nodes and spleens separately in a cell sieve, put them into 15 mL centrifuge tubes, and centrifuge at 4°C and 1500 rmp for 7 min. Discard the supernatant. Resuspend the lymph with 1 mL of PBS buffer. After lysing the spleen with 1 mL of red blood cell lysate on ice for 15 min, centrifuge at 4°C and 1500 rmp for 7 min, discard the supernatant, and resuspend with 1 mL of PBS to obtain a single-cell suspension.

[0148] Cell surface molecule staining: Transfer the single-cell suspension to a flow tube, add surface molecule staining antibodies, and after staining for 30 min, add 1 mL of PBS to terminate the staining. Then centrifuge at 4°C and 1500 rmp for 7 min, discard the supernatant, and resuspend with 200 μL of PBS to prepare for flow cytometry detection.

[0149] The staining protocol is as follows: CD19-BV421, CD38-FITC, IgD-APC.

[0150] The results of Western Blot experiments, ELISA experiments, and flow cytometry detection are shown in Figure 5 as follows.

[0151] Western Blot detection showed that: The sera of mice immunized with RBD, RBD-Ferritin, and XCL1-RBD-Ferritin could all have obvious cross-reactions with RBD, RBD-Ferritin, and XCL1-RBD-Ferritin proteins ( Figure 5 A).

[0152] The results of ELISA experiments showed that: The final titer of RBD IgG antibodies in the sera of the XCL1-RBD-Ferritin group was 1:1.5 million, that of the RBD-Ferritin group was 1:1 million, and that of the RBD group was 1:0.5 million. By comparison, it was found that the level of specific antibodies in the XCL1-RBD-Ferritin group was significantly higher than that in the RBD-Ferritin group (*p < 0.05) and the RBD group (***p < 0.001), and the immunized groups were all significantly higher than the Ferritin group and the Control group (***p < 0.001)( Figure 5 B).

[0153] Flow cytometry analysis results showed that: 6 weeks after the first immunization, the proportion of Memory B cells in the Control group among the total B cells was 29%, in the Ferritin group was 31%, in the RBD group was 32%, in the RBD-Ferritin group was 36.5%, and in the XCL1-RBD-Ferritin group was 39%. The XCL1-RBD-Ferritin group was significantly higher than the RBD-Ferritin group (*p < 0.05), and the XCL1-RBD-Ferritin group and the RBD-Ferritin group were significantly higher than the RBD group, Ferritin group, and Control group (**p < 0.01), while there was no difference among the RBD group, Ferritin group, and Control group (*p > 0.1) (see Figure 5 C).

[0154] The above results indicate that compared with RBD-Ferritin nanoparticles and RBD monomer protein antigens, XCL1-RBD-Ferritin targeted nanoparticles can induce a higher level of humoral immune response in the body.

[0155] Example 6 Analysis of the ability of XCL1-RBD-Ferritin nanoparticles to induce cellular immune response

[0156] The immunization protocol was the same as in Example 5. The specific grouping, immunization protocol, and sampling time are shown in Table 5.

[0157] Table 5. Immunization protocol and sampling time of experimental animals

[0158]

[0159] 1. Analysis of the levels of immune cells related to cellular immunity by flow cytometry

[0160] (1) Preparation of single-cell suspension: Take the spleens of mice 6 weeks after the first immunization, grind the lymph and spleen separately in a cell sieve, put them into a 15 mL centrifuge tube, and centrifuge at 4°C and 1500 rmp for 7 min. Discard the supernatant, and resuspend the lymph with 1 mL of PBS buffer. After lysing the spleen with 1 mL of erythrocyte lysate on ice for 15 min, centrifuge at 4°C and 1500 rmp for 7 min, discard the supernatant, and resuspend the cells with 1 mL of PBS to obtain a single-cell suspension.

[0161] (2) Staining of cell surface molecules: Aliquot the single-cell suspension into different flow cytometry tubes, 100 μL per tube, add surface molecule staining antibodies, after staining for 30 min, add 1 mL of PBS to terminate the staining, then centrifuge at 4°C and 1500 rmp for 7 min, discard the supernatant, and resuspend with 200 μL of PBS, and use a flow cytometer to detect the levels of immune cells.

[0162] The staining protocols include: CD69-BV421, CD4-PECY7, CD8-APC, CD62L-PECY5.5, CD44-PE; CD3-BV421, CD45-APCCY7, CD4-PECY7, CD8-APC, CD62L-PECY5.5, CD44-PE.

[0163] (3) Intracellular cytokine staining: Centrifuge the single-cell suspension at 4°C and 1500 rmp for 7 min, discard the supernatant, resuspend it completely with 1 ml of PRIM-1640. Take 100 μL of the cell suspension into a 96-well plate, add cytokine stimulants (PMA (phorbol ester) and ionomycin) and Golgi blocker (Monensin) according to a 1:100 ratio, and incubate for 8 h. Then blow down the cells and collect them into a flow cytometry tube. Centrifuge at 4°C and 1500 rmp for 7 min, discard the supernatant, add 100 μL of PBS to resuspend the cells, and then add surface staining antibodies and stain for 30 min. Add 1 ml of PBS to terminate the staining, then centrifuge at 4°C and 1500 rmp for 7 min and discard the supernatant. Add 200 μL of Fix / Perm to permeabilize the cells for 45 min, and then add 1 mL of Perm / wash to terminate the permeabilization. Centrifuge at 4°C and 1800 rmp for 7 min, discard the supernatant, add 100 μL of Perm / wash to resuspend the cells, add intracellular staining antibodies and stain for 30 min, and then add 500 μL of Perm / wash to terminate the staining.

[0164] The staining protocols include: CD3-FITC, CD4-PECY5.5, CD8-APCCY7, IL2-PECY7, TNFα-APC, IFNγ-PE; CD4-PEcy5.5, IFNγ-PE, IL4-PECy7.

[0165] (4) Centrifuge at 4°C and 1800 rmp for 7 min, discard the supernatant, add 1 mL of PBS to resuspend, centrifuge again and discard the supernatant. Add 200 μL of PBS to resuspend the cells, and then use a flow cytometer to detect the levels of relevant immune cells, including IFNγ in the spleen + CD8 + T cells, TNFα + CD8 + T cells, Th1 cells, Th2 cells and CD8 + TCM cells and IFNγ + T cell levels.

[0166] Figure 6 are the results of the analysis of cellular immune response levels; 6 weeks after the first immunization, A is IFNγ in different antigen-immunized groups + CD8 +T cell level; B is TNFα + CD8 + T cell level; C is Th1 cell level; D is Th2 cell level; E is CD8 + T cell level; F is IFNγ analyzed by ELISpot technology + T cell level.

[0167] The specific results are shown in Table 6 and Figure 6 as follows. The results show that: 6 weeks after the first immunization, the XCL1-RBD-Ferritin targeted nanoparticle antigen can induce a high level of IFNγ + CD8 + T cells ( Figure 6 A), TNFα + CD8 + T cells ( Figure 6 B), Th1 cells ( Figure 6 C), CD8 + TCM cells and IFNγ + T( Figure 6 E) cells, and are significantly higher than those in the other antigen immunization groups and the control group, while the Th2 level ( Figure 6 D) has no difference (related to the occurrence of vaccine immune pathological phenomena), indicating that the XCL1-RBD-Ferritin targeted nanoparticle antigen can induce a high level of cellular immune response in the body and has high safety.

[0168] Table 6. Levels of immune cells related to cellular immune response (%)

[0169] Animal grouping <![CDATA[IFNγ + CD8 + T]]> <![CDATA[TNFα + CD8 + T]]> Th1 Th2 <![CDATA[CD8 + TCM]]> PBS Control group 1.63 1.74 0.62 ＜0.02 2.1 Ferritin group 1.92 2.11 0.73 ＜0.02 7.9 RBD group 1.71 2.23 0.86 ＜0.03 8.5 RBD-Ferritin group 2.09 3.34 1.19 ＜0.02 9.4 XCL1-RBD-Ferritin group 4.12 4.76 1.79 ＜0.02 21.5

[0170] 2. Detection of specific IFNγ by ELISpot + Situation of

[0171] T cells + Using ELISpot technology to analyze the IFNγ

[0172] (1) Activation of the ELISpot plate: In a sterile environment, add 200 μL of ELISpot-specific serum-free medium to each well to activate the ELISpot plate, and let it stand at room temperature for 10 min.

[0173] (2) Cell seeding: Count the single-cell suspension of the spleen of mice 10 days after immunization, add ELISpot serum-free medium to prepare a suitable cell concentration, and add 100 μL of cell suspension to each well.

[0174] (3) Stimulation: Add 10 μL of positive stimulant to the positive control wells, 10 μL of serum-free medium to the negative control wells, and 10 μL of the synthesized SARS-CoV-2 RBD protein peptide pool to the experimental wells.

[0175] (4) Cell incubation: Incubate the ELISpot plate in the dark at 37 °C in a 5% CO2 incubator for 20 - 22 h.

[0176] (5) Cell lysis: Drain the liquid and residual cells in the wells, add 200 μL of ice-cold ultrapure water to each well, and place it in a 4 °C refrigerator for lysis for 10 min.

[0177] (6) Plate washing: Drain the liquid, add 280 μL of 1×Washing buffer to each well, let it stand in the well for 1 min, and repeat the plate washing 6 times.

[0178] (7) Incubation with biotinylated antibody: Drain the washing solution, add 100 μL of the biotinylated antibody prepared to the working concentration to each well, and react in a 37 °C, 5% CO2 incubator for 1 h.

[0179] (8) Plate washing: Drain the liquid, add 280 μL of 1×Washing buffer to each well, let the liquid stand in the well for 1 min each time, and repeat the plate washing 6 times.

[0180] (9) Incubation with enzyme-linked avidin: Add 100 μL of the enzyme-linked avidin prepared to the working concentration to each well, and incubate in a 37 °C, 5% CO2 incubator for 1 h.

[0181] (10) Plate washing: Discard the enzyme-linked avidin, add 280 μL of 1×Washing buffer to each well, let it stand in the well for 1 min, and repeat the plate washing 5 times. Thoroughly wash the bottom membrane and the bottom plate with tap water, cover the bottom plate, add 280 μL of 1×Washing buffer to each well, and pat dry after standing for 1 min.

[0182] (11) Termination of color development: After pouring out the liquid, uncover the bottom plate, rinse the front and back of the membrane 5 times with tap water to terminate the color development, and dry in the dark.

[0183] (12) Reading: Use a fluorescence enzyme-linked immunosorbent spot (ELISPOT) analyzer to count the number of cell spots.

[0184] The ELISpot test results showed that: 6 weeks after the first immunization, the IFNγ in the XCL1-RBD-Ferritin group +The average number of spots in the T cell group was 223, which was significantly higher than that in the RBD-Ferritin group (67) (*p < 0.05). The nanoparticle groups were all significantly higher than the RBD group (0), the Ferritin group (1), and the Control group (0) (**p < 0.01) (as Figure 6 shown in Figure F).

[0185] The above results indicate that compared with the RBD-Ferritin nanoparticles and the RBD monomer protein antigen, the XCL1-RBD-Ferritin targeted nanoparticles can induce a higher level of humoral immune response in the body.

[0186] Example 7 Analysis of the maintenance of immune memory of adaptive immune responses induced by XCL1-RBD-Ferritin nanoparticles

[0187] The immunization protocol was the same as in Example 5. The specific grouping, immunization dose, and sampling time are shown in Table 7.

[0188] Table 7. Immunization protocol and sampling time of experimental animals

[0189]

[0190] The ELISA experiment was used to analyze the titer changes of RBD-specific antibodies induced by XCL1-RBD-Ferritin nanoparticles in the body, as well as the memory B cells (Memory B) and CD8 + memory T cells (CD8 + TCM) levels related to the maintenance of humoral and cellular immune memory, so as to comprehensively evaluate the maintenance of immune memory of the adaptive immune response induced by this antigen.

[0191] The ELISA method was used to detect at different time points (as shown in Table 7 and Figure 7A) The levels of RBD-specific IgG in the collected mouse sera showed that: in the XCL1-RBD-Ferritin group, the serum RBD IgG antibody reached the highest titer of 1:1,500,000 at 6 weeks after the first immunization, the antibody titer was 1:1,250,000 at 10 weeks, and the antibody titer remained at 1:1,000,000 from 14 to 18 weeks; in the RBD-Ferritin group, the serum RBD IgG antibody titer reached the highest of 1:1,000,000 at 6 weeks after the first immunization, the antibody titer was 1:750,000 from 10 to 14 weeks, and the antibody titer was 1:500,000 at 18 weeks; in the RBD group, the serum RBD IgG antibody reached the highest titer of 1:500,000 at 6 weeks after the first immunization, and the antibody titer remained at 1:250,000 from 10 to 18 weeks. Compared with the RBD-Ferritin nanoparticles and the RBD monomer antigen, the XCL1-RBD-Ferritin nanoparticles could induce a higher level of RBD IgG antibody in the body, and still maintained a relatively high level 18 weeks after the first immunization( Figure 7 B).

[0192] Flow cytometry was used to analyze the Memory B cells and CD8 + TCM levels in the spleens of mice. The specific method was the same as the flow cytometry staining and experimental operation steps in Examples 5 and 6. The analysis results of Memory B cells showed that: at 18 weeks after the first immunization, the XCL1-RBD-Ferritin group was 28.8%, the RBD-Ferritin group was 21.5%, the RBD group was 21.3%, and the Ferritin group was 20.6%. The level of Memory B cells in the XCL1-RBD-Ferritin group was significantly higher than that in other antigen-immunized groups (**p < 0.01), while there was no difference among the RBD-Ferritin group, the RBD group and the Ferritin group( Figure 7 C). The analysis results of the CD8 + TCM cell levels showed that: the average level of CD8 + TCM cells in the XCL1-RBD-Ferritin group was 29.3%, the RBD-Ferritin group was 24.1%, the RBD group was 22.9%, and the Ferritin group was 16.9%. The level of CD8 + TCM cells in the XCL1-RBD-Ferritin group was significantly higher than that in the RBD-Ferritin group (*p < 0.05) and the RBD group (***p < 0.001). There was no difference between the RBD-Ferritin group and the RBD group (*p > 0.1), while the antigen-immunized groups were all significantly higher than the Ferritin control group (***p < 0.001, **p < 0.01, *p < 0.05)( Figure 7 D).

[0193] The above results indicate that the XCL-RBD-Ferritin targeted nanoparticles can induce cellular and humoral immune responses with more persistent immune memory in the body.

[0194] In summary, the XCL1-RBD-Ferritin fusion protein may be an effective COVID-19 candidate vaccine, providing a new design scheme and idea for the development of SARS-CoV-2 vaccines. The strategy of the present invention can also be extended to various infectious diseases and become a promising technical platform for vaccine development.

[0195] The above is a further detailed description of the present invention and should not be regarded as a limitation on the specific implementation of the present invention. For those of ordinary skill in the technical field to which the present invention pertains, simple deductions or substitutions without departing from the concept of the present invention are within the protection scope of the present invention.

Claims

1. A fusion protein, characterized in that: The fusion protein includes the amino acid sequence of the RBD domain of the novel coronavirus. The N-terminus of the amino acid sequence of the RBD domain is connected to XCL1 through a flexible linker GSG, and the C-terminus of the RBD domain sequence is connected to the Ferritin protein through a flexible linker GSG; The amino acid sequence of the RBD domain is shown in SEQ ID NO.1, the amino acid sequence of XCL1 is shown in SEQ ID NO.2, and the amino acid sequence of the Ferritin protein is shown in SEQ ID NO.

3.

2. The fusion protein according to claim 1, wherein: The amino acid sequence of the fusion protein is shown in SEQ ID NO.

4.

3. A gene encoding the fusion protein according to any one of claims 1-2.

4. The gene according to claim 3, wherein The nucleotide sequence encoding the RBD domain of the novel coronavirus is shown in SEQ ID NO.5, the nucleotide sequence encoding XCL1 is shown in SEQ ID NO.6, the nucleotide sequence encoding the Ferritin protein is shown in SEQ ID NO.7, the nucleotide sequence encoding the flexible linker GSG between XCL1 and RBD is GGTAGCGGT, and the nucleotide sequence encoding the flexible linker GSG between RBD and Ferritin is GGCAGTGGT.

5. The gene according to claim 4, characterized in that, The nucleotide sequence encoding the fusion protein is shown in SEQ ID NO.

9.

6. A vaccine, characterized in that the active ingredient of the vaccine is the fusion protein according to any one of claims 1-2.

7. The vaccine according to claim 6, characterized in that, The preparation form of the vaccine is an aqueous solution or a freeze-dried preparation.

8. A method for constructing a fusion protein expression vector, characterized in that, It includes the following steps: (1) Connect the N-terminus of the nucleotide sequence encoding the RBD domain of the novel coronavirus to the nucleotide sequence encoding the flexible linker, and then connect it to the nucleotide sequence encoding XCL1; (2) Connect the C-terminus of the nucleotide sequence encoding the RBD domain of the novel coronavirus to the nucleotide sequence encoding the flexible linker, and then connect it to the nucleotide sequence encoding the Ferritin protein; to obtain the sequence shown in SEQ ID NO.9; (3) Transfer the sequence obtained above into an expression vector to obtain a fusion protein expression vector.

9. A fusion protein expression vector, characterized in that, It includes an expression vector, a nucleotide sequence expressing XCL1, a nucleotide sequence expressing the Ferritin protein, and a nucleotide sequence encoding the RBD domain of the novel coronavirus. Among them, the N-terminus of the nucleotide sequence encoding the RBD domain of the novel coronavirus is connected to the nucleotide sequence expressing XCL1, and the C-terminus is connected to the nucleotide sequence expressing the Ferritin protein. The obtained sequence shown in SEQ ID NO.9 is then connected to the expression vector.

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