Targeted synergistic novel coronavirus nano vaccine

Abstract

The invention discloses a novel targeted synergistic coronavirus nano vaccine, and belongs to the field of vaccines. The active component of the vaccine is fusion protein, the fusion protein comprises a novel coronavirus RBD structural domain amino acid sequence, the N terminal of the RBD structural domain amino acid sequence is connected with XCL1 through a flexible joint GSG, and the C terminal of the RBD structural domain sequence is connected with Ferritin protein through a flexible joint GSG. The purified XCL1-RBD-Ferritin can be assembled into 24-polymer nanoparticles, so that the efficiency of activating DCs, especially DC1s, by an RBD antigen is improved, and the immune response of body fluid and cells is efficiently started.

Description

Technical Field

[0001] The present invention belongs to the field of vaccines, and specifically relates to a targeted and synergistic novel coronavirus nanovaccine. Background Art

[0003] The pathogen of COVID-19 is the new coronavirus (severe acute respiratory syndrome coronavirus 2, SARS-CoV-2). The virus gene is very easy to mutate, which brings great difficulties to the prevention and control of the disease. At present, there are six major types of mutated new coronaviruses: Alpha, Beta, Gamma, Delta, Lambda and Omicron. The severity of Omicron is much higher than influenza and other previous new coronavirus variants in terms of transmission speed and mortality rate.

[0004] At present, the COVID-19 vaccines that have been launched in my country include: inactivated vaccines, adenovirus vector vaccines, and recombinant subunit vaccines; many companies have also actively carried out research on mRNA vaccines. Inactivated vaccines have the advantages of short preparation cycle and high safety, but their immunogenicity is low, and they mainly induce humoral immunity. Adenovirus vector vaccines use harmless adenoviruses that have been modified as vectors, load the S protein gene of the new coronavirus, and stimulate the human body to produce antibodies, but the fact that some people have been infected with adenovirus may affect the immune effect of the vaccine. The mRNA vaccine has good immunogenicity and can effectively stimulate the body to produce high levels of neutralizing antibodies, but because the technology is relatively new, its safety still needs further clinical follow-up to determine. The main component of the recombinant subunit vaccine is the large-scale expression of S protein or RBD antigen through bioengineering methods. This vaccine has a single component, very good safety, and a relatively mature mass production process, but due to its relatively weak immunogenicity, it is necessary to optimize the vaccine components and construction methods.

[0005] The frequent mutations of SARS-CoV-2 pose a great challenge to the effectiveness of existing vaccines. Clinical follow-up studies have shown that the protective effect of mRNA vaccines (ChAdOx-1 and BNT162b2) against the Delta variant has 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 serum neutralizing antibodies of recovered COVID-19 patients against the Delta variant has decreased by an average of 6 times, and against the Omicron variant by 10.14 times. Existing studies have shown that cellular immune responses are not only correlated with the severity of COVID-19, but also that early CD8 +T cell immune response is also very important for the body's defense and inhibition of the spread of SARS-CoV-2. Long-term studies on patients who recovered from SARS in 2003 showed that cellular immune memory lasts longer, some even up to 17 years, while humoral immune memory of recovered patients is relatively short. However, compared with attenuated vaccines, other types of vaccines induce CD8 + The ability of T cell immune response is limited, and the attenuated vaccine itself has a potential risk of spreading the virus and is not suitable for the prevention and control of the new coronavirus.

[0006] Therefore, new research strategies are needed to improve the CD8 + The ability of T cell immune response and immune memory to maintain in order to cope with the possibility of repeated infection of the human body and severe illness caused by continuous mutation of the virus. Summary of the invention

[0007] The purpose of the present invention is to overcome at least one of the shortcomings of the prior art and provide a targeted and enhanced novel coronavirus nano-vaccine (antigen). After the antigen is immunized, it is expected to stimulate the body's high level of humoral immunity and CD8 + T cell immune response and immune memory are maintained for a longer period of time.

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

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

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

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

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

[0013] A gene encoding any of the above-mentioned fusion proteins.

[0014] In some of the embodiments, 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, and the nucleotide sequence encoding the Ferritin protein is shown in SEQ ID NO.7. The present invention obtains the XCL1, RBD, and Ferritin nucleotide sequences after optimizing the preference of the E. Coli expression system.

[0015] In some 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 E. coli has a variety of preferences for GSG linkers. The inventors optimized and achieved the best combination to effectively increase protein expression.

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

[0018] A vaccine, the active ingredient of which is the fusion protein described above.

[0019] In some embodiments, the vaccine is in the form of an aqueous solution or a lyophilized preparation.

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

[0021] In some 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 to play a key neutralizing role against antibodies in the RBD region of the new coronavirus S protein. For people who have been vaccinated with the S protein vaccine, more than 90% of the neutralizing activity is caused by antibodies in the RBD region; existing studies have shown that after clearing the RBD antibodies, the titer of the remaining neutralizing antibodies drops directly from the original antibody titer >1000 to <25. There are several potential advantages to choosing RBD as an antigen: the neutralizing antigenic epitopes of the S protein are mainly concentrated in the RBD region, and the neutralizing epitopes in other regions are less and there are a certain number of non-neutralizing epitopes. Therefore, under the same vaccination quality, the RBD antigen has a stronger ability to stimulate the body to produce neutralizing antibodies and a lower possibility of inducing non-neutralizing antibodies, so it is safer.

[0023] Subunit vaccines are highly safe and have mature production technology, but their immunogenicity is low. They require optimized antigen design or adjuvants and multiple immunizations to improve humoral immunity. However, based on the above optimization, they can induce the body to produce CD8 + The ability of T cell immune response is still extremely limited. In order to overcome the problem of poor immunogenicity of RBD monomer protein obtained from the Escherichia coli expression system, this application first fuses the RBD amino acid sequence of the new coronavirus Omicron strain and the Helicobacter pylori non-hemoglobin iron Ferritin amino acid sequence using the flexible Linker GSG, in order to use the self-assembly ability of Ferritin to obtain a polymerized RBD nanoparticle antigen. After expressing it using the Escherichia coli expression system, it was found that the following problems existed in the preparation of the RBD-Ferritin antigen: ① The loss of the target protein RBD-Ferritin is large during the harvesting of protein inclusion bodies; ② During the concentration process after the purification of the target protein RBD-Ferritin, the protein is prone to aggregation and loss is large. In the immune effect evaluation, it was found that compared with the RBD monomer, RBD-Ferritin nanoparticles can effectively improve the level of humoral immune response, but enhance CD8 + The ability of T cell immune response still needs to be further improved. Therefore, it is crucial to find a soluble small protein molecule that can be fused with RBD-Ferritin to improve the solubility of RBD-Ferritin recombinant protein and further improve the ability of RBD-Ferritin nanoparticles to induce adaptive immune response, which is crucial for the development of a new SARS-CoV-2 vaccine with a simple production process and high immunogenicity. Dendritic cells (DCs) are key antigen presenting cells (APCs) that initiate adaptive immunity. The activation of DCs determines the ability of T cells and the direction of immune response. DCs can be divided into two subgroups: classical DCs (cDCs) and plasmacytoid DCs (pDCs). cDCs can be further divided into classical type 1 DCs (cDC1s) and classical type 2 DCs (cDC2s). cDC1s subgroup cells have the ability to cross-present antigens and induce cytotoxic T cell responses, playing a key role in antiviral immunity. Chemokine receptor 1 (XCR1) is specifically expressed on cDC1s of mice and humans. Chemokine ligand 1 (XCL1) is a specific ligand of XCR1, with a size of about 13KDa. It is a small molecule secretory protein with strong solubility. Therefore, based on the targeting effect of XCL1, the inventors can improve the recognition and presentation efficiency of cDC1s to target antigens after fusion with target antigens, and promote CD8 +The occurrence of T cell immune response; based on its high solubility and small molecular weight, it can improve the solubility of the fusion-expressed target antigen during the expression process and has little effect on the formation of its natural spatial structure.

[0024] Ferritin is an endogenous protein present in many organisms. It can be generally divided into four subfamilies based on its structural characteristics, namely classical ferritin, bacterial ferritin, DNA-binding protein of starving cells, and encapsulated ferritin. Helicobacter pylori ferritin (Ferritin for short) belongs to the classical ferritin, which is composed of 24 subunits, forming a symmetrical hollow spherical structure. It can efficiently display target antigens on its surface and improve the recognition efficiency of the target antigen by the immune system. It also has low autoimmunogenicity, high safety, and good stability.

[0025] Therefore, the present application fuses the amino acid sequence of human XCL1, the amino acid sequence of RBD of novel coronavirus Omicron strain, and the amino acid sequence of Helicobacter pylori non-hemoglobin iron Ferritin using flexible linker GSG. The loss of XCL1-RBD-Ferritin recombinant protein is low during protein purification and concentration, and can be assembled into a 24-mer nanoparticle structure. The nanoparticle antigen can effectively activate cDC1s, efficiently induce humoral and CD8 + T cell immune response and immune memory are maintained more persistently.

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

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

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

[0029] (3) The sequence obtained above is transferred into an expression vector to obtain a fusion protein expression vector.

[0030] The antigen protein may be a full-length sequence of a structural protein of a certain pathogen, or may be a partial fragment of the pathogen that carries a neutralizing epitope.

[0031] After the fusion protein expression vector is transferred into cells, it is expressed to obtain a fusion protein. The fusion protein is a targeted nanoparticle antigen that can self-assemble into a 24-mer nanostructure and has a targeting effect on DC1s cells. It can be expected that the nanoparticle antigen can effectively activate cDC1s and efficiently induce body fluid and CD8 + T cell immune response and immune memory are maintained more persistently.

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

[0033] In the field of vaccine design and biotechnology, a "flexible linker" refers to an 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 as needed.

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

[0035] Expression vectors include prokaryotic expression vectors and eukaryotic expression vectors, and those skilled in the art can make adjustments based on actual conditions.

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

[0037] The beneficial effects of this application are:

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

[0039] In order to overcome the poor immunogenicity of RBD protein monomer, the low efficiency of RBD-Ferritin purification and concentration, and the induction of CD8 + In order to solve the problem that the level of T cell immune response is still limited, the inventors fused the human XCL1 amino acid sequence, the RBD amino acid sequence of the novel coronavirus Omicron strain, and the Helicobacter pylori african hemoglobin ferritin amino acid sequence using the flexible Linker GSG, optimized and synthesized the XCL1-RBD-Ferritin nucleotide sequence according to the codon preference of the Escherichia coli (E. coli) expression system, cloned it into the pET28a(+) prokaryotic expression plasmid, and expressed the XCL1-RBD-Ferritin fusion protein using the E. coli BL21(DE3) expression strain. The purified XCL1-RBD-Ferritin can be assembled into 24-mer nanoparticles, which improves the efficiency of RBD antigen in activating DCs, especially DC1s, thereby efficiently initiating humoral and cellular immune responses.

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

[0041] The expression of the fusion protein using the Escherichia coli prokaryotic expression system in the present application is through the prokaryotic expression system. The addition of XCL1 molecules improves the expression, purification and concentration efficiency of the XCL1-RBD-Ferritin protein, effectively increases the yield of XCL1-RBD-Ferritin nanoparticles, and is beneficial to the mass production of vaccine antigens.

[0042] 3. High humoral immune response and lasting immune memory

[0043] It can effectively activate TFH, induce high levels of humoral immune response, make the immune memory more lasting, and provide long-term and effective immune protection.

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

[0045] XCL1 molecules can target antigens to DC1s with cross-presentation function, achieve antigen cross-presentation, and thus induce the body to produce efficient humoral and CD8 + T cell immune response. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 are the identification results of the target protein expression strain; 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 are the results of purification and identification of the target protein; A is the result of purification and renaturation of RBD protein; B is the result of purification and renaturation of RBD-Ferritin protein; C is the result of purification and renaturation of XCL1-RBD-Ferritin protein.

[0048] Figure 3 The results of nanoparticle assembly analysis; A is a simulation 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 Flow cytometry was used to analyze the adaptive immune response initiated by nanoparticles, where A is the schematic diagram of the experimental principle; B is the DCs level; and C is the DC1s level.

[0050] Figure 5These are the analysis results of the humoral immune response levels induced by targeted nanoparticle antigens; A is the Western Blot experiment analyzing the reactivity of serum in different antigen immunization groups with antigens; B is the RBD-specific IgG antibody level in different immunization groups 6 weeks after the first immunization; C is the Memory B cell level in different immunization groups 6 weeks after the first immunization.

[0051] Figure 6 Results of the analysis of the cellular immune response induced by targeted nanoparticle antigens; 6 weeks after the first immunization, A is the IFNγ level in different antigen immunization 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 the analysis of IFNγ by ELSpot technology + T cell level.

[0052] Figure 7 The results of immune memory maintenance analysis; A is the blood sample collection time; B is the dynamic analysis results of serum RBD-specific IgG antibodies in different antigen immunization groups; C is the level of Memory B cells in different immunization groups 18 weeks after the first immunization; D is the level of CD8 in different immunization groups 18 weeks after the first immunization + TCM cell level. DETAILED DESCRIPTION

[0053] The experimental methods in the following examples of the present invention, for which specific conditions are not specified, are generally carried out under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or under conditions recommended by the manufacturer. The various commonly used chemical reagents used in the examples are all commercially available products.

[0054] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art of the present invention. 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 "including" and "having" and any variations thereof of the present invention are intended to cover non-exclusive inclusions. For example, a process, method, device, product or equipment comprising a series of steps is not limited to the listed steps or modules, but may optionally include steps not listed, or may optionally include other steps inherent to these processes, methods, devices, products or equipment.

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

[0057] At present, commercialized 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 using traditional methods. The antibodies induced by inactivated vaccines target all viral structural proteins, most of which are not related to the production of neutralizing antibodies. However, due to the inactivation of the virus, the natural spatial structure of the protein is damaged in the spike S protein or the receptor binding domain RBD region, resulting in reduced immunogenicity and limited ability to induce the body to produce neutralizing antibodies. Studies have shown that, except for live attenuated vaccines, other types of vaccines induce the body to produce CD8 + The immune response capacity of T cells is extremely limited, so new research strategies should be explored to improve vaccine-induced CD8 + T cell immune response capacity.

[0058] In order to improve the immunogenicity of RBD, this application pioneered the fusion of DC1s targeting factor XCL1, SARS-CoV-2 RBD domain and Ferritin protein with self-assembly ability, and developed a SARS-CoV-2 nanoparticle vaccine with targeting ability. The self-assembly property of Ferritin was used to display RBD protein on the surface of nanoparticles, thereby improving the efficiency of RBD protein neutralizing antigen epitopes recognized 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 response. At the same time, the addition of XCL1 molecules 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] Experimental design flow chart Figure 1 shown.

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

[0061] SFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFDEVFNATRFASVYAWNRKRISNCVADYSVLYNLAPFFTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKVS GNYNYLYRLFRKSNLKPFERDISTEIYQAGNKPCNGVAGFNCYFPLRSYSFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLKGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEIL(SEQ ID No.1).

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

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

[0064] Ferritin amino acid sequence, selected from ferritin [Helicobacter pylori] (NCBI Reference Sequence: WP_021436151.1), the specific sequence is 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] MRLLILALLGICSLTAYIVEGVGSEVSDKRTCVSLTTQRLPVSRIKTYTITEGSLRAVIFITKRGLKVCADPQATWVRDVVRSMDRKSNTRNMIQTKPTGTQQSTNTAVTLTG GSG SFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFDEVFNATRFASVYAWNRKRISNCVADYSVLYNLAPFFTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSK VSGNYNYLYRLFRKSNLKPFERDISTEIYQAGNKPCNGVAGFNCYFPLRSYSFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLKGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEIL GSG MLSKEIIKLLNEQVNKEMNSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYDHAKKLIVFLNENNVPVQLNSISAPEHKFESLTQIFQKAYKHEQDISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDILDKIELIGNENHGLYLADQYIKGIAKSKKSHHHHHHH (SEQ ID No. 4).

[0069] The nucleotide sequences of XCL1, RBD and Ferritin were further optimized according to the preference of the E. Coli expression system.

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

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

[0072] The optimized XCL1 nucleotide sequence is as follows:

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

[0074] The optimized Ferritin nucleotide sequence is as follows:

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

[0076] The nucleotide sequence of the GSG amino acid fragment of the flexible linker 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] 6×His Tag nucleotide sequence: CATCACCACCACCACCAC (SEQ ID No. 8).

[0079] The XCL1-RBD-Ferritin nucleotide sequence obtained after optimization of the 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 human XCL1 amino acid sequence, RBD amino acid sequence and Helicobacter pylori ferritin amino acid sequence were fused using the flexible linker GSG. According to the codon preference 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 Co., Ltd.

[0083] 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 the bacteria and culturing overnight for 12 hours, the bacterial solution was inoculated into the LB medium containing kanamycin resistance at a ratio of 1:100. When the OD value reached between 0.6-0.8, the IPTG inducer with a final concentration of 0.8M was added. After culturing for 5 hours, the bacterial solution was harvested and the expression of the XCL1-RBD-Ferritin recombinant protein was analyzed by SDS electrophoresis. 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] At the same time, in order to carry out comparative experiments, the pET28a recombinant plasmids pET28a-RBD and pET28a-RBD-Ferritin carrying RBD and RBD-Ferritin nucleotide sequences were simultaneously transformed into E. coli BL21 (DE3) expression strains, and the bacterial solution was inoculated in LB medium containing kanamycin resistance at a ratio of 1:100. When the OD value reached between 0.6-0.8, IPTG inducer with a final concentration of 0.8M was added. After culturing for 5 hours, the bacterial solution was harvested and the expression of RBD and RBD-Ferritin recombinant proteins was analyzed by SDS electrophoresis. The RBD and RBD-Ferritin expression strains were mixed with 40% glycerol at a ratio of 1:1 and stored at -80°C for later use.

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

[0086] Example 3 Recombinant protein preparation

[0087] 1. Identification of expression strains

[0088] 1.1 The XCL1-RBD-Ferritin expression strain was inoculated into LB medium containing kanamycin resistance at a ratio of 1:1000 and cultured 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, and culture 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 0.8M, and continue to culture at 37°C and 200 rpm for 5 hours. After the culture is completed, centrifuge at 6000 rpm, 4°C, and 10 minutes to harvest all the bacteria.

[0090] According to steps 1.1 and 1.2, the RBD and RBD-Ferritin plasmids were propagated and induced 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 Use 6mL non-denaturing lysis buffer to resuspend all the cells and transfer them to a 10mL centrifuge tube, place it in an ice-water mixture, and use an ultrasonic crusher (parameter setting power: 30%, time: 60min, ultrasound on 3s and off 3s) with the ultrasonic probe placed 1 / 2 below the liquid surface;

[0093] 2.2 After the ultrasonic treatment, transfer the bacterial suspension to three 2 mL centrifuge tubes, centrifuge at 4°C and 12,000 rpm for 25 min, discard the supernatant completely, add 1 mL of 0.1 M PBS buffer to each tube to resuspend and wash the precipitate, centrifuge again at 4°C and 12,000 rpm for 5 min, 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 DTT solution with a final concentration of 1 mM, mix and use an ultrasonic crusher to promote the dissolution of the precipitate (parameter setting power: 10%, time: 20 min, ultrasound on 3 s and off 3 s), the whole process should be completed in an ice-water mixture, and the ultrasonic probe should be kept 1 / 2 below the liquid surface;

[0095] 2.4 Divide the ultrasonic product equally and transfer it to a 2 mL centrifuge tube. Centrifuge again at 4°C, 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. Recombinant protein purification process

[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, and equilibrate the purification column;

[0099] 3.2 Add the inclusion body lysate to the Ni-TED protein purification column, repeat the flow-through 3 times, and collect the flow-through 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 (repeat this step 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 (repeat this step twice);

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

[0103] 4. Protein refolding and concentration

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

[0105] 4.2 Transfer the dialysis bag containing the purified protein solution into 1L of 2M urea dialysis solution at 4°C for 4 hours;

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

[0107] 4.4 Take out the purified protein solution after renaturation, centrifuge it in a low-temperature centrifuge at 4°C and 12000rpm for 10 minutes, collect the supernatant and remove the protein precipitate precipitated by denaturation.

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

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

[0110] The protein purification and concentration were analyzed by SDS-PAGE electrophoresis. Figure 2 As shown: Using Ni-TED protein purification column, RBD can be effectively purified under denaturing conditions ( Figure 2 A) RBD-Ferritin ( Figure 2 B) XCL1-RBD-Ferritin ( Figure 2C) recombinant protein. However, during the purification process, it was found that the RBD-Ferritin expression strain had a low recovery of inclusion bodies after the induction expression was completed and the protein expressed in the form of inclusion bodies was collected. In addition, during the protein purification, renaturation and concentration process, RBD-Ferritin showed low solubility and easy precipitation. After XCL1 and RBD-Ferritin were fused and expressed, the recovery rate of inclusion bodies was high, and a large amount of XCL1-RBD-Ferritin recombinant protein with good solubility could be obtained after protein purification, renaturation and concentration. The protein concentration was detected using an ultra-trace nucleic acid protein detector. The results showed that for every 200 mL of activated RBD, RBD-Ferritin, and XCL1-RBD-Ferritin expression strains, when the bacterial concentration OD value 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 after purification, renaturation, and concentration (the results are shown in Table 1).

[0111] Table 1. Protein yield

[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 show that although the addition of XCL1 molecules increases the molecular weight of the antigen to be expressed, it improves the solubility of the fusion protein.

[0114] Ferritin protein that can self-assemble into nanoparticles stored in the laboratory was used as a control. Ferritin, RBD-Ferritin and XCL1-RBD-Ferritin were dropped on a carbon-coated 400-mesh copper grid at a concentration of 0.3 mg / mL, and then stained with 0.5% phosphotungstate for 30 seconds after 2 minutes. All samples were imaged at 80 kV using a Hitachi H-7650 electron microscope equipped with an Olympus Cantega G2 bottom-mounted CCD TEM camera. TEM images were processed and analyzed using iTEM (Olympus). Results are shown in Figure 3 , where A is a simulation diagram of XCL1-RBD-Ferritin nanoparticle assembly; B is the transmission electron microscopy analysis result of Ferrtin / RBD-Ferritin / XCL1-RBD-Ferrtin nanoparticle assembly. Figure 3 It was shown that both RBD-Ferritin and XCL1-RBD-Ferrtin could be assembled into 80nm polymerized nanoparticle structures similar to the Ferritin nanoparticle structure.

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

[0116] Six-week-old female BALB / c mice were used as animal models and divided into five groups (three mice in each group). Freund's adjuvant was used as an immune adjuvant and mixed with antigen and PBS in 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 performed on 0d and 28d, for a total of two immunizations, both of which were performed by subcutaneous injection in the abdomen. For each immunization, the blank control group was injected with 50 μL PBS buffer, the Ferritin group was injected with 6.45 μg antigen, the RBD group was injected with 10 μg antigen, the RBD-Ferritin group was injected with 16.6 μg antigen, and the XCL1-RBD-Ferritin group was injected with 21 μg antigen to ensure that the total amount of RBD antigen received by each group of experimental animals was consistent (immunization procedure is shown in Table 2).

[0117] Table 2. Immunization procedures 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] Take the inguinal lymph nodes and spleen of mice, grind the lymph and spleen separately in a cell sieve, put them into a 15mL centrifuge tube, and centrifuge them at 4℃ and 1500rmp for 7min. Discard the supernatant, and resuspend the lymph in 1mL PBS buffer. Lyse the spleen on ice with 1mL red blood cell lysis buffer for 15min, centrifuge at 4℃ and 1500rmp for 7min, discard the supernatant, and resuspend it in 1mL PBS.

[0122] 2. Cell Surface Molecular Staining

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

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

[0125] Flow cytometry was used to analyze the activation levels of DCs and cDC1s cells, that is, the analysis of the adaptive immune response initiated by nanoparticles. The results are shown in Figure 4 , where A is the experimental principle diagram; B is the DCs level; C is the DC1s level. Specific data are shown in Table 3.

[0126] 4 hours 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 immunization groups (*p<0.05, **p<0.01, ***p<0.001), and all immunization groups were 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 the total DCs in the control group was 29.3%, 33.3% in the Ferritin group, 36.8% in the RBD group, 37.8% in the RBD-Ferritin group, and 48.0% in the XCL1-RBD-Ferritin group. The XCL1-RBD-Ferritin group was higher than that in the RBD and RBD-Ferritin immune groups (**p<0.01) (see Figure 4 C).

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

[0131] Six-week-old female BALB / c mice were used as animal models and divided into five groups (five mice in each group) to be 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 performed on 0d and 28d, for a total of two times, all of which were performed by subcutaneous immunization in the abdomen. For each immunization, the blank control group was injected with 50μL PBS buffer, the Ferritin group was injected with 6.45μg antigen, the RBD group was injected with 10μg antigen, the RBD-Ferritin group was injected with 16.6μg antigen, and the XCL1-RBD-Ferritin group was injected with 21μg antigen to ensure that the total amount of RBD antigen received by each group of experimental animals was consistent (see Table 4). Six weeks after the first immunization, the ability of XCL1-RBD-Ferritin nanoparticles to induce humoral immune response was analyzed by Western Blot technology, ELISA experiment and flow cytometry.

[0132] Table 4. Experimental animal immunization procedures and sampling time

[0133]

[0134] 1. Western Blot

[0135] SDS-PAGE electrophoresis: Ferritin, RBD, RBD-Ferritin and XCL1-RBD-Ferrtin protein purification products were separated by SDS-PAGE electrophoresis. The following steps were included:

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

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

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

[0139] (4) Washing the membrane: Recover the primary antibody and wash the NC membrane with 1× TBST at 90 rpm for 10 min at room temperature, three times.

[0140] (5)1.5 Incubation with secondary antibody: Add 10 mL of goat anti-mouse-HRP IgG antibody diluted 1:2000 and incubate on a horizontal shaker at room temperature at 60 rpm for 1 h.

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

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

[0143] 2. ELISA method to determine the titer of RBD protein antibodies in mouse serum

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

[0145] (1) Take blood from the tail vein of BALB / c mice after immunization, let it stand for 1 hour, centrifuge it at 4℃ and 2500rmp for 10 minutes, and take the supernatant; (2) Coat each well of the antigen adsorption plate with 100μl of 4ug / mL RBD protein and place it at 4℃ for 12 hours; (3) After discarding the liquid in the well, add 150μl / well blocking solution and place it at 37℃ for 90 minutes; (4) Add 300μL / well PBST and wash 3 times; (5) Add 100μL of the test serum of appropriate dilution to each well and incubate at 37℃ for 1 hour; (6) Add 300μL / well PBST and wash 3 times; (7) Add 100μL HRP-labeled goat anti-mouse secondary antibody (1:5000 dilution) to each well and incubate at 37℃ for 1 hour; (8) Add 300μL / well PBST and wash 3 times; (9) Add 200μL to each well TMB substrate solution was reacted at 37°C in the dark for 10 min; 50 μL 1MH2SO4 was added to each well to terminate the reaction; the OD value at 450 nm absorbance was detected by an enzyme-labeled instrument. The results were statistically analyzed.

[0146] 3. Analysis of Memory B Cell Levels Related to Humoral Immune Memory by Flow Cytometry

[0147] Preparation of single cell suspension: Take the inguinal lymph nodes and spleen of mice 6 weeks after the first immunization, grind the lymph and spleen separately in a cell sieve, put them into a 15mL centrifuge tube, and centrifuge them at 4℃ and 1500rmp for 7min. Discard the supernatant, and resuspend the lymph in 1mL PBS buffer. Lyse the spleen on ice with 1mL red blood cell lysis buffer for 15min, centrifuge at 4℃ and 1500rmp for 7min, discard the supernatant, and resuspend in 1mL PBS to obtain a single cell suspension.

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

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

[0150] Western Blot experiment, ELISA experiment results and flow cytometry results are shown in Figure 5 shown.

[0151] Western Blot analysis showed that the sera of mice immunized with RBD, RBD-Ferritin and XCL1-RBD-Ferritin could significantly cross-react with RBD, RBD-Ferritin and XCL1-RBD-Ferritin proteins ( Figure 5 A).

[0152] The results of ELISA experiments showed that the final titer of serum RBD IgG antibodies in the XCL1-RBD-Ferritin group was 1:1.5 million, 1:1 million in the RBD-Ferritin group, and 1:500,000 in the RBD group. By comparison, it was found that the specific antibody level in the XCL1-RBD-Ferritin group was significantly higher than that in the RBD-Ferritin group (*p<0.05) and the RBD-Ferritin group (***p<0.001), and the immune group was significantly higher than the Ferritin group and the Control group (***p<0.001) ( Figure 5 B).

[0153] The results of flow cytometric analysis showed that 6 weeks after the first immunization, memory B cells accounted for 29% of the total B cells in the control group, 31% in the Ferritin group, 32% in the RBD group, 36.5% in the RBD-Ferritin group, and 39% in the XCL1-RBD-Ferritin group. 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, and the Control group (**p<0.01), while there was no difference between the RBD group, Ferritin, and the 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 procedure was the same as in Example 5. The specific grouping, immunization procedure and sampling time are shown in Table 5.

[0157] Table 5. Experimental animal immunization procedures and sampling time

[0158]

[0159] 1. Flow cytometry analysis of immune cell levels related to cellular immunity

[0160] (1) Preparation of single cell suspension: Take the spleen of mice 6 weeks after the first immunization, grind the lymph and spleen separately in a cell sieve, put them into a 15mL centrifuge tube, and centrifuge them at 4℃ and 1500rpm for 7min. Discard the supernatant and resuspend the lymph in 1mL PBS buffer. Lyse the spleen with 1mL red blood cell lysis buffer on ice for 15min, centrifuge at 4℃ and 1500rpm for 7min, discard the supernatant, and resuspend the cells in 1mL PBS to obtain a single cell suspension.

[0161] (2) Cell surface molecule staining: The single cell suspension was dispensed into different flow tubes with 100 μL per tube, and surface molecule staining antibodies were added. After staining for 30 min, 1 mL of PBS was added to terminate the staining. The cells were centrifuged at 4°C and 1500 rpm for 7 min, the supernatant was discarded, and the cells were resuspended in 200 μL PBS. The immune cell levels were detected using a flow cytometer.

[0162] The staining scheme includes: 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: Single cell suspension was centrifuged at 4°C and 1500 rpm for 7 min, the supernatant was discarded, and 1 ml of PRIM-1640 was used to completely resuspend the suspension. 100 μL of the cell suspension was transferred to a 96-well plate, and cytokine stimulators (PMA (phorbol ester) and lonomycin (ionomycin)) and Golgi blocker (Monensin) were added at a ratio of 1:100. After stimulation for 8 h, the cells were blown off and collected in a flow tube. Centrifuged at 4°C and 1500 rpm for 7 min, the supernatant was discarded, 100 μL of PBS was added to resuspend the cells, and then the surface staining antibody was added and stained for 30 min. After adding 1 ml of PBS to terminate the staining, the cells were centrifuged at 4°C and 1500 rpm for 7 min, and the supernatant was discarded. 200 μL of Fix / Perm was added to permeabilize the membrane for 45 min, and then 1 mL of Perm / wash was added to terminate the membrane permeabilization. Centrifuge at 4°C and 1800 rpm for 7 min, discard the supernatant, add 100 μL Perm / wash to resuspend the cells, add cell staining antibody for staining, and add 500 μL Perm / wash to terminate staining after staining for 30 min.

[0164] The staining scheme includes: 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 rpm for 7 min, discard the supernatant, add 1 mL of PBS to resuspend, centrifuge again, discard the supernatant, add 200 μL of PBS to resuspend the cells, and use flow cytometry to detect the levels of related immune cells, including spleen IFNγ + CD8 + T cells, TNFα + CD8 + T cells, Th1 cells, Th2 cells and CD8 + TCM cells and IFNγ + T cell level.

[0166] Figure 6 The results of the analysis of cellular immune response levels; 6 weeks after the first immunization, A is the IFNγ in different antigen immunization 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 ELISpot analysis of IFNγ + T cell level.

[0167] The specific results are shown in Table 6 and Figure 6 The results showed that 6 weeks after the first immunization, XCL1-RBD-Ferritin targeted nanoparticle antigen could induce the body to produce high levels 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 were significantly higher than those in other antigen immunization groups and the control group, while the Th2 level ( Figure 6 D) No difference (related to the occurrence of vaccine immunopathology), indicating that XCL1-RBD-Ferritin targeted nanoparticle antigen can induce the body to produce a high level of cellular immune response and has a high safety.

[0168] Table 6. Cellular immune response-related immune cell levels (%)

[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. ELISpot detection of specific IFNγ + T cell status

[0171] ELISpot analysis of IFNγ in mouse spleen cells 6 weeks after the first immunization + T cell level, in order to further clarify the ability of various antigens to activate the body's cellular immunity. The specific steps are as follows:

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

[0173] (2) Cell plating: Count the single cell suspension of the spleen of mice 10 days after immunization, add ELISpot serum-free medium to prepare the appropriate 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 well, 10 μL of serum-free medium to the negative control well, and 10 μL of synthetic SARS-CoV-2 RBD protein peptide pool to the experimental well.

[0175] (4) Cell incubation: The ELISpot plate was incubated in a 37°C, 5% CO2 incubator in the dark for 20-22 hours.

[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 in a 4°C refrigerator for 10 min for lysis.

[0177] (6) Washing: Drain the liquid and add 280 μL 1× Washing buffer to each well. Let it stand in the well for 1 min. Repeat the washing process 6 times.

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

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

[0180] (9) Incubation of enzyme-linked avidin: Add 100 μL of enzyme-linked avidin prepared to the working concentration to each well and incubate in a 37° C., 5% CO 2 incubator for 1 h.

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

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

[0183] (12) Reading: Count the number of cell spots using a fluorescent enzyme-linked immunosorbent assay (ELISPOT) analyzer.

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

[0185] 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.

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

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

[0188] Table 7. Experimental animal immunization procedures and sampling time

[0189]

[0190] ELISA experiments were used to analyze the changes in the titer of RBD-specific antibodies induced by XCL1-RBD-Ferritin nanoparticles, as well as the memory B cells and CD8 + Memory T cells (CD8 + The TCM level was used to comprehensively evaluate the maintenance of immune memory of the adaptive immune response induced by the antigen.

[0191] The ELISA method was used to detect the different time points (as shown in Table 7 and Figure 7A) The levels of RBD-specific IgG in the serum of mice collected showed that the serum RBD IgG antibody titer of the XCL1-RBD-Ferritin group reached a maximum of 1:1.5 million 6 weeks after the first immunization, and the antibody titer was 1:1.25 million at 10 weeks, and the antibody titer was maintained at 1:1 million from 14 to 18 weeks; the serum RBD IgG antibody titer of the RBD-Ferritin group reached a maximum of 1:1 million 6 weeks after the first immunization, and the antibody titer was 1:750,000 from 10 to 14 weeks, and the antibody titer was 1:500,000 at 18 weeks; the serum RBD IgG antibody titer of the RBD group reached a maximum of 1:500,000 6 weeks after the first immunization, and the antibody titer was maintained at 1:250,000 from 10 to 18 weeks. Compared with RBD-Ferritin nanoparticles and RBD monomer antigens, XCL1-RBD-Ferritin nanoparticles can induce the body to produce higher levels of RBD IgG antibodies, and the antibody titer is still maintained at a high level 18 weeks after the first immunization ( Figure 7 B).

[0192] Analysis of Memory B Cells and CD8 in Mouse Spleen by Flow Cytometry + TCM level, the specific method is the same as the flow cytometry staining and experimental operation steps in Examples 5 and 6. The results of memory B cell analysis showed that 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 memory B cell level in the XCL1-RBD-Ferritin group was significantly higher than that in the other antigen immunization groups (**p < 0.01), while there was no difference between the RBD-Ferritin group, the RBD group, and the Ferritin group ( Figure 7 C) CD8 + The results of TCM cell level analysis showed that: XCL1-RBD-Ferritin group CD8 + The mean cell level in TCM group was 29.3%, that in RBD-Ferritin group was 24.1%, that in RBD group was 22.9%, that in Ferritin group was 16.9%, and that in XCL1-RBD-Ferritin group was CD8 + The level of TCM cells was significantly higher than that of the RBD-Ferritin group (*p<0.05) and the RBD group (***p<0.001), and there was no difference between the RBD-Ferritin group and the RBD group (*p>0.1), while the antigen immunization group was significantly higher than that of the Ferritin control group (***p<0.001, **p<0.01, *p<0.05) ( Figure 7 D).

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

[0194] In summary, the XCL1-RBD-Ferritin fusion protein may be an effective COVID-19 vaccine candidate, 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 technology platform for vaccine development.

[0195] The above is a further detailed description of the present invention, which should not be regarded as a limitation on the specific implementation of the present invention. For ordinary technicians in the technical field to which the present invention belongs, simple deduction or replacement without departing from the concept of the present invention is 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, wherein the N-terminus of the RBD domain amino acid sequence is connected to XCL1 via a flexible linker GSG, and the C-terminus of the RBD domain sequence is connected to the Ferritin protein via a flexible linker GSG.

2. The fusion protein according to claim 1, characterized in that: 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.

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

4.

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

5. The gene according to claim 4, characterized in that The nucleotide sequence encoding the RBD domain of the new 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.

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

9.

7. A vaccine, characterized in that the active ingredient of the vaccine is the fusion protein according to any one of claims 1 to 3.

8. The vaccine according to claim 7, characterized in that The vaccine is in the form of an aqueous solution or a lyophilized preparation.

9. A method for constructing a fusion protein expression vector, characterized in that: The following steps are involved: (1) connecting the N-terminus of the nucleotide sequence of the antigen protein to be expressed to the nucleotide sequence encoding the flexible linker, and then connecting to XCL1; (2) connecting the C-terminus of the nucleotide sequence of the antigen protein to be expressed to the nucleotide sequence encoding the flexible linker, and then connecting to the nucleotide sequence encoding the Ferritin protein; (3) The sequence obtained above is transferred into an expression vector to obtain a fusion protein expression vector.

10. A fusion protein expression vector, characterized in that: It includes an expression vector, a nucleotide sequence expressing XCL1, a nucleotide sequence expressing Ferritin protein, and a nucleotide sequence encoding a target protein to be expressed, wherein the N-terminus of the nucleotide sequence encoding the target protein to be expressed is connected to the nucleotide sequence expressing XCL1, and the C-terminus is connected to the nucleotide sequence expressing Ferritin protein, and the resulting sequence is then connected to the expression vector.

Images