A viral polypeptide-protein subunit combination vaccine based on DNA nanotechnology and its preparation method and application

Abstract

The present invention provides a virus polypeptide-protein subunit combination vaccine based on DNA nanotechnology, and a preparation method and application thereof, belonging to the technical field of biological products. A virus polypeptide-protein subunit combination vaccine based on DNA nanotechnology uses a tetrahedral framework nucleic acid formed by DNA chain assembly as a carrier, a protein antigen that causes virus-specific T cell activation is coupled to one edge of the tetrahedral framework nucleic acid, and a virus B cell epitope peptide is coupled to the four vertices of the tetrahedral framework nucleic acid. Among all virus subunit-peptide vaccines with framework nucleic acids, coupling SARS-Cvo-2N protein to the tetrahedral framework can induce CD4 in a variety of animal models. + T cell activation can induce higher antibody titers and an increase in the ratio of memory B cells to T cells, and can also induce stronger CD8 + T cell activation provides a new idea for constructing efficient viral peptide vaccines based on protein carriers.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological products, and specifically relates to a viral polypeptide-protein subunit combination vaccine based on DNA nanotechnology, and a preparation method and application thereof. Background Art

[0002] Peptide vaccines constructed with pathogen antigen epitopes can induce a more precise immune response against pathogenic microorganisms. In addition, peptide vaccines are prepared by chemical means, with low production costs, and the vaccine composition can be quickly adjusted according to the pathogen mutation information, thereby quickly responding to the hazards caused by pathogen mutations. Since the difference in B cell receptors is at the cellular level, for B cell epitopes, a B cell epitope of a pathogen may cause B cell activation in different individuals. However, due to individual differences in the human major histocompatibility complex, which is responsible for presenting T cell epitopes, it is difficult to screen T cell epitopes (including CD4 T cell epitopes) from pathogens that can effectively induce antigen-specific T lymphocyte activation in most human bodies. + and CD8 + T cell epitopes). Therefore, peptide vaccines are currently mainly constructed using B cell epitopes. However, it is difficult to induce efficient humoral immune responses with B cell epitopes alone. This is because the activation of B cells into antibody-secreting cells requires not only multivalent antigens to trigger B cell receptor cross-linking, but also activated TH2-type helper CD4-positive T cells to provide CD40 ligands for B cells, while B cell epitopes usually cannot induce T cell activation.

[0003] At present, in order to make B cell epitopes immunogenic, B cell epitopes are generally coupled to exogenous carrier proteins (such as OVA, BSA, KLH) through cross-linking agents, and the T cell epitopes in the carrier protein are used to activate CD4 positive T cells, thereby inducing effective humoral immunity. Studies have also shown that combining the B cell epitopes of pathogens with a so-called universal T cell epitope can effectively enhance humoral immunity. Although the peptide vaccine constructed in the above manner may induce a broad humoral immune response, it cannot induce pathogen antigen-specific CD4 + T cell activation, which results in the inability to generate memory CD4 + T lymphocytes affect the vaccine's preventive effect. In addition, pathogen-specific cytotoxic CD8 + T cells can effectively inhibit viral replication by killing virus-infected cells, and are also critical for resisting viral transmission. Some studies have even shown that Th1-biased immunity (helper CD8 + T cell activation) response is more beneficial for resisting viruses. However, current peptide vaccines are also unable to induce a wide range of pathogen-specific CD8 +T cell activation. To address the above issues, a possible strategy is to integrate the B cell epitopes of pathogens with protein subunits rich in T cell epitopes. However, if the B cell epitopes and pathogen protein antigens are directly cross-linked, on the one hand, the T cell epitopes of the antigen may be destroyed, and on the other hand, the lack of a delivery vector will affect the T cell epitope presentation efficiency of the protein antigen, especially the cross-presentation of the antigen, which is not conducive to CD8 + T cell activation is crucial. Summary of the invention

[0004] In view of this, the purpose of the present invention is to provide a viral polypeptide-protein subunit combination vaccine based on DNA nanotechnology, using framework nucleic acid as a delivery vector to precisely reorganize B cell epitopes and protein subunits from the virus, and to construct a universally applicable new vaccine that can simultaneously induce efficient humoral immunity and cellular immunity.

[0005] The present invention provides a viral polypeptide-protein subunit combination vaccine based on DNA nanotechnology, which uses a tetrahedral framework nucleic acid formed by assembling DNA chains as a carrier, a protein antigen that causes virus-specific T cell activation is coupled to one edge of the tetrahedral framework nucleic acid, and viral B cell epitope peptides are coupled to the four vertices of the tetrahedral framework nucleic acid.

[0006] Preferably, the nucleotide sequence of the DNA chain for tetrahedral framework nucleic acid assembly is shown in SEQ ID NO:1 to SEQ ID NO:8.

[0007] Preferably, the B cell epitope peptide of the virus is hybridized with the single-stranded DNA extending from the vertices of the tetrahedral framework nucleic acid through the thiol-modified DNA chain;

[0008] The nucleotide sequence of the thiol-modified DNA chain is shown in SEQ ID NO:9.

[0009] Preferably, the protein antigen that causes virus-specific T cell activation hybridizes with the single-stranded DNA extending from the edge of the tetrahedral framework nucleic acid through the coupled DNA chain;

[0010] The nucleotide sequence of the coupled DNA chain is shown in SEQ ID NO:10.

[0011] Preferably, the protein antigen and DNA chain that cause virus-specific T cell activation are coupled via Halo tag and Halo ligand.

[0012] Preferably, the virus comprises at least one of the following viruses: coronavirus, human immunodeficiency virus polypeptide vaccine, respiratory syncytial virus polypeptide vaccine and influenza virus.

[0013] Preferably, the coronavirus includes the novel coronavirus and its variants;

[0014] The protein antigens in the novel coronavirus or its variants that cause virus-specific T cell activation include N protein;

[0015] B cell epitope peptides of the new coronavirus or its variants include S3 404-412 and / or S4 440-445 .

[0016] The present invention provides a method for preparing the viral polypeptide-protein subunit combination vaccine based on DNA nanotechnology, comprising the following steps:

[0017] Performing a first hybridization reaction on a DNA chain for assembling a tetrahedral framework nucleic acid to obtain a tetrahedral framework nucleic acid;

[0018] The thiol-modified DNA chain and the maleimide-modified viral B cell epitope peptide are mixed for coupling reaction to obtain the DNA-coupled B cell epitope peptide;

[0019] Covalently couple the protein antigen modified by Halo tag that causes virus-specific T cell activation and the DNA chain modified by Halo ligand to obtain the protein antigen coupled to DNA;

[0020] Tetrahedral framework nucleic acid, DNA-coupled B cell epitope peptide and DNA-coupled protein antigen

[0021] A second hybridization reaction is performed to obtain a viral polypeptide-protein subunit combination vaccine.

[0022] Preferably, in the first hybridization reaction, the DNA chains for tetrahedral framework nucleic acid assembly are mixed in an equimolar ratio, treated at 95°C for 10 min, and then kept at 4°C for 30 min; the final concentration of each DNA chain of the tetrahedral framework nucleic acid assembly DNA chain is independently 0.8 to 1.2 μM;

[0023] In the coupling reaction, the molar ratio of the thiol-modified DNA chain to the maleimide-modified viral B cell epitope peptide is 1:(2-10);

[0024] In the covalent coupling, the molar ratio of the protein antigen modified by the Halo tag that causes virus-specific T cell activation and the DNA chain modified by the Halo ligand is (0.9-1.1): (0.9-1.1);

[0025] In the second hybridization reaction, the molar ratio of the tetrahedral framework nucleic acid, the DNA-coupled B cell epitope peptide and the DNA-coupled protein antigen is (0.9-1.1): (3.9-4.1): (0.9-1.1).

[0026] The present invention provides the use of the viral polypeptide-protein subunit combination vaccine based on DNA nanotechnology or the viral polypeptide-protein subunit combination vaccine obtained by the preparation method in preparing an immune animal model;

[0027] Preferably, the immune animal model comprises at least one of: rats, mice and rabbits.

[0028] The present invention provides a virus polypeptide-protein subunit combination vaccine based on DNA nanotechnology, which uses a tetrahedral framework nucleic acid formed by DNA chain assembly as a carrier, a protein antigen that causes virus-specific T cell activation is coupled to one edge of the tetrahedral framework nucleic acid, and a virus B cell epitope peptide is coupled to the four vertices of the tetrahedral framework nucleic acid. Through systemic immune evaluation, the results show that coupling SARS-Cvo-2 N protein to the tetrahedral framework nucleic acid can induce CD4 in a variety of animal models, including C57, Babl / c mice, rats and rabbits. + T cell activation, thereby effectively improving the immune effect of B cell epitopes, inducing higher antibody titers, and increasing the proportion of memory B cells and T cells. In addition, we were pleasantly surprised to find that integrating N protein into tetrahedrons can induce stronger CD8-positive T cell activation compared to free N protein, which is probably because tetrahedrons promote antigen cross-presentation. It can be seen that the viral polypeptide-protein subunit combination vaccine provided by the present invention uses tetrahedral framework nucleic acid as a delivery carrier to effectively deliver N protein to T cells, enhance antigen-specific B cell-T cell binding, and enable B cells to obtain more CD40L stimulation, providing a new idea for the construction of efficient viral peptide vaccines based on protein carriers. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 A schematic diagram of the construction principle of the viral polypeptide-protein subunit combination vaccine based on DNA nanotechnology provided by the present invention;

[0030] Figure 2 Schematic diagram of the preparation of tetrahedral framework nucleic acid structure (a) and electrophoresis identification results (b);

[0031] Figure 3 The results of the coupling experiment between thiol-modified SH-DNA (CpG) and the B cell epitope peptide of the new coronavirus;

[0032] Figure 4 This is the electrophoresis result of the coupling experiment between N protein carrying Halo-tag and DNA-ligand;

[0033] Figure 5These are the test results of the ability of viral polypeptide-protein subunit vaccines to stimulate B cells; a is a flow cytometric analysis diagram of in vitro B cell proliferation; b is statistical data, c is a flow cytometric analysis diagram of in vitro B cell proliferation; d is statistical data.

[0034] Figure 6 These are the antibody titer measurement results of live viral polypeptide-protein subunit vaccines, where a is a schematic diagram of the immunization scheme and b is the antibody titer results induced by different groups of vaccines.

[0035] Figure 7 These are the measurement results of live helper T cells after vaccination with the new coronavirus polypeptide vaccine, where a is the immunization timeline; b is the flow cytometric analysis result of activated helper T cells after immunization of mice with the vaccine; and c is the statistical data.

[0036] Figure 8 These are the measurement results of live cytotoxic T cells after vaccination with the new coronavirus polypeptide vaccine, where a is the immunization timeline; b is the flow cytometry analysis result of activated cytotoxic T cells after vaccine immunization of mice; and c is statistical data.

[0037] Fig. 9 The results of the antibody virus neutralization experiment are as follows:

[0038] Fig.10 These are the measurement results of memory B cells in mice on the 60th day after the first immunization, where a is the immunization timeline; b is the flow cytometric analysis result of memory B cells after vaccine immunization of mice; and c is the statistical data.

[0039] Fig.11 These are the measurement results of memory T cells in mice on the 60th day after the first immunization, where a is the immunization timeline; b is the flow cytometry analysis results of memory T cells after vaccine immunization of mice; and c is statistical data.

[0040] Fig.12 These are the results of antibody titer measurement after Balb / c mice were immunized with the new coronavirus polypeptide vaccine, where a is a schematic diagram of the immunization scheme, and b is the antibody titer results induced by different groups of vaccines.

[0041] Fig.13 These are the results of antibody titer measurement after SD rats were immunized with the new coronavirus polypeptide vaccine, where a is a schematic diagram of the immunization scheme, and b is the antibody titer results induced by different groups of vaccines.

[0042] Fig.14 These are the results of antibody titer measurement after rabbits were immunized with the new coronavirus polypeptide vaccine, where a is a schematic diagram of the immunization scheme, and b is the antibody titer results induced by different groups of vaccines. DETAILED DESCRIPTION

[0043] The present invention provides a viral polypeptide-protein subunit combination vaccine based on DNA nanotechnology, which uses a tetrahedral framework nucleic acid formed by assembling DNA chains as a carrier, a protein antigen that causes virus-specific T cell activation is coupled to one edge of the tetrahedral framework nucleic acid, and viral B cell epitope peptides are coupled to the four vertices of the tetrahedral framework nucleic acid.

[0044] The present invention utilizes self-adjuvant tetrahedral framework nucleic acid to precisely reorganize B cell epitopes from the virus and protein subunits that cause virus-specific T cell activation, thereby constructing a universally applicable new vaccine that can simultaneously induce highly effective humoral immunity and cellular immunity. In order not to damage T cell epitopes, the present invention uses a DNA single labeling method to modify the protein subunits that cause virus-specific T cell activation, that is, the recombinantly expressed protein subunits that cause virus-specific T cell activation carrying Halo-tag are covalently coupled to the DNA chain modified with Halo ligand, and then the N protein is assembled onto an edge of the tetrahedral framework nucleic acid with a side length of 37 base pairs through DNA hybridization. At the same time, the B cell epitopes coupled to DNA through a small molecule cross-linking agent are assembled at the vertices of the tetrahedral framework nucleic acid (see Figure 1 ).

[0045] In the present invention, the nucleotide sequence of the DNA chain for tetrahedral framework nucleic acid assembly is preferably as shown in SEQ ID NO: 1 to SEQ ID NO: 8. In the embodiment of the present invention, the 1st to 37th bases in A37.1 are complementary to the 1st to 37th bases in D37.1; the 40th to 57th bases in A37.1 are complementary to the 20th to 37th bases in B37.1; the 1st to 19th bases in A37.2 are complementary to the 1st to 19th bases in B37.1; the 23rd to 40th bases in A37.2 are complementary to the 1st to 18th bases in C37.2; the 41st to 59th bases in A37.2 are complementary to the 41st to 59th bases in C37.1; Complementary pairing; bases 41-58 in B37.1 are complementary paired with bases 41-58 in D37.2; bases 1-19 in B37.2 are complementary paired with bases 22-40 in D37.2; bases 23-59 in B37.2 are complementary paired with bases 22-58 in C37.2; bases 1-18 in C37.1 are complementary paired with bases 1-18 in D37.2; bases 19-37 in C37.1 are complementary paired with bases 41-59 in D37.1.

[0046] In the present invention, the B cell epitope peptide of the virus is preferably hybridized with the single-stranded DNA extending from the vertices of the tetrahedral framework nucleic acid through a thiol-modified DNA chain; the nucleotide sequence of the thiol-modified DNA chain is shown in SEQ ID NO: 7. The B cell epitope peptide of the virus is covalently cross-linked through a chemical reaction between the thiol groups on the DNA chain and a small molecule cross-linking agent. The small molecule cross-linking agent is preferably maleimide (see Figure 1 ). The small molecule cross-linking agent is modified on the B cell epitope peptide of the virus. The present invention has no special restrictions on the modification method, and the modification method known in the art can be used. The single-stranded DNA extending from the vertices of the tetrahedral framework nucleic acid is the 60th to 74th bases in A37.2, the 60th to 74th bases in B37.2, the 59th to 73rd bases in C37.2, and the 59th to 73rd bases in C37.2.

[0047] In the present invention, the protein antigen that causes virus-specific T cell activation is preferably hybridized with the single-stranded DNA on the edge of the tetrahedral framework nucleic acid through a coupled DNA chain; the nucleotide sequence of the coupled DNA chain is shown in SEQ ID NO: 8. The protein antigen that causes virus-specific T cell activation and the DNA chain are preferably coupled through Halo tag and Haloligand. The single-stranded DNA extending from the edge of the tetrahedral framework nucleic acid is the 59th to 73rd bases in A37.1.

[0048] In the present invention, the virus preferably includes coronavirus, human immunodeficiency virus polypeptide vaccine, respiratory syncytial virus polypeptide vaccine, influenza virus. The coronavirus preferably includes the novel coronavirus and its variants; the protein antigen that causes virus-specific T cell activation in the novel coronavirus or its variants includes N protein. The amino acid sequence of the N protein is as shown in SEQ ID NO: 13 (MSDNGPQNQRNAPRITFGGPSDSTGSNQNGERSGARSKQRRPQGLPNN TASWFTALTQHGKEDLKFPRGQGVPINTNSSPDDQIGYYRRATRRIRGGDGKMKDLSPRWYFYYLGTGPEAGLPYGANKDGIIWVATEGALNTPKDHIGTRNPANNAAIVLQLPQGTTLPKGFYAEGSRGGSQASSRSSSRSRNSSRNSTPGSSRGTSPARMAGNGGDAALALLLLDRLNQLESKMS GKGQQQQGQTVTKKSAAEASKKPRQKRTATKAYNVTQAFGRRGPEQTQGNFGDQELIRQGTDYKHWPQIAQFAPSASAFFGMSRIGMEVTPSGTWLTYTGAIKLDDKDPNFKDQVILLNKHIDAYKTFPPTEPKKDKKKKADETQALPQRQKKQQTVTLLPAADLDDFSKQLQQSMSSADSTQA). B cell epitope peptides of the new coronavirus or its variants include S3 404-412 and / or S4 440-445 . S3 404-412 The amino acid sequence of S4 is shown in SEQ ID NO: 11. 440-445 The amino acid sequence is shown in SEQ ID NO:12.

[0049] In the embodiment of the present invention, in order to verify the immune effect of T cell epitope peptides and proteins that cause T cell activation in dealing with different individual differences, a composite vaccine prepared by replacing N protein with ORF3a was constructed, specifically, DNA-ORF3a was formed by covalent coupling of ORF3a-linker-3 chain through thiol and maleimide, and then the DNA chain was hybridized with the single-stranded DNA extending from the edge of the tetrahedral framework nucleic acid to achieve coupling. The results of the immune experiment showed that due to individual differences in the major histocompatibility complex of different individuals, ORF3a may not be an effective inducing antigen-specific CD4 in Balb / c mice, rats, and rabbits. +T cell epitopes for T lymphocyte activation, such as TH37-S3-ORF3a & TH37-S4-ORF3a, have little effect on further promoting the production of RBD-specific antibodies in mice, rats, and rabbits, while coupling SARS-Cvo-2 N protein to tetrahedral framework nucleic acids can induce higher antibody titers in a variety of animal models, including C57 or Babl / c mice, rats, and rabbits.

[0050] The present invention provides a method for preparing the viral polypeptide-protein subunit combination vaccine based on DNA nanotechnology, comprising the following steps:

[0051] Performing a first hybridization reaction on a DNA chain for assembling a tetrahedral framework nucleic acid to obtain a tetrahedral framework nucleic acid;

[0052] The thiol-modified DNA chain and the maleimide-modified viral B cell epitope peptide are mixed for coupling reaction to obtain the DNA-coupled B cell epitope peptide;

[0053] Covalently couple the protein antigen modified by Halo tag that causes virus-specific T cell activation and the DNA chain modified by Halo ligand to obtain the protein antigen coupled to DNA;

[0054] The tetrahedral framework nucleic acid, DNA-coupled B cell epitope peptide and DNA-coupled protein antigen are subjected to a second hybridization reaction to obtain a virus polypeptide-protein subunit combination vaccine.

[0055] In the present invention, in the first hybridization reaction, the DNA chains for tetrahedral framework nucleic acid assembly are preferably mixed in an equimolar ratio, treated at 95°C for 10 minutes, and then kept at 4°C for 30 minutes. The final concentration of each DNA chain of the tetrahedral framework nucleic acid assembly DNA chain is independently 0.8 to 1.2 μM, more preferably 1 μM. The solvent of the first hybridization reaction is preferably 1×TAE buffer containing 10 mM magnesium chloride.

[0056] In the present invention, in the coupling reaction, the molar ratio of the thiol-modified DNA chain to the maleimide-modified viral B cell epitope peptide is 1:(2-10), more preferably 1:3-5. The present invention has no special restrictions on the preparation method of the maleimide-modified viral B cell epitope peptide, and the preparation method known in the art can be used. The temperature of the coupling reaction is preferably 18-27°C, more preferably 20-25°C. The time of the coupling reaction is preferably 10-14h, more preferably 12h. After the coupling reaction is completed, the reaction product is preferably purified by ultrafiltration.

[0057] In the present invention, in the covalent coupling, the molar ratio of the protein antigen modified by Halo tag that causes virus-specific T cell activation and the DNA chain modified by Halo ligand is (0.9-1.1): (0.9-1.1), and more preferably 1:1. During the covalent coupling, the reaction temperature is preferably 36-38°C, and more preferably 37°C. The reaction time is preferably 10-14h, and more preferably 12h. The present invention is effective for causing virus-specific

[0058] The method for modifying Halo tag with protein antigen for virus-specific T cell activation is to construct plasmid to express fusion protein. The DNA chain modified with Halo ligand is preferably completed by using Halo Tag Succinimidyl Ester (O2) Ligand Kit. The reaction solvent for the covalent coupling is preferably 1×PBS.

[0059] In the present invention, in the second hybridization reaction, the molar ratio of the tetrahedral framework nucleic acid, the DNA-coupled B cell epitope peptide and the DNA-coupled protein antigen is preferably (0.9-1.1): (3.9-4.1): (0.9-1.1), more preferably 1:4: 1. The temperature of the second hybridization reaction is preferably 18-27° C., more preferably 20-25° C. The time of the second hybridization reaction is preferably 20 min to 40 min, more preferably 30 min.

[0060] The present invention provides the use of the viral polypeptide-protein subunit combination vaccine based on DNA nanotechnology or the viral polypeptide-protein subunit combination vaccine obtained by the preparation method in preparing an immune animal model;

[0061] In the present invention, the immune animal model preferably includes at least one of: rats, mice and rabbits.

[0062] In the present invention, both tetrahedral framework nucleic acid coupled B cell epitopes can effectively induce B cell proliferation, while free peptides and free CpG show very limited ability to stimulate B cell proliferation. It is worth noting that vaccines targeting C57 mouse T cell epitope peptide (ORF3a) and universal N protein group have stronger stimulation ability. This is attributed to CD4 + T cell activation, thereby effectively improving the immune effect of B cell epitopes. Animals immunized with the virus polypeptide-protein subunit combination vaccine (TH37-S3 / S4-N pro) prepared by the present invention can produce RBD-specific antibodies. Compared with TH37+free S3 / S4, TH37-S3 / S4, TH37-S3 / S4+free N protein, and TH37-S3 / S4-ORF3a, the antibody secretion amount is significantly different, and the antibody titer is as high as 10 4This may be due to the activation of helper CD4 T cells by N protein, which effectively promotes the activation and differentiation of B cells. Compared with the free N protein vaccine group, the framework nucleic acid can effectively deliver N protein to T cells, enhance antigen-specific B cell T cell binding, and enable B cells to obtain more CD40-L stimulation. Compared with TH37+free S3 / S4, TH37-S3 / S4, TH37-S3 / S4+free N protein, and TH37-S3 / S4-ORF3a, after immunization with TH37-S3 / S4-N pro, the CD4 + T cells are activated to the greatest extent. And the results of the antibody neutralization experiment show that the antibodies in the serum of mice introduced with the N protein vaccine group showed the most effective ability to neutralize the virus. The new coronavirus subunit-peptide vaccine constructed by the present invention can effectively activate B cells, thereby inducing B cell-mediated humoral immune response, promoting the production of specific neutralizing antibodies, and protecting mice from viral infection. In addition, the new coronavirus subunit-peptide vaccine constructed by the present invention can also induce high levels of memory B cells and memory T cells. In addition, the present invention surprisingly found that integrating N protein into tetrahedrons can induce stronger CD8-positive T cell activation compared with free N protein, which may be because tetrahedrons promote antigen cross-presentation.

[0063] The following is a detailed description of a viral polypeptide-protein subunit combination vaccine based on DNA nanotechnology and its preparation method and application provided by the present invention in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0064] Example 1

[0065] A method for preparing a viral polypeptide-protein subunit combination vaccine based on DNA nanotechnology

[0066] Experimental reagents: DNA chains used to construct extended chain framework nucleic acids for assembling vaccines are shown in Table 1; other DNA chains used to construct viral polypeptide-protein subunit vaccines are shown in Table 2. All DNA primers were synthesized and purified by Sangon Biotech Co., Ltd.

[0067] B cell epitope peptides in the RBD region of the SARS-CoV-2 spike glycoprotein: S3, S4; CD4 T cell epitope peptide: ORF3a. The amino acid sequences of all peptides are shown in Table 3. They were all synthesized and purified by GenScript Biotech Co., Ltd.

[0068] Table 1

[0069]

[0070]

[0071] Table 2

[0072]

[0073] * represents phosphorothioate diester bond modification, which is used to improve the stability of the DNA chain.

[0074] Table 3

[0075]

[0076] 1. Preparation of framework nucleic acid structure

[0077] Each DNA strand designed in Table 1 was mixed in an equimolar ratio in 1×TAE (containing 10 mM magnesium chloride) buffer (final concentration 1 μM), heated to 95° C. for 10 minutes, and then cooled to 4° C. for annealing. The reaction product was identified by polyacrylamide gel electrophoresis.

[0078] Results Figure 2 b. The electrophoresis diagram shows that the tetrahedral framework nucleic acid structure (TH37) was successfully synthesized.

[0079] 2. Construction of viral polypeptide-protein subunit combination vaccine based on framework nucleic acid

[0080] In order to construct a novel coronavirus polypeptide vaccine based on the tetrahedral framework nucleic acid TH37, the thiol-modified DNA-SH was first coupled to the viral polypeptide.

[0081] 1) For the coupling of thiol-modified SH-DNA (CpG) with the SARS-CoV-2 B cell epitope peptide S3 or S4, SH-DNA (CpG) (100 μM) was mixed with maleimide Mal-modified S3 and S4 (5 mM) in PBS at a molar ratio of 1:3, shaken at room temperature overnight, and ultrafiltration was used to purify DNA-S3 and DNA-S4. Other experiments with different molar ratios were also set up according to the same method, including mixing SH-DNA and S3 at a molar ratio of 1:1 or 1:2; mixing SH-DNA and S4 at a molar ratio of 1:1, 1:5 and 1:10. The ultrafiltration purified product was subjected to electrophoresis. The electrophoresis results are shown in Figure 3 .Depend on Figure 3 It can be seen that when SH-DNA and S3 or S4 react at a molar ratio of 1:1, no single band can be formed, indicating that the coupling effect between the two is poor. When SH-DNA and S3 or S4 are mixed at a molar ratio of 1:2, 1:3, 1:5 and 1:10, good coupling can be achieved. From an economic point of view, SH-DNA and S3 or S4 are preferably mixed at a molar ratio of 1:2 to 1:5, and 1:3 is most preferred.

[0082] 2) For the coupling of thiol-modified ORF3a-linker-3 and the SARS-CoV-2 CD4 T cell epitope peptide ORF3a, ORF3a-linker-3 (100 μM) and maleimide Mal-modified ORF3a (5 mM) were mixed in PBS at a molar ratio of 1:10, shaken at room temperature overnight, and ultrafiltration was used to purify DNA-ORF3a.

[0083] 3) The recombinantly expressed N protein carrying the Halo-tag is covalently coupled to the DNA modified with the Halo ligand.

[0084] Plasmid construction:

[0085] The DNA sequence encoding the severe acute respiratory syndrome coronavirus 2 (2019-nCoV) nucleocapsid protein (YP_009724397.2 (335Gly / Ala)) (Met1-Ala419) was synthesized by Transheep (Shanghai, China). The N-terminal TPA signal peptide for secretion was first incorporated into the pcDNA3.1(+) vector. The N sequence, Halo-tag sequence (GenBank ADN27525.1) and 8×His-tag were then subcloned downstream of the TPA signal peptide according to standard molecular techniques.

[0086] Cell culture and transfection:

[0087] Expi293F cells (Thermo Fisher) were grown in OPM-293CD05 medium (OPM Biosciences) at 120 rpm, 8% CO2, 37°C. To produce N Halo, cells were adjusted to 2×106 / ml. Per ml culture, 1 μg of plasmid and 3 μg of polyethyleneimine (PEI, Polysciences) were mixed in 100 μl OPM-293CD05 medium (OPM Biosciences). Transfection ProFeed (OPM Biosciences) was added 20 hours after transfection, and cells were cultured for 3-6 days. Cells were removed by centrifugation at 1000×g for 3 minutes, and the culture medium was used for the following protein purification.

[0088] Purification of N Halo Protein:

[0089] The culture supernatant was injected through a Ni-NTA column (Smart Lifesciences) at 4°C. The His8-tagged protein was eluted with a stepwise elution of PBS supplemented with 20mM, 50mM and 250mM imidazole pH 8. The relevant imidazole fractions were combined and concentrated with a 30kD MWCO centrifugal device (MilliporeSigma). Proteins were separated using PBS as a running buffer on a Superdex 200Increase 16 / 600GL column (GE Healthcare). After rapid freezing in liquid nitrogen, the peak fractions were combined, concentrated and stored at -80°C.

[0090] Add 40mM Halo Tag Succinimidyl Ester(O 2 ) The dimethyl sulfoxide solution of Halo ligand and the amino-modified DNA-ligand were shaken at room temperature for 5 hours in a 1×PBS buffer system. After the coupling was successfully characterized by mass spectrometry, the prepared Halo ligand-coupled DNA was mixed with the N protein carrying Halo-tag in a 1×PBS buffer system at a molar ratio of 1:1 and incubated at 37°C overnight to obtain DNA-N pro.

[0091] The prepared DNA-N pro (N-DNA) and Halo ligand coupled DNA (DNA-ligand) were subjected to electrophoresis detection. The results are shown in Figure 4 .Depend on Figure 4 It can be seen that the band formed by the DNA coupled with the Halo ligand is far away from the spotting well, while the band of N-DNA is close to the spotting well, which indicates that the DNA-ligand is successfully coupled with the N protein.

[0092] 4) Assembly of CD4 T cell epitope peptide ORF3a on TH37

[0093] Tetrahedral framework nucleic acid TH37 and DNA-S3 were mixed at a molar ratio of 1:4, and hybridized and complementary paired at room temperature to obtain TH37-S3.

[0094] Tetrahedral framework nucleic acid TH37 and DNA-S4 were mixed at a molar ratio of 1:4, and hybridized and complementary paired at room temperature to obtain TH37-S4.

[0095] The tetrahedral framework nucleic acid TH37 was mixed with DNA-S3 and DNA-ORF3a at a molar ratio of 1:4:1, and hybridization and complementary pairing were performed at room temperature to obtain TH37-S3-ORF3a.

[0096] The tetrahedral framework nucleic acid TH37 was mixed with DNA-S4 and DNA-ORF3a at a molar ratio of 1:4:1, respectively, and hybridization and complementary pairing were performed at room temperature to obtain TH37-S4-ORF3a.

[0097] 5) Assembly of N protein on TH37

[0098] Tetrahedral framework nucleic acid TH37 was mixed with DNA-S3 and DNA-N pro at a molar ratio of 1:4:1, and hybridization and complementary pairing were obtained at room temperature to obtain TH37-S3-N pro.

[0099] The tetrahedral framework nucleic acid TH37 was mixed with DNA-S4 and DNA-N pro in a molar ratio of 1:4:1 to obtain TH37-S4-Npro.

[0100] Example 2

[0101] The ability of viral peptide-protein subunit vaccines based on framework nucleic acids to stimulate B cells

[0102] A C57 mouse was killed, the spleen was removed and placed in a cell culture dish, PBS was added and ground, the crushed spleen suspension was centrifuged at 1500rpm for 3 minutes to collect the precipitate, and then 2mL of red blood cell lysis buffer was added. After lysis for 3 minutes, an equal volume of PBS was added to terminate the lysis. After centrifugation (1500rpm, 3min), the spleen cells were filtered with gauze, resuspended in 2mL PBS and counted with a cell counter. Carboxyfluorescein diacetate succinimidyl ester (CFSE) was used for staining, and 1×10 7 Splenocytes were suspended in 5 mL PBS and 0.25 μL 10 mM CFSE in dimethyl sulfoxide was added. The cell suspension was then placed in a cell culture incubator at 37°C for 15 minutes. Afterwards, CFSE-stained splenocytes were collected by centrifugation (1500 rpm, 5 min) and resuspended in 1640 medium. For the B cell activation capacity assay, 5×10 5 CFSE-stained spleen cells were suspended in 200 μL 1640 medium and incubated with viral polypeptide-protein subunit vaccine samples based on framework nucleic acid in round-bottom 96-well plates (final concentration: framework nucleic acid 60 nM). After 3 days of culture, spleen cells were collected by centrifugation (1500 rpm, 3 min) and B cells (CD19 + ) proliferation, and the cell proliferation was analyzed by CFSE fluorescence dilution method.

[0103] Results Figure 5The results showed that the vaccine groups with B cell epitopes could effectively induce B cell proliferation, while free peptides and free CpG showed very limited ability to stimulate B cell proliferation. It is worth noting that the vaccines targeting C57 mouse T cell epitopes and universal N protein groups had stronger stimulation ability. This is attributed to CD4 + T cell activation, thereby effectively improving the immune effect of B cell epitopes.

[0104] Example 3

[0105] Determination of antibody titers in vivo after vaccination with viral polypeptide-protein subunit vaccines based on framework nucleic acid

[0106] For the immunization of mice with the viral peptide-protein subunit vaccine constructed by TH37, 30 female C57BL / 6 mice were randomly divided into 6 groups and immunized subcutaneously on the back of the mice on day 0 and day 14 using the following vaccination schemes:

[0107] Plan I: No vaccination;

[0108] Scheme II: TH37 (500 pmol) + free S3 and TH37 (500 pmol) + free S4, denoted as TH37 + free S3 / S4 group;

[0109] Scheme III: TH37-S3 (500 pmol) + TH37-S4 (500 pmol), denoted as TH37-S3 / S4 group;

[0110] Scheme IV: TH37-S3-ORF3a (500 pmol) + TH37-S4-ORF3a (500 pmol), denoted as TH37-S3 / S4-ORF3a group;

[0111] Scheme V: TH37-S3+free N pro (500 pmol) and TH37-S4+free N pro (500 pmol), denoted as TH37-S3 / S4+freeNpro group;

[0112] Scheme VI: TH37-S3-Npro (500 pmol) and TH37-S4-Npro (500 pmol), denoted as TH37-S3 / S4-Npro group.

[0113] For the determination of specific antibody titers in mouse serum after vaccination, peptides or RBD proteins of the SARS-CoV-2 S protein were coated on high-absorption 96-well Costar plates and incubated overnight at 4°C. After blocking with blocking buffer, serum samples at different dilutions on the 21st day were added to the plate for incubation. Subsequently, they were detected with goat anti-mouse IgG conjugated to horseradish peroxidase (HRP). Finally, TMB substrate was added for color development, and the absorbance at 450nm and 620nm was detected using a microplate reader. The antibody titer of each vaccine-immunized group was measured compared with that of the unvaccinated group, that is, if the absorbance value was greater than twice the average value of the absorbance of the unvaccinated group, the signal was considered to be detectable.

[0114] Results Figure 6 Compared with the control group, RBD-specific antibodies were detected in mice immunized with the framework nucleic acid-based viral subunit-peptide vaccine. In contrast, mice immunized with the free peptide produced only extremely limited specific antibodies. It is noteworthy that among all the framework nucleic acid-based viral subunit-peptide vaccines, the TH37-S3-N pro and TH37-S4-N pro groups showed the strongest induction of specific antibodies in vivo, with antibody titers as high as 10 4 The above results can be attributed to the effect of N protein on helper CD4 + Activation of T cells, thereby effectively promoting the activation and differentiation of B cells. Compared with the free N protein vaccine group, the framework nucleic acid can effectively deliver N protein to T cells, enhance antigen-specific B cell-T cell binding, and enable B cells to obtain more CD40L stimulation.

[0115] Example 4

[0116] Activation of live helper T cells after vaccination with the new coronavirus peptide vaccine

[0117] The following vaccination scheme was used to inoculate mice with framework nucleic acid-based new coronavirus subunit-peptide vaccines (TH37-S3 / S4-N pro, TH37-S3 / S4+free N pro), framework nucleic acid-based new coronavirus peptide vaccines (TH37-S3 / S4-ORF3a, TH37-S3 / S4) and TH37+free S3 / S4. The specific scheme is as follows:

[0118] Plan I: No vaccination;

[0119] Scheme II: TH37 (500 pmol) + free S3 and TH37 (500 pmol) + free S4, denoted as TH37 + free S3 / S4 group;

[0120] Scheme III: TH37-S3 (500 pmol) + TH37-S4 (500 pmol), denoted as TH37-S3 / S4 group;

[0121] Scheme IV: TH37-S3-ORF3a (500 pmol) + TH37-S4-ORF3a (500 pmol), denoted as TH37-S3 / S4-ORF3a group;

[0122] Scheme V: TH37-S3+free N pro (500 pmol) and TH37-S4+free N pro (500 pmol), denoted as TH37-S3 / S4+free N pro group;

[0123] Scheme VI: TH37-S3-N pro (500 pmol) and TH37-S4-N pro (500 pmol), denoted as TH37-S3 / S4-N pro group.

[0124] Determination of CD4 in mice + T cell activation level: mice were immunized on days 0 and 14, and peripheral blood was collected on day 21. The single cell suspension obtained after lysing red blood cells was detected by flow cytometry to detect activated CD4 + T cells (IL-4 + in CD3 + CD4 + ) ratio.

[0125] Results Figure 7 From the flow cytometry results and the corresponding statistical results, it can be seen that after TH37-S3 / S4-N pro treatment, the CD4 + T cells were activated to the greatest extent, which was consistent with the above results on B cell proliferation.

[0126] Example 5

[0127] Activation of live killer T cells after vaccination with the new coronavirus peptide vaccine

[0128] CD8 + The activation level of T cells was determined by immunizing mice on days 0 and 14, and collecting peripheral blood on day 21. The single cell suspension obtained after lysing red blood cells was detected by flow cytometry. + T cells (IFN-γ + in CD3 + CD8 + ) ratio.

[0129] Results Figure 8We were pleasantly surprised to find that, compared with free N protein, N protein integrated on tetrahedrons could induce stronger CD8-positive T cell activation, which was probably because tetrahedrons promoted antigen cross-presentation.

[0130] Example 6

[0131] Antibody virus neutralization assay

[0132] HEK-293T cells transfected with the SARS-CoV-2 S protein RBD region binding receptor ACE2 (HEK-293T-ACE2) used in the antibody virus neutralization experiment were cultured in DMEM high-glucose medium containing 10% FBS and 1% double antibody at 37°C and 5% CO 2 The cells were cultured under standard conditions. The expression of GFP in pseudovirus-SARS-COV-2 was observed and analyzed by flow cytometry. The specific protocol is as follows: 5×10 4 HEK-293T-ACE2 cells were inoculated in a 24-well plate, and then 1 μL of the new crown pseudovirus pseudovirus-SARS-CoV-2 and the serum sample on the 21st day of immunization in Example 3 were incubated at 37°C for 1 hour and added to the well plate to incubate with the cells. After 12 hours, the sample was washed away, and fresh DMEM high-glucose medium was added to continue culturing for 24 hours. Finally, the supernatant was centrifuged and discarded, and the cells were resuspended in DMEM high-glucose medium, and the green fluorescence expression of GFP was observed under flow cytometry.

[0133] Results Fig. 9 . It is worth noting that the antibodies in the serum of mice introduced with the N protein vaccine group showed the most effective ability to neutralize the virus. The above results collectively prove that the constructed TH37-based new coronavirus subunit-peptide vaccine can effectively activate B cells, thereby inducing B cell-mediated humoral immune responses, promoting the production of specific neutralizing antibodies, and protecting mice from viral infection.

[0134] Example 7

[0135] Evaluation of immune memory effect

[0136] The immune memory effect in mice was detected on the 60th day after the first immunization. The mice were killed and the spleen was taken and processed as above to obtain a single cell suspension. After flow cytometry antibody staining, memory T cells (CD3 + CD4 + CD44highCD62Lhigh) and memory B cells (CD19 + CD27 + ) level.

[0137] Results Figure 10-11Memory B cells and memory T cells were produced in mice immunized with the vaccine, and the proportion of memory cells in mice immunized with CD4 T epitope peptides and universal N protein was further increased.

[0138] Example 8

[0139] Comparison of advantages of viral polypeptide-protein subunit vaccines constructed with framework nucleic acids

[0140] In order to demonstrate the advantages of the constructed framework nucleic acid virus polypeptide-protein subunit vaccine, Balb / c mice were immunized. The experimental animals were randomly divided into 5 groups and immunized subcutaneously on the back on days 0 and 14 with (I) no vaccination, (II) TH37+free S3 (500 pmol TH37) and TH37+free S4 (500 pmol), (III) TH37-S3 (500 pmol) and TH37-S4 (500 pmol), (IV) TH37-S3-ORF3a (500 pmol) and TH37-S4-ORF3a (500 pmol), (V) TH37-S3-Npro (500 pmol) and TH37-S4-N pro (500 pmol), respectively.

[0141] Rats and rabbits were immunized with TH37-S3-ORF3a&TH37-S4-ORF3a, and TH37-S3-N pro&TH37-S4-N pro, respectively. The experimental animals were randomly divided into 3 groups and immunized subcutaneously on the back on days 0 and 14 with (I) no vaccination, (II) TH37-S3-ORF3a&TH37-S4-ORF3a, and (III) TH37-S3-N pro&TH37-S4-N pro, respectively.

[0142] The serum RBD-specific antibody titer after vaccination was determined according to the method described in Example 3. ORF3a is specific for CD4 + However, due to individual differences in major histocompatibility complexes, ORF3a may not be an effective T cell epitope for inducing antigen-specific T lymphocyte activation in Balb / c mice, rats, and rabbits.

[0143] Results Figure 12 to Figure 14The results showed that TH37-S3-ORF3a & TH37-S4-ORF3a had little effect on further promoting the production of RBD-specific antibodies in mice, rats and rabbits, while coupling SARS-Cvo-2N protein to tetrahedral framework nucleic acids could induce higher antibody titers in multiple animal models, including C57, Babl / c, rats and rabbits.

[0144] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A viral polypeptide-protein subunit combination vaccine based on DNA nanotechnology, characterized in that: A tetrahedral framework nucleic acid formed by assembling DNA chains is used as a vector, a protein antigen causing virus-specific T cell activation is coupled to one edge of the tetrahedral framework nucleic acid, and a viral B cell epitope peptide is coupled to the four vertices of the tetrahedral framework nucleic acid; The nucleotide sequence of the DNA chain for tetrahedral framework nucleic acid assembly is shown in SEQ ID NO: 1 to SEQ ID NO:

8.

2. The viral polypeptide-protein subunit combination vaccine based on DNA nanotechnology according to claim 1, characterized in that: The B cell epitope peptide of the virus is hybridized with the single-stranded DNA extending from the vertices of the tetrahedral framework nucleic acid through the thiol-modified DNA chain; The nucleotide sequence of the thiol-modified DNA chain is shown in SEQ ID NO:

9.

3. The viral polypeptide-protein subunit combination vaccine based on DNA nanotechnology according to claim 1, characterized in that: The protein antigen that causes virus-specific T cell activation is hybridized with the single-stranded DNA extending from the edge of the tetrahedral framework nucleic acid through the coupled DNA chain; The nucleotide sequence of the coupled DNA chain is shown in SEQ ID NO:

10.

4. The viral polypeptide-protein subunit combination vaccine based on DNA nanotechnology according to claim 3, characterized in that: The protein antigen and DNA chain that cause virus-specific T cell activation are coupled through Halo tag and Halo ligand.

5. The viral polypeptide-protein subunit combination vaccine based on DNA nanotechnology according to claim 1, characterized in that: The virus comprises at least one of the following viruses: coronavirus, human immunodeficiency virus, respiratory syncytial virus and influenza virus.

6. The viral polypeptide-protein subunit combination vaccine based on DNA nanotechnology according to claim 5, characterized in that: The coronavirus includes the new coronavirus and its variants; The protein antigens in the novel coronavirus or its variants that cause virus-specific T cell activation include N protein; B cell epitope peptides of the new coronavirus or its variants include S3 404-412 and / or S4 440-445 .

7. The method for preparing a viral polypeptide-protein subunit combination vaccine based on DNA nanotechnology according to any one of claims 1 to 6, characterized in that: The following steps are involved: Performing a first hybridization reaction on a DNA chain for assembling a tetrahedral framework nucleic acid to obtain a tetrahedral framework nucleic acid; The thiol-modified DNA chain and the maleimide-modified viral B cell epitope peptide are mixed for coupling reaction to obtain the DNA-coupled B cell epitope peptide; Covalently couple the protein antigen modified by Halo tag that causes virus-specific T cell activation and the DNA chain modified by Halo ligand to obtain the protein antigen coupled to DNA; The tetrahedral framework nucleic acid, DNA-coupled B cell epitope peptide and DNA-coupled protein antigen are subjected to a second hybridization reaction to obtain a virus polypeptide-protein subunit combination vaccine.

8. The preparation method according to claim 7, characterized in that: In the first hybridization reaction, the DNA chains for tetrahedral framework nucleic acid assembly are mixed in an equal molar ratio, treated at 95° C. for 10 min, and kept at 4° C. for 30 min; the final concentration of each DNA chain for tetrahedral framework nucleic acid assembly is independently 0.8 to 1.2 μM; In the coupling reaction, the molar ratio of the thiol-modified DNA chain to the maleimide-modified viral B cell epitope peptide is 1:(2-10); In the covalent coupling, the molar ratio of the protein antigen modified by Halo tag that causes virus-specific T cell activation and the DNA chain modified by Haloligand is (0.9-1.1): (0.9-1.1); In the second hybridization reaction, the molar ratio of the tetrahedral framework nucleic acid, the DNA-coupled B cell epitope peptide and the DNA-coupled protein antigen is (0.9-1.1): (3.9-4.1): (0.9-1.1).

9. Use of the viral polypeptide-protein subunit combination vaccine based on DNA nanotechnology according to any one of claims 1 to 6 or the viral polypeptide-protein subunit combination vaccine obtained by the preparation method according to claim 7 or 8 in preparing an immune animal model.

10. The use according to claim 9, characterized in that: The immune animal model includes at least one of: rats, mice and rabbits.

Images