Multi-epitope peptide of novel coronavirus, vaccine as well as preparation method and application of multi-epitope peptide and vaccine

Abstract

The invention discloses a multi-epitope peptide of novel coronavirus, a vaccine as well as a preparation method and application of the multi-epitope peptide, and belongs to the technical field of preparation of polypeptide vaccines. The multi-epitope peptide of the novel coronavirus not only contains T cell epitopes, but also contains B cell epitopes, and can induce humoral immunity and cellular immunity responses of an organism at the same time; the vaccine comprises epitopes from four antigenic proteins of novel coronavirus, and can induce comprehensive immune response. According to the multi-epitope peptide vaccine based on the novel coronavirus, the nano-carrier KFE8 serves as a delivery carrier, different antigen epitopes of the same pathogen or antigen epitopes of different pathogens can be presented in a mixed mode, safety is good, large-scale rapid preparation is achieved in an emergency state, and the vaccine is suitable for large-scale popularization and application. The coronavirus vaccine is a novel coronavirus universal vaccine which can cover various pathogen subtypes and induce broad-spectrum and long-term immune effects.

Description

Technical Field

[0001] The present invention belongs to the technical field of polypeptide vaccine preparation, and specifically relates to multi-epitope peptides, vaccines, and preparation methods and applications of novel coronaviruses. Background Art

[0002] The 2019 coronavirus (COVID-19) genome encodes four structural proteins: spike protein (S protein), envelope protein (E protein), membrane glycoprotein (M protein), and nucleocapsid protein (N protein). The S protein is a large, trimeric transmembrane glycoprotein that forms a distinctive corolla structure on the viral surface. Based on its structure and function, the S protein can be divided into two functional units: S1 and S2. S1 contains the receptor binding domain (RBD), responsible for recognizing and binding to cellular receptors; S2 contains essential components required for membrane fusion, anchoring the S protein to the viral membrane and mediating fusion between the viral and host cell membranes. Therefore, the S protein is the primary protein that mediates viral entry into host cells and is the primary target for neutralizing antibodies produced by the host immune system after infection. The M and E proteins are primarily involved in viral assembly and assembly. The M protein maintains the intact viral form and is linked to the N protein. The E protein has functions related to viral aggregation and release. The N protein plays a role in protecting the genome. Both the N-terminus and the C-terminus of the N protein can bind to the viral RNA. The N protein is mainly responsible for the replication function of RNA. It is often used as a diagnostic detection tool for coronavirus and is the core raw material for rapid immunological diagnostic reagents.

[0003] The novel coronavirus (SARS-CoV-2) is the pathogen that causes COVID-19. It belongs to the β-coronavirus genus and is highly contagious and variable. Under external conditions and evolutionary pressure, SARS-CoV-2 can evade the host immune system through rapid and high-frequency mutations. In this regard, vaccination has played a key role in the control of the global Sars-CoV-2 pandemic. However, due to the continuous evolution of the virus, viruses with stronger transmission capabilities will emerge, thereby escaping the immunity of vaccines against early epidemic strains. Therefore, it is necessary to design a universal novel coronavirus vaccine that can cover multiple pathogen subtypes and induce broad-spectrum and long-term immune effects.

[0004] The immune effect of the vaccine depends on the immunogenicity of the antigenic epitope. The antigenic epitope or antigenic determinant is the smallest immunogenic part of a specific antigen that can induce a specific immune response. Epitope vaccines are made by genetic engineering to express or artificially synthesize pathogen antigenic epitopes in vitro. Epitope vaccines have shown good results in immune experiments infected with various pathogens. The survival time of animals in the vaccine group was significantly prolonged and the cellular and humoral immune responses were significantly enhanced. Therefore, by predicting, screening and optimizing the antigenic epitopes of multiple subtypes of the new coronavirus pathogen, the epitopes with the greatest immune protection potential and relatively conservative are selected to construct a universal multi-epitope vaccine, which can induce a comprehensive immune response in the body and effectively improve the immune effect. Summary of the Invention

[0005] In order to overcome the shortcomings of the above-mentioned prior art, the purpose of the present invention is to provide a multi-epitope peptide, vaccine and preparation method and application of the new coronavirus, so as to solve the technical problem that existing vaccines cannot cover multiple pathogen subtypes and induce broad-spectrum and long-term immune effects.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] The first aspect of the present invention discloses a multi-epitope peptide of a novel coronavirus, wherein the multi-epitope peptide is obtained by linking a novel coronavirus vaccine epitope with a nano-delivery vector;

[0008] Among them, the new coronavirus vaccine epitope is one or more combinations of amino acid sequences shown in SEQ ID NO.1 to SEQ ID NO.3, and SEQ ID NO.5 to SEQ ID NO.12.

[0009] Preferably, the multi-epitope peptide is obtained by connecting the novel coronavirus vaccine epitope and the nano delivery vector through a flexible linker, and the amino acid sequence of the flexible linker is SGSG.

[0010] Preferably, the nano delivery vector is KFE8, and the amino acid sequence is shown in SEQ ID NO.13.

[0011] Preferably, the amino acid sequence of the multi-epitope peptide is shown as SEQ ID NO.16 to SEQ ID NO.26.

[0012] The second aspect of the present invention discloses a multi-epitope vaccine for the new coronavirus, which includes multi-epitope peptides of the above-mentioned new coronavirus.

[0013] Preferably, a nano delivery carrier is also included.

[0014] Further preferably, the nano delivery vector is KFE8, and the amino acid sequence is shown in SEQ ID NO.13.

[0015] Preferably, the molar concentration ratio of the multi-epitope peptide of the new coronavirus and the nano delivery vector is 1:0.95.

[0016] Preferably, an adjuvant is also included.

[0017] The third aspect of the present invention discloses a method for preparing a multi-epitope vaccine for the new coronavirus, comprising vortexing a multi-epitope peptide of the new coronavirus according to any one of claims 1 to 3 and a single nano-delivery vector with an aqueous solution of a PADRE-nano-delivery vector to form a fiber-like strip structure, thereby obtaining a multi-epitope vaccine for the new coronavirus;

[0018] Among them, the PADRE-nano delivery vector is formed by connecting PADRE and a nano delivery vector, and the amino acid sequence of PADRE is shown in SEQ ID NO.14.

[0019] Preferably, the molar concentration ratio of the multi-epitope peptide of the new coronavirus, the PADRE-nano delivery vector and the single nano delivery vector is 1:0.05:0.95.

[0020] The fourth aspect of the present invention discloses an anti-new coronavirus pharmaceutical composition, including the above-mentioned new coronavirus multi-epitope vaccine.

[0021] Preferably, the novel coronavirus multi-epitope vaccine KP1 and the novel coronavirus multi-epitope vaccine KP3 are included, wherein the novel coronavirus multi-epitope vaccine KP1 is assembled from the multi-epitope peptide KP1, the PADRE-nano delivery vector and a single nano delivery vector, and the novel coronavirus multi-epitope vaccine KP3 is assembled from the multi-epitope peptide KP3, the PADRE-nano delivery vector and a single nano delivery vector;

[0022] Among them, the multi-epitope peptide KP1 is obtained by connecting the new coronavirus vaccine epitope P1 with a nano-delivery vector, and the amino acid sequence of the new coronavirus vaccine epitope P1 is shown in SEQ ID NO.1; the multi-epitope peptide KP3 is obtained by connecting the new coronavirus vaccine epitope P3 with a nano-delivery vector, and the amino acid sequence of the new coronavirus vaccine epitope P3 is shown in SEQ ID NO.3; the PADRE-nano-delivery vector is formed by connecting PADRE with a nano-delivery vector, and the amino acid sequence of PADRE is shown in SEQ ID NO.14.

[0023] Preferably, it further comprises any one or a combination of at least two of a pharmaceutically acceptable carrier, excipient or diluent.

[0024] The fifth aspect of the present invention discloses the use of the multi-epitope peptides of the above-mentioned new coronavirus, the multi-epitope vaccine of the new coronavirus or the anti-new coronavirus pharmaceutical composition in the preparation of preventive drugs and / or therapeutic drugs for the new coronavirus.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] The multi-epitope peptides of the novel coronavirus provided by the present invention are obtained by connecting novel coronavirus vaccine epitopes with nano-delivery vectors. 1) The novel coronavirus vaccine epitopes contain both T-cell epitopes and B-cell epitopes, which can simultaneously induce the body's humoral and cellular immune responses; they contain epitopes from four antigenic proteins of the novel coronavirus, which can be made into vaccines to induce a comprehensive immune response; 2) by connecting to the nano-delivery vector, they help form nanofibers, protecting the antigens from premature degradation. The multi-epitope peptides all exhibited nanofiber-like structures under electron microscopy, with an assembly efficiency of over 95%, and have the potential to be made into vaccines to prevent and / or treat novel coronavirus-related diseases.

[0027] Furthermore, using the nanocarrier KFE8 as a delivery vehicle can granulate the antigens and present them at high frequency, thereby enhancing the immune response of the vaccine and presenting different antigenic epitopes of the same pathogen or mixed antigenic epitopes of different pathogens.

[0028] The novel coronavirus multi-epitope vaccine provided by the present invention, the multi-epitope peptide can simultaneously induce the body's humoral immunity and cellular immune response, contains epitopes from four antigenic proteins of the novel coronavirus, and can induce a comprehensive immune response. The immune effect of the novel coronavirus multi-epitope vaccine was detected by animal experiments. The results showed that the novel coronavirus multi-epitope vaccine prepared by this method 1) has immunogenicity, can efficiently induce the production of specific antibodies, has strong T cell immune efficacy, and the virus blocking effect is better than the Zhikeweide positive control as a whole. 2) It is highly safe and no adverse reactions were found. Therefore, the novel coronavirus multi-epitope vaccine is a universal vaccine for the novel coronavirus that can cover multiple pathogen subtypes and induce broad-spectrum and long-term immune effects. In addition, the novel coronavirus multi-epitope vaccine can be expressed or synthesized in advance. In an emergency, it is only necessary to express or synthesize the tag part with the antigenic epitope, and the vaccine can be made by mixed assembly, which can achieve large-scale rapid preparation and has rapid response capabilities. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a simulated diagram of the distribution of P1, P2, P3, P5, and P6 in the S protein structure of the present invention;

[0030] Figure 2 This is a simulated diagram of the positional distribution of P7 and P8 in the M protein structure of the present invention;

[0031] Figure 3 This is a simulated diagram of the distribution of P10, P11, and P12 in the N protein structure of the present invention;

[0032] Figure 4 This is a diagram showing the morphological test results of the novel coronavirus multi-epitope vaccine of the present invention;

[0033] Figure 5 This is a graph showing the specific antibody detection in mice immunized with the novel coronavirus multi-epitope vaccine of the present invention;

[0034] Figure 6 This is a graph showing the detection of neutralizing antibodies in mice immunized with the novel coronavirus multi-epitope vaccine of the present invention;

[0035] Figure 7 This is a graph showing the T cell activity detection of mice immunized with the novel coronavirus multi-epitope vaccine of the present invention; wherein A represents data statistics and B represents the reaction results;

[0036] Figure 8 This is a monitoring chart of the organ conditions of mice immunized with the new coronavirus multi-epitope vaccine of the present invention. DETAILED DESCRIPTION

[0037] To facilitate understanding of the features and effects of the present invention by those skilled in the art, the following provides a general description and definition of the terms and expressions used in the specification and claims. Unless otherwise indicated, all technical and scientific terms used herein have the ordinary meanings as understood by those skilled in the art regarding the present invention. In the event of conflict, the definitions in this specification shall prevail.

[0038] The theories or mechanisms described and disclosed herein, whether correct or incorrect, should not limit the scope of the present invention in any way, that is, the present invention can be implemented without being limited by any specific theory or mechanism.

[0039] Herein, all features such as values, amounts, amounts, and concentrations defined in numerical ranges or percentage ranges are for brevity and convenience only. Accordingly, the description of numerical ranges or percentage ranges should be considered to include and specifically disclose all possible subranges and individual values ​​within the range (including integers and fractions).

[0040] In this document, unless otherwise specified, “include,” “including,” “contains,” “has” or similar terms cover the meanings of “consisting of” and “mainly consisting of,” for example, “A includes a” covers the meanings of “A includes a and other” and “A only includes a.”

[0041] In this document, for the sake of brevity, not all possible combinations of the various technical features in each embodiment or example are described. Therefore, as long as there are no contradictions in the combination of these technical features, the various technical features in each embodiment or example can be combined in any way, and all possible combinations should be considered to be within the scope of this specification.

[0042] Herein, the novel coronavirus multi-epitope vaccine includes but is not limited to connection, preparation and expression with KFE8, Q11 (QQKFQFQFEQQ) or other vectors or fusion proteins.

[0043] Herein, the flexible linker includes but is not limited to SGSG, and may also be GGGGS or EAAAK.

[0044] In this article, the "PADRE" mentioned refers to the pan-DR helper T cell epitope (Pan HLA-DR-binding Epitope; PADRE), an artificially designed helper T cell (Th cell) epitope, the amino acid sequence of which is shown in SEQ ID NO.14 in Table 2, and is mainly used to enhance the immune response of vaccines or immunotherapy.

[0045] Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiments are only used to illustrate the present invention and are not used in limiting the scope of the present invention.In addition, should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall equally within the scope limited by the appended claims of the application.

[0046] Conventional instruments and equipment in this field are used in the following examples. The experimental methods for which specific conditions are not specified in the following examples are generally carried out under conventional conditions or under the conditions recommended by the manufacturer. In the following examples, PADRE-KFE8 and KFE8 used were synthesized by Chengdu Shengnuo Biopharmaceutical Co., Ltd. The BCA kit (BCA ProteinAssay Kit, 381475) used was purchased from Thermo Scientific. The Zhikeweide vaccine used was purchased from Anhui Zhifei Longcoma Biopharmaceutical Co., Ltd. The S protein (TMPY-06615), N protein (TMPY-06626), hACE2-Fc recombinant protein (TMPK-00383) and recombinant new crown S protein (TMPY-06615) used were all purchased from TargetMol. The pseudoviruses OmicronBQ.1.1, Omicron EG.5.1, Omicron JN.1 and Omicron XBB.1.5 used were all provided by the China National Institute for the Control of Biological Products. All other raw materials used, unless otherwise specified, are conventional commercially available products, and their specifications are conventional in the field.

[0047] Example 1 Prediction and Screening of Novel Coronavirus Vaccine Epitopes

[0048] 1. Sequence download and selection

[0049] All COVID-19 protein sequences from April 2023 were downloaded from the National Center for Biotechnology Information (NCBI) database. BLAST (Basic Local Alignment Search Tool) analysis revealed a sequence similarity of over 98%. For the four core proteins (S, E, M, and N proteins) encoded by the five prevalent COVID-19 strains, including BA5.2, BF7, BQ1.1, CH1.1, and XBB1.5, the most conserved sequences were selected and clustered to identify the parent sequence.

[0050] 2. T cell and B cell epitope prediction

[0051] To obtain T cell epitopes with high population coverage, T cell epitope prediction is primarily performed within the HLA class, screening for epitope sequences with strong binding to HLA class I (10 types) and HLA-DR (6 types) (covering over 91% of the global population), and further screening for overlapping epitope sequences between the two classes (detailed information is as follows):

[0052] HLA class I (HLA-A*01:01, -A*02:01, -A*03:01, -A*11:01, -A*24:02, -B*07:02, -B*08:01, -B*15:01, -B*40:01 and -C*07:02) covers 91.7% of the world's population.

[0053] HLA-DR (HLA-DRB1*01:01, -DRB1*03:01, -DRB1*04:01, -DRB1*07:01, -DRB1*11:01 and -DRB1*15:01) can cover 70.6% of the world's population.

[0054] The BepiPred algorithm was used to predict B cell epitopes, identifying two B cell epitopes located on the S protein. Overlapping T cell and B cell epitopes were also screened to identify candidate epitopes that could induce both cellular and humoral immunity. As shown in Table 1, a total of 12 novel coronavirus vaccine epitopes were synthesized for subsequent experimental validation.

[0055] Table 1 Epitope sequences of novel coronavirus vaccines

[0056]

[0057]

[0058] 3. Simulation of the position distribution of the screened vaccine epitopes in the protein structure

[0059] Downloaded from the Protein Structure Database (RCSB PDB) data, S protein (PDB ID: 6VYB), M protein (PDB ID: 8CTK), N protein (PDB ID: 8FD5), and E protein do not have crystal structures.

[0060] The 12 vaccine epitopes screened in step 2 were analyzed and located in the protein structure using Molecular Operating Environment (MOE) software ( Figures 1 to 3 It was observed that, with the exception of P4, all other epitopes were located on the surface of the protein structure, making them easily captured and recognized by antigen-presenting cells, indicating good antigenic potential. Therefore, subsequent experiments were conducted using vaccine epitopes P1-P3 and P5-P12.

[0061] Example 2 Preparation of a novel coronavirus multi-epitope vaccine

[0062] 1. Preparation of multi-epitope peptide KP based on nanocarrier KFE8

[0063] The nanocarrier KFE8 (sequence shown in SEQ ID NO.13 in Table 2) is composed of alternating positively charged lysine (K) and negatively charged glutamic acid (E), with a hydrophobic phenylalanine (F) inserted in the middle. Acetylation modification (Ac) enhances molecular stability and regulates assembly dynamics. It has the ability to form nanofibers and good stability, which can protect antigens from premature degradation.

[0064] As shown in Tables 2 and 3, using nanocarrier KFE8 as the delivery vector, the vaccine epitopes P1 to P3 and P5 to P12 screened in Example 1 were connected in series to one end of the nanocarrier KFE8 through the flexible linker SGSG, respectively, to obtain multi-epitope peptides KP1 to KP12, respectively.

[0065] Table 2 Amino acid sequence table

[0066] name sequence Serial number Nanocarrier KFE8 <![CDATA[Ac-FKFEFKFE-NH2]]> SEQ ID NO.13 PADRE <![CDATA[Ac-AKFVAAWTLKAAA-NH2]]> SEQ ID NO.14 PADRE-KFE8 <![CDATA[Ac-AKFVAAWTLKAAASGSGFKFEFKFE-NH2]]> SEQ ID NO.15

[0067] Table 3 Sequence list of multi-epitope peptide KP based on nanocarrier KFE8

[0068]

[0069]

[0070] Note: In the sequence listing, Ac represents acetylation, NH2 represents amidation, and SGSG represents a flexible linker.

[0071] 2. Preparation of a novel coronavirus multi-epitope vaccine

[0072] 1) PADRE-KFE8 was dissolved in sterile water to obtain a 0.2 mM PADRE-KFE8 aqueous solution; 4 mM multi-epitope peptide KP (KP1-KP12) and 3.8 mM KFE8 were then vortexed with the PADRE-KFE8 aqueous solution for 30 minutes to obtain a mixed polypeptide solution with a total concentration of 8 mM; the solution was incubated at 4°C overnight to promote fibril formation;

[0073] 2) Dilute the mixed polypeptide solution obtained in step 1) 4-fold with sterile water and 10× phosphate buffered saline (PBS) so that the final concentration of the polypeptides (multi-epitope peptides KP, KFE8, and PADRE-KFE8) in the mixed polypeptide solution is 2 mM, and dissolve in a 1× PBS buffer system and incubate at room temperature for at least 3 h;

[0074] 3) Take 2 mM of the sample solution after incubation in step 2) for negative staining (dilute with 1× PBS if necessary), and then observe it under a transmission electron microscope (TEM) at a field of view of 200 nm. The sample is clearly observed to have a fiber-like strip structure, thereby obtaining a new coronavirus multi-epitope vaccine.

[0075] TEM observation results are as follows Figure 4 As shown, each group of samples showed a fiber-like structure under TEM, indicating that the multi-epitope peptide KP, nanocarrier KFE8 and PADRE-KFE8 were successfully assembled to form a nanofiber-like structure.

[0076] 3. Assembly efficiency test of novel coronavirus multi-epitope vaccine

[0077] Use the BCA kit to detect the protein concentration of the novel coronavirus multi-epitope vaccine sample obtained in step 2 to detect its assembly efficiency. The experimental steps refer to the BCA kit instructions. The specific steps are as follows:

[0078] 1) Add Reagent A and Reagent B (50:1) to each well of a 96-well plate, then add 200 μL of BCA working solution (prepared immediately before use) and mix thoroughly.

[0079] 2) Standard Curve: Prepare a gradient of protein standards (2, 1, 0.5, 0.25, 0.125, 0 mg / mL). Take 100 μL of the 2019-nCoV multi-epitope vaccine sample obtained in step 2 and centrifuge to obtain the supernatant of the test sample. Add 10 μL of the standard (6 concentration gradients) and the supernatant of the test sample to each well of the 96-well plate containing Reagent A, Reagent B, and BCA working solution in step 1) and gently mix.

[0080] 3) Incubation: Incubate at 37°C for 30 min. Measure the absorbance at a wavelength of 562 nm using a microplate reader.

[0081] 4) According to the formula obtained from the standard curve, the protein content in the supernatant of the test sample was calculated to obtain the assembly efficiency of the new coronavirus multi-epitope vaccine, as shown in Table 4.

[0082] Table 4 Assembly efficiency of novel coronavirus multi-epitope vaccine

[0083] KP1 KP2 KP3 KP5 KP6 KP7 KP8 KP9 KP10 KP11 KP12 Assembly efficiency 99.9％ 96.8％ 95.9％ 96.8％ 99.5％ 95.4％ 100％ 95.2％ 100％ 96.4％ 100％

[0084] As can be seen from Table 4, the assembly efficiency of the new coronavirus multi-epitope vaccine obtained by this method is above 95%, indicating that efficient assembly occurs between the multi-epitope peptide KP, nanocarrier KFE8 and PADRE-KFE8.

[0085] Example 3 Preliminary Pharmacodynamic Evaluation of the Novel Coronavirus Multi-epitope Vaccine (Animal Experimental Immune Effect Detection)

[0086] 1. Immunity

[0087] As shown in Table 5, 18-22 g female BALB / c mice were divided into 13 groups of 5 mice each. Each group was immunized three times, with the corresponding dose of vaccine (as shown in Tables 6-16) injected subcutaneously at multiple sites on the back. Seven days after the fourth immunization, eye blood was collected. The blood was incubated at 4°C for several hours and centrifuged at 12,000 g for 10 minutes. The supernatant, representing the mouse antiserum, was stored at -20°C.

[0088] Table 5 Immunization groups

[0089]

[0090]

[0091] Table 6 Novel Coronavirus Multi-epitope Vaccine KP1

[0092]

[0093] Table 7 Novel Coronavirus Multi-epitope Vaccine KP2

[0094]

[0095] Table 8 Novel Coronavirus Multi-epitope Vaccine KP3

[0096]

[0097] Table 9 Novel Coronavirus Multi-epitope Vaccine KP5

[0098]

[0099]

[0100] Table 10 Novel Coronavirus Multi-epitope Vaccine KP6

[0101]

[0102] Table 11 Novel Coronavirus Multi-epitope Vaccine KP7

[0103]

[0104] Table 12 Novel Coronavirus Multi-epitope Vaccine KP8

[0105]

[0106] Table 13 Novel Coronavirus Multi-epitope Vaccine KP9

[0107]

[0108] Table 14 Novel Coronavirus Multi-epitope Vaccine KP10

[0109]

[0110] Table 15 Novel Coronavirus Multi-epitope Vaccine KP11

[0111]

[0112]

[0113] Table 16 Novel Coronavirus Multi-epitope Vaccine KP12

[0114]

[0115] 2. Detection of specific antibodies by enzyme-linked immunosorbent assay (ELISA)

[0116] 1) Coating: Take the required ELISA plate strips, dilute the S protein and N protein to 100 ng / 100 μL / well with coating solution, add to the ELISA plate wells, place in a humidified chamber, and refrigerator at 4°C overnight.

[0117] 2) Blocking: On the next day, remove the ELISA plate, discard the liquid, and wash it three times with washing solution. After the last wash, pat it dry, add 1% blocking solution (100 μL / well), and incubate at 37°C for 2 h.

[0118] 3) Add antiserum: After incubation, discard the solution and wash three times with washing solution. Dilute the serum to be tested with antibody diluent (1:100, 1:1000, 1:10000), 100 μL / well, and incubate at 37°C for 1 hour.

[0119] 4) Secondary Antibody: After incubation, discard the solution, wash three times with washing solution, add 1:6000 diluted HRP-labeled anti-mouse secondary antibody, 100 μL / well, and incubate at 37°C for 1 hour.

[0120] 5) Color development: After incubation, discard the solution and wash the plate five times with washing solution. After the final wash, pat dry. Add 100 μL / well of color development solution and develop the color for ~30 min. Then, add 50 μL / well of stop solution to stop the color development.

[0121] 6) Detect the absorbance at 490 nm using an enzyme-labeled instrument.

[0122] Test results such as Figure 5 As shown in the figure, compared with the negative control, all vaccine groups induced specific antibodies, indicating that they are immunogenic. Among them, the antibody titers of vaccine groups KP1, KP5, KP8, KP10 and KP11 were significantly higher than those of the positive control group, indicating that they can effectively induce the production of specific antibodies.

[0123] 3. Detection of neutralizing antibody titer by further ACE2 competitive binding ELISA

[0124] 1) Add 200 ng / 100 μL / well of hACE2-Fc recombinant protein to coat the ELISA plate at 4°C overnight;

[0125] 2) The next day, the cells were blocked with 3% bovine serum albumin (BSA), 100 μL / well, at room temperature for 2 h.

[0126] 3) Prepare serial dilutions (1:100, 1:1000, 1:10000) of immune mouse serum (duplicate wells) and pre-incubate with recombinant SARS-CoV-2 S protein 100 ng / 100 μL / well at 37°C for 1 hour;

[0127] 4) After blocking, PBST (Phosphate-Buffered Saline with ) Wash the ELISA plate thoroughly, add 100 μL / well of the first-step incubation solution to the ELISA plate, and incubate at 37°C for 1 h;

[0128] 5) After incubation, wash with PBST and add 100 μL / well of 1:500 diluted rabbit anti-COVID-19 S1-RBD polyclonal antibody (Ab) and incubate for 1 hour;

[0129] 6) After incubation, wash with PBST; add enzyme-labeled secondary antibody diluted 1:5000, incubate at room temperature for 1 hour, and wash with PBST;

[0130] 7) Add 100 μL of color development solution to each well and develop the color for 30 min. Then, add 50 μL of stop solution to terminate the color development reaction. Measure the absorbance of each well at 490 nm using a microplate reader.

[0131] Test results such as Figure 6 As shown, the ACE2 competitive ELISA results showed that the antibody titer of the multi-epitope combination vaccine KP1+KP3 was higher, indicating that it induced neutralizing antibodies.

[0132] 4. T cell immune response effect detection (ELISPOT)

[0133] 1) Isolation of mouse spleen lymphocytes

[0134] Mice were killed by cervical dislocation, immersed in 75% ethanol (~5min), dissected in a clean bench, and the spleen was removed and placed in a 6cm culture dish with PBS; a 70μm cell mesh was placed in a 50mL centrifuge tube, first rinsed with 2mL of PBS, and the spleen was placed in the mesh for grinding. The mesh was rinsed with PBS to obtain a single cell suspension, and centrifuged at 1500r / 5min. After the centrifugation, the liquid was discarded, and ~2mL of red blood cell lysis buffer was added to lyse on ice for 5min, then PBS was added to 10mL to stop lysis, and centrifuged at 1500r / 5min. After the centrifugation, the liquid was discarded, and 10mL of PBS was added to wash the cells, and centrifuged at 1500r / 5min. After the centrifugation, the liquid was discarded, and 1mL of 1640 culture medium was added to resuspend the cells. The cells were counted and the number of cells diluted was 5×10 5 pcs / 100μL.

[0135] 2) ELISpot (sterile procedure)

[0136] Take the required strips, add 200 μL of 1640 culture medium to each well, let it stand at room temperature for 5 to 10 minutes, then remove the culture medium, and then add cells to the ELISpot plate, 5×10 5 Each well was added with 100 μL of stimuli P1, P3, P5, P8, and P10 (6 μg / well). Phytohemagglutinin (PHA) was added to the positive control wells, and the negative control wells and background wells were added. After addition, the plate was covered and placed in a cell culture incubator for 24 hours. The plate was wrapped with tin foil to prevent evaporation of the liquid.

[0137] 3) ELISpot (no sterile procedure required)

[0138] After the incubation, remove the ELISpot plate, discard the liquid in the well, add 200 μL / well of ice-cold deionized water, and place in a 4°C refrigerator for 10 minutes to hypotonic lyse the cells; shake out the liquid in the well, add 1× washing buffer, 260 μL / well, stay for 1 minute, discard, repeat washing 6 times, and pat dry the liquid in the well for the last time; incubate with detection antibodies: add 1× Biotinylated Antibody working solution to each well, 100 μL / well, incubate at 37°C for 1 hour; after the incubation, shake out the liquid in the well, add 1× washing buffer, 260 μL / well, stay for 1 minute, discard, repeat washing 6 times, and pat dry the liquid in the well for the last time; add 1× streptavidin-HRP working solution to each well, 100 μL / well, and incubate at 37°C for 1 hour; after the incubation, shake out the liquid in the well, add 1× washing buffer, 260 μL / well, stay for 1 minute, discard, repeat washing 6 times, and pat dry the liquid in the well for the last time Buffer, 260μL / well, stay for 1 minute, discard, repeat washing 6 times, and pat the liquid in the well dry for the last time; add the freshly prepared ACE color development solution to each well, 100μL / well, and stand at 37℃ in the dark for 5-10 minutes, which can also be extended to 30 minutes; after the color development is completed, discard the liquid in the well, wash the front and back of each well 3-5 times with deionized water (remove the back plate) to stop the color development; place the plate in a cool place at room temperature to dry, and take pictures to count the number of spots.

[0139] ELISPOT test results of T cell immune response Figure 7 As shown, it can be seen that each group of vaccines can induce a certain T cell response in mice. Among them, the levels of INF-γ factors produced by T cell responses induced by KP1, KP5, KP6, KP8, KP10, and KP11 groups are higher, and have stronger T cell immune efficacy.

[0140] Example 4 Coronavirus Neutralizing Antibody Detection (Pseudovirus Experiment)

[0141] 1) Remove reagents (trypsin, Dulbecco's Modified Eagle Medium (DMEM) complete culture medium) stored at 2-8°C and equilibrate to room temperature for at least 30 minutes.

[0142] 2) In a 96-well plate, refer to Table 17. Add 150 μL / well of DMEM complete medium (1% double-antibody, 25 mM HEPES (4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid), 10% fetal bovine serum (FBS)) to column 2 (cell control CC). Add 100 μL / well of DMEM complete medium to column 3 (virus control VC). Add 100 μL / well of DMEM complete medium to wells C4-G11, and 142.5 μL / well of DMEM complete medium to wells B4-B11. Add 7.5 μL / well of sample 1 (30-fold initial dilution) to wells B4 and B5. Similarly, add 7.5 μL / well of sample 4 (30-fold initial dilution) to wells B10 and B11.

[0143] 3) Sample Dilution: Adjust the multichannel pipette to 50 μL. Gently pipette up and down 6-8 times to mix thoroughly in wells B4-B11. Then, transfer 50 μL of the liquid to the corresponding wells C4-C11. Gently pipette up and down 6-8 times to transfer to wells D4-D11, and so on. Finally, aspirate and discard 50 μL of the liquid from wells G4-G11.

[0144] 4) Pseudovirus dilution: Dilute the pseudovirus (Omicron BQ.1.1, Omicron EG.5.1, Omicron JN.1, Omicron XBB.1.5) to 1.3×10 4 (1×10 4 ~2×10 4 ) TCID50 / mL (dilute according to the provided dilution factor) and add 50 μL to each well in columns 3-11 to a pseudovirus load of 650 (500-1000) / well. Dilute BA.4 / 5 10-fold, BQ.1.1 13-fold, EG.5.1 14-fold, JN.1 5-fold, and XBB.1.5 10-fold.

[0145] Table 17 96-well plate sample information

[0146]

[0147] 5) Place the 96-well plate from step 4) in a cell culture incubator (37°C, 5% CO2) and incubate for 1 hour. When the incubation time reaches 0.5 hours, remove the cells prepared in advance from the incubator (the confluence rate reaches 80% to 90%). Taking a T75 culture flask as an example, aspirate the culture medium in the flask and add 5 mL of PBS buffer to wash the cells. After decanting the PBS, add 3 mL of 0.25% trypsin-EDTA and submerge the cells to digest for 1 minute. Decant the trypsin and place in a cell culture incubator to digest for 5 minutes. Gently tap the side of the culture flask to remove the cells. Add 10 mL of culture medium to neutralize the trypsin. After pipetting several times, count the cells and dilute the cells to 2 × 10 with DMEM complete medium. 5 After incubation for 1 hour, add 100 μL of cells to each well of a 96-well plate to make each well contain 2×10 cells. 4 Gently shake the 96-well plate back and forth and left and right to evenly distribute the cells in the wells. Place the 96-well plate in a cell culture incubator at 37°C and 5% CO2 for 20-28 hours.

[0148] 6) Remove the 96-well plate from the cell culture incubator and use a multichannel pipette to aspirate 150 μL of supernatant from each well. Then, add 100 μL of luciferase assay reagent and incubate at room temperature in the dark for 2 minutes. After the reaction is complete, pipette the liquid in the wells 6-8 times to fully lyse the cells. Aspirate 150 μL of liquid from each well and add it to the corresponding 96-well chemiluminescence assay plate. Read the luminescence value in a chemiluminescence analyzer.

[0149] 7) Calculation of Neutralization Inhibition Rate: If the ratio of the virus control (VC) to the cell control (CC) is less than 1000, the experiment is invalid and the 96-well plate needs to be retested. If the test results are reversed, with the infection inhibition rate at the high dilution being greater than or equal to 50% and the infection inhibition rate at the low dilution being less than 50%, the sample test is invalid and requires retesting. A positive control and a negative control should be added to each experiment. If the positive control test result is less than 30, or the negative control test result is greater than or equal to 30, the experiment is invalid and the experiment needs to be retested. Inhibition rate = [1 - (mean luminescence intensity of the sample group - mean luminescence intensity of the blank control (CC)) / (mean luminescence intensity of the negative group (VC) - mean luminescence intensity of the blank control (CC))] × 100%. Calculate the EC50 based on the neutralization inhibition rate results. The test results are shown in Tables 18 and 19.

[0150] Table 18 Sera from mice immunized with the novel coronavirus multi-epitope vaccine (KP1+KP3 group) - pseudovirus blocking results

[0151] Serum dilution multiple Omicron BQ.1.1 Omicron EG.5.1 Omicron JN.1 Omicron XBB.1.5 1:30 51.4％ 70.6％ 98.0％ 58.7％ 1:90 39.6％ 58.0％ 94.0％ 45.5％ 1:270 38.5％ 45.2％ 93.5％ 45.3％ 1:810 36.1％ 34.5％ 79.7％ 42.8％ 1:2430 34.7％ 27.6％ 70.1％ 40.0％ 1:7290 30.0％ 20.4％ 67.9％ 37.4％

[0152] Table 19 Positive control Zhikeweide mouse immune serum pseudovirus blocking results

[0153]

[0154]

[0155] It can be seen from Tables 18 and 19 that the pseudovirus inhibition rates of mouse serum in the new coronavirus multi-epitope vaccine KP1+KP3 group against the new coronavirus mutant strains BQ1.1, EG.5.1, JN.1 and XBB.1.5 were 51.4%, 70.6%, 98% and 58.7% respectively, and the overall blocking effect was better than that of the Zhikeweide positive control.

[0156] Example 5 Preliminary Safety Assessment of Novel Coronavirus Epitope Vaccine

[0157] 1) BALB / c female mice weighing 18-22 g were randomly divided into 7 groups, with 5 mice in each group. The mice in each group were immunized with the corresponding dose of vaccine by multiple subcutaneous injections at the back for a total of three immunizations, once every two weeks.

[0158] 2) During the immunization period, weigh the mice every two weeks. First, adjust the scale to zero, place the mouse in the beaker, and record the accurate value when the weighing balance reading is relatively stable. Monitor the mice's food intake weekly: subtract the remaining amount from the last added feed to obtain the total food intake of the mice in the cage, and divide it by the number of mice to obtain the average food intake per mouse. Monitor the mice's body temperature weekly: After coating the rectal thermometer probe with glycerin, insert it horizontally into the mouse's anus and record the temperature.

[0159] 3) After immunization, the mouse heart, liver, spleen, lung, and fresh tissue blocks were removed and cut into small pieces with a sharp scalpel and fixed in fixative. The pieces were routinely dehydrated, transparentized, paraffin-embedded, and sectioned. After hematoxylin-eosin staining, the pieces were dehydrated, transparentized, and mounted with gum for histopathological examination under a light microscope and photographed.

[0160] The experimental results are as follows Figure 8 As shown, there were no obvious changes in HE staining of the heart, liver, spleen, lung, and kidney of the immunized mice, indicating that the new coronavirus multi-epitope vaccines KP1, KP3, KP5, KP8, KP10 and KP1+KP3 are highly safe and no adverse reactions were found.

[0161] The above content is only for explaining the technical idea of ​​the present invention and cannot be used to limit the protection scope of the present invention. Any changes made on the basis of the technical solution in accordance with the technical idea proposed by the present invention shall fall within the protection scope of the claims of the present invention.

Claims

1. A multi-epitope peptide of a novel coronavirus, characterized in that: The multi-epitope peptide is obtained by connecting the novel coronavirus vaccine epitope with a nano-delivery vector; Among them, the new coronavirus vaccine epitope is one or more combinations of amino acid sequences shown in SEQ ID NO.1 to SEQ ID NO.3, and SEQ ID NO.5 to SEQ ID NO.

12.

2. The multi-epitope peptide of the novel coronavirus according to claim 1, characterized in that The nano delivery vector is KFE8, and the amino acid sequence is shown in SEQ ID NO.

13.

3. The multi-epitope peptide of the novel coronavirus according to claim 1 or 2, characterized in that The amino acid sequences of the multi-epitope peptides are shown in SEQ ID NO.16 to SEQ ID NO.

26.

4. A novel coronavirus multi-epitope vaccine, characterized in that: The novel coronavirus multi-epitope vaccine comprises the multi-epitope peptide of the novel coronavirus according to any one of claims 1 to 3.

5. The novel coronavirus multi-epitope vaccine according to claim 4, characterized in that Also included are nanodelivery vehicles.

6. The novel coronavirus multi-epitope vaccine according to claim 5, characterized in that The nano delivery vector is KFE8, and the amino acid sequence is shown in SEQ ID NO.

13.

7. A method for preparing a multi-epitope vaccine of a novel coronavirus, characterized in that: Vortexing the multi-epitope peptide of the novel coronavirus according to any one of claims 1 to 3 and a single nano-delivery vector with an aqueous solution of the PADRE-nano-delivery vector until a fiber-like strip structure is formed to obtain a novel coronavirus multi-epitope vaccine; Among them, the PADRE-nano delivery vector is formed by connecting PADRE and a nano delivery vector, and the amino acid sequence of PADRE is shown in SEQ ID NO.

14.

8. An anti-novel coronavirus pharmaceutical composition, characterized in that: Including the new coronavirus multi-epitope vaccine according to any one of claims 5 to 7.

9. The anti-new coronavirus pharmaceutical composition according to claim 8, characterized in that Including the novel coronavirus multi-epitope vaccine KP1 and the novel coronavirus multi-epitope vaccine KP3. The novel coronavirus multi-epitope vaccine KP1 is assembled by the multi-epitope peptide KP1, the PADRE-nano delivery vector and a single nano delivery vector. The novel coronavirus multi-epitope vaccine KP3 is assembled by the multi-epitope peptide KP3, the PADRE-nano delivery vector and a single nano delivery vector. Among them, the multi-epitope peptide KP1 is obtained by connecting the new coronavirus vaccine epitope P1 with a nano-delivery vector, and the amino acid sequence of the new coronavirus vaccine epitope P1 is shown in SEQ ID NO.1; the multi-epitope peptide KP3 is obtained by connecting the new coronavirus vaccine epitope P3 with a nano-delivery vector, and the amino acid sequence of the new coronavirus vaccine epitope P3 is shown in SEQ ID NO.3; the PADRE-nano-delivery vector is formed by connecting PADRE with a nano-delivery vector, and the amino acid sequence of PADRE is shown in SEQ ID NO.

14.

10. Use of the multi-epitope peptide of the new coronavirus according to any one of claims 1 to 3, the multi-epitope vaccine of the new coronavirus according to any one of claims 4 to 6, or the anti-new coronavirus pharmaceutical composition according to any one of claims 8 to 9 in the preparation of preventive and / or therapeutic drugs for the new coronavirus.

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