Antigen epitope peptide combination of broad-spectrum novel coronavirus vaccine and application of antigen epitope peptide combination

Abstract

The invention belongs to the technical field of biomedicine, and particularly relates to an antigen epitope peptide combination of a broad-spectrum novel coronavirus vaccine and application of the antigen epitope peptide combination. The antigen epitope peptide combination comprises a virus spike protein Epi-I antigen epitope peptide combination, a virus spike protein Epi-II antigen epitope peptide combination and a virus spike protein Epi-III antigen epitope peptide combination. The antigen epitope peptide combination is obtained by screening spike protein epitopes of three novel coronavirus broad-spectrum neutralizing antibodies, can induce a host to generate broad-spectrum neutralizing antibody response, and is used for preparing broad-spectrum novel coronavirus vaccines. The antigen epitope peptide combination is used for screening to obtain the antigen epitope fusion protein L9, the antigen epitope fusion protein L9 is prepared into a vaccine, and the result of the embodiment shows that the neutralizing effect of the L9 vaccine on a new coronavirus variant strain is superior to that of a new coronavirus Wuhan strain poison spike protein RBD control group, and a good cross neutralizing effect is achieved.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to an antigenic epitope peptide combination for a broad-spectrum novel coronavirus vaccine and its application. Background Technology

[0002] The novel coronavirus (SARS-CoV-2) poses a significant challenge to global public health. While multiple vaccines have been successfully deployed and played a crucial role in reducing severe illness and mortality rates, the protective efficacy of existing vaccines is gradually weakening due to continuous viral mutations, making them ineffective against emerging variants. The emergence of these variants not only accelerates viral transmission but also significantly increases the risk of immune escape after vaccination, introducing new uncertainties into epidemic prevention and control.

[0003] To achieve broader-spectrum immune protection, developing broad-spectrum novel coronavirus vaccines that are independent of specific variants and can target multiple viral lineages has become a crucial goal in vaccine development. The S protein is a key structure for the novel coronavirus to invade host cells, containing multiple important antigenic epitopes recognized by neutralizing antibodies. Some regions of these epitopes can induce broad-spectrum neutralizing antibodies with neutralizing efficacy against different variants. These important epitope regions provide important targets for the design of broad-spectrum vaccines. However, most current vaccines struggle to effectively induce broad-spectrum neutralizing antibody responses targeting these key sites, limiting their broad-spectrum protective efficacy.

[0004] Therefore, designing novel antigens that can precisely activate broad-spectrum neutralizing antibody immune responses based on the key epitope structure of the S protein has become one of the effective strategies to enhance the broad-spectrum efficacy of novel coronavirus vaccines. This strategy is expected to break the limitations imposed by variant strains on vaccine efficacy and lay the foundation for achieving sustained immune protection against multiple novel coronavirus variants. Summary of the Invention

[0005] The purpose of this invention is to provide an antigenic epitope peptide combination for a broad-spectrum novel coronavirus vaccine and its application. The antigenic epitope peptide combination described in this invention can induce a variety of broad-spectrum COVID-19 neutralizing antibody responses and can be used to prepare a broad-spectrum novel coronavirus vaccine.

[0006] This invention provides an antigenic epitope peptide combination for a broad-spectrum novel coronavirus vaccine, the antigenic epitope peptide combination comprising a viral spike protein Epi-I antigenic epitope peptide combination, a viral spike protein Epi-II antigenic epitope peptide combination, and a viral spike protein Epi-III antigenic epitope peptide combination. The viral spike protein Epi-I antigenic epitope peptide combination includes a first antigenic epitope peptide and a second antigenic epitope peptide; the amino acid sequence of the first antigenic epitope peptide is shown in SEQ ID NO.1, and the amino acid sequence of the second antigenic epitope peptide is shown in SEQ ID NO.2; The viral spike protein Epi-II antigenic epitope peptide assembly includes a third antigenic epitope peptide and a fourth antigenic epitope peptide; the amino acid sequence of the third antigenic epitope peptide is TR; the amino acid sequence of the fourth antigenic epitope peptide is shown in SEQ ID NO. 3; The viral spike protein Epi-III antigenic epitope peptide combination includes a fifth antigenic epitope peptide and a sixth antigenic epitope peptide; the amino acid sequence of the fifth antigenic epitope peptide is shown in SEQ ID NO.4, and the amino acid sequence of the sixth antigenic epitope peptide is shown in SEQ ID NO.5.

[0007] As a preferred embodiment, from the N-terminus to the C-terminus, the viral spike protein Epi-I antigenic epitope peptide assembly comprises a first antigenic epitope peptide and a second antigenic epitope peptide connected sequentially but not contiguously; the first antigenic epitope peptide and the second antigenic epitope peptide are linked by amino acids or polypeptides. From the N-terminus to the C-terminus, the viral spike protein Epi-II antigenic epitope peptide assembly includes a third antigenic epitope peptide and a fourth antigenic epitope peptide connected sequentially but not contiguously; the third antigenic epitope peptide and the fourth antigenic epitope peptide are linked by amino acids or polypeptides. From the N-terminus to the C-terminus, the viral spike protein Epi-III antigenic epitope peptide assembly includes a fifth antigenic epitope peptide and a sixth antigenic epitope peptide connected sequentially but not contiguously; the fifth antigenic epitope peptide and the sixth antigenic epitope peptide are linked by amino acids or polypeptides.

[0008] As a preferred embodiment, the amino acid sequence of the viral spike protein Epi-I antigenic epitope peptide combination is shown in SEQ ID NO.6; the amino acid sequence of the viral spike protein Epi-II antigenic epitope peptide combination is shown in SEQ ID NO.7; and the amino acid sequence of the viral spike protein Epi-III antigenic epitope peptide combination is shown in SEQ ID NO.8.

[0009] As a preferred embodiment, the viral spike protein Epi-I antigenic epitope peptide combination, the viral spike protein Epi-II antigenic epitope peptide combination, and the viral spike protein Epi-III antigenic epitope peptide combination are linked by peptides.

[0010] As a preferred embodiment, the amino acid sequence of the antigen epitope peptide combination is shown in SEQ ID NO.9.

[0011] The present invention also provides a nucleotide sequence encoding the antigen epitope peptide combination described in the above scheme.

[0012] As a preferred embodiment, the nucleotide sequence is shown in SEQ ID NO.10.

[0013] The present invention also provides the application of the antigen epitope peptide combination or the nucleotide sequence described above in the preparation of a drug for preventing novel coronavirus infection.

[0014] The present invention also provides the application of the antigen epitope peptide combination or the nucleotide sequence described above in the preparation of a broad-spectrum novel coronavirus vaccine.

[0015] The present invention also provides a broad-spectrum novel coronavirus vaccine, which comprises an adjuvant and the antigen epitope peptide combination described in the above-described scheme.

[0016] This invention provides a broad-spectrum novel coronavirus vaccine epitope peptide combination, comprising a viral spike protein Epi-I epitope peptide combination, a viral spike protein Epi-II epitope peptide combination, and a viral spike protein Epi-III epitope peptide combination; the viral spike protein Epi-I epitope peptide combination includes a first epitope peptide and a second epitope peptide; the amino acid sequence of the first epitope peptide is shown in SEQ ID NO.1, and the amino acid sequence of the second epitope peptide is shown in SEQ ID NO.2; the viral spike protein Epi-II epitope peptide combination includes a third epitope peptide and a fourth epitope peptide; the amino acid sequence of the third epitope peptide is TR; the amino acid sequence of the fourth epitope peptide is shown in SEQ ID NO.3; the viral spike protein Epi-III epitope peptide combination includes a fifth epitope peptide and a sixth epitope peptide; the amino acid sequence of the fifth epitope peptide is shown in SEQ ID NO.4, and the amino acid sequence of the sixth epitope peptide is shown in SEQ ID NO.5. The antigenic epitope peptide combination described in this invention was obtained by screening spike protein epitopes from three broad-spectrum neutralizing antibodies against SARS-CoV-2. It can induce a broad-spectrum neutralizing antibody response in the host and is used to prepare a broad-spectrum novel coronavirus vaccine. Using the antigenic epitope peptide combination described in this invention, the antigenic epitope fusion protein L9 was obtained and prepared into a vaccine. The results of the examples show that the L9 vaccine has a better neutralizing effect against SARS-CoV-2 variants than the control group containing the spike protein RBD of the Wuhan strain of SARS-CoV-2, exhibiting good cross-neutralization. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0018] Figure 1 This is a three-dimensional structural diagram of an antigenic epitope fragment; where A is the EPI-Ⅰ antigenic epitope fragment, and the arrows in the diagram indicate the beta fold structure of the protein; B is the EPI-Ⅱ antigenic epitope fragment; and C is the EPI-Ⅲ antigenic epitope fragment. Figure 2 The diagram shows the three-dimensional support structure for the antigenic epitope fragments; where A is the three-dimensional support structure for the EPI-Ⅰ antigenic epitope fragment; B is the three-dimensional support structure for the EPI-Ⅱ antigenic epitope fragment; and C is the three-dimensional support structure for the EPI-Ⅲ antigenic epitope fragment. The gray area in the diagram represents the support structure. Figure 3 Predict the three-dimensional structure of the L9 protein using AlphaFold software; Figure 4 The results of SDS-PAGE electrophoresis of L9 protein; Figure 5 The results are for L9 protein plate ELISA assay; *** represents... P <0.001; ns is P >0.05; Figure 6 The results are for RBD protein plate ELISA assay; *** represents... P <0.001; ns is P >0.05; Figure 7 The assay was performed to detect the pseudovirus inhibition rate in mouse immune serum; where A represents Omicron strain BF.7; B represents Omicron strain BQ.1; C represents XBB strain; and D represents the original Wuhan strain of SARS-CoV-2. Detailed Implementation

[0019] This invention provides a broad-spectrum novel coronavirus vaccine epitope peptide combination, comprising a viral spike protein Epi-I epitope peptide combination, a viral spike protein Epi-II epitope peptide combination, and a viral spike protein Epi-III epitope peptide combination; the viral spike protein Epi-I epitope peptide combination includes a first epitope peptide and a second epitope peptide; the amino acid sequence of the first epitope peptide is shown in SEQ ID NO.1, and the amino acid sequence of the second epitope peptide is shown in SEQ ID NO.2; the viral spike protein Epi-II epitope peptide combination includes a third epitope peptide and a fourth epitope peptide; the amino acid sequence of the third epitope peptide is TR; the amino acid sequence of the fourth epitope peptide is shown in SEQ ID NO.3; the viral spike protein Epi-III epitope peptide combination includes a fifth epitope peptide and a sixth epitope peptide; the amino acid sequence of the fifth epitope peptide is shown in SEQ ID NO.4, and the amino acid sequence of the sixth epitope peptide is shown in SEQ ID NO.5.

[0020] This invention screens and obtains three viral spike protein epitope sequences recognized by three broad-spectrum neutralizing antibodies against the novel coronavirus (CV38-142 antibody, C135 antibody, and S309 antibody), which are named the Epi-I antigenic epitope peptide combination, the Epi-II antigenic epitope peptide combination, and the Epi-III antigenic epitope peptide combination, respectively. The CV38-142 antibody selected in this invention can provide broad-spectrum neutralizing protection against Alpha, Beta, Gamma, and Delta variants of SARS-CoV-2; the C135 antibody can provide broad-spectrum neutralizing protection against wild-type, B.1.1.7 (Alpha), and B.1.351 (Beta) variants of SARS-CoV-2; and the S309 antibody can provide broad-spectrum neutralizing protection against BA.1 (Omicron), BA.1.1 (Omicron), BA.2 (Omicron), B.1.617.2 (Delta), B.1.621 (Mu), and B.1.1.529 (Omicron) variants of SARS-CoV-2. The antigenic epitope peptide combination described in this invention can induce a broad-spectrum neutralizing antibody response in the host and can be used to prepare a broad-spectrum novel coronavirus vaccine.

[0021] In one embodiment, from the N-terminus to the C-terminus, the viral spike protein Epi-I antigenic epitope peptide assembly comprises a first antigenic epitope peptide and a second antigenic epitope peptide connected sequentially but discontinuously; the first and second antigenic epitope peptides are linked by amino acids or polypeptides. In a specific embodiment of the present invention, the first and second antigenic epitope peptides are linked by GGSGGPLGDEGAELAKDGEYAKWLKLAKAIGLDAD (SEQ ID NO. 11); the N-terminus of the first antigenic epitope peptide is also connected to a P-terminus; and the C-terminus of the second antigenic epitope peptide is also connected to an S-terminus.

[0022] From the N-terminus to the C-terminus, the viral spike protein Epi-II antigenic epitope peptide assembly comprises a third and a fourth antigenic epitope peptide connected sequentially but discontinuously; the third and fourth antigenic epitope peptides are linked by amino acids or polypeptides. In a specific embodiment of the present invention, the third and fourth antigenic epitope peptides are linked by IAKGSVASQTSDRYEDWKDWLKKWLEYLLRGGEGLP (SEQ ID NO. 12); the N-terminus of the third antigenic epitope peptide is also connected to G; and the C-terminus of the fourth antigenic epitope peptide is also connected to IYW.

[0023] From the N-terminus to the C-terminus, the viral spike protein Epi-III antigenic epitope peptide assembly includes a fifth and a sixth antigenic epitope peptide connected sequentially but not contiguously; the fifth and sixth antigenic epitope peptides are linked by amino acids or polypeptides. In a specific embodiment of the present invention, the fifth and sixth antigenic epitope peptides are linked by GTEDNLGAQKELDIWWKQVGKSGGDGEEDTKKATKYGGEAKNGAP (SEQ ID NO.13); the N-terminus of the fifth antigenic epitope peptide is also connected to an N; and the C-terminus of the sixth antigenic epitope peptide is also connected to an MA.

[0024] In one embodiment, the amino acid sequence of the viral spike protein Epi-I antigenic epitope peptide combination is shown in SEQ ID NO.6; the amino acid sequence of the viral spike protein Epi-II antigenic epitope peptide combination is shown in SEQ ID NO.7; and the amino acid sequence of the viral spike protein Epi-III antigenic epitope peptide combination is shown in SEQ ID NO.8.

[0025] In one embodiment, the viral spike protein Epi-I antigenic epitope peptide combination, the viral spike protein Epi-II antigenic epitope peptide combination, and the viral spike protein Epi-III antigenic epitope peptide combination are linked by peptides. In a specific embodiment of the present invention, the viral spike protein Epi-I antigenic epitope peptide combination, the viral spike protein Epi-II antigenic epitope peptide combination, and the viral spike protein Epi-III antigenic epitope peptide combination are linked by PPPP.

[0026] As one embodiment, the amino acid sequence of the antigen epitope peptide combination is shown in SEQ ID NO.9, and it is named L9 protein.

[0027] The present invention also provides a nucleotide sequence encoding the antigenic epitope peptide combination described in the above-described scheme. As one embodiment, the nucleotide sequence is shown in SEQ ID NO. 10. The sequence of SEQ ID NO. 10 of the present invention can increase the expression level of L9 protein.

[0028] The present invention also provides the application of the antigen epitope peptide combination or the nucleotide sequence described above in the preparation of a drug for preventing novel coronavirus infection.

[0029] This invention also provides the application of the antigenic epitope peptide combination or the nucleotide sequence described above in the preparation of a broad-spectrum novel coronavirus vaccine. The antigenic epitope peptide combination of this invention can induce a targeted response of three broad-spectrum neutralizing antibodies, achieving broad-spectrum immune protection against the novel coronavirus.

[0030] This invention also provides a broad-spectrum novel coronavirus vaccine, comprising an adjuvant and the antigen epitope peptide combination described in the above-described scheme. This invention does not impose any particular limitation on the type of adjuvant; any vaccine adjuvant well-known to those skilled in the art can be used. In a specific embodiment of this invention, the adjuvant is aluminum hydroxide adjuvant (CRODA); aluminum hydroxide adjuvant can adsorb antigens, prolong the retention time of antigens in the body, and enhance the recognition by the immune system.

[0031] The present invention also provides a drug for preventing novel coronavirus infection, the drug comprising the antigen epitope peptide combination described in the above scheme.

[0032] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0033] Example 1: Obtaining the Epitope Sequence and Three-Dimensional Structure of the Novel Coronavirus Spike Protein Information on three broad-spectrum neutralizing antibodies against the novel coronavirus (CV38-142 antibody, C135 antibody, and S309 antibody) was retrieved from the coronavirus antibody database CoV-AbDab (https: / / opig.stats.ox.ac.uk / webapps / covabdab / ). High-resolution crystal structures of the complexes of the three broad-spectrum neutralizing antibodies (CV38-142, C135, and S309) with the viral spike protein were obtained from the protein database PDB (https: / / www.rcsb.org / ) (PDB ID: 7LM9, 7K8Z, 6WPT). Among them, the CV38-142 antibody provides broad-spectrum neutralizing protection against the Alpha, Beta, Gamma, and Delta variants of the novel coronavirus; the C135 antibody provides broad-spectrum neutralizing protection against the wild-type, B.1.1.7 (Alpha), and B.1.351 (Beta) variants of the novel coronavirus; and the S309 antibody provides broad-spectrum neutralizing protection against the BA.1 (Omicron), BA.1.1 (Omicron), BA.2 (Omicron), B.1.617.2 (Delta), B.1.621 (Mu), and B.1.1.529 (Omicron) variants of the novel coronavirus. Analysis of the antigen complexes with these three antibodies using IBEIP (Intelligent Batch Epitope Identification and Partitioning) software yielded the viral spike protein epitope sequences and three-dimensional structures recognized by the three antibodies. After structural simplification, the spike protein epitope Epi-I recognized by the CV38-142 antibody comprises two discontinuous sequence segments from the amino terminus to the carboxyl terminus: FNATK (SEQ ID NO.1) and WNTRNIDATSTGNHN (SEQ ID NO.2). The three-dimensional structure of the Epi-I epitope fragment is shown below. Figure 1 As shown in Figure A, the spike protein epitope Epi-II recognized by the C135 antibody comprises two discontinuous sequence segments from the amino terminus to the carboxyl terminus: TR, NSNNLDSKVGGNYN (SEQ ID NO. 3). The three-dimensional structure of the Epi-II epitope fragment is shown in Figure A. Figure 1 As shown in Figure B, the spike protein epitope Epi-III recognized by the S309 antibody comprises two discontinuous sequence segments from the amino terminus to the carboxyl terminus: TNLCPFGEVFNATR (SEQ ID NO.4) and NRKRISNC (SEQ ID NO.5). The three-dimensional structure of the Epi-III epitope fragment is shown in Figure B. Figure 1 As shown in C.

[0034] Example 2: Design of a three-dimensional supporting protein structure for antigen epitopes Three-dimensional supporting protein structures were designed for discontinuous antigenic epitope fragments using TopoBuilder software (by adding different numbers or layers of α-helices and β-sheets), ultimately yielding 16 Epi-I, 22 Epi-II, and 43 Epi-III epitope three-dimensional supporting protein structures. To select the optimal epitope three-dimensional supporting protein structure, 50 amino acid sequences were designed for each epitope three-dimensional supporting protein structure using Rosetta software. The 300 amino acid sequences with the lowest energy fraction for each epitope were selected for statistical analysis, and the epitope three-dimensional supporting protein structure with the highest sequence number and the lowest average protein energy fraction was identified as the optimal structure.

[0035] The optimal three-dimensional supporting protein structure for Epi-I epitope is the three-dimensional supporting protein structure with sequence number 1 in Table 1. This structure contains 45 sequences and has the lowest average protein energy fraction of 188. The structure is as follows: Figure 2 As shown in Figure A. The optimal three-dimensional supporting protein structure for Epi-II epitopes is the three-dimensional supporting protein structure with sequence number 1 in Table 2. This structure contains 50 sequences and has the lowest average protein energy fraction of 106. The structure is shown in Figure B of Structure 2. The optimal three-dimensional supporting protein structure for Epi-III epitopes is the three-dimensional supporting protein structure with sequence number 1 in Table 3. This structure contains 50 sequences and has the lowest average protein energy fraction of 159. The structure is shown in Figure A. Figure 2 As shown in C.

[0036] Table 1. Statistical analysis of the lowest protein energy distribution sequences of 300 Epi-I epitopes.

[0037] Table 2. Statistical analysis of the lowest protein energy distribution sequences of 300 Epi-II epitopes.

[0038] Table 3. Statistical analysis of the lowest protein energy distribution sequences of 300 Epi-III epitopes.

[0039] Example 3: Design of three-dimensional supporting protein sequences for antigen epitopes Two thousand different protein sequences were designed using Rosetta software to create optimal 3D support structures for Epi-I, Epi-II, and Epi-III epitopes. The 500 protein support structure sequences with the lowest energy fractions were selected for 3D protein structure prediction using AlphaFold software. For each epitope, the sequence with the best conformational reduction (Root Mean Square Deviation value) and structural stability (protein energy fraction) was selected.

[0040] The optimal sequence for the Epi-I epitope is Epi-I_3 (RMSD: 0 Å, protein energy fraction: -2.47). The Epi-I epitope fragment and supporting protein sequence (in bold in parentheses) are named the Epi-I_3 protein sequence (SEQ ID NO.6): (P)FNATK(GGSGGPLGDEGAELAKDGEYAKWLKLAKAIGLDAD)WNTRNIDATSTGNHN(S).

[0041] The optimal sequence for the Epi-II epitope is Epi-II_1 (RMSD: 0.80 Å, protein energy fraction: -15.64). The Epi-II epitope fragment and supporting protein sequence (in bold in parentheses) are named the Epi-II_1 protein sequence (SEQ ID NO.7): (G)TR(IAKGSVASQTSDRYEDWKDWLKKWLEYLLRGGEGLP)NSNNLDSKVGGNYN(IYW).

[0042] The optimal sequence for the Epi-III epitope is Epi-III_1 (RMSD: 0.35 Å, protein energy fraction: 46.18). The Epi-III epitope fragment and supporting protein sequence (in bold in parentheses) are named the Epi-III_1 protein sequence (SEQ ID NO. 8): (N)TNLCPFGEVFNATR(GTEDNLGAQKELDIWWKQVGKSGGDGEEDTKKATKYGGEAKNGAP)NRKRISNC(MA).

[0043] Example 4: Tandem of antigen protein sequences Epi-I_3, Epi-II_1, and Epi-III_1 sequences were tandemly linked into a single antigen protein sequence using three different protein linker sequences: linker1: GGGG; linker2: PPPP; and linker3: PPPPPP. A total of 18 different tandem protein sequences were obtained, as shown in Table 4. The three-dimensional structures of all tandem protein sequences were predicted using AlphaFold software. The tandem sequence L9, with the highest epitope structural reduction (average RMSD: 1.04 Å), was selected. The tandem sequence of the three epitopes was Epi-II_1 / Epi-III_1 / Epi-I_3, and the protein linker sequence was "PPPP". The three-dimensional structure of the L9 protein predicted by AlphaFold software is shown below. Figure 3 As shown.

[0044] Table 4 Epitope Serialization Statistics

[0045] The L9 protein sequence (SEQ ID NO. 9) is: GTRIAKGSVASQTSDRYEDWKDWLKKWLEYLLRGGEGLPNSNNLDSKVGGNYNIYW(PPPP)NTNLCPFGEVFNATRGTEDNLGAQKELDIWWKQVGKSGGDGEEDTKKATKYGGEAKNGAPNRKRISNCMA(PPPP)PFNATKGGSGGPLGDEGAELAKDGEYAKWLKLAKAIGLDADWNTRNIDATSTGNHNS, where the bold text in parentheses is the protein linker sequence.

[0046] Example 5: Preparation of L9 novel coronavirus vaccine The L9 protein was synthesized and expressed by Suzhou Hongxun Biotechnology Co., Ltd.

[0047] The L9 protein synthesis gene sequence (SEQ ID NO.10) is 5′--3′. 1–18 bp encodes a His tag, used for affinity purification of the synthesized protein, as well as subsequent detection and analysis. A plasmid expressing the L9 protein was synthesized and constructed, transfected into Expi293F cells, and after 4 days of expression, the supernatant was collected by centrifugation for protein purification. The purified L9 protein was analyzed by SDS-PAGE electrophoresis, performing both reduction and non-reduction electrophoresis. Reduction electrophoresis involves adding a reducing agent to disrupt the protein's three-dimensional structure, flattening it into a linear structure; non-reduction electrophoresis does not involve adding a reducing agent, preserving the protein's native conformation. The detection results are as follows: Figure 4 As shown.

[0048] according to Figure 4 It is known that the molecular weight of L9 protein is 25~35kDa, and the protein purity is greater than 90%.

[0049] Example 6: Evaluation of Immunization and Virus Neutralization of L9 Novel Coronavirus Vaccine (1) Animal immunization: The immunization subjects were 6-8 week old BALB / c female rats, immunized three times, two weeks apart, intramuscularly, with aluminum hydroxide adjuvant (purchased from CRODA), and raised in a sterile environment, with 6 rats in each group. Currently, most novel coronavirus vaccines are developed based on the Wuhan strain of novel coronavirus. The RBD region of the novel coronavirus S protein plays a key role in mediating viral infection of host cells and is the core antigenic region of the novel coronavirus. Therefore, the RBD antigen of the Wuhan strain of novel coronavirus (Institute of Medical Biology, Chinese Academy of Medical Sciences) was selected as the vaccine control group.

[0050] Animal experiment grouping: L9 group: L9 protein 10 μg / mouse, aluminum adjuvant 35 μg / mouse; RBD group: RBD antigen protein 10 μg / mouse, aluminum adjuvant 35 μg / mouse; All animal experiments were approved by the Animal Experiment Ethics Committee of the Institute of Medical Biology, Chinese Academy of Medical Sciences, and were conducted in strict accordance with the regulations of the Yunnan Provincial Laboratory Animal Welfare and Ethics Committee.

[0051] (2) ELISA detection of L9 vaccine immunogenicity: Twelve days after the second and third doses of immunization, blood was collected from the tail vein of mice, and the endpoint titer of immune serum antibodies was detected by ELISA. The experimental procedure is as follows: 1. Antigen coating: Dilute the antigen to a concentration of 5 µg / mL with coating solution, add 100 µL to each reaction well of a polystyrene ELISA plate, incubate overnight at 4°C, and discard the solution in the well the next day.

[0052] 2. Sealing: Add sealing solution at 250 µL / well, incubate in a 37°C oven for 2 hours, then discard the sealing solution in the wells and wash 3 times (plate washer settings: liquid volume 250 µL, washing time 3.5s, soaking time 5s, the same below).

[0053] 3. Sample loading (primary antibody): The primary antibody is the serum to be tested. Dilute it 400 times with antibody diluent. Take 100µL of the sample to be tested into the above-coated reaction wells, place it in a 37℃ oven for 1 hour, and then wash it three times.

[0054] 4. Add enzyme-labeled antibody (secondary antibody): Prepare the enzyme-labeled antibody according to the product instructions (dilution concentration is 1:40000), add 100µL / well, incubate in a 37℃ oven for 1 hour, and then wash three times.

[0055] 5. Add TMB substrate solution for color development: Prepare TMB color development solution fresh and add 100 µL / well. Develop at 37°C in the dark for 10 minutes.

[0056] 6. Termination of reaction: Add 100 µL of stop solution to each well, and then read the values ​​on a microplate reader. Measure the OD value at 450 nm and 630 nm, respectively; the difference is the OD value of that well. If the OD value is greater than 2.1 times that of the negative control well, it is considered positive.

[0057] Targeting the L9 protein antigen, the endpoint antibody titer (log10) in the L9 group significantly increased from 1.65 after the second dose to 3.16. In the RBD group, the endpoint antibody titers (log10) after the second and third doses were 1.53 and 1.42, respectively, showing no significant change. The results are as follows: Figure 5 As shown in the figure. Regarding the RBD protein antigen, the endpoint antibody titer (log10) of the L9 group significantly increased from 0 in the second dose to 1.30 in the third dose. The endpoint antibody titers (log10) of the RBD group in the second and third doses were 4.68 and 4.80, respectively, showing no significant change. The results are as follows. Figure 6 As shown.

[0058] ELISA results showed that the third dose significantly enhanced the immune response level of L9 antibody; Figure 5 and Figure 6 The results showed that both the L9 group and the RBD group immune serum antibodies could bind to the L9 antigen and the RBD antigen, indicating that the L9 antigen targeted and induced antibody responses that could bind to the Epi-I, Epi-II, and Epi-III epitopes of the novel coronavirus S protein.

[0059] (3) Pseudovirus Neutralization Assay: Tail vein blood was collected from mice 12 days after the third injection. The inhibition rate of the novel coronavirus pseudovirus was detected using the 293T-ACE2 cell line. To test the broad-spectrum neutralizing efficacy of the L9 novel coronavirus vaccine, the original novel coronavirus strains (Wuhan), XBB, and Omicron (BF.7, BQ.1) were selected. Mouse serum was diluted 50-12150 times, and the pseudovirus inhibition rate was detected. All pseudovirus strains were provided by the Institute of Medical Biology, Chinese Academy of Medical Sciences. The test results are as follows: Figure 7 As shown in Table 5.

[0060] The L9 group serum showed a higher inhibition rate against the Omicron strain of the novel coronavirus than the RBD group. Specifically, the inhibition rate of BF.7 strain was 1.28–3.37 times that of the RBD group, and the inhibition rate of BQ.1 strain (except for the 50-fold dilution) was 1.46–15.23 times that of the RBD group. The results are as follows: Figure 7 As shown in Figures A through B. The inhibition rates of the L9 group and the RBD group against the XBB strain were not significantly different, as shown in the results. Figure 7 As shown in Figure C. Since the RBD group originated from the original Wuhan strain of the novel coronavirus, its inhibition rate against the Wuhan strain was 1.76–2.72 times that of the L9 group, as shown in the results. Figure 7As shown in Figure D, the pseudovirus neutralization experiment demonstrates that our designed L9 vaccine has a better neutralizing effect against the novel coronavirus variant than the RBD control group, exhibiting a good cross-neutralization effect.

[0061] Table 5. Neutralization inhibition rate of pseudovirus (%)

[0062] In summary, the L9 protein can be obtained by combining the antigenic epitope peptide combination of the broad-spectrum novel coronavirus vaccine described in this invention with a suitable supporting protein. Using this protein as a vaccine can induce multiple broad-spectrum neutralizing antibody responses and exhibit superior cross-neutralizing efficacy against novel coronavirus variants.

[0063] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A combination of antigenic epitope peptides for a broad-spectrum novel coronavirus vaccine, characterized in that, The antigenic epitope peptide combination includes the viral spike protein Epi-I antigenic epitope peptide combination, the viral spike protein Epi-II antigenic epitope peptide combination, and the viral spike protein Epi-III antigenic epitope peptide combination. The viral spike protein Epi-I antigenic epitope peptide combination includes a first antigenic epitope peptide and a second antigenic epitope peptide; the amino acid sequence of the first antigenic epitope peptide is shown in SEQ ID NO.1, and the amino acid sequence of the second antigenic epitope peptide is shown in SEQ ID NO.2; The viral spike protein Epi-II antigenic epitope peptide assembly includes a third antigenic epitope peptide and a fourth antigenic epitope peptide; the amino acid sequence of the third antigenic epitope peptide is TR; the amino acid sequence of the fourth antigenic epitope peptide is shown in SEQ ID NO.3; The viral spike protein Epi-III antigenic epitope peptide combination includes a fifth antigenic epitope peptide and a sixth antigenic epitope peptide; the amino acid sequence of the fifth antigenic epitope peptide is shown in SEQ ID NO.4, and the amino acid sequence of the sixth antigenic epitope peptide is shown in SEQ ID NO.

5.

2. The antigen epitope peptide combination according to claim 1, characterized in that, From the N-terminus to the C-terminus, the viral spike protein Epi-I antigenic epitope peptide assembly includes a first antigenic epitope peptide and a second antigenic epitope peptide connected sequentially but not contiguously; the first antigenic epitope peptide and the second antigenic epitope peptide are linked by amino acids or polypeptides. From the N-terminus to the C-terminus, the viral spike protein Epi-II antigenic epitope peptide assembly includes a third antigenic epitope peptide and a fourth antigenic epitope peptide connected sequentially but not contiguously; the third antigenic epitope peptide and the fourth antigenic epitope peptide are linked by amino acids or polypeptides. From the N-terminus to the C-terminus, the viral spike protein Epi-III antigenic epitope peptide assembly includes a fifth antigenic epitope peptide and a sixth antigenic epitope peptide connected sequentially but not contiguously; the fifth antigenic epitope peptide and the sixth antigenic epitope peptide are linked by amino acids or polypeptides.

3. The antigen epitope peptide combination according to claim 2, characterized in that, The amino acid sequence of the viral spike protein Epi-I antigenic epitope peptide combination is shown in SEQ ID NO.6; the amino acid sequence of the viral spike protein Epi-II antigenic epitope peptide combination is shown in SEQ ID NO.7; and the amino acid sequence of the viral spike protein Epi-III antigenic epitope peptide combination is shown in SEQ ID NO.

8.

4. The antigen epitope peptide combination according to claim 3, characterized in that, The viral spike protein Epi-I antigenic epitope peptide combination, the viral spike protein Epi-II antigenic epitope peptide combination, and the viral spike protein Epi-III antigenic epitope peptide combination are linked by peptides.

5. The antigen epitope peptide combination according to claim 4, characterized in that, The amino acid sequence of the antigen epitope peptide combination is shown in SEQ ID NO.

9.

6. A nucleotide sequence encoding the combination of antigenic epitope peptides according to any one of claims 1 to 5.

7. The nucleotide sequence according to claim 6, characterized in that, The nucleotide sequence is shown in SEQ ID NO.

10.

8. The use of the antigenic epitope peptide combination according to any one of claims 1 to 5 or the nucleotide sequence according to claim 6 or 7 in the preparation of a medicament for preventing novel coronavirus infection.

9. The use of the antigenic epitope peptide combination according to any one of claims 1 to 5 or the nucleotide sequence according to claim 6 or 7 in the preparation of a broad-spectrum novel coronavirus vaccine.

10. A broad-spectrum novel coronavirus vaccine, characterized in that, The broad-spectrum novel coronavirus vaccine comprises an adjuvant and a combination of antigenic epitope peptides as described in any one of claims 1 to 5.

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