A polypeptide composition of a novel coronavirus, a prepared vaccine and application thereof

Abstract

The present application belongs to the technical field of biological pharmacy, and particularly relates to a polypeptide composition of a novel coronavirus, a vaccine prepared therefrom and application thereof. The polypeptide composition of the novel coronavirus comprises four polypeptides of GY, VV, VS and DL. Experiments show that the polypeptide or the composition thereof can effectively induce T cell immune response and specific humoral immunity against SARS-CoV-2 and its mutant strains, stimulate the body to produce antigen-specific antibodies, provide a basis for constructing SARS-CoV2 preventive and / or therapeutic antibodies with high affinity, high quality and strong specificity, and have high application value for prevention and treatment of the novel coronavirus.

Description

Technical Field

[0001] This invention belongs to the field of biopharmaceutical technology, and in particular relates to a polypeptide composition of a novel coronavirus, a vaccine prepared therefrom, and its application. Technical Background

[0002] The novel coronavirus (SARS-CoV-2, hereinafter referred to as SARS2) causes infection (COVID-19), which is highly contagious. Although various types of COVID-19 vaccines, such as inactivated vaccines, mRNA vaccines, and recombinant protein vaccines, have been approved and widely used, the continuous evolution of the SARS-CoV-2 virus and the emergence of variants of concern (VOCs) pose a serious challenge to the protective efficacy of existing vaccines.

[0003] Multiple studies have demonstrated that T-cell immunity plays a crucial role in combating SARS-CoV-2 infection. SARS-CoV-2-specific T-cell immune responses have been detected in COVID-19 convalescents, SARS survivors, and uninfected controls. Virus-specific T cells induced by β-coronavirus infection can persist in vivo for up to 17 years and exhibit significant cross-reactivity. Furthermore, the strength of the virus-specific T-cell response is not directly correlated with the severity or presence of symptoms; and for patients with humoral immunodeficiency, T-cell immunity provides significant protection. These findings strongly suggest that T-cell immunity plays a vital role in long-term memory protection after SARS-CoV-2 infection and is a key determinant of achieving long-term immune protection against the virus.

[0004] Currently, scientists have completed the identification of protein epitopes across the entire genome of the SARS-CoV-2 virus using high-throughput analysis. The spike protein of SARS-CoV-2 has two key regions: the RBD region, which exhibits high variability, and the S2 fusion subunit, which shows high conservation among different variants, particularly in regions crucial for cell fusion such as the fusion peptide (FP), heptacap Repeat 1 (HR1), and heptacap Repeat 2 (HR2). Specifically, the S2 subunit shows over 85% sequence homology among different coronaviruses, while the FP, HR1, and HR2 regions have shown almost no mutations in any known variants of interest. Furthermore, these highly conserved regions are enriched with experimentally validated T-cell and B-cell epitopes.

[0005] Peptide vaccines are developed by precisely screening short peptide sequences from viral antigen proteins that can be recognized by the immune system. Once formulated, these vaccines can be directly recognized by immune cells, inducing an immune response. Currently, although research on peptide vaccines against the novel coronavirus has made some progress, several challenges remain: limited epitope selection, with most vaccines still focusing on the easily mutated RBD region; insufficiently comprehensive immune response, as a single peptide cannot effectively activate both humoral and cellular immunity simultaneously; insufficient broad-spectrum efficacy, making it difficult to effectively combat emerging variants; and a lack of systematic optimization research regarding peptide combinations, adjuvant systems, and immunization procedures.

[0006] Given the ongoing evolution of the SARS-CoV-2 virus, developing new SARS-CoV-2 vaccines that are safer, more effective, and have broad-spectrum protective effects, especially peptide combination vaccines that can simultaneously induce long-acting T-cell immunity and highly effective humoral immunity and are effective against multiple variants, is of great scientific significance and application value. Summary of the Invention

[0007] In order to solve the problems in the prior art, one of the objectives of the present invention is to provide a polypeptide composition for the novel coronavirus.

[0008] The present invention adopts the following technical solution: A polypeptide composition for a novel coronavirus, comprising the following four polypeptides: The first polypeptide, named GY, has the amino acid sequence shown in SEQ ID NO:1; The second polypeptide, named VV, has the amino acid sequence shown in SEQ ID NO:2; The third polypeptide, named VS, has the amino acid sequence shown in SEQ ID NO:3; The fourth polypeptide, named DL, has the amino acid sequence shown in SEQ ID NO:4.

[0009] A second objective of the present invention is to provide a vaccine composition comprising the polypeptide composition described above and an immunologically or pharmaceutically acceptable adjuvant.

[0010] Preferably, the adjuvant is selected from any one or more of inorganic adjuvants, oil emulsions, and TLR agonists.

[0011] Preferably, the inorganic adjuvant includes, but is not limited to, any one or more combinations of aluminum hydroxide, aluminum phosphate, aluminum sulfate, aluminum hydroxyphosphate, ammonium aluminum sulfate, and potassium aluminum sulfate; the oil emulsion includes, but is not limited to, any one or more combinations of MF59 and AS03; and the TLR agonist includes, but is not limited to, any one or more combinations of CpG-ODN, CpG1018, and MPL.

[0012] Preferably, the adjuvant is a compound adjuvant, including but not limited to any one or more combinations of AS01, AS04, Matrix-M, and 720VG.

[0013] More preferably, the adjuvant is 720VG.

[0014] A third objective of this invention is to provide the use of the polypeptide composition described above in the preparation of an agent for the prevention or treatment of novel coronavirus infection.

[0015] Preferably, the formulation is a pharmaceutically acceptable effective dose, and the dosage form of the formulation is selected from pharmaceutically acceptable dosage forms, including but not limited to injections, lyophilized powder injections, suspensions, etc.

[0016] Preferably, the formulation is a vaccine enhancer.

[0017] Preferably, the polypeptide can also be a derived polypeptide formed by selectively adding, substituting, or deleting one or more amino acids of the GY, VV, VS, and DL polypeptides as described above, and the derived polypeptide has the same or substantially the same function as the GY, VV, VS, and DL polypeptides.

[0018] The fourth objective of this invention is to provide the nucleic acid encoding the polypeptide as described above.

[0019] The fifth objective of this invention is to provide an expression vector comprising the encoded nucleic acid as described above.

[0020] The sixth object of the present invention is to provide a host cell comprising the expression vector described above.

[0021] The beneficial effects of this invention are as follows: This invention screened four highly conserved and potentially immunogenic peptides from the SARS-CoV-2 spike protein, named GY, VV, VS, and DL, respectively. Experiments showed that these peptides or combinations thereof can effectively induce T-cell immune responses and specific humoral immunity against SARS-CoV-2 and its variants, stimulating the body to produce antigen-specific antibodies. This provides a foundation for constructing high-affinity, high-quality, and highly specific SARS-CoV-2 preventive and / or therapeutic antibodies, and has high application value for the prevention and treatment of the novel coronavirus. Attached Figure Description

[0022] Figure 1 This is a screening map of antigenic epitopes for the novel coronavirus spike protein. In the map, A represents the SARS-CoV-2 spike protein neutralizing antibody epitope map, and B represents the epitope map targeting HLA-A. 02:01 allele of the spike protein CD8+ T-cell epitope atlas.

[0023] Figure 2 The diagram shows the positions and sequence conservation analysis of four candidate peptides in the spike protein. Figure A is a schematic diagram of the spike protein domain and the distribution of candidate peptide positions. The upper half of Figure BE shows the amino acid sequence alignment results of the four long peptides GY, VV, VS, and DL in the original SARS-CoV-2 strain and various variant strains. The lower half of Figure BE shows the sequence homology alignment analysis of the four long peptides GY, VV, VS, and DL in SARS-CoV-2 and other human coronaviruses.

[0024] Figure 3 The figure shows the inhibitory activity of different long peptides against coronavirus-mediated cell fusion. In the figure, AD represents the inhibitory results of four long peptides: DL, VV, VS, and GY, respectively.

[0025] Figure 4 The results show the detection of the immune response induced in mice by the tetrapeptide combination vaccine. In the figure, A represents the specific IgG antibody levels of the four long peptides GY, VV, VS, and DL in the serum of immunized mice, B represents the cell fusion inhibition rate of different coronaviruses in the serum of immunized mice, and C represents the neutralization rate of different pseudoviruses in the serum of mice detected by the pseudovirus neutralization experiment.

[0026] Figure 5 The results show the detection of local activation and functional differentiation of T cells induced by the tetrapeptide vaccine composition. In the figure, A represents CD4 in lung tissue. + T cells, CD8 + T cell activation rate, B represents CD4+ in spleen tissue. + T cells, CD8 + T cell activation percentage, C represents CD4+ after peptide pool stimulation. + Analysis of functional factor secretion in T cells, D represents CD8 after peptide pool stimulation. + Analysis of functional factor secretion from T cells; in the figure, 4-1BB + OX40 + %CD4 + T cells, 4-1BB + CD40L + %CD4 + T cells, 4-1BB + CD69 + %CD8 + T cells represent the proportion of T cells expressing the corresponding activating or co-stimulatory molecules, reflecting the antigen-specific activation level of T cells; among them, IFN-γ + %CD4 + T cells, TNF-α + %CD4 + T cells, IL-2+ %CD4 + T cells are CD4 cells that secrete IFN-γ, TNF-α, and IL-2. + The proportion of T cells, IFN-γ + %CD8 + T cells, TNF-α + %CD8 + T cells, IL-2 + %CD8 + T cells are CD8 cells that secrete IFN-γ, TNF-α, and IL-2. + The proportion of T cells.

[0027] Figure 6 The results show the detection of T cell activation and germinal center response induced by the tetrapeptide combination vaccine. In the figure, A from left to right represents the percentage of B cells and CD3+ in the bronchoalveolar lavage fluid. + T cell percentage, CD4 + T cells account for CD3 + The percentage of T cells, CD8 + T cells account for CD3 + The percentage of T cells is shown in Figure 1. B represents the proportion of tissue-resident memory T cell subsets in lung tissue, C represents the proportion of tissue-resident memory T cell subsets in spleen tissue, and D represents the proportion of germinal center-related immune cells in the spleen. The vertical axis represents CD62L. - CD69 + CD44 + %CD4 + CD4 representing lung / spleen tissue + Activated, resident, and functionally active memory CD4+ cells in T cells + T cell percentage; CD62L - CD103 + %CD8 + CD69 + CD44 + Represents memory CD8 cells that are anchored and reside in lung / spleen tissues and have cytotoxic functions. + T cell percentage; GL-7 + Fas + %CD19 + Bcells represent CD19 in the spleen. + The percentage of germinal center B cells in the activated and maturing stage among B cells; PD-1 + CXCR5 + %CD4 + Tcells represent spleen CD4 + The percentage of follicular helper T cells (Tfh) that assist B cell maturation among T cells.

[0028] Figure 7 The figures show the results of the challenge protection effect of the tetrapeptide combination vaccine booster immunization. In the figure, A is a schematic diagram of the immunization and challenge process of the SARS-CoV-2KP.2 challenge experiment, B is the result of the viral load detection in the lung tissue of mice after challenge, C is the result of H&E staining in the lung tissue of mice after challenge, D is the pathological damage score of the lung tissue of mice after challenge, E is a schematic diagram of the HCoV-229E challenge experiment, and FH is the result of the HCoV-229E challenge experiment, with the same meaning as BD.

[0029] Figure 8 This figure shows the results of a comparative assay of the immunogenicity of a tetrapeptide combination and a single peptide. Figure A represents the concentration of CD4 in the spleen of different immunization groups. + T cells and CD8 + The proportion of T cells that produce IFN-γ positive cells after stimulation with different peptide pools, where B represents the cell fusion inhibition rate of immune serum against different coronaviruses.

[0030] In the figure, asterisks indicate statistical differences. express P < 0.01、 express P < 0.05 express P < 0.001 P < 0.0001. Detailed Implementation

[0031] To facilitate understanding, the technical solution of the present invention will be described in more detail below with reference to the embodiments.

[0032] Example 1

[0033] Analysis of peptide synthesis and conservation

[0034] Download the currently reported linear neutralizing antibody epitopes of SARS-CoV-2 Spike detected using cell experiments from the IEDB database (https: / / www.iedb.org / ). Then, use ImmunomeBrowser (http: / / tools.iedb.org / immunomebrowser / ) to perform systematic analysis and summarization of these cellular epitopes, constructing a linear neutralizing antibody epitope map of the Spike protein, such as... Figure 1 As shown in Figure A.

[0035] See Figure 1In the A region, three experimentally validated functional regions were identified: S440-519 (RBD region), S799-837, and S1137-1170. Due to the high mutation rate in the S440-519 region, the more conserved S799-837 and S1137-1170 regions were selected for further research.

[0036] Furthermore, considering the MHC restriction of T cell responses, targeting CD8... + T-cell epitopes: This application focuses on HLA-A, which is ubiquitous globally. 02:01 alleles were used to construct a Spike T cell epitope map targeting the HLA0201 subtype, such as... Figure 1 As shown in B. In HLA-A 02:01 Restricted CD8 + In the T-cell epitope atlas, two functional validation regions, S269-277 and S976-1008, were identified. Among them, S976-1008 was included in subsequent analysis due to its higher conservation.

[0037] In addition, based on previously reported strong fusion inhibition activity, the HR2-derived peptide HR2P (amino acids 1168–1203) was included in the candidate antigen range.

[0038] Based on the above screening and HR2P peptide analysis, the conservation of the original SARS-CoV-2 strain and some previously circulating variants (including Alpha, Beta, Gamma, Delta, BA.5, BF.7.1, XBB.1, CH.1, JN.1, and KP.2) and seven human-infecting coronaviruses (SARS, MERS, HKU1, OC43, NL63, and 229E) was compared with the screened peptide sequences using MEGA 7.0, ClustalX 2.1, and the ESPript 3.0 website (http: / / espript.ibcp.fr / ESPript / ESPript / ). Ultimately, four long peptides with high conservation and potential immunogenicity were successfully screened from the SARS-CoV-2 spike protein and named GY, VV, VS, and DL based on their flanking residues. Figure 2 (A), the information is as follows: The amino acid sequence of GY (799–837aa): GFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQY (SEQ ID NO: 1); The amino acid sequence of VV (976-1008 aa): VLNDILSRLDKVEAEVQIDRLITGRLQSLQTYV (SEQ ID NO:2); The amino acid sequence of VS (1137-1170 aa): VYDPLQPELDSFKEELDKYFKNHTSPDVDLGDIS (SEQ ID NO:3); The amino acid sequence of DL (1168–1203 aa): DISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL (SEQ ID NO: 4).

[0039] These four peptides are highly conserved in circulating SARS-CoV-2 strains. Among other coronaviruses, VV shows the highest conservation, followed by DL, while VS and GY show relatively low conservation. Figure 2 (BE).

[0040] Example 2

[0041] Peptide-cell fusion inhibition experiment

[0042] The four peptides GY, VV, VS, and DL are all located within the S2 subunit of the spike protein, a key domain mediating viral-cell membrane fusion. To evaluate the inhibitory effect of the screened peptides on coronavirus-mediated cell fusion, the following experimental procedure was used to assess their ability to inhibit cell fusion: HEK293T cells (human kidney epithelial cell line) were co-transfected for 24 hours with different coronavirus spike protein particles (pcDNA3.1-SARS-CoV-2-S, -Omicron-S, or -NL63-S-ΔC18) and pN-1-eGFP plasmid (using Beyotime Lipo8000 transfection reagent, catalog number C0533). Cells were then digested with 0.125% trypsin and counted. The cell density was adjusted to 1 × 10⁶ cells per well. 4 Afterwards, long peptides GY, VV, VS, and DL (20 μM, 2 μM, or 0.2 μM) were added to the culture medium and incubated for 1 hour at 5% CO2 and 37°C. GY, VV, VS, and DL were synthesized by Hanyu Pharmaceutical under commission, with a purity greater than 95%. VS was dissolved in 1×PBS containing 10% DMSO, DL was dissolved in DMSO, and GY and VV were dissolved in 1×PBS. HEK293T-hACE2 cells (HEK293T cells stably expressing hACE2) were pre-filled at 2×10⁶ cells per well. 4Seeds were incubated at a density of [number] cells per cell, and allowed to adhere for 6 hours. After incubation, pretreated HEK293T cells were added to HEK293T-hACE2 monolayer cells and co-cultured for another 8 hours. Cell fusion was observed using a fluorescence microscope. Five random fields of view were selected from each experimental group for analysis, and the proportion of fused cells was calculated. The cell fusion inhibition rate was calculated using the formula: Inhibition rate (%) = [1 - (average fusion rate of experimental groups / average fusion rate of untreated control groups)] × 100.

[0043] Experimental results showed that peptide DL exhibited significant and dose-dependent inhibitory activity in inhibiting cell fusion mediated by the SARS-CoV-2 spike protein. In a cell fusion model expressing the SARS-CoV-2 prototype spike protein, peptide DL achieved approximately 80% fusion inhibition at a concentration of 2 μM, and even at concentrations as low as 0.2 μM, it maintained an inhibitory effect of approximately 40–60%, demonstrating excellent inhibitory efficacy and good low-dose activity. Figure 3 (A). Similar inhibitory effects were also observed in the cell fusion model of the Omeprone mutant strain B.1.1.529, indicating that the DL peptide has a stable and effective fusion inhibition ability against different SARS-CoV-2 mutant strains. In contrast, although GY, VV, and VS long peptides also showed some cell fusion inhibition effects, they failed to reach an inhibitory level comparable to that of the DL peptide. Figure 3 (BD), suggesting that DL peptides have unique advantages in structure and function.

[0044] In experimental models involving other human coronavirus spike proteins, the DL peptide also exhibited a certain degree of fusion inhibition activity, with the inhibition efficiency varying depending on the spike protein sequence. These results collectively indicate that the DL peptide is a highly efficient fusion inhibition candidate molecule against SARS-CoV-2 and its variants, possessing good development potential in applications related to anti-coronavirus infection.

[0045] Example 3

[0046] Vaccine immunogenicity test

[0047] Flow cytometry antibodies were purchased from Biolegend, HPR-labeled goat anti-mouse IgG was purchased from Sangon Biotech, 6-8 week old female C57BL / 6 mice were purchased from Jiangsu Jicui Pharmaceutical Co., Ltd., and the immune adjuvant Montanide ISA 720VG was a stable water-in-oil emulsion provided by Hanyu Pharmaceutical Co., Ltd.

[0048] 1. Specific IgG antibody ELISA detection

[0049] To evaluate the level of specific IgG antibodies induced in mice after immunization with the tetrapeptide vaccine, the following experimental procedure was used: Female C57BL / 6 mice aged 6-8 weeks were intramuscularly injected with a reagent containing a polypeptide mixture on days 0, 14, and 28, with a total injection volume of 100 μL per mouse. This polypeptide mixture was prepared by emulsifying a polypeptide mixture (50 µg each of GY, VV, VS, and DL) with Montanide ISA 720VG adjuvant at a 3:7 volume ratio. The control group was injected with either 720VG-PBS emulsion or PBS alone. On day 14 after the third immunization, blood was collected for ELISA testing. Whole blood was coagulated at room temperature for 2 hours, followed by centrifugation at 5000×g for 10 minutes to separate serum. Serum was aliquoted and stored at -80℃ for later use. During the experiment, 1 μg of each of the four polypeptides was spread onto microplates and incubated at room temperature for 2 hours. After washing three times with PBS, the wells were blocked with PBS solution containing 5% skim milk for 2 hours. After washing again, serum samples diluted with 5% skim milk were added, and the plate was incubated at room temperature with shaking for 1 hour (80 rpm). After washing the plate, HRP-labeled goat anti-mouse IgG secondary antibody (1:10,000 diluted in 5% skim milk, Sangon Biotech, catalog number D110087) was added, and the plate was incubated for another hour under the same shaking conditions. After three final washes, 100 μL of TMB substrate (Beyotime, catalog number P0209) was added to each well, and the plate was incubated in the dark for 8 minutes. After the reaction was terminated, 50 μL of 1 M H2SO4 stop solution was added, and the absorbance at 450 nm was immediately measured to analyze the level of specific IgG antibodies.

[0050] See results Figure 4 The results showed that specific IgG antibodies against the GY, VS, and DL peptides were present in the serum, with the DL peptide showing the highest antibody titer. Since the VV long peptide only possesses T-cell epitopes and can only induce cellular immunity, it failed to induce antibody production. These results indicate that the vaccine containing the four peptides can effectively induce a specific humoral immune response in mice targeting the vaccine antigen.

[0051] 2. Mouse serum cell fusion inhibition experiment

[0052] To evaluate the inhibitory effect of mouse serum immunized with the tetrapeptide on coronavirus-mediated cell fusion, the following experimental procedure was used to assess its ability to inhibit cell fusion: HEK293T cells (human kidney epithelial cell line) were co-transfected for 24 hours with different coronavirus spike protein particles (pcDNA3.1-SARS-CoV-2-S, -Omicron-S, or -NL63-S-ΔC18) and pN-1-eGFP plasmid (using Beyotime Lipo8000 transfection reagent, catalog number C0533). Cells were then digested with 0.125% trypsin and counted. The cell density was adjusted to 1 × 10⁶ cells per well. 4 Afterwards, 10% complement-inactivated immunized mouse serum was added, and the cells were incubated with the culture medium at 5% CO2 and 37°C for 1 hour. HEK293T-hACE2 cells (HEK293T cells stably expressing hACE2) were pre-filled at 2 × 10⁶ cells per well. 4 Seeds were incubated at a density of [number] cells per cell, and allowed to adhere for 6 hours. After incubation, pretreated HEK293T cells were added to HEK293T-hACE2 monolayer cells and co-cultured for another 8 hours. Cell fusion was observed using a fluorescence microscope. Five random fields of view were selected from each experimental group for analysis, and the proportion of fused cells was calculated. The cell fusion inhibition rate was calculated using the formula: Inhibition rate (%) = [1 - (average fusion rate of experimental groups / average fusion rate of untreated control groups)] × 100.

[0053] See Figure 4 Functional evaluation by cell fusion inhibition assay showed that, compared with the control group containing only adjuvant, 10-fold dilution of immunized mouse serum significantly inhibited cell fusion mediated by multiple coronaviruses (including SARS-CoV-2 prototype, Delta, Omicron B.1.1.529 and HCoV-NL63 spike protein).

[0054] 3. Pseudovirus neutralization experiment

[0055] To evaluate the neutralizing effect of mouse serum immunized with tetrapeptide on pseudoviruses expressing the coronavirus S protein, the following experimental procedure was used: HEK293T cells were seeded at 70% confluence in 10 cm culture dishes and cultured using Lipo8000. TM Transfection reagent (Beyotime, C0533) was used to co-transfect pNL4-3-Luc-RE plasmid and spike protein encoding plasmid (pcDNA3.1-SARS-CoV-2-S-Delta or Omicron-S). Supernatant containing pseudovirus was collected 48 hours later. In the neutralization experiment, 10% diluted mouse serum was mixed with an equal volume of pseudovirus supernatant containing 5 μg / mL Polybrene (Yisheng, 40804ES76) and incubated at 37°C and 5% CO2 for 1 hour. HEK293T-hACE2 cells were pre-transfected 24 hours prior to the experiment with 1×10⁻⁶ cells per well. 4Cells were seeded at a density of [insert density here] in 96-well plates. After neutralization, the mixture was added dropwise to the cell surface and incubated for 48 hours. The supernatant was then removed, and the cells were gently washed with PBS. Britelite Plus luciferase reagent (PerkinElmer) was added, and after incubation in the dark, the relative optical units (RLU) were measured using a PE Ensight multimode microplate reader.

[0056] The neutralization percentage is calculated as follows: Neutralization rate (%) = [1 - (Experimental group RLU / Virus control group RLU)] × 100%, where the experimental group RLU represents the serum-containing wells and the virus control group RLU is the average value of the wells containing only pseudovirus.

[0057] See Figure 4 In the C-test, the pseudovirus neutralization experiment showed that although the overall neutralizing antibody level induced by the vaccine was moderate, the serum of the vaccine-treated mice showed significant neutralizing effects against both Delta and Omicron B.1.1.529 variants, demonstrating that the specific antibodies induced by the vaccine have functional virus-neutralizing activity.

[0058] In summary, the serum of the vaccine group not only contains specific antibodies that can recognize vaccine antigens, but also effectively inhibits viral fusion and neutralizes multiple SARS-CoV-2 variants.

[0059] 4. Flow cytometry

[0060] To verify the technical efficacy of the polypeptide immunotherapy composition described in this invention in inducing antigen-specific cellular immunity and local respiratory immunity, an adjuvant-only immunotherapy group was used as a control group. T cell activation status, effector function, and tissue residency characteristics were systematically analyzed using antigen stimulation combined with multi-parameter flow cytometry. All operations were performed on a CytoFLEX flow cytometer (Beckman Coulter), and data were analyzed using FlowJo software (version 10).

[0061] Two weeks after the final immunization, mice were euthanized, and bronchoalveolar lavage fluid, lung tissue, and spleen were collected. Bronchoalveolar lavage fluid was centrifuged at 500×g for 5 minutes to collect cells. Lung tissue was digested for 1 hour at 37°C and 100 rpm in RPMI 1640 solution containing 5% fetal bovine serum and 1 mg / mL type IV collagenase (Sigma-Aldrich, C5138-5G). The digested lung tissue was further homogenized by pipetting and filtered through a 70 μm filter. Red blood cells were then lysed using erythrocyte lysis buffer (Beyotime, catalog number C3702). After several minutes of reaction, the cells were centrifuged at 500 g for 5 minutes, the supernatant was discarded, and the cells were resuspended in 1×PBS to obtain a single-cell suspension. The spleen was ground and then processed into a single-cell suspension using the same method via red blood cell lysis.

[0062] (1) Detection of antigen stimulation and activation-inducible marker (AIM)

[0063] To detect antigen-specific T cell activation, single-cell suspensions from the spleen and lungs were co-incubated with the antigen peptide pool (10 μg / mL for each peptide) in Table 1 for 24 hours to stimulate the expression of activation-inducing markers. After stimulation, the expression of activation-inducing markers was analyzed by flow cytometry. During the assay, cells were first used to block the Fc receptor with anti-CD16 / 32 antibody (catalog number 101302). After incubation on ice for 15 minutes, cell viability was marked using the Zombie Red™ Fixable Viability Kit (catalog number 423109). Subsequently, surface antibodies against CD3 (PerCP / Cy5.5, 100328), CD4 (APC / Cy7, 100x525), CD8 (BV510, 100752), OX40 (PE, 119409), CD69 (FITC, 104505), and 4-1BB (APC, 106109) were used for staining, followed by incubation on ice in the dark for 1 hour. After two washes with PBS (500×g, 5 minutes each), cells were resuspended in 100 μL PBS for flow cytometry analysis. The analysis was performed by detecting CD4+. + Co-expression of OX40 and CD69 in T cells, and CD8 + Co-expression of 4-1BB and CD69 in T cells was used to assess the activation level of antigen-specific T cells.

[0064] The results showed that, compared with the control group, the number of AIM-positive CD4+ cells in the lung tissue of mice in the peptide-immunized group was significantly higher. + T cells and CD8 + The proportion of T cells increased significantly ( Figure 5 (A), and AIM-positive CD8 in the spleen + The number of T cells also increased significantly. Figure 5 (See section B), indicating that the polypeptide immunization strategy of the present invention can effectively induce antigen-specific T cell activation, and its performance is particularly outstanding in local lung immunity.

[0065] (2) Cytokine function detection

[0066] Each polypeptide (long peptide) was segmented into multiple peptide segments to form a peptide pool. See Table 1 for details.

[0067] To evaluate T cell effector function, spleen single-cell suspensions were stimulated in vitro using different long peptide pools, with protein secretion inhibitors added during stimulation to enrich intracellular cytokines. After stimulation, cells were surface stained, fixed, and subjected to transmembrane treatment, followed by intracellular staining using fluorescently labeled antibodies against IFN-γ, TNF-α, and IL-2. CD4 counts were analyzed by flow cytometry. + and CD8 + The expression levels of cytokines in T cells.

[0068] Spleen single-cell suspensions were co-cultured with the peptide pool shown in Table 1 (10 µg / mL for each peptide) for 24 hours to stimulate cytokine production. After 18 hours, brefeldin A (BFA) (Biolegend, 420701) and monensin (Biolegend 420601) were added to inhibit cytokine secretion. Cells were then collected for staining.

[0069] Table 1 Peptide Pool Information

[0070] Cells were blocked on ice for 15 minutes with anti-mouse CD16 / 32 antibody. Zombie Red was then used. TM Immobilized live cell assay kit (423109) was used for staining on ice in the dark for 1 hour, with surface antibodies against CD3 (APC / Cy7, 100222), CD4 (FITC, 100406), and CD8 (PerCP / Cy5.5, 100734) added simultaneously. After washing with PBS, cells were fixed and permeabilized using a BDBiosciences kit (554714). After centrifugation, intracellular staining was performed on ice in the dark for 1 hour with IFN-γ (BV421, 505830), TNF-α (APC, 506308), and IL-2 (PE, 503808; all purchased from BioLegend) antibodies diluted with permeabilization buffer. Finally, cells were washed twice with PBS and resuspended in 100 μL PBS for flow cytometry analysis.

[0071] The results showed that the VS, VV, and GY peptide pools could all significantly enhance CD4. + The expression levels of IFN-γ, TNF-α, and IL-2 in T cells were analyzed, indicating that they can induce CD4+, a cell with multifunctional characteristics. + T-cell immune response. (In CD8) + In T cells, VS peptide stimulation significantly increased the proportion of IFN-γ positive cells, while VV peptide stimulation significantly enhanced the expression intensity of IFN-γ in single cells. Furthermore, VS and GY peptide stimulation also promoted CD8 expression. +The increasing trend of TNF-α and IL-2 expression levels in T cells ( Figure 5 (C and D) further demonstrate that the polypeptide composition of the present invention can enhance the effector function of cytotoxic T cells.

[0072] (3) Establishment of pulmonary mucosal immunity

[0073] To evaluate the induction effect of the polypeptide composition of the present invention on local respiratory tract immunity, immune cell subset analysis was performed on mouse bronchoalveolar lavage fluid, lung tissue and spleen samples.

[0074] Flow cytometry was used to detect B cells and CD3 in bronchoalveolar lavage fluid. + T cells, CD4 + T cells and CD8 + The proportion of T cells was used to evaluate vaccine-induced recruitment of pulmonary mucosal immune cells. Further analysis was conducted by detecting markers such as CD62L, CD69, CD44, and CD103 to determine tissue-resident memory CD4+ in lung tissue. + Cells (CD4) + T RM cells) and tissue-resident memory CD8 + T cells (CD8) + T RM The proportion of respiratory tract local immunity and tissue-resident memory T cell responses was used to assess the ratio of these cells.

[0075] Cells were first incubated on ice with anti-mouse CD16 / 32 antibody (catalog number 101302) for 15 min to block Fc receptors; then, dead and live cells were stained using the Zombie Red™ Fixable Viability Kit; and finally, the cells were stained with the following antibody mixture for 1 h under light-protected conditions. For cells in bronchoalveolar lavage fluid, anti-mouse CD3 (APC / Cy7, catalog number 557596), CD4 (FITC, catalog number 100406), CD8 (BV510, catalog number 100752), and B220 (PE, catalog number B33718) were used. For lung and spleen cells, anti-mouse CD3 (PerCP / Cy5.5, catalog number 100328), CD4 (APC / Cy7, catalog number 100525), CD8 (BV510, catalog number 100752), CD44 (PE, catalog number 103007), CD62L (BV421, catalog number 104436), CD69 (FITC, catalog number 104505), and CD103 (APC, catalog number 110905; all purchased from BioLegend) were used. After staining, the cells were washed twice by centrifugation at 500×g for 5 min with PBS buffer, then resuspended in 100 µL PBS and flow cytometry data were acquired.

[0076] See results Figure 6 Analysis of bronchoalveolar lavage fluid showed that, compared with the 720VG adjuvant control group, the levels of B cells and CD3+ in the bronchoalveolar lavage fluid of mice in the peptide-immunized group were significantly higher. + The proportion of T cells all showed an increasing trend, among which CD4 + T cells in CD3 + The proportion of T cells increased significantly, while CD8 cells... + The proportion of T cells also showed an increasing trend. Figure 6 (A) suggests that this immunization strategy can promote the recruitment of local immune cells in the respiratory tract, especially beneficial for lung CD4 cells. + The establishment of T cell responses.

[0077] In lung tissue, tissue-resident memory CD4 + The proportion of T cells was significantly increased, and CD8+ + T RM The number of cells also showed an upward trend. Figure 6 (B in the middle); while no corresponding T was observed in the spleen. RM Significant increase in cells ( Figure 6 The C indicates that this immunization strategy can preferentially induce tissue-resident immunity in the local respiratory tract.

[0078] (4) Evaluation of germinal center response and T cell helper function

[0079] In spleen samples, follicular helper T cells (Tfh cells) were identified using CXCR5 and PD-1 markers, and the proportion of germinal center B cells (GC B cells) was detected using GL7 and Fas markers to assess relevant indicators of T cell-assisted humoral immune responses.

[0080] Cells were first incubated on ice with anti-mouse CD16 / 32 antibody (catalog number 101302) for 15 minutes to block Fc receptors, Zombie Red. TM Cell viability was determined by staining with a Fixable Viability Kit, followed by staining for 1 hour with the following antibody mixtures under light-protected conditions: anti-mouse CD19 (APC / Cy7, catalog number 152411), CD4 (FITC, catalog number 100406), CXCR5 (PE, catalog number 145503), PD-1 (APC, catalog number 109110), GL7 (PE / Cy7, catalog number 144620), and Fas (PerCP / Cy5.5, catalog number 152609). After staining, cells were washed twice by centrifugation at 500×g for 5 minutes with PBS buffer, and then resuspended in 100 µL PBS before data acquisition.

[0081] See results Figure 6Compared with the control group, the proportion of follicular helper T cells in the spleen of mice in the polypeptide immunization group was significantly increased, and the proportion of germinal center B cells showed an upward trend. Figure 6 The results (D) suggest that this immunization strategy can effectively promote germinal center response and is beneficial to the formation and maturation of humoral immune response.

[0082] In summary, the polypeptide composition provided in this application can significantly enhance lung tissue resident memory immunity while inducing a potent antigen-specific T cell response, and synergistically promote the formation of humoral immune responses. It can induce a comprehensive immune response covering humoral and cellular immunity in vivo, and exert its effects simultaneously at the levels of systemic immunity and local respiratory immunity, demonstrating good immunogenicity and providing experimental evidence for its application as a coronavirus vaccine.

[0083] Example 4

[0084] Virus challenge protection experiment

[0085] Female BALB / c mice aged 6-8 weeks were purchased from Jiangsu Jicui Pharmaceutical Biotechnology Co., Ltd., and K18-hACE2 transgenic mice were kindly provided by Professor Zhao's laboratory at the Beijing Institute of Microbiology and Epidemiology. The inactivated vaccine was provided by Shenzhen Kangtai Biological Products Co., Ltd. The SARS-CoV-2 KP.2 variant and HCoV-229E virus strain were kindly provided by the ABSL-3 laboratory of the University of Science and Technology of China and Professor Sandra Chiu's research team, respectively.

[0086] Grouped according to the type of virus strain, K18-hACE2 transgenic mice were used for SARS-CoV-2 KP.2 variant infection experiments, and BALB / c mice were used for HCoV-229E infection experiments. Each virus model had three groups: control group (PBS), inactivated vaccine immunization group, and peptide combination vaccine booster immunization group (initial immunization with inactivated vaccine + booster immunization with peptide vaccine).

[0087] Immunization regimen: Mice were initially immunized on day 0 with an inactivated vaccine (20 units / dose), followed by booster immunizations on days 14 and 28 with a vaccine containing four polypeptides (GY+VV+VS+DL) as described in Example 3. Figure 7 A, E).

[0088] After immunization was completed, a viral challenge experiment was conducted 14 days after the last immunization.

[0089] 1. Detection of the protective effect of SARS-CoV-2 KP.2 variant strain against virus challenge

[0090] Given the continued prevalence of the Omecjung subtype variant, this embodiment focuses on investigating the protective effect of the polypeptide combination vaccine described in this invention against the SARS-CoV-2 KP.2 variant.

[0091] In the SARS-CoV-2 KP.2 mutant strain challenge experiment, K18-hACE2 transgenic mice were infected with the SARS-CoV-2 KP.2 mutant strain via intranasal drip on day 14 after the last immunization, with an infection dose of 5 × 10⁻⁶. 4.25 TCID 50 Lung tissue samples were collected on the fourth day post-infection and processed in two parts. One part was used to extract RNA, and the expression level of the SARS-CoV-2 ORF1ab gene was detected by real-time quantitative PCR using the Vazyme RM501 and Q221 kits, with primers 5′-ccctgtggggttttacacttaa-3′ (forward primer) and 5′-acgattgtgcatcagctga-3′ (reverse primer). The other part was fixed in 4% paraformaldehyde, then embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E) to assess the pathological damage to the lungs.

[0092] The results showed that the viral load in lung tissue was measured 4 days after infection. Figure 7 Compared with the PBS control group and the inactivated vaccine-only group, mice receiving a booster immunization with the peptide combination vaccine showed a significant reduction in viral load in the lungs. Histopathological examination of H&E-stained lung tissue sections revealed significantly reduced tissue damage in the booster immunization group. Compared with the control group, mice immunized with the peptide vaccine showed better preservation of alveolar structure, reduced bronchial hemorrhage, reduced exudate, and decreased inflammatory cell infiltration. Figure 7 (C, D)

[0093] 2. HCoV-229E challenge and protection efficacy detection

[0094] To further evaluate the broad-spectrum protective potential of the polypeptide combination vaccine described in this invention, its cross-protective effect against α-coronavirus HCoV-229E was assessed.

[0095] On day 14 following the final immunization, BALB / c mice were infected with HCoV-229E virus via intranasal instillation. Due to the low pathogenicity of HCoV-229E, a two-dose intranasal administration regimen with a 48-hour interval was used, with each dose being 5 × 10⁻⁶. 5 TCID 50 ( Figure 7E). Lung tissue samples were collected 48 hours after the second infection and processed in two parts. One part was used for RNA extraction, and the expression level of viral RNA-dependent RNA polymerase gene was detected by qRT-PCR to assess viral load. The kits used were Vazyme RM501 and Q221 kits, and the primers used were 5′-ttgactgttacgaggggtggc-3′ (forward) and 5′-ggccaaccagcgcttttatt-3′ (reverse). The other part of the lung tissue samples was fixed in 4% paraformaldehyde, then embedded in paraffin, sectioned, and stained with H&E to assess the degree of lung inflammation and damage.

[0096] The results showed that, compared with the PBS control group and the inactivated vaccine-only group, booster immunization with peptide vaccine significantly reduced viral load in the lungs of challenged mice (F in Figure 7). Histopathological evaluation showed that lung injury was significantly reduced in the peptide vaccine-immunized group, manifested as reduced inflammatory cell infiltration, reduced bronchial exudate, and reduced alveolar destruction (G and H in Figure 7).

[0097] In summary, the peptide vaccine booster immunization regimen significantly reduced viral load in the lungs and alleviated lung pathological changes after infection with both SARS-CoV-2 KP.2 and HCoV-229E, demonstrating its potential as a cross-protective vaccine strategy.

[0098] Example 5

[0099] Immunological effects of peptide compositions versus single peptides

[0100] The applicant's previous patents disclosed single peptides VV and VS, as well as a recombinant protein VV-HR2P-VS containing DL, as detailed in patents CN118561967A, CN118638195A, and CN118834278A. These technical solutions demonstrate the application potential of related single peptides or fusion proteins in the prevention and treatment of coronaviruses. However, single peptide immunization strategies typically only cover a limited number of antigenic epitopes, resulting in a relatively insufficient breadth of the induced immune response. This may lead to limited protection against viral mutations and different coronavirus strains. Especially for pathogens like coronaviruses that are constantly evolving and capable of cross-species transmission, relying solely on a single epitope or functional region for immune intervention makes it difficult to simultaneously ensure immune strength, immune breadth, and cross-protection capabilities.

[0101] To evaluate the immunization effect of the polypeptide combination vaccine described in this invention and to verify its advantages over the single peptide immunization strategy, a comparative experiment was conducted between the tetrapeptide combination immunization group and the single peptide immunization group.

[0102] Female C57BL / 6 mice aged 6-8 weeks were randomly divided into four groups of five mice each: a tetrapeptide combination immunization group (GY+VV+VS+DL), a VV single-peptide immunization group, a VS single-peptide immunization group, and an adjuvant control group. Montanide ISA720VG adjuvant was used as the immunizing agent, with the peptides and adjuvant emulsified at a 3:7 volume ratio. In the tetrapeptide combination immunization group, the immunization dose of each peptide was 50 μg; in the VV or VS single-peptide immunization group, the corresponding single-peptide immunization dose was 50 μg. The total injection volume per mouse was 100 μL. Maintaining a consistent peptide dose (50 μg / peptide) throughout the immunization experiments ensured that each candidate antigen adequately induced an immune response. Immunization was performed on days 0, 14, and 28 via intramuscular injection, with an injection volume of 100 μL per mouse. Serum and spleen samples were collected 14 days after the third immunization for subsequent immunological assays.

[0103] 1. Flow cytometry detection of T cell immune response

[0104] To verify the synergistic enhancing effect of the peptide combination in inducing antigen-specific cellular immunity, an immunization group using only adjuvant (PBS) (Mock) was used as a control. The immunogenicity differences between the single peptide group (VV or VS) and the tetrapeptide combination group (GY+DL+VV+VS) were compared. All flow cytometry data were analyzed on a CytoFLEX flow cytometer and processed using FlowJo software.

[0105] To evaluate the effect of the tetrapeptide combination on T cell effector function, single-cell suspensions of spleen cells from mice in each group were collected and co-cultured with the VV and VS peptide pools (10 µg / mL of each peptide) shown in Table 1 for 24 hours. After 18 hours of culture, brefedipine A (BFA) and monensin were added to inhibit cytokine secretion.

[0106] Subsequently, the cells were used to block the Fc receptor with anti-mouse CD16 / 32 antibody, and the cells were stained for live and dead cells using Zombie Red™ reagent. Then, surface antibodies against CD3 (APC / Cy7), CD4 (FITC), and CD8 (PerCP / Cy5.5) were added for staining. After fixation and permeabilization, intracellular staining was performed using an antibody against IFN-γ (BV421), and flow cytometry was used for analysis.

[0107] The results show that ( Figure 8 A) in CD4 + T cells and CD8 + In T cells, the proportion of IFN-γ-positive cells produced by the tetrapeptide combination immunotherapy group was significantly higher than that of the single peptide group after stimulation with either VV or VS peptide pools. This was especially true for CD8 cells. +In T cell responses, the tetrapeptide combination induced a highly significant fold increase in IFN-γ expression levels. p The value of < 0.001 indicates that the combined immunization with the tetrapeptide is not a simple additive effect, but rather a significant enhancement of the strength and quality of the body's cellular immunity through epitope synergistic stimulation.

[0108] 2. Cell-cell fusion inhibition experiments mediated by multiple coronaviruses

[0109] To verify the technological advantages of the tetrapeptide composition in generating broad-spectrum functional antibodies, this experiment evaluated the inhibitory effects of single-component (VV, VS) and tetrapeptide combination (GY+VV+VS+DL) immune sera on cell fusion mediated by different coronavirus spike proteins. The experimental methods are the same as those described in Example 3 regarding the mouse serum cell fusion inhibition experiment.

[0110] The results show that ( Figure 8 In the case of SARS-CoV-2 prototype and variant strains (Omicron), VV or VS single-peptide immunization groups showed only limited fusion inhibitory activity (approximately 20%-40%); while tetrapeptide combination immunized serum showed stronger inhibitory ability, with an inhibition rate of over 75%. p < 0.0001). Furthermore, only the tetrapeptide combination immunization group induced highly efficient neutralizing activity (approximately 70%) against the cross-species coronavirus HCoV-NL63, while the single component showed weak inhibitory effects. These results indicate that the tetrapeptide combination has a significant advantage over the single component in inhibiting coronavirus spike protein-mediated cell fusion and demonstrates broader potential in combating viral variants and cross-species coronaviruses.

[0111] The above comparative experiments demonstrate that the polypeptide composition (GY+VV+VS+DL) provided by this invention is significantly superior to single components in inducing cellular immunity through IFN-γ secretion and in inhibiting cell fusion mediated by broad-spectrum coronaviruses.

[0112] Analysis of the experimental results suggests that the enhanced immune response primarily stemmed from an increase in the number of antigenic epitopes, rather than an increase in the total amount of antigen. The four peptides in this invention originate from the fusion peptide (FP), the central helix (CH), and the HR2-related region, respectively. These domains play crucial roles in viral membrane fusion, and their combination can cover more functional epitopes, thereby enhancing the breadth and stability of the immune response. Therefore, based on the synergistic effect among the components, the tetrapeptide combination vaccine of this invention exhibits stronger immunogenicity and a broader potential protective spectrum.

[0113] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A polypeptide composition for a novel coronavirus, characterized in that, It contains the following four peptides: The first polypeptide, named GY, has the amino acid sequence shown in SEQ ID NO:1; The second polypeptide, named VV, has the amino acid sequence shown in SEQ ID NO:2; The third polypeptide, named VS, has the amino acid sequence shown in SEQ ID NO:3; The fourth polypeptide, named DL, has the amino acid sequence shown in SEQ ID NO:

4.

2. The use of the polypeptide composition of claim 1 in the preparation of an agent for the prevention or treatment of novel coronavirus infection.

3. A vaccine composition, characterized in that, It comprises the polypeptide composition as described in claim 1 and an immunologically or pharmaceutically acceptable adjuvant.

4. The vaccine composition according to claim 3, characterized in that, The adjuvant is selected from any one or more of inorganic adjuvants, oil emulsions, and TLR agonists.

5. The vaccine composition according to claim 4, characterized in that, The inorganic adjuvants include, but are not limited to, any one or more combinations of aluminum hydroxide, aluminum phosphate, aluminum sulfate, aluminum hydroxyphosphate, ammonium aluminum sulfate, and potassium aluminum sulfate; the oil emulsions include, but are not limited to, any one or more combinations of MF59 and AS03; and the TLR agonists include, but are not limited to, any one or more combinations of CpG-ODN, CpG1018, and MPL.

6. The vaccine composition according to claim 3, characterized in that, The adjuvant is a compound adjuvant, including but not limited to any one or more combinations of AS01, AS04, Matrix-M, and 720VG.

7. The vaccine composition according to claim 6, characterized in that, The adjuvant is 720VG.

8. The nucleic acid encoded by the polypeptide as described in claim 1.

9. An expression vector comprising the encoded nucleic acid as described in claim 8.

10. A host cell comprising the expression vector as described in claim 9.

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