Multiple epitope-based SARS-cov-2 vaccine composition

Abstract

The present application relates to a vaccine composition comprising peptides isolated from structural proteins of SARS-CoV-2.

Description

Multi-epitope-based SARS-CoV-2 vaccine composition

[0001] The present invention relates to a multi-epitope-based SARS-CoV-2 vaccine composition.

[0002] Understanding the immune response to the SARS-CoV-2 virus and developing COVID-19 vaccines hinges on the role of B and T cell epitopes. Epitopes are the portions of an antigen specifically recognized by lymphocytes.

[0003] B-cell epitopes are divided into linear epitopes and conformational epitopes. Linear epitopes are located contiguously in the primary sequence of a protein, whereas conformational epitopes are composed of amino acids that are adjacent to each other in the three-dimensional structure. The B-cell receptor (BCR) binds to these epitopes and induces antibody production.

[0004] T-cell epitopes are recognized by the T-cell receptor (TCR) as peptides bound to major histocompatibility complex (MHC) molecules. A single peptide can bind to multiple HLA allele variants, a phenomenon called a promiscuous epitope. Furthermore, structurally similar HLA variants are classified into HLA supertypes.

[0005] Immunodominance (ID) and immunoprevalence (IP) are concepts that indicate how strongly or frequently a specific antigen is recognized. These are influenced by various factors, including antigen expression level, stability, HLA binding capacity, and antigen processing.

[0006] In the context of SARS-CoV-2, studies have utilized proteomic data to infer the relative abundance of viral proteins. Furthermore, HLA-bound SARS-CoV-2-derived peptides have been isolated, but whether these are actually recognized by TCRs remains to be confirmed. It is well known that HLA binding is a necessary but not sufficient condition for T-cell recognition.

[0007] Therefore, it is necessary to accurately understand the characteristics of B cell and T cell epitopes and select epitopes with high immunodominance and immunoprevalence to develop an effective vaccine against SARS-CoV-2.

[0008] The background technology of this application, Republic of Korea Publication Patent No. 10-2023-0025670, relates to a SARS-COV-2 vaccine.

[0009] The present invention aims to solve the problems of the above-mentioned conventional technology and provides a vaccine composition comprising a peptide isolated from a structural protein of SARS-CoV-2.

[0010] In addition, a composition for preventing or treating SARS-CoV-2 comprising the above vaccine composition is provided.

[0011] In addition, a kit for preventing or treating SARS-CoV-2 comprising the above vaccine composition is provided.

[0012] However, the technical tasks to be achieved by the embodiments of the present invention are not limited to the technical tasks described above, and other technical tasks may exist.

[0013] As a technical means for achieving the above-mentioned technical task, the first aspect of the present invention provides a vaccine composition comprising a peptide isolated from structural proteins of SARS-CoV-2.

[0014] According to one embodiment of the present invention, the peptide may include, but is not limited to, one or more B cell epitopes.

[0015] According to one embodiment of the present invention, the B cell epitope may be linear or three-dimensional, but is not limited thereto.

[0016] According to one embodiment of the present invention, the structural protein of the SARS-CoV-2 may include, but is not limited to, a protein selected from the group consisting of a spike protein, a nucleocapsid protein, a membrane, and combinations thereof.

[0017] According to one embodiment of the present invention, the peptide isolated from the spike protein of the SARS-CoV-2 may include, but is not limited to, one or more sequences selected from the group consisting of sequence numbers 1 to 11.

[0018] According to one embodiment of the present invention, the peptide isolated from the nucleocapsid protein of the SARS-CoV-2 may include, but is not limited to, one or more sequences selected from the group consisting of SEQ ID NOs: 12 to 19.

[0019] According to one embodiment of the present invention, the peptide isolated from the membrane of the SARS-CoV-2 may include, but is not limited to, one or more sequences selected from the group consisting of SEQ ID NOs: 20 to 26.

[0020] According to one embodiment of the present invention, the vaccine composition may induce neutralizing antibodies against a variant selected from the group consisting of, but not limited to, Wuhan (Wu01), alpha, beta, gamma, Omicron (BA.1) and combinations thereof of SARS-CoV-2.

[0021] In addition, the second aspect of the present invention provides a composition for preventing or treating SARS-CoV-2 infection, comprising a vaccine composition according to the first aspect of the present invention.

[0022] In addition, the third aspect of the present invention provides a kit for preventing or treating SARS-CoV-2 infection, comprising a vaccine composition according to the first aspect of the present invention.

[0023] The above-described problem-solving methods are merely exemplary and should not be construed as limiting the present invention. In addition to the exemplary embodiments described above, additional embodiments may be included in the drawings and detailed description of the invention.

[0024] The vaccine composition according to the present invention can induce a strong immune response by including an immunodominant and highly immunoprevalent epitope isolated from the structural protein of SARS-CoV-2.

[0025] Additionally, the above epitopes can exhibit varying degrees of neutralizing efficacy against various mutations such as Wu01, alpha, beta, gamma, delta, and omicron, and by combining different epitopes, improved neutralizing efficacy can be exhibited against immune evasion mutations such as delta and omicron.

[0026] However, the effects that can be obtained from this center are not limited to the effects described above, and other effects may exist.

[0027] Figure 1a is a schematic diagram of the epitope selection and design strategy.

[0028] Figure 1b shows the clustering of monoclonal antibodies (mAbs) around the spike protein.

[0029] Figure 1c shows clustering around the spike protein RBD of FDA-approved and other clinically active SARS-CoV-2 neutralizing monoclonal antibodies.

[0030] Figure 1d shows B cell epitope selection based on RBM motifs.

[0031] Figure 1e shows the design of a SARS-CoV-2 N protein epitope via monoclonal antibody-antigen recognition.

[0032] Figure 2a shows the anti-peptide antibody titers of SARS-CoV-2 vaccinated individuals.

[0033] Figure 2b shows the immunodominance of spike and nucleocapsid-derived peptides.

[0034] Figure 2c is a structural superposition showing the cross-reactive immune response potential of the stem helix.

[0035] Figure 2d is a structural superposition diagram showing the potential for cross-reactive immune responses of fusion peptides.

[0036] Figure 2e shows the immunization scheme of selected B cell epitopes in BALB / c mice.

[0037] Figures 2f to 2i show the anti-peptide antibody titers and their cross-reactivity in immunized mice.

[0038] Figures 2j and 2k show that antibodies from immunized mouse sera inhibit infection against all major variants (VOCs) of the SARS-CoV-2 pseudovirus.

[0039] Figure 2l shows the effect of S1 antibodies in mouse sera immunized with S1 protein on various spike pseudovirus infections.

[0040] Figure 3a is a schematic diagram of a T cell epitope selection and design strategy.

[0041] Figures 3b and 3c are simplified heatmaps showing multiple HLA binding of MHC-I peptides.

[0042] Figure 3d shows the complex structure of the 0105-Ajou2-LA peptide with A02:01 and A11:01 HLA and the DSF results.

[0043] Figure 3e shows the complex structure of the 0082-Ajou2-NV peptide with A02:01 and B51:01 HLA and the DSF results.

[0044] Figure 3f shows the complex structure of the 0092-Ajou2-KF peptide and B51:01, B07:02 HLA and the DSF results.

[0045] Figure 3g is a graph showing the structure of AlphaFold 2-generated p-MHC complexes of the 0094-Ajou2-KY peptide and the HLA-C07:01 vs HLA-C07:02 and HLA-C07:01 vs HLA-B*15:01 haplotypes (top) and the results of DSF analysis of the same complexes (bottom).

[0046] Figure 4a is a bar graph showing the affinity of epitopes with HLA-A02:06 and HLA-A02:01.

[0047] Figure 4b is a bar graph showing the affinity of HLA-A*03:01 and four epitopes and a model showing the differences in binding pockets.

[0048] Figure 4c is a bar graph showing the affinity of peptides with HLA-A11:01 and HLA-A24:02.

[0049] Figures 4d and 4e show the binding affinity of HLA-B and the epitope peptide and the similarity of the anchor residues.

[0050] Figures 5a to 5f show HLA-II binding affinity and promiscuity modeled through AlphaFold 2.

[0051] Figures 6a and 6b show T cell responses to spike protein and peptide candidates for nine HLA class I and five HLA class II.

[0052] Figure 6c is a schematic representation of the mouse immunization protocol.

[0053] Figures 6d and 6e show IFN-γ secretion by class I and class II T lymphocytes in the spleen of immunized mice.

[0054] Figure 7 shows models predicted by AlphaFold 2 for the B cell peptides in Table 1.

[0055] Below, with reference to the attached drawings, embodiments of the present invention are described in detail to facilitate easy implementation by those skilled in the art. However, the present invention can be implemented in various different forms and is not limited to the embodiments described herein. In the drawings, irrelevant parts have been omitted for clarity, and similar reference numerals have been used throughout the specification to indicate similar elements.

[0056] Throughout this specification, when a part is said to be "connected" to another part, this includes not only cases where it is "directly connected" but also cases where it is "electrically connected" with another element in between.

[0057] Throughout this specification, when it is said that a member is located “on,” “above,” “upper,” “lower,” “lower” or “lower” another member, this includes not only cases where the member is in contact with the other member, but also cases where another member exists between the two members.

[0058] Throughout this specification, whenever a part is said to "include" a component, this does not mean that it excludes other components, but rather that it may include other components, unless otherwise specifically stated.

[0059] The terms "about," "substantially," and the like, as used herein, are used to mean at or near the numerical value when manufacturing and material tolerances inherent to the meanings referred to are presented, and are used to prevent unscrupulous infringers from unfairly exploiting disclosures that contain precise or absolute numerical values ​​to aid understanding of the present disclosure. Furthermore, throughout the present disclosure, the terms "step of ~" or "step of ~" do not mean "step for ~."

[0060] Throughout this specification, the term "combination thereof" included in the expressions in the Makushi format means one or more mixtures or combinations selected from the group consisting of the components described in the expressions in the Makushi format, and means including one or more selected from the group consisting of said components.

[0061] Throughout this specification, references to “A and / or B” mean “A, B, or A and B.”

[0062] Hereinafter, the vaccine composition of this application will be described in detail with reference to implementation examples, examples, and drawings. However, this application is not limited to these implementation examples, examples, and drawings.

[0063]

[0064] As a technical means for achieving the above-mentioned technical task, the first aspect of the present invention provides a vaccine composition comprising a peptide isolated from structural proteins of SARS-CoV-2.

[0065] The vaccine composition according to the present invention can induce a strong immune response by including an immunodominant and highly immunoprevalent epitope isolated from the structural protein of SARS-CoV-2.

[0066] Additionally, the above epitopes can exhibit varying degrees of neutralizing efficacy against various mutations such as Wu01, alpha, beta, gamma, delta, and omicron, and by combining different epitopes, improved neutralizing efficacy can be exhibited against immune evasion mutations such as delta and omicron.

[0067] According to one embodiment of the present invention, the peptide may include, but is not limited to, one or more B cell epitopes.

[0068] According to one embodiment of the present invention, the B cell epitope may be linear or three-dimensional, but is not limited thereto.

[0069] According to one embodiment of the present invention, the structural protein of the SARS-CoV-2 may include, but is not limited to, a protein selected from the group consisting of a spike protein, a nucleocapsid protein, a membrane, and combinations thereof.

[0070] According to one embodiment of the present invention, the peptide isolated from the spike protein of the SARS-CoV-2 may include, but is not limited to, one or more sequences selected from the group consisting of sequence numbers 1 to 11.

[0071] According to one embodiment of the present invention, the peptide isolated from the nucleocapsid protein of the SARS-CoV-2 may include, but is not limited to, one or more sequences selected from the group consisting of SEQ ID NOs: 12 to 19.

[0072] According to one embodiment of the present invention, the peptide isolated from the membrane of the SARS-CoV-2 may include, but is not limited to, one or more sequences selected from the group consisting of SEQ ID NOs: 20 to 26.

[0073] According to one embodiment of the present invention, the vaccine composition may induce neutralizing antibodies against a variant selected from the group consisting of, but not limited to, Wuhan (Wu01), alpha, beta, gamma, Omicron (BA.1) and combinations thereof of SARS-CoV-2.

[0074] In addition, the second aspect of the present invention provides a composition for preventing or treating SARS-CoV-2 infection, comprising a vaccine composition according to the first aspect of the present invention.

[0075] Regarding the composition for preventing or treating SARS-CoV-2 infection according to the second aspect of the present invention, detailed descriptions of parts overlapping with the first aspect of the present invention have been omitted. However, even if the descriptions are omitted, the contents described in the first aspect of the present invention can be equally applied to the second aspect of the present invention.

[0076] In addition, the third aspect of the present invention provides a kit for preventing or treating SARS-CoV-2 infection, comprising a vaccine composition according to the first aspect of the present invention.

[0077] Regarding the kit for preventing or treating SARS-CoV-2 infection according to the third aspect of this application, detailed descriptions of parts overlapping with the first aspect of this application have been omitted. However, even if the descriptions are omitted, the contents described in the first aspect of this application may be equally applied to the third aspect of this application.

[0078] The present invention will be described in more detail through the following examples; however, the following examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

[0079]

[0080] [Example 1] B cell epitope screening

[0081] The full-length structure of the SARS-CoV-2 protein was retrieved from the RSCPDB and AlphaFold databases, or, if unavailable, built using the AlphaFold monomer module. Approximately 300 spike (S)-binding and SARS-CoV-2-neutralizing antibodies were selected from the SAbDab and CoV-AbDab databases and downloaded from RSCB-PDB. If unavailable, antibody models were built using MOE 2022.2 or the AlphaFold multimer module. Similarly, nucleocapsid (N) and membrane (M)-bound antibodies were collected for epitope analysis. A two-step strategy was used to identify conserved and immunogenic B-cell epitopes on the SARS-CoV-2 structure and accessory proteins (Fig. 1a).

[0082] First, epitopes of structural proteins, including S, N, and M, were identified by clustering available antibodies against each antigen (Fig. 1b). For this purpose, structural superposition, AlphaFold multimer modeling, and the MOE epitope-paratope identification module were implemented.

[0083] Second, B-cell epitopes for all antigens (structural and nonstructural) were identified using Discotope and Ellipro, and verified through the IEDB resource. For common antigens (S, N, M), common or overlapping epitopes were selected by both methods, while for non-shared antigens, conserved epitopes were selected by the second strategy. For short, linear epitopes with a likely unstable structure, adjacent secondary structures were concatenated after epitope annotation within the full-length protein.

[0084]

[0085] [Example 2] HLA type selection

[0086] Fourteen HLA-I alleles, consisting of five HLA-A, six HLA-B, and three HLA-C variants, were selected based on expression and frequency as determined in a study of 5,802 Korean individuals. The expression ratios of these alleles were as follows: A02:01 (16%), A02:06 (10.2%), A03:01 (1.6%), A11:01 (10%), A24:02 (20.4%), B07:02 (3%), B15:01 (9.2%), B40:01 (3.7%), B44:03 (10%), B51:01 (9.4%), B58:01 (7%), C01:02 (17%), C07:01 (3.2%), and C07:02 (7.8%).

[0087] HLA-DRB1 haplotypes were selected based on separate studies and showed the following frequencies: DRB101:01 (5.9%), DRB101:03 (2.1%), DRB103:01 (2.7%), DRB104:05 (8.2%), DRB107:01 (6%), DRB109:01 (10.2%), DRB111:01 (5.5%), DRB113:01 (2.7%), DRB113:02 (7.4%), and DRB115:01 (7%).

[0088] Additionally, HLA-DQ and HLA-DP alpha / beta pairs were selected based on their prevalence in previous studies. These included HLA-DQA101:02 (17%) / HLA-DQB105:01 (8.5%), HLA-DQA105:01 (3%) / HLA-DQB103:01 (14.3%), HLA-DPA101:03 (44%) / HLA-DPB102:01 (25%), and HLA-DPA101:03 (44%) / HLA-DPB104:01 (7%). Notably, haplotypes with a frequency of less than 5% in the Korean population, as recorded in the Allele Frequency Net Database, were also considered due to their high global prevalence.

[0089]

[0090] [Example 3] Selection of MHC I and II ligands

[0091] For HLA-I, 9-13-mer peptides were screened using NetMHCpan EL 4.1, NetMHCCon, and IEDB consensus methods. Shared or overlapping epitopes were selected from these modules and further sorted based on ANN 4.0 and SMM scores, and only those with p-MHC binding affinity of IC50 < 500 nM were selected.

[0092] For human HLA-II, relatively longer ligands (14- to 22-mer peptides) were screened using the NetMHCIIpan EL 4.3 and IEDB consensus 2.22 methods. Ligands with an affinity of IC50 < 500 nM and the best combined scores of NN-align IC50, SMM-align IC50, and NetMHCIIpan IC50 were selected. The step-by-step procedure is summarized in Figure 3a.

[0093] Next, the selected candidates were virtually screened using the MOE module with the derivative-conjugation-docking module, which identified high-affinity epitopes that bind broadly to both HLA-I and HLA-II haplotypes. Candidate peptides showing high HLA affinity (based on docking scores) were modeled using the AlphaFold multimer module.

[0094] Finally, seven final selection criteria were applied to filter out poor quality peptides from the final candidate pool (Fig. 3a).

[0095]

[0096] [Example 4] Peptide synthesis and characterization

[0097] B-cell and HLA II epitope peptides with no issues in peptide manufacturability were synthesized at ≥90% purity by GenScript (Piscataway, NJ, USA). HLA I peptides were synthesized at ≥90% purity by GeneCust (Boynes, France). The purity of the HLA I peptide was confirmed by HPLC using a ChromCore120 C18 Naco Chrom 5 μM, 4.6 × 250 mm column. An Inertsil ODS-SP 4.6 × 250 mm column was used to confirm the purity of the HLA II and B-cell peptides. For the preparation of stock solutions, lyophilized peptides were suspended in dimethyl sulfoxide or distilled water at a working concentration of 10 mg / ml and stored at -20°C.

[0098]

[0099] [Example 5] Peptide-MHC binding analysis

[0100] To measure the binding affinity between predicted class I peptides and HLA class I, differential scanning fluorescence (DSF) analysis was performed on an Imusyn (Hannover, Germany). Briefly, synthetic peptides were added in a 10-fold molar excess to HLA class I containing several endogenous peptides with unique melting temperatures for the measurement.

[0101] Peptide binding affinity was measured by the change in native protein fluorescence during a temperature ramp. A relatively steep temperature gradient of 3°C / min was implemented during the measurement. Three types of samples were measured: HLA alone, peptide alone, and HLA + peptide. For the negative control, the value measured only with HLA containing the endogenous peptide was considered. A clear shift in the HLA + peptide curve compared to the negative control was indicative of binding. Certain peptides containing one or more tyrosines and / or tryptophans did not provide clear DSF measurements. To overcome this discrepancy, the peptide measurements were subtracted from the HLA + peptide measurements. Otherwise, the complexes were loaded on native PAGE and HLA-peptide binding was confirmed by comparing the results with the HLA-native peptide PAGE results.

[0102]

[0103] [Example 6] Blood sample collection and PBMC isolation

[0104] Blood samples collected from healthy adult volunteers who gave informed consent in accordance with an IRB protocol (AJOUIRB-EX-2023-123) were provided by the Ajou University Biobank (AHBB, Suwon) under the guidelines of the Environmental Health and Biosafety Program. The collected blood samples had received at least two doses of a World Health Organization (WHO) recommended vaccine, including additional booster doses. However, recovery from COVID-19 was not disclosed at the time of sample collection. The Human SARS-CoV-2 Spike IgG ELISA Kit (Thermo Fisher Scientific, BMS2325, MA, USA) was used to detect the presence of anti-Spike antibodies in the blood samples. However, this kit only detects anti-Spike antibodies and does not confirm recovery from COVID-19. PBMCs were isolated by Ficoll-Paque (Amersham Biosciences, Uppsala, Sweden) gradient centrifugation. PBMCs were collected as non-adherent cells and cultured in complete medium (RPMI1640, supplemented with 10% FBS, 100 U / ml penicillin, and 100 μg / ml streptomycin). Due to limited sample quantity, only 3 out of 10 samples contained sufficient PBMCs (4 × 10 6 -1.5x10 7 cells) could be created.

[0105]

[0106] [Example 7] Estimation of anti-peptide antibody titer (peptide ELISA)

[0107] For peptide ELISA, 96-well immunoplates (SPL Life Science, Korea) were coated with 10 μg / ml peptide and 100 ng / ml S1 protein (AcroBiosystems, S1N-C52H3-100UG, USA) as a positive control and incubated overnight at 4°C. After washing with PBS containing 0.1 TritonX-100, the plates were blocked with protein-free blocking buffer (protein-free compound in phosphate-buffered saline, pH 7.4) for 1 h at room temperature and reacted with 100 μl of various dilutions of human or mouse serum for 2 h at 37°C. The reacted sera were detected using HRP-conjugated goat anti-human IgG and the chromogenic substrate TMB (ThermoFisher, Waltham, MA, USA). The cutoff value for a positive peptide-serum reaction was set using the mean value of the negative control sample plus three times the standard deviation in each case. All ELISA assays were performed in duplicate at least twice.

[0108]

[0109] [Example 8] Analysis of T lymphocyte proliferation and IFN-γ secretion (cytokine staining)

[0110] To promote T lymphocyte proliferation, PBMCs were cultured in 24-well culture plates with 2 mL of complete medium supplemented with 1 μg / mL of CD3 / CD28 antibodies (catalog numbers 14-0037-82 and 14-0289-82, Thermo Fisher Scientific, 81 Wyman Street, Waltham, MA, USA). The plates were placed in a humidified 37°C incubator with 5% CO2 and cultured for 9 to 14 days until cell proliferation was confirmed. After T lymphocyte proliferation, cells were seeded at 2–7 × 10 in 96-well plates. 5Cells were seeded in 0.1 ml complete medium at a final working concentration of 10 cells / well and cultured for 24 h. The following day, each sample was treated with 1 μg / well of peptide and 100 ng / well of S1 protein. After 2 days, the supernatant from each well was used to quantify IFN-γ levels using the Human IFN-gamma Quantikine ELISA Kit (R&D Systems, catalog no. DIF50C, MN, USA).

[0111]

[0112] [Example 9] Isolation of splenocytes from mice immunized with T-cell peptides

[0113] The spleen of each mouse was gently homogenized with the plunger of a 10-mL syringe and aseptically filtered through a cell strainer. After dissociating the spleen from the mouse, spleen cells and PBMCs were isolated using a density gradient centrifugation method using Ficoll-Paque (Amersham Biosciences, Uppsala, Sweden) as previously described. The same method was used in the study by Dehghan et al. IFN-γ analysis isolated from the spleen or blood of mice immunized with T-cell peptides was performed using the Mouse IFN-gamma Quantikine ELISA Kit (R&D Systems, catalog no. MIF00, MN, USA).

[0114]

[0115] [Example 10] Mouse immunization

[0116] Six-week-old female Balb / c mice (Orient Bio, Korea) were housed under standard specific pathogen-free (SPF) conditions. Animals were group-housed (n = 4 per cage, n = 4 per group) at an ambient temperature of 18 to 24°C and a humidity of 40 to 60%, fed a 20% protein diet ad libitum, and maintained on a 12-h light / dark cycle. For B-cell vaccine immunization, mice were divided into groups: PBS, S1 (subunit vaccine), or four B-cell peptide groups (CSNP4, SGp_7, SGp_10, SGp_11). B-cell peptides (100 μg / peptide / mouse), S1 (50 μg / mouse), or PBS mixed with Freud's complete adjuvant (F5881, Sigma-Aldrich, USA) were administered subcutaneously. Mice in each group received the first injection on day 0 and booster injections on days 14 and 28. Serum samples were collected 3 days after each immunization. Blood samples were collected for peptide ELISA and SARS-CoV-2 spike pseudovirus neutralization. For T cell vaccine immunization, mice were administered 100 μg / peptide / mouse at 2-week intervals with T cell peptides (TC-I-1, TC-I-7, TC-II-1, TC-II-5) and sacrificed 3 days after the last vaccination, under the same conditions as B cell peptide injection.

[0117]

[0118] [Example 11] Pseudovirus neutralization assay

[0119] For luciferase assay, hACE2-293T cells (631289, Takara, USA) were treated with CSNP4, PEP7, and PEP10 along with serum dilutions (0, 1:100, 1:50, and 1:25), and 100 μl of various pseudoviruses (Wild, Alpha, Beta, Gamma, Delta, and Omicron) were added to the medium. Luciferase activity was measured using the ONE-Glo^TM^ Luciferase Assay System (Promega, USA, E6120) according to the manufacturer's instructions using a Synergy HTX Fluorometer (BioTek, USA) without attenuation. For mCherry fluorescence assay, hACE2-293T cells were treated with pseudoviruses in 100 μl of medium and cultured for 48 h. Images were then visualized using an Axiovert 200 fluorescence microscope (Carl Zeiss, Gφttingen, Germany). To evaluate the combined effect (synergy) of the two peptide sera, neutralization against Wild, Delta, and Omicron pseudoviruses was demonstrated by luciferase activity. Cells were treated with each sera (CSNP4-PEP7, PEP7-PEP10, and CSNP4-PEP10) at dilutions of 1:100, 1:50, or 1:25.

[0120]

[0121] [Experimental Example 1] Design of an immunodominant B cell epitope

[0122] To select effective and structurally resilient immunodominant B-cell epitopes, the epitope-paratope interface-based selection strategy is unique in that it explores immunogenic residues that overlap with nearby epitopes but are present sporadically. Antibody clustering around overlapping epitopes is a key step in identifying immunodominant motifs within the functional domains of an antigen. Leveraging the availability of abundant structural information, we identified functionally active epitopes in the N-terminal domain (NTD), receptor-binding domain (RBD), S2 domain of the S protein, and the RNA-binding domain (RBDn) of the N protein. Antibody clustering, Discotope and Ellipro screening, and conservation filtering within the Sarbecovirus family identified 10 and 8 epitopes in the S and N proteins, respectively, some of which overlapped partially or completely. Seven ligands were selected from the Discotope and Ellipro results for ORF1ab and membrane proteins (Table 1).

[0123] [Table 1]

[0124]

[0125] Of the ten ligands located in S, three were annotated in the RBD, two in the vicinity of the fusion peptide, and two in the stem helix (Fig. 1b). The ligands surrounding the fusion peptide and the stem helix partially overlapped with previously reported short, linear epitopes (Fusion peptide: PSKRSFIEDLLFNK, Stem helix epitope: FKEELDKYF). However, combined immunogenicity predictions from Discotope and Ellipro suggest the presence of highly immunogenic residues outside the core region of these peptides, which was confirmed by human serum reactivity to these epitopes. The receptor binding motif (RBM) of the RBD has been recognized as the most efficient epitope in terms of neutralization. However, the spatial distribution of immunogenic residues in the RBM is important for its immunodominance. To date, dozens of highly effective RBM-binding SARS-CoV-2 neutralizing antibodies have been identified and approved by the FDA (Fig. 1c). Antibody-antigen clustering suggests that one of the two beta sheets of the RBM is the most abundant binding epitope. However, no previous studies have reported this motif as a linear epitope, likely due to its unstable folding. Considering this, we combined two structurally adjacent but sequence-distant motifs via a linker peptide (LIGRGP) to create a conformational epitope (CSNP4, Figure 1d). Knowing that CSNP4 can potentially bind to the spike protein and block the upward and downward movement of the RBD, we previously evaluated its SARS-CoV-2 pan-variant inhibitory activity. The flexible linker of the RBM was also selected as a potential linear epitope, SGp_2, based on the combined immunogenicity scores of Discotope and Ellipro (Figure 1d). Furthermore, we identified a structurally flexible and highly conserved epitope, SGp_7, in the S2 domain (Figure 1b). Moderna mRNA vaccine was reported to induce antibodies to an epitope overlapping with SGp_7.Antibody clustering identified a single structural epitope, subdivided into three highly flexible loops within the RNA-binding domain (RBDn) of the N protein (Fig. 1e, middle). After several unsuccessful peptide modeling attempts, we selected the structural epitope “Np_1” proposed by Discotop and Ellipro to collect the available immunogenic residues suggested by antibody clustering onto a structurally flexible peptide scaffold. Just as in CSNP4, two beta sheets of the RBM directly contact ACE2, here too, two inverted beta sheets of Np_1 directly contact the bound RNA (Fig. 1e, right). In addition, seven other epitopes were selected in the N protein, of which Np_7 and Np_8 partially overlap with the “RQKKQQT” motif located in the C-terminal helix of the N protein (Fig. 1e, Table 1).

[0126]

[0127] [Experimental Example 2] Screening of immunodominant and immunopredisposed (ID / IP) B cell epitopes

[0128] To investigate ID / IP candidates among the selected B-cell epitopes, human sera from 30 individuals who had received the COVID-19 vaccine or who may have been infected with SARS-CoV-2 were tested for potential antibody responses to each peptide. The presence of anti-S antibodies in these samples was verified using ELISA. All 30 samples exceeded the median control value and were classified as anti-S seropositive. Serum #7 was found to be highly reactive in most reactions, including the S1 protein as an antigen, whereas serum #8 was found to be non-reactive (Fig. 2a). Epitopes that reacted with at least three sera with the same intensity and with an OD value ≥ 0.3, excluding #7 and #8, were considered IP / ID.

[0129] Anti-N antibodies in Pfizer and AstraZeneca vaccine recipients were due to natural COVID-19 infection, not vaccination. However, Sinopharm induces a deep anti-N response with its inactivated COVID-19 vaccine. Because the subjects in this study received mRNA COVID-19 vaccines (AstraZeneca for the first dose, and AstraZeneca or BNT162b2 for the second dose), sera reactive with at least two peptides derived from the non-S antigen were designated as convalescent because their COVID-19 infectivity was unknown at the time of blood sample collection. Based on the cutoff value, a total of 10 / 30 samples were designated as convalescent (Figure 2a).

[0130] The S1 subunit is bulky and contains both the NTD and RBD, reacting with 27 / 30 sera. Six epitopes, specifically SGp_7, SGp_10, and CSNP4, were identified as ID / IP in the S protein, while Np_11, Np_12, Np_17, and Np_18 were identified as ID / IP in the N protein. Np_11 and Np_17 of the N protein stood out as ID / IP candidates, reacting with more than three sera (Fig. 2a). No ID / IP epitopes were identified in the M protein and ORF1ab.

[0131] Based on overlap and domain sharing, we identified three pairs of S-derived epitopes. The first pair is CSNP4 and SGp_2 located within the RBM, the second pair is SGp_8 and SGp_10, which overlap in the stem helix peptide, and the third pair, SGp_5 and SGp_6, overlap by eight residues; SGp_6 contains the fusion peptide "PSKRSFIEDLLFN", whereas SGp_5 does not contain this motif (Figures 1b and 1d).

[0132] The immunodominant and hypervariable nature of the RBM motif within the RBD has been well established. Nevertheless, this motif has garnered significant attention for the development of SARS-CoV-2 neutralizing mAbs, including broad-spectrum neutralizing antibodies, with numerous mAbs, such as RGN10985, receiving FDA approval (Fig. 1c). Remarkably, CSNP4 reacted with ~50% of sera, while SGp_2 reacted with only three sera (Fig. 2b).

[0133] However, both Discotope and Ellipro identified the SGp_2 ligand "458-KSNLKPFERDISTE-472" as a highly immunogenic epitope. At this point, antibody clustering and the application of structural constraints, such as replacing the unstable P2 loop with a shorter "LIGRGP" linker, may be alternatives to traditional epitope design strategies. Furthermore, after the S1 protein, CSNP4 was identified as the ligand with the broadest and most frequent serum reactivity (Fig. 2b).

[0134] SGp_8 and SGp_10 share an 11-mer "FKEELDKYFKN" motif within their stem helix, which has been previously shown to induce broadly neutralizing antibodies due to its highly conserved nature. A recent study of antibody responses in COVID-19-infected and vaccinated individuals reported that "antibody responses to linear peptides derived from the conserved region of the SARS virus in infected individuals were significantly increased in the S2 C-terminal region compared to vaccinated individuals." Since most samples in this study were vaccinated only, SGp_8, which contains the conserved stem helix motif, did not react with any sera. However, SGp_10, a 38-mer peptide with a central stem helix motif, acquired a helical structure and reacted strongly with three or more sera (OD > 0.3) ( Figures 1A and 1B ).

[0135] Using the AlphFold multimer module, we verified whether SGp_10 could encompass the antibody response induced by "FKEELDKYFKN." CC25.106, a monoclonal antibody (mAb) targeting this motif, was able to bind perfectly to the epitope residues of SGp_10 with minimal root-mean-square deviation (RMSD) ( Figure 2C ). This further suggests that the additional N- and C-terminal residues enhance the immunogenicity (ID / IP) of this parental peptide, while the core region retains its function. Unlike SGp_10, where the core helix is ​​flanked by additional residues, the fusion peptide "PSKRSFIEDLLFN" with a broad neutralizing antibody response was located at the N-terminus of SGp_6 (Table 1). Surprisingly, SGp_5 reacted strongly with three or more sera, particularly sera 9, 21, 25, and 26, whereas SGp_6 reacted moderately with two sera (Fig. 2a). Like SGp_10, we found that SGp_6 could potentially induce broadly neutralizing antibodies because it shares a fusion peptide with the VN01H1 mAb paratope (Fig. 2d).

[0136]

[0137] [Experimental Example 3] S-derived ID / IP epitopes induce highly specific antibody responses.

[0138] Based on the ID / IP responses in human serum, three peptides, CSNP4, SGp_7, and SGp_10, from S and Np_1 from N protein, were selected to determine the immune responses in BALB / c mice. The B cell vaccine immunization regimen is illustrated in Fig. 2e.

[0139] Antibody titer analysis was performed in 4-week-old vaccinated mice that received three doses of the vaccine. S1, used as a control, induced a robust immune response because it contains the entire NTD and RBD domains of the S protein. This is consistent with previous studies using S1 as a subunit vaccine. Surprisingly, CSNP4 induced antibody titers similar to those of the S1 subunit, followed by other peptides (Fig. 2f). To investigate possible cross-reactive antibody responses, sera from peptide-immunized mice were cross-assayed against the S1 subunit and the peptide itself. All peptide-immunized sera reacted with the S1 subunit (Fig. 2g).

[0140] Because CSNP4 is derived from the RBD, these results suggest that CSNP4 induces an S1- or perhaps RBD-specific immune response. However, the cross-reactivity of S2-derived and N-derived peptides with S1 antibody titers requires further confirmation. Furthermore, sera vaccinated with SGp_7, SCp_10, and to some extent Np_1 also cross-reacted with CSNP4 (Fig. 2h).

[0141] Compared to CSNP4, antibody responses to S2-derived epitopes SGp_7 and SGp_10 were more specific. Serum immunized with SGp_11 also showed some cross-reactivity with S-derived peptides (Fig. 2i). To explain this cross-reactivity, we investigated the sequence similarity of Np_1 in the S protein, but no similarity could be found to explain this discrepancy. In conclusion, these data suggest that CSNP4 provides a strong immune response, while SGp_7 responses may be more effective against mutant SARS-CoV-2 strains, as SGp_7 is highly conserved not only among SARS-CoV-2 variants but also among SARS-CoV-2 strains.

[0142]

[0143] [Experimental Example 4] S-derived ID / IP epitopes induce broadly neutralizing antibodies against SARS-CoV-2 variants.

[0144] To investigate the neutralizing effects of antibodies induced by the peptide vaccine, we used a well-documented and previously implemented pseudovirus neutralization assay. Sera from peptide-immunized mice were assayed against several SARS-CoV-2 variants, including Wuhan (Wu01), alpha, beta, gamma, and Omicron (BA.1), in three serial dilutions under three different conditions:

[0145] Condition 1) A single serum was analyzed for all variants.

[0146] Condition 2) Serum from three immunized mice was pooled and analyzed for all variants.

[0147] Condition 3) Two high titer sera from mice vaccinated with different peptides were mixed and analyzed in the pseudovirus system.

[0148] SGp_7 single serum inhibited Wu01 and the alpha variant but did not show a significant response against other strains. Nevertheless, deep neutralization was achieved against the delta and omicron variants after pooling the three sera (Fig. 2j). SGp_10 neutralized Wu01, delta, and gamma variants with some variability. However, when the three sera immunized with SGp_10 were pooled, the delta and omicron variants were significantly neutralized depending on the serum dilution (Fig. 2j). CSNP4-immunized serum neutralized all variants regardless of serum pooling. However, the neutralization response was more profound when the three sera were pooled (Fig. 2j).

[0149] Next, we examined whether cross-pooling sera from mice immunized with different peptides could enhance neutralizing efficacy and suggest a potential multi-epitope vaccination regimen for these vaccine candidates in the future. We prepared three serum cocktails, mixing CSNP4 / SGp_7, SGp_7 / SGp_10, and CSNP4 / SGp_10 sera (one serum from each vaccine group) and assayed them at three different dilutions against Wu01, Delta, and Omicron variants. We found that pooling two sera from different immunized groups profoundly neutralized the immune-evading SARS-CoV-2 variants Delta and Omicron, compared to pooling three sera from the same group ( Fig. 2k ). The Delta variant was neutralized by ~80% at all three dilutions, and the Omicron variant was neutralized by >50% at dilutions of 1:25 and 1:50. These results offer a novel opportunity for cocktail (multi-epitope) immunization with these candidates against novel SARS-CoV-2 variants. Nevertheless, further validation is warranted to assess actual virus neutralization following immunization with the candidates in animal models requiring controlled settings. We also assessed pseudovirus neutralization with S1 immune serum. However, limited serum quantities allowed analysis only for Wu01, Delta, and Omicron variants. While greater than 95% of the pseudoparticles were neutralized at a 1:25 dilution, neutralization efficiency against Delta and Omicron variants decreased with further dilutions (Fig. 2l).

[0150] These results suggest that the neutralization response is related to antibody titers in immune sera. CSNP4, which induced antibody responses similar to S1, was able to neutralize the variants even without pooling. However, because the antibody titers of SGp_7 and SGp_10 sera were lower than those of CSNP4 (Fig. 2f), neutralization was not subtle when treated with a single serum. We also inferred that the relatively less conserved CSNP4 peptide may induce higher antibody responses than the highly conserved SGp_7 and SGp_10 peptides. Both serum pooling neutralization and antibody titers suggest that SGp_7 and SGp_10 may require booster doses to induce effective and broad immune responses.

[0151]

[0152] [Experimental Example 5] Promiscuity of HLA-I binding peptides

[0153] DSF was used to verify the binding affinity between class I peptides and each HLA haplotype. In DSF, the melting temperature (Tm) or inflection point temperature (Ti) indicates the thermal shift and the point of p-MHC dissociation / denaturation. The stability of the selected peptides was compared to the well-characterized N-derived HLA-A / *02:01-restricted peptide “LLLDRLNQL.” In this experiment, the Ti of this peptide was recorded as 75.5°C. A total of 27 selected peptides (Table 2) were tested against 14 HLA-I types from the proposed 49 combinations to select high-affinity broad-spectrum candidates.

[0154] [Table 2]

[0155]

[0156] Thirty-three reliable p-MHC pairs were identified based on moderate, good, and very good criteria (Ti range 60°C to 80°C) (Fig. 2b, top). Eleven p-MHC binders suggested by the in silico epitope selection process showed no binding (Fig. 3a), and five p-MHC pairs were very unstable (Ti < 60°C). At least 10 candidates showed MHC-affinity similar to or better than "LLLDRLNQL." Eight candidates bound to more than one HLA-I allele, demonstrating a possible broad affinity (Fig. 2b, bottom).

[0157] Class I MHC molecules contain a peptide-binding cleft between the α1 and α2 helices of the α chain. This pocket is important for anchoring residues of the binding peptide. 0105-Ajou2-LA was tested against four HLA-A haplotypes, among which *02:01 and *11:01 showed the best affinity, while *03:01 showed no binding in DSF (Fig. 3d, right). We modeled the p-MHC complex using the AlphaFold multimer module to determine how these haplotypes differ in peptide binding. Tyr3 of 'LSYYKLGASQRVA' fits into the D pocket of *02:01 and *11:01, but not in the D pocket of *24:02 and *03:01, which is 180 o We found that the D pocket of these haplotypes likely determines peptide affinity in that it rotates (Fig. 3d).

[0158] The reliability of the proposed AlphaFold model was further validated by differences in the E pocket between *24:02 and *03:01. HLA-A*24:02 contains Glu152, which forms a hydrogen bond with Gln10 of 'LSYYKLGASQRVA' in the α2 helix of the E pocket. This explains why *24:02 showed Ti = 47.5°C, whereas *03:01 showed no binding in DSF (Fig. 3d, right). Similarly, 0082-Ajou2-NV showed very good binding to A*02:01 and B*51:01, but failed to bind to B*44:03. Here, the A pocket of these haplotypes was observed to play a pivotal role in peptide binding, where the N-terminal Asn1 of the peptide can interact with different residues in A*02:01 and B*51:01 (Fig. 3e). 0092-Ajou2-KF showed broad binding to HLA-B haplotypes B*51:01 and B*15:01. However, B*07:02 was designated as a poor binder (Ti = 55°C). Interestingly, while B*07:02 differed from B*51:01 and B*15:01 within the B pocket, it shared identity with most other pockets (Fig. 3f). Both B*51:01 and B*15:01 contain Asn70, which interacts with the backbone nitrogen of Val5 of "KAYNVTQAF," whereas B*07:02 contains Gln70, which is distant from Val5. The broad binding of 0094-Ajou2-KY appears to be determined by the A and F pockets within C*07:01, C*07:02, and B*15:01. Ti of C*07:01 and C*07:02 were 80.4°C and 78°C, respectively, while Ti of B*15:01 was recorded as 66°C. While C*07:01 and C*07:02 share the same pocket, particularly the F pocket, B*15:01 has Tyr8 instead of Asp8 in the same pocket, resulting in the loss of a strong salt bridge with Arg2 of the peptide "KRVDWTIEY" (Fig. 3g).In silico screening suggested 0094-Ajou2-KY as a potential binder to B*51:01. However, DSF analysis showed borderline binding. Native PAGE analysis revealed no band shift, confirming the negative result (Fig. 3g, bottom).

[0159]

[0160] [Experimental Example 6] HLA supertype and peptide anchor points

[0161] HLA supertypes are associated with broad antigenic specificity, meaning that HLA molecules within the same supertype can bind and present similar peptides. This can influence the range of antigens recognized by T cells, thereby influencing the immune response. Each supertype can be characterized by a supermotif, which represents a comprehensive set of key, fixed motifs recognized by molecules within that supertype. For example, A2-supertype molecules exhibit specificity for peptides containing aliphatic hydrophobic residues at position 2 and the C-terminus. Conversely, A3-supertype molecules recognize peptides containing small or aliphatic residues at position 2 and basic residues at the C-terminus.

[0162] Combining DSF, AlphaFold multimer modeling, and sequence alignment, we investigated various peptides for HLA-I haplotypes to determine which types can react / bind to multiple peptides with shared motifs. Confirming the importance of position 2 for the A2 haplotype, we found that A*02:01 and A*02:06 responded well to 9-mer peptides containing Leu, Val, or Ala at position 2 (Figure 4a). Furthermore, the last residue of the peptides that showed high-affinity binding in DSF was the major contributing anchor residue. However, in 0105-Ajou2-LA (a 13-mer peptide), we found that the second residue was not the anchor residue, but Val12, the penultimate residue of 'LSYYKLGASQRVA', contributed as an atypical anchor residue. To further validate that both the second and last positions in the 9-mer peptide serve as anchor points, we investigated the A*02:01 haplotype against the HLA-A*02:01-restricted peptide “LLLDRLNQL” for 0079-Ajou2-DV and 0083-Ajou2-HW. Both peptides failed to bind to A*02:01 in DSF analysis. A closer look at the 3D model proposed by AlphaFold revealed that the B and F pockets of A*02:01 are hydrophobic, best suited to accommodate aliphatic hydrophobic residues (LIVMQ), but not acidic and bulky aromatic residues such as aspartic acid in 0079-Ajou2-DV and tryptophan in 0083-Ajou2-HW (Fig. 4a).

[0163] The notion that the second and last residues in a 9-mer peptide are important was further confirmed by DSF analysis of four different peptides against A*03:01. As discussed above, "peptides with a small or aliphatic residue at position 2 and a basic residue at the C-terminus" were identified as the best binders for A*03:01, whereas 0105-Ajou2-LA, which lacks such an anchoring point, failed to bind to A*03:01 (Fig. 4b). A closer look revealed that the F pocket of A*03:01 contains the acidic Asp116 residue, suggesting the importance of the C-terminal basic residue (Fig. 4b, top). Based on the second and C-terminal anchoring, A*11:01 responded to all four tested peptides containing Serine and Threonine at position 2 and a basic residue at the C-terminus (Fig. 4c, left). This anchoring preference has been confirmed by previous studies, where Threonine and aliphatic hydrophobic residues Val, Ile, and Leu at position 2 form stronger anchors, and Lys at the C-terminus forms stronger anchors. HLA-A*24:02 prefers bulky aromatic residues in the B and F pockets due to its deep hydrophobic cavity. We found that the 13-mer peptide 0105-Ajou2-LA, which does not contain hydrophobic aromatic residues at the C-terminus, exhibited a Ti of ~47°C, leaving both the B and F pockets empty (Fig. 4c, right). In addition, the 9-mer peptide 081-Ajou2-LL, which contains Leu at position 2 but Phe8 near the C-terminus, exhibited a Ti of 64. Three peptides satisfying previously reported anchoring criteria, 090-Ajou2-QI, 091-Ajou2-VF, and 091-Ajou2-QF, showed the highest binding affinity (Fig. 4c, right).

[0164] For HLA-B haplotypes, previous studies have suggested anchoring preferences for the following peptides: B*15:01 prefers 9-mer peptides containing aromatic residues, particularly Phe or Tyr, at the C-terminus. For B*40:01, two main anchoring points were previously proposed: Glu or Asp at position 2 and an aliphatic amino acid at the C-terminus. HLA-B*51:01 prefers Ala Gly or Pro at position 2 and Phe, Ile, or Val at the C-terminus. HLA-B*58:01 mainly prefers peptides containing Trp or Phe at the C-terminus of the F pocket and serine or Ala at position 2 of the A pocket (Fig. 4). Considering these preferences, we found that peptides with the anchor residues mentioned above preferentially bind with high affinity to each allele, whereas differences in the anchor residues can lower Ti or completely abolish p-MHC binding (Figures 4d and 4e).

[0165] These results suggest that vaccine development targeting common motifs present in relatively conserved epitopes within specific HLA supertypes recognized by T cells could potentially provide broad coverage across diverse human populations.

[0166]

[0167] [Experimental Example 7] Broad spectrum and IFN-γ induction by T cell peptides in human PBMCs isolated from humans

[0168] Eleven class II peptides, ranging in length from 15 to 22 amino acids, were selected as illustrated in Figure 3a. Nine of the class I peptides were selected for IFN-γ analysis based on their confirmed broad association with multiple HLA-I haplotypes (Figures 3b and 3c) and their frequent expression in HLA. Additionally, five of the 11 class II peptides were selected for IFN-γ analysis given their proposed broad association with multiple HLA-DRB1 haplotypes and, in some cases, HLA-DQ (Table 3) (see the multi-HLA affinity step in Figure 3a).

[0169] [Table 3]

[0170]

[0171] To assess the efficacy of selected class II peptides as IFN-γ inducers, we initially investigated their HLA-II binding affinity using the AlphaFold multimer module. Variation across HLA-DRB1 haplotypes was analyzed to determine potential broad-spectrum conserved regions. Box 1, encompassing peptide-binding pockets 6, 7, and 9, is largely conserved across the DRB103:01, DRB113:01, and DRB1*13:02 haplotypes. However, *03:01 exhibits variation in box 2, which also contributes to this pocket. Other haplotypes, including *01:03, *04:05, *09:01, *07:01, and *15:01, exhibit varying degrees of variation in these pockets. Nevertheless, Leu67, Glu68, and Arg71 are conserved across all haplotypes in box 2.

[0172] CII-1 was modeled using AlphaFold multimers with *09:01, *13:02, and *15:01, and superimposed to highlight pocket residues and potential anchors. Typically, peptides bind to the HLA-II pocket from N- to C-terminus, with the N-terminal residue anchored in pocket 1 (P1). However, we observed that the CII-1 peptide did not follow this pattern with *15:01, but instead anchored at four points with *09:01 and *13:02 (Fig. 5a). For the CII-2 peptide, *13:02 was not considered a peptide conjugate, whereas *09:01 and *15:01 were bound by aliphatic hydrophobic leucines at P1, P4, P6, and P9 (Fig. 5b). CII-3 was found to bind to *09:01 in HLA-DRB1, whereas CII-4 was identified as a potential binding site for *09:01 and *15:01 (Figs. 5c and 5d). CII-5 and CII-6, which contain Arg at positions 7 and 12, respectively, were identified to bind to *09:01, *13:01, *13:02, and *15:01 via P6 containing Asp27 and Glu10, respectively (Figs. 5e and 5f). Among HLA-DQs, binding of CII-1 and CII-2 to QA101:02 / QB105:01 and binding of CII-1 and CII-4 to QA105:01 / QB103:01 were evaluated. CII-1 bound to QA105:01 / QB103:01, whereas CII-2 bound to QA101:02 / QB105:01. Overall, these models suggest multi-HLA binding affinities for all five peptides, but these structure-based predictions require further experimental evaluation.

[0173]

[0174] [Experimental Example 8] Induction of IFN-γ by T cell peptide in human PBMC

[0175] Upon activation, T cells undergo clonal expansion and differentiation to produce effector T cells, including IFN-γ-secreting T cells. IFN-γ release is crucial for initiating and regulating immune responses to infection. IFN-γ production by T cells can be assessed experimentally using ELISA, intracellular cytokine staining by flow cytometry, or ELISPOT assays.

[0176] Due to the limited cell numbers and failure of most cell proliferation in the limited-size PBMC samples, only three PBMC samples were sufficient to perform IFN-γ assays for five class II peptides and nine class I peptides. Based on the B-cell peptide ELISA results, samples 3, 4, and 29 were found to react strongly with S1 but moderately with S- and N-derived B-cell peptides (Fig. 2a). However, we expected that T-cell peptides would activate T cells within the isolated PBMCs. We used ELISA to quantify the levels of IFN-γ released by peptide-activated PBMCs. PBMCs were stimulated with CD3 and CD28 antibodies and expanded for 9 days. Up to 14 days were allowed between the start of expansion and the assessment of immunoreactivity to prevent loss of sensitivity due to tolerance and cell senescence. Cell counts and spheroid formation rates increased rapidly from day 4 of expansion, and by day 9, each sample had undergone 20-100-fold cell amplification. Spike pseudovirus and RBD-derived peptides have previously been reported to induce IFN-γ in T cells. Furthermore, the spike protein has been considered a more potent and sustained T cell responder in terms of IFN-γ. Therefore, S1 was used as a reference to compare the IFN-γ levels induced by the selected peptides. Of the nine HLA-I peptides, four (CI-1, 3, 4, and 6) were spike-derived, CI-8 and CI-9 were derived from ORF1ab, CI-5 was derived from ORF7a, and CI-2 and CI-7 were derived from the N protein. Sample 29 was found to be more reactive than Sample 3 when activated with HLA-I and HLA-II peptides, which in turn were found to be more reactive than Sample 4 (Fig. 6a). All MHC-I peptides, except CI-6 and CI-9, induced IFN-γ in all three samples. However, CI-1, CI-7, and CI-8 consistently induced IFN-γ in all samples.Among the HLA-II peptides, CII-1 and CII-5 consistently induced IFN-γ in all three PBMC samples (Fig. 6b). Notably, in two of the three PBMC samples, CI-1, CI-7, CI-8, CII-1, and CII-5 surpassed the IFN-γ levels induced by S1.

[0177]

[0178] [Experimental Example 9] Immunization in BALB / c Mice: Cross-Reactivity and IFN-γ Release

[0179] DSF and AlphaFold multimeric modeling suggest that CI-1 (0082-Ajou2-NV) binds HLA-A*02:01 and HLA-B*51:01, and CI-7 binds HLA-B*15:01, HLA-B*51:01, and to some extent HLA-B*07:02. Similarly, the HLA-II peptides CII-1 and CII-5 potentially bind more than one HLA-DRB1 haplotype (Figures 5A and 5E). Because transgenic mice expressing these HLAs in identical combinations were not available, the four peptides were instead evaluated for T cell activation and IFN-γ responses in BALB / c mice. Although there are differences between human and mouse MHC molecules, some T cell epitopes may be conserved across species and recognized by pan-HLA (e.g., PADRE (AKFVAAWTLKAAA)). PADRE is recognized by human and mouse MHC molecules and induces a strong T cell response. Furthermore, testing human HLA-restricted peptides in mice can determine whether the peptide cross-reacts with mouse MHC molecules. For example, when a peptide derived from human RGFR was vaccinated with CpG and PADRE in BALB / c mice, it induced a stronger IFN-γ response compared to peptide administration alone. Furthermore, peptide immunogenicity in mice can provide information on whether the peptide can stimulate the mouse immune system similar to that observed in humans. For example, non-glycosylated or GalNAc-glycosylated MUC1-derived peptides were evaluated for immune responses in BALB / c or HLA-A2 transgenic mice. Both GalNAc-glycosylated and MUC1 peptides stimulated T cell proliferation in BALB / c and HLA-A2 transgenic mice.

[0180] Before immunization, the mouse MHC binding affinity of four peptides for class I and class II MHC molecules of mice was predicted using SYFPEITHI and NetMHCIIpan 4.1, NetMHCpan 4.1. CI-1 was predicted as H2-K in NetMHCpan 4.1 El and SYFPEITHI predictions. k showed the best affinity for H2-K in the SYFPEITHI model. d and also showed binding (Table 4). CI-2 showed the best performance for H2-K^b^ in both prediction models (Table 4). For class II mouse MHC, the 15-mer overlapping fragment of CII-1 was the best predictor for H2-E k and H2-A d showed the best combination with H2-D in the SYFPEITHI scoring model. Similarly, CII-5 b For , the 10-mer derivative of CII-5 is H2-E d showed the best performance for alleles. However, these alleles were not available in the NetMHCIIpan 4.1 El interface.

[0181] [Table 4]

[0182]

[0183] To determine whether these peptides induce T cell responses in mice, BALB / c mice were immunized with these peptides for 4 weeks at 2-week intervals (Fig. 5c). Splenocytes from immunized and non-immunized mice were then stimulated with each peptide to assess IFN-γ responses. Among the MHC-I peptides, CI-1 induced significant cytokine responses in all three splenocyte samples. However, CI-7 showed a less promising response (Fig. 5d). In contrast, both MHC-II peptides induced significant IFN-γ responses in all three mice (Fig. 5e). Because MHC-I peptides are all 9-mers, they are thought to have a greater potential to induce CD8 T cell responses than CD4 T cell responses. In contrast, MHC-II peptides are 15-mers or longer, and due to possible MHA-anchor-point overlap, these peptides can activate both CD8 and CD4 cells. However, due to limited sample size, specific T cell responses could not be estimated, which may require further validation. Splenocytes and PBMCs isolated from adjuvanted mice were also activated by S1 and T cell peptides, but neither ligand induced an IFN-γ response in splenocytes or PBMCs.

[0184] Overall, these results indicate that immunizing BALB / c mice with human HLA-restricted peptides provides valuable insights into T cell activation and IFN-γ responses, even in the absence of transgenic mice expressing specific human HLA combinations. Through predictive modeling and subsequent immunizations, we demonstrate the potential cross-reactivity and immunogenicity of these peptides in a mouse model, laying the foundation for further investigation as potential Sarbecovirus vaccine agents.

[0185] The above description of the present invention is for illustrative purposes only, and those skilled in the art will readily appreciate that the present invention can be readily modified into other specific forms without altering the technical spirit or essential characteristics of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive. For example, each component described as a single entity may be implemented in a distributed manner, and similarly, components described as distributed may be implemented in a combined manner.

[0186] The scope of the present invention is indicated by the claims described below rather than the detailed description above, and all changes or modifications derived from the meaning and scope of the claims and their equivalent concepts should be interpreted as being included in the scope of the present invention.

Claims

1. Containing a peptide isolated from the structural proteins of SARS-CoV-2. Vaccine composition.

2. In paragraph 1, wherein the peptide comprises one or more B cell epitopes; Vaccine composition.

3. In paragraph 2, The above B cell epitope is linear or three-dimensional in structure, Vaccine composition.

4. In paragraph 1, The structural protein of the above SARS-CoV-2 comprises a protein selected from the group consisting of a spike protein, a nucleocapsid protein, a membrane, and combinations thereof. Vaccine composition.

5. In paragraph 4, The peptide isolated from the spike protein of the SARS-CoV-2 comprises at least one sequence selected from the group consisting of sequence numbers 1 to 11. Vaccine composition.

6. In paragraph 4, The peptide isolated from the nucleocapsid protein of the above SARS-CoV-2 comprises at least one sequence selected from the group consisting of SEQ ID NOs: 12 to 19. Vaccine composition.

7. In paragraph 4, The peptide isolated from the membrane of the above SARS-CoV-2 comprises at least one sequence selected from the group consisting of SEQ ID NOs: 20 to 26. Vaccine composition.

8. In paragraph 1, The above vaccine composition induces neutralizing antibodies against variants selected from the group consisting of Wuhan (Wu01), alpha, beta, gamma, Omicron (BA.1) and combinations thereof of SARS-CoV-2. Vaccine composition.

9. A vaccine composition comprising any one of claims 1 to 8, A composition for preventing or treating SARS-CoV-2 infection.

10. A vaccine composition comprising any one of claims 1 to 8, A kit for the prevention or treatment of SARS-CoV-2 infection.

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