SARS-CoV-2 vaccine composition

Abstract

Coronavirus (CoV) spike (S) polypeptides, including natural and non-natural polypeptides, as well as nanoparticles and immunogenic compositions containing them, are disclosed herein. The nanoparticles are surrounded by a surfactant core and present pathogen-derived antigens associated thereto, resulting in enhanced stability and good immunogenicity. Dosages, formulations, and methods for preparing vaccines and nanoparticles are also disclosed.

Description

Technical Field

[0001] Description of an Electronically Filed Text File This application claims priority from U.S. Provisional Patent Application No. 63 / 497,986, filed on April 24, 2023; U.S. Provisional Patent Application No. 63 / 507,079, filed on June 8, 2023; U.S. Provisional Patent Application No. 63 / 507,412, filed on June 9, 2023; U.S. Provisional Patent Application No. 63 / 508,088, filed on June 14, 2023; U.S. Provisional Patent Application No. 63 / 579,438, filed on August 29, 2023; and U.S. Provisional Patent Application No. 63 / 580,596, filed on September 5, 2023, the entire contents of which are incorporated herein by reference.

[0002] The content of the electronic sequence listing (1450_103WO1_Sequence_Listing_04_24_2024.xml, size: 817,457 bytes and creation date: April 24, 2024) is incorporated herein by reference in its entirety.

[0003] The present disclosure generally relates to coronavirus (CoV) spike (S) polypeptides, including natural and non-natural polypeptides, nanoparticles containing the same, and immunogenic compositions that are useful for stimulating an immune response. The nanoparticles provide antigens, such as glycoprotein antigens, optionally associated with a surfactant core, and are typically produced using recombinant techniques. The nanoparticles have improved stability and enhanced epitope presentation. The present disclosure also provides compositions containing the nanoparticles, methods of making the same, and methods of stimulating an immune response.

Background Art

[0004] Infectious diseases remain a global problem. While progress is being made in developing vaccines against some pathogens, many still pose a threat to human health. The SARS-CoV-2 pandemic killed 6.5 million people worldwide. The SARS-CoV-2 coronavirus belongs to the same viral family as the severe acute respiratory syndrome coronavirus (SARS-CoV) and the Middle East respiratory syndrome coronavirus (MERS-CoV), which have killed hundreds of people over the past 17 years. SARS-CoV-2 causes the disease COVID-19. [Overview of the project] [Problems that the invention aims to solve]

[0005] The development of vaccines that prevent or reduce the severity of life-threatening infectious diseases like SARS-CoV-2 coronavirus is desirable. However, human vaccine development remains a challenge due to the highly sophisticated evasion mechanisms of the pathogen and the difficulty in stabilizing vaccines. The development of vaccines that induce protection against current and future SARS-CoV-2 strains is desirable. [Means for solving the problem]

[0006] This disclosure provides CoV S polypeptides, including natural and non-natural polypeptides, and compositions containing them, which are suitable for inducing an immune response to SARS-CoV-2. This disclosure also provides nanoparticles and compositions containing glycoproteins, as well as methods for stimulating an immune response. The nanoparticles and compositions described herein are also suitable for inducing cross-neutralizing immune responses to Middle East Respiratory Syndrome (MERS) and Severe Acute Respiratory Syndrome (SARS). [Brief explanation of the drawing]

[0007] [Figure 1A] The primary structure of the wild-type SARS-CoV-2 S polypeptide containing the signal peptide, numbered for SEQ ID NO: 1, is shown. [Figure 1B] The primary structure of the wild-type SARS-CoV-2 S polypeptide without the signal peptide, numbered for SEQ ID NO: 2, is shown. [Figure 2] The administration regimen for Example 7 is shown. [Figure 3A-3C] The results of Example 3 show that Composition 3 (Figures 3A, 3D), Composition 2 (Figures 3B, 3E), and Composition 1 (Figures 3C, 3F) neutralized pseudoviruses expressing S proteins from various heterologous SARS-CoV-2 strains, respectively. In Figures 3D-3F, the white circles represent individual data points, the solid bars represent the geometric mean titer of the group, the error bars represent the 95% confidence interval, and the horizontal dashed lines represent the limit of detection (LOD) of the assay. [Figure 3D-3F] Same as above. [Figure 4A-4B] The results show that administration of two doses of Composition 2, a monovalent composition containing CoV S glycoprotein of SEQ ID NO: 274 (Figures 4A and 4C), elicits a superior immune response in mice compared to administration of two doses of Composition 4, a bivalent composition containing CoV S glycoprotein of SEQ ID NO: 274 and CoV S glycoprotein of SEQ ID NO: 87 (Figures 4B and 4D). In Figures 4C-4D, white circles represent individual data points, solid bars represent the geometric mean titer of the group, error bars represent the 95% confidence interval, and horizontal dashed lines represent the limit of detection (LOD) of the assay. [Figure 4C-4D] Same as above. [Figures 5A-5C] The immune response induced by boost doses containing composition 2 (Figures 5B, 5E) or composition 3 (Figures 5C, 5F) in Example 3 is shown. Boost doses of either composition 2 (Figures 5B, 5E) or composition 1 (Figures 5C, 5F) resulted in an improved immune response to pseudoviruses expressing S proteins from various heterologous SARS-CoV-2 strains compared to mice that did not receive a boost dose (Figures 5A, 5D). [Figure 5D-5F] Same as above. [Figure 6A-6C]The immune response induced by boost doses containing composition 2 (Figure 6B) or composition 3 (Figure 6C) in Example 3 is shown. Boost doses of either composition 2 (Figure 6B) or composition 1 (Figure 6C) resulted in an improved immune response to pseudoviruses expressing S proteins from various heterologous SARS-CoV-2 strains compared to mice that did not receive a boost dose (Figure 6A). [Figure 7A-7C] This shows the pseudovirus neutralizing ability of rhesus macaque serum administered with various immunogenic compositions. Figure 7A shows the pseudovirus neutralizing ability of rhesus macaque serum administered with two doses of composition 3 and a booster dose of composition 2. Figure 7B shows the pseudovirus neutralizing ability of rhesus macaque serum administered with two doses of composition 6 and a booster dose of composition 2. Figure 7A shows the pseudovirus neutralizing ability of rhesus macaque serum administered with two doses of composition 5 and a booster dose of composition 2. [Figure 7D-7E] Same as above. [Figure 8] A dosing regimen comprising administering two doses of composition 5 followed by a boost dose of composition 2 demonstrates the ability to induce an immune response that blocks the binding of hACE2 to the SARS-CoV-2 S glycoprotein. Compositions 2 and 5 are described in Example 3. [Figures 9A-9C] Various dosing regimens demonstrate the ability to induce an immune response in rhesus monkeys by blocking the binding of hACE2 to the SARS-CoV-2 S glycoprotein. Figure 9A shows the immune response induced when two doses of composition 3 are administered, followed by a boost dose of composition 2. Figure 9B shows the immune response induced when two doses of composition 6 are administered, followed by a boost dose of composition 2. Figure 9C shows the immune response induced when two doses of composition 5 are administered, followed by a boost dose of composition 2. Compositions 2, 3, 5, and 6 are described in Example 3. [Figure 10A-10B]Figure 10A shows the ratio of Th1 / Th2 cytokines produced by CD4+ T cells in mice administered two doses of composition 3 followed by a boost dose of composition 2, or two doses of composition 5 followed by a boost dose of composition 2 (Figure 10B). Compositions 2, 3, and 5 are described in Example 3. [Figure 11] The CD4+ T cell response in rhesus monkeys administered two doses of composition 5, followed by a boost dose of composition 2, is shown. Compositions 5 and 2 are described in Example 3. [Figure 12] The administration regimen for Example 8 is shown. [Figure 13A] The image shows anti-S IgG antibodies in mice after administration of two doses of an immunogenic composition containing SARS-CoV-2 S glycoprotein (SEQ ID NO: 274). Further experimental details can be found in Example 8. [Figure 13B] The image shows anti-S IgG antibodies in mice after administration of two doses of an immunogenic composition containing SARS-CoV-2 S glycoprotein (SEQ ID NO: 260). Further experimental details can be found in Example 8. [Figure 14A] This study demonstrates that administration of two doses of an immunogenic composition containing the SARS-CoV-2 S glycoprotein of SEQ ID NO: 274 results in the acquisition of antibodies that block the binding of SARS-CoV-2 to hACE2. Further experimental details are found in Example 8. [Figure 14B] This study demonstrates that administration of two doses of an immunogenic composition containing SARS-CoV-2 S glycoprotein (SEQ ID NO: 260) results in the acquisition of antibodies that block the binding of SARS-CoV-2 to hACE2. Further experimental details are found in Example 8. [Figure 15A] This study demonstrates that administration of two doses of an immunogenic composition containing SARS-CoV-2 S glycoprotein (SEQ ID NO: 274) neutralizes pseudoviruses expressing SARS-CoV-2 S glycoprotein. Further experimental details are found in Example 8. [Figure 15B]This study demonstrates that administration of two doses of an immunogenic composition containing SARS-CoV-2 S glycoprotein (SEQ ID NO: 260) neutralizes pseudoviruses expressing SARS-CoV-2 S glycoprotein. Further experimental details are found in Example 8. [Figure 16A-16C] Figure 16A shows the neutralizing antibody titers resulting from the administration of two doses of an immunogenic composition containing SARS-CoV-2 S glycoprotein of SEQ ID NO: 87. Further experimental details can be found in Example 8. Figure 16B shows that pseudoviruses expressing SARS-CoV-2 S glycoprotein are neutralized as a result of the administration of two doses of an immunogenic composition containing SARS-CoV-2 S glycoprotein of SEQ ID NO: 274. Further experimental details can be found in Example 8. Figure 16C shows that pseudoviruses expressing SARS-CoV-2 S glycoprotein are neutralized as a result of the administration of two doses of an immunogenic composition containing SARS-CoV-2 S glycoprotein of SEQ ID NO: 260. Further experimental details can be found in Example 8. [Figure 17] The administration regimen for Example 9 is shown. [Figure 18A] The image shows anti-S IgG antibodies in the serum of mice after administration of two doses of an immunogenic composition containing SARS-CoV-2 S glycoprotein (SEQ ID NO: 274). Further experimental details can be found in Example 9. [Figure 18B] This shows the neutralizing antibodies present in the serum of mice after administration of two doses of an immunogenic composition containing SARS-CoV-2 S glycoprotein (SEQ ID NO: 274). Further experimental details can be found in Example 9. [Figures 19A-19C]Figure 19A shows the antigen map of the neutralization response induced by immunizing with two doses of an immunogenic composition containing the SARS-CoV 2 S glycoprotein of SEQ ID NO: 274. Details of further experiments are found in Example 9. Figure 19B shows the antigen map of the neutralization reaction induced by boosting with an immunogenic composition containing the SARS-CoV 2 S glycoprotein of SEQ ID NO: 274. Details of further experiments are found in Example 9. Figure 19C shows the antigen map of the neutralization reaction induced by boosting with an immunogenic composition containing the SARS-CoV 2 S glycoprotein of SEQ ID NO: 260. Details of further experiments are found in Example 9. [Figures 20A-20C] Figure 20A shows the pseudovirus neutralization titers induced by administering the SARS-CoV-2 S glycoprotein of SEQ ID NO: 274 in a primary series. Details of further experiments are found in Example 9. Figure 20B shows the pseudovirus neutralization titers induced by administering the SARS-CoV-2 S glycoprotein of SEQ ID NO: 274 with a 1-month boost. Details of further experiments are found in Example 9. Figure 20C shows the pseudovirus neutralization titers induced by administering the SARS-CoV-2 S glycoprotein of SEQ ID NO: 260 with a 1-month boost. Details of further experiments are found in Example 9. [Figure 21] Shows the dosing regimen of Example 10. [Figure 22A] Shows anti-S IgG antibodies in the serum of macaques after administration of the first and second doses containing the SARS-CoV 2 S glycoprotein of SEQ ID NO: 87 and the third dose containing the SARS-CoV 2 S glycoprotein of SEQ ID NO: 274. Details of further experiments are found in Example 10. [Figure 22B] Shows anti-S IgG antibodies in the serum of macaques after administration of the first and second doses containing the SARS-CoV 2 S glycoprotein of SEQ ID NO: 222 and the third dose containing the SARS-CoV 2 S glycoprotein of SEQ ID NO: 274. Details of further experiments are found in Example 10. [Figure 22C]The images show anti-S IgG antibodies in macaque serum after administration of first and second doses containing SARS-CoV-2 S glycoprotein of SEQ ID NO: 87 and SARS-CoV-2 S glycoprotein of SEQ ID NO: 222, and a third dose containing SARS-CoV-2 S glycoprotein of SEQ ID NO: 274. Further experimental details can be found in Example 10. [Figure 23] This shows anti-S IgG antibodies in macaque serum after administration of a first and second dose containing SARS-CoV-2 S glycoprotein (SEQ ID NO: 274). [Figure 24] This shows the pseudovirus neutralizing titer induced by administration of SARS-CoV-2 S glycoprotein SEQ ID NO: 274 in a primary series. Further experimental details can be found in Example 10. [Figure 25] The CD4+ T cell responses of macaques administered with first and second doses containing the SARS-CoV 2 S glycoprotein of SEQ ID NO: 274 are shown. Further experimental details can be found in Example 10. [Figure 26] This shows the multifunctional antigen-specific CD4+ T cell response in macaques administered with a first and second dose containing the SARS-CoV 2 S glycoprotein of SEQ ID NO: 274. Further experimental details can be found in Example 10. [Figure 27] The administration regimen for Example 11 is shown below. [Figures 28A-28B] Figure 28A shows anti-rS IgG antibodies in the serum of mice after administration of two doses of an immunogenic composition containing SARS-CoV 2 S glycoprotein (SEQ ID NO: 274). Further experimental details can be found in Example 11. Figure 28B shows neutralizing antibodies present in the serum of mice after administration of two doses of an immunogenic composition containing SARS-CoV 2 S glycoprotein (SEQ ID NO: 329). Further experimental details can be found in Example 11. [Figures 29A-29B]Figure 29A shows the pseudovirus neutralizing titer induced by administration of SARS-CoV-2 S glycoprotein of SEQ ID NO: 274 in a primary series. Further experimental details can be found in Example 11. Figure 29B shows the pseudovirus neutralizing titer induced by administration of SARS-CoV-2 S glycoprotein of SEQ ID NO: 329 in a primary series. Further experimental details can be found in Example 11. [Figure 30] The pseudovirus neutralizing titers and antigenic distances induced by administering SARS-CoV-2 S glycoprotein SEQ ID NO: 274 to different SARS-CoV-2 variant strains in a primary series are shown, with lower titers for JN.1 and the JN.1 subvariant. Further experimental details can be found in Example 11. [Figure 31] The primary series administration of SARS-CoV-2 S glycoprotein SEQ ID NO: 329 to different SARS-CoV-2 variant strains induced pseudovirus neutralization titers and antigenic distances, with high titers observed for JN.1 and the JN.1 subvariant. Further experimental details can be found in Example 11. [Figures 32A-32D] Figure 32A shows the anti-rS IgG antibodies in mouse serum after administration of composition 2 in the primary series, compared to the anti-rS IgG antibodies in mouse serum before administration of the boost dose (Figure 32B), with the anti-rS IgG antibodies induced in mouse serum after administration of a boost dose containing composition 2 (Figure 32C) or composition 8 (Figure 32D). Further experimental details can be found in Example 11. [Figure 33A-33C]Example 11 shows the pseudovirus neutralizing titer and antigenic distance induced by administration of SARS-CoV-2 S glycoprotein of Sequence ID No. 274 in the primary series, followed by administration of a boost dose containing Composition 8 (Figure 33C). Boost administration of Composition 8 (Figure 33C) resulted in improved immune response to pseudoviruses expressing S proteins from various SARS-CoV-2 heterologous strains compared to mice before boost administration (Figure 33B) that had received primary inoculation with Composition 2 (Figure 33A). Further experimental details can be found in Example 11. [Figures 34A-34B] The pseudovirus neutralizing titers and antigenic distances induced by administration of composition 2 in the primary series (Figure 34A) and a boost dose containing composition 8 two months later (Figure 34B) are shown in Example 11. Boost dose with composition 8 (Figure 34B) induced an improved immune response against pseudoviruses expressing S proteins from various heterologous SARS-CoV-2 strains (Figure 34A). Further experimental details can be found in Example 11. [Figures 35A-35B] Figure 35A shows the pseudovirus neutralizing titer and antigenic distance induced by administration of composition 7 in the primary series, followed two months later by a boost dose containing composition 8 from Example 11 (Figure 35B). The boost dose with composition 8 (Figure 35B) induced an improved immune response against pseudoviruses expressing S proteins from various heterologous SARS-CoV-2 strains (Figure 35A). Further experimental details can be found in Example 11. [Figure 36] The ratio of Th1 / Th2 cytokines produced by CD4+ T cells in mice administered a dose of Composition 2, followed by a boost dose of Composition 2 over two months, is shown. Further experimental details can be found in Example 11. [Figure 37] The ratio of Th1 / Th2 cytokines produced by CD4+ T cells in mice administered a dose of Composition 2, followed by a boost dose of Composition 8 over two months, is shown. Further experimental details can be found in Example 11. [Figures 38A-38B]Figure 38A shows the Triple Th1 CD4+ T cell response in mice administered a dose of Composition 2 followed by a boost dose of Composition 2 over two months, and in mice administered a dose of Composition 2 followed by a boost dose of Composition 8 over two months (Figure 38B). Further experimental details can be found in Example 11. [Figure 39A-39B] Figure 39A shows the CD4+ T cell IFN-γ response in mice administered a dose of Composition 2 followed by a boost dose of Composition 2 over two months, and in mice administered a dose of Composition 2 followed by a boost dose of Composition 8 over two months (Figure 39B). Further experimental details can be found in Example 11. [Figures 40A-40B] Figure 40A shows the CD8+ T cell Th1 response in mice administered a dose of Composition 2 followed by a boost dose of Composition 2 over two months, and in mice administered a dose of Composition 2 followed by a boost dose of Composition 8 over two months (Figure 40B). Further experimental details can be found in Example 11. [Figure 41A-41B] Figure 41A shows T follicular helper cell levels (Tfh) in mice administered a dose of Composition 2 followed by a boost dose of Composition 8 over two months, compared to mice administered a dose of Composition 2 followed by a boost dose of Composition 8 over two months. Figure 41B shows germinal center (GC) B cell levels (Tfh) in mice administered a dose of Composition 2 followed by a boost dose of Composition 8 over two months, compared to mice administered a dose of Composition 2 followed by a boost dose of Composition 8 over two months. [Figure 42] The administration regimen for Example 12 is shown. [Figures 43A-43B]Figure 43A shows the anti-rS response in primed rhesus monkeys before administration of a second booster dose of the immunogenic composition containing SARS-CoV 2 S glycoprotein of SEQ ID NO: 329. Further experimental details can be found in Example 12. Figure 43B shows the anti-rS response in primed rhesus monkeys after administration of a second booster dose of the immunogenic composition containing SARS-CoV 2 S glycoprotein of SEQ ID NO: 329. Further experimental details can be found in Example 12. [Figure 44A-44B] Figure 44A shows the pseudovirus neutralizing titer and antigenic distance in primed rhesus monkeys before administration of a second booster dose of the immunogenic composition containing SARS-CoV 2 S glycoprotein of SEQ ID NO: 329. Further experimental details can be found in Example 12. Figure 44B shows the pseudovirus neutralizing titer and antigenic distance in primed rhesus monkeys after administration of a second booster dose of the immunogenic composition containing SARS-CoV 2 S glycoprotein of SEQ ID NO: 329. The second booster dose of the immunogenic composition containing SARS-CoV 2 S glycoprotein of SEQ ID NO: 329 induced pseudovirus neutralizing antibodies against different SARS-CoV-2 variant strains. Further experimental details can be found in Example 12. [Figure 45] This image shows the CD4+ T cell IFN-γ response in primed rhesus monkeys after administration of a second booster dose of an immunogenic composition containing SARS-CoV 2 S glycoprotein of SEQ ID NO: 329. The second booster dose of the immunogenic composition containing SARS-CoV 2 S glycoprotein of SEQ ID NO: 329 induced a Th1-biased cell response and maintained T cell epitopes recognized for all variants tested. Further experimental details can be found in Example 12. [Figure 46] This shows the multifunctional antigen-specific CD4+ T cell response in primed rhesus monkeys after administration of a second booster dose of an immunogenic composition containing SARS-CoV-2 S glycoprotein of SEQ ID NO: 329. Further experimental details can be found in Example 12. [Modes for carrying out the invention]

[0008] definition As used herein and in the appended claims, the singular forms “one (a),” “one (an),” and “it” include multiple references unless otherwise clearly indicated by the context. Thus, for example, a reference to “protein” may refer to one protein or a mixture of such proteins, and a reference to “method” may include references to equivalent steps and / or methods known to those skilled in the art.

[0009] As used herein, the term “adjuvant” means a compound that, when used in combination with an immunogen, enhances, otherwise alters, or modifies the immune response induced to an immunogen. Modification of the immune response may include enhancing or expanding the specificity of either or both the antibody and / or cellular immune response.

[0010] As used herein, the terms “about” or “approximately” when preceding a number indicate a range of plus or minus 10%. For example, “about 100” includes 90 and 110.

[0011] As used herein, the terms “immunogen,” “antigen,” and “epitope” refer to substances, such as proteins and peptides, including glycoproteins, that are capable of inducing an immune response.

[0012] As used herein, “immunogenic composition” is a composition comprising an antigen that, as a result of administration of the composition to a subject, causes the subject to develop a humoral and / or cellular immune response to the antigen.

[0013] As used herein, a “subunit” composition, such as a vaccine, contains one or more selected antigens, but does not contain all antigens derived from a pathogen. Such compositions are substantially free of intact viruses or lysates of such cells or particles, and are typically prepared from immunogenic polypeptides that are at least partially purified, and often substantially purified, from a pathogen. The antigens in the subunit compositions disclosed herein are typically prepared by recombinant means, and often using baculovirus systems.

[0014] As used herein, “substantially” means the isolation of a substance (e.g., a compound, polynucleotide, or polypeptide) such that it forms the majority of the sample in which it is contained. For example, in a sample, substantially purified components constitute 85%, preferably 85% to 90%, more preferably at least 95% to 99.5%, and most preferably at least 99% of the sample. If components are substantially replaced, the remainder in the sample is about 0.5% to about 10% or less, preferably about 0.5% to less than 1.0%.

[0015] The terms “to treat,” “treatment,” and “to treat” as used herein refer to methods for obtaining beneficial or desired results, such as clinical outcomes. For the purposes of this disclosure, beneficial or desired results may include inhibiting or suppressing the onset or progression of an infection or disease, improving or reducing the symptoms of an infection or disease, or a combination thereof.

[0016] As used herein, “prevention” is interchangeable with “prophylaxis” and may mean the complete prevention of an infection or disease or the prevention of the onset of symptoms of such infection or disease, the delay of the onset of an infection or disease or symptoms thereof, or a reduction in the severity of any subsequent infection or disease or symptoms thereof.

[0017] As used herein, “effective dose” or “effective amount” refers to the amount of immunogen sufficient to induce an immune response that alleviates at least one symptom of a pathogen infection. The effective dose or effective amount may be determined, for example, by measuring the amount of neutralizing secretion and / or serum antibodies by plaque neutralization, complement fixation, enzyme-linked immunosorbent assay (ELISA), or microneutralization assay.

[0018] As used herein, the term “vaccine” refers to an immunogenic composition, such as an immunogen derived from a pathogen, which is used to induce an immune response to a pathogen that provides protective immunity (for example, immunity that protects an object from infection by the pathogen and / or reduces the severity of a disease or condition caused by infection by the pathogen). The protective immune response may include antibody formation and / or cell-mediated reactions. Depending on the context, the term “vaccine” may also refer to a suspension or solution of an immunogen administered to an object to produce protective immunity.

[0019] As used herein, the term “Subject” includes humans and other animals. Typically, the subject is human. For example, the subject may be an adult, a teenager, a child (2 to 14 years of age), an infant (from birth to 2 years of age), or a newborn (within 2 months of birth). In certain embodiments, the subject may be up to 4 months of age or up to 6 months of age. In embodiments, an adult may be an elderly person about 65 years of age or older or about 60 years of age or older. In embodiments, the subject may be a pregnant woman or a woman intending to become pregnant. In other embodiments, the subject may not be human, but a non-human primate, such as a baboon, chimpanzee, gorilla, or macaque. In certain embodiments, the subject may be a pet, such as a dog or cat.

[0020] In one embodiment, the subject is immunocompromised. In another embodiment, the immunocompromised subject is administered an immunosuppressant drug. Non-limiting examples of immunosuppressant drugs include corticosteroids (e.g., prednisone), alkylating agents (e.g., cyclophosphamide), antimetabolites (e.g., azathioprine or 6-mercaptopurine), transplant-related immunosuppressants (e.g., cyclosporine, tacrolimus, sirolimus or mycophenolate mofetil), mitoxantrone, chemotherapeutic agents, methotrexate, and tumor necrosis factor (TNF) blockers (e.g., etanercept, adalimumab, infliximab). In yet another embodiment, the immunocompromised subject is infected with a virus (e.g., human immunodeficiency virus or Epstein-Barr virus). In yet another embodiment, the virus is a respiratory virus such as respiratory syncytial virus, influenza, parainfluenza, adenovirus, or picornavirus. In one embodiment, the immunocompromised subject has acquired immunodeficiency syndrome (AIDS). In another embodiment, the immunocompromised subject is a person infected with human immunodeficiency virus (HIV). In yet another embodiment, the immunocompromised subject is an immunocompromised state resulting from a treatment regimen designed to prevent inflammation or transplant rejection. In yet another embodiment, the immunocompromised subject is a person who has previously undergone a transplant. In yet another embodiment, the immunocompromised subject has previously undergone radiotherapy or splenectomy.In the embodiments, the target of immunodeficiency is cancer, autoimmune disease, tuberculosis, substance use disorder (e.g., alcohol, opioid, or cocaine use disorder), stroke or cerebrovascular disease, solid organ or hematopoietic stem cell transplantation, sickle cell disease, thalassemia, autoimmune lymphoproliferative syndrome (ALPS), polyglandular autoimmune syndrome type 1 (APS-1), B cell proliferation disorder with NF-κB and T cell anergy (BENTA), caspase-8 deficiency (CEDS), chronic granulomatous disease (CGD), unclassified immunodeficiency (CVID), congenital neutropenia, cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) deficiency, DOCK8 deficiency, GATA2 deficiency, glycosylation disorders with immunodeficiency, hyperimmune globulin E syndrome (HIES), and high The subjects have been diagnosed with immunoglobulin M syndrome, diabetes mellitus, type 1 diabetes mellitus, type 2 diabetes mellitus, interferon-gamma deficiency, interleukin-12 deficiency, interleukin-23 deficiency, leukocyte adhesion deficiency, lipopolysaccharide-responsive beige-like anchor (LRBA) deficiency, PI3 kinase disease, PLCG2-related antibody deficiency and immunodysregulation (PLAID), severe combined immunodeficiency (SCID), STAT3 dominant-negative disease, STAT3 gain-of-function disease, verrucae, hypogammaglobulinemia, infection and myeloid cell retention (WHIM) syndrome, Wiscott-Aldrich syndrome (WAS), X-linked agammaglobulinemia (XLA), X-linked lymphoproliferative disorder (XLP), uremia, malnutrition, or X-Men disease. In the embodiments, the immunocompromised subjects were currently or formerly tobacco smokers. In the embodiment, the immunocompromised subject has B cell deficiency, T cell deficiency, macrophage deficiency, cytokine deficiency, phagocyte deficiency, phagocyte dysfunction, complement deficiency, or a combination thereof.

[0021] In the embodiments, the subjects are overweight or obese. In the embodiments, the body mass index (BMI) of overweight subjects is ≥25 kg / m2 and <30 kg / m2. In the embodiments, the BMI of obese subjects is ≥30 kg / m2. In the embodiments, the subjects have a mental health condition. In the embodiments, the mental health condition is depression, schizophrenia, or anxiety.

[0022] As used herein, the term “pharmaceutically acceptable” means that it is approved by a U.S. federal or state regulatory authority for use in mammals and more specifically in humans, or that it is listed in the United States Pharmacopeia, the European Pharmacopoeia, or other generally accepted pharmacopoeias. These compositions may be useful as vaccines and / or antigenic compositions for inducing a protective immune response in vertebrates.

[0023] As used herein, the term "NVX-CoV2373" refers to a vaccine composition comprising the BV2373 spike glycoprotein (SEQ ID NO: 87) and fractions A and C iscom matrix (e.g., MATRIX-M®).

[0024] As used herein, the term “modification” refers to a mutation, deletion, or addition of one amino acid in a CoV S polypeptide, when referring to a CoV S polypeptide. The location of a modification within a CoV S polypeptide may be determined by aligning the polypeptide sequence with SEQ ID NO: 1 (a CoV S polypeptide containing a signal peptide) or SEQ ID NO: 2 (a mature CoV S polypeptide lacking a signal peptide).

[0025] The terms "heterogeneous SARS-CoV-2 strain" and "SARS-CoV-2 variant" as used interchangeably herein refer to a SARS-CoV-2 virus comprising a CoV S polypeptide having one or more modifications compared to the SARS-CoV S polypeptide having the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. For example, a SARS-CoV-2 variant may have at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, at least about 25, at least about 26, at least about 27, at least about 28, at least about 29, at least about 30, at least about 31, at least about 32, at least about 33, at least about 34, or at least about 35 modifications compared to a CoV S polypeptide having the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. Compared to S polypeptide, at least 1 and at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 21, at most 22, at most 23, at most 24, at most 25, at most 26, at most 27, at most 28, at most 29, may have a maximum of 30, 31, 32, 33, 34, 35 modifications, a maximum of 40 modifications, a maximum of 45 modifications, a maximum of 50 modifications, a maximum of 55 modifications, a maximum of 60 modifications, a maximum of 65 modifications, a maximum of 70 modifications, a maximum of 75 modifications, a maximum of 80 modifications, a maximum of 85 modifications, a maximum of 90 modifications, a maximum of 95 modifications, or a maximum of 100 modifications.In some embodiments, the SARS-CoV-2 variant may have approximately 2 to approximately 35 modifications, approximately 5 to approximately 10 modifications, approximately 5 to approximately 20 modifications, approximately 10 to approximately 20 modifications, approximately 15 to approximately 25 modifications, approximately 20 to approximately 30 modifications, approximately 20 to approximately 40 modifications, approximately 25 to approximately 45 modifications, approximately 25 to approximately 100 modifications, approximately 25 to approximately 45 modifications, and approximately 35 to approximately 100 modifications compared to the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

[0026] In one embodiment, the heterologous SARS-CoV-2 strain is a SARS-CoV-2 virus containing a CoV S polypeptide having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity with the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In another embodiment, the heterologous SARS-CoV-2 strain is a SARS-CoV-2 virus containing a CoV S polypeptide having about 70% to about 99.9% identity with the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In yet another embodiment, the heterologous SARS-CoV-2 strain is a SARS-CoV-2 virus containing a CoV S polypeptide having about 70% to about 99.5% identity with the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In one embodiment, the heterologous SARS-CoV-2 strain is a SARS-CoV-2 virus containing a CoV S polypeptide having approximately 90% to approximately 99.9% identity with the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In another embodiment, the heterologous SARS-CoV-2 strain is a SARS-CoV-2 virus containing a CoV S polypeptide having approximately 90% to approximately 99.8% identity with the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In yet another embodiment, the heterologous SARS-CoV-2 strain is a SARS-CoV-2 virus containing a CoV S polypeptide having approximately 95% to approximately 99.9% identity with the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In yet another embodiment, the heterologous SARS-CoV-2 strain is a SARS-CoV-2 virus containing a CoV S polypeptide having approximately 95% to approximately 99.8% identity with the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In this embodiment, the heterologous SARS-CoV-2 strain is a SARS-CoV-2 virus that contains a CoV S polypeptide having approximately 95% to 99% identity with the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.In the embodiment, the heterologous SARS-CoV-2 strain has the World Health Organization label alpha, beta, gamma, delta, epsilon, etha, iota, kappa, zeta, mu, or omicron. In embodiments, heterologous SARS-CoV-2 strains are PANGO strains selected from the group consisting of B.1.1.529;BA.1,BA.1.1,BA.2,BA.3,BA.4,BA.5,B.1.1.7,B.1.351,P.1,B.1.617.2,AY,B.1.427,B.1.429,B.1.525,B.1.526,B.1.617.1,B.1.617.3,P.2,B.1.621, or B.1.621.1. The following literature explains the naming of Pango strains, and is incorporated herein by reference in its entirety: O'Toole et al. BMC Genomics, 23, 121 (2022).

[0027] In the embodiment, the heterologous SARS-CoV-2 strain has the Omicron World Health Organization label. In the embodiment, the heterologous SARS-CoV-2 strain with the Omicron World Health Organization label has at least 35 modifications compared to the wild-type SARS-CoV-2 S polypeptide of SEQ ID NO: 2. In the embodiment, the heterologous SARS-CoV-2 strain with the Omicron World Health Organization label has 35-55, 35-65, 35-75, 35-85, 35-95, or 35-105 modifications compared to the wild-type SARS-CoV-2 S polypeptide of SEQ ID NO: 2. In this embodiment, the modifications are T6I, T6R, A14S, A54V, V70A, T82I, G129D, H133Q, K134E, W139R, E143G, F144L, Q170E, I197V, L199I, V200E, V200G, G239V, G244S, G326D, G326H, R333T, L355I, S3 58F, S358L, S360P, S362F, T363A, D392N, R395S, K404N, N427K, K431T, V432P, G433S, L4 39R, L439Q, N447K, S464N, T465K, E471A, F473V, F473S, F477S, Q480R, G483S, Q485R, N4 The following are selected from the group consisting of 88Y, Y492H, T534K, T591I, D601G, G626V, H642Y, N645S, N666K, P668H, S691L, N751K, D783Y, N843K, Q941H, N956K, L968F, D1186N, deletion of amino acid 11, deletion of amino acid 12, deletion of amino acid 13, deletion of amino acid 56, deletion of amino acid 57, deletion of amino acid 130, deletion of amino acid 131, deletion of amino acid 132, deletion of amino acid 144, deletion of amino acid 145, deletion of amino acid 198, and insertion of a tripeptide having the amino acid sequence of EPE between amino acids 214 and 215, as well as combinations thereof.

[0028] In this embodiment, the variant CoV S polypeptide is (i)A54V, T82I, G129D, L199I, G326D, S358L, S360P, S362F, K404N, N427K, G433S, S464 N, T465K, E471A, Q480R, G483S, Q485R, N488Y, Y492H, T534K, D601G, H642Y, N666K, P66 8H, N751K, D783Y, N843K, Q941H, N956K, L968F, deletion of amino acid 56, deletion of amino acid 57, deletion of amino acid 130, deletion of amino acid 131, deletion of amino acid 132, deletion of amino acid 198, and insertion of a tripeptide having the amino acid sequence of EPE between amino acids 214 and 215. (ii) T6I, A14S, G129D, V200G, G326D, S358F, S360P, S362F, T363A, D392N, R395S, K404N, N427K, S464N, T465K, E471A, Q480R, Q485R, N488Y, Y492H, D601G, H642Y, N666K, P668H, N751K, D783Y, Q941H, N956K, deletion of amino acid 11, deletion of amino acid 12 and deletion of amino acid 13, (iii) T6R, A14S, T82I, G129D, E143G, L199I, G326D, S358L, S360P, K404N, N427K, G433S, S464N, T465K, E471A, Q480R, G483S, Q485R, N488Y, Y492H, T534K, D601G, H642Y, N666K, P668H, N751K, D783Y, N843K, Q941H, N956K, L968F, deletion of amino acid 144, deletion of amino acid 145, deletion of amino acid 198, and insertion of a tripeptide having the amino acid sequence of EPE between amino acids 214 and 215. (iv) T6I, A14S, G129D, V200G, G326D, S358F, S360P, S362F, T363A, D392N, K404N, N427K, L439Q, S464N, T465K, E471A, Q480R, Q485R, N488Y, Y492H, D601G, H642Y, N666K, P668H, S691L, N751K, D783Y, Q941H, N956K, deletion of amino acid 11, deletion of amino acid 12 and deletion of amino acid 13, (v)T6I, A14S, G129D, V200G, G326D, S358F, S360P, S362F, T363A, D392N, S464N, T465K, E471A, Q480R, Q485R, N488Y, Y492H, D601G, H642Y, N666K, P668H, D783Y, Q941H, N956K, deletion of amino acid 11, deletion of amino acid 12 and deletion of amino acid 13, (vi) T6I, A14S, G129D, V200G, G326D, S358F, S360P, S362F, T363A, D392N, R395S, K404N, D601G, H642Y, N645S, N666K, N751K, D783Y, Q941H, N956K, deletion of amino acid 11, deletion of amino acid 12, deletion of amino acid 13, deletion of amino acid 56 and deletion of amino acid 57, (vii) V3G, T6I, A14S, G129D, V200G, G326D, S358F, S360P, S362F, T363A, D392N, R395S, K404N, L439R, S464N, T465K, E471A, F473V, Q485R, N488Y, Y492H, D601G, G626V, H642Y, N666K, P668H, N751K, D783Y, Q941H, N956K, deletion of amino acid 11, deletion of amino acid 12, deletion of amino acid 13, deletion of amino acid 56 and deletion of amino acid 57, (viii) V3G, T6I, A14S, G129D, V200G, G326D, S358F, S360P, S362F, T363A, D392N, R395S, K404N, N427K, L439R, S464N, T465K, E471A, F473V, Q485R, N488Y, Y492H, D601G, H642Y, N666K, P668H, N751K, D783Y, Q941H, N956K, deletion of amino acid 11, deletion of amino acid 12, deletion of amino acid 13, deletion of amino acid 56 and deletion of amino acid 57, (ix) T6I, A14S, G129D, V200G, G326D, S358F, S360P, S362F, T363A, D392N, R395S, K404N, N427K, L439R, S464N, T465K, E471A, F473V, Q485R, N488Y, Y492H, D601G, H642Y, N666K, P668H, N751K, D783Y, Q941H, N956K, deletion of amino acid 11, deletion of amino acid 12, deletion of amino acid 13, deletion of amino acid 56 and deletion of amino acid 57, (x)T6I, A14S, G129D, K134E, W139R, F144L, I197V, V200G, G244S, G326H, S358F, S360P, S362F, T363A, D392N, R395S, K404N, N427K, G433S, N447K, S464N, T465K, E471A, Q485R, N488Y, Y492H, D601G, H642Y, N666K, P668H, N751K, D783Y, Q941H, N956K, deletion of amino acid 11, deletion of amino acid 12 and deletion of amino acid 13, (xi)T6I, A14S, G129D, K134E, W139R, F144L, I197V, V200G, G244S, G326H, R333T , S358F, S360P, S362F, T363A, D392N, R395S, K404N, N427K, G433S, L439R, N447K, S464N, T465K, E471A, F473S, Q485R, N488Y, Y492H, T591I, D601G, H642Y, N666K, P668H, N751K, D783Y, Q941H, N956K, D1186N, deletion of amino acid 11, deletion of amino acid 12 and deletion of amino acid 13, (xii)T6I, A14S, G129D, V200G, G326D, R333T, S358F, S360P, S362F, T363A, D392N, R395S, K404N, N427K, L439R, S464N, T465K, E471A, F473V, Q485R, N488Y, Y492H, D601G, H642Y, N645S, N666K, P668H, N751K, D783Y, Q941H, N956K, deletion of amino acid 11, deletion of amino acid 12, deletion of amino acid 13, deletion of amino acid 56 and deletion of amino acid 57, (xiii)T6I, A14S, G129D, V200G, G326D, R333T, S358F, S360P, S362F, T363A, D392N, R395S, K404N, N427K, L439R, S464N, T465K, E471A, F473V, Q485R, N488Y, Y492H, D601G, H642Y, N666K, P668H, N751K, D783Y, Q941H, N956K, deletion of amino acid 11, deletion of amino acid 12, deletion of amino acid 13, deletion of amino acid 56 and deletion of amino acid 57, (xiv)T6I, A14S, V70A, G129D, H133Q, Q170E, V200E, G239V, G326H, R333T, L355I, S358F, S360P, S362F, T363A, D392N, R395S, K404N, N427K, V432P, G433S, N447K, S4 64N, T465K, E471A, F473S, F477S, Q485R, N488Y, Y492H, D601G, H642Y, N666K, P668H, N751K, D783Y, Q941H, N956K, deletion of amino acid 11, deletion of amino acid 12, deletion of amino acid 13 and deletion of amino acid 131, (xv)T6I, A14S, G129D, H133Q, Q170E, V200E, G326H, R333T, L355I, S358F, S360P, S3 62F, T363A, D392N, R395S, K404N, N427K, V432P, G433S, N447K, S464N, T465K, E471A, F473S, F477S, Q485R, N488Y, Y492H, D601G, H642Y, N666K, P668H, N751K, D783Y, Q941H, N956K, deletion of amino acid 11, deletion of amino acid 12, deletion of amino acid 13, deletion of amino acid 56, deletion of amino acid 57 and deletion of amino acid 131, (xvi)T6I, A14S, G129D, V200G, G326D, R333T, S358F, S360P, S362F, T363A, D392N, R395S, K404N, N427K, K431T, L439R, N447K, S464N, T465K, E471A, F473V, Q485R, N488Y, Y492H, D601G, H642Y, N666K, P668H, N751K, D783Y, Q941H, N956K, deletion of amino acid 11, deletion of amino acid 12, deletion of amino acid 13, deletion of amino acid 56 and deletion of amino acid 57, (xvii)T6I, A14S, G129D, V200G, G326D, S358F, S360P, S362F, T363A, D392N, R395S, K404N, N427K, K431T, L439R, N447K, S464N, T465K, E471A, F473V, Q485R, N488Y, Y492H, D601G, H642Y, N666K, P668H, N751K, D783Y, Q941H, N956K, deletion of amino acid 11, deletion of amino acid 12, deletion of amino acid 13, deletion of amino acid 56 and deletion of amino acid 57, and (xviii)T6I, A14S, G129D, V200G, G326D, R333T, S358F, S360P, S362F, T363A, D392N, R395S, K404N, N427K, L439R, S464N, T465K, E471A, F473V, Q485R, N488Y, Y492H, D601G, H642Y, N666K, P668H, N751K, D783Y, Q941H, N956K, deletion of amino acid 11, deletion of amino acid 12, deletion of amino acid 13, deletion of amino acid 56, deletion of amino acid 57 and deletion of amino acid 131, (xix) Deletion of amino acid 56, deletion of amino acid 57 and deletion of amino acid 131, N488Y, A557D, D601G, P668H or P668R, T703I, S969A and D1105H, (xx)D67A, K404N, E471K, N488Y, D601G and A688V, (xxi) D67A, D202G, L229H, K404N, E471K, N488Y, D601G and A688V, (xxii)D67A, D202G, deletion of 1, 2, or 3 amino acids between amino acids 228 and 230, K404N, E471K, N488Y, D601G, and A688V, (xxiii) D67A, L229H, R233I, N488Y, K404N, E471K, D601G and A688V, (xxiv) L5F, T7N, P13S, D125Y, R177S, K404T, E471K, N488Y, D601G, H642Y, T1014I and V1163F, (xxv)W139C and L439, (xxvi) Deletion of amino acid 144, deletion of amino acid 145, T6R, E143G, L439R, T465K, D601G, P668R and D937N, (xxvii) Deletion of amino acid 144, deletion of amino acid 145, T6R, G129D, E143G, L439R, T465K, D601G, P668R and D937N, (xxviii) Deletion of amino acid 144, deletion of amino acid 145, T6R, T82I, G129D, Y132H, E143G, A209V, K404N, L439R, T465K, D601G, P668R and D937N, (xxix) Deletion of amino acid 144, deletion of amino acid 145, T6R, G129D, E143G, W245I, K404N, N426K, L439R, T465K, E471K, N488Y, D601G, P668R and D937N, (xxx) Deletion of amino acid 144, deletion of amino acid 145, T6R, W51H, H53W, G129D, E143G, D200V, L201R, W245I, K404N, N426K, L439R, T465K, E471K, N488Y, D601G, P668R and D937N, (xxxi) Deletion of amino acid 144, deletion of amino acid 145, T6R, G129D, E143G, K404N, L439R, T465K, E471Q, D601G, P668R and D937N, (xxxii) Deletion of one, two, three or four amino acids Q39R, A54V, E471K, D601G, Q664H, F875L, and amino acids 56, 57, 131, 132. (xxxiii)T82I, D240G, E471K, D601G and A688V, (xxxiv) L439R, E471Q, D601G, P668R and Q1058H, (xxxv)G62V, T63I, R233N, L439Q, F477S, D601G, T846N and 1, 2, 3, 4, 5, or 6 deletions of amino acids 234-240, (xxxvi)T82I, Y131S, Y132N, R333K, E471K, N488Y, D601G, P668H and D937N, and (xxxvii) G129D, G326D, S360P, S362F, K404N, N427K, T465K, E471A or E471K, Q480K or Q480R, Q485R, N488Y, Y492H, D601G, H642Y, N666K, P668H, N751K, D783Y, Q941H and N953K, (xxxviii)F456L, T572I, F59S, R346T, A1087S or A475V The amino acids of the CoV S glycoprotein are numbered relative to the polypeptide having the sequence of SEQ ID NO: 1 or SEQ ID NO: 2, and the amino acids are selected from the group consisting of the following:

[0029] The term "efficacy" of the immunogenic composition or vaccine composition described herein refers to the percentage reduction in disease (e.g., COVID-19) in the group administered the immunogenic composition compared to the group not administered the immunogenic composition. In embodiments, efficacy (E) is calculated using the following formula: E(%) = (1 - RR) × 100 (where RR = relative risk of incidence between the group administered the immunogenic composition and the group not administered the immunogenic composition). In embodiments, the immunogenic compositions described herein have an efficacy of at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, It has efficacy against SARS-CoV-2 virus or heterologous SARS-CoV-2 strains with efficacy rates of approximately 50% to 99%, 50% to 98%, 60% to 99%, 60% to 98%, 70% to 98%, 70% to 95%, 70% to 99%, 80% to 99%, 80% to 98%, 80% to 95%, 85% to 99%, 85% to 98%, 85% to 95%, 90% to 95%, 90% to 98%, or 90% to 99%.

[0030] Composition containing coronavirus (CoV) spike (S) protein This disclosure provides coronavirus (CoV) spike (S) polypeptides, including natural and non-natural polypeptides, nanoparticles containing CoV S polypeptides, and immunogenic compositions and vaccine compositions containing either CoV S polypeptides or nanoparticles containing CoV S polypeptides. In embodiments, methods for using CoV S polypeptides, nanoparticles, immunogenic compositions and vaccine compositions to stimulate an immune response to the SARS-CoV-2 virus or heterologous SARS-CoV-2 strains are provided herein. In embodiments, the heterologous SARS-CoV-2 strain has a PANGO lineage selected from the group consisting of B.1.1.529;BA.1,BA.1.1,BA.2,BA.3,BA.4,BA.5, B.1.1.7, B.1.351, P.1, B.1.617.2, AY, B.1.427, B.1.429, B.1.525, B.1.526, B.1.617.1, B.1.617.3, P.2, B.1.621, or B.1.621.1. In embodiments, the heterologous SARS-CoV-2 strain has the World Health Organization label alpha, beta, gamma, delta, epsilon, etha, iota, kappa, zeta, mu, or omicron.

[0031] Methods for producing nanoparticles and vaccine compositions are also provided herein. An advantage of this method is that it provides nanoparticles that are substantially free from contamination by other proteins, such as proteins associated with the recombinant expression of proteins in insect cells. In embodiments, expression is carried out using a baculovirus / Sf9 or baculovirus / Sf22a system.

[0032] CoV S polypeptide antigen The immunogenic compositions of this disclosure contain about 1 to about 15, about 2 to about 15, about 3 to about 15, about 3 to about 12, about 4 to about 6, about 3 to about 7, about 4 to about 12, about 5 to about 8, about 6 to about 9, or about 5 to 10 non-natural CoV S polypeptides. In embodiments, the immunogenic composition contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 different CoV S polypeptides. In embodiments, the immunogenic composition contains 5 non-natural CoV S polypeptides. The CoV S polypeptides may be derived from coronaviruses including, but not limited to, SARS-CoV-2, such as SARS-CoV-2, MERS-CoV, and SARS-CoV.

[0033] In the embodiments, the CoV S polypeptide is derived from SARS-CoV-2. In the embodiments, the CoV S polypeptide is derived from a heterologous SARS-CoV-2 strain. The SARS-CoV-2 S protein has four amino acid insertions at the S1 / S2 cleavage site, resulting in a polybasic RRAR furin-like cleavage motif. The SARS-CoV-2 S protein is synthesized as an inactive precursor (S0) and proteolytically cleaved at the furin cleavage site to form non-covalently bonded S1 and S2 subunits, forming a prefusion trimer. The S2 domain of the SARS-CoV-2 S protein contains a fusion peptide (FP), two 7-repeats (HR1 and HR2), a transmembrane (TM) domain, and a cytoplasmic tail (CT). The S1 domain of the SARS-CoV-2 S protein folds to form a C-terminal domain containing four distinct domains: an N-terminal domain (NTD) and a receptor-binding domain (RBD) and two subdomains SD1 and SD2. The pre-fusion SARS-CoV-2 S protein trimer undergoes structural reconfiguration from its pre-fusion to its post-fusion structure upon binding to and cleavage of the S protein receptor.

[0034] In embodiments, the CoV S polypeptide is a glycoprotein resulting from post-translational glycosylation. The glycoprotein comprises one or more domains, including a signal peptide, an S1 subunit, an S2 subunit, an NTD, an RBD, two subdomains (SD1 and SD2, labeled SD1 / 2 in Figures 1A-B and referred to herein as "SD1 / 2"), an intact or modified fusion peptide, an HR1 domain, an HR2 domain, a TM, and a CD. In embodiments, the amino acids for each domain are given in Figure 1A (shown according to SEQ ID NO: 1) and Figure 1B (shown according to SEQ ID NO: 2).

[0035] In the embodiments, the immunogenic composition comprises (i) a first CoV S polypeptide derived from SARS-CoV-2 or a heterologous SARS-CoV-2 strain (the first CoV S polypeptide contains an inactive furin cleavage site and mutations at amino acids 973 and 974, and the CoV S polypeptides are numbered according to SEQ ID NO: 2) and (ii) a second CoV S polypeptide having about 1 to about 50 modifications compared to the first CoV S polypeptide. In the embodiments, the immunogenic composition comprises a third, fourth, fifth, sixth, seventh, eighth, ninth or tenth CoV S polypeptide, each CoV S polypeptide containing about 1 to about 50 modifications compared to the first CoV S polypeptide.

[0036] In the embodiment, the amino acid sequences of the S1 subunit, S2 subunit, NTD, RBD, SD1 / 2, fusion peptide, HR1 domain, HR2 domain, TM domain, or CD of the second, third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth CoV S polypeptide are independently identical to the amino acid sequences of the respective domains of the first polypeptide by at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5%.

[0037] In the embodiments, the amino acid sequences of the S1 subunit, S2 subunit, NTD, RBD, SD1 / 2, fusion peptide, HR1 domain, HR2 domain, TM domain, or CD of the second, third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth CoV S polypeptide contain up to about 1, up to about 2, up to about 3, up to about 4, up to about 5, up to about 6, up to about 7, up to about 8, up to about 9, up to about 10, up to about 15, up to about 20, up to about 25, up to about 30, 1 to about 5, about 2 to about 4, about 3 to about 5, about 3 to about 10, about 5 to about 10, about 6 to about 12, about 8 to about 12, about 10 to about 15, about 15 to about 20, about 12 to about 18, or about 18 to about 25 modifications compared to the amino acid sequences of each domain of the first polypeptide.

[0038] In this embodiment, the amino acid sequences of the second, third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth CoV S polypeptide are at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% identical to the amino acid sequence of the first CoV S polypeptide, but the identity with the amino acid sequence of the first polypeptide is 99.92% or less.

[0039] In embodiments, the compositions described herein may be used to stimulate an immune response to SARS-CoV-2 or a heterologous SARS-CoV-2 strain.

[0040] In embodiments, the CoV S polypeptide described herein exists in its pre-fusion conformation. In embodiments, the CoV S polypeptide described herein includes a flexible HR2 domain. Unless otherwise specified, the flexibility of the domain is determined by transmission electron microscopy (TEM) and 2D class averaging. A decrease in electron density corresponds to a flexible domain.

[0041] CoV S polypeptide antigen - modification of the S1 subunit In the embodiment, the CoV S polypeptide contains one or more modifications to the S1 subunit having the amino acid sequence of SEQ ID NO: 121.

[0042] The amino acid sequence of the S1 subunit (sequence number 121) is shown below. [ka]

[0043] In embodiments, the CoV S polypeptide described herein includes an S1 subunit having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity with the S1 subunit of SEQ ID NO: 1 or SEQ ID NO: 2. The S1 subunit may have up to about 1, up to about 2, up to about 3, up to about 4, up to about 5, up to about 10, up to about 15, up to about 20, up to about 25, up to about 30 amino acids, up to about 35 amino acids, up to about 40 amino acids, up to about 45 amino acids, or up to about 50 amino acids deleted, inserted, or mutated compared to the amino acid sequence of the S1 subunit of SEQ ID NO: 1 or SEQ ID NO: 2. The S1 subunit may have deletions, insertions, or mutations of approximately 1 to 5 amino acids, 3 to 10 amino acids, 5 to 10 amino acids, 8 to 12 amino acids, 10 to 15 amino acids, 12 to 17 amino acids, 15 to 20 amino acids, 18 to 23 amino acids, 20 to 25 amino acids, 22 to 27 amino acids, or 25 to 30 amino acids compared to the amino acid sequence of the S1 subunit of SEQ ID NO: 1 or SEQ ID NO: 2.

[0044] In the embodiment, the S1 subunit may contain any combination of the modifications shown in Table 1A.

[0045] [Table 1]

[0046] Table 2

[0047] Table 3

[0048] Table 4

[0049] Table 5

[0050] Table 6

[0051] Table 7

[0052] Table 8

[0053] Table 9

[0054] Table 10

[0055] Table 11

[0056] Table 12

[0057] Table 13

[0058] Table 14

[0059] Table 15

[0060] Table 16

[0061] Table 17

[0062] Table 18

[0063] Table 19

[0064] Table 20

[0065] Table 21

[0066] Table 22

[0067] [Table 23]

[0068] [Table 24]

[0069] [Table 25]

[0070] CoV S polypeptide antigen - S1 subunit - Modification of NTD In the embodiment, the CoV S polypeptide contains one or more modifications to the NTD. In the embodiment, the NTD has the amino acid sequence of SEQ ID NO: 118, which corresponds to amino acids 14-305 of SEQ ID NO: 1 or amino acids 1-292 of SEQ ID NO: 2.

[0071] The amino acid sequence of NTD (sequence number 118) is shown below. [ka]

[0072] In this embodiment, the NTD has the amino acid sequence of SEQ ID NO: 45, which corresponds to amino acids 14-331 of SEQ ID NO: 1 or amino acids 1-318 of SEQ ID NO: 2. The amino acid sequence of the NTD (SEQ ID NO: 45) is shown below. [ka]

[0073] In this embodiment, the NTD and RBD overlap by a maximum of approximately 1 amino acid, a maximum of approximately 5 amino acids, a maximum of approximately 10 amino acids, or a maximum of approximately 20 amino acids.

[0074] In embodiments, the NTDs provided herein may be extended by up to 5, up to 10, up to 15, up to 20, up to 25, or up to 30 amino acids at the C-terminus.

[0075] In embodiments, the CoV S polypeptide described herein includes an NTD having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity with the NTD of SEQ ID NO: 1 or SEQ ID NO: 2. The NTD may have up to about 1, up to about 2, up to about 3, up to about 4, up to about 5, up to about 10, up to about 15, up to about 20, up to about 25, or up to about 30 amino acid deletions, insertions, or mutations compared to the amino acid sequence of the NTD of SEQ ID NO: 1 or SEQ ID NO: 2. NTDs may have deletions, insertions, or mutations of approximately 1 to 5 amino acids, 3 to 10 amino acids, 5 to 10 amino acids, 8 to 12 amino acids, 10 to 15 amino acids, 12 to 17 amino acids, 15 to 20 amino acids, 18 to 23 amino acids, 20 to 25 amino acids, 22 to 27 amino acids, or 25 to 30 amino acids compared to NTDs of SEQ ID NO: 1 or SEQ ID NO: 2.

[0076] In embodiments, the CoV S polypeptide contains one or more amino acid deletions from the N-terminal domain (NTD) (corresponding to amino acids 1-292 of SEQ ID NO: 2). In embodiments, the CoV S polypeptide contains up to approximately 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 292 amino acid deletions in the NTD.

[0077] In the embodiment, the CoV S polypeptide contains the deletion of one or more amino acids from the NTD (corresponding to amino acids 1-318 of SEQ ID NO: 2). In the embodiment, the CoV S polypeptide contains the deletion of amino acids 1-318 of the NTD of SEQ ID NO: 2. In the embodiment, the NTD deletion promotes the protein expression of the CoV spike (S) polypeptide. In the embodiment, the CoV S polypeptide with the NTD deletion has the amino acid sequence represented by SEQ ID NOs: 46, 48, 49, 51, 52, and 54. In the embodiment, the CoV S polypeptide with the NTD deletion is encoded by an isolated nucleic acid sequence selected from the group consisting of SEQ ID NOs: 47, SEQ ID NOs: 50, and SEQ ID NOs: 53.

[0078] In embodiments, the NTD may contain any combination of the modifications shown in Table 1B. The modifications are shown with respect to Sequence ID No. 2, which is a mature S polypeptide sequence for reference.

[0079] [Table 26]

[0080] [Table 27]

[0081] [Table 28]

[0082] [Table 29]

[0083] [Table 30]

[0084] [Table 31]

[0085] [Table 32]

[0086] [Table 33]

[0087] [Table 34]

[0088] [Table 35]

[0089] CoV S polypeptide antigen - S1 subunit - Modification of RBD In the embodiment, the CoV S polypeptide contains one or more modifications to RBD.

[0090] In this embodiment, RBD has the amino acid sequence of SEQ ID NO: 126, which corresponds to amino acids 331-527 of SEQ ID NO: 1 or amino acids 318-514 of SEQ ID NO: 2.

[0091] The amino acid sequence of RBD (sequence number 126) is shown below. [ka]

[0092] In this embodiment, RBD has the amino acid sequence of SEQ ID NO: 116, which corresponds to amino acids 335-530 of SEQ ID NO: 1 or amino acids 322-517 of SEQ ID NO: 2.

[0093] The amino acid sequence of RBD (SEQ ID NO: 116) is shown below. [ka]

[0094] In embodiments, the RBDs provided herein may be extended by up to 1 amino acid, up to 5 amino acids, up to 10 amino acids, up to 15 amino acids, up to 20 amino acids, up to 25 amino acids, or up to 30 amino acids at the N-terminus or C-terminus.

[0095] In embodiments, the CoV S polypeptide described herein includes an RBD having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity with the RBD of SEQ ID NO: 1 or SEQ ID NO: 2. The RBD may have up to about 1, up to about 2, up to about 3, up to about 4, up to about 5, up to about 10, up to about 15, up to about 20, up to about 25, or up to about 30 amino acid deletions, insertions, or mutations compared to the amino acid sequence of the RBD of SEQ ID NO: 1 or SEQ ID NO: 2. RBD may have deletions, insertions, or mutations of approximately 1 to 5 amino acids, 3 to 10 amino acids, 5 to 10 amino acids, 8 to 12 amino acids, 10 to 15 amino acids, 12 to 17 amino acids, 15 to 20 amino acids, 18 to 23 amino acids, 20 to 25 amino acids, 22 to 27 amino acids, or 25 to 30 amino acids compared to RBD of SEQ ID NO: 1 or SEQ ID NO: 2.

[0096] In one embodiment, the CoV S polypeptide has at least 1, at least 2, at least 3, at least 4, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 mutations in the RBD. In another embodiment, the RBD may contain any combination of modifications as shown in Table 1C.

[0097] [Table 36]

[0098] [Table 37]

[0099] [Table 38]

[0100] [Table 39]

[0101] [Table 40]

[0102] [Table 41]

[0103] Modification of CoV S polypeptide antigen - SD1 / 2 In the embodiment, the CoV S polypeptide contains one or more modifications to SD1 / 2 having the amino acid sequence of SEQ ID NO: 122, corresponding to amino acids 542-681 of SEQ ID NO: 1 or amino acids 529-668 of SEQ ID NO: 2.

[0104] The amino acid sequence of SD1 / 2 (SEQ ID NO: 122) is shown below. [ka]

[0105] In embodiments, the CoV S polypeptide described herein comprises an SD1 / 2 having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity with the SD1 / 2 of SEQ ID NO: 1 or SEQ ID NO: 2. The SD1 / 2 may have up to about 1, up to about 2, up to about 3, up to about 4, up to about 5, up to about 10, up to about 15, up to about 20, up to about 25, or up to about 30 amino acid deletions, insertions, or mutations compared to the amino acid sequence of the SD1 / 2 of SEQ ID NO: 1 or SEQ ID NO: 2. SD1 / 2 may have deletions, insertions, or mutations of approximately 1 to 5 amino acids, 3 to 10 amino acids, 5 to 10 amino acids, 8 to 12 amino acids, 10 to 15 amino acids, 12 to 17 amino acids, 15 to 20 amino acids, 18 to 23 amino acids, 20 to 25 amino acids, 22 to 27 amino acids, or 25 to 30 amino acids compared to SD1 / 2 of SEQ ID NO: 1 or SEQ ID NO: 2.

[0106] In one embodiment, the CoV S polypeptide has at least 1, at least 2, at least 3, at least 4, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 mutations in SD1 / 2. In one embodiment, SD1 / 2 may contain any combination of modifications as shown in Table 1D.

[0107] [Table 42]

[0108] [Table 43]

[0109] [Table 44]

[0110] CoV S polypeptide antigen - Modification of the furin cleavage site In the embodiments, the CoV S polypeptide contains a furin site (RRAR) corresponding to amino acids 682-685 of SEQ ID NO: 1 or amino acids 669-672 of SEQ ID NO: 2, which is inactivated by one or more mutations. Inactivation of the furin cleavage site prevents furin from cleaving the CoV S polypeptide. In the embodiments, the CoV S polypeptide described herein, containing the inactivated furin cleavage site, is expressed as a single chain.

[0111] In the embodiment, one or more amino acids containing the native furin cleavage site are mutated to any natural amino acid. In the embodiment, the amino acid is an L-amino acid. Non-limiting examples of amino acids include alanine, arginine, glycine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, serine, threonine, histidine, lysine, methionine, proline, valine, isoleucine, leucine, tyrosine, tryptophan, and phenylalanine.

[0112] In the embodiment, one or more amino acids containing the native furin cleavage site are mutated to glutamine. In the embodiment, one, two, three, or four amino acids may be mutated to glutamine. In the embodiment, one arginine molecule containing the native furin cleavage site is mutated to glutamine. In the embodiment, two arginine molecules containing the native furin cleavage site are mutated to glutamine. In the embodiment, three arginine molecules containing the native furin cleavage site are mutated to glutamine.

[0113] In the embodiment, one or more amino acids containing the native furin cleavage site are mutated to alanine. In the embodiment, one, two, three, or four amino acids may be mutated to alanine. In the embodiment, one arginine molecule containing the native furin cleavage site is mutated to alanine. In the embodiment, two arginine molecules containing the native furin cleavage site are mutated to alanine. In the embodiment, three arginine molecules containing the native furin cleavage site are mutated to alanine.

[0114] In the embodiment, one or more amino acids containing the native furin cleavage site are mutated to glycine. In the embodiment, one, two, three, or four amino acids may be mutated to glycine. In the embodiment, one arginine molecule containing the native furin cleavage site is mutated to glycine. In the embodiment, two arginine molecules containing the native furin cleavage site are mutated to glycine. In the embodiment, three arginine molecules containing the native furin cleavage site are mutated to glycine.

[0115] In the embodiment, one or more amino acids containing the native furin cleavage site are mutated to asparagine. For example, one, two, three, or four amino acids may be mutated to asparagine. In the embodiment, one arginine molecule containing the native furin cleavage site is mutated to asparagine. In the embodiment, two arginine molecules containing the native furin cleavage site are mutated to asparagine. In the embodiment, three arginine molecules containing the native furin cleavage site are mutated to asparagine.

[0116] Non-restrictive examples of amino acid sequences of inactivated furin sites contained within CoV S polypeptides can be found in Table 1E.

[0117] [Table 45]

[0118] [Table 46]

[0119] [Table 47]

[0120] In the embodiments, instead of an active furin cleavage site (SEQ ID NO: 6), the CoV S polypeptide described herein contains an inactivated furin cleavage site. In the embodiments, the amino acid sequence of the inactivated furin cleavage site is represented by any one of SEQ ID NOs: 7-34 or SEQ ID NO: 97. In the embodiments, the amino acid sequence of the inactivated furin cleavage site is QQAQ (SEQ ID NO: 7). In the embodiments, the amino acid sequence of the inactivated furin cleavage site is GSAS (SEQ ID NO: 97). In the embodiments, the amino acid sequence of the inactivated furin cleavage site is GSGA (SEQ ID NO: 111). In the embodiments, the amino acid sequence of the inactivated furin cleavage site is GG, GGG (SEQ ID NO: 127), GGGG (SEQ ID NO: 128), or GGGGG (SEQ ID NO: 129).

[0121] CoV S polypeptide antigen - modification of the S2 subunit In the embodiment, the CoV S polypeptide contains one or more modifications to the S2 subunit having the amino acid sequence of SEQ ID NO: 120, which corresponds to amino acids 686-1273 of SEQ ID NO: 1 or amino acids 673-1260 of SEQ ID NO: 2.

[0122] The amino acid sequence of the S2 subunit (sequence number 120) is shown below. [ka]

[0123] In embodiments, the CoV S polypeptide described herein includes an S2 subunit having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity with the S2 subunit of SEQ ID NO: 1 or SEQ ID NO: 2. The S2 subunit may have up to about 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20, at least 25, or at least 30 amino acid deletions, insertions, or mutations compared to the amino acid sequence of the S2 subunit of SEQ ID NO: 1 or SEQ ID NO: 2. The S2 subunit may have deletions, insertions, or mutations of approximately 1 to 5 amino acids, 3 to 10 amino acids, 5 to 10 amino acids, 8 to 12 amino acids, 10 to 15 amino acids, 12 to 17 amino acids, 15 to 20 amino acids, 18 to 23 amino acids, 20 to 25 amino acids, 22 to 27 amino acids, or 25 to 30 amino acids compared to the S2 subunit of SEQ ID NO: 1 or SEQ ID NO: 2.

[0124] In the embodiment, the S2 subunit may contain any combination of modifications as shown in Table 1F.

[0125] [Table 48]

[0126] [Table 49]

[0127] [Table 50]

[0128] [Table 51]

[0129] [Table 52]

[0130] In the embodiment, the CoV S polypeptide contains deletions corresponding to one or more deletions in amino acids 676-685 of the native CoV spike (SEQ ID NO: 2). In the embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids are deleted in amino acids 676-685 of the native CoV spike (SEQ ID NO: 2). In the embodiment, the deletions of amino acids 676-685 are consecutive, for example, amino acids 676 and 677 are deleted, or amino acids 680 and 681 are deleted. In the embodiment, the deletions of amino acids 676-685 are discontinuous, for example, amino acids 676 and 680 are deleted, or amino acids 677 and 682 are deleted. In the embodiment, the CoV S polypeptide containing deletions corresponding to one or more deletions in amino acids 676-685 has an amino acid sequence selected from the group consisting of SEQ ID NO: 62 and SEQ ID NO: 63.

[0131] In one embodiment, the CoV S polypeptide contains deletions corresponding to one or more deletions in amino acids 702-711 of the native CoV spike (S) polypeptide (SEQ ID NO: 2). In one embodiment, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids are deleted in amino acids 702-711 of the native SARS-CoV-2 spike (SEQ ID NO: 2). In one embodiment, the one or more deletions in amino acids 702-711 are contiguous, for example, amino acids 702 and 703 are deleted, or amino acids 708 and 709 are deleted. In one embodiment, the deletions in amino acids 702-711 are discontinuous, for example, amino acids 702 and 704 are deleted, or amino acids 707 and 710 are deleted. In one embodiment, the CoV S polypeptide containing deletions corresponding to one or more deletions in amino acids 702-711 has an amino acid sequence selected from the group consisting of SEQ ID NO: 64 and SEQ ID NO: 65.

[0132] In the embodiment, the CoV S polypeptide contains deletions corresponding to one or more deletions in amino acids 775-793 of the native CoV S polypeptide (SEQ ID NO: 2). In the embodiment, up to about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 amino acids are deleted in amino acids 775-793 of the native SARS-CoV-2 spike (SEQ ID NO: 2). In the embodiment, one or more deletions of amino acids in amino acids 775-793 are contiguous, for example, amino acids 776 and 777 are deleted, or amino acids 780 and 781 are deleted. In the embodiment, the deletions of amino acids in amino acids 775-793 are discontinuous, for example, amino acids 775 and 790 are deleted, or amino acids 777 and 781 are deleted.

[0133] In one embodiment, the CoV S polypeptide contains a deletion in the fusion peptide (SEQ ID NO: 104) corresponding to amino acids 806-815 of SEQ ID NO: 2. In another embodiment, the CoV spike (S) polypeptide (SEQ ID NO: 2) has one, two, three, four, five, six, seven, eight, nine, or ten amino acids deleted from the fusion peptide. In one embodiment, the amino acid deletions in the fusion peptide are consecutive, for example, amino acids 806 and 807 are deleted, or amino acids 809 and 810 are deleted. In another embodiment, the amino acid deletions in the fusion peptide are discontinuous, for example, amino acids 806 and 808 are deleted, or amino acids 810 and 813 are deleted. In yet another embodiment, the CoV S polypeptide containing a deletion corresponding to one or more amino acids in the fusion peptide has an amino acid sequence selected from SEQ ID NOs: 66, 77, and 105-108.

[0134] In the embodiments, the CoV S polypeptide contains a mutation at Lys-973 of the native CoV spike (S) polypeptide (SEQ ID NO: 2). In the embodiments, Lys-973 is mutated to any native amino acid. In the embodiments, Lys-973 is mutated to proline. In the embodiments, Lys-973 is mutated to glycine. In the embodiments, the CoV S polypeptide containing the mutation at amino acid 973 is selected from the group consisting of SEQ ID NOs: 84-89, 105-106, and 109-110.

[0135] In the embodiments, the CoV S polypeptide contains a mutation at Val-974 of the native CoV spike (S) polypeptide (SEQ ID NO: 2). In the embodiments, Val-974 is mutated to any native amino acid. In the embodiments, Val-974 is mutated to proline. In the embodiments, Val-974 is mutated to glycine. In the embodiments, the CoV S polypeptide containing the mutation at amino acid 974 is selected from the group consisting of SEQ ID NOs: 84-89, 105-106, and 109-110.

[0136] In one embodiment, the CoV S polypeptide contains mutations in Lys-973 and Val-974 of the native CoV spike (S) polypeptide (SEQ ID NO: 2). In another embodiment, Lys-973 and Val-974 are mutated to any native amino acid. In yet another embodiment, Lys-973 and Val-974 are mutated to proline. In yet another embodiment, the CoV S polypeptide containing mutations in amino acids 973 and 974 includes SEQ ID NOs: 84-89, 105-106, 109-110, 175, 220, and 217-228.

[0137] CoV S polypeptide antigen - S2 subunit - Modification of the HR1 domain In this embodiment, the CoV S polypeptide contains one or more modifications to the HR1 domain having the amino acid sequence of SEQ ID NO: 119, which correspond to amino acids 912-984 of SEQ ID NO: 1 or amino acids 889-971 of SEQ ID NO: 2.

[0138] The amino acid sequence of the HR1 domain (SEQ ID NO: 119) is shown below. MAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRL

[0139] In embodiments, the CoV S polypeptide described herein comprises an HR1 domain having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity with the HR1 domain of SEQ ID NO: 1 or SEQ ID NO: 2. The HR1 domain may have up to about 1, up to about 2, up to about 3, up to about 4, up to about 5, up to about 10, up to about 15, up to about 20, up to about 25, or up to about 30 amino acid deletions, insertions, or mutations compared to the amino acid sequence of the HR1 domain of SEQ ID NO: 1 or SEQ ID NO: 2. The HR1 domain may have deletions, insertions, or mutations of approximately 1 to 5 amino acids, 3 to 10 amino acids, 5 to 10 amino acids, 8 to 12 amino acids, 10 to 15 amino acids, 12 to 17 amino acids, 15 to 20 amino acids, 18 to 23 amino acids, 20 to 25 amino acids, 22 to 27 amino acids, or 25 to 30 amino acids compared to the HR1 domain of SEQ ID NO: 1 or SEQ ID NO: 2.

[0140] In the embodiment, the HR1 domain may contain any combination of modifications as shown in Table 1G.

[0141] [Table 53]

[0142] CoV S polypeptide antigen - S2 subunit - Modification of the HR2 domain In this embodiment, the CoV S polypeptide contains one or more modifications to the HR2 domain having the amino acid sequence of SEQ ID NO: 125, which correspond to amino acids 1163-1213 of SEQ ID NO: 1 or amino acids 1150-1200 of SEQ ID NO: 2.

[0143] The amino acid sequence of the HR2 domain (SEQ ID NO: 125) is shown below. DVDLGDISGINASVVNIQKEIDRNEVAKNLNESLIDLQELGKYEQYIKWP

[0144] In embodiments, the CoV S polypeptide described herein comprises an HR2 domain having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity with the HR2 domain of SEQ ID NO: 1 or SEQ ID NO: 2. The HR2 domain may have up to about 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20, at least 25, or at least 30 amino acid deletions, insertions, or mutations compared to the amino acid sequence of the HR2 domain of SEQ ID NO: 1 or SEQ ID NO: 2. The HR2 domain may have deletions, insertions, or mutations of approximately 1 to 5 amino acids, 3 to 10 amino acids, 5 to 10 amino acids, 8 to 12 amino acids, 10 to 15 amino acids, 12 to 17 amino acids, 15 to 20 amino acids, 18 to 23 amino acids, 20 to 25 amino acids, 22 to 27 amino acids, or 25 to 30 amino acids compared to the HR2 domain of SEQ ID NO: 1 or SEQ ID NO: 2.

[0145] CoV S polypeptide antigen - Modification of the TM domain In the embodiment, the CoV S polypeptide contains one or more modifications to the TM domain having the amino acid sequence of SEQ ID NO: 123, corresponding to amino acids 1214-1237 of SEQ ID NO: 1 or amino acids 1201-1224 of SEQ ID NO: 2.

[0146] The amino acid sequence of the TM domain (SEQ ID NO: 123) is shown below. WYIWLGFIAGLIAIVMVTIMLCCM

[0147] In embodiments, the CoV S polypeptide described herein includes a TM domain having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity with the TM domain of SEQ ID NO: 1 or SEQ ID NO: 2. The TM domain may have up to about 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20, at least 25, or at least 30 amino acid deletions, insertions, or mutations compared to the amino acid sequence of the TM domain of SEQ ID NO: 1 or SEQ ID NO: 2. The TM domain may have deletions, insertions, or mutations of approximately 1 to 5 amino acids, 3 to 10 amino acids, 5 to 10 amino acids, 8 to 12 amino acids, 10 to 15 amino acids, 12 to 17 amino acids, 15 to 20 amino acids, 18 to 23 amino acids, 20 to 25 amino acids, 22 to 27 amino acids, or 25 to 30 amino acids compared to the TM domain of SEQ ID NO: 1 or SEQ ID NO: 2.

[0148] In some embodiments, the CoV S polypeptide described herein lacks the entire TM domain. In some embodiments, the CoV S polypeptide includes the TM domain.

[0149] Modification of CoV S polypeptide antigen - CT In the embodiment, the CoV S polypeptide contains one or more modifications to the CT having the amino acid sequence of SEQ ID NO: 124, which corresponds to amino acids 1238-1273 of SEQ ID NO: 1 or amino acids 1225-1260 of SEQ ID NO: 2.

[0150] The amino acid sequence of CT (sequence number 124) is shown below. TSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT

[0151] In embodiments, the CoV S polypeptide described herein includes a CT having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identity with the CT of SEQ ID NO: 1 or SEQ ID NO: 2. The CT may have up to about 1, up to about 2, up to about 3, up to about 4, up to about 5, up to about 10, up to about 15, up to about 20, up to about 25, or up to about 30 amino acid deletions, insertions, or mutations compared to the amino acid sequence of the CT of SEQ ID NO: 1 or SEQ ID NO: 2. CT may have deletions, insertions, or mutations of approximately 1 to 5 amino acids, 3 to 10 amino acids, 5 to 10 amino acids, 8 to 12 amino acids, 10 to 15 amino acids, 12 to 17 amino acids, 15 to 20 amino acids, 18 to 23 amino acids, 20 to 25 amino acids, 22 to 27 amino acids, or 25 to 30 amino acids compared to CT of SEQ ID NO: 1 or SEQ ID NO: 2.

[0152] In the embodiments, the CoV S polypeptide described herein lacks CT. In the embodiments, the CoV S polypeptide contains CT.

[0153] In the embodiment, the CoV S polypeptide comprises TM and CT. In the embodiment, the CoV spike(S) polypeptide contains one or more amino acid deletions from the transmembrane and cytoplasmic tail (TMCT) (corresponding to amino acids 1201-1260). The amino acid sequence of TMCT is represented by SEQ ID NO: 39. In the embodiment, the CoV S polypeptide having one or more deletions from TMCT exhibits enhanced protein expression. In the embodiment, the CoV spike(S) polypeptide having one or more deletions from TMCT has an amino acid sequence selected from the group consisting of SEQ ID NOs: 40, 41, 42, 52, 54, 59, 61, 88, and 89. In the embodiment, the CoV S polypeptide having one or more deletions from TM-CD is encoded by an isolated nucleic acid sequence selected from the group consisting of SEQ ID NOs: 39, 43, 53, and 60.

[0154] CoV S polypeptide antigen - non-limited combination of mutations In an embodiment, the CoV S polypeptide contains deletions of amino acids 56 and 57 of the native CoV spike (S) polypeptide (SEQ ID NO: 2).

[0155] In an embodiment, the CoV S polypeptide contains deletions of amino acids 131 and 132 of the native CoV spike (S) polypeptide (SEQ ID NO: 2).

[0156] In an embodiment, the CoV S polypeptide contains deletions of amino acids 56 and 131 of the native CoV spike (S) polypeptide (SEQ ID NO: 2). In an embodiment, the CoV S polypeptide contains deletions of amino acids 57 and 131 of the native CoV spike (S) polypeptide (SEQ ID NO: 2).

[0157] In an embodiment, the CoV S polypeptide contains deletions of amino acids 56, 57 and 131 of the native CoV spike (S) polypeptide (SEQ ID NO: 2).

[0158] In an embodiment, the CoV S polypeptide contains deletions of amino acids 56 and 132 of the native CoV spike (S) polypeptide (SEQ ID NO: 2).

[0159] In an embodiment, the CoV S polypeptide contains deletions of amino acids 57 and 132 of the native CoV spike (S) polypeptide (SEQ ID NO: 2).

[0160] In an embodiment, the CoV S polypeptide contains deletions of amino acids 56, 57 and 132 of the native CoV spike (S) polypeptide (SEQ ID NO: 2).

[0161] In an embodiment, the CoV S polypeptide contains deletions of amino acids 56, 57, 131 and 132 of the native CoV spike (S) polypeptide (SEQ ID NO: 2).

[0162] In embodiments, the CoV S polypeptide contains a mutation that stabilizes the pre-fusion structure of the CoV S polypeptide. In embodiments, the CoV S polypeptide contains a proline or glycine substitution that stabilizes the pre-fusion structure. This strategy has been used to develop pre-fusion-stabilized MERS-CoV S proteins as described in the following literature, each of which is incorporated herein by reference in its entirety: Proc Natl Acad Sci USA. 2017 Aug 29;114(35):E7348-E7357; Sci Rep. 2018 Oct 24;8(1):15701; U.S. Patent Application Publication No. 2020 / 0061185 and PCT Application PCT / US2017 / 058370.

[0163] In one embodiment, the CoV S polypeptide contains mutations at Lys-973 and Val-974 and an inactivated furin cleavage site. In another embodiment, the CoV S polypeptide contains mutations at Lys-973 and Val-974 to proline and an inactivated furin cleavage site having the amino acid sequence QQAQ (SEQ ID NO: 7) or GSAS (SEQ ID NO: 96). In yet another embodiment, the CoV S polypeptide containing mutations at Lys-973 and Val-974 to proline and an inactivated furin cleavage site has the amino acid sequence of SEQ ID NO: 86 or 87 and the nucleic acid sequence of SEQ ID NO: 96.

[0164] In one embodiment, the CoV S polypeptide contains mutations at Lys-973 and Val-974, an inactivating furin cleavage site, and the deletion of one or more amino acids in the fusion peptide. In another embodiment, the CoV S polypeptide contains mutations at Lys-973 and Val-974 to proline, an inactivating furin cleavage site having the amino acid sequence QQAQ (SEQ ID NO: 7) or GSAS (SEQ ID NO: 96), and the deletion of one or more amino acids in the fusion peptide. In yet another embodiment, the CoV S polypeptide contains mutations at Lys-973 and Val-974 to proline, an inactivating furin cleavage site, and the deletion of one or more amino acids in the fusion peptide having the amino acid sequence of SEQ ID NO: 105 or 106. In the embodiment, the CoV S polypeptide contains, compared to the native CoV spike (S) polypeptide (SEQ ID NO: 2), a mutation to phenylalanine at Leu-5, a mutation to asparagine at Thr-7, a mutation to serine at Pro-13, a mutation to tyrosine at Asp-125, a mutation to serine at Asp-177, a mutation to threonine at Lys-404, a mutation to lysine at Glu-471, a mutation to tyrosine at Asn-488, a mutation to tyrosine at His-642, a mutation to isoleucine at Thr-1014, mutations to proline at Lys-973 and Val-974, and an inactivated furin cleavage site having the amino acid sequence QQAQ (SEQ ID NO: 7) or GSAS (SEQ ID NO: 96).

[0165] In the embodiment, the CoV S polypeptide contains a mutation to cysteine ​​at TRP-139, a mutation to arginine at Leu-439, a mutation to proline at Lys-973 and Val-974, and an inactivated furin cleavage site having the amino acid sequence QQAQ (SEQ ID NO: 7) or GSAS (SEQ ID NO: 96), compared to the native CoV spike (SEQ ID NO: 2). In the embodiment, the CoV S polypeptide contains a mutation to cysteine ​​at Trp-152, a mutation to arginine at Leu-452, a mutation to isoleucine at Ser-13, a mutation to proline at Lys-986 and Val-987, and an inactivated furin cleavage site having the amino acid sequence QQAQ (SEQ ID NO: 7) or GSAS (SEQ ID NO: 96), compared to the native CoV spike (SEQ ID NO: 1).

[0166] In the embodiment, the CoV S polypeptide contains, compared to the native CoV spike (S) polypeptide (SEQ ID NO: 2), a mutation to threonine or asparagine at Lys-404, a mutation to lysine at Glu-471, a mutation to tyrosine at Asn-488, a mutation to phenylalanine at Leu-5, a mutation to alanine at Asp-67, a mutation to glycine at Asp-202, one or more deletions of amino acids 229-231, a mutation to isoleucine at Arg-233, mutations to proline at Lys-973 and Val-974, and an inactivated furin cleavage site having the amino acid sequence QQAQ (SEQ ID NO: 7) or GSAS (SEQ ID NO: 96).

[0167] In the embodiment, the CoV S polypeptide contains a tyrosine mutation at Asn-488, proline mutations at Lys-973 and Val-974, and an inactivated furin cleavage site having the amino acid sequence QQAQ (SEQ ID NO: 7) or GSAS (SEQ ID NO: 96), compared to the native CoV spike (SEQ ID NO: 2). In the embodiment, the CoV S polypeptide having a tyrosine mutation at Asn-488, proline mutations at Lys-973 and Val-974, and an inactivated furin cleavage site having the amino acid sequence QQAQ (SEQ ID NO: 7) or GSAS (SEQ ID NO: 96) contains the amino acid sequence of SEQ ID NO: 112.

[0168] In the embodiment, the CoV S polypeptide contains a mutation to glycine at Asp-601, a mutation to tyrosine at Asn-488, a mutation to proline at Lys-973 and Val-974, and an inactivated furin cleavage site having the amino acid sequence QQAQ (SEQ ID NO: 7) or GSAS (SEQ ID NO: 96), compared to the native CoV spike (SEQ ID NO: 2). In the embodiment, the CoV S polypeptide having a mutation to tyrosine at Asn-488, a mutation to proline at Lys-973 and Val-974, and an inactivated furin cleavage site having the amino acid sequence QQAQ (SEQ ID NO: 7) or GSAS (SEQ ID NO: 96) contains the amino acid sequence of SEQ ID NO: 113.

[0169] In the embodiment, the CoV S polypeptide contains, compared to the native CoV spike (S) polypeptide (SEQ ID NO: 2), deletions of amino acids 56, 57 and 131, a mutation to tyrosine at Asn-488, a mutation to aspartic acid at Ala-557, a mutation to glycine at Asp-601, a mutation to histidine at Pro-668, a mutation to isoleucine at Thr-703, a mutation to alanine at Ser-969, a mutation to histidine at Asp-1105, mutations to proline at Lys-973 and Val-974, and an inactivated furin cleavage site having the amino acid sequence QQAQ (SEQ ID NO: 7), GSAS (SEQ ID NO: 96), or GG. In this embodiment, the CoV S polypeptide having an inactivated furin cleavage site with the amino acid sequences QQAQ (SEQ ID NO: 7) or GSAS (SEQ ID NO: 96) contains the amino acid sequence of SEQ ID NO: 114. In this embodiment, the CoV S polypeptide having an inactivated furin cleavage site with the amino acid sequences QQAQ (SEQ ID NO: 7), GSAS (SEQ ID NO: 96), or GG includes the amino acid sequence of SEQ ID NO: 136.In some embodiments, a CoV S polypeptide having an inactivated furin cleavage site with the amino acid sequences GG, 56, 57, and 131, a mutation to tyrosine at Asn-488, a mutation to aspartic acid at Ala-557, a mutation to glycine at Asp-601, a mutation to histidine at Pro-668, a mutation to isoleucine at Thr-703, a mutation to alanine at Ser-969, a mutation to histidine at Asp-1105, a mutation to proline at Lys-973 and Val-974, and the amino acid sequence GG comprises the amino acid sequence of SEQ ID NO: 137 or SEQ ID NO: 138. In some embodiments, a CoV S polypeptide having the amino acid sequence of SEQ ID NO: 114 or SEQ ID NO: 136 is encoded by a nucleic acid having the nucleic acid sequence of SEQ ID NO: 135. In some embodiments, a CoV S polypeptide having the amino acid sequence of SEQ ID NO: 137 or SEQ ID NO: 138 is encoded by a nucleic acid having the sequence of SEQ ID NO: 139.

[0170] In the embodiment, compared to the native CoV spike (S) polypeptide (SEQ ID NO: 2), the CoV S polypeptide contains deletions of amino acids 56, 57 and 132, a mutation to tyrosine at Asn-488, a mutation to aspartic acid at Ala-557, a mutation to glycine at Asp-601, a mutation to histidine at Pro-668, a mutation to isoleucine at Thr-703, a mutation to alanine at Ser-969, a mutation to histidine at Asp-1105, mutations to proline at Lys-973 and Val-974, and an inactivated furin cleavage site having the amino acid sequence QQAQ (SEQ ID NO: 7) or GSAS (SEQ ID NO: 96). In this embodiment, the CoV S polypeptide having an inactivated furin cleavage site with the amino acid sequences QQAQ (SEQ ID NO: 7) or GSAS (SEQ ID NO: 96) contains the amino acid sequence of SEQ ID NO: 114.

[0171] In the embodiment, the CoV S polypeptide contains, compared to the native CoV spike (S) polypeptide (SEQ ID NO: 2), a mutation to tyrosine at Asn-488, a mutation to alanine at Asp-67, a mutation to histidine at Leu-229, a mutation to glycine at Asp-202, a mutation to asparagine at Lys-404, a mutation to lysine at Glu-471, a mutation to valine at Ala-688, a mutation to glycine at Asp-601, a mutation to proline at Lys-973 and Val-974, and an inactivated furin cleavage site having the amino acid sequence QQAQ (SEQ ID NO: 7) or GSAS (SEQ ID NO: 96). In this embodiment, the CoV S polypeptide having an inactivated furin cleavage site with an amino acid sequence of QQAQ (SEQ ID NO: 7) or GSAS (SEQ ID NO: 96) includes the amino acid sequence of SEQ ID NO: 115.

[0172] In the embodiments, the CoV S polypeptide contains one or more modifications selected from the following: K973P, V974P, an inactivated furin cleavage site, deletion of amino acid 56, deletion of amino acid 57, deletion of amino acid 131, N488Y, A557D, D601G, P668H, T703I, S969A, and D1105H (amino acids are numbered relative to the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 2). In the embodiments, the inactivated furin cleavage site has the amino acid sequence QQAQ (SEQ ID NO: 7). In the embodiments, the inactivated furin cleavage site has the amino acid sequence GG.

[0173] In the embodiments, the CoV S polypeptide contains one or more modifications selected from the following: K973P, V974P, an inactivated furin cleavage site, D67A, D202G, L229H, K404N, E471K, N488Y, D601G, and A688V (amino acids are numbered relative to the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 2). In the embodiments, the inactivated furin cleavage site has the amino acid sequence QQAQ (SEQ ID NO: 7). In the embodiments, the inactivated furin cleavage site has the amino acid sequence GG.

[0174] In the embodiment, the CoV S polypeptide contains one or more modifications selected from the following: K973P, V974P, an inactivated furin cleavage site, deletion of amino acids 229-231, D67A, D202G, K404N, E471K, N488Y, D601G, and A688V (the amino acids are numbered relative to the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 2).

[0175] In the embodiment, the CoV S polypeptide contains one or more modifications selected from the following: an inactivated furin cleavage site having the amino acid sequence K973P, V974P, QQAQ (SEQ ID NO: 7), deletion of amino acids 229-231, L5F, D67A, D202G, K404N, E471K, N488Y, D601G, and A688V (the amino acids are numbered relative to the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 2). In the embodiment, a CoV S polypeptide having an inactivated furin cleavage site with the amino acid sequences K973P, V974P, QQAQ (SEQ ID NO: 7), deletion of amino acids 229-231, and one or more modifications selected from L5F, D67A, D202G, K404N, E471K, N488Y, D601G, and A688V (amino acids are numbered relative to the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 2) contains the amino acid sequence of SEQ ID NO: 144. In the embodiment, a CoV S polypeptide having the amino acid sequence of SEQ ID NO: 144 is encoded by a nucleic acid having the sequence of SEQ ID NO: 145.

[0176] In the embodiment, the CoV S polypeptide contains one or more modifications selected from the following: an inactivated furin cleavage site having the amino acid sequence K973P, V974P, GG; deletion of amino acids 229-231; L5F, D67A, D202G, K404N, E471K, N488Y, D601G, and A688V (amino acids are numbered relative to the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 2). In the embodiment, the CoV S polypeptide having one or more modifications selected from the following: an inactivated furin cleavage site having the amino acid sequence K973P, V974P, GG; deletion of amino acids 229-231; L5F, D67A, D202G, K404N, E471K, N488Y, D601G, and A688V (amino acids are numbered relative to the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 2) contains the amino acid sequence of SEQ ID NO: 144. In this embodiment, the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 144 is encoded by a nucleic acid having the sequence of SEQ ID NO: 145.

[0177] In the embodiment, the CoV S polypeptide contains one or more modifications selected from the following: K973P, V974P, inactivated furin cleavage site, L5F, T7N, P13S, D125Y, R177S, K404T, E471K, N488Y, D601G, H642Y, T1014I, and V1163F (amino acids are numbered relative to the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 2). In the embodiment, a CoV S polypeptide containing one or more modifications selected from K973P, V974P, an inactivated furin cleavage site, L5F, T7N, P13S, D125Y, R177S, K404T, E471K, N488Y, D601G, H642Y, T1014I, and V1163F (amino acids are numbered relative to the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 2) has the amino acid sequence of SEQ ID NO: 151. In the embodiment, the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 151 is encoded by a nucleic acid having the sequence of SEQ ID NO: 150.

[0178] In an embodiment, the CoV S polypeptide contains one or more modifications selected from the following: K973P, V974P, an inactivated furin cleavage site, deletion of amino acids 229-231, L5F, D67A, D202G, L229H, K404N, E471K, N488Y, D601G, and A688V (the amino acids are numbered relative to the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 2).

[0179] In an embodiment, the CoV S polypeptide contains one or more modifications selected from the following: K973P, V974P, an inactivated furin cleavage site, K404N, E471K, N488Y, L5F, D67A, D202G, L229H, D601G, A688V, and deletion of amino acids 229-231 (the amino acids are numbered relative to the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 2). In an embodiment, the inactivated furin cleavage site has the amino acid sequence of QQAQ (SEQ ID NO: 7). In an embodiment, the inactivated furin cleavage site has the amino acid sequence of GG.

[0180] In the embodiment, the CoV S polypeptide contains one or more modifications selected from the following: K973P, V974P, an inactivated furin cleavage site, K404N, E471K, and N488K (amino acids are numbered relative to the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 2). In the embodiment, the CoV S polypeptide contains one or more modifications selected from the following: K973P, V974P, an inactivated furin cleavage site, K404N, E471K, and N488Y. In the embodiment, the CoV S polypeptide is an RBD of the CoV S polypeptide having one or more modifications selected from K973P, V974P, an inactivated furin cleavage site, K404N, E471K, and N488K (amino acids are numbered relative to the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 2). In this embodiment, the CoV S polypeptide is an RBD of the CoV S polypeptide having one or more modifications selected from K973P, V974P, an inactivating furin cleavage site, K404N, E471K, and N488Y (amino acids are numbered relative to the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 2).

[0181] In the embodiment, the CoV S polypeptide contains one or more modifications selected from the following: an inactivated furin cleavage site having the amino acid sequence K973P, V974P, GG, D601G, E404N, E471K, and N488Y. In the embodiment, the CoV S polypeptide contains one or more modifications selected from the following: an inactivated furin cleavage site having the amino acid sequence K973P, V974P, GG, and the D601G mutation (the amino acids are numbered relative to the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 2). In the embodiment, the CoV S polypeptide containing modifications selected from an inactivated furin cleavage site having the amino acid sequence K973P, V974P, GG, and the D601G mutation has the amino acid sequence of SEQ ID NO: 133.

[0182] In the embodiment, the CoV S polypeptide contains one or more modifications selected from the following: K973P, V974P, an inactivated furin cleavage site which is optionally QQAQ (SEQ ID NO: 7) or GG, K404N, E471K, N488K, D67A, D202G, L229H, D601G, and A688V (the amino acids are numbered relative to the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 2). In the embodiment, the CoV S polypeptide containing one or more modifications selected from K973P, V974P, an inactivated furin cleavage site which is optionally QQAQ (SEQ ID NO: 7) or GG, K404N, E471K, N488K, D67A, D202G, L229H, D601G, and A688V has the amino acid sequence of SEQ ID NO: 132 or SEQ ID NO: 141. In one embodiment, the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 132 is encoded by a nucleic acid having the nucleic acid sequence of SEQ ID NO: 131. In another embodiment, the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 132 is encoded by a nucleic acid having the nucleic acid sequence of SEQ ID NO: 142.

[0183] In the embodiment, the CoV S polypeptide contains one or more modifications selected from the following: K973P, V974P, an inactivated furin cleavage site, W139C, and L439R (amino acids are numbered relative to the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 2). In the embodiment, the CoV S polypeptide containing the K973P, V974P, an inactivated furin cleavage site, W139C, and L439R modifications is expressed together with a signal peptide having the amino acid sequence of SEQ ID NO: 117 or SEQ ID NO: 5. In the embodiment, the CoV S polypeptide contains one or more modifications selected from the following: K973P, V974P, an inactivated furin cleavage site, D601G, W139C, and L439R (amino acids are numbered relative to the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 2). In this embodiment, the CoV S polypeptide comprises K973P, V974P, an inactivating furin cleavage site, D601G, W139C, and L439R modifications, and is expressed together with a signal peptide having the amino acid sequence of SEQ ID NO: 117 or SEQ ID NO: 5.

[0184] In this embodiment, the CoV S polypeptide includes one or more modifications selected from the following: K973P, V974P, an inactivated furin cleavage site, D601G, L5F, D67A, D202G, deletion of amino acids 229-231, R233I, K404N, E471K, N488Y, and A688V (the amino acids are numbered relative to the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 2).

[0185] In the embodiment, the CoV S polypeptide contains one or more modifications selected from the following: K973P, V974P, an inactivated furin cleavage site which is optionally QQAQ (SEQ ID NO: 7), W139C, S481P, D601G, and L439R (amino acids are numbered relative to the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 2). In the embodiment, the CoV S polypeptide contains one or more modifications selected from the following: K973P, V974P, an inactivated furin cleavage site which is optionally QQAQ (SEQ ID NO: 7), W139C, D601G, and L439R (amino acids are numbered relative to the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 2). In the embodiment, the CoV S polypeptide contains one or more modifications selected from the following: K973P, V974P, an inactivated furin cleavage site which is optionally QQAQ (SEQ ID NO: 7), W139C, S481P, and D601G (amino acids are numbered relative to the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 2). In the embodiment, the CoV S polypeptide containing one or more modifications selected from K973P, V974P, an inactivated furin cleavage site which is optionally QQAQ (SEQ ID NO: 7), W139C, S481P, D601G, and L439R has the amino acid sequence of SEQ ID NO: 153. In the embodiment, the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 153 contains a signal peptide having the amino acid sequence of SEQ ID NO: 117. In the embodiment, the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 153 contains a signal peptide having the amino acid sequence of SEQ ID NO: 5.

[0186] In the embodiment, the CoV S polypeptide contains one or more modifications selected from the following: K973P, V974P, an inactivated furin cleavage site which is optionally QQAQ (SEQ ID NO: 7), T82I, D240G, E471K, D601G, and A688V (amino acids are numbered relative to the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 2). In the embodiment, the CoV S polypeptide containing one or more modifications selected from K973P, V974P, an inactivated furin cleavage site which is optionally QQAQ (SEQ ID NO: 7), T82I, D240G, E471K, D601G, and A688V (amino acids are numbered relative to the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 2) has the amino acid sequence of SEQ ID NO: 156. In the embodiment, a CoV S polypeptide containing one or more modifications selected from K973P, V974P, an inactivated furin cleavage site which is optionally QQAQ (SEQ ID NO: 7), T82I, D240G, E471K, D601G, and A688V (amino acids are numbered relative to the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 2) includes a signal peptide having the amino acid sequence of SEQ ID NO: 154 or SEQ ID NO: 5.

[0187] In the embodiment, the CoV S polypeptide contains one or more modifications selected from the following: K973P, V974P, an inactivated furin cleavage site which is optionally QQAQ (SEQ ID NO: 7), T82I, D240G, S464N, D601G, and A688V (amino acids are numbered relative to the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 2). In the embodiment, the CoV S polypeptide containing one or more modifications selected from K973P, V974P, an inactivated furin cleavage site which is optionally QQAQ (SEQ ID NO: 7), T82I, D240G, S464N, D601G, and A688V (amino acids are numbered relative to the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 2) has the amino acid sequence of SEQ ID NO: 158. In the embodiment, a CoV S polypeptide containing one or more modifications selected from K973P, V974P, an inactivating furin cleavage site which is optionally QQAQ (SEQ ID NO: 7), T82I, D240G, S464N, D601G, and A688V (amino acids are numbered relative to the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 2) contains the signal peptide of SEQ ID NO: 154.

[0188] In the embodiment, the CoV S polypeptide contains one or more modifications selected from the following: K973P, V974P, an inactivated furin cleavage site which is optionally QQAQ (SEQ ID NO: 7), a deletion of amino acid 56, a deletion of amino acid 57, a deletion of amino acid 131, an N488Y mutation, an A557D mutation, a D601G mutation, a P668H mutation, a T703I mutation, an S969A mutation, and a D1105H mutation (the CoV S polypeptide is numbered relative to the wild-type SARS-CoV-2 S polypeptide having the amino acid sequence of SEQ ID NO: 2). In the embodiment, the CoV S polypeptide contains one or more modifications selected from the following: K973P, V974P, an inactivated furin cleavage site which is optionally QQAQ (SEQ ID NO: 7), a deletion of amino acid 56, a deletion of amino acid 57, a deletion of amino acid 132, an N488Y mutation, an A557D mutation, a D601G mutation, a P668H mutation, a T703I mutation, an S969A mutation, and a D1105H mutation (the CoV S polypeptide is numbered relative to the wild-type SARS-CoV-2 S polypeptide having the amino acid sequence of SEQ ID NO: 2).

[0189] In the embodiment, the CoV S polypeptide contains one or more modifications selected from the following: K973P, V974P, an inactivated furin cleavage site which is optionally QQAQ (SEQ ID NO: 7), a D67A mutation, a L229H mutation, a R233I mutation, an A688V mutation, a N488Y mutation, a K404N mutation, an E471K mutation, and a 601G mutation (the CoV S polypeptide is numbered relative to the wild-type SARS-CoV-2 S polypeptide having the amino acid sequence of SEQ ID NO: 2).

[0190] In the embodiment, the CoV S polypeptide contains one or more modifications selected from the following: K973P, V974P, an inactivated furin cleavage site which is optionally QQAQ (SEQ ID NO: 7), an L5F mutation, a T7N mutation, a P13S mutation, a D125Y mutation, a R177S mutation, a K404T mutation, an E471K mutation, a N488Y mutation, a D601G mutation, a H642Y mutation, a T1014I mutation, and a T1163F mutation (the CoV S polypeptide is numbered relative to the wild-type SARS-CoV-2 S polypeptide having the amino acid sequence of SEQ ID NO: 2).

[0191] In the embodiment, the CoV S polypeptide contains one or more modifications selected from the following: K986P, V987P, an inactivated furin cleavage site which is optionally QQAQ (SEQ ID NO: 7), an S13I mutation, a W152C mutation, and an L452R mutation (the CoV S polypeptide is numbered relative to the wild-type SARS-CoV-2 S polypeptide having the amino acid sequence of SEQ ID NO: 1). In the embodiment, the CoV S polypeptide contains one or more modifications selected from K986P, V987P, an inactivated furin cleavage site which is optionally QQAQ (SEQ ID NO: 7), an S13I mutation, a W152C mutation, and an L452R mutation (the CoV S polypeptide is numbered relative to the wild-type SARS-CoV-2 S polypeptide having the amino acid sequence of SEQ ID NO: 1), and lacks an N-terminal signal peptide.

[0192] In the embodiment, the CoV S polypeptide contains one or more modifications selected from the following: K986P, V987P, A67V, T95I, G142D, L212I, G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T54 7K, D614G, H655Y, H679K, P681H, N764K, D796Y, N856K, Q954H, N969K, L981F, deletion of amino acids 69, 70, 143, 144, 145 and 211, and insertion of amino acid EPE between amino acids 214 and 215 (optionally, the inactivating furin cleavage site is QQAQ (SEQ ID NO: 7), and the CoV S polypeptide is numbered relative to the wild-type SARS-CoV-2 S polypeptide having the amino acid sequence of SEQ ID NO: 1). In embodiments, CoV S polypeptides having one or more of the above modifications lack an N-terminal signal peptide. In embodiments, the CoV S polypeptide has the amino acid sequence of SEQ ID NO: 159.

[0193] In this embodiment, the CoV S polypeptide contains one or more modifications selected from the following: K986P, V987P, A67V, T95I, G142D, L212I, G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, H679K, P681H, N764K, D796Y, N856K, Q954H, N969K, L981F (optionally, the inactivating furin cleavage site is QQAQ (SEQ ID NO: 7), and CoV The S polypeptide is numbered relative to the wild-type SARS-CoV-2 S polypeptide having the amino acid sequence of SEQ ID NO: 1. In the embodiment, the CoV S polypeptide having one or more of the aforementioned modifications lacks an N-terminal signal peptide. In the embodiment, the CoV S polypeptide has the amino acid sequence of SEQ ID NO: 160.

[0194] In one embodiment, the CoV S polypeptide is either SEQ ID NO: 159 or 167. In another embodiment, the amino acid sequence of the CoV S polypeptide is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to either SEQ ID NO: 159 or 167. In yet another embodiment, the CoV S polypeptide is either SEQ ID NO: 160 or 170. In yet another embodiment, the amino acid sequence of the CoV S polypeptide is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to either SEQ ID NO: 160 or 170.

[0195] In one embodiment, the CoV S polypeptide is encoded by any one of the nucleic acids of SEQ ID NOs: 161, 162, 163, 164, 165, 166, 168, 169, 171, and 172. In another embodiment, any one of the CoV S polypeptides of SEQ ID NOs: 160, 170, 159, or 167 lacks an N-terminal signal peptide. For example, the CoV S polypeptide comprises the polypeptide sequence of SEQ ID NOs: 160, 170, 159, or 167, which is the C-terminus for MFVFLVLLPLVSS (SEQ ID NO: 5).

[0196] In the embodiment, the CoV S polypeptide contains a series of modifications as shown in the table below, the modifications are numbered relative to SEQ ID NO: 1. In the embodiment, the CoV S polypeptide contains a series of modifications as shown in the table below, an inactivating furin cleavage site (optionally, the furin cleavage site is QQAQ (SEQ ID NO: 7)) and K986P and V987P modifications, the modifications are numbered relative to SEQ ID NO: 1.

[0197] In the embodiment, the CoV S polypeptide contains one or more modifications to amino acids 180, 252, 253, 444, 478, and 521, and the CoV S polypeptide is numbered according to the CoV S polypeptide of SEQ ID NO: 1.

[0198] In embodiments, modified CoV S polypeptides are provided herein compared to a CoV S polypeptide having an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identical to any one of the SEQ ID NOs. 245, 250, 255, 260, 265, 269, 273, and 277. In embodiments, the modification occurs at one or more amino acid positions 180, 252, 253, 444, 478, 486, and 521 (where the modifications are numbered according to the CoV S polypeptide having the amino acid sequence of SEQ ID NO. 1).

[0199] [Table 54]

[0200] [Table 55]

[0201] In some embodiments, the CoV spike(S) polypeptide comprises a polypeptide linker. In some embodiments, the polypeptide linker contains glycine and serine. In some embodiments, the linker has about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100% glycine.

[0202] In the embodiment, the polypeptide linker has a repeat of (SGGG)n (SEQ ID NO: 91), where n is an integer from 1 to 50 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50). In the embodiment, the polypeptide linker has an amino acid sequence corresponding to SEQ ID NO: 90.

[0203] In the embodiment, the polypeptide linker has a repeating sequence of (GGGGS)n (SEQ ID NO: 93), where n is an integer from 1 to 50 (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50).

[0204] In the embodiment, the polypeptide linker has a repeat of (GGGS)n (SEQ ID NO: 92), where n is an integer from 1 to 50 (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50).

[0205] In one embodiment, the polypeptide linker is a poly-(Gly)n linker, where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 16, 17, 18, 19, or 20. In another embodiment, the linker is selected from the group consisting of dipeptides, tripeptides, and quadripeptides. In an embodiment, the linker is a dipeptide selected from the group consisting of alanine-serine (AS), leucine-glutamic acid (LE), and serine-arginine (SR).

[0206] In the embodiment, the polypeptide linker comprises 1 to 100 consecutive amino acids of a natural CoV S polypeptide or a CoV S polypeptide disclosed herein. In the embodiment, the polypeptide linker has an amino acid sequence corresponding to SEQ ID NO: 94.

[0207] In embodiments, the CoV spike(S) polypeptide comprises foldon. In embodiments, TMCT is replaced with foldon. In embodiments, foldon induces trimerization of the CoV spike(S) polypeptide. In embodiments, foldon is an amino acid sequence known in the art. In embodiments, foldon has the amino acid sequence of SEQ ID NO: 68. In embodiments, foldon is a T4 fibrintin trimerizing motif. In embodiments, the T4 fibrintin trimerizing domain has the amino acid sequence of SEQ ID NO: 103. In embodiments, foldon is separated in amino acid sequence from the CoV spike(S) polypeptide by a polypeptide linker. Non-limiting examples of polypeptide linkers are found throughout this disclosure.

[0208] In embodiments, the disclosure provides CoV S polypeptides comprising fragments of coronavirus S protein, nanoparticles comprising the same, and vaccines. In embodiments, the coronavirus S protein fragments are 10 to 1500 amino acid lengths (e.g., about 10, about 20, about 30, about 40, about 50 amino acids, about 60, about 70, about 80, about 90, about 100, about 150, about 200, about 250, about 300, about 350, about 400, about 450, about 500, about 550, about 600, about 650, about 700, about 750, about 800, about 850, about 900, about 950, about 1000, about 1050, about 1100, about 1150, about 1200, about 1250, about 1300, about 1350, about 1400 amino acids, about 1450, or about 1500 amino acid lengths). In the embodiment, the coronavirus S protein fragment is selected from the group consisting of a receptor-binding domain (RBD), subdomain 1, subdomain 2, upper helix, fusion peptide, connecting region, 7-amino acid repeat 1, central helix, 7-amino acid repeat 2, NTD, and TMCT.

[0209] In the embodiment, the CoV S polypeptide comprises RBD and subdomain 1. In the embodiment, the CoV S polypeptide comprising RBD and subdomain 1 is amino acids 319-591 of SEQ ID NO: 1.

[0210] In this embodiment, the CoV S polypeptide contains a fragment of the coronavirus S protein, and the fragment of the coronavirus S protein is an RBD. Non-limiting examples of RBDs include the SARS-CoV-2 RBD (amino acid sequence = SEQ ID NO: 69), the SARS RBD (amino acid sequence = SEQ ID NO: 70), and the MERS RBD (amino acid sequence = SEQ ID NO: 71).

[0211] In some embodiments, the CoV S polypeptide contains two or more RBDs linked by a polypeptide linker. In some embodiments, the polypeptide linker has the amino acid sequence of SEQ ID NO: 90 or SEQ ID NO: 94.

[0212] In the embodiment, the CoV S polypeptide contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 RBDs.

[0213] In some embodiments, the CoV S polypeptide contains two or more SARS-CoV-2 RBDs linked by a polypeptide linker. In embodiments, the antigen containing two or more SARS-CoV-2 RBDs has an amino acid sequence corresponding to one of SEQ ID NOs. 72-75.

[0214] In the embodiment, the CoV S polypeptide contains SARS-CoV-2 RBD and SARS RBD. In the embodiment, the CoV S polypeptide contains SARS-CoV-2 RBD and SARS RBD, and each RBD is separated by a polypeptide linker. In the embodiment, the CoV S polypeptide containing SARS-CoV-2 RBD and SARS RBD has an amino acid sequence selected from the group consisting of SEQ ID NOs. 76 to 79.

[0215] In the embodiment, the CoV S polypeptide contains SARS-CoV-2 RBD and MERS RBD. In the embodiment, the CoV S polypeptide contains SARS-CoV-2 RBD and MERS RBD, and each RBD is separated by a polypeptide linker.

[0216] In this embodiment, the CoV S polypeptide comprises SARS RBD and MERS RBD, and each RBD is separated by a polypeptide linker.

[0217] In the embodiment, the CoV S polypeptide contains SARS-CoV-2 RBD, SARS RBD, and MERS RBD. In the embodiment, the CoV S polypeptide contains SARS-CoV-2 RBD, SARS RBD, and MERS RBD, and each RBD is separated by a polypeptide linker. In the embodiment, the CoV S polypeptide containing SARS-CoV-2 RBD, SARS RBD, and MERS RBD has an amino acid sequence selected from the group consisting of SEQ ID NOs: 80 to 83.

[0218] In embodiments, the CoV S polypeptide described herein is expressed together with an N-terminal signal peptide. In embodiments, the N-terminal signal peptide has the amino acid sequence of SEQ ID NO: 5 (MFVFLVLLPLVSS). In embodiments, the N-terminal signal peptide has the amino acid sequence of SEQ ID NO: 117 (MFVFLVLLPLVSI). In embodiments, the N-terminal signal peptide has the amino acid sequence of SEQ ID NO: 154 (MFVFFVLLPLVSS). In embodiments, the N-terminal signal peptide has the amino acid sequence of SEQ ID NO: 193 (MFGFLVLLPLVSS). In embodiments, the signal peptide may be replaced with any signal peptide that enables the expression of the CoV S protein. In embodiments, one or more amino acids of the CoV S protein signal peptide may be deleted or mutated. An initiating methionine residue is maintained to initiate expression. In this embodiment, the CoV S polypeptide is encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 35, 37, 95, 43, 47, 50, 53, 55, 57, 96, 60, 131, 135, 142, 145, 148, 150, 196, 197, 198, 199, 201, 202, 204, 206, 208, 210, 212, 214, 216, 329, 330, 331, 332, 333, and 334. In this embodiment, the N-terminal signal peptide of the CoV S polypeptide contains a mutation at Ser-13 compared to the native CoV spike (S) signal polypeptide (SEQ ID NO: 5). In this embodiment, Ser-13 is mutated to any native amino acid. In the embodiment, Ser-13 is mutated to alanine, methionine, isoleucine, leucine, threonine, or valine. In the embodiment, Ser-13 is mutated to isoleucine.

[0219] After CoV S protein expression in host cells, the N-terminal signal peptide is cleaved to form the mature CoV protein sequence (SEQ ID NOs: 2, 4, 38, 41, 44, 48, 51, 54, 58, 61, 63, 65, 67, 73, 75, 78, 79, 82, 83, 85, 87, 89, 106, 110, 132, 133, 114, 138, 141, 144, 147, 151, 153, 156, 158, 174, 175 The following are provided: 176, 181-184, 186, 188, 190, 195, 217-228, 233-236, 243, 255-264, 273-280, 283-284, 287-288, 291-292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 236 and 328). In one embodiment, the signal peptide is cleaved by a host cell protease. In another embodiment, the full-length protein may be isolated from the host cell, and the signal peptide is subsequently cleaved.

[0220] During expression and purification, SEQ ID NOs: 1, 3, 36, 40, 42, 46, 49, 52, 56, 59, 62, 64, 66, 72, 74, 76, 77, 80, 81, 84, 86, 87, 105, 107, 88, 109, 130, 134, 136, 137, 140, 143, 146, 149, 152, 155, 157, 159, 160, 173, 177~180, 185, 189, 191, 194, 200, 203, 205, 207 , has an amino acid sequence corresponding to any one of 209, 211, 213, 215, 229~232, 242, 245~254, 265~272, 281~282, 285~286, 289~290 and 293, 295, 297, 299, 301, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 330, 331, 332, 333 and 334. After cleaving the signal peptide from the CoV spike (S) polypeptide, SEQ ID NOs: 2, 4, 38, 41, 44, 48, 51, 54, 58, 61, 63, 65, 67, 73, 75, 78, 79, 82, 83, 85, 106, 108, 89 and 110, 112-115, 132, 133, 114, 138, 141, 144, 147, 151, 153, 156, 158, 174, 175, 176, 181-184, 1 A mature polypeptide having an amino acid sequence selected from the group consisting of 86, 188, 190, 195, 217-228, 233-236, 243, 255-264, 273-280, 283, 284, 287, 288, 291, 292 and 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 236 and 328 is obtained and used to produce a CoV S nanoparticle vaccine or CoV S nanoparticles.

[0221] Advantageously, the disclosed CoV S polypeptide may exhibit enhanced protein expression and stability compared to the native CoV spike (S) protein.

[0222] In embodiments, the CoV S polypeptide described herein contains further modifications from the native coronavirus S protein (SEQ ID NO: 2). In embodiments, the coronavirus S protein described herein exhibits at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% identity with the native coronavirus S protein. Those skilled in the art can use known techniques to calculate the percentage identity of recombinant coronavirus S protein to either the native protein or the CoV S polypeptide described herein. For example, percentage identity can be calculated using the online tool CLUSTALW2. The following default parameters can be used for CLUSTALW2 pairwise alignment: protein weight matrix = Gonnet, gap open = 10, gap elongation = 0.1.

[0223] In embodiments, the CoV S polypeptide described herein is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to a CoV S polypeptide having any one amino acid sequence of SEQ ID NOs: 87, 174, 175, 176, 181-184, 186, 188, 190, 195, 217-228, 233-236, and 243, 255-328, 329-333, or 334. Compared to the amino acid sequence of a CoV S polypeptide having any one of the amino acid sequences of SEQ ID NOs: 87, 174, 175, 176, 181-184, 186, 188, 190, 195, 217-228, 233-236, 243, and 255-328, 329-333, or 334, the CoV S polypeptide may have up to approximately 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acid deletions, insertions, or mutations. Compared to the amino acid sequence of a CoV S polypeptide having any one of the following amino acid sequences: SEQ ID NO: 87, SEQ ID NO: 174, SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 181-184, SEQ ID NO: 186, SEQ ID NO: 188, SEQ ID NO: 190, SEQ ID NO: 195, SEQ ID NO: 217-228, SEQ ID NO: 233-236, SEQ ID NO: 243, and SEQ ID NO: 255-302, SEQ ID NO: 329-333, and SEQ ID NO: 334, the CoV S polypeptide may have deletions, insertions, or mutations of approximately 1-5 amino acids, approximately 3-10 amino acids, approximately 5-10 amino acids, approximately 8-12 amino acids, approximately 10-15 amino acids, approximately 12-17 amino acids, approximately 15-20 amino acids, approximately 18-23 amino acids, approximately 20-25 amino acids, approximately 22-27 amino acids, approximately 25-30 amino acids, approximately 30-35 amino acids, approximately 35-40 amino acids, approximately 40-45 amino acids, or approximately 45-50 amino acids.In embodiments, the CoV S polypeptide described herein is approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 18, 19, 195, 217, 228, 233, 236, 243, and 255, 328, 329, 333, and 334 amino acid sequences compared to coronavirus S proteins having approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, and 18 amino acids. , including approximately 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, or 75 deletions, insertions, or mutations.

[0224] In embodiments, the coronavirus S polypeptide is extended at the N-terminus, C-terminus, or both the N-terminus and C-terminus. In embodiments, the extension is a tag useful for functions such as purification or detection. In embodiments, the tag contains an epitope. For example, the tag may be a polyglutamate tag, FLAG tag, HA tag, polyHis tag (having about 5-10 histidines) (SEQ ID NO: 101), hexahistidine tag (SEQ ID NO: 100), 8X-His tag (having 8 histidines) (SEQ ID NO: 102), Myc tag, glutathione-S-transferase tag, green fluorescent protein tag, maltose-binding protein tag, thioredoxin tag, or Fc tag. In other embodiments, the extension may be an N-terminal signal peptide fused to the protein to enhance expression. Such signal peptides are often cleaved during expression in cells, but some nanoparticles may contain antigens with intact signal peptides. Therefore, if the nanoparticles contain an antigen, the antigen may contain an extension and, if incorporated into the nanoparticles, may be a fusion protein. The extension is not included in order to calculate identity with respect to the sequence. In embodiments, the tag is a protease cleavage site. Non-limiting examples of protease cleavage sites include the HRV3C protease cleavage site, chymotrypsin, trypsin, elastase, endopeptidase, caspase-1, caspase-2, caspase-3, caspase-4, caspase-5, caspase-6, caspase-7, caspase-8, caspase-9, caspase-10, enterokinase, factor Xa, granzyme B, TEV protease, and thrombin. In embodiments, the protease cleavage site is the HRV3C protease cleavage site. In embodiments, the protease cleavage site includes the amino acid sequence of SEQ ID NO: 98.

[0225] In the embodiment, the CoV S glycoprotein includes a fusion protein. In the embodiment, the CoV S glycoprotein includes an N-terminal fusion protein. In the embodiment, the CoV S glycoprotein includes a C-terminal fusion protein. In the embodiment, the fusion protein includes a tag useful for protein expression, purification, or detection. In the embodiment, the tag is a polyHis tag (having about 5-10 histidines), a Myc tag, a glutathione-S-transferase tag, a green fluorescent protein tag, a maltose-binding protein tag, a thioredoxin tag, a Strep tag, a Twin-Strep tag, or an Fc tag. In the embodiment, the tag is an Fc tag. In the embodiment, the Fc tag is a monomer, a dimer, or a trimer. In the embodiment, the tag is a hexahistidine tag, for example, a polyHis tag (SEQ ID NO: 100) containing 6 histidines. In the embodiment, the tag is a Twin-Strep tag having the amino acid sequence of SEQ ID NO: 99.

[0226] In the embodiment, the CoV S polypeptide is a fusion protein containing another coronavirus protein. In the embodiment, the other coronavirus protein is derived from the same coronavirus. In the embodiment, the other coronavirus protein is derived from a different coronavirus.

[0227] In some embodiments, the CoV S protein can be cleaved. For example, the N-terminus can be truncated by approximately 10, 30, 50, 75, 100, or 200 amino acids. The C-terminus can be truncated instead of, or in addition to, the N-terminus. For example, the C-terminus can be truncated by approximately 10, 30, 50, 75, 100, or 200 amino acids. To calculate identity for a protein having truncation, identity is measured across the rest of the protein.

[0228] Nanoparticles containing CoV spike(S) polypeptide In the embodiments, a vaccine containing coronavirus S nanoparticles is prepared using a mature CoV S polypeptide antigen. In the embodiments, the nanoparticles of the Disclosure contain the CoV S polypeptide described herein. In the embodiments, the nanoparticles of the Disclosure contain the CoV S polypeptide associated with a surfactant core. The presence of the surfactant facilitates the formation of the nanoparticles by forming a core that organizes and presents the antigen. In the embodiments, the nanoparticles may contain the CoV S polypeptide assembled into polyoligomeric glycoprotein surfactant (e.g., PS80) nanoparticles having a PS80 surfactant that forms a central core surrounded by an outwardly projecting head region and a hydrophobic region and a glycoprotein. In the embodiments, the CoV S polypeptide is essentially contained or configured to contain a transmembrane domain to facilitate the association of the protein to the surfactant core. In the embodiments, the CoV S polypeptide contains a head domain. Primarily, the transmembrane domain of the CoV S polypeptide trimer associates with the surfactant, but other parts of the polypeptide may also interact. An advantage is that nanoparticles have improved resistance to environmental stress, resulting in enhanced stability and / or improved presentation to the immune system, due to the organization of multiple copies of proteins around the surfactant.

[0229] In the embodiment, the surfactant core is a nonionic surfactant core. In the embodiment, the CoV S polypeptide is associated with the nonionic surfactant core. In the embodiment, the surfactant is selected from the group consisting of polysorbate-20 (PS20), polysorbate-40 (PS40), polysorbate-60 (PS60), polysorbate-65 (PS65), and polysorbate-80 (PS80).

[0230] In this embodiment, the surfactant is PS80.

[0231] In embodiments, the CoV S polypeptide forms trimers. In embodiments, the CoV S polypeptide nanoparticles consist of multiple polypeptide trimers surrounding a nonionic surfactant core. In embodiments, the nanoparticles contain at least about one trimer or more. In embodiments, the nanoparticles contain at least about five to about 30 trimers of the spike protein. In embodiments, each nanoparticle may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 15, 20, 25 or 30 trimers (including all values ​​and ranges in between). The compositions disclosed herein may contain nanoparticles having different numbers of trimers. For example, a composition may contain nanoparticles with a number of trimers ranging from 2 to 9, and in embodiments, the nanoparticles in the composition may contain 2 to 6 trimers. In embodiments, a composition contains a heterogeneous population of nanoparticles having 2 to 6 trimers per nanoparticle or 2 to 9 trimers per nanoparticle. In embodiments, the composition may contain a substantially homogeneous collection of nanoparticles. For example, the collection may contain about 95% nanoparticles having five trimers.

[0232] In embodiments, nanoparticles having 1 to 50 CoV S polypeptides are provided herein, each CoV S polypeptide having a different amino acid sequence. In embodiments, the nanoparticles contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 different CoV S polypeptides.

[0233] The nanoparticles disclosed herein take on a range of particle sizes. In embodiments, the nanoparticles disclosed herein have particle sizes ranging from Z-ave sizes of approximately 20 nm to 60 nm, 20 nm to 50 nm, 20 nm to 45 nm, 20 nm to 35 nm, 20 nm to 30 nm, 25 nm to 35 nm, 25 nm to 45 nm, 30 nm to 120 nm, 30 nm to 80 nm, 30 nm to 60 nm, 30 nm to 65 nm, or 30 nm to 50 nm. The particle size (Z-ave) is measured by dynamic light scattering (DLS) using Zetasizer NanoZS (Malvern, UK) unless otherwise specified.

[0234] In embodiments, nanoparticles containing the CoV S polypeptide disclosed herein have a smaller particle size compared to nanoparticles containing wild-type CoV S polypeptide. In embodiments, the CoV S polypeptide has a particle size that is at least about 40% smaller, for example, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, or at least about 85% smaller.

[0235] Nanoparticles containing the CoV S polypeptide disclosed herein are more uniform in size, shape, and mass than nanoparticles containing the wild-type CoV S polypeptide. The polydispersity index (PDI), a measure of heterogeneity, is measured by dynamic light scattering using a Malvern Setasizer unless otherwise specified. In embodiments, the particles measured herein have a PDI of about 0.1 to about 0.45, for example, about 0.1, about 0.2, about 0.25, about 0.29, about 0.3, about 0.35, about 0.40, or about 0.45. In embodiments, the nanoparticles measured herein have a PDI that is at least about 25% smaller than the PDI of nanoparticles containing the wild-type CoV S polypeptide of SEQ ID NO: 2, for example, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, or at least about 60% smaller.

[0236] CoV S polypeptides and nanoparticles containing them exhibit improved thermal stability compared to wild-type CoV S polypeptides or their nanoparticles. The thermal stability of CoV S polypeptides is measured using differential scanning calorimetry (DSC) unless otherwise specified. Transition enthalpy (ΔHcal) is the energy required to unfold the CoV S polypeptide. In embodiments, the CoV S polypeptide exhibits increased ΔHcal compared to wild-type CoV S polypeptides. In embodiments, the ΔHcal of the CoV S polypeptide is approximately 2, 3, 4, 5, 6, 7, 8, 9, or 10 times greater than that of the wild-type CoV S polypeptide.

[0237] The vaccine compositions disclosed herein may include several nanoparticle types. In some embodiments, the nanoparticle type is in the form of anisotropic rods, which may be dimers or monomers. In other embodiments, the nanoparticle type is a spherical oligomer. In yet another embodiment, the nanoparticles may be described as intermediate nanoparticles having sedimentation properties intermediate between the first two types. The formation of the nanoparticle type can be controlled by controlling the surfactant and protein concentrations during the manufacturing process. The nanoparticle type can be determined by measuring the sedimentation coefficient.

[0238] Preparation of nanoparticles containing CoV S polypeptide antigen The nanoparticles disclosed herein are non-natural products, and their components do not exist together in nature. Generally, the methods disclosed herein employ a surfactant exchange technique, which involves isolating a protein using a first surfactant and then exchanging the first surfactant with a second surfactant to form nanoparticles.

[0239] Antigens contained in nanoparticles are typically produced by recombinant expression in host cells. Standard recombination techniques may be used. In embodiments, CoV S polypeptides are expressed in insect host cells using a baculovirus system. In embodiments, 1 to 50 CoV S polypeptides are co-expressed in host cells. In embodiments, the baculoviruses are cathepsin-L knockout baculoviruses and chitinase knockout baculoviruses. Optionally, the baculoviruses are double knockout for both cathepsin-L and chitinase. High levels of expression can be obtained in insect cell expression systems. Non-limiting examples of insect cells include Spodoptera frugiperda (SF) cells, e.g., Sf9, Sf21, Trichoplusiani cells, e.g., High Five cells, and Drosophila S2 cells. In embodiments, the CoV S polypeptides described herein are produced in any suitable host cell. In embodiments, the host cell is an insect cell. In this embodiment, the insect cells are Sf9 cells.

[0240] Cells can be cultured using typical gene transfer and cell proliferation methods. Vectors, such as those containing polynucleotides encoding fusion proteins, can be transferred into host cells according to methods well known in the art. For example, the introduction of nucleic acids into eukaryotic cells can be achieved by calcium phosphate coprecipitation, electroporation, microinjection, lipofection, and gene transfer using polyamine gene transfer reagents. In one embodiment, the vector is a recombinant baculovirus.

[0241] Methods for growing host cells include, but are not limited to, batch cell culture, batch feed cell culture, continuous cell culture, and perfusion cell culture techniques. Cell culture refers to the growth and proliferation of cells in a bioreactor (fermentation chamber) where cells are grown and express proteins (e.g., recombinant proteins) for purification and isolation. Typically, cell culture is carried out in a bioreactor under sterile, controlled temperature and atmospheric conditions. A bioreactor is a chamber used to culture cells where environmental conditions such as temperature, atmosphere, agitation, and / or pH can be monitored. In one embodiment, the bioreactor is a stainless steel chamber. In another embodiment, the bioreactor is a pre-sterilized plastic bag (e.g., Cellbag®, Wave Biotech, Bridgewater, NJ). In yet another embodiment, the pre-sterilized plastic bag is a bag of about 50L to 3500L.

[0242] Extraction and purification of nanoparticles containing CoV spike (S) protein antigen. After host cell proliferation, proteins can be recovered from the host cells using a surfactant and purification protocol. In embodiments, multiple CoV S proteins are purified simultaneously. In embodiments, host cells expressing multiple CoV S proteins are pooled together. After growing the host cells for 48–96 hours, the cells are isolated from the culture medium, and a surfactant-containing solution is added to solubilize the cell membrane and release the proteins into the surfactant extract. TERGITOL® nonylphenol ethoxylate, also known as Triton X-100 and NP-9, are preferred surfactants for extraction. The surfactant may be added to a final concentration of about 0.1% to about 1.0%. For example, the concentration may be about 0.1%, about 0.2%, about 0.3%, about 0.5%, about 0.7%, about 0.8%, or about 1.0%. The range may be about 0.1% to about 0.3%. In embodiments, the concentration is about 0.5%.

[0243] In other embodiments, proteins can be isolated from host cells using different first surfactants. For example, the first surfactants are bis(polyethylene glycol bis[imidazoylcarbonyl]), nonoxynol-9, bis(polyethylene glycol bis[imidazoylcarbonyl]), BRIJ® polyethylene glycol dodecyl ether 35, BRIJ® polyethylene glycol (3) cetyl ether 56, BRIJ® alcohol ethoxylate 72, BRIJ® polyoxyl 2 stearyl ether 76, BRIJ® polyethylene glycol monoolelyl ether 92V, BRIJ® polyoxyethylene (10) oleyl ether 97, BRIJ® polyethylene glycol hexadecyl ether 58P, and CREMOPHOR® EL macrogolglycerol ricinoleic acid Decaethylene glycol monododecyl ether, N-decanoyl-N-methylglucamine, n-decyl alpha-D-glucopyranoside, decyl β-D-maltopyranoside, n-dodecanoyl-N-methylglucamide, n-dodecyl alpha-D-maltoside, n-dodecyl beta-D-maltoside, n-dodecyl beta-D-maltoside, heptaethylene glycol monodecyl ether, heptaethylene glycol monododecyl ether, heptaethylene glycol monotetradecyl ether, n-hexadecyl β-D-maltoside, hexaethylene glycol monododecyl ether, hexaethylene glycol monohexadecyl ether, hexaethylene glycol monooctadecyl ether, hexaethylene glycol monotetradecyl ether, Igepal CA-630, Igepal CA-630, Methyl-6-0-(N-heptylcarbamoyl)-alpha-D-glucopyranoside, Nonaethylene glycol monododecyl ether, N-nonanoyl-N-methylglucamine, N-nonanoyl-N-methylglucamine, Octaethylene glycol monodecyl ether, Octaethylene glycol monododecyl ether, Octaethylene glycol monohexadecyl ether, Octaethylene glycol monooctadecyl ether, Octaethylene glycol monotetradecyl ether, Octyl-beta-D-glucopyranoside,Pentaethylene glycol monodecyl ether, pentaethylene glycol monododecyl ether, pentaethylene glycol monohexadecyl ether, pentaethylene glycol monohexyl ether, pentaethylene glycol monooctadecyl ether, pentaethylene glycol monooctyl ether, polyethylene glycol diglycidyl ether, polyethylene glycol ether W-1, polyoxyethylene 10 tridecyl ether, polyoxyethylene 100 stearat, polyoxyethylene 20 isohexadecyl ether, polyoxyethylene 20 oleyl ether, polyoxyethylene 40 stearat, polyoxyethylene 50 stearat, polyoxyethylene 8 stearat, polyoxyethylene bis(imidazoyl carbonyl), polyoxyethylene 25 propylene glycol stearat, saponin from quillaja bark, SPAN(registered trademark) 20 sorbitan laurate, SPAN(registered trademark) 40 sorbitan monopalmitate, SPAN(registered trademark) 60 sorbitan stearat, SPAN(registered trademark) 65 sorbitan tristearat, SPAN(registered trademark) 80 Sorbitan monooleate, SPAN(registered trademark) 85 sorbitan trioleate, TERGITOL(registered trademark) secondary alcohol ethoxylate type 15-S-12, TERGITOL(registered trademark) secondary alcohol ethoxylate type 15-S-30, TERGITOL(registered trademark) secondary alcohol ethoxylate type 15-S-5, TERGITOL(registered trademark) secondary alcohol ethoxylate type 15-S-7, TERGITOL(registered trademark) secondary alcohol ethoxylate type 15-S-9, TERGITOL(registered trademark) nonilph Nonylphenol ethoxylate type NP-10, TERGITOL® Nonylphenol ethoxylate type NP-4, TERGITOL® Nonylphenol ethoxylate type NP-40, TERGITOL® Nonylphenol ethoxylate type NP-7, TERGITOL® Nonylphenol ethoxylate type NP-9, TERGITOL® Branched secondary alcohol ethoxylate type TMN-10, TERGITOL® Branched secondary alcohol ethoxylate type TMN-6,TRITON™ X-100 polyethylene glycol, tert-octylphenyl ether, or a combination thereof.

[0244] The nanoparticles can then be isolated from the cell debris using centrifugation. In embodiments, gradient centrifugation may be used, for example, with cesium chloride, sucrose, and iodixanol. Other techniques may be used alternatively or additionally, such as standard purification techniques including ion exchange, affinity, and gel filtration chromatography.

[0245] For example, the first column may be an ion exchange chromatography resin such as FRACTOGEL® EMD methacrylate polymer beads TMAE (EMD Millipore), the second column may be a lentil (Lens culinaris) lectin affinity resin, and the third column may be a cation exchange column such as FRACTOGEL® EMD methacrylate polymer beads SO3 (EMD Millipore) resin. In other embodiments, the cation exchange column may be an MMC column or a Nuvia C Prime column (Bio-Rad Laboratories, Inc.). Preferably, the methods disclosed herein do not use surfactant extraction columns, such as hydrophobic interaction columns. Such columns are often used to remove surfactants during purification, but they may adversely affect the methods disclosed herein.

[0246] Surfactant exchange of nanoparticles containing CoV S polypeptide antigen To form nanoparticles, the first surfactant used to extract proteins from host cells is substantially replaced by a second surfactant to reach the nanoparticle structure. NP-9 is a preferred extraction surfactant. Typically, the nanoparticles do not contain detectable NP-9 when measured by HPLC. The second surfactant is typically selected from the group consisting of PS20, PS40, PS60, PS65, and PS80. Preferably, the second surfactant is PS80.

[0247] In certain embodiments, surfactant exchange is performed using affinity chromatography, which binds to glycoproteins via the carbohydrate moiety. For example, affinity chromatography can use legume lectin columns. Legume lectins are proteins originally identified in plants and are known to interact specifically and reversibly with carbohydrate residues. See, for example, Sharon and Lis, “Legume Lectins—A large family of homologous proteins,” FASEB J.1990 Nov;4(14):3198-208; Liener, “The Lectins: Properties, Functions, and Applications in Biology and Medicine,” Elsevier, 2012. Suitable lectins include concanavalin A (con A), pea lectin, sinfoin lect, and lentil lectin. Lentil lectin is a preferred column for surfactant exchange due to its binding properties. Lectin columns are commercially available; for example, Capto Lentil Lectin is available from GE Healthcare. In certain embodiments, the lentil lectin column may use recombinant lectins. At the molecular level, it is thought that the carbohydrate moiety binds to the lentil lectin, releasing the amino acids of the protein and fusing them around the surfactant, resulting in the formation of a surfactant core that provides nanoparticles having glycoprotein oligomers, which may be multiple copies of the antigen, for example, dimers, trimers, or tetramers immobilized on the surfactant. In embodiments, the CoV S polypeptide forms trimers. In embodiments, the CoV S polypeptide trimers are immobilized on the surfactant. In embodiments, each CoV S polypeptide nanoparticle contains at least one trimer associated with a nonionic core.

[0248] Surfactants can be present at up to about 0.1% (w / v) during the initial purification step when incubated with proteins to form nanoparticles during surfactant exchange, and this amount is reduced to achieve final nanoparticles with optimal stability. For example, nonionic surfactants (e.g., PS80) can be present at about 0.005% (v / v) to about 0.1% (v / v), e.g., about 0.005% (v / v), about 0.006% (v / v), about 0.007% (v / v), about 0.008% (v / v), about 0.009% (v / v), about 0.01% (v / v), about 0.015% (v / v), about 0.02% (v / v), about 0.025% (v / v), about 0.03% (v / v), about 0 The PS80 content may be 0.035%(v / v), approximately 0.04%(v / v), approximately 0.045%(v / v), approximately 0.05%(v / v), approximately 0.055%(v / v), approximately 0.06%(v / v), approximately 0.065%(v / v), approximately 0.07%(v / v), approximately 0.075%(v / v), approximately 0.08%(v / v), approximately 0.085%(v / v), approximately 0.09%(v / v), approximately 0.095%(v / v), or approximately 0.1%(v / v). In one embodiment, the nanoparticles contain approximately 0.03% to approximately 0.05% PS80. In another embodiment, the nanoparticles contain approximately 0.01%(v / v) PS80.

[0249] In the embodiment, the purified CoV S polypeptide is dialyzed. In the embodiment, dialyzed is performed after purification. In the embodiment, the CoV S polypeptide is dialyzed in a solution containing sodium phosphate, NaCl and PS80. In the embodiment, the dialyzed solution containing sodium phosphate contains about 5 mM to about 100 mM of sodium phosphate, for example, about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM, about 80 mM, about 85 mM, about 90 mM, about 95 mM or about 100 mM of sodium phosphate. In the embodiment, the pH of the solution containing sodium phosphate is about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, or about 7.5. In the embodiment, the dialysis solution containing sodium chloride is about 50 mM NaCl to about 500 mM NaCl, for example about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, about 100 mM, about 110 mM, about 120 mM, about 130 mM, about 140 mM, about 150 mM, about 160 mM, about 170 mM, about 180 mM, about 190 mM, about 200 mM, about 210 mM, about 220 mM, about 230 mM, about 240 mM, about 25 Contains 0 mM, approximately 260 mM, approximately 270 mM, approximately 280 mM, approximately 290 mM, approximately 300 mM, approximately 310 mM, approximately 320 mM, approximately 330 mM, approximately 340 mM, approximately 350 mM, approximately 360 mM, approximately 370 mM, approximately 380 mM, approximately 390 mM, approximately 400 mM, approximately 410 mM, approximately 420 mM, approximately 430 mM, approximately 440 mM, approximately 450 mM, approximately 460 mM, approximately 470 mM, approximately 480 mM, approximately 490 mM, or approximately 500 mM of NaCl.In this embodiment, the dialysis solution containing PS80 is approximately 0.005% (v / v), approximately 0.006% (v / v), approximately 0.007% (v / v), approximately 0.008% (v / v), approximately 0.009% (v / v), approximately 0.01% (v / v), approximately 0.015% (v / v), approximately 0.02% (v / v), approximately 0.025% (v / v), approximately 0.03% (v / v), approximately 0.035% (v / v), and approximately 0.04 The solution contains %(v / v), approximately 0.045%(v / v), approximately 0.05%(v / v), approximately 0.055%(v / v), approximately 0.06%(v / v), approximately 0.065%(v / v), approximately 0.07%(v / v), approximately 0.075%(v / v), approximately 0.08%(v / v), approximately 0.085%(v / v), approximately 0.09%(v / v), approximately 0.095%(v / v), or approximately 0.1%(v / v) of PS80. In one embodiment, the dialysis solution contains approximately 25 mM sodium phosphate (pH 7.2), approximately 300 mM NaCl, and approximately 0.01%(v / v) of PS80.

[0250] Surfactant exchange can be carried out with purified proteins as discussed above, and the proteins may be purified, frozen for storage, and then thawed for surfactant exchange.

[0251] The stability of the compositions disclosed herein can be measured in various ways. One method involves preparing peptide maps to determine the integrity of antigen proteins after various treatments designed to stress the nanoparticles by mimicking severe storage conditions. Thus, the measure of stability is the relative abundance of the antigen peptide in the stressed sample compared to a control sample. For example, the stability of nanoparticles containing CoV S polypeptides can be assessed by exposing the nanoparticles to various pH levels, proteases, salts, and oxidizing agents, including but not limited to hydrogen peroxide, various temperatures, freeze / thaw cycles, and stirring. The position of glycoproteins immobilized on a surfactant core is thought to enhance stability by reducing undesirable interactions. For example, improved protection against protease-based degradation can be achieved by immobilizing glycoproteins on the core in the molar ratios disclosed herein, by a shielding effect where steric hindrance blocks protease access. Stability can also be measured by monitoring intact proteins.

[0252] Immunogenic composition containing CoV S polypeptide antigen In embodiments, immunogenic compositions comprising 1-50, 2-50, 3-50, 4-50, 5-50, 1-25, 2-25, 3-25, 4-25, 5-25, 1-10, 2-10, 3-10, 4-10, 5-10, or 3-8 CoV S glycoproteins are provided herein. In embodiments, immunogenic compositions comprising five different CoV S glycoproteins are provided herein. In embodiments, the CoV S glycoproteins are in the form of nanoparticles. In embodiments, the nanoparticles comprise 1-50, 2-50, 3-50, 4-50, 5-50, 1-25, 2-25, 3-25, 4-25, 5-25, 1-10, 2-10, 3-10, 4-10, 5-10, or 3-8 CoV S glycoproteins. In the embodiment, the nanoparticles contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 CoV S glycoproteins.

[0253] In one embodiment, the immunogenic composition comprises a CoV S glycoprotein having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the CoV S glycoprotein of SEQ ID NO: 260. In another embodiment, the immunogenic composition further comprises 1 to 10 additional CoV S glycoproteins. In yet another embodiment, the immunogenic composition comprises a CoV S glycoprotein having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the CoV S glycoprotein of SEQ ID NO: 261. In one embodiment, the immunogenic composition contains a CoV S glycoprotein having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the CoV S glycoprotein of SEQ ID NO: 262. In another embodiment, the immunogenic composition contains a CoV S glycoprotein having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the CoV S glycoprotein of SEQ ID NO: 263. In one embodiment, the immunogenic composition contains a CoV S glycoprotein having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the CoV S glycoprotein of SEQ ID NO: 264. In another embodiment, the immunogenic composition contains a CoV S glycoprotein having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the CoV S glycoprotein of SEQ ID NO: 87.In one embodiment, the immunogenic composition comprises a CoV S glycoprotein having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with any one of the CoV S glycoproteins of SEQ ID NOs.

[0254] In one embodiment, the immunogenic composition comprises a CoV S glycoprotein having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the CoV S glycoprotein of SEQ ID NO: 274. In another embodiment, the immunogenic composition further comprises 1 to 10 additional CoV S glycoproteins. In yet another embodiment, the immunogenic composition comprises a CoV S glycoprotein having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the CoV S glycoprotein of SEQ ID NO: 276. In one embodiment, the immunogenic composition contains a CoV S glycoprotein having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the CoV S glycoprotein of SEQ ID NO: 278. In another embodiment, the immunogenic composition contains a CoV S glycoprotein having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the CoV S glycoprotein of SEQ ID NO: 280. In one embodiment, the immunogenic composition contains a CoV S glycoprotein having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the CoV S glycoprotein of SEQ ID NO: 87. In another embodiment, the immunogenic composition contains a CoV S glycoprotein having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with any one of the CoV S glycoproteins of SEQ ID NOs: 244 to 328.

[0255] In one embodiment, the immunogenic composition comprises a CoV S glycoprotein having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the CoV S glycoprotein of SEQ ID NO: 284. In another embodiment, the immunogenic composition further comprises 1 to 10 additional CoV S glycoproteins. In yet another embodiment, the immunogenic composition comprises a CoV S glycoprotein having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the CoV S glycoprotein of SEQ ID NO: 288. In one embodiment, the immunogenic composition contains a CoV S glycoprotein having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the CoV S glycoprotein of SEQ ID NO: 292. In another embodiment, the immunogenic composition contains a CoV S glycoprotein having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the CoV S glycoprotein of SEQ ID NO: 87. In one embodiment, the immunogenic composition comprises a CoV S glycoprotein having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with any one of the CoV S glycoproteins of SEQ ID NOs.

[0256] In one embodiment, the immunogenic composition contains a CoV S glycoprotein having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the CoV S glycoprotein of SEQ ID NO: 260. In another embodiment, the immunogenic composition contains a CoV S glycoprotein having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the CoV S glycoprotein of SEQ ID NO: 274. In one embodiment, the immunogenic composition includes a CoV S glycoprotein having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the CoV S glycoprotein of SEQ ID NO: 222. In another embodiment, the immunogenic composition includes a CoV S glycoprotein having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the CoV S glycoprotein of SEQ ID NO: 87 and a CoV S glycoprotein having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the CoV S glycoprotein of SEQ ID NO: 274.In one embodiment, the immunogenic composition includes a CoV S glycoprotein having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the CoV S glycoprotein of SEQ ID NO: 87, and a CoV S glycoprotein having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the CoV S glycoprotein of SEQ ID NO: 222. In one embodiment, the immunogenic composition comprises a CoV S glycoprotein having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with any one of the CoV S glycoproteins of SEQ ID NOs.

[0257] In one embodiment, the immunogenic composition is a vaccine composition. In another embodiment, the immunogenic composition may contain nanoparticles having antigens from two or more virus strains of the same species of virus. In yet another embodiment, the disclosure provides a pharmaceutical pack or kit comprising one or more containers filled with one or more components of the immunogenic composition.

[0258] The compositions disclosed herein may be used prophylactically or therapeutically, but are typically prophylactic. Accordingly, this disclosure includes methods for treating or preventing infection. The methods include administering a therapeutic or prophylactic dose of the immunogenic composition of this disclosure to a subject. Preferably, the pharmaceutical composition is a vaccine composition that provides a protective effect. In other embodiments, the prophylactic effect may include improvement of symptoms associated with infection in a percentage of the exposed population. For example, the composition may prevent or reduce one or more symptoms of viral disease selected from symptoms of fever, fatigue, muscle pain, headache, sore throat, vomiting, diarrhea, rash, impaired renal and hepatic function, internal bleeding and external bleeding, compared to an untreated subject.

[0259] Nanoparticles can be formulated for administration as vaccines in the presence of various excipients, buffers, etc. For example, a vaccine composition may contain sodium phosphate, sodium chloride, and / or histidine. Sodium phosphate may be present at about 10 mM to about 50 mM, about 15 mM to about 25 mM, or about 25 mM, and in certain cases, about 22 mM of sodium phosphate. Histidine may be present at about 0.1% (w / v), about 0.5% (w / v), about 0.7% (w / v), about 1% (w / v), about 1.5% (w / v), about 2% (w / v), or about 2.5% (w / v). Sodium chloride, if present, may be at about 150 mM. In certain compositions, sodium chloride may be present at higher concentrations, for example, about 200 mM to about 500 mM. In the embodiment, sodium chloride is present in high concentrations, including but not limited to about 200 mM, about 250 mM, about 300 mM, about 350 mM, about 400 mM, about 450 mM, or about 500 mM.

[0260] In embodiments, the nanoparticles described herein are stable at slightly acidic pH levels, in embodiments where stability is improved at a specific pH level. For example, nanoparticles that are stable at slightly acidic pH, e.g., pH 5.8 to pH 7.0. In embodiments, nanoparticles and compositions containing nanoparticles may be stable at pH levels in the range of approximately pH 5.8 to approximately pH 7.0, e.g., approximately pH 5.9 to approximately pH 6.8, approximately pH 6.0 to approximately pH 6.5, approximately pH 6.1 to approximately pH 6.4, approximately pH 6.1 to approximately pH 6.3, or approximately pH 6.2H. In embodiments, the nanoparticles and compositions described herein are stable at neutral pH levels, including approximately pH 7.0 to approximately pH 7.4. In embodiments, the nanoparticles and compositions described herein are stable at slightly alkaline pH levels, e.g., approximately pH 7.0 to approximately pH 8.5, approximately pH 7.0 to approximately pH 8.0, or approximately pH 7.0 to approximately pH 7.5 (including all values ​​and the ranges in between).

[0261] Adjuvant In embodiments, the compositions disclosed herein may be combined with one or more adjuvants to enhance the immune response. In embodiments, the compositions are prepared without adjuvants and are therefore administerable as adjuvant-free compositions. Advantageously, the adjuvant-free compositions disclosed herein may provide a protective immune response when administered as a single dose. Alum-free compositions that induce a robust immune response are particularly useful in adults approximately 60 years of age or older.

[0262] Aluminum-based adjuvants In embodiments, the adjuvant may be alum (e.g., AlPO4 or Al(OH)3). In embodiments, the nanoparticles are substantially bound to the alum. In embodiments, the nanoparticles may be bound to at least 80%, at least 85%, at least 90%, or at least 95% of the alum. In embodiments, the nanoparticles are bound to 92% to 97% of the alum in the composition. The amount of alum present per dose is typically in the range of about 400 μg to about 1250 μg. For example, alum may be present in doses of about 300 μg to about 900 μg, about 400 μg to about 800 μg, about 500 μg to about 700 μg, about 400 μg to about 600 μg, or about 400 μg to about 500 μg. Typically, there is about 400 μg of alum for a dose of 120 μg of protein nanoparticles.

[0263] Saponin adjuvant Saponin-containing adjuvants may also be combined with immunogens disclosed herein. Saponins are glycosides derived from the bark of Quillaya Saponaria Molina. Typically, saponins are prepared using a multi-step purification process that yields multiple fractions. As used herein, the term "saponin fraction derived from Quillaya Saponaria Molina" is generally used to refer to a semi-purified or defined saponin fraction of Quillaya saponaria or a substantially pure fraction thereof.

[0264] Saponin fraction Several methods are preferred for generating the saponin fraction. Fractions A, B, and C are described in U.S. Patent No. 6,352,697 and can be prepared as follows: The lipophilic fraction from Quil A, a crude aqueous extract of Quillaja Saponaria Molina, is separated by chromatography and recovered by elution with 70% acetonitrile in water. This lipophilic fraction is then separated by half-isolated HPLC using a gradient of 25% to 60% acetonitrile in acidic water. The fraction referred to herein as “Fraction A” or “QH-A” is or corresponds to the fraction eluted with about 39% acetonitrile. The fraction referred to herein as “Fraction B” or “QH-B” is or corresponds to the fraction eluted with about 47% acetonitrile. The fraction referred to herein as “Fraction C” or “QH-C” is or corresponds to the fraction eluted with about 49% acetonitrile. Additional information regarding the purification of the fractions can be found in U.S. Patent No. 5,057,540. When prepared as described herein, fractions A, B, and C of Quillaya Saponaria Molina correspond to groups or families of chemically closely related molecules with definable characteristics. The chromatographic conditions under which they are obtained are such that batch-to-batch reproducibility in terms of elution profiles and biological activity is very consistent.

[0265] Other saponin fractions are described. Fractions B3, B4, and B4b are described in European Patent No. 0436620. Fractions QA1 to QA22 are described in European Patent No. 03632279B2, Q-VAC (Nor-Feed, AS Denmark), and Quillaya saponaria (Molina Spikoside) (Isconova AB, Ultunaallen 2B, 756 51 Uppsala, Sweden). Fractions QA-1, QA-2, QA-3, QA-4, QA-5, QA-6, QA-7, QA-8, QA-9, QA-10, QA-11, QA-12, QA-13, QA-14, QA-15, QA-16, QA-17, QA-18, QA-19, QA-20, QA-21 and QA-22 of European Patent No. 03632279B2, particularly QA-7, QA-17, QA-18 and QA-21, may be used. These are obtained in Example 1, particularly on pages 6, 8 and 9, as described in European Patent No. 03632279B2.

[0266] The saponin fractions described herein and used to form adjuvants are often substantially pure fractions, i.e., the fractions substantially free from the presence of contamination from other materials. In certain embodiments, a substantially pure saponin fraction may contain up to 40% by weight, up to 30% by weight, up to 25% by weight, up to 20% by weight, up to 15% by weight, up to 10% by weight, up to 7% by weight, up to 5% by weight, up to 2% by weight, up to 1% by weight, up to 0.5% by weight, or up to 0.1% by weight of other compounds, such as other saponins or other adjuvant materials.

[0267] ISCOM structure The saponin fraction may be administered in the form of cage-like particles called ISCOMs (immunostimulatory complexes). ISCOMs may be prepared as described in European Patent Nos. 0109942B1, 0242380B1, and 0180546B1. In embodiments, transport antigens and / or passenger antigens may be used as described in European Patent No. 9600647-3 (PCT / SE97 / 00289).

[0268] Matrix adjuvant In embodiments, ISCOM is an ISCOM matrix complex. The ISCOM matrix complex comprises at least one saponin fraction and a lipid. The lipid is at least a sterol, such as cholesterol. In embodiments, the ISCOM matrix complex also contains phospholipids. The ISCOM matrix complex may also contain one or more other immunomodulatory (adjuvant) substances, not necessarily glycosides, and may be prepared as described in European Patent No. 0436620B1, which is incorporated herein by reference in whole.

[0269] In other embodiments, ISCOM is an ISCOM complex. The ISCOM complex contains at least one saponin, at least one lipid, and at least one antigen or epitope. The ISCOM complex contains an antigen that has been associated by surfactant treatment such that a portion of the antigen is incorporated into the particles. In contrast, the ISCOM matrix is ​​formulated as a mixture with the antigen, and electrostatic and / or hydrophobic interactions are mediated in the association between the ISCOM matrix particles and the antigen.

[0270] In the embodiment, the saponin fraction is incorporated into the ISCOM matrix complex or the ISCOM complex, or is incorporated into or mixed with ISCOM or the ISCOM matrix complex, and at least one further adjuvant is selected from fraction A, fraction B, or fraction C of Quillaya saponaria, a semi-purified preparation of Quillaya saponaria, a purified preparation of Quillaya saponaria, or some purified sub-fraction, e.g., QA1-21.

[0271] In certain embodiments, each ISCOM particle may contain at least two saponin fractions. Any combination of weight percent of different saponin fractions may be used. Any combination of weight percent of any two fractions may be used. For example, each particle may contain any weight percent of fraction A and any weight percent of another saponin fraction, such as crude saponin fraction or fraction C. Accordingly, in certain embodiments, each ISCOM matrix particle or each ISCOM complex particle may contain one saponin fraction, e.g., fraction A, in amounts of 0.1–99.9 wt, 5–95 wt%, 10–90 wt%, 15–85 wt%, 20–80 wt%, 25–75%, 30–70 wt%, 35–65 wt%, 40–60 wt%, 45–55 wt%, 40–60 wt%, or 50 wt%, in each case, the remainder until 100% is another saponin, e.g., any crude fraction or any other fraction, e.g., fraction C. The weight is calculated as the total weight of the saponin fractions. Examples of ISCOM matrix complexes and ISCOM complex adjuvants are disclosed in U.S. Patent Application Publication No. 2013 / 0129770, which is incorporated herein by reference in its entirety.

[0272] In the embodiment, the ISCOM matrix or ISCOM complex contains one fraction, e.g., fraction A, in the proportion of 5 to 99% by weight, and the remainder until it reaches 100% by weight is another fraction, e.g., crude saponin fraction or fraction C. The weight is calculated as the total weight of the saponin fraction.

[0273] In the embodiment, the ISCOM matrix or ISCOM complex comprises one fraction of 40% to 99% by weight, e.g., fraction A, and another fraction of 1% to 60% by weight, e.g., crude saponin fraction or fraction C. The weight is calculated as the total weight of the saponin fraction.

[0274] In some embodiments, the ISCOM matrix or ISCOM complex comprises 70% to 95% by weight of one fraction, e.g., fraction A, and 30% to 5% by weight of another fraction, e.g., crude saponin fraction or fraction C. The weight is calculated as the total weight of the saponin fraction. In other embodiments, the saponin fraction derived from Quillaya Saponaria Molina is selected from any one of QA1 to QA21.

[0275] In addition to particles containing a mixture of saponin fractions, ISCOM matrix particles and ISCOM complex particles may each be formed using only one saponin fraction. The compositions disclosed herein may comprise multiple particles, each containing only one saponin fraction. That is, a particular composition may comprise one or more different types of ISCOM matrix complex particles and / or one or more different types of ISCOM complex particles, each individual particle containing one saponin fraction from Quillaya Saponaria Molina, and the saponin fraction in one complex may differ from the saponin fractions in other complex particles.

[0276] In embodiments, one type of saponin fraction or crude saponin fraction may be incorporated into one ISCOM matrix complex or particle, and another type of substantially pure saponin fraction or crude saponin fraction may be incorporated into another ISCOM matrix complex or particle. The composition or vaccine may comprise at least two types of complexes or particle types, each having one type of saponin incorporated into physically different particles.

[0277] In this composition, a mixture of ISCOM matrix complex particles and / or ISCOM complex particles may be used, in which one saponin fraction of Quillaya Saponaria Molina and another saponin fraction of Quillaya Saponaria Molina are separately incorporated into different ISCOM matrix complex particles and / or ISCOM complex particles.

[0278] ISCOM matrix or ISCOM complex particles, each having one saponin fraction, may be present in the composition in any combination of weight percent. In certain embodiments, the composition may contain ISCOM matrix or complexes containing a first saponin fraction in amounts of 0.1% to 99.9% by weight, 5% to 95% by weight, 10% to 90% by weight, 15% to 85% by weight, 20% to 80% by weight, 25% to 75% by weight, 30% to 70% by weight, 35% to 65% by weight, 40% to 60% by weight, 45% to 55% by weight, 40% to 60% by weight, or 50% by weight, with the remainder consisting of ISCOM matrix or complexes containing different saponin fractions. In other embodiments, the remainder consists of one or more ISCOM matrix or complexes, each matrix or complex particle containing only one saponin fraction. In other embodiments, the ISCOM matrix or complex particles may contain two or more saponin fractions.

[0279] In a particular composition, the sole saponin fraction in the first ISCOM matrix or ISCOM complex particles is fraction A, and the sole saponin fraction in the second ISCOM matrix or ISCOM complex particles is fraction C.

[0280] In the embodiment, in the adjuvant, fraction A of Quillaya Saponaria Molina accounts for at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% by weight of the sum of the weights of fraction A and fraction C of Quillaya Saponaria Molina, and fraction C of Quillaya Saponaria Molina accounts for the remainder.

[0281] A preferred composition comprises a first ISCOM matrix containing fraction A and a second ISCOM matrix containing fraction C, where the fraction A ISCOM matrix constitutes about 70% / weight of the total saponin adjuvant and the fraction C ISCOM matrix constitutes about 30% / weight of the total saponin adjuvant. In another preferred composition, the fraction A ISCOM matrix constitutes about 85% / weight of the total saponin adjuvant and the fraction C ISCOM matrix constitutes about 15% / weight of the total saponin adjuvant. In yet another preferred composition, the fraction A ISCOM matrix constitutes about 92% / weight of the total saponin adjuvant and the fraction C ISCOM matrix constitutes about 8% / weight of the total saponin adjuvant. Thus, in a particular composition, the fraction A ISCOM matrix is ​​present in the range of about 70% to about 85% of the total weight of saponin adjuvant in the composition, and the fraction C ISCOM matrix is ​​present in the range of about 15% to about 30%. In certain compositions, fraction A ISCOM matrix is ​​present in the range of about 70% to about 92% of the total weight of the saponin adjuvant in the composition, and fraction C ISCOM matrix is ​​present in the range of about 8% to about 30%. In embodiments, in the adjuvant, fraction A ISCOM matrix accounts for 50 to 96% by weight of the combined weight of fraction A ISCOM matrix and fraction C ISCOM, respectively, and fraction C ISCOM matrix accounts for the remainder. In a particularly preferred composition referred herein as Matrix-M®, fraction A ISCOM matrix is ​​present in the range of about 85% of the total weight of the saponin adjuvant in the composition, and fraction C ISCOM matrix is ​​present in the range of about 15%. Matrix-M® may be interchangeably referred to as Matrix-M1.

[0282] Exemplary QS-7 and QS-21 fractions, their production, and their uses are described in U.S. Patents No. 5,057,540, No. 6,231,859, No. 6,352,697, No. 6,524,584, No. 6,846,489, No. 7,776,343, and No. 8,173,141, which are incorporated herein by reference.

[0283] In embodiments, other adjuvants may be added or used as substitutes. The inclusion of any adjuvants described in Vogel et al., “A Compendium of Vaccine Adjuvants and Excipients (2nd Edition),” which is incorporated herein by reference in whole for all purposes, is assumed within the scope of this disclosure. Other adjuvants include complete Freund's adjuvant (a nonspecific stimulant of the immune response containing dead Mycobacterium tuberculosis), incomplete Freund's adjuvant, and aluminum hydroxide adjuvant. Other adjuvants include GMCSP, BCG, MDP compounds such as thur-MDP and nor-MDP, CGP (MTP-PE), Lipid A and monophosphoryl lipid A (MPL), MF-59, RIBI containing three components extracted from bacteria, MPL, trehalose dimycolate (TDM), and cell wall skeletons (CWS) in 2% squalene / TWEEN® polysorbate 80 emulsion. In embodiments, the adjuvant may be pouch lamellar lipid vesicles, such as NOVASOMES®. NOVASOMES® are pouch lamellar nonphospholipid vesicles in the range of about 100 nm to about 500 nm. They contain BRIJ® alcohol ethoxylate 72, cholesterol, oleic acid, and squalene. NOVASOMES® has been shown to be an effective adjuvant (see U.S. Patent Nos. 5,629,021, 6,387,373, and 4,911,928).

[0284] Dosage and Administration In embodiments, the present disclosure provides a method for inducing an immune response to one or more coronaviruses. In embodiments, the response is to one or more SARS-CoV-2 viruses, MERS, and SARS. In embodiments, the response is to heterologous SARS-CoV-2 strains. In embodiments, the heterologous SARS-CoV-2 strains have the World Health Organization labels alpha, beta, gamma, delta, epsilon, eta, iota, kappa, zeta, mu, or omicron. In embodiments, the heterologous SARS-CoV-2 strain has a PANGO lineage selected from the group consisting of B.1.1.529;BA.1,BA.1.1,BA.2,BA.3,BA.4,BA.5, B.1.1.7, B.1.351, P.1, B.1.617.2, AY, B.1.427, B.1.429, B.1.525, B.1.526, B.1.617.1, B.1.617.3, P.2, B.1.621, or B.1.621.1. The method involves administering an immunologically effective amount of a composition containing nanoparticles or recombinant CoV spike(S) polypeptide to a target. Advantageously, the proteins disclosed herein induce one or more particularly useful anti-coronavirus responses.

[0285] In one embodiment, the nanoparticles or CoV S polypeptide are administered together with an adjuvant. In another embodiment, the nanoparticles or CoV S polypeptide are administered without an adjuvant. In yet another embodiment, the adjuvant may be bound to the nanoparticles by non-covalent interactions or the like. In yet another embodiment, the adjuvant is administered co-administered with the nanoparticles, but the adjuvant and the nanoparticles do not interact substantially.

[0286] In embodiments, nanoparticles or CoV S polypeptides may be used for the prevention and / or treatment of one or more SARS-CoV-2 infection, heterologous SARS-CoV-2 strain infection, SARS infection, or MERS infection. Accordingly, this disclosure provides a method for inducing an immune response to one or more SARS-CoV-2 virus, heterologous SARS-CoV-2 virus, MERS, and SARS. This method involves administering an immunologically effective amount of a composition containing nanoparticles or CoV S polypeptides to a target. Advantageously, the proteins disclosed herein induce particularly useful anti-coronavirus responses.

[0287] In embodiments, the nanoparticles or CoV S polypeptides described herein have efficacy against SARS-CoV-2 virus or heterologous SARS-CoV-2 strains in a proportion of about 50% to about 99%, about 80% to about 99%, about 75% to about 99%, about 80% to about 95%, about 90% to about 98%, about 75% to about 95%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%.

[0288] The compositions disclosed herein may be administered systemically, via mucosal, or transdermal routes, or directly to specific tissues. As used herein, the term “systemic administration” includes parenteral administration routes. Parenteral administrations include, in particular, subcutaneous, intraperitoneal, intravenous, intra-arterial, intramuscular, or intrasternal injection, and intravenous or renal dialysis infusion techniques. Typically, systemic parenteral administration is intramuscular injection. As used herein, the term “mucosal administration” includes oral, intranasal, intravaginal, intrarectal, intratracheal, enteral, and ophthalmic administration. Preferably, administration is intramuscular.

[0289] The composition may be a single-dose schedule or a multi-dose schedule. Multi-dose schedules may be used in primary immunization schedules and / or booster immunization schedules. In embodiments, approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 doses are administered. In multi-dose schedules, various doses may be administered via the same or different routes, such as parenteral prime and mucosal boost, mucosal prime and parenteral boost, etc. In some embodiments, boost doses are administered approximately 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months (1 year), 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, or 10 years after the initial dose. In some embodiments, boost doses are administered annually after the initial dose. In some embodiments, subsequent boost doses are administered 3 or 4 weeks after the previous dose. In some embodiments, the first dose is administered on day 0 and the boost dose is administered on day 21. In some embodiments, the first dose is administered on day 0 and the boost dose is administered on day 28. In this embodiment, the first dose is administered on day 0, the boost dose on day 21, and the second boost dose is administered approximately 6 months after the administration of the first or second dose. In this embodiment, the first dose is administered on day 0, the boost dose on day 28, and the second boost dose is administered approximately 6 months after the administration of the first dose. In this embodiment, the first dose is administered on day 0, the boost dose on day 21, and the second boost dose is administered approximately 6 months after the second dose. In this embodiment, the first dose is administered on day 0, the boost dose on day 28, and the second boost dose is administered approximately 6 months after the administration of the second dose.

[0290] In one embodiment, the first dose is administered on day 0, the boost dose on day 21, and the second boost dose is administered approximately one year after the first dose or the first boost dose. In another embodiment, the first dose is administered on day 0, the boost dose on day 28, and the second boost dose is administered approximately one year after the first dose. In yet another embodiment, the first dose is administered on day 0, the first boost dose on day 21, and the second boost dose is administered approximately one year after the second dose. In yet another embodiment, the first dose is administered on day 0, the first boost dose on day 28, and the second boost dose is administered approximately one year after the second dose. In yet another embodiment, the second boost dose is administered 6 to 24 months or 12 to 24 months after the first boost dose.

[0291] In the embodiment, the boost dose contains the same immunological composition as the initial dose. In the embodiment, the boost dose contains a different immunological composition from the initial dose. In the embodiment, the different immunological composition is SARS-CoV-2 spike glycoprotein, mRNA encoding SARS-CoV-2 spike glycoprotein, plasmid DNA encoding SARS-CoV-2 spike glycoprotein, a viral vector encoding SARS-CoV-2 spike glycoprotein, or an inactivated SARS-CoV-2 virus. In the embodiment, the boost dose contains the initial composition. In the embodiment, the initial dose contains SARS-CoV-2 S glycoprotein (e.g., SARS-CoV-2 S glycoprotein having the amino acid sequence of SEQ ID NO: 87), and the boost dose contains the same SARS-CoV-2 S glycoprotein (e.g., SARS-CoV-2 S glycoprotein having the amino acid sequence of SEQ ID NO: 87). In this embodiment, the initial dose comprises SARS-CoV-2 S glycoprotein (e.g., SARS-CoV-2 S glycoprotein having the amino acid sequence of SEQ ID NO: 87), and the boost dose comprises a different SARS-CoV-2 S glycoprotein (e.g., SARS-CoV-2 S glycoprotein having the amino acid sequence of SEQ ID NO: 132).

[0292] In the embodiment, the initial dose comprises a combination of SARS-CoV-2 S glycoproteins (for example, SARS-CoV-2 S glycoprotein having the amino acid sequence of SEQ ID NO: 87 and SARS-CoV-2 S glycoprotein having the amino acid sequence of SEQ ID NO: 132). In the embodiment, the boost dose comprises a combination of SARS-CoV-2 S glycoproteins (for example, SARS-CoV-2 S glycoprotein having the amino acid sequence of SEQ ID NO: 87 and SARS-CoV-2 S glycoprotein having the amino acid sequence of SEQ ID NO: 132). In the embodiment, the initial dose comprises SARS-CoV-2 S glycoprotein, plasmid DNA encoding SARS-CoV-2 S glycoprotein, a viral vector encoding SARS-CoV-2 spike glycoprotein, or an inactivated SARS-CoV-2 virus. In the embodiment, the initial dose comprises SARS-CoV-2 spike glycoprotein, plasmid DNA encoding SARS-CoV-2 spike glycoprotein, a viral vector encoding SARS-CoV-2 spike glycoprotein, or an inactivated SARS-CoV-2 S virus, and the boost dose comprises one or more SARS-CoV-2 S glycoproteins.

[0293] In the embodiments, the dose, when measured in units of μg, may be the total weight of the dose including the solute, the weight of the CoV S polypeptide nanoparticles, or the weight of the CoV S polypeptide alone. The dose is measured using either an A280 or ELISA protein concentration assay.

[0294] The antigen dose, including for pediatric administration, may range from approximately 5 μg to approximately 25 μg, approximately 1 μg to approximately 300 μg, approximately 90 μg to approximately 270 μg, approximately 100 μg to approximately 160 μg, approximately 110 μg to approximately 150 μg, approximately 120 μg to approximately 140 μg, or approximately 140 μg to approximately 160 μg. In one embodiment, the dose is approximately 120 μg and is administered together with alum. In another embodiment, the pediatric dose may range from approximately 1 μg to approximately 90 μg. In the embodiment, the doses of CoV spike(S) polypeptide are approximately 1 μg, 2 μg, 3 μg, 4 μg, 5 μg, 6 μg, 7 μg, 8 μg, 9 μg, 10 μg, 11 μg, 12 μg, 13 μg, 14 μg, 15 μg, 16 μg, 17 μg, 18 μg, 19 μg, 20 μg, 21, 22, 23, 24, 25 μg, 26 μg, 27 μg, 28 μg, 29 μg, 30 μg, and The doses are 40 μg, approximately 50, approximately 60, approximately 70, approximately 80, approximately 90, approximately 100 μg, approximately 110 μg, approximately 120 μg, approximately 130 μg, approximately 140 μg, approximately 150 μg, approximately 160 μg, approximately 170 μg, approximately 180 μg, approximately 190 μg, approximately 200 μg, approximately 210 μg, approximately 220 μg, approximately 230 μg, approximately 240 μg, approximately 250 μg, approximately 260 μg, approximately 270 μg, approximately 280 μg, approximately 290 μg, or approximately 300 μg (including all values ​​and ranges in between). In one embodiment, the dose of CoV S polypeptide is 5 μg. In another embodiment, the dose of CoV S polypeptide is 25 μg. In yet another embodiment, the dose of CoV S polypeptide is the same for both the initial dose and the boost dose. In this embodiment, the dose of CoV S polypeptide differs between the initial dose and the boost dose.

[0295] In the embodiment, the amount of the first CoV S glycoprotein in the composition is in the range of about 1 μg to about 100 μg. In the embodiment, the amounts of the second, third, fourth, and fifth CoV S glycoproteins are less than the amount of the first CoV S glycoprotein in the composition. In the embodiment, the immunogenic composition contains about 1 ng to about 5 μg of the second CoV S glycoprotein. In the embodiment, the immunogenic composition contains about 1 ng to about 5 μg of the third CoV S glycoprotein. In the embodiment, the immunogenic composition contains about 1 ng to about 5 μg of the fourth CoV S glycoprotein. In the embodiment, the immunogenic composition contains about 1 ng to about 5 μg of the fifth CoV S glycoprotein. In the embodiment, the total amount of CoV S glycoprotein in the immunogenic composition is greater than 1 μg, less than about 5 μg, less than about 10 μg, less than about 15 μg, less than about 20 μg, or less than about 25 μg.

[0296] Certain populations may be administered with or without an adjuvant. In certain embodiments, the composition may not contain the added adjuvant. In such circumstances, the dose may be increased by approximately 10%.

[0297] In embodiments, the immunogenic compositions described herein are provided in pre-filled syringes. When preparing the immunogenic compositions in pre-filled syringes, the CoV S polypeptide and adjuvant are combined before administration.

[0298] In the embodiment, the dose of the adjuvant administered with the CoV S polypeptide, which includes natural and non-natural polypeptides, is approximately 1 μg to approximately 100 μg, for example, approximately 1 μg, approximately 2 μg, approximately 3 μg, approximately 4 μg, approximately 5 μg, approximately 6 μg, approximately 7 μg, approximately 8 μg, approximately 9 μg, approximately 10 μg, approximately 11 μg, approximately 12 μg, approximately 13 μg, approximately 14 μg, approximately 15 μg, approximately 16 μg, approximately 17 μg, approximately 18 μg, approximately 19 μg, approximately 20 μg, approximately 21 μg, approximately 22 μg , about 23, about 24, about 25μg, about 26μg, about 27μg, about 28μg, about 29μg, about 30μg, about 31μg, about 32μg, about 33μg, about 34μg, about 35μg, about 36μ g, about 37μg, about 38μg, about 39μg, about 40μg, about 41μg, about 42μg, about 43μg, about 44μg, about 45μg, about 46μg, about 47μg, about 48μg, about 49μg, Approximately 50μg, approximately 51μg, approximately 52μg, approximately 53μg, approximately 54μg, approximately 55μg, approximately 56μg, approximately 57μg, approximately 58μg, approximately 59μg, approximately 60μg, approximately 61μg, approximately 62μg, approximately 63μg, about 64μg, about 65μg, about 66μg, about 67μg, about 68μg, about 69μg, about 70μg, about 71μg, about 72μg, about 73μg, about 74μg, about 75μg, about 76 The adjuvant is approximately μg, about 77 μg, about 78 μg, about 79 μg, about 80 μg, about 81 μg, about 82 μg, about 83 μg, about 84 μg, about 85 μg, about 86 μg, about 87 μg, about 88 μg, about 89 μg, about 90 μg, about 91 μg, about 92 μg, about 93 μg, about 94 μg, about 95 μg, about 96 μg, about 97 μg, about 98 μg, about 99 μg, or about 100 μg. In the embodiment, the dose of the adjuvant is about 50 μg. In the embodiment, the adjuvant is a saponin adjuvant, for example, MATRIX-M (trademark).

[0299] In some embodiments, the dose is administered in a volume of about 0.1 mL to about 1.5 mL, for example, about 0.1 mL, about 0.2 mL, about 0.25 mL, about 0.3 mL, about 0.4 mL, about 0.5 mL, about 0.6 mL, about 0.7 mL, about 0.8 mL, about 0.9 mL, about 1.0 mL, about 1.1 mL, about 1.2 mL, about 1.3 mL, about 1.4 mL, or about 1.5 mL. In some embodiments, the dose is administered in a volume of 0.25 mL. In some embodiments, the dose is administered in a volume of 0.5 mL. In some embodiments, the dose is administered in a volume of 0.6 mL.

[0300] In certain embodiments of vaccines against MERS, SARS, or SARS-CoV-2 coronavirus, the dose may contain CoV S polypeptide concentrations of approximately 1 μg / mL to approximately 50 μg / mL, 10 μg / mL to approximately 100 μg / mL, approximately 10 μg / mL to approximately 50 μg / mL, approximately 175 μg / mL to approximately 325 μg / mL, approximately 200 μg / mL to approximately 300 μg / mL, approximately 220 μg / mL to approximately 280 μg / mL, or approximately 240 μg / mL to approximately 260 μg / mL.

[0301] In another embodiment, the Disclosure provides a method for formulating a vaccine composition to induce immunity to an infection or at least one disease symptom thereof in a mammal, comprising adding an effective dose of nanoparticles or CoV S polypeptide to the composition. The disclosed CoV S polypeptides and nanoparticles are useful for preparing compositions that stimulate an immune response to confer immunity or substantial immunity to an infectious pathogen. Accordingly, in one embodiment, the Disclosure provides a method for inducing immunity to an infection or at least one disease symptom thereof in a subject, comprising administering at least one effective dose of nanoparticles and / or CoV S polypeptide.

[0302] In the embodiment, the CoV S polypeptide or nanoparticles containing the same are administered in combination with an additional immunogenic composition. In the embodiment, the additional immunogenic composition induces an immune response to SARS-CoV-2. In the embodiment, the additional immunogenic composition is administered to the disclosed CoV S polypeptide or nanoparticles containing the same at approximately 1 minute, 5 minutes, 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, and 21 hours. The drug is administered within approximately 22 hours, 23 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, or 31 days. In embodiments, the additional composition is administered together with a first dose of the composition comprising CoV S polypeptide or nanoparticles containing the same. In embodiments, the additional composition is administered together with a boost dose of the composition comprising CoV S polypeptide or nanoparticles containing the same.

[0303] In the embodiment, the additional immunogenic composition comprises mRNA encoding the SARS-CoV-2 spike glycoprotein, plasmid DNA encoding the SARS-CoV-2 spike glycoprotein, a viral vector encoding the SARS-CoV-2 spike glycoprotein, or an inactive SARS-CoV-2 virus.

[0304] In the embodiments, the additional immunogenic composition includes mRNA encoding a CoV S polypeptide. In the embodiments, the mRNA encodes a CoV S polypeptide with proline substitutions at positions 986 and 987 of SEQ ID NO: 1. In the embodiments, the mRNA encodes a CoV S polypeptide with intact furin cleavage sites. In the embodiments, the mRNA encodes a CoV S polypeptide with proline substitutions at positions 986 and 987 of SEQ ID NO: 1 and intact furin cleavage sites. In the embodiments, the mRNA encodes a CoV S polypeptide with proline substitutions at positions 986 and 987 of SEQ ID NO: 1 and inactive furin cleavage sites. In the embodiments, the mRNA encodes a CoV S polypeptide having the amino acid sequence of SEQ ID NO: 87. In the embodiments, the mRNA encoding the CoV S polypeptide is encapsulated in lipid nanoparticles. Exemplary immunogenic compositions containing mRNA encoding a CoV S polypeptide are described in Jackson et al. N.Eng.J.Med. 2020 An mRNA Vaccine against SARS-CoV-2 - preliminary report, which is incorporated herein by reference in its entirety. In the embodiment, the composition containing mRNA encoding the CoV S polypeptide is administered in doses of 25 μg, 100 μg, or 250 μg.

[0305] In the embodiment, the additional immunogenic composition comprises an adenovirus vector encoding a CoV S polypeptide. In the embodiment, the AAV vector encodes a wild-type CoV S polypeptide. In the embodiment, the AAV vector encodes a CoV S polypeptide including proline substitutions at positions 986 and 987 of SEQ ID NO: 1 and an intact furin cleavage site. In the embodiment, the AAV vector encodes a CoV S polypeptide including proline substitutions at positions 986 and 987 of SEQ ID NO: 1 and an inactive furin cleavage site. In the embodiment, the AAV vector encodes a CoV S polypeptide having the amino acid sequence of SEQ ID NO: 87. The following publications describe immunogenic compositions containing adenovirus vectors encoding CoV S polypeptides, each incorporated herein by reference in its entirety: van Doremalen N. et al. A single dose of ChAdOx1 MERS provides protective immunity in rhesus macaques. Science Advances, 2020; van Doremalen N. et al. ChAdOx1 nCoV-19 vaccination prevents SARS-CoV-2 pneumonia in rhesus macaques. bioRxiv, (2020).

[0306] In the embodiment, the additional immunogenic composition comprises deoxyribonucleic acid (DNA). In the embodiment, the additional immunogenic composition comprises plasmid DNA. In the embodiment, the plasmid DNA encodes a CoV S polypeptide. In the embodiment, the DNA encodes a CoV S polypeptide comprising proline substitutions at positions 986 and 987 of SEQ ID NO: 1 and intact furin cleavage sites. In the embodiment, the DNA encodes a CoV S polypeptide comprising proline substitutions at positions 986 and 987 of SEQ ID NO: 1 and inactive furin cleavage sites. In the embodiment, the DNA encodes a CoV S polypeptide having the amino acid sequence of SEQ ID NO: 87. In the embodiment, the DNA encodes a CoV S polypeptide having the amino acid sequence of SEQ ID NO: 174 or SEQ ID NO: 175.

[0307] In this embodiment, the additional immunogenic composition includes an inactivated viral vaccine.

[0308] In the embodiments, the CoV S polypeptide or nanoparticles containing the CoV S polypeptide are administered to patients who have been confirmed to have an infection caused by SARS-CoV-2 or a heterologous SARS-CoV-2 strain, or who have been previously confirmed to have an infection. Infection by SARS-CoV-2 or a heterologous SARS-CoV-2 strain can be confirmed by nucleic acid amplification tests (e.g., polymerase chain reaction) or serological tests (e.g., testing for antibodies against SARS-CoV-2 viral antigens). In the embodiments, the CoV S polypeptide or nanoparticles containing the CoV S polypeptide are administered to patients at least about 3 days, at least about 1 week, at least about 2 weeks, at least about 3 weeks, and at least about 4 weeks after the patient is diagnosed with COVID-19. In the embodiment, CoV S polypeptide or nanoparticles containing CoV S polypeptide are administered to patients from one week to one year after diagnosis of COVID-19, for example, about one week, two weeks, three weeks, four weeks, five weeks, six weeks, one month, two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, eleven months, or one year after the patient's diagnosis. In the embodiment, CoV S polypeptide or nanoparticles containing CoV S polypeptide are administered to the patient 1 week to 20 years after diagnosis of COVID-19, for example, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 1 year, about 2 years, about 3 years, about 4 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, about 10 years, about 11 years, about 12 years, about 13 years, about 14 years, about 15 years, about 16 years, about 17 years, about 18 years, about 19 years, or about 20 years after diagnosis of COVID-19.

[0309] In embodiments, the CoV S polypeptide or nanoparticles containing the same are administered to the patient after administration of a first immunogenic composition. Non-limiting examples of the first immunogenic composition include SARS-CoV-2 spike glycoprotein, mRNA encoding SARS-CoV-2 spike glycoprotein, plasmid DNA encoding SARS-CoV-2 spike glycoprotein, viral vector encoding SARS-CoV-2 spike glycoprotein, or inactivated SARS-CoV-2 virus. In embodiments, the CoV S polypeptide or nanoparticles containing the same are administered about 1 week to 1 year, about 1 week to 1 month, about 3 weeks to 4 weeks, about 1 week to 5 years, about 1 year to 5 years, about 1 year to 3 years, about 3 years to 5 years, about 5 years to 10 years, about 1 year to 10 years, or about 1 year to 2 years after administration of the first immunogenic composition. In the embodiment, CoV S polypeptide or nanoparticles containing the same are administered about 1 week to about 1 year after administration of the first immunogenic composition, for example, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, or about 1 year after administration.

[0310] In embodiments, CoV S protein or nanoparticles containing CoV S protein are useful for preparing immunogenic compositions to stimulate an immune response that confers immunity or substantial immunity to one or more MERS, SARS, SARS-CoV-2, and heterologous SARS-CoV-2 strains. Both mucosal and cellular immunity can contribute to immunity against infection and disease. Antibodies secreted locally in the upper respiratory tract are a major factor in resistance to innate infection. Secretory immunoglobulin A (sIgA) is involved in upper respiratory tract protection and serum IgG in the lower respiratory tract. The immune response induced by infection protects against reinfection with the same virus or antigenically similar virus strains. Antibodies produced in a host after immunization with the nanoparticles disclosed herein can be administered to another person, thereby providing passive administration to the subject.

[0311] In the embodiment, the CoV S protein or nanoparticles containing the CoV S protein have one or more amino acid deletions selected from the group consisting of amino acids 11-14, 56, 57, 130, 131, 132, 144, 145, 198, 199, 228, 229, 230, 231, 234, 235, 236, 237, 238, 239, 240, 676-685, 676-702, 702-711, 775-793, 806-815 and combinations thereof. (b) Amino acids 5, 6, 7, 11, 12, 13, 14, 51, 53, 54, 56, 57, 62, 63, 67, 70, 82, 125, 129, 131, 132, 133, 134, 139, 143, 144, 145, 170, 177, 197, 198, 199, 200, 201, 202, 209, 229, 233, 239, 240, 244, 245, 326, 333, 355, 358, 360, 362, 363, 392, 395, 404, 419, 426, 427, 431, 432, 433, 439, 440, 447, 464, 465, 471, 473, 477, 480, 481, 483, 485, 488, 492, 534, 557, 591, 600, 601, 626, 642, 645, 664, 666, 668, 688, 691, 703, 751, 783, 843, 846, 875, 937, 94 Cross-neutralizing antibodies against the SARS-CoV-2 virus are induced, containing an S protein having one or more amino acid mutations selected from the group consisting of 1, 956, 968, 969, 1014, 1058, 1105, 1163, 1186 and combinations thereof, or (c) one or more modifications selected from the insertion of a tripeptide having the amino acid sequence of EPE between amino acids 214 and 215, and the amino acids of the CoV S glycoprotein are numbered relative to the polypeptide having the sequence of SEQ ID NO: 2.

[0312] In the embodiment, the CoV S protein or nanoparticles containing the CoV S protein induce cross-neutralizing antibodies against the SARS-CoV-2 virus, which contain an S protein having one or more modifications selected from the following: deletion of amino acid 56, deletion of amino acid 57, deletion of amino acid 131, N488Y, A557D, D601G, P668H, T703I, S969A, D1105H, N426K, and Y440F (the amino acids are numbered relative to the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 2).

[0313] In the embodiment, the CoV S protein or nanoparticles containing the CoV S protein induce cross-neutralizing antibodies against the SARS-CoV-2 virus containing an S protein having one or more modifications selected from amino acid 56 deletion, amino acid 57 deletion, amino acid 131, N488Y, A557D, D601G, P668H, T703I, S969A and D1105H (amino acids are numbered relative to the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 2).

[0314] In the embodiment, CoV S protein or nanoparticles containing CoV S protein induce cross-neutralizing antibodies against the SARS-CoV-2 virus containing S proteins with one or more modifications selected from D67A, D202G, L229H, K404N, E471K, N488Y, D601G, and A688V (amino acids are numbered relative to the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 2).

[0315] In the embodiment, the CoV S protein or nanoparticles containing the CoV S protein induce cross-neutralizing antibodies against the SARS-CoV-2 virus, which contain an S protein having one or more modifications selected from the deletion of amino acids 229-231, D67A, D202G, K404N, E471K, N488Y, D601G, and A688V (the amino acids are numbered relative to the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 2).

[0316] In the embodiment, the CoV S protein or nanoparticles containing the CoV S protein induce cross-neutralizing antibodies against the SARS-CoV-2 virus, which contain an S protein having one or more modifications selected from the deletion of amino acids 229-231, L5F, D67A, D202G, K404N, E471K, N488Y, D601G, and A688V (the amino acids are numbered relative to the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 2).

[0317] In the embodiment, CoV S protein or nanoparticles containing CoV S protein induce cross-neutralizing antibodies against the SARS-CoV-2 virus containing S proteins with one or more modifications selected from L5F, T7N, P13S, D125Y, R177S, K404T, E471K, N488Y, D601G, H642Y, T1014I and V1163F (amino acids are numbered relative to the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 2).

[0318] In one embodiment, the CoV S protein or nanoparticles containing the CoV S protein induce cross-neutralizing antibodies against the SARS-CoV-2 virus having an S protein containing one or more modifications selected from W139C and L439R (amino acids are numbered relative to the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 2). In another embodiment, the CoV S protein containing the W139C and L439R modifications is expressed together with a signal peptide having the amino acid sequence of SEQ ID NO: 117 or SEQ ID NO: 5. In yet another embodiment, the CoV S protein or nanoparticles containing the CoV S protein induce cross-neutralizing antibodies against the SARS-CoV-2 virus having one or more modifications selected from D601G, W139C, and L439R (amino acids are numbered relative to the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 2). In yet another embodiment, the CoV S protein or nanoparticles containing D601G, W139C, and L439R modifications are expressed together with a signal peptide having the amino acid sequence of SEQ ID NO: 117 or SEQ ID NO: 5.

[0319] In the embodiment, CoV S protein or nanoparticles containing CoV S protein induce cross-neutralizing antibodies against SARS-CoV-2 virus having one or more modifications selected from D601G, L5F, D67A, D202G, deletion of amino acids 229-231, R233I, K404N, E471K, N488Y, and A688V (wherein the amino acids are numbered relative to the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 2).

[0320] In embodiments, the disclosure provides a method for producing one or more high-affinity anti-MERS-CoV, anti-SARS-CoV, and anti-SARS-CoV-2 virus antibodies. The high-affinity antibodies produced by immunization with nanoparticles disclosed herein are produced by administering an immunogenic composition containing S CoV polypeptide or nanoparticles containing S CoV polypeptide to an animal, recovering serum and / or plasma from the animal, and purifying the antibodies from the serum and / or plasma. In embodiments, the animal is human. In embodiments, the animal is chicken, mouse, guinea pig, rat, rabbit, goat, human, horse, sheep, or cattle. In one embodiment, the animal is cattle or horse. In another embodiment, the cattle or horse is transgenic. In yet another embodiment, the transgenic cattle or horse produces human antibodies. In embodiments, the animal produces monoclonal antibodies. In embodiments, the animal produces polyclonal antibodies. In one embodiment, the method further comprises the administration of an adjuvant or immunostimulatory compound. In further embodiments, the purified high-affinity antibody is administered to a human subject. In one embodiment, the human subject is at risk of infection with one or more of MERS, SARS, and SARS-CoV-2.

[0321] In embodiments, the CoV S protein or nanoparticles are administered co-administered with influenza glycoprotein or nanoparticles containing influenza glycoprotein or respiratory syncytial virus (RSV) fusion (F) glycoprotein. In embodiments, the CoV S protein or nanoparticles are formulated simultaneously with RSV F glycoprotein, influenza glycoprotein, or a combination thereof. Suitable glycoproteins and nanoparticles are described in U.S. Patent Application Publication 2018 / 0133308 and U.S. Patent Application Publication 2019 / 0314487, each of which is incorporated herein by reference in whole. In embodiments, the CoV S protein or nanoparticles are administered co-administered with: (a) surfactant core nanoparticles containing recombinant influenza hemagglutinin (HA) glycoprotein derived from influenza B strain; and (b) hemagglutinin saponin matrix nanoparticles (HaSMaN) containing recombinant influenza HA glycoprotein derived from influenza A strain and an ISCOM matrix adjuvant. In the embodiment, the CoV S protein or nanoparticles are administered simultaneously with nanoparticles containing a nonionic surfactant core and influenza HA glycoprotein, wherein the influenza HA glycoprotein contains a head region protruding outward from the nonionic surfactant core and a transmembrane domain associated with the nonionic surfactant core, the influenza HA glycoprotein is HA0 glycoprotein, and the amino acid sequence of the influenza HA glycoprotein is 100% identical to the amino acid sequence of the native influenza HA protein. In the embodiment, the influenza glycoprotein or nanoparticles are formulated simultaneously with the CoV S protein or nanoparticles.

[0322] In some embodiments, this disclosure provides a co-formulation (i.e., pre-filled syringe or premix) strategy for an immunogenic composition comprising CoV S glycoprotein and an adjuvant (e.g., a saponin adjuvant). A typical vaccine administration strategy currently in use is bedside mixing; that is, the vaccine composition and adjuvant are stored separately and mixed before administration. Premix, co-formulation, or pre-filled syringe strategies for vaccines are less common due to concerns about the stability of the antigen (e.g., CoV S glycoprotein) and its subsequent immunogenicity. This disclosure provides immunogenic compositions that can be pre-mixed and stored. The disclosed vaccination strategies and formulations may improve the efficiency of vaccination and reduce the risk of errors during bedside mixing, while maintaining overall safety and immunogenicity.

[0323] Premix formulations can be stored and transported using a variety of containers, including syringes and plastic ampoules for single-dose administration. In some cases, plastic ampoules can be manufactured using concurrent molding and filling (BFS) manufacturing techniques or methods. Generally, concurrent molding and filling (BFS) manufacturing methods involve extruding a plastic material (e.g., resin) to form a parison, then placing it in a mold and cutting it to size. The plastic is then inflated using a filling needle or mandrel, which produces a hollow ampoule that substantially conforms to the shape of the mold. Once inflated, a desired volume of liquid can be injected into the ampoule, the filling needle or mandrel can be removed, and the ampoule can be sealed. Thus, BFS can be an automated process that can be performed in a sterile environment without direct human intervention.

[0324] In some cases, the ability to aseptically produce sterile ampoules containing the desired liquid makes BFS-manufactured ampoules particularly suitable for the pharmaceutical industry. However, BFS technology is not suitable for all pharmaceutical liquids, products, etc. For example, some known BFS manufacturing methods involve delivering the liquid or product into the ampoule while the plastic is still relatively hot, which can adversely affect temperature-sensitive liquids and / or products, such as vaccines and biologics. However, advances in cooled BFS technology have increased the diversity of suitable products and liquids, making it possible for some vaccines, biologics, and / or other temperature-sensitive pharmaceuticals to be contained in BFS ampoules.

[0325] In some cases, BFS ampoules may have a size, shape, and / or configuration that is at least partially based on the desired use and / or the desired liquid or dosage that the ampoule is configured to contain. For example, some known BFS ampoules may include tops such as puncture tops, twist-off tops, male Luer or female Luer connectors. Some known BFS ampoules may have a size and / or shape based on the volume of the liquid or the dosage that is configured to be placed therein. In addition, some known BFS ampoules may be manufactured in strips of multiple temporarily connected ampoules, which may improve manufacturing, packaging, and / or storage efficiency, etc.

[0326] In embodiments, the immunogenic compositions described herein are provided in pre-filled syringes. When the immunogenic compositions are prepared in pre-filled syringes, the antigen and adjuvant are combined before administration. In embodiments, the pre-filled syringes contain CoV S glycoprotein and an adjuvant (e.g., a saponin adjuvant). In this embodiment, the pre-filled syringe contains CoV S glycoprotein and a saponin adjuvant, the adjuvant comprising at least two iscom particles, the first iscom particle comprising fraction A of Quillaya Saponaria Molina and not fraction C of Quillaya Saponaria Molina, and the second iscom particle comprising fraction C of Quillaya Saponaria Molina and not fraction A of Quillaya Saponaria Molina, the Quillaya Saponaria Molina fraction A and the Quillaya Saponaria Molina fraction C in the adjuvant It accounts for approximately 85% by weight and approximately 15% by weight, respectively, of the total weight of fraction A of Saponaria Molina and fraction C of Quillaya Saponaria Molina.In this embodiment, the pre-filled syringe contains CoV S glycoprotein and a saponin adjuvant, the adjuvant comprising at least two iscom particles, the first iscom particle comprising fraction A of Quillaya Saponaria Molina and not fraction C of Quillaya Saponaria Molina, and the second iscom particle comprising fraction C of Quillaya Saponaria Molina and not fraction A of Quillaya Saponaria Molina, the Quillaya Saponaria Molina fraction A and the Quillaya Saponaria Molina fraction C in the adjuvant It accounts for approximately 92% by weight and approximately 8% by weight, respectively, of the total weight of fraction A of Saponaria Molina and fraction C of Quillaya Saponaria Molina.In this embodiment, the pre-filled syringe contains CoV S glycoprotein and a saponin adjuvant, the adjuvant containing at least two iscom particles, the first iscom particle containing fraction A of Quillaya Saponaria Molina and fraction C of Quillaya Saponaria Molina, the second iscom particle containing fraction C of Quillaya Saponaria Molina and not fraction A of Quillaya Saponaria Molina, the fraction A of Quillaya Saponaria Molina in the adjuvant accounting for at least about 75% by weight, and Quillaya Saponaria Molina Fraction C of Quillaya Saponaria Molina accounts for the remainder of the combined weight of Fraction A and Fraction C of Quillaya Saponaria Molina. [Examples]

[0327] Example 1 Expression and purification of pre-fusion coronavirus spike (S) polypeptide nanoparticles CoV spike polypeptides having amino acid sequences corresponding to native coronavirus (CoV) spike (SEQ ID NO: 1 and 2) and SEQ ID NOs: 3, 4, 38, 41, 44, 48, 51, 54, 58, 61, 63, 65, 67, 73, 75, 78, 79, 82, 83, 85, 87, 106, 108, 89, 112-115, 132, 133, 114, 138, 141, 144, 147, 151, 153, 156, 158, 174-176, 186, 188, 190, 195, 217-228, 233-236, and 245-292 were expressed in a baculovirus expression system, and recombinant plaques expressing CoV S polypeptide were selected and confirmed. In all cases, the signal peptide was SEQ ID NOs. 5, 154, 193, or 117. Furthermore, CoV S polypeptides encoded by one of the nucleic acids from SEQ ID NOs. 161, 162, 163, 164, 165, 166, 168, 169, 171, 172, 196-199, 201, 202, 204, 206, 208, 210, 212, 214, 216, and 237-292 were constructed. Furthermore, CoV S polypeptides were created that contained the polypeptide sequences of SEQ ID NOs. 87, 159, 167, 160, 170, 174, 175, 186, 188, 190, 195, 217-228, 233-236, 243, 255-264, 273-280, 283, 284, 287, 288, 291, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 329, 330, 331, 332, 333, and 334 at the C-terminus of a signal peptide having any one of the amino acid sequences of SEQ ID NOs. 5, 154, 193, or 117.

[0328] Table 2A shows the sequence characteristics of the aforementioned CoV spike polypeptide. Table 2B shows the sequences of additional CoV spike polypeptides that have been constructed. The CoV S polypeptides in Table 2B have approximately 1 to 50 modifications compared to the CoV S polypeptides in Table 2A. Modifications can be located in the RBD, NTD, SD1 / 2, S1 subunit, or S2 subunit. Modifications can also be located near the furin cleavage domain.

[0329] [Table 56]

[0330] [Table 57]

[0331] [Table 58]

[0332] [Table 59]

[0333] [Table 60]

[0334] [Table 61]

[0335] [Table 62]

[0336] [Table 63]

[0337] Production of proteins and nanoparticles Recombinant baculoviruses were amplified by infection with Sf9 or rhabdovirus-free Sf22a insect cells. Baculoviruses were infected with insect cell cultures at approximately 0.6 MOI (infection multiplicity = virus ffu or pfu / cell). Baculoviruses expressing different CoV S glycoproteins can be used to infect insect cell cultures. For example, five different baculoviruses expressing CoV S glycoproteins can be used to infect insect cell cultures. The cultures and supernatants were collected 48-72 hours after infection. Approximately 30 mL of the crude cell recovery was clarified by centrifugation at approximately 800 xG for 15 minutes. The resulting crude cell recovery containing coronavirus spike (S) protein was purified into nanoparticles as described below.

[0338] To produce nanoparticles, the nonionic surfactant TERGITOL® nonylphenol ethoxylate NP-9 is used in the membrane protein extraction protocol. The crude extract is further purified by anion exchange chromatography, lentil lectin affinity / HIC, and cation exchange chromatography. Washed cells are lysed with surfactant and then subjected to low pH treatment, which leads to the precipitation of BV and Sf9 host cell DNA and proteins. Before the second low pH treatment, the neutralized low pH lysate is clarified and further purified by anion exchange and affinity chromatography.

[0339] Affinity chromatography was used to remove Sf9 / BV protein, DNA, and NP-9, and to concentrate the coronavirus spike (S) protein. Briefly, lentil lectin is a metalloprotein containing calcium and manganese that reversibly binds to glycosylated proteins containing polysaccharides and glucose or mannose. The anion exchange flow-through fraction containing coronavirus spike (S) protein was loaded onto a lentil lectin affinity chromatography resin (Capto Lentil Lectin, GE Healthcare). Glycosylated coronavirus spike (S) protein selectively bound to the resin, while unglycosylated proteins and DNA were removed in the column flow-through. Weakly binding glycoproteins were removed by a buffer containing high salt and low molar concentration of methyl alpha-D-mannopyranoside (MMP).

[0340] Column washing is also used to replace the NP-9 surfactant with the surfactant polysorbate 80 (PS80). Coronavirus spike (S) polypeptide is eluted from the lentil lectin column in a nanoparticle structure with high concentrations of MMP. After elution, the coronavirus spike (S) protein trimer is assembled into nanoparticles consisting of the coronavirus spike (S) protein trimer contained in the surfactant core and PS80.

[0341] Binding of CoV S polypeptide to hACE2 The binding ability of CoV S polypeptides to hACE2 is evaluated by biolayer interferometry and ELISA.

[0342] Biolayer Interferometry (BLI) BLI experiments were performed using the Octet QK384 system (Pall Forte Bio, Fremont, CA). His-tagged human ACE2 (2 μg mL-1) was immobilized on a nickel-charged Ni-NTA biosensor chip. After baseline, SARS-CoV-2 S protein-containing samples were serially diluted 2-fold, associated for 600 seconds, and then dissociated for a further 900 seconds. Data were analyzed using global curve fitting with Octet software HT 101:1.

[0343] The CoV S polypeptides in Table 2A retained their ability to bind to hACE2. Dissociation kinetics showed that the CoV S polypeptides remained firmly bound, as evidenced by minimal or no dissociation over 900 seconds of observation in the absence of fluid-phase S protein. The ability of the CoV S polypeptides in Table 2B to bind to hACE2 was evaluated by BLI.

[0344] ELISA The specificity of the CoV S polypeptide to hACE2 was confirmed by ELISA. 96-well plates were coated overnight at 4°C with 100 μL of SARS-CoV-2 spike protein (2 μg / mL). The plates were washed with phosphate-buffered saline in 0.05% Tween (PBS-T) buffer and blocked with TBS Startblock blocking buffer (ThermoFisher, Scientice). His-tagged hACE2 and hDPP4 receptors were added to the wells coated at room temperature for 2 hours in 3-fold serial dilutions (5-0.0001 μg / mL-1). The plates were washed with PBS-T. Optimally diluted horseradish peroxidase (HRP) complex antihistidine was added, and color development was induced by adding 3,3',5,5'-tetramethylbenzidine peroxidase substrate (TMB, T0440-IL, Sigma, St. Louis, MO, USA). Plate readings were performed at a 450nm OD using a SpectraMax Plus plate reader (Molecular Devices, Sunnyvale, CA, USA), and the data was analyzed using SoftMax software. EC50 values ​​were calculated using 4-parameter fitting with GraphPad Prism 7.05 software.

[0345] Example 2 Formulation of pentavalent COVID-19 immunogenic composition A pentavalent COVID-19 immunogenic composition is formulated. The pentavalent COVID-19 immunogenicity contains five CoV S glycoproteins. Each of the first, second, third, fourth, and fifth CoV S glycoproteins may have an inactivating furin cleavage site. The inactivating furin cleavage site may contain the amino sequence QQAQ (SEQ ID NO: 7). Each of the first, second, third, fourth, and fifth CoV S glycoproteins may also have modifications at amino acids 986 and 987, and each CoV S glycoprotein is numbered relative to the CoV S glycoprotein of SEQ ID NO: 1. One or more amino acids 986 and 987 of the first, second, third, fourth, and fifth CoV S glycoproteins may be proline.

[0346] Table 2C1 shows the identities of the five CoV S glycoproteins in each immunogenic composition. The second, third, fourth, and fifth CoV S glycoproteins have one or more modifications compared to the first CoV S glycoprotein. Each of the first, second, third, fourth, and fifth CoV S glycoproteins in the immunogenic composition has a different amino acid sequence. Table 2C2 shows the possible first, second, third, fourth, and fifth CoV S glycoproteins in the composition and their modifications compared to the CoV S glycoprotein of SEQ ID NO: 1.

[0347] Compared to the first CoV S glycoprotein in the composition, one or more of the second, third, fourth, and fifth CoV S glycoproteins may have modifications to the receptor-binding domain, N-terminal domain, S1 subunit, S2 subunit, or SD1 / 2. The modifications may occur at positions 180, 252, 253, 444, 478, 486, 521, or combinations thereof, and these modifications are numbered relative to the CoV S polypeptide of SEQ ID NO: 1. Position 180 of the CoV S polypeptide may be glutamic acid or valine, and the CoV S polypeptide is numbered relative to SEQ ID NO: 1. Position 252 of the CoV S polypeptide may be glycine or valine, and the CoV S polypeptide is numbered relative to SEQ ID NO: 1. Position 253 of the CoV S polypeptide may be aspartic acid or glycine, and the CoV S polypeptide is numbered relative to SEQ ID NO: 1. Position 444 of the CoV S polypeptide may be threonine or lysine, and the CoV S polypeptide is numbered relative to SEQ ID NO: 1. Position 478 of the CoV S polypeptide can be threonine, arginine, or lysine, and the CoV S polypeptide is numbered relative to SEQ ID NO: 1. Position 486 of the CoV S polypeptide can be threonine or serine, and the CoV S polypeptide is numbered relative to SEQ ID NO: 1. Position 521 of the CoV S polypeptide can be proline or serine, and the CoV S polypeptide is numbered relative to SEQ ID NO: 1.

[0348] [Table 64]

[0349] [Table 65]

[0350] [Table 66]

[0351] Five CoV S polypeptide nanoparticles are prepared according to the procedure of Example 1 and suspended in a pharmaceutically acceptable buffer to form an immunogenic composition. An adjuvant may be added to the composition. The adjuvant may be a saponin adjuvant. For example, a saponin adjuvant may contain two iscom particles, the first iscom particle containing fraction A of Quillaya Saponaria Molina but not fraction C of Quillaya Saponaria Molina, and the second iscom particle containing fraction C of Quillaya Saponaria Molina but not fraction A of Quillaya Saponaria Molina. Fractions A and C account for 85% and 15% respectively of the total weight of Quillaya Saponaria Molina fraction A and Quillaya Saponaria Molina fraction C in the adjuvant.

[0352] [Table 67]

[0353] [Table 68]

[0354] [Table 69]

[0355] [Table 70]

[0356] [Table 71]

[0357] Example 3 Immunogenicity of monovalent and bivalent COVID-19 immunogenic compositions A monovalent COVID-19 immunogenic composition containing one CoV S glycoprotein was prepared. A bivalent COVID-19 immunogenic composition containing two CoV S glycoproteins was also prepared. The immunogenicity of the following immunogenic compositions was evaluated. Table C3 shows the identity of the CoV S glycoprotein in each composition.

[0358] [Table 72]

[0359] CoV S glycoprotein nanoparticles were prepared according to the procedure of Example 1 and suspended in a pharmaceutically acceptable buffer to form immunogenic compositions. Each composition contained a saponin adjuvant containing two ISCOM particles, the first ISCOM particle containing fraction A of Quillaya Saponaria Molina but not fraction C of Quillaya Saponaria Molina, and the second ISCOM particle containing fraction C of Quillaya Saponaria Molina but not fraction A of Quillaya Saponaria Molina. Fractions A and C account for 85% and 15% respectively of the total weight of Quillaya Saponaria Molina fraction A and Quillaya Saponaria Molina fraction C in the adjuvant.

[0360] Administration of a monovalent composition A group of mice (n=10) was inoculated intramuscularly with two doses of composition 3, two doses of composition 2, or two doses of composition 1. Figures 3A-3C show that composition 3 (Figures 3A, 3D), composition 2 (Figures 3B, 3E), and composition 1 (Figures 3C, 3F) each neutralized pseudoviruses expressing S proteins derived from various heterologous SARS-CoV-2 strains. The humoral responses after administration of composition 2 and composition 1 were equivalent, inducing pseudovirus-neutralizing antibodies against SARS-CoV-2 omicron BA.5, XBB.1.5, XBB.1.16, XBB.2.3, EG.5.1, and XBB.1.16.6, indicating very high immunogenicity (Figures 3E, 3F). Following the primary vaccination series, homologous variant responses to vaccination with Composition 2 (GMT=4148, 95%CI:2494-6900) and Composition 1 (GMT=7622, ​​95%CI:4451-13053) were slightly higher than heterologous responses to Composition 2 (GMT=3473, 95%CI:2282-5287) against pseudoviruses expressing the SARS-CoV-2 S glycoprotein of Composition 1, and to Composition 1 (GMT=5846, 95%CI:2863-11940) against SARS-CoV-2 pseudoviruses expressing the SARS-CoV-2 S glycoprotein of Composition 2 (Figures 3E and 3F). Compared to homologous variant responses, the magnitude of the EG.5.1 neutralization response was comparable after immunization with XBB.1.5 (GMT=6478, 95%CI:3464-12112) and XBB.1.16 (GMT=5770, 95%CI:1918-17358) (Figure 3E). The XBB.1.16.6 variant neutralization response was similar after immunization with XBB.1.5 (GMT=5548, 95%CI:1960-15704) and XBB.1.16 (GMT=10781, 95%CI:5413-21472) (Figure 3E). The magnitude of the EG.5.1 and XBB sub-variant responses was comparable to the levels achieved in homologous responses to the two-dose primary series of the prototype vaccine, suggesting that appropriate antibody levels were achieved.

[0361] Immunogenicity induced by divalent composition 4 compared to monovalent composition 2 Mice (n=10 per group) were intramuscularly inoculated with either Composition 2 (1 μg) or Composition 4, a bivalent composition containing 0.5 μg of prototype CoV S glycoprotein (SEQ ID NO: 87) and 0.5 μg of XBB.1.5 (SEQ ID NO: 274), on day 0 and day 14. Serum was collected on day 21 (one week after the second dose). Figures 4A-4B show that administration of two doses of Composition 2, a monovalent composition containing CoV S glycoprotein SEQ ID NO: 274 (Figures 4A and 4C), resulted in a superior immune response in mice compared to administration of two doses of Composition 4, a bivalent composition containing CoV S glycoprotein SEQ ID NO: 274 and CoV S glycoprotein SEQ ID NO: 87 (Figures 4B and 4D). Specifically, the monovalent composition induced cross-reactive pseudovirus neutralizing antibodies against SARS-CoV-2 variant strains Omicron XBB.1.5 (GMT=4554, 95%CI:3285~6313), XBB.1.16 (GMT=4156, 95%CI:2568~6724), and XBB.2.3 (GMT=2058, 95%CI:1019~4157). In contrast, the bivalent composition induced approximately 50% or lower responses against XBB.1.5 (GMT=981, 95%CI:625~1541), XBB.1.16 (GMT=2168, 95%CI:1387~3389), and XBB.2.3 (GMT=524, 95%CI:224~1227).

[0362] Effect of administration of composition 5 and composition 2 or composition 1 booster as a primary series on immunogenicity against SARS-CoV-2 variants. A primary immunization series of the bivalent vaccine (composition 5) was administered intramuscularly to groups of mice (n=10 per group) on days 0 and 14, followed by a single booster dose of either composition 2 or composition 1 on day 47. Serum was collected on day 21 (one week after the second dose) and day 61 (two weeks after the booster dose).

[0363] Figures 5A-5C and 6A-6C show the immune response induced by boost doses containing composition 2 (Figures 5B, 6B, 5E) or composition 1 (Figures 5C, 5F, 6C). Boost administration of either composition 2 (Figures 5B, 6B, 5E) or composition 1 (Figures 5C, 5F, 6C) resulted in an improved immune response to pseudoviruses expressing S proteins from various heterologous SARS-CoV-2 strains compared to unboosted mice (Figures 5A, 5D, 6A). Specifically, boost administration of composition 2 or composition 1 induced a more than 35-fold increase in pseudovirus neutralizing antibodies against SARS-CoV-2 pseudoviruses expressing the SARS-CoV-2 S glycoprotein of composition 2 or composition 1 (Figures 5D-5F). Pseudovirus neutralizing antibodies were higher against XBB.2.3 after immunization with Composition 2 (GMT=4561, 95%CI:2219~9375) compared to after immunization with Composition 1 (GMT=2938, 95%CI:1495~5774).

[0364] The pseudovirus neutralizing antibody response in mice was further analyzed by antigen mapping. Priming with composition 5 resulted in an immune response to omicron XBB.1.5 and XBB.1.16 variants, which was 30 times stronger than priming with composition 3 (Figure 6A). Boost administration of composition 2 to primed mice induced a well-matched response to XBB.1.16 with an antigenic distance of 0.691 (Figure 6B). Similarly, a boost with composition 1 induced a well-matched response to XBB.1.5 with a fold-difference of 0.750 (Figure 6C). Antigenic distances with a fold-difference of less than 2x are considered to be well-matched responses.

[0365] Figure 8 shows the ability of a dosing regimen, which includes administering two doses of composition 5 followed by a boost dose of composition 2, to induce an immune response that blocks the binding of hACE 2 to the SARS-CoV-2 S glycoprotein.

[0366] CD4+ T cell response in mice: Mice were administered two doses of composition 3 containing the CoV S glycoprotein of SEQ ID NO: 87, or two doses of composition 5 containing the CoV S glycoprotein of SEQ ID NO: 87 and the CoV S glycoprotein of SEQ ID NO: 222. Mice were also given a boost dose of composition 2.

[0367] In spleen cells isolated two weeks after booster administration, Th1 (IFN-γ, IL-2, and TNF-α) and Th2 (IL-4) cytokine-producing CD4+ T cells were measured. Regardless of the priming vaccine (i.e., composition 3 or composition 5), boosting with composition 2 reproduced a robust CD4+ T cell response at a comparable level after boosting (Figure 10A, Figure 10B).

[0368] Results from primary vaccine regimens followed by boosts in rhesus monkeys: Rhesus macaques (Macaca mulatta, n=5 per group) were administered two doses of composition 3 containing CoV S glycoprotein SEQ ID NO: 87, two doses of composition 6 containing CoV S glycoprotein SEQ ID NO: 222, or two doses of composition 5 containing CoV S glycoprotein SEQ ID NO: 87 and CoV S glycoprotein SEQ ID NO: 222 on days 0 and 21. These doses were "priming" doses. Macacas were boosted on day 246 with composition 2 containing CoV S glycoprotein SEQ ID NO: 274. All macaques were monitored twice daily to assess vaccine safety, and no local or systemic adverse events were reported after primary series or booster vaccination.

[0369] Boost administration of Composition 2 resulted in comparable neutralization responses against pseudoviruses expressing XBB1.5, XBB1.16S, XBB.2.3, and EG.5.1 glycoproteins. Priming with compositions containing the CoV S glycoprotein of SEQ ID NO: 222 (Compositions 5 and 6) resulted in superior neutralization compared to priming with a composition lacking the CoV S glycoprotein of SEQ ID NO: 222 (Composition 3) (Figures 7A-7E). Specifically, priming with compositions containing the CoV S glycoprotein of SEQ ID NO: 222 (e.g., Compositions 5 and 6) resulted in higher neutralizing antibody titers against omicron XBB variants than priming with Composition 3. Boosting with Composition 2 also resulted in the induction of an immune response that blocked the interaction between hACE2 and the CoV S glycoprotein (Figures 9A-9C). Figure 11 shows the CD4+ T cell response in rhesus monkeys administered two doses of Composition 5 followed by a boost dose of Composition 2. Macacas primed with the bivalent vaccine (composition 5) and boosted with XBB.1.5 (composition 2) induced a Th1-biased cell response with cytokine-positive cells of comparable magnitude for all variants tested (Figure 11).

[0370] Example 4 Immunogenicity of the immunogenic compositions of Examples 2 and 3 in mice The immunogenic compositions described in Examples 2 and 3 were evaluated for immunogenicity and toxicity in a mouse model using female BALB / c mice (7-9 weeks old, Harlan Laboratories Inc., Frederick, MD). The compositions were evaluated in the presence of an adjuvant (e.g., the saponin adjuvant of Example 2). The compositions may contain 1 μg to approximately 200 μg of adjuvant and 0.01 μg to approximately 100 μg of CoV S polypeptide. The immunogenic compositions were administered intramuscularly as a single dose (also called a single priming dose) (Day 14 of the study) or as two doses (also called a prime / boost dose) with an interval of 14-21 days. The placebo group served as a non-immunizing control. Serum was collected for analysis on Days 1, 13, 21, 28, and 35 of the study. Serum was analyzed for the presence of anti-spike antibodies.

[0371] To evaluate the induction of protective immunity, immunized mice were loaded with SARS-CoV-2. Since mice do not support replication of wild-type SARS-CoV-2 virus, they were induced into tolerance by intranasal infection with an adenovirus expressing hACE2 (Ad / hACE2) 52 days after the initial vaccination. SARS-CoV-2 was loaded intranasally into immunized and control animals 42 days after one (single dose) or two (double dose) immunization. The body weight of loaded mice was measured daily on the day of infection and for up to 7 days post-infection. Five mice were sacrificed from each vaccination and control group 4 and 7 days after infection, and their lungs were collected and prepared for lung histology.

[0372] Viral titer is quantified by a plaque assay. Briefly, lung samples collected in PBS are homogenized using 1.0 mm glass beads (Sigma Aldrich) and a Beadruptor (Omini International Inc.). The homogenate is added to Vero E6 cultures near confluence, and SARS-CoV-2 viral titer is determined by counting plaque-forming units (pfus) using a 6-point dilution curve.

[0373] The effect of immunogenic compositions on inducing cellular immunity will also be evaluated. Spleens will be collected 7 days after the second immunization (day 28 of the study). A non-vaccinated group (N=3) will be used as a control.

[0374] T cell response The effect of immunogenic compositions on T cell responses will be evaluated. Antigen-specific T cell responses will be measured from spleens collected 7 days after the second immunization (day 28 of the study) using the ELISPOT® enzyme-linked immunosorbent assay and intracellular cytokine staining (ICCS). The frequency of IFN-γ+, TNF-α+, and IL-2+ cytokine-secreting CD4+ and CD8+ T cells will be assessed. The secretion of type 2 cytokines IL-4 and IL-5 from CD4+ T cells will also be determined by ICCS and ELISPOT®, respectively. Finally, the effect of immunization on germinal center formation will be evaluated by measuring the frequency of CD4+ T follicular helper (TFH) cells and germinal center (GC) B cells in the spleen.

[0375] Example 5 Immunogenicity of the immunogenic compositions of Examples 2 and 3 in Anubis baboons and cynomolgus monkeys The immunogenicity of the immunogenic compositions of Examples 2 and 3 will be evaluated in baboons and cynomolgus monkeys. Adult olive baboons and / or cynomolgus monkeys will be immunized with one of the immunogenic compositions of Example 2 and / or 3, with and without an adjuvant, in two doses 21 days apart. The anti-S protein IgG titer will be measured daily after immunization. The level of hACE2 receptor blocking antibody will also be evaluated.

[0376] The effect of immunogenic compositions on cellular immunity will also be evaluated. PBMCs will be collected 7 days after the second immunization (day 28), and the T cell response will be measured by the ELISPOT assay.

[0377] Example 6 Immunogenicity of the immunogenic compositions of Examples 2 and 3 in humans The safety and immunogenicity of the immunogenic compositions of Examples 2 and 3 will be evaluated in a Phase 1 / 2 study in adults. The first CoV S glycoprotein will be present in the composition at a concentration of approximately 3 μg to 25 μg. The second, third, fourth, and fifth CoV S glycoproteins will be present in the composition at lower concentrations. The total amounts of CoV S glycoproteins to be evaluated will be 5, 10, and 25 μg. The immunogenicity of the compositions will be evaluated in and out of the presence of an adjuvant (e.g., the saponin adjuvant in Examples 2A and 2B). Approximately 50 μg of adjuvant will be present in the composition.

[0378] Example 7 Immunogenicity of the immunogenic compositions of Examples 2 and 3 in mice The pentavalent immunogenic composition described in Example 2 and the divalent and monovalent immunogenic compositions of Example 1 will be evaluated for immunogenicity and toxicity in a BALB / c mouse model (10 mice per group). This composition contains 5 μg of saponin adjuvant. This composition contains approximately 1 μg of S protein. As a control, mice will be administered a composition containing 1 μg of one CoV S glycoprotein and 5 μg of saponin adjuvant.

[0379] The saponin adjuvant contains two iscom particles: the first iscom particle contains fraction A of Quillaya Saponaria Molina but does not contain fraction C of Quillaya Saponaria Molina, and the second iscom particle contains fraction C of Quillaya Saponaria Molina but does not contain fraction A of Quillaya Saponaria Molina. Fractions A and C account for 85% and 15% by weight, respectively, of the total weight of fractions A and C of Quillaya Saponaria Molina in the adjuvant.

[0380] Three doses of the immunogenic composition are administered to mice. The first dose is administered on day 0. The second dose is administered 14 days after the administration of the second dose (day 14). The third dose is administered 3 to 4 months after the administration of the first dose (days 104 to 134). Figure 2 shows a schematic diagram of the administration regimen.

[0381] Serum is collected from mice on day 0, day 14, day 35, between day 35 and days 104-134, and on one day between days 118-148.

[0382] The serum will be analyzed for the presence of anti-spike antibodies.

[0383] We will also evaluate the pseudovirus neutralizing ability of serum cells expressing CoV S glycoprotein.

[0384] To evaluate the induction of protective immunity, immunized mice were loaded with SARS-CoV-2. Since mice do not support replication of wild-type SARS-CoV-2 virus, they were intranasally infected with an adenovirus expressing hACE2 (Ad / hACE2) 52 days after the initial vaccination to induce tolerance. SARS-CoV-2 was intranasally loaded into both immunized and control animals 42 days after one (single dose) or two (two doses) immunization. The body weight of loaded mice was measured daily on the day of infection and for up to 7 days post-infection. Five mice from each vaccinated and control group were sacrificed 4 and 7 days after infection, and their lungs were collected and prepared for lung histology.

[0385] Viral titer is quantified by a plaque assay. Briefly, lung samples collected in PBS are homogenized using 1.0 mM glass beads (Sigma Aldrich) and a Beadruptor (Omini International Inc.). The homogenate is added to Vero E6, which is close to a confluent culture state, and SARS-CoV-2 viral titer is determined by counting plaque-forming units (pfus) using a 6-point dilution curve.

[0386] The effect of immunogenic compositions on inducing cellular immunity will also be evaluated. Spleens will be collected 7 days after the second immunization (day 28 of the experiment). A non-vaccinated group (N=3) will be used as a control.

[0387] T cell response The effect of the composition on T cell response will be evaluated. Antigen-specific T cell response will be measured by ELISPOT® enzyme-linked immunosorbent assay and intracellular cytokine staining (ICCS) from spleens collected 7 days after the second immunization (day 28 of the study). The frequency of IFN-γ+, TNF-α+, and IL-2+ cytokine-secreting CD4+ and CD8+ T cells will be assessed. Type 2 cytokines IL-4 and IL-5 secretion from CD4+ T cells will also be determined by ICCS and ELISPOT®, respectively. Finally, the effect of immunization on germinal center formation will be evaluated by measuring the frequency of CD4+ T follicular helper (TFH) cells and germinal center (GC) B cells in the spleen.

[0388] Example 8 Immunogenicity of immunogenic compositions in mice An immunogenic composition containing SARS-CoV-2 S glycoprotein and a saponin adjuvant was administered to BALB / c mice (n=10). The saponin adjuvant contained two iscom particles: the first iscom particle contained fraction A of Quillaya Saponaria Molina but not fraction C of Quillaya Saponaria Molina, and the second iscom particle contained fraction C of Quillaya Saponaria Molina but not fraction A of Quillaya Saponaria Molina. Fractions A and C accounted for 85% and 15% by weight, respectively, of the total weight of Quillaya Saponaria Molina fraction A and Quillaya Saponaria Molina fraction C in the adjuvant. The SARS-CoV-2 S glycoproteins were (i) SARS-CoV-2 S glycoprotein of SEQ ID NO: 260 (referred to as XBB.1.16), (ii) SARS-CoV-2 S glycoprotein of SEQ ID NO: 274 (referred to as XBB.1.5), (iiii) SARS-CoV-2 S glycoprotein of SEQ ID NO: 87 (referred to as the prototype), or (iv) SARS-CoV-2 S glycoprotein of SEQ ID NO: 284 (referred to as XBB.2.3). The immunogenic composition was administered according to the schedule in Figure 12. Each immunogenic composition contained 1 μg of SARS-CoV-2 S glycoprotein and 5 μg of saponin adjuvant.

[0389] Figure 13A shows anti-S IgG antibodies in mice after administration of two doses of an immunogenic composition containing SARS-CoV-2 S glycoprotein (SEQ ID NO: 274). Figure 13B shows anti-S IgG antibodies in mice after administration of two doses of an immunogenic composition containing SARS-CoV-2 S glycoprotein (SEQ ID NO: 260). Figure 14A shows that administration of two doses of an immunogenic composition containing SARS-CoV-2 S glycoprotein (SEQ ID NO: 274) results in the acquisition of antibodies that block the binding of SARS-CoV-2 to hACE2. Figure 14B shows that administration of two doses of an immunogenic composition containing SARS-CoV-2 S glycoprotein (SEQ ID NO: 260) results in the acquisition of antibodies that block the binding of SARS-CoV-2 to hACE2. Figures 15A and 16B show that administration of two doses of an immunogenic composition containing SARS-CoV-2 S glycoprotein (SEQ ID NO: 274) neutralizes pseudoviruses expressing SARS-CoV-2 S glycoprotein. Figures 15B and 16C show that administration of two doses of an immunogenic composition containing SARS-CoV-2 S glycoprotein (SEQ ID NO: 260) neutralizes pseudoviruses expressing SARS-CoV-2 S glycoprotein. Figure 16A shows the neutralizing antibody titer obtained from administration of two doses of an immunogenic composition containing SARS-CoV-2 S glycoprotein (SEQ ID NO: 87).

[0390] Example 9 Immunogenicity of immunogenic compositions in mice An immunogenic composition containing one or two SARS-CoV-2 S glycoproteins and a saponin adjuvant was administered to BALB / c mice (n=10). The saponin adjuvant contained two iscom particles: the first iscom particle contained fraction A of Quillaya Saponaria Molina but not fraction C of Quillaya Saponaria Molina, and the second iscom particle contained fraction C of Quillaya Saponaria Molina but not fraction A of Quillaya Saponaria Molina. Fractions A and C account for 85% and 15% by weight, respectively, of the total weight of Quillaya Saponaria Molina fraction A and Quillaya Saponaria Molina fraction C in the adjuvant. The immunogenic compositions were administered according to the schedule in Figure 17. On days 0 and 14, mice were administered the first and second doses of the first immunogenic composition (referred to as the "primary series"). On day 44, mice were administered a boost dose of the second immunogenic composition (referred to as the "1-month booster").

[0391] Each of the first immunogenic compositions contained 1 μg of total SARS-CoV-2 S glycoprotein and 5 μg of saponin adjuvant. When two glycoproteins were present in the first immunogenic composition, each glycoprotein was present in equal amounts in the composition. The first composition contained saponin adjuvant and (i) SARS-CoV-2 S glycoprotein of SEQ ID NO: 227 (referred to as BQ.1.1), (ii) SARS-CoV-2 S glycoprotein of SEQ ID NO: 274 (referred to as XBB.1.5), (iii) the first SARS-CoV-2 S glycoprotein of SEQ ID NO: 227 and the second SARS-CoV-2 S glycoprotein of SEQ ID NO: 274 (referred to as BQ.1.1 + XBB.1.5), or (iv) the first SARS-CoV-2 S glycoprotein of SEQ ID NO: 222 and the second SARS-CoV-2 S glycoprotein of SEQ ID NO: 274 (referred to as BA.5 + XBB.1.5). The one-month booster contained a saponin adjuvant and either (i) SARS-CoV-2 S glycoprotein (XBB.1.5) of SEQ ID NO: 274 or (ii) SARS-CoV-2 S glycoprotein (XBB.1.16) of SEQ ID NO: 260, and the immunogenic composition was administered according to the schedule in Figure 17.

[0392] Figure 18A shows anti-S IgG antibodies in the serum of mice after administration of two doses of an immunogenic composition containing SARS-CoV 2 S glycoprotein of SEQ ID NO: 274. Figure 18B shows neutralizing antibodies present in the serum of mice after administration of two doses of an immunogenic composition containing SARS-CoV 2 S glycoprotein of SEQ ID NO: 274. Figure 19A shows the antigen map of the neutralization reaction induced by immunization with two doses of an immunogenic composition containing SARS-CoV 2 S glycoprotein of SEQ ID NO: 274. Figure 19B shows the antigen map of the neutralization reaction induced by boost administration of an immunogenic composition containing SARS-CoV 2 S glycoprotein of SEQ ID NO: 274. Figure 19C shows the antigen map of the neutralization reaction induced by boost administration of an immunogenic composition containing SARS-CoV 2 S glycoprotein of SEQ ID NO: 260. Figure 20A shows the pseudovirus neutralizing titer induced by administration of SARS-CoV-2 S glycoprotein of SEQ ID NO: 274 in a primary series. Figure 20B shows the pseudovirus neutralizing titer induced by administration of SARS-CoV-2 S glycoprotein of SEQ ID NO: 274 in a 1-month boost regimen. Figure 20C shows the pseudovirus neutralizing titer induced by administration of SARS-CoV-2 S glycoprotein of SEQ ID NO: 260 in a 1-month boost regimen.

[0393] Example 10 Immunogenicity of immunogenic compositions in rhesus monkeys An immunogenic composition containing one or two SARS-CoV-2 S glycoproteins and a saponin adjuvant was administered to rhesus monkeys (n=5). The saponin adjuvant contained two iscom particles, the first of which contained fraction A of Quillaya Saponaria Molina but not fraction C of Quillaya Saponaria Molina, and the second of which contained fraction C of Quillaya Saponaria Molina but not fraction A of Quillaya Saponaria Molina. Fractions A and C account for 85% and 15% by weight, respectively, of the total weight of Quillaya Saponaria Molina fraction A and Quillaya Saponaria Molina fraction C in the adjuvant. The immunogenic compositions were administered according to the schedule in Figure 21. The first and second doses of the first immunogenic composition were administered to macaques on days 0 and 21 (referred to as the "primary series"). A boost dose of the second immunogenic composition was administered to macaques on day 246 (referred to as the "booster").

[0394] The first and second immunogenic compositions each contained 5 μg of SARS-CoV-2 S glycoprotein and 50 μg of saponin adjuvant. The first composition contained saponin adjuvant and (i) SARS-CoV-2 S glycoprotein of SEQ ID NO: 87 (referred to as prototype), (ii) SARS-CoV-2 S glycoprotein of SEQ ID NO: 222 (referred to as BA.5), (iii) SARS-CoV-2 S glycoprotein of SEQ ID NO: 87 and SARS-CoV-2 S glycoprotein of SEQ ID NO: 222 (referred to as "prototype + BA.5"), or (iv) SARS-CoV-2 S glycoprotein of SEQ ID NO: 274 (referred to as XBB.1.5). The second immunogenic composition contained a saponin adjuvant and (i) SARS-CoV-2 S glycoprotein of Sequence ID No. 274 (referred to as XBB.1.5") or (ii) SARS-CoV-2 S glycoprotein of Sequence ID No. 227 (referred to as BQ.1.1).

[0395] Figure 22A shows anti-S IgG antibodies in macaque serum after administration of a first and second dose containing SARS-CoV-2 S glycoprotein of SEQ ID NO: 87 and a third dose containing SARS-CoV-2 S glycoprotein of SEQ ID NO: 274. Figure 22B shows anti-S IgG antibodies in macaque serum after administration of a first and second dose containing SARS-CoV-2 S glycoprotein of SEQ ID NO: 222 and a third dose containing SARS-CoV-2 S glycoprotein of SEQ ID NO: 274. Figure 22C shows anti-S IgG antibodies in macaque serum after administration of a first and second dose containing SARS-CoV-2 S glycoprotein of SEQ ID NO: 87 and SARS-CoV-2 S glycoprotein of SEQ ID NO: 222 and a third dose containing SARS-CoV-2 S glycoprotein of SEQ ID NO: 274. Figure 23 shows anti-S IgG antibodies in macaque serum after administration of first and second doses containing SARS-CoV-2 S glycoprotein of SEQ ID NO: 274. Figure 24 shows the pseudovirus neutralizing titer induced by administration of SARS-CoV-2 S glycoprotein of SEQ ID NO: 274 in a primary series. Figure 25 shows the CD4+ T cell response of macaques administered with first and second doses containing SARS-CoV-2 S glycoprotein of SEQ ID NO: 274. Figure 26 shows the multifunctional antigen-specific CD4+ T cell response of macaques administered with first and second doses containing SARS-CoV-2 S glycoprotein of SEQ ID NO: 274.

[0396] Example 11 Immunogenicity of immunogenic compositions in mice A monovalent COVID-19 immunogenic composition containing one CoV S glycoprotein was prepared. A trivalent COVID-19 immunogenic composition containing three CoV S glycoproteins was also prepared. The immunogenicity of the following immunogenic compositions was evaluated. Table C4 shows the identity of the CoV S glycoprotein in each composition.

[0397] [Table 73]

[0398] Each monovalent COVID-19 immunogenic composition (compositions 2, 8-9) was prepared with 20 μg / mL CoV S rS + 100 μg / mL MATRIX-M, and 1 μg of CoV S rS was delivered with a 5 μg MATRIX-M dose in 50 μL. A trivalent COVID-19 immunogenic composition (composition 7) was prepared with 20 μg / mL / each CoV S rS (60 μg / mL total CoV S rS) + 100 μg / mL MATRIX-M, and 1 μg of CoV S rS / strain (total 3 μg rS) was delivered with a 5 μg Matrix-M dose in 50 μL.

[0399] Intramuscular inoculation was performed on a group of mice (n=20) in the following manner, as shown in Figure 27.

[0400] [Table 74]

[0401] Following administration of composition 2, composition 7, or composition 8, a booster dose of composition 8 was administered, which induced cross-reactive pseudovirus neutralizing antibodies against SARS-CoV-2 variant strains XBB.1.5, HV.1, JN.1, JN4, JN.1.11.1, JN.1.7, JN.1.16, and JN.1.13.1 (Figures 31, 32D, 33C, 34D, and 35B).

[0402] Figure 28A shows anti-rS IgG antibodies in the serum of mice after administration of two doses of an immunogenic composition containing SARS-CoV-2 S glycoprotein of SEQ ID NO: 274. Figure 28B shows neutralizing antibodies present in the serum of mice after administration of two doses of an immunogenic composition containing SARS-CoV-2 S glycoprotein of SEQ ID NO: 329. Figure 29A shows the pseudovirus neutralizing titer induced by administration of SARS-CoV-2 S glycoprotein of SEQ ID NO: 274 in a primary series. Figure 29B shows the pseudovirus neutralizing titer induced by administration of SARS-CoV-2 S glycoprotein of SEQ ID NO: 329 in a primary series.

[0403] Figure 30 shows the pseudovirus neutralizing titers and antigenic distances induced by administering SARS-CoV-2 S glycoprotein of Sequence ID No. 274 to different SARS-CoV-2 variant strains in a primary series at low titers for JN.1 and JN.1 subvariants. Specifically, the pseudovirus neutralizing titers and antigenic distances after the primary vaccination series with Composition 2 were JN.1 (pVNT50=55, AU=6.582), JN.4 (pVNT50=51, AU=6.684), JN.1.11.1 (pVNT50=58, AU=6.515), JN.1.7 (pVNT50=56, AU=6.568), and JN.1.13.1 (pVNT50=58, AU=6.500). Figure 31 shows the pseudovirus neutralizing titers and antigenic distances induced by administering SARS-CoV-2 S glycoprotein SEQ ID NO: 329 in a primary series to different SARS-CoV-2 variant strains, with higher titers for JN.1 and the JN.1 subvariant. Specifically, the pseudovirus neutralizing titers and antigenic distances after the primary vaccination series with composition 8 were lower for JN.1 (pVNT50=11,215, AU=-) and JN.4 (pVNT50=11,215), compared to the lower responses for the XBB.1.5 (pVNT50=50, AU=7.81) and HV.1 (pVNT50=56, AU=7.65) subvariants. The results were as follows: 50=7,185, AU=0.642), JN.1.11.1 (pVNT50=7,650, AU=0.552), JN.1.7 (pVNT50=7,001, AU=0.680), JN.1.13.1 (pVNT50=14,583, AU=-0.379), and JN.1.16 (pVNT50=8,140, ​​AU=0.462) (Figure 31).

[0404] Figures 32A-32D show the anti-rS IgG antibodies in the serum of mice after administration of composition 2 in the primary series (Figure 32A), compared to the anti-rS IgG antibodies in the serum of mice before administration of the boost dose (Figure 32B), with the anti-rS IgG antibodies in the serum of mice induced after administration of a boost dose containing composition 2 (Figure 32C) or composition 8 (Figure 32D).

[0405] Figures 33A–33C show the pseudovirus neutralizing titers and antigenic distances induced by the administration of SARS-CoV-2 S glycoprotein of SEQ ID NO: 274 in the primary series in Example 11, followed by the administration of a boost dose containing Composition 8 (Figure 33C). The boost dose of Composition 8 (Figure 33C) induced an improved immune response to pseudoviruses expressing S proteins from various heterologous SARS-CoV-2 strains compared to pre-boosted mice (Figure 33B) administered with the primary dose of Composition 2 (Figure 33A).

[0406] Figures 34A-34B show the pseudovirus neutralizing titers and antigenic distances induced by administration of composition 2 in the primary series in Example 11 (Figure 34A), followed by a boost dose containing composition 8 at two months later (Figure 34B). The boost with composition 8 induced an improved immune response against pseudoviruses expressing S proteins from various heterologous SARS-CoV-2 strains (Figure 34B). Specifically, the pseudovirus neutralizing titers and antigenic distances after the primary vaccination series with composition 2, followed by a booster dose of composition 8 at 2 months, were JN.1 (pVNT50=2839, AU=-), JN.1.11.1 (pVNT50=2204, AU=0.365), JN.1.7 (pVNT50=2118, AU=0.423), JN.1.13.1 (pVNT50=2131, AU=0.414), XBB.1.5 (pVNT50=46668, AU=-4.04), and HV.1 (pVNT50=31899, AU=-3.49) (Figure 34B).

[0407] Figures 35A-35B show the pseudovirus neutralizing titers and antigenic distances induced by administration of composition 7 (Figure 35A) in a primer series in Example 11, followed by administration of a boost dose containing composition 8 two months later (Figure 35B). The boost dose of composition 8 (Figure 35B) induced an improved immune response against pseudoviruses expressing S proteins from various heterologous SARS-CoV-2 strains. Specifically, the pseudovirus neutralizing titers and antigenic distances after a primary vaccination series with composition 7, followed by a 2-month booster dose of composition 8, were JN.1 (pVNT50=1219, AU=-), JN.1.11.1 (pVNT50=1836, AU=-0.591), JN.1.7 (pVNT50=1434, AU=-0.234), XBB.1.5 (pVNT50=30035, AU=-4.62), and HV.1 (pVNT50=18341, AU=-3.91).

[0408] Figure 36 shows the Th1 / Th2 cytokine ratio produced by CD4+ T cells in mice administered with the dose of Composition 2, followed by a boost dose of Composition 2 over two months. Figure 37 shows the Th1 / Th2 cytokine ratio produced by CD4+ T cells in mice administered with the dose of Composition 2, followed by a boost dose of Composition 8 over two months. Th1 (IFN-γ, IL-2, and TNF-α) and Th2 (IL-4) cytokine-producing CD4+ T cells were measured in spleen cells isolated two months after booster doses of either Composition 2 or Composition 8. Robust CD4+ T cell responses reproduced equivalent post-boosted levels with the boost of Composition 8.

[0409] The effect of immunogenic compositions on T cell responses will be evaluated. Antigen-specific T cell responses will be measured by ELISPOT® enzyme-linked immunosorbent assay and intracellular cytokine staining (ICCS) from spleens collected after a booster dose of composition 2 or composition 8 (day 90 of the study). The frequency of IFN-γ+, TNF-α+, and IL-2+ cytokine-secreting CD4+ and CD8+ T cells will be evaluated (Figures 38A-38B, 39A-39B, 40A-40B). Finally, the effect of immunization on germinal center formation will be evaluated by measuring the frequency of CD4+ T follicular helper (TFH) cells and germinal center (GC) B cells in the spleen (Figures 41A-41B).

[0410] Example 12 Immunogenicity of immunogenic compositions in rhesus monkeys An immunogenic composition containing one or two SARS-CoV-2 S glycoproteins and MATRIX-M was administered to rhesus monkeys (n=5). The immunogenic compositions were administered according to the schedule in Figure 42. For macaques, the first and second doses of the first immunogenic composition were administered on days 0 and 21, respectively (referred to as the "primary series"). For macaques, a boost dose of the second immunogenic composition was administered on day 211 (referred to as the "first booster"). Subsequently, the second boost dose of the third immunogenic composition was administered to the primed macaques on day 346 (referred to as the "second booster").

[0411] The immunogenic composition for intramuscular administration contained 10 μg / mL of SARS-CoV-2 S glycoprotein and 100 μg / mL of MATRIX-M w, respectively, to deliver 5 μg of rS along with a 50 μg dose of Matrix-M in a 500 μL injection via the intramuscular (IM) pathway. The immunogenic composition for IN administration contained 50 μg / mL of SARS-CoV-2 S glycoprotein and 100 μg / mL of MATRIX-M, respectively, to deliver 25 μg of rS along with a 50 μg dose of Matrix-M in a 500 μL injection via the IN pathway. The first and second compositions contained Matrix-M and SARS-CoV-2 S glycoprotein of SEQ ID NO: 274 (referred to as XBB.1.5). The third immunogenic composition contained Matrix-M and SARS-CoV-2 S glycoprotein of SEQ ID NO: 329 (referred to as JN.1).

[0412] Figure 43A shows the anti-rS response in primed rhesus monkeys before administration of a second booster dose of the immunogenic composition containing SARS-CoV 2 S glycoprotein of SEQ ID NO: 329. Figure 43B shows the anti-rS response in primed rhesus monkeys after administration of a second booster dose of the immunogenic composition containing SARS-CoV 2 S glycoprotein of SEQ ID NO: 329.

[0413] Figure 44A shows the pseudovirus neutralizing titer and antigenic distance in primed rhesus monkeys before administration of a second booster dose of the immunogenic composition containing SARS-CoV 2 S glycoprotein of SEQ ID NO: 329. Figure 44B shows the pseudovirus neutralizing titer and antigenic distance in primed rhesus monkeys after administration of a second booster dose of the immunogenic composition containing SARS-CoV 2 S glycoprotein of SEQ ID NO: 329. The second booster dose of the immunogenic composition containing SARS-CoV 2 S glycoprotein of SEQ ID NO: 329 induced pseudovirus neutralizing antibodies against different SARS-CoV-2 variant strains.

[0414] Figure 45 shows the CD4+ T cell IFN-γ response in primed rhesus monkeys after administration of a second booster dose of the immunogenic composition containing SARS-CoV 2 S glycoprotein of SEQ ID NO: 329. The second booster dose of the immunogenic composition containing SARS-CoV 2 S glycoprotein of SEQ ID NO: 329 induced a recognized Th1-biased cell response and conserved T cell epitopes for all variants tested. Figure 46 shows the multifunctional antigen-specific CD4+ T cell response in primed rhesus monkeys after administration of a second booster dose of the immunogenic composition containing SARS-CoV 2 S glycoprotein of SEQ ID NO: 329.

[0415] Enumeration of embodiments of this disclosure The specific enumerated embodiments provided below <1> ~ <199> These are illustrative examples only and do not otherwise limit the scope of the subject matter of this disclosure as defined by the claims. These listed embodiments encompass all combinations, partial combinations and multiple referenced (e.g., multiple dependent) combinations described herein. 1. An immunogenic composition comprising a first CoV S glycoprotein having an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with any of SEQ ID NOs. 329 to 333. 2. The first CoV S glycoprotein comprises an immunogenic composition of Embodiment 1, as listed above, including an inactive furin cleavage site. 3.0.An immunogenic composition according to any one of the listed embodiments 1 and 2, comprising 1 μg to 25 μg, 0.1 μg to 10 μg, 0.001 μg to 10 μg, 0.001 μg to 25 μg, or 0.1 μg to 5 μg of a first CoV S glycoprotein. 4. The immunogenic composition according to Embodiment 1, as listed above, comprising nanoparticles containing a first CoV S glycoprotein and a nonionic surfactant core. 5. The immunogenic composition according to Embodiment 4, wherein the nonionic surfactant is selected from the group consisting of polysorbate-20 (PS20), polysorbate-40 (PS40), polysorbate-60 (PS60), polysorbate-65 (PS65), and polysorbate-80 (PS80). 6. The immunogenic composition according to Embodiment 1, further comprising an adjuvant and a pharmaceutically acceptable carrier. 7. The immunogenic composition according to Embodiment 8, wherein the adjuvant is a saponin adjuvant. 8. The saponin adjuvant contains at least two iscom particles. The first iscom particle contains fraction A of Quillaya Saponaria Molina, but does not contain fraction C of Quillaya Saponaria Molina, and The immunogenic composition according to the enumerated embodiment 7, wherein the second iscom particle comprises fraction C of Quillaya Saponaria Molina and does not contain fraction A of Quillaya Saponaria Molina. 9. The immunogenic composition according to Embodiment 8, wherein fraction A of Quillaya Saponaria Molina and fraction C of Quillaya Saponaria Molina constitute about 85% by weight and about 15% by weight, respectively, of the total weight of fraction A of Quillaya Saponaria Molina and fraction C of Quillaya Saponaria Molina in the saponin adjuvant. 10. The immunogenic composition according to Embodiment 8, wherein fraction A of Quillaya Saponaria Molina and fraction C of Quillaya Saponaria Molina constitute about 92% by weight and about 8% by weight, respectively, of the total weight of fraction A of Quillaya Saponaria Molina and fraction C of Quillaya Saponaria Molina in the saponin adjuvant. 11. The immunogenic composition according to the enumerated Embodiment 8, wherein, of the total weight of fraction A and fraction C of Quillaya Saponaria Molina in the saponin adjuvant, fraction A of Quillaya Saponaria Molina accounts for at least about 85% by weight, and fraction C of Quillaya Saponaria Molina accounts for the remainder. 12. The immunogenic composition according to the enumerated Embodiment 8, wherein, of the total weight of fraction A and fraction C of Quillaya Saponaria Molina in the saponin adjuvant, fraction A of Quillaya Saponaria Molina accounts for 50-96% by weight, and fraction C of Quillaya Saponaria Molina accounts for the remainder. 13. An immunogenic composition according to Embodiment 6, comprising approximately 25 μg to approximately 100 μg of an adjuvant. 14. An immunogenic composition according to the listed embodiment 13, comprising approximately 50 μg or 75 μg of an adjuvant. 15. The immunogenic composition according to Embodiment 1, wherein the first CoV S glycoprotein comprises one or more modifications at amino acids 59, 346, 456, 475, 572 and 1087 compared to SEQ ID NO: 329, and the one or more modifications are numbered according to SEQ ID NO: 1. The amino acid in 16.(i)59 is phenylalanine or serine. (ii) The amino acid in 346 is arginine or threonine. (iii) The amino acid in 456 is phenylalanine or leucine. (iv) The amino acid in 475 is alanine or valine. The amino acid in (v)572 is threonine or isoleucine, and (vi) The immunogenic composition according to the listed embodiment 15, wherein the amino acid in 1087 is alanine or serine. 17. An immunogenic composition according to any of the listed embodiments 1 to 16, contained in a syringe. 18. A method for stimulating an immune response in a human to SARS-CoV-2 or a heterologous SARS-CoV-2 strain, comprising administering to a human an immunogenic composition described in any one of the listed embodiments 1 to 16. 19. A method for enhancing the immune response to SARS-CoV-2 or a heterologous SARS-CoV-2 strain in humans, comprising administering a first immunogenic composition comprising (a) one or more first SARS-CoV-2 S glycoproteins having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 329, and (b) a pharmaceutically acceptable buffer. A method comprising administering an immunogenic composition after administration of another immunogenic composition intended to induce an immunogenic response in humans to SARS-CoV-2 or a heterologous SARS-CoV-2 strain. 20. The method according to the listed embodiment 19, comprising administering a first dose of the first immunogenic composition at least 21 days after administration of another immunogenic composition. 21. The method according to one of the listed embodiments 20, comprising administering the first immunogenic composition at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 months after administration of another immunogenic composition. 22. The method according to any one of the listed embodiments 19 to 21, comprising administering the first immunogenic composition about 1 to 18 months, about 6 to 18 months, about 9 to 18 months, about 12 to 15 months, or about 12 to 18 months after administration of another immunogenic composition. 23. The method according to any one of the listed embodiments 19 to 22, wherein the other immunogenic composition comprises mRNA encoding the SARS-CoV-2 spike glycoprotein, plasmid DNA encoding the SARS-CoV-2 spike glycoprotein, a viral vector encoding the SARS-CoV-2 spike glycoprotein, an inactivated SARS-CoV-2 virus, or a protein subunit vaccine. 24. The method according to the enumerated embodiment 23, wherein the protein subunit vaccine of another immunogenic composition comprises at least one second SARS-CoV-2 S glycoprotein having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with any of SEQ ID NOs. 25. The method according to the listed embodiment 24, wherein at least one second SARS-CoV-2 S glycoprotein comprises at least two SARS-CoV-2 S glycoproteins having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with any of SEQ ID NOs: 87, 260, 222, 227, 274, and 284 and 329. 26. A method for amplifying the immune response to SARS-CoV-2 or a heterologous SARS-CoV-2 strain in humans, comprising administering a first immunogenic composition comprising (a) one or more first SARS-CoV-2 S glycoproteins having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 329, and (b) a pharmaceutically acceptable buffer. A method comprising administering an immunogenic composition after administration of another immunogenic composition intended to induce an immunogenic response to SARS-CoV-2 or a heterologous SARS-CoV-2 strain in humans, wherein the other immunogenic composition does not contain SARS-CoV-2 S glycoprotein having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 329. 27. The method according to the enumerated embodiment 26, wherein the other immunogenic composition comprises one of the mRNA vaccine and one of the protein subunit vaccine. 28. The method according to the enumerated embodiment 27, wherein the other immunogenic composition comprises a second CoV S glycoprotein having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with any one of SEQ ID NOs: 87, 260, 222, 227, 274, and 284. 29. A method for inducing an immune response in a human to SARS-CoV-2 or a heterologous SARS-CoV-2 strain thereof, comprising administering a first immunogenic composition comprising (a) one or more first SARS-CoV-2 S glycoproteins having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 329, and (b) a pharmaceutically acceptable buffer. 30.(a) The method according to the enumerated embodiment 29, further comprising administering a second immunogenic composition comprising one or more second SARS-CoV-2 S glycoproteins having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with any of SEQ ID NOs: 87, 260, 222, 227, 274, and 284 and 329. 31. The method according to the listed embodiment 30, comprising administering a second immunogenic composition at least 21 days after administration of a first immunogenic composition. 32. The method according to one of the listed embodiments 31, comprising administering a second immunogenic composition at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 months after administration of a first immunogenic composition. 33. The method according to any one of the listed embodiments 30 to 32, comprising administering a second immunogenic composition about 1 to 18 months, about 6 to 18 months, about 9 to 18 months, about 12 to 15 months, or about 12 to 18 months after administration of a first immunogenic composition. 34. (i) The first coronavirus spike (CoV S) glycoprotein, (ii) A second CoV S glycoprotein having 1 to about 2, 1 to about 3, 1 to about 4, 1 to about 5, 2 to about 5, 1 to about 6, 1 to about 7, 1 to about 8, 1 to about 9, 1 to about 10, 1 to about 11, 1 to about 12, 1 to about 13, 1 to about 14, 1 to about 15, 1 to about 16, 1 to about 17, 1 to about 18, 1 to about 18, 1 to about 19 or 1 to about 20, 1 to about 25, 1 to about 30, 1 to about 35, 1 to about 40, 1 to about 45 or 1 to about 50 modifications compared to the first CoV S glycoprotein, (iii) A third CoV S glycoprotein having 1 to about 2, 1 to about 3, 1 to about 4, 1 to about 5, 2 to about 5, 1 to about 6, 1 to about 7, 1 to about 8, 1 to about 9, 1 to about 10, 1 to about 11, 1 to about 12, 1 to about 13, 1 to about 14, 1 to about 15, 1 to about 16, 1 to about 17, 1 to about 18, 1 to about 18, 1 to about 19 or 1 to about 20, 1 to about 25, 1 to about 30, 1 to about 35, 1 to about 40, 1 to about 45 or 1 to about 50 modifications compared to the first CoV S glycoprotein. An immunogenic composition comprising the second and third CoV S glycoproteins having different amino acid sequences. 35. The immunogenic composition according to the listed embodiment 34, wherein the first CoV S glycoprotein contains an inactive furin cleavage site and mutations at amino acids 973 and 974, and the amino acid sequence of the CoV S glycoprotein is numbered according to SEQ ID NO: 2. 36. A fourth CoV S glycoprotein containing 1 to approximately 2, 1 to approximately 3, 1 to approximately 4, 1 to approximately 5, 2 to approximately 5, 1 to approximately 6, 1 to approximately 7, 1 to approximately 8, 1 to approximately 9, 1 to approximately 10, 1 to approximately 11, 1 to approximately 12, 1 to approximately 13, 1 to approximately 14, 1 to approximately 15, 1 to approximately 16, 1 to approximately 17, 1 to approximately 18, 1 to approximately 18, approximately 1 to approximately 19 or 1 to approximately 20, 1 to approximately 25, 1 to approximately 30, 1 to approximately 35, 1 to approximately 40, 1 to approximately 45 or 1 to approximately 50 modifications compared to the first CoV S glycoprotein. The second, third, and fourth CoV S glycoproteins are immunogenic compositions according to the listed embodiments 34 or 35, having different amino acid sequences. 37. A fifth CoV S glycoprotein containing 1 to approximately 2, 1 to approximately 3, 1 to approximately 4, 1 to approximately 5, 2 to approximately 5, 1 to approximately 6, 1 to approximately 7, 1 to approximately 8, 1 to approximately 9, 1 to approximately 10, 1 to approximately 11, 1 to approximately 12, 1 to approximately 13, 1 to approximately 14, 1 to approximately 15, 1 to approximately 16, 1 to approximately 17, 1 to approximately 18, 1 to approximately 18, approximately 1 to approximately 19 or 1 to approximately 20, 1 to approximately 25, 1 to approximately 30, 1 to approximately 35, 1 to approximately 40, 1 to approximately 45 or 1 to approximately 50 modifications compared to the first CoV S glycoprotein. The second, third, fourth, and fifth CoV S glycoproteins have different amino acid sequences, and the immunogenic composition is as described in any one of the listed embodiments 34 to 36. 38. An immunogenic composition according to any one of the listed embodiments 34 to 37, wherein one or more of the second, third, fourth, and fifth CoV S glycoproteins include modifications to the receptor-binding domain (RBD). 39. An immunogenic composition according to any one of the listed embodiments 34 to 37, wherein one or more of the second, third, fourth, and fifth CoV S glycoproteins comprises one or more modifications to the S1 subunit. 40. An immunogenic composition according to any one of the listed embodiments 34 to 39, wherein one or more of the second, third, fourth, and fifth CoV S glycoproteins comprises one or more modifications to the S2 subunit. 41. An immunogenic composition according to any one of the listed embodiments 34 to 40, wherein one or more of the second, third, fourth, and fifth CoV S glycoproteins have one or more modifications to their N-terminal domain (NTD). 42. An immunogenic composition according to any one of the listed embodiments 34 to 41, wherein one or more of the second, third, fourth, and fifth CoV S glycoproteins comprises one or more modifications to any one of amino acids 630 to 710, any one of amino acids 640 to 700, any one of amino acids 650 to 690, or any one of amino acids 660 to 680, and the amino acids are numbered according to Sequence ID No. 2. 43. The immunogenic composition according to any one of the listed embodiments 35 to 42, wherein the inactive furin cleavage site of the first CoV S glycoprotein comprises the amino acid sequence QQAQ (SEQ ID NO: 7), and amino acids 973 and 974 are proline. 44. An immunogenic composition according to any one of the listed embodiments 34 to 43, wherein one or more of the second, third, fourth, or fifth CoV S glycoproteins comprises an inactive furin cleavage site and mutations in amino acids 973 and 974, the amino acid sequences of the second, third, fourth, and fifth CoV S glycoproteins are numbered according to SEQ ID NO: 2, and optionally, the inactive furin cleavage site comprises the amino acid sequence QQAQ (SEQ ID NO: 7), and amino acids 973 and 974 are proline. 45. The first CoV S glycoprotein is at least 90%, at least 91%, at least 92%, and less than one of the following sequence numbers: 86, 87, 88, 89, 105, 106, 109, 110, 112, 113, 115, 130, 132-134, 140-141, 143-144, 146, 147, 149, 151-153, 155-158, 174, 175, 186, 188, 190, 195, 217-228, 233-236, 243, and 245-292. An immunogenic composition according to any one of the listed embodiments 34 to 44, comprising an amino acid sequence that is at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% identical. 46. ​​The first CoV S glycoprotein is at least 90%, at least 91%, at least 92%, and less than one of the following sequence numbers: 86, 87, 88, 89, 105, 106, 109, 110, 112, 113, 115, 130, 132-134, 140-141, 143-144, 146, 147, 149, 151-153, 155-158, 174, 175, 186, 188, 190, 195, 217-228, 233-236, 243, and 245-292. An immunogenic composition according to any one of the listed embodiments 34 to 45, comprising an amino acid sequence that is at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% identical. 47. The second CoV S glycoprotein is at least 90%, at least 91%, at least 92%, and less than one of the following sequence numbers: 86, 87, 88, 89, 105, 106, 109, 110, 112, 113, 115, 130, 132-134, 140-141, 143-144, 146, 147, 149, 151-153, 155-158, 174, 175, 186, 188, 190, 195, 217-228, 233-236, 243, and 245-292. An immunogenic composition according to any one of the listed embodiments 34 to 46, comprising an amino acid sequence that is at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% identical. 48. The third CoV S glycoprotein is at least 90%, at least 91%, at least 92%, and less than one of the following sequence numbers: 86, 87, 88, 89, 105, 106, 109, 110, 112, 113, 115, 130, 132-134, 140-141, 143-144, 146, 147, 149, 151-153, 155-158, 174, 175, 186, 188, 190, 195, 217-228, 233-236, 243, and 245-292. An immunogenic composition according to any one of the listed embodiments 34 to 47, comprising an amino acid sequence that is at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% identical. 49. The fourth CoV S glycoprotein is at least 90%, at least 91%, at least 92%, and less than one of the following sequence numbers: 86, 87, 88, 89, 105, 106, 109, 110, 112, 113, 115, 130, 132-134, 140-141, 143-144, 146, 147, 149, 151-153, 155-158, 174, 175, 186, 188, 190, 195, 217-228, 233-236, 243, and 245-292. An immunogenic composition according to any one of the listed embodiments 36 to 48, comprising an amino acid sequence that is at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% identical. 50. The fifth CoV S glycoprotein is at least 90%, at least 91%, at least 92%, and less than one of the following sequence numbers: 86, 87, 88, 89, 105, 106, 109, 110, 112, 113, 115, 130, 132-134, 140-141, 143-144, 146, 147, 149, 151-153, 155-158, 174, 175, 186, 188, 190, 195, 217-228, 233-236, 243, and 245-292. An immunogenic composition according to any one of the listed embodiments 37 to 49, comprising an amino acid sequence that is at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% identical. 51. An immunogenic composition according to any one of the listed embodiments 37 to 50, wherein each of the second, third, fourth, and fifth CoV S glycoproteins contains one modification to the RBD compared to the first CoV S glycoprotein. 52. The immunogenic composition according to the listed embodiment 51, wherein the modification in RBD is one or more of the P508S mutation, K431T mutation, F473S mutation, or T465K mutation, and the amino acid sequence of each CoV S glycoprotein is numbered according to SEQ ID NO: 2. 53. An immunogenic composition according to any one of the listed embodiments 34 to 52, comprising approximately 3 μg to approximately 7 μg of a first CoV S glycoprotein. 54. An immunogenic composition according to any one of the listed embodiments 34 to 53, comprising approximately 1 ng to approximately 1 μg or approximately 3 μg to approximately 7 μg of a second CoV S glycoprotein. 55. An immunogenic composition according to any one of the listed embodiments 34 to 54, comprising approximately 1 ng to approximately 1 μg or approximately 3 μg to approximately 7 μg of a third CoV S glycoprotein. 56. An immunogenic composition according to any one of the listed embodiments 37 to 53, comprising approximately 1 ng to approximately 1 μg or approximately 3 μg to approximately 7 μg of the fourth CoV S glycoprotein. 57. An immunogenic composition according to any one of the listed embodiments 37 to 53, comprising approximately 1 ng to approximately 1 μg or approximately 3 μg to approximately 7 μg of the fifth CoV S glycoprotein. 58. (i) A first nanoparticle containing the first CoV S glycoprotein, (ii) A second nanoparticle comprising a second CoV S glycoprotein and a third CoV S glycoprotein, wherein each of the second CoV S glycoprotein and the third CoV S glycoprotein contains 1 to about 2, 1 to about 3, 1 to about 4, 1 to about 5, 2 to about 5, 1 to about 6, 1 to about 7, 1 to about 8, 1 to about 9, 1 to about 10, 1 to about 11, 1 to about 12, 1 to about 13, 1 to about 14, 1 to about 15, 1 to about 16, 1 to about 17, 1 to about 18, 1 to about 18, 1 to about 19 or 1 to about 20, 1 to about 25, 1 to about 30, 1 to about 35, 1 to about 40, 1 to about 45 or 1 to about 50 modifications compared to the first CoV S glycoprotein. An immunogenic composition containing [a specific substance]. 59. The immunogenic composition according to the listed embodiment 25, wherein the first CoV S glycoprotein contains an inactive furin cleavage site and mutations at amino acids 973 and 974, and the amino acid sequence of the CoV S glycoprotein is numbered according to SEQ ID NO: 2. 60. A fourth CoV S glycoprotein containing 1 to approximately 2, 1 to approximately 3, 1 to approximately 4, 1 to approximately 5, 2 to approximately 5, 1 to approximately 6, 1 to approximately 7, 1 to approximately 8, 1 to approximately 9, 1 to approximately 10, 1 to approximately 11, 1 to approximately 12, 1 to approximately 13, 1 to approximately 14, 1 to approximately 15, 1 to approximately 16, 1 to approximately 17, 1 to approximately 18, 1 to approximately 18, 1 to approximately 19 or 1 to approximately 20, 1 to approximately 25, 1 to approximately 30, 1 to approximately 35, 1 to approximately 40, 1 to approximately 45 or 1 to approximately 50 modifications compared to the first CoV S glycoprotein. The second, third, and fourth CoV S glycoproteins are immunogenic compositions according to the listed embodiments 58 or 59, having different amino acid sequences. 61. A fifth CoV S glycoprotein containing 1 to approximately 2, 1 to approximately 3, 1 to approximately 4, 1 to approximately 5, 2 to approximately 5, 1 to approximately 6, 1 to approximately 7, 1 to approximately 8, 1 to approximately 9, 1 to approximately 10, 1 to approximately 11, 1 to approximately 12, 1 to approximately 13, 1 to approximately 14, 1 to approximately 15, 1 to approximately 16, 1 to approximately 17, 1 to approximately 18, 1 to approximately 18, 1 to approximately 19 or 1 to approximately 20, 1 to approximately 25, 1 to approximately 30, 1 to approximately 35, 1 to approximately 40, 1 to approximately 45 or 1 to approximately 50 modifications compared to the first CoV S glycoprotein. The second, third, fourth, and fifth CoV S glycoproteins have different amino acid sequences, and the immunogenic composition is as described in any one of the listed embodiments 58 to 60. 62. An immunogenic composition according to any one of the listed embodiments 58 to 61, wherein one or more of the second, third, fourth, and fifth CoV S glycoproteins include modifications to the receptor-binding domain (RBD). 63. An immunogenic composition according to any one of the listed embodiments 58 to 62, wherein one or more of the second, third, fourth, and fifth CoV S glycoproteins comprises one or more modifications to the S1 subunit. 64. An immunogenic composition according to any one of the listed embodiments 58 to 63, wherein one or more of the second, third, fourth, and fifth CoV S glycoproteins comprises one or more modifications to the S2 subunit. 65. An immunogenic composition according to any one of the listed embodiments 58 to 64, wherein one or more of the second, third, fourth, and fifth CoV S glycoproteins have one or more modifications to their N-terminal domain (NTD). 66. An immunogenic composition according to any one of the listed embodiments 58 to 65, wherein one or more of the second, third, fourth, and fifth CoV S glycoproteins comprises one or more modifications to any one of amino acids 630 to 710, any one of amino acids 640 to 700, any one of amino acids 650 to 690, or any one of amino acids 660 to 680, and the amino acids are numbered according to SEQ ID NO: 2. 67. The immunogenic composition according to any one of the listed embodiments 59 to 66, wherein the inactive furin cleavage site of the first CoV S glycoprotein comprises the amino acid sequence QQAQ (SEQ ID NO: 7), and amino acids 973 and 974 are proline. 68. An immunogenic composition according to any one of the listed embodiments 58 to 67, wherein one or more of the second, third, fourth, or fifth CoV S glycoproteins include an inactive furin cleavage site and mutations at amino acids 973 and 974, and the amino acid sequences of the second, third, fourth, and fifth CoV S glycoproteins are numbered according to SEQ ID NO: 2. 69. The immunogenic composition according to the listed embodiment 68, wherein the inactive furin cleavage site comprises the amino acid sequence QQAQ (SEQ ID NO: 7), and amino acids 973 and 974 are proline. 70. The first CoV S glycoprotein is at least 90%, at least 91%, at least 92%, and less than one of the following sequence numbers: 86, 87, 88, 89, 105, 106, 109, 110, 112, 113, 115, 130, 132-134, 140-141, 143-144, 146, 147, 149, 151-153, 155-158, 174, 175, 186, 188, 190, 195, 217-228, 233-236, 243, and 245-292. An immunogenic composition according to any one of the listed embodiments 58 to 69, comprising an amino acid sequence that is at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% identical. 71. An immunogenic composition according to any one of the listed embodiments 58 to 70, wherein the first CoV S glycoprotein comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% identical to any one of SEQ ID NOs. 245, 250, 255, and 260. 72. An immunogenic composition according to any one of the listed embodiments 58 to 71, wherein the second CoV S glycoprotein comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% identical to any one of SEQ ID NOs. 246, 251, 256, and 261. 73. An immunogenic composition according to any one of the listed embodiments 58 to 72, wherein the third CoV S glycoprotein comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% identical to any one of SEQ ID NOs. 247, 252, 257, and 262. 74. An immunogenic composition according to any one of the listed embodiments 60 to 73, wherein the fourth CoV S glycoprotein comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% identical to any one of SEQ ID NOs. 248, 253, 258, and 263. 75. An immunogenic composition according to any one of the listed embodiments 61 to 74, wherein the fifth CoV S glycoprotein comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% identical to any one of SEQ ID NOs. 249, 254, 259, and 264. 76. An immunogenic composition according to any one of the listed embodiments 61 to 75, wherein each of the second, third, fourth, and fifth CoV S glycoproteins contains one modification to the RBD compared to the first CoV S glycoprotein. 77. The immunogenic composition according to the enumerated embodiment 76, wherein the modification is one or more of the P508S mutation, K431T mutation, F473S mutation, or T465K mutation, and the amino acid sequence of each CoV S glycoprotein is numbered according to SEQ ID NO: 2. 78. An immunogenic composition according to any one of the listed embodiments 58 to 77, comprising approximately 3 μg to approximately 7 μg of a first CoV S glycoprotein. 79. An immunogenic composition according to any one of the listed embodiments 58 to 78, comprising approximately 1 ng to approximately 1 μg or approximately 3 μg to approximately 7 μg of a second CoV S glycoprotein. 80. An immunogenic composition according to any one of the listed ...

Claims

1. An immunogenic composition comprising a first CoV S glycoprotein having an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with any of the sequence numbers 329.

2. The immunogenic composition according to claim 1, wherein the first CoV S glycoprotein includes an inactive furin cleavage site.

3. The immunogenic composition according to claim 1 or 2, comprising 0.1 μg to 25 μg, 0.1 μg to 10 μg, 0.001 μg to 10 μg, 0.001 μg to 25 μg, or 0.1 μg to 5 μg of the first CoV S glycoprotein.

4. The immunogenic composition according to claim 1, comprising nanoparticles containing the first CoV S glycoprotein and a nonionic surfactant core.

5. The immunogenic composition according to claim 4, wherein the nonionic surfactant is selected from the group consisting of polysorbate-20 (PS20), polysorbate-40 (PS40), polysorbate-60 (PS60), polysorbate-65 (PS65), and polysorbate-80 (PS80).

6. The immunogenic composition according to claim 1, further comprising an adjuvant and a pharmaceutically acceptable carrier.

7. The immunogenic composition according to claim 8, wherein the adjuvant is a saponin adjuvant.

8. The saponin adjuvant comprises at least two iscom particles, The first iscom particle contains fraction A of Quillaya Saponaria Molina, but does not contain fraction C of Quillaya Saponaria Molina, and The immunogenic composition according to claim 7, wherein the second iscom particle contains fraction C of Quillaja saponaria molina and does not contain fraction A of Quillaja saponaria molina.

9. The immunogenic composition according to claim 8, wherein fraction A of Quillaja saponaria molina and fraction C of Quillaja saponaria molina account for about 85% by weight and about 15% by weight, respectively, of the total weight of fraction A of Quillaja saponaria molina and fraction C of Quillaja saponaria molina in the saponin adjuvant.

10. The immunogenic composition according to claim 8, wherein fraction A of Quillaja saponaria molina and fraction C of Quillaja saponaria molina account for about 92% by weight and about 8% by weight, respectively, of the total weight of fraction A of Quillaja saponaria molina and fraction C of Quillaja saponaria molina in the saponin adjuvant.

11. The immunogenic composition according to claim 8, wherein, in the saponin adjuvant, fraction A of Quillaja saponaria molina and fraction C of Quillaja saponaria molina account for at least about 85% by weight of fraction A and fraction C of Quillaja saponaria molina, respectively.

12. The immunogenic composition according to claim 8, wherein, in the saponin adjuvant, fraction A of Quillaja saponaria molina and fraction C of Quillaja saponaria molina account for 50 to 96% by weight of fraction A, and fraction C of Quillaja saponaria molina account for the remainder.

13. The immunogenic composition according to claim 6, comprising approximately 25 μg to approximately 100 μg of adjuvant.

14. The immunogenic composition according to claim 13, comprising approximately 50 μg or 75 μg of adjuvant.

15. The immunogenic composition according to claim 1, wherein the first CoV S glycoprotein comprises one or more modifications at amino acids 59, 346, 456, 475, 572 and 1087 compared to SEQ ID NO: 329, and the one or more modifications are numbered according to SEQ ID NO:

1.

16. (i) The amino acid in 59 is phenylalanine or serine. (ii) The amino acid in 346 is arginine or threonine. The amino acid in (iii) 456 is phenylalanine or leucine. The amino acid in (iv) 475 is alanine or valine. (v) The amino acid in 572 is threonine or isoleucine, and The immunogenic composition according to claim 15, wherein the amino acid in (vi) 1087 is alanine or serine.

17. An immunogenic composition according to any one of claims 1 to 16, which is contained in a syringe.

18. A method for stimulating an immune response in a human to SARS-CoV-2 or a heterologous SARS-CoV-2 strain, comprising administering to the human a human an immunogenic composition according to any one of claims 1 to 16.

19. A method for enhancing the immune response to SARS-CoV-2 or a heterologous SARS-CoV-2 strain in humans, comprising administering a first immunogenic composition comprising (a) one or more first SARS-CoV-2 S glycoproteins having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 329, and (b) a pharmaceutically acceptable buffer. A method wherein the immunogenic composition is administered after administration of another immunogenic composition intended to induce an immunogenic response in a human to SARS-CoV-2 or a heterologous SARS-CoV-2 strain.

20. The method according to claim 19, comprising administering a first dose of the first immunogenic composition at least 21 days after administration of the other immunogenic composition.

21. The method according to claim 20, comprising administering the first immunogenic composition at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 months after administration of the other immunogenic composition.

22. The method according to any one of claims 19 to 21, comprising administering the first immunogenic composition about 1 to 18 months, about 6 to 18 months, about 9 to 18 months, about 12 to 15 months, or about 12 to 18 months after administration of the other immunogenic composition.

23. The method according to any one of claims 19 to 22, wherein the other immunogenic composition comprises mRNA encoding SARS-CoV-2 spike glycoprotein, plasmid DNA encoding SARS-CoV-2 spike glycoprotein, a viral vector encoding SARS-CoV-2 spike glycoprotein, an inactivated SARS-CoV-2 virus, or a protein subunit vaccine.

24. The method according to claim 23, wherein the protein subunit vaccine of the other immunogenic composition comprises at least one second SARS-CoV-2 S glycoprotein having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with any of SEQ ID NOs: 87, 260, 222, 227, 274, and 284 and 329.

25. The method according to claim 24, wherein the at least one second SARS-CoV-2 S glycoprotein comprises at least two SARS-CoV-2 S glycoproteins having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with any of SEQ ID NOs: 87, 260, 222, 227, 274, and 284 and 329.

26. A method for amplifying the immune response to SARS-CoV-2 or a heterologous SARS-CoV-2 strain in humans, comprising administering a first immunogenic composition comprising (a) one or more first SARS-CoV-2 S glycoproteins having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 329, and (b) a pharmaceutically acceptable buffer. A method wherein the immunogenic composition is administered after administration of another immunogenic composition intended to induce an immunogenic response in a human to SARS-CoV-2 or a heterologous SARS-CoV-2 strain thereof, wherein the other immunogenic composition does not contain SARS-CoV-2 S glycoprotein having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO:

329.

27. The method according to claim 26, wherein the other immunogenic composition comprises one mRNA vaccine and one protein subunit vaccine.

28. The method according to claim 27, wherein the other immunogenic composition comprises a second CoV S glycoprotein having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with any one of SEQ ID NOs: 87, 260, 222, 227, 274, and 284.

29. A method for inducing an immune response in a human to SARS-CoV-2 or a heterologous SARS-CoV-2 strain thereof, comprising administering a first immunogenic composition comprising (a) one or more first SARS-CoV-2 S glycoproteins having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO: 329, and (b) a pharmaceutically acceptable buffer.

30. The method according to claim 29, further comprising (a) administering a second immunogenic composition comprising one or more second SARS-CoV-2 S glycoproteins having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with any of SEQ ID NOs: 87, 260, 222, 227, 274, and 284 and 329.

31. The method according to claim 30, comprising administering the second immunogenic composition at least 21 days after administering the first immunogenic composition.

32. The method according to claim 31, comprising administering the second immunogenic composition at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 months after administration of the first immunogenic composition.

33. The method according to any one of claims 30 to 32, comprising administering the second immunogenic composition about 1 to 18 months, about 6 to 18 months, about 9 to 18 months, about 12 to 15 months, or about 12 to 18 months after administration of the first immunogenic composition.

34. (i) The first coronavirus spike (CoV S) glycoprotein, (ii) A second CoV S glycoprotein having 1 to about 2, 1 to about 3, 1 to about 4, 1 to about 5, 2 to about 5, 1 to about 6, 1 to about 7, 1 to about 8, 1 to about 9, 1 to about 10, 1 to about 11, 1 to about 12, 1 to about 13, 1 to about 14, 1 to about 15, 1 to about 16, 1 to about 17, 1 to about 18, 1 to about 18, 1 to about 19 or 1 to about 20, 1 to about 25, 1 to about 30, 1 to about 35, 1 to about 40, 1 to about 45 or 1 to about 50 modifications compared to the first CoV S glycoprotein, (iii) A third CoV S glycoprotein having 1 to about 2, 1 to about 3, 1 to about 4, 1 to about 5, 2 to about 5, 1 to about 6, 1 to about 7, 1 to about 8, 1 to about 9, 1 to about 10, 1 to about 11, 1 to about 12, 1 to about 13, 1 to about 14, 1 to about 15, 1 to about 16, 1 to about 17, 1 to about 18, 1 to about 18, 1 to about 19 or 1 to about 20, 1 to about 25, 1 to about 30, 1 to about 35, 1 to about 40, 1 to about 45 or 1 to about 50 modifications compared to the first CoV S glycoprotein. An immunogenic composition comprising the second and third CoV S glycoproteins having different amino acid sequences.

35. The immunogenic composition according to claim 34, wherein the first CoV S glycoprotein contains an inactive furin cleavage site and mutations at amino acids 973 and 974, and the amino acid sequence of the CoV S glycoprotein is numbered according to Sequence ID No.

2.

36. The fourth CoV S glycoprotein comprises, compared to the first CoV S glycoprotein, 1 to about 2, 1 to about 3, 1 to about 4, 1 to about 5, 2 to about 5, 1 to about 6, 1 to about 7, 1 to about 8, 1 to about 9, 1 to about 10, 1 to about 11, 1 to about 12, 1 to about 13, 1 to about 14, 1 to about 15, 1 to about 16, 1 to about 17, 1 to about 18, 1 to about 18, about 1 to about 19 or 1 to about 20, 1 to about 25, 1 to about 30, 1 to about 35, 1 to about 40, 1 to about 45 or 1 to about 50 modifications. The immunogenic composition according to claim 34 or 35, wherein the second, third, and fourth CoV S glycoproteins have different amino acid sequences.

37. The fifth CoV S glycoprotein comprises, compared to the first CoV S glycoprotein, 1 to about 2, 1 to about 3, 1 to about 4, 1 to about 5, 2 to about 5, 1 to about 6, 1 to about 7, 1 to about 8, 1 to about 9, 1 to about 10, 1 to about 11, 1 to about 12, 1 to about 13, 1 to about 14, 1 to about 15, 1 to about 16, 1 to about 17, 1 to about 18, 1 to about 18, 1 to about 19 or 1 to about 20, 1 to about 25, 1 to about 30, 1 to about 35, 1 to about 40, 1 to about 45 or 1 to about 50 modifications. The immunogenic composition according to any one of claims 34 to 36, wherein the second, third, fourth, and fifth CoV S glycoproteins have different amino acid sequences.

38. The immunogenic composition according to any one of claims 34 to 37, wherein one or more of the second, third, fourth, and fifth CoV S glycoproteins include modifications to the receptor-binding domain (RBD).

39. The immunogenic composition according to any one of claims 34 to 37, wherein one or more of the second, third, fourth, and fifth CoV S glycoproteins comprises one or more modifications to the S1 subunit.

40. The immunogenic composition according to any one of claims 34 to 39, wherein one or more of the second, third, fourth, and fifth CoV S glycoproteins comprises one or more modifications to the S2 subunit.

41. The immunogenic composition according to any one of claims 34 to 40, wherein one or more of the second, third, fourth, and fifth CoV S glycoproteins include one or more modifications to the N-terminal domain (NTD).

42. The immunogenic composition according to any one of claims 34 to 41, wherein one or more of the second, third, fourth, and fifth CoV S glycoproteins comprises one or more modifications to any one of amino acids 630 to 710, any one of amino acids 640 to 700, any one of amino acids 650 to 690, or any one of amino acids 660 to 680, and the amino acids are numbered according to Sequence ID No.

2.

43. The immunogenic composition according to any one of claims 35 to 42, wherein the inactive furin cleavage site of the first CoV S glycoprotein comprises the amino acid sequence QQAQ (SEQ ID NO: 7), and amino acids 973 and 974 are proline.

44. The immunogenic composition according to any one of claims 34 to 43, wherein one or more of the second, third, fourth, or fifth CoV S glycoproteins include an inactive furin cleavage site and mutations in amino acids 973 and 974, the amino acid sequences of the second, third, fourth, and fifth CoV S glycoproteins are numbered according to SEQ ID NO: 2, and optionally, the inactive furin cleavage site includes the amino acid sequence QQAQ (SEQ ID NO: 7), and amino acids 973 and 974 are proline.

45. The first CoV S glycoprotein is at least 90%, at least 91%, and at least 92% of any one of sequence numbers 86, 87, 88, 89, 105, 106, 109, 110, 112, 113, 115, 130, 132-134, 140-141, 143-144, 146, 147, 149, 151-153, 155-158, 174, 175, 186, 188, 190, 195, 217-228, 233-236, 243, and 245-292. An immunogenic composition according to any one of claims 34 to 44, comprising amino acid sequences that are identical in percentages of %, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99%.

46. The first CoV S glycoprotein is at least 90%, at least 91%, and at least 92% of any one of sequence numbers 86, 87, 88, 89, 105, 106, 109, 110, 112, 113, 115, 130, 132-134, 140-141, 143-144, 146, 147, 149, 151-153, 155-158, 174, 175, 186, 188, 190, 195, 217-228, 233-236, 243, and 245-292. An immunogenic composition according to any one of claims 34 to 45, comprising amino acid sequences that are identical in percentages of %, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99%.

47. The second CoV S glycoprotein is at least 90%, at least 91%, and at least 92% of any one of sequence numbers 86, 87, 88, 89, 105, 106, 109, 110, 112, 113, 115, 130, 132-134, 140-141, 143-144, 146, 147, 149, 151-153, 155-158, 174, 175, 186, 188, 190, 195, 217-228, 233-236, 243, and 245-292. An immunogenic composition according to any one of claims 34 to 46, comprising amino acid sequences that are identical in percentages of %, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99%.

48. The third CoV S glycoprotein is at least 90%, at least 91%, and at least 92% of any one of sequence numbers 86, 87, 88, 89, 105, 106, 109, 110, 112, 113, 115, 130, 132-134, 140-141, 143-144, 146, 147, 149, 151-153, 155-158, 174, 175, 186, 188, 190, 195, 217-228, 233-236, 243, and 245-292. An immunogenic composition according to any one of claims 34 to 47, comprising amino acid sequences that are identical in percentages of %, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99%.

49. The fourth CoV S glycoprotein is at least 90%, at least 91%, and at least 92% of any one of sequence numbers 86, 87, 88, 89, 105, 106, 109, 110, 112, 113, 115, 130, 132-134, 140-141, 143-144, 146, 147, 149, 151-153, 155-158, 174, 175, 186, 188, 190, 195, 217-228, 233-236, 243, and 245-292. An immunogenic composition according to any one of claims 36 to 48, comprising an amino acid sequence that is identical in percentages of %, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99%.

50. The fifth CoV S glycoprotein is at least 90%, at least 91%, and at least 92% of any one of sequence numbers 86, 87, 88, 89, 105, 106, 109, 110, 112, 113, 115, 130, 132-134, 140-141, 143-144, 146, 147, 149, 151-153, 155-158, 174, 175, 186, 188, 190, 195, 217-228, 233-236, 243, and 245-292. An immunogenic composition according to any one of claims 37 to 49, comprising amino acid sequences that are identical in percentages of %, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99%.

51. The immunogenic composition according to any one of claims 37 to 50, wherein each of the second, third, fourth, and fifth CoV S glycoproteins comprises one modification to the RBD compared to the first CoV S glycoprotein.

52. The immunogenic composition according to claim 51, wherein the modification in the RBD is one or more of the P508S mutation, K431T mutation, F473S mutation, or T465K mutation, and the amino acid sequence of each CoV S glycoprotein is numbered according to Sequence ID No.

2.

53. An immunogenic composition according to any one of claims 34 to 52, comprising approximately 3 μg to approximately 7 μg of the first CoV S glycoprotein.

54. An immunogenic composition according to any one of claims 34 to 53, comprising approximately 1 ng to approximately 1 μg or approximately 3 μg to approximately 7 μg of the second CoV S glycoprotein.

55. An immunogenic composition according to any one of claims 34 to 54, comprising approximately 1 ng to approximately 1 μg or approximately 3 μg to approximately 7 μg of the third CoV S glycoprotein.

56. An immunogenic composition according to any one of claims 37 to 53, comprising approximately 1 ng to approximately 1 μg or approximately 3 μg to approximately 7 μg of the fourth CoV S glycoprotein.

57. An immunogenic composition according to any one of claims 37 to 53, comprising approximately 1 ng to approximately 1 μg or approximately 3 μg to approximately 7 μg of the fifth CoV S glycoprotein.

58. (i) First nanoparticles containing the first CoV S glycoprotein, (ii) A second nanoparticle comprising a second CoV S glycoprotein and a third CoV S glycoprotein, wherein each of the second CoV S glycoprotein and the third CoV S glycoprotein contains 1 to about 2, 1 to about 3, 1 to about 4, 1 to about 5, 2 to about 5, 1 to about 6, 1 to about 7, 1 to about 8, 1 to about 9, 1 to about 10, 1 to about 11, 1 to about 12, 1 to about 13, 1 to about 14, 1 to about 15, 1 to about 16, 1 to about 17, 1 to about 18, 1 to about 18, 1 to about 19 or 1 to about 20, 1 to about 25, 1 to about 30, 1 to about 35, 1 to about 40, 1 to about 45 or 1 to about 50 modifications compared to the first CoV S glycoprotein. An immunogenic composition containing [a specific substance].

59. The immunogenic composition according to claim 25, wherein the first CoV S glycoprotein contains an inactive furin cleavage site and mutations at amino acids 973 and 974, and the amino acid sequence of the CoV S glycoprotein is numbered according to Sequence ID No.

2.

60. The fourth CoV S glycoprotein comprises, compared to the first CoV S glycoprotein, 1 to about 2, 1 to about 3, 1 to about 4, 1 to about 5, 2 to about 5, 1 to about 6, 1 to about 7, 1 to about 8, 1 to about 9, 1 to about 10, 1 to about 11, 1 to about 12, 1 to about 13, 1 to about 14, 1 to about 15, 1 to about 16, 1 to about 17, 1 to about 18, 1 to about 18, 1 to about 19 or 1 to about 20, 1 to about 25, 1 to about 30, 1 to about 35, 1 to about 40, 1 to about 45 or 1 to about 50 modifications. The immunogenic composition according to claim 58 or 59, wherein the second, third, and fourth CoV S glycoproteins have different amino acid sequences.

61. The fifth CoV S glycoprotein comprises, compared to the first CoV S glycoprotein, 1 to about 2, 1 to about 3, 1 to about 4, 1 to about 5, 2 to about 5, 1 to about 6, 1 to about 7, 1 to about 8, 1 to about 9, 1 to about 10, 1 to about 11, 1 to about 12, 1 to about 13, 1 to about 14, 1 to about 15, 1 to about 16, 1 to about 17, 1 to about 18, 1 to about 18, 1 to about 19 or 1 to about 20, 1 to about 25, 1 to about 30, 1 to about 35, 1 to about 40, 1 to about 45 or 1 to about 50 modifications. The immunogenic composition according to any one of claims 58 to 60, wherein the second, third, fourth, and fifth CoV S glycoproteins have different amino acid sequences.

62. The immunogenic composition according to any one of claims 58 to 61, wherein one or more of the second, third, fourth, and fifth CoV S glycoproteins include modification of the receptor-binding domain (RBD).

63. The immunogenic composition according to any one of claims 58 to 62, wherein one or more of the second, third, fourth, and fifth CoV S glycoproteins comprises one or more modifications to the S1 subunit.

64. The immunogenic composition according to any one of claims 58 to 63, wherein one or more of the second, third, fourth, and fifth CoV S glycoproteins comprises one or more modifications to the S2 subunit.

65. The immunogenic composition according to any one of claims 58 to 64, wherein one or more of the second, third, fourth, and fifth CoV S glycoproteins comprises one or more modifications to the N-terminal domain (NTD).

66. The immunogenic composition according to any one of claims 58 to 65, wherein one or more of the second, third, fourth, and fifth CoV S glycoproteins comprises one or more modifications to any one of amino acids 630 to 710, any one of amino acids 640 to 700, any one of amino acids 650 to 690, or any one of amino acids 660 to 680, and the amino acids are numbered according to Sequence ID No.

2.

67. The immunogenic composition according to any one of claims 59 to 66, wherein the inactive furin cleavage site of the first CoV S glycoprotein comprises the amino acid sequence QQAQ (SEQ ID NO: 7), and amino acids 973 and 974 are proline.

68. The immunogenic composition according to any one of claims 58 to 67, wherein one or more of the second, third, fourth, or fifth CoV S glycoproteins include an inactive furin cleavage site and mutations at amino acids 973 and 974, and the amino acid sequences of the second, third, fourth, and fifth CoV S glycoproteins are numbered according to Sequence ID No.

2.

69. The immunogenic composition according to claim 68, wherein the inactive furin cleavage site comprises the amino acid sequence QQAQ (SEQ ID NO: 7), and amino acids 973 and 974 are proline.

70. The first CoV S glycoprotein is at least 90%, at least 91%, and at least 92% of any one of sequence numbers 86, 87, 88, 89, 105, 106, 109, 110, 112, 113, 115, 130, 132-134, 140-141, 143-144, 146, 147, 149, 151-153, 155-158, 174, 175, 186, 188, 190, 195, 217-228, 233-236, 243, and 245-292. An immunogenic composition according to any one of claims 58 to 69, comprising an amino acid sequence that is identical in percentages of %, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99%.

71. The immunogenic composition according to any one of claims 58 to 70, wherein the first CoV S glycoprotein comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% identical to any one of SEQ ID NOs. 245, 250, 255, and 260.

72. The immunogenic composition according to any one of claims 58 to 71, wherein the second CoV S glycoprotein comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% identical to any one of SEQ ID NOs. 246, 251, 256, and 261.

73. The immunogenic composition according to any one of claims 58 to 72, wherein the third CoV S glycoprotein comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% identical to any one of SEQ ID NOs. 247, 252, 257, and 262.

74. The immunogenic composition according to any one of claims 60 to 73, wherein the fourth CoV S glycoprotein comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% identical to any one of SEQ ID NOs. 248, 253, 258, and 263.

75. The immunogenic composition according to any one of claims 61 to 74, wherein the fifth CoV S glycoprotein comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% identical to any one of SEQ ID NOs. 249, 254, 259, and 264.

76. The immunogenic composition according to any one of claims 61 to 75, wherein each of the second, third, fourth, and fifth CoV S glycoproteins comprises one modification of the RBD compared to the first CoV S glycoprotein.

77. The immunogenic composition according to claim 76, wherein the modification is one or more of the P508S mutation, K431T mutation, F473S mutation, or T465K mutation, and the amino acid sequence of each CoV S glycoprotein is numbered according to Sequence ID No.

2.

78. An immunogenic composition according to any one of claims 58 to 77, comprising approximately 3 μg to approximately 7 μg of the first CoV S glycoprotein.

79. An immunogenic composition according to any one of claims 58 to 78, comprising approximately 1 ng to approximately 1 μg or approximately 3 μg to approximately 7 μg of the second CoV S glycoprotein.

80. An immunogenic composition according to any one of claims 58 to 79, comprising approximately 1 ng to approximately 1 μg or approximately 3 μg to approximately 7 μg of the third CoV S glycoprotein.

81. An immunogenic composition according to any one of claims 60 to 80, comprising approximately 1 ng to approximately 1 μg or approximately 3 μg to approximately 7 μg of the fourth CoV S glycoprotein.

82. An immunogenic composition according to any one of claims 61 to 81, comprising approximately 1 ng to approximately 1 μg or approximately 3 μg to approximately 7 μg of the fifth CoV S glycoprotein.

83. An immunogenic composition according to any one of claims 58 to 82, comprising a pharmaceutically acceptable buffer.

84. An immunogenic composition according to any one of claims 58 to 83, comprising an adjuvant.

85. The immunogenic composition according to claim 84, wherein the adjuvant is a saponin adjuvant.

86. The saponin adjuvant comprises at least two iscom particles, The first iscom particle contains fraction A of Quillaya Saponaria Molina, but does not contain fraction C of Quillaya Saponaria Molina, and The immunogenic composition according to claim 85, wherein the second iscom particle contains fraction C of Quillaja saponaria molina and does not contain fraction A of Quillaja saponaria molina.

87. The immunogenic composition according to claim 86, wherein fraction A of Quillaja saponaria molina and fraction C of Quillaja saponaria molina account for about 85% by weight and about 15% by weight, respectively, of the total weight of fraction A of Quillaja saponaria molina and fraction C of Quillaja saponaria molina in the saponin adjuvant.

88. The immunogenic composition according to claim 86, wherein fraction A of Quillaja saponaria molina and fraction C of Quillaja saponaria molina account for approximately 92% by weight and approximately 8% by weight, respectively, of the total weight of fraction A of Quillaja saponaria molina and fraction C of Quillaja saponaria molina in the saponin adjuvant.

89. The immunogenic composition according to claim 86, wherein, of the total weight of fraction A and fraction C of Quillaja saponaria molina in the saponin adjuvant, fraction A of Quillaja saponaria molina accounts for at least about 85% by weight, and fraction C of Quillaja saponaria molina accounts for the remainder.

90. The immunogenic composition according to claim 86, wherein, in the saponin adjuvant, fraction A of Quillaja saponaria molina and fraction C of Quillaja saponaria molina account for 50 to 96% by weight of fraction A and fraction C of Quillaja saponaria molina, respectively.

91. An immunogenic composition according to any one of claims 84 to 90, comprising approximately 25 μg to approximately 100 μg of adjuvant.

92. An immunogenic composition according to any one of claims 84 to 90, comprising approximately 50 μg of adjuvant.

93. An immunogenic composition according to any one of claims 1 to 92, comprising mRNA encoding SARS-CoV-2 spike glycoprotein, plasmid DNA encoding SARS-CoV-2 spike glycoprotein, a viral vector encoding SARS-CoV-2 spike glycoprotein, or an inactivated SARS-CoV-2 virus.

94. An immunogenic composition according to any one of claims 1 to 93, comprising at least one, at least two, at least three or at least four hemagglutinin (HA) glycoproteins, each HA glycoprotein derived from a different influenza strain.

95. An immunogenic composition according to any one of claims 1 to 94, comprising respiratory syncytial virus (RSV) fusion (F) glycoprotein.

96. The immunogenic composition according to any one of claims 1 to 95, wherein the total amount of CoV S glycoprotein in the composition is about 3 μg to about 10 μg.

97. A pre-filled syringe comprising the immunogenic composition according to any one of claims 34 to 96 or 125 to 196.

98. A method for stimulating an immune response to SARS-CoV-2 or a heterologous SARS-CoV-2 strain, comprising administering an immunogenic composition according to any one of claims 1 to 17, 34 to 96, or 125 to 196 to a subject.

99. The method according to claim 98, comprising administering the immunogenic composition in doses of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

100. The method according to claim 98 or 99, comprising administering a first dose of the immunogenic composition and a second dose of the immunogenic composition approximately three weeks after the first dose.

101. The method according to any one of claims 98 to 100, comprising administering a first dose of the immunogenic composition and a second dose of the immunogenic composition about 21 days after the first dose.

102. The method according to any one of claims 98 to 101, comprising administering at least three doses of the immunogenic composition, wherein the third dose of the immunogenic composition is administered at least six months, at least seven months, at least eight months, at least nine months, at least ten months, at least eleven months, at least twelve months, at least thirteen months, at least fourteen months, at least fifteen months, at least sixteen months, at least seventeen months, at least eighteen months, at least nineteen months, at least twenty months, at least twenty months, at least twenty-one months, at least twenty-two months, at least twenty-three months, or at least twenty-four months after the first or second dose.

103. The method according to any one of claims 98 to 102, comprising administering a second immunogenic composition different from the first immunogenic composition.

104. The method according to claim 103, wherein the second immunogenic composition comprises mRNA encoding SARS-CoV-2 spike glycoprotein, plasmid DNA encoding SARS-CoV-2 spike glycoprotein, a viral vector encoding SARS-CoV-2 spike glycoprotein, or an inactivated SARS-CoV-2 virus.

105. The method according to claim 103, wherein the second immunogenic composition comprises at least one, at least two, at least three or at least four hemagglutinin (HA) glycoproteins, each HA glycoprotein derived from a different influenza strain.

106. The method according to claim 103, wherein the second immunogenic composition comprises RSV F glycoprotein.

107. The method according to claim 103, wherein the second immunogenic composition comprises a CoV S glycoprotein different from that of the first immunogenic composition.

108. The method according to any one of claims 98 to 107, wherein the immunogenic composition is administered intramuscularly.

109. The method according to any one of claims 98 to 108, comprising administering the immunogenic composition in a pre-filled syringe.

110. After administration of the immunogenic composition, for at least about 2 months, at least about 2.5 months, at least about 3 months, at least about 3.5 months, at least about 4 months, at least about 4.5 months, at least about 5 months, at least about 5.5 months, at least about 6 months, at least about 6.5 months, at least about 7 months, at least about 7.5 months, at least about 8 months, at least about 8.5 months, at least about 9 months, at least about 9.5 months, at least about 10 months, at least about 10.5 months, at least about 11 months, at least about 11.5 months, at least about 12 months, at least 13 months, at least 14 months, at least 15 months, at least 16 months, at least 17 months, at least 18 months, at least 19 months, and at least 20 months. Months, over a period of at least 21 months, at least 22 months, at least 23 months or at least 24 months, approximately 50% to approximately 99%, approximately 50% to approximately 95%, approximately 50% to approximately 90%, approximately 50% to approximately 85%, approximately 50% to approximately 80%, approximately 60% to approximately 99%, approximately 65% ​​to approximately 95%, approximately 65% ​​to approximately 90%, approximately 65% ​​to approximately 85%, approximately 69% to approximately 81%, approximately 60% to approximately 95%, approximately 60% to approximately 9 The method according to any one of claims 98 to 109, for preventing COVID-19 with an effectiveness of 0%, approximately 60% to approximately 85%, approximately 60% to approximately 80%, approximately 40% to approximately 99%, approximately 40% to approximately 95%, approximately 40% to approximately 90%, approximately 40% to approximately 85%, approximately 40% to approximately 80%, approximately 40% to approximately 75%, approximately 40% to approximately 70%, approximately 40% to approximately 65%, approximately 40% to approximately 55%, or approximately 40% to approximately 50%.

111. After administration of the immunogenic composition, the effects last for up to approximately 2 months, up to approximately 2.5 months, up to approximately 3 months, up to approximately 3.5 months, up to approximately 4 months, up to approximately 4.5 months, up to approximately 5 months, up to approximately 5.5 months, up to approximately 6 months, up to approximately 6.5 months, up to approximately 7 months, up to approximately 7.5 months, up to approximately 8 months, up to approximately 8.5 months, up to approximately 9 months, up to approximately 9.5 months, up to approximately 10 months, up to approximately 10.5 months, up to approximately 11 months, up to approximately 11.5 months, up to approximately 12 months, up to 13 months, up to 14 months, up to 15 months, up to 16 months, up to 17 months, up to 18 months, up to 19 months, up to 20 months, up to 21 months, up to 22 months, and up to 23 months. Or for a maximum of 24 months, approximately 50% to 99%, approximately 50% to 95%, approximately 50% to 90%, approximately 50% to 85%, approximately 50% to 80%, approximately 60% to 99%, approximately 65% ​​to 95%, approximately 65% ​​to 90%, approximately 65% ​​to 85%, approximately 69% to 81%, approximately 60% to 95%, approximately 60% to 90%, approximately 60% to 85%, approximately 60% The method according to any one of claims 98 to 110, for preventing COVID-19 with an effectiveness of % to approximately 80%, approximately 40% to approximately 99%, approximately 40% to approximately 95%, approximately 40% to approximately 90%, approximately 40% to approximately 85%, approximately 40% to approximately 80%, approximately 40% to approximately 75%, approximately 40% to approximately 70%, approximately 40% to approximately 65%, approximately 40% to approximately 55%, or approximately 40% to approximately 50%.

112. A method for stimulating an immune response in a subject to SARS-CoV-2, a heterologous SARS-CoV-2 strain, influenza virus, or a combination thereof, comprising administering the immunogenic composition described in claim 94.

113. A method for stimulating an immune response in a subject to SARS-CoV-2, a heterologous SARS-CoV-2 strain, influenza virus, respiratory syncytial virus (RSV), or a combination thereof, comprising administering the immunogenic composition described in claim 95.

114. A method for preparing CoV S nanoparticles, (i) A step of binding a protein extract containing a first surfactant and a first CoV S glycoprotein to a protein purification column, wherein the column is bound to the CoV S glycoprotein, (ii) A step of performing surfactant exchange by substantially replacing the first surfactant with a second surfactant, (iii) The step of eluting the bound CoV S glycoprotein from the column in the presence of the second surfactant to provide the nanoparticles. A method that includes this.

115. The method according to claim 114, wherein the protein extract further comprises a second CoV S glycoprotein, the second CoV S glycoprotein comprising 1 to about 2, 1 to about 3, 1 to about 4, 1 to about 5, 2 to about 5, 1 to about 6, 1 to about 7, 1 to about 8, 1 to about 9, 1 to about 10, 1 to about 11, 1 to about 12, 1 to about 13, 1 to about 14, 1 to about 15, 1 to about 16, 1 to about 17, 1 to about 18, 1 to about 18, 1 to about 19 or 1 to about 20, 1 to about 25, 1 to about 30, 1 to about 35, 1 to about 40, 1 to about 45 or 1 to about 50 modifications compared to the first CoV S glycoprotein.

116. The method according to claim 115, wherein the protein extract further comprises a third CoV S glycoprotein, the third CoV S glycoprotein comprising 1 to about 2, 1 to about 3, 1 to about 4, 1 to about 5, 2 to about 5, 1 to about 6, 1 to about 7, 1 to about 8, 1 to about 9, 1 to about 10, 1 to about 11, 1 to about 12, 1 to about 13, 1 to about 14, 1 to about 15, 1 to about 16, 1 to about 17, 1 to about 18, 1 to about 18, 1 to about 19 or 1 to about 20, 1 to about 25, 1 to about 30, 1 to about 35, 1 to about 40, 1 to about 45 or 1 to about 50 modifications compared to the first CoV S glycoprotein.

117. The method according to claim 116, wherein the protein extract further comprises a fourth CoV S glycoprotein, the fourth CoV S glycoprotein comprising 1 to about 2, 1 to about 3, 1 to about 4, 1 to about 5, 2 to about 5, 1 to about 6, 1 to about 7, 1 to about 8, 1 to about 9, 1 to about 10, 1 to about 11, 1 to about 12, 1 to about 13, 1 to about 14, 1 to about 15, 1 to about 16, 1 to about 17, 1 to about 18, 1 to about 18, 1 to about 19 or 1 to about 20, 1 to about 25, 1 to about 30, 1 to about 35, 1 to about 40, 1 to about 45 or 1 to about 50 modifications compared to the first CoV S glycoprotein.

118. The method according to claim 117, wherein the protein extract further comprises a fifth CoV S glycoprotein, the fifth CoV S glycoprotein comprising 1 to about 2, 1 to about 3, 1 to about 4, 1 to about 5, 2 to about 5, 1 to about 6, 1 to about 7, 1 to about 8, 1 to about 9, 1 to about 10, 1 to about 11, 1 to about 12, 1 to about 13, 1 to about 14, 1 to about 15, 1 to about 16, 1 to about 17, 1 to about 18, 1 to about 18, 1 to about 19 or 1 to about 20, 1 to about 25, 1 to about 30, 1 to about 35, 1 to about 40, 1 to about 45 or 1 to about 50 modifications compared to the first CoV S glycoprotein.

119. The protein extract is (a) Transforming host cells with nucleic acids encoding the first CoV S glycoprotein including a transmembrane domain, (b) Culturing the host cells under conditions that promote the production of the CoV S glycoprotein The method according to any one of claims 114 to 118, prepared by [the specified method].

120. The method according to claim 119, comprising transforming the host cells with a nucleic acid encoding a second CoV S glycoprotein comprising a transmembrane domain, wherein the second CoV S glycoprotein comprises 1 to about 2, 1 to about 3, 1 to about 4, 1 to about 5, 2 to about 5, 1 to about 6, 1 to about 7, 1 to about 8, 1 to about 9, 1 to about 10, 1 to about 11, 1 to about 12, 1 to about 13, 1 to about 14, 1 to about 15, 1 to about 16, 1 to about 17, 1 to about 18, 1 to about 18, 1 to about 19 or 1 to about 20, 1 to about 25, 1 to about 30, 1 to about 35, 1 to about 40, 1 to about 45 or 1 to about 50 modifications compared to the first CoV S glycoprotein.

121. The method according to claim 120, comprising transforming the host cells with a nucleic acid encoding a third CoV S glycoprotein comprising a transmembrane domain, wherein the third CoV S glycoprotein comprises 1 to about 2, 1 to about 3, 1 to about 4, 1 to about 5, 2 to about 5, 1 to about 6, 1 to about 7, 1 to about 8, 1 to about 9, 1 to about 10, 1 to about 11, 1 to about 12, 1 to about 13, 1 to about 14, 1 to about 15, 1 to about 16, 1 to about 17, 1 to about 18, 1 to about 18, 1 to about 19 or 1 to about 20, 1 to about 25, 1 to about 30, 1 to about 35, 1 to about 40, 1 to about 45 or 1 to about 50 modifications compared to the first CoV S glycoprotein.

122. The method according to claim 121, comprising transforming the host cells with a nucleic acid encoding a fourth CoV S glycoprotein comprising a transmembrane domain, wherein the fourth CoV S glycoprotein comprises 1 to about 2, 1 to about 3, 1 to about 4, 1 to about 5, 2 to about 5, 1 to about 6, 1 to about 7, 1 to about 8, 1 to about 9, 1 to about 10, 1 to about 11, 1 to about 12, 1 to about 13, 1 to about 14, 1 to about 15, 1 to about 16, 1 to about 17, 1 to about 18, 1 to about 18, 1 to about 19 or 1 to about 20, 1 to about 25, 1 to about 30, 1 to about 35, 1 to about 40, 1 to about 45 or 1 to about 50 modifications compared to the first CoV S glycoprotein.

123. The method according to claim 122, comprising transforming the host cells with a nucleic acid encoding a fifth CoV S glycoprotein comprising a transmembrane domain, wherein the fifth CoV S glycoprotein comprises 1 to about 2, 1 to about 3, 1 to about 4, 1 to about 5, 2 to about 5, 1 to about 6, 1 to about 7, 1 to about 8, 1 to about 9, 1 to about 10, 1 to about 11, 1 to about 12, 1 to about 13, 1 to about 14, 1 to about 15, 1 to about 16, 1 to about 17, 1 to about 18, 1 to about 18, 1 to about 19 or 1 to about 20, 1 to about 25, 1 to about 30, 1 to about 35, 1 to about 40, 1 to about 45 or 1 to about 50 modifications compared to the first CoV S glycoprotein.

124. The method according to any one of claims 119 to 123, wherein the host cell is an insect cell, and optionally the insect cell is an Sf9 or Sf22a cell.

125. An immunogenic composition comprising the first, second, third, fourth, and fifth CoV S glycoproteins.

126. The immunogenic composition according to claim 125, wherein the first CoV S glycoprotein has an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 245, 250, 255, 260, 265, 269, 273, 274, 277, and 284.

127. The immunogenic composition according to claim 125 or 126, wherein the second, third, fourth, and fifth CoV S glycoproteins contain 1 to 60 modifications compared to the first CoV S glycoprotein.

128. The immunogenic composition according to claim 127, wherein the second, third, fourth, and fifth CoV S glycoproteins each contain 1 to 10 modifications compared to the first CoV S glycoprotein.

129. The immunogenic composition according to claim 127 or 128, wherein the modification occurs in one or more amino acids 180, 252, 253, 444, 478, 486, and 521, and the modification is numbered according to the CoV S polypeptide having the amino acid sequence of SEQ ID NO:

1.

130. An immunogenic composition according to any one of claims 125 to 129, comprising 0.1 μg to 10 μg of each CoV S polypeptide.

131. An immunogenic composition according to any one of claims 125 to 130, comprising 0.1 μg to 10 μg of the first CoV S glycoprotein.

132. An immunogenic composition according to any one of claims 125 to 131, comprising 0.1 μg to 5 μg of the first CoV S glycoprotein.

133. The immunogenic composition according to any one of claims 125 to 132, wherein the amount of the second CoV S glycoprotein in the composition is less than the amount of the first CoV S glycoprotein in the composition.

134. The immunogenic composition according to any one of claims 125 to 133, wherein the amount of the third CoV S glycoprotein in the composition is less than the amount of the first CoV S glycoprotein in the composition.

135. The immunogenic composition according to any one of claims 125 to 134, wherein the amount of the fourth CoV S glycoprotein in the composition is less than the amount of the first CoV S glycoprotein in the composition.

136. The immunogenic composition according to any one of claims 125 to 135, wherein the amount of the fifth CoV S glycoprotein in the composition is less than the amount of the first CoV S glycoprotein in the composition.

137. An immunogenic composition according to any one of claims 125 to 136, comprising an adjuvant.

138. The immunogenic composition according to claim 137, wherein the adjuvant is a saponin adjuvant.

139. The saponin adjuvant comprises at least two iscom particles, The first iscom particle contains fraction A of Quillaya Saponaria Molina, but does not contain fraction C of Quillaya Saponaria Molina, and The immunogenic composition according to claim 138, wherein the second iscom particle contains fraction C of Quillaja saponaria molina and does not contain fraction A of Quillaja saponaria molina.

140. The immunogenic composition according to claim 139, wherein fraction A of Quillaja saponaria molina and fraction C of Quillaja saponaria molina account for about 85% by weight and about 15% by weight, respectively, of the total weight of fraction A of Quillaja saponaria molina and fraction C of Quillaja saponaria molina in the saponin adjuvant.

141. The immunogenic composition according to claim 139, wherein fraction A of Quillaja saponaria molina and fraction C of Quillaja saponaria molina account for about 92% by weight and about 8% by weight, respectively, of the total weight of fraction A of Quillaja saponaria molina and fraction C of Quillaja saponaria molina in the saponin adjuvant.

142. The immunogenic composition according to claim 139, wherein, in the saponin adjuvant, fraction A of Quillaja saponaria molina and fraction C of Quillaja saponaria molina account for at least about 85% by weight of fraction A and fraction C of Quillaja saponaria molina, respectively.

143. The immunogenic composition according to claim 139, wherein, in the saponin adjuvant, fraction A of Quillaja saponaria molina and fraction C of Quillaja saponaria molina account for 50 to 96% by weight of fraction A, and fraction C of Quillaja saponaria molina account for the remainder.

144. An immunogenic composition according to any one of claims 137 to 143, comprising approximately 25 μg to approximately 100 μg of an adjuvant.

145. An immunogenic composition according to any one of claims 137 to 144, comprising approximately 50 μg of adjuvant.

146. The immunogenic composition according to any one of claims 139 to 1145, wherein the second, third, fourth, or fifth CoV S glycoprotein comprises one or more modifications at amino acids 180, 252, 253, 444, 478, 486, or 521 compared to the first CoV S glycoprotein, and the one or more modifications are numbered according to the CoV S glycoprotein of SEQ ID NO:

1.

147. (i) Amino acid 180 is glutamic acid or valine, (ii) Amino acid 252 is glycine or valine, (iii) Amino acid 253 is aspartic acid or glycine, (iv) Amino acid 444 is lysine or threonine, (v) Amino acid 478 is threonine, arginine, or lysine. (vi) Amino acid 486 is phenylalanine, proline or serine, and (vii) The immunogenic composition according to claim 146, wherein amino acid 521 is proline or serine.

148. An immunogenic composition comprising at least two CoV S glycoproteins.

149. The immunogenic composition according to claim 148, wherein one CoV S glycoprotein has an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:

87.

150. The immunogenic composition according to claim 148 or 149, wherein one CoV S glycoprotein has an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs. 260, 274, and 284.

151. The immunogenic composition according to any one of claims 148 to 150, wherein one CoV S glycoprotein comprises an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with any one of SEQ ID NOs: 245, 250, 255, 260, 265, 269, 273, 274, 277, and 284.

152. An immunogenic composition according to any one of claims 148 to 151, comprising two CoV S glycoproteins.

153. An immunogenic composition according to any one of claims 148 to 151, comprising three CoV S glycoproteins.

154. An immunogenic composition according to any one of claims 148 to 151, comprising four CoV S glycoproteins.

155. An immunogenic composition according to any one of claims 148 to 151, comprising five CoV S glycoproteins.

156. The immunogenic composition according to any one of claims 148 to 155, wherein the second CoV S glycoprotein comprises 1 to 60 modifications compared to the first CoV S glycoprotein.

157. The immunogenic composition according to any one of claims 153 to 156, wherein the third CoV S glycoprotein comprises 1 to 60 modifications compared to the first CoV S glycoprotein.

158. The immunogenic composition according to any one of claims 154 to 157, wherein the fourth CoV S glycoprotein comprises 1 to 60 modifications compared to the first CoV S glycoprotein.

159. The immunogenic composition according to any one of claims 155 to 158, wherein the fifth CoV S glycoprotein comprises 1 to 60 modifications compared to the first CoV S glycoprotein.

160. The immunogenic composition according to any one of claims 148 to 155, wherein the second CoV S glycoprotein comprises 1 to 10 modifications compared to the first CoV S glycoprotein.

161. The immunogenic composition according to any one of claims 153 to 156, wherein the third CoV S glycoprotein comprises 1 to 10 modifications compared to the first CoV S glycoprotein.

162. The immunogenic composition according to any one of claims 154 to 157, wherein the fourth CoV S glycoprotein comprises 1 to 10 modifications compared to the first CoV S glycoprotein.

163. The immunogenic composition according to any one of claims 155 to 158, wherein the fifth CoV S glycoprotein comprises 1 to 10 modifications compared to the first CoV S glycoprotein.

164. The immunogenic composition according to any one of claims 148 to 163, wherein at least one of the CoV S glycoproteins includes an inactive furin cleavage site.

165. The immunogenic composition according to claim 164, wherein the inactive furin cleavage site comprises the amino acid sequence QQAQ (SEQ ID NO: 7).

166. The immunogenic composition according to any one of claims 148 to 165, wherein at least one of the CoV S glycoproteins comprises mutations to proline at amino acids 986 and 987, and the CoV S glycoprotein sequence is numbered according to Sequence ID No.

1.

167. The immunogenic composition according to any one of claims 148 to 166, wherein all of the CoV S glycoproteins in the composition include inactive furin cleavage sites.

168. The immunogenic composition according to claim 167, wherein the inactive furin cleavage site comprises the amino acid sequence QQAQ (SEQ ID NO: 7).

169. The immunogenic composition according to any one of claims 148 to 168, wherein all of the CoV S glycoproteins contain mutations to proline at amino acids 986 and 987, and the CoV S glycoprotein sequences are numbered according to Sequence ID No.

1.

170. An immunogenic composition according to any one of claims 148 to 169, comprising 0.1 μg to 25 μg, 0.1 μg to 10 μg, 0.001 μg to 10 μg, 0.001 μg to 25 μg, or 0.1 μg to 5 μg of each CoV S polypeptide.

171. The immunogenic composition according to any one of claims 148 to 170, wherein a first CoV S glycoprotein is present in the composition in an amount of about 1 μg to about 10 μg, and other CoV S glycoproteins are present in an amount less than 1 / 2, less than 1 / 3, less than 1 / 4, less than 1 / 5, less than 1 / 6, less than 1 / 7, less than 1 / 8, less than 1 / 9, less than 1 / 10, less than 1 / 25, less than 1 / 50, less than 1 / 75, or less than 1 / 100 of the first CoV S glycoprotein.

172. The immunogenic composition according to any one of claims 148 to 171, wherein the amount of the second CoV S glycoprotein in the composition is less than the amount of the first CoV S glycoprotein in the composition.

173. The immunogenic composition according to any one of claims 153 to 172, wherein the amount of the third CoV S glycoprotein in the composition is less than the amount of the first CoV S glycoprotein in the composition.

174. The immunogenic composition according to any one of claims 154 to 173, wherein the amount of the fourth CoV S glycoprotein in the composition is less than the amount of the first CoV S glycoprotein in the composition.

175. The immunogenic composition according to any one of claims 155 to 174, wherein the amount of the fifth CoV S glycoprotein in the composition is less than the amount of the first CoV S glycoprotein in the composition.

176. An immunogenic composition according to any one of claims 148 to 175, comprising an adjuvant.

177. The immunogenic composition according to claim 176, wherein the adjuvant is a saponin adjuvant.

178. The saponin adjuvant comprises at least two iscom particles, The first iscom particle contains fraction A of Quillaya Saponaria Molina, but does not contain fraction C of Quillaya Saponaria Molina, and The immunogenic composition according to claim 177, wherein the second iscom particle contains fraction C of Quillaja saponaria molina and does not contain fraction A of Quillaja saponaria molina.

179. The immunogenic composition according to claim 178, wherein fraction A of Quillaja saponaria molina and fraction C of Quillaja saponaria molina account for about 85% by weight and about 15% by weight, respectively, of the total weight of fraction A of Quillaja saponaria molina and fraction C of Quillaja saponaria molina in the saponin adjuvant.

180. The immunogenic composition according to claim 178, wherein fraction A of Quillaja saponaria molina and fraction C of Quillaja saponaria molina account for approximately 92% by weight and approximately 8% by weight, respectively, of the total weight of fraction A of Quillaja saponaria molina and fraction C of Quillaja saponaria molina in the saponin adjuvant.

181. The immunogenic composition according to claim 178, wherein, in the saponin adjuvant, fraction A of Quillaja saponaria molina and fraction C of Quillaja saponaria molina account for at least about 85% by weight of fraction A and fraction C of Quillaja saponaria molina, respectively.

182. The immunogenic composition according to claim 178, wherein, in the saponin adjuvant, fraction A of Quillaja saponaria molina and fraction C of Quillaja saponaria molina account for 50 to 96% by weight of fraction A, and fraction C of Quillaja saponaria molina account for the remainder.

183. An immunogenic composition according to any one of claims 148 to 182, comprising approximately 25 μg to approximately 100 μg of an adjuvant.

184. An immunogenic composition according to any one of claims 148 to 183, comprising approximately 50 μg or 75 μg of an adjuvant.

185. The immunogenic composition according to any one of claims 148 to 184, wherein one or more of the second, third, fourth, or fifth CoV S glycoproteins comprises one or more modifications at amino acids 180, 252, 253, 444, 478, 486, or 521 compared to the first CoV S glycoprotein, and the one or more modifications are numbered according to the CoV S glycoprotein of SEQ ID NO:

1.

186. (i) Amino acid 180 is glutamic acid or valine, (ii) Amino acid 252 is glycine or valine, (iii) Amino acid 253 is aspartic acid or glycine, (iv) Amino acid 444 is lysine or threonine, (v) Amino acid 478 is threonine, arginine, or lysine. (vi) Amino acid 486 is phenylalanine, proline or serine, and (vii) The immunogenic composition according to claim 185, wherein amino acid 521 is proline or serine.

187. (i) One or more non-natural SARS-CoV-2 S glycoproteins having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with any one of sequence numbers 87, 260, 222, 227, 274, and 284, (ii) A pharmaceutically acceptable buffer and An immunogenic composition containing [a specific substance].

188. The immunogenic composition according to claim 187, comprising a non-natural SARS-CoV-2 S glycoprotein having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with SEQ ID NO:

274.

189. The immunogenic composition according to claim 187 or 188, comprising approximately 1 μg to approximately 25 μg of SARS-CoV-2 S glycoprotein.

190. An immunogenic composition according to any one of claims 187 to 189, comprising a saponin adjuvant.

191. An immunogenic composition according to any one of claims 187 to 190, comprising approximately 25 to approximately 100 μg of saponin adjuvant.

192. An immunogenic composition according to any one of claims 187 to 190, comprising approximately 25 to approximately 50 μg of saponin adjuvant.

193. The immunogenic composition according to any one of claims 187 to 192, comprising nanoparticles containing one or more SARS-CoV-2 S glycoproteins and a nonionic surfactant core.

194. The immunogenic composition according to claim 193, wherein the nonionic surfactant is selected from the group consisting of polysorbate-20 (PS20), polysorbate-40 (PS40), polysorbate-60 (PS60), polysorbate-65 (PS65), and polysorbate-80 (PS80).

195. The aforementioned saponin adjuvant is (i) A first iscom particle containing fraction A of Quillaya Saponaria Molina and not containing fraction C of Quillaya Saponaria Molina, (ii) A second iscom particle containing fraction C of Quillaya Saponaria Molina and not containing fraction A of Quillaya Saponaria Molina An immunogenic composition according to any one of claims 187 to 194, comprising the above.

196. The immunogenic composition according to claim 195, wherein fraction A of Quillaja saponaria molina and fraction C of Quillaja saponaria molina account for about 85% by weight and about 15% by weight, respectively, of the total weight of fraction A of Quillaja saponaria molina and fraction C of Quillaja saponaria molina in the saponin adjuvant.

197. A method for stimulating an immune response in a human to SARS-CoV-2 or a heterologous SARS-CoV-2 strain, comprising administering an immunogenic composition according to any one of claims 187 to 196.

198. The immunogenic composition according to claim 1, wherein the first CoV S glycoprotein includes an inactive furin cleavage site.

199. The immunogenic composition according to claim 198, wherein the inactive furin cleavage site comprises the amino acid sequence QQAQ (SEQ ID NO: 7).

Images