SARS-COV 2 vaccine compositions and methods of using same

Abstract

Disclosed herein arc vaccine compositions, in particular, polyvalent icosahedral compositions for presentation of a SARS-CoV-2 antigen. The disclosed compositions may contain an S particle comprising a norovirus (NoV) S domain protein and a SARS-CoV-2 antigen, which may be linked via a linker protein domain operatively connected to the norovirus S domain protein and the SARS- CoV-2 antigen. Methods of using the disclosed vaccine composition are also provided.

Description

SARS-COV 2 VACCINE COMPOSITIONS AND METHODS OF USING SAMECROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is an international application which claims priority to and benefit of U.S. Patent Application Serial No. 63 / 472,958, filed June 14, 2023, the contents of which are incorporated in their entirety for all purposes.REFERENCE TO SEQUENCE LISTING

[0002] A Sequence Listing submitted as an XML text file via Patent Center is hereby incorporated by reference. The name of the XML file for the Sequence Listing is CHMC_2022- 0301b_SequenceListing.xml, the date of the creation of the XML file is June 13, 2024, and the size of the XML file is 14,400 bytes.BACKGROUND

[0003] The newly emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes the coronavirus disease 2019 (COVID- 19), is a member of the beta-CoV genus in the family Coronaviridae. It is a single- stranded, positive-sense RNA virus covered by a lipid envelope with trimcric spikes decorating the viral surface. COVID-19 symptoms may include cough, headache, dyspnea, myalgia, fever, and pneumonia, which can be mild, severe, or fatal (1-3). As of this writing, the continued CO VID- 19 pandemic has resulted in over 676 million confirmed cases with at least 6.7 million deaths globally (4). In the USA alone, more than 103 million cases have been diagnosed, and the pandemic has claimed over one million lives. Hence, COVID-19 remains a serious global public health threat, calling urgently for improved medical countermeasures.

[0004] The SARS-CoV-2 viral spikes are assembled as trimeric fusion spike proteins that consists of two sections, the head region SI and the stalk moiety S2, playing important roles in viral receptor binding and host cell attachment, as well as viral fusion and entry, respectively (5). The exterior SI contains the receptor binding domains (RBDs) that recognize a membrane -bound protein named angiotensin-converting enzyme 2 (ACE2) (5-8). The RBD has also been shown to bind heparin / heparan sulfate glycans (9) and oligosaccharides containing sialic acid, withpreference for monosialylated gangliosides (10), to facilitate SARS-CoV-2 infection (11 , 12). Accordingly, many RBD-spccific neutralizing monoclonal antibodies (mAbs) have been identified (13-16), supporting the RBD as an excellent vaccine target. In fact, several recombinant RBD- based subunit COVID vaccines have been under development, with the most advanced one consisting of RBD dimers (ZF2001) (17, 18) being approved for use in China (19). Another well- developed RBD-based vaccine contains RBD monomers formulated with 3M-053-alum adjuvant (20). In addition, several studies have been performed to display the RBDs using polyvalent nanoparticle or virus-like particle platforms to enhance the immune response of the RBD-based vaccine candidates [reviewed in (21, 22)].

[0005] As a single-stranded RNA virus, SARS-CoV-2 evolves rapidly with frequent emergence of new variants (23, 24). Since the beginning of the COVID- 19 pandemic in 2019, many new variants have been identified and several have been closely monitored, including alpha, beta, gamma, delta, epsilon, eta, iota, kappa, mu, zeta, and omicron (25). The delta variant with specific mutations in the RBD antigen showed higher infectivity with higher mortality than the ancestral Wuhan strain and was predominant globally (26), before the emergence of the omicron variant. The Omicron variant, on the other hand, with further mutation of the RBD antigen has been reported for its higher infectivity, and reduced virulence than the delta variant (27).

[0006] The currently approved COVID- 19 vaccines, including mRNA vaccines, viral vectored vaccines, inactivated vaccines, and recombinant protein vaccines, have been shown to be effective and have saved millions of lives. However, issues and concerns associated with these vaccines have also been noted. For example, they show significantly lower efficacy toward the heterologous delta variant (28-31), and vaccine-elicited immunity wanes rapidly over time (32-35). As SARS- CoV-2 will constantly evolve and new variants will continue to emerge, new vaccine strategies to circumvent these issues are warranted.BRIEF SUMMARY

[0007] Disclosed herein are vaccine compositions, in particular, polyvalent icosahedral compositions for presentation of a SARS-CoV-2 antigen. The disclosed compositions may contain an S particle comprising a norovirus (NoV) S domain protein and a SARS-CoV-2 antigen, which may be linked via a linker protein domain operatively connected to the norovirus S domain proteinand the SARS-CoV-2 antigen. Methods of using the disclosed vaccine composition are also provided.BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Those of skill in the art will understand that the drawings, described below, are for illustrative purposes only. The drawings are not intended to limit the scope of the present teachings in any way.

[0009] FIGS. 1A-1F. Production and characterization of the S-RBD protein. (1A) Schematic construct of the Hisx6 tagged S-RBD fusion protein. S, modified norovirus (NoV) S domain; hinge / linker, the hinge region of NoV VP1 and an added linker; RBD, the receptor binding domain of SARS-CoV-2. (IB) SDS-PAGE of purified S-RBD protein. M, protein standards with indicated molecular weights. (1C) A gel-filtration elution curve of the S-RBD protein through a size exclusion column (Superdex 200). The single major PVNP elution peak and the minimal S-RBD monomer peak (arrow) are indicated. The two elution peaks were calibrated using the P24-VP8* nanoparticles (1,290.8 kDa) (67) and GST dimers (52 kDa) (42), which are shown by a red dashed line with indications of the elution positions of the two proteins (star symbols). Y-axis shows UV (A280) absorbances (mAU), while X-axis indicates elusion volume (mL). (ID and IE) CsCl density gradient centrifugation of the S-RBD protein. (ID) A photograph of the CsCl density gradient after centrifugation showing a protein band (arrow). (IE) 23 fractions of the gradient with their relative amounts of S-RBD protein determined by EIA assays showing the peak value in fraction 16 (Fl 6). X-axis indicates the fraction numbers from the bottom to the top of the gradient, while Y-axis shows signal intensities in optical density (OD450) with a red dashed line indicating the cut-off signal at OD = 0.1. (IF and 1G) Two TEM micrographs showing the self-assembled S- RBD PVNPs from the major peak fraction of the gel filtration (IF) and from the peak fraction (Fl 6) of the CsCl density gradient centrifugation (1G).

[0010] FIGS. 2A-2H. Production and characterization of CHO cell expressed S-RBD PVNPs. (2A) Schematic construct of the tag-free S-RBD protein. Sig, a secretory signal peptide; S, modified norovirus (NoV) S domain; hinge / linker, the hinge region of NoV VP1 with an added linker; RBD, the receptor binding domain of SARS-CoV-2. (2B) Purification of the S-RBD PVNP by a sucrose gradient centrifugation. After centrifugation, the gradient was fractionated into 29portions and their relative amounts of the S-RBD protein were measured by ETA assays using commercial antibody against the RBD. X-axis indicates the fraction numbers from bottom to top of the gradient, while Y-axis shows signal intensity of the S-RBD protein in optical density (OD450). (2C and 2D) SDS-PAGE (2C) and western blot (2D) analyses of the two peak fractions, F6 and F9, of the sucrose gradient. The harvested culture medium (CM) was included in (2C) for comparison. M, protein standard with indicated molecular weights. (E to H) Two negative staining TEM micrographs of PVNPs from the two peak fractions, F6 (2E) and F9 (2F), respectively of the sucrose gradient. Typical S-RBD PVNPs that are focused on the surface showing the protrusions [indicated by dark (red) arrows in (2E) and (2F)] are enlarged in (2G), while other images that are focused in the center showing both the inner shell and the outward protrusions [indicated by light (yellow) arrows in (2E) and (2F)] are enlarged in (2H).

[0011] FIGS. 3A-3L Structural modeling of the S-RBD PVNPs and characterization of their antigenic, receptor binding, and immunogenic properties. (3 A to 3F) A 3D structural model of the S60-RBD PVNP. The model was built based on the previously solved cryoEM map of the S60- VP8* PVNP (36) using the known RBD crystal structure (6) with help of the UCSF ChimeraX software. (3A) Surface representations of the S60 nanoparticle in full view. The 60 exposed S domain termini are shown in green. (3B and 3C) Surface representations of the S60-RBD PVNP in full (3B) and section view (3C), respectively. (3D to 3F) Cartoon representations of above three structures, including the S60 nanoparticle (3D), the full (3E) and section view (3F) of the S60- RBD PVNP. The S60 nanoparticles in all images are shown in orange, while the RBDs are shown in cyan in (3B and 3C) or green in (3E and 3F). All images are shown at the five-fold axes. (3G to 31) Authentic antigenic reactivity, receptor binding function, and improved immune response of the PVNP displayed RBDs. (3G) Antigenic reactivity of the S60-RBD PVNPs to commercial RBD-specific antibody in EIA assays. X-axis shows serially diluted PVNPs at indicated concentrations before (S60-RBD) and after (heated S60-RBD) heat treatment, using the S60 nanoparticle (S60) as a negative control, while Y-axis shows EIA signal intensity in optical density (OD450). (H) Binding of the S60-RBD PVNPs to its ACE2 receptor in EIAs. X-axis shows serially diluted PVNPs (S60-RBD) at indicated concentrations, using the S60 nanoparticle (S60) as a negative control, while Y-axis shows the binding signals in optical density (OD450). (31) Serum IgG response of the S60-RBD PVNPs mice. X-axis indicates the three immunogens, the S60-RBDPVNP (S60-RBD), free RBD protein (RBD), and the S60 nanoparticle (S60). Y-axis indicates RBD-spccific IgG titers.

[0012] FIGS. 4A-4L Generation and characterization of the S-NTD PVNPs. (4A) Schematic construct of Hisx6 tagged S-NTD protein. S, modified norovirus S domain; hinge / linker, the hinge of norovirus VP1 with an added linker; NTD, the N-terminal domain of SARS-CoV-2. (4B) SDS- PAGE of purified S-NTD protein (~58 kDa, arrow). M, protein standards with indicated molecular weights. (4C) A gel-filtration elution curve of the S-NTD protein through a size exclusion column. The single major PVNP elution peak and a minor S-NTD monomer peak (arrow) are indicated. The elution peaks were calibrated using the P24-VP8* nanoparticles (1,290.8 kDa) (67) and GST dimers (52 kDa) (42) , which is shown by a red dashed line with indications of the elution positions of the two proteins (star symbols). Y-axis reveals UV (A280) absorbances (mAU), while X-axis shows elusion volume (m ). (4D) A TEM micrograph of the major elution peak of the gel filtration (4C) showing self-assembled the S-NTD PVNPs. (4E and 4F) CsCl density gradient centrifugation of the S-NTD protein. After centrifugation a photo of the CsCl density gradient (4E) shows a protein band (arrow). Relative amounts of the S-NTD protein in the 23 fractions of the gradient were shown with a peak centered at fraction F17 (4F). X-axis indicates the fraction numbers from the bottom to the top of the gradient, while Y-axis shows the signal intensities of the S-NTD protein in optical density (OD). A red dashed line indicates the cut-off signal at OD = 0.1. (4G) A TEM micrograph of the peak fraction (Fl 7) of the CsCl density gradient revealing typical S60-NTD PVNPs. (4H) Antigenic reactivity of the S-NTD PVNPs to commercial antibody against SARS- CoV 2 spike protein in EIA. X-axis displays serially diluted S60-NTD PVNPs (S60-NTD, green columns) at indicated concentrations, using the S60 nanoparticle as a negative control (S60, red columns). Y-axis shows EIA signal intensity in OD. (41) Immune responses of the S60-NTD PVNPs in balb / c mice. X-axis shows two immunogens, the S60-NTD PVNP (S60-NTD) and the S60 nanoparticle (S60). Y-axis indicates NTD-specific IgG titers.

[0013] FIGS. 5A-5F. Immune responses and protective efficacies of the S60-RBD and S60- NTD PVNPs in the K18-hACE2 transgenic mice. (5A) Schematic illustration of the immunization, blood / serum collection, and virus challenge schedules. Mice were vaccinated (V) at nine, seven, and four weeks before SARS-CoV-2 challenge (Ch). Blood / serum samples (S) were collected at five different time points after S60-RBD and S60-NTD PVNP immunizations, including twoweeks post-prime; one- and three-weeks post-boost 1 ; as well as one- and four-weeks post-boost 2. (5B and 5C) Immune responses of scrum IgG (B) and scrum neutralizing antibody (nAb) (5C) after PVNP immunizations. Y-axis indicates RBD / NTD specific IgG titers (B), or SARS-CoV-2 neutralizing antibody titer (5C). X-axis indicates the five serum collection time points after immunizations. The two vaccine candidates, the S60-RBD PVNP (S60-RBD, red) and S60-NTD PVNP (S60-NTD, blue), and the platform control, the S60 nanoparticle (S60, green) are shown by different colors. Limit of detection (LOD) was indicated by dashed lines. Statistic differences between data groups were shown by “ns” (non- significant) for P-values >0.05, (significant) for P-values <0.05, “**” (highly significant) for P-values <0.01, or “***” (extremely significant) for P-values < 0.001. (5D to 5F) Protective efficacies of the S60-RBD and S60-NTD PVNPs in K18-hACE2 mice challenged with SARS-CoV-2 virus. (5D and 5E) Survival curves (5D) and body weight change curves (5E) of mice after immunization with the S60-RBD PVNPs (S60- RBD, red lines), S60-NTD PVNPs (S60-NTD, blue lines), or S60 nanoparticle (S60, green lines), followed by challenge with SARS-CoV-2 WA1 / 2020 strain at lethal dose. X-axis shows days post infection (DPI), whereas Y-axis indicates survival rates (5D) or % change compared to 0 DPI (E). Each data point in (5E) was the mean value of all survival mice. The standard deviations of each data point are indicated in one direction. * (P < 0.05) indicates a statistically significant difference. (5F) organ virus loads of mice after PVNP immunization and viral challenge. At 4 DPI three mice per group were euthanized to assess viral loads in lung and brains. Y-axis indicates loglO viral titers in pfu / g. X-axis shows three experiment groups that was vaccinated with S60-RBD PVNP (red columns), S60-NTD PVNP (blue columns), and S60 nanoparticle (green columns), respectively.DETAILED DESCRIPTIONDEFINITIONS

[0014] Unless otherwise noted, terms are to be understood according to conventional usage by those of ordinary skill in the relevant art. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein may be used in practice or testing of the present invention. All publications, patent applications, patents and other references mentionedherein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein arc illustrative only and not intended to be limiting. The methods may comprise, consist of, or consist essentially of the elements of the compositions and / or methods as described herein, as well as any additional or optional element described herein or otherwise useful in S ARS- CoV-2 vaccine compositions and methods of using same.

[0015] As used herein and in the appended claims, the singular forms “a,” "‘and,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a method” includes a plurality of such methods and reference to “a dose” includes reference to one or more doses and equivalents thereof known to those skilled in the art, and so forth.

[0016] The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, “about” may mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, “about” may mean a range of up to 20%, or up to 10%, or up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term may mean within an order of magnitude, preferably within 5-fold, and more preferably within 2- fold, of a value. Where particular values are described in the application and claims, unless otherwise stated the term “about” meaning within an acceptable error range for the particular value should be assumed.

[0017] As used herein, the term “effective amount” means the amount of one or more active components that is sufficient to show a desired effect. This includes both therapeutic and prophylactic effects. When applied to an individual active ingredient, administered alone, the term refers to that ingredient alone. When applied to a combination, the term refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, serially or simultaneously.

[0018] The terms “individual,” “host,” “subject,” and “patient” are used interchangeably to refer to an animal that is the object of treatment, observation and / or experiment. Generally, the term refers to a human patient, but the methods and compositions may be equally applicable tonon-human subjects such as other mammals. In some embodiments, the terms refer to humans. In further embodiments, the terms may refer to children.

[0019] As used herein, the term “antigen” may be used interchangeably with the terms “immunogen” and “immunogenic antigen”, as defined below. Technically speaking, an antigen is a substance that is able to combine with the products of an immune response once they are made, but is not necessarily able to induce an immune response (i.e. while all immunogens are antigens, the reverse is not true); however, the antigens that are discussed herein as the subject of the present invention are assumed to be immunogenic antigens, even when referred to as antigens.

[0020] The term “fusion protein” means a protein created through translation of a fusion gene, resulting in a single polypeptide with functional properties derived from each of the original proteins.

[0021] The term “immunity” means the state of having sufficient biological defenses to avoid infection, disease, or other biological invasion by a disease-causing organism.

[0022] The term “immunogenicity” means the ability of an immunogen to elicit a humoral and / or cell-mediated immune response.

[0023] The terms “immunogen” and “immunogenic antigen” mean a specific type of antigen that is able to induce or provoke an adaptive immune response in the form of the production of one or more antibodies.

[0024] The terms “immunogenic response” and “immune response” mean an alteration in the reactivity of an organisms' immune system in response to an immunogen. This can involve antibody production, induction of cell-mediated immunity, complement activation or development of acquired immunity or immunological tolerance to a certain disease or pathogen.

[0025] The terms “immunization” and “vaccination” mean the deliberate induction of an immune response and involve effective manipulation of the immune system's natural specificity, as well as its inducibility. The principle behind immunization is to introduce an antigen, derived from a disease-causing organism, which stimulates the immune system to develop protective immunity against that organism, but wherein the antigen itself does not cause the pathogeniceffects of that organism. The desired outcome of a prophylactic or therapeutic immune response resulting from an immunization may vary according to the disease. For example, an immune response against a pathogen may inhibit or prevent colonization and replication of the pathogen, effecting protective immunity and the absence or reduction of any disease symptoms. A vaccine against pathogens may also be considered effective if it reduces the number, severity, or duration of symptoms; if it reduces the number of individuals in a population with symptoms; or even if it merely reduces the transmission of an infectious pathogen.

[0026] The term “infection” means the invasion of an animal or plant host's body tissues by a pathogen, as well as the multiplication of the pathogen within the body and the body's reaction to the pathogen and any toxins that it may produce.

[0027] The terms “Norovirus,” “NoV”, “Norwalk-like virus,” or “NLV” refer to any virus of the Norovirus genus in the Calicivirus family, and includes, without limitation, the following: Norwalk Virus (“NV”), MOH, Mexico, VA 207, VA 387, 02-1419, C59, VA 115, Hawaii, Snow Mountain, Hillington, Toronto, Leeds, Amsterdam, Idaho Falls, Lordsdale, Grimsby, Southampton, Desert Shield, Birmingham, and White Rivercap. NoVs cause acute gastroenteritis in humans.

[0028] As used herein, the letter “S” means “S domain” when used in the context of the described particles, for example, in S69A-VP4c, which means the S-VP4c protein with an R69A mutation. In other aspects, the nomenclature used may be, for example, S with “69A” denoted as a superscript.

[0029] The term “vaccine” means a biological preparation or composition that improves immunity to a particular disease. Vaccines are examples of immunogenic antigens intentionally administered to induce an immune response in the recipient.

[0030] Noroviruses (NoVs) are members of the Norovirus genus in the family Caliciviridae, causing epidemic acute gastroenteritis in humans with significant morbidity and mortality [4, 5]. Structurally, NoV virions are encapsulated by a protein capsid that is composed of a single major structural protein, the capsid protein or viral protein 1 (VP1). The crystal structures of NoV capsids revealed that NoV VP1 contains two principal domains, the N-terminal shell (S) and the C-terminalprotruding (P) domains, linked by a short hinge. The S domain builds the interior, icosahedral shell supporting the basic scaffold of a NoV virion, while the P domain constitutes the dimeric protrusions to stabilize NoV capsid and recognize cell surface glycans as the host attachment factors or receptors.

[0031] Homotypic interactions of viral capsid proteins are common, driving viral capsid selfformation. By taking advantage of such interactions of the norovirus shell (S) domain that naturally builds the interior shells of norovirus capsids, Applicant has developed methods for the production of 60-valent, icosahedral S60 particles using E. coli as an expression system. The S60 particles include several modifications to the S domain such as an R69A mutation to destroy an exposed proteinase cleavage site. The polyvalent S60 particle with 60 exposed S domain C-termini provides a platform for antigen presentation, leading to enhanced immunogenicity to the displayed antigen for vaccine development.

[0032] Disclosed herein are methods and compositions for immunization of an individual against a SARS-CoV-2 infection. In one aspect, a P particle platform displaying a SARS-CoV-2 antigen is disclosed. In one aspect, a P particle platform displaying a SARS-CoV-2 RBD antigen is disclosed. In a further aspect, a fusion protein comprising a P domain and a SARS-CoV-2 antigen, for example an RBD antigen, is disclosed. Such fusion proteins may be used in the manufacture of the P particle platforms that display the SARS-CoV-2 antigen, which may then be used as the basis of a vaccine composition to illicit an immune response to the selected antigen. In a further aspect, vaccine compositions are disclosed. Such vaccine compositions may comprise the P particles displaying the SARS-CoV-2 antigen, for example the RBD antigen. Monovalent and polyvalent vaccine composition are disclosed. In further aspects, the P platform and vaccine composition may employ a modified norovirus S particle. Yet further, methods of using the P particles displaying a SARS-CoV-2 antigen, and vaccine compositions comprising the P particles displaying a SARS-CoV-2 antigen are disclosed, for example, for the purpose of immunizing an individual against a SARS-CoV-2 infection.

[0033] In aspects, a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine is disclosed, the vaccine comprising a non-replicating pseudovirus nanoparticle (PVNP). The PVNP may comprise (display) a receptor-binding domain of SARS-CoV-2 spike (S-RBD). ThePVNP may comprise an icosahedral particle comprising a norovirus (NoV) shell (S) domain protein. In aspects, the S domain protein comprises 60 S domain proteins. The S domain protein may comprise a sequence having at least 90% sequence identity to SEQ ID NO: 1, or at least 95% sequence identity to SEQ ID NO: 1, or at least 99% sequence identity to SEQ ID NO: 1. In other aspects, the S domain protein comprises or consists of SEQ ID NO: 1. In aspects, the S-RBD may comprise a sequence having at least 90% sequence identity to SEQ ID NO: 2, or sequence having at least 95% sequence identity to SEQ ID NO: 2, or a sequence having at least 99% sequence identity to SEQ ID NO: 2. 1. In other aspects, the S RBD antigen may comprise or consist of SEQ ID NO: 2. The S-RBD may be fused to the NoV S domain protein. In aspects, the S-RBD retains angiotensin-converting enzyme 2 (ACE2) binding function. In aspects, the SARS-CoV-2 vaccine elicits S-RBD-specific IgG and neutralizing antibodies in an individual administered the SARS- CoV-2 vaccine. In aspects, the SARS-CoV-2 vaccine may be produced via both a prokaryotic and eukaryotic system.

[0034] In a further aspect, a pseudovirus nanoparticle (PVNP) composition comprising an aggregate of fusion proteins forming an icosahedral structure and a nanoparticle shell is disclosed, wherein the fusion protein comprises a modified norovirus (NoV) S domain protein; a receptor binding domain (RBD) antigen of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2); and a peptide linker, the peptide linker connecting the C-terminus of the NoV S domain to the RBD antigen; wherein the modified NoV S domain protein forms the interior of the nanoparticle shell of the PVNP composition; wherein the nanoparticle shell displays 60 exposed C-termini of the S domain in a triangular pattern; wherein 60 RBD antigens are displayed on the surface of the nanopaiticle shell.

[0035] In aspects, the NoV S domain is wildtype at one or more of positions V57, Q58, or S136, and M140. In aspects, the the NoV S domain is wildtypc at all positions V57, Q58, or S136, and M140. In aspects, the the linker comprises or consists of a sequence selected from HHHH (SEQ ID NO: 8), GGGG (SEQ ID NO: 9), GSGS (SEQ ID NO: 10), HHHHHH (SEQ ID NO: 11) and SSRVDGGGGG (SEQ ID NO: 12). In aspects, the linker comprises or consists of GGGG (SEQ ID NO: 9). In aspects, the linker comprises or consists of SSRVDGGGGG (SEQ ID NO: 12). In aspects, the PVNP composition induces substantial immunity to SARS-CoV-2 infection or at least one symptom thereof when administered to a subject.

[0036] In one aspect, a non-replicating SARS-CoV-2 pseudovirus nanoparticle, wherein the SARS-CoV-2 antigen is RBD (S60-RBD-PVNP) is disclosed. The S60-RBD PVNP may comprise a fusion protein, which in turn comprises a modified NoV shell (S) domain, a SARS-CoV-2 RBD antigen as described herein, and a hinge region. In one aspect, the non-replicating S60-RBD PVNP may comprise an S domain 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% sequence identity to the Modified S Domain, Norovirus, genogroup II, genotype 4 sequence, as follows:MKMASNDASPSDGSTANLVPEVNNEVMALEPVVGAAIAAPVAGQQNVIDPWIRNNFV QAPGGEFTVSPANAPGEILWSAPLGPDLNPYLSHLARMYNGYAGGFEVQVILAGNAFTA GKVIFAAVPPNFPTEGLSPSQVTMFPHIIVDVRQLEPVLIPLPDVRNNFYHYNQSNDSTIK LIAMLYTPLRANNAGDDVFTVSCRVLTRPSPDFDFIFLVPPTVE (Modified Shell (S) Domain, Norovirus, genogroup II, genotype 4, SEQ ID NO: 1).

[0037] The SARS-CoV-2 antigen of the S60-RBD PVNP may comprise an RBD antigen as described herein. For example, a suitable SARS-CoV-2 antigen sequence useful for the S60-RBD- PVNP vaccine composition may comprise the RBD domain, GenBank ID: QHR63250.1, as follows:RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFK CYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWN SNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGF QPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF (SEQ ID NO: 2). In one aspect, the SARS-CoV-2 antigen may comprise a sequence having at least about 90%, at least91 %, 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% sequence identity sequence identity to SEQ ID NO: 2.

[0038] In other aspects, the SARS-CoV-2 antigen of the S60-PVNP may comprise an NTD antigen as described herein. For example, a suitable SARS-CoV-2 antigen sequence useful for the S60-PVNP vaccine composition may comprise the N-terminal domain (NTD) GenBank ID: QKM76786.1, spanning from S13 to L303, 291 residues, as follows:

[0039] SQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWF HAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATN VVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQ GNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFS ALEPL VDLPIGINITRFQTLLALH RSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTL (SEQ ID NO: 3).

[0040] For example, the S60-NTD-PVNP fusion protein may comprise a sequence having at least about 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% sequence identity sequence identity to the following sequence, wherein the modified S domain is in plain text, the hinge region is in bold / italic, the NTD region is italicized, and the linker is bold:MKMASNDASPSDGSTANLVPEVNNEVMALEPVVGAAIAAPVAGQQNVIDPWIRNNFV QAPGGEFTVSPRNAPGEILWSAPLGPDLNPYLSHLARMYNGYAGGFEVQVILAGNAFTA GKVIFAAVPPNFPTEGLSPSQVTMFPHIIVDVRQLEPVLIPLPDVRNNFYHYNQSNDSTIK LIAMLYTPLRANNAGDDVFTVSCRVLTRPSPDFDFIFLVPPTVERGSSRVDGGGGGSeCV NLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRF DNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGV YYHKNNKS'WMESEFRVYSSANNCTFEYVSQPFEMDLEGKQGNFKNLREFVFKNIDGYFKIYS KHTPINLVRDLPQGFSALEPEVDLPIGINITRFQTLEALHRSYLTPGDSSSGWTAGAAAYYVGYL QPRTFELKYNENGTITDAVDCALDPESETKCTEHHHHHR (SEQ ID NO: 4).

[0041] In one aspect, the S60-RBD PVNP is a monovalent S60-RBD PVNP displaying a SARS-CoV-2 antigen, for example the RBD antigen. In another aspect, the S60-RBD PVNP maybe a bivalent or polyvalent S60-RBD PVNP displaying more than one variant of the S ARS-CoV- 2 antigen, in which the more than one SARS-CoV-2 antigen comprises at least two, or at least three, or at least four, or more than four different SARS-CoV-2 antigens. In aspects, the different SARS-CoV-2 antigens may be variants of the same antigen, for example RBD antigens in which one or more amino acids are different.

[0042] In one aspect, the S60-RBD PVNP may comprise a fusion protein as disclosed herein. For example, the S60-RBD PVNP may comprise any one or more fusion protein sequence, the fusion protein 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% sequence identity to the following sequence, wherein the modified S domain is in plain text, the hinge region is in bold / italic, the RBD region is italicized, and the linker is bold:

[0043] MKMASNDASPSDGSTANLVPEVNNEVMALEPVVGAAIAAPVAGQQNVIDP WIRNNFVQAPGGEFTVSPRNAPGEILWSAPLGPDLNPYLSHLARMYNGYAGGFEVQVIL AGNAFTAGKVIFAAVPPNFPTEGLSPSQVTMFPHIIVDVRQLEPVLIPLPDVRNNFYHYN QSNDSTlKLlAMLYTPLRANNAGDDVFTVSCRVLTRPSPDFDFI LVPPrVE^GSSRVDGG GGGRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKC YGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCV1AWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEPYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGY QPYRWVLSFELLHAPATVCGPKKSTNLVKNKCVNFHHHHHH (SEQ ID NO: 5).

[0044] In one aspect, the S60-RBD PVNP may be tag-free. In a further aspect, the S60-RBD PVNP may comprise a tag, for example, a HIS tag.

[0045] In one aspect, the S60-RBD PVNP may have a diameter of between about 20 and about 40 nm.

[0046] Further disclosed are fusion proteins. The fusion proteins may comprise an SARS- CoV-2 antigen, a linker region, a hinge region, and an S-domain.

[0047] For example, the fusion proteins may comprise the S-domain, which comprises the hinge region of norovirus, a linker, and a SARS-CoV-2 antigen sequence. The hinge region may comprise the sequence FLVPPTVE (SEQ ID NO: 6).

[0048] In general, the fusion protein may comprise, from the N-terminus to the C-terminus direction, an S domain, a linker, and a SARS-CoV-2 antigen sequence.

[0049] In one aspect, a polyvalent icosahedral particle for antigen presentation, wherein the antigen is a SARS-CoV-2 antigen, is disclosed. The particle may be an S particle, wherein the S particle may comprise a recombinant fusion protein comprising a norovirus (NoV) S domain protein; a linker protein domain operatively connected to the norovirus S domain protein; and an antigen protein domain operatively connected to the linker. The particle may have an icosahedral symmetry structure. In one aspect, the S60-RBD PVNP comprises 60 sites for antigen presentation. In one aspect, the norovirus S domain protein is that of a calicivirus. The calicivirus may be characterized by having 180 copies of a single capsid protein.

[0050] In one aspect, the norovirus S domain protein may comprise a mutation in a proteinase cleavage site of the NoV S domain protein, wherein the mutation renders the site resistant to trypsin cleavage. One or more mutations may be made to the site, provided the mutation effectively destroys the trypsin cleavage site. Modifications to the site that achieve such effect may include a mutation at position 69 or position 70. In one aspect, the mutation may occur at position R69. In certain aspects, the mutation may be a change to any amino acid other than K (lysine), which is sufficient to destroy the proteinase cleavage site. In certain aspects, the mutation is R69A. In other aspects, the mutation may occur at position N70, for example, the mutation may be any amino acid other than P (proline) sufficient to destroy the proteinase cleavage site. In one aspect, the norovirus S domain protein may comprise a wildtype sequence at one more, or two or more, or three or more, or all four of the amino acids at position 57, 58, 136, and 140, with reference to the wild type P Domain sequence, SEQ ID NO: 7.SRTKPFTVPILTVEEMSNSRFPIPLEKLYTGPSSAFVVQPQNGRCTTDGVLLGTTQLSAVN ICTFRGDVTHIAGSHDYIMNLASQNWNNYDPTEEIPAPLGTPDFVGKIQGMLTQTTREDG STRAHKATVSTGSVHFTPKLGSVQYTTDTNNDLQTGQNTKFTPVGVIQDGNNHQNEPQ QWVLPNYSGRTGHNVHLAPAVAPTFPGEQLLFFRSTMPGCSGYPNMNLDCLLPQEWVQ HFYQEAAPAQSDVALLRFVNPDTGRVLFECKLHKSGYVTVAHTGPHDLVIPPNGYFRFD SWVNQFYTLAPMGNGAGRRRAL (SEQ ID NO: 7).

[0051] In one aspect, the norovirus S domain protein is that of a calicivirus, wherein the calicivirus is characterized by having 180 copies of a single capsid protein.

[0052] In one aspect, the linker may comprise an amino acid sequence of a length sufficient to provide space and certain flexibility between the S domain protein particle and the displayed antigens. The linker is typically a short peptide of one to ten amino acid units, or three to six amino acids, that connect the C-terminus of the S domain to the displayed antigens. The linker provides space and certain flexibility between the S60 particle and the displayed antigens, which helps the independent folding of the S domain and the displayed antigens. A longer linker may be used as necessary. The amino acid length of the linker should be sufficient to allow flexibility of the protein domains to form the claimed compositions. In one aspect, the non-replicating S-RBD-PVNP may comprise a linker having a sequence selected from HHHH (SEQ ID NO: 8), GGGG (SEQ ID NO: 9), GSGS (SEQ ID NO: 10), HHHHHH (SEQ ID NO: 11) and SSRVDGGGGG (SEQ ID NO: 12). In one aspect, the linker may comprise the sequence GGGG (SEQ ID NO: 9). In one aspect, the linker may comprise the sequence SSRVDGGGGG (SEQ ID NO: 12).

[0053] The disclosed compositions may be used for presentation of an antigen, in particular, a SARS-CoV-2 antigen. In certain aspects, the antigen may encode for a SARS-CoV-2 antigen having a size of from 8 amino acids up to about 300 amino acids, or from 8 amino acids up to about 400 amino acids, or from 8 amino acids up to about 500 amino acids.

[0054] In one aspect, the polyvalent icosahedral composition may comprise an antigen protein domain that is a SARS-CoV-2 antigen. It will be understood that antigen sequences used to generate the antigen peptide may have at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the reference nucleic acid sequence, provided that the resulting antigen elicits at least a partial immune response in an individual administered the composition having the antigen.

[0055] The recombinant fusion protein may be a subunit of the disclosed vaccine compositions. Further disclosed are recombinant fusion proteins that may form the basis of the polyvalent icosahedral compositions. The fusion protein may comprise a norovirus (NoV) S domain protein having a mutation to the trypsin site as described above; a linker protein domainoperatively connected to the norovirus S domain protein having the aforementioned mutations; and an antigen protein domain operatively connected to the linker.

[0056] In addition to the S particle described above, the disclosed compositions may further comprise one or more pharmaceutically-acceptable carriers, for example, a solvent, dispersion media, coating, stabilizing agent, diluent, preservative, antibacterial and / or antifungal agent, isotonic agent, adsorption delaying agent, adjuvant, or combinations thereof. In one aspect, the disclosed S particles may be provided in physiological saline. Optionally, a protectant may be included, for example, an anti-microbiological active agent, such as for example Gentamycin, Merthiolate, and the like. Stabilizing agents which may be used include saccharides, trehalose, mannitol, saccharose and the like, which may be added in an amount sufficient to increase and / or maintain product shelf-life. The disclosed herein may include known injectable, physiologically acceptable sterile solutions. For preparing a ready-to-use solution for parenteral injection or infusion, aqueous isotonic solutions, such as, for example, saline or a corresponding plasma protein solution may be used. Exemplary diluents may include water, saline, dextrose, ethanol, glycerol, and the like. Exemplary isotonic agents may include sodium chloride, dextrose, mannitol, sorbitol, and lactose, among others. Exemplary stabilizers may include albumin and alkali salts of ethylenediaminetetraacetic acid, among others.

[0057] In one aspect, disclosed is a container, for example a container suited for delivery to an individual in need thereof, for example a capsule, a vial, or a syringe, comprising at least one dose of the immunogenic composition as disclosed herein. The container may comprise, for example, 1 to 250 doses of the immunogenic composition, or in other aspects, 1, 10, 25, 50, 100, 150, 200, or 250 doses of the immunogenic composition. In one aspect, each of the containers may comprise a single dose of the vaccine composition, or more than one dose of the vaccine composition and may further comprises an anti-microbiological active agent. Those agents may include, for example, antibiotics such as Gentamicin and Merthiolate and the like.

[0058] A further aspect relates to a kit. The kit may comprise any of the containers described above and an instruction manual, including the information for the delivery of the immunogenic composition disclosed above. For example, instructions related to intramuscular application of at least one dose may be provided for lessening the severity of clinical symptoms associated with aninfection of an antigen as disclosed here. The kits and / or compositions may further include an immune stimulant such as keyhole limpet hemocyanin (KLH), or incomplete Freund's adjuvant (KLH / ICFA). Any other immune stimulant known to a person skilled in the art may also be used. In one aspect, the adjuvant is aluminum hydroxide.

[0059] In one aspect, a method of making the disclosed polyvalent icosahedral structures is disclosed. The method may comprise the steps of a) making a first region comprising a modified NoV S domain protein, wherein the modification comprises a mutation sufficient to destruct an exposed protease cleavage site (wherein the mutation prevents protein degradation), preferably an R69A mutation, and b) recombinantly expressing the first region having a modified NoV S domain protein with a linker and an antigen. In certain aspects, the composition may be effectively produced in E.coli.

[0060] In one aspect, a method of immunizing an individual in need thereof against a SARS- CoV-2 infection is disclosed. For example, in one aspect, a method of inducing an antigen specific immune response in an individual is disclosed, the method comprising administering any of the vaccine compositions described herein to an individual in an amount effective to produce an antigen specific immune response. In some aspects, the antigen specific immune response may comprise a T cell response. In some aspects, the antigen specific immune response may comprise a B cell response. In some aspects, the method may comprise a single administration of the vaccine composition. In some aspects, the method may further comprise administering a booster dose of the vaccine. In some aspects, the vaccine composition may be administered to the subject by intradermal or intramuscular injection. The method may comprise administering a RV-PNVP or vaccine composition containing an RV-PVNP as disclosed here. In one aspect, the administration may be sufficient to induce a neutralizing antibody titer against the antigen. The disclosed RV- PVNPs may be used to elicit an immune response in an individual in need thereof, comprising administering a RV-PVNP or composition disclosed herein, to the individual. In some aspects, the vaccine is administered to the individual via a route selected from intramuscular administration, intradermal administration and subcutaneous administration. In some aspects, the administering step comprises contacting a muscle tissue of the individual with a device suitable for injection of the composition. In some aspects, the administering step comprises contacting a muscle tissue ofthe subject with a device suitable for injection of the composition in combination with electroporation.

[0061] In aspects, the method of reducing the severity of a SARS-Co-V 2 infection and / or eliciting an immune response in a subject against SARS-Co-V 2 in an individual in need thereof is disclosed, the method comprising administering a SARS-CoV-2 vaccine according to any preceding claim to the individual. In aspects, the administration comprises one dose, or two doses, or three doses of the SARS-CoV-2 vaccine. In aspects, the composition is administered to an individual via a route selected from one or more of intradermal injection, intramuscular injection, or by intranasal administration. In aspects, the administered composition comprises a pharmaceutically acceptable carrier. In aspects, the administered composition comprises one or more of an adjuvant and a preservative.

[0062] Dosing

[0063] In one aspect, provided are methods comprising administering the vaccine compositions to a subject in need thereof. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the disease, the particular composition, its mode of administration, its mode of activity, and the like. Compositions may be formulated in dosage unit form for ease of administration and uniformity of dosage.

[0064] In certain aspects, the compositions may be administered at dosage levels sufficient to deliver from about 0.0001 mg / kg to about 100 mg / kg, from about 0.001 mg / kg to about 0.05 mg / kg, from about 0.005 mg / kg to about 0.05 mg / kg, from about 0.001 mg / kg to about 0.005 mg / kg, from about 0.05 mg / kg to about 0.5 mg / kg, from about 0.01 mg / kg to about 50 mg / kg, from about 0.1 mg / kg to about 40 mg / kg, from about 0.5 mg / kg to about 30 mg / kg, from about 0.01 mg / kg to about 10 mg / kg, from about 0.1 mg / kg to about 10 mg / kg, or from about 1 mg / kg to about 25 mg / kg, of subject body weight per day, one or more times a day, to obtain the desired therapeutic or prophylactic effect. The dosage may be delivered three times a day, two times a day, once a day, every other day, every third day, every week, every two weeks, every three weeks, or every four weeks. In certain aspects, the desired dosage may be delivered using multiple administrations (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen,fourteen, or more administrations). When multiple administrations are employed, split dosing regimens may be used. As used herein, a “split dose” is the division of single unit dose or total daily dose into two or more doses, e.g, two or more administrations of the single unit dose. As used herein, a “single unit dose” is a dose of any therapeutic administered in one dose / at one time / single route / single point of contact, i.e., single administration event. As used herein, a “total daily dose” is an amount given or prescribed in 24 hr period. It may be administered as a single unit dose.

[0065] Dosage Forms

[0066] The vaccine composition described herein may be formulated into a dosage form described herein, such as an intranasal, intratracheal, or injectable (e.g., intravenous, intraocular, intravitreal, intramuscular, intracardiac, intraperitoneal, subcutaneous).

[0067] Liquid Dosage Forms

[0068] Liquid dosage forms for parenteral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and / or elixirs. In addition to active ingredients, liquid dosage forms may comprise inert diluents commonly used in the art including, but not limited to, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, com, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. In certain aspects for parenteral administration, compositions may be mixed with solubilizing agents, alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and / or combinations thereof.

[0069] Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated and may include suitable dispersing agents, wetting agents, and / or suspending agents. Sterile injectable preparations may be sterile injectable solutions, suspensions, and / or emulsions in nontoxic parenterally acceptable diluents and / or solvents, for example, a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employedinclude, but are not limited to, water, Ringer's solution, U.S.P., and isotonic sodium chloride solution. Sterile, fixed oils arc conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides. Fatty acids such as oleic acid can be used in the preparation of injectables.

[0070] Injectable formulations can be sterilized, for example, by filtration through a bacterial- retaining filter, and / or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

[0071] Pulmonary

[0072] Formulations described herein as being useful for pulmonary delivery may also be used for intranasal delivery of a pharmaceutical composition. Another formulation suitable for intranasal administration may be a coarse powder comprising the active ingredient and having an average particle from about 0.2 pm to 500 pm. Such a formulation may be administered in the manner in which snuff is taken, i.e. by rapid inhalation through the nasal passage from a container of the powder held close to the nose.

[0073] Formulations suitable for nasal administration may, for example, comprise from about as little as 0.1 % (w / w) and as much as 100% (w / w) of active ingredient, and may comprise one or more of the additional ingredients described herein. A pharmaceutical composition may be prepared, packaged, and / or sold in a formulation suitable for buccal administration. Such formulations may, for example, be in the form of tablets and / or lozenges made using conventional methods, and may, for example, contain about 0.1% to 20% (w / w) active ingredient, where the balance may comprise an orally dissolvable and / or degradable composition and, optionally, one or more of the additional ingredients described herein. Alternately, formulations suitable for buccal administration may comprise a powder and / or an aerosolized and / or atomized solution and / or suspension comprising active ingredient. Such powdered, aerosolized, and / or aerosolized formulations, when dispersed, may have an average particle and / or droplet size in the range from about 0.1 nm to about 200 nm, and may further comprise one or more of any additional ingredients described herein.

[0074] Coatings or Shells

[0075] Solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating ait. They may optionally comprise opacifying agents and can be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions which can be used include polymeric substances and waxes. Solid compositions of a similar type may be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.

[0076] Multi-Dose and Repeat-Dose Administration

[0077] In some aspects, the vaccine compositions may be administered in two or more doses (referred to herein as “multi-dose administration”). Such doses may comprise the same components or may comprise components not included in a previous dose. Such doses may comprise the same mass and / or volume of components or an altered mass and / or volume of components in comparison to a previous dose. In some aspects, multi-dose administration may comprise repeat-dose administration. As used herein, the term “repeat-dose administration” refers to two or more doses administered consecutively or within a regimen of repeat doses comprising substantially the same components provided at substantially the same mass and / or volume. In some aspects, subjects may display a repeat-dose response. As used herein, the term “repeat-dose response” refers to a response in a subject to a repeat-dose that differs from that of another dose administered within a repeat-dose administration regimen. In some aspects, such a response may be the expression of a protein in response to a repeat-dose comprising the vaccine composition.

[0078] In one aspect, a method of eliciting an immune response, in particular, an immune response to a SARS-CoV-2 antigen, sufficient to deter, prevent, decrease the likelihood of obtaining, reduce the severity of, and / or mitigate a SARS-CoV-2 infection, in an individual in need thereof is disclosed. In this aspect, the method may include the step of administering a vaccine composition as disclosed above to an individual in need thereof. The disclosed compositions may be administered to an individual according to any method known in the art. The vaccine compositions may be administered prophylactically to an individual suspected of having a future exposure to the antigen incorporated into the vaccine composition. In certain aspects, provided isa method of providing an immune response that protects an individual receiving the composition from infection, or reduces or lessens the severity of the clinical symptoms associated from an infection. Dosage regimen may be a single dose schedule or a multiple dose schedule (e.g., including booster doses) with a unit dosage form of the composition administered at different times. The term “unit dosage form,” as used herein, refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of the antigenic compositions disclosed herein in an amount sufficient to produce the desired effect, which compositions are provided in association with a pharmaceutically acceptable excipient (e.g., pharmaceutically acceptable diluent, carrier or vehicle). The vaccine may be administered in conjunction with other immunoregulatory agents.

[0079] The following non-limiting examples are provided to further illustrate embodiments of the invention disclosed herein. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent approaches that have been found to function well in the practice of the invention, and thus may be considered to constitute examples of modes for its practice. However, those of skill in the ail should, in light of the present disclosure, appreciate that many changes may be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.Example 1

[0080] The COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused considerable morbidity and mortality worldwide. Although authorized COVID-19 vaccines have been shown highly effective, their significantly lower efficacy against heterologous variants, and the rapid decrease of vaccine-elicited immunity raises serious concerns, calling for improved vaccine tactics. To this end, disclosed are pseudovirus nanoparticles (PVNPs) displaying the receptor-binding domains (RBDs) of SARS-CoV-2 spike, named S-RBD for vaccination against COVID-19. PVNPs have been described, see, for example, U.S. Patent No. 11,833,198, “Norovirus S particle based vaccines and methods of making and using same,” WO2022177942A1, “Influenza vaccine compositions and methods of using same,” and WO2024102697A1, “Vp4-based trivalent pseudovirus nanoparticle vaccine for rotavirus and methods of using same,” each of which is incorporated in their entirety herein.

[0081] The S-RBD PVNP was produced using both prokaryotic and eukaryotic systems. A three-dimensional structural model of the S-RBD PVNPs was built based on the known structures of the S60 particle and RBDs, revealing an S60 particle-based icosahedral symmetry with multiple surface-displayed RBDs that retain authentic conformations and receptor-binding functions. The PVNP is highly immunogenic, eliciting high titers of RBD-specific IgG and neutralizing antibodies in mice. The S-RBD PVNP demonstrated exceptional protective efficacy, and fully (100%) protected K18-hACE2 mice from mortality and weight loss after a lethal SARS-CoV-2 challenge, supporting the S-RBD PVNPs as a potent CO VID-19 vaccine candidate. By contrast, a PVNP displaying the N-terminal domain (NTD) of SARS-CoV-2 spike exhibited only 50% protective efficacy. Since the RBD antigens of the disclosed PVNP vaccine are adjustable as needed to address the emergence of future variants, and various S-RBD PVNPs can be combined as a cocktail vaccine for broad efficacy, for example the RBD sequences of future emergent variants, as well as other variants such as those from Alpha, Omicron, or other Sars-CoV-2 variants. Thus, these non-replicating PVNPs offer a flexible platform for a safe, effective CO VID- 19 vaccine with minimal manufacturing cost and time.

[0082] Recently, Applicant has established a technology to generate pseudovirus nanoparticles (PVNPs) (36, 37). This was achieved by using the S60 nanoparticle that is self-assembled by 60 norovirus (NoV) shell (S) domain proteins. This 20 nm, T=1 icosahedral particle is produced by expressing modified NoV S proteins in vitro and each S60 nanoparticle contains 60 exposed S domain C-termini. These polyvalent C-termini make the S60 nanoparticle a versatile platform to display antigens, resulting in self-assembled PVNPs with enhanced immunogenicity toward the displayed antigens for novel vaccine development (36, 37). This principle has been proven by generating two PVNPs displaying receptor binding VP8* antigens of rotavirus and the HA1 antigens of influenza (flu) virus as promising rotavirus and flu vaccine candidates, respectively (36-39).

[0083] A new PVNP was created to display the RBD antigens of SARS-CoV-2, referred to as S-RBD. The PVNP can be generated by bacterial and mammalian cell systems, and the displayed RBDs retain original antigenic reactivity and receptor binding function. The three-dimensional (3D) structural models of the PVNPs were constructed using the known structures of the S60 particle and RBDs, showing an icosahedral symmetry with multiple RBD antigens being displayedon the surface. The PVNP is highly immunogenic, inducing high titers of RBD-specific antibodies in mice. A thrcc-dosc vaccination regimen of the PVNP fully (100%) protected hACE2 mice from mortality and weight loss caused by SARS-CoV-2 challenge, supporting efficacy of the S-RBD PVNP as a COVID-19 vaccine candidate.

[0084] Materials and methods

[0085] Plasmid constructs for protein expression in bacteria. Two DNA fragments, each encoding the RBD domain (GenBank ID: QHR63250.1, spanning from R319 to F541, 223 residues, SEQ ID NO: 2), or the N-terminal domain (NTD, GenBank ID: QKM76786.1, spanning from S13 to L3O3, 291 residues, SEQ ID NO: 3) of the SARS-CoV-2 spike protein of the Wuhan strain, were codon optimized for Escherichia coli (E. coli) and synthesized via GenScript (Piscataway, NJ). DNA fragments were cloned into the previously made, pET-24b (Novagen)- based plasmid that was used for the expression of SR69A-VP8* fusion protein (36) by replacing the VP8*-encoding sequences, respectively. Specifically, the RBD or NTD sequences were fused to the C-terminus of the modified NoV S domain, via a linker of SSRVDGGGGG (SEQ ID NO: 12) in between and HHHHHH (SEQ ID NO: 11) at the end, resulting in the S-RBD or S-NTD fusion proteins with a Hisx6 tag at the C-terminus (FIG. 1A and FIG. 4A).

[0086] Protein production using the E. coli system. His-tagged, insoluble S-RBD and S-NTD fusion proteins were expressed using E. coli strain BL21 / DE3 and purified using a denaturing protocol according to HisPurTM Cobalt resin manual (Thermo Fisher Scientific). After overnight induction with 0.4 mM IPTG (isopropyl B-D-l-thiogalactoyranoside), bacteria were collected and suspended in denaturing buffer containing 6M guanidine hydrochloride (Sigma) (200 mM Tributylphosphine; 0.5 M lodoacetamide; 100 mM Ammonium Bicarbonate; 0.2 mg / ml Proteomics Grade Trypsin; pH 8.5). The bacteria were then lysed by sonication and denatured target proteins were purified using the Cobalt resin. The purified proteins were dialyzed overnight serially against phosphate buffer (50 mM Na-PO4, 300 mM NaCl, pH 7.4) containing 6M, 4M, 2M and IM urea, respectively to remove guanidine hydrochloride buffer. For refolding, the proteins in phosphate buffer with IM urea were dialyzed against Redox buffer (100 mM Tris, 400 mM L-arginine, 2 mM EDTA, 5 mM reduced glutathione, 1 mmol / L oxidized glutathione, pH 8.0) for 16 hours.

[0087] Plasmid constructs for mammalian cell expression. The S-RBD fusion protein encoding DNA sequences (sec above) with a secretory signal peptide (MKWVTFISLLFLFSSAYS, (SEQ ID NO: 13) encoding sequence at N-terminus was codon optimized to Chinese hamster ovary (CHO) cell and synthesized de novo (GenScript). Sequences were cloned into the pcDNA 3.4-TOPO vector (Thermo Fisher Invitrogen) with the Kozak consensus sequences at and upstream of the start codon, as suggested by the manufacturer. The plasmid DNA construct free from phenol and sodium chloride was filtered through a 0.22 pm filter to ensure sterility for transfection.

[0088] Protein expression by CHO cells. This was carried out by transient transfection using the ExpiCHO Expression System (Gibco / Thermo Fisher Scientific) based on the manufacturer’s instruction. Briefly, ExpiCHO-S cells were diluted with ExpiCHO Expression Medium to a density of 6xl06viable cells / mL with a viability of >95%. Plasmid DNA was diluted with OptiPRO medium, and ExpiFectamine CHO Reagent was diluted with OptiPRO medium. Then the diluted ExpiFectamine CHO Reagent was mixed with diluted plasmid. After a short (1-5 minutes) incubation, mixture was slowly transferred to the diluted cells with swirling. The cells were then incubated at 37 °C with 8% CO2 on an orbital shaker. Twenty-four hours post transfection, ExpiFectamine CHO Enhancer and ExpiCHO Feed were added to the cells. Eight to ten days post transfection, cell culture was harvested by centrifugation at 5000 rpm (3,450 x g) at 4°C for 20 minutes by the Avanti J-26XP centrifuge (Beckman Coulter) using an JA 14 rotor to separate cells and cell debris from the culture medium.

[0089] Gel filtration chromatography. Gel filtration was conducted to monitor protein complex formation as described elsewhere (40-42) using a Fast Performance Liquid Chromatography system (FPLC, AKTATM pure 25L, GE Healthcare Life Sciences) using a size exclusion column (Superdex 200, 10 / 300 GL, 25 mL bed volume, GE Healthcare Life Sciences). The size exclusion column was calibrated using gel filtration calibration kits (GE Healthcare Life Sciences), whereas the major elution peak of the purified S-RBD / S-NTD protein was compared with that of the formerly prepared SR60R-VP8* nanoparticle (-3.4 mDa

[0033] and GST-dimer (-52 kDa). Relative concentrations of the proteins in the effluents were shown by AR280R absorbance.

[0090] Sucrose gradient centrifugation. PVNPs in culture medium were purified as previously described with NoV VLPs (43). Briefly, 20 mL clarified culture medium with PVNPs was centrifuged at 25,000 rpm (82,700 x g) at 4 °C for two hours using the Optima L-90K ultracentrifuge and the SW28 rotor. The PVNPs in the pellet were resuspended in 1.5 mL lx phosphate buffer saline (PBS, pH 7.4) and was loaded on the top of a step sucrose gradient consisting of 10%, 20%, 30%, 40%, and 50% sucrose in PBS (from top to bottom). After centrifugation at 27,500 rpm (100,000 x g) at 4 °C for two hours using the same the ultracentrifuge and rotor, the gradient was fractionated and the PVNPs in the fractions were detected by EIA assays (see below) using an antibody specific to NoV VLPs (44), followed by an SDS-PAGE and western blot analyses, as well as transmission electron microscopy (TEM) inspection (see below).

[0091] Cesium chloride (CsCl) density gradient ultracentrifugation. This approach was utilized to further characterize the S-RBD and S-NTD PVNPs as described previously (36, 37). Briefly, 0.5 mL of the resin purified S-RBD or S-NTD PVNP was mixed with 10 mL of CsCl solution with a density of 1.3630. The mixture was loaded in a tube and centrifuged at 4 °C for 45 hours at 41,000 rpm (207,500 x g) utilizing the Optima L-90K ultracentrifuge (Beckman Coulter). Through bottom puncture of the tube, the CsCl gradient was fractionated into 23 fractions with about ~0.5 mL each. Sample from each fraction was diluted 200-fold in PBS and coated on microtiter plates (Thermo Scientific). The relative amounts of the PVNP were measured utilizing in-house antibody against NoV VLP (44) or commercially available antibody against SARS-CoV- 2 spike (Sino Biological, Cat: 40150-D006). The CsCl fraction densities were measured based on refractive index.

[0092] Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blot. Purified recombinant proteins were analyzed by SDS-PAGE using 10% separating gels. For western blot, the proteins after SDS-PAGE were transferred to a nitrocellulose membrane, on which the S-RBD protein was detected using guinea pig antibody against NoV VLPs (44). Fluorescence labeled donkey antibody against guinea pig IgG (IRdye 680RD, LLCOR Biosciences) was used as secondary antibody to show the S-RBD protein specific signals that were recorded using an Odyssey CLx imager (LI-COR Biosciences).

[0093] Transmission electron microscopy (TEM). Negative stain TEM was used to examine the morphology of the S-RBD / S-NTD PVNPs. PVNPs in samples from gel filtration, CsCl density gradient, or sucrose gradient in 6.0 F volume were absorbed to a grid (FCF200-CV-50, Electron Microscopy Sciences) for 20 minutes in a humid chamber and were then negatively stained with 1% ammonium molybdate. After washed and air dried, the grids were inspected using a Hitachi microscope (model H-7650) at 80 kV for a magnification between 15,000x and 70,000x as described previously (36).

[0094] Structural modeling of S-RBD PVNPs. Two 3D structural models of S-RBD PVNPs were built with help of the UCSF ChimeraX software (version E4) (45) based on the known cryoEM (cryogenic electron-microscopy) density map of the S60-VP8* nanoparticle (36) using the crystal structures of the inner shell of the 60-valent VLPs of feline calicivirus (FCV) (PDB code: 4PB6) (46) and GII.4 NoV (to be published), as well as the RBD of SARS-CoV-2 (PDB code: 6M17) (6).

[0095] Enzyme immunoassays (EIAs). EIAs were performed for two purposes, 1) to examine the antigenic reactivity of the S-RBD / S-NTD PVNPs to specific antibodies, and 2) to determine mouse antibody titers elicited by S-RBD / S-NTD PVNPs as immunogens, using previously described methods (47, 48). For the first purpose, purified PVNPs were coated on 96-well microtiter plates (Thermo Scientific) at 1 pg / mL overnight. After blocking using 5% (w / v) skim milk, the coated PVNPs were incubated with related commercial antibodies specific to SARS- CoV-2 spike protein (Sino Biological, Cat: 40150-D006), or in-house antibody against NoV VLPs (44). The bound antibodies were detected by corresponding secondary antibody conjugated with horse radish peroxidase (HRP) (ThermoFisher Scientific). The HRP conjugates were shown by tetramethylbenzidine (TMB) substrate (ThermoFisher Scientific) using 0.16M sulfuric acid as stopping reagent. The positive signals were defined as OD450 = 0.15. For the second purpose, baculovirus expressed spike proteins (provided by Dr. Dave Hildeman at Cincinnati Children’s Hospital Medical Center (49)) were coated on plates at 1 pg / mL overnight. The plates were then incubated with mouse sera at serial dilutions. The bound antibodies were detected by goat- antimouse IgG-HRP conjugate (ThermoFisher Scientific) IgG titers were defined as end-point dilutions with cut-off signals of OD450 = 0.15.

[0096] EIA-based receptor binding assay. EIAs were also used to measure binding of the PVNP-displaycd RBD to host receptors. To this end, the commercial recombinant ACE2 protein (Sino Biological, Cat: 1O1O8-HO8H) was used as the SARS-CoV-2 receptor. The ACE2 protein at 1 ng / pL was coated on microtiter plates overnight. After blocking with 5% nonfat milk, the coated wells were incubated with purified S60-RBD PVNPs at ~2 ng / pL. The bound PVNPs were detected by antibody against NoV VLPs, followed by an incubation with the goat-anti-rabbit IgG- HRP conjugate (ThermoFisher Scientific) at 1:5000 dilution. The HRP conjugates were shown by tetramethylbenzidine (TMB) substrate (ThermoFisher Scientific) using 0.16M sulfuric acid as stopping reagent. Signals were shown in optical density (OD) at wavelength 450 nm.

[0097] Immunization of PVNP in mice. Specific pathogen free BALB / c mice at about 6 weeks of age from Charles River Laboratories were maintained under specific pathogen-free conditions at the Division of Veterinary Services of Cincinnati Children’s Hospital Medical Center. Mice were randomly divided into five groups with 6 to 8 mice each (N=6-8). Animals in each group were administered with each of the following immunogens: 1) S-RBD PVNPs, 2) S-NTD PVNPs; (3) recombinant RBD protein (Sino Biological) as a free antigen control; (4) S60 nanoparticle as a platform control (S60); (5) phosphate buffer saline (PBS, pH 7.4) as immunogen diluent control (PBS); Immunogens were administered with Alum adjuvant (Thermo Scientific, 25 pL / dose with 1:1 of antigen diluents) mixed with antigens at 15 pg / mouse / dose. Immunogens in ~50 pL were administered by intramuscular injection in the thigh muscle three times at two-week intervals. Blood samples were collected two weeks after the final injection via exsanguination for sera preparation (50).

[0098] Evaluating vaccine efficacy in a K18-hACE2 murine model. The K18-hACE2 transgenic murine model of SARS-CoV-2 infection and disease (51, 52) was employed to explore the efficacy of the PVNP vaccines. Mice (n-9 / group) were intramuscularly immunized with S- RBD PVNPs or S-NTD PVNPs at 15 pg / mouse / dose with Alum adjuvant (Thermo Scientific, 50 pL / dose with antigen diluents at 1:1 ratio) nine, seven, and four weeks prior to challenge. The S60 nanoparticle at the same dose with the same adjuvant was used as a control for comparison. Mice were bled two weeks post-prime, one- and three- weeks post-boost 1, as well as one- and four- weeks post-boost 2 for sera (FIG. 5A) to measure serum IgG and neutralizing antibody (nAb) titers. nAb titers were measured by 50% PRNT (PRNT50) on Vero E6 cells as previously described(53). Mice were intranasally challenged with 2 x 105 PFU of SARS-CoV-2 (WA 1 / 2020 strain) in each narc and monitored daily for survival, weight loss, and signs of disease.

[0099] Statistical analysis. Statistical differences between two data groups were calculated in GraphPad Prism 7.0 (GraphPad Software, Inc.) using unpaired t test. Log-rank (Mantel-Cox) test (Chi square) was used to analyze differences between survival curves. Differences were considered non-significant (ns) for P-values > 0.05 (marked as ns), significant for P-values < 0.05 (marked as *), highly significant for P-values < 0.01 (marked as **), and extremely significant for P-values < 0.001 (marked as ***) or P-values < 0.0001 (marked as ****), respectively.

[0100] Results

[0101] Generation and characterization of S-RBD fusion protein from the E. coli system. The RBD antigen of SARS-CoV-2 Wuhan strain was fused to NoV S domain, forming the S-RBD fusion protein with a C-terminal Hisx6-tag (FIG. 1A) with a calculated molecular weight (MW) of 50.8 kDa. The fusion protein was expressed using the E. coli system and purified using the Cobalt resin by a denaturing method. After refolding, the purified protein showed a band at ~54 kDa in SDS-PAGE, reaching a yield of ~30 mg / L of bacterial culture (FIG. IB). Gel-filtration chromatography of the protein using a size exclusion column Superdex 200 revealed a single major peak in void volume with high MW (FIG. 1C), indicating that the S-RBD protein self-assembled into particles or large complexes efficiently. Further analysis of the protein by a CsCl density gradient centrifugation revealed a single protein band (FIG. ID) at a density of 1.32 g / mL that was detected to be the S-RBD protein by EIA assays using antibodies against NoV VLPs (FIG. IE) and commercial antibody against SARS-CoV-2 spike protein (data not shown), respectively. Negative staining TEM inspection of the S-RBD proteins from the major peaks of the gel-filtration (FIG. 1C) and the CsCl density gradient (FIG. IE) indicated typical PVNPs in diameter ~25 nm with some size variations (FIG. IF and FIG. 1G).

[0102] Production of S-RBD PVNPs by the mammalian cell system. Tag-free S-RBD PVNPs were also generated using the mammalian CHO cell system for glycosylation feature of the RBD protein. To this end, a secreting signal peptide was added to the N-terminus of the S-RBD protein (FIG. 2A) to promote secretion of the target protein. After expression, the S-RBD protein in the culture medium was collected by ultracentrifugation. The S-RBD PVNPs were then purified usinga sucrose step gradient consisting of 10-50% sucrose. ETA assays using NoV VLP-specific antibody showed that most S-RBD PVNPs distributed in the gradient containing 40% and 30% sucrose (FIG. 2B). SDS-PAGE of two representative fractions (F6 and F9) indicated a band at ~54 kDa (FIG. 2C), which was shown to be the S-RBD protein by western blot using NoV VLP- specific antibody (FIG. 2D). The method yielded about 100 pg / 100 mL of cell culture was clearly lower than that of the bacterially expressed protein (compare FIG. IB with FIG. 2C). Inspection of the proteins from fractions 6 and 9 of the sucrose gradient by TEM revealed typical PVNPs, mostly in sizes of -25 nm (FIG. 2E , FIG. 2F), similar to those produced by the bacterial expression system (compare FIGS. IF and 1G, with FIGS. 2E and 2F).

[0103] Structural features of the S-RBD PVNPs. The PVNP images revealed by the negative staining TEM micrographs, particularly those from the CHO cell-expressed ones (FIG. 2E and FIG. 2SF), indicated the basic structural features of S-RBD PVNPs. Two types of PVNP images were noted, some PVNPs were apparently focused on the surface, showing multiple exterior protrusions (FIG. 2G), while others were obviously focused at the centers, showing an inner shell or core and multiple protrusions extending toward the surface (FIG. 2H). In both cases, the PVNPs exhibited wheel-shaped structures. According to the previously solved 3D structures of the S60-VP8* (36) and the S60-HA1 (37) PVNPs, the inner shell recorded by the TEM micrographs should represent the S60 nanoparticle, while the outward protrusions should consist of the displayed RBD antigens. Since most PVNPs shares similar diameters of -25 nm like the S60-VP8* (36) and the S60-HA1 (37) PVNPs, they should also be made by 60 S-RBD proteins, organizing in the same T=1 icosahedral symmetry, forming the S60-RBD PVNPs. Accordingly, the larger PVNPs at size of -35 nm may be organized in a T=3 icosahedral symmetry, while the symmetries of the smaller PVNPs remain elusive.

[0104] 3D structural model of the S60-RBD PVNP. Applicant’s previously solved cryoEM structure of the S60-VP8* PVNP (36) provides an excellent basis to make a 3D structural model of the S60-RBD PVNP to further elucidate its structure features. To this end, an S60-RBD PVNP model was created (FIG. 3) using the known RBD crystal structure (6) with help of the UCSF ChimeraX software. The N-terminal ends of 60 RBDs were docked to the 60 exposed C-terminal ends of S domains of the S60 nanoparticle (FIG. 3A) based on the S60-VP8* PVNP density map (36), resulting in 60 RBDs standing on the surface of the S60 nanoparticle with their receptorbinding sites on the distal ends (FIG. 3B). Using this S60-RBD PVNP model, images representing the full and section views of the S60-RBD PVNP in both surface and cartoon representations were made (FIGS. 3B, 3C, 3E and 3F). Comparing these structures with the TEM PVNP images appeared to match each other well in general, in which FIG. 2G represents their full view images, while FIG. 2H represents the section view images of the PVNPs. A major discrepancy was also noted, the inner shells and their central lumens recorded by the TEM micrographs (FIG. 2H) appeared to be smaller than expected, suggesting that S60 nanoparticle underwent certain distortion during the formation of the PVNP, going inward to become a more compacted S particle with a smaller center lumen.

[0105] The authentic antigenic and functional features of the PVNP-displayed RBDs. The antigenic reactivity of the RBD antigens on the bacterially expressed PVNP was examined by EIA assays using commercial antibody specific to the RBD domain of SARS-CoV-2 (Sino Biological) (54). The results revealed that the antibody reacted well with the S60-RBD PVNP in a doseresponse manner, and the EIA signals decreased significantly after the PVNP was heated to 100 °C for 10 minutes to destroy the conformational epitopes (FIG. 3G, Ps <0.001). As expected, the S60 nanoparticle did not react with the RBD protein-specific antibody. In addition, ElA-based binding assays were performed to test binding function of the PVNP displayed RBDs to recombinant ACE2 receptor (Sino Biological). The data showed that the PVNPs bound the ACE2 protein, whereas the S60 nanoparticle without the RBD components did not bind (FIG. 3H, P <0.001), indicating that the PVNP-displayed RBDs retained their receptor binding function. These data support that the PVNP displayed RBDs preserved their authentic structure, conformational epitopes, and function.

[0106] Improved immune response toward the PVNP-displayed RBD antigens. Applicant’s previous studies indicated that polyvalent antigens with pathogen associated molecular patterns (PAMPs) exhibited significantly higher immunogenicity than those induced by the mono- or low- valent antigens (55-58). A similar scenario for the S60-RBD PVNP was anticipated. To prove this, in balb / c mice were immunized with the S60-RBD PVNP and free recombinant RBD antigen (Sino Biological), respectively. After three immunizations with Alum adjuvant, scrum samples were collected for RBD-specific IgG titers. The S60-RBD PVNP induced higher RBD-specific IgG titers to 46,050 which was 8.9-fold greater than that (5,180) elicited by the free RBD protein (FIG.31, P <0.0001). As a control, immunization with the S60 nanoparticle did not induce RBD specific antibody responses.

[0107] Immune response and protective efficacy of the S60-RBD PVNP vaccine in the KI 8- hACE2 mice. Further immune response and protective efficacy of the S60-RBD PVNP vaccine were evaluated using the K18-hACE2 transgenic murine model of SARS-CoV-2 infection and disease. Mice (n=9 / group) were immunized with the S60-RBD PVNP plus Alum adjuvant (referred as S60-RBD) nine, seven, and four weeks prior to challenge using the S60 nanoparticle as a control (referred as S60). Sera were collected at five different time points for IgG antibody and neutralizing antibody (nAb) responses (FIG. 5A). Applicant’s data showed that S60-RBD PVNP elicited high IgG response in the K18-hACE2 mice along with each boost immunization, reaching 3,911 at three weeks after boost 1 and 52,088 at four weeks post-boost 2 (FIG. 5B). Accordingly, S60-RBD PVNP immunized mouse sera showed strong nAb response, continually increasing after each booster immunization, to the strongest PRNT50 titer of 2.2 Log 10 at 4 weeks after the third immunization (FIG. 5C). As expected, the mouse sera after immunization with the S60 nanoparticle did not show detectable RBD specific IgG or nAb titers.

[0108] The immunized mice were intranasally challenged with 2 x 105PFU of SARS-CoV-2 WA 1 / 2020 strain at four weeks post-boost 2. Results showed that the S60-RBD PVNP vaccinated mice were completely protected from mortality, while the S60 immunized mice showed 66.7% mortality (FIG. 5D, P = 0.0201). Additionally, none of the S60-RBD PVNP immunized mice showed weight loss in comparison to the S60 control mice, which showed significant weight loss starting 5 days post infection (DPI, FIG. 5E). The rapid weight increases of the S60 control mice at 8 DPI was due to removal of sick animals at 7 DPI (see FIG. 5D). At 4 DPI, three mice per group were euthanized to assess viral loads in lung and brains. SARS-CoV-2 was detected in the brain and lung of only one S60-RBD vaccinated mouse (FIG. 5F). In contrast, a high viral load was detected in the lungs and brains of animals immunized with the S60 nanoparticle. These data indicated that the S60-RBD PVNP is a promising COVID- 19 vaccine candidate.

[0109] Generation and characterization of S-NTD PVNPs. The N-terminal domain (NTD) is believed to play an important role in SARS-CoV-2 infection with several neutralizing and protective mAbs being identified against this domain (59, 60). To explore the potential of NTD asa vaccine target, a new PVNP displaying the NTD antigen (291 residues, see Materials and methods) was generated and characterized. The bactcrially expressed S-NTD protein was produced at a high yield of 20 mg / L of bacteria culture (FIG. 4A and FIG. 4B). Gel filtration chromatography of the S-NTD protein revealed a single major peak with high molecular weight in the void volume, with a minor peak at the position corresponding to S-NTD monomers (FIG. 4C). These data suggested that vast majority of the S-NTD protein self-assembled into S-NTD PVNPs, which were confirmed by negative staining TEM inspection of the protein in the major elution peak, showing typical S60-NTD PVNPs, mostly at ~25 nm in diameter (FIG. 4D).

[0110] The S-NTD PVNPs were analyzed by CsCl density gradient centrifugation, followed by EIA analysis and TEM inspection. After ultracentrifugation, a protein band at the top third of the gradient was recognized (FIG. 4E), which was confirmed to be the S-NTD protein by EIA using an S domain specific antibody (FIG. 4F). Typical S60-NTD PVNPs, mostly at 25 nm in diameter (FIG. 4G), were observed in the peak fraction (F17) of the gradient with a density of 1.3049 mg / mL. The S60-NTD PVNP reacted with commercial antibody against the spike protein of SARS-CoV-2 (Sino Biological) in a dose dependent manner (FIG. 4H), indicating that the PVNP displayed NTD retain authentic epitopes. After three immunizations, the S60-NTD PVNP elicited high titer (40,000) of NTD specific IgG in balb / c mice (FIG. 41).

[0111] Low protective efficacy of the S60-NTD PVNP. The S60-NTD PVNPs (referred as S60-NTD) were further assessed for their immune response and protective efficacy using the same K18-hACE2 murine model of SARS-CoV-2 infection and disease in parallel with the S60-RBD PVNP (FIG. 5). S60-NTD appeared to induce similar level of IgG response compared with that elicited by the S60-RBD after one and two immunizations (FIG. 5B, Ps>0.05). However, the IgG titers induced by the S60-NTD reached 78,222 and 133,689 at one and four weeks after the second booster respectively, which were significantly higher than those elicited by the S60-RBD after the third immunization. By contrast, nAb titers after immunization of the S60-NTD PVNP were significantly lower compared with those after immunization of the S60-RBD (FIG. 5C, Ps<0.05), although the titers continually increased after each boost immunization, peaked to 1.5 Log 10 at four weeks post boost 2. These data indicated that the S60-NTD induced less neutralizing antibody against SARS-CoV 2 compared with the S60-NTD PVNP.

[0112] After SARS-CoV-2 challenge, the S60-NTD PVNP provided only 50% protection against viral caused mortality, which was lower than that (100%) conferred by the S60-RBD PVNP (FIG. 5D, P=0.0555). Accordingly, half of the S60-NTD immunized mice showed significant weight loss before succumbing to infection starting at 5 DPI (FIG. 5E, P<0.05), in stark contrast to the S60-RBD immunized mice, which showed no weight loss after challenge. At 4 DPI, three animals per group were euthanized to assess viral loads in lungs and brains. SARS-CoV-2 was not detected in the brains of any S60-NTD immunized mice but was detected at high titers in the lungs of these three mice (FIG. 5F). Overall, the S60-NTD vaccine candidate showed moderate efficacy in this challenge model, although its IgG response appeared to be high.

[0113] Discussion

[0114] Based on Applicant’s established S60 nanoparticle technology (36, 37), the S60-RBD PVNP was designed, generated, and characterized with a long-term goal to develop the PVNP into a safe, effective, and adaptable COVID-19 vaccine platform. As a proof of concept, this study provided evidence showing that 1) the S60-RBD PVNP can be produced by both prokaryotic E. coli or the eukaryotic mammalian cell systems; 2) the S60-RBD PVNP consists of typically an inner shell made by the S60 nanoparticle and 60 surface-displayed RBD antigens; 3) the exposed RBD antigens preserved their authentic ACE2 receptor-binding function and antigenic epitopes; and 4) the polyvalent S60-RBD PVNP was highly immunogenic, inducing elevated RBD-specific IgG titers in mice, as well as high nAb titers against SARS-CoV-2. Most importantly, immunizations of the S60-RBD PVNP protected K18-hACE2 mice completely (100%) from mortality and body weight loss caused by SARS-CoV-2 challenge. These data support the S60- RBD PVNP as an effective COVID vaccine.

[0115] The bacterially expressed S60-RBD PVNP showed a higher yield compared with that produced by the mammalian CHO cell system. The E. coli system is known for its fast production of a recombinant protein at low-cost, but the resulting target protein lacks post-translational modification of glycosylation. A previous study revealed only two glycosylation sites (N331 and N343) in the RBD region (61) and they both are located at the base of the RBD protein, far away from the host receptor binding site that is located at the distal end of the RBD antigen. Applicant hypothesized that lacking the glycosylation may not affect receptor-binding function and antigenicfeature of the RBD antigens significantly. This hypothesis is apparently correct, as the bacterially expressed S60-RBD PVNP retained authentic receptor binding function and strong antigenic reactivity to the RBD antibody, supporting its potential to be used as a COVID vaccine. Indeed, the bacterially generated S60-RBD PVNP demonstrated 100% protective efficacy against SARS- CoV-2 challenge in K18-hACE2 mouse model. Similarly, two previous reports showed that bacterially expressed RBD proteins can induce SARS-COVID-2 neutralizing antibodies (62, 63), consistent with the observations in this study. On the other hand, since a mammalian cell system offers better folding conditions and the ability of glycosylation of the target proteins, future studies on mammalian cell expressed S60-RBD PVNP would help to clarify whether the glycosylated S60-RBD PVNP could further increase its protective efficacy against SARS-CoV-2.

[0116] A further advantage of the disclosed PVNP vaccine is that its RBD antigen components are adjustable as needed to address the emergence of future SARS-CoV-2 variants. In case more than one variant are co-circulating simultaneously, two or more S60-RBD PVNPs, each displaying the RBD antigens of a co-circulating variant, can be combined as a bi- or multivalent vaccine for broad efficacy. In addition, the recombinant, non-replicating PVNP technology offers a flexible platform for a safe, effective CO VID vaccine with minimized manufacturing cost and time.

[0117] The NTD of SARS-CoV-2 spike plays a crucial role in viral infection and a few neutralizing and protective mAbs were found in this region (59, 60), suggesting that the NTD may be another vaccine target. To test this hypothesis, another PVNP displaying the NTD antigens as a vaccine candidate was generated. Typical S60-NTD PVNPs were produced by the E. coli system and elevated NTD-specific antibody titers were observed after S60-NTD immunization in mice. However, a challenge study using the same K18-hACE2 murine model revealed only 50% protective efficacy, lower than that (100%) of the S60-RBD PVNP vaccine, implying that the NTD antigen might not be an ideal vaccine target against SARS-CoV-2 compared with the RBD antigen. NTD is known for its heavy glycosylation, with eight known glycosylation sites (N17, N61, N74, N122, N149, N165, N234, and N285) (61). Thus, the observed lower protective efficacy of the E. coli generated S60-NTD PVNP could be due to the lack of these glycosylations.

[0118] Most known VLP platforms based on the full VLPs showed relatively low capacity of antigen display, because the full VLPs themselves are large and they are in their evolutionarystable forms. As a result, the displayed antigens serve as a negative factor in their stability. By contrast, the disclosed S60 nanoparticlc platform comprises the inner shell of norovirus capsid with removal of protruding (P) domain of the capsid. The displayed antigens, such as the RBD domain of the SARS-CoV-2 in this study, serve as the protruding domain of the norovirus, allowing the S-RBD protein to self-assemble into stable pseudovirus nanoparticles resembling norovirus VLPs. Therefore, the norovirus inner shell based S60 nanoparticle generally confers a larger capacity for antigen presentation.

[0119] Negative staining TEM micrographs revealed some larger and smaller particles in addition to the typical ~25 nm S60-RBD PVNPs. The larger particles could be made by 180 S- RBD fusion proteins organizing in T=3 icosahedrons, instead of the typical T=1 S60-RBD PVNPs, as they are in the authentic norovirus capsid (64). On the other hand, the smaller particles could be made by 24 and / or even 12 S-RBD fusion proteins that organized in a octahedral or tetrahedral symmetry, as they are observed in the norovirus P and small P particles, respectively (65, 66). In all these cases, the larger and smaller particles share the basic structures as the S60-RBD PVNP, consisting of an inner S particle shell or core and multiple displayed RBD antigens on the surface. In other words, the observed structural heterogeneity should not change the safety, immunogenicity, or protective efficacy of the disclosed PVNPs as a COVID vaccine candidate. In conclusion, the data presented in this study demonstrated collectively that the RBD antigen is an excellent vaccine target and the S-RBD PVNPs are a promising COVID vaccine platform.Abbreviations and References

[0120] SARS-CoV-2, severe acute respiratory syndrome coronavirus 2; COVID-19, coronavirus disease 2019; PVNP, pseudovirus nanoparticle; RBD, receptor binding domain; NTD, N-terminal domain; ACE2, angiotensin-converting enzyme 2; 3D, three-dimensional; DNA, Deoxyribonucleic acid; E. coli, Escherichia coli; S, shell domain; IPTG, isopropyl B-D-l- thiogalactoyranoside; CHO cell, Chinese hamster ovary cells; FPLC, Fast Performance Liquid Chromatography system; PBS, phosphate buffer saline ; VLP, virus-like particle; NoV, norovirus; SDS-PAGE, Sodium dodecyl sulfate polyacrylamide gel electrophoresis; TEM, transmission electron microscopy; EIA, enzyme immunoassay; CsCl, Cesium chloride; HRP, horse-radish peroxidase; NIH, National Institute of Health; IACUC, Institutional Animal Care and UseCommittee; Ns, non-significant; FCV, feline calicivirus; PDB, Protein Database; IgG, immunoglobulin G; nAb, neutralizing antibody; PRNT50, 50% plaque reduction neutralization test; PFU, plaque-forming unit; MW, molecular weight.

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[0190] All percentages and ratios are calculated by weight unless otherwise indicated.

[0191] All percentages and ratios are calculated based on the total composition unless otherwise indicated.

[0192] It should be understood that every maximum numerical limitation given throughout this specification includes every lower numerical limitation, as if such lower numerical limitations were expressly written herein. Every minimum numerical limitation given throughout this specification will include every higher numerical limitation, as if such higher numerical limitations were expressly written herein. Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.

[0193] The dimensions and values disclosed herein are not to be understood as being strictly limited to the exact numerical values recited. Instead, unless otherwise specified, each such dimension is intended to mean both the recited value and a functionally equivalent range surrounding that value. For example, a dimension disclosed as “20 mm” is intended to mean “about 20 mm.”

[0194] Every document cited herein, including any cross referenced or related patent or application, is hereby incorporated herein by reference in its entirety unless expressly excluded or otherwise limited. All accessioned information (e.g., as identified by PUBMED, PUBCHEM, NCBI, UNIPROT, or EBI accession numbers) and publications in their entireties are incorporated into this disclosure by reference in order to more fully describe the state of the art as known to those skilled therein as of the date of this disclosure. The citation of any document is not an admission that it is prior art with respect to any invention disclosed or claimed herein or that it alone, or in any combination with any other reference or references, teaches, suggests or discloses any such invention. Further, to the extent that any meaning or definition of a term in this document conflicts with any meaning or definition of the same term in a document incorporated by reference, the meaning or definition assigned to that term in this document shall govern.

[0195] While particular embodiments of the present invention have been illustrated and described, it would be obvious to those skilled in the art that various other changes and modifications may be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.

Claims

CLAIMSWhat is claimed is:

1. A severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccine comprising a non-replicating pseudovirus nanoparticle (PVNP), the PVNP displaying a receptor-binding domain of SARS-CoV-2 spike (S-RBD).

2. The SARS-CoV-2 vaccine of claim 1, wherein the PVNP comprises an icosahedral particle comprising a norovirus (NoV) shell (S) domain protein.

3. The SARS-CoV-2 vaccine of claim 2, the S domain protein comprising 60 S domain proteins.

4. The SARS-CoV-2 vaccine of claim 2, the S domain protein comprising a sequence having at least 90% sequence identity to SEQ ID NO: 1.MKMASNDASPSDGSTANLVPEVNNEVMALEPVVGAAIAAPVAGQQNVIDPWIR NNFVQAPGGEFTVSPANAPGEILWSAPLGPDLNPYLSHLARMYNGYAGGFEVQV ILAGNAFTAGKVIFAAVPPNFPTEGLSPSQVTMFPHIIVDVRQLEPVLIPLPDVRNN FYHYNQSNDSTIKLIAMLYTPLRANNAGDDVFTVSCRVLTRPSPDFDFIFLVPPTV E5. The SARS-CoV-2 vaccine of claim 2, the S domain protein comprising a sequence having at least 95% sequence identity to SEQ ID NO: 1.

6. The SARS-CoV-2 vaccine of claim 2, the S domain protein comprising a sequence having at least 99% sequence identity to SEQ ID NO: 1.

7. The SARS-CoV-2 vaccine of claim 2, the S domain protein comprising SEQ ID NO: 1 .

8. The SARS-CoV-2 vaccine of any preceding claim, wherein the S-RBD comprises a sequence having at least 90% sequence identity to SEQ ID NO: 2.

9. The SARS-CoV-2 vaccine of any preceding claim, wherein the S-RBD comprises a sequence having at least 95% sequence identity to SEQ ID NO: 2.

10. The SARS-CoV-2 vaccine of any preceding claim, wherein the S-RBD comprises a sequence having at least 99% sequence identity to SEQ ID NO: 2.

11. The SARS-CoV-2 vaccine of any preceding claim, wherein the S-RBD is fused to the NoV S domain protein.

12. The SARS-CoV-2 vaccine of any preceding claim, wherein the S-RBD retains angiotensinconverting enzyme 2 (ACE2) binding function.

13. The SARS-CoV-2 vaccine of any preceding claim, wherein the SARS-CoV-2 vaccine elicits S-RBD-specific IgG and neutralizing antibodies in an individual administered the SARS-CoV-2 vaccine.

14. The SARS-CoV-2 vaccine of any preceding claim, the SARS-CoV-2 vaccine being produced via both a prokaryotic and eukaryotic system.

15. A pseudovirus nanoparticle (PVNP) composition comprising an aggregate of fusion proteins forming an icosahedral structure and a nanoparticle shell, the fusion protein comprising a) a modified norovirus (NoV) S domain protein;b) a receptor binding domain (RBD) antigen of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2); and c) a peptide linker, the peptide linker connecting the C-terminus of the NoV S domain to the RBD antigen; wherein the modified NoV S domain protein forms the interior of the nanoparticle shell of the PVNP composition; wherein the nanoparticle shell displays 60 exposed C-termini of the S domain in a triangular pattern; wherein 60 RBD antigens are displayed on the surface of the nanoparticle shell.

16. The PVNP composition of claim 15, wherein the NoV S domain is wildtype at one or more of positions V57, Q58, or S136, and M140.

17. The PVNP composition of claim 15, wherein the NoV S domain is wildtype at all positions V57, Q58, or S136, and M140.

18. The PVNP composition of any of claims 15 through 17, wherein the linker comprises or consists of a sequence selected from HHHH (SEQ ID NO: 8), GGGG (SEQ ID NO: 9), GSGS (SEQ ID NO: 10), HHHHHH (SEQ ID NO: 1 1) and SSRVDGGGGG (SEQ ID NO: 12).

19. The PVNP composition of any of claims 15 through 17, wherein the linker comprises or consists of GGGG (SEQ ID NO: 9).

20. The PVNP composition of any of claims 15 through 17, wherein the linker comprises or consists of SSRVDGGGGG (SEQ ID NO: 12).

21. The PVNP composition of any preceding claim, wherein the PVNP composition induces substantial immunity to SARS-CoV-2 infection or at least one symptom thereof when administered to a subject.

22. A method of reducing the severity of a SARS-Co-V 2 infection and / or eliciting an immune response in a subject against SARS-Co-V 2 in an individual in need thereof, comprising administering a SARS-CoV-2 vaccine according to any preceding claim to the individual.

23. The method of claim 22, wherein the administration comprises one dose, or two doses, or three doses of the SARS-CoV-2 vaccine.

24. The method of claim 22 or 23, wherein the composition is administered to an individual via a route selected from one or more of intradermal injection, intramuscular injection, or by intranasal administration.

25. The method of any of claims 22 through 24, the composition comprising a pharmaceutically acceptable carrier.

26. The method of any of claims 22 through 25, the composition comprising one or more of an adjuvant and a preservative.

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