Coronavirus vaccine

Abstract

The present invention relates to chimeric and fusion proteins and compositions thereof, and the use of such proteins and compositions in the prevention and / or treatment of coronavirus infection, or a respiratory disease or condition associated with coronavirus infection.

Description

[Technical field]

[0001] Related Applications This application claims the benefit of priority to Australian Provisional Application No. 2020904820, filed December 23, 2020, and Australian Provisional Application No. 2021903911, filed December 2, 2021, both of which are incorporated by reference in their entireties.

[0002] The present invention relates to chimeric and fusion proteins and compositions thereof, and the use of such proteins and compositions in the prevention and / or treatment of coronavirus infection, or a respiratory disease or condition associated with coronavirus infection. [Background technology]

[0003] Emerging respiratory coronaviruses pose a major threat to human health and economies worldwide. Coronaviruses (CoVs) constitute a phylogenetically diverse group of enveloped viruses that encode large positive-stranded RNA genomes and replicate efficiently in the majority of mammals. Human CoV (HCoVs-229E, OC43, NL63, and HKU1) infections typically cause mild to severe upper and lower respiratory tract disease.

[0004] Coronaviruses belong to the family Coronaviridae of the order Nidovirales, are minute in size (80-200 nm in diameter) and contain a single-stranded RNA of 26-32 kb in length as the core material. The subgroups of the Coronaviridae family are the alpha (α), beta (β), gamma (γ), and delta (δ) coronaviruses. The betacoronaviruses are of greatest clinical importance in humans. These include the lineage A betacoronaviruses OC43 and HKU1 (which can cause the common cold). Lineage B betacoronaviruses include the severe acute respiratory syndrome coronaviruses SARS-CoV and SARS-CoV-2 (which cause the disease COVID-19). Middle East Respiratory Syndrome Coronavirus (MERS-CoV) is a betacoronavirus from lineage C. These viruses can cause acute lung injury (ALI) and acute respiratory distress syndrome (ARDS), leading to lung failure and death.

[0005] Severe acute respiratory syndrome coronavirus (SARS-CoV) emerged in 2002–2003 and caused ARDS with an overall mortality rate of 10% and up to 50% in the elderly.

[0006] Middle East Respiratory Syndrome-Coronavirus (MERS-CoV) emerged in the Middle East in April 2012 and manifested as severe pneumonia, ARDS, and acute renal failure.

[0007] In 2020, the world faced an extreme situation in which immunologically naive people around the world encountered a highly infectious coronavirus (2019-nCoV; SARS-CoV-2), manifesting as a disease called "COVID-19." SARS-CoV-2 infection has so far caused more than 50 million confirmed cases worldwide and more than 1 million deaths in more than 200 countries, territories, or areas. Symptoms of COVID-19 range from mild to severe and life-threatening, with a significant mortality rate. Summary of the Invention [Problem to be solved by the invention]

[0008] There is a need for new or improved treatments for coronavirus infections and / or diseases associated with or caused by coronaviruses.

[0009] The reference to prior art herein is not an admission or suggestion that this prior art forms part of common general knowledge in any jurisdiction, or that this prior art could reasonably be expected to be understood, considered relevant, and / or incorporated with other pieces of prior art by those skilled in the art. [Means for solving the problem]

[0010] In one aspect, the present invention provides a chimeric or fusion protein for inducing an immune response against coronavirus, the protein comprising two or more polypeptides comprising or consisting of the amino acid sequence of a receptor binding domain (RBD) from a coronavirus spike protein linked to an Fc region of an antibody.

[0011] In another aspect, the present invention provides a chimeric or fusion protein for inducing an immune response against a coronavirus, the protein comprising two or more polypeptides, each comprising or consisting of the amino acid sequence of a receptor binding domain from the spike protein of a coronavirus linked to a polypeptide comprising an Fc receptor binding domain.

[0012] In one aspect, the present invention provides a chimeric or fusion protein for inducing an immune response against coronavirus, the protein comprising a dimer of a receptor binding domain from the spike protein of coronavirus linked to the Fc region of an antibody.

[0013] In another aspect, the present invention provides a chimeric or fusion protein for inducing an immune response against coronavirus, comprising a dimer of a receptor binding domain from the spike protein of coronavirus linked to a polypeptide comprising an Fc receptor binding domain, in one embodiment, the chimeric or fusion protein is a single chain dimer comprising two RBD chain sequences covalently linked in a contiguous polypeptide chain.

[0014] In any embodiment, the receptor binding domain from the coronavirus spike protein comprises, consists essentially of, or consists of an amino acid sequence of one or more of SEQ ID NOs: 15 or 28, or an amino acid sequence having 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to one of SEQ ID NOs: 15 or 28, and in one embodiment, the amino acid sequence may include one or more of the mutations as shown in Figures 6 or 7.

[0015] In any aspect, the receptor binding domain may be derived from a SARS-CoV-2 variant. SARS-CoV-2 variants have evolved from the original (Wuhan-Hu-1) ancestral or ("Wuhan") strain of the virus (Genomic Reference Sequence: GenBank Accession NC_045512.2) and are referred to herein as wild-type (WT) strains. As described herein, the receptor binding domain may be derived from a WT ("Wuhan"), alpha, beta, gamma, kappa, or delta strain, or any other strain defined herein, including Table 3. In any embodiment, the receptor binding domain may be N334-P527 of a WT, alpha, beta, gamma, kappa, or delta strain. The alpha variant carries one mutation in the RBD, N501Y, compared to the original WT strain. The beta variant carries three mutations in the RBD, N501Y, E484K, and K417N, compared to the original WT strain. The gamma mutant harbors three mutations in the RBD compared to the original WT strain: K417T, E484K, and N501Y. The kappa mutant harbors two mutations in the RBD compared to the original WT strain: L452R and E484Q. The delta mutant harbors two mutations in the RBD compared to the original WT strain: L452R and T478K.

[0016] In any aspect, the receptor binding domain from coronavirus spike protein comprises, consists essentially of, or consists of one or more amino acid sequences of either SEQ ID NO: 15 or 28, having 0-8 amino acid insertions, deletions, substitutions or additions (or combinations thereof). In some embodiments, the relevant amino acid sequence may have 0-7, preferably 0-6, preferably 0-5, preferably 0-4, preferably 0-3, preferably 0-2, preferably 0-1 amino acid insertions, deletions, substitutions or additions (or combinations thereof), where the amino acid insertions, deletions, substitutions or additions (or combinations thereof) are located at the N-terminus and / or C-terminus.

[0017] In any embodiment, the receptor binding domain from coronavirus spike protein comprises, consists essentially of, or consists of one or more amino acid sequences of either SEQ ID NO: 15 or 28 with 0-8 amino acid insertions, deletions, substitutions or additions (or combinations thereof) and an antibody Fc region comprising, consisting essentially of, or consisting of the amino acid sequence of any one of SEQ ID NO: 18 with 0-8 amino acid insertions, deletions, substitutions or additions (or combinations thereof). In some embodiments, the relevant amino acid sequence may have 0-7, preferably 0-6, preferably 0-5, preferably 0-4, preferably 0-3, preferably 0-2, preferably 0-1 amino acid insertions, deletions, substitutions or additions (or combinations thereof), where the amino acid insertions, deletions, substitutions or additions (or combinations thereof) are located at the N-terminus and / or C-terminus.

[0018] In any embodiment, the amino acid sequence of the chimeric or fusion protein of the invention comprises, consists essentially of or consists of the sequence set forth in any one of SEQ ID NOs: 2, 3, 5, 6, 22 or 23. Furthermore, the amino acid sequence of the chimeric or fusion protein of the invention comprises, consists essentially of or consists of the receptor binding domain N334-P527 from the spike protein of a SARS-CoV-2 variant strain described herein, in particular a WT, alpha, beta, gamma, kappa or delta strain, directly or indirectly linked to a human IgG1 Fc, preferably the linker comprises or consists of the amino acid sequence of SEQ ID NO: 17 and the human IgG1 Fc comprises or consists of the amino acid sequence of SEQ ID NO: 18.

[0019] In any aspect, the antibody Fc region of the chimeric or fusion protein is an IgG, more preferably an IgG1, more preferably a human IgG1 Fc region. In some embodiments, the IgG Fc region is a mouse IgG1.

[0020] In any embodiment, one or more of the two or more polypeptides, or one or both of the receptor binding domains in a dimer of a chimeric or fusion protein, may be fused to an Fc region at the C-terminus. Alternatively, one or more of the two or more polypeptides, or one or both of the receptor binding domains in a dimer of a chimeric or fusion protein, may be fused to an Fc region via a C-terminal linker.

[0021] Preferably, the Fc region of the chimeric or fusion protein comprises two heavy chain fragments, more preferably the CH2 and CH3 domains of said heavy chains, in one embodiment the heavy chain fragments are linked via disulfide bonds.

[0022] In any embodiment, the Fc region of the antibody comprises, consists essentially of, or consists of the amino acid sequence of either one of SEQ ID NOs: 16 or 18, or an amino acid sequence having 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to either one of SEQ ID NOs: 16 or 18.

[0023] In any aspect, the antibody Fc region comprises, consists essentially of, or consists of the amino acid sequence of either SEQ ID NO: 16 or 18, having 0 to 8 amino acid insertions, deletions, substitutions, or additions (or a combination thereof). In some embodiments, the relevant amino acid sequence may have 0 to 7, preferably 0 to 6, preferably 0 to 5, preferably 0 to 4, preferably 0 to 3, preferably 0 to 2, preferably 0 to 1 amino acid insertions, deletions, substitutions, or additions (or a combination thereof).

[0024] In any embodiment, the receptor binding domain from the coronavirus spike protein comprises or is an amino acid sequence of any one or more of SEQ ID NOs: 15 or 28, or an amino acid sequence having 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NOs: 15 or 28. and the Fc region of the antibody comprises, consists essentially of or consists of the amino acid sequence of any one of SEQ ID NOs: 16 or 18, or an amino acid sequence having 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NOs: 16 or 18. In one embodiment, the amino acid sequence of the receptor binding domain may include one or more of the mutations as shown in Figure 6 or as described herein in connection with SARS-CoV-2 variants such as the alpha, beta, gamma, kappa, delta, delta plus, tamda, mu, iota, or omicron strains.

[0025] The chimeric or fusion proteins of the invention may be isolated, purified, substantially purified, enriched, synthetic, recombinant, hi one embodiment, purification is mediated by the use of a poly-His tag attached to the chimeric or fusion protein.

[0026] In another aspect of the invention, the invention provides a composition comprising the chimeric or fusion protein of the invention as described herein and an adjuvant. Preferably, the composition further comprises a pharma- ceutically acceptable carrier, diluent or excipient.

[0027] In any embodiment, the adjuvant is any described herein, preferably a TLR2-agonist, more preferably a Pam-2-Cys containing molecule such as PEG-4 arginine(R4)-Pam-2-Cys (PEG-R4-Pam-2-Cys), or preferably a stimulator of NKT cells, more preferably any glycolipid capable of stimulating NKT cells, such as α-galactosylceramide (also referred to herein as "α-GalCer"), α-glucosylceramide, α-glucosyldiacylglycerol, α-galactosyldiacylglycerol, β-mannosylceramide, and analogs thereof, comprising a diversity of acyl and sphingosine chain lengths, degrees of saturation, and a diversity of polar head group compositions.

[0028] In some embodiments, the TLR2-agonist may be selected from the group consisting of Pam-3-CSK4, PEG-R4-Pam-2-Cys, MALP-2, lipoteichoic acid, OspA, Porin, LcrV, lipomannan, lysophosphatidylserine, lipophosphoglycan (LPG), glycophosphatidylinositol (GPI), and zymosan.

[0029] In some embodiments, the adjuvant is Poly-I:C, CpG, Poly-ICLC, 1018 ISS, aluminum salts, Amplivax, AS03, AS04, AS15, BCG, CP-870,893, CpG7909, CyaA, dSLIM, GM-CSF, IC30, IC31, Imiquimod, ImuFact IMP321, IS Patch, ISS, ISCOMATRIX, JuvImmune, LipoVac, Matrix M, MF59®, AddaVax™, monophosphoryl lipid A, Montanide IMS1312, Montanide ISA206, Montanide ISA V, Montanide ISA-51, OK-432, OM-174, OM-197-MP-EC, ONTAK, PEPTEL, Vector Systems, PLGA microparticles, resiquimod, SRL172, virosomes and other virus-like particles, YF-17D, VEGF trap, R848, β-glucan, Pam-3-Cys, and Aquila's QS21 Stimulon.

[0030] In some embodiments, the adjuvant comprises a metabolizable oil and an emulsifier, such as a detergent or surfactant. Preferably, the oil and emulsifier are in the form of an oil-in-water emulsion, with substantially all of the oil droplets being less than 1 micron in diameter. Exemplary metabolizable oils and emulsifiers are described in U.S. Pat. Nos. 6,299,884 and 6,086,901. In one embodiment, the adjuvant comprises an oil-in-water emulsion. Preferably, the oil is squalene. Preferably, the aqueous phase is a citrate buffer (e.g., 10 mM, pH 6.5).

[0031] In one embodiment, the adjuvant comprises squalene in an oil-in-water emulsion. Preferably, the adjuvant further comprises Tween® 80 (polyoxyethylene sorbitan monooleate) and Span® 85 (sorbitan trioleate). The adjuvant may comprise 4.3% squalene, 0.5% Tween® 80 (polyoxyethylene sorbitan monooleate), 0.5% Span® 85 (sorbitan trioleate), and optionally 400 pg / ml MTP-PE (N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-[1,2-dipalmitoyl-sn-glycero-3-3(hydroxyphosphoryl-oxy)]ethylamide).

[0032] In one embodiment, the composition comprises 50% vol / vol of an adjuvant, preferably the adjuvant is MF59.

[0033] In one embodiment, the composition is lyophilized.

[0034] In another embodiment, the present invention provides a lyophilized composition comprising the chimeric or fusion protein of the present invention as described herein and an adjuvant, preferably a TLR2-agonist, more preferably a Pam-2-Cys containing molecule such as PEG-4 arginine (R4)-Pam-2-Cys (PEG-R4-Pam-2-Cys).

[0035] The lyophilized composition may be stable for at least one month at a temperature range of -20° C. to 40° C. Preferably, the immunogenicity and protective efficacy of the reconstituted composition after storage in lyophilized form at a temperature range of -20° C. to 40° C. for one month is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of that of the reconstituted composition not stored at a temperature range of -20° C. to 40° C. for one month. Preferably, the antibody titer, preferably the neutralizing antibody titer, induced by the reconstituted composition after storage in lyophilized form at a temperature range of -20°C to 40°C for one month is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of that of a reconstituted composition that has not been stored at a temperature range of -20°C to 40°C for one month. Preferably, the neutralizing activity of the reconstituted composition after storage in lyophilized form for one month at a temperature range of -20°C to 40°C against multiple coronavirus variants, e.g., the original (Wuhan-Hu-1) ancestral or ("Wuhan") strain of SARS-CoV-2 and beta variants of SARS-CoV-2, is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of that of a reconstituted composition that has not been stored for one month at a temperature range of -20°C to 40°C.

[0036] In any embodiment, the chimera or fusion of the invention, or the composition of the invention induces an immune response against coronavirus by inducing antibodies, preferably antibodies that neutralize coronavirus particles or virions. More preferably, the chimera or fusion of the invention, or the composition of the invention induces immunoglobulins that specifically bind to the RBD of coronavirus and inhibit the binding of the RBD to ACE2.

[0037] In any embodiment of the invention, the chimeric or fusion protein or composition of the invention induces an immune response against coronavirus by inducing a CD4+ T cell response that promotes the production of antibodies by B cells.

[0038] In any embodiment of the invention, the chimeric or fusion protein of the invention, or the composition of the invention induces an immune response against coronavirus by inducing a CD8 T cell response that mediates cytotoxicity against coronavirus-infected cells.

[0039] In any embodiment of the invention, the chimeric or fusion protein of the invention, or the composition of the invention induces an immune response against coronavirus by inducing an NKT cell response.

[0040] In any embodiment of the invention, the chimeric or fusion protein of the invention, or the composition of the invention, is administered as a single dose.

[0041] In any aspect of the present invention, the chimeric or fusion protein of the present invention or the composition of the present invention is administered as a prime boost regime. The boost may be the first boost (i.e., the second dose) or the second boost (i.e., the third dose), or even the third or subsequent boost. The regime may be, for example, a two-dose regimen or a three-dose regimen, or a four-, five-, and more-boosting regimen, as described herein, including in the examples. Preferably, the prime and boost doses are administered at different times. In some embodiments, the time interval between the prime and boost is 1 week, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, 3 months, 6 months, or 1 year.

[0042] In any aspect of the invention, the chimeric or fusion protein of the invention, or the composition of the invention, is administered as two or more doses, hi one embodiment, the chimeric or fusion protein of the invention, or the composition of the invention, is administered as three separate doses.

[0043] In any embodiment of the invention where prophylaxis or preventative therapy is intended or desired, the chimeric or fusion protein of the invention, or the composition of the invention, is administered to a subject prior to clinical or biochemical detectable symptoms of viral infection, preferably coronavirus infection. The subject may be identified as being at risk for coronavirus infection.

[0044] In any embodiment of the present invention, the chimeric or fusion protein of the present invention or the composition of the present invention is administered to the respiratory tract. Typically, the chimeric or fusion protein of the present invention or the composition of the present invention is administered to the upper and / or lower respiratory tract. For example, the chimeric or fusion protein of the present invention or the composition of the present invention may be administered to a subject via inhalation or intranasally.

[0045] In any embodiment of the present invention, administration of the chimeric or fusion protein of the present invention or the composition of the present invention to a subject reduces the viral load in the subject. Preferably, the viral load is reduced in the respiratory tract, such as the upper and / or lower respiratory tract. More preferably, the viral load is reduced in the upper and lower respiratory tract. Preferably, the viral load is reduced in the lungs.

[0046] In any aspect, the compositions of the present invention may be formulated or adapted for administration to the respiratory tract, for example the upper and / or lower respiratory tract. Preferably, the compositions are formulated or adapted for inhalation or intranasal administration. In one embodiment, the compositions are formulated as a spray, mist, or aerosol.

[0047] In a preferred embodiment, the composition is formulated as a nasal spray or as nasal drops.

[0048] In any embodiment, the compositions may be formulated or adapted for administration subcutaneously, intramuscularly, or via any other route described herein.

[0049] Thus, in another aspect, the present invention further provides a vaccine or immunostimulatory composition for inducing an immune response against a coronavirus in a subject, the composition comprising: an immunogen in the form of a chimeric or fusion protein as described herein, and -An adjuvant for enhancing the immune response to an immunogen in a subject.

[0050] Preferably, the only immunogen provided in the composition, vaccine or immunostimulatory composition of the invention is a chimeric or fusion protein as described herein.

[0051] In another aspect, the present invention provides a pharmaceutical composition for the treatment and / or prevention of (a) a disease associated with or caused by a coronavirus, or (b) a coronavirus infection, comprising as an active ingredient a chimeric or fusion protein of the present invention and a pharma- ceutical acceptable diluent, excipient or carrier. In one embodiment, the only active ingredient present in the composition is the chimeric or fusion protein of the present invention.

[0052] In another aspect, the present invention provides a pharmaceutical composition for the treatment and / or prevention of (a) a disease associated with or caused by a coronavirus, or (b) a coronavirus infection, comprising as a major component a polypeptide or fusion protein of the present invention and a pharma- ceutical acceptable diluent, excipient or carrier. In one embodiment, the only active component present in the composition is the chimeric or fusion protein of the present invention.

[0053] In another aspect, the present invention also provides a method of eliciting an immune response in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a chimeric or fusion protein of the present invention, or a pharmaceutical composition of the present invention, thereby eliciting an immune response in the subject.

[0054] The present invention also provides a method for inducing an immune response against coronavirus in a subject, comprising administering to a subject in need thereof a chimeric or fusion protein, vaccine or immunostimulatory composition described herein.

[0055] The present invention also provides a method of inducing a humoral immune response against coronavirus in a subject comprising administering to the subject a chimeric or fusion protein, composition, vaccine or immunostimulatory composition as defined herein. Preferably, the induced immune response comprises a "balanced" Th1-Th2 response.

[0056] The present invention also provides a method of immunizing a subject against coronavirus infection comprising administering to the subject a chimeric or fusion protein, composition, vaccine or immunostimulatory composition as defined herein.

[0057] The present invention provides a method for reducing or minimizing the severity of symptoms associated with infection by a coronavirus comprising administering to an individual in need thereof a chimeric or fusion protein, composition, vaccine or immunostimulatory composition as defined herein, wherein the symptoms are selected from the group consisting of fever, dry cough, fatigue, aches and pains, sore throat, diarrhea, nausea and vomiting, conjunctivitis, headache, loss of taste or smell, skin rash or discoloration of the fingers or toes, respiratory distress or shortness of breath, chest pain or pressure, and loss of speech or movement.

[0058] In another aspect, the present invention also provides a method for treating and / or preventing (a) a disease associated with or caused by a coronavirus, or (b) a coronavirus infection in a subject in need thereof, the method comprising administering a therapeutically effective amount of a chimeric or fusion protein of the invention or a composition of the invention to the subject, thereby treating and / or preventing (a) a disease associated with or caused by a coronavirus, or (b) a coronavirus infection in the subject.

[0059] In any embodiment, the method of the invention further comprises identifying a subject at risk for coronavirus infection.

[0060] In another aspect, the present invention provides the use of a chimeric or fusion protein of the invention, or a composition of the invention, in the preparation of a medicament for the treatment and / or prevention of (a) a disease associated with or caused by a coronavirus, or (b) a coronavirus infection in a subject in need thereof. Preferably, the medicament is adapted for administration to the upper respiratory tract.

[0061] In any embodiment, the present invention also provides for the use of the chimeric or fusion protein, or the composition of the present invention, to suppress or reduce the amount of coronavirus particles in the upper respiratory tract or a region thereof, or the lower respiratory tract or a region thereof, of an individual. The region of the upper respiratory tract or the region of the lower respiratory tract may be a region as described herein (e.g., the lung is a region of the lower respiratory tract). Preferably, the present invention also provides for the use of the chimeric or fusion protein, or the composition of the present invention, to suppress or reduce the amount of coronavirus particles in any tissue or organ in which coronavirus is detected. Preferably, the chimeric or fusion protein, or the composition of the present invention is adapted for use in the upper respiratory tract.

[0062] In some embodiments, the chimeric or fusion protein of the invention is administered only to the upper respiratory tract, in other words, the chimeric or fusion protein of the invention, or the composition of the invention is not administered to the lower respiratory tract, or to both the upper and lower respiratory tract (i.e., administration to all respiratory tract).

[0063] In a further aspect, the invention provides a method of inhibiting or reducing the amount of coronavirus particles in the lungs of an individual comprising inhibiting or reducing the amount of coronavirus particles in the lungs of the individual by administering a chimeric or fusion protein, or a composition of the invention to the upper respiratory tract of the individual.

[0064] In another aspect, the invention provides a method for inhibiting, slowing or reducing the progression of coronavirus particles from the upper respiratory tract to the lungs of an individual, comprising inhibiting, slowing or reducing the progression of coronavirus particles from the upper respiratory tract to the lungs of the individual by administering an inventive chimeric or fusion protein, or a composition of the invention to the upper respiratory tract of the individual.

[0065] The invention further provides the use of a chimeric or fusion protein of the invention in the preparation of a medicament for inhibiting, slowing or reducing the progression of coronavirus particles from the upper respiratory tract to the lungs of an individual, preferably the medicament is adapted for administration to the upper respiratory tract.

[0066] In any embodiment, the invention provides for the use of a chimeric or fusion protein of the invention, or a composition of the invention, to inhibit, slow or reduce progression of coronavirus particles from the upper respiratory tract to the lungs of an individual. Preferably, the chimeric or fusion protein of the invention, or a composition of the invention is adapted for use in the upper respiratory tract.

[0067] In any of the methods or uses of the invention, the composition is a lyophilized composition, and the method or use further comprises the step of reconstituting the lyophilized composition, and preferably administering the reconstituted composition to an individual.

[0068] Preferably, either method reduces or prevents the spread of coronavirus from the upper respiratory tract to the lungs. Preferably, the chimeric or fusion protein or composition of the present invention is retained in the upper respiratory tract. In other words, administration of the chimeric or fusion protein or composition of the present invention to the upper respiratory tract prevents or reduces the spread of the virus to the lungs.

[0069] As used herein, the upper respiratory tract may include any one or more of the following areas: nose and nasal meatus, nasal turbinates, paranasal sinuses, pharynx, and portions of the larynx above the vocal folds (vocal cords).

[0070] Typically, the lower respiratory tract includes any one or more of the portions of the larynx below the vocal folds, the trachea, the bronchi, and the bronchioles. The lungs may be included in the lower respiratory tract, including the respiratory bronchioles, alveolar ducts, alveolar sacs, and alveoli.

[0071] In any embodiment of the invention, administration to the upper respiratory tract may be administration to any one or more of the nose and nasal passages, paranasal sinuses, pharynx, and portions of the larynx above the vocal folds (vocal cords). Thus, the chimeric or fusion protein or composition of the invention may be administered (a) to the nose and nasal passages, (b) to the nose, nasal passages, and paranasal sinuses, (c) to the nose, nasal passages, paranasal sinuses, and pharynx, or (d) to the nose, nasal passages, paranasal sinuses, pharynx, and portions of the larynx above the vocal folds (vocal cords).

[0072] In any embodiment, the disease associated with or caused by a coronavirus is a respiratory disease.

[0073] In one aspect, the invention provides a chimeric or fusion protein of the invention, or a composition of the invention, for use in raising an immune response in a subject.

[0074] In one aspect, the invention provides a chimeric or fusion protein of the invention, or a composition of the invention for use in the treatment and / or prevention of (a) a disease associated with or caused by a coronavirus, or (b) a coronavirus infection in a subject in need thereof.

[0075] In any embodiment of the invention, the coronavirus infection may be an infection with a coronavirus from any of the genera Alphacoronavirus, Betacoronavirus, Gammacoronavirus, or Deltacoronavirus. Preferably, the coronavirus is from one of Alphacoronavirus subgroup clusters 1a and 1b, or one of Betacoronavirus subgroup clusters 2a, 2b, 2c, and 2d. The coronavirus may be any coronavirus that infects humans. Exemplary coronaviruses are SARS-CoV, MERS-CoV, SARS-CoV-2, HCoV-NL63, HCoV-229E, HCoV-OC43, and HKU1, although the coronavirus may be any of those described herein. Most preferably, the coronavirus infection is an infection with SARS-CoV-2.

[0076] In another aspect, the present invention also provides a method for obtaining antibodies directed against a coronavirus comprising generating antibodies directed against a coronavirus in a non-human animal by administering to the animal a chimeric or fusion protein, composition, vaccine or immunostimulatory composition of the invention. Preferably, the method further comprises isolating the antibodies from the animal.

[0077] In another aspect, the present invention also provides an antibody preparation comprising antibodies directed against a coronavirus, wherein the antibody preparation is obtained by administering a chimeric or fusion protein, composition, vaccine or immunostimulatory composition of the invention to a non-human animal to generate antibodies directed against the coronavirus in the animal and isolating the antibodies from the animal.

[0078] In another aspect, the present invention also provides a nucleic acid molecule encoding a chimeric or fusion protein of the present invention. The nucleic acid may be DNA or RNA (e.g., mRNA).

[0079] Preferably, the nucleic acid has a nucleotide sequence that encodes any one or more of the amino acid sequences corresponding to SEQ ID NO: 2, 5, 8, 12, 15, 22 or 23.

[0080] In any embodiment, the nucleic acid molecule encoding the chimeric or fusion protein comprises a nucleotide sequence corresponding to SEQ ID NO: 1, 4, 29, 30, 31 or 32.

[0081] In any embodiment, such a nucleic acid is included in an expression construct in which the nucleic acid is operably linked to a promoter. Such an expression construct may be a vector, for example, a plasmid.

[0082] In another aspect, the present invention also provides a vector comprising a nucleic acid molecule of the present invention.

[0083] In another aspect, the invention also provides a cell comprising the vector or nucleic acid of the invention. Examples of cells of the invention include bacterial cells, yeast cells, insect cells or mammalian cells. Preferably, the cells are isolated, substantially purified, or recombinant.

[0084] In another aspect, the present invention also provides an animal, or a tissue derived therefrom, comprising a cell of the present invention.

[0085] As used herein, unless the context otherwise requires, the term "comprise" and variations of terms such as "comprising," "comprises," and "comprised" are not intended to exclude additional additives, components, integers or steps.

[0086] Further aspects of the invention and further embodiments of the aspects described in the preceding paragraphs will become apparent from the following description, given by way of example only, with reference to the accompanying drawings, in which: [Brief description of the drawings]

[0087] [Figure 1] Generation of mutant RBD proteins. (A) Cartoon of the SARS-CoV-2 virus, whose surface is covered by spike proteins, highlighting the receptor binding domain (RBD) that binds ACE2 and initiates viral entry. (B) Cartoon of the various RBD proteins. (CF) FPLC elution profile from gel filtration size exclusion chromatography. Collected fractions are shown. RBD monomer was eluted from the Superdex-75 column, whereas RBD dimer and RBD-Fc protein were eluted from the Superdex-200 column. (G) SDS-PAGE gel of the four RBD mutants after gel filtration. (H) Flow cytometry contour plots showing HEK293T cells transiently transfected to express human ACE2 or T cell receptor (TCR) as a negative control, stained with RBD-Fc (mouse IgG1) or anti-TCR antibody. [Diagram 2]RBD-specific antibody responses in primary and secondary sera from mice vaccinated with RBD mouse IgG1-Fc dimer (RBD). BALB / c mice (n=5 per group) were vaccinated with 0.1 nmoles of RBD dimer administered without adjuvant (RBD) or in the presence of 0.3 nmoles of PEG-R4-Pam-2-Cys (RBD P2C) or 0.2 μg of α-GC (RBD a-GC) via either the subcutaneous route at the base of the tail or the intranasal route into the URT. Mice were primed on day 0 and boosted on day 28. Sera taken immediately before the first injection (prebleed), immediately before the second injection (1°), and 2 weeks after the second injection (2°) were tested for RBD-specific antibodies by ELISA. Antibody titers were calculated as the reciprocal of the highest dilution (log10) of serum giving an OD of 0.3. Antibody titers from individual animals are expressed as the mean for each group, indicated by the horizontal line. [Diagram 3] SARS-CoV-2 neutralizing antibody titers in primary and secondary sera from mice vaccinated with RBD mouse IgG1-Fc dimer (RBD). Sera from BALB / c mice shown in Figure 2 were also evaluated for SARS-CoV-2 neutralizing antibodies. Sera taken immediately before the second injection (1°) and 2 weeks after the second injection (2°) were tested for neutralizing antibodies using a microneutralization assay. Serial dilutions of individual serum samples were incubated with 100 TCID50 of SARS-CoV-2 Wuhan indicator strain VIC01 for 1 h and residual viral infectivity was assessed using Vero cell monolayers set up in 96-well plates. Viral cytopathic effect was measured on day 5. Neutralizing antibody titers were calculated using the Reed / Muench method (Reed, LJ and Muench, H. (1938) A Simple Method of Estimating Fifty Percent Endpoints. American Journal of Hygiene, 27, 493-497). Neutralizing antibody titers from individual animals are represented by the mean values ​​for each group, indicated by horizontal lines. [Figure 4]A surrogate virus neutralization assay assesses the neutralizing activity of sera from mice vaccinated with RBD mouse IgG1-Fc dimer (RBD). Secondary sera from BALB / c mice shown in Figure 2 were also evaluated for SARS-CoV-2 neutralizing antibodies using the surrogate virus neutralization assay. The ability of serum antibodies to inhibit binding of horseradish peroxidase-conjugated SARS-CoV-2 RBD protein (HRP-RBD) to wells coated with immobilized ACE2 receptor was tested in an ELISA format at a serum dilution of 1:10. The degree to which sera inhibited binding of HRP-RBD to ACE2 receptor was determined compared to control serum by optical density readings, with ≥20% inhibition considered a positive result. Percent inhibition of binding of RBD to ACE-2 for individual animals is shown, with the mean value for each group indicated by a horizontal line. [Diagram 5] Serological responses to SARS-CoV-2 RBD in human convalescent samples (hCov) compared with serum samples from mice vaccinated with RBD mouse IgG1-Fc dimer (RBD). RBD-specific antibody responses and neutralizing antibody responses assessed by microneutralization assays and surrogate virus neutralization tests were compared between serum from RBD-vaccinated mice and plasma samples from hCov patients. Mouse serum samples and hCov plasma samples were tested in separate experiments. [Figure 6]Mutations in the SARS-CoV-2 spike protein and neutralization of SARS-CoV-2 variants by human convalescent plasma samples (hCov) and by sera from mice vaccinated with RBD mouse IgG1-Fc dimer (RBD). (A) Spike coding mutations and (frequency) >10 local cases, mutations in the RBD region are shown in red. (B) SARS-CoV-2 neutralization assay showing that three convalescent plasma samples (DD2, DD3, and DD4) are unable to neutralize or have reduced neutralizing ability of the 501Y and 477N variants. (C) SARS-CoV-2 neutralization assay showing that sera from mice immunized with the RBD mouse IgG1-Fc dimer vaccine neutralize the 501Y and 477N variants (VIC2089 and VIC4881) and the original VIC01 strain in the presence of P2C or a-GC adjuvants. [Figure 7] Breadth of binding of antibodies in serum from mice vaccinated with RBD mouse IgG1-Fc dimer with or without adjuvant to RBD variants. Using the RBD multiplex bead array assay, antibodies in serum collected from mice 4 weeks after the second subcutaneous dose of RBD mouse IgG1-Fc dimer vaccine administered in the presence of PEG-R4-Pam-2-Cys (RBD P2C) or α-GC (RBD a-GC) adjuvants, or administered without adjuvant (RBD), were tested for binding to 18 RBD variants. Binding is shown as the half maximal effective concentration (EC50) of each serum sample against each variant. [Figure 8] A SARS-CoV-2 mouse challenge model was established using VIC2089(N501Y / D614G). BALB / c and C57BL / 6 mice were aerosol challenged with either the index SARS-CoV-2 strain VIC01 or the VIC2089(N501Y / D614G) mutant. Mice were sacrificed 3 days after challenge, right lung lobes were harvested and homogenized, and supernatants were collected and stored at -80°C. Infectious virus titers (TCID50; 50% tissue culture infectious dose) were determined by titrating supernatants on Vero cell monolayers and assessing CPE 5 days later. [Figure 9] Comparison of neutralization of the index SARS-CoV-2 strains VIC01 (WT) and VIC2089 (D614G / N501Y) mutant by sera from mice vaccinated with RBD mouse IgG1-Fc dimer. Sera collected 4 weeks following the second injection of RBD dimer administered without adjuvant (RBD) or in the presence of PEG-R4-Pam-2-Cys (RBD P2C) or α-GC (RBD a-GC) were tested for neutralizing antibodies against VIC01 (WT) and VIC2089 (D614G / N501Y) mutant using a microneutralization assay. Viral CPE was measured on day 5. Neutralizing antibody titers were calculated using the Reed / Muench method. Neutralizing antibody titers from individual animals are represented as the mean of each group, indicated by a horizontal line. [Figure 10] Viral titers in lungs of mice vaccinated with RBD mouse IgG1-Fc dimer and challenged with the VIC2089(D614G / N501Y) variant. Mice immunized with RBD mouse IgG1-Fc vaccine alone (RBD) or with PEG-R4-Pam-2-Cys (RBD P2C) or α-GC (RBD a-GC) adjuvants were challenged with the VIC2089(D614G / N501Y) variant 103 days after the second vaccination. Age-matched unvaccinated control mice were challenged at the same time. Mice were sacrificed 3 days after challenge, right lung lobes were harvested and homogenized, and supernatants were collected and stored at -80°C. Infectious virus titers (TCID50) in lungs were determined by titrating supernatants on Vero cell monolayers and assessing CPE 5 days later. [Figure 11]Viral titers in nasal turbinates as a measure of upper respiratory tract protection in mice vaccinated with RBD mouse IgG1-Fc dimer and challenged with the VIC2089(D614G / N501Y) variant. In addition to assessing viral titers in the lungs of mice immunized with RBD mouse IgG1-Fc vaccine alone (RBD) or with PEG-R4-Pam-2-Cys (RBD P2C) or α-GC (RBD a-GC) adjuvants and challenged with the VIC2089(D614G / N501Y) variant, viral titers were also assessed in the nasal turbinates of the same mice as a measure of upper respiratory tract protection. Mice were sacrificed 3 days after challenge, nasal turbinates were harvested and homogenized, and supernatants were collected and stored at -80°C. Infectious virus titers (TCID50) in nasal turbinates were determined by titrating supernatants on Vero cell monolayers and assessing CPE after 5 days. [Figure 12] Evaluation of the protective efficacy of intramuscular administration of RBD mouse IgG1-Fc (RBD) dimer vaccine by virus challenge. Groups of 10 BALB / c mice were vaccinated intramuscularly with 0.1 nmoles of RBD dimer administered with 0.3 nmoles of PEG-R4-Pam-2-Cys (RBD P2C). Mice were primed on day 0, boosted on day 28, and challenged 4 weeks later with the VIC2089 (D614G / N501Y) mutant strain. Five age-matched unvaccinated control BALB / c mice were also challenged at the same time. Mice were sacrificed 3 days after challenge, right lung lobes and nasal turbinates were harvested and homogenized, and supernatants were collected and stored at -80°C. Titers of infectious virus (TCID50) in tissues were determined by titrating supernatants on Vero cell monolayers and assessing CPE 5 days later. [Figure 13]Evaluation of protective efficacy of intramuscular administration of RBD mouse IgG1-Fc (RBD) dimer vaccine in C57BL / 6 mice following viral challenge. Groups of five C57BL / 6 mice were vaccinated intramuscularly with 0.1 nmoles of RBD dimer administered with 0.3 nmoles of PEG-R4-Pam-2-Cys (RBD P2C). Mice were primed on day 0, boosted on day 14, and challenged 3 months later with the VIC2089 (D614G / N501Y) mutant strain. Five age-matched unvaccinated control C57BL / 6 mice were also challenged at the same time. Mice were sacrificed 3 days after challenge, right lung lobes and nasal turbinates were harvested and homogenized, and supernatants were collected and stored at -80°C. Infectious virus titers (TCID50) in tissues were determined by titrating supernatants on Vero cell monolayers and assessing CPE after 5 days. [Figure 14] Comparison of RBD-specific antibody responses in primary and secondary sera from mice vaccinated with RBD mouse Fc dimer, RBD human Fc dimer, or RBD tandem dimer. (A) Schematic of the three RBD dimer antigens used for vaccination. (B) C57BL / 6 mice (5 per group) were vaccinated intramuscularly with either 0.1 nmoles of RBD mouse Fc dimer, RBD human Fc dimer, or single chain dimer, all administered with 0.3 nmoles of PEG-R4-Pam-2-Cys. Mice were primed on day 0 and boosted on day 28. Sera collected immediately before the first injection (prebleed), immediately before the second injection (1°), and 2 weeks after the second injection (2°) were tested for RBD-specific antibodies by ELISA. Antibody titers were calculated as the reciprocal of the highest dilution (log10) of serum giving an OD of 0.3. Antibody titers from individual animals are expressed as the mean for each group, indicated by the horizontal line. [Figure 15]Durability of RBD-specific antibody responses elicited by each RBD antigen administered with PEG-R4-Pam-2-Cys as adjuvant. RBD-specific antibody responses in serum of C57BL / 6 mice shown in FIG. 14 were measured on days 42, 63, and 84 to assess the durability of antibody responses induced by three different RBD antigens administered with PEG-R4-Pam-2-Cys (P2C). Antibody titers were calculated as the reciprocal of the highest serum dilution (log10) that gave an OD of 0.3. Antibody titers from individual animals are represented as the mean values ​​for each group, indicated by the horizontal lines. [Figure 16] Comparison of RBD-specific antibody responses in serum of mice vaccinated with RBD mouse IgG1-Fc dimer formulated with either MF59® or PEG-R4-Pam-2-Cys. C57BL / 6 mice were immunized intramuscularly with 0.1 nmol RBD mouse Fc dimer and 0.3 nmol PEG-R4-Pam-2-Cys, or 0.1 nmol RBD mouse Fc dimer and MF59® (50% vol / vol). Mice were bled prior to the first immunization (pre-bleed) and on day 14 (1°), boosted either on day 14 or day 28, and in both cases rebled 2 weeks after the second immunization (2°). Total RBD-specific antibody titers were determined by ELISA. Antibody titers were calculated as the reciprocal of the highest dilution (log10) of serum giving an OD of 0.3. Antibody titers from individual animals are expressed as the mean for each group, indicated by the horizontal line. [Figure 17]Comparison of RBD-specific antibody responses in serum of mice vaccinated with RBD mouse IgG1-Fc dimer or RBD single chain dimer formulated with either MF59® or PEG-R4-Pam-2-Cys. C57BL / 6 mice were immunized intramuscularly with 10 μg of RBD mouse Fc dimer or 10 μg of RBD single chain dimer, both formulated with either 0.3 nmol of PEG-R4-Pam-2-Cys (P2C) or MF59® (50% vol / vol). Mice were primed on day 0 and boosted on day 14. Sera collected immediately before the first injection (prebleed), immediately before the second injection (1°), and 4 weeks after the second injection (2°) were tested for RBD-specific antibodies by ELISA. Antibody titers were calculated as the reciprocal of the highest dilution of serum giving an OD of 0.3 (log10). Antibody titers from individual animals are expressed as the mean for each group, indicated by the horizontal line. [Figure 18] Comparison of RBD-specific antibody responses in serum of mice vaccinated with RBD monomer or RBD mouse IgG1-Fc dimer formulated with PEG-R4-Pam-2-Cys. Groups of 10 BALB / c mice were immunized intramuscularly with either 0.2 nmoles of RBD monomer formulated with 0.3 nmoles of PEG-R4-Pam-2-Cys (P2C) or 0.1 nmoles of RBD mouse Fc dimer formulated with 0.3 nmoles of PEG-R4-Pam-2-Cys (P2C). Mice were primed on day 0 and boosted on day 28. Sera collected immediately before the first injection (prebleed), immediately before the second injection (1°), and 2 weeks after the second injection (2°) were tested for RBD-specific antibodies by ELISA. Antibody titers were calculated as the reciprocal of the highest dilution (log10) of serum giving an OD of 0.3. Antibody titers from individual animals are expressed as the mean for each group, indicated by the horizontal line. [Figure 19]Comparison of protective efficacy induced by RBD mouse IgG1-Fc dimer formulated with PEG-R4-Pam-2-Cys and RBD monomer formulated with PEG-R4-Pam-2-Cys. Groups of 10 BALB / c mice shown in Figure 18 were challenged with the VIC2089 (D614G / N501Y) mutant 4 weeks after the boost on day 28. Five age-matched unvaccinated control BALB / c mice were also challenged at the same time. Mice were sacrificed 3 days after challenge, right lung lobes and nasal turbinates were harvested and homogenized, and supernatants were collected and stored at -80°C. Infectious virus titers (TCID50) in tissues were determined by titrating supernatants on Vero cell monolayers and assessing CPE 5 days later. [Figure 20]Total and nAb responses in mice vaccinated intramuscularly with WT RBD-mFc and either PEG-R4-Pam-2-Cys or MF59®. (A-D) Total mouse Ig reacting to WT RBD monomer. (A and B) Total WT RBD antibody titers in primary (days 8, 15, 22, 41, 62, and 90) and secondary (day 128) sera of mice vaccinated with the two-dose regime on days 0 and 112. ELISA titers are expressed as the reciprocal (log10) of the antibody dilution that gives an absorbance of 0.3. This corresponds to at least 5 times the background level of binding. (C and D) Total WT RBD antibody titers in primary (days 8, 15, and 22), secondary (days 41, 62, 90, and 111) and tertiary (day 132) sera of mice vaccinated with the two-dose regime on days 0, 22, and 112. ELISA titers are expressed as the reciprocal (log10) of the antibody dilution giving an absorbance of 0.3. This corresponds to at least 5 times the background level of binding. (E) Neutralization capacity by microneutralization assay using the WT indicator strain (VIC01). Neutralization titers of secondary (days 62 and 111) and tertiary (day 132) sera from mice vaccinated intramuscularly with WT RBD-mFc combined with MF59®. The half maximal inhibitory concentration (IC50) was calculated based on the reciprocal dilution of sera that completely prevented CPE in 50% of the wells and was calculated by the Reed-Muench formula. LOD: limit of detection. [Figure 21]Total and nAb responses in mice vaccinated intramuscularly with WT RBD-mFc combined with PEG-R4-Pam-2-Cys or WT RBD-nFc combined with PEG-R4-Pam-2-Cys. (A-B) Total mouse Ig reacting to WT RBD monomer. Total WT RBD antibody titers in primary (day 27) and secondary sera (days 41, 55, 76, and 115) from BALB / c (A) and C57BL / 6 (B) mice vaccinated intramuscularly with WT RBD mouse Fc or WT RBD human Fc combined with PEG-R4-Pam-2-Cys. ELISA titers are expressed as the reciprocal (log10) of the antibody dilution giving an absorbance of 0.3. This corresponds to at least 5-fold background levels of binding. (C) Neutralization capacity by microneutralization assay using the WT indicator strain (VIC01). Neutralization titers of secondary (day 41) sera from C57BL / 6 mice vaccinated intramuscularly with WT RBD mouse Fc or WT RBD human Fc in combination with PEG-R4-Pam-2-Cys. Half maximal inhibitory concentrations (IC50) were calculated based on the inverse dilution of serum that completely prevented CPE in 50% of the wells and calculated by the Reed-Muench equation. [Figure 22] Viral titers in lungs of C57BL / 6 mice challenged with VIC2089 (N501Y / D614G). Mice vaccinated intramuscularly with WT RBD mouse Fc with PEG-R4-Pam-2-Cys or WT RBD human Fc with PEG-R4-Pam-2-Cys on days 0 and 28 were aerosol challenged with VIC2089 on day 139 (111 days after the second immunization). Age- and sex-matched unvaccinated control C57BL / 6 mice were also challenged at the same time. Mice were sacrificed 3 days after challenge and infectious virus titers (TCID50) in the lungs of individual mice were determined by titrating lung supernatants on Vero cell monolayers and measuring viral CPE 5 days later. [Figure 23]Total and nAb responses in mice vaccinated intramuscularly with WT RBD-mFc combined with MF59®. (A) Total WT RBD antibody titers in primary (1°, day 20) and secondary (2°, day 33) sera from mice vaccinated intramuscularly with 10 μg WT RBD-hFc formulated with MF59®. ELISA titers are expressed as the reciprocal (log10) of the antibody dilution giving an absorbance of 0.3. This corresponds to at least 5 times the background level of binding. (B) Neutralization of WT indicator strain VIC01 or beta mutant strain B.1.351 by microneutralization assay. Neutralization titers of secondary sera (day 45) from mice vaccinated intramuscularly with WT RBD-hFc combined with MF59®. The half maximal inhibitory concentration (IC50) was calculated based on the reciprocal dilution of serum that completely prevented CPE in 50% of the wells and was calculated by the Reed-Muench formula. [Figure 24] Viral titers in lungs and nasal turbinates of mice challenged with VIC2089 (N501Y / D614G). Mice vaccinated intramuscularly with 10 μg WT RBD-hFc formulated with MF59® on days 0 and 21 were aerosol challenged with VIC2089 on day 60 after the second immunization. Age- and sex-matched unvaccinated control mice were also challenged at the same time. Mice were sacrificed 3 days after challenge and infectious virus titers (TCID50) in the lungs of individual mice were determined by titrating lung supernatants on Vero cell monolayers and measuring viral CPE 5 days later. [Diagram 25]Total and nAb responses in mice vaccinated intramuscularly with betaRBD-mFc combined with MF59®. (A) Total WT antibody titers in primary (1°, day 20) and secondary (2°, day 33) sera from mice vaccinated intramuscularly with 10 μg betaRBD-hFc or 3 μg betaRBD-hFc formulated with MF59®. ELISA titers are expressed as the reciprocal (log10) of the antibody dilution giving an absorbance of 0.3. This corresponds to at least 5 times background levels of binding. (B) Neutralization of WT indicator strain VIC01 or beta mutant strain B.1.351 by microneutralization assay. Neutralization titers of secondary sera (day 33) from mice vaccinated intramuscularly with 10 μg betaRBD-hFc or 3 μg betaRBD-hFc formulated with MF59®. The half maximal inhibitory concentration (IC50) was calculated based on the inverse dilution of serum that completely prevented CPE in 50% of the wells and was calculated by the Reed-Muench formula. [Figure 26] Total Ab responses in mice immunized with WT-RBD-hFC combined with MF59® or betaRBD-hFc. Total WT RBD antibody titers in primary (1°, day 20; A and C) and secondary (2°, day 37; B and D) sera from C57BL / 6 mice vaccinated intramuscularly with (10, 3, 1 or 0.3 μg) WT RBD-hFc (A and B), or betaRBD-hFc (C and D) in the presence of MF59® on days 0 and 21. ELISA titers are expressed as the reciprocal antibody dilution giving an absorbance of 0.3 (log10). This corresponds to at least 5-fold background levels of binding. [Figure 27]Ab responses to WT RBD monomer and betaRBD monomer in mice immunized with WT-RBD-hFC combined with MF59® or betaRBD-hFc combined with MF59®. WT RBD-specific antibody titers (A) and betaRBD-specific antibody titers (B) in secondary serum (day 56) of C57BL / 6 mice vaccinated intramuscularly with (10, 3, 1 or 0.3 μg) WT RBD-hFc or betaRBD-hFc in the presence of MF59® on days 0 and 21. ELISA titers are expressed as the reciprocal antibody dilution giving an absorbance of 0.3 (log10). This corresponds to at least 5-fold background levels of binding. [Figure 28] Virus titers in lungs of C57BL / 6 mice immunized with WT-RBD-hFC combined with MF59® or betaRBD-hFc combined with MF59® and challenged with VIC2089 (N501Y / D614G) or beta variant B.1.351. Mice vaccinated intramuscularly with 10, 3, 1 or 0.3 mg WT RBD-hFc or betaRBD-hFc in the presence of MF59® on days 0 and 21 were aerosol challenged with VIC2089 (A) or beta variant B.1.351 (B), respectively, 55 or 62 days after the second immunization. Age- and sex-matched unvaccinated control C57BL / 6 mice were also challenged on the corresponding days, respectively. Mice were sacrificed 3 days after challenge and lung and nasal supernatants were titrated on Vero cell monolayers to determine infectious virus titers (TCID50; tissue culture infectious dose 50%) in the lungs of individual mice by measuring viral cytopathic effect (CPE) 5 days later. LOD: limit of detection. [Figure 29] Anti-RBD antibody titers in hamsters. Anti-RBD antibody titers in hamsters calculated as the reciprocal of the highest dilution of serum giving an OD value of 0.3. PBS control data on day 14 was not included in the study. [Diagram 30]CSU hamster study: Plaque reduction neutralization titer (PRNT) testing day 78 sera taken 14 days after the third dose against the WT strain. [Diagram 31] Viral titers in oropharyngeal swabs and lungs of hamsters immunized with WT RBD-hFc combined with MF59® or beta RBD-hFc combined with MF59® and challenged with WT (WA-01 / USA) or beta mutant (B.1.351) viruses. Hamsters were vaccinated intramuscularly with 30 or 10 μg WT RBD-hFc or 30, 10 or 3 μg beta RBD-hFc formulated with MF59® on days 0, 21, and 64, and challenged on day 85 by intranasal instillation of 1×104 pfu of virus in a volume of 100 μl under ketamine-xylazine anesthesia. SARS-CoV-2 challenge strains used were WA-01 / USA (WT) and beta mutant (B.1.351), and hamsters were sacrificed 3 days after challenge. Viral loads were measured (A) in oropharyngeal swabs taken 3 days after challenge, just prior to euthanasia of the animals, and (B) in cranial lung homogenates prepared 3 days after challenge. Viral titers were determined on confluent monolayers of Vero cells by performing serial 10-fold dilutions and counting plaques the following day. Titers are expressed as plaque forming units (pfu). LoD: limit of detection. [Diagram 32] Rat antibody titers specific to WT SARS-CoV-2 spike antigen. IgG antibody titers to WT spike in serum from groups of 30 rats vaccinated intramuscularly with saline, 50 μg betaRBD-hFc, or 50 μg betaRBD-hFc combined with MF59® adjuvant on days 0, 22, and 43 are shown below each row. At the end of toxicity on days 44-45 or 56, terminal blood samples were taken from each rat and processed to serum to assess SARS-CoV-2 spike-specific IgG antibody responses by ELISA. [Diagram 33]Total mouse Ig reacting to WT RBD monomer. Total WT RBD antibody titers in (A) primary sera (day 21) and (B) secondary sera (day 43) from mice vaccinated intranasally with freshly prepared formulations of RBD-hFc with PEG-R4-Pam-2-Cys or lyophilized formulations of RBD-hFc with PEG-R4-Pam-2-Cys stored at different temperatures. ELISA titers are expressed as the reciprocal (log10) of the antibody dilution giving an absorbance of 0.3, which corresponds to at least 5-fold background levels of binding. [Diagram 34] Neutralization capacity. (A) Neutralization of WT indicator strain VIC01 by microneutralization assay. Neutralization titers of secondary sera (day 70) from mice intranasally vaccinated with freshly prepared RBD-hFc formulations combined with PEG-R4-Pam-2-Cys or RBD-hFc lyophilized formulations combined with PEG-R4-Pam-2-Cys stored at different temperatures. The half inhibitory dilution (ID50) was calculated based on the reciprocal dilution of sera that completely prevented CPE in 50% of the wells and calculated by the Reed-Muench formula. (B) Percentage inhibition of binding of RBD to ACE2 assessed using the GenScript surrogate virus neutralization test (sVNT). sVNT titers of secondary sera (day 43) from mice intranasally vaccinated with freshly prepared RBD-hFc / PEG-R4-Pam-2-Cys formulations or lyophilized RBD-hFc / PEG-R4-Pam-2-Cys formulations stored at different temperatures. [Diagram 35]Viral titers in lungs and nasal turbinates of mice challenged with VIC2089 (N501Y / D614G). Mice vaccinated intranasally on days 0 and 30 with WT RBD-hFc combined with PEG-R4-Pam-2-Cys or lyophilized WT RBD-hFc combined with PEG-R4-Pam-2-Cys stored at 40°C for 1 month were aerosol challenged with VIC2089 82 days after the second immunization. Age- and sex-matched unvaccinated control mice were also challenged at the same time. Mice were sacrificed 3 days after challenge and infectious virus titers (TCID50) in lungs and nasal turbinates of individual mice were determined by titrating lung (A) and nasal turbinate (B) supernatants on Vero cell monolayers and measuring viral CPE 5 days later. [Diagram 36] Neutralization capacity. Neutralization of WT indicator strain VIC01 and beta mutant strain B1.351 by microneutralization assay. Neutralization titers of secondary sera (day 70) from mice vaccinated intranasally with WT RBD-hFc combined with freshly prepared PEG-R4-Pam-2-Cys or lyophilized WT RBD-hFc combined with PEG-R4-Pam-2-Cys stored at 40°C for 1 month. The half inhibitory dilution (ID50) was calculated based on the reciprocal dilution of sera that completely prevented CPE in 50% of the wells and was calculated by the Reed-Muench formula. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

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[0106] It will be understood that the invention disclosed and defined herein extends to all alternative combinations of two or more individual features mentioned or evident from the text or drawings, all these different combinations constituting various alternative embodiments of the invention.

[0107] Reference will now be made in detail to certain embodiments of the invention. While the invention will be described in conjunction with the embodiments, it will be understood that it is not intended to limit the invention to those embodiments. On the contrary, the invention is intended to cover all alternatives, modifications, and equivalents included within the scope of the present invention as defined by the claims.

[0108] Those skilled in the art will recognize many methods and materials similar or equivalent to those described herein that could be used in the practice of the present invention. The present invention is not limited in any way to the methods and materials described. It will be understood that the invention disclosed and defined herein extends to all alternative combinations of two or more individual features mentioned or evident from the text or drawings. All these different combinations constitute various alternative embodiments of the invention.

[0109] All patents and publications referenced herein are incorporated by reference in their entirety.

[0110] For purposes of interpreting this specification, terms used in the singular will also include the plural and vice versa.

[0111] General chemical terms used in the formulae herein have their ordinary meanings.

[0112] The inventors have developed novel recombinant RBD chimeric or fusion protein vaccines that, when combined with adjuvants (e.g., α-GalCer or PEG-R4-Pam-2-Cys), elicit strong neutralizing antibody responses that are broadly protective against a range of SARS-CoV-2 strains. Remarkably, the inventors have shown that vaccination with a vaccine composition containing an adjuvant (e.g., α-GalCer or PEG-R4-Pam-2-Cys) via various routes, including the upper respiratory tract via intranasal administration, elicits strong neutralizing antibody responses that protect against viral replication in both the upper respiratory tract (nasal cavity) and the lower respiratory tract. The broad protection provides protection against disseminated pulmonary infections that can lead to potentially fatal complications. Furthermore, the inventors have found that vaccination-induced immunity lasts for at least 3 months, and broad protection is maintained to avoid mutant strains.

[0113] coronavirus The term "coronavirus" or "CoV" refers to any virus of the coronavirus family, including, but not limited to, SARS-CoV-2, MERS-CoV, and SARS-CoV; and endemic coronaviruses such as HCoV-NL63, HCoV-229E, HCoV-OC43, and HKU1. SARS-CoV-2 refers to a newly emerged coronavirus identified as the cause of a severe pandemic that began in Wuhan, China, and has spread rapidly to other parts of the world. SARS-CoV-2 is also known as 2019-nCoV and Wuhan coronavirus. It binds to the human host cell receptor angiotensin-converting enzyme 2 (ACE2) through the viral spike protein. The spike protein also binds and is cleaved by TMPRSS2, activating the spike protein for viral membrane fusion.

[0114] Subfamily Coronaviridae and order Nidovirales (International Committee on Taxonomy of Viruses). This subfamily consists of four genera: Alphacoronavirus, Betacoronavirus, Gammacoronavirus, and Deltacoronavirus, based on their phylogenetic relationships and genome structure. The subfamily clusters are labeled Alphacoronavirus 1a and 1b, and Betacoronavirus 2a, 2b, 2c, and 2d. Alphacoronaviruses and Betacoronaviruses infect only mammals. Gammacoronaviruses and Deltacoronaviruses infect birds, although some of them can also infect mammals. Alphacoronaviruses and Betacoronaviruses usually cause respiratory disease in humans and gastroenteritis in animals. Three highly pathogenic viruses, SARS-CoV, MERS-CoV, and SARS-CoV-2, cause severe respiratory syndromes in humans, and four other human coronaviruses (HCoV-NL63, HCoV-229E, HCoV-OC43, and HKU1) cause only mild upper respiratory tract disease in immunocompetent hosts, although some of them can cause severe infections in infants, young children, and the elderly. Alphacoronaviruses and betacoronaviruses can pose a large disease burden in livestock; these viruses include porcine transmissible gastroenteritis virus, porcine enteric diarrhea virus (PEDV), and the recently emerged porcine acute diarrhea syndrome coronavirus (SADS-CoV). Based on current sequence databases, all human coronaviruses are of animal origin: SARS-CoV, MERS-CoV, SARS-CoV-2, HCoV-NL63, and HCoV-229E are believed to be of bat origin, and HCoV-OC43 and HKU1 are believed to be of rodent origin.

[0115] Coronaviruses include antigenic groups I, II, and III. Non-limiting examples of coronaviruses include SARS coronavirus, MERS coronavirus, transmissible gastroenteritis virus (TGEV), human respiratory coronavirus, porcine respiratory coronavirus, canine coronavirus, feline enteric coronavirus, feline infectious peritonitis virus, rabbit coronavirus, mouse hepatitis virus, rat salivary gland dacryoadenitis virus, porcine hemagglutinating encephalomyelitis virus, bovine coronavirus, avian infectious bronchitis virus, and turkey coronavirus, as well as any others described herein, and those mentioned in Cui, et al. Nature Reviews Microbiology volume 17, pages 181-192 (2019); and Shereen et al. Journal of Advanced Research, Volume 24, July 2020 (published online 16 March 2020), Pages 91-98.

[0116] Non-limiting examples of subgroup 1a coronaviruses include FCov.FIPV.79.1146.VR.2202 (GenBank accession number NV_007025), transmissible gastroenteritis virus (TGEV) (GenBank accession number NC_002306; GenBank accession number Q811789.2; GenBank accession number DQ811786.2; GenBank accession number DQ811788.1; GenBank accession number DQ811785.1; GenBank accession number X52157.1; GenBank accession number AJ011482.1; GenBank accession number KC962433.1; GenBank accession number AJ271965.2; GenBank accession number JQ693060.1; GenBank accession number KC60937 1.1; GenBank Accession No. JQ693060.1; GenBank Accession No. JQ693059.1; GenBank Accession No. JQ693058.1; GenBank Accession No. JQ693057.1; GenBank Accession No. JQ693052.1; GenBank Accession No. JQ693051.1; GenBank Accession No. JQ693050.1), porcine reproductive and respiratory syndrome virus (PRRSV) (GenBank Accession No. NC_001961.1; GenBank Accession No. DQ811787), and any other subgroup 1a coronavirus currently known (e.g., that can be found in the GenBank® database) or later identified, and any combination thereof.

[0117] Non-limiting examples of subgroup 1b coronaviruses include BtCoV.1A.AFCD62 (GenBank accession number NC_010437), BtCoV.1B.AFCD307 (GenBank accession number NC_010436), BtCov.HKU8.AFCD77 (GenBank accession number NC_010438), BtCoV.512.2005 (GenBank accession number DQ648858), porcine epidemic diarrhea virus PEDV.CV777 (GenBank accession number NC_003436, GenBank accession number DQ355224.1, GenBank accession number DQ355224.2, GenBank accession number DQ355224.3, GenBank accession number DQ355224.4, GenBank accession number DQ355224.5, GenBank accession number DQ355224.6, GenBank accession number DQ355224.7, GenBank accession number DQ355224.9, GenBank accession number DQ355224.1, GenBank accession number DQ355224.8, GenBank accession number DQ355224.9, GenBank accession number DQ355224.1 ... k Accession number DQ355223.1, GenBank accession number DQ355221.1, GenBank accession number JN601062.1, GenBank accession number N601061.1, GenBank accession number JN601060.1, GenBank accession number JN601059.1, GenBank accession number JN601058.1, GenBank accession number JN601057.1, GenBank accession number JN601056.1, GenBank accession number JN601055.1, GenBank accession number JN601054.1, GenBank accession number JN601053.1, GenBank accession number JN601052.1, GenBank accession number JN400902.1, GenBank accession number JN547395.1, GenBank accession number FJ687473.1, GenBank accession number FJ687472.1, GenBank accession number FJ687471.1, GenBank accession number FJ687470.1, GenBank accession number FJ687469.1, GenBank accession number FJ687468.1, GenBank accession number FJ687467.1, GenBank accession number FJ68 7466.1, GenBank accession number FJ687465.1, GenBank accession number FJ687464.1, GenBank accession number FJ687463.1, GenBank accession number FJ687462.1, GenBank accession number FJ687461.1, GenBank accession number FJ687460.1, GenBank accession number FJ687459.1, GenBank accession number FJ687458.1, GenBank accession number FJ687457.1, GenBank accession number FJ687456.1, GenBank accession number FJ687455.1, GenBank accession number FJ687454.1, GenBank accession number FJ687453 GenBank accession number FJ687452.1, GenBank accession number FJ687451.1, GenBank accession number FJ687450.1, GenBank accession number FJ687449.1, GenBank accession number AF500215.1, GenBank accession number KF476061.1, GenBank accession number KF476060.1, GenBank accession number KF476059.1, GenBank accession number KF476058 ... 76057.1, GenBank accession number KF476056.1, GenBank accession number KF476055.1, GenBank accession number KF476054.1, GenBank accession number KF476053.1, GenBank accession number KF476052.1, GenBank accession number KF476051.1, GenBank accession number KF476050.1, GenBank accession number KF476049.1, GenBank accession number KF476048.1, GenBank accession number KF177258.1, GenBank accession number KF177257.1, GenBank accession number KF177256.1, GenBank accession number KF177255.1), HCoV.229E (GenBank accession number NC_002645), HCoV.NL63.Amsterdam.I (GenBank accession number NC_005831), BtCoV.HKU2.HK.298.2006 (GenBank accession number EF203066), BtCoV.HKU2. Examples of coronaviruses that may be identified include BtCoV.HKU2.HK.33.2006 (GenBank Accession No. EF203067), BtCoV.HKU2.HK.46.2006 (GenBank Accession No. EF203065), BtCoV.HKU2.GD.430.2006 (GenBank Accession No. EF203064), and any other subgroup 1b coronaviruses currently known (e.g., that may be found in the GenBank® database) or later identified, and any combination thereof.

[0118] Non-limiting examples of subgroup 2a coronaviruses include HCoV.HKU1.C.N5 (GenBank Accession No. DQ339101), MHV.A59 (GenBank Accession No. NC 001846), PHEV.VW572 (GenBank Accession No. NC 007732), HCoV.OC43.ATCC.VR.759 (GenBank Accession No. NC_005147), bovine enteric coronavirus (BCoV.ENT) (GenBank Accession No. NC_003045), and any other subgroup 2a coronaviruses currently known (e.g., that can be found in the GenBank® database) or later identified, and any combination thereof.

[0119] Non-limiting examples of subgroup 2b coronaviruses include bat SARSCoV (GenBank accession number FJ211859), SARSCoV (GenBank accession number FJ211860), SARS-CoV-2 (GenBank accession number NC_045512.2), BtSARS.HKU3.1 (GenBank accession number DQ022305), BtSARS.HKU3.2 (GenBank accession number DQ084199), BtSARS.HKU3.3 (GenBank accession number DQ0842 00), BtSARS.Rm1 (GenBank accession number DQ412043), BtCoV.279.2005 (GenBank accession number DQ648857), BtSARS.Rf1 (GenBank accession number DQ412042), BtCoV.273.2005 (GenBank accession number DQ648856), BtSARS.Rp3 (GenBank accession number DQ071615), SARSCoV.A022 (GenBank accession number AY686863), SARSCoV.CUHK-W 1 (GenBank accession number AY278554), SARSCoV.GDO1 (GenBank accession number AY278489), SARSCoV.HC.SZ.61.03 (GenBank accession number AY515512), SARSCoV.SZ16 (GenBank accession number AY304488), SARSCoV.Urbani (GenBank accession number AY278741), SARSCoV.civet010 (GenBank accession number AY572035), and SARSCoV.MA.1 5 (GenBank Accession No. DQ497008), RsSHC014 (GenBank® Accession No. KC881005), Rs3367 (GenBank® Accession No. KC881006), WiV1S (GenBank® Accession No. KC881007), and any other subgroup 2b coronaviruses currently known (e.g., that can be found in the GenBank® database) or later identified, and any combination thereof.

[0120] Non-limiting examples of subgroup 2c coronaviruses include Middle East respiratory syndrome coronavirus isolate Riyadh_2_2012 (GenBank accession number KF600652.1), Middle East respiratory syndrome coronavirus isolate Al-Hasa_18_2013 (GenBank accession number KF600651.1), Middle East respiratory syndrome coronavirus isolate Al-Hasa_17_2013 (GenBank accession number KF600647.1), Middle East respiratory syndrome coronavirus isolate Al-Hasa_15_2013 (GenBank accession number KF600645.1), and the like. .1), Middle East respiratory syndrome coronavirus isolate Al-Hasa_16_2013 (GenBank accession number KF600644.1), Middle East respiratory syndrome coronavirus isolate Al-Hasa_21_2013 (GenBank accession number KF600634), Middle East respiratory syndrome coronavirus isolate Al-Hasa_19_2013 (GenBank accession number KF600632), Middle East respiratory syndrome coronavirus isolate Buraidah_1_2013 (GenBank accession number KF600630.1), Middle East respiratory syndrome coronavirus single isolate Hafr-Al-Batin_1_2013 (GenBank accession number KF600628.1), Middle East respiratory syndrome coronavirus isolate Al-Hasa_12_2013 (GenBank accession number KF600627.1), Middle East respiratory syndrome coronavirus isolate Bisha_1_2012 (GenBank accession number KF600620.1), Middle East respiratory syndrome coronavirus isolate Riyadh_3_2013 (GenBank accession number KF600613.1), Middle East respiratory syndrome coronavirus isolate Riyadh_1_2012 ( GenBank accession number KF600612.1), Middle East respiratory syndrome coronavirus isolate Al-Hasa_3_2013 (GenBank accession number KF186565.1), Middle East respiratory syndrome coronavirus isolate Al-Hasa_1_2013 (GenBank accession number KF186567.1), Middle East respiratory syndrome coronavirus isolate Al-Hasa_2_2013 (GenBank accession number KF186566.1), and Middle East respiratory syndrome coronavirus isolate Al-Hasa_4_2013 (GenBank accession number KF186564.1), Middle East respiratory syndrome coronavirus (GenBank accession number KF192507.1), Betacoronavirus England 1-N1 (GenBank accession number NC_019843), MERS-CoV_SA-N1 (GenBank accession number KC667074), and the following isolates of Middle East respiratory syndrome coronavirus (GenBank accession number KF600656.1, GenBank accession number KF600655.1, GenBank accession number KF600654.1, GenBank accession number KF600649.1, Gen ... 600648.1, GenBank accession number KF600646.1, GenBank accession number KF600643.1, GenBank accession number KF600642.1, GenBank accession number KF600640.1, GenBank accession number KF600639.1, GenBank accession number KF600638.1, GenBank accession number KF600637.1, GenBank accession number KF600636.1, GenBank accession number No. KF600635.1, GenBank accession no. KF600631.1, GenBank accession no. KF600626.1, GenBank accession no. KF600625.1, GenBank accession no. KF600624.1, GenBank accession no. KF600623.1, GenBank accession no. KF600622.1, GenBank accession no. KF600621.1, GenBank accession no. KF600619.1, GenBank Accession number KF600618.1, GenBank accession number KF600616.1, GenBank accession number KF600615.1, GenBank accession number KF600614.1, GenBank accession number KF600641.1, GenBank accession number KF600633.1, GenBank accession number KF600629.1, GenBank accession number KF600617.1), Coronavirus Neoromicia / PML-PHE1 / RSA / 2011 GenBank accession number KC869678.2, Bat coronavirus Taper / CII_KSA_287 / Bisha / Saudi Arabia / GenBank accession number KF493885.1, Bat coronavirus Rhhar / CII_KSA_003 / Bisha / Saudi Arabia / 2013 GenBank accession number KF493888.1, Bat coronavirus Pikuh / CII_KSA_001 / Riyadh / Saudi Arabia / 2013 GenBank accession number KF493887.1, Bat coronavirus Rhhar / CII_KSA_002 / Bisha / Saudi Arabia / 2013 GenBank accession number KF493886.1, Bat coronavirus Rhhar / CII_KSA_004 / Bisha / Saudi Arabia / 2013 GenBank accession number KF493884.1, BtCoV.HKU4.2 (GenBank accession number EF065506), BtCoV.HKU4.1 (GenBank accession number NC_009019), BtCoV.HKU4.3 (GenBank accession number EF065507), BtCoV.HKU4.4 (GenBank accession number EF065508), BtCoV 133.2005 (GenBank accession number NC 008315), BtCoV.HKU5.5 (GenBank accession number EF065512); BtCoV.HKU5.1 (GenBank accession number NC_009020), BtCoV.HKU5.2 (GenBank accession number EF065510), BtCoV.HKU5.3 (GenBank accession number EF065511), human betacoronavirus 2c Jordan-N3 / 2012 (GenBank accession number KC776174.1; human betacoronavirus 2c EMC / 2012 (GenBank accession number JX869059.2), pipistrella bat coronavirus HKU5 isolate (GenBank accession number KC522089.1, GenBank accession number KC522088.1, GenBank accession number KC522087.1, GenBank accession number KC522086.1, GenBank accession number KC522085.1, GenBank accession number KC522084.1, GenBank accession number KC522083.1, GenBank accession number KC522082.1, GenBank accession number KC522081.1, GenBank accession number KC522080.1, GenBank accession number KC522079.1, GenBank accession number KC522078.1, GenBank accession number KC522077.1, GenBank accession number KC522076.1, GenBank accession number KC522075.1, GenBank accession number KC522104.1, GenBank accession number KC522104.1, GenBank accession number KC522103.1, GenBank accession number KC522102.1, GenBank accession number KC522101.1, GenBank accession number KC522100.1, GenBank accession number KC522099. 1, GenBank accession number KC522098.1, GenBank accession number KC522097.1, GenBank accession number KC522096.1, GenBank accession number KC522095.1, GenBank accession number KC522094.1, GenBank accession number KC522093.1, GenBank accession number KC522092.1, GenBank accession number KC522091.1, GenBank accession number KC522090.1, GenBank accession number KC522119.1 GenBank Accession Number KC522118.1 GenBank Accession Number KC522117.1 GenBank Accession Number KC522116.1 GenBank Accession Number KC522115.1 GenBank Accession Number KC522114.1 GenBank Accession Number KC522113.1 GenBank Accession Number KC522112.1 GenBank Accession Number KC522111.1 GenBank Accession Number KC522110.1 GenBank Accession Number KC522109.1 GenBank accession number KC522108.1, GenBank accession number KC522107.1, GenBank accession number KC522106.1, GenBank accession number KC522105.1), and the pipistrelle coronavirus HKU4 isolate (GenBank accession number KC522048.1, GenBank accession number KC522047.1, GenBank accession number KC522046.1, GenBank accession number KC522045.1, GenBank accession number KC522044.1, GenBank accession number KC522043.1, GenBank accession number KC522042.1, GenBank accession number KC522041.1, GenBank accession number KC522040.1 and GenBank accession number KC522039).1, GenBank accession number KC522038.1, GenBank accession number KC522037.1, GenBank accession number KC522036.1, GenBank accession number KC522048.1 GenBank accession number KC522047.1 GenBank accession number KC522046.1 GenBank accession number KC522045.1 GenBank accession number KC522044.1 GenBank accession number KC522043.1 GenBank accession number KC522042.1 GenBank accession number KC522041.1 GenBank accession number KC522040.1, GenBank accession number KC522039.1 GenBank accession number KC522038.1 GenBank accession number KC522037.1 GenBank accession number KC522036.1, GenBank accession number KC522061.1 GenBank accession number KC522060.1 GenBank accession number KC522059.1 GenBank accession number KC522058.1 GenBank accession number KC522057.1 GenBank accession number KC522056.1 GenBank accession number KC522055.1 GenBank accession number KC522054.1 GenBank accession number KC522053.1 GenBank accession number KC522052.1 GenBank accession number KC522051.1 GenBank accession number KC522050.1 GenBank accession number KC522049.1 GenBank accession number KC522074.1, GenBank accession number KC522073.1 GenBank Accession Number KC522072.1 GenBank Accession Number KC522071.1 GenBank Accession Number KC522070.1 GenBank Accession Number KC522069.1 GenBank Accession Number KC522068.1 GenBank Accession Number KC522067.1, GenBank Accession Number KC522066.1 GenBank Accession Number KC522065.1 GenBank Accession Number KC522064.1, GenBank Accession Number KC522063.1, or GenBank Accession Number KC522062.1, as well as any other subgroup 2b coronavirus currently known (e.g., that may be found in the GenBank® database) or later identified, and any combination thereof.

[0121] Non-limiting examples of subgroup 2d coronaviruses include BtCoV.HKU9.2 (GenBank Accession No. EF065514), BtCoV.HKU9.1 (GenBank Accession No. NC_009021), BtCoV.HkU9.3 (GenBank Accession No. EF065515), BtCoV.HKU9.4 (GenBank Accession No. EF065516), and any other subgroup 2d coronaviruses currently known (e.g., that can be found in the GenBank® database) or later identified, and any combination thereof.

[0122] Non-limiting examples of subgroup 3 coronaviruses include IBV.Beaudette.IBV.p65 (GenBank Accession No. DQ001339), as well as any other subgroup 3 coronaviruses currently known (e.g., that can be found in the GenBank® database) or later identified, and any combination thereof.

[0123] The coronavirus may be any virus that comprises the receptor binding domain of the spike protein that contains one or more of the mutations as shown in FIG.

[0124] A subject or individual in need of treatment according to any aspect of the invention or in need of administration of any composition described herein may be an individual exhibiting symptoms of coronavirus infection or an individual diagnosed with coronavirus infection. Further, the subject or individual may have been clinically or biochemically determined to be infected with coronavirus.

[0125] The subject may be at a stage of coronavirus infection before developing end-stage organ failure. The subject in need thereof may be anyone who has had coronavirus infection from the beginning of clinical progression before developing end-stage organ failure. In one embodiment, the subject has coronavirus infection symptoms for 12 days or less and does not have life-threatening organ dysfunction or organ failure. Preferably, the subject is early in the course of the disease, for example, before day 14 from symptom onset or in the viremia and seronegative stage.

[0126] A "subject" or "individual" may be any animal susceptible to infection by coronavirus and / or to a disease or disorder caused by coronavirus infection. The subject of the present invention may be a mammal, and in certain embodiments is a human, which may be an infant, a child, an adult, or the elderly. A "subject at risk of infection by coronavirus" or a "subject at risk of coronavirus infection" is any subject that may be or has been exposed to coronavirus. A "subject" or "individual" includes any human or non-human animal. Thus, in addition to being useful for human treatment, the compounds of the present invention may be useful for veterinary treatment of mammals, including companion animals and livestock, such as, but not limited to, cats, horses, cows, sheep, and pigs, or any animal that may be infected with coronavirus.

[0127] Those at risk include, but are not limited to, immunocompromised individuals, older adults (over 65 years of age), children under the age of 2, healthcare workers, adults or children in close contact with someone with confirmed or suspected coronavirus infection, and people with underlying medical conditions such as lung infections, heart disease or diabetes, or primary or secondary immunodeficiencies.

[0128] Proteins and Polypeptides "Isolated," as used to describe the various polypeptides disclosed herein, refers to a polypeptide that has been identified and separated and / or recovered from a component of its natural environment. Contaminant components of the natural environment are typically substances that would interfere with diagnostic or therapeutic uses of the polypeptide and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In a preferred embodiment, the polypeptide is (1) purified to a sufficient extent to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (2) purified to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or, preferably, silver staining. Isolated proteins also include in situ polypeptides in recombinant cells, since at least one component of the polypeptide's natural environment is absent. Ordinarily, however, isolated polypeptides are prepared by at least one purification step.

[0129] A "fragment" is a portion of a polypeptide of the present invention that retains substantially similar functional activity, or substantially the same biological function or activity, as the polypeptide, which can be determined using the assays described herein.

[0130] "Percent (%) amino acid sequence identity" or "percent identity" with respect to a polypeptide sequence, i.e., a polypeptide of the invention as defined herein, is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in a particular polypeptide of the invention, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, without taking into account conservative substitutions as part of the sequence identity.

[0131] Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms (non-limiting examples described below) required to achieve maximum alignment over the entire length of the sequences being compared. When amino acid sequences are aligned, the percent amino acid sequence identity of a given amino acid sequence A with, or with respect to, or to a given amino acid sequence B (which can alternatively be expressed as a given amino acid sequence A having or comprising a particular percent amino acid sequence identity with, or with respect to, or to a given amino acid sequence B) can be calculated as follows: percent amino acid sequence identity=X / Y100, where X is the number of amino acid residues scored as perfect matches by the alignment of A and B by a sequence alignment program or algorithm, and Y is the total number of amino acid residues in B. If the length of amino acid sequence A is not equal to the length of amino acid sequence B, the percent amino acid sequence identity of A to B will not be equal to the percent amino acid sequence identity of B to A.

[0132] In calculating percent identity, typically, exact matches are counted. The determination of percent identity between two sequences can be achieved using a mathematical algorithm. A non-limiting example of a mathematical algorithm that can be used to compare two sequences is the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264 (modified according to Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877). Such an algorithm is incorporated into the BLASTN and BLASTX programs of Altschul et al. (1990) J. MoI. Biol. 215:403. To obtain gapped alignment, Gapped BLAST (BLAST 2.0) can be used as described in Altschul et al. (1997) Nucleic Acids Res. 25:3389. Alternatively, PSI-Blast can be used to perform an iterative search to detect distant relationships between molecules. See Altschul et al. (1997), supra. When using BLAST, Gapped BLAST, and PSI-BLAST programs, the default parameters of the respective programs (e.g., BLASTX and BLASTN) can be used. Alignment can also be performed manually by inspection. Another non-limiting example of a mathematical algorithm utilized for sequence comparison is the ClustalW algorithm (Higgins et al. (1994) Nucleic Acids Res. 22:4673-4680). ClustalW compares sequences to align entire amino acid or DNA sequences, and thus can provide data on sequence conservation throughout the entire amino acid sequence. The ClustalW algorithm is used in several commercially available DNA / amino acid analysis software packages, such as the ALIGNX module of the Vector NTI Program Suite (Invitrogen Corporation, Carlsbad, CA). After aligning amino acid sequences with ClustalW, amino acid identity sequences can be evaluated.A non-limiting example of a software program useful for analyzing ClustalW alignments is GENEDOC™ or JalView (http: / / www.jalview.org / ). GENEDOC™ allows the assessment of amino acid (or DNA) similarity and identity between multiple proteins. Another non-limiting example of a mathematical algorithm utilized for sequence comparison is the algorithm of Myers and Miller, 1988, CABIOS 4:11-17. Such an algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG Wisconsin Genetics software package, version 10 (available from Accelrys, Inc., 9685 Scranton Rd., San Diego, CA, USA). When using the ALIGN program to compare amino acid sequences, a PAM 120 weight residue table, gap length penalty=12, and gap penalty=4 may be used.

[0133] The polypeptide desirably comprises an amino terminus and a carboxyl terminus. The polypeptide may comprise D-amino acids, L-amino acids or a mixture of D- and L-amino acids. However, the D-form of amino acids is particularly preferred, since polypeptides composed of D-amino acids are expected to have greater retention of biological activity in vivo.

[0134] Polypeptides may be prepared by any of a number of conventional techniques. Polypeptides may be isolated or purified from naturally occurring sources or from recombinant sources. Recombinant production is preferred. For example, in the case of recombinant polypeptides, a DNA fragment encoding the desired peptide may be subcloned into an appropriate vector using well-known molecular genetic techniques (see, e.g., Maniatis et al., Molecular Cloning: A Laboratory Manual, 2nd ed. (Cold Spring Harbor Laboratory, 1982); Sambrook et al., Molecular Cloning A Laboratory Manual, 2nd ed. (Cold Spring Harbor Laboratory, 1989). The fragment may be transcribed and the polypeptide may subsequently be translated in vitro. Commercially available kits may also be employed (such as those manufactured by, e.g., Clontech, Palo Alto, Calif.; Amersham Pharmacia Biotech Inc., Piscataway, NJ; InVitrogen, Carlsbad, Calif.). The polymerase chain reaction may optionally be employed for nucleic acid manipulation.

[0135] The term "conservative substitution," as used herein, refers to the replacement of an amino acid present in the native sequence of a peptide with a natural or non-natural amino acid, or a peptidomimetic having similar steric properties. If the side chain of the substituted natural amino acid is either polar or hydrophobic, the conservative substitution must be with a natural or non-natural amino acid, or a peptidomimetic moiety that is also polar or hydrophobic (in addition to having the same steric properties as the side chain of the substituted amino acid).

[0136] Conservative amino acid substitution tables providing functionally similar amino acids are well known to those of skill in the art. The following six groups are examples of amino acids that are considered to be conservative substitutions for one another: 1) Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid (D), glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

[0137] Naturally occurring amino acids are typically grouped according to their properties, so that conservative substitutions with naturally occurring amino acids can be determined, bearing in mind the fact that the substitution of a charged amino acid with a sterically similar uncharged amino acid is considered a conservative substitution. It is also possible to use amino acid analogs (synthetic amino acids) well known in the art to generate conservative substitutions with non-natural amino acids. Peptide mimetics of natural amino acids are well documented in the literature known to those skilled in the art, and non-natural or unnatural amino acids are described in more detail below. When affecting a conservative substitution, the substituting amino acid should have the same or similar functional group in the side chain as the original amino acid.

[0138] The phrase "non-conservative substitution" or "non-conserved residue" as used herein refers to the replacement of an amino acid present in a parent sequence with another natural or non-natural amino acid having different electrochemical and / or steric properties. Thus, the side chain of the substituted amino acid may be significantly larger (or smaller) than the side chain of the natural amino acid being replaced and / or may have a functional group with significantly different electronic properties than the amino acid being replaced. Examples of this type of non-conservative substitution include the replacement of alanine with phenylalanine or cyclohexylmethylglycine, the replacement of isoleucine with glycine, or the replacement of aspartic acid with -NH-CH[(-CH2)5-COOH]-CO-. Non-conservative substitutions include any mutation that is not considered conservative.

[0139] Non-conservative amino acid substitutions can arise from changes in: (a) the structure of the amino acid backbone in the area of ​​substitution; (b) the charge or hydrophobicity of the amino acid; or (c) the bulk of the amino acid side chain. In general, the substitutions expected to produce the greatest changes in the properties of a protein are those in which: (a) a hydrophilic residue is replaced by (or by) a hydrophobic residue; (b) proline is replaced by (or by) another residue; (c) a residue having a bulky side chain, e.g., phenylalanine, is replaced by (or by) a residue without a bulky side chain, e.g., glycine; (d) a residue having an electropositive side chain, e.g., lysyl, arginyl, or histidyl, is replaced by (or by) an electronegative residue, e.g., glutamyl or aspartyl.

[0140] Modification of the native amino acid sequence to generate mutant polypeptides, for example by insertion, deletion and / or substitution, can be carried out by various means known to those skilled in the art. For example, site-specific mutations can be introduced by ligating synthetic oligonucleotides comprising the modification site into an expression vector. Alternately, oligonucleotide-directed site-specific mutagenesis procedures such as those disclosed in Walder et al., Gene 42:133 (1986); Bauer et al., Gene 37:73 (1985); Craik, Biotechniques, 12-19 (January 1995); and U.S. Pat. Nos. 4,518,584 and 4,737,462 can be used. A preferred means for introducing mutations is the QuikChange site-directed mutagenesis kit (Stratagene, LaJolla, Calif.).

[0141] Any suitable expression vector (e.g., as described in Pouwels et al., Cloning Vectors: A Laboratory Manual (Elsevier, NY: 1985)) and corresponding suitable host can be employed for the production of recombinant polypeptides. Expression hosts include, but are not limited to, bacterial species within the genera Escherichia, Bacillus, Pseudomonas, Salmonella; mammalian or insect host cell systems, including baculovirus systems (e.g., as described by Luckow et al., Bio / Technology 6:47 (1988)); and established cell lines such as COS-7, C127, 3T3, CHO, HeLa, and BHK cell lines. One of skill in the art will recognize that the choice of expression host will affect the type of polypeptide produced. For example, the glycosylation of a polypeptide produced in yeast cells or mammalian cells (e.g., COS-7 cells) will differ from the glycosylation of a polypeptide produced in bacterial cells such as Escherichia coli.

[0142] Alternatively, the polypeptides of the invention can be synthesized using standard peptide synthesis techniques well known to those skilled in the art (e.g., as reviewed in Bodanszky, Principles of Peptide Synthesis (Springer-Verlag, Heidelberg: 1984)). In particular, the polypeptides can be synthesized using solid-phase synthesis procedures (see, e.g., Merrifield, J. Am. Chem. Soc. 85:2149-54 (1963); Barany et al., Int. J. Peptide Protein Res. 30:705-739 (1987); and U.S. Pat. No. 5,424,398). If desired, this can be carried out using an automated peptide synthesizer. Removal of t-butyloxycarbonyl (t-BOC) or 9-fluorenylmethyloxycarbonyl (Fmoc) amino acid blocking groups and separation of the polypeptide from the resin can be accomplished, for example, by acid treatment at low temperature. The synthesized polypeptide may then be extracted from the resin powder (e.g., with about 25% w / v acetic acid), for example by extraction with dimethyl ether to remove non-peptide organic compounds. Following synthesis of the polypeptide, optional further purification (e.g., using high performance liquid chromatography (HPLC)) may be performed to remove incomplete polypeptides or free amino acids. Amino acid analysis and / or HPLC analysis may be performed on the synthesized polypeptide to verify its identity. For other applications according to the invention, it may be preferable to produce the polypeptide as part of a larger fusion protein, such as by methods described herein or other genetic means, or as part of a larger complex, such as via physical or chemical conjugation, as known to those of skill in the art and described herein.

[0143] The polypeptides of the invention may also be modified, conjugated or fused with other moieties to facilitate purification or to increase the in vivo half-life of the polypeptides, or to be used in immunoassays using methods known in the art. For example, the polypeptides of the invention may be modified by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, conjugation to cellular ligands or other proteins, and the like.

[0144] A "peptidomimetic" is a synthetic compound having substantially the same structural and / or functional characteristics as a polypeptide of the invention, the latter being described in more detail herein. Typically, a peptidomimetic has the same or similar structure as a polypeptide of the invention, e.g., the same or similar sequence of SEQ ID NO: 6 or 23, or a fragment thereof, with a reduced ability to form dimers. A peptidomimetic generally comprises at least one residue that is not naturally synthesized. The non-natural component of a peptidomimetic compound may conform to one or more of the following: a) a residue linkage other than the natural amide bond ("peptide bond") linkage; b) a non-natural residue in place of a natural amino acid residue; or c) a residue that induces a secondary structure mimic, i.e., a residue that induces or stabilizes a secondary structure, such as, for example, a β-turn, γ-turn, β-sheet, α-helical conformation.

[0145] Peptide mimetics can be synthesized using a variety of procedures and methodologies described in the scientific and patent literature, e.g., Organic Syntheses Collective Volumes, Gilman et al. (Eds) John Wiley & Sons, Inc., NY, al-Obeidi (1998) Mol. Biotechnol. 9:205-223; Hruby (1997) Curr. Opin. Chem. Biol. 1:114-119; Ostergaard (1997) Mol. Divers. 3:17-27; Ostresh (1996) Methods Enzymot. 267:220-234.

[0146] Modifications contemplated herein include, but are not limited to, side chain modifications, incorporation of unnatural amino acids and / or derivatives thereof during polypeptide synthesis, and the use of crosslinkers and other methods that impose conformational constraints on the polypeptides of the invention. Any modification is contemplated herein, including post-translational modifications that reduce the ability of the molecule to form dimers. Examples include modifications incorporated by click chemistry as known in the art. Exemplary modifications include pegylation and glycosylation.

[0147] Examples of side chain modifications contemplated by the present invention include modification of amino groups, such as by reductive alkylation by reaction with aldehydes followed by reduction with NaBH4; amidation with methylacetimidic acid; acylation with acetic anhydride; carbamoylation of amino groups with cyanates; trinitrobenzylation of amino groups with 2,4,6-trinitrobenzenesulfonic acid (TNBS); acylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; and pyridoxylation of lysine with pyridoxal-5-phosphate followed by reduction with NaBH4.

[0148] The guanidine group of arginine residues may be modified by the formation of heterocyclic condensation products with reagents such as 2,3-butanedione, phenylglyoxal and glyoxal.

[0149] The carboxyl group may be modified by carbodiimide activation via O-acylisourea formation followed by subsequent derivatization, for example, to a corresponding amide.

[0150] Sulfhydryl groups may be modified by methods such as carboxymethylation with iodoacetic acid or iodoacetamide; oxidation to cysteic acid with performic acid; formation of mixed disulfides with other thiol compounds; reaction with maleimide, maleic anhydride or other substituted maleimides; formation of mercury derivatives using 4-chloromercuribenzoic acid, 4-chloromercuriphenylsulfonic acid, phenylmercuric chloride, 2-chloromercuri-4-nitrophenol, and other mercuries; and carbamoylation with cyanate at alkaline pH.

[0151] Tryptophan residues may be modified by, for example, oxidation with N-bromosuccinimide or alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or sulphenyl halides, while tyrosine residues may be modified by nitration with tetranitromethane to form 3-nitrotyrosine derivatives.

[0152] Modification of the imidazole ring of a histidine residue may be accomplished by alkylation with iodoacetic acid derivatives or N-carboethoxylation with diethylpyrocarbonate.

[0153] Examples of incorporation of unnatural amino acids and derivatives during protein synthesis include, but are not limited to, the use of norleucine, 4-aminobutyric acid, 4-amino-3-hydroxy-5-phenylpentanoic acid, 6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine, ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienylalanine, and / or D-isomers of amino acids. A list of unnatural amino acids contemplated herein is provided in Table 2.

[0154] TIFF2024502783000018.tif95170

[0155] TIFF2024502783000019.tif212170

[0156] TIFF2024502783000020.tif228170

[0157] TIFF2024502783000021.tif62170

[0158] Crosslinkers can be used to stabilize the 3D conformation using, for example, homobifunctional crosslinkers such as bifunctional imide esters with (CH2)n spacer groups, n=1 to n=6, heterobifunctional reagents that typically contain an amino-reactive moiety and another group-specific reactive moiety, such as glutaraldehyde, N-hydroxysuccinimide esters, and N-hydroxysuccinimide.

[0159] The term "antibody" refers to a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, and multispecific antibodies. The term "antibody" may be used interchangeably with the term "immunoglobulin."

[0160] A "human antibody" is an antibody that possesses an amino acid sequence that corresponds to that of an antibody produced by a human or human cell, or an antibody derived from a non-human source that utilizes a human antibody repertoire or other human antibody coding sequence. This definition of a human antibody specifically excludes humanized antibodies that comprise non-human antigen-binding residues. In particular, the term "recombinant human antibody" includes all human sequence antibodies that are prepared, expressed, produced, or isolated by recombinant means, such as antibodies isolated from animals (e.g., mice) that are genetically modified for human immunoglobulin genes; antibodies expressed using recombinant expression vectors transfected into host cells; antibodies isolated from recombinant combinatorial human antibody libraries; and antibodies prepared, expressed, produced, or isolated by any other means that involve splicing of human immunoglobulin gene sequences to other DNA sequences. Thus, such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, such antibodies may be subjected to in vitro mutagenesis (or in vivo somatic mutagenesis, when animals recombinant with human Ig sequence genes are used) such that the amino acid sequences of the VH and VL regions of the recombinant antibodies, while derived from and related to human germline VH and VL sequences, may not naturally occur within the human antibody germline repertoire in vivo.

[0161] Spike protein and RBD Coronavirus virions are spherical with a diameter of approximately 80-200 nm. The most striking feature of coronaviruses are the club-like projections that protrude from the surface of the virion. These spikes are characteristic of virions, giving them a solar corona-like appearance and giving them the name coronavirus. Within the virion envelope is the nucleocapsid. Coronaviruses have a helical symmetry nucleocapsid, which is rare in positive-stranded RNA viruses but much more common in negative-stranded RNA viruses. SARS-CoV-2, MERS-CoV, and SARS-CoV belong to the Coronaviridae family.

[0162] The term "CoV-S", also referred to as coronavirus "S" or "S protein" or "spike protein", may refer to specific S proteins, such as SARS-CoV-2-S, MERS-CoVS, and SARS-CoVS, or other members of the Coronaviridae family. The SARS-CoV-2 spike protein is a 1273 amino acid type I membrane glycoprotein that assembles into trimers that constitute spikes or peplomers on the surface of enveloped coronavirus particles. The protein has two important functions: (1) host receptor binding and (2) membrane fusion, which are ascribed to the N-terminal (S1) and C-terminal (S2) halves of the S protein. CoV-S binds to its cognate receptor via a receptor binding domain (RBD) present in the S1 subunit. The amino acid sequence of the full-length SARS-CoV-2 spike protein is exemplified by the amino acid sequence set forth in SEQ ID NO:21. The term "CoV-S" includes protein variants of the CoV spike protein isolated from different CoV spike proteins or fragments thereof. The term also encompasses CoV spike proteins or fragments thereof linked to a signal sequence, such as, for example, a histidine tag, mouse or human Fc, or ROR1.

[0163] In any embodiment, the spike protein may be derived from a variant or strain described herein, including in Table 3 below.

[0164] In any aspect, the receptor binding domain may be derived from a SARS-CoV-2 variant such as those listed in Table 3 below. SARS-CoV-2 variants have evolved from the original WT strain of the virus (Genome Reference Sequence: GenBank Accession NC_045512.2). As described herein, the receptor binding domain may be derived from a WT, alpha, beta, gamma, kappa, delta, lambda, iota, mu, or Omicron strain (Table 3), and other strains that are expected to emerge with mutations in the RBD. In any embodiment, the receptor binding domain may be N334-P527 of a WT, alpha, beta, gamma, kappa, delta, lambda, iota, mu, or Omicron strain, and other strains that may emerge such as delta plus, e.g., Y.1, AY.2, AY.3, and AY.4.2. The alpha variant carries one mutation, N501Y, in the RBD compared to the original WT strain. The beta mutant has three mutations in the RBD, N501Y, E484K, and K417N, compared to the original WT strain. The gamma mutant has three mutations in the RBD, K417T, E484K, and N501Y, compared to the original WT strain. The kappa mutant has two mutations in the RBD, L452R and E484Q, compared to the original WT strain. The delta mutant has two mutations in the RBD, L452R and T478K, compared to the original WT strain. The lambda mutant has two mutations in the RBD, L452Q and F490S, compared to the original WT strain. The iota mutant has one mutation in the RBD, E484K, compared to the original WT strain. The mu mutant has two mutations in the RBD, E484K and N501Y, compared to the original WT strain. The Omicron mutant strain harbors 15 mutations in the RBD compared to the original WT strain: G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, and Y505H.

[0165] The betaRBD, and thus a chimeric or fusion protein of the invention comprising a betaRBD (e.g., SEQ ID NO:22) (with mutations (N501Y, K417N, and E484K)), is closer to omicron than to wild type RBD, since it shares with omicron the RBD mutations N501Y and K417N, two important mutations known to affect binding to ACE2 (N501Y) and immune evasion (K417N). For similar reasons, the betaRBD, and thus a chimeric or fusion protein of the invention comprising a betaRBD, is closer to gamma (N501Y, E484K (immune evasion), and K417T), mu (N501Y and E484K), and iota (E484K). The chimeric or fusion protein of the invention, or a composition of the invention comprising a betaRBD (e.g., SEQ ID NO:2), should drive superior responses to gamma, mu, iota, and omicron.

[0166] TIFF2024502783000022.tif159170

[0167] In any embodiment, the receptor binding domain from the coronavirus spike protein comprises, consists essentially of, or consists of an amino acid sequence of any one or more of SEQ ID NOs: 15 or 28, or an amino acid sequence having 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NOs: 15 or 28. In one embodiment, the amino acid sequence may include one or more of the mutations shown in Figures 6 or 7, or the mutations described herein in connection with SARS-CoV-2 variants, such as alpha, beta, gamma, kappa, or delta strains.

[0168] In any aspect, the receptor binding domain from coronavirus spike protein comprises, consists essentially of, or consists of one or more amino acid sequences of either SEQ ID NO: 15 or 28, having 0-8 amino acid insertions, deletions, substitutions or additions (or combinations thereof). In some embodiments, the relevant amino acid sequence may have 0-7, preferably 0-6, preferably 0-5, preferably 0-4, preferably 0-3, preferably 0-2, preferably 0-1 amino acid insertions, deletions, substitutions or additions (or combinations thereof), where the amino acid insertions, deletions, substitutions or additions (or combinations thereof) are located at the N-terminus and / or C-terminus.

[0169] Antibody Fc Regions and Fc Receptor Binding Domains The term "Fc region" is used herein to define the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. In other words, the Fc region contains two heavy chain fragments comprising the CH2 and CH3 domains of an antibody. In the context of the present invention, the Fc region comprises two heavy chain fragments, preferably the CH2 and CH3 domains of said heavy chain. The two heavy chain fragments are held together by two or more disulfide bonds and hydrophobic interactions of the CH3 domain. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, μ.

[0170] In some embodiments, the RBD-Fc dimer does not exhibit any effector function or any detectable effector function. "Effector function" or "effector activity" refers to the biological activity attributed to the Fc region of an antibody and varies with antibody isotype. Examples of antibody effector functions include C1q binding and complement-dependent cytotoxicity (CDC); antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (such as B cell receptors); and B cell activation. In vitro and / or in vivo cytotoxicity assays can be performed to confirm the reduction / depletion of CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that the antibody lacks FcγR binding (and therefore likely lacks ADCC activity). NK cells, the primary cells for mediating ADCC, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays for assessing ADCC activity of a molecule of interest are described in U.S. Patent Nos. 5,500,362 (see, e.g., Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)); and Hellstrom, I et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); 5,821,337 (see, Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assay methods may be used (see, e.g., ACTI™ non-radioactive cytotoxicity assay for flow cytometry (Cell Technology, Inc. Mountain View, CA; and CytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, WI)).Useful effector cells in such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively, or in addition, the ADCC activity of a molecule of interest may be assessed in vivo in an animal model, for example as disclosed in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). A C1q binding assay may be performed to confirm that the antibody is unable to bind C1q and therefore lacks CDC activity. See, for example, the C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, a CDC assay may be performed (see, e.g., Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, MS et al., Blood 101:1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance / half-life determinations may also be performed using methods known in the art (see, e.g., Petkova, SB et al., Int'l. Immunol. 18(12):1759-1769 (2006); WO 2013 / 120929 A1).

[0171] Antibodies with reduced effector function include those with substitutions at one or more of Fc region residues 238, 265, 269, 270, 297, 327, and 329 (US Pat. No. 6,737,056). Such Fc variants include Fc variants with substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc variant in which residues 265 and 297 are substituted with alanine (US Pat. No. 7,332,581). For example, the antibody variant may comprise an Fc region with one or more amino acid substitutions that reduce FcγR binding, such as, for example, substitutions at positions 234 and 235 of the Fc region (EU numbering of residues). For example, the substitutions are L234A and L235A (LALA) (see, for example, WO 2012 / 130831). Additionally, changes may be made to the Fc region that result in altered (i.e., decreased) C1q binding and / or complement dependent cytotoxicity, e.g., as described in U.S. Pat. No. 6,194,551, WO 99 / 51642, and Idusogie et al. J. Immunol. 164:4178-4184 (2000).

[0172] In some embodiments, the Fc region comprises mutations to the complement (C1q) and / or Fc gamma receptor (FcγR) binding sites. In some embodiments, such mutations can render the fusion protein incapable of antibody-directed cytotoxicity (ADCC) and complement-directed cytotoxicity (CDC).

[0173] The term "Fc region" also includes native sequence Fc regions and variant Fc regions. The Fc region may include the carboxyl terminus of the heavy chain. The antibody produced by the host cell may undergo post-translational truncation of one or more, particularly one or two, amino acids from the C-terminus of the heavy chain. Thus, an antibody produced in a host cell by expression of a particular nucleic acid molecule encoding a full-length heavy chain may include a full-length heavy chain or it may include a truncated variant of the full-length heavy chain. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also referred to as the EU guidelines as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991. Amino acid sequence variants of the Fc region of the antibody may be envisaged. Amino acid sequence variants of the Fc region of the antibody may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletions from, and / or insertions into, and / or substitutions of, residues within the amino acid sequences of the Fc region of the antibody. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired properties, such as, for example, inducing or supporting an anti-inflammatory response.

[0174] The Fc region of an antibody may be the Fc region of any of the antibody classes, such as IgA, IgD, IgE, IgG, and IgM. The "class" of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and some of these may be further classified into subclasses (isotypes), for example, IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. Thus, as used in the context of the present invention, an antibody may be an IgG Fc region. For example, an antibody Fc region may be an IgG1, anIgG2, anIgG2b, anIgG3, or anIgG4 Fc region. In some embodiments, the fusion protein of the present invention comprises an IgG of an antibody Fc region. In the context of the present invention, an antibody Fc region is an IgG, preferably an IgG1 Fc region.

[0175] The Fc region is made up of two heavy chains that contribute two or three constant domains depending on the class of the antibody. The Fc region ensures that each antibody mounts an appropriate immune response against a given antigen by binding to a specific protein.

[0176] The Fc receptor binding domain is any protein or polypeptide that binds to the Fc receptor on the cell surface.The Fc receptor binding domain may be the antigen binding domain of an antibody.The Fc receptor binding domain also binds to various cell receptors, such as Fc receptors, and other immune molecules, such as complement proteins.

[0177] Linker Furthermore, the fusion proteins provided herein may comprise a linker (or "spacer"). In the context of the present invention, two or more polypeptides comprising or consisting of the amino acid sequence of a receptor binding domain or a dimer of receptor binding domains from the spike protein of a coronavirus are fused at their C-termini to an Fc region or an Fc receptor binding domain via a linker.

[0178] The linker is usually a peptide having a length of up to 20 amino acids. The terms "linked", "linked to" or "fused to" refer to a covalent bond formed between two moieties, such as a peptide bond. Thus, in the context of the present invention, the linker may have a length of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22 amino acids. For example, the fusion proteins provided herein may comprise a linker between two or more polypeptides comprising or consisting of the amino acid sequence of a receptor binding domain or a dimer of receptor binding domains from a coronavirus spike protein and an Fc region of an antibody, e.g., a linker between the N-terminus of the Fc region / FcR binding domain and the C-terminus of a receptor binding domain polypeptide. Such linkers have the advantage that they can increase the likelihood that the different polypeptides of the fusion protein will fold independently and behave as expected.

[0179] Thus, in the context of the present invention, two or more polypeptides comprising or consisting of the amino acid sequence of a receptor binding domain or a dimer of a receptor binding domain from a coronavirus spike protein and an Fc region or an Fc receptor binding domain of an antibody may be comprised in a single covalently linked multifunctional polypeptide.

[0180] In some embodiments, the fusion proteins of the invention include a peptide linker. In some embodiments, the peptide linker may include the amino acid sequence Gly-Gly-Ser (GGS), Gly-Gly-Gly-Ser (GGGS), or Gly-Gly-Gly-Gly-Ser (GGGGS). In some embodiments, the peptide linker may include the amino acid sequence GGGGS (a linker of 6 amino acids in length) or a longer amino acid sequence. The linker may be a series of repeating glycine and serine residues (GS) of different lengths, i.e., (GS)n, where n is any number from 1 to 15 or more. For example, the linker may be (GS)3 (i.e., GSGSGS) or more, or (GS)11 or more. It will be understood that n can be any number, including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or more. Fusion proteins with linkers of such lengths are included within the scope of the invention. Additionally, fusion proteins without a linker are also included within the scope of the invention.

[0181] nucleic acid Nucleic acid molecules encoding any of the polypeptides of the invention are also within the scope of the invention. Nucleic acids are useful, for example, in the manufacture of the polypeptides of the invention and as therapeutic agents. They may be administered to cells in culture or in vivo and may contain secretion signals that induce or promote secretion of the polypeptides of the invention from the cells. Expression vectors and host cells that contain or include the nucleic acids of the invention are also within the scope of the invention (described in more detail below). While the nucleic acids of the invention may be referred to as "isolated" by definition, the polypeptides of the invention are not wild-type polypeptides and therefore will not be encoded by naturally occurring nucleic acids. Thus, the polypeptides and nucleic acids of the invention may be, but need not be, "purified," "substantially purified," "isolated," "recombinant," or "synthetic" in order to be distinct from naturally occurring substances.

[0182] An "isolated" nucleic acid molecule is a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule with which it is normally associated in the natural source of the nucleic acid encoding a polypeptide, typically a spike protein. An isolated nucleic acid molecule is different from the form or setting in which it is found in nature. Thus, an isolated nucleic acid molecule is distinct from a nucleic acid molecule present in a natural cell. However, an isolated nucleic acid molecule also includes a nucleic acid molecule that is normally contained in an infected cell that expresses a spike protein, where, for example, the nucleic acid molecule is in a different chromosomal location than in a natural cell.

[0183] The terms "nucleic acid molecule" and "polynucleotide" are used interchangeably herein and refer to a polymer of nucleotides of any length, either deoxyribonucleotides or ribonucleotides or analogs thereof. Non-limiting examples of polynucleotides include genes, gene fragments, messenger RNA (mRNA), cDNA, recombinant polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. The polynucleotides of the invention may be provided in isolated or purified form. A nucleic acid sequence that "encodes" a selected polypeptide is a nucleic acid molecule that is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus. For purposes of the present invention, such nucleic acid sequences can include, but are not limited to, cDNA from viral, prokaryotic or eukaryotic mRNA, genomic sequences from viral or prokaryotic DNA or RNA, as well as synthetic DNA sequences. A transcription termination sequence may be located 3' to the coding sequence.

[0184] Polynucleotides of the invention can be synthesized according to methods well known in the art, for example, as described in Sambrook et al. (1989, Molecular Cloning - a laboratory manual; Cold Spring Harbor Press).

[0185] The polynucleotide molecules of the invention may be provided in the form of expression cassettes comprising control sequences operably linked to the insert sequence, thus allowing expression of the polypeptides of the invention in vivo in the target subject. These expression cassettes are then typically provided within vectors (e.g., plasmids or recombinant viral vectors) suitable for use as reagents for nucleic acid immunization. Such expression cassettes may be administered directly to the host subject. Alternatively, a vector comprising the polynucleotides of the invention may be administered to the host subject. Preferably, the polynucleotides are prepared and / or administered using a genetic vector. A suitable vector may be any vector capable of carrying a sufficient amount of genetic information and allowing expression of the polypeptides of the invention.

[0186] The present invention therefore includes expression vectors comprising such polynucleotide sequences.The present invention thus provides a vector for use in the prevention or treatment of an inflammatory disease or condition comprising a polynucleotide sequence encoding a polypeptide of the present invention and, optionally, one or more further polynucleotide sequences encoding a different polypeptide as defined herein.

[0187] It will further be appreciated that the compositions and products of the invention may comprise a mixture of polypeptides and polynucleotides.Thus, the invention provides a composition or product as defined herein, in which, instead of any one polypeptide, there is a polynucleotide capable of expressing said polypeptide.

[0188] Expression vectors are routinely constructed in the art of molecular biology and may involve, for example, the use of plasmid DNA and appropriate initiators, promoters, enhancers, and other elements such as polyadenylation signals, which are necessary and positioned in the correct orientation, to allow expression of the peptides of the invention. Other suitable vectors will be apparent to those skilled in the art. As further examples in this regard, we refer to Sambrook et al. (Molecular Cloning A Laboratory Manual, 2nd ed. (Cold Spring Harbor Laboratory, 1989).

[0189] Thus, a polypeptide of the invention may be provided by delivering such a vector to a cell and allowing transcription from the vector to occur. Preferably, a polynucleotide of the invention, or for use in the invention in a vector, is operably linked to a control sequence which is capable of providing for the expression of the coding sequence by the host cell, i.e. the vector is an expression vector.

[0190] "Operably linked" refers to an arrangement of elements configured so that the components described perform their normal functions. Thus, a given regulatory sequence, such as a promoter, operably linked to a nucleic acid sequence can result in the expression of that sequence when the appropriate enzymes are present. The promoter need not be adjacent to the sequence, so long as it functions to induce its expression. Thus, for example, a promoter sequence can still be considered "operably linked" to a coding sequence even if an intervening untranslated but transcribed sequence is present between the promoter sequence and the nucleic acid sequence.

[0191] Several expression systems have been described in the art, each of which typically consists of a vector containing a gene or nucleotide sequence of interest operably linked to expression control sequences. These control sequences include a transcription promoter sequence, a transcription start sequence, and a transcription termination sequence. The vector of the invention may be, for example, a plasmid, virus or phage vector, equipped with an origin of replication, optionally a promoter for the expression of said polynucleotide, and optionally a regulator of the promoter. A "plasmid" is a vector in the form of an extrachromosomal genetic element. For example, in the case of a bacterial plasmid, the vector may contain one or more selectable marker genes, such as an ampicillin resistance gene, or in the case of a fungal vector, a resistance gene. Vectors may be used in vitro, for example for the production of DNA or RNA, or may be used to transfect or transform a host cell, for example a mammalian host cell. Vectors may also be adapted to be used in vivo, for example to allow in vivo expression of a polypeptide.

[0192] A "promoter" is a nucleotide sequence that initiates and controls transcription of a polynucleotide encoding a polypeptide. Promoters can include inducible promoters (where expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), repressible promoters (where expression of a polynucleotide sequence operably linked to the promoter is repressed by an analyte, cofactor, regulatory protein, etc.), and constitutive promoters. The term "promoter" or "regulatory element" is intended to include full-length promoter regions and functional (e.g., transcription or translation controlling) segments of these regions.

[0193] The polynucleotide, expression cassette or vector according to the invention may further comprise a signal peptide sequence, which is generally inserted in operable linkage with a promoter such that the signal peptide is expressed and promotes secretion of a polypeptide encoded by a coding sequence that is also operably linked to the promoter.

[0194] Typically, a signal peptide sequence encodes a peptide of 10-30 amino acids, for example 15-20 amino acids. Often, the amino acids are predominantly hydrophobic. In a typical situation, the signal peptide targets a growing polypeptide chain carrying the signal peptide to the endoplasmic reticulum of the expressing cell. The signal peptide is cleaved in the endoplasmic reticulum, allowing the polypeptide to be secreted via the Golgi apparatus. Thus, the peptides of the invention may be provided to an individual by expression from cells within the individual and secretion from those cells.

[0195] Immunogenic and Vaccine Compositions The present invention further provides compositions comprising chimeric or fusion proteins as defined herein, and the use of such chimeric or fusion proteins in immunogenic or vaccine compositions in the treatment or prevention of coronavirus infection.

[0196] The term "vaccine composition" as used herein is defined as a composition used to elicit an immune response against an antigen (immunogen) within the composition in order to protect or treat an organism against disease.

[0197] As used herein, the terms "immunostimulatory composition," "vaccine composition," and "immunogenic composition" may generally be used interchangeably.

[0198] The immunostimulatory composition or vaccine of the present invention may suitably contain, in addition to one or more peptides of the present invention as therapeutic or prophylactic active ingredients, a pharma- ceutically acceptable carrier, excipient, diluent, adjuvant, vehicle, buffer or stabilizer, including, but not limited to, saline, buffered saline, dextrose, liposomes, water, glycerol, polyethylene glycol, ethanol, and combinations thereof.

[0199] The immunostimulatory or vaccine composition may be adapted for administration by any suitable route, such as, for example, parenteral (including subcutaneous, intramuscular, intravenous or intradermal, or by injection into the cerebrospinal fluid), oral (including buccal or sublingual), nasal, topical (including buccal, sublingual or transdermal), vaginal or rectal. Such compositions may be prepared by any method known in the pharmaceutical art, for example, by mixing the peptide with a carrier or excipient under sterile conditions. Typically, the vaccine composition is adapted for administration by subcutaneous, intramuscular, intravenous or intradermal route, typically by injection. Alternatively, the vaccine composition may be adapted for oral or nasal administration.

[0200] Immunostimulatory or vaccine compositions adapted for parenteral administration may be aqueous and non-aqueous sterile injection solutions, which may contain antioxidants, buffers, bacteriostats, and solutes that render the formulation substantially isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions, which may contain suspending agents and thickening agents. Excipients that may be used for injection solutions include, for example, water, alcohols, polyols, glycerin, and vegetable oils. The compositions may be presented in unit-dose or multi-dose containers, for example, sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) state, requiring only the addition of a sterile liquid carrier, for example, water for injection, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets.

[0201] Immunostimulatory or vaccine compositions adapted for oral administration may be presented as discrete units such as capsules or tablets; as a powder or granules; as a solution, syrup or suspension (in an aqueous or non-aqueous liquid; or an edible foam or whip; or as an emulsion).

[0202] Suitable excipients for tablets or hard gelatin capsules include lactose, corn starch or derivatives thereof, stearic acid or its salts. Suitable excipients for use with soft gelatin capsules include, for example, vegetable oils, waxes, fats, semi-solid or liquid polyols, etc.

[0203] For the preparation of solutions and syrups, excipients that can be used include, for example, water, polyols, and sugars. For the preparation of suspensions, oils (e.g. vegetable oils) can be used to provide oil-in-water or water-in-oil suspensions.

[0204] Immunostimulatory or vaccine compositions adapted for nasal administration, where the carrier is a solid, include a coarse powder having a particle size, for example, in the range 20 to 500 microns, which is administered in the manner in which snuff is sniffed, i.e. rapid inhalation through the nasal passage from a container of the powder held close to the nose. Compositions suitable for administration as a nasal spray or nasal drops, where the carrier is a liquid, may comprise aqueous or oily solutions of the active ingredient.

[0205] Compositions adapted for administration by inhalation include fine particle dusts or mists, which may be generated by means of various types of metered dose pressurized aerosols, nebulizers or insufflators.

[0206] Immunostimulatory or vaccine compositions adapted for transdermal administration may be presented as discrete patches intended to remain in intimate contact with the epidermis of the recipient for a prolonged period of time. For example, the active ingredient may be delivered from the patch by iontophoresis, as generally described in Pharmaceutical Research. 3(6):318 (1986).

[0207] Compositions adapted for topical administration may be formulated as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols or oils. In the case of infections of the eye or other external tissues, such as the mouth and skin, the compositions may be applied as topical ointments or creams. When formulated in an ointment, the active ingredient may be used with either a paraffinic or a water-miscible ointment base. Alternatively, the active ingredient may be formulated in a cream with an oil-in-water or water-in-oil cream base. Pharmaceutical compositions adapted for topical administration to the eye may comprise eye drops, in which the active ingredient is dissolved or suspended in a suitable carrier, in particular an aqueous solvent. Pharmaceutical compositions adapted for topical administration in the mouth may comprise lozenges, pastilles or mouthwashes.

[0208] The immunostimulatory or vaccine composition may contain preservatives, solubilizers, stabilizers, wetting agents, emulsifiers, sweeteners, colorants, flavorings, salts (the substances of the invention themselves may be provided in the form of pharma- ceutically acceptable salts), buffers, coating agents, or antioxidants.

[0209] The vaccine composition of the present invention may also contain, in addition to the chimeric or fusion protein as defined herein, one or more other prophylactically or therapeutically active agents.

[0210] Chimeric or fusion proteins for use in the vaccine compositions of the present invention may or may not be lyophilized.

[0211] Adjuvants The vaccine composition of the present invention may also comprise, in addition to the peptides defined herein, a pharma- ceutically acceptable adjuvant, which is added to enhance the immunogenicity of the vaccine composition.

[0212] Suitable adjuvants are known in the art and include any of those described herein, preferably TLR2 agonists, more preferably Pam-2-Cys-containing molecules such as PEG-R4-Pam-2-Cys, or preferably stimulators of NKT cells, more preferably α-galactosylceramide (also referred to herein as "α-GalCer"), α-glucosylceramide, α-glucosyldiacylglycerol, α-galactosyldiacylglycerol, β-mannosylceramide, and analogs thereof comprising a variety of acyl and sphingosine chain lengths, degrees of saturation, and polar head group compositions.

[0213] In some embodiments, the TLR2-agonist may be selected from the group consisting of Pam3CSK4, PEG-R4-Pam-2-Cys, MALP-2, lipoteichoic acid, OspA, Porin, LcrV, lipomannan, lysophosphatidylserine, lipophosphoglycan (LPG), glycophosphatidylinositol (GPI), and zymosan.

[0214] In some embodiments, the adjuvant is Poly-I:C, CpG, Poly-ICLC, 1018ISS, aluminum salts, Amplivax, AS15, B(C, CP-870,893, CpG7909, CyaA, dSLIM, GM-CSF, IC30, IC31, Imiquimod, ImuFact IMP321, IS Patch, ISS, ISCOMATRIX, JuvImmune, LipoVac, MF59®, AddaVax™, monophosphoryl lipid A, Montanide IMS1312, Montanide ISA206, Montanide ISAV, Montanide ISA-51, OK-432, OM-174, OM-197-MP-EC, ONTAK, PEPTEL, Vector System, PLGA microparticles, resiquimod, SRL172, virosomes and other virus-like particles, YF-17D, VEGF trap, R848, β-glucan, Pam3Cys, and Aquila's QS21 Stimulon.

[0215] In some embodiments, the adjuvant comprises a metabolizable oil and an emulsifier, such as a detergent or surfactant. Preferably, the oil and emulsifier are in the form of an oil-in-water emulsion, with substantially all of the oil droplets being less than 1 micron in diameter. Exemplary metabolizable oils and emulsifiers are described in 6,299,884 and 6,086,901. In one embodiment, the adjuvant comprises an oil-in-water emulsion. Preferably, the oil is squalene. Preferably, the aqueous phase is a citrate buffer (e.g., 10 mM, pH 6.5).

[0216] In one embodiment, the adjuvant comprises squalene in an oil-in-water emulsion. Preferably, the adjuvant further comprises Tween® 80 (polyoxyethylene sorbitan monooleate) and Span® 85 (sorbitan trioleate). The adjuvant may comprise 4.3% squalene, 0.5% Tween® 80 (polyoxyethylene sorbitan monooleate), 0.5% Span® 85 (sorbitan trioleate), and optionally 400pig / ml MTP-PE (N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-[1,2-dipalmitoyl-sn-glycero-3-3(hydroxyphosphoryl-oxy)]ethylamide).

[0217] In one embodiment, the composition comprises 50% vol / vol of an adjuvant, preferably the adjuvant is MF59®. MF59® is an oil-in-water emulsion of squalene oil. Squalene is a naturally occurring substance found in humans, animals and plants, and is highly purified for the vaccine manufacturing process. MF59® adjuvant (MF59C.1) is an oil-in-water emulsion with a squalene internal oil phase and a citrate buffer external aqueous phase. See, e.g., U.S. Pat. Nos. 6,299,884 and 6,086,901; Ott et al. "MF59-Design and Evaluation of a Safe and Potent Adjuvant for Human Vaccines," Vaccine Design: The Subunit and Adjuvant Approach (Powell, MF and Newman, MJ eds.) Plenum Press, New York, pp. 277-296 (1995). Two non-ionic surfactants, sorbitan trioleate and polysorbate 80, help stabilize the emulsion. The safety of MF59® adjuvant has been demonstrated in animals and humans in combination with several antigens. See, e.g., Higgins et al., "MF59 Adjuvant Enhances the Immunogenicity of Influenza Vaccine in Both Young and Old Mice," Vaccine 14(6):478-484 (1996). MF59® is 4.3% squalene, 0.5% Tween® 80 (polyoxyethylene sorbitan monooleate), 0.5% Span® 85 (sorbitan trioleate), and optionally contains 400 pg / ml MTP-PE (N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-[1,2-dipalmitoyl-sn-glycero-3-3(hydroxyphosphoryl-oxy)]ethylamide).An exemplary composition of MF59 comprises citrate buffer pH 6.5 (10 mM citric acid, 140 mM NaCl, 0.02% PS80, pH 6.5) and 39 mg / ml squalene, 4.7 mg / ml polysorbate 80, 4.7 mg / ml sorbitan trioleate, 2.65 mg / ml sodium citrate, 0.17 mg / ml citric acid monohydrate.

[0218] In any embodiment, the adjuvant is any one described herein, preferably PEG-R4-Pam-2-Cys, α-galactosylceramide (also referred to herein as "α-GalCer"), or MF59 (registered trademark).

[0219] Dosage and route of administration As used herein, the upper respiratory tract (URT) may include the areas of the nose and nasal passages, the paranasal sinuses, the pharynx, and the portion of the larynx above the vocal folds (vocal cords).

[0220] Typically, the lower respiratory tract (LRT) includes the portion of the larynx below the vocal folds, the trachea, the bronchi, and the bronchioles. The lungs may be included in the lower respiratory tract, including the respiratory bronchioles, the alveolar ducts, the alveolar sacs, and the alveoli. In any embodiment of the invention, administration to the URT may be administration to the nose and nasal passages, the paranasal sinuses, the pharynx, and the portion of the larynx above the vocal folds (vocal cords). Administration to any one or more regions of the URT is also contemplated, provided that the compound is retained within the URT or does not contact a region of the LRT.

[0221] The phrase "therapeutically effective amount" generally refers to an amount of one or more polypeptides or polynucleotides of the invention that (i) treats a particular disease, condition, or disorder, (ii) reduces, ameliorates, or eliminates one or more symptoms of a particular disease, condition, or disorder, or (iii) delays the onset of one or more symptoms of a particular disease, condition, or disorder described herein. Undesirable effects, e.g., side effects, may manifest along with the desired therapeutic effect; thus, a practitioner balances the potential risks against the potential benefits in determining an appropriate "effective amount."

[0222] In some embodiments, an effective amount of a chimeric or fusion protein of the invention for a human subject is in the range of about 0.25 nmoles / kg body weight / dose to 0.0001 nmoles / kg body weight / dose. Preferably, the range is about 0.25 nmoles / kg body weight / dose to 0.0001 nmoles / kg body weight / dose. More preferably, the range is about 0.002 nmoles / kg body weight / dose to 0.001 nmoles / kg body weight / dose. In some embodiments, the body weight / dose range is about 0.25 nmoles / kg to 0.001 nmoles / kg, about 0.1 nmoles / kg to 0.001 nmoles / kg, about 0.025 nmoles / kg to 0.001 nmoles / kg, about 0.01 nmoles / kg to 0.001 nmoles / kg, or about 0.005 nmoles / kg to 0.001 nmoles / kg body weight / dose. In some embodiments, the amount is on the order of 0.25 nmol, 0.1 nmol, 0.05 nmol, 0.01 nmol, 0.005 nmol, 0.002 nmol, or 0.001 nmol / kg body weight / dose of chimeric or fusion protein of the invention. Dosage regimes are adjusted according to the exigencies of the situation and adjusted to provide the optimal therapeutic dosage.

[0223] In some embodiments, the effective amount of the chimeric or fusion protein of the present invention for a human subject is in the range of about 1 μg to 100 μg per dose. Preferably, the range is about 1 μg to 50 μg per dose. More preferably, the range is about 1 μg to 20 μg per dose. In some embodiments, the dose is about 5 μg to 100 μg, about 5 μg to 50 μg, about 10 μg to 45 μg, about 10 μg to 25 μg, or about 10 μg to 20 μg per dose. In some embodiments, the dose is about 0.1 μg, 0.2 μg, 0.5 μg, 1 μg, 5 μg, 10 μg, 15 μg, 25 μg, 30 μg, 35 μg, 40 μg, 45 μg, 50 μg, or the like. Preferably, the dose is 15 μg.

[0224] The exact amount required will vary from subject to subject, depending on the subject's species, age and general condition, mode of administration, etc. Thus, it may not be possible to specify an exact "effective amount". However, the appropriate "effective amount" in each case can be determined by one of ordinary skill in the art using only routine experimentation. In one aspect, the dose administered to the subject is any dose that reduces the viral load.

[0225] The chimeric or fusion protein or vaccine composition thereof may be administered using immunization schemes known to those skilled in the art to induce a protective immune response. These include a single immunization or multiple immunizations in a prime-boost strategy. The boost immunization may be administered days, weeks, months, or even years after the initial immunization (prime immunization). In certain embodiments, the boost immunization is administered 2 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months or more after the initial prime immunization. Furthermore, multiple boost immunizations may be administered weekly, biweekly, monthly, bimonthly, every 3 months, or more. In other embodiments, the boost immunization is administered every 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, or 12 weeks. In certain embodiments, the boost immunizations may be continued until protective anti-SARS-CoV-2 antibody titers are observed in the serum of the subject. In certain embodiments, the subject receives one boost immunization, two boost immunizations, three boost immunizations, or four or more boost immunizations, as needed, to obtain protective antibody titers. In further embodiments, the adjuvant in the first prime immunization is different from the adjuvant in the boost immunization. Alternatively, the adjuvant in the prime immunization is the same as the adjuvant in the boost immunization.

[0226] This administration can be repeated as frequently as necessary, for example, an initial dose of the vaccine can be administered followed by a booster at a later date.

[0227] The compositions of the present invention may be administered by any convenient route as described herein, such as intramuscular, intranasal, subcutaneous, intravenous, intraperitoneal or oral routes.

[0228] The compositions of the present invention are formulated or adapted for administration by any convenient route described herein, such as intramuscular, intranasal, subcutaneous, intravenous, intraperitoneal or oral routes.

[0229] The compositions of the invention may be formulated for administration only to the URT. The limitation to the URT may be achieved by the amount, particularly by the form, i.e., the particle size, volume and composition of the physical form of the composition that would otherwise be administered to the LRT or TRT, whether as a dry powder or liquid droplets. Alternatively, the vaccine composition of the invention may be administered via a device that ensures retention only within the URT.

[0230] The compositions described herein may be formulated for intranasal administration, including as a dry powder, spray, mist, or aerosol, which may be particularly preferred for the treatment of coronavirus infections.

[0231] Using intranasal delivery, there are several options that can be employed to confine delivery to the URT: (1) ensuring that the droplet / particle size is large enough (>10 μm) to prevent access to the LRT; (2) in the case of liquids, limiting the dose to minimize spillage / drainage; (3) administering in a head-inverted position, also minimizing spillage / drainage; (4) incorporating viscosity enhancers / mucoadhesives to promote retention in the nasal cavity and prevent spillage / drainage; (5) using a nasal device (such as the Optinose bidirectional delivery device) to completely eliminate the possibility of LRT exposure. One or a combination of these methods may be applied.

[0232] The selection of a suitable carrier will depend on the particular type of administration envisaged. For example, for administration via the upper respiratory tract, such as the nasal mucosal surface, the compound may be formulated into a solution, e.g., water or isotonic saline (buffered or unbuffered), or suspension, and administered intranasally as drops or spray. Preferably, such a solution or suspension is isotonic with respect to nasal secretions and has approximately the same pH, e.g., in the range of about pH 4.0 to about pH 7.4, or pH 6.0 to pH 7.0. The buffer should be physiologically compatible, and by way of example only, phosphate buffers are mentioned. For example, a representative nasal decongestant is described as being buffered to a pH of about 6.2 (Remington's, supra, p. 1445). Of course, one of ordinary skill in the art can easily determine the saline content and pH appropriate for a non-toxic aqueous carrier for nasal and / or upper respiratory tract administration.

[0233] Other ingredients, such as preservatives, colorants, lubricants or viscous mineral or vegetable oils, flavors, natural or synthetic plant extracts such as aromatic oils, and humectants and viscosity enhancers, such as glycerol, may also be included to provide additional viscosity, moisture retention, and a pleasant texture and odor to the formulation, as known in the art. For nasal administration of solutions or suspensions according to the invention, a variety of devices are available in the art for generating droplets, droplets, and sprays. For example, the vaccine compositions described herein may be administered intranasally by means of a simple dropper (or pipette), including a glass, plastic, or metal dispensing tube, from which the contents are expelled dropwise by air pressure provided by a hand-operated pump, such as a flexible rubber bulb, attached to one end.

[0234] Since tear secretions from the eye drain from the orbit into the nasal passages, suitable pharma- ceutically acceptable eye drops can be readily provided by one of skill in the art as a carrier for delivering the vaccine compositions described herein, if desired, and can be administered into the orbit in the form of eye drops to provide both intraocular and intranasal administration.

[0235] In one embodiment, a pre-measured unit dose dispenser, including a dropper or spray device containing a solution or suspension for delivery as drops or a spray, is prepared containing one or more doses of the drug to be administered. The invention also includes kits containing one or more dehydrated unit doses of the vaccine composition, along with any necessary salts and / or buffers, preservatives, colorants, etc., ready to be prepared as a solution or suspension by adding an appropriate amount of water. The water may be sterile or non-sterile, although sterile water is generally preferred.

[0236] The compositions of the present invention may be provided in unit dosage form, generally in a sealed container, and may be provided as part of a kit. Such a kit will usually (but not necessarily) include instructions for use. It may include a plurality of said unit dosage forms.

[0237] Prevention and Treatment Methods In any embodiment, the chimeric or fusion protein of the invention, or the composition of the invention, generates antibodies, preferably neutralizing antibodies, against any one or more of the coronavirus strains described herein, particularly those in Table 3. In any embodiment, the chimeric or fusion protein of the invention, or the composition of the invention, provides therapeutic or prophylactic treatment against any one or more of the coronavirus strains described herein, particularly those in Table 3. In the aspects and embodiments defined below, reference to "coronavirus" also includes reference to one or more strains of coronavirus described herein, including but not limited to those described in Table 3, e.g., WT, alpha and beta strains; WT, alpha, beta, and VIC2089 strains; WT, alpha, beta, delta strains; WT, alpha, beta, delta, delta plus, gamma, lambda, mu, iota, kappa, and micron strains. The chimeric or fusion protein of the invention, or the composition of the invention, may provide therapeutic or prophylactic benefit against a SARS-CoV-2 strain different from that from which the RBD amino acid sequence in the chimeric or fusion protein is derived.

[0238] Exemplary strains for which a chimeric or fusion protein of the invention, or a composition of the invention, provides a therapeutic or prophylactic benefit are provided in the Examples.

[0239] In one aspect, the invention provides a method of treating and / or preventing a coronavirus associated disease comprising administering to a subject in need thereof a chimeric or fusion protein, composition, vaccine or immunostimulatory composition as described herein, thereby treating and / or preventing a coronavirus associated disease.

[0240] In one aspect, the invention provides a method of treating and / or preventing a disease associated with or caused by a coronavirus, comprising administering to a subject in need thereof a chimeric or fusion protein, composition, vaccine or immunostimulatory composition as described herein, thereby treating and / or preventing the disease associated with or caused by a coronavirus.

[0241] In another aspect, the present invention provides a method of treating and / or preventing a respiratory disease or condition associated with a coronavirus infection, comprising administering to a subject in need thereof a chimeric or fusion protein, composition, vaccine or immunostimulatory composition as described herein, thereby treating and / or preventing the respiratory disease or condition associated with a coronavirus infection.

[0242] In another aspect, the invention provides a method of reducing airway inflammation associated with or caused by coronavirus, comprising reducing airway inflammation associated with or caused by coronavirus by administering to a subject in need thereof a chimeric or fusion protein, composition, vaccine or immunostimulatory composition as described herein.

[0243] In another aspect, the present invention also provides a method of improving a subject's ability to control a respiratory disease or condition during a coronavirus infection, comprising improving the subject's ability to control a respiratory disease or condition during a coronavirus infection by administering to a subject in need thereof a chimeric or fusion protein, composition, vaccine or immunostimulant as described herein.

[0244] In another aspect, the present invention provides the use of a chimeric or fusion protein, composition, vaccine or immunostimulatory composition as described herein in the preparation of a medicament for eliciting an innate immune response in a subject diagnosed with or suspected of having a coronavirus infection.

[0245] In another aspect, the present invention provides the use of a chimeric or fusion protein, composition, vaccine or immunostimulatory composition as described herein in the preparation of a medicament for the treatment and / or prevention of a disease caused by a coronavirus.

[0246] In another aspect, the present invention further provides the use of a chimeric or fusion protein, composition, vaccine or immunostimulatory composition as described herein in the preparation of a medicament for treating and / or preventing a respiratory disease or condition associated with a coronavirus infection in a subject.

[0247] In another aspect, the present invention further provides the use of a chimeric or fusion protein, composition, vaccine or immunostimulatory composition as described herein in the preparation of a medicament for treating and / or preventing a coronavirus infection in a subject.

[0248] In another aspect, the present invention provides the use of a chimeric or fusion protein, composition, vaccine or immunostimulatory composition as described herein in the preparation of a medicament for reducing airway inflammation in a subject diagnosed with or suspected of having a coronavirus infection.

[0249] In another aspect, the present invention further provides the use of a chimeric or fusion protein, composition, vaccine or immunostimulatory composition as described herein in the preparation of a medicament for improving a subject's ability to control a respiratory disease or condition during coronavirus infection.

[0250] In one aspect, the invention provides a chimeric or fusion protein, composition, vaccine or immunostimulatory composition as described herein for use in eliciting an innate immune response in a subject diagnosed with or suspected of having a coronavirus infection.

[0251] In another aspect, the present invention provides a chimeric or fusion protein, composition, vaccine or immunostimulatory composition as described herein for use in the treatment and / or prevention of a disease caused by a coronavirus in a subject.

[0252] In another aspect, the present invention provides a chimeric or fusion protein, composition, vaccine or immunostimulatory composition as described herein for use in the treatment and / or prevention of a respiratory disease or condition associated with a coronavirus infection in a subject.

[0253] In another aspect, the invention provides a chimeric or fusion protein, composition, vaccine or immunostimulatory composition as described herein for use in reducing airway inflammation in a subject diagnosed with or suspected of having a coronavirus infection.

[0254] In another aspect, the invention provides a chimeric or fusion protein, composition, vaccine or immunostimulatory composition as described herein for use in controlling a respiratory disease or condition during coronavirus infection in a subject.

[0255] In any aspect of the invention where prophylaxis or prophylactic therapy is intended or required, the chimeric or fusion protein, composition, vaccine or immunostimulatory composition described herein is administered to a subject prior to clinical or biochemical detectable symptoms of viral infection.

[0256] In any aspect of the invention, administration of the chimeric or fusion protein, composition, vaccine or immunostimulatory composition described herein to a subject reduces the viral load in the subject. Preferably, the viral load is reduced in the respiratory tract, e.g., the upper and / or lower respiratory tract. Preferably, the viral load is reduced in the nasal cavity and pharynx (i.e., throat). In alternative embodiments, the viral load may be present in the gastrointestinal tract, peripheral circulation, heart, liver, kidneys, spleen, or other organs known to be susceptible to infection with coronaviruses, preferably SARS-CoV-2.

[0257] The term "coronavirus infection" or "CoV infection" as used herein refers to an infection caused by a coronavirus, such as SARS-CoV-2, MERS-CoV, or SARS-CoV. The term includes respiratory tract infections caused by coronaviruses, most often affecting the lower respiratory tract. Symptoms can include high fever, dry cough, shortness of breath, pneumonia, gastrointestinal symptoms such as diarrhea, organ failure (kidney failure and renal insufficiency), septic shock, and in severe cases, death.

[0258] The reduction in coronavirus infection may be determined using any method known in the art or described herein, including measuring the viral load in a sample from a subject after treatment and comparing it to the viral load in a sample from the same subject before treatment. The sample may be any biological sample obtained from the subject, including blood, saliva, urine, feces, nasal washings, sputum, and mucous secretions. The sample may be taken from the respiratory tract, preferably the upper respiratory tract, such as the nose or pharynx (i.e., throat). The term "respiratory disease" or "respiratory condition" refers to any one of several illnesses that involve inflammation and affect components of the respiratory system, including the upper respiratory tract (including the nasal cavity, pharynx, and larynx) and the lower respiratory tract (including the trachea, bronchi, and lungs). Inflammation of the upper and lower respiratory tract may be associated with or caused by a viral infection.

[0259] Symptoms of respiratory illness may include coughing, excessive phlegm production, shortness of breath, or tightness in the chest accompanied by audible wheezing.

[0260] The presence, amelioration, treatment or prevention of respiratory disease may be determined by a clinically or biochemically relevant method in a subject or a biopsy therefrom. For example, parameters measured may be the presence and degree of pulmonary function, signs and symptoms of obstruction, exercise tolerance, nighttime awakenings, days missed from school or work, bronchodilator use, dose of inhaled corticosteroids (ICS), oral glucocorticoid (GC) use, need for other medications, need for medical treatment, or hospitalization.

[0261] As used herein, the term respiratory infection refers to an infection anywhere in the respiratory tract. Examples of respiratory infections include, but are not limited to, cold, sinusitis, throat infection, tonsillitis, laryngitis, bronchitis, pneumonia, or bronchiolitis. Preferably, in any embodiment of the present invention, the respiratory infection is a cold. An individual may be identified as having a respiratory tract infection by a viral test and may exhibit systems such as itchy watery eyes, runny nose, nasal congestion, sneezing, sore throat, cough, headache, fever, fatigue, nausea, vomiting, fatigue, weakness, etc. In one aspect, a subject with a respiratory infection may not have other respiratory diseases. Detection of the presence or amount of virus, preferably coronavirus, may be performed by PCR / sequencing of RNA isolated from clinical samples (nasal wash, sputum, BAL) or serology.

[0262] As used herein, the term respiratory infection refers to an infection with a coronavirus anywhere in the respiratory tract, preferably SARS-CoV-2.

[0263] Individuals may be identified as having a respiratory tract infection by viral testing and may exhibit symptoms such as itchy watery eyes, runny nose, nasal congestion, sneezing, sore throat, cough, headache, fever, fatigue, nausea, vomiting, malaise, weakness, etc. In one embodiment, subjects with a respiratory infection may not have other respiratory diseases. Detection of the presence or amount of virus may be performed by PCR / sequencing of RNA isolated from clinical samples (nasal wash, sputum, BAL) or serology. The term "treatment" or "treating" of a subject includes the application or administration of the chimeric or fusion protein, composition, vaccine or immunostimulatory composition of the invention to a subject (or the application or administration of the chimeric or fusion protein, composition, vaccine or immunostimulatory composition of the invention described herein to a cell or tissue derived from a subject) for the purpose of delaying, slowing, stabilizing, curing, curing, palliating, alleviating, altering, correcting, lessening the deterioration, ameliorating, or affecting a disease or condition, a symptom or condition of a disease, or the risk (or susceptibility) of a disease or condition. "Treating" refers to any measure of success in treating or ameliorating an injury, condition, or condition, including any objective or subjective parameter, such as alleviation; remission; reducing the rate of deterioration; reducing the severity of a disease; stabilizing, reducing symptoms, or making the injury, condition, or condition more tolerable to the subject; slowing the rate of degeneration or decline; reducing the debilitating end point of degeneration; or improving the physical or mental health of the subject.

[0264] Given the wide range of symptoms caused by coronaviruses, including SARS-CoV-2, it will be understood that a positive response to vaccination with the methods described herein may include recovery or amelioration of any symptoms experienced by the subject.

[0265] For example, a positive response to vaccination may be a reduction in the overall level of fatigue, muscle pain, headache, and / or lethargy in the subject. A positive response may also include a reduction in fever and a return of the subject to an afebrile state.

[0266] A positive response to vaccination may prevent or attenuate respiratory symptoms following respiratory virus infection. This may be assessed by comparing the mean change in disease score from baseline to the end of the study period, for example based on a questionnaire, and the lower respiratory tract symptom score (LRSS - symptoms of chest tightness, wheezing, shortness of breath, and cough) may be assessed daily following the onset of infection / cold symptoms. The change from baseline lung function (peak expiratory flow PEF) may also be assessed, with a positive response to treatment being a significant attenuation of the decrease in PEF. For example, the placebo treatment group shows a significant decrease of 15% in morning PEF at the peak of exacerbation, while the treatment group shows a non-significant decrease with a change in PEF from baseline of less than 15%.

[0267] A positive response to vaccination may also be a reduction in the presence of ground glass opacities around the lungs or near the pleura (eg, as determined using chest CT imaging techniques).

[0268] A positive response to vaccination may also include an increase in blood oxygen levels or a return to normal levels.

[0269] A positive response to vaccination may also include improvement in cardiovascular disorders such as changes in blood pressure and increased presence of clotting factors.

[0270] Protective immune responses can include humoral and cellular immune responses. Protection against SARS-CoV-2 is thought to be conferred by serum neutralizing antibodies (humoral immune response) directed against the spike protein, with mucosal IgA antibodies and cell-mediated immune responses also playing a role. Cellular immune responses are useful in protecting against SARS-CoV-2 infection, with CD4+ and CD8+ T cell responses and memory B cell responses being particularly important. CD8+ immunity is particularly important in killing virus-infected cells. Natural killer cells and NKT cells may also be important in killing and / or eliminating virus-infected cells.

[0271] kit In another embodiment, there is provided a kit or article of manufacture comprising one or more proteins, polypeptides or polynucleotides of the invention and / or immunogenic compositions as described above.

[0272] In yet another aspect, the present invention provides a kit of parts comprising a vaccine composition of the invention and one or more adjuvants for separate, subsequent or simultaneous administration to a subject.

[0273] In other embodiments, a kit is provided for use in the above therapeutic or prophylactic applications, the kit comprising: - a container holding a protein, polypeptide, polynucleotide or immunogenic composition of the invention; - A label or insert containing instructions for use.

[0274] In any embodiment, the kit may contain one or more additional effective or active ingredients for eliciting an immune response against coronavirus in a subject.

[0275] The kit or "article of manufacture" may comprise a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, blister packs, and the like. The container may be formed from a variety of materials such as glass or plastic. The container holds a therapeutic composition effective for treating a medical condition and may have a sterile access port (e.g., the container may be an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle). The label or package insert indicates that the therapeutic composition is used to treat a selected medical condition. In one embodiment, the label or package insert includes instructions for use indicating that the therapeutic or prophylactic composition can be used to treat an inflammatory disease or condition described herein.

[0276] The kit may comprise (a) a therapeutic or prophylactic composition; and (b) a second container containing a second active ingredient or component. The kit in this embodiment of the invention may further comprise a package insert indicating that the composition and other active ingredients may be used to treat or prevent complications resulting from an inflammatory disease or condition described herein. Alternatively, or in addition, the kit may further comprise a second (or third) container comprising a pharmacologically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution, and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0277] In any embodiment, the therapeutic composition may be provided in the form of a disposable or reusable device that includes a container for holding the therapeutic, prophylactic, or immunogenic composition. In one embodiment, the device is a syringe. The device may hold 1-2 mL of the therapeutic or immunogenic composition. The therapeutic or prophylactic composition may be provided in the device ready to use or requiring mixing or addition of additional components.

[0278] It will be understood that the invention disclosed and defined herein extends to all alternative combinations of two or more individual features mentioned or evident from the text or drawings, all these different combinations constituting various alternative embodiments of the invention.

[0279] The following examples are presented to more fully illustrate some embodiments of the present invention. However, they should not be construed as limiting the broad scope of the present invention in any way. Those skilled in the art can easily devise many variations and modifications of the principles disclosed herein without departing from the scope of the present invention. EXAMPLES

[0280] Example 1 - Generation of RBD-IgG1 Fc fusion protein Materials and Methods RBD Mouse IgG1 Fc Fusion: To generate the recombinant RBD mouse IgG1 Fc fusion protein (SEQ ID NO:3), recombinant DNA fragments encoding the truncated receptor binding domain (RBD) of ancestral SARS-CoV-2 (N334-P527; GenBank accession NC_045512.2; SEQ ID NO:15) were synthesized between the 5'AgeI and 3'BamHI cloning sites (GeneArt Gene Strings, Thermo Scientific). These were then cloned into the mammalian expression vector pHLSec, which contains the mouse IgG1 Fc domain (SEQ ID NO:16) from the core hinge region to the C-terminal lysine and includes an N-terminal GSGSG linker (SEQ ID NO:17), thereby fusing the RBD to mIgG1-Fc.

[0281] RBD human IgG1 Fc fusion: To generate the recombinant RBD human IgG1 Fc fusion protein (SEQ ID NO:25), a recombinant DNA fragment encoding ancestral SARS-CoV-2 (N334-P527; GenBank accession NC_045512.2; SEQ ID NO:15) fused to the Fc domain of human IgG1 from the core hinge region to the C-terminal lysine (SEQ ID NO:18) via a GSGSG linker (SEQ ID NO:17), followed by a stop codon, was codon-optimized for mammalian expression (GeneArt Gene Strings, Thermo Scientific) and synthesized between the 5'NheI and 3'XhoI cloning sites (IDT). This was then cloned into the mammalian expression vector pHLSec (SEQ ID NO:4).

[0282] RBD Monomer: To generate the recombinant RBD monomer, a recombinant DNA fragment encoding the truncated receptor binding domain (RBD) of ancestral SARS-CoV-2 (N334-P527; GenBank accession NC_045512.2; SEQ ID NO: 15) was codon-optimized for mammalian expression (GeneArt Gene Strings, Thermo Scientific) and synthesized between the 5'NheI and 3'XhoI cloning sites (IDT). It was then cloned into the mammalian expression vector pHLSec, incorporating a 6-HIS tag followed by a C-terminal stop codon (SEQ ID NO: 8). The RBD tandem dimer is also referred to herein as a single-stranded RBD dimer.

[0283] RBD tandem dimer: To generate the recombinant RBD tandem dimer, a recombinant DNA fragment encoding two truncated receptor binding domains (RBDs) of ancestral SARS-CoV-2 (R319-K537; GenBank accession NC_045512.2, SEQ ID NOs: 19 and 20) in tandem repeats based on Dai et al. (Cell, 2020) was codon-optimized for mammalian expression (GeneArt Gene Strings, Thermo Scientific) and synthesized between the 5'NheI and 3'BamHI cloning sites (IDT). It was then cloned into the mammalian expression vector pHLSec to incorporate a 6-HIS tag followed by a C-terminal stop codon (SEQ ID NO: 11).

[0284] RBD beta human IgG1 Fc fusion PCR primers were designed to allow cloning of a recombinant DNA fragment encoding the truncated RBD of ancestral SARS-CoV-2 (N334-P527; GenBank accession NC_045512; SEQ ID NO: 15) fused to the Fc domain of human IgG1 (SEQ ID NO: 18) from the core-hinge region to the C-terminal lysine via a GSGSG linker (SEQ ID NO: 17), followed by a stop codon from the mammalian expression vector pHLSec, into the mammalian expression vector pXC-17.4. The resulting PCR product was cloned into the mammalian expression vector pXC-17.4 using the restriction enzyme sites HindIII and EcoRI to generate the polynucleotide set forth in SEQ ID NO: 29. PCR mutagenesis was used to introduce the mutations K417N, E484K, and N501Y. This was the construct used for the generation of DoCo-Pro-RBD-1 ADP.

[0285] Protein Expression: All RBD proteins, except for the RBD-β human IgG1 Fc fusion protein, were expressed by transient transfection of Expi293S cells using the ExpiFectamine 293 transfection kit according to the manufacturer's instructions (ThermoFisher Scientific). Proteins were harvested on day 6. RBD-Fc protein was purified from the supernatant by protein A sepharose (CL-4B, Cytiva), and RBD monomer and RBD tandem dimer were purified by Ni-NTA resin (HisPur, Thermo Scientific). All proteins were further purified using gel filtration size exclusion chromatography, RBD-Fc and RBD tandem dimer were purified using a Superdex-200 column (Cytiva), and RBD monomer was purified using a SuperdexS75 column (Cytiva). All proteins were sterile filtered and stored at -80°C before use.

[0286] A research cell bank (RCB) for a stable pool expressing beta-RBD-hFc protein was developed at the National Biologics Facility (QLD Node). Cells from a single vial were revived and expanded in shake flasks as recommended by Lonza GSv9™, and protein was produced in a fed-batch shake flask production over 12 days. Conditioned media was harvested by depth filtration and 0.2 μm filtration. Antigen was captured on Protein A resin, MabSelect PrismA (Cytiva), and eluted at pH 4.0. Antigen was acidified to pH 3.5 and held for >60 min, then neutralized to pH 5.5 and subjected to low pH viral inactivation. Antigen was buffer exchanged into citrate buffer pH 6.5, formulated with PS80 at 0.02%, and diluted to 1.06 mg / mL before aseptically filling vials.

[0287] AF647-conjugation of proteins: Purified RBD-mFc and mouse IgG1 isotype control (clone MOPC-21, Biolegend) were then labeled with an Alexa Fluor 647 antibody labeling kit (Thermo Scientific) according to the manufacturer's instructions.

[0288] Results and Discussion DNA encoding the isolated form of the SARS-CoV-2 spike receptor binding domain (RBD) (Figure 1A) was cloned into the mammalian expression vector pHLSec for expression in three different forms: RBD monomer, RBD tandem dimer, and RBD-IgG1 Fc dimer (Figure 1B). Both mouse and human RBD-IgG1 Fc dimers were generated. These proteins were expressed by transiently transfecting mammalian Expi-293S cells, and after 6 days the proteins were isolated and purified by gel filtration size exclusion chromatography (Figure 1C-G), followed by sterile filtration and storage at -80°C.

[0289] The activity of the purified RBD-Fc dimer protein was verified by staining 293T cells transfected to express human ACE2, the natural receptor for RBD (Figure 1H). A control cell line transfected with an unrelated protein (T cell receptor) did not stain with the RBD-Fc dimer but did stain with an anti-TCR antibody as expected.

[0290] Example 2 - Comparison of subcutaneous and intranasal vaccination routes of RBD-Fc dimer with or without adjuvant Materials and Methods Vaccination and blood collection system BALB / c mice (n=5 per group) were vaccinated with 0.1 nmoles of RBD dimer administered without adjuvant or in the presence of 0.3 nmoles of PEG-R4-Pam-2-Cys or 0.2 μg of α-GC via either the subcutaneous route at the base of the tail (100 μl) or the intranasal route into the URT (15 μl). Mice were primed on day 0 and boosted on day 28. Mice were bled immediately before the first injection (pre-bleed), immediately before the second injection (1°), and 2 weeks after the second injection (2°).

[0291] Enzyme-linked immunosorbent assay (ELISA) for measuring RBD-specific antibody responses RBD-specific antibody titers were determined by ELISA. Flat-bottom 96-well maxisorp plates (ThermoFisher Scientific) were coated with RBD monomer at a concentration of 2 μg / ml in Dulbecco's phosphate-buffered saline (DPBS; Gibco Life Technologies), 50 μl / well. Plates were incubated overnight at 4 °C in a humidified atmosphere. Unbound antibodies were removed and wells were blocked with 100 μl / well of 1% bovine serum albumin (BSA fraction V, Invitrogen Corporation, Gibco) in PBS for 1–2 h, followed by washing three times with PBS containing 0.05% v / v Tween-20 (PBST). Serial dilutions of mouse sera were added to the wells and incubated overnight at room temperature. After washing, bound Abs were detected using horseradish peroxidase (HRP)-conjugated rabbit anti-mouse Ig Ab (Dako, Denmark). The detection antibodies were incubated for 1 h at room temperature in a humidified atmosphere, and then the plates were washed five times with PBST. Then, 100 μl of tetramethylbenzidine substrate (TMB, BD Biosciences, Cat. No. 555214) was added to each well, and the reaction was stopped after 5–7 min by the addition of 100 μl / well of 1 M orthophosphoric acid (BDH Chemicals, Australia). The optical density (OD) of each well was measured at wavelengths of 450 nm and 540 nm using a Labsystems Multiskan microplate reader (Labsystems, Finland). Ab titers are expressed as the reciprocal of the highest dilution of serum required to achieve an OD of 0.3.

[0292] Microneutralization assay SARS-CoV-2 isolates used in the microneutralization assay were propagated in Vero cell cultures and stored at -80°C. The day before the assay, Vero cells were plated in flat-bottom 96-well plates at 2 × 10 4 Cells / well were seeded. Serial two-fold dilutions of heat-inactivated serum were added at 100 TCID 50(50% tissue culture infectious dose) of SARS-CoV-2 for 1 h and residual viral infectivity was assessed in quadruplicate wells of Vero cells. Plates were incubated at 37 °C and viral cytopathic effect was measured on day 5. Neutralizing antibody titers were calculated using the Reed / Muench method.

[0293] SARS-CoV-2 surrogate virus neutralization assay Detection of circulating antibodies directed against the spike protein receptor-binding domain (RBD), based on antibody-mediated blockade of the interaction between the ACE2 receptor protein and RBD, was measured in mouse serum using the SARS-CoV-2 surrogate virus neutralization test (GenScript, USA) according to the manufacturer's instructions. Briefly, 10 μl of mouse serum was diluted in 90 μl of sample dilution buffer and incubated with horseradish peroxidase-conjugated SARS-CoV-2 RBD protein (HRP-RBD); the test solution was added to wells coated with immobilized ACE2 receptor. The extent to which serum inhibited the binding of HRP-RBD to the ACE2 receptor was determined compared to control serum by optical density readings, with ≥20% inhibition considered a positive result.

[0294] Results and Discussion Groups of five BALB / c mice were immunized with RBD murine IgG1-Fc dimer (RBD) without adjuvant or mixed with PEG-R4-Pam-2-Cys (P2C) or α-galactosylceramide (α-GC) adjuvant. Two immunization routes were tested: subcutaneous and intranasal (upper respiratory tract, URT).

[0295] The first injection (day 0) was followed 4 weeks later by an identical boost injection. Mice were bled immediately before the first injection (pre-bleed), immediately before the second injection (1°), and 2 weeks after the second injection (2°). For each mouse in each group, total anti-RBD IgG antibody titers were determined using an RBD-specific ELISA assay (Figure 2).

[0296] These results showed that RBD without adjuvant induced little response above background as determined by prebleed samples. However, both adjuvants (P2C and α-GC) induced very strong antibody titers (10 3 ~10 4 ) and after the second injection, 10 5 It induced antibody titers of 100 or more.

[0297] These serum samples were also tested in a microneutralization assay using SARS-CoV-2 virus, and neutralization titers were determined by the titer required to inhibit infection of Vero cells. This assay demonstrated that despite the high antibody titers after primary immunization, little to no antibodies were induced. However, after secondary immunization, very strong neutralizing antibody responses were detected in all groups that received RBD combined with adjuvant, with antibody titers ranging from 200 to 2000 (Figure 3).

[0298] As an independent validation of the neutralizing capacity of these serum samples, a separate assay known as the surrogate virus neutralization test (sVNT) (Tan et al. (2020) Nature Biotechnology) was also employed. This test measures the ability of antibody-containing samples to prevent RBD from binding to ACE2. Due to the limited number of tests that could be performed, only secondary serum samples were tested, which revealed that the adjuvanted RBD vaccine inhibited RBD-ACE-2 binding by more than 95% in this assay. Serum from mice immunized with the non-adjuvanted RBD vaccine failed to record a neutralizing antibody response in this assay (Figure 4).

[0299] These data are presented in relation to similar data obtained from human convalescent samples (hCov), not from the same experiment but collected and assayed over the same time frame (Figure 5). These data are provided as a comparison only.

[0300] Example 3 - Testing of spike protein variants Human convalescent plasma sample (hCov) Human convalescent plasma samples (hCov) DD1, DD2, DD3, and DD4 were provided by Prof. Katherine Kedzierska (Peter Doherty Institute for Infection and Immunity, Department of Microbiology and Immunology). These patients were infected with the SARS-CoV-2 Wuhan indicator strain VIC01 in April 2020. Sera were tested for neutralizing activity against the 501Y and 477N mutants (VIC2089 and VIC4881), and the original VIC01 strain, using a microneutralization assay.

[0301] Mouse serum sample BALB / c mice (n=5 per group) were vaccinated via subcutaneous route at the base of the tail with 0.1 nmoles of RBD dimer administered without adjuvant or in the presence of 0.3 nmoles of PEG-R4-Pam-2-Cys or 0.2 μg α-GC adjuvant. Mice were primed on day 0 and boosted on day 28. Sera collected 4 weeks after the second dose were tested for neutralizing activity against the 501Y and 477N mutants (VIC2089 and VIC4881) and the original VIC01 strain using a microneutralization assay. These sera were also tested for binding to 18 RBD mutant proteins (GenScript, USA) using a multiplex bead array.

[0302] Results and Discussion With the emergence of two SARS-CoV-2 variants in Australia, (D614G / N501Y) and (D614G / S477N), we tested the ability of these virus isolates (VIC2089 and VIC4881) to be neutralized by three different convalescent plasma samples (DD2, DD3, and DD4) compared to the original VIC01 strain (Figure 6B). DD1 was included as a patient sample that did not contain measurable neutralizing antibody titers. Samples DD2, DD3, and DD4 were impaired in their ability to neutralize the variants (VIC2089 and VIC4881). This suggests that patients infected with the original VIC01 strain may have limited immunity to these new variants, and furthermore, as the S protein used in the vaccine is based on the Wuhan index (VIC01) strain, the emergence of these variants may limit the efficacy and longevity of vaccine-induced immunity.

[0303] Next, we tested whether sera from mice immunized with the RBD-Fc dimer vaccine in the presence of P2C or α-GC adjuvant were impaired in their ability to neutralize these two SARS-CoV-2 mutant strains (Figure 6C). The results clearly show that these immunized mouse serum samples are able to neutralize these mutant virus strains at least as well as or better than the original VIC01 strain when α-GC is used as an adjuvant. To investigate a broader range of RBD mutants, we used an RBD multiplex assay that allows us to compare binding to 18 different RBD mutants detected so far during the pandemic (Figure 7). The binding capacity (half-maximal effective concentration (EC)) of serum samples from mice immunized with the RBD vaccine combined with either P2C or α-GC adjuvant was measured. 50 The weakest binding (EC 50 The mutant strain with the lowest EC value was T478I, but this mutant still had a high EC value of over 300. 50 The reading was shown.

[0304] Taken together, vaccination with RBD-Fc dimers combined with adjuvants achieves comparable levels of neutralization in microneutralization assays against different virus strains (Figure 5). This may be because the vaccine focuses all immune responses on the RBD epitopes, providing comprehensive coverage of this target, making it less likely that mutations of individual amino acid residues will escape vaccine-induced immunity. This may be less likely to happen if the vaccine is based on the whole spike or whole virus, as there are more epitopes for the immune system to cover.

[0305] Example 4 - Testing of vaccines in a SARS-CoV-2 mouse challenge model Materials and Methods Establishment of a SARS-CoV-2 mouse challenge model using VIC2089 (N501Y / D614G) Groups of four BALB / c and C57BL / 6 mice were aerosol challenged with either the index SARS-CoV-2 strain VIC01 or the VIC2089 (N501Y / D614G) mutant strain. Three days after challenge, mice were sacrificed, right lung lobes were harvested and homogenized, and supernatants were collected and stored at -80°C. Infectious virus titers (TCID 50 ; 50% tissue culture infective dose) was added at 2 × 10 4 Supernatants were titrated and determined on Vero cell monolayers seeded in flat-bottom 96-well plates at a concentration of 1000 cells / well. Ten-fold serial dilutions of SARS-CoV-2 were assessed in quadruplicate wells of Vero cells; viral cytopathic effect (CPE) was measured after 5 days.

[0306] Assessment of protective effects in the upper respiratory tract and lungs of vaccinated mice At the indicated times after vaccination, mice were challenged with the VIC2089 (N501Y / D614G) mutant by aerosol. Three days after challenge, mice were sacrificed and blood was collected by cardiac puncture. The left lung was harvested and fixed in 4% paraformaldehyde for histology. To determine the virus titer in the lungs and nasal turbinates of challenged mice, the right lung lobe and nasal turbinates were harvested, homogenized by bead beating in 500 μl of medium, and centrifuged at 2000×g for 5 min. The supernatant was then collected and stored at −80° C. until testing. Infectious virus titers (TCID 50 ; 50% tissue culture infective dose) was added at 2 × 10 4 Supernatants were titrated and determined on Vero cell monolayers seeded in flat-bottom 96-well plates at a concentration of 1000 cells / well. Ten-fold serial dilutions of SARS-CoV-2 were assessed in quadruplicate wells of Vero cells; viral cytopathic effect (CPE) was measured after 5 days.

[0307] Results and Discussion The VIC2089 strain of SARS-CoV-2 carries two key mutations, D614G and N501Y, compared to the original VIC01 strain. The latter mutation is particularly important since this amino acid substitution has been observed in previous mouse-adapted SARS-CoV-2 viruses. This is because the N501Y mutant RBD is able to bind to mouse ACE2, in contrast to the original ancestral SARS-CoV-2 virus that has the 501N residue. Therefore, we tested isolates of the naturally occurring mutant strain in two commonly used mouse strains (C57BL / 6 and BALB / c) (Figure 8). The results showed that the N501Y mutant strain infected these mice and showed high virus titers that peaked around day 3, whereas the original wild-type strain (N501) was unable to infect these mice. We therefore developed a small animal model to challenge immunized mice with the virus.

[0308] To confirm that serum samples from immunized mice could neutralize the VIC2089 strain, Fig. 9 shows side-by-side neutralization titers of WT and VIC2089 strains, and no significant differences were observed.

[0309] We tested mice that had been pre-immunized (approximately 100 days prior) with the RBD mouse IgG1-Fc vaccine alone or with PEG-R4-Pam-2-Cys or α-GalCer adjuvants. Despite the long period between the second immunization and the virus challenge, mice immunized with RBD combined with either adjuvant were completely protected from lung infection with no detectable virus in lung samples. This was seen regardless of whether the vaccine was administered subcutaneously or intranasally. No protection was afforded by the unadjuvanted RBD vaccine (Figure 10). We also tested samples from the nasal turbinates of mice as a measure of upper respiratory tract protection. While low levels of virus were still detectable in some of these immunized mice, viral titers were reduced by at least 99%, and 4 / 5 mice immunized via the subcutaneous route with RBD combined with α-GalCer had no detectable virus (Figure 11).

[0310] Example 5 - Efficacy evaluation of intramuscular administration of RBD vaccine Materials and Methods Vaccination and challenge regime Groups of 10 BALB / c mice were inoculated intramuscularly with 50 μl of 0.1 nmoles of RBD dimer administered with 0.3 nmoles of PEG-R4-Pam-2-Cys. Mice were primed on day 0 and boosted on day 28. Four weeks later, mice were aerosol challenged with the VIC2089 (D614G / N501Y) mutant strain. Five age-matched unvaccinated control BALB / c mice were also challenged at the same time.

[0311] Groups of five C57BL / 6 mice were vaccinated intramuscularly with 0.1 nmoles of RBD dimer administered with 0.3 nmoles of PEG-R4-Pam-2-Cys. Mice were primed on day 0, boosted on day 14, and challenged 3 months later with the VIC2089 (D614G / N501Y) mutant strain. Five age-matched unvaccinated control C57BL / 6 mice were also challenged at the same time.

[0312] Assessment of protective effects in the upper respiratory tract and lungs of vaccinated mice Three days after challenge in both BALB / c and C57BL / 6 mice, the animals were sacrificed, the right lung lobes and nasal turbinates were harvested and homogenized, and the supernatants were collected and stored at -80°C. The titers of infectious virus in tissues (TCID 50 ) was determined by titrating the supernatants on Vero cell monolayers and assessing CPE after 5 days.

[0313] Results and Discussion Since the injection route in clinical settings is usually intramuscular, we also tested mice immunized via the intramuscular route (quadracept) in a mouse virus challenge essay. These data impressively showed that virus was undetectable in both the lungs and nasal turbinates of 10 / 10 mice immunized with the RBD vaccine combined with PEG-R4-Pam-2-Cys (Figure 13). Similar results were observed in 5 / 5 C57BL / 6 mice, suggesting that the vaccine was fully protective across two different laboratory mouse strains.

[0314] Example 6 - Comparison of RBD mouse Fc dimer, RBD human Fc dimer, and single chain RBD dimer Materials and Methods Vaccination and blood collection system C57BL / 6 mice (5 per group) were vaccinated intramuscularly with 50 μl of either 0.1 nmoles of RBD mouse Fc dimer, RBD human Fc dimer, or single chain dimer, all administered with 0.3 nmoles of PEG-R4-Pam-2-Cys. Mice were primed on day 0 and boosted on day 28. Mice were bled immediately prior to the first injection (prebleed), immediately prior to the second injection (42°), and 2 weeks after the second injection (2°). To assess the durability of antibody responses induced by the three different RBD antigens administered with PEG-R4-Pam-2-Cys, mice were also bled on days 63 and 82. All serum samples were tested for RBD-specific antibodies by ELISA.

[0315] Results and Discussion Next, we tested whether there was a difference in the efficacy of the RBD vaccine depending on whether mouse or human Fc regions were used to generate the dimers, and whether a simple single-chain dimer would work as well. We compared RBD fused to mouse IgG1 Fc region, human IgG1 Fc region, or single-chain RBD dimers, using PEG-R4-Pam-2-Cys as the adjuvant, respectively (Figure 14a). Mouse Fc dimers showed slightly stronger responses than human Fc dimers after primary and secondary immunizations, while RBD single-chain dimers induced a very weak response after primary immunization that increased to a similar level to Fc dimers after boosting (Figure 14b). Mouse Fc dimers performed better than human Fc dimers in mice, most likely due to the better fit of mouse Fc to mouse Fc receptors. However, because humans will eventually be immunized with the vaccine, it is encouraging that the human Fc dimer performed nearly as well in mice, and is likely to perform better in humans.

[0316] We measured the durability of the response to each RBD formulation (using PEG-R4-Pam-2-Cys as adjuvant) (Figure 15). Both mouse and human dimers demonstrated stable levels of RBD-specific antibody titers for up to 84 days after the first injection. In contrast, antibody levels in mice immunized with the RBD single chain dimer showed a progressive decline over the 84 day time frame, suggesting that the response with this dimer was less durable. This suggests that when immunizing humans, the RBD human IgG1 Fc dimer is the optimal formulation to provide a strong and durable response.

[0317] Example 7 - Testing of RBD Mouse IgG1-Fc Dimer with MF59® Adjuvant Materials and Methods Vaccination and blood collection system To assess RBD-specific antibody responses in mice vaccinated with RBD mouse IgG1-Fc dimer formulated with either MF59® or PEG-R4-Pam-2-Cys, groups of 10 C57BL / 6 mice were immunized intramuscularly with 50 μl of 0.1 nmoles RBD mouse Fc dimer and 0.3 nmoles PEG-R4-Pam-2-Cys, or 0.1 nmoles RBD mouse Fc dimer and MF59® (50% vol / vol). Mice were bled prior to the first immunization (pre-bleed) and on day 14 (1°), boosted on either day 14 or day 28, and in both cases re-bled 2 weeks after the second immunization (2°).

[0318] To assess RBD-specific antibody responses in mice vaccinated with RBD mouse IgG1-Fc dimer or RBD single chain dimer formulated with either MF59® or PEG-R4-Pam-2-Cys, C57BL / 6 mice were immunized intramuscularly with 10 μg of RBD mouse Fc dimer or 10 μg of RBD single chain dimer, both formulated with 0.3 nmoles of PEG-R4-Pam-2-Cys or MF59® (50% vol / vol). Mice were primed on day 0 and boosted on day 14. Mice were bled immediately before the first injection (pre-bleed), immediately before the second injection (1°), and 4 weeks after the second injection (2°). All serum samples were tested for RBD-specific antibodies by ELISA.

[0319] Results and Discussion Since MF59® is a relatively well-established adjuvant, we next compared it to the PEG-R4-Pam-2-Cys adjuvant (Figure 16). Mice were immunized with RBD mouse Fc dimer along with either PEG-R4-Pam-2-Cys or MF59® adjuvant. Mice were bled prior to the first immunization (pre-bleed), boosted either 2 or 4 weeks later, and in each case re-bled 2 weeks after the secondary immunization to determine total anti-RBD specific IgG titers. This experiment revealed that the MF59® adjuvant was equally good or better at inducing high titers of RBD specific antibodies.

[0320] We also compared the RBD murine Fc dimer to the RBD single chain dimer in the presence of MF59 adjuvant® or PEG-R4-Pam-2-Cys adjuvant (Figure 17). After both primary and secondary immunizations, antibody titers were higher when the RBD Fc dimer was used as a vaccine compared to the RBD single chain dimer, regardless of which adjuvant was used.

[0321] Example 8 - Comprehensive analysis of responses to RBD m-Fc dimer administered with PEG-R4-Pam-2-Cys versus RBD monomer administered with PEG-R4-Pam-2-Cys Materials and Methods Vaccination and blood collection system Groups of 10 BALB / c mice were immunized intramuscularly with 50 μl of 0.2 nmoles of RBD monomer formulated with 0.3 nmoles of PEG-R4-Pam-2-Cys or 0.1 nmoles of RBD mouse Fc dimer formulated with 0.3 nmoles of PEG-R4-Pam-2-Cys. Mice were primed on day 0 and boosted on day 28. Mice were bled immediately before the first injection (pre-bleed), immediately before the second injection (1°), and 2 weeks after the second injection (2°). Sera were tested for RBD-specific antibodies by ELISA.

[0322] Assessment of protective effects in the upper respiratory tract and lungs of vaccinated mice Four weeks after the boost on day 28, mice were challenged with the VIC2089 (D614G / N501Y) mutant strain. Five age-matched unvaccinated control BALB / c mice were also challenged at the same time. Mice were sacrificed 3 days after challenge, the right lung lobe and nasal turbinates were harvested and homogenized, and the supernatants were collected and stored at -80°C. Titers of infectious virus in tissues (TCID 50 ) was determined by titrating the supernatants on Vero cell monolayers and assessing CPE after 5 days.

[0323] Results and Discussion The next experiment was designed to examine whether the RBD monomer could induce a similar response as the RBD Fc dimer in the presence of adjuvant (Figure 18). Groups of 10 BALB / c mice were pre-bled and then immunized intramuscularly with either the RBD monomer and PEG-R4-Pam-2-Cys vaccine formulation or the RBD m-Fc dimer and PEG-R4-Pam-2-Cys vaccine formulation. Mice were bled 4 weeks after the primary immunization and boosted 2 weeks after the secondary immunization. Serum samples were tested for total anti-RBD antibody titers, revealing that the RBD Fc dimer produced a much stronger response after primary immunization than the RBD monomer. The titers of anti-RBD antibodies in the serum increased significantly after the boost, but still did not reach the same levels as the RBD Fc dimer vaccine.

[0324] Four weeks after the boost, these mice were tested in the N501Y mouse challenge model (Figure 19). As before, mice immunized with RBD dimer were fully protected, with no virus detected in the lungs or nasal turbinates. In contrast, 8 / 10 mice immunized with RBD monomer showed no detectable virus in the lungs, but all mice had persistent moderate to high virus titers in the nasal turbinates.

[0325] Example 9 - Comparison of immunogenicity of WT RBD-mFc antigen formulation with MF59® or PEG-R4-Pam-2-Cys adjuvants and the effect of dose regimen and frequency Materials and Methods The clinically approved squalene oil-in-water adjuvant MF59® (Seqirus, Holly Springs, USA) was selected for testing with the WT RBD-mFc antigen due to its long-standing good safety record in the Fluad® Quad vaccine.

[0326] C57BL / 6 mice were compared with WT RBD-mFc dimer vaccine (10 μg) administered intramuscularly with 0.3 nmol PEG-R4-Pam-2-Cys adjuvant and WT RBD-mFc dimer vaccine (10 μg) with 50% vol / vol MF59® adjuvant. Mice were inoculated according to either a two-dose regimen on days 0 and 112, or a three-dose regimen on days 0, 22, and 112. Serum was collected pre- and post-vaccination.

[0327] Results and Discussion Intramuscular administration of WT RBD-mFc dimer with either MF59® or PEG-R4-Pam-2-Cys resulted in comparable total RBD-specific antibody responses. Enhanced total RBD-specific Ab responses were observed in both groups following both primary and secondary immunization with a two dose schedule (Figure 20A-B) and a three dose schedule (Figure 20C-D).

[0328] The extended period between primary and secondary immunization with WT RBD-mFc dimer combined with MF59® in either the two-dose or three-dose regimen did not appear to affect the total RBD antibody response following booster immunization with similar titers observed 16 and 12 days after secondary immunization in the two-dose regimen (Figure 20A) and three-dose regimen (Figure 20C), respectively. Total antibody levels appeared to reach a plateau over the 112 days between the two doses of the two-dose regimen (Figure 20A). For the total antibody response in the three-dose regimen, total RBD-specific Ab responses increased from the secondary immunization (day 22) until day 90, and tended to decrease slightly on day 111 (day 89 from secondary immunization). A third dose administered on day 111 reversed the downward trend and total RBD-specific Ab responses increased again by day 132 (Figure 20C).

[0329] Virus neutralization titer results from mice inoculated with a three-dose regimen of WT RBD-mFc dimer combined with MF59® demonstrated increased levels of SARS-CoV-2-specific nAbs beginning 40 days after the secondary immunization (day 62) and further increasing 21 days after the tertiary immunization (day 132) (Figure 20E).

[0330] These results indicate that the immunogenicity of the WT RBD-mFc dimer is maintained when used with the clinically approved MF59® adjuvant, compared to the PEG-R4-Pam-2-Cys adjuvant. Furthermore, antibody levels appear to be similar whether the booster is administered 22 or 112 days after the priming immunization. A third booster immunization appears to be able to reverse the decline in total antibody levels and further increase nAb levels. This reflects the ability of the WT RBD-mFc dimer to provide a boost that raises total and nAb levels higher than after the second injection.

[0331] Experiment 10 - Evaluation of immunogenicity and protective efficacy of increasing doses of WT RBD-mFc in combination with MF59® after one dose Materials and Methods To determine whether greater than 10 μg of WT RBD-mFc, with or without MF59® adjuvant, would provide improved protective immunity after a single vaccination, C57BL / 6 mice were immunized via the intramuscular route using a prime-only regimen with 10, 30 or 50 μg of WT RBD-mFc in the presence or absence of MF59® (50% v / v).

[0332] Results and Discussion Results showed that a single dose of either 10 μg or 30 μg of WT RBD-mFc combined with MF59® was not sufficient to provide protective immunity, with virus titers detectable in the lungs of all animals challenged. Furthermore, compared to the 10 μg dose of WT RBD-mFc combined with MF59®, the higher 30 μg dose did not provide superior protective immunity against SARS-CoV-2 virus challenge, with virus titers higher and comparable to those observed in unvaccinated controls.

[0333] These results indicate that increasing the dose of the priming vaccine of WT RBD-mFc combined with MF59® does not overcome the need for a second vaccination for the induction of protective immune responses in naive mice.

[0334] Experiment 11 - Comparison of immunogenicity and protective efficacy of RBD human IgG1 Fc dimer and RBD mouse IgG1 Fc dimer Materials and Methods To further advance the WT RBD-Fc vaccine candidate into human clinical trials, a WT RBD human Fc dimer protein (using the human IgG1 Fc region) was generated. Intramuscular administration of 10 μg of WT RBD-hFc dimer combined with PEG-R4-Pam-2-Cys was compared to intramuscular administration of 10 μg of WT RBD-mFc dimer combined with PEG-R4-Pam-2-Cys in both BALB / c and C57BL / 6 mouse strains using a prime-boost regime.

[0335] Results and Discussion RBD-specific antibody analysis demonstrated that the human Fc form of the RBD-Fc vaccine induced similar levels and patterns of total anti-RBD antibodies as the mouse Fc form, although slightly lower in C57BL / 6 mice. In both mouse strains, the mouse and human Fc forms of the RBD vaccine induced sustained antibody responses that were generally maintained at constant levels until day 115 (87 days post-boost) (Figures 21A-B).

[0336] When neutralizing antibody (nAb) titers were assessed in C57BL / 6 mice harvested 13 days after the second immunization, all animals demonstrated elevated nAbs, and as observed with total RBD-specific antibodies, the human Fc form of the RBD vaccine induced similar or slightly lower levels of nAbs than the murine Fc form of the vaccine (Figure 21C).

[0337] A mouse SARS-CoV-2 challenge model was applied on day 139 (11 days after booster immunization) and the results demonstrated that in the presence of the PEG-R4-Pam-2-Cys adjuvant, both the mouse and human forms of the RBD-Fc vaccine were able to fully protect against lower respiratory tract infection in 5 / 5 mice immunized with RBD human Fc and 3 / 5 mice immunized with RBD mouse Fc (Figure 22).

[0338] These results demonstrate that the WT RBD human Fc dimer protein provides similar immunogenicity and protective efficacy as the WT RBD murine Fc dimer in the presence of the PEG-R4-Pam-2-Cys adjuvant.

[0339] Experiment 12 - Immunogenicity and protective efficacy of WT RBD-hFc combined with MF59® Next, the immunogenicity and protective efficacy of WT RBD human FC combined with MF59® adjuvant was evaluated in C57BL / 6 mice via the intramuscular route using a prime-boost regimen.

[0340] The WT RBD-hFc vaccine candidate combined with MF59® provided high levels of RBD-specific total Abs 20 days after primary immunization (day 20), which were further elevated 12 days after boost immunization (day 33) (Figure 23A). High nAb levels were also observed, capable of neutralizing both WT and beta mutant viruses (Figure 23B). As expected, higher nAbs against the WT virus were observed compared to the beta virus.

[0341] A murine SARS-CoV-2 challenge model was applied on day 81 (60 days after secondary immunization) and showed complete protection against both upper and lower respiratory tract infection (Figure 24).

[0342] These results indicate that WT RBD-hFc dimer combined with MF59® adjuvant can provide protective immunity to SARS-CoV-2 in mice when administered intramuscularly in a prime / boost regimen.

[0343] Experiment 13 - Immunogenicity of betaRBD-hFc vaccine candidate combined with MF59® Materials and Methods Since the outbreak of the pandemic, mutant strains of SARS-CoV-2 have replaced the original WT strain of the virus. Some of these mutant strains, including the beta and delta strains, appear to be partially resistant to vaccine-induced immunity mediated by WT-based vaccines. The beta mutant is the most evasive of the mutants reported so far and carries three mutations in the RBD (N501Y, E484K, and K417N).

[0344] To demonstrate the suitability of the RBD-hFc vaccine in combination with MF59®, a betaRBD version of the vaccine was generated (betaRBD-hFc). Subsequently, two doses (10 μg and 3 μg) of the betaRBD-hFc vaccine in combination with MF59® were examined in mice via the intramuscular route in a prime-boost regimen.

[0345] Results and Discussion In mice immunized with betaRBD-hFc combined with MF59® at doses of either 10 μg or 3 μg, following a prime-boost regimen, a robust antibody response against WT RBD monomer was observed (FIG. 25A).

[0346] Neutralizing antibody titers specific for the beta variant were observed in 19 / 20 mice immunized with betaRBD-hFc combined with either 3 μg or 10 μg of MF59® (Figure 25B). Interestingly, mice receiving a prime-boost at a dose of 3 μg appeared to have higher nAbs against the beta variant compared to mice receiving a prime-boost at a dose of 10 μg. Furthermore, nAbs capable of neutralizing the WT variant were observed in 16 / 20 mice, albeit at lower effective titers (Figure 25B).

[0347] These results suggest that intramuscular administration of betaRBD-hFc dimers in combination with MF59® in a prime-boost regimen to mice generates robust WT-RBD-specific antibody responses and nAbs capable of neutralizing both WT and beta mutant strains of SARS-CoV-2.

[0348] Experiment 14 - Evaluation of immunogenicity and protective efficacy of different doses of WT RBD-hFc antigen and beta mutant RBD-hFc antigen administered with MF59® Materials and Methods To determine the minimal effective dose of WT RBD-hFc dimer and beta RBD-hFc dimer, C57BL / 6 mice were immunized intramuscularly with a range of doses (10, 3, 1 and 0.3 μg) of either dimer in the presence of MF59® using a prime boost regimen.

[0349] Results and Discussion immunogenicity Total anti-WT RBD antibody titers in primary and secondary sera of mice vaccinated intramuscularly with 10, 3, 1, or 0.3 μg of either WT RBD-hFc or betaRBD-hFc in the presence of MF59® were measured using an RBD-specific ELISA (Figure 14). Primary RBD antibody responses were relatively consistent across all doses of WT RBD-hFc (Figure 26) and betaRBD-hFc (Figure 26) evaluated, with slightly lower overall titers in mice immunized with betaRBD-hFc formulations. Secondary RBD antibody titers were boosted across all groups, with a slight trend for lower boost levels as the dose of antigen was reduced (Figure 26C,D). Similarly, after boost, mice immunized with betaRBD-hFc formulations had slightly lower overall RBD antibody titers, with greater variability also observed within each betaRBD-hFc vaccinated group. Similar results were observed when secondary sera (day 56) from mice vaccinated with WT RBD-hFc combined with MF59® or betaRBD-hFc combined with MF59® were analyzed for Ab responses to both WT RBD monomer and betaRBD monomer (Figure 27)).

[0350] Defensive Efficiency Protective efficacy against lower respiratory tract infection was evaluated in a mouse SARS-CoV-2 challenge model using VIC2089 (N501Y / D614G) and beta mutant strain B.1.351 as challenge strains. On day 76 of the study (day 55 post-boost), 5 of 10 mice from each vaccination group were immunized with either 10, 3, 1 or 0.3 μg of WT RBD-hFc or beta RBD-hFc in the presence of MF59® and aerosol-challenged with VIC2089 (Figure 28A). On day 83 of the study (day 62 post-boost), the remaining 5 mice from each group were aerosol-challenged with B.1.351 (Figure 28B).

[0351] Experiment 15 - Evaluation of immunogenicity and protective efficacy of RBD-hFc dimer vaccine in a hamster model Materials and Methods Hamsters infected with SARS-CoV-2 show symptoms similar to those of upper and lower respiratory tract infection in humans. Hamsters also lose weight, become lethargic, unkempt, hunched, and breathe rapidly. High levels of SARS-CoV-2 have been found in hamster lungs and intestines, and these tissues are studded with ACE2, which can bind to the SARS-CoV-2 virus. For this reason, hamsters have been used as a model to evaluate COVID vaccines in development.

[0352] Hamsters were administered 30 μg or 10 μg of WT RBD-hFc dimer formulated with either MF59® or PEG-R4-Pam-2-Cys on days 0 and 21. SARS-CoV-2 challenge was performed according to the method in “Syrian Hamster Experiments” by Zhou et al., Cell Host & Microbe, 2021, 29:551-563.

[0353] Results and Discussion Total RBD-specific antibody responses All hamsters seroconverted after the second injection, but the response in some hamsters was lower and more variable than that observed in previous mouse studies. Therefore, on day 49, the hamsters were administered a third dose of the corresponding vaccine. This resulted in much higher antibody production in all hamsters, as shown in Figure 29. Interestingly, after the second vaccine dose, the 30 μg WT RBD-hFc dimer group combined with MF59® showed lower antibody titers than the 10 μg WT RBD-hFc dimer group combined with MF59®. The reverse response was also observed in the mouse study.

[0354] Experiment 16 - Evaluation of immunogenicity and protective efficacy of RBD vaccine against beta variants in a hamster model Materials and Methods Hamsters were vaccinated with 30, 10 or 3 μg of WT RBD-hFc dimer combined with MF59®, or betaRBD-hFc combined with MF59®, administered intramuscularly twice on days 0 and 21. A third dose was administered on day 63, challenge was performed on day 85, and tissues were harvested on day 88 to measure viral load in the lungs and nasal turbinates of each hamster. Neutralizing antibody (nAb) titers were assessed as described elsewhere herein.

[0355] Results and Discussion Figure 30 shows the individual nAb titers for hamsters in each group. At this time point, 7 of 10 hamsters immunized with WT RBD-hFc dimer combined with MF59® had detectable nAb responses, and 15 of 30 mice immunized with WT RBD-hFc dimer combined with MF59® had detectable nAb responses. These nAb responses were measured against WT virus.

[0356] These hamsters were then challenged with either WT (WA-01 / USA) or beta mutant (B.1.351) SARS-CoV-2 virus on day 85, and oropharyngeal swabs were collected daily for 3 days post-challenge. Hamsters were sacrificed on day 88 for lung viral titer assessment. Data presented are day 3 swabs and viral loads per mg of lung tissue at the end of the experiment. Consistent with the moderate and highly variable nAb responses observed (Figure 30), immunized hamsters exhibited a moderate and variable degree of resistance to viral challenge (Figure 31). Upon WT SARS-CoV-2 virus challenge, viral loads in PBS (placebo)-treated hamsters in day 3 swabs were low (average ~80 PFU), below the detection limit of 5 PFU in one control hamster, and little room for obvious reduction in vaccinated hamsters. Nevertheless, the mean viral load in swabs on day 3 was lower (13 PFU) in hamsters immunized with 30 μg WT RBD-hFc combined with MF59®, with three hamsters having PFU below the limit of detection (5 PFU) and two hamsters having low but detectable virus. All hamsters treated with 10 μg WT RBD-hFc combined with MF59® had detectable virus, and the mean viral load in swabs on day 3 (42 PFU) was only moderately lower than in hamsters treated with PBS. Hamsters immunized with 30, 10, and 3 μg beta SARS-CoV-2 RBD-hFc combined with MF59® showed only moderate reductions in mean PFU (29, 18, 61 PFU) (Figure 31A).

[0357] Hamsters challenged with beta (B.1.351) SARS-CoV-2 virus had higher viral loads in the PBS-treated group (mean 1200 PFU) and better evidence of vaccine-induced protection in swabs on day 3. All hamsters immunized with beta RBD-hFc combined with MF59®, except for one that received the lowest 3 μg dose, had lower viral loads that did not overlap with the PBS group (mean PFU of 110, 32, and 250 for the 30, 10, and 3 μg treatment groups, respectively) (Figure 31A).

[0358] Results from cranial lung tissue samples were similar, with some notable differences from the day 3 swabs (Figure 31B). Thus, the 10μg WT RBD-hFc group combined with MF59® provided the best protection against WT (WA-01 / USA) SARS-CoV-2 challenge (9.1x10^4 vs. 9.2x10^6 PFU / 100mg tissue, PBS-treated group), with the 10μg and 3μg betaRBD-hFc groups combined with MF59® providing lower mean titers (2.7x10^5 and 1.1x10^6) than the 30μg betaRBD group combined with MF59® (1x10^7 PFU / 100mg tissue). In the betavirus challenge groups, the 10μg betaRBD-hFc immunized group with MF59® had the lowest mean PFU (9.4×10^5) compared to the PBS treated group which had a mean of 1.7×10^7 PFU / 100mg tissue. The group immunized with 30μg betaRBD-hFc with MF59® showed a greater spread, with three hamsters having much lower PFUs than the PBS group (over 100-fold) and two similar to the PBS group, with a mean PFU value of 2.3×10^5. 6 The lowest was the 3 μg betaRBD-hFc immunization group combined with MF59® (7.9 × 10 6 PFU) hamsters showed little evidence of protection.

[0359] Taken together, the RBD-hFc vaccine in combination with MF59® was shown to be immunogenic in hamster studies (Figures 29-30).

[0360] Experiment 17 - Immunogenicity of Beta-RBD Vaccine in a Rat Model Materials and Methods As part of the toxicology study, the immunogenicity of the betaRBD-hFc vaccine with or without MF59® adjuvant was evaluated in a rat model (Figure 32). Three groups of 30 Sprague-Dawley rats were vaccinated intramuscularly on days 0, 22, and 43 with saline, 50 μg betaRBD-hFc, or betaRBD-hFc combined with 50 μg MF59® adjuvant. At the end of toxicity on days 44-45 or 56, terminal blood samples were taken from each rat and processed into serum to assess WT SARS-CoV-2 spike-specific IgG antibody responses by ELISA.

[0361] Results and Discussion As shown in FIG. 32, the betaRBD-hFc vaccine administered with MF59® was highly immunogenic, and of 30 rats immunized with the betaRBD-hFc vaccine with MF59®, all but one rat showed a 10% immunization rate of 100%. 4 All rats immunized with betaRBD hFc without MF59® adjuvant showed spike-reactive IgG antibody titers of 100 or greater. One rat developed a lower antibody titer, but this was still clearly above background levels (titer 100) in saline-injected control animals. In contrast, of rats immunized with betaRBD hFc without MF59® adjuvant, only 3 of 30 produced detectable IgG anti-spike antibodies.

[0362] Experiment 18 - Evaluation of the stability of lyophilized RBD human Fc / PEG-R4-Pam-2-Cys vaccine formulation Methods and Materials We evaluated the stability of lyophilized RBD human Fc dimers formulated with PEG-R4-Pam-2-Cys. These formulations were stored at 40° C., room temperature (RT), 4° C. or −20° C. for one month before being reconstituted and used for intranasal vaccination of BALB / c mice. Responses to the stored lyophilized formulations were compared to responses elicited by freshly prepared RBD human Fc dimers formulated with PEG-R4-Pam-2-Cys and by lyophilized formulations reconstituted 24 hours after lyophilization.

[0363] Groups of five BALB / c mice were vaccinated intranasally twice with either freshly prepared vaccine, reconstituted 24 hours after lyophilization, or lyophilized vaccine reconstituted after 4 weeks of storage at 40°C, RT, 4°C, or -20°C. Vaccines were administered on days 0 and 30. Lyophilized vaccines for administration of the first dose were prepared 32 days prior to the experiment, and lyophilized vaccines for administration of the second dose were prepared on day 0 of the experiment. This allowed storage of both the first and second doses of vaccine for one month under the specified conditions. Mice were bled immediately before the first injection (prebleed), 21 days after the first immunization (1° bleed), and at various time points after the second immunization (2° bleed).

[0364] Humoral responses were assessed after the first and second immunizations, including anti-RBD antibody responses by ELISA, as well as assessment of SARS-CoV-2 neutralizing antibodies by in vitro microneutralization assay and surrogate virus neutralization test (GenScript, USA).

[0365] On day 112 of the study (82 days after the second dose), mice vaccinated with the freshly prepared RBD-hFc / PEG-R4-Pam-2-Cys formulation and mice vaccinated with the lyophilized RBD-hFc / PEG-R4-Pam-2-Cys formulation stored at 40°C for 1 month were challenged with VIC2089(D614G / N501Y).

[0366] Results and Discussion Total immunoglobulin (Ig) and neutralizing Ab responses to WT RBD monomer Primary and secondary WT RBD-specific antibody responses were evaluated in the serum of mice vaccinated with either the freshly prepared PEG-R4-Pam-2-Cys / RBD-hFc formulation or the lyophilized PEG-R4-Pam-2-Cys / RBD-hFc formulation. All mice administered the lyophilized formulation stored under different conditions showed strong antibody titers that were comparable to those generated in mice vaccinated with the freshly prepared formulation (Figure 33).

[0367] Similarly, when comparing neutralizing antibody responses in the serum of mice administered freshly prepared RBD-hFc in combination with PEG-R4-Pam-2-Cys with the neutralizing responses of animals vaccinated with RBD-hFc in combination with lyophilized PEG-R4-Pam-2-Cys formulations using both a microneutralization assay (FIG. 34A) and a surrogate virus neutralization test (FIG. 34B), comparable neutralizing responses were observed among all mice.

[0368] Defensive Efficiency Protective efficacy against upper and lower respiratory tract infection was evaluated in a murine SARS-CoV-2 challenge model using VIC2089 (N501Y / D614G). Mice vaccinated intranasally on days 0 and 30 with WT RBD-hFc+PEG-R4-Pam-2-Cys or lyophilized WT RBD-hFc+PEG-R4-Pam-2-Cys stored at 40°C for 1 month were aerosol-challenged with VIC2089 82 days after the second immunization. Age- and sex-matched unvaccinated control mice were also challenged at the same time. Mice were sacrificed 3 days after challenge and infectious virus titers (TCID50) were determined in the lungs and nasal turbinates of individual mice by titrating lung and nasal turbinate supernatants on Vero cell monolayers and measuring viral CPE 5 days later.

[0369] Complete protection against pulmonary infection was observed in all BALB / c mice vaccinated with either the freshly prepared vaccine or the lyophilized PEG-R4-Pam-2-Cys / RBD-mFc vaccine stored at 40° C. (FIG. 35A). Furthermore, no virus was detected in the nasal turbinates of 4 / 5 mice that received the freshly prepared vaccine and 5 / 5 mice immunized with the lyophilized vaccine (FIG. 35B).

[0370] From these results, the inventors concluded that the PEG-R4-Pam-2-Cys+RBD-mFc formulation maintained its immunogenicity and protective efficacy when lyophilized and stored at temperatures ranging from -20°C to 40°C for up to one month.

[0371] Neutralizing activity against Wuhan indicator strain VIC01 and beta variant B1.351 Secondary sera (day 70) from mice vaccinated intranasally with freshly prepared WT RBD-hFc combined with PEG-R4-Pam-2-Cys or lyophilized WT RBD-hFc combined with PEG-R4-Pam-2-Cys stored at 40° C. were evaluated for their ability to neutralize both the Wuhan indicator strain VIC01 and the beta variant B.1.351 by microneutralization assay. As shown in FIG 36, both formulations elicited potent neutralizing activity against both the homologous Wuhan indicator strain VIC01 and the beta variant B.1.351 despite containing the Wuhan indicator strain RBD antigen.

Claims

1. A chimeric or fusion protein for inducing an immune response against coronavirus, comprising: the chimeric or fusion protein comprises a dimer of a receptor binding domain from the spike protein of a coronavirus linked to an Fc region of an antibody; or A chimeric or fusion protein and an adjuvant, wherein the chimeric or fusion protein comprises a dimer of a receptor binding domain derived from a coronavirus spike protein linked to a polypeptide comprising an Fc receptor binding domain.

2. 2. The chimeric or fusion protein of claim 1, wherein the receptor binding domain comprises an amino acid sequence derived from a WT, alpha, beta, gamma, kappa, or delta SARS-CoV-2 strain or variant, or any other strain or variant defined in Table 3.

3. 3. The chimeric or fusion protein of claim 1 or 2, wherein the receptor binding domain comprises an amino acid sequence derived from a WT SARS-CoV-2 strain.

4. 3. The chimeric or fusion protein of claim 1 or 2, wherein the receptor binding domain comprises an amino acid sequence derived from a beta SARS-CoV-2 strain.

5. The chimeric or fusion protein of any one of claims 1 to 4, wherein the receptor binding domain comprises an amino acid sequence from N334 to P527 (numbering based on the full-length spike protein).

6. The chimeric or fusion protein of any one of claims 1 to 4, wherein the receptor binding domain consists of an amino acid sequence from N334 to P527 (numbering based on the full-length spike protein).

7. 5. The chimeric or fusion protein of any one of claims 1 to 4, wherein the receptor binding domain derived from a coronavirus spike protein comprises, consists essentially of or consists of an amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 15 or 28.

8. 8. The chimeric or fusion protein of claim 7, wherein the amino acid sequence comprises one or more of the mutations shown in Figures 6 or 7 or Table 3.

9. 5. The chimeric or fusion protein according to any one of claims 1 to 4, wherein the receptor binding domain derived from coronavirus spike protein comprises, consists essentially of or consists of one or more amino acid sequences of either SEQ ID NO: 15 or 28 having 0-8 amino acid insertions, deletions, substitutions or additions (or a combination thereof), preferably 0-7, preferably 0-6, preferably 0-5, preferably 0-4, preferably 0-3, preferably 0-2, preferably 0-1 amino acid insertions, deletions, substitutions or additions (or a combination thereof), wherein the amino acid insertions, deletions, substitutions or additions (or a combination thereof) are located at the N-terminus and / or C-terminus.

10. A chimeric or fusion protein described in any one of claims 1 to 4, wherein the receptor binding domain derived from the coronavirus spike protein consists of one or more amino acid sequences of either SEQ ID NO: 15 or 28.

11. The chimeric or fusion protein according to any one of claims 1 to 10, wherein the Fc region of the antibody is an IgG Fc region.

12. The chimeric or fusion protein of claim 11 , wherein the IgG is IgG1.

13. 13. The chimeric or fusion protein of claim 11 or 12, wherein the IgG is human.

14. 14. The chimeric or fusion protein of any one of claims 1 to 13, wherein the Fc region of said chimeric or fusion protein comprises two heavy chain fragments, more preferably the CH2 and CH3 domains of said heavy chain.

15. 15. The chimeric or fusion protein of any one of claims 1 to 14, wherein the Fc region of the antibody comprises, consists essentially of or consists of an amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 16 or 18.

16. 16. The chimeric or fusion protein according to any one of claims 1 to 15, wherein the antibody Fc region comprises, consists essentially of or consists of the amino acid sequence of either one of SEQ ID NOs: 16 or 18, with 0-8 amino acid insertions, deletions, substitutions or additions (or a combination thereof), preferably 0-7, preferably 0-6, preferably 0-5, preferably 0-4, preferably 0-3, preferably 0-2, preferably 0-1 amino acid insertions, deletions, substitutions or additions (or a combination thereof).

17. 16. The chimeric or fusion protein of any one of claims 1 to 15, wherein the chimeric or fusion protein comprises, consists essentially of or consists of any one of the sequences set forth in SEQ ID NO: 3, 22 or 23.

18. A nucleic acid molecule encoding a chimeric or fusion protein according to any one of claims 1 to 17.

19. A nucleic acid molecule described in claim 18, comprising any one of the nucleic acid sequences set forth in SEQ ID NO: 1, 4, 29, 30, 31 or 32.

20. A chimeric or fusion protein according to any one of claims 1 to 17, or a nucleic acid molecule according to claim 18 or 19, and Adjuvants A composition comprising:

21. The composition of claim 20, wherein the adjuvant is a TLR2 agonist.

22. The composition of claim 21, wherein the TLR2 agonist is a Pam-2-Cys containing molecule such as PEG-4 Arginine (R4)-Pam-2-Cys (PEG-R4-Pam-2-Cys), or an NKT cell stimulator.

23. The composition of claim 20, wherein the adjuvant is selected from the group consisting of Pam-3-CSK4, PEG-R4-Pam-2-Cys, MALP-2, lipoteichoic acid, OspA, Porin, LcrV, lipomannan, lysophosphatidylserine, lipophosphoglycan (LPG), glycophosphatidylinositol (GPI), and zymosan.

24. The method according to claim 24, wherein the adjuvant is selected from the group consisting of poly-I:C, CpG, poly-ICLC, 1018 ISS, aluminum salts, Amplivax, AS15, B(C, CP-870,893, CpG7909, CyaA, dSLIM, GM-CSF, IC30, IC31, imiquimod, ImuFact IMP321, IS Patch, ISS, ISCOMATRIX, JuvImmune, LipoVac, MF59®, AddaVax™, monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA 206, Montanide ISA 21. The composition of claim 20, wherein the active ingredient is selected from the group consisting of V, Montanide ISA-51, OK-432, OM-174, OM-197-MP-EC, ONTAK, PEPTEL, Vector Systems, PLGA microparticles, resiquimod, SRL172, virosomes and other virus-like particles, YF-17D, VEGF trap, R848, β-glucan, Pam-3-Cys, and Aquila's QS21 stimulon.

25. The composition of claim 20, wherein the adjuvant comprises a metabolizable oil and an emulsifier.

26. The composition of claim 20, wherein the oil is MF59 (registered trademark).

27. A pharmaceutical composition for inducing an immune response in a subject against a coronavirus, comprising a chimeric or fusion protein described in any one of claims 1 to 17, a nucleic acid molecule described in claim 18 or 19, or a composition described in any one of claims 20 to 26.

28. A pharmaceutical composition for reducing or minimizing the severity of symptoms associated with infection by a coronavirus, comprising a chimeric or fusion protein according to any one of claims 1 to 17, a nucleic acid molecule according to claim 18 or 19, or a composition according to any one of claims 20 to 26.

29. A pharmaceutical composition for the treatment and / or prevention of (a) a disease associated with or caused by a coronavirus, or (b) a coronavirus infection, comprising a chimeric or fusion protein according to any one of claims 1 to 17, a nucleic acid molecule according to claim 18 or 19, or a composition according to any one of claims 20 to 26.

30. A pharmaceutical composition for inhibiting or reducing the amount of coronavirus particles in the lungs of an individual, comprising a chimeric or fusion protein according to any one of claims 1 to 17, a nucleic acid molecule according to claim 18 or 19, or a composition according to any one of claims 20 to 26.

31. A pharmaceutical composition described in any one of claims 27 to 30, wherein the composition is administered to the respiratory tract.

32. The pharmaceutical composition described in any one of claims 27 to 31, wherein the coronavirus is SARS-CoV.

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