CORONAVIRUS VACCINES COMPRISING A TLR9 AGONIST

Abstract

The present disclosure relates to immunogenic compositions comprising a severe acute respiratory syndrome coron-avirus 2 (SARS-CoV-2) antigen, and a toll-like receptor 9 (TLR9) agonist, such as an oligonucleotide comprising an unmethylated cytidine-phospho-guanosine (CpG) motif. The immunogenic compositions are suitable for stimulating an immune response against a SARS-CoV-2 in an individual in need thereof.

Claims

1. An immunogenic composition for stimulating an immune response against a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), comprising a SARS-CoV-2 antigen and a toll-like receptor 9 (TLR9) agonist, wherein the TLR9 agonist is an oligonucleotide of from 10 to 35 nucleotides in length comprising an unmethylated cytidine-phospho-guanosine (CpG) motif, and the SARS-CoV-2 antigen and the oligonucleotide are present in the immunogenic composition in amounts effective to stimulate an immune response against the SARS-CoV-2 antigen in a mammalian subject.

2. The composition of claim 1, wherein the oligonucleotide comprises the sequence TABLE-US-00003 5’-AACGTTCGAG-3’ (SEQ ID NO:3).

3. The composition of claim 1, wherein the oligonucleotide comprises the sequence of TABLE-US-00004 5’-TGACTGTGAA CGTTCGAGAT GA-3’ (SEQ ID NO: 1).

4. The composition of claim 1, wherein the oligonucleotide comprises a modified nucleoside, optionally wherein the modified nucleoside is selected from the group consisting of 2′-deoxy-7-deazaguanosine, 2′-deoxy-6-thioguanosine, arabinoguanosine, 2′-deoxy-2’substituted-arabinoguanosine, and 2′-O-substituted-arabinoguanosine.

5. The composition of claim 4, wherein the oligonucleotide comprises the sequence TABLE-US-00005 5’-TCG.sub.1AACG.sub.1TTCG.sub.1-3’ (SEQ ID NO:2) in which G.sub.1 is 2′-deoxy-7-deazaguanosine, optionally wherein the oligonucleotide comprises the sequence TABLE-US-00006 5′-TCG.sub.1AACG.sub.1TTCG.sub.1-X-G.sub.1CTTG.sub.1CAAG.sub.1CT-5′, and in which G.sub.1 is 2′-deoxy-7-deazaguanosine and X is glycerol (5′-SEQ ID NO:2-3′-X-3′-SEQ ID NO:2-5′).

6. The composition of claim 3, wherein the oligonucleotide comprises at least one phosphorothioate linkage, or wherein all nucleotide linkages are phosphorothioate linkages.

7. The composition of claim 6, wherein the oligonucleotide is a single-stranded oligodeoxynucleotide.

8. The composition of claim 7, wherein a 0.5 ml dose of the immunogenic composition comprises from about 750 to about 3000 .Math.g of the oligonucleotide, or wherein the immunogenic composition comprises about 750 .Math.g, about 1500 .Math.g, or about 3000 .Math.g of the oligonucleotide.

9. The composition of claim 8, wherein the SARS-CoV-2 antigen is an inactivated whole SARS-CoV-2.

10. The composition of claim 9, wherein the SARS-CoV-2 is inactivated by treatment with one or both of formalin and ultraviolet light.

11. The composition of claim 8, wherein the SARS-CoV-2 antigen comprises the receptor-binding domain (RBD) of the SARS-CoV-2 spike (S) protein.

12. The composition of claim 11, wherein the SARS-CoV-2 antigen comprises a truncated, recombinant S protein devoid of signal peptide, transmembrane, and cytoplasmic domains of a full length S protein.

13. The composition of claim 3, wherein the SARS-CoV-2 antigen further comprises one or more of the SARS-CoV-2 membrane (M) protein, nucleocapsid (N) protein, and envelope (E) protein.

14. The composition of any one of claims 1-13, further comprising an aluminum salt adjuvant.

15. The composition of claim 14, wherein the aluminum salt adjuvant comprises one or more of the group consisting of amorphous aluminum hydroxyphosphate sulfate, aluminum hydroxide, aluminum phosphate, and potassium aluminum sulfate.

16. The composition of claim 14, wherein the aluminum salt adjuvant comprises aluminum hydroxide.

17. The composition of claim 15, wherein a 0.5 ml dose of the immunogenic composition comprises from about 0.25 to about 0.50 mg Al.sup.3+.

18. The composition of claim 17, wherein the mammalian subject is a human subject.

19. A kit comprising: i) the immunogenic composition of claim 14, and ii) instructions for administration of the composition to stimulate an immune response against the SARS-CoV-2 antigen in the mammalian subject.

20. The kit of claim 19, further comprising iii) a syringe and needle for intramuscular injection of the immunogenic composition.

21. A method for stimulating an immune response against a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in a mammalian subject, comprising administering the immunogenic composition of claim 14 to a mammalian subject so as to stimulate an immune response against the SARS-CoV-2 antigen in the mammalian subject.

22. The method of claim 21, wherein the mammalian subject is a human subject and/or the immunogenic composition is administered by intramuscular injection.

23. Use of the immunogenic composition of claim 14 for stimulating an immune response against a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in a mammalian subject, the method comprising administering to the subject an effective amount of the immunogenic composition.

24. Use of the immunogenic composition of claim 14 for protecting a mammalian subject from infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the method comprising administering to the subject an effective amount of the immunogenic composition.

25. Use of the immunogenic composition of claim 14 for preventing a mammalian subject from contracting COVID-19 disease, the method comprising administering to the subject an effective amount of the immunogenic composition.

26. The use of any one of claims 23-25, wherein the mammalian subject is a human subject and/or the immunogenic composition is administered by intramuscular injection.

Description

DETAILED DESCRIPTION

[0022] The present disclosure relates to immunogenic compositions comprising a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antigen and a toll-like receptor 9 (TLR9) agonist, such as an oligonucleotide comprising an unmethylated cytidine-phospho-guanosine (CpG) motif. The immunogenic compositions are suitable for stimulating an immune response against a SARS-CoV-2 in an individual in need thereof.

I. Immunogenic Compositions and Kits

[0023] The present disclosure relates to immunogenic compositions for stimulating an immune response against a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), comprising a SARS-CoV-2 antigen and a toll-like receptor 9 (TLR9) agonist, wherein the TLR9 agonist is an oligonucleotide of from 8 to 35 nucleotides in length comprising an unmethylated cytidine-phospho-guanosine (also referred to as CpG or cytosine-phosphate-guanosine) motif, and the SARS-CoV-2 antigen and the oligonucleotide are present in the immunogenic composition in amounts effective to stimulate an immune response against the SARS-CoV-2 antigen in a mammalian subject, such as a human subject in need thereof.

A. Toll-Like Receptor 9 (TLR9) Agonists

[0024] Toll-like receptors (TLRs) are expressed on dendritic cells and other innate immune cells and are among the most important receptors for stimulating a response to the presence of invading pathogens. Humans have multiple types of TLRs that are similar in structure but recognize different parts of viruses or bacteria. By activating specific TLRs, it is possible to stimulate and control specific types of innate immune responses that can be harnessed to enhance adaptive responses.

[0025] TLR9 (CD289) recognizes unmethylated cytidine-phospho-guanosine (CpG) motifs found in microbial DNA, which can be mimicked using synthetic CpG-containing oligodeoxynucleotides (CpG-ODNs). CpG-ODNs are known to enhance antibody production and to stimulate T helper 1 (Th1) cell responses (Coffman et al., Immunity, 33:492-503, 2010). Based on structure and biological function, CpG-ODNs have been divided into three general classes: CpG-A, CpG-B, and CpG-C (Campbell, Methods Mol Biol, 1494:15-27, 2017). The degree of B cell activation varies between the classes with CpG-A ODNs being weak, CpG-C ODNs being good, and CpG-B ODNs being strong B cell activators. Oligonucleotide TLR9 agonists of the present disclosure are preferably good B cell activators (CpG-C ODN) or more preferably strong (CpG-B ODN) B cell activators.

[0026] Optimal oligonucleotide TLR9 agonists often contain a palindromic sequence following the general formula of: 5′-purine-purine-CG-pyrimidine-pyrimidine-3′, or 5′-purine-purine-CG-pyrimidine-pyrimidine-CG-3′ (U.S. Pat. No. 6,589,940). TLR9 agonism is also observed with certain non-palindromic CpG-enriched phosphorothioate oligonucleotides, but may be affected by changes in the nucleotide sequence. Additionally, TLR9 agonism is abolished by methylation of the cytosine within the CpG dinucleotide. Accordingly in some embodiments, the TLR9 agonist is an oligonucleotide of from 8 to 35 nucleotides in length comprising the sequence 5′-AACGTTCG-3′. In some embodiments, the oligonucleotide is greater than 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length, and the oligonucleotide is less than 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, or 24 nucleotides in length. In some embodiments, the TLR9 agonist is an oligonucleotide of from 10 to 35 nucleotides in length comprising the sequence 5′-AACGTTCGAG-3′ (SEQ ID NO:3). In some embodiments, the oligonucleotide is greater than 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length, and the oligonucleotide is less than 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, or 24 nucleotides in length.

[0027] Researchers at Dynavax Technologies Corporation (Emeryville, CA) have identified a 22-mer phosphorothioate linked oligodeoxynucleotide, CpG 1018, which contains specific sequences that can substantially enhance the immune response to co-administered antigens across species (Campbell, Methods Mol Biol, 1494:15-27, 2017). CpG 1018 (5′-TGACTGTGAA CGTTCGAGAT GA-3′, set forth as SEQ ID NO:1) was chosen after screening a broad panel of oligonucleotides for immunostimulatory activity in vitro and in vivo. CpG 1018 is a CpG-B ODN that is active in mice, rabbits, dogs, baboons, cynomolgus monkeys, and humans. Thus in some preferred embodiments, the TLR9 agonist is an oligonucleotide comprising the sequence of SEQ ID NO:1.

[0028] Although the exemplary oligonucleotide TLR9 agonist, CpG 1018, is a CpG-ODN, the present disclosure is not restricted to fully DNA molecules. That is, in some embodiments, the TLR9 agonist is a DNA/RNA chimeric molecule in which the CpG(s) and the palindromic sequence are deoxyribonucleic acids and one or more nucleic acids outside of these regions are ribonucleic acids. In some embodiments, the CpG oligonucleotide is linear. In other embodiments, the CpG oligonucleotide is circular or includes hairpin loop(s). The CpG oligonucleotide may be single stranded or double stranded.

[0029] In some embodiments, the CpG oligonucleotide may contain modifications. Modifications include but are not limited to, modifications of the 3′OH or 5′OH group, modifications of the nucleotide base, modifications of the sugar component, and modifications of the phosphate group. Modified bases may be included in the palindromic sequence of the CpG oligonucleotide as long as the modified base(s) maintains the same specificity for its natural complement through Watson-Crick base pairing (e.g., the palindromic portion is still self-complementary). In some embodiments, the CpG oligonucleotide comprises a non-canonical base. In some embodiments, the CpG oligonucleotide comprises a modified nucleoside. In some embodiments, the modified nucleoside is selected from the group consisting of 2′-deoxy-7-deazaguanosine, 2′-deoxy-6-thioguanosine, arabinoguanosine, 2′-deoxy-2′substituted-arabinoguanosine, and 2′-O-substituted-arabinoguanosine. In some embodiments, the TLR9 agonist is an oligonucleotide comprising the sequence 5′-TCG.sub.1AACG.sub.1TTCG.sub.1-3′ (SEQ ID NO:2), in which G.sub.1 is 2′-deoxy-7-deazaguanosine. In some embodiments, the oligonucleotide comprises the sequence 5′-TCG.sub.1AACG.sub.1TTCG.sub.1-X-G.sub.1CTTG.sub.1CAAG.sub.1CT-5′, and in which G.sub.1 is 2′-deoxy-7-deazaguanosine and X is glycerol (5′-SEQ ID NO:2-3′-X-3′-SEQ ID NO:2-5′).

[0030] The CpG oligonucleotide may contain a modification of the phosphate group. For example, in addition to phosphodiester linkages, phosphate modifications include, but are not limited to, methyl phosphonate, phosphorothioate, phosphoramidate (bridging or non-bridging), phosphotriester and phosphorodithioate and may be used in any combination. Other non-phosphate linkages may also be used. In some embodiments, the oligonucleotides comprise only phosphorothioate backbones. In some embodiments, the oligonucleotides comprise only phosphodiester backbones. In some embodiments, the oligonucleotide comprises a combination of phosphate linkages in the phosphate backbone such as a combination of phosphodiester and phosphorothioate linkages. Oligonucleotides with phosphorothioate backbones can be more immunogenic than those with phosphodiester backbones and appear to be more resistant to degradation after injection into the host (Braun et al., J Immunol, 141:2084-2089, 1988; and Latimer et al., Mol Immunol, 32:1057-1064, 1995). The CpG oligonucleotides of the present disclosure include at least one, two or three internucleotide phosphorothioate ester linkages. In some embodiments, when a plurality of CpG oligonucleotide molecules are present in a pharmaceutical composition comprising at least one excipient, both stereoisomers of the phosphorothioate ester linkage are present in the plurality of CpG oligonucleotide molecules. In some embodiments, all of the internucleotide linkages of the CpG oligonucleotide are phosphorothioate linkages, or said another way, the CpG oligonucleotide has a phosphorothioate backbone.

[0031] A unit dose of the immunogenic composition, which is typically a 0.5 ml dose, may comprises from about 500 .Math.g to about 5000 .Math.g of the CpG oligonucleotide, preferably from about 750 .Math.g to about 3000 .Math.g of the CpG oligonucleotide. In some embodiments, a 0.5 ml dose of the immunogenic composition comprises greater than about 500, 750, 1000, or 1250 .Math.g of the CpG oligonucleotide, and less than about 3250, 3000, 2750, 2500, 2250, 2000, or 1750 .Math.g of the CpG oligonucleotide. In some embodiments, a 0.5 ml dose of the immunogenic composition comprises about 750, 1500, or 3000 .Math.g of the CpG oligonucleotide. In some embodiments, a 0.5 ml dose of the immunogenic composition comprises about 250 .Math.g of the CpG oligonucleotide. In some embodiments, a 0.5 ml dose of the immunogenic composition comprises about 500 .Math.g of the CpG oligonucleotide. In some embodiments, a 0.5 ml dose of the immunogenic composition comprises about 750 .Math.g of the CpG oligonucleotide. In some embodiments, a 0.5 ml dose of the immunogenic composition comprises about 1000 .Math.g of the CpG oligonucleotide. In some embodiments, a 0.5 ml dose of the immunogenic composition comprises about 1500 .Math.g of the CpG oligonucleotide. In some embodiments, a 0.5 ml dose of the immunogenic composition comprises about 3000 .Math.g of the CpG oligonucleotide.

[0032] The CpG oligonucleotides described herein are in their pharmaceutically acceptable salt form unless otherwise indicated. Exemplary basic salts include ammonium salts, alkali metal salts such as sodium, lithium, and potassium salts, alkaline earth metal salts such as calcium and magnesium salts, zinc salts, salts with organic bases (for example, organic amines) such as N-Me-D-glucamine, N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride, choline, tromethamine, dicyclohexylamines, t-butyl amines, and salts with amino acids such as arginine, lysine and the like. In some embodiment, the CpG oligonucleotides are in the ammonium, sodium, lithium, or potassium salt form. In one preferred embodiment, the CpG oligonucleotides are in the sodium salt form.

B. SARS-CoV-2 Antigens

[0033] A SARS-CoV-2 antigen of the immunogenic compositions of the present disclosure comprises at least one SARS-CoV-2 protein or fragment thereof. In preferred embodiments, the SARS-CoV-2 antigen is recognized by SARS-CoV-2 reactive antibodies and/or T cells. In some embodiments, the SARS-CoV-2 antigen is an inactivated whole virus (COVID-19 virus). In other embodiments, the SARS-CoV-2 antigen is a subunit of the virus. In some embodiments, the SARS-CoV-2 antigen comprises a structural protein of SARS-CoV-2 or a fragment thereof. In some embodiments, the structural protein of SARS-CoV-2 comprises one or more of the group consisting of the spike (S) protein, the membrane (M) protein, nucleocapsid (N) protein, and envelope (E) protein. In some embodiments, the SARS-CoV-2 antigen comprises or further comprises a non-structural protein of SARS-CoV-2 or a fragment thereof. The nucleotide sequence of a representative SARS-CoV-2 isolate (Wuhan-Hu-1) is set forth as GenBank No. MN908947.3 (Wu et al., Nature, 579:265-269, 2020).

[0034] The amino acid sequence of a SARS-CoV-2 S protein is set forth as SEQ ID NO:4:

TABLE-US-00001 MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHS TQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNI IRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNK SWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGY FKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLT PGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETK CTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASV YAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSF VIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYN YLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPT NGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTG VLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITP GTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCL IGAEHVNNSYECDIPIGAGICASYQTQTNSPRRARSVASQSIIAYTMSLG AENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECS NLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGF NFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLI CAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAM QMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQD VVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGR LQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLM SFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGT HWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKE ELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDL QELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSC GSCCKFDEDDSEPVLKGVKLHYT. The signal peptide extend s from residues 1-13, the extracellular region

extends from residues 14-1213, the transmembrane domain extends from residues 1214-1236, and the cytoplasmic domain extends from residues 1237-1273.

[0035] In some preferred embodiments, the SARS-CoV-2 antigen comprises the receptor-binding domain (RBD) of the S protein, which is set forth as SEQ ID NO:5:

TABLE-US-00002 NSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGF NCYFPLQSYGFQPTNGVGYQPYR.

In some embodiments, the SARS-CoV-2 antigen comprises a variant of the RBD of the S protein having an amino acid sequence that it at least 75%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:5. In some preferred embodiments, the SARS-CoV-2 antigen comprises the extracellular region of the S protein extending from residues 14-1213 of SEQ ID NO:4, or an amino acid sequence that it at least 75%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to residues 14-1213 of SEQ ID NO:4. That is, in some embodiments, the SARS-CoV-2 antigen comprises a truncated, S protein devoid of signal peptide, transmembrane and cytoplasmic domains of a full length S protein. In some embodiments, the SARS-CoV-2 antigen is a recombinant protein, while in other embodiments, the SARS-CoV-2 antigen is purified from virions. In some preferred embodiments, the SARS-CoV-2 antigen is an isolated antigen.

[0036] A unit dose of the immunogenic composition, which is typically a 0.5 ml dose, may comprise from about 10 .Math.g to about 100 .Math.g of the SARS-CoV-2 antigen, preferably from about 25 .Math.g to about 75 .Math.g of the SARS-CoV-2 antigen, preferably from about 40 .Math.g to about 60 .Math.g of the SARS-CoV-2 antigen, or about 50 .Math.g of the SARS-CoV-2 antigen. In some embodiments, the dose contains from about 3 .Math.g to about 30 .Math.g of the SARS-CoV-2 antigen, or about 3 .Math.g, or about 9 .Math.g, or about 30 .Math.g of the SARS-CoV-2 antigen. In some embodiments, the dose contains from about 5 .Math.g to about 25 .Math.g of the SARS-CoV-2 antigen, or about 5 .Math.g, or about 15 .Math.g, or about 25 .Math.g of the SARS-CoV-2 antigen. In some embodiments, the dose contains from about 0.25 .Math.g to about 25 .Math.g of the SARS-CoV-2 S antigen. In some embodiments, the dose contains from about 15 .Math.g to about 50 .Math.g of the SARS-CoV-2 antigen, or about 15 .Math.g, or about 25 .Math.g, or about 50 .Math.g of the SARS-CoV-2 antigen.

C. Additional Components

[0037] The immunogenic compositions of the present disclosure may comprise one or more additional components, such as one or more excipients, another adjuvant, and/or additional antigens.

1. Excipients

[0038] Pharmaceutically acceptable excipients of the present disclosure include for instance, solvents, bulking agents, buffering agents, tonicity adjusting agents, and preservatives (Pramanick et al., Pharma Times, 45:65-77, 2013). In some embodiments the immunogenic compositions may comprise an excipient that functions as one or more of a solvent, a bulking agent, a buffering agent, and a tonicity adjusting agent (e.g., sodium chloride in saline may serve as both an aqueous vehicle and a tonicity adjusting agent).

[0039] In some embodiments, the immunogenic compositions comprise an aqueous vehicle as a solvent. Suitable vehicles include for instance sterile water, saline solution, phosphate buffered saline, and Ringer’s solution. In some embodiments, the composition is isotonic.

[0040] The immunogenic compositions may comprise a buffering agent. Buffering agents control pH to inhibit degradation of the active agent during processing, storage and optionally reconstitution. Suitable buffers include for instance salts comprising acetate, citrate, phosphate or sulfate. Other suitable buffers include for instance amino acids such as arginine, glycine, histidine, and lysine. The buffering agent may further comprise hydrochloric acid or sodium hydroxide. In some embodiments, the buffering agent maintains the pH of the composition within a range of 6 to 9. In some embodiments, the pH is greater than (lower limit) 6, 7 or 8. In some embodiments, the pH is less than (upper limit) 9, 8, or 7. That is, the pH is in the range of from about 6 to 9 in which the lower limit is less than the upper limit.

[0041] The immunogenic compositions may comprise a tonicity adjusting agent. Suitable tonicity adjusting agents include for instance dextrose, glycerol, sodium chloride, glycerin and mannitol.

[0042] The immunogenic compositions may comprise a bulking agent. Bulking agents are particularly useful when the pharmaceutical composition is to be lyophilized before administration. In some embodiments, the bulking agent is a protectant that aids in the stabilization and prevention of degradation of the active agents during freeze or spray drying and/or during storage. Suitable bulking agents are sugars (mono-, di- and polysaccharides) such as sucrose, lactose, trehalose, mannitol, sorbital, glucose and raffinose.

[0043] The immunogenic compositions may comprise a preservative. Suitable preservatives include for instance antioxidants and antimicrobial agents. However, in preferred embodiments, the immunogenic composition is prepared under sterile conditions and is in a single use container, and thus does not necessitate inclusion of a preservative.

2. Additional Adjuvants

[0044] Adjuvants are known in the art and include, but are not limited to, alum (aluminum salts), oil-in-water emulsions, water-in-oil emulsions, liposomes, and microparticles, such as poly(lactide-co-glycolide) microparticles (Shah et al., Methods Mol Biol, 1494:1-14, 2017). In some embodiments, the immunogenic compositions further comprises an aluminum salt adjuvant to which the SARS-CoV-2 antigen is adsorbed. In some embodiments, the aluminum salt adjuvant comprises one or more of the group consisting of amorphous aluminum hydroxyphosphate sulfate, aluminum hydroxide, aluminum phosphate, and potassium aluminum sulfate. In some embodiments, the aluminum salt adjuvant comprises one or both of aluminum hydroxide and aluminum phosphate. In some embodiments, the aluminum salt adjuvant consists of aluminum hydroxide. In some embodiments, a unit dose of the immunogenic composition, which is typically a 0.5 ml dose, comprises from about 0.25 to about 0.50 mg Al.sup.3+, or about 0.35 mg Al.sup.3+, or about 0.375 mg Al.sup.3+. In some embodiments, a 0.5 ml unit dose of the immunogenic composition comprises from about 0.05 to about 0.25 mg Al.sup.3+. In some embodiments, a 0.5 ml dose of the immunogenic composition comprises greater than about 0.050, 0.075, 0.100, 0.125, 0.150, 0.175, 0.200, 0.225, or 0.250 mg Al.sup.3+, and less than about 0.50, 0.45, 0.40, 0.35, 0.30 or 0.25 mg Al.sup.3+, provided that the minimum is lower than the maximum.

[0045] In other embodiments, the immunogenic composition further comprises an additional adjuvant. Other suitable adjuvants include, but are not limited to, squalene-in-water emulsion (e.g., MF59 or AS03), TLR3 agonists (e.g., poly-IC or poly-ICLC), TLR4 agonists (e.g., bacterial lipopolysaccharide derivatives such monophosphoryl lipid A (MPL), and/or a saponin such as Quil A or QS-21, as in AS01 or AS02), a TLR5 agonist (bacterial flagellin), and TLR7 and/or TLR8 agonists (imidazoquinoline derivatives such as imiquimod, and resiquimod)(Coffman et al., Immunity, 33:492-503, 2010). In some embodiments, the additional adjuvant comprises MPL and alum (e.g., AS04). For veterinary use and for production of antibodies in non-human animals, mitogenic components of Freund’s adjuvant (both complete and incomplete) can be used.

D. Kits

[0046] The present disclosure also provides kits comprising: i) an immunogenic composition comprising a SARS-CoV-2 antigen and a toll-like receptor 9 (TLR9) agonist, such as a CpG oligonucleotide; and ii) a set of instructions for administration of the immunogenic composition to stimulate an immune response against the SARS-CoV-2 antigen in a mammalian subject, such as a human subject in need thereof. Additionally, the present disclosure provides kits comprising: i) a first composition comprising a SARS-CoV-2 antigen; ii) a second composition comprising a TLR9 agonist, such as a CpG oligonucleotide; iii) instructions for mixing the first composition with the second composition to prepare an immunogenic composition; and optionally iv) a further set of instructions for administration of the immunogenic composition to stimulate an immune response against the SARS-CoV-2 antigen in a mammalian subject, such as a human subject in need thereof. In some embodiments, the CpG oligonucleotide comprises the sequence 5′-AACGTTCG-3′. In some embodiments, the CpG oligonucleotide comprises the sequence 5′-AACGTTCGAG-3′ (SEQ ID NO:3). In some preferred embodiments, the CpG oligonucleotide comprises the sequence of 5′-TGACTGTGAA CGTTCGAGAT GA-3′ (SEQ ID NO: 1).

[0047] The kits may comprise an immunogenic composition packaged appropriately. For example, if the immunogenic composition is a freeze-dried power, a vial with a resilient stopper is normally used so that the powder may be easily resuspended by injecting fluid (e.g., sterile water, saline, etc.) through the resilient stopper. In some embodiments, the kits comprise a device for administration (e.g., syringe and needle for intramuscular injection). The instructions relating to the use of the immunogenic composition generally include information as to dosage, schedule and route of administration for the intended methods of use.

II. Methods Of Use

[0048] The present disclosure relates to methods for stimulating an immune responses against a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), comprising: administering an immunogenic composition comprising a SARS-CoV-2 antigen and a toll-like receptor 9 (TLR9) agonist, such as a CpG oligonucleotide, to a mammalian subject so as to stimulate an immune response against the SARS-CoV-2 antigen in the mammalian subject. The immunogenic compositions of the present disclosure are intended for active immunization against COVID-19. In preferred embodiments, the immunogenic compositions are to be administered by intramuscular injection, optionally in a volume of about 0.5 mL (e.g., unit dose). In some embodiments, the intramuscular injection is into the deltoid muscle of the upper arm of a human subject in need thereof. In some embodiments, one dose of the immunogenic composition is administered. In other embodiments, a first dose and a second dose of the immunogenic composition are administered, with the second dose administered from about 2 weeks to 8 weeks after the first dose, or from about 3 weeks to 6 weeks after the first dose, or about 4 weeks after the first dose. In some preferred embodiments, the second dose is administered about 3 or 4 weeks after the first dose. In some embodiments, the second dose is administered about 3 weeks after the first dose. In some embodiments, the second dose is administered about 4 weeks after the first dose.

[0049] “Stimulating” an immune response, means increasing the immune response, which can arise from eliciting a de novo immune response (e.g., as a consequence of an initial vaccination regimen) or enhancing an existing immune response (e.g., as a consequence of a booster vaccination regimen). In some embodiments, stimulating an immune response includes but is not limited to one or more of the group consisting of: stimulating cytokine production; stimulating B lymphocyte proliferation; stimulating antibody production; stimulating interferon pathway-associated gene expression; stimulating chemoattractant-associated gene expression; and stimulating plasmacytoid dendritic cell (pDC) maturation. In some preferred embodiments, stimulating an immune response comprises increasing an antigen-specific antibody response in the subject.

ENUMERATED EMBODIMENTS

[0050] Embodiment 1. An immunogenic composition for stimulating an immune response against a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), comprising a SARS-CoV-2 antigen and a toll-like receptor 9 (TLR9) agonist, wherein the TLR9 agonist is an oligonucleotide of from 10 to 35 nucleotides in length comprising an unmethylated cytidine-phospho-guanosine (CpG) motif, and the SARS-CoV-2 antigen and the oligonucleotide are present in the immunogenic composition in amounts effective to stimulate an immune response against the SARS-CoV-2 antigen in a mammalian subject.

[0051] Embodiment 2. The composition of embodiment 1, wherein the oligonucleotide comprises the sequence 5′-AACGTTCGAG-3′ (SEQ ID NO:3).

[0052] Embodiment 3. The composition of embodiment 1, wherein the oligonucleotide comprises the sequence of 5′-TGACTGTGAA CGTTCGAGAT GA-3′ (SEQ ID NO: 1).

[0053] Embodiment 4. The composition of any one of embodiments 1-3, wherein the oligonucleotide comprises a modified nucleoside, optionally wherein the modified nucleoside is selected from the group consisting of 2′-deoxy-7-deazaguanosine, 2′-deoxy-6-thioguanosine, arabinoguanosine, 2′-deoxy-2′substituted-arabinoguanosine, and 2′-O-substituted-arabinoguanosine.

[0054] Embodiment 5. The composition of embodiment 4, wherein the oligonucleotide comprises the sequence 5′-TCG.sub.1AACG.sub.1TTCG.sub.1-3′ (SEQ ID NO:2) in which G.sub.1 is 2′-deoxy-7-deazaguanosine, optionally wherein the oligonucleotide comprises the sequence 5′-TCG.sub.1AACG.sub.1TTCG.sub.1-X-G.sub.1CTTG.sub.1CAAG.sub.1CT-5′, and in which G.sub.1 is 2′-deoxy-7-deazaguanosine and X is glycerol (5′-SEQ ID NO:2-3′-X-3′-SEQ ID NO:2-5′).

[0055] Embodiment 6. The composition of any one of embodiments 1-5, wherein the oligonucleotide comprises at least one phosphorothioate linkage, or wherein all nucleotide linkages are phosphorothioate linkages.

[0056] Embodiment 7. The composition of any one of embodiments 1-6, wherein the oligonucleotide is a single-stranded oligodeoxynucleotide.

[0057] Embodiment 8. The composition of any one of embodiments 1-7, wherein a 0.5 ml dose of the immunogenic composition comprises from about 750 to about 3000 .Math.g of the oligonucleotide, or wherein the immunogenic composition comprises about 750 .Math.g, about 1000 .Math.g, about 1500 .Math.g, or about 3000 .Math.g of the oligonucleotide.

[0058] Embodiment 9. The composition of any one of embodiments 1-8, wherein the SARS-CoV-2 antigen is an inactivated whole SARS-CoV-2.

[0059] Embodiment 10. The composition of embodiment 9, wherein the SARS-CoV-2 is inactivated by treatment with one or both of formalin and ultraviolet light.

[0060] Embodiment 11. The composition of any one of embodiments 1-8, wherein the SARS-CoV-2 antigen comprises the receptor-binding domain (RBD) of the SARS-CoV-2 spike (S) protein.

[0061] Embodiment 12. The composition of embodiment 11, wherein the SARS-CoV-2 antigen comprises a truncated, recombinant S protein devoid of signal peptide, transmembrane and cytoplasmic domains of a full length S protein.

[0062] Embodiment 13. The composition of embodiment 11 or 12, wherein the SARS-CoV-2 antigen further comprises one or more of the SARS-CoV-2 membrane (M) protein, nucleocapsid (N) protein, and envelope (E) protein.

[0063] Embodiment 14. The composition of any one of embodiments 1-13, further comprising an aluminum salt adjuvant.

[0064] Embodiment 15. The composition of embodiment 14, wherein the aluminum salt adjuvant comprises one or more of the group consisting of amorphous aluminum hydroxyphosphate sulfate, aluminum hydroxide, aluminum phosphate, and potassium aluminum sulfate

[0065] Embodiment 16. The composition of embodiment 14, wherein the aluminum salt adjuvant comprises aluminum hydroxide.

[0066] Embodiment 17. The composition of any one of embodiments 14-16, wherein a 0.5 ml dose of the immunogenic composition comprises from about 0.25 to about 0.50 mg Al.sup.3+, or about 0.250 mg Al.sup.3+, or about 0.375 mg Al.sup.3+, or about 0.05 to about 0.50 mg Al.sup.3+, or about 0.075 to about 0.175 mg Al.sup.3+.

[0067] Embodiment 18. The composition of any one of embodiments 1-17, wherein the mammalian subject is a human subject.

[0068] Embodiment 19. A kit comprising: [0069] i) the immunogenic composition of any one of embodiments 1-18, and [0070] ii) instructions for administration of the composition to stimulate an immune response against the SARS-CoV-2 antigen in the mammalian subject.

[0071] Embodiment 20. The kit of embodiment 19, further comprising iii) a syringe and needle for intramuscular injection of the immunogenic composition.

[0072] Embodiment 21. A method for stimulating an immune response against a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in a mammalian subject, comprising administering the immunogenic composition of any one of embodiments 1-18 to a mammalian subject in an amount effective to stimulate an immune response against the SARS-CoV-2 antigen in the mammalian subject.

[0073] Embodiment 22. The method of embodiment 21, wherein the mammalian subject is a human subject and/or the immunogenic composition is administered by intramuscular injection.

[0074] Embodiment 23. Use of the immunogenic composition of any one of embodiments 1-18 for stimulating an immune response against a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in a mammalian subject, the method comprising administering to the subject an effective amount of the immunogenic composition.

[0075] Embodiment 24. Use of the immunogenic composition of any one of embodiments 1-18 for protecting a mammalian subject from infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the method comprising administering to the subject an effective amount of the immunogenic composition.

[0076] Embodiment 25. Use of the immunogenic composition of any one of embodiments 1-18 for preventing a mammalian subject from contracting COVID-19 disease, the method comprising administering to the subject an effective amount of the immunogenic composition.

[0077] Embodiment 26. The use of any one of embodiments 23-25, wherein the mammalian subject is a human subject and/or the immunogenic composition is administered by intramuscular injection.