Anti-coronavirus vaccine

Abstract

The present invention provides stable coronavirus spike proteins, immunogenic compositions containing the same, and methods of using these immunogenic compositions are also provided.

Description

[Technical Field]

[0001] The present invention relates to recombinant coronavirus spike protein and immunogenic compositions containing the same.

[0002] Sequence List This application includes a sequence listing submitted via EFS-Web as an ASCII-compliant text file (.txt), the entire listing of which is incorporated herein by reference. The ASCII file was created on 7 October 2020, is named "Sequence_Listing_000360_ST25", and has a size of 37,849 bytes. This sequence listing serves as a sequence listing required under U.S. Patent Code Section 1.821(c) and a paper copy in computer-readable format (CRF) required under U.S. Patent Code Section 1.821(e). No statement under U.S. Patent Code Section 1.821(f) is required. [Background technology]

[0003] Coronaviruses are a large group of viruses that can cause illnesses ranging in severity. The first known serious illness caused by a coronavirus emerged in China in 2003 with the outbreak of Severe Acute Respiratory Syndrome (SARS). In Saudi Arabia, a second outbreak of severe illness, Middle East Respiratory Syndrome (MERS), began in 2012.

[0004] On December 31, 2019, Chinese authorities warned the World Health Organization of the outbreak of a novel coronavirus causing severe illness, which was subsequently named SARS-CoV-2. SARS-CoV-2 is the virus that causes the disease known as COVID-19. As of July 16, 2020, approximately 13.6 million cases of COVID-19 had been reported worldwide, but many more mild cases may have gone undiagnosed. The virus has killed more than 585,000 people.

[0005] Shortly after the outbreak began, Chinese scientists sequenced the SARS-CoV-2 genome and provided the data to researchers worldwide. The number of COVID-19 cases is increasing due to human-to-human transmission following a single human introduction.

[0006] The SARS-CoV-2 spike protein is located on the outside of the virus. This virus uses its spike protein to grasp and penetrate the outer walls of human and animal cells. Scientists are focusing on two characteristic features of the SARS-CoV-2 spike protein: the receptor-binding domain (RBD) portion that binds to cells, and the cleavage site that opens the virus and allows it to enter host cells. The S1 and S2 subunits of the spike protein are involved in receptor recognition and membrane fusion, respectively.

[0007] Scientists are still learning about the virus, but it is thought that it may, in some cases, spread from humans to animals, especially after close contact with a person infected with COVID-19.

[0008] Based on information available on the Centers for Disease Control and Prevention (CDC) website as of June 22, 2020, it is known that cats, dogs, and several other types of animals can be infected with SARS-CoV-2, but not all potentially infected animals are yet known. Cases of animals being infected with this virus have been reported worldwide.

[0009] In several countries, including the United States, a small number of pet cats and dogs have been reported to be infected with the virus. Most of these pets became ill after coming into contact with a human infected with COVID-19. Several lions and tigers at the New York Zoo were diagnosed with SARS-CoV-2 after showing signs of respiratory illness. Public health officials believe these large cats became ill after coming into contact with a zoo employee who was infected with SARS-CoV-2.

[0010] SARS-CoV-2 was recently discovered in mink (a close relative of ferrets) on several farms in the Netherlands. The mink showed signs of respiratory and gastrointestinal problems, and there was also an increase in mink deaths on the farms. Since some workers on these farms had shown symptoms of COVID-19, it is likely that infected farm workers were the source of the mink infection. Some cats on several mink farms have developed antibodies against the virus, suggesting that the cats were exposed to the virus at some point.

[0011] The CDC, the U.S. Department of Agriculture (USDA), and state public health and animal health authorities are conducting active surveillance for SARS-CoV-2 in pets, including cats, dogs, and other small mammals, that have come into contact with individuals infected with COVID-19 in some states. These animals are being tested for SARS-CoV-2 infection and to determine whether the pet will develop antibodies against the virus. This research is being conducted to better understand the likelihood of SARS-CoV-2 infection in pets and the potential role pets may play in the spread of the virus. The U.S. Department of Agriculture maintains a list of confirmed cases of SAR-CoV-2 (the same virus that causes COVID-19 in humans) in animals within the United States, as confirmed by the USDA's National Veterinary Services Laboratory.

[0012] While the development of a human vaccine against SARS-CoV-2 is underway, a veterinary vaccine is also needed. [Overview of the project]

[0013] In a first aspect, the present invention provides a composition comprising a coronavirus, a spike protein of the coronavirus or an immunogenic fragment of the spike protein, and an adjuvant comprising saponin, sterol, and a CpG-containing immunostimulatory oligonucleotide. In certain embodiments of this first aspect, the adjuvant consists essentially of or consists of saponin, sterol, and a CpG-containing immunostimulatory oligonucleotide. In any of the above embodiments, the saponin can be a triterpenoid saponin, preferably extracted from the bark of Quillaia Saponaria, and the sterol can be selected from the group consisting of β-sitosterol, stigmasterol, ergosterol, ergocalciferol, and cholesterol. In any of the above embodiments, the saponin can be present in an amount of about 20 μg per dose, and the sterol can be present in an amount of about 20 μg per dose.

[0014] In a second aspect, the present invention provides a composition comprising a coronavirus, a spike protein from the coronavirus or an immunogenic fragment of the spike protein, and an adjuvant comprising (or consisting essentially of or consisting of) a CpG-containing immunostimulatory oligonucleotide and a glycolipid according to Formula I,

Chemical formula

[0015] In certain embodiments of this second aspect, the glycolipid is N-(2-deoxy-2-L-leucylamino-β-D-glucopyranosyl)-N-octadecyldodecanoylamide or a salt thereof, such as its acetate. In any of the embodiments of this second aspect of the invention, the glycolipid may be present in an amount of about 250 μg per dose.

[0016] In any of the embodiments of the first and / or second aspects of the invention as described above, the immunostimulatory oligonucleotide can be a P-class immunostimulatory oligonucleotide characterized by the presence of one or more TLR-9 activating motifs and two palindromes or two complementary regions. Preferably, the P-class immunostimulatory oligonucleotide is 5'-modified, and more preferably, the P-class immunostimulatory oligonucleotide contains at least 22 consecutive nucleotides of SEQ ID NO: 8. In certain embodiments of the first and / or second aspects of the invention, the CpG-containing immunostimulatory oligonucleotide is present in an amount of about 20 to about 50 μg per dose.

[0017] In a third aspect, the present invention provides a composition comprising a coronavirus, a spike protein or immunogenic fragment of the spike protein from the coronavirus, and an adjuvant comprising a saponin, a sterol, a quaternary ammonium compound, and a polyacrylic acid polymer. In a particular embodiment of this third aspect of the present invention, the saponin is a triterpenoid saponin, preferably Quil A, extracted from the bark of Quillia Saponaria; the sterol is selected from the group consisting of β-sitosterol, stigmasterol, ergosterol, ergocalciferol, and cholesterol; and the quaternary ammonium compound is DDAB. In a particular embodiment, the Quil A is present in an amount of about 20 μg per dose; the sterol is cholesterol, present in an amount of about 20 μg per dose; the DDAB is present in an amount of about 10 μg per dose; and the polyacrylic acid polymer is present in an amount of about 0.05% v / v.

[0018] In certain embodiments of the first, second, or third aspects of the present invention, the coronavirus is SARS-2 coronavirus, and the antigen is the spike protein or an immunogenic fragment thereof. In certain embodiments applicable to the first, second, and third aspects of the present invention, the spike protein is at least 90% identical to SEQ ID NO: 13, provided that the protein is in a pre-fusion state. In certain embodiments, the pre-fusion state is conferred by the substitution of amino acids at positions 973 and / or 974 of SEQ ID NO: 13. In certain preferred embodiments, the amino acids at positions 973 and 974 are substituted with proline.

[0019] In a particular embodiment applicable to any of the embodiments described above, the spike protein or fragment thereof includes a mutation in SEQ ID NO: 15, preferably a mutation in which SEQ ID NO: 15 is replaced by SEQ ID NO: 16. In a further embodiment applicable to all of the spike proteins or fragments described above, the protein may further include a Foldon sequence, such as SEQ ID NO: 12. In a particular embodiment, a composition according to the first, second, or third aspect of the present invention includes a spike protein or immunogenic fragment thereof in an amount of about 20 μg per dose.

[0020] A fourth aspect of the present invention provides a method for inducing an immune response in a subject requiring induction of an immune response, the method comprising administering to the subject a composition described in any one of the above embodiments.

[0021] In a particular embodiment of this fourth aspect, the immunogenic composition is administered to the subject in a prime dose and a boost dose, with the boost dose occurring approximately 14 to 42 days after the prime dose.

[0022] Preferably, the immune response is a protective immune response, and in certain embodiments, this protective immune response is maintained for 6 or 12 months after prime vaccination.

[0023] In certain embodiments of this fourth aspect, the subject is a canid, and the adjuvant in the immunogenic composition comprises a saponin, a sterol, and a CpG-containing immunostimulatory oligonucleotide. In other embodiments, the subject is a feline, and the adjuvant in the immunogenic composition comprises a CpG-containing immunostimulatory oligonucleotide and a glycolipid of formula I. In yet another embodiment, the subject is a feline, and the adjuvant in the immunogenic composition comprises a sterol, a saponin, a quaternary ammonium compound, and a polyacrylic acid polymer. [Modes for carrying out the invention]

[0024] definition When used in relation to a measurable numerical variable, "approximately" or "about" refers to the stated value of the variable and all values ​​of the variable within the experimental error of the stated value (e.g., within the 95% confidence interval of the mean) or within 10 percent of the stated value, whichever is greater. With respect to time, the term "approximately" refers to a range within 10 percent of the stated value (e.g., "approximately 8 months" includes 8 months and 8 months plus or minus 10%), except that "approximately 11 months" has an upper limit of 12 months, and "approximately 12 months" has an upper limit of 12.5 months.

[0025] An "adjuvant" refers to any substance that increases the humoral or cellular immune response to an antigen. Adjuvants are generally used to achieve two objectives: controlled release of the antigen from the injection site and stimulation of the immune system.

[0026] An "antibody" refers to an immunoglobulin molecule that can bind to a specific antigen as a result of an immune response to that antigen. Immunoglobulins are serum proteins composed of "light" and "heavy" polypeptide chains with "constant" and "variable" regions, and are classified into classes (e.g., IgA, IgD, IgE, IgG, and IgM) based on the composition of their constant regions.

[0027] An "antigen" or "immunogen" refers to any substance recognized by an animal's immune system that generates an immune response. This term includes dead, inactivated, attenuated, or modified live bacteria, viruses, or parasites. The term "antigen" also includes polynucleotides, polypeptides, recombinant proteins, synthetic peptides, protein extracts, cells (including tumor cells), tissues, polysaccharides, or lipids, individually or in any combination thereof. The term "antigen" also includes antibodies, such as anti-idiotype antibodies or fragments thereof, and synthetic peptide mimotopes that can mimic an antigen or antigenic determinant (epitope).

[0028] A "buffer" refers to a chemical system that prevents changes in the concentration of another chemical substance, such as a proton donor and acceptor system that acts as a buffer to prevent large changes in hydrogen ion concentration (pH). Further examples of buffers are solutions containing a weak acid and its salt (conjugate base) or a mixture of a weak base and its salt (conjugate acid).

[0029] A "conservative substitution" refers to the substitution of one amino acid with another, where the substituted amino acid and the substituted amino acid have similar structures. For example, a change resulting from the substitution of one negatively charged residue, such as aspartate for glutamate, or another positively charged residue, such as lysine for arginine, can also be expected to produce a protein with substantially the same functional activity.

[0030] The following six groups contain amino acids that are typical conservative substitutions of each other: [1] alanine (A), serine (S), threonine (T), [2] aspartic acid (D), glutamic acid (E), [3] asparagine (N), glutamine (Q), [4] arginine (R), lysine (K), histidine (H), [5] isoleucine (I), leucine (L), methionine (M), valine (V), and [6] phenylalanine (F), tyrosine (Y), tryptophan (W) (see, for example, U.S. Patent Publication No. 2010 / 0291549).

[0031] In the context of adjuvant formulations, "essentially composed" means a formulation in which the drug exerts a measurable adjuvant or immunomodulatory effect and does not contain any additional, unlisted adjuvants or immunomodulators. Preferably, if present, such additional, unlisted adjuvants or immunomodulators are present in amounts below the detection threshold.

[0032] "Dosage" refers to the vaccine or immunogenic composition administered to a subject in a single dose.

[0033] In this context, "immune response" refers to the occurrence of a humoral immune response, a cellular immune response, or both humoral and cellular immune responses to an antigen. The immune response can typically be determined using standard immunoassays, cell-based assays, and neutralization assays known in the art.

[0034] An "immunologically effective amount" or "effective amount to produce an immune response" of an antigen is an effective amount to induce an immunogenic response in a recipient. The immunogenic response may be sufficient for diagnostic purposes or other tests, or sufficient to prevent signs or symptoms of a disease, including adverse health effects or complications resulting from infection by a disease-causing agent. Either humoral immunity or cell-mediated immunity, or both, may be induced. An animal's immunogenic response to an immunogenic composition may be assessed indirectly, for example, through antibody titer measurement, cytokine assays, lymphocyte proliferation assays, or directly, through monitoring signs and symptoms after challenge with wild-type strains. On the other hand, protective immunity conferred by a vaccine may be assessed by measuring, for example, a reduction in clinical signs such as mortality, morbidity, body temperature, overall physical condition, and the overall health and capacity of the subject. The immune response may include, but is not limited to, the induction of cellular and / or humoral immunity.

[0035] "Immunogenicity" means that it elicits an immune or antigenic response. Therefore, an immunogenic composition can be any composition that induces an immune response.

[0036] "Pharmacologically acceptable" refers to a substance that is within the bounds of reasonable medical judgment, suitable for use in contact with the target tissue without excessive toxicity, irritation, or allergic reactions, has a reasonable benefit-to-risk ratio, and is effective for its intended use.

[0037] The term "protective immune response" refers to an immune response induced in a subject by an immunogenic composition or vaccine, in which, upon challenge with an immunized coronavirus, the subject either does not become infected (complete protection) or exhibits symptoms of a smaller magnitude and / or duration compared to an unimmunized animal (partial protection). In a particularly preferred embodiment of partial protection, both the immunized and challenged subjects do not excrete the coronavirus, or the magnitude and / or duration of excretion is reduced. Thus, the protective immune response prevents infection and / or reduces the symptoms and / or duration of infection.

[0038] The term "sequence identity" refers to the identity between two sequences within a comparison window. Protein sequence identity can be assessed using any of the various sequence comparison algorithms and programs known in the art. For sequence comparison, one sequence typically serves as a reference sequence (e.g., a sequence disclosed herein), against which the test sequence is compared. The sequence comparison algorithm then calculates the sequence identity percentage of the test sequence relative to the reference sequence, based on program parameters.

[0039] The percentage of identity between two amino acid sequences can be determined, for example, by comparing sequence information using the computer program GAP, i.e., the Genetics Computer Group (GCG; Madison, WI) Wisconsin package version 10.0 program, GAP (Devereux et al. (1984), Nucleic Acids Res. 12:387-95). When calculating the percentage of identity, the sequences being compared are typically aligned in a way that gives the greatest match between them. The preferred default parameters for the GAP program include: (1) a GCG implementation of a unary comparison matrix for nucleotides (including values ​​of 1 for identity and 0 for non-identity), as well as the Gribskov and Burgess weighted amino acid comparison matrix described in Atlas of Polypeptide Sequence and Structure, Schwartz and Dayhoff, eds., National Biomedical Research Foundation, pp. 353-358 (1979), ((1986) Nucleic Acids Res. 14: 6745), or other equivalent comparison matrices; (2) a penalty of 8 for each gap and an additional penalty of 2 for each symbol in each gap for amino acid sequences, or a penalty of 50 for each symbol in each gap and an additional penalty of 3 for each symbol in each gap for nucleotide sequences; (3) no penalty for end gaps; and (4) no maximum penalty for long gaps.

[0040] Sequence identity and / or similarity can also be determined by the local sequence identity algorithm of Smith and Waterman, 1981, Adv.Appl.Math.2:482, the sequence identity alignment algorithm of Needleman and Wunsch, 1970, J.Mol.Biol.48:443, the similarity search method of Pearson and Lipman, 1988, Proc.Nat.Acad.Sci.USA85:2444, and by computer implementation of these algorithms (BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package by Genetics Computer Group, 575 Science Dr., Madison, WI).

[0041] Another example of a useful algorithm is PILEUP. PILEUP creates a large number of sequence alignments from a group of related sequences using progressive pairwise alignment. A genealogy showing the clustering relationships used to create the alignments can also be plotted. PILEUP uses a simplified progressive alignment method from Feng & Doolittle, 1987, J.Mol.Evol.35:351-360; this method is similar to the method described by Higgins and Sharp, 1989, CABIOS 5:151-153. Useful PILEUP parameters include a default gap weight of 3.00, a default gap length weight of 0.10, and a weighted end gap.

[0042] Another example of a useful algorithm is the BLAST algorithm described in Altschul et al., 1990, J.Mol.Biol.215:403-410, Altschul et al., 1997, Nucleic Acids Res.25:3389-3402, and Karin et al., 1993, Proc.Natl.Acad.Sci.USA90:5873-5787. A particularly useful BLAST program is WU-BLAST-2, derived from Altschul et al., 1996, Methods in Enzymology 266:460-480. WU-BLAST-2 uses several search parameters, most of which are set to their default values. The adjustable parameters are set to the values ​​of overlap span=1, overlap portion=0.125, and word threshold(T)=II. The HSP S and HSP S2 parameters are dynamic values, determined by the program itself depending on the composition of a particular sequence and the configuration of the particular database against which the target sequence is being searched; however, these values ​​can be adjusted to increase sensitivity.

[0043] A further useful algorithm is gapped BLAST, as reported by Altschul et al., 1993, Nucl. Acids Res. 25:3389-3402. Gapped BLAST uses a BLOSUM-62 substitution score, i.e., a threshold T parameter set to 9, a two-hit method to induce non-gapped extension, a weighted gap length of k with a cost of 10+k, and X set to 16. u X is set to 40 for the database search phase and to 67 for the algorithm output phase. g This is used. Gap alignment is caused by a score that corresponds to approximately 22 bits.

[0044] The term "target" refers to an organism susceptible to a given coronavirus, and may be represented by different species of birds and mammals, including but not limited to humans and non-human mammals. For example, poultry are susceptible to avian infectious bronchitis, pigs are susceptible to porcine infectious diarrhea, and cats, dogs, Mustelllidae (ferrets, saber-toothed ferrets, minks, weasels), and humans are susceptible to SARS-CoV-2.

[0045] The term "treating" refers to reducing or mitigating the severity and / or duration of at least one symptom of an existing coronavirus infection.

[0046] The term "vaccine" refers to an immunogenic composition that induces a protective immune response in a subject, and when administered to a subject, induces or stimulates a protective immune response. Vaccines can immunize an organism against a specific disease, in this case coronavirus infection, more specifically SARS-CoV-2 infection. Therefore, the vaccine of the present invention induces an immune response in a subject that is protective against subsequent SARS-CoV-2 challenge. The vaccine comprising the antigen and adjuvant of the present invention may be capable of inducing cross-protective immune responses against multiple coronavirus genotypes.

[0047] antigen The antigens used in the compositions described herein are inactivated coronaviruses or coronavirus spike proteins, or immunogenic fragments of said spike proteins.

[0048] Several coronaviruses are suitable for use in the compositions described herein. These coronaviruses include, but are not limited to, porcine epidemic diarrhea virus (PEDV), porcine delta coronavirus (CoV), feline infectious peritonitis virus, feline enteritis CoV, avian infectious bronchitis virus, turk CoV, canine CoV, canine respiratory CoV, bovine CoV, equine CoV, TGEV, porcine respiratory CoV, and porcine hemagglutinating encephalomyelitis virus.

[0049] In certain embodiments, the recombinant spike protein antigen comprises a wild-type 2019-nCoVS protein having the amino acid sequence of SEQ ID NO: 11, or a sequence that is at least 80% identical thereto (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to SEQ ID NO: 11), provided that the protein is in a pre-fusion conformation. Sequence identity should be determined without considering the N-terminal signal peptide "MFVFLVLLPLVSS" (SEQ ID NO: 14).

[0050] In certain embodiments, the pre-fusion conformation is achieved by introducing a mutation between Heptad Repeat 1 and the central helix of SEQ ID NO: 11 (or a sequence that is at least 80% identical to it as considered above). The amino acids at positions 986 and 987 of SEQ ID NO: 11 are particularly suitable for mutation. In certain embodiments, both amino acids 986 and 987 are replaced with proline.

[0051] In certain embodiments, the fulin cleavage site PRRARS (SEQ ID NO: 15), which is commonly located between the S1 and S2 domains of the spike protein, is mutated so that fulin does not cleave the antigen. In certain embodiments, SEQ ID NO: 15 is mutated to SEQ ID NO: 16 (PGSASS).

[0052] In certain embodiments, the recombinant spike protein includes a C-terminal T4 fibrintin foldon motif such as "GYIPEAPRGDQAYVRKDGEWVLLSTFL" (SEQ ID NO: 12), and optionally a purified tag such as a C-terminal polyhistidine tag.

[0053] In a preferred embodiment, the amino acids of the recombinant spike protein according to the present invention corresponding to the amino acids at positions 986 and 987 of SEQ ID NO: 11 are proline residues, the furin cleavage site is mutated to SEQ ID NO: 16, and the protein contains the Foldon sequence of SEQ ID NO: 12 and a C-terminal polyhistidine purified tag.

[0054] In certain embodiments, the amino acids that differ between recombinant spike proteins are conserved substitutions.

[0055] In another embodiment, the antigen is a fragment of the wild-type 2019-nCoVS protein, as described above, provided that this fragment contains both the S1 and S2 domains.

[0056] In certain embodiments, the fragment corresponds to residues 14-1208 of the wild-type 2019-nCoVS protein of SEQ ID NO: 11, as provided in SEQ ID NO: 13, or sequences that are at least 80% identical to them (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to SEQ ID NO: 13), provided that the protein is in a pre-fusion conformation, and optionally further provided that the fulin cleavage site of the fragment is non-functional. As described above, the pre-fusion conformation can be achieved by substituting residues 973 and / or 974 of SEQ ID NO: 13 (corresponding to residues 986 and 987 of SEQ ID NO: 11). Preferred substitutions involve proline residues at both positions.

[0057] The fragment may further include a Foldon and / or immunopurification tag, as described above. In a particular embodiment, the coronavirus spike protein fragment is a conservatively substituted variant of SEQ ID NO: 13. In the most preferred embodiment, the antigen includes (or consists of) SEQ ID NO: 17.

[0058] Methods for preparing antigens according to the present invention are well known. For example, genetic engineering techniques and recombinant DNA expression systems can be used.

[0059] A nucleic acid molecule encoding the amino acid sequence of an antigen according to any embodiment of the present invention may also be inserted into one or more vectors, such as nonviral and / or viral vectors (e.g., recombinant vectors). Nonviral vectors may include, for example, plasmid vectors (e.g., those suitable for bacterial, insect, and / or mammalian host cells). Exemplary vectors include, for example, PCR-ii, PCR3, and pcDNA3.1 (Invitrogen, San Diego, Calif.), pBSii (Stratagene, La Jolla, Calif.), pet15 (Novagen, Madison, Wis.), pGEX (Pharmacia Biotech, Piscataway, NJ), pEGFp-n2 (Clontech, Palo Alto, Calif.), pET1 (Bluebacii, Invitrogen), pDSR-alpha (PCT Publication WO90 / 14363) and pFASTBACdual (Gibco-BRL, Grand Island, NY), as well as Bluescript plasmid derivatives (high copy number COLe1-based phagemids, Stratagene Cloning Systems, La Jolla, Calif.), and TAQ-amplified PCR products (e.g., TOPO® TA). This may include the Cloning® kit, PCR2.1® plasmid derivatives, and PCR cloning plasmids designed for cloning Invitrogen, Carlsbad, and Calif. Bacterial vectors, including, for example, Shigella, Vibrio cholerae, Lactobacillus, Bacille Calmette Guerin (BCG), and Streptococcus (see, for example, WO88 / 6626, WO90 / 0594, WO91 / 13157, WO92 / 1796, and WO92 / 21376), may also be used. The vectors may be constructed using standard recombination techniques widely available to those skilled in the art. Many other nonviral plasmid expression vectors and systems are known in the art and may be used.

[0060] Various viral vectors successfully used to introduce nucleic acids into a host include, among others, retroviruses, adenoviruses, adeno-associated viruses (AAVs), herpesviruses, baculoviruses, and poxviruses. Viral vectors can be constructed using standard recombinant techniques widely available to those skilled in the art. See, for example, Molecular cloning: a laboratory manual (Sambrook & Russell: 2000, Cold Spring Harbor Laboratory Press; ISBN: 0879695773) and Current protocols in molecular biology (Ausubel et al., 1988+ updates, Greene Publishing Assoc., New York; ISBN: 0471625949). Vectors can be used to infect host cells, such as bacteria, yeast cells (e.g., Pichia cells), insect cells, or mammalian cells (e.g., CHO cells), and the expressed proteins can be collected and purified according to methods known in the art.

[0061] The expression of amino acid sequences of antigens listed herein can also be carried out in so-called cell-free expression systems. Such systems include all the essential factors for the expression of the nucleic acid encoding the antigen, and the antigen is operably linked to a promoter that can be expressed in that particular system. Examples include the E. coli lysate system (Roche, Basel, Switzerland) or the rabbit reticulocyte lysate system (Promega corp., Madison, USA).

[0062] In a particular embodiment, SEQ ID NO: 17 is prepared by expressing the amino acid sequence containing SEQ ID NO: 17 and the signal peptide of SEQ ID NO: 14, which is upstream of SEQ ID NO: 17. SEQ ID NO: 14 is cleaved during processing.

[0063] The antigen according to any of the embodiments may be present in the immunogenic compositions listed herein in an immunologically effective amount sufficient to elicit an immune response, preferably a protective immune response. Generally, an immunologically effective amount is about 1 μg to 1 mg per dose. In embodiments where the antigen is a recombinant spike protein or a fragment thereof, the protein or fragment may be present in an amount of about 1 μg to about 500 μg, or about 1 μg to about 200 μg, or about 2 μg to about 100 μg, or about 5 μg to about 50 μg, or about 10 μg to about 25 μg, or about 20 μg per dose.

[0064] Adjuvant Several adjuvant compounds are known in the art, including, but not limited to, saponins, sterols, quaternary ammonium compounds, glycolipids that stimulate the Th2 response, polymers, particularly polymers of polyacrylic acid, and immunostimulatory oligonucleotides. saponin

[0065] Suitable saponins include triterpenoid saponins. These triterpenoids are a group of plant-derived surface-active glycosides that share a common chemical core consisting of a hydrophilic region (usually several sugar chains) associated with either a hydrophobic region of the steroid or triterpenoid structure. Due to these similarities, saponins sharing this chemical core are likely to have similar adjuvant properties. Suitable triterpenoids for use in adjuvant compositions can be derived from many sources, either plant-derived or synthetic equivalents, and include, but are not limited to, Quillaja saponaria, tomatine, ginseng extract, mushrooms, and alkaloid glycosides structurally similar to steroid saponins.

[0066] When saponins are used, the adjuvant composition generally contains an immunoactive saponin fraction from the bark of Quillaja saponaria. The saponin may be, for example, Quil A or another commercially available purified or partially purified saponin preparation. Thus, the saponin extract may be used as a mixture or as purified individual components such as QS-7, QS-10, QS-17, QS-18, and QS-21. In one embodiment, Quil A is at least 85% pure. In other embodiments, Quil A is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% pure.

[0067] Where triterpenoid saponins (e.g., Quil A or the purified fractions herein) may be present in the vaccine, in certain embodiments, a single dose of the vaccine may contain 1 to 1000 μg of such triterpenoid saponins per dose, or 10 to 100, or 5 to 50, or 1 to 25, or 25 to 300, or 50 to 200, or 50 to 100 μg. For neonatal small animals (e.g., dogs, cats, or minks), this amount may be about 1 to about 100 μg per dose (e.g., about 5 to about 50 μg per dose, or about 10 to about 25 μg per dose, or about 15 to about 20 μg per dose), and for large animals (e.g., horses, pigs, or cattle), this amount may be about 50 to about 1000 μg per dose. Sterols

[0068] Sterols share a common chemical core, which is a steroid ring structure and typically has a hydroxyl (OH) group bonded to carbon-3. The hydrocarbon chains of fatty acid substituents vary in length and typically have 16 to 20 carbon atoms and can be saturated or unsaturated. Sterols generally contain one or more double bonds in the ring structure and also contain various substituents bonded to the ring. Sterols and their fatty acid esters are essentially water-insoluble. Given these chemical similarities, sterols sharing this chemical core are likely to have similar properties when used in the vaccine composition of the present invention. Sterols are well known in the art and can be commercially available. For example, cholesterol is disclosed in Merck Index, 12th Ed., p. 369. Preferred sterols include, but are not limited to, β-sitosterol, stigmasterol, ergosterol, ergocalciferol, and cholesterol.

[0069] If sterols may be present in the vaccine, in certain embodiments, a single dose of the vaccine may contain 1 to 1000 μg of such sterols. In different embodiments, the amount of sterols is 10 to 100, or 5 to 50, or 1 to 25, or 25 to 300, or 50 to 200, or 50 to 100 μg per dose. For neonates or small animals (e.g., dogs, cats, or minks), this amount may be about 1 to about 100 μg per dose (e.g., about 5 to about 50 μg per dose, or about 10 to about 25 μg per dose, or about 15 to about 20 μg per dose), and for large animals (e.g., horses, pigs, or cattle), this amount may be about 50 to about 1000 μg per dose. CpG-containing immunostimulatory oligonucleotides

[0070] The adjuvant component of the vaccine also includes an immunomodulatory oligonucleotide. The immunomodulatory oligonucleotide according to the present invention contains CpG (also referred to as "CpG-containing immunomodulatory oligonucleotide," "CpG oligonucleotide," or simply "CpG"). The effects of CpG-containing oligonucleotides on the immune system have been known for more than 20 years.

[0071] Generally, CpGs suitable for the present invention are 15 to 100 base pairs long, for example, 15 to 50 base pairs, or 18 to 40 base pairs, or 20 to 30 base pairs, or 20 to 24 base pairs.

[0072] Several classes of CpGs are described, including Class A CpGs, Class B CpGs, Class C CpGs, and Class P CpGs. In certain embodiments, the CpG-containing immunostimulatory oligonucleotide is a Class P CpG. A Class P CpG is characterized by the presence of one or more TLR-9 activating motifs and two palindromes or two complementary regions. Preferably, one or more TLR-9 activating motifs are located at the 5' of the oligonucleotide and may be fully or partially incorporated into the 5' palindrome region or 5' complementary region. Known TLR-9 activating motifs include, but are not limited to, TCG, TTCG, TTTCG, TYpR, TTYpR, TTTYpR, UCG, UUCG, UUUCG, TTT, or TTTT. The 5' palindrome or 5' complementary region is at least 6 nucleotides long. The 3' palindrome or 3' complementary region is at least 8 nucleotides long and is generally rich in C and G. These structural features of P-class CpG confer the ability to spontaneously self-assemble into concatemers, either in vitro or in vivo.

[0073] To increase the lipophilicity of CpG oligonucleotides, at least one lipophilic substitution nucleotide analog may be included, preferably at the 5' end of the oligonucleotide. P-class immunostimulatory oligonucleotides can be modified according to techniques known in the art. For example, J modification refers to iodine-modified nucleotides. E modification refers to ethyl-modified nucleotides. Thus, an E-modified P-class immunostimulatory oligonucleotide is a P-class immunostimulatory oligonucleotide in which at least one nucleotide (preferably the 5' nucleotide) is ethylated. Additional modifications include attachment of 6-nitro-benzimidazole, O-methylation, modification with propynyl-dU, inosine modification, and 2-bromovinyl attachment (preferably to uridine).

[0074] Oligonucleotides modified by the addition of lipophilic moieties are generally described in US2010 / 0166780.

[0075] In certain embodiments, the CpG according to the present invention includes, but is not limited to, modified skeletons comprising phosphorothioate modification, halogenation, alkylation (e.g., ethyl or methyl modification), and phosphodiester modification.

[0076] A selection of suitable, non-limiting examples of modified P-class immunostimulatory oligonucleotides is provided below (where "*" indicates a phosphorothioate bond, "-" indicates a phosphodiester bond, "JU" indicates 5'-iodo-2'-deoxyuridine, and "EU" indicates 5-ethyl-2'-deoxyuridine). Sequence ID 1 5'T*CG*T*CG*A*CG*A*T*CG*G*C*G*CG*C*G*C*C*G 3' Sequence ID 2 5'T*CG*A*C*G*T*C*G*A*T*C*G*G*C*G*C*G*C*G*C*C*G 3' Sequence number 3 5'T*C*G*A*C*G*T*C*G*A*T*C*G*G*C*G*C*G*C*G*C*C*G*T 3' Sequence No. 4 5'JU*CG*A*C*G*T*C*G*A*T*C*G*G*C*G*C*G*C*G*C*G 3' Sequence No. 5 5'JU*CG*A*C*G*T*C*G*A*T*C*G*G*C*G*C*G*C*G*C*G*C*G*T 3' Sequence number 6 5'JU*C*G*A*C*G*T*C*G*A*T*C*G*G*C*G*C*G*C*G*C*C*G*T 3' Sequence No. 7 5'EU*CG*A*C*G*T*C*G*A*T*C*G*G*C*G*C*G*C*G*C*G 3' Sequence ID 8 5'JU*CG*T*C*G*A*C*G*A*T*C*G*G*C*G*G*C*C*G*C*C*G*T 3' Sequence number 9 5'JU*C*G*T*C*G*A*C*G*A*T*C*G*G*C*G*G*C*C*G*C*C*G*T 3' Sequence ID 10 5'T*CG*T*CG*A*CG*A*T*CG*G*C*G*CG*C*G*C*C*G 3'

[0077] In a particular embodiment, the CpG oligonucleotide according to the present invention comprises an oligonucleotide comprising any one of SEQ ID NOs: 1 to 10, or at least 15 consecutive bases of any one of SEQ ID NOs: 1 to 10. In the most preferred embodiment, the vaccine comprises an oligonucleotide comprising at least 15 consecutive bases of SEQ ID NO: 8 (for example, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, or at least 23).

[0078] CpG oligonucleotides may be present in the vaccine in amounts of 10 to 400 μg per dose, or 25 to 300 μg, 50 to 200 μg, or 50 to 100 μg per dose. In neonates or small animals (e.g., dogs, cats, or minks), this amount may be about 0.5 μg to 70 μg per dose (e.g., about 2 μg to 40 μg per dose, or about 5 μg to 30 μg per dose, or about 10 μg to 25 μg per dose, or about 20 μg per dose), and in large animals (e.g., horses, pigs, or cattle), this amount may be about 50 μg to 400 μg per dose (e.g., about 100 μg to 300 μg per dose, or about 150 μg to 250 μg per dose).

[0079] Polyacrylic polymer Polyacrylic acid polymers are also suitable adjuvant compounds. For example, CARBOPOL® polymer is a polymer of acrylic acid crosslinked with polyalkenyl ether or divinyl glycol. CARBOPOL® is used in many vaccines.

[0080] Polyacrylic polymers may be present in vaccines in amounts ranging from 0 to about 30% v / v, for example, about 0.001% v / v to about 25% v / v, about 0.005% v / v to about 15% v / v, about 0.01% v / v to about 10% v / v, about 0.05% v / v to about 1% v / v, and about 0.05% v / v to about 0.25% v / v.

[0081] Glycolipid Suitable glycolipids are generally those that activate the Th2 response. Examples of glycolipids include, but are not limited to, those included in Formula I and generally described in U.S. Patent Publication No. 2007 / 0196384 (Ramasamy et al). [ka] Equation I In the formula, R 1is hydrogen, or a saturated alkyl radical having up to 20 carbon atoms, where X is -CH2-, -O-, or -NH-, and R 2 However, R is hydrogen, or a saturated or unsaturated alkyl radical having up to 20 carbon atoms. 3 , R 4 , and R 5 However, independently, hydrogen, -SO4 2- , -PO4 2- ,-COC 1-10 It is alkyl, R 6 These are L-alanyl, L-alpha-aminobutyl, L-arginyl, L-asparginyl, L-aspartyl, L-cysteinyl, L-glutamyl, L-glycyl, L-histidyl, L-hydroxyprolyl, L-isoleucyl, L-leucyl, L-lucyl, L-methionyl, L-ornithinyl, L-phenyalany, L-prolyl, L-ceryl, L-threonyl, L-tyrosyl, L-tryptophanyl, and L-valyl, or their D-isomers.

[0082] Examples of glycolipids include, but are not limited to, N-(2-deoxy-2-L-leucylamino-β-D-glucopyranosyl)-N-octadecyldodecanoylamide (Bay® 1005, or R1005) or its salts (e.g., acetate).

[0083] If glycolipids may be present in the vaccine, in different embodiments, one dose of the vaccine may contain 0.01 mg to about 10 mg of glycolipids per dose mg. Therefore, for example, glycolipids may be present in amounts of about 0.05 to 2 mg per dose, or about 1 to 5 mg per dose, or about 4 to 8 mg per dose, or about 5 to 10 mg per dose. In the case of neonates or small animals (e.g., dogs or cats or minks or weasels), this amount may be about 0.1 to 1 mg per dose (e.g., about 0.25 to 0.75 mg per dose, or about 0.2 mg to 0.4 mg per dose), and in the case of large animals (e.g., horses, pigs or cattle), this amount may be about 1 to 10 mg per dose.

[0084] Quaternary ammonium compounds Quaternary ammonium compounds are ammonium compounds having four hydrocarbon groups. In practice, the hydrocarbon groups are generally limited to alkyl or aryl groups. In a series of embodiments, a quaternary ammonium compound consists of four alkyl chains, two of which are C10-C20 alkyl and the remaining two are C1-C4 alkyl. In certain embodiments, the quaternary ammonium is dimethyldioctadecylammonium (DDA) bromide, chloride, or another pharmaceutically acceptable counterion.

[0085] If quaternary ammonium compounds are present in the vaccine, one dose of the vaccine may contain quaternary ammonium in amounts of 1 to 1000 μg, or 1 to 1000 μg, 1 to 500 μg, or 10 to 100 μg, or 5 to 50 μg, or 1 to 25 μg, or 25 to 300 μg, or 50 to 200 μg, or 50 to 100 μg. For neonates or small animals (e.g., dogs, cats, or minks), this amount may be about 1 mg to about 100 μg per dose (e.g., about 5 μg to about 50 μg per dose, or about 10 μg to about 25 μg per dose, or about 15 μg to about 20 μg per dose), and for large animals (e.g., horses, pigs, or cattle), this amount may be about 50 μg to about 1000 μg per dose.

[0086] In certain embodiments, the adjuvant generally comprises (or consists of) a combination of triterpenoid saponins, sterols, and CpG-containing immunostimulatory oligonucleotides. Optionally, the adjuvant may further comprise an effective amount of a quaternary ammonium compound, glycolipid, and / or polyacrylic acid polymer (e.g., CARBOPOL®). In certain embodiments, the adjuvant comprises an effective amount (or any detectable amount) of a quaternary ammonium compound, e.g., abridine or DDAB, a polyacrylic acid polymer, e.g., CARBOPOL®, and / or lack thereof. In certain embodiments, one dose of this adjuvant contains about 10 μg to about 300 μg of Quil A, about 10 μg to about 300 μg of cholesterol, and 10 μg to 250 μg of CpG-containing immunostimulatory oligonucleotide. Preferably, the CpG-containing immunostimulatory oligonucleotide comprises or includes SEQ ID NO: 8.

[0087] In embodiments particularly suitable for dogs, Quil A may be present in an amount of 10 μg to 50 μg (preferably about 15 μg to 25 μg, or about 20 μg), cholesterol may be present in an amount of 10 μg to 50 μg (preferably about 15 μg to 25 μg, or about 20 μg), and CpG-containing immunostimulatory oligonucleotides may be present in an amount of about 10 μg to about 50 μg (preferably about 15 μg to about 25 μg, or about 20 μg).

[0088] In other embodiments, the adjuvant comprises (or consists of) a combination of CpG-containing immunostimulatory oligonucleotides and glycolipids according to formula I. In certain embodiments, the adjuvant lacks an effective amount (or any detectable amount) of quaternary ammonium compounds, e.g., abridine or DDAB, polyacrylic acid polymers, e.g., CARBOPOL® and / or triterpenoid saponins, particularly saponins from Q. Saponaria (including Quil A and its fraction). In certain embodiments, one dose of the adjuvant contains about 15 to about 100 μg of a CpG-containing immunostimulatory oligonucleotide and about 100 to about 1000 μg per dose (e.g., about 250 to about 750 μg per dose, or about 200 to 400 μg per dose) of the glycolipid according to formula I described above, which is preferably N-(2-deoxy-2-L-leucylamino-β-D-glucopyranosyl)-N-octadecyldodecanoylamide or a salt thereof (e.g., acetate). Preferably, the CpG-containing immunostimulatory oligonucleotide consists of or includes SEQ ID NO: 8.

[0089] In further embodiments, the adjuvant contains Quil A, cholesterol, DDAB, and CARBOPOL®. In certain embodiments, which are particularly suitable for felines and Mustellidae animals, Quil A may be present in amounts of 10 μg to 50 μg (preferably about 15 μg to 25 μg, or about 20 μg), cholesterol may be present in amounts of 10 μg to 50 μg (preferably about 15 μg to 25 μg, or about 20 μg), DDAB may be present in amounts of about 5 μg to 20 μg, or about 10 μg to about 15 μg, or about 10 μg, and CARBOPOL® may be present in amounts of about 0.01% to about 0.1%, or about 0.05% v / v.

[0090] Excipients Other components of the composition may include carriers, solvents, and pharmaceutically acceptable excipients such as diluents, isotonic agents, buffers, stabilizers, preservatives, antimicrobial agents, and antifungal agents. Typical carriers, solvents, and diluents include water, physiological saline, dextrose, ethanol, glycerol, and oil. Typical isotonic agents include sodium chloride, dextrose, mannitol, sorbitol, and lactose. Useful stabilizers include gelatin and albumin. The composition may also contain antibiotics or preservatives, such as gentamicin, methylthiolate, or chlorocresol. Various classes of antibiotics or preservatives for selection are well known to those skilled in the art.

[0091] Method of vaccine administration The compositions described herein are suitable for inducing an immune response against the coronavirus spike protein. The compositions described herein are also suitable for use as vaccines, i.e., administration of the immunogenic compositions disclosed herein results in the induction of a protective immune response against coronavirus, thereby preventing a subject in need from becoming infected with the coronavirus, or, if the subject is still infected, reducing the number and / or severity of symptoms of the coronavirus infection.

[0092] In certain embodiments, the subjects requiring vaccination are cattle, sheep, pigs, horses, or birds (e.g., chickens, turkeys, geese, or ducks). In certain embodiments, the subjects are dogs, cats, or animals of the Mustellidae family (including minks, ferrets, sables, and weasels). In other embodiments, the subjects are monkeys or humans.

[0093] The immunogenic composition according to the present invention may be administered according to a regimen: prime dose, followed by a boost (or booster) dose approximately 14 to 42 days after the prime dose. In different embodiments, the booster dose is administered approximately 14 to 28 days, or approximately 21 days, after the prime dose. In certain embodiments, this regimen provides an immunization period of at least 6 months after the booster dose, preferably at least 12 months (e.g., immunization periods of 6, 7, 8, 9, 10, or 11 months). Thus, in certain embodiments, semi-annual or annual revaccination is intended.

[0094] The immunogenic compositions according to the present invention may be formulated and administered to subjects by any known route, including oral, intranasal, mucosal, topical, transdermal, and parenteral (e.g., intravenous, intraperitoneal, intradermal, subcutaneous, or intramuscular). Administration may also be achieved using needle-free delivery devices. Administration may be achieved using a combination of routes, for example, a prime administration using a parent route and a boost administration using a mucosal route. Description of each claim in the patent claims at the time of international filing [Section 1] A composition comprising a coronavirus, the spike protein of the coronavirus or an immunogenic fragment of the spike protein, and an adjuvant comprising a saponin, a sterol, and a CpG-containing immunostimulatory oligonucleotide. [Section 2] The composition is essentially free of quaternary ammonium compounds and essentially free of glycolipids according to formula I. [ka] Equation I In the formula, R 1 is hydrogen, or a saturated alkyl radical having up to 20 carbon atoms, where X is -CH2-, -O-, or -NH-, and R 2 However, R is hydrogen, or a saturated or unsaturated alkyl radical having up to 20 carbon atoms. 3 , R 4 , and R 5However, independently, hydrogen, -SO4 2- , -PO4 2- ,-COC 1-10 It is alkyl, R 6 The composition according to claim 1, wherein the is L-alanyl, L-alpha-aminobutyl, L-arginyl, L-asparginyl, L-aspartyl, L-cysteinyl, L-glutamyl, L-glycyl, L-histidyl, L-hydroxyprolyl, L-isoleucyl, L-leucyl, L-lucyl, L-methionyl, L-ornithinyl, L-phenyalany, L-prolyl, L-ceryl, L-threonyl, L-tyrosyl, L-tryptophanyl, and L-valyl, or their D-isomers. [Section 3] The composition according to claim 1 or 2, wherein the adjuvant comprises the saponin, the sterol, and the CpG-containing immunostimulatory oligonucleotide. [Section 4] The composition according to any one of claims 1 to 3, wherein the saponin is Quil A, and the sterol is selected from the group consisting of β-sitosterol, stigmasterol, ergosterol, ergocalciferol, and cholesterol. [Section 5] The composition according to any one of claims 1 to 4, wherein the saponin is present in an amount of about 20 μg per dose, and the sterol is present in an amount of about 20 μg per dose. [Section 6] A composition comprising a coronavirus, a spike protein from the coronavirus or an immunogenic fragment of the spike protein, and an adjuvant comprising a CpG-containing immunostimulatory oligonucleotide and a glycolipid according to formula I, [ka] Equation I In the formula, R 1 is hydrogen, or a saturated alkyl radical having up to 20 carbon atoms, where X is -CH2-, -O-, or -NH-, and R 2However, R is hydrogen, or a saturated or unsaturated alkyl radical having up to 20 carbon atoms. 3 , R 4 , and R 5 However, independently, hydrogen, -SO4 2- , -PO4 2- ,-COC 1-10 It is alkyl, R 6 A composition in which the active ingredient is L-alanyl, L-alpha-aminobutyl, L-arginyl, L-asparginyl, L-aspartyl, L-cysteinyl, L-glutamyl, L-glycyl, L-histidyl, L-hydroxyprolyl, L-isoleucyl, L-leucyl, L-lucyl, L-methionyl, L-ornithinyl, L-phenyalany, L-prolyl, L-ceryl, L-threonyl, L-tyrosyl, L-tryptophanyl, and L-valyl, or their D-isomers. [Section 7] The composition according to claim 6, wherein the glycolipid is N-(2-deoxy-2-L-leucylamino-β-D-glucopyranosyl)-N-octadecyldodecanoylamide or a salt thereof. [Section 8] The composition according to claim 7, wherein the salt is an acetate salt. [Section 9] The composition according to any one of claims 6 to 8, wherein the composition is essentially free of saponins. [Section 10] The composition according to any one of claims 6 to 9, wherein the composition essentially does not contain a quaternary ammonium compound. [Section 11] The composition according to any one of claims 6 to 10, wherein the adjuvant comprises the glycolipid and the CpG-containing immunostimulatory oligonucleotide. [Section 12] The composition according to any one of claims 6 to 11, wherein the glycolipid is present in an amount of about 250 μg per dose. [Section 13] The composition according to any one of claims 1 to 12, wherein the immunostimulatory oligonucleotide is a P-class immunostimulatory oligonucleotide characterized by the presence of one or more TLR-9 activating motifs and two palindromes or two complementary regions. [Section 14] The composition according to claim 13, wherein the P-class immunostimulatory oligonucleotide is 5' modified. [Section 15] The composition according to claim 14, wherein the P-class immunostimulatory oligonucleotide comprises at least 22 consecutive nucleotides of SEQ ID NO: 8. [Section 16] The composition according to any one of claims 1 to 15, wherein the CpG-containing immunostimulatory oligonucleotide is present in an amount of about 20 to about 50 μg per dose. [Section 17] A composition comprising a coronavirus, a spike protein from the coronavirus or an immunogenic fragment of the spike protein, and an adjuvant comprising a saponin, a sterol, a quaternary ammonium compound, and a polyacrylic acid polymer. [Section 18] The composition according to claim 17, wherein the saponin is Quil A, the sterol is selected from the group consisting of β-sitosterol, stigmasterol, ergosterol, ergocalciferol, and cholesterol, and the quaternary ammonium compound is DDAB. [Section 19] The composition according to claim 18, wherein Quil A is present in an amount of about 20 μg per dose, the sterol is cholesterol and is present in an amount of about 20 μg per dose, DDAB is present in an amount of about 10 μg per dose, and the polyacrylic acid polymer is present in an amount of about 0.05% v / v. [Section 20] The composition according to any one of claims 1 to 19, wherein the coronavirus is SARS-2 coronavirus and the antigen is the spike protein or an immunogenic fragment thereof. [Section 21] The composition according to claim 20, wherein the spike protein is at least 90% identical to SEQ ID NO: 13, provided that the protein is in a pre-fusion state. [Section 22] The composition according to claim 21, wherein the amino acids at positions 973 and 974 are substituted with proline residues. [Section 23] The composition according to any one of claims 20 to 22, comprising the mutation of Sequence ID No. 15. [Section 24] The composition according to claim 23, wherein sequence number 15 is replaced with sequence number 16. [Section 25] The composition according to any one of claims 20 to 24, wherein the spike protein or immunogenic fragment thereof further comprises Sequence ID No. 12. [Section 26] The composition according to any one of claims 20 to 25, wherein the spike protein comprises sequence number 17 or a sequence that is at least 99% identical thereto. [Section 27] The composition according to any one of claims 1 to 26, wherein the spike protein of the coronavirus or an immunogenic fragment of the spike protein is present in an amount of about 20 μg per dose. [Section 28] A method for inducing an immune response in a subject requiring induction of an immune response, the method comprising administering to the subject a composition according to any one of claims 1 to 27. [Section 29] The method according to claim 28, wherein the subject is a canid, and the adjuvant in the immunogenic composition comprises the saponin, the sterol, and the CpG-containing immunostimulatory oligonucleotide. [Section 30] The subject is a feline, and the adjuvant in the immunogenic composition comprises the CpG-containing immunostimulatory oligonucleotide and a glycolipid according to formula I, as described in claim 28. method. [Section 31] The method according to claim 28, wherein the subject is a feline, and the adjuvant in the immunogenic composition comprises the sterol, the saponin, the quaternary ammonium compound, and the polyacrylic acid polymer. [Section 32] The method according to any one of claims 28 to 31, wherein the immunogenic composition is administered to the subject in a prime dose and a boost dose, the boost dose occurring approximately 14 to 42 days after the prime dose. [Section 33] The method according to any one of claims 28 to 31, wherein the immune response is a protective immune response. [Section 34] The method according to claim 32, wherein the protective immune response is maintained for at least 6 months after the boost administration. [Section 35] The method according to claim 34, wherein the protective immune response is maintained for at least 12 months after the boost administration.

[0095] The present invention is described below in the following non-limiting embodiments. [Examples]

[0096] Example 1 The objective of this study is to evaluate the efficacy of a recombinant SARS-CoV-2 trimer spike protein vaccine in dogs by generating antibodies capable of neutralizing SARS-CoV-2 in vitro. The vaccine protein is recognized as a target for antibody-mediated binding. This protein is similar to those used in human SARS and MERS vaccines.

[0097] This study used male (neutered) and female beagle dogs aged 6–11 months. Nine of these dogs had previously been exposed to canine parvovirus (orally administered MLV vaccine and CPV2c challenge), canine parainfluenza virus (orally administered MLV vaccine and CPIV challenge strain D008), and canine distemper virus (orally administered MLV vaccine). Six of these dogs had previously been exposed to canine distemper virus (orally administered MLV vaccine). The dogs were healthy and negative for SARS-CoV-2 via PCR using oral-pharyngeal swabs 0 days prior to the study.

[0098] The animals were kept in appropriate living conditions to meet the guidelines of the USDA Animal Welfare Code (9 Federal Codes of Regulation, Chapter 1, Section A - Animal Welfare), AAALAC (Association for the Evaluation and Accreditation of Laboratory Animal Care), and the Institutional Animal Care and Use Committee (IACUC). The dogs were given dry food appropriate to their age and nutritional requirements, moistened as needed, and provided freely at least once a day throughout the course of the study. Canned food or non-medicinal nutritional supplements were given as needed. Water was freely available at all times.

[0099] The dogs were randomly assigned to one of three groups, as provided in Table 1. [Table 1]

[0100] Blood samples (approximately 3.0–6.0 mL) were collected for pre-screening and 0 days prior for titer screening. Blood samples were collected from all animals in SST tubes.

[0101] Blood samples for serological testing (approximately 6.0–12.0 mL, or according to the individual dog's weight and blood collection guidelines) were collected from all animals in SST tubes on days 0 and 21, either the day before vaccination (i.e., days -1 and 20) or on the day of vaccination, but prior to the vaccination itself. On day 42 (end of the study), the maximum blood volume for each animal was calculated based on the individual animal's weight and IACUC guidelines. Blood was collected in SST tubes from all animals.

[0102] All animals were observed once on day -1, twice on day 0 (before and 3-6 hours after vaccination), once daily from day 1 to day 5, twice on day 21 (before and 3-6 hours after vaccination), and once daily from day 22 to day 26. Each clinical observation session lasted approximately 30 minutes.

[0103] Observations of the injection sites were recorded on day 0 of the study (before vaccination and 3-6 hours after vaccination), and on days 1-5 for the first injection site (left shoulder). Observations of the injection sites were also recorded on day 21 of the study (before vaccination and 3-6 hours after vaccination), and on days 22-26 (right shoulder).

[0104] The vaccine showed good resistance in dogs. No pain or swelling at the injection site was observed during the study. A slight increase in tympanic membrane temperature was observed in all study groups after both vaccinations. No abnormal clinical signs were observed in any of the animals.

[0105] To measure SN titers, known amounts of virus were combined with different dilutions of inactivated serum from test animals. After incubating cells with the virus mixture and different serum dilutions, SN titers were measured by assessing the viability of Vero E6 cells. See Tan et al., Nat Biotech 38:1073-78 (September 2020) and Wang et al., J Immunol. Methods 301:21-30 (2005).

[0106] To determine ELISA titer, plates were coated with 100 μl / well of 250 ng / ml protein (SEQ ID NO: 13) in coating buffer.

[0107] Peroxidase-conjugated rabbit anti-canine IgG (H+L), polyclonal antibody (Jackson ImmunoResearch #304-035-003, lot 135618) was used as the secondary antibody, and TMB microwell peroxidase substrate DAKO True Blue #1601 was used as the substrate. Serum was first diluted 1:300, and then serially diluted 1:3. The secondary antibody was diluted 1:30,000 in PBST (PBS + 0.05% (w / v) TWEEN®-20). 100 μl / well of the sample serum dilution was added to the plate, and the plate was incubated at room temperature for 60 minutes. The secondary antibody was diluted 1:30,000 in dilution buffer, and 100 μl / well of this solution was added to the wells, and the reaction was allowed to proceed for 30 minutes. The plates were washed (four times using PBST) after sample incubation and after incubation with the secondary antibody.

[0108] The lateral flow test is a semi-quantitative test. Generally, it is a binary test used to determine whether an animal has or lacks antibodies against SARS-CoV-2 based on the presence or absence of a visible band indicating the presence of antibodies. However, because lateral flow devices can also measure the intensity of the band, it is possible to semi-quantitatively measure the amount of antibodies. For convenience, this semi-quantitative measure is sometimes referred to as the LF titer, or "titer measured by the LF." However, it should be understood that, when applied to lateral flow measurements, the term "titer" is not strictly titer.

[0109] Lateral flow titer was measured by filling the sample wells of a lateral flow device, where they were absorbed by a pad, with a sample containing BSA, a buffer to maintain pH, Tween 20, sodium azide, and blocker proteins such as polyethylene glycol (PEG) 8000, along with a chase buffer. The sample and buffer were drawn in via capillary action through a colloidal gold deposit conjugated with SEQ ID NO: 13.

[0110] A saturated amount of protein was added to gold, incubated for 10 minutes, and then a BSA blocker and a stabilizing buffer containing BSA and sucrose were added to prepare recombinant spike protein-colloidal gold conjugates.

[0111] The antibody-gold complex continues to move down the specimen until it crosses the line of deposition reagent (protein A or G) to immobilize the antibody. Crosslinking of the antibody-gold complex to the reagent on this line results in the accumulation of colloidal gold on the line, forming a visible red line.

[0112] The SN titers, lateral flow measurements, and ELISA results for each animal are summarized in Tables 2, 3, and 4, respectively. [Table 2] [Table 3] [Table 4]

[0113] All animals in group T01 were negative, while all animals in groups T02 and T03 were positive on visual observation at days 127, 155, and 187. [Table 5]

[0114] As expected, no serum conversion occurred in the control animals. In contrast, all animals in groups T02 and T03 underwent serum conversion. All animals in groups T02 and T03 had protective titers on day 42.

[0115] Example 2 The objective of this study is to evaluate the efficacy of a recombinant SARS-CoV-2 trimer spike protein vaccine in cats by generating antibodies capable of neutralizing SARS-CoV-2 in vitro.

[0116] Healthy domestic shorthair cats, approximately 10 months old, were used in the study. The cats were allowed to acclimate for at least 14 days before being used in the study.

[0117] All animals in the study were healthy prior to day 0. All animals in the study were negative for SARS-CoV-2 by PCR via nasal swab and serological testing prior to day 0. The animals were kept in appropriate living environments to meet USDA animal welfare regulations. Environmental conditions and floor space were consistent with standard practices at the laboratory. The animals were fed age-appropriate diets and provided with free access to water.

[0118] Randomization was performed using a SAS program (SAS release 9.4 or later, SAS Institute, Cary, NC) specifically developed for research using the ranuni function, which is used to generate random numbers.

[0119] The animals were treated as summarized in Table 5 below. [Table 6]

[0120] The animals were observed, and tympanic membrane temperature was measured twice a day on day 0 (before vaccination and 3-6 hours after vaccination), once a day on day -1 and days 1-5, and twice on day 21 (before vaccination and 3-6 hours after vaccination, and once a day on days 22-26). Clinical observations were conducted for approximately 30 minutes per session.

[0121] The injection site was observed daily for 5 days after each vaccination (days 1-5 and 22-26), or until no reaction was observed with the Callcat test.

[0122] The vaccine showed good resistance. No pain or swelling at the injection site was observed during the study. No abnormal clinical observations or temperature increases were observed during the study.

[0123] Antibody responses to vaccination were measured by serum neutralizing titer measurement, lateral flow assay, and ELISA.

[0124] As described in Example 1, the SN titer and LF titer were measured.

[0125] ELISA titers were measured as described in Example 1, except that the initial dilution was 1:100 (not 1:300) and the secondary antibody was diluted to 1:40,000 instead of 1:30,000. Results for individual animals are provided in Tables 6 (SN titers), 7 (lateral flow), and 8 (ELISA). [Table 7] [Table 8] [Table 9] N / A - Samples were not analyzed.

[0126] All animals in group T01 were negative, while all animals in groups T02 and T03 were positive on day 181 and day 265, respectively, as observed visually. [Table 10] N / A - Samples were not analyzed. [Table 11]

[0127] These data demonstrate that the vaccine according to the present invention elicits a robust immune response against the COVID-19 spike protein, and that this immune response lasts for at least 265 days, for example, 12 months or more, or 13 months or more, or 419 days.

[0128] All publications, both patent and non-patent publications, cited herein represent the level of skill of those skilled in the art to which the present invention relates. All of these publications are incorporated herein by reference to the same extent as each individual publication is specifically and individually indicated as being incorporated by reference.

Claims

1. An immunogenic composition comprising a protein having an amino acid sequence of sequence number 17, and an adjuvant comprising a saponin, a sterol, and a CpG-containing immunostimulatory oligonucleotide, wherein the immunogenic composition is for inducing an immune response in a subject requiring induction of an immune response.

2. The immunogenic composition according to claim 1, wherein the saponin is a triterpenoid saponin extracted from the bark of Quillia saponia, and the sterol is selected from the group consisting of β-sitosterol, stigmasterol, ergosterol, ergocalciferol, and cholesterol.

3. The immunogenic composition according to claim 2, wherein the saponin is present in an amount of 20 μg per dose, and the sterol is present in an amount of 20 μg per dose.

4. The immunogenic composition according to claim 1, wherein the immunostimulating oligonucleotide is a P-class immunostimulating oligonucleotide characterized by the presence of one or more TLR-9 activating motifs and two palindromes or two complementary regions.

5. The immunogenic composition according to claim 4, wherein the P-class immunostimulatory oligonucleotide is 5' modified.

6. The immunogenic composition according to claim 5, wherein the P-class immunostimulatory oligonucleotide comprises at least 22 consecutive nucleotides of SEQ ID NO:

8.

7. The immunogenic composition according to claim 1, wherein the CpG-containing immunostimulatory oligonucleotide is present in an amount of 20 to 50 μg per dose.

8. A method for inducing an immune response in a subject other than a human, wherein the method comprises administering to the subject an immunogenic composition according to any one of claims 1 to 7.

9. The method according to claim 8, wherein the subject is a canid.

10. The method according to claim 8, wherein the immunogenic composition is administered to the subject in a prime dose and a boost dose, the boost dose occurring 14 to 42 days after the prime dose.

11. The method according to claim 10, wherein the immune response is a protective immune response.

12. The method according to claim 11, wherein the protective immune response is maintained for at least six months after the boost administration.

13. The method according to claim 12, wherein the protective immune response is maintained for at least 12 months after the boost administration.

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