PHARMACEUTICAL COMPOSITION COMPRISING POLYNUCLEOTIDES AND USE THEREOF FOR PREVENTION OR TREATMENT OF COVID-19

Abstract

The present application relates to a pharmaceutical composition comprising polynucleotides and use thereof for prevention or treatment of COVID-19. More specifically, disclosed in the present application is a composition used for prevention or treatment of COVID-19, comprising a polyriboinosinic-polyribocytidylic acid, an antibiotic or polyamino compound, a positive ion, and an optional antigen derived from novel coronavirus SARS-CoV-2. Also provided is use of the composition in preparation of a drug or vaccine for prevention or treatment of novel coronavirus SARS-CoV-2.

Claims

1. An immune composition, containing: polyriboinosinic-polyribocytidylic acid (PIC), an antibiotic or a polyamino compound, a positive ion, and optionally, an antigen derived from coronavirus, wherein the coronavirus is SARS-CoV-2 or a variant thereof; preferably, the antibiotic is one selected from the group consisting of tobramycin, anthracycline, butyrosin sulfate, gentamicin, hygromycin, amikacin, kanamycin, nebramycin, β-lactam, metrzamide, neomycin, puromycin, streptomycin and streptozotocin, or a combination therefrom; preferably, the polyamino compound is one selected from the group consisting of spermidine acid salt, spermidine, N-(3-aminopropyl), N-(3-aminopropyl)-1,4-butanediamine, spermine, spermine, OS-dimethylamidothiophosphate, polylysine and aminoglycoside, or a combination therefrom; preferably, the positive ion is a divalent positive ion, and more preferably, the positive ion is one selected from the group consisting of calcium, cadmium, lithium, magnesium, cerium, cesium, chromium, cobalt, deuterium, gallium, iodine, iron and zinc, or a combination therefrom; and most preferably, the positive ion is a calcium ion; and preferably, the antigen derived from coronavirus is one selected from the group consisting of an inactivated virus SARS-CoV-2, an attenuated virus SARS-CoV-2, a virus SARS-CoV-2 that cannot be propagated in a subject, an S protein of SARS-CoV-2 or an immunogenic fragment thereof, an M protein or an immunogenic fragment thereof, an N protein or an immunogenic fragment thereof, an E protein or an immunogenic fragment thereof, and a protein, a polypeptide, RNA, and DNA designed according to an SARS-CoV-2 structure, or a combination therefrom.

2. The immune composition according to claim 1, wherein when the antigen derived from coronavirus is an inactivated virus SARS-CoV-2, an attenuated virus SARS-CoV-2, or a virus SARS-CoV-2 that cannot be propagated in a subject, a ratio of the antigen derived from coronavirus to the PIC is one selected from the group consisting of: 1 active unit/50 μg, 1 active unit/60 μg, 1 active unit/70 μg, 1 active unit/80 μg, 1 active unit/90 μg, 1 active unit/100 μg, 1 active unit/125 μg, 1 active unit/200 μg, 1 active unit/250 μg, 1 active unit/300 μg, 1 active unit/350 μg, 1 active unit/400 μg, 1 active unit/450 μg, 1 active unit/500 μg, 1 active unit/550 μg, 1 active unit/600 μg, 1 active unit/700 μg, 1 active unit/800 μg, 1 active unit/1000 μg, 1 active unit/1500 μg, 1 active unit/2000 μg, 1 active unit/2500 μg, 1 active unit/3000 μg, 1 active unit/4000 μg, 1 active unit/5000 μg, 1 active unit/6000 μg, 1 active unit/7000 μg, 1 active unit/8000 μg, 1 active unit/9000 μg 1 active unit/10000 μg and a range between any two values of the foregoing; or when the antigen derived from coronavirus is one selected from the group consisting of an S protein of SARS-CoV-2 or an immunogenic fragment thereof, an M protein or an immunogenic fragment thereof, an N protein or an immunogenic fragment thereof, an E protein or an immunogenic fragment thereof, and a protein, a polypeptide, DNA, and RNA designed according to a SARS-CoV-2 structure, a ratio of the antigen to the PIC is one selected from the group consisting of: 1 μg/10 μg, 1 μg/20 μg, 1 μg/30 μg, 1 μg/40 μg, 1 μg/50 μg, 1 μg/60 μg, 1 μg/70 μg, 1 μg/80 μg, 1 μg/90 μg, 1 μg/100 μg, 1 μg/125 μg, 1 μg/200 μg, 1 μg/250 μg, 1 μg/300 μg, 1 μg/350 μg, 1 μg/400 μg, 1 μg/450 μg, 1 μg/500 μg, 1 μg/550 μg, 1 μg/600 μg, 1 μg/700 μg, 1 μg/800 μg, 1 μg/1000 μg, 1 μg/1500 μg, 1 μg/2000 μg, 1 μg/2500 μg, 1 μg/3000 μg, 1 μg/4000 μg, 1 μg/5000 μg, 1 μg/6000 μg, 1 μg/7000 μg, 1 μg/8000 μg, 1 μg/9000 μg, 1 μg/10000 μg and a range between any two values of the foregoing.

3. The immune composition according to claim 1, wherein a concentration of the PIC in the immune composition is 250 μg/unit dose to 5000 μg/unit dose; and preferably, a concentration of the PIC in the immune composition is one selected from the group consisting of 250 μg/unit dose, 500 μg/unit dose, 1000 μg/unit dose, 1500 μg/unit dose, 2000 μg/unit dose, 3000 μg/unit dose, 4000 μg/unit dose and 5000 μg/unit dose.

4. The immune composition according to claim 1, wherein when the antigen derived from coronavirus is an inactivated virus SARS-CoV-2, an attenuated virus SARS-CoV-2, or a virus SARS-CoV-2 that cannot be propagated in a subject, a concentration of the antigen derived from coronavirus in the immune composition is 0.1 active unit/unit dose to 100.0 active unit/unit dose, preferably, a concentration of the antigen derived from coronavirus in the immune composition is one selected from the group consisting of 0.5 active units/unit dose, 1.0 active unit/unit dose, 1.5 active units/unit dose, 2.0 active units/unit dose, 2.5 active units/unit dose, 3.0 active units/unit dose, 3.5 active units/unit dose, 4.0 active units/unit dose, 5.0 active units/unit dose, 6.0 active units/unit dose, 7.0 active units/unit dose, 8.0 active units/unit dose, 9.0 active units/unit dose, 10.0 active units/unit dose, 15.0 active units/unit dose, 20.0 active units/unit dose, 30.0 active units/unit dose, 40.0 active units/unit dose, 50.0 active units/unit dose, 60.0 active units/unit dose, 70.0 active units/unit dose, 80.0 active units/unit dose, 90.0 active units/unit dose, 100.0 active units/unit dose and a range between any two values of the foregoing; or when the antigen derived from coronavirus is one selected from the group consisting of an S protein of SARS-CoV-2 or an immunogenic fragment thereof, an M protein or an immunogenic fragment thereof, an N protein or an immunogenic fragment thereof, an E protein or an immunogenic fragment thereof, and a protein, a polypeptide, DNA, and RNA designed according to a SARS-CoV-2 structure, a concentration of the antigen derived from coronavirus in the immune composition is 0.1 μg/unit dose to 1000.0 μg/unit dose, preferably, a concentration of the antigen derived from coronavirus in the immune composition is one selected from the group consisting of 0.5 μg/unit dose, 1.0 μg/unit dose, 2.0 μg/unit dose, 3.0 μg/unit dose, 4.0 μg/unit dose, 5.0 μg/unit dose, 6.0 μg/unit dose, 7.0 μg/unit dose, 8.0 μg/unit dose, 9.0 μg/unit dose, 10.0 μg/unit dose, 15.0 μg/unit dose, 20.0 μg/unit dose, 30.0 μg/unit dose, 40.0 μg/unit dose, 50.0 μg/unit dose, 60.0 μg/unit dose, 70.0 μg/unit dose, 80.0 μg/unit dose, 90.0 μg/unit dose, 100.0 μg/unit dose, 200.0 μg/unit dose, 300.0 μg/unit dose, 400.0 μg/unit dose, 500.0 μg/unit dose, 600.0 μg/unit dose, 700.0 μg/unit dose, 800.0 μg/unit dose, 900.0 μg/unit dose, 1000.0 μg/unit dose and a range between any two values of the foregoing.

5. The immune composition according to claim 3, wherein the unit dose is one selected from the group consisting of 0.1 ml, 0.15 ml, 0.2 ml, 0.5 ml, 1.0 ml, 1.5 ml, 2.0 ml, 2.5 ml, 3.0 ml, 4.0 ml, 5.0 ml, 10.0 ml, 20.0 ml, 30.0 ml, 40.0 ml, 50.0 ml, 60.0 ml, 70.0 ml, 80.0 ml, 90.0 ml, 100.0 ml, 150.0 ml, 200.0 ml, 250.0 ml and a range between any two values of the foregoing.

6. The immune composition according to claim 1, further containing one selected from the group consisting of: gelatin, sucrose, white granulated sugar, lactose, maltose, trehalose, glucose, low molecular dextran, sorbitol, polysorbate 20, polysorbate 80, arginine hydrochloride, mannitol polyethylene glycol, human serum albumin, recombinant albumin, sodium caprylate, urea, aluminium hydroxide, aluminium phosphate, squalene, saponin, oligonucleotide, phenol red, magnesium chloride, potassium chloride, sodium chloride, sodium thiosulfate, potassium dihydrogen phosphate, ascorbic acid, chloroform, phenol and thimerosal, or a combination therefrom.

7. The immune composition according to claim 1, further containing a physiologically acceptable buffer solution, which is one selected from the group consisting of acetate, trishydroxymethylaminomethane, bicarbonate, carbonate and phosphate buffer solution, or a combination therefrom; preferably, the buffer solution has a concentration of 5 mM to 50 mM, preferably 5 mM to 20 mM; and preferably, a pH value of the immune composition is 6 to 9.

8. The immune composition according to claim 1, wherein the immune composition can be prepared in a solid dosage form or a liquid dosage form, wherein the liquid dosage form is one selected from the group consisting of injectable solution, suspension, spray, aerosol, naristillae, ointment, emulsion, drop, syrup and gel; and the solid dosage form is one selected from the group consisting of dry powder, lyophilized powder, tablet, capsule, suppository, granule and sugar-coated lozenge.

9. The immune composition according to claim 1, wherein the PIC is heterogeneous in molecular weight, and has a molecular weight of 50,000 daltons to 1,200,000 daltons; or the PIC is heterogeneous in molecular weight, and has a sedimentation coefficient unit of 5.0 to 24.0.

10. The immune composition according to claim 1, wherein a concentration of the antibiotic or polyamino compound in the immune composition is 400 U/unit dose to 1200 U/unit dose, preferably, 400 U/unit dose, 500 U/unit dose, 600 U/unit dose, 700 U/unit dose, 800 U/unit dose, 900 U/unit dose, 1000 U/unit dose, 1100 U/unit dose, and 1200 U/unit dose; and a concentration of the positive ion in the immune composition is 0.01 mg/unit dose to 0.1 mg/unit dose, preferably, 0.01 mg/unit dose, 0.02 mg/unit dose, 0.03 mg/unit dose, 0.04 mg/unit dose, 0.05 mg/unit dose, 0.06 mg/unit dose, 0.07 mg/unit dose, 0.08 mg/unit dose, 0.09 mg/unit dose and 0.1 mg/unit dose.

11. The immune composition according to claim 1, wherein the antigen derived from coronavirus is one selected from the group consisting of: a SARS-CoV-2 spike protein of 2.5 μg/unit dose to 20 μg/unit dose, a SARS-CoV-2 spike protein multimer of 2.5 μg/unit dose to 20 μg/unit dose, an immunogenic fragment of the SARS-CoV-2 spike protein of 2.5 μg/unit dose to 20 μg/unit dose, a SARS-CoV-2 spike protein RBD region of 2.5 μg/unit dose to 20 μg/unit dose, and inactivated SARS-CoV-2 virion of 300 active units/unit dose to 1000 active units/unit dose, wherein preferably, the multimer is dimer, trimer, or tetramer; and preferably, the multimer is homomultimer.

12. The immune composition according to claim 1, containing: an antigen derived from coronavirus, 0.5 mg/ml to 2 mg/ml PIC, 400 units/ml to 1200 units/ml kanamycin, 0.01 mg/ml to 0.1 mg/ml calcium chloride, 100 mM to 200 mM sodium chloride, and 5 mM to 20 mM phosphate buffer solution, and optionally, further containing one selected from the group consisting of 100 mM to 200 mM arginine hydrochloride, 0.005% w/v to 0.05% w/v polysorbate 80, and 0.1% w/w to 1.0% w/w aluminum phosphate, or a combination therefrom.

13. Use of the immune composition according to claim 1 in preparation of a vaccine or medicament for preventing or treating coronavirus infection, wherein preferably, the coronavirus is SARS-CoV-2 or a variant thereof; and preferably, the vaccine or medicament is prepared in a dosage form suitable for any one of a route of administration selected form the group consisting of intramuscular, intraperitoneal, intravenous, subcutaneous, transdermal, intradermal, oral, sublingual and respiratory tract (nose, pharynx, trachea and bronchus) administration.

14. A method for preventing or treating coronavirus SARS-CoV-2 infection, comprising: administering to a subject a prophylactically or therapeutically effective amount of the immune composition according to claim 1, wherein preferably, the immune composition is administered to the subject at a following frequency: 1 to 4 times every four years, 1 to 3 times every three years, 1 to 2 times every two years, once a year, twice a year, 3 times a year, 4 times a year, 5 times a year, 6 times a year, once a month, twice a month, 3 times a month, 4 times a month, 5 times a month, 6 times a month, 7 times a month, 8 times a month, once a week, twice a week, 3 times a week, 4 times a week, 5 times a week, 6 times a week, once every 3 days, twice every 3 days, 3 times every 3 days, once every 2 days, twice every 2 days, once a day, and twice a day; a time interval between the administering is the same or different; and a route of the administering is one selected from the group consisting of intramuscular, intraperitoneal, intravenous, subcutaneous, transdermal, intradermal, oral, sublingual and respiratory tract (nose, pharynx, trachea and bronchus) administration.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0077] FIG. 1 shows a pharmaceutical composition of the present disclosure activating body DC to upregulate costimulatory molecule production.

[0078] FIG. 2A to FIG. 2B show that the concentration of cytokines in the lungs can be effectively increased after administration of the pharmaceutical composition of the present disclosure to mice by nasal drip.

[0079] FIG. 3: Titers of IgG subtype antibodies.

[0080] FIG. 4: Titers of IgG antibodies against novel coronavirus S protein in rabbit serum.

[0081] FIG. 5: Neutralizing antibodies induced by different pharmaceutical compositions have different titers.

[0082] FIG. 6A to FIG. 6D: Proportion of cells expressing IL-2, IFN-γ.

[0083] FIG. 7: Virus load in lungs after challenge of mice.

[0084] FIG. 8: Virus load in lungs after challenge of mice.

DETAILED DESCRIPTION OF EMBODIMENTS

[0085] “Antigen” refers to any substance (e.g. protein, peptide, cancer cell, glycoprotein, glycolipid, live virus, killed virus, and DNA) that can be recognized by a host's immune system and induce an immune response when it invades the host. The antigen can be provided in a purified or unpurified form. The antigen in the present disclosure includes protein of pathogen, recombinant protein, peptide, polysaccharide, glycoprotein, glycolipid and polynucleotide, live virus, recombinant virus, attenuated or inactivated virus.

[0086] Immunogenic fragment refers to a portion of the foregoing “antigen” (e.g. when the antigen is a protein or polypeptide, the immunogenic fragment is a non-full-length peptide fragment of the protein or polypeptide), which still retains (in whole or in part) the ability to be recognized by the host's immune system and induce the immune response.

[0087] Inactivation refers to removing the pathogenic ability and propagation ability of the virus, but still retain the ability to stimulate the human body to produce the immune response. The methods for inactivating viruses are well known in the art. Any commonly used method can be used to inactivate viruses, and can be selected as appropriate according to virus types. These methods for inactivating viruses include, but are not limited to, using a photoreactive compound, an oxidant, radiation (e.g. UV rays; γ-rays), a combination of riboflavin and UV rays, solvent-detergent treatment (e.g. treatment with organic solvent tri-N-butyl-phosphate and Tween 80 detergent), polyethylene glycol treatment, pasteurization (heat treatment), low pH treatment, enzyme treatment (pepsin or trypsin), methylene blue-light treatment, treatment with dimethylmethylene blue and visible light, and treatment with psoralen derivative S-59 and UVA irradiation.

[0088] Attenuation: Attenuated viruses are still alive, while the viruses lose pathogenicity during preparation, but still retain the propagation capacity and the ability to stimulate the human body to produce an immune response.

[0089] Herein, the term spike protein (Spike or S protein) covers variants thereof. The variant of spike protein refers to a polypeptide that is substantially homologous to natural spike polypeptide, but has an amino acid sequence different from the natural spike protein due to the deletion, insertion or substitution of one or more amino acids. The variant may contain a sequence of conservative substitution, wherein the conservative substitution refers to substitution of a given amino acid residue by a residue having similar biochemical properties. Such conservative substitution is known, and examples of conservative substitution include substitution of aliphatic residues with each other, such as substitution of Ile, Val, Leu, or Ala with each other; or substitution of polar residues with each other, for example, between Lys and Arg; between Glu and Asp; and between Gln and Asn. The present disclosure also includes naturally occurring spike polypeptide variants. Examples of such variants are proteins resulting from alternative mRNA splicing events or protease cleavage of the spike polypeptide. Variations that may be attributable to proteolysis include, for example, variations having differences in terminal upon expression in different types of host cells, due to proteolytic removal of one or more terminal amino acids from the spike polypeptide.

[0090] When the SARS-CoV-2 antigen of the present disclosure is a protein or polypeptide, it is possible to prepare it into a form of multimer (homomultimer or heteromultimer), for example, but not limited to, dimer, trimer, tetramer, and pentamer, thereby providing more antigen contact opportunities. In a specific example, the skilled person fuses a segment of fold at the N- or C-terminus of the SARS-CoV-2 antigen, this segment of fold facilitates the antigen in forming the multimer form. Such fold is well known in the art and does not affect the correct folding of the antigen in the present disclosure or affect the immunogenicity. As an example, folds that can be used for trimerization allow for the formation of trimeric structures when attached (also referred to as fused) to proteins or peptides. In the present disclosure, any known trimerization fold/domain can be used. Examples of trimerization folds/domains include, but are not limited to: HIV-1gp41 trimerization domain, SIV gp41 trimerization domain, Ebola virus gp-2 trimerization domain, HTLV-1gp-21 trimerization domain, T4 fibritin trimerization domain, yeast heat shock transcription factor trimerization domain, and human collagen trimerization domain. For another example, in CN111560074A, a twenty-four-polymer nano antigen can be formed by HP_Ferritin self-assembly function; and in CN111607605A, a pentamer of fusion protein is obtained by means of the characteristic that LTB26 can be self-assembled to form a pentamer.

[0091] The term “unit dose” as used in the present disclosure refers to a unit that is physically isolated, and suitable as a unit dose used for a subject individual. Each unit contains a predetermined amount of the composition of the present disclosure, wherein the predetermined amount is an amount sufficient to produce the desired effect together with a pharmaceutically/physiologically acceptable diluent, carrying agent or carrier.

[0092] The term “effective amount” of a composition herein refers to an amount of the composition that is non-toxic but sufficient to provide a desired reaction (e.g. an immunoreaction) and produce a corresponding preventive/therapeutic effect. The exact amount required varies from subject to subject, depending on their species, ages, overall conditions of the subjects, the severity of the condition to be treated, and particular macromolecules of interest, the manner of administration, etc. The suitable “effective amount” in a particular example can be determined by one of ordinary skill in the art using conventional experimental methods, for example, determining an effective amount of a human subject through animal studies.

[0093] The term “treatment” as used in the present description refers to obtaining a desired pharmacological and/or physiological effect. The effect may be preventive from the perspective of completely and/or partially preventing diseases or symptoms thereof, and/or the effect may be medical from the perspective of completely and/or partially stabilizing or curing diseases and/or negative effects caused by the diseases.

[0094] In the present description, when a numerical range is described, the expressions “ . . . to . . . ”, “within a range” or “between ranges” used include endpoint values.

[0095] In the context of the present description, pharmaceutical composition, medicine (drug), immune composition, and vaccine may be used interchangeably.

EXAMPLES

[0096] Unless otherwise specified, the operation for test animals of the present disclosure follows Regulations for the Administration of Laboratory Animals, Guidelines for the Humane Treatment of Laboratory Animals, and National Standard GB/14925.

Example 1: Preparation Method of Pharmaceutical Composition 1 of the Present Disclosure

[0097] 1. Antigen: recombinant novel coronavirus S-trimer protein.

[0098] 2. Preparation of the composition of the present disclosure according to the following ingredients:

[0099] the composition 1 (with a unit dose of 1 ml) of the present disclosure contains: [0100] recombinant novel coronavirus S-trimer protein (2.5 μg, 5 μg, 6 μg, 10 μg, and 20 μg), [0101] PIC (1 mg/ml), [0102] kanamycin (800 units/ml), and [0103] calcium chloride (0.0472 mg/ml).

[0104] The above composition was formulated in a physiologically acceptable buffer solution (sodium chloride 150 mM, disodium hydrogen phosphate 0.0043 M, and sodium dihydrogen phosphate 0.0017 M) under sterile conditions.

TABLE-US-00001 TABLE 1 Ingredients Dosage (1 ml) Concentration recombinant novel 2.5 μg, 2.5 μg/ml, coronavirus S-trimer 5 μg, 5 μg/ml, protein 6 μg, 6 μg/ml, 10 μg, 10 μg/ml, 20 μg, 20 μg/ml, PIC 1 mg 1 mg/ml kanamycin 800 units 800 units/ml calcium chloride 0.0472 mg 0.0472 mg/ml sodium chloride 8.77 mg 150 mM disodium hydrogen 0.61 mg 0.0043M phosphate sodium dihydrogen 0.204 mg 0.0017M phosphate

Example 2: Preparation Method of Pharmaceutical Composition 2 of the Present Disclosure

[0105] 1. Antigen: recombinant novel coronavirus S-trimer protein.
2. Preparation of the composition of the present disclosure according to the following ingredients:

[0106] the composition 2 (with a unit dose of 1 ml) of the present disclosure contains: [0107] recombinant novel coronavirus S-trimer protein (2.5 μg, 5 μg, 10 μg, and 20 μg), [0108] PIC (1 mg/ml), [0109] kanamycin (800 units/ml), [0110] calcium chloride (0.0472 mg/ml), [0111] arginine hydrochloride (140 mM), and [0112] polysorbate 80 (0.01% w/v).

[0113] The above composition was formulated in a physiologically acceptable buffer solution (sodium chloride 150 mM, disodium hydrogen phosphate 0.0043 M, and sodium dihydrogen phosphate 0.0017 M) under sterile conditions.

TABLE-US-00002 TABLE 2 Ingredients Dosage (1 ml) Concentration recombinant novel 2.5 μg, 2.5 μg/ml, coronavirus S-trimer protein 5 μg, 5 μg/ml, 10 μg, 10 μg/ml, 20 μg, 20 μg/ml, PIC 1 mg 1 mg/ml kanamycin 800 units 800 units/ml calcium chloride 0.0472 mg 0.0472 mg/ml arginine hydrochloride 29.492 mg 140 mM polysorbate 80 0.1 mg 0.01% w/v sodium chloride 8.77 mg 150 mM disodium hydrogen 0.61 mg 0.0043M phosphate sodium dihydrogen 0.204 mg 0.0017M phosphate

Example 3: Preparation Method of Pharmaceutical Composition 3 of the Present Disclosure

[0114] 1. Antigen: recombinant novel coronavirus S1 protein.
2. Preparation of the composition of the present disclosure according to the following ingredients:

[0115] the composition 3 (with a unit dose of 1 ml) of the present disclosure contains: [0116] recombinant novel coronavirus S1 protein (2.5 μg, 5 μg, 6 μg, 10 μg, and 20 μg), [0117] PIC (1 mg/ml), [0118] kanamycin (800 units/ml), and [0119] calcium chloride (0.0472 mg/ml).

[0120] The above composition was formulated in a physiologically acceptable buffer solution (sodium chloride 150 mM, disodium hydrogen phosphate 0.0043 M, and sodium dihydrogen phosphate 0.0017 M) under sterile conditions.

TABLE-US-00003 TABLE 3 Ingredients Dosage (1 ml) Concentration recombinant novel 2.5 μg, 2.5 μg/ml, coronavirus S1 protein 5 μg, 5 μg/ml, 6 μg, 6 μg/ml, 10 μg, 10 μg/ml, 20 μg, 20 μg/ml, PIC 1 mg 1 mg/ml kanamycin 800 units 800 units/ml calcium chloride 0.0472 mg 0.0472 mg/ml sodium chloride 8.77 mg 150 mM disodium hydrogen 0.61 mg 0.0043M phosphate sodium dihydrogen 0.204 mg 0.0017M phosphate

Example 4: Preparation Method of Pharmaceutical Composition 4 of the Present Disclosure

[0121] 1. Antigen: recombinant novel coronavirus RBD protein (i.e., spike protein RBD region of SARS-CoV-2).
2. Preparation of the composition of the present disclosure according to the following ingredients:

[0122] the composition 4 (with a unit dose of 1 ml) of the present disclosure contains: [0123] recombinant novel coronavirus RBD protein (2.5 μg, 5 μg, 6 μg, 10 μg, and 20 μg), [0124] PIC (1 mg/ml), [0125] kanamycin (800 units/ml), and [0126] calcium chloride (0.0472 mg/ml).

[0127] The above composition was formulated in a physiologically acceptable buffer solution (sodium chloride 150 mM, disodium hydrogen phosphate 0.0043 M, and sodium dihydrogen phosphate 0.0017 M) under sterile conditions.

TABLE-US-00004 TABLE 4 Ingredients Dosage (1 ml) Concentration Recombinant novel 2.5 μg, 2.5 μg/ml, coronavirus RBD 5 μg, 5 μg/ml, protein 6 μg, 6 μg/ml, 10 μg, 10 μg/ml, 20 μg, 20 μg/ml, PIC 1 mg 1 mg/ml kanamycin 800 units 800 units/ml calcium chloride 0.0472 mg 0.0472 mg/ml sodium chloride 8.77 mg 150 mM disodium hydrogen 0.61 mg 0.0043M phosphate sodium dihydrogen 0.204 mg 0.0017M phosphate

Example 5: Preparation Method of Pharmaceutical Composition 5 of the Present Disclosure

[0128] Composition 5 (with a unit dose of 1 ml) of the present disclosure contains: [0129] PIC (1 mg/ml), [0130] kanamycin (800 units/ml), and [0131] calcium chloride (0.0472 mg/ml).

[0132] The above composition was formulated in a physiologically acceptable buffer solution (sodium chloride 150 mM, disodium hydrogen phosphate 0.0043 M, and sodium dihydrogen phosphate 0.0017 M) under sterile conditions.

TABLE-US-00005 TABLE 5 Ingredients Dosage (1 ml) Concentration PIC 1 mg 1 mg/ml kanamycin 800 units 800 units/ml calcium chloride 0.0472 mg 0.0472 mg/ml sodium chloride 8.77 mg 150 mM disodium hydrogen 0.61 mg 0.0043M phosphate sodium dihydrogen 0.204 mg 0.0017M phosphate

Example 6: Preparation Method of Pharmaceutical Composition 6 of the Present Disclosure

[0133] 1. Antigen: inactivated and purified novel coronavirus.

[0134] 2. Preparing the composition 6 (with a unit dose of 1 ml) of the present disclosure according to the following ingredients, containing: [0135] inactivated and purified novel coronavirus (600 active units), [0136] PIC (1 mg/ml), [0137] kanamycin (800 units/ml), [0138] calcium chloride (0.0472 mg/ml), and [0139] aluminium phosphate (with an aluminium content of 0.5% w/w).

[0140] The above composition was formulated in a physiologically acceptable buffer solution (sodium chloride 150 mM, disodium hydrogen phosphate 0.0043 M, and sodium dihydrogen phosphate 0.0017 M) under sterile conditions.

TABLE-US-00006 TABLE 6 Ingredients Dosage (1 ml) Concentration inactivated and 600 600 purified novel active units active units/ml coronavirus PIC 1 mg 1 mg/ml kanamycin 800 units 800 units/ml calcium chloride 0.0472 mg 0.0472 mg/ml aluminium 0.005 ml 0.5% w/w phosphate sodium chloride 8.77 mg 150 mM disodium 0.61 mg 0.0043M hydrogen phosphate sodium 0.204 mg 0.0017M dihydrogen phosphate

Test Example 1: The Pharmaceutical Composition 5 of the Present Disclosure Activates Dendritic Cells of the Body to Upregulate Costimulatory Molecules

[0141] Method: C57BL/6 mouse myeloid-derived dendritic cells (BMDC) were in vitro co-incubated with the pharmaceutical composition 5 of the present disclosure for 18 hours. The proportions and mean fluorescence intensities of CD80+, CD86+, and CD40+ cells were detected with flow cytometry.

[0142] Result: As shown in FIG. 1, after the pharmaceutical composition 5 of the present disclosure interacted with the mouse BMDC, the proportion of CD80+ cells was increased from 59.82% to 90.66%, the proportion of CD86+ cells was increased from 11.70% to 38.66%, and the proportion of CD40+ cells was increased from 16.98% to 58.62%, significantly upregulating the production of costimulatory molecules.

[0143] Conclusion: The present experiment proves that the pharmaceutical composition 5 of the present disclosure can significantly induce dendritic cells of the body to upregulate costimulatory molecules, and promote activation of the dendritic cells.

Test Example 2: After Nasal Delivery, the Pharmaceutical Composition 5 of the Present Disclosure can Effectively Increase the Concentration of Cytokines in the Lungs

[0144] Method: The mice were intranasally administered with PBS or the pharmaceutical composition 5 of the present disclosure, respectively, and sacrificed at specified time. Lung tissues were collected, and the TNF-α, IFN-γ, and KC of the sample were repeatedly tested by a Bio-plex protein array system; and IFN-β was detected with an ELISA kit.

TABLE-US-00007 TABLE 7 Laboratory Animals Grouping Administration Administration Samples Dosage Mode PBS 50 μl per i.n. mouse pharmaceutical 50 μl per i.n. composition 5 of the mouse present disclosure Result: See FIG. 2A to FIG. 2B.

[0145] Conclusion: After intranasally administering the mice with the pharmaceutical composition 5 of the present disclosure, the cytokines TNF-α, IFN-γ, KC, and IFN-β in the mouse lungs were significantly increased, indicating that nasally administering the mice can effectively activate the intrinsic immunity of the respiratory tract, causing the immune cells to produce cytokines and chemokines. These cytokines and chemokines are associated with inhibition of the respiratory virus replication of mice.

Test Example 3: The Pharmaceutical Composition 1 of the Present Disclosure can Effectively Induce the Mice to Produce IgG Antibodies (IgG1, IgG2a, IgG3) Against the Novel Coronavirus S1 Protein

[0146] Method: The pharmaceutical composition 1 of the present disclosure was used to immunize the mice. The blood was taken, and the serum was isolated, Titers of IgG antibody subtypes against the novel coronavirus S1 protein in mouse serum was detected by the ELISA method.

TABLE-US-00008 TABLE 8 Laboratory Animals Grouping Administration Administration Immunization Sample Dosage Mode Time Number pharmaceutical 0.1 ml i.m. immunizing once on 8 composition 1 of the per mouse days 0, 7, and 14 present disclosure (antigen of 10 μg)

[0147] Conclusion: The pharmaceutical composition of the present disclosure can significantly increase the titers of IgG subtype antibodies against the novel coronavirus S1 protein in serum (FIG. 3).

Test Example 4: The Pharmaceutical Composition 1 of the Present Disclosure can Effectively Induce Rabbits to Produce IgG Antibodies Against the Novel Coronavirus S Protein

[0148] The rabbits were immunized with the pharmaceutical composition 1 of the present disclosure. The blood was taken, and the serum was isolated. Titers of the IgG antibodies against the novel coronavirus S protein in the rabbit serum was detected by the ELISA method.

TABLE-US-00009 TABLE 9 Laboratory Animals Grouping Administration Administration Immunization Sample Dosage Mode Time Number pharmaceutical 1 ml per i.m. immunizing 5 composition 1 rabbit once on days (antigen of 5 μg) 0 and 7 of the present disclosure

[0149] Conclusion: The pharmaceutical composition of the present disclosure can significantly increase the titers of IgG antibodies in the rabbit serum against the novel coronavirus S protein (FIG. 4).

Test Example 5: The Pharmaceutical Compositions 1 and 3-4 of the Present Disclosure can Effectively Induce Rabbits to Produce Neutralizing Antibodies Against the Novel Coronavirus

[0150] Method: The pharmaceutical compositions 1 and 3-4 of the present disclosure were used respectively to immunize the rabbits. The blood was taken, and the serum was isolated. Titers of neutralizing antibodies in the rabbit serum was detected through pseudovirus neutralizing test.

TABLE-US-00010 TABLE 10 Laboratory Animals Grouping Administration Administration Immunization Samples Dosage Mode Time Number pharmaceutical 1 ml per rabbit i.m. immunizing once on 3 composition 1 (antigen of days 0, 7, and 14 6 μg) of the present disclosure pharmaceutical 1 ml per rabbit i.m. immunizing once on 3 composition 3 (antigen of days 0, 7, and 14 6 μg) of the present disclosure 1 ml per rabbit i.m. immunizing once on 3 pharmaceutical days 0, 7, and 14 composition 4 (antigen of 6 μg) of the present disclosure

[0151] Conclusion: The pharmaceutical compositions 1 and 3-4 of the present disclosure can significantly increase the titers of neutralizing antibodies in rabbit serum, and titers of neutralizing antibodies induced and produced by different pharmaceutical compositions are different (FIG. 5).

Test Example 6: The Pharmaceutical Composition 2 of the Present Disclosure Effectively Induces the Mice to Produce Novel Coronavirus-Specific Cellular Immunoreaction

[0152] Method: The pharmaceutical composition 2 of the present disclosure was used to immunize the mice. Spleen was taken, and a spleen cell suspension was isolated. Antigen-specific cellular immunoreaction was detected by the Elispot method.

TABLE-US-00011 TABLE 11 Laboratory Animals Grouping Administration Administration Immunization Sample Dosage Mode Time Number pharmaceutical 0.1 ml per i.m. immunizing once on 8 composition 2 mouse days 0, 7, and 14 (antigen: low dosage 2.5 μg, medium dosage: 5 μg, and high dosage: 10 μg) of the present disclosure

[0153] Conclusion: The pharmaceutical composition 2 of the present disclosure can significantly improve the mouse cellular immunoreaction, and significantly increase the proportions of cells expressing IL-2 and IFN-γ (see FIG. 6A to FIG. 6D for the results).

Test Example 7: The Pharmaceutical Composition 1 of the Present Disclosure Effectively Reduces the Novel Coronavirus Load in hACE2 Mice

[0154] Method: The hACE2 transgenic mice (6-week old, 18-26 g, SARS-CoV-2 infected hACE2 transgenic mouse models) were immunized with the pharmaceutical composition 1 of the present disclosure, and were subjected to challenge by nasal drip with SARS-CoV-2 14 days after primary immunization, and 5 days after the challenge, lung tissues were taken to detect the lung virus load.

TABLE-US-00012 TABLE 12 Laboratory Animals Grouping Challenge Administration Administration Immunization Dosage and Samples Dosage Mode Time Challenge Time Volume Number pharmaceutical 200 μl per i.m. immunizing challenge 14 days 10.sup.5 TCID.sub.50 6 composition 1 mouse once on days 0 after the primary per mouse (antigen of 5 μg) and 7 immunization 50 μl of the present disclosure

[0155] Conclusion: The pharmaceutical composition 1 of the present disclosure can significantly reduce the lung virus load of the mice after challenge, relieve the pulmonary inflammation to some extent, and have a good preventive effect (see FIG. 7 for the results).

Test Example 8: The Pharmaceutical Composition 2 of the Present Disclosure Effectively Reduces the Novel Coronavirus Load in hACE2 Mice

[0156] Method: The hACE2 mice were subjected to intranasal challenge with SARS-CoV-2, and simultaneously administered with the pharmaceutical composition 2 of the present disclosure. The lung tissues were taken 5 days after the challenge to detect the virus load in lungs.

TABLE-US-00013 TABLE 13 Laboratory Animals Grouping Challenge Administration Administration Immunization Challenge Dosage and Sample Dosage Mode Time Time Volume Number pharmaceutical 200 μl per i.m. administering the 6th day 0.5 * 10.sup.6 TCID.sub.50 6 composition 2 mouse continuously 5 after the first per mouse (antigen of 10 days before time of 50 μl μg) of the challenge, immunization present and stopping disclosure immunization on the 4th day after challenge

[0157] Conclusion: The pharmaceutical composition 2 of the present disclosure can significantly reduce the lung virus load of the mice after challenge, and have a good therapeutic effect (see FIG. 8 for the results).

Test Example 9: The Pharmaceutical Composition 2 of the Present Disclosure can Effectively Induce Machin to Produce IgG Antibodies Against the Novel Coronavirus S Protein

[0158] Method: The machins were immunized with the pharmaceutical composition 2 of the present disclosure. The blood was taken, and the serum was isolated. Titers of IgG antibodies against novel coronavirus S protein in machin serum were detected by the ELISA method.

TABLE-US-00014 TABLE 14 Laboratory Animals Grouping Administration Administration Immunization Samples Dosage Mode Time Number pharmaceutical 1 ml per i.m. immunizing once on 5 composition 2 machin days 1, 8, 15, and 29 (low dosage antigen 10 μg) of the present disclosure pharmaceutical 3 ml per i.m. immunizing once on 5 composition 2 machin days 1, 8, 15, and 29 (high dosage antigen 30 μg) of the present disclosure

[0159] Conclusion: The pharmaceutical composition of the present disclosure can significantly increase the titers of IgG antibodies in machin serum against novel coronavirus S protein. The specific IgG antibodies can be detected 1 week after the second time of immunization of the animals in the groups of the low dosage of the pharmaceutical composition 2 of the present disclosure and the high dosage of the pharmaceutical composition 2 of the present disclosure (D15, before the third time of administration), and the occurrence rates are 10/10 and 10/10, respectively, and the antibody titer ranges from 1:1600 to >1:102400. As the number of times of administration increases, the titers of the specific IgG antibodies of animals in the low-dosage and high-dosage groups both are visibly increased. After 4 weeks of drug discontinuance and recovery (D57), no titers of specific IgG antibodies of all animals in both the low-dosage and high-dosage groups are decreased.

Test Example 10: The Pharmaceutical Composition 6 of the Present Disclosure can Effectively Induce Rabbits to Produce Neutralizing Antibodies Against the Novel Coronavirus

[0160] Method: The rabbits were immunized with the pharmaceutical composition 6 of the present disclosure. The blood was taken, and the serum was isolated. Titers of neutralizing antibodies in the rabbit serum were detected by neutralizing test of SARS-CoV-2 wild strain.

TABLE-US-00015 TABLE 15 Laboratory Animals Grouping Administration Administration Immunization Sample Dosage Mode Time Number pharmaceutical 1 ml per rabbit i.m. immunizing once 3 composition 6 on days 0 and 7 (antigen: 600 active units) of the present disclosure

[0161] The pharmaceutical composition 6 of the present disclosure can induce the titer of neutralizing antibodies in the rabbit serum.

[0162] The pharmaceutical compositions for preventing and treating the novel coronavirus SARS-CoV-2 infection provided by the present disclosure can achieve the following effects.

[0163] 1. The pharmaceutical effect is better and the efficacy is significant. The ingredients of the pharmaceutical compositions of the present disclosure can activate interferon regulatory factor 3 and NF-kB, upregulate the expression of I-type interferon and proinflammatory cytokines, activate natural immunity and acquired immunity of the human body, and play an important role in resisting virus. A plurality of animal test results show that the ingredients of the pharmaceutical compositions of the present disclosure can effectively reduce the titer and load of the respiratory tract viruses of animals.

[0164] 2. The pharmaceutical compositions of the present disclosure can simultaneously achieve the preventive and therapeutic effects.

[0165] 3. The dosage form is flexible, the injection dosage form may be adopted, the action is rapid and reliable, the medicine liquid can be directly injected into tissues, without being affected by pH, enzyme, food, etc., and without first pass effect, and the drug content is not easy to lose, therefore, the pharmaceutical compositions of the present disclosure have reliable efficacy and can be used to rescue critical patients. The spray preparation also can be adopted, which is convenient for administration, easy to be accepted by patients, and rapidly absorbed through mucosa, takes effect quickly, and has good patient compliance.