SARS-COV-2 VACCINE

Abstract

Disclosed is a SARS-CoV-2 vaccine, wherein the S protein of SARS-CoV-2 serves as the antigen, and the vaccine comprises an adenoviral vector, and the vaccine induces an improved protective immune response through mucosal immunity, thus preventing SARS-CoV-2 infection. Specifically, when atomized by an appropriate apparatus, the vaccine generates particles of improved uniformity, which can reach the lungs after being inhaled via the nasal cavity or the oral cavity, thus producing a protective immune response with respect to the entire respiratory tract and the lungs, enhancing the effective utilization rate of the vaccine, and increasing the effect of the vaccine.

Claims

1-16. (canceled)

17. A SARS-CoV-2 recombinant adenoviral vector vaccine, wherein the vaccine is a transmucosal drug delivery system, and the vaccine comprises an S protein gene of SARS-CoV-2 which is inserted into a recombinant adenoviral vector.

18. The vaccine according to claim 17, wherein the vaccine induces mucosal immune, and the vaccine further comprises pharmaceutically acceptable auxiliary materials.

19. The vaccine according to claim 18, wherein the pharmaceutically acceptable auxiliary materials are selected from a buffer, a protective agent, a stabilizer, a surfactant and an osmotic pressure regulator.

20. The vaccine according to claim 17, wherein the adenoviral vector comprises a human adenovirus type 5 or a chimpanzee adenoviral vector.

21. The vaccine according to claim 20, wherein the human adenovirus is AdHu2 type or AdHu5 type, and the chimpanzee adenoviral vector is selected from AdC6 type, AdC7 type, AdC63 type and AdC68 type.

22. The vaccine according to claim 17, wherein the transmucosal drug delivery system is selected from: a nasal drop, an aerosol, a spray, a powder spray, a powder, gel, a microsphere agent, a liposome, a membrane, and a suspension.

23. The vaccine according to claim 17, wherein the recombinant adenovirus further comprises structural protein genes of SARS-CoV-2.

24. The vaccine according to claim 23, wherein the recombinant adenovirus comprises an S protein gene and an M protein gene of SARS-CoV-2; or, the recombinant adenovirus comprises an S protein gene and an E protein gene of SARS-CoV-2; or, the recombinant adenovirus comprises an S protein gene, an M protein gene and an E protein gene of SARS-CoV-2.

25. The vaccine according to claim 18, wherein the vaccine comprises: a recombinant adenoviral vector, sucrose, mannitol, sodium chloride, HEPES, magnesium chloride, Tween 80 and gelatin.

26. The vaccine according to claim 18, wherein the vaccine comprises: a recombinant adenoviral vector vaccine, sucrose, mannitol, sodium chloride, glycerin, HEPES, magnesium chloride, Tween 80, His, EDTA, human serum albumin and gelatin.

27. The vaccine according to claim 25, wherein the vaccine is a liquid dosage form, comprising: 1×10.sup.9-5×10.sup.11VP/ml of a recombinant adenovirus, 10-50 mg/ml of sucrose, 15-75 mg/ml of mannitol, 40-60 mM of sodium chloride, 1-5 mM of HEPES, 1-5 mM of magnesium chloride, 0.05-0.5 mg/ml of Tween 80, and 5-15 mg/ml of gelatin.

28. The vaccine according to claim 26, wherein the vaccine is a liquid dosage form, comprising: 1×10.sup.9-5×10.sup.11VP/ml of a recombinant adenovirus, 10-50 mg/ml of sucrose, 15-75 mg/ml of mannitol, 40-60 mM of sodium chloride, 0.5-5 mg/ml of glycerin, 1-5 mm of HEPES, 1-5 mm of magnesium chloride, 0.05-0.5 mg/ml of Tween 80, 3-7 mM of His, 0.05-0.5 mM of EDTA, 0.1%-1% of HSA, and 5-15 mg/ml of gelatin.

29. The vaccine according to claim 25, wherein the vaccine is a liquid dosage form, comprising: 1×10.sup.11VP/ml of a recombinant adenovirus, 25 mg/ml of sucrose, 50 mg/ml of mannitol, 50 mM of sodium chloride, 2.5 mM of HEPES, 2 mM of magnesium chloride, 0.1 mg/ml of Tween 80, and 10 mg/ml of gelatin.

30. The vaccine according to claim 26, wherein the vaccine is a liquid dosage form, comprising:1×10.sup.11VP/ml of a recombinant adenovirus, 25 mg/ml of sucrose, 50 mg/ml of mannitol, 50 mM of sodium chloride, 1.5 mg/ml of glycerin, 2.5 mM of HEPES, 2 mM of magnesium chloride, 0.1 mg/ml of Tween 80, 5 mM of His, 0.1 mM of EDTA, 0.60% of HSA, and 10 mg/ml of gelatin.

31. The vaccine according to claim 17, wherein the vaccine is a freeze-dried preparation.

32. A method for preventing SARS-CoV-2 infection, comprising administering a vaccine, wherein the vaccine comprises an S protein gene of SARS-CoV-2, and the vaccine is a transmucosal drug delivery system.

33. A method for preventing SARS-CoV-2 infection, comprising administering a recombinant adenovirus vaccine, wherein the recombinant adenovirus is inserted with an S protein gene of SARS-CoV-2, and the vaccine is a transmucosal drug delivery system.

34. The method according to claim 32, wherein the transmucosal drug delivery system is selected from: a nasal drop, an aerosol, a spray, a powder spray, a powder, gel, a microsphere agent, a liposome, a membrane, and a suspension.

35. The method according to claim 32, wherein the transmucosal drug delivery system is a spray.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0070] FIG. 1 shows an electron micrograph of the recombinant SARS-CoV-2 adenoviral vector.

[0071] FIG. 2 shows the results of antibody titers in serum after intramuscular and aerosolized administration of the recombinant SARS-CoV-2 adenoviral vector vaccine.

[0072] FIG. 3 shows the results of antibody titers in alveolar lavage fluid after intramuscular and aerosolized administration of the recombinant SARS-CoV-2 adenoviral vector vaccine.

[0073] FIG. 4 shows cellular immunity in serum after intramuscular and aerosolized administration of the recombinant SARS-CoV-2 adenoviral vector vaccine.

[0074] FIG. 5 shows cellular immunity in alveolar lavage after intramuscular and aerosolized administration of the recombinant SARS-CoV-2 adenoviral vector vaccine.

DETAILED DESCRIPTION

[0075] Unless defined otherwise, all scientific and technical terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this invention relates.

[0076] The “adenoviral vector vaccine” refers to the vaccine made by recombining the target antigen gene (in the present invention, for example, the S protein gene of SARS-CoV-2) into the adenovirus genome by using the adenovirus as the vector, and using the recombinant adenovirus that can express the antigen gene. Specifically, the gene of SARS-CoV-2 and its various structural proteins can be retrieved through well-known technologies in the art. For example, the gene of SARS-CoV-2 can be shown as GenBank: MT419849.1, and the genes of its various structural proteins: S protein, E protein, M protein can be shown as 21387-25208, 26069-26296, 26347-27015 of GenBank: MT419849.1.

[0077] The technical solution of the invention will be described clearly and completely in combination with the embodiments of the invention. Obviously, the described embodiments are only part of the embodiments of the invention, not all of them. Based on the embodiments of the invention, all other embodiments obtained by those of ordinary skill in the art without creative work belong to the scope of the invention.

[0078] The human type 5 adenoviral vector and the chimpanzee virus vector belong to the same viral vector vaccine, and the transmucosal drug delivery systems developed by two different vectors have similar effects on immunizing animals. In this embodiment, human type 5 adenoviral vector is taken as an example to explain.

EMBODIMENT 1: PACKAGING OF THE RECOMBINANT NOVEL CORONAVIRUS (SARS-COV-2) ADENOVIRAL VECTOR VACCINE

[0079] The S antigen gene of SARS-CoV-2 is connected to the shuttle plasmid of the adenovirus, and then the shuttle plasmid co-transfects 293 cells with the skeleton plasmid carrying most of the adenovirus genome. After the shuttle plasmid and the skeleton plasmid co transfect the cells, the Cre/loxP system is used to achieve recombination, thus producing the recombinant adenovirus.

[0080] For the successfully packaged recombinant adenovirus, single plaques are serially picked for three times, and those with strong infectivity, high expression and fast assembly speed are selected as the original seeds for the development of production technology and preparation technology. The recombinant adenovirus is observed by an electron microscope, and the result is shown in FIG. 1. As shown in FIG. 1, the typical adenovirus structure is observed under the electron microscope.

EMBODIMENT 2: PREPARATION STUDY OF THE RECOMBINANT SARS-COV-2 ADENOVIRAL VECTOR VACCINE

[0081] The liquid preparation formulation of the recombinant SARS-CoV-2 adenoviral vector vaccine is shown in Table 1 and Table 2:

TABLE-US-00001 TABLE 1 Liquid preparation formulation of recombinant SARS-CoV-2 adenoviral vector vaccine Formulation Content Recombinant adenovirus 1 × 10.sup.11 VP/ml (preparation of Embodiment 1) Sucrose (mg/ml) 25 Mannitol (mg/ml) 50 Sodium chloride (mM) 50 HEPES (mM) 2.5 Magnesium chloride (mM) 2 Tween 80 (mg/ml) 0.1 Gelatin (mg/ml) 10

TABLE-US-00002 TABLE 2 Liquid preparation formulation of recombinant SARS-CoV-2 adenoviral vector vaccine Formulation Content Recombinant adenovirus 1 × 10.sup.11 VP/ml (preparation of Embodiment 1) Sucrose (mg/ml) 25 Mannitol (mg/ml) 50 Sodium chloride (mM) 50 Glycerin (mg/ml) 1.5 HEPES (mM) 2.5 Magnesium chloride (mM) 2 Tween 80 (mg/ml) 0.1 His(mM) 5 EDTA(mM) 0.1 HSA 0.60% Gelatin (mg/ml) 10

[0082] The changes of VP and IFU of the liquid preparation of the recombinant SARS-CoV-2 adenoviral vector vaccine in Table 2 are determined after multiple doses of atomization. The results are shown in Table 3. The results show that the recombinant SARS-CoV-2 adenoviral vector vaccine has good stability, and its specific activity does not change significantly after repeated atomization.

TABLE-US-00003 TABLE 3 Specific activity changes of recombinant SARS-CoV-2 adenoviral vector vaccine after aerosol administration IFU/ VP number/ Specific Name mL, ×10{circumflex over ( )}9 mL, ×10{circumflex over ( )}11 activity % Before spraying 14.5 2.69 5.39 After one time 12.5 2.01 6.22 of atomization - 1 After two times 12.0 2.33 5.15 of atomization - 2 After three times 11.0 1.71 6.43 of atomization - 3 After four times 11.3 2.00 5.65 of atomization - 4

[0083] The particle size range of the aerosolized spray of the recombinant SARS-CoV-2 adenoviral vector vaccine in the formulation of Table 2 above is determined and the result shows that the particle size is 1-6 μm with an average of 4 μm.

[0084] The atomization time of the recombinant SARS-CoV-2 adenoviral vector vaccine with different administration volumes is measured for three consecutive times, and the results are shown in Table 4. The results show that the liquid preparation of the recombinant SARS-CoV-2 adenoviral vector vaccine is suitable for atomization, and the CV variation of different atomization time is not more than 10%.

TABLE-US-00004 TABLE 4 Atomization rate of recombinant SARS-CoV-2 adenoviral vector vaccine with different volumes Volume Atomization Atomization Atomization Average (μL) time 1 (S) time 2 (S) time 3 (S) value (S) CV 200 26 27 24 26 5.88 300 32 32 32 32 0.00 400 42 44 43 43 2.33 500 54 53 54 54 1.07 600 67 68 70 68 2.25 1000 107 106 106 106 0.54 3000 304 301 303 303 0.50

EMBODIMENT 3: STUDY ON IMMUNOGENICITY OF RECOMBINANT SARS-COV-2 ADENOVIRAL VECTOR VACCINE

[0085] According to Table 2, the recombinant SARS-CoV-2 adenoviral vector vaccine with the same preparation formulation contains 1×10.sup.11VP per ml and is used to immunize cynomolgus monkeys by intramuscular injection and aerosol inhalation, and determine IgG in serum and IgA in alveolar lavage fluid.

[0086] Experimental animal: cynomolgus monkeys, 2 in each group;

[0087] Immunization dose: 1 dose, each dose is 0.5 ml, containing 5×10.sup.10 VP virus particles;

[0088] Immunization mode: intramuscular injection and aerosol inhalation, with the same immunization dose;

[0089] Single needle immunization is adopted, blood is collected 4 weeks after immunization, the alveolar lavage fluid is collected, and IgG in serum and IgA in alveolar lavage fluid are determined by ELISA.

[0090] The detection results of IgG in serum are shown in FIG. 2. The results show that the above recombinant SARS-CoV-2 adenoviral vector vaccine could immunize the cynomolgus monkeys in two means, intramuscular injection and aerosol inhalation. At the same dose, the IgG antibody titers produced in serum are basically the same.

[0091] The detection results of IgA in alveolar lavage fluid are shown in FIG. 3. The results show that the above recombinant SARS-CoV-2 adenoviral vector vaccine could immunize the cynomolgus monkeys in two means, intramuscular injection and aerosol inhalation. The muscle immunization do not effectively produce IgA, but the transmucosal immunization of aerosol inhalation realizes detection of high-titer IgA in alveolar lavage.

EMBODIMENT 4: STUDY ON CELLULAR IMMUNITY OF RECOMBINANT SARS-COV-2 ADENOVIRAL VECTOR VACCINE

[0092] According to Table 2, the recombinant SARS-CoV-2 adenoviral vector vaccine with the same preparation formulation contains 1×10.sup.11VP per ml and is used to immunize the cynomolgus monkeys by intramuscular injection and aerosol inhalation, and the cellular immune level in serum and alveolar lavage fluid is measured.

[0093] Experimental animal: cynomolgus monkeys, 2 in each group; Immunization dose: 1 dose, each dose is 0.5 ml, containing 5×10.sup.10 VP virus particles; Immunization method: intramuscular injection and aerosol inhalation; Single needle immunization is adopted, blood is collected 4 weeks after immunization, and cellular immunity is determined.

[0094] The detection results of IgG in serum are shown in FIG. 4. The results show that the level of cellular immunity induced by atomized inhalation of the recombinant COVID-19 vaccine is similar to that induced by intramuscular injection.

[0095] The cellular immunity in the alveolar lavage fluid is measured, and the results are shown in FIG. 5. The results show that the atomized inhalation of the recombinant COVID-19 vaccine could effectively stimulate the cellular immunity level of the alveolar lavage fluid, and the cellular immunity could not be detected in the alveolar lavage fluid of intramuscular injection.

EMBODIMENT 5: STUDY ON PROTECTIVE EFFECT OF RECOMBINANT SARS-COV-2 ADENOVIRAL VECTOR VACCINE

[0096] According to Table 2, the recombinant SARS-CoV-2 adenoviral vector vaccine with the same preparation formulation contains 1×10.sup.11VP per ml and is used to immunize cynomolgus monkeys by intramuscular injection and aerosol inhalation, and the neutralization antibody titer is determined.

[0097] Experimental animal: cynomolgus monkeys, 2 in each group;

[0098] Immunization dose: 1 dose, each dose is 0.5 ml, containing 5×10.sup.10 VP virus particles;

[0099] Immunization methods: intramuscular injection and aerosol inhalation;

[0100] Single needle immunization is adopted, blood is collected 4 weeks after immunization, and the neutralization antibody titer is determined. The results are shown in the following table:

TABLE-US-00005 TABLE 5 Neutralizing antibody of recombinant SARS-CoV-2 adenoviral vector vaccine in serum after intramuscular injection and aerosol inhalation Neutralizing antibody No.1 monkey No. 2 monkey Average value Intramuscular 550 580 565 injection Transmucosal 630 560 595 immunity - inhalation

[0101] The results show that the above recombinant SARS-CoV-2 adenoviral vector vaccine could produce the neutralizing antibody level in the cynomolgus monkeys immunized with intramuscular injection and aerosol inhalation, and the neutralizing antibody levels produced by the two administration means are similar.

[0102] The above is only preferred embodiments of the invention, and does not limit the invention. Any modification, equivalent replacement, etc. made within the spirit and principle of the invention should be included in the protection scope of the invention.

[0103] The aforementioned embodiments and methods described in the invention may be different based on the abilities, experiences and preferences of those skilled in the art.

[0104] In the invention, only listing the steps of the method in a certain order does not constitute any restriction on the sequence of the steps of the method.