TW202300514A - Microneedle vaccine against severe acute respiratory syndrome coronavirus 2 (sars-cov-2) - Google Patents

Abstract

The present invention relates to a microneedle vaccine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), especially to a microneedle vaccine having a recombinant SARS-CoV-2 S protein and providing a sustained release of the recombinant SARS-CoV-2 S protein in a subject.

Description

Microneedle vaccine against novel coronavirus

CROSS-REFERENCE TO RELATED APPLICATIONS: This case asserts priority and benefit to U.S. Provisional Application No. 63/191,777, filed May 21, 2021, the disclosure of which is incorporated herein by reference in its entirety.

The present invention relates to a microneedle vaccine against severe acute respiratory syndrome coronavirus 2 (severe acute respiratory syndrome coronavirus 2, SARS-CoV-2, also known as novel coronavirus), especially a recombinant SARS-CoV-2 spike protein A microneedle vaccine for sustained release of the recombinant SARS-CoV-2 spike protein in a subject is also provided.

The World Health Organization (WHO) was alerted on December 31, 2019, of several cases of pneumonia in Wuhan, Hubei Province, China. The viral pathogen did not match any other known virus, and the virus was later officially named "Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2, also known as novel coronavirus)". The official name of the disease caused by SARS-CoV-2 is coronavirus disease 2019 (COVID-19). Common symptoms of COVID-19 include fever, dry cough, fatigue, tiredness, muscle or body aches, sore throat, diarrhea, conjunctivitis, headache, loss of taste or smell, skin rash, and shortness of breath. Although most cases are mild, some patients develop acute respiratory distress syndrome (ARDS), which is caused by a cytokine storm, multiple organ failure, septic shock, and blood clots. The first confirmed death from a novel coronavirus infection occurred on January 9, 2020, and as of May 13, 2022, WHO has received notifications of 517,648,631 confirmed cases of COVID-19, including 6,261,708 deaths. And those numbers are still growing rapidly.

Although several SARS-CoV-2 vaccines are currently available on the market, average global vaccination rates remain low. In addition, all SARS-CoV-2 vaccines currently on the market are intramuscular injections, which must be administered by professionals and trained personnel, which reduces the vaccination rate and increases the workload of medical staff.

The present invention is based, at least in part, on the discovery that antigenicity is modulated by, for example, controlled and/or sustained release compositions and devices (e.g., microneedles, such as silk-based microneedles and microneedle patches) comprising an immunogenic recombinant protein. Presents kinetics that can drive a more effective immune response against SARS-CoV-2 in a subject, wherein the immunogenic recombinant protein Basically from the 14th to 1208th residues of the SARS-CoV-2 spike protein, the 986th and 987th residues are replaced by proline, and the 682nd to 685th residues are replaced by "Glycine-serine-alanine-serine (GSAS)" and a T4 fibrin trimerization domain at its C-terminus. In certain embodiments, the SARS-CoV-2 spike protein described herein is substantially composed of residues 14 to 1208 of the SARS-CoV-2 spike protein, wherein residues 986 and 987 are replaced by proline , the 682nd to 685th residues are replaced by "glycine-serine-alanine-serine (GSAS)", and it has a T4 fibrin trimerization domain at its C-terminus Controlled and/or sustained release of the immunogenic recombinant protein of the present invention can be used to achieve broad immunity in a subject.

In certain embodiments, the microneedles and microneedle devices described herein exhibit a sequence consisting essentially of residues 14 to 1208 of the SARS-CoV-2 spike protein, wherein residues 986 and 987 residues were replaced by proline, residues 682 to 685 were replaced by "glycine-serine-alanine-serine (GSAS)", and a T4 Controlled and/or sustained release of an immunogenic recombinant protein composed of a fibrin trimerization domain for at least about 1-2 weeks (e.g., at least about 1, 2, 3, 4, 5, 6, 7, 8, 9 , 10, 11, 12, 13 or 14 days), resulting in one or more of increased immunogenicity, enhanced immune response, and/or broad immunity.

In other embodiments, it is also disclosed that the 14th to 1208th residues of the SARS-CoV-2 spike protein are used for administering, wherein the 986th and 987th residues are replaced by proline , the 682nd to 685th residues are replaced by "glycine-serine-alanine-serine (GSAS)", and it has a T4 fibrin trimerization domain at its C-terminus Methods, formulations, compositions, articles of manufacture, devices, and/or formulations of immunogenic recombinant proteins provide improved immunogenicity, enhanced immune response, and/or broad immunity in a subject. Accordingly, disclosed herein are compositions, formulations, devices (eg, microneedles and microneedle patches), kits, and methods of making and using the same for controlled and/or sustained release of a vaccine in a subject.

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be covered by the specific examples below.

Specific embodiments 1. A microneedle vaccine against novel coronavirus (SARS-CoV-2), comprising at least one microneedle, wherein the at least one microneedle comprises (i) a backing, (ii) A soluble base comprising components other than poly acrylic acid (PAA), for example, other than a solution of about 35% PAA. In certain embodiments, the soluble base comprises a composition (eg, one or more water-soluble components) that has improved biocompatibility in a subject, eg, compared to PAA. In certain embodiments, the dissolvable base comprises a composition (e.g., a water-soluble composition) having a pH similar to that of the biological barrier to be dissolved therein, such as having a pH of about 4.0-8 pH. In certain embodiments, the soluble base comprises at least one (e.g., one, two, three, four, five, six, seven, eight, or more (e.g., all)) of : Gelatin, polyethylene glycol (PEG), sucrose, carboxymethylcellulose (CMC), polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA), hyaluronic acid salt, maltose , and methylcellulose. In certain embodiments, the soluble base does not comprise an immunogenic recombinant protein as described herein. In certain embodiments, the dissolvable base is applied to the backing, (iii) a microneedle tip, such as an implantable sustained-release tip, comprising a strand of fibrin and an immunogenic recombinant protein consisting essentially of: one having the following SEQ ID NO: The 14th to 1208th residues of the SARS-CoV-2 spike protein of the amino acid sequence shown in 1, wherein the 986th and 987th residues are replaced by proline, and the 682nd to 685th residues residues are replaced by "glycine-serine-alanine-serine (GSAS)", and has a T4 fibrin trimerization structure as shown in SEQ ID NO: 2 at its C-terminus domain; in certain embodiments, the tip is applied to the soluble base, Wherein the microneedle is configured to implant the tip into a biological barrier, e.g., the skin of a subject (e.g., a human subject), e.g., to a depth of between about 100 µm and about 600 µm ( For example, the maximum penetration depth of the distal portion of the tip), Wherein the tip comprises a strand of fibrin, eg, a regenerated silk fibrin and/or a recombinant silk fibrin.

In some embodiments, the immunogenic recombinant protein has the amino acid sequence shown in SEQ ID NO: 5.

Specific embodiment 2. A microneedle vaccine against SARS-CoV-2, comprising at least one microneedle, wherein the at least one microneedle comprises (i) a backing, (ii) a dissolvable base applied to the backing comprising at least one of the following (e.g., one, two, three, four, five, six, seven, eight, or more (e.g., All)): gelatin, polyethylene glycol (PEG), sucrose, carboxymethylcellulose (CMC), polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA), hyaluronate, maltose, and methylcellulose, (iii) a microneedle tip (eg, an implantable sustained release tip) applied to the soluble base, comprising a strand of fibrin and an immunogenic recombinant protein consisting essentially of Composition: the 14th to 1208th residues of the SARS-CoV-2 spike protein having the amino acid sequence shown in SEQ ID NO: 1, wherein the 986th and 987th residues are replaced by pro Amino acid, the 682nd to 685th residues are replaced by "glycine-serine-alanine-serine (GSAS)", and at its C-terminus there is a The T4 fibrin trimerization domain shown, Wherein the microneedle is configured to implant the tip into the skin of a subject (e.g., a human subject), e.g., to a depth between about 100 μm and about 600 μm (e.g., the distal end of the tip maximum penetration depth of the end portion), Wherein the tip comprises a strand of fibrin, eg, a regenerated silk fibrin and/or a recombinant silk fibrin.

In some embodiments, the immunogenic recombinant protein has the amino acid sequence shown in SEQ ID NO: 5.

Specific embodiment 3. The microneedle vaccine according to specific embodiment 1 or 2, wherein the soluble base comprises gelatin, polyethylene glycol (PEG), sucrose, carboxymethylcellulose (CMC), polyvinylpyrrolidone (PVP ), polyvinyl alcohol (PVA), hyaluronate, maltose, and one of methylcellulose.

Specific embodiment 4. The microneedle vaccine according to specific embodiment 1 or 2, wherein the soluble base is composed of two kinds of gelatin, PEG, sucrose, CMC, PVP, PVA, hyaluronic acid salt, maltose, and methyl cellulose composed of.

Embodiment 5. The microneedle vaccine according to Embodiment 1 or 2, wherein the soluble base comprises three of gelatin, PEG, sucrose, CMC, PVP, PVA, hyaluronic acid salt, maltose, and methylcellulose.

Specific embodiment 6. The microneedle vaccine according to specific embodiment 1 or 2, wherein the soluble base comprises four kinds of gelatin, PEG, sucrose, CMC, PVP, PVA, hyaluronic acid salt, maltose, and methyl cellulose .

Specific embodiment 7. The microneedle vaccine according to specific embodiment 1 or 2, wherein the soluble base includes five kinds of gelatin, PEG, sucrose, CMC, PVP, PVA, hyaluronic acid salt, maltose, and methyl cellulose .

Specific embodiment 8. The microneedle vaccine according to specific embodiment 1 or 2, wherein the soluble base comprises six kinds of gelatin, PEG, sucrose, CMC, PVP, PVA, hyaluronic acid salt, maltose, and methyl cellulose .

Specific embodiment 9. The microneedle vaccine according to specific embodiment 1 or 2, wherein the soluble base comprises seven kinds of gelatin, PEG, sucrose, CMC, PVP, PVA, hyaluronic acid salt, maltose, and methyl cellulose .

Specific embodiment 10. The microneedle vaccine according to specific embodiment 1 or 2, wherein the soluble base comprises eight kinds of gelatin, PEG, sucrose, CMC, PVP, PVA, hyaluronic acid salt, maltose, and methyl cellulose .

Embodiment 11. The microneedle vaccine according to Embodiment 1 or 2, wherein the soluble base comprises gelatin, PEG, sucrose, CMC, PVP, PVA, hyaluronic acid salt, maltose, and methylcellulose.

Embodiment 12. The microneedle vaccine according to any one of the foregoing embodiments, wherein the soluble base comprises gelatin and sucrose.

Embodiment 13. The microneedle vaccine according to any one of the preceding embodiments, wherein the soluble base comprises CMC.

Embodiment 14. The microneedle vaccine according to any one of the preceding embodiments, wherein the soluble base comprises PVP.

Embodiment 15. The microneedle vaccine according to any one of the preceding embodiments, wherein the soluble base comprises PVA.

Embodiment 16. The microneedle vaccine according to any one of the foregoing embodiments, wherein the soluble base comprises PVP and PVA.

Embodiment 17. The microneedle vaccine according to any one of the foregoing embodiments, wherein the soluble base comprises PVP, PVA, and sucrose.

Embodiment 18. The microneedle vaccine according to any one of the preceding embodiments, wherein the soluble base does not comprise polyacrylic acid (PAA).

Embodiment 19. The microneedle vaccine according to any one of the preceding embodiments, wherein the soluble base comprises PEG.

Embodiment 20. The microneedle vaccine according to any one of the preceding embodiments, wherein the soluble base comprises gelatin (e.g., hydrolyzed gelatin) between about 10% and about 70% (w/v) ( For example, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, or about 70% (w/v) gelatin).

Embodiment 21. The microneedle vaccine according to any one of the preceding embodiments, wherein the soluble base comprises between about 1% and about 35% (w/v) sucrose (e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, about 20%, about 25%, about 30% or about 35% (w/v) sucrose).

Embodiment 22. The microneedle vaccine according to any one of the preceding embodiments, wherein the soluble base comprises about 1% to about 35% (w/v) of CMC (for example, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, about 20%, about 25%, about 30% , or about 35% (w/v) of CMC).

Embodiment 23. The microneedle vaccine according to any one of the preceding embodiments, wherein the soluble base comprises PVP between about 10% and about 70% (w/v) (for example, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, or about 70% (w/v) of PVP).

Embodiment 24. The microneedle vaccine according to any one of the preceding embodiments, wherein the soluble base comprises between about 1% and about 35% (w/v) PVA (for example, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, about 20%, about 25%, about 30% or about 35% (w/v) of PVA).

Embodiment 25. The microneedle vaccine according to any one of the preceding embodiments, wherein the soluble base comprises about 40% (w/v) hydrolyzed gelatin and about 10% (w/v) sucrose.

Embodiment 26. The microneedle vaccine according to any one of the preceding embodiments, wherein the soluble base comprises at most about 50% (w/v) PVP (eg, PVP with a molecular weight of 10 kD).

Embodiment 27. The microneedle vaccine according to any one of the preceding embodiments, wherein the soluble base comprises at most about 20% (w/v) of PVA (e.g., 87% (w/v) of 13 kD Molecular weight hydrolyzed PVA).

Embodiment 28. The microneedle vaccine according to any one of the preceding embodiments, wherein the soluble base comprises at most about 10% (w/v) CMC.

Embodiment 29. The microneedle vaccine according to any one of the preceding embodiments, wherein the soluble base comprises about 1% (w/v) CMC (eg, low-viscosity CMC).

Embodiment 30. The microneedle vaccine according to any one of the preceding embodiments, wherein the soluble base comprises about 30% (w/v) PVP and about 10% (w/v) PVA.

Embodiment 31. The microneedle vaccine according to any one of the preceding embodiments, wherein the soluble base comprises about 37% (w/v) of PVP, about 5% (w/v) of PVA, and about 15% (w/v) sucrose.

Embodiment 32. The microneedle vaccine according to any one of the preceding embodiments, wherein the soluble base comprises between about 1% and about 75% (w/v) of hyaluronate (e.g., about 1%, About 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, about 20%, about 25%, about 30% %, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, or about 75% (w/v) of hyaluronate).

Embodiment 33. The microneedle vaccine according to any one of the preceding embodiments, wherein the soluble base comprises between about 1% and about 75% (w/v) maltose (e.g., about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, about 20%, about 25%, about 30% , about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, or about 75% (w/v) maltose).

Embodiment 34. The microneedle vaccine according to any one of the preceding embodiments, wherein the soluble base comprises between about 1% and about 75% (w/v) methylcellulose (e.g., about 1 %, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, about 20%, about 25%, About 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, or about 75% (w/v) methylcellulose ).

Embodiment 35. The microneedle vaccine according to any one of the preceding embodiments, wherein the soluble base comprises about 1% to about 70% (w/v) of PEG (eg, about 1%, about 2% , about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, or about 70% (w/v) of PEG).

Embodiment 36. The microneedle vaccine according to any one of the preceding embodiments, wherein the implantable sustained-release tip is configured to last for at least about 4 days (e.g., about 1, 2, 3, 4 , 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days or longer, for example about 4 days to about 14 days, for example about 1-2 weeks, about 1-3 weeks or about 1-4 weeks) to release the immunogenic recombinant protein into the skin of the subject.

Embodiment 37. The microneedle vaccine according to any one of the preceding embodiments, wherein the implantable sustained-release tip is configured for a period ranging from about 1 week to about 2 weeks (e.g., about 1, 2, 3 , 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days) to release the immunogenic recombinant protein into the skin of the subject.

Embodiment 38. The microneedle vaccine according to any one of the preceding embodiments, wherein the implantable sustained release tip comprises about 0.1% (w/v) to about 10% (w/v) (eg , about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10% (w/v)) of silk fibers protein, or a silk fibroin having a molecular weight distribution according to Table 1, or comprising (eg, per 121 microneedle arrays) about 2 μg to about 245 μg of silk fibroin).

Embodiment 39. The microneedle vaccine according to any one of the preceding embodiments, wherein the implantable sustained-release tip comprises the immunogenic recombinant protein formulated at about 0.1% (w/v) to about 10% (w/v) (e.g., about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10% (w/v)) in a 10 MB silk fibroin solution, or a silk fibroin solution according to Table 1.

Embodiment 40. The microneedle vaccine according to any one of the preceding embodiments, wherein the implantable sustained-release tip comprises the immunogenic recombinant protein formulated at about 0.1% (w/v) to about 10% (w/v) (e.g., about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10% (w/v)) in a 60 MB silk fibroin solution, or a silk fibroin solution according to Table 1, e.g., a 100 kDa to 200 kDa (e.g., about 153 kDa) in the silk fibroin solution.

Embodiment 41. The microneedle vaccine according to any one of the preceding embodiments, wherein the implantable sustained-release tip comprises the immunogenic recombinant protein formulated at about 0.1% (w/v) to about 10% (w/v) (e.g., about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10% (w/v)) in 120 MB silk fibroin solution, or a silk fibroin solution according to Table 1, for example, a 70 kDa to 150 kDa (for example, about 100 kDa) in the silk fibroin solution.

Embodiment 42. The microneedle vaccine according to any one of the preceding embodiments, wherein the implantable sustained-release tip comprises the immunogenic recombinant protein formulated at about 0.1% (w/v) to about 10% (w/v) (e.g., about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10% (w/v)) in 180 MB silk fibroin solution, or a silk fibroin solution according to Table 1, for example, a 36 kDa to 100 kDa (for example, about 71 kDa) in the silk fibroin solution.

Embodiment 43. The microneedle vaccine according to any one of the preceding embodiments, wherein the implantable sustained-release tip comprises the immunogenic recombinant protein formulated at about 0.1% (w/v) to about 10% (w/v) (e.g., about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10% (w/v)) in 480 MB silk fibroin solution, or a silk fibroin solution according to Table 1, for example, a 1 kDa to 60 kDa (for example, about 16 kDa) in the silk fibroin solution.

Embodiment 44. The microneedle vaccine according to embodiment 40, wherein the implantable sustained release tip comprises immunogenic recombinant protein formulated in about 1% (w/v) silk fibroin solution.

Embodiment 45. The microneedle vaccine according to any one of the preceding embodiments, wherein the implantable sustained release tip further comprises about 0.1% (w/v) to about 10% (w/v) ( For example, about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10% (w/v)) of the interface Active agent (eg, polysorbate 20, polysorbate 80, Tween® 20, Tween® 80, or a combination thereof).

Embodiment 46. The microneedle vaccine according to any one of the preceding embodiments, wherein the implantable sustained-release tip comprises the immunogenic recombinant protein formulated at about 0.1% (w/v) to about 10% (w/v) (e.g., about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10% (w/v)) of 10 MB silk fibroin, or a silk fibroin according to Table 1, and about 0.1% (w/v) to about 10% (w/v) (e.g., about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10% (w/v)) In a solution of a surfactant (eg, polysorbate 20, polysorbate 80, Tween® 20, Tween® 80, or a combination thereof).

Embodiment 47. The microneedle vaccine according to any one of the preceding embodiments, wherein the implantable sustained-release tip comprises the immunogenic recombinant protein formulated at about 0.1% (w/v) to about 10% (w/v) (e.g., about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10% (w/v)) of 60 MB silk fibroin, or a silk fibroin according to Table 1 (e.g., a silk fiber of 100 kDa to 200 kDa (e.g., about 153 kDa) protein), and about 0.1% (w/v) to about 10% (w/v) (e.g., about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4 , 5, 6, 7, 8, 9, or 10% (w/v)) of surfactant (for example, polysorbate 20, polysorbate 80, Tween® 20, Tween® 80, or one of them combination) solution.

Embodiment 48. The microneedle vaccine according to any one of the preceding embodiments, wherein the implantable sustained-release tip comprises the immunogenic recombinant protein formulated at about 0.1% (w/v) to about 10% (w/v) (e.g., about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10% (w/v)) of 120 MB silk fibroin, or a silk fibroin according to Table 1 (e.g., a silk fiber of 70 kDa to 150 kDa (e.g., about 100 kDa) protein), and about 0.1% (w/v) to about 10% (w/v) (e.g., about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4 , 5, 6, 7, 8, 9, or 10% (w/v)) of surfactant (for example, polysorbate 20, polysorbate 80, Tween® 20, Tween® 80, or one of them combination) solution.

Embodiment 49. The microneedle vaccine according to any one of the preceding embodiments, wherein the implantable sustained-release tip comprises the immunogenic recombinant protein formulated at about 0.1% (w/v) to about 10% (w/v) (e.g., about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10% (w/v)) of 180 MB silk fibroin, or a silk fibroin according to Table 1 (e.g., a silk fiber of 36 kDa to 100 kDa (e.g., about 71 kDa) protein), and about 0.1% (w/v) to about 10% (w/v) (e.g., about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4 , 5, 6, 7, 8, 9, or 10% (w/v)) of surfactant (for example, polysorbate 20, polysorbate 80, Tween® 20, Tween® 80, or one of them combination) solution.

Embodiment 50. The microneedle vaccine according to any one of the preceding embodiments, wherein the implantable sustained-release tip comprises the immunogenic recombinant protein formulated at about 0.1% (w/v) to about 10% (w/v) (e.g., about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10% (w/v)) of 480 MB silk fibroin, or a silk fibroin according to Table 1 (e.g., a silk fiber of 1 kDa to 60 kDa (e.g., about 16 kDa) protein), and about 0.1% (w/v) to about 10% (w/v) (e.g., about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4 , 5, 6, 7, 8, 9, or 10% (w/v)) of surfactant (for example, polysorbate 20, polysorbate 80, Tween® 20, Tween® 80, or one of them combination) solution.

Embodiment 51. The microneedle vaccine according to embodiment 45, wherein the implantable sustained-release tip comprises the immunogenic recombinant protein formulated at about 1% (w/v ) in a solution of silk fibroin and about 0.5% (w/v) surfactant (eg, polysorbate 20, polysorbate 80, Tween® 20, Tween® 80, or a combination thereof).

Embodiment 52. The microneedle vaccine according to any one of the preceding embodiments, wherein the implantable sustained release tip comprises a human standard dose of the immunogenic recombinant protein.

Embodiment 53. The microneedle vaccine according to any one of the preceding embodiments, wherein the standard dose of the immunogenic recombinant protein comprises about 0.1 μg to about 65 μg, such as 0.2 μg to about 50 μg (for example, About 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 , 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, or 65 μg each dose).

Embodiment 54. The microneedle vaccine according to embodiment 52 or 53, wherein the implantable sustained release tip comprises at least about 1%, 2%, 3%, 4%, 5%, 6%, 7% %, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25% or more of that standard dose.

Embodiment 55. The microneedle of any one of embodiments 52-54, wherein the implantable sustained release tip comprises from about 0.1 μg to about 65 μg of the immunogenic recombinant protein (eg, about 0.1 μg, about 0.2 μg, about 0.3 μg, about 0.4 μg, about 0.5 μg, about 0.6 μg, about 0.7 μg, about 0.8 μg, about 0.9 μg, about 1 μg, about 1 μg to about 10 μg, about 10 μg to about 20 μg, about 20 μg to about 30 μg, about 30 μg to about 40 μg, about 40 μg to about 50 μg, about 50 μg to about 65 μg of immunogenic recombinant protein).

Specific embodiment 56. The microneedle vaccine according to any one of the preceding specific embodiments, wherein the length of the microneedle is between about 350 μm and about 1500 μm (for example, about 350 μm, about 400 μm, about 450 μm , about 500 μm, about 550 μm, about 600 μm, about 650 μm, about 700 μm, about 750 μm, about 800 μm, about 850 μm, about 900 μm, about 950 μm, about 1000 μm, about 1050 μm, about 1100 μm, about 1150 μm, about 1200 μm, about 1250 μm, about 1300 μm, about 1350 μm, about 1400 μm, about 1450 μm, about 1500 μm).

Embodiment 57. The microneedle vaccine according to any one of the preceding embodiments, wherein the height of the implantable sustained release tip extends to about half of the full length of the microneedle.

Embodiment 58. The microneedle vaccine according to any one of the preceding embodiments, wherein the implantable sustained release tip has a height between about 75 μm and about 475 μm (eg, about 75 μm, about 100 μm, about 125 μm, about 150 μm, about 175 μm, about 200 μm, about 225 μm, about 250 μm, about 275 μm, about 300 μm, about 325 μm, about 375 μm, about 400 μm, about 425 μm , or about 475 μm).

Embodiment 59. The microneedle vaccine according to any one of the preceding embodiments, wherein the implantable sustained release tip comprises a tip radius between about 0.5 μm and about 25 μm (eg, about 0.5, 0.6 , 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 , 23, 24, or 25 µm).

Embodiment 60. The microneedle vaccine according to any one of the preceding embodiments, wherein the implantable sustained release tip comprises a tip radius between about 5 μm and about 10 μm (eg, about 5, 6 , 7, 8, 9, or 10 µm).

Embodiment 61. The microneedle vaccine according to any one of the preceding embodiments, wherein the implantable sustained release tip comprises an angle between about 5 degrees and about 45 degrees (e.g., about 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, or 45 degrees).

Embodiment 62. The microneedle vaccine according to any one of the preceding embodiments, wherein the backing is selected from a solid support, for example, a paper-based material, a plastic material, a polymeric material, or a Polyester-based material (eg, Whatman 903 paper, a polymer tape, a plastic tape, an adhesive backed polyester tape, or other medical tape).

Embodiment 63. A device (e.g., an array or patch) comprising a plurality of microneedles (e.g., two or more microneedles as described herein), e.g., a plurality of microneedles according to embodiment 1- The microneedle described in any one of 62.

Embodiment 64. The device according to Embodiment 63, wherein the implantable microneedle tip comprises about 0.1 μg to about 65 μg of immunogenic recombinant protein (eg, about 0.1 μg, about 0.2 μg, about 0.3 μg μg, about 0.4 μg, about 0.5 μg, about 0.6 μg, about 0.7 μg, about 0.8 μg, about 0.9 μg, about 1 μg, about 1 μg to about 10 μg, about 10 μg to about 20 μg, about 20 μg to about 30 μg, about 30 μg to about 40 μg, about 40 μg to about 50 μg, about 50 μg to about 65 μg of the immunogenic recombinant protein described herein).

Embodiment 65. A method of providing immunity (e.g., broad immunity) to SARS-CoV-2 in a subject in need thereof, comprising exposing the subject's skin to a The microneedle vaccine described in any one of 1-62, or the device contacted according to any one of specific embodiments 63-64.

Embodiment 66. A method of providing controlled or sustained release of an anti-SARS-CoV-2 vaccine in a subject in need thereof, comprising exposing the subject's skin to any one of embodiments 1-62. One of the microneedle vaccines, or the device contact according to any one of the specific embodiments 63-64.

Embodiment 67. A method of enhancing an immune response to SARS-CoV-2 in a subject in need thereof, comprising exposing the subject's skin to the skin according to any one of embodiments 1-62. The microneedle vaccine described above, or the device contacted according to any one of the specific embodiments 63-64.

Embodiment 68. The method according to any one of embodiments 65-67, wherein the implantable sustained release tip is configured to last for at least about 4 days (e.g., about 1, 2, 3, 4 , 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 days or more days, such as about 4 days to about 14 days, such as about 1-2 weeks, about 1-3 weeks or about 1 -4 weeks) to release a vaccine into the skin of the subject.

Embodiment 69. The method according to embodiment 68, wherein the implantable sustained release tip is configured for a period ranging from about 1 week to about 2 weeks (e.g., about 1, 2, 3, 4, 5, 6 , 7, 8, 9, 10, 11, 12, 13, or 14 days) release a vaccine into the skin of the subject.

Specific embodiment 70. The method according to any one of specific embodiments 65-69, further comprising contacting the microneedle vaccine according to any one of specific embodiments 1-62 or according to specific embodiments for the first time After the device described in any one of 63-64, the subject's skin was mixed with another microneedle vaccine according to any one of specific embodiments 1-62 or according to any one of specific embodiments 63-64. The other device of one aspect is contacted between about 3 weeks to about 12 weeks (e.g., about 3-4 weeks, about 3-5 weeks, about 3-6 weeks, about 3-7 weeks, about 3-8 weeks, about 3-9 weeks, about 3-10 weeks, about 3-11 weeks, about 3-12 weeks).

Embodiment 71. The microneedle vaccine according to any one of embodiments 1-62 or the device according to any one of embodiments 63-64 in a subject in need thereof Use in a method of eliciting an immune response against SARS-CoV-2.

Embodiment 72. The microneedle vaccine according to any one of embodiments 1-62 or the device according to any one of embodiments 63-64 in a subject in need thereof Use in a method of controlling or sustaining release of an anti-SARS-CoV-2 vaccine is provided.

Embodiment 73. The microneedle according to any one of embodiments 1-62 or the device according to any one of embodiments 63-64 augmented in a subject in need thereof Use in a method of immune response to SARS-CoV-2.

Embodiment 74. The microneedle according to any one of embodiments 1-62 or the device according to any one of embodiments 63-64 provided in a subject in need thereof As a medicament in a method for immunity (eg, broad immunity) to SARS-CoV-2.

Embodiment 75. The microneedle according to any one of embodiments 1-62 or the device according to any one of embodiments 63-64 provided in a subject in need thereof A method of sustained release of an anti-SARS-CoV-2 vaccine as a drug.

Embodiment 76. The microneedle according to any one of embodiments 1-62 or the device according to any one of embodiments 63-64 augmented in a subject in need thereof As a drug in the method of immune response to SARS-CoV-2.

These and other aspects will become apparent from the following description of preferred embodiments in conjunction with the accompanying drawings.

The present invention is based at least in part on the discovery that modulating the kinetics of antigen presentation to mimic natural infection (e.g., SARS-CoV-2 viral infection) can drive a more effective immune response (e.g., a more effective humoral immune response) (see, e.g., Tam et al., PNAS. 113:E6639-E6648, 2016; and Schipper et al., J. Control Release. 242:141-147, 2016). Without wishing to be bound by theory, the microneedles and microneedle devices described herein can be used to release (e.g., controlled or sustained release) an immunogenic recombinant protein to a subject (e.g., a skin layer of a subject) , to mimic the natural process of antigen presentation (e.g., viral antigen presentation), wherein the immunogenic recombinant protein consists essentially of the 14th to 1208th residues of the SARS-CoV-2 spike protein, wherein the 986th and 1208th residues 987 residues were replaced by proline, the 682nd to 685th residues were replaced by "glycine-serine-alanine-serine (GSAS)", and a T4 fibrin trimerization domain. Compared with single dose or bolus administration of the SARS-CoV-2 spike protein consisting essentially of the 14th to 1208th residues, wherein the 986th and 987th residues were replaced by proline, the 1st The 682nd to 685th residues were replaced by "glycine-serine-alanine-serine (GSAS)", and there was a T4 fibrin trimerization domain at its C-terminus. Controlled or sustained release of native recombinant proteins from the formulations, compositions, articles, devices, and formulations, microneedles, and microneedle devices described herein can induce greater immunogenicity in a subject , an enhanced immune response (eg, a more effective humoral immune response), and/or broad immunity.

In certain embodiments, the microneedles and microneedle devices described herein can include an implantable silk-based controlled or sustained release microneedle tip that encapsulates and/or stabilizes the From the 14th to 1208th residues of -2 spinin, the 986th and 987th residues were replaced by proline, and the 682nd to 685th residues were replaced by "glycine-silk amino acid-alanine-serine (GSAS)", and an immunogenic recombinant protein consisting of a T4 fibrin trimerization domain at its C-terminus; and a soluble base supporting the tip of the distal microneedle . Upon application of a microneedle or microneedle device as described herein to a biological barrier of a subject, the base dissolves and the silk-based microneedle tip is implanted into the biological barrier (e.g., dermis of skin, For example, to a predetermined depth (eg, the maximum penetration depth of the distal portion of the tip) within a depth of between about 100 μm and about 800 μm. In certain embodiments, the entire tip is not embedded within, eg, the dermis of the skin (eg, to a depth between about 100 μm and about 800 μm). The implanted tip will then be given a period of time sufficient to generate immunity (e.g., for a period of at least about 4 days (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 or more days, e.g., between about 4 days and about 14 days, e.g., about 1-2 weeks, about 1-3 weeks, or about 1-4 weeks , for example, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, or about 6 weeks or more)) slowly releasing the 14th protein that is substantially composed of the SARS-CoV-2 spike protein To the 1208th residue, the 986th and 987th residues were replaced by proline, and the 682nd to 685th residues were replaced by "glycine-serine-alanine-serine Acid (GSAS)", and an immunogenic recombinant protein composed of a T4 fibrin trimerization domain at its C-terminus. Various properties (including, for example, crystallinity, β-sheet content, and molecular weight) of the silk fibroin base comprising the microneedle tip of the implantable controlled or sustained release can be adjusted to adjust (for example, change and/or modify ) the kinetics (e.g., release rate) of the immunogenic recombinant protein released from the microneedle tip, wherein the immunogenic recombinant protein consists essentially of residues 14 to 1208 of the SARS-CoV-2 spike protein , wherein the 986th and 987th residues were replaced by proline, and the 682nd to 685th residues were replaced by "glycine-serine-alanine-serine (GSAS)", and a T4 fibrin trimerization domain at its C-terminus. In certain embodiments, the implantable controlled or sustained release microneedle tip comprises about 10% to about 60% (e.g., about 10%, about 20%, about 30%, about 40%, about 50% , about 60%), for example, based on a "crystallinity index", for example, a "crystallinity index" known in the art.

The formulation, composition, product, device, and preparation for controlled or sustained release comprise an immunogenic recombinant spike protein of SARS-CoV-2, which is basically composed of an amine group as shown in SEQ ID NO: 1 The 14th to 1208th residues of the SARS-CoV-2 spike protein in acid sequence, wherein the 986th and 987th residues were replaced by proline, and the 682nd to 685th residues were replaced by "Glycine-serine-alanine-serine (GSAS)" and a T4 fibrin trimerization domain as shown in SEQ ID NO: 2 at its C-terminus. In some embodiments, the immunogenic recombinant protein has an amino acid sequence as shown in SEQ ID NO:5. In certain embodiments, the formulations, compositions, articles, devices, and formulations described herein for controlled and/or sustained release are administered for a period of time sufficient to confer immunity (e.g., for a period of at least about 1 days to about 14 days (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 or more days, e.g., about 4 Between days and about 14 days, e.g., about 1-2 weeks, about 1-3 weeks, or about 1-4 weeks, e.g., about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks , or about 6 weeks or more)) releases the substantially 14th to 1208th residues of the SARS-CoV-2 spike protein, wherein the 986th and 987th residues are replaced by proline , the 682nd to 685th residues are replaced by "glycine-serine-alanine-serine (GSAS)", and it has a T4 fibrin trimerization domain at its C-terminus immunogenic recombinant proteins. Accordingly, controlled or sustained release formulations, compositions, articles, devices, and formulations, microneedles, microneedle devices, kits, and methods of making and using the same are disclosed.

definition

Unless defined otherwise below, all scientific and technical terms used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art. References to techniques used herein are intended to indicate techniques as commonly understood in the art, including variations or equivalents of those techniques or later developed alternatives as would be apparent to those skilled in the art. In addition, in order to more clearly and concisely describe the subject matter of the present invention, the following definitions are provided for certain terms used in the specification and appended claims.

The articles "a" and "an" are used herein to refer to one or more than one (ie, at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

As used herein, the term "about" means +/- 10% of the recited value.

The phrase "and/or", as used herein in the specification and claims, should be understood to mean "one or both" of the elements so combined, that is, in some cases present in combination, and In other cases detaches existing elements. Multiple elements listed with "and/or" should be construed in the same fashion, ie, "one or more" of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the "and/or" clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, references to "A and/or B" in one embodiment may refer to A only (optionally including elements other than B); in another embodiment, only B (optionally including elements other than A); in yet another embodiment, both A and B (optionally including other element); etc.

As used herein in the description and scope of claims, "or" should be understood as having the same meaning as "and/or" as defined above. For example, "or" or "and/or" when separating the items of a list should be construed as being inclusive, i.e., containing a plurality of elements or at least one (but also including more than one) of a list of elements. ), and optionally include other items not listed. "Consisting of" will refer to a list comprising a plurality of elements or the exact an element. In general, the term "or" as used herein when preceded by an exclusive term such as "either", "one of", "only one", or "exactly one" should be construed only to indicate an exclusive alternative options (ie, "one or the other, but not both").

As used herein in the description and claims, with respect to a list of one or more elements, the phrase "at least one" should be understood to mean at least one element selected from any one or more elements in the list of elements, but At least one of every element specifically listed in the list of elements is not necessarily included, and any combination of elements in the list of elements is not excluded. This definition also allows that elements may optionally be present other than the elements specifically identified in the list of elements to which the phrase "at least one" refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, in an embodiment, "at least one of A and B" (or equivalently, "at least one of A or B", or equivalently "A and/or B At least one of ") can refer to at least one, optionally including more than one (A), not including B (and optionally including elements other than B); in another specific embodiment, refers to at least one , optionally including more than one (B), not including A (and selectively including elements other than A); in yet another specific embodiment, refers to at least one, optionally including more than one (A ), and at least one, optionally including more than one (B) (and optionally including other elements); and so on.

It should also be understood that, unless expressly indicated to the contrary, in any method claimed herein comprising more than one step or action, the order of the steps or actions of the method is not necessarily limited to the order in which the steps or actions of the method are recited .

As used herein, the term "microneedle" refers to a structure having at least two, and more typically, three components (e.g., three layers) for transporting or delivering a therapeutic agent (e.g., a vaccine, an antigen, and/or an immunogen) across a biological barrier (eg, skin, tissue, or cell membrane). In certain embodiments, a microneedle comprises a base (eg, the soluble base described herein), a tip (eg, the implantable tip described herein), and optionally, a backing material. The microneedles described herein can be of any shape and/or geometry suitable for piercing a biological barrier (e.g., a layer of skin) to enable release (e.g., controlled or sustained release) of a vaccine in a subject. ). Non-limiting examples of the shape and/or geometry of the microneedles include: a cylinder, a wedge, a cone, a pyramid, and/or an irregular shape, or any combination thereof.

In specific embodiments, a microneedle has a height dimension between about 350 μm and about 1500 μm (eg, between about 350 μm and about 1500 μm, such as about 350 μm, about 400 μm, about 450 μm, about 500 μm, about 550 μm, about 600 μm, about 650 μm, about 700 μm, about 750 μm, about 800 μm, about 850 μm, about 900 μm, about 950 μm, about 1000 μm, about 1050 μm, about 1100 μm , about 1150 μm, about 1200 μm, about 1250 μm, about 1300 μm, about 1350 μm, about 1400 μm, about 1450 μm, about 1500 μm). In certain embodiments, the height of the implantable tip can extend to about half of the full height of the microneedle, and the height of the implantable tip is between about 75 μm and about 475 μm (e.g., about 75 μm , about 100 μm, about 125 μm, about 150 μm, about 175 μm, about 200 μm, about 225 μm, about 250 μm, about 275 μm, about 300 μm, about 325 μm, about 375 μm, about 400 μm, about 425 μm, or about 475 μm). In certain embodiments, the implantable tip comprises a tip radius ranging from about 0.5 μm to about 25 μm (e.g., about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4 , 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 μm), preferably at about Between 5 μm and about 10 μm (eg, about 5, 6, 7, 8, 9, or 10 μm). In certain embodiments, the implantable tip comprises an angle between about 5 degrees and about 45 degrees (e.g., about 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35 , 40, or 45 degrees). In certain embodiments, the microneedles are fabricated to have any size and/or geometry capable of deploying a microneedle at a depth between about 100 μm and about 900 μm (e.g., at a depth of about 800 μm). Sustained release implants to enter the dermis of the skin for controlled or sustained release of a vaccine.

As used herein, the terms "microneedle patch" and "microneedle array" refer to a device comprising a plurality of microneedles, such as silk fibrin-based microneedles, for example, arranged in a random or predetermined pattern, such as a array. In some embodiments, the microneedle patch or microneedle array may include about 121 microneedles arranged in a square grid of 11 x 11 with a pitch of about 0.75 mm. A single microneedle is a cone with a length of about 0.65 mm, a diameter of about 0.35 mm at the bottom, and an included angle of about 30 degrees. Microneedle tips must be sharp to penetrate the skin. The radius of curvature of the tip should ideally not exceed 0.01 mm.

As used herein, the term "backing" refers to a material suitable for bonding and/or adhering to a microneedle assembly. In certain embodiments, the backing material is suitable for bonding and/or adhering to the dissolvable bases of the microneedles described herein.

As used herein, the term "soluble base" refers to the layer that forms the base of the microneedle (e.g., as a support for a distal implantable filament tip loaded with a vaccine, antigen, or immunogen), and/or can also serve as Layers of adjacent microneedles are joined to form a continuous microneedle array or microneedle patch. In certain embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more of the base is applied to a biological barrier (e.g., skin or mucous surface or buccal cavity) after dissolution. In certain embodiments, the soluble base comprises at least one (e.g., one, two, three, four, five, six, seven, eight, or more (e.g., all)) of : Gelatin, polyethylene glycol (PEG), sucrose, carboxymethylcellulose (CMC), polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA), hyaluronic acid salt, maltose, and methylcellulose. In certain embodiments, the soluble base comprises between about 10% and about 70% (w/v) gelatin (e.g., hydrolyzed gelatin), between about 1% and about 35% (w/v) sucrose, about 1% to about 35% (w/v) of CMC, about 10% to about 70% (w/v) of PVP, about 1% to about 35% (w/v) of Between about 1% to about 75% (w/v) of PVA, about 1% to about 75% (w/v) of hyaluronate, about 1% to about 75% (w/v) of maltose, about 1% to about 75% (w/v ), and between about 1% and about 70% (w/v) of polyethylene glycol. In certain embodiments, the soluble base comprises about 40% (w/v) hydrolyzed gelatin and about 10% (w/v) sucrose. In certain embodiments, the soluble base comprises about 30% (w/v) PVP and about 10% (w/v) PVA. In certain embodiments, the soluble base comprises about 37% (w/v) PVP, about 5% (w/v) PVA, and about 15% (w/v) sucrose.

As used interchangeably herein, the term "implantable sustained-release tip" or "releasable tip" refers to a tip capable of piercing a biological barrier (e.g., skin, mucous surface, or buccal cavity) of a subject and depositing The distal ends (eg, tips) of the microneedles within the biological barrier, skin layer (eg, dermis). In particular embodiments, the tip comprises a strand of fibrin in an amount sufficient to maintain the release of a therapeutic agent (e.g., vaccine, antigen, and/or immunogen) for an extended period of time, such as at least about 4 days (e.g., about 1, 2 , 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 or more days, e.g., between about 4 days and about 14 days, e.g., about 1-2 weeks , about 1-3 weeks, or about 1-4 weeks). In certain embodiments, the implantable sustained release tip comprises an immunogenic recombinant protein consisting essentially of residues 14 to 1208 of the SARS-CoV-2 spike protein, wherein residue 986 With residue 987 replaced by proline, residues 682 to 685 replaced by "glycine-serine-alanine-serine (GSAS)", and at its C-terminal It consists of a T4 fibrin trimerization domain.

As used herein, the term "gelatin" refers to a water-soluble protein derived from collagen. In certain embodiments, the term "gelatin" refers to a sterile pyrogen-free protein preparation produced by partial acid hydrolysis (type A gelatin) or by partial alkaline hydrolysis (type B gelatin) of animal collagen (eg, grade points), the most common sources are cattle, pigs, and fish. Gelatin is available in different molecular weight ranges. Gelatin from recombinant sources may also be used.

As used herein, the term "polyethylene glycol (PEG)" refers to an oligomer or polymer of ethylene oxide. PEG is also known as polyethylene oxide (polyethylene oxide, PEO) or polyoxyethylene (polyoxyethylene, POE). The structure of PEG is generally represented as H-(O- _CH2 - _CH2 ) _n -OH.

As used herein, the term "silk fibroin" includes silk fibroin as well as insect or spider silk protein. According to aspects described herein, any type of silk fibroin may be used. Silk fibroin produced by silkworms (eg, Bombyx mori ) is the most common and is an environmentally friendly and renewable resource. For example, silk fibroin for use in microneedles (eg, implantable controlled or sustained release tips of microneedles) can be obtained by removing sericin from cocoons of silkworms. In certain embodiments, the silk fibroin is a regenerated silk fibroin, for example, sericin is extracted from cocoons of Bombyx mori and silk fibroin is obtained after additional processing such as a boiling step. Organic cocoons are also available for purchase. However, many different kinds of silk can be used, including spider silk (e.g., obtained from Nephila clavipes ), transgenic silk, recombinant and/or genetically engineered silk, e.g., from bacteria, yeast, mammalian cells, Silk of transgenic animals, or transgenic plants (see, eg, WO 97/08315; US Patent No. 5,245,012) and variants thereof.

Exemplary silk fibroin (eg, regenerated silk fibroin) solutions can have various molecular weight distributions as determined by size exclusion chromatography (SEC) methods (see, eg, Table 1). In certain embodiments, silk fibroin solutions may, for example, be prepared according to established methods. In certain embodiments, cocoon fragments from Bombyx mori were first boiled in 0.02 M Na ₂ CO ₃ to remove sericin present in raw natural silk, and then subjected to size sieve chromatography (SEC) analyze. In certain embodiments, the silk fibroin composition can be obtained by degumming silkworm cocoons from Bombyx mori for about 480 minutes or less (e.g., less than 480 minutes, less than 400 minutes, less than 300 minutes) at atmospheric boiling temperature. minutes, less than 200 minutes, less than 180 minutes, less than 120 minutes, less than 100 minutes, less than 60 minutes, less than 50 minutes, less than 40 minutes, less than 30 minutes, less than 20 minutes, less than 10 minutes minutes or less) resulting compositions or mixtures. In one embodiment, the silk fibroin composition can be obtained by degumming silk cocoons in an aqueous solution of sodium carbonate at atmospheric boiling temperature for about 480 minutes or less (e.g., less than 480 minutes, less than 400 minutes , less than 300 minutes, less than 200 minutes, less than 180 minutes, less than 120 minutes, less than 100 minutes, less than 60 minutes, less than 50 minutes, less than 40 minutes, less than 30 minutes, less than 20 minutes , less than 10 minutes or less) resulting compositions or mixtures.

In some specific embodiments, the silk fibroin solution can be boiled for 10 minutes (10-minute boil, 10 MB), boiled for 60 minutes (60 MB), boiled for 120 minutes (120 MB), boiled for 180 minutes (180 MB) MB), or a silk fibroin solution boiled for 480 minutes (480 MB) (see, for example, Table 1). In some specific embodiments, the 14th to 1208th residues of the SARS-CoV-2 spike protein are basically composed, wherein the 986th and 987th residues are replaced by proline, and the 682nd The 685th residue was replaced by "glycine-serine-alanine-serine (GSAS)" and has an immunogenicity composed of a T4 fibrin trimerization domain at its C-terminus Recombinant proteins can be formulated at about 0.1% (w/v) to about 10% (w/v) (e.g., about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8% , 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% (w/v)) in 10 MB silk fibroin solution. In some specific embodiments, the 14th to 1208th residues of the SARS-CoV-2 spike protein are basically composed, wherein the 986th and 987th residues are replaced by proline, and the 682nd The 685th residue was replaced by "glycine-serine-alanine-serine (GSAS)" and has an immunogenicity composed of a T4 fibrin trimerization domain at its C-terminus Recombinant proteins can be formulated at about 0.1% (w/v) to about 10% (w/v) (e.g., about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8% , 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% (w/v)) in a 60 MB silk fibroin solution. In some specific embodiments, the 14th to 1208th residues of the SARS-CoV-2 spike protein are basically composed, wherein the 986th and 987th residues are replaced by proline, and the 682nd The 685th residue was replaced by "glycine-serine-alanine-serine (GSAS)" and has an immunogenicity composed of a T4 fibrin trimerization domain at its C-terminus Recombinant proteins can be formulated at about 0.1% (w/v) to about 10% (w/v) (e.g., about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8% , 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% (w/v)) in 120 MB silk fibroin solution. In some specific embodiments, the 14th to 1208th residues of the SARS-CoV-2 spike protein are basically composed, wherein the 986th and 987th residues are replaced by proline, and the 682nd The 685th residue was replaced by "glycine-serine-alanine-serine (GSAS)" and has an immunogenicity composed of a T4 fibrin trimerization domain at its C-terminus Recombinant proteins can be formulated at about 0.1% (w/v) to about 10% (w/v) (e.g., about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8% , 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% (w/v)) in 180 MB silk fibroin solution. In some specific embodiments, the 14th to 1208th residues of the SARS-CoV-2 spike protein are basically composed, wherein the 986th and 987th residues are replaced by proline, and the 682nd The 685th residue was replaced by "glycine-serine-alanine-serine (GSAS)" and has an immunogenicity composed of a T4 fibrin trimerization domain at its C-terminus Recombinant proteins can be formulated at about 0.1% (w/v) to about 10% (w/v) (e.g., about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8% , 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% (w/v)) in 480 MB silk fibroin solution.

Without being bound by theory, the main tunability of the implantable sustained release tip is its crystallinity, as measured by β-sheet content (intermolecular and intramolecular β-sheets). This affects the solubility of the filament tip matrix and the ability to retain antigen. As the β-sheet content increases, the mechanical strength of the tip also becomes stronger. A specific vaccine release profile is achieved by adjusting the crystallinity and diffusivity of the silk matrix. This is achieved through silk input materials and formulations and post-treatments to increase crystallinity (eg, water annealing, methanol/solvent annealing). In certain embodiments, the implantable controlled or sustained release microneedle tip comprises about 10% to about 60% (e.g., about 10%, about 20%, about 30%, about 40%, about 50% %, about 60%), e.g., based on the β-sheet content of the "crystallinity index" (e.g., the "crystallinity index" known in the art). In certain embodiments, the implantable controlled or sustained release microneedle tip can be formulated as a particle (eg, a microparticle and/or a nanoparticle).

Table 1. Comparison of 4 Molecular Weight Groups (Typical Values) Boil for 60 minutes (60 MB) molecular weight range range number High Molecular Weight Limitation [ Daltons ] Low Molecular Weight Limitation [ Daltons ] % range full range: (Maximum) 6,551,088 (minimum) 52 100 1 6,551,088 200,000 14.33 2 200,000 116,300 9.94 3 116,300 66,300 13.09 Total 71.67 4 66,300 36,500 15.63 5 36,500 3,500 42.95 6 3,500 52 4.06 Boil for 10 minutes (10 MB) molecular weight range range number High Molecular Weight Limitation [ Daltons ] Low Molecular Weight Limitation [ Daltons ] % range full range: (maximum) 34,079,621 (minimum) 128 100 1 34,079,621 200,000 4.96 2 200,000 116,300 3.66 3 116,300 66,300 6.34 Sum 80.17 4 66,300 36,500 10.54 5 36,500 3,500 63.29 6 3,500 128 11.22 Boil for 180 minutes (180 MB) molecular weight range range number High Molecular Weight Limitation [ Daltons ] Low Molecular Weight Limitation [ Daltons ] % range full range: (Max) 3,495,391 (minimum) 71 100 1 3,495,391 200,000 7.12 2 200,000 116,300 6.07 3 116,300 66,300 9.66 Total 79.55 4 66,300 36,500 14.07 5 36,500 3,500 55.82 6 3,500 71 7.26 Boil for 480 minutes (480 MB) molecular weight range range number High Molecular Weight Limitation [ Daltons ] Low Molecular Weight Limitation [ Daltons ] % range full range: (Max) 552,130 (minimum) 128 100 1 552,130 200,000 0.26 2 200,000 116,300 0.75 3 116,300 66,300 2.26 Total 82.57 4 66,300 36,500 5.97 5 36,500 3,500 74.34 6 3,500 128 16.42

20、Tween

40、Tween

60、Tween

65、Tween

80、Tween

85、Laureth-4、Ceteth-2、Ceteth-20、Steareth-2、PEG40、PEG100、PEG150、PEG200、PEG600、Span

20、Span

40、Span

60、Span

65、Span

80。 As used herein, the term "surfactant" refers to any molecule having both a polar head group energetically inclined to be water-solubilized, and a hydrophobic tail group which is not water-solubilizable. The term "cationic surfactant" refers to a surfactant having a cationic head group. The term "anionic surfactant" refers to a surfactant having an anionic head group. The term "nonionic surfactant" refers to a surfactant having an uncharged head group. The term "zwitterionic surfactant" refers to a surfactant having both cationic and anionic head groups. Exemplary surfactants include, but are not limited to, cationic surfactants, anionic surfactants, zwitterionic surfactants, and nonionic surfactants, preferably including, but not limited to, Tween

20. Tween

40、Tween

60、Tween

65、Tween

80、Tween

85, Laureth-4, Ceteth-2, Ceteth-20, Steareth-2, PEG40, PEG100, PEG150, PEG200, PEG600, Span

20. Span

40、Span

60、Span

65、Span

80.

As used herein, the term "controlled or sustained release" refers to a period of time, such as at least about 1-14 days (for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, or 14 days or more, e.g., between about 4 days and about 14 days, e.g., about 1-2 weeks, about 1-3 weeks, or about 1-4 weeks) release a therapeutic agent (e.g., from the microneedles, microneedle devices, formulations, compositions, articles, devices, and formulations described herein, e.g., from silk fibroin-based microneedle tips as described herein), such as a vaccine, antigen , and/or immunogens. In certain embodiments, the controlled or sustained release of a vaccine from the microneedles, microneedle devices, formulations, compositions, articles, devices, or formulations described herein is, for example, over a period of about 1 to about 14 days ( For example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days) can result in, for example, broad immunity in a subject. In certain embodiments, vaccine formulations and formulations comprising silk fibroin have controlled or sustained release properties (e.g., are formulated and/or configured to release a vaccine, e.g., to the skin of the subject over a period of time). For example, within a period of at least 1, 5, 10, 15, 30, 45 minutes; or within a period of at least 1, 2, 3, 4, 5, 10, 24 hours; or at least 1, 2, 3 , 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days; or at least 1, 2, 3, 4, 5, 6, 7, 8 weeks ; or for a period of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 months; or for a period of at least 1, 2, 3, 4, 5 years or longer .

As used herein, an "adjuvant" is a substance that promotes or amplifies the cascade of immune events, ultimately resulting in an enhanced immune response, eg, the overall body response to an antigen, including cellular and/or humoral immune responses. Non-limiting examples of adjuvants include: aluminum (e.g., aluminum gel and/or aluminum salts, such as aluminum hydroxide, aluminum phosphate, and aluminum potassium sulfate), lipids (e.g., squalene, monophosphoryl lipid A ( monophosphoryl lipid A, MPL)), AS03 (for example, containing D,L-α-tocopherol (vitamin E), squalene, and an adjuvant of polysorbate 80), AS04 (for example, containing aluminum hydroxide and MPL combination adjuvant), and MF59® (for example, an adjuvant containing squalene).

As used herein, the term "immunity" or "protective immunity" refers to an immune response elicited by a vaccine or immunization regimen (e.g., vaccination regimen), e.g., humoral and/or cellular responses, when the vaccine or Immunization regimens, when administered to a subject in need thereof (e.g., a subject described herein), can prevent, delay the development of an infection caused by a virus described herein, and/or reduce the risk of infection caused by a virus described herein. The severity of the infection caused by the virus. In certain embodiments, the immunity or protective immunity reduces or completely eliminates the symptoms of the viral infection. In certain embodiments, immunity or protective immunity is characterized by the presence of one or more of: circulating antibodies (e.g., humoral immunity), presence of sensitized T lymphocytes (e.g., cellular immunity), presence of Secretory IgA (eg, mucosal immunity), or a combination thereof.

As used herein, the phrase "broad immunity" refers to immunity against at least one (e.g., against at least two, at least three, at least four, at least five, or more) viruses (e.g., SARS-CoV-2 ) strains and/or variants, such as humoral and/or cellular responses (e.g., immunity or protective immunity), wherein at least one strain and/or variant is absent (e.g., as described herein method, microneedle, and microneedle device) administered to a subject in a vaccine.

As used herein, the term "dose" refers to the amount of a vaccine, antigen, and/or immunogen administered (e.g., vaccinated) to elicit an immune response (e.g., humoral and/or cellular immune response) in an organism .

As used herein, a "standard dose" refers to the amount of antigen in a typical human dose of a vaccine, as approved for marketing by national or international regulatory agencies (eg, U.S. FDA, EMA).

As used herein, the term "immunogenicity" refers to the ability of a substance (eg, an antigen or epitope) to elicit a humoral and/or cell-mediated immune response in a subject. Immunogenicity of a substance can be readily measured by one skilled in the art. Following administration of an immunogen, the presence of a cell-mediated immune response can be determined by any art-recognized method, for example, proliferation assays (CD4 ⁺ T cells), cytotoxic T lymphocyte (CTL) assays, Or perform immunohistochemical analysis on a tissue section of a subject to determine the presence of activated cells such as monocytes and macrophages. A person skilled in the art can readily determine whether a humoral-mediated immune response exists in a subject by any recognized method. For example, the amount of antibodies produced in a biological sample (eg, blood) can be measured by Western blot analysis, ELISA, or other known methods for antibody detection (eg, pseudovirus-based neutralizing antibody assays).

As used herein, a "subject" refers to a human or animal. Typically, the animal is a vertebrate, eg, a primate, rodent, livestock, or game animal. Primates include chimpanzees, cynomolgus monkeys, spider monkeys, and rhesus monkeys (eg, rhesus monkeys). Rodents include mice, rats, woodchucks, ferrets, rabbits, and hamsters. Livestock and game animals include cattle, horses, pigs, deer, bison, buffalo, felines (e.g., domestic cats), canines (e.g., dogs, foxes, wolves), birds (e.g., chickens, emu, ostriches ), and fish (eg, trout, catfish and salmon). In certain embodiments of the aspects described herein, the subject is a mammal (eg, a primate, such as a human). Subjects can be male or female. In certain embodiments, the subject is a mammal. The mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but is not limited to these examples. In addition, the methods and formulations described herein can be used to treat domesticated animals and/or pets.

冠狀病毒屬( Betacoronavirus)的成員。SARS-CoV-2的RNA序列長度約為30,000個鹼基。每個SARS-CoV-2病毒顆粒的直徑為50-200奈米。與其他冠狀病毒一樣，SARS-CoV-2具有四種結構蛋白，分別為棘蛋白(spike, S)、包膜蛋白(envelope, E)、膜蛋白(membrane, M)，以及核殼蛋白(nuicleocapsid, N)。N蛋白抓住RNA基因組，而S、E及M蛋白共同形成病毒包膜。S蛋白為負責讓病毒附著在宿主細胞膜上並與之融合的蛋白質。 As used herein, the term "severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)" refers to the strain of coronavirus that causes coronavirus disease 2019 (COVID-19) and variants thereof. SARS-CoV-2 is a positive-sense single-stranded RNA virus belonging to the family Coronavirinae ,

A member of the genus Betacoronavirus . The RNA sequence of SARS-CoV-2 is approximately 30,000 bases in length. Each SARS-CoV-2 virus particle is 50-200 nanometers in diameter. Like other coronaviruses, SARS-CoV-2 has four structural proteins, namely spike protein (spike, S), envelope protein (envelope, E), membrane protein (membrane, M), and nucleocapsid protein (nucleocapsid). , N). The N protein grabs the RNA genome, while the S, E, and M proteins together form the viral envelope. The S protein is the protein responsible for allowing the virus to attach to and fuse with the host cell membrane.

As used herein, the terms "spike protein", "S polypeptide", "S protein", "SARS-CoV-2 spike protein", or "SARS-CoV-2 S protein" used interchangeably refer to SARS CoV The surface structural glycoprotein on -2 is responsible for allowing the virus to attach to and fuse with the membrane of the host cell. Each monomer of the trimeric S protein is approximately 180 kDa and contains two subunits, S1 and S2, which regulate attachment and membrane fusion, respectively. Spiny protein enters human cells mainly by binding to the receptor angiotensin converting enzyme 2 (ACE2).

Mutants have been detected regularly since the start of the COVID-19 global pandemic. The emergence of variant strains increases the risk of global public health and prompts special attention to specific variant strains (Variants of Interest, VOIs), high concern variant strains (Variants of Concern, VOCs), and variant strains under monitoring (Variants Under Monitoring, VUMs) for characterization to identify variants for priority global surveillance and research, and ultimately to inform the ongoing response to the COVID-19 pandemic (https://www.who.int/en/activities/tracking-SARS- CoV-2-variants/).

As used herein, the term "Variants of Very High Concern (VOCs)" refers to variants of SARS-CoV-2 that meet the definition of a Variant of Attention (VOI) and, through comparative evaluation, have been shown to have global Changes of public health significance related to: (i) increased transmissibility or deleterious changes in the epidemiology of COVID-19; or (ii) increased virulence or changes in clinical disease manifestations; or (iii) public health and social measures or existing diagnoses Methods, vaccines, treatments are less effective. Likewise, these definitions may be adjusted periodically as the virus that causes COVID-19 continues to evolve, and as understanding of the effects of variant strains evolves.

As used herein, the term "Alpha variant" also known as lineage B.1.1.7 refers to a variant of SARS-CoV-2, the virus that causes COVID-19. Compared with the S protein of the SARS-CoV-2 Wuhan-Hu-1 strain (wild type), the amino acid sequence of the S protein of the SARS-CoV-2 Alpha variant contains the following substitutions: 69del (deletion), 70del, 144del , (E484K*), (S494P*), N501Y, A570D, D614G, P681H, T716I, S982A, D1118H (K1191N*) (https://www.cdc.gov/coronavirus/2019-ncov/variants/variant-info .html) and (https://www.acep.org/corona/covid-19-field-guide/characteristics-of-covid-19-variants-and-mutants/characteristics-of-covid-19-variants-and -mutants/).

As used herein, the term "Beta variant" is also known as the B.1.351 or 501Y.V2 lineage, which refers to a variant of the virus SARS-CoV-2 that causes COVID-19. Compared with the S protein of the SARS-CoV-2 Wuhan-Hu-1 virus strain (wild type), the S protein amino acid sequence of the SARS-CoV-2 Beta variant contains the following substitutions: D80A, D215G, 241del, 242del, 243del , K417N, E484K, N501Y, D614G, A701V (https://www.cdc.gov/coronavirus/2019-ncov/variants/variant-info.html), (https://www.acep.org/corona/ covid-19-field-guide/characteristics-of-covid-19-variants-and-mutants/characteristics-of-covid-19-variants-and-mutants/).

An "effective amount" or a "sufficient amount" of a substance is an amount sufficient to produce beneficial or desired results (including clinical results), thus an "effective amount" depends on the context of its use. In the case of administering an immunogenic composition, an effective amount comprises sufficient adjuvant and SARS-CoV-2 S-2P recombinant protein to elicit an immune response. One or more doses may be administered to achieve an effective amount.

The anti-SARS-CoV-2 microneedle vaccine of the present invention can be prepared through the method comprising the following steps: providing a mold comprising a mold body having an array of needle cavities formed therein having a predetermined shape (e.g., pyramidal and/or conical needle cavities); An immunogenic recombinant protein is provided, which basically consists of the 14th to 1208th residues of the SARS-CoV-2 spike protein, wherein the 986th and 987th residues are replaced by proline, and the 682nd The 685th residue was replaced by "glycine-serine-alanine-serine (GSAS)", and it has a T4 fibrin trimerization domain at its C-terminus; filling the tip of the needle lumen with a composition consisting of a strand of fibrin and the immunogenic recombinant protein; drying the tip of the filled needle cavity to produce a releasable tip, and optionally annealing the needle tip; filling the needle cavity of the mold with a soluble base solution; drying the soluble base solution to form the base layer of the releasable tip; and A backing layer is applied to the base layer to make a microneedle vaccine.

In some embodiments, the method further comprises the step of removing the microneedle vaccine from the mold.

In some embodiments, the microneedle vaccine is removed by bending the mold away from the microneedle vaccine.

In some embodiments, the method further includes the step of packaging the microneedle vaccine in a container with a low moisture vapor transmission rate and a desiccant, so that the relative humidity inside the package is maintained at about 0% to Between about 50% (e.g., between about 0% to 10%, between about 10% to about 20%, between about 20% to about 30%, between about 30% to about 40%, or about 40% % to 50%, such as about 25%).

In certain embodiments, the silk fibroin, antigen solution is dispensed into each needle cavity in the mold by nanoliter printing.

In certain embodiments, the step of filling the lumen tips includes dispensing a solution (eg, an antigen-silk formulation) into each lumen.

In some embodiments, the step of drying the filling tip of the lumen includes a first drying step and a second drying step.

In certain embodiments, the step of filling the needle cavity of the mold with a soluble base solution comprises a deionized water (deionized) containing 40% (w/v) hydrolyzed gelatin and 10% (w/v) sucrose water DIW) solution.

In some embodiments, the step of filling the soluble base solution includes centrifuging the mold at 3900 rpm for 2 minutes, and filling the needle cavity with 50 μL of the base solution.

In some embodiments, the method further includes an annealing step after filling the tip of the needle cavity (eg, before filling the base).

In some embodiments, the method further includes a step of water annealing after filling the tip of the needle lumen (eg, before filling the base).

In some embodiments, the backing layer includes one of a paper backing layer and an adhesive plastic tape.

The invention is further illustrated by the following examples, which are provided for purposes of illustration and not limitation. Those of skill in the art should, in light of the teachings of the present invention, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

Example

Example 1 anti- SARS-CoV-2 Preparation of the immunogenic composition of

1. The construct of the S-2P recombinant protein (S-2P) derived from the Wuhan strain of SARS-CoV-2.

A plastid with a polynucleotide encoding residues 14-1208 of the SARS-CoV-2 S protein (Wuhan-Hu-1 strain; GenBank: MN908947), wherein the 986th and 987th residues are replaced by proline and was substituted at the furin cleavage site (residues 682 to 685) by "glycine-serine-alanine-serine (GSAS)" (SEQ ID NO: 1 ), and has a T4 fibrin trimerization domain (SEQ ID NO: 2), a HRV3C protease cleavage site (SEQ ID NO: 3), an 8x His tag, and a Twin-Strep tag at its C-terminus (SEQ ID NO: 4), the plasmid was transfected into CHO cells obtained from the American Type Culture Collection (ATCC).

Cell cultures were harvested after 6 days and proteins were purified from the supernatant using Strep-Tactin resin (IBA Lifesciences, Göttingen, Germany). HRV3C protease (1%, by weight) was added to the protein and reacted overnight at 4°C. The cleaved protein was further purified using Superose 6 16/70 tubes (GE Healthcare Biosciences, Chicago, IL, USA). The purified SARS-CoV-2 S-2P recombinant protein (SEQ ID NO: 5) was then stored in phosphate-buffered saline (PBS) for future use.

2. Controlled or sustained release microneedle formulation and fabrication

Then the purified SARS-CoV-2 S-2P recombinant protein (S-2P) (SEQ ID NO: 5) was mixed with silk fibroin (180 MB), Tween® 20, and Milli-Q deionized (deionized, DI) Water was mixed to produce a formulation containing 1% (w/v) silk fibroin, 0.5% (w/v) Tween® 20, and about 2.067 mg/ml (equivalent to 5 µg/microneedle patch) of S-2P protein solution, which will be printed into the microneedle mold. The microneedle mold was used to produce a microneedle patch containing 121 microneedles arranged in a square grid of 11 x 11 at approximately 0.75 mm pitch, and each microneedle was approximately 0.65 mm long A cone with a base diameter of approximately 0.35 mm and an included angle of approximately 30 degrees.

Tip filling: 20 nL of the formulation was printed into a polydimethylsiloxane (PDMS) microneedle mold using vision-guided dispensing (Biodot AD3420).

Tip Fill Inspection: Visually evaluate prints under a stereo microscope for defects, including misaligned prints, incompletely filled needle cavities, and foreign objects.

Tip Drying: The filled microneedle molds were dried overnight (14-20 hours) under controlled 20% relative humidity.

Tip Annealing: Dried tips were annealed in water at 37°C for 4 hours by placing the mold in a vacuum desiccator filled with Milli-Q water, applying vacuum for 5 minutes, then closing the vacuum valve and moving the desiccator to 37° C incubator.

Tip Drying: After annealing, the tips are dried again overnight (14-20 hours) under controlled 20% relative humidity.

Base filling: A solution containing 40% (w/v) hydrolyzed gelatin (Gelita) and 10% (w/v) sucrose (Sigma-Aldrich) was pipetted onto the microneedle mold and centrifuged at 3,900 rpm for 2 minutes to fill.

Base Filling Inspection: Base fillings were also visually assessed through a stereomicroscope for the appearance of incompletely filled needle lumens. If underfilling is observed, refill and re-centrifuge.

Base Drying: The base solution is dried overnight (14-20 hours) at a controlled 20% relative humidity.

Backing application: Whatman 903 cardboard was punched into 12 mm rounds and the rounds were applied to a dried gelatin base pre-moistened with 10 µL of Milli-Q water.

Backing Drying: Devices were dried for 2 hours under controlled 20% relative humidity conditions before demoulding.

Demolding: The device was manually removed from the microneedle mold by carefully bending the mold away from the device while holding the device stationary.

Release check: Check devices under a stereomicroscope for complete release; discard devices that are not fully released.

Example 2. SARS-CoV-2 S-2P Recombinant protein (S-2P) Sustained intradermal delivery enhances antibody response

1. Immunization of mice

The purpose of the S-2P microinjection study in this example is to evaluate the immunogenicity of SARS-CoV-2 S-2P recombinant protein (S-2P) (SEQ ID NO: 5 or 6) without adjuvant. Immunogenicity testing differentiates between intramuscular (IM) and intradermal (ID) delivery. In addition, the humoral immune response to continuous intradermal (ID) delivery for 10 consecutive days was assessed by immunization with 1 mcg and 5 mcg S-2P. To assess dose dependence, immunogenicity was tested in single and double sustained release immunoassays.

The experimental design of this embodiment is shown in Table 2 . Five (5) BALB/c mice (n = 5) were used per group. The first group was an unvaccinated control group. Groups 2 and 3 were inoculated twice with 1 mcg per dose of S-2P via intradermal (ID) administration at 28-day intervals (Day 0 and Day 28). However, Group 2 was administered as a bolus injection compared to Group 3, which was administered with intradermal (ID) delivery for 10 consecutive days. The design of groups 4 and 5 was similar to that of groups 2 and 3, except that the dose of S-2P was increased from 1 mcg to 5 mcg per injection. Group 6 was similar in design to Group 5, but Group 6 was vaccinated once at day 0 (D0) with 5 mcg of S-2P. Group 7 was injected intramuscularly (IM) with 5 mcg of S-2P twice at 28-day intervals (Day 0 and Day 28). Group 7 was used to compare the effect of intramuscular (IM) and intradermal (ID) delivery on the immune response. Antisera were collected on day 29 (D29), day 49 (D49), and day 64 (D64). Each sample was analyzed for spinin-specific IgG by ELISA.

The experimental design of table 2 embodiment 2 group delivery route Dose of S-2P per immunization (µg) delivery method Number of immunizations Total dose (µg) immune schedule blood collection 1 ID - - - - - D29 D49 D64 2 ID 1 Bolus 2 2 D0, D28 3 ID 1 10 consecutive days 2 2 D0, D28 4 ID 5 Bolus 2 10 D0, D28 5 ID 5 10 consecutive days 2 10 D0, D28 6 ID 5 10 consecutive days 1 5 D0 7 IM 5 Bolus 2 10 D0, D28

2. Statistical analysis

Statistical analysis was performed with Prism 6.01 (GraphPad Software Inc., San Diego, CA, USA). Multiple T tests were performed with the false discovery rate (FDR) of Benjamini, Krieger, and Yekutieli, one-way ANOVA plus Tukey's multiple comparison method for log-transformed values, and repeated measures two-way ANOVA plus Log-transformed values were tested by Tukey's multiple comparison method, and significance was calculated where appropriate using these statistics. * p <0.05, ** p <0.01, *** p <0.001.

3. Results

The results are shown in Figs. 1A to 1C . Sustained intradermal (ID) delivery of slow-release S-2P antigen for 10 consecutive days ( Groups 3, 5, and 6) increase the content of spinin-specific IgG. As shown in Figure 1A , sustained delivery of S-2P protein (Groups 3, 5, 6) resulted in a significant increase in antibody responses at all three time points for the 1 μg and 5 μg doses. As shown in Figure 1B , after the second immunization, the antibody potency obtained by continuous intradermal delivery of S-2P protein (group 5) was 30 times higher than that of traditional intramuscular (IM) injection (group 7) ( p < 0.001). As shown in Figure 1C , the antibody potency produced by single sustained release immunization of S-2P protein (group 6) was more than 5 times higher than that of traditional intramuscular (IM) injection (group 7), and the antibody potency of group 6 The dose was only half of the total dose in group 7 ( p < 0.001 or p < 0.05). Taken together, these results show that continuous intradermal injection of an anti-SARS-CoV-2 vaccine produces a stronger humoral immune response compared with equal or double doses delivered by traditional intramuscular injection.

Example 3. SARS-CoV-2 S-2P Recombinant protein (S-2P) Sustained intradermal delivery enhances anti- SARS-CoV-2 Neutralizing antibody responses of mutant strains

1. Immunization of mice

The purpose of the S-2P microinjection study in this example was to evaluate the immunogenicity of the unadjuvanted S-2P protein (SEQ ID NO: 5 or 6). Immunogenicity testing differentiates between intramuscular (IM) and intradermal (ID) delivery. In addition, the humoral immune response to continuous intradermal (ID) delivery for 10 consecutive days was assessed by immunization with 1 mcg and 5 mcg S-2P. To assess dose dependence, immunogenicity was tested in single and two sustained release immunizations.

The experimental design of this embodiment is shown in Table 3 . Five (5) BALB/c mice (n = 5) were used per group. The first group was an unvaccinated control group. Groups 2 and 3 were inoculated twice with 1 mcg per dose of S-2P via intradermal (ID) administration at 28-day intervals (Day 0 and Day 28). However, Group 2 was administered as a bolus injection compared to Group 3, which was administered with intradermal (ID) delivery for 10 consecutive days. The design of groups 4 and 5 was similar to that of groups 2 and 3, except that the dose of S-2P was increased from 1 mcg to 5 mcg per injection. Group 6 was similar in design to Group 5, but Group 6 was vaccinated once at day 0 (D0) with 5 mcg of S-2P. Group 7 was injected intramuscularly (IM) with 5 mcg of S-2P twice at 28-day intervals (Day 0 and Day 28). Group 7 was used to compare the effect of intramuscular (IM) and intradermal (ID) delivery on the immune response. Blood samples were collected on day 28 (D28) and day 49 (D49). Each sample was analyzed for spinin-specific IgG by ELISA. And test the neutralizing antibody of each sample with different SARS-CoV-2 virus strains (Wuhan strain and D614G, B.1.1.7, and 501Y.V2 variants).

The experimental design of table 3 embodiment 3 group delivery route Dose of S-2P per immunization (µg) delivery method Number of immunizations Total dose (µg) immune schedule blood collection 1 ID - - - - - D28 D49 2 ID 1 Bolus 2 2 D0, D28 3 ID 1 10 consecutive days 2 2 D0, D28 4 ID 5 Bolus 2 10 D0, D28 5 ID 5 10 consecutive days 2 10 D0, D28 6 ID 5 10 consecutive days 1 5 D0 7 IM 5 Bolus 2 10 D0, D28

2. Neutralization assay based on pseudovirus

a. Production and quantification of pseudoviruses

8.91及報導質體pLAS2w.FLuc.Ppuro (RNA技術平台，中央研究院，台灣)共轉染至HEK293T細胞中。該質體表現以下SARS-CoV-2病毒株/變異株之一的全長 SARS-CoV-2棘蛋白：武漢-Hu-1病毒株(野生型；GenBank登錄號MN908947)、D614G (相較於野生型的S蛋白，D614G變異株具有D614G置換)、B.1.1.7 (Alpha變異株，相較於野生型的S蛋白具有以下置換：69del、70del、144del、(E484K*)、(S494P*)、N501Y、A570D、D614G、P681H、T716I、S982A、D1118H (K1191N*))，以及501Y.V2 (亦即B.1.351，β變異株，相較於野生型的S蛋白具有以下置換：D80A、D215G、241del、242del、243del、K417N、E484K、N501Y、D614G、A701V)。模擬假病毒係透過省略p2019-nCoV棘蛋白(野生行)所產生的。轉染後72小時，收集、過濾並於-80 °C下冷凍上清液。以細胞對慢病毒極限稀釋的反應進行細胞活性分析來估算SARS-CoV-2假型慢病毒的轉導單位 (transduction unit, TU)。簡言之，在進行慢病毒轉導的前1天，將穩定表現人類 ACE2基因的HEK-293 T細胞接種在96孔盤上。為了定量假病毒，將不同量的假病毒加入含有聚凝胺的培養基中。將96孔盤於37°C下以1100 xg進行離心感染30分鐘。於37°C下培養細胞16小時後，移除含有病毒及聚凝胺的培養基，並以含有2.5 µg/ml嘌呤黴素的新鮮完整DMEM培養基代替。以嘌呤黴素處理48小時後，移除培養基並根據製造商的說明使用10%阿爾瑪藍(Alarma Blue)試劑檢測細胞活性。將未被感染的細胞(未經嘌呤黴素處理)的存活率設為100%。以存活細胞對稀釋的病毒劑量做圖來確定病毒力價(轉導單位)。 To produce SARS-CoV-2 pseudoviruses, pCMV plastids expressing the full-length SARS-CoV-2 spike protein were combined with the packaging plastids using TransIT-LT1 transfection reagent (Mirus Bio)

8.91 and reporter plasmid pLAS2w.FLuc.Ppuro (RNA Technology Platform, Academia Sinica, Taiwan) were co-transfected into HEK293T cells. The plastid expresses the full-length SARS-CoV-2 spike protein of one of the following SARS-CoV-2 strains/mutants: Wuhan-Hu-1 strain (wild type; GenBank accession number MN908947), D614G (compared to wild type Type S protein, D614G mutant strain has D614G substitution), B.1.1.7 (Alpha mutant strain, compared with wild type S protein has the following substitutions: 69del, 70del, 144del, (E484K*), (S494P*) , N501Y, A570D, D614G, P681H, T716I, S982A, D1118H (K1191N*)), and 501Y.V2 (that is, B.1.351, the β mutant strain, which has the following substitutions in the S protein compared to the wild type: D80A, D215G , 241del, 242del, 243del, K417N, E484K, N501Y, D614G, A701V). The mock pseudovirus was generated by omitting the p2019-nCoV spike protein (wild line). 72 hours after transfection, the supernatant was collected, filtered and frozen at -80 °C. Cell viability analysis was performed based on the response of cells to the limiting dilution of lentivirus to estimate the transduction unit (TU) of SARS-CoV-2 pseudotyped lentivirus. Briefly, HEK-293 T cells stably expressing the human ACE2 gene were seeded in 96-well plates 1 day before lentiviral transduction. To quantify pseudoviruses, different amounts of pseudoviruses were added to polybrene-containing media. Infect the 96-well plate by centrifugation at 1100 x g for 30 min at 37 °C. After incubating the cells for 16 hours at 37°C, the medium containing the virus and polybrene was removed and replaced with fresh complete DMEM medium containing 2.5 µg/ml puromycin. After 48 hours of puromycin treatment, the medium was removed and cell viability was assayed using 10% Alarma Blue reagent according to the manufacturer's instructions. The viability of uninfected cells (not treated with puromycin) was set as 100%. Virus potency (transducing units) was determined by plotting surviving cells versus diluted virus dose.

b. Neutralization test based on pseudovirus

HEK293-hAce2 cells (2 x ¹⁰⁴ cells/well) were seeded in 96-well white cell culture dishes and cultured overnight. Serum was heated at 56°C for 30 minutes to inactivate complement, and the serum was diluted with MEM medium supplemented with 2% FBS, with an initial dilution factor of 100, followed by a two-fold serial dilution (a total of 8 dilution steps, the final dilution concentration is 1:25,600). Diluted serum was mixed with an equal volume of pseudovirus (1000 TU) and incubated at 37°C for 1 hour before adding to the culture dish with cells. After 1 hour of incubation, replace the medium with 50 µL of fresh medium. Replace the medium with 100 µL of fresh medium every other day. Cells were lysed 72 hours post infection and relative luciferase units (RLU) were measured. Luciferase activity was detected with Tecan i-control (Infinite 500). The uninfected cells were regarded as 100% neutralization, and the cells transduced with virus only were regarded as 0% neutralization, and the dilution multiple (namely ID ₅₀ ) when 50% inhibitory effect was achieved was calculated. Confirm that the reciprocal of the geometric mean power price (GMT) of ID ₅₀ is ID ₅₀ power price.

3. Statistical Analysis

Statistical analysis was as described in Example 2.

4. Results

The results are shown in Figures 2A to 2J . Sustained intradermal (ID) delivery of slow-release S-2P antigen for 10 consecutive days ( Groups 3, 5 and 6) increased spikin-specific IgG content ( Figure 2A and Figure 2B ) and neutralizing antibody responses ( Figure 2C to Figure 2J ). On day 28, anti-wild-type (Wuhan strain) ( FIG. 2C ) and high Pay attention to the neutralizing antibody titers of variant strains (VOCs), including D614G ( Figure 2E ), B.1.1.7 ( Figure 2G ) and 501Y.V2 ( Figure 2I ). On day 49, mice (Groups 3 and 5) immunized with 2 doses of S-2P delivered continuously intradermally (ID) developed strong resistance to wild-type as well as the three tested variants of high concern (VOCs ) neutralizing antibody potency ( Figure 2D , Figure 2F , Figure 2H , and Figure 2J ). The spinin-specific IgG and neutralizing antibody responses of the single-dose sustained-release group (group 6) were better than those of the second intramuscular (IM) injection of unadjuvanted S-2P group (group 7) ( Fig . 2B , Fig. 2D , Figure 2F , Figure 2H ). Taken together, these results show that continuous intradermal injection of an anti-SARS-CoV-2 vaccine produces a stronger neutralizing antibody response compared to equal or double doses delivered by traditional intramuscular injection.

Example 4. Immunoenhancement of antimicrobial agents via controlled or sustained release silk microneedles SARS-CoV-2 Neutralizing antibody responses of mutant strains

1. Immunization of mice

The purpose of the study in Example 4 was to evaluate the immunogenicity of the microneedle vaccine of the present invention compared to traditional intramuscular (IM) injection, and to compare single-dose and two-dose intradermal (ID) delivery with two-dose intramuscular (IM) delivery. ) injection regimen.

The experimental design of this embodiment is shown in Table 4 . Five BALB/c mice were used per group. One mouse died in group 4, but it was not related to the vaccine. The tip of the microneedle vaccine is formulated with 1% (w/v) silk fibroin and 0.5% (w/v) Tween® 20 with concentrated S-2P antigen. Store the microneedle vaccine at room temperature for 1 week prior to immunization. Group 1 mice were inoculated twice with 5 mcg of S-2P protein by intramuscular injection at 28-day intervals (Day 0 and Day 28). Concentrated antigen was used in group 2 compared to pure unconcentrated antigen used in group 1. Concentrated S-2P protein has passed through a concentration procedure prior to formulation, while pure S-2P protein remains unconcentrated. By comparing Group 1 and Group 2, the immunogenicity of the S-2P antigen after the enrichment process can be confirmed. Groups 3 and 4 used concentrated S-2P antigen and delivered it through a microneedle patch. The dose for groups 3 and 4 was 5 mcg per injection, which was the same as that for groups 1 and 2. However, mice in group 3 were vaccinated only once, while mice in group 4 were vaccinated twice. Blood samples were collected on day 28 (D28) and day 52 (D52). Each sample was analyzed for spinin-specific IgG by ELISA. And test the neutralizing antibody of each sample with different SARS-CoV-2 virus strains (Wuhan strain and D614G, B.1.1.7, and 501Y.V2 variants). Statistical analysis and neutralization test method based on pseudovirus are as described in embodiment 2 and embodiment 3 respectively.

The experimental design of table 4 embodiment 4 group number of mice antigen dose per dose delivery route delivery method Number of immunizations total dose 1 5 Unconcentrated S-2P 5 µg IM Bolus 2 10 µg 2 5 Concentrated S-2P 5 µg IM Bolus 2 10 µg 3 5 Concentrated S-2P 5 µg ID 10 consecutive days 1 5 µg 4 5* Concentrated S-2P 5 µg ID 10 consecutive days 2 10 µg * n = 4 due to the death of one of the mice (unrelated to the vaccine).

2. Results

The results are shown in Figure 3A to Figure 3E . The results of intradermal (ID) delivery of unadjuvanted S-2P (groups 3 and 4) showed stronger spikes compared to conventional intramuscular (IM) administration (groups 1 and 2). Protein-specific IgG ( FIG. 3A ) and neutralizing antibody responses ( FIGS . 3B to 3E ). Intradermal ( ID ) delivery of two doses of unadjuvanted S - 2P protein showed enhanced resistance to wild-type ( Fig . and antibody titers. While a single-dose intradermal (ID) immunization produced a strong spinin-specific IgG response, a neutralizing antibody response was only observed with a second-dose intradermal (ID) immunization. Taken together, these results show that controlled or sustained-release anti-SARS-CoV-2 vaccines delivered by silk microneedles generate stronger neutralizing antibody responses than equivalents delivered by traditional intramuscular injection.

equivalent

Although the present invention has been particularly shown and described with reference to preferred embodiments thereof, those skilled in the art will appreciate that changes may be made in its form and scope without departing from the spirit and scope of the invention as defined by the appended claims. Various changes were made to the details. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention specifically described herein. Such equivalents are intended to be included within the scope of the appended claims.

The drawings illustrate one or more specific embodiments of the invention and, together with the description, serve to explain the principles of the invention. Wherever possible, the same reference numbers are used throughout the drawings to refer to the same or like elements of specific embodiments.

Figures 1A to 1C are a series of graphs showing the enhanced antibody response generated by sustained intradermal delivery of SARS-CoV-2 S-2P recombinant protein (S-2P). BALB/c mice (n = 5/group) were immunized twice at 28-day intervals (day 0 and day 28): each intramuscular injection of 5 mcg S-2P (IM bolus, 2 doses, group 7) , Intradermal injection of 1 mcg S-2P each time (ID injection, 2 doses, group 2), continuous intradermal injection of 1 mcg S-2P for 10 consecutive days (ID daily, 2 doses, group 3) , Intradermal injection of 5 mcg S-2P each time (ID injection, 2 doses, group 4), continuous intradermal injection of 5 mcg S-2P for 10 consecutive days (ID daily, 2 doses, group 5) , or only immunized once with continuous intradermal injection of 5 mcg S-2P for 10 consecutive days (ID daily, 1 dose, group 6), or not immunized (initial immune status, group 1). Antisera were harvested on day 29 (D29), day 49 (D49), and day 64 (D64). Anti-SARS-CoV-2 spike protein IgG potency was measured by enzyme-linked immunosorbent assay (ELISA). Each point represents the IgG titer of a single serum sample. Bars represent geometric mean titers (GMT), error bars represent 95% confidence intervals, and statistical significance was calculated by Benjamini, Krieger and Yekutieli FDR = 1% multiple T-test. The dashed line represents the lower limit of detection. * p < 0.05, *** p < 0.001.

Figures 2A to 2J are a series of graphs showing that sustained intradermal delivery of S-2P produces enhanced neutralizing antibody responses against SARS-CoV-2 variants. BALB/c mice (n = 5/group) were immunized twice at 28-day intervals (day 0 and day 28): each intramuscular injection of 5 mcg S-2P (IM bolus, 2 doses, group 7) , Intradermal injection of 1 mcg S-2P each time (ID injection, 2 doses, group 2), continuous intradermal injection of 1 mcg S-2P for 10 consecutive days (ID daily, 2 doses, group 3) , Intradermal injection of 5 mcg S-2P each time (ID injection, 2 doses, group 4), continuous intradermal injection of 5 mcg S-2P for 10 consecutive days (ID daily, 2 doses, group 5) , or only immunized once with continuous intradermal injection of 5 mcg S-2P for 10 consecutive days (ID daily, 1 dose, group 6), or not immunized (initial immune status, group 1). The antiserum was harvested on the 28th day (D28) and the 49th day (D49), and the anti-SARS-CoV-2 spike protein IgG was titrated by ELISA ( Figure 2A and Figure 2B ), and the SARS-CoV-2 Wuhan strain ( Wild type) ( Figure 2C and Figure 2D ), D614G ( Figure 2E and Figure 2F ), B.1.1.7 (Alpha variant) ( Figure 2G and Figure 2H ), and 501Y.V2 (Beta variant) ( Figure 2I and Figure 2J ) for neutralizing antibody analysis of pseudoviruses. Each point represents the IgG titer or neutralizing antibody titer of a single serum sample. Bars represent geometric mean valency (GMT) and statistical significance was calculated with Benjamini, Krieger and Yekutieli FDR = 1% multiple t-test. The lower dotted line indicates the lower detection limit, and the upper dotted line indicates the upper detection limit. * p < 0.05, ** p < 0.01, **** p < 0.0001.

3A to 3E are a series of graphs showing that immunization by controlled or sustained release silk microneedles produces enhanced neutralizing antibody responses against SARS-CoV-2 variants. BALB/c mice (n = 5/group) were immunized twice at intervals of 28 days (day 0 and day 28): each intramuscular injection of 5 mcg non-concentrated S-2P (IM bolus, non-concentrated, 2 doses , group 1), 5 mcg concentrated S-2P per intramuscular injection (IM bolus, concentrated, 2 doses, group 2), 5 mcg S-2P per microneedle patch (MIMIX, sustained release ( SR), 2 doses, group 4), or 5 mcg S-2P (MIMIX, sustained release (SR), 1 dose, group 3) administered once as a microneedle patch. Antiserum was harvested on day 28 (D28) and day 52 (D52), and anti-SARS-CoV-2 spike protein IgG was titrated by ELISA ( Figure 3A ), and SARS-CoV-2 Wuhan strain (wild type) ( Figure 3B ), D614G ( Figure 3C ), B.1.1.7 (Alpha variant) ( Figure 3D ), and 501Y.V2 (Beta variant) ( Figure 3E ) pseudoviruses were analyzed for neutralizing antibodies. Each point represents the IgG titer or neutralizing antibody titer of a single serum sample. Bars represent geometric mean valency (GMT) and statistical significance was calculated with Benjamini, Krieger and Yekutieli FDR = 1% multiple t-test. The lower dotted line indicates the lower detection limit, and the upper dotted line indicates the upper detection limit. * p <0.05, ** p <0.01, *** p <0.001.

Claims (16)

A microneedle vaccine against novel coronavirus (severe acute respiratory syndrome coronavirus 2, SARS-CoV-2), comprising at least one microneedle, wherein the at least one microneedle includes (i) a backing; (ii) a soluble base applied to the backing comprising gelatin, polyethylene glycol (PEG), sucrose, carboxymethylcellulose (CMC), polyvinylpyrrolidone (PVP), At least one of polyvinyl alcohol (PVA), hyaluronate, maltose, and methylcellulose; and (iii) an implantable sustained release tip applied to the soluble base, comprising a strand of fibrin and an immunogenic recombinant protein consisting essentially of: a : The 14th to 1208th residues of the SARS-CoV-2 spike protein of the amino acid sequence shown in 1, wherein the 986th and 987th residues are replaced by proline, and the 682nd to 1st 685 residues were replaced by "glycine-serine-alanine-serine (GSAS)", and at its C-terminal has a T4 fibrin trimerization as shown in SEQ ID NO: 2 Domain; Wherein the microneedle is configured to implant the sustained release tip into the skin of a subject to a depth of between about 100 μm and about 600 μm. According to the microneedle vaccine described in Claim 1, wherein the immunogenic recombinant protein has the amino acid sequence shown in SEQ ID NO: 5. The microneedle vaccine according to claim 1 or 2, wherein the implantable sustained-release tip contains about 0.1 μg to about 65 μg of the immunogenic recombinant protein. The microneedle vaccine according to claim 1 or 2, wherein the implantable sustained-release tip further comprises at least one adjuvant. The microneedle vaccine according to claim 1 or 2, wherein the implantable sustained-release tip further comprises about 0.1% (w/v) to about 10% (w/v) of a surfactant. According to the microneedle vaccine described in claim 5, wherein the surfactant is selected from polysorbate 20, polysorbate 80, Tween® 20, Tween® 80, or a combination thereof. The microneedle vaccine according to claim 1 or 2, wherein the silk fibroin is a regenerated silk fibroin and/or a recombinant silk fibroin. According to the microneedle vaccine described in claim 1 or 2, wherein the tip of the implantable sustained release comprises the immunogenic recombinant protein, the immunogenic recombinant protein is formulated in a solution containing about 1% (w/v) In a solution of silk fibroin and about 0.5% (w/v) surfactant. The microneedle vaccine as claimed in item 1 or 2, wherein the soluble base comprises at least one of the following: hydrolyzed gelatin between about 10% and about 70% (w/v), about 1% to about 35% (w Sucrose between /v), carboxymethylcellulose (CMC) between about 1% and about 35% (w/v), polyvinylpyrrolidone between about 10% and about 70% (w/v) (PVP), about 1% to about 35% (w/v) of polyvinyl alcohol (PVA), about 1% to about 75% (w/v) of hyaluronate, about 1% to about 75% % (w/v) of maltose, between about 1% and about 75% (w/v) of methylcellulose, and between about 1% and about 70% (w/v) of polyethylene glycol Alcohol (PEG). The microneedle vaccine according to claim 1 or 2, wherein the soluble base comprises one of the following combinations: (i) about 40% (w/v) hydrolyzed gelatin and about 10% (w/v) sucrose, (ii ) about 30% (w/v) polyvinylpyrrolidone (PVP) and about 10% (w/v) polyvinyl alcohol (PVA), and (iii) about 37% (w/v) polyvinylpyrrolidone (PVP), About 5% (w/v) polyvinyl alcohol (PVA), and about 15% (w/v) sucrose. According to the microneedle vaccine according to claim 1 or 2, wherein the backing is selected from a paper-based material, a plastic material, a polymeric material, or a polyester-based material. A patch comprising a plurality of microneedles according to claim 1. The patch according to claim 12, wherein the implantable microneedle tip comprises about 0.1 μg to about 65 μg of immunogenic recombinant protein. The preparation of the microneedle vaccine according to claim 1 or the patch according to claim 12 provides broad immunity to novel coronavirus (SARS-CoV-2) in a subject in need thereof Use in medicine. The microneedle vaccine according to claim 1 or the patch according to claim 12 provides sustained release of an anti-new group coronavirus (SARS-CoV-2) vaccine in a subject in need thereof use in medicines. The microneedle vaccine according to claim 1 or the patch according to claim 12 in the preparation of a drug for enhancing the immune response against novel coronavirus (SARS-CoV-2) in a subject in need thereof the purpose of.

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