CORONAVIRUS 2 (SARS-COV-2)

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and the benefit of U.S. Provisional Application No. 63/191,777, filed on May 21, 2021, the disclosure of which is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

1. FIELD OF THE INVENTION

[0002] The present invention generally 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.

2. DESCRIPTION OF THE PRIOR ART

[0003] On 31 December 2019, the World Health Organization (WHO) was alerted to several cases of pneumonia in Wuhan City, Hubei Province of China. The viral pathogen did not match any other known virus and was later officially named “severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).” 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, a rash on skin, and shortness of breath. While the majority of cases result in mild symptoms, some progress to acute respiratory distress syndrome (ARDS), precipitated by cytokine storm, multi-organ failure, septic shock, and blood clots. The first confirmed death from the coronavirus infection occurred on January 9, and as of 13 May 2022, 517,648,631 confirmed cases of COVID-19, including 6,261,708 deaths, have been reported to the WHO

(h tip s : Z/c o v i d 19. w ho i nt) . The numbers are still growing fast.

[0004] Although several SARS-CoV-2 vaccines are available, the average worldwide vaccination rate is still low. Besides that, all of the currently available SARS-CoV-2 vaccines are intramuscular vaccines, which require experts and trained people for administration, and therefore, slow down the vaccination rate and increase workload among healthcare workers.

SUMMARY OF THE INVENTION

[0005] The present invention is based, at least in part, on the discovery that modulating the kinetics of antigen presentation via, e.g., controlled- and/or sustained release compositions and devices (e.g., microneedles, e.g., silk-based microneedles, and microneedles patches) comprising an immunogenic recombinant protein substantially consisting of residues 14-1208 of SARS-CoV- 2 S protein with proline substitutions at residues 986 and 987 and a "GSAS" substitution at residues 682 - 685 and a C-terminal T4 fibritin trimerization domain can drive a more potent immune response against SARS-CoV-2 in a subject, e.g., as compared to the administration of single-dose or bolus administration of the recombinant protein. In some embodiments, controlled- or sustained-release of the immunogenic recombinant protein substantially consisting of residues 14-1208 of SARS-CoV-2 S protein with proline substitutions at residues 986 and 987 and a “GSAS” substitution at residues 682 - 685 and a C-terminal T4 fibritin trimerization domain as described herein can be used to achieve broad spectrum immunity in a subject.

[0006] In some embodiments, the microneedles and microneedles devices described herein demonstrate controlled- or sustained-release of the immunogenic recombinant protein substantially consisting of residues 14-1208 of SARS-CoV-2 S protein with proline substitutions at residues 986 and 987 and a "GSAS" substitution at residues 682 - 685 and a C-terminal T4 fibritin trimerization domain for at least about 1-2 weeks (e.g., for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 days), which results in one or more of improved immunogenicity, an enhanced immune response, and/or broad-spectrum immunity.

[0007] In other embodiments, methods, formulations, compositions, articles, devices, and/or preparations for administering the immunogenic recombinant protein substantially consisting of residues 14-1208 of SARS-CoV-2 S protein with proline substitutions at residues 986 and 987 and a "GSAS" substitution at residues 682 - 685 and a C-terminal T4 fibritin trimerization domain that provide improved immunogenicity, an enhanced immune response, and/or a broadspectrum immunity to a subject are also disclosed. Accordingly, disclosed herein are compositions, preparations, devices (e.g., microneedles and microneedles patches), kits for controlled- and/or sustained release of a vaccine, in a subject, as well as methods of making and using the same.

[0008] 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 encompassed by the following embodiments.

[0009] Embodiment 1. 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 comprising a component other than poly acrylic acid (PAA), e.g., other than a solution of about 35% PAA. In some embodiments, the dissolvable base comprises a component, e.g., one or more water-soluble components, having improved biocompatibility e.g., in a subject, compared to PAA. In some embodiments, the dissolvable base comprises a component, e.g., a water soluble components, that has a pH similar to that of a biological barrier into which it will be dissolved, e.g., has a pH of about 4.0-8. In some embodiments, the dissolvable 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), hyaluronate, maltose, and methyl cellulose. In some embodiments, the dissolvable base does not comprise an immunogenic recombinant protein, as described herein. In some embodiments, the dissolvable base is applied to the backing,

(iii) a microneedle tip, e.g., an implantable sustained-release tip, comprising a silk fibroin and an immunogenic recombinant protein substantially consisting of residues 14-1208 of SARS-CoV-2 sipke (S) protein with proline substitutions at residues 986 and 987 and a “GSAS” substitution at residues 682 - 685 having an amino acid sequence of SEQ ID NO: 1, and a C-terminal T4 fibritin trimerization domain having an amino acid sequence of SEQ ID NO: 2. In some embodiments, the tip is applied to the dissolvable 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., at a depth (e.g., a max penetration depth of the distal part of tip) of between about 100 pm and about 600 pm, wherein the tip comprises a silk fibroin, e.g., a regenerated silk fibroin and/or a recombinant silk fibroin.

[0010] In some embodiments, the immunogenic recombinant protein has an amino acid sequence of SEQ ID NO: 5.

[0011] 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 and comprising 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), hyaluronate, maltose, and methyl cellulose,

(iii) a microneedle tip, e.g., an implantable sustained-release tip, applied to the dissolvable base and comprising a silk fibroin and an immunogenic recombinant protein substantially consisting of residues 14-1208 of SARS-CoV-2 S protein with proline substitutions at residues 986 and 987 and a “GSAS” substitution at residues 682 - 685 having an amino acid sequence of SEQ ID NO: 1, and a C-terminal T4 fibritin trimerization domain having an amino acid sequence of SEQ ID NO: 2, wherein the microneedle is configured to implant the tip into a skin of a subject, e.g., a human subject, e.g., at a depth (e.g., a max penetration depth of the distal part of tip) of between about 100 pm and about 600 pm, wherein the tip comprises a silk fibroin, e.g., a regenerated silk fibroin and/or a recombinant silk fibroin.

[0012] In some embodiments, the immunogenic recombinant protein has an amino acid sequence of SEQ ID NO: 5.

[0013] Embodiment 3. The microneedle vaccine of embodiment 1 or 2, wherein the dissolvable base comprises one of gelatin, polyethylene glycol (PEG), sucrose, carboxymethylcellulose (CMC), polyvinylpyrrolidone (PVP), polyvinyl alcohol (PVA), hyaluronate, maltose, and methyl cellulose.

[0014] Embodiment 4. The microneedle vaccine of embodiment 1 or 2, wherein the dissolvable base is comprised of two of gelatin, PEG, sucrose, CMC, PVP, PVA, hyaluronate, maltose, and methyl cellulose.

[0015] Embodiment 5. The microneedle vaccine of embodiment 1 or 2, wherein the dissolvable base comprising three of gelatin, PEG, sucrose, CMC, PVP, PVA, hyaluronate, maltose, and methyl cellulose.

[0016] Embodiment 6. The microneedle vaccine of embodiment 1 or 2, wherein the dissolvable base comprising four of gelatin, PEG, sucrose, CMC, PVP, PVA, hyaluronate, maltose, and methyl cellulose.

[0017] Embodiment 7. The microneedle vaccine of embodiment 1 or 2, wherein the dissolvable base comprising five of gelatin, PEG, sucrose, CMC, PVP, PVA, hyaluronate, maltose, and methyl cellulose.

[0018] Embodiment 8. The microneedle vaccine of embodiment 1 or 2, wherein the dissolvable base comprising six of gelatin, PEG, sucrose, CMC, PVP, PVA, hyaluronate, maltose, and methyl cellulose.

[0019] Embodiment 9. The microneedle vaccine of embodiment 1 or 2, wherein the dissolvable base comprising seven of gelatin, PEG, sucrose, CMC, PVP, PVA, hyaluronate, maltose, and methyl cellulose.

[0020] Embodiment 10. The microneedle vaccine of embodiment 1 or 2, wherein the dissolvable base comprising eight of gelatin, PEG, sucrose, CMC, PVP, PVA, hyaluronate, maltose, and methyl cellulose.

[0021] Embodiment 11. The microneedle vaccine of embodiment 1 or 2, wherein the dissolvable base comprising gelatin, PEG, sucrose, CMC, PVP, PVA, hyaluronate, maltose, and methyl cellulose.

[0022] Embodiment 12. The microneedle vaccine of any one of the preceding embodiments, wherein the dissolvable base comprising gelatin and sucrose.

[0023] Embodiment 13. The microneedle vaccine of any one of the preceding embodiments, wherein the dissolvable base comprises CMC.

[0024] Embodiment 14. The microneedle vaccine of any one of the preceding embodiments, wherein the dissolvable base comprises PVP.

[0025] Embodiment 15. The microneedle vaccine of any one of the preceding embodiments, wherein the dissolvable base comprises PVA.

[0026] Embodiment 16. The microneedle vaccine of any one of the preceding embodiments, wherein the dissolvable base comprises about PVP and PVA.

[0027] Embodiment 17. The microneedle vaccine of any one of the preceding embodiments, wherein the dissolvable base comprises PVP, PVA, and sucrose.

[0028] Embodiment 18. The microneedle vaccine of any one of the preceding embodiments, wherein the dissolvable base does not comprise poly(acrylic acid) (PAA).

[0029] Embodiment 19. The microneedle vaccine of any one of the preceding embodiments, wherein the dissolvable base comprises PEG.

[0030] Embodiment 20. The microneedle vaccine of any one of the preceding embodiments, wherein the dissolvable base comprises between about 10% and about 70% (w/v) gelatin (e.g., hydrolyzed gelatin) (e.g., about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, or about 70% (w/v) gelatin).

[0031] Embodiment 21. The microneedle vaccine of any one of the preceding embodiments, wherein the dissolvable 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).

[0032] Embodiment 22. The microneedle vaccine of any one of the preceding embodiments, wherein the dissolvable base comprises between about 1% and about 35% (w/v) CMC (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) CMC).

[0033] Embodiment 23. The microneedle vaccine of any one of the preceding embodiments, wherein the dissolvable base comprises between about 10% and about 70% (w/v) PVP (e.g., about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, or about 70% (w/v) PVP).

[0034] Embodiment 24. The microneedle vaccine of any one of the preceding embodiments, wherein the dissolvable base comprises between about 1% and about 35% (w/v) PVA (e.g., 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) PVA).

[0035] Embodiment 25. The microneedle vaccine of any one of the preceding embodiments, wherein the dissolvable base comprises about 40% (w/v) hydrolyzed gelatin and about 10% (w/v) sucrose.

[0036] Embodiment 26. The microneedle vaccine of any one of the preceding embodiments, wherein the dissolvable base comprises up to about 50% (w/v) of PVP (e.g., PVP of 10 kD MW).

[0037] Embodiment 27. The microneedle vaccine of any one of the preceding embodiments, wherein the dissolvable base comprises up to about 20% (w/v) PVA (e.g., 87% (w/v) hydrolyzed PVA at 13 kD MW).

[0038] Embodiment 28. The microneedle vaccine of any one of the preceding embodiments, wherein the dissolvable base comprises CMC at up to about 10% (w/v).

[0039] Embodiment 29. The microneedle vaccine of any one of the preceding embodiments, wherein the dissolvable base comprises about 1% (w/v) CMC (e.g., low-viscosity CMC).

[0040] Embodiment 30. The microneedle vaccine of any one of the preceding embodiments, wherein the dissolvable base comprises about 30% (w/v) PVP and about 10% (w/v) PVA.

[0041] Embodiment 31. The microneedle vaccine of any one of the preceding embodiments, wherein the dissolvable base comprises about 37% (w/v) PVP, about 5% (w/v) PVA, and about 15% (w/v) sucrose.

[0042] Embodiment 32. The microneedle vaccine of any one of the preceding embodiments, wherein the dissolvable base comprises between about 1% and about 75% (w/v) 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%, or about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, or about 75% (w/v) hyaluronate).

[0043] Embodiment 33. The microneedle vaccine of any one of the preceding embodiments, wherein the dissolvable 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%, or about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, or about 75% (w/v) maltose).

[0044] Embodiment 34. The microneedle vaccine of any one of the preceding embodiments, wherein the dissolvable comprises between about 1% and about 75% (w/v) methyl cellulose (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%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, or about 75% (w/v) methyl cellulose).

[0045] Embodiment 35. The microneedle vaccine of any one of the preceding embodiments, wherein the dissolvable base comprises between about 1% and about 70% (w/v) PEG (e.g., 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) PEG).

[0046] Embodiment 36. The microneedle vaccine of any one of the preceding embodiments, wherein the implantable sustained-release tip is configured to release the immunogenic recombinant protein into the skin of the subject over a period of time comprising 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., between about 1-2 weeks, about 1-3 weeks, or about 1-4 weeks).

[0047] Embodiment 37. The microneedle vaccine of any one of the preceding embodiments, wherein the implantable sustained-release tip is configured to release the immunogenic recombinant protein into the skin of the subject over a period of time comprising between 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).

[0048] Embodiment 38. The microneedle vaccine of any one of the preceding embodiments, wherein the implantable sustained-release tip comprises silk fibroin 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)), or a silk fibroin having a molecular weight distribution according to Table 1, or, comprises silk fibroin in an amount between about 2 pg to about 245 pg, e.g., per 121 microneedle array).

[0049] Embodiment 39. The microneedle vaccine of any one of the preceding embodiments, wherein the implantable sustained-release tip comprises the immunogenic recombinant protein formulated in an 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 solution, or a silk fibroin solution according to Table 1.

[0050] Embodiment 40. The microneedle vaccine of any one of the preceding embodiments, wherein the implantable sustained-release tip comprises the immunogenic recombinant protein formulated in an 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 solution, or a silk fibroin solution according to Table 1, e.g., a 100 kDa to 200 kDa (e.g., about 153 kDa) silk fibroin solution.

[0051] Embodiment 41. The microneedle vaccine of any one of the preceding embodiments, wherein the implantable sustained-release tip comprises the immunogenic recombinant protein formulated in an 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 solution, or a silk fibroin solution according to Table 1, e.g., a 70 kDa to 150 kDa (e.g., about 100 kDa) silk fibroin solution.

[0052] Embodiment 42. The microneedle vaccine of any one of the preceding embodiments, wherein the implantable sustained-release tip comprises the immunogenic recombinant protein formulated in an 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 solution, or a silk fibroin solution according to Table 1, e.g., a 36 kDa to 100 kDa (e.g., about 71 kDa) silk fibroin solution.

[0053] Embodiment 43. The microneedle vaccine of any one of the preceding embodiments, wherein the implantable sustained-release tip comprises the immunogenic recombinant protein formulated in an 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 solution, or a silk fibroin solution according to Table 1, e.g., a 1 kDa to 60 kDa (e.g., about 16 kDa) silk fibroin solution.

[0054] Embodiment 44. The microneedle vaccine of embodiment 40, wherein the implantable sustained-release tip comprises the immunogenic recombinant protein formulated in an about 1% (w/v) of silk fibroin solution.

[0055] Embodiment 45. The microneedle vaccine of any one of the preceding embodiments, wherein the implantable sustained-release tip further comprises 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 (e.g., polysorbate 20, polysorbate 80, Tween® 20, Tween® 80, or a combination thereof).

[0056] Embodiment 46. The microneedle vaccine of any one of the preceding embodiments, wherein the implantable sustained-release tip comprises the immunogenic recombinant protein formulated in a solution of 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)) of surfactant (e.g., polysorbate 20, polysorbate 80, Tween® 20, Tween® 80, or a combination thereof).

[0057] Embodiment 47. The microneedle vaccine of any one of the preceding embodiments, wherein the implantable sustained-release tip comprises the immunogenic recombinant protein formulated in a solution of 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 100 kDa to 200 kDa (e.g., about 153 kDa) silk fibroin 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 (e.g., polysorbate 20, polysorbate 80, Tween® 20, Tween® 80, or a combination thereof). [0058] Embodiment 48. The microneedle vaccine of any one of the preceding embodiments, wherein the implantable sustained-release tip comprises the immunogenic recombinant protein formulated in a solution of 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 70 kDa to 150 kDa (e.g., about 100 kDa) silk fibroin, 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 (e.g., polysorbate 20, polysorbate 80, Tween® 20, Tween® 80, or a combination thereof).

[0059] Embodiment 49. The microneedle vaccine of any one of the preceding embodiments, wherein the implantable sustained-release tip comprises the immunogenic recombinant protein formulated in a solution of 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 36 kDa to 100 kDa (e.g., about 71 kDa) silk fibroin, 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 (e.g., polysorbate 20, polysorbate 80, Tween® 20, Tween® 80, or a combination thereof).

[0060] Embodiment 50. The microneedle vaccine of any one of the preceding embodiments, wherein the implantable sustained-release tip comprises the immunogenic recombinant protein formulated in a solution of 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 1 kDa to 60 kDa (e.g., about 16 kDa) silk fibroin, 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 (e.g., polysorbate 20, polysorbate 80, Tween® 20, Tween® 80, or a combination thereof).

[0061] Embodiment 51. The microneedle vaccine of embodiment 45, wherein the implantable sustained-release tip comprises the immunogenic recombinant protein formulated in a solution of about 1% (w/v) of silk fibroin and about 0.5% (w/v) of surfactant (e.g., polysorbate 20, polysorbate 80, Tween® 20, Tween® 80, or a combination thereof).

[0062] Embodiment 52. The microneedle vaccine of any one of the preceding embodiments, wherein the implantable sustained-release tip comprises a standard human dose of the immunogenic recombinant protein.

[0063] Embodiment 53. The microneedle vaccine of any one of the preceding embodiments, wherein the standard dose of the immunogenic recombinant protein comprises between about 0.1 pg and about 65 pg, e.g., 0.2 pg and about 50 pg (e.g., about each of 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 pg).

[0064] Embodiment 54. The microneedle vaccine of 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 the standard dose.

[0065] Embodiment 55. The microneedle of any one of embodiment 52-54, wherein the implantable sustained-release tip comprises about 0.1 pg to about 65 pg of the immunogenic recombinant protein (e.g., about 0.1 pg, about 0.2 pg, about 0.3 pg, about 0.4 pg, about 0.5 pg, about 0.6 pg, about 0.7 pg, about 0.8 pg, about 0.9 pg, about 1 pg, about 1 pg to about 10 pg, about 10 pg to about 20 pg, about 20 pg to about 30 pg, about 30 pg to about 40 pg, about 40 pg to about 50 pg, about 50 pg to about 65 pg of the immunogenic recombinant protein).

[0066] Embodiment 56. The microneedle vaccine of any one of the preceding embodiments, wherein the length of the microneedle is between about 350 pm to about 1500 pm ((e.g., about 350 pm, about 400 pm, about 450 pm, about 500 pm, about 550 pm, about 600 pm, about 650 pm, about 700 pm, about 750 pm, about 800 pm, about 850 pm, about 900 pm, about 950 pm, about 1000 pm, about 1050 pm, about 1100 pm, about 1150 pm, about 1200 pm, about 1250 pm, about 1300 pm, about 1350 pm, about 1400 pm, about 1450 pm, about 1500 pm).

[0067] Embodiment 57. The microneedle vaccine of any one of the preceding embodiments, wherein the height of the implantable sustained-release tip may extend to approximately half of the full height of the microneedle.

[0068] Embodiment 58. The microneedle vaccine of any one of the preceding embodiments, wherein the height of the implantable sustained-release tip is between about 75 pm to about 475 pm (e.g., about 75, about 100 pm, about 125 pm, about 150 pm, about 175 pm, about 200 pm, about 225 m, about 250 pm, about 275 pm, about 300 pm, about 325 pm, about 375 pm, about 400 pm, about 425 pm, or about 475 pm).

[0069] Embodiment 59. The microneedle vaccine of any one of the preceding embodiments, wherein the implantable sustained-release tip comprises a tip radius between about 0.5 pm to about 25 pm (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 pm).

[0070] Embodiment 60. The microneedle vaccine of any one of the preceding embodiments, wherein the implantable sustained-release tip comprises a tip radius between about 5 pm to about 10 pm (e.g., about 5, 6, 7, 8, 9, or 10 pm).

[0071] Embodiment 61. The microneedle vaccine of 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).

[0072] Embodiment 62. The microneedle vaccine of any one of the preceding embodiments, wherein the backing is chosen from a solid support, e.g., a paper-based material, a plastic material, a polymeric material, or a polyester-based material (e.g., a Whatman 903 paper, a polymeric tape, a plastic tape, an adhesive-backed polyester tape, or other medical tape).

[0073] 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 any one of embodiments 1-62.

[0074] Embodiment 64. The device of embodiment 63, wherein the implantable microneedle tip comprises about 0.1 pg to about 65 pg of the immunogenic recombinant protein (e.g., about 0.1 pg, about 0.2 pg, about 0.3 pg, about 0.4 pg, about 0.5 pg, about 0.6 pg, about 0.7 pg, about 0.8 pg, about 0.9 pg, about 1 pg, about 1 pg to about 10 pg, about 10 pg to about 20 pg, about 20 pg to about 30 pg, about 30 pg to about 40 pg, about 40 pg to about 50 pg, about 50 pg to about 65 pg of the immunogenic recombinant protein described herein).

[0075] Embodiment 65. A method of providing immunity, e.g., broad-spectrum immunity, to SARS-CoV-2, in a subject in need thereof, comprising contacting a skin of the subject with the microneedle vaccine of any one of embodiments 1-62, or the device of any one of embodiments 63-64. [0076] Embodiment 66. A method of providing a controlled- or sustained-release of a vaccine against SARS-CoV-2 in a subject in need thereof, comprising contacting a skin of the subject with the microneedle vaccine of any one of embodiments 1-62, or the device of any one of embodiments 63-64.

[0077] Embodiment 67. A method of enhancing an immune response to SARS-CoV-2 in a subject in need thereof, comprising contacting a skin of the subject with a microneedle vaccine of any one of embodiments 1-62, or the device of any one of embodiments 63-64.

[0078] Embodiment 68. The method of any one of embodiments 65-67, wherein the implantable sustained-release tip is configured to release a vaccine into the skin of the subject over a period of time comprising 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., between about 1-2 weeks, about 1-3 weeks, or about 1-4 weeks).

[0079] Embodiment 69. The method of embodiment 68, wherein the implantable sustained- release tip is configured to release a vaccine into the skin of the subject over a period of time comprising between 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).

[0080] Embodiment 70. The method of any one of embodiments 65-69, further comprising contacting the skin of the subject with another microneedle vaccine according to any one of embodiments 1-62, or the device of any one of embodiments 63-64 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) after the first contacting of the microneedle vaccine according to any one of embodiments 1-62, or the device of any one of embodiments 63-64.

[0081] Embodiment 71. The use of a microneedle vaccine of any one of embodiments 1-62 or a device according to any one of embodiments 63-64 in a method of el eciting an immune response against SARS-CoV-2 in a subject in need thereof.

[0082] Embodiment 72. The use of a microneedle vaccine of any one of embodiments 1-62 or a device according to any one of embodiments 63-64 in a method of providing a controlled- or sustained-release of a vaccine against SARS-CoV-2 in a subject in need thereof. [0083] Embodiment 73. The use of a microneedle of any one of embodiments 1-62 or a device according to any one of embodiments 63-64 in a method of enhancing an immune response to SARS-CoV-2 in a subject in need thereof.

[0084] Embodiment 74. The microneedle vaccine of any one of embodiments 1-62 or the device according to any one of embodiments 63-64 for use as a medicament in a method of providing immunity, e.g., broad-spectrum immunity, to SARS-CoV-2 in a subject in need thereof.

[0085] Embodiment 75. The microneedle vaccine of any one of embodiments 1-62 or the device according to any one of embodiments 63-64 for use as a medicament in a method of providing a sustained-release of a vaccine against SARS-CoV-2 in a subject in need thereof.

[0086] Embodiment 76. The microneedle vaccine of any one of embodiments 1-62 or the device according to any one of embodiments 63-64 for use as a medicament in a method of enhancing an immune response to SARS-CoV-2 in a subject in need thereof.

[0087] These and other aspects will become apparent from the following description of the preferred embodiment taken in conjunction with the following drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

[0088] The accompanying drawings illustrate one or more embodiments of the invention and, together with the written 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 an embodiment.

[0089] Figures 1A to 1C are a series of graphs showing that the sustained intradermal delivery of SARS-CoV-2 S-2P recombinant protein (S-2P) generates improved antibody responses.

BALB/c mice (n=5/group) were immunized twice 28 days apart (Days 0 and 28) with 5 mcg of S-2P by intramuscular injection (IM Blous, 2 doses, Group 7), 1 mcg of S-2P by intradermal bolus injection (ID Bolus, 2 doses, Group 2), 1 mcg of S-2P by 10-day sustained ID injection (ID Daily, 2 doses, Group 3), 5 mcg of S-2P by ID bolus injection (ID Bolus, 2 doses, Group 4), 5 mcg of S-2P by 10-day sustained ID injection (ID Daily, 2 doses, Group 5), or were immunized once with 5 mcg of S-2P by 10-day sustained ID injection (ID Daily, 1 dose, Group 6), or were unimmunized (Naive, Group 1). The antisera were harvested on days 29 (D29), 49 (D49), and 64 (D64). Anti-SARS-CoV-2 spike IgG titers were measured by enzyme-linked immunosorbent assay (ELISA). Each dot represents individual serum sample IgG titer. Bars indicate geometric mean titers (GMT) and error bars indicate 95% confidence intervals, and statistical significance was calculated with multiple T-tests with Benjamini, Krieger and Yekutieli FDR = 1%. Dotted lines represent lower limits of detection. *p < 0.05, ***/? < 0.001.

[0090] Figures 2A to 2J are a series of graphs showing that the sustained intradermal delivery of S-2P generates improved neutralization antibody responses against SARS-CoV-2 variants. BALB/c mice (n=5/group) were immunized twice 28 days apart (Days 0 and 28) with 5 mcg of S-2P by intramuscular injection (IM Blous, 2 doses, Group 7), 1 mcg of S-2P by intradermal bolus injection (ID Bolus, 2 doses, Group 2), 1 mcg of S-2P by 10-day sustained ID injection (ID Daily, 2 doses, Group 3), 5 mcg of S-2P by ID bolus injection (ID Bolus, 2 doses, Group 4), 5 mcg of S-2P by 10-day sustained ID injection (ID Daily, 2 doses, Group 5), or were immunized once with 5 mcg of S-2P by 10-day sustained ID injection (ID Daily, 1 dose, Group 6), or were unimmunized (Naive, Group 1). The antisera were harvested on days 28 (D28) and 49 (D49) and subject to anti-SARS-CoV-2 spike IgG titration by ELISA (Figures 2A and 2B) and neutralization assays with pseudoviruses of SARS-CoV-2 Wuhan strain (wild type) (Figures 2C and 2D), D614G (Figures 2E and 2F), B.1.1.7 (Alpha) (Figures 2G and 2H), and 501Y.V2 (Beta) (Figures 21 and 2J). Each dot represents individual serum sample IgG titer or neutralizing titer. Bars indicate geometric mean titers (GMT), and statistical significance was calculated with multiple T-tests with Benjamini, Krieger and Yekutieli FDR = 1%. The lower dotted line represents the lower limit of detection, and the upper dotted line represents the high limit of detection. * p < 0.05, ** p < 0.01, **** p < 0.0001.

[0091] Figures 3 A to 3E are a series of graphs showing that immunization via controlled- or sustained-release silk microneedles generates improved neutralization antibody responses against SARS-CoV-2 variants. BALB/c mice (n=5/group) were immunized twice 28 days apart (Days 0 and 28) with 5 mcg of unconcentrated S-2P by intramuscular injection (IM Blous, Neat, 2 doses, Group 1), 5 mcg of concentrated S-2P by intramuscular injection (IM Bolus, Cone., 2 doses, Group 2), 5 mcg of S-2P by application of microneedle patches (MIMIX, Sustained-Release (SR), 2 doses, Group 4), or were immunized once with 5 mcg of S-2P by application of microneedle patches (MIMIX, SR, 1 dose, Group 3). The antisera were harvested on days 28 (D28) and 52 (D52) and subject to anti-SARS-CoV-2 spike IgG titration by ELISA (Figure 3A) and neutralization assays with pseudoviruses of SARS-CoV-2 Wuhan strain (wild type) (Figure 3B), D614G (Figure 3C), B. l.1.7 (Alpha) (Figure 3D), and 501Y.V2 (Beta) (Figure 3E). Each dot represents individual serum sample IgG titer or neutralizing titer. Bars indicate geometric mean titers (GMT), and statistical significance was calculated with multiple T-tests with Benjamini, Krieger and Yekutieli FDR = 1%. The lower dotted line represents the lower limit of detection, and the upper dotted line represents the high limit of detection. * p < 0.05, ** p < 0.01, *** p < 0.001.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0092] The present invention is based, at least in part, on the discovery that modulating the kinetics of antigen presentation to mimic that of a natural infection (e.g., a SARS-CoV-2 virus infection) can drive a more potent immune response (e.g., a more potent humoral immune response) (see, e.g., Tam et al. PNAS. 113:E6639-E6648, 2016; and Schipper at al. J. Control Release. 242: 141-147, 2016). Without wishing to be bound by theory, the microneedles and microneedle devices described herein can mimic the natural process of antigen presentation (e.g., viral antigen presentation) by enabling the release, e.g., controlled- or sustained-release, of an immunogenic recombinant protein substantially consisting of residues 14-1208 of SARS-CoV-2 S protein with proline substitutions at residues 986 and 987 and a "GSAS" substitution at residues 682 - 685 and a C-terminal T4 fibritin trimerization domain into a subject, e.g., into the dermis skin layer of a subject. The controlled- or sustained-release enabled by the formulations, compositions, articles, devices, and preparations, microneedles, and microneedle devices described herein can induce greater immunogenicity, an enhanced immune response (e.g., a more potent humoral immune response), and/or broad-spectrum immunity in a subject, as compared to the administration of single-dose or bolus administration of the immunogenic recombinant protein substantially consisting of residues 14-1208 of SARS-CoV-2 S protein with proline substitutions at residues 986 and 987 and a "GSAS" substitution at residues 682 - 685 and a C- terminal T4 fibritin trimerization domain.

[0093] In some embodiments, the microneedles and microneedle devices described herein can comprise an implantable controlled- or sustained-release silk-based microneedle tip that encapsulates and/or stabilizes the immunogenic recombinant protein substantially consisting of residues 14-1208 of SARS-CoV-2 S protein with proline substitutions at residues 986 and 987 and a “GSAS” substitution at residues 682 - 685 and a C-terminal T4 fibritin trimerization domain; and a dissolving base layer that supports the distal microneedle tip. Upon application of a microneedle or microneedle device, as described herein, to a biological barrier of a subject, the base layer dissolves and the silk-based microneedle tips are implanted at a predetermined depth (e.g., a max penetration depth of the distal part of tip) within the biological barrier (e.g., the dermis layer of the skin, e.g., at a depth of between about 100 pm and about 800 pm). In some embodiments, the whole tip is not embedded within, e.g., the dermis layer of the skin, e.g., at a depth of between about 100 pm and about 800 pm. The implanted tip then slowly releases the immunogenic recombinant protein substantially consisting of residues 14-1208 of SARS-CoV-2 S protein with proline substitutions at residues 986 and 987 and a “GSAS” substitution at residues 682 - 685 and a C-terminal T4 fibritin trimerization domain over a time period sufficiently long enough to enable immunity (e.g., over a time 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., between about 1-2 weeks, about 1-3 weeks, or about 1-4 weeks, e.g., about one week, about two weeks, about three weeks, about four weeks, about five weeks, or about six weeks or more weeks). Various properties of the silk fibroin matrix comprising the implantable controlled- or sustained-release microneedle tip, including, for example, crystallinity, beta-sheet content, and molecular weight, can be modulated to tune (e.g., alter and/or modify) the release kinetics (e.g., rate of release) of the immunogenic recombinant protein substantially consisting of residues 14-1208 of SARS-CoV-2 S protein with proline substitutions at residues 986 and 987 and a “GSAS” substitution at residues 682 - 685 and a C-terminal T4 fibritin trimerization domain from the microneedle tip. In some embodiments, the implantable controlled- or sustained-release microneedle tip comprises a beta-sheet content of between about 10% and about 60% (e.g., about 10%, about 20%, about 30%, about 40%, about 50%, about 60%), e.g., as based on a “crystallinity index,” e.g., a “crystallinity index” known in the art.

[0094] The controlled- or sustained-release formulations, compositions, articles, devices, and preparations, comprise an immunogenic recombinant spike protein of SARS-CoV-2 substantially consisting of residues 14-1208 of SARS-CoV-2 S protein with proline substitutions at residues 986 and 987 and a “GSAS” substitution at residues 682 - 685 having an amino acid sequence of SEQ ID NO: 1, and a C-terminal T4 fibritin trimerization domain having an amino acid sequence of SEQ ID NO: 2. In some embodiments, the immunogenic recombinant protein has an amino acid sequence of SEQ ID NO: 5. In some embodiments, the formulations, compositions, articles, devices, and preparations for controlled- and/or sustained release described herein release the immunogenic recombinant protein substantially consisting of residues 14-1208 of SARS-CoV-2 S protein with proline substitutions at residues 986 and 987 and a “GSAS” substitution at residues 682 - 685 and a C-terminal T4 fibritin trimerization domain over a time period sufficiently long enough to enable immunity (e.g., over a time period of at least about 1 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., between about 4 days and about 14 days, e.g., between about 1-2 weeks, about 1-3 weeks, or about 1-4 weeks, e.g., about one week, about two weeks, about three weeks, about four weeks, about five weeks, or about six weeks or more weeks). Accordingly controlled- or sustained-release formulations, compositions, articles, devices, and preparations, microneedles, microneedle devices, kits, as well as methods of making and using the same are disclosed.

[0095] Definitions

[0096] All scientific and technical terms used herein, unless otherwise defined below, are intended to have the same meaning as commonly understood by one of ordinary skill in the art. References to techniques employed herein are intended to refer to the techniques as commonly understood in the art, including variations on those techniques or substitutions of equivalent or later-developed techniques which would be apparent to one of skill in the art. In addition, in order to more clearly and concisely describe the subject matter which is the invention, the following definitions are provided for certain terms which are used in the specification and appended claims.

[0097] The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

[0098] As used herein, the term “about” means+/-10% of the recited value.

[0099] The phrase “and/or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and/or” should be construed in the same fashion, i.e., “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, a reference to “A and/or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

[0100] As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and/or” as defined above. For example, when separating items in a list, “or” or “and/or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of’ or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e., “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.”

[0101] As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within 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, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and/or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, 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); etc.

[0102] It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

[0103] As used herein, the term “microneedle” refers to a structure having at least two, more typically, three components, e.g., layers, for transport or delivery of a therapeutic agent, such as a vaccine, an antigen, and/or an immunogen, across a biological barrier, such as the skin, tissue, or cell membrane. In some embodiments, a microneedle comprises a base (e.g., a dissolvable base as described herein), a tip (e.g., an implantable tip as described herein), and optionally, a backing material. The microneedles described herein can be in any shape and/or geometry suitable for use in piercing a biological barrier, e.g., a layer of the skin, to enable release, e.g., controlled- or sustained-release, of a vaccine within a subject. Non-limiting examples of the shape and/or geometry of the microneedles include: a cylindrical shape, a wedge-shape, a cone-shape, a pyramid-shape, and/or an irregular-shape, or any combinations thereof.

[0104] In embodiments, a microneedle has dimension of between about 350 pm to about 1500 pm in height (e.g., between about 350 pm to about 1500 pm, e.g., about 350 pm, about 400 pm, about 450 pm, about 500 pm, about 550 pm, about 600 pm, about 650 pm, about 700 pm, about 750 pm, about 800 pm, about 850 pm, about 900 pm, about 950 pm, about 1000 pm, about 1050 pm, about 1100 pm, about 1150 pm, about 1200 pm, about 1250 pm, about 1300 pm, about 1350 pm, about 1400 pm, about 1450 pm, about 1500 pm)). In some embodiments, the height of the implantable tip may extend to approximately half of the full height of the microneedle, and the height of the implantable tip is between about 75 pm to about 475 pm (e.g., about 75, about 100 pm, about 125 pm, about 150 pm, about 175 pm, about 200 pm, about 225 pm, about 250 pm, about 275 pm, about 300 pm, about 325 pm, about 375 pm, about 400 pm, about 425 pm, or about 475 pm). In some embodiments, the implantable tip comprises a tip radius between about 0.5 pm to about 25 pm (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 pm) , preferably between about 5 pm to about 10 pm (e.g., about 5, 6, 7, 8, 9, or 10 pm). In some 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 some embodiments, the microneedle is fabricated to have any dimension and/or geometry to enable the deployment of an implantable sustained-release at a depth between about 100 pm and about 900 pm (e.g., at a depth of about 800 pm) into the dermis layer of the skin for controlled- or sustained-release of a vaccine.

[0105] As used herein, the term “microneedle patch” and “microneedle array” refers to a device comprising a plurality of microneedles, e.g., silk fibroin-based microneedles, e.g., arranged in a random or predefined pattern, such as an array. In some embodiments, the microneedle patch or microneedle array may comprise about 121 needles in an 11 x 11 square grid with approximately 0.75 mm pitch. Individual needles are cones approximately 0.65 mm long with base diameter approximately 0.35 mm and included angle of approximately 30°. The tip of the needle must be sharp in order to penetrate the skin. The radius of curvature of the tip should ideally be no more than 0.01 mm.

[0106] As used herein, the term “backing” refers to a material that is suitable for bonding to and/or adhering to a component of a microneedle. In some embodiments, a backing material is suitable for bonding to and/or adhering to the dissolvable base of a microneedle described herein.

[0107] As used herein, the term “dissolvable base” refers to the layer that forms the base of the microneedles (e.g., functions as the support for the distal implantable silk tips that are loaded with a vaccine, an antigen, or an immunogen), and/or can also serve as a layer connecting adjacent microneedles to form a continuous microneedle array or microneedle patch. In some embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the base is dissolved after application to a biological barrier, e.g., skin or mucous surface, or buccal cavity. In some embodiments, the dissolvable 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), hyaluronate, maltose, and methyl cellulose. In some embodiments, the dissolvable base comprises at least one of between about 10% and about 70% (w/v) gelatin (e.g., hydrolyzed gelatin), between about 1% and about 35% (w/v) sucrose, between about 1% and about 35% (w/v) CMC, between about 10% and about 70% (w/v) PVP, between about 1% and about 35% (w/v) PVA, between about 1% and about 75% (w/v) hyaluronate, between about 1% and about 75% (w/v) maltose, between about 1% and about 75% (w/v) methyl cellulose, and between about 1% and about 70% (w/v) PEG. In some embodiments, the dissolvable base comprises about 40% (w/v) hydrolyzed gelatin and about 10% (w/v) sucrose. In some embodiments, the dissolvable base comprises about 30% (w/v) PVP and about 10% (w/v) PVA. In some embodiments, the dissolvable base comprises about 37% (w/v) PVP, about 5% (w/v) PVA, and about 15% (w/v) sucrose.

[0108] As used interchangeably herein, the terms “implantable sustained-release tip” or “releasable tip” refers to the distal end, e.g., tip, of a microneedle capable of piercing a biological barrier, e.g., the skin, mucous surface, or buccal cavity, of a subject and being deposited within the biological barrier, a skin layer (e.g., the dermis). In embodiments, the tip comprises a silk fibroin protein in an amount sufficient to sustain the release of a therapeutic agent, such as a vaccine, antigen, and/or immunogen for a prolonged period of time, e.g., for 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., between about 1-2 weeks, about 1-3 weeks, or about 1-4 weeks). In some embodiments, the implantable sustained-release tip comprises an immunogenic recombinant protein substantially consisting of residues 14-1208 of SARS-CoV-2 S protein with proline substitutions at residues 986 and 987 and a “GSAS” substitution at residues 682 - 685 and a C- terminal T4 fibritin trimerization domain.

[0109] As used herein, the term “gelatin” refers to a water-soluble protein derived from collagen. In some embodiments, the term “gelatin” refers to a sterile nonpyrogenic protein preparation (e.g., fractions) produced by partial acid hydrolysis (type A gelatin) or by partial alkaline hydrolysis (type B gelatin) of animal collagen, most commonly derived from cattle, pig, and fish sources. Gelatin can be obtained in varying molecular weight ranges. Recombinant sources of gelatin may also be used.

[0110] As used herein, the term “polyethylene glycol (PEG)” refers to an oligomer or polymer of ethylene oxide. PEG is also known as polyethylene oxide (PEO) or polyoxyethylene (POE). The structure of PEG is commonly expressed as H — (O — CEE — CtEf, — OH.

[0111] As used herein, the term “silk fibroin” includes silkworm fibroin and insect or spider silk protein. Any type of silk fibroin can be used according to various aspects described herein. Silk fibroin produced by silkworms, such as Bombyx mori, is the most common and represents an earth-friendly, renewable resource. For instance, silk fibroin used in a microneedle (for example, an implantable controlled- or sustained-release tip of a microneedle) may be obtained by removing sericin from the cocoons of B. mori. In some embodiments, the silk fibroin is a regenerated silk fibroin, for example, a silk fibroin obtained after extraction of sericin from the cocoons of B. mori, and an additional processing such as via a boiling step. Organic silkworm cocoons are also commercially available. There are many different silks, however, including spider silk (for example, obtained from Nephila clavipes), transgenic silks, recombinant and/or genetically engineered silks, such as silks from bacteria, yeast, mammalian cells, transgenic animals, or transgenic plants (see, e.g., WO 97/08315; U.S. Pat. No. 5,245,012), and variants thereof, that can be used.

[0112] Exemplary silk fibroin (e.g., regenerated silk fibroin) solutions may have different molecular weight profiles are shown as determined by size exclusion chromatography (SEC) methods (see, e.g., Table 1). In some embodiments, the silk fibroin solutions can be prepared, e.g., according to established methods. In some embodiments, pieces of cocoons from the silkworm Bombyx mori were first boiled in 0.02 M Na2COs to remove sericin protein which is present in unprocessed, natural silk, prior to analysis by SEC. In some embodiments, silk fibroin composition can be a composition or mixture produced by degumming cocoons from the silkworm Bombyx mori at an 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 shorter. In one embodiment, the silk fibroin composition can be a composition or mixture produced by degumming silk cocoon at an atmospheric boiling temperature in an aqueous sodium carbonate solution 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 shorter.

[0113] In some embodiments, the silk fibroin solution may be a 10-minute boil (10 MB), a 60- minute boil (60 MB), a 120-minute boil (120 MB), a 180-minute boil (180 MB), or a 480-minute boil (480 MB) silk fibroin solution (see, e.g., Table 1). In some embodiments, an immunogenic recombinant protein substantially consisting of residues 14-1208 of SARS-CoV-2 S protein with proline substitutions at residues 986 and 987 and a “GSAS” substitution at residues 682 - 685 and a C-terminal T4 fibritin trimerization domain can be formulated in an 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)) 10 MB silk fibroin solution. In some embodiments, an immunogenic recombinant protein substantially consisting of residues 14-1208 of SARS-CoV-2 S protein with proline substitutions at residues 986 and 987 and a “GSAS” substitution at residues 682 - 685 and a C-terminal T4 fibritin trimerization domain can be formulated in an 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)) 60 MB silk fibroin solution. In some embodiments, an immunogenic recombinant protein substantially consisting of residues 14-1208 of SARS-CoV-2 S protein with proline substitutions at residues 986 and 987 and a “GSAS” substitution at residues 682 - 685 and a C-terminal T4 fibritin trimerization domain can be formulated in an 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)) 120 MB silk fibroin solution. In some embodiments, an immunogenic recombinant protein substantially consisting of residues 14-1208 of SARS-CoV-2 S protein with proline substitutions at residues 986 and 987 and a “GSAS” substitution at residues 682 - 685 and a C-terminal T4 fibritin trimerization domain can be formulated in an 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)) 180 MB silk fibroin solution. In some embodiments, an immunogenic recombinant protein substantially consisting of residues 14-1208 of SARS-CoV-2 S protein with proline substitutions at residues 986 and 987 and a “GSAS” substitution at residues 682 - 685 and a C-terminal T4 fibritin trimerization domain can be formulated in an 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)) 480 MB silk fibroin solution.

[0114] Without being bound by theory, the primary tunability of the implantable sustained- release tip is its crystallinity, measured via beta-sheet content (intermolecular and intramolecular P-sheet). This impacts the solubility of the silk tip matrix and the ability of antigen to be retained. With the increased P-sheet content, the tip also becomes more mechanically strong. Specific vaccine release profiles are achieved through modulation of the crystallinity and the diffusivity of the silk matrix. This is accomplished through both silk input material and formulation as well as post-treatment to increase crystallinity (e.g. water annealing, methanol/solvent annealing). In some embodiments, the implantable controlled- or sustained-release microneedle tip comprises a beta-sheet content of between about 10% and about 60% (e.g., about 10%, about 20%, about 30%, about 40%, about 50%, about 60%), e.g., as based on a “crystallinity index,” e.g., a “crystallinity index” known in the art. In some embodiments, the implantable controlled- or sustained-release microneedle tip can be formulated as a particle (e.g., a microparticle and/or a nanoparticle).

[0115] Table 1 Comparison of 4 Molecular Weight Groups (Typical Values)

Sum

71.67

Sum

80.17

Sum

79.55

Sum 82.57

[0116] As used herein, the term “surfactant” refers to any molecule having both a polar head group, which energetically prefers solvation by water, and a hydrophobic tail which is not well solvated by water. The term “cationic surfactant” refers to a surfactant with a cationic head group. The term “anionic surfactant” refers to a surfactant with an anionic head group. The term “nonionic surfactant: refers to a surfactant with uncharged head groups. The term “zwitterioninc surfactant refers to a surfactant with 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.

[0117] As used herein, the term “controlled- or sustained-release” refers to the release of a therapeutic agent (e.g., from a microneedle, microneedle device, formulation, composition, article, device, and preparation described herein, e.g., from a silk fibroin-based microneedle tip as described herein), such as a vaccine, antigen, and/or immunogen over a period of time, e.g., for at least about 1-14 days (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 or more days, e.g., between about 4 days and about 14 days, e.g., between about 1-2 weeks, about 1-3 weeks, or about 1-4 weeks). In some embodiments, the controlled- or sustained-release of a vaccine, e.g., over a time period of about 1 to about 14 days, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days, by a microneedle, microneedle device, formulation, composition, article, device, or preparation as described herein can result, e.g., in broad-spectrum immunity in a subject. In some embodiments, the vaccine formulations and preparations comprising silk fibroin have controlled- or sustained-release properties (e.g., are formulated and/or configured to release a vaccine, e.g., into the skin of the subject, over a period of, or at least 1, 5, 10, 15, 30, 45 minutes; a period of, or at least, 1, 2, 3, 4, 5, 10, 24 hours; a period of, or at least, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days; a period of, or at least, 1, 2, 3, 4, 5, 6, 7, 8 weeks; a period of, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 months; a period of, or at least, 1, 2, 3, 4, 5 years, or longer. [0118] As used herein, an “adjuvant” is a substance that is able to favor or amplify the cascade of immunological events, ultimately leading to an increased immunological response, e.g., the integrated bodily response to an antigen, including cellular and/or humoral immune responses. Non-limiting examples of adjuvants include: aluminum (e.g., aluminum gels and/or aluminum salts, such as aluminum hydroxide, aluminum phosphate, and aluminum potassium sulfate), lipids (e.g., squalene, monophosphoryl lipid A (MPL)), AS03 (e.g., an adjuvant comprising D,L- alpha-tocopherol (vitamin E), squalene, and polysorbate 80), AS04 (e.g., an adjuvant comprising a combination of aluminum hydroxide and MPL), and MF59® (e.g., an adjuvant comprising squalene).

[0119] As used herein, the term “immunity” or “protective immunity” refers to an immune response, e.g., a humoral and/or cellular response, elicited by a vaccine or immunization schedule (e.g., vaccination regimen) that when administered to a subject in need thereof (e.g., a subject described herein), that prevents, retards the development of, and/or reduces the severity of a viral infection that is caused by a virus described herein. In some embodiments, immunity or protective immunity diminishes or altogether eliminates the symptoms of the viral infection. In some embodiments, immunity or protective immunity is characterized by the presence of one or more of: circulating antibodies (e.g., humoral immunity), the presence of sensitized T lymphocytes (e.g., cellular immunity), the presence of secretory IgA on mucosal surfaces (e.g., mucosal immunity), or a combination thereof.

[0120] As used herein, the phrase “broad-spectrum immunity” refers to an immune response, e.g., a humoral and/or cellular response (e.g., immunity or protective immunity), against at least one (e.g., against at least two, at least three, at least four, at least five, or more) strains and/or variants of a virus (e.g., SARS-CoV-2), wherein the at least one strain and/or variant is not present in a vaccine administered to a subject, e.g., according to the methods, microneedles, and microneedle devices described herein.

[0121] As used herein, the term “dose” means the amount of a vaccine, antigen, and/or immunogen which is administered (e.g., in a vaccination) to elicit an immune response (e.g., a humoral and/or a cellular immune response) in an organism.

[0122] As used herein, a “standard dose” means the amount of antigen in a typical human dose of a vaccine, as approved for marketing by national or international regulatory authorities (e.g., U.S. FDA, EMEA).

[0123] As used herein, the term “immunogenic” refers to the ability of a substance, such as an antigen or epitope, to provoke humoral and/or cell-mediated immunological response in a subject. A skilled artisan can readily measure immunogenicity of a substance. The presence of a cell-mediated immunological response can be determined by any art-recognized methods, e.g., proliferation assays (CD4+ T cells), CTL (cytotoxic T lymphocyte) assays, or immunohistochemistry with tissue section of a subject to determine the presence of activated cells such as monocytes and macrophages after the administration of an immunogen. One of skill in the art can readily determine the presence of humoral-mediated immunological response in a subject by any well-established methods. For example, the level of antibodies produced in a biological sample such as blood can be measured by western blot, ELISA or other methods (e.g., pseudovirus-based neutralization assay) known for antibody detection.

[0124] As used herein, a “subject” refers to a human or animal. Usually the animal is a vertebrate such as a primate, rodent, domestic animal or game animal. Primates include chimpanzees, cynomolgus monkeys, spider monkeys, and macaques (e.g., Rhesus). Rodents include mice, rats, woodchucks, ferrets, rabbits and hamsters. Domestic and game animals include cows, horses, pigs, deer, bison, buffalo, feline species (e.g., domestic cat), canine species (e.g., dog, fox, wolf), avian species (e.g., chicken, emu, ostrich), and fish (e.g., trout, catfish and salmon). In certain embodiments of the aspects described herein, the subject is a mammal (e.g., a primate, e.g., a human). A subject 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 are not limited to these examples. In addition, the methods and formulations described herein can be used to treat domesticated animals and/or pets.

[0125] As used herein, the term “severe acute respiratory syndrome coronavirus 2 (SARS- CoV-2)” refers to the strains and variants of coronavirus that cause coronavirus disease 2019 (COVID-19). SARS-CoV-2 is a positive-sense single- stranded RNA virus that is a member of the genus Betacoronavirus of the family Coronavirinae . The RNA sequence of SARS-CoV-2 is approximately 30,000 bases in length. Each SARS-CoV-2 virion is 50-200 nanometres in diameter. Like other coronaviruses, SARS-CoV-2 has four structural proteins, known as the S (spike), E (envelope), M (membrane), and N (nucleocapsid) proteins. The N protein holds the RNA genome, and the S, E, and M proteins together create the viral envelope. The S protein is the protein responsible for allowing the virus to attach to and fuse with the membrane of a host cell.

[0126] As used herein, the term “spike protein,” “S polypeptide,” “S protein,” “SARS-CoV-2 spike,” or “SARS-CoV-2 S protein,” which can be used interchangeably, refers to a surface structure glycoprotein on SARS CoV-2 and is responsible for allowing the virus to attach to and fuse with the membrane of a host cell. Each monomer of trimeric S protein is about 180 kDa, and contains two subunits, SI and S2, mediating attachment and membrane fusion, respectively. Spike protein mainly enters human cells by binding to the receptor angiotensin converting enzyme 2 (ACE2).

[0127] Since the beginning of the CO VID-19 pandemic, mutants have been detected periodically. The emergence of variants that posed an increased risk to global public health prompted the characterization of specific Variants of Interest (VOIs), Variants of Concern (VOCs), and Variants Under Monitoring (VUMs), in order to prioritize global monitoring and research, and ultimately to inform the ongoing response to the COVID-19 pandemic

[0128] As used herein, the term “Variant of Concern (VoC)” refers to a SARS-CoV-2 variant that meets the definition of a VOI and, through a comparative assessment, has been demonstrated to be associated with one or more of the following changes at a degree of global public health significance: (i) increase in transmissibility or detrimental change in COVID-19 epidemiology; or (ii) increase in virulence or change in clinical disease presentation; or (iii) decrease in effectiveness of public health and social measures or available diagnostics, vaccines, therapeutics. Again, given the continuous evolution of the virus that leads to SARS-CoV-2 and the constant developments in people’s understanding of the impacts of variants, these definitions may be periodically adjusted. Currently (September, 2021) designated VoCs by WHO include Alpha variant, Beta variant, Gamma variant, and Delta variant.

(https://www.who.int/en/activities/tracking-SARS-CoV-2-va riants/).

[0129] 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. The amino acid sequence of S protein of SARS-CoV-2 Alpha variant has the following substitutions compared to the S protein of SARS-CoV-2 Wuhan-Hu-1 strain (wild type): 69del, 70del, 144del, (E484K*), (S494P*), N501 Y, A570D, D614G, P681H, T716I, S982A, D1118H (K1191N*)

(https://www.cdc. gov/coronavirus/2019-ncov/variants/vari ant-info.html) (https://www.acep.org/corona/covid-19-field-guide/characteri stics-of-covid-19-variants-and- mutants/characteristics-of-covid-19-variants-and-mutants/).

[0130] As used herein, the term “Beta variant” also known as lineage B.1.351 or 501Y.V2, refers to a variant of SARS-CoV-2, the virus that causes C0VID-19. The amino acid sequence of S protein of SARS-CoV-2 Beta variant has the following substitutions compared to the S protein of SARS-CoV-2 Wuhan-Hu- 1 strain (wild type): D80A, D215G, 241del, 242del, 243del, K417N, E484K, N501Y, D614G, A701V (https://www.cdc. ov/coronavirus/2019-ncov/variants/vari ant- info.html) (https://www.acep.org/corona/covid-19-field-guide/characteri stics-of-covid-19- variants-and-mutants/characteristics-of-covid-19-variants-an d-mutants/).

[0131] An “effective amount” or a “sufficient amount” of a substance is that amount sufficient to effect beneficial or desired results, including clinical results, and, as such, an “effective amount” depends upon the context in which it is being applied. In the context of administering an immunogenic composition or a vaccine, an effective amount contains sufficient SARS-CoV-2 S- 2P recombinant protein to elicit an immune response. An effective amount can be administered in one or more doses.

[0132] The microneedle vaccine against SARS-CoV-2 of the present invention can be produced by a method comprising the following steps: providing a mold including a mold body with an array of needle cavities having a predefined shape, e.g., pyramid- shaped and/or conical-shaped needle cavities, formed therein; providing an immunogenic recombinant protein substantially consists of residues 14-1208 of SARS-CoV-2 S protein with proline substitutions at residues 986 and 987 and a “GSAS” substitution at residues 682 - 685 and a C-terminal T4 fibritin trimerization domain; filling tips of the needle cavities with a composition consisting of a silk fibroin and the immunogenic recombinant protein; drying the filled tips of the needle cavities to create releasable tips, and optionally annealing the needle tips; filling the needle cavities of the mold with a dissolvable base solution; drying the dissolvable base solution to create base layers for the releasable tips; and applying a backing layer to the base layers to create a microneedle vaccine.

[0133] In some embodiments, the method further comprises a step of removing the microneedle vaccine from the mold.

[0134] In some embodiments, the microneedle vaccine is removed by bending the mold away from the microneedle vaccine.

[0135] In some embodiments, the method further comprises a step of packaging microneedle vaccine in a container with low moisture vapor transmission rate with a desiccant to maintain between about 0% and about 50% (e.g., between about 0% and 10%, between about 10% and about 20%, between about 20% and about 30%, between about 30% and about 40%, or between about 40% and 50%, e.g., about 25%) relative humidity inside the package.

[0136] In some embodiments, the silk fibroin, antigen solution is dispensed into each needle cavity in the mold via nanoliter printing.

[0137] In some embodiments, the step of filling the tips of the needle cavities includes dispensing a solution, e.g., an antigen-silk formulation into each needle cavity.

[0138] In some embodiments, the step of drying the filled tips of the needle cavities includes a primary drying step and a secondary drying step.

[0139] In some embodiments, the step of filling the needle cavities of the mold with a dissolvable base solution includes a solution of 40% (w/v) Hydrolyzed Gelatin and 10% (w/v) Sucrose in deionized water (DIW).

[0140] In some embodiments, the step of filling the dissolvable base solution includes subjecting the mold to a centrifuge at 3900 rpm for 2 minutes and topping off the needle cavities with 50 pL of base solution.

[0141] In some embodiments, the method further comprises a step of an annealing step (e.g., before filling the base) after the filling the tips of the needle cavities.

[0142] In some embodiments, the method further comprises a step of a water annealing step (e.g., before filling the base) after the filling the tips of the needle cavities.

[0143] In some embodiments, the backing layer includes one of a paper backing layer and an adhesive plastic tape.

[0144] The present invention is further illustrated by the following examples, which are provided for the purpose of demonstration rather than limitation. Those of skill in the art should, in light of the present disclosure, 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.

[0145] EXAMPLES

[0146] Example 1. Preparation of Immunogenic Compositions Against SARS-CoV-2

[0147] 1. Construct of S-2P recombinant protein derived from SARS-CoV-2 Wuhan strain (S-

2P).

[0148] A plasmid having a polynucleotide encoding the residues 14-1208 of SARS-CoV-2 S protein (Wuhan-Hu- 1 strain; GenBank: MN908947) with proline substitutions at residues 986 and 987, a “GSAS” substitution at the furin cleavage site (residues 682 - 685) (SEQ ID NO: 1) and a C-terminal T4 fibritin trimerization domain (SEQ ID NO: 2), an HRV3C protease cleavage site (SEQ ID NO: 3), an 8x His Tag, and a Twin-Strep Tag (SEQ ID NO: 4) was transfected into CHO cells obtained from ATCC.

[0149] Cell culture was harvested after 6 days, and protein was purified from the supernatant using Strep-Tactin resin (IB A Lifesciences, Gottingen, Germany). HRV3C protease (1%, w/w) was added to the protein and the reaction was incubated overnight at 4 °C. The digested protein was further purified using a Superose 6 16/70 column (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 further use.

[0150] 2. Controlled- or Sustained-Release Microneedle Formulation and Fabrication

[0151] The purified SARS-CoV-2 S-2P recombinant protein (S-2P) (SEQ ID NO: 5) was then mixed with silk fibroin (180 MB), Tween® 20, and Milli-Q deionized (DI) water to generated a 1% (w/v) silk fibroin, 0.5% (w/v) Tween® 20, and around 2.067 mg/ml (equals to 5 pg/microneedle patch) S-2P protein solution to be printed into microneedle molds. The microneedle mold is a mold for a microneedle patch having 121 needles in an 11 x 11 square grid with approximately 0.75 mm pitch, and the individual needles are cones approximately 0.65 mm long with base diameter approximately 0.35 mm and included angle of approximately 30°.

[0152] Tip Filling: 20 nL of formulation was printed using vision-guided dispensing (Biodot AD3420) into a polydimethylsiloxane (PDMS) microneedle mold.

[0153] Tip Fill Inspection: Printing was visually assessed under stereomicroscope for defects, including misaligned prints, incompletely filled needle cavities, and foreign debris.

[0154] Tip Dry: Filled microneedle molds were dried under controlled 20% RH conditions overnight (14-20 hours).

[0155] Tip Anneal: Dried tips were water annealed at 37 °C for 4 hours, through placement of molds in a vacuum desiccator filled with Milli-Q water, applying vacuum for 5 minutes, then closing vacuum valve and moving desiccator to 37 °C incubator.

[0156] Tip Dry: after annealing tips were again dried under controlled 20% RH conditions overnight (14-20 hours).

[0157] Base Filling: A solution of 40% (w/v) hydrolyzed gelatin (Gelita) and 10% (w/v) sucrose (Sigma-Aldrich) was pipetted onto microneedle molds and filled via centrifugation at 3,900 rpm for 2 minutes.

[0158] Base Fill Inspection: Base filling is also assessed visually by stereomicroscope for the appearance of needle cavities that were not entirely filled. Re-filling and re-centrifugation are performed if lack of fill is observed.

[0159] Base Drying: Base solution is dried under controlled 20% RH conditions overnight (14- 20 hours).

[0160] Backing Apply: Whatman 903 cards were punched into 12 mm discs and applied to pre-wetted (10 uL Milli-Q water) dried gelatin base.

[0161] Backing Dry: Devices were dried under controlled 20% RH conditions for 2 hours before demolding. [0162] Demolding: Devices were manually removed from microneedle molds by carefully bending the mold away from the device while holding device stationary.

[0163] Demold Inspect: Devices were inspected for complete demolding under stereomicroscope; incompletely demolded devices were discarded.

[0164] Example 2. Sustained Intradermal Delivery of SARS-CoV-2 S-2P Recombinant Protein (S-2P) Improves Antibody Responses

[0165] 1. Mouse Immunizations

[0166] The goal of S-2P microinjection study in this Example was to evaluate the immunogenicity of unadjuvanted SARS-CoV-2 S-2P recombinant protein (S-2P) (SEQ ID NO: 5 or 6). The immunogenicity test would be differentiated between intramuscular (IM) and intradermal (ID) delivery. Also, the humoral immune response of 10-day sustained ID delivery was evaluated by immunization with 1 mcg and 5 mcg of S-2P. The immunogenicity was tested in single and 2 doses of sustained release immunization in order to evaluate dose dependency.

[0167] Study design of this Example is shown in Table 2. Five (5) BALB/c mice were used in each group (n = 5). The first group was the control group without vaccination. Groups 2 and 3 were vaccinated twice with 1 mcg of S-2P per dose 28 days apart (Day 0 and Day 28) by ID administration. However, group 2 was administrated in bolus to compare with group 3, which was administrated in 10-day sustained ID delivery. Groups 4 and 5 were designed similarly to groups 2 and 3 along with dosage per shot escalated from 1 mcg to 5 mcg of S-2P. Group 6 was designed similarly to group 5, but was vaccinated once with 5 mcg of S-2P at day 0 (DO). Group 7 was vaccinated intramuscularly twice with 5 mcg of S-2P per dose 28 days apart (DO and D28). Group 7 was to compare the impact on immune response of IM delivery with that of ID delivery. The antisera were harvested on day 29 (D29), day 49 (D49), and day 64 (D64). Spike-specific IgG in each sample was analyzed by ELISA.

[0168] Table 2 Study design of Example 2

[0169] 2. Statistical analysis

[0170] Prism 6.01 (GraphPad Software Inc., San Diego, CA, USA) was used for statistical analysis. Multiple T-tests with Benjamini, Krieger and Yekutieli’s false discovery rate (FDR), one-way ANOVA with Tukey's multiple comparison test on log-transformed values, and repeated-measure two-way ANOVA with Tukey's multiple comparisons test on log-transformed values were used to calculate significance where appropriate. * p < 0.05, ** p < 0.01, *** p < 0.001.

[0171] 3. Results

[0172] The results are shown in Figures lA to 1C. Sustained ID delivery (Groups 3, 5, and 6), which released slowly S-2P antigen in 10 days, improves spike-specific IgG, compared with IM (Group 7) or ID bolus injection (Groups 2 and 4). As shown in Figure 1A, sustained delivery of S-2P protein (Groups 3, 5, 6) led to significantly higher antibody responses at all three timepoints for both 1 and 5 pg doses. As shown in Figure IB, after 2 immunizations, sustained intradermal delivery of S-2P protein (Group 5) results in antibody titers 30 times higher than traditional IM injection (Group 7) (p < 0.001). As shown in Figure 1C, a single sustained release immunization of S-2P protein (Group 6) results in antibody titers greater than 5 times higher than traditional IM injection (Group 7), at half the total dose (p < 0.001 orp < 0.05). Taken together, these results indicate that sustained intradermal delivery of a vaccine against SARS-CoV-2 results in stronger humoral responses than equivalent or double dose delivered by conventional intramuscular injections. [0173] Example 3. Sustained Intradermal Delivery of SARS-CoV-2 S-2P Recombinant Protein (S-2P) Improves Neutralization Antibody Responses against SARS-CoV-2 Variants

[0174] 1. Mouse Immunizations

[0175] The goal of S-2P microinjection study in this Example was to evaluate the immunogenicity of unadjuvanted S-2P protein (SEQ ID NO: 5 or 6). The immunogenicity test would be differentiated between intramuscular (IM) and intradermal (ID) delivery. Also, the humoral immune response of 10-day sustained ID delivery was evaluated by immunization with 1 mcg and 5 mcg of S-2P. The immunogenicity was tested in single and 2 doses of sustained release immunization in order to evaluate dose dependency.

[0176] Study design of this Example is shown in Table 3. Five (5) BALB/c mice were used in each group (n = 5). The first group was the control group without vaccination. Groups 2 and 3 were vaccinated twice with 1 mcg of S-2P per dose 28 days apart (DO and D28) by ID administration. However, group 2 was administrated in bolus to compare with group 3, which was administrated in 10-day sustained ID delivery. Groups 4 and 5 were designed similarly to groups 2 and 3 along with dosage per shot escalated from 1 mcg to 5 mcg of S-2P. Group 6 was designed similarly to group 5, but was vaccinated once with 5 mcg of S-2P at day 0 (DO). Group 7 was vaccinated intramuscularly twice with 5 mcg of S-2P per dose 28 days apart (DO and D28). Group 7 was to compare the impact on immune response of IM delivery with that of ID delivery. Blood samples were collected on day 28 (D28) and day 49 (D49). Spike-specific IgG in each sample was analyzed by ELISA. Neutralization antibodies in each sample were also test against different SARS-CoV-2 strains (Wuhan strain and D614G, B.l.1.7, and 501Y.V2 variants).

[0177] Table 3 Study design of Example 3

[0178] 2. Pseudovirus-based neutralization assay

[0179] a. Pseudovirus production and titration.

[0180] To produce SARS-CoV-2 pseudovirus, a plasmid expressing full-length SARS-CoV-2 spike protein was co-transfected into HEK293T cells with packaging and reporter plasmids pCMVA8.91 and pLAS2w.FLuc.Ppuro (RNAi Core, Academia Sinica), using TransIT-LTl transfection reagent (Minis Bio). The plasmid expresses the full-length SARS-CoV-2 spike protein of one of the following SARS-CoV-2 strains/variants: Wuhan-Hu-1 strain (wild-type; GenBank Accession No. MN908947), D614G (having the D614G substitution compared to the S protein of the wild-type), B.1.1.7 (Alpha variant, having the following substitutions compared to the S protein of the wild-type: 69del, 70del, 144del, (E484K*), (S494P*), N501Y, A570D, D614G, P681H, T716I, S982A, D1118H (K1191N*)), and 501Y.V2 (i.e., B.1.351, Beta variant, having the following substitutions compared to the S protein of the wild-type: D80A, D215G, 241del, 242del, 243del, K417N, E484K, N501Y, D614G, A701V). Mock pseudoviruses were produced by omitting the p2019-nCoV spike (WT). Seventy -two (72) hours post-transfection, supernatants were collected, filtered, and frozen at -80 °C. The transduction unit (TU) of SARS- CoV-2 pseudotyped lentivirus was estimated by using cell viability assay in response to the limited dilution of lentivirus. In brief, HEK-293 T cells stably expressing human ACE2 gene were plated on 96-well plate 1 day before lentivirus transduction. For the titration of pseudovirus, different amounts of pseudovirus were added into the culture medium containing polybrene. Spin infection was carried out at 1100 xg in 96-well plate for 30 minutes at 37 °C. After incubating cells at 37 °C for 16 hours, the culture media containing virus and polybrene were removed and replaced with fresh complete DMEM containing 2.5 pg/ml puromycin. After treating with puromycin for 48 hours, the culture media were removed and cell viability was detected by using 10% AlarmaBlue reagents according to manufacturer’s instruction. The survival rate of uninfected cells (without puromycin treatment) was set as 100%. The virus titer (transduction units) was determined by plotting the survival cells versus diluted viral dose. [0181] b. Pseudovirus-based neutralization assay

[0182] HEK293-hAce2 cells (2 x io ⁴ cells/well) were seeded in 96-well white isoplates and incubated for overnight. Sera were heated at 56 °C for 30 minutes to inactivate complement and diluted in MEM supplemented with 2% FBS at an initial dilution factor of 100, and then twofold serial dilutions were carried out (for a total of 8 dilution steps to a final dilution of 1 :25,600). The diluted sera were mixed with an equal volume of pseudovirus (1000 TU) and incubated at 37 °C for 1 hour before adding to the plates with cells. After the 1-hour incubation, the culture medium was replaced with 50 pL of fresh medium. On the following day, the culture medium was replaced with 100 pL of fresh medium. Cells were lysed at 72 hours post infections and relative luciferase units (RLU) were measured. The luciferase activity was detected by Tecan i-control (Infinite 500). The 50% inhibition dilution titers (ID50) were calculated considering uninfected cells as 100% neutralization and cells transduced with only virus as 0% neutralization. Reciprocal ID50 geometric mean titers (GMT) were determined as ID50 titer.

[0183] 3. Statistical analysis

[0184] Statistical analysis is the same as described in Example 2.

[0185] 4. Results

[0186] The results are shown in Figures 2 A to 2 J. Compared with IM (Group 7) or ID bolus injection (Groups 2 and 4), sustained ID delivery (Groups 3, 5, and 6), which released slowly S- 2P antigen in 10 days, improves spike-specific IgG (Figures 2A and 2B) and neutralizing antibody response (Figures 2C to 2J). At day 28, neutralizing titers of serum sample from vaccinated mice were observed only in sustained ID delivery (Groups 3 and 5) groups against the wildtype (Wuhan strain) (Figure 2C) and Variants of Concern (VoC), including D614G (Figure 2E), B. l.1.7 (Figure 2G) and 501Y.V2 (Figure 21). At day 49, strong neutralizing titers were generated in mice immunized with sustained ID delivery of 2 doses of S-2P against the wildtype and the three VoC tested (Groups 3 and 5) (Figures 2D, 2F, 2H, and 2J). Spike-specific IgG and neutralizing antibody response of the single dose sustained release group (Group 6) outperforms 2 bolus IM injections of unadjuvanted S-2P (Group 7) (Figures 2B, 2D, 2F, 2H). Taken together, these results indicate that sustained intradermal delivery of a vaccine against SARS-CoV-2 results in stronger neutralization antibody responses than equivalent or double dose delivered by conventional intramuscular injections. [0187] Example 4. Immunization Via Controlled- or Sustained-Release Silk Microneedles Improves Neutralization Antibody Responses against SARS-CoV-2 Variants

[0188] 1. Mouse Immunizations

[0189] The objectives of this study in Example 4 is to evaluate immunogenicity of the microneedle vaccine of the present invention compared with traditional IM injection, and to compare single and 2 doses ID to 2 doses IM regimen.

[0190] Study design of this Example is shown in Table 4. Five (5) BALB/c mice were used in each group. There was one animal death in group 4, but not deemed to be vaccine-related. Tips of the microneedle vaccine were formulated by 1% (w/v) silk fibroin and 0.5% (w/v) Tween® 20. The microneedle vaccine was stored at room temperature for 1 week prior to immunization. Group 1 was vaccinated intramuscularly twice with 5 mcg of S-2P per dose 28 days apart (DO and D28). The antigen used in group 2 was concentrated compared with that used in group 1, which was neat. Concentrated S-2P has been through a concentration procedure prior to the formulation, while neat S-2P remained unconcentrated. By comparing group 1 with group 2, the immunogenicity of S-2P antigen after concentration process could be confirmed. The S-2P antigen used in groups 3 and 4 was concentrated, and was delivered by microneedle patches. Dosages of groups 3 and 4 were 5 mcg per shot, the same as those of groups 1 and 2. However, the mice of group 3 were vaccinated once while those of group 4 were vaccinated twice. Blood samples were collected on day 28 (D28) and day 52 (D52). Spike-specific IgG in each sample was analyzed by ELISA. Neutralization antibodies in each sample were also test against different SARS-CoV-2 strains (Wuhan, D614G, B.l.1.7, and 501Y.V2). Statistical analysis and the method of pseudovirus-based neutralization assay are the same as described in Example 2 and Example 3, respectively.

[0191] Table 4 Study design of Example 4

* n = 4 due to one animal death (not deemed to be vaccine related).

[0192] 2. Results

[0193] The results are shown in Figures 3A to 3E. The result of ID delivery of unadjuvanted S-2P (Groups 3 and 4) shows stronger spike-specific IgG (Figure 3A) and neutralizing antibody responses (Figures 3B to 3E), compared with conventional IM administration (Groups 1 and 2). The result of two doses ID delivery of unadjuvanted S-2P shows enhanced neutralizing titers against the wildtype (Figure 3B) and the three VoC (Figures 3C to 3E) tested. Although singledose ID immunization leads to strong spike-specific IgG responses, neutralizing responses are only observed with 2-dose ID immunization. Taken together, these results indicate that controlled- or sustained-release silk microneedle delivery of a vaccine against SARS-CoV-2 results in stronger neutralization antibody responses than equivalent delivered by conventional intramuscular injections.

[0194] EQUIVALENTS

[0195] While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. 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 specifically herein. Such equivalents are intended to be encompassed in the scope of the appended claims.